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Sample records for bacteriophage-mediated hemocyte agglutination

  1. Partial inhibition of hemocyte agglutination by Lathyrus odoratus lectin in Crassotrea virginica infected with Perkinsus marinus

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    Thomas C. Cheng

    1995-06-01

    Full Text Available Quantitative determinations of agglutination of hemocytes from oysters, Crassostrea virginica, by the Lathyrus odoratus lectin at five concentrations revealed that clumping of hemocytes from oysters infected with Perkinsus marinus is partially inhibited. Although the nature of the hemocyte surface saccharide, which is not D(+-glucose, D(+mannose, or alpha-methyl-D-mannoside, remains to be determined, it may be concluded that this molecule also occurs on the surface of P. marinus. It has been demonstrated that the panning technique (Ford et al. 1990 is qualitatively as effective for determining the presence of P. marinus in C. virginica as the hemolymph assay method (Gauthier & Fisher 1990.

  2. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

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    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  3. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  4. Eicosanoids mediate insect hemocyte migration

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    Hemocyte chemotaxis toward infection and wound sites is an essential component of insect defense reactions, although the biochemical signal mechanisms responsible for mediating chemotaxis in insect cells are not well understood. Here we report on the outcomes of experiments designed to test the hyp...

  5. Spelling Correction in Agglutinative Languages

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    Oflazer, K

    1994-01-01

    This paper presents an approach to spelling correction in agglutinative languages that is based on two-level morphology and a dynamic programming based search algorithm. Spelling correction in agglutinative languages is significantly different than in languages like English. The concept of a word in such languages is much wider that the entries found in a dictionary, owing to {}~productive word formation by derivational and inflectional affixations. After an overview of certain issues and relevant mathematical preliminaries, we formally present the problem and our solution. We then present results from our experiments with spelling correction in Turkish, a Ural--Altaic agglutinative language. Our results indicate that we can find the intended correct word in 95\\% of the cases and offer it as the first candidate in 74\\% of the cases, when the edit distance is 1.

  6. Agglutination of Mouse Erythrocytes by Eperythrozoon coccoides

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    Iralu, Vichazelhu; Ganong, Kevin D.

    1983-01-01

    Erythrocytes from blood of mice infected with Eperythrozoon coccoides for 3 or 4 days agglutinated spontaneously. Washed E. coccoides particles agglutinated washed erythrocytes of uninfected mice. E. coccoides-mediated agglutination of normal mouse erythrocytes would be an excellent system for studies of bacterial adhesion.

  7. Agglutination of Mouse Erythrocytes by Eperythrozoon coccoides

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    Iralu, Vichazelhu; Ganong, Kevin D.

    1983-01-01

    Erythrocytes from blood of mice infected with Eperythrozoon coccoides for 3 or 4 days agglutinated spontaneously. Washed E. coccoides particles agglutinated washed erythrocytes of uninfected mice. E. coccoides-mediated agglutination of normal mouse erythrocytes would be an excellent system for studies of bacterial adhesion. Images PMID:6832825

  8. Agglutination of Mouse Erythrocytes by Eperythrozoon coccoides

    OpenAIRE

    Iralu, Vichazelhu; Ganong, Kevin D.

    1983-01-01

    Erythrocytes from blood of mice infected with Eperythrozoon coccoides for 3 or 4 days agglutinated spontaneously. Washed E. coccoides particles agglutinated washed erythrocytes of uninfected mice. E. coccoides-mediated agglutination of normal mouse erythrocytes would be an excellent system for studies of bacterial adhesion.

  9. Plasmatocyte spreading peptide influences hemocyte behavior via eicosanoids

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    Insect innate immunity is composed of humoral and hemocytic (or cellular) responses. Humoral responses involve induced biosynthesis of anti-microbial proteins and cellular responses are characterized by direct interactions between hemocytes (mainly granulocytes and plasmatocytes) and invading micro...

  10. Manufacturing High-Fidelity Lunar Agglutinate Simulants

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    Gutafson, R. J.; Edmunson, J. E.; Rickman, D. L.

    2010-01-01

    The lunar regolith is very different from many naturally occurring material on Earth because it forms in the unique, impact-dominated environment of the lunar surface. Lunar regolith is composed of five basic particle types: mineral fragments, pristine crystalline rock fragments, breccia fragments, glasses of various kinds, and agglutinates (glass-bonded aggregates). Agglutinates are abundant in the lunar regolith, especially in mature regoliths where they can be the dominant component.This presentation will discuss the technical feasibility of manufacturing-simulated agglutinate particles that match many of the unique properties of lunar agglutinates.

  11. High Fidelity, High Volume Agglutinate Manufacturing Process Project

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    National Aeronautics and Space Administration — Up to 65% of the lunar soils are comprised of agglutinates. Although the importance of agglutinate in simulants is often debated, the fact is that agglutinates...

  12. Agglutination of Helicobacter pylori coccoids by lectins

    Institute of Scientific and Technical Information of China (English)

    Mar Mar Khin; Jie Song Hua; Hah Cong Ng; Bow Ho; Torkel Wadstrorr

    2000-01-01

    AIM To study the agglutination pattern of Helicobacter pylori coccoid and spiral forms.METHODS Assays of agglutination and agglutination inhibition were applied using fifteen commercial lectins. RESULTS Strong agglutination was observed with mannose-specific Concanavalin A (Con A ),fucose-specific Tetragonolobus purpureas ( Lotus A ) and N-acetyl glucosamine-specific Triticum vulgaris (WGA) lectins. Mannose and fucose specific lectins were reactive with all strains of H. pylori coccoids as compared to the spirals. Specific carbohydrates, glycoproteins and mucin were shown to inhibit H. pylori lectin-agglutination reactions. Pre-treatment of the bacterial cells with formalin and sulphuric acid did not alter the agglutination patterns with lectins. However, sodium periodate treatment of bacterial cells were shown to inhibit agglutination reaction with Con A, Lotus A and WGA lectins. On the contrary, enzymatic treatment of coccoids and spirals did not show marked inhibition of H. pylori-lectin agglutination. Interestingly, heating of H.pylori cells at 60℃ for 1 hour was shown to augment the agglutination with all of the lectins tested. CONCLUSION The considerable differences in lectin agglutination patterns seen among the two differentiated forms of H. pylori might be attributable to the structural changes during theevents of morphological transformation,resulting in exposing or masking some of the sugar residues on the cell surface. Possibility of various sugar residues on the cell wall of the coccoids may allow them to bind to different carbohydrate receptors on gastric mucus and epithelial cells. The coccoids with adherence characteristics like the spirals could aid in the pathogenic process of Helicobacter infection.This may probably lead to different clinical outcome of H. pylori associated gastroduodenal disease.

  13. Activation of Drosophila hemocyte motility by the ecdysone hormone

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    Christopher J. Sampson

    2013-11-01

    Drosophila hemocytes compose the cellular arm of the fly's innate immune system. Plasmatocytes, putative homologues to mammalian macrophages, represent ∼95% of the migratory hemocyte population in circulation and are responsible for the phagocytosis of bacteria and apoptotic tissues that arise during metamorphosis. It is not known as to how hemocytes become activated from a sessile state in response to such infectious and developmental cues, although the hormone ecdysone has been suggested as the signal that shifts hemocyte behaviour from quiescent to migratory at metamorphosis. Here, we corroborate this hypothesis by showing the activation of hemocyte motility by ecdysone. We induce motile behaviour in larval hemocytes by culturing them with 20-hydroxyecdysone ex vivo. Moreover, we also determine that motile cell behaviour requires the ecdysone receptor complex and leads to asymmetrical redistribution of both actin and tubulin cytoskeleton.

  14. DNA & Protein detection based on microbead agglutination

    KAUST Repository

    Kodzius, Rimantas

    2012-06-06

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microparticles in the presence of a specific analyte thus enabling the macroscopic observation. Agglutination-based tests are most often used to explore the antibody-antigen reactions. Agglutination has been used for mode protein assays using a biotin/streptavidin two-component system, as well as a hybridization based two-component assay; however, as our work shows, two-component systems are prone to self-termination of the linking analyte and thus have a lower sensitivity. Three component systems have also been used with DNA hybridization, as in our work; however, their assay requires 48 hours for incubation, while our assay is performed in 5 minutes making it a real candidate for POC testing. We demonstrate three assays: a two-component biotin/streptavidin assay, a three-component hybridization assay using single stranded DNA (ssDNA) molecules and a stepped three-component hybridization assay. The comparison of these three assays shows our simple stepped three-component agglutination assay to be rapid at room temperature and more sensitive than the two-component version by an order of magnitude. An agglutination assay was also performed in a PDMS microfluidic chip where agglutinated beads were trapped by filter columns for easy observation. We developed a rapid (5 minute) room temperature assay, which is based on microbead agglutination. Our three-component assay solves the linker self-termination issue allowing an order of magnitude increase in sensitivity over two–component assays. Our stepped version of the three-component assay solves the issue with probe site saturation thus enabling a wider range of detection. Detection of the agglutinated beads with the naked eye by trapping in microfluidic channels has been shown.

  15. Honey bee hemocyte profiling by flow cytometry.

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    Marringa, William J; Krueger, Michael J; Burritt, Nancy L; Burritt, James B

    2014-01-01

    Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure.

  16. [Latex agglutination test in amebic liver abscess].

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    Gómez Maganda y Silva, T; García Carrizosa, R; Torres Valadez, F; Ortiz Ramírez, E; Villaseñor de la Parra, C; Flores González, A; Gómez García, E

    1978-01-01

    Amebic hepatic abscesses are one of the most frequent and serious complications of intestinal amibiasis. Although many methods exists with which the diagnosis can be made, frequently problems do arise. Serologic reactions play an important role in the diagnosis of amebic hepatic abscess. Among the most useful of the serological tests, is that which evaluates agglutination with latex particles. Latex agglutination was positive in 98.5% of 200 cases of proved amebic hepatic abscess. The pros and cons of the utility of this test compared with other serological tests are discussed. It is concluded that or the especialist as well as the general practicioner latex agglutination can be extremely useful in the diagnosis of amebic hepatic abscess.

  17. Process to create simulated lunar agglutinate particles

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    Gustafson, Robert J. (Inventor); Gustafson, Marty A. (Inventor); White, Brant C. (Inventor)

    2011-01-01

    A method of creating simulated agglutinate particles by applying a heat source sufficient to partially melt a raw material is provided. The raw material is preferably any lunar soil simulant, crushed mineral, mixture of crushed minerals, or similar material, and the heat source creates localized heating of the raw material.

  18. On the origin of the Biomphalaria glabrata hemocytes

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    Samaly dos Santos Souza

    2006-10-01

    Full Text Available A histologic, morphometric and ultrastructural study performed on Biomphalaria glabrata submitted to infection with Schistosoma mansoni miracidia failed to provide significant evidences that the so-called amebocyte-producing organ (APO is really the central organ for hemocyte production. In infected snails no general reactive changes appeared in the APO, the mitoses were seen only occasionally, and the possibility of cellular hyperplasia was ruled out by morphometric measurements. Under the electron microscope the APO cells presented an essentially epithelial structure, without features indicative of transition toward hemocytes. On the other hand, the present findings pointed to a multicentric origin for the mollusck hemocytes, as earlier studies had indicated. Dense foci of hemocyte collections appeared sometimes around disintegrating sporocysts and cercariae in several organs and tissues of the infected snails, including a curious accumulation of such cells inside the ventricular cavity of the heart. In the heart and other sites, features suggestive of transformation of vascular space endothelial lining cells into hemocytes were apparent. To some extent, the postulated multicentric origin for B. glabrata hemocytes recapitulates earlier embryologic findings in vertebrates, when mesenchymal vascular spaces generate the circulating and phagocytic blood cells.

  19. Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors.

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    Qi, Yi-Xiang; Huang, Jia; Li, Meng-Qi; Wu, Ya-Su; Xia, Ren-Ying; Ye, Gong-Yin

    2016-03-14

    Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. We report that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B-null Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution.

  20. Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti.

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    Castillo, J C; Robertson, A E; Strand, M R

    2006-12-01

    Hemocytes are an essential component of the mosquito immune system but current knowledge of the types of hemocytes mosquitoes produce, their relative abundance, and their functions is limited. Addressing these issues requires improved methods for collecting and maintaining mosquito hemocytes in vitro, and comparative data that address whether important vector species produce similar or different hemocyte types. Toward this end, we conducted a comparative study with Anopheles gambiae and Aedes aegypti. Collection method greatly affected the number of hemocytes and contaminants obtained from adult females of each species. Using a collection method called high injection/recovery, we concluded that hemolymph from An. gambiae and Ae. aegypti adult females contains three hemocyte types (granulocytes, oenocytoids and prohemocytes) that were distinguished from one another by a combination of morphological and functional markers. Significantly more hemocytes were recovered from An. gambiae females than Ae. aegypti. However, granulocytes were the most abundant cell type in both species while oenocytoids and prohemocytes comprised less than 10% of the total hemocyte population. The same hemocyte types were collected from larvae, pupae and adult males albeit the absolute number and proportion of each hemocyte type differed from adult females. The number of hemocytes recovered from sugar fed females declined with age but blood feeding transiently increased hemocyte abundance. Two antibodies tested as potential hemocyte markers (anti-PP06 and anti-Dox-A2) also exhibited alterations in staining patterns following immune challenge with the bacterium Escherichia coli.

  1. Characterization of hemocytes from the yellow fever mosquito, Aedes aegypti.

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    Hillyer, Julián F; Christensen, Bruce M

    2002-05-01

    Mosquitoes are the most important arthropod disease vectors, transmitting a broad range of pathogens that cause diseases such as malaria, lymphatic filariasis, and yellow fever. Mosquitoes and other insects are able to mount powerful cellular and humoral immune responses against invading pathogens. To date, most studies have concentrated on the humoral response. In the current study we describe the hemocytes (blood cells) of the yellow fever mosquito, Aedes aegypti, by means of morphology, lectin binding, and enzyme activity and immunocytochemistry. Our light and electron microscopic studies suggest the presence of four distinct hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. We believe granulocytes and oenocytoids are true circulating hemocytes, but adipohemocytes and thrombocytoids are likely adhered to fixed tissues. Granulocytes, the most abundant cell type, have acid phosphatase and alpha-naphthyl acetate esterase activity, and bind the exogenous lectins WGA, HPA, and GNL. Phenoloxidase, an essential enzyme in the melanotic encapsulation immune response, was detected inside oenocytoids. This is, to our knowledge, the first report that has detected phenoloxidase inside mosquito hemocytes at the ultrastructural level. These results have begun to form a knowledge base for our ongoing studies on the function of Ae. aegypti hemocytes, and their involvement in controlling infections.

  2. Morphology and classification of hemocytes in Pinctada fucata and their responses to ocean acidification and warming.

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    Li, Shiguo; Liu, Yangjia; Liu, Chuang; Huang, Jingliang; Zheng, Guilan; Xie, Liping; Zhang, Rongqing

    2015-07-01

    Hemocytes play important roles in the innate immune response and biomineralization of bivalve mollusks. However, the hemocytes in pearl oysters are poorly understood. In the present study, we investigated the morphology and classification of hemocytes in the pearl oyster, Pinctada fucata. Three types of hemocytes were successfully obtained by light microscopy, electron microscopy and flow cytometry methods: small hyalinocytes, large hyalinocytes and granulocytes. The small hyalinocytes are the major hemocyte population. Morphological analyses indicated that these hemocytes have species-specific characterizations. In addition, we assessed the potential effects of ocean acidification (OA) and ocean warming (OW) on the immune parameters and calcium homeostasis of the hemocytes. OA and OW (31 °C) altered pH value of hemolymph, increased the total hemocyte count, total protein content, and percentage of large hyalinocytes and granulocytes, while it decreased the neutral red uptake ability, suggesting active stress responses of P. fucata to these stressors. Exposure to OW (25 °C) resulted in no significant differences, indicating an excellent immune defense to heat stress at this level. The outflow of calcium from hemocytes to hemolymph was also determined, implying the potential impact of OA and OW on hemocyte-mediated biomineralization. This study, therefore, provides insight into the classification and characterization of hemocyte in the pearl oyster, P. fucata, and also reveals the immune responses of hemocytes to OA and OW, which are helpful for a comprehensive understanding of the effects of global climate change on pearl oysters.

  3. Mass-based readout for agglutination assays

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    Chunara, Rumi; Godin, Michel; Knudsen, Scott M.; Manalis, Scott R.

    2007-11-01

    We present a mass-based readout for agglutination assays. The suspended microchannel resonator (SMR) is used to classify monomers and dimers that are formed during early stage aggregation, and to relate the total count to the analyte concentration. Using a model system of streptavidin functionalized microspheres and biotinylated antibody as the analyte, we obtain a dose-response curve over a concentration range of 0.63-630nM and show that the results are comparable to what has been previously achieved by image analysis and conventional flow cytometry.

  4. Penicillamine prevents ram sperm agglutination in media that support capacitation.

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    Leahy, T; Rickard, J P; Aitken, R J; de Graaf, S P

    2016-02-01

    Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7 ± 2.7% to 2.8 ± 1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

  5. Comparison of latex agglutination and co-agglutination for the diagnosis and prognosis of cryptococcal meningitis

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    Khyriem A

    2003-01-01

    Full Text Available PURPOSE: To compare a commercially available Latex agglutination test and an in house co-agglutination test for the detection of cryptococcal antigen in cases of chronic meningitis. METHODS: One hundred and fifty cerebrospinal fluid (CSF samples from 150 cases of chronic meningitis were tested for the presence of Cryptococcus neoformans by modified India ink, culture and antigen detection by latex agglutination test (LAT and co-agglutination (Co-A test. RESULTS: Thirty-nine cases were positive by one or more tests employed. Antigen detection in CSF by LAT and Co-A was found to be most sensitive (94.9% while culture was the least (25.6%. Of the two antigen detection methods, Co-A was found to be more sensitive than the LAT, the difference being statistically significant. Initial CSF antigen titres did not have any prognostic significance. CONCLUSIONS: Co-A for antigen detection is an inexpensive and useful adjunct to direct microscopy and culture for the diagnosis of cryptococcal meningitis, though its usefulness in prognosis needs to be evaluated further.

  6. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas

    2013-10-22

    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

  7. Classification of Homarus americanus hemocytes and the use of differential hemocyte counts in lobsters infected with Aerococcus viridans var. homari (Gaffkemia).

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    Battison, Andrea; Cawthorn, Richard; Horney, Barbara

    2003-11-01

    Hemocytes of the American lobster (Homarus americanus H. Milne Edwards) were classified after examination of Wright-Giemsa stained cytocentrifuge preparations by brightfield light microscopy. Eleven hemocyte types were identified using morphologic criteria. The classification system was then used to monitor changes in the differential hemocyte count (DHC) of lobsters infected with the Gram positive coccus Aerococcus viridans var. homari, etiologic agent of gaffkemia. The appearance of less mature hemocytes in the DHCs of lobsters in the late stages of infection was similar to the 'left shift' of vertebrate inflammation. Results from this study suggest that DHCs can be used to assess and characterize inflammation in H. americanus and possibly other crustaceans.

  8. Hemocyte-hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit.

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    Lapointe, Jason F; Dunphy, Gary B; Mandato, Craig A

    2012-01-01

    Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell-bacteria adhesion and hemocyte-hemocyte adhesion (microaggregation). Hemocyte-microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte-hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2 nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6-120 nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2-120 nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2-6 nM) cause reduced hemocyte adhesion, while high levels (12-120 nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6-120 nM) and in vivo (12-120 nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the hemocytes, suggesting

  9. Effects of Destruxin A on Hemocytes Morphology of Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    FAN Ji-qiao; CHEN Xiu-run; HU Qiong-bo

    2013-01-01

    Destruxin A (DA), a kind of cyclo-hexadepsipeptide isolated from entomopathogenic fungus, Metarhizium anisopliae, is an inhibitor of insect’s immunity. But its mechanism has not been clarified yet. In this study, the effects of DA on morphologic changes of in vivo and in vitro hemocytes of silkworm, Bombyx mori, were investigated by means of inverted phase contrast microscopy (IPCM), fluorescence microscopy (FCM) and environmental scanning electron microscopy (ESEM). The results indicated that DA was cytotoxic to granulohemocytes (GR) and plasmatocytes (PL). The LC50 values of DA against in vitro GR and PL of silkworm were 68.77 and 84.11μg mL-1, respectively. However, the hemocytes in vivo were more susceptible to DA, although at the extremely low dose of 10μL of 12.5μg mL-1 for each insect (i.e., 0.036μg g-1 body weight, or approximately 0.25μg mL-1 hemolymph), DA could induce obviously morphologic alterations of hemocytes in vivo. The results imply that there might be some factors in silkworm’s hemolymph, which influence the interaction of DA and hemocytes.

  10. Systems, devices, and methods for agglutination assays using sedimentation

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    Schaff, Ulrich Y.; Sommer, Gregory J.; Singh, Anup K.

    2016-01-26

    Embodiments of the present invention include methods for conducting agglutination assays using sedimentation. Aggregates may be exposed to sedimentation forces and travel through a density medium to a detection area. Microfluidic devices, such as microfluidic disks, are described for conducting the agglutination assays, as are systems for conducting the assays.

  11. High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans.

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    Cerenius, Lage; Liu, Haipeng; Zhang, Yanjiao; Rimphanitchayakit, Vichien; Tassanakajon, Anchalee; Gunnar Andersson, M; Söderhäll, Kenneth; Söderhäll, Irene

    2010-01-01

    Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.

  12. Serotype assignment by sero-agglutination, ELISA, and PCR

    Science.gov (United States)

    For assessing isolates of Listeria monocytogenes serotype designation is the foremost subtyping method used. Traditionally serotyping has been done with agglutination reactions. In the last decade alternative serotyping methods were described using Enzyme Linked Immunosorbent Assay(ELISA)and Polymer...

  13. Surgical management of vulvovaginal agglutination due to lichen planus.

    Science.gov (United States)

    Fairchild, Pamela S; Haefner, Hope K

    2016-02-01

    Lichen planus is a rare dermatological disorder that is often associated with painful and disfiguring vulvovaginal effects. At the University of Michigan Center for Vulvar Diseases, we see many women with vulvovaginal lichen planus each year, with marked scarring and vulvovaginal agglutination that precludes vaginal intercourse and causes difficulty with urination. Through our experience, we developed a protocol for the operative management and postoperative care for severe vulvovaginal agglutination. Our objective is to share this protocol with a wider audience so that providers who see patients with these devastating effects of lichen planus can benefit from our experience to better serve this patient population. The figure represents a case of erosive lichen planus with early vaginal agglutination. The video reviews the pathophysiology and presentation of lichen planus. We then present a case of scarring and agglutination in a young woman, including our surgical management and postoperative care recommendations.

  14. Detection of Legionella antigenuria by reverse passive agglutination.

    OpenAIRE

    Tang, P W; Savigny, D. de; Toma, S

    1982-01-01

    A reverse passive agglutination method was developed to detect soluble antigens of Legionella spp. By this method Legionella antigens were detected in urine specimens from 14 of 15 antigenuric patients with clinically diagnosed Legionnaires disease and in none of 263 urine samples from healthy subjects or patients with urinary tract infections. Intra-genus cross-reactivity was observed only between L. pneumophila serogroups 2, 3, and 6. The Legionella reverse passive agglutination method was ...

  15. Antibody blocks acquisition of bacterial colonization through agglutination.

    Science.gov (United States)

    Roche, A M; Richard, A L; Rahkola, J T; Janoff, E N; Weiser, J N

    2015-01-01

    Invasive infection often begins with asymptomatic colonization of mucosal surfaces. A murine model of bacterial colonization with Streptococcus pneumoniae was used to study the mechanism for mucosal protection by immunoglobulin. In previously colonized immune mice, bacteria were rapidly sequestered within large aggregates in the nasal lumen. To further examine the role of bacterial agglutination in protection by specific antibodies, mice were passively immunized with immunoglobulin G (IgG) purified from antipneumococcal sera or pneumococcal type-specific monoclonal human IgA (hIgA1 or hIgA2). Systemically delivered IgG accessed the mucosal surface and blocked acquisition of colonization and transmission between littermates. Optimal protection by IgG was independent of Fc fragment and complement and, therefore, did not involve an opsonophagocytic mechanism. Enzymatic digestion or reduction of IgG before administration showed that protection required divalent binding that maintained its agglutinating effect. Divalent hIgA1 is cleaved by the pneumococcal member of a family of bacterial proteases that generate monovalent Fabα fragments. Thus, passive immunization with hIgA1 blocked colonization by an IgA1-protease-deficient mutant (agglutinated) but not the protease-producing wild-type parent (not agglutinated), whereas protease-resistant hIgA2 agglutinated and blocked colonization by both. Our findings highlight the importance of agglutinating antibodies in mucosal defense and reveal how successful pathogens evade this effect.

  16. Effects of Plant Lectins on Human Erythrocyte Agglutination

    Directory of Open Access Journals (Sweden)

    Zubcevic Nadja

    2016-09-01

    Full Text Available Plant lectins are carbohydrate binding proteins or phytohaemagglutinins present in most plants, especially seeds and tubers, which include cereals, potatoes and beans. Lectins have great significance in the diet because of their involvement in gastrointestinal difficulties and erythrocyte agglutination. Blood agglutination activity against A, B, AB and O groups was shown after exposing blood to extracts obtained from 55% of tested plants, while in 45% of plants, agglutination was absent. The results of our study have shown that in humans, 40% of plant extracts exhibited activity against A, 40% of plant extracts exhibited activity against B, and 50% of plant extracts exhibited activity against AB and O groups in humans. The concentration of plant lectins depends on the part of the plant. Lectins from the seeds of certain plants cause the greatest percentage of erythrocyte agglutination, while the lowest agglutination was caused by plant bulbs and leaves. However, lectins derived from all plant species of the family Fabaceae agglutinated erythrocytes of all blood types to some extent.

  17. Demonstration of expression of a neuropeptide-encoding gene in crustacean hemocytes.

    Science.gov (United States)

    Wu, Su-Hua; Chen, Yan-Jhou; Huang, Shao-Yen; Tsai, Wei-Shiun; Wu, Hsin-Ju; Hsu, Tsan-Ting; Lee, Chi-Ying

    2012-04-01

    Crustacean hyperglycemic hormone (CHH) was originally identified in a neuroendocrine system-the X-organ/sinus gland complex. In this study, a cDNA (Prc-CHH) encoding CHH precursor was cloned from the hemocyte of the crayfish Procambarus clarkii. Analysis of tissues by a CHH-specific enzyme-linked immunosorbent assay (ELISA) confirmed the presence of CHH in hemocytes, the levels of which were much lower than those in the sinus gland, but 2 to 10 times higher than those in the thoracic and cerebral ganglia. Total hemocytes were separated by density gradient centrifugation into layers of hyaline cell (HC), semi-granular cell (SGC), and granular cell (GC). Analysis of extracts of each layer using ELISA revealed that CHH is present in GCs (202.8±86.7 fmol/mg protein) and SGCs (497.8±49.4 fmol/mg protein), but not in HCs. Finally, CHH stimulated the membrane-bound guanylyl cyclase (GC) activity of hemocytes in a dose-dependent manner. These data for the first time confirm that a crustacean neuropeptide-encoding gene is expressed in cells essential for immunity and its expression in hemocytes is cell type-specific. Effect of CHH on the membrane-bound GC activity of hemocyte suggests that hemocyte is a target site of CHH. Possible functions of the hemocyte-derived CHH are discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. FACTORS INFLUENCING IN VITRO KILLING OF BACTERIA BY HEMOCYTES OF THE EASTERN OYSTER (CRASSOSTREA VIRGINICA)

    Science.gov (United States)

    A tetrazolium dye reduction assay was used to study factors governing killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains...

  19. Modulation by Biogenic Amines for the Hemocyte Count and Prophenoloxidase Exocytosis via Receptors in Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    PAN Luqing; LIU Huijie; YU Jinhong

    2011-01-01

    Hemocyte counts and phenoloxidase (PO) activity were examined after hemolymph being incubated in dopamine (DA),noradrenaline (NE) and serotonin (5-HT).Results showed that all the three biogenic amines (BAs) had a significant impact on total hemocyte count (THC),differential hemocyte count (DHC),and intracelluar and extracelluar phenoloxidase (PO) activity.Among these Bas,DA had the strongest effect on the above parameters,whereas 5-HT had the least effect.Preincubation with D1 receptor antagonist SCH23390,D2 receptor antagonist Sulpiride and 1∶1 admixture of the two could significantly inhibit the effect of DA on these parameters.SCH23390 showed a stronger inhibitory effect than Sulpiride,and the admixture exhibited the strongest effect.These results suggested that the change of hemocyte count and activation of prophenoloxidase (proPO) system in Litopenaeusvannamei hemocyte can be regulated by BAs,and DA modulates the two parameters via its receptors.

  20. Stress-based modulation of the immune response in molluscan hemocytes: a two-receptor model

    Directory of Open Access Journals (Sweden)

    R Barcia

    2011-03-01

    Full Text Available In molluscs, hemocytes perform the molecular mechanisms related to immunity. These cells have the ability to respond to the different varieties of stress by modulating their responses. The stressors may be bacterial toxins, cytokines or growth factors, and even physical agents such as changes in temperature or oxygen partial pressure. In the first place, hemocytes synthesise catecholamines, which, in turn, modify the immune response in terms of phagocytosis or nitric oxide synthesis. According to studies on the hemocytes of the mussel Mytilus galloprovincialis, we propose a model for a sequential action where the IL-2 receptor and its wide agonist specificity play an important role. Also, α and β-adrenergic receptors suggest the functioning of a return-to-hemocyte mechanism. The model is proposed taking into account the possible relationship between the pathways mediated by cAMP-activated protein kinase and protein kinase C in hemocytes.

  1. Capsular Serotyping of Streptococcus pneumoniae by latex agglutination.

    Science.gov (United States)

    Porter, Barbara D; Ortika, Belinda D; Satzke, Catherine

    2014-09-25

    Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the 'gold standard' Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. This method of production and quality control may also be suitable for other testing purposes.

  2. Reactive oxygen species in unstimulated hemocytes of the pacific oyster Crassostrea gigas: a mitochondrial involvement.

    Directory of Open Access Journals (Sweden)

    Ludovic Donaghy

    Full Text Available The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster's hemocytes have been reported to produce reactive oxygen species (ROS, even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψ(m of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψ(m in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor had no effect on ROS production. Incubation with antimycin A (complex III inhibitor resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψ(m and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.

  3. Involvement of Pacific oyster CgPGRP-S1S in bacterial recognition, agglutination and granulocyte degranulation.

    Science.gov (United States)

    Iizuka, Masao; Nagasaki, Toshihiro; Takahashi, Keisuke G; Osada, Makoto; Itoh, Naoki

    2014-03-01

    Peptidoglycan recognition protein (PGRP) recognizes invading bacteria through their peptidoglycans (PGN), a component of the bacterial cell wall. Insect PGRPs contribute to effective immune systems as inducers of other host defense responses, while this function has not been reported from PGRP of bivalves. In this study, recombinant CgPGRP-S1S (rCgPGRP-S1S), produced in the mantle and the gill, was synthesized and used to elucidate the immunological function of CgPGRP-S1S. rCgPGRP-S1S bound specifically to DAP-type PGN and to Escherichia coli cells, but not to other DAP-type PGN-containing bacterial species, Vibrio anguillarum, or Bacillus subtilis. Antibacterial activity was not detected, but E. coli cells were agglutinated. Moreover, in addition to these direct interactions with bacterial cells, rCgPGRP-S1S induced secretion of granular contents by hemocyte degranulation. Taken together, these results suggest for the first time that a PGRP of bivalves is, just as in insects, involved in host defense, not only by direct interaction with bacteria, but also by triggering other defense pathways.

  4. Evolution of Shock Melt Compositions in Lunar Agglutinates

    Science.gov (United States)

    Vance, A. M.; Christoffersen, R.; Keller, L. P.

    2015-01-01

    Lunar agglutinates are aggregates of regolith grains fused together in a glassy matrix of shock melt produced during smaller-scale (mostly micrometeorite) impacts. Agglutinate formation is a key space weathering process under which the optically-active component of nanophase metallic Fe (npFe(sup 0)) is added to the lunar regolith. Here we have used energy-dispersive X-ray (EDX) compositional spectrum imaging in the SEM to quantify the chemical homogeneity of agglutinitic glass, correlate its homogeneity to its parent soil maturity, and identify the principle chemical components contributing to the shock melt compositional variations.

  5. Detection of Legionella antigenuria by reverse passive agglutination.

    Science.gov (United States)

    Tang, P W; de Savigny, D; Toma, S

    1982-06-01

    A reverse passive agglutination method was developed to detect soluble antigens of Legionella spp. By this method Legionella antigens were detected in urine specimens from 14 of 15 antigenuric patients with clinically diagnosed Legionnaires disease and in none of 263 urine samples from healthy subjects or patients with urinary tract infections. Intra-genus cross-reactivity was observed only between L. pneumophila serogroups 2, 3, and 6. The Legionella reverse passive agglutination method was also evaluated with reference to reagent concentrations, test conditions, and subjectivity of reading test results. The method is rapid and does not require special equipment.

  6. Innate hemocyte responses of Malacosoma disstria larvae (C. Insecta) to antigens are modulated by intracellular cyclic AMP.

    Science.gov (United States)

    Gulii, Vladislav; Dunphy, Gary B; Mandato, Craig A

    2009-08-01

    Invertebrate intracellular hemocyte signaling pathways affecting cellular-antigen responses, although defined for molluscs and some arthropods including dipteran insects, is less known for lepidopterans. Hemocytic-antigen responses of the arboreal pest lepidopteran Malacosoma disstria are linked to cAMP-dependent protein kinase A implicating cAMP in cellular hemocyte immune responses. The purpose in the present study was to determine intracellular cAMP effects on larval M. disstria hemocytes adhering to slides and bacteria. Altering adenylate cyclase and phosphodiesterase activities as well as cAMP levels in vitro and in vivo changed hemocyte responses to antigens. Quiescent hemocytes had high cAMP levels due to adenylate cyclase activity and possibly low phosphodiesterase (type 4) activity. Antigen contact diminished hemocytic cAMP levels. Inhibiting adenylate cyclase increased hemocyte-antigen and hemocyte-hemocyte adhesion, the latter producing nodules in vivo without bacterial antigens. Inhibiting phosphodiesterase type 4 produced the reverse effects. Pharmacologically increasing intracellular cAMP in attached hemocytes caused many of the cells to detach. Diminished intracellular cAMP changed hemograms in vivo in bacteria-free larvae comparable to changes induced by the bacterium, Bacillus subtilis, by producing nodules. Lowering cAMP enhanced also the removal of Xenorhabdus nematophila and B. subtilisin vivo.

  7. The relative abundance of hemocyte types in a polyphagous moth larva depends on diet.

    Science.gov (United States)

    Vogelweith, Fanny; Moret, Yannick; Monceau, Karine; Thiéry, Denis; Moreau, Jérôme

    2016-05-01

    Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae's investment into the different types of hemocytes.

  8. Hemocyte types and total and differential counts in unparasitized and parasitized Anastrepha obliqua (Diptera, Tephritidae larvae

    Directory of Open Access Journals (Sweden)

    J. E. B. SILVA

    Full Text Available The hemocyte types, in addition to total and differential hemocyte counts were studied in parasitized and unparasitized Anastrepha obliqua larvae at the beginning and at the end of the third instar. In both developmental phases, in parasitized and unparasitized larvae, prohemocytes, plasmatocytes, granulocytes, adipohemocytes, spherulocytes and oenocytoids cells were observed. Mitotic figures indicate prohemocytes as stem cells. Prohemocytes, plasmatocytes and granulocytes are the most numerous cells in the hemolymph of A. obliqua. Difference in the total number of hemocytes was observed between unparasitized and parasitized larvae at the end of the third instar, but not at the beginning.

  9. Synthesis for Lunar Simulants: Glass, Agglutinate, Plagioclase, Breccia

    Science.gov (United States)

    Weinstein, Michael; Wilson, Stephen A.; Rickman, Douglas L.; Stoeser, Douglas

    2012-01-01

    The video describes a process for making glass for lunar regolith simulants that was developed from a patented glass-producing technology. Glass composition can be matched to simulant design and specification. Production of glass, pseudo agglutinates, plagioclase, and breccias is demonstrated. The system is capable of producing hundreds of kilograms of high quality glass and simulants per day.

  10. Rapid identification of Mycobacterium species by lectin agglutination.

    Science.gov (United States)

    Athamna, Abed; Cohen, Dani; Athamna, Muhammad; Ofek, Itzhak; Stavri, Henriette

    2006-05-01

    The purpose of the present study is to explore the possibility that plant lectins can be used for the development of rapid and inexpensive technique for differentiation of mycobacterial species. The method is based on interaction between mycobacteria and lectins as visualized by agglutination in a microtiter plate. We employed 18 mycobacterium species and determined the minimal lectin concentration (MLC) of 23 different lectins. For some of the bacteria as a high as 1000 microg/ml of one or more lectins were required to induce agglutination, while for other strains as low as 1.95 microg/ml of the lectin were needed. A unique pattern of agglutination was observed for each species over a range of 62-1000 microg/ml lectin concentrations. There were little or no variations in MLC within strains (intraspecies) of each of two species tested. In contrast, there were marked interspecies variations in MLC. Analysis of the MLC showed that the highest score of interspecies differences with 23 lectins was obtained at 125 microg/ml lectin concentration. At this concentration it was found that the pattern of agglutinations with only two lectins was sufficient to differentiate mycobacterium species from each other. Because the bacteria-lectin interaction is adaptable to various methods of visualization, our findings may set the stage for developing a rapid and reliable tool to differentiate mycobacterium species.

  11. Multivalent dendritic molecules as broad spectrum bacteria agglutination agents.

    Science.gov (United States)

    Xiao, Shuzhang; Abu-Esba, Lica; Turkyilmaz, Serhan; White, Alexander G; Smith, Bradley D

    2013-01-01

    This study reports the first set of synthetic molecules that act as broad spectrum agglutination agents and thus are complementary to the specific targeting of antibodies. The molecules have dendritic architecture and contain multiple copies of zinc(II)-dipicolylamine (ZnDPA) units that have selective affinity for the bacterial cell envelope. A series of molecular structures were evaluated, with the number of appended ZnDPA units ranging from four to thirty-two. Agglutination assays showed that the multivalent probes rapidly cross-linked ten different strains of bacteria, regardless of Gram-type and cell morphology. Fluorescence microscopy studies using probes with four ZnDPA units indicated a high selectivity for bacteria agglutination in the presence of mammalian cells and no measurable effect on the health of the cells. The high bacterial selectivity was confirmed by conducting in vivo optical imaging studies of a mouse leg infection model. The results suggest that multivalent ZnDPA molecular probes with dendritic structures have great promise as selective, broad spectrum bacterial agglutination agents for infection imaging and theranostic applications.

  12. Synthesis for Lunar Simulants: Glass, Agglutinate, Plagioclase, Breccia

    Science.gov (United States)

    Weinstein, Michael; Wilson, Stephen A.; Rickman, Douglas L.; Stoeser, Douglas

    2012-01-01

    The video describes a process for making glass for lunar regolith simulants that was developed from a patented glass-producing technology. Glass composition can be matched to simulant design and specification. Production of glass, pseudo agglutinates, plagioclase, and breccias is demonstrated. The system is capable of producing hundreds of kilograms of high quality glass and simulants per day.

  13. TROPLE-LUX-B: Phagocytosis in mussel hemocytes

    Science.gov (United States)

    Hansen, P. D.; Unruh, E.

    2005-08-01

    The TRIPLELUX-B Experiment contributes to risk assessment concerning immunotoxicity under space flight conditions. The assay system of the TRIPLELUX-B Experiment will be performed with a well defined quantification and evaluation of the immune function phagocytosis. The indicator cells are the hemocytes of blue mussels (Mytilus edulis) and oysters (Crassostrea gigas). The signals of the immuno cellular responses are translated into luminescence as a rapid optical reporter system. The results expected will allow to conclude wether the observed responses are caused by microgravity and/or radiation. The immune system of invertebrates has not been studied so far in space.The choice of the phagocytes from invertebrates is justified by the claim to study the universal validity of innate immune responses. The components of the phagocytosis test system for the BIOLAB are now established under terrestrial conditions. The next step for adaptation to the BIOLAB conditions and hardware is in progress.

  14. Lectin-like molecules in transcriptome of Littorina littorea hemocytes.

    Science.gov (United States)

    Gorbushin, Alexander M; Borisova, Elena A

    2015-01-01

    The common periwinkle Littorina littorea was introduced in the list of models for comparative immunobiology as a representative of phylogenetically important taxon Caenogastropoda. Using Illumina sequencing technology, we de novo assembled the transcriptome of Littorina littorea hemocytes from 182 million mRNA-Seq pair-end 100 bp reads into a total of 15,526 contigs clustered in 4472 unigenes. The transcriptome profile was analyzed for presence of carbohydrate-binding molecules in a variety of architectural contexts. Hemocytes' repertoire of lectin-like proteins bearing conserved carbohydrate-recognition domains (CRDs) is highly diversified, including 11 of 15 lectin families earlier described in animals, as well as the novel members of lectin family found for the first time in mollusc species. The new molluscan lineage-specific domain combinations were confirmed by cloning and sequencing, including the fuco-lectin related molecules (FLReMs) composed of N-terminal region with no sequence homology to any known protein, a middle Fucolectin Tachylectin-4 Pentaxrin (FTP) domain, and a C-terminal epidermal growth factor (EGF) repeat region. The repertoire of lectin-like molecules is discussed in terms of their potential participation in the receptor phase of immune response. In total, immune-associated functions may be attributed to 70 transcripts belonging to 6 lectin families. These lectin-like genes show low overlap between species of invertebrates, suggesting relatively rapid evolution of immune-associated genes in the group. The repertoire provides valuable candidates for further characterization of the gene functions in mollusc immunity.

  15. Carbohydrate Moieties and Cytoenzymatic Characterization of Hemocytes in Whiteleg Shrimp Litopenaeus vannamei

    Directory of Open Access Journals (Sweden)

    Norma Estrada

    2016-01-01

    Full Text Available Hemocytes represent one of the most important defense mechanisms against foreign material in Crustacea and are also involved in a variety of other physiological responses. Fluorescent lectin-binding assays and cytochemical reactions were used to identify specificity and distribution of carbohydrate moieties and presence of several hydrolytic enzymes, in hemocytes of whiteleg shrimp Litopenaeus vannamei. Two general classes of circulating hemocytes (granular and agranular exist in L. vannamei, which express carbohydrates residues for FITC-conjugated lectins WGA, LEA, and PNA; UEA and Con-A were not observed. Enzymatic studies indicated that acid phosphatase, nonspecific esterase, and specific esterases were present; alkaline phosphatase was not observed. The enzymes and carbohydrates are useful tools in hemocyte classification and cellular defense mechanism studies.

  16. Carbohydrate Moieties and Cytoenzymatic Characterization of Hemocytes in Whiteleg Shrimp Litopenaeus vannamei

    Science.gov (United States)

    Velázquez, Edwin; Rodríguez-Jaramillo, Carmen; Ascencio, Felipe

    2016-01-01

    Hemocytes represent one of the most important defense mechanisms against foreign material in Crustacea and are also involved in a variety of other physiological responses. Fluorescent lectin-binding assays and cytochemical reactions were used to identify specificity and distribution of carbohydrate moieties and presence of several hydrolytic enzymes, in hemocytes of whiteleg shrimp Litopenaeus vannamei. Two general classes of circulating hemocytes (granular and agranular) exist in L. vannamei, which express carbohydrates residues for FITC-conjugated lectins WGA, LEA, and PNA; UEA and Con-A were not observed. Enzymatic studies indicated that acid phosphatase, nonspecific esterase, and specific esterases were present; alkaline phosphatase was not observed. The enzymes and carbohydrates are useful tools in hemocyte classification and cellular defense mechanism studies. PMID:27833641

  17. IN VITRO KILLING OF PERKINSUS MARINUS BY HEMOCYTES OF OYSTERS CRASSOSTREA VIRGINICA

    Science.gov (United States)

    A colorimetric microbicidal assay was adapted, optimized and applied in experiments to characterize the in vitro capacity of eastern oyster (Crassostrea virginica) hemocytes to kill cultured isolates of Perkinsus marinus, a protozoan parasite causing a highly destructive disease...

  18. INFLUENCE OF SEASONAL FACTORS ON OYSTER HEMOCYTE KILLING OF VIBRIO PARAHEMOLYTICUS

    Science.gov (United States)

    Seasonal variation of cellular defenses of oyster Crassostrea virginica against Vibrio parahaemolyticus were examined from June 1997 to December 1998 using a recently developed bactericidal assay that utilizes a tetrazolium dye. Mean hemocyte numbers, plasma lysozyme, and P. mari...

  19. Effects and toxicity of phthalate esters to hemocytes of giant freshwater prawn, Macrobrachium rosenbergii

    Energy Technology Data Exchange (ETDEWEB)

    Sung, H.-H.; Kao, W.-Y.; Su, Y.-J

    2003-06-19

    Phthalate esters (PAEs) have been considered as environmental pollutants and have been subject to control in the United States of America and Japan. The aim of this study was to investigate the effects and toxicity of eight PAEs to hemocytes and the defense functions of giant freshwater prawn (Macrobrachium rosenbergii), including hemocytic adhesion, pseudopodia formation, phenoloxidase (PO) activity, and superoxide anion (O{sub 2}{sup -}) production, by means of in vitro exposure experiments. After hemocytes were treated separately with eight PAEs at concentrations of 100 {mu}g/ml, the results showed that two PAEs (dipropyl phthalate, DPrP and diethyl phthalate, DEP) increased cells with pseudopodia formation, but decreased adhesive cells; reduction in the percentages of both pseudopodia formation and adhesive cells were detected in the dihexyl phthalate (DHP) and diphenyl phthalate (DPP) experiment groups; and di-(2-ethyl hexyl) phthalate (DEHP) decreased pseudopodia formation, but did not affect the adhesion. In addition, both PO activity and O{sub 2}{sup -} production were decreased after hemocytes were treated with five PAEs (benzyl butyl phthalate (BBP), di-n-butyl phthalate (DBP), DEP, DHP and DPrP), respectively. At the same time, microscopy showed that both DPrP and DHP altered morphology of the cell nucleus and led to the presence of vacuoles in cytosol of hemocytes. Using the annexin assay, and after analysis of DNA fragmentation and transmission electron microscopy (TEM), it was found that hemocytes exposed to DHP and DPrP for more than 10 min would primarily die via apoptosis, the fatality correlates with increasing treatment time; and hemocytes treated with either BBP, dicyclohexyl phthalate (DCP), DEP or DPP would primarily die via necrosis. According to these results, we suggest that all eight PAEs examined could damage hemocytes and further influence the defense mechanism of prawns. This study reveals an important precaution for prawn cultivation.

  20. Identification and characterization of three novel hemocyte-specific promoters in silkworm Bombyx mori.

    Science.gov (United States)

    Zhang, Kui; Yu, Shuang; Su, Jingjing; Xu, Man; Tan, Peng; Zhang, Yajun; Xiang, Zhonghuai; Cui, Hongjuan

    2015-05-22

    Insect hemocytes play essential roles in the metabolism, metamorphosis and immunity, which are closely related events of growth and development. Here, four novel hemocyte-specific genes were obtained and conformed in our study, namely, Bmintβ2, Bmintβ3, BmCatO, and BmSw04862, respectively. Subsequently, their promoter sequences were cloned, and their activity in hemocytes, fat body, and silk gland were analyzed using recombinant AcNPV vector system in vivo. Our results showed that Bmintβ2, Bmintβ3, and BmCatO were hemocyte-specific promoters in the silkworm, Bombyx mori. Interestingly, Bmintβ2, and Bmintβ3 promoter regions were both located in their first intron. Further analysis of a series of BmCatO promoter truncations showed that a 254 bp region could function as a promoter element in the tissue-specificity expression. In summary, the results of this study revealed that we have identified three hemocyte-specific promoters in silkworm that will not only great significance for better understanding of hemocyte-specific gene, but also has potential applications in insect hematopoiesis and innate immunity research.

  1. A cytokine-like factor astakine accelerates the hemocyte production in Pacific oyster Crassostrea gigas.

    Science.gov (United States)

    Li, Yiqun; Jiang, Shuai; Li, Meijia; Xin, Lusheng; Wang, Lingling; Wang, Hao; Qiu, Limei; Song, Linsheng

    2016-02-01

    Astakine has been reported to be a hematopoietic growth factor of prokineticin homolog firstly found in arthropods freshwater crayfish Pacifastacus leniusculus. In the present study, an astakine homologous gene was identified from Pacific oyster Crassostrea gigas (designated CgAstakine). The full length cDNA of CgAstakine encoded a polypeptide of 103 amino acids containing a prokineticin (PK) domain homologous to that in astakine from freshwater crayfish P. leniusculus. The deduced amino acid sequence of CgAstakine shared higher similarity with those of other invertebrate astakines than prokineticins from vertebrates. The mRNA of CgAstakine was highly expressed in hepatopancreas and adductor muscle of oyster, while the CgAstakine protein was mainly distributed in hepatopancreas, gill and hemocytes. The mRNA expression of CgAstakine in hemocytes was significantly increased (p oyster hemocytes were incubated with 5 μg/mL recombinant CgAstakine protein (rCgAstakine) for 24 h in vitro, the proliferation of hemocytes was significantly increased to 1.89 fold of that in control group (p oyster hemocytes was significantly upregulated (2.45 fold of that in control group, p oysters were received an injection of rCgAstakine (0.5 μg/g). These results collectively indicated that CgAstakine could modulate the hemocytes proliferation both in vitro and in vivo, and probably involved in the hematopoietic process fighting against the invasion of foreign pathogens.

  2. Crayfish hematopoietic tissue cells but not hemocytes are permissive for white spot syndrome virus replication.

    Science.gov (United States)

    Wu, Junjun; Li, Fang; Huang, Jiajun; Xu, Limei; Yang, Feng

    2015-03-01

    Hemocytes are the major immune cells of crustaceans which are believed to be essential for the pathogenesis of white spot syndrome virus (WSSV) infection. Crayfish hemocytes and hematopoietic tissue (HPT) cells have been found to be susceptible to WSSV infection, but the procedure of WSSV infection to both cell types has not yet been carefully investigated. In this study, we analyzed the infection and proliferation of WSSV in crayfish hemocytes as well as HPT cells in detail through transmission electronic microscopy (TEM). The results showed that WSSV could enter both hemocytes and HPT cells through endocytosis, but the production of progeny virus was only achieved in HPT cells. Further investigation demonstrated that although WSSV could transcribe its genes in both cell types, viral genome replication and structural protein expression were unsuccessful in hemocytes, which may be responsible for the failure of progeny production. Therefore, we propose that both hemocytes and HPT cells are susceptible to WSSV infection but only HPT cells are permissive to WSSV replication. These findings will extend our knowledge of the interaction between WSSV and the host immune system.

  3. Disseminated cryptococcal lymphadenitis with negative latex agglutination test

    Institute of Scientific and Technical Information of China (English)

    XU Xiao-guang; BI Xin-ling; WU Jian-hua; XU Hong; LIAO Wan-qing

    2012-01-01

    We reported an unusual case of disseminated cryptococcal lymphadenitis in an immunocompetent host who presented with fever and lymphadenopathy,which were the only two symptoms and signs.Latex agglutination test of serum and cerebrospinal fluid (CSF) were negative,while lymph node biopsy showed Cryptococcus neoformans.A diagnosis of disseminated cryptococcal lymphadenitis was made.Then the patient was treated with amphotericin B for 15 days as initial therapy and itraconazole for 6 months as maintenance therapy respectively.The patient received re-examination per 6 months and was followed up for 2 years.Swollen lymph nodes diminished gradually,and no fever or other symptoms were found.Latex agglutination test of serum and CSF were negative throughout the follow-up period,and anti-HIV,syphilis and tuberculosis antibody were all negative.

  4. Dopamine modulates hemocyte phagocytosis via a D1-like receptor in the rice stem borer, Chilo suppressalis.

    Science.gov (United States)

    Wu, Shun-Fan; Xu, Gang; Stanley, David; Huang, Jia; Ye, Gong-Yin

    2015-07-16

    Dopamine (DA) is a signal moiety bridging the nervous and immune systems. DA dysregulation is linked to serious human diseases, including addiction, schizophrenia, and Parkinson's disease. However, DA actions in the immune system remain incompletely understood. In this study, we found that DA modulates insect hemocyte phagocytosis using hemocytes prepared from the rice stem borer (RSB), Chilo suppressalis. We investigated whether insect hemocytes are capable of de novo DA production. Here we show that exposing hemocytes to lipopolysaccharide (LPS) led to induction of DA-generating enzymes. Exogenous DA induced rapid phosphorylation of extracellular signal-regulated kinase (ERK) in naïve hemocytes. Activation of ERK was inhibited by preincubating with a DOP1 receptor antagonist. Thus, DA signaling via the DOP1 receptor may contribute to early hemocyte activation. DA synthesized and released from hemocytes may act in an autocrine mechanism to stimulate or maintain phagocytic activity. Consistent with this hypothesis, we found that inhibition of DA synthesis with α-methyl-DL-tyrosine methyl ester hydrochloride or blockage of DOP1 receptor with antagonist SCH23390 impaired hemocyte phagocytosis. Topical DA application also significantly decreased RSB mortality following challenge with the insect pathogenic fungus, Beauveria bassiana. We infer that a DA-dependent signaling system operates in hemocytes to mediate phagocytotic functions.

  5. Ocean Acidification Affects Hemocyte Physiology in the Tanner Crab (Chionoecetes bairdi)

    Science.gov (United States)

    Meseck, Shannon L.; Alix, Jennifer H.; Swiney, Katherine M.; Long, W. Christopher; Wikfors, Gary H.; Foy, Robert J.

    2016-01-01

    We used flow cytometry to determine if there would be a difference in hematology, selected immune functions, and hemocyte pH (pHi), under two different, future ocean acidification scenarios (pH = 7.50, 7.80) compared to current conditions (pH = 8.09) for Chionoecetes bairdi, Tanner crab. Hemocytes were analyzed after adult Tanner crabs were held for two years under continuous exposure to acidified ocean water. Total counts of hemocytes did not vary among control and experimental treatments; however, there were significantly greater number of dead, circulating hemocytes in crabs held at the lowest pH treatment. Phagocytosis of fluorescent microbeads by hemocytes was greatest at the lowest pH treatment. These results suggest that hemocytes were dying, likely by apoptosis, at a rate faster than upregulated phagocytosis was able to remove moribund cells from circulation at the lowest pH. Crab hemolymph pH (pHe) averaged 8.09 and did not vary among pH treatments. There was no significant difference in internal pH (pHi) within hyalinocytes among pH treatments and the mean pHi (7.26) was lower than the mean pHe. In contrast, there were significant differences among treatments in pHi of the semi-granular+granular cells. Control crabs had the highest mean semi-granular+granular pHi compared to the lowest pH treatment. As physiological hemocyte functions changed from ambient conditions, interactions with the number of eggs in the second clutch, percentage of viable eggs, and calcium concentration in the adult crab shell was observed. This suggested that the energetic costs of responding to ocean acidification and maintaining defense mechanisms in Tanner crab may divert energy from other physiological processes, such as reproduction. PMID:26859148

  6. Characterization of a tyramine receptor type 2 from hemocytes of rice stem borer, Chilo suppressalis.

    Science.gov (United States)

    Wu, Shun-Fan; Xu, Gang; Ye, Gong-Yin

    2015-04-01

    Calcium acts as a second messenger in many cell types, including insect hemocytes. Intracellular calcium level has a definite role in innate and adaptive immune signaling. Biogenic amines such as octopamine (OA), tyramine (TA), dopamine (DA) and serotonin (5-HT) play various important physiological roles in insects by activating distinct G-protein-coupled receptors (GPCRs) that share a putative seven transmembrane domain structure. OA and 5-HT have been shown that can mediate insect hemocytic immune reactions to infections and invasions. Here, we showed that TA increase hemocyte spreading in the rice stem borer, Chilo suppressalis. Furthermore, we cloned a cDNA encoding a tyramine receptor type 2 from the hemocytes in the C. suppressalis, viz., CsTA2, which shares high sequence similarity to members of the invertebrate tyramine receptor family. The CsTA2 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with TA induced a dose-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in cells, with an EC50 value of 18.7±5.3 nM, whereas OA, DA, 5-HT and other potential agonists did not have this response. The mRNA is present in various tissues including nerve cord, hemocytes, fat body, midgut, Malpighian tubules, and epidermis in the larval stage. Western blot analysis and immunohistochemistry assay displayed that CsTA2 was detected and presented on hemocytes. We also showed that TA induced Ca(2+) release from the hemocytes of C. suppressalis.

  7. Measurement of RBC agglutination with microscopic cell image analysis in a microchannel chip.

    Science.gov (United States)

    Cho, Chi Hyun; Kim, Ju Yeon; Nyeck, Agnes E; Lim, Chae Seung; Hur, Dae Sung; Chung, Chanil; Chang, Jun Keun; An, Seong Soo A; Shin, Sehyun

    2014-01-01

    Since Landsteiner's discovery of ABO blood groups, RBC agglutination has been one of the most important immunohematologic techniques for ABO and RhD blood groupings. The conventional RBC agglutination grading system for RhD blood typings relies on macroscopic reading, followed by the assignment of a grade ranging from (-) to (4+) to the degree of red blood cells clumping. However, with the new scoring method introduced in this report, microscopically captured cell images of agglutinated RBCs, placed in a microchannel chip, are used for analysis. Indeed, the cell images' pixel number first allows the differentiation of agglutinated and non-agglutinated red blood cells. Finally, the ratio of agglutinated RBCs per total RBC counts (CRAT) from 90 captured images is then calculated. During the trial, it was observed that the agglutinated group's CRAT was significantly higher (3.77-0.003) than that of the normal control (0). Based on these facts, it was established that the microchannel method was more suitable for the discrimination between agglutinated RBCs and non-agglutinated RhD negative, and thus more reliable for the grading of RBCs agglutination than the conventional method.

  8. Role of hemocytes in invertebrate adult neurogenesis and brain repair

    Directory of Open Access Journals (Sweden)

    PG Chaves da Silva

    2015-05-01

    Full Text Available The repair of lesions of the central nervous system (CNS varies widely throughout the animal kingdom. At the level of neuronal replacement lie the major differences in CNS regeneration. At one extreme are the amniote vertebrates (reptile, avian and mammalian groups, which have very limited capacity for neuronal replacement, and therefore for neural regeneration; at the other extreme, animals such as planarians (flatworms and colonial tunicates can repair their entire CNS after major injuries. These differences can be attributed to the abundance of multipotent and/or pluripotent stem cells and/or undifferentiated precursors among the general cell population. In this review we discuss recent advancements in knowledge of regeneration of the CNS of invertebrates. We focus on ascidians, which are a sister group of vertebrates, but we also address other invertebrate groups. Because neurogenesis is central to the events that allow regeneration of the adult CNS, we address this issue focusing on crustaceans, which have provided a paradigm to study the mechanisms underlying this phenomenon. The attraction of hemocytes toward a neurogenic niche and respecification of these cells toward a neural fate has been strongly suggested. Based on recent and emerging research, we suggest that cells of the blood lineage are not only associated with the roles that are generally attributed to them, but are the cells that either signal other cell types to differentiate into neural cells, or even eventually themselves transdifferentiate into neural cells.

  9. Characterization of the hemocytes in Larvae of Protaetia brevitarsis seulensis: involvement of granulocyte-mediated phagocytosis.

    Directory of Open Access Journals (Sweden)

    Hyojung Kwon

    Full Text Available Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe, are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in P. brevitarsis seulensis. The circulating hemocytes were classified based on their size, morphology, and dye-staining properties into six types, including granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. The percentages of circulating hemocyte types were as follows: 13% granulocytes, 20% plasmatocytes, 1% oenocytoids, 5% spherulocytes, 17% prohemocytes, and 44% adipohemocytes. Next, we identified the professional phagocytes, granulocytes, which mediate encapsulation and phagocytosis of pathogens. The granulocytes were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo. In addition, we showed that the phagocytosis by granulocytes is associated with autophagy, and that the activation of autophagy could be an efficient way to eliminate pathogens in this system. We also observed a high accumulation of autophagic vacuoles in activated granulocytes, which altered their shape and led to autophagic cell death. Finally, the granulocytes underwent mitotic division thus maintaining their number in vivo.

  10. Characterization of the hemocytes in Larvae of Protaetia brevitarsis seulensis: involvement of granulocyte-mediated phagocytosis.

    Science.gov (United States)

    Kwon, Hyojung; Bang, Kyeongrin; Cho, Saeyoull

    2014-01-01

    Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in P. brevitarsis seulensis. The circulating hemocytes were classified based on their size, morphology, and dye-staining properties into six types, including granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. The percentages of circulating hemocyte types were as follows: 13% granulocytes, 20% plasmatocytes, 1% oenocytoids, 5% spherulocytes, 17% prohemocytes, and 44% adipohemocytes. Next, we identified the professional phagocytes, granulocytes, which mediate encapsulation and phagocytosis of pathogens. The granulocytes were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo. In addition, we showed that the phagocytosis by granulocytes is associated with autophagy, and that the activation of autophagy could be an efficient way to eliminate pathogens in this system. We also observed a high accumulation of autophagic vacuoles in activated granulocytes, which altered their shape and led to autophagic cell death. Finally, the granulocytes underwent mitotic division thus maintaining their number in vivo.

  11. Interpretation of microscopic agglutination test for leptospirosis diagnosis and seroprevalence

    Institute of Scientific and Technical Information of China (English)

    Chintana Chirathaworn; Rajada Inwattana; Yong Poovorawan; Duangjai Suwancharoen

    2014-01-01

    Determination of antibody titer by microscopic agglutination test (MAT) has been used as a tool for leptospirosis diagnosis. Four fold or greater rise in antibody titers between acute and convalescent sera suggests recent Leptospira infection. In addition, results obtained by MAT have been used to predict infecting serovars. However, cross reactivity among various Leptospira serovars have been reported when patient sera were tested with a battery of Leptospira serovars. This study demonstrates cross- reactivity among several Leptospira serovars when MAT was performed on leptospirosis sera. The data support a role of MAT as a tool for diagnosis. However, for information on infecting serovars, Leptospira isolation and molecular identification should be performed.

  12. Latex agglutination test (LAT) for the diagnosis of typhoid fever.

    Science.gov (United States)

    Sahni, Gopal Shankar

    2013-06-01

    The efficacy of latex agglutination test in the rapid diagnosis of typhoid fever was studied and the result compared with that of blood culture. This study included 80 children suffering from typhoid fever, among which 40 were confirmed by blood culture isolation and 40 had possible typhoid fever based on high Widal's titre (a four-fold rise in the titre of antibody to typhi "O" and "H" antigen was considered as a positive Widal's test result). Eighty children, 40 with febrile illness confirmed to be other than typhoid and 40 normal healthy children were used as negative controls. The various groups were: (i) Study group ie, group I had 40 children confirmed by culture isolation of Salmonella typhi(confirmed typhoid cases). (ii) Control groups ie, (a) group II with 40 febrile controls selected from paediatrics ward where cause other than S typhi has been established, (b) group III with 40 afebrile healthy controls that were siblings of the children admitted in paediatric ward for any reason with no history of fever and TAB vaccination in the last one year, and (c) group IV with 40 children with high Widal's titre in paired sera sample. Widal's test with paired sera with a one week interval between collections were done in all 40 patients. Latex aggtutination test which could detect 900 ng/ml of antigen as observed in checker board titration, was positive in all 40 children from group I who had positive blood culture and in 30 children from group IV who had culture negative and had high Widal's titre positive. Latex agglutination test was positive in 4 children in group II and none in group III. Using blood culture positive cases as true positive and children in groups II and III as true negative, the test had a sensitivity of 100% and specificity of 96%. Latex agglutination test was found to be significantly sensitive (100%) and specific (96%) and could detect 75% more cases in group IV (possible typhoid cases). Thus latex agglutination test can be used for rapid

  13. The Effects of Morphine Sulfate on Agglutination, Clot Formation and Hemolysis in Packed Red Blood Cells

    Science.gov (United States)

    2007-11-02

    THE EFFECTS OF MORPHINE SULFATE ON AGGLUTINATION , CLOT FORMATION AND HEMOLYSIS IN PACKED RED BLOOD CELLS 6. AUTHOR(S) CAPT ESTAVILLO BRIAN K 7...ANSI Std8 239.18 Designed using Perform Pro, WHS/DIOR, Oct 94 THE EFFECTS OF MORPHINE SULFATE ON AGGLUTINATION , CLOT FORMATION AND HEMOLYSIS IN PACKED...that the use of any copyrighted material in the thesis entitled: "THE EFFECTS OF MORPHINE SULFATE ON AGGLUTINATION , CLOT FORMATION AND HEMOLYSIS IN

  14. Diagnosis of myocardial infarction based on lectin-induced erythrocyte agglutination: a feasibility study

    Science.gov (United States)

    Bocsi, József; Nieschke, Kathleen; Mittag, Anja; Reichert, Thomas; Laffers, Wiebke; Marecka, Monika; Pierzchalski, Arkadiusz; Piltz, Joachim; Esche, Hans-Jürgen; Wolf, Günther; Dähnert, Ingo; Baumgartner, Adolf; Tarnok, Attila

    2014-03-01

    Myocardial infarction (MI) is an acute life-threatening disease with a high incidence worldwide. Aim of this study was to test lectin-carbohydrate binding-induced red blood cell (RBC) agglutination as an innovative tool for fast, precise and cost effective diagnosis of MI. Five lectins (Ricinus communis agglutinin (RCA), Phaseolus vulgaris erythroagglutinin (PHA), Datura stramonium agglutinin (DSA), Artocarpus agglutinin (ArA), Triticum agglutinin (TA)) were tested for ability to differentiate between agglutination characteristics in patients with MI (n = 101) or angina pectoris without MI (AP) (n = 34) and healthy volunteers (HV) as control (n =68) . RBC agglutination was analyzed by light absorbance of a stirred RBC suspension in the green to red light spectrum in an agglutimeter (amtec, Leipzig, Germany) for 15 min after lectin addition. Mean cell count in aggregates was estimated from light absorbance by a mathematical model. Each lectin induced RBC agglutination. RCA led to the strongest RBC agglutination (~500 RBCs/aggregate), while the others induced substantially slower agglutination and lead to smaller aggregate sizes (5-150 RBCs/aggregate). For all analyzed lectins the lectin-induced RBC agglutination of MI or AP patients was generally higher than for HV. However, only PHA induced agglutination that clearly distinguished MI from HV. Variance analysis showed that aggregate size after 15 min. agglutination induced by PHA was significantly higher in the MI group (143 RBCs/ aggregate) than in the HV (29 RBC-s/aggregate, p = 0.000). We hypothesize that pathological changes during MI induce modification of the carbohydrate composition on the RBC membrane and thus modify RBC agglutination. Occurrence of carbohydrate-lectin binding sites on RBC membranes provides evidence about MI. Due to significant difference in the rate of agglutination between MI > HV the differentiation between these groups is possible based on PHA-induced RBC-agglutination. This novel assay

  15. Nuclear morphology and lysosomal stability of molluskan hemocytes as possible biomarkers of arsenic toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Sudipta [Post Graduate Department of Zoology, Parasitology and Medical Entomology Laboratory, Darjeeling Government College, Darjeeling (India); Ray, Sajal [Department of Zoology, Aquatic Toxicology Laboratory, University of Calcutta, Kolkata (India)

    2009-10-15

    The frequency of nuclear aberrations and neutral red retention time of hemocytes in the mollusk Lamellidens marginalis were recorded under exposure to sublethal concentrations of sodium arsenite in order to examine the sensitivity and effectiveness of these inexpensive assays for screening the toxicity of As{sup 3+}in a freshwater ecosystem. A dose and time dependent increase in the density of micronucleated and binucleated hemocytes and gill cells was indicative of the pronounced genotoxic effect of arsenic on this animal. The disruption of intrahemocyte homeostasis imposed by this natural toxicant was evident from a dose and time dependent reduction in the lysosomal stability of the hemocytes of the animal. The tested parameters are indicative of arsenic toxicity in L. marginalis in the freshwater systems of the arsenic affected geographical areas of West Bengal, India. (Abstract Copyright [2009], Wiley Periodicals, Inc.)

  16. Quantitative analysis of hemocyte morphological abnormalities associated with Campoletis sonorensis parasitization

    Directory of Open Access Journals (Sweden)

    Matthew W. Turnbull

    2004-04-01

    Full Text Available Endoparasitoids of arthropods evoke host cellular immune responses that result in hemocytic encapsulation of the endoparasitoid, unless these responses are disrupted by the parasite. Our interest has focused on mutualistic viruses found in some hymenopteran endoparasitoids that disrupt hemocyte function and prevent encapsulation. Specifically, the Campoletis sonorensis polydnavirus interacts with wasp factors to suppress immunity via expression of intracellular and secreted viral proteins. To study the roles of specific parasitization-associated factors on immunocyte morphology, fluorescence microscopy was used to visualize the actin cytoskeleton in infected and uninfected cells, or after treatment with C. sonorensis ovarian proteins or plasma from infected larvae. The titer and distribution of F- and G-actin were altered in hemocytes from parasitized insects relative to control cells, with plasma from parasitized larvae having an intermediate effect. This suggests that intracellular and secreted factors contribute to suppression of cellular immune responses in C. sonorensis.

  17. Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

    Science.gov (United States)

    Castellanos-Martínez, S; Prado-Alvarez, M; Lobo-da-Cunha, A; Azevedo, C; Gestal, C

    2014-05-01

    The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes.

  18. The immune response of hemocytes of the insect Oncopeltus fasciatus against the flagellate Phytomonas serpens.

    Science.gov (United States)

    Alves e Silva, Thiago L; Vasconcellos, Luiz R C; Lopes, Angela H; Souto-Padrón, Thaïs

    2013-01-01

    The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites.

  19. Quantitative Determination of Fibrinogen of Patients with Coronary Heart Diseases through Piezoelectric Agglutination Sensor

    Directory of Open Access Journals (Sweden)

    Ming Chen

    2010-03-01

    Full Text Available Fibrinogen can transform fibrin through an agglutination reaction, finally forming fibrin polymer with grid structure. The density and viscosity of the reaction system changes drastically during the course of agglutination. In this research, we apply an independently-developed piezoelectric agglutination sensor to detect the fibrinogen agglutination reaction in patients with coronary heart diseases. The terminal judgment method of determining plasma agglutination reaction through piezoelectric agglutination sensor was established. In addition, the standard curve between plasma agglutination time and fibrinogen concentration was established to determinate fibrinogen content quantitatively. The results indicate the close correlation between the STAGO paramagnetic particle method and the method of piezoelectric agglutination sensor for the detection of Fibrinogen. The correlation coefficient was 0.91 (γ = 0.91. The determination can be completed within 10 minutes. The fibrinogen concentration in the coronary heart disease group was significantly higher than that of the healthy control group (P < 0.05. The results reveal that high fibrinogen concentration is closely correlated to the incurrence, development and prognosis of coronary heart diseases. Compared with other traditional methods, the method of piezoelectric agglutination sensor has some merits such as operation convenience, small size, low cost, quick detecting, good precision and the common reacting agents with paramagnetic particle method.

  20. Quantitative determination of fibrinogen of patients with coronary heart diseases through piezoelectric agglutination sensor.

    Science.gov (United States)

    Chen, Qinghai; Hua, Xing; Fu, Weiling; Liu, Dongbo; Chen, Ming; Cai, Guoru

    2010-01-01

    Fibrinogen can transform fibrin through an agglutination reaction, finally forming fibrin polymer with grid structure. The density and viscosity of the reaction system changes drastically during the course of agglutination. In this research, we apply an independently-developed piezoelectric agglutination sensor to detect the fibrinogen agglutination reaction in patients with coronary heart diseases. The terminal judgment method of determining plasma agglutination reaction through piezoelectric agglutination sensor was established. In addition, the standard curve between plasma agglutination time and fibrinogen concentration was established to determinate fibrinogen content quantitatively. The results indicate the close correlation between the STAGO paramagnetic particle method and the method of piezoelectric agglutination sensor for the detection of Fibrinogen. The correlation coefficient was 0.91 (γ = 0.91). The determination can be completed within 10 minutes. The fibrinogen concentration in the coronary heart disease group was significantly higher than that of the healthy control group (P high fibrinogen concentration is closely correlated to the incurrence, development and prognosis of coronary heart diseases. Compared with other traditional methods, the method of piezoelectric agglutination sensor has some merits such as operation convenience, small size, low cost, quick detecting, good precision and the common reacting agents with paramagnetic particle method.

  1. Morphofunctional study of hemocytes from lions-paw scallop Nodipecten subnodosus.

    Science.gov (United States)

    Estrada, Norma; Velázquez, Edwin; Rodríguez-Jaramillo, Carmen; Ascencio, Felipe

    2013-08-01

    Hemocytes play an important role in internal defense in mollusk bivalves; they are generally divided into granular and hyaline types, where the granules possess molecules to combat foreign particles. We investigated the morphology, staining reactions, and immune-related activities of hemocytes from the pectinid lions-paw scallop Nodipecten subnodosus. We showed that, in contrast to the conditions in most bivalves and similar to other pectinids, no granular cells were observed; only very few semi-granular cells were present and the large majority of cells were hyalinocytes and blast-like cells. The cytoplasm in many hyalinocytes showed vesicles of various sizes, but none with the staining characteristics of typical granules. When hemolymph was removed from these scallops, the plasma did not clot and the hemocytes rapidly adhered to one another in suspension. When living hemocytes were placed on a glass surface, they clustered into groups of various sizes; within minutes, pseudopods extended from the cells and outward migration began. Many cells spread to form extensive networks of flat cells, and other undifferentiated cells did not spread and kept a rounded morphology. Some hemocytes were immunologically active because they phagocytize Escherichia coli bacteria. Hemocytes expressed the carbohydrates residues N-acetyl-D-glucosamine, α-D-glucose, α-D-mannose, and α-L-fucose moieties, and cytochemical assays showed activity of acid phosphatase, specific esterase, and non-specific esterase, enzymes commonly associated with lysosomes and immunologic activity. These observations serve as a useful tool for further investigation of morphofunctional studies and physiological status of scallops. Copyright © 2013 Elsevier GmbH. All rights reserved.

  2. Recognition between symbiotic Vibrio fischeri and the hemocytes of Euprymna scolopes

    Science.gov (United States)

    Nyholm, Spencer V.; Stewart, Jennifer J.; Ruby, Edward G.; McFall-Ngai, Margaret J.

    2008-01-01

    Summary The light-organ crypts of the squid Euprymna scolopes permit colonization exclusively by the luminous bacterium Vibrio fischeri. Because the crypt interior remains in contact with seawater, the squid must not only foster the specific symbiosis but also continue to exclude other bacteria. Investigation of the role of the innate immune system in these processes revealed that macrophage-like hemocytes isolated from E. scolopes recognized and phagocytosed V. fischeri less than other closely related bacterial species common to the host’s environment. Interestingly, phagocytes isolated from hosts that had been cured of their symbionts bound five-times more V. fischeri cells than those from uncured hosts. No such change in the ability to bind other species of bacteria was observed, suggesting that the host adapts specifically to V. fischeri. Deletion of the gene encoding OmpU, the major outer membrane protein of V. fischeri, increased binding by hemocytes from uncured animals to the level observed for hemocytes from cured animals. Co-incubation with wild-type V. fischeri reduced this binding, suggesting they produce a factor that complements the mutant’s defect. Analyses of the phagocytosis of bound cells by fluorescence-activated cell sorting (FACS) indicated that, once binding to hemocytes had occurred, V. fischeri cells are phagocytosed as effectively as other bacteria. Thus, discrimination by this component of the squid immune system occurs at the level of hemocyte binding, and this response: (i) is modified by previous exposure to the symbiont and, (ii) relies on outer membrane and/or secreted components of the symbionts. These data suggest that regulation of host hemocyte binding by the symbiont may be one of many factors that contribute to specificity in this association. PMID:19196278

  3. Understanding the Role of Host Hemocytes in a Squid/Vibrio Symbiosis Using Transcriptomics and Proteomics

    Directory of Open Access Journals (Sweden)

    Andrew J. Collins

    2012-05-01

    Full Text Available The symbiosis between the squid, Euprymna scolopes, and the bacterium, Vibrio fischeri, serves as a model for understanding interactions between beneficial bacteria and animal hosts. The establishment and maintenance of the association is highly specific and depends on the selection of V. fischeri and exclusion of non-symbiotic bacteria from the environment. Current evidence suggests that the host’s cellular innate immune system, in the form of macrophage-like hemocytes, helps to mediate host tolerance of V. fischeri. To begin to understand the role of hemocytes in this association, we analyzed these cells by high-throughput 454 transcriptomic and liquid chromatography/ tandem mass spectrometry (LC-MS/MS proteomic analyses. 454 high-throughput sequencing produced 650,686 reads totaling 279.9 Mb while LC-MS/MS analyses of circulating hemocytes putatively identified 702 unique proteins. Several receptors involved with the recognition of microbial associated molecular patterns (MAMPs were identified. Among these was a complete open reading frame (ORF to a putative peptidoglycan recognition protein (EsPGRP5 that has conserved residues for amidase activity. Assembly of the hemocyte transcriptome showed EsPGRP5 had high coverage, suggesting it is among the 5% most abundant transcripts in circulating hemocytes. Other transcripts and proteins identified included members of the conserved NFκB signaling pathway, putative members of the complement pathway, the carbohydrate binding protein galectin, and cephalotoxin. Quantitative PCR of complement-related genes, cephalotoxin, EsPGRP5, and a nitric oxide synthase showed differential expression in circulating hemocytes isolated from adult squid with colonized light organs compared to those for which the symbionts were removed. These data suggest that the presence of the symbiont influences gene expression of the cellular innate immune system of the host.

  4. A new fibrinogen-related protein from Argopecten irradians (AiFREP-2) with broad recognition spectrum and bacteria agglutination activity.

    Science.gov (United States)

    Yang, Chuanyan; Wang, Leilei; Zhang, Huan; Wang, Lingling; Huang, Mengmeng; Sun, Zhibin; Sun, Ying; Song, Linsheng

    2014-05-01

    Fibrinogen-related proteins (FREPs) are a kind of pattern recognition receptors (PRRs) containing fibrinogen-like (FBG) domains, and they play curial roles in the innate immune response. In the present study, a new FREP protein was identified from bay scallop Argopecten irradians (designated as AiFREP-2). The full-length cDNA of AiFREP-2 was of 1299 bp with an open reading frame of 762 bp encoding a polypeptide of 253 amino acids, including a signal sequence and an FBG domain. The FBG domain in AiFREP-2 was highly similar to those of ficolins, tenascins and other FREPs. The mRNA expression of AiFREP-2 could be detected in all the examined tissues with the highest level in gill. The mRNA expression of AiFREP-2 in hemocytes was significantly up-regulated post the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan (GLU) (P agglutination activity towards Gram-negative bacteria V. anguillarum and Gram positive bacteria S. aureus. The results indicated that AiFREP-2 was involved in the immune response against Gram-negative bacteria, Gram-positive bacteria and fungus as a PRR in bay scallop, and the information was helpful to understand the innate immune defense mechanisms of mollusks.

  5. Development of a slide agglutination assay for detection of blastomycosis.

    Science.gov (United States)

    Hatch, Wayne O; Scalarone, Gene M

    2013-11-01

    Blastomycosis, caused by the thermally dimorphic fungus Blastomyces dermatitides, which is endemic to eastern regions of the USA, is commonly misdiagnosed as a viral or bacterial infection and therefore treated improperly. Over the years, many immunodiagnostic assays to aid in the diagnosis of blastomycosis have been developed; however, a reliable assay for use in local clinics still remains elusive. Procedures for a slide agglutination assay for detection of antibody in serum from rabbits immunized with B. dermatitidis were evaluated with antigenic preparations from B. dermatitidis adsorbed to polystyrene microparticles. Yeast-phase lysates from five isolates of B. dermatitides: namely ER-593 (Eagle River, WI, USA), ER-598 (Eagle River, WI, USA), 48938 (India), B5896 (Mt. Iron, MN, USA), and T-58 (TN, USA) were evaluated for their sensitivity and specificity. Sensitivities of the lysates ranged from 29% to 83% whereas specificities ranged from 13% to 100%. Lysate ER-593 provided the most promising results with a sensitivity of 82% and specificity of 100%. This study provides suggests that a simple rapid slide agglutination assay for detecting blastomycosis may be used for screening patients with suspected B. dermatitidis infection.

  6. Latex agglutination test for field diagnosis of haemorrhagic septicaemia

    Directory of Open Access Journals (Sweden)

    Lily Natalia

    2002-10-01

    Full Text Available Pasteurella multocida is bacterial pathogens that cause haemorrhagic septicaemia (HS in cattle and buffaloes. Various tests have been used to differentiate types of P. multocida, as well as to diagnose this specific disease. A latex agglutination test has been developed for the detection of P. multocida B:2 which is the causal agent of HS. This test is a rapid and simple test suitable for local laboratorium to diagnose HS cases in the field. A heat stable antigen consisting of mainly lipopolysaccharide (LPS extract of formalin killed P. multocida 0019 was used to produce specific antibody against P. multocida B:2. The antiboy was then used to sensitise latex particles. Latex agglutination test have been used to screen some P. multocida field isolates and this test have been proven to be specific, simple and easy to be used in detecting P. multocida B:2. The specificity is due to antibodies recognising LPS or LPS protein complexes. Sensitised latex was stable at 4° C for at least12 months. This test should be used as an aid to diagnosis and employed principally to confirm and support clinical and post mortem findings of HS.

  7. A protocol for collecting and staining hemocytes from the yellow fever mosquito Aedes aegypti.

    Science.gov (United States)

    Qayum, Amina A; Telang, Aparna

    2011-05-16

    Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens(1, 2). The innate immune system of mosquitoes consists of several lines of defense (3). Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut (4) enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens (5, 6). Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages (7). The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis (8), and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen (9, 10). These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively (11). More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues (11). While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are

  8. Agglutinated foraminifera from the Ludlow (Silurian) of Ireland

    Science.gov (United States)

    Kaminski, Michael; Ferretti, Annalisa; Messori, Fabio; Papazzoni, Cesare Andrea; Sevastopulo, George

    2017-04-01

    Agglutinated foraminifera are one of the most primitive groups of foraminifera, possibly already appearing in the Cryogenian but usually rare in lower Paleozoic rocks. Their mean standing diversity slowly increased during Cambrian and Ordovician times, reaching a stable value of about 50 genera in the mid-Silurian which remained fairly constant up to the Triassic. An assemblage of agglutinated foraminifera was unexpectedly found in conodont residue from material collected in the Dingle Peninsula, County Kerry, southwestern Ireland. This material comes from rare calcareous occurrences in volcanoclastics previously known for their rich trilobite and conodont assemblages. The limestones are trilobite-crinoidal silty wackestone to packstone, with local brachiopod concentrations, documenting brachiopod-trilobite-crinoidal dominated communities of shallow and well-ventilated water that might have periodically colonized the bottom intercalating with volcanic events and then successively redeposited in deeper waters. The conodont fauna indicates an early Ludlow (Gorstian-earliest Ludfordian) age (Kaminski et al., 2016). The foraminiferal assemblage has limited potential for stratigraphical correlation as long-range taxa are present, but it represents the first record from the Silurian of Ireland. The assemblage is dominated by tubothalamids (Rectoammodiscus and rare Sansabaina), with less abundant monothalamids (Psammosiphonella and Psammosphaera). The assemblage displays low diversity compared with other assemblages described from the British Isles (Kircher & Brasier, 1989). At the species level, this assemblage is identical to those described previously from the Silurian of North America but with lower diversity. Only Rectoammodiscus diai had apparently a wider geographic distribution, including not only the central USA (Oklahoma and Kansas) but also the Welsh Borderlands and Senegal. The affinities with the assemblages reported at several localities in the central

  9. Hemocyte characterization of Nasutitermes coxipoensis (Holmgren) (Isoptera: Termitidae) workers and hemocyte evaluation after parasitism by Metarhizium anisopliae; Caracterizacao dos hemocitos de operarios de Nasutitermes coxipoensis (Holmgren) (Isoptera: Termitidae) e avaliacao hemocitaria apos parasitismo por Metarhizium anisopliae

    Energy Technology Data Exchange (ETDEWEB)

    Cunha, Franklin M.; Wanderley-Teixeira, Valeria; Albuquerque, Auristela C. [Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE (Brazil). Programa de Pos-Graduacao em Entomologia Agricola], e-mail: ukento@yahoo.com.br; Teixeira, Alvaro A.C. [Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE (Brazil). Dept. de Morfologia e Fisiologia Animal], e-mail: valeria@dmfa.ufrpe.br, e-mail: auritermes@yahoo.com.br; Alves, Luiz C. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Lab. de Imunopatologia Keizo Asami (LIKA); Lima, Elza A.L.A. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Micologia. Lab. de Controle Biologico

    2009-03-15

    We aimed to characterize the morphology and ultrastructure of hemocytes of Nasutitermes coxipoensis (Holmgren) workers and to quantify the cell types 24h, 48h and 72h after inoculation with Metarhizium anisopliae. Six hemocytes types were identified, plasmatocyte, granulocyte, spherulocyte, prohemocyte, adipohemocyte and eonocytoid Hemocytes did not present any morphological alteration at the several observation periods, but they did have a change in their abundance, as observed for spherulocytes, adipohemocytes and eonocytoids at all intervals, and for plasmatocytes and granulocytes at 48h after host inoculation. We argue on the possible reasons and implications of the observed changes. (author)

  10. An early Cambrian agglutinated tubular lophophorate with brachiopod characters

    Science.gov (United States)

    Zhang, Z.-F.; Li, G.-X.; Holmer, L. E.; Brock, G. A.; Balthasar, U.; Skovsted, C. B.; Fu, D.-J.; Zhang, X.-L.; Wang, H.-Z.; Butler, A.; Zhang, Z.-L.; Cao, C.-Q.; Han, J.; Liu, J.-N.; Shu, D.-G.

    2014-05-01

    The morphological disparity of lophotrochozoan phyla makes it difficult to predict the morphology of the last common ancestor. Only fossils of stem groups can help discover the morphological transitions that occurred along the roots of these phyla. Here, we describe a tubular fossil Yuganotheca elegans gen. et sp. nov. from the Cambrian (Stage 3) Chengjiang Lagerstätte (Yunnan, China) that exhibits an unusual combination of phoronid, brachiopod and tommotiid (Cambrian problematica) characters, notably a pair of agglutinated valves, enclosing a horseshoe-shaped lophophore, supported by a lower bipartite tubular attachment structure with a long pedicle with coelomic space. The terminal bulb of the pedicle provided anchorage in soft sediment. The discovery has important implications for the early evolution of lophotrochozoans, suggesting rooting of brachiopods into the sessile lophotrochozoans and the origination of their bivalved bauplan preceding the biomineralization of shell valves in crown brachiopods.

  11. Interpretation of microscopic agglutination test for leptospirosis diagnosis and seroprevalence

    Directory of Open Access Journals (Sweden)

    Chintana Chirathaworn

    2014-05-01

    Full Text Available Determination of antibody titer by microscopic agglutination test (MAT has been used as a tool for leptospirosis diagnosis. Four fold or greater rise in antibody titers between acute and convalescent sera suggests recent Leptospira infection. In addition, results obtained by MAT have been used to predict infecting serovars. However, cross reactivity among various Leptospira serovars have been reported when patient sera were tested with a battery of Leptospira serovars. This study demonstrates cross-reactivity among several Leptospira serovars when MAT was performed on leptospirosis sera. The data support a role of MAT as a tool for diagnosis. However, for information on infecting serovars, Leptospira isolation and molecular identification should be performed.

  12. Genesis of Hematopoietic Tissue and Its Relation with Hemocytes of Litopenaeus Vannamei (Boone, 1931 (Crustacea: Decapoda

    Directory of Open Access Journals (Sweden)

    Faiz Muhammad§, Zhi-Feng Zhang*, Ming-Yu Shao, Xiao-Li Shi and Muhammad Shafi

    2013-01-01

    Full Text Available The Litopenaeus vannamei is a prime aquaculture species and has a worth market value all over the world. In this study, we investigated the genesis and morphology of hematopoietic tissue (HPT and types of hemocyte based on its morphology and cytochemical feature using histological and histochemical methods. The results revealed that HPT in L. vannamei is not visible histologically in mysis and early two post larvae (P1 & P2, and emerges in P3 which locates in epigastric region. The HPT showed a continuous propagation of cells in successive post larval stages. Four types of cells can be identified in the HPT from adult specimen and mitotic activity is visible in the HPT. The hemocytes have been differentiated into five types and some of these types are co-related with the HPT cells. The cytochemical studies suggested that type I and II cells of hemocytes are PAS positive while scanty presence of prophenoloxidase was observed in type I cells. Similarly the type I cells are Sudan Black B positive and rest of the cells showed weak activity against lipid detective stain. This is the first effort towards genesis of HPT and its relation with circulating hemocytes in L. vannamei.

  13. New insights from the oyster Crassostrea rhizophorae on bivalve circulating hemocytes.

    Directory of Open Access Journals (Sweden)

    Mauro de Freitas Rebelo

    Full Text Available Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size as it degranulates in the event of an environmental challenge.

  14. New insights from the oyster Crassostrea rhizophorae on bivalve circulating hemocytes.

    Science.gov (United States)

    Rebelo, Mauro de Freitas; Figueiredo, Eliane de Souza; Mariante, Rafael M; Nóbrega, Alberto; de Barros, Cintia Monteiro; Allodi, Silvana

    2013-01-01

    Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size) as it degranulates in the event of an environmental challenge.

  15. Induction of micronuclei in hemocytes of Mytilus edulis and statistical analysis

    DEFF Research Database (Denmark)

    Wrisberg, M. N.; Bilbo, Carl M.; Spliid, Henrik

    1992-01-01

    A genotoxicity test focusing on micronucleus production in the blood cells (hemocytes) of blue mussel M. edulis has been developed as a possible indicator for marine pollution. A linear dose-response relationship was found when M. edulis was exposed to low concentrations (0, 12.5, and 25 mg...

  16. Oyster hemocyte mobilization and increased adhesion activity after beta glucan administration

    Science.gov (United States)

    In the eastern oyster (Crassostrea virginica) hemocytes are important effector cells for maintenance of defense against pathogenic microorganisms. Various forms of ß-glucans have been suggested for use in shrimp and fish aquaculture because of their potential to enhance disease resistance via hemoc...

  17. New Insights from the Oyster Crassostrea rhizophorae on Bivalve Circulating Hemocytes

    Science.gov (United States)

    Mariante, Rafael M.; Nóbrega, Alberto; de Barros, Cintia Monteiro; Allodi, Silvana

    2013-01-01

    Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size) as it degranulates in the event of an environmental challenge. PMID:23451217

  18. Effects of cadmium on aneuploidy and hemocyte parameters in the Pacific oyster, Crassostrea gigas

    Energy Technology Data Exchange (ETDEWEB)

    Bouilly, Karine [IFREMER, Laboratoire de Genetique et Pathologie, La Tremblade 17390 (France); Gagnaire, Beatrice [IFREMER, Laboratoire de Genetique et Pathologie, La Tremblade 17390 (France); Bonnard, Marc [IFREMER, Laboratoire de Genetique et Pathologie, La Tremblade 17390 (France); Thomas-Guyon, Helene [Laboratoire de Biologie et Environnement Marins, FRE-CNRS, 2727, Universite de La Rochelle, 22 Avenue Michel Crepeau, La Rochelle 17042 (France); Renault, Tristan [IFREMER, Laboratoire de Genetique et Pathologie, La Tremblade 17390 (France); Miramand, Pierre [Laboratoire de Biologie et Environnement Marins, FRE-CNRS, 2727, Universite de La Rochelle, 22 Avenue Michel Crepeau, La Rochelle 17042 (France); Lapegue, Sylvie [IFREMER, Laboratoire de Genetique et Pathologie, La Tremblade 17390 (France)]. E-mail: slapegue@ifremer.fr

    2006-06-15

    Pacific oysters, Crassostrea gigas, are commonly reared in estuaries where they are exposed to anthropogenic pollution. Much research has been made on the toxicity of cadmium to aquatic organisms because the compound recurrently contaminates their environment. Our study examined the influence of cadmium on aneuploidy level (lowered chromosome number in a percentage of somatic cells) and hemocyte parameters in C. gigas at different stages of life. Adults and juveniles were exposed to two different concentrations of cadmium. The first concentration applied was equivalent to a peak value found in Marennes-Oleron bay (Charente-Maritime, France; 50 ng L{sup -1}) and the second was 10 times higher (500 ng L{sup -1}). Exposure to 50 ng L{sup -1} cadmium caused a significant decrease in the survival time of C. gigas, but exposure to 500 ng L{sup -1} surprisingly affected the survival time positively. Significant differences in aneuploidy level were observed between the cadmium treatments and the control in adults but not in juveniles or the offspring of the adult groups. The effects of cadmium on hemocyte parameters were analyzed by flow cytometry. Several hemocyte parameters increased significantly after 21 days of cadmium exposure and subsequently decreased. Phenoloxidase-like activity, evaluated by spectrophotometry, varied over the time of the experiment and increased after 66 days of contact with 500 ng L{sup -1} cadmium. Taken together, cadmium at environmentally relevant concentrations seems to have only moderate effects on aneuploidy and hemocyte parameters.

  19. Latex agglutination: diagnose the early cryptococcus neoformans test of capsular polysaccharide antigen.

    Science.gov (United States)

    Wang, Huanrong; Yuan, Xueqian; Zhang, Lifeng

    2015-01-01

    This paper aims to discuss the early diagnosis value of latex agglutination test in Cryptococcal meningitis. The cerebrospinal fluid (CSF) of 112 patients with definite Cryptococcal meningitis and 26 patients with tubercular meningitis and virus meningitis were collected, latex agglutination test is adopted to detect Cryptococcal capsular polysaccharide antigen. Then it was compared with fungal culture and direct microscopy method for evaluating the sensitivity and specificity of the diagnosis. The sensitivity of three methods including latex agglutination test, fungal culture and direct microscopy was 91.1%,69.6% and 73.2% respectively. The specificity of latex agglutination test was 96.0%, 100% and 100% respectively. That latex agglutination test to detect Cryptococcal capsular polysaccharide antigen could be taken as the early diagnostic method of Cryptococcus neoformans meningitis.

  20. Sperm-agglutinating antibodies and testicular morphology in fifty-nine men with azoospermia or cryptozoospermia.

    Science.gov (United States)

    Friberg, J; Kjessler, B

    1975-04-01

    The relationship between the state of the germinal epithelium and the type and titer of circulating sperm-agglutinating antibodies has been investigated in a series of 59 azoospermic or occasionally cryptozoospermic men. The patients were grouped according to the condition of the germinal epithelium as observed from testicular biopsy specimens, as well as to type and titer of circulating sperm-agglutinating antibodies investigated by a previously described microagglutination technique. Evidence is presented to suggest that the presence of mature spermatozoa in the testicular structures may be a prerequisite for the spontaneous production of circulating sperm-agglutinating antibodies, at least of the head-to-tail (H-T) agglutinating type. Furthermore, these circulating H-T sperm-agglutinating antibodies, once they are formed, do not seem to interfere adversely with the germinal epithelium of the carrier.

  1. Deep transcriptome sequencing of Pecten maximus hemocytes: a genomic resource for bivalve immunology.

    Science.gov (United States)

    Pauletto, Marianna; Milan, Massimo; Moreira, Rebeca; Novoa, Beatriz; Figueras, Antonio; Babbucci, Massimiliano; Patarnello, Tomaso; Bargelloni, Luca

    2014-03-01

    Pecten maximus, the king scallop, is a bivalve species with important commercial value for both fisheries and aquaculture, traditionally consumed in several European countries. Major problems in larval rearing, however, still limit hatchery-based seed production. High mortalities during early larval stages, likely related to bacterial pathogens, represent the most relevant bottleneck. To address this issue, understanding host defense mechanisms against microbes is extremely important. In this study next-generation RNA-sequencing was carried on scallop hemocytes. To enrich for immune-related transcripts, cDNA libraries from hemocytes challenged in vivo with inactivated-Vibrio anguillarum and in vitro with pathogen-associated molecular patterns, as well as unchallenged controls, were sequenced yielding 216,444,674 sequence reads. De novo assembly of the scallop hemocyte transcriptome consisted of 73,732 contigs (31% annotated). A total of 934 contigs encoded proteins with a known immune function, grouped into several functional categories. Particular attention was reserved to Toll-like receptors (TLRs), a family of pattern recognition receptors (PRRs) involved in non-self recognition. Through mining the scallop hemocyte transcriptome, at least four TLRs could be identified. The organization of canonical TLR domains demonstrated that single cysteine cluster and multiple cysteine cluster TLRs co-exist in this species. In addition, preliminary data concerning their mRNA level following bacterial challenge suggested that different members of this family could exhibit opposite responses to pathogenic stimuli. Finally, a global analysis of differential expression comparing gene-expression levels in in vitro and in vivo stimulated hemocytes against controls provided evidence on a large set of transcripts involved in the great scallop immune response.

  2. Immune responses and ultrastructural changes of hemocytes in freshwater crab Sinopotamon henanense exposed to elevated cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Qin Qin [College of Life Science, Shanxi University, Taiyuan, Shanxi, 030006 (China); Central Laboratory, Shanxi Provincial People' s Hospital, Taiyuan, Shanxi, 030012 (China); Qin Shengjuan [College of Life Science, Shanxi University, Taiyuan, Shanxi, 030006 (China); Wang Lan, E-mail: lanwang@sxu.edu.cn [College of Life Science, Shanxi University, Taiyuan, Shanxi, 030006 (China); Lei Wenwen [College of Life Science, Shanxi University, Taiyuan, Shanxi, 030006 (China)

    2012-01-15

    Cadmium (Cd) is one of the most toxic heavy metals that can impact immunological parameters in aquatic animals. To investigate the immunotoxicity and ultrastructural changes of hemocytes, specimens of Sinopotamon henanense were exposed to different concentrations of cadmium and the differences in immunologic parameters between Cd exposure groups and control groups were investigated. Total hemocyte count (THC) in Cd-exposure groups were decreased significantly when compared with the control groups, especially in the groups treated with higher Cd concentrations and longer exposure time, while no significant differences were observed in the proportions of the three types of hemocytes. Phenoloxidase (PO) activities were significantly higher in Cd-exposure groups than the control groups. Superoxide dismutase (SOD) activities gradually increased in 7.25 and 14.5 mg L{sup -1} Cd groups, but in other higher Cd groups, they showed first increase and following decrease with the exposure time prolonged. Acid phosphatase (ACP) activities were induced at 48 h, and then decreased, while alkaline phosphatase (AKP) activities increased gradually until 96 h. Electron microscopic results showed that nucleus, mitochondria and rough endoplasm recutulum (rER) of three types of hemocytes were sensitive to acute Cd toxicity. In Cd-exposed groups, chromatin condensation, nucleus deformation and nucleus envelope rupture were noted. Additionally, mitochondrial dilation and rER degranulation were observed in Cd-treated crabs. These results suggested that immune response and organelles of hemocyte of S. henanense were impacted by Cd exposure, and the changes of these immunologic parameters reflect changes in crab immune response capability consequent to Cd exposure.

  3. Imaging based agglutination measurement of magnetic micro-particles on a lab-on-a-disk platform

    DEFF Research Database (Denmark)

    Wantiya, P.; Burger, Robert; Alstrøm, Tommy Sonne;

    2014-01-01

    In this work we present a magnetic micro beads based agglutination assay on a centrifugal microfluidic platform. An imaging based method is used to quantify bead agglutination and measure the concentration of antibodies or C-reactive protein in solution.......In this work we present a magnetic micro beads based agglutination assay on a centrifugal microfluidic platform. An imaging based method is used to quantify bead agglutination and measure the concentration of antibodies or C-reactive protein in solution....

  4. Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata).

    Science.gov (United States)

    Yan, Fang; Zhang, Yueling; Jiang, Ruiping; Zhong, Mingqi; Hu, Zhong; Du, Hong; Lun, Jingsheng; Chen, Jiehui; Li, Yuanyou

    2011-01-01

    Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 μg/ml. Agglutination was inhibited by 50-200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ▵OmpA and ▵OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.

  5. Parasitism of Pieris rapae (Lepidoptera: Pieridae) by the endoparasitic wasp Pteromalus puparum (Hymenoptera:Pteromalidae): Effects of parasitism on differential hemocyte counts, micro-and ultra-structures of host hemocytes

    Institute of Scientific and Technical Information of China (English)

    Qian-Qian Zhang; Jia Huang; Jia-Ying Zhu; Gong-Yin Ye

    2012-01-01

    Parasitism by the endoparasitic wasp Pteromalus puparum (Hymenoptera:Pteromalidae) by using only its associated venom,can suppress the immunal responses of Pieris rapae (Lepidoptera:Pieridae).However,up to now,current knowledge of the mechanisms has been limited.The response of host hemocytes to parasitism was investigated using a combination of light and transmission electron microscopy (TEM).Five hemocyte types,prohemocytes (PRs),granulocytes (GRs),plasmatocytes (PLs),oenocytoids (OEs) and coagulocytes (COs),were observed and characterized from both unparasitized and parasitized Pieris rapae pupae.Light microscopy showed that both GRs and PLs became more round and spread abnormally after parasitism,whereas the shape of other types of hemocytes remained unaffected.In addition,the size of PRs and PLs became larger while OEs became smaller.The proportion of PRs significantly increased after parasitism and that of PLs decreased by 43.9%,but there was no significant increase of GRs and OEs.TEM showed that all types of hemocytes except COs were damaged to various degrees after parasitism,especially resulting in electron opaque cytoplasm and nucleus,fewer cell organelles of rough endoplasmic reticulum,mitochondria and vesicles.Our results indicate that parasitism by P.puparum affects differential hemocyte counts and structures of host hemocytes,particularly for GRs and PLs,which may be the main cause of the parasitoid suppressing host cellular immune responses.

  6. The Involvement of Hemocyte Prophenoloxidase in the Shell-Hardening Process of the Blue Crab, Callinectes sapidus.

    Directory of Open Access Journals (Sweden)

    Javier V Alvarez

    Full Text Available Cuticular structures of arthropods undergo dramatic molt-related changes from being soft to becoming hard. The shell-hardening process of decapod crustaceans includes sclerotization and mineralization. Hemocyte PPO plays a central role in melanization and sclerotization particularly in wound healing in crustaceans. However, little is known about its role in the crustacean initial shell-hardening process. The earlier findings of the aggregation of heavily granulated hemocytes beneath the hypodermis during ecdysis imply that the hemocytes may be involved in the shell-hardening process. In order to determine if hemocytes and hemocyte PPO have a role in the shell-hardening of crustaceans, a knockdown study using specific CasPPO-hemo-dsRNA was carried out with juvenile blue crabs, Callinectes sapidus. Multiple injections of CasPPO-hemo-dsRNA reduce specifically the levels of CasPPO-hemo expression by 57% and PO activity by 54% in hemocyte lysate at the postmolt, while they have no effect on the total hemocyte numbers. Immunocytochemistry and flow cytometry analysis using a specific antiserum generated against CasPPO show granulocytes, semigranulocytes and hyaline cells as the cellular sources for PPO at the postmolt. Interestingly, the type of hemocytes, as the cellular sources of PPO, varies by molt stage. The granulocytes always contain PPO throughout the molt cycle. However, semigranulocytes and hyaline cells become CasPPO immune-positive only at early premolt and postmolt, indicating that PPO expression in these cells may be involved in the shell-hardening process of C. sapidus.

  7. The Involvement of Hemocyte Prophenoloxidase in the Shell-Hardening Process of the Blue Crab, Callinectes sapidus.

    Science.gov (United States)

    Alvarez, Javier V; Chung, J Sook

    2015-01-01

    Cuticular structures of arthropods undergo dramatic molt-related changes from being soft to becoming hard. The shell-hardening process of decapod crustaceans includes sclerotization and mineralization. Hemocyte PPO plays a central role in melanization and sclerotization particularly in wound healing in crustaceans. However, little is known about its role in the crustacean initial shell-hardening process. The earlier findings of the aggregation of heavily granulated hemocytes beneath the hypodermis during ecdysis imply that the hemocytes may be involved in the shell-hardening process. In order to determine if hemocytes and hemocyte PPO have a role in the shell-hardening of crustaceans, a knockdown study using specific CasPPO-hemo-dsRNA was carried out with juvenile blue crabs, Callinectes sapidus. Multiple injections of CasPPO-hemo-dsRNA reduce specifically the levels of CasPPO-hemo expression by 57% and PO activity by 54% in hemocyte lysate at the postmolt, while they have no effect on the total hemocyte numbers. Immunocytochemistry and flow cytometry analysis using a specific antiserum generated against CasPPO show granulocytes, semigranulocytes and hyaline cells as the cellular sources for PPO at the postmolt. Interestingly, the type of hemocytes, as the cellular sources of PPO, varies by molt stage. The granulocytes always contain PPO throughout the molt cycle. However, semigranulocytes and hyaline cells become CasPPO immune-positive only at early premolt and postmolt, indicating that PPO expression in these cells may be involved in the shell-hardening process of C. sapidus.

  8. Apoptosis of Spodoptera litura larval hemocytes induced by heavy metal zinc

    Institute of Scientific and Technical Information of China (English)

    XIA Qiang; SUN Hongxia; HU Xinjun; SHU Yinghua; GU Dexiang; ZHANG Guren

    2005-01-01

    By adding different amount of zinc into the artificial medium of the insect larvae, the zinc-induced apoptosis of the larvae haemocytes of the herbivorous insect Spodoptera litura Fabricius was investigated with flow cytometer. The results showed that the increase of zinc dose in the artificial feed led to the accumulations of zinc in the larval hemolymph and fat body, and more zinc was accumulated in fat body than in hemolymph. The apoptosis of hemocytes was significantly induced at high zinc concentration (1000 mg·kg-1) in the insect diet, and the apoptosis rate was 63.63%, which was remarkably higher than that at control and lower concentrations (50-500 mg·kg-1). This suggests that the high dose of zinc in the artificial diet of S. Litura larvae could induce the apoptosis of the larval hemocytes of S. Litura.

  9. Halocyntin and papillosin, two new antimicrobial peptides isolated from hemocytes of the solitary tunicate, Halocynthia papillosa.

    Science.gov (United States)

    Galinier, Richard; Roger, Emmanuel; Sautiere, Pierre-Eric; Aumelas, Andre; Banaigs, Bernard; Mitta, Guillaume

    2009-01-01

    We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram-positive and Gram-negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP. Copyright 2008 European Peptide Society and John Wiley & Sons, Ltd.

  10. Nuclease enhancement of specific cell agglutination in a serodiagnostic test for Neisseria gonorrhoeae.

    Science.gov (United States)

    Arko, R J; Wong, K H; Peacock, W L

    1979-01-01

    Antiserum to a purified type R lipopolysaccharide antigen isolated from Neisseria gonorrhoeae was used in a slide agglutination test and compared with conventional carbohydrate utilization and fluorescent antibody tests to confirm the identity of laboratory cultures classified as typical or "atypical" N. gonorrhoeae. Cultures of Corynebacterium vaginalis, N. meningitidis, N. catarrhalis, N. sicca, and N. lactamicus were also tested in the slide agglutination procedure. The addition of both deoxyribonuclease and ribonuclease (1 mg/ml) to the cell suspension medium of phosphate-buffered saline improved the sensitivity and specificity of the agglutination reaction for N. gonorrhoeae. Problems relating to the agglutination test as an aid in identification of N. gonorrhoeae are discussed. PMID:110830

  11. A study of the incubation of microbead agglutination assays in a microfluidic system

    KAUST Repository

    Castro, David

    2016-12-19

    This work reports on a quantitative study of the incubation of a microbead-based agglutination assay inside a microfluidic system. In this system, a droplet (1.25µL) consisting of a mixture of functionalized microbeads and analyte is flowed through a 0.51mm internal diameter silicone tube. Hydrodynamic forces alone produce a very efficient mixing of the beads within the droplet. We tested the agglutination at different speeds and show a robust response at the higher range of speeds (150 – 200µL/min), while also reaching a completion in the agglutination process. At these velocities, a length of 180cm is shown to be sufficient to confidently measure the agglutination assay, which takes between 2.5 – 3 minutes. This high throughput quantification method has the potential of accelerating the measurements of various types of biomarkers, which can greatly benefit the fields of biology and medicine.

  12. A Microtitration Agglutination Test for Detecting Group E Streptococcus Infection in Swine

    OpenAIRE

    1982-01-01

    A microtitration agglutination test was developed and evaluated for detecting infection of swine with group E streptococci type IV, the most common causative agent of streptococcic lymphadenitis of swine.

  13. The Cenozoic Diversity of Agglutinated Foraminifera - Evidence for a late Oligocene to early Miocene diversification event

    Science.gov (United States)

    Kaminski, Michael; Setoyama, Eiichi; Kender, Sev; Cetean, Claudia

    2014-05-01

    The agglutinated foraminifera are among the most abundant micro-organisms in the deep marine environment and have a diversity record extending back to the late Precambrian. We present an updated diversity curve for agglutinated foraminiferal genera based on the stratigraphic ranges of all the agglutinated genera recognized as valid in the classification of Kaminski (2014). The data set for this analysis is based on the stratigraphic ranges of agglutinated genera published in Foraminiferal Genera and their Classification, which has been subsequently updated based on published studies and our new observations. The mean standing diversity of agglutinated foraminiferal genera was compiled by counting the number of boundary crossers rather than the number of genera in each stage. In this study, we report the stratigraphic and geographical occurrence of a benthic foraminiferal diversification event that has previously received little attention. In the latest Oligocene to earliest Miocene a number of trochospiral agglutinated genera with alveolar or canaliculate walls first appeared in the fossil record. Our studies of late Oligocene of the Congo fan, offshore Angola (Kender et al., 2008; Cetean and Kaminski, 2011) have revealed a diverse assemblage that includes new taxa of deep-water agglutinated foraminifera. In a biostratigraphic study of the Miocene foraminiferal assemblages Kender et al. (2008) noted steadily increasing diversity and proportions of infaunal agglutinated foraminiferal morphotypes over the lower Miocene interval. The proportion of infaunal agglutinated foraminifera assigned to the order Textularida increased dramatically in the lower mid-Miocene, suggesting expansion of the oxygen minimum zone into deeper waters. In addition to the trochospiral alveolar genera, several species of Reticulophragmium and Cyclammina display rapid diversification into numerous separate lineages that are at present not reflected in our generic diversity record owing to

  14. Venom of the ectoparasitoid, Nasonia vitripennis, influences gene expression in Musca domestica hemocytes.

    Science.gov (United States)

    Qian, Cen; Liu, Yang; Fang, Qi; Min-Li, Yan; Liu, Shu-Sheng; Ye, Gong-Yin; Li, Yan-Min

    2013-08-01

    Insect hosts have evolved potent innate immunity against invasion by parasitoid wasps. Host/parasitoids live in co-evolutionary relationships. Nasonia vitripennis females inject venom into their dipteran hosts just prior to laying eggs on the host's outer integument. The parasitoid larvae are ectoparasitoids because they feed on their hosts within the puparium, but do not enter the host body. We investigated the influence of N. vitripennis venom on the gene expression profile of hemocytes of their hosts, pupae of the housefly, Musca domestica. We prepared venom by isolating venom glands and treated experimental host pupae with venom. We used suppression subtractive hybridization (SSH) to determine the influence of venom on hemocyte gene expression. At 1 h post treatment, we recorded decreases in transcript levels of 133 EST clones derived from forward a subtractive library of host hemocytes and upregulation in transcript levels of 111 EST clones from the reverse library. These genes are related to immune and stress response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, and transcription/translation regulation. We verified the reliability of our data with reverse transcription quantitative real-time PCR analysis of randomly selected genes, and with assays of enzyme activities. These analyses showed that the expression level of all selected genes were downregulated after venom treatment. Outcomes of our experiments support the hypothesis that N. vitripennis venom influences the gene expression in host hemocytes. We conclude that the actions of venom on host gene expression influence host biology in ways that benefit the development and emergence of the next generation of parasitoids.

  15. Blood feeding induces hemocyte proliferation and activation in the African malaria mosquito, Anopheles gambiae Giles

    OpenAIRE

    Bryant, William B.; Michel, Kristin

    2014-01-01

    Malaria is a global public health problem, especially in sub-Saharan Africa, where the mosquito Anopheles gambiae Giles serves as the major vector for the protozoan Plasmodium falciparum Welch. One determinant of malaria vector competence is the mosquito's immune system. Hemocytes are a critical component as they produce soluble immune factors that either support or prevent malaria parasite development. However, despite their importance in vector competence, understanding of their basic biolo...

  16. Proteomic analysis of differentially expressed proteins in Fenneropenaeus chinensis hemocytes upon white spot syndrome virus infection.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available To elucidate molecular responses of shrimp hemocytes to white spot syndrome virus (WSSV infection, two-dimensional gel electrophoresis was applied to investigate differentially expressed proteins in hemocytes of Chinese shrimp (Fenneropenaeus chinensis at 24 h post infection (hpi. Approximately 580 protein spots were detected in hemocytes of healthy and WSSV-infected shrimps. Quantitative intensity analysis revealed 26 protein spots were significantly up-regulated, and 19 spots were significantly down-regulated. By mass spectrometry, small ubiquitin-like modifier (SUMO 1, cytosolic MnSOD, triosephosphate isomerase, tubulin alpha-1 chain, microtubule-actin cross-linking factor 1, nuclear receptor E75 protein, vacuolar ATP synthase subunit B L form, inositol 1,4,5-trisphosphate receptor, arginine kinase, etc., amounting to 33 differentially modulated proteins were identified successfully. According to Gene Ontology annotation, the identified proteins were classified into nine categories, consisting of immune related proteins, stimulus response proteins, proteins involved in glucose metabolic process, cytoskeleton proteins, DNA or protein binding proteins, proteins involved in steroid hormone mediated signal pathway, ATP synthases, proteins involved in transmembrane transport and ungrouped proteins. Meanwhile, the expression profiles of three up-regulated proteins (SUMO, heat shock protein 70, and arginine kinase and one down-regulated protein (prophenoloxidase were further analyzed by real-time RT-PCR at the transcription level after WSSV infection. The results showed that SUMO and heat shock protein 70 were significantly up-regulated at each sampling time point, while arginine kinase was significantly up-regulated at 12 and 24 hpi. In contrast, prophenoloxidase was significantly down-regulated at each sampling time point. The results of this work provided preliminary data on proteins in shrimp hemocytes involved in WSSV infection.

  17. Miocene deep water agglutinated foraminifera from Viosca Knoll, offshore Louisiana (Gulf of Mexico)

    OpenAIRE

    Green, R C; Kaminski, M.A.; Sikora, P. J.

    2004-01-01

    An exploration well from the Gulf of Mexico, Amoco Viosca Knoll-915, has been studied in order to document the Neogene foraminiferal assemblages. Ditch cuttings samples from the Amoco V.K. 915 well yielded diverse assemblages of agglutinated and calcareous benthic foraminifera over a stratigraphic interval of 2940 m. Three species associations can be identified in the studied interval; the stratigraphical location of these associations is evident when total agglutinated species...

  18. Problems with rapid agglutination methods for identification of Staphylococcus aureus when Staphylococcus saprophyticus is being tested.

    Science.gov (United States)

    Gregson, D B; Low, D E; Skulnick, M; Simor, A E

    1988-01-01

    Six rapid agglutination tests for identification of Staphylococcus aureus were evaluated by using 62 strains of S. aureus, 63 strains of S. saprophyticus, and 67 strains of other coagulase-negative staphylococci. S. saprophyticus was responsible for 19 of 26 false-positive results and 20 uninterpretable reactions. Thus, urinary staphylococcal isolates that are positive by rapid agglutination tests may require other confirmatory tests for the identification of possible S. saprophyticus. PMID:3410950

  19. Problems with rapid agglutination methods for identification of Staphylococcus aureus when Staphylococcus saprophyticus is being tested.

    OpenAIRE

    Gregson, D B; Low, D E; Skulnick, M; Simor, A. E.

    1988-01-01

    Six rapid agglutination tests for identification of Staphylococcus aureus were evaluated by using 62 strains of S. aureus, 63 strains of S. saprophyticus, and 67 strains of other coagulase-negative staphylococci. S. saprophyticus was responsible for 19 of 26 false-positive results and 20 uninterpretable reactions. Thus, urinary staphylococcal isolates that are positive by rapid agglutination tests may require other confirmatory tests for the identification of possible S. saprophyticus.

  20. Baculovirus Induced Transcripts in Hemocytes from the Larvae of Heliothis virescens

    Directory of Open Access Journals (Sweden)

    Holly J.R. Popham

    2011-10-01

    Full Text Available Using RNA-seq digital difference expression profiling methods, we have assessed the gene expression profiles of hemocytes harvested from Heliothis virescens that were challenged with Helicoverpa zea single nucleopolyhedrovirus (HzSNPV. A reference transcriptome of hemocyte-expressed transcripts was assembled from 202 million 42-base tags by combining the sequence data of all samples, and the assembled sequences were then subject to BLASTx analysis to determine gene identities. We used the fully sequenced HzSNPV reference genome to align 477,264 Illumina sequence tags from infected hemocytes in order to document expression of HzSNPV genes at early points during infection. A comparison of expression profiles of control insects to those lethally infected with HzSNPV revealed differential expression of key cellular stress response genes and genes involved in lipid metabolism. Transcriptional regulation of specific insect hormones in baculovirus-infected insects was also altered. A number of transcripts bearing homology to retroviral elements that were detected add to a growing body of evidence for extensive invasion of errantiviruses into the insect genome. Using this method, we completed the first and most comprehensive gene expression survey of both baculoviral infection and host immune defense in lepidopteran larvae.

  1. The immune response of hemocytes of the insect Oncopeltus fasciatus against the flagellate Phytomonas serpens.

    Directory of Open Access Journals (Sweden)

    Thiago L Alves e Silva

    Full Text Available The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites.

  2. CD63 Promotes Hemocyte-Mediated Phagocytosis in the Clam, Paphia undulata

    Directory of Open Access Journals (Sweden)

    Mingjia Yu

    2016-01-01

    Full Text Available As one of the surface membrane proteins of tetraspanin family, CD63 plays a crucial role in cellular trafficking and endocytosis, which also is associated with activation of a wide variety of immune cells. Here, the homolog of CD63 was characterized from one marine mollusk, Paphia undulata, which is designated as Pu-CD63. The complete cDNA of Pu-CD63 is 1,738 bp in length with an open reading frame (ORF of 849 bp, encoding a 282 amino acid protein with four putative hydrophobic transmembrane helixes. Bioinformatic analysis revealed that Pu-CD63 contains one putative YXXØ consensus motif of “110-YVII-113” and one N-glycosylation site “155-NGT-157” within the large extracellular loop (LEL region, supporting its conserved function in plasma membrane and endosomal/lysosomal trafficking. Moreover, temporal expression profile analysis demonstrates a drastic induction in the expression of CD63 in hemocytes after pathogenic challenge with either V. parahaemolyticus or V. alginolyticus. By performing dsRNA-mediate RNAi knockdowns of CD63, a dramatic reduction in hemocytes phagocytic activity to pathogenic Vibrio is recorded by flow cytometry, revealing the definite role of Pu-CD63 in promoting hemocyte-mediated phagocytosis. Therefore, our work has greatly enhanced our understanding about primitive character of innate immunity in marine mollusk.

  3. Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophila

    Directory of Open Access Journals (Sweden)

    Margherita Anna Barracco

    1993-03-01

    Full Text Available The blood cells of the pulmonate snail Biomphalaria tenagophila, an important transmiter of the trematode Schistosoma mansoni in Brazil, were examined by ligth and transmission electron microscopy (TEM. Two hemocyte types were identified: hyalinocytes and granulocytes. Hyalinocytes are small young (immature, poorly spreading cells, which have a high nucleocytoplasmic ratio and are especially rich in free ribosomes. They do not appear to contain lysosome-like bodies and represent less than 10% of the circulating hemocytes. Granulocytes are larger hemocytes which readily spread on glass surface and which strongly react to the Gomori substrate, indicating the enzyme acid phosphatase usually found in lysosomes. Ultra-structurally, they contain a well-developed rough endoplasmic reticulum, dictyosomes and some some lysosome-like dense bodies. Granulocytes do not exhibit a characteristic granular aspect and the few granules observed in the cytoplasm should correspond to a lysosome system. They were named granulocytes instead of amoebocytes to use the same terminology adopted for Biomphalaria glabrata in order to make easier comparative studies. This is a preface study for more specific investigations on the functional activities of the blood cells of B. tenagophila and their interactions with the trematode parasite.

  4. Effect of gamma irradiation on the hemocyte-mediated immune response of Aedes aegypti against microfilariae

    Energy Technology Data Exchange (ETDEWEB)

    Christensen, B.M.; Huff, B.M.; Li, J. (Univ. of Wisconsin, Madison (USA))

    1990-07-01

    The effect of gamma irradiation on the melanotic encapsulation response of Aedes aegypti black eye Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 2, 3, and 6 days postinoculation (PI). Mosquitoes received 6000 rad from a 137Cs source (Shepard Mark I irradiator) at 3 days postemergence and were inoculated with 15-20 mff 24 hr later. These mosquitoes were compared to nonirradiated controls that also were inoculated with 15-20 mff at 3 days postemergence. The immune response was significantly reduced in irradiated mosquitoes as compared with controls at all days PI. Although the response was significantly inhibited compared with controls, irradiated mosquitoes were still capable of eliciting a response against 69% of recovered mff at 6 days PI. External gamma irradiation did not significantly affect the proliferation of hemocytes associated with the melanotic encapsulation response of A. aegypti. The number of circulating hemocytes increased in irradiated mosquitoes in response to inoculated mff in a manner similar to nonirradiated, inoculated controls. Hemocyte monophenol oxidase activity, however, was significantly reduced in gamma-irradiated mosquitoes at 12 hr PI as compared with controls. The reduced immunological capacity of irradiated mosquitoes might be related to an interference with gene activity required for the synthesis or activation of enzymes that are directly or indirectly involved in the biochemical processes associated with the production of melanotic substances that sequester mff.

  5. Comprehensive Study of the Influence of Altered Gravity on the Oxidative Burst of Mussel ( Mytilus edulis) Hemocytes

    Science.gov (United States)

    Unruh, E.; Brungs, S.; Langer, S.; Bornemann, G.; Frett, T.; Hansen, P.-D.

    2016-06-01

    Microgravity induces alterations in the functioning of immune cell; however, the underlying mechanisms have not yet been identified. In this study, hemocytes (blood cells) of the blue mussel Mytilus edulis were investigated under altered gravity conditions. The study was conducted on the ground in preparation for the BIOLAB TripleLux-B experiment, which will be performed on the International Space Station (ISS). On-line kinetic measurements of reactive oxygen species (ROS) production during the oxidative burst and thus cellular activity of isolated hemocytes were performed in a photomultiplier (PMT)-clinostat (simulated microgravity) and in the 1 g operation mode of the clinostat in hypergravity on the Short-Arm Human Centrifuge (SAHC) as well as during parabolic flights. In addition to studies with isolated hemocytes, the effect of altered gravity conditions on whole animals was investigated. For this purpose, whole mussels were exposed to hypergravity (1.8 g) on a multi-sample incubator centrifuge (MuSIC) or to simulated microgravity in a submersed clinostat. After exposure for 48 h, hemocytes were taken from the mussels and ROS production was measured under 1 g conditions. The results from the parabolic flights and clinostat studies indicate that mussel hemocytes respond to altered gravity in a fast and reversible manner. Hemocytes (after cryo-conservation) exposed to simulated microgravity ( μ g), as well as fresh hemocytes from clinorotated animals, showed a decrease in ROS production. Measurements during a permanent exposure of hemocytes to hypergravity (SAHC) show a decrease in ROS production. Hemocytes of mussels measured after the centrifugation of whole mussels did not show an influence to the ROS response at all. Hypergravity during parabolic flights led to a decrease but also to an increase in ROS production in isolated hemocytes, whereas the centrifugation of whole mussels did not influence the ROS response at all. This study is a good example how

  6. Conventional rapid latex agglutination in estimation of von Willebrand factor: method revisited and potential clinical applications.

    Science.gov (United States)

    Mahat, Marianor; Abdullah, Wan Zaidah; Hussin, Che Maraina Che

    2014-01-01

    Measurement of von Willebrand factor antigen (VWF : Ag) levels is usually performed in a specialised laboratory which limits its application in routine clinical practice. So far, no commercial rapid test kit is available for VWF : Ag estimation. This paper discusses the technical aspect of latex agglutination method which was established to suit the purpose of estimating von Willebrand factor (VWF) levels in the plasma sample. The latex agglutination test can be performed qualitatively and semiquantitatively. Reproducibility, stability, linearity, limit of detection, interference, and method comparison studies were conducted to evaluate the performance of this test. Semiquantitative latex agglutination test was strongly correlated with the reference immunoturbidimetric assay (Spearman's rho = 0.946, P agglutination test and the reference assay. Using the scoring system for the rapid latex test, no agglutination is with 0% VWF : Ag (control negative), 1+ reaction is equivalent to 150% VWF : Ag (when comparing with immunoturbidimetric assay). The findings from evaluation studies suggest that latex agglutination method is suitable to be used as a rapid test kit for the estimation of VWF : Ag levels in various clinical conditions associated with high levels and low levels of VWF : Ag.

  7. Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood.

    Science.gov (United States)

    McAdow, Molly; Kim, Hwan Keun; Dedent, Andrea C; Hendrickx, Antoni P A; Schneewind, Olaf; Missiakas, Dominique M

    2011-10-01

    Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci--coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA)--were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans.

  8. A Novel Viral Responsive Protein Is Involved in Hemocyte Homeostasis in the Black Tiger Shrimp, Penaeus monodon*

    Science.gov (United States)

    Prapavorarat, Adisak; Vatanavicharn, Tipachai; Söderhäll, Kenneth; Tassanakajon, Anchalee

    2010-01-01

    A novel viral responsive protein, namely hemocyte homeostasis-associated protein (HHAP), was characterized for its role in the response of shrimp to white spot syndrome virus infection. The full-length cDNAs of HHAP from the black tiger shrimp (PmHHAP), Penaeus monodon, and the fresh water crayfish (PlHHAP), Pacifastacus leniusculus, were obtained and showed high sequence identity to a hypothetical protein from various organisms, with the highest identity to the hypothetical protein TcasGA2_TC006773 from the red flour beetle, Tribolium castaneum (54% amino acid sequence identity). Transcripts of PmHHAP were expressed in various shrimp tissues with the highest expression in hematopoietic tissue, whereas the transcripts of PlHHAP were found in the hematopoietic and nerve tissues. Upon white spot syndrome virus infection, a high up-regulation level of shrimp hemocytic HHAP mRNA and protein was observed by real-time reverse transcription-PCR and immunofluorescence microscopy, respectively. Gene silencing of PmHHAP by RNA interference resulted in a significant decrease in the number of circulating hemocytes and 100% shrimp mortality within 30 h of the double-stranded PmHHAP RNA injection (but not in control shrimp), indicating that HHAP is essential for shrimp survival. Interestingly, severe damage of hemocytes was observed in vivo in the PmHHAP knockdown shrimp and in vitro in shrimp primary hemocyte cell culture, suggesting that PmHHAP plays an important role in hemocyte homeostasis. Thus, it is speculated that the up-regulation of PmHHAP is an important mechanism to control circulating hemocyte levels in crustaceans during viral infection. PMID:20444692

  9. Hemiuroid trematode sporocysts are undetected by hemocytes of their intermediate host, the ark cockle Anadara trapezia: potential role of surface carbohydrates in successful parasitism.

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    Kawasaki, Minami; Delamare-Deboutteville, Jerome; Dang, Cecile; Barnes, Andrew C

    2013-12-01

    In order to establish a successful relationship with their hosts, parasites must subvert or evade immune defences. Cockle Anadara trapezia and Sydney Rock oyster (SRO) Saccostrea glomerata live in the same location but only ark cockles are infected by sporocysts of hemiuroid trematode. This provides an opportunity to explore differing interactions between the parasite and the immune system of susceptible and refractive hosts. Rapid migration and encapsulation of sporocysts was observed by SRO hemocytes but not by cockle hemocytes. This migration/encapsulation was inhibited by N-acetylglucosamine or N-acetylgalactosamine but not by the other sugars, implicating specific surface carbohydrates in immune detection. Effector responses of hemocytes were investigated in vitro in terms of production of reactive oxygen production (ROS). Hemocytes of both species strongly reacted to Zymosan, but only SRO hemocytes responded to live sporocysts. Neither species' hemocytes produced ROS in the presence of dead/fixed sporocysts, and there was no suppression of Zymosan-induced respiratory burst by sporocysts. This suggests that immune escape is mediated by avoiding encapsulation, perhaps through molecular mimicry. Membrane-shaving with proteases indicated that sporocyst surface proteins are not a key factors in hemocytic detection. Surface carbohydrates of SRO and cockle hemocytes and of sporocysts were profiled with a panel of biotinylated lectins. This revealed substantial differences between cockle and SRO hemocytes, but greater similarity between cockle hemocytes and sporocysts. Results suggest that surface carbohydrates play an integral role in hemocyte immunorecognition and that surface carbohydrate molecular mimicry is a potential strategy for immune evasion in cockles by hemiuroid trematode sporocysts.

  10. Simultaneous detection of pathogenic bacteria using agglutination test based on colored silica nanoparticles.

    Science.gov (United States)

    Yu, Hui; Zhao, Guangying; Dou, Wenchao

    2015-01-01

    Aimed to explore an agglutination test which can simultaneously detect two pathogenic bacteria, an agglutination test based on colored silica nanoparticles (colored-SiNps) was established in this work. Monodisperse colored-SiNps were used as agglutination test carriers; red-SiNps and blue-SiNps were prepared by reverse microemulsion with C.I. Reactive red 136 and C.I. Reactive Blue 14. Then the red-SiNps were sensitized with antibodies against E. sakazaki and denoted as IgG-red-SiNps; The blue-SiNps were coated with antibodies against S. pullorum and S. Gallinarum and denoted as IgGblue- SiNps. The mixture solution of IgG-red-SiNps and IgG-blue-SiNps could simultaneously agglutinate with E. sakazakii and S. pullorum and S. gallinarum on glass slide. The E. sakazakii and S. pullorum and S. gallinarum could be simultaneously detected by agglutination test with obvious agglutination phenomena. The E. sakazakii and S. pullorum and S. gallinarum could both be detected in a range from 4×10(3) to 4×10(9) CFU/mL. The pullorum and S. gallinarum and E. sakazakii in the infected food sample were detected by mixture solution of IgG-red-SiNps and IgG-blue-SiNps too. This agglutination test was easy and rapid, it might be useful for in situ rapid detection method for simultaneously screening different pathogenic microorganisms of foods and feeds in the field.

  11. Cerebrospinal Fluid Treponema pallidum Particle Agglutination Assay for Neurosyphilis Diagnosis.

    Science.gov (United States)

    Marra, Christina M; Maxwell, Clare L; Dunaway, Shelia B; Sahi, Sharon K; Tantalo, Lauren C

    2017-06-01

    Limited data suggest that the cerebrospinal fluid Treponema pallidum particle agglutination assay (CSF-TPPA) is sensitive and a CSF Treponema pallidum hemagglutination assay (CSF-TPHA) titer of ≥1:640 is specific for neurosyphilis diagnosis. CSF-TPPA reactivity and titer were determined for a convenience sample of 191 CSF samples from individuals enrolled in a study of CSF abnormalities in syphilis (training data set). The sensitivity of a reactive test and the specificity for reactivity at serial higher CSF dilutions were determined. Subsequently, CSF-TPPA reactivity at a 1:640 dilution was determined for all available samples from study participants enrolled after the last training sample was collected (validation data set, n = 380). Neurosyphilis was defined as (i) a reactive CSF Venereal Disease Research Laboratory test (CSF-VDRL), (ii) detection of T. pallidum in CSF by reverse transcriptase PCR, or (iii) new vision loss or hearing loss. In the training data set, the diagnostic sensitivities of a reactive CSF fluorescent treponemal antibody absorption test (CSF-FTA-ABS) and a reactive CSF-TPPA did not differ significantly (67 to 98% versus 76 to 95%). The specificity of a CSF-TPPA titer of ≥1:640 was significantly higher than that of lower dilutions and was not significantly different from that of CSF-VDRL. In the validation data set, the diagnostic specificity of a CSF-TPPA titer of ≥1:640 was high and did not differ significantly from that of CSF-VDRL (93 to 94% versus 90 to 91%). Ten CSF samples with a nonreactive CSF-VDRL had a CSF-TPPA titer of ≥1:640. If a CSF-TPPA titer of ≥1:640 was used in addition to a reactive CSF-VDRL, the number of neurosyphilis diagnoses would have increased from 47 to 57 (21.3%). A CSF-TPPA titer cutoff of ≥1:640 may be useful in identifying patients with neurosyphilis when CSF-VDRL is nonreactive. Copyright © 2017 American Society for Microbiology.

  12. Exposure to the Neurotoxic Dinoflagellate, Alexandrium catenella, Induces Apoptosis of the Hemocytes of the Oyster, Crassostrea gigas

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    Walid Medhioub

    2013-12-01

    Full Text Available This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7 at 29 h, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD and initiation-phase (caspase-2 was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70 inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster.

  13. Flow cytometry studies on the Macrobrachium rosenbergii hemocytes sub-populations and immune responses to novel pathogen spiroplasma MR-1008.

    Science.gov (United States)

    Du, Jie; Zhu, Huanxi; Ren, Qian; Liu, Peng; Chen, Jing; Xiu, Yunji; Yao, Wei; Meng, Qingguo; Gu, Wei; Wang, Wen

    2012-10-01

    Flow cytometry provides rapid and reproducible methods for analyzing crustacean cellular immune responses to pathogens. We used this method to investigate the hemocytes sub-populations of freshwater prawn Macrobrachium rosenbergii and their immune responses to a novel pathogen spiroplasma MR-1008. M. rosenbergii inoculated with 100 μl spiroplasma strain MR-1008 in logarithmic phase (10(8) spiroplasmas ml(-1)) were examined for total hemocytes count (THC) and changes in differential involvement of hemocytes sub-populations during 1-28 d after inoculation. The results showed that THC was dramatically lowered 1 d after inoculation, and it obviously increased at the 5 d after inoculation; thereafter, a high level of THC was maintained to 15 d. Three morphologically distinct hemocytes sub-populations including granular cells (GC), semigranular cells (SGC) and hyaline cells (HC) could be identified by flow cytometry, and the proportions of the 3 kinds of cell categories varied obviously during the infection of spiroplasma suggesting differential involvement according to the pathogen. The flow cytometry used in this study confirmed that the semigranular cells were the main hemocytes involved in the cellular defense against spiroplasma in the M. rosenbergii.

  14. Hypoxia drives apoptosis independently of p53 and metallothionein transcript levels in hemocytes of the whiteleg shrimp Litopenaeus vannamei.

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    Felix-Portillo, Monserrath; Martínez-Quintana, José A; Arenas-Padilla, Marina; Mata-Haro, Verónica; Gómez-Jiménez, Silvia; Yepiz-Plascencia, Gloria

    2016-10-01

    The cellular mechanisms used by the shrimp Litopenaeus vannamei to respond to hypoxia have been studied from the energetic metabolism and antioxidant angles. We herein investigated the participation of p53 and metallothionein (MT) in the apoptotic process in response to hypoxia in shrimp hemocytes. The Lvp53 or LvMT genes were efficiently silenced by injection of double stranded RNA for p53 or MT. The effects of silencing on apoptosis were measured as caspase-3 activity and flow cytometry in hemocytes after 24 and 48 h of hypoxia (1.5 mg DO L(-1)). Hemocytes from unsilenced animals had significantly higher apoptosis levels upon both times of hypoxia. The apoptotic levels were diminished but not suppressed in dsp53-silenced but not dsMT-silenced hemocytes after 24 h of hypoxia, indicating a contribution of Lvp53 to apoptosis. Apoptosis in normoxia was significantly higher in dsp53-and dsMT-silenced animals compared to the unsilenced controls, pointing to a possible cytoprotective role of LvMT and Lvp53 during the basal apoptotic program in normoxia. Overall, these results indicate that hypoxia augments apoptosis in shrimp hemocytes and high mRNA levels of Lvp53 and LvMT are not necessary for this response.

  15. Cellular and Transcriptional Responses of Crassostrea gigas Hemocytes Exposed in Vitro to Brevetoxin (PbTx-2

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    Alcir Luiz Dafre

    2012-03-01

    Full Text Available Hemocytes mediate a series of immune reactions essential for bivalve survival in the environment, however, the impact of harmful algal species and their associated phycotoxins upon bivalve immune system is under debate. To better understand the possible toxic effects of these toxins, Crassostrea gigas hemocytes were exposed to brevetoxin (PbTx-2. Hemocyte viability, monitored through the neutral red retention and MTT reduction assays, and apoptosis (Hoechst staining remained unchanged during 12 h of exposure to PbTx-2 in concentrations up to 1000 µg/L. Despite cell viability and apoptosis remained stable, hemocytes incubated for 4 h with 1000 µg/L of PbTx-2 revealed higher expression levels of Hsp70 (p < 0.01 and CYP356A1 ( p < 0.05 transcripts and a tendency to increase FABP expression, as evaluated by Real-Time quantitative PCR. The expression of other studied genes (BPI, IL-17, GSTO, EcSOD, Prx6, SOD and GPx remained unchanged. The results suggest that the absence of cytotoxic effects of PbTx-2 in Crassostrea gigas hemocytes, even at high concentrations, allow early defense responses to be produced by activating protective mechanisms associated to detoxification (CYP356A1 and possibly FABP and stress (Hsp70, but not to immune or to antioxidant (BPI, IL-17, EcSOD, Prx6, GPx and SOD related genes.

  16. Control of sperm concentration is necessary for standardization of sperm cryopreservation in aquatic species: evidence from sperm agglutination in oysters.

    Science.gov (United States)

    Dong, Qiaoxiang; Huang, Changjiang; Tiersch, Terrence R

    2007-02-01

    A lack of standardization in sperm cryopreservation of aquatic organisms is one of the main reasons for inconsistency observed among various studies. In particular, there have been few attempts to standardize sperm concentration during procedural optimization. This study was intended to call attention to sperm concentration standardization through research of sperm agglutination in Pacific oysters Crassostrea gigas. Sperm agglutination after thawing is a relatively frequent phenomenon observed for various aquatic species, especially when sub-optimal cryopreservation protocols are used; however, no systematic attempts have been made to explain this phenomenon. The present study evaluated various factors affecting sperm agglutination of thawed samples from diploid and tetraploid Pacific oysters, and is the first detailed report addressing the sperm agglutination phenomenon of thawed samples from any aquatic organism. Agglutination of oyster sperm was classified into six levels with a scale ranging from 0 (homogenous suspension) to 5 (well-developed "noodles"). It was found that agglutination in thawed samples was mainly due to the lack of sufficient cryoprotectant for a specific sperm concentration. Interestingly, high levels of agglutination did not necessarily lead to low fertilization. On the contrary, some sperm cells appeared to gain protection from the formation of peripheral agglutination within 0.5-ml French straws. The exact mechanism of sperm agglutination remains unclear. However, morphological examination of cross sections of the noodles (agglutination level 5) indicated at least two forms of agglutination (formed with and without cryoprotectant) which could be used as a tool to understand the cryopreservation process within the micro-environment of the straw. Furthermore, the fact that the level of sperm agglutination was directly determined by sperm concentration, in addition to the type of cryoprotectant, cryoprotectant concentration, and cooling and

  17. Microplate Agglutination Test for Canine Brucellosis Using Recombinant Antigen-Coated Beads.

    Science.gov (United States)

    Castillo, Yussaira; Tachibana, Masato; Kimura, Yui; Kim, Suk; Ichikawa, Yasuaki; Endo, Yasuyuki; Watanabe, Kenta; Shimizu, Takashi; Watarai, Masahisa

    2014-01-01

    Brucella canis, a facultative intracellular pathogen, is the causative agent of canine brucellosis. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods, including agglutination and gel diffusion tests. In this study, four recombinant antigens, heat shock protein 60, rhizopine-binding protein, Cu-Zn superoxide dismutase, and hypothetical protein (Ag 4), were constructed. These antigens were coated on latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. All recombinant antigens showed specific reaction with sera from B. canis-infected dogs in Western blotting. In a microplate agglutination test, mixing sera from B. canis-infected dogs, but not sera from B. canis-free dogs, with single or multiple antigens-coated latex beads produced clear agglutination. Moreover, the antigen-coated latex beads did not show nonspecific agglutination in hemolyzed serum samples. A survey of canine serum samples conducted by the microplate agglutination test using single antigen-coated latex beads showed that this method would be useful in the serological diagnosis of canine brucellosis. Further investigations using more serum samples are required to confirm the usefulness of our method.

  18. Dodecamer is required for agglutination of Litopenaeus vannamei hemocyanin with bacterial cells and red blood cells.

    Science.gov (United States)

    Pan, Jian-yi; Zhang, Yue-ling; Wang, San-ying; Peng, Xuan-xian

    2008-01-01

    Hemocyanins are multi-functional proteins, although they are well known to be respiratory proteins of invertebrate to date. In the present study, the agglutination ability of two oligomers of hemocyanin, hexamer and dodecamer, with pathogenic bacteria and red blood cells (RBCs) is investigated in pacific white shrimp, Litopenaeus vannamei. Hexameric hemocyanin exhibits an extremely high stability even in the absence of Ca(2+) and in alkaline pH. Dodecamer (di-hexamer) is easily dissociated into hexamers in unphysiological conditions. Hexamer and dodecamer are interchanged reciprocally with environmental conditions. Both oligomers can bind to bacteria and RBCs, but agglutination is observed only using dodecamer but not using hexamer in agglutination assay. However, the agglutination is detected when hexamer is utilized in the presence of antiserum against hemocyanin. These results indicate that dodecamer of hemocyanin is required for agglutination with bacteria and RBCs. It can be logically inferred that there is only one carbohydrate-binding site to bacterial cells and RBCs in the hexamer, while at least two sites in the dodecamer. Our finding has provided new insights into structural-functional relationship of hemocyanin.

  19. Rheologic characterization of vegetal lectins by dissociation of induced erythrocyte agglutinates.

    Science.gov (United States)

    Rasia, R J; Valverde, J R; Gentils, M; Cauchois, C; Stoltz, J F

    1997-01-01

    Energy evolved from hemagglutination reaction or spent in dissociating erythrocyte agglutinates has been proved to be an excellent parameter for analyzing cell-cell interactions mediated by bridging molecules such as antibodies or lectins. We developed a new rheo-optical method to estimate the energy of dissociation of red blood cell agglutinates. In a Couette shear field agglutinates can be dissociated until a suspension of monodispersed cells is obtained. Intensity of light backscattered by suspended agglutinates increases during their mechanical dissociation. Variation of backscattered light intensity correlates with the energy spent in the process. The adhesive energy of erythrocyte agglutination induced by lectins has been estimated by applying this method. Two specific lectins (Dolichus Biflorus agglutinin and Ulex Europaeus agglutinin) and a new lectin obtained from Amarantus Cruentus seeds which specificity is unknown were studied. Results obtained in this work for Dolichus Biflorus lectin are comparable with values published by other authors. An asymptotic decrease of adhesive energy was observed when the mechanical dissociation was applied several times on the same sample. Our results suggest that the cell detachment is accompanied by the extraction of membrane receptors. This finding is consistent with results obtained by other authors.

  20. Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae).

    Science.gov (United States)

    Giannoulis, Paschalis; Brooks, Cory L; Dunphy, Gary B; Mandato, Craig A; Niven, Donald F; Zakarian, Robert J

    2007-01-01

    Malacosoma disstria larvae are a pest of deciduous trees. Little is known on the interaction of bacteria with the immediate hemocytic antimicrobial responses of these insects. Incubating dead Xenorhabdus nematophila and Bacillus subtilis with a mixture of serum-free granular cells and plasmatocytes in vitro revealed differential bacterial-hemocyte adhesion and differential discharge of lysozyme and phenoloxidase but not total protein. Although active phenoloxidase adhered equally to both bacterial species, X. nematophila limited enzyme activation whereas B. subtilis enhanced activation. Serum with active phenoloxidase (as opposed to tropolone-inhibited phenoloxidase) and purified insect lysozyme increased bacterial-hemocyte adhesion of both bacterial species. An apolipophorin-III-like protein when incubated with hemocytes, limited their responses to glass slides and bacterial adhesion. However, initial binding of the protein to both bacteria increased granular cell levels with bacteria while lowering the plasmatocyte levels with adhering procaryotes. The protein also increased lysozyme and phenoloxidase activities. Although B. subtilis in vivo elicited a nodulation-based decline in total hemocyte counts and did not affect hemocyte viability, dead X. nematophila elevated hemocyte counts and damaged the hemocytes as lipopolysaccharide levels increased and X. nematophila emerged into the hemolymph. Apolipophorin-III-like protein once bound to the bacteria slowed their removal from the hemolymph.

  1. Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies.

    Science.gov (United States)

    Duressa, Tewodros Firdissa; Huybrechts, Roger

    2016-01-01

    Insect cell cultures played central roles in unraveling many insect physiological and immunological processes. Regardless, despite imminent needs, insect cell lines were developed primarily from Dipteran and Lepidopteran orders, leaving many important insects such as Orthopteran locusts under-represented. Besides the lack of cell lines, the slow progress in development of in vitro techniques is attributed to poor communications between different laboratories regarding optimized primary cell cultures. Therefore, we report here about methods developed for primary cell culture of Locusta migratoria hemocyte and phagocytic tissue cells by which we could maintain viable hemocytes in vitro for over 5 d and phagocytic tissue cells for over 12 d. 2-Mercaptoethanol and phenyl-thiourea supplements in Grace's medium together with addition of fetal bovine serum 30 min after cell seeding resulted in a successful setup of the primary cell cultures and a week-long survival of the hemocytes and phagocytic tissue cells in vitro.

  2. A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections

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    Rosa Rafael

    2012-06-01

    Full Text Available Abstract Background The complex balance between environmental and host factors is an important determinant of susceptibility to infection. Disturbances of this equilibrium may result in multifactorial diseases as illustrated by the summer mortality syndrome, a worldwide and complex phenomenon that affects the oysters, Crassostrea gigas. The summer mortality syndrome reveals a physiological intolerance making this oyster species susceptible to diseases. Exploration of genetic basis governing the oyster resistance or susceptibility to infections is thus a major goal for understanding field mortality events. In this context, we used high-throughput genomic approaches to identify genetic traits that may characterize inherent survival capacities in C. gigas. Results Using digital gene expression (DGE, we analyzed the transcriptomes of hemocytes (immunocompetent cells of oysters able or not able to survive infections by Vibrio species shown to be involved in summer mortalities. Hemocytes were nonlethally collected from oysters before Vibrio experimental infection, and two DGE libraries were generated from individuals that survived or did not survive. Exploration of DGE data and microfluidic qPCR analyses at individual level showed an extraordinary polymorphism in gene expressions, but also a set of hemocyte-expressed genes whose basal mRNA levels discriminate oyster capacity to survive infections by the pathogenic V. splendidus LGP32. Finally, we identified a signature of 14 genes that predicted oyster survival capacity. Their expressions are likely driven by distinct transcriptional regulation processes associated or not associated to gene copy number variation (CNV. Conclusions We provide here for the first time in oyster a gene expression survival signature that represents a useful tool for understanding mortality events and for assessing genetic traits of interest for disease resistance selection programs.

  3. Proteomic analysis of differentially expressed proteins in Penaeus monodon hemocytes after Vibrio harveyi infection

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    Fang Lo Chu

    2010-07-01

    Full Text Available Abstract Background Viral and bacterial diseases can cause mass mortalities in commercial shrimp aquaculture. In contrast to studies on the antiviral response, the responses of shrimps to bacterial infections by high throughput techniques have been reported only at the transcriptional level and not at the translational level. In this study, a proteomic analysis of shrimp hemocytes to identify differentially expressed proteins in response to a luminous bacterium Vibrio harveyi was evaluated for its feasibility and is reported for the first time. Results The two-dimensional gel electrophoresis (2-DE patterns of the hemocyte proteins from the unchallenged and V. harveyi challenged shrimp, Penaeus monodon, at 24 and 48 h post infection were compared. From this, 27 differentially expressed protein spots, and a further 12 weakly to non-differentially regulated control spots, were selected for further analyses by the LC-ESI-MS/MS. The 21 differentially expressed proteins that could be identified by homologous annotation were comprised of proteins that are directly involved in the host defense responses, such as hemocyanin, prophenoloxidase, serine proteinase-like protein, heat shock protein 90 and alpha-2-macroglobulin, and those involved in signal transduction, such as the14-3-3 protein epsilon and calmodulin. Western blot analysis confirmed the up-regulation of hemocyanin expression upon bacterial infection. The expression of the selected proteins which were the representatives of the down-regulated proteins (the 14-3-3 protein epsilon and alpha-2-macroglobulin and of the up-regulated proteins (hemocyanin was further assessed at the transcription level using real-time RT-PCR. Conclusions This work suggests the usefulness of a proteomic approach to the study of shrimp immunity and revealed hemocyte proteins whose expression were up regulated upon V. harveyi infection such as hemocyanin, arginine kinase and down regulated such as alpha-2-macroglobulin

  4. Jack bean (Canavalia ensiformis) urease induces eicosanoid-modulated hemocyte aggregation in the Chagas' disease vector Rhodnius prolixus.

    Science.gov (United States)

    Defferrari, M S; da Silva, R; Orchard, I; Carlini, C R

    2014-05-01

    Ureases are multifunctional proteins that display biological activities independently of their enzymatic function, such as induction of exocytosis and insecticidal effects. Rhodnius prolixus, a major vector of Chagas' disease, is a model for studies on the entomotoxicity of jack bean urease (JBU). We have previously shown that JBU induces the production of eicosanoids in isolated tissues of R. prolixus. In insects, the immune response comprises cellular and humoral reactions, and is centrally modulated by eicosanoids. Cyclooxygenase products signal immunity in insects, mainly cellular reactions, such as hemocyte aggregation. In searching for a link between JBU's toxic effects and immune reactions in insects, we have studied the effects of this toxin on R. prolixus hemocytes. JBU triggers aggregation of hemocytes after injection into the hemocoel and when applied to isolated cells. On in vitro assays, the eicosanoid synthesis inhibitors dexamethasone (phospholipase A2 indirect inhibitor) and indomethacin (cyclooxygenase inhibitor) counteracted JBU's effect, indicating that eicosanoids, more specifically cyclooxygenase products, are likely to mediate the aggregation response. Contrarily, the inhibitors esculetin and baicalein were inactive, suggesting that lipoxygenase products are not involved in JBU's effect. Extracellular calcium was also necessary for JBU's effect, in agreement to other cell models responsive to ureases. A progressive darkening of the medium of JBU-treated hemocytes was observed, suggestive of a humoral response. JBU was immunolocalized in the cultured cells upon treatment along with cytoskeleton damage. The highest concentration of JBU tested on cultured cells also led to nuclei aggregation of adherent hemocytes. This is the first time urease has been shown to affect insect hemocytes, contributing to our understanding of the entomotoxic mechanisms of action of this protein.

  5. Exposure to the toxic dinoflagellate Alexandrium catenella modulates juvenile oyster Crassostrea gigas hemocyte variables subjected to different biotic conditions.

    Science.gov (United States)

    Lassudrie, Malwenn; Soudant, Philippe; Nicolas, Jean-Louis; Miner, Philippe; Le Grand, Jacqueline; Lambert, Christophe; Le Goïc, Nelly; Hégaret, Hélène; Fabioux, Caroline

    2016-04-01

    The Pacific oyster Crassostrea gigas is an important commercial species cultured throughout the world. Oyster production practices often include transfers of animals into new environments that can be stressful, especially at young ages. This study was undertaken to determine if a toxic Alexandrium bloom, occurring repeatedly in French oyster beds, could modulate juvenile oyster cellular immune responses (i.e. hemocyte variables). We simulated planting on commercial beds by conducting a cohabitation exposure of juvenile, "specific pathogen-free" (SPF) oysters (naïve from the environment) with previously field-exposed oysters to induce interactions with new microorganisms. Indeed, toxic Alexandrium spp. exposures have been reported to modulate bivalve interaction with specific pathogens, as well as physiological and immunological variables in bivalves. In summary, SPF oysters were subjected to an artificial bloom of Alexandrium catenella, simultaneously with a cohabitation challenge. Exposure to A. catenella, and thus to the paralytic shellfish toxins (PSTs) and extracellular bioactive compounds produced by this alga, induced higher concentration, size, complexity and reactive oxygen species (ROS) production of circulating hemocytes. Challenge by cohabitation with field-exposed oysters also activated these hemocyte responses, suggesting a defense response to new microorganism exposure. These hemocyte responses to cohabitation challenge, however, were partially inhibited by A. catenella exposure, which enhanced hemocyte mortality, suggesting either detrimental effects of the interaction of both stressors on immune capacity, or the implementation of an alternative immune strategy through apoptosis. Indeed, no infection with specific pathogens (herpesvirus OsHV-1 or Vibrio aesturianus) was detected. Additionally, lower PST accumulation in challenged oysters suggests a physiological impairment through alteration of feeding-related processes. Overall, results of this

  6. Hemocyte quantitative changes in Anticarsia gemmatalis (Lepidoptera: Noctuidae larvae infected by AgMNPV

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    Fábio Goulart de Andrade

    2010-04-01

    Full Text Available The initial effects of the infection by AgMNPV in the total and differential counts of the hemocytes in Anticarsia gemmatalis (Lepidoptera: Noctuidae larvae were studied. The total number of the hemocytes did not decrease in infected larvae, as it occurred in non infected larvae. In infected larvae, the hemocyte types showed the following frequencies: plasmatocytes - 47.8%, esferulocytes - 25.9%, granulocytes - 15.8%, oenocytoids - 7.2%, prohemocytes - 2.8%, vermicytes - 0,5%. Only the percentage of the granulocytes was different among infected and non infected larvae, indicating that these cells responded quickly to the initial viral infection. These results showed the effective role of the hemocytes in the response of the A. gemmatalis to the infection by AgMNPV. The comprehension of the immunological mechanisms of this insect is an important tool to understand its biological control.Os efeitos iniciais da infecção por AgMNPV nas contagens total e diferencial dos hemócitos em Anticarsia gemmatalis (Lepidoptera: Noctuidae foram estudados. O número total de hemócitos não diminuiu nas larvas infectadas, como ocorreu nas larvas não infectadas. Nas larvas infectadas, os tipos de hemócitos apresentaram as seguintes freqüências: plasmatócitos - 47,8%, esferulócitos - 25,9%, granulócitos - 15,8%, oenocitóides - 7,2%, prohemócitos - 2,8%, vermiformes - 0,5%. Apenas a porcentagem de granulócitos foi diferente entre larvas infectadas e não infectadas, indicando que estas células responderam rapidamente à infecção viral inicial. Estes resultados mostraram o papel efetivo que dos hemócitos na resposta de A. gemmatalis à infecção por AgMNPV. A compreensão dos mecanismos imunológicos deste inseto é uma ferramenta importante para compreender seu controle biológico.

  7. Crustacean hyperglycemic hormones directly modulate the immune response of hemocytes in shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Wang, Lin; Chen, Hao; Xu, Jianchao; Xu, Qingsong; Wang, Mengqiang; Zhao, Depeng; Wang, Lingling; Song, Linsheng

    2017-03-01

    A robust immune response against invading pathogens is crucial for host to survive, which depends greatly on the well balance of metabolism. Increasing evidence has indicated that some metabolic hormones, such as insulin, could modulate immune responses directly. Crustacean hyperglycemic hormone (CHH) family is a group of ecdysozoans-specific peptide hormone involved in glucose metabolism and other biological events. In the present study, two members of CHH family (designated as LvCHH I and LvCHH II) in shrimp Litopenaeus vannamei with one and two crustacean neurohormone domains respectively were chosen to investigate their putative modulatory roles in both glucose metabolism and immune response. LvCHH I and LvCHH II were both expressed in the sinus gland and lamina ganglionalis of eyestalks and were significantly induced after white spot syndrome virus (WSSV) infection. Meanwhile, significant increases of hemolymph glucose levels were observed in shrimp at 12 and 24 h after WSSV infection while the glucose inside the hemocytes decreased at 6 h and then increased at 12 h. Gain-of-function of rLvCHHs was subsequently conducted in vivo by injecting the recombinant proteins (rLvCHH I and rLvCHH II). The hemolymph glucose increased significantly from 0.5 h to 3 h after the shrimps received an injection of rLvCHH I, while it decreased at 0.5 h and increased afterward at 3 h post rLvCHH II injection. At the meantime, significant decreases of reactive oxygen species level in hemocytes were observed at 3 h and 6 h post rLvCHH I injection, while it remained unchanged in rLvCHH II injection group. rLvCHH I and rLvCHH II could bind to the cytomembrane of primary shrimp hemocytes in vitro, and the expressions of superoxide dismutase and LvRelish increased when the hemocytes were incubated with rLvCHH I for 3 h. Meanwhile, the expression of antimicrobial peptides, crustin and penaeidin-4, were also induced by rLvCHH I and rLvCHH II. These results demonstrated that

  8. Induction of micronuclei in hemocytes of Mytilus edulis and statistical analysis

    DEFF Research Database (Denmark)

    Wrisberg, M. N.; Bilbo, Carl M.; Spliid, Henrik

    1992-01-01

    A genotoxicity test focusing on micronucleus production in the blood cells (hemocytes) of blue mussel M. edulis has been developed as a possible indicator for marine pollution. A linear dose-response relationship was found when M. edulis was exposed to low concentrations (0, 12.5, and 25 mg...... biological variation, emphasizing the importance of application of a correct statistical method. A systematic approach to the statistical evaluation of the mussel MN test is outlined. The statistical model includes three different situations: (a) estimation of parameters of a single sample, (b) estimation...

  9. Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

    Science.gov (United States)

    Skazina, M A; Gorbushin, A M

    2016-07-01

    Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits.

  10. Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae: Phagocytic Hemocytes in the Circulation and the Kidney.

    Directory of Open Access Journals (Sweden)

    Juan A Cueto

    Full Text Available Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy and transmission electron microscopy (TEM. Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules. Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets' occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently and may mean that

  11. Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32.

    Science.gov (United States)

    Balbi, T; Fabbri, R; Cortese, K; Smerilli, A; Ciacci, C; Grande, C; Vezzulli, L; Pruzzo, C; Canesi, L

    2013-12-01

    Marine bivalves can accumulate large numbers of bacteria, in particular Vibrio species, whose persistence in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating hemocytes and hemolymph soluble factors. The interactions between vibrios and hemolymph have been investigated, in particular in bivalve species susceptible to infection by certain Vibrio spp. and strains. In this work, the effects of two bivalve pathogens, Vibrio splendidus LGP32 (V.s.) and Vibrio aestuarianus 01/032 (V.a.), isolated from oyster mortality outbreaks, on the hemocytes of Mytilus galloprovincialis were investigated. In vitro, V.s., but not V.a., induced a dramatic decrease in lysosomal membrane stability-LMS in the hemocytes; both vibrios induced a moderate lysozyme release, with V.s. > V.a.. The V.s.-induced decrease in LMS was mediated by activation of PI-3Kinase, as shown by use of different kinase inhibitors. TEM analysis showed rapid internalization of both vibrios; however, V.s. lead to cellular and lysosomal damage and was able to survive within the hemocytes, whereas significant killing of V.a. was observed. In vivo, in mussels challenged with either vibrio and sampled at 6, 24 and 96 h post-injection, transient decreases in hemocyte LMS and progressive increases in serum lysozyme activity were observed, with V.s. > V.a.. Moreover, whereas V.a. was efficiently cleared from hemolymph, V.s. showed significant growth, that was maximal at 24 h p.i. when lowest LMS values were recorded in the hemocytes. Both vibrios also induced significant decreases in LMS in the digestive gland, again with V.s. > V.a.. The results indicate distinct interactions between mussel hemocytes and the two vibrio strains tested. The effects of V.s. may be due to the capacity of this strain to interfere with the signaling pathways involved in hemocyte function, thus escaping the bactericidal activity of the host cell, as observed for certain mammalian pathogens

  12. [Evaluation of latex agglutination test for anti-treponemal antibody in comparison with chemical luminescence tests].

    Science.gov (United States)

    Watanabe, Naomi; Nagatomo, Ritsuko; Okubo, Shigeo; Yokota, Hiromitsu; Ikeda, Hitoshi; Yatomi, Yutaka

    2011-02-01

    The performance of a latex agglutination test (Mediace TPLA) in the detection of anti-treponemal antibody was evaluated in comparison with chemical luminescence tests (LumipulsII-N and Architect TPAb) in 346 cases. Anti-treponemal antibody was further determined by immunochromatography and immunoblotting tests and additionally evaluated by a serological test for syphilis with lipoidal antigens. The total concordance rate between the latex agglutination test and chemical luminescence tests ranged from 96% to 97%: the positive concordance rate ranged from 96% to 97%, and the negative concordance rate, from 97% to 98%. The latex agglutination test showed two false positive cases, and each chemical luminescence test showed two false positive cases, respectively. In eight cases, only the latex agglutination test showed negative results; all specimens contained anti-treponemal antibodies. However, none of these was considered to be a false positive and each was treated as syphilis based on the results of confirmatory analysis with immunochromatography and immunoblotting tests and a serological test for syphilis. The discordant results in the latex agglutination test and chemical luminescence tests may be caused by the different antigenisity of each test. With detailed analysis of those sera treated as syphilis, each specimen was found to contain various antibodies against syphilitic antigens, suggesting that there was a different specificity of native and recombinant antigens. Based on the present results for the comparison between the latex agglutination test and chemical luminescence tests, it was considered that further investigation is necessary to clarify the anti-treponemal antibody profile of syphilis at the disease stage.

  13. Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis.

    Science.gov (United States)

    Nagalingam, Mohandoss; Thirumalesh, Sushma Rahim Assadi; Kalleshamurthy, Triveni; Niharika, Nakkala; Balamurugan, Vinayagamurthy; Shome, Rajeswari; Sengupta, Pinaki Prasad; Shome, Bibek Ranjan; Prabhudas, Krishnamsetty; Rahman, Habibur

    2015-10-01

    This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

  14. Comparative evaluation of slide agglutination and Widal tube agglutination test in detecting enteric fever among patients attending a tertiary care hospital in North India

    OpenAIRE

    Noor Jahan; Razia Khatoon; Amrita,; Sudhir Mehrotra; Swatantra Kumar

    2016-01-01

    Background: Enteric fever is a major public health problem with significant morbidity and mortality in developing countries. Although, isolation of causative organism from blood is the standard laboratory method, but due to frequent use of self-medication by patients, and its long turnaround time, it is seldom used, and enteric fever is usually diagnosed by using serological methods. Widal tube agglutination test is the standard serological test used, which is now a days replaced by slide agg...

  15. A simple system for in-droplet incubation and quantification of agglutination assays

    KAUST Repository

    Castro, David

    2013-10-28

    This work reports on a simple system for quantitative sensing of a target analyte based on agglutination in micro-channels. Functionalized microbeads and analyte with no prior incubation are flowed in droplets (~2μL) through a thin silicone tube filled with mineral oil at a flow rate of 150 μL/min. Hydrodynamic forces alone produce a highly efficient mixing of the beads within the droplet, without the need of complex mixing structures or magnetic actuation. The setup allows rapid observation of agglutination (<2 min), which is quantified using image analysis, and has potential application to high-throughput analysis.

  16. Discovery of agglutinated foraminifers from the Longzhaogou Group in eastern Heilongjiang Province

    Institute of Scientific and Technical Information of China (English)

    LI Gang; YU Shanmao

    2004-01-01

    The low diversity agglutinated foraminifers are recovered from the Qihulin Formation of the Longzhaogou Group in eastern Heilongjiang, China. The foraminiferal fauna consists of 9 species of 5 genera. The common members are Cribrostomoides nonioninoides (Reuss), Haplophragmoides concavus (Chapman), H. gigas minor Nauss. Although the diagnostic zonal taxa are absent in the agglutinated fauna, according to the global stratigraphic distribution of the above-mentioned species, and the associated Pseudohaploceras ammonite fauna, the foraminiferal fauna may be of a Barremian-Aptian (Early Cretaceous) age.

  17. [Relationship between the sensitivity of the delayed agglutination test and synthetic detergents].

    Science.gov (United States)

    Iovchev, E; Vodas, K

    1977-01-01

    Residual amounts of detergents (Losk, Bio-73, Alka-lux, Bourgas, Bourgaslux, and Vero) in a concentration of 10-5 to 10-7 in physiologic saline can inhibit the agglutination titers by 3 to 5 degrees. This could mislead in the assessment of the reaction with regard to its diagnostic value. It is admitted that the inhibition produced is due to changes in the antibodies--drop in the total protein and light variations in all protein fractions as well as in the probable surface deterioration of the antigen, leading to its defective agglutinability. It is suggested to rinse more than five times all glassware that has been cleaned with detergents.

  18. Seasonal variation in agglutination of Plasmodium falciparum-infected erythrocytes

    DEFF Research Database (Denmark)

    Giha, H A; Theander, T G; Staalsø, T;

    1998-01-01

    place in the absence of disease, presumably as a consequence of subclinical infection. This is the first demonstration of marked seasonal fluctuations in the capacity of individuals' sera to agglutinate parasitized red blood cells. Possible explanations for this effect include a decrease in the levels...... malaria infection samples taken from five of the cohort members. Our data show that the capacity of donor plasma samples to agglutinate parasitized cells depended largely on the time of sampling relative to the transmission season, at least within this epidemiologic setting. Thus, although less than half...

  19. Hemocyte–hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit

    Science.gov (United States)

    Lapointe, Jason F.; Dunphy, Gary B.; Mandato, Craig A.

    2012-01-01

    Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell–bacteria adhesion and hemocyte–hemocyte adhesion (microaggregation). Hemocyte–microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte–hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2 nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6–120 nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2–120 nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2–6 nM) cause reduced hemocyte adhesion, while high levels (12–120 nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6–120 nM) and in vivo (12–120 nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the

  20. Transcriptome analysis of the unfolded protein response in hemocytes of Litopenaeus vannamei.

    Science.gov (United States)

    Chen, Yi-Hong; Yuan, Feng-Hua; Bi, Hai-Tao; Zhang, Ze-Zhi; Yue, Hai-Tao; Yuan, Kai; Chen, Yong-Gui; Wen, Shao-Ping; He, Jian-Guo

    2016-07-01

    In this study, Litopenaeus vannamei was injected with double-stranded RNA (dsRNA) against L. vannamei immunoglobulin heavy chain binding protein (LvBip) to activating UPR in the hemocytes, shirmps injected dsRNA against enhanced green fluorescence protein (eGFP) as control group. And genes expression in hemocytes of then were analyzed using Illumina Hiseq 2500 (PE100). By comparing the analyzed results, 1418 unigenes were significantly upregulated, and 596 unigenes were significantly down-regulated upon UPR. Analysis of the differentially expressed genes against known databases indicated that the distribution of gene pathways between the upregulated and down-regulated genes were substantially different. A total of 208 genes of UPR system were obtained, and 69 of them were differentially expressed between the two groups. Results also showed that L. vannamei UPR was involved in various metabolic processes, such as glycometabolism, lipid metabolism, amino acid metabolism, and nucleic acid metabolism. In addition, UPR was emgaged in immune-assicoated signaling pathways, such as NF-κB signaling pathway, NOD-like receptor signaling pathway, Hippo signaling pathway, p38 MAPK signaling pathway and Wnt signaling pathway in L. vannamei. These results improved our current understanding of the L. vannamei UPR, and highlighted its importance in cell homeostasis upon environmental stress.

  1. Real time observation and automated measurement of red blood cells agglutination inside a passive microfluidic biochip containing embedded reagents.

    Science.gov (United States)

    Huet, Maxime; Cubizolles, Myriam; Buhot, Arnaud

    2016-09-20

    The process of agglutination is commonly used for the detection of biomarkers like proteins or viruses. The multiple bindings between micrometer sized particles, either latex beads or red blood cells (RBCs), create aggregates that are easily detectable and give qualitative information about the presence of the biomarkers. In most cases, the detection is made by simple naked-eye observation of agglutinates without any access to the kinetics of agglutination. In this study, we address the development of a real-time time observation of RBCs agglutination. Using ABO blood typing as a proof-of-concept, we developed i) an integrated biological protocol suitable for further use as point-of-care (POC) analysis and ii) two dedicated image processing algorithms for the real-time and quantitative measurement of agglutination. Anti-A or anti-B typing reagents were dried inside the microchannel of a passive microfluidic chip designed to enhance capillary flow. A blood drop deposit at the tip of the biochip established a simple biological protocol. In situ agglutination of autologous RBCs was achieved by means of embedded reagents and real time agglutination process was monitored by video recording. Using a training set of 24 experiments, two real-time indicators based on correlation and variance of gray levels were optimized and then further confirmed on a validation set. 100% correct discrimination between positive and negative agglutinations was performed within less than 2min by measuring real-time evolution of both correlation and variance indicators.

  2. Comparison of the Denka Seiken slide agglutination method to the quellung test for serogrouping of Streptococcus pneumoniae isolates.

    Science.gov (United States)

    Shutt, Cheryl K; Samore, Matthew; Carroll, Karen C

    2004-03-01

    This study compared a slide agglutination test (Denka Seiken, Tokyo, Japan) to the "gold standard" quellung reaction (Pneumotest; Statens Serum Institut, Copenhagen, Denmark) for the serogrouping of pneumococci. Two hundred clinical isolates of Streptococcus pneumoniae were used for the comparison. Each assay was performed according to the manufacturer's instructions. There was an overall agreement of 95.7% between the two methods. Only 4 of 10 isolates of serogroup 22 were detected with the slide agglutination assay. Two isolates that were untypeable by the Pneumotest method were typed as serogroups 6 and 31 by the slide agglutination method. The Pneumotest method was unable to type 22 isolates, and the slide agglutination method was unable to type 16 isolates. The slide agglutination method compares favorably with the Pneumotest method and is easier to perform and to interpret.

  3. Single agglutinates: A comparative study of compositions of agglutinitic glass, whole-grain, bulk soil, and FMR

    Science.gov (United States)

    Basu, A.; Robinson, R.; Mckay, D. S.; Blanchard, D. P.; Morris, R. V.; Wentworth, Susan J.

    1994-01-01

    Previous workers on single agglutinates have variously interpreted the composition of agglutinitic glass to represent impact melts of (1) bulk soil, (2) mixed components in finer sizes, and (3) microtargets. Separately, Papike has argued in favor of fusion of the finest fraction of bulk soils. Thirty-four single agglutinates were hand-picked from the mature Apollo 16 soil 61181 (I(sub s)/FeO = 82) and the FMR and chemical composition (INAA for Fe, Sc, Sm, Co, Ni, and Cr) of each agglutinate particle were measured. Thirteen of these single agglutinates were selected for electron beam microanalysis and imaging. Less than 1 micron spots were analyzed (for Na, Mg, Al, Si, P, S, K, Ca, Ti, Cr, Mn, Fe, Ni, and Ba) on pure glassy areas (approximately ten in each particle) selected on the basis of optical and BSE images (avoiding all clasts and inclusions) with an electron microprobe to obtain average glass compositions of each single agglutinate.

  4. Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus.

    Science.gov (United States)

    Trinh, Dai Quang; Ogawa, Haruko; Bui, Vuong Nghia; Nguyen, Tham Thi Hong; Gronsang, Dulyatad; Baatartsogt, Tugsbaatar; Kizito, Mugimba Kahoza; AboElkhair, Mohammed; Yamaguchi, Shigeo; Nguyen, Viet Khong; Imai, Kunitoshi

    2015-09-01

    A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology.

  5. Reading Development in Agglutinative Languages: Evidence from Beginning, Intermediate, and Adult Basque Readers

    Science.gov (United States)

    Acha, Joana; Laka, Itziar; Perea, Manuel

    2010-01-01

    Do typological properties of language, such as agglutination (i.e., the morphological process of adding affixes to the lexeme of a word), have an impact on the development of visual word recognition? To answer this question, we carried out an experiment in which beginning, intermediate, and adult Basque readers (n = 32 each, average age = 7, 11,…

  6. [The latex agglutination with video digital registration: the enhancement of diagnostic significance of conventional technique].

    Science.gov (United States)

    Starovoĭtova, T A; Steriopolo, N A; Zaĭko, V V; Vengerov, Iu Iu

    2012-02-01

    The rapid semiquantitative latex-tests, because of their analytic characteristics and convenient application, became widespread in the practice of laboratory diagnostics. Though, in spite of high sensitivity and specificity, their diagnostic effectiveness is lower that it could be mainly because of the impossibility to document the results of latex agglutinative re4actions and to manage the objective quality control. The application of systems of video digital registration permits to enhance the clinical significance of these analyses. By means of scanner systems (control and program complex "Expert Lab") the image of analytic objects is received with the results of latex agglutination reaction. The application of program techniques (the programs "Expert Lab - Agglutination" and "Expert Lab - Agglutination - Micros") in data processing permits to get the precise qualitative characteristics of active reactions, to ensure the automatic interpretation of results and gives an opportunity to proceed with the internal laboratory quality control. The saving of analytic object image in computer memory after termination of reaction favors the formation of data base, the implementation of retrospective evaluation of obtained results, additional consultations in dubious cases, including on-line. The application of complex "Expert Lab" permitted to develop the miniaturizes matrix systems permitting to decrease the withdrawal of latex reagents, to increase the productivity of analytical stage of operation preserving all analytical characteristics of method.

  7. The Classroom-Friendly ABO Blood Types Kit: Blood Agglutination Simulation

    Science.gov (United States)

    Arnold, Savittree Rochanasmita; Kruatong, Tussatrin; Dahsah, Chanyah; Suwanjinda, Duongdearn

    2012-01-01

    The classroom-friendly ABO blood type kit was developed by combining advantages of modelling and a simulation laboratory to teach the topics of ABO blood types and blood transfusion. Teachers can easily simulate the agglutination reaction on a blood type testing plate in the classroom, and show the students how this reaction occurs by using the…

  8. Development and evaluation of a latex agglutination test for the rapid serodiagnosis of tularemia.

    Science.gov (United States)

    Rastawicki, Waldemar; Rokosz-Chudziak, Natalia; Chróst, Anna; Gierczyński, Rafał

    2015-05-01

    A latex agglutination test (LAT) was developed for a rapid detection of antibodies against Francisella tularensis. The assay is performed by mixing serum with antigen-coated latex beads and read within 5 min. Developed LAT has been proved to be a specific, sensitive, fast, easy-to-perform and cost-efficient tool for the routine diagnosis of tularemia.

  9. Conventional Rapid Latex Agglutination in Estimation of von Willebrand Factor: Method Revisited and Potential Clinical Applications

    Directory of Open Access Journals (Sweden)

    Marianor Mahat

    2014-01-01

    Full Text Available Measurement of von Willebrand factor antigen (VWF : Ag levels is usually performed in a specialised laboratory which limits its application in routine clinical practice. So far, no commercial rapid test kit is available for VWF : Ag estimation. This paper discusses the technical aspect of latex agglutination method which was established to suit the purpose of estimating von Willebrand factor (VWF levels in the plasma sample. The latex agglutination test can be performed qualitatively and semiquantitatively. Reproducibility, stability, linearity, limit of detection, interference, and method comparison studies were conducted to evaluate the performance of this test. Semiquantitative latex agglutination test was strongly correlated with the reference immunoturbidimetric assay (Spearman’s rho = 0.946, P150% VWF : Ag (when comparing with immunoturbidimetric assay. The findings from evaluation studies suggest that latex agglutination method is suitable to be used as a rapid test kit for the estimation of VWF : Ag levels in various clinical conditions associated with high levels and low levels of VWF : Ag.

  10. Agglutination of pYV+ Yersinia enterocolitica strains by agglutinin from Mangifera indica.

    OpenAIRE

    Wauters, Georges; Charlier, Jacqueline; Janssens, Michèle

    1995-01-01

    Agglutination of 271 strains of Yersinia enterocolitica and related species grown at 37 degrees C by a 0.01% dilution of the agglutinin from Mangifera indica was correlated with the presence of the virulence plasmid. The study of YadA mutants suggested that the YadA protein is the target of the plant agglutinin.

  11. Obtaining hemocytes from the Hawaiian bobtail squid Euprymna scolopes and observing their adherence to symbiotic and non-symbiotic bacteria.

    Science.gov (United States)

    Collins, Andrew J; Nyholm, Spencer V

    2010-02-11

    Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

  12. cDNA microarray analysis of disk abalone genes in gills and hemocytes after viral hemorrhagic septicemia virus (VHSV) challenge.

    Science.gov (United States)

    De Zoysa, Mahanama; Nikapitiya, Chamilani; Oh, Chulhong; Whang, Ilson; Shin, Hyun-Jin; Lee, Jehee

    2012-06-01

    A disk abalone Haliotis discus discus 4.2 K cDNA microarray was designed by selecting abalone expressed sequence tags (ESTs). Transcriptional profiles in gills and hemocytes were analyzed upon abalone challenged with viral hemorrhagic septicemia virus (VHSV) in order to select candidates for screening of immune response genes. Among the 4188 genes analyzed, 280 (6.6%) transcripts were changed their expression level in gills and hemocytes against VHSV challenge compared to control animals. Total of 88 and 65 genes were up-regulated in gills and hemocytes, respectively. These genes can be grouped under various immune-functional categories such as transcription factors (Krüppell-like factor; ETS-family transcription factor), inflammatory and apoptosis related genes (TNF superfamily members, Fas ligand), IFN regulatory proteins (IFN-44 like, interferon gamma-inducible lysosomal thiol reductase) and detoxification proteins (glutathione peroxidase). In contrast, 25 and 102 genes were shown down-regulation in gills and hemocytes, respectively. Among the differentially expressed transcripts, considerably higher numbers of ESTs were represented as either hypothetical (unknown) proteins or no GenBank match suggesting those may be novel genes associated with internal defense of abalone.

  13. Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis.

    Science.gov (United States)

    Wang, Youji; Hu, Menghong; Chiang, M W L; Shin, P K S; Cheung, S G

    2012-03-01

    The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis. Copyright © 2011. Published by Elsevier Ltd.

  14. TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

    Science.gov (United States)

    Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

    2012-06-01

    Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

  15. Hemocyte-specific responses to the peroxidizing herbicide fomesafen in the pond snail Lymnaea stagnalis (Gastropoda, Pulmonata)

    Energy Technology Data Exchange (ETDEWEB)

    Russo, Jacqueline [UMR 6553 Ecobio CNRS Universite de Rennes 1, Campus de Beaulieu, F-35042 Rennes cedex 35042 (France); Lefeuvre-Orfila, Luz [UMR 985 INRA-Agrocampus Ecobiologie et Qualite des Hydrosystemes Continentaux, Equipe Ecotoxicologie et Qualite des Milieux aquatiques, 65 rue de Saint-Brieuc CS 84215, F-35042 Rennes cedex 35042 (France); Lagadic, Laurent [UMR 985 INRA-Agrocampus Ecobiologie et Qualite des Hydrosystemes Continentaux, Equipe Ecotoxicologie et Qualite des Milieux aquatiques, 65 rue de Saint-Brieuc CS 84215, F-35042 Rennes cedex 35042 (France)]. E-mail: laurent.lagadic@rennes.inra.fr

    2007-03-15

    Responses of circulating hemocytes were studied in Lymnaea stagnalis exposed to 10, 30, 90, and 270 {mu}g/L fomesafen for 24 and 504 h. Flow cytometry was used to quantify fomesafen-induced production of reactive oxygen species (ROS), phagocytic activity on Escherichia coli, and oxidative burst when hemocytes were challenged by E. coli or phorbol 12-myristate-13-acetate (PMA). Lysosomal membrane damage was assessed, using the neutral-red retention time (NRRT) assay. Exposure to fomesafen for 24 h resulted in increase in ROS levels and decreases in phagocytosis and the oxidative burst in PMA-stimulated hemocytes. After 504 h, intracellular levels of ROS returned to normal, but phagocytosis of E. coli was still inhibited and the associated oxidative burst significantly reduced. After both durations of exposure, decreases of NRRT indicated that lysosome membrane fragility increased with fomesafen concentration. Potential implications for the health and survival of the snails and consequences on populations are discussed. - Fomesafen inhibited phagocytosis and the associated oxidative burst, and increased lysosome fragility in L. stagnalis hemocytes.

  16. Sorption and agglutination phenomenon of nanofluids on a plain heating surface during pool boiling

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhen-hua; Liao, Liang [School of Mechanical and Power Engineering, Shanghai Jiaotong University, 200030 Shanghai (China)

    2008-05-15

    The pool nucleate boiling heat transfer experiments of water (H{sub 2}O) based and alcohol (C{sub 2}H{sub 5}OH) based nanofluids and nanoparticles-suspensions on the plain heated copper surface were carried out. The study was focused on the sorption and agglutination phenomenon of nanofluids on a heated surface. The nanofluids consisted of the base liquid, the nanoparticles and the surfactant. The nanoparticles-suspensions consisted of the base liquid and nanoparticles. The both liquids of water and alcohol and both nanoparticles of CuO and SiO{sub 2} were used. The surfactant was sodium dodecyl benzene sulphate (SDBS). The experimental results show that for nanofluids, the agglutination phenomenon occurred on the heated surface when the wall temperature was over 112{sup o}C and steady nucleated boiling experiment could not be carried out. The reason was that an unsteady porous agglutination layer was formed on the heated surface. However, for nanoparticles-suspensions, no agglutination phenomenon occurred on the heating surface and the steady boiling could be carried out in the whole nucleate boiling region. For the both of alcohol based nanofluids and nano-suspensions, no agglutination phenomenon occurred on the heating surface and steady nucleate boiling experiment could be carried out in the whole nucleate boiling region whose wall temperature did not exceed 112{sup o}C. The boiling heat transfer characteristics of the nanofluids and nanoparticles-suspensions are somewhat poor compared with that of the base fluids, since the decrease of the active nucleate cavities on the heating surface with a very thin nanoparticles sorption layer. The very thin nanoparticles sorption layer also caused a decrease in the solid-liquid contact angle on the heating surface which leaded to an increase of the critical heat flux (CHF). (author)

  17. A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

    Science.gov (United States)

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

    2014-01-10

    Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to

  18. Hemocytes and Enzyme-based Route to Evaluate the Impact of Seed Cocoons Preservation Conditions on Antheraea mylitta Pupae

    Directory of Open Access Journals (Sweden)

    B.C. Prasad

    2012-01-01

    Full Text Available Tropical tasar silkworm, Antheraea mylitta Drury DABA-ecorace is commercially exploited in India for tasar silk production. Generally, its cocoons used to preserve in outdoor and indoor conditions. But in recent past, due to global warming, tasar silk zone day time temperature (May-June shoots up and it goes up to 35 to 45C or even more. It cause adverse impact on the survival of tasar silkworm pupae and invite heavy loss to the tasar silk industry. In the present study, hemocyte and enzyme-based methods have been used to evaluate the impact of outdoor and indoor seed cocoon preservation on A. mylitta pupae. Interestingly, differences in Total Hemocyte Count (THC, hemocyte contour/morphology, protein concentration, fat body and hemolymph acid phosphatase (Acp activity of outdoor and indoor preserved pupae have been observed. Marked variation in THC was observed among high, low and median weight pupae. Although, no significant variation was observed in THC of indoor and outdoor preserved female pupae but significant difference was found in male pupae of both the preservation conditions. Differential profile of hemocytes was differing in outdoor and indoor conditions with change in cell-contour. Elevation in immune cell plasmatocytes (PLs numbers was recorded in indoor preserved cocoon. Acp activity in fat body of indoor preserved pupae was higher than outdoor but reverse trend was found in hemolymph. Activity profile Acp vary in male and female with higher, lower and median weight pupae. Protein concentration in hemolymph of outdoor preserved pupae was more in comparison to indoor but this trend was found usually reverse in fat body. In addition, less protein concentration was recorded in fat body and hemolymph of male pupae in contrast to female. It is expected that, based on hemocyte and enzyme-based method impact of cocoon preservation conditions on A. mylitta pupae can be evaluated/observed.

  19. Immunomodulation by different types of N-oxides in the hemocytes of the marine bivalve Mytilus galloprovincialis.

    Directory of Open Access Journals (Sweden)

    Caterina Ciacci

    Full Text Available The potential toxicity of engineered nanoparticles (NPs for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO(2, n-SiO(2, n-ZnO, n-CeO(2 chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may

  20. High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster.

    Science.gov (United States)

    Kacsoh, Balint Z; Schlenke, Todd A

    2012-01-01

    Among the most common parasites of Drosophila in nature are parasitoid wasps, which lay their eggs in fly larvae and pupae. D. melanogaster larvae can mount a cellular immune response against wasp eggs, but female wasps inject venom along with their eggs to block this immune response. Genetic variation in flies for immune resistance against wasps and genetic variation in wasps for virulence against flies largely determines the outcome of any fly-wasp interaction. Interestingly, up to 90% of the variation in fly resistance against wasp parasitism has been linked to a very simple mechanism: flies with increased constitutive blood cell (hemocyte) production are more resistant. However, this relationship has not been tested for Drosophila hosts outside of the melanogaster subgroup, nor has it been tested across a diversity of parasitoid wasp species and strains. We compared hemocyte levels in two fly species from different subgroups, D. melanogaster and D. suzukii, and found that D. suzukii constitutively produces up to five times more hemocytes than D. melanogaster. Using a panel of 24 parasitoid wasp strains representing fifteen species, four families, and multiple virulence strategies, we found that D. suzukii was significantly more resistant to wasp parasitism than D. melanogaster. Thus, our data suggest that the relationship between hemocyte production and wasp resistance is general. However, at least one sympatric wasp species was a highly successful infector of D. suzukii, suggesting specialists can overcome the general resistance afforded to hosts by excessive hemocyte production. Given that D. suzukii is an emerging agricultural pest, identification of the few parasitoid wasps that successfully infect D. suzukii may have value for biocontrol.

  1. Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm.

    Science.gov (United States)

    Yang, D H; McMillan, A G; Standley, N T; Shannon, P; Xu, Z Z

    2012-10-15

    Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citrate diluent without streptomycin. The objectives were to investigate causes of sperm agglutination and the underlying mechanism. Aliquots of bull semen were diluted in a base diluent (BD) supplemented with various test components and the percentage of agglutinated sperm (% AggSp) was quantified at 1, 5, 24, 48, and 72 h of incubation. When sperm were incubated at 22 °C, no agglutination was observed in BD for up to 72 h, whereas the % AggSp was 5.0, 41.7, 72.2, 91.1, and 92.8% in BD + 5% egg yolk (BD + EY) at 1, 5, 24, 48 and 72 h, respectively. However, no sperm agglutination was observed in BD + EY if incubation temperature was 37 °C. Addition of 5 or 10 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid to BD + EY reduced the % AggSp from 95% to sperm agglutination in the absence of egg yolk, implicating calcium and other factors in egg yolk. Addition of the citrate-soluble fraction (CSF) of egg yolk to BD induced sperm agglutination similar to whole egg yolk, whereas water- and saline-soluble fractions of egg yolk were ineffective. The sperm-agglutinating efficacy of CSF (the % AggSp = 95% at 72 h) was reduced by dialysis (20%; P sperm agglutination, whereas addition of 2 mM of a cAMP analogue, dbcAMP, to a nonagglutinating diluent failed to induce sperm agglutination. Agglutination status had no effect on sperm plasma membrane/acrosome status and mitochondrial membrane potential. In conclusion, calcium and other component(s) in the CSF of egg yolk induced head-to-head agglutination of bull sperm in a time- and temperature-dependent manner. Although the mechanism of agglutination was not determined, the cAMP- protein kinase A signaling pathway was not involved.

  2. Gene expression profile of Bombyx mori hemocyte under the stress of destruxin A.

    Directory of Open Access Journals (Sweden)

    Liang Gong

    Full Text Available Destruxin A (DA is a cyclo-peptidic mycotoxin from the entomopathogenic fungus Metarhizium anisopliae. To uncover potential genes associated with its molecular mechanisms, a digital gene expression (DGE profiling analysis was used to compare differentially expressed genes in the hemocytes of silkworm larvae treated with DA. Ten DGE libraries were constructed, sequenced, and assembled, and the unigenes with least 2.0-fold difference were further analyzed. The numbers of up-regulated genes were 10, 20, 18, 74 and 8, as well as the numbers of down-regulated genes were 0, 1, 8, 13 and 3 at 1, 4, 8, 12 and 24 h post treatment, respectively. Totally, the expression of 132 genes were significantly changed, among them, 1, 3 and 12 genes were continually up-regulated at 4, 3 and 2 different time points, respectively, while 1 gene was either up or down-regulated continually at 2 different time points. Furthermore, 68 genes were assigned to one or multiple gene ontology (GO terms and 89 genes were assigned to specific Kyoto Encyclopedia of Genes and Genomes (KEGG Orthology. In-depth analysis identified that these genes putatively involved in insecticide resistance, cell apoptosis, and innate immune defense. Finally, twenty differentially expressed genes were randomly chosen and validated by quantitative real-time PCR (qRT-PCR. Our studies provide insights into the toxic effect of this microbial insecticide on silkworm's hemocytes, and are helpful to better understanding of the molecular mechanisms of DA as a biological insecticide.

  3. Effect of acclimatization on hemocyte functional characteristics of the Pacific oyster (Crassostrea gigas) and carpet shell clam (Ruditapes decussatus).

    Science.gov (United States)

    Hurtado, Miguel Ángel; da Silva, Patricia Mirella; Le Goïc, Nelly; Palacios, Elena; Soudant, Philippe

    2011-12-01

    Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 ± 1 °C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 ± 1 °C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed

  4. Elemental X-ray mapping of agglutinated foraminifer tests: a non- destructive technique for determining compositional characteristics.

    Science.gov (United States)

    Commeau, R.F.; Reynolds, Leslie A.; Poag, C.W.

    1985-01-01

    The composition of agglutinated foraminiferal tests vary remarkably in response to local substrate characteristics, physiochemical properties of the water column and species- dependant selectivity of test components. We have employed a technique that combines a scanning electron microscope with an energy dispersive X-ray spectrometer system to identify major and minor elemental constituents of agglutinated foraminiferal walls. As a sample is bombarded with a beam of high energy electrons, X-rays are generated that are characteristic of the elements present. As a result, X- ray density maps can be produced for each of several elements present in the tests of agglutinated foraminifers. -Authors

  5. Comparison of Rose Bengal Plate Agglutination, Standard tube agglutination and Indirect ELISA tests for detection of Brucella antibodies in Cows and Buffaloes

    Directory of Open Access Journals (Sweden)

    S. N. Ghodasara

    2010-04-01

    Full Text Available A total of 180 serum samples (107 cows, 73 buffaloes from cases of abortion and various reproductive disorders were collected for detection of Brucella antibody by Rose Bengal Plate Agglutination Test (RBPT, Serum Tube Agglutination Test (STAT and indirect- ELISA (i-ELISA. The overall prevalence of brucellosis by RBPT, STAT and i-ELISA were 11.21%, 16.00% and 24.30% in cows 9.59%, 12.33% and 26.03% in buffaloes respectively. Overall seroprevalence of Brucellosis in cases of abortion, R.O.P. by RBPT, STAT and i-ELISA were 11.32%, 16.04% and 32.08% respectively. When three serological tests were compared, seropositivity was found highest by i-ELISA (25%, followed by STAT (14.45% and RBPT (10.56%. The results shows higher prevalence of brucellosis in cases of abortion and R.O.P., while at lower level from various reproductive disorders as detected serologically indicating endemicity of the infection in villages around Anand city, Gujarat. [Vet. World 2010; 3(2.000: 61-64

  6. MEMS reagent and sample handling procedure: Feasibility of viral antibody detection by passive immune agglutination

    Science.gov (United States)

    Bailey, G. D.; Tenoso, H. J.

    1975-01-01

    An attempt was made to develop a test requiring no preadsorption steps for the assessment of antibodies to rubella and mumps viruses using the passive immune agglutination (PIA) method. Both rubella and mumps antigens and antibodies were prepared. Direct PIA tests, using rubella antigen-coated beads, and indirect PIA tests, using rubella antibody-coated beads, were investigated. Attempts, using either method, were unsuccessful. Serum interference along with nonspecific agglutination of beads by the rubella antigen resulted in no specific response under the test conditions investigated. A new, highly sensitive approach, the enzyme immunoassay (EIA) test system, is recommended to overcome the nonspecificity. This system is a logical outgrowth of some of the solid phase work done on MEMS and represents the next generation tests system that can be directly applied to early disease detection and monitoring.

  7. Mapping key interactions in the dimerization process of HBHA from Mycobacterium tuberculosis, insights into bacterial agglutination.

    Science.gov (United States)

    Esposito, Carla; Cantisani, Marco; D'Auria, Gabriella; Falcigno, Lucia; Pedone, Emilia; Galdiero, Stefania; Berisio, Rita

    2012-03-23

    HBHA is a cell-surface protein implicated in the dissemination of Mycobacterium tuberculosis (Mtb) from the site of primary infection. Its N-terminal coiled-coil region is also involved in bacterial agglutination. However, despite the importance of HBHA dimerization in agglutination, protein regions involved in dimerization are hitherto not known. Here, we mapped these regions by coupling peptide synthesis, biochemical and computational analyses, and identified structural determinants for HBHA monomer-monomer recognition. Importantly, we obtained the first molecule able to induce HBHA dimer disaggregation at 37°C, the typical growth temperature of Mtb. This result provides new opportunities towards the development of Mtb anti-aggregation molecules with therapeutic interest.

  8. Dimerisation and structural integrity of Heparin Binding Hemagglutinin A from Mycobacterium tuberculosis: implications for bacterial agglutination.

    Science.gov (United States)

    Esposito, Carla; Carullo, Paola; Pedone, Emilia; Graziano, Giuseppe; Del Vecchio, Pompea; Berisio, Rita

    2010-03-19

    Heparin Binding Hemagglutinin A (HBHA) is hitherto the sole virulence factor associated with tuberculosis dissemination from the lungs, the site of primary infection, to epithelial cells. We have previously reported the solution structure of HBHA, a dimeric and elongated molecule. Since oligomerisation of HBHA is associated with its ability to induce bacterial agglutination, we investigated this process using experimental and modelling techniques. We here identified a short segment of HBHA whose presence is mandatory for the stability of folded conformation, whose denaturation is a reversible two-state process. Our data suggest that agglutination-driven cell-cell interactions do not occur via association of HBHA monomers, nor via association of HBHA dimers and open the scenario to a possible trans-dimerisation process.

  9. Serotyping of Actinobacillus pleuropneumoniae serotype 5 strains using a monoclonal-based polystyrene agglutination test

    DEFF Research Database (Denmark)

    Dubreuil, J.D.; Letellier, A.; Stenbæk, Eva

    1996-01-01

    A polystyrene agglutination test has been developed for serotyping Actinobacillus pleuropneumoniae serotype 5a and 5b strains. Protein A-coated polystyrene microparticles were sensitized with a murine monoclonal antibody recognizing an epitope on serotype 5 LPS-O chain as shown by SDS......-PAGE and Western blotting, A total of 205 A. pleuropneumoniae, strains including all 12 serotype reference strains and 13 strains representing 8 common bacterial species associated with swine or related to A, pleuropneumoniae, were tested by mixing 25 mu L of polystyrene reagent with the same volume of a dense...... suspension of bacterial cells grown for 18 h. All A, pleuropneumoniae strains had been previously serotyped using standard procedures, The polystyrene agglutination test was rapid (less than 3 min) and easy to perform. Overall a very good correlation (97.3%) with the standard techniques was found...

  10. Lead isotopic studies of lunar soils - Their bearing on the time scale of agglutinate formation

    Science.gov (United States)

    Church, S. E.; Tilton, G. R.; Chen, J. H.

    1976-01-01

    Fines (smaller than 75 microns) and bulk soil were studied to analyze loss of volatile lead; losses of the order of 10% to 30% radiogenic lead during the production of agglutinates are assessed. Lead isotope data from fine-agglutinate pairs are analyzed for information on the time scale of micrometeorite bombardment, from the chords generated by the data in concordia diagrams. Resulting mean lead loss ages were compared to spallogenic gas exposure ages for all samples. Labile parentless radiogenic Pb residing preferentially on or in the fines is viewed as possibly responsible for aberrant lead loss ages. Bulk soils plot above the concordia curve (in a field of excess radiogenic Pb) for all samples with anomalous ages.

  11. [Isolation of an agglutinating anti-E. coli K 88+ serum].

    Science.gov (United States)

    Petkov, M; Belchev, K; Ganovski, D

    1981-01-01

    An agglutinating anti-K 88+ serum was obtained through immunizing rabbits with geometrically rising amounts of cell-free K 88 antigenic extraction. Use was made of bacterial suspensions cultured in Minka agar and homogenized at 8000 r. p. m. to remove the K 88 pili. The cell depot was removed by centrifugation at 15 000 r. p. m., and the protein in the supernatant was determined by the method of Kingsey. The titer of the K88 serum was within the 1:320-1:640 range. The specificity and activity of the serum was evaluated by the hemagglutination test, immunoelectrophoresis, and immunodiffusion. The serum is highly specific and yields positive agglutination results with all K 88+ Escherichia coli strains. It does not react with antigen - K 88-negative E. coli organisms as well as with the O antigen of the investigated strains.

  12. Evaluation of a latex agglutination assay for the identification of Burkholderia pseudomallei and Burkholderia mallei.

    Science.gov (United States)

    Duval, Brea D; Elrod, Mindy G; Gee, Jay E; Chantratita, Narisara; Tandhavanant, Sarunporn; Limmathurotsakul, Direk; Hoffmaster, Alex R

    2014-06-01

    Cases of melioidosis and glanders are rare in the United States, but the etiologic agents of each disease (Burkholderia pseudomallei and Burkholderia mallei, respectively) are classified as Tier 1 select agents because of concerns about their potential use as bioterrorism agents. A rapid, highly sensitive, and portable assay for clinical laboratories and field use is required. Our laboratory has further evaluated a latex agglutination assay for its ability to identify B. pseudomallei and B. mallei isolates. This assay uses a monoclonal antibody that specifically recognizes the capsular polysaccharide produced by B. pseudomallei and B. mallei, but is absent in closely related Burkholderia species. A total of 110 B. pseudomallei and B. mallei were tested, and 36 closely related Burkholderia species. The latex agglutination assay was positive for 109 of 110 (99.1% sensitivity) B. pseudomallei and B. mallei isolates tested.

  13. Serotyping of Actinobacillus pleuropneumoniae serotype 5 strains using a monoclonal-based polystyrene agglutination test

    DEFF Research Database (Denmark)

    Dubreuil, J.D.; Letellier, A.; Stenbæk, Eva;

    1996-01-01

    A polystyrene agglutination test has been developed for serotyping Actinobacillus pleuropneumoniae serotype 5a and 5b strains. Protein A-coated polystyrene microparticles were sensitized with a murine monoclonal antibody recognizing an epitope on serotype 5 LPS-O chain as shown by SDS......-PAGE and Western blotting, A total of 205 A. pleuropneumoniae, strains including all 12 serotype reference strains and 13 strains representing 8 common bacterial species associated with swine or related to A, pleuropneumoniae, were tested by mixing 25 mu L of polystyrene reagent with the same volume of a dense...... suspension of bacterial cells grown for 18 h. All A, pleuropneumoniae strains had been previously serotyped using standard procedures, The polystyrene agglutination test was rapid (less than 3 min) and easy to perform. Overall a very good correlation (97.3%) with the standard techniques was found...

  14. [The mechanism of change in speed of agglutination of human erythrocytes under the influence of adrenaline].

    Science.gov (United States)

    Volodchenko, A I; Tsirkin, V I; Kostiaev, A A

    2014-01-01

    In the study of red blood cells of 80 men found that adrenaline (10(-10) - 10(-6) g/mL) and phenylephrine (10-(10) - 10(-6) g/mL) dose-dependently increase the speed of agglutination of red blood cells, according to the decrease in agglutination of the start time and ginipral (10(-10) - 10(-7) g/mL), on the contrary, decreases it. The effect of adrenaline and phenylephrine is blocked by nicergoline (10(-6) g/mL), increased obzidan (10(-6) g/mL) and does not change under the action ofyohimbine (10(-6) g/mL) and atenolol (10(-6) g/mL). These data indicate that the speed of agglutination increases with activation alpha1-adrenergic receptor (AR) and decreases in the activation of beta2-AR, while the activation of alpha2- and beta1-AR does not affect it. Trifluoperazine (10(-6) g/mL) as the calmodulin antagonist, barium chloride (10(-6) g/mL) as a blocked of Ca(2+)-dependent K(+)-channels and indomethacine (10(-6) g/mL) as an inhibitor of cyclooxygenase and phospholipase A2 inhibit the ability of adrenaline to increases the speed of agglutination of red blood cells. This suggests that the effect of adrenaline caused an increase in erythrocyte entry of Ca2+, activation of calmodulin, cyclooxygenase, phospholipase A2 and the release of K+ from red blood cell through the Ca(2+)-dependent K+ channels, which is regarded as a manifestation of eryptosis. Indirectly, this means that more efficient activation of alpha1-AR and beta2-AR, respectively, increases or, conversely, decreases the rate of eryptosis.

  15. A rapid ultrasound particle agglutination method for HIV antibody detection: Comparison with conventional rapid HIV tests.

    Science.gov (United States)

    Bystryak, Simon; Ossina, Natalya

    2017-08-24

    We present the results of the feasibility and preliminary studies on analytical performance of a rapid test for detection of human immunodeficiency virus (HIV) antibodies in human serum or plasma that is an important advance in detecting HIV infection. Current methods for rapid testing of antibodies against HIV are qualitative and exhibit poor sensitivity (limit of detection). In this paper, we describe an ultrasound particle agglutination (UPA) method that leads to a significant increase of the sensitivity of conventional latex agglutination tests for HIV antibody detection in human serum or plasma. The UPA method is based on the use of: 1) a dual mode ultrasound, wherein a first single-frequency mode is used to accelerate the latex agglutination process, and then a second swept-frequency mode of sonication is used to disintegrate non-specifically bound aggregates; and 2) a numerical assessment of results of the agglutination process. The numerical assessment is carried out by optical detection and analysis of moving patterns in the resonator cell during the swept-frequency mode. The single-step UPA method is rapid and more sensitive than the three commercial rapid HIV test kits analyzed in the study: analytical sensitivity of the new UPA method was found to be 510-, 115-, and 80-fold higher than that for Capillus™, Multispot™ and Uni-Gold™ Recombigen HIV antibody rapid test kits, respectively. The newly developed UPA method opens up additional possibilities for detection of a number of clinically significant markers in point-of-care settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. A Comparison of Immuncapture Agglutination and ELISA Methods in Serological Diagnosis of Brucellosis

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    Mehmet Özdemir, Bahadır Feyzioğlu, Muhammed Güzel Kurtoğlu, Metin Doğan, Hatice Türk Dağı, Şerife Yüksekkaya, Recep Keşli, Bülent Baysal

    2011-01-01

    Full Text Available Background: Different serological tests are used in serologic diagnosis of brucellosis. The most widely used of these are Standard Tube Agglutination and Coombs anti-brucella tests. Whereas ELISA Ig M and Ig G tests have been in use for a long time, immuncapture agglutination test has been recently introduced and used in serological diagnosis. The aim of this study was to compare diagnostic values of ELISA Ig M and Ig G and immuncapture agglutination tests with Coombs anti-brucella test.Methods: Sera from 200 patients with presumptive diagnosis of brucellosis were included into the study. Coombs anti-brucella test, ELISA Ig M and Ig G tests and Immuncapture test were investigated in these sera. Then, sensitivity, specificity, negative predictive and positive predictive values were calculated.Results: Sensitivity, specificity, negative predictive and positive predictive values were found to be 90,6 %, 76,3 %, 94,2 %, and 65,9 % respectively for the Immuncapture test, whereas they were found to be 73,7 %, 58,9 %, 84,2 %, and 42,8 % for Ig G and 72,2 %, 67,8 %, 85,2 %, and 48,7 % for Ig M. The Immuncapture test was found to be compatible with ELISA Ig M and Ig G tests but it was statistically incompatible with Coombs anti-brucella test.Conclusions: Immuncapture agglutination test yields similar results to those of Coombs anti-brucella test. This test is a useful test by virtue of the fact that it determines blocking antibodies in the diagnosis and follow-up of brucellosis.

  17. Comparison of local Salmonella pullorum antigen with imported product in whole blood agglutination test

    Directory of Open Access Journals (Sweden)

    Priyani Medewewa

    Full Text Available Background: Salmonellosis is considered as one of the most important diseases in poultry as it causes devastating losses in chicken industry. Proper identification of the infected and carrier birds is required to control the disease among chickens. In field situation whole blood agglutination test is performed in order to identify carriers of Pullorum and Fowl typhoid particularly, in breeder operations. In this test, serum antibodies are detected by using a specially made antigen for this purpose. In Sri Lanka, three antigen products are used commonly in whole blood agglutination test. Objectives: This study was carried out to compare these two locally available S. Pullorum antigen products and to determine any difference in the efficacy. Methods:“Shaver Brown” commercial layer birds (70 in number were used in the experiment. Birds were inoculated orally with 1.8X109cfu/ml of S. Pullorum at 16 weeks age. After Three weeks post inoculation, blood was collected from each bird and Whole blood agglutination test was performed using both antigen products. Fifteen (15 inoculated hens were selected randomly and cloacal swabs were cultured on cultured Agar on same day of serum collected. Results: In this study, there was no significant difference observed between two antigens to detect carrier birds by whole blood agglutination test. Salmonella was not isolated from cloacal swabs since no observed excretion of Salmonella Pullorum through faces. All cloacal swabs gave negative results, when cultured on artificial Agar. Conclusion: Both antigen can be used effectively to detect carrier birds under the control program in country. [Vet World 2012; 5(9.000: 546-548

  18. Evaluation of a new latex agglutination test for detection of streptolysin O antibodies.

    OpenAIRE

    Gerber, M. A.; Caparas, L S; Randolph, M F

    1990-01-01

    Acute- and convalescent-phase serum specimens were collected from 50 patients with group A streptococcal pharyngitis. The anti-streptolysin O (ASO) titer for each serum specimen was determined by using both the standard neutralization assay and the latex agglutination (LA) test (Rheumagen ASO; Biokit Inc., New Britain, Conn.). When the ASO titers derived by the two methods were compared, the correlation coefficient was 0.93. When the ability of the LA test to demonstrate a significant ASO tit...

  19. Enhancement of Leptospira hardjo agglutination titers in sheep and goat serum by heat inactivation.

    Science.gov (United States)

    Malkin, K

    1984-04-01

    Heat inactivation of sheep serum samples resulted in the detection of an additional 9% reactors to Leptospira hardjo that were negative on the initial test of fresh samples. Treatment with EDTA gave results generally similar to heat inactivation suggesting that complement was responsible for the inhibition of agglutination. Tests on heat inactivated serum from experimentally infected sheep and goats revealed enhanced titers or reactions which were not detected in fresh serum.

  20. Use of commercial extenders and alternatives to prevent sperm agglutination for cryopreservation of brown bear semen.

    Science.gov (United States)

    Gomes-Alves, S; Alvarez, M; Nicolas, M; Lopez-Urueña, E; Martínez-Rodríguez, C; Borragan, S; de Paz, P; Anel, L

    2014-08-01

    The objective of this study was to evaluate different bovine and canine commercial semen extenders for cryopreservation of brown bear ejaculates and the effect of semen collection directly into extender on sperm agglutination. Semen samples were obtained by electroejaculation from 13 adult males. In experiment 1, eleven ejaculates from eight bears were used to evaluate Bioxcell and Andromed as extenders, whereas in experiment 2, nine ejaculates from six bears were used to evaluate Triladyl canine, CaniPro, and Extender 2 as extenders. An extender specifically developed for brown bears (Test-Tris-fructose-egg yolk-glycerol, TTF-ULE/bear) served as a control extender in both experiments. After thawing, total and progressive sperm motility and sperm viability were greater (P agglutination in fresh samples (score 0.5 ± 0.2 vs. 1.8 ± 0.4 in diluted and control samples, respectively) with no effect on pre-freeze and post-thawing semen quality. In conclusion, TTF-ULE/bear is the most suitable extender for brown bear semen cryopreservation, but comparable results can be obtained with the commercial extender Andromed. In addition, collection of ejaculates directly in TTF-H extender decreases sperm agglutination in fresh samples.

  1. Automated point-of-care testing for ABO agglutination test: proof of concept and validation.

    Science.gov (United States)

    El Kenz, H; Corazza, F

    2015-07-01

    ABO-incompatible red blood cell transfusions still represent an important hazard in transfusion medicine. Therefore, some countries have introduced a systematic bedside ABO agglutination test checking that the right blood is given to the right patient. However, this strategy requires an extremely time-consuming learning programme and relies on a subjective interpretation of ABO test cards agglutination. We developed a prototype of a fully automated device performing the bedside agglutination test that could be completed by reading of a barcoded wristband. This POCT checks the ABO compatibility between the patient and the blood bag. Proof of concept and analytical validation of the prototype has been completed on 451 blood samples: 238 donor packed red blood cells, 137 consecutive unselected patients for whom a blood group determination had been ordered and on 76 patient samples selected with pathology that could possibly interfere with or impair performances of the assay. We observed 100% concordance for ABO blood groups between the POCT and the laboratory instrument. These preliminary results demonstrate the feasibility of ABO determination with a simple POCT device eliminating manipulation and subjective interpretation responsible for transfusion errors. This device should be linked to the blood bank system allowing all cross-check of the results. © 2015 International Society of Blood Transfusion.

  2. [Yes, we should keep ABO agglutination test within bedside transfusion checks].

    Science.gov (United States)

    Daurat, G

    2008-11-01

    ABO incompatible transfusions are still a frequent cause of serious adverse transfusion reactions. Bedside check is intended to detect patient errors and prevent ABO mismatch. France is one of the few countries that includes ABO agglutination test for red blood cells in bedside checks. Evaluation of this ABO agglutination test, performed with a special card, shows that, on the field, despite frequent users' mishandling, it can detect up to 93% of ABO incompatibilities. This is not enough to rely on this sole test for bedside checks. But, linking it with an another test, currently, checks that the right blood is given to the right patient, rises the sensitivity of the whole bedside procedure up to an estimated 99.65%, for detection of ABO incompatibilities. This linkage has been introduced in the French regulation in 2003. Since then, the incidence of ABO incompatible transfusions has decreased dramatically and faster than in any other country, so France has now, probably, the lowest rate of ABO incompatible transfusions. The investigation of the few ABO accidents that still occur, shows that professionals have always bypassed this linkage. On the other hand, introducing bedside recipient and blood products barcode or radio-chip checks in all the 1500 French hospitals, though technically possible, would provide very little enhancement and lead to major difficulties and expenses. Linkage of ABO agglutination test to patient and blood checks within the bedside procedure has proved to be efficient and should be kept.

  3. Lack of chemical fractionation in major and minor elements during agglutinate formation. [in lunar soil

    Science.gov (United States)

    Hu, H.-N.; Taylor, L. A.

    1977-01-01

    Rhodes et al. (1975, 1976) and Adams et al. (1975) have reported that the agglutinate fraction of the soils on the lunar surface displays a marked enrichment in Fe, Mg, Ti, K, and La, and a depletion in Ca, Na, Al, and Eu, relative to the bulk soils. The reported investigation is concerned with a testing of the theory of chemical fractionation involving magnetic separation which was developed in connection with these findings. Soils 64421 and 71501 were sieved and the magnetic fractions separated according to the method developed by Adams and McCord (1973). Analyses of agglutinitic glass did not indicate any appreciable chemical fractionation for the major and minor elements accompanying the agglutination process. It was found that most, if not all fractionations reported can be accounted for completely by the magnetic nonagglutinate impurities in the agglutinate fraction. It is, therefore, concluded that there appears to be no reason to make use of any chemical fractionation theory, whose validity remains to be demonstrated.

  4. D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein.

    Science.gov (United States)

    Leahy, T; Rickard, J P; Aitken, R J; de Graaf, S P

    2016-05-01

    Head-to-head agglutination of ram spermatozoa is induced by dilution in the Tyrode's capacitation medium with albumin, lactate and pyruvate (TALP) and ameliorated by the addition of the thiol d-penicillamine (PEN). To better understand the association and disassociation of ram spermatozoa, we investigated the mechanism of action of PEN in perturbing sperm agglutination. PEN acts as a chelator of heavy metals, an antioxidant and a reducing agent. Chelation is not the main mechanism of action, as the broad-spectrum chelator ethylenediaminetetraacetic acid and the copper-specific chelator bathocuproinedisulfonic acid were inferior anti-agglutination agents compared with PEN. Oxidative stress is also an unlikely mechanism of sperm association, as PEN was significantly more effective in ameliorating agglutination than the antioxidants superoxide dismutase, ascorbic acid, α-tocopherol and catalase. Only the reducing agents cysteine and DL-dithiothreitol displayed similar levels of non-agglutinated spermatozoa at 0 h compared with PEN but were less effective after 3 h of incubation (37 °C). The addition of 10 µM Cu(2+) to 250 µM PEN + TALP caused a rapid reversion of the motile sperm population from a non-agglutinated state to an agglutinated state. Other heavy metals (cobalt, iron, manganese and zinc) did not provoke such a strong response. Together, these results indicate that PEN prevents sperm association by the reduction of disulphide bonds on a sperm membrane protein that binds copper. ADAM proteins are possible candidates, as targeted inhibition of the metalloproteinase domain significantly increased the percentage of motile, non-agglutinated spermatozoa (52.0% ± 7.8) compared with TALP alone (10.6% ± 6.1).

  5. The Staphylococcus aureus ArlRS two-component system is a novel regulator of agglutination and pathogenesis.

    Science.gov (United States)

    Walker, Jennifer N; Crosby, Heidi A; Spaulding, Adam R; Salgado-Pabón, Wilmara; Malone, Cheryl L; Rosenthal, Carolyn B; Schlievert, Patrick M; Boyd, Jeffrey M; Horswill, Alexander R

    2013-01-01

    Staphylococcus aureus is a prominent bacterial pathogen that is known to agglutinate in the presence of human plasma to form stable clumps. There is increasing evidence that agglutination aids S. aureus pathogenesis, but the mechanisms of this process remain to be fully elucidated. To better define this process, we developed both tube based and flow cytometry methods to monitor clumping in the presence of extracellular matrix proteins. We discovered that the ArlRS two-component system regulates the agglutination mechanism during exposure to human plasma or fibrinogen. Using divergent S. aureus strains, we demonstrated that arlRS mutants are unable to agglutinate, and this phenotype can be complemented. We found that the ebh gene, encoding the Giant Staphylococcal Surface Protein (GSSP), was up-regulated in an arlRS mutant. By introducing an ebh complete deletion into an arlRS mutant, agglutination was restored. To assess whether GSSP is the primary effector, a constitutive promoter was inserted upstream of the ebh gene on the chromosome in a wildtype strain, which prevented clump formation and demonstrated that GSSP has a negative impact on the agglutination mechanism. Due to the parallels of agglutination with infective endocarditis development, we assessed the phenotype of an arlRS mutant in a rabbit combined model of sepsis and endocarditis. In this model the arlRS mutant displayed a large defect in vegetation formation and pathogenesis, and this phenotype was partially restored by removing GSSP. Altogether, we have discovered that the ArlRS system controls a novel mechanism through which S. aureus regulates agglutination and pathogenesis.

  6. Schistosoma mansoni in susceptible and resistant snail strains Biomphalaria tenagophila: in vivo tissue response and in vitro hemocyte interactions.

    Directory of Open Access Journals (Sweden)

    Rafael Nacif-Pimenta

    Full Text Available Schistosomiasis is a parasitic disease that is highly prevalent, especially in developing countries. Biomphalaria tenagophila is an important invertebrate host of Schistosoma mansoni in Brazil, with some strains (e.g. Cabo Frio being highly susceptible to the parasite, whereas others (e.g. Taim are completely resistant to infection. Therefore, B. tenagophila is an important research model for studying immune defense mechanisms against S. mansoni. The internal defense system (IDS of the snail comprises hemocytes and hemolymph factors acting together to recognize self from non-self molecular patterns to eliminate the threat of infection. We performed experiments to understand the cellular defenses related to the resistance and/or susceptibility of B. tenagophila to S. mansoni. During the early stages of infection, fibrous host cells of both snail strains were arranged as a thin layer surrounding the sporocysts. However, at later stages of infection, the cellular reactions in resistant snails were increasingly more intense, with thicker layers surrounding the parasites, in contrast to susceptible strains. All parasites were damaged or destroyed inside resistant snails after 10 h of infection. By contrast, parasites inside susceptible snails appeared to be morphologically healthy. We also performed experiments using isolated hemocytes from the two strains interacting with sporocysts. Hemocyte attachment started as early as 1 h after initial infection in both strains, but the killing of sporocysts was exclusive to hemocytes from the resistant strain and was time course dependent. The resistant strain was able to kill all sporocysts. In conclusion, our study revealed important aspects of the initial process of infection related to immune defense responses of strains of B. tenagophila that were resistant to S. mansoni compared with strains that were susceptible. Such information is relevant for the survival or death of the parasites and so is important

  7. A thymosin beta-4 is involved in production of hemocytes and immune defense of Hong Kong oyster, Crassostrea hongkongensis.

    Science.gov (United States)

    Li, Jun; Zhang, Yuehuan; Liu, Ying; Zhang, Yang; Xiang, Zhiming; Qu, Fufa; Yu, Ziniu

    2016-04-01

    Thymosin beta-4 (Tβ4) is a ubiquitous protein with multiple and diverse intracellular and extracellular functions in vertebrates. In this study, the full-length cDNA of Tβ4 was cloned and identified in Crassostrea hongkongensis, designated as ChTβ4. The full-length cDNA of ChTβ4 consists of 530 bp with an open reading frame of 126 bp encoding a 41 amino acid polypeptide. SMART analysis indicated that there is one thymosin domain and a highly conserved actin-binding motif (18LKKTET23) in ChTβ4. In vivo injection of recombinant ChTβ4 protein could significantly increase total hemocytes count in oysters, and knockdown of the expression of ChTβ4 resulted in a significant decrease in the circulating hemocytes. Tissue distribution analysis revealed a ubiquitous presence of ChTβ4, with the highest expression in hemocytes. The upregulated transcripts of ChTβ4 in response to bacterial challenge and tissue injury suggest that ChTβ4 is involved in both innate immunity against pathogen infection and wound healing. Moreover, bacteria-clearance experiment showed ChTβ4 could facilitate the clearance of injected bacteria in oysters. In vivo injection with ChTβ4 resulted in reduction of the intracellular ROS in hemocytes, which was associated with increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD), Catalase, and Glutathione Peroxidase (GPX) by pre-treatment with ChTβ4. These results suggest that ChTβ4 is a thymosin beta-4 homolog and plays a vital role in the immune defense of C. hongkongensis.

  8. Differential actions of proteinases and neuraminidase on mammalian erythrocyte surface and its impact on erythrocyte agglutination by concanavalin A.

    Science.gov (United States)

    Sharma, Savita; Gokhale, Sadashiv M

    2012-12-01

    Action of proteinases viz. trypsin and chymotrypsin, and neuraminidase on intact erythrocyte membrane proteins and glycophorins (sialoglycoproteins) exposed to cell surface and its impact on lectin (concanavalin A)-mediated agglutination were studied in Homo sapiens (human), Capra aegagrus hircus (goat) and Bubalus bubalis (buffalo). Membrane proteins and glycophorins analysis by SDS-PAGE as visualized by coomassie brilliant blue and periodic acid-schiff stains, respectively, and agglutination behaviour revealed marked differences: 1) there were prominent dissimilarities in the number and molecular weights of glycophorins in human, goat and buffalo erythrocyte membranes; 2) proteinase action(s) on human and buffalo erythrocyte surface membrane proteins and glycophorins showed similarity but was found different in goat; 3) significant differences in erythrocyte agglutinability with concanavalin A can be attributed to differences in membrane composition and alterations in the surface proteins after enzyme treatment; 4) a direct correlation was found between degradation of glycophorins and concanavalin A agglutinability; 5) action of neuraminidase specifically indicated the negative role of cell surface sialic acids in determining concanavalin A agglutinability of goat and buffalo erythrocytes, similar to human. Present studies clearly indicate that there are some basic differences in human, goat and buffalo erythrocyte membrane proteins, especially with respect to glycophorins, which determine the concanavalin A-mediated agglutination in enzyme treated erythrocytes.

  9. Concanavalin A-mediated cell agglutinability induced by Vaccinia virions. [Uv radiation, /sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Mbuy, G.; Bubel, H.C.

    1978-12-01

    The induction of enhanced concanavalin A (Con A)-mediated cellular agglutinability by purified vaccinia virus was examined quantitatively. Increased HEp-2 cell agglutinability by the lectin occurred within the first hour of infection and persisted without further change throughout the virus infectious cycle. Ultraviolet, but not heat-inactivated, virus was as effective as infectious virus in causing increased Con A agglutinability. Inhibition of viral and host cell protein synthesis by Streptovitacin A failed to alter the lectin response to vaccinia virus infection. Fluorescein-labeled Con A was observed to form clusters and large fluorescent patches on the infected cell surface during the earliest stage of infection. Studies with /sup 125/I-labeled Con A revealed an early but minimal increase in lectin binding to infected cells. After the first hour of infection, no further increase in Con A binding was observed. When cells were exposed to purified vaccinia virus surface tubules increased Con A agglutinability comparable to that obtained with native virus was demonstrated. Con A-mediated agglutinability of cells was temperature-dependent and displayed a higher temperature transition in infected cells. These data suggest that upon contact with the host cell, vaccinia virions or surface tubules induce alterations in the plasma membrane which are reflected in an enhanced agglutinability by Con A.

  10. Comparison of antibody titers using conventional tube technique versus column agglutination technique in ABO blood group incompatible renal transplant.

    Science.gov (United States)

    Bhangale, Amit; Pathak, Amardeep; Pawar, Smita; Jeloka, Tarun

    2017-01-01

    Measurement of alloantibody titer to a red cell antigen (ABO titers) is an integral part of management of ABO incompatible kidney transplants (ABOiKT). There are different methods of titer estimation. Alloantibody detection by tube titration and Gel agglutination columns are accepted methodologies. It is essential to find the difference in titers between the two methods so as to set the 'cut-off' titer accordingly, depending upon the method used. We did a prospective observational study to compare and correlate the ABO titers using these two different techniques - conventional tube technique (CTT) and the newer column agglutination technique (CAT). A total of 67 samples were processed in parallel for anti-A/B antibodies by both tube dilution and column agglutination methods. The mean titer by conventional tube method was 38.5 + 96.6 and by the column agglutination test was 96.4 + 225. The samples correlated well with Spearman rho correlation coefficient of 0.94 (P = 0.01). The column agglutination method for anti A/B titer estimation in an ABO incompatible kidney transplant is more sensitive, with the column agglutination results being approximately two and half fold higher (one more dilution) than that of tube method.

  11. Combined effects of seawater acidification and high temperature on hemocyte parameters in the thick shell mussel Mytilus coruscus.

    Science.gov (United States)

    Wu, Fangli; Lu, Weiqun; Shang, Yueyong; Kong, Hui; Li, Lisha; Sui, Yanming; Hu, Menghong; Wang, Youji

    2016-09-01

    In this work, flow cytometry was used to examine the immune responses of hemocytes in the thick shell mussel Mytilus coruscus exposed to six combinations of pH (7.3, 7.7, and 8.1) and temperature (25 °C and 30 °C) for 14 days. Temperature showed significant effects on all immune parameters throughout the experiment. Generally, the total hemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) significantly decreased at high temperature. By contrast, the hemocyte mortality (Hm) and reactive oxygen species (ROS) levels increased at high temperature. Moreover, pH significantly influenced all the immune parameters, but its effects are not as strong as those of temperature; only Hm, Est, and THC were negatively affected by pH throughout the experiment. After 7 days, low pH resulted in decreased Lyso and increased Hm and ROS levels. Significant interactions between temperature and pH in most measured parameters from 7 days suggested that long-term combined stress, i.e., low pH and high temperature, would cause more severe effects on mussel health than an individual stressor in the field.

  12. Insight into the messenger role of reactive oxygen intermediates in immunostimulated hemocytes from the scallop Argopecten purpuratus.

    Science.gov (United States)

    Oyanedel, Daniel; Gonzalez, Roxana; Brokordt, Katherina; Schmitt, Paulina; Mercado, Luis

    2016-12-01

    Reactive oxygen intermediates (ROI) are metabolites produced by aerobic cells which have been linked to oxidative stress. Evidence reported in vertebrates indicates that ROI can also act as messengers in a variety of cellular signaling pathways, including those involved in innate immunity. In a recent study, an inhibitor of NF-kB transcription factors was identified in the scallop Argopecten purpuratus, and its functional characterization suggested that it may regulate the expression of the big defensin antimicrobial peptide ApBD1. In order to give new insights into the messenger role of ROI in the immune response of bivalve mollusks, the effect of ROI production on gene transcription of ApBD1 was assessed in A. purpuratus. The results showed that 48 h-cultured hemocytes were able to display phagocytic activity and ROI production in response to the β-glucan zymosan. The immune stimulation also induced the transcription of ApBD1, which was upregulated in cultured hemocytes. After neutralizing the ROI produced by the stimulated hemocytes with the antioxidant trolox, the transcription of ApBD1 was reduced near to base levels. The results suggest a potential messenger role of intracellular ROI on the regulation of ApBD1 transcription during the immune response of scallops.

  13. Hemocytes of zebra mussels (Dreissena polymorpha are relevant cells for the monitoring of environmental genotoxicity by the comet assay.

    Directory of Open Access Journals (Sweden)

    Marc Bonnard

    2015-06-01

    Full Text Available The measure of DNA integrity by the single cell gel electrophoresis (SCGE or comet assay is especially recommended for its sensitivity and its capacity for detecting different types of damages. Therefore, it has been applied in environmental genotoxicity in a variety of organisms. It appears today necessary to define both reference and threshold levels of DNA damage, for their application in in situ biomonitoring. However, little is known about the influence of both biological (sex, reproduction status or external (temperature… confounding factors on the measure of DNA damage by the comet assay. These variables need to be taken into account if the robustness of the assay is to be established (Jha, 2008. In the zebra mussel Dreissena polymorpha (recommended as a sentinel species in the evaluation of freshwater quality the measure of DNA damage by the comet assay is mainly performed on hemocytes, which are circulating cells involved in key physiological functions such as immunity, homeostasis, detoxication…. This communication will present and discuss results from an innovative study about the variability of the baseline level of DNA damage in hemocytes of mussels encaged for one year in the canal de l’Aisne à la Marne (Reims, according to their sex and their reproductive status. The sensitivity and the suitability of hemocytes in the evaluation of environmental genotoxicity will also be discussed, referring to observations during a 6 month-exposure of mussels in mesocosms to environmentally realistic concentrations of carbamazepine.

  14. Apoptosis of hemocytes from lions-paw scallop Nodipecten subnodosus induced with paralyzing shellfish poison from Gymnodinium catenatum.

    Science.gov (United States)

    Estrada, Norma; Ascencio, Felipe; Shoshani, Liora; Contreras, Rubén G

    2014-12-01

    The toxic dinoflagellate Gymnodinium catenatum produces paralyzing shellfish poisons (PSPs) that are consumed and accumulated by bivalves. Previously, we recorded a decrease in hemocytes 24h after injection of PSPs (gonyautoxin 2/3 epimers, GTX2/3) in the adductor muscle in the lions-paw scallop Nodipecten subnodosus. In this work, qualitative and quantitative analyses, in in vivo and in vitro experiments, revealed that the lower count of hemocytes results from cells undergoing typical apoptosis when exposed to GTX 2/3 epimers. This includes visible morphological alterations of the cytoplasmic membrane, damage to the nuclear membrane, condensation of chromatin, DNA fragmentation, and release of DNA fragments into the cytoplasm. Induction of apoptosis was accompanied by phosphatidylserine exposure to the outer cell membrane and activation of cysteine-aspartic proteases, caspase 3 and caspase 8. Addition of an inhibitor of caspase to the medium suppressed activation in hemocytes exposed to the toxins, suggesting that cell death was induced by a caspase-dependent apoptotic pathway. The results are important for future investigation of the scallop's immune system and should provide new insights into apoptotic processes in immune cells of scallops exposed to PSPs. Copyright © 2014 Elsevier GmbH. All rights reserved.

  15. Different Effects of Metarhizium anisopliae Strains IMI330189 and IBC200614 on Enzymes Activities and Hemocytes of Locusta migratoria L.

    Science.gov (United States)

    Cao, Guangchun; Jia, Miao; Zhao, Xia; Wang, Lei; Tu, Xiongbing; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-01-01

    Background Metarhizium is an important class of entomopathogenic fungi in the biocontrol of insects, but its virulence is affected by insect immunity. To clarify the mechanism in virulence of Metarhizium, we compared the immunological differences in Locusta migratoria L. when exposed to two strains of Metarhizium anisopliae (Ma). Results The virulence of Ma IMI330189 was significantly higher than that of Ma IBC200614 to locust, and IMI330189 overcame the hemocytes and began destroying the hemocytes of locust at 72 h after spray, while locust is immune to IBC200614. IMI330189 could overcome the humoral immunity of locust by inhibiting the activities of phenol oxidase (PO), esterases, multi-function oxidases (MFOs) and acetylcholinesterases in locust while increasing the activities of glutathione-S-transferases (GSTs), catalase and aryl-acylamidase (AA). However IBC200614 inhibit the activities of GSTs and AA in locust and increase the activities of MFOs, PO, superoxide dismutase, peroxidase and chitinase in locust. The changes of enzymes activities in period of infection showed that the time period between the 2nd and the 5th day after spray is critical in the pathogenic process. Conclusion These results found the phenomenon that Ma initiatively broke host hemocytes, revealed the correlation between the virulence of Ma and the changes of enzymes activities in host induced by Ma, and clarified the critical period in the infection of Ma. So, these results should provide guidance for the construction of efficient biocontrol Ma strains. PMID:27227835

  16. Recent advances in crayfish hematopoietic stem cell culture: a model for studies of hemocyte differentiation and immunity.

    Science.gov (United States)

    Söderhäll, Irene

    2013-10-01

    Hematopoiesis is the process by which blood cells (hemocytes) mature and subsequently enter the circulation and we have developed a new technique to culture the hematopoietic progenitor cells in vitro. The reason for the successful culture was the isolation of a plasma protein that turned out to be a novel cytokine, astakine 1 (Ast1) containing a domain present in several vertebrates, so-called prokineticins. Now we have detected several astakines from other invertebrate species. Depending on our discovery of the cytokine Ast1 we have an opportunity to study in detail the differentiation of cells in the hematopoietic tissue of a crustacean, a tissue of evolutionary interest for studies of the connection between the vascular system and the nervous system. We have been able to isolate the entire hematopoietic tissue and for the first time detected a link between this tissue and the brain. We have further localized a proliferation center in the tissue and characterized its different parts. We have also used this system to isolate a new hematopoietic factor CHF that is important in the crossroad between apoptosis and hemocyte differentiation. Our technique for culture of crayfish hematopoietic stem cells provides a simple tool for studying the mechanism of hematopoiesis, but also enables detailed studies of immune defense reactions. Further, the culture system has been used for studies of viral defense and the system is suitable for gene silencing which allows functional characterization of different molecules involved in host defense as well as in hemocyte differentiation.

  17. Effect of Dopamine Injection on the Hemocyte Count and Prophenoloxidase System of the White Shrimp Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    PAN Luqing; HU Fawen; ZHENG Debin

    2011-01-01

    Effects of dopamine injection on the hemocyte count,phenoloxidase activity,serine proteinase activity,proteinase inhibitor activity and α2-macroglobulin-like activity in L.vannamei were studied.Results showed that dopamine injection resulted in a significant effect on the parameters measured (P<0.05),while no significant difference was observed in the control group (0.85% NaCl).In the experimental groups,the hemocyte count reached the minimum in 3 h; granular and semi-granular cells became stable after 12 h and hyaline cells and the total hemocyte count became stable after 18 h.Phenoloxidase activity reached the minimum in 6 h,and then became stable after 9h.Serine protease activity and proteinase inhibitor activity reached the minimum in 3 h,and α2-macroglobulin-like activity reached the maximum in 3 h,and all the three parameters became stable after 12 h.The results suggest that the activating mechanisms of the proPO system triggered by dopamine are different from those triggered by invasive agents or spontaneously activated under a normal physical condition.

  18. Functional and metabolic characterization of hemocytes of the green mussel, Perna viridis: in vitro impacts of temperature.

    Science.gov (United States)

    Donaghy, Ludovic; Volety, Aswani K

    2011-12-01

    The green mussel, Perna viridis, is a bivalve mollusk native to Asia and was recently introduced to Florida, USA. Since its first observation in 1999 in Tampa Bay, Florida, green mussel population has expanded considerably, to reach the Atlantic coast of Florida, Georgia and South Carolina. Most of currently available studies about the ecology and biology of green mussels were performed in the Indian and Pacific oceans. Very recently, it has been suggested that due to a weak low temperature resistance, green mussels might have already reached the Northern edge of their distribution in the USA. However, there is currently an obvious lack of data about the adaptation capacities of Perna viridis to environmental conditions in Florida, especially at the physiological and cellular levels. In the present work, we determined and characterized the populations of circulating hemocytes, and the cellular components of hemolymph involved in various physiological functions, including immunity. Two main populations were characterized, hyalinocytes and granulocytes. Granulocytes accounted for 60% of circulating cells, and displayed higher phagocytic capacities, lysosomal content and basal oxidative metabolism than hyalinocytes. Hemocyte parameters were not influenced by the size of green mussels. In addition, hemocytes were subjected to acute temperature challenges (10, 20 and 30 °C) and their immune-related functions and metabolism analyzed. Our results showed that 10 °C represent a stressful condition for the Floridian green mussels, as depicted by a low phagocytosis capacity and an increase of oxidative metabolism. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Down-regulation apoptosis signal-regulating kinase 1 gene reduced the Litopenaeus vannamei hemocyte apoptosis in WSSV infection.

    Science.gov (United States)

    Yuan, Feng-Hua; Chen, Yong-Gui; Zhang, Ze-Zhi; Yue, Hai-Tao; Bi, Hai-Tao; Yuan, Kai; Weng, Shao-Ping; He, Jian-Guo; Chen, Yi-Hong

    2016-03-01

    Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is crucial in various cellular responses. In the present study, we identified and characterized an ASK1 homolog from Litopenaeus vannamei (LvASK1). The full-length cDNA of LvASK1 was 5400 bp long, with an open reading frame encoding a putative 1420 amino acid protein. LvASK1 was highly expressed in muscle, hemocyte, eyestalk and heart. Real-time RT-PCR analysis showed that the expression of the LvASK1 was upregulated during the white spot syndrome virus (WSSV) challenge. The knocked-down expression of LvASK1 by RNA interference significantly reduced the apoptotic ratio of the hemocytes collected from WSSV-infected L. vannamei. Furthermore, the down-regulation of LvASK1 also decreased the cumulative mortality of WSSV-infected L. vannamei. These results suggested that down-regulation of LvASK1 decreased the apoptotic rate of hemocytes in WSSV-infected shrimp, and that it could contribute to the reduction of cumulative mortality in WSSV-infected L. vannamei.

  20. Different Effects of Metarhizium anisopliae Strains IMI330189 and IBC200614 on Enzymes Activities and Hemocytes of Locusta migratoria L.

    Directory of Open Access Journals (Sweden)

    Guangchun Cao

    Full Text Available Metarhizium is an important class of entomopathogenic fungi in the biocontrol of insects, but its virulence is affected by insect immunity. To clarify the mechanism in virulence of Metarhizium, we compared the immunological differences in Locusta migratoria L. when exposed to two strains of Metarhizium anisopliae (Ma.The virulence of Ma IMI330189 was significantly higher than that of Ma IBC200614 to locust, and IMI330189 overcame the hemocytes and began destroying the hemocytes of locust at 72 h after spray, while locust is immune to IBC200614. IMI330189 could overcome the humoral immunity of locust by inhibiting the activities of phenol oxidase (PO, esterases, multi-function oxidases (MFOs and acetylcholinesterases in locust while increasing the activities of glutathione-S-transferases (GSTs, catalase and aryl-acylamidase (AA. However IBC200614 inhibit the activities of GSTs and AA in locust and increase the activities of MFOs, PO, superoxide dismutase, peroxidase and chitinase in locust. The changes of enzymes activities in period of infection showed that the time period between the 2nd and the 5th day after spray is critical in the pathogenic process.These results found the phenomenon that Ma initiatively broke host hemocytes, revealed the correlation between the virulence of Ma and the changes of enzymes activities in host induced by Ma, and clarified the critical period in the infection of Ma. So, these results should provide guidance for the construction of efficient biocontrol Ma strains.

  1. Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies.

    Science.gov (United States)

    Canesi, Laura; Lorusso, Lucia Cecilia; Ciacci, Caterina; Betti, Michele; Rocchi, Marco; Pojana, Giulio; Marcomini, Antonio

    2007-02-15

    Endocrine disrupting compounds (EDCs) are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus the natural estrogen 17beta-estradiol (E2) and different EDCs have been recently demonstrated to affect the function of the immune cells, the hemocytes. The effects were Tamoxifen-sensitive and were mediated by rapid modulation of kinase-mediated transduction pathways. In this work we compared the in vitro effects of individual estrogenic chemicals (E2, EE: 17alpha-ethynyl estradiol; MES: mestranol; NP: nonylphenol; NP1EC: nonylphenol monoethoxylate carboxylate; BPA: bisphenol A; BP: benzophenone) on hemocyte parameters: lysosomal membrane stability (LMS), phagocytosis, lysozyme release. LMS was the most sensitive effect parameter, showing a decreasing trend at increasing concentrations of estrogens. EC50 values obtained from LMS data were utilized to calculate the estradiol equivalency factor (EEF) for each compound; these EEFs allowed for an estimation of the estrogenic potential of a synthetic mixture with a composition very similar to that previously found in waters of the Venice lagoon. Concentrated mixtures significantly affected hemocyte parameters in vitro and the effects were prevented by Tamoxifen. Significant effects of the mixture were also observed in vivo, at longer exposure times and at concentrations comparable with environmental exposure levels. The results indicate that Mytilus immune parameters can be suitably utilized to evaluate the estrogenic potential of environmental samples.

  2. RNA-Seq Study of Microbially Induced Hemocyte Transcripts from Larval Heliothis virescens (Lepidoptera: Noctuidae

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    Kent S. Shelby

    2012-08-01

    Full Text Available Larvae of the tobacco budworm are major polyphagous pests throughout the Americas. Development of effective microbial biopesticides for this and related noctuid pests has been stymied by the natural resistance mediated innate immune response. Hemocytes play an early and central role in activating and coordinating immune responses to entomopathogens. To approach this problem we completed RNA-seq expression profiling of hemocytes collected from larvae following an in vivo challenge with bacterial and fungal cell wall components to elicit an immune response. A de novo exome assembly was constructed by combination of sequence tags from all treatments. Sequence tags from each treatment were aligned separately with the assembly to measure expression. The resulting table of differential expression had > 22,000 assemblies each with a distinct combination of annotation and expression. Within these assemblies > 1,400 were upregulated and > 1,500 downregulated by immune activation with bacteria or fungi. Orthologs to innate immune components of other insects were identified including pattern recognition, signal transduction pathways, antimicrobial peptides and enzymes, melanization and coagulation. Additionally orthologs of components regulating hemocytic functions such as autophagy, apoptosis, phagocytosis and nodulation were identified. Associated cellular oxidative defenses and detoxification responses were identified providing a comprehensive snapshot of the early response to elicitation.

  3. Cloning of prophenoloxidase from hemocytes of the blue crab, Callinectes sapidus and its expression and enzyme activity during the molt cycle.

    Science.gov (United States)

    Alvarez, Javier V; Chung, J Sook

    2013-11-01

    The arthropods cuticle undergoes dramatic morphological and biochemical changes from being soft to hardness through each molting process. Prophenoloxidase (PPO) known as a key enzyme in the arthropod innate immune system involved in the melanization reaction, has been related with the initial shell-hardening process, specifically in the sclerotization of the protein matrix in the new cuticle. Since hemocytes have been reported as the main PPO source in arthropods, the transport of hemocyte PPO into the newly laid, soft cuticle has been proposed for shell-hardening occurring during and immediately after ecdysis. In order to define the role of hemocyte PPO in the shell-hardening of crustaceans, the full-length cDNA sequence (2806 nt) of hemocytes PPO of the blue crab Callinectes sapidus (CasPPO-hemo) is isolated using degenerate PCR and 5'-3' RACE. CasPPO-hemo encodes a putative PPO (672 aa) showing three hemocyanin domains: N, M, and C in order and two copper binding sites (CuA & CuB). The sequence analysis identifies the putative CasPPO-hemo as zymogen which requires the cleavage at the N-terminus for its activation. Hemocyte extract (CasHLS) contains the PO, the activity of which depends on the in vitro activation of trypsin. The expression levels of CasPPO-hemo are kept constant during the molt cycle. The increase in the number of hemocytes at early premolt correlates with the elevated PO activity, while at late premolt, the increment in hemocyte numbers does not reflect on the PO activity. The functional importance of the changes in the levels of CasHLS-PO activity during molt cycle is discussed in relation to cuticle hardening process. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Evaluation of agglutination strength by a flow-induced cell movement assay based surface plasmon resonance (SPR) technique.

    Science.gov (United States)

    Sudprasert, Krisda; Peungthum, Patjaree; Vongsakulyanon, Apirom; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Kitpoka, Pimpun; Kunakorn, Mongkol; Srikhirin, Toemsak

    2015-02-07

    A flow-induced cell movement assay combined with a surface plasmon resonance (SPR) technique was developed to quantify the agglutination strength, derived from the standard tube-agglutination test. Red blood cells (RBCs), based on the ABO blood group system, were specifically captured by anti-A and/or anti-B antibodies immobilized on a sensor surface. The agglutination strength corresponds to the amount of antigen-antibody interactions or the strength of RBC adhesion. Under a shear flow, the adherent RBCs were forced to move out of the region of interest with different average cell velocities (vc) depending upon the adhesion strength and wall shear stress (WSS). That is, a higher adhesion strength (higher agglutination strength) or lower WSS represents a lower vc or vice versa. In this work, the agglutination strength was derived from the vc that was calculated from the time derivative of the relative SPR signal by using a simple model of cell movement response, whose validity was verified. The vc values of different samples were correlated with their agglutination strengths at a given WSS and antibody surface density. The vc decreased as the agglutination strength increased, which can be considered as a linear regression. The coefficient of variation of the calculated vc decreased to 0.1 as vc increased to 30 μm min(-1). The sensitivity of this assay can be controlled by optimizing the antibody surface density or the WSS. This assay has the capability to resolve the antigen density of A1 and B RBCs from that of A1B RBCs.

  5. Sepsis and Hemocyte Loss in Honey Bees (Apis mellifera) Infected with Serratia marcescens Strain Sicaria.

    Science.gov (United States)

    Burritt, Nancy L; Foss, Nicole J; Neeno-Eckwall, Eric C; Church, James O; Hilger, Anna M; Hildebrand, Jacob A; Warshauer, David M; Perna, Nicole T; Burritt, James B

    2016-01-01

    Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies.

  6. Hemoglobins Likely Function as Peroxidase in Blood Clam Tegillarca granosa Hemocytes.

    Science.gov (United States)

    Wang, Sufang; Yu, Xiaopei; Lin, Zhihua; Zhang, Shunqin; Xue, Liangyi; Xue, Qinggang; Bao, Yongbo

    2017-01-01

    Hemoglobins are a group of respiratory proteins principally functioning in transport of oxygen and carbon dioxide in red blood cells of all vertebrates and some invertebrates. The blood clam T. granosa is one of the few invertebrates that have hemoglobin-containing red hemocytes. In the present research, the peroxidase activity of T. granosa hemoglobins (Tg-Hbs) was characterized and the associated mechanism of action was deciphered via structural comparison with other known peroxidases. We detected that purified Tg-Hbs catalyzed the oxidation of phenolic compounds in the presence of exogenous H2O2. Tg-Hbs peroxidase activity reached the maximum at pH 5 and 35°C and was inhibited by Fe(2+), Cu(2+), SDS, urea, and sodium azide. Tg-Hbs shared few similarities in amino acid sequence and overall structural characteristics with known peroxidases. However, the predicted structure at their heme pocket was highly similar to that of horseradish peroxidase (HRP) and myeloperoxidase (MPO). This research represented the first systemic characterization of hemoglobin as a peroxidase.

  7. Effects of Several Immunostimulants on Phenoloxidase and Hemocytes of the Crab Charybdis japonica

    Institute of Scientific and Technical Information of China (English)

    FAN Tingjun; YU Miaomiao; YANG Lingling; SHI Zhenping; SUN Wenjie; CONG Rishan; YANG Xiuxia; JIANG Guojian

    2009-01-01

    To investigate the stimulating effects of immunostimulants on the autogenous immunocompetence of crabs and the possible mechanisms involved, the immunostimulating effects of β-1,3-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on phenoloxidase (PO) and hemocytes of Charybdis japonica were investigated in this study. It was found that the yields and the enzymatic activities of purified PO in C. japonica increased significantly after the crabs were treated with immunostimulants, while the unit enzymatic activities remained almost the same. After treatment with β-1,3-glucan and LPS, the amount of rough endoplasmic reticulum (RER) and the number of mitochondria in both semigranular cells and granular cells increased greatly, and the number of cytoplasmic granules decreased but with enlarged volume. However, the corresponding characteristics of hyaline cells remained almost the same. On the other hand, the number of granules in semigranular cells decreased greatly, and the number of mitochondria of hyaline cells increased greatly, after treatment with inactivated vibrios. It may be concluded that the effect of polysaccharide immunostimulants on the innate immune system of C. japonica is different from that of inactivated vibrio immunostimulants. The immunity-enhancing mechanism of polysaccharides in crab autogenous immunocompetence is probably accomplished by the increased yields of PO and total PO activities, while that of inactivated vibrios is probably accomplished by the partially increased yields of PO and total PO activities as well as the significantly improved phagocytotic abilities of semigranular cells and hyaline cells.

  8. Hemoglobins Likely Function as Peroxidase in Blood Clam Tegillarca granosa Hemocytes

    Science.gov (United States)

    Yu, Xiaopei; Lin, Zhihua; Xue, Liangyi

    2017-01-01

    Hemoglobins are a group of respiratory proteins principally functioning in transport of oxygen and carbon dioxide in red blood cells of all vertebrates and some invertebrates. The blood clam T. granosa is one of the few invertebrates that have hemoglobin-containing red hemocytes. In the present research, the peroxidase activity of T. granosa hemoglobins (Tg-Hbs) was characterized and the associated mechanism of action was deciphered via structural comparison with other known peroxidases. We detected that purified Tg-Hbs catalyzed the oxidation of phenolic compounds in the presence of exogenous H2O2. Tg-Hbs peroxidase activity reached the maximum at pH 5 and 35°C and was inhibited by Fe2+, Cu2+, SDS, urea, and sodium azide. Tg-Hbs shared few similarities in amino acid sequence and overall structural characteristics with known peroxidases. However, the predicted structure at their heme pocket was highly similar to that of horseradish peroxidase (HRP) and myeloperoxidase (MPO). This research represented the first systemic characterization of hemoglobin as a peroxidase. PMID:28182094

  9. Transcriptome analysis of the pearl oyster (Pinctada fucata) hemocytes in response to Vibrio alginolyticus infection.

    Science.gov (United States)

    Wang, Zhongliang; Wang, Bei; Chen, Gang; Jian, Jichang; Lu, Yishan; Xu, Youhou; Wu, Zaohe

    2016-01-10

    The pearl oyster Pinctada fucata is cultured widely for production of marine pearls in China, while mass mortalities, likely related to pathogenic infections, have occurred frequently in juvenile, mother and operated oysters. To address this issue, understanding host defense mechanisms of P. fucata against pathogenic challenge is extremely important. In the present study, a comparative analysis of hemocyte transcriptomes of P. fucata before and after Vibrio alginolyticus infection was conducted using the Illumina/Hiseq-2000 RNA-Seq technology. A total of 56,345,139 clean reads were generated and then assembled into 74,007 unigenes with an average length of 680 bp and an N50 of 1197 bp. Unigenes were annotated by comparing against non-redundant protein sequence (nr), non-redundant nucleotide (nt), Swiss-Prot, Pfam, Gene Ontology database (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and 29,615 unigenes (40.01%) were annotated in at least one database. There were 636 genes (518 up-regulated and 118 down-regulated) that were significantly differentially expressed after bacterial challenge, and among which 369 were associated with 122 pathways, including classical immune-related pathways, such as 'MAPK signaling pathway', 'Chemokine signaling pathway', 'Apoptosis' and 'Wnt signaling pathway'. These findings provide information on the pearl oyster innate immunity and may contribute to developing strategies for management of diseases and long-term sustainability of P. fucata culture. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Nanoscale Mineralogy and Composition of Experimental Regolith Agglutinates Produced under Asteroidal Impact Conditions

    Science.gov (United States)

    Christoffersen, Roy; Cintala, M. J.; Keller, L. P.; See, T. H.; Horz, F.

    2013-01-01

    On the Moon, the energetics of smaller impactors and the physical/chemical characteristics of the granular regolith target combine to form a key product of lunar space weathering: chemically reduced shock melts containing optically-active nanophase Fe metal grains (npFe0) [1]. In addition to forming the optically dark glassy matrix phase in lunar agglutinitic soil particles [1], these shock melts are becoming increasingly recognized for their contribution to optically active patina coatings on a wide range of exposed rock and grain surfaces in the lunar regolith [2]. In applying the lessons of lunar space weathering to asteroids, the potential similarities and differences in regolith-hosted shock melts on the Moon compared to those on asteroids has become a topic of increasing interest [3,4]. In a series of impact experiments performed at velocities applicable to the asteroid belt [5], Horz et al. [6] and See and Horz [7] have previously shown that repeated impacts into a gabbroic regolith analog target can produce melt-welded grain aggregates morphologically very similar to lunar agglutinates [6,7]. Although these agglutinate-like particles were extensively analyzed by electron microprobe and scanning electron microscopy (SEM) as part of the original study [7], a microstructural and compositional comparison of these aggregates to lunar soil agglutinates at sub-micron scales has yet to be made. To close this gap, we characterized a representative set of these aggregates using a JEOL 7600 field-emission scanning electron microscope (FE-SEM), and JEOL 2500SE field-emission scanning transmission electron microscope (FE-STEM) both optimized for energy dispersive X-ray spectroscopy (EDX) compositional spectrum imaging at respective analytical spatial resolutions of 0.5 to 1 micron, and 2 to 4 nm.

  11. Preliminary evaluation of a gel tube agglutination major cross-match method in dogs.

    Science.gov (United States)

    Villarnovo, Dania; Burton, Shelley A; Horney, Barbara S; MacKenzie, Allan L; Vanderstichel, Raphaël

    2016-09-01

    A major cross-match gel tube test is available for use in dogs yet has not been clinically evaluated. This study compared cross-match results obtained using the gel tube and the standard tube methods for canine samples. Study 1 included 107 canine sample donor-recipient pairings cross-match tested with the RapidVet-H method gel tube test and compared results with the standard tube method. Additionally, 120 pairings using pooled sera containing anti-canine erythrocyte antibody at various concentrations were tested with leftover blood from a hospital population to assess sensitivity and specificity of the gel tube method in comparison with the standard method. The gel tube method had a good relative specificity of 96.1% in detecting lack of agglutination (compatibility) compared to the standard tube method. Agreement between the 2 methods was moderate. Nine of 107 pairings showed agglutination/incompatibility on either test, too few to allow reliable calculation of relative sensitivity. Fifty percent of the gel tube method results were difficult to interpret due to sample spreading in the reaction and/or negative control tubes. The RapidVet-H method agreed with the standard cross-match method on compatible samples, but detected incompatibility in some sample pairs that were compatible with the standard method. Evaluation using larger numbers of incompatible pairings is needed to assess diagnostic utility. The gel tube method results were difficult to categorize due to sample spreading. Weak agglutination reactions or other factors such as centrifuge model may be responsible. © 2016 American Society for Veterinary Clinical Pathology.

  12. LEPINOCONUS CHIOCCHINII GEN. N., N. SP., A CONICAL AGGLUTINATED FORAMINIFERA FROM THE UPPER CRETACEOUS OF ITALY

    Directory of Open Access Journals (Sweden)

    ERZIKA CRUZ-ABAD

    2017-04-01

    Full Text Available A new conical agglutinated foraminifer, Lepinoconus chiocchinii gen n., n. sp. from the lower Campanian shallow-water platform deposits of the Lepini Mountains (central Apennines, Italy, is described. It has a pseudo-keriothecal wall structure, uniserial arrangement of the adult chambers and multiple apertures. The exoskeleton is constituted by beams (main and intercalary continuous from one chamber to the next, while the endoskeleton bears pillars. The new taxon is included in the Coskinolinidae family. Lepinoconus chiocchinii gen. n., n. sp. is known from southern Italy, Greece and Albania.

  13. Estudio de la quitosana cubana como agente aglutinante Study of Cuban quitosana as agglutination agent

    Directory of Open Access Journals (Sweden)

    Diamelva del Campo Portales

    2007-08-01

    Full Text Available Para evaluar la capacidad como agente aglutinante de la quitosana cubana, derivada de quitina de langosta, se realizó un diseño experimental cuyas variables fueron la relación entre la masa de quitosana y granulado base, así como la concentración de ácido láctico en la solución aglutinante. Los resultados muestran que el porcentaje de ácido láctico no tiene influencia en las propiedades de los granulados elaborados con quitosana. La relación quitosana/granulado base influye sobre el tamaño de partícula promedio de los granulados estudiados. Las propiedades de los granulados elaborados con quitosana, independientemente de la relación de quitosana/granulado base y el porcentaje de ácido láctico, tienen un comportamiento intermedio entre la polivinilpirrolidona y la gelatina. Con el fármaco modelo sulfato de cloroquina, al emplear la quitosana cubana como agente aglutinante se obtuvieron tabletas con adecuadas propiedades. Adicionalmente se comparó su efectividad como aglutinante con respecto a excipientes de uso tradicional: gelatina, polivinilpirrolidona y celacol, con el clorhidrato de propranolol como fármaco modelo. Los granulados obtenidos muestran que la quitosana al 1 % tiene un comportamiento próximo al polivinilpirrolidona, celacol y gelatina, mientras que para las tabletas, independientemente del valor de la concentración de la quitosana, es similar al polivinilpirrolidona.To assess ability of Cuban quitosona as agglutination agent, derived from lobster quitine, we performed an experimental design of which he variable were: relationship between quitosana mass and granular base as well as lactic acid concentration in agglutination solution. Results shows that lactic acid percentage hasn’t influence on properties of granules processed using quitosana. Above mentioned relationship influences on average particle size of study granules. Properties of quitosana-processed granules, regardless of whether quitosana

  14. Evolution of magma feeding system in Kumanodake agglutinate activity, Zao Volcano, northeastern Japan

    Science.gov (United States)

    Takebe, Yoshinori; Ban, Masao

    2015-10-01

    The Kumanodake agglutinate of Zao Volcano in northeastern Japan consists of pyroclastic surge layers accumulated during the early part of the newest stage of activity (ca. 33 ka to present). Our petrologic study of this agglutinate based on systematically collected samples aims to reveal the evolution of magma feeding system. To understand the magma evolution, we have examined samples from the agglutinate by using petrologic data including, petrography, analysis of minerals (plagioclase, pyroxene, and olivine), glass compositions, and whole rock major element and trace element (Ba, Sr, Cr, Ni, V, Rb, Zr, Nb, and Y) compositions. Agglutinate are mixed, medium-K, calc-alkaline olv-cpx-opx basaltic andesite (55.2-56.2% SiO2). Results show that the magma feeding system comprised a shallow felsic chamber injected by mafic magma from depth. The felsic magma (59-62% SiO2, 950-990 °C), which was stored at a shallower depth, had orthopyroxene (Mg# = 60-69), clinopyroxene (Mg# = 65-71), and low-An plagioclase (Anca. 58-70). The mafic magma is further divisible into two types: less-differentiated and more-differentiated, designed respectively as an initial mafic magma-1 and a second mafic magma-2. The original mafic magma-1 was olivine (Fo 84) basalt (ca. 48-51% SiO2, 1110-1140 °C). The second mafic magma-2, stored occasionally at 4-6 km depth, was basalt (1070-1110 °C) having Foca. 80 olivine and high-An (Anca. 90) plagioclase phenocrysts. These two magmas mixed (first mixing) to form hybrid mafic magma. The forced injections of the hybrid mafic magmas activated the felsic magma, and these two were mixed (second mixing) shortly before eruptions. The explosivity is inferred to have increased over time because the abundance of large scoria increased. Furthermore, the erupted magma composition became more mafic, which reflects increased percentage of the hybrid mafic magma involved in the second mixing. At the beginning of activity, the mafic magma also acted as a heat

  15. Comparative evaluation of coagglutination and latex agglutination test (Rotalex kit for detection of rota virus.

    Directory of Open Access Journals (Sweden)

    Mathur M

    1993-07-01

    Full Text Available Coagglutination test was compared with commercially available latex agglutination test (Rotalex kit for detection of rota virus in faecal samples from clinically suspected cases of viral gastroenteritis. Out of 80 test samples 16 (20% and 20 (25.3% were positive for rota virus antigen by Rotalex kit and coagglutination test respectively. All the 40 controls were negative for viral antigen by Rotalex kit and only one gave positive result by coagglutination test. Coagglutination test was found to be economical, sensitive and specific for screening and rapid diagnosis of Rota virus diarrhoea.

  16. Pliocene and Pleistocene chronostratigraphy and paleoenvironment of the Central Arctic Ocean, using deep water agglutinated foraminifera

    OpenAIRE

    Evans, J. R.; Kaminski, M.A

    1998-01-01

    Deep-water agglutinated foraminifera (DWAF) were studied from Cores PS2177-5, PS2200-5, PS2212-3 and PS2185-6; from the R/V POLARSTERN ARK-VIII/3 Cruise in the central Arctic Ocean. The sediments were non-calcareous containing a sparse assemblage of eleven DWAF species. A chronostratigraphic framework is presented for Cores PS2200-5 and PS2185-6. Paleoenvironmental data suggests a bathyal environment (2000-4000m) affected by water masses in the Arctic Ocean. The taxonomy of all of the DWAF fo...

  17. Elevated water temperature increases the levels of reo-like virus and selected innate immunity genes in hemocytes and hepatopancreas of adult female blue crab, Callinectes sapidus.

    Science.gov (United States)

    Chung, J Sook; Pitula, J S; Schott, E; Alvarez, J V; Maurer, L; Lycett, K A

    2015-11-01

    Seasonal changes in water temperature directly affect the aquatic ecosystem. The blue crab, Callinectes sapidus, inhabiting the Chesapeake Bay has been adapted to seasonal changes of the environmental conditions. In this, the animals halt their physiological process of the growth and reproduction during colder months while they resume these processes as water temperatures increase. We aimed to understand the effect of the elevated temperatures on a disease progression of reo-like virus (CsRLV) and innate immunity of adult female C. sapidus. Following a rise in water temperature from 10 to 23 °C, CsRLV levels in infected crabs rose significantly in hemocytes and multiple organs. However, in hemocytes, the elevated temperature had no effect on the levels of three innate immune genes: Cas-ecCuZnSOD-2, CasPPO and CasLpR three carbohydrate metabolic genes: CasTPS, CasGlyP; and CasTreh and the total hemocyte counts (THC). Interestingly, the hemocytes of CsRLV infected animals exposed to 23 °C for 10 days had significantly elevated levels of Cas-ecCuZnSOD-2 and CasTPS, compared to those of the uninfected ones also exposed to the same condition and compared to hatchery-raised females kept at 23 °C. Despite the lack of changes in THC, the types of hemocytes from the animals with high CsRLV levels differed from those of uninfected ones and from hatchery animals kept at 23 °C: CsRLV-infected crabs had hemocytes of smaller size with less cytosolic complexity than uninfected crabs. It therefore appears that the change in temperature influences rapid replication of CsRLV in all internal tissues examined. This implies that CsRLV may have broad tissue tropism. Interestingly, the digestive tract (mid- and hindgut) contains significantly higher levels of CsRLV than hemocytes while hepatopancreas and ovary have lower levels than hemocytes. Innate immune responses differ by tissue: midgut and hepatopancreas with upregulated Cas-ecCuZnSOD-2 similar to that found in hemocytes. By

  18. A trehalose 6-phosphate synthase gene of the hemocytes of the blue crab, Callinectes sapidus: cloning, the expression, its enzyme activity and relationship to hemolymph trehalose levels.

    Science.gov (United States)

    Chung, J Sook

    2008-12-12

    Trehalose in ectoderms functions in energy metabolism and protection in extreme environmental conditions. We structurally characterized trehalose 6-phosphate synthase (TPS) from hemocytes of the blue crab, Callinectes sapidus. C. sapidus Hemo TPS (CasHemoTPS), like insect TPS, encodes both TPS and trehalose phosphate phosphatase domains. Trehalose seems to be a major sugar, as it shows higher levels than does glucose in hemocytes and hemolymph. Increases in HemoTPS expression, TPS enzyme activity in hemocytes, and hemolymph trehalose levels were determined 24 h after lipopolysaccharide challenge, suggesting that both TPS and TPP domains of CasHemoTPS are active and functional. The TPS gene has a wide tissue distribution in C. sapidus, suggesting multiple biosynthetic sites. A correlation between TPS activity in hemocytes and hemolymph trehalose levels was found during the molt cycle. The current study provides the first evidence of presence of trehalose in hemocytes and TPS in tissues of C. sapidus and implicates its functional role in energy metabolism and physiological adaptation.

  19. Antimicrobial action and cell agglutination by the eosinophil cationic protein are modulated by the cell wall lipopolysaccharide structure.

    Science.gov (United States)

    Pulido, David; Moussaoui, Mohammed; Andreu, David; Nogués, M Victòria; Torrent, Marc; Boix, Ester

    2012-05-01

    Antimicrobial proteins and peptides (AMPs) are essential effectors of innate immunity, acting as a first line of defense against bacterial infections. Many AMPs exhibit high affinity for cell wall structures such as lipopolysaccharide (LPS), a potent endotoxin able to induce sepsis. Hence, understanding how AMPs can interact with and neutralize LPS endotoxin is of special relevance for human health. Eosinophil cationic protein (ECP) is an eosinophil secreted protein with high activity against both Gram-negative and Gram-positive bacteria. ECP has a remarkable affinity for LPS and a distinctive agglutinating activity. By using a battery of LPS-truncated E. coli mutant strains, we demonstrate that the polysaccharide moiety of LPS is essential for ECP-mediated bacterial agglutination, thereby modulating its antimicrobial action. The mechanism of action of ECP at the bacterial surface is drastically affected by the LPS structure and in particular by its polysaccharide moiety. We have also analyzed an N-terminal fragment that retains the whole protein activity and displays similar cell agglutination behavior. Conversely, a fragment with further minimization of the antimicrobial domain, though retaining the antimicrobial capacity, significantly loses its agglutinating activity, exhibiting a different mechanism of action which is not dependent on the LPS composition. The results highlight the correlation between the protein's antimicrobial activity and its ability to interact with the LPS outer layer and promote bacterial agglutination.

  20. Enzymatic cleavage of cell surface proteins of pig and cow erythrocytes and its effect on concanavalin-mediated agglutinability.

    Science.gov (United States)

    Sharma, Savita; Gokhale, Sadashiv M

    2014-10-01

    Study was carried out to understand and compare architecture of the proteins of erythrocyte cell surface of some mammals viz., Homo sapiens (human), Sus scorfa domestica (pig) and Bos taurus domestica (cow). In this study, we investigated the action of proteinases viz., trypsin and chymotrypsin and neuraminidase on the erythrocyte surface proteins and erythrocyte agglutination tendency with a lectin (concanavalin A). The electrophoretic pattern of membrane proteins and glycophorins (analyzed by SDS-PAGE and visualized by Coomassie brilliant blue and periodic acid-schiff stains, respectively) and concanavalin A (Con A) agglutinability revealed that: (i) There were variations in the number and molecular weights of glycophorins in human, pig and cow, (ii) trypsin action on pig and cow erythrocyte membrane proteins was similar, unlike human, (iii) glycophorins degradation by trypsin and chymotrypsin was not similar in pig, as compared to that of human and cow, (iv) erythrocytes agglutination with Con A was significantly different due to differences in membrane composition and alterations in the surface proteins after enzyme treatment, (v) a direct correlation was found between degradation of glycophorins and Con A agglutinability, and (vi) removal of erythrocyte surface sialic acids by neuraminidase specifically indicated an increase in Con A agglutinability of pig and cow erythrocytes, similar to human.

  1. Use of the Directigen Latex Agglutination Test for Detection of Haemphilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis Antigens in Cerebrospinal Fluid from Meningitis Patients,

    Science.gov (United States)

    Reprint: Use of the Directigen Latex Agglutination Test for Detection of Haemphilus influenzae, Streptococcus pneumoniae , and Neisseria meningitidis Antigens in Cerebrospinal Fluid from Meningitis Patients.

  2. A Piezoelectric Immunosensor Based on Agglutination Reaction with Amplification of Silica Nanoparticles

    Institute of Scientific and Technical Information of China (English)

    JIN,Xiao-Yong; JIN,Xue-Fang; DING,Yan-Jun; JIANG,Jian-Hui; SHEN,Guo-Li; Ru-Qin

    2008-01-01

    A simple piezoelectric immunoagglutination assay technique with antibody-modified nanoparticles has been developed for direct quantitative detection of protein. The proposed technique is based on the specific agglutination of goat anti-hlgG-coated silica (or gold) nanoparticles in the presence of human immunoglobulin G (hlgG), which causes a frequency change and is monitored by a piezoelectric device. The antibody modified on the probe surface would combine with antibody-coated nanoparticles in the presence of antigen (hlgG) when the surface agglutination reaction took place, which couples both the mass effect and viscoelastic effect acting on the probe. The results indi-cate that the background interference can be substantially minimized and the probe signal can be observably multi-plied. In addition, the surface of the modified probe and that after combining the complex of immunoagglutination were imaged by scanning electronic microscopy (SEM). Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination enhancer and sodium chloride to control the ion-strength was investigated. The frequency responses of the immunoagglutination assay were found to correlate well with the hlgG concentration with a detection limit of 84 ng.mL-1.

  3. Detection of acute childhood meningitis by PCR, culture and agglutination tests in Tabriz, Iran.

    Science.gov (United States)

    Ghotaslou, Reza; Farajnia, Safar; Yeganeh, Fatemeh; Abdoli-Oskouei, Shahram; Ahangarzadeh Rezaee, Mohammad; Barzegar, Mohammad

    2012-01-01

    Meningitis is one of the hazardous and life threatening infections and is associated with mortality and morbidity. The aim of this study was to determine etiological agents of childhood bacterial meningitis. The culture, Gram staining, agglutination and PCR assays were used to examine CSF specimens from 277 patients with presumed bacterial meningitis for the occurrence of 4 most common infectious agents consist of N. meningitis, H. influnsae, S. pneumoniae and S. agalactiae between 2008 and 2009 at different wards of the Children Hospital of Tabriz. The mean age of patients was 35 ± 2 (Mean ± SEM) month, (minimum 11 days maximum 14 years), of all cases 59.6% male and 40.4% female. Overall the diagnosis was confirmed with a CSF culture in 11/277 (3.97%), by agglutination test in 14/277 (5.05%). The isolated bacteria included S. pneumoniae 5 cases, H. influnsae 2 cases, N. meningitis 3 cases and P. aeroginusae 1 case. A positive PCR assay allowed us to diagnose bacterial meningitis in 19 patients (6.8%). In the present study, we found PCR to be a useful and sensitive method for the detection of bacterial DNA in the CSF samples from suspected meningitis patients. Furthermore, to maximize management of meningitis cases, a combination of culture and PCR is necessary.

  4. Comparison of PCR, Wright agglutination test and blood culture for diagnosis of brucellosis in suspected patients.

    Science.gov (United States)

    Hekmatimoghaddam, Seyedhosssein; Sadeh, Maryam; Khalili, Mohammad Bagher; Mollaabedin, Mansour; Sazmand, Alireza

    2013-11-15

    Brucellosis has long been prevalent in Iran, with considerable medical and economic importance. Timely diagnosis is needed for early management and effective prevention of its consequences in human beings and animals. Current diagnostic methods impose peculiar challenges in terms of analytical method performance. This study compares diagnostic sensitivity, specificity, predictive Value of Positive (PVP) and Predictive Value of Negative (PVN) for Polymerase Chain Reaction (PCR), Wright agglutination test and blood culture used for patients suspected of brucellosis. In 120 patients clinically suspected of brucellosis and referred by physicians to the Yazd central Medical Laboratory, some relevant demographic, occupational, nutritional and clinical data were collected. Also, venous blood samples were drawn for diagnosis of brucellosis using PCR, Wright agglutination test and blood culture techniques. The most frequent symptom of patients was arthralgia (82 cases, 68.3%). PCR was positive in 25 cases (20.8%), wright test in 21 patients (17.5%) and blood culture in 6 cases (5%). In 20 out of 21 wright-positive cases, PCR was positive and all of the culture-positive patients had positive PCR. Sensitivity, specificity, PVP and PVN of blood culture compared to PCR (as the gold standard test) were 24, 100, 100 and 86%, respectively, but the above parameters when PCR is compared with blood culture (as gold standard) were 100, 83, 24 and 95%, respectively. PCR has better analytical performances than blood culture for diagnosis of brucellosis and is suitable for confirmation of Wright-positive cases.

  5. Diagnostic value of latex agglutination test in diagnosis of acute bacterial meningitis.

    Science.gov (United States)

    Mohammadi, Syeda Fasiha; Patil, Asha B; Nadagir, Shobha D; Nandihal, Namrata; Lakshminarayana, S A

    2013-10-01

    To know the incidence of bacterial meningitis in children below five years of age. To compare conventional culture and antigen detection methods (Latex agglutination test). 100 CSF samples of clinically suspected meningitis cases in children below 5 years of age were included. The samples were subjected to cell count, Gram stain, culture and LAT. The organisms isolated in the study were characterized according to standard procedures. Of the 100 cases studied, 31 cases were diagnosed as ABM by Gram stain, culture and latex agglutination test as per WHO criteria. The hospital frequency of ABM was 1.7%. 15 (48.38) cases were culture positive. Gram stain was positive in 22(70.96) cases and LAT in 17(54.83) cases. Haemophilus influenzae was the most common causative agent of acute bacterial meningitis followed by S.pneumoniae. Case fatality rate was 45.16%. The sensitivity and specificity of LAT was 66.66% and 87.91% respectively. Bacterial meningitis is a medical emergency and early diagnosis and treatment is life saving and reduces chronic morbidity. LAT was more sensitive compared to conventional Gram stain and Culture technique in identifying the fastidious organisms like H.influenzae, S.pneumoniae and Group B Streptococcus. However, the combination of Gram stain, Culture and LAT proved to be more productive than any of the single tests alone.

  6. Amyloidogenic amyloid-β-peptide variants induce microbial agglutination and exert antimicrobial activity

    Science.gov (United States)

    Spitzer, Philipp; Condic, Mateja; Herrmann, Martin; Oberstein, Timo Jan; Scharin-Mehlmann, Marina; Gilbert, Daniel F.; Friedrich, Oliver; Grömer, Teja; Kornhuber, Johannes; Lang, Roland; Maler, Juan Manuel

    2016-01-01

    Amyloid-β (Aβ) peptides are the main components of the plaques found in the brains of patients with Alzheimer’s disease. However, Aβ peptides are also detectable in secretory compartments and peripheral blood contains a complex mixture of more than 40 different modified and/or N- and C-terminally truncated Aβ peptides. Recently, anti-infective properties of Aβ peptides have been reported. Here, we investigated the interaction of Aβ peptides of different lengths with various bacterial strains and the yeast Candida albicans. The amyloidogenic peptides Aβ1-42, Aβ2-42, and Aβ3p-42 but not the non-amyloidogenic peptides Aβ1-40 and Aβ2-40 bound to microbial surfaces. As observed by immunocytochemistry, scanning electron microscopy and Gram staining, treatment of several bacterial strains and Candida albicans with Aβ peptide variants ending at position 42 (Aβx-42) caused the formation of large agglutinates. These aggregates were not detected after incubation with Aβx-40. Furthermore, Aβx-42 exerted an antimicrobial activity on all tested pathogens, killing up to 80% of microorganisms within 6 h. Aβ1-40 only had a moderate antimicrobial activity against C. albicans. Agglutination of Aβ1-42 was accelerated in the presence of microorganisms. These data demonstrate that the amyloidogenic Aβx-42 variants have antimicrobial activity and may therefore act as antimicrobial peptides in the immune system. PMID:27624303

  7. Systematic updates of the agglutinated foraminiferal genus Colominella Popescu, 1998: insights from sectioned specimens

    Science.gov (United States)

    Mancin, Nicoletta; Kaminski, Michael A.

    2017-04-01

    The occurrence of agglutinated foraminiferal specimens belonging to the Badenian (middle Miocene) genus Colominella Popescu, 1998 was recently documented for the first time in a lower Pliocene succession of the western Mediterranean area. Direct comparisons with topotype specimens of Colominella paalzowi (Cushman 1936), sampled in the Badenian type section of Lăpugiu de Sus (Transylvania), show that the Pliocene individuals from the western Mediterranean morphologically resemble the type species C. paalzowi, but they also differ in possessing a longer biserial chamber arrangement with a higher number of internal chamber partitions, in lacking a clear early triserial stage and in having a more complex microstructure of the agglutinated wall, thereby supporting the idea that the Pliocene Mediterranean specimens represent a new, more highly evolved species. The fact that the Pliocene individuals from the Mediterranean appear to be more evolved with respect to the Badenian specimens from Paratethys represents an interesting evolutionary development of the genus Colominella that also permits the known stratigraphical and geographical range of the genus, previously limited to the middle Miocene (Badenian) of the Paratethys, to be extended.

  8. Diagnostic value of latex agglutination test in diagnosis of acute bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Syeda Fasiha Mohammadi

    2013-01-01

    Full Text Available Objectives: To know the incidence of bacterial meningitis in children below five years of age. To compare conventional culture and antigen detection methods ( Latex agglutination test. Materials and Methods: 100 CSF samples of clinically suspected meningitis cases in children below 5 years of age were included. The samples were subjected to cell count, Gram stain, culture and LAT. The organisms isolated in the study were characterized according to standard procedures. Results: Of the 100 cases studied, 31 cases were diagnosed as ABM by Gram stain, culture and latex agglutination test as per WHO criteria. The hospital frequency of ABM was 1.7%. 15 (48.38 cases were culture positive. Gram stain was positive in 22(70.96 cases and LAT in 17(54.83 cases. Haemophilus influenzae was the most common causative agent of acute bacterial meningitis followed by S.pneumoniae. Case fatality rate was 45.16%.The sensitivity and specificity of LAT was 66.66% and 87.91% respectively. Conclusion : Bacterial meningitis is a medical emergency and early diagnosis and treatment is life saving and reduces chronic morbidity. LAT was more sensitive compared to conventional Gram stain and Culture technique in identifying the fastidious organisms like H.influenzae, S.pneumoniae and Group B Streptococcus. However, the combination of Gram stain, Culture and LAT proved to be more productive than any of the single tests alone.

  9. Bacteriophage-mediated toxin gene regulation in Clostridium difficile.

    Science.gov (United States)

    Govind, Revathi; Vediyappan, Govindsamy; Rolfe, Rial D; Dupuy, Bruno; Fralick, Joe A

    2009-12-01

    Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by PhiCD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in PhiCD119 lysogens. During this study we found that repR, a putative repressor gene of PhiCD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in PhiCD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen.

  10. Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile▿

    Science.gov (United States)

    Govind, Revathi; Vediyappan, Govindsamy; Rolfe, Rial D.; Dupuy, Bruno; Fralick, Joe A.

    2009-01-01

    Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by ΦCD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in ΦCD119 lysogens. During this study we found that repR, a putative repressor gene of ΦCD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in ΦCD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen. PMID:19776116

  11. Substantial changes in hemocyte parameters of Manila clam Ruditapes philippinarum two years after the Hebei Spirit oil spill off the west coast of Korea.

    Science.gov (United States)

    Hong, Hyun-Ki; Donaghy, Ludovic; Kang, Chang-Keun; Kang, Hyun-Sil; Lee, Hee-Jung; Park, Heung-Sik; Choi, Kwang-Sik

    2016-07-15

    Two years after the Hebei Spirit oil spill occurred off the west coast of Korea, we determined sub-lethal effects of the spilled oil on hemocyte parameters of Ruditapes philippinarum in the damaged areas. Clams in the spilled sites displayed unusually high proportion of granulocytes, which may result in higher phagocytosis capacity and reactive oxygen species production. Hemocytes in clams from the polluted sites also displayed less DNA damage and mortality than in the control site, possibly due to a faster phagocytosis of the impaired cells. Glycogen, the major energetic reserve, was depleted in clams from the spilled sites, potentially due to energetic consumption for maintenance of a large pool of granulocytes, detoxification processes and oxidative stress. Modified hemocyte parameters in clams in the spilled area, may reflect sub-lethal physiological stresses caused by the residual oils in the sediment, in conjunction with environmental modifications such as food availability and pathogens pattern.

  12. Suppression of cell-spreading and phagocytic activity on nano-pillared surface: in vitro experiment using hemocytes of the colonial ascidian Botryllus schlosseri

    Directory of Open Access Journals (Sweden)

    L Ballarin

    2015-02-01

    Full Text Available Nano-scale nipple array on the body surface has been described from various invertebrates including endoparasitic and mesoparasitic copepods, but the functions of the nipple array is not well understood. Using the hydrophilized nanopillar sheets made of polystyrene as a mimetic material of the nipple arrays on the parasites’ body surface, we assayed the cell spreading and phagocytosis of the hemocytes of the colonial ascidian Botryllus schlosseri. On the pillared surface, the number of spreading amebocytes and the number of phagocytizing hemocytes per unit area were always smaller than those on the flat surface (Mann-Whitney test, p < 0.05 - 0.001, probably because the effective area for the cell attachment on the pillared surface is much smaller than the area on the flat sheet. The present results supports the idea that the nipple array on the parasites' body surface reduces the innate immune reaction from the host hemocytes.

  13. Analysis of digital gene expression profiling in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress.

    Science.gov (United States)

    Guo, Hui; Xian, Jian-An; Wang, An-Li

    2016-09-01

    Accumulation of nitrite in water is highly toxic to aquatic animals. To understand immune responses in shrimp under such environmental stress, a digital gene expression (DGE) technology was applied to detect the gene expression profile of the Litopenaeus vannamei hemocytes in response to nitrite for 48 h. A total of 1922 differently expressed unigenes were generated. Of these transcripts, 1269 and 653 genes were up- or down-regulated respectively. Functional categorization and pathways of the differentially expressed genes revealed that immune defense, xenobiotics biodegradation and metabolism, amino acid and nucleobase metabolic process, apoptosis were the differentially regulated processes occurring during nitrite stress. We selected 19 differential expression transcripts (DETs) to validate the sequencing results by real time quantitative PCR (qPCR). The Pearson's correlation coefficient (R) of the 19 DETs was 0.843, which confirmed the consistency and accuracy between these two approaches. Subsequently, we screened 10 genes to examine the changes in the time course of gene expression in more detail. The results indicated that expressions of ATP-binding cassette transporter (ABC transporter), caspase10, QM protein, C type lectin 4 (CTL4), protein disulfide isomerase (PDI), serine protease inhibitor 8 (SPI8), transglutaminase (TGase), chitinase1, inhibitors of apoptosis proteins (IAP) and cytochrome P450 enzyme (CYP450) were induced to participate in the anti-stress defense against nitrite. These results will provide a reference for follow-up study of molecular toxicology and valuable gene information for better understanding of immune response in L. vannamei under environmental stress.

  14. Death associated protein 1 (DAP 1) positively regulates virus replication and apoptosis of hemocytes in shrimp Marsupenaeus japonicus.

    Science.gov (United States)

    Xia, Wen-Li; Kang, Li-Hua; Liu, Chang-Bin; Kang, Cui-Jie

    2017-04-01

    Death-associated protein 1 (DAP1) is a small proline-rich cytoplasmic protein that functions both in the apoptosis and autophage process of mammalian and in the clinical cancer of human. However, little knowledge is known about the homologue gene of DAP1 and its roles in the physiological process of invertebrates. In this paper, we report a novel function of DAP1 in the antivirus immunity of shrimp. A homologue gene of DAP1 was cloned from Marsupenaeus japonicus and named as Mjdap-1. The full-length of Mjdap-1 was 1761 bp with a 309 bp open reading frame that encoded 102 amino acids. Reverse transcription-PCR results showed that Mjdap-1 was expressed in all tested tissues, including hemocytes, gills, intestines, stomach, heart, hepatopancreas, testes, and ovaries. In shrimp, Mjdap-1 transcripts were up-regulated by white spot syndrome virus (WSSV) infection; Mjdap-1 knockdown decreased the virus copy in vivo and the mortality of M. japonicus to WSSV challenge. Conversely, injecting the purified recombinant MjDAP1 protein promoted the amplification of virus in shrimp. Flow cytometric assay showed, the virus infection-induced apoptosis of hemocytes was enhanced by MjDAP1 protein injection and inhibited in MjDAP1 knockdown shrimp. Furthermore, the expression of apoptosis-inducing factor (AIF) was regulated by Mjdap-1, but the caspase transcripts were not affected. Our results suggested that MjDAP1 facilitated the amplification of virus in shrimp, which may be attributed to the promotion of hemocyte apoptosis in an AIF-dependent manner. These results provided a new insight into the function of this protein that may be used for virus disease control. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Evidence for immunomodulation and apoptotic processes induced by cationic polystyrene nanoparticles in the hemocytes of the marine bivalve Mytilus.

    Science.gov (United States)

    Canesi, L; Ciacci, C; Bergami, E; Monopoli, M P; Dawson, K A; Papa, S; Canonico, B; Corsi, I

    2015-10-01

    Polymeric nanoparticles can reach the marine environment from different sources as weathering of plastic debris and nanowaste. Nevertheless, few data are available on their fate and impact on marine biota. Polystyrene nanoparticles (PS NPs) can be considered as a model for studying the effects of nanoplastics in marine organisms: recent data on amino-modified PS NPs (PS-NH2) toxicity in sea urchin embryos underlined that marine invertebrates can be biological targets of nanoplastics. Cationic PS NPs have been shown to be toxic to mammalian cells, where they can induce apoptotic processes; however, no information is available on their effects and mechanisms of action in the cells of marine organisms. In this work, the effects of 50 nm PS-NH2 were investigated in the hemocytes of the marine bivalve Mytilus galloprovincialis. Hemocytes were exposed to different concentrations (1, 5, 50 μg/ml) of PS-NH2 suspension in ASW. Clear signs of cytoxicity were evident only at the highest concentrations (50 μg/ml). On the other hand, a dose dependent decrease in phagocytic activity and increase in lysozyme activity were observed. PS-NH2 NPs also stimulated increase in extracellular ROS (reactive oxygen species) and NO (nitric oxide) production, with maximal effects at lower concentrations. Moreover, at the highest concentration tested, PS-NH2 NPs induced apoptotic process, as evaluated by Flow cytometry (Annexin V binding and mitochondrial parameters). The results demonstrate that in marine invertebrates the immune function can represent a significant target for PS-NPs. Moreover, in Mytilus hemocytes, PS-NH2 NPs can act through mechanisms similar to those observed in mammalian cells. Further research is necessary on specific mechanisms of toxicity and cellular uptake of nanoplastics in order to assess their impact on marine biota.

  16. Bombyx mori and Aedes aegypti form multi-functional immune complexes that integrate pattern recognition, melanization, coagulants, and hemocyte recruitment

    Science.gov (United States)

    Phillips, Dennis R.

    2017-01-01

    The innate immune system of insects responds to wounding and pathogens by mobilizing multiple pathways that provide both systemic and localized protection. Key localized responses in hemolymph include melanization, coagulation, and hemocyte encapsulation, which synergistically seal wounds and envelop and destroy pathogens. To be effective, these pathways require a targeted deposition of their components to provide protection without compromising the host. Extensive research has identified a large number of the effectors that comprise these responses, but questions remain regarding their post-translational processing, function, and targeting. Here, we used mass spectrometry to demonstrate the integration of pathogen recognition proteins, coagulants, and melanization components into stable, high-mass, multi-functional Immune Complexes (ICs) in Bombyx mori and Aedes aegypti. Essential proteins common to both include phenoloxidases, apolipophorins, serine protease homologs, and a serine protease that promotes hemocyte recruitment through cytokine activation. Pattern recognition proteins included C-type Lectins in B. mori, while A. aegypti contained a protein homologous to Plasmodium-resistant LRIM1 from Anopheles gambiae. We also found that the B. mori IC is stabilized by extensive transglutaminase-catalyzed cross-linking of multiple components. The melanization inhibitor Egf1.0, from the parasitoid wasp Microplitis demolitor, blocked inclusion of specific components into the IC and also inhibited transglutaminase activity. Our results show how coagulants, melanization components, and hemocytes can be recruited to a wound surface or pathogen, provide insight into the mechanism by which a parasitoid evades this immune response, and suggest that insects as diverse as Lepidoptera and Diptera utilize similar defensive mechanisms. PMID:28199361

  17. Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes.

    Science.gov (United States)

    Park, Youngjin; Stanley, David W; Kim, Yonggyun

    2015-08-01

    Eicosanoids mediate cellular immune responses in insects, including phagocytosis of invading microbes. Phagocytosis entails two major steps, the internalization of microbes and the subsequent killing of them via formation of reactive oxygen species (ROS). Here, we posed the hypothesis that eicosanoids mediate ROS production by activating NADPH-dependent oxidase (NOX) and tested the idea in the model insect, Spodoptera exigua. A NOX gene (we named SeNOX4) was identified and cloned, yielding a full open reading frame encoding 547 amino acid residues with a predicted molecular weight of 63,410Da and an isoelectric point at 9.28. A transmembrane domain and a large intracellular domain containing NADPH and FAD-binding sites were predicted. Phylogenetic analysis indicated SeNOX4 clusters with other NOX4 genes. SeNOX4 was expressed in all life stages except eggs, and exclusively in hemocytes. Bacterial challenge and, separately, arachidonic acid (AA, a precursor of eicosanoid biosynthesis) injection increased its expression. The internalization step was assessed by counting hemocytes engulfing fluorescence-labeled bacteria. The phagocytic behavior was inhibited by dsRNA suppression of SeNOX4 expression and, separately by dexamethasone (DEX, a specific inhibitor of eicosanoid biosynthesis) treatments. However, injecting AA to dsSeNOX4-treated larvae did not rescue the phagocytic activity. Hemocytic ROS production increased following bacterial challenge, which was sharply reduced in dsSeNOX4-treated, and separately, in DEX-treated larvae. AA partially reversed the suppressed ROS production in dsSeNOX4-treated larvae. Treating larvae with either the ROS-suppressing dsSeNOX4 construct or DEX rendered experimental larvae unable to inhibit bacterial proliferation in their hemocoels. We infer that eicosanoids mediate ROS production during phagocytosis by inducing expression of SeNOX4.

  18. Bombyx mori and Aedes aegypti form multi-functional immune complexes that integrate pattern recognition, melanization, coagulants, and hemocyte recruitment.

    Science.gov (United States)

    Phillips, Dennis R; Clark, Kevin D

    2017-01-01

    The innate immune system of insects responds to wounding and pathogens by mobilizing multiple pathways that provide both systemic and localized protection. Key localized responses in hemolymph include melanization, coagulation, and hemocyte encapsulation, which synergistically seal wounds and envelop and destroy pathogens. To be effective, these pathways require a targeted deposition of their components to provide protection without compromising the host. Extensive research has identified a large number of the effectors that comprise these responses, but questions remain regarding their post-translational processing, function, and targeting. Here, we used mass spectrometry to demonstrate the integration of pathogen recognition proteins, coagulants, and melanization components into stable, high-mass, multi-functional Immune Complexes (ICs) in Bombyx mori and Aedes aegypti. Essential proteins common to both include phenoloxidases, apolipophorins, serine protease homologs, and a serine protease that promotes hemocyte recruitment through cytokine activation. Pattern recognition proteins included C-type Lectins in B. mori, while A. aegypti contained a protein homologous to Plasmodium-resistant LRIM1 from Anopheles gambiae. We also found that the B. mori IC is stabilized by extensive transglutaminase-catalyzed cross-linking of multiple components. The melanization inhibitor Egf1.0, from the parasitoid wasp Microplitis demolitor, blocked inclusion of specific components into the IC and also inhibited transglutaminase activity. Our results show how coagulants, melanization components, and hemocytes can be recruited to a wound surface or pathogen, provide insight into the mechanism by which a parasitoid evades this immune response, and suggest that insects as diverse as Lepidoptera and Diptera utilize similar defensive mechanisms.

  19. Changes of gill and hemocyte-related bio-indicators during long term maintenance of the vent mussel Bathymodiolus azoricus held in aquaria at atmospheric pressure.

    Science.gov (United States)

    Bettencourt, Raul; Dando, Paul; Rosa, Domitília; Riou, Virginie; Colaço, Ana; Sarrazin, Jozée; Sarradin, Pierre-Marie; Santos, Ricardo Serrão

    2008-05-01

    The deep-sea hydrothermal vent mussel Bathymodiolus azoricus has been the subject of several studies aimed at understanding the physiological adaptations that vent animals have developed in order to cope with the particular physical and chemical conditions of hydrothermal environments. In spite of reports describing successful procedures to maintain vent mussels under laboratory conditions at atmospheric pressure, few studies have described the mussel's physiological state after a long period in aquaria. In the present study, we investigate changes in mucocytes and hemocytes in B. azoricus over the course of several months after deep-sea retrieval. The visualization of granules of mucopolysaccharide or glycoprotein was made possible through their inherent auto-fluorescent property and the Alcian blue-Periodic Acid Schiff staining method. The density and distribution of droplets of mucus-like granules was observed at the ventral end of lamellae during acclimatization period. The mucus-like granules were greatly reduced after 3 months and nearly absent after 6 months of aquarium conditions. Additionally, we examined the depletion of endosymbiont bacteria from gill tissues, which typically occurs within a few weeks in sea water under laboratory conditions. The physiological state of B. azoricus after 6 months of acclimatization was also examined by means of phagocytosis assays using hemocytes. Hemocytes from mussels held in aquaria up to 6 months were still capable of phagocytosis but to a lesser extent when compared to the number of ingested yeast particles per phagocytic hemocytes from freshly collected vent mussels. We suggest that the changes in gill mucopolysaccharides and hemocyte glycoproteins, the endosymbiont abundance in gill tissues and phagocytosis are useful health criteria to assess long term maintenance of B. azoricus in aquaria. Furthermore, the laboratory set up to which vent mussels were acclimatized is an applicable system to study physiological

  20. Function of an anti-lipopolysaccharide factor (ALF) isoform isolated from the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii in protecting against bacterial infection.

    Science.gov (United States)

    Liu, Chia-Chen; Chung, Chien-Pang; Lin, Chang-Yi; Sung, Hung-Hung

    2014-02-01

    In this study, a 780-bp full-length cDNA encoding Macrobrachium rosenbergii anti-lipopolysaccharide factor (MrALF) from hemocytes was cloned and identified. The ALF isoform exhibited immune activities, and its concentration in hemolymph was determined. An in vivo expression study showed that the ALF mRNA level of hemocytes could be induced by lipopolysaccharides (LPSs) in an exposure time-dependent manner. Purified recombinant MrALF (rMrALF) expressed in the yeast Pichia pastoris SMD1168 eukaryotic protein expression system demonstrated antibacterial activity against the Gram-negative prawn pathogen Aeromonas hydrophila (minimum inhibitory concentration (MIC)=0.806μM, minimum bactericidal concentration (MBC)=1.606μM) but not the Gram-positive pathogen Lactococcus garvieae exposed to 25.696μM of rALF. However, rMrALF can bind to Gram-negative and -positive bacteria. An in vivo expression study demonstrated that the ALF concentrations in prawn hemocytes and plasma were 0.176μM and 0.168μM, respectively; following LPS treatment for 6h, the corresponding concentrations were 0.133μM in hemocytes and 0.272μM in plasma. Furthermore, the percentage of hemocytes phagocytosing bacteria cells was higher in hemoyctes previously treated with MrALF than those treated with sterile medium. These results suggest that in the innate immune response of M. rosenbergii, the MrALF from hemocytes may play an opsonin during a bacterial invasion.

  1. BAX, a novel cell pro-apoptotic protein, involved in hemocytes early antiviral immune response in fresh water crayfish, Procambarus clarkii.

    Science.gov (United States)

    du, Zhi-Qiang

    2016-08-01

    Apoptosis plays an important role in various biological processes and acts as a host defending mechanism by which infected cells are eliminated to restrict the virus propagation scale. Bax is a crucial pro-apoptotic protein, which mediates the release of cytochrome c from mitochondrion to cytosol in mammalian. However, its role in invertebrate is still obscure. Here, a novel pro-apoptotic protein gene was identified from hemocytes of red swamp crayfish. There was a Bcl-2 domain in the C-terminus of Pc-Bax, which possessed 497 amino acids residues. And an important transmembrane region existed in the C-terminus of Pc-Bax, which implied that Pc-Bax located in mitochondrial membrane. Besides, Pc-Bax was expressed at a relative high level in hemocytes, and a relative low expression levels in hepatopancreas, gills, and intestine. In hemocytes, Pc-Bax transcript was rapidly up-regulated from 12 h to 36 h after WSSV infection. And there was the same trend for Pc-Bax protein expression level in hemocytes after WSSV infection. Results of qRT-PCR testing for VP28 gene showed WSSV replication was obviously enhanced after Pc-Bax knockdown. Meantime, hemocytes apoptosis was suppressed in Pc-Bax knockdown crayfish after WSSV injection, compared with the dsGFP injection group and normal group. Taken together, these results revealed that crayfish hemocytes apoptosis scale was enhanced to suppress WSSV replication by up-regulating Bax protein expression level after WSSV infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Collagen study and regulation of the de novo synthesis by IGF-I in hemocytes from the gastropod mollusc, Haliotis tuberculata.

    Science.gov (United States)

    Serpentini, A; Ghayor, C; Poncet, J M; Hebert, V; Galéra, P; Pujol, J P; Boucaud-Camou, E; Lebel, J M

    2000-09-01

    To evidence a collagen synthesis and identify which type(s) of collagen is present in hemocytes from the mollusc Haliotis tuberculata, we have performed three separate approaches, namely, de novo synthesis by cultured cells, immunological approaches, and northern blot analysis. We demonstrated first that after 40-hr labeling, the de novo synthesis of collagen in the cell layer of cultured hemocytes represents 9.48 +/- 1.25% with respect to the total [(3)H]proline-labeled protein synthesis. In addition, IGF-I elicited a significant stimulation of collagen synthesis in cultured hemocytes in a dose-dependent manner from 10(-10) to 10(-8) M. The maximal stimulation (10(-9) M) induced an increase of 286 +/- 56% with respect to 100% control. By immunocytochemistry and immunoblotting, we showed that hemocytes present immunoreactive molecules to antibodies directed against the type I fibrillar collagen. In addition, using as a probe Hf 677 corresponding to a human pro alpha1(I) collagen cDNA and which encompasses the (Gly-X-Y) repeated sequence found in all Metazoa, four collagen transcripts of approximately 6.4, 5, 2.2, and 2 kb in length have been detected. These data suggest the presence of fibrillar type I collagen in hemocytes and are compatible with the concept that these cells are involved in the extracellular matrix deposition, a cardinal function in tissue repair as well as in developmental processes. Our model may appear as an excellent system to study the role of growth factors on the regulation of collagen synthesis by molluscan hemocytes. J. Exp. Zool. 287:275-284, 2000.

  3. Effect of dopamine injection on the hemocyte count and prophenoloxidase system of the white shrimp Litopenaeus vannamei

    Science.gov (United States)

    Pan, Luqing; Hu, Fawen; Zheng, Debin

    2011-09-01

    Effects of dopamine injection on the hemocyte count, phenoloxidase activity, serine proteinase activity, proteinase inhibitor activity and α2-macroglobulin-like activity in L. vannamei were studied. Results showed that dopamine injection resulted in a significant effect on the parameters measured ( P proteinase inhibitor activity reached the minimum in 3 h, and α2-macroglobulin-like activity reached the maximum in 3 h, and all the three parameters became stable after 12 h. The results suggest that the activating mechanisms of the proPO system triggered by dopamine are different from those triggered by invasive agents or spontaneously activated under a normal physical condition.

  4. Hemocyte responses of Dreissena polymorpha following a short-term in vivo exposure to titanium dioxide nanoparticles: Preliminary investigations

    Energy Technology Data Exchange (ETDEWEB)

    Couleau, Nicolas; Techer, Didier [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), CNRS UMR 7146, IUT Thionville-Yutz, Espace Cormontaigne, Yutz, F-57970 (France); Pagnout, Christophe [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), UMR 7146, Campus Bridoux, rue du General Delestraint, Metz, F-57070 (France); International Consortium for the Environmental Implications of Nanotechnology, iCEINT, http://www.i-ceint.org (France); Jomini, Stephane [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), UMR 7146, Campus Bridoux, rue du General Delestraint, Metz, F-57070 (France); Foucaud, Laurent; Laval-Gilly, Philippe; Falla, Jairo [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), CNRS UMR 7146, IUT Thionville-Yutz, Espace Cormontaigne, Yutz, F-57970 (France); Bennasroune, Amar, E-mail: amar.bennasroune@univ-metz.fr [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), CNRS UMR 7146, IUT Thionville-Yutz, Espace Cormontaigne, Yutz, F-57970 (France)

    2012-11-01

    The widespread use of titanium-based nanoparticles and their environmental release may pose a significant risk to aquatic organisms within freshwater ecosystems. Suspension-feeder invertebrates like bivalve molluscs represent a unique target group for nanoparticle toxicology. The aim of this work was to investigate the short-term responses of Dreissena polymorpha hemocytes after in vivo exposure to titanium dioxide nanoparticles (TiO{sub 2} NP). For this purpose, freshwater mussels were exposed to P25 TiO{sub 2} NP at the concentrations of 0.1, 1, 5 and 25 mg/L during 24 h. Viability, phagocytosis activity and mitogen activated protein kinase (MAPK) phosphorylation level of ERK 1/2 and p38 in hemocytes extracted from exposed mussels were compared to those from control specimens. Results demonstrated an inhibition of the phagocytosis activity after exposure to TiO{sub 2} NP at 0.1 and 1 mg/L. Similar trends, albeit less pronounced, were reported for higher concentrations of NP. Transmission electron microscopy showed for the first time the internalization of TiO{sub 2} NP into Dreissena polymorpha hemocytes. Besides, exposure to NP increased the ERK 1/2 phosphorylation levels in all treatments. Concerning the phosphorylation level of p38, only exposures to 5 and 25 mg/L of NP induced significant p38 activation in comparison to that of the control. Finally, these short-term effects observed at environmentally relevant concentrations highlighted the need for further studies concerning ecotoxicological evaluation of nanoparticle release into an aquatic environment. -- Highlights: Black-Right-Pointing-Pointer Phagocytosis inhibition at TiO{sub 2} NP exposure concentrations of 0.1 and 1 mg/L. Black-Right-Pointing-Pointer Internalization of TiO{sub 2} NP in freshwater mussel hemocytes. Black-Right-Pointing-Pointer Increased phosphorylation level of p38 and ERK 1/2 after in vivo exposure to TiO{sub 2} NP.

  5. Comparison of optomagnetic and AC susceptibility readouts in a magnetic nanoparticle agglutination assay for detection of C-reactive protein

    DEFF Research Database (Denmark)

    Fock, Jeppe; Parmvi, Mattias; Strömberg, Mattias

    2017-01-01

    conjugated with CRP antibodies. Both methods detect agglutination as a shift to lower frequencies in measurements of the dynamics in response to an applied oscillating magnetic field. The magnetic susceptibility method probes the magnetic response whereas the optomagnetic technique probes the modulation...... of laser light transmitted through the sample. The two techniques provided highly correlated results upon agglutination when they measure the decrease of the signal from the individual MNPs (turn-off detection strategy), whereas the techniques provided different results, strongly depending on the read...

  6. Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

    Science.gov (United States)

    Lapointe, Jason F; Dunphy, Gary B; Giannoulis, Paschalis; Mandato, Craig A; Nardi, James B; Gharib, Osama H; Niven, Donald F

    2011-11-01

    The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in

  7. Agglutinated foraminifera from the Northern Tarcău Nappe (Eastern Carpathians, Romania

    Directory of Open Access Journals (Sweden)

    Raluca Bindiu

    2011-10-01

    Full Text Available The Tarcău Nappe is the most important unit of the Carpathian flysch due to its size, stratigraphic, and tectonic complexity. Our purpose was to identify the major types of foraminifera assemblages in relation to the paleoenvironmental settings and their biostratigraphic potential. The identified assemblages are characteristic to the Cretaceous and Paleogene, consisting mostly of benthic agglutinated and, in lower proportions, benthic calcareous and planktonic species. Local abundances of Glomospira specimens allowed the correlation of the examined strata to the early Eocene “Glomospira event” described from the Carpathians in Poland, Morocco, and Labrador. Rzehakina fissistomata (Grzybowski identified at Palma makes possible the correlation of these deposits to the Paleocene Rzehakina fissistomata Zone. Paleoenvironmental conditions (depth, amount of oxygen, nutrients could be inferred based on specific assemblages and compared to the already described types of facies from the Carpathians.

  8. Multivalent nanofibers of a controlled length: regulation of bacterial cell agglutination.

    Science.gov (United States)

    Lee, Dong-Woo; Kim, Taehoon; Park, Il-Soo; Huang, Zhegang; Lee, Myongsoo

    2012-09-12

    Control of the size and shape of molecular assemblies on the nanometer scale in aqueous solutions is very important for the regulation of biological functions. Among the well-defined supramolecular structures of organic amphiphiles, one-dimensional nanofibers have attracted much attention because of their potential applications in biocompatible materials. Although much progress has been made in the field of self-assembled nanofibers, the ability to control the fiber length remains limited. The approach for control of the fiber length presented herein overcomes this limitation through the coassembly of amphiphilic rod-coil molecules in which the crystallinity of the aromatic segment can be regulated by π-π stacking interactions. The introduction of carbohydrate segments into the fiber exterior endows the nanofibers with the ability to adhere to bacterial cells. Notably, the fiber length systematically regulates the agglutination and proliferation of bacterial cells exposed to these fibers.

  9. The clinical significance comparison of a latex agglutination based syphilis screening test at low antibody titer.

    Science.gov (United States)

    Wang, Hua-Cheng; Chen, Cha; Wang, Li-Na; Long, Yi-Fei; Zhang, Wei-Zheng; Li, You-Qiang; Xiao, Qian; Yuan, Hui

    2013-01-01

    The rapid increase of syphilis underscores a tremendous need to carefully evaluate many new serological tests for syphilis and choose efficient and economical strategies for syphilis screening, especially in the case of primary infection with low antibody titer. Between 2011 and 2012, 73 patients' sera samples were included in this retrospective study. They were either TRUST or TPPA reactive, either LA (latex agglutination) based auto3 TP or CLIA (chemiluminescence assay) based Architect Syphilis TP assay reactive. The contradictory weak response samples were further examined by FTA-Abs method. TPPA could not give reactive results in samples with antibody concentration less than 10 mIU. Auto3 TP reagent shows good linearity at low antibody titers and was more sensitive than TPPA, while the former does not show significant superiority compared to the Architect Syphilis TP assay at low antibody titer, except that it is suitable for adaptation on diverse automated chemistry analyzers.

  10. Prevalence of agglutinating antibodies to Toxoplasma gondii and Sarcocystis neurona in beavers (Castor canadensis) from Massachusetts

    Science.gov (United States)

    Jordan, C.N.; Kaur, T.; Koenen, K.; DeStefano, S.; Zajac, A.M.; Lindsay, D.S.

    2005-01-01

    The present study examined the seroprevalence of Toxoplasma gondii and Sarcocystls neurona in a population of beavers (Castor canadensis) from Massachusetts. Sixty-two blood samples were collected during the field seasons over 3 consecutive years from different animals. Blood was collected onto filter paper and shipped to the Department of Biomedical Sciences, Virginia Tech, Blacksburg, Virginia, for parasite testing. The samples were tested at dilutions of 1:25, 1:50, and 1:100 against each parasite antigen by modified agglutination tests to determine whether antibodies to either parasite were present in the blood. Six of 62 samples (10%) were positive for T. gondii, with 2 samples having titers of 1:25 and 4 having titers of 1:50. Four of 62 samples (6%) were positive for S. neurona, with 2 samples having titers of 1:25 and 2 having titers of 1:50. ?? American Society of Pathologists 2005.

  11. Seroprevalence of bovine leptospiral antibodies by microscopic agglutination test in Southeast of Iran

    Institute of Scientific and Technical Information of China (English)

    Mohammad Khalili; Ehsanollah Sakhaee; Mohammad Reza Aflatoonian; Gholamreza Abdollahpour; Saeed Sattari Tabrizi; Elham Mohammadi Damaneh; Sajad Hossini-nasab

    2014-01-01

    Objective:To evaluate serological findings of bovine leptospirosis which is a zoonotic disease with worldwide distribution caused by Leptospira interrogans. Methods: One hundred and sixty seven sera were collected from 9 commercial dairy herds in jiroft suburbs, from July to October 2011. Microscopic agglutination test (MAT) was used to evaluates serological findings of bovine leptospirosis in Jiroft suburb dairy farms, Kerman province, Iran. Results:Antibodies were found by MAT at least against one serovar of Leptospira interrogans in 29 samples (17.36%) among 167 sera at a dilution 1:100 or higher, and Leptospira pomona was the most prevalent serovar. Positive titers against more than one serovar were detected in 6 sera of the positive samples. Conclusion:This study is the first report of leptospirosis in Southeast Iran and showed that Leptospira pomona was the most and Leptospira icterohaemorrhagiae the least prevalent serovars in Southeast Iran.

  12. Similarity Between Turkish & Akkadian Based on Rules of Inflective & Agglutinative Languages

    Directory of Open Access Journals (Sweden)

    Elşad Allili

    2014-08-01

    Full Text Available Akkadian, although a dead language, has left deep imprints on Semitic and some Indo-European languages, and has played an important role in the history of mankind. It is accepted as the ancestor of all the Semitic languages. Beginning from the era of Sargon I, it became the official language in a vast area from Anatolia to Egypt and to India. Akkadian was the “Lingua Franca” of the ancient world, and has passed on many words to other languages such as Persian, Sanskrit and Greek. Although, Assyriologists at present ignore it, the language spoken in the very early days of Akkad, in BCE XXVIII-XXIV, may have been an agglutinative language like today’s Turkish or Magyar, rather than an inflective language like today’s Arabic and all Syriac languages. Thus it may show parallelism with Turkish.

  13. Latex particle agglutination test as an adjunct to the diagnosis of bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Surinder K

    2007-01-01

    Full Text Available The present study aimed to review the results of microscopic examination, routine culture and antigen detection by latex particle agglutination test (LPAT, in order to evaluate the diagnostic value of the LPAT in establishing the aetiological diagnosis of bacterial meningitis. LPAT was done in 65 clinically suspected meningitis cases ranging from 5 days to 60 years of age and was compared with culture and Gram stain. Using LPAT, an aetiological diagnosis could be done in 10 out of 65 (15.4% cases of bacterial meningitis. In contrast, Gram stain and culture showed 16.9 and 23.1% positivity, respectively. LPAT correlated well with Gram stain and culture and can be recommended as an adjunct laboratory test for rapid aetiological diagnosis of bacterial meningitis for prompt institution of proper antibiotics.

  14. [Identification of mosquitoes' human food source by using the co-agglutination technique].

    Science.gov (United States)

    Castex, M; Fachado, A; Fonte, L

    1997-01-01

    The utilization of a coagglutination technique for the identification of a human source for feeding mosquitoes is described. The dilution of ingested blood samples in filter paper was performed in 2 mL of a sodium chloride solution at 0.85%. It was used a suspension of sensibilized Staphylococcus aureus with rabbit's serum, human plasmatic anti-proteins, and human anti-IgG rabbit's serum discriminated well between human and non human blood. No agglutination was observed with the negative control. This technique proved to be sensitive to identify 100% of the human blood samples taken to the paper 24 hours after the mosquitoes completed their feeding at a temperature of 26 to 28 degrees C. Among mosquitoes fed and collected in the fields the test had a satisfactory result. Therefore, it may be used in routine work in the fields. The results showed the sensitivity and specificity of this method for identifying human blood ingested by mosquitoes.

  15. Phoneme-level speech and natural language intergration for agglutinative languages

    CERN Document Server

    Lee, G; Kim, K; Lee, Geunbae; Lee, Jong-Hyeok; Kim, Kyunghee

    1994-01-01

    A new tightly coupled speech and natural language integration model is presented for a TDNN-based large vocabulary continuous speech recognition system. Unlike the popular n-best techniques developed for integrating mainly HMM-based speech and natural language systems in word level, which is obviously inadequate for the morphologically complex agglutinative languages, our model constructs a spoken language system based on the phoneme-level integration. The TDNN-CYK spoken language architecture is designed and implemented using the TDNN-based diphone recognition module integrated with the table-driven phonological/morphological co-analysis. Our integration model provides a seamless integration of speech and natural language for connectionist speech recognition systems especially for morphologically complex languages such as Korean. Our experiment results show that the speaker-dependent continuous Eojeol (word) recognition can be integrated with the morphological analysis with over 80\\% morphological analysis s...

  16. Differentially expressed immune-related genes in hemocytes of the pearl oyster Pinctada fucata against allograft identified by transcriptome analysis.

    Science.gov (United States)

    Wei, Jinfen; Liu, Baosuo; Fan, Sigang; Li, Haimei; Chen, Mingqiang; Zhang, Bo; Su, Jiaqi; Meng, Zihao; Yu, Dahui

    2017-03-01

    The pearl oyster Pinctada fucata is commonly cultured for marine pearls in China. To culture pearls, a mantle piece from a donor pearl oyster is grafted with a nucleus into a receptor. This transplanted mantle piece may be rejected by the immune system of the recipient oyster, thus reducing the success of transplantation. However, there have been limited studies about the oyster's immune defense against allograft. In this study, hemocyte transcriptome analysis was performed to detect the immune responses to allograft in P. fucata at 0 h and 48 h after a transplant. The sequencing reaction produced 92.5 million reads that were mapped against the reference genome sequences of P. fucata. The Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to identify all immune-related differentially expressed genes (DEGs). Compared with patterns at 0 h, a total of 798 DEGs were identified, including 410 up-regulated and 388 down-regulated genes at 48 h. The expression levels of interleukin receptor and toll-like receptor in hemocytes were increased significantly 48 h post-transplant, indicating that the oyster immune response was induced. Finally, altered levels of 18 randomly selected immune-related DEGs were confirmed by quantitative real-time PCR (qRT-PCR). Our results provide the basis for further analysis of the immune rejection of allotransplantation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Agglutination of Trypanosoma cruzi in infected cells treated with serum from chronically infected mice.

    Science.gov (United States)

    Wendelken, Jennifer L; Rowland, Edwin C

    2009-04-01

    The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. The chronic stage of infection is characterized by a production of neutralizing antibodies in the vertebrate host. A polyclonal antibody, anti-egressin, has been found to inhibit egress of parasites from the host cell late in the intracellular cycle, after the parasites have transformed from the replicative amastigote into the trypomastigote. It has also been found that BALB/c mouse fibroblasts in the late stages of parasite infection become permeable to molecules as large as antibodies, leading to the possibility that anti-egressin affects the intracellular parasites. This project addresses the fate of the intracellular trypomastigotes that have been inhibited from egressing the host cell. Extended cultures of infected fibroblasts treated with chronic mouse serum reduced parasite egress at all time points measured. Parasites released from infected fibroblasts treated with chronic serum had a reduced ability to infect fibroblasts in culture, yet did not lose infectivity entirely. Absorption of chronic serum with living trypomastigotes removed the anti-egressin effect. The possibility that the target of anti-egressin is a parasite surface component is further indicated by the agglutination of extracellular trypomastigotes by chronic serum. The possibility that cross-linking by antibody occurs intracellularly, thus inhibiting egress, was reinforced by cleaving purified IgG into Fab fragments, which did not inhibit egress when added to infected cultures. From this work, it is proposed that the current, best explanation of the mechanism of egress inhibition by anti-egressin is intracellular agglutination, preventing normal parasite-driven egress.

  18. Physical, morphological and dosimetric characterization of the Teflon agglutinator to thermoluminescent dosimetry

    Energy Technology Data Exchange (ETDEWEB)

    D' Amorim, R.A.P.O., E-mail: raquelalinepe@gmail.com [Departamento de Física-CCET, Universidade Federal de Sergipe, 49100-000 São Cristóvão, SE (Brazil); Teixeira, M.I., E-mail: miteixeira@ipen.br [Instituto de Pesquisas Energéticas e Nucleares-IPEN-CNEN/SP, Av. Professor Lineu Prestes, 2242, 05508-000 São Paulo (Brazil); Caldas, L.V.E., E-mail: lcaldas@ipen.br [Instituto de Pesquisas Energéticas e Nucleares-IPEN-CNEN/SP, Av. Professor Lineu Prestes, 2242, 05508-000 São Paulo (Brazil); Souza, S.O., E-mail: susanasouzalalic@gmail.com [Departamento de Física-CCET, Universidade Federal de Sergipe, 49100-000 São Cristóvão, SE (Brazil)

    2013-04-15

    In preparing of thermoluminescent dosimeters (TLD) it is common to use as agglutinator the polytetrafluoroethylene (PTFE), called Teflon{sup ®}. In this paper the physical, morphological and dosimetric characteristics of Teflon{sup ®} were evaluated aiming its application in thermoluminescent dosimetry. The differential thermal analysis (DTA) and thermogravimetry (TG) results showed that the Teflon glass transition and melting points are of about 48 °C and 340 °C, respectively. By means of the X-ray diffraction technique, the crystallinity index K{sub c} was estimated as 94%. Micrographs of Scanning Electron Microscopy (SEM) showed a cohesive surface in spodumene–Teflon pellets, as required for thermoluminescent dosimeters (TLD), leading to the conclusion that Teflon acts as binder, providing greater mechanical resistance to the TL pellets. However, Teflon may influence high doses dosimetry when it is applied as an agglutinator. Preliminary results of Teflon pellets dosimetric properties, with their dose–response curve between 50 Gy and 60 kGy, TL response reproducibility and minimum detectable dose, indicate the possibility of use of pure Teflon TLD in high-dose dosimetry. -- Highlights: ► Pure Teflon{sup ®} pellets can be exploited for high-dose dosimetry. ► Pure Teflon{sup ®} pellets showed two TL peaks. ► Low dose limits of Teflon{sup ®} pellets were 7.0 and 4.0 Gy for the first and second TL peaks respectively. ► The reproducibility of TL response of Teflon{sup ®} pellets is 2.9%.

  19. An integrin from shrimp Litopenaeus vannamei mediated microbial agglutination and cell proliferation.

    Directory of Open Access Journals (Sweden)

    Ying Zhang

    Full Text Available BACKGROUND: Integrins are a family of adhesion receptors which regulate cell proliferation, differentiation, leukocyte migration, and complement receptor-dependent phagocytosis. In invertebrates, as a cell adhesion receptor, β integrins play an important role for the balanced activation of immune defense responses especially during the encounter of infections. The present study attempts to characterize the immune functions of shrimp integrin (LvIntegrin to have better understanding on the immune system and its regulation mechanisms in shrimps. METHODOLOGY: A shrimp integrin was identified from the Pacific white shrimp Litopenaeus vannamei (designated as LvIntegrin. Its full-length cDNA was of 2621 bp with an open reading frame (ORF of 2439 bp encoding a polypeptide of 812 amino acids. The mRNA expression of LvIntegrin was significantly up-regulated at 3, 6 and 12 h after Listonella anguillarum challenge. The cDNA fragment encoding β integrin domains (βA and hybrid domain of LvIntegrin was recombined and expressed in Escherichia coli BL21(DE3-pLysS. The recombinant protein (rLvIntegrin could significantly agglutinate the tested microbe including E. coli JM109, L. anguillarum, Micrococcus luteus and Candida dattiladattila in the presence of divalent cations. Moreover, when NIH3T3 cells were cultured with rLvIntegrin, the proliferation rate increased significantly in a dose-dependent manner. CONCLUSIONS: LvIntegrin, a shrimp β integrin was identified from L. vannamei, shared several highly conserved features. LvIntegrin exhibited broad-spectrum agglutination activity towards both bacteria and fungi and could improve the proliferation of NIH3T3 cells, indicating that LvIntegrin is involved in the immune response against microbe challenge and regulation of cell proliferation as a cell adhesion receptor in shrimp.

  20. The Expression and Characterization of a Bifunctional Protein in E. coli for Autologous Erythrocyte Agglutination Test

    Institute of Scientific and Technical Information of China (English)

    Changli Shao; Jingang Zhang

    2008-01-01

    H antigen, the precursor of A and B antigens, belongs to Hh blood system in which it is the only antigen. H antigen distributes on all the human RBC surface except for Bombay phenotype and the copy number of H antigen on the surface of an adult RBC is approximately 1.7 x 106. These characteristics made H antigen the potential target molecule for the immunoassay and immunotherapy. A monoclonal antibody 2E8 against H antigen on the surface of erythrocyte had been prepared in previous work. Based on this antibody, the variable region genes of heavy and light chains (VH and VL) from 2E8 had been cloned by 5' RACE. The two variable region genes were spliced by overlap extension and assembled ScFv (VH-linker-VL) gene encoding the anti-H antigen named ScFv2EB. According to the prediction of the three-dimension structure of ScFv2EB and CH1 fragment from 2E8 and HIV-1 gp41 antigen peptide, we further constructed the ScFv2EBCH1-gp41 fusion molecule. The recombinant ScFv2EB-CH1-gp41 gene was cloned into pET-his vector and expressed in BL21(DE3)plysS cells. The fusion protein was purified from the inclusion bodies. In a series of subsequent analyses, this fusion protein showed identical antigen binding site and activity with the parent antibody. Meanwhile, in mimic test, as the main ingredient of reagent for autologous erythrocyte agglutination test, the bifunctional protein could agglutinate the RBCs in the presence of HIV-1 gp41 antibodies using sera from HIV-infected individuals. Cellular & Molecular Immunology. 2008;5(4):299-306.

  1. Modeling of Virion Collisions in Cervicovaginal Mucus Reveals Limits on Agglutination as the Protective Mechanism of Secretory Immunoglobulin A.

    Science.gov (United States)

    Chen, Alex; McKinley, Scott A; Shi, Feng; Wang, Simi; Mucha, Peter J; Harit, Dimple; Forest, M Gregory; Lai, Samuel K

    2015-01-01

    Secretory immunoglobulin A (sIgA), a dimeric antibody found in high quantities in the gastrointestinal mucosa, is broadly associated with mucosal immune protection. A distinguishing feature of sIgA is its ability to crosslink pathogens, thereby creating pathogen/sIgA aggregates that are too large to traverse the dense matrix of mucin fibers in mucus layers overlying epithelial cells and consequently reducing infectivity. Here, we use modeling to investigate this mechanism of "immune exclusion" based on sIgA-mediated agglutination, in particular the potential use of sIgA to agglutinate HIV in cervicovaginal mucus (CVM) and prevent HIV transmission. Utilizing reported data on HIV diffusion in CVM and semen, we simulate HIV collision kinetics in physiologically-thick mucus layers-a necessary first step for sIgA-induced aggregation. We find that even at the median HIV load in semen of acutely infected individuals possessing high viral titers, over 99% of HIV virions will penetrate CVM and reach the vaginal epithelium without colliding with another virion. These findings imply that agglutination is unlikely to be the dominant mechanism of sIgA-mediated protection against HIV or other sexually transmitted pathogens. Rather, we surmise that agglutination is most effective against pathogens either present at exceedingly high concentrations or that possess motility mechanisms other than Brownian diffusion that significantly enhance encounter rates.

  2. Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-screen latex agglutination test

    NARCIS (Netherlands)

    W.B. van Leeuwen (Willem); C. van Pelt (Cindy); A. Luijendijk (Ad); H.A. Verbrugh (Henri); W.H.F. Goessens (Wil)

    1999-01-01

    textabstractThe slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR ("gold standard") for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a

  3. Good agreement of conventional and gel-based direct agglutination test in immune-mediated haemolytic anaemia

    NARCIS (Netherlands)

    Piek, C.J.; Teske, E.; van Leeuwen, M.W.; Day, M.J.

    2012-01-01

    Abstract Background The aim of this study was to compare a gel-based test with the traditional direct agglutination test (DAT) for the diagnosis of immune-mediated haemolytic anaemia (IMHA). Methods Canine (n = 247) and feline (n = 74) blood samples were submitted for DAT testing to two

  4. Good agreement of conventional and gel-based direct agglutination test in immune-mediated haemolytic anaemia

    NARCIS (Netherlands)

    Piek, C.J.; Teske, E.; van Leeuwen, M.W.; Day, M.J.

    2012-01-01

    Abstract Background The aim of this study was to compare a gel-based test with the traditional direct agglutination test (DAT) for the diagnosis of immune-mediated haemolytic anaemia (IMHA). Methods Canine (n = 247) and feline (n = 74) blood samples were submitted for DAT testing to two laboratories

  5. Carbon nanotube/biocompatible bola-amphiphile supramolecular biohybrid materials: preparation and their application in bacterial cell agglutination.

    Science.gov (United States)

    Yu, Guocan; Li, Jinying; Yu, Wei; Han, Chengyou; Mao, Zhengwei; Gao, Changyou; Huang, Feihe

    2013-11-26

    Supramolecular biohybrid materials were successfully constructed driven by non-covalent interactions between three biocompatible bolaform amphiphiles and single walled carbon nanotubes (SWNTs). The existence of galactoses in these supramolecular systems endowed the hybrid materials with interesting bio-function. By introducing the SWNTs as semi-flexible platforms, these supramolecular biohybrid materials display excellent agglutination ability for E. coli.

  6. Detection of Salmonella enterica serovar Enteritidis (SE) Antibodies in Serum Using A Polystyrene Bead/SE Flagella Agglutination Assay

    Science.gov (United States)

    Serologic screening of flocks can be an important method to detect Salmonella enteritidis (SE) infections but can be labor intensive or lack specificity. Our goal was to develop a rapid agglutination assay using SE flagella adsorbed to polystyrene beads as a simple, relatively specific test to dete...

  7. Hemocyte parameters of the Pacific oyster Crassostrea gigas a year after the Hebei Spirit oil spill off the west coast of Korea

    Science.gov (United States)

    Donaghy, Ludovic; Hong, Hyun-Ki; Lee, Hee-Jung; Jun, Je-Cheon; Park, Young-Je; Choi, Kwang-Sik

    2010-12-01

    In marine bivalves, hemocytes support various physiological functions, including immune defense, nutrient transport, shell repair, and homeostatic maintenance. Although the effects of marine contaminants on the immunological functions of bivalves have been extensively investigated, the impacts of oil spills are not well understood. Therefore, we investigated hemocyte parameters in the Pacific oyster Crassostrea gigas 13 months after the Hebei Spirit oil spill (December 2007) off the west coast of Korea. The parameters studied included hemocyte concentration and mortality, relative proportion of hemocyte populations, and immunological functions such as phagocytosis and oxidative activity using flow cytometry. These immune-related parameters in oysters damaged by the oil spill were also compared to control oysters that were collected from an area unaffected by the spill. The flow cytometry study indicated that granulocyte population, phagocytic capacity, and reactive oxygen species production in oysters exposed to crude oil 13 months prior were depressed compared to the unexposed control oysters. Our data suggest that immunocompetence in oysters affected by the oil spill had not fully recovered 1 year after the accident, although more detailed studies on the physiology and disease resistance should be performed.

  8. A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

    Science.gov (United States)

    Teste, Bruno; Ali-Cherif, Anaïs; Viovy, Jean Louis; Malaquin, Laurent

    2013-06-21

    Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

  9. Cytotoxicity and cellular mechanisms involved in the toxicity of CdS quantum dots in hemocytes and gill cells of the mussel Mytilus galloprovincialis

    Energy Technology Data Exchange (ETDEWEB)

    Katsumiti, A. [CBET Research Group, Dept. Zoology and Animal Cell Biology, Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE, University of the Basque Country UPV/EHU, Basque Country (Spain); Gilliland, D. [EU Commission–Joint Research Centre, Institute of Health and Consumer Protection, NSB Unit, Ispra (Italy); Arostegui, I. [Department of Applied Mathematics, Statistics and Operations Research, Faculty of Science and Technology, University of the Basque Country UPV/EHU, Leioa (Spain); Cajaraville, M.P., E-mail: mirenp.cajaraville@ehu.es [CBET Research Group, Dept. Zoology and Animal Cell Biology, Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE, University of the Basque Country UPV/EHU, Basque Country (Spain)

    2014-08-15

    Highlights: • CdS QDs were cytotoxic for mussel hemocytes and gill cells in vitro. • Ionic Cd was the most toxic form, followed by CdS QDs and bulk CdS. • CdS QDs altered oxidative balance and caused DNA damage in mussel cells. • CdS QDs caused a particle-specific immunostimulation on phagocytosis of hemocytes. • Conceptual models for cellular handling and toxicity of CdS QDs are proposed. - Abstract: CdS quantum dots (QDs) show a great promise for treatment and diagnosis of cancer and for targeted drug delivery, due to their size-tunable fluorescence and ease of functionalization for tissue targeting. In spite of their advantages it is important to determine if CdS QDs can exert toxicity on biological systems. In the present work, cytotoxicity of CdS QDs (5 nm) at a wide range of concentrations (0.001–100 mg Cd/L) was screened using neutral red (NR) and thiazolyl blue tetrazolium bromide (MTT) assays in isolated hemocytes and gill cells of mussels (Mytilus galloprovincialis). The mechanisms of action of CdS QDs were assessed at sublethal concentrations (0.31–5 mg Cd/L) in the same cell types through a series of functional in vitro assays: production of reactive oxygen species (ROS), catalase (CAT) activity, DNA damage, lysosomal acid phosphatase (AcP) activity, multixenobiotic resistance (MXR) transport activity, Na-K-ATPase activity (only in gill cells) and phagocytic activity and damage to actin cytoskeleton (only in hemocytes). Exposures to CdS QDs lasted for 24 h and were performed in parallel with exposures to bulk CdS and ionic Cd. Ionic Cd was the most toxic form to both cell types, followed by CdS QDs and bulk CdS. ROS production, DNA damage, AcP activity and MXR transport were significantly increased in both cell types exposed to the 3 forms of Cd. CAT activity increased in hemocytes exposed to the three forms of Cd while in gill cells only in those exposed to ionic Cd. No effects were found on hemocytes cytoskeleton integrity. Effects on

  10. Studying longterm effects of micro gravity on basic immune functions - The development of an application based on the measuring of phagocytosis activity of Blue Mussel hemocytes

    Science.gov (United States)

    Unruh, Eckehardt

    The immunsystem of astronauts exposed to microgravity is declining. Whether this effect is caused by microgravity or in combination with cosmic radiation is so far not clear. The immune system of vertebrates has several defence strategies but the basic immune response (Phagocytosis) is present as well in invertebrates. Phagocytotic cells are drawn by chemotaxis to the origin of an infection. By adhesion, ingestion and phagosome formation foreign particles, bacteria etc are transported inside of a cell were they are destroyed by native powerful biocides. Related to this biocide production is the formation of Reactive Oxygen Species (ROS). ROS can be measured by luminescence. The effects of microgravity will be simultaneously tested by exposure of phagocytotic hemocytes on orbit under microgravity, artificial gravity and, on ground under natural gravity. To address this purpose defined pools of Blue Mussel (Mytilus edulis) hemocytes will be launched frozen to the ISS. References for all batches will stay on ground. Shortly after arrival and then in three-month intervals batches of the same pool will be thawed and reconstituted. The phagocytosis related production of ROS will be stimulated with opsonized Zymosan. Luminescence will be measured and the data will be sent to ground. The experiment is scheduled for the Columbus Biolab early 2009. In preparation of this flight experiments the following procedures were investigated and the results will be presented: - a protocol for the cryoconservation and reconstituton of blue mussel hemocytes. - preliminary results of phagocytosis activity by reconstituted hemocytes after cryo-conservation and hemocytes without cryo-conservation treatment. The TRIPLELUX-B Experiment contributes to risk assessment concerning longterm immunotoxicity under space flight conditions. The immune system of invertebrates has not been studied so far in space. The choice of the phagocytes from invertebrates is justified by the claim to study the

  11. An Insight into the proteome of Crithidia fasciculata choanomastigotes as a comparative approach to axenic growth, peanut lectin agglutination and differentiation of Leishmania spp. promastigotes.

    Science.gov (United States)

    Alcolea, Pedro J; Alonso, Ana; García-Tabares, Francisco; Toraño, Alfredo; Larraga, Vicente

    2014-01-01

    The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein. The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation. As L. major and L. infantum agglutinate with peanut lectin and non-agglutinating parasites are more infective, the agglutination properties were evaluated in C. fasciculata. The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated. As a difference with L. infantum, the non-agglutinating subpopulation over-expresses the whole machinery for maintenance of redox homeostasis and the translation factors eIF5a, EF1α and EF2, what suggests a relationship between the lack of agglutination and a differentiation process.

  12. Typhoid fever in a Tertiary Hospital in Nigeria: Another look at the Widal agglutination test as a preferred option for diagnosis.

    Science.gov (United States)

    Enabulele, Osahon; Awunor, Simeon Nyemike

    2016-01-01

    Single Widal agglutination test rather than blood culture, is commonly employed to diagnose typhoid fever in Nigeria. We took another look at the Widal agglutination test as a preferred option for diagnosis of typhoid fever by determining the specificity and sensitivity of Widal agglutination test in febrile adult patients. Two hundred and seventy-one blood samples from consecutive adults (>18 years) with febrile illness attending the General Practice Clinic of the University of Benin Teaching Hospital were tested using the Widal agglutination test, blood culture, and malaria parasite test on each sample to establish the diagnosis of typhoid fever. Of the 271 blood samples 124 (45.76%) were positive following a Widal agglutination test, 60 (22.10%) blood samples grew Salmonella organisms on blood culture while 55 (20.29%) blood samples showed a co-infection of typhoid fever and malaria. A sensitivity of 35%, specificity of 51%, positive predictive value of 17%, and a negative predictive value of 73% were observed for Widal agglutination test as a diagnostic modality for typhoid fever infection. A single Widal agglutination test is not a valid diagnostic option for typhoid fever while co-infection with malaria parasite is the preponderant microbiological finding in typhoid fever infections. The severity of malaria parasitemia is associated with positive titers on Widal test.

  13. Agglutination of human erythrocytes by the interaction of Zn(2+)ion with histidine-651 on the extracellular domain of band 3.

    Science.gov (United States)

    Kiyotake, Kento; Ochiai, Hideharu; Yamaguchi, Takeo

    2016-05-01

    Clustering of band 3, chloride/bicarbonate exchanger, has been reported in Zn(2+)-treated human erythrocytes. However, the agglutination of human erythrocytes is also induced by the interaction of Zn(2+)ion with histidine on band 3. Identification of histidine that interacts with Zn(2+)ion remains to be determined. The Zn(2+)-induced agglutination of human erythrocytes was unaffected by chymotrypsin cleavage of the small loop region containing His-547 in the extracellular domain of band 3. On the other hand, papain digestion of the large loop region containing His-651 in band 3 inhibited such Zn(2+)-induced agglutination. Moreover, Zn(2+)-induced erythrocyte agglutination was inhibited by the peptide (ARGWVIHPLG) containing His-651, but not by the peptide such as ARGWVIRPLG, which His-651 was substituted by arginine. Among 10 kinds of animal erythrocytes tested, interestingly, no agglutination by Zn(2+)ions was observed in cow cells only that the forth amino acid in the upstream from His-669 on the large loop of cow band 3 is aspartate (Asp-665) instead of glycine. As expected, the agglutination of human erythrocytes by Zn(2+) ions was inhibited in the presence of aspartate. These data indicate that the interaction of Zn(2+) ion with His-651 residue of band 3 plays an important role in the Zn(2+)-induced agglutination of human erythrocytes.

  14. Digital gene expression analysis in hemocytes of the white shrimp Litopenaeus vannamei in response to low salinity stress.

    Science.gov (United States)

    Zhao, Qun; Pan, Luqing; Ren, Qin; Hu, Dongxu

    2015-02-01

    The white shrimp Litopenaeus vannamei has been greatly impacted by low salinity stress. To gain knowledge on the immune response in L. vannamei under such stress, we investigated digital gene expression (DEG) in L. vannamei hemocytes using the deep-sequencing platform Illumina HiSeq 2000. In total, 38,155 high quality unigenes with average length 770 bp were generated; 145 and 79 genes were identified up- or down-regulated, respectively. Functional categorization and pathways of the differentially expressed genes revealed that immune signaling pathways, cellular immunity, humoral immunity, apoptosis, cellular protein synthesis, lipid transport and energy metabolism were the differentially regulated processes occurring during low salinity stress. These results will provide a resource for subsequent gene expression studies regarding environmental stress and a valuable gene information for a better understanding of immune mechanisms of L. vannamei under low salinity stress.

  15. A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay

    Directory of Open Access Journals (Sweden)

    Anju Mohan

    2016-07-01

    Full Text Available Aim: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. Materials and Methods: The antibody titers were measured by standard tube agglutination test (STAT, microtiter plate agglutination test (MAT, indirect hemagglutination assay (IHA, and indirect enzyme-linked immunosorbent assay (iELISA as per standard protocols. Results: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001. The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005. The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002. However, the difference in mean iELISA titers of infected cattle (1.3678±0.014 and healthy vaccinated cattle (1.367±0.014 was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms. Conclusion: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals.

  16. Mechanisms of Toxicity of Ag Nanoparticles in Comparison to Bulk and Ionic Ag on Mussel Hemocytes and Gill Cells.

    Science.gov (United States)

    Katsumiti, Alberto; Gilliland, Douglas; Arostegui, Inmaculada; Cajaraville, Miren P

    2015-01-01

    Silver nanoparticles (Ag NPs) are increasingly used in many products and are expected to end up in the aquatic environment. Mussels have been proposed as marine model species to evaluate NP toxicity in vitro. The objective of this work was to assess the mechanisms of toxicity of Ag NPs on mussel hemocytes and gill cells, in comparison to ionic and bulk Ag. Firstly, cytotoxicity of commercial and maltose stabilized Ag NPs was screened in parallel with the ionic and bulk forms at a wide range of concentrations in isolated mussel cells using cell viability assays. Toxicity of maltose alone was also tested. LC50 values were calculated and the most toxic Ag NPs tested were selected for a second step where sublethal concentrations of each Ag form were tested using a wide array of mechanistic tests in both cell types. Maltose-stabilized Ag NPs showed size-dependent cytotoxicity, smaller (20 nm) NPs being more toxic than larger (40 and 100 nm) NPs. Maltose alone provoked minor effects on cell viability. Ionic Ag was the most cytotoxic Ag form tested whereas bulk Ag showed similar cytotoxicity to the commercial Ag NPs. Main mechanisms of action of Ag NPs involved oxidative stress and genotoxicity in the two cell types, activation of lysosomal AcP activity, disruption of actin cytoskeleton and stimulation of phagocytosis in hemocytes and increase of MXR transport activity and inhibition of Na-K-ATPase in gill cells. Similar effects were observed after exposure to ionic and bulk Ag in the two cell types, although generally effects were more marked for the ionic form. In conclusion, results suggest that most observed responses were due at least in part to dissolved Ag.

  17. Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model.

    Science.gov (United States)

    Rajendran, Ranjith; Borghi, Elisa; Falleni, Monica; Perdoni, Federica; Tosi, Delfina; Lappin, David F; O'Donnell, Lindsay; Greetham, Darren; Ramage, Gordon; Nile, Christopher

    2015-08-01

    Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

  18. Transcriptome analysis of hemocytes and hepatopancreas in red swamp crayfish, Procambarus clarkii, challenged with white spot syndrome virus

    Directory of Open Access Journals (Sweden)

    X-Z Shi

    2010-04-01

    Full Text Available Red swamp crayfish Procambarus clarkii is used for the innate immune defense of crustaceans due to its convenience for laboratory culture and study. To know more about the transcriptome of the crayfish, we constructed and sequenced a cDNA library from a mixture of hemocytes and hepatopancreas from white spot syndrome virus (WSSV-infected crayfish. By random sequencing, we obtained 9115 high-quality expressed sequence tags with a mean length of 370 bp, representing 3033 unigenes. Most of the unigenes are first reports for the red swamp crayfish. Besides the metabolic genes, many genes that may be involved in the innate immune system of the crayfish are also obtained from the library, such as antimicrobial peptides, pattern recognition receptors, proteases and protease inhibitors, signal transduction proteins, apoptosis-, antioxidant-, and RNA interference-related proteins. We chose ten immune-related genes to analyze their expression pattern by quantitative real time polymerase chain reaction (qRT-PCR from the hemocytes of normal and WSSV-challenged crayfish. Seven of them, including anti-lipopolysaccharide factor, astacidin, crustin 1, H3 histone family 3A, serine/threonine protein kinase, TGF beta-inducible nuclear protein, and tar RNA binding protein, were upregulated after WSSV injection, but the mRNA expression levels of crustin 2, a lectin, and a digestive cysteine protease decreased after WSSV infection. Our results showed that the transcriptome analysis provides a useful resource for identification of immune related genes and understanding the immune responses of the crayfish.

  19. The effects of ocean acidification on hemocyte of crab species in Alaska from laboratory experiment studies from 2011-07-01 to 2013-07-06 (NODC Accession 0123400)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This archival package contains laboratory experiment data that were collected to examine the effects of ocean acidification on hemocyte of crab species in Alaska....

  20. Identification and characterization of a putative agglutination/immobilization antigen on the surface of Cryptocaryon irritans.

    Science.gov (United States)

    Hatanaka, A; Umeda, N; Yamashita, S; Hirazawa, N

    2007-08-01

    The ciliated protozoan Cryptocaryon irritans, a parasite of seawater fishes, was found to express an antigen that elicits antibodies in rabbits and tiger puffer (Takifugu ruburipes). Serum from rabbits and fish immunized with theronts had agglutination/immobilization activity against theronts in vitro; fish serum antibody levels (measured by enzyme-linked immunosorbent assays: ELISA) correlated with this activity. Anti-theront antibody levels in fish were significantly higher in the immunized group as compared with control fish at 2 weeks after booster immunization (injection of bovine serum albumin; Student's t-test, Pagglutination/immobilization antigen. Indirect immunofluorescence staining of theronts suggested that this 32 kDa antigen was expressed on the surface of cilia. The full-length 32 kDa antigen cDNA contained 1147 basepairs, encoding a 328-amino acid protein including hydrophobic N- and C-termini. As with Tetrahymena and Paramecium spp., TAA and TAG appear to be used as glutamine codons in the 32 kDa antigen gene.

  1. The comparison of Brucella gel agglutination test with other Brucella tests

    Directory of Open Access Journals (Sweden)

    N. Mine Turhanoğlu

    2015-12-01

    Full Text Available Objective: In this study, it was aimed to compare the sensitivity of diagnostic tests in patients with a preliminary diagnosis of brucellosis. Methods: We have compared the serological methods, standard tube agglutination test (STA, Coombs Test (CT, Rose Bengal (RBT, and the gel centrifugation test. In patients with a preliminary diagnosis of brucellosis, subjects with a positive test result of RBT has been included in the research and other diagnostic tests STA, CT and Coombs Gel centrifugation tests were performed within the range of same titration. Results: Total 132 patient’s serums were studied. In RBT positive 92 patients’ serums, negative test results were found in 11 with STA, in 9 with CT and in 6 with gel test. While 35 patients were identified to be positive by using Brucella gel test at 1/5120 titer, no positive test results were seen with STA and CT at the same titer. Generally, CT results were one titration below the gel centrifugation test results. Conclusion: In conclusion, RBT and STA were not always adequate to determine the diagnosis of brucellosis. Low titer STA results should be supported by tests such as CT or gel centrifugation and the seroconversion must be monitored. Due to giving fast results, gel centrifugation test can be preferred in diagnosis of Brucellosis.

  2. Evaluation of glycerin as preserving agent of chicken serum for plate agglutination test

    Directory of Open Access Journals (Sweden)

    ES de Freitas

    2014-09-01

    Full Text Available Serum is widely used for the purpose of monitoring and diagnosis support for most of poultry diseases. In the case of the serum plate agglutination test (SPA, commonly used to detect antibodies for Salmonella Pullorum (SP, Mycoplasma gallisepticum (MG and Mycoplasma synoviae (MS, serum cannot be frozen because it may result in false positive. Without freezing, serum can last only for a few days. In this experiment, glycerin was evaluated as a serum preservering agent. About 50 samples for each disease and analyzed by SPA test previously were separated. Glycerin was added to serum from commercial chickens, with and without antibodies for SP, MG and MS, in the proportion of 1:1 (serum:glycerin and kept at refrigerated conditions (2 to 8 ºC. For four years they were tested by the SPA, initially weekly, afterward monthly and then annually. The results show that serum with glycerin give consistent and valid results according to the kind of antibodies present for the period tested. Sera that glycerin was not added to, the results were valid only for the first week. From the second week on, microbial growth affected the test results of the sera without glycerin. Our investigation shows that glycerin can be used to preserve chicken serum for SPA under refrigerated conditions. It is an easy, simple and cheap procedure that can extend serum shelf life, useful mainly for control sera.

  3. Development of a Specific Latex Agglutination Test to Detect Antibodies of Enterovirus 71.

    Science.gov (United States)

    Qin, Bo; Zhang, Jianhua; Xie, Wenhao; Liu, Xuehong; He, Tingting; Chen, Jinkun; Dong, Xuejun

    2015-10-01

    A latex agglutination test (LAT) was developed for the rapid detection of antibodies against the VP1 or VP1 proteins of Enterovirus 71 (EV71). The proteins of interest including prokaryotically expressed VP1 and two strains of anti-VP1 monoclonal antibody (McAb) against EV71 were covalently linked to carboxylated latex using ethyl-dimethyl-amino-propyl carbodiimide (EDC) to prepare sensitized latex beads. LAT was evaluated by an enzyme-linked immunosorbent assay (ELISA) as a reference test. The VP1-LAT showed a sensitivity of 87.0%, specificity of 88.9%, and an agreement ratio of 90.0% in detecting VP1 in 100 serum samples from experimentally infected mice, whereas these values were 86.8, 96.7, and 93.3%, respectively, for 608 clinical human serum samples. The VP1-LAT has advantages over other assays in terms of low cost, rapidity, chemical stability, high sensitivity, repeatability, and specificity. The LAT established in the present study is a rapid and simple test suitable for field monitoring of antibodies against VP1-EV71.

  4. Detection of leptospiral antibodies by microscopic agglutination test in north-east of Iran

    Institute of Scientific and Technical Information of China (English)

    Ehsanollah Sakhaee; Gholam Reza Abdollah pour

    2011-01-01

    Objective: To detect leptospiral antibodies by microscopic agglutination test (MAT) in north-east of Iran. Methods: This study was conducted to evaluate prevalence of human leptospiral infections by MAT, using six current reference strains of Leptospira interrogans in north-east of Iran. A total of 285 serum samples were collected from three north-east provinces of Iran, from December, 2009 to June, 2010. Results: Antibodies were detected at least against one serovar of Leptospira interrogans in 45 sera (15.79 %) among 285 samples at a dilution 1:100 or greater. Positive titers against more than one serovar were detected in 24 sera of the positive samples. Therefore, there were 75 positive reactions against different serovar of Leptospira interrogans. Positive titers were recorded against serovar icterohaemorrhagiae (31 samples), hardjo (26 samples), grippotyphosa (7 samples), pomona (5 samples), canicola (4 samples) and ballum (2 sample).Conclusions:In present study the most prevalent (Leptospira icterohaemorrhagiae) and the least prevalent (Leptospira ballum) serovar are different from previous studies. Maybe, species and prevalence of serovars change during the time in one area and between regions.

  5. The evolution of pretransfusion testing: from agglutination to solid-phase red cell adherence tests.

    Science.gov (United States)

    Plapp, F V; Sinor, L T; Rachel, J M

    1989-01-01

    Hospital transfusion services and blood centers still use manual hemagglutination tests for most of their serological procedures. Automation of hemagglutination reactions has proven to be difficult, primarily because hemagglutination lacks an objective endpoint which can be easily interpreted by inexpensive instruments. Alternatively, solid-phase red cell adherence assays for ABO cell and serum grouping, Rh typing, red cell and platelet antibody screening, red cell and platelet crossmatching, IgA deficiency screening, hepatitis B surface antigen, and HIV antibody screening have been developed. The performance of these assays compares favorably with current hemagglutination and enzyme immunoassay methods. All of these tests share a common objective endpoint of adherence or nonadherence of indicator red cells. This uniformity allows easy interpretation of results visually, spectrophotometrically, or by image analysis. The latter technique has the potential to revolutionize the reading and interpretation of all agglutination tests. Solid-phase red cell adherence tests in microplates are ideal for batch processing large numbers of specimens. However, adherence tests are not restricted to this format. Therefore, blood grouping dipsticks have been produced, which permit testing of individual blood samples even outside of the laboratory.

  6. Counterimmunoelectrophoresis and latex particle agglutination in the etiologic diagnosis of presumed bacterial pneumonia in pediatric patients.

    Science.gov (United States)

    Rusconi, F; Rancilio, L; Assael, B M; Bonora, G; Cerri, M; Pietrogrande, M C; Razon, S; Serafini, L; Torti, G; Vaggi, D

    1988-11-01

    A commercial latex agglutination (LA) kit (Wellcogen, Wellcome Diagnostics) used to detect bacterial polysaccharide antigens (Haemophilus influenzae type b and Streptococcus pneumoniae) was compared with a modified counterimmunoelectrophoresis technique and blood culture for etiologic diagnosis of presumptive bacterial pneumonia requiring hospitalization in 60 infants and children. Serum, urine and either sputum or nasopharyngeal secretions were collected during the first 5 days of therapy for antigen detection. Blood culture was positive in 6 of 52 (11.5%) of cases. Antigens were detected by counterimmunoelectrophoresis and/or LA in 13 of 60 (21.7%) serum samples, 2 of 16 (12.5%) unconcentrated urine samples, 19 of 42 (45.2%) urine samples concentrated 25-fold and 21 of 45 (46.7%) sputum or nasopharyngeal secretions. Antibiotic treatment for 5 days did not affect the antigen detection rate. Counter-immunoelectrophoresis was more sensitive than LA in serum and urine but not in sputum. However, because false positive reactions were frequently obtained with LA on nasopharyngeal secretions of an age-matched control group, this test appears unreliable.

  7. Simple solutions to false results with plate/slide agglutination tests in diagnosis of infectious diseases of man and animals.

    Science.gov (United States)

    Saxena, Hari Mohan; Chothe, Shubhada; Kaur, Paviter

    2015-01-01

    We have developed a new Superagglutination test for serodiagnosis of infectious diseases. It differs from conventional plate/slide agglutination tests (PAT/SAT) by three additional steps: prior staining of serum antibody by adding a dye and addition of diluted biotinylated antiglobulin and avidin in sequence after mixing the antigen with the test serum. The new steps circumvent the problems of false positive and false negative results of PAT/SAT. In serodiagnosis of brucellosis, Superagglutination test had higher positive predictive value and specificity than Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT) and higher negative predictive value and sensitivity than RBPT, STAT, ELISA and Complement Fixation Test (CFT).•Superagglutination is a simple, accurate and economic screening test for infections.•More specificity, sensitivity, positive & negative predictive value than RBPT, STAT.•More sensitivity, negative predictive value than ELISA and Complement Fixation Test.

  8. THE INVESTIGATION OF BRUCELLA ANTIBODY WITH MILK RING TEST AND AGGLUTINATION TEST IN MILK COLLECTED FROM SAMSUN REGION

    Directory of Open Access Journals (Sweden)

    Goknur TERZI

    2006-06-01

    Full Text Available In this study Brucella antibodies were investigated with agglutination test (Whey-AT and Milk Ring Test (MRT in a total of 100 milk samples as 50 of cow milk and 50 of goat milk collected from center and villages of Samsun. According to MRT Brucella antibodies was positive at 10 samples (20 % of cow milk and 6 samples (12 % of goat milk. In cow milk, 4 (8 % positive, 3 (6 % suspicious and 43 (86 % negative samples; in goat milk 3 (6 % positive, 2 (4 % suspicious and 45 (90 % negative samples were determined according to antibodies titre of serum agglutination test (Whey-AT. [TAF Prev Med Bull 2006; 5(3.000: 196-203

  9. An acousto-optical method for registration of erythrocytes' agglutination reaction—sera color influence on the resolving power

    Science.gov (United States)

    Doubrovski, V. A.; Medvedeva, M. F.; Torbin, S. O.

    2016-01-01

    The absorption spectra of agglutinating sera were used to determine blood groups. It was shown experimentally that the sera color significantly affects the resolving power of the acousto-optical method of blood typing. In order to increase the resolving power of the method and produce an invariance of the method for sera color, we suggested introducing a probing light beam individually for different sera. The proposed technique not only improves the resolving power of the method, but also reduces the risk of false interpretation of the experimental results and, hence, error in determining the blood group of the sample. The latter is especially important for the typing of blood samples with weak agglutination of erythrocytes. This study can be used in the development of an instrument for instrumental human blood group typing based on the acousto-optical method.

  10. New approach for serological testing for leptospirosis by using detection of leptospira agglutination by flow cytometry light scatter analysis.

    Science.gov (United States)

    Yitzhaki, S; Barnea, A; Keysary, A; Zahavy, E

    2004-04-01

    Leptospirosis is considered an important reemerging infectious disease worldwide. The standard and most widespread method for the diagnosis of leptospirosis is the microscopic agglutination test (MAT). This test is laborious and time-consuming, and the interpretation of the results is subjective. In the present work we describe an application of flow cytometry (FCM) as a tool for the serological diagnosis of leptospirosis. The analysis is based on the sensitivity of FCM to the size and shape of the bacteria analyzed by measurement of light scatter parameters: forward scatter (FSC) and side scatter (SSC). The addition of positive serum to an infecting leptospiral serovar results in a shift of the light scatter parameter to a different location with higher FSC and SSC values, indicating the formation of leptospiral aggregates. By using immunofluorescent staining, we have shown that the large particles formed are the agglutinated leptospires. Quantification of the agglutination process has been achieved by calculating an agglutination factor (Af), based on changes in the light scatter parameters measured by FCM. Af enables us to determine the specificity of the serological reaction of the patient serum with each leptospiral serovar. In this work, 27 serum samples from 18 leptospirosis patients were tested by both the MAT and the FCM techniques, in which each serum sample was tested against 13 serovars. Twenty-six human serum samples derived from patients with a variety of other defined illnesses were used as negative controls and enabled us to define the Af threshold value as < 9.3 for negative patients, while any value higher than that would be a positive result for leptospirosis. Compared to MAT, the FCM technique was found to be more specific and sensitive, especially in identifying the serogroup in the acute phase of the disease. The whole process was found to be rapid and took less than 1.5 h. Moreover, FCM analysis is objective and can be automated for the

  11. Evaluation of latex agglutination tests for fibrin-fibrinogen degradation products in the forensic identification of menstrual blood.

    Science.gov (United States)

    Akutsu, Tomoko; Watanabe, Ken; Motani, Hisako; Iwase, Hirotaro; Sakurada, Koichi

    2012-01-01

    The identification of menstrual blood is important when discriminating menstruation from vaginal trauma in sexual assault cases. The aim of this study was to evaluate two fibrin-fibrinogen degradation product (FDP)-latex agglutination test kits, FDPL® Test (FDP-L) and FDP Plasma "RD" (FDP-P), for their ability to forensically identify menstrual blood. Sensitivity and specificity of the two kits were compared for menstrual blood and various body fluids, and the sensitivity of the FDP-latex agglutination test kit was also compared with that of an immunochromatographic test for human hemoglobin. The robustness of the FDP-latex agglutination test was compared with that of gene expression analysis of menstrual blood specific markers. The FDP-L kit was more sensitive than the FDP-P kit, but it cross-reacted with peripheral bloodstains from healthy volunteers. The FDP-P kit was specific for menstrual blood, with the exception of postmortem blood samples, and was not affected by other body fluids. In an FDP-negative menstrual blood sample, the sensitivity of human hemoglobin detection was lower than for FDP-positive samples and peripheral blood stains, suggesting that determination of human hemoglobin could be useful in interpreting negative results in the FDP-latex agglutination test. In menstrual blood samples incubated in wet conditions, FDP was found to be a robust marker in the identification of menstrual blood compared with mRNA markers. FDP-P testing was shown to be a suitable and highly efficient rapid screening test for the laboratory identification of menstrual blood.

  12. Modern agglutinated Foraminifera from the Hovgaard Ridge, Fram Strait, west of Spitzbergen: Evidence for a deep bottom current

    OpenAIRE

    Kaminski, M.A.; Niessen, Frank; Bazhenova, E.; De La Guardia, L C.; Coakley, B.; de Vernal, A.; Eagles, Graeme; Eisermann, Hannes; Forwick, Matthias; Gebhardt, Catalina; Geissler, Wolfram; Horner, T.; Jensen, Laura; Jin, H.; Jokat, W.

    2015-01-01

    Deep-water agglutinated foraminifera on the crest of the Hovgaard Ridge, west of Spitsbergen, consist mostly of large tubular astrorhizids. At a boxcore station collected from the crest of Hovgaard Ridge at a water depth of 1169 m, the sediment surface was covered with patches of large (1 mm diameter) tubular forms, be longing mostly to the species Astrorhiza crassatina Brady, with smaller numbers of Saccorhiza, Hyperammina, and Psammosiphonella. Non-tubular species consisted mainly of ...

  13. Agglutination of human O erythrocytes by influenza A(H1N1) viruses freshly isolated from patients.

    Science.gov (United States)

    Murakami, T; Haruki, K; Seto, Y; Kimura, T; Minoshiro, S; Shibe, K

    1991-04-01

    The hemagglutinin titers of 10 influenza A (H1N1) viruses were examined using the erythrocytes of several species. Human O erythrocytes showed the highest agglutination titer to the viruses, whereas chicken erythrocytes showed a low titer. These findings were noted for at least 10 passages by serial dilutions of the viruses in Madin-Darby canine kidney (MDCK) cells. All influenza A(H1N1) viruses, plaque-cloned directly from throat-washing specimens of patients, also agglutinated human O but not chicken erythrocytes. The results of a hemadsorption test indicated that chicken erythrocytes possess less affinity to MDCK cells infected with the A/Osaka City/2/88(H1N1) stain than to those infected with the A/Yamagata/120/86(H1N1) strain which is used as an inactivated influenza vaccine in Japan. However, there were no significant differences between the A/Osaka City/2/88 and the A/Yamagata/120/86 strains in the hemagglutination inhibition test. Since human O erythrocytes have high agglutination activity to influenza A(H1N1) and also to A(H3N2) and B viruses in MDCK cells, these erythrocytes may be useful for the serological diagnosis of influenza.

  14. Evaluation of a C-reactive protein latex agglutination detection test with sera from patients with sexually transmitted diseases.

    Science.gov (United States)

    Schalla, W O; Arko, R J; Thompson, S E

    1984-01-01

    A total of 149 sera, including 79 pre- and posttreatment sera from 33 patients with disseminated gonococcal infections, 18 from patients with uncomplicated gonococcal infections, 6 from patients with pelvic inflammatory disease, 4 from patients with genital Chlamydia trachomatis infections, and 42 from normal volunteers, were examined for C-reactive protein with a latex agglutination C-reactive protein detection kit (Difco Laboratories, Detroit, Mich.). Results were quantitated with LC-Partigen C-reactive protein radial immuno-diffusion plates (Calbiochem-Behring, La Jolla, Calif.). Positive latex agglutination results were observed in all of the pretreatment sera and some of the posttreatment sera of patients with disseminated gonococcal infections and in two sera from patients with pelvic inflammatory disease, which corresponded to quantitative C-reactive protein levels in the radial immunodiffusion plates. C-reactive protein levels were not detectable in the serum samples from normal volunteers or patients with uncomplicated gonococcal infections or genital chlamydial infections. Positive latex agglutination occurred as early as 20 s in sera with high C-reactive protein levels, and all positive results were observed within 90 s of the 3-min test limit. Positive latex test results were obtained with C-reactive protein levels as low as 1 mg/dl (10 micrograms/ml). PMID:6440907

  15. R and G color component competition of RGB image decomposition as a criterion to register RBC agglutinates for blood group typing.

    Science.gov (United States)

    Doubrovski, Valeri A; Ganilova, Yuliya A; Zabenkov, Igor V

    2014-03-01

    A new approach of the criterion assignment for registration of erythrocyte agglutinates to instrumentally determine blood group type is suggested. The criterion is based on comparison of R and G components of RGB decomposition of microscopy digital image taken for the blood-serum mixture sample. For the chosen experimental conditions, the minimal size (area) of RBC agglutinate to be registered by the criterion suggested is estimated theoretically. The proposed method was tested experimentally on the example of monitoring agglutinates in flow. The encouraging experimental results were obtained for improvement of the resolving power of the method; the optimal experimental conditions were revealed for maximum resolution. Though the suggested method was realized for dynamic (flow) blood group determination, it could also be applied for diagnostics in a stationary environment. This approach increases the reliability of RBC agglutinates registration and, hence, blood group typing. The results may be used to develop the apparatus for automated determination of human blood group.

  16. Ammolagena clavata (Jones and Parker, 1860), an agglutinated benthic foraminiferal species - first report from the Recent sediments, Arabian Sea, Indian Ocean region

    Digital Repository Service at National Institute of Oceanography (India)

    Nigam, R.; Mazumder, A.; Saraswat, R.

    The rare presence of the agglutinated foraminiferal species Ammolagena clavata is presented for the first time from the Recent sediments of the Indian Ocean region. This species has previously been reported in Recent sediments from all other oceans...

  17. Comparison of the latex agglutination test with the hemagglutination inhibition test, enzyme-linked immunosorbent assay, and neutralization test for detection of antibodies to rubella virus.

    OpenAIRE

    Meegan, J M; Evans, B. K.; Horstmann, D. M.

    1982-01-01

    The ability of a rapid, latex agglutination test to diagnose rubella infection and to measure immune status was evaluated by comparison with the hemagglutination-inhibition (HAI) test, enzyme-linked immunosorbent assay (ELISA), and the neutralization (NT) test. The latex agglutination test accurately detected serological conversions in 74 pairs of sera representing 21 natural infections and 53 immunizations. The antibody levels of 276 sera from the general population were determined by latex ...

  18. A hemocyte-expressed fibrinogen-related protein gene (LvFrep) from the shrimp Litopenaeus vannamei: Expression analysis after microbial infection and during larval development.

    Science.gov (United States)

    Coelho, Jaqueline da Rosa; Barreto, Cairé; Silveira, Amanda da Silva; Vieira, Graziela Cleusa; Rosa, Rafael Diego; Perazzolo, Luciane Maria

    2016-09-01

    Fibrinogen-related proteins (FREPs) comprise a large family of microbial recognition proteins involved in many biological functions in both vertebrate and invertebrate animals. By taking advantage of publicly accessible databases, we have identified a FREP-like homolog in the most cultivated penaeid shrimp, Litopenaeus vannamei (LvFrep). The obtained sequence showed a conserved fibrinogen-related domain (FReD) and displayed significant similarities to FREP-like proteins from other invertebrates and to ficolins from crustaceans. The expression of LvFrep appeared to be limited to circulating hemocytes. Interestingly, LvFrep gene expression was induced in shrimp hemocytes only in response to a Vibrio infection but not to the White spot syndrome virus (WSSV). Moreover, LvFrep transcript levels were detected early in fertilized eggs, suggesting the participation of this immune-related gene in the antimicrobial defenses during shrimp development.

  19. Effects of salinity on the accumulation of hemocyte aggregates and bacteria in the gills of Callinectes sapidus, the Atlantic blue crab, injected with Vibrio campbellii.

    Science.gov (United States)

    Ikerd, Jennifer L; Burnett, Karen G; Burnett, Louis E

    2015-05-01

    In addition to respiration and ion regulation, crustacean gills accumulate and eliminate injected particles, along with hemocyte aggregates that form in response to those particles. Here we report that the dose of Vibrio campbellii previously shown to induce a decrease in respiration and hemolymph flow across the gill in the Atlantic blue crab, Callinectes sapidus, also triggered the formation of aggregates containing four or more hemocytes in the gills, compared with saline-injected controls. More bacteria were trapped and rendered non-culturable per unit weight by anterior respiratory gills than posterior gills specialized for ion regulation. Further, more bacteria accumulated in the anterior gills of animals held at 30 ppt than those at 10 ppt. Thus, the role of the gills in immune defense comes at an energetic cost to this and likely to other crustaceans; this cost is influenced by acclimation salinity and the position and specialized function of individual gills. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Dynamics of hemocyte subsets from ascidian Halocynthia aurantium in response to tissue damage: a comparative analysis of flow cytometry vs confocal microscopy data

    Directory of Open Access Journals (Sweden)

    AN Sukhachev

    2016-03-01

    Full Text Available The current study was aimed at investigating the dynamics of circulatory ascidian hemocyte subsets from Halocynthia aurantium in response to tunic damage. By using flow cytometry and confocal microscopy, it was demonstrated that the relative amount of hemoblasts and hyaline amebocytes was increased 24 h after cutting ascidian tunic and subjacent muscle layer. By applying a broad panel of fluorescently labeled monoclonal antibodies against human adhesion molecules expressed by lymphoid and stem cells both assays allowed to detect two cross-reactive epitopes (CD54 and CD90 on the surface of ascidian hemocytes. Upon that, the expression of CD54-like epitope was found to be downregulated on ascidian cells after tissue damage, whereas binding to CD90-like epitope was upregulated in all examined cell subsets.

  1. Adhesion, invasion, and agglutination mediated by two trimeric autotransporters in the human uropathogen Proteus mirabilis.

    Science.gov (United States)

    Alamuri, Praveen; Löwer, Martin; Hiss, Jan A; Himpsl, Stephanie D; Schneider, Gisbert; Mobley, Harry L T

    2010-11-01

    Fimbriae of the human uropathogen Proteus mirabilis are the only characterized surface proteins that contribute to its virulence by mediating adhesion and invasion of the uroepithelia. PMI2122 (AipA) and PMI2575 (TaaP) are annotated in the genome of strain HI4320 as trimeric autotransporters with "adhesin-like" and "agglutinating adhesin-like" properties, respectively. The C-terminal 62 amino acids (aa) in AipA and 76 aa in TaaP are homologous to the translocator domains of YadA from Yersinia enterocolitica and Hia from Haemophilus influenzae. Comparative protein modeling using the Hia three-dimensional structure as a template predicted that each of these domains would contain four antiparallel beta sheets and that they formed homotrimers. Recombinant AipA and TaaP were seen as ∼28 kDa and ∼78 kDa, respectively, in Escherichia coli, and each also formed high-molecular-weight homotrimers, thus supporting this model. E. coli synthesizing AipA or TaaP bound to extracellular matrix proteins with a 10- to 60-fold-higher level of affinity than the control strain. Inactivation of aipA in P. mirabilis strains significantly (P < 0.01) reduced the mutants' ability to adhere to or invade HEK293 cell monolayers, and the functions were restored upon complementation. A 51-aa-long invasin region in the AipA passenger domain was required for this function. E. coli expressing TaaP mediated autoagglutination, and a taaP mutant of P. mirabilis showed significantly (P < 0.05) more reduced aggregation than HI4320. Gly-247 in AipA and Gly-708 in TaaP were indispensable for trimerization and activity. AipA and TaaP individually offered advantages to P. mirabilis in a murine model. This is the first report characterizing trimeric autotransporters in P. mirabilis as afimbrial surface adhesins and autoagglutinins.

  2. Seroprevalence of Mycoplasma gallisepticum antibody by ELISA and serum plate agglutination test of laying chicken

    Directory of Open Access Journals (Sweden)

    Md. Zulfekar Ali

    2015-01-01

    Full Text Available Aim: Mycoplasma gallisepticum (MG is important avian pathogen responsible for chronic respiratory disease of chicken and turkeys, which result in large economic loss for the poultry industry. The objectives of this study were determination of seroprevalence of MG antibody of commercial layer chicken at laying period in selected areas of Bangladesh. Materials and Methods: A total of 563 blood samples were collected randomly from selected commercial layer chickens at laying period during the period from July to December, 2013. Indirect enzyme linked immunosorbent assay (iELISA and serum plate agglutination (SPA test were performed to detect the presence of antibodies against MG. Results: Of 563 samples, 64.47% and 56.13% showed an overall prevalence of MG antibodies in iELISA and SPA test respectively. Prevalence of MG was recorded the highest (69.63% at 50-55 weeks of age compared with lowest (53.26% at 56-61 weeks of age (p<0.05. Significant (p<0.05 effect of breed were observed in the seroprevalence of MG infection in layer birds in the present study. The overall, 68.77%, 63.74% and 59.37% prevalence were found respectively in sonali, ISA Brown and White leg horn. The prevalence of MG antibodies was the highest (70.13% in December followed by November (68%, October (65.67%, August (63.46%, September (58.54% and July (51.78% month. The seroprevalence of MG antibodies was higher (69.63% in most of the large flocks and lower (56.82% in small flocks. Conclusion: Therefore, might be suggested that the commercial layer farms should be routinely checked to monitor MG infection and the reactor birds should be culled since MG organism has the potential to transmit vertically. The correlation between MG antibody in month and flock size was not significant (p=0.359 and p=0.868, respectively.

  3. Validation studies of the latex agglutination test for the detection of Trichinella larvae in meat products.

    Science.gov (United States)

    Gayda, Jennifer; Reckinger, Sabine; Thaben, Nora; Nöckler, Karsten; Mayer-Scholl, Anne

    2016-11-15

    Human trichinellosis is a foodborne disease caused by ingestion of meat infected with Trichinella muscle larvae. To control Trichinella spp. infection in the European Union, all slaughtered pigs from holdings that are not officially recognized as applying controlled housing conditions and other animals susceptible to Trichinella infection and intended for human consumption should be examined by one of the approved digestion methods described in Regulation (EU) No. 2015/1375. In the past, Trichinella outbreaks due to the consumption of cured wild boar or pork products have been described in several European countries, making the identification of the larvae from these products relevant for Trichinella control. Therefore, this study aimed to validate the newly approved latex agglutination test (Trichin-L) for routine testing of cured meat products. The test was validated based on the OIE Guidelines using pork products spiked with Trichinella larvae. The sensitivity of the method varied greatly depending on the investigated meat product and was usually lower than for the gold standard, the magnetic stirrer method. The detection rate reached 80% for three larvae and 60% for one larva in cured pork sausages. A detection rate of 100% for three larvae and 50% for one larva was found in bacon. For frozen samples (-20°C) the Trichin-L kit is similarly sensitive as for cured samples. Further, to determine the performance of the test under field conditions, pork products from regions with known high Trichinella prevalences confiscated by customs authorities at two German international airports were analyzed. Problems associated with the Trichin-L test were incomplete digestion due to fatty ingredients, spices and very dry meat products, resulting in data which could not be evaluated. Therefore, the test is currently not suitable for the detection of Trichinella larvae in cured meat products and needs further adaptation steps to increase both usability and sensitivity

  4. Agglutinating secretory IgA preserves intestinal epithelial cell integrity during apical infection by Shigella flexneri.

    Science.gov (United States)

    Mathias, Amandine; Longet, Stéphanie; Corthésy, Blaise

    2013-08-01

    Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-κB nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion.

  5. An unusual presentation of brucellosis, involving multiple organ systems, with low agglutinating titers: a case report

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    Khorvash Farzin

    2007-07-01

    Full Text Available Abstract Background Brucellosis is a multi-system disease that may present with a broad spectrum of clinical manifestations. While hepatic involvement in brucellosis is not rare, it may rarely involve the kidney or display with cardiac manifestations. Central nervous system involvement in brucellosis sometimes can cause demyelinating syndromes. Here we present a case of brucella hepatitis, myocarditis, acute disseminated encephalomyelitis, and renal failure. Case presentation A 26-year-old man presented with fever, ataxia, and dysarthria. He was a shepherd and gave a history of low grade fever, chilly sensation, cold sweating, loss of appetite, arthralgia and 10 Kg weight loss during the previous 3 months. He had a body temperature of 39°C at the time of admission. On laboratory tests he had elevated level of liver enzymes, blood urea nitrogen, Creatinine, Creatine phosphokinase (MB, and moderate proteinuria. He also had abnormal echocardiography and brain MRI. Enzyme-linked immunosorbent assay for IgG and IgM was negative. Standard tube agglutination test (STAT and 2-mercaptoethanol (2-ME titers were 1:80 and 1:40 respectively. Finally he was diagnosed with brucellosis by positive blood culture and the polymerase chain reaction for Brucella mellitensis. Conclusion In endemic areas clinicians should consider brucellosis in any unusual presentation involving multiple organ systems, even if serology is inconclusive. In endemic areas low STAT and 2-ME titers should be considered as an indication of brucellosis and in these cases additional testing is recommended to rule out brucellosis.

  6. A soro-aglutinação das Leishmanias Agglutination of Leishmanias

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    A. M. da Cunha

    1942-01-01

    Full Text Available The first agglutination experiments (Tables 1 and 2 showed that the serum obtained with any one strain of Leishmania, agglutinates all the others even of another species. This finding reveals the existence of a common antigen. However as the titre of agglutination did not permit a sharp differentiation of species we tried the adsorption method. The first adsorption tests made demonstrated differences in antigenic constitution between a strain of. L. donovani on one hand and strains of L. tropica or L. brasiliensis on the other. Further experiments in which L. chagasi was tested against the other species revealed that the former was antigenically different from the others. These tests were performed by adsorbing an anti-chagasi serum with organisms belonging to the other species or, conversely, adsorbing with L. chagasi sera prepared against the other species (See Tables 9 to 24. On the other hand, the adsorption of a serum prepared against one strain of l. chagasi by another of the same species showed that they had identifical antigenie constitution. These findings suggested the possibility of separating different species of Leishmania by this method. However, tests to separate the other species from one to another gave inconclusive results. (See Tables 27 to 35. It was soon observed that all the strains of L. chagasi were of recent isolation while all the others had been maintained in artificial culture media for a long time. We were led to believe that this condition was responsible for the differences in behaviour encountered. Accordingly, recently isolated strains of L. brasiliensis and L. donovani were tested and shown to be antigenically similar to strains of L. chagasi also recently isolated. The conclusion may be drawn that all strains have the same antigenic constitution when freshly isolated. It has been noted that when a serum which has been prepared against a freshly isolated is adsorbed with an old strain, the amount of agglutinins

  7. Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays.

    Science.gov (United States)

    Jose, Seena; Mohandas, A; Philip, Rosamma; Bright Singh, I S

    2010-11-01

    Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 gl⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2'-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24h. Susceptibility of the cells to WSSV was confirmed by immunofluorescence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT(50)/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC(50). The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.

  8. Immune toxicity of TiO₂ under hypoxia in the green-lipped mussel Perna viridis based on flow cytometric analysis of hemocyte parameters.

    Science.gov (United States)

    Wang, Youji; Hu, Menghong; Li, Qiongzhen; Li, Jiale; Lin, Daohui; Lu, Weiqun

    2014-02-01

    The combined effects of DO and TiO2 (mixed rutile/anatase phase, 7/3) on immune responses in Perna viridis were examined. Mussels were exposed to six combinations of oxygen levels (hypoxia: 1.5 mg O2l(-1), normoxia: 6.0 mg O2 l(-1)) and TiO2 concentrations (0, 2.5 mg l(-1) and 10 mg l(-1)) for 216 h. Mussels were sampled after 24h, 48h, 120 h and 216 h, and immune parameters of hemocytes, including mortality, phagocytosis, non-specific esterase, ROS production, lysosomal content and total hemocyte count were investigated using flow cytometric assay. Hemocyte mortality was higher under hypoxia than normoxia, and increased with TiO2 concentrations, but no interaction was found between DO and TiO2. Phagocytosis was reduced under hypoxia and decreased with TiO2 exposure, and the interactive effect between time and TiO2 was observed. The percentage of hemocytes showing non-specific esterase activity was lower under hypoxia, and decreased as TiO2 concentration increased with the significant interactive effect of DO and TiO2. ROS production and lysosomal content were lower under hypoxia and reduced as concentration of TiO2 increased, and interactive effect of DO and TiO2 on ROS was evident. THC was significantly affected by the interactive effect between TiO2 and DO, with higher values under normoxia in the presence of TiO2. The present study demonstrated that immune functions of P. viridis were influenced by both nano-TiO2 and hypoxia with some synergistic effects between the two stressors. This implies that DO has to be considered in the evaluation of the toxicity of nano-materials to bivalves. © 2013.

  9. Comparison of hemocytes of V-instar nymphs of Rhodniusprolixus (Stål and Rhodnius robustus (Larousse 1927, before and after molting

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    Elizabeth Ruiz

    2015-01-01

    Full Text Available Background. The immune response of insects involves humoral factors and cellular elements known as hemocytes. There are different reports that explore the response of hemocytes to infections, but the effect that molting has on this response has not been explored so far. We hypothesized that there would be a change in the percentage of hemocytes as a response to the molting process. Objective. The aim of this work was to compare the hemogram (CBC, the formula, and the differential count of hemocytes in IV instar before molting and in V instar nymphs 24 hours after molting in two species: Rhodnius prolixus and Rhodnius robustus. Materials and methods. We assayed different staining methodologies including Giemsa, Alcian Blue pH 2.0, Alcian Blue pH 2.6, Gomori substrate, PAS (Schiff, Sudan Black and Papanicolau with positive controls for each one. In the Gomori staining, we observed lysosomes in the granulocytes and plasmatocytes, but the differentiation was better detected using Giemsa staining. Results. There were no statistically significant differences between the two species studied in plasmatocytes (p=0,053 or even in granulocytes (p=0,5. However, differences were significant in the prohemocytes (p=0,001 during the molting process in both Rhodnius prolixus and Rhodnius robustus. Conclusions. Significant differences in prohemocytes between nymphs of IV and V instar were detected. No significant differences in the amount of cells were observed between the two species and the two stages. These findings may be explained due to their role as precursor cell of prohemocytes.

  10. Proteomic analysis of differentially expressed protein in hemocytes of wild giant freshwater prawn Macrobrachium rosenbergii infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV

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    T. Alinejad

    2015-09-01

    Full Text Available Epizootic diseases cause huge mortality and economical loses at post larvae stages in freshwater prawn aquaculture industry. These prawns seem less susceptible to viral diseases except for infectious hypodermal and hematopoietic necrosis virus (IHHNV. During viral infection in prawns, hemocytes are the primary organ that shows immunological response within the early stages of infection. We applied proteomic approaches to understand differential expression of the proteins in hemocytes during the viral disease outbreak. To aid the goal, we collected Macrobrachium rosenbergii broodstocks from the local grow out hatchery which reported the first incidence of IHHNV viral outbreak during larvae stage. Primarily, application of the OIE primer targeting 389 bp fragments of IHHNV virus was used in identification of the infected and non-infected samples of the prawn breeding line. Analysis of two-dimensional gel electrophoresis showed specific down-regulation of Arginine kinase and Sarcoplasmic calcium-binding protein and up/down-regulation of Prophenoloxidase1 and hemocyanin isoforms. These proteins were validated using semi quantitative RT-PCR and gene transcripts at mRNA level. These identified proteins can be used as biomarkers, providing a powerful approach to better understanding of the immunity pathway of viral disease with applications in analytic and observational epidemiology diagnosis. Proteomic profiling allows deep insight into the pathogenesis of IHHNV molecular regulation and mechanism of hemocyte in freshwater prawns.

  11. Proteomic analysis of differentially expressed protein in hemocytes of wild giant freshwater prawn Macrobrachium rosenbergii infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV).

    Science.gov (United States)

    Alinejad, T; Bin, Kwong Q; Vejayan, J; Othman, R Y; Bhassu, S

    2015-09-01

    Epizootic diseases cause huge mortality and economical loses at post larvae stages in freshwater prawn aquaculture industry. These prawns seem less susceptible to viral diseases except for infectious hypodermal and hematopoietic necrosis virus (IHHNV). During viral infection in prawns, hemocytes are the primary organ that shows immunological response within the early stages of infection. We applied proteomic approaches to understand differential expression of the proteins in hemocytes during the viral disease outbreak. To aid the goal, we collected Macrobrachium rosenbergii broodstocks from the local grow out hatchery which reported the first incidence of IHHNV viral outbreak during larvae stage. Primarily, application of the OIE primer targeting 389 bp fragments of IHHNV virus was used in identification of the infected and non-infected samples of the prawn breeding line. Analysis of two-dimensional gel electrophoresis showed specific down-regulation of Arginine kinase and Sarcoplasmic calcium-binding protein and up/down-regulation of Prophenoloxidase1 and hemocyanin isoforms. These proteins were validated using semi quantitative RT-PCR and gene transcripts at mRNA level. These identified proteins can be used as biomarkers, providing a powerful approach to better understanding of the immunity pathway of viral disease with applications in analytic and observational epidemiology diagnosis. Proteomic profiling allows deep insight into the pathogenesis of IHHNV molecular regulation and mechanism of hemocyte in freshwater prawns.

  12. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

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    Gi Won Seo

    2016-08-01

    Full Text Available The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

  13. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

    Science.gov (United States)

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-08-20

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

  14. Hemomucin, an O-glycosylated protein on embryos of the wasp Macrocentrus cingulum that protects it against encapsulation by hemocytes of the host Ostrinia furnacalis.

    Science.gov (United States)

    Hu, Jian; Xu, Qiuyun; Hu, Shengfeng; Yu, Xiaoqiang; Liang, Zhikun; Zhang, Wenqing

    2014-01-01

    It is unclear how endoparasites passively evade their host's immune reactions in most parasite-host systems. Hemomucin from the parasitoid wasp Macrocentrus cingulum (McHEM) is a 97-kDa transmembrane protein containing 51 potential O-glycosylation sites that can be specifically recognized by Arachis hypogaea lectin. Mchem mRNA is highly expressed in M. cingulum eggs, morulae and secondary embryos, and McHEM protein is mainly located on the extraembryonic membrane of embryos. When secondary embryos of M. cingulum were transplanted into naïve larvae of their host, Ostrinia furnacalis, the embryos proliferated to generate dozens of embryos. However, more than 90% of these embryos were encapsulated by host hemocytes after blocking with anti-McHEM serum. Similarly, following knockdown of Mchem expression using double-stranded RNA encoding Mchem (dshem), many more embryos were encapsulated by host hemocytes after transplantation compared to controls (p embryos were encapsulated by host hemocytes following digestion with O-glycosidase, which specifically digests β-gal (1→3) linkages between GalNAc and Ser/Thr of proteins. Western blotting results showed that O-glycosidase digested McHEM into a smaller product. These results indicate that McHEM may protect embryos from being encapsulated by their host and that the McHEM sugar chains play an important role.

  15. CLASSIFICATION AND COMPARISON ON HEMOCYTES AMONG FOUR COMMON CEPHALOPOD SPECIES%4种常见头足类动物的血细胞分类及比较

    Institute of Scientific and Technical Information of China (English)

    宋微微; 王春琳; 励迪平; 母昌考; 李荣华

    2013-01-01

    以长蛸(Octopus variabilis)、短蛸(Octopus ocellatus)、真蛸(Octopus vulgaris)和曼氏无针乌贼(Sepiella maindroni)的血细胞为研究对象,通过光镜和电镜观察对血液中的血细胞形态结构进行比较.根据观察结果,将头足类血细胞分成3种类型:透明细胞,小颗粒细胞和颗粒细胞,但在真蛸血细胞中有一定数量的血细胞其细胞核呈马蹄形,因此将此类细胞独立划为马蹄形核细胞.通过比较发现,曼氏无针乌贼的血细胞与其它3个种存在明显的差异,其胞质内的颗粒数量与性质均与其它3个种有较明显的区别差异.从总体来看,头足类血细胞内含颗粒数量较少,其颗粒的数量与免疫力的强弱无直接关联.通过对比,推断真蛸血细胞中的马蹄形核细胞为透明细胞到小颗粒细胞的中间类型,从而得出此4个类型细胞有从透明细胞→马蹄形核细胞→小颗粒细胞→颗粒细胞的发育进程.%Blood from four cephalopod species,namely,Octopus variabilis,O.ocellatus,O.vulgaris and Sepiella maindroni,were investigated in this study.Hemocyte configuration was observed under a microscope and a transmission electron microscope.The hemocytes were classified according to cytoplasm granules and size,consistency,proportion,and so on,and were divided into three types,namely,hyaline hemocytes,small granular hemocytes,and granular hemocytes.A number of O.vulgaris hemocyte nucleoli were U-shaped,which were classified as an individual type named U-shaped nuclear homocytes.These four hemocyte types were systematically described and compared.The quantity and quality of S.maindroni hemocyte granules were obviously different from those of the other species.In general,the cephalopod hemocyte granules were few.The number of granules is not directly related to its immunity.By comparison,we conclude that the U-shaped nuclear hemocytes of O.vulgaris were the intergradation between the hyaline hemocytes and the small granular

  16. Cytotoxicity and cellular mechanisms involved in the toxicity of CdS quantum dots in hemocytes and gill cells of the mussel Mytilus galloprovincialis.

    Science.gov (United States)

    Katsumiti, A; Gilliland, D; Arostegui, I; Cajaraville, M P

    2014-08-01

    CdS quantum dots (QDs) show a great promise for treatment and diagnosis of cancer and for targeted drug delivery, due to their size-tunable fluorescence and ease of functionalization for tissue targeting. In spite of their advantages it is important to determine if CdS QDs can exert toxicity on biological systems. In the present work, cytotoxicity of CdS QDs (5 nm) at a wide range of concentrations (0.001-100 mg Cd/L) was screened using neutral red (NR) and thiazolyl blue tetrazolium bromide (MTT) assays in isolated hemocytes and gill cells of mussels (Mytilus galloprovincialis). The mechanisms of action of CdS QDs were assessed at sublethal concentrations (0.31-5 mg Cd/L) in the same cell types through a series of functional in vitro assays: production of reactive oxygen species (ROS), catalase (CAT) activity, DNA damage, lysosomal acid phosphatase (AcP) activity, multixenobiotic resistance (MXR) transport activity, Na-K-ATPase activity (only in gill cells) and phagocytic activity and damage to actin cytoskeleton (only in hemocytes). Exposures to CdS QDs lasted for 24h and were performed in parallel with exposures to bulk CdS and ionic Cd. Ionic Cd was the most toxic form to both cell types, followed by CdS QDs and bulk CdS. ROS production, DNA damage, AcP activity and MXR transport were significantly increased in both cell types exposed to the 3 forms of Cd. CAT activity increased in hemocytes exposed to the three forms of Cd while in gill cells only in those exposed to ionic Cd. No effects were found on hemocytes cytoskeleton integrity. Effects on phagocytosis were found in hemocytes exposed to bulk CdS and to CdS QDs at concentrations equal or higher than 1.25 mg Cd/L but not in those exposed to ionic Cd, indicating a particle-specific effect on phagocytosis. In conclusion, cell-mediated immunity and gill cell function represent significant targets for CdS QDs toxicity.

  17. Evaluation of an Immunocapture-Agglutination Test (Brucellacapt) for Serodiagnosis of Human Brucellosis

    Science.gov (United States)

    Orduña, Antonio; Almaraz, Ana; Prado, Ana; Gutierrez, M. Purificación; Garcia-Pascual, Agustina; Dueñas, Ana; Cuervo, Milagros; Abad, Ramon; Hernández, Beatriz; Lorenzo, Belen; Bratos, Miguel A.; Torres, Antonio Rodriguez

    2000-01-01

    We evaluated the validity and the usefulness of a new test for the diagnosis of human brucellosis based on an immunocapture-agglutination technique. A total of 315 sera from 82 patients with a diagnosis of brucellosis, 157 sera from patients in whom brucellosis was suspected but not confirmed, and 412 sera from people living in rural areas with endemic brucellosis were studied. The seroagglutination test (SAT), Coombs anti-Brucella test, and Brucellacapt test were evaluated. All the initial sera from the 82 patients proved to be positive in Brucellacapt and Coombs tests, while only 75 (91.4%) were positive in the SAT. If a ≥1/160 diagnostic threshold titer was defined for the Brucellacapt test, Coombs test, and SAT, the sensitivities were 95.1, 91.5, and 65.8%, respectively. Taking the same diagnostic threshold titer for the 157 sera from the unconfirmed but suspected patients, the specificities of the Brucellacapt, Coombs, and SAT were 81.5, 96.2, and 100%, respectively; for the 412 control sera, the specificities were 99.0, 99.8, and 100%. The diagnostic efficiency (area below the receiver operating characteristic curve) of Brucellacapt was 0.987852 (95% confidence interval [CI], 0.95109 to 0.99286), very similar to the diagnostic efficiency of the Coombs test (0.97611; 95% CI, 0.94781 to 0.99146) and higher than that of SAT (0.91013; 95% CI, 0.86649 to 0.94317). The results of the Brucellacapt test were compared with those of the Coombs test (correlation coefficient, 0.956; P = 0.000) and SAT (correlation coefficient, 0.866; P = 0.000). The study shows very good correlation between the Brucellacapt and Coombs tests, with a high concordance between titers obtained in the two tests. Nevertheless, lower correlation and concordance were found between the Brucellacapt and Coombs tests when the results for titers of ≥1/160 were compared (0.692; P = 0.000). In acute brucellosis, the Brucellacapt and Coombs tests render positive titers of ≥1/160. When the titers

  18. The Life Cycle of Entzia, an Agglutinated Foraminifer from the Salt Marshes in Transylvania

    Science.gov (United States)

    Kaminski, Michael; Telespan, Andreea; Balc, Ramona; Filipescu, Sorin; Varga, Ildiko; Görög, Agnes

    2013-04-01

    The small salt marshes associated with Miocene salt domes in Transylvania are host to a variety of marine organisms, including communities of halophytic plants as well as an agglutinated foraminifer that is normally found in coastal salt marshes worldwide. Originally described as the species Entzia tetrastoma by Daday (1884), the foraminifer is more widely known by the name Jadammina macrescens (Brady, 1870). Because the genus name Entzia has priority over Jadammina, the valid name of this taxon is Entzia macrescens (Brady, 1870). In 2007, we discovered a living population of Entzia inhabiting a small salt marsh just outside the town of Turda in central Transylvania, only a kilometer from the famous Maria Theresa Salt Mine. This is the first discovery of a living population of Entzia in Transylvania since the species was originally described in 1884. To determine whether or not the specimens we found represent a breeding population, samples were collected from the marsh on a monthly basis over the span of a year. This species can be found among the roots of the halophytic plants, in the uppermost one or two centimeters of the mud. Sediment samples were preserved in Vodka with Rose Bengal to distinguish living and dead specimens, and examined quantitatively. To document the life cycle of the species the following metrics were carried out: test size, abundance, number of chambers, ratio between live and dead specimens, and the diameter of the proloculus. An increase in the mean diameter of specimens was found from October to December. However the mean diameter decreased again in January, which suggests that asexual reproduction had apparently taken place. Small specimens again appeared in March, when sexual reproduction is presumed to have taken place. The median proloculus diameter was smallest in April and May, but the monthly changes in mean proloculus size within the population over the span of a year are not significant. However, specimens with largest

  19. Seroepidemiological detection of antibodies against Leptospira spp using microscopic agglutination test in Urmia cows and sheep

    Directory of Open Access Journals (Sweden)

    Ramin Ag

    2013-01-01

    Full Text Available The study was designed to determine the level of incidence, titer and various serovars of leptospira in 203 cows and 166 sheep at Urmia abattoir in 2011. Blood samples were collected during the slaughter of animals and sera were separated to evaluate the serological reaction to Leptospira spp by Microscopic Agglutination Test (MAT using live antigens representing Leptospira interrogans serogroups: pomona, grippotyphosa, canicola, hardjo, icterrohaemoragiae, and ballum. Overall, 36% of cows and 19.3% of sheep including 33.8% of bulls, 40.5% of female cows, 18.3% of rams and 25% of ewes had a positive reaction to at least one of the leptospira serovars. The most prevalent serovars in cows were pomona (22.7%, grippotyphosa (13.8%, and hardjo (8.4%, and in sheep were grippotyphosa (66.7%, pomona (26.2% and canicola (7.1%. Other serovars were not detected in cows and sheep. The most prevalent serological titers of 1:100 and 1:200 in cows was 18.2% and 26.6%, and for sheep were 13.5% and 8%, respectively, and of 1:400 in sheep was 2.3%. Cows with a positive reaction to one, two and three serovars were 28.6%, 5.9%, and 1.5% and sheep positive to one and two serovars were 13.3% and 6%, respectively. Age comparison in seropositive cows and sheep showed a significantly increased infection (p<0.05 from young to adult ruminants, while no differences were seen regarding gender. The main mixed serovars were between grippotyphosa/pomona, grippotyphosa/canicola and canicola/pomona. The gender comparison of the serovars' distribution revealed that the pomona and grippotyphosa were predominant among other leptospiral serovars in cows and sheep, respectively. In conclusion, the rate of leptospirosis in Urmia cows was about 2 fold in sheep. The most current serovars in cows and sheep were pomona and grippotyphosa, respectively. The majority of animals was infected with one serovar, but polyserovars, are also possible. The highest titer (1:200 was observed in cows

  20. Expression Profile of Antioxidant Enzymes in Hemocytes from Freshwater Prawn Macrobrachium rosenbergii Exposed to an Elevated Level of Copper.

    Science.gov (United States)

    Guo, Hui; Miao, Yu-Tao; Xian, Jian-An; Qian, Kun; Wang, An-Li

    2015-10-01

    This study evaluated the expression level of antioxidant enzymes Cu, Zn-superoxide dismutase (Cu, Zn-SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) in hemocytes of Macrobrachium rosenbergii exposed to copper by real-time PCR (qRT-PCR). Results showed that the mRNA expression of Cu, Zn-SOD increased to reach a peak at 6 h, then recovered to its normal level at 48 h. CAT expression level was significantly increased at 12 h and reached a peak at 24 h, but recovered to its normal level later. GPx expression level was significantly increased at 6 h and reached the peak at 12 h. GST expression level was significantly induced from 12 to 24 h and then dropped to its normal level at 48 h. These results indicated that antioxidant enzymes were inducible, possibly for removing excessive reactive oxygen species to protect prawn from oxidative stress.

  1. THE INVESTIGATION OF BRUCELLA ANTIBODY WITH MILK RING TEST AND AGGLUTINATION TEST IN MILK COLLECTED FROM SAMSUN REGION

    OpenAIRE

    Goknur TERZI

    2006-01-01

    In this study Brucella antibodies were investigated with agglutination test (Whey-AT) and Milk Ring Test (MRT) in a total of 100 milk samples as 50 of cow milk and 50 of goat milk collected from center and villages of Samsun. According to MRT Brucella antibodies was positive at 10 samples (20 %) of cow milk and 6 samples (12 %) of goat milk. In cow milk, 4 (8 %) positive, 3 (6 %) suspicious and 43 (86 %) negative samples; in goat milk 3 (6 %) positive, 2 (4 %) suspicious and 45 (90 %) negativ...

  2. A new type of pseudothrombocytopenia: EDTA-mediated agglutination of platelets bearing Fab fragments of a chimaeric antibody.

    Science.gov (United States)

    Christopoulos, C G; Machin, S J

    1994-07-01

    In vitro agglutination of platelets leading to low automated platelet counts was observed in EDTA-anticoagulated blood from human volunteers receiving infusions of Fab fragments of a chimaeric monoclonal antibody to platelet glycoprotein IIb-IIIa. This pseudothrombocytopenia depended on the presence of chimaeric Fab on the platelet surface and was not seen when sodium citrate was used as anticoagulent. Preliminary evidence suggests that this phenomenon might be mediated by immunoglobulin G reactive with the human component of the chimaeric Fab. It is important to exclude pseudothrombocytopenia when low automated platelet counts are reported in association with the administration of chimaeric anti-platelet antibodies.

  3. Construction of supported lipid membrane modified piezoelectric biosensor for sensitive assay of cholera toxin based on surface-agglutination of ganglioside-bearing liposomes.

    Science.gov (United States)

    Chen, Huan; Hu, Qing-Yuan; Yue-Zheng; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2010-01-11

    A novel piezoelelctric biosensor has been developed for cholera toxin (CT) detection based on the analyte-mediated surface-agglutination of ganglioside (GM1)-functionalized liposomes. To achieve a CT-specific agglutination at the surface, the gold electrode is modified by a GM1-functionalized supported lipid membrane via spontaneous spread of the liposomes on a self-assembled monolayer of a long-chain alkanethiol. In the presence of CT, the GM1-incorporated liposomes in assay medium will rapidly specifically agglutinate at the electrode surface through the binding of CT to GM1 on the electrode surface and the liposome interface. This results in an enormous mass loading on the piezoelelctric crystal as well as a significant increase of density and viscosity at the interface, thereby generating a decrease in frequency of the piezoelelctric crystal. The combination of mass loading with interfacial change in the surface-agglutination reaction allows the developed piezoelelctric biosensor to show substantial signal amplification in response to the analyte CT. The detection limit can be achieved as low as 25 ng mL(-1) CT. This is the first demonstration on CT detection based on specific surface-agglutination of GM1-modified liposomes. The supported lipid layer based sensing interface can be prepared readily and renewably, making the developed technique especially useful for simple, reusable and sensitive determination of proteins.

  4. Induction of Staphylococcus aureus-specific IgA and agglutination potency in milk of cows by mucosal immunization.

    Science.gov (United States)

    Tempelmans Plat-Sinnige, Marjan J; Verkaik, Nelianne J; van Wamel, Willem J B; de Groot, Nanda; Acton, Dennis S; van Belkum, Alex

    2009-06-19

    Lactating cows were immunized with inactivated Staphylococcus aureus strains and concentrated culture supernatants. Application of a repeated mucosal immunization scheme resulted in significant levels of S. aureus-specific IgA in milk of dairy cows. Average IgA titers against whole cell S. aureus increased during the first 10 weeks of immunization after which a plateau level was reached and maintained during lactation. Immune whey agglutinated both bovine and human S. aureus strains including methicillin-resistant S. aureus (MRSA) strains and recognized extracted S. aureus proteins on Western blot. ELISAs to quantify milk IgA reactive with a number of S. aureus virulence proteins (e.g. enterotoxins, microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) and immune modulating proteins) and cell wall components, demonstrated the polyclonality of the IgA. Correlations observed between agglutination and specific IgA titers for whey and for purified IgA suggested functionality of the induced antibodies. Milk from immunized cows may provide a way of producing potentially therapeutic polyclonal antibodies against S. aureus colonization and infection.

  5. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation.

    Science.gov (United States)

    Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

    2016-04-13

    Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1-45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance.

  6. Monounsaturated fatty acid ether oligomers formed during heating of virgin olive oil show agglutination activity against human red blood cells.

    Science.gov (United States)

    Patrikios, Ioannis S; Mavromoustakos, Thomas M

    2014-01-29

    The present work focuses on the characterization of molecules formed when virgin olive oil is heated at 130 °C for 24 h open in air, which are found to be strong agglutinins. The hemagglutinating activity of the newly formed molecule isolated from the heated virgin olive oil sample was estimated against human red blood cells (RBCs). Dimers and polymers (high molecular weight molecules) were identified through thin layer chromatography (TLC) of the oil mixture. (1)H and (13)C nuclear magnetic resonance (NMR) and gas chromatography-mass spectroscopy (GC-MS) were the methods used for structural characterization. Among others, oligomerization of at least two monounsaturated fatty acids (FA) by an ether linkage between the hydrocarbon chains is involved. Light microscopy was used to characterize and visualize the agglutination process. Agglutination without fusion or lysis was observed. It was concluded that the heating of virgin olive oil open in air, among other effects, produces oligomerization as well as polymerization of unsaturated FA, possibly of monohydroxy, monounsaturated FA that is associated with strong hemagglutinating activity against human RBCs. The nutritional value and the effects on human health of such oligomers are not discussed in the literature and remain to be investigated.

  7. siRNA delivery targeting to the lung via agglutination-induced accumulation and clearance of cationic tetraamino fullerene

    Science.gov (United States)

    Minami, Kosuke; Okamoto, Koji; Doi, Kent; Harano, Koji; Noiri, Eisei; Nakamura, Eiichi

    2014-05-01

    The efficient treatment of lung diseases requires lung-selective delivery of agents to the lung. However, lung-selective delivery is difficult because the accumulation of micrometer-sized carriers in the lung often induces inflammation and embolization-related toxicity. Here we demonstrate a lung-selective delivery system of small interfering RNA (siRNA) by controlling the size of carrier vehicle in blood vessels. The carrier is made of tetra(piperazino)fullerene epoxide (TPFE), a water-soluble cationic tetraamino fullerene. TPFE and siRNA form sub-micrometer-sized complexes in buffered solution and these complexes agglutinate further with plasma proteins in the bloodstream to form micrometer-sized particles. The agglutinate rapidly clogs the lung capillaries, releases the siRNA into lung cells to silence expression of target genes, and is then cleared rapidly from the lung after siRNA delivery. We applied our delivery system to an animal model of sepsis, indicating the potential of TPFE-based siRNA delivery for clinical applications.

  8. Prevalence of agglutinating antibodies to Sarcocystis neurona in skunks (Mephitis Mephitis), raccoons (Procyon lotor), and opossums (Didelphis Virginiana) from Connecticut.

    Science.gov (United States)

    Mitchell, Sheila M; Richardson, Dennis J; Cheadle, M Andy; Zajac, Anne M; Lindsay, David S

    2002-10-01

    Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural cases of encephalitis caused by S. neurona have been reported in skunks (Mephitis mephitis) and raccoons (Procyon lotor). Opossums (Didelphis spp.) are the only known definitive host. Sera from 24 striped skunks, 12 raccoons, and 7 opossums (D. virginiana) from Connecticut were examined for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. The SAT was validated for skunk sera using pre- and postinfection serum samples from 2 experimentally infected skunks. Of the 24 (46%) skunks 11 were positive, and all 12 raccoons were positive for S. neurona antibodies. None of the 7 opossums was positive for antibodies to S. neurona. These results suggest that exposure to sporocysts of S. neurona by intermediate hosts is high in Connecticut. The absence of antibodies in opossums collected from the same areas is most likely because of the absence of systemic infection in the definitive host.

  9. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation

    Science.gov (United States)

    Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

    2016-01-01

    Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1–45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance. PMID:27089320

  10. Study of polycation effects on erythrocyte agglutination mediated by anti-glycophorins using microscopic image digital analysis

    Science.gov (United States)

    Riquelme, B.; Dumas, D.; Relancio, F.; Fontana, A.; Alessi, A.; Foresto, P.; Grandfils, C.; Stoltz, J.; Valverde, J.

    2006-04-01

    The aim of this work was to study synthetic polycation effects on erythrocyte agglutination mediated by anti-glycophorin using image digital analysis. Polycations are oligomers or polymers of natural or synthetic origin, which bear a great number of positive charges at pH 7.4. Several of these polycations are nowadays used in clinic for human and veterinary purposes. New applications of polycations to the development of new drug delivery systems are investigated, in order to promote the drug absorption through the gastro-intestinal and blood brain barriers. However, up to now, there are no clear relationships between macromolecular features of polycations (molecular weight, mean charge density, charge repartition, etc.) and their interactions with blood elements (which bear superficial negative charges). The interaction on the red blood cell membrane with synthetic polycations having well-controlled macromolecular features and functionalized with pendent polyethylene glycol segments was investigated. The alterations over stationary and dynamic viscoelastic properties of erythrocyte membranes were analyzed through laser diffractometry. Image digital analysis was used to study erythrocyte agglutination mediated by anti-glycophorin. Results show different reactivities of the polycations on the erythrocyte membrane. These findings could provide more information about the mechanisms of polycation interaction on erythrocyte membranes. We consider that this work could provide useful tools to understand and improve the haemocompatibility of polycations and enlarge their potential in clinic.

  11. Coexistence of erythrocyte agglutination and EDTA-dependent platelet clumping in a patient with thymoma and plasmocytoma.

    Science.gov (United States)

    Bizzaro, N; Fiorin, F

    1999-02-01

    For 8 years, EDTA-dependent pseudothrombocytopenia was observed in a 55-year-old woman with a history of rheumatoid arthritis who had undergone surgery for lymphoepithelial thymoma 11 years earlier. The clinical picture was characterized by the presence of platelet clumps and antiplatelet antibodies of the IgM class. With the recent appearance of a solitary extramedullary plasmocytoma in the right retrobulbar region and the detection of an IgGlambda monoclonal gammopathy, blood examination also revealed erythrocyte agglutinates alongside the platelet clumps and the presence of a cold IgG antibody with antiI specificity. Both phenomena were observed in vitro when the sample temperature declined to 20 degrees C to 25 degrees C, but not at 37 degrees C. While the EDTA-dependent antiplatelet antibodies did not appear to be chronologically correlated with the patient's diseases, the cold antierythrocyte autoantibodies were strictly related to the plasmocytoma and the IgGlambda monoclonal component in serum. To our knowledge, this is the first description of an association between EDTA-dependent platelet and erythrocyte agglutinates, with a clinical picture of pseudothrombocytopenia and pseudoerythrocytopenia due to cold agglutinins.

  12. A prototype of the direct agglutination test kit (DAT-Canis) for the serological diagnosis of canine visceral leishmaniasis.

    Science.gov (United States)

    Oliveira, Edward; Saliba, Juliana Wilke; Oliveira, Diana; Dias, Edelberto Santos; Paz, Gustavo Fontes

    2016-05-15

    This report describes the stege I/II development of a new direct agglutination test (DAT) for the diagnosis of canine visceral leishmaniasis (CVL) using freeze-dried antigen produced Coomassie blue-stained Leishmania (Leishmania) infantum promastigotes. In stage I, 16 canine serum samples, collected from eight dogs carrying CVL and eight healthy dogs, were assessed with the DAT using 2-mercaptoethanol (2-ME), N-acetyl-cysteine (NAC), kaolin or NAC plus urea (NAC+U) to improve the assay conditions. Stage II assessed the diagnostic accuracy with 100 serum samples collected from dogs with symptomatic CVL and clinically healthy dogs, comparing the four different sample diluents. The CVL-DAT prototype kit showed equivalent performances when 2-ME, NAC or NAC+U were used: 97.1% sensitivity (CI: 83-99.8%), 97% specificity (CI: 88.5-99.5%) and a 97% diagnostic accuracy (CI: 90.8-99.2). With kaolin, a 94.1% sensitivity (CI: 79-99%), 97% specificity (CI: 88.5-99.5%) and 96% diagnostic accuracy were observed (CI: 89.5-98.7), with no statistically significant differences among the four reagents (p=1.0). The NAC plus urea in sample diluent decreased non-specific agglutination, promoted a better defined sharp-edged blue spot and was thus chosen as a component for the new DAT prototype to diagnose canine VL, designated DAT-Canis.

  13. Jacalin domain in wheat jasmonate-regulated protein Ta-JA1 confers agglutinating activity and pathogen resistance.

    Science.gov (United States)

    Ma, Qing-Hu; Zhen, Wei-Bo; Liu, Yun-Chao

    2013-02-01

    Ta-JA1 is a jacalin-like lectin from wheat (Triticum aestivum) plants. To date, its homologs are only observed in the Gramineae family. Our previous experiments have demonstrated that Ta-JA1 contains a modular structure consisting of an N-terminal dirigent domain and a C-terminal jacalin-related lectin domain (JRL) and this protein exhibits a mannose-specific lectin activity. The over-expression of Ta-JA1 gene provides transgenic plants a broad-spectrum resistance to diseases. Here, we report the differential activities of the dirigent and JRL domains of Ta-JA1. In vitro assay showed that the recombinant JRL domain could effectively agglutinate rabbit erythrocytes and pathogen bacteria Pseudomonas syringe pv tabaci. These hemagglutination activities could be inhibited by mannose but not by galactose. In contrast, the recombinant dirigent domain did not show agglutination activity. Corresponding to these differentiations of activities, similar to full-length of Ta-JA1, the over-expression of JRL domain in transgenic plants also increased resistance to the infection of P. syringe. Unlike JRL, the over-expression of dirigent domain in transgenic plants led to alteration of the seedling sensitivity to salts. In addition, a d(N)/d(S) ratio analysis of Ta-JA1 and its related proteins showed that this protein family functionally limited to a few crop plants, such as maize, rice and wheat.

  14. Correlation Between the Mobility of Inner Plasma Membrane Structure and Agglutination by Concanavalin A in Two Cell Lines of MOPC 173 Plasmocytoma Cells

    Science.gov (United States)

    Guérin, Claudine; Zachowski, Alain; Prigent, Bernadette; Paraf, Alain; Dunia, Irène; Diawara, Marie-Aline; Benedetti, E. L.

    1974-01-01

    Both the distribution of the concanavalin A-binding sites and the rearrangement of the intramembranous particles revealed by the freeze-etching technique, have been studied by means of two variants of the same cell line issued from MOPC 173 murine plasmocytoma. One variant does not agglutinate even in presence of high lectin concentration. It has been shown that the number of binding sites and affinity are almost the same in the two variants. The clustered distribution of intramembranous particles is induced by the interaction of the concanavalin A and the cell surface only in the variant which is agglutinable. From these results it became apparent that the clustered distribution of the membrane particulate components is an acquired feature of the plasma membrane accompanying cell agglutination. Images PMID:4521044

  15. Lab-on-a-disc agglutination assay for protein detection by optomagnetic readout and optical imaging using nano- and micro-sized magnetic beads

    DEFF Research Database (Denmark)

    Uddin, Rokon; Burger, Robert; Donolato, Marco;

    2016-01-01

    We present a biosensing platform for the detection of proteins based on agglutination of aptamer coated magnetic nano- or microbeads. The assay, from sample to answer, is integrated on an automated, low-cost microfluidic disc platform. This ensures fast and reliable results due to a minimum...... of manual steps involved. The detection of the target protein was achieved in two ways: (1) optomagnetic readout using magnetic nanobeads (MNBs); (2) optical imaging using magnetic microbeads (MMBs). The optomagnetic readout of agglutination is based on optical measurement of the dynamics of MNB aggregates...... whereas the imaging method is based on direct visualization and quantification of the average size of MMB aggregates. By enhancing magnetic particle agglutination via application of strong magnetic field pulses, we obtained identical limits of detection of 25 pM with the same sample-to-answer time (15 min...

  16. Sequencing and de novo analysis of the hemocytes transcriptome in Litopenaeus vannamei response to white spot syndrome virus infection.

    Directory of Open Access Journals (Sweden)

    Shuxia Xue

    Full Text Available BACKGROUND: White spot syndrome virus (WSSV is a causative pathogen found in most shrimp farming areas of the world and causes large economic losses to the shrimp aquaculture. The mechanism underlying the molecular pathogenesis of the highly virulent WSSV remains unknown. To better understand the virus-host interactions at the molecular level, the transcriptome profiles in hemocytes of unchallenged and WSSV-challenged shrimp (Litopenaeus vannamei were compared using a short-read deep sequencing method (Illumina. RESULTS: RNA-seq analysis generated more than 25.81 million clean pair end (PE reads, which were assembled into 52,073 unigenes (mean size = 520 bp. Based on sequence similarity searches, 23,568 (45.3% genes were identified, among which 6,562 and 7,822 unigenes were assigned to gene ontology (GO categories and clusters of orthologous groups (COG, respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG mapped 14,941 (63.4% unigenes to 240 KEGG pathways. Among all the annotated unigenes, 1,179 were associated with immune-related genes. Digital gene expression (DGE analysis revealed that the host transcriptome profile was slightly changed in the early infection (5 hours post injection of the virus, while large transcriptional differences were identified in the late infection (48 hpi of WSSV. The differentially expressed genes mainly involved in pattern recognition genes and some immune response factors. The results indicated that antiviral immune mechanisms were probably involved in the recognition of pathogen-associated molecular patterns. CONCLUSIONS: This study provided a global survey of host gene activities against virus infection in a non-model organism, pacific white shrimp. Results can contribute to the in-depth study of candidate genes in white shrimp, and help to improve the current understanding of host-pathogen interactions.

  17. Transcriptomics of in vitro immune-stimulated hemocytes from the Manila clam Ruditapes philippinarum using high-throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Rebeca Moreira

    Full Text Available BACKGROUND: The Manila clam (Ruditapes philippinarum is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. METHODOLOGY AND PRINCIPAL FINDINGS: High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. CONCLUSIONS: This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum.

  18. White spot syndrome virus induces metabolic changes resembling the warburg effect in shrimp hemocytes in the early stage of infection.

    Science.gov (United States)

    Chen, I-Tung; Aoki, Takashi; Huang, Yun-Tzu; Hirono, Ikuo; Chen, Tsan-Chi; Huang, Jiun-Yan; Chang, Geen-Dong; Lo, Chu-Fang; Wang, Han-Ching

    2011-12-01

    The Warburg effect is an abnormal glycolysis response that is associated with cancer cells. Here we present evidence that metabolic changes resembling the Warburg effect are induced by a nonmammalian virus. When shrimp were infected with white spot syndrome virus (WSSV), changes were induced in several metabolic pathways related to the mitochondria. At the viral genome replication stage (12 h postinfection [hpi]), glucose consumption and plasma lactate concentration were both increased in WSSV-infected shrimp, and the key enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PDH), showed increased activity. We also found that at 12 hpi there was no alteration in the ADP/ATP ratio and that oxidative stress was lower than that in uninfected controls. All of these results are characteristic of the Warburg effect as it is present in mammals. There was also a significant decrease in triglyceride concentration starting at 12 hpi. At the late stage of the infection cycle (24 hpi), hemocytes of WSSV-infected shrimp showed several changes associated with cell death. These included the induction of mitochondrial membrane permeabilization (MMP), increased oxidative stress, decreased glucose consumption, and disrupted energy production. A previous study showed that WSSV infection led to upregulation of the voltage-dependent anion channel (VDAC), which is known to be involved in both the Warburg effect and MMP. Here we show that double-stranded RNA (dsRNA) silencing of the VDAC reduces WSSV-induced mortality and virion copy number. For these results, we hypothesize a model depicting the metabolic changes in host cells at the early and late stages of WSSV infection.

  19. A second copper zinc superoxide dismutase (CuZnSOD) in the blue crab Callinectes sapidus: cloning and up-regulated expression in the hemocytes after immune challenge.

    Science.gov (United States)

    Sook Chung, J; Bachvaroff, T R; Trant, J; Place, A

    2012-01-01

    The full-length cDNA (1362 nucleotides, GenBank JF736621) encoding an extracellular copper zinc superoxide dismutase initially isolated from an EST library of the blue crab Callinectes sapidus was characterized using 3' RACE and named Cas-ecCuZnSOD-2. The open reading frame of Cas-ecCuZnSOD-2 contains 203 deduced amino acids with the conserved active catalytic center for copper and zinc binding and the post-translational modification at two putative N-glycosylation and nine phosphorylation sites. Overall, the deduced amino acids of Cas-ecCuZnSOD-2 shared only 35% sequence identity with that of Cas-ecCuZnSOD (GenBank AF264031) which was previously found in C. sapidus, while it showed ∼75% sequence identity to Scylla paramamosain ecCuZnSOD (GenBank FJ774661). The expression profile of Cas-ecCuZnSOD-2 and the other three C. sapidus SODs: ecCuZn, cytMn- and mitMn SODs was largely ubiquitous among the tested tissues obtained from a juvenile female at intermolt: brain, eyestalk ganglia, pericardial organs, and thoracic ganglia complex (nervous system); hepatopancreas (digestive system); heart, artery and hemocytes (circulatory system); gill and antennal gland (excretory system), hypodermis, and Y-organ (endocrine organ). Our study reports, for the first time in the crustaceans, expression analyses for all four Cas-SODs in hemocytes after immune challenges. Crabs challenged with lipopolysaccharides (LPS) injection had a remarkable induction of Cas-ecCuZnSOD-2 expression along with three other SODs in hemocytes, suggesting that Cas-SODs including Cas-ecCuZnSOD-2 are involved in the defense system, possibly innate immunity and immunocompetency of C. sapidus. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. M-cholinoreactivity of erythrocytes of non-pregnant and pregnant women evaluated by changes in the rate of erythrocyte agglutination under the influence of acetylcholine.

    Science.gov (United States)

    Strelnikova, A I; Tsirkin, V I; Krysova, A V; Hlybova, S V; Dmitrieva, S L

    2012-12-01

    Acetylcholine (5.5×10(-10)-5.5×10(-6)M) accelerated erythrocyte agglutination in men, non-pregnant women in follicular phase of the menstrual cycle, and pregnant women in the first trimester. The effect was blocked with atropine (5.5×10(-6)M). Acetylcholine had no effect on the rate of erythrocyte agglutination in non-pregnant women in the luteal phase and pregnant women in the second and third trimesters, which coincided with the development of myometrium refractoriness to acetylcholine in pregnant women. The results indicate that erythrocytes can reflect M-cholinoreactivity of internal organs.

  1. The Puzzle of the False Positve Reactions in Cr. Neoformanse’s Capsular Antigen Slide Lates Agglutination Test by Sera of Patients with Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    F. Zaini

    1987-07-01

    Full Text Available Different examination have shown that rheumatoid factor is not responsible for false positive reaction (F.P.R. in Cr. Neoformanse’s free capsular antigen latex agglutination test, whereas high levels of iron in sera of patients with rheumatoid arthritis as well as other sera was responsible not only for this F.P.R. , but also had an important role in the production of F.P.R. in many slide latex agglutination tests. This is because of Iron’s Ion reaction with reagent’s preservative: sodium azid and/or negative charge of the antigens fixed to the latex particles.

  2. Prozone effects in microscopic agglutination tests for leptospirosis in the sera of mice infected with the pathogenic Leptospira interrogans serovar Canicola

    Directory of Open Access Journals (Sweden)

    Fabio Hiroto Shimabukuro

    2013-08-01

    Full Text Available Mice experimentally infected with a pathogenic strain of Leptospira interrogans serovar Canicola produced false negative results (prozone effect in a microscopic agglutination test (MAT. This prozone effect occurred in several serum samples collected at different post-infection times, but it was more prominent in samples collected from seven-42 days post-infection and for 1:50 and 1:100 sample dilutions. This phenomenon was correlated with increased antibody titres in the early post-infection phase. While prozone effects are often observed in serological agglutination assays for the diagnosis of animal brucellosis and human syphilis, they are not widely reported in leptospirosis MATs.

  3. De Novo Transcriptome Sequencing of the Octopus vulgaris Hemocytes Using Illumina RNA-Seq Technology: Response to the Infection by the Gastrointestinal Parasite Aggregata octopiana

    Science.gov (United States)

    Castellanos-Martínez, Sheila; Arteta, David; Catarino, Susana; Gestal, Camino

    2014-01-01

    Background Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus’ well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. Results A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. Conclusion The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers

  4. Coronatin-1 isolated from entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella hemocytes in vitro and forms potassium channels in planar lipid membrane.

    Science.gov (United States)

    Wieloch, Wioletta; Boguś, Mieczysława I; Ligęza, Marta; Koszela-Piotrowska, Izabela; Szewczyk, Adam

    2011-09-15

    Entomopathogenic fungi are important natural regulatory factors of insect populations and have potential as biological control agents of insect pests. The cosmopolitan soil fungus Conidiobolus coronatus (Entomopthorales) easily attacks Galleria mellonella (Lepidoptera) larvae. Prompt death of invaded insects is attributed to the action of toxic metabolites released by the invader. Effect of fungal metabolites on hemocytes, insect blood cells involved in innate defense response, remains underexplored to date. C. coronatus isolate 3491 inducing 100% mortality of G. mellonella last instar larvae exposed to sporulating colonies, was cultivated at 20 °C in minimal medium. Post-incubation filtrates were used as a source of fungal metabolites. A two-step HPLC (1 step: Shodex KW-803 column eluted with 50 mM KH(2)PO(4) supplemented with 0.1 M KCl, pH 6.5; 2 step: ProteinPak™ CM 8HR column equilibrated with 5 mM KH(2)PO(4), pH 6.5, proteins eluted with a linear gradient of 0.5 M KCl) allowed the isolation of coronatin-1, an insecticidal 36 kDa protein showing both elastolytic and chitinolytic activities. Addition of coronatin-1 into primary in vitro cultures of G. mellonella hemocytes resulted in rapid disintegration of spherulocytes freely floating in culture medium and shrinkage of plasmatocytes adhering to the bottom of culture well. Coronatin-1 stimulated pseudopodia atrophy and, in consequence, disintegration of nets formed by cultured hemocytes. After incorporation of coronatin-1 into planar lipid membrane (PLM) ion channels selective for K(+) ions in 50/450 mM KCl solutions were observed. Potassium current flows were recorded in nearly 70% of experiments with conductance from 300 pS up to 1 nS. All observed channels were active at both positive and negative membrane potentials. Under experimental conditions incorporated coronatin-1 exhibited a zero current potential (E(rev)) of 47.7 mV, which indicates K(+)-selectivity of this protein. The success of the

  5. De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

    Directory of Open Access Journals (Sweden)

    Sheila Castellanos-Martínez

    Full Text Available BACKGROUND: Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus' well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. RESULTS: A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. CONCLUSION: The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to

  6. Oxidative stress parameters induced by exposure to either cadmium or 17β-estradiol on Mytilus galloprovincialis hemocytes. The role of signaling molecules

    Energy Technology Data Exchange (ETDEWEB)

    Koutsogiannaki, Sophia [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece); Franzellitti, Silvia [University of Bologna, Interdepartment Centre for Environmental Science Research, via S. Alberto 163, 48123 Ravenna (Italy); Fabbri, Elena [University of Bologna, Interdepartment Centre for Environmental Science Research, via S. Alberto 163, 48123 Ravenna (Italy); University of Bologna, Department of Biological, Geological, and Environmental Sciences, via Selmi 3, 40100 Bologna (Italy); Kaloyianni, Martha, E-mail: kaloyian@bio.auth.gr [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece)

    2014-01-15

    Highlights: •Oxidative parameters in Mytilus galloprovincialis hemocytes were measured. •Comparison between cadmium and 17β-estradiol cytotoxicity is discussed. •NHE, PKC, PI3-K, NADPH oxidase, NO synthase, JNK involvement was observed. •Protective role of cAMP is suggested. •Signaling molecules studied could constitute novel biomarkers. -- Abstract: The aim of the present study was to determine and compare the possible effects of exposure to an estrogen, 17β-estradiol and to a metal, cadmium on oxidative parameters of Mytilus galloprovincialis hemocytes and to elucidate the signaling pathways that probably mediate the studied effects exerted by these two chemicals. In addition, it was of interest to investigate if the studied parameters could constitute biomarkers for aquatic pollution monitoring. Our results suggest that micromolar concentrations of either cadmium or 17β-estradiol affected the redox status of mussels by modulating oxidative parameters and antioxidant enzymes gene expression in mussel M. galloprovincialis hemocytes. In particular, our results showed that treatment of hemocytes with either 5 μM of cadmium chloride or with 25 nM of 17β-estradiol for 30 min caused significant increased ROS production; this led to oxidative damage exemplified by significant increased DNA damage, protein carbonylation and lipid peroxidation, as well as increased mRNA levels of the antioxidant enzymes catalase (CAT), superoxide dismoutase (SOD) and glutathione S-transferase (GST). Furthermore, our results suggest that either cadmium or 17β-estradiol signal is mediated either through one of the already known pathways initiated by photatidyl-inositol 3-kinase (PI3 K) and reaching Na{sup +}/H{sup +} exchanger (NHE) probably through protein kinase C (PKC) or a kinase-mediated signaling pathway that involves in most of the cases NHE, PKC, Ca{sup 2+}-dependent PKC isoforms, PI3-K, NADPH oxidase, nitric oxide (NO) synthase, c-Jun N-terminal kinase (JNK) and

  7. [The development of polymer immunoglobulin preparations to identify different serovars legionella pneumophilia in reaction of slide-agglutination].

    Science.gov (United States)

    Karbyshev, G L; Narkevich, A N; Kochetkova, A P; Larionova, L V; Simakova, D I; Liukshina, E Iu; Lysova, L K; Terent'ev, A N; Shelokhovich, A I; Sokirkina, O G

    2013-03-01

    The article deals with the results of study targeted to develop polymer diagnostic preparation to identify epidemically significant serogroups Legionella pneumophilia. The preparation combines rate of record (1-5 min) of reaction of paragglutinining preparations with color visualization and demonstrative of reaction of volume agglomeration with polymer diagnosticums. The specially synthesized polymer microspheres were sensibilized with serums enriched with antibodies to lipopolysaccharide of corresponding serovar L. pneumophilia. The derived immunoglobulin diagnostic preparations detect agent of legionellesis in the reaction of slide-agglutination on glass during 1-5 min. The polymer diagnostic preparations provide positive reaction with culture of corresponding serovar and no reaction with other gomologic and geterologic agents of infectious diseases.

  8. Good agreement of conventional and gel-based direct agglutination test in immune-mediated haemolytic anaemia

    Directory of Open Access Journals (Sweden)

    Piek Christine J

    2012-02-01

    Full Text Available Abstract Background The aim of this study was to compare a gel-based test with the traditional direct agglutination test (DAT for the diagnosis of immune-mediated haemolytic anaemia (IMHA. Methods Canine (n = 247 and feline (n = 74 blood samples were submitted for DAT testing to two laboratories. A subset of canine samples was categorized as having idiopathic IMHA, secondary IMHA, or no IMHA. Results The kappa values for agreement between the tests were in one laboratory 0.86 for canine and 0.58 for feline samples, and in the other 0.48 for canine samples. The lower agreement in the second laboratory was caused by a high number of positive canine DATs for which the gel test was negative. This group included significantly more dogs with secondary IMHA. Conclusions The gel test might be used as a screening test for idiopathic IMHA and is less often positive in secondary IMHA than the DAT.

  9. A rapid latex agglutination test for the detection of anti-cysticercus antibodies in cerebrospinal fluid (CSF

    Directory of Open Access Journals (Sweden)

    ROCHA Sérgio M.

    2002-01-01

    Full Text Available Simple and rapid latex-based diagnostic tests have been used for detecting specific antigens or antibodies in several diseases. In this article, we present the preliminary results obtained with a latex agglutination test (LAT for diagnosing neurocysticercosis by detection of antibodies in CSF. A total of 43 CSF samples were assayed by the LAT: 19 CSF samples from patients with neurocysticercosis and 24 CSF samples from patients with other neurologic disorders (neurosyphilis, n = 8; neurotoxoplasmosis, n = 3; viral meningitis, n = 4, chronic headache, n = 9. The LAT exhibited 89.5% sensitivity and 75% specificity. The use of LAT seems to be an additional approach for the screening of neurocysticercosis with advantage of simplicity and rapidity. Further studies could be performed using purified antigens and serum samples.

  10. Gelatin particle indirect agglutination and enzyme-linked immunosorbent assay for diagnosis of strongyloidiasis using Strongyloides venezuelensis antigen.

    Science.gov (United States)

    Huaman, Maria Cecilia; Sato, Yoshiya; Aguilar, Jose Luis; Terashima, Angelica; Guerra, Humberto; Gotuzzo, Eduardo; Kanbara, Hiroji

    2003-01-01

    Routine microscopical examination of stool specimens for diagnosis of strongyloidiasis is insensitive and serological methods using Strongyloides stercoralis antigen are at present not available for field studies. We evaluated 2 techniques, enzyme-linked immunosorbent assay (ELISA) and gelatin particle indirect agglutination (GPIA), using an antigen obtained from the rodent parasite, S. venezuelensis. Fifty-four Peruvian patients with different clinical forms of strongyloidiasis were studied: 12 asymptomatic, 31 symptomatic, and 11 hyperinfection cases. Our results demonstrate that both ELISA and GPIA using S. venezuelensis antigen are useful for diagnosis of strongyloidiasis, with sensitivities of 74.1% and 98.2%, respectively and a specificity of 100% for both techniques. We found that GPIA is a highly sensitive test for patients with suspected chronic infection and/or hyperinfection. In the hyperinfection cases, significantly lower concentrations of specific immunoglobulin antibodies and eosinophils (P < 0.001) were found compared with the asymptomatic and symptomatic cases.

  11. Aptamers that bind to the hemagglutinin of the recent pandemic influenza virus H1N1 and efficiently inhibit agglutination.

    Science.gov (United States)

    Gopinath, Subash C B; Kumar, Penmetcha K R

    2013-11-01

    Influenza virus hemagglutinin (HA) mediates both receptor (glycan) binding and membrane fusion for cell entry and has been the basis for typing influenza A viruses. In this study we have selected RNA aptamers (D-12 and D-26) that specifically target the HA protein of the recent pandemic influenza virus pdmH1N1 (A/California/07/2009). Among the selected aptamers the D-26 aptamer showed higher affinity for the HA of pdmH1N1 and was able to distinguish HA derived from other sub-types of influenza A viruses. The affinity of the D-26 aptamer was further improved upon incorporation of 2'-fluoropyrimidines to a level of 67 fM. Furthermore, the high affinity D-12 and D-26 aptamers were tested for their ability to interfere with HA-glycan interactions using a chicken red blood cell (RBC) agglutination assay. At a concentration of 200 nM the D-26 aptamer completely abolished the agglutination of RBCs, whereas D-12 only did so at 400 nM. These studies suggest that the selected aptamer D-26 not only has a higher affinity and specificity for the HA of pdmH1N1 but also has a better ability to efficiently interfere with HA-glycan interactions compared with the D-12 aptamer. The D-26 aptamer warrants further study regarding its application in developing topical virucidal products against the pdmH1N1 virus and also in surveillance of the pdmH1N1 influenza virus.

  12. Combined albumin and bicarbonate induces head-to-head sperm agglutination which physically prevents equine sperm-oviduct binding.

    Science.gov (United States)

    Leemans, Bart; Gadella, Bart M; Stout, Tom A E; Sostaric, Edita; De Schauwer, Catharina; Nelis, Hilde; Hoogewijs, Maarten; Van Soom, Ann

    2016-04-01

    In many species, sperm binding to oviduct epithelium is believed to be an essential step in generating a highly fertile capacitated sperm population primed for fertilization. In several mammalian species, this interaction is based on carbohydrate-lectin recognition. D-galactose has previously been characterized as a key molecule that facilitates sperm-oviduct binding in the horse. We used oviduct explant and oviduct apical plasma membrane (APM) assays to investigate the effects of various carbohydrates; glycosaminoglycans; lectins; S-S reductants; and the capacitating factors albumin, Ca(2+) and HCO3(-) on sperm-oviduct binding in the horse. Carbohydrate-specific lectin staining indicated that N-acetylgalactosamine, N-acetylneuraminic acid (sialic acid) and D-mannose or D-glucose were the most abundant carbohydrates on equine oviduct epithelia, whereas D-galactose moieties were not detected. However, in a competitive binding assay, sperm-oviduct binding density was not influenced by any tested carbohydrates, glycosaminoglycans, lectins or D-penicillamine, nor did the glycosaminoglycans induce sperm tail-associated protein tyrosine phosphorylation. Furthermore, N-glycosidase F (PNGase) pretreatment of oviduct explants and APM did not alter sperm-oviduct binding density. By contrast, a combination of the sperm-capacitating factors albumin and HCO3(-) severely reduced (>10-fold) equine sperm-oviduct binding density by inducing rapid head-to-head agglutination, both of which events were independent of Ca(2+) and an elevated pH (7.9). Conversely, neither albumin and HCO3(-) nor any other capacitating factor could induce release of oviduct-bound sperm. In conclusion, a combination of albumin and HCO3(-) markedly induced sperm head-to-head agglutination which physically prevented stallion sperm to bind to oviduct epithelium.

  13. ELISA Cut-off Point for the Diagnosis of Human Brucellosis; a Comparison with Serum Agglutination Test

    Directory of Open Access Journals (Sweden)

    Anahita Sanaei Dashti

    2012-03-01

    Full Text Available Background: Brucellosis is a world-wide disease, which has a diverse clinical manifestation, and its diagnosis has to be proven by laboratory data. Serum agglutination test (SAT is the most-widely used test for diagnosing brucellosis. The enzyme linked immunosorbent assay (ELISA can also determine specific antibody classes against brucella. It is a sensitive, simple and rapid test, which could be an acceptable alternative to SAT with fewer limitations, however, like any other new test it should be further evaluated and standardized for various populations. This study was planned to determine an optimal cut-off point, for ELISA which would offer maximum sensitivity and specificity for the test when compared to SAT.Methods: Four hundred and seven patients with fever and other compatible symptoms of brucellosis were enrolled in the study. Serum agglutination test, 2-Mercaptoethanol test, and ELISA were performed on their sera. Results: The cut-off point of 53 IU/ml of ELISA-IgG yielded the maximal sensitivity and specificity comparing to the other levels of ELISA-IgG, and was considered the best cut off-point of ELISA-IgG to diagnose acute brucellosis. At this cut-off, the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio were 84.09%, 85.38%, 62.20, 94.90, 5.75, 0.18, respectively.Conclusion: The best cut-off point of ELISA-IgG is 53 IU/ml, which yields the maximal sensitivity and specificity to diagnose acute brucellosis.

  14. Evaluation of the MycoAKT latex agglutination test for rapid diagnosis of Mycobacterium avium complex infections.

    Science.gov (United States)

    Olano, J P; Holmes, H; Woods, G L

    1998-01-01

    Rapid diagnosis of Mycobacterium avium complex (MAC) bacteremia is important for management of patients with the acquired immunodeficiency syndrome who have disseminated MAC. The purpose of this study was to determine the reliability of the MycoAKT latex agglutination test for direct detection of MAC in positive mycobacterial blood cultures. First, colonies of isolates of previously identified mycobacteria, including 35 MAC, were tested. Of the 55 isolates evaluated, 33 were identified as MAC by the latex test, including 31 of the known MAC and 2 M. chelonae (sensitivity, 88.6%; specificity, 90.0%). Second, broth from 20 ESP II and 20 BACTEC 12B bottles seeded with isolates of MAC were tested. Aliquots from 19 (95%) ESP II cultures and 16 (80%) 12B cultures were positive by the latex test. In phase 3, broth from 115 signal-positive ESP II blood cultures were tested by latex agglutination. Forty-three subcultures from these bottles grew mycobacteria (41 MAC and 2 Mycobacterium tuberculosis complex); the remainder grew no organisms. Broth from 40 of the blood cultures (39 that grew MAC and 1 from which no organisms were recovered) were latex positive; thus, the sensitivity, specificity, and positive and negative predictive values of the latex test for direct identification of MAC in ESP II blood cultures were 95.1, 98.6, 97.5, and 97.3%, respectively. The mean time to detection of MAC was 14.6 days (range, 6-34 days) with the direct latex test, compared with 18.3 days (range, 9-36 days) with subculture and probe (p < 0.05).

  15. Integration of agglutination assay for protein detection in microfluidic disc using Blu-ray optical pickup unit and optical fluid scanning

    DEFF Research Database (Denmark)

    Uddin, Rokon; Burger, Robert; Donolato, Marco;

    2015-01-01

    We present a novel strategy for thrombin detection by combining a magnetic bead based agglutination assay and low-cost microfluidic disc. The detection method is based on an optomagnetic readout system implemented using a Blu-ray optical pickup unit (OPU) as main optoelectronic component. The assay...

  16. [Evaluation of the Fundamental Performance of 4 Latex Agglutination Reagents to Measure Anti-TP Antibody Concentration and Detailed Investigation of Decision-Mismatched Samples].

    Science.gov (United States)

    Ito, Atsushi; Niizeki, Noriyasu; Kurose, Hitomi; Yonezawa, Takatoshi; Sasaki, Rie; Tachibana, Mineji; Tomoda, Yutaka; Kino, Shuichi; Fujii, Satoshi

    2015-01-01

    Serological diagnosis of syphilis can be made by using the serological test for syphilis (STS) method for detecting a lipid antibody and Treponema pallidum (TP) method for detecting the anti-TP-specific antibody. In STS and TP methods, the basis using latex agglutination reaction has been used in many facilities. However, in latex agglutination, false-positive results due to non-specific reaction have sometimes been obtained in reactions of a routine laboratory test reagent detecting the anti-TP antibody used in our medical laboratory. We evaluated the fundamental performance of 4 reagents to measure anti-TP antibody concentration using latex agglutination: Reagents A, B, C and D produced by SEKISUI MEDICAL, FUJI REBIO, DENKA SEIKEN and SHINO TEST, respectively. We examined the correlations between Reagent A (routine laboratory test reagent) and Reagents B, C, and D in sera from 68 patients, and we performed additional investigation by using a neutralization test, immunochromatography, Western blotting, FTA-ABS (IgG), and STS method by an automatic analyzer for 13 decision-mismatched samples. The fundamental performance of each reagent was as good as that previously reported. Eight of the 13 decision-mismatched samples were false positives due to non-specific reaction of Reagent A. In latex agglutination non-specific reaction is inevitable. However, this study strongly suggests that using a neutralization test and immunochromatography that can be performed quickly is sufficient to verify whether positive reactions are true or false.

  17. Bayesian estimation of sensitivity and specificity of the modified agglutination test and bioassay for detection of Toxoplasma gondii in free-range chickens

    Science.gov (United States)

    Toxoplasma gondii infects virtually all warm-blooded animals worldwide. Serological tests, including the modified agglutination test (MAT), are often used to determine exposure to the parasite. The MAT can be used for all hosts because it does not need species-specific reagents and has been shown to...

  18. Integration of agglutination assay for protein detection in microfluidic disc using Blu-ray optical pickup unit and optical fluid scanning

    DEFF Research Database (Denmark)

    Uddin, Rokon; Burger, Robert; Donolato, Marco;

    2015-01-01

    We present a novel strategy for thrombin detection by combining a magnetic bead based agglutination assay and low-cost microfluidic disc. The detection method is based on an optomagnetic readout system implemented using a Blu-ray optical pickup unit (OPU) as main optoelectronic component. The ass...

  19. Comparison of Brucella immunoglobulin M and G flow assays with serum agglutination and 2-mercaptoethanol tests in the diagnosis of brucellosis

    NARCIS (Netherlands)

    A. Zeytinoglu; A. Turhan; I. Altuglu; A. Bilgic; T.H. Abdoel; H.L. Smits

    2006-01-01

    The diagnostic value of Brucella IgM/IgG flow assays was evaluated in comparison with serum agglutination and 2-mercaptoethanol tests by testing a selection of serum samples submitted to the laboratory because of clinical suspicion of brucellosis. All 39 admission and 11 follow-up samples that agglu

  20. Evaluation of latex agglutination test and oxacillin resistant screening agar base (ORSAB medium for the detection of oxacillin resistant coagulase negative Staphylococci (ORCoNS (Preliminary study

    Directory of Open Access Journals (Sweden)

    Sjoekoer M. Dzen

    2007-12-01

    Full Text Available Coagulase negative Staphylococci (CoNS are recognized as an important cause of nosocomial infection, especially in neonates and patients with indwelling prosthetic devices. The CoNS resistance rate to oxacillin has been increasing. Therefore, rapid and accurate detection of oxacillin resistance is essential in order to determine the most appropriate antimicrobial therapy. This study aimed to prove that latex agglutination test and oxacillin resistant screening agar base (ORSAB medium can be used for rapid detection of oxacilllin resistant CoNS (ORCoNS. Latex agglutination test and ORSAB medium compared with the conventional method was conducted in this study toward 30 clinical isolates of CoNS for the detection of ORCoNS. Mc Nemar test was used to analyze the data. The study result revealed that there was no significant difference (P>0.05 in terms of ORCoNS detection between the latex agglutination test and ORSAB medium on the one hand, and the conventional method on the other. It is concluded that latex agglutination test and ORSAB medium can be used for rapid detection of ORCoNS. (Med J Indones 2007; 16:228-32Keywords: nosocomial infection, rapid detection, mecA gene

  1. Antiviral Activity of Ctn[15-34], A Cathelicidin-Derived Eicosapeptide, Against Infectious Myonecrosis Virus in Litopenaeus vannamei Primary Hemocyte Cultures.

    Science.gov (United States)

    Vieira-Girão, P R N; Falcão, C B; Rocha, I R C B; Lucena, H M R; Costa, F H F; Rádis-Baptista, G

    2017-02-16

    The shrimp farming has been converted into a mature aquaculture industry dealing with over millions of metric tonnes of processed commodities. Nevertheless, the global shrimp productions are constantly threatened by disease outbreaks, mainly triggered by rapidly disseminating viruses. Infectious myonecrosis virus (IMNV) is one of these epizootic agents affecting shrimp production in Brazil, of which no treatment exists. Herein, the antiviral activity against IMNV of an eicosapeptide, named Ctn[15-34], derived from a member of the cathelicidin family of antimicrobial peptides, was demonstrated. Cultures of hemocytes from Litopenaeus vannamei were established that support IMNV replication and infectivity titration. The cytotoxic effect of IMNV in culture and the in vitro anti-IMNV activity of Ctn[15-34] were assessed using a high-sensitive fluorescent-based method in combination with quantitative PCR. The Ctn[15-34] (shrimp hemocytes. This study reported for the first time the replication of IMNV in vitro and the employment of a straightforward methodology to assess cell viability and viral/antiviral activities. In addition, it provided the basis for the development of the anti-infective multi-effector Ctn[15-34] eicosapeptide and analogs as components of antiviral formulations against shrimp viral diseases.

  2. Evaluation of radiosensitivity hemocytes of Biomphalaria glabrata exposed to gamma radiation; Avaliacao da radiossensibilidade de hemocitos de Biomphalaria glabrata expostos a radiacao gama

    Energy Technology Data Exchange (ETDEWEB)

    Silva, L.R.S.; Amaral, A.J., E-mail: luannaribeiro_lua@hotmail.com [Universidade Federal de Pernambuco (UFPE), Recife-PE (Brazil). Dept. de Energia Nuclear; Silva, E.B. [Universidade Federal de Pernambuco (UFPE), Vitoria de Santo Antao, PE (Brazil). Centro Academico de Vitoria; Amancio, F.F.; Melo, A.M.M.A. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Biofisica e Radiobiologia

    2013-06-15

    The mollusc Biomphalaria glabrata have characteristics that allow them to be identified as an animal model ideal for monitoring areas exposed to chemical agents and physical. This study evaluated the effect of ionizing radiation from Cobalt-60 in haemocytes present in the hemolymph of Biomphalaria glabrata, with the goal of using these cells as indicators of the presence of radiation in aquatic environments. The mollusks were divided into five groups: one control and four subjected doses of 25, 35, 45 and 55 Gy of gamma radiation. After 48 hours of irradiation, the clam hemolymph was collected and slides were prepared and stained with Giemsa for analyses under a light microscope. Statistical analysis was performed using ANOVA and Tukey's test, p <0.05. The results showed that the total number of cells after irradiation reduced compared to control except at a dose of 55 Gy. During data analysis, morphological changes were observed in haemocytes of mollusks subjected to doses of 35, 45 and 55 Gy. These modifications consisted of nucleus bilobulated and nucleo plasmatic bridges. Another change was exclusively observed in the cellular exposure of 55 Gy, where hemocytes showed misshapen nuclei and cytoplasm vacuolisation, suggestive of apoptosis. It is concluded that hemocytes are sensitive to radiation and can be used as indicators of the presence of high doses of ionizing radiation in aquatic environments. (author)

  3. A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination.

    Science.gov (United States)

    Mu, Changkao; Song, Xiaoyan; Zhao, Jianmin; Wang, Lingling; Qiu, Limei; Zhang, Huan; Zhou, Zhi; Wang, Mengqiang; Song, Linsheng; Wang, Chunlin

    2012-05-01

    C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin.

  4. Impacts of papain and neuraminidase enzyme treatment on electrohydrodynamics and IgG-mediated agglutination of type A red blood cells.

    Science.gov (United States)

    Hyono, Atsushi; Gaboriaud, Fabien; Mazda, Toshio; Takata, Youichi; Ohshima, Hiroyuki; Duval, Jérôme F L

    2009-09-15

    The stability of native and enzyme-treated human red blood cells of type A (Rh D positive) against agglutination is investigated under conditions where it is mediated by immunoglobuline G (IgG) anti-D antibody binding. The propensity of cells to agglutinate is related to their interphasic (electrokinetic) properties. These properties significantly depend on the concentration of proteolytic papain enzyme and protease-free neuraminidase enzyme that the cells are exposed to. The analysis is based on the interpretation of electrophoretic data of cells by means of the numerical theory for the electrokinetics of soft (bio)particles. A significant reduction of the hydrodynamic permeability of the external soft glycoprotein layer of the cells is reported under the action of papain. This reflects a significant decrease in soft surface layer thickness and a loss in cell surface integrity/rigidity, as confirmed by nanomechanical AFM analysis. Neuraminidase action leads to an important decrease in the interphase charge density by removing sialic acids from the cell soft surface layer. This is accompanied by hydrodynamic softness modulations less significant than those observed for papain-treated cells. On the basis of these electrohydrodynamic characteristics, the overall interaction potential profiles between two native cells and two enzyme-treated cells are derived as a function of the soft surface layer thickness in the Debye-Hückel limit that is valid for cell suspensions under physiological conditions (approximately 0.16 M). The thermodynamic computation of cell suspension stability against IgG-mediated agglutination then reveals that a decrease in the cell surface layer thickness is more favorable than a decrease in interphase charge density for inducing agglutination. This is experimentally confirmed by agglutination data collected for papain- and neuraminidase-treated cells.

  5. Características físicas de dietas para peixes confeccionadas com diferentes aglutinantes Influence of agglutinants on physical stability of fish diets

    Directory of Open Access Journals (Sweden)

    Margarida Maria Barros

    2002-04-01

    Full Text Available Este experimento teve por objetivo avaliar a eficiência de diferentes aglutinantes, a seco e na água, por meio da estabilidade física do pélete. Foram avaliadas duas técnicas de processamento (com ou sem vapor e seis aglutinantes (carboximetilcelulose, polimetilcarbamida, amido de mandioca, alginato de sódio, polivinilpirrolidona, goma guar, através da análise de variância no esquema fatorial (2x6, além de um controle, ao qual não foi acrescido aglutinante. Concluiu-se que o aglutinante melhora significativamente a resistência física do grânulo, independente da técnica de processamento; que o vapor determina grânulos mais estáveis quando em contato com a água e, que o alginato de sódio proporciona grânulos fisicamente mais estáveis, em ambas as técnicas de processamento, enquanto a goma guar a pior.The aim of this paper was to determine the influence of different dry and water agglutinants, through physical stability of pellets. The agglutinants were sodium alginate, guar gum, polymetylcarbamide, polyvinylpirrolidone, carboxymetilcellulose, and cassava starch. The manufacturing processes were two: with and without steam and extrusion. These treatments were evaluated through the variance analysis technique with the factorial scheme 2 x 6 (two processes and six agglutinants, and one control where no extra agglutinants was added. Results showed that, independently of processing technique, the presence the agglutinants improves the physical resistance of the pellets significantly, giving the whole pellets more stability while in contact with water, and that the sodium alginate gives pellets the highest aggregated characteristic, in both processes, while that guar gum gives the lowest.

  6. [Detection of mutans streptococci by latex agglutination test and its application as a caries-activity test].

    Science.gov (United States)

    Takei, T

    1990-06-01

    The number of mutans streptococci in saliva and dental plaque has been reported to correlate with the incidence of dental caries. This report describes a simple and rapid diagnostic method for the detection of mutans streptococci in dental plaque using latex agglutination (LA) test. Latex particles (0.876 microns, diameter) were sensitized with partially purified antibodies against whole cells of Streptococcus mutans MT8148 (c), MT703R (e) and OMZ175 (f) and Streptococcus sobrinus B13 (d) and 6715 (g). Whole cells of mutans streptococci or dental plaque was extracted with a mixture of 8M sodium nitrite (5 microliters) and 2M acetic acid (5 microliters) for five minutes and neutralized with 2M sodium hydroxide (10 microliters), and the extract and the sensitized latex suspension (20 microliters) were mixed and the grade of agglutination reaction was read macroscopically after ten minutes standing at room temperature. The LA tests could detect 1.0 10ng of purified serotype antigen and 10(5)-10(6) CFU of live cells of mutans streptococci and specifically distinguish not only the mutans streptococci from the other streptococci but also S. mutans from S. sobrinus. However, cross-reactions were still observed among the serotypes c, e and f of S. mutans or between the serotypes d and g of S. sobrinus. Plaque samples were collected from 168 children (2 to 12 years of age) and the 0.1 mg (wet weight) were applied to the LA tests. At the same time, the total number of mutans streptococci in plaque and the serotypes of each isolate were determined. The results of LA reaction correlated significantly with the number of mutans streptococci in plaque (chi-square analysis; p less than 0.0001). The LA tests discriminated between S. mutans and S. sobrinus in dental plaque. It was found that the latex particles sensitized with anti-serotype c and/or e S. mutans antibodies were most effective in demonstrating mutans streptococci, and they were used in the following studies. The

  7. 改进试管凝集试验法检测布鲁氏菌病抗体%Detection of Anti-Brucella abortus Sera Agglutination Titers with Modified Tube Agglutination Test

    Institute of Scientific and Technical Information of China (English)

    李翠; 关孚时; 戴志红; 蒋卉; 温芳; 陆连寿; 王在时

    2012-01-01

    In order to prove the use value of the modified tube agglutination test,six anti-Brucella abortus sera were detected with MSAT and SAT referred to the international standard.The results showed that the sera titers were almost consistent detected with SAT and MSAT and the advantages of MSAT could be list as follows: The dilution method was simpler and more accurate.We could save time and effort,and save the materials such as serum,antigen etc.The result was easier to determine and with good reproducibility.%为证实改进的试管凝集试验(SAT)的使用价值,分别用微量试管凝集试验(MSAT)和SAT以国际标准血清为参比检测6份牛布鲁氏菌病阳性血清的抗体效价。结果表明:MSAT和SAT试验结果一致,而且MSAT具有稀释方法更简便、精确,省时省力,可节约血清、抗原等试验原材料,试验结果容易判定,重现性良好等优越性。

  8. 猪血球粉中血红素提取工艺的研究%Study on the extraction of hemoglobin from pig hemocyte podwer

    Institute of Scientific and Technical Information of China (English)

    皮桃英; 陈博; 马宇峰; 张革; 谢伟全; 何光富; 余蓉

    2011-01-01

    探索猪血球粉中血红素的提取方法.方法采用单因素设计考察不同影响因素对猪血球粉中血红素提取的影响,在此基础上再采用正交设计筛选,优化得到最佳工艺为:8.0 g猪血球粉经4mol·L-1脲溶液溶解,用体积比2.0-2.5:1的60mL丙酮水溶液沉淀,用70%的丙酮水溶液分次洗涤沉淀后,采用盐酸丙酮法进行提取得到血红素.结果每克猪血球粉中可获得22.3 mg产品,收率为93.9%,血红素的纯度为96.1%±0.1%,其血红素中铁的含量为7.67% ±0.13%.结论工艺简单,生产周期短,血红素的提取率高,血红素中铁的含量明显提升,为实际生产中直接从血球粉中提取血红素和实现猪血浆中有效成分的联产提供了一条有价值的提取工艺.%OBJECTIVE To study the extraction of hemoglobin from pig hemocyte powder. METHODS The effects of different factors on the extraction of hemoglobin from pig hemocyte powder were studied by single factor investigation. On the basis of single factor investigation, the process was optimized by orthogonal design resulting in the optimal process as follows ;8.0 g pig hemocyte powder were dissolved in 4 mol·L-1 urea solution understirring. Then heme was ostained by precipitation of the solution using a mixture of acetone and water( 2.0 - 2.5 : 1) , washing the precipitate for two times with 70% acetone - water solution, and by extraction from the precipitate using Hcl - acetone method. RESULTS The heme obtained from each gram dried pig hemocyte powder was 22.3 mg. The yield of heme was 93.9% with purity and heme iron content of 96.1% ±0. 1% and 7.67% ±0. 13% .respectively. CONCLUSION This extraction process was simple,short in production period,could enhanced the yield and the heme iron content. It has realized cogenera-tion of heme and the effective component from pig blood plasma and provided a reference craft to withdraws the heme for the practical production from the blood cell powder.

  9. A latex agglutination test for the field determination of abnormal vitellogenin production in male fishes contaminated by estrogen mimics

    Energy Technology Data Exchange (ETDEWEB)

    Magalhaes, Ilizabete [Laboratoire d' Immunologie-Microbiologie (LIM/ESE-CNRS, unite FRE2635), IUT de Thionville-Yutz, 1, Impasse A Kastler, F-57970 Yutz (France); Pihan, Jean-Claude [Laboratoire de Production des Ecosystemes et Ecotoxicologie (LBFE), UFR SciFa, campus Bridoux, rue du G Delestraint, F-57070 Metz (France); Falla, Jairo [Laboratoire d' Immunologie-Microbiologie (LIM/ESE-CNRS, unite FRE2635), IUT de Thionville-Yutz, 1, Impasse A Kastler, F-57970 Yutz (France)

    2004-06-09

    Estrogen mimics are pollutants present in the aquatic environment. These compounds induce abnormalities in the reproductive system of male fishes, which lead to a total or partial male feminization, or to their demasculinization. Ultimately, these alterations could lead to a disappearance of the total contaminated fish population. Moreover, these toxic substances possess the capacity to mimic endogenous estrogens and to induce the abnormal production of vitellogenin (VTG) in male and immature fishes. The purpose of this research was to develop an easy, specific, cheap and fast method for diagnosing the contamination of male fishes by estrogen mimics, using VTG as biomarker. The selected method is based on a reverse latex agglutination test (rLAT), developed with monoclonal antibodies specific of this biomarker. The development of this VTG-rLAT has involved, firstly, the purification of carp VTG to produce monoclonal antibodies, specifics of this protein. One of these antibodies was selected to recover latex particles (diameter: 1 {mu}m). Finally, the immunoreactivity of the VTG-rLAT was verified with different fish plasma samples from males treated with 17{beta}-estradiol and non-treated males or females in vitellogenesis.

  10. Establishment Brucella micro agglutination test%布鲁氏菌微量凝集试验的建立

    Institute of Scientific and Technical Information of China (English)

    张双宅

    2012-01-01

    This paper expounded the development of Brucella micro agglutination test methods. At first, we used 96 hole V type blood coagulation plate, 12 tip needle connecting latex drop head for pipetting and then carried out test Now, we used sophisticated 12 adjustable micropipettor, one -time suction head and 96 bore hole type U reaction plate for setting up a convenience, shortcut, time-saving, labor-saving, specimen -saving, reagent - saving brucellosis surveillance and inspection method.%本文阐述了布鲁氏菌微量凝集试验的演变过程,从最初应用96孔V型血凝板、12号去尖针头接乳胶滴头为移液器进行试验,到应用精密的12道可调微量移液器、一次性吸头和96孔一次性U型反应板,建立了一种操作方便、快捷,省时、省力、省标本、省试剂的布鲁氏菌病监测检验方法.

  11. Sero-epidemiology of equine toxoplasmosis using a latex agglutination test in the three metropolises of Punjab, Pakistan.

    Science.gov (United States)

    Saqib, M; Hussain, M H; Sajid, M S; Mansoor, M K; Asi, M N; Fadya, A A K; Zohaib, A; Sial, A U R; Muhammad, G; Ullah, I

    2015-06-01

    Toxoplasmosis is a serious threat for livestock in addition to being of zoonotic significance. In this study, serodiagnosis of equine toxoplasmosis was conducted in a randomly selected population from the 3 metropolises of Punjab, Pakistan. To this end, 272 draught equines were screened using a commercial latex agglutination assay kit. Association of probable risk factors of equine toxoplasmosis was also documented. A total of 91 (33.5%) equines were found sero-positive for Toxoplama (T.) gondii having antibody titers ranging between 1:32 to 1:612. The highest rates of seropositive cases were observed in donkeys (58.7%) followed by mules (28.6%) and horses (23.5%). Age, sex and species of draught equines were found not to be statistically (p>0.05) associated with the distribution of T. gondii antibodies. The results of the study provided a baseline data for the exposure of equine population in this area. In addition, it is recommended that the contiguous population of domestic ruminants and possible reservoirs such as feral cats should be screened in order to explore the potential risk for the human population in Pakistan.

  12. Comparative evaluation of Rose Bengal plate agglutination test, mallein test, and some conventional serological tests for diagnosis of equine glanders.

    Science.gov (United States)

    Naureen, Abeera; Saqib, Muhammad; Muhammad, Ghulan; Hussain, Muhammad H; Asi, Muhammad N

    2007-07-01

    The Rose Bengal plate agglutination test (RBT) was evaluated for the diagnosis of equine glanders, and its diagnostic efficiency was compared with that of mallein and other serological tests, including indirect hemagglutination test (IHAT), complement fixation test (CFT), and modified counter immunoelectrophoresis test (mCIET). Sera from 70 naturally infected culture-positive, 96 potentially exposed cohorts, and 110 healthy equines were tested. All tests but mCIET showed 100% specificity when testing the sera from glanders-negative equines. The calculated sensitivities of RBT, IHAT, CFT, mCIET, and mallein test when testing culture-positive equines were 90.0, 97.1, 91.4, 81.4, and 75.7%, respectively. The RBT was significantly (P glandered and nonglandered animals, the highest agreement (0.987) was found between RBT and CFT followed by RBT and IHAT (0.940), RBT and mallein test (0.871), and RBT and mCIET (0.852). Because the RBT is simpler and rapid to perform, the inclusion of the test as a supplementary test for the diagnosis of glanders in field conditions is recommended.

  13. Safety studies of sperm agglutinating factor produced by Staphylococcus aureus as a vaginal contraceptive: in vivo studies.

    Science.gov (United States)

    Kaur, Siftjit; Prabha, Vijay; Kaur, Kiranjeet

    2011-11-01

    Sperm agglutinating factor (SAF) isolated from Staphylococcus aureus when applied at concentration 10 μg before mating completely prevented conception in the mouse. The objective of the present study was to evaluate its safety, as safety is an important concern to be addressed before a compound is selected for contraceptive use. Our results showed that SAF has a very high safety profile. Vaginal application of SAF at 10 μg to the mouse for 14 consecutive days caused no systemic toxicity and vaginal irritation as indicated by lack of effect on organ weights and histology. Moreover, no adverse effect was observed on the subsequent reproductive capability, perinatal outcome and growth and development of the offspring. SAF (10 μg) did not irritate the skin or penile mucosa. Oral administration of 2 mg/kg body weight of SAF did not show any toxicity to reproductive and non-reproductive organs. Therefore, SAF with spermicidal activity and lack of toxicity may have the potential to become the active ingredient of a vaginal contraceptive.

  14. Use of RBC-O and S-MCV parameters of SYSMEX XE-2100 in a patient with RBC cold agglutination.

    Science.gov (United States)

    Wang, Hong; Lu, Lin; Zhou, Yun; Liu, Jian; Qian, Min; Tang, Weiming; Jie, Zhang; Pan, Shiyang

    2013-01-01

    Sometimes EDTA blood of erythrocyte agglutination cannot be well resolved by incubation at 37 degrees C. In this case report, however, such a specimen was detected from a lymphoma patient at room temperature by using RBC-O and S-MCV parameters of the SYSMEX XE-2100 hematology analyzer. The specimen was diluted with 0.9% NaCL solution at 1:1 before measurement. HCT, MCV, and MCHC, corrected by RBC-O, HGB and S-MCV, were all in their normal ranges. This case indicates that RBC-O and S-MCV parameters of XE-2100 can be used in the routine blood examination of erythrocyte agglutination specimen at room temperature.

  15. COMPARATIVE STUDY ON EFFICACY OF ROSE BENGAL PLATE TEST (I{BPT AND SERUM AGGLUTINATION TEST (SAT FOR DETECTING THE INCIDENE OF BRUCELLOSIS IN BUFFALOES (Bubolus bubolis

    Directory of Open Access Journals (Sweden)

    Idrees Ali Zahid, Ishtiaq Ahmad and Umer Hayat

    2002-03-01

    Full Text Available Rose Bengal Plate Test (RBPT and Serum Agglutination Test (SAT were applied for the diagnosis of brucellosis in 240 buffaloes maintained at organized livestock farms in Punjab, to measure their comparative efficacy. Based on RBPT and SAT, 11.25 (n=27 and 10.42 percent (n=25 buffaloes were found seropositive, 11.67 (n 28 and 4.58 percent (n= 11 animals showed doubtful results, while 77.08 (n= 185 and 85 percept (n= 204 animals were found seronegative, respectively. Rose Bengal Plate Test detected higher percentages of seropositive, doubtful and seronegative cases than those detected by Serum Agglutination Test, which showed lower percentages or seropositive, doubtful and seronegative cases. This study indicated that SAT is more sensitive and reliable diagnostic test for the detection of Brucella aborlus, antibodies in buffaloes.

  16. Rapid effects of a protective O-polysaccharide-specific monoclonal IgA on Vibrio cholerae agglutination, motility, and surface morphology.

    Science.gov (United States)

    Levinson, Kara J; De Jesus, Magdia; Mantis, Nicholas J

    2015-04-01

    2D6 is a dimeric monoclonal immunoglobulin A (IgA) specific for the nonreducing terminal residue of Ogawa O-polysaccharide (OPS) of Vibrio cholerae. It was previously demonstrated that 2D6 IgA is sufficient to passively protect suckling mice from oral challenge with virulent V. cholerae O395. In this study, we sought to define the mechanism by which 2D6 IgA antibody protects the intestinal epithelium from V. cholerae infection. In a mouse ligated-ileal-loop assay, 2D6 IgA promoted V. cholerae agglutination in the intestinal lumen and limited the ability of the bacteria to associate with the epithelium, particularly within the crypt regions. In vitro fluorescence digital video microscopy analysis of antibody-treated V. cholerae in liquid medium revealed that 2D6 IgA not only induced the rapid (5- to 10-min) onset of agglutination but was an equally potent inhibitor of bacterial motility. Scanning electron microscopy showed that 2D6 IgA promoted flagellum-flagellum cross-linking, as well as flagellar entanglement with bacterial bodies, suggesting that motility arrest may be a consequence of flagellar tethering. However, monovalent 2D6 Fab fragments also inhibited V. cholerae motility, demonstrating that antibody-mediated agglutination and motility arrest are separate phenomena. While 2D6 IgA is neither bactericidal nor bacteriostatic, exposure of V. cholerae to 2D6 IgA (or Fab fragments) resulted in a 5-fold increase in surface-associated blebs, as well an onset of a wrinkled surface morphotype. We propose that the protective immunity conferred by 2D6 IgA is the result of multifactorial effects on V. cholerae, including agglutination, motility arrest, and possibly outer membrane stress.

  17. Rapid Effects of a Protective O-Polysaccharide-Specific Monoclonal IgA on Vibrio cholerae Agglutination, Motility, and Surface Morphology

    Science.gov (United States)

    Levinson, Kara J.; De Jesus, Magdia

    2015-01-01

    2D6 is a dimeric monoclonal immunoglobulin A (IgA) specific for the nonreducing terminal residue of Ogawa O-polysaccharide (OPS) of Vibrio cholerae. It was previously demonstrated that 2D6 IgA is sufficient to passively protect suckling mice from oral challenge with virulent V. cholerae O395. In this study, we sought to define the mechanism by which 2D6 IgA antibody protects the intestinal epithelium from V. cholerae infection. In a mouse ligated-ileal-loop assay, 2D6 IgA promoted V. cholerae agglutination in the intestinal lumen and limited the ability of the bacteria to associate with the epithelium, particularly within the crypt regions. In vitro fluorescence digital video microscopy analysis of antibody-treated V. cholerae in liquid medium revealed that 2D6 IgA not only induced the rapid (5- to 10-min) onset of agglutination but was an equally potent inhibitor of bacterial motility. Scanning electron microscopy showed that 2D6 IgA promoted flagellum-flagellum cross-linking, as well as flagellar entanglement with bacterial bodies, suggesting that motility arrest may be a consequence of flagellar tethering. However, monovalent 2D6 Fab fragments also inhibited V. cholerae motility, demonstrating that antibody-mediated agglutination and motility arrest are separate phenomena. While 2D6 IgA is neither bactericidal nor bacteriostatic, exposure of V. cholerae to 2D6 IgA (or Fab fragments) resulted in a 5-fold increase in surface-associated blebs, as well an onset of a wrinkled surface morphotype. We propose that the protective immunity conferred by 2D6 IgA is the result of multifactorial effects on V. cholerae, including agglutination, motility arrest, and possibly outer membrane stress. PMID:25667263

  18. Latex agglutination using the periplasmic proteins antigen of Brucella melitensis is a successful, rapid, and specific serodiagnostic test for ovine brucellosis.

    Science.gov (United States)

    Ismael, Alaa Bassuny; Swelum, Ayman Abdel-Aziz; Mostafa, Salama A-H; Alhumiany, Abdel-Rahman A

    2016-09-01

    Brucellosis, especially caused by Brucella melitensis, is considered the most-widespread zoonosis in the world, particularly in developing countries. This study was planned to develop an accurate test for diagnosis of ovine brucellosis using a specific hot saline extracted soluble Brucella melitensis periplasmic proteins (SBPPs). The efficacy of the latex agglutination test (LAT) using SBPPs compared to the Rose Bengal test (RBT), buffered plate agglutination test (BPAT), serum agglutination test (SAT), and an indirect enzyme-linked immunosorbent assay (i-ELISA) was evaluated in the field diagnosis of ovine brucellosis. The test performance was evaluated by estimating sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), disease prevalence (DP), positive likelihood ratio (PLR), and negative likelihood ratio (NLR) using test agreement and bacteriological culture in 1777 samples. The false-positive result was significantly (P ⩽0.05) lower in LAT than RBT, BPAT, SAT, and i-ELISA. With reference to test agreement, the Se, Sp, PPV, and PLR were highest (P ⩽0.05) in LAT 99.33%, 99.88%, 98.68%, and 827.25%, respectively. With reference to bacteriological culture, the LAT and i-ELISA tests showed a significant difference in Se with SAT. However, no significant difference in specificity was detected. The DP was 8.44% in the five tests. In conclusion, LAT using SBPPs of B. melitensis could be a suitable serodiagnostic field test for ovine brucellosis, with high sensitivity and specificity.

  19. Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies.

    Science.gov (United States)

    Klaus, Tomasz; Bzowska, Monika; Kulesza, Małgorzata; Kabat, Agnieszka Martyna; Jemioła-Rzemińska, Małgorzata; Czaplicki, Dominik; Makuch, Krzysztof; Jucha, Jarosław; Karabasz, Alicja; Bereta, Joanna

    2016-08-03

    Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab')2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the μ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool.

  20. Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies

    Science.gov (United States)

    Klaus, Tomasz; Bzowska, Monika; Kulesza, Małgorzata; Kabat, Agnieszka Martyna; Jemioła-Rzemińska, Małgorzata; Czaplicki, Dominik; Makuch, Krzysztof; Jucha, Jarosław; Karabasz, Alicja; Bereta, Joanna

    2016-01-01

    Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab′)2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the μ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool. PMID:27484487

  1. Lab-on-a-disc agglutination assay for protein detection by optomagnetic readout and optical imaging using nano- and micro-sized magnetic beads.

    Science.gov (United States)

    Uddin, Rokon; Burger, Robert; Donolato, Marco; Fock, Jeppe; Creagh, Michael; Hansen, Mikkel Fougt; Boisen, Anja

    2016-11-15

    We present a biosensing platform for the detection of proteins based on agglutination of aptamer coated magnetic nano- or microbeads. The assay, from sample to answer, is integrated on an automated, low-cost microfluidic disc platform. This ensures fast and reliable results due to a minimum of manual steps involved. The detection of the target protein was achieved in two ways: (1) optomagnetic readout using magnetic nanobeads (MNBs); (2) optical imaging using magnetic microbeads (MMBs). The optomagnetic readout of agglutination is based on optical measurement of the dynamics of MNB aggregates whereas the imaging method is based on direct visualization and quantification of the average size of MMB aggregates. By enhancing magnetic particle agglutination via application of strong magnetic field pulses, we obtained identical limits of detection of 25pM with the same sample-to-answer time (15min 30s) using the two differently sized beads for the two detection methods. In both cases a sample volume of only 10µl is required. The demonstrated automation, low sample-to-answer time and portability of both detection instruments as well as integration of the assay on a low-cost disc are important steps for the implementation of these as portable tools in an out-of-lab setting.

  2. Traces of dissolved particles, including coccoliths, in the tests of agglutinated foraminifera from the Challenger Deep (10,897 m water depth, western equatorial Pacific)

    Science.gov (United States)

    Gooday, A. J.; Uematsu, K.; Kitazato, H.; Toyofuku, T.; Young, J. R.

    2010-02-01

    We examined four multilocular agglutinated foraminiferan tests from the Challenger Deep, the deepest point in the world's oceans and well below the depth at which biogenic and most detrital minerals disappear from the sediment. The specimens represent undescribed species. Three are trochamminaceans in which imprints and other traces of dissolved agglutinated particles are visible in the orange or yellowish organic test lining. In Trochamminacean sp. A, a delicate meshwork of organic cement forms ridges between the grain impressions. The remnants of test particles include organic structures identifiable as moulds of coccoliths produced by the genus Helicosphaera. Their random alignment suggests that they were agglutinated individually rather than as fragments of a coccosphere. Trochamminacean sp. C incorporates discoidal structures with a central hole; these probably represent the proximal sides of isolated distal shields of another coccolith species, possibly Hayaster perplexus. Imprints of planktonic foraminiferan test fragments are also present in both these trochamminaceans. In Trochamminacean sp. B, the test surface is densely pitted with deep, often angular imprints ranging from roughly equidimensional to rod-shaped. The surfaces are either smooth, or have prominent longitudinal striations, probably made by cleavage traces. We presume these imprints represent mineral grains of various types that subsequently dissolved. X-ray microanalyses reveal strong peaks for Ca associated with grain impressions and coccolith remains in Trochamminacean sp. C. Minor peaks for this element are associated with coccolith remains and planktonic foraminiferan imprints in Trochamminacean sp. A. These Ca peaks possibly originate from traces of calcite remaining on the test surfaces. Agglutinated particles, presumably clay minerals, survive only in the fourth specimen (' Textularia' sp.). Here, the final 4-5 chambers comprise a pavement of small, irregularly shaped grains with flat

  3. Outbreak of uncommon O4 non-agglutinating Salmonella typhimurium linked to minced pork, Saxony-Anhalt, Germany, January to April 2013.

    Directory of Open Access Journals (Sweden)

    Katja Alt

    Full Text Available In January 2013, the National Reference Centre for Salmonella (NRC detected a salmonellosis cluster in Saxony-Anhalt, Germany, caused by uncommon O4 non-agglutinating, monophasic Salmonella (S. Typhimurium DT193. Circulating predominant monophasic S. Typhimurium DT193 clones typically display resistance phenotype ASSuT. We investigated common exposures to control the outbreak, and conducted microbiological investigations to assess the strains' phenotype.We conducted a case-control study defining cases as persons living or working in Saxony-Anhalt diagnosed with the O4 non-agglutinating strain between January and March 2013. We selected two controls contemporarily reported with norovirus infection, frequency-matched on residence and age group, per case. We interviewed regarding food consumption, especially pork and its place of purchase. We calculated odds ratios (ORs with 95% confidence intervals (95% CI using logistic regression. The NRC investigated human and food isolates by PCR, SDS-PAGE, MLST, PFGE, MLVA and susceptibility testing.Altogether, 68 O4 non-agglutinating human isolates were confirmed between January and April 2013. Of those, 61 were assigned to the outbreak (median age 57 years, 44% female; 83% cases ≥ 60 years were hospitalized. Eating raw minced pork from butcheries within 3 days was associated with disease (31 cases, 28 controls; OR adjusted for sex: 3.6; 95% CI: 1.0-13. Phage type DT193 and MLST ST34 were assigned, and isolates' lipopolysaccharide (LPS matched control strains. Isolates linked to Saxony-Anhalt exhibited PFGE type 5. ASSuT- and ACSSuT phenotype proportions were 34 and 39% respectively; 54% were resistant to chloramphenicol. Three pork isolates matched the outbreak strain.Raw minced pork was the most likely infection vehicle in this first reported outbreak caused by O4 non-agglutinating, mostly chloramphenicol-resistant S. Typhimurium DT193. High hospitalization proportions demand awareness on the risk of

  4. High influx of carbon in walls of agglutinated foraminifers during the Permian-Triassic transition in global oceans

    Science.gov (United States)

    Nestell, Galina P.; Nestell, Merlynd K.; Ellwood, Brooks B.; Wardlaw, Bruce R.; Basu, Asish R.; Ghosh, Nilotpal; Phuong Lan, Luu Thi; Rowe, Harry D.; Hunt, Andrew G.; Tomkin, Jonathan H.; Ratcliffe, Kenneth T.

    2015-01-01

    The Permian–Triassic mass extinction is postulated to be related to the rapid volcanism that produced the Siberian flood basalt (Traps). Unrelated volcanic eruptions producing several episodes of ash falls synchronous with the Siberian Traps are found in South China and Australia. Such regional eruptions could have caused wildfires, burning of coal deposits, and the dispersion of coal fly ash. These eruptions introduced a major influx of carbon into the atmosphere and oceans that can be recognized in the wallstructure of foraminiferal tests present in survival populations in the boundary interval strata. Analysis of free specimens of foraminifers recovered from residues of conodont samples taken at aPermian–Triassic boundary section at Lung Cam in northern Vietnam has revealed the presence of a significant amount of elemental carbon, along with oxygen and silica, in their test wall structure, but an absence of calcium carbonate. These foraminifers, identified as Rectocornuspira kalhori, Cornuspira mahajeri, and Earlandia spp. and whose tests previously were considered to be calcareous, are confirmed to be agglutinated, and are now referred to as Ammodiscus kalhori and Hyperammina deformis. Measurement of the 207Pb/204Pb ratios in pyrite clusters attached to the foraminiferal tests confirmed that these tests inherited the Pb in their outer layer from carbon-contaminated seawater. We conclude that the source of the carbon could have been either global coal fly ash or forest fire-dispersed carbon, or a combination of both, that was dispersed into the Palaeo-Tethys Ocean immediately after the end-Permian extinction event.

  5. Application of Direct Agglutination Test (DAT for the Diagnosis and Seroepide-miological Studies of Visceral Leishmaniasis in Iran

    Directory of Open Access Journals (Sweden)

    S Charehdar

    2006-08-01

    Full Text Available Visceral leishmaniasis (VL is one of the most important parasitic diseases which is endemic in different parts of Iran. Serological studies were conducted by direct agglutination test (DAT on 12144 human serum samples, collected from four geographical zones of Iran. Sero prevalence, geographical distribution, clinical signs and symptoms for human visceral leishmaniasis based on DAT for the period of 2002 through 2005 were determined. From 516 kala-azar cases detected: 50.6% were from Meshkin-shahr and Moghan districts in Ardabil Province, northwest of Iran and 49.4% were detected from other areas of Iran. In physical examination of seropositive cases, which were detected by DAT with anti-leishmanial antibodies at titers of 1: 3200 to 1: 102400, almost 50% of suspected individuals showed the classical kala-azar signs and symptoms. Predominant signs and symptoms in 233 hospitalized patients with anti-Leishmania antibodies at 1:3200 and higher, were fever (88.0% and splenomegaly (84.5%. Statistically significant difference was found between males (58% and females (42% (P< 0.01. Moreover, 93.6% of the VL patients were < 5 yr of age, and 6.4% were older than 5 yr that this difference was statistically significant (P< 0.01. From 1383 serum samples collected from domestic dogs in the villages that are known as endemic foci of human leishmaniasis, 152 (11.0% were positive by DAT (≥ 1:320. Parasitological and serological examinations that were performed in 30 wild canines showed that 10% of these animals were infected by L. infantum. L. infantum Lon49 is the principal agent of the disease in human as well as animal reservoir hosts in different parts of Iran. For the first time in Iran, L. tropica isolated from both skin lesions in the face and bone marrow aspiration in a HIV+ man who co-infected with VL as well as in an infected dog from Ardabil Province.

  6. Effects of Avermectin on DNA Variation in Hemocytes of Silkworm%阿维菌素对家蚕血细胞DNA变异的影响

    Institute of Scientific and Technical Information of China (English)

    沈卫锋; 孟智启; 王强; 牛宝龙; 何丽华; 刘岩; 翁宏飚; 陈玉银

    2011-01-01

    利用RAPD标记技术检测杀虫剂阿维菌素对家蚕血细胞DNA的损伤,作为评价其对蚕体毒性的依据.分别用1、2、4、8 μg/L阿维菌素药液处理的桑叶给4龄起蚕添食,96 h后对家蚕血细胞的基因组DNA进行RAPD分析.24条RAPD引物对5个样品基因组DNA扩增产生的清晰条带数为143条,其中多态性片段19条,多态性带数比率为13.29%.用Ntsyspc 2.1软件计算出不同处理样品血细胞DNA间的遗传相似系数分布在0.867~0.993,树状聚类图显示正常样品与1、2、4μg/L阿维菌素药液处理的3个样品聚为一类,而8 μg/L阿维菌素药液处理的样品单独聚为一类,说明该样品血细胞DNA的变异最大.研究结果提示,RAPD标记作为杀虫剂对家蚕的毒性检测标志技术,可准确预警蚕区农药使用对养蚕生产存在的潜在危害.%DNA damage of silkworm (Bombyx mori) hemocytes detected by RAPD marker technology could be used as a reference for evaluating the toxicity of avermectin to silkworm. The 4th instar newly exuviated silkworm larvae were fed with mulberry leaves treated with 1,2,4, and 8 μg/L avermectin. After 96 hours, the genomic DNAs of silkworm hemocytes were isolated for RAPD assay. By using 24 RAPD primers to amplify the genomic DNAs of five samples, 143 clearly distinguishable bands were obtained, among which 19 bands were polymorphic, showing a polymorphism percentage of 13. 29%. Calculation with Ntsyspc 2. 1 software displayed that the genetic similarity coefficients between hemocytic DNAs from different treatments were in the range of 0. 867 to 0. 993. The result of dendrogram showed that the samples treated with 1,2, and 4 μg/L avermectin and the normal sample were clustered into one group, and the sample treated with 8 μg/L avermectin was in a separate group, indicating that 8 μg/L of avermectin caused the most serious effect on DNA integrality. These results showed that RAPD molecular marker technology, as a measure of

  7. Microbead agglutination based assays

    KAUST Repository

    Castro, David

    2013-11-28

    A method for detecting the presence of an analyte in a sample can include adding a plurality of microparticles of a first-type to the sample, where each microparticle of the first-type includes a first binding partner configured to interact with at least a first portion of the analyte, adding a plurality of microparticles of a second-type to the sample, where each microparticle of the second-type includes a second binding partner configured to interact with at least a second portion of the analyte, the first portion of the analyte being different from the second portion of the analyte, and identifying an aggregate including at least one microparticle of the first-type, at least one microparticle of the second-type and the analyte, where the aggregate indicates the presence of the analyte.

  8. Effects of Heavy Metal Ions (Cu2+, Pb2+ and Cd2+) on DNA Damage of the Gills, Hemocytes and Hepatopancreas of Marine Crab, Charybdis japonica

    Institute of Scientific and Technical Information of China (English)

    PAN Luqing; LIU Na; ZHANG Hongxia; WANG Jing; MIAO Jingjing

    2011-01-01

    There are rising concerns about the hazardous effects of heavy metals on the environment. In this study, comet assay and DNA alkaline unwinding assay were conducted on the tissues (gills, hepatopancreas, and hemocytes) of Charybdis japonica in order to illustrate genotoxicity of three heavy metal ions (Cu2+, Pb2+, and Cd2+) on the marine crabs C. japonica. The crabs were exposed to Cu2+ (10, 50, and 100 μgL-1), Pb2+ (50, 250, and 500 μgL-1) and Cd2+ (5, 25, and 50μgL-1), and the tissues were sampled at days 0.5,1, 3, 6, 9, and 15. DNA alkaline unwinding assay was used for testing the DNA single strand break in gills and hepatopancreas and comet assay was employed for testing the DNA damage in hemocytes. The results showed that the DNA damage (F-value) of gills in the crabs exposed to the three heavy metals was decreased gradually during the exposure periods and there was a dose-time response relationship in certain time, suggesting that the levels of DNA single strand break in all the experimental groups increased significantly compared to the controls. Changes of F-value in hepatopancreas of the crabs exposed to the three heavy metals were similar to those in gills except that the peak values were found in the 500 μg L-1 Pb2+ treatment group at day 3 and the 50 μg L-1 Cd2+treatment group at day 9. The ranks of DNA damage in gills and hepatopancreas induced by the three heavy metal ions (50μgL-1,day 15) were Cd2+ >Pb2+ >Cu2+ and Pb2+ >Cu2+ >Cd2+. The levels of DNA damage in gills were higher than those in hepatopancreas in the same experimental group. It can be concluded that indices of DNA damage can be used as the potential biomarkers of heavy metal pollution in marine environment.

  9. Gene expression profiling of Spodoptera frugiperda hemocytes and fat body using cDNA microarray reveals polydnavirus-associated variations in lepidopteran host genes transcript levels

    Directory of Open Access Journals (Sweden)

    Feyereisen R

    2006-06-01

    Full Text Available Abstract Background Genomic approaches provide unique opportunities to study interactions of insects with their pathogens. We developed a cDNA microarray to analyze the gene transcription profile of the lepidopteran pest Spodoptera frugiperda in response to injection of the polydnavirus HdIV associated with the ichneumonid wasp Hyposoter didymator. Polydnaviruses are associated with parasitic ichneumonoid wasps and are required for their development within the lepidopteran host, in which they act as potent immunosuppressive pathogens. In this study, we analyzed transcriptional variations in the two main effectors of the insect immune response, the hemocytes and the fat body, after injection of filter-purified HdIV. Results Results show that 24 hours post-injection, about 4% of the 1750 arrayed host genes display changes in their transcript levels with a large proportion (76% showing a decrease. As a comparison, in S. frugiperda fat body, after injection of the pathogenic JcDNV densovirus, 8 genes display significant changes in their transcript level. They differ from the 7 affected by HdIV and, as opposed to HdIV injection, are all up-regulated. Interestingly, several of the genes that are modulated by HdIV injection have been shown to be involved in lepidopteran innate immunity. Levels of transcripts related to calreticulin, prophenoloxidase-activating enzyme, immulectin-2 and a novel lepidopteran scavenger receptor are decreased in hemocytes of HdIV-injected caterpillars. This was confirmed by quantitative RT-PCR analysis but not observed after injection of heat-inactivated HdIV. Conversely, an increased level of transcripts was found for a galactose-binding lectin and, surprisingly, for the prophenoloxidase subunits. The results obtained suggest that HdIV injection affects transcript levels of genes encoding different components of the host immune response (non-self recognition, humoral and cellular responses. Conclusion This analysis of the

  10. 东亚飞蝗血细胞形态学研究%Morphological study on hemocytes of Locusta migratoria manilensis

    Institute of Scientific and Technical Information of China (English)

    贾雷坡; 崔智芳; 王强; 吴昊; 张小民

    2011-01-01

    通过Wright's染色和光学显微镜对不同地区东亚飞蝗的血细胞进行了观察,发现东亚飞蝗Locusta migratoria manilensis(Meyen)的血细胞包括原血胞、浆血胞、粒血胞和类绛血胞4种类型.原血胞的细胞核为红色,而其它3种血细胞的细胞核均被Wright's染液染成红色和蓝色2种类型.故认为浆血胞是由原血胞分裂而来;浆血胞进一步分化为蓝核浆血胞和红核浆血胞,蓝核浆血胞进一步演化为蓝核粒血胞和蓝核类绛血胞;红核浆血胞进一步演化为红核粒血胞和红核类绛血胞.粒血胞又分化为大颗粒粒血胞和小颗粒粒血胞,小颗粒粒血胞又进一步分化为密集小颗粒粒血胞和稀少小颗粒粒血胞,由此将东亚飞蝗的血细胞分为4类11种.该研究不仅为蝗虫形态学提供依据,同时也为蝗虫生理、蝗虫免疫,以及防蝗治蝗提供依据.%Light microscopy and the Wright stain method revealed four types of hemocytes; prohaemocytes,plasmatocytes, granulocytes and oenocytoids, in Locusta migratoria manilensis (Meyen). Prohaemocytes' had a red nucleus, whereas the nucleus of the other three haemocytes was dyed red and blue by Wright' s dye. Plasmatocytes are considered to be disintegrated prohaemocytes and can be further differentiated into red-nucleus plasmatocytes and bluenucleus plasmatocytes. Blue-nucleus plasmatocytes evolve into blue-nucleus granulocyte and oenocytoids whereas rednucleus plasmatocytes evolve into red-nucleus granulocyte and ocnocytoids. Granulocytes are differentiated into largegranular granulocytes and small-granular granulocytes, the latter are differentiated into exiguous and dense small granular granulocytes. Hemocytes of L. m. manilersi can therefore be divided into four main types with eleven subtypes. This study not only provides a basis for grasshopper morphology, but also grasshopper physiology, immunity and locust control.

  11. Genetic and mechanistic evaluation for the mixed-field agglutination in B3 blood type with IVS3+5G>A ABO gene mutation.

    Directory of Open Access Journals (Sweden)

    Ding-Ping Chen

    Full Text Available BACKGROUND: The ABO blood type B(3 is the most common B subtype in the Chinese population with a frequency of 1/900. Although IVS3+5G>A (rs55852701 mutation of B gene has been shown to associate with the development of B(3 blood type, genetic and mechanistic evaluation for the unique mixed-field agglutination phenotype has not yet been completely addressed. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we analyzed 16 cases of confirmed B(3 individuals and found that IVS3+5G>A attributes to all cases of B(3. RT-PCR analyses revealed the presence of at least 7 types of aberrant B(3 splicing transcripts with most of the transcripts causing early termination and producing non-functional protein during translation. The splicing transcript without exon 3 that was predicted to generate functional B(3 glycosyltransferase lacking 19 amino acids at the N-terminal segment constituted only 0.9% of the splicing transcripts. Expression of the B(3 cDNA with exon 3 deletion in the K562 erythroleukemia cells revealed that the B(3 glycosyltransferase had only 40% of B(1 activity in converting H antigen to B antigen. Notably, the typical mixed-field agglutination of B(3-RBCs can be mimicked by adding anti-B antibody to the K562-B(3 cells. CONCLUSIONS/SIGNIFICANCE: This study thereby demonstrates that both aberrant splicing of B transcripts and the reduced B(3 glycosyltransferase activity contribute to weak B expression and the mixed-field agglutination of B(3, adding to the complexity for the regulatory mechanisms of ABO gene expression.

  12. Misidentification of Vibrio cholerae O155 isolated from imported shrimp as O serogroup O139 due to cross-agglutination with commercial O139 antisera

    DEFF Research Database (Denmark)

    Dalsgaard, A.; Mazur, J.; Dalsgaard, Inger

    2002-01-01

    Fish and shellfish products imported into Denmark are routinely analyzed for pathogenic Vibrio spp., particularly Vibrio cholerae, if products originate from subtropical or tropical areas. A V. cholerae strain that agglutinated commercial O139 antiserum but not the O1, Inaba, or Ogawa antisera...... was isolated from imported raw frozen shrimp. The toxigenicity of the strain was analyzed, and the results of a polymerase chain reaction showed that the V. cholerae strain did not contain the virulence genes ctx, tcp9, and zot, which are normally found in V. cholerae O1 and O139. The strain was resistant...

  13. Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

    Science.gov (United States)

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Lee, Yong Seok; Kang, Sang Sun; Han, Yeon Soo

    2015-07-01

    Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor.

  14. A small ciliary surface glycoprotein of the monogenean parasite Neobenedenia girellae acts as an agglutination/immobilization antigen and induces an immune response in the Japanese flounder Paralichthys olivaceus.

    Science.gov (United States)

    Hatanaka, A; Umeda, N; Yamashita, S; Hirazawa, N

    2005-11-01

    The capsalid monogenean Neobenedenia girellae, a parasite of seawater fishes, was found to express an antigen that elicits antibodies in rabbits, and these antibodies had agglutination/immobilization activity against N. girellae larvae (oncomiracidia) in vitro. Indirect immunofluorescence staining of N. girellae oncomiracidia showed that this agglutination/immobilization antigen was expressed on the surface of cilia. An intraperitoneal injection of ciliary proteins (either sonicated or intact) with adjuvant also elicited agglutinizing/immobilizing antibodies in sera from Japanese flounder, Paralichthys olivaceus. These antisera showed a clear correlation between anti-ciliary antibody levels (measured by enzyme-linked immunosorbent assays) and their agglutination/immobilization activity. Anti-ciliary antibody levels in Japanese flounder reached a plateau at 39 days after booster immunization and were significantly higher in the two immunized groups (injection of sonicated or intact cilia) as compared with control fish (injection of bovine serum albumin; ANOVA, Tukey's test, P agglutination/immobilization antigen based on SDS-polyacrylamide gel electrophoresis and immunoblot analyses with rabbit and fish antisera.

  15. Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

    Directory of Open Access Journals (Sweden)

    Amanda Donnelly

    2015-12-01

    Full Text Available The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i provides enhanced phage-mediated gene transfer; (ii is applicable for laboratory transfection processes and (iii shows promise within industry for large-scale gene transfer applications.

  16. Bacteriophage-mediated acquisition of antibiotic resistance by Staphylococcus aureus type 88.

    OpenAIRE

    Schaefler, S.

    1982-01-01

    Antibiotic-resistant Staphylococcus aureus strains of phage type 88, lysogenic for phage 188, when grown in mixed culture with a nonlysogenic novobiocin-resistant strain, acquired novobiocin resistance at a high rate from the nonlysogenic strain. With most strains of phage type 88, there was no detectable transfer of resistance from lysogenic to nonlysogenic cells. Lysogenization with phage 188 of phage-sensitive strains conferred on the lysogenized cells the ability to acquire chromosome and...

  17. The secret life of the anthrax agent Bacillus anthracis: bacteriophage-mediated ecological adaptations.

    Science.gov (United States)

    Schuch, Raymond; Fischetti, Vincent A

    2009-08-12

    Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.

  18. The secret life of the anthrax agent Bacillus anthracis: bacteriophage-mediated ecological adaptations.

    Directory of Open Access Journals (Sweden)

    Raymond Schuch

    Full Text Available Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.

  19. Analysis of 1926 Samples of Brucellosis Serological Agglutination Test%1926份布鲁氏菌病血清学凝集试验结果分析

    Institute of Scientific and Technical Information of China (English)

    姜勇波; 李晓红; 孙宗祥; 孙力; 冯波

    2015-01-01

    In this paper,through the comparison of Rose Bengal plate agglutination test(RBPT)and serum agglutination test(SAT)in the diagnosis ofbrucel osis sensitivity and accuracy,draw a conclusion:Brucel a diagnosis in the application of two methods,two methods used at the same time can improve the detection rate of brucel osis,not a single confirmed by a method,and contact must be combined with epidemiological and clinical symptoms and signs seriously analysis,careful judgment.%本文通过比较虎红平板凝集试验(RBPT)和试管凝集试验(SAT)在诊断布鲁氏菌病的敏感性和准确性,得出结论:在应用两种方法进行布病诊断时,两种方法同时使用可提高布鲁氏菌病的检出率,不能单一通过一种方法就确诊,必须结合流行病学接触史和临床症状体征认真分析谨慎判断。

  20. [Agglutination and phagocytosis of foreign abiotic particles by bluebottle Calliphora vicina haemocytes in vivo. II. Influence of the previous septic immune induction on haemocytic activity].

    Science.gov (United States)

    Kind, T V

    2010-01-01

    The rate of Calliphora vicina haemocytic defense reaction to foreign particles injection depends on the larval age and on the previous bacterial immunization. Immunization of crop-empting larvae induces an evident increase in particles phagocytosis by juvenile plasmatocytes in 24 h after injection. Both the hemogram and the pattern of cellular defense reaction change significantly after crop-empting. Immunized larvae start intensive adhesion of foreign particles to plasmatocytes surface and formation of great aggregations of plasmatocytes (morules) no longer than in 34 min after injection. The period of particle-haemocyte adhesion is short-termed and no more than after 30 min cell aggregates dissociate and adhered charcoal particles pass to thrombocydoidal agglutinates. Unimmunized control larvae of the same age have shown no adhesion and morules formation. In immunized wadering and diapausing larvae, formation of capsules consisting of central thrombocydoidal agglutinate filled with alien particles and adherent plasmatocytes I is intensified. In contrast to moru-les, this capsule formation is not accompanied by charcoal particles adhesion to plasmatocytes. Immunization of mature larvae of C. vicina shown no prominent influence on both the rate of phagocytosis and the hyaline cells differentiation. It might be supposed that the receptors system is complex and the immunization both the mechanisms of foreigners recognition (adhesion, morulation and incapsulation) and the far more lately occurring phagocytosis.

  1. 牛布氏杆菌试管凝集试验不确定度研究%Study in Uncertainty of Tube Agglutination Assay of Brucellosis

    Institute of Scientific and Technical Information of China (English)

    李冰玲; 马贵平; 刘会英; 谷强; 刘全国; 李炎鑫; 史喜菊

    2012-01-01

    This study made an attempt to evaluate the uncertainty of tube agglutination assay.Transformation of the result from semi-quantitative to quantitative(turbidity) was made,so that the evaluation for uncertainty of a semi-quantitative or qualitative result is possible.The applied way of uncertainty of tube agglutination assay was proposed,so that the expression of the result is more reasonable and authentic.%对牛布氏杆菌病试管凝集试验的测量不确定度进行了评估。通过将定性的检测结果转化成浊度进行量化,从而实现对试管凝集试验检测结果不确定度的评估,并提出了试管凝集试验检测结果不确定度的应用方法,使得检测结果的表述更加科学,同时也是对该类试验测量结果不确定度评估的有益探索。

  2. Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India.

    Science.gov (United States)

    Deneke, Yosef; Sabarinath, T; Gogia, Neha; Lalsiamthara, Jonathan; Viswas, K N; Chaudhuri, Pallab

    2014-08-01

    Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira, causing febrile infection characterized by multi-organ failure in humans and animals. Leptospiral Ig-like protein B (LigB) is a surface-expressed antigen that mediates host cell invasion or attachment. In this study, N-terminal conserved region of LigB protein (46 kDa) was evaluated for its diagnostic potential to detect anti-leptospiral antibodies in the sera of various animal species. Dot blot analysis revealed immunoreactivity of Leptospira-positive sera of cattle, buffalo, dog, sheep and goat to purified LigB protein. We have analyzed 1126 bovine serum samples, collected from Northern and Eastern part of India, by microscopic agglutination test (MAT) and recombinant LigB (rLigB) based ELISA and latex agglutination test (LAT). The sensitivity of rLigB based ELISA for 554 MAT positive sera was 96.9% and the specificity with 572 MAT negative sera was 91.08% whereas LAT showed sensitivity and specificity of 93.68% and 92.31%, respectively. Kappa values of 0.879 and 0.860 for recombinant antigen based ELISA and LAT indicate excellent agreement with the gold standard serological test, MAT, for the detection of anti-leptospiral antibodies in sera. Further, LAT based on rLigB antigen is a simple and rapid test, suitable for serodiagnosis of leptospirosis under field conditions, owing to its portability and longer shelf life.

  3. A novel method for negating cold agglutination interference by dithiothreitol during complete blood count and peripheral blood smear: a case study

    Directory of Open Access Journals (Sweden)

    Hamit Yasar Ellidag

    2017-02-01

    Full Text Available Cold agglutinin disease is an autoimmune disorder that is characterized by antibodies attacking polysaccharide antigens on one’s own erythrocytes. In cold agglutinin disease, hemolysis occurs due to degeneration of erythrocyte membranes and autoagglutination of erythrocytes. Both autoagglutination and hemolysis alter many laboratory test results of the patients, especially complete blood count (CBC and peripheral blood smear analyses. In our laboratory, we have encountered a blood sample of a 65-years-old man, who was independently diagnosed with cold agglutinin disease, and failed to produce meaningful CBC or peripheral blood smear analysis. Fresh blood samples were taken from the patient, and CBC and peripheral blood smear analyses were repeated with and without water bath incubation at 37 °C. Also, varying amounts of dithio­threitol (DTT were added to fresh samples without heat treatment prior to blood analysis. Heat treatment at 37 °C for varying lengths of time failed to improve CBC and peripheral blood smear analysis. On the other hand, addition of DTT into a blood sample of 2 ml in a K3-EDTA tube was sufficient to negate cold agglutination interference during both CBC and peripheral blood smear analyses. The presented method supports the potential for DTT to be used in negating cold agglutination interference during CBC and peripheral blood smear analyses of patients with cold agglutinin disease. The method we describe is very easy and quick with remarkable results.

  4. COMPARISON STUDY BETWEEN IN-HOUSE IGM DOT-ELISA AND THE MICROSCOPIC AGGLUTINATION TEST (MAT FOR THE DIAGNOSIS OF HUMAN LEPTOSPIROSIS

    Directory of Open Access Journals (Sweden)

    Ganesh Kumar A

    2012-04-01

    Full Text Available An indirect enzyme-linked immunosorbent assay (ELISA was compared with the microscopic agglutination test (MAT for the diagnosis of bovine leptospirosis. Blood samples from a total number of 319 HBsAg negative suspected leptospirosis case’s were received from Government Hospital and from a few private hospitals of Salem district, Tamilnadu, India. The serum samples were examined for the presence of anti leptospiral antibodies using a commercial qualitative method of an in-house Dot-ELISA assay and the results were compared with WHO standard Microscopic Agglutination Test (MAT. The following interesting results were noted, 132 (41.7 % serum samples were positive to Dot-ELISA, while 130 (40.7 % were positive to MAT. All samples positive to MAT were positive to Dot-ELISA, on of the samples were positive for MAT and negative to Dot-ELISA. The Dot-ELISA showed 100% sensitivity compared to MAT. The current diagnostic Dot-ELISA appears as a rapid, non hazardous and better alternative to MAT for the diagnosis of human Leptospirosis.

  5. Survey of False-positive Reactivity of Latex Agglutination Test for Kala-azar (Katex) without Urine Sample Boiling Process in Autoimmune Patients.

    Science.gov (United States)

    Ghatee, Mohammad Amin; Kanannejad, Zahra; Sharifi, Iraj; Askari, Asma; Bamorovat, Mehdi

    2017-06-01

    Latex agglutination test for Kala-azar (KAtex) is an easy, inexpensive, and field-applicable antigen detection test. However, the main drawback of this method is the boiling step applied to remove false positivity of the test. This study was conducted to survey false positivity results of latex agglutination test for KAtex without boiling process in urine of some autoimmune patients. Ninety-two urine samples from autoimmune patients including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), scleroderma, autoimmune vasculitis, vitiligo, pemphigus and Wagner cases and 20 urine samples from healthy individuals were collected from Kerman Province in Southeastern Iran in 2010-2011. All urine samples were checked by KAtex after boiling for 5 min false positivity rate of the test was surveyed in different healthy and patients groups while boiling process was removed. Rheumatoid factor (RF) then was checked in sera of all cases to evaluate the relationship between RF and KAtex false positivity. All samples represented negative results with KAtex when boiling was performed (100% specificity). Then, 20% positivity was evident in healthy cases. False-positive reactivity was more prominent observed in patient groups than healthy individuals, except in vitiligo. However, a significant difference was only observed in RA group (Pboiling process removal.

  6. Evaluation of Western blot, ELISA and latex agglutination tests to detect Toxoplasma gondii serum antibodies in farmed red deer.

    Science.gov (United States)

    Patel, Kandarp Khodidas; Howe, Laryssa; Heuer, Cord; Asher, Geoffery William; Wilson, Peter Raymond

    2017-09-15

    Abortion due to Toxoplasma gondii has been suspected in New Zealand farmed red deer. However, knowledge around the epidemiology and prevalence of T. gondii in farmed red deer is limited. The aim of this study was to firstly, assess the sensitivity and specificity of two commercially available assays, ELISA and latex agglutination test (LAT), for use in deer and secondly, to estimate the sero-prevalence of T. gondii in red deer. A total of 252 sera from rising 2-year-old and adult hinds from 17 New Zealand red deer herds at early and late pregnancy scanning and from known aborted and/or non-aborted hinds were tested for the presence of T. gondii antibodies. Each assays' sensitivity and specificity was evaluated by both the Western Blot (WB) as a gold standard method and Bayesian latent class (BLC) analysis in the absence of a gold standard. The sensitivity and specificity for WB were 95.8% (95% credible interval: 89.5-99.2%) and 95.1% (95% credible interval: 90.6-98.1%), respectively. For the LAT at the manufacturer's recommended ≥1:32 cut-off titre, the sensitivity (88.7%, 95% credible interval: 80.8-94.7%) and specificity (74.3%, 95% credible interval: 67.5-80.5%) were lower and higher than the sensitivity (76.2%, 95% credible interval: 66.7-84.5%) and specificity (89.7%, 95% credible interval: 84.5-93.9%) at a ≥1:64 cut-off, using (BLC) analysis. Sensitivity and specificity of the LAT at cut-off titre of 1:32 were estimated to be 84.4% (95% CI: 74.9-90.9%) and 73.5% (95% CI: 65.8-79.9%) against WB. The LAT had better agreement with WB at cut-off titre of ≥1:64 than ≥1:32 (Kappa=0.63 vs 0.54). At optimised cut-off S/P of 15.5%, the sensitivity (98.8%, 95% credible interval 96.1-99.8%) and specificity (92.8%, 95% credible interval 88.9-95.7%) of the ELISA were higher and lower, respectively, than the sensitivity (85.1%, 95% credible interval 76.2-91.9%) and specificity (98.5%, 95% credible interval 96.9-99.4%) at manufacturer's cut-off S/P of 30%, from BLC

  7. The Preliminary Analysis of Agglutinating Activity of Seven Kinds of Genuine Medicinal Materials in Gansu%7种甘肃道地药材凝血活性初步分析

    Institute of Scientific and Technical Information of China (English)

    袁毅君; 赵丽; 焦成瑾; 陈荃

    2016-01-01

    Objective: To test the agglutinating activity about seven kinds of Genuine Medicinal Materials in Gansu react with the red blood cells of chicken and rabbit respectively. Methods: Selected seven medicinal herbs were ex⁃tracted with PBS, then extracts agglutinating activity were detected with the red blood cells from rabbit and chicken respectively, determined the lowest titer of the herbs. Results: The agglutination activities of the lectins varied in the seven herbs extracts, and the higher agglutinating activity of the herb lectins in the two different blood cells are fol⁃lows: the titer of extracts from Radix Saposhnikoviae with the rabbit red blood cells is 23; but the titer of extracts from Radix et Rhizoma Rhel with chicken red blood cells is 25. Conclusion: Seven kinds of Chinese herbs present better agglutinating activity, the same Chinese herbs has different agglutinating activity with different erythrocyte samples.%目的:检测7种甘肃道地药材对鸡、兔红细胞的凝集活性。方法:将7种药材分别用PBS (磷酸缓冲液)浸提,浸提液分别对兔和鸡红细胞进行凝集活性测定,以检测7种药材的最低效价。结果:7种中药浸提液对红细胞表现不同的凝集活性,具有较高凝集活性的为防风对兔红细胞效价23;大黄对鸡红细胞效价25.结论:7种中药均有不同的凝集活性,同种中药对不同红细胞的凝集活性不同。

  8. 对虾血蓝蛋白多聚结构与凝集活性%Differences in the agglutination activity of two oligomers of hemocyanin from Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    潘建义; 吴海港; 王浩波

    2011-01-01

    目的 分析凡纳对虾血蓝蛋白六聚体和十二聚体两种结构形式的凝集活性差异. 方法 制备凡纳对虾血清,采用阴离子交换层析法分离血蓝蛋白,SDS-PAGE、Native-PAGE和质谱分析其多聚结构,玻片法红细胞凝集试验测定血蓝蛋白的凝集作用. 结果 离子交换层析得到2个明显的蛋白质峰,经电泳和质谱分析,二者分别为血蓝蛋白的六聚体和十二聚体,且十二聚体可凝集人红细胞,而六聚体无凝集活性. 结论 凡纳对虾血蓝蛋白主要以十二聚体形式存在,且具有凝集活性.%Objective To investigate differences in the agglutination activity of two oligomers, a hexamer and a dodecamer, of hemocyanin from Litopenaeus vannamei.Methods Prepared shrimp sera were isolated by anion exchange chromatography, and obtained oligomers were identified by combined SDS-PAGE, Native-PAGE, and mass spectrometry analysis.A red blood cell agglutination test was used to analyze differences in the agglutination ability of the two oligomers.Results Two distinct protein peaks were obtained in ion exchange chromatography, and these peaks were identified as a hexamer and dodecamer of hemocyanin.The shrimp sera and dodecamer caused human red blood cells to agglutinate while the hexamer was not found to have agglutination activity.Conclusion The dodecamer of hemocyanin from L.vannamei is active under physiological conditions and has agglutination activity.

  9. Validation of reference genes for RT-qPCR in marine bivalve ecotoxicology: Systematic review and case study using copper treated primary Ruditapes philippinarum hemocytes.

    Science.gov (United States)

    Volland, Moritz; Blasco, Julián; Hampel, Miriam

    2017-04-01

    The appropriate selection of reference genes for the normalization of non-biological variance in reverse transcription real-time quantitative PCR (RT-qPCR) is essential for the accurate interpretation of the collected data. The use of multiple validated reference genes has been shown to substantially increase the robustness of the normalization. It is therefore considered good practice to validate putative genes under specific conditions, determine the optimal number of genes to be employed, and report the method or methods used. Under this premise, we assessed the current state of reference gene based normalization in RT-qPCR bivalve ecotoxicology studies (post 2011), employing a systematic quantitative literature review. A total of 52 papers met our criteria and were analysed for genes used, the use of multiple reference genes, as well as the validation method employed. We further critically discuss methods for reference gene validation based on a case study using copper exposed primary hemocytes from the marine bivalve Ruditapes philippinarum; including the established algorithms geNorm, NormFinder and BestKeeper, as well as the popular online tool RefFinder. We identified that RT-qPCR normalization is largely performed using single reference genes, while less than 40% of the studies attempted to experimentally validate the expression stability of the genes used. 18s rRNA and β-Actin were the most popular genes, yet their un-validated use did introduce artefactual variance that altered the interpretation of the resulting data. Our findings further suggest that combining the results from multiple individual algorithms and calculating the overall best-ranked gene, as computed by the RefFinder tool, does not by default lead to the identification of the most suitable reference genes. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam Ruditapes philippinarum challenged by the protozoan parasite Perkinsus olseni

    Science.gov (United States)

    Kang, Hyun-Sil; Hong, Hyun-Ki; Park, Kyung-Il; Cho, Moonjae; Youn, Seok-Hyun; Choi, Kwang-Sik

    2017-02-01

    Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.

  11. New functions of arthropod bursicon: inducing deposition and thickening of new cuticle and hemocyte granulation in the blue crab, Callinectes sapidus.

    Directory of Open Access Journals (Sweden)

    J Sook Chung

    Full Text Available Arthropod growth requires molt-associated changes in softness and stiffness of the cuticle that protects from desiccation, infection and injury. Cuticle hardening in insects depends on the blood-borne hormone, bursicon (Burs, although it has never been determined in hemolymph. Whilst also having Burs, decapod crustaceans reiterate molting many more times during their longer life span and are encased in a calcified exoskeleton, which after molting undergoes similar initial cuticle hardening processes as in insects. We investigated the role of homologous crustacean Burs in cuticular changes and growth in the blue crab, Callinectes sapidus. We found dramatic increases in size and number of Burs cells during development in paired thoracic ganglion complex (TGC neurons with pericardial organs (POs as neurohemal release sites. A skewed expression of Burs β/Burs α mRNA in TGC corresponds to protein contents of identified Burs β homodimer and Burs heterodimer in POs. In hemolymph, Burs is consistently present at ∼21 pM throughout the molt cycle, showing a peak of ∼89 pM at ecdysis. Since initial cuticle hardness determines the degree of molt-associated somatic increment (MSI, we applied recombinant Burs in vitro to cuticle explants of late premolt or early ecdysis. Burs stimulates cuticle thickening and granulation of hemocytes. These findings demonstrate novel cuticle-associated functions of Burs during molting, while the unambiguous and constant presence of Burs in cells and hemolymph throughout the molt cycle and life stages may implicate further functions of its homo- and heterodimer hormone isoforms in immunoprotective defense systems of arthropods.

  12. New functions of arthropod bursicon: inducing deposition and thickening of new cuticle and hemocyte granulation in the blue crab, Callinectes sapidus.

    Science.gov (United States)

    Chung, J Sook; Katayama, Hidekazu; Dircksen, Heinrich

    2012-01-01

    Arthropod growth requires molt-associated changes in softness and stiffness of the cuticle that protects from desiccation, infection and injury. Cuticle hardening in insects depends on the blood-borne hormone, bursicon (Burs), although it has never been determined in hemolymph. Whilst also having Burs, decapod crustaceans reiterate molting many more times during their longer life span and are encased in a calcified exoskeleton, which after molting undergoes similar initial cuticle hardening processes as in insects. We investigated the role of homologous crustacean Burs in cuticular changes and growth in the blue crab, Callinectes sapidus. We found dramatic increases in size and number of Burs cells during development in paired thoracic ganglion complex (TGC) neurons with pericardial organs (POs) as neurohemal release sites. A skewed expression of Burs β/Burs α mRNA in TGC corresponds to protein contents of identified Burs β homodimer and Burs heterodimer in POs. In hemolymph, Burs is consistently present at ∼21 pM throughout the molt cycle, showing a peak of ∼89 pM at ecdysis. Since initial cuticle hardness determines the degree of molt-associated somatic increment (MSI), we applied recombinant Burs in vitro to cuticle explants of late premolt or early ecdysis. Burs stimulates cuticle thickening and granulation of hemocytes. These findings demonstrate novel cuticle-associated functions of Burs during molting, while the unambiguous and constant presence of Burs in cells and hemolymph throughout the molt cycle and life stages may implicate further functions of its homo- and heterodimer hormone isoforms in immunoprotective defense systems of arthropods.

  13. New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam Ruditapes philippinarum challenged by the protozoan parasite Perkinsus olseni

    Science.gov (United States)

    Kang, Hyun-Sil; Hong, Hyun-Ki; Park, Kyung-Il; Cho, Moonjae; Youn, Seok-Hyun; Choi, Kwang-Sik

    2017-03-01

    Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.

  14. A novel C-type lectin, Nattectin-like protein, with a wide range of bacterial agglutination activity in large yellow croaker Larimichthys crocea.

    Science.gov (United States)

    Lv, Changhuan; Zhang, Dongling; Wang, Zhiyong

    2016-03-01

    C-type lectins (CTLs) are generally recognized as a superfamily of Ca(2+)-dependent carbohydrate-binding proteins, which serve as pattern recognition receptors (PRRs) in innate immunity of vertebrates. In this study, the molecular characterization and immune roles of a novel CTL from Larimichthys crocea (designated as LcNTC) were investigated. LcNTC is a novel protein that shared 33%-49% homology with other teleosts CTLs. The full-length cDNA of LcNTC was composed of 859 bp with a 465 bp open reading frame encoding a putative protein of 154 residues. LcNTC contained a single CRD with four conserved disulfide-bonded cysteine residues (Cys(57)-Cys(148), Cys(126)-Cys(140)) and EPN/AND motifs instead of invariant EPN/WND motifs required for carbohydrate-binding specificity and constructing Ca(2+)-binding sites. LcNTC mRNA was detected in all examined tissues with the most abundant in the gill. After challenged with poly I:C and Vibrio parahaemolyticus, the temporal expression of LcNTC was significantly up-regulated in the liver, spleen and head-kidney. LcNTC transcripts were also induced in the gill, skin, spleen and head-kidney post-infection with Cryptocaryon irritans. The recombinant LcNTC (rLcNTC) purified from Escherichia coli BL21 (DE3) exhibited strong agglutination activity against erythrocytes from human, rabbit and large yellow croaker in a Ca(2+)-dependent manner, and the agglutination could be inhibited by D-Mannose, D-Glucose, D-Fructose, α-Lactose, D-Maltose and LPS. Positive microbial agglutination activities of rLcNTC were observed against all tested bacteria in the presence of Ca(2+), including Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus and Micrococcus lysoleikticus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila). These findings collectively indicated that LcNTC might be involved in the innate immunity of L. crocea as a PRR.

  15. A novel putative lipoprotein receptor (CasLpR) in the hemocytes of the blue crab, Callinectes sapidus: cloning and up-regulated expression after the injection of LPS and LTA.

    Science.gov (United States)

    Tsutsui, Naoaki; Chung, J Sook

    2012-03-01

    The full-length cDNA encoding a putative lipoprotein receptor (CasLpR) was isolated from the hemocytes of Callinectes sapidus using 5' and 3' RACEs. The open reading frame for CasLpR contains a precursor of putative CasLpR consisting of 1710 amino acid residues including 22 amino acid residues of the signal peptide (22 amino acids). Mature CasLpR (1688 amino acids with 5.6% of phosphorylation sites) has multiple, putative functional domains: five low-density lipoprotein receptor domains in the N-terminus, and a G-protein-coupled receptor proteolysis site domain and a 7 transmembrane receptor (secretin family) domain in the C-terminus. To date, there are no proteins with a similar domain structure in the GenBank. The expression pattern of CasLpR was exclusive in hemocytes among all tested tissues obtained from a juvenile female at intermolt stage: brain, eyestalk ganglia, pericardial organs, and thoracic ganglia complex (nervous system); hepatopancreas (digestive system); heart, artery and hemocytes (circulatory system); gill and antennal gland (excretory system), hypodermis; and Y-organ (endocrine organ). There was no CasLpR expression in the ovary of an adult female. A putative function of CasLpR was examined after challenges of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in vivo using qRT-PCR assays. Animals at 24 h after injection of LPS or LTA up-regulated the expression of CasLpR in hemocytes by ∼3.5 and 1.4 folds, respectively, compared to the controls that received saline injection. LPS challenge also caused the greatest increment (∼55 folds) of heat shock protein 90 (Hsp90) expression in these samples. These data indicate that putative CasLpR and CasHsp90 may be involved in the defense system or the stress response of C. sapidus. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Function of Agglutination in the Forming of Chinese Polysyllables%论黏合在汉语复音词形成中的作用

    Institute of Scientific and Technical Information of China (English)

    唐子恒

    2011-01-01

    索绪尔在中把"两个或几个原来分开的但常在句子内部的句段里相遇的要素互相熔合成为一个绝对的或者难于分析的单位"的演变过程称为"黏合"(agglutination).在汉语复音词中,由原来的两个或更多的词构成的词组或原来不在同一个结构层次上但在句段中相邻的成分,经过历时的发展渐渐熔合形成的占大多数,在这些复音词形成的过程中,黏合起了十分重要的作用.

  17. Low yield of screening for cryptococcal antigen by latex agglutination assay on serum and cerebrospinal fluid from Danish patients with AIDS or ARC

    DEFF Research Database (Denmark)

    Hoffmann, S; Stenderup, J; Mathiesen, Lars Reinhardt

    1991-01-01

    From July 1, 1989 to September 5, 1990, 530 serum specimens and 50 cerebrospinal fluid (CSF) specimens from 334 HIV-1 infected patients, most of whom had AIDS or ARC, were analysed in a cryptococcal antigen latex agglutination assay, and all were negative. Three cases of meningitis due...... to Cryptococcus neoformans diagnosed by microscopy and culture in 3 HIV-1 infected patients are presented. Stored specimens of serum and CSF from these patients were assayed for cryptococcal antigen, and in all 3 the onset of meningitis was preceded by the presence of cryptococcal antigen in serum....... It is concluded that the low occurrence of cryptococcosis in our patient population does not justify a routine serum screening for cryptococcal antigen....

  18. Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

    Science.gov (United States)

    Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

    2015-01-01

    In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene.

  19. Performance of CHROMagar Staph aureus and CHROMagar MRSA for detection of Staphylococcus aureus in seawater and beach sand--comparison of culture, agglutination, and molecular analyses.

    Science.gov (United States)

    Goodwin, K D; Pobuda, M

    2009-11-01

    Beach seawater and sand were analyzed for Staphylococcus aureus and methicillin resistant S. aureus (MRSA) for samples collected from Avalon, and Doheny Beach, CA. Membrane filtration followed by incubation on CHROMagar Staph aureus (SCA) and CHROMagar MRSA (C-MRSA) was used to enumerate S. aureus and MRSA, respectively. Media performance was evaluated by comparing identification via colony morphology and latex agglutination tests to PCR (clfA, 16S, and mecA genes). Due to background color and crowding, picking colonies from membrane filters and streaking for isolation were sometimes necessary. The specificity of SCA and C-MRSA was improved if colony isolates were identified by the presence of a matte halo in addition to mauve color; however routine agglutination testing of isolates did not appear warranted. Using the appearance of a colony on the membrane filter in conjunction with isolate appearance, the positive % agreement, the negative % agreement, and the % positive predictive accuracy for SCA was 84%, 95%, and 99% respectively, and for C-MRSA it was 85%, 98%, and 92%, respectively. Sensitivity and specificity of SCA and C-MRSA with membrane-filtered beach samples were optimized through identification experience, control of filter volume and incubation time, and isolation of colonies needing further identification. With optimization, SCA and C-MRSA could be used for enumeration of S. aureus and MRSA from samples of beach water and sand. For the sites studied here, the frequency of detection of S. aureus ranged from 60 to 76% and 53 to 79% for samples of beach seawater and sand, respectively. The frequency of detection of MRSA ranged from 2 to 9% and 0 to 12% for samples of seawater and sand, respectively.

  20. [Acute bacterial meningitis with soluble antigen detected by latex particle agglutination tests at the Sourô-Sanou University Hospital of Bobo-Dioulasso (Burkina Faso)].

    Science.gov (United States)

    Ouédraogo, S M; Yaméogo, T M; Kyelem, C G; Poda, G E A; Ouédraogo, N F; Millogo, A; Ouédraogo, A; Ouédraogo-Traoré, A; Drabo, Y J

    2012-01-01

    Acute bacterial meningitis constitutes a major public health problem in Burkina Faso, in part because of its high lethality rate, estimated in 2004 at 17.5%. Failure to confirm suspected cases of meningitis results in overestimating reported cases and incorrectly treating false positives. The latex particle agglutination test is a diagnostic alternative that overcomes these limitations. Determine the bacteriological and therapeutic profile as well as the course of cases of acute meningitis confirmed by the latex agglutination test at Sourô-Sanou University Hospital. This prospective longitudinal study took place over a one-year period (2008 to 2009). Data were collected from clinical and laboratory records. The diagnosis of meningitis was confirmed by testing for specific soluble antigens in the spinal fluid. We used the Pastorex(™) Meningitis Kit for that purpose. The threshold of significance selected for our study was 0.05. In all, 457 samples of spinal fluid from patients with suspected acute bacterial meningitis were analyzed and the latex test was performed in 438 of these samples: 154 (35.2%) were positive. The average age of our cases confirmed by the latex test was 13.2 ± 4.2 years old. This test confirmed more cases than any other method of identification. The therapeutic strategy used from one to four treatment agents. Streptococcus pneumoniae was the most virulent and the most lethal pathogen, with a 64.7% lethality rate. The earliness of the consultation and the treatment of the bacterial meningitis seem to have a positive effect on the course of disease.

  1. Glycodendrimersomes from Sequence-Defined Janus Glycodendrimers Reveal High Activity and Sensor Capacity for the Agglutination by Natural Variants of Human Lectins.

    Science.gov (United States)

    Zhang, Shaodong; Xiao, Qi; Sherman, Samuel E; Muncan, Adam; Ramos Vicente, Andrea D M; Wang, Zhichun; Hammer, Daniel A; Williams, Dewight; Chen, Yingchao; Pochan, Darrin J; Vértesy, Sabine; André, Sabine; Klein, Michael L; Gabius, Hans-Joachim; Percec, Virgil

    2015-10-21

    A library of eight amphiphilic Janus glycodendrimers (Janus-GDs) presenting D-lactose (Lac) and a combination of Lac with up to eight methoxytriethoxy (3EO) units in a sequence-defined arrangement was synthesized via an iterative modular methodology. The length of the linker between Lac and the hydrophobic part of the Janus-GDs was also varied. Self-assembly by injection from THF solution into phosphate-buffered saline led to unilamellar, monodisperse glycodendrimersomes (GDSs) with dimensions predicted by Janus-GD concentration. These GDSs provided a toolbox to measure bioactivity profiles in agglutination assays with sugar-binding proteins (lectins). Three naturally occurring forms of the human adhesion/growth-regulatory lectin galectin-8, Gal-8S and Gal-8L, which differ by the length of linker connecting their two active domains, and a single amino acid mutant (F19Y), were used as probes to study activity and sensor capacity. Unpredictably, the sequence of Lac on the Janus-GDs was demonstrated to determine bioactivity, with the highest level revealed for a Janus-GD with six 3EO groups and one Lac. A further increase in Lac density was invariably accompanied by a substantial decrease in agglutination, whereas a decrease in Lac density resulted in similar or lower bioactivity and sensor capacity. Both changes in topology of Lac presentation of the GDSs and seemingly subtle alterations in protein structure resulted in different levels of bioactivity, demonstrating the presence of regulation on both GDS surface and lectin. These results illustrate the applicability of Janus-GDs to dissect structure-activity relationships between programmable cell surface models and human lectins in a highly sensitive and physiologically relevant manner.

  2. Kinetics and time dependence of the differential agglutination of acetone [AC]- and formalin [HS]-fixed Toxoplasma tachyzoites by serum of mice with experimental toxoplasmosis.

    Science.gov (United States)

    Ali, Nehad Mahmoud; Habib, Khaled Sayed Mohamed

    2012-04-01

    Researches to specify a serologic diagnostic test capable of determining the stage of toxoplasmosis, whether recent or latent, have been hampered by lack of knowing the real time of infection. Studying the precise kinetics of the differential agglutination of acetone [AC]-fixed versus that of formalin [HS]-fixed tachyzoites (differential agglutination test or AC/HS test) by sera of mice during the course of toxoplasmosis and assessment of its value in the differentiation between recent and latent infections in mice were the aims of the present work. Experimental toxoplasmosis was induced in mice, sera were collected sequentially and AC/HS test, FAST-ELISA to determine levels of IgM and IgG and microscopic examination of brain for Toxoplasma cysts were done. Both AC and HS specific patterns in the AC/HS test were noted to be dependent on the time from the onset of infection. Acute patterns of the AC/HS test were observed early in infection till before the appearance of brain cysts. Non-acute patterns were obtained late on 28th day post infection coinciding with the disappearance of IgM, persistence of IgG and presence of cysts in brains. The AC antibody was high in the recent phase of infection, and then it declined to be replaced by high sustained level of HS antibody late in infection. In conclusion, in the presence of both IgM and IgG, the appearance of either equivocal pattern or the non-acute pattern in the AC/HS test is significant in ruling out acute infection in mice.

  3. 地稔凝集素的提取及凝血活性的研究%Study on the Extraction and Agglutination of Lectin from Melastoma dodecandrum Lour

    Institute of Scientific and Technical Information of China (English)

    邓政东; 程爱芳; 李秀丽

    2015-01-01

    为研究和利用地稔的促凝血活性物质,以地稔为试材,提取地稔凝集素,研究pH、温度和糖等理化因素对凝血活性的影响。结果表明:经硫酸铵沉淀及透析提取的地稔凝集素具有红细胞凝集活性;碱性条件下,抑制作用明显,而酸性条件有较强促进作用;温度对凝集素凝血活性影响很小;半乳糖有明显促进作用。%In order to study and utilize the procoagulant activity substances from Melastoma dodecandrum Lour ,taking Melastoma dodecandrum Lour as material ,extraction of lectin and the effect of physical and chem‐ical agents on agglutinating activity were studied ,such as pH ,temperature and sugar .The results showed that the lectin extracted by ammonium sulfate precipitation and dialysis had agglutinating activity .Alkaline condi‐tions had significantly inhibited the agglutinating activity .While acidic conditions had a strong role in promo‐ting .The effect of temperature on agglutinating activity was little .Galactose had an obvious effect on promo‐ting .

  4. Purification of Soybean Agglutinin and Its Agglutination Activity Toward Different Cancer Cell Lines%大豆凝集素的纯化及其凝集不同肿瘤细胞的探讨

    Institute of Scientific and Technical Information of China (English)

    荆剑; 赵翔; 张页

    2003-01-01

    A novel and efficient method for purification of soybean agglutinin(SBA) from soybean was reported.The method was characterized by selective extraction of SBA from soybean homogenate with barbiturate buffer(pH 6.2) ,removal of impurity by hydroxyapatite,and the final purification of SBA by guaran affinity chromatography.The purified SBA showed a single band of 27.5kD by SDS-PAGE.The lowest concentration of SBA that caused agglutination of the rabbit red blood cells was 0.31 mg/L.Agglutination of different cancer cell lines by the purified SBA was examined.Strong agglutination of the human nasopharyngeal CNE cells.mouse Lewis lung carcinoma cells.and rat mammary adenocarcinoma R3230AC cells was observed.However,SBA could not agglutinate the human hepatocellular carcinoma BEL-7402cells,suggesting that unlike the above-mentioned three cell lines,the BEL-7402 cells may not express N-acetylgalactosamine(GalNAc) or galactose(Gal) residues in significant amount at the non-reducing terminals of their cell surface glycans.

  5. Comparison of the H7 latex agglutination test with a fliCh7 real-time PCR assay for confirmation of the H type of Escherichia coli O157:H7

    Science.gov (United States)

    Escherichia coli O157:H7 is a food-borne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. Positive identification of E. coli O157:H7 is made using biochemical tests and latex agglutination using specific antisera. However, under certain conditions, some E. coli O157:H7 isolate...

  6. Latex agglutination assays for detection and of non-O157 Shiga toxin-producing E. coli serogroups O26, O45, O103, O111, O121 and O145

    Science.gov (United States)

    Latex agglutination assays were developed for the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups utilizing polyclonal antibodies. Rabbit antisera were affinity purified through Protein A/G columns and the isolated immunoglobulins (IgG) were covalently immobilized onto pol...

  7. Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas.

    Directory of Open Access Journals (Sweden)

    Rafael D Rosa

    Full Text Available BACKGROUND: Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. FINDINGS: Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3 that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. CONCLUSIONS: We provide here the first report showing that big defensins form a family

  8. Cytotoxicity of TiO2 nanoparticles to mussel hemocytes and gill cells in vitro: Influence of synthesis method, crystalline structure, size and additive.

    Science.gov (United States)

    Katsumiti, Alberto; Berhanu, Deborah; Howard, Kieren T; Arostegui, Inmaculada; Oron, Miriam; Reip, Paul; Valsami-Jones, Eugenia; Cajaraville, Miren P

    2015-01-01

    Increasing the production and applications of TiO2 nanoparticles (NPs) has led to grow concerns about the consequences for the environment. In this study, we investigated the effects of a set of TiO2 NPs on the viability of mussel hemocytes and gill cells using neutral red and thiazolyl tetrazolium bromide assays. For this, we compared the cytotoxicity of TiO2 NPs (0.1-100 mg Ti/L) produced by different techniques: rutile NPs (60 nm) produced by milling and containing disodium laureth sulfosuccinate (DSLS), rutile NPs (10, 40 and 60 nm) produced by wet chemistry and anatase/rutile NPs (∼100 nm) produced by plasma synthesis. The commercially available P25 anatase/rutile NPs (10-20 nm) were also tested. Exposures were performed in parallel with their respective bulk forms and the cytotoxicity of the additive DSLS was also tested. Z potential values in distilled water indicated different stabilities depending on the NP type and all NPs tested formed agglomerates/aggregates in cell culture media. In general, TiO2 NPs showed a relatively low and dose-dependent toxicity for both cell models with the two assays tested. NPs produced by milling showed the highest effects, probably due to the toxicity of DSLS. Size-dependent toxicity was found for NPs produced by wet chemistry (10 nm > 40 nm and 60 nm). All TiO2 NPs tested were more toxic than bulk forms excepting for plasma produced ones, which were the least toxic TiO2 tested. The mixture bulk anatase/rutile TiO2 was more toxic than bulk rutile TiO2. In conclusion, the toxicity of TiO2 NPs varied with the mode of synthesis, crystalline structure and size of NPs and can also be influenced by the presence of additives in the suspensions.

  9. Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity.

    Science.gov (United States)

    Fernandez-del-Carmen, Asun; Juárez, Paloma; Presa, Silvia; Granell, Antonio; Orzáez, Diego

    2013-02-20

    The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25 mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins.

  10. Validation of the modified agglutination test for the detection of Toxoplasma gondii in free-range chickens by using cat and mouse bioassay.

    Science.gov (United States)

    Dubey, J P; Laurin, E; Kwowk, O C H

    2016-03-01

    The modified agglutination test (MAT) is one of the most commonly used tests for the detection of antibodies to Toxoplasma gondii in animal and human sera. The objective of the present study was to evaluate the diagnostic accuracy of the MAT and bioassay in free-range/backyard (FR) chickens (Gallus domesticus). Previously-published T. gondii test results from 2066 chickens from 19 countries were compiled for the present study. The frequency of isolation of T. gondii increased for MAT titres between 1:5 and 1:160, and ranged from 61 to 75% for antibody titres of 1:160, 1:320, and ⩾1:640. Twenty-three cats fed pooled hearts from a total of 802 FR seronegative (MAT, <1:5) chickens from several countries did not excrete oocysts, indicating a high negative predictive value of MAT because FR chickens would have been exposed to many microbes; cats are the most sensitive indicators of T. gondii infection in tissues and can excrete millions of oocysts after ingesting even a few bradyzoites. Of the 29 cats in this study, six cats, fed hearts pooled from 15-122 FR chickens, excreted oocysts; but these identifications were likely related to misidentification or prozone. Results of the present study support the validity of MAT for the detection of T. gondii infection in chickens.

  11. Evaluation of the Widal tube agglutination test for the diagnosis of typhoid fever among children admitted to a rural hdospital in Tanzania and a comparison with previous studies

    Directory of Open Access Journals (Sweden)

    Malahiyo Rajabu

    2010-06-01

    Full Text Available Abstract Background The diagnosis of typhoid fever is confirmed by culture of Salmonella enterica serotype Typhi (S. typhi. However, a more rapid, simpler, and cheaper diagnostic method would be very useful especially in developing countries. The Widal test is widely used in Africa but little information exists about its reliability. Methods We assessed the performance of the Widal tube agglutination test among febrile hospitalized Tanzanian children. We calculated the sensitivity, specificity, positive predictive value (PPV, and negative predictive value (NPV of various anti-TH and -TO titers using culture-confirmed typhoid fever cases as the "true positives" and all other febrile children with blood culture negative for S. typhi as the "true negatives." Results We found that 16 (1% of 1,680 children had culture-proven typhoid fever. A single anti-TH titer of 1:80 and higher was the optimal indicator of typhoid fever. This had a sensitivity of 75%, specificity of 98%, NPV of 100%, but PPV was only 26%. We compared our main findings with those from previous studies. Conclusion Among febrile hospitalized Tanzanian children with a low prevalence of typhoid fever, a Widal titer of ≥ 1:80 performed well in terms of sensitivity, specificity, and NPV. However a test with improved PPV that is similarly easy to apply and cost-efficient is desirable.

  12. An evaluation of the relative sensitivities of the venereal disease research laboratory test and the Treponema pallidum particle agglutination test among patients diagnosed with primary syphilis.

    Science.gov (United States)

    Creegan, Linda; Bauer, Heidi M; Samuel, Michael C; Klausner, Jeffrey; Liska, Sally; Bolan, Gail

    2007-12-01

    Because definitive methods for diagnosing primary syphilis are limited, it is important to optimize the sensitivity of serodiagnosis. To determine the most sensitive testing approach to the diagnosis of primary syphilis, using the commonly available serologic tests: the Venereal Disease Research Laboratory (VDRL) test and the Treponema pallidum particle agglutination (TP-PA) test. Sensitivities of 2 serologic testing strategies for primary syphilis were compared among 106 darkfield-confirmed cases treated in San Francisco from January 2002 through December 2004. The sensitivity of the diagnostic strategy using VDRL confirmed by TP-PA was 71% (95% CI, 61%-79%). Substituting Rapid Plasma Reagin test for VDRL in a subset of 51 patients produced the same sensitivity (71%; 95% CI, 56%-83%). The sensitivity of TP-PA as the first-line diagnostic test was 86% (95% CI, 78%-92%). The sensitivity of the former approach was significantly lower among HIV-positive patients, compared with HIV-negative patients (55% vs. 77%, P = 0.05). The TP-PA test as the first-line diagnostic test yielded higher sensitivity for primary syphilis than did the use of the currently recommended strategy.

  13. Development of a rapid agglutination latex test for diagnosis of enteropathogenic and enterohemorrhagic Escherichia coli infection in developing world: defining the biomarker, antibody and method.

    Directory of Open Access Journals (Sweden)

    Letícia B Rocha

    2014-09-01

    Full Text Available Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco's modified Eagle's medium (DMEM or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT was developed and tested with the same collection of bacterial isolates.EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.

  14. Prevalence of agglutinating antibodies to Toxoplasma gondii in small ruminants of the Madrid region, Spain, and identification of factors influencing seropositivity by multivariate analysis.

    Science.gov (United States)

    Mainar, R C; de la Cruz, C; Asensio, A; Domínguez, L; Vázquez-Boland, J A

    1996-01-01

    A seroepidemiological survey of Toxoplasma gondii infection in sheep and goats was conducted in the Madrid region of Spain. Sera were collected from 60 herds, for which farming management information and other relevant data for their characterization were also obtained through a questionnaire. The seroprevalence was 11.8% (64 out of 541), using the modified (2-mercaptoethanol) direct agglutination technique with a 1:64 cut-off titre. The relationship between seropositivity and the variables in the questionnaire was assessed by multivariate analysis. Four variables were found to be significantly associated with seroprevalence. Two of them, the presence of cats and a previous history of abortion outbreaks in the farm, were factors known to be linked with toxoplasmosis, indicating the validity of the serological data. Seropositivity was also related to a lack of replacements in the preceding year. Proximity to other farms appeared to be a protective factor negatively associated with seropositivity, probably because it was an indicator of proximity to an urban area and the availability of local sanitary facilities.

  15. Comparison of indirect fluorescent antibody test and the modified agglutination test for the detection of Toxoplasma gondii antibodies in stray dogs from Southern Brazil.

    Science.gov (United States)

    de Almeida, Jonatas Campos; Frehse, Michelle Salmon; Navarro, Italmar Teodorico; Garcia, João Luis; Biondo, Alexander Welker; Freire, Roberta Lemos

    2016-12-01

    The aim of the present study was to determine the prevalence of antibodies to Toxoplasma gondii by two serological techniques in sera of 364 stray dogs from Brazil by immunofluorescence antibody test (IFAT, cut off point 1:16) and to the modified agglutination test (MAT, cut-off points 1:25 and 1:50). A total of 175/364 (48.07%) sera were positive by IFAT, and 108/364 (29.67%) and 85/364 (23.35%) were positive by MAT with cutoff points 1:25 and 1:50, respectively were positive by MAT. Cohen's Kappa Coefficient between IFAT and MAT was 0.81 (excellent) and 0.66 (substantial) with cutoff points 1:25 and 1:50, respectively. Using IFAT as gold standard, MAT sensitivity and specificity were 78% and 99% for 1:25 and 61% and 99% for 1:50, respectively. The results document of the usefulness of MAT for serological diagnosis because it does not require species-specific conjugate.

  16. 中华圆田螺血细胞的分类和吞噬活性%Classification and phagocytosis property of hemocytes from Viviparus chinensis

    Institute of Scientific and Technical Information of China (English)

    裴鹏祖; 胡宝庆; 谢彦海; 文春根

    2011-01-01

    血细胞作为主要的免疫效应细胞在贝类的免疫和防御中有重要作用.本文通过相差显微镜和透射电子显微镜对中华圆田螺(Viviparus chinensis)血细胞的形态与类型以及血细胞对枯草芽孢杆菌的吞噬观察.结果发现壳高4~6 cm的田螺血细胞浓度为(2.17±0.38)×106个/mL.根据血细胞大小,形态和胞质内颗粒的有无可以将血细胞分为颗粒细胞、无颗粒细胞、透明细胞、淋巴样细胞四种类型.Wrighte-Giemsa染色显示,颗粒细胞表现为嗜酸性,其余3类细胞均呈现嗜碱性.另外,通过对颗粒细胞形态的连续观察,发现透明细胞可能是颗粒细胞脱颗粒而形成的衰老细胞.血细胞对枯草芽孢杆菌的吞噬实验结果表明,颗粒细胞吞噬杆菌活性最强,吞噬率和吞噬指数分别是47.73%和1.44,其次无颗粒细胞,吞噬率和吞噬指数分别是30.44%和1.12,透明细胞和淋巴样细胞无吞噬活性.%Haemocytes, as the main immune effector cells, play a vital roles in the immune defense of molluscs.The morphology, type, and phagocytotic characteristics of haemocytes from Viviparus chinensis were studied by light microscope,vital staining and electron microscopic techniques.The results showed that the total hemocyte cotent of Viviparus chinensis was about(2.17±0.38) × 106/mL from 4 to 6 centimeters in the shell length.The haemocytes were classified as 4 types based on the size, morphological characterization, presence or absence of granules in the cytoplasm.They were granulocyte, agranulocyte, hyalinocyte, lymphoid cell.It observed that the snile granulocyte formed hyalinocyte through observe of the dynamic variation in vivo of the granulocyte.After Wrighte - Giemsa's staining, granulocyte were acidophils.The remaining three types were basophils.The results of bacillus phagocytosis assay indicated that the phagocytic activity of granulocyte was the stongest, the phagocytic rate and index were 47.73% and 1.44,the

  17. Latex agglutination assays for detection of non-O157 Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145.

    Science.gov (United States)

    Medina, Marjorie B; Shelver, Weilin L; Fratamico, Pina M; Fortis, Laurie; Tillman, Glenn; Narang, Neelam; Cray, William C; Esteban, Emilio; Debroy, Andchitrita

    2012-05-01

    Latex agglutination assays utilizing polyclonal antibodies were developed for the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups. Rabbit antisera were affinity purified through protein A/G columns, and the isolated immunoglobulins (IgGs) were covalently immobilized onto polystyrene latex particles. The resulting latex-IgG complex had a protein (IgG) load of 0.20 to 0.28 mg/ml in a 1% latex suspension. Optimum conditions for the agglutination assay consisted of utilizing 20 μm l of latex-IgG reagent containing 2.0 to 2.8 μm g IgG in a 0.5% latex suspension. Agglutination or flocculation was observed almost instantly after mixing the colonies with the latex-IgG, indicating STEC strains. More than 100 target and nontarget strains were tested in more than 3,000 test replicates. All target organisms produced positive results, but three antisera (anti-O26, anti-O103, and anti-O145) cross-reacted with some other STECs. The anti-O103 and anti-O145 latex reagents cross-reacted with O26 strains, and the anti-O26 cross-reacted with O103 strains. The latex-IgG reagents are stable for at least 1 year and are easy to prepare. These agglutination assays can be used for identification of presumptive non-O157 STEC colonies from agar media. The techniques used to prepare the latex reagents also can be utilized for testing other STEC serogroups, other E. coli serotypes, or other pathogens to ensure safe foods to consumers.

  18. H-deficient Bombay and para-Bombay red blood cells are most strongly agglutinated by the galactophilic lectins of Aplysia and Pseudomonas aeruginosa that detect I and P1 antigens.

    Science.gov (United States)

    Gilboa-Garber, N; Sudakevitz, D; Levene, C; Rahimi-Levene, N; Yahalom, V

    2006-01-01

    The galactophilic lectins Aplysia gonad lectin (AGL) and Pseudomonas aeruginosa lectin (PA-IL), which detect human I and P1 RBC antigens, were examined for hemagglutination of H+ (group O and B) and H-deficient (Bombay and para-Bombay phenotype) RBCs. The results were compared with those obtained using two other galactophilic lectins, Maclura pomifera lectin (MPL) and Arachis hypogaea (peanut) agglutinin (PNA), which share T-antigen affinity, and two fucose-binding H-specific lectins, Ulex europaeus (UEA-I) and Pseudomonas aeruginosa lectin (PA-IIL), as well as with those achieved with anti-I serum. The results revealed that, in contrast to UEA-I and PA-IIL, which preferentially agglutinated H+ RBCs, and to MPL and PNA, which similarly agglutinated all examined RBCs, AGL, PA-IL, and the anti-I serum agglutinated the H-deficient RBCs more strongly than did the H+ RBCs. These findings could be attributed to increased levels of I and P1 antigens on those RBCs resulting from the use of the free common H-type 2 precursor for their synthesis. Since both PA-IL and PA-IIL are regarded as potential pathogen adhesins, it would be interesting to statistically compare the sensitivities of individuals of H+ and H-deficient RBC populations to P. aeruginosa infections.

  19. A comparative study of Toxoplasma gondii seroprevalence in mink using a modified agglutination test, a Western blot, and enzyme-linked immunosorbent assays.

    Science.gov (United States)

    Gu, Yi; Wang, Zedong; Cai, Yufeng; Li, Xiaoxing; Wei, Feng; Shang, Limin; Li, Jiping; Liu, Quan

    2015-09-01

    Toxoplasma gondii can infect almost all warm-blooded animals, and many serological methods have been developed to detect T. gondii infection in a variety of animal species. In the present study, the seroprevalence of T. gondii infection in farmed mink in northeast China was determined using the modified agglutination test (MAT), a Western blot (WB), and 3 enzyme-linked immunosorbent assays (ELISAs) with protein A/G conjugate, using either of 2 recombinant dense granule antigens, GRA1 and GRA7, or Toxoplasma soluble antigens (TSA). There was no significant difference between the detection results of the GRA1-, GRA7-, and TSA-ELISAs and WB (McNemar chi-square, P > 0.05), but a significant difference was observed between MAT and WB (P < 0.05). A near perfect agreement (97.0%) was found between the GRA7-ELISA and WB (κ = 0.83), and a substantial agreement (92.4-93.1%) was observed in the TSA- and GRA1-ELISAs (κ = 0.68-0.73). The GRA7-ELISA showed the highest sensitivity and specificity, and the lowest false-positive and negative rates, while the MAT gave both a low sensitivity and frequent false positives in comparison to the WB. Receiver operating characteristic analysis revealed the largest area under curve of 0.85 (95% confidence interval: 0.74-0.96), and the highest relative sensitivity (72.7%) and specificity (99.0%) for a cutoff value of 0.19 in the GRA7-ELISA. These results indicate that the GRA7-ELISA is suitable for detection of T. gondii infection in mink and that MAT should be used with caution.

  20. Campylobacter jejuni carbon starvation protein A (CstA) is involved in peptide utilization, motility and agglutination, and has a role in stimulation of dendritic cells.

    Science.gov (United States)

    Rasmussen, J J; Vegge, C S; Frøkiær, H; Howlett, R M; Krogfelt, K A; Kelly, D J; Ingmer, H

    2013-08-01

    Campylobacter jejuni is the most frequent cause of severe gastroenteritis in the developed world. The major symptom of campylobacteriosis is inflammatory diarrhoea. The molecular mechanisms of this infection are poorly understood compared to those of less frequent disease-causing pathogens. In a previous study, we identified C. jejuni proteins that antibodies in human campylobacteriosis patients reacted with. One of the immunogenic proteins identified (Cj0917) displays homology to carbon starvation protein A (CstA) from Escherichia coli, where this protein is involved in the starvation response and peptide uptake. In contrast to many bacteria, C. jejuni relies on amino acids and organic acids for energy, but in vivo it is highly likely that peptides are also utilized, although their mechanisms of uptake are unknown. In this study, Biolog phenotype microarrays have been used to show that a ΔcstA mutant has a reduced ability to utilize a number of di- and tri-peptides as nitrogen sources. This phenotype was restored through genetic complementation, suggesting CstA is a peptide uptake system in C. jejuni. Furthermore, the ΔcstA mutant also displayed reduced motility and reduced agglutination compared to WT bacteria; these phenotypes were also restored through complementation. Murine dendritic cells exposed to UV-killed bacteria showed a reduced IL-12 production, but the same IL-10 response when encountering C. jejuni ΔcstA compared to the WT strain. The greater Th1 stimulation elicited by the WT as compared to ΔcstA mutant cells indicates an altered antigenic presentation on the surface, and thus an altered recognition of the mutant. Thus, we conclude that C. jejuni CstA is important not only for peptide utilization, but also it may influence host-pathogen interactions.

  1. Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic Escherichia coli Infection in Developing World: Defining the Biomarker, Antibody and Method

    Science.gov (United States)

    Munhoz, Danielle D.; Cardoso, Lucas T. A.; Luz, Daniela E.; Andrade, Fernanda B.; Horton, Denise S. P. Q.; Elias, Waldir P.; Piazza, Roxane M. F.

    2014-01-01

    Background Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens. Methodology First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates. Principal findings EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world. Conclusion RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency. PMID:25254981

  2. Stability of Freeze-Dried Sera Stored at Different Temperatures for the Detection of Anti-Leishmania infantum Antibodies Using Direct Agglutination Test.

    Directory of Open Access Journals (Sweden)

    Zahra Kakooei

    2014-11-01

    Full Text Available This study aimed to evaluate freeze-dried sera as an alternative to non-freeze dried for detection of anti-Leishmania infantum antibodies over the course of 11 months using the direct agglutination test (DAT.Altogether, 60 serum samples (30 from humans and 30 from dogs were collected from various geographical locations in Iran. All the collected sera were pooled and each pooled serum sample contained 10 different sera. In the beginning, the human and dog pooled sera were categorized as positive (weak and strong and negative based on anti-L. infantum antibodies using the DAT. All the freeze-dried and non-freeze-dried sera were stored at -70°C, -20°C, 4°C, 22-28°C and 56°C for 11 months. The positive and negative human and dog pooled sera were separately tested using the DAT each month and the results were compared to non-freeze-dried sera kept under the same conditions.We found strong agreement (100% between the results obtained from freeze-dried human and dog in strong DAT positive sera kept at -70°C, -20°C, 4°C and 22-28°C during this study. The human and dog pooled sera stored at 56°C were corrupted after 2 weeks. The DAT results were highly reproducible using freeze-dried human pooled sera in the beginning and month 11 of this study (CV = 0.036.Freeze-dried human and dog strong DAT positive sera are highly stable under different temperature conditions, are easy to transport and are safe for use as positive and negative serum controls in laboratories.

  3. Development and Evaluation of a New Creatine Kinase MB Mass Determination Assay Using a Latex Agglutination Turbidimetric Immunoassay with an Automated Analyzer.

    Science.gov (United States)

    Hoshino, Tadashi; Hanai, Kazuma; Tanimoto, Kazuhito; Nakayama, Tomohiro

    2016-01-01

    The diagnosis of myocardial infraction (MI) in patients presenting to the emergency department represents a clinical challenge. It is known that creatine kinase-MB isoenzyme (CK-MB) is present in soluble cell fractions of cardiac muscle, and injury to those cells results in an increase of CK-MB in the blood. Therefore, CK-MB is a suitable clinical biomarker of myocardial infraction. To measure CK-MB mass rapidly and easily, we developed the new reagent 'L-type Wako CK-MB mass' (L-CK-MB mass) for the latex agglutination turbidimetric immunoassay method. Using a Hitachi LABOSPECT 008, we evaluated the performance of this assay as a method for quantifying CK-MB mass, and we compared the measurement of the serum CK-MB mass concentration with this assay to that obtained using an electrochemiluminescence immunoassay (ECLIA). A dilution test showed linearity from 5 μg/L to 190 μg/L, and the limit of quantification of the L-CK-MB mass assay was 3.0 μg/L. The within-run CV and between-day CV were 1.0 - 4.5% and 1.8 - 4.4%, respectively. Serum CK-MB mass concentration determined using the L-CK-MB mass assay was reliably and strongly correlated with that determined using ECLIA (n = 163, r = 0.999, y = 0.977x + 0.307). The L-CK-MB mass assay is able to specifically determine CK-MB mass and is a very useful method for the accurate measurement of CK-MB mass for routine clinical analyses.

  4. Evaluation of chromogenic medium and direct latex agglutination test for detection of group B streptococcus in vaginal specimens from pregnant women in Lebanon and Kuwait.

    Science.gov (United States)

    Ghaddar, Nahed; Alfouzan, Wadha; Anastasiadis, Elie; Al Jiser, Tamima; Itani, Saad Eddine; Dernaika, Racha; Eid, Toufic; Ghaddar, Ali; Charafeddine, Adib; Dhar, Rita; El Hajj, Hiba

    2014-10-01

    This study was undertaken to evaluate chromogenic medium and a direct latex agglutination test (DLA) for detection of Group B Streptococcus (GBS) in the vaginal specimens of pregnant women, and to ascertain the prevalence of GBS in this population in Kuwait and Lebanon. Vaginal swabs, collected from women at 35-37 weeks of gestation, were cultured on 5 % sheep blood agar (SBA), colistin nalidixic acid agar (CNA), Strept B Select chromogenic agar (SBS) as well as Lim enrichment broth in 168 cases in Lebanon while only SBA was used for 1391 samples in Kuwait. In addition, vaginal samples from 102 GBS-positive and 20 GBS-negative women near the time of delivery were collected in Kuwait for evaluation of the DLA test. During the study period, the prevalence of GBS colonization was determined to be 20.7 % (288/1391) in Kuwait while 18.4 % (31) of 168 pregnant women in Lebanon had vaginal cultures positive for GBS. By direct plating of vaginal swabs on the three media used, the isolation rates of GBS were 51.6, 64.5 and 77.4 % on SBA, CNA and SBS, respectively, which increased to 90.35, 93.1 and 96.8 %, respectively, following subculture in Lim broth after 18 h of incubation. The sensitivity of the DLA test was found to be dependent on the density of GBS colonization, resulting in 100 % sensitivity and 100 % specificity for heavy (>10(2) c.f.u. per swab) and moderately heavy (50-100 c.f.u. per swab) growth of GBS. However, for vaginal specimens yielding <50 c.f.u. per swab, the sensitivity, specificity, positive and negative predictive values of the DLA test were 100, 55.5, 63.6 and 100 %, respectively. In conclusion, a chromogenic agar, such as SBS, and a DLA test can be used for rapid detection of GBS in pregnant women. The DLA test, in particular, could prove to be a useful tool for immediate detection of GBS in women near delivery so that intrapartum antibiotic prophylaxis can be initiated.

  5. Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection.

    Science.gov (United States)

    Mohammadiha, A; Haghighi, A; Mohebali, M; Mahdian, R; Abadi, A R; Zarei, Z; Yeganeh, F; Kazemi, B; Taghipour, N; Akhoundi, B; Barati, M; Mahmoudi, M R

    2013-02-18

    Canine visceral leishmaniasis (CVL) is endemic in northwestern Iran. This study aimed to compare real-time PCR, conventional PCR, and the direct agglutination test (DAT) for the diagnosis Leishmania infantum infection in 167 serum samples of domestic dog. Bone marrow was used for parasitological examination (smears and/or culture) in symptomatic visceral leishmaniasis, and serum was used for detection of L. infantum kinetoplast DNA (kDNA) by both conventional PCR and real-time PCR, while anti-L. infantum antibodies in sera were measured by DAT. The sera were collected from 37 symptomatic and 112 asymptomatic dogs during April to May 2011. Eighteen presumed negative samples were obtained from healthy dogs kept in non-endemic areas with no history of CVL and used as controls. All 18 samples were negative by DAT and Dipstick rK39. DAT confirmed previous exposure to L. infantum for all 149 serum samples collected from symptomatic and asymptomatic dogs in CVL endemic areas of Iran. Among the 37 symptomatic dogs, 20 (54%), 25 (67.6%), 36 (97.3%), and 37 (100%) showed L. infantum infection by parasitological methods, conventional PCR, real-time PCR, and DAT (≥ 1:80), respectively. Of 112 asymptomatic dogs, 79 (70.5%), 111 (99.1%), and 112 (100%) were shown to be positive by conventional PCR, and DAT (≥ 1:80), respectively. For ethical reasons, no asymptomatic or healthy control dogs were examined by parasitological methods. Three (16.7%) control dogs were positive by real-time PCR, but were negative by DAT, dipstick rK39, and conventional PCR methods. Parasitemia levels were measured by real-time PCR targeting kDNA using SYBR(®) green assay. This quantitative technique detected infection in 89.9% (150/167) of the domestic dogs that harbored L. infantum kDNA, ranging from 0.01 49 to 310.1 parasites/ml. The average was 16.60 parasites/ml. A good agreement (0.97) was found between real-time PCR and DAT at ≥ 1:80 titer, used as cut-off value by Kappa analysis. Thus

  6. Agglutination and Determination of Red Blood Cell Debris Interference Analysis of the Blood Analyzer%冷凝集和红细胞碎片对血液分析仪测定干扰分析

    Institute of Scientific and Technical Information of China (English)

    张颜粉

    2015-01-01

    Objective Analysis of erythrocyte agglutination and debris on the determination of the blood analyzer interference. MethodsRetrospective analysis of January 2014 January 2015 in our hospital 30 cases of mycoplasma pneumonia patient data, performed at 37℃ after extraction of venous blood, and blood analyzer blood indicators, analysis erythrocyte agglutination and debris.ResultsHematology analyzer results patient specimens at 37℃before the red blood cell count was significantly associated with inconsistent results, and abnormal red blood cell distribution histogram showed multiple peaks. ConclusionAgglutination of red blood cells and debris may cause falsely elevated or lowered blood indicators.%目的:分析冷凝集和红细胞碎片对血液分析仪测定结果的干扰性。方法回顾分析2014年1月~2015年1月本院30例支原体肺炎患者的资料,抽取患者静脉血后进行37℃水浴,并以血液分析仪测定血常规指标,分析冷凝集和红细胞碎片影响。结果患者标本37℃水浴前的血液分析仪的结果与红细胞计数结果不符,且红细胞直方图呈多峰异常分布。结论冷凝集和红细胞碎片可能会引起血常规指标假性升高或者降低。

  7. 草履蚧血细胞的显微形态与超微结构观察%The microscopic morphology and ultrastructure of hemocytes in the scale insect Drosicha corpulenta

    Institute of Scientific and Technical Information of China (English)

    魏丽芳; 谢映平

    2011-01-01

    为研究蚧虫血淋巴中血细胞的种类及其结构特征,采用相差显微镜、荧光显微镜和扫描电镜与透射电镜技术,观察了草履蚧雌成虫血细胞的显微形态与超微结构特征.结果显示,在荧光显微镜和扫描电镜下观察识别出草履蚧血淋巴中的5种血细胞,即:原血胞、浆血胞、粒血胞、囊血胞和类绛色血细胞.在透射电镜下可见,原血胞的细胞核明显,表现出高的细胞质密度;浆血胞最典型的特征是细胞质中有大量的囊泡;粒血胞的细胞质中有发达的粗面内质网和许多玫瑰形的细颗粒.类绛色血细胞最典型的特征是有许多结晶,并在细胞质的空泡区内分布的更多.囊血胞透明性强,具有围核空间,膨大成潴泡状.%For studying the types and structures of blood cells in the haemolymph of scale insects , the morphology and ultrastructure of hemocytes collected form the adult Drosicha corpulenta , Drosicha corpulenta( Hemiptera : Coccoidea : Monophlebidae) were observed by phase contrast microscope, fluorescence microscope, scanning electron microscopy and transmission electron microscopy. The result showed that five kinds of hemocytes such as prophemocytes , graulocytes, plasmatocytes , cystocytes and oenocytoid were observed in the haemolymph of the scale insect. Their typical characteristics were as follow : prophemocyte was the smallest one in the five types of the blood cells and it was with a clear cell nucleus and a high dense cytoplasmic; plasmatocyte was featured with a large number of vesicles in the cytoplasm; the cytoplasm of graulocytes has developed rough endoplasmic reticulum and many rose-shaped fine particles; oenocytoid was characterized by many crystalline in the cell; cystocytes has a circumference of nuclear space and swelling into a bubble retention.

  8. 虎红平板凝集试验确诊布鲁杆菌病截断值的探讨%Cut-off value of rose bengal plate agglutination test in rapid diagnosis of brucellosis

    Institute of Scientific and Technical Information of China (English)

    马志东; 刘云霞; 李艳红; 李建云

    2013-01-01

    Objective To find out the cut-off value of rose bengal plate agglutination test(RBPT) in rapid diagnosis of brucellosis.Methods From May to June 2009,398 people who came to the outpatient department of Inner Mongolia Center for Endemic Disease Control and Research were diagnosed brucellosis by RBPT and tube agglutination test (SAT).Tube agglutination test as a gold standard,rose bengal plate agglutination test,its authenticity and reliability were evaluated.Results Taking positive predictive value 100.0% as the selection standard,the cut-off value of RBPT was "++" which could be used to diagnose brucellosis.The sensitivity was 83.3%; the specificity was 100.0%; the Youden index was 0.832; and the compliance rote was 89.9%.Conclusion The cut-off value "++" of RBPT to diagnosis brucellosis is worthy of clinical promotion.%目的 探讨虎红平板凝集试验确诊布鲁杆菌病凝集度的截断值.方法 内蒙古地方病防治研究中心门诊部选择2009年5月至2009年6月,进行布鲁杆菌病检查的398人,进行试管凝集试验和虎红平板凝集试验检测,以试管凝集试验为金标准,探讨虎红平板凝集试验诊断布鲁杆菌病的截断值,并对可靠性和真实性进行评价.结果 以阳性预测值100.0%作为筛选标准,虎红平板凝集试验快速确诊布鲁杆菌病的截断值为“++”,以该凝集度强度作为截断值进行布鲁杆菌病诊断,灵敏度为83.3%,特异度为100.0%,约登指数为0.823,符合率为89.9%.结论 以虎红平板凝集试验的“++”作为截断值诊断布鲁杆菌病,值得临床推广.

  9. Caracterização citológica dos hemócitos de Anticarsia gemmatalis (Lepidoptera, Noctuidae em larvas resistentes ao vírus AgMNPV Citological characterization of the Anticarsia gemmatalis (Lepidoptera, Noctuidae hemocytes in resistant larvae to the virus AgMNPV

    Directory of Open Access Journals (Sweden)

    Maria C. C. de Negreiro

    2009-03-01

    Full Text Available A ocorrência de larvas de Anticarsia gemmatalis (Hübner, 1932 resistentes ao vírus AgMNPV em laboratório levou ao estudo dos hemócitos deste inseto para avaliar sua participação nos mecanismos que possibilitam a resistência ao vírus. As larvas resistentes com 6 - 11 dias de desenvolvimento (3º a 5º instar foram anestesiadas por resfriamento e rapidamente limpas em álcool 70%. A hemolinfa foi coletada através de punção abdominal, a análise morfológica foi realizada em contraste de fase e esfregaços corados com solução de Seller. A contagem total de hemócitos (CTH foi realizada em câmara de Neubauer com hemolinfa não diluída. Para a contagem diferencial de hemócitos (CDH, utilizou-se hemolinfa diluída em solução anticoagulante para insetos. Foram identificados seis tipos de hemócitos: plasmatócitos (38,5%, granulócitos (22,6%, oenocitóides (20,4%, esferulócitos (14,5%, prohemócitos (2,3% e vermiformes (1,5%. O número total de hemócitos mostrou um aumento significativo durante o período larval estudado.The occurrence of Anticarsia gemmatalis (Hübner, 1932 larvae resistant to the virus AgMNPV in laboratory led to the study of the hemocytes of this insect, in order to evalue its participation in the mechanisms which enables the resistance to the virus. The resistant larvae with 6 to 11 developmental days old (3rd to 5th instars were anesthetized by cold and after that were quickly cleaned in 70% alcohol. The hemolymph was collected through abdominal puncturing; the morphological analysis was made in phase contrast and colored smears with Seller solution. The total hemocytes counting (THC, was made in modified Neubauer chamber with not diluted hemolymph. For the differential hemocytes counting (DHC, was used diluted hemolymph in anticoagulant solution for insects. Six hemocytes types were identified: plasmatocytes (38.5%, granulocytes (22.6%, oenocytoids (20.4%, spherulocytes (14.5%, prohemocytes (2.3% and

  10. Protection of mice against Chlamydophila abortus infection with a bacteriophage-mediated DNA vaccine expressing the major outer membrane protein.

    Science.gov (United States)

    Ling, Yong; Liu, Wei; Clark, Jason R; March, John B; Yang, Junjing; He, Cheng

    2011-12-15

    A bacteriophage-delivered DNA vaccine against Chlamydophila abortus was constructed by cloning a eukaryotic cassette containing the ompA gene (which expresses the Major Outer Membrane Protein) into a bacteriophage lambda vector. Four groups, each of 20 BALB/c mice were inoculated separately with the phage vaccine, a conventional DNA vaccine based on the same ompA expression cassette, a live attenuated vaccine (strain 1B) or the empty phage vector. The phage and DNA vaccines and empty phage vector were administered intramuscularly on days 0, 14 and 28; the attenuated vaccine was given once on day 0. Half the animals in each group were challenged on day 42 by intraperitoneal injection of live C. abortus and sacrificed on day 49. Phage-vaccinated mice developed moderate antibody levels against C. abortus and yielded higher levels of IFN-γ and IL-2 compared with the attenuated live vaccine group. Clearance of chlamydiae from spleens was significantly better in the attenuated vaccine group compared with the phage vaccine group, while both groups were significantly superior to the DNA vaccine and control groups (p<0.01). Although levels of protection in the mouse model were lower in phage-vaccinated animals, than in 1B vaccinated animals, phage vaccines offer several other advantages, such as easier handling and safety, potentially cheaper production and no chance of reversion to virulence. Although these are preliminary results in a model system, it is possible that with further optimisation immunization with phage vaccines may provide a novel way to improve protection against C. abortus infection and trials in large animals are currently being initiated.

  11. A Constitutively Mannose-Sensitive Agglutinating Salmonella enterica subsp. enterica Serovar Typhimurium Strain, Carrying a Transposon in the Fimbrial Usher Gene stbC, Exhibits Multidrug Resistance and Flagellated Phenotypes

    Directory of Open Access Journals (Sweden)

    Kuan-Hsun Wu

    2012-01-01

    Full Text Available Static broth culture favors Salmonella enterica subsp. enterica serovar Typhimurium to produce type 1 fimbriae, while solid agar inhibits its expression. A transposon inserted in stbC, which would encode an usher for Stb fimbriae of a non-flagellar Salmonella enterica subsp. enterica serovar Typhimurium LB5010 strain, conferred it to agglutinate yeast cells on both cultures. RT-PCR revealed that the expression of the fimbrial subunit gene fimA, and fimZ, a regulatory gene of fimA, were both increased in the stbC mutant when grown on LB agar; fimW, a repressor gene of fimA, exhibited lower expression. Flagella were observed in the stbC mutant and this phenotype was correlated with the motile phenotype. Microarray data and RT-PCR indicated that the expression of three genes, motA, motB, and cheM, was enhanced in the stbC mutant. The stbC mutant was resistant to several antibiotics, consistent with the finding that expression of yhcQ and ramA was enhanced. A complementation test revealed that transforming a recombinant plasmid possessing the stbC restored the mannose-sensitive agglutination phenotype to the stbC mutant much as that in the parental Salmonella enterica subsp. enterica serovar Typhimurium LB5010 strain, indicating the possibility of an interplay of different fimbrial systems in coordinating their expression.

  12. Usefulness of the rK39-Immunochromatographic Test, Direct Agglutination Test, and Leishmanin Skin Test for Detecting Asymptomatic Leishmania Infection in Children in a New Visceral Leishmaniasis Focus in Amhara State, Ethiopia

    Science.gov (United States)

    Gadisa, Endalamaw; Custodio, Estefanía; Cañavate, Carmen; Sordo, Luis; Abebe, Zelalem; Nieto, Javier; Chicharro, Carmen; Aseffa, Abraham; Yamuah, Lawrence; Engers, Howard; Moreno, Javier; Cruz, Israel

    2012-01-01

    In areas where visceral leishmaniasis is anthroponotic, asymptomatically infected patients may play a role in transmission. Additionally, the number of asymptomatic patients in a disease-endemic area will also provide information on transmission dynamics. Libo Kemkem and Fogera districts (Amhara State, Ethiopia) are now considered newly established areas to which visceral leishmaniasis is endemic. In selected villages in these districts, we conducted a study to assess the usefulness of different approaches to estimate the asymptomatic infection rate. Of 605 participants, the rK39 immunochromatographic test was able to detect asymptomatic infection in 1.5% (9 of 605), direct agglutination test in 5.3% (32 of 605), and leishmanin skin test in 5.6% (33 of 589); the combined use of serologic methods and leishmanin skin test enabled detecting asymptomatic infection in 10.1% (61 of 605). We conclude that the best option to detect asymptomatic infection in this new visceral leishmaniasis–endemic focus is the combined use of the direct agglutination test and the leishmanin skin test. PMID:22556076

  13. Direct appraisal of latex agglutination testing, a convenient alternative to enzyme immunoassay for the detection of rotavirus in childhood gastroenteritis, by comparison of two enzyme immunoassays and two latex tests.

    Science.gov (United States)

    Sambourg, M; Goudeau, A; Courant, C; Pinon, G; Denis, F

    1985-04-01

    During February and March 1984, 207 fecal samples from infants and children with gastroenteritis were tested for rotavirus with four techniques: two enzyme immunoassays (Rotazyme; Abbott Laboratories, North Chicago, Ill., and Enzygnost-Rotavirus; Calbiochem-Behring, La Jolla, Calif.) and two latex agglutination tests (Rotalex; Orion Research, Inc., Cambridge, Mass., and Slidex Rota-Kit; Biomérieux). All stool samples were also tested for yeasts and bacterial pathogens. Electron microscopy was used to investigate discrepant results. We found 47% positive samples with Enzygnost-Rotavirus, 38% with Rotazyme, 37% with Slidex Rota-Kit, and 34% with Rotalex. No specimen was found positive by Rotazyme only or Slidex Rota-Kit only. On the contrary, 12 samples which were positive with Enzygnost-Rotavirus only and 3 which were positive with Rotalex only were not confirmed as positive by electron microscopy. Both enzyme immunoassays gave 6% equivocal results; Slidex Rota-Kit gave significantly fewer equivocal results than did Rotalex: 2.9% versus 9.7% (P less than 0.01). The sensitivity and specificity of latex tests compared favorably with that of enzyme immunoassays. Latex agglutination tests can be performed by unskilled personnel and are rapid and relatively cheap. They appear to be very suitable for routine laboratory work and may prove useful for large-scale screening in developing countries.

  14. Usefulness of the rK39-immunochromatographic test, direct agglutination test, and leishmanin skin test for detecting asymptomatic Leishmania infection in children in a new visceral leishmaniasis focus in Amhara State, Ethiopia.

    Science.gov (United States)

    Gadisa, Endalamaw; Custodio, Estefanía; Cañavate, Carmen; Sordo, Luis; Abebe, Zelalem; Nieto, Javier; Chicharro, Carmen; Aseffa, Abraham; Yamuah, Lawrence; Engers, Howard; Moreno, Javier; Cruz, Israel

    2012-05-01

    In areas where visceral leishmaniasis is anthroponotic, asymptomatically infected patients may play a role in transmission. Additionally, the number of asymptomatic patients in a disease-endemic area will also provide information on transmission dynamics. Libo Kemkem and Fogera districts (Amhara State, Ethiopia) are now considered newly established areas to which visceral leishmaniasis is endemic. In selected villages in these districts, we conducted a study to assess the usefulness of different approaches to estimate the asymptomatic infection rate. Of 605 participants, the rK39 immunochromatographic test was able to detect asymptomatic infection in 1.5% (9 of 605), direct agglutination test in 5.3% (32 of 605), and leishmanin skin test in 5.6% (33 of 589); the combined use of serologic methods and leishmanin skin test enabled detecting asymptomatic infection in 10.1% (61 of 605). We conclude that the best option to detect asymptomatic infection in this new visceral leishmaniasis-endemic focus is the combined use of the direct agglutination test and the leishmanin skin test.

  15. 比浊法血小板聚集试验的影响因素研究%Analysis of influencing factors of turbidimetry for platelet agglutination test

    Institute of Scientific and Technical Information of China (English)

    石红婷; 周伯荣; 王融; 邓燕华; 关海涛; 刘子凡

    2012-01-01

    目的 在不同的实验条件下,观察影响比浊法血小板聚集试验的因素.方法 选择30例健康对照组和204例病例组,观察不同的血小板计数及诱导剂浓度对糖尿病患者餐前与餐后、采血后的检测时间、诱导剂的种类互补等不同试验条件下的血小板聚集率.结果 随着血小板计数的减少或增加,血小板聚集率相应的减少或增加;二磷酸腺苷(ADP)、胶原(COL)、花生四烯酸(AA)浓度增加,血小板最大聚集率增大,相应的阿司匹林抵抗(AR)或氯吡格雷抵抗(CR)的检出率增高;服用不同的抗血小板药物,糖尿病患者对以ADP、COL、AA为诱导剂测定的血小板聚集率,其影响不同;未空腹及检测时间超过3 h重复性较差;服药2周后,COL、AA诱导的血小板聚集率下降不明的患者,随着时间的延长,6个月后易重新出现CR.结论 血小板计数、诱导剂浓度、糖尿病疾病、空腹状态、检测时间与比浊法检测血小板聚集率相关,以ADP、COL、AA作诱导剂检测血小板聚集率较单一的ADP更全面、准确.%Objective To investigate the influencing factors of turbidimetry for platelet agglutination test under different experi -mental conditions .Methods 30 cases of healthy subjects and 204 cases of patents were enrolled . The rate of platelet aggregation (RPA ) was detected under different conditions , including platelet counts ,inducer concentration and types , diabetes mellitus (DM ), fasting state and detection time .Results With the increase or decrease of platelet counts , RPA varied correspondingly . With the increase of the concentration of adenosine diphosphate(ADP), collagen (COL) and arachidonic acid(AA), the maxim RPA and the detection rate of aspirin resistance (AR) or clopidogre resistance(CR) increased . For patients with DM , taking different antiplatelet drugs (aspirin or clopidogrel), inducers of ADP, COL and AA were with different influence on RPA . None-fasting and

  16. Títulos de anticorpos aglutinantes induzidos por vacinas comerciais contra leptospirose bovina Agglutinating antibody titers induced by commercial vaccines against bovine leptospirosis

    Directory of Open Access Journals (Sweden)

    Gabriela de Godoy Cravo Arduino

    2009