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Sample records for acanthamoeba castellanii encystment

  1. Francisella tularensis type A Strains Cause the Rapid Encystment of Acanthamoeba castellanii and Survive in Amoebal Cysts for Three Weeks post Infection

    El-Etr, S H; Margolis, J; Monack, D; Robison, R; Cohen, M; Moore, E; Rasley, A

    2009-07-28

    Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown, and the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype (REP) is caused by factor(s) secreted by amoebae and/or F. tularensis into the co-culture media. Further, our results indicate that in contrast to LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks post infection and that induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that the interactions between F. tularensis strains and amoeba may play a role in the environmental persistence of F. tularensis.

  2. Acanthamoeba castellanii STAT protein.

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  3. Acanthamoeba castellanii STAT protein.

    Anna Kicinska

    Full Text Available STAT (signal transducers and activators of transcription proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil, a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups.

  4. Gene discovery in the Acanthamoeba castellanii genome

    Anderson, Iain J.; Watkins, Russell F.; Samuelson, John; Spencer,David F.; Majoros, William H.; Gray, Michael W.; Loftus, Brendan J.

    2005-08-01

    Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit adversity of environments. Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptorserine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.

  5. Acanthamoeba encystment: multifactorial effects of buffers, biocides, and demulcents present in contact lens care solutions

    Kovacs CJ

    2015-10-01

    Full Text Available Christopher J Kovacs, Shawn C Lynch, Marjorie J Rah, Kimberly A Millard, Timothy W Morris Bausch & Lomb Incorporated, Rochester, NY, USA Purpose: To determine whether agents which are purportedly capable of inducing encystment of Acanthamoeba can recapitulate the signal when tested in differing formulations. Methods: In accordance with the International Standard ISO 19045, Acanthamoeba castellanii ATCC 50370 trophozoites were cultured in antibiotic-free axenic medium, treated with test solutions, and encystment rates plus viability were measured via bright field and fluorescent microscopy. Test solutions included phosphate-buffered saline (PBS, borate-buffered saline, biguanide- and hydrogen peroxide (H2O2-based biocides, propylene glycol (PG and povidone (POV ophthalmic demulcents, and one-step H2O2-based contact lens disinfection systems. Results: Only PBS solutions with 0.25 ppm polyaminopropyl biguanide (PAPB and increasing concentrations of PG and POV stimulated A. castellanii encystment in a dose-dependent manner, whereas PBS solutions containing 3% H2O2 and increasing concentrations of PG and POV did not stimulate encystment. Borate-buffered saline and PBS/citrate solutions containing PG also did not stimulate encystment. In addition, no encystment was observed after 24 hours, 7 days, or 14 days of exposures of trophozoites to one-step H2O2 contact lens disinfection products or related solutions. Conclusion: The lack of any encystment observed when trophozoites were treated with existing or new one-step H2O2 contact lens care products, as well as when trophozoites were exposed to various related test solutions, confirms that Acanthamoeba encystment is a complex process which depends upon simultaneous contributions of multiple factors including buffers, biocides, and demulcents. Keywords: propylene glycol, contact lens care system, hydrogen peroxide disinfecting solution

  6. Anaerobic respiration: In vitro efficacy of Nitazoxanide against mitochondriate Acanthamoeba castellanii of the T4 genotype.

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Farooq, Maria; Khan, Naveed Ahmed

    2015-10-01

    Acanthamoeba is an opportunistic protist pathogen that is responsible for serious human and animal infection. Being one of the most frequently isolated protists from the environment, it is likely that it readily encounters microaerophilic environments. For respiration under anaerobic or low oxygen conditions in several amitochondriate protists, decarboxylation of pyruvate is catalyzed by pyruvate ferredoxin oxidoreductase instead of pyruvate dehydrogenase. In support, Nitazoxanide, an inhibitor of pyruvate ferredoxin oxidoreductase, is effective and non-mutagenic clinically against a range of amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The overall aim of the present study was to determine in vitro efficacy of Nitazoxanide against Acanthamoeba castellanii. At micromolar concentrations, the findings revealed that Nitazoxanide neither affected A. castellanii growth or viability nor amoeba-mediated host cell monolayer damage in vitro or extracellular proteolytic activities. Similarly, microaerophilic conditions alone had no significant effects. In contrast, microaerophilic conditions together with Nitazoxanide showed amoebicidal effects and inhibited A. castellanii-mediated host cell monolayer damage as well as extracellular proteases. Using encystation assays, it was observed that Nitazoxanide inhibited trophozoite transformation into cysts both under aerophilic and microaerophilic conditions. Furthermore, pre-treatment of cysts with Nitazoxanide inhibited A. castellanii excystation. These findings are important in the identification of potential targets that could be useful against parasite-specific respiration as well as to understand the basic biology of the life cycle of Acanthamoeba. PMID:26297676

  7. Purification and characterization of transcription factor IIIA from Acanthamoeba castellanii

    Polakowski, Nicholas; Paule, Marvin R.

    2002-01-01

    TFIIIA is required to activate RNA polymerase III transcription from 5S RNA genes. Although all known TFIIIA homologs harbor nine zinc fingers that mediate DNA binding, very limited sequence homology is found among these proteins, which reflects unique properties of some TFIIIA homologs. For example, the Acanthamoeba castellanii homolog directly regulates 5S RNA transcription. We have purified and characterized A.castellanii TFIIIA (AcTFIIIA) as a step toward obtaining a clearer understanding...

  8. Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51)

    Lamb, David C.; Warrilow, Andrew G. S.; Rolley, Nicola J.; Parker, Josie E.; Nes, W. David; Smith, Stephen N; Kelly, Diane E.; Kelly, Steven L.

    2015-01-01

    In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole e...

  9. Temperature-induced membrane-lipid adaptation in Acanthamoeba castellanii.

    Jones, A L; Hann, A C; Harwood, J L; Lloyd, D

    1993-02-15

    A method has been developed for the separation of the major membrane fractions of Acanthamoeba castellanii after growth at different temperatures. The acyl-lipid compositions of individual membrane fractions, microsomal membranes, plasma membrane and mitochondria were analysed after a shift in culture temperature from 30 degrees C to 15 degrees C. The major change in lipid composition observed was an alteration in the relative proportions of oleate and linoleate. This reciprocal change was seen in all the membrane fractions, but occurred most rapidly in the phosphatidylcholine of the microsomal fraction. Thus, there appears to be a rapid induction of delta 12-desaturase activity in A. castellanii after a downward shift in growth temperature. Changes were also seen in the proportions of the n-6 C20 fatty acids, with a decrease in the proportions of icosadienoate and increases of icosatrienoate and arachidonate. However, unlike the alteration in oleate/linoleate ratios, this change was not seen in all the individual lipids of each membrane fraction. PMID:8439295

  10. Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii Trophozoites and Cysts

    Matin, Abdul; Jung, Suk-Yul

    2011-01-01

    The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. ...

  11. Artemether Exhibits Amoebicidal Activity against Acanthamoeba castellanii through Inhibition of the Serine Biosynthesis Pathway

    Deng, Yihong; Ran, Wei; Man, Suqin; Li, Xueping; Gao, Hongjian; Tang, Wei; Tachibana, Hiroshi; Cheng, Xunjia

    2015-01-01

    Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner a...

  12. Acanthamoeba castellanii induces host cell death via a phosphatidylinositol 3-kinase-dependent mechanism

    Sissons, James; Kim, Kwang Sik; Stins, Monique; Jayasekera, Samantha; Alsam, Selwa; Khan, Naveed Ahmed

    2005-01-01

    Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent as...

  13. Acanthamoeba castellanii Induces Host Cell Death via a Phosphatidylinositol 3-Kinase-Dependent Mechanism

    Sissons, James; Kim, Kwang Sik; Stins, Monique; Jayasekera, Samantha; Alsam, Selwa; Khan, Naveed Ahmed

    2005-01-01

    Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase. PMID:15845472

  14. Superdiffusion dominates intracellular particle motion in the supercrowded space of pathogenic Acanthamoeba castellanii

    Reverey, J F; Bao, H; Leippe, M; Metzler, R; Selhuber-Unkel, C

    2015-01-01

    Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements, and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin, and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved ...

  15. Effects of pesticides, polychlorinated biphenyls and metals on the growth and reproduction of Acanthamoeba castellanii

    Prescott, L.M.; Kubovec, M.K.; Tryggestad, D.

    1977-07-01

    The effects of pollutants (pesticides, PCB and metals) were studied in the free-living amoeba, Acanthamoeba castellanii. Eight pesticides were used--the insecticides dieldrin, aldrin and sevin, and the herbicides linuron, stam F-34, IPC, atrazine and simazine. It was shown that the sensitivity of A. castellanii to pesticides varied greatly. The population growth was inhibited by linuron, stam F-34, IPC, sevin and atrazine at a level of 10 mg/l. The polychlorinated biphenyl, Arochor 1254, had no significant effect at a concentration of 0.01 mg/l (10 ppb). The studies with metal ions showed that A. castellanii was unaffected by moderately high levels of Cu and Zn, but was sensitive to the presence of Pb and mercuric ions.

  16. The role of Src kinase in the biology and pathogenesis of Acanthamoeba castellanii

    Siddiqui Ruqaiyyah

    2012-06-01

    Full Text Available Abstract Background Acanthamoeba species are the causative agents of fatal granulomatous encephalitis in humans. Haematogenous spread is thought to be a primary step, followed by blood–brain barrier penetration, in the transmission of Acanthmaoeba into the central nervous system, but the associated molecular mechanisms remain unclear. Here, we evaluated the role of Src, a non-receptor protein tyrosine kinase in the biology and pathogenesis of Acanthamoeba. Methods Amoebistatic and amoebicidal assays were performed by incubating amoeba in the presence of Src kinase-selective inhibitor, PP2 (4-amino-5-(4-chlorophenyl-7-(t-butylpyrazolo[3,4-d]pyrimidine and its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyrimidine. Using this inhibitor, the role of Src kinase in A. castellanii interactions with Escherichia coli was determined. Zymographic assays were performed to study effects of Src kinase on extracellular proteolytic activities of A. castellanii. The human brain microvascular endothelial cells were used to determine the effects of Src kinase on A. castellanii adhesion to and cytotoxicity of host cells. Results Inhibition of Src kinase using a specific inhibitor, PP2 (4-amino-5-(4 chlorophenyl-7-(t-butylpyrazolo [3,4-d] pyrimidine but not its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d] pyrimidine, had detrimental effects on the growth of A. castellanii (keratitis isolate, belonging to the T4 genotype. Interestingly, inhibition of Src kinase hampered the phagocytic ability of A. castellanii, as measured by the uptake of non-invasive bacteria, but, on the contrary, invasion by pathogenic bacteria was enhanced. Zymographic assays revealed that inhibition of Src kinases reduced extracellular protease activities of A. castellanii. Src kinase inhibition had no significant effect on A. castellanii binding to and cytotoxicity of primary human brain microvascular endothelial cells, which constitute the blood–brain barrier

  17. Cellular Response of the Amoeba Acanthamoeba castellanii to Chlorine, Chlorine Dioxide, and Monochloramine Treatments ▿

    Mogoa, Emerancienne; Bodet, Charles; Morel, Franck; Rodier, Marie-Hélène; Legube, Bernard; Héchard, Yann

    2011-01-01

    Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested o...

  18. Azole Antifungal Agents To Treat the Human Pathogens Acanthamoeba castellanii and Acanthamoeba polyphaga through Inhibition of Sterol 14α-Demethylase (CYP51).

    Lamb, David C; Warrilow, Andrew G S; Rolley, Nicola J; Parker, Josie E; Nes, W David; Smith, Stephen N; Kelly, Diane E; Kelly, Steven L

    2015-08-01

    In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kd of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 μM), whereas inhibition by fluconazole was weak (IC50, 30 μM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections. PMID:26014948

  19. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii

    Shanan, Salah; Bayoumi, Magdi; Saeed, Amir; Sandström, Gunnar; Abd, Hadi

    2016-01-01

    Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11,...

  20. Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila

    Yousuf, Farzana Abubakar; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2013-01-01

    Background Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts. Methods Here, we used a keratitis isolate of A. castellanii of the T4 genotyp...

  1. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii

    Salah Shanan

    2016-04-01

    Full Text Available Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 with A. castellanii. The interaction between A. castellanii and V. cholerae strains was studied by means of amoeba cell counts, viable counts of the bacteria in the absence or presence of amoebae, and of the intracellularly growing bacteria, visualised by electron microscopy. These results show that all V. cholerae can grow and survive outside and inside the amoebae, disclosing that V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 all can be considered as facultative intracellular bacteria.

  2. Caspase-like proteins: Acanthamoeba castellanii metacaspase and Dictyostelium discoideum paracaspase, what are their functions?

    Entsar Saheb; Wendy Trzyna; John Bush

    2014-12-01

    Caspases are cysteine proteases that are important regulators of programmed cell death in animals. Two novel relatives to members of the caspase families metacaspases and paracaspase have been discovered. Metacaspase type-1 was identified in Acanthamoeba castellanii, an opportunistic protozoan parasite that causes severe diseases in humans. Paracaspase was found in the non-pathogenic protozoan Dictyostelium discoideum. Since their discovery in Acanthamoeba and Dictyostelium, metacaspases and paracaspases have remained poorly characterized. At present we do not have sufficient data about the molecular function of these caspase-like proteins or their role, if any, in programmed cell death. How these caspase proteins function at the molecular level is an important area of study that will provide insight into their potential for treatment therapies against Acanthamoeba infection and other similar parasitic protozoan. Additionally, finding the molecular functions of these caspase-like proteins will provide information concerning their role in more complex organisms.The aim of this article was to review recent discoveries about metacaspases and paracaspases as regulators of apoptotic and non-apoptotic processes.

  3. Rapid induction of microsomal delta 12(omega 6)-desaturase activity in chilled Acanthamoeba castellanii.

    Jones, A L; Lloyd, D; Harwood, J L

    1993-11-15

    The activity of microsomal delta 12-desaturase in Acanthamoeba castellanii was increased after growing cultures were chilled from the optimal growth temperature (30 degrees C) to 15 degrees C. This increase was detectable in microsomes isolated from organisms subjected to only 10 min chilling. The mechanism of induction was investigated. The increase in activity on chilling was greatly reduced when protein synthesis was blocked before the temperature shift. Thus the major mechanism for the induction of delta 12-desaturase is increased protein synthesis. delta 12-Desaturase activity was higher when assayed at 20 degrees C than when assayed at 30 degrees C, but these changes were not due to the increased solubility of O2 at 20 degrees C. The major substrate of delta 12-desaturase was found to be 1-acyl-2-oleoyl phosphatidylcholine. PMID:8250841

  4. Oxygen induces fatty acid (n-6)-desaturation independently of temperature in Acanthamoeba castellanii.

    Thomas, K; Rutter, A; Suller, M; Harwood, J; Lloyd, D

    1998-03-20

    Induction of a microsomal oleate delta12 (n-6) desaturase which is mainly responsible for an increase in membrane lipid unsaturation at low temperature has been observed in the free-living amoeba Acanthamoeba castellanii. In this study we show that the enzyme can also be regulated by oxygen independently of temperature in batch cultures grown to O2-limitation. Raising the oxygen concentration from below the lower limit of detection (< 0.1 microM) to approximately air-saturation (230 microM), whilst maintaining the growth temperature constant (30 degrees C), increased lipid unsaturation and elevated n-6-desaturase activity 2.3-fold. Addition of the protein synthesis inhibitor, anisomycin, showed that increased desaturase activity was due to new protein synthesis rather than activation of pre-existing enzyme. These observations are important for future studies of the mechanism of temperature adaptation in poikilotherms. PMID:9541030

  5. Molecular Characterization and Sequencing of a Gene Encoding Mannose Binding Protein in an Iranian Isolate of Acanthamoeba castellanii as a Major Agent of Acanthamoeba Keratitis

    SH Farnia

    2008-07-01

    Full Text Available Background: Acanthamoeba castellanii is the important cause of amoebic keratitis in Iran. The key molecule in pathogenesis of Acanthamoeba keratitis is Mannose Binding Protein (MBP led to adhesion of amoeba to corneal epithelium. Subsequent to adhesion other cytopathic effects occur. The goal of this study was to identify the molecular characterization of a gene encoding MBP in an Iranian isolate of A.castellanii in order to pave the way for further investigations such as new therapeutic advances or immunization. Methods: A.castellanii was cultured on non nutrient agar. Extraction of DNA was performed by phenol-chloroform method. After designing a pair of primer for the gene encoding MBP, PCR analysis was performed. Finally, the PCR product has been sequenced and the result submitted to the gene data banks. Results: An MBP gene of 1081 nucleotides was sequenced. This fragment contained three introns and encodes a protein with 194 amino acids. Homology search by Blast program showed a significant homology with the MBP gene in gene data banks (96%. Besides, the identity of amino acids with the other MBPs in gene data banks was about 86%. Conclusion: We isolated and sequenced a gene fragment encoding MBP in an Iranian isolate of A.castellanii. Molecular characterization of this important gene is the first step in pursuing researches such as developing better therapeutic agents, immunization of population at risk or even developing a diagnostic tool by PCR techniques.

  6. Survival of Campylobacter jejuni in co-culture with Acanthamoeba castellanii: role of amoeba-mediated depletion of dissolved oxygen

    Bui, Thanh Xuan; Winding, Anne; Qvortrup, Klaus;

    2012-01-01

    Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation...... of intracellular C. jejuni in A. castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A. castellanii promoted the extracellular growth of C. jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae – bacteria contact. The growth....... Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae – bacteria contact were much lower than those observed with culture media or with C. jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C. jejuni under microaerophilic...

  7. Tracking amino acid's uptake into the protozoan Acanthamoeba castellanii by stable-isotope labelling and Raman spectral imaging

    Naemat, Abida; Elsheikha, Hany M.; Notingher, Ioan

    2016-04-01

    The capacity of pathogens to acquire nutrients from their host cells is one of the most fundamental aspects of infection biology. Hence, measuring the patterns of nutrients' uptake by pathogens is essential for understanding the interactions of pathogens with eukaryotic host cells. In this study, we optimized a technique that allows fast and non-destructive measurement of the amino acid Phenylalanine (Phe) acquired by the trophozoite stage of the protozoan Acanthamoeba castellanii (A. castellanii) as they engage with individual human retinal pigment epithelial cells (ARPE-19). ARPE-19 host cells were pre-saturated with Deuterated Phe (L-Phe(D8)) to replace the native substrate Phe (L-Phe). The uptake of L-Phe(D8) by A. castellanii trophozoites was measured by Raman microspectroscopy. This approach allowed us to characterize the uptake patterns of this essential amino acid into A. castellanii trophozoites at a single cell level. At 24 hours post infection (PI) A. castellanii trophozoites are capable of salvaging L-Phe(D8) from host cells. The uptake pattern was time-dependent during the first 24 hours of infection and complete substitution with L-Phe(D8) in all parasites was detected at 48 hours PI. On the other hand, isolated A. castellanii trachyzoites (grown without host cells) did not show significant uptake for L-Phe(D8) from the media; only achieved an uptake ratio of 16-18% of L-Phe(D8) from the culture medium after 24 hours. These findings demonstrate the potential of combining Raman microspectroscopy and stable isotope labelling approaches to elucidate the role of metabolism in mediating A. castellanii interaction with host cells.

  8. Oxygen induction of a novel fatty acid n-6 desaturase in the soil protozoon, Acanthamoeba castellanii.

    Rutter, Andrew J; Thomas, Katie L; Herbert, Derek; Henderson, R James; Lloyd, David; Harwood, John L

    2002-11-15

    Induction of fatty acid desaturation is very important for the temperature adaptation of poikilotherms. However, in oxygen-limited late-exponential-phase Acanthamoeba castellanii cultures, oxygen alone was able to induce increased activity of a fatty acid desaturase that converts oleate into linoleate and which has been implicated in the temperature adaptation of this organism. Experiments with Delta(10)-nonadecenoate showed that the enzyme is an n -6 desaturase rather than a Delta(12)-desaturase. It also used preferentially 1-acyl-2-oleoyl-phosphatidylcholine as substrate and NAD(P)H as electron donor. The involvement of cytochrome b (5) as an intermediate electron carrier was shown by difference spectra measurements and anti-(cytochrome b (5)) antibody experiments. Of the three protein components of the desaturase complex, oxygen only increased the activity of the terminal (cyanide-sensitive) protein during n -6 desaturase induction. The induction of this terminal protein paralleled well the increase in overall oleate n -6 desaturation. The ability of oxygen to induce oleate desaturase independently of temperature in this lower eukaryotic animal model is of novel intrinsic interest, as well as being important for the design of future experiments to determine the molecular mechanism of temperature adaptation in poikilotherms. PMID:12153399

  9. In Vitro Efficacies of Clinically Available Drugs against Growth and Viability of an Acanthamoeba castellanii Keratitis Isolate Belonging to the T4 Genotype

    Baig, Abdul Mannan; Iqbal, Junaid; Khan, Naveed Ahmed

    2013-01-01

    The effects of clinically available drugs targeting muscarinic cholinergic, adrenergic, dopaminergic, and serotonergic receptors; intracellular calcium levels and/or the function of calcium-dependent biochemical pathways; ion channels; and cellular pumps were tested against a keratitis isolate of Acanthamoeba castellanii belonging to the T4 genotype. In vitro growth inhibition (amoebistatic) assays were performed by incubating A. castellanii with various concentrations of drugs in the growth ...

  10. The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum.

    Wojtkowska, Małgorzata; Buczek, Dorota; Stobienia, Olgierd; Karachitos, Andonis; Antoniewicz, Monika; Slocinska, Małgorzata; Makałowski, Wojciech; Kmita, Hanna

    2015-07-01

    Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle. PMID:26074248

  11. The role of Src kinase in the biology and pathogenesis of Acanthamoeba castellanii

    Siddiqui Ruqaiyyah; Iqbal Junaid; Maugueret Marie-josée; Khan Naveed

    2012-01-01

    Abstract Background Acanthamoeba species are the causative agents of fatal granulomatous encephalitis in humans. Haematogenous spread is thought to be a primary step, followed by blood–brain barrier penetration, in the transmission of Acanthmaoeba into the central nervous system, but the associated molecular mechanisms remain unclear. Here, we evaluated the role of Src, a non-receptor protein tyrosine kinase in the biology and pathogenesis of Acanthamoeba. Methods Amoebistatic and amoebicidal...

  12. A bifunctional Delta12,Delta15-desaturase from Acanthamoeba castellanii directs the synthesis of highly unusual n-1 series unsaturated fatty acids.

    Sayanova, Olga; Haslam, Richard; Guschina, Irina; Lloyd, David; Christie, William W; Harwood, John L; Napier, Johnathan A

    2006-12-01

    The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist. PMID:16950768

  13. Effect of environmental stress factors on the uptake and survival of Campylobacter jejuni in Acanthamoeba castellanii

    Bui, Thanh Xuan; Qvortrup, Klaus; Wolff, Anders;

    2012-01-01

    Background: Campylobacter jejuni is a major cause of bacterial food-borne illness in Europe and North America. The mechanisms allowing survival in the environment and transmission to new hosts are not well understood. Environmental free-living protozoa may facilitate both processes. Pre-exposure to...... heat, starvation, oxidative or osmotic stresses encountered in the environment may affect the subsequent interaction of C. jejuni with free-living protozoa. To test this hypothesis, we examined the impact of environmental stress on expression of virulence-associated genes (ciaB, dnaJ, and htrA) of C...... could be recovered at this time point. Overall, pre-exposure of C. jejuni to environmental stresses did not promote intracellular survival in A. castellanii. Conclusions: Together, these findings suggest that the stress response in C. jejuni and its interaction with A. castellanii are complex and...

  14. Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival of Vibrio cholerae and Suppresses Viability of Acanthamoeba castellanii

    Soni Priya Valeru

    2014-01-01

    Full Text Available Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive inside Acanthamoeba castellanii. It has been shown that V. cholerae expresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA and outer membrane vesicles (OMVs in survival of V. cholerae alone and during its interaction with A. castellanii. The results showed that an OmpA mutant of V. cholerae survived longer than wild-type V. cholerae when cultivated alone. Cocultivation with A. castellanii enhanced the survival of both bacterial strains and OmpA protein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of the OmpA mutant of V. cholerae decreased the viability of A. castellanii and this bacterial strain released more OMVs than wild-type V. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from the OmpA mutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule for OmpA in survival of V. cholerae and OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment.

  15. Acanthamoeba and Fusarium interactions: A possible problem in keratitis.

    Nunes, Thais Esther Teixeira; Brazil, Nathalya Tesch; Fuentefria, Alexandre Meneghello; Rott, Marilise Brittes

    2016-05-01

    The incidence of Acanthamoeba and Fusarium species has increased in contact lens-related infectious keratitis. They share several environments and cases of co-infection have been reported. The interaction between the amoebae and other microorganisms may result in significant changes for both, like increased virulence in mammalian hosts. In this study, we evaluated the interaction of three Acanthamoeba castellanii strains with Fusarium conidia and the possible implications on keratitis. F. conidia were internalized by A. castellanii strains and were able to germinate inside the amoebae. The co-culture with the live amoebae, as well as the amoebal culture supernatant and lysate, increased the fungal growth significantly. Moreover, live F. solani and its culture supernatant enhanced the survival of amoebae, but in a different way in each amoebal strain. The encystment of the A. castellanii strain re-isolated from rat lung was increased by the fungus. These results show that A. castellanii and F. solani interaction may have an important influence on survival of both, and specially indicate a possible effect on virulence characteristics of these microorganisms. These data suggest that the A. castellanii-F. solani interaction may cause severe impacts on keratitis. PMID:26851515

  16. Use of In Vitro Assays To Determine Effects of Human Serum on Biological Characteristics of Acanthamoeba castellanii

    Sissons, James; Alsam, Selwa; Stins, Monique; Rivas, Antonio Ortega; Morales, Jacob Lorenzo; Faull, Jane; Khan, Naveed Ahmed

    2006-01-01

    Normal human serum inhibits Acanthamoeba (encephalitis isolate) binding to and cytotoxicity of human brain microvascular endothelial cells, which constitute the blood-brain barrier. Zymographic assays revealed that serum inhibits extracellular protease activities of acanthamoebae. But it is most likely that inhibition of specific properties of acanthamoebae is a consequence of the initial amoebicidal-amoebistatic effects induced by serum. For example, serum exhibited amoebicidal effects; i.e., up to 50% of the exposed trophozoites were killed. The residual subpopulation, although viable, remained static over longer incubations. Interestingly, serum enhanced the phagocytic ability of acanthamoebae, as measured by bacterial uptake. Overall, our results demonstrate that human serum has inhibitory effects on Acanthamoeba growth and viability, protease secretions, and binding to and subsequent cytotoxicity for brain microvascular endothelial cells. Conversely, Acanthamoeba phagocytosis was stimulated by serum. PMID:16825391

  17. Use of In Vitro Assays To Determine Effects of Human Serum on Biological Characteristics of Acanthamoeba castellanii

    Sissons, James; Alsam, Selwa; Stins, Monique; Rivas, Antonio Ortega; Morales, Jacob Lorenzo; Faull, Jane; Khan, Naveed Ahmed

    2006-01-01

    Normal human serum inhibits Acanthamoeba (encephalitis isolate) binding to and cytotoxicity of human brain microvascular endothelial cells, which constitute the blood-brain barrier. Zymographic assays revealed that serum inhibits extracellular protease activities of acanthamoebae. But it is most likely that inhibition of specific properties of acanthamoebae is a consequence of the initial amoebicidal-amoebistatic effects induced by serum. For example, serum exhibited amoebicidal effects; i.e....

  18. A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii.

    Iwamoto, M; Pi, M; Kurihara, M; Morio, T; Tanaka, Y

    1998-04-01

    We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11 gene (rpl11) in the mitochondrial DNA (54+/-2 kb) of the cellular slime mold Dictyostelium discoideum. Sequence analysis revealed that eleven ribosomal protein genes and six open reading frames (ORFs) formed a cluster arranged in the order: rpl11-orf189-rps12-rps7-rpl2-rps19-+ ++orf425-orf1740-rpl16-rpl14-orf188- rps14-rps8-rpl6-rps13-orf127-orf796. This order was very similar to that of homologous genes in Acanthamoeba castellanii mitochondrial DNA. The N-terminal region of ORF425 and the C-terminal region of ORF1740 had partial similarities to the S3 ribosomal protein of other organisms. The termination codons of rpl16 and orf188 were UGA, which has not hitherto been found in genes encoded in D. discoideum mitochondrial DNA. PMID:9560439

  19. Localization of myosin IC and myosin II in Acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy

    1990-01-01

    Polyclonal antisera have been raised against purified Acanthamoeba myosin II and to a synthetic 26 amino acid peptide that corresponds in sequence to the phosphorylation site of Acanthamoeba myosin IC. These antisera are specific for their respective antigens as determined by immunoblotting after SDS-PAGE of total cell lysates. By using the antisera, localization studies were performed by indirect immunofluorescence and by immunogold electron microscopy. Myosin II occurred in the cell cytopla...

  20. Establishment and validation of whole-cell based fluorescence assays to identify anti-mycobacterial compounds using the Acanthamoeba castellanii-Mycobacterium marinum host-pathogen system.

    Sébastien Kicka

    Full Text Available Tuberculosis is considered to be one of the world's deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches.

  1. Photochemotherapeutic strategies against Acanthamoeba keratitis

    Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2012-01-01

    Here, we determined the potential of photochemotherapy, namely the application of photodynamic compounds followed by exposure to a suitable source of UV-visible radiation against corneal pathogen, Acanthamoeba. Organometallic macromolecule, tin porphyrin [Sn(IV)porphyrin] was synthesized and purity confirmed using nuclear magnetic resonance spectroscopy. The Sn(IV)porphyrin was tested against a keratitis isolate of Acanthamoeba castellanii belonging to the T4 genotype using growth and viabili...

  2. Interactions of Neuropathogenic Escherichia coli K1 (RS218) and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

    2014-01-01

    Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity. PMID:24818136

  3. Interactions of Neuropathogenic Escherichia coli K1 (RS218 and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

    Farzana Abubakar Yousuf

    2014-01-01

    Full Text Available Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin, adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin, protein secretion system (T1SS for hemolysin, invasins (IbeA, CNF1, metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin, protein secretion system (T1SS for hemolysin, invasins (CNF1, metabolism (D-serine catabolism abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity.

  4. SJL mice infected with Acanthamoeba castellanii develop central nervous system autoimmunity through the generation of cross-reactive T cells for myelin antigens

    Massilamany, Chandirasegaran; Marciano-Cabral, Francine; Rocha-Azevedo, Bruno da;

    2014-01-01

    SJL mice reminiscent of the diseases induced with their corresponding cognate peptides. We now demonstrate that mice infected with ACA also show the generation of cross-reactive T cells, predominantly for PLP 139-151, as evaluated by T cell proliferation and IAs/dextramer staining. We verified that......, suggesting that ACA infection can trigger CNS autoimmunity in the presence of preexisting repertoire of autoreactive T cells. Taken together, the data provide novel insights into the pathogenesis of Acanthamoeba infections, and the potential role of infectious agents with mimicry epitopes to self-antigens in...

  5. Biophysical Studies of Processes Involved in Acanthamoeba Infections : Contact Lens Contamination, Adhesion, and Intracellular Dynamics

    Reverey, Julia Franziska

    2014-01-01

    Acanthamoeba castellanii can cause a severe and painful, acanthamoeba keratitis. In this disease, the acanthamoebae reach the eye via contaminated contact lenses. In the eye, they invade cornea cells and kill them. The first step in this process is the formation of a close contact between the acanthamoeba and the target-cell. This contact is mainly mediated by mannose. Subsequently, cytolytic factors move to the contact site and are released leading to the death of the target-cell. The single...

  6. Pathogenic strains of Acanthamoeba are recognized by TLR4 and initiated inflammatory responses in the cornea.

    Hassan Alizadeh

    Full Text Available Free-living amoebae of the Acanthamoeba species are the causative agent of Acanthamoeba keratitis (AK, a sight-threatening corneal infection that causes severe pain and a characteristic ring-shaped corneal infiltrate. Innate immune responses play an important role in resistance against AK. The aim of this study is to determine if Toll-like receptors (TLRs on corneal epithelial cells are activated by Acanthamoeba, leading to initiation of inflammatory responses in the cornea. Human corneal epithelial (HCE cells constitutively expressed TLR1, TLR2, TLR3, TLR4, and TLR9 mRNA, and A. castellanii upregulated TLR4 transcription. Expression of TLR1, TLR2, TLR3, and TLR9 was unchanged when HCE cells were exposed to A. castellanii. IL-8 mRNA expression was upregulated in HCE cells exposed to A. castellanii. A. castellanii and lipopolysaccharide (LPS induced significant IL-8 production by HCE cells as measured by ELISA. The percentage of total cells positive for TLR4 was higher in A. castellanii stimulated HCE cells compared to unstimulated HCE cells. A. castellanii induced upregulation of IL-8 in TLR4 expressing human embryonic kidney (HEK-293 cells, but not TLR3 expressing HEK-293 cells. TLR4 neutralizing antibody inhibited A. castellanii-induced IL-8 by HCE and HEK-293 cells. Clinical strains but not soil strains of Acanthamoeba activated TLR4 expression in Chinese hamster corneas in vivo and in vitro. Clinical isolates but not soil isolates of Acanthamoeba induced significant (P< 0.05 CXCL2 production in Chinese hamster corneas 3 and 7 days after infection, which coincided with increased inflammatory cells in the corneas. Results suggest that pathogenic species of Acanthamoeba activate TLR4 and induce production of CXCL2 in the Chinese hamster model of AK. TLR4 may be a potential target in the development of novel treatment strategies in Acanthamoeba and other microbial infections that activate TLR4 in corneal cells.

  7. Cysteine Protease Inhibitor (AcStefin) Is Required for Complete Cyst Formation of Acanthamoeba

    Lee, Jung-Yub; Song, Su-Min; Moon, Eun-Kyung; Lee, Yu-Ran; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Cha, Hee-Jae; Yu, Hak Sun; Kong, Hyun-Hee; Chung, Dong-Il; Hong, Yeonchul

    2013-01-01

    The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protect Acanthamoeba from intra...

  8. Acanthamoeba species in Swimming Pools of Cairo, Egypt.

    Ahmad Al-Herrawy

    2014-06-01

    Full Text Available The free-living amoebae Acanthamoeba spp. have been recognized as etiologic agents of amoebic encephalitis, keratitis, otitis, lung lesions and other skin infections mainly in immuno-compromised individuals. The purpose of this study is to detect the presence of Acanthamoeba in swimming pools in Egypt using a polymerase chain reaction (PCR method.Water samples were collected from 10 different swimming pools in Cairo, Egypt. Samples were cultured on non-nutrient agar for the detection of Acanthamoeba isolates that were confirmed by PCR amplification using genus specific primers. The molecularly confirmed Acanthamoeba isolates were morphologically identified to the species level.Members of genus Acanthamoeba were detected in 49.2% of the examined swimming-pool water samples. Morphologically, six Acanthamoeba species were isolated from the examined swimming pool water namely A. polyphaga, A.castellanii, A. rhysodes, A. mauritaniensis, A. royreba and A. triangularis. All the identified species of Acanthamoeba were molecularly confirmed to be related to the genus Acanthamoeba.The isolated species of Acanthamoeba could provoke variable degrees of infections to the swimmers. The culture method is cheaper and easier than PCR techniques that are faster for the detection of free-living amoebae.

  9. Acanthamoeba meningoencephalitis

    S R Chandra

    2014-01-01

    Full Text Available Report of a case of young immunocompetent male adult with autopsy proven acanthamoeba meningoencephalitis. The patient presented with a protracted febrile illness of 3 months duration with features of meningoencephalitis, this was followed by rapid deterioration while on anti tuberculous therapy and steroids and ended fatally. His magnetic resonance imaging showed features of hemorrhagic meningoencephalitis and magnetic resonance spectroscopy showed choline peak. Autopsy revealed necrotizing meningoencephalitis and intraocular colonization due to acanthamoeba.

  10. F. novicida-Infected A. castellanii Does Not Enhance Bacterial Virulence in Mice

    Ozanic, Mateja; Gobin, Ivana; Brezovec, Martin; Marecic, Valentina; Trobonjaca, Zlatko; Abu Kwaik, Yousef; Santic, Marina

    2016-01-01

    Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia. PMID:27242974

  11. Metabolic profiling of the protozoan parasite Entamoeba invadens revealed activation of unpredicted pathway during encystation.

    Ghulam Jeelani

    Full Text Available Encystation, which is cellular differentiation from the motile, proliferative, labile trophozoite form to the dormant, resistant cyst form, is a crucial process found in parasitic and free-living protozoa such as Entamoeba, Giardia, Acanthamoeba, and Balamuthia. Since encystation is an essential process to deal with the adverse external environmental changes during the life cycle, and often integral to the transmission of the diseases, biochemical understanding of the process potentially provides useful measures against the infections caused by this group of protozoa. In this study, we investigated metabolic and transcriptomic changes that occur during encystation in Entamoeba invadens, the reptilian sibling of mammal-infecting E. histolytica, using capillary electrophoresis-tandem mass spectrometry-based metabolite profiling and DNA microarray-based expression profiling. As the encystation progressed, the levels of majority of metabolites involved in glycolysis and nucleotides drastically decreased, indicating energy generation is ceased. Furthermore, the flux of glycolysis was redirected toward chitin wall biosynthesis. We found remarkable temporal increases in biogenic amines such as isoamylamine, isobutylamine, and cadaverine, during the early period of encystation, when the trophozoites form large multicellular aggregates (precyst. We also found remarkable induction of γ-aminobutyric acid (GABA during encystation. This study has unveiled for the first time the dynamics of the transcriptional and metabolic regulatory networks during encystation, and should help in better understanding of the process in pathogenic eukaryotes, and further development of measures controlling infections they cause.

  12. Survival of taylorellae in the environmental amoeba Acanthamoeba castellanii

    Allombert, Julie; Vianney, Anne; Laugier, Claire; Petry, Sandrine; Hébert, Laurent

    2014-01-01

    Background Taylorella equigenitalis is the causative agent of contagious equine metritis, a sexually-transmitted infection of Equidae characterised in infected mares by abundant mucopurulent vaginal discharge and a variable degree of vaginitis, cervicitis or endometritis, usually resulting in temporary infertility. The second species of the Taylorella genus, Taylorella asinigenitalis, is considered non-pathogenic, although mares experimentally infected with this bacterium can develop clinical...

  13. Failure of chemotherapy in the first reported cases of Acanthamoeba keratitis in Pakistan

    Siddiqui, Ruqaiyyah; Chaudhry, Tanveer; Lakhundi, Sahreena; Ahmad, Khabir; Khan, Naveed Ahmed

    2014-01-01

    Acanthamoeba keratitis is a painful and progressive infection of the cornea that can result in loss of vision. Here, for the first time in Pakistan, we report two cases of Acanthamoeba keratitis. The first patient was a 37-year-old female who presented with severe itching, redness, pain, along with loss of vision. The patient was a regular soft contact lens wearer. The second patient was a 25-year-old female who had been using soft contact lenses for the past two years. She presented with a burning sensation and extreme pain, along with loss of vision. Both patients were treated for a possible microbial keratitis with topical moxifloxacin hydrochloride drops, vancomycin drops, propamidine isethionate ointment, amphotericin B drops, and amikacin drops. However, the response was inadequate and both patients were referred for corneal transplant. Acanthamoeba castellanii was isolated by placing contact lenses and contact lens cases on non-nutrient agar plates containing a lawn of non-invasive Escherichia coli K-12 HB101 bacteria. The polymerase chain reaction (PCR) using genus-specific probes confirmed the identity of Acanthamoeba spp., whereas the morphological characteristics of trophozoites and cysts were suggestive of A. castellanii in both cases. With growing use of contact lenses for vision correction/cosmetic use coupled with sub-standard lens care in this region and the possibility of non-contact lens-associated Acanthamoeba keratitis, a need for increased awareness of this sight-threatening infection is discussed further. PMID:24548160

  14. Effect of microwave treatment to acanthamoeba: a possibility of contact lens storage case sterilization?

    Microbially contaminated contact lens storage cases are considered to be a predisposing risk factor for Acanthamoeba keratitis. What kind of effect does microwave irradiation have on contact lens cases contaminated with Acanthamoeba cysts and trophozoites? Different types of contact lens cases were contaminated with trophozoites and cysts of three different Acanthamoeba species (A. hatchetti, A. castellanii, A. comandoni) and exposed to microwave irradiation for 3, 5 and 8 minutes respectively. One of the three test series was run with dehydrated cysts. After an irradiation period of 3 minutes under humid conditions cysts and trophozoites of all three strains were completely destroyed. Dehydrated cysts of A. hatchetti and A. castellanii were not killed by irradiation. There were no negative effects of irradiation on the contact lens cases themselves. Acanthamoeba cysts and trophozoites are effectively killed by microwave irradiation on condition that irradiation takes place in humid conditions. This can be easily achieved by filling the contact lens storage cases with tap water. We recommend microwave irradiation as a cheap and save method for the sterilization of contact lens cases in order to avoid a possible Acanthamoeba infection of the eye. (author)

  15. Regulation of carbohydrate metabolism during Giardia encystment

    Vliegenthart, J.F.G.; Jarroll, E.L.; Macechko, P.T.; Steimle, P.A.; Bulik, D.; Karr, C.D.; Keulen, Harry van; Paget, P.A.

    2001-01-01

    Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-d-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzy

  16. Cellulose degradation: a therapeutic strategy in the improved treatment of Acanthamoeba infections

    Lakhundi, Sahreena; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2015-01-01

    Acanthamoeba is an opportunistic free-living amoeba that can cause blinding keratitis and fatal brain infection. Early diagnosis, followed by aggressive treatment is a pre-requisite in the successful treatment but even then the prognosis remains poor. A major drawback during the course of treatment is the ability of the amoeba to enclose itself within a shell (a process known as encystment), making it resistant to chemotherapeutic agents. As the cyst wall is partly made of cellulose, thus cel...

  17. Inactivation of Acanthamoeba spp. and Other Ocular Pathogens by Application of Cold Atmospheric Gas Plasma.

    Heaselgrave, Wayne; Shama, Gilbert; Andrew, Peter W; Kong, Michael G

    2016-05-15

    Currently there are estimated to be approximately 3.7 million contact lens wearers in the United Kingdom and 39.2 million in North America. Contact lens wear is a major risk factor for developing an infection of the cornea known as keratitis due to poor lens hygiene practices. While there is an international standard for testing disinfection methods against bacteria and fungi (ISO 14729), no such guidelines exist for the protozoan Acanthamoeba, which causes a potentially blinding keratitis most commonly seen in contact lens wearers, and as a result, many commercially available disinfecting solutions show incomplete disinfection after 6 and 24 h of exposure. Challenge test assays based on international standard ISO 14729 were used to determine the antimicrobial activity of cold atmospheric gas plasma (CAP) against Pseudomonas aeruginosa, Candida albicans, and trophozoites and cysts of Acanthamoeba polyphaga and Acanthamoeba castellanii P. aeruginosa and C. albicans were completely inactivated in 0.5 min and 2 min, respectively, and trophozoites of A. polyphaga and A. castellanii were completely inactivated in 1 min and 2 min, respectively. Furthermore, for the highly resistant cyst stage of both species, complete inactivation was achieved after 4 min of exposure to CAP. This study demonstrates that the CAP technology is highly effective against bacterial, fungal, and protozoan pathogens. The further development of this technology has enormous potential, as this approach is able to deliver the complete inactivation of ocular pathogens in minutes, in contrast to commercial multipurpose disinfecting solutions that require a minimum of 6 h. PMID:26994079

  18. Biology and pathogenesis of Acanthamoeba

    Siddiqui Ruqaiyyah; Khan Naveed

    2012-01-01

    Abstract Acanthamoeba is a free-living protist pathogen, capable of causing a blinding keratitis and fatal granulomatous encephalitis. The factors that contribute to Acanthamoeba infections include parasite biology, genetic diversity, environmental spread and host susceptibility, and are highlighted together with potential therapeutic and preventative measures. The use of Acanthamoeba in the study of cellular differentiation mechanisms, motility and phagocytosis, bacterial pathogenesis and ev...

  19. Photochemotherapeutic Strategy against Acanthamoeba Infections

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Anwar, Ayaz; Shah, Muhammad Raza; Khoja, Shahrukh; Khan, Naveed Ahmed

    2015-01-01

    Acanthamoeba is a protist pathogen that can cause serious human infections, including blinding keratitis and a granulomatous amoebic encephalitis that almost always results in death. The current treatment for these infections includes a mixture of drugs, and even then, a recurrence can occur. Photochemotherapy has shown promise in the treatment of Acanthamoeba infections; however, the selective targeting of pathogenic Acanthamoeba has remained a major concern. The mannose-binding protein is a...

  20. Biology and pathogenesis of Acanthamoeba

    Siddiqui Ruqaiyyah

    2012-01-01

    Full Text Available Abstract Acanthamoeba is a free-living protist pathogen, capable of causing a blinding keratitis and fatal granulomatous encephalitis. The factors that contribute to Acanthamoeba infections include parasite biology, genetic diversity, environmental spread and host susceptibility, and are highlighted together with potential therapeutic and preventative measures. The use of Acanthamoeba in the study of cellular differentiation mechanisms, motility and phagocytosis, bacterial pathogenesis and evolutionary processes makes it an attractive model organism. There is a significant emphasis on Acanthamoeba as a Trojan horse of other microbes including viral, bacterial, protists and yeast pathogens.

  1. The proteome landscape of Giardia lamblia encystation.

    Carmen Faso

    Full Text Available Giardia lamblia is an intestinal protozoan parasite required to survive in the environment in order to be transmitted to a new host. To ensure parasite survival, flagellated trophozoites colonizing the small intestine differentiate into non-motile environmentally-resistant cysts which are then shed in the environment. This cell differentiation process called encystation is characterized by significant morphological remodeling which includes secretion of large amounts of cyst wall material. Although much is known about the transcriptional regulation of encystation and the synthesis and trafficking of cyst wall material, the investigation of global changes in protein content and abundance during G. lamblia encystation is still unaddressed. In this study, we report on the quantitative analysis of the G. lamblia proteome during encystation using tandem mass spectrometry. Quantification of more than 1000 proteins revealed major changes in protein abundance in early, mid and late encystation, notably in constitutive secretory protein trafficking. Early stages of encystation were marked by a striking decrease of endoplasmic reticulum-targeted variant-specific surface proteins and significant increases in cytoskeleton regulatory components, NEK protein kinases and proteins involved in protein folding and glycolysis. This was in stark contrast to cells in the later stages of encystation which presented a surprisingly similar proteome composition to non-encysting trophozoites. Altogether these data constitute the first quantitative atlas of the Giardia proteome covering the whole process of encystation and point towards an important role for post-transcriptional control of gene expression in Giardia differentiation. Furthermore, our data provide a valuable resource for the community-based annotation effort of the G. lamblia genome, where almost 70% of all predicted gene models remains "hypothetical".

  2. The proteome landscape of Giardia lamblia encystation.

    Faso, Carmen; Bischof, Sylvain; Hehl, Adrian B

    2013-01-01

    Giardia lamblia is an intestinal protozoan parasite required to survive in the environment in order to be transmitted to a new host. To ensure parasite survival, flagellated trophozoites colonizing the small intestine differentiate into non-motile environmentally-resistant cysts which are then shed in the environment. This cell differentiation process called encystation is characterized by significant morphological remodeling which includes secretion of large amounts of cyst wall material. Although much is known about the transcriptional regulation of encystation and the synthesis and trafficking of cyst wall material, the investigation of global changes in protein content and abundance during G. lamblia encystation is still unaddressed. In this study, we report on the quantitative analysis of the G. lamblia proteome during encystation using tandem mass spectrometry. Quantification of more than 1000 proteins revealed major changes in protein abundance in early, mid and late encystation, notably in constitutive secretory protein trafficking. Early stages of encystation were marked by a striking decrease of endoplasmic reticulum-targeted variant-specific surface proteins and significant increases in cytoskeleton regulatory components, NEK protein kinases and proteins involved in protein folding and glycolysis. This was in stark contrast to cells in the later stages of encystation which presented a surprisingly similar proteome composition to non-encysting trophozoites. Altogether these data constitute the first quantitative atlas of the Giardia proteome covering the whole process of encystation and point towards an important role for post-transcriptional control of gene expression in Giardia differentiation. Furthermore, our data provide a valuable resource for the community-based annotation effort of the G. lamblia genome, where almost 70% of all predicted gene models remains "hypothetical". PMID:24391747

  3. Molecular Phylogeny of Acanthamoeba

    Kong, Hyun Hee

    2009-01-01

    After morphological grouping of Acanthamoeba by Pussard and Pons, phylogeny of the genus has been always a big topic to the researchers. Because of the variability of morphological characteristics, unchangeable and stable characters have been investigated for phylogenic criteria. Isoenzyme and mitochondrial DNA RFLP (Mt DNA RFLP) analyses revealed different patterns among strains assigned to a same species. Therefore, these characteristics would be considered as tools for strain discriminatio...

  4. Evaluation of Ozone Application in Dental Unit Water Lines Con¬taminated with Pathogenic Acanthamoeba

    Wafaa HIKAL

    2015-10-01

    Full Text Available Background: In this study morphological and molecular characterization of Acan­thamoeba strains, isolated from dental unit waterlines (DUWLs were surveyed and the levels of disinfection achievable in vitro by the application of ozone disinfect­ant to DUWLs were evaluate.Methods: Water samples were collected from air-water syringes, cup fillers and tap water before and at the end of the working day. They were cultured on non-nutrient agar (NNA plates. Species identification was carried out with a PCR assay based on sequence analysis of the 18S rRNA gene. The cellular response to ozone was tested on Acanthamoeba cyst with different doses at different contact time in vitro twice.Results: Prevalence rates for Acanthamoeba contamination were 100, 100 and 72% for air-water syringes, cup fillers and tap water, respectively. The morphological analysis revealed the presence of A. castellanii, A. griffin, A. hatchitti and A. lenticulata. Phylogenetic analysis of the sequences showed the four strains to be closely related to a sequence type (T3, T4, T5 and T11. Acanthamoeba cells were stained with try­pan blue, which revealed killed of Acanthamoeba instantaneously after 10 minutes in ozonized water. There was no growth of Acanthamoeba occurred after ozone treat­ment in water bottles for 5 minutes with a flow rate of 500 mg/hour.Conclusion: Ozone can play an important role in controlling the problem of contami­nation of DUWLs as a potent disinfectant.

  5. Analysis of Giardin expression during encystation of Giardia lamblia

    The present study analyzed giardin transcription in trophozoites and cysts during encystation of Giardia lamblia. Encystment was induced using standard methods, and the number of trophozoites and cysts were counted at various time-points during encystation. At all time points, RNA from both stages...

  6. Acanthamoeba feature a unique backpacking strategy to trap and feed on Listeria monocytogenes and other motile bacteria.

    Doyscher, Dominik; Fieseler, Lars; Dons, Lone; Loessner, Martin J; Schuppler, Markus

    2013-02-01

    Despite its prominent role as an intracellular human pathogen, Listeria monocytogenes normally features a saprophytic lifestyle, and shares many environmental habitats with predatory protozoa. Earlier studies claimed that Acanthamoeba may act as environmental reservoirs for L. monocytogenes, whereas others failed to confirm this hypothesis. Our findings support the latter and provide clear evidence that L. monocytogenes is unable to persist in Acanthamoeba castellanii and A. polyphaga. Instead, external Listeria cells are rapidly immobilized on the surface of Acanthamoeba trophozoites, forming large aggregates of densely packed bacteria that we termed backpacks. While the assembly of backpacks is dependent on bacterial motility, flagellation alone is not sufficient. Electron micrographs showed that the aggregates are held together by filaments of likely amoebal origin. Time-lapse microscopy revealed that shortly after the bacteria are collected, the amoeba can change direction of movement, phagocytose the backpack and continue to repeat the process. The phenomenon was also observed with avirulent L. monocytogenes mutants, non-pathogenic Listeria, and other motile bacteria, indicating that formation of backpacks is not specific for L. monocytogenes, and independent of bacterial pathogenicity or virulence. Hence, backpacking appears to represent a unique and highly effective strategy of Acanthamoeba to trap and feed on motile bacteria. PMID:22925311

  7. Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis.

    Einarsson, Elin; Troell, Karin; Hoeppner, Marc P; Grabherr, Manfred; Ribacke, Ulf; Svärd, Staffan G

    2016-03-01

    Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the intestinal protozoan parasite Giardia intestinalis. Hitherto the majority of studies have focused on the early events, leaving late encystation poorly defined. In order to further study encystation, focusing on the later events, we developed a new encystation protocol that generates a higher yield of mature cysts compared to standard methods. Transcriptome changes during the entire differentiation from trophozoites to cysts were thereafter studied using RNA sequencing (RNA-seq). A high level of periodicity was observed for up- and down-regulated genes, both at the level of the entire transcriptome and putative regulators. This suggests the trajectory of differentiation to be coordinated through developmentally linked gene regulatory activities. Our study identifies a core of 13 genes that are consistently up-regulated during initial encystation. Of these, two constitute previously uncharacterized proteins that we were able to localize to a new type of encystation-specific vesicles. Interestingly, the largest transcriptional changes were seen in the late phase of encystation with the majority of the highly up-regulated genes encoding hypothetical proteins. Several of these were epitope-tagged and localized to further characterize these previously unknown genetic components of encystation and possibly excystation. Finally, we also detected a switch of variant specific surface proteins (VSPs) in the late phase of encystation. This occurred at the same time as nuclear division and DNA replication, suggesting a potential link between the processes. PMID:27015092

  8. The pathogenesis of Acanthamoeba infections: current status and future implications

    Khan, Naveed Ahmed

    2004-01-01

    Acanthamoeba are opportunistic protozoan parasites that can cause painful, visionthreatening keratitis. However the pathogenesis and pathophysiology of Acanthamoeba keratitis remain incompletely understood. Most cases of Acanthamoeba keratitis develop as a result of poor hygiene in contact lens care but it is unclear how amoebae transmigrate from the environment into the cornea leading to inflammation, photophobia and blindness. Acanthamoeba keratitis has become increasingly...

  9. Ceratite bilateral por Acanthamoeba: relato de caso Bilateral Acanthamoeba keratitis: case report

    Wilson Nahmatallah Obeid

    2003-12-01

    Full Text Available A ceratite por Acanthamoeba é uma infecção ocular grave que, apesar dos recentes progressos no diagnóstico e tratamento, ainda provoca prolongada morbidade e perda da acuidade visual. Relatamos um caso de ceratite bilateral por Acanthamoeba em usuário de lentes de contato, que é o primeiro caso descrito na literatura brasileira.Acanthamoeba keratitis is a severe ocular infection which even with recent progress in diagnosis and treatment still causes long morbidity and loss of visual acuity. We describe a case of bilateral Acanthamoeba keratitis in a user of contact lenses, which is the first case discussed in Brazilian literature.

  10. The immunological aspects of Acanthamoeba infections

    Naveed A.  Khan

    2005-01-01

    Full Text Available Acanthamoeba is a protozoan pathogen that is responsible for two recognized diseases; i a blinding keratitis, most commonly associated with contact lens wear, and ii a rare but fatal granulomatous encephalitis that usually is limited to immunocompromised patients. The fact that Acanthamoeba infections are generally limited to avascular cornea or immunocompromised patients suggests that the normal immune responses may be sufficient to control and/or eradicate these pathogens. Here we describe our current understanding of the immune responses to Acanthamoeba infections.

  11. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of 3H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei

  12. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    Das, S.; Gillin, F.D.

    1987-05-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of /sup 3/H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei.

  13. Development of a new oxygen consumption rate assay in cultures of Acanthamoeba (Protozoa: Lobosea) and its application to evaluate viability and amoebicidal activity in vitro.

    Heredero-Bermejo, I; Criado-Fornelio, A; Soliveri, J; Díaz-Martín, J A; Matilla-Fuentes, J; Sánchez-Arias, J A; Copa-Patiño, J L; Pérez-Serrano, J

    2015-08-01

    A new fluorometric method has been developed for measuring the oxygen consumption rate (OCR) of Acanthamoeba cultures in microplates and for screening molecules with amoebicidal activity against this microorganism. The use of a biofunctional matrix (containing an oxygen-sensitive fluorogenic probe) attached to the microplate wells allowed continuous measurement of OCR in the medium, hence assessment of amoebic growth. The new OCR method applied to cell viability yielded a linear relationship and monitoring was much quicker than with indirect viability assays previously used. In addition, two drugs were tested in a cytotoxicity assay monitored by the new OCR viability test. With this procedure, the standard amoebicidal drug chlorhexidine digluconate showed an IC50 of 3.53 + 1.3 mg/l against Acanthamoeba polyphaga and 3.19 + 1.2 mg/l against Acanthamoeba castellanii, whereas a cationic dendrimer [G1Si(NMe3+)4] showed an IC50 of 6.42 + 1.3 mg/l against A. polyphaga. These data agree with previous studies conducted in our laboratory. Therefore, the new OCR method has proven powerful and quick for amoebicidal drug screening and is likely to be applied in biochemical studies concerning protozoa respiration and metabolism. PMID:25956947

  14. Acanthamoeba induces cell-cycle arrest in host cells

    Sissons, J.; Alsam, S.; Jayasekera, S.; Kim, K S; Stins, M; Khan, Naveed Ahmed

    2004-01-01

    Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed seve...

  15. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment

    Lorenzo-Morales Jacob; Khan Naveed A.; Walochnik Julia

    2015-01-01

    Free-living amoebae of the genus Acanthamoeba are causal agents of a severe sight-threatening infection of the cornea known as Acanthamoeba keratitis. Moreover, the number of reported cases worldwide is increasing year after year, mostly in contact lens wearers, although cases have also been reported in non-contact lens wearers. Interestingly, Acanthamoeba keratitis has remained significant, despite our advances in antimicrobial chemotherapy and supportive care. In part, this is due to an inc...

  16. Is there evidence of sexual reproduction (meiosis) in Acanthamoeba?

    Khan, Naveed A.; Siddiqui, Ruqaiyyah

    2015-01-01

    Evolution of independently breeding species into males and females (gametes) has remained a puzzle. Given the significant advantages of sexual reproduction over asexual reproduction as a long-term species survival strategy; here, we pose the question whether there is some form of meiosis in Acanthamoeba species, which represents our ancient lineage. The recently available Acanthamoeba genome revealed several genes implicated in meiosis in sexual eukaryotes such as Spo11, Mre11, Rad50, Rad51, Rad52, Mnd1, Dmc1, Msh, and Mlh, suggesting that Acanthamoeba is capable of some form of meiosis, inferring the presence of sexual reproduction in Acanthamoeba, and that meiosis evolved early in eukaryotic evolution. PMID:25800982

  17. Heat Shock Protein 90 regulates encystation in Entamoeba

    Meetali eSingh

    2015-10-01

    Full Text Available Enteric protozoan Entamoeba histolytica is a major cause of debilitating diarrheal infection worldwide with high morbidity and mortality. Even though the clinical burden of this parasite is very high, this infection is categorized as a neglected disease. Parasite is transmitted through feco-oral route and exhibit two distinct stages namely – trophozoites and cysts. Mechanism and regulation of encystation is not clearly understood. Previous studies have established the role of Heat shock protein 90 (Hsp90 in regulating stage transition in various protozoan parasites like Giardia, Plasmodium, Leishmania and Toxoplasma. Our study for the first time reports that Hsp90 plays a crucial role in life cycle of Entamoeba as well. We identify Hsp90 to be a negative regulator of encystation in Entamoeba. We also show that Hsp90 inhibition interferes with the process of phagocytosis in Entamoeba. Overall, we show that Hsp90 plays an important role in virulence and transmission of Entamoeba.

  18. Encystation commitment in Giardia duodenalis: a long and winding road

    Argüello-Garciá R.

    2009-12-01

    Full Text Available Cholesterol and bile salts are relevant modulators of Giardia encystation. Although several molecules within signaling cascades have been identified, and changes in their expression observed during giardial encystation, their underlying interactions leading to expression of cyst wall markers (CWPs and precursors of the GalNAc homopolymer are not well defined. Recent experimental data and the completion of the Giardia Genome Project Database (GiardiaDB allow us now to consider the role of bile salts as “natural stimuli” and the potential involvement of a Raf/MEK/ERK pathway mediating cholesterol-regulated expression of cyst-specific genes. These new findings may provide promising targets for diagnostics, drug design and prophylactic intervention against giardiasis.

  19. Prevalence of encysted Toxoplasma gondii in raptors from Alabama.

    Lindsay, D S; Smith, P C; Hoerr, F J; Blagburn, B L

    1993-12-01

    Little is known about the prevalence of encysted Toxoplasma gondii in wild birds. We examined the hearts and breast muscles from 101 raptors for encysted T. gondii. All of the raptors had been submitted for necropsy to the State Veterinary Diagnostic Laboratory, Auburn, Alabama. Tissues were digested in acid-pepsin solution and inoculated into groups of 3-5 laboratory mice. Toxoplasma gondii was isolated from 27 of 101 (26.7%) raptors: 8 of 12 (66.7%) red-shouldered hawks (Buteo lineatus), 13 of 27 (41.1%) red-tailed hawks (Buteo jamaicensis), 1 of 4 (25%) Cooper's hawks (Accipiter cooperi), 1 of 5 (20%) great horned owls (Bubo virginianus), 4 of 15 (26.7%) barred owls (Strix varia), and 1 of 3 (33.3%) kestrels (Falco sparverius). Toxoplasma gondii was not isolated from 3 broad-winged hawks (Buteo platypterus), 3 sharp-shinned hawks (Accipiter striatus), 6 barn owls (Tyto alba), 9 screech owls (Asio otus), a Mississippi kite (Ictinia misisippiensis), 2 golden eagles (Aquila chrysaetos), a bald eagle (Haliaeetus leucocephalus), 4 ospreys (Pandion haliaetus), 4 turkey vultures (Cathartes aura), or 2 black vultures (Coragyps atratus). No significant difference (P > 0.05) in prevalence was detected based on sex using chi-square analysis. Chi-square analysis of the data demonstrated that adult raptors had encysted stages of T. gondii significantly (P < 0.05) more often than did immature raptors. PMID:8277379

  20. Current Status of Acanthamoeba in Iran: A Narrative Review Article.

    Maryam Niyyati

    2015-06-01

    Full Text Available Free-living amoebae belonging to the genus Acanthamoeba have an environmental distribution. Amoebic keratitis due to these protozoan parasites continue to rise in Iran and worldwide. In Iran, there are various researches regarding both morphological and molecular identification of Acanthamoeba spp. in environmental and clinical samples. However, there is no thorough review about Acanthamoeba genotypes and their distribution in environmental sources such as water, dust and biofilm in Iran. Besides, according to increasing cases of Amoebic keratitis in the region awareness regarding the pathogenic potential of these sight-threatening amoebae is of utmost importance.We conducted a thorough review based on the database sources such as MEDLINE, PubMed and Google scholar. No restrictions were placed on study date, study design or language of publication. We searched all valuable and relevant information considering the occurrence of the Acanthamoeba in both environmental and clinical samples.According to our thorough review Acanthamoeba belonging to T4 genotype is the most prevalent type strain in environmental and clinical samples in several regions in Iran and worldwide, however, there are reports regarding Acanthamoeba belonging to other genotypes such as T2, T3, T5, T6 and T11 and the mentioned point could leads us to more researches with the goal of presenting the real genotype dominance of Acanthamoeba and related disease in the country.Overall, the present review will focus on present status of genotypes of Acanthamoeba in Iran during recent years.

  1. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment

    Lorenzo-Morales Jacob

    2015-01-01

    Full Text Available Free-living amoebae of the genus Acanthamoeba are causal agents of a severe sight-threatening infection of the cornea known as Acanthamoeba keratitis. Moreover, the number of reported cases worldwide is increasing year after year, mostly in contact lens wearers, although cases have also been reported in non-contact lens wearers. Interestingly, Acanthamoeba keratitis has remained significant, despite our advances in antimicrobial chemotherapy and supportive care. In part, this is due to an incomplete understanding of the pathogenesis and pathophysiology of the disease, diagnostic delays and problems associated with chemotherapeutic interventions. In view of the devastating nature of this disease, here we present our current understanding of Acanthamoeba keratitis and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be potential targets for improved diagnosis, therapeutic interventions and/or for the development of preventative measures. Novel molecular approaches such as proteomics, RNAi and a consensus in the diagnostic approaches for a suspected case of Acanthamoeba keratitis are proposed and reviewed based on data which have been compiled after years of working on this amoebic organism using many different techniques and listening to many experts in this field at conferences, workshops and international meetings. Altogether, this review may serve as the milestone for developing an effective solution for the prevention, control and treatment of Acanthamoeba infections.

  2. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment

    Lorenzo-Morales, Jacob; Khan, Naveed A.; Walochnik, Julia

    2015-01-01

    Free-living amoebae of the genus Acanthamoeba are causal agents of a severe sight-threatening infection of the cornea known as Acanthamoeba keratitis. Moreover, the number of reported cases worldwide is increasing year after year, mostly in contact lens wearers, although cases have also been reported in non-contact lens wearers. Interestingly, Acanthamoeba keratitis has remained significant, despite our advances in antimicrobial chemotherapy and supportive care. In part, this is due to an incomplete understanding of the pathogenesis and pathophysiology of the disease, diagnostic delays and problems associated with chemotherapeutic interventions. In view of the devastating nature of this disease, here we present our current understanding of Acanthamoeba keratitis and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be potential targets for improved diagnosis, therapeutic interventions and/or for the development of preventative measures. Novel molecular approaches such as proteomics, RNAi and a consensus in the diagnostic approaches for a suspected case of Acanthamoeba keratitis are proposed and reviewed based on data which have been compiled after years of working on this amoebic organism using many different techniques and listening to many experts in this field at conferences, workshops and international meetings. Altogether, this review may serve as the milestone for developing an effective solution for the prevention, control and treatment of Acanthamoeba infections. PMID:25687209

  3. Acanthamoeba: epidimiology, pathogenicity and evaluation of effectiveness of recent drugs

    To study the epidimiology of Acanthamoeba and to evaluate the effectiveness of some recent drugs against parasite. The study was carried out from March to May 2005 at the ophthalmic clinic of King Fahad Hospital, Saudi Arabia. Samples of rinsing solutions and saline of contact lens, tap water, Swimming pool water and Soil from Hufof city Saudi Arabia were tested. Mice were used to infect them via intranasal inoculation from isolated culture strain for confirming pathogenicity. Rokitamycin, polymixin B, suramin and chloropromazin were used to study their effects on Acanthamoeba growth in vitro. Acanthamoeba were detected in 20% of solution of contact lens, 20% of tap water, 50% of swimming pool samples and 40% of soil samples. All animals died or were sacrificed and had Acanthamoeba isolated from their organs. Higher percentage of growth inhibition of Acanthamoeba cultured was shown by chloropromazine and rokitamycin after 21 days (100%), while Polymyin B and Suramin showed 83% and 64% inhibition respectively. Acanthamoeba isolated in significant percent of environmental sources. Pathogenicity of organism was confirmed in mice. Contact lens wearers should be aware of the risks associated with Acanthamoeba. Rokitamycin and chlorpromazine showed good inhibition in vitro. (author)

  4. Diversity and Seasonal Impact of Acanthamoeba Species in a Subtropical Rivershed

    Po-Min Kao

    2013-01-01

    Full Text Available This study evaluated the presence of Acanthamoeba species in the Puzih River watershed, which features typical subtropical monsoon climate and is located just above the Tropic of Cancer in Taiwan. The relationship between the seasonal and geographical distributions of Acanthamoeba species in this rivershed was also investigated. Acanthamoeba species were detected in water samples using the amoebal enrichment culture method and confirmed by PCR. A total of 136 water samples were included in this study, 16 (11.7% of which contained Acanthamoeba species. Samples with the highest percentage of Acanthamoeba (32.4% were obtained during the summer season, mainly from upstream areas. The identified species in the four seasons included Acanthamoeba palestinensis (T2, Acanthamoeba sp. IS2/T4 (T4, Acanthamoeba lenticulata (T5, Acanthamoeba hatchetti (T11, Acanthamoeba healyi (T12, and Acanthamoeba jacobsi (T15. The most frequently identified Acanthamoeba genotype was T4 (68.7%. Acanthamoeba genotype T4 is responsible for Acanthamoeba keratitis and should be considered for associated human health risk potential in the rivershed.

  5. Acanthamoeba encephalitis: A Case Report and Review of Therapy

    A Zamora

    2014-01-01

    Full Text Available Background: Acanthamoeba is a rare cause of encephalitis yet is associated with high mortality. Treatment protocols vary greatly and generally include combination therapy across a wide spectrum of antiinfective classes. Case Description: A 63-year-old male who underwent renal transplantation presented 6 months after transplantation with depressed level of consciousness. Imaging of the head with computerized tomography showed an enhancing lesion suspicious for brain abscess. Biopsy of the lesion showed Acanthamoeba cysts. The patient was treated with sulfadiazine, fluconazole, flucytosine, azithromycin, and miltefosine but without success. We review recently published cases of Acanthamoeba encephalitis with an emphasis on treatment protocols and outcomes. Conclusion: Free-living protozoans such as Acanthamoeba are ubiquitous in the environment and should be suspected in immunosuppressed persons who present with central nervous system findings and brain abscess. Biopsy is critical to establish the etiology so that appropriate combination therapy can be deployed.

  6. Is there evidence of sexual reproduction (meiosis) in Acanthamoeba?

    Khan, Naveed A.; Siddiqui, Ruqaiyyah

    2015-01-01

    Evolution of independently breeding species into males and females (gametes) has remained a puzzle. Given the significant advantages of sexual reproduction over asexual reproduction as a long-term species survival strategy; here, we pose the question whether there is some form of meiosis in Acanthamoeba species, which represents our ancient lineage. The recently available Acanthamoeba genome revealed several genes implicated in meiosis in sexual eukaryotes such as Spo11, Mre...

  7. The immunological aspects of Acanthamoeba infections

    Naveed A.  Khan

    2005-01-01

    Acanthamoeba is a protozoan pathogen that is responsible for two recognized diseases; i) a blinding keratitis, most commonly associated with contact lens wear, and ii) a rare but fatal granulomatous encephalitis that usually is limited to immunocompromised patients. The fact that Acanthamoeba infections are generally limited to avascular cornea or immunocompromised patients suggests that the normal immune responses may be sufficient to control and/or eradicate these pathogens. Here we describ...

  8. Proteases as Markers for Differentiation of Pathogenic and Nonpathogenic Species of Acanthamoeba

    Khan, Naveed A.; Jarroll, Edward L.; Panjwani, Noorjahan; Cao, Zhiyi; Paget, Timothy A.

    2000-01-01

    Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenic Acanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoeba isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with Acanthamoeba-conditioned medium as well as with Acanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic Acanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5. PMID:10921939

  9. Azasterols impair Giardia lamblia proliferation and induces encystation

    The effects of sterol biosynthesis inhibitors on growth and fine structure of Giardia lamblia P1 strain cultures were analyzed. Azasterols demonstrated high efficacy in killing cells. The IC50 values for 22,26-azasterol and 24(R,S),25-epiminolanosterol were 7 μM and 170 nM, respectively. Morphological analysis showed that azasterols induced changes in G. lamblia ultrastructure. The most significant alterations were: (a) considerable increase of the size of the peripheral vesicles, which are part of the parasite endosomal-lysosomal system; (b) appearance of autophagosomal structures; and (c) induction of differentiation, followed by an abnormal enlargement of encystation secretory vesicles. We propose that azasterols are effective chemotherapeutic drugs against Giardia lamblia in vitro and may have another target in cells besides sterol biosynthesis

  10. Proteases as Markers for Differentiation of Pathogenic and Nonpathogenic Species of Acanthamoeba

    Khan, Naveed A.; Jarroll, Edward L.; Panjwani, Noorjahan; Cao, Zhiyi; Paget, Timothy A.

    2000-01-01

    Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using azo-l...

  11. Identification of the Major Cysteine Protease of Giardia and Its Role in Encystation*S⃞

    DuBois, Kelly N.; Abodeely, Marla; Sakanari, Judy; Craik, Charles S.; Lee, Malinda; McKerrow, James H.; Sajid, Mohammed

    2008-01-01

    Giardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using qu...

  12. A case of radial keratoneuritis in non-Acanthamoeba keratitis

    Mutoh T

    2012-09-01

    Full Text Available Tetsuya Mutoh, Yukihiro Matsumoto, Makoto ChikudaDepartment of Ophthalmology, Dokkyo Medical University Koshigaya Hospital, Saitama, JapanAbstract: A case of non-Acanthamoeba keratitis with radial keratoneuritis, which is thought to be pathognomonic for Acanthamoeba keratitis, is reported. A healthy 32-year-old woman with a history of frequent replacement of her contact lenses due to wear was examined at Dokkyo Medical University Koshigaya Hospital (Saitama, Japan and found to have a slight corneal opacity that was accompanied by radial keratoneuritis. Based on both the patient’s clinical findings and past history, the presence of Acanthamoeba keratitis was highly suspected. However, direct light microscopy of corneal scrapings stained by the Parker ink–potassium hydroxide method only found Acanthamoeba-type material in the specimen collected at her initial visit. In all other specimens collected from the patient, no Acanthamoeba was found either when using the same method or when performing cultures of the surgical debridement of the corneal lesion. In addition, topical antifungal eye drops, systemic antifungal drugs, and surgical debridement were also not effective in this case. Since a precise diagnosis could not be made, the patient was treated with topical 0.1% betamethasone sodium, which ultimately resulted in a dramatic improvement of her corneal inflammation. At 23 days after initiation of topical administration of 0.1% betamethasone sodium, visual acuity was 20/250, with a slight corneal opacity noted at the original site of infection. The outcome of the current case suggests that radial keratoneuritis is not always pathognomonic for Acanthamoeba keratitis.Keywords: radial keratoneuritis, non-Acanthamoeba keratitis, topical corticosteroid

  13. IDENTIFICATION OF Pseudomonas spp. AS AMOEBA-RESISTANT MICROORGANISMS IN ISOLATES OF Acanthamoeba

    Vinicius José Maschio

    2015-02-01

    Full Text Available Acanthamoeba is a “Trojan horse” of the microbial world. The aim of this study was to identify the presence of Pseudomonas as an amoeba-resistant microorganism in 12 isolates of Acanthamoeba. All isolates showed the genus Pseudomonas spp. as amoeba-resistant microorganisms. Thus, one can see that the Acanthamoeba isolates studied are hosts of Pseudomonas.

  14. Actin, RhoA, and Rab11 Participation during Encystment in Entamoeba invadens

    M. Herrera-Martínez

    2013-01-01

    Full Text Available In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

  15. The Transcriptional Response to Encystation Stimuli in Giardia lamblia Is Restricted to a Small Set of Genes ▿†

    Morf, Laura; Spycher, Cornelia; Rehrauer, Hubert; Fournier, Catharine Aquino; Morrison, Hilary G.; Hehl, Adrian B.

    2010-01-01

    The protozoan parasite Giardia lamblia undergoes stage differentiation in the small intestine of the host to an environmentally resistant and infectious cyst. Encystation involves the secretion of an extracellular matrix comprised of cyst wall proteins (CWPs) and a β(1-3)-GalNAc homopolymer. Upon the induction of encystation, genes coding for CWPs are switched on, and mRNAs coding for a Myb transcription factor and enzymes involved in cyst wall glycan synthesis are upregulated. Encystation in vitro is triggered by several protocols, which call for changes in bile concentrations or availability of lipids, and elevated pH. However, the conditions for induction are not standardized and we predicted significant protocol-specific side effects. This makes reliable identification of encystation factors difficult. Here, we exploited the possibility of inducing encystation with two different protocols, which we show to be equally effective, for a comparative mRNA profile analysis. The standard encystation protocol induced a bipartite transcriptional response with surprisingly minor involvement of stress genes. A comparative analysis revealed a core set of only 18 encystation genes and showed that a majority of genes was indeed upregulated as a side effect of inducing conditions. We also established a Myb binding sequence as a signature motif in encystation promoters, suggesting coordinated regulation of these factors. PMID:20693303

  16. The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes.

    Morf, Laura; Spycher, Cornelia; Rehrauer, Hubert; Fournier, Catharine Aquino; Morrison, Hilary G; Hehl, Adrian B

    2010-10-01

    The protozoan parasite Giardia lamblia undergoes stage differentiation in the small intestine of the host to an environmentally resistant and infectious cyst. Encystation involves the secretion of an extracellular matrix comprised of cyst wall proteins (CWPs) and a β(1-3)-GalNAc homopolymer. Upon the induction of encystation, genes coding for CWPs are switched on, and mRNAs coding for a Myb transcription factor and enzymes involved in cyst wall glycan synthesis are upregulated. Encystation in vitro is triggered by several protocols, which call for changes in bile concentrations or availability of lipids, and elevated pH. However, the conditions for induction are not standardized and we predicted significant protocol-specific side effects. This makes reliable identification of encystation factors difficult. Here, we exploited the possibility of inducing encystation with two different protocols, which we show to be equally effective, for a comparative mRNA profile analysis. The standard encystation protocol induced a bipartite transcriptional response with surprisingly minor involvement of stress genes. A comparative analysis revealed a core set of only 18 encystation genes and showed that a majority of genes was indeed upregulated as a side effect of inducing conditions. We also established a Myb binding sequence as a signature motif in encystation promoters, suggesting coordinated regulation of these factors. PMID:20693303

  17. In vitro Effect of Monosaccharides on the Virulence of Acanthamoeba Isolated from Patients with Amoebic Keratitis

    Y. Maroofi

    2007-04-01

    Full Text Available Introduction & Objective: Acanthamoeba is free-living amoeba that is found in soil, water, air as well as in human pharynx. Acanthamoeba is causative agent of granulomatose amoebic encephalitis (GAE in immunosuppressed and AIDS individuals and amoebic keratitis in people who use the lens. Pathogenic species of Acanthamoeba have protein receptors named mannose binding protein (MBP. Acanthamoeba via MBP adhere to the glycoproteins included mannose. Acanthamoeba adhesion to the target cells induces a protease secretion is called mannose inducing protein-133 (MIP-133. Exogense mannose can inhibit the adherence of Acanthamoeba; also, it can increase the cytopathatic effect (CPE through increase the secretion of MIP-133. In the present work, the effect of monosaccharides on the virulance of Acanthamoeba isolated from patient with amoebic keratitis, in HeLa cell culture was investigated.Materials & Methods: The isolates were cultured in HeLa cell culture, then 100, 50, 10, 1 and 0.1 mM of galactose, glucose and mannose were added to plates. Plates were observed with invert microscope in 8, 16, 32, 48, and 72 hours after culture.Results: Data implicated that mannose (100 mM showed the highest effect on increasing cytopathy of Acanthamoeba in HeLa cell culture. Meanwhile, galactose (100 mM increased the virulence of Acanthamoeba in the cell culture after 32 hours.Conclusion: Adding mannose and galactose to HeLa cell culture contain Acanthamoeba can increase the virulence of the parasite significantly.

  18. A retrospective study of nine cases of Acanthamoeba keratitis

    Tetsuya Mutoh

    2010-10-01

    Full Text Available Tetsuya Mutoh, Isao Ishikawa, Yukihiro Matsumoto, Makoto ChikudaDokkyo Medical University Koshigaya Hospital, Saitama, JapanPurpose: To evaluate the clinical features of Acanthamoeba keratitis in nine patients diagnosed at Dokkyo Medical University Koshigaya Hospital, Saitama, Japan.Methods: In nine eyes of nine patients, Acanthamoeba keratitis was diagnosed by direct light microscopy of corneal scrapings stained by the Parker ink-potassium hydroxide method between September 2006 and September 2009. Their clinical features and course were studied retrospectively. Antifungal eye drops, systemic antifungal therapy, and surgical debridement of the corneal lesions were performed in all patients.Results: At presentation, the clinical stage was initial in six cases, transient in one case, and complete in two cases. The patients were all contact lens wearers who had washed their lens storage cases with tap water. After treatment, final visual acuity was improved in six cases, unchanged in one case, and worse in two cases. The patient with the worst final vision (hand motion had rheumatoid arthritis and was taking oral prednisolone, which led to corneal perforation and prevented adequate debridement from being done.Conclusion: Acanthamoeba keratitis is closely related to wearing contact lenses and washing the lens storage case with tap water. Although final visual acuity improved after treatment in most patients, insufficient surgical debridement resulted in a poor visual prognosis.Keywords: surgical debridement, Acanthamoeba keratitis, contact lens wearers

  19. Salmonella pneumonia complicated with encysted empyema in an immunocompromised youth: Case report and literature Review.

    Saeed, Nermin Kamal

    2016-01-01

    In this case report we described a Bahraini male patient of twenty years of age, a smoker and diagnosed with stage IV B Hodgkin lymphoma. He presented with fever, nonproductive cough, upper back pain and shortness of breath due to right upper lobe pneumonia with right encysted pleural effusion. Salmonella enterica serotype Enteritidis was isolated from the sputum. He was successfully treated with 2 weeks of ceftriaxone followed by 2 weeks of oral cefixime. This was the first case of encysted empyema caused by Salmonella enterica serotype Enteritidis reported in the Kingdom of Bahrain. The different aspects of pulmonary Salmonella infections were discussed and the literature was reviewed. PMID:27131011

  20. Isolation of Acanthamoeba-Specific Antibodies from a Bacteriophage Display Library

    Khan, Naveed A.; Greenman, John; Topping, Katherine P.; Victoria C. Hough; Temple, Graham S.; Paget, Timothy A.

    2000-01-01

    Acanthamoeba causes opportunistic eye infections in humans, which can lead to severe keratitis and may ultimately result in blindness. Current methods for identifying this organism rely on culture and microscopy. In this paper, we describe the isolation of antibody fragments that can be used for the unequivocal identification of Acanthamoeba. A bacteriophage antibody display library was used to isolate antibody fragments that bind specifically to Acanthamoeba. Individual clones were studied b...

  1. Acanthamoeba spp. in Contact Lenses from Healthy Individuals from Madrid, Spain

    Gomes, Thiago dos Santos; Magnet, Angela; Izquierdo, Fernando; Vaccaro, Lucianna; Redondo, Fernando; Bueno, Sara; Sánchez, Maria Luisa; Angulo, Santiago; Fenoy, Soledad; Hurtado, Carolina; del Aguila, Carmen

    2016-01-01

    Purpose Acanthamoeba keratitis (AK) is a painful and potentially blinding corneal infection caused by Acanthamoeba spp. In Madrid, environmental studies have demonstrated a high presence of these free-living amoebae in tap water. Since most of AK cases occur in contact lenses (CL) wearers with inadequate hygiene habits, the presence of Acanthamoeba in discarded CL has been studied and compared with other common etiological agents of keratitis, such as Pseudomonas aeruginosa and Staphylococcus...

  2. A Case of Non-Contact Lens related Acanthamoeba keratitis in Malaysia

    Mohamed Kamel, A. G.; Faridah, H.; Yusof, S; Norazah, A.; Nakisah, M. A.

    2005-01-01

    Acanthamoeba is a ubiquitous free-living amoeba and is responsible for an uncommon yet increasingly diagnosed keratitis in humans. Acanthamoeba keratitis is perhaps the most challenging ocular infection to manage successfully and it can result in permanently impaired vision or blindness. Although contact lens use is the principal risk factor, about 10% of cases occur following trauma and exposure to contaminated soil or water. Cases of Acanthamoeba keratitis involving contact lens wearers hav...

  3. Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

    Jarroll Edward L; Lightfoot Mary; Goldsworthy Graham; Alsam Selwa; Sissons James; Khan Naveed

    2006-01-01

    Abstract Background Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. Results Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAG...

  4. Isolation of Acanthamoeba-Specific Antibodies from a Bacteriophage Display Library

    Khan, Naveed A.; Greenman, John; Topping, Katherine P.; Hough, Victoria C.; Temple, Graham S.; Paget, Timothy A.

    2000-01-01

    Acanthamoeba causes opportunistic eye infections in humans, which can lead to severe keratitis and may ultimately result in blindness. Current methods for identifying this organism rely on culture and microscopy. In this paper, we describe the isolation of antibody fragments that can be used for the unequivocal identification of Acanthamoeba. A bacteriophage antibody display library was used to isolate antibody fragments that bind specifically to Acanthamoeba. Individual clones were studied by enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence. Four antibody clones that specifically bind to Acanthamoeba spp. were identified. PMID:10835006

  5. Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

    Mi Kyung Park

    Full Text Available Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25 in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

  6. Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

    Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

    2007-01-01

    Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment.

  7. Taken to the limit--Is desiccation stress causing precocious encystment of trematode parasites in snails?

    O'Dwyer, Katie; Poulin, Robert

    2015-12-01

    When hosts experience environmental stress, the quantity and quality of resources they provide for parasites may be diminished, and host longevity may be decreased. Under stress, parasites may adopt alternative strategies to avoid fitness reductions. Trematode parasites typically have complex life cycles, involving asexual reproduction in a gastropod first intermediate host. A rare phenomenon, briefly mentioned in the literature, and termed 'precocious encystment' involves the next stage in the parasites' life cycle (metacercarial cyst) forming within the preceding stage (redia), while still inside the snail. In the trematode Parorchis sp. NZ using rocky shore snails exposed to long periods outside water, we hypothesised that this might be an adaptive strategy against desiccation, preventing parasite emergence from the snail. To test this, we first investigated the effect of prolonged desiccation on the survival of two species of high intertidal snails. Secondly, we measured the reproductive output (cercarial production) of the parasite under wet and dry conditions. Finally, we quantified the influence of desiccation stress on the occurrence of precocious encystment. Snail mortality was higher under dry conditions, indicating stress, and it was somewhat exacerbated for infected snails. Parasite reproductive output differed between wet and dry conditions, with parasites of snails kept in dry conditions producing more cercariae when placed in water. Little variation was observed in the occurrence of precocious encystment, although some subtle patterns emerged. Given the stresses associated with living in high intertidal environments, we discuss precocious encystment as a possible stress response in this trematode parasite. PMID:26344863

  8. ENCYSTATION AND EXPRESSION OF CYST ANTIGENS BY 'GIARDIA LAMBLIA' IN VITRO

    The cyst form of Giardia lamblia is responsible for transmission of giardiasis, a major waterborne intestinal disease. These studies demonstrate for the first time expression of cyst antigens and encystation of G. lamblia in vitro by both morphologic and immunologic criteria. The...

  9. Viability of Acanthamoeba after exposure to a multipurpose disinfecting contact lens solution and two hydrogen peroxide systems

    Hiti, K; Walochnik, J.; Haller-Schober, E M; Faschinger, C; Aspöck, H.

    2002-01-01

    Background/aim: Contact lens cases contaminated with Acanthamoeba are a major risk factor for an infection of the eye. In this study the anti-Acanthamoeba activity of three different contact lens storage solutions was tested.

  10. The generation gap: Proteome changes and strain variation during encystation in Giardia duodenalis.

    Emery, Samantha J; Pascovi, Dana; Lacey, Ernest; Haynes, Paul A

    2015-05-01

    The prevalence of Giardia duodenalis in humans is partly owed to its direct and simple life cycle, as well as the formation of the environmentally resistant and infective cysts. Proteomic and transcriptomic studies have previously analysed the encystation process using the well-characterised laboratory genomic strain, WB C6. This study presents the first quantitative study of encystation using pathogenically relevant and alternative assemblage A strains: the human-derived BRIS/82/HEPU/106 (H-106)and avian-derived BRIS/95/HEPU/2041 (B-2041). We utilised tandem MS/MS with a label-free quantitative approach to compare cysts and trophozoite life stages for strain variation, as well as confirm universal encystation markers of assemblage A. A total of 1061 non-redundant proteins were identified from both strains, including trophozoite- and cyst-specific proteomes and life-stage differentially expressed proteins. Additionally, 24 proteins previously classified in the literature as encystation-specific were confirmed as strain-independent markers of encystation. Functional cluster analysis of differentially expressed proteins saw significant overlap between strains, including protein trafficking and localisation in cysts, NEK kinase function, and carbohydrate metabolism in trophozoites. Two significant points of strain specific adaptations in cysts were also identified. B-2041 possessed major up-regulation of the ankyrin repeat protein 21.1 family compared to H-106. Furthermore, cysts of B-2041 retained near-complete VSP variant diversity between cysts and trophozoites, while H-106 lost 45% of its VSP variant diversity between life cycle stages, a constriction previously observed in studies of WB C6. This is the first report of strain variation in the cyst stage in G. duodenalis, and highlights cyst variation and its impacts on reinfection and life cycle success. PMID:26045354

  11. Acanthamoeba on Sabouraud's agar from a patient with keratitis

    Baradkar, Vasant; Samal, Badhuli; Mali, Swapna A; Kulkarni, Ketaki; Shastri, Jayanthi

    2011-01-01

    A 25-year-old transgender patient came with complaints of watery discharge, red eye and photophobia in the left eye since 2 days. The patient had a history of wearing colored contact lenses since 4 years and cleaning the lens with tap water. Culture of lenses on Mac Conkey and blood agar yielded Klebsiella pneumoniae and Pseudomonas aeruginosa. Sabouroud's agar showed yeast cells and double-walled cysts of Acanthamoeba species. On further incubation of Sabouroud's agar, the cysts transformed ...

  12. Uptake and Replication of Salmonella enterica in Acanthamoeba rhysodes

    Tezcan-Merdol, Dilek; Ljungström, Marianne; Winiecka-Krusnell, Jadwiga; Linder, Ewert; Engstrand, Lars; Rhen, Mikael

    2004-01-01

    The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internal...

  13. Optimized methods for Legionella pneumophila release from its Acanthamoeba hosts

    Dietersdorfer, Elisabeth; Cervero-Aragó, Sílvia; Sommer, Regina; Kirschner, Alexander K.; Walochnik, Julia

    2016-01-01

    Background Free-living amoebae (FLA) and particularly acanthamoebae serve as vehicles and hosts for Legionella pneumophila, among other pathogenic microorganisms. Within the amoebae, L. pneumophila activates a complex regulatory pathway that enables the bacteria to resist amoebal digestion and to replicate. Moreover, the amoebae provide the bacteria protection against harsh environmental conditions and disinfectants commonly used in engineered water systems. To study this ecological relations...

  14. The Isolation and Detection of Acanthamoeba Keratitis in Rural Water Sources of Arak, Iran

    Mosayebi, M. (PhD

    2014-05-01

    Full Text Available Background and Objective: Acanthamoeba species are free-living protozoa that can be isolated from all environments. They can bring about different diseases in healthy individuals and immune suppressed patients, for example, Granulomatous Amoebic Encephalitis (GAE, Acanthamoeba Keratitis (AK, Cutaneous and Nasopharyngeal infections. The aim of this study was to evaluate the Acanthamoeba prevalence in rural water sources of Markazi province. Material and Methods: In this cross sectional study, 54 water samples were collected from 36 villages of Markus province. First, the Samples were filtered by filter paper (watchman 42. Next, the filtered paper were placed in page saline solution and centrifuged. Then, the obtained sediment was cultured on non-nutrient agar (NNA plates overlaid with heat-killed Escherichia coli. After that, the provided smear (after 4 - 7 days stained with Geimsa. Results: The samples were positive (33; 61.11% and negative (21; 38.89% for Acanthamoeba cyst. The best result for isolation of Acanthamoeba cysts was obtained after shaking of filter paper. Conclusion: A high percentage of rural water sources have been contaminated with Acanthamoeba, which can be the major factor in causing human infections. Therefore, some effective methods are required to prevent from water sources contamination. Keywords: Acanthamoeba; Acanthamoeba Keratitis; Markazi Province; Rural; Water Sources

  15. Influence of cadmium exposure on the incidence of first intermediate host encystment by Echinoparyphium recurvatum cercariae in Lymnaea peregra

    Morley, Neil; Crane, M.; Lewis, J W

    2006-01-01

    The effect of cadmium exposure of the snail first intermediate host Lymnaea peregra on the incidence of encystment of Echinoparyphium recurvatum (Digenea: Echinostomatidae) cercariae without emergence from the snail was investigated. Exposure to 100mg l21 Cd for 72 h caused a significant increase in the incidence of first host encystment when compared to controls. In addition, autometallographic staining of E. recurvatum daughter rediae and developing cercariae showed that there was metal acc...

  16. Rab11 and actin cytoskeleton participate in Giardia lamblia encystation, guiding the specific vesicles to the cyst wall.

    Araceli Castillo-Romero

    Full Text Available BACKGROUND: Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs and the transport of CWPs into encystation-specific vesicles (ESVs. Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. CONCLUSIONS AND SIGNIFICANCE: Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and

  17. Encysted hydrocele of the cord presenting as a groin swelling in an adult: a case report

    Bibekananda Mahapatra

    2015-09-01

    Full Text Available The hydrocele of the cord presents as a translucent swelling in the inguinal canal or scrotum, separately above the testis. It is more commonly seen in childhood and is rare in adults. It comprises of two varieties based on their communication with the peritoneal cavity. The encysted hydrocele of the cord does not communicate with the peritoneal cavity above or the tunica vaginalis below. The other variety known as the funicular hydrocele communicates with the peritoneal cavity above but does not communicate with the tunica vaginalis below. This case report presents the case of a 22 year old young male with an inguinal swelling. Clinical differential diagnoses were of inguinal lymphadenitis, encysted hydrocele of the cord and irreducible hernia. The aim of this report is to highlight the role of ultrasonogram in early diagnosis and surgical excision as mainstay of treatment. [Int J Res Med Sci 2015; 3(9.000: 2475-2477

  18. Effect of Antimicrobial Compounds on Balamuthia mandrillaris Encystment and Human Brain Microvascular Endothelial Cell Cytopathogenicity▿

    Siddiqui, Ruqaiyyah; Matin, Abdul; Warhurst, David; Stins, Monique; Khan, Naveed Ahmed

    2007-01-01

    Cycloheximide, ketoconazole, or preexposure of organisms to cytochalasin D prevented Balamuthia mandrillaris-associated cytopathogenicity in human brain microvascular endothelial cells, which constitute the blood-brain barrier. In an assay for inhibition of cyst production, these three agents prevented the production of cysts, suggesting that the biosynthesis of proteins and ergosterol and the polymerization of actin are important in cytopathogenicity and encystment. PMID:17875991

  19. Toxicity of cadmium and zinc to encystment and in vitro excystment of Parorchis acanthus (Digenea: Philophthalmidae)

    Morley, N.J.; Crane, M.; Lewis, J W

    2001-01-01

    The toxicity of cadmium, zinc and cadmium}zinc mixtures at concentrations ranging from 1000 to 50000 lg}l were investigated against cercariae and metacercariae of Parorchis acanthus obtained from the dog whelk Nucella lapillus. Cercarial encystment at concentrations of 25000 lg}l or higher was signi®cantly impaired by all test metals; however, at lower concentrations only zinc demonstrated toxicity. Mixtures of cadmium and zinc had a synergistic effect compared with single metal toxicity but ...

  20. Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion

    Krtková, Jana; Thomas, Elizabeth B.; Alas, Germain C. M.; Schraner, Elisabeth M.; Behjatnia, Habib R.; Hehl, Adrian B.

    2016-01-01

    ABSTRACT Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-RacCA) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-RacCA-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis. PMID:27555307

  1. A small heat shock/α-crystallin protein from encysted Artemia embryos suppresses tubulin denaturation

    Day, Rossalyn M.; Gupta, Jagdish S.; MacRae, Thomas H.

    2003-01-01

    Small heat shock/α-crystallin proteins function as molecular chaperones, protecting other proteins from irreversible denaturation by an energy-independent process. The brine shrimp, Artemia franciscana, produces a small heat shock/α-crystallin protein termed p26, found in embryos undergoing encystment, diapause, and metabolic arrest. These embryos withstand long-term anoxia and other stresses normally expected to cause death, a property likely dependent on molecular chaperone activity. The as...

  2. Encysted hydrocele of the cord presenting as a groin swelling in an adult: a case report

    Bibekananda Mahapatra; Anuradha Panchal; Abhishek Mahadik; Qurratulain Chougle

    2015-01-01

    The hydrocele of the cord presents as a translucent swelling in the inguinal canal or scrotum, separately above the testis. It is more commonly seen in childhood and is rare in adults. It comprises of two varieties based on their communication with the peritoneal cavity. The encysted hydrocele of the cord does not communicate with the peritoneal cavity above or the tunica vaginalis below. The other variety known as the funicular hydrocele communicates with the peritoneal cavity above but does...

  3. Genotypic, physiological, and biochemical characterization of potentially pathogenic Acanthamoeba isolated from the environment in Cairo, Egypt.

    Tawfeek, Gihan Mostafa; Bishara, Sawsan Abdel-Hamid; Sarhan, Rania Mohammad; ElShabrawi Taher, Eman; ElSaady Khayyal, Amira

    2016-05-01

    Acanthamoebae are the most common opportunistic amphizoic protozoa that cause life-threatening granulomatous amoebic encephalitis in immunocompromised individuals and sight-threatening amoebic keratitis (AK) in contact lens wearers. The present work aimed to determine the presence of Acanthamoeba isolates in different environmental sources: water, soil, and dust in Cairo, Egypt and to characterize the pathogenic potential of the isolated Acanthamoeba using physiological and biochemical assays as well as determination of the genotypes in an attempt to correlate pathogenicity with certain genotypes. The study included the collection of 22 corneal scrapings from patients complaining of symptoms and signs indicative of acanthamoeba keratitis (AK) and 75 environmental samples followed by cultivation on non-nutrient agar plates preseeded with E. coli. Positive samples for Acanthamoeba were subjected to osmo- and thermo-tolerance assays and zymography analysis. Potentially pathogenic isolates were subjected to PCR amplification using genus-specific primer pair. Isolates were classified at the genotype level based on the sequence analysis of Acanthamoeba 18S rRNA gene (diagnostic fragment 3). The total detection rate for Acanthamoeba in environmental samples was 33.3 %, 31.4 % in water, 40 % in soil, and 20 % in dust samples. Three and two Acanthamoeba isolates from water and soil sources, respectively, had the potential for pathogenicity as they exhibited full range of pathogenic traits. Other 12 isolates were designated as weak potential pathogens. Only ten of the environmental isolates were positive in PCR and were classified by genotype analysis into T4 genotype (70 %), T3 (10 %) and T5 (20 %). Potential pathogens belonged to genotypes T4 (from water) and T5 (from soil) while weak potential pathogens belonged to genotypes T3 (from water) and T4 (from water and soil). Additionally, T7 genotype was isolated from keratitis patients. There is a considerable

  4. A Case of Non-Contact Lens related Acanthamoeba keratitis in Malaysia

    Mohamed Kamel, A. G.

    2005-01-01

    Full Text Available Acanthamoeba is a ubiquitous free-living amoeba and is responsible for an uncommon yet increasingly diagnosed keratitis in humans. Acanthamoeba keratitis is perhaps the most challenging ocular infection to manage successfully and it can result in permanently impaired vision or blindness. Although contact lens use is the principal risk factor, about 10% of cases occur following trauma and exposure to contaminated soil or water. Cases of Acanthamoeba keratitis involving contact lens wearers have previously been reported in Malaysia but this is the first time, a non-contact lens relatedAcanthamoeba keratitis is reported. The case involved a 28 year old Indonesian male construction worker who had a trauma of the right eye. While working his eye was struck by some sand and dust particles and he quickly washed his eye with water from an open tank at the construction site. He then experienced pain, redness, glaring and blurring of vision of the right eye. The diagnosis was missed at the initial presentation but subsequent culture of the corneal scraping demonstrated Acanthamoeba as the aetiological agent. The history, clinical findings, diagnosis and treatment of non-contact lens related Acanthamoeba keratitis are briefly discussed in this communication. We hope to create awareness especially among the medical and paramedical staff about the existence of this infection in the country and fully support the consideration of Acanthamoeba keratitis as part of the differential diagnosis of most cases of presumed microbial keratitis.

  5. Isolation and genotyping of Acanthamoeba spp. from recreational soil of parks in Tehran, Iran

    M Niyyati

    2013-11-01

    Full Text Available Background & aim: Acanthamoeba is a genus of free-living amoebae found in environmental sources. These amphizoic amoebae can lead to severe human disease such as encephalitis and keratitis. Acanthamoeba transmits to humans through contact with soil and dust from scratching the skin. The aim of the present study was to identify the genotypes of Acanthamoeba in parks of the city of Tehran using molecular and morphological - based methods. Methods: In this study, 52 samples of soil were collected from 17 parks in Tehran. Samples were then filtered and cultured on 1.5% non-nutrient agar. DNA extraction and PCR amplification was performed using genus specific primers. Sequencing analysis and BLAST search were done for genotype identification. Results: Out of 52 soil samples, 14 strain (26.9% were positive for Acanthamoeba amoebae by microscopic observation. Out Of 14 positive isolates, 9 (17.3% were positive for Acanthamoeba using genus specific primer pairs. Of 14 strains, 9 were sequenced successfully. Genotype identification was revealed that all strains were belonged to T4 type. T4 genotypes among strains are human pathogens. Conclusions: Identification of pathogenic Acanthamoeba belonging to T4 genotype in recreational parks could be of utmost importance. Results of this study show that soil contamination, particularly in parks where children play and assemble is a sanitary risk for them. Key words: Acanthamoeba, Genotypes, Soil, Park

  6. Acanthamoeba spp. in Contact Lenses from Healthy Individuals from Madrid, Spain

    Gomes, Thiago dos Santos; Magnet, Angela; Izquierdo, Fernando; Vaccaro, Lucianna; Redondo, Fernando; Bueno, Sara; Sánchez, Maria Luisa; Angulo, Santiago; Fenoy, Soledad; Hurtado, Carolina; del Aguila, Carmen

    2016-01-01

    Purpose Acanthamoeba keratitis (AK) is a painful and potentially blinding corneal infection caused by Acanthamoeba spp. In Madrid, environmental studies have demonstrated a high presence of these free-living amoebae in tap water. Since most of AK cases occur in contact lenses (CL) wearers with inadequate hygiene habits, the presence of Acanthamoeba in discarded CL has been studied and compared with other common etiological agents of keratitis, such as Pseudomonas aeruginosa and Staphylococcus aureus. Methods One hundred and seventy-seven healthy individuals from Madrid contributed their discarded CL and answered a questionnaire on hygiene habits. DNA was extracted from the CL solution and analyzed by real-time PCR for Acanthamoeba, Pseudomonas aeruginosa and Staphylococcus aureus. These CL and their solutions were also cultured on non-nutrient agar to isolate Acanthamoeba. Results Among the 177 samples, Acanthamoeba DNA was detected in 87 (49.2%), P. aeruginosa DNA in 14 (7.9%) and S. aureus DNA in 19 (10.7%). Cultivable amoebae, however, were observed in only one sample (0.6%). This isolate was genotyped as T4. The habits reported by this CL owner included some recognized risk factors for AK, but in this study only the practice of “not cleaning the CL case” presented some statistical significant association with Acanthamoeba DNA presence. Detection of the investigated bacterial DNA did not demonstrate statistical significant association with the studied practices, but the presence of P. aeruginosa revealed a possible inhibition of Acanthamoeba in these samples. Conclusions The PCR results suggest a high presence of Acanthamoeba spp. in healthy CL wearers from Madrid, but we can assume that CL solutions are properly disinfecting the CL since only 1.1% of the positive PCR samples correspond to viable amoebae and, after four years, only one participant reported stronger ocular problems. Nevertheless, more studies are necessary to corroborate this hypothesis. PMID

  7. Evaluation of immunoperoxidase staining technique in the diagnosis of Acanthamoeba keratitis

    Sharma Savitri

    2001-01-01

    Full Text Available Purpose: We describe a simple procedure of Immunoperoxidase (IP technique, using indigenously raised antibody, to screen corneal scrapings for Acanthamoeba cysts and trophozoites. This study sought to determine the utility of this test in the diagnosis of Acanthamoeba keratitis. Methods: A high titre polyclonal antibody against a local clinical isolate (axenic of Acanthamoeba species (trophozoite lysate antigen was raised in rabbits and used for standardization of IP technique for corneal scrapings. Twenty two smears of corneal scrapings, collected from patients showing Acanthamoeba cysts in corneal scrapings stained with calcofluorwhite (pool-1 and patients showing no cysts in similar scrapings (pool-2, were coded and stained by IP technique by a masked technician. All 22 patients had also been tested for bacteria, fungus, and Acanthamoeba in their corneal scrapings by smears and cultures. IP stained smears were examined for organisms including cysts and trophozoites of Acanthamoeba and background staining by two observers masked to the results of other smears and cultures. The validity of the IP test in detection of Acanthamoeba cysts and trophozoites was measured by sensitivity, specificity, positive predictive value and negative predictive value in comparison (McNemar test for paired comparison with calcofluor white staining and culture. Results: Based on the readings of observer 1 and compared to calcofluor white staining, the IP test had a sensitivity of 100%, a specificity of 94%, positive predictive value of 80% and negative predictive value of 100%. When compared to culture, the values were 83%, 100%, 100% and 94% respectively. Trophozoites missed in calcofluor white stained smears, were detected in 2 out of 6 cases of culture-positive Acanthamoeba keratitis. The Kappa coefficient of interobserver agreement was determined as fair (30.4%. Conclusion: The immunoperoxidase technique is a simple and useful test in the diagnosis of

  8. Acanthamoeba keratitis in a non-contact lens wearer with human immunodeficiency virus

    Hansen, Birgitte Rønde; Kronborg, Gitte

    2003-01-01

    Acanthamoeba keratitis is potentially blinding and often associated with contact lens wearing. A human immunodeficiency virus (HIV)-positive patient, a non-contact lens wearer, presented with keratitis. She experienced a protracted course of disease, characterized by exacerbations and remissions......, and was treated with various topical antibiotics and steroids. 13 months after symptom onset the eye was removed owing to serious scarring of cornea and unbearable pain. Microbiological and histopathological examination of the cornea showed Acanthamoeba. In non-contact lens wearers suffering from...... Acanthamoeba keratitis the diagnosis is delayed, pathognomonic features are often not seen and visual outcome is usually poor. There is no known relation between HIV infection and Acanthamoeba keratitis....

  9. Proteomic approach to reveal the proteins associated with encystment of the ciliate Euplotes encysticus.

    Jiwu Chen

    Full Text Available In order to identify and reveal the proteins related to encystment of the ciliate Euplotes encysticus, we analyzed variation in the abundance of the proteins isolated from the resting cyst comparing with proteins in the vegetative cell. 2-D electrophoresis, MALDI-TOF MS techniques and Bioinformatics were used for proteome separation, quantification and identification. The comparative proteomics studies revealed 26 proteins with changes on the expression in the resting cysts, including 12 specific proteins and 14 differential proteins. 12 specific proteins and 10 out of the 14 differential proteins were selected and identified by MALDI-TOF MS. The identified specific proteins with known functions included type II cytoskeletal 1, keratin, Nop16 domain containing protein, protein arginine n-methyltransferase, epsilon-trimethyllysine hydroxylase and calpain-like protein. The identified differential proteins with known functions included Lysozyme C, keratinocyte growth factor, lysozyme homolog AT-2, formate acetyltransferase, alpha S1 casein and cold-shock protein. We discussed the functions of these proteins as well as their contribution in the process of encystment. These identified proteins covered a wide range of molecular functions, including gene regulation, RNA regulation, proteins degradation and oxidation resistance, stress response, material transport and cytoskeleton organization. Therefore, differential expression of these proteins was essential for cell morphological and physiological changes during encystment. This suggested that the peculiar proteins and differential proteins might play important roles in the process of the vegetative cells transforming into the resting cysts. These observations may be novel findings that bring new insights into the detailed mechanisms of dormancy.

  10. Differentiation of Naegleria fowleri from Acanthamoeba species by using monoclonal antibodies and flow cytometry.

    Flores, B M; Garcia, C A; Stamm, W E; Torian, B E

    1990-01-01

    Monoclonal antibodies to Naegleria fowleri and Acanthamoeba polyphaga were analyzed by enzyme-linked immunosorbent assay, indirect immunofluorescence microscopy, and fluorescence flow cytometry to assess specificity and cross-reactivity with axenically cultured N. fowleri and Acanthamoeba spp. Four monoclonal antibodies to N. fowleri were specific for N. fowleri and had no reactivity to A. polyphaga. Similarly, four monoclonal antibodies to A. polyphaga did not react with N. fowleri. Two of t...

  11. Distribution of Acanthamoeba Genotypes Isolated from Recreational and Therapeutic Geothermal Water Sources in Southwestern Iran

    Niyyati, Maryam; Saberi, Reza; Latifi, Alireza; LASJERDI, Zohreh

    2016-01-01

    A comprehensive survey was conducted along 10 km of geothermal rivers in southwestern Iran. A total of 40 water samples were tested for the presence of Acanthamoeba spp., and genotypes were determined by targeting the diagnostic fragment 3 region of the 18S rRNA gene. The pathogenic potential of all positive isolates was also identified using tolerance ability test. High occurrences of Acanthamoeba (50%) were detected in the sampling areas. Based on sequencing analysis, isolates belonging to ...

  12. Detection of Acanthamoeba and Toxoplasma in River Water Samples by Molecular Methods in Iran.

    Mohammad Reza Mahmoudi

    2015-06-01

    Full Text Available Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples.A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively.Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites.The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oocystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran.

  13. Presence of Acanthamoeba spp.in water purification plants in southern England

    Shanmuganathan V; Khan NA

    2009-01-01

    Objective:To identify the prevalence of Acanthamoeba in drinking water treatment plants during the course of the purification processes.Methods:Samples were taken from two drinking water purification plants and moni-tored for the presence of Acanthamoeba in order to estimate the removal capacity of treatment methods em-ployed.Water samples were collected at each step in the purification,during the one year survey,and ana-lysed for the presence of Acanthamoeba spp.by plating on bacterial-seeded plates.Results:The results showed that amoebae were present in surface raw waters in 100 % of the samples tested.Acanthamoeba spp.were iso-lated from 71 % and 57 % of the water samples collected from post flat-bottom clarifier 1 and post-sedimenta-tion plant respectively.Considering the outflow drinking waters,the removal capacity was 100 % in both puri-fication plants monitored.The occurrence of Acanthamoeba was not associated with seasonality.Conclusion:These findings confirm that water purification plants employing methods of flocculation,sedimentation,and fil-tration in combination with activated charcoal filtration,ozonisation and chlorination exhibited sufficient Acan-thamoeba removal capacity and the presence of amoebae in the tap water may be due to older plumbing,water storage tanks,tap water hygiene,and /or environmental settings.

  14. Isolation of Acanthamoeba isolates belonging to T2, T3, T4 and T7 genotypes from environmental samples in Ankara, Turkey

    Kiliç, A.; Tanyuksel, M.; Sissons, J.; Jayasekera, S.; Khan, Naveed Ahmed

    2004-01-01

    Acanthamoeba keratitis is a blinding infection that is becoming increasingly important in human health. Early diagnosis is a prerequisite for successful treatment and requires identification of Acanthamoeba at the genotypic level. The genus Acanthamoeba consists of both pathogenic and non-pathogenic species and has been recently classified into 13 different genotypes, T1-T12 and T14. More importantly, 95% of Acanthamoeba isolates that produce keratitis belong to T4 genotypes. In this study, w...

  15. [Use of the presence of cellulose in cellular wall of Acanthamoeba cysts for diagnostic purposes].

    Derda, Monika; Hadaś, Edward

    2009-01-01

    Species identification within the genus Acanthamoeba is based predominantly on morphological and biochemical features. It is labor-intensive and requires cloning and axenization. We described a novel immunocytochemical method for the identification of Acanthamoeba spp. based on selective binding of Clostridium cellulovorans cellulase to protozoan cyst wall cellulose. Free-living amoebae isolated from different water sources by filtration and subsequent cultivation on non-nutrient agar were assigned to genera Acanthamoeba, Naegleria or Hartmannella using morphological taxonomic criteria. Tissues samples from experimentally infected mice were fixed in formalin and for sectioning embedded in paraffin or snap frozen. The Cellulose-Binding Domain of C. cellulovorans cellulase (CBD) obtained as a recombinant protein, were coupled to the fluorescent dye using Alexa Fluor350, 488, 568 - Protein Labelling Kit or labelled with the biotin using EZ-Link Sulfo-NHS-Biotin. All coupling procedures were performed according to the methods provided by manufacturers. For staining with CBD conjugate, slides containing cysts collected from the agar plates or tissue sections were immersed with PBS and incubated with CBD for 30 min at room temperature, washed 3 times with PBS. For staining with CBD-biotin slides containing cysts were incubated with biotinylated CBD for 30 min at room temperature. Subsequent washings in changes of PBS were followed by the incubation with Strept ABComplex/HRP, for 30 min at room temperature, than 3,3 diaminobenzidine tetrahydrochloride was added for 15 min. Slides were rinsed with water, dried and examined in the light microscope. We showed that cellulose could be easily detected by immunofluorescence using conjugated CBD in the inner cyst wall of Acanthamoeba spp. The reference strains of Acanthamoeba spp. and all Acanthamoeba strains isolated from water and from tissues of infected animals gave positive reaction. CBD prepared as a biotynylated protein

  16. Acanthamoeba Keratitis Presenting as Dendritic Keratitis in a Soft Contact Lens Wearer

    Edward Yip Yeung

    2002-03-01

    Full Text Available Acanthamoeba keratitis is a rare cause of corneal infection in Taiwan, which can resultin devastating visual outcomes. A 37-year-old woman, who wore soft contact lenses, sufferedfrom severe pain in her left eye. Biomicroscopy revealed dendritic keratitis, radialkeratoneuritis, and fine keratic precipitates on her cornea. Culture, using non-nutrient agarplate seeded with Escherichia coli, resulted in heavy growth of Acanthamoeba. The inpatienttreatment, including topical neomycin-polymyxin B and metronidazole (0.5% eyedrops,oral ketoconazole, and then oral prednisolone, successfully controlled the cornealinfection. The best-corrected visual acuity was 0.9 without any evidence of recurrence ofinfection after 21 months of follow up. Acanthamoeba keratitis can present as dendritic keratitis,which mimics herpes simplex infection, thus, delays appropriate treatment. Early diagnosisand judicious treatment are essential for restoring the vision and avoiding the subsequentneed of penetrating keratoplasty.

  17. Failure of chemotherapy in the first reported cases of Acanthamoeba keratitis in Pakistan

    Siddiqui, Ruqaiyyah; Chaudhry, Tanveer; Lakhundi, Sahreena; Ahmad, Khabir; Khan, Naveed Ahmed

    2014-01-01

    Acanthamoeba keratitis is a painful and progressive infection of the cornea that can result in loss of vision. Here, for the first time in Pakistan, we report two cases of Acanthamoeba keratitis. The first patient was a 37-year-old female who presented with severe itching, redness, pain, along with loss of vision. The patient was a regular soft contact lens wearer. The second patient was a 25-year-old female who had been using soft contact lenses for the past two years. She presented with a b...

  18. Validation of Real-Time PCR for Laboratory Diagnosis of Acanthamoeba Keratitis▿

    Thompson, Paul P.; Kowalski, Regis P.; Shanks, Robert M. Q.; Gordon, Y. Jerold

    2008-01-01

    Confirmation of Acanthamoeba keratitis by laboratory diagnosis is the first step in the treatment of this vision-threatening disease. Two real-time PCR TaqMan protocols (the Rivière and Qvarnstrom assays) were developed for the detection of genus-specific Acanthamoeba DNA but lacked clinical validation. We have adapted these assays for the Cepheid SmartCycler II system (i) by determining their real-time PCR limits of detection and amplification efficiencies, (ii) by determining their ability ...

  19. Eukaryotic cell encystation and cancer cell dormancy: is a greater devil veiled in the details of a lesser evil?

    Baig, Abdul Mannan; Khan, Naveed Ahmed; Abbas, Farhat

    2015-01-01

    Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy as dormant cells evade not only the anticancer drugs but also the host immune system. These dormant cells veil themselves from detection by imaging and/or using biomarkers, which imposes an additional problem in targeting such cells. A similar form of hibernation process known as encystation is studied in detail for pathogenic unicellular eukaryotic microorganisms. By examination using microarray gene expressio...

  20. Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates

    A.H. Maghsood; Sissons, J.; Rezaian, M.; Nolder, D; Warhurst, D.; Khan, Naveed Ahmed

    2005-01-01

    The majority of the keratitis-causing Acanthamoeba isolates are genotype T4. In an attempt to determine whether predominance of T4 isolates in Acanthamoeba keratitis is due to greater virulence or greater prevalence of this genotype, Acanthamoeba genotypes were determined for 13 keratitis isolates and 12 environmental isolates from Iran. Among 13 clinical isolates, eight (61.5 %) belonged to T4, two (15.3 %) belonged to T3 and three (23 %) belonged to the T2 genotype. In contrast, the majorit...

  1. Eukaryotic cell encystation and cancer cell dormancy: is a greater devil veiled in the details of a lesser evil?

    Baig, Abdul Mannan; Khan, Naveed Ahmed; Abbas, Farhat

    2015-01-01

    Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy as dormant cells evade not only the anticancer drugs but also the host immune system. These dormant cells veil themselves from detection by imaging and/or using biomarkers, which imposes an additional problem in targeting such cells. A similar form of hibernation process known as encystation is studied in detail for pathogenic unicellular eukaryotic microorganisms. By examination using microarray gene expression profiles, immunocytochemistry tools, and siRNAs during the process of encystation, understanding the covert features of cancer cell dormancy as proposed could be possible. This knowledge can be extended to dormant cancer cells to uncover the mechanisms that underlie this ghost, yet dangerous state of human cancers. We propose a strategy to induce dormancy and exit this state by application of knowledge gained from the encystation induction and retrieval processes in pathogenic eukaryotic microorganisms. Given that early detection and characterization of dormant malignant tumor cells is important as a general strategy to monitor and prevent the development of overt metastatic disease, this homology may enable the design of therapies that could either awake the dormant cell from dormancy to make it available for therapies or prolong such a phase to make cancer appear as a chronic disease. PMID:25859414

  2. Acanthamoeba keratitis in a non-contact lens wearer with human immunodeficiency virus

    Hansen, Birgitte Rønde; Kronborg, Gitte

    2003-01-01

    , and was treated with various topical antibiotics and steroids. 13 months after symptom onset the eye was removed owing to serious scarring of cornea and unbearable pain. Microbiological and histopathological examination of the cornea showed Acanthamoeba. In non-contact lens wearers suffering from...

  3. Isolation and identification of Acanthamoeba spp. from thermal swimming pools and spas in Southern Brazil.

    Fabres, Laura Fuhrich; Rosa Dos Santos, Sayonara Peixoto; Benitez, Lisianne Brittes; Rott, Marilise Brittes

    2016-06-01

    Free-living amoebae (FLA) are widely distributed in soil and water. A few number of them are implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Species of Acanthamoeba can cause keratitis and brain infections. In this study, 72 water samples were taken from both hot tubs and thermal swimming pools in the city of Porto Alegre, RS, Brazil, to determine the presence of Acanthamoeba in the water as well as perform the phenotypic and genotypic characterization of the isolates. The identification of the isolates was based on the cysts morphology and PCR amplification using genus-specific oligonucleotides. When the isolates were submitted to PCR reaction only 8 were confirmed as belonging to the genus Acanthamoeba. The sequences analysis when compared to the sequences in the GenBank, showed genotype distribution in group T3 (12,5%), T5 (12,5%), T4 (25%) and T15 (50%). The results of this study confirmed the presence of potentially pathogenic isolates of free living amoebae in hot swimming pool and spas which can present risks to human health. PMID:27078644

  4. Molecular Identification and Sequencing of Mannose Binding Protein (MBP Gene of Acanthamoeba palestinensis

    M Rezaeian

    2010-02-01

    Full Text Available "nBackground: Acanthamoeba keratitis develops by pathogenic Acanthamoeba such as A. pal­es­tinen­sis. Indeed this species is one of the known causative agents of amoebic keratitis in Iran. Mannose Binding Protein (MBP is the main pathogenicity factors for developing this sight threatening disease. We aimed to characterize MBP gene in pathogenic Acanthamoeba isolates such as A. palestinensis."nMethods: This experimental research was performed in the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran during 2007-2008.  A. palestinensis was grown on 2% non-nutrient agar overlaid with Escherichia coli. DNA extraction was performed using phenol-chloroform method. PCR reaction and amplification were done using specific primer pairs of MBP. The amplified fragment were purified and sequenced. Finally, the obtained fragment was deposited in the gene data bank."nResults: A 900 bp PCR-product was recovered after PCR reaction. Sequence analysis of the purified PCR product revealed a gene with 943 nucleotides. Homology analysis of the ob­tained sequence showed 81% similarity with the available MBP gene in the gene data bank. The fragment was deposited in the gene data bank under accession number EU678895"nConclusion: MBP is known as the most important factor in Acanthamoeba pathogenesis cas­cade. Therefore, characterization of this gene can aid in developing better therapeutic agents and even immunization of high-risk people.

  5. Free-living Amoebae (FLA: morphological and molecular identification of Acanthamoeba in dental unit water

    Trabelsi H.

    2010-03-01

    Full Text Available The aim of our study was to detect free-living Amoebae (FLA by morphological methods and to identify Acanthamoeba spp. by PCR in the dental unit water lines (DUWL. Materials and methods: it was a prospective study dealing with 196 water samples collected from DUWL (94 samples taken in the early morning before materials flush and patient consultations and 102 samples taken after consultations. At the same time, 39 samples from tap water were realized. Results: 135 (69 % samples were positives by the morphological study with morphotypical diversity. The predominant morphotype was the monopodial (39.2 %. 18 strains of Acanthamoeba spp. were detected in DUW (13.3 % and three strains in tap water (10 %. The amplification of 18S rDNA gene of these strains of Acanthamoeba spp. was positive for all samples. Conclusion: the FLA and Acanthamoeba were isolated both in tap water and in dental unit. The amoeba pathogenicity has not been demonstrated after oral or dental contamination; but the presence of intracellular and pathogenic bacteria in the amoeba could be a source of microbiological risks for patients in case of deep dental care or immunodepression. The improvement of this dental unit was necessary by putting a filter of 0.2 microns porosity before the arrival of the water in hand-pieces allowing the limitation of FLA passage.

  6. Fatal Granulomatous Amoebic Encephalitis Caused by Acanthamoeba in a Patient With Kidney Transplant: A Case Report.

    Salameh, Ahmad; Bello, Nancy; Becker, Jennifer; Zangeneh, Tirdad

    2015-09-01

    Granulomatous amoebic encephalitis (GAE) due to Acanthamoeba is almost a uniformly fatal infection in immune-compromised hosts despite multidrug combination therapy. We report a case of GAE in a female who received a deceased donor kidney graft. She was treated with a combination of miltefosine, pentamidine, sulfadiazine, fluconazole, flucytosine, and azithromycin. PMID:26280011

  7. Habitat diversity and adaptation to environmental stress in encysted embryos of the crustacean Artemia

    Joshua A Tanguay; Reno C Reyes; James S Clegg

    2004-12-01

    Encysted embryos (cysts) of the brine shrimp, Artemia, provide excellent opportunities for the study of biochemical and biophysical adaptation to extremes of environmental stress in animals. Among other virtues, this organism is found in a wide variety of hypersaline habitats, ranging from deserts, to tropics, to mountains. One adaptation implicated in the ecological success of Artemia is p26, a small heat shock protein that previous evidence indicates plays the role of a molecular chaperone in these embryos. We add to that evidence here. We summarize recently published work on thermal tolerance and stress protein levels in embryos from the San Francisco Bay (SFB) of California inoculated into experimental ponds in southern Vietnam where water temperatures are much higher. New results on the relative contents of three stress proteins (hsp70, artemin and p26) will be presented along with data on cysts of A. tibetiana collected from the high plateau of Tibet about 4.5 km above sea level. Unpublished results on the stress protein artemin are discussed briefly in the context of this paper, and its potential role as an RNA chaperone. Interestingly, we show that the substantial tolerance of A. franciscana embryos to ultraviolet (UV) light does not seem to result from intracellular biochemistry but, rather, from their surrounding thick shell, a biophysical adaptation of considerable importance since these embryos receive heavy doses of UV in nature.

  8. Comparative efficacy of moxidectin and ivermectin against hypobiotic and encysted cyathostomes and other equine parasites.

    Xiao, L; Herd, R P; Majewski, G A

    1994-05-01

    Efficacies of moxidectin and ivermectin were compared in four groups of eight ponies with natural parasite infections: placebo (Control), oral moxidectin gel at 0.3 mg kg-1 of body weight (Mox 0.3), oral moxidectin gel at 0.4 mg kg-1 of body weight (Mox 0.4), and oral ivermectin paste at 0.2 mg kg-1 of body weight (Ivermectin). Fecal samples were taken 0 and 2 weeks after treatment. Animals were necropsied and worms were collected 2 weeks after treatment. Moxidectin and ivermectin showed similar efficacy (99%) against adult cyathostomes, Strongylus spp., Triodontophorus spp. and Habronema muscae. Both drugs were also more than 98% effective against luminal cyathostome and Oxyuris equi fourth stage larvae (L4). Neither drug was effective (0-10.1%) against hypobiotic early third stage cyathostome larvae (EL3). Moxidectin was moderately effective (62.6-79.1%) in removing encysted cyathostome late third stage larvae (LL3) and L4, whereas ivermectin was ineffective (0%) against these stages. By contrast, ivermectin was 95.4% effective against Gasterophilus spp. third instar stage, whereas moxidectin was only 0-20.4% effective. PMID:8091622

  9. Acanthamoeba genotypes T2, T4, and T11 in soil sources from El Hierro island, Canary Islands, Spain.

    Reyes-Batlle, María; Zamora-Herrera, Jonadab; Vargas-Mesa, Alejandro; Valerón-Tejera, Marco Antonio; Wagner, Carolina; Martín-Navarro, Carmen Ma; López-Arencibia, Atteneri; Sifaoui, Ines; Martínez-Carretero, Enrique; Valladares, Basilio; Piñero, José E; Lorenzo-Morales, Jacob

    2016-08-01

    The genus Acanthamoeba includes pathogenic strains which are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, forty soil samples were collected in the island of El Hierro, Canary Islands, Spain, and checked for the presence of Acanthamoeba. Samples were cultivated onto 2 % non-nutrient agar plates seeded with a layer of heat killed Escherichia coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 47.5 % of soil samples. Moreover, genotypes T2, T4, and T11 were identified in these samples. To the best of our knowledge, this is the first study to establish genotypes T2, T4, and T11 in soil sources from El Hierro island. PMID:27075307

  10. Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

    Carolina De Marco Verissimo

    2013-11-01

    Full Text Available Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

  11. Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City.

    Bonilla-Lemus, Patricia; Ramírez-Bautista, Gerardo A; Zamora-Muñoz, Claudia; Ibarra-Montes, María Del Rocío; Ramírez-Flores, Elizabeth; Hernández-Martínez, María Dolores

    2010-09-01

    A survey was carried out in the metropolitan area of Mexico City to determine the presence of Acanthamoeba in the tap water of houses of contact lens wearers. Water samples were taken from the mains water entry, bathroom sinks and storage containers (roof tanks, cisterns) of 27 houses; and from the solution contained in the contact lens cases. Samples were filtered and cultured onto NNE medium. The isolates were identified based on their morphological features and pathogenicity. Total and fecal coliforms, water temperature, pH, dissolved oxygen and residual free-chlorine were measured by standard methods. Forty five isolates of Acanthamoeba from 200 water samples were obtained. The highest number of amoebae was isolated from cisterns and roof tanks. Most Acanthamoeba isolates were non-pathogenic, however, their presence in tap water is a potential hazard since some species can cause Acanthamoeba keratitis and granulomatous amoebic encephalitis. PMID:19995560

  12. Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

    Pan, Hong [Department of Ophthalmology, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012 (China); The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012 (China); Wu, Xinyi, E-mail: xywu8868@163.com [Department of Ophthalmology, Qilu Hospital, Shandong University, 107, Wenhua Xi Road, Jinan 250012 (China)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-{beta}. Black-Right-Pointing-Pointer Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. Black-Right-Pointing-Pointer Hypoxia inhibits Acanthamoeba-induced the activation of NF-{kappa}B and ERK1/2 in HCECs. Black-Right-Pointing-Pointer Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. Black-Right-Pointing-Pointer LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-{beta} (IFN-{beta}) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-{beta}. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-{kappa}B and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88

  13. Reporting of T4 Genotype of Acanthamoeba Isolates in Recreational Water Sources of Gilan Province, Northern Iran

    Maryam Niyyati

    2015-02-01

    Full Text Available Background: Acanthamoeba spp. is the causative agent of blindness keratitis and fatal encephalaitis. Presence of Acanthamoeba spp. in a wide variety of niches such as different water types can lead to exposure of high risk people such as contact lens wearers. The main aim of the present study was to explore the occurrence of Acanthamoeba genotypes in the recreational water sources using both morphological and molecular approaches in Gilan province, Iran.Materials and Methods: Overall, 50 samples were collected from recreational water sources including man- made and natural waters in Gilan province. Filtration and cultivation of samples was performed using non-nutrient agar. Cloning of Acanthamoeba spp. was done to eliminate bacterial and fungi contamination. PCR amplification and sequencing were performed using genus-specific primer pair. Genotype identification was based on homology analysis of 18S rRNA gene (DF3 of the obtained sequences with the available genes in the gene bank data base.Results: Out of 50 water samples, 15 (30% were positive for Acanthamoeba trophozoites and cysts according to morphological criteria. Cloning of 13 isolates (26% was done successfully. Molecular analysis of 13 Acanthamoeba strain revealed that all isolates were belonged to potentially pathogenic T4 genotype.Conclusion: T4 genotype is the main cause of Acanthamoeba-related infections. Presence of Acanthamoeba belonged to T4 genotype in recreational water sources is of concern for high risk people. Alarming sign and education to high risk people is of utmost importance to prevent such infections.

  14. Acanthamoeba produces disseminated infection in locusts and traverses the locust blood-brain barrier to invade the central nervous system

    Kirk Ruth; Goldsworthy Graham; Mortazavi Parisa N; Khan Naveed A.

    2010-01-01

    Abstract Background Many aspects of Acanthamoeba granulomatous encephalitis remain poorly understood, including host susceptibility and chronic colonization which represent important features of the spectrum of host-pathogen interactions. Previous studies have suggested locusts as a tractable model in which to study Acanthamoeba pathogenesis. Here we determined the mode of parasite invasion of the central nervous system (CNS). Results Using Acanthamoeba isolates belonging to the T1 and T4 gen...

  15. Acanthamoeba produces disseminated infection in locusts and traverses the locust blood-brain barrier to invade the central nervous system

    Kirk Ruth

    2010-07-01

    Full Text Available Abstract Background Many aspects of Acanthamoeba granulomatous encephalitis remain poorly understood, including host susceptibility and chronic colonization which represent important features of the spectrum of host-pathogen interactions. Previous studies have suggested locusts as a tractable model in which to study Acanthamoeba pathogenesis. Here we determined the mode of parasite invasion of the central nervous system (CNS. Results Using Acanthamoeba isolates belonging to the T1 and T4 genotypes, the findings revealed that amoebae induced sickness behaviour in locusts, as evidenced by reduced faecal output and weight loss and, eventually, leading to 100% mortality. Significant degenerative changes of various tissues were observed by histological sectioning. Both isolates produced disseminated infection, with viable amoebae being recovered from various tissues. Histological examination of the CNS showed that Acanthamoeba invaded the locust CNS, and this is associated with disruption of the perineurium cell/glial cell complex, which constitutes the locust blood-brain barrier. Conclusions This is the first study to demonstrate that Acanthamoeba invades locust brain by modulating the integrity of the insect's blood-brain barrier, a finding that is consistent with the human infection. These observations support the idea that locusts provide a tractable model to study Acanthamoeba encephalitis in vivo. In this way the locust model may generate potentially useful leads that can be tested subsequently in mammalian systems, thus replacing the use of vertebrates at an early stage, and reducing the numbers of mammals required overall.

  16. Macronuclear chromatin structure dynamics in Colpoda inflata (Protista, Ciliophora) resting encystment.

    Tiano, L; Chessa, M G; Carrara, S; Tagliafierro, G; Delmonte Corrado, M U

    1999-01-01

    The chromatin structure dynamics of the Colpoda inflata macronucleus have been investigated in relation to its functional condition, concerning chromatin body extrusion regulating activity. Samples of 2- and 25-day-old resting cysts derived from a standard culture, and of 1-year-old resting cysts derived from a senescent culture, were examined by means of histogram analysis performed on acquired optical microscopy images. Three groups of histograms were detected in each sample. Histogram classification, clustering and matching were assessed in order to obtain the mean histogram of each group. Comparative analysis of the mean histogram showed a similarity in the grey level range of 25-day- and 1-year-old cysts, unlike the wider grey level range found in 2-day-old cysts. Moreover, the respective mean histograms of the three cyst samples appeared rather similar in shape. All this implies that macronuclear chromatin structural features of 1-year-old cysts are common to both cyst standard cultures. The evaluation of the acquired images and their respective histograms evidenced a dynamic state of the macronuclear chromatin, appearing differently condensed in relation to the chromatin body extrusion regulating activity of the macronucleus. The coexistence of a chromatin-decondensed macronucleus with a pycnotic extrusion body suggests that chromatin unable to decondense, thus inactive, is extruded. This finding, along with the presence of chromatin structural features common to standard and senescent cyst populations, supports the occurrence of 'rejuvenated' cell lines from 1-year-old encysted senescent cells, a phenomenon which could be a result of accomplished macronuclear renewal. PMID:10439214

  17. Citologia de impressão no diagnóstico de infecção corneana por Acanthamoeba: relato de caso Diagnosis of Acanthamoeba corneal infection by impression cytology: case report

    Jeison de Nadai Barros

    2007-03-01

    Full Text Available Relatamos três casos de infecção corneana por Acanthamoeba sp em que foi possível detectar cistos do microorganismo com a técnica de citologia de impressão. Três pacientes encaminhados ao Laboratório de Doenças Externas Oculares em 2004 com alterações superficiais da córnea foram submetidos ao exame de citologia de impressão para investigação da presença de cistos de Acanthamoeba sp. Duas amostras foram obtidas da córnea de cada paciente e coradas com PAS, hematoxilina e Papanicolaou. Investigação microbiológica de rotina e cultura também foram realizadas após raspado da córnea. O cultivo das amostras e a citologia de impressão foram positivas para Acanthamoeba sp em todos os pacientes, ao passo que os raspados corados com Giemsa foram positivos em dois casos. A citologia de impressão revelou cistos de Acanthamoeba sp entre feixe de células epiteliais corneanas e como células isoladas. Foram observados cistos no epitélio de um dos pacientes com a citologia de impressão após três meses de tratamento, enquanto o raspado foi negativo. No exame anatomopatológico observaram-se cistos no epitélio e estroma de uma córnea receptora de um dos pacientes após transplante. Neste estudo, a citologia de impressão detectou com sucesso cistos de Acanthamoeba sp em pacientes com acometimento epitelial. Por tratar-se de método não invasivo, a técnica pode ser usada para facilitar o diagnóstico mais precoce da infecção por Acanthamoeba, sendo útil também no acompanhamento do tratamento da doença.To describe three cases of corneal infection due to Acanthamoeba sp in which was possible to detect Acanthamoeba sp cysts by the corneal impression cytology technique. Three patients referred to the External Eye Disease Laboratory in 2004 with superficial corneal alterations were submitted to corneal specimen collection by impression cytology filter paper to investigate the presence of Acanthamoeba sp cysts. Two impression

  18. Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

    Naveed Ahmed Khan

    2015-01-01

    Full Text Available In the face of harsh conditions and given a choice, a cell may (i undergo programmed cell death, (ii transform into a cancer cell, or (iii enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes.

  19. Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

    Naveed Ahmed Khan; Junaid Iqbal; Ruqaiyyah Siddiqui

    2015-01-01

    In the face of harsh conditions and given a choice, a cell may (i) undergo programmed cell death, (ii) transform into a cancer cell, or (iii) enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the ...

  20. Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

    Khan, Naveed Ahmed; Iqbal, Junaid

    2015-01-01

    In the face of harsh conditions and given a choice, a cell may (i) undergo programmed cell death, (ii) transform into a cancer cell, or (iii) enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes. PMID:25648302

  1. Distribution of Acanthamoeba Genotypes Isolated from Recreational and Therapeutic Geothermal Water Sources in Southwestern Iran.

    Niyyati, Maryam; Saberi, Reza; Latifi, Alireza; Lasjerdi, Zohreh

    2016-01-01

    A comprehensive survey was conducted along 10 km of geothermal rivers in southwestern Iran. A total of 40 water samples were tested for the presence of Acanthamoeba spp., and genotypes were determined by targeting the diagnostic fragment 3 region of the 18S rRNA gene. The pathogenic potential of all positive isolates was also identified using tolerance ability test. High occurrences of Acanthamoeba (50%) were detected in the sampling areas. Based on sequencing analysis, isolates belonging to T4 (93.7%) and T2 (6.25%) genotypes were reported. Thermo- and osmotolerance tests revealed that five strains are highly pathogenic. Since every collection site of this study was associated with high human activity, posting of warning signs, monitoring of recreational water sources, and awareness of high-risk people are of utmost importance. To the best of our knowledge, the present research is the first to report T2 genotype from geothermal water sources in Iran. PMID:27127409

  2. Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

    Sissons, James; Alsam, Selwa; Goldsworthy, Graham; Lightfoot, Mary; Jarroll, Edward L; Khan, Naveed Ahmed

    2006-01-01

    Background Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. Results Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood-brain barrier perturbations. Conclusion Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae. PMID:16672059

  3. Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis

    Jarroll Edward L

    2006-05-01

    Full Text Available Abstract Background Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. Results Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM, which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM, elastin (elastic fibrils of ECM, plasminogen (involved in proteolytic degradation of ECM, as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC. Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like suggesting a role in blood-brain barrier perturbations. Conclusion Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s in invasion of the brain tissue by amoebae.

  4. Effect of Parachlamydia acanthamoebae on pulmonary function parameters in a bovine respiratory model.

    Lohr, M; Prohl, A; Ostermann, C; Diller, R; Greub, G; Reinhold, P

    2016-07-01

    The aim of this study was to evaluate pulmonary dysfunction induced by experimental infection with Parachlamydia acanthamoebae in calves. Intrabronchial inoculation with P. acanthamoebae was performed in 31 calves aged 2-3 months old at two different challenge doses of 10(8) and 10(10) inclusion-forming units (IFU) per animal. Control animals received heat inactivated bacteria. The effects on pulmonary gas exchange were determined by arterial blood gas analysis and haemoximetry during the 7 days post inoculation (DPI). For pulmonary function testing (PFT), impulse oscillometry, capnography, and measurement of O2 uptake were undertaken in spontaneously breathing animals 7 and 3 days before inoculation and were repeated until 10 DPI. In the early phase after challenge (1-3 DPI), mild hypoxaemia occurred, which was accompanied by a significant reduction in both tidal and alveolar volumes (each related to bodyweight, BW). In parallel, expiratory flow rate and specific ventilation (i.e. minute ventilation related to O2 uptake) were significantly increased. Minute and alveolar ventilations (each related to metabolic BW) increased significantly due to higher respiratory rates, lasting until 4 and 5 DPI, respectively. Oxygen uptake was slightly reduced during the first 2 days after challenge, but increased significantly during the recovery phase, from 4 to 8 DPI. No deterioration in respiratory mechanics or acid-base balance was observed. Respiratory infection with 10(10) IFU P. acanthamoebae per calf induced mild respiratory dysfunction, mainly characterised by hypoxaemia. The study's findings do not indicate severe pathophysiological consequences of P. acanthamoebae infection on pulmonary function in the bovine host. PMID:27240907

  5. Prevalence of Acanthamoeba and other naked amoebae in South Florida domestic water.

    Shoff, M E; Rogerson, A; Kessler, K; Schatz, S; Seal, D V

    2008-03-01

    The purpose was to identify the prevalence of naked amoebae in tap water in south Florida to ascertain the risk of amoebal infections of the cornea in contact lens wearers. Over the course of a 2-year period, water samples were collected from sites throughout Broward, Palm Beach, and Dade counties, Florida. The presence of amoebae in samples was based on an enrichment cultivation method appropriate for Acanthamoeba. Amoebae were identified using diagnostic features discernable by light microscopy. A total of 283 water samples were processed and amoebae were noted in 80 of these. Acanthamoeba were found on 8 occasions (2.8%). The genera Hartmannella and Vahlkampfia, rarely involved in keratitis cases, were found in 3.5% and 2.8% of samples, respectively. A total of 19 different naked amoebae were recorded and amoebae (regardless of genus) were present in 19.4% of all samples. Previous surveys in England and Korea have shown that acanthamoebae are found in 15 to 30% of tap water samples in the home and have been associated with corneal infection in contact lens wearers. The incidence of acanthamoebae infection in the USA (2.8%) has been found to be lower than that in the UK and it has been postulated that this is related to the lack of a storage water tank in the roof loft space. However, the level of treatment of municipal water is clearly not effective at killing amoebal cysts (or trophozoites) as evidenced by the high occurrence of amoebae (19.4%) in this study. PMID:17998610

  6. Isolation of Acanthamoeba Spp. from Drinking Waters in Several Hospitals of Iran

    HR Bagheri

    2010-06-01

    Full Text Available Background: Acanthamoeba is an opportunistic amphizoic protozoan found in different wa­ter sources including swimming pool as well as in sewage. The aim of this study was to in­vestigate the prevalence of Acanthamoeba in tap-water samples in Iran.Method: In this descriptive cross-sectional study, 94 samples of cold and warm tap-water were collected from different wards of hospitals in 13 cities of Iran in 2007-2008. Free resi­dual chlorine, pH, and temperature of samples were measured. After filtration through multi­pore nylon membrane, samples were cultured on non-nutrient agar. Then we investigated ex­istence of Acanthamoeba by reverse contrast phase microscope.Results: Acanthamoeba was found in 45 samples (48%. Thirty-four and 11 positive samples were collected from cold and warm tap water, respectively. The samples belonged to the cate­gory of 20-30 °C temperature with 0-2 ppm free residual chlorine and pH 6-7.4 showed the most coincidence to the positive cases. The greatest proportion of positive samples was ob­tained from Mashhad hospitals, while all samples collected from Arak and Semnan hospitals were negative.Conclusion: considering the results of this study and the pathogenic role of this protozoan on pa­tients with immunodeficiency, as well as capability of this microorganism in carrying other pathogens such as Legionella, further studies are needed. What is more important, potable water in hospitals should follow the procedure of treatment and sanitation, in order to prevent the relevant nosocomial infections.

  7. Experimental infection of T4 Acanthamoeba genotype determines the pathogenic potential.

    Alves, Daniella de Sousa Mendes Moreira; Moraes, Aline Silva; Alves, Luciano Moreira; Gurgel-Gonçalves, Rodrigo; Lino Junior, Ruy de Souza; Cuba-Cuba, César Augusto; Vinaud, Marina Clare

    2016-09-01

    T4 is the Acanthamoeba genotype most related to cases of granulomatous amoebic encephalitis (GAE) in immunocompromised patients and of keratitis in contact lens wearers. The determination of the pathogenic potential of Acanthamoeba clinical and environmental isolates using experimental models is extremely important to elucidate the capacity of free-living organisms to establish and cause disease in hosts. The aim of this study was to compare and evaluate the histopathology and culture between two different routes of experimental infection of T4 Acanthamoeba isolated from environmental and clinical source in mice (intracranial and intraperitoneal). Swiss isogenic healthy mice were inoculated with 10(4) trophozoites by intracranial (IC) and intraperitoneal (IP) routes and observed during 21 days. The brains from animals inoculated by the IC route were collected and from the animals of the IP inoculation group, the brains, livers, kidneys, spleens, and lungs were removed. The organs were prepared and appropriately divided to be evaluated with histopathology and culture. There was no significant difference between the inoculation routes in terms of isolates recovery (χ(2) = 0.09; p = 0.76). In the IC group, isolate recovery rate was significantly higher in histopathology than the one achieved by culture (χ(2) = 6.45; p protozoa, improving the knowledge on free-living amoebae isolates. PMID:27164833

  8. High occurrence of Acanthamoeba genotype T4 in soil sources from Bolívar State, Venezuela.

    Wagner, Carolina; Reyes-Batlle, María; Hernán, Aurora; Rojas, Elsy; Pérez, Gladymar; López-Arencibia, Atteneri; Sifaoui, Ines; Martínez-Carretero, Enrique; Piñero, José E; Valladares, Basilio; Lorenzo-Morales, Jacob

    2016-09-01

    Pathogenic strains of Acanthamoeba are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, twenty-seven soil samples were collected in the Bolivar State in Venezuela and checked for the presence of Acanthamoeba. Samples were cultivated onto 2% non-nutrient agar plates seeded with a layer of heat killed E. coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 51.8% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotype T4 in all samples. To the best of our knowledge, this is the first report of genotype T4 in soil sources from Venezuela. Further studies should be carried out in this State and in the country in order to determine the current occurrence of Acanthamoeba in Venezuelan environments. PMID:27447209

  9. Incidence and histopathology of encysted progenetic metacercaria of Clinostomum complanatum (Digenea:Clinostomidae) in Channa punctatus and its development in experimental host

    PA Ahammed Shareef; SMA Abidi

    2012-01-01

    Objective: To study the incidence of encysted progenetic metacercariae of Clinostomum complanatum (C. complanatum) in Channa punctatus (C. punctatus), associated histopathology and the experimental infection to laboratory chicken to obtain ovigerous adult worms. Methods:Live C. punctatus were brought from local fish market of Aligarh, India, dissected and examined on a monthly basis for the presence of C. complanatum cysts. For histochemistry, infected tissue sections with attached cysts were processed for haematoxylene and eosin staining. Cysts were aseptically fed to 4 day old leghorn chicken to obtain adult worms. Mechanically excysted metacercaria and the ovigerous adult worms were stained in carmine to prepare permanent slides. Results: One year survey for the infection of encysted progenetic metacercaria of C. complanatum in C. punctatus revealed the prevalence, intensity and abundance of 24.7%, 2.27 and 0.608, respectively. Histopathology showed heavy infiltration of immune cells at the site of cyst attachment and some tissue damage was also evident. Following feeding to experimental chicken, about 41.07%of the encysted metacercariae were able to excyst and migrate back to bucco-pharyngeal region where they tenaciously attached and fed on blood, and transformed into ovigerous adult worms from 62 hours onwards of post infection. Conclusions:The parasite is potentially pathogenic to the host, and the availability of a suitable intermediate host can be a contributing factor for the occurrence of C. complanatum metacercaria either in the excysted or encysted form, indicating loose host specificity and zoonotic potential.

  10. In vitro anti-Acanthamoeba synergistic effect of chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst forms.

    Heredero-Bermejo, I; Sánchez-Nieves, J; Soliveri, J; Gómez, R; de la Mata, F J; Copa-Patiño, J L; Pérez-Serrano, J

    2016-07-25

    Acanthamoeba sp. are the causative agents of severe illnesses in humans such as Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Medical therapy is not yet well established. Treatments of AK last for several months and generate toxicity, resistances appear due to the cysts stage and recurrences can occur. In this study has been demonstrated that the combination of chlorhexidine digluconate (CLX) and carbosilane dendrimers containing ammonium or guanidine moieties has in vitro synergistic effect against Acanthamoeba polyphaga. This synergy provokes an important reduction in the minimal trophozoite amoebicidal concentration (MTAC) of CLX, which means a reduction of their toxic effects on human cells. Moreover, some CLX/dendrimer combinations show important activity against the cyst resistance stage. PMID:27173821

  11. Isolation, morphologic, serologic and molecular identification of Acanthamoeba T4 genotype from the liver of a Temminck's tragopan (Tragopan temminckii).

    Visvesvara, Govinda S; Shoff, Megan E; Sriram, Rama; Booton, Gregory C; Crary, Monica; Fuerst, Paul A; Hanley, Christopher S; Garner, Michael M

    2010-06-24

    Members of the genus Acanthamoeba are usually free-living amoebae that are found in a variety of ecological niches including soil, fresh and brackish water, dust in the air, heating, ventilating, and air conditioning filters, swimming pools and hot tubs. Occasionally they are also known to cause central nervous system infections in humans and animals. We isolated into culture an amoeba from the liver of a Temminck's tragopan (horned pheasant) (Tragopan temminckii) that died of amoebic infection. We identified the infecting amoeba as Acanthamoeba sp. based on culture characteristics, cyst morphology and immunofluorescence assays. Additionally, we identified the amoeba as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene. PMID:20347228

  12. Random amplified polymorphic DNA profiles as a tool for the characterization of Brazilian keratitis isolates of the genus Acanthamoeba

    Alves J.M.P.

    2000-01-01

    Full Text Available The genus Acanthamoeba comprises free-living amebae identified as opportunistic pathogens of humans and other animal species. Morphological, biochemical and molecular approaches have shown wide genetic diversity within the genus. In an attempt to determine the genetic relatedness among isolates of Acanthamoeba we analyzed randomly amplified polymorphic DNA (RAPD profiles of 11 Brazilian isolates from cases of human keratitis and 8 American type culture collection (ATCC reference strains. We found that ATCC strains belonging to the same species present polymorphic RAPD profiles whereas strains of different species show very similar profiles. Although most Brazilian isolates could not be assigned with certainty to any of the reference species, they could be clustered according to pattern similarities. The results show that RAPD analysis is a useful tool for the rapid characterization of new isolates and the assessment of genetic relatedness of Acanthamoeba spp. A comparison between RAPD analyses and morphological characteristics of cyst stages is also discussed.

  13. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 00C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain

  14. Isolation and molecular characterization of Acanthamoeba strains isolated from the oral cavity of immunosuppressed individuals in Tehran, Iran.

    Memari, Fatemeh; Niyyati, Maryam; Lorenzo-Morales, Jacob; Jonaydi, Zaynab

    2016-09-01

    Acanthamoeba spp. is an opportunistic protozoan parasite which is the causative agent of granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK). GAE usually occurs in immunocompromised patients which in most cases is fatal. The present study was conducted to determine the genotypes of Acanthamoeba isolated from patients with compromised immunological status. For this purpose, 90 samples from the oral cavity of these individuals were collected in different hospitals of Tehran, Iran using sterile cotton swabs. Samples were cultured in 2% Non-Nutrient Agar (NNA) plates in order to check for the presence of amoebae. Identification of isolates was carried out using both morphological and molecular tools. The pathogenic potential of the obtained strains was assessed by performing osmo- and thermotolerance assays as previously described. Genotyping of the isolates was carried out by PCR/sequencing of the DF3 region of the 18S rDNA gene of Acanthamoeba. From the 90 collected samples, 11 (13.4%) were positive for Acanthamoeba genus. Molecular analysis revealed the presence of genotypes T3, T4 and T11, although most of the isolates belonged to genotype T4. Only 3 of the isolates genotyped as T4 were positive for the pathogenic potential assays. To this end if the immunological status is considered as one of the key factors for the development of GAE due to Acanthamoeba in the previous reported cases, individuals suffering from the conditions mentioned in this study should be considered as a high risk group of population in Iran and worldwide. PMID:27447206

  15. Compositions and methods for pathogen transport

    El-Etr, Sahar; Farquar, George R.

    2016-01-26

    This disclosure provides a method for transporting a pathogen under ambient conditions, by culturing the pathogen with an amoeba under conditions that favor the incorporation of the pathogen into a trophozoite, starving the amoeba until it encysts, then culturing under conditions that favor conversion of the amoeba back to a trophozoite. In one aspect, the conditions that favor incorporation of the pathogen into the cyst of the amoeba comprises contacting the pathogen with the amoeba in an iron rich environment. Virus and/or bacteria are pathogens that can be transported by the disclosed method. Amoeba that are useful in the disclosed methods include, without limitation Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria gruberi. The disclosed methods have utility in: transporting pathogens from military field hospitals and clinics to the laboratory; transporting pathogens from global satellite laboratories to clinical laboratories; long term storage of pathogens; enriching contaminated patient samples for pathogens of interest; biosurveillance and detection efforts.

  16. Results of case-control studies support the association between contact lens use and Acanthamoeba keratitis

    Pacella E

    2013-05-01

    Full Text Available Elena Pacella,1 Giuseppe La Torre,2 Maria De Giusti,2 Chiara Brillante,1 Anna Maria Lombardi,2 Gianpaolo Smaldone,1 Tommaso Lenzi,1 Fernanda Pacella11Department of Sense Organs, Faculty of Medicine and Dentistry, Sapienza University of Rome, Italy; 2Department of Public Health and Infectious Diseases, Faculty of Pharmacy and Medicine, Sapienza University of Rome, ItalyBackground: Acanthamoeba keratitis (AK is ever more frequently reported in industrialized countries. The loss of the corneal surface integrity consequent to secondary microtrauma produced by the use of contact lens (CL favors the penetration of the parasite into the corneal tissue.Objectives: A scientific review was performed to investigate the association of CL wear as an Acanthamoeba keratitis (AK risk factor.Methods: A computerized screening of 7834 Medline articles (4623 from PubMed; 3211 from Scopus used a strict selection criteria of case-control studies involving CL wear and/or trauma.Results: The search yielded five case-control studies published from 1995 to 2012. All studies included showed a statistically significant positive association between AK and CL use, with a combined odds ratio (OR of 10.21 (95%, confidence intervals [CI]; 3.57–27.64.Statistical analysis: All studies included showed a statistically significant positive association between AK and CL use, though with differing OR values.Conclusion: Though rare, AK should be held in higher consideration when ophthalmologists are faced with CL users exhibiting simplex-like lesions associated with circular stromal infiltrates and disproportionate ocular pain in respect to the objective clinical picture.Keywords: keratitis, contact lens, Acanthamoeba

  17. A novel antiamoebic agent against Acanthamoeba sp. - A causative agent for eye keratitis infection

    Kusrini, Eny; Hashim, Fatimah; Azmi, Wan Nor Nadhirah Wan Noor; Amin, Nakisah Mat; Estuningtyas, Ari

    2016-01-01

    The terbium trinitrate.trihydrate.18-crown ether-6, Tb(NO3)3(OH2)3.(18C6) complex has been characterized by elemental analysis, photoluminescence and single X-ray diffraction. The IC50 values were determined based on MTT assay while light and fluorescence microscopy imaging were employed to evaluate the cellular morphological changes. Alkaline comet assay was performed to analyze the DNA damage. The photoluminescence spectrum of the Tb complex excited at 325 nm displayed seven luminescence peaks corresponding to the 5D4 → 7F0, 1, 2, 3, 4, 5, 6 transitions. The cytotoxicity and genotoxicity studies indicated that the Tb(NO3)3(OH2)3.(18C6) complex and its salt form as well as the 18C6 molecule have excellent anti-amoebic activity with very low IC50 values are 7, 2.6 and 1.2 μg/mL, respectively, with significant decrease (p < 0.05) in Acanthamoeba viability when the concentration was increased from 0 to 30 μg/mL. The mode of cell death in Acanthamoeba cells following treatment with the Tb complex was apoptosis. This is in contrast to the Tb(NO3)3.6H2O salt- and 18C6 molecule-treated Acanthamoeba, which exhibited necrotic type cells. The percentage of DNA damage following treatment with all the compounds at the IC25 values showed high percentage of type 1 with the % nuclei damage are 14.15 ± 2.4; 46.00 ± 4.2; 36.36 ± 2.4; 45.16 ± 0.6%, respectively for untreated, treated with Tb complex, Tb salt and 18C6 molecule. The work features promising potential of Tb(NO3)3(OH2)3.(18C6) complex as anti-amoebic agent, representing a therapeutic option for Acanthamoeba keratitis infection.

  18. Acanthamoeba Species Keratitis in a Soft Contact Lens Wearer Molecularly Linked to Well Water

    Samira Mubareka

    2006-01-01

    Full Text Available Acanthamoeba species keratitis has been associated with soft contact lens wear. In the present report, an epidemiological link was established between the patient's isolate and well water from the home using molecular methods. To the authors' knowledge, this is the first case in Canada where such a link has been established. Primary care practitioners and specialists, including ophthalmologists and infectious diseases specialists, must maintain a high degree of clinical suspicion in soft contact lens wearers with keratitis unresponsive to conventional topical and systemic treatment.

  19. In vitro cytotoxicity of Acanthamoeba spp. isolated from contact lens containers in Korea by crystal violet staining and LDH release assay

    Shin, Ho-Joon; Cho, Myung-Soo; Jung, Suk-Yul; Kim, Hyung-Il; Im, Kyung-il

    2000-01-01

    In order to observe the cytotoxicity of Acanthamoeba spp., which were isolated from contact lens containers as ethiological agents for the probable amoebic keratitis in Korea, the crystal violet staining method and LDH release assay were carried out. In the crystal violet staining method, among eight contact lens container isolates, isolate 3 (Acanthamoeba KA/LS5) showed 83.6% and 81.8% of cytotoxicity, and isolate 7 (Acanthamoeba KA/LS37) showed 28.2% and 25.1% of cytotoxicity, in 1 mg/ml an...

  20. Eukar yotic cell encystation and cancer cell dormancy:is a greater devil veiled in the details of a lesser evil?

    Abdul Mannan Baig; Naveed Ahmed Khan; Farhat Abbas

    2015-01-01

    Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy as dormant cells evade not only the anticancer drugs but also the host immune system. hTese dormant cells veil themselves from detection by imaging and/or using biomarkers, which imposes an additional problem in targeting such cells. A similar form of hibernation process known as encystation is studied in detail for pathogenic unicellular eukaryotic microorganisms. By examination using microarray gene expression proifles, immunocytochemistry tools, and siRNAs during the process of encystation, understanding the covert features of cancer cell dormancy as proposed could be possible. hTis knowledge can be extended to dormant cancer cells to uncover the mechanisms that underlie this ghost, yet dangerous state of human cancers. We propose a strategy to induce dormancy and exit this state by application of knowledge gained from the encystation induction and retrieval processes in pathogenic eukaryotic microorganisms. Given that early detection and characterization of dormant malignant tumor cells is important as a general strategy to monitor and prevent the development of overt metastatic disease, this homology may enable the design of therapies that could either awake the dormant cell from dormancy to make it available for therapies or prolong such a phase to make cancer appear as a chronic disease.

  1. A Case of Medication-Resistant Acanthamoeba Keratitis Treated by Corneal Crosslinking in Turkey

    Goktug Demirci

    2013-01-01

    Full Text Available Purpose. To report a case of medication-resistant acanthamoeba keratitis (AK treated successfully by corneal crosslinking (CXL. Methods. A 26-year-old male with medication-resistant AK underwent a standard CXL procedure with local anesthesia, followed by central corneal epithelial debridement, application of riboflavin 0.1%, and UV-A irradiation. Results. The patient experienced a dramatic symptomatic improvement within 24 hours. At two months, keratitis was healed with a semitransparent paracentral scar that did not affect visual acuity. Conclusions. Our experience, considered in the context of recent studies, suggests that CXL may be an option for selected patients with medication-resistant AK and corneal melting. CXL allows patients to avoid emergency keratoplasty and experience rapid symptomatic relief.

  2. Progress on excretory-secretory antigens of Trichinella spiralis pre-encysted larva%旋毛虫成囊前期幼虫排泄分泌抗原的研究进展

    董明治; 申丽洁

    2009-01-01

    在旋毛虫感染过程中,成囊前期幼虫(PEL)是旋毛虫侵入宿主肌肉的侵入期.旋毛虫PEL比成囊期幼虫(EL)在宿主体内约早2周出现.成囊前期幼虫抗原(PELA)对旋毛虫病的早期免疫学诊断具有较高的敏感性.而旋毛虫排泄分泌抗原具双重免疫功能,即有良好的抗原性和免疫原性.因此,本文就旋毛虫成囊前期幼虫ES抗原研究进展做一综述.%The pre-encysted larvae is the period of invasive hosts muscle in the development of Trichinella spiralis. T. spiralis pre-encysted larvae than encysted larva appeared about two weeks early in hosts. The pre-encysted larva antigens of T. spiralis have high sensitivity in the eary diagnosis of immunization. And excretory-secretory antigen of T. spiralis has double immune function, that is good antigenicity and immunogenicity. Therefore, this article introduced the progress on excretory-secretory antigens of T. spiralis pre-encysted larva.

  3. Lens Epithelial Cell Death Secondary to Acanthamoeba Keratitis: Absence of Capsular Bag Opacification Six Years after Cataract Surgery

    Moreno-Montañes, J. (Javier); Barrio-Barrio, J. (Jesús); E. Nova; Werner, L.

    2011-01-01

    Purpose: To show the evolution of anterior chamber structures 6 years after cataract surgery in a case with Acanthamoeba keratitis (AK). Methods: A 37-year-old woman with AK receiving long-term treatment with chlorhexidine, propamidine isethionate and steroids developed a white cataract and iris atrophy. Penetrating keratoplasty and cataract surgery were performed with subsequent intraocular pressure elevation requiring Molteno shunt implantation. Two years after the last surgery, endothel...

  4. A study of the spectrum of Acanthamoeba keratitis: A three-year study at a tertiary eye care referral center in South India

    Bharathi Jayahar

    2007-01-01

    Full Text Available Purpose: To determine the epidemiological and clinical characteristics of Acanthamoeba keratitis and also to determine the sensitivity and specificity of smears in the detection of Acanthamoeba . Materials and Methods: A retrospective review of all culture-positive cases of Acanthamoeba keratitis seen between October 1999 and August 2002 was performed. Corneal scrapes were subjected to culture and microscopy using standard protocols. Results: Out of 3183 consecutive patients with clinically diagnosed corneal ulcers evaluated, 33 (1.04% were found to be due to Acanthamoeba . Twenty-four out of 33 (72.72% were less than 51 years of age ( P < 0.001. All patients were from rural areas ( P < 0.001 and 26 (78.79% of them were agricultural workers ( P = 0.031. All 33 had history of corneal injury ( P < 0.001 and 28 (84.85% patients had injury with mud ( P < 0.001. All 33 (100% patients had previous medical treatment ( P =0.009 and 10 (30.3% had used traditional eye medicines ( P =0.183. A clinical pattern of ring infiltrate was characteristic in 15 (45.45% patients. The diameter of the corneal ulcer was more than 6mm in 27 (81.82% eyes ( P < 0.001. Twenty-six (78.79% patients had visual acuity of perception of light on initial presentation ( P < 0.001 and 24 (72.73% had the same as their final visual outcome. The sensitivity of 10% potassium hydroxide (KOH preparation was found to be higher ( P < 0.001 in the detection of Acanthamoeba cysts. Conclusion: The incidence of Acanthamoeba keratitis amongst the corneal ulcer patients was 1% in this setting and it was mainly due to corneal injury by mud. The KOH preparation is a sensitive diagnostic tool for the detection of Acanthamoeba . Delayed diagnosis or misdiagnosis and inappropriate antimicrobial therapy results in poor visual outcome.

  5. Collection of pre-encysted larvae of Trichinella spiralis%旋毛虫成囊前期幼虫的收集

    董明治; 申丽洁

    2010-01-01

    目的 探索旋毛虫成囊前期幼虫的收集时间与方法.方法 20只成年大鼠,每只大鼠经口感染3000条旋毛虫脱囊幼虫,分别于第14、15、16、17、18、19、20天将大鼠处死,用人工胃蛋白酶消化肌肉收集旋毛虫.结果 感染后第14、15、16大膈肌均未见旋毛虫,第17、18、19、20天膈肌有旋毛虫的侵入并逐渐增多;第14、15、16天均未收集到旋毛虫,第17、18、19、20天平均每只大鼠分别收集5000、8000、10000、30000条旋毛虫.结论 旋毛虫在感染后第20天是旋毛虫成囊前期幼虫收集的最佳时期.人工胃蛋白酶消化肌肉可以收集成囊前期幼虫.%Objective To explore the collection time and method of the pre-encysted larvae of Trichinella spiralis. Methods Twenty adult rats were inoculated with 3 000 encysted larva of Trichinella spir-alis per rat by the oral route. At the 14th, 15th, 16th, 17th, 18th, 19th, 20th day after infection, the rats were killed and the muscles were digested with pepsin to collect Trichinella spiralis. Results At the 14th,15th, 16th day after infection, no Trichinella spiralis was found in diaphragm. At the 17th, 18th, 19th, 20th day after infection, Trichinella spiralis could be seen in the diaphragm and the number of Trichinella spiralis in-creased with time passing. At the 14th, 15th, 16th day, no Trichinella spiralis was obtained. At the 17th,18th, 19th, 20th day, the mean number of Trichinella spirulis collected were 5000, 8000, 10000, 30000per rat respectively. Conclusion The 20th day after Trichinella spiralis infection is the best time to collect the pre-encysted larvae of Trichinella spiralis. Pepsin digestion of muscles can be the choice of methods for col-lecting the pre-encysted larvae of Trichinella spirulis.

  6. Comparison of moxidectin oral gel and ivermectin oral paste against a spectrum of internal parasites of ponies with special attention to encysted cyathostome larvae.

    Monahan, C M; Chapman, M R; Taylor, H W; French, D D; Klei, T R

    1996-06-01

    Two dosages of moxidectin oral gel were evaluated and compared to a therapeutic dose of ivermectin oral paste in the control of a spectrum of gastrointestinal parasites of ponies naturally infected in southern Louisiana or Mississippi. Thirty-two mixed-breed ponies ranging in age from one to 21 years were used in this controlled test. Eight weeks prior to the experiment, ponies grazing on contaminated pasture were moved to a paddock and fed a pelleted ration, thus reducing or eliminating the potential for additional infection and ensuring the existence of a population of encysted larvae. Ponies were then allocated to replicates of four animals based on values of fecal strongyle egg counts and percent strongyle larvae composition determined from Baermann sedimentations of fecal cultures. Members of replicates were allocated to one of four treatment groups: moxidectin oral gel administered at 300 micrograms kg-1 body weight, moxidectin oral gel at 400 micrograms kg-1, the oral gel vehicle as negative control, and ivermectin oral paste at 200 micrograms kg-1. Prior to treatment, ponies were confined in pairs to covered concrete runs by treatment group. Two weeks following treatment, necropsy examinations of all animals were performed. Parasites were recovered from the lumen of the stomach, the intestinal tract, the cranial mesenteric artery and its major branches, the peritoneal body wall and from pepsin digests of mucosal scrapings taken from the cecum and large colon. Encysted cyathostome larval burdens were also compared using mural transillumination of segments of the large colon for visualization of the encysted forms. Control ponies were not uniformly infected with the spectrum of parasites; however, moxidectin, at either dosage, compared favorably with ivermectin in the control of the adults of Strongylus vulgaris, Strongylus edentatus, Triodontophorus spp., Oesophagodontus robustus, Trichostrongylus axei, Oxyuris equi, Parascaris equorum, Habronema muscae, as

  7. Isolation and Genotyping of Acanthamoeba Strains from Envi-ron¬mental Sources in Ahvaz City, Khuzestan Province, South-ern Iran

    M Rahdar

    2012-12-01

    Full Text Available Background: Acanthamoeba spp. are free-living amoebae commonly found in the environmental sources such as water, soil, and air. This ubiquitous amoeba is the causative agent of amoebic kerati­tis (AK. The objective of the present study was to investigate the presence of Acanthamoeba spp. in water and soil sources in Ahvaz City, Khuzestan Province, southern Iran.Methods: In general, 110 samples of water and soil were taken from different localities of Ahvaz including agricultural canals, rivers, and swimming pools. Filtration and cultivation were carried out on non-nutrient agar medium (NNA. Axenic cultivation was performed for all of positive isolates. PCR analysis was conducted on positive samples. Sequencing was done for 15 PCR products. Geno­types were identified by Blast search and homology analysis.Result: Acanthamoeba spp. was found in 43 (71.6% of samples of water and 13 (26% soil samples. Genotyping of 15 samples proved that Acanthamoeba belonged to T4 (86.6%, T2 (6.6%, and T5 (6.6% genotypes.Conclusion: TYI-S-33 medium could be better than PYG medium for Acanthamoeba axenic culture.

  8. In vitro culture, serologic and molecular analysis of Acanthamoeba isolated from the liver of a keel-billed toucan (Ramphastos sulfuratus).

    Visvesvara, Govinda S; Booton, Gregory C; Kelley, Darryl J; Fuerst, Paul; Sriram, Rama; Finkelstein, Ariana; Garner, Michael M

    2007-01-19

    Members of the genus Acanthamoeba are usually free-living amebae and are found in a variety of ecological niches including soil, fresh and brackish water, dust in air, filters of heating, ventilating, and air conditioning units, swimming pools and hot tubs, etc. Occasionally, they are also known to cause central nervous system infections in humans and other animals. We isolated into culture an amoeba from the liver tissue of a keel-billed toucan and identified it as Acanthamoeba sp. based on culture characteristics and immunofluorescent analysis. Further, we characterized the cultured amoeba and also the amoeba in the liver tissue as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene. PMID:16962705

  9. Acanthamoeba and other free-living amoebae in bat guano, an extreme habitat.

    Mulec, Janez; Dietersdorfer, Elisabeth; Üstüntürk-Onan, Miray; Walochnik, Julia

    2016-04-01

    Several representatives of the so-called free-living amoebae (FLA) are of medical relevance, not only as facultative pathogens but also as vehicles for pathogenic bacteria. Some FLA can survive and even grow under extreme environmental conditions. Bat guano is an exceptional habitat, the conditions becoming gradually more extreme with aging. In the current study, samples of bat guano of different ages from five caves in Slovenia were screened for the presence of FLA. FLA were isolated from almost all guano samples, including guano with a pH of 3.5. Only the two samples that had been drawn from >20-year-old guano were negative for FLA. Generally, FLA diversity correlated to high concentrations of cultivable bacteria (∼10(8) CFU/g) and fungi (∼10(5) CFU/g). Interestingly, the absence of FLA in seasoned guanos was mirrored by the presence of dictyostelid slime moulds. The isolated amoebae were identified as belonging to the genera Acanthamoeba, Copromyxa, Naegleria, Sappinia, Tetramitus, Thecamoeba, Vahlkampfia, Vannella and Vermamoeba. To the best of our knowledge, this is the first study on the diversity of FLA in guano. PMID:26678653

  10. Comparison of a PCR-Based Method with Culture and Direct Examination for Diagnosis of Acanthamoeba keratitis

    S Farnia

    2009-05-01

    Full Text Available "nBackground: The aim was to compare three different methods (direct examination, culture and PCR meth­ods for the diagnosis of Acanthamoeba keratitis (AK in corneal scrapes."nMethods: Twenty eight corneal scrapes and contact lenses were collected from keratitis patients and re­ferred to the De­partment of Medical Parasitology and Mycology, School of Public Health, Tehran Univer­sity of Medical Sci­ences. Corneal scrapes were divided in three parts for direct examination, culture on non-nutrient agar and PCR analysis. PCR analysis was also performed using a 18S rRNA gene primer pair (DF3 region. DF3 (Diagnostic frag­ment 3 is a region of the nuclear small subunit ribosomal RNA gene which is specific for detecting Acan­thamoeba strains."nResults:  Acanthamoeba was the causative agent of keratitis in 50% of the patients. Direct smear of all pre­pared corneal scrapes in AK patients was negative and culture was positive in only 14.3% of the isolates. PCR analysis was positive in 71.4% of AK patients. These three methods were negative in corneal scrapes of non-AK patients. The sensitivity and specificity of PCR technique for the detection of Acanthamoeba sp. were calculated as 71.4% and 100%, respectively."nConclusion: According to high sensitivity and specificity of PCR-based method, this study confirmed that PCR using 18S rRNA gene primers (DF3 region is more useful for detecting AK cases compare to culture and direct microscopy methods.

  11. Ether-type moieties in the lipid part of glycoinositolphospholipids of Acanthamoeba rhysodes.

    Karaś, Magdalena A; Russa, Ryszard

    2014-04-01

    Ether lipids were identified among components liberated with HF and nitrous acid deamination from Acanthamoeba rhysodes whole cells and its membrane glycoinositolphospholipids (GIPL). Liberated ether glycerols were converted to various derivatives that served characterization thereof. These included TMS and isopropylidene derivatives, oxidation with sodium periodate to aldehyde followed by reduction with NaBH4 to alcohol, and reaction of the alcohol with acetic anhydrite to form acetate derivatives. Periodate sensitivity demonstrated that the alkyl side chains were linked to the sn-1 position of glycerol. Combined information from TLC, GC-MS analysis, MALDI-TOF spectrometry, and chemical degradation experiments indicated the presence of ether-linked saturated normal and branched hydrocarbons with a length of C20-23 in the phospholipid fraction, C20-24 in free GPI, and C21-23 in the LPG polymer. The distribution of particular classes of alkylglycerols was similar for phospholipid and GPI fractions, and amounted to 2.62% (±0.04-0.28) 1-O-eicosanyl-sn-glycerol, 16.66% (±0.32-1.1) 1-O-uncosanyl-sn-glycerol, 9.18% (±0.33-1.37) anteiso-1-O-docosanyl-sn-glycerol, 47.56% (±0.32-2.14) 1-O-docosanyl-sn-glycerol, 20.56% (±0.58-1.67) anteiso-1-O-tricosanyl-sn-glycerol, and 2.34% (±0.12-0.63) 1-O-tricosanyl-sn-glycerol. For LPG preparation, the most abundant were anteiso-1-O-tricosanyl-sn-glycerol (57.26%) and 1-O-docosanyl-sn-glycerol (30.12%). The data from TLC and GC-MS analysis showed that ether lipids from phospholipids probably represent the lyso-alkylglycerol type, while those derived from GIPL are alkylacylglycerol moieties. PMID:24535098

  12. The fate of Helicobacter pylori phagocytized by Acanthamoeba polyphaga demonstrated by fluorescent in situ hybridization and quantitative polymerization chain reaction tests

    Helicobacter pylori able to express green fluorescent protein, as well as an ATCC strain, and a clinical isolate of this pathogen were evaluated for their ability to survive predation by Acanthamoeba polyphaga. Ingestion was evaluated by microscopic observation of the GFP-H. pyl...

  13. The GacS/A-RsmA Signal Transduction Pathway Controls the Synthesis of Alkylresorcinol Lipids that Replace Membrane Phospholipids during Encystment of Azotobacter vinelandii SW136

    Romero, Yanet; Guzmán, Josefina; Moreno, Soledad; Cocotl-Yañez, Miguel; Vences-Guzmán, Miguel Ángel; Castañeda, Miguel; Espín, Guadalupe; Segura, Daniel

    2016-01-01

    Azotobacter vinelandii is a soil bacterium that undergoes a differentiation process that forms cysts resistant to desiccation. During encystment, a family of alkylresorcinols lipids (ARs) are synthesized and become part of the membrane and are also components of the outer layer covering the cyst, where they play a structural role. The synthesis of ARs in A. vinelandii has been shown to occur by the activity of enzymes encoded in the arsABCD operon. The expression of this operon is activated by ArpR, a LysR-type transcriptional regulator whose transcription occurs during encystment and is dependent on the alternative sigma factor RpoS. In this study, we show that the two component response regulator GacA, the small RNA RsmZ1 and the translational repressor protein RsmA, implicated in the control of the synthesis of other cysts components (i.e., alginate and poly-ß-hydroxybutyrate), are also controlling alkylresorcinol synthesis. This control affects the expression of arsABCD and is exerted through the regulation of arpR expression. We show that RsmA negatively regulates arpR expression by binding its mRNA, repressing its translation. GacA in turn, positively regulates arpR expression through the activation of transcription of RsmZ1, that binds RsmA, counteracting its repressor activity. This regulatory cascade is independent of RpoS. We also show evidence suggesting that GacA exerts an additional regulation on arsABCD expression through an ArpR independent route. PMID:27055016

  14. Efficacy of hand held, inexpensive UV light sources on Acanthamoeba, causative organism in amoebic keratitis

    Ivan Cometa

    2010-01-01

    Full Text Available Ivan Cometa1, Andrew Rogerson1, Scott Schatz21Department of Biology, California State University Fresno, Fresno, CA, USA; 2Arizona College of Optometry, Midwestern University, Glendale, AZ, USAAbstract: Multipurpose lens cleaning solutions (MPS fail to consistently kill or inactivate Acanthamoeba cysts and UV irradiation, while effective at high doses, can damage contact lenses. The present study considered synergy of action between MPS and hand-held inexpensive (ie, relatively weak UV irradiation units. Regardless of disinfection method recently formed cysts (<10 days were far more susceptible to treatment than mature cysts (>14 days. This has important implications for future protocols on testing methods for killing amoebae. The study also showed that cysts of different strains (two tested, FLA2 and P120 are variable in their response to MPS, presumably reflecting differences in cyst wall structure and thus permeability to the disinfectant. On the other hand, the effect of UV irradiation was not wall structure dependent. A 6-hour treatment with MPS alone killed trophic amoebae but failed to kill any mature cysts. Cysts of strain FLA2 were killed after 24 hours with MPS but cysts of strain P120 survived. UV irradiation with the larger 4 W unit killed all cysts after 7 minutes and was more effective than the smaller battery-powered unit (after 10 minutes about 50% of cysts were killed. When the larger unit was used with the MPS disinfection, all trophozoites were killed using UV for 3 minutes and MPS for 1 hour. The resistant P120 cysts remained a challenge but a 2- to 4-minute UV treatment followed by MPS for 3 or 6 hours reduced mature cyst survival by about 50%. The small unit in combination with MPS was less effective but did reduce the time required to kill trophic amoebae in MPS (6 hours MPS alone versus 3 hours MPS with a 1-minute UV treatment. In short, inexpensive UV units do enhance MPS disinfection and future lens cleaning systems

  15. Acanthamoeba feature a unique backpacking strategy to trap and feed on Listeria monocytogenes and other motile bacteria

    Doyscher, Dominik; Fieseler, Lars; Dons, Lone Elisabet;

    2013-01-01

    , forming large aggregates of densely packed bacteria that we termed backpacks. While the assembly of backpacks is dependent on bacterial motility, flagellation alone is not sufficient. Electron micrographs showed that the aggregates are held together by filaments of likely amoebal origin. Time......-lapse microscopy revealed that shortly after the bacteria are collected, the amoeba can change direction of movement, phagocytose the backpack and continue to repeat the process. The phenomenon was also observed with avirulent L.?monocytogenes mutants, non-pathogenic Listeria, and other motile bacteria, indicating...... that formation of backpacks is not specific for L.?monocytogenes, and independent of bacterial pathogenicity or virulence. Hence, backpacking appears to represent a unique and highly effective strategy of Acanthamoeba to trap and feed on motile bacteria....

  16. Selective Requirement of the Shikimate Pathway of Legionella pneumophila for Intravacuolar Growth within Human Macrophages but Not within Acanthamoeba

    Jones, Snake C.; Price, Christopher T. D.; Santic, Marina

    2015-01-01

    Legionella pneumophila utilizes the Dot/Icm type IV translocation system to proliferate within a vacuole in a wide variety of natural amoebal hosts and in alveolar macrophages of the human accidental host. Although L. pneumophila utilizes host amino acids as the main sources of carbon and energy, it is not known whether de novo synthesis of amino acids by intravacuolar L. pneumophila contributes to its nutrition. The aroB and aroE genes encode enzymes for the shikimate pathway that generates the aromatic amino acids Phe, Trp, and Tyr. Here we show the aroB and aroE mutants of L. pneumophila to be defective in growth in human monocyte-derived macrophages (hMDMs) but not in Acanthamoeba spp. The aroB and aroE mutants are severely attenuated in intrapulmonary proliferation in the A/J mouse model of Legionnaires' disease, and the defect is fully complemented by the respective wild-type alleles. The two mutants grow normally in rich media but do not grow in defined media lacking aromatic amino acids, and the growth defect is rescued by inclusion of the aromatic amino acids, which are essential for production of the pyomelanin pigment. Interestingly, supplementation of infected hMDMs with the three aromatic amino acids or with Trp alone rescues the intramacrophage defect of the aroE but not the aroB mutant. Therefore, the shikimate pathway of L. pneumophila is differentially required for optimal growth within human macrophages, which are auxotrophic for Trp and Phe, but is dispensable for growth within the Acanthamoeba spp. that synthesize the aromatic amino acids. PMID:25847958

  17. Comparison of molecular diagnostic methods for the detection of Acanthamoeba spp. from clinical specimens submitted for keratitis.

    Khairnar, Krishna; Tamber, Gurdip S; Ralevski, Filip; Pillai, Dylan R

    2011-08-01

    Acanthamoeba spp. are responsible for a significant annual number of keratitis (AK) cases leading to vision-threatening disease worldwide. Current methods rely on direct examination of specimens by microscopy and/or culture. The former lacks sensitivity and the latter suffers from a poor turnaround time. We undertook a comparison of all published molecular methods, evaluating performance characteristics such as analytical sensitivity, specificity, limit of detection (LOD), reproducibility, accuracy, and cost of test. The study population comprised 128 patients. Eligible specimens were tested prospectively between April 2007 and May 2010 by microscopy and/or culture. Eleven different specimen types were used including corneal scrapings (51.5%), corneal swab (17.9%), and contact lens material (10.9%). Results of 2 published gel-based polymerase chain reaction (PCR) and 2 published real-time quantitative (Q) PCR methods were compared in a blinded manner to direct microscopic examination and/or culture for the detection of Acanthamoeba in clinical specimens. QPCR (Riviere method) had the highest sensitivity at 89.3%, excellent accuracy using ROC analysis (AUC ∼0.90), lowest LOD down to 0.1 organism per microliter, and superior linear correlation with parasite density (R(2) = 0.9965) when compared with microscopy, culture, and other molecular methods. Phylogenetic analysis using a sequence-based typing method revealed that clinical isolates in this population with AK were genetically distinct from granulomatous amebic encephalitis or environmental isolates. The QPCR method was more expensive ($14.80) than traditional methods such as culture ($2.50) or microscopy ($2.50). However, 13 culture- and microscopy-negative specimens were positive by QPCR during the study period, suggesting that detection using QPCR may result in reduced complications and health care costs associated with misdiagnosed AK. PMID:21658877

  18. Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: Predominance of heterotypic encystment and implications for mating interactions and biogeography

    Brosnahan, Michael L.; Kulis, David M.; Solow, Andrew R.; Erdner, Deana L.; Percy, Linda; Lewis, Jane; Anderson, Donald M.

    2010-02-01

    We report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic Group III isolates. However, hypnozygotes produced in Group I/III co-cultures complete no more than three divisions after germinating. Previous reports have suggested a mate recognition mechanism whereby hypnozygotes produced in co-cultures could arise from either homotypic (inbred) or heterotypic (outbred) gamete pairs. To determine the extent to which each occurs, a nested PCR assay was developed to determine parentage of individual hypnozygotes. The vast majority of hypnozygotes from pairwise Group I/III co-cultures were outbred, so that inviability was a result of hybridization, not inbreeding. These findings support the assertion that complete speciation underlies the phylogenetic structure of the Alexandrium tamarense species complex. Additionally, the ribosomal DNA (rDNA) copy numbers of both hybrid and single ribotype hypnozygotes were reduced substantially from those of haploid motile cells. The destruction of rDNA loci may be crucial for the successful mating of genetically distant conjugants and appears integral to the process of encystment. The inviability of Group I/III hybrids is important for public health because the presence of hybrid cysts may indicate ongoing displacement of a nontoxic population by a toxic one (or vice versa). Hybrid inviability also suggests a bloom control strategy whereby persistent, toxic Group I blooms could be mitigated by introduction of nontoxic Group III cells. The potential for hybridization in nature was investigated by applying the nested PCR assay to hypnozygotes from Belfast Lough, Northern Ireland, a region where Group I and III populations co-occur. Two hybrid cysts were identified in 14 successful assays, demonstrating that Group I and III populations do interbreed in that region. However, an analysis of mating data

  19. Reevaluating the Role of Acanthamoeba Proteases in Tissue Invasion: Observation of Cytopathogenic Mechanisms on MDCK Cell Monolayers and Hamster Corneal Cells

    Maritza Omaña-Molina; Arturo González-Robles; Lizbeth Iliana Salazar-Villatoro; Jacob Lorenzo-Morales; Ana Ruth Cristóbal-Ramos; Verónica Ivonne Hernández-Ramírez; Patricia Talamás-Rohana; Adolfo René Méndez Cruz; Adolfo Martínez-Palomo

    2013-01-01

    The morphological analysis of the cytopathic effect on MDCK cell monolayers and hamster cornea and qualitative and quantitative analyses of conditioned medium and proteases were evaluated and compared between two strains of Acanthamoeba genotype T4. Further than highlighting the biological differences found between both strains, the most important observation in this study was the fact that proteases both in total extracts and in conditioned medium are apparently not determinant in tissue d...

  20. Free-living amoebae (FLA: detection, morphological and molecular identification of Acanthamoeba genus in the hydraulic system of an haemodialysis unit in Tunisia

    Dendana F.

    2008-06-01

    Full Text Available The free-living amoebae (FLA are ubiquitous and opportunistic protozoa. They can induce human and animal diseases. The aim of our study was to detect the FLA and Acanthamoeba genus in the hydraulic system of an hemodialysis unit. It was a prospective study of 46 water samples. The first collect (23 was before cleaning and after the haemodialysis sessions and the second (23 after cleaning and before the hemodialysis sessions. Results: the morphological study enabled us to detect morphotypic diversity. The predominant morphotypes were the acanthopodial forms (29%. At the entrance of hemodialysis unit there were acanthopodial (44% and monotactic (25% forms; at the outlet, acanthopodial and fan-shaped forms (25% each. In addition, Acanthamoeba genus was present in 39% (1st collect and 18% (2nd collect. The amplification of the FLA 18S rDNA gene was negative in only one sample localized in the last stage of water treatment unit (WTU. The amplification of the 18S rDNA (ASA.A1 Acanthamoeba gene was positive in15 samples. Conclusions: we noted that, in the hemodialysis unit, the purification techniques used in the WTU were effective, but there is a problem of water stagnation in the drain, which constitutes an appropriate condition for the biofilms formation. It is then necessary to use a filter with a low porosity (0.2 μm at the entrance of the hemodialysis unit and if possible to change the drain

  1. Emerging Threats for Human Health in Poland: Pathogenic Isolates from Drug Resistant Acanthamoeba Keratitis Monitored in terms of Their In Vitro Dynamics and Temperature Adaptability

    Lidia Chomicz

    2015-01-01

    Full Text Available Amphizoic amoebae generate a serious human health threat due to their pathogenic potential as facultative parasites, causative agents of vision-threatening Acanthamoeba keratitis (AK. Recently, AK incidences have been reported with increasing frequency worldwide, particularly in contact lens wearers. In our study, severe cases of AK in Poland and respective pathogenic isolates were assessed at clinical, morphological, and molecular levels. Misdiagnoses and the unsuccessful treatment in other ophthalmic units delayed suitable therapy, and resistance to applied chemicals resulted in severe courses and treatment difficulties. Molecular assessment indicated that all sequenced pathogenic corneal isolates deriving from Polish patients with AK examined by us showed 98–100% homology with Acanthamoeba genotype T4, the most prevalent genotype in this human ocular infection worldwide. In vitro assays revealed that the pathogenic strains are able to grow at elevated temperature and have a wide adaptive capability. This study is our subsequent in vitro investigation on pathogenic Acanthamoeba strains of AK originating from Polish patients. Further investigations designed to foster a better understanding of the factors leading to an increase of AK observed in the past years in Poland may help to prevent or at least better cope with future cases.

  2. The role of contact lenses and ocular TRAUMA in determining Acanthamoeba Keratitis: a case-control study in Italy.

    Elena Pacella

    2012-03-01

    Full Text Available

    Abstract:
    Background: acanthamoeba keratitis (a.k. is a rare secondary infection due to acanthamoeba (a invading the cornea. cofactors of risk may include TrauMa and contact lenses (cL. The objectives of this observational study are to evaluate the occurrence of a.k. in patients accessing the department of Special Senses of the Teaching Hospital “Policlinico umberto I" of rome with ocular infections dur- ing the period from 2005 to 2011, and the role played by risk factors which included contact lenses, TrauMa, sex and age.
    Methods: a case-control study involving 714 subjects was conducted (350 male and 364 female. all of the patients underwent a thorough ocular examination with confocal microscopy and a review of their medical history to outline relevant variables (contact lens and TrauMa. Statistical analysis calculated the frequency distribution and the odds ratio (or (95%cI for the risk factors "contact lenses" (cL and "TrauMa". The odds ratio (95%cI was adjusted for age and sex trough Logistic regression. Results: out of 714 patients, 11 patients (7 male and 4 female with a mean age of 34.09 years (19 to 51 years old were found to be affected by a.k. The age group most afflicted ranged from 30 to 51 years old. Statistical analysis produced a crude or equal to 17.68 (95%cI 2.25-138.89 for cL-a.k. and or equal to 7.71 (95%cI 0.89-66.75 for ocular TrauMa.-a.k. Logistic regression performed to adjust or for age and sex showed the following values, respectively: or*=82.54 (95%cI 3.04-2239.58 and or*=11.52 (95%cI 1.19-111.76.
    Conclusions: the data highlights an increase in a.k. cases during the 6 year study period. The association between this pathology and the use of contact lenses and/or TrauMa has been statisti- cally proven. The strength of this association increases when the odds ratio (or* is adjusted for the confounding risk factors of

  3. Host-dependent variations in the seasonal prevalence and intensity of heterophyid encysted metacercariae (Digenea: Heterophyidea) in brackish water fish in Egypt.

    Elsheikha, Hany M; Elshazly, Attef M

    2008-05-01

    The prevalence of heterophyid (Digenea: Heterophyidae) encysted metacercariae (EMC) in its second intermediate host, the fish Mugil spp. and Tilapia spp. was studied in a subtropical permanent Lake in northeastern Egypt. Seasonal changes in the occurrence of the EMC in different fish hosts were monitored in a longitudinal field survey lasting 12 months from June 2006 to May 2007. This study tested two hypotheses; (i) prevalence and intensity of heterophyid EMC fluctuate seasonally throughout the year and (ii) variation in the prevalence and intensity of heterophyid EMC is host-dependent. A total of 832 fish specimens comprising 5 species collected from Manzala Lake, northeastern Egypt were examined by artificial gastric juice digestion for EMC. All five species of brackish water fish examined were found to harbor the EMC of the family Heterophyidae in their muscles. The overall infection prevalence of EMC over 12 months was 23.2%. The adult flukes recovered from puppies experimentally infected with morphologically different metacercariae from different fish species were compatible with six species belong to five genera of Heterophyidae, namely, Heterophyes heterophyes, Heterophyes aequalis, Pygidiopsis genata, Phagicola sp., Haplorchis sp. and Stictodora sp. EMC of H. heterophyes were most abundant, detected in 56% of the total fish examined. P. genata was ranked second, followed by Phagicola sp., H. aequalis, Haplorchis sp., and Stictodora sp., respectively. Seasonal differences in infection were observed for all heterophyid species studied in all fish species examined. Heterophyid infections reached peak prevalences during the summer season 38.2% followed by spring 26.6% and autumn 19.3% seasons, whereas the lowest prevalence was recorded in the winter 8.7%. Intensity of heterophyid EMC followed the same seasonal pattern, being high during summer months and low in winter months. All fish species were infected with all the heterophyid digeneans, but with different

  4. Morphological and Molecular Identification of Acanthamoeba Spp from Surface Waters in Birjand, Iran, During 2014-2015

    Mahmoodreza Behravan

    2016-04-01

    Full Text Available Background & Aims of the Study: Free-living amoebae (FLA are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections including a fatal encephalitis, a blinding keratitis, and pneumonia. So, due to their medical importance, the identification of free living amoeba in water resources, as a source of human infection, is necessary. The objective of this study was to isolate the Acanthamoebaspp from the surface waters of Birjand, Iran, during 2014-2015 by Morphological and molecular method. Materials and Methods:  In a cross-sectional study, 50 samples were collected from different localities of Birjand city including the surface waters, pools and fountains in parks,squares and water stations from the October 2014 to the January 2015.Each sample was filtered through a nitrocellulose membrane filters and cultured on non-nutrient agar (NNA with Escherichia coli suspension and incubated for 1 week to 2 months at room temperature.The plates were examined by the microscopy to morphologically identify Acanthamoeba species. Following DNA extraction, PCR specific primers was used to confirm the identification morphologically. Results:  Out of 50 water samples, 19 (38% were positive for Acanthamoebatrophozoites and cysts according to the morphological criteria. In addition, Acanthamoebaspp was identified by PCR method, using genus specific primers pairs in 15 (78.9% cases of positive cultures, showing anearly 500bp band. Conclusion: According to the prevalent of Acanthamoebaspp in the surface stagnant waters of Birjand, more attention to the potential role of such waters in transmission of infection by the regional clinicians and health practitioners is necessary.

  5. Queratitis por Acanthamoeba sp.: primer caso confirmado por aislamiento y tipificación molecular en Bahía Blanca, provincia de Buenos Aires, Argentina Acanthamoeba sp. keratitis: first case confirmed by isolation and molecular typification in Bahía Blanca, Buenos Aires Province, Argentina

    M. L. Gertiser; E. Giagante; E. Sgattoni; N. Basabe; Rivero, F.; H. Luján; M. Occhionero; L. Paniccia; E. Visciarelli; S.R. Costamagna

    2010-01-01

    Algunas especies del género Acanthamoeba provocan queratitis amebiana (QA), una infección de la córnea muy dolorosa, generalmente unilateral, con compromiso importante de la visión. Se presenta el caso de una mujer de 31 años usuaria de lentes de contacto con hábitos inadecuados de higiene y conservación de las lentes. La paciente consultó por enrojecimiento y dolor intenso en el ojo derecho, de dos meses de evolución. Luego del examen oftalmológico y ante la sospecha de una etiología parasit...

  6. Protozoa: a novel Campylobacter reservoir?

    In previous in vitro studies we found that Campylobacter jejuni remained viable for longer periods of time when they were cultivated in the presence of Tetrahymena pyriformis (ciliate) and Acanthamoeba castellanii (amoeba) than when they were in an independent planktonic state. Increased survival t...

  7. AcEST: BP911838 [AcEST

    Full Text Available (Hsp70/Hs... 65 2e-09 tr|P90647|P90647_ACACA Transformation-sensitive protein homolog ... 64 4e-09 tr|B0JN3... 107 >tr|P90647|P90647_ACACA Transformation-sensitive protein homolog OS=Acanthamoeba castellanii PE=2 SV=1

  8. The killing efficiency of arilin and contact lens solutions on Acanthamoeba cultured in vitro%甲硝唑与护理液对棘阿米巴原虫体外杀伤效果的评估

    车成业; 赵桂秋; 张丽丽

    2008-01-01

    目的:分析6种角膜接触镜多功能护理液和加入甲硝唑滴跟液的护理液对自生生活性棘阿米巴原虫的杀伤效果.方法:将6种多功能护理液分别加入96孔板中,每种护理液占用48孔,其中24孔滴入阿米巴悬液,另外24孔先滴入甲硝唑滴眼液后再滴入阿米巴悬液,室温静置8h后在倒置显微镜下观察残存阿米巴的形态变化和数量.将残存的棘阿米巴原虫分别在PYG培养液中培养5d,观察其形态、活性与增殖能力的变化.结果:单纯护理液组1-6号阿米巴检出率分别为0%、80.3%、29.1%、41.7%、62.5%、79.2%,加入甲硝唑滴眼液后护理液1-6号阿米巴检出率分别为0%、0%、4.2%、8.3%、16.7%、16.7%,3-6号护理液加与不加甲硝唑滴眼液杀伤阿米巴的效力差异有统计学意义(3-6组的X2值分别为3.75、7.11、10.54和18.78,P<0.05).残存的棘阿米巴原虫经培养后活力与增殖力减弱.结论:部分多功能护理液对棘阿米巴原虫的杀伤效果不佳,添加甲硝唑滴眼液后杀伤效果明显提高.%AIM:To analyze the killing efficiency of six kinds of contact lens solutions and solutions with arilin on free living Acanthamoeba culturedin vitroMETHODS:Six kinds of contact lens solutions were added into 96-well microtiter plates,respectively,with each care solutions used 48 holes of them.Suspension of Acanthamoeba were added into 24 of these holes.and arilin gutta and suspension of Acanthamoeba were added into the other 24 holes.After standing in room ternperature for 8 hours,the morphologic change and quantity of the remnant Acanthamoeba were observed under the jnverted microscope.The remnant Acanthamoeba were cultivanted in peptone-yeast extract-glucose (PYG)-culture medium for 5 days.Their variation of appearance,activity and reproductive activity were observed.RESULTS:In the six experimental groups using contact lens solutions only.the detection rate of Acanthamoeba of were 0%,80.3%,29.1%,41.7%,62.5% and

  9. Bilateral Acanthamoeba ulcer in a user of disposable soft contact lenses: a tragic incident or a consequence of the aggressive policy of soft contact lens trading? Úlcera bilateral por Acanthamoeba em usuária de lentes de contato gelatinosas descartáveis: um incidente trágico ou uma conseqüência da política agressiva de venda de lentes de contato gelatinosas?

    Sidney Júlio de Faria e Sousa; Vanderson Glerian Dias; Luís Antonio Gorla Marcomini

    2008-01-01

    This is the report of a case of bilateral Acanthamoeba keratitis in a 19-year-old woman who bought a pair of disposable soft contact lenses in a boutique. She wore this same pair of lenses for 3 months daily without the appropriate care. This led to bilateral corneal transplantation with cataract extraction and also trabeculectomy in the right eye. When last seen, both grafts were crystal clear but the visual acuities were far from satisfactory. She also had bilateral secondary glaucoma, bare...

  10. Temperature stress: reacting and adapting: lessons from poikilotherms.

    Harwood, John L

    2007-10-01

    Acanthamoeba castellanii (A. castellanii) is a common soil- or water-borne protozoon that feeds on bacteria by phagocytosis. A. castellanii can grow between 4 and 32 degrees C and has to adapt quickly to chilling in order to survive. We have identified a Delta12-fatty acid desaturase as key to low temperature adaptation. The activity of this enzyme is mainly increased through gene expression and new protein synthesis. Interestingly, the activity can also be altered independently by dissolved oxygen levels. In addition, we have identified a gene for the Delta12-desaturase, which, when expressed in yeast, catalyses Delta15-desaturation also. Moreover, it is also capable of producing very unusual n-1 polyunsaturated products. PMID:17584990

  11. The free-living amoeba Willaertia magna, is particularly resistant to infection by the pathogenic bacteria Legionella pneumophila

    Dey, Rafik; Cavalié, Laurent; Vernet, Christine; Bodennec, Jacques; Pernin, Pierre

    2008-01-01

    Legionella pneumophila, the causative agent of Legionnaire's disease, is well characterized as a bacteria surviving and developing, almost exclusively, as intracellular parasite within freshwater protozoa. Several species of protozoa and ciliae have been shown to support the growth of the pathogenic bacteria. In the present study, we report for the first time the behaviour of the protozoan Willaertia magna towards L. pneumophila and compared it with Acanthamoeba castellanii and Hartmannella v...

  12. Plasma membrane biogenesis in eukaryotic cells: translocation of newly synthesized lipid.

    Mills, J T; Furlong, S T; Dawidowicz, E A

    1984-01-01

    We examined the transfer of sterols and phospholipids from their site of synthesis to the plasma membrane of Acanthamoeba castellanii. Cells were labeled with [3H]acetate, and plasma membrane fractions were isolated under conditions that minimize the nonspecific exchange of lipids between subcellular membrane fractions. Sterols and phospholipids were purified from both whole-cell homogenates and isolated plasma membrane. In whole cells, 3H-labeled lipids were formed, with no apparent time lag...

  13. Legionella Pneumophila Transcriptome during Intracellular Multiplication in Human Macrophages

    Faucher, Sébastien P.; Mueller, Catherine A.; Shuman, Howard A.

    2011-01-01

    Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot type IVB secretion system, which is used to translocate more than 150 effector proteins into host cells. While the transcriptional response of Legionella to the intracellular envir...

  14. Characterization of giardin protein expression during encystation of Giardia duodenalis

    Giardia duodenalis trophozoites attach to the gut surface by means of a ventral disk that contains various giardin proteins that appear to be important to VD structural integrity. One approach to preventing giardiasis is to stimulate giardin-specific antibodies and thereby block trophozoite attachme...

  15. Dicty_cDB: AFF514 [Dicty_cDB

    Full Text Available arhtkasneaaeyaklvaqrqqakakrfrqsfkqkscxpsk Frame B: *verwyivyyylkks**tllkqfnkrkfieveh*mvqpkvii*tsntinklttrqtmsak...*kscssh*sqq*srrir*trcpktts*sqeipskfqakkllxk* Homology vs CSM-cDNA Score E Sequences producing significant...own update 2004.12.25 Homology vs DNA Score E Sequences producing significant alignment...80 4e-26 9 U03732 |U03732.1 Acanthamoeba castellanii Neff mitochondrion rRNA larg...9, full insert sequence. 58 5e-22 5 AF416765 |AF416765.1 Ehrlichia chaffeensis strain Arkansas 23S ribosomal RNA an

  16. Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa

    Rowe Michael T

    2006-07-01

    Full Text Available Abstract Background Interactions between Mycobacterium avium subsp. paratuberculosis (Map and free-living protozoa in water are likely to occur in nature. The potential impact of ingestion of Map by two naturally occurring Acanthamoeba spp. on this pathogen's survival and chlorine resistance was investigated. Results Between 4.6 and 9.1% of spiked populations of three Map strains (NCTC 8578, B2 and ATCC 19698, which had been added at a multiplicity of infection of 10:1, were ingested by Acanthamoeba castellanii CCAP 1501/1B and A. polyphaga CCAP 1501/3B during co-culture for 3 h at 25°C. Map cells were observed to be present within the vacuoles of the amoebae by acid-fast staining. During extended co-culture of Map NCTC 8578 at 25°C for 24 d with both A. castellanii and A. polyphaga Map numbers did not change significantly during the first 7 days of incubation, however a 1–1.5 log10 increase in Map numbers was observed between days 7 and 24 within both Acanthamoeba spp. Ingested Map cells were shown to be more resistant to chlorine inactivation than free Map. Exposure to 2 μg/ml chlorine for 30 min resulted in a log10 reduction of 0.94 in ingested Map but a log10 reduction of 1.73 in free Map (p Conclusion This study demonstrated that ingestion of Map by and survival and multiplication of Map within Acanthamoeba spp. is possible, and that Map cells ingested by amoebae are more resistant to inactivation by chlorine than free Map cells. These findings have implications with respect to the efficacy of chlorination applied to Map infected surface waters.

  17. Isolation of Free-Living Amoebae from Sarein Hot Springs in Ardebil Province, Iran

    A Badirzadeh

    2011-06-01

    Full Text Available Background: Free-living amoebae (FLA are a group of ubiquitous protozoan, which are distrib­uted in the natural and artificial environment sources. The main aim of the current study was to identify the presence of FLA in the recreational hot springs of Sarein in Ardebil Province of Iran.Methods: Seven recreational hot springs were selected in Sarein City and 28 water samples (four from each hot spring were collected using 500 ml sterile plastic bottles during three month. Filtra­tion of water samples was performed, and culture was done in non-nutrient agar medium enriched with Escherichia coli. Identification of the FLA was based on morphological criteria of cysts and trophozoites. Genotype identification of Acanthamoeba positive samples were also per­formed using sequencing based method.Results: Overall, 12 out of 28 (42.9% samples were positive for FLA which Acanthamoeba and Vahlkampfiid amoebae were found in one (3.6% and 11 (39.3% samples, respectively. Se­quence analysis of the single isolate of Acanthamoeba revealed potentially pathogenic T4 geno­type corresponding to A. castellanii.Conclusion: Contamination of hot springs to FLA, such as Acanthamoeba T4 genotype (A. castel­lanii and Vahlkampfiid amoebae, could present a sanitary risk for high risk people, and health authorities must be aware of FLA presence.

  18. Free-living freshwater amoebae differ in their susceptibility to the pathogenic bacterium Legionella pneumophila.

    Dey, Rafik; Bodennec, Jacques; Mameri, Mouh Oulhadj; Pernin, Pierre

    2009-01-01

    Legionella pneumophila is known as a facultative intracellular parasite of free-living soil and freshwater amoebae, of which several species have been shown to support the growth of the pathogenic bacteria. We report for the first time the behaviour of two strains (c2c and Z503) of the amoeba Willaertia magna towards different strains of L. pneumophila serogroup 1 and compared it with Acanthamoeba castellanii and Hartmannella vermiformis, known to be L. pneumophila permissive. In contrast to the results seen with other amoebae, W. magna c2c inhibited the growth of one strain of Legionella (L. pneumophila, Paris), but not of others belonging to the same serogroup (L. pneumophila, Philadelphia and L. pneumophila, Lens). Also, the different L. pneumophila inhibited cell growth and induced cell death in A. castellanii, H. vermiformis and W. magna Z503 within 3-4 days while W. magna c2c strain remained unaffected even up to 7 days. Electron microscopy demonstrated that the formation of numerous replicative phagosomes observed within Acanthamoeba and Hartmannella is rarely seen in W. magna c2c cocultured with L. pneumophila. Moreover, the morphological differences were observed between L. pneumophila cultured either with Willaertia or other amoebae. These observations show that amoebae are not all equally permissive to L. pneumophila and highlight W. magna c2c as particularly resistant towards some strains of this bacterium. PMID:19016880

  19. Sterility and the disinfection potential of Indian contact lens solutions

    Gopinathan Usha

    1994-01-01

    Full Text Available Ocular infection associated with microbial contamination of contact lens care products is a major problem in contact lens wearers. The sterility and the antimicrobial activity of contact lens care systems reflect their suitability for disinfection of contact lenses. These factors remain to be evaluated for the various newer contact lens care products manufactured in India. In this study, 35 bottles of contact lens solutions marketed by different manufacturing units in India were tested for sterility. Seven solutions were tested for antimicrobial effectiveness employing the D value method of analysis. The D value is defined as the time required to reduce a population of organisms by 90% (one log unit. A standard inoculum of the ocular isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Serratia marcescens, Aspergillus fumigatus, Fusarium solani, and Acanthamoeba castellanii were used as challenge organisms. Bacterial contamination was detected in 20 (57.1% solution bottles and none yielded fungus or Acanthamoeba. Pseudomonas species were the most commonly encountered contaminant (11/20; 55%. Only sterile solutions were analyzed for antimicrobial activity. D values ranging between 12 and 20 minutes were demonstrated by six of the seven solutions against bacterial challenge. Good antifungal activity was noticed in five solutions against Fusarium solani though results varied with Aspergillus flavus and Candida albicans. All solutions were adequately effective against Acanthamoeba.

  20. Acanthamoeba and bacteria produce antimicrobials to target their counterpart

    Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2014-01-01

    Background In the microbial ecosystem, microbes compete for space and nutrients. Consequently, some have developed the ability to kill or inhibit the growth of other competing microbes by producing antimicrobial substances. As the ‘producer’ species are generally immune to these substances, their compounds act on the competing microbial species and give the producer more space and access to nutrients for growth. Many currently used antibiotics were developed by exploiting this potential of ce...

  1. Species specificity of a monoclonal antibody produced to Naegleria fowleri and partial characterization of its antigenic determinant.

    Réveiller, F L; Marciano-Cabral, F; Pernin, P; Cabanes, P A; Legastelois, S

    2000-08-01

    Monoclonal antibody (Mab) 5D12 against Naegleria fowleri was analyzed for species specificity. Mab 5D12 reacted with a ubiquitous epitope present on the membrane of N. fowleri but not with soluble antigens. The Mab did not react with N. lovaniensis, N. gruberi, N. australiensis, or Acanthamoeba castellanii. The decreased reactivity of Mab 5D12 with N. fowleri observed after periodate oxidation, after digestion of carbohydrate moieties by three glycosidases, or after treatment of amebas with tunicamycin strongly suggests that the antigenic determinant has a polysaccharide component. Inhibition of the reactivity of Mab 5D12 by soluble saccharides supports the idea that N-acetyl or amino groups may play an important role in the recognition of the carbohydrate component of the epitope by the Mab. The specificity of Mab 5D12 makes this an ideal reagent for the identification of N. fowleri in environmental samples or in clinical specimens. PMID:10952262

  2. Isolation and complete genome sequencing of Mimivirus bombay, a Giant Virus in sewage of Mumbai, India.

    Chatterjee, Anirvan; Ali, Farhan; Bange, Disha; Kondabagil, Kiran

    2016-09-01

    We report the isolation and complete genome sequencing of a new Mimiviridae family member, infecting Acanthamoeba castellanii, from sewage in Mumbai, India. The isolated virus has a particle size of about 435 nm and a 1,182,200-bp genome. A phylogeny based on the DNA polymerase sequence placed the isolate as a new member of the Mimiviridae family lineage A and was named as Mimivirus bombay. Extensive presence of Mimiviridae family members in different environmental niches, with remarkably similar genome size and genetic makeup, point towards an evolutionary advantage that needs to be further investigated. The complete genome sequence of Mimivirus bombay was deposited at GenBank/EMBL/DDBJ under the accession number KU761889. PMID:27330993

  3. Fate of pathogenic Bacillus cereus spores after ingestion by protist grazers

    Winding, Anne; Santos, Susana; Hendriksen, Niels Bohse; Jakobsen, Hans

    evolution of Bacillus cereus group bacteria (e.g. B. cereus, B. anthracis, B. thuringiensis) as a pathogen. It has been hypothesized that the spore stage protects against digestion by predating protists. Indeed, B. thuringiensis spores have been shown to be readily ingested by ciliated protists but failed...... to be digested (Manasherob et al 1998 AEM 64:1750-). Here we report how diverse protist grazers grow on both vegetative cells and spores of B. cereus and how the bacteria survive ingestion and digestion, and even proliferate inside the digestive vacuoles of ciliated protists. The survival ability of...... B. cereus was initially investigated in microcosms inoculated with pure cultures of the protists Acanthamoeba castellanii, Tetrahymena pyriformis and Cercomonas sp. as grazers. Individual protist cultures were fed with fluorescently labelled (CellTracker™RedCMTPX) B. cereus spores or vegetative...

  4. UV inactivation of pathogenic and indicator microorganisms

    Chang, J.C.; Ossoff, S.F.; Lobe, D.C.; Dorfman, M.H.; Dumais, C.M.; Qualls, R.G.; Johnson, J.D.

    1985-06-01

    Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts.

  5. UV inactivation of pathogenic and indicator microorganisms

    Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts

  6. Survival and Virulence of Campylobacter spp. in the Environment

    Bui, Thanh Xuan

    : Escherichia coli and Enterococcus spp., in raw slurry, in the liquid fraction of separated slurry, and in the liquid fraction after ozonation to ground water using intact soil columns models. I observed that solid-liquid separation of slurry increased the redistribution of contaminants in liquid fraction...... in the soil compared to raw slurry, and the recovery of E. coli and Enterococcus spp. was higher for liquid fraction after the four leaching events. The liquid fraction also resulted in a higher leaching of all contaminants except Enterococcus spp. than raw slurry while the Ozonation reduced E. coli leaching....... Interestingly, I identified the depletion of dissolved oxygen by A. castellanii as the major contributor for the observed amoeba-mediated growth enhancement. To test whether another protozoan rather than Acanthamoeba has similar impacts on survival of C. jejuni as well as other food-borne pathogens S...

  7. srRNA evolution and phylogenetic relationships of the genus Naegleria (Protista: Rhizopoda).

    Baverstock, P R; Illana, S; Christy, P E; Robinson, B S; Johnson, A M

    1989-05-01

    A rapid RNA sequencing technique was used to partially sequence the small-subunit ribosomal RNA (srRNA) of four species of the amoeboid genus Naegleria. The extent of nucleotide sequence divergence between the two most divergent species was roughly similar to that found between mammals and frogs. However, the pattern of variation among the Naegleria species was quite different from that found for those species of tetrapods characterized to date. A phylogenetic analysis of the consensus Naegleria sequence showed that Naegleria was not monophyletic with either Acanthamoeba castellanii or Dictyostelium discoideum, two other amoebas for which sequences were available. It was shown that the semiconserved regions of the srRNA molecule evolve in a clocklike fashion and that the clock is time dependent rather than generation dependent. PMID:2622334

  8. Black cobra (Naja naja karachiensis) lysates exhibit broad-spectrum antimicrobial activities

    Sagheer, Mehwish; Siddiqui, Ruqaiyyah; Iqbal, Junaid; Khan, Naveed Ahmed

    2014-01-01

    It is hypothesized that animals living in polluted environments possess antimicrobials to counter pathogenic microbes. The fact that snakes feed on germ-infested rodents suggests that they encounter pathogenic microbes and likely possess antimicrobials. The venom is used only to paralyze the rodent, but the ability of snakes to counter potential infections in the gut due to disease-ridden rodents requires robust action of the immune system against a broad range of pathogens. To test this hypothesis, crude lysates of different organs of Naja naja karachiensis (black cobra) were tested for antimicrobial properties. The antimicrobial activities of extracts were tested against selected bacterial pathogens (neuropathogenic Escherichia coli K1, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Streptococcus pneumonia), protist (Acanthamoeba castellanii), and filamentous fungus (Fusarium solani). The findings revealed that plasma and various organ extracts of N. n. karachiensis exhibited antimicrobial activity against E. coli K1, MRSA, P. aeruginosa, S. pneumoniae, A. castellanii, and F. solani in a concentration-dependent manner. The results of this study are promising for the development of new antimicrobials. PMID:24625321

  9. Optimized DNA extraction methods for encysted embryos of the endangered fairy shrimp, Branchinecta sandiegonensis

    Steele, A.N.; Simovich, M.A.; Pepino, D.; Schroeder, K.M.; Vandergast, A.G.; Bohonak, A.J.

    2009-01-01

    The San Diego fairy shrimp Branchinecta sandiegonensis is a federally endangered species endemic to vernal pools in southern California, USA. Filling events in these habitats are highly variable, with some pools failing to hold water long enough for reproduction over many successive years. Studies of this species are thus hindered by the relatively rare appearance of aquatically active life history phases. Because diapausing cysts are abundant and present at all times, they provide an underutilized opportunity for both species identification and genetic studies. However, methods for extracting DNA from cysts are technically challenging because of their structure and size. Here we present a protocol for extracting DNA from B. sandiegonensis cysts in sufficient quantities for "DNA Barcoding", microsatellite analysis and other genotyping and sequencing applications. The technique will aid in population genetic studies and species identification (since taxonomic keys only distinguish among adults), and will be applicable to other crustaceans with similar diapausing cysts. ?? Springer Science+Business Media B.V. 2008.

  10. Encysted Tenia solium larva of oral cavity: Case report with review of literature

    Bhuvana Krishnamoorthy

    2012-01-01

    Full Text Available Cysticercosis is caused by the larvae of the pig tapeworm, Tenia solium. Oral cysticercosis is a rare event and is often a diagnostic challenge to the clinician. We report a 12-year-old girl who presented with a single, painless, nodule on the lower lip that was diagnosed as cysticercosis. Current literature on the clinical presentations, investigations, and treatment of the condition has been reviewed in this article. We have also proposed a set of criteria for the diagnosis of oral cysticercosis.

  11. Encysted Tenia solium larva of oral cavity: Case report with review of literature

    Bhuvana Krishnamoorthy; N Suma Gundareddy; Manu Dhillon; Siddharth Srivastava; Manisha Lakhanpal Sharma; Sangeeta Singh Malik

    2012-01-01

    Cysticercosis is caused by the larvae of the pig tapeworm, Tenia solium. Oral cysticercosis is a rare event and is often a diagnostic challenge to the clinician. We report a 12-year-old girl who presented with a single, painless, nodule on the lower lip that was diagnosed as cysticercosis. Current literature on the clinical presentations, investigations, and treatment of the condition has been reviewed in this article. We have also proposed a set of criteria for the diagnosis of oral cysticer...

  12. The type II secretion system of Legionella pneumophila elaborates two aminopeptidases, as well as a metalloprotease that contributes to differential infection among protozoan hosts.

    Rossier, Ombeline; Dao, Jenny; Cianciotto, Nicholas P

    2008-02-01

    Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysine- and arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts. PMID:18083880

  13. Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs.

    Weissenmayer, Barbara A

    2011-01-01

    Second generation sequencing has prompted a number of groups to re-interrogate the transcriptomes of several bacterial and archaeal species. One of the central findings has been the identification of complex networks of small non-coding RNAs that play central roles in transcriptional regulation in all growth conditions and for the pathogen\\'s interaction with and survival within host cells. Legionella pneumophila is a gram-negative facultative intracellular human pathogen with a distinct biphasic lifestyle. One of its primary environmental hosts in the free-living amoeba Acanthamoeba castellanii and its infection by L. pneumophila mimics that seen in human macrophages. Here we present analysis of strand specific sequencing of the transcriptional response of L. pneumophila during exponential and post-exponential broth growth and during the replicative and transmissive phase of infection inside A. castellanii. We extend previous microarray based studies as well as uncovering evidence of a complex regulatory architecture underpinned by numerous non-coding RNAs. Over seventy new non-coding RNAs could be identified; many of them appear to be strain specific and in configurations not previously reported. We discover a family of non-coding RNAs preferentially expressed during infection conditions and identify a second copy of 6S RNA in L. pneumophila. We show that the newly discovered putative 6S RNA as well as a number of other non-coding RNAs show evidence for antisense transcription. The nature and extent of the non-coding RNAs and their expression patterns suggests that these may well play central roles in the regulation of Legionella spp. specific traits and offer clues as to how L. pneumophila adapts to its intracellular niche. The expression profiles outlined in the study have been deposited into Genbank\\'s Gene Expression Omnibus (GEO) database under the series accession GSE27232.

  14. RELATIONSHIP OF TADPOLE STAGE TO LOCATION OF ECHINOSTOME CERCARIAE ENCYSTMENT AND THE CONSEQUENCES FOR TADPOLE SURVIVAL. (R825867)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  15. Experimental study on pathogenic potential of six Acanthamoeba strains isolated from fish

    Veverková, Marie; Dyková, Iva; Pecková, Hana

    2002-01-01

    Roč. 49, č. 3 (2002), s. 243-245. ISSN 0015-5683 R&D Projects: GA AV ČR KSK6005114 Institutional research plan: CEZ:AV0Z6022909 Keywords : amoeba * fish * pathology Subject RIV: EA - Cell Biology Impact factor: 0.515, year: 2002

  16. Structure and function of a unique pore-forming protein from a pathogenic acanthamoeba

    Michalek, M.; Sönnichsen, F.D.; Wechselberger, R.W.; Wienk, H.L.J.; Leippe, M.; et al., [No Value

    2013-01-01

    Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acantha

  17. Acanthamoeba polyphaga mimivirus stability in environmental and clinical substrates: implications for virus detection and isolation.

    Fábio P Dornas

    Full Text Available Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water and hospital (ventilator plastic device tube substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

  18. Acanthamoeba polyphaga mimivirus Stability in Environmental and Clinical Substrates: Implications for Virus Detection and Isolation

    Dornas, Fábio P.; Silva, Lorena C. F.; de Almeida, Gabriel M.; Rafael K Campos; Boratto, Paulo V.M.; Ana P M Franco-Luiz; La Scola, Bernard; Ferreira, Paulo C. P.; Kroon, Erna G.; Abrahão, Jônatas S.

    2014-01-01

    Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions betwee...

  19. Amoebozoa possess lineage-specific globin gene repertoires gained by individual horizontal gene transfers.

    Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

    2014-01-01

    The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

  20. The CpxRA two-component system contributes to Legionella pneumophila virulence.

    Tanner, Jennifer R; Li, Laam; Faucher, Sébastien P; Brassinga, Ann Karen C

    2016-06-01

    The bacterium Legionella pneumophila is capable of intracellular replication within freshwater protozoa as well as human macrophages, the latter of which results in the serious pneumonia Legionnaires' disease. A primary factor involved in these host cell interactions is the Dot/Icm Type IV secretion system responsible for translocating effector proteins needed to establish and maintain the bacterial replicative niche. Several regulatory factors have been identified to control the expression of the Dot/Icm system and effectors, one of which is the CpxRA two-component system, suggesting essentiality for virulence. In this study, we generated cpxR, cpxA and cpxRA in-frame null mutant strains to further delineate the role of the CpxRA system in bacterial survival and virulence. We found that cpxR is essential for intracellular replication within Acanthamoeba castellanii, but not in U937-derived macrophages. Transcriptome analysis revealed that CpxRA regulates a large number of virulence-associated proteins including Dot/Icm effectors as well as Type II secreted substrates. Furthermore, the cpxR and cpxRA mutant strains were more sodium resistant than the parental strain Lp02, and cpxRA expression reaches maximal levels during postexponential phase. Taken together, our findings suggest the CpxRA system is a key contributor to L. pneumophila virulence in protozoa via virulence factor regulation. PMID:26934669

  1. A conserved OmpA-like protein in Legionella pneumophila required for efficient intracellular replication.

    Goodwin, Ian P; Kumova, Ogan K; Ninio, Shira

    2016-08-01

    The OmpA-like protein domain has been associated with peptidoglycan-binding proteins, and is often found in virulence factors of bacterial pathogens. The intracellular pathogen Legionella pneumophila encodes for six proteins that contain the OmpA-like domain, among them the highly conserved uncharacterized protein we named CmpA. Here we set out to characterize the CmpA protein and determine its contribution to intracellular survival of L. pneumophila Secondary structure analysis suggests that CmpA is an inner membrane protein with a peptidoglycan-binding domain at the C-teminus. A cmpA mutant was able to replicate normally in broth, but failed to compete with an isogenic wild-type strain in an intracellular growth competition assay. The cmpA mutant also displayed significant intracellular growth defects in both the protozoan host Acanthamoeba castellanii and in primary bone marrow-derived macrophages, where uptake into the cells was also impaired. The cmpA phenotypes were completely restored upon expression of CmpA in trans The data presented here establish CmpA as a novel virulence factor of L. pneumophila that is required for efficient intracellular replication in both mammalian and protozoan hosts. PMID:27421957

  2. Short-Term and Long-Term Survival and Virulence of Legionella pneumophila in the Defined Freshwater Medium Fraquil.

    Nilmini Mendis

    Full Text Available Legionella pneumophila (Lp is the etiological agent responsible for Legionnaires' disease, a potentially fatal pulmonary infection. Lp lives and multiplies inside protozoa in a variety of natural and man-made water systems prior to human infection. Fraquil, a defined freshwater medium, was used as a highly reproducible medium to study the behaviour of Lp in water. Adopting a reductionist approach, Fraquil was used to study the impact of temperature, pH and trace metal levels on the survival and subsequent intracellular multiplication of Lp in Acanthamoeba castellanii, a freshwater protozoan and a natural host of Legionella. We show that temperature has a significant impact on the short- and long-term survival of Lp, but that the bacterium retains intracellular multiplication potential for over six months in Fraquil. Moreover, incubation in Fraquil at pH 4.0 resulted in a rapid decline in colony forming units, but was not detrimental to intracellular multiplication. In contrast, variations in trace metal concentrations had no impact on either survival or intracellular multiplication in amoeba. Our data show that Lp is a resilient bacterium in the water environment, remaining infectious to host cells after six months under the nutrient-deprived conditions of Fraquil.

  3. The ClpP protease homologue is required for the transmission traits and cell division of the pathogen Legionella pneumophila

    Zhang Qin-fen

    2010-02-01

    Full Text Available Abstract Background Legionella pneumophila, the intracellular bacterial pathogen that causes Legionnaires' disease, exhibit characteristic transmission traits such as elevated stress tolerance, shortened length and virulence during the transition from the replication phase to the transmission phase. ClpP, the catalytic core of the Clp proteolytic complex, is widely involved in many cellular processes via the regulation of intracellular protein quality. Results In this study, we showed that ClpP was required for optimal growth of L. pneumophila at high temperatures and under several other stress conditions. We also observed that cells devoid of clpP exhibited cell elongation, incomplete cell division and compromised colony formation. Furthermore, we found that the clpP-deleted mutant was more resistant to sodium stress and failed to proliferate in the amoebae host Acanthamoeba castellanii. Conclusions The data present in this study illustrate that the ClpP protease homologue plays an important role in the expression of transmission traits and cell division of L. pneumophila, and further suggest a putative role of ClpP in virulence regulation.

  4. Short-Term and Long-Term Survival and Virulence of Legionella pneumophila in the Defined Freshwater Medium Fraquil

    Mendis, Nilmini; McBride, Peter; Faucher, Sébastien P.

    2015-01-01

    Legionella pneumophila (Lp) is the etiological agent responsible for Legionnaires’ disease, a potentially fatal pulmonary infection. Lp lives and multiplies inside protozoa in a variety of natural and man-made water systems prior to human infection. Fraquil, a defined freshwater medium, was used as a highly reproducible medium to study the behaviour of Lp in water. Adopting a reductionist approach, Fraquil was used to study the impact of temperature, pH and trace metal levels on the survival and subsequent intracellular multiplication of Lp in Acanthamoeba castellanii, a freshwater protozoan and a natural host of Legionella. We show that temperature has a significant impact on the short- and long-term survival of Lp, but that the bacterium retains intracellular multiplication potential for over six months in Fraquil. Moreover, incubation in Fraquil at pH 4.0 resulted in a rapid decline in colony forming units, but was not detrimental to intracellular multiplication. In contrast, variations in trace metal concentrations had no impact on either survival or intracellular multiplication in amoeba. Our data show that Lp is a resilient bacterium in the water environment, remaining infectious to host cells after six months under the nutrient-deprived conditions of Fraquil. PMID:26406895

  5. Host - parasite interactions: : the relationship between encystment load of the freshwater pearl mussel (Margaritifera margaritifera) and genetic diversity of its host (Salmo trutta)

    Winser, Helena

    2015-01-01

    The freshwater pearl mussel (FPM, Margaritifera margaritifera) is an endangered bivalve mollusc with an obligatory larval parasitic phase on brown trout (Salmo trutta). The FPM has declined throughout its entire range due to causes such as habitat degradation, eutrophication, acidification, changed hydrology and lack of host fish. This study aimed to investigate if heterozygosity, allelic richness, number of alleles, inbreeding and differentiation of brown trout (Salmo trutta) are related to ...

  6. Surviving within the amoebal exocyst: the Mycobacterium avium complex paradigm

    Drancourt Michel

    2010-04-01

    Full Text Available Abstract Background Most of environmental mycobacteria have been previously demonstrated to resist free-living amoeba with subsequent increased virulence and resistance to antibiotics and biocides. The Mycobacterium avium complex (MAC comprises of environmental organisms that inhabit a wide variety of ecological niches and exhibit a significant degree of genetic variability. We herein studied the intra-ameobal location of all members of the MAC as model organisms for environmental mycobacteria. Results Type strains for M. avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium arosiense, Mycobacterium marseillense, Mycobacterium timonense and Mycobacterium bouchedurhonense were co-cultivated with the free-living amoeba Acanthamoeba polyphaga strain Linc-AP1. Microscopic analyses demonstrated the engulfment and replication of mycobacteria into vacuoles of A. polyphaga trophozoites. Mycobacteria were further entrapped within amoebal cysts, and survived encystment as demonstrated by subculturing. Electron microscopy observations show that, three days after entrapment into A. polyphaga cysts, all MAC members typically resided within the exocyst. Conclusions Combined with published data, these observations indicate that mycobacteria are unique among amoeba-resistant bacteria, in residing within the exocyst.

  7. The abundant free-living amoeba, Acanthamoeba polyphaga, increases the survival of Campylobacter jejuni in milk and orange juice

    Olofsson, Jenny; Griekspoor Berglund, Petra; Olsen, Björn; Ellström, Patrik; Axelsson-Olsson, Diana

    2015-01-01

    Background: Campylobacter jejuni is a common cause of human bacterial diarrhea in most parts of the world. Most C. jejuni infections are acquired from contaminated poultry, milk, and water. Due to health care costs and human suffering, it is important to identify all possible sources of infection. Unpasteurized milk has been associated with several outbreaks of C. jejuni infection. Campylobacter has been identified on fresh fruit, and other gastrointestinal pathogens such as Salmonella, E. co...

  8. Genetic Characterization of Clinical Acanthamoeba Isolates Using Gene Loci of Nuclear and Mitochondrial Small Subunit Ribosomal RNA

    ラフマン, モハメド, モシウル

    2013-01-01

    以下に掲載:The Korean Journal of Parasitology 51(4) pp.401-411 2013. Korean Society for Parasitology and Tropical Medicine. 共著者:Md Moshiur Rahman, Kenji Yagita, Akira Kobayashi, Yosaburo Oikawa, Amjad I.A. Hussein, Takahiro Matsumura and Masaharu Tokoro

  9. Genetic Characterization of Clinical Acanthamoeba Isolates Using Gene Loci of Nuclear and Mitochondrial Small Subunit Ribosomal RNA

    ラフマン, モハメド, モシウル

    2013-01-01

    要旨Abstract 以下に掲載:The Korean Journal of Parasitology. 51(4) pp.401-411 2013. Korean Society for Parasitology and Tropical Medicine. 共著者:Md Moshiur Rahman, Kenji Yagita, Akira Kobayashi, Yosaburo Oikawa, Amjad I.A. Hussein, Takahiro Matsumura and Masaharu Tokoro

  10. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes.

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506