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Sample records for echinococcus granulosus tegumental

  1. Depolarization of the tegument precedes morphological alterations in Echinococcus granulosus protoscoleces incubated with ivermectin.

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    Pérez-Serrano, J; Grosman, C; Urrea-París, M A; Denegri, G; Casado, N; Rodríguez-Caabeiro, F

    2001-10-01

    The nematocidal activity of ivermectin (IVM) largely arises from its activity as a potent agonist of muscular and neuronal glutamate-gated chloride channels. A cestocidal effect has also been suggested following in vitro treatments, but the molecular basis of this activity is not clear. We studied the effect of IVM on the metacestode stage of the tapeworm Echinococcus granulosus by assessing the viability, ultrastructure, and tegumental membrane potential as a function of drug concentration and incubation time. Concentrations of 0.1 and 1.0 microg/ml of IVM had no effect on any of these three parameters for up to 6 days of treatment. A concentration of 10 microg/ml, however, elicited a sequence of alterations that started with a approximately 20-mV depolarization of the tegumental membrane, and was followed by rostellar disorganization, rigid paralysis and, eventually, loss of viability. It is likely that the IVM-induced depolarization of the tegument acts as the signal that initiates the cascade of degenerative processes that leads to the parasite's death. This would place the tegument as the primary target of action of IVM on cestodes. As an appropriate chemotherapy for the hydatid disease is still lacking, the cestocidal effect of IVM reported here is worth considering.

  2. Osmoregulatory and tegumental ultrastructural damages to protoscoleces of hydatid cysts Echinococcus granulosus induced by fungal endophytes.

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    Verma, Vijay C; Gangwar, Mayank; Nath, Gopal

    2014-12-01

    Characteristic ultrastructural changes were observed when protoscoleces of hydatid cysts Echinococcus granulosus was treated with extract of endophytic fungi Eupenicillium and Chaetomium sp. isolated from Azadirachta indica and Piper longum plants respectively. A sharp decrease in viability of protoscoleces was observed after 6 h of incubation with fungal extracts. The ultrastructural changes included rosteller disorganization, loss of hooks and shedding of the microtriches of scolex region. The formation of digitiform projections on tegument layer which, increased in size as prolong incubation with extract and get burst, leading to a osmoregulatory damage into tegumental layers of parasite. This osmoregulatory damages caused the loss of turgidity due to leakage of cell contents, which might be the major cause of the mortality in treated parasites. It is remarkable, since very similar type of ultrastructural changes were observed with some pyrazinoisoquinoline derivatives, as praziquantel. Our initial results indicate that extract of endophytic Eupenicillium and Chaetomium spp. are having significant anti-cestodal activity and have selective activity on tegument layer. Further chemical prospection is required through rigorous bioassay guided fractionation coupled with robust high resolution mass spectrometric analysis to get final stereo-structures responsible for the parasiticidal activity. This initial strain selection outcome will serve a platform for isolation and characterization of new drug lead that can be useful in tailoring novel, safe and effective anthelmintics.

  3. Freeze-etch characterization of the teguments of three metacestodes: Echinococcus granulosus, Taenia crassiceps, and Taenia taeniaeformis.

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    Conder, G A; Marchiondo, A A; Williams, J F; Andersen, F L

    1983-06-01

    The objective of this study was to characterize the teguments of metacestodes of Echinococcus granulosus, Taenia crassiceps, and Taenia taeniaeformis using the freeze-etch technique. Metacestodes of E. granulosus (19 mo old), T. crassiceps (28 days old), and T. taeniaeformis (34 days old) from gerbils, mice and rats, respectively, were fixed for 2 hr in 3% glutaraldehyde and then prepared for freeze-etching and thin sectioning by standard techniques. Freeze-etch replicas of the teguments of all three species displayed morphologic characteristics that were generally in agreement with previous ultrastructural work, although some new features and interpretations arose from use of this technique. For each species there was a concentric ring structure within the microthrix base, and cytoplasmic extensions of the perikarya into the distal tegument were membrane-bound rather than confluent bridges; these extensions frequently branched within the tegument. In addition, channels running from the proximal tegumental membrane to, and opening at the distal surface of, the tegument were seen in thin sections.

  4. Echinococcus granulosus tegumental enzymes as in vitro markers of pharmacological damage: a biochemical and molecular approach.

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    Cumino, Andrea C; Nicolao, M Celeste; Loos, Julia A; Denegri, Guillermo; Elissondo, M Celina

    2012-12-01

    Cystic echinococcosis is a chronic, complex, and neglected disease. Novel therapeutical tools are needed to optimize human treatment. A number of compounds have been investigated, either using in vitro cultured parasites and/or applying in vivo rodent models. Although some of these compounds showed promising activities in vitro, and to some extent also in the rodent models, they have not been translated into clinical applications. Membrane enzyme activities in culture supernatants of treated protoscoleces with calcium modulator drugs and anthelmintic drugs were measured and provided an indication of compound efficacy. This work describes for the first time the detection of alkaline phosphatase, gamma-glutamyl-transpeptidase and acetylcholinesterase activities in supernatants of in vitro treated Echinococcus granulosus protoscoleces. Marked differences on the enzymatic activities in supernatants from drug treated cultures were detected. We demonstrated that those genes that show the highest degree of conservation when compared to orthologs, are constitutively and highly expressed in protoscoleces and metacestodes. Due to high sensibility and the lack of activity in supernatants of intact protoscoleces, gamma-glutamyl-transpeptidase is proposed as the ideal viability marker during in vitro pharmacological studies against E. granulosus protoscoleces.

  5. 用抗虫体表膜抗原抗体测定犬细粒棘球绦虫粪抗原%Using Antibodies against Tegument Antigens for Copro-ELISA to Detect Echinococcus granulosus in Dogs

    Institute of Scientific and Technical Information of China (English)

    古努尔·吐尔逊; 金映红; 张文宝; 米晓云; 张壮志; 石保新; 吐尔洪·依米提; 张旭; 巫剑; 赵莉; 阿曼古丽·马木提

    2011-01-01

    The study was aimed at determining whether antibodies against worm tegument antigens can be used for copro-ELISA to detect Echinococcus granulosus in dogs. In order to produce specific antibodies used for sandwich (copro)-ELISA, we isolated antigens from E. granulosus adult worm tegument. The proteins were used to immunize mice, rabbits and sheep to generate antibodies. ELISA and Western blot analysis were used to identify antigenicity and quality of the serum antibodies. Immunohistochemistry was used to indentify the localization of the proteins in the parasite. Copro-ELISA was used to determine the specificity of the immunodiagnosis for detecting E. granulosus. infection in dogs. Scrum titer analysis showed that E. granulosus tegument antigens were antigenic with serum titers reaching to 1 : (80000-320000) by ELISA. The antibodies recognized antigens from different stages of the parasite. Immunolocalization analysis revealed that the antigens located in the tegument of adult E. granulosus. The antibodies specifically recognized the antigens from E. granulosus with no reaction to the proteins isolated from Multiceps multicepes and T. hydatigena. The results suggest that E. granulosus tegument antigens are a source of antigens for generating antibodies for copro-ELISA to detect the worm antigens in dog feces.%利用Echinococcus granulosus(Eg)成虫表膜抗原抗体夹心ELISA方法检测犬细粒棘球绦虫感染的粪抗原.首先提取Eg成虫表膜抗原,制备抗Eg成虫表膜抗原的血清,然后用间接ELISA和Western blotting方法检测其抗原抗体反应及免疫原性,用间接免疫组化方法对Eg成虫表膜蛋白进行组织定位.利用双抗体夹心ELISA方法检测感染Eg的犬粪抗原.ELISA结果表明Eg成虫表膜抗原具有良好的抗原性并能产生高效价抗体,抗体不与多头绦虫和泡状带绦虫抗原反应;Western blotting结果表明Eg成虫表膜抗原与原头蚴、不成熟Eg有共同蛋白成分.免疫组化结

  6. Efficacy of thymol against Echinococcus granulosus protoscoleces.

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    Elissondo, M Celina; Albani, Clara M; Gende, Liesel; Eguaras, Martín; Denegri, Guillermo

    2008-06-01

    The aim of the present work was to determine the in vitro protoscolicidal effect of thymol against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with thymol at concentrations of 10, 5 and 1 mug/ml. The first signs of thymol-induced damage were observed between 1 and 4 days post-incubation. The maximum protoscolicidal effect was found with thymol at 10 microg/ml, viability reduced to 53.5+/-11.9% after 12 days of incubation. At day 42, viability was 11.5+/-15.3% and, reached 0% after 80 days. Thymol at concentrations of 5 and 1 microg/ml provoked a later protoscolicidal effect. Results of viability tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of thymol against E. granulosus protoscoleces.

  7. Proteomics analysis of Echinococcus granulosus protoscolex stage.

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    Hidalgo, Christian; García, María Pía; Stoore, Caroll; Ramírez, Juan Pablo; Monteiro, Karina Mariante; Hellman, Ulf; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer; Galanti, Norbel; Landerer, Eduardo; Paredes, Rodolfo

    2016-03-15

    Echinococcus granulosus protoscolex proteins were separated using two-dimensional electrophoresis and then identified using mass spectrometry; we identified 61 proteins, 28 which are newly described of which 4 could be involved in hydatid cyst fertility molecular mechanisms.

  8. [In vitro cultivation of Echinococcus granulosus].

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    Bölükbaş, Cenk Soner; Doğanay, Ahmet

    2008-01-01

    Hydatidosis is a zoonotic disease that is caused by larva of Echinococcus species. It affects animals and humans and is very important from the aspect of health and economy. There have been many studies concerning the biology, physiology and biochemistry of Echinococcus granulosus which is responsible for hydatidosis in both humans and animals in Turkey. Frequently in vitro culture methods have been used in antigen production, vaccine and drug development. In this article, the in vitro culture of E. granulosus has been examined under various headings.

  9. In vitro effects of flubendazole on Echinococcus granulosus protoscoleces.

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    Elissondo, M; Dopchiz, M; Ceballos, L; Alvarez, L; Sánchez Bruni, S; Lanusse, C; Denegri, G

    2006-03-01

    The aim of the present work was to determine the in vitro protoscolicidal effect of flubendazole (FLBZ) against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with FLBZ at concentrations of 10, 5 and 1 microg/ml. The first signs of FLBZ-induced damage were observed 3 days post-incubation. A clear protoscolicidal effect, reducing the vitality of protoscoleces to 35.6+/-0.7%, was observed after 18 days of incubation. After 25 days of FLBZ incubation (5 microg/ml), the percentage of vital protoscoleces was 13.9+/-5.9%. Protoscolex mortality was 100% (10 and 1 microg/ml) and 0.7+/-0.7% (5 microg/ml) after FLBZ incubation for 30 days. Results of vitality tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of FLBZ against E. granulosus protoscoleces.

  10. In vitro effect of sodium arsenite on Echinococcus granulosus protoscoleces.

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    Xing, Guoqiang; Wang, Bo; Lei, Ying; Liu, Chunli; Wang, Zhuo; Shi, Hongjuan; Yang, Rentan; Qin, Wenjuan; Jiang, Yufeng; Lv, Hailong

    2016-06-01

    Cystic echinococcosis (CE) caused by the metacestodes of Echinococcus granulosus is an important cosmopolitan zoonosis. Surgery is the main treatment option for CE. Meanwhile, chemotherapy is used as an significant adjunct to surgery. However, the benzimidazole carbamate group and the existing scolicidal agents may not be as effective as hoped. In this study, we aimed to explore the in vitro effect of sodium arsenite (NaAsO2) on Echinococcus granulosus protoscoleces, the causative agents of CE. Protoscoleces of E. granulosus were incubated in vitro with 4, 8, 12, 16, and 20μM NaAsO2. Viability and changes in morphology were investigated by 0.1% eosin staining. The ultrastructural alterations were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Additionally, caspase-3 activity was measured by colorimetric assay. Obvious protoscolicidal effect was seen with NaAsO2 at concentrations of 16μM and 20μM. Protoscolex mortality was 83.24% (16μM) and 100% (20μM) after 6 days post-incubation. SEM showed that the primary site of drug damage was the tegument of the protoscoleces. TEM analysis demonstrated that the internal tissues were severely affected and revealed an increase in the number of lipid droplets and vacuoles after treatment with 16μM NaAsO2. Meanwhile, the caspase-3 activity significantly increased in protoscoleces after 24h of NaAsO2 incubation compared to the untreated controls. Our study demonstrated the clear in vitro scolicidal effect of NaAsO2 against E. granulosus protoscoleces. However, the in vivo efficacy, specific mechanism, and any possible side effects of NaAsO2 remain to be investigated.

  11. Detection of Echinococcus granulosus infection in dogs

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    Dalimi, A.

    2010-01-01

    Full Text Available Cystic Echinococcosis is an important zoonosis in the sheep rising areas of Iran. To develop a simple andreliable diagnostic method for Echinococcus infection in definitive hosts, E. granulosus polyoclonalantibodies (PolyAbs were prepared from adult worm in rabbit. A selected PoAb was used for coproantigendetection in faecal samples obtained from animals naturally infected with Echinococcus and compared withnecropsy method. Finally, the sensitivity and specificity of the test were evaluated. The results indicated E.granulosus worms was detected in 36 (43% of small intestine contents of dogs. The results obtained by CpAg- ELISA test showed 30 (36.14% positive and 53 (63.86% negative cases. The sensitivity andspecificity of CpAg-ELISA test were evaluated 83.33% and 100% respectively. In conclusion, the present result suggests that, CpAg-ELISA is a valid test for detection of E. granulosus infection in living dogs. Thus it is appropriate to apply for epidemiological study.

  12. Arsenic trioxide negatively affects Echinococcus granulosus.

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    Wang, Bo; Jiang, Yufeng; Wang, Zhuo; Li, Fangfang; Xing, Guoqiang; Peng, Xinyu; Zhang, Shijie; Lv, Hailong

    2015-11-01

    Spillage of cyst contents during surgery is the major cause of recurrences of hydatidosis, also called cystic echinococcosis (CE). Currently, many scolicidal agents are used for inactivation of the cyst contents. However, due to complications in the use of those agents, new and more-effective treatment options are urgently needed. The aim of this study was to investigate the in vitro efficacy of arsenic trioxide (ATO) against Echinococcus granulosus protoscolices. Protoscolices of E. granulosus were incubated in vitro with 2, 4, 6, and 8 μmol/liter ATO; viability of protoscolices was assessed daily by microscopic observation of movements and 0.1% eosin staining. A small sample from each culture was processed for scanning and transmission electron microscopy. ATO demonstrated a potent ability to kill protoscolices, suggesting that ATO may represent a new strategy in treating hydatid cyst echinococcosis. However, the in vivo efficacy and possible side effects of ATO need to be explored.

  13. Effect of praziquantel on adult Echinococcus granulosus in vitro: scanning electron microscopy.

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    Conder, G A; Marchiondo, A A; Andersen, F L

    1981-01-01

    The effect of praziquantel in vitro at concentrations of 5, 50 and 500 ppm for 1 h resulted in the progressive breakdown of the tegument and in morphologic distortion of adult Echinococcus granulosus when compared to controls. Scanning electron microscopy of all specimens treated in the various concentrations of praziquantel showed loss of most, if not all of the rostellar hooks and changes in the structure of the suckers. Many of the tapeworms immediately detached from the host's gut upon being placed in the drug, and all treated cestodes exhibited contraction or swelling, particularly in the penultimate proglottid. Intense contraction was apparent in the worms exposed to the higher drug concentrations. Characteristic conical microtriches on the terminal proglottid, as observed in the control specimens, became fused and matted when exposed to 5 ppm of praziquantel. At a drug concentration of 50 ppm, the tegumental surface developed grooves or furrows between clumps of fused microtriches, while 500 ppm caused production of holes within the denuded tegument of the parasite. Ovoid bodies, presumed to be eggs, were observed on the outer surfaces and just below the tegument of tapeworms treated with concentrations of 50 ppm. These structures also appeared to adhere to the outer surfaces of specimens exposed to 500 ppm. In view of the foregoing, special care should be taken in handling and disposing of feces from infected or suspect dogs after praziquantel treatment, since the breakdown in the tegumental surface of E. granulosus presumably results in the release of potentially infective eggs.

  14. Molecular identification of Echinococcus granulosus isolates from ruminants in Greece.

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    Roinioti, Erifylli; Papathanassopoulou, Aegli; Theodoropoulou, Ioanna; Simsek, Sami; Theodoropoulos, Georgios

    2016-08-15

    Cystic echinococcosis is a parasitic disease caused by Echinococcus granulosus, a cestode with worldwide distribution. Data on the circulating Echinococcus granulosus genotypes in Greek livestock is scant. The aim of the present study was to conduct a genetic analysis of 82 Echinococcus granulosus isolates from ruminants in Greece, including areas which until today have not been the subject of studies. The analysis relied on a PCR assay targeting cytochrome c oxidase, subunit 1 gene (CO1), followed by bidirectional sequence analysis of the amplification product. Eighty (n=80) of the 82 (97.6%) isolates were allocated to Echinococcus granulosus sensu stricto (G1-G3) and were classified in 13 distinct haplotypes (9 common and 4 novel) with 12 polymorphic sites. The presence of the dominant haplotype EG1 as was documented in the European populations, was indicated in the country. Almost all regions shared the same common haplotype. In comparison to this predominant haplotype, the number of the nucleotide changes in all the other haplotypes ranged from 1 to 5. All nucleotide changes proved to be transitions (A↔G or C↔T). Two fertile hydatid cysts of sheep origin in different areas (Arkadia, Ilia) of the Peloponnese were identified as Echinococcus canadensis (G7 genotype).

  15. The Larval Stage of Echinococcus Granulosus

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    Mohsen Sokouti

    2015-01-01

    Full Text Available Throughout the life cycle of Echinococcus granulosus, eggs of the parasite cannot mature into adult worms without first passing through the larval stage. Regarding the fact that this stage cannot take place in the definitive host, the eggs must look for an intermediate host, such as humans which are considered accidental intermediate hosts, in order to undergo their vital metamorphosis. In the upper gastrointestinal tract of the intermediate host, including humans (but not that of definitive host, the outer chitinous shells of the hexacanth embryos become lysed, enabling the embryos to penetrate the mucosa of the duodenum and upper jejunum to enter mesentric venule and be carried in the portal stream to the liver. Theoretically, a few of the embryos can enter the lymphatics of the intestinal wall and bypassing the liver through the cisterna chyli (1-3. It is believed that the larger amount of deoxycholic acid in the bile of herbivores and humans conjugated principally with glycine is responsible for lysis of the larva‘s protective center cuticle. On the other hand bile salts of carnivores such as dog are relatively poor in deoxy cholic acid which is linked with urine and have no effect on the cuticle of the larvae, which remain in the bowel lumen and developing into adult worms. Thus unlike what is mostly believed, humans do not serve as definitive hosts for the parasite; yet they carry only the larval forms which later penetrate into the villi of small bowel and form hydatid cyst in any organ of body (4-6.

  16. Development of a cell line from Echinococcus granulosus germinal layer.

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    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  17. Taxonomy and molecular epidemiology of Echinococcus granulosus sensu lato.

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    Romig, T; Ebi, D; Wassermann, M

    2015-10-30

    Echinococcus granulosus, formerly regarded as a single species with a high genotypic and phenotypic diversity, is now recognised as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. This diversity is reflected in the mitochondrial and nuclear genomes and has led to the construction of phylogenetic trees and hypotheses on the origin and geographic dispersal of various taxa. Based on phenotypic characters and gene sequences, E. granulosus (sensu lato) has by now been subdivided into E. granulosus sensu stricto (including the formerly identified genotypic variants G1-3), Echinococcus felidis (the former 'lion strain'), Echinococcus equinus (the 'horse strain', genotype G4), Echinococcus ortleppi (the 'cattle strain', genotype G5) and Echinococcus canadensis. The latter species, as recognised here, shows the highest diversity and is composed of the 'camel strain', genotype G6, the 'pig strain', genotype G7, and two 'cervid strains', genotypes G8 and G10. There is debate whether the closely related G6 and G7 should be placed in a separate species, but more morphological and biological data are needed to support or reject this view. In this classification, the application of rules for zoological nomenclature led to the resurrection of old species names, which had before been synonymised with E. granulosus. This nomenclatural subdivision of the agents of cystic echinococcosis (CE) may appear inconvenient for practical applications, especially because molecular tools are needed for identification of the cyst stage, and because retrospective data on 'E. granulosus' are now difficult to interpret without examination of voucher specimens. However, the increased awareness for the diversity of CE agents - now emphasised by species names rather than genotype numbers - has led to a large number of recent studies on this issue and a rapid increase of knowledge

  18. molecular characterisation of echinococcus granulosus species ...

    African Journals Online (AJOL)

    2013-07-01

    Jul 1, 2013 ... Hohenheim, 70599 Stuttgart, Germany, P. Kern, PhD, Centre for Internal Medicine, ... Conclusion: This study corroborates previous reports that E. canadensis G6/7 strain is ... E. granulosus s.l cyst material/protoscolices.

  19. Molecular characterization of a signal-regulated kinase homolog from Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    LI Jing; ZHANG Chuan-shan; L(U) Guo-dong; WANG Jun-hua; WEN Hao; YAN Gen-qiang; WEI Xu-fa; LIN Ren-yong

    2011-01-01

    Background Cystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs ofthe host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage.Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK)mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK.Methods DNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence.Results We cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices.Conclusions We have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and

  20. Microdiversity of Echinococcus granulosus sensu stricto in Australia.

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    Alvarez Rojas, C A; Ebi, D; Gauci, C G; Scheerlinck, J P; Wassermann, M; Jenkins, D J; Lightowlers, M W; Romig, T

    2016-07-01

    Echinococcus granulosus (sensu lato) is now recognized as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. One of these species, E. granulosus sensu stricto (s.s.), is now clearly identified as the principal agent causing cystic echinococcosis in humans. Previous studies of a small section of the cox1 and nadh1 genes identified two variants of E. granulosus s.s. to be present in Australia; however, no further work has been carried out to characterize the microdiversity of the parasite in its territory. We have analysed the sequence of the full length of the cox1 gene (1609 bp) from 37 isolates of E. granulosus from different hosts and geographic regions of Australia. The analysis shows that seven haplotypes of E. granulosus s.s. not previously described were found, together with five haplotypes known to be present in other parts of the world, including the haplotype EG01 which is widespread and present in all endemic regions. These data extend knowledge related to the geographical spread and host range of E. granulosus s.s. in a country such as Australia in which the parasite established around 200 years ago.

  1. In vitro effect of praziquantel and albendazole combination therapy on the larval stage of Echinococcus granulosus.

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    Urrea-París, M A; Moreno, M J; Casado, N; Rodriguez-Caabeiro, F

    2000-12-01

    Protoscolices of Echinococcus granulosus were incubated in vitro with praziquantel (PZ), albendazole (ABZ), or a combination of both (PZ + ABZ). PZ and ABZ displayed slower protoscolicidal activity when applied separately than when used in combination. Despite the low PZ + ABZ concentrations used, protoscolex viability dropped rapidly (within 15 days). At this time, cysts did not develop following their inoculation into mice. The ultrastructural changes induced in the protoscolices by PZ + ABZ were (a) the loss of sucker concavity, (b tegumental contraction of the soma region, (c) the formation of digitiform tegumental extensions, (d) destruction of the tegument, and (e) the degeneration of parenchyma cells as reflected by the presence of numerous lamellar bodies. The PZ + ABZ treatment was effective only against small cysts, which had collapsed at 10 days postinoculation (p.i.). This treatment caused the following alterations: (a) loss of cyst turgidity at 6 days p.i.; (b) separation of the laminated and germinal layers; (c) loss of microtriches; (d) the appearance of numerous lipid droplets in the inner region of the germinal layer, (e) vacuolation of the cyton cytoplasm; and (f) the formation of abundant autophagosomes, which finally led to loss of the integrity of the germinal layer.

  2. Mutation scan screening of Echinococcus granulosus isolates of Indian origin.

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    Bhattacharya, D; Pan, D; Bera, A K; Konar, A; Das, S K

    2008-08-01

    During the present investigation a total of forty Indian animal isolates were screened by single strand conformation polymorphism (SSCP) collected from sheep, goat, cattle and buffalo. The result of the study indicated that nuclear variants of Echinococcus granulosus were present in both small and large ruminants. SSCP phenotypes of AgB, intron of actin II and Hbx-2 have been deduced. Presence of nuclear variants due to mutation of E. granulosus has been discussed depending on hypotheses imparted earlier in literature. High polymophism of AgB demands further investigation because the gene is related with immune evasion and infectivity. This communication reports for the first time the comparative profile of Indian goat, sheep, cattle and buffalo isolates of E. granulosus complex.

  3. Comparison of two procedures for labelling the surface of the hydatid disease organism, Echinococcus granulosus, with /sup 125/I

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    McManus, D.P.; McLaren, D.J.; Clark, N.W.T.; Parkhouse, R.M.E.

    1987-03-01

    Living, intact protoscoleces of the British horse and sheep strains of Echinococcus granulosus were subjected to surface radioiodination procedures using /sup 125/I and Iodogen and /sup 125/I-Bolton Hunter reagent. Subsequent combined electron microscopy and autoradiography revealed specific surface membrane labelling with the Iodogen procedure, but significant tegumental labelling with the Bolton-Hunter reagent. The two parasite strains yielded different profiles of electrophoretically separated labelled proteins; the Iodogen method, not surprisingly, resulted in a less complex pattern of labelled polypeptides than the Bolton and Hunter reagent.

  4. Molecular characterization of Echinococcus granulosus sensu stricto and Echinococcus canadensis in humans and livestock from Algeria.

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    Zait, Houria; Kouidri, Mokhtaria; Grenouillet, Florence Elisabeth; Umhang, Gérald; Millon, Laurence; Hamrioui, Boussad; Grenouillet, Frédéric

    2016-06-01

    In Algeria, previous studies investigated genotypes of Echinococcus granulosus sensu lato in animals and identified E. granulosus sensu stricto (s.s.) genotypes G1 and G3 whereas Echinococcus canadensis genotype G6 was only reported from dromedary cysts. Molecular data on human cystic echinococcosis (CE) were limited. We implemented a large genotyping study of hydatid cysts from humans and livestock animals to specify CE's molecular epidemiology and the genetic diversity in Algeria. Fifty-four human CE cysts from patients predominantly admitted in surgical units from Mustapha Hospital, Algiers, and 16 cysts from livestock animals gathered in two geographically distinct slaughterhouses, Tiaret and Tamanrasset, were collected. Molecular characterization was performed using sequencing of two mitochondrial genes, cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit I (NDI). In humans, G1 of E. granulosus s.s. was the main genotype (90.7 %); four samples (7.4 %) were characterized as E. granulosus s.s. G3 and one cyst as E. canadensis G6 (1.8 %). This molecular confirmation of E. canadensis G6 human infection in Algeria was observed in a Tuareg female living in a desertic area in Tamanrasset. All cysts from sheep, cattle, and goat were identified as E. granulosus s.s. G1 and the two cysts originating from dromedary as E. canadensis G6. Twenty concatenated haplotypes (COI + NDI) were characterized. Among E. granulosus s.s., one haplotype (HL1) was highly predominant in both humans and animals cysts (71.6 %). This study revealed main occurrence of E. granulosus s.s. in humans and livestock animals, with description of a predominant shared haplotype corresponding to the main worldwide observed haplotype E.granulosus s.s. G1. E. canadensis G6 was limited to South Algeria, in dromedary as well as in human.

  5. Echinococcus equinus and Echinococcus granulosus sensu stricto from the United Kingdom: genetic diversity and haplotypic variation.

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    Boufana, Belgees; Lett, Wai San; Lahmar, Samia; Buishi, Imad; Bodell, Anthony J; Varcasia, Antonio; Casulli, Adriano; Beeching, Nicholas J; Campbell, Fiona; Terlizzo, Monica; McManus, Donald P; Craig, Philip S

    2015-02-01

    Cystic echinococcosis is endemic in Europe including the United Kingdom. However, information on the molecular epidemiology of Echinococcus spp. from the United Kingdom is limited. Echinococcus isolates from intermediate and definitive animal hosts as well as from human cystic echinococcosis cases were analysed to determine species and genotypes within these hosts. Echinococcus equinus was identified from horse hydatid isolates, cysts retrieved from captive UK mammals and copro-DNA of foxhounds and farm dogs. Echinococcus granulosus sensu stricto (s.s.) was identified from hydatid cysts of sheep and cattle as well as in DNA extracted from farm dog and foxhound faecal samples, and from four human cystic echinococcosis isolates, including the first known molecular confirmation of E. granulosus s.s. infection in a Welsh sheep farmer. Low genetic variability for E. equinus from various hosts and from different geographical locations was detected using the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), indicating the presence of a dominant haplotype (EQUK01). In contrast, greater haplotypic variation was observed for E. granulosus s.s. cox1 sequences. The haplotype network showed a star-shaped network with a centrally placed main haplotype (EgUK01) that had been reported from other world regions.

  6. In vitro metacestodicidal activities of genistein and other isoflavones against Echinococcus multilocularis and Echinococcus granulosus.

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    Naguleswaran, Arunasalam; Spicher, Martin; Vonlaufen, Nathalie; Ortega-Mora, Luis M; Torgerson, Paul; Gottstein, Bruno; Hemphill, Andrew

    2006-11-01

    Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.

  7. Echinococcus granulosus fatty acid binding proteins subcellular localization.

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    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes.

  8. Surveillance of Echinococcus granulosus in dogs with arecoline hydrobromide

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    Gemmell, M. A.

    1973-01-01

    Arecoline hydrobromide has been used for almost half a century for the treatment of Echinococcus granulosus in dogs. Trials in New Zealand showed that it had real limitations for this purpose. Its main value lies in its use as a diagnostic agent for detecting infections in dogs on a group basis. The data so obtained can be used in educating dog owners as well as for providing base-line data and an index of progress in a continuing control programme. The drug's limitations for treatment and value as a diagnostic agent in a field trial are assessed. PMID:4544776

  9. Echinococcus granulosus Prevalence in Dogs in Southwest Nigeria

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    Oyeduntan Adejoju Adediran

    2014-01-01

    Full Text Available Echinococcosis is a public health parasitic disease that is cosmopolitan (Echinococcus granulosus in its distribution. Domestic dogs (Canis familiaris have been recognised as the definitive host of the parasite. The present study was carried out to determine the prevalence of canine echinococcosis in Southwest Nigeria using direct enzyme linked immunosorbent assay (ELISA to detect sera antigen. Two hundred and seventy-three (273 canine sera were tested for the presence of Echinococcus antigen. Purpose of keeping (hunting or companion, age (young or adult, and sex of each dog were considered during sampling. Total prevalence recorded was 12.45% (34/273. There was significant difference (P0.05 between young and adult dogs. There was no association between sex and prevalence of canine echinococcosis. The result of this study established the presence of canine echinococcosis in Southwest Nigeria; thus there is the possibility of occurrence of zoonotic form of the disease (human cystic hydatid diseases in the region.

  10. Echinococcus canadensis (G7) and Echinococcus granulosus sensu stricto (G1) in swine of southern Brazil.

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    Monteiro, D U; Botton, S A; Tonin, A A; Azevedo, M I; Graichen, D A S; Noal, C B; de la Rue, M L

    2014-05-28

    The cystic echinococcosis (CE) is an important zoonotic disease caused by the parasite Echinococcus spp. In Brazil, this parasite is present in Rio Grande do Sul (RS) state, border with Argentina and Uruguay, causing several damages to human and animal health. This study aimed to identify Echinococcus spp. in hydatid cysts of swine and evaluate the similarity of the genotypes through the phylogenetic analysis. A total of 3,101,992 swine were slaughtered in the central/northern region of RS/Brazil, during 2008-2012. Five isolates were characterized as hydatid cyst by molecular analysis, based on the mitochondrial gene cytochrome c oxidase subunit I (cox-I). The genotypes E. granulosus sensu stricto (G1) (n=2) and E. canadensis (G7) (n=3) were identified in the hydatid cysts. The swine represents a potential intermediate host for different genotypes of Echinococcus spp., besides it can contribute to the perpetuation of the parasite's life cycle in rural areas.

  11. Anticestodal Activity of Endophytic Pestalotiopsis sp. on Protoscoleces of Hydatid Cyst Echinococcus granulosus

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    Vijay C. Verma

    2013-01-01

    Full Text Available Surgery is still the main treatment in hydatidosis caused by Echinococcus, which is a global health problem in human and animals. So, there is need for some natural protoscolicidal agents for instillation to prevent their reoccurrence at therapeutic doses. In this present investigation, anticestodal activity of one of the endophytic fungi Pestalotiopsis sp. from Neem plant was observed on protoscoleces of hydatid cysts of Echinococcus granulosus. Viability of protoscoleces was confirmed by 0.1% aqueous eosin red stain method, where mortality was observed at different concentrations with respect to time. An average anticestodal activity was observed with different endophytic fungal strains, that is, Nigrospora (479 ± 2.9, Colletotrichum (469 ± 25.8, Fusarium (355 ± 14.5, and Chaetomium (332 ± 28.3 showing 64 to 70% protoscolicidal activity, except Pestalotiopsis sp. (581 ± 15.0, which showed promising scolicidal activity up to 97% mortality just within 30 min of incubation. These species showed significant reduction in viability of protoscoleces. This is the first report on the scolicidal activity of endophytic Pestalotiopsis sp. We conclude that ultrastructural changes in protoscoleces were due to endophytic extract suggesting that there may be some bioactive compounds that have selective action on the tegument layer of protoscoleces. As compared with that of standard drug used, endophytic species of Neem plant shows significant anticestodal activity.

  12. Genetic diversity of Echinococcus granulosus in center of Iran.

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    Pestechian, Nader; Hosseini Safa, Ahmad; Tajedini, Mohammadhasan; Rostami-Nejad, Mohammad; Mousavi, Mohammad; Yousofi, Hosseinali; Haghjooy Javanmard, Shaghayegh

    2014-08-01

    Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.

  13. Gene cloning, expression, and localization of antigen 5 in the life cycle of Echinococcus granulosus.

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    Li, Yuzhe; Xu, Hongxu; Chen, Jiajia; Gan, Wenjia; Wu, Weihua; Wu, Weiping; Hu, Xuchu

    2012-06-01

    Antigen 5 (Ag5) has been identified as a dominant component of cyst fluid of Echinococcus granulosus and is considered as a member of serine proteases family, which in other helminth, plays an important role in the egg hatch and larva invasion. However, whether Ag5 is expressed and secreted in all life stages is unknown. In this study, according to the sequence in GenBank, we cloned and sequenced the open reading frame (ORF) of Ag5 gene from the protoscolices of E. granulosus isolated from the sheep in Qinhai Province of China, and found several substitutions and a base insert and deletion in a short region near the stop code, leading to a frameshift mutation which is conserved with the homologue of other cestode. The ORF is 1,455 bp in length, encoding 484 amino acids with a secretory signal peptide. Bioinformatics analysis predicted several phosphorylation and myristoylation sites and a N-glycosylation site and a species-specific linear B epitope in the protein. The ORF was cloned into the plasmid pET28a(+) vector and expressed in Escherichia coli . The recombinant protein was purified by affinity chromatography. Anti-rEgAg5 antiserum was prepared in rats and used to analyze the localization of Ag5 in protoscolex and adult worm by immunofluorescence technique. Results demonstrated that the Ag5 is strongly expressed in the tegument of protoscolex and the embryonic membrane of egg and surface of oncosphere; meanwhile, it is also weakly expressed in tegument of the adult. This study showed that Ag5 is expressed in all stages of life cycle, secreted from the surface of the worm and may be anchored in membrane by its myristoylation sites; these characteristics make it a candidate antigen for diagnosis and vaccine for both intermediate and definitive hosts.

  14. Characterization of a Secretory Annexin in Echinococcus granulosus.

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    Song, Xingju; Hu, Dandan; Zhong, Xiuqin; Wang, Ning; Gu, Xiaobin; Wang, Tao; Peng, Xuerong; Yang, Guangyou

    2016-03-01

    Cystic echinococcosis, caused by Echinococcus granulosus, is a widespread parasitic zoonosis causing economic loss and public health problems. Annexins are important proteins usually present in the plasma membrane, but previous studies have shown that an annexin B33 protein of E. granulosus (Eg-ANX) could be detected in the excretory/secretory products and cyst fluid. In this study, we cloned and characterized Eg-ANX. In silico analysis showed that the amino acid sequence of Eg-ANX was conserved and lacked any signal peptides. The phospholipid-binding activity of recombinant Eg-ANX (rEg-ANX) was tested; liposomes could bind to rEg-ANX only in the presence of Ca(2+). In addition, we performed western blotting and immunohistochemical analyses to further validate the secretory properties of Eg-ANX. The protein could be detected in the cyst fluid of E. granulosus and was also present in the intermediate host tissues, which suggested that Eg-ANX might play an important role in parasite-host interaction.

  15. Different protein of Echinococcus granulosus stimulates dendritic induced immune response.

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    Wang, Yana; Wang, Qiang; Lv, Shiyu; Zhang, Shengxiang

    2015-06-01

    Cystic echinococcosis is a chronic infectious disease that results from a host/parasite interaction. Vaccination with ferritin derived from Echinococcus granulosus is a potential preventative treatment. To understand whether ferritin is capable of inducing a host immune response, we investigated the response of dendritic cells (DCs) to both recombinant ferritin protein and the hydatid fluid (HF) of E. granulosus. We evaluated the immunomodulatory potential of these antigens by performing, immunocytochemistry, electron microscopy and in vivo imaging of monocyte-derived murine DCs. During antigen stimulation of DCs, ferritin cause DCs maturation and induced higher levels of surface marker expression and activated T-cell proliferation and migration. On contrary, HF failed to induce surface marker expression and to stimulate T-cell proliferation. In response to HF, DCs produced interleukin-6 (IL-6), but no IL-12 and IL-10. DCs stimulated with ferritin produced high levels of cytokines. Overall, HF appears to induce host immunosuppression in order to ensure parasite survival via inhibits DC maturation and promotes Th2-dependent secretion of cytokines. Although ferritin also promoted DC maturation and cytokine release, it also activates CD4+T-cell proliferation, but regard of the mechanism of the Eg.ferritin induce host to eradicate E. granulosus were not clear.

  16. Mucin-like peptides from Echinococcus granulosus induce antitumor activity.

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    Noya, Verónica; Bay, Sylvie; Festari, María Florencia; García, Enrique P; Rodriguez, Ernesto; Chiale, Carolina; Ganneau, Christelle; Baleux, Françoise; Astrada, Soledad; Bollati-Fogolín, Mariela; Osinaga, Eduardo; Freire, Teresa

    2013-09-01

    There is substantial evidence suggesting that certain parasites can have antitumor properties. We evaluated mucin peptides derived from the helminth Echinococcus granulosus (denominated Egmuc) as potential inducers of antitumor activity. We present data showing that Egmuc peptides were capable of inducing an increase of activated NK cells in the spleen of immunized mice, a fact that was correlated with the capacity of splenocytes to mediate killing of tumor cells. We demonstrated that Egmuc peptides enhance LPS-induced maturation of dendritic cells in vitro by increasing the production of IL-12p40p70 and IL-6 and that Egmuc-treated DCs may activate NK cells, as judged by an increased expression of CD69. This evidence may contribute to the design of tumor vaccines and open new horizons in the use of parasite-derived molecules in the fight against cancer.

  17. In vitro effects of SB202190 on Echinococcus granulosus.

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    Lv, Hailong; Li, Siyuan; Zhang, Jing; Liang, Weihua; Mu, Xiaoling; Jiang, Yufeng

    2013-04-01

    Spillage of cyst contents during surgical operation is the major cause of recurrence after hydatid cyst surgery. Instillation of a scolicidal agent into a hepatic hydatid cyst is the most commonly employed measure to prevent this complication. SB202190 is a pyridinyl imidazole derivative and is known to be a specific inhibitor of p38 MAPK. In the present study, the scolicidal effect of SB202190 was investigated. Freshly isolated Echinococcus granulosus protoscolices were subjected to SB202190 treatment (10, 20, 40, and 80 µM), and the effects on parasite viability were monitored by trypan blue staining. Corresponding effects were visualized by scanning and transmission electron microscopy. Dose-dependent protoscolex death within a few days of SB202190 treatment was observed. Although the in vitro scolicidal effect of SB202190 was satisfactory, the in vivo efficacy of this drug and also possible side effects remain to be further investigated.

  18. Fructose-bisphosphate aldolase and enolase from Echinococcus granulosus: genes, expression patterns and protein interactions of two potential moonlighting proteins.

    Science.gov (United States)

    Lorenzatto, Karina Rodrigues; Monteiro, Karina Mariante; Paredes, Rodolfo; Paludo, Gabriela Prado; da Fonsêca, Marbella Maria; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer

    2012-09-10

    Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon-intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory-secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host-parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host-parasite interplay.

  19. Flubendazole and ivermectin in vitro combination therapy produces a marked effect on Echinococcus granulosus protoscoleces and metacestodes.

    Science.gov (United States)

    Elissondo, M Celina; Ceballos, L; Alvarez, L; Sánchez Bruni, S; Lanusse, C; Denegri, G

    2009-09-01

    The aim of the present work was to evaluate the in vitro efficacy of the flubendazole (FLBZ) and ivermectin (IVM) combination against Echinococcus granulosus protoscoleces and metacestodes. Protoscoleces and groups of ten peritoneal cysts obtained from BALB/c mice were incubated with the two drugs, either separately or in combination, at the following final concentrations: 10 microg/mL FLBZ, 1 microg/mL FLBZ, 1 microg/mL IVM, 10 microg/mL FLBZ + 1 microg/mL IVM, and 1 microg/mL FLBZ + 1 microg/mL IVM. The maximum protoscolicidal effect was found with the combination 10 microg/mL FLBZ + 1 microg/mL IMV. After 1 day of incubation, the presence of numerous blebs in the tegument of protoscoleces was observed. Ultrastructural studies revealed that the primary site of damage was the tegument of the parasite. The effect of the two drugs on hydatid cysts obtained from mice was more rapidly detected in cysts treated with the combination of FLBZ + IVM than when drugs were used separately. Ultrastructural studies revealed that the germinal layer of treated cysts lost the multicellular structure feature and underwent considerable degenerative changes after in vitro treatment. The outcomes obtained demonstrated the favorable effect of the combination of FLBZ and IVM against E. granulosus.

  20. First molecular evidence of the simultaneous human infection with two species of Echinococcus granulosus sensu lato: Echinococcus granulosus sensu stricto and Echinococcus canadensis.

    Science.gov (United States)

    Oudni-M'rad, Myriam; M'rad, Selim; Ksia, Amine; Lamiri, Rachida; Mekki, Mongi; Nouri, Abdellatif; Mezhoud, Habib; Babba, Hamouda

    2016-03-01

    Cystic echinococcosis is a widespread zoonotic parasitic disease especially in Tunisia which is one of the most endemic countries in the Mediterranean area. The etiological agent, Echinococcus granulosus sensu lato, implies dogs and other canids as definitive hosts and different herbivore species as intermediate hosts. Human contamination occurs during the consumption of parasite eggs passed in the environment through canid feces. Hydatid cysts coming from a child operated for multiple echinococcosis were collected and analyzed in order to genotype and to obtain some epidemiological molecular information. Three targets, ribosomal DNA ITS1 fragment, NADH dehydrogenase subunit 1 (nad1), and mitochondrial cytochrome c oxydase subunit 1 (CO1) genes, were amplified and analyzed by RFLP and sequencing approach. This study presents the first worldwide report in human of a simultaneous infection with Echinococcus granulosus sensu stricto (genotype G1) and Echinococcus canadensis (genotype G6) species. This is also the first report of the presence of E. canadensis in the Tunisian population which argues in favor of a greater importance of this species in human infestation in Tunisia than previously believed.

  1. Natural infection of the ground squirrel (Spermophilus spp. with Echinococcus granulosus in China.

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    Yu Rong Yang

    Full Text Available BACKGROUND: Echinococcus granulosus is usually transmitted between canid definitive hosts and ungulate intermediate hosts. METHODOLOGY/PRINCIPAL FINDINGS: Lesions found in the livers of ground squirrels, Spermophilus dauricus/alashanicus, trapped in Ningxia Hui Autonomous Region, an area in China co-endemic for both E. granulosus and E. multilocularis, were subjected to molecular genotyping for Echinococcus spp. DNA. One of the lesions was shown to be caused by E. granulosus and subsequently by histology to contain viable protoscoleces. CONCLUSIONS/SIGNIFICANCE: This is the first report of a natural infection of the ground squirrel with E. granulosus. This does not provide definitive proof of a cycle involving ground squirrels and dogs or foxes, but it is clear that there is active E. granulosus transmission occurring in this area, despite a recent past decline in the dog population in southern Ningxia.

  2. Identification and characterization of functional Smad8 and Smad4 homologues from Echinococcus granulosus.

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    Zhang, Chuanshan; Wang, Limin; Wang, Hui; Pu, Hongwei; Yang, Le; Li, Jing; Wang, Junhua; Lü, Guodong; Lu, Xiaomei; Zhang, Wenbao; Vuitton, Dominique A; Wen, Hao; Lin, Renyong

    2014-10-01

    Smad family proteins are essential cellular mediators of the transforming growth factor-β superfamily. In the present study, we identified two members of the Smad proteins, Smad8 and Smad4 homologues (termed as EgSmadE and EgSmadD, respectively), from Echinococcus granulosus, the causative agent of cystic echinococcosis (CE). Phylogenetic analysis placed EgSmadE in the Smad1, 5, and 8 subgroup of the R-Smad sub-family and EgSmadD in the Co-Smad family. Furthermore, EgSmadE and EgSmadD attained a high homology to EmSmadE and EmSmadD of E. multilocularis, respectively. Both EgSmadE and EgSmadD were co-expressed in the larval stages and exhibited the highest transcript levels in activated protoscoleces, and their encoded proteins were co-localized in the sub-tegumental and tegumental layer of the parasite. As shown by yeast two-hybrid and pull-down analysis, EgSmadE displayed a positive binding interaction with EgSmadD. In addition, EgSmadE localized in the nuclei of Mv1Lu cells (mink lung epithelial cells) upon treatment with human TGF-β1 or human BMP2, indicating that EgSmadE is capable of being translocated into nucleus, in vitro. Our study suggests that EgSmadE and EgSmadD may take part in critical biological processes, including echinococcal growth, development, and parasite-host interaction.

  3. P-glycoprotein expression and pharmacological modulation in larval stages of Echinococcus granulosus.

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    Nicolao, María Celeste; Denegri, Guillermo M; Cárcamo, Juan Guillermo; Cumino, Andrea C

    2014-02-01

    P-glycoprotein (Pgp) is an ATP-dependent transporter involved in the efflux of a wide variety of lipophilic substrates, such as toxins and xenobiotics, out of cells. Pgp expression level is associated with the ineffective therapeutic treatment of cancer cells and microbial pathogens which gives it high clinical importance. Research on these transporters in helminths is limited. This work describes for the first time the Echinococcus granulosus Pgp (Eg-Pgp) expression, in a model cestode parasite and an important human pathogen. Based on calcein efflux assays in the presence of common Pgp modulators, we demonstrated the occurrence of active Eg-Pgp in protoscoleces and metacestodes. Eg-Pgp, which showed a molecular mass of ~130 kDa in western blots, is localized in the suckers and the tegument of control protoscoleces as well as in the subtegument or all parenchymatous cells of protoscoleces treated with Pgp-interfering agents. We also identified five genes encoding Pgp which are constitutively expressed in protoscoleces and metacestodes. We showed that the Eg-pgp1 and Eg-pgp2 transcripts were up-regulated in response to in vitro drug treatment with amiodarone and loperamide, in agreement with the increased polypeptide levels. Finally, in vitro treatment of protoscoleces and metacestodes with trifluoperazine and loperamide was lethal to the parasites. This indicates that both drugs as well as cyclosporine A negatively modulate the E. granulosus Pgp efflux activity, favoring the retention of these drugs in the larval tissue. These events could be associated with the reduction in protoscolex and metacestode viability.

  4. Biochemical and molecular characterization of the calcineurin in Echinococcus granulosus larval stages.

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    Nicolao, María Celeste; Cumino, Andrea C

    2015-06-01

    Calcineurin (CaN) is a Ca(2+)-calmodulin activated serine-threonine protein phosphatase that couples the local or global calcium signals, thus controlling important cellular functions in physiological and developmental processes. The aim of this study was to characterize CaN in Echinococcus granulosus (Eg-CaN), a human cestode parasite of clinical importance, both functionally and molecularly. We found that the catalytic subunit isoforms have predicted sequences of 613 and 557 amino acids and are substantially similar to those of the human counterpart, except for the C-terminal end. We also found that the regulatory subunit consists of 169 amino acids which are 87% identical to the human ortholog. We cloned a cDNA encoding for one of the two catalytic subunit isoforms of CaN (Eg-can-A1) as well as the only copy of the Eg-can-B gene, both constitutively transcribed in all Echinococcus larval stages and responsible for generating a functionally active heterodimer. Eg-CaN native enzyme has phosphatase activity, which is enhanced by Ca(2+)/Ni(2+) and reduced by cyclosporine A and Ca(2+) chelators. Participation of Eg-CaN in exocytosis was demonstrated using the FM4-64 probe and Eg-CaN-A was immunolocalized in the cytoplasm of tegumental cells, suckers and excretory bladder of protoscoleces. We also showed that the Eg-can-B transcripts were down-regulated in response to low Ca(2+) intracellular level, in agreement with decreased enzyme activity. Confocal microscopy revealed a striking pattern of Eg-CaN-A in discrete fluorescent spots in the protoscolex posterior bladder and vesicularized protoscoleces beginning the vesicular differentiation. In contrast, Eg-CaN-A was undetectable during the pre-microcyst closing stage while a high DDX-like RNA helicase expression was evidenced. Finally, we identified and analyzed the expression of CaN-related endogenous regulators.

  5. A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

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    Ghalia Boubaker

    Full Text Available Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10 and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3, E. equinus (G4, E. ortleppi (G5, and E. canadensis (G6-G10. The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR allowing three levels of discrimination: (i Echinococcus genus, (ii E. granulosus complex in common, and (iii the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20 and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13. The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%. Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

  6. A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

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    Boubaker, Ghalia; Macchiaroli, Natalia; Prada, Laura; Cucher, Marcela A; Rosenzvit, Mara C; Ziadinov, Iskender; Deplazes, Peter; Saarma, Urmas; Babba, Hamouda; Gottstein, Bruno; Spiliotis, Markus

    2013-01-01

    Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (Echinococcus genus.

  7. Echinococcus granulosus infection dynamics in livestock of Greece.

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    Chaligiannis, I; Maillard, S; Boubaker, G; Spiliotis, M; Saratsis, A; Gottstein, B; Sotiraki, S

    2015-10-01

    An epidemiological and molecular survey on the occurrence of Echinococcus hydatid cysts in livestock was conducted in Greece. In total 898 sheep, 483 goats, 38 buffaloes, 273 wild boars and 15 deer were examined and 30.2% (6.45% cyst fertility), 7.86% (3.2% cyst fertility), 42% (7.9% cyst fertility), 1.1% (0% cyst fertility), 0% of them were found infected, respectively. Infection rate in different geographical regions varied between 26.1 and 53.8% (cyst fertility 2.04 and 34.6%) in sheep, 7.33 and 13.3% (cyst fertility 0 and 3.2%) in goats. Genotyping, based on cox1 and nad1 analyses, demonstrated the predominance of E. granulosus s.s. (G1 genotype). The presence of one single genotype-complex within a relatively large spectrum of intermediate host species in Greece indicates the presence of a dominant transmission dog-sheep cycle involving additional host species which may act as disease reservoir for human infections.

  8. Echinococcus granulosus: praziquantel treatment against the metacestode stage.

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    Urrea-París, M A; Moreno, M J; Casado, N; Rodriguez-Caabeiro, F

    1999-12-01

    The efficacy of praziquantel against the metacestode of Echinococcus granulosus was studied by means of in vitro incubations or in vivo experiments. The results of in vitro incubations indicated that the effectiveness of praziquantel was higher when the parasite material comprised cysts from cyst masses than in the case of intact cysts that retained their adventitial layer. Ultrastructural alterations in the germinal layer of collapsed cysts incubated in vitro were detected. The results obtained in mice after 4 months of treatment demonstrated no significant difference between the control and treated groups with regard to the number and wet weight of developed cysts. However, ultrastructural alterations were detected in the cyst tissue that were similar to those described in the in vitro experiment. In contrast, the effect of chemoprophylaxis on the number and the wet weight of developed cysts was extremely significant as compared with the control value, the efficacy being 99.41% and 98.32%, respectively. Moreover, ultrastructural observations of the cyst tissue revealed loss of its integrity, and no intact cyton was observed in the germinal layer of the developed cyst.

  9. Echinococcus granulosus: protoscolicidal effect of high intensity focused ultrasound.

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    Zou, Xiaoyi; Wang, Junan; Zhao, Hailong; Zhang, Jing; Wu, Weihua; Ye, Bin

    2009-04-01

    High intensity focused ultrasound (HIFU) is a new non-invasive technique which can cause cell death and tissue necrosis by focusing high-energy ultrasonic waves on a single location. The aim of our work is to investigate the damaging effect of HIFU on Echinococcus granulosus protoscolices, as well as its inhibitory effect on growth of hydatid cysts derived from protoscolices. The damaging effect of HIFU on protoscolices was investigated by following parasite mortality after irradiation, while the inhibitory effect was investigated by infection experiments in vivo. The results demonstrated that HIFU was able to damage protoscolices and the protoscolicidal effect was dose-dependent and showed late-onset. The growth of protoscolices that survived the exposure to HIFU was obviously suppressed in vitro, and the mean weight of hydatid cysts resulting from such protoscolices in the experimental group was less than that in controls. Evidences including the protoscolicidal effect, fragmentized protoscolices and low post exposure temperatures, suggest that cavitation may contribute to the protoscolicidal effect of HIFU. In addition, the structure of the germinal membrane in cysts developing from the irradiated protoscolices was not as normal or intact as that from non-irradiated ones, and morphological changes related to degeneration were observed, suggesting that HIFU could prevent protoscolices from developing normal germinal membrane and consequently stop the proliferation of secondary hydatid cysts. HIFU demonstrated damaging effect on protoscolices, inhibited the growth of protoscolices in vitro and in vivo, and could be a possible therapeutic option for cystic echinococcosis.

  10. A novel zoonotic genotype related to Echinococcus granulosus sensu stricto from southern Ethiopia.

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    Wassermann, Marion; Woldeyes, Daniel; Gerbi, Banchwosen Mechal; Ebi, Dennis; Zeyhle, Eberhard; Mackenstedt, Ute; Petros, Beyene; Tilahun, Getachew; Kern, Peter; Romig, Thomas

    2016-09-01

    Complete mitochondrial and two nuclear gene sequences of a novel genotype (GOmo) related to Echinococcus granulosus sensu stricto are described from a metacestode isolate retrieved from a human patient in southwestern Ethiopia. Phylogenetically, the genotype is positioned within the E. granulosus sensu stricto/Echinococcus felidis cluster, but cannot easily be allocated to either species. Based on different mitochondrial DNA markers, it is closest to the haplotype cluster that currently defines the species E. granulosus sensu stricto (which includes variants showing the widely cited G1, G2 and G3 sequences), but is clearly not part of this cluster. Pairwise distances between GOmo and E. granulosus sensu stricto are in the range of those between the most distant members of the Echinococcus canadensis complex (G6-10) that were recently proposed as separate species. At this stage, we prefer to list GOmo informally as a genotype rather than giving it any taxonomic rank because our knowledge rests on a single isolate from a dead-end host (human), and its lifecycle is unknown. According to data on molecularly characterised Echinococcus isolates from this region, GOmo has never been found in the usual livestock species that carry cystic echinococcosis and the possibility of a wildlife source of this newly recognised zoonotic agent cannot be excluded. The discovery of GOmo adds complexity to the already diverse array of cystic echinococcosis agents in sub-Saharan Africa and challenges hypotheses on the biogeographical origin of the E. granulosus sensu stricto clade.

  11. Efficacy of osthole for Echinococcus granulosus in vitro and Echinococcus multilocularis in vivo.

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    Yuan, Miaomiao; Luo, Yanping; Xin, Qi; Gao, Haijun; Zhang, Guochao; Jing, Tao

    2016-08-15

    Echinococcosis is a zoonotic infection caused by cestode species of the genus Echinococcus; in addition, this zoonosis has long been neglected as a parasitic disease and has limited treatment options. Clinical drugs such as benzimidazole derivatives have limited treatment efficacy. The current study evaluated a novel drug, osthole, with low toxicity and high activity against Echinococcus in vitro and in vivo. The results in vitro indicated that the viability of Echinococcus granulosus protoscoleces in the group treated with osthole (120μM) decreased by 100% within 3days. In vivo experiments were conducted using parasite-infected mice. For this purpose, three groups of infected mice were treated daily for 6 weeks with albendazole (ABZ, 100mg/kg, positive control group), osthole (100mg/kg, experimental group), or honey/PBS (100mg/kg, negative control group), respectively. The osthole- and ABZ-treated groups presented a significant reduction in wet weight of metacestodes, increase in the level of interleukin (IL)-4 and the percentage of eosinophils compared with the control group. Osthole exhibited a high activity against echinococcosis in vivo. In addition, the toxicity of osthole was evaluated via an in vitro 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, as well as via morphological observation and calculation of liver and kidney function indexes in vivo. No obvious toxic effects of osthole were observed in our study. Therefore, this novel drug may be a promising alternative to benzimidazole in anti-echinococcosis chemotherapy.

  12. Comparative analysis of the 14-3-3 gene and its expression in Echinococcus granulosus and Echinococcus multilocularis metacestodes.

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    Siles-Lucas, M; Nunes, C P; Zaha, A

    2001-03-01

    It was suggested that the unlimited proliferative capacity of the Echinococcus multilocularis metacestode may be related to overproduction of the 14-3-3 protein. As is known, the proliferative capacities of E. granulosus and E. multilocularis metacestodes are very different. By comparing the expression levels of the 14-3-3 gene between in vitro-obtained E. granulosus and E. multilocularis metacestodes, we were able to provide experimental evidence of the potential relation between 14-3-3 over-expression and tumour-like growth in E. multilocularis metacestodes. RT-PCR and Northern blot experiments indicated that 14-3-3 expression level is about 4-fold higher in the E. multilocularis metacestode. This differential expression was confirmed both by immunoblotting and immunocytochemistry experiments, which allowed detection of the protein in the cyst wall from E. multilocularis but not in the cyst wall from E. granulosus. The alignment of the Echinococcus 14-3-3 cDNA sequence with known 14-3-3 isoforms from other organisms, grouped the parasite sequence into the tumour growth-related isoforms. The known relation between over-expression of some 14-3-3 isoforms and tumour-related processes, together with the present results, suggest that the Echinococcus 14-3-3 protein could be one of the molecules responsible for the differences between E. granulosus and E. multilocularis metacestode growth behaviour.

  13. Echinococcus granulosus sensu stricto (s.s.) from the critically endangered antelope Addax nasomaculatus in Tunisia.

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    Boufana, Belgees; Saïd, Yousra; Dhibi, Mokhtar; Craig, Philip S; Lahmar, Samia

    2015-12-01

    Echinococcus granulosus sensu lato (s.l.) is a zoonotic disease highly endemic in Tunisia. Canids including stray and semi-stray dogs, jackals and foxes are known as definitive hosts and a wide range of ungulates have been shown to harbour the metacestode hydatid stage and may serve as intermediate hosts. Fertile hydatid cysts of Echinococcus equinus and E. granulosus sensu stricto (s.s.) were recently molecularly identified for the first time from Tunisian donkeys. E. granulosus (s.s.) was also identified from wild boars in Tunisia. Here we report the confirmation of hydatid cysts caused by E. granulosus (s.s.) in the critically endangered antelope, Addax nasomaculatus in Tunisia. DNA-based molecular analysis revealed that A.nasomaculatus was infected with E. granulosus (s.s.) which had a 100% identity with the main globally distributed E. granulosus (s.s.) (EgTu01) haplotype. Cysts of Taenia hydatigena (n=33) were also observed on the liver and in the body cavity. Due to their endangered status and their relatively small numbers, it is unlikely that hydatid infection of A. nasomaculatus will form a major contribution to the epidemiology and transmission of E. granulosus in Tunisia, but infection may result in pathology, morbidity and early mortality, and may still play a role in the perpetuation of the parasite in wildlife cycles.

  14. Evidence that the Echinococcus granulosus G6 genotype has an affinity for the brain in humans.

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    Sadjjadi, S M; Mikaeili, F; Karamian, M; Maraghi, S; Sadjjadi, F S; Shariat-Torbaghan, S; Kia, E B

    2013-10-01

    The present study investigates the molecular characteristics of cerebral Echinococcus cysts. A total of 10 specimens of cerebral Echinococcus cysts, including six formalin-fixed paraffin blocks and four intact cerebral cysts, were used for this study. The target DNA was successfully amplified from eight samples and sequenced. BLAST analysis indicated that sequenced isolates belong to the Echinococcus granulosus (G6) genotype. All of the eight sampled brain cysts belonged to the G6 genotype, while all of the eight liver cysts belonged to G1. This is a strong indication that G6 has a higher affinity for the human brain than G1.

  15. Scolicidal activity of biosynthesized silver nanoparticles against Echinococcus granulosus protoscolices.

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    Rahimi, Mohammad Taghi; Ahmadpour, Ehsan; Rahimi Esboei, Bahman; Spotin, Adel; Kohansal Koshki, Mohammad Hasan; Alizadeh, Ahad; Honary, Soheila; Barabadi, Hamed; Ali Mohammadi, Milad

    2015-07-01

    Cystic hydatid disease (CHD), a helminth infection with various clinical complications caused by the larval stage of the dog tapeworm Echinococcus granulosus is considered as a public health problem in different regions of the world. To date, scolicidal agents have been broadly applied for inactivation of the fertile cysts and these scolicidal agents have several side effects on patients. Therefore, the objective of this study was to evaluate the scolicidal efficacies of synthesized silver nanoparticles (AgNPs) derived from the aqueous aerial extract of Penicillium aculeatum against protoscolices of CHD in-vitro. Protoscolices were aseptically aspirated from infected livers of sheep with CHD. Various concentrations (0.025, 0.05, 0.1 and 0.15 mg/mL) of green synthesis of Ag-NPs and different exposure times (10, 30, 60 and 120 min) were used against protoscolices of CHD. Viability of protoscolices was confirmed by 0.1% eosin staining. The findings showed that the Ag-NPs at all concentrations have high scolicidal effects. The concentrations 0.1 and 0.15 mg/mL after 120 min of exposure times showed 83% and 90% mortality rate, respectively. The least scolicidal activity of biosynthesized Ag-NPs was 40% (0.025 mg/mL and 10 min). The current investigation indicated that applying biogenic Ag-NPs may be considered as a potential scolicidal agent for CHD surgery due to being economical, safer and non-toxic compared to the used chemical materials. However, further studies are required to evaluate the efficacy of Ag-NPs in vivo.

  16. Direct interaction between EgFABP1, a fatty acid binding protein from Echinococcus granulosus, and phospholipid membranes.

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    Jorge L Porfido

    Full Text Available BACKGROUND: Growth and maintenance of hydatid cysts produced by Echinococcus granulosus have a high requirement for host lipids for biosynthetic processes, membrane building and possibly cellular and developmental signalling. This requires a high degree of lipid trafficking facilitated by lipid transporter proteins. Members of the fatty acid binding protein (FABP family have been identified in Echinococcus granulosus, one of which, EgFABP1 is expressed at the tegumental level in the protoscoleces, but it has also been described in both hydatid cyst fluid and secretions of protoscoleces. In spite of a considerable amount of structural and biophysical information on the FABPs in general, their specific functions remain mysterious. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the way in which EgFABP1 may interact with membranes using a variety of fluorescence-based techniques and artificial small unilamellar vesicles. We first found that bacterial recombinant EgFABP1 is loaded with fatty acids from the synthesising bacteria, and that fatty acid binding increases its resistance to proteinases, possibly due to subtle conformational changes induced on EgFABP1. By manipulating the composition of lipid vesicles and the ionic environment, we found that EgFABP1 interacts with membranes in a direct contact, collisional, manner to exchange ligand, involving both ionic and hydrophobic interactions. Moreover, we observed that the protein can compete with cytochrome c for association with the surface of small unilamellar vesicles (SUVs. CONCLUSIONS/SIGNIFICANCE: This work constitutes a first approach to the understanding of protein-membrane interactions of EgFABP1. The results suggest that this protein may be actively involved in the exchange and transport of fatty acids between different membranes and cellular compartments within the parasite.

  17. Effect of cyclosporin A on the survival and ultrastructure of Echinococcus granulosus protoscoleces in vitro.

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    Colebrook, A L; Jenkins, D J; Jones, M K; Tatarczuch, L; Lightowlers, M W

    2004-10-01

    Surgical treatment of human hydatidsosis involves the use of various scolicidal agents to kill infective Echinococcus granulosus protoscoleces that may disseminate into the peritoneal cavity during surgery and potentially re-infect the patient. Currently, no scolicidal agent is completely effective in killing intracystic protoscoleces in humans. Cyclosporin A (CsA) has previously been found to be lethal for E. granulosus protoscoleces in vitro. In this study, we further assessed the effectiveness of CsA as a scolicidal agent by testing the toxic effect of CsA at higher doses over various time-periods. Experiments were performed on activated and unactivated protoscoleces cultured in nutrient medium or sheep hydatid cyst fluid. All activated protoscoleces were killed following culture in 100 microg/ml of CsA for 3 days and 50 or 20 microg/ml for 5 days. The lethal effect of CsA on unactivated protoscoleces varied but reached 100% over 15 days in culture with 100 or 50 microg/ml of CsA. Pulse treatment of protoscoleces with 50, 20 or 10 microg/ml of CsA for 5 min or 72 h killed all parasites by day 10 and day 5 respectively. Untreated protoscoleces remained greater than 95 % viable for the duration of experiments. Changes in protoscolex ultrastructure induced by treatment with 10 microg/ml of CsA over 10 days in in vitro culture was assessed by TEM. Protoscolex alterations observed in treated parasites included an increase in cellular vacuolization, swelling of mitochondria, rounding of cells, damage to the tegument, decrease in glycogen, a breakdown of the extracellular matrix and an increase in lipid globules. The untreated protoscoleces, by comparison, had few changes during the 10-day culture period with the exception of large amounts of extracellular glycogen observed in the protoscoleces at culture days 7 and 10. From these results, CsA is clearly an effective scolicidal agent in vitro that may have potential application as a new therapeutic agent in the

  18. First insights into the genetic diversity of Echinococcus granulosus sensu stricto (s.s.) in Serbia.

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    Debeljak, Zoran; Boufana, Belgees; Interisano, Maria; Vidanovic, Dejan; Kulisic, Zoran; Casulli, Adriano

    2016-06-15

    Cystic echinococcosis (CE) caused by Echinococcus granulosus sensu stricto (s.s.) is a cosmopolitan zoonotic infection which is endemic in Serbia where it is subject to mandatory reporting. However, information on the incidence of the disease in humans and prevalence of hydatid infection in livestock remains limited. We used sequenced data of the cytochrome c oxidase subunit 1 (cox 1) mitochondrial gene to examine the genetic diversity and population structure of E. granulosus (s.s.) from intermediate hosts from Serbia. We also compared our generated nucleotide sequences with those reported for neighbouring European countries. Echinococcus canadensis was molecularly confirmed from pig and human hydatid isolates. E. granulosus (G1) was confirmed from sheep and cattle hydatid isolates as well as the first molecular confirmation in Serbia of E. granulosus G2 in sheep and E. granulosus G3 in sheep and cattle hydatid isolates. The Serbian E. granulosus (s.s.) parsimony network displayed 2 main haplotypes (SB02 and SB05) which together with the neutrality indices were suggestive of bottleneck and/or balancing selection. Haplotype analysis showed the presence of the common E. granulosus haplotype described from other worldwide regions. Investigation of the pairwise fixation (Fst) index suggested that Serbian populations of E. granulosus (s.s.) from sheep and cattle hosts showed moderate genetic differentiation. Six of the Serbian haplotypes (SB02-SB07) were shared with haplotypes from Bulgaria, Hungary and/or Romania. Further studies using a larger number of hydatid isolates from various locations across Serbia will provide more information on the genetic structure of E. granulosus (s.s.) within this region.

  19. Detection of Echinococcus granulosus G3 in a Wild Boar ( Sus scrofa ) in Central Italy Using PCR and Sequencing.

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    Di Paolo, Antonella; Piseddu, Toni; Sebastianelli, Martina; Manuali, Elisabetta; Corneli, Sara; Paniccià, Marta; Papa, Paola; Viali, Selina; Mazzone, Piera

    2017-01-24

    We report cystic echinococcosis in a free-living wild boar ( Sus scrofa ) in Europe. Parasites were identified by histopathology and molecular techniques, revealing Echinococcus granulosus of the G3 genotype.

  20. Identification and pharmacological induction of autophagy in the larval stages of Echinococcus granulosus: an active catabolic process in calcareous corpuscles.

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    Loos, Julia A; Caparros, Pedro A; Nicolao, María Celeste; Denegri, Guillermo M; Cumino, Andrea C

    2014-06-01

    Autophagy is a fundamental catabolic pathway conserved from yeast to mammals, but which remains unknown in parasite cestodes. In this work, the pharmacological induction of autophagy was cellularly and molecularly analysed in the larval stages of Echinococcus granulosus. Metacestode sensitivity to rapamycin and TORC1 expression in protoscoleces and metacestodes were shown. Ultrastructural studies showed that treated parasites had an isolation membrane, autophagosomes and autolysosomes, all of which evidenced the autophagic flux. Genes coding for key autophagy-related proteins were also identified in the Echinococcus genome. These genes were involved in autophagosome formation and transcriptional over-expression of Eg-atg5, Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16 and Eg-atg18 was shown in presence of rapamycin or arsenic trioxide. Thus, Echinococcus autophagy could be regulated by non-transcriptional inhibition through TOR and by transcription-dependent up-regulation via FoxO-like transcription factors and/or TFEB proteins. An increase in the punctate pattern and Eg-Atg8 polypeptide level in the tegument, parenchyma cells and excretory system of protoscoleces and in vesicularised parasites was detected after rapamycin treatment. This suggests the occurrence of basal autophagy in the larval stages and during vesicular development. In arsenic-treated protoscoleces, high Eg-Atg8 polypeptide levels within the free cytoplasmic matrix of calcareous corpuscles were observed, thus verifying the occurrence of autophagic events. These experiments also confirmed that the calcareous corpuscles are sites of arsenic trioxide accumulation. The detection of the autophagic machinery in this parasite represents a basic starting point to unravel the role of autophagy under both physiological and stress conditions which will allow identification of new strategies for drug discovery against neglected parasitic diseases caused by cestodes.

  1. Echinococcus granulosus genomics: a new dawn for improved diagnosis, treatment, and control of echinococcosis.

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    Zhang, Wenbao; Wang, Shengyue; McManus, Donald P

    2014-01-01

    Cystic echinococcosis (CE) is a cosmopolitan disease caused by the dog tapeworm Echinococcus granulosus. The disease is difficult to diagnose, treat, and control and is responsible for considerable human morbidity and mortality globally. There is an urgent need for new diagnostic tests and new drugs for treatment of CE and the development of a vaccine against adult worms of E. granulosus in dogs. We recently presented a draft genomic sequence for the worm comprising 151.6 Mb encoding 11,325 proteins. We undertook an extensive comparative analysis of the E. granulosus transcriptome using representative life stages (protoscoleces, cyst germinal cells and membranes, adult worms, and oncospheres) to explore different aspects of tapeworm biology and parasitism. The genome and transcriptome of E. granulosus provide a unique platform for post-genomic research and to facilitate the development of new, effective treatments and interventions for echinococcosis control.

  2. Prevalence survey and first molecular characterization of Echinococcus granulosus in France.

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    Umhang, G; Richomme, C; Boucher, J-M; Hormaz, V; Boué, F

    2013-04-01

    Human cystic echinococcosis (hydatid disease) caused by the Echinococcus granulosus tapeworm continues to be a substantial cause of morbidity and mortality in many parts of the world. France is still considered as endemic area, but the current infestation by E. granulosus of intermediate hosts in France remains currently unknown due to the absence of official data reporting for the last 20 years. A 1-year prevalence survey was conducted in the 24 slaughterhouses of ten departments of the South of France. We demonstrate that the E. granulosus parasite is still currently present at low prevalence at slaughterhouses in the study area (4 cases for 100,000 sheep and 3 cases for 100,000 cattle). In addition, we assess the presence of genotype G1 in infected animals and identify for the first time in France genotypes G2 and G3 of E. granulosus sensu stricto.

  3. Canine echinococcosis: genetic diversity of Echinococcus granulosus sensu stricto (s.s.) from definitive hosts.

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    Boufana, B; Lett, W; Lahmar, S; Griffiths, A; Jenkins, D J; Buishi, I; Engliez, S A; Alrefadi, M A; Eljaki, A A; Elmestiri, F M; Reyes, M M; Pointing, S; Al-Hindi, A; Torgerson, P R; Okamoto, M; Craig, P S

    2015-11-01

    Canids, particularly dogs, constitute the major source of cystic echinococcosis (CE) infection to humans, with the majority of cases being caused by Echinococcus granulosus (G1 genotype). Canine echinococcosis is an asymptomatic disease caused by adult tapeworms of E. granulosus sensu lato (s.l.). Information on the population structure and genetic variation of adult E. granulosus is limited. Using sequenced data of the mitochondrial cytochrome c oxidase subunit 1 (cox1) we examined the genetic diversity and population structure of adult tapeworms of E. granulosus (G1 genotype) from canid definitive hosts originating from various geographical regions and compared it to that reported for the larval metacestode stage from sheep and human hosts. Echinococcus granulosus (s.s) was identified from adult tapeworm isolates from Kenya, Libya, Tunisia, Australia, China, Kazakhstan, United Kingdom and Peru, including the first known molecular confirmation from Gaza and the Falkland Islands. Haplotype analysis showed a star-shaped network with a centrally positioned common haplotype previously described for the metacestode stage from sheep and humans, and the neutrality indices indicated population expansion. Low Fst values suggested that populations of adult E. granulosus were not genetically differentiated. Haplotype and nucleotide diversities for E. granulosus isolates from sheep and human origin were twice as high as those reported from canid hosts. This may be related to self-fertilization of E. granulosus and/or to the longevity of the parasite in the respective intermediate and definitive hosts. Improved nuclear single loci are required to investigate the discrepancies in genetic variation seen in this study.

  4. Molecular characterization of Echinococcus granulosus sensu lato from farm animals in Egypt.

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    Said Amer

    Full Text Available Little is known on the diversity and public health significance of Echinococcus species in livestock in Egypt. In this study, 37 individual hydatid cysts were collected from dromedary camels (n=28, sheep (n=7 and buffalos (n=2. DNA was extracted from protoscoleces/germinal layer of individual cysts and amplified by PCR targeting nuclear (actin II and mitochondrial (COX1 and NAD1 genes. Direct sequencing of amplicons indicated the presence of Echinococcus canadenesis (G6 genotype in 26 of 28 camel cysts, 3 of 7 sheep cysts and the 2 buffalo derived cysts. In contrast, Echinococcus granulosus sensu stricto (G1 genotype was detected in one cyst from a camel and 4 of 7 cysts from sheep, whereas Echinococcus ortleppi (G5 genotype was detected in one cyst from a camel. This is the first identification of E. ortleppi in Egypt.

  5. Molecular characterization of Echinococcus granulosus sensu lato from farm animals in Egypt.

    Science.gov (United States)

    Amer, Said; Helal, Ibrahim B; Kamau, Evelyne; Feng, Yaoyu; Xiao, Lihua

    2015-01-01

    Little is known on the diversity and public health significance of Echinococcus species in livestock in Egypt. In this study, 37 individual hydatid cysts were collected from dromedary camels (n=28), sheep (n=7) and buffalos (n=2). DNA was extracted from protoscoleces/germinal layer of individual cysts and amplified by PCR targeting nuclear (actin II) and mitochondrial (COX1 and NAD1) genes. Direct sequencing of amplicons indicated the presence of Echinococcus canadenesis (G6 genotype) in 26 of 28 camel cysts, 3 of 7 sheep cysts and the 2 buffalo derived cysts. In contrast, Echinococcus granulosus sensu stricto (G1 genotype) was detected in one cyst from a camel and 4 of 7 cysts from sheep, whereas Echinococcus ortleppi (G5 genotype) was detected in one cyst from a camel. This is the first identification of E. ortleppi in Egypt.

  6. Echinococcus granulosus pig strain (G7 genotype) protoscoleces did not develop secondary hydatid cysts in mice.

    Science.gov (United States)

    Cucher, M; Mourglia-Ettlin, G; Prada, L; Costa, H; Kamenetzky, L; Poncini, C; Dematteis, S; Rosenzvit, M C

    2013-03-31

    Echinococcus granulosus, the aetiological agent of cystic hydatid disease, exists as a series of strains or genotypes which differ in biological features. Pig strain (G7 genotype) has been shown to differ from sheep strain (G1 genotype) in phenotypical characters such as intermediate host range, geographical distribution and rate of development of the adult worm. Since in vivo studies of different parasite genotypes can provide insights into host-parasite relationship we analysed for the first time the behaviour of E. granulosus G7 genotype protoscoleces in the murine experimental model. Our results show that G7 protoscoleces were unable to establish a regular infection in mice in contrast to G1 protoscoleces which developed intraperitoneal hydatid cysts. This inability was observed in co-infection experiments, i.e. even in the presence of a controlled immune response that allows G1 genotype protoscoleces establishment. In addition, the implantation of in vitro obtained E. granulosus G7 genotype microcysts resulted in a low percentage of hydatid cysts establishment. These results show a difference in the biological ability of both E. granulosus strains to develop secondary hydatid cysts in mice. We suggest that the comparison of infective and non infective genotypes of E. granulosus in the experimental host can be regarded as a new model to study the mechanisms of infection of Echinococcus spp. This knowledge could provide helpful information for the development of therapies, drugs and/or vaccines against cystic hydatid disease.

  7. Molecular identification of Echinococcus granulosus on the Tibetan Plateau using mitochondrial DNA markers.

    Science.gov (United States)

    Hu, D; Song, X; Wang, N; Zhong, X; Wang, J; Liu, T; Jiang, Z; Dawa, T; Gu, X; Peng, X; Yang, G

    2015-10-30

    Cystic echinococcosis (CE) is an important worldwide zoonotic disease that causes large economic losses and human suffering. Echinococcus granulosus, the causative agent of CE, exhibits different genotypes in different locations. In order to identify its genotypes and analyze its genetic structure on the Tibetan Plateau, we collected 72 hydatid cysts from different intermediate hosts and amplified and sequenced their mitochondrial cytochrome c oxidase subunit 2 (cox2) genes. Seventy isolates were identified as the E. granulosus G1 genotype, while two isolates belonged to the G6 genotype. There were 18 haplotypes among the 70 E. granulosus isolates, which exhibited a star-like network pattern and shared a common haplotype (H1). There was little difference between geographical sub-populations. Our results suggest that a recent E. granulosus population expansion occurred on the Tibetan Plateau, suggesting that E. granulosus was introduced into China. This study increases the basic molecular data needed for the molecular diagnosis, epidemiology, prevention, and control of Echinococcus diseases.

  8. Emergence of Sylvatic Echinococcus granulosus as a Parasitic Zoonosis of Public Health Concern in an Indigenous Community in Canada

    Science.gov (United States)

    Himsworth, Chelsea G.; Jenkins, Emily; Hill, Janet E.; Nsungu, Mandiangu; Ndao, Momar; Andrew Thompson, R. C.; Covacin, Catherine; Ash, Amanda; Wagner, Brent A.; McConnell, Athena; Leighton, Frederick A.; Skinner, Stuart

    2010-01-01

    Within a remote Canadian Indigenous community, at least 11* of people had antibodies against Echinococcus granulosus and E. granulosus eggs were detected in 6* of environmentally collected canine fecal samples. Dog ownership, hunting, and trapping were not risk factors for seropositivity, suggesting that people are most likely exposed to E. granulosus through indirect contact with dog feces in the environment. In this situation, human exposure could be most effectively curtailed by preventing consumption of cervid viscera by free-roaming dogs. PMID:20348513

  9. Cases of Echinococcus granulosus Sensu Stricto Isolated from Polish Patients: Imported or Indigenous?

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    Monika Dybicz

    2015-01-01

    Full Text Available The cases of nine Polish patients with diagnosed cystic echinococcosis (CE were examined. A total of nine isolates obtained postoperatively were investigated using PCR and sequencing. The mitochondrial region of nad1 gene was amplified. This PCR and sequencing analysis revealed the presence of Echinococcus canadensis G7 in seven patients and E. granulosus G1 in two patients. These data demonstrate that E. canadensis is the predominant causative agent of human cystic echinococcosis in Poland. E. granulosus G1 detection in Polish patients suggests that the parasite was imported; however it does not exclude the possibility that these cases could have been of Polish origin.

  10. Cases of Echinococcus granulosus Sensu Stricto Isolated from Polish Patients: Imported or Indigenous?

    Science.gov (United States)

    Dybicz, Monika; Borkowski, Piotr Karol; Dąbrowska, Julia; Chomicz, Lidia

    2015-01-01

    The cases of nine Polish patients with diagnosed cystic echinococcosis (CE) were examined. A total of nine isolates obtained postoperatively were investigated using PCR and sequencing. The mitochondrial region of nad1 gene was amplified. This PCR and sequencing analysis revealed the presence of Echinococcus canadensis G7 in seven patients and E. granulosus G1 in two patients. These data demonstrate that E. canadensis is the predominant causative agent of human cystic echinococcosis in Poland. E. granulosus G1 detection in Polish patients suggests that the parasite was imported; however it does not exclude the possibility that these cases could have been of Polish origin.

  11. Incidence of Echinococcus granulosus in Domestic Dogs in Palestine as Revealed by Copro-PCR.

    Science.gov (United States)

    Al-Jawabreh, Amer; Dumaidi, Kamal; Ereqat, Suheir; Nasereddin, Abedelmajeed; Al-Jawabreh, Hanan; Azmi, Kifaya; Al-Laham, Nahed; Abdeen, Ziad

    2015-01-01

    Hydatidosis or echinococcosisis considered a neglected zoonotic disease despite its high burden in the livestock industry and the high risk of infection by humans in endemic areas. In a cross-sectional study we estimated the copro-Incidence and also genotyped Echinococcus granulosus isolates from domestic dogs using polymerase chain reaction (PCR). Medical archives in nine major hospitals in Palestine were reviewed to determine incidence of E. granulosus infection detected in humans during surgery. Faecal samples were collected from 93 domestic dogs in three districts with the highest number of human cases: Al-Khalil (Hebron), Tubas and Jenin. Genomic DNA was extracted from dog faecal samples and amplified by PCR targeting the repeat DNA sequence (EgG1 Hae III) followed by sequencing of five positive samples. Genotyping was determined by sequencing and BLAST searching of mitochondrial cytochrome c oxidase subunit (CO1). The incidence of E. granulosus infection detected in humans at surgery was 1.2 per 100,000 in the West Bank and 1.0 per 100,000 in Gaza Strip. Seventeen of 93 domestic dogs (18%) were positive, based upon comparison with the Echinococcus DNA control. The five sequenced samples were confirmed to be E. granulosus. Successfully genotyped sample belonged to E.granulosus sensu stricto (formerly G1-G3 complex, sheep strain). For domestic dogs, age group (13-24 months) and sex were identified as two risk factors for contracting E. granulosus. The study identified the high incidence of E. granulosus sensu stricto in dogs in Palestine.

  12. Incidence of Echinococcus granulosus in Domestic Dogs in Palestine as Revealed by Copro-PCR.

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    Amer Al-Jawabreh

    Full Text Available Hydatidosis or echinococcosisis considered a neglected zoonotic disease despite its high burden in the livestock industry and the high risk of infection by humans in endemic areas. In a cross-sectional study we estimated the copro-Incidence and also genotyped Echinococcus granulosus isolates from domestic dogs using polymerase chain reaction (PCR. Medical archives in nine major hospitals in Palestine were reviewed to determine incidence of E. granulosus infection detected in humans during surgery. Faecal samples were collected from 93 domestic dogs in three districts with the highest number of human cases: Al-Khalil (Hebron, Tubas and Jenin. Genomic DNA was extracted from dog faecal samples and amplified by PCR targeting the repeat DNA sequence (EgG1 Hae III followed by sequencing of five positive samples. Genotyping was determined by sequencing and BLAST searching of mitochondrial cytochrome c oxidase subunit (CO1. The incidence of E. granulosus infection detected in humans at surgery was 1.2 per 100,000 in the West Bank and 1.0 per 100,000 in Gaza Strip. Seventeen of 93 domestic dogs (18% were positive, based upon comparison with the Echinococcus DNA control. The five sequenced samples were confirmed to be E. granulosus. Successfully genotyped sample belonged to E.granulosus sensu stricto (formerly G1-G3 complex, sheep strain. For domestic dogs, age group (13-24 months and sex were identified as two risk factors for contracting E. granulosus. The study identified the high incidence of E. granulosus sensu stricto in dogs in Palestine.

  13. The anthelmintic effect of Urtica dioica and Tanacetum vulgare L. on Protoscoleces of Echinococcus granulosus

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    Omer lokman Omer

    2013-04-01

    Full Text Available The current study evaluated the effects of alcoholic (Ethanol extracts and aquaus extract of leaves Urtica dioica and extract of both leaves and flowers of Tanacetum vulgare on the viability of Echinococcus granulosus protoscolices in vitro. Three different concentrations of each extract (I, 2, 4 micrograms /ml were used. The mortality of Protoscoleces of Echinococcus granulosus by using aqueous extract of two plants was increased with the increasing the concentration and duration of exposure; reach 96.2% and 97.8% at concentration of 4 micrograms/ml for 30 minutes for Urtica dioica and Tanacetum volgare respectively. However, it was found that the effec of these plants by ethanol were decreased with the same concentration when the time of exposure is increased. The mortality rate of protocoleces decreased from 69% to 4.2% when exposed to Urtica dioica extract for 10 and 30 min respectively at 1mg/ml.

  14. In vitro assessment of praziquantel and a novel nanomaterial against protoscoleces of Echinococcus granulosus.

    Science.gov (United States)

    De, S; Pan, D; Bera, A K; Sreevatsava, V; Bandyopadhyay, S; Chaudhuri, D; Kumar, S; Rana, T; Das, S; Das, S K; Suryanaryana, V V; Singh, M Norjit; Bhattacharya, D

    2012-03-01

    The present study describes the activity of a nanomaterial on protoscoleces of Echinococcus granulosus, which exhibited morphological changes and apoptosis. Apoptotic changes were deduced on the basis of effector caspase activation and nucleosomal laddering. Invaginated protoscoleces maintained in vitro became evaginated and had hooks, presumptive suckers and stalks. Degenerative changes of protoscoleces were evidenced after treatment with praziquantel and nano-combination. Protoscoleces treated with praziquantel had distinct attestation of necrosis and nano-combination-treated protoscoleces had signatures of apoptosis.

  15. Effect of gamma irradiation on the protoscoleces of Echinococcus granulosus of sheep origin

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    Singh, B.P.; Dhar, D.N.

    1988-06-01

    In vitro and in vivo effects of varying levels of gamma irradiation on protoscoleces of Echinococcus granulosus of sheep origin were studied. Radiation doses of 100 Gy onwards caused a decrease in the viability of protoscoleces in vitro. However, infectivity of protoscoleces was not affected at radiation doses of 300 Gy in golden hamsters and 200 Gy in mice although number and size of cysts developing from infections with irradiated protoscoleces in these animals was small in comparison to cysts developing from infections with normal protoscoleces. Four hundred E. granulosus protoscoleces, normal or 100 Gy irradiated, proved fatal for mice. A significant progressive decline in worm establishment was observed in pups given an infection of E. granulosus protoscoleces exposed to increasing levels of gamma irradiation from 100 to 600 Gy. No worms established in pups infected with protoscoleces irradiated at 400 and 600 Gy, respectively. Worms developing from irradiated infections in pups were stunted and showed developmental abnormalities.

  16. Serological monitoring of protection of sheep against Echinococcus granulosus induced by the EG95 vaccine.

    Science.gov (United States)

    Heath, D D; Koolaard, J

    2012-01-01

    Although immunity to Echinococcus granulosus in sheep has been shown to be antibody-mediated and complement-dependent and can be passively transferred in colostrum, in animals vaccinated with EG95, the relationship between protection against an oral challenge infection with E. granulosus eggs and anti-EG95 IgG ELISA absorbance values at the time of challenge has not been satisfactorily proven. Using a combination of results from three EG95 vaccination trials, we have found that the IgG ELISA absorbance at the time of challenge infection explains approximately 50% (P ≤ 0·001) of the variability in the percentage protection against an oral challenge with E. granulosus eggs (transformed with arcsin).

  17. Molecular genotyping of Echinococcus granulosus from dromedaries (Camelus dromedarius) in eastern Iran.

    Science.gov (United States)

    Moghaddas, E; Borji, H; Naghibi, A; Shayan, P; Razmi, G R

    2015-01-01

    With the aim of genotyping Echinococcus granulosus cysts found in Iranian dromedaries (Camelus dromedarius), 50 cysts of E. granulosus were collected from five geographical regions in Iran. Cysts were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer 1 (ITS1) gene and sequencing fragments of the genes coding for mitochondrial cytochrome c oxidase subunit 1 (cox1). Morphological criteria using rostellar hook dimensions were also undertaken. The present results have shown that 27 out of 50 E. granulosus cysts (54%) were determined as the G1 strain, and the other (46%) were determined as the G6 strain. The molecular analysis of the ITS1 region of ribosomal DNA corresponded with the morphological findings. Because of its recognized infectivity in humans, the G1 genotype is a direct threat to human health and its presence in Iranian dromedaries is of urgent public health importance.

  18. Molecular characterization of the horse isolate of Echinococcus granulosus in Turkey.

    Science.gov (United States)

    Utuk, A E; Simsek, S

    2013-09-01

    Cystic echinococcosis is one of the most important helminthozoonoses, affecting various species of intermediate hosts and humans. In this report, we present Echinococcus granulosus infection in a horse and its molecular characterization. Polymerase chain reaction (PCR) amplification of mitochondrial 12S rRNA (mt-12S rRNA) and partial sequencing of mitochondrial cytochrome c oxidase subunit 1 (mt-CO1) genes were performed. According to the mt-12S rRNA-PCR result, the horse isolate was grouped with E. granulosus sensu stricto (G1-G3) and the partial mt-CO1 sequence corresponded to the G1 strain. This is the first study of the molecular characterization of the horse isolate of E. granulosus in Turkey.

  19. In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

    Science.gov (United States)

    Wang, Fen; Ye, Bin

    2016-10-01

    Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

  20. First report of Echinococcus granulosus (genotype G6) in a dog in Bamako, Mali.

    Science.gov (United States)

    Mauti, S; Traoré, A; Crump, L; Zinsstag, J; Grimm, F

    2016-02-15

    Cystic echinococcosis is one of the most widespread and important helminthic zoonoses, caused by the larval stage of Echinococcus granulosus sensu lato. However, to date there is little information about the disease in West Africa. Faecal and fur samples from 193 dogs, the main final hosts, were collected in 2010 and 2011 in Bamako, Mali. Taeniid eggs were found microscopically in 28/118 (24%) and 80/223 (36%) faecal and fur samples, respectively. One faecal and one fur sample from the same dog were positive for E. granulosus s. l. DNA. In the remaining 27 faecal (96%) and 77 fur samples (96%) only Taenia DNA was detected. Three microscopically positive fur samples were negative by PCR. Sequence analysis of part of the NADH dehydrogenase subunit 1 gene identified the parasite as E. granulosus (genotype G6; Echinococcus canadensis). This is the first study to focus on the final host of E. granulosus s. l. in Mali and the first report of E. canadensis in Mali.

  1. Identification of Echinococcus granulosus strains using polymerase chain reaction-restriction fragment length polymorphism amongst livestock in Moroto district, Uganda.

    Science.gov (United States)

    Chamai, Martin; Omadang, Leonard; Erume, Joseph; Ocaido, Michael; Oba, Peter; Othieno, Emmanuel; Bonaventure, Straton; Kitibwa, Annah

    2016-07-29

    A descriptive study was conducted to identify the different strains of Echinococcus granulosus occurring in livestock in Moroto district, Uganda. Echinococcus cysts from 104 domestic animals, including cattle, sheep, goats and camels, were taken and examined by microscopy, polymerase chain reaction with restriction fragment length polymorphism and Sanger DNA sequencing. Echinococcus granulosus genotypes or strains were identified through use of Bioinformatics tools: BioEdit, BLAST and MEGA6. The major finding of this study was the existence of a limited number of E. granulosus genotypes from cattle, goats, sheep and camels. The most predominant genotype was G1 (96.05%), corresponding to the common sheep strain. To a limited extent (3.95%), the study revealed the existence of Echinococcus canadensis G6/7 in three (n = 3) of the E. granulosus-positive samples. No other strains of E. granulosus were identified. It was concluded that the common sheep strain of Echinococcus sensu stricto and G6/7 of E. canadensis were responsible for echinococcal disease in Moroto district, Uganda.

  2. Association between Echinococcus granulosus infection and cancer risk - a pilot study in Cyprus.

    Science.gov (United States)

    Oikonomopoulou, Katerina; Yu, Herbert; Wang, Zhanwei; Vasiliou, Stella K; Brinc, Davor; Christofi, Georgios; Theodorou, Marilena; Pavlou, Pavlos; Hadjisavvas, Andreas; Demetriou, Christiana A; Kyriacou, Kyriacos; Diamandis, Eleftherios P

    2016-12-01

    Infections from microorganisms and parasites have been connected with either increased or decreased cancer risk. The objective of this study was to investigate whether infection by Echinococcus granulosus is associated with cancer risk. We assembled a pilot retrospective cohort of patients who were diagnosed as being infected by E. granulosus in Cyprus between 1930 and 2011. Age/gender-matched non-infected family members and neighbors were selected as references. Medical history was ascertained from each study subject through in-person interview. Cox proportional hazards regression analysis was performed to assess the association of being infected by E. granulosus with cancer risk. Individuals with prior infection by E. granulosus (n=249) were more likely to have cancer compared to those without infection (n=753), 11.65% vs. 8.37% (p=0.0492). Survival analysis also showed that subjects with prior infection had a higher risk for developing cancer. The hazards ratio (HR) was 1.595, [95% confidence interval (CI) between 1.008 and 2.525]. The risk ratio did not change significantly (HR=1.536; 95% CI: 0.965-2.445) after adjusting for gender, year of birth, smoking status, alcohol consumption, and family history of cancer. Our study suggests that infection by E. granulosus may increase cancer risk. If this observation can be confirmed independently, further investigation of the mechanisms underlying the association is warranted.

  3. Molecular characterization of Echinococcus granulosus in a hyperendemic European focus, the Republic of Moldova.

    Science.gov (United States)

    Umhang, Gérald; Chihai, Oleg; Boué, Franck

    2014-12-01

    Cystic echinococcosis is a zoonosis caused by the tapeworm Echinococcus granulosus sensu lato. The lifecycle of the parasite is mainly domestic, requiring dogs as definitive hosts and livestock species as intermediate hosts. Although human cystic echinococcosis is a high public health priority in the Republic of Moldova, the rare animal data available concerns only infection in cattle. A preliminary slaughterhouse survey was conducted to assess prevalence and perform the first molecular characterization of E. granulosus sensu lato in sheep and cattle. For the survey, 40 sheep and 19 cattle were inspected. Very high prevalence in sheep (82.5%) and in cattle (78.9%) was found. Molecular analyses identified genotypes G1 and G3 of E. granulosus sensu stricto in all the liver and lung samples. Based on the concatenated sequences of cox1 + nad3 (701 bp), 23 different haplotypes were obtained. Mixed infections by different haplotypes/genotypes were frequently identified in both sheep and cattle. The relatively high (20.0%) cyst fertility observed in cattle argues for the potential contribution of cattle to the lifecycle of E. granulosus sensu stricto, unlike previous observations in Europe. The hyperendemic situation of Moldova can be explained by a high majority of animals slaughtered at home usually without veterinary inspection. Further extensive slaughterhouse surveys with molecular identification also involving pigs and goats are needed to obtain a better overview of the epidemiological situation of E. granulosus sensu lato in this hyperendemic focus in the Republic of Moldova.

  4. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    Science.gov (United States)

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

  5. Genetic variability of Echinococcus granulosus based on the mitochondrial 16S ribosomal RNA gene.

    Science.gov (United States)

    Wang, Ning; Wang, Jiahai; Hu, Dandan; Zhong, Xiuqin; Jiang, Zhongrong; Yang, Aiguo; Deng, Shijin; Guo, Li; Tsering, Dawa; Wang, Shuxian; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

    2015-06-01

    Echinococcus granulosus is the etiological agent of cystic echinococcosis, a major zoonotic disease of both humans and animals. In this study, we assessed genetic variability and genetic structure of E. granulosus in the Tibet plateau, using the complete mitochondrial 16 S ribosomal RNA gene for the first time. We collected and sequenced 62 isolates of E. granulosus from 3 populations in the Tibet plateau. A BLAST analysis indicated that 61 isolates belonged to E. granulosus sensu stricto (genotypes G1-G3), while one isolate belonged to E. canadensis (genotype G6). We detected 16 haplotypes with a haplotype network revealing a star-like expansion, with the most common haplotype occupying the center of the network. Haplotype diversity and nucleotide diversity were low, while negative values were observed for Tajima's D and Fu's Fs. AMOVA results and Fst values revealed that the three geographic populations were not genetically differentiated. Our results suggest that a population bottleneck or population expansion has occurred in the past, and that this explains the low genetic variability of E. granulosus in the Tibet Plateau.

  6. Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN.

    Science.gov (United States)

    Sharbatkhori, Mitra; Tanzifi, Asal; Rostami, Sima; Rostami, Masoomeh; Fasihi Harandi, Majid

    2016-01-01

    Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

  7. In vitro and in vivo treatments of Echinococcus granulosus with Huaier aqueous extract and albendazole liposome.

    Science.gov (United States)

    Lv, Hailong; Jiang, Yufeng; Liao, Min; Sun, Hongli; Zhang, Shijie; Peng, Xinyu

    2013-01-01

    The aim of this study was to investigate the in vitro and in vivo efficacies of chemotherapy employing albendazole liposome (L-ABZ), Huaier aqueous extract, and a Huaier aqueous extract/L-ABZ combination against Echinococcus granulosus. Protoscolices of E. granulosus were incubated in vitro with the two drugs, either separately or in combination, at the following final concentrations: 2 mg/mL Huaier aqueous extract, 10 μg/mL L-ABZ, and 2 mg/mL Huaier aqueous extract + 10 μg/mL L-ABZ. Huaier aqueous extract and L-ABZ displayed slower protoscolicidal activity when applied separately than when used in combination. The maximum protoscolicidal effect was found with the combination Huaier aqueous extract + L-ABZ. Despite the low Huaier aqueous extract + L-ABZ concentrations used, protoscolex viability dropped rapidly. In vivo studies were performed on mice injected with protoscolices of E. granulosus. Huaier aqueous extract and L-ABZ were administered three times a week for a period of 4 months by the oral route. Huaier aqueous extract in E. granulosus-infected mice was effective. Combined application of both drugs did increase the treatment efficacy. In conclusion, the outcomes obtained clearly demonstrated that in vitro and in vivo treatment with Huaier aqueous extract and L-ABZ is effective against E. granulosus.

  8. Myophilin of Echinococcus granulosus: isoforms and phosphorylation by protein kinase C.

    Science.gov (United States)

    Martin, R M; Csar, X F; Gasser, R B; Felleisen, R; Lightowlers, M W

    1997-08-01

    Myophilin is a muscle-associated antigen of the taeniid cestode Echinococcus granulosus. This protein shows a high amino acid sequence homology with calponins and calponin-like proteins, which are proposed to be associated with the regulation of smooth muscle contraction. In order to provide supportive evidence for a relationship between these proteins, we characterized myophilin using electrophoretic, biochemical and molecular biological approaches. Two-dimensional protein electrophoretic separation of E. granulosus larval proteins defined 4 isoelectric isoforms of myophilin (alpha, beta, gamma and delta), which appeared to be a consequence of post-translational modification of a single gene product. It was also demonstrated biochemically that E. granulosus myophilin undergoes specific phosphorylation in vitro by protein kinase C (PKC). Finally, myophilin homologues were identified in extracts of Taenia hydatigena and T. ovis by immunoblot. A partial cDNA of the closely related species, E. multilocularis, was isolated by cloning procedures and showed 99% homology with the E. granulosus myophilin gene. The similarities of E. granulosus myophilin with calponins in their tissue localization, protein isoforms patterns, ability to be phosphorylated in vitro by PKC, and the relatively conserved nature of the protein among related parasites suggest that myophilin may be associated with smooth muscle contraction.

  9. Fisiopatología y respuesta inmune de ovinos experimentalmente infectados con Echinococcus granulosus Pathophysiology and immune response in sheep experimentally infected with Echinococcus granulosus

    Directory of Open Access Journals (Sweden)

    Larrieu Edmundo

    2009-06-01

    Full Text Available La respuesta inmune a la infección por Echinococcus granulosus en el ovino ha sido poco estudiada. El objetivo del presente trabajo fue aportar información sobre la fisiopatología y la respuesta inmune a la infección experimental con E. granulosus en ovinos. Se inocularon experimentalmente ovinos con tres dosis distintas de huevos de E. granulosus, evaluándose la repuesta inmune por seguimiento mediante enzimo inmuno ensayo con tres preparaciones antigénicas (líquido hidatídico total, fracción purificada de líquido hidatídico total y fracción lipoproteica purificada durante 500 días. Se sacrificaron animales en forma escalonada para observar macroscópica y microscópicamente el desarrollo del parásito. La respuesta inmune se detectó a partir de los 10 días y se mantuvo durante el período de observación, resultando inicialmente proporcional a la carga de huevos inoculados, y disminuyendo las diferencias con el tiempo. Se identificaron quistes fértiles a los 10 meses post inoculación y oncósferas vivas 500 días post inoculación. La respuesta de anticuerpos en el ovino a la infección por E. granulosus fue anterior a la formación de líquido hidatídico y resultó generada por la movilidad de la oncósfera. La temprana fertilidad identificada histológicamente indica que la alimentación de canes con vísceras de ovinos jóvenes puede producir ciclos de infección. La presencia de oncósferas vivas en el hígado, por su parte, aporta información sobre la patogenia de la enfermedad y permite expresar hipótesis sobre las causas de nuevas operaciones en el hombre luego de la extirpación de un quiste hidatídico lo que podría liberar el freno inmunitario sobre dichas oncósferas.The immune response to Echinococcus granulosus in sheep has not been extensively investigated. The objective of this study was to increase the information on the physiopathology of E. granulosus and the immune response elicited in sheep. Animals

  10. Echinococcus ortleppi (G5) and Echinococcus granulosus sensu stricto (G1) loads in cattle from Southern Brazil.

    Science.gov (United States)

    Balbinotti, Helier; Santos, Guilherme B; Badaraco, Jeferson; Arend, Ana C; Graichen, Daniel Ângelo S; Haag, Karen L; Zaha, Arnaldo

    2012-09-10

    Echinococcus granulosus sensu stricto (G1) and Echinococcus ortleppi (G5) are haplotypes of the parasite formerly known as Echinococcus granulosus sensu lato, which in its larval stage causes cystic hydatid disease, endemic in Southern Brazil. Epidemiological and molecular knowledge about the haplotypes occurring in a region is essential to control the spread of the disease. The aim of this work was to analyze the haplotype frequency and fertility of hydatid cysts in cattle from the state of Rio Grande do Sul. Cysts were collected and classified according to their fertility status. DNA was extracted from protoscoleces and germinal layers and then used as template for the amplification of the cytochrome c oxidase subunit 1 gene by PCR. Amplicons were purified and sequenced, and the sequences were analyzed for haplotype identification. A total of 638 fertile cysts collected in the last ten years were genotyped. On average, G1 (56.6%) was more frequent than G5 (43.4%). In lungs, the G5 haplotype exhibited a higher parasite load (52.8%), whereas in the liver, G1 was more frequent (90.4%). The analysis revealed an increase in the frequency of G5 haplotype cysts during the period of sampling, and an increase in the abundance of fertile cysts has also been observed in the last several years. Most infertile cysts were genotyped as G1. The possible factors involved in the increase in the proportion of E. ortleppi (G5) and the consequences of this increase are discussed. This study suggests that the proportion of E. ortleppi (G5) loads in cattle may be increasing overtime.

  11. Occurrence and genetic characterization of Echinococcus granulosus in naturally infected adult sheep and cattle in Romania.

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    Mitrea, Ioan Liviu; Ionita, Mariana; Costin, Irina Ioana; Predoi, Gabriel; Avram, Eugeniu; Rinaldi, Laura; Maurelli, Maria Paola; Cringoli, Giuseppe; Genchi, Claudio

    2014-12-15

    An epidemiological and molecular study was conducted to investigate the occurrence and genetic diversity of Echinococcus granulosus isolates from adult sheep and cattle in Romania. Overall, 642 sheep (aged over 3 years) and 1878 cattle (aged over 5 years) from 16 counties were examined for hydatid cysts. Of them, 421 (65.6%) sheep and 754 (40.1%) cattle were found infected by cystic echinococcosis (CE). Germinal layers were collected from 98 individual cysts (one cyst per animal; 31 from sheep and 67 from cattle), DNA was extracted and two different mitochondrial DNA genes, namely cytochrome c oxidase subunits 1 (CO1) and 12S ribosomal DNA (12S rDNA) were used as genetic markers. The assessment of the genetic diversity of the Echinococcus strains showed the presence of the E. granulosus sensu stricto complex and disclosed an apparent dominance of the G1 genotype within the G1–G3 complex. Furthermore, several mitochondrial variants were identified for the G1 and G2 genotypes of E. granulosus s.s. complex. Overall, the findings were of epidemiological relevance and highlighted a high potential risk of zoonotic infection.

  12. Morphological and biological characterization of cell line developed from bovine Echinococcus granulosus.

    Science.gov (United States)

    Echeverría, Claudia I; Isolabella, Dora M; Prieto Gonzalez, Elio A; Leonardelli, Araceli; Prada, Laura; Perrone, Alina; Fuchs, Alicia G

    2010-10-01

    The taeniid tapeworm Echinococcus granulosus is the causative agent of echinococcal disease, a major zoonosis with worldwide distribution. Several efforts to establish an in vitro model of E. granulosus have been undertaken; however, many of them have been designed for Echinococcus multilocularis. In the present study, we have described and characterized a stable cell line obtained from E. granulosus bovine protoscoleces maintained 3 yr in vitro. Growth characterization, morphology by light, fluorescent and electronic microscopy, and karyotyping were carried out. Cell culture origin was confirmed by immunofluorescent detection of AgB4 antigen and by PCR for the mitochondrial cytochrome c-oxidase subunit 1 (DCO1) gene. Cells seeded in agarose biphasic culture resembled a cystic structure, similar to the one formed in secondary hosts. This cell line could be a useful tool to research equinococcal behavior, allowing additional physiological and pharmacological studies, such as the effect of growth factors, nutrients, and antiparasitic drugs on cell viability and growth and on cyst formation.

  13. Echinococcus granulosus Sensu Stricto in Dogs and Jackals from Caspian Sea Region, Northern Iran

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    Shirzad GHOLAMI

    2016-10-01

    Full Text Available Background: The aim of the present study was genotyping of Echinococcus granulosus isolates from dogs and jackals in Mazandaran Province, northern Iran, and using partial sequence of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1.Methods: E. granulosus isolates (n = 15 were collected from 42 stray dogs and 16 jackals found in south of the Caspian Sea in northern Iran. After morphological study, the isolates were genetically characterized using consensus sequences (366bp of the cox1 gene. Phylogenetic analysis of cox1 nucleotide sequence data was performed using a Bayesian Inference approach.Results: Four different sequences were observed among the isolates. Two genotypes [G1 (66.7% and G3 (33.3%] were identified among the isolates. The G1 sequences indicated three sequence profiles. One profile (Maz1 had 100% homology with reference sequence (AN: KP339045. Two other profiles, designated Maz2 and Maz3, had 99% homology with the G1 genotype (ANs: KP339046 and KP339047. A G3 sequence designated Maz4 showed 100% homology with a G3 reference sequence (AN: KP339048.Conclusion: The occurrence of the G1 genotype of E. granulosus sensu stricto as a frequent genotype in dogs is emphasized. This study established the first molecular characterization of E. granulosus in the province.

  14. Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage.

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    Lorenzatto, Karina R; Kim, Kyunggon; Ntai, Ioanna; Paludo, Gabriela P; Camargo de Lima, Jeferson; Thomas, Paul M; Kelleher, Neil L; Ferreira, Henrique B

    2015-11-01

    Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.

  15. In vitro effect of 5-fluorouracil and paclitaxel on Echinococcus granulosus larvae and cells.

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    Pensel, P E; Albani, C; Gamboa, G Ullio; Benoit, J P; Elissondo, M C

    2014-12-01

    Human cystic echinococcosis is a zoonosis caused by the metacestode stage of the tapeworm Echinococcus granulosus. Although benzimidazole compounds such as albendazole and mebendazole have been the cornerstone of chemotherapy for the disease, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of 5-fluorouracil (5-FU) and paclitaxel (PTX) against E. granulosus germinal cells, protoscoleces and cysts. 5-FU or PTX inhibited the growth of E. granulosus cells in a time dependent manner. Although both treatments had a protoscolicidal effect, 5-FU had a considerably stronger effect than PTX. 5-FU produced a dose- and time-dependent effect, provoking the complete loss of viability after 24 days of incubation. Moreover, cysts did not develop following the inoculation of treated protoscoleces into mice. The loss of viability was slower in PTX treated protoscoleces, reaching to approximately 60% after 30 days. The results of the in vitro treatment with 5-FU and PTX were similar in secondary murine cysts. The employment of SEM and TEM allowed us to examine, at an ultrastructural level, the effects induced by 5-FU and PTX on E. granulosus germinal cells, protoscoleces and murine cysts. In conclusion, the data obtained clearly demonstrated that 5-FU and PTX at clinically achievable concentrations inhibit the survival of larval cells, protoscoleces and metacestodes. In vivo studies to test the antiparasitic activities of 5-FU and PTX are currently being undertaken on the murine model of cystic echinococcosis.

  16. Sequence analysis of cytb gene in Echinococcus granulosus from Western China.

    Science.gov (United States)

    Zhong, Xiuqin; Wang, Ning; Hu, Dandan; Wang, Jiahai; Liu, Tianyu; Gu, Xiaobin; Wang, Shuxian; Peng, Xuerong; Yang, Guangyou

    2014-04-01

    Echinococcus granulosus is the causative agent of cystic echinococcosis with medical and veterinary importance in China. Our main objective was to discuss the genotypes and genetic diversity of E. granulosus present in domestic animals and humans in western China. A total of 45 hydatid cyst samples were collected from sheep, humans, and a yak and subjected to an analysis of the sequences of mitochondrial cytochrome b (cytb) gene. The amplified PCR product for all samples was a 1,068 bp band. The phylogenetic analysis showed that all 45 samples were identified as E. granulosus (genotype G1). Ten haplotypes were detected among the samples, with the main haplotype being H1. The haplotype diversity was 0.626, while the nucleotide diversity was 0.001. These results suggested that genetic diversity was low among our samples collected from the west of China based on cytb gene analysis. These findings may provide more information on molecular characteristics of E. granulosus from this Chinese region.

  17. Analysis of the chemical components of hydatid fluid from Echinococcus granulosus

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    Li Juyi

    2013-10-01

    Full Text Available Introduction The aim of this study was to explore the environment of Echinococcus granulosus (E. granulosus protoscolices and their relationship with their host. Methods Proteins from the hydatid-cyst fluid (HCF from E. granulosus were identified by proteomics. An inductively coupled plasma atomic emission spectrometer (ICP-AES was used to determine the elements, an automatic biochemical analyzer was used to detect the types and levels of biochemical indices, and an automatic amino acid analyzer was used to detect the types and levels of amino acids in the E. granulosus HCF. Results I Approximately 30 protein spots and 21 peptide mass fingerprints (PMF were acquired in the two-dimensional gel electrophoresis (2-DE pattern of hydatid fluid; II We detected 10 chemical elements in the cyst fluid, including sodium, potassium, calcium, magnesium, copper, and zinc; III We measured 19 biochemical metabolites in the cyst fluid, and the amount of most of these metabolites was lower than that in normal human serum; IV We detected 17 free amino acids and measured some of these, including alanine, glycine, and valine. Conclusions We identified and measured many chemical components of the cyst fluid, providing a theoretical basis for developing new drugs to prevent and treat hydatid disease by inhibiting or blocking nutrition, metabolism, and other functions of the pathogen.

  18. Genetic differences between Tunisian camel and sheep strains of the cestode Echinococcus granulosus revealed by SSCP

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    Oudni-M’rad M.

    2006-06-01

    Full Text Available Ovine and dromedary Echinococcus granulosus isolates from Tunisia were identified as G1 and G6 strains based on polymorphism of the mitochondrial cytochrome C oxydase CO1. Single strand conformation polymorphism (SSCP was used in order to examine the genetic variation within and between Tunisian G1 and G6 strains and to estimate the extent of selfing. The dromedary isolates are genetically distinct from sheep isolates (high value of genetic variation between populations: Fst = 0.46. No significant deficiency in heterozygotes was found in sheep isolates, whereas heterozygote deficiency (suggesting selfing was found in a limited number of camel isolates.

  19. Stressor-induced changes to the protoscoleces of Echinococcus granulosus of Indian buffalo origin.

    Science.gov (United States)

    Pan, D; Bhattacharya, D; Bera, A K; Gudewar, J; De, S; Das, S K

    2008-12-01

    In the present study viable protoscoleces of Echinococcus granulosus were exposed to in vitro anthelmintic treatment to observe efficacy against Indian buffalo isolates. Evaluation criteria included morphological changes, viability scores and expression of peptides as a product of prestressed protoscoleces. Protoscolex changes included presence of bladder-like structure and morphological distortion. Two peptides of relative molecular weight (Mr) 40 and 70 kDa were visualized when proteins were separated by discontinuous gel electrophoresis. These two peptides seemed to be products of prestressed protoscoleces.

  20. Echinococcus granulosus: DNA extraction from germinal layers allows strain determination in fertile and nonfertile hydatid cysts.

    Science.gov (United States)

    Kamenetzky, L; Canova, S G; Guarnera, E A; Rosenzvit, M C

    2000-06-01

    A method for the isolation of Echinococcus granulosus DNA from germinal layers of hydatid cysts is described. The method includes a hexadecyltrimethylammonium bromide/chloroform extraction and an adsorption to diatomaceous earth suspension. DNA suitable for polymerase chain reaction was obtained and used for parasite strain determination by mitochondrial cytochrome c oxidase I gene sequencing. Fertile and nonfertile cyst isolates from sheep, cattle, pigs, and humans were characterized. Hitherto, no direct parasite strain characterization has been made on nonfertile hydatid cysts, whereas here we report that nonfertile hydatid cysts were produced by sheep strain (G1 genotype) in sheep, cattle, and humans and by pig strain (G7 genotype) in pigs.

  1. Reprint of "Echinococcus granulosus sensu stricto (s.s.) from the critically endangered antelope Addax nasomaculatus in Tunisia".

    Science.gov (United States)

    Boufana, Belgees; Saïd, Yousra; Dhibi, Mokhtar; Craig, Philip S; Lahmar, Samia

    2017-01-01

    Echinococcus granulosus sensu lato (s.l.) is a zoonotic disease highly endemic in Tunisia. Canids including stray and semi-stray dogs, jackals and foxes are known as definitive hosts and a wide range of ungulates have been shown to harbour the metacestode hydatid stage and may serve as intermediate hosts. Fertile hydatid cysts of Echinococcus equinus and E. granulosus sensu stricto (s.s.) were recently molecularly identified for the first time from Tunisian donkeys. E. granulosus (s.s.) was also identified from wild boars in Tunisia. Here we report the confirmation of hydatid cysts caused by E. granulosus (s.s.) in the critically endangered antelope, Addax nasomaculatus in Tunisia. DNA-based molecular analysis revealed that A. nasomaculatus was infected with E. granulosus (s.s.) which had a 100% identity with the main globally distributed E. granulosus (s.s.) (EgTu01) haplotype. Cysts of Taenia hydatigena (n=33) were also observed on the liver and in the body cavity. Due to their endangered status and their relatively small numbers, it is unlikely that hydatid infection of A. nasomaculatus will form a major contribution to the epidemiology and transmission of E. granulosus in Tunisia, but infection may result in pathology, morbidity and early mortality, and may still play a role in the perpetuation of the parasite in wildlife cycles.

  2. Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus.

    Science.gov (United States)

    Ranasinghe, Shiwanthi L; Fischer, Katja; Zhang, Wenbao; Gobert, Geoffrey N; McManus, Donald P

    2015-12-01

    The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic.

  3. Characterization of the eg95 gene family in the G6 genotype of Echinococcus granulosus.

    Science.gov (United States)

    Alvarez Rojas, Cristian A; Gauci, Charles G; Nolan, Matthew J; Harandi, Majid Fasihi; Lightowlers, Marshall W

    2012-06-01

    Cystic echinococcosis in humans and livestock animals is caused by infection with the cestode parasite Echinococcus granulosus. A number of genotypes of the parasite (designated G1-G10) are known to exist, with the genotype cluster G1-G3 and genotype G6 being responsible for the majority of humans infections. A recombinant vaccine has been developed for use in livestock to prevent infection with E. granulosus. The vaccine is based on the antigen EG95 which is expressed in the early larval stage (oncosphere) of the parasite. The EG95 antigen was originally cloned from the G1 genotype of E. granulosus and the protein has been found to be encoded by members of a small family of related genes in this genotype. Reliable information has not been available about the likely efficacy of the EG95 vaccine against genotypes other than G1. In this study, genomic DNA cloning techniques were used to characterize seven eg95-related gene fragments from the G6 genotype of E. granulosus. Three proteins appear to be encoded by these genes. Considerable differences were found between the EG95 related proteins from the G6 genotype compared with the EG95 protein from the G1 genotype. These differences suggest that the EG95-related proteins from the G6 genotype may have different antigenic epitopes compared with the current vaccine antigen. Data presented in this study have implications for future vaccine design and provide the information that would enable a G6 genotype-specific vaccine to be developed against E. granulosus, should this be considered a desirable addition to the available tools for control of cystic echinococcosis transmission.

  4. Morphological and molecular characterisation of Echinococcus granulosus from goat isolates in Iran.

    Science.gov (United States)

    Rajabloo, Mohammad; Hosseini, Seyed Hossein; Jalousian, Fatemeh

    2012-08-01

    Hydatidosis is considered to be an important economic and human public health problem in Iran, where a variety of animals act as intermediate hosts. There is limited information about the genotypes of Echinococcus granulosus in goats. In this study, 20 isolates of E. granulosus obtained from goats were characterised by mitochondrial DNA sequencing and morphology of the metacestode. The mitochondrial cytochrome oxidase 1 sequences were evaluated, and the sequence analysis indicated two genotypes within the isolates. 17 samples were identified as G1 strain, and 3 isolates were identified as G6 strain. The results of the morphological studies support the findings of the molecular studies. Two types of rostellar hooks were observed in the goat isolates, in agreement with the strain identification. Type 1 hooks were morphologically similar to those of the common sheep strains, whereas the dimensions of the hooks in type 2 were similar to those normally found in the camel strain. The morphological results suggest that Echinococcus of goat origin is phenotypically similar to either the sheep (G1) or the camel (G6) strains. Further, these results suggest that the transmission of the G1 genotype between sheep and goats seems to be the predominant mode of transmission, but further work is required to verify this.

  5. Echinococcus granulosus sensu lato genotypes infecting humans--review of current knowledge.

    Science.gov (United States)

    Alvarez Rojas, Cristian A; Romig, Thomas; Lightowlers, Marshall W

    2014-01-01

    Genetic variability in the species group Echinococcus granulosus sensu lato is well recognised as affecting intermediate host susceptibility and other biological features of the parasites. Molecular methods have allowed discrimination of different genotypes (G1-10 and the 'lion strain'), some of which are now considered separate species. An accumulation of genotypic analyses undertaken on parasite isolates from human cases of cystic echinococcosis provides the basis upon which an assessment is made here of the relative contribution of the different genotypes to human disease. The allocation of samples to G-numbers becomes increasingly difficult, because much more variability than previously recognised exists in the genotypic clusters G1-3 (=E. granulosus sensu stricto) and G6-10 (Echinococcus canadensis). To accommodate the heterogeneous criteria used for genotyping in the literature, we restrict ourselves to differentiate between E. granulosus sensu stricto (G1-3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and E. canadensis (G6-7, G8, G10). The genotype G1 is responsible for the great majority of human cystic echinococcosis worldwide (88.44%), has the most cosmopolitan distribution and is often associated with transmission via sheep as intermediate hosts. The closely related genotypes G6 and G7 cause a significant number of human infections (11.07%). The genotype G6 was found to be responsible for 7.34% of infections worldwide. This strain is known from Africa and Asia, where it is transmitted mainly by camels (and goats), and South America, where it appears to be mainly transmitted by goats. The G7 genotype has been responsible for 3.73% of human cases of cystic echinococcosis in eastern European countries, where the parasite is transmitted by pigs. Some of the samples (11) could not be identified with a single specific genotype belonging to E. canadensis (G6/10). Rare cases of human cystic echinococcosis have been identified as having been caused by

  6. Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels.

    Science.gov (United States)

    Martinez, J; Perez-Serrano, J; Bernadina, W E; Rodriguez-Caabeiro, F

    1999-11-01

    Martinez, J., Perez-Serrano, J., Bernadina, W. E., Rodriguez-Caabeiro, F. 1999 Echinococcus granulosus: In vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels. Experimental Parasitology93, 171-180. Organisms or cells exposed to injurious stresses such as heat shock or chemicals respond by increased (or altered) expression of heat-shock proteins (HSPs). Conversely, an earlier exposure to stress can prepare cells to cope with a subsequent more severe stress. In the present study, protoscolices of Echinococcus granulosus were subjected to several anthelmintic treatments, involving storage of the protoscolices for 18, 30, and 50 h with 0.1 mg/ml of ivermectin (IV), praziquantel (PZ), and a combination of each with albendazole (ALB). The organisms were analyzed for the effects of drug treatment on cell integrity and on levels of hsp60 and hsp70 production. Drug efficacy was evaluated by microscopy and by protein content measurement. Hsp60 and hsp70 were detected by Western blotting and incubation with anti-hsp60 and anti-hsp70 antibody, respectively, and quantitation of these proteins was obtained using image analysis. Incubation with IV alone produced the most damage to the protoscolices as indicated by viability loss, decreased protein content, and altered hsp60 and hsp70 levels; incubation with IV + ALB produced less damage as manifested by fewer changes in the aforementioned damage parameters but PZ and PZ + ALB, in this context, were poor anthelmintics. Exposure of protoscolices to thermal stress prior to anthelmintic treatment, in most cases, increased drug efficacy. It is concluded that in the E. granulosus model system drug efficacy is associated with decreased levels of hsp70 expression and increased levels of hsp60 expression.

  7. Genetic variability of Echinococcus granulosus from the Tibetan plateau inferred by mitochondrial DNA sequences.

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    Yan, Ning; Nie, Hua-Ming; Jiang, Zhong-Rong; Yang, Ai-Guo; Deng, Shi-Jin; Guo, Li; Yu, Hua; Yan, Yu-Bao; Tsering, Dawa; Kong, Wei-Shu; Wang, Ning; Wang, Jia-Hai; Xie, Yue; Fu, Yan; Yang, De-Ying; Wang, Shu-Xian; Gu, Xiao-Bin; Peng, Xue-Rong; Yang, Guang-You

    2013-09-01

    To analyse genetic variability and population structure, 84 isolates of Echinococcus granulosus (Cestoda: Taeniidae) collected from various host species at different sites of the Tibetan plateau in China were sequenced for the whole mitochondrial nad1 (894 bp) and atp6 (513 bp) genes. The vast majority were classified as G1 genotype (n=82), and two samples from human patients in Sichuan province were identified as G3 genotype. Based on the concatenated sequences of nad1+atp6, 28 different haplotypes (NA1-NA28) were identified. A parsimonious network of the concatenated sequence haplotypes showed star-like features in the overall population, with NA1 as the major haplotype in the population networks. By AMOVA it was shown that variation of E. granulosus within the overall population was the main pattern of the total genetic variability. Neutrality indexes of the concatenated sequence (nad1+atp6) were computed by Tajima's D and Fu's Fs tests and showed high negative values for E. granulosus, indicating significant deviations from neutrality. FST and Nm values suggested that the populations were not genetically differentiated.

  8. In vitro and in vivo effects of tamoxifen against larval stage Echinococcus granulosus.

    Science.gov (United States)

    Nicolao, María Celeste; Elissondo, María Celina; Denegri, Guillermo M; Goya, Alejandra B; Cumino, Andrea C

    2014-09-01

    Cystic echinococcosis is a zoonotic infection caused by the larval stage of the cestode Echinococcus granulosus. Chemotherapy currently employs benzimidazoles; however, 40% of cases do not respond favorably. With regard to these difficulties, novel therapeutic tools are needed to optimize treatment in humans. The aim of this work was to explore the in vitro and in vivo effects of tamoxifen (TAM) against E. granulosus. In addition, possible mechanisms for the susceptibility of TAM are discussed in relation to calcium homeostasis, P-glycoprotein inhibition, and antagonist effects on a putative steroid receptor. After 24 h of treatment, TAM, at a low micromolar concentration range (10 to 50 μM), inhibited the survival of E. granulosus protoscoleces and metacestodes. Moreover, we demonstrated the chemotherapeutic and chemopreventive pharmacological effects of the drug. At a dose rate of 20 mg/kg of body weight, TAM induced protection against the infection in mice. In the clinical efficacy studies, a reduction in cyst weight was observed after the administration of 20 mg/kg in mice with cysts developed during 3 or 6 months, compared to that of those collected from control mice. Since the collateral effects of high TAM doses have been largely documented in clinical trials, the use of low doses of this drug as a short-term therapy may be a novel alternative approach for human cystic echinococcosis treatment.

  9. Delivery of Echinococcus granulosus antigen EG95 to mice and sheep using recombinant vaccinia virus.

    Science.gov (United States)

    Dutton, S; Fleming, S B; Ueda, N; Heath, D D; Hibma, M H; Mercer, A A

    2012-06-01

    The tapeworm Echinococcus granulosus is the causative agent of hydatid disease and affects sheep, cattle, dogs and humans worldwide. It has a two-stage life cycle existing as worms in the gut of infected dogs (definitive host) and as cysts in herbivores and humans (intermediate host). The disease is debilitating and can be life threatening where the cysts interfere with organ function. Interruption of the hydatid life cycle in the intermediate host by vaccination may be a way to control the disease, and a protective oncosphere antigen EG95 has been shown to protect animals against challenge with E. granulosus eggs. We explored the use of recombinant vaccinia virus as a delivery vehicle for EG95. Mice and sheep were immunized with the recombinant vector, and the result monitored at the circulating antibody level. In addition, sera from immunized mice were assayed for the ability to kill E. granulosus oncospheres in vitro. Mice immunized once intranasally developed effective oncosphere-killing antibody by day 42 post-infection. Antibody responses and oncosphere killing were correlated and were significantly enhanced by boosting mice with either EG95 protein or recombinant vector. Sheep antibody responses to the recombinant vector or to EG95 protein mirrored those in mice.

  10. In Vitro and In Vivo Effects of Tamoxifen against Larval Stage Echinococcus granulosus

    Science.gov (United States)

    Nicolao, María Celeste; Elissondo, María Celina; Denegri, Guillermo M.; Goya, Alejandra B.

    2014-01-01

    Cystic echinococcosis is a zoonotic infection caused by the larval stage of the cestode Echinococcus granulosus. Chemotherapy currently employs benzimidazoles; however, 40% of cases do not respond favorably. With regard to these difficulties, novel therapeutic tools are needed to optimize treatment in humans. The aim of this work was to explore the in vitro and in vivo effects of tamoxifen (TAM) against E. granulosus. In addition, possible mechanisms for the susceptibility of TAM are discussed in relation to calcium homeostasis, P-glycoprotein inhibition, and antagonist effects on a putative steroid receptor. After 24 h of treatment, TAM, at a low micromolar concentration range (10 to 50 μM), inhibited the survival of E. granulosus protoscoleces and metacestodes. Moreover, we demonstrated the chemotherapeutic and chemopreventive pharmacological effects of the drug. At a dose rate of 20 mg/kg of body weight, TAM induced protection against the infection in mice. In the clinical efficacy studies, a reduction in cyst weight was observed after the administration of 20 mg/kg in mice with cysts developed during 3 or 6 months, compared to that of those collected from control mice. Since the collateral effects of high TAM doses have been largely documented in clinical trials, the use of low doses of this drug as a short-term therapy may be a novel alternative approach for human cystic echinococcosis treatment. PMID:24936598

  11. Morphometric differentiation between camel and sheep strains of Echinococcus granulosus using computer image analysis system (CIAS)

    Institute of Scientific and Technical Information of China (English)

    Gholamreza Mowlavi; Mitra Salehi; Mohammadreza Eshraghian; Mohammad Bagher Rokni; Majid Fasihi-Harandi; Ehsan Mohajeran; Abdoreza Salahi-Moghaddam

    2012-01-01

    Objective:To find importance of morphometric criterion of larval rostellar hook of Echinococcus granulosus (E. granulosus) and the easy and reliable method for distinguish sheep and camel strains in epidemiologic studies. Methods:Larval rostellar hooks (n=1860) of 31 camel and sheep isolates in Iran, which already had been characterized by PCR, were carefully processed by computerized imagime analysis system (CIAS) and acquired data about rostellar hooks were analyzed using software SPSS. Results:Measurement analysis of rostellar hooks [mean length (24.23±3.12) μm] indicated that length of the large hook was a remarkable parameter for strain differentiation. Data analysis demonstrated that CIAS could be used as a reliable tool to distinguish camel from sheep strains with high sensitivity (95.2%) and specificity (91.5%). Conclusions:CIAS as a specific, sensitive, economic, fast, and reliable means might be used for differentiation of E. granulosus strains. Although perimeter and area were measured by digital technology, they were not shown as discriminative criterion as total hook length did.

  12. Shared and non-shared antigens from three different extracts of the metacestode of Echinococcus granulosus

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    David Carmena

    2005-12-01

    Full Text Available Hydatid cyst fluid (HCF, somatic antigens (S-Ag and excretory-secretory products (ES-Ag of Echinococcus granulosus protoscoleces are used as the main antigenic sources for immunodiagnosis of human and dog echinococcosis. In order to determine their non-shared as well as their shared antigenic components, these extracts were studied by ELISA-inhibition and immunoblot-inhibition. Assays were carried out using homologous rabbit polyclonal antisera, human sera from individuals with surgically confirmed hydatidosis, and sera from dogs naturally infected with E. granulosus. High levels of cross-reactivity were observed for all antigenic extracts, but especially for ES-Ag and S-Ag. Canine antibodies evidenced lesser avidity for their specific antigens than antibodies from human origin. The major antigenic components shared by HCF, S-Ag, and ES-Ag have apparent molecular masses of 4-6, 20-24, 52, 80, and 100-104 kDa, including doublets of 41/45, 54/57, and 65/68 kDa. Non-shared polypeptides of each antigenic extract of E. granulosus were identified, having apparent masses of 108 and 78 kDa for HCF, of 124, 94, 83, and 75 kDa for S-Ag, and of 89, 66, 42, 39, 37, and 35 kDa for ES-Ag.

  13. A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries.

    Science.gov (United States)

    Chaâbane-Banaoues, Raja; Oudni-M'rad, Myriam; M'rad, Selim; Amani, Hizem; Mezhoud, Habib; Babba, Hamouda

    2016-10-01

    Cystic echinococcosis, due to Echinococcus granulosus sensu lato (s. l.), currently affects three million people, especially in low-income countries and results in high livestock production loss. DNA-based methods demonstrated genetic variability of E. granulosus s. l., and five species were recognized to belong to the complex, including E. granulosus sensu stricto (s.s) (genotypes G1-G3), Echinococcus equinus (genotype G4), Echinococcus ortleppi (genotype G5), Echinococcus canadensis (genotypes G6-G10), and the lion strain Echinococcus felidis. The characterization of Echinococcus species responsible for human and animal echinococcosis is crucial to adapt the preventive measures against this parasitic disease. The sequencing approach is the gold standard for genotyping assays. Unfortunately, developing countries do not often have access to these techniques. Based on in silico RFLP tools, we described an accurate PCR-RFLP method for Echinococcus spp. characterization. The double digestion with the HaeIII and HinfI restriction enzymes of the PCR product from nad1 gene (1071 bp) led to a clear discrimination between E. granulosus s. l. and most closely related species (Echinococcus shiquicus and Echinococcus multilocularis).Molecular procedures and phylogenetic analysis confirmed the efficiency and the reproducibility of this simple and fast PCR-RFLP method. This technique is proved useful for fresh/unfixed and FF-PET tissues and enables large-scale molecular epidemiological screening in developing countries.

  14. Echinococcus granulosus genotypes circulating in alpacas (Lama pacos and pigs (Sus scrofa from an endemic region in Peru

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    Elizabeth Sánchez

    2012-03-01

    Full Text Available The identification of the genotypes of Echinococcus granulosus present in livestock and wild animals within regions endemic for cystic echinococcosis (CE is epidemiologically important. Individual strains display different biological characteristics that contribute to outbreaks of CE and that must be taken into account in the design of intervention programs. In this study, samples of hydatid cysts due to E. granulosus were collected from alpacas (4 in Puno and pigs (8 in Ayacucho in Peru, an endemic region for CE. Polymerase chain reaction amplification and DNA sequencing of specific regions of the mitochondrial cytochrome C oxidase subunit 1 and NADH dehydrogenase subunit 1 genes confirmed the presence of a strain common to sheep, the G1 genotype, in alpacas. Two different strains of E. granulosus were identified in pigs: the G1 and the G7 genotypes. This is the first report of the G1 genotype of E. granulosus in alpacas in endemic regions of CE in Peru.

  15. The optimum cut-off value to differentiate Echinococcus granulosus sensu stricto from other species of E. granulosus sensu lato using larval rostellar hook morphometry.

    Science.gov (United States)

    Soriano, S V; Pierangeli, N B; Pianciola, L A; Mazzeo, M; Lazzarini, L E; Debiaggi, M F; Bergagna, H F J; Basualdo, J A

    2015-01-01

    Cystic echinococcosis caused by Echinococcus granulosus sensu lato is one of the most important helminth zoonoses in the world; it affects both humans and livestock. The disease is endemic in Argentina and highly endemic in the province of Neuquén. Considerable genetic and phenotypic variation has been demonstrated in E. granulosus, and ten different genotypes (G1-G10) have been identified using molecular tools. Echinococcus granulosus sensu lato may be considered a species complex, comprised of E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-G10). In endemic areas, the characterization of cystic echinococcosis molecular epidemiology is important in order to apply adequate control strategies. A cut-off value for larval large hook total length to distinguish E. granulosus sensu stricto isolates from those produced by other species of the complex was defined for the first time. Overall, 1780 larval hooks of 36 isolates obtained from sheep (n= 11, G1), goats (n= 10, G6), cattle (n= 5, G6) and pigs (n= 10, G7) were analysed. Validation against molecular genotyping as gold standard was carried out using the receiver operating characteristic (ROC) curve analysis. The optimum cut-off value was defined as 26.5 μm. The proposed method showed high sensitivity (97.8%) and specificity (91.1%). Since in most endemic regions the molecular epidemiology of echinococcosis includes the coexistence of the widely distributed E. granulosus sensu stricto G1 strain and other species of the complex, this technique could be useful as a quick and economical tool for epidemiological and surveillance field studies, when fertile cysts are present.

  16. Molecular characterization and seroprevalence of Echinococcus granulosus in wild boars (Sus scrofa) in south-western Iran.

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    Sarkari, Bahador; Mansouri, Majid; Khabisi, Samaneh Abdolahi; Mowlavi, Gholamreza

    2015-01-01

    This study presents the first molecular and serological evaluation of Echinococcus granulosus infections in wild boars in Iran. Twenty five wild boars were collected in south-western Iran, during authorized hunting program, from March to October 2013, necropsied and examined for E. granulosus infection. Furthermore, seroprevalence of cystic echinococcosis in hunted boars was evaluated by an ELISA system. A fertile hydatid cyst due to E. granulosus was detected in the lung of one of the animals. Genotype analysis of the isolate was determined by analyzing a mitochondrial gene, cytochrome C oxidase subunit 1 (co1). DNA was extracted from the cyst sample and polymerase chain reaction amplification and DNA sequencing of the specific region of the co1 gene was performed. Molecular evaluation confirmed the presence of a sheep strain, the G1 genotype, in the wild boar in south-western Iran. This is the first report of the presence of G1 genotype of E. granulosus in wild boar in Iran. Serological evaluation of hydatid cyst by antigen-B ELISA revealed E. granulosus antibodies in 5 (20%) of 25 wild boars. A statistically significant difference was observed between the prevalence of E. granulosus antibodies and gender while the difference between the seroprevalence of E. granulosus and age was insignificant. Findings of this study might have important implications for the prevention and control of cystic echinococcosis.

  17. Echinococcus granulosus: the potential use of specific radiolabelled antibodies in diagnosis by immunoscintigraphy

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    Rogan, M.T.; Morris, D.L.; Pritchard, D.I.; Perkins, A.C. (Nottingham Univ. (UK))

    1990-05-01

    Diagnosis of hydatid disease in man is frequently dependent on the imaging of cysts in situ by techniques such as ultrasonography and CAT scans. Such methods are useful but are not specific and can lead to errors in diagnosis. The present work reports preliminary experiments on the development of a specific imaging technique for hydatid cysts using radiolabelled antibodies. A purified preparation of antigen B of hydatid fluid was used to raise polyclonal antisera in rabbits and the resulting affinity-purified IgG labelled with {sup 131}I. Gerbils with an established Echinococcus granulosus infection were injected intraperitoneally with the labelled antibody and imaged 48 h later with a gamma camera. Hydatid cysts could be identified within the peritoneal cavity and post-mortem assessment of activity showed the cysts to contain approximately four times as much activity as the surrounding organs thereby indicating successful targeting of the antibody to the cysts. (author).

  18. Echinococcus granulosus antigen B: a Hydrophobic Ligand Binding Protein at the host-parasite interface.

    Science.gov (United States)

    Silva-Álvarez, Valeria; Folle, Ana Maite; Ramos, Ana Lía; Zamarreño, Fernando; Costabel, Marcelo D; García-Zepeda, Eduardo; Salinas, Gustavo; Córsico, Betina; Ferreira, Ana María

    2015-02-01

    Lipids are mainly solubilized by various families of lipid binding proteins which participate in their transport between tissues as well as cell compartments. Among these families, Hydrophobic Ligand Binding Proteins (HLBPs) deserve special consideration since they comprise intracellular and extracellular members, are able to bind a variety of fatty acids, retinoids and some sterols, and are present exclusively in cestodes. Since these parasites have lost catabolic and biosynthetic pathways for fatty acids and cholesterol, HLBPs are likely relevant for lipid uptake and transportation between parasite and host cells. Echinococcus granulosus antigen B (EgAgB) is a lipoprotein belonging to the HLBP family, which is very abundant in the larval stage of this parasite. Herein, we review the literature on EgAgB composition, structural organization and biological properties, and propose an integrated scenario in which this parasite HLBP contributes to adaptation to mammalian hosts by meeting both metabolic and immunomodulatory parasite demands.

  19. Echinococcus granulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein.

    Science.gov (United States)

    Cortez-Herrera, E; Yamamoto, R R; Rodrigues, J J; Farias, S E; Ferreira, H B; Zaha, A

    2001-04-01

    We report the isolation and characterization of an Echinococcus granulosus gene that codes for a protein with actin filament fragmenting and nucleating activities (EgAFFP). The genomic region corresponding to the EgAFFP gene presents a coding sequence of 1110 bp that is interrupted by eight introns. The EgAFFP deduced amino acid sequence is about 40% homologous to those of several members of the gelsolin family, such as Physarum polycephalum fragmin, Dictyostelium discoideum severin, and Lumbricus terrestris actin modulator. As do other proteins of the same family, EgAFFP presents three repeated domains, each one characterized by internal conserved amino acid motifs. Assays with fluorescence-labeled actin showed that the full-length recombinant EgAFFP effectively binds actin monomers in both a calcium-dependent and calcium-independent manner and also presents actin nucleating and severing activities.

  20. In Vitro Study of Nitric Oxide Metabolites Effects on Human Hydatid of Echinococcus granulosus.

    Science.gov (United States)

    Zeghir-Bouteldja, Razika; Amri, Manel; Aitaissa, Saliha; Bouaziz, Samia; Mezioug, Dalila; Touil-Boukoffa, Chafia

    2009-01-01

    Hydatidosis is characterized by the long-term coexistence of larva Echinococcus granulosus and its host without effective rejection. Previous studies demonstrated nitric oxide (NO) production (in vivo and in vitro) during hydatidosis. In this study, we investigated the direct in vitro effects of NO species: nitrite (NO(2) (-)), nitrate (NO(3) (-)) and peroxynitrite (ONOO(-)) on protoscolices (PSCs) viability and hydatid cyst layers integrity for 24 hours and 48 hours. Our results showed protoscolicidal activity of NO(2) (-) and ONOO(-) 24 hours and 3 hours after treatment with 320 muM and 80 muM respectively. Degenerative effects were observed on germinal and laminated layers. The comparison of the in vitro effects of NO species on the PSCs viability indicated that ONOO(-) is more cytotoxic than NO(2) (-). In contrast, NO(3) (-) has no effect. These results suggest possible involvement of NO(2) (-) and ONOO(-) in antihydatic action and point the efficacy of these metabolites as scolicidal agents.

  1. Effect of immunization with protoscolices antigens of hydatid cysts on growth of Echinococcus granulosus in dogs

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    M. A. Aljawady

    2010-01-01

    Full Text Available The study was designed to evaluate the immune response of puppies, injected by different protein fractions extracted from protoscolieces, obtained from ovine hydatid cyst. Indirect heamagglutination revealed a remarkable increase in the antibody titers for the immunized groups (A1.5, A3, B1.5, and B3 before and after challenge when compared with the control. Biological variations showed decline in numbers of adult Echinococcus granulosus in the immunized groups when compared with the control. Other variations proved dropping in numbers of the worms within the same immunized groups. Subsequent reductions of cestodes were reported which were 83.8%, 81.3%, 78.2% and 74.6% for the groups A3, A1.5, B3, and B1.5, respectively.

  2. Molecular characterization of Echinococcus granulosus s.l. cysts from cattle, camels, goats and pigs in Ethiopia.

    Science.gov (United States)

    Tigre, Worku; Deresa, Benti; Haile, Adane; Gabriël, Sarah; Victor, Bjorn; Pelt, Jani Van; Devleesschauwer, Brecht; Vercruysse, Jozef; Dorny, Pierre

    2016-01-15

    Cystic Echinococcosis (CE) caused by Echinococcus granulosus sensu lato (s.l.) is a neglected helminth zoonosis affecting humans and various animal species. Human CE has been reported in almost all countries of sub-Saharan Africa but its prevalence and public health impact are subject to large geographical variations. The reasons for these differences are not well understood; among other factors, occurrence of different species/genotypes of E. granulosus s.l. has been suggested. CE is very common in all livestock species in Ethiopia; human CE is poorly documented in the country. The aim of this study was to assess the fertility and molecularly characterize hydatid cysts collected from cattle, camels, goats and pigs from different parts of the country. From the 137 samples characterized by PCR-RFLP and sequencing, 115 (83.9%) were identified as E. granulosus s.s. (G1, common sheep strain), 6 (4.4%) as Echinococcus ortleppi (G5, cattle strain) and 16 (11.7%) as Echinococcus intermedius (G6/7, camel strain). In cattle, E. granulosus s.s. and E. ortleppi were found; in camels and goats, E. granulosus s.s. and E. intermedius; two cysts found in pigs were identified as E. granulosus s.s. and E. ortleppi, respectively. All cysts recovered from goats and pigs were sterile, while fertility was 34% and 50% in cysts from cattle and camels, respectively. In cattle, 31% of E. granulosus s.s. cysts were fertile, showing the importance of cattle in the transmission of the "sheep strain". Next to E. granulosus s.s., E. intermedius (camel strain) was the predominant species: 34.4% of the cysts collected from camels and 62.5% from goats were identified as E. intermedius. These animals originated from the drier Central, Eastern and Southern parts of the country. For the first time, we showed the presence of CE in pigs in Ethiopia. The presence of these strains and especially the fact that the zoonotic E. granulosus s.s. and E. intermedius are dominant, make CE an important public

  3. Bioinformatics analysis and construction of phylogenetic tree of aquaporins from Echinococcus granulosus.

    Science.gov (United States)

    Wang, Fen; Ye, Bin

    2016-09-01

    Cyst echinococcosis caused by the matacestodal larvae of Echinococcus granulosus (Eg), is a chronic, worldwide, and severe zoonotic parasitosis. The treatment of cyst echinococcosis is still difficult since surgery cannot fit the needs of all patients, and drugs can lead to serious adverse events as well as resistance. The screen of target proteins interacted with new anti-hydatidosis drugs is urgently needed to meet the prevailing challenges. Here, we analyzed the sequences and structure properties, and constructed a phylogenetic tree by bioinformatics methods. The MIP family signature and Protein kinase C phosphorylation sites were predicted in all nine EgAQPs. α-helix and random coil were the main secondary structures of EgAQPs. The numbers of transmembrane regions were three to six, which indicated that EgAQPs contained multiple hydrophobic regions. A neighbor-joining tree indicated that EgAQPs were divided into two branches, seven EgAQPs formed a clade with AQP1 from human, a "strict" aquaporins, other two EgAQPs formed a clade with AQP9 from human, an aquaglyceroporins. Unfortunately, homology modeling of EgAQPs was aborted. These results provide a foundation for understanding and researches of the biological function of E. granulosus.

  4. Echinococcus granulosus in Portugal: the first report of the G7 genotype in cattle.

    Science.gov (United States)

    Beato, Sílvia; Parreira, Ricardo; Roque, Cláudio; Gonçalves, Matilde; Silva, Liliana; Maurelli, Maria Paola; Cringoli, Giuseppe; Grácio, Maria Amélia

    2013-11-15

    Although cystic echinococcosis (CE) has been a recognized public health problem in Portugal, molecular data regarding the types and prevalence of infecting strains of its etiological agent (Echinococcus granulosus) are still scarce. Over the last years we have been evaluating the prevalence of CE in the country, and in this report we determined the parasite genotypes infecting sheep, goats, cattle and human in Portugal, based on 209 hydatid cysts recovered from liver (n=96), lung (n=95), pancreas (n=17) and kidney (n=1) samples obtained between 2008 and 2011. Protoscoleces or germinal layers were collected from individual cysts, DNA was extracted, and a part of the mitochondrial DNA encoding the cytochrome c oxidase subunit 1 was amplified by PCR. Overall, the results confirm the overall dominance of the G1-G3 cluster of strains, which are particularly prevalent in southern Portugal in livestock ruminants. Unexpectedly, one parasite sequence with cattle origin was found to correspond to E. granulosus G7 genotype (also known as E. intermedius), here reported for the first time in bovine, in Portugal.

  5. DNA extraction and a cost-effective detection method for Echinococcus granulosus protoscoleces.

    Science.gov (United States)

    Petrigh, R S; Fugassa, M H

    2013-12-01

    Most methods of DNA purification from protoscoleces of Echinococcus granulosus involve the use of expensive kits and may also require a second step after extraction for an effective purification. The present work describes an optimized cost-effective method that is fast and simple. This method is based on a chemical lysis with proteinase K with a subsequent one-step PCR detection. In this study we used already available primers and newly designed primers to amplify two fragments of different size corresponding to the mitochondrial cytochrome C oxidase subunit 1 gene. By one-step PCR, both fragments were successfully amplified from even a single protoscolex. This result demonstrates that this method of extraction is efficient even with small amounts of sample and that PCR is highly sensitive. The major advantage of this lysis-PCR method is that it avoids a second step of purification resulting in a simpler and more economical method. Our research will serve as a base for future studies on E. granulosus genotyping, mainly with wild mammals with a low number of cysts.

  6. Genotyping of Echinococcus granulosus from domestic animals and humans from Ardabil Province, northwest Iran.

    Science.gov (United States)

    Pezeshki, A; Akhlaghi, L; Sharbatkhori, M; Razmjou, E; Oormazdi, H; Mohebali, M; Meamar, A R

    2013-12-01

    Cystic echinococcosis is endemic in Iran, particularly in Ardabil Province, where it causes health and economic problems. The genetic pattern of Echinococcus granulosus has been determined in most parts of Iran, except in this area. In the present investigation, 55 larval isolates were collected from humans (11), sheep (19), goats (4) and cattle (21). For analysis of the genetic characteristics of E. granulosus isolates, DNA sequencing of mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes was applied. Fifty isolates were successfully analysed, with 92% (46) and 8% (4) identified as G1 and G3 genotypes, respectively. The sequence analyses of the isolates displayed nine characteristic profiles in cox1 sequences and eight characteristic profiles in nad1 sequences. Based on these results, the sheep strain (G1 genotype) was the most prevalent in humans, sheep, goats and cattle. The buffalo strain (G3 genotype) was not only demonstrated in sheep (1 isolate) and cattle (1 isolate), but also for the first time in two human isolates. These findings will provide information for local control of echinococcosis.

  7. Construction of In Vivo Fluorescent Imaging of Echinococcus granulosus in a Mouse Model

    Science.gov (United States)

    Wang, Sibo; Yang, Tao; Zhang, Xuyong; Xia, Jie; Guo, Jun; Wang, Xiaoyi; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

    2016-01-01

    Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation. PMID:27417083

  8. Construction of In Vivo Fluorescent Imaging of Echinococcus granulosus in a Mouse Model.

    Science.gov (United States)

    Wang, Sibo; Yang, Tao; Zhang, Xuyong; Xia, Jie; Guo, Jun; Wang, Xiaoyi; Hou, Jixue; Zhang, Hongwei; Chen, Xueling; Wu, Xiangwei

    2016-06-01

    Human hydatid disease (cystic echinococcosis, CE) is a chronic parasitic infection caused by the larval stage of the cestode Echinococcus granulosus. As the disease mainly affects the liver, approximately 70% of all identified CE cases are detected in this organ. Optical molecular imaging (OMI), a noninvasive imaging technique, has never been used in vivo with the specific molecular markers of CE. Thus, we aimed to construct an in vivo fluorescent imaging mouse model of CE to locate and quantify the presence of the parasites within the liver noninvasively. Drug-treated protoscolices were monitored after marking by JC-1 dye in in vitro and in vivo studies. This work describes for the first time the successful construction of an in vivo model of E. granulosus in a small living experimental animal to achieve dynamic monitoring and observation of multiple time points of the infection course. Using this model, we quantified and analyzed labeled protoscolices based on the intensities of their red and green fluorescence. Interestingly, the ratio of red to green fluorescence intensity not only revealed the location of protoscolices but also determined the viability of the parasites in vivo and in vivo tests. The noninvasive imaging model proposed in this work will be further studied for long-term detection and observation and may potentially be widely utilized in susceptibility testing and therapeutic effect evaluation.

  9. Susceptibility and resistance to Echinococcus granulosus infection: Associations between mouse strains and early peritoneal immune responses.

    Science.gov (United States)

    Mourglia-Ettlin, Gustavo; Merlino, Alicia; Capurro, Rafael; Dematteis, Sylvia

    2016-03-01

    In helminth infections, there are no easy associations between host susceptibility and immune responses. Interestingly, immunity to cestodes - unlike most helminths - seems to require Th1-type effectors. In this sense, we reported recently that Balb/c and C57Bl/6 mice are high and low susceptible strains, respectively, to experimental infection by Echinococcus granulosus. However, the role of the early cellular peritoneal response in such differential susceptibility is unknown. Here, we analyzed the kinetics of cytokines expression and cellular phenotypes in peritoneal cells from infected Balb/c and C57Bl/6 mice. Additionally, Principal Components Analysis (PCA) were conducted to highlight the most relevant differences between strains. Finally, the anti-parasite activities of peritoneal cells were assessed through in vitro systems. PCAs clustered C57Bl/6 mice by their early mixed IL-5/TNF-α responses and less intense expression of Th2-type cytokines. Moreover, they exhibited lower counts of eosinophils and higher numbers of macrophages and B cells. Functional studies showed that peritoneal cells from infected C57Bl/6 mice displayed greater anti-parasite activities, in accordance with higher rates of NO production and more efficient ADCC responses. In conclusion, mild Th2-responses and active cellular mechanisms are key determinants in murine resistance to E. granulosus infection, supporting the cestode immune exception among helminth parasites.

  10. Identification of novel glutathione transferases in Echinococcus granulosus. An evolutionary perspective.

    Science.gov (United States)

    Iriarte, Andrés; Arbildi, Paula; La-Rocca, Silvana; Musto, Héctor; Fernández, Verónica

    2012-09-01

    Glutathione transferase enzymes (GSTs) constitute a major detoxification system in helminth parasites and have been related to the modulation of host immune response mechanisms. At least three different GSTs classes have been described in Platyhelminthes: Mu, Sigma and Omega. Mining the genome of Echinococcus multilocularis and the ESTs databases of Taenia solium and E. granulosus identified two new GSTs from the cestode E. granulosus, named EgGST2 and EgGST3. It also revealed that the Omega class of GSTs was absent from the Taenidae family. EgGST2 and EgGST3 are actively expressed in the parasite. In order to know the origin of these new GSTs, in silico analyses were performed. While EgGST2 is classified as belonging to the Sigma class, the data obtained for EgGST3 allowed a less clear interpretation. The study of the evolutionary relatedness based on the C-terminal domain sequence, gene structure conservation and three-dimensional structure predictions, suggests that EgGST3 is derived from the Platyhelminthes' Sigma-class cluster. Interestingly, the N-terminal domain displays some characteristic Omega-class residues, including a Cys residue that is likely to be involved in the catalytic mechanism. We discuss different evolutionary scenarios that could explain the observed patterns.

  11. Identification of Newly Synthesized Proteins by Echinococcus granulosus Protoscoleces upon Induction of Strobilation.

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    João Antonio Debarba

    2015-09-01

    Full Text Available The proteins responsible for the key molecular events leading to the structural changes between the developmental stages of Echinococcus granulosus remain unknown. In this work, azidohomoalanine (AHA-specific labeling was used to identify proteins expressed by E. granulosus protoscoleces (PSCs upon the induction of strobilar development.The in vitro incorporation of AHA with different tags into newly synthesized proteins (NSPs by PSCs was analyzed using SDS-PAGE and confocal microscopy. The LC-MS/MS analysis of AHA-labeled NSPs by PSCs undergoing strobilation allowed for the identification of 365 proteins, of which 75 were differentially expressed in comparison between the presence or absence of strobilation stimuli and 51 were expressed exclusively in either condition. These proteins were mainly involved in metabolic, regulatory and signaling processes.After the controlled-labeling of proteins during the induction of strobilar development, we identified modifications in protein expression. The changes in the metabolism and the activation of control and signaling pathways may be important for the correct parasite development and be target for further studies.

  12. Biochemical analysis of a recombinant glutathione transferase from the cestode Echinococcus granulosus.

    Science.gov (United States)

    Harispe, Laura; García, Gabriela; Arbildi, Paula; Pascovich, Leticia; Chalar, Cora; Zaha, Arnaldo; Fernandez, Cecilia; Fernandez, Veronica

    2010-04-01

    Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 micromol min(-1)mg(-1)), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism.

  13. Activity in mice of recombinant BCG-EgG1Y162 vaccine for Echinococcus granulosus infection.

    Science.gov (United States)

    Ma, Xiumin; Zhao, Hui; Zhang, Fengbo; Zhu, Yuejie; Peng, Shanshan; Ma, Haimei; Cao, Chunbao; Xin, Yan; Yimiti, Delixiati; Wen, Hao; Ding, Jianbing

    2016-01-01

    Cystic hydatid disease is a zoonotic parasitic disease caused by Echinococcus granulosus which is distributed worldwide. The disease is difficult to treat with surgery removal is the only cure treatment. In the high endemic areas, vaccination of humans is believed a way to protect communities from the disease. In this study we vaccinated BALB/c mice with rBCG-EgG1Y162, and then detected the level of IgG and IgE specifically against the recombinant protein by ELISA, rBCG-EgG1Y162 induced strong and specific cellular and humoral immune responses. In vitro study showed that rBCG-EgG1Y162 vaccine not only promote splenocytes proliferation but also active T cell. In addition, the rBCG-EgG1Y162 induced a protection in the mice against secondary infection of Echinococcus granulosus.

  14. The diagnostic importance of species specific and cross-reactive components of Taenia solium, Echinococcus granulosus, and Hymenolepis nana.

    Science.gov (United States)

    Montenegro, T; Gilman, R H; Castillo, R; Tsang, V; Brandt, J; Guevara, A; Sanabria, H; Verastegui, M; Sterling, C; Miranda, E

    1994-01-01

    Sera from patients infected with Taenia solium, Hymenolepis nana and Echinococcus granulosus were tested against homologous and heterologous parasite antigens using an ELISA assay, and a high degree of cross-reactivity was verified. To identify polypeptides responsible for this cross reactivity, the Enzyme Linked Immunoelectro Transfer Blot (EITB) was used. Sera from infected patients with T.solium, H.nana, and E.granulosus were assessed against crude, ammonium sulphate precipitated (TSASP), and lentil-lectin purified antigens of T.solium and crude antigens of H.nana and E.granulosus. Several bands, recognized by sera from patients with T.solium, H.nana, and E.granulosus infections, were common to either two or all three cestodes. Unique reactive bands in H.nana were noted at 49 and 66 K-Da and in E.granulosus at 17-21 K-Da and at 27-32 K-Da. In the crude cysticercosis extract, a specific non glycoprotein band was present at 61-67 K-Da in addiction to specific glycoprotein bands of 50, 42, 24, 21, 18, 14, and 13 K-Da. None of the sera from patients with H.nana or E.granulosus infection cross reacted with these seven glycoprotein bands considered specific for T.solium infection.

  15. Th9/IL-9 profile in human echinococcosis: their involvement in immune response during infection by Echinococcus granulosus.

    Science.gov (United States)

    Pang, Nannan; Zhang, Fengbo; Ma, Xiumin; Zhang, Zhaoxia; Zhao, Hui; Xin, Yan; Wang, Song; Zhu, Yuejie; Wen, Hao; Ding, Jianbing

    2014-01-01

    Th9 cells have been reported to contribute to immune responses; however, the role of Th9 cells in Echinococcus granulosus infection is unknown. This study is to determine whether Th9 cells and IL-9 are involved in human Echinococcus granulosus infection. Compared with healthy controls (HC group), the mRNA levels of PU.1, IL-9, and GATA-3 were significantly increased in patients before therapy (CE group), as revealed by qRT-PCR. Flow cytometry analysis showed that the percentages of Th9 and Th2 cells in CE group were significantly higher. The levels of IL-9, IL-4, IL-10, and TGF- β in CE group were also significantly increased, as detected by CBA assay. The percentages of Th9 and Th2 cells in CE group were positively correlated. After treatments of surgery in combination with albendazole, the PU.1 and GATA-3 mRNA levels were significantly decreased in patients after therapy (PCE group) compared with CE group. The numbers of Th9 and Th2 cells and levels of IL-9, IL-4, IL-10, and TGF- β were also significantly decreased in PCE group. In conclusion, the ratios of Th9 cells and IL-9 levels were significantly decreased after treatment, suggesting that Th9/IL-9 may be involved in immune response induced by Echinococcus granulosus infection.

  16. Th9/IL-9 Profile in Human Echinococcosis: Their Involvement in Immune Response during Infection by Echinococcus granulosus

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    Nannan Pang

    2014-01-01

    Full Text Available Th9 cells have been reported to contribute to immune responses; however, the role of Th9 cells in Echinococcus granulosus infection is unknown. This study is to determine whether Th9 cells and IL-9 are involved in human Echinococcus granulosus infection. Compared with healthy controls (HC group, the mRNA levels of PU.1, IL-9, and GATA-3 were significantly increased in patients before therapy (CE group, as revealed by qRT-PCR. Flow cytometry analysis showed that the percentages of Th9 and Th2 cells in CE group were significantly higher. The levels of IL-9, IL-4, IL-10, and TGF-β in CE group were also significantly increased, as detected by CBA assay. The percentages of Th9 and Th2 cells in CE group were positively correlated. After treatments of surgery in combination with albendazole, the PU.1 and GATA-3 mRNA levels were significantly decreased in patients after therapy (PCE group compared with CE group. The numbers of Th9 and Th2 cells and levels of IL-9, IL-4, IL-10, and TGF-β were also significantly decreased in PCE group. In conclusion, the ratios of Th9 cells and IL-9 levels were significantly decreased after treatment, suggesting that Th9/IL-9 may be involved in immune response induced by Echinococcus granulosus infection.

  17. Cloning,expression,and protective immunity in mice of a gene encoding the diagnostic antigen P-29 of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    Zhiyun Shi; Yana Wang; Zongji Li; Zhaoyu Li; Yang Bo; Rui Ma; Wei Zhao

    2009-01-01

    Taeniid tapeworm Echinococcus granulosus is the causative agent of Echinococcosis,an important zoonosis with worldwide distribution.In this study,a diagnostic antigen P-29 was cloned from E.granulosus and expressed in Escherichia coli.Sequence analysis showed that EgP-29 contains 717-bp open reading frame and encodes a protein of 238 amino acid residues with a predicted molecular weight of 27.1 kDa.The recombinant EgP-29(rEgP-29)could be recognized with antimice sera in Western blotting.The specific antibody was detected by enzyme-linked immunosorbent assay.Mice vaccinated with rEgP-29 and challenged intraperitoneally with E.granulosus protoscoleces revealed significant protective immunity of 96.6%(P<0.05),compared with the control group.Thus,rEgP-29protein is a promising candidate for an effective vaccine to prevent secondary echinococcosis.

  18. A transcriptomic analysis of Echinococcus granulosus larval stages: implications for parasite biology and host adaptation.

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    John Parkinson

    Full Text Available BACKGROUND: The cestode Echinococcus granulosus--the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide--is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages. METHODOLOGY/PRINCIPAL FINDINGS: We generated ~10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces, and pepsin/H(+-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep that could either be active molecular species or represent precursors of small RNAs (like piRNAs; (ii an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii highly expressed thiol- and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv candidate apomucins for the external layer of the tissue-dwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development. CONCLUSIONS/SIGNIFICANCE: This survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the

  19. Molecular Cloning and Characterization of a P38-Like Mitogen-Activated Protein Kinase from Echinococcus granulosus

    Science.gov (United States)

    Lü, Guodong; Li, Jing; Zhang, Chuanshan; Li, Liang; Bi, Xiaojuan; Li, Chaowang; Fan, Jinliang; Lu, Xiaomei; Vuitton, Dominique A.; Wen, Hao; Lin, Renyong

    2016-01-01

    Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus. We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus. Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens p38α, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for p38α. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with TGF-β1 effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus, as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human TGF-β1. PMID:28095661

  20. Genetic diversity of Echinococcus granulosus in southwest China determined by the mitochondrial NADH dehydrogenase subunit 2 gene.

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    Wang, Jiahai; Wang, Ning; Hu, Dandan; Zhong, Xiuqin; Wang, Shuxian; Gu, Xiaobin; Peng, Xuerong; Yang, Guangyou

    2014-01-01

    We evaluated genetic diversity and structure of Echinococcus granulosus by analyzing the complete mitochondrial NADH dehydrogenase subunit 2 (ND2) gene in 51 isolates of E. granulosus sensu stricto metacestodes collected at three locations in this region. We detected 19 haplotypes, which formed a distinct clade with the standard sheep strain (G1). Hence, all 51 isolates were identified as E. granulosus sensu stricto (G1-G3). Genetic relationships among haplotypes were not associated with geographical divisions, and fixation indices (Fst) among sampling localities were low. Hence, regional populations of E. granulosus in the southwest China are not differentiated, as gene flow among them remains high. This information is important for formulating unified region-wide prevention and control measures. We found large negative Fu's Fs and Tajima's D values and a unimodal mismatch distribution, indicating that the population has undergone a demographic expansion. We observed high genetic diversity among the E. granulosus s. s. isolates, indicating that the parasite population in this important bioregion is genetically robust and likely to survive and spread. The data from this study will prove valuable for future studies focusing on improving diagnosis and prevention methods and developing robust control strategies.

  1. In vitro and in vivo effects of flubendazole on Echinococcus granulosus metacestodes.

    Science.gov (United States)

    Elissondo, M; Ceballos, L; Dopchiz, M; Andresiuk, V; Alvarez, L; Bruni, S Sánchez; Lanusse, C; Denegri, G

    2007-04-01

    The aim of the present work was to determine the efficacy of flubendazole (FLBZ) against Echinococcus granulosus metacestodes by using in vitro and in vivo models. Groups of 50 microcysts developed in vitro, and groups of 10 peritoneal cysts were obtained from Balb C mice with experimental secondary infections of 8 months. The cysts were placed in Leighton tubes containing 10 ml of culture medium. FLBZ was added to the medium resulting in final concentrations of 5 and 1 microg/ml for mycrocysts treatment and 10, 5, and 1 microg/ml for murine cysts treatment. In vivo treatment was performed on 20 mice that developed an experimental secondary hydatid disease over a period of 11 months. FLBZ was given (1.5 mg/kg) by the oral route once a day for 50 days. A loss of turgidity was detected in all in vitro drug treated cysts irrespective of the drug concentration or parasite origin. Inspection of treated cysts by scanning electron microscopy (SEM) revealed that the germinal layer lost it characteristic multicelular structure. These results were confirmed on the ultrastructural level by transmission electron microscopy (TEM), treated metacestodes had undergone considerable degenerative changes after the in vitro treatment. The results obtained after the in vivo treatment with FLBZ showed no significant difference between the control and treated groups related to the weight of cyst masses. However, the ultrastructural study at TEM of cysts that developed in mice from the treated group revealed alterations in the germinal layer with the presence of numerous vacuoles. With regard to the ultrastructural study at SEM, only cellular debris of the germinal layer could be seen. In conclusion, the data obtained clearly demonstrate that in vitro and in vivo treatment with FLBZ is effective against E. granulosus metacestodes.

  2. DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts.

    Science.gov (United States)

    Cabrera, Gonzalo; Cabrejos, María Eugenia; Morassutti, Alessandra Loureiro; Cabezón, Carolina; Orellana, Juana; Hellman, Ulf; Zaha, Arnaldo; Galanti, Norbel

    2008-08-01

    Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.

  3. In vitro viability test for the eggs of Echinococcus granulosus: a rapid method.

    Science.gov (United States)

    Moazeni, Mohammad; Rakhshandehroo, Ehsan

    2012-02-01

    In this study an attempt was made to develop an efficient, rapid, simple, and reproducible method for the in vitro viability test of Echinococcus granulosus eggs. The eggs were obtained from an experimentally infected dog and kept at 4°C until use. To prepare the dead or damaged eggs, the eggs were heated in hot water (69-72°C for 10 min), preserved in 70% ethyl alcohol (16 days) or exposed to direct sunlight (18 h). Sodium hypochlorite (0.5-0.7%) was used for the hatching process, and the hatched oncospheres were stained with 0.1% eosin for the viability test. With 0.5% sodium hypochlorite, the hatching rates for viable eggs and eggs killed or damaged by heat (69°C), 70% ethyl alcohol, and direct sunlight were 96%, 97.5%, 91.5%, and 94.6% respectively and there was no significant difference between the hatching rate for viable and dead or damaged eggs (p > 0.05). After staining with 0.1% eosin, the rates of the viable oncospheres hatched from viable eggs and the eggs killed or damaged by heat (69°C), 70% ethyl alcohol, and direct sunlight were 97.5% 3.6%, 7%, and 10.5%, respectively. The difference between the rates of viable oncospheres hatched from viable and dead or damaged eggs was extremely significant (P < 0.0001). With 0.7% sodium hypochlorite, the hatching rates for viable and dead eggs (killed by 72°C for 10 min) were 99.1% and 99.9%, respectively. In this condition, the rate of viable oncospheres was an average of 98.5% for viable eggs and 0.0% for dead ones. The results of this study showed that hatching of eggs by 0.7% sodium hypochlorite and staining of hatched oncospheres by 0.1% eosin are practical methods for the differentiation of viable and nonviable (dead) eggs of Echinococcus granulosus.

  4. Anti-parasitic effect of cyclosporin A on Echinococcus granulosus and characterization of the associated cyclophilin protein.

    Science.gov (United States)

    Colebrook, A L; Jenkins, D D; Lightowlers, M W

    2002-11-01

    Cyclophilins are a family of proteins found ubiquitously in eukaryotes, many of which bind to the immunosuppressive drug cyclosporin A (CsA). CsA has been found to have anti-parasitic effects against a variety of helminth and protozoan parasites and this activity could be mediated via cyclophilin. In this study we characterize a full length cyclophilin gene from Echinococcus granulosus, the associated natural gene and expression pattern, and investigate the functional properties of the recombinant E. granulosus cyclophilin protein. In addition, the effects of CsA were investigated on E. granulosus protoscoleces in in vitro culture. The full length E. granulosus cyclophilin cDNA encodes a protein of 20 kDa and is encoded by a single gene (EGCyP-1) comprising 2 exons separated by a 31 bp intron. The gene is expressed constitutively in all E. granulosus life-cycle stages examined. Recombinant E. granulosus cyclophilin (egCyP-l) exhibited functional enzyme activity as an isomerase. Treatment of in vitro cultures of E. granulosus protoscoleces with CsA was found to be lethal to the parasites. No protoscoleces survived treatment with 10 microg/ml of CsA over 7 culture days, as determined by observing motility and the uptake of toluidine blue dye. Untreated protoscoleces remained viable for the duration of experiments. The survival of protoscoleces was CsA dose dependent. A concentration of 10 microg/ml CsA was 100% lethal while doses of 8 microg/ml and 5 microg/ml resulted in 82% and 32% killing, respectively, after 7 days in culture. The anti-parasitic activity of CsA may have the potential to be developed as a new therapeutic agent for treatment of cystic hydatidosis in humans.

  5. Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes.

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    Cucher, M; Prada, L; Mourglia-Ettlin, G; Dematteis, S; Camicia, F; Asurmendi, S; Rosenzvit, M

    2011-03-01

    The aetiological agent of cystic hydatid disease, the platyhelminth parasite Echinococcus granulosus, undergoes a series of metamorphic events during its complex life cycle. One of its developmental stages, the protoscolex, shows a remarkable degree of heterogeneous morphogenesis, being able to develop either into the vesicular or strobilar direction. Another level of complexity is added by the existence of genotypes or strains that differ in the range of intermediate hosts where they can develop and form fertile cysts. These features make E. granulosus an interesting model for developmental studies. Hence, we focused on the study of the regulation of gene expression by microRNAs (miRNAs), one of the key mechanisms that control development in metazoans and plants and which has not been analysed in E. granulosus yet. In this study, we cloned 38 distinct miRNAs, including four candidate new miRNAs that seem to be specific to Echinococcus spp. Thirty-four cloned sequences were orthologous to miRNAs already described in other organisms and were grouped in 16 metazoan miRNA families, some of them known for their role in the development of other organisms. The expression of some of the cloned miRNAs differs according to the parasite life cycle stage analysed, showing differential developmental expression. We did not detect differences in the expression of the analysed miRNAs between protoscoleces of two parasite genotypes. This work sets the scene for the study of gene regulation mediated by miRNAs in E. granulosus and provides a new approach to study the molecules involved in its developmental plasticity and intermediate host specificity. Understanding the developmental processes of E. granulosus may help to find new strategies for the control of cystic hydatid disease, caused by the metacestode stage of the parasite.

  6. Polyfunctional Specific Response to Echinococcus Granulosus Associates to the Biological Activity of the Cysts.

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    Linda Petrone

    2015-11-01

    Full Text Available Cystic echinococcosis (CE is a complex disease caused by Echinococcus granulosus (E.granulosus, and its immunophatogenesis is still not clearly defined. A peculiar feature of chronic CE is the coexistence of Th1 and Th2 responses. It has been suggested that Th1 cytokines are related to disease resistance, whereas Th2 cytokines are related to disease susceptibility and chronicity. The aim of this study was to evaluate, by multi-parametric flow cytometry (FACS, the presence of CE specific immune signatures.We enrolled 54 subjects with suspected CE; 42 of them had a confirmed diagnosis, whereas 12 were classified as NO-CE. Based on the ultrasonography images, CE patients were further categorized as being in "active stages" (25 and "inactive stages" (17. The ability of CD4+ T-cells to produce IFN-γ, IL-2, TNF-α, Th2 cytokines or IL-10 was assessed by FACS on antigen-specific T-cells after overnight stimulation with Antigen B (AgB of E.granulosus. Cytokine profiles were evaluated in all the enrolled subjects. The results show that none of the NO-CE subjects had a detectable AgB-specific response. Among the CE patients, the frequency and proportions of AgB-specific CD4+ T-cells producing IL-2+TNF-α+Th2+ or TNF-α+Th2+ were significantly increased in the "active stages" group compared to the "inactive stages" group. Moreover, an increased proportion of the total polyfunctional subsets, as triple-and double-functional CD4 T-cells, was found in CE patients with active disease. The response to the mitogen, used as a control stimulus to evaluate the immune competence status, was characterized by the same cytokine subsets in all the subjects enrolled, independent of CE.We demonstrate, for the first time to our knowledge, that polyfunctional T-cell subsets as IL-2+TNF-α+Th2+ triple-positive and TNF-α+Th2+ double-positive specific T-cells associate with cyst biological activity. These results contribute to increase knowledge of CE immunophatogenesis and

  7. Identification and bioinformatics analysis of lactate dehydrogenase genes fromEchinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    Gang Lu; Yajun Lu; Lihua Li; Lixian Wu; Zhigang Fan; Dazhong Shi; Hu Wang; Xiumin Han

    2010-01-01

    Objective:To identify full length cDNA sequence of lactate dehydrogenase(LDH) from adultEchinococcus granulosus (E. granulosus) and to predict the structure and function of its encoding protein using bioinformatics methods.Methods: With the help ofNCBI, EMBI, Expasy and other online sites, the open reading frame (ORF), conserved domain, physical and chemical parameters, signal peptide, epitope, topological structures of the protein sequences were predicted and a homology tertiary structure model was created; VectorNTI software was used for sequence alignment, phylogenetic tree construction and tertiary structure prediction. Results: The target sequence was1 233 bp length with a996 bp biggestORFencoding331 amino acids protein with typicalL-LDH conserved domain. It was confirmed as full length cDNA of LDH fromE. granulosus and named asEgLDH (GenBank accession number:HM748917). The predicted molecular weight and isoelectric point of the deduced protein were3 5516.2Da and6.32 respectively. Compared withLDHs fromTaenia solium, Taenia saginata asiatica, Spirometra erinaceieuropaei, Schistosoma japonicum, Clonorchis sinensis and human, it showed similarity of 86%, 85%, 55%, 58%, 58% and 53%, respectively. EgLDH contained3putative transmembrane regions and4 major epitopes (54aa-59aa,81aa-87aa,97aa-102aa,307aa-313aa), the latter were significant different from the corresponding regions of humanLDH. In addition, someNAD and substrate binding sites located on epitopes54aa-59aa and97aa-102aa, respectively. Tertiary structure prediction showed that3 key catalytic residues105R, 165D and192H forming a catalytic center near the epitope97aa-102aa, mostNAD and substrate binding sites located around the center.Conclusions: The full length cDNA sequences of EgLDH were identified. It encoded a putative transmembrane protein which might be an ideal target molecule for vaccine and drugs.

  8. In Vitro Scolicidal Effects of Salvadora persica Root Extract against Protoscolices of Echinococcus granulosus.

    Science.gov (United States)

    Abdel-Baki, Abdel-Azeem S; Almalki, Esam; Mansour, Lamjed; Al-Quarishy, Saleh

    2016-02-01

    It has been known that Arak, Salvadora persica, has a number of medicinal properties. We tried to investigate in vitro scolicidal effect of root extracts of this plant against protoscolices from hydatid cysts of Echinococcus granulosus. Protoscolices were aseptically collected from sheep livers containing hydatid cysts. S. persica root extract was used in 10, 30, and 50 mg/ml concentration for 10, 20, and 30 min. The viability of protoscolices was ascertained by 0.1% eosin staining. Scolicidal activity of S. persica extract at a concentration of 10 mg/ml was 36.3%, 50.3%, and 70.8% after 10, 20, and 30 min of exposure, respectively. The scolicidal effect of this extract at a concentration of 30 mg/ml was 52.9%, 86.7%, and 100% after 10, 20, and 30 min of exposure, respectively. S. persica extract at a concentration of 50 mg/ml, meanwhile, killed 81.4%, 100%, and 100% of protoscolices after 10, 20, and 30 min, respectively. Also, the cytotoxic potential of S. persica was assessed on human liver cells (HepG2) using trypan blue exclusion test. No cytotoxic effect was observed on HepG2 cell line. The present study confirmed for the first time that the ethanolic extract of S. persica has high scolicidal power in vitro. However, in vivo effect of this material remains to be studied for treatment of echinococcosis in humans and herbivorous animals.

  9. Excretory/secretory products from in vitro-cultured Echinococcus granulosus protoscoleces.

    Science.gov (United States)

    Virginio, Veridiana G; Monteiro, Karina M; Drumond, Fernanda; de Carvalho, Marcos O; Vargas, Daiani M; Zaha, Arnaldo; Ferreira, Henrique B

    2012-05-01

    Cystic hydatid disease (CHD) is caused by infection with Echinococcus granulosus metacestodes and affects humans and livestock. Proteins secreted or excreted by protoscoleces, pre-adult worms found in the metacestode, are thought to play fundamental roles in the host-parasite relationship. In this work, we performed an LC-MS/MS proteomic analysis of the excretory-secretory products obtained from the first 48 h of an in vitro culture of the protoscoleces. We identified 32 proteins, including 18 that were never detected previously in metacestode proteomic studies. Among the novel identified excretory-secretory products are antigenic proteins, such as EG19 and P-29 and a calpain protease. We also identified other important protoscolex excretory-secretory products, such as thioredoxin peroxidase and 14-3-3 proteins, which are potentially involved in evasion mechanisms adopted by parasites to establish infection. Several intracellular proteins were found in the excretory-secretory products, revealing a set of identified proteins not previously thought to be exposed at the host-parasite interface. Additionally, immunological analyses established the antigenic profiles of the newly identified excretory-secretory products and revealed, for the first time, the in vitro secretion of the B antigen by protoscoleces. Considering that the excretory-secretory products obtained in vitro might reflect the products released and exposed to the host in vivo, our results provide valuable information on parasite survival strategies in adverse host environments and on the molecular mechanisms underpinning CHD immunopathology.

  10. Assessment of in vivo complement activation on the Echinococcus granulosus hydatid cyst wall.

    Science.gov (United States)

    Ferreira, A M; Diaz, A; Fernandez, C; Sim, R B

    2001-12-01

    The larval stage of the parasite Echinococcus granulosus causes hydatid disease. The hydatid cyst is potentially capable of activating host complement, since it is a large, persistent, carbohydrate-rich structure, coated with host immunoglobulins, and localized in the host's internal organs. Nonetheless, in vitro studies have suggested that the cyst surface, the hydatid cyst wall (HCW), is a poor complement activator. In this study, we assessed the occurrence of in vivo complement activation on the hydatid cyst by measuring the levels of two complement activation products, C3d and complexes bearing a C9 activation neoepitope (TCC/MAC), in extracts from HCW of human origin. Low amounts of C3d and TCC/MAC were found in HCW in comparison with their levels in normal human plasma and activated human sera, suggesting that in vivo complement activation on HCW is efficiently down-regulated. This regulation may contribute to limit host inflammation which has been observed to correlate with parasite degeneration and death.

  11. Bovine IgG subclasses and fertility of Echinococcus granulosus hydatid cysts.

    Science.gov (United States)

    Riesle, Silke; García, María Pía; Hidalgo, Christian; Galanti, Norbel; Saenz, Leonardo; Paredes, Rodolfo

    2014-09-15

    Hydatidosis is an important zoonotic disease of worldwide distribution, causing important health problems to humans and major economical losses in infected livestock. Echinococcus granulosus, the etiological agent of hydatid disease, induces a humoral immune response in the intermediate host (human and herbivorous) against hydatid cyst antigens. Specifically, IgGs are found in the laminar and germinal layers and inside the lumen of fertile and infertile hydatid cysts. In the germinal layer of infertile cysts IgGs are found in an order of magnitude greater than in the germinal layer of fertile cysts; a fraction of those IgGs are associated with high affinity to germinal layer proteins, suggesting their binding to specific parasite antigens. We have previously shown that those immunoglobulins, bound with high affinity to the germinal layer of hydatid cysts, induce apoptosis leading to cyst infertility. In the present work the presence of IgG1 and IgG2 subclasses in the germinal layer of both fertile and infertile hydatid cysts is reported. IgG1 is the most relevant immunoglobulin subclass present in the germinal layer of infertile cysts and bound with high affinity to that parasite structure. Contrarily, though the IgG2 subclass was also found in the germinal and adventitial layers, those immunoglobulins show low affinity to parasite antigens. We propose that the binding of an IgG1 subclass to parasite antigens present in the germinal layer is involved in the mechanism of cyst infertility.

  12. In Vitro Study of Nitric Oxide Metabolites Effects on Human Hydatid of Echinococcus granulosus

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    Razika Zeghir-Bouteldja

    2009-01-01

    Full Text Available Hydatidosis is characterized by the long-term coexistence of larva Echinococcus granulosus and its host without effective rejection. Previous studies demonstrated nitric oxide (NO production (in vivo and in vitro during hydatidosis. In this study, we investigated the direct in vitro effects of NO species: nitrite (NO2−, nitrate (NO3− and peroxynitrite (ONOO− on protoscolices (PSCs viability and hydatid cyst layers integrity for 24 hours and 48 hours. Our results showed protoscolicidal activity of NO2− and ONOO− 24 hours and 3 hours after treatment with 320 μM and 80 μM respectively. Degenerative effects were observed on germinal and laminated layers. The comparison of the in vitro effects of NO species on the PSCs viability indicated that ONOO− is more cytotoxic than NO2−. In contrast, NO3− has no effect. These results suggest possible involvement of NO2− and ONOO− in antihydatic action and point the efficacy of these metabolites as scolicidal agents.

  13. In Vitro Anti-Echinococcal and Metabolic Effects of Metformin Involve Activation of AMP-Activated Protein Kinase in Larval Stages of Echinococcus granulosus.

    Science.gov (United States)

    Loos, Julia A; Cumino, Andrea C

    2015-01-01

    Metformin (Met) is a biguanide anti-hyperglycemic agent, which also exerts antiproliferative effects on cancer cells. This drug inhibits the complex I of the mitochondrial electron transport chain inducing a fall in the cell energy charge and leading 5'-AMP-activated protein kinase (AMPK) activation. AMPK is a highly conserved heterotrimeric complex that coordinates metabolic and growth pathways in order to maintain energy homeostasis and cell survival, mainly under nutritional stress conditions, in a Liver Kinase B1 (LKB1)-dependent manner. This work describes for the first time, the in vitro anti-echinococcal effect of Met on Echinococcus granulosus larval stages, as well as the molecular characterization of AMPK (Eg-AMPK) in this parasite of clinical importance. The drug exerted a dose-dependent effect on the viability of both larval stages. Based on this, we proceeded with the identification of the genes encoding for the different subunits of Eg-AMPK. We cloned one gene coding for the catalytic subunit (Eg-ampkɑ) and two genes coding for the regulatory subunits (Eg-ampkβ and Eg-ampkγ), all of them constitutively transcribed in E. granulosus protoscoleces and metacestodes. Their deduced amino acid sequences show all the conserved functional domains, including key amino acids involved in catalytic activity and protein-protein interactions. In protoscoleces, the drug induced the activation of AMPK (Eg-AMPKɑ-P176), possibly as a consequence of cellular energy charge depletion evidenced by assays with the fluorescent indicator JC-1. Met also led to carbohydrate starvation, it increased glucogenolysis and homolactic fermentation, and decreased transcription of intermediary metabolism genes. By in toto immunolocalization assays, we detected Eg-AMPKɑ-P176 expression, both in the nucleus and the cytoplasm of cells as in the larval tegument, the posterior bladder and the calcareous corpuscles of control and Met-treated protoscoleces. Interestingly, expression of Eg

  14. In Vitro Anti-Echinococcal and Metabolic Effects of Metformin Involve Activation of AMP-Activated Protein Kinase in Larval Stages of Echinococcus granulosus.

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    Julia A Loos

    Full Text Available Metformin (Met is a biguanide anti-hyperglycemic agent, which also exerts antiproliferative effects on cancer cells. This drug inhibits the complex I of the mitochondrial electron transport chain inducing a fall in the cell energy charge and leading 5'-AMP-activated protein kinase (AMPK activation. AMPK is a highly conserved heterotrimeric complex that coordinates metabolic and growth pathways in order to maintain energy homeostasis and cell survival, mainly under nutritional stress conditions, in a Liver Kinase B1 (LKB1-dependent manner. This work describes for the first time, the in vitro anti-echinococcal effect of Met on Echinococcus granulosus larval stages, as well as the molecular characterization of AMPK (Eg-AMPK in this parasite of clinical importance. The drug exerted a dose-dependent effect on the viability of both larval stages. Based on this, we proceeded with the identification of the genes encoding for the different subunits of Eg-AMPK. We cloned one gene coding for the catalytic subunit (Eg-ampkɑ and two genes coding for the regulatory subunits (Eg-ampkβ and Eg-ampkγ, all of them constitutively transcribed in E. granulosus protoscoleces and metacestodes. Their deduced amino acid sequences show all the conserved functional domains, including key amino acids involved in catalytic activity and protein-protein interactions. In protoscoleces, the drug induced the activation of AMPK (Eg-AMPKɑ-P176, possibly as a consequence of cellular energy charge depletion evidenced by assays with the fluorescent indicator JC-1. Met also led to carbohydrate starvation, it increased glucogenolysis and homolactic fermentation, and decreased transcription of intermediary metabolism genes. By in toto immunolocalization assays, we detected Eg-AMPKɑ-P176 expression, both in the nucleus and the cytoplasm of cells as in the larval tegument, the posterior bladder and the calcareous corpuscles of control and Met-treated protoscoleces. Interestingly

  15. Echinococcus P29 antigen: molecular characterization and implication on post-surgery follow-up of CE patients infected with different species of the Echinococcus granulosus complex.

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    Boubaker, Ghalia; Gottstein, Bruno; Hemphill, Andrew; Babba, Hamouda; Spiliotis, Markus

    2014-01-01

    The protein P29 is a potential serological marker for post-treatment monitoring of cystic echinococcosis (CE) especially in young patients. We now have demonstrated that P29 is encoded in the Echinococcus genus by a single gene consisting of 7 exons spanning 1.2 kb of DNA. Variability of the p29 gene at inter- and intra-species level was assessed with 50 cDNA and 280 genomic DNA clones isolated from different E. granulosus s.l. isolates (E. granulosus sensu stricto (G1), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6), E. canadensis (G7) and E. canadensis (G10)) as well as four E. multilocularis isolates. Scarce interspecies polymorphism at the p29 locus was observed and affected predominantly E. granulosus s.s. (G1), where we identified two alleles (A1 and A2) coding for identical P29 proteins and yielding in three genotypes (A1/A1, A2/A2 and A1/A2). Genotypic frequencies expected under Hardy-Weinberg equilibrium revealed a high rate of heterozygosity (47%) that strongly supports the hypothesis that E. granulosus s.s. (G1) is predominantly outbreeding. Comparative sequence analyses of the complete p29 gene showed that phylogenetic relationships within the genus Echinococcus were in agreement with those of previous nuclear gene studies. At the protein level, the deduced P29 amino acid (AA) sequences exhibited a high level of conservation, ranging from 97.9% AA sequence identity among the whole E. granulosus s.l. group to 99.58% identity among E. multilocularis isolates. We showed that P29 proteins of these two species differ by three AA substitutions without implication for antigenicity. In Western-blot analyses, serum antibodies from a human CE patient infected with E. canadensis (G6) strongly reacted with recombinant P29 from E. granulosus s.s. (G1) (recEg(G1)P29). In the same line, human anti-Eg(G1)P29 antibodies bound to recEcnd(G6)P29. Thus, minor AA sequence variations appear not to impair the prognostic serological use of P29.

  16. A loop-mediated isothermal amplification (LAMP) method for the identification of species within the Echinococcus granulosus complex.

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    Wassermann, Marion; Mackenstedt, Ute; Romig, Thomas

    2014-02-24

    To facilitate the specific identification of Echinococcus spp. isolates in endemic countries, a LAMP (loop-mediated isothermal amplification) assay was developed to detect the various agents known to cause cystic echinococcosis (E. granulosus s.s., E. equinus, E. ortleppi, E. canadensis and E. felidis). The infectivity of the different species and the severity of the disease in humans and livestock vary significantly among those species, and correct molecular identification of large numbers of field isolates is crucial to understand their epidemiology. However, funding constraints in many CE endemic countries often prevent PCR-based screening of field isolates. The LAMP method allows the amplification of DNA fragments under isothermal conditions which can be achieved using an ordinary waterbath, and the detection of amplification products only requires a UV light source. In the present study a LAMP assay was developed which allows the detection and differentiation of the 5 CE causing Echinococcus species. The diagnostic power was adjusted to species level, i.e. intraspecific strains (G1-3 within E. granulosus s.s., G6-10 within E. canadensis) are not discriminated. Wherever this would be necessary for epidemiological purposes, the method can be adjusted according to local requirements. The sensitivity of the assay was tested down to one fiftieth of a single protoscolex or egg, respectively. The present study describes a fast and simple method for the differentiation of CE causing Echinococcus species which can facilitate epidemiological studies in endemic countries.

  17. [Detection of Echinococcus granulosus and Echinococcus multilocularis in cyst samples using a novel single tube multiplex real-time polymerase chain reaction].

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    Can, Hüseyin; İnceboz, Tonay; Caner, Ayşe; Atalay Şahar, Esra; Karakavuk, Muhammet; Döşkaya, Mert; Çelebi, Fehmi; Değirmenci Döşkaya, Aysu; Gülçe İz, Sultan; Gürüz, Yüksel; Korkmaz, Metin

    2016-04-01

    Cystic echinococcosis (CE) and alveolar echinococcosis (AE) caused by Echinococcus granulosus and Echinococcus multilocularis, respectively, are important helminthic diseases worldwide as well as in our country. Epidemiological studies conducted in Turkey showed that the prevalence of CE is 291-585/100.000. It has also been showed that the seroprevalence of AE is 3.5%. For the diagnosis of CE and AE, radiological (ultrasonography, computed tomography, magnetic resonance) and serological methods, in addition to clinical findings, are being used. The definitive diagnosis relies on pathological examination When the hydatid cysts are sterile or does not contain protoscolex, problems may occur during pathological discrimination of E.granulosus and E.multilocularis species. In this study, we aimed to develop a novel multiplex real-time polymerase chain reaction (M-RT-PCR) targeting mitochondrial 12S rRNA gene of E.granulosus and E.multilocularis using Echi S (5'-TTTATGAATATTGTGACCCTGAGAT-3') and Echi A (5'-GGTCTTAACTCAACTCATGGAG-3') primers and three different probes; Anchor Ech (5'-GTTTGCCACCTCGATGTTGACTTAG-fluoroscein-3'), Granulosus (5'-LC640-CTAAGGTTTTGGTGTAGTAATTGATATTTT-phosphate-3') and Multilocularis (5'-LC705-CTGTGATCTTGGTGTAGTAGTTGAGATT-phosphate-3') that will enable the diagnosis of CE and AE in same assay. During M-RTR-PCR, plasmids containing E.granulosus (GenBank: AF297617.1) and E.multilocularis (GenBank: NC_000928.2) mitochondrial 12S rRNA regions were used as positive controls. Cysts samples of patients which were pathologically confirmed to be CE (n: 10) and AE (n: 15) and healthy human DNA samples (n: 25) as negative control as well as DNA samples of 12 different parasites (Taenia saginata, Hymenolepis nana, Trichuris trichiura, Fasciola hepatica, Enterobius vermicularis, Toxoplasma gondii, Pneumocystis jirovecii, Trichomonas vaginalis, Cryptosporidium hominis, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax) were used to develop M

  18. A protective effect of the laminated layer on Echinococcus granulosus survival dependent on upregulation of host arginase.

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    Amri, Manel; Touil-Boukoffa, Chafia

    2015-09-01

    The role of nitric oxide (NO) in host defense against Echinococcus granulosus larvae was previously reported. However, NO production by NOS2 (inducible NO synthase) is counteracted by the expression of Arginase. In the present study, our aim is to evaluate the involvement of the laminated layer (external layer of parasitic cyst) in Arginase induction and the protoscoleces (living and infective part of the cyst) survival. Our in vitro results indicate that this cystic compound increases the Arginase activity in macrophages. Moreover, C-type lectin receptors (CLRs) with specificity for mannan and the TGF-β are implicated in this effect as shown after adding Mannan and Anti-TGFβ. Interestingly, the laminated layer increases protoscoleces survival in macrophages-parasite co-cultures. Our results indicate that the laminated layer protects E. granulosus against the NOS2 protective response through Arginase pathway, a hallmark of M2 macrophages.

  19. Boiling sheep liver or lung for 30 minutes is necessary and sufficient to kill Echinococcus granulosus protoscoleces in hydatid cysts

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    Li Jun

    2014-01-01

    Full Text Available Proper disposal of carcasses and offal after home slaughter is difficult in poor and remote communities and therefore dogs readily have access to hydatid cysts containing offal from livestock, thus completing the parasite cycle of Echinococcus granulosus and putting communities at risk of cystic echinococcosis. Boiling livers and lungs which contain hydatid cysts could be a simple, efficient and energy- and time-saving way to kill the infectious protoscoleces. The aim of this study was to provide precise practical recommendations to livestock owners. Our results show that boiling the whole sheep liver and/or lung, with single or multiple hydatid cysts, for 30 min is necessary and sufficient to kill E. granulosus protoscoleces in hydatid cysts. Advertising on this simple rule in at-risk communities would be an efficient and cheap complement to other veterinary public health operations to control cystic echinococcosis.

  20. Echinococcus granulosus infection and options for control of cystic echinococcosis in Tibetan communities of Western Sichuan Province, China.

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    Yu Rong Yang

    Full Text Available BACKGROUND: Human cystic echinococcosis (CE is highly endemic in the Tibetan regions of Sichuan where most families keep guard dogs and where there are considerable numbers of ownerless/stray dogs. Strong Buddhist beliefs do not allow for elimination of stray dogs, and many strays are actually fed and adopted by households or monasteries. On account of the high altitude (3900-5000 m, pasturage is the major agricultural activity in this area. The harsh mountainous climate often leads to many grazing animals dying on the pasture at the end of a hard winter. The skin and some meat are taken, and the rest of the animal is left for scavenging birds and animals. The poor sanitation and hygiene, the Buddhist doctrine of allowing old livestock to die naturally, plus the unrestricted disposal of animal viscera post-slaughter may be responsible for the high prevalence of human CE in this setting. METHODS AND FINDINGS: As part of a large collaborative control program for CE in Ganzi County, situated in the west of Sichuan Province, surveillance for Echinococcus infection in domestic dogs using a coproantigen method and necropsy of unwanted dogs was carried out prior to (in 2000 and after (in 2005 dog anthelminthic treatment (5 mg/kg oral praziquantal at 6 month intervals to determine the efficacy of the treatment for control. The prevalence of E. granulosus only in dogs by necropsy was 27% and 22%, and prevalence of both Echinococcus spp. by necropsy was 63% and 38%; prevalence of both Echinococcus spp. by coproantigen analysis was 50% and 17%. Necropsy of sheep/goats (age <1 to 12 years (prevalence of E. granulosus in 1-6-year-old animals was 38% and in 10-12-year-old animals was 70% and yaks (age 4 years (prevalence of E. granulosus was 38% was undertaken to determine the baseline transmission pressure. Protoscoleces were only found in very old sheep/goats and yaks. Necropsy of dogs in the Datangma district indicated that there was no apparent

  1. Genetic diversity and population genetic structure analysis of Echinococcus granulosus sensu stricto complex based on mitochondrial DNA signature.

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    Monika Sharma

    Full Text Available The genetic diversity and population genetics of the Echinococcus granulosus sensu stricto complex were investigated based on sequencing of mitochondrial DNA (mtDNA. Total 81 isolates of hydatid cyst collected from ungulate animals from different geographical areas of North India were identified by sequencing of cytochrome c oxidase subunit1 (coxi gene. Three genotypes belonging to E. granulosus sensu stricto complex were identified (G1, G2 and G3 genotypes. Further the nucleotide sequences (retrieved from GenBank for the coxi gene from seven populations of E. granulosus sensu stricto complex covering 6 continents, were compared with sequences of isolates analysed in this study. Molecular diversity indices represent overall high mitochondrial DNA diversity for these populations, but low nucleotide diversity between haplotypes. The neutrality tests were used to analyze signatures of historical demographic events. The Tajima's D test and Fu's FS test showed negative value, indicating deviations from neutrality and both suggested recent population expansion for the populations. Pairwise fixation index was significant for pairwise comparison of different populations (except between South America and East Asia, Middle East and Europe, South America and Europe, Africa and Australia, indicating genetic differentiation among populations. Based on the findings of the present study and those from earlier studies, we hypothesize that demographic expansion occurred in E. granulosus after the introduction of founder haplotype particular by anthropogenic movements.

  2. Characterisation of the native lipid moiety of Echinococcus granulosus antigen B.

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    Gonzalo Obal

    Full Text Available Antigen B (EgAgB is the most abundant and immunogenic antigen produced by the larval stage (metacestode of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5. In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40-50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters and polar (mainly phosphatidylcholine lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9 are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding

  3. Characterisation of the native lipid moiety of Echinococcus granulosus antigen B.

    Science.gov (United States)

    Obal, Gonzalo; Ramos, Ana Lía; Silva, Valeria; Lima, Analía; Batthyany, Carlos; Bessio, María Inés; Ferreira, Fernando; Salinas, Gustavo; Ferreira, Ana María

    2012-01-01

    Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40-50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid

  4. Characterisation of the Native Lipid Moiety of Echinococcus granulosus Antigen B

    Science.gov (United States)

    Obal, Gonzalo; Ramos, Ana Lía; Silva, Valeria; Lima, Analía; Batthyany, Carlos; Bessio, María Inés; Ferreira, Fernando; Salinas, Gustavo; Ferreira, Ana María

    2012-01-01

    Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40–50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid

  5. Anthelmintic effect of Mentha spp. essential oils on Echinococcus granulosus protoscoleces and metacestodes.

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    Maggiore, Marina A; Albanese, Adriana A; Gende, Liesel B; Eguaras, Martín J; Denegri, Guillermo M; Elissondo, María Celina

    2012-03-01

    The aim of the present work was to determine the in vitro effect of Mentha piperita and Mentha pulegium essential oils against Echinococcus granulosus and to compare the effectiveness of both oils according to the exposure time and concentration. Although both treatments had a protoscolicidal effect, M. pulegium had a considerably stronger effect than M. piperita. Essential oil of M. pulegium produced dose- and time-dependent effects. Maximal protoscolicidal effect was observed after 12 days of incubation and reached 0% after 18 days. This lack of viability was proved during the determination of infectivity into mice. Essential oil of M. piperita produced only a time-dependent effect. At 24 days p.i., the viability of protoscoleces decreased to approximately 50%. Scanning and transmission electron microscopy (SEM and TEM) demonstrated the drug-induced ultrastructural damage. On the other hand, a loss of turgidity was detected in all M. pulegium-treated cysts respective of the drug concentration. There was a correlation between the intensity of damage and the concentration of the essential oil assayed. Studies by SEM revealed that the germinal layer of treated cysts lost the feature multicellular structure. M. pulegium essential oil showed piperitone oxide as main compound in their composition, and we suggest that this component could be responsible of the markedly anthelmintic effect detected. Our data suggest that essential oils of Mentha spp. can be a promising source of potential protoscolicidal agents. The isolation of active anthelmintic constituents is in progress and may lead to the discovery of compounds with improved therapeutic value.

  6. Still and Moving Image Evidences for Mating of Echinococcus granulosus Reared in Culture Media.

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    Tahereh Mohammadzadeh

    2014-03-01

    Full Text Available Echinococcus granulosus cultivation is very important for improvement of different aspect of medical and veterinary researches. Despite many advances in this case, there is a missing link for in vitro life cycle of adult worms and it is fertilization. Regarding the researchers' observations, self-fertilization can be done in worms living in dog intestine, but despite all sorts of experimental techniques, this phenomenon has never been observed in reared worms in culture media. Furthermore, cross fertilization has not been observed in vitro and even in parasites with dog intestinal origin; although it theoretically is possible. During a follow-up of cultivated adult worms, evidences of behaviors similar to self-mating (Type 2 and cross-mating were observed in our lab which will be presented here.Protoscoleces were aseptically removed from sheep hydatid cysts, washed twice with PBS and then cultivated in S.10E.H culture medium. The stages of parasite growth were observed using an inverted microscope for two months and all stages and behaviors were microscopically photographed. Different movies have also been made from these behavioral features.After around 55 days post cultivation, some evidences of behaviors similar to self-mating (Type 2 and cross-mating were observed in some of the mature adult worms. However, fertile eggs in these parasites have never been observed.Regarding the above observations, these parasites show tendency to unsuccessful self-mating/fertilization (type 2 which failure could be due to anatomical position and physiological maturation. Also lack of suitable conditions for self-fertilization causes the worms try to do unsuccessful cross- mating/fertilization in culture media.

  7. Unconventional maturation of dendritic cells induced by particles from the laminated layer of larval Echinococcus granulosus.

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    Casaravilla, Cecilia; Pittini, Alvaro; Rückerl, Dominik; Seoane, Paula I; Jenkins, Stephen J; MacDonald, Andrew S; Ferreira, Ana M; Allen, Judith E; Díaz, Alvaro

    2014-08-01

    The larval stage of the cestode parasite Echinococcus granulosus causes hydatid disease in humans and livestock. This infection is characterized by the growth in internal organ parenchymae of fluid-filled structures (hydatids) that elicit surprisingly little inflammation in spite of their massive size and persistence. Hydatids are protected by a millimeter-thick layer of mucin-based extracellular matrix, termed the laminated layer (LL), which is thought to be a major factor determining the host response to the infection. Host cells can interact both with the LL surface and with materials that are shed from it to allow parasite growth. In this work, we analyzed the response of dendritic cells (DCs) to microscopic pieces of the native mucin-based gel of the LL (pLL). In vitro, this material induced an unusual activation state characterized by upregulation of CD86 without concomitant upregulation of CD40 or secretion of cytokines (interleukin 12 [IL-12], IL-10, tumor necrosis factor alpha [TNF-α], and IL-6). When added to Toll-like receptor (TLR) agonists, pLL-potentiated CD86 upregulation and IL-10 secretion while inhibiting CD40 upregulation and IL-12 secretion. In vivo, pLL also caused upregulation of CD86 and inhibited CD40 upregulation in DCs. Contrary to expectations, oxidation of the mucin glycans in pLL with periodate did not abrogate the effects on cells. Reduction of disulfide bonds, which are known to be important for LL structure, strongly diminished the impact of pLL on DCs without altering the particulate nature of the material. In summary, DCs respond to the LL mucin meshwork with a "semimature" activation phenotype, both in vitro and in vivo.

  8. Environmental Contamination by Echinococcus granulosus sensu lato Eggs in Relation to Slaughterhouses in Urban and Rural Areas in Tunisia.

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    Chaâbane-Banaoues, Raja; Oudni-M'rad, Myriam; M'rad, Selim; Mezhoud, Habib; Babba, Hamouda

    2016-02-01

    Hydatidosis has become a real concern for health care institutions and animal rearers in Tunisia. The Tunisian endemicity is aggravated by the growing number of dogs and the difficulty of getting rid of contaminated viscera because of the lack of equipment in most slaughterhouses. Therefore, microscopic and molecular tools were applied to evaluate the role of slaughterhouses in canine infection and Echinococcus granulosus sensu lato (s. l.) egg dissemination. Exposure risk to E. granulosus s. l. eggs in urban and rural areas was explored in order to implant preventive and adapted control strategies. Microscopic examinations detected taeniid eggs in 152 amongst 553 fecal samples. The copro-PCR demonstrated that 138 of 152 taeniid samples analyzed were positive for E. granulosus s. l. DNA. PCR-RFLP demonstrated that all isolated samples belonged to E. granulosus sensu stricto (s. s.). An important environmental contamination index (25.0%) by E. granulosus s. l. eggs was demonstrated. The average contamination index from the regions around slaughterhouses (23.3%; 95% CI: 17.7-28.9%) was in the same range as detected in areas located far from slaughterhouses (26.0%, 95% CI: 21.3-30.8%). Echinococcosis endemic areas were extended in both rural (29.9%, 95% CI: 24.8-34.9%) and urban locations (18.1%, 95% CI: 13.0-22.9%). The pathogen dissemination is related neither to the presence/absence of slaughterhouses nor to the location in urban or rural areas, but is probably influenced by human activities (home slaughtering) and behavior towards the infected viscera.

  9. Construction of pEGFP-ChEgTrp as DNA model for multi-epitope vaccine against Echinococcus granulosus

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    M. Ahmadzadeh

    2015-12-01

    Full Text Available Background: Infection with Echinococcus granulosus causes hydatidosis in human and ruminants. With regards to the high prevalence of hydatidosis in Iran, dealing with this disease is important in terms of public health. Objective: The aim of this study was to construct pEGFP-ChEgTrp as DNA model for multi-epitope vaccine against Echinococcus granulosus Methods: This experimental study was conducted in the Razi Vaccine & Serum Research Institute, Karaj in 2013. Initially, epitopes stimulating the host immune response were predicted by IEDB Database and the coding sequences were made. The sequences were amplified by PCR. The PCR products were cloned into pEGFP-N1 vector after digestion with XhoI restriction enzyme. The bacteria containing recombinant plasmid were evaluated using Colony PCR, agarose gel electrophoresis and sequencing methods. Findings: Four peptides with 10 linear epitopes were predicted in EgTrp antigen. The nucleotide sequence coding ChEgTrp was amplified by PCR using specific primers and a 270 bp fragment was obtained. This fragment was cloned into pEGFP-N1 vector and the recombinant plasmid was confirmed by Colony PCR and agarose gel electrophoresis. For final confirmation, the recombinant plasmid was sequenced and the pEGFP-ChEgTrp was constructed. Conclusion: The ChEgTrp was successfully cloned into the pEGFP-N1 vector and this plasmid can be used to design DNA vaccines.

  10. Use of FTA(®) card methodology for sampling and molecular characterization of Echinococcus granulosus sensu lato in Africa.

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    Boué, Franck; El Berbri, Ikhlass; Hormaz, Vanessa; Boucher, Jean-Marc; El Mamy, Ahmed Bezeid; Traore, Abdallah; Fihri, Ouafaa Fassi; Petavy, Anne-Françoise; Dakkak, Allal; Umhang, Gérald

    2017-02-01

    Cystic Echinococcosis is a parasitic disease caused by the cestode Echinococcus granulosus widely distributed in Africa. Monitoring of this parasite requires access to cyst samples on intermediate hosts observed at the slaughterhouse. In order to facilitate sampling in the field and analysis, the French National Reference Laboratory for Echinococcus spp. has developed a tissue derived from DNA sampling with FTA(®) card technology. The DNA samples were taken by applying the FTA(®) paper on the germinal layer after opening the cysts. The sampling technique was validated using frozen cysts (n = 76) stored in the laboratory and from field samples (n = 134) taken at the slaughterhouse by veterinarian technicians during meat inspection in Morocco, Mali and Mauritania. DNA was extracted after several weeks of storage at room temperature. PCR assays were performed using primers for generic cestode (cox1) and amplified fragments were sequenced. All samples taken in the lab and 80% of field samples were capable of molecular characterization. Cyst-derived DNA from FTA(®) samples can be useful for easy sampling, storage and rapid, safe and cheap shipment. The use of the FTA methodology will facilitate studies in the field to investigate the presence and genetic characterization of E. granulosus sensu lato in African countries.

  11. Negligible elongation of mucin glycans with Gal β1-3 units distinguishes the laminated layer of Echinococcus multilocularis from that of Echinococcus granulosus.

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    Del Puerto, Lucía; Rovetta, Romina; Navatta, Marco; Fontana, Carolina; Lin, Gerardo; Moyna, Guillermo; Dematteis, Sylvia; Brehm, Klaus; Koziol, Uriel; Ferreira, Fernando; Díaz, Alvaro

    2016-05-01

    The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O-glycans in which the core Galpβ1-3 residue can initiate a chain comprising one to three additional Galpβ1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Galpα1-4 residue, and can be ramified with GlcNAcpβ1-6 residues. These, as well as the GlcNAcpβ1-6 residue in core 2, can be decorated with the Galpα1-4Galpβ1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis, showing that the non-decorated cores, together with Galpβ1-3(Galpα1-4Galpβ1-4GlcNAcpβ1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Galpβ1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc β1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis.

  12. The impact of socio-cultural factors on transmission of Taenia spp. and Echinococcus granulosus in Kosovo.

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    Alishani, M; Sherifi, K; Rexhepi, A; Hamidi, A; Armua-Fernandez, M T; Grimm, F; Hegglin, D; Deplazes, P

    2017-08-11

    Echinococcus granulosus sensu latu (s.l.) and Taenia hydatigena are common parasites of ruminant intermediate hosts in the Balkans. Transmission is linked mainly to home slaughtering and the feeding of infected organs to dogs. In Kosovo, many old sheep are slaughtered particularly during Eid al-Adha (Feast of Sacrifice). To determine whether this tradition could affect parasite transmission, we compared the probability of 504 dogs to contract taenid infections after deworming during one period before Eid al-Adha and a similar period beginning with this event. Initially, taeniid eggs were detected in 6·2% (CI 4·2-8·6) of the dogs. The prevalence before Eid al-Adha was significantly lower (1·2%, CI 0·4-2·6) as compared with the prevalence after the event (4·3%, CI 2·6-6·3). A comparable trend was apparent at species level for T. hydatigena and E. granulosus. These results indicate that the pronounced increase of taeniid infections, including E. granulosus s.l., after Eid al-Adha is linked to traditional home slaughtering that occurs during this celebration. This particular epidemiological situation provides an opportunity for implementing focussed control activities.

  13. Antigenic differences between the EG95-related proteins from Echinococcus granulosus G1 and G6 genotypes: implications for vaccination.

    Science.gov (United States)

    Alvarez Rojas, C A; Gauci, C G; Lightowlers, M W

    2013-02-01

    Cystic echinococcosis caused by Echinococcus granulosus remains an important and neglected issue in public health. The study of the likely efficacy of the currently available EG95 vaccine against other genotypes of the parasite is important to improve the vaccine as a potential tool to be used in control programmes. The recombinant vaccine EG95-1G1 was developed based on the G1 genotype of E. granulosus. Characterization of the eg95 gene family in the G6 genotype by genomic DNA cloning previously produced the first unequivocal information about the composition of the gene family in a different genotype. The information was used in this study to predict and express two EG95-related proteins from the G6 genotype as recombinants, for assessment of their capacity to bind antibodies raised in sheep vaccinated with the EG95-1G1 vaccine. The proteins (EG95-1G6 and EG95-5G6) from the G6 genotype of E. granulosus were unable to bind all the antibodies raised by sheep vaccinated with EG95-1G1. Differences in the amino acid sequence of EG95-related proteins from G6 and likely the differences in the encoded FnIII domain may be responsible for changes in the conformation of these epitopes.

  14. Molecular characterization of Echinococcus granulosus from Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene.

    Science.gov (United States)

    Sánchez, Elizabeth; Cáceres, Omar; Náquira, César; Garcia, David; Patiño, Gladys; Silvia, Herrera; Volotão, Aline C; Fernandes, Octavio

    2010-09-01

    Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosus worldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  15. Caracterización y análisis de la actividad antigénica de proteínas de la membrana germinal de Echinococcus granulosus Characterization and antigenic activity of Echinococcus granulosus germinal layer proteins

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    L.H. Prieto

    2006-12-01

    Full Text Available Proteínas presentes en un extracto de membrana germinal de Echinococcus granulosus fueron separadas en una fase acuosa y en una fase detergente mediante el tratamiento con Triton X-114. La caracterización mediante electroforesis en gel de poliacrilamida en presencia de dodecil sulfato de sodio, mostró que proteínas con pesos moleculares aproximados de 102, 45, 28.5-28 y 15.5-15 kDa se presentaron en el extracto y en la fase acuosa (proteínas hidrofílicas. La caracterización inmunológica de proteínas del extracto mediante inmunotransferencia frente a suero ovino no inmunizado, frente a suero ovino inmunizado con antígenos de E. granulosus y frente a suero anti-inmunoglobulina ovina, mostró patrones de reactividad similares. El ensayo inmunodetección en un punto, de proteínas del extracto previamente tratadas con Triton X-114 frente a sueros humanos incubados con el detergente iónico dodecil sulfato de sodio, mostró reactividad frente a sueros humanos con equinococosis quística y no mostró reactividad frente a sueros humanos sin equinococosis quística ni frente a un suero humano infectado con la filaria Mansonella ozzardi.Proteins present in an extract of Echinococcus granulosus germinal layer were separated in hydrophobic and hydrophilic phases using Triton X-114. The characterization carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that proteins of apparent molecular weights of 102, 45, 28.5-28 and 15.5-15 kDa were present in the extract and in the aqueous phase (hydrophilic proteins. The immunological characterization of extract proteins performed by immunoblotting against to no immunized sheep serum, against to immunized with E. granulosus antigens sheep serum and against to anti-sheep immunoglobulin serum, showed similar reaction patterns. The dot immunobinding assay of extract proteins after Triton X-114 treatment against to human sera incubated in the presence of sodium dodecil sulphate

  16. Production of Recombinant Echinococcus granulosus Antigen B Subunits, In Order to Using Of Them in Serodiagnostic Tests of Hydatidosis

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    R Pazoki

    2007-06-01

    Full Text Available Background: Hydatidosis is one of the most important helminthiasis, and is a public health problem in many regions of the world. Methods: With the aim of production of recombinant subunits of antigen B, two different sequences of Echinococcus granulosus Antigen B, acquired from Gene Bank and amplified with specific primers via RT-PCR reaction. The amplified fragments (HI, HII cloned into pTZ57R T.vector, and then subcloned into pGEMEX-1 expression vector. Resaults: The SDS-PAGE performed after induction of cloned genes, and production of about 35 K.Da recombinant fusion proteins were confirmed for either two cloned genes. The immunogenicity of the recombinant fusion proteins were tested using double diffusion and immunoblotting. Both recombinant fusion proteins derived from lysate of transformed bacteria, were reactive for antibodies in serum of cystic hydatid patient. Conclusion: The produced recombinant antigen B subunits can be use in seroldiagnostic tests of hydatidosis, after purification.

  17. Perioperative Prophylactic Chemotherapy of Echinococcus granulosus: Determination of Minimum Effective Length of Albendazole Therapy in In Vitro Protoscolex Culture

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    D. H. Taylor

    1990-01-01

    Full Text Available Protoscoleces of Echinococcus granulosus were cultured in vitro in 500, 250 or 100 μg/1 albendazole sulphoxide for 1, 3, 7, 10, 14d and then ‘recued’ (R into drug-free medium for the remainder ofthe culture period. Successful minimum lengths of therapy were much longer than for praziquantel, and only at 500μg/1 was the 10dR treatment as effective as continuous therapy for 28d. Treatment with 100 μg/1 both in continuous culture and in the ‘R’ experiments was ineffective over a 35d period. The results are compared with those from similar experiments using praziquantel.

  18. Echinococcus granulosus: pre-culture of protoscoleces in vitro significantly increases development and viability of secondary hydatid cysts in mice.

    Science.gov (United States)

    Zhang, Wen-Bao; Jones, Malcolm K; Li, Jun; McManus, Donald P

    2005-05-01

    We describe a method for obtaining improved secondary infections of Echinococcus granulosus that involves culturing protoscolex larvae in vitro prior to inoculation into mice. This approach provides a far superior method for obtaining secondary echinococcosis infections in mice compared with the traditional method of direct inoculation of protoscoleces (PSC) where the majority of parasites are killed by the host. We obtained a high rate of recovery both in terms of secondary cyst numbers and their viability. After 50 weeks post-infection (p.i.), brood capsules were formed and the first PSC developed in each of the capsules. After 56 weeks p.i., the fastest developing brood capsule contained four PSC. The approach will prove valuable for investigating parasite development and the host-parasite interaction in secondary echinococcosis.

  19. Genetic variability of Echinococcus granulosus complex in various geographical populations of Iran inferred by mitochondrial DNA sequences.

    Science.gov (United States)

    Spotin, Adel; Mahami-Oskouei, Mahmoud; Harandi, Majid Fasihi; Baratchian, Mehdi; Bordbar, Ali; Ahmadpour, Ehsan; Ebrahimi, Sahar

    2017-01-01

    To investigate the genetic variability and population structure of Echinococcus granulosus complex, 79 isolates were sequenced from different host species covering human, dog, camel, goat, sheep and cattle as of various geographical sub-populations of Iran (Northwestern, Northern, and Southeastern). In addition, 36 sequences of other geographical populations (Western, Southeastern and Central Iran), were directly retrieved from GenBank database for the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The confirmed isolates were grouped as G1 genotype (n=92), G6 genotype (n=14), G3 genotype (n=8) and G2 genotype (n=1). 50 unique haplotypes were identified based on the analyzed sequences of cox1. A parsimonious network of the sequence haplotypes displayed star-like features in the overall population containing IR23 (22: 19.1%) as the most common haplotype. According to the analysis of molecular variance (AMOVA) test, the high value of haplotype diversity of E. granulosus complex was shown the total genetic variability within populations while nucleotide diversity was low in all populations. Neutrality indices of the cox1 (Tajima's D and Fu's Fs tests) were shown negative values in Western-Northwestern, Northern and Southeastern populations which indicating significant divergence from neutrality and positive but not significant in Central isolates. A pairwise fixation index (Fst) as a degree of gene flow was generally low value for all populations (0.00647-0.15198). The statistically Fst values indicate that Echinococcus sensu stricto (genotype G1-G3) populations are not genetically well differentiated in various geographical regions of Iran. To appraise the hypothetical evolutionary scenario, further study is needed to analyze concatenated mitogenomes and as well a panel of single locus nuclear markers should be considered in wider areas of Iran and neighboring countries.

  20. Increased Expression of TGF-β1 in Correlation with Liver Fibrosis during Echinococcus granulosus Infection in Mice

    Science.gov (United States)

    Liu, Yumei; Abudounnasier, Gulizhaer; Zhang, Taochun; Liu, Xuelei; Wang, Qian; Yan, Yi; Ding, Jianbing; Wen, Hao; Yimiti, Delixiati; Ma, Xiumin

    2016-01-01

    To investigate the potential role of transforming growth factor (TGF)-β1 in liver fibrosis during Echinococcus granulosus infection, 96 BALB/c mice were randomly divided into 2 groups, experimental group infected by intraperitoneal injection with a metacestode suspension and control group given sterile physiological saline. The liver and blood samples were collected at days 2, 8, 30, 90, 180, and 270 post infection (PI), and the expression of TGF-β1 mRNA and protein was determined by real-time quantitative RT-PCR and ELISA, respectively. We also evaluated the pathological changes in the liver during the infection using hematoxylin and eosin (H-E) and Masson staining of the liver sections. Pathological analysis of H-E stained infected liver sections revealed liver cell edema, bile duct proliferation, and structural damages of the liver as evidenced by not clearly visible lobular architecture of the infected liver, degeneration of liver cell vacuoles, and infiltration of lymphocytes at late stages of infection. The liver tissue sections from control mice remained normal. Masson staining showed worsening of liver fibrosis at the end stages of the infection. The levels of TGF-β1 did not show significant changes at the early stages of infection, but there were significant increases in the levels of TGF-β1 at the middle and late stages of infection (Pgranulosus infection may play a significant role in liver fibrosis associated with E. granulosus infection. PMID:27658605

  1. Cytokine response and outcome of infection depends on the infective dose of parasites in experimental infection by Echinococcus granulosus.

    Science.gov (United States)

    Dematteis, Sylvia; Rottenberg, Martin; Baz, Adriana

    2003-04-01

    We here analysed whether the cytokine responses in early and late experimental infection with Echinococcus granulosus depend on the dose of parasites to which the host is exposed. To this purpose Balb/c mice were inoculated intraperitoneally (i.p.) with either 500 or 2000 protoscoleces. Splenocytes of mice were obtained at days 3, 7, 14 and 21 and also on week 37 post-infection and cultured in vitro with protoscolex antigens. Type-1 and type-2 cytokines were analysed in supernatants by ELISA. Results showed that the inoculation of 500 protoscoleces induced an early type-0 and a late type-2 cytokine response, whereas the inoculation of 2000 protoscoleces induced an early type-2 and a late type-0 cytokine response. Parasite growth was lower in the group inoculated with the low infective dose. These results indicate that the cytokine response during the infection by the helminth E. granulosus depends on the dose of parasites to which the host has been exposed.

  2. In-vitro scolicidal activity of Mallotus philippinensis (Lam.) Muell Arg. fruit glandular hair extract against hydatid cyst Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    Mayank Gangwar; Vijay C Verma; Tryambak D Singh; Sushil K Singh; RK Goel; Gopal Nath

    2013-01-01

    Objective: To investigate new scolicidal agent from natural resources to cope with the side effects associated with synthetic drugs in Echinococcosis. Methods:The scolicidal potential of methanolic fruit powder extract (10 and 20 mg/mL) of Mallotus philippinensis (M. philippinensis) was investigated. Viability of protoscoleces was confirmed by trypan blue exclusion method, where mortality was observed at concentration of 10 and 20 mg/mL in 60 min treatment against Echinococcus granulosus (E. granulosus), under in-vitro conditions with reference to the known standard drug Praziquantel®. Results: At concentration 10 and 20 mg/mL, the mortality rate was observed 97%and 99%respectively for 60 min treatment;while up to 93%mortality was observed with 20 mg/mL for only 10 min treatment. The concentration above 20 mg/mL for above 2 h showed 100%mortality, irrespective of further incubation. Conclusions: As compared with the standard anti-parasitic drug Praziquantel our extract has significant scolicidal activity with almost no associated side effects.

  3. An oral recombinant vaccine in dogs against Echinococcus granulosus, the causative agent of human hydatid disease: a pilot study.

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    Anne-Francoise Petavy

    Full Text Available Dogs are the main source of human cystic echinococcosis. An oral vaccine would be an important contribution to control programs in endemic countries. We conducted two parallel experimental trials in Morocco and Tunisia of a new oral vaccine candidate against Echinococcus granulosus in 28 dogs. The vaccine was prepared using two recombinant proteins from adult worms, a tropomyosin (EgTrp and a fibrillar protein similar to paramyosin (EgA31, cloned and expressed in a live attenuated strain of Salmonella enterica serovar typhimurium.In each country, five dogs were vaccinated with the associated EgA31 and EgTrp; three dogs received only the vector Salmonella; and six dogs were used as different controls. The vaccinated dogs received two oral doses of the vaccine 21 d apart, and were challenged 20 d later with 75,000 living protoscoleces. The controls were challenged under the same conditions. All dogs were sacrificed 26-29 d postchallenge, before the appearance of eggs, for safety reasons.We studied the histological responses to both the vaccine and control at the level of the duodenum, the natural localization of the cestode. Here we show a significant decrease of parasite burden in vaccinated dogs (70% to 80% and a slower development rate in all remaining worms. The Salmonella vaccine EgA31-EgTrp demonstrated a high efficacy against E. granulosus promoting its potential role in reducing transmission to humans and animals.

  4. Co-existence of Echinococcus granulosus infection and cancer metastasis in the liver correlates with reduced Th1 immune responses.

    Science.gov (United States)

    Turhan, N; Esendagli, G; Ozkayar, O; Tunali, G; Sokmensuer, C; Abbasoglu, O

    2015-01-01

    A possible relationship between cancer and Echinococcus granulosus infection has been postulated. As T cells are critical players in immune responses against both infections and malignancies, in an experimental model of secondary echinococcosis and breast cancer, this study aims to observe the progression of cancer and to determine the characters of T-cell responses. 4T1 breast tumour cells were subcutaneously injected into mammary region, whereas protoscoleces were intraperitoneally inoculated into the mice. Hydatid cysts, tumours and metastases were determined with macroscopic and histopathological evaluation. T cells found in spleen, liver and tumour were characterised by flow cytometric analysis of CD3, CD4, CD8, CD25, CCR5, CCR3, IL-4 and IFN-γ. In the mice inoculated both with protoscoleces and with breast tumour cells, increased frequency of cancer metastasis was observed in the liver. The amount of CD4(+) T cells was increased in the liver and in the spleen of mice infected with E. granulosus. However, co-existence of echinococcosis and metastatic lesions in the liver was associated with significant reduction in the IFN-γ(+) and CCR5(+) Th1 cells and increase in the CD25(+) T cells. Our results may indicate an immunological link between cystic echinococcosis and cancer that allows tumour metastasis to flourish in the liver.

  5. Unique precipitation and exocytosis of a calcium salt of myo-inositol hexakisphosphate in larval Echinococcus granulosus.

    Science.gov (United States)

    Irigoín, Florencia; Casaravilla, Cecilia; Iborra, Francisco; Sim, Robert B; Ferreira, Fernando; Díaz, Alvaro

    2004-12-15

    The ubiquitous intracellular molecule myo-inositol hexakisphosphate (IP6) is present extracellularly in the hydatid cyst wall (HCW) of the parasitic cestode Echinococcus granulosus. This study shows that extracellular IP6 is present as its solid calcium salt, in the form of deposits that are observed, at the ultrastructural level, as naturally electron dense granules some tens of nanometers in diameter. The presence of a calcium salt of IP6 in these structures was determined by two different electron microscopy techniques: (i) the analysis of the spatial distribution of phosphorus and calcium in the outer, acellular layer of the HCW (the laminated layer, LL) through electron energy loss spectroscopy, and (ii) the observation, by transmission electron microscopy, of HCW that were selectively depleted of IP6 by treatment with EGTA or phytase, an enzyme that catalyses the dephosphorylation of IP6. The deposits of the IP6-Ca(II) salt are also observed inside membrane vesicles in cells of the germinal layer (the inner, cellular layer of the HCW), indicating that IP6 precipitates with calcium within a cellular vesicular compartment and is then secreted to the LL. Thus, much as in plants (that produce vesicular IP6 deposits), the existence of transporters for IP6 or its precursors in internal membranes is needed to explain the compound's cellular localisation in E. granulosus.

  6. Genotyping of Echinococcus granulosus from goats and sheep indicating G7 genotype in goats in the Northeast of Iran.

    Science.gov (United States)

    Fadakar, Bahman; Tabatabaei, Nasim; Borji, Hassan; Naghibi, Abolghasem

    2015-11-30

    Although cystic echinococcosis (CE) has been a human public health problem in the Northeast of Iran, molecular data regarding the genotypes of Echinococcus granulosus in goats and sheep in these regions are still scarce. In the present study, we determined the genotypes of E. granulosus infecting sheep and goats in northeast of Iran. During April 2013-June 2014, 50 and 30 hydatid cysts were recovered from liver tissue of sheep and goats, respectively,. Protoscoleces or germinal layers were collected from individual cysts, DNA was extracted, and the ribosomal DNA internal transcribed spacer 1 (ITS1) gene was amplified by PCR. The results of PCR-RFLP and the sequence analysis showed that all the samples isolated from sheep (n=50) and most of samples in goats (n=24) were G1 strain, the most prevalent strain in livestock ruminants of Iran. Furthermore, six parasites isolated from goats were found to correspond to E. intermedius (G7 genotype), here reported for the first time from Iran. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Permeability studies on taenid metacestodes: I. Uptake of proteins by larval stages of Taenia taeniaeformis, T. crassiceps, and Echinococcus granulosus.

    Science.gov (United States)

    Hustead, S T; Williams, J F

    1977-04-01

    Host immunoglobulins of several different classes were detected within the bladder fluids of Taenia taeniaeformis, T. crassiceps, and Echinococcus granulosus. Radioiodinated proteins were taken up in vitro by larvae of both T. taeniaeformis and T. crassiceps and were shown to retain their physicochemical and antigenic characteristics. Rates of uptake were similar in the 2 species and were not related to the molecular weight of the proteins. Immunoglobulins were taken up both in vitro and in vivo by larvae of T. taeniaeformis. Absorbed immunoglobulins were shown to retain both antigen binding capacity and biologic functions associated with the Fc portion of the molecules. Not all cysts of E. granulosus contained detectable host proteins. Uptake of 125I occurred when hydatid cysts were exposed to labeled proteins in vitro, but it appeared that rapid degradation of the labeled carrier led to the appearance of dialysable fragments. This may be attributable to proteolysis within the bladder fluid. We conclude that taeniid metacestodes are capable of absorbing a variety of proteins, and that these macromolecules can retain their structural and functional integrity following transport. This absorptive capacity accounts for the presence of host serum components within bladder fluids.

  8. Increased Expression of TGF-β1 in Correlation with Liver Fibrosis during Echinococcus granulosus Infection in Mice.

    Science.gov (United States)

    Liu, Yumei; Abudounnasier, Gulizhaer; Zhang, Taochun; Liu, Xuelei; Wang, Qian; Yan, Yi; Ding, Jianbing; Wen, Hao; Yimiti, Delixiati; Ma, Xiumin

    2016-08-01

    To investigate the potential role of transforming growth factor (TGF)-β1 in liver fibrosis during Echinococcus granulosus infection, 96 BALB/c mice were randomly divided into 2 groups, experimental group infected by intraperitoneal injection with a metacestode suspension and control group given sterile physiological saline. The liver and blood samples were collected at days 2, 8, 30, 90, 180, and 270 post infection (PI), and the expression of TGF-β1 mRNA and protein was determined by real-time quantitative RT-PCR and ELISA, respectively. We also evaluated the pathological changes in the liver during the infection using hematoxylin and eosin (H-E) and Masson staining of the liver sections. Pathological analysis of H-E stained infected liver sections revealed liver cell edema, bile duct proliferation, and structural damages of the liver as evidenced by not clearly visible lobular architecture of the infected liver, degeneration of liver cell vacuoles, and infiltration of lymphocytes at late stages of infection. The liver tissue sections from control mice remained normal. Masson staining showed worsening of liver fibrosis at the end stages of the infection. The levels of TGF-β1 did not show significant changes at the early stages of infection, but there were significant increases in the levels of TGF-β1 at the middle and late stages of infection (PTGF-β1 mRNA was low and comparable with that in control mice at the early stages of infection, and that it was significantly increased at day 30 PI and remained at high levels until day 270 PI (PTGF-β1 during E. granulosus infection may play a significant role in liver fibrosis associated with E. granulosus infection.

  9. Coproantigen detection in dogs experimentally and naturally infected with Echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Malgor, R; Nonaka, N; Basmadjian, I; Sakai, H; Carámbula, B; Oku, Y; Carmona, C; Kamiya, M

    1997-12-01

    A sandwich ELISA for the detection of Echinococcus granulosus coproantigen in formalin and heat-treated faecal supernatants of dogs was developed. The assay used affinity-purified polyclonal antibodies obtained from rabbits hyperimmunised with E. granulosus excretory/secretory antigens and biotinylated monoclonal antibody EmA9 produced against adult E. multilocularis somatic extract. The test was sensitive to 7 ng and 2.3 ng of E. granulosus protein and carbohydrate/ml of faecal supernatant, respectively. Thirteen helminth-free dogs were infected with different amounts of E. granulosus protoscoleces and the presence of coproantigen was monitored during the prepatent period until day 35 post-infection, when they were necropsied. Faecal antigen levels started to rise above the normal range between days 10 and 20 post-infection, and typically peaked at the end of the experiment. All the dogs, bearing from 3 to 67,700 worms, showed positive values in the ELISA during the prepatent period. One dog experimentally infected with Taenia hydatigena metacestode and harbouring three worms, tested positive only after the prepatent period at day 52. The test was applied to 98 stray dogs. The ELISA detected all of four dogs naturally infected with E. granulosus, two dogs with patent infections of T. hydatigena and two dogs with no cestode infections, showing a sensitivity of 100% and a specificity of 96%.

  10. Molecular characterization of Echinococcus granulosus cysts in north Indian patients: identification of G1, G3, G5 and G6 genotypes.

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    Monika Sharma

    Full Text Available BACKGROUND: Cystic echinococcosis (CE caused by the Echinococcus granulosus, is a major public health problem worldwide, including India. The different genotypes of E. granulosus responsible for human hydatidosis have been reported from endemic areas throughout the world. However, the genetic characterization of E. granulosus infecting the human population in India is lacking. The aim of study was to ascertain the genotype(s of the parasite responsible for human hydatidosis in North India. METHODOLOGY/PRINCIPAL FINDINGS: To study the transmission patterns of E. granulosus, genotypic analysis was performed on hydatid cysts obtained from 32 cystic echinococcosis (CE patients residing in 7 different states of North India. Mitochondrial cytochrome c oxidase subunit1 (cox1 sequencing was done for molecular identification of the isolates. Most of the CE patients (30/32 were found to be infected with hydatid cyst of either G3 (53.1% or G1 (40.62% genotype and one each of G5 (cattle strain and G6 (camel strain genotype. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate the zoonotic potential of G1 (sheep strain and G3 (buffalo strain genotypes of E. granulosus as these emerged as predominant genotypes infecting the humans in India. In addition to this, the present study reports the first human CE case infected with G5 genotype (cattle strain in an Asian country and presence of G6 genotype (camel strain in India. The results may have important implications in the planning of control strategies for human hydatidosis.

  11. Preliminary study of the presence of antibodies against excretory-secretory antigens from protoscoleces of Echinococcus granulosus in dogs with intestinal echinococcosis.

    Science.gov (United States)

    Carmena, David; Benito, Aitziber; Martínez, Jorge; Guisantes, Jorge A

    2005-05-01

    The aim of the present study was to analyze the antibody response against excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, using sera from dogs infected with E. granulosus and other helminths. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. Immunochemical characterization was performed by immunoblotting with sera from dogs naturally infected with E. granulosus (n = 12), sera from dogs infected with helminths other than E. granulosus (n = 30), and helminth-free dog sera (n = 20). These findings were compared to those obtained from a somatic extract of protoscoleces (S-Ag). ES-Ag only showed four cross-reacting proteins of 65, 61, 54, and 45-46 kDa. Antigens with apparent masses of 89 and 50 kDa in ES-Ag and of 130 and 67 kDa in S-Ag were identified by sera of dogs infected with E. granulosus only, whereas a protein of 41-43 kDa was recognised by the majority of the sera from dogs with non-echinococcal infection. Employing ELISA to study the same sera, S-Ag revealed higher immunoreactivity than ES-Ag, but also showed higher cross-reactivity levels when sera from dogs with non-echinococcal infection were assayed in immunoblotting.

  12. Preliminary study of the presence of antibodies against excretory-secretory antigens from protoscoleces of Echinococcus granulosus in dogs with intestinal echinococcosis

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    David Carmena

    2005-05-01

    Full Text Available The aim of the present study was to analyze the antibody response against excretory-secretory antigens (ES-Ag from Echinococcus granulosus protoscoleces, using sera from dogs infected with E. granulosus and other helminths. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. Immunochemical characterization was performed by immunoblotting with sera from dogs naturally infected with E. granulosus (n = 12, sera from dogs infected with helminths other than E. granulosus (n = 30, and helminth-free dog sera (n = 20. These findings were compared to those obtained from a somatic extract of protoscoleces (S-Ag. ES-Ag only showed four cross-reacting proteins of 65, 61, 54, and 45-46 kDa. Antigens with apparent masses of 89 and 50 kDa in ES-Ag and of 130 and 67 kDa in S-Ag were identified by sera of dogs infected with E. granulosus only, whereas a protein of 41-43 kDa was recognised by the majority of the sera from dogs with non-echinococcal infection. Employing ELISA to study the same sera, S-Ag revealed higher immunoreactivity than ES-Ag, but also showed higher cross-reactivity levels when sera from dogs with non-echinococcal infection were assayed in immunoblotting.

  13. Molecular characterization of Echinococcus granulosus cysts in north Indian patients: identification of G1, G3, G5 and G6 genotypes.

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    Monika Sharma

    Full Text Available BACKGROUND: Cystic echinococcosis (CE caused by the Echinococcus granulosus, is a major public health problem worldwide, including India. The different genotypes of E. granulosus responsible for human hydatidosis have been reported from endemic areas throughout the world. However, the genetic characterization of E. granulosus infecting the human population in India is lacking. The aim of study was to ascertain the genotype(s of the parasite responsible for human hydatidosis in North India. METHODOLOGY/PRINCIPAL FINDINGS: To study the transmission patterns of E. granulosus, genotypic analysis was performed on hydatid cysts obtained from 32 cystic echinococcosis (CE patients residing in 7 different states of North India. Mitochondrial cytochrome c oxidase subunit1 (cox1 sequencing was done for molecular identification of the isolates. Most of the CE patients (30/32 were found to be infected with hydatid cyst of either G3 (53.1% or G1 (40.62% genotype and one each of G5 (cattle strain and G6 (camel strain genotype. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate the zoonotic potential of G1 (sheep strain and G3 (buffalo strain genotypes of E. granulosus as these emerged as predominant genotypes infecting the humans in India. In addition to this, the present study reports the first human CE case infected with G5 genotype (cattle strain in an Asian country and presence of G6 genotype (camel strain in India. The results may have important implications in the planning of control strategies for human hydatidosis.

  14. Prevalence and risk factors of Echinococcus granulosus infection in dogs in Moroto and Bukedea districts in Uganda.

    Science.gov (United States)

    Oba, Peter; Ejobi, Francis; Omadang, Leonard; Chamai, Martin; Okwi, Andrew Livex; Othieno, Emmanuel; Inangolet, Francis Olaki; Ocaido, Michael

    2016-02-01

    A cross sectional study was conducted in Moroto and Bukedea districts of Uganda from May to September 2013 to determine the prevalence and risk factors of Echinococcus granulosus infection in dogs. Fresh dog faecal samples were collected, preserved in 70 % ethanol, and later screened for presence of taeniid eggs using zinc chloride floatation method. Positive samples were confirmed by a copro-PCR (polymerase chain reaction) for E. granulosus using NADH dehydrogenase sub-unit 1 gene (NADH1) as a target molecular marker. Structured questionnaires and focus group discussions were used to collect quantitative and qualitative data for risk factor identification. Study sub-counties were selected by simple random sampling. Overall apparent prevalence of taeniid infection in dogs of 14.9 % (39/261, confidence interval 10.6-19.2) in both districts was recorded using the faecal floatation test. The sensitivity of the faecal floatation test was found to be 78 % (25/32), while the specificity was 93 % (215/229). Copro-PCR results revealed a true prevalence of 14.4 % (9.91-19.0, 95 % CI) in dogs in Moroto district and 7.4 % (2.14-12.60, 95 % CI) in Bukedea district. The overall true prevalence of cystic echinococcosis (CE) was 12.2 % (8.70-15.76, 95 % CI) in both districts. The major risk factors identified using logistic regression were uncontrolled access of dogs to animal slaughter facilities, higher cattle herd sizes and lack of knowledge about the disease. It was recommended that restricting dog access to infected tissues and public health education about epidemiology of CE should be done.

  15. Molecular Cloning and Expression an 8-kDa Subunit of Antigen B from G1 strain of Echinococcus granulosus

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    Hakim AZIZI

    2015-10-01

    Full Text Available Background: Echinococcosis or hydatidosis is a chronic, zoonotic worldwide infection caused by the larval stage of the dog taeniid tapeworm Echinococcus granulosus. Vaccination has been considered as one of the ways to prevent of hy-datidosis in recent decades. The aim of this study was to construct a pcDNA3.1 eukaryotic expression vector contain-ing the subunit 8-kDa antigen B (Hyd1 of E. granulosus (G1 strain and investigate its capability to induce protein ex-pression in mammalian cell line, as a basis toward developing a DNA vaccine against hydatidosis.Methods: The coding sequence of HydI was amplified by PCR with the specific PCR primers from pQE/HydI, and then was sub-cloned into pcDNA3.1 plasmid as expression vector. The pcHyd1 plasmid was digested by restriction enzymes and amplified with the specific PCR primers to confirm cloning of this gene in pcDNA3 plasmid. In last step, the sub-cloned gene was expressed in mammalian cell line (NIH 3T3 cells.Result: The subunit 8-kDa antigen B (Hyd1 was successfully sub-cloned in pcDNA3.1 and Hyd1 protein was ex-pressed in eukaryotic cell confirmed by SDS-PAGE and Western blot.Conclusion: Recombinant plasmid of pcDNA3.1 was successfully constructed and express of recombinant Hyd1 protein was confirmed. That is promising step for forthcoming measures on providing vaccine against human and animal hydatidosis.

  16. Genetic diversity and phylogeography of highly zoonotic Echinococcus granulosus genotype G1 in the Americas (Argentina, Brazil, Chile and Mexico) based on 8279bp of mtDNA.

    Science.gov (United States)

    Laurimäe, Teivi; Kinkar, Liina; Andresiuk, Vanessa; Haag, Karen Luisa; Ponce-Gordo, Francisco; Acosta-Jamett, Gerardo; Garate, Teresa; Gonzàlez, Luis Miguel; Saarma, Urmas

    2016-11-01

    Echinococcus granulosus is a taeniid cestode and the etiological agent of an infectious zoonotic disease known as cystic echinococcosis (CE) or hydatid disease. CE is a serious public health concern in many parts of the world, including the Americas, where it is highly endemic in many regions. Echinococcus granulosus displays high intraspecific genetic variability and is divided into multiple genotypes (G1-G8, G10) with differences in their biology and etiology. Of these, genotype G1 is responsible for the majority of human and livestock infections and has the broadest host spectrum. However, despite the high significance to the public and livestock health, the data on genetic variability and regional genetic differences of genotype G1 in America are scarce. The aim of this study was to evaluate the genetic variability and phylogeography of G1 in several countries in America by sequencing a large portion of the mitochondrial genome. We analysed 8279bp of mtDNA for 52 E. granulosus G1 samples from sheep, cattle and pigs collected in Argentina, Brazil, Chile and Mexico, covering majority of countries in the Americas where G1 has been reported. The phylogenetic network revealed 29 haplotypes and a high haplotype diversity (Hd=0.903). The absence of phylogeographic segregation between different regions in America suggests the importance of animal transportation in shaping the genetic structure of E. granulosus G1. In addition, our study revealed many highly divergent haplotypes, indicating a long and complex evolutionary history of E. granulosus G1 in the Americas.

  17. Genetic variation of the 8-kDa glycoprotein family from Echinococcus granulosus, Taenia multiceps and Taenia hydatigena

    Institute of Scientific and Technical Information of China (English)

    JIA Wan-zhong; YAN Hong-bin; LOU Zhong-zi; NI Xing-wei; LIU Hong-xia; LI Hong-min; GUO Ai-jiang; FU Bao-quan

    2011-01-01

    Background Echinococcosis, coenurosis and cysticercosis are debilitating diseases which prevail in China.Immunological diagnosis of metacestodosis is important in disease control. The 8-kDa glycoproteins from taeniid cestodes have successfully been used for diagnosis of human cysticercosis in immunological assays. The aim of the present study was to investigate genetic variations and phylogenetic relationships of the 8-kDa proteins for evaluating the possibility of utilizing these proteins as diagnostic antigens for other metacestode infections.Methods The genes and complementary DNAs (cDNAs) encoding the 8-kDa proteins from Echinococcus (E.)granulosus, Taenia (T.) multiceps and T. hydatigena were amplified using PCR method. Their amplicons were cloned into the vector pMD18 and the positive clones were sequenced. Sequence data were analyzed with the SeqMan program,and sequence homology searches were performed using the BLAST program. Alignments were conducted using the ClustalX program, and the phylogenetic analyses were performed with the Protein Sequences Program and the Puzzle Program using the Neighbor-joining method.Results Fifteen, 18 and 22 different genomic DNA sequences were identified as members of the 8-kDa protein gene family from E. granulosus, T. multiceps and T. hydatigena, respectively. Eight, four and six different cDNA clones respectively from E. granulosus, T. multiceps and T. hydatigena were characterized. Analysis of these sequences revealed 54 unique 8-kDa protein sequences. Phylogenetic trees demonstrated that the taeniid 8-kDa proteins are clustered into eight clades at least: Ts18, Ts14, TsRS1, TsRS2, T8kDa-1, T8kDa-2, T8kDa-3 and T8kDa-4.Conclusion We found that the gene family encoding for the taeniid 8-kDa antigens is comprised of many members with high diversity, which will provide molecular evidence for cross-reaction or specific reaction among metacestode infections and may contribute to the development of promising immunological

  18. Efficacy of Essential Oils of Thymus vulgaris and Origanum vulgare on Echinococcus granulosus

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    P. E. Pensel

    2014-01-01

    Full Text Available The aim of the present work was to determine the in vitro effect of T. vulgaris and O. vulgare essential oils against E. granulosus protoscoleces and cysts. Essential oils were added to the medium resulting in thymol final concentrations of 10 μg/mL. The essential oils had a time-dependent effect provoking the complete loss of protoscolex viability after 72 days of postincubation. The results were confirmed at the ultrastructure level. Loss of infectivity in protoscoleces incubated with O. vulgare after 60 days was observed. On the other hand, the weight of cysts recorded in mice inoculated with T. vulgaris treated protoscoleces was significantly lower than that obtained in control group. Gamma-glutamyl-transpeptidase activity was readily detected in the culture supernatant of protoscoleces treated either with the essential oils or thymol. T. vulgaris and O. vulgare essential oils and thymol can induce cell apoptosis of protoscoleces after short incubation times. The efficacy of T. vulgaris and O. vulgare essential oils was also demonstrated in vitro on E. granulosus murine cysts. Our data suggest that essential oils of T. vulgaris and O. vulgare have anthelmintic effect against protoscoleces and cysts of E. granulosus.

  19. Efficacy of Essential Oils of Thymus vulgaris and Origanum vulgare on Echinococcus granulosus.

    Science.gov (United States)

    Pensel, P E; Maggiore, M A; Gende, L B; Eguaras, M J; Denegri, M G; Elissondo, M C

    2014-01-01

    The aim of the present work was to determine the in vitro effect of T. vulgaris and O. vulgare essential oils against E. granulosus protoscoleces and cysts. Essential oils were added to the medium resulting in thymol final concentrations of 10 μg/mL. The essential oils had a time-dependent effect provoking the complete loss of protoscolex viability after 72 days of postincubation. The results were confirmed at the ultrastructure level. Loss of infectivity in protoscoleces incubated with O. vulgare after 60 days was observed. On the other hand, the weight of cysts recorded in mice inoculated with T. vulgaris treated protoscoleces was significantly lower than that obtained in control group. Gamma-glutamyl-transpeptidase activity was readily detected in the culture supernatant of protoscoleces treated either with the essential oils or thymol. T. vulgaris and O. vulgare essential oils and thymol can induce cell apoptosis of protoscoleces after short incubation times. The efficacy of T. vulgaris and O. vulgare essential oils was also demonstrated in vitro on E. granulosus murine cysts. Our data suggest that essential oils of T. vulgaris and O. vulgare have anthelmintic effect against protoscoleces and cysts of E. granulosus.

  20. Efficacy of Essential Oils of Thymus vulgaris and Origanum vulgare on Echinococcus granulosus

    Science.gov (United States)

    Pensel, P. E.; Maggiore, M. A.; Gende, L. B.; Eguaras, M. J.; Denegri, M. G.; Elissondo, M. C.

    2014-01-01

    The aim of the present work was to determine the in vitro effect of T. vulgaris and O. vulgare essential oils against E. granulosus protoscoleces and cysts. Essential oils were added to the medium resulting in thymol final concentrations of 10 μg/mL. The essential oils had a time-dependent effect provoking the complete loss of protoscolex viability after 72 days of postincubation. The results were confirmed at the ultrastructure level. Loss of infectivity in protoscoleces incubated with O. vulgare after 60 days was observed. On the other hand, the weight of cysts recorded in mice inoculated with T. vulgaris treated protoscoleces was significantly lower than that obtained in control group. Gamma-glutamyl-transpeptidase activity was readily detected in the culture supernatant of protoscoleces treated either with the essential oils or thymol. T. vulgaris and O. vulgare essential oils and thymol can induce cell apoptosis of protoscoleces after short incubation times. The efficacy of T. vulgaris and O. vulgare essential oils was also demonstrated in vitro on E. granulosus murine cysts. Our data suggest that essential oils of T. vulgaris and O. vulgare have anthelmintic effect against protoscoleces and cysts of E. granulosus. PMID:25180033

  1. Two haplotype clusters of Echinococcus granulosus sensu stricto in northern Iraq (Kurdistan region) support the hypothesis of a parasite cradle in the Middle East.

    Science.gov (United States)

    Hassan, Zuber Ismael; Meerkhan, Azad Abdullah; Boufana, Belgees; Hama, Abdullah A; Ahmed, Bayram Dawod; Mero, Wijdan Mohammed Salih; Orsten, Serra; Interisano, Maria; Pozio, Edoardo; Casulli, Adriano

    2017-08-01

    Human cystic echinococcosis (CE) caused by Echinococcus granulosus s.s. is a major public health problem in Iraqi Kurdistan with a reported surgical incidence of 6.3 per 100,000 Arbil inhabitants. A total of 125 Echinococcus isolates retrieved from sheep, goats and cattle were used in this study. Our aim was to determine species/genotypes infecting livestock in Iraqi Kurdistan and examine intraspecific variation and population structure of Echinococcus granulosus s.s. in this region and relate it to that of other regions worldwide. Using nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox 1) we identified E. granulosus s.s. as the cause of hydatidosis in all examined animals. The haplotype network displayed a double-clustered topology with two main E. granulosus s.s. haplotypes, (KU05) and (KU33). The 'founder' haplotype (KU05) confirmed the presence of a common lineage of non-genetically differentiated populations as inferred by the low non-significant fixation index values. Overall diversity and neutrality indices indicated demographic expansion. We used E. granulosus s.s. nucleotide sequences from GenBank to draw haplotype networks for the Middle East (Iran, Jordan and Turkey), Europe (Albania, Greece, Italy, Romania and Spain), China, Mongolia, Russia, South America (Argentina, Brazil, Chile and Mexico) and Tunisia. Networks with two haplotype clusters like that reported here for Iraqi Kurdistan were seen for the Middle East, Europe, Mongolia, Russia and Tunisia using both 827bp and 1609bp cox1 nucleotide sequences, whereas a star-like network was observed for China and South America. We hypothesize that the double clustering seen at what is generally assumed to be the cradle of domestication may have emerged independently and dispersed from the Middle East to other regions and that haplotype (KU33) may be the main haplotype within a second cluster in the Middle East from where it has spread into Europe, Mongolia, Russia and North

  2. Modelling a 3D structure for EgDf1 from shape Echinococcus granulosus: putative epitopes, phosphorylation motifs and ligand

    Science.gov (United States)

    Paulino, M.; Esteves, A.; Vega, M.; Tabares, G.; Ehrlich, R.; Tapia, O.

    1998-07-01

    EgDf1 is a developmentally regulated protein from the parasite Echinococcus granulosus related to a family of hydrophobic ligand binding proteins. This protein could play a crucial role during the parasite life cycle development since this organism is unable to synthetize most of their own lipids de novo. Furthermore, it has been shown that two related protein from other parasitic platyhelminths (Fh15 from Fasciola hepatica and Sm14 from Schistosoma mansoni) are able to confer protective inmunity against experimental infection in animal models. A three-dimensional structure would help establishing structure/function relationships on a knowledge based manner. 3D structures for EgDf1 protein were modelled by using myelin P2 (mP2) and intestine fatty acid binding protein (I-FABP) as templates. Molecular dynamics techniques were used to validate the models. Template mP2 yielded the best 3D structure for EgDf1. Palmitic and oleic acids were docked inside EgDf1. The present theoretical results suggest definite location in the secondary structure of the epitopic regions, consensus phosphorylation motifs and oleic acid as a good ligand candidate to EgDf1. This protein might well be involved in the process of supplying hydrophobic metabolites for membrane biosynthesis and for signaling pathways.

  3. Echinococcus granulosus-specific T-cell lines derived from patients at various clinical stages of cystic echinococcosis.

    Science.gov (United States)

    Riganò, R; Buttari, B; De Falco, E; Profumo, E; Ortona, E; Margutti, P; Scottà, C; Teggi, A; Siracusano, A

    2004-01-01

    To investigate the role of T lymphocytes in the immune response to Echinococcus granulosus, using sheep hydatid fluid (SHF) and antigen B (AgB), we generated T-cell lines from patients with active, transitional and inactive hydatid cysts. We established 16 T-cell lines, eight specific to SHF and eight specific to AgB. At surface phenotyping 88-98% of cells displayed the helper/inducer CD4 antigen. In all patients, at all clinical stages of hydatid cyst disease, T-cell stimulation with SHF and AgB invariably amplified a large number of almost identical Vbeta subfamily fragments. Irrespective of antigen-specificity, the two cell lines from the patient with an inactive cyst had a Th1 profile, because they exclusively expressed and produced IFN-gamma. Conversely, the T-cell lines derived from the seven patients with active and transitional hydatid cysts had mixed Th1/Th2 and Th0 clones. The functional characteristics of the 16 T-cell lines differed markedly in the various clinical stages of cystic echinococcosis, thus providing new in vitro evidence that Th1 lymphocytes contribute decisively to the inactive stage of hydatid disease, Th2 lymphocytes in the active and transitional stages. The parasite-specific T-cell lines, especially the two Th1 lines from the patient with an inactive cyst, may help identify Th1 protective epitopes on SHF and AgB.

  4. [A case of hydatid cyst caused by Echinococcus granulosus in Puebla, Mexico, that resulted in successful surgical treatment].

    Science.gov (United States)

    Orea-Martínez, J G; Pérez-Corro, M A; Contreras-Vera, R A; Bretón-Márquez, J H

    2013-01-01

    We present herein the case of a 16-year-old female from the southern portion of the State of Puebla, Mexico. When gathering her past medical history, it was revealed that she had grown up with pet dogs and that her family raised sheep. Because the patient presented with few symptoms, a benign lesion was suspected, and after laparoscopic exploration, the possibility of surgical management for a non-parasitic cyst was considered. A dull pain in the right hypochondrium persisted and open surgical exploration was performed in which a 6cm young, active, uncomplicated hydatid cyst was discovered. Its surgical removal was successful and the pathologist provided the definitive diagnosis. The three layers characteristic of a parasitic cyst were present and it was histologically consistent with Echinococcus granulosus. Postoperative progression was unremarkable and the control ultrasound study revealed complete restitution of the hepatic parenchyma. Copyright © 2012 Asociación Mexicana de Gastroenterología. Published by Masson Doyma México S.A. All rights reserved.

  5. Evaluation of rabbit antibody response against 8 and 16 kDa recombinant subunits of antigen B fromEchinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    Jahangir Abdi; Bahram Kazemi; Mohammad Hasan Karimfar; Mohammad Bagher Rokni

    2012-01-01

    ABSTRACT Objective:To immunize rabbits with12 and16 kDa recombinant subunits of antigenB from Echinococcus granulosus (E. granulosus) and measuring polyclonal antibody and humoral immune response usingELISA and gel diffusion.Methods:Two mentioned antigens were cloned and expressed in expression vector and purified by affinity chromatography.Four young rabbits were selected and challenged intradermally with yielded recombinant antigens.Rabbits’ sera were collected post infection and were tested usingELISA and gel diffusion for polyclonal antibody detection10 days after last injection.Results:The specific antibody against the recombinant peptides was efficiently produced within4 weeks post infection.Conclusions:Produced recombinants proteins could induce the immune response of the rabbits successfully. This process might improve the clarification of diagnosis and vaccination as regards hydatidosis.

  6. Application of a cDNA microarray for profiling the gene expression of Echinococcus granulosus protoscoleces treated with albendazole and artemisinin.

    Science.gov (United States)

    Lü, Guodong; Zhang, Wenbao; Wang, Jianhua; Xiao, Yunfeng; Zhao, Jun; Zhao, Jianqin; Sun, Yimin; Zhang, Chuanshan; Wang, Junhua; Lin, Renyong; Liu, Hui; Zhang, Fuchun; Wen, Hao

    2014-12-01

    Cystic echinoccocosis (CE) is a neglected zoonosis that is caused by the dog-tapeworm Echinococcus granulosus. The disease is endemic worldwide. There is an urgent need for searching effective drug for the treatment of the disease. In this study, we sequenced a cDNA library constructed using RNA isolated from oncospheres, protoscoleces, cyst membrane and adult worms of E. granulosus. A total of 9065 non-redundant or unique sequences were obtained and spotted on chips as uniEST probes to profile the gene expression in protoscoleces of E. granulosus treated with the anthelmintic drugs albendazole and artemisinin, respectively. The results showed that 7 genes were up-regulated and 38 genes were down-regulated in the protoscoleces treated with albendazole. Gene analysis showed that these genes are responsible for energy metabolism, cell cycle and assembly of cell structure. We also identified 100 genes up-regulated and 6 genes down-regulated in the protoscoleces treated with artemisinin. These genes play roles in the transduction of environmental signals, and metabolism. Albendazole appeared its drug efficacy in damaging cell structure, while artemisinin was observed to increase the formation of the heterochromatin in protoscolex cells. Our results highlight the utility of using cDNA microarray methods to detect gene expression profiles of E. granulosus and, in particular, to understand the pharmacologic mechanism of anti-echinococcosis drugs.

  7. Effect of Different Terpene-Containing Essential Oils on the Proliferation of Echinococcus granulosus Larval Cells

    Science.gov (United States)

    Albani, Clara María; Denegri, Guillermo María; Elissondo, María Celina

    2014-01-01

    Human cystic echinococcosis remains a major public health problem on several countries and the treatment strategies are not solved. The aim of the present work was to determine the in vitro effect of thymol and Mentha piperita, M. pulegium, and Rosmarinus officinalis essential oils on the proliferation of E. granulosus larval cells. Isolated cells and cellular aggregates were obtained from hydatid cyst's germinal layer and exposed to 1, 5, and 10 μg/ml of thymol and the different essential oils for 7 days. Drug effect was evaluated using test viability and scanning electron microscopy. Control cell culture viability was 2.1 x 106 (100%) after 7 days of incubation. At day 7, thymol 5 μg/ml caused a reduction in cell viability of 63% and the essential oils of M. piperita 10 μg/ml, M. pulegium 10 μg/ml, and R. officinalis 10 μg/ml produced a reduction in the viability of 77, 82, and 71%, respectively. Moreover essential oils caused reduction in cell number, collapsed cells, and loss of normal tridimensional composition of the aggregates. Due to the inhibitory effect caused by essential oils on E. granulosus cells we suggested that it would be an effective means for suppression of larval growth. PMID:25328517

  8. Effect of Different Terpene-Containing Essential Oils on the Proliferation of Echinococcus granulosus Larval Cells

    Directory of Open Access Journals (Sweden)

    Clara María Albani

    2014-01-01

    Full Text Available Human cystic echinococcosis remains a major public health problem on several countries and the treatment strategies are not solved. The aim of the present work was to determine the in vitro effect of thymol and Mentha piperita, M. pulegium, and Rosmarinus officinalis essential oils on the proliferation of E. granulosus larval cells. Isolated cells and cellular aggregates were obtained from hydatid cyst’s germinal layer and exposed to 1, 5, and 10 μg/ml of thymol and the different essential oils for 7 days. Drug effect was evaluated using test viability and scanning electron microscopy. Control cell culture viability was 2.1 x 106 (100% after 7 days of incubation. At day 7, thymol 5 μg/ml caused a reduction in cell viability of 63% and the essential oils of M. piperita 10 μg/ml, M. pulegium 10 μg/ml, and R. officinalis 10 μg/ml produced a reduction in the viability of 77, 82, and 71%, respectively. Moreover essential oils caused reduction in cell number, collapsed cells, and loss of normal tridimensional composition of the aggregates. Due to the inhibitory effect caused by essential oils on E. granulosus cells we suggested that it would be an effective means for suppression of larval growth.

  9. Effect of Different Terpene-Containing Essential Oils on the Proliferation of Echinococcus granulosus Larval Cells.

    Science.gov (United States)

    Albani, Clara María; Denegri, Guillermo María; Elissondo, María Celina

    2014-01-01

    Human cystic echinococcosis remains a major public health problem on several countries and the treatment strategies are not solved. The aim of the present work was to determine the in vitro effect of thymol and Mentha piperita, M. pulegium, and Rosmarinus officinalis essential oils on the proliferation of E. granulosus larval cells. Isolated cells and cellular aggregates were obtained from hydatid cyst's germinal layer and exposed to 1, 5, and 10 μg/ml of thymol and the different essential oils for 7 days. Drug effect was evaluated using test viability and scanning electron microscopy. Control cell culture viability was 2.1 x 10(6) (100%) after 7 days of incubation. At day 7, thymol 5 μg/ml caused a reduction in cell viability of 63% and the essential oils of M. piperita 10 μg/ml, M. pulegium 10 μg/ml, and R. officinalis 10 μg/ml produced a reduction in the viability of 77, 82, and 71%, respectively. Moreover essential oils caused reduction in cell number, collapsed cells, and loss of normal tridimensional composition of the aggregates. Due to the inhibitory effect caused by essential oils on E. granulosus cells we suggested that it would be an effective means for suppression of larval growth.

  10. First case of peritoneal cystic echinococcosis in a domestic cat caused by Echinococcus granulosus sensu stricto (genotype 1) associated to feline immunodeficiency virus infection.

    Science.gov (United States)

    Armua-Fernandez, Maria Teresa; Castro, Oscar F; Crampet, Alejandro; Bartzabal, Álvaro; Hofmann-Lehmann, Regina; Grimm, Felix; Deplazes, Peter

    2014-04-01

    A new cystic echinococcosis case in a cat in Uruguay is reported herein. The cat was taken to a veterinary clinic in Rocha city, Uruguay, due to dyspnea, constipation and abdominal enlargement. During surgery a large quantity of cysts was retrieved from the abdominal cavity. The cysts were morphologically studied and confirmed as Echinococcus granulosus sensu stricto (genotype 1) by molecular tools using cytochrome oxidase submit 1 and small subunit ribosomal RNA gene as target genes. Moreover, for the first time a coinfection with feline immunodeficiency virus (FIV) was detected. FIV-induced immunosuppression could be a determining factor in the development of cystic echinococcosis in cats.

  11. Comparison of excretory-secretory antigen and positive faecal supernatant antigen in the detection of Echinococcus granulosus infection in dogs by CIEP

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    P. R. Prathiush

    Full Text Available Coproantigen detection of Echinococcosis in dogs by counter immunoelectrophoresis was standardized. Adult Echinococcus granulosus worms were obtained from intestine of a necropsied positive dog. Excretory-secretory antigen was prepared by culturing adult worms in Medium 199 (pH 7.4. Faeces of positive dog were collected and fecal supernatant was prepared and used for coproantigen detection. CIEP was carried out using tris-borate buffer (pH 8.0 at a constant current of 8mA/slide for 60 minutes. CIEP detected infection with both the antigens. [Vet World 2009; 2(11.000: 421-422

  12. Strain characterization of Echinococcus granulosus protoscoleces of cattle origin using the in vitro vesicular development.

    Science.gov (United States)

    Elissondo, M C; Dopchiz, M C; Zanini, F; Pérez, H; Brasesco, M; Denegri, G

    2005-06-01

    The aim of this work was to characterize the strain of protoscoleces of E. granulosus of cattle origin using the in vitro vesicular development. The in vitro development of these samples was compared to samples of sheep origin determined previously by genetic analyses as common sheep strain (G1). There were similarities between sheep and cattle samples not only in the time of microcysts formation, but also in the development process. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. In the sheep samples, microcysts were observed between 19 and 20 days. In the cattle samples, microcysts appeared between 20 and 23 days. The coincidence between the development times and physiological characteristics found in the present study may indicate that the parasites from cattle and sheep were of the same strain.

  13. Strain characterization of Echinococcus granulosus protoscoleces of cattle origin using the in vitro vesicular development

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    Elissondo M.C.

    2005-06-01

    Full Text Available The aim of this work was to characterize the strain of protoscoleces of E. granulosus of cattle origin using the in vitro vesicular development. The in vitro development of these samples was compared to samples of sheep origin determined previously by genetic analyses as common sheep strain (G1. There were similarities between sheep and cattle samples not only in the time of microcysts formation, but also in the development process. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. In the sheep samples, microcysts were observed between 19 and 20 days. In the cattle samples, microcysts appeared between 20 and 23 days. The coincidence between the development times and physiological characteristics found in the present study may indicate that the parasites from cattle and sheep were of the same strain.

  14. Therapeutic effects of Sophora moorcroftiana alkaloids in combination with albendazole in mice experimentally infected with protoscolices of Echinococcus granulosus.

    Science.gov (United States)

    Ma, X M; Bao, G Sh; Wan, J M; Liao, D J; Yin, Sh F; Meng, X Q; Zhou, G K; Lu, X M; Li, H Y

    2007-10-01

    The objective of the present study was to determine if the combination of alkaloids from Sophora moorcroftiana seeds and albendazole might be effective in the treatment of experimental echinococcosisin female NIH mice (6 weeks old and weighing 18-20 g, N = 8 in each group) infected withprotoscolices of Echinococcus granulosus. Viable protoscolices (N = 6 x 10(3)) were cultured in vitro in 1640 medium and mortality was calculated daily. To determine the in vivo efficacy, mice were inoculated intraperitoneally with viable protoscolices and then treated once daily by gavage for three months with the alkaloids (50 mg kg-1 day-1) and albendazole (50 mg kg-1 day-1), separately and in combination (both alkaloids at 25 mg kg-1 day-1 and albendazole at 25 mg kg-1 day-1). Next, the hydatid cysts collected from the peritoneal cavity of the animals were weighed and serum IL-4, IL-2, and IgE levels were analyzed. Administration of alkaloids to cultured protoscolices showed significant dose- and time-dependent killing effects. The weight of hydatid cysts was significantly decreased upon treatment with each drug (P < 0.01), but the decrease was more prominent and the rate of hydatid cyst growth inhibition was much higher (76.1%) in the group receiving the combined treatments (18.3 +/- 4.6 mg). IL-4 and total IgE were decreased (939 +/- 447 pg/mL and 2.03 +/- 0.42 IU/mL, respectively) in serum from mice treated with alkaloids and albendazole compared with the untreated control (1481 +/- 619 pg/mL and 3.31 +/- 0.37 IU/mL; P < 0.01). These results indicate that S. moorcroftiana alkaloids have protoscolicidal effects and the combination of alkaloids and albendazole has significant additive effects.

  15. Functional expression and characterization of Echinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage.

    Science.gov (United States)

    Li, Jun; Zhang, Wen-Bao; Loukas, Alex; Lin, Ren-Yong; Ito, Akira; Zhang, Li-Hua; Jones, Malcolm; McManus, Donald P

    2004-02-01

    A full-length cDNA sequence coding for Echinococcus granulosus thioredoxin peroxidase (EgTPx) was isolated from a sheep strain protoscolex cDNA library by immunoscreening using a pool of sera from mice infected with oncospheres. EgTPx expressed as a fusion protein with glutathione S-transferase (GST) exhibited significant thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Furthermore, the suggested antioxidant role for EgTPx was reinforced in an in vivo assay, whereby its expression in BL21 bacterial cells markedly increased the tolerance and survival of the cells to high concentrations of H2O2 compared with controls. Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules. Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces. The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection. Only a portion of the sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated. Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease. Moreover, given that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of antiparasite TPx inhibitors that do not react with host TPx might be feasible.

  16. Therapeutic effects of Sophora moorcroftiana alkaloids in combination with albendazole in mice experimentally infected with protoscolices of Echinococcus granulosus

    Directory of Open Access Journals (Sweden)

    X.M. Ma

    2007-10-01

    Full Text Available The objective of the present study was to determine if the combination of alkaloids from Sophora moorcroftiana seeds and albendazole might be effective in the treatment of experimental echinococcosisin female NIH mice (6 weeks old and weighing 18-20 g, N = 8 in each group infected withprotoscolices of Echinococcus granulosus. Viable protoscolices (N = 6 x 103 were cultured in vitro in 1640 medium and mortality was calculated daily. To determine the in vivo efficacy, mice were inoculated intraperitoneally with viable protoscolices and then treated once daily by gavage for three months with the alkaloids (50 mg kg-1 day-1 and albendazole (50 mg kg-1 day-1, separately and in combination (both alkaloids at 25 mg kg-1 day-1 and albendazole at 25 mg kg-1 day-1. Next, the hydatid cysts collected from the peritoneal cavity of the animals were weighed and serum IL-4, IL-2, and IgE levels were analyzed. Administration of alkaloids to cultured protoscolices showed significant dose- and time-dependent killing effects. The weight of hydatid cysts was significantly decreased upon treatment with each drug (P < 0.01, but the decrease was more prominent and the rate of hydatid cyst growth inhibition was much higher (76.1% in the group receiving the combined treatments (18.3 ± 4.6 mg. IL-4 and total IgE were decreased (939 ± 447 pg/mL and 2.03 ± 0.42 IU/mL, respectively in serum from mice treated with alkaloids and albendazole compared with the untreated control (1481 ± 619 pg/mL and 3.31 ± 0.37 IU/mL; P < 0.01. These results indicate that S. moorcroftiana alkaloids have protoscolicidal effects and the combination of alkaloids and albendazole has significant additive effects.

  17. Modulation of the cellular immune response by a carbohydrate rich fraction from Echinococcus granulosus protoscoleces in infected or immunized Balb/c mice.

    Science.gov (United States)

    Dematteis, S; Pirotto, F; Marqués, J; Nieto, A; Orn, A; Baz, A

    2001-01-01

    Infection of Balb/c mice with Echinococcus granulosus protoscoleces constitutes the model for secondary hydatid infection. The immune response of Balb/c mice infected with E. granulosus is characterized by secretion of antibodies specific for carbohydrate epitopes and production of type-2 cytokines. A role for glycoconjugates in the induction of type-2 responses has been suggested in other host--parasite systems. Although glycoconjugates are immunogenic in E. granulosus infection, the role of these molecules in the establishment of the type-2 response has never been analysed. In this study, a carbohydrate rich fraction (E4+) from E. granulosus protoscoleces was obtained using the monoclonal antibody E492/G1 specific for the moiety Galalpha(1,4)Gal which is widely represented in protoscoleces and other E. granulosus antigenic preparations. The results showed that E4+ was immunogenic in Balb/c mice evoking an antibody response mainly directed against carbohydrate epitopes. In addition, splenocytes from E4+-immunized mice showed suppressed proliferative responses to Con A and E4+ induced IL-10 secretion by E4+-primed and naive splenocytes. The fraction E4+ also was immunogenic in infected mice during early infection. In this case also, splenocytes from infected mice as well as peritoneal cells from infected or naive mice, when stimulated in vitro with E4+, secreted IL-10. Collectively, these results suggest that E4+ may be involved in immunosuppression phenomena and, by stimulating IL-10 secretion, may contribute to the induction and sustaining of the type-2 cytokine response established in early experimental infection.

  18. Identification and analysis of miRNAs in Echinococcus granulosus%细粒棘球绦虫miRNAs的鉴定及分析

    Institute of Scientific and Technical Information of China (English)

    郝力力; 李锐; 李必富

    2012-01-01

    There exists complicated pattern of genes regulation in Echinococcus granulosus, which have distinct characteristics in definitive and intermediate hosts. miRNAs play important role of genes regulation in eukaryotic animals. There are one hundred and eight miRNAs were identified in this experiment and among of them twenty miRNAs belong to conserved miRNA families and eighty two are novel miRNAs in Echinococcus granulosus G1 strain.%细粒棘球绦虫具有复杂的基因调控模式,在中间宿主和终末宿主体内具有截然不同的生物学特性.miRNA是真核生物内存在的非常重要的调控因子,本研究采用Solexa高通量测序技术,对G1株原头蚴microRNAs进行了鉴定和分析,总共发现了108条miRNAs,其中26条属于保守家族,82条是细粒棘球绦虫特有.

  19. [EFFICACY OF A NEW MEBENDAZOLE FORMULATION FOR EXPERIMENTAL ECHINOCOCCUS GRANULOSUS LARVAL INVASION IN ALBINO MICE].

    Science.gov (United States)

    Kovalenko, F P; Kukhaleva, I V; Shkolyar, N A; Legonkov, Yu A; Musaev, G Kh; Bulanova, T E; Samochatova, E I

    2015-01-01

    The problem of echinococcosis has acquired special urgency in Russia in the last 10 years. The dramatically worse epidemiological situation of echinococcosis in the country is suggested by just frequent cases of cystic echinococcosis in the indigenous population of Moscow and its region, including children. Currently, albendazole that is less toxic than mebendazole remains the drug of choice, However, some authors note that E. granulosus larval cysts are moresusceptible to mebendazole than to albendazole. Both drugs mainly show parasitological activity and have no larvicidal effect particularly in larval alveococcosis. Analysis of the results of chemotherapy, with a group of benzimidazole carbamates for echinococcosis in 6 clinical centers from 5 European countries (Italy, Bulgaria, Romania, Greece, and Turkey) over the past 30 years showed that the evaluation of therapeutic effectiveness was overestimated; thus, 40% of all parasitic larval cysts that were considered dead became active again after, 2 years after the treatment. The original oil micronized mebendazole suspension tested by us in albino mice with late-stage larval cystic echinococcosis showed the properties of a highly effective and safe systemic larvicide that caused prompt death in the entire parasite population in the treated animals even in extreme hyperinvasion when the baseline parasite weight was greater than the host's one.

  20. High-resolution melting analysis (HRM) for differentiation of four major Taeniidae species in dogs Taenia hydatigena, Taenia multiceps, Taenia ovis, and Echinococcus granulosus sensu stricto.

    Science.gov (United States)

    Dehghani, Mansoureh; Mohammadi, Mohammad Ali; Rostami, Sima; Shamsaddini, Saeedeh; Mirbadie, Seyed Reza; Harandi, Majid Fasihi

    2016-07-01

    Tapeworms of the genus Taenia include several species of important parasites with considerable medical and veterinary significance. Accurate identification of these species in dogs is the prerequisite of any prevention and control program. Here, we have applied an efficient method for differentiating four major Taeniid species in dogs, i.e., Taenia hydatigena, T. multiceps, T. ovis, and Echinococcus granulosus sensu stricto. High-resolution melting (HRM) analysis is simpler, less expensive, and faster technique than conventional DNA-based assays and enables us to detect PCR amplicons in a closed system. Metacestode samples were collected from local abattoirs from sheep. All the isolates had already been identified by PCR-sequencing, and their sequence data were deposited in the GenBank. Real-time PCR coupled with HRM analysis targeting mitochondrial cox1 and ITS1 genes was used to differentiate taeniid species. Distinct melting curves were obtained from ITS1 region enabling accurate differentiation of three Taenia species and E. granulosus in dogs. The HRM curves of Taenia species and E .granulosus were clearly separated at Tm of 85 to 87 °C. In addition, double-pick melting curves were produced in mixed infections. Cox1 melting curves were not decisive enough to distinguish four taeniids. In this work, the efficiency of HRM analysis to differentiate four major taeniid species in dogs has been demonstrated using ITS1 gene.

  1. The diagnostic importance of species specific and cross-reactive components of Taenia solium, Echinococcus granulosus, and Hymenolepis nana Importância diagnóstica da reação cruzada espécie-específica de componentes da Taenia solium, Echinococcus granulosus e Hymenolepis nana

    Directory of Open Access Journals (Sweden)

    Teresa Montenegro

    1994-08-01

    Full Text Available Sera from patients infected with Taenia solium, Hymenolepis nana and Echinococcus granulosus were tested against homologous and heterologous parasite antigens using an ELISA assay, and a high degree of cross-reactivity was verified. To identify polypeptides responsible for this cross reactivity, the Enzyme Linked Immunoelectro Transfer Blot (EITB was used. Sera from infected patients with T.solium, H.nana, and E.granulosus were assessed against crude, ammonium sulphate precipitated (TSASP, and lentil-lectin purified antigens of T.solium and crude antigens of.H.nana and E.granulosus. Several bands, recognized by sera from patients with T.solium, H.nana, and E.granulosus infections, were common to either two or all three cestodes. Unique reactive bands in H.nana were noted at 49 and 66 K-Da and in E.granulosus at 17-21 K-Da and at 27-32 K-Da. In the crude cysticercosis extract, a specific non glycoprotein band was present at 61-67 K-Da in addiction to specific glycoprotein bands of 50, 42, 24, 21, 18, 14, and 13 K-Da. None of the sera from patients with H.nana or E.granulosus infection cross reacted with these seven glycoprotein bands considered specific for T.solium infection.Soros de pacientes infectados com Taenia solium, Hymenolepis nana e Echinococcus granulosus foram testados contra antígenos parasitários homólogos e heterólogos usando o teste de ELISA e foi verificado alto grau de reatividade cruzada. Para identificar os polipetídeos responsáveis por esta reatividade cruzada foi utilizado o teste "Enzyme Linked Immunoelectro Transfer Blot (EITB". Soros de pacientes infectados por T.solium, H.nana, e E.granulosus foram colocados em contato com precipitado de sulfato de amônia e antígenos não purificados de T.solium e os de H.nana e E.granulosus. Várias bandas reconhecidas pelos soros de pacientes com infecção por T.solium, H.nana e E.granulosus foram comuns a dois ou três destes cestódeos. Uma única banda foi notada em H

  2. A pharmacology-based comparison of the activity of albendazole and flubendazole against Echinococcus granulosus metacestode in sheep.

    Science.gov (United States)

    Ceballos, L; Virkel, G; Elissondo, C; Canton, C; Canevari, J; Murno, G; Denegri, G; Lanusse, C; Alvarez, L

    2013-09-01

    Cyst echinococcosis (CE) is a zoonotic disease caused by the larval stage of the Echinococcus granulosus helminth parasite. The work reported here aimed to compare the efficacy of albendazole (ABZ) and flubendazole (FLBZ) against CE in naturally infected sheep. Additionally, their comparative pharmacokinetic behaviour and the assessment of serum liver enzymes activities were studied. Twelve (12) naturally infected sheep were allocated to the following experimental groups: unmedicated control group, FLBZ-treated and ABZ-treated. Treatments were orally performed every 48 h, over 55 days at dose rate of 10 (FLBZ) and 8.5 (ABZ) mg/kg (equimolar dose rates). The efficacy of the drug treatments was based on protoscoleces' vitality/viability. The kinetic disposition assessment included the Initial and Final Kinetic Studies which implicated the collection of blood samples after both the first and the last drug administration. Blood samples were processed to measure drug concentrations by HPLC. The protoscoleces' vitality observed in the untreated control group (98%) was significantly reduced in the presence of both ABZ and FLBZ. 90% of mice inoculated with protoscoleces in the control group developed hydatid cysts in their peritoneal cavity (viability study). However, only 25% (FLBZ) and 33% (ABZ) of mice inoculated with protoscoleces recovered from treated sheep, developed hydatid cysts in their abdominal cavity. Reduced FLBZ (R-FLBZ) was the main metabolite recovered in the bloodstream after oral administration of FLBZ to sheep. Low plasma concentrations of FLBZ parent drug were measured up to 48 h post-administration. ABZ was not detected in plasma at any time post-treatment, being its metabolites ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO₂) recovered in plasma. Hepatotoxicity due to the continued treatment with either ABZ or FLBZ was not observed. A 3-fold increase ethoxyresorufin O-deethylase activity, a cytochrome P450 1A (CYP1A)-dependent enzyme reaction, was

  3. Molecular and biochemical mining of heat-shock and 14-3-3 proteins in drug-induced protoscolices of Echinococcus granulosus and the detection of a candidate gene for anthelmintic resistance.

    Science.gov (United States)

    Pan, D; Das, S; Bera, A K; Bandyopadhyay, S; Bandyopadhyay, S; De, S; Rana, T; Das, S K; Suryanaryana, V V; Deb, J; Bhattacharya, D

    2011-06-01

    Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus is a disease that affects both humans and animals. In humans the disease is treated by surgery with a supplementary option of chemotherapy with a benzimidazole compound. During the present study heat-shock protein 60 (HSP 60) was identified as one of the most frequently expressed biomolecules by E. granulosus after albendazole treatment. Data were correlated with 14-3-3 protein signature, and overexpression of this molecule after albendazole induction was an indicator of cell survival and signal transduction during in vitro maintenance of E. granulosus for up to 72 h. This observation was further correlated with a uniform expression pattern of a housekeeping gene (actin II). Out of three β-tubulin gene isoforms of E. granulosus, β-tubulin gene isoform 2 showed a conserved point mutation indicative of benzimidazole resistance.

  4. ADVANCE IN GENOTYPE AND ANTIGEN OF ECHINOCOCCUS GRANULOSUS%细粒棘球绦虫基因型与抗原研究进展

    Institute of Scientific and Technical Information of China (English)

    王颖旺; 杨光友; 蒋忠荣; 邓世金

    2010-01-01

    细粒棘球绦虫(Echinococcus granulosus,Eg)是一种重要的人兽共患寄生虫.现已报道细粒棘球绦虫有10个基因型(G1~G10),我国仅发现G1和G6.细粒棘球绦虫六钩蚴阶段的Eg95,原头蚴阶段的EgFABP,成虫阶段的EgM9和Eg31分别被认为是最有前途的用于免疫保护的抗原.其中Eg95基因工程疫苗已经商业化生产,对羊免疫保护率达到了95%以上.

  5. In-vitro susceptibility of hydatid cysts of Echinococcus granulosus to nitric oxide and the effect of the laminated layer on nitric oxide production.

    Science.gov (United States)

    Steers, N J; Rogan, M T; Heath, S

    2001-08-01

    Murine hydatid cysts of Echinococcus granulosus were incubated in vitro in the presence of nitric oxide produced from S-nitroso-N-acetylpenicillamine (SNAP) or interferon-gamma activated peritoneal macrophages. In both situations, evidence of cyst damage and death was observed by microscopy in over 77% of cysts after 3 days, indicating that intact hydatid cysts could be susceptible to a Th1 driven macrophage attack. A crude extract of the laminated layer from cysts was found to be able to reduce the production of nitric oxide from activated macrophages in vitro and in vivo and this may have been due to phagocytosis of laminated layer fragments by the macrophages. The results indicate that, although cysts may be susceptible to the effects of nitric oxide, the laminated layer may be involved in downregulating nitric oxide production.

  6. Identification of functional FKB protein in Echinococcus granulosus: its involvement in the protoscolicidal action of rapamycin derivates and in calcium homeostasis.

    Science.gov (United States)

    Cumino, Andrea C; Lamenza, Pamela; Denegri, Guillermo M

    2010-05-01

    FK506 (tacrolimus) and polyketide macrolides such as rapamycin and its derivates bind to FK506-binding proteins (FKBPs). These proteins display a peptidyl-prolyl rotamase function that is believed to catalyze protein folding and they are well-validated anti-proliferative drug targets in certain pathogenic microorganisms, and their functions have been characterized in parasitic protozoa. However, much less is known in helminths and trials with rapalogs on cestoda have not yet been reported. Due to a growing need for new treatment options for human cystic echinococcosis, the in vitro efficacy of rapalogs in Echinococcus granulosus was investigated. We determined the effect of ramapycin, FK506 and everolimus against this cestode, demonstrating their protoscolicidal ability. Also, we observed synergic scolicidal actions during combined therapy with rapalogs plus cyclosporine A, proposing dual administration of drugs to improve pharmacological effects in vivo. We have identified an E. granulosus (Eg)-fkb1 gene that encodes Eg-FKBP, an archetypal protein of the FKBP family, which includes all residues implicated in the binding of pharmacological ligands, in the enzymatic activity and in interactions with possible target proteins. Levels of Eg-fkb1 mRNA are over-expressed by acid but not rapalog treatment. We also described the presence of receptor-operated calcium channels in the larval stage, suggesting that exogenous ligands may dissociate the interaction of Eg-FKBP from these intracellular channels, enhancing the activity of the Ca(2+) release and interfering with their normal regulatory functions. As rapamycin sensitivity is the major criterion used to detect targets of rapamycin kinase, we identified and analyzed in silico critical residues of putative homologs in the Echinococcus genome. These preliminary results will allow us to continue subsequent studies that could reveal the precise intracellular functions of Eg-FKBP, providing greater knowledge for further

  7. Identification of Echinococcus Granulosus Strains in Isolated Hydatid Cyst Specimens from Animals by PCR-RFLP Method in West Azerbaijan – Iran

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    Haleh Hanifian

    2013-09-01

    Full Text Available Background: The aim of this study was DNA extraction from protosco­lecses of Echinococcus granulosus and identification of these strains in West-Azerbai­jan Province, north western Iran.Methods: Thirty one livestock isolates from sheep and cattle were collected from abattoirs of the province. To investigate the genetic variation of the isolates, after DNA extraction by Glass beads-phenol chloroform method; PCR-RLFP analysis of rDNA-ITS1 was performed using three different restric­tion enzymes of Taq 1, Rsa 1 and Alu 1.Result: Amplified PCR products for all isolates were 1000bp band which is expected band in sheep strains (G1-G3 complex. The results of RFLP analy­sis also were the same for all isolates. PCR-RFLP patterns restriction en­zymes were identical as follows, Rsa1 bands under UV showed two bands approximately 655bp and 345bp. Alu1 bands were as follows: two approx­imately 800bp and 200bp and Taq1 did not cut any region and bands were approximately 1000 bp in all samples.Conclusions: Based on PCR-RFLP patterns of ITS1 fragment produced with endonucleases enzyme digestion in animal isolates, it can be concluded that a single strain of E. granulosus (sheep strain or G1-G3 complex is domi­nant genotype in this province

  8. Characterization of excretory-secretory products from protoscoleces of Echinococcus granulosus and evaluation of their potential for immunodiagnosis of human cystic echinococcosis.

    Science.gov (United States)

    Carmena, D; Martínez, J; Benito, A; Guisantes, J A

    2004-09-01

    This study describes, for the first time, the characterization of excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, evaluating their usefulness in the immunodiagnosis of human cystic echinococcosis. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. This preparation contained over 20 major protein components which could be distinguished by 1-dimensional SDS-PAGE with apparent masses between 9 and 300 kDa. The culture of of protoscoleces from liver produced a greater variety of excretory-secretory protein components than those from lung. Determination of enzymatic activities of secreted proteins revealed the presence of phosphatases, lipases and glucosidases, but no proteases. These findings were compared to those obtained from somatic extracts of protoscoleces and hydatid cyst fluid products. Immunochemical characterization was performed by immunoblotting with sera from individuals infected by cystic echinococcosis (n = 15), non-hydatidic parasitoses (n = 19), various liver diseases (n = 24), lung neoplasia (n = 16), and healthy donors (n = 18). Antigens with apparent masses of 89, 74, 47/50, 32, and 20 kDa showed specificity for immunodiagnosis of human hydatidosis. The 89 and 74 kDa components corresponded to antigens not yet described in E. granulosus, whereas proteins of 41-43 kDa and 91-95 kDa were recognized by the majority of the non-hydatid sera studied.

  9. MicroRNA profiling of the intestinal tissue of Kazakh sheep after experimental Echinococcus granulosus infection, using a high-throughput approach

    Science.gov (United States)

    Jiang, Song; Li, Xin; Wang, Xuhai; Ban, Qian; Hui, Wenqiao; Jia, Bin

    2016-01-01

    Cystic echinococcosis (CE), caused by infection with the larval stage of the cestode Echinococcus granulosus, is a chronic zoonosis, to which sheep are highly susceptible. Previously, we found that Kazakh sheep with different MHC haplotypes differed in CE infection. Sheep with haplotype MHCMvaIbc-SacIIab-Hin1Iab were resistant to CE infection, while their counterparts without this haplotype were not. MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at the post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. To identify microRNA controlling resistance to CE in the early stage of infection, microRNA profiling was conducted in the intestinal tissue of sheep with resistant and non-resistant MHC haplotypes after peroral infection with E. granulosus eggs. A total of 351 known and 186 novel miRNAs were detected in the resistant group, against 353 known and 129 novel miRNAs in the non-resistant group. Among these miRNAs, 83 known miRNAs were significantly differentially expressed, including 75 up-regulated and 8 down-regulated miRNAs. Among these known microRNAs, miR-21-3p, miR-542-5p, miR-671, miR-134-5p, miR-26b, and miR-27a showed a significantly higher expression in CE-resistant sheep compared to the CE-non-resistant library, with the FC > 3. Functional analysis showed that they were NF-kB pathway-responsive miRNAs, which are involved in the inflammation process. The results suggest that these microRNAs may play important roles in the response of intestinal tissue to E. granulosus. PMID:27235195

  10. Interleukin-17A correlates with interleukin-6 production in human cystic echinococcosis: a possible involvement of IL-17A in immunoprotection against Echinococcus granulosus infection.

    Science.gov (United States)

    Mezioug, Dalila; Touil-Boukoffa, Chafia

    2012-01-01

    Hydatidosis is a parasitic disease caused by the development, in humans and other mammals, of the larval form of Taenia, Echinococcus granulosus. It is one of the world's major zoonotic infections. This study aimed to examine interleukin-6 (IL-6), interferon-γ (IFN-γ) and interleukin-17A (IL-17A) production in patients with cystic echinococcosis (CE), and the role of IL-17A in the modulation of the immune response against the extracellular parasite, E. granulosus. A relationship between IL-6, IL-17A production and C reactive Protein (CRP) levels was also assessed. IL-6, IFN-γ, IL-17A and CRP production were determined in serum from Algerian hydatid patients. Cytokine production was also measured in supernatants from cultures of peripheral blood mononuclear cells (PBMCs) from hydatid patients stimulated by a major parasitic antigen (antigen-5). The increased activity of IL-6, IFN-γ and IL-17A were observed in most serum samples from patients. In contrast, healthy controls showed only minor levels. Similarly, high levels of CRP were detected. Our in vitro results indicate a positive correlation between IL-6, IFN-γ and IL-17A production in PBMC culture supernatants. However, IL-6, IFN-γ and IL-17A activity was low in serum and supernatants of PBMC cultures from relapsing patients, and there was no evidence of an immune response against parasitic antigen. Collectively, our results show that IL-17A was produced during human cystic echinococcosis, and was involved in the host defense mechanisms against the extracellular parasite E. granulosus. Our data suggest that IL-17A plays an immunoprotective role in this parasitic, helminth infection.

  11. MicroRNA profiling of the intestinal tissue of Kazakh sheep after experimental Echinococcus granulosus infection, using a high-throughput approach

    Directory of Open Access Journals (Sweden)

    Jiang Song

    2016-01-01

    Full Text Available Cystic echinococcosis (CE, caused by infection with the larval stage of the cestode Echinococcus granulosus, is a chronic zoonosis, to which sheep are highly susceptible. Previously, we found that Kazakh sheep with different MHC haplotypes differed in CE infection. Sheep with haplotype MHCMvaIbc-SacIIab-Hin1Iab were resistant to CE infection, while their counterparts without this haplotype were not. MicroRNAs (miRNAs, a class of small non-coding RNAs, are key regulators of gene expression at the post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. To identify microRNA controlling resistance to CE in the early stage of infection, microRNA profiling was conducted in the intestinal tissue of sheep with resistant and non-resistant MHC haplotypes after peroral infection with E. granulosus eggs. A total of 351 known and 186 novel miRNAs were detected in the resistant group, against 353 known and 129 novel miRNAs in the non-resistant group. Among these miRNAs, 83 known miRNAs were significantly differentially expressed, including 75 up-regulated and 8 down-regulated miRNAs. Among these known microRNAs, miR-21-3p, miR-542-5p, miR-671, miR-134-5p, miR-26b, and miR-27a showed a significantly higher expression in CE-resistant sheep compared to the CE-non-resistant library, with the FC > 3. Functional analysis showed that they were NF-kB pathway-responsive miRNAs, which are involved in the inflammation process. The results suggest that these microRNAs may play important roles in the response of intestinal tissue to E. granulosus.

  12. Echinococcus granulosus: first report of microcysts formation from protoscoleces of cattle origin using the in vitro vesicular culture technique.

    Science.gov (United States)

    Elissondo, M C; Dopchiz, M C; Brasesco, M; Denegri, G

    2004-12-01

    The aim of this work was the achievement of microcysts formation from protoscoleces of E. granulosus of cattle origin using the in vitro vesicular culture technique. Vesiculated protoscoleces and protoscoleces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoleces. On day 20, some microcysts with a complete laminated layer were observed. By day 48, microcysts completely developed could be observed. This is the first study where microcysts formation was obtained using protoscoleces of E. granulosus of cattle origin.

  13. Echinococcus granulosus: first report of microcysts formation from protoscoleces of cattle origin using the in vitro vesicular culture technique

    Directory of Open Access Journals (Sweden)

    Elissondo M.C.

    2004-12-01

    Full Text Available The aim of this work was the achievement of microcysts formation from protoscoieces of E. granulosus of cattle origin using the in vitro vesicular culture technique. Vesiculated protoscoieces and protoscoieces with posterior bladders appeared during the first week of incubation. After 14 days of culture, a laminated layer appeared like a fine membrane in one of the extremes of the protoscoieces. On day 20, some microcysts with a complete laminated layer were observed. By day 48, microcysts completely developed could be observed. This is the first study where microcysts formation was obtained using protoscoieces of E. granulosus of cattle origin.

  14. Development of three PCR assays for the differentiation between Echinococcus shiquicus, E. granulosus (G1 genotype), and E. multilocularis DNA in the co-endemic region of Qinghai-Tibet plateau, China.

    Science.gov (United States)

    Boufana, Belgees; Umhang, Gérald; Qiu, Jiamin; Chen, Xingwang; Lahmar, Samia; Boué, Franck; Jenkins, David; Craig, Philip

    2013-04-01

    To investigate echinococcosis in co-endemic regions, three polymerase chain reaction (PCR) assays based on the amplification of a fragment within the NADH dehydrogenase subunit 1 (ND1) mitochondrial gene were optimized for the detection of Echinococcus shiquicus, Echinococcus granulosus G1, and Echinococcus multilocularis DNA derived from parasite tissue or canid fecal samples. Specificity using parasite tissue-derived DNA was found to be 100% except for E. shiquicus primers that faintly detected E. equinus DNA. Sensitivity of the three assays for DNA detection was between 2 and 10 pg. Ethanol precipitation of negative PCR fecal samples was used to eliminate false negatives and served to increase sensitivity as exemplified by an increase in detection from 0% to 89% of E. shiquicus coproDNA using necropsy-positive fox samples.

  15. Prevalência de Echinococcus granulosus (Batsch, 1786 em cães urbanos errantes do município de Dom Pedrito (RS, Brasil Prevalence of Echinococcus granulosus (Batsch, 1786 in urban stray dogs from Dom Pedrito in the State of Rio Grande do Sul, Brazil

    Directory of Open Access Journals (Sweden)

    Adriane Nunes Hoffmann

    2001-10-01

    Full Text Available Echinococcus granulosus é um dos parasitas mais importantes envolvidos em zoonoses de municípios próximos a fronteira do Rio grande do Sul com a Argentina e Uruguai. Amostras de 65 cães urbanos errantes do município de Dom Pedrito foram analisadas por meio de três técnicas: purgação pelo bromidrato de arecolina para visualização da presença do parasito; ensaio de imunoadsorção enzimática (ELISA, para detecção de coproantígenos, imunofluorescência indireta (IFI, para detecção de anticorpos séricos contra E. granulosus. Destes cães, 7,7% (5/65 apresentaram o parasita nas fezes, 10,8 (7/65 coproantígenos e 13,8% (9/65 anticorpos séricos contra o cestódeo. Conclui-se que a equinococose canina, no meio urbano, pode representar um sério problema à saúde pública, devido ao risco de hidatidose humana.Echinococcus granulosus is the one of the most important parasites involved in zoonosis in the State of Rio Grande do Sul, in cities near the Argentinian and Uruguayian border. Sixty-five samples of urban stray dogs from Dom Pedrito county were analyzed by three techniques: purgation by arecoline hydrobromide, to verify the presence of the parasite; enzyme-linked immunosorbent assay test (ELISA, to detect the coproantigen and indirect immunofluorescence antibody test (IFA to identify serum antibodies against E. granulosus. From the analyzed dogs 7.7% (5/65 have presented the parasite in feces, 10.8% (7/65 had coproantigens and 13.8% (9/65 serum antibodies against this cestode. It was concluded that canine echinococcosis in the urban zone may represent a serious problem to public health, due to hidatidosis risk for humans.

  16. Preliminary study on detection of Echinococcus granulosus DNA by loop-mediated isothermal amplification%环介导等温扩增技术检测细粒棘球绦虫DNA的初步研究

    Institute of Scientific and Technical Information of China (English)

    徐祥珍; 金小林; 李健; 江文才; 蒋岗

    2011-01-01

    Objective To evaluate the sensitivity of loop-mediated isothermal amplification (LAMP) on the detection of Echi-nococcus granulosus. Methods The DNAs were extracted from Echinococcus granulosus eggs and adults. According to Echinococcus mitochondrial 12S rRNA sequences and the mechanism of LAMP, 4 Echinococcus specific primers were designed and used for LAMP assay, and Bubble taenia and the blank were used as the negative control for evaluation of the specificity. The LAMP products were stained by SYBR Green I and analyzed by electrophoresis, and 1000, 100, 10, 1 eggs of Echinococcus granulosus per 200 μl were amplified by LAMP for evaluating the sensitivity. Results The LAMP products of Echinococcus granulosus adult DNA became turbid and green after staining while the products of control DNA kept clarify and brown after staining. Electrophoresis analysis showed that the LAMP products of Echinococcus granulosus eggs presented characteristic ladders, but the products of control did not. The detection limit of LAMP assay was 1 egg of Echinococcus granulosus per reaction. Conclusions LAMP assay is a simple, sensitive and specific method for detection of hydatid disease pathogens and could be used for the disease surveillance.%目的 评价环介导等温扩增技术(LAMP)检测细粒棘球绦虫的敏感性.方法 提取细粒棘球绦虫虫卵及成虫DNA,根据棘球绦虫线粒体12SrRNA基因序列及LAMP法原理,设计4条细粒棘球绦虫特异性引物,进行LAMP反应,反应产物经SYBR Green Ⅰ显色及1.5%琼脂糖电泳鉴定,同时设置泡状带绦虫及空白对照.用1000、100、10、1个虫卵/200μl细粒棘球绦虫虫卵DNA进行LAMP反应,评价其敏感性.结果 细粒棘球绦虫检测管反应液呈混浊沉淀,显色后为绿色;对照组澄清,显色后为棕色.虫卵产物电泳后呈LAMP特征性梯状条带,对照组无扩增产物.LAMP可检测到的虫卵最低数量为1个.结论 LAMP法敏感、特异、简便,可用于棘

  17. Green chemical synthesis of gold nanoparticles by using Penicillium aculeatum and their scolicidal activity against hydatid cyst protoscolices of Echinococcus granulosus.

    Science.gov (United States)

    Barabadi, Hamed; Honary, Soheila; Ali Mohammadi, Milad; Ahmadpour, Ehsan; Rahimi, Mohammad Taghi; Alizadeh, Ahad; Naghibi, Farzaneh; Saravanan, Muthupandian

    2017-02-01

    Hydatid disease is a helminth infection with various clinical complications caused by the larval stage of the dog tapeworm Echinococcus granulosus. The scolicidal agents have been broadly applied for inactivation of the fertile cysts up to now, but these scolicidal agents have several side effects on patients. Therefore, this study aimed to explore the scolicidal activity of green synthesized gold nanoparticles (AuNPs) utilizing mycelia-free culture filtrate of Penicillium aculeatum against hydatid cyst protoscolices of E. granulosus. The size and morphology of AuNPs were affirmed by UV-visible spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), and dynamic light scattering (DLS) analysis. The Fourier transform infrared (FT-IR) analysis of AuNPs showed the presence of possible functional groups responsible for the bioreduction and capping. The AuNPs were formed relatively uniform with spherical shape and superior monodispersity with the average diameter of 60 nm. Consequently, various concentrations (0.05, 0.1, 0.2, and 0.3 mg/mL) of green synthesized AuNPs and different exposure times (10, 30, 60, and 120 min) were used against hydatid cyst protoscolices. Statistically, the difference between the scolicidal effects of AuNPs were seen extremely significant for all four concentrations and at various exposure times in comparison to the control group (P < 0.0001). The most mean protoscolex elimination ratio was 94% (0.3 mg/mL AuNPs and 120-min exposure time). The current investigation indicated that applying biogenic AuNPs may be considered as a potential scolicidal agent for cystic hydatid disease. However, further studies are required to evaluate the efficacy of AuNPs in vivo.

  18. Serological Evaluation of EgAgB16 kDa, a Recombinant Antigen from Echinococcus Granulosus for Diagnosis of Human Hydatidosis

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    L Maghen

    2010-09-01

    Full Text Available Background: Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits (EgAgB16 kDa from Echinococcus granulosus (Iranian G1 strain and its evaluation by ELISA test.Methods: DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus (Iranian G1 strain protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individu­als (n=72, healthy individual (n=48, toxoplasmosis (n=4, strongyloidosis (n=4, kala-azar (n=5 and tuberculosis (n=5 were examined using this recombinant antigen.Results: Recombinant protein was purified by affinity chromatography using His-Tag column. After purifica­tion, recombinant protein was confirmed by western blot analysis using His Tag monoclonal anti­body or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16. Conclusion: While the produced recombinant AgB16 kDa showed promising results in diagnosing human hy­datidosis, but more investigations should be implemented to reach an accurate gold standard.

  19. In vitro effects of p38MAPK inhibitor SB202190 on Echinococcus granulosus protoscoleces%p38MAPK抑制剂SB202190体外对细粒棘球蚴原头节的作用

    Institute of Scientific and Technical Information of China (English)

    张晶; 吕海龙; 王成华; 孙冯; 雷颖; 彭心宇; 姜玉峰

    2013-01-01

    目的 探讨p38MAPK抑制剂SB202190体外抑制细粒棘球蚴原头节生长的作用.方法 将体外培养的细粒棘球蚴原头节分别加入12.5、25、50、100 μmol/L的SB202190中体外孵育.利用伊红染色的方法,在光镜下观察原头节的活力变化.实验重复3次;扫描电子显微镜下(SEM)下观察SB202190作用后原头节表面超微结构改变;不同浓度SB202190作用24 h后,半胱氨酸天冬氨酸蛋白酶-3(caspase-3)活性检测试剂盒检测caspase-3酶活性.结果 50 μmol/L和100 μmol/L的SB202190作用1d后,头节活力开始下降.作用14d后,100 μmol/LSB202190组无存活的头节,50 μmol/L SB202190组的头节活力仅为13.8%.超微结构显示100 μmol/LSB202190作用6d后,原头节顶突外翻、变形,顶突界面缺损,吸盘变形,体表出现虫蛀样损害.不同浓度SB202190作用24 h后,与正常对照组比较,SB202190高浓度组原头节的caspase-3表达明显增高.结论 p38MAPK抑制剂SB202190在体外有明显的抑制细粒棘球蚴原头节生长的作用.%The aim of this study was to investigate the in vitro efficacy of the p38MAPK inhibitor SB202190 against Echinococcus granulosus protoscoleces.Protoscolices of Echinococcusgranulosus were incubated with SB202190 at concentrations of 12.5,25,50,100μmol/L,and the effects on protoscoleces viability were monitored by 0.1% eosin staining and light microscopic inspection.Each experiment was repeated for three times.Starting from day 1 of incubation in the presence of 50 μmol/L and 100μmol/L SB202190,viability of protoscoleces started to decline depending on the concentration of the inhibitors.After 14 days of incubation in 100μmol/L of SB202190,no survival protoscolece remained but 13.8% survival proto scoleces were found in 50μmol/L of SB202190 after 14 days.At the same time,ultrastructural effects of protoscoleces were observed by scanning electron microscopy (SEM),the morphological changes included contraction of the soma

  20. Study on Glutathione S-Transferase (GST Inhibition Assay by Triclabendazole. І: Protoscoleces (Hydatid Cyst; Echinococcus granulosus and Sheep Liver Tissue

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    MA Seyyedi

    2005-06-01

    Full Text Available Hydatid disease is a term used to refer infection with the methacestode of Echinococcus granulosus parasite in humans, and echinococcusis is restricted to infection with the adult stage in carnivores.Glutathione S-Transferase (GST represents the major class of detoxification enzymes from helminth parasites such as Echinococcus protoscoleces (PSC and it is candidate for chemotherapeutic and vaccine design. Therefore, GST of protoscoleces could be a target for evaluation of drug effect as triclabendazole in hydatid cyst. For this purpose, GST enzymes were purified from protoscoleces of hydatid cyst and sheep liver tissue by glutathione affinity chromatography using a wash-batch method and subsequently detected their SDS-PAGE pattern. Afterward, GST specific activity levels were assayed in the whole extract and purified solutions spectrophotometrically at 30°C with reduced glutathione (GSH and 1-chloro-2, 4-dinitrobenzen (CDNB substrate. Finally, GST inhibition assay was investigated in the solutions by powder and bolus of triclabendazole. GST fraction as a 26 kDa (MW band was obtained on SDS-PAGE. The level of GST specific activity in purified solutions was detected 10.24 µmol/min/mg proteins for protoscoleces and 37.84 µmol/min/mg protein for liver tissue. Comparison of the effect of powder and bolus of triclabendazole in solutions revealed inhibition concentration (IC50 8.71 and 11.16 µg/ml for protoscoleces GST and 8.65 and 9.70 µg/ml for liver tissue GSTs, respectively. These findings suggest the possibility of selective inhibition of protoscoleces. GSTs by triclabendazol in vitro and use of these results for understanding of its molecular effect in vivo.

  1. Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

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    Majid Esmaelizad

    2013-06-01

    Full Text Available In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3 were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST. The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL, but interleukin (IL-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6% was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

  2. The Effects of LBP on Lymphocyte Subsets in Echinococcus Granulosus Mice%枸杞多糖对细粒棘球蚴小鼠T淋巴细胞亚群的影响

    Institute of Scientific and Technical Information of China (English)

    刘丽华; 王元; 王娅娜; 赵巍; 赵嘉庆

    2012-01-01

    Objective To explore effects of LBP (Lycium barbarum polysaccharide) on lymphocyte subsets in echinococcus granulosus mice and to disscuss the immune role of LBP in echinococcus infected body. Methods LBP solution was injected subcutaneous to BALB/c mice 3 times at 2w interval. Mice were infected with E chinococcus granulosus larva at 8th w. IFN - γ + , IL - 4 + and CD4 +/CD8 + in mice were detected with Flow cytometry before and after the LBP administration and Echinococcus granulosus infection. Results compared with no LBP group,LBP made IFN - "y increase,IL -4 reduce,CD4 +/CD8 + increase significantly in BALB/c mice's lymphocyte's T cell. Compared with Echinococcus granulosus mice, LBP made IFN - "y increase (2,15 w) ,IL-4 reduce(2, lOw) ,CD4 +/CD8 + increase(2, lOw) ,then reduce( 15 ,20w) in Echinococcus granulosus mouse models lymphocyte's T cell. Conclusion it shows that LBP has the positive role of immune regulation by promoting T lymphocytes in Echinococcus granulosus infected mice to Thl differentiation , inhibiting Th2 differentiation in BALB/c mouse.%目的 研究枸杞多糖(Lycium Barbarum Polysaccharides,LBPs)对细粒棘球蚴小鼠脾脏T细胞亚群的影响,初步探讨LBP对细粒棘球蚴感染后机体免疫功能变化的影响.方法 枸杞多糖溶液皮下给药3次,每次间隔2周,于第3次给药后2周,用细粒棘球蚴原头蚴攻击感染BALB/c小鼠,使用流式细胞仪动态检测给药前后、感染前后不同时间点小鼠脾脏IFN-γ+CD3+T细胞、IL-4+CD3+T细胞占总T细胞的比例以及CD4+/CD8+T细胞的比值变化.结果 与给药前比较,枸杞多糖给药后7周,小鼠脾脏分泌IFN-γ的T细胞比例增加,分泌IL-4的T细胞比例减少,CD4+/CD8+的比例明显增加;与感染对照组相比,枸杞多糖能使细粒棘球蚴感染小鼠脾脏分泌IFN-γ的T细胞比例增加(感染后2、15周);分泌IL-4的T细胞比例减少(感染后2、10周),CD4+/CD8+的比例先升高(2、10周),后降低(15、20

  3. Echinococcus granulosus: biological comparison of cattle isolates from endemic regions of Argentina and Spain Echinococcus granulosus: comparación biológica de aislados de bovinos de regiones endémicas de Argentina y España

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    M. V. Andresiuk

    2009-12-01

    Full Text Available In the present study we have compared cattle isolates of Echinococcus granulosus from Argentina and Spain. The aim was to compare and determine if there exist phenotypic and genetic differences within E. granulosus cattle isolates between an endemic area of Spain (where the disease is mainly restricted to a sheep-dog cycle and an endemic area of Argentina (where cattle are the most abundant intermediate hosts. The Spanish samples were previously identified as G1 genotype. The Argentinean samples were also identified as G1, but some variants were found for the cytochrome c oxidase-1 (CO1 and NADH dehydrogenase-1 (ND1 mitochondrial genes. When comparing the cyst features and the morphology of the larval rostellar hooks in both regions, some differences were found. The morphometric analyses of the larval rostellar hooks showed the existence of two distinct clearly separated groups (one corresponding to the Argentinean samples and the other to the Spanish ones. In conclusion, there are some genetic and phenotypic differences within E. granulosus cattle isolates from Argentina and Spain. Probably these differences, more important from an epidemiological point of view, are related to different steps in the disease control in both countries. Further studies involving other epidemiological, morphometric and molecular data, including other types of livestock, would contribute to clarify and expand the present work.El objetivo del presente trabajo fue determinar si existen diferencias fenotípicas y genéticas entre los aislados de Echinococcus granulosus de origen bovino provenientes de dos regiones geográficas donde la hidatidosis es endémica, una de España (donde predomina el ciclo perro-oveja y una de Argentina (donde el bovino es el hospedador intermediario más importante. Las muestras españolas fueron previamente identificadas como pertenecientes al genotipo G1. Las muestras argentinas también correspondían al genotipo G1, pero entre ellas se

  4. Research on immunology of Echinococcus granulosus infections%宿主感染细粒棘球蚴免疫反应的研究进展

    Institute of Scientific and Technical Information of China (English)

    郑宏; 徐志新; 汪师贞; 温浩

    2002-01-01

    @@ 棘球绦虫的中绦期寄生于人体和动物,可导致棘球蚴病(也称为包虫病).由细粒棘球蚴绦虫(Echinococcus granulosus, E.g.)所致的包虫病称为囊型包虫病,它是一种严重危害人畜健康的人兽共患寄生虫病.棘球蚴在宿主体内发育缓慢,人往往在感染数月甚至数年后因出现临床症状而被发现.细粒棘球蚴感染激发宿主产生免疫应答的机制较为复杂.细粒棘球蚴抗原成分多且可在特定的生活史阶段(期)表达特定的抗原,产生不同的特异性免疫应答[1].机体感染E.g.后的初期,尤其是以前曾有过接触的患者对免疫杀伤作用较为明显,而一旦包囊在宿主体内完全形成,其免疫应答则明显减弱.针对细粒棘球蚴感染和其它带科寄生虫感染后其宿主免疫应答的机制,各国学者已做了大量工作,认为在包囊形成前期和后期会诱发宿主产生不同的免疫应答[2].

  5. Evaluation of Immunogenicity of Novel Isoform of EG95 (EG95-5G1 From Echinococcus granulosus in BALB/C Mice.

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    Majid Pirestani

    2014-12-01

    Full Text Available Echinococcosis is a zoonotic parasitic disease of humans and various herbivorous domestic animals transmitted by the contact with domestic and wild carnivores, mainly dogs and foxes. The aim of this study is the production, purification and evaluation immunogenicity of new construction of EG95 protein.The recombinant plasmid pET32-a+ used for Eg95 expression was constructed with the EG95 gene of Echinococcus granulosus fused with the thioredoxin tag. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies in cell lysate. The purification was performed under denaturing conditions in the presence of 8M urea by Ni-NTA column and dialysis. The purified recombinant proteins were confirmed with western blot analysis using polyclonal antiserum. To find out the immunogenicity of the purified protein, the BALB/c mice (10 mice/group were immunized by injecting 20 μg rEG95 protein formulated in Freund's and alum adjuvant.Immunization of mice with rEG95 using CFA/IFA and alum adjuvant generated high level of total antibody. In proliferation assay, the lymphocytes were able to mount a strong proliferative response with related production of IFN-γ, IL-12 and TNF-α but with low secretion of either IL-4 or IL-10. The humoral and cellular immune responses against rEG95 suggested a mixed Th1/Th2 response with high intensity toward Th1.Our findings suggest that new construct of rEG95 formulated with CFA/IFA and alum adjuvant elicited strong cellular and humoral responses supporting further development of this vaccine candidate.

  6. Genetic characterization of human-derived hydatid cysts of Echinococcus granulosus sensu lato in Heilongjiang Province and the first report of G7 genotype of E. canadensis in humans in China.

    Science.gov (United States)

    Zhang, Tiemin; Yang, Dong; Zeng, Zhaolin; Zhao, Wei; Liu, Aiqin; Piao, Daxun; Jiang, Tao; Cao, Jianping; Shen, Yujuan; Liu, Hua; Zhang, Weizhe

    2014-01-01

    Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus sensu lato (s.l.) is one of the most important zoonotic parasitic diseases worldwide and 10 genotypes (G1-G10) have been reported. In China, almost all the epidemiological and genotyping studies of E. granulosus s.l. are from the west and northwest pasturing areas. However, in Heilongjiang Province of northeastern China, no molecular information is available on E. granulosus s.l. To understand and to speculate on possible transmission patterns of E. granulosus s.l., we molecularly identified and genotyped 10 hydatid cysts from hepatic CE patients in Heilongjiang Province based on mitochondrial cytochrome c oxidase subunit I (cox1), cytochrome b (cytb) and NADH dehydrogenase subunit 1 (nad1) genes. Two genotypes were identified, G1 genotype (n = 6) and G7 genotype (n = 4). All the six G1 genotype isolates were identical to each other at the cox1 locus; three and two different sequences were obtained at the cytb and nad1 loci, respectively, with two cytb gene sequences not being described previously. G7 genotype isolates were identical to each other at the cox1, cytb and nad1 loci; however, the cytb gene sequence was not described previously. This is the first report of G7 genotype in humans in China. Three new cytb gene sequences from G1 and G7 genotypes might reflect endemic genetic characterizations. Pigs might be the main intermediate hosts of G7 genotype in our investigated area by homology analysis. The results will aid in making more effective control strategies for the prevention of transmission of E. granulosus s.l.

  7. Genetic characterization of human-derived hydatid cysts of Echinococcus granulosus sensu lato in Heilongjiang Province and the first report of G7 genotype of E. canadensis in humans in China.

    Directory of Open Access Journals (Sweden)

    Tiemin Zhang

    Full Text Available Cystic echinococcosis (CE caused by the larval stage of Echinococcus granulosus sensu lato (s.l. is one of the most important zoonotic parasitic diseases worldwide and 10 genotypes (G1-G10 have been reported. In China, almost all the epidemiological and genotyping studies of E. granulosus s.l. are from the west and northwest pasturing areas. However, in Heilongjiang Province of northeastern China, no molecular information is available on E. granulosus s.l. To understand and to speculate on possible transmission patterns of E. granulosus s.l., we molecularly identified and genotyped 10 hydatid cysts from hepatic CE patients in Heilongjiang Province based on mitochondrial cytochrome c oxidase subunit I (cox1, cytochrome b (cytb and NADH dehydrogenase subunit 1 (nad1 genes. Two genotypes were identified, G1 genotype (n = 6 and G7 genotype (n = 4. All the six G1 genotype isolates were identical to each other at the cox1 locus; three and two different sequences were obtained at the cytb and nad1 loci, respectively, with two cytb gene sequences not being described previously. G7 genotype isolates were identical to each other at the cox1, cytb and nad1 loci; however, the cytb gene sequence was not described previously. This is the first report of G7 genotype in humans in China. Three new cytb gene sequences from G1 and G7 genotypes might reflect endemic genetic characterizations. Pigs might be the main intermediate hosts of G7 genotype in our investigated area by homology analysis. The results will aid in making more effective control strategies for the prevention of transmission of E. granulosus s.l.

  8. Cultivation and identification of the transgenic alfalfa containing Echinococcus granulosus Eg95 gene%细粒棘球绦虫转Eg95基因苜蓿疫苗的培育及鉴定

    Institute of Scientific and Technical Information of China (English)

    叶艳菊; 李文桂; 周辉

    2009-01-01

    Objective To cultivate and identify the transgenic affalfa containing Echinococcus granulosus Eg95 gene. Methods The alfalfa plants were transformed by co-cultivating alfalfa cotyledons via recombinant Agrobacterium tumefaciens LBA4404 harboring pBI-Eg95. The transgenic alfalfa explants were selected by kanamyein after calli formation, shoots and roots regeneration in the selective medium, the seedlings of transgenic plants were obtained which were finally transplanted into pots containing nutrient soil. After 2-3 months growth, the complete transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene were obtained. To identify the transgenic alfalfa plants, the total DNA, RNA and leaf protein were extracted from fresh leaf tissue of the transgenic alfalfa plants and confirmed by PCR, RT-PCR, SDS-PAGE and Western blot assay. Results A specific band around 471 bp was amplified by PCR with total DNA, and the same band was obtained by RT-PCR with total RNA, which confirmed that the Eg95 gene was stably integrated into the transformed alfalfa genome. SDS-PAGE analysis showed that the relative molecular mass(Mr) of the expressed protein was about 16.5×103, consistent with the Eg95 protein, and the level of Eg95 expression was up to 0.06% of total soluble leaf protein by Bio-Rad Quantity one assay. Western blot verified the expressed protein was reactive with the sera of mice infected with Echinococcus granulosus. Conclusion The transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene are successfully cultivated.%目的 培育并鉴定细粒棘球绦虫转Eg95基因苜蓿疫苗.方法 利用转pBI-Eg95质粒的根癌农杆菌LBA4404株介导的苜蓿子叶浸染法,将Eg95基因导人紫花苜蓿基因组,转Eg95基因苜蓿外植体在含有卡那霉素的选择培养基上经愈伤、出芽和生根阶段生长出小苗,最后移栽到装有营养土的花盆中,生长2~3个月,获得完整的转Eg95基因苜蓿疫苗.提取转Eg95基因苜

  9. Establishment of laboratory animal models of hydatid disease through inoculating mice and rabbits with echinococcus granulosus protoscolices%异源接种建立小鼠和兔包虫病动物模型的初步探讨

    Institute of Scientific and Technical Information of China (English)

    张静; 叶彬; 邹晓毅; 武卫华; 韩秀敏

    2011-01-01

    目的 采用人工接种羊源细粒棘球绦虫原头蚴感染法建立小鼠与兔包虫病动物模型.方法 6周龄昆明小鼠经皮穿刺腹腔内接种羊源细粒棘球绦虫原头蚴悬液,新西兰大白兔于腹部手术后肝脏接种羊源细粒棘球绦虫原头蚴悬液.接种原头蚴6个月后剖检动物,观察小鼠腹腔和新西兰大白兔肝脏内包虫囊生长情况.结果 接种羊源细粒棘球绦虫原头蚴6个月后,小鼠腹腔内包囊生成率为95%,新西兰大白兔肝内包囊生成率为50%.光镜观察见在小鼠腹腔和兔肝脏形成的囊泡具有与羊肝脏棘球蚴囊壁类似的类上皮细胞层和板层状结构.3种动物体内棘球蚴均有原头蚴.结论 以羊源细粒棘球绦虫原头蚴悬液接种昆明小鼠腹腔和新西兰大白兔肝脏,可以建立小鼠和兔包虫病动物模型.%Objective To establish laboratory animal models of hydatid disease in mice and rabbits with sheep-derived Echinococcus granulosus protoscolices. Methods Kunming mice at 6 weeks of age were inoculated with the suspension of sheep-derived Echinococcus granulosus protoscolices in the abdomen. New Zealand rabbits were inoculated with the suspension of sheep-derived Echinococcus granulosus protoscolices in the liver after surgical operation in the abdominal region. The hydatid cysts in the animals were observed by naked eyes and optical microscopy in 6 months after inoculation. Results At the observation time point, the hydatid cysts were found in the abdominal cavities of 95% experimental mice (19/20) and in the liver of 50% experimental rabbits (4/8). Compared with those derived from the sheep liver, the hydatid cysts had similar epithelioid cell layer and lamellar structure but thinner cyst wall. Protoscolices were found in the hydatid cysts derived from all animals. Conclusion The animal models of hydatid disease can be established by inoculating mice and rabbits with the suspension of Echinococcus granulosus

  10. Determination of the date of LAMP diagnosis of canine Echinococcus granulosus infection%犬细粒棘球绦虫感染LAMP检出日期的确定

    Institute of Scientific and Technical Information of China (English)

    陈璐; 吾拉木·马木提; 陈洁; 古力帕丽·麦曼提依明

    2011-01-01

    目的 确定犬细粒棘球绦虫感染环介导等温扩增技术(LAMP)最早检出日期.方法 家犬8只,口服喂饲细粒棘球绦虫原头节,建立细粒棘球绦虫感染动物模型.感染后40 d收集犬粪,用LAMP检测粪便中细粒棘球绦虫目的基因并与传统的PCR比较.结果 8只犬均获得细粒棘球绦虫感染,感染后第18dLAMP法检查犬粪中细粒棘球绦虫目的基因均阳性,较普通PCR法提早2d.结论 LAMP法简便、快捷、灵敏,有望成为早期诊断犬细粒棘球绦虫感染的重要手段.%Objective To determine the earliest point for LAMP detection of infection in dogs infected with Echinococcus granulosus. Methods Eight dogs were artificially infected with E. Granulosus by oral intake of live protoscoleces. Fe-ces were collected for 40 days post-infection, and target gene fragments were detected using LAMP and compared to results using conventional PCR. Results All eight dogs developed severe infections. LAMP detected target genes in dog feces on day 18 post-infection, which is 2 days earlier than conventional PCR. Conclusion Due to its rapidity, simplicity, and sensitivity, the LAMP assay is an important and promising tool for early detection of infection with E. Granulosus.

  11. Comparison of ex vivo harvested and in vitro cultured materials from Echinococcus granulosus by measuring expression levels of five genes putatively involved in the development and maturation of adult worms.

    Science.gov (United States)

    Dezaki, Ebrahim Saedi; Yaghoubi, Mohammad Mehdi; Spiliotis, Markus; Boubaker, Ghalia; Taheri, Elham; Almani, Pooya Ghaseminejad; Tohidi, Farideh; Harandi, Majid Fasihi; Gottstein, Bruno

    2016-11-01

    Parts of the natural life cycle of Echinococcus granulosus can be retraced in vitro such as the development of protoscoleces into semiadult worms with three or more proglottids, or the redifferentiation of in vitro cultured protoscoleces into metacestode-like cystic structures. Most in vitro generated samples share-at the microscopical level-high similarities with those naturally grown, but developmental differences have also been documented, such as missing egg production in in vitro grown adults or unusual bladder/vesicle formation in protoscoleces cultured into the metacestode direction. The aim of the present study was to explore how far different in vitro generated stage-specific materials/structures match the natural situation on the transcriptome level, based on testing five exemplarily chosen different genes: the frizzled receptor eg-fz4 (posterior marker), the FGF receptor-like factor eg-fgfrl (anterior association), the cell differentiation protein eg-rcd1 (part of the CCR4-NOT complex, a key regulator of eukaryotic gene expression), the rapidly accelerated fibrosarcoma serin/threonin kinase eg-braf (part of the MAPK pathway involved, e.g., in EGF signaling) and the co-smad eg-smadD (downstream factor of TGFβ/BMP2/activin signaling). These genes-tested via qPCR-were selected such as to allow a discussion on their potential role in the development of E. granulosus into the adult stage. Thus, testing took place with three ex vivo isolated samples, namely (i) egg-containing adult worms, (ii) invaginated protoscoleces, and (iii) protoscolex-free germinal layer tissue. Respective data were compared (a) with in vitro generated metacestode-like microcysts developed from protoscolices, and (b) different development stages of protoscoleces in vitro cultured toward adult maturation. As a finding, only eg-smadD and partially eg-fz4 showed high expression similarities between ex vivo harvested and in vitro cultured E. granulosus, thus suggesting a putative role in

  12. Position Analysis of Three Recombinant Proteins of Echinococcus granulosus Protoscolex with Two-dimensional Electrophoresis%细粒棘球蚴原头节3种重组抗原的双向电泳定位分析

    Institute of Scientific and Technical Information of China (English)

    朱明星; 李君良; 巨艳; 高鹏; 赵巍

    2013-01-01

    Objective To obtain specific antibodies of the three recombinant antigens obtained previously,rEgZW-5,rEg14-3-3 and rEgP-29,for identifiying the corresponding proteins in two-dimensional electrophoretogram of Echinococcus granulosus protoscolex.Methods The distribution of proteins from E.granulosus protoscoleces was judged by SDS-PAGE previously.Two-dimensional electrophoresis was used to separate proteins from E.granulosus protoscoleces,and the result was scanned and analyzed by the PDquest software to get the information about the quantity of proteins as well as their isoelectric point (IP) and relative molecular mass (Mr).Rabbits were immunized with the 3 recombinant antigens and antibodies were purified from antisera.Western blotting was used to identify the protein as marker in two-dimensional electrophoretogram of protoscolex.Results SDS-PAGE displayed that the proteins separated from Echinococcus granulosus protoscoleces mainly distributed in the Mr region of 18 000-90000.240proteins were obtained by two-dimensional electrophoresis with Mr 15790-117050 and IP 4.0-9.5,and 85.8% (206/241) of the proteins showed the IP ranged from 5 to 9.Western blotting showed that the specific antibody of rEg14-3-3 identified the 14-3-3 protein in two-dimensional electrophoretogram of protoscolex with Mr 33 000 and IP 4.86,the specific antibody of rEgZW-5 identified the ZW-5 protein with Mr 23 000 and IP 4.98,and the specific antibody of rEg P-29 identified the P-29 protein with Mr 29 000 and IP 5.65.Conclusion The antibodies against the three recombinant proteins from Echinococcus granulosus protoscoleces can identify corresponding proteins in the two-dimensional electrophotoregrams.%目的 利用已有的细粒棘球蚴(Echinococcus granulosus)原头节3种重组抗原rEgZW-5、rEg14-3-3和rEgP-29获得特异性抗体,以识别双向电泳图谱中相应的蛋白位点. 方法 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)初步分离细粒棘球蚴原头节

  13. In vitro effects of the ERK inhibitor PD98059 on Echinococcus granulosus protoscoleces%ERK抑制剂PD98059体外抗细粒棘球绦虫原头蚴作用的研究

    Institute of Scientific and Technical Information of China (English)

    王成华; 吕海龙; 张晶; 彭心宇; 姜玉峰

    2012-01-01

    Objective The aim of the present work was to determine the in vitro protoscolicidal effect of ERK inhibitor PD98059 against Echhiococcus granulosus protoscoleces. Methods Protoscoleces of E. granulosus were incubated with PD98059 at concentrations of 100, 50, 25, and 12. 5 μmol/L. Viability of protoscoleces was assessed microscopically u-sing 0. 1% eosin staining. Each experiment was repeated three times, and viability was assessed daily. Ultrastructural effects of PD98O59 on E. granulosus protoscoleces in vitro were determined with SEM. Results The first signs of PD98059-induced damage were observed 1 day post-incubation. After 7 days of PD98059 incubation (100μmol/L), the percentage of vital protoscoleces was (12. 4 ± 0. 9) %. Results of vitality tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks, and destruction of microtriches. Conclusion The data obtained demonstrate a clear in vitro effect of PD98059 on E. granulosus protoscoleces, and this may represent a new strategy to treat hydatid cyst echinococcosis.%目的 探讨ERK1/2抑制剂PD98059体外抗细粒棘球绦虫原头蚴作用.方法 体外培养细粒棘球绦虫原头蚴,分组后分别加入100、50、25、12.5 μmol/L的PD98059后继续培养7d,利用伊红染色方法在倒置显微镜下观察原头蚴的活力,实验重复3次,绘制原头蚴活力曲线;扫描电子显微镜(SEM)下观察不同浓度PD98059组原头节表面结构改变.结果 PD98059作用1d对细粒棘球蚴原头节具有杀伤作用,第7 d 100 μmol/L PD98059组原头节的活力仅为(12.4±0.9)%.超微结构显示原头节顶突外翻、变形,顶突界面缺损,吸盘变形,甚至出现虫蛀样损害.结论 PD98059在体外有显著的抗细粒棘球蚴原头节的作用,可认为

  14. Induction of T helper 1 response by immunization of BALB/c mice with the gene encoding the second subunit of Echinococcus granulosus antigen B (EgAgB8/2

    Directory of Open Access Journals (Sweden)

    Boutennoune H.

    2012-05-01

    Full Text Available A pre-designed plasmid containing the gene encoding the second subunit of Echinococcus granulosus AgB8 (EgAgB8/2 was used to study the effect of the immunization route on the immune response in BALB/c mice. Mice were immunized with pDRIVEEgAgB8/ 2 or pDRIVE empty cassette using the intramuscular (i.m., intranasal (i.n. or the epidermal gene gun (g.g. routes. Analysis of the antibody response and cytokine data revealed that gene immunization by the i.m. route induced a marked bias towards a T helper type 1 (Th1 immune response as characterized by high IFN-γ gene expression and a low IgG1/IgG2a reactivity index (R.I. ratio of 0.04. The i.n. route showed a moderate IFN-γ expression but a higher IgG1/IgG2a R.I. ratio of 0.25 indicating a moderate Th1 response. In contrast, epidermal g.g. immunization induced a Th2 response characterized by high IL-4 expression and the highest IgG1/IgG2a R.I. ratio of 0.58. In conclusion, this study showed the advantage of genetic immunization using the i.m. route and i.n. over the epidermal g.g. routes in the induction of Th1 immunity in response to E. granulosus AgB gene immunization.

  15. A possible Echinococcus granulosus calcified cyst found in a medieval adult female from the churchyard of Santo Domingo de Silos (Prádena del Rincón, Madrid, Spain).

    Science.gov (United States)

    Monge Calleja, Álvaro M; Sarkic, Natasa; López, Jesus Herrerín; Antunes, Wilson D T; Pereira, Manuel F C; Matos, António Pedro Alves de; Santos, Ana Luísa

    2017-03-01

    Calcification, or mineralisation, can occur as part of a natural process, or by pathological processes. The purpose of this work is to examine an unidentified semi-spherical and perforate hollow mass, found near the pelvis of an adult female, dated 12th-13th century AD, exhumed of the Church of Santo Domingo de Silos (Prádena del Ricón, Madrid, Spain). The mass was examined by SEM and Energy Dispersive X-Ray Spectroscopy. These procedures revealed a heterogeneous inner surface with both smooth and irregular areas. A larger spherical and several smaller crescent-shaped perforations were noticed. X-ray microanalysis revealed the presence of the elements C, K, P, Ca, Al, Si, Fe, and Mg. The co-localisation of Ca and P suggests that they may be combined in a mineral matrix, likely formed in vivo. Other minerals probably came from the soil, although Fe could be related to the presence of blood. The macroscopic and microscopic appearances, chemical composition, and location of the calcified mass point to a possible hydatid cyst from Echinococcus granulosus, common in agricultural populations. This study used a suite of analytical techniques that are useful in the diagnosis of unknown calcified masses and can, therefore, be recommended for use in future analytical work.

  16. 细粒棘球绦虫重组pCD-Eg95质粒构建及鉴定%Construction and identification of the recombinant pCD-Eg95 plasmid of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    周辉; 李文桂

    2009-01-01

    目的:构建并鉴定细粒棘球绦虫(Echinococcus granulosus,Eg)重组pCD-Eg95质粒.方法:从细粒棘球蚴包囊中分离原头节,超声粉碎后抽提总RNA,采用RT-PCR方法扩增Eg95编码基因,将该基因定向克隆到真核表达载体pCDNA3.1中构建pCD-Eg95重组质粒;电穿孔转化大肠杆菌BIA21(DE3)株,抽提质粒进行酶切及PCR鉴定.结果:电泳及测序证实471 bpEg95基因克隆成功.经酶切及PCR证实重组质粒pCD-Eg95成功转入BL21(DE3).结论:成功构建了Eg重组pCD-Eg95质粒.

  17. Expression, Purification and Immunologic Identification of the Recombinant HSP70 Gene From Echinococcus Granulosus%细粒棘球蚴重组HSP70基因的表达、纯化及免疫学鉴定

    Institute of Scientific and Technical Information of China (English)

    于晶晶; 于辛酉; 王娅娜; 师志云; 马锐; 卜阳; 罗永云; 赵巍

    2008-01-01

    目的 构建细粒棘球蚴(Echinococcus granulosus,Eg)热休克蛋白70(Heat shock protein,HSP70)基因的重组质粒并原核表达、纯化及对其免疫特性进行鉴定.方法 从重组质粒HSP70/pGEM-T中酶切HSP70目的片段,亚克隆入表达载体pET-28a,转化入大肠杆菌E.coli BL21(DE3)plysS进行融合表达,经His-band树脂纯化试剂盒小量纯化,SDS-PAGE 和Western blot方法鉴定表达产物.结果 成功构建Eg.HSP70/ pET-28a/ E.coli BL21基因工程菌株,诱导表达重组蛋白和纯化分离得到14kDa Eg.HSP70均能被细粒棘球蚴天然抗原免疫的兔多克隆抗血清识别.结论 初步证实该重组蛋白具有较好的抗原性.

  18. 细粒棘球绦虫14-3-3zeta蛋白的生物信息学分析%Application of bioinformatic analysis in 14-3-3zeta protein of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    符瑞佳; 吕刚; 尹飞飞; 梁培

    2015-01-01

    目的:应用生物信息学技术对细粒棘球绦虫(Echinococcus granulosus)14-3-3zeta蛋白的结构和功能进行预测和分析,为进一步的实验研究提供依据。方法利用美国国家生物技术信息中心(NCBI,http://www.ncbi.nlm.nih.gov/)和瑞士生物信息学研究所的蛋白分析专家系统(ExPASY,http://expasy.org/)提供的各种有关基因和蛋白序列、结构信息分析的工具,并结合其它生物信息学分析软件,对该蛋白质的结构和功能进行预测和分析。结果该基因全长为771 bp ,编码256个氨基酸,其编码的蛋白相对分子量理论预测值和等电点分别是29.4 kDa和5.04。预测该蛋白无信号肽和跨膜区,二级结构含8个α-螺旋和12个β-折叠股,氨基酸序列中有9个潜在抗原表位。结论初步认识了细粒棘球绦虫14-3-3zeta蛋白的基本特征,为深入研究该蛋白的生物学功能奠定了基础。%Objective To predict and analyze the structure and function of 14-3-3zeta protein from Echinococcus granulosus by bioinformatics technology. Methods The structure and function of Eg14-3-3zeta protein was identified from two biological information sites, USA National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/), and Expert System for analysis of protein of the Swiss Institute of bioinformatics (ExPASY,http://expasy.org/), which offer the analysis of various related gene and protein sequence, structure information tools, and other bioinformatics analysis software. Results The full-length cDNA sequence encoding Eg14-3-3zeta included a complete open reading frame (ORF) of 771 bp coding to a putative protein with 256 amino acids. Molecular weight of Eg14-3-3zeta was predicted to be 29.4 kDa and its isoelectric point was 5.04. The protein had no signal peptide site and transmembrane do-main. Secondary structure of Eg14-3-3zeta contained 8 alpha-helices and 12 beta-strands.There were

  19. 细粒棘球蚴细胞系培育及其免疫研究%The Cell Line Establishment and Immunogenic Study of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    陆家海; 郭中敏; 余新炳; 冯德元; 李德昌; 程维兴

    2001-01-01

    The germinal layer and protoscoleces of larval Echinococcusgranulosus excised surgically from a patient with liver hydatid disease were isolated and grew in the modified DMEM in collagen-coated culture flasks. The optimal condition for larval E.granulosus growth in vitro was established, and effect factors for larval E.granulosus growth were analyzed. The cells were continuously grew in the modified DMEM over 140 days and past 21 passages. The cells grew with the fibroblast-like cells dominating and attached to the glass surface. The results indicated that the cell line of larval E.granulosus, 13G-5, was established. The Km mice were inoculated intraperitoneally with cells of 13G-5 and cyst-like structures which proved to be cyst under microscope. The mice produced specific antibody against hydatid cyst-fluid antigens. The specific antigen was found in the excretory products of 13G-5 by ELISA. The same EST isoenzyme band as that of E.granulosus protoscoleces was identified in 13G-5. After the mice were vaccinated with the crude antigen, the excretory products of 13G-5, 60% Km mice have no cyst formed when infected with E.granulosus. When excretory products of 13G-5 were used as diagnostic antigen, the sensitivity is 43.33%~77.42%. The chromosome of larval E.granulosus were numbered 14 to 18 and G-band and C-band were analyzed for the first time.%从细粒棘球蚴病人体内获取生发层和原头节作为培养材料,采用改良DMEM培养液和鼠胶原蛋白包被的培养瓶进行细胞培养,建立了适合人源细粒棘球蚴细胞体外培养的方法,并探讨棘球蚴细胞培养的制约因素。首次成功培育出一株人源细粒棘球蚴细胞系(13G-5),至1997年3月1日为止该细胞系已培养了140d,其间传了21代。该细胞系主要以成纤维型细胞为主,呈贴壁生长;用该细胞系细胞接种于昆明(Km)小鼠腹腔内,能形成具包囊样结构的类似包囊;接种鼠产生对囊液抗原的特异性抗

  20. 应用荧光定量PCR对细粒棘球绦虫miRNAs在虫体不同发育阶段表达量的研究%Quantification Analysis of miRNAs Expression Level in Different Developing Stages of Echinococcus granulosus Using Real-time PCR

    Institute of Scientific and Technical Information of China (English)

    郝力力; 李必富

    2012-01-01

    为确定通过Solexa高通量测序鉴定出的大量细粒棘球绦虫miRNAs序列在不同生活史阶段的表达量.采用SYBR染料法和Race试剂盒相结合的荧光定量方法对原头节和生发层阶段的miRNAs表达进行了检测分析.结果表明:两阶段miRNAs表达差异较大,其中mir-71、mir-7、mir-1和mir-19在生发层阶段表达量较高,而let-7则在原头节阶段表达较高.说明这些miRNAs可能在细粒棘球绦虫发育阶段的基因转录调控中起着重要作用.%In this experiment, many new miRNAs in Echinococcus granulosus were discovered by high-throughout sequencing technology Solexa. To investigate the expression level of these miRNAs in different life stages of Echinococcus granulosus, Real-time PCR was used to relatively quantify the dominantly expressed miRNAs in protoscolex and germinal layer by combination of SYBR Green PCR and Race kit. It was found that mir-71, rnir-7, mir-land mir-19 were highly expressed at germinal layer other than protoscolex and let-7 was more aboudunt in protoscolex than germinal layer. The results suggested that miNRAs might play an important role in regulation of genes transcription in different developing stages of Echinococcus-granulosus.

  1. 细粒棘球绦虫分子生物学研究进展%Advanced on the study of molecular biology of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    朱佑明; 李文桂

    2005-01-01

    细粒棘球蚴病(Echinococcosis)又称囊型包虫病(Cystic echinococcosis,CE),是由细粒棘球绦虫(Echinococcus granulosus,Eg)幼虫引起的一种人畜共患寄生虫病。Eg幼虫称棘球蚴(Hydatid cyst),寄生于人体的肝、肺和脑等不同脏器,表现出不同的临床症状。若囊肿破裂人胸腔和腹腔可引起继发性感染、过敏性休克,甚至死亡。CE已成为我国新五大寄生虫病之一。该病呈全球分布,我国新疆、甘肃、宁夏、青海、内蒙古、

  2. Cloning of linear T-cell epitops of EG95 antigen of Echinococcus granulosus into pGEX4t1 vector and expression analysis by SDS-PAGE

    Directory of Open Access Journals (Sweden)

    Roghayeh Mesri

    2016-12-01

    Full Text Available Background & Objectives: Echinococcus granulosus causes a common disease between humans and animals that called hydatid cyst or hydatidosis. The recombinant EG95 vaccine based on cloning of the 25 KD oncospheral antigen-Eg95- in pGEX stimulated significant humoral and cellular immunity in sheep, but regarding the effect of TH1cell immunity against this parasite and the influence of the linear T-cell epitopes in stimulating this immunity, only the coding sequence of linear T Cell epitopes of EG95 was cloned in pGEX and analyzed it's expression to optimize the qualification of this available vaccine in this study. Materials & Methods: The coding sequence of EG95 linear epitopes by IEDB software were predicted and synthesized. After PCR, the amplicon and pGEX4T1 were digested by xhoI restriction enzyme, the fragment was cloned into pGEX4T1 by heat shock method, and Positive colonies were selected by direct PCR with specific primers. The recombinant protein expression was evaluated in BL21 cells by 10% SDS-PAGE. Results: The coding sequence of EG95 linear T-cell epitopes was amplified by PCR and cloned into pGEX4T1 vector.  The recombinant plasmid was selected with Colony PCR and size difference between intact and recombinant purificated vectors. Recombinant protein expression with high significant concentration was recognized by SDS-PAGE. Conclusion: The EG95 linear T-cell epitopes coding sequence has been successfully cloned into pGEX4T1 vector and expressed into BL21 cells.

  3. Initial Study of Anaphylactic Models Induced by Echinococcus granulosus Cystic Fluid in Mice%细粒棘球蚴致过敏反应小白鼠模型复制的初步报告

    Institute of Scientific and Technical Information of China (English)

    张文胜; 郑宏; 温浩; 尹极峰

    1999-01-01

    本实验研究利用细粒棘球蚴(Echinococcus granulosus)囊内容物及其提纯抗原复制过敏反应模型.将100只昆明种小白鼠,根据决定性注射致敏原的不同,随机分为羊源囊液抗原组、羊源囊液部分纯化抗原组、人源囊液抗原组和人源囊液部分纯化抗原组,并设立生理盐水对照组.以腹腔注射方式进行攻击,复制出小白鼠过敏反应模型.结果表明,四个致敏原组过敏反应发生率分别为94.4%、89.5%、95%和94.4%,而对照组则无一例发生过敏反应.同时,各组决定性注射前后分别进行血嗜酸性粒细胞直接计数,并与健康小白鼠对照,发现过敏反应发生后血中嗜酸性粒细胞计数明显升高(P<0.05).提示该模型症状典型,方法简单,有较高的应用价值.

  4. Gene expression of Echinococcus granulosus under H2O2 stress using SSH technology%应用SSH技术研究H2O2胁迫下细粒棘球蚴基因的表达

    Institute of Scientific and Technical Information of China (English)

    侯秋莲; 王慧; 张壮志; 李江伟; 张富春; 张文宝

    2008-01-01

    目的 构建H2O2胁迫下细粒棘球蚴(Echinococcus granulosus)与正常组织差异表达的消减cDNA文库.方法 以H2O2胁迫细粒棘球蚴cDNA为试验方(tester),正常生长的细粒棘球蚴cDNA为驱动方(driver),应用抑制性消减杂交技术(suppression subtractive hybridization,SSH)研究H2O2胁迫下细粒棘球蚴基因的表达.结果 文库扩增后得到124个阳性克隆,菌落PCR分析,均得到200~1 000 bp插入片段.将整个文库克隆进行测序,测得序列结果 利用BLAST在线软件与GenBank数据库进行同源序列比对分析和BlastX分析.结果 获得重要基因的cDNA序列,如氧化还原酶、蛋白激酶、生长因子等.另有部分克隆在GenBank中无法查到对应的同源基因,可能代表了新基因.结论 成功构建了H2O2胁迫与正常组织差异表达的消减cDNA文库,为研究细粒棘球蚴在抗氧化过程中的相关靶基因筛选奠定基础.

  5. 高强度聚焦超声波对细粒棘球绦虫原头节的杀伤效应%Inhibiting effects of high intensity focused ultrasound on Echinococcus granulosus protoscolices in vitro

    Institute of Scientific and Technical Information of China (English)

    邹晓毅; 王俊安; 周潜涛; 叶彬; 张成武; 赵发生; 韩秀敏

    2008-01-01

    目的 探索高强度聚焦超声波(HIFU)对体外分离的细粒棘球绦虫原头节的急、慢性杀灭作用.方法 实验对象为感染细粒棘球蚴病的羊肝原头节.选择声功率为0(对照组)、25、50、100、200、250 W的超声波辐照离体原头节,每种功率辐照时间分别为5、10、20、30、40、50、60 s,观察HIFU对离体原头节的即刻杀灭作用.以不致即刻杀伤的超声剂量作用原头节后,观察HIFU对原头节的迟发性生长抑制作用.光镜下,根据台盼蓝排斥法染色结果及观察原头节形态变化计算原头节死亡率.结果 HIFU对原头节有明显的急性杀伤作用,在一定的功率和时间范围内有剂量-效应关系,不同功率和辐照时间对原头节死亡率的影响,组间比较差异均有统计学意义(F值分别为5201.59、1865.65,P<0.05),且功率与辐照时间之间存在交互效应(F=214.50,P<0.05).随着超声照射剂量增大,其生物学效应越明显,声功率≥200 W的短时照射即可致原头节全部即刻死亡,部分原头节被打碎.以不致原头节即刻杀伤的超声照射后,体外培养2~7 d的原头节死亡率均较对照组明显增高(P<0.05),随超声剂量的增大抑制原头节生长作用增强,培养第2天开始,50 W×10 s组强于25 W×20 s组(P<0.05).结论 HIFU能够即刻杀灭原头节并能抑制原头节在体外的生长.%Objective To evaluate the acute and delayed killing effect of high intensity focused ultrasound (HIFU) on Echinococcus granulosus(E. granulosus)protoscolices in vitro.Methods E. granulosus protoscolices were treated with different dosage of effective power(0,25,50,100,200,250 W)and time(5,10,20,30,40,50,60 s)of HIFU in vitro to obtain the dosage-effect curves.Then the survival pmtoscolices were incubated,and the mortality of each group was counted daily.The protoscolicidal effects were investigated by trypan blue exclusion assay.Results Compared with the untreated group,the Vitality of E.granulosus

  6. Sequencing and bioinformatic analysis of mRNA from Echinococcus granulosus protoscolex%细粒棘球蚴原头节mRNA测序及生物信息学初步分析

    Institute of Scientific and Technical Information of China (English)

    朱明星; 王娅娜; 巨艳; 王志昇; 朱佳佳; 赵巍

    2014-01-01

    Objective To reveal the transcriptomic information and biological characteristics of Echinococcus granulosus protoscolex by using RNA-Seq technique and through bioinformatic analysis of sequencing data.Methods Total RNA was isolated from Echinococcus granulosus protoscolex using TRIzol.mRNA kit with Oligo(dT) magnetic beads was used to isolate poly(A) mRNA.Illumina HiSeq 2000 was applied for sequencing.Data from sequeced were assembled into unigene by using mapping-first approaches based on the genome which uploaded in the Wellcome Trust Sanger Institute.Alignment between unigenes and protein databases,such as NCBI non-redundant protein (Nr) database,UniProt database,the gene ontology (GO) database,the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database,was performed.Results A total of 132 007 609 clean reads and 91 342 unigenes were generated.The average length of unigene was 419 bp.Through analysed with GO,26 552 unigenes were mapped.Further biological process categories of GO prominently represented in biological process,molecular functions and cellular components including 48 categories.Six thousand six hundred and sixty-four unigenes were mapped to 227 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways,including 6 first level categories and 34 second level categories.Conclusion Extensive transcriptome data from sequencing of Echinococcus granulosus protoscolex were obtained.The information has provided a database for further study on the hydatid disease.%目的 利用RNA-seq技术对细粒棘球蚴原头节的转录组进行测序并对测序数据进行生物信息学分析,以揭示细粒棘球蚴原头节mRNA所包含的信息. 方法 用Trizol法提取原头节总RNA,分离mRNA,利用Illumina公司的HiSeq2000高通量测序仪对mRNA序列进行测序.参考英国桑格研究院(Wellcome Trust Sanger Institute)公布的细粒棘球绦虫基因组数据,对测序数据进行拼接组装,将获得的unigene与非冗余的蛋白序列数

  7. Proline modulates the effect of bisphosphonate on calcium levels and adenosine triphosphate production in cell lines derived from bovine Echinococcus granulosus protoscoleces.

    Science.gov (United States)

    Fuchs, A G; Echeverría, C I; Pérez Rojo, F G; Prieto González, E A; Roldán, E J A

    2014-12-01

    Bisphosphonates have been proposed as pharmacological agents against parasite and cancer cell growth. The effect of these compounds on helminthic cell viability and acellular compartment morphology, however, has not yet been studied. The effects of different types of bisphosphonates, namely etidronate (EHDP), pamidronate (APD), alendronate (ABP), ibandronate (IB) and olpadronate (OPD), and their interaction with amiloride, 1,25-dihydroxycholecalciferol (D3) and proline were evaluated on a cell line derived from bovine Echinococcus granulousus protoscoleces (EGPE) that forms cystic colonies in agarose. The EGPE cell line allowed testing the effect of bisphosphonates alone and in association with other compounds that could modulate calcium apposition/deposition, and were useful in measuring the impact of these compounds on cell growth, cystic colony formation and calcium storage. Decreased cell growth and cystic colony formation were found with EHDP, IB and OPD, and increased calcium storage with EHDP only. Calcium storage in EGPE cells appeared to be sensitive to the effect of amiloride, D3 and proline. Proline decreased calcium storage and increased colony formation. Changes in calcium storage may be associated with degenerative changes of the cysts, as shown in the in vitro colony model and linked to an adenosine triphosphate (ATP) decrease. In conclusion, bisphosphonates could be suitable tempering drugs to treat cestode infections.

  8. Cloning, Prokaryotic Expression of Echinococcus granulosus Heat Shock Protein 70 and Preparation of It's Antiserum%细粒棘球绦虫Hsp70基因的克隆、表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    赵莉; 薛晶; 石保新; 陈皓斐; 张文宝; 马正海; 张壮志; 张旭; 古努尔·吐尔逊; 米晓云; 金映红

    2012-01-01

    [Objective] The objective of the experiment is to express and purify E. granulosus (Eg) heat shock protein 70 (EgHsp70) in E. coli and prepare the antibody against E. granulosus. [ Methods] EgHsp70 gene was amplified by PCR and cloned into prokaryotic expression vector pMAL-p2x, and the recombinant plasmid was transformed into E. coil BL-21. The soluble expression conditions of fusion protein were optimized by induction with different concentrations, of IPTG different temperatures and cultivation times. The expressed fusion protein was purified by Mal-tag Magnetic Beads. To prepare the anti-serum, New Zealand white rabbits were immunized with purified EgHsp70 protein via hypodermic and volar. Western blot was used to determine the serum's specificity against EgHsp70 and native proteins. The serum titers were analyzed by ELISA. [Results] Full-length of EgHsp70 gene had an open reading frame of 765 bps encoding a protein mass of 68.6 kD. Restriction endonuclease analysis and DNA sequencing showed that EgHsp70 was cloned into the plasmid pMAL-p2x. Based on the optimization experiments, it was concluded that the best soluble expression conditions for the EgHsp70 protein are using 0.3 mmol· L-1 IPTG when bacterial cells growing to OD600 0.6 and induced for 4 h at 30℃. ELISA and Western blotting showed that the titers of the anti-serum were above 1 : 256 000, and the anti-serum could specifically bind with EgHsp70 protein and native proteins. [Conclusion] The EgHsp70 fusion protein was obtained by expressing in E.coli and purifying, and the antibody against EgHsp70 was prepared with the fusion protein immunized New Zealand white rabbits. This work will provide an antigen and detection antibody for further study on the EgHsp70 function. The protein is immunogenic and can be a vaccine candidate against Echinococcus infection.%[目的]克隆细粒棘球绦虫(Echinococcus granulosus,E.g)热休克蛋白家族基因Hsp70,在原核细胞中表达、纯化Hsp70蛋白并制

  9. An Observation in Vitro of Recombinant Ferritin Protein Antiserum Impaired the Protoscolex of Echinococcus Granulosus%Eg.ferritin抗血清介导的体外杀伤细粒棘绦虫原头节作用的观察

    Institute of Scientific and Technical Information of China (English)

    王娅娜; 张炎; 朱佳

    2012-01-01

    目的 体外观察细粒棘球绦虫重组铁蛋白(Eg.ferritin)抗血清对细粒棘球蚴原头节(PSC)的杀伤作用.方法 用纯化的Eg.ferritin免疫新西兰家兔制备抗血清,以不同浓度的抗血清与细粒棘球绦虫原头蚴体外共培养,光镜观察培养不同时间点细粒棘球原头节的变化,透射电子显微镜观察原头节超微结构的改变.结果 抗血清组的原头蚴的死亡率及破坏程度与抗血清的浓度呈正相关,与对照组相比,抗血清对原头节有杀伤作用(P<0.01).在20%抗血清组中,原头节内部被大量空泡及髓样结构代替或无结构.结论 细粒棘球蚴重组铁蛋白抗体对细粒棘球绦虫有明显的杀伤作用.%Objective To observe the impaired the effect of recombinant ferritin protein antiserum on the pro-toscolex (PSC) of Echinococcus granulosus in vitro. Methods Antiserum was made from New Zealand rabbits with purified Eg. ferritin immunization. Antiserum was divided into three different group with 10% ,15% , 20% concentration. PSC were cultured in the three antiserum groups. Living situation and mortality of PSC was investigated through microscope in different time points and sub structure of PSC was observed through transmission electronic microscope( TEM) . Results Impairment of PSC was positively related to concentration of anti - serum( P < 0. 01) , Lots of vacuole and myelinfigures were found inside of PSC in 20% anti - serum group. Conclusion The results confirmed that Eg. ferritin could impaire PSC in vitro.

  10. 定量PCR研究细粒棘球蚴抗氧化相关基因的差异表达%The differential expression of the anti-oxidation related genes in Echinococcus granulosus by real-time PCR

    Institute of Scientific and Technical Information of China (English)

    侯秋莲; 张富春; 张文宝; 吾拉木·马木提; 张壮志

    2010-01-01

    目的 在已构建的氧化胁迫下细粒棘球蚴(Echinococcus granulosus)与正常组织差异表达的消减cDNA文库中,筛选细粒棘球蚴在抗氧化过程中差异表达的重要基因.方法 将前期研究中应用抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建氧化胁迫下细粒棘球蚴差异表达基因的消减cDNA文库进行蓝白斑筛选和菌落PCR分析后测序分析.测序结果利用BLAST在线软件与GenBank数据库进行同源序列比对分析和BlastX分析.从文库中随机挑选4个未知新序列和抗氧化密切相关的TPx基因片段,利用定量PCR法研究氧化胁迫下,差异表达基因片段在mRNA水平上的变化情况.结果 整个文库克隆测序结果获得重要基因的cDNA序列,如氧化还原酶、蛋白激酶、生长因子等.另有部分克隆在GenBank中无法查到对应的同源基因,可能代表了新基因.定量PCR结果显示:S88、H32-1 两个基因在0.8 mmol/L H_2O_2氧化胁迫的原头蚴中表达量分别上调为未经氧化胁迫原头蚴中的2.0和2.3倍,TPx基因片段当H_2O_2浓度大于0.8 mmol/L时,其表达量增高.结论 上述基因的上调表达很可能与细粒棘球蚴在抗氧化过程中的相关功能有密切的联系,可以作为研究细粒棘球蚴抗氧化的候选基因.

  11. Construction and Expression of the Echinococcus granulosus Recombinant BCG-EgG1Y162%细粒棘球绦虫重组BCG-EgG1Y162菌株的构建和表达

    Institute of Scientific and Technical Information of China (English)

    祖力皮也·吐尔逊; 德力夏提·依米提; 曹春宝; 马海梅; 李玉娇; 周晓涛; 朱明; 马秀敏; 温浩

    2013-01-01

    Objective To construct and express Echinococcus granulosus recombinant bacille Calmette-Guerin (BCG) strain rBCG-EgG1Y162. Methods The encoding gene of the antigen EgG1Y162 of E. granulosus was recombined with E. coli-Mycobacterium shuttle expression plasmid vector pMV361 by genetic engineering technique, and transformed into E. coll for amplification. The recombinant plasmid rpMV-EgG1Y162 was identified by PCR, double digestion with restriction enzymes, and sequence analysis. The confirmed rpMV-EgG1Y162 was transformed into BCG strain via electroporation technique to construct the recombinant rBCG-EgG1Y162. After identification by PCR and double digestion with restriction enzymes, the recombinant strain was cultured for about 2 weeks. In order to induce the expression of target protein, the rBCG was placed in 45℃ for 30 min. SDS-PAGE and Western blotting were used to analyze the expressive protein. Results The product of recombinant plasmid rpMV-EgG1Y162 was approximately 360 bp by PCR amplification and double digestion with restriction enzymes, consistent with the expected fragment length. Sequencing results showed that the inserted sequence was correct. The rBCG-EgG1Y162 grew well and the identification of PCR and enzyme digestion revealed accuracy. The results of SDS-PAGE and Western blotting showed that the relative molecular weight (Mr) of the protein was about 71 000. Conclusion The E. granulosus rBCG-EgG1Y162 strain is constructed and expressed.%目的 构建和表达细粒棘球绦虫重组卡介苗(BCG)菌株rBCG-EgG1Y162.方法 通过基因工程技术将细粒棘球绦虫抗原EgG1Y162的编码基因与大肠埃希菌(E.coli)-分枝杆菌穿梭表达质粒载体pMV361重组,并转化E.coli后进行扩增.重组质粒pMV-EgG1Y162经PCR和双酶切鉴定后,进行测序.将鉴定正确的rpMV-EgG1Y162通过电穿孔技术转化至感受态BCG菌株中,构建rBCG-EgG1Y162.经PCR和双酶切鉴定正确后,扩增培养2周,并于45℃放置30 min,诱导

  12. 细粒棘球蚴感染大鼠过敏反应中IL-10水平的初步研究%IL-10 Level in Allergic Rats Infected by Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    安然; 黄谋; 张秦; 郑宏

    2013-01-01

    Forty Wistar rats were randomly divided into 4 groups with 10 in each group. Group A served as normal control. The other 3 groups were injected intraperitoneally with Echinococcus granulosus protoscoleces 5×104 each. Six months later, group D was injected intraperitoneally with antibody against IL-10 2.0 μg/time, twice a day for 2 d. Two days later, rats in groups C and D were injected intraperitoneally with cyst fluid 5 ml each to induce allergic reaction. 30 min later, all the rats were sacrificed to observe the infection status and obtain peripheral blood. The level of IL-10, IgE and histamine in the sera was detected by ELISA. The results showed that 5, 6 and 5 rats in groups B, C and D were infected successfully, respectively, with 2 deaths in group A. The rats in groups C and D appeared nasal itching, sneezing and declined activity. 30 min later, the symptoms in group C got improved, but not for group D. The levels of IL-10 and IgE in groups C and D increased significantly compared to group B (P0.05).%将40只Wistar大鼠随机均分为A、B、C和D4组(每组10只),A组为健康对照组,B、C和D组分别腹腔注射细粒棘球蚴原头节5×104/只.感染后6个月,D组大鼠腹腔注射白细胞介素10(IL-10)抗体每鼠2.0 μg/次,2次/d,连续2d.2d后,C组和D组大鼠分别腹腔注射羊源细粒棘球蚴囊液每鼠5 ml,激发过敏.观察各组大鼠的过敏反应.30min后,剖杀大鼠,观察各组大鼠感染情况,腹主动脉取血,制备血清.ELISA检测各组血清中IL-10、免疫球蛋白E (IgE)和组胺水平.结果显示,B、C和D组大鼠分别有5、6和5只感染成功,A组有2只死亡.C组和D组大鼠均出现了抓鼻、喷嚏和活动力下降等过敏症状.在激发过敏后30 min,C组大鼠过敏症状均好转,D组大鼠仍对刺激反应较弱,活动力下降.C组和D组大鼠血清的IL-10和IgE水平均显著高于未激发过敏的B组(P<0.05),其中D组的IL-10和IgE水平分别为(142.61±43.58) pg/ml和(20.67±1

  13. Cloning and Bioinformatics Analysis of TSP1 and TSP6 Gene of Echinococcus granulosus%细粒棘球蚴TSP1和TSP6基因的克隆及生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    刘田莉; 孟庆玲; 乔军; 陈诚; 马玉; 胡政香; 才学鹏; 陈创夫

    2015-01-01

    In order to study the function of two important antigen genes tetraspanin 1-TSP1( TSP1 )and tet-raspanin 1-TSP6(TSP6),primers derived from Echinococcus granulosus genome database in GenBank were designed and the open reading frame( ORF)sequences of TSP1 and TSP6 were cloned by RT-PCR from hydatid protoscolex. Then they were cloned into pMD19-T vector for bioinformatics analysis. The results indicated that the TSP1 cDNA contains 792 nucleotides. The deduced protein consisted of 263 amino acids and has three N-glycosylation sites,two N-acylation sites. The gene sequence showed about 98. 99% identity with the TSP1(EG 11043)reported and the induced amino acid sequence showed about 98. 48% identity. The TSP6 cDNA contains 666 nucleotides. The de-duced protein consisted of 221 amino acids and has five N-acylation sites,one Tyrosine kinase phosphorylation sites. The gene sequence showed about 98. 18% identity with the TSP6(EG 00715)reported and the induced ami-no acid sequence showed about 85. 07% identity. The study carried out bioinformatics analysis of the TSP1 and TSP6 gene of Eg by molecular biology software to predict the structure and epitope of protein antigens known and laid a good foundation for the preparation of developing a vaccine.%为了研究绵羊细粒棘球蚴重要抗原基因 Tetraspanin 1-TSP1( TSP1)和 Tetraspanin 1-TSP6( TSP6)的功能,对GenBank中 Eg基因组数据库检索,获得 TSP1和 TSP6的 cDNA序列并设计特异性引物。以 Eg头节为总 RNA 模板,进行 RT-PCR,将 PCR产物克隆到 pMD19-T载体后测序并进行生物信息学分析。TSP1 cDNA 全长792个核苷酸,编码263个氨基酸,该多肽含有3个潜在的 N端糖基化位点,2个 N 端酰基化位点,与已登录的标准株 TSP1基因序列(EG 11043)同源性为98.99%,其推导的氨基酸序列同源性为98.48%;TSP6 cDNA 全长666个核苷酸,编码221个氨基酸,该多肽含有5个 N 端酰基化位点,1个酪氨酸激酶磷

  14. Genetic Polymorphisms of Echinococcus granulosus in Qinghai Based on the Mitochondrial DNA 12S Gene%基于线粒体12S基因对青海地区细粒棘球蚴种群遗传多态性研究

    Institute of Scientific and Technical Information of China (English)

    延宁; 杨德英; 郝桂英; 古小彬; 杨光友; 聂华明; 蒋忠荣; 邓世金; 余华; 严玉宝; 达瓦次仁; 谢跃; 付彦

    2012-01-01

    The aim of the current study was to investigate the genetic polymorphisms of Echinococcus granulosus obtained from sheep in Qinghai Province, and provide the foundational data for prevention and control of echinococcus disease. The complete mitochondrial DNA 12S genes of 42 isolates(33 were from liver and 9 were from lung) were sequenced, and phylogenetic analyses were conducted using neighbor-joining tree-building methods. There were five haplotypes (Hi to H5) detected among the samples, and H5 was the main type. Phylogenetic analysis supported these observations. The haplotype diversity (H) and nucleotide diversity (Pi) were 0. 418 and 0. 000 66, respectively. It was demonstrated that the genetic homology of these isolates with the 12S gene of E. granulosus Gl genotype(AF297617)was over 99. 86%. All 42 samples were identified as E. granulosus sensu stricto (G1-G3 clusters). In this study,genotypes H1 to H4 has not been identified elsewhere.%本研究旨在分析青海地区细粒棘球蚴的种群基因多态性,为细粒棘球蚴病的防控提供基础资料.对采自青海地区的42株细粒棘球蚴(33株采自绵羊肝脏,9株采自绵羊肺脏)进行了线粒体12S基因的全序列测序并构建了NJ系统发生树.结果显示:在本研究样品的线粒体12S基因序列中共检测出5种单倍型(即H1~H5),其中以单倍型H5为主(占32株),并且系统发生树分析支持这一结果.单倍型多样性(H)和核苷酸多样性(Pi)分别为0.418、0.000 66,与E granulosus Gl(AF297617)的12S基因序列的核苷酸相似性达到99.86%以上.采自青海地区的42株细粒棘球蚴均鉴定为E.granulosus sensu stricto(基因型G1-G3),在检测出的5种单倍型中,单倍型H1~H4是本地区特有的单倍型.

  15. 细粒棘球蚴感染中阻断TGF-β1受体对淋巴细胞的影响%Effect of inhibition of TGF-β1 receptors on lymphocytes during infection of Echi-nococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    印双红; 杨文才; 张俊波; 陈小林; 徐芳洁; 侯隽; 吴向未; 陈雪玲; 姚元勇; 邢明明

    2015-01-01

    Objective:To investigate the effects of TGF-β1 on T lymphocytes of BALB/c mice infected with Echinococcus granulosus( E.granulosus ) in vitro.Methods: The inhibitor group:the spleen cells of BALB/c mouse were co-cultured with E.granulosus and SB525334.The control group:the spleen cells of BALB/c mouse were co-cultured with E.granulosus and PBS.The blank group:the spleen cells of BALB/c mouse were co-cultured with RPMI-1640 medium and SB525334.The lymphocytes were collected at 48 h post-infection.The T lymphocyte subsets, the number of CD4+CD25+T cells, the number of NK cells, and the expression of NKG2D receptor were detected by flow cytometry.The NK cell activity was determined with the lactate dehydrogenase leakage assay(LDH).Results:The inhibition the TGF-β1 receptors resulted in the increase of in the number of CD4+T cells,the decrease in the number of CD8+T cells,the increase of in the ratio CD4+/CD8+T cells,the decrease of in the number of CD4+CD25+T cells,the increase in the expression of the NKG2D receptors,the increase in the lysis rate of Yac-1 cells by NK cells,and a positive cor-relation between the expression of activity receptor NKG2D and killing activity of NK, which were mediated by E.granulosus.Conclusion: The inhibition of TGF-β1 receptors can enhance the immune response of T lymphocytes against E.granulosus infection in vitro.%目的:观察TGF-β1对细粒棘球蚴体外与BALB/c小鼠T淋巴细胞培养的影响。方法:阻断剂组:BALB/c小鼠脾细胞+细粒棘球蚴+SB525334共培养;对照组:BALB/c小鼠脾细胞+细粒棘球蚴+PBS共培养;空白对照:BALB/c小鼠脾细胞+RPMI-1640培养基+SB525334共培养。方法:48 h收集淋巴细胞,流式细胞仪检测T淋巴细胞亚群、CD4+CD25+T细胞的数量、NK细胞的数量及其活性受体NKG2D的表达;乳酸脱氢酶法检测NK细胞的杀伤活性。结果:体外阻断TGF-β1受体后,引起细粒棘球蚴介导的CD4

  16. The pilot study on the influence of T-lymphocyte from mouse spleen cells treated with echinococcus granulosus fluid in vitro%细粒棘球蚴囊液对体外培养小鼠T淋巴细胞影响的初步研究

    Institute of Scientific and Technical Information of China (English)

    陈小林; 印双红; 吴向未; 陈雪玲

    2013-01-01

    目的:观察细粒棘球蚴囊液对体外培养的BABL/c小鼠T淋巴细胞的影响.方法:BABL/c小鼠脾细胞与100μl、200 μl、300 μl、500μl不同浓度的细粒棘球蚴囊液共培养,流式细胞仪检测CD3+CD4+/CD3+CD8+T淋巴细胞亚群和CD4+CD25+T细胞,RT-PCR法检测Foxp3基因的表达量;相同浓度的囊液和BABL/c小鼠脾细胞悬液共培养,在0、12、24、36、48小时收集细胞,检测T淋巴细胞亚群和Foxp3基因表达情况.结果:随着囊液浓度的增加,CD4+T细胞比例逐渐下降,CD8+T细胞比例上升,CD4+/CD8+的比值下降;CD4+CD25+T细胞则没有随着囊液量的增加而上升,而是在100μl组达到高峰值.RT-PCR的结果显示,在低浓度100μl和200μl组的时候Foxp3 mRNA的相对表达量最高.结论:细粒棘球蚴囊液促使CD4+/CD8+的比值下降,Treg细胞增加,可能在包虫免疫逃逸过程中发挥一定作用.%Objective:To observe the influence of T-lymphocyte subsets from mouse spleen cells treated with echinococcus granulosus fluid in vitro. Methods: After 72 h co-culture of spleen cells from BABL/c mouse and Hydatid fluid with different concentrations : 100, 200, 300, 500 μl, percentage of CD3 + CD4 + /CD3 + CD8 + T-lymphocyte subsets and CD4 + CD25 + T were detected by FCM, and gene expression of Foxp3 was measured by RT-PCR. The percentage of T-lymphocyte subsets of spleen cells were also detected at different time points (0, 12, 24, 36, 48 h). Results: With the increasing of the concentration of Hydatid fluid, CD4 + T cells to scale down gradually, while the CD8 + T cells ratio slowly rose, leading to the decreased ratio of CD4+ /CD8+. The percentage of CD4 + CD25 + T cells were not rise as the cyst fluid volume increases, but gels peak when in 100 μl Hydatid fluid group. RT-PCR showed Foxp3 mRNA relative expression had the highest value at low concentrations of 100 μl and 200 μl HF group. Conclusion: Echinococcus granulosus cyst fluid can regulate the ratio of CD4VCD8

  17. 细粒棘球绦虫转基因植物载体重组pBI-Eg95质粒构建及鉴定%Construction and identification of the transgenic plant vector recombinant pBI-Eg95 plasmid of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    周辉; 李文桂

    2008-01-01

    Objective To construct and identify the transgenic plant vector recombinant pBI-Eg95 plasmid of Echinococcus granulosus. Methods Total RNA was extracted from hydatid cyst protoscoleces of Echinococcus granulosus after sonication. A couple of specific primers were designed on the basis of known sequences of Eg95 gene. The desired gene was amplified by PCR technique from the cDNA, and then was cloned into the plant expression vector pBI121 to construct the recombinant pBI-Eg95 plasmid. The recombinant plasmid was electroporated into Agrobocterium tumefaciens (At) LBA4404 strain. The positive recombinant clones were confirmed by restriction endonuclease digestion and characterized by PCR. Results For RT-PCR, a specific band around 471 bp was amplified. The result of DNA sequencing of Eg95 showed the identity with the published sequence. The same band was obtained by restriction endonuclease digestion and PER from the plasmids of positive recombinant At(rAt). Conclusions The recombinant pBI-Eg95 plasmid was successfully constructed, and it provides the basis to further research of the transgenic plant vaccine of Echinococcus granulosus.%目的 构建并鉴定细粒棘球绦虫(Eg)转基因植物载体重组pBI-Eg95质粒.方法 从细粒棘球蚴包囊中分离原头节.经超声粉碎后抽提总RNA,反转录成eDNA,设计合成引物,以eDNA为模板.通过PCR从cDNA中扩增出目的 基因Eg95,经电泳及测序鉴定后,将该基因定向克隆到植物表达载体pBI121中构建pBI-Eg95重组质粒,电穿孔转化根癌农杆菌(At)LBA4404株;从转化的At阳性株中抽提质粒进行双酶切和以抽提的质粒为模板进行PCR鉴定.结果 RT-PCR扩增出1条约471 bp的特异性条带,DNA序列分析与GenBank知的序列同源性为100%.从转化的At中抽提的质粒,双酶切及PCR测定的结果与预期相符.结论 成功构建了细粒棘球绦虫转基因植物载体重组pBI-Eg95质粒,为进一步构建细粒棘球绦虫转基因植物疫苗奠定了基础.

  18. 细粒棘球蚴自发荧光观察及光谱特征分析%Autofluorescence and spectrum characteristics of Echinococcus granulosus culturing in vitro

    Institute of Scientific and Technical Information of China (English)

    杨宁; 张雪; 张传山; 吕国栋; 李亮; 刘欢元; 王慧

    2015-01-01

    目的:研究细粒棘球蚴的自发荧光现象及共聚焦λ扫描特点,丰富细粒棘球蚴光谱学和生物学信息,为后期免疫荧光及医学光子学研究奠定基础。方法采集自然感染细粒棘球蚴的绵羊肝脏,在无菌条件下收集包囊内的原头蚴进行体外培养,亚甲基蓝染色确定原头蚴活力。激光扫描共聚焦显微镜下分别以405 nm、488 nm、514 nm、561 nm等不同激发光激发观察虫体自发荧光,并分别对这5种激发光激发的细粒棘球蚴做自发荧光λ扫描分析,同时采用全波长多功能酶标仪下分别以405 nm、488 nm、514 nm、563 nm、633 nm等5种激发光激发细粒棘球蚴,绘制其自发荧光曲线。结果经亚甲基蓝染色测定原头蚴活力为100%。共聚焦显微镜观察不同激发光照射下细粒棘球蚴虫体能发出多种不同颜色的自发荧光。在相同的激发强度及探测器电压的情况下,以405 nm激光激发的蓝色荧光效果最佳,虫体大体结构清晰;λ扫描分别以405 nm、488 nm、514 nm、561 nm、633 nm等5种激发光激发样品,相应敏感的发射波长分别为490~520 nm、520 nm及580 nm左右、580~600 nm、610~630 nm和650 nm左右,其中405 nm、488 nm和561 nm激发效果较好,514 nm、633 nm激发效果欠佳。全波长多功能酶标仪检测细粒棘球蚴做自发荧光曲线与共聚焦显微镜结果基本一致,但所测定的虫体自发荧光强度较共聚焦显微镜偏低。结论激光扫描共聚焦显微镜下可观察到细粒棘球蚴虫体自发荧光,其中以405 nm激发的蓝色荧光最强;细粒棘球蚴虫体自发荧光光谱较宽,490~520 nm、610~630 nm ,405 nm、488 nm和561 nm为较适宜的激发波长。%The objective of this study was to illustrate the autofluorescence phenomenon and scanning confocal λcharacteris‐tics of Echinococcus granulosus (Eg) cultured in vitro ,which can not only rich the spectroscopy and

  19. Production and immunoanalytical application of 32 monoclonal antibodies against metacestode somatic antigens of Echinococcus multilocularis.

    Science.gov (United States)

    Wang, Xin; Lu, Rui; Liu, Qiao-Feng; Chen, Jian-Ping; Deng, Qiang; Zhang, Ya-Lou; Zhang, Bing-Hua; Xu, Jia-Nan; Sun, Lei; Niu, Qin-Wang; Liang, Quan-Zeng

    2010-06-01

    Alveolar echinococcosis is a rare but potentially fatal disease. Immunodiagnosis based on antibodies or antigens plays an important role in its diagnosis. In this study, metacestode somatic antigens of Echinococcus multilocularis were used to immunize BALB/c mice, and hybridomas were formed by cell fusion. Making use of the inherent effect of monoclonal antibody techniques to isolate different epitopes, we obtained a repertoire of 32 monoclonal antibodies against the metacestode somatic antigens. These monoclonal antibodies were used to investigate the specificity and localization of the metacestode antigens by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Nine antibodies specifically reacted with E. multilocularis, while 14 and ten cross-reacted with Echinococcus granulosus and Taenia saginata, respectively. Twenty-five antibodies stained the laminated layer. Eight reacted with the tegument of the protoscolex. Fourteen antibodies recognized the germinal layer. Most of the monoclonal antibodies can react with the antigen Em2. One antibody can react with antigen Em2 and Em10. One antibody that cross-reacted with T. saginata stained the germinal layer and protoscolex, especially its hooklets and suckers, but could not react with Em2 and Em10 antigens. It detected protein bands at 26 and 52 kDa. Two E. multilocularis-specific monoclonal antibodies stained both the germinal and laminated layers and could be used not only to purify specific antigens but also for immunohistochemical studies of E. multilocularis. In summary, these 32 monoclonal antibodies could have potential applications as useful tools in further studies of E. multilocularis antigen profiles.

  20. 新疆家犬体内存在细粒棘球绦虫G1(羊)株和G6(骆驼)株的分子证据%MOLECULAR EVIDENCE OF SHEEP(G1) AND CAMEL(G6) STRAINS OF ECHINOCOCCUS GRANULOSUS IN XINJIANG, CHINA

    Institute of Scientific and Technical Information of China (English)

    张亚楼; Jean-Mathieu BART; 温浩; 马旭东; 苗玉清; 林仁勇; 王星; 卢晓梅

    2005-01-01

    Objective 17 dogs' adult worms of Echinococcus granulosus were collected in order to establish some epidemiological molecular information in Xinjiang. Methods The sequencing of the mitochondrial cytochrome c oxidaseⅠ( COⅠ) gene of several adult worm from dogs was performed to confirm genotype of the E. granulosus strains. Results The most part (17) of adult worms' genotype was sheep strain (G1). Especially, the mixed infection of the camel strain G6 and sheep strain G1 were uniquely found in one dog. Conclusion The results confirm the importance of the prophylaxis measures in order to disrupt the cycle of sheep-dog and camel-dog transmission in Xinjiang. Nevertheless, the supervision of dog infection should be reinforced because this definitive host constitutes an important link with human contamination.%目的收集家犬体内的细粒棘球绦虫成虫以建立新疆棘球蚴病的分子流行病学资料. 方法对家犬体内成虫细胞色素c氧化酶Ⅰ基因序列进行测定以确定其亚株型. 结果所有感染犬体内成虫的基因型为G1型.特别是1条家犬体内发现存在细粒棘球绦虫G1(羊)株和G6(骆驼)株混合感染的情况. 结论在新疆阻断羊犬和羊骆驼循环圈是预防棘球蚴病的重要措施.作为终末宿主的家犬与人类的感染密切相关,对感染犬的管理应该加强.

  1. 细粒棘球绦虫(中国大陆株)谷胱甘肽s-转移酶重组蛋白(rEgGST)的免疫保护性研究%Immune protection of recombinant protein Glutathione s-transferase (rEgGST) of Echinococcus granulosus(Chinese mainland strain)

    Institute of Scientific and Technical Information of China (English)

    高鹏; 雄英; 杜娟; 李居怡; 王娅娜; 李宗吉; 赵巍

    2011-01-01

    The aim of the present study was to investigate the protective immunity against Echinococcus granulosus and its potential as vaccine candidate by using rEgGST immunizes in mice.ICR mice were randomly divided into the immunized group and the control group, and subcutaneously immunized three times.Mice were infected with protoscolices 4 weeks after immunization and sacrificed 20 weeks after infection to calculate immune protection and examine the levels in serum.Results showed that compared with the adjuvant control group, protein immunized mice's protective immunity was 89.39%.The levels of IgG, IgG's subtypes and IgE increased in varying degrees(P<0.05).It's suggested that rEgGST could induce some protective immunity, indicating that rEgGST is a promising candidate vaccine against echinococcosis.%目的 应用细粒棘球蚴(Echinococcus granulosus,Eg)诊断抗原GST重组蛋白进行动物免疫,探讨GST免疫保护性及其作为候选疫苗的潜在价值.方法 ICR小鼠随机分为蛋白免疫组和对照组,每隔2周皮下免疫1次,在第3次免疫后4周,用Eg原头蚴进行攻击感染,感染后20周剖杀小鼠,检获棘球蚴包囊,计算免疫保护力,并用ELISA法测定血清中IgG及其亚型和IgE水平.结果 与佐剂对照组比较,蛋白免疫组小鼠的免疫保护力为89.39%;与免疫前比较,免疫后和攻击感染后蛋白免疫组小鼠血清IgG及其亚型和IgE均有不同成程度的升高(P<0.05).结论 rEgGST能诱导小鼠产生部分免疫保护力,表明rEgGST是具有发展前途的抗包虫病候选疫苗.

  2. Phylogenetic systematics of the genus Echinococcus (Cestoda: Taeniidae).

    Science.gov (United States)

    Nakao, Minoru; Lavikainen, Antti; Yanagida, Tetsuya; Ito, Akira

    2013-11-01

    Echinococcosis is a serious helminthic zoonosis in humans, livestock and wildlife. The pathogenic organisms are members of the genus Echinococcus (Cestoda: Taeniidae). Life cycles of Echinococcus spp. are consistently dependent on predator-prey association between two obligate mammalian hosts. Carnivores (canids and felids) serve as definitive hosts for adult tapeworms and their herbivore prey (ungulates, rodents and lagomorphs) as intermediate hosts for metacestode larvae. Humans are involved as an accidental host for metacestode infections. The metacestodes develop in various internal organs, particularly in liver and lungs. Each metacestode of Echinococcus spp. has an organotropism and a characteristic form known as an unilocular (cystic), alveolar or polycystic hydatid. Recent molecular phylogenetic studies have demonstrated that the type species, Echinococcus granulosus, causing cystic echinococcosis is a cryptic species complex. Therefore, the orthodox taxonomy of Echinococcus established from morphological criteria has been revised from the standpoint of phylogenetic systematics. Nine valid species including newly resurrected taxa are recognised as a result of the revision. This review summarises the recent advances in the phylogenetic systematics of Echinococcus, together with the historical backgrounds and molecular epidemiological aspects of each species. A new phylogenetic tree inferred from the mitochondrial genomes of all valid Echinococcus spp. is also presented. The taxonomic nomenclature for Echinococcus oligarthrus is shown to be incorrect and this name should be replaced with Echinococcus oligarthra. Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  3. Análise dos efeitos de protoescólices e AgB de Echinococcus granulosus em possíveis mecanismos de evasão da resposta imune do hospedeiro

    OpenAIRE

    Veridiana Gomes Virginio

    2007-01-01

    Os fatores que afetam a susceptibilidade a infecção com E. granulosus são pouco conhecidos. Neste trabalho, foi avaliada a interação de protoescólices e um antígeno imunodominante secretado (AgB) de E. granulosus com neutrófilos humanos saudáveis. A intensidade de expressão de CD11b e produção de H2O2 foram avaliadas por citometria de fluxo usando sangue total. Os neutrófilos isolados também foram incubados com protoescólices ou AgB, e a interleucina 8 (IL-8) foi determinada por ELISA de capt...

  4. Expression,purification and immunologic identification of the recombinant EF-1 gene from Echinococcus granulosus%细粒棘球蚴重组延伸因子-1基因的表达、纯化及免疫学鉴定

    Institute of Scientific and Technical Information of China (English)

    卜阳; 李昭宇; 师志云; 马锐; 于晶晶; 于辛酉; 赵巍

    2008-01-01

    目的 对构建细粒棘球蚴(Echinococcus granulosus,Eg)延伸因子-1(Elongation factor 1,EF-1)基因的重组质粒,并原核表达、纯化及对其免疫特性进行鉴定.方法 从重组质粒EF-1/pGEM-T中酶切出EF-1目的片段,亚克隆入表达载体pET28a,转化入大肠杆菌E.coli BL21(DE3)plysS进行融合表达,经His-band树脂纯化试剂盒小量纯化,SDS-PAGE和Western blot方法鉴定表达产物.结果 成功构建Eg.EF-1/pET28a/E.coliBL21基因工程菌株,诱导表达重组蛋白和纯化分离得到31KDa Eg.EF-1均能被细粒棘球蚴天然抗原免疫的兔多克隆抗血清识别.结论 初步证实该重组蛋白具有较好的抗原性.

  5. Expression and purification of gene encoding the diagnostic antigen P-29 of Echinococcus granulosus and the preliminary analysis on its immunogenicity%细粒棘球绦虫(中国大陆株)诊断抗原P-29基因的表达、纯化及免疫原性初步分析

    Institute of Scientific and Technical Information of China (English)

    师志云; 李昭宇; 卜阳; 王娅娜; 李宗吉; 马锐; 赵巍

    2009-01-01

    目的 对细粒棘球绦虫(Echinococcus granulosus,Eg)诊断抗原P-29 (diagnostic antigen P-29)重组质粒进行原核表达、纯化,并初步分析重组蛋白的免疫原性.方法 从重组质粒EgP-29/pGEM-T中获取诊断抗原P-29基因,亚克隆于表达载体pET-28a构建基因工程菌株,并表达、纯化重组蛋白,经Western-blot、ELISA分析重组蛋白的免疫原性.结果 成功构建原核重组表达载体EgP-29/pET-28a/BL21(DE3)plysS,并纯化出浓度较高的重组蛋白.ELISA检测显示,用表达、纯化的重组蛋白免疫小鼠,诱导产生了特异性抗体IgG,Western-blotting鉴定该抗体能识别重组抗原及天然抗原原头蚴.结论 重组蛋白具有良好的免疫原性.

  6. Molecular differentiation of cryptic stage ofEchinococcus granulosusand Taenia species from faecal and environmental samples

    Institute of Scientific and Technical Information of China (English)

    Diganta Pan; Sumanta De; Asit Kumar Bera; Subhashis Bandyopadhyay; Subrata Kumar Das; Debasis Bhattacharya

    2010-01-01

    Objective:To differentiate cryptic stage ofEchinococcus granulosus(E. granulosus) andTaenia by PCR-RFLP and sequence information of amplicon.Methods: DNA were isolated from metacestodes stage ofTaenia andE. granulosus using DNA isolation kit (Q-BIOgene kit, USA), the amplified and purified DNA product was then cloned and sent for sequencing. The generating sequence information was used for amplicons identification.Results:Out of 112 faecal and environmental samples, 16 exhibited positive result. The product size of amplicon positive for E. granulosus was 310 bp; whereas, forTaenia spp. sizes varied from 379 to 388 bp. Restriction profile of actin II with Csp61 also differedTaenia spp. andE. granulosus.Conclusions: The result of the study indicated that, the primers were useful to differentiate cryptic stage of the two genera which is yet to be reported earlier.

  7. 3种助溶剂对阿苯达唑和阿苯达唑亚砜体外抗包虫活性的影响%Effects of albendazole and albendazole sulfoxide dissolved with different cosolvents on Echinococcus granulosus protoscoleces and vesicles in vitro

    Institute of Scientific and Technical Information of China (English)

    于春洋; 商少华; 张瑞妮; 高惠静; 吕国栋; 赵军; 肖云峰; 吕顺忠; 温浩

    2013-01-01

    目的 评估二甲基亚砜(DMSO)、吐温80和二者混合溶液等3种助溶剂溶解阿苯达唑(ABZ)和阿苯达唑亚砜(ABZSX)对其体外抗细粒棘球绦虫幼虫作用的影响.方法 采用高效液相色谱法测定吐温80、DMSO及二者混合溶液对阿苯达唑的助溶作用,并比较抗包虫效果.分别将DMSO、吐温80及其混合溶液溶解的ABZ和ABZSX饱和浓度药物溶液加入RPMI 1640培养基中,使DMSO、吐温80及其混合溶液的浓度达到1.0%、0.1%和1.0%+0.1%.用上述含药培养液体外培养细粒棘球蚴原头节和囊泡,并设空白对照组以及适当浓度的助溶剂对照组,隔天观察原头蚴死亡率,周期为10 d;每5d观察囊泡的形态及塌陷率,周期为20 d.结果 各药物作用至第10d和20 d时,联用DMSO及吐温80溶解的ABZ组和ABZSX组原头蚴死亡率及囊泡塌陷率分别为(57.9±6.1)%、(49.32±8.5)%和(58.56±5.34)%、(80.74±1.58)%,均显著高于单用助溶剂组(P<0.01);空白对照组和助溶剂组原头蚴死亡率及囊泡塌陷率均低于9%.结论 当药物相同时,DMSO和吐温80混合助溶剂组的抗原头蚴及囊泡作用优于DMSO助溶剂组,DMSO助溶剂组优于吐温80助溶剂组;助溶剂一致时,抗原头蚴效果ABZ组优于ABZSX组,抗囊泡效果ABZSX组优于ABZ组.%Objective Albendazole (ABZ) and albendazole sulfoxide (ABZSX) were dissolved with 1% dimethyl sulfoxide (DMSO),0.1 % Tween80,or a mixture of DMSO and Tween80 to investigate their anti-hydatid action on Echinococcus granulosus protoscoleces and E.granulosus vesicles.Methods High-performance liquid chromatography (HPLC)was used to examine dissolution of ABZ by Tween 80,DMSO,and a mixture of DMSO and Tween80.RPMI1640 culture medium with saturated concentrations of ABZ and ABZSX was used to culture E.granulosus protoscoleces and vesicles.ABZ and ABZSX were dissolved with DMSO,Tween80,or a mixture of DMSO and Tween80.The concentrations of DMSO,Tween80,and the

  8. 细粒棘球绦虫未知功能基因384p02i04的克隆及生物信息学分析%Molecular cloning and bioinformatics analysis of a novel gene384p02i04 in Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    刘辉; 肖云峰; 赵军; 王建华; 吕国栋

    2016-01-01

    目的:克隆细粒棘球绦虫与药物应激相关的未知功能基因384p02i04序列,并进行生物信息学分析。方法提取细粒棘球绦虫原头蚴总 RNA,采用实时荧光定量聚合酶链反应(RT-PCR)和 cDNA 末端快速扩增技术(RACE)法扩增384p02i04基因片段,测序,并进行生物信息学分析。采用定量反转录聚合酶连锁反应(qRT-PCR)法分析青蒿素、阿苯达唑和吡喹酮等药物干预对384p02i04基因表达的影响。结果384p02i04基因全长449 bp,通过各种生物数据库对该基因进行预测,发现384p02i04基因存在1个编码109个氨基酸的蛋白质开放阅读框,利用 BLAST 数据库将其核酸及氨基酸序列进行比对,并未发现相似度较高的基因及氨基酸,利用 RNA structure软件发现384p02i04基因具有非常低的自由能,其二级结构非常稳定。在青蒿素、阿苯达唑和吡喹酮等药物干预后384p02i04基因均明显下调(P <0.05)。结论克隆获得细粒棘球绦虫的一种与药物应激反应相关的未知功能基因,为研发以该基因为靶点的抗包虫病药物奠定基础。%Objective To do the clone and bioinformatics analysis of a novel drug stress-related gene in Echinococcus granulosus .Methods The gene sequence of 384p02i04 was cloned by RT-PCR and RACE methods from protoscolex of E .Granulosus and analyzed by bioinformatics.The gene expressions of 384p02i04 in the protoscoleces of E .Granulosus treated by artemisinin,albendazole and praziquantel were detected by qPCR.Results The size of 384p02i04 gene was 449 bp and there was a protein with open read-ing frame encoded by 109 amino acids.The comparison with the data in BLAST database showed that the gene had low similarity to the known gene.The results in RNA structure analysis showed that the free en-ergy of the gene was very low,and its secondary structure was very stable.Artemisinin,albendazole and praziquantel could down-regulate the

  9. p38MAPK抑制剂SB202190体外抑制细粒棘球蚴原头节生长的研究%Effects of p38MAPK inhibitor SB202190 on the viability of Echinococcus granulosus protoscoleces in vitro

    Institute of Scientific and Technical Information of China (English)

    张晶; 吕海龙; 王成华; 孙冯; 雷颖; 彭心宇; 姜玉峰

    2013-01-01

    Objective The aim of this study was to investigate the in vitro efficacy of the p38MAPK inhibitor SB202190 against Echinococcus granulosus protoscoleces.Methods Protoscoleces of E.granulosus were incubated in vitro with SB202190 at concentrations of 12.5,25,50,and 100 μmol/L,and the viability of protoscoleces was assessed on a daily basis by microscopic observation of movements,flame cell activity,and 0.1% eosin staining.Each experiment was repeated three times.Results Control E.granulosus protoscoleces incubated in RPMI 1640 medium or in RPMI 1640 medium+DMSO remained viable after 14 days of incubation.From day 1,the viability of protoscoleces started to decline due to the 50 μmol/L or 100 μmol/L of SB202190.After 14 days,viable protoscoleces were no longer observed in cultures treated with 100 μmol/L of SB202190 but survival of 13.8% of protoscoleces was observed when they were treated with 50 μmol/L of SB202190.Although 12.5μmol/L and 25 μmol/L of SB202190 also had an effect on protoscoleces,the effect was not as pronounced as that of 100 μmol/L of SB202190.Conclusion Results indicated that SB202190 has satisfactory action against E.granulosus protoscoleces in vitro.%目的 探讨p38MAPK抑制剂SB202190体外对细粒棘球蚴原头节生长的作用. 方法 将体外培养的细粒棘球蚴原头节分别加入12.5、25、50、100 μmol/L的SB202190中体外孵育,在光镜下观察原头节的形态变化,同时通过伊红染色显示原头节活力.实验重复3次. 结果 孵育14d后,正常和DMSO对照组原头节的活力几乎没有改变.50 μmol/L和100μmol/L SB202190作用1d后,细粒棘球蚴原头节的活力开始下降;作用14d后,100μmol/L SB202190组无存活的头节,50 μmol/L SB202190组的头节活力仅为13.8%.12.5 μmol/L和25 μmol/L SB202190对原头节的活力也有影响,但不及高浓度组显著. 结论 p38MAPK抑制剂SB202190在体外有抗细粒棘球蚴原头节的作用.

  10. Protective immunity of Eg14-3-3 against Echinococcus granulosus in mice%细粒棘球绦虫(中国大陆株)14-3-3重组蛋白的免疫保护力

    Institute of Scientific and Technical Information of China (English)

    李宗吉; 雄英; 孙俊峰; 赵巍

    2012-01-01

    Objective To investigate the protective immunity against Echinococcus granulosus in mice immunized with rEgl4-3-3. Methods ICR mice were subcutaneously immunized three times with rEgl4-3-3, followed by the challenge with Echinococcus granulosus protoscoleces intraperitoneally, and then sacrificed in the sixth month post-challenge to detect the proliferation of splenocytes with MTT assay and to measure the secretion of IL-2, IL-4, IL-10 and IFN-γ with ELISA. The rate of reduced hydatid cyst and the levels of IgE, IgG and IgG subclasses in sera were examined. Results Compared with the control group, mice vaccinated with rEgl4-3-3 and challenged intraperitoneally with E. granulosus protoscoleces revealed significant protective immunity of 84. 47% (P<0. 05). Enzyme-linked immunosorbent assay and Western blot analysis indicated that immunized mice generated specific high level of IgG against rEgl4-3-3. The prevailing isotypes of IgG induced by rEgl4-3-3 in mice were IgGl and IgG2a. Spleen lymphocytes from mice immunized with rEgl4-3-3 showed a significant proliferation response to rEgl4-3-3. The culture of spleen cells showed that secretion of IFN-y and IL-2 increased significantly in the vaccinated mice whereas IL-4 and IL-10 levels did not differ significantly between vaccinated and control mice. Conclusion The results indicate that rEgl4-3-3 vaccination elicit significant levels of protective immunity against Echinococcus granulosus infection. Thus, rEgl4-3-3 protein is a promising candidate as an effective vaccine to prevent cystic echinococcosis.%目的 探讨细粒棘球蚴Eg14-3-3重组蛋白的免疫保护性及其伴随的免疫反应.方法 ICR小鼠随机分为rEg14-3-3蛋白免疫组和PBS佐剂对照组,每隔2周背部皮下免疫1次,连续免疫3次.在第3次免疫后6周,用细粒棘球蚴活的原头蚴进行攻击感染,感染后24周杀鼠取脾,分离培养脾细胞,MTT检测淋巴细胞增殖率,用试剂盒检测脾细胞培养上清液的IL-2

  11. 细粒棘球蚴细胞外信号调节激酶基因克隆、序列分析及功能的初步鉴定%Molecular cloning, sequencing and function of extracellular signal regulated kinase of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    吕国栋; 纪静; 王俊华; 李亮; 王红丽; 卢晓梅; 王星; 温浩; 林仁勇

    2010-01-01

    Objective To perform molecular cloning and sequencing, bioinformatics analysis,protein expression and function of extracellular signal regulated kinase (EgERK1) of Echinococcus granulosus in Xinjiang. Methods The specific primers of EgERK1 were designed and total RNA was extracted from Echinococcus granulosus in Xinjiang. EgERK1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and prokaryotic expression plasmid pET28a-EgERK1 was constructed and sequenced. The sequences were analyzed by DNA sequencing and bioinformatics technology. The recombinant EgERK1 protein was induced and expressed. The biological function was detected using sodium dodecyl sulfate polyacrylamide gel electropheresis and Western blot. Results The sequence of RT-PCR product was 1125 bp, encoding 374 amino acids with isoelectric point of 6.34.This gene was a new ERK-homologues gene indicated by BLAST, named EgERK1(EU701008).Homology comparisons indicated that the homology of EgERK1 and EmMPK1from Echinococcus multilocularis was 95.45%, and was 43.04%-61.88% to ERK from Caenorhabditis elegans, S. cerevisiae, D. melanogaster and human. Phylogenetic analysis showed that EgERK1 clustered with EmMPK1. Bioinformatics analysis predicted that EgERK1 contained a highly conserved T-X-Y motif and activation loop segment of ERK-like kinase.Western blot results showed the EgERK1 recombinant protein could reacted specifically with anti-human ERK monoclonal antibody. Conclusion A new EgERK1 gene of Echinococcus granulosus is successfully cloned and its recombinant protein could reacted specifically with ERK1/2 antibody, which provides the basis for further study of EgERK1 function in the host-parasite interaction.%目的 从新疆株细粒棘球蚴中克隆细胞外信号调节激酶(EgERK1)基因,进行序列测定、生物信息学分析,蛋白表达及功能初步鉴定.方法 设计特异性引物,从新疆株细粒棘球蚴中提取总RNA,RT-PCR法扩增EgERK1基因,构建pET28

  12. Selection, Recombination and History in a Parasitic Flatworm (Echinococcus) Inferred from Nucleotide Sequences

    OpenAIRE

    KL Haag; Araújo AM; Gottstein B; Zaha A

    1998-01-01

    Three species of flatworms from the genus Echinococcus (E. granulosus, E. multilocularis and E. vogeli) and four strains of E. granulosus (cattle, horse, pig and sheep strains) were analysed by the PCR-SSCP method followed by sequencing, using as targets two non-coding and two coding (one nuclear and one mitochondrial) genomic regions. The sequencing data was used to evaluate hypothesis about the parasite breeding system and the causes of genetic diversification. The calculated recombination ...

  13. New data on Echinococcus spp. in Southern Brazil Novos dados sobre Echinococcus spp. no sul do Brasil

    Directory of Open Access Journals (Sweden)

    Mário L. de La Rue

    2006-04-01

    Full Text Available 40 Echinococcus isolates from sheep and cattle in Southern Brazil were genetically analysed in order to obtain further data on the presence of different taxa of the Echinococcus granulosus complex. Differentiation was done using a PCR technique and sequencing of mitochondrial cytochrome c oxidase subunit 1 (CO1. Most samples (38 could be allocated to the sheep strain (G1 of E. granulosus, while two samples belonged to E. ortleppi, previously known as cattle strain (G5 of E. granulosus. Due to the shorter prepatent period in dogs of the latter taxon, this records have important implications for the design of control measures in this endemic region.Quarenta isolados de Echinococcus provenientes de ovinos e bovinos do sul do Brasil foram analisados geneticamente com o objetivo de obter dados a respeito das diferentes cepas dentro do gênero Echinococcus granulosus. A diferenciação foi feita empregando-se a técnica de PCR a o seqüenciamento da subunidade 1 da citocromo c oxidase (CO1. A maior parte das amostras (38 pôde ser alocada na cepa ovina (G1 enquanto duas amostras pertenceram ao gênero E. ortleppi, anteriormente conhecido como cepa bovina (G5 do E. granulosus. Devido ao menor período pré-patente em cães deste último gênero ressalta-se a importância do presente registro devido às implicações no delineamento de medidas de controle nesta região endêmica.

  14. In vitro and in vivo treatments of echinococcus protoscoleces and metacestodes with artemisinin and artemisinin derivatives.

    Science.gov (United States)

    Spicher, Martin; Roethlisberger, Carole; Lany, Catharina; Stadelmann, Britta; Keiser, Jennifer; Ortega-Mora, Luis M; Gottstein, Bruno; Hemphill, Andrew

    2008-09-01

    In vitro treatment of Echinococcus multilocularis and Echinococcus granulosus larval stages with the antimalarials dihydroartemisinin and artesunate (10 to 40 microM) exhibited promising results, while 6 weeks of in vivo treatment of mice infected with E. multilocularis metacestodes (200 mg/kg of body weight/day) had no effect. However, combination treatments of both drugs with albendazole led to a substantial but statistically not significant reduction in parasite weight compared to results with albendazole alone.

  15. 绵羊细粒棘球蚴全血金标渗滤检测方法的建立%Development of Dot Immunogold Filtration Assay (DIGFA) for Rapid Detection of Sheep Echinococcus granulosus Antidody with Whole Blood Sample

    Institute of Scientific and Technical Information of China (English)

    余华; 聂华明; 严玉宝; 杨光友; 陈世界; 胡娟; 崔鹏博

    2011-01-01

    In order to establish a rapid and high-performance immunoassay assay in whole blood of livestock for diagnosis of hydatidosis, the cyst fluid antigen was extracted from fresh sheep Echinococcus cysts and salted-out by ammonium sulphate.The soluble cyst fluid used as antigen was dotted on nitro-cellulose and colliodal gold-donkey anti-goat IgG and colliodal gold-rabbit anti-mice IgG were employed as the probe, and a filtration device with vertical flow was applied to detect the specific IgG antibodies of sheep and mice infected with protoscoleces of Echinococcus granulosus. By using the new immunoassay, it was found that 90.91%-94.4% of whole blood and serum from sheep and 100% of whole blood and serum from mice were positive for hydatidosis, and 5% of health individuals had false positive results.However, partially high percentages of cross reactions could be demonstrated in serum of sheep with Taenia multiceps.The results showed that the dot immune gold filtration assay (DIGFA) could be used for diagnosis and quarantine inspection of livestock hydatidosis.%为建立一种快速、高效的诊断家畜细粒棘球蚴病方法,提取绵羊细粒棘球蚴包囊新鲜囊液,盐析囊液抗原,点样于硝酸纤维膜,以胶体金-驴抗羊IgG和胶体金-兔抗鼠IgG为检测标记物,采用垂直流渗滤装置检测绵羊与人工感染细粒棘球蚴小鼠血清和全血特异性抗体。患病绵羊阳性血清及全血检出率在90.91%~94.4%,细粒棘球蚴感染小鼠血清及全血检出率均为100%;细粒棘球蚴阴性羊血清和全血假阳性率为4.00%-4.59%;与脑多头蚴病血清交叉反应率为28.57%(2/7)。研究结果表明细粒棘球蚴全血金标渗滤法(DIGFA)可应用于绵羊棘球蚴病的诊断与检疫。

  16. 细粒棘球绦虫Eg18基因的克隆表达和重组抗原免疫检测的初步评价%Cloning and expression of Echinococcus granulosus Eg18 and preliminary evaluation of immunological assay with recombinant antigen

    Institute of Scientific and Technical Information of China (English)

    王永顺; 韩秀敏; 王虎

    2011-01-01

    Objective To clone and express Echinococcus granulosus Egl8 gene and evaluate the immunoreactivity of the re-combinant protein. Methods Egl8 coding sequence was amplified by RT-PCR using primers designed according to the sequence of Egl8 in GenBank from total RNA extracted from Echinococcus granulosus protoscoleces isolated from infected sheep in Qinhai Province and cloned into the prokaryotic expression vector pET-28a( + ). The recombinant expression vector was transformed to Escherichia coli BL21 (DE) and induced to express by IPTG. The expressed products were analyzed by SDS-PAGE and purified with Ni-IDA agarose affinity chromatography. rEgl8 was evaluated for its reactivity with the sera from the patients infected with hy-datid and other helminthes by Western blotting and ELISA. Results The sequence of cloned Egl8 was completely identical with the original sequences of Egl8 and Em 18 deposited in GenBank. The recombinant protein strongly reacted to the sera from the patients with alveolar echinococcosis, cystic echinococcosis, and cysticercosis, and weakly reacted to the sera from the patients with nematodiasis, schistosomiasis and clonorchiasis. The detection of specific IgG4 was much more specific than that of IgG. Conclusion Egl8/Eml8 is the common antigen of cestode and its specific IgG4 is a serum marker of alveolar echinococcosis.%目的 克隆、表达细粒棘球绦虫Eg18基因,评价重组蛋白的免疫反应性.方法 根据已知Eg18基因序列,利用RT-PCR方法从青海绵羊肝棘球蚴原头节提取的总RNA中扩增目的基因,克隆到原核表达质粒pET-28a(+)中,转化大肠埃希菌BI21 (DE),IPTG诱导表达.表达产物经亲和层析纯化,用免疫印迹试验(Western blot)和酶联免疫吸附试验(ELISA)初步评价其与棘球绦虫及其他蠕虫感染血清的反应性.结果 RT-PCR扩增产物编码的蛋白质序列与GenBank中的Eg18和Em18完全相同.重组蛋白与多种蠕虫病人血清中的IgG和IgG4均

  17. Characterization of Echinococcus granulosus thioredoxin peroxidase expressed in Pichia pastoris%细粒棘球绦虫硫氧还蛋白过氧化物酶基因在毕赤酵母中的分泌表达及生物学功能鉴定

    Institute of Scientific and Technical Information of China (English)

    王慧; 李军; 张富春; 张文宝; 温浩

    2014-01-01

    目的 在毕赤酵母中分泌表达细粒棘球绦虫硫氧还蛋白过氧化物酶(Echinococcus granulosus thioredoxin per-oxidase,EgTPx),并检测其抗氧化活性. 方法 从包囊中分离原头蚴,抽提总RNA,采用RT-PCR获取EgTPx的cD-NA片段并克隆至分泌型表达载体pPIC9K,构建重组表达质粒pPIC9K-EgTPx,电穿孔法转化毕赤酵母菌株GSll5,在MD平板上筛选His+克隆,采用梯度浓度G418抗性筛选高拷贝转化子,提取酵母基因组DNA,采用PCR鉴定Mut表型,阳性克隆经甲醇诱导表达EgTPx蛋白,表达产物经SDS-PAGE和Western blot鉴定,并检测其抗氧化活性. 结果 酶切和测序证实重组表达质粒pPIC9K-EgTPx构建正确,表达的目的蛋白分子质量单位约为22 ku,该蛋白可被鼠抗EgTPx多抗识别,且具有较强的抗氧化活性. 结论 在毕赤酵母表达系统中成功表达了EgTPx,该蛋白具有免疫反应性和抗氧化活性.

  18. The changes in blood electrolytes and blood gases during anaphylactic shock induced by Echinococcus granulosus in sheep%细粒棘球蚴感染绵羊诱发过敏性休克期间电解质及血气的动态变化

    Institute of Scientific and Technical Information of China (English)

    郑宏; 杨戈雄; 李亦梅; 徐志新

    2003-01-01

    目的以细粒棘球蚴(Echinococcus granulosus,EG)感染绵羊并诱发过敏性休克动物模型为基础,观察休克期间电解质、血气的动态变化趋势,为其防治提供理论依据.方法人工感染EG绵羊15只,用EG囊液粗制抗原攻击发敏复制过敏性休克动物模型,分别于抗原攻击前后各时点,经右颈内动脉采血,测定电解质及血气各指标.结果抗原攻击后3.5 min血K+显著增高(P<0.01),30min后降至基础值水平,其它电解质无显著变化;动脉氧分压(PaO2)在抗原攻击后即持续性降低(P<0.01),动脉血二氧化碳分压(PaCO2)在8min时有显著升高(P<0.01),以后逐渐恢复,pH值明显下降;血细胞容积(Hct)及血红蛋白(Hb)在抗原攻击后3.5 min增高显著(P<0.05).结论 EG诱发的过敏性休克电解质及血气变化剧烈,表现为明显的高血钾、酸中毒、PaO2的持续降低以及PaCO2的增高,且反应迅速,消退较快.

  19. Application of Bioinformatics in the Study of HSP70 Recombinant Vaccine of Echinococcus Granulosus%生物信息学技术在细粒棘球蚴热休克蛋白70重组疫苗研究中的应用

    Institute of Scientific and Technical Information of China (English)

    丁淑琴; 刘宏鹏; 张爱君; 张彩芳; 赵巍

    2009-01-01

    目的 通过基因序列分析,寻找细粒棘球蚴(Echinococcus granulosus,Eg)热休克蛋白基因(Heat shock pm-tein7,HSP70),为包虫病防治筛选新的候选疫苗抗原.方法 ①登录 GenBank 公共数据库,检索 EgHSP70目的基因序列.②对克隆基因进行测序,结果输入DNAStax、BioSun分析软件和互联网 SWISS-MODEI 数据库,对重组 EgHSP70 的特性、抗原表位及构象进行预测分析.结果 ①获得了具有完整开放阅读框的 EgHSP70核苷酸序列.②DNAStar分析软件推算氨基酸序列结果显示,EgHSP70由133个理论氨基酸组成,分子量约为14.53kDa.DNAStar、BioSun分析软件确定了重组 EgHSP70 可能的抗原表位.与其他动物热休克蛋白70氨基酸的同源性分析结果显示EgHSP70具有高度的保守性.结论 生物信息学技术在 EgHSP70 重组疫苗研究中有一定的理论和应用价值.

  20. 藏羊源细粒棘球蚴抗原B8/2基因的克隆表达和免疫学鉴定%Cloning and Expression of Tibetan Sheep-origin Echinococcus granulosus Antigen B Gene and Protein Identification Using Immunological Method

    Institute of Scientific and Technical Information of China (English)

    朵红; 李伟; 付永; 彭毛; 郭志宏; 沈秀英; 牛建章; 尼玛; 童延军

    2016-01-01

    目的 克隆、表达青海省藏羊源细粒棘球蚴(Echinococcus granulosus)抗原B8/2(EgAgB8/2)基因,并进行免疫学鉴定.方法 用RT-PCR扩增EgAgB8/2基因cDNA,构建原核表达载体pET-EgAgB8/2,转化至大肠埃希菌BL21 (DE3)中诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)对诱导表达后的蛋白进行鉴定.结果 克隆的EgAgB8/2基因长335 bp,序列分析表明与已报道的EgAgB8/2 cDNA序列的同源性为98%~ 100%,原核表达载体pET-EgAgB8/2构建正确.诱导表达后,通过SDS-PAGE检测,上清中有大量目的蛋白表达.纯化的蛋白经Western blotting鉴定为目的蛋白.结论 成功克隆了青海省藏羊源EgAgB8/2基因,构建了原核表达载体,诱导表达后的蛋白为目的蛋白.

  1. Protective immunity of recombinant Eg myophilin against Echinococcus granulosus (Chinese mainland strain) in mice%细粒棘球绦虫(中国大陆株)亲肌肉抗原重组蛋白的免疫保护性研究

    Institute of Scientific and Technical Information of China (English)

    马锐; 师志云; 李昭宇; 王娅娜; 李宗吉; 赵巍

    2010-01-01

    目的 探讨细粒棘球蚴(Echinococcus granulosus,Eg)亲肌肉抗原重组蛋白的免疫保护性及其作为候选疫苗的潜在价值.方法 ICR小鼠随机分为蛋白免疫组和佐剂对照组,每隔2w皮下免疫1次,在第3次免疫后2周,用Eg原头蚴进行攻击感染,感染后20w剖杀小鼠,检获棘球蚴包囊,计算免疫保护力,并用ELISA法测定血清中IgG及其亚型和IgE水平.结果 与佐剂对照组比较,蛋白免疫组小鼠的免疫保护力为94.46%;与免疫前比较,免疫后和攻击感染后蛋白免疫组小鼠血清IgG、IgG1、IgG3和IgE水平均明显升高(P<0.05),IgG2b降低(P<0.05).结论 细粒棘球绦虫亲肌肉抗原重组蛋白能诱导小鼠产生一定的保护性免疫,是潜在的疫苗候选抗原分子.

  2. 细粒棘球绦虫亲肌肉抗原重组蛋白诱导小鼠免疫应答的研究%Study on Immune Response in ICR Mice by Immunization with Recombinant Myophilin Vaccine against Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    马锐; 师志云; 王娅娜; 李宗吉; 孙俊峰; 赵巍

    2012-01-01

    目的 探讨细粒棘球绦虫(Echinococcus granulosus,Eg)亲肌肉抗原重组蛋白诱导ICR小鼠产生的免疫应答及其对Eg原头节攻击感染的保护性作用.方法 36只雄性ICR小鼠随机分为A(重组蛋白免疫组)、B(空质粒蛋白免疫组)和C (PBS对照组)等3组,每组12只.3组小鼠分别经皮下注射重组蛋白(10μg/只)、空质粒蛋白(10μg/只)和PBS(100 μl/只),第2和4周同法加强免疫2次.末次免疫后2周,每只小鼠腹腔接种细粒棘球蚴原头节50个进行攻击感染.感染后20周,剖杀小鼠,分离并称重细粒棘球蚴组织,计算囊重减少率.取脾脏,分离脾细胞,流式细胞仪检测脾脏CD4+T和CD8+T淋巴细胞亚群百分比.脾细胞体外经脾细胞悬液、Eg粗抗原(EgAg)和伴刀豆球蛋白A(ConA)刺激培养4~5 h后,四甲基偶氮唑盐比色法(MTT法)检测T淋巴细胞增殖情况. 结果 感染后20周,A组小鼠包囊质量[(0.019±0.036)g]明显低于C组[(0.058±0.057)g](P<0.05),囊重减少率为69.1%; CD4+T和CD8+T细胞亚群百分比分别为(29.7±0.9)%和(9.7±0.8)%,均高于C组[(11.6±1.4)%和(7.8±0.2)%](P<0.01和P<0.05),CD4+/CD8+比值(3.061±0.015)也显著高于C组(1.487±0.106) (P<0.01).未经刺激时,A组小鼠脾T淋巴细胞增殖水平(0.237±0.009)高于C组(0.159±0.005)(P<0.01);用EgAg和ConA刺激后,A组小鼠脾T淋巴细胞增殖水平[(0.283±0.008)和(0.325±0.025)]均高于C组[(0.203±0.002)和(0.244±0.006)] (P<0.01).结论 棘球蚴亲肌肉抗原重组蛋白可诱导免疫小鼠脾脏T淋巴细胞增殖,CD4+T细胞亚群在重组蛋白诱导的小鼠抗Eg原头节攻击感染的保护性免疫机制中起一定作用.%Objective To investigate the immune response and the protection in mice induced by the recombi-nant myophilin protein of Echinococcus granulosus. Methods Thirty-six male ICR mice of 6-8 weeks old were randomly divided into groups A, B and C each with 12. The mice in the 3 groups were subcutaneously

  3. 细粒棘球蚴重组抗原rEgferritin诱导宿主抗感染免疫应答的初步研究%An initial investigation on effects of rEgferritin on immune response of mice infected with hydatid cysts of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    王娅娜; 王洁; 王淑静; 张焱; 赵巍

    2011-01-01

    In order to initially investigate the effects of rEgferritin on the immune response of mice infected with hydatid cysts of Echinococcus granulosus. ICR mice were immunized with rEgferritin by thrice and challenged by protoscoleces of E. Granulosus at 12 week. Mice were killed at 20 weeks after infection, and blood of mice were obtain at 0 week, 12 week and 32 week prepare serum, The splenocytes were isolated and the percentages of the splenic CD4+ and CD8+ T lymphocytes were measured by FCM . In every groups splenocytes were cultivated with stimulation of rEgferritin native antigen, ConA and normal nutrient. The levels of the supernatant of cultures were determined by ELISA. The adjuvant group and blank group were set up for comparison. It was found that the OD values of IgG, IgG2a and IgG2b of immunized group were all higher than that of the control group ( P<0. 01), and the OD value of IgG3 was no significant difference among the three groups. Mice vaccinated with rEgferritin and challenged intraperitoneally with E. Granulosus protoscoleces revealed significant protective efficacy up to 85. 6%. The percentage of the splenic CD4+ and CD8+ T lymphocytes were all increased in the immunized group of mice, but CD4+ T lymphocytes of immunized group were obviously higher than adjuvant group and control group( P<0. 01), splenic lymphocytes cell of immunized rEgferritin group of mice were growthed obviously by induced with rEgferritin protein and native antigen of Echinococcus g ranulosus. These data indicated that rEgferritin protein immunized could can induce humoral immunity of mice against the hydatid cyst of E. G ranulosus. The rEgferritin might be a valuable candidate vaccine for against hydatid disease.%目的 初步探讨细粒棘球蚴重组铁蛋白rEgferritin免疫小鼠产生的抗细粒棘球蚴感染的免疫机制.方法 rEgferritin免疫ICR小鼠3次、同时设立佐剂组和对照组,第12w用细粒棘球蚴原头蚴对3组小鼠进行攻击感染,5个

  4. Biochemical and molecular characterization of the tegument protein RT10 from Raillietina tetragona.

    Science.gov (United States)

    Chen, Li; Li, Haiyun

    2014-03-01

    Tegument antigens of tapeworm play an important role in modulation of host response and parasite survival. Characterizing appropriate antigens for parasite infection diagnosis and vaccination is rational and could have both economic and epidemiological significance in poultry industry. In the present study, a major protoscolex homologue (named RT10) of Echinococcus and Taenia spp. was amplified from Raillietina tetragona cestode. The RT10 cDNA was 1,877 bp long containing an open reading frame of 1,683 bp nucleotides, which encoded a deduced protein of 560 amino acids with an isoelectric point of 6.33. Secondary structure analysis demonstrated that RT10 was both hydrophilic and antigenic, and possessed N-terminal FERM domain and C-terminal ERM domain, respectively. With the same structural properties of previously reported antigens from Echinococcus and Taenia spp., RT10 tegument antigen had a more than 82% similarity in nucleotide level with initially reported antigens from Echinococcus and Taenia spp., and a more than 83% similarity in protein level, with the highest similarity of 85.2% to Taenia antigen H17g. In addition, phylogenetic analysis illustrated a high consistency between different genus antigens and evolutionary branching. Although the detailed function of RT10 is still unknown, the high sequence conservation and structural similarity to formerly identified tegument antigens from Echinococcus and Taenia spp. suggested that RT10 may play a similar role as the previous reported antigens between cestode and host. It is significant to clarify the antigenic and serodiagnostic characteristics in the subsequent work.

  5. Current status of the genetics and molecular taxonomy of Echinococcus species.

    Science.gov (United States)

    McManus, D P

    2013-11-01

    The taxonomy of Echinococcus has long been controversial. Based mainly on differences in morphology and host-parasite specificity characteristics, 16 species and 13 subspecies were originally described. Subsequently, most of these taxa were regarded as synonyms for Echinococcus granulosus and only 4 valid species were recognised: E. granulosus; E. multilocularis; E. oligarthrus and E. vogeli. But, over the past 50 years, laboratory and field observations have revealed considerable phenotypic variability between isolates of Echinococcus, particularly those of E. granulosus, which include differences in: morphology in both larval and adult stages, development in vitro and in vivo, host infectivity and specificity, chemical composition, metabolism, proteins and enzymes, pathogenicity and antigenicity. The application of molecular tools has revealed differences in nucleic acid sequences that reflect this phenotypic variation and the genetic and phenotypic characteristics complement the previous observations made by the descriptive parasitologists many years ago. The fact that some of these variants or strains are poorly or not infective to humans has resulted in a reappraisal of the public health significance of Echinococcus in areas where such variants occur. A revised taxonomy for species in the Echinococcus genus has been proposed that is generally accepted, and is based on the new molecular data and the biological and epidemiological characteristics of host-adapted species and strains.

  6. Changes of splenocyte lymphokines in ICR mice induced by recombinant Egmyophilin(rEgmyophilin)vaccine against Echinococcus granulosus%细粒棘球绦虫重组Egmyophilin蛋白诱导免疫小鼠脾细胞增殖及细胞因子水平的变化

    Institute of Scientific and Technical Information of China (English)

    马锐; 师志云; 王娅娜; 李宗吉; 孙俊峰; 赵巍

    2012-01-01

    To investigate the immunologic mechanism in mice immunized with recombinant Egmyophilin vaccine of E. Granulosus and against challenge with Egprotoscolecs, ICR mice were subcutaneously and intranasally vaccinated by the vaccine, following by the challenge with Egprotoscolices on the 8th week of vaccination and killed on the 20th week of infection to obtai spleen. Spleen cells were separated and the proliferation was determined by MTT method. Then the cells were collected, stained by PI Annexin V-FITC. The supernatant was collected and measure the level of IL-2, IL-10, IFN-γ and TNF-α by ELISA, and the blank vector and PBS were served as control. As demonstrated by the MTT assay, the recombinant Egmyophilin vaccine could stimulate specifically the proliferation of splenic T lymphocytes in mice. ELISA assay indicated that the level of IL-2, IFN-y and TNF-a in immunity group were significantly increased after stimulus with ConA or recombinant Egmyophilin. The results indicated that the recombinant Egmyophilin vaccination induces Thl cellular response in the mice a-gainst the challenge of Egprotoscoleces.%目的 探讨细粒棘球蚴绦虫(Echinococcus granulosus,Eg)亲肌肉抗原重组疫苗(Egmyophilin)诱导ICR小鼠产生免疫保护机制.方法 将亲肌肉抗原重组疫苗皮下注射免疫ICR小鼠,同时设空质粒组及PBS对照组.末次免疫后8 w,用棘球绦虫原头节腹腔注射进行攻击感染,感染后20 w剖杀小鼠,取脾,脾细胞悬液加入EgAg、ConA(刀豆蛋白A)或脂多糖(LPS)刺激培养后,四甲基偶氮唑盐比色法(MTT法)检测免疫小鼠T淋巴细胞增殖情况;Annexin V-FITC和碘化丙啶(PI)双染色法检测脾细胞的凋亡发生率;常规ELISA法检测脾细胞培养上清液中IL-2、IL-10、IFN-γ和TNF-α水平.结果 与空质粒组及PBS对照组相比,脾细胞凋亡发生率明显降低,脾T淋巴细胞增殖水平显著升高,脾细胞培养上清液中IL-2、IFN-γ和TNF-α水平显著增高,IL-10

  7. 细粒棘球绦虫重组Bb-Eg95疫苗诱导小鼠体液免疫应答的动态观察%Dynamic observation of humoral immune response in mice immunized with a recombinant Bb-Eg95 vaccine of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    周必英; 李文桂

    2012-01-01

    Objective To dynamically observe humoral immune response in mice immunized with a recombinant Bifidohacteria bifidum (Bb)-Eg95 vaccine against Echinococcus granulosus. Methods BALB/c mice were immunized orally or intranasally with the vaccine. In the 2nd, 4th , 6th , 8th, 10th, 12th, 14th , 16th, 18th, and 20th week after vaccination, sera were collected to determine levels of IgG, its subclass, and IgE using an enzyme-linked immunosorbent assay (ELISA) Results In the oral administration group, the level of IgG, IgG2a, IgG2b, IgGI, IgG3, and IgE peaked in the 8th, 6th, 6th, 6th , 6th , and 10th week after vaccination. In the intranasal vaccination group, the level of IgG, IgG2a, IgG2b, IgGl , IgG3, and IgE peaked in the 8th, 8th, 10th , 8th, 6th, and 10th week after vaccination. Conclusion A recombinant Bb-Eg95 vaccine against E. granulosus induced a specific humoral immune response in mice.%目的 观察细粒棘球绦虫(Eg)重组双歧杆菌(Bb)- Eg95疫苗免疫小鼠体液免疫应答的动态变化.方法 将疫苗分别采用经口灌胃和鼻内接种法免疫BALB/c鼠,分别于免疫后0、2、4、6、8、10、12、14、1 6、18和20周采血,分离血清,用ELISA法测定IgG及其亚类和IgE水平.结果 经口灌胃免疫组小鼠血清IgG、IgG2a、IgG2b、IgG1、IgG3和IgE水平分别在免疫后8、6、6、6、6和10周达最高水平;鼻内接种组小鼠血清IgG、IgG2a、IgG2b、IgG1、IgG3和IgE水平分别在免疫后8、8、10、8、6和10周达最高水平.结论 细粒棘球绦虫重组Bb-Eg95疫苗可诱导小鼠产生特异性体液免疫应答.

  8. Echinococcus granulosus 2 heat shock protein 70 gene: construction of recombinant plasmid, prokaryotic expression, purification and characterization%细粒棘球蚴2HSP70重组质粒的构建、原核表达、纯化和初步鉴定

    Institute of Scientific and Technical Information of China (English)

    丁淑琴; 王洁; 王淑静; 张焱; 赵巍

    2006-01-01

    目的 构建细粒棘球蚴(Echinococcus granulosus,EG)2热休克蛋白70(heat shock protein,2HSP70)表达重组质粒,原核表达及纯化重组蛋白,并对重组蛋白进行免疫学特性的初步鉴定.方法 从EG2HSP70/pGEM-T/JM109质粒中分离EG2HSP70目的基因,将此片段重组入表达载体pGEX-6P-1后转化入大肠杆菌BL21,鉴定后以IPTG诱导表达,通过SDS-PAGE检测其表达水平,用亲和层析纯化并分离重组蛋白,利用Western blot来鉴定重组蛋白的免疫学特性.结果 酶切鉴定及测序分析表明,成功构建细粒棘球蚴pGEX-6P-1/EG2HSP70重组质粒;IPTG诱导表达后,融合蛋白占菌体总蛋白的39%,占裂解物上清总蛋白的70.4%,表明重组的EG2HSP70基因能在BL21中高效表达,表达的蛋白大部分以可溶性状态存在.亲和层析法纯化重组蛋白EG2HSP70,通过Western blot证实该重组蛋白能被细粒棘球蚴囊壁免疫兔血清识别.结论 成功构建细粒棘球蚴中国大陆株pGEX-6P-1/EG2HSP70重组质粒,重组的EG2HSP70能够高效表达,表达的EG2HSP70具有免疫学活性.

  9. 细粒棘球绦虫硫氧还蛋白过氧化物酶基因的克隆、表达及鉴定%Cloning, fusion expression and identification of gene encoding the thioredoxin peroxidase from Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    王慧; 侯秋莲; 张壮志; 张富春; 张文宝

    2008-01-01

    目的 克隆细粒棘球绦虫(Echinococcus granulosus, Eg)硫氧还蛋白过氧化物酶(thioredoxin peroxidase TPx)基因,构建原核表达载体,通过诱导表达,纯化融合蛋白抗原,免疫小鼠制备抗血清.方法 从包囊中分离原头蚴, 抽提总RNA,采用RT-PCR的方法 扩增EgTPx基因,克隆到原核表达载体pET-41b中,转化大肠杆菌BL21,经过IPTG诱导目的基因表达,SDS-PAGE对表达蛋白进行分析,通过谷胱甘肽Sepharose4B层析柱分离纯化获得重组蛋白并免疫小鼠,制备抗血清.Western blot鉴定抗血清的特异性,ELISA测定抗体的效价.结果 用PCR方法扩增获得的EgTPx基因长度为582 bp,经酶切鉴定和序列测定,插入到原核表达载体的基因片段为EgTPx基因,SDS-PAGE结果显示表达融合蛋白相对分子质量约为54 kDa,鼠抗EgTPx抗体能与融合蛋白和天然原头蚴抗原发生特异性反应;间接ELISA法测定该抗体效价为1∶256 000.结论 成功地制备了鼠抗EgTPx抗体,并能够特异识别原核表达的融合蛋白EgTPx和天然原头蚴抗原,为研制有效的包虫病疫苗及相关诊断试剂盒奠定了基础.

  10. 细粒棘球蚴抗原B对体外培养人外周血单个核细胞中Th17细胞的影响%The influence of echinococcus granulosus antigen B on the proportion of Th17 cells in human peripheral blood mononuclear cells in vitro

    Institute of Scientific and Technical Information of China (English)

    金一帮; 周洋; 王俊华; 吐尔洪江·吐逊; 张雪; 温浩

    2012-01-01

    目的 观察细粒棘球蚴抗原B(AgB)对体外培养人外周血单个核细胞(PBMC)中CD4+ IL-17+T细胞(Th17)细胞的影响.方法 Ficoll法分离健康人群外周血中的PBMC并接种于24孔培养板,分为阴性对照组、低剂量抗原B(2 mg/L)实验组和高剂量抗原B实验组(5 mg/L)进行培养.分别在培养96、120和144 h后取样,通过流式细胞术(FCM)检测培养液中Th17细胞的阳性表达率.所得结果采用两组配对t检验.结果 Th17细胞的阳性表达率在低剂量和高剂量抗原B干预培养96 h后分别为0.433±0.115和0.500±0.265,较阴性对照组1.067±0.473均降低,差异有统计学意义(P<0.05),而在120 h和144h虽有降低趋势,但差异无统计学意义(P>0.05).结论 细粒棘球蚴抗原B对健康人外周血单个核细胞中Th17细胞的表达具有抑制作用,它可能与细粒棘球蚴所致免疫逃避机制有关.%Objective To observe the change of Thl7 cells in human peripheral blood mononuclear cells (PBMCs) stimulated by antigen B (AgB) in vitro.Methods The PBMCs were separated from blood by Ficoll density gradient centrifugation,cultured in a 24-well plate and randomly divided into low AgB concentration experimental group (2 mg/L),high AgB concentration experimental group (5 mg/L) and the control group.All the groups were collected at 96th,120th and 144th h and then detected by flow cytometry (FCM) to observe the expression of Th17.The results were analyzed by two sets of paired T test.Results According to the results,the proportion of Thl7 cells was decreased in human PBMCs stimulated by AgB (2 mg/L and 5 mg/L) at 96th,120th and 144th h.There was significant difference between experimental groups (0.433 ± 0.115 and 0.500 ± 0.265 ) and control group ( 1.067 ± 0.473 ) at 96th h ( P < 0.05 ).However,there was no statistically significant difference at 120th and 140th h ( P > 0.05 ).Conclusion Echinococcus granulosus AgB could inhibit human Th17 cells,suggesting that it may be

  11. High-throughput characterization of Echinococcus spp. metacestode miRNomes.

    Science.gov (United States)

    Cucher, Marcela; Macchiaroli, Natalia; Kamenetzky, Laura; Maldonado, Lucas; Brehm, Klaus; Rosenzvit, Mara Cecilia

    2015-03-01

    Echinococcosis is a worldwide zoonosis of great public health concern, considered a neglected disease by the World Health Organisation. The cestode parasites Echinococcus granulosus sensu lato (s. l.) and Echinococcus multilocularis are the main aetiological agents. In the intermediate host, these parasites display particular developmental traits that lead to different patterns of disease progression. In an attempt to understand the causes of these differences, we focused on the analysis of microRNAs (miRNAs), small non-coding regulatory RNAs with major roles in development of animals and plants. In this work, we analysed the small RNA expression pattern of the metacestode, the stage of sanitary relevance, and provide a detailed description of Echinococcus miRNAs. Using high-throughput small RNA sequencing, we believe that we have carried out the first experimental identification of miRNAs in E. multilocularis and have expanded the Echinococcus miRNA catalogue to 38 miRNA genes, including one miRNA only present in E. granulosus s. l. Our findings show that although both species share the top five highest expressed miRNAs, 13 are differentially expressed, which could be related to developmental differences. We also provide evidence that uridylation is the main miRNA processing mechanism in Echinococcus spp. These results provide detailed information on Echinococcus miRNAs, which is the first step in understanding their role in parasite biology and disease establishment and/or progression, and their future potential use as drug or diagnostic targets.

  12. 新疆阿尔泰山和西部天山牛源细粒棘球蚴原头节感染不同品系小鼠后发育情况的比较%A Comparative Observation on Infection of Different Strains of Mice with Protoscoleces of Bovine Origin Echinococcus granulosus From Altai Mountain Region and Western Tianshan Mountain Region of Xinjiang,China

    Institute of Scientific and Technical Information of China (English)

    伊斯拉音·乌斯曼; 亚德卡·吐尔逊; 焦伟; 严雷; 波拉提; 瞿群; 汤旭; 张文林; 柴君杰

    2001-01-01

    用新疆西部天山区域和阿尔泰山区域牛源细粒棘球蚴(Echinococcus granulosus E.g)原头节实验感染不同品系小鼠后的不同时期剖检,比较观察细粒棘球蚴在其体内的发育情况.结果为四种小鼠的继发性细粒棘球蚴囊在雌性鼠中的发育较雄性间的为快.在四种小鼠中,西部天山牛源E.g比阿尔泰山牛源E.g的发育较好,生长亦较快.感染后10个月,西部天山牛源E.g在鼠体内出现了发育成熟的原头节,而阿尔泰山牛源E.g到第12个月时仅有一只鼠出现发育成熟的原头节.表明西部天山牛源E.g与阿尔泰山牛源E.g在四种小鼠中的发育情况存在着明显的差别.

  13. Tegument Assembly and Secondary Envelopment of Alphaherpesviruses

    Directory of Open Access Journals (Sweden)

    Danielle J. Owen

    2015-09-01

    Full Text Available Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei.

  14. 细粒棘球蚴囊液对体外培养小鼠脾细胞Foxp3及Smad4基因表达的影响%The influence of Echinococcus granulosus fluid on the expression of Foxp3 and Smad4 genes of mouse spleen cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    周洋; 温浩; 金一帮; 王俊华; 吐尔洪江·吐逊; 魏晓丽; 张志; 张传山; 李亮; 林仁勇

    2011-01-01

    目的:观察细粒棘球蚴囊液对体外培养BABL/c小鼠脾脏细胞中调节性T细胞(Treg细胞)相对特异分子Foxp3(叉头蛋白3)及TGF-β1(转化生长因子-β1)下游信号通路Smad4表达的影响.方法:以BABL/c小鼠作为脾细胞供者,用研磨法分离脾脏细胞,随机分为实验组(与细粒棘球蚴囊液共同培养)和对照组,取1×10(5)个细胞接种于96孔板上,分别在1、3、6和12h提取RNA.实时荧光定量PCR(qRT-PCR)检测Foxp3以及Smad4的基因表达.结果:qRT-PCR显示,细粒棘球蚴囊液处理1、3和12h,实验组Foxp3表达量分别为4.577d±0.317、8.517±0.978和7.406±0.822,对照组分别为9.274±0.451、3.297±0.408和2.464±0.328,差异均有统计学意义(P<0.05);细粒棘球蚴囊液处理1h和12h,实验组Smad4表达量分别为3.862±1.417和1.690±0.248,对照组分别为1.689±0.221和3.600±1.081,差异有统计学意义(P<0.05),且Foxp3与Smad4二者之间存在负相关(γ=-0.991,P<0.05).结论:细粒棘球蚴囊液对小鼠脾脏细胞中Treg相对特异分子Foxp3有上调作用.而对TGF-pl的下游信号通路Smad4有下调的作用,二者之间存在负相关,提示细粒棘球蚴侵染宿主的过程中可能通过负调控TGF-β信号通路而造成宿主Treg细胞的表达升高,可能参与细粒棘球蚴感染过程中的免疫逃避.%Objective To observe the expression of Foxp3 and Smad4 in the spleens of BABL/c mice treated with Echinococcus granulosus fluid (EgF). Methods Spleen cells of BABL/c mice were isolated by grinding and randomly divided into an experimental group (treated with EgF) and a control group. Total RNA was extracted at 1, 3, 6, and 12 h, and the gene expression of Foxp3 and Smad4 was detected by real-time quantitative PCR (qRT-PCR). Results qRT-PCR results showed that the expression of Foxp3 at 1 h, 3 h, and 12 h was 4. 577±0. 317, 8. 517±0. 978, and 7. 406±0. 822 for the experimental group while expression of Foxp3 was 9.274±0.451, 3.297 ± 0.408, and 2

  15. Factors Influencing the Serological Response in Hepatic Echinococcus granulosus Infection

    Science.gov (United States)

    Lissandrin, Raffaella; Tamarozzi, Francesca; Piccoli, Luca; Tinelli, Carmine; De Silvestri, Annalisa; Mariconti, Mara; Meroni, Valeria; Genco, Francesca; Brunetti, Enrico

    2016-01-01

    Knowledge of variables influencing serology is crucial to evaluate serology results for the diagnosis and clinical management of cystic echinococcosis (CE). We analyzed retrospectively a cohort of patients with hepatic CE followed in our clinic in 2000–2012 to evaluate the influence of several variables on the results of commercial enzyme-linked immunosorbent assay (ELISA) and indirect hemagglutination (IHA) tests. Sera from 171 patients with ≥ 1 hepatic CE cyst, and 90 patients with nonparasitic cysts were analyzed. CE cysts were staged according to the WHO-IWGE classification and grouped by activity. A significant difference in ELISA optical density (OD) values and percentage of positivity was found among CE activity groups and with controls (P < 0.001). The serological response was also influenced by age (P < 0.001) and cyst number (P = 0.003). OD values and cyst size were positively correlated in active cysts (P = 0.001). IHA test showed comparable results. When we analyzed the results of 151 patients followed over time, we found that serology results were significantly influenced by cyst activity, size, number, and treatment ≤ 12 months before serum collection. In conclusion, serological responses as assessed by commercial tests depend on CE cyst activity, size and number, and time from treatment. Clinical studies and clinicians in their practice should take this into account. PMID:26503271

  16. Grading of liver lesions caused by Echinococcus granulosus

    Energy Technology Data Exchange (ETDEWEB)

    Rozanes, I. [Dept. of Radiology, Istanbul Medical Faculty, Istanbul (Turkey); Acunas, B. [Dept. of Radiology, Istanbul Medical Faculty, Istanbul (Turkey); Celik, L. [Dept. of Radiology, Haydarpasa Numune Hospital, Istanbul (Turkey); Acarli, K. [Hepatobiliary Surgery Unit, Dept. of General Surgery, Istanbul Medical Faculty (Turkey); Sayi, I. [Dept. of Radiology, Istanbul Medical Faculty, Istanbul (Turkey); Minareci, Oe. [Dept. of Radiology, Istanbul Medical Faculty, Istanbul (Turkey); Alper, A. [Hepatobiliary Surgery Unit, Dept. of General Surgery, Istanbul Medical Faculty (Turkey); Emre, A. [Hepatobiliary Surgery Unit, Dept. of General Surgery, Istanbul Medical Faculty (Turkey); Ariogul, O. [Hepatobiliary Surgery Unit, Dept. of General Surgery, Istanbul Medical Faculty (Turkey); Goekmen, E. [Dept. of Radiology, Istanbul Medical Faculty, Istanbul (Turkey)

    1993-10-01

    We have proposed a new scheme in which lesions are graded 1-5 according to the morphology of the cyst and the presence of findings in favour of cyst rupture. Ultrasound (US), CT and cholangiographic findings in 214 hydatid liver lesions in 152 patients who underwent surgery within 15 days of their radiological examinations were evaluated retrospectively. Results of the radiological classification were correlated with surgical data. We found that purely cystic unilocular lesions never ruptured, that dilatation of biliary canaliculi around a lesion with a complex morphological appearance is a non-specific finding and that there was no reliable radiological sign for a simple biliary communication. US, CT and cholangiography were reliable in diagnosing intra-biliary and direct ruptures. (orig.)

  17. A case of primary osseous pelvic hydatid disease (echinococcus granulosus)

    Energy Technology Data Exchange (ETDEWEB)

    Sinner, W.N. von (King Faisal Specialist Hospital and Research Centre, Riyadh (Saudi Arabia). Dept. of Radiology)

    1991-07-01

    Primary bone involvement in hydatid disease (HD) is rare. Sporadic reports estimate its prevalence to 1%. Only a few cases have been reported where CT has been used. Magnetic resonance imaging (MRI) of primary pelvic skeletal hydatid disease has, to my best knowledge, previously not been published. This report deals with a case of primary hydatid disease of the right ilium and the sacrum examined by plain film radiography. CT and MRI confirmed by pathology. (orig.).

  18. Echinococcus granulosus: in vitro effectiveness of warm water on protoscolices.

    Science.gov (United States)

    Moazeni, Mohammad; Alipour-Chaharmahali, Mohammad-Reza

    2011-01-01

    Hydatid disease is one of the most important helminthic diseases worldwide. Hydatid cysts may be found anywhere in the body. The most effective treatment of hydatid cyst is surgical operation. Spillage of live protoscolices during the operation is the major cause of recurrence. Instillation of scolicidal agent into hydatid cyst is the most commonly employed measure to prevent this complication. To date, many scolicidal agents have been used for inactivation of the hydatid cyst content, however, most common scolicidal agents may cause unacceptable side-effects, limiting their use. In this study the scolicidal effect of warm water (45, 50, 55, and 60 °C) at different exposure times (1, 2, 3, 4, 5, 6, 8, 10, 12, and 15 min) is investigated. Protoscolices were collected aseptically from sheep livers containing hydatid cyst. Viability of protoscolices was determined by 0.1% eosin staining. Even though the highest scolicidal activity of warm water at 45 °C was 40.4% at the end of 15 min, the best scolicidal effect (100%) of warm water at 50, 55, and 60 °C was obtained after 5, 2, and 1 min, respectively. The results of this in vitro study showed that warm water at 50-60 °C can be regarded as an effective scolicidal agent. Warm water is commonly available, easily prepared, and inexpensive. In vivo scolicidal activity of warm water and also the possible side effects need further investigation.

  19. Echinococcus and Taenia spp. from captive mammals in the United Kingdom.

    Science.gov (United States)

    Boufana, B; Stidworthy, M F; Bell, S; Chantrey, J; Masters, N; Unwin, S; Wood, R; Lawrence, R P; Potter, A; McGarry, J; Redrobe, S; Killick, R; Foster, A P; Mitchell, S; Greenwood, A G; Sako, Y; Nakao, M; Ito, A; Wyatt, K; Lord, B; Craig, P S

    2012-11-23

    Taeniid tapeworms which include Echinococcus and Taenia spp. are obligatory parasites of mammals with pathogenicity usually related to the larval stages of the life cycle. Two species (or genotypes) of Echinococcus, E. granulosus sensu stricto and E. equinus, as well as several Taenia spp. are endemic in the UK. Here we report on the occurrence of larval cystic stages of Echinococcus and Taenia spp. in captive mammals in the UK. Using molecular techniques we have identified E. granulosus (G1 genotype) in a guenon monkey and a Philippine spotted deer; E. equinus in a zebra and a lemur; E. ortleppi in a Philippine spotted deer; E. multilocularis in a macaque monkey and Taenia polyacantha in jumping rats. To the best of our knowledge this is the first report of E. multilocularis in a captive primate translocated to the UK. As far as we know these are the first reports of E. equinus in a primate (lemur) and in a zebra; as well as E. granulosus (G1 genotype) and E. ortleppi in a cervid translocated to the UK. These infections and implications of the potential establishment of exotic species of cestodes are discussed.

  20. Selection, Recombination and History in a Parasitic Flatworm (Echinococcus Inferred from Nucleotide Sequences

    Directory of Open Access Journals (Sweden)

    Haag KL

    1998-01-01

    Full Text Available Three species of flatworms from the genus Echinococcus (E. granulosus, E. multilocularis and E. vogeli and four strains of E. granulosus (cattle, horse, pig and sheep strains were analysed by the PCR-SSCP method followed by sequencing, using as targets two non-coding and two coding (one nuclear and one mitochondrial genomic regions. The sequencing data was used to evaluate hypothesis about the parasite breeding system and the causes of genetic diversification. The calculated recombination parameters suggested that cross-fertilisation was rare in the history of the group. However, the relative rates of substitution in the coding sequences showed that positive selection (instead of purifying selection drove the evolution of an elastase and neutrophil chemotaxis inhibitor gene (AgB/1. The phylogenetic analyses revealed several ambiguities, indicating that the taxonomic status of the E. granulosus horse strain should be revised

  1. Dynamic observation of the expression of indoleamine 2,3-dioxygenase on the surface of dendritic cells induced by Echinococcus granulosus antigens%细粒棘球蚴抗原诱导树突状细胞表达吲哚胺2,3-双加氧酶的动态观察

    Institute of Scientific and Technical Information of China (English)

    单骄宇; 李海涛; 吐尔洪江·吐逊; 肖晋; 李亮; 林仁勇; 温浩

    2012-01-01

    Objective To dynamically observe the expression of indoleamine 2, 3-dioxygenase(IDO) on the surface of dendritic cells (DCs) using different Echinococcus granulosus antigens. Methods Bone marrow DCs (BMDCs) from C57BL/6 mice were obtained in vitro. BMDCs were then treated with 15 μg/ml rAgB, 5 mg/ml MHF or 1 000 U/ml IFN-γ (as a positive control. The relative expression of IDO mRNA and IL-10 mRNA was dynamically measured with FQ-RT-PCR and the expression of IDO protein was determined with Western blot at 6 , 18, 24, 48, and 60 h. Results FQ-RT-PCR analysis showed that IDO was up-regulated 26. 8-fold at 24 h while IL-10 was markedly up-regulated 65. 1-fold at 48 h in rAgB-treated DCs. In MHF-treated DCs, IDO and IL-10 were up-regulated 12. 6-fold and 3. 9-fold at 24 h, respectively. In IDO protein detection, the expression of IDO increased in DCs treated with rAgB and MHF. In rAgB-treated DCs, the expression of IDO became evident at 24 h; in MHF-treated DCs, the expression of IDO became evident at 48 h. Conclusion rAgB and MHF up-regulate the expression of IDO. rAgB was better able to up-regulate IDO than was MHF. IDO is a molecule that regulates the immune response and can play a key role in the Th2 immune responsc. Results suggest that IDO may be involved in immune evasion and inhibition of the inflammatory response by echinococco-sis.%目的 动态检测细粒棘球蚴不同抗原体外诱导树突状细胞表达吲哚胺2,3-双加氧酶(IDO). 方法 在体外实验的条件下,获得C57BL/6小鼠骨髓来源的树突状细胞(BMDCs),分别应用15 μg/ml重组抗原B(rAgB)、5 mg/ml小鼠囊型包虫囊液(MHF)、1 000 U/ml IFN-γ(阳性对照)刺激BMDCs,在6、18、24、48、60h采用实时荧光定量RT-PCR动态监测IDO、IL-10 mRNA相对表达情况;在不同时间点收集各组DCs,应用Western blot检测IDO蛋白的表达.结果 FQ-RT-PCR显示,rAgB处理组IDO mRNA在24 h时上调26.8倍,IL-10 mRNA在48 h时上调65.1倍,MHF处理组干预24 h

  2. 细粒棘球蚴重组抗原B诱导小鼠骨髓源树突状细胞表达吲哚胺2,3-双加氧酶的研究%Echinococcus granulosus Recombinant Antigen B Induced IDO Expression in Mouse Bone Marrow-derived Dendritic Cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    单骄宇; 纪卫政; 吐尔洪江·吐逊; 李亮; 张传山; 林仁勇; 温浩

    2011-01-01

    目的 观察细粒棘球蚴重组抗原B (rAgB)体外诱导小鼠骨髓源树突状细胞表达吲哚胺2,3-双加氧酶(IDO)的情况.方法 从小鼠股骨中分离出骨髓细胞,进行小鼠重组巨噬细胞集落刺激因子(rmGM-CSF)的诱导,培养并获得CD11c+ 树突状细胞.应用倒置显微镜和扫描电镜观察树突状细胞形态,采用流式细胞术检测其表面标志物;用混合淋巴细胞反应(MLR)观察树突状细胞对T淋巴细胞的增殖能力.培养至第6天,收集未成熟树突状细胞进行流式细胞术检测;另向部分未成熟树突状细胞中加脂多糖(LPS)刺激24 h后,收集成熟树突状细胞,进行流式细胞术检测.在获得的未成熟树突状细胞中分别加入RPMI 1640完全培养液(为阴性对照组)、重组小鼠γ干扰素(rmIFN-γ,1 000 U/ml,为IFN-γ组)和rAgB(终浓度15 *9滋g/ml,为rAgB组),培养24 h后,通过细胞免疫组织化学和蛋白质印迹(Western blotting)检测各组树突状细胞IDO的表达情况.结果 获得纯度为80%的CD11c+ 树突状细胞,在倒置显微镜和扫描电镜下均观察到典型树突状细胞.经LPS刺激的成熟树突状细胞的CD40、CD80和主要组织相容性复合体(MHC)Ⅱ类分子I-A/I-E的阳性表达率与未成熟树突状细胞的相比,差异均有统计学意义(均P0.05).结论 细粒棘球蚴重组抗原B在体外具有诱导树突状细胞表达吲哚胺2,3-双加氧酶的功能.%Objective To observe the expression of indoleamine 2,3-dioxygenase (IDO) in mouse bone marrowderived dendritic cells (DCs) after adding Echinococcus granulosus recombinant antigen B (rAgB) in vitro. Methods CD11c+ DCs generated from bone marrow precursor cells of C57BL/6 mice and cultured in the presence of recombinant mouse GM-CSF (rmGM-CSF). The morphology of DCs was observed by inverted microscope and scanning electronic microscope. The level of I-A/I-E, CD40, CD80, and CD86 on DCs were determined by flow cytometry. T cell proliferation induced by

  3. Immunological response to recombinant Eg myophilin to protect against Echinococcus granulosus in mice%细粒棘球绦虫(中国大陆株)亲肌肉抗原重组蛋白诱导小鼠免疫应答的研究

    Institute of Scientific and Technical Information of China (English)

    于晶晶; 王娅娜; 赵巍

    2012-01-01

    目的 研究细粒棘球绦虫(中国大陆株)重组亲肌肉抗原(rEg.myophilin)的免疫保护作用及其机制,为包虫病分子疫苗的研制提供新的候选分子. 方法 (1)选取111只ICR小鼠随机分为A、B对照组和C实验组,分别于皮下注射PBS、PBS+佐剂和PBS+佐剂+候选疫苗,之后分别用同样的试剂进行两次加强免疫;(2)用Eg原头蚴进行攻击感染,感染后25周剖杀小鼠,检获棘球蚴包囊,计算免疫保护力;(3)按不同的时间点分批次对小鼠采血、取脾,用ELISA法测定血清中IgG及其亚型和IgE水平,并检测其细胞因子IFN-γ、IL-4和IL-10水平. 结果 rEg.myophilin能诱导小鼠产生≥86.11%的免疫保护力;免疫小鼠血清IgG、IgG1、IgG2a、IgE和细胞因子IFN-γ、IL-4水平均增高,对照组攻击感染后IL-10增高,实验组未增高. 结论 rEg.myophilin免疫后IgG及其亚类IgG1和IgE介导的体液免疫在抗细粒棘球蚴感染过程中发挥重要作用.IL-4在IgE亚型类别转换中起关键作用,这一机制可能直接影响IgE参与的嗜酸性粒细胞相关的抗寄生虫感染.%Objectives This study sought to investigate immunoprotection provided by recombinant Echinococcus granu-losus myophilin (rEg. myophilin) and tentatively analyze the mechanism of this protection. Methods One hundred and eleven male ICR mice were randomly divided into 3 groups, control groups A and B and experimental group C. Mice were subcutaneously injected with PBS; PBS and adjuvant; or PBS, adjuvant, and rEg. myophilin. After two boosters, mice were intraperitoneally challenged with Eg protoscoleces (PSCs), and then their humoral immune response was determined based on specific IgG, IgGl, IgG2a, IgG2b, IgG3, and IgE. Cellular immunity was also determined using IFN-γ, IL-4, and IL-10. Results Mice immunized with rEg. myophilin had effective immunoprotection (reduction in cyst load of more than 86. 11%). Mice immunized with rEg. myophilin had specific IgG, IgGl, IgG2a

  4. Synthesising 30 years of mathematical modelling of Echinococcus transmission.

    Directory of Open Access Journals (Sweden)

    Jo-An M Atkinson

    Full Text Available BACKGROUND: Echinococcosis is a complex zoonosis that has domestic and sylvatic lifecycles, and a range of different intermediate and definitive host species. The complexities of its transmission and the sparse evidence on the effectiveness of control strategies in diverse settings provide significant challenges for the design of effective public health policy against this disease. Mathematical modelling is a useful tool for simulating control packages under locally specific transmission conditions to inform optimal timing and frequency of phased interventions for cost-effective control of echinococcosis. The aims of this review of 30 years of Echinococcus modelling were to discern the epidemiological mechanisms underpinning models of Echinococcus granulosus and E. multilocularis transmission and to establish the need to include a human transmission component in such models. METHODOLOGY/PRINCIPAL FINDINGS: A search was conducted of all relevant articles published up until July 2012, identified from the PubMED, Web of Knowledge and Medline databases and review of bibliographies of selected papers. Papers eligible for inclusion were those describing the design of a new model, or modification of an existing mathematical model of E. granulosus or E. multilocularis transmission. A total of 13 eligible papers were identified, five of which described mathematical models of E. granulosus and eight that described E. multilocularis transmission. These models varied primarily on the basis of six key mechanisms that all have the capacity to modulate model dynamics, qualitatively affecting projections. These are: 1 the inclusion of a 'latent' class and/or time delay from host exposure to infectiousness; 2 an age structure for animal hosts; 3 the presence of density-dependent constraints; 4 accounting for seasonality; 5 stochastic parameters; and 6 inclusion of spatial and risk structures. CONCLUSIONS/SIGNIFICANCE: This review discusses the conditions under

  5. Detection of Echinococcus multilocularis in carnivores in Razavi Khorasan province, Iran using mitochondrial DNA.

    Directory of Open Access Journals (Sweden)

    Molouk Beiromvand

    2011-11-01

    Full Text Available BACKGROUND: Echinococcus multilocularis is the source of alveolar echinococcosis, a potentially fatal zoonotic disease. This investigation assessed the presence of E. multilocularis infection in definitive hosts in the Chenaran region of Razavi Khorasan Province, northeastern Iran. METHODOLOGY/PRINCIPAL FINDINGS: Fecal samples from 77 domestic and stray dogs and 14 wild carnivores were examined using the flotation/sieving method followed by multiplex PCR of mitochondrial genes. The intestinal scraping technique (IST and the sedimentation and counting technique (SCT revealed adult Echinococcus in the intestines of five of 10 jackals and of the single wolf examined. Three jackals were infected only with E. multilocularis but two, and the wolf, were infected with both E. multilocularis and E. granulosus. Multiplex PCR revealed E. multilocularis, E. granulosus, and Taenia spp. in 19, 24, and 28 fecal samples, respectively. Echinococcus multilocularis infection was detected in the feces of all wild carnivores sampled including nine jackals, three foxes, one wolf, one hyena, and five dogs (6.5%. Echinococcus granulosus was found in the fecal samples of 16.9% of dogs, 66.7% of jackals, and all of the foxes, the wolf, and the hyena. The feces of 16 (21.8% dogs, 7 of 9 (77.8% jackals, and all three foxes, one wolf and one hyena were infected with Taenia spp. CONCLUSIONS/SIGNIFICANCE: The prevalence of E. multilocularis in wild carnivores of rural areas of the Chenaran region is high, indicating that the life cycle is being maintained in northeastern Iran with the red fox, jackal, wolf, hyena, and dog as definitive hosts.

  6. The laminated layer: Recent advances and insights into Echinococcus biology and evolution.

    Science.gov (United States)

    Díaz, Álvaro; Fernández, Cecilia; Pittini, Álvaro; Seoane, Paula I; Allen, Judith E; Casaravilla, Cecilia

    2015-11-01

    The laminated layer is the unique mucin-based extracellular matrix that protects Echinococcus larvae, and thus to an important extent, shapes host-parasite relationships in the larval echinococcoses. In 2011, we published twin reviews summarizing what was known about this structure. Since then, important advances have been made. Complete genomes and some RNAseq data are now available for E. multilocularis and E. granulosus, leading to the inference that the E. multilocularis LL is probably formed by a single type of mucin backbone, while a second apomucin subfamily additionally contributes to the E. granulosus LL. Previously suspected differences between E. granulosus and E. multilocularis in mucin glycan size have been confirmed and pinned down to the virtual absence of Galβ1-3 chains in E. multilocularis. The LL carbohydrates from both species have been found to interact selectively with the Kupffer cell receptor expressed in rodent liver macrophages, highlighting the ancestral adaptations to rodents as intermediate hosts and to the liver as infection site. Finally, LL particles have been shown to possess carbohydrate-independent mechanisms profoundly conditioning non-liver-specific dendritic cells and macrophages. These advances are discussed in an integrated way, and in the context of the newly determined phylogeny of Echinococcus and its taenid relatives.

  7. Different Echinococcus granulosus Antigens Induced Indoleamine 2,3-dioxygenase Expression in Dendritic Cells%不同棘球蚴抗原诱导树突状细胞表达吲哚胺2,3-双加氧酶的实验研究

    Institute of Scientific and Technical Information of China (English)

    单骄宇; 李海涛; 李春燕; 肖晋; 李亮; 张雪; 林仁勇; 温浩

    2013-01-01

    Objective To observe the expression of indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) via different Echinococcus granulosus antigens in vitro.Methods Bone Marrow DCs generated from bone marrow precursor cells of C57BL/6 mice and cultured in the presence of recombinant mouse GM-CSF (rmGM-CSF).Then,DCs were induced with 15 μg/ml recombinant antigen B (rAgB),5 mg/ml mouse hydatid fluid (MHF),1 000 U/ml IFN-γ (as positive control),and RPMI 1640 complete medium (as negative control),respectively.Meanwhile,the treated DCs and cell supernatants were collected at 18,24 and 48 h after induction.The positive expressions of D40,CD80,CD86 and I-A/I-E on DCs were determined by flow cytometry.By real-time fluorescent quantitative reverse-transcription polymerase chain reaction (FQ-RT-PCR),the expression level of IDO mRNA in DCs was measured.Concentrations of tryptophan (Try) were tested by high-performance liquid chromatography (HPLC) assay in cell supernatant.Results The data from flow cytometry showed that the positive expressions of CD40,CD80,CD86,I-A/I-E were decreased after stimulated by rAgB and MHF.At 24 h after induction,there was significant difference in the level of CD40,CD86 and I-A/I-E among rAgB-treated group [(22.60±2.69)%,(35.50±4.38)%,(57.30±4.38)%],MHF-treated group [(38.00±3.54)%,(53.00± 3.39)%,(77.10±1.70)%] and negative control [(37.95±3.61) %,(19.55±1.06) % and (85.45±1.63) %](P<0.05).At 18,24 and 48h after induction,the levels of IDO mRNA in rAgB-treated group [(9.20t0.01),(29.44±0.02),(16.48±0.04)] and MHF-treated group [(9.67±0.02),(17.52±0.01),(16.81±0.01)] was higher than that of negative control group[(2.46±0.01),(7.77±0.01),and(10.56±0.01)] (P<0.01).And significant difference was found between rAgB-treated group and MHF-treated group (P<0.05).At 18,24 and 48 h after induction,the concentrations of Try were lowest in rAgB-treated group [(23.65±0.64),(13.95±1.06),(19.05±0.64) μmol/L].At 24h

  8. 细粒棘球绦虫重组Bb-Eg95-EgA31疫苗免疫小鼠后Th1/Th2型免疫应答的动态观察%Dynamic observation on Th1/Th2 type immune response in mice immunized with recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    周必英; 陈雅棠; 李文桂; 杨梅

    2010-01-01

    目的:动态观察细粒棘球绦虫(Echinococcus granulosus,Eg)重组双歧杆菌(Bb)-Eg95-EgA31疫苗免疫小鼠后Th1/Th2型免疫应答的变化.方法:将5×108克隆形成单位(CFU)和5×105CFU疫苗分别采用口服灌胃和鼻腔内接种免疫BALB/c鼠,在免疫后2、4、6、8、10、12、14、16、18和20周各剖杀4只小鼠,收集血清,用ELISA法测定血清IgG及其亚类和IgE水平;无菌取脾,用ELISA法检测脾淋巴细胞培养上清液中IFN-γ、IL-12、TNF-α和IL-10水平.结果:口服免疫组小鼠血清IgG、IgG2a 、IgG2b、IgG1、IgG3和IgE水平分别在免疫后8~10周、2~20周、2~20周、4~8周、6~12周和10周显著升高,脾淋巴细胞培养上清液中IFN-γ、IL-12、TNF-α和IL-10水平分别在免疫后2~16周、2~12周、2~6周和4~12周显著升高;鼻腔内接种组小鼠血清IgG、IgG2a 、IgG2b、IgG1、IgG3和IgE水平分别在免疫后4~10周、4~20周、2~20周、2~12周、4~12周和10~12周显著升高,脾淋巴细胞培养上清液中IFN-γ、IL-12、TNF-α和IL-10水平分别在免疫后2~8周、2~12周、2~8周和6~16周显著升高.结论:细粒棘球绦虫重组Bb-Eg95-EgA31疫苗可诱导小鼠产生1个Th1/Th2混合型免疫应答.口服免疫和鼻腔内接种是两种较好的免疫途径,且前者优于后者.

  9. Echinococcus multilocularis and Echinococcus canadensis in wolves from western Canada.

    Science.gov (United States)

    Schurer, Janna M; Gesy, Karen M; Elkin, Brett T; Jenkins, Emily J

    2014-02-01

    Echinococcus species are important parasites of wildlife, domestic animals and people worldwide; however, little is known about the prevalence, intensity and genetic diversity of Echinococcus tapeworms in Canadian wildlife. Echinococcus tapeworms were harvested from the intestines of 42% of 93 wolves (Canis lupus) from five sampling regions in the Northwest Territories, Manitoba and Saskatchewan, and visually identified to genus level by microscopic examination. Genetic characterization was successful for tapeworms from 30 wolves, and identified both Echinococcus canadensis and Echinococcus multilocularis in all sampling locations. Mixed infections of E. canadensis/E. multilocularis, as well as the G8/G10 genotypes of E. canadensis were observed. These findings suggest that wolves may be an important definitive host for both parasite species in western Canada. This represents the first report of wolves naturally infected with E. multilocularis in North America, and of wolves harbouring mixed infections with multiple species and genotypes of Echinococcus. These observations provide important information regarding the distribution and diversity of zoonotic species of Echinococcus in western North America, and may be of interest from public health and wildlife conservation perspectives.

  10. Surveillance for Echinococcus canadensis genotypes in Canadian ungulates.

    Science.gov (United States)

    Schurer, Janna; Shury, Todd; Leighton, Frederick; Jenkins, Emily

    2013-12-01

    The geographic and host distribution, prevalence and genotypes of Echinococcus canadensis in wild ungulates in Canada are described to better understand the significance for wildlife and public health. We observed E. canadensis in 10.5% (11/105) of wild elk (wapiti; Cervus canadensis) in Riding Mountain National Park, Manitoba, examined at necropsy, over two consecutive years (2010-2011). Molecular characterization of hydatid cyst material from these elk, as well as three other intermediate wildlife host species, was based on sequence of a 470 bp region of the NADH dehydrogenase subunit 1 (NAD1) mitochondrial gene. In moose [Alces alces], elk, and caribou [Rangifer tarandus] from northwestern Canada, the G10 genotype was the only one present, and the G8 genotype was detected in a muskox (Ovibos moschatus) from northeastern Canada. On a search of the national wildlife health database (1992-2010), cervids with hydatid cysts were reported in all provinces and territories except the Atlantic provinces, from which wolves [Canis lupis] are historically absent. Of the 93 cervids with records of hydatid cysts, 42% were elk, 37% were moose, 14% were caribou, and 6% were white-tailed and mule deer [Odocoileus virginianus and Odocoileus hemonius]. In these animals, 83% of cysts were detected in lungs alone, 8% in both lungs and liver, 3% in liver alone, and 6% in other organs. These observations can help target surveillance programs and contribute to a better understanding of ecology, genetic diversity, and genotype pathogenicity in the Echinococcus granulosus species complex.

  11. Mitochondrial phylogeny of the genus Echinococcus (Cestoda: Taeniidae) with emphasis on relationships among Echinococcus canadensis genotypes.

    Science.gov (United States)

    Nakao, Minoru; Yanagida, Tetsuya; Konyaev, Sergey; Lavikainen, Antti; Odnokurtsev, Valeriy A; Zaikov, Vladimir A; Ito, Akira

    2013-11-01

    The mitochondrial genomes of the genus Echinococcus have already been sequenced for most species and genotypes to reconstruct their phylogeny. However, two important taxa, E. felidis and E. canadensis G10 genotype (Fennoscandian cervid strain), were lacking in the published phylogeny. In this study, the phylogeny based on mitochondrial genome sequences was completed with these taxa. The present phylogeny highly supports the previous one, with an additional topology showing sister relationships between E. felidis and E. granulosus sensu stricto and between E. canadensis G10 and E. canadensis G6/G7 (closely related genotypes referred to as camel and pig strains, respectively). The latter relationship has a crucial implication for the species status of E. canadensis. The cervid strain is composed of two genotypes (G8 and G10), but the present phylogeny clearly suggests that they are paraphyletic. The paraphyly was also demonstrated by analysing the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) of E. canadensis genotypes from various localities. A haplotype network analysis using the short cox1 sequences from worldwide isolates clearly showed a close relatedness of G10 to G6/G7. Domestic and sylvatic life cycles based on the host specificity of E. canadensis strains have been important for epidemiological considerations. However, the taxonomic treatment of the strains as separate species or subspecies is invalid from a molecular cladistic viewpoint.

  12. Echinococcus metacestodes as laboratory models for the screening of drugs against cestodes and trematodes.

    Science.gov (United States)

    Hemphill, A; Stadelmann, B; Scholl, S; Müller, J; Spiliotis, M; Müller, N; Gottstein, B; Siles-Lucas, M

    2010-03-01

    Among the cestodes, Echinococcus granulosus, Echinococcus multilocularis and Taenia solium represent the most dangerous parasites. Their larval stages cause the diseases cystic echinococcosis (CE), alveolar echinococcosis (AE) and cysticercosis, respectively, which exhibit considerable medical and veterinary health concerns with a profound economic impact. Others caused by other cestodes, such as species of the genera Mesocestoides and Hymenolepis, are relatively rare in humans. In this review, we will focus on E. granulosus and E. multilocularis metacestode laboratory models and will review the use of these models in the search for novel drugs that could be employed for chemotherapeutic treatment of echinococcosis. Clearly, improved therapeutic drugs are needed for the treatment of AE and CE, and this can only be achieved through the development of medium-to-high throughput screening approaches. The most recent achievements in the in vitro culture and genetic manipulation of E. multilocularis cells and metacestodes, and the accessability of the E. multilocularis genome and EST sequence information, have rendered the E. multilocularis model uniquely suited for studies on drug-efficacy and drug target identification. This could lead to the development of novel compounds for the use in chemotherapy against echinococcosis, and possibly against diseases caused by other cestodes, and potentially also trematodes.

  13. Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

    Science.gov (United States)

    Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

    2014-01-01

    In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the

  14. Taxonomy, phylogeny and molecular epidemiology of Echinococcus multilocularis: From fundamental knowledge to health ecology.

    Science.gov (United States)

    Knapp, Jenny; Gottstein, Bruno; Saarma, Urmas; Millon, Laurence

    2015-10-30

    Alveolar echinococcosis, caused by the tapeworm Echinococcus multilocularis, is one of the most severe parasitic diseases in humans and represents one of the 17 neglected diseases prioritised by the World Health Organisation (WHO) in 2012. Considering the major medical and veterinary importance of this parasite, the phylogeny of the genus Echinococcus is of considerable importance; yet, despite numerous efforts with both mitochondrial and nuclear data, it has remained unresolved. The genus is clearly complex, and this is one of the reasons for the incomplete understanding of its taxonomy. Although taxonomic studies have recognised E. multilocularis as a separate entity from the Echinococcus granulosus complex and other members of the genus, it would be premature to draw firm conclusions about the taxonomy of the genus before the phylogeny of the whole genus is fully resolved. The recent sequencing of E. multilocularis and E. granulosus genomes opens new possibilities for performing in-depth phylogenetic analyses. In addition, whole genome data provide the possibility of inferring phylogenies based on a large number of functional genes, i.e. genes that trace the evolutionary history of adaptation in E. multilocularis and other members of the genus. Moreover, genomic data open new avenues for studying the molecular epidemiology of E. multilocularis: genotyping studies with larger panels of genetic markers allow the genetic diversity and spatial dynamics of parasites to be evaluated with greater precision. There is an urgent need for international coordination of genotyping of E. multilocularis isolates from animals and human patients. This could be fundamental for a better understanding of the transmission of alveolar echinococcosis and for designing efficient healthcare strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Effect of Echinococcus granulosus cyst fluid on mitogen-activated protein kinases and the cell cycle of host cells in vitro%细粒棘球蚴囊液对HepG2肝癌细胞系细胞周期的影响

    Institute of Scientific and Technical Information of China (English)

    李朝旺; 赵晋明; 张传山; 吕国栋; 王俊华; 李静; 范金亮; 温浩; 林仁勇

    2012-01-01

    目的 探讨细粒棘球蚴(Eg)囊液(HCF)在感染急性期对宿主肝细胞MAPK信号通路及细胞周期的影响.方法 常规方法培养HepG2细胞,以HCF为刺激物,于不同时间点用Western blot检测p-ERK、增殖细胞核抗原PCNA、细胞周期蛋白cyclin-D1、A、B1和E的表达水平,并与无FCS组比较. 结果 Western blot检测显示,15%、30%HCF组p-ERK分别在换液培养3h和30 min高表达(P<0.05),PCNA分别在3h和12h高表达(P<0.05),cyclin-D1在3h高表达(P<0.05),cyclin-A分别在3h和12h高表达(P<0.05),15%HCF组cyclin-E在3h高表达(P<0.05),15%、30% HCF组cyclin-B1在各时间点表达量微弱(P<0.05). 结论 HCF对体外培养的HepG2细胞无毒害,且对其增殖有一定的促进作用.%Objective To discuss the effect of Echinocuccus granulosus cyst fluid on mitogen-activated protein kinases and the cell cycle of host cells in the acute phase of infection. Methods HepG2 cells were cultured normally and stimulated with hydatid cyst fluid (HCF). Western blot indicated the expression of p-ERK, PCNA, cyclin-A, cyclinBl, cyc-lin Dl, and cyclin-E at different times and results were compared to those of cells not in the presence of FCS. Results Western blot showed that cells stimulated with 15% HCF and 30% HCF had a high level of p-ERK expression at 3 h and 30 min (P<0. 05), a high level of PCNA expression at 3 h and 12 h (P<0. 05), a high level of cyclin-Dl expression at 3 h (P<0. 05), and a high level of cyclin-A expression at 3 h and 12 h (P<0. 05). Cells stimulated with 15%HCF had a high level of cyclin-E expression at 3 h (P<0. 05). Cells stimulated with 15%HCF and 30%HCF had a low level of cy-clin-Bl expression at every test lime (P<0. 05). Conclusion HCF did not inhibit primary cultured host cells but did promote the proliferation of host cells.

  16. A sylvatic lifecycle of Echinococcus equinus in the Etosha National Park, Namibia

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    Wassermann, Marion; Aschenborn, Ortwin; Aschenborn, Julia; Mackenstedt, Ute; Romig, Thomas

    2014-01-01

    Various species of Echinococcus have been described in the past from wild mammals of sub-Saharan Africa. However, it is only recently, that a few isolates have become available for molecular identification; therefore, the involvement of wildlife in the lifecycles of the various cryptic species within Echinococcus granulosus sensu lato is still only partially known. A preliminary survey was undertaken in Etosha National Park, Namibia, from August to October 2012. Faecal samples were obtained from 34 individual wild carnivores, and metacestodes were collected from carcasses of 18 culled herbivores. Single eggs and metacestode tissue were lysed and identified from sequences of the mitochondrial nad1 gene. In case of metacestodes, the cox1 gene was additionally sequenced and haplotype networks were constructed. Echinococcus equinus was found in lions (4 of 6), black-backed jackals (2 of 7) and Burchell's zebras (11 of 12). The frequency of this parasite in the absence of domestic dogs, horses and donkeys strongly indicates its transmission in a wildlife cycle. Further, a variety of sequences were obtained from eggs and cysticerci from lions, cheetahs, caracals, spotted hyenas and oryx, which most closely clustered with species of Taenia. Only 3 of them, 2 of lion and 1 of hyena origin, could be allocated to Hydatigera (=Taenia) taeniaeformis (lion), Taenia regis (lions and oryx) and Taenia cf. crocutae (spotted hyena and oryx). PMID:25830103

  17. Investigation on the occurrence of Echinococcus multilocularis in Central Italy

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    Grifoni Goffredo

    2009-11-01

    Full Text Available Abstract Background Recent studies on geographic distribution of Echinococcus multilocularis in Europe show that it has a wider range than previously thought. It is unclear, however, if the wider distribution is due to its recent spreading or to a lack of previous data from the new areas. Italy, previously considered E. multilocularis-free, is now part of these new areas: infected foxes (the main definitive host of the tapeworm have been observed in a Northern Alpine territory. Thus, more surveys need to be done in other Italian regions in order to monitor the spreading of E. multilocularis. The aim of the present study was to look for this parasite in 283 foxes collected in an Apennine area of Central Italy by different diagnostic methods. Results The foxes were heavily parasitized by 11 helminthic genera, but none of the animals was infected by E. multilocularis neither by E. granulosus (harboured adult worms or their DNA. Low specificity was observed in commercially available ELISA kits for the detection of E. multilocularis antigens in the faeces. Molecular diagnostics were sensitive and specific for the detection and identification of tapeworm eggs in faeces, but less sensitive, although specific, to adult tapeworms in the intestinal content. Conclusion Preliminarily, we can say that no E. multilocularis could be found in the study area. These data will enable us to follow temporal changes of the spatial distribution of the parasite in the study area of the Central Apennines. Due to its low specificity the ELISA kit for E. multilocularis coproantigens is not suitable for epidemiological surveys, whereas molecular diagnostics applied to faecal samples give useful results. Finally, absence of E. granulosus in foxes living in the endemic areas studied confirms the thought that this tapeworm prefers a different definitive host.

  18. Surveillance for Echinococcus canadensis genotypes in Canadian ungulates☆

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    Schurer, Janna; Shury, Todd; Leighton, Frederick; Jenkins, Emily

    2013-01-01

    The geographic and host distribution, prevalence and genotypes of Echinococcus canadensis in wild ungulates in Canada are described to better understand the significance for wildlife and public health. We observed E. canadensis in 10.5% (11/105) of wild elk (wapiti; Cervus canadensis) in Riding Mountain National Park, Manitoba, examined at necropsy, over two consecutive years (2010–2011). Molecular characterization of hydatid cyst material from these elk, as well as three other intermediate wildlife host species, was based on sequence of a 470 bp region of the NADH dehydrogenase subunit 1 (NAD1) mitochondrial gene. In moose [Alces alces], elk, and caribou [Rangifer tarandus] from northwestern Canada, the G10 genotype was the only one present, and the G8 genotype was detected in a muskox (Ovibos moschatus) from northeastern Canada. On a search of the national wildlife health database (1992–2010), cervids with hydatid cysts were reported in all provinces and territories except the Atlantic provinces, from which wolves [Canis lupis] are historically absent. Of the 93 cervids with records of hydatid cysts, 42% were elk, 37% were moose, 14% were caribou, and 6% were white-tailed and mule deer [Odocoileus virginianus and Odocoileus hemonius]. In these animals, 83% of cysts were detected in lungs alone, 8% in both lungs and liver, 3% in liver alone, and 6% in other organs. These observations can help target surveillance programs and contribute to a better understanding of ecology, genetic diversity, and genotype pathogenicity in the Echinococcus granulosus species complex. PMID:24533321

  19. Echinococcus multilocularis laminated-layer components and the E14t 14-3-3 recombinant protein decrease NO production by activated rat macrophages in vitro.

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    Andrade, M Amparo; Siles-Lucas, Mar; Espinoza, Elsa; Pérez Arellano, José Luis; Gottstein, Bruno; Muro, Antonio

    2004-05-01

    Echinococcus multilocularis and Echinococcus granulosus cause alveolar and cystic (unilocular) echinococcosis, respectively, in humans and animals. It is known that these parasites can affect, among other molecules, nitric oxide (NO) production by periparasitic host cells. Nevertheless, detailed dissection of parasite components specifically affecting cell NO production has not been done to date. We compare the effect of E. granulosus and E. multilocularis defined metacestode structural (laminated-layer associated) and metabolic (14-3-3 protein, potentially related with E. multilocularis metacestode tumor-like growth) components on the NO production by rat alveolar macrophages in vitro. Our results showed that none of these antigens could stimulate macrophage NO production in vitro. However, a reversed effect of some Echinococcus antigens on NO in vitro production was found when cells were previously exposed to LPS stimulation. This inhibitory effect was found when E. multilocularis laminated-layer (LL) or cyst wall (CW) soluble components from both species were used. Pre-stimulation of cells with LPS also resulted in a strong, dose-dependent reduction of NO and iNOS mRNA production after incubation of cells with the E14t protein. Thus, the E. multilocularis 14-3-3 protein appears to be one of the components accounting for the suppressive effect of the CW and LL metacestode extracts.

  20. [First report of Echinococcus vogeli in a paca in Misiones province, Argentina].

    Science.gov (United States)

    Vizcaychipi, Katherina A; Helou, Marcia; Dematteo, Karen; Macchiaroli, Natalia; Cucher, Marcela; Rosenzvit, Mara; D'Alessandro, Antonio

    2013-01-01

    We report the first finding of Echinococcus vogeli in a paca, Cuniculus paca, in the tropical forest of Misiones, in the north of Argentina. The presence of the bush dog, Speothos venaticus, E. vogelís only natural definitive host, was also reported. The polycystic hydatids, 2 to 3 cm in diameter, were only found in the liver of an adult paca. The size range of the hooks and the relative proportion blade/handle did not show significant differences with respect to the ones reported for E. vogeli. The size of E. granulosus hooks, measured for comparison purposes, was significantly smaller (p E. vogeli in Argentina. The probability of finding neotropical echinococcosis in humans reinforces the need to expand the search for E. vogeli in Argentina. Echinococcosis due to E. vogeli is very aggressive and may cause death in about a third of the human population affected.

  1. Latent-class methods to evaluate diagnostics tests for Echinococcus infections in dogs.

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    Sonja Hartnack

    Full Text Available BACKGROUND: The diagnosis of canine echinococcosis can be a challenge in surveillance studies because there is no perfect gold standard that can be used routinely. However, unknown test specificities and sensitivities can be overcome using latent-class analysis with appropriate data. METHODOLOGY: We utilised a set of faecal and purge samples used previously to explore the epidemiology of canine echinococcosis on the Tibetan plateau. Previously only the purge results were reported and analysed in a largely deterministic way. In the present study, additional diagnostic tests of copro-PCR and copro-antigen ELISA were undertaken on the faecal samples. This enabled a Bayesian analysis in a latent-class model to examine the diagnostic performance of a genus specific copro-antigen ELISA, species-specific copro-PCR and arecoline purgation. Potential covariates including co-infection with Taenia, age and sex of the dog were also explored. The dependence structure of these diagnostic tests could also be analysed. PRINCIPLE FINDINGS: The most parsimonious result, indicated by deviance-information criteria, suggested that co-infection with Taenia spp. was a significant covariate with the Echinococcus infection. The copro-PCRs had estimated sensitivities of 89% and 84% respectively for the diagnoses of Echinococcus multilocularis and E. granulosus. The specificities for the copro-PCR were estimated at 93 and 83% respectively. Copro-antigen ELISA had sensitivities of 55 and 57% for the diagnosis of E. multilocularis and E. granulosus and specificities of 71 and 69% respectively. Arecoline purgation with an assumed specificity of 100% had estimated sensitivities of 76% and 85% respectively. SIGNIFICANCE: This study also shows that incorporating diagnostic uncertainty, in other words assuming no perfect gold standard, and including potential covariates like sex or Taenia co-infection into the epidemiological analysis may give different results than if the

  2. First detection of Echinococcus multilocularis in dogs in a highly endemic area of Poland.

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    Karamon, Jacek; Samorek-Pierog, Malgorzata; Kochanowski, Maciej; Dabrowska, Joanna; Sroka, Jacek; Golab, Elzbieta; Umhang, Gerald; Cencek, Tomasz

    2016-06-02

    The aim of the investigation was to estimate the epizootic situation concerning infection by the cestode Echinococcus multilocularis Leuckart, 1863 in dogs (Canis lupus familiaris Linnaeus) from a Polish region where this parasite is highly prevalent in red foxes. Faecal samples (n = 148) were collected from rural dogs in Podkarpackie Province. Samples were examined through nested PCR (for E. multilocularis), multiplex PCR (E. multilocularis, species of Taenia Linnaeus, 1758) and PCR [E. granulosus (Batsch, 1786)]. Specific products were sequenced. Faeces were also examined coproscopically. In samples from two dogs (1.4%), there were positive PCR results for E. multilocularis. Taenia-specific PCR products were found in nine dogs (6.1%). Sequencing identified Taenia serialis (Gervais, 1847), T. hydatigena Pallas, 1766, T. pisiformis (Bloch, 1780) and Hydatigera taeniaeformis (Batsch, 1786). One sample (0.7%) was identified as Mesocestoides litteratus (Batsch, 1786). All samples were negative for E. granulosus with PCR. Taking into account coproscopic and PCR results, 28% of dogs were infected with helminths (8% with tapeworms). It should be stressed that one of the infected with E. multilocularis dogs shed eggs of the Taenia type and had a habit of preying on rodents. This investigation revealed the presence of E. multilocularis in dogs for the first time in Poland.

  3. Impact of anthropogenic and natural environmental changes on Echinococcus transmission in Ningxia Hui Autonomous Region, the People’s Republic of China

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    Yang Yu

    2012-07-01

    Full Text Available Abstract Echinococcus transmission is known to be affected by various environmental factors, which may be modified by human influence or natural events including global warming. Considerable population growth in the last fifty years in Ningxia Hui Autonomous Region (NHAR, the People’s Republic of China (PRC, has led to dramatic increases in deforestation and modified agricultural practices. In turn, this has resulted in many changes in the habitats for the definitive and intermediate hosts of both Echinococcus granulosus and E. multilocularis, which have increased the risks for transmission of both parasites, affecting echinococcosis prevalence and human disease. Ecological environmental changes due to anthropogenic activities and natural events drive Echinococcus transmission and NHAR provides a notable example illustrating how human activity can impact on a parasitic infection of major public health significance. It is very important to continually monitor these environmental (including climatic factors that drive the distribution of Echinococcus spp. and their impact on transmission to humans because such information is necessary to formulate reliable future public health policy for echinococcosis control programs and to prevent disease spread.

  4. In vitro treatments of Echinococcus granulosus with fungal chitosan, as a novel biomolecule

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    Bahman Rahimi-Esboei; Mahdi Fakhar; Aroona Chabra; Mahboobeh Hosseini

    2013-01-01

    Objective: To determined the antiparasitic activity of the isolated chitosan from Penicillium viridicatum, Penicillium aurantiogriseum and commercial chitosan against protoscolicidal of hydatid cysts were determined. Methods:After isolating chitosan from fungal cell walls, four concentrations (50, 100, 200, 400μg/mL) of each type of prepared chitosan and commercial chitosan were used for 10, 30, 60, and 180 min, respectively. Results: Among different type of chitosan, commercial chitosan with the highest degree of deacetylation showed high scolicidal activity in vitro. Fungal chitosan could be recommended, as good as commercial chitosan, for hydatic cysts control. Conclusions:It seems to be a good alternative to synthetic and chemical scolicidal.

  5. Purification of a Recombinant Glutathione Transferase from the Causative Agent of Hydatidosis, "Echinococcus granulosus"

    Science.gov (United States)

    Fleitas, Andrea L.; Randall, Lía M.; Möller, Matías N.; Denicola, Ana

    2016-01-01

    This practical class activity was designed to introduce students to recombinant protein expression and purification. The principal goal is to shed light on basic aspects concerning recombinant protein production, in particular protein expression, chromatography methods for protein purification, and enzyme activity as a tool to evaluate purity and…

  6. Histochemistry and histoenzymology of the hydatid cyst (Echinococcus granulosus Batsch, 1786). II. Scolices and brood capsules.

    Science.gov (United States)

    Reissenweber, N J; Vercelli-Retta, J; Siri, A M; Lozano, W

    1975-12-11

    Scolices and brood capsules of healthy hydatid cysts from lungs of human patients were studied with histochemical and histoenzymatic methods. The subtegumental and flame cells were sepcially rich in glycogen, RNA and some dehydrogenases such as SDH, MDH, NADH-reductase and G-6-PDH. The rostellar zone or invaginated pole, an area of marked contractile movements, showed intense activity in ATP'ase and simple esterase. The so-called excretory pole shows strong activity in simple esterases, lipase, beta-HBH, alpha-GDH and NADPH-reductase. Lipids are also abundant in this zone implying the important role of this metabolic path in the development of the parasite. Intense activity in alkaline phosphatase was observed in cells associated to the calcereous corpuscles. The largest corpuscles were devoid of enzymatic activity. The enzyme could play some role in the calcification of the corpuscles. Wide enzymatic variations are described according to morphology being orthoscolices the most rich in enzyme activity. Accumulations of small cells surrounded by specialized cells on the germinal membrane are interpreted as the origin or "embryo" of brood capsules. Some enzymes detected in the wall of mature brood capsules depicted alternating types of cells. Some of them are positive for ATP'ase that may be related to active transport of substances across the brood capsule wall. The intenst ATP'ase activity at the stalks of scolices may be similarly interpreted. However, a miosine-like activity is a more feasible explanation since this area showed striking contractile movements in vivo.

  7. Echinococcus granulosus infection in humans and livestock in the Coquimbo region, north-central Chile.

    Science.gov (United States)

    Acosta-Jamett, Gerardo; Cleaveland, Sarah; Cunningham, Andrew A; Bronsvoort, Barend M deC; Craig, Philip S

    2010-04-19

    Cyst echinococcosis (CE) is one of the most important zoonosis in Chile, where studies have focussed mainly in moist southern regions. The present study was conducted to determine the prevalence of cystic echinococcosis (CE) in livestock and humans in the semiarid Coquimbo region in north-central Chile. A review of all surgical cases of CE in humans reported in the Elqui, Limarí and Choapa provinces in Coquimbo region for the period comprising 1995-2006 was obtained. In addition, a retrospective study of CE covering condemnation records from slaughterhouses of these provinces from the same period was carried out. The surgical incidence of CE in humans ranged between 2.3 and 8.5 cases per 10(5) people, with more cases reported in provinces with a higher percentage of rural inhabitants (Limarí and Choapa). A total of 174,034 cattle, 22,208 goats, 35,404 sheep, 25,355 swine and 9391 equines were examined during the period. Higher prevalence of CE was detected in cattle (24%), followed by swine (14%), sheep (11%), goats (6%), and equines (9%). More cases of CE in livestock were also found in provinces with higher rural population. The overall trend of the prevalence of CE for each livestock species across years was a significant downward one, except for swine in Elqui and sheep and swine in Choapa. Cattle showed higher prevalence of CE in liver in Elqui, in kidney in Limarí and in lungs in Choapa. Swine showed similar prevalence by organs in all provinces. Sheep showed higher prevalence of CE in liver in Elqui and Limarí and lower prevalence in Choapa. Goats presented higher prevalence of CE in kidney in all provinces, and equines had higher prevalence of CE in liver in the provinces where animals were slaughtered. Further studied are needed to assess whether these differences are due to different strains affecting these species or due to ecological factors. When analyzing the CE prevalence of each species within each province, a negative correlation between prevalence of CE in goats and rainfall in the Limarí province was found. This could be attributed to changes in management practices and/or ecological factors. This study shows that surveillance of CE at slaughterhouses combined with the analyses of incidence in humans can be used to detect areas with a higher risk of infection. Improvements in record-keeping of infected animals at slaughterhouses are proposed in order to trace back farms where infection was most likely acquired.

  8. Proteomic analysis of the shistosome tegument and its surface membranes

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    Simon Braschi

    2006-10-01

    Full Text Available The tegument surface of the adult schistosome, bounded by a normal plasma membrane overlain by a secreted membranocalyx, holds the key to understanding how schistosomes evade host immune responses. Recent advances in mass spectrometry (MS, and the sequencing of the Schistosoma mansoni transcriptome/genome, have facilitated schistosome proteomics. We detached the tegument from the worm body and enriched its surface membranes by differential extraction, before subjecting the preparation to liquid chromatography-based proteomics to identify its constituents. The most exposed proteins on live worms were labelled with impearmeant biotinylation reagents, and we also developed methods to isolate the membranocalyx for analysis. We identified transporters for sugars, amino acids, inorganic ions and water, which confirm the importance of the tegument plasma membrane in nutrient acquisition and solute balance. Enzymes, including phosphohydrolases, esterases and carbonic anhydrase were located with their catalytic domains external to the plasma membrane, while five tetraspanins, annexin and dysferlin were implicated in membrane architecture. In contrast, few parasite proteins could be assigned to the membranocalyx but mouse immune response proteins, including three immunoglobulins and two complement factors, were detected, plus host membrane proteins such as CD44, integrin and a complement regulatory protein, testifying to the acquisitive properties of the secreted bilayer.

  9. [Tegumental ultrastructures of Paragonimus iloktsuenensis according to the developmental stages].

    Science.gov (United States)

    Lee, S H; Kim, S J; Chai, J Y; Sohn, W M

    1989-03-01

    A scanning electron microscopic study was performed to observe the tegumental ultrastructures of Paragonimus iloktsuenensis according to its developmental stages. The metacercariae were obtained from the liver of the brackish water crab, Sesarma dehaani. Juvenile and adult P. iloktsuenensis were recovered from the experimental rats on 2, 4 and 8 weeks after infection. The findings were summarized as follows: 1. The excysted metacercariae were characteristically gourd-shape, with their whole body surface beset with numerous spade-shape spines. The large, type II sensory papillae (non-ciliated round swellings) were arranged along the rim of the oral and ventral suckers, 11-12 and 6-8 in numbers respectively. 2. Two-week old juvenile worms, recovered chiefly from the liver of the experimental rats, were slender in body shape, with their ventral sucker near the anterior one-third level. The distribution of tegumental spines was less dense than in the excysted metacercariae. The spines were with 1-2 pointed tips and 3-4 longitudinal splits. Numerous ciliated knob-like, type I papillae were observed in both sides of the oral sucker, and 6 large, type II papillae were arranged along the rim of the ventral sucker. 3. Four-week old worms, recovered from the thoracic cavity and/or lung parenchyme of the experimental rats, were thicker than wide in body configuration, and their ventral sucker was located near the anterior one-fourth level. The tegumental spines at ventral surface were grouped, each group with 3-5 aggregated ones. The type I and type II papillae (small-sized) were distributed chiefly around the rim of two suckers. 4. Adult (eight-week old) worms, recovered from the capsules in the lung parenchyme, were very stout, and covered densely with bearfoot-like spines. At dorsal surface, cobblestone-like cytoplasmic processes were well-developed, with many tegumental spines embedded in them. It was observed in this study that the tegument of P. iloktsuenesis continued

  10. Ultrastructure of the tegument of Prosorhynchoides arcuatus (Linton, 1900 Bray, 1984 (Trematoda, Bucephalidae

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    Simone C Cohen

    1996-08-01

    Full Text Available The tegument of the adult form of Prosorhynchoides arcuatus (Linton, 1900 (Trematoda, Bucephalidae from the intestine of Pomatomus saltator (L. from the Atlantic coast of the State of Rio de Janeiro, Brazil was studied by transmission electron microscopy. The tegument consists of two layers: an external, constituted by a syncytium, containing spines, unicilliated papillae, inclusion bodies and mitochondria and an internal, consisting of a muscular layer and mononucleated tegumental cells.

  11. Molecular characterization of Echinococcus isolates indicates goats as reservoir for Echinococcus canadensis G6 genotype in Neuquén, Patagonia Argentina.

    Science.gov (United States)

    Soriano, S V; Pierangeli, N B; Pianciola, L; Mazzeo, M; Lazzarini, L E; Saiz, M S; Kossman, A V; Bergagna, H F J; Chartier, K; Basualdo, J A

    2010-12-01

    Human cystic echinococcosis is a highly endemic zoonotic disease in the province of Neuquén, Patagonia Argentina, although a hydatid control programme has been carried out since 1970. Human infection due to Echinococcus canadensis (G6 genotype) is frequent in Neuquén. However, the reservoir for this species remains undetermined in a region where camels are absent. We investigated the fertility, viability and molecular epidemiology of hydatid cysts obtained from local goats, pigs and sheep in order to identify the possible reservoirs of E. canadensis (G6). We also analyzed isolates from infected dogs. A total of 67 isolates were identified by the DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene. Cysts from sheep (n=16), goats (n=23) and pigs (n=18) and adult worms from 10 infected dogs were analyzed. The fertility of the hydatid cysts was 78.6%; 90.4% and 94.4% for sheep, goats and pigs, respectively. We detected E. canadensis (G6) in 21 of 23 goat samples and in 1 dog isolate, E. canadensis (G7) in all the pig isolates, E. granulosus sensu stricto (G3) in 1 sheep and the G1 genotype in 15 sheep, 2 goats and 9 dog samples. The G1 haplotypes included the common sheep strain sequence and 2 microvariants of this sequence. E. granulosus sensu stricto (G3) is described for the first time in South America. We conclude that goats act as reservoir for E. canadensis (G6) in Neuquén, and that control strategies may have to be adapted to local molecular epidemiology to improve the control of parasite transmission. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  12. Establishment of a Modified in Vitro Cultivation of Protoscoleces to Adult Echinococcus Ganulosus; an Important Way for New Investigations on Hydatidosis

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    HR Rahimi

    2012-02-01

    Full Text Available Background: Echinococcus granulosus, a zoonotic cestode parasite, causative agent of hydatid cyst is endemic in many parts of the world including the Middle East. Study on different aspects of this parasite is very important and valuable. However, working with adult worms which their habitat situated in the small intestine of canids, is dangerous and risky. Achieving such risky situation needs a controlled condition which is cultivation of the organisms in the laboratory. In this regard, cultivation of E. granulosus protoscoleces leading to adult worms was established in the laboratory for the first time in Iran.Methods: Under aseptic conditions a number of protoscoleces were cultivated in diphasic S.10E.H medium using CO2 incubator to produce adult worms.Results: Different forms of parasites including pre-segmentation stages (PS1 - PS4 and segmentation stages (S5-S8 and developing stages in segmented worms (S10-S11 were observed and evaluated in these medium. Finally adult worms contained four proglottids with a large and distinct genital pore were observed 50-55 days post cultivation. These parasites do not produce fertile eggs and conclusively do not have risk of hydatid disease transmission to the researchers.Conclusion: The mentioned method for producing E. granulosus adult worms can open a new window for researches and facilitate working on different aspects of hydatidosis especially for diagnosis, protection and treatment studies.

  13. Epidemiology and investigation of Echinococcus infection in domestic animal in six counties, Xinjiang area%新疆地区6县棘球蚴病流行病学调查

    Institute of Scientific and Technical Information of China (English)

    马力克·艾则孜

    2012-01-01

    :In order to study the; prevalence situation of Echinococcus granulosus in cattle , sheep , and dog in Xinjiang area, the infection of hydatidosis in animal in 6 different districts of Xinjiang was surveyed from March to November in 2010 . Echinococcus granulosus in the livers and lungs of cattle and sheep were detected directly in the slaughterhouse . Dogs' feces were detected by enzyme-linked immnnoabsorbent assay . It was found that the numbers of detected samples were 604 (sheep) , 582 (cattle) and 1 080 (dog) , respectively . The infection rate was 45 .58% (sheep) , 20 .83% (cattle;) and 17% (dog) , respectively . The investigation results suggest that the Echinococcus granulosus is widespread and high in infection rates in Xinjiang .%目的 为掌握新疆地区家畜棘球蚴病的流行病学情况,2010年4月至2010年11月对其6个县进行了牛、羊、犬包虫感染情况调查.方法 通过在屠宰场直接检查牛、羊肝脏和肺脏的棘球蚴数量,采用酶联免疫吸附试验方法检测犬的粪便.结果 共检查羊604只,感染率为45.58%;共检查牛582头,感染率为20.83%;共检测1 080只犬的粪样,虫体阳性率为17%.结论 棘球蚴病在新疆的家畜中流行广泛、感染率高,应引起高度重视.

  14. Echinococcus

    Science.gov (United States)

    ... in dogs, and livestock such as sheep, pigs, goats, and cattle. These tapeworms are around 2 to ... ed. Philadelphia, PA: Elsevier Saunders; 2015:chap 291. Parasites-echinococcosis. Centers of Disease Control and Prevention Web ...

  15. Tegumental histological effects of Mirazid® and myrrh volatile oil on adult Fasciola gigantica

    Institute of Scientific and Technical Information of China (English)

    Ahmad Mohamed Massoud; Hatem Abdel Mawgoud Shalaby; Rabab Mohamed El Khateeb; Mona Said Mahmoud; Mohamed Abdel Aziz Kutkat

    2013-01-01

    To evaluated the histological changes within the tegument of adult Fasciola gigantica (F. gigantica) that led to the gross changes that were visible externally. Methods: The effects of oleoresin extract of myrrh (Mirazid ® ), myrrh volatile oil and triclabendazole sulphoxide (reference drug) on the tegumental structure of adult F. gigantica following treatment in vitro had been determined by light microscopy. Results: The internal changes in the tegument observed in this study were compatible with surface changes seen in the previous scanning electron microscopy study, using the same drugs. The swelling of tegumental syncytium was a particular feature of their action, but its level was much greater with myrrh volatile oil, in which vacuolization of the tegument and loss of spines were observed. Conclusions: The present study demonstrated the fasciocidal properties of Mirazid ® oleoresin extract, and it might be possible to reinforce its fasciocidal activity by increasing its content of myrrh volatile oil.

  16. Molecular characterization of Echinococcus granulosusfrom Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

    Directory of Open Access Journals (Sweden)

    Elizabeth Sánchez

    2010-09-01

    Full Text Available Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10, have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44, sheep (41 and humans (14 from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4. The G1 genotype is considered the most widespread and infectious form of E. granulosusworldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

  17. The effects of Fasciola hepatica tegumental antigens on mast cell function.

    Science.gov (United States)

    Vukman, Krisztina V; Adams, Paul N; Dowling, David; Metz, Martin; Maurer, Marcus; O'Neill, Sandra M

    2013-06-01

    Fasciola hepatica infection is associated with T helper 2/T regulatory immune responses and increased mast cell numbers. The aim of this study was to examine the interaction between F. hepatica tegumental coat antigen and mast cells in vivo and in vitro. Firstly, BALB/C, C57BL/6 or STAT6(-/-) mice were infected with F. hepatica metacercarie or mice were treated with F. hepatica tegumental coat antigen and then mast cells numbers in the peritoneal cavity and/or the liver were quantified. Also, the proliferation, chemotaxis, degranulation and cytokine secretion of mast cells from bone marrow or from peritoneal exudate cells stimulated with F. hepatica tegumental coat antigen were measured. Finally, we tested whether F. hepatica tegumental coat antigen inhibits degranulation of mast cells in vivo in a passive cutaneous and systemic anaphylaxis mouse model. Mast cell numbers increased in the peritoneal cavity and liver of F. hepatica infected mice, and this was mimicked by injection of F. hepatica tegumental coat antigen in a STAT6(-/-) independent manner. The increase in mast cell number was not the result of F. hepatica tegumental coat antigen-induced proliferation; rather F. hepatica tegumental coat antigen indirectly induces mast cell migration by dendritic cell-derived chemokines. Fasciola hepatica tegumental coat antigen interactions with mast cells do not drive T helper 2 or T regulatory immune responses. These studies on mast cell and F. hepatica tegumental coat antigen interaction may help us to understand the function of mast cells in immunity against F. hepatica and the immunomodulatory effect of F. hepatica tegumental coat antigen on these cells.

  18. Advance research of molecular biology of Echinococcus%棘球蚴分子生物学研究现状

    Institute of Scientific and Technical Information of China (English)

    王昌源; 陈雅棠

    2002-01-01

    @@ 能感染人类的棘球属绦虫有4种,即细粒棘球绦虫、多房棘球绦虫、少节棘球绦虫和福氏棘球绦虫.目前在我国仅发现2种,即细粒棘球绦虫(Echinococcus granulosus)和多房棘球绦虫(Echinococcus multilocularis).前者引起囊型棘球蚴病(cystic hydatid disease),亦即囊型包虫病(cystic echinococcosis),后者引起泡型棘球蚴病(alveolar hydatid disease),俗称泡型包虫病(alveolar echinococcosis).棘球属绦虫的终宿主为狗、猫、狐等食肉类动物,成虫寄生于小肠,人和其它中间宿主被终宿主粪便中的虫卵所感染.

  19. Analysis on the transmission dynamic factors of echinococcosis granulosus%影响细粒棘球绦虫病传播动力学的因素分析

    Institute of Scientific and Technical Information of China (English)

    杨诗杰; 伍卫平

    2009-01-01

    Life cycle of Echinococcus granulosus includes definitive hosts,intermediate hosts and a free egg stage.Its transmission dynamics depends on Echinococcus granulosus,the two hosts,the ecological environ-ment and their interactions,including biological potential of Echinococctu granulosus,aggregation,activation of host immune capability,the eggs spread,distribution and activity,environmental temperature,humidity and geographical landscape,quarantine for meat,dealing with the dead animals and their internal organs,helmin-thicide for dogs,life styles and habits of the crowd.h is essential for development and evaluation of control measures to understand their mutual relations and the influence Oil transmission dynamics.This review mainly focused Oil biological factors ofEchinococcus granulosus,the two hosts,the ecological environment and means of prevention and treatment.%细粒棘球绦虫的生活史包括两种宿主和一个自由牛存的卵期,其传播动力学取决于细粒棘球绦虫、两种宿主、生态环境等三者的相互作用,主要包括细粒棘球绦虫的生物潜能、聚集性,宿主的免疫激活能力,虫卵的散播、分布、活力,环境温度、湿度、地理景观,肉类检疫、死亡动物及其内脏处理、犬驱虫和流浪宿主处理,人群生活卫生习惯和生产方式等.了解它们的相互关系及其对传播动态的影响,对制定和评价防治措施至关重要.该文重点就细粒棘球绦虫的生物因素、两种宿主、生态环境和防治手段埘其传播动力学的影响进行讨论.

  20. Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype

    Science.gov (United States)

    Folle, Ana Maite; Kitano, Eduardo S.; Lima, Analía; Gil, Magdalena; Cucher, Marcela; Mourglia-Ettlin, Gustavo; Iwai, Leo K.; Rosenzvit, Mara; Batthyány, Carlos

    2017-01-01

    The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in

  1. Ultrastructural observations on the tegument and associated structures of the monogenean Cichlidogyrus halli typicus (Price & Kirk, 1967) Paperna, 1979.

    Science.gov (United States)

    el-Naggar, M M; Khidr, A A; Kearn, G C

    1991-10-01

    The tegument of the ancyrocephaline monogenean gill parasite Cichlidogyrus halli typicus is basically similar to that of other monogeneans. However, there are some tegumental features shared with relatively few other monogeneans, namely the presence in the surface tegument of extensive regions of homogeneous granular cytoplasm, a close association between myofibres and the basal region of the tegument and the production of two different tegumental secretory bodies by the same cyton. It is suggested that the regions of homogeneous granular cytoplasm found in the troughs between the circular muscle fibres provide some kind of support for the tegument, perhaps related to contraction of the tegumental circular muscle fibres and body shape changes. Three kinds of transtegumental sensory structures are recognized, namely a uniciliated bulb, a bulb bearing a non-ciliary process and a non-ciliated bulb. The latter two kinds are reported for the first time in monogenans. Possible functions of these sensory structures are discussed.

  2. Nrf2信号通路抑制剂对细粒棘球蚴作用的研究%Influence of Nrf2 Signaling Pathway Inhibitor on Echinococcus Granul osus

    Institute of Scientific and Technical Information of China (English)

    王卓; 姜玉峰; 邢国强; 吕海龙

    2015-01-01

    Objective To investigate the in vitro effect of Nrf2 signaling pathway inhibitor DRB on the activity of echinococcus granulosus protoscoleces. Methods We obtained fresh echinococcus granulosus protoscoleces from lamb liver naturally infected with echinococcosis granulose and conducted in vitro culture in RPMI 1640 medium. The samples were divided into four groups:DMSO control group,25 g/ L DRB group,50 g/ L DRB group and 75 g/ L DRB group. By eosin staining,the activity and morphological changes of echinococcus granulosus protoscoleces were observed under inverted microscope,and activity curves were made. The ultrastructure of protoscoleces was observed under scanning electron microscope. Results There were not significantly different in vitro effect of control group and 25 g/ L DRB group on the activity of echinococcus granulosus protoscoleces in different time(Fcontrol = 0. 542,P ﹥ 0. 05;F25 g/ L DRB = 0. 658,P ﹥ 0. 05). There were significantly different in vitro effect of 50 g/ L DRB group and 75 g/ L DRB group on the activity of echinococcus granulosus protoscoleces in different time (F 50 g/ L DRB = 6. 686,P ﹤ 0. 01;F75 g/ L DRB = 10. 756,P ﹤ 0. 01). With the lengthening of medication time and the increase of drug concentration,the inhibiting effect of DRB on protoscoleces vitality was strengthened,and the ultrastructure was changed. Conclusion High concentrations of DRB inhibitors can all inhibit in vitro cultured echinococcus granulosus protoscoleces,the dependence on concentration and time length exists. Nrf2 signaling pathway is a key factor for antioxidant stress. The study on the Nrf2 signaling pathway of echinococcus granulosus may provide a new thought for the treatment of echinococcus granulosus.%目的:研究 Nrf2信号通路抑制剂 DRB 对体外培养的细粒棘球蚴原头节活力的影响。方法从自然感染细粒棘球蚴病的羊肝中获取新鲜细粒棘球蚴原头节,并于 RPMI 1640培养基

  3. The Pseudorabies Virus VP1/2 Tegument Protein Is Required for Intracellular Capsid Transport†

    OpenAIRE

    Luxton, G.W. Gant; Lee, Joy I-Hsuan; Haverlock-Moyns, Sarah; Schober, Joseph Martin; Smith, Gregory Allan

    2006-01-01

    Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was nec...

  4. Role of a reducing environment in disassembly of the herpesvirus tegument

    Energy Technology Data Exchange (ETDEWEB)

    Newcomb, William W. [Department of Microbiology Immunology and Cancer Biology, University of Virginia Health System, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave. Charlottesville, VA 22908 (United States); Jones, Lisa M. [Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130 (United States); Dee, Alexander [Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030 (United States); Chaudhry, Farid [Department of Microbiology Immunology and Cancer Biology, University of Virginia Health System, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave. Charlottesville, VA 22908 (United States); Brown, Jay C., E-mail: JCB2G@VIRGINIA.EDU [Department of Microbiology Immunology and Cancer Biology, University of Virginia Health System, Box 800734, University of Virginia Health System, 1300 Jefferson Park Ave. Charlottesville, VA 22908 (United States)

    2012-09-15

    Initiation of infection by herpes family viruses involves a step in which most of the virus tegument becomes detached from the capsid. Detachment takes place in the host cell cytosol near the virus entry site and it is followed by dispersal of tegument proteins and disappearance of the tegument as a distinct entity. Here we describe the results of experiments designed to test the idea that the reducing environment of the cytosol may contribute to tegument detachment and disassembly. Non-ionic detergent was used to remove the membrane of purified herpes simplex virus under control and reducing conditions. The effects on the tegument were then examined by SDS-PAGE and electron microscopy. Protein analysis demonstrated that most major tegument proteins were removed under both oxidizing and reducing conditions except for UL49 which required a reducing environment. It is proposed therefore that the reducing conditions in the cytosol are involved in removal of UL49 protein. Electron microscopic analysis revealed that capsids produced under oxidizing conditions contained a coating of protein that was absent in reduced virions and which correlated uniquely with the presence of UL49. This capsid-associated layer is suggested to be the location of UL49 in the extracted virion.

  5. Anti-inflammatory polysaccharides of Azadirachta indica seed tegument

    Directory of Open Access Journals (Sweden)

    Lívia de Paulo Pereira

    2012-06-01

    Full Text Available Azadirachta indica A. Juss., Meliaceae, or Indian neem is a plant used to treat inûammatory disorders. Total polysaccharide (TPL and FI (fractioned by ion exchange chromatography from the seed tegument of A. indica were evaluated in models of acute inflammation (paw edema/peritonitis using Wistar rats. Paw edema (measured by hydroplethysmometry was induced s.c. by Λ-carrageenan (300 µg, histamine (100 µg, serotonin (20 µg, compound 48/80 (10 µg, prostaglandin (PGE2 30 µg or L-arginine (15 µg. Peritonitis (analyzed for leukocyte counts/protein dosage was induced i.p. by carrageenan (500 mg or N-formyl-methionyl-leucyl-phenylalanine (fMLP 50 ng. Animals were treated i.v. with TPL (1 mg/kg or FI (0.01, 0.1, 1 mg/kg 30 min before stimuli. FI toxicity (at 0.1 mg/kg, i.v. for seven days was analyzed by the variation of body/organ mass and hematological/biochemical parameters. TPL extraction yielded 1.3%; FI, presenting high carbohydrate and low protein content, at 0.1 mg/kg inhibited paw edema induced by carrageenan (77%, serotonin (54%, PGE2 (69% and nitric oxide (73%, and the peritonitis elicited by carrageenan (48% or fMLP (67%, being well tolerated by animals. FI exhibited potent anti-inflammatory activity, revealing to be important active component in traditionally prepared remedies to treat inflammatory states.

  6. 青海省藏犬和野生狐狸棘球绦虫感染调查及虫种分子鉴定%SURVEY AND MOLECULAR IDENTIFICATION OF ECHINOCOCCUS SPECIES IN TIBETAN DOGS AND WILD FOXES IN QINGHAI PROVINCE

    Institute of Scientific and Technical Information of China (English)

    朵红; 郭志宏; 更求久乃; 尼玛; 河生德; 常明华; 李伟; 吴润; 黄福强; 冯凯; 刁小龙; 付永; 沈秀英; 彭毛; 赵海龙; 野中成晃

    2012-01-01

    为了解青海省藏犬和野生狐狸的棘球绦虫 Echinococcus 感染状况及感染种类,本研究在青海省7个地区调查了175条藏犬和28只野生狐狸,对感染藏犬和野生狐狸的21株棘球绦虫分离株,线粒体DNA(mtDNA)的COⅠ、NDⅠ基因种属特异性序列进行了研究.结果表明,16条藏犬感染棘球绦虫,感染率为9.14%,5只野生狐狸感染棘球绦虫,感染率17.86%.测定的棘球绦虫mtDNA的COⅠ、NDⅠ基因片段长度分别为285、507、510 bp,通过基因序列分析,发现青海省存在3种棘球绦虫混合感染,藏犬存在Echinococcus granulosus genotype 1(E.g1)和Echinococcus multilocularis(E.m)感染,部分藏犬存在E.g1和E.m混合感染,野生狐狸存在E.m和Echinococcus shiquicus(E.s)感染.检测的21株棘球绦虫分离株中,E.g1 11株,占52.4%,E.m 6株,占28.6%,E.s4株,占19.0%.COⅠ基因存在15个不同的基因序列,种内变异率小于2.5%,种间变异率大于8.2%.NDⅠ基因存在17个不同的基因序列,种内变异率小于1.6%,种间变异率大于17.3%.%To understand the Echinococcus species and its infections, 28 wild foxes and 175 Tibetan dogs were surveyed in seven different regions of Qinghai Province, by using 2 mitochondrial target genes (CO I and ND I ) . 5 strains were isolated from foxes and 16 from dogs. The results showed that the infection rate of Echirwcoccosis infected with wild foxes and Tibetan dogs were 17. 86% and 9. 14% , respectively. The length of the fragment of CO I and ND I of Echinococcus strains was 285 and 507, 510 bp. And it appeared that three species of Echinococcus were prevalent mixedly infection in Qinghai Province. Among the three species of Echinococcus, Tibetan dogs can be infected with Echinococcus granulosus genotype 1 (E. g 1) or Echinococcus multilocularis (E. m) , part of the dogs can be infected mixedly by E. g\\ and E. m, and wild foxes were found only infected with E. m or Echinococcus shiquicus (E.s). In 21 Echinococcus

  7. Scanning electron microscopy of the tegumental surface of adult Schistosoma spindale.

    Science.gov (United States)

    Kruatrachue, M; Riengrojpitak, S; Upatham, E S; Sahaphong, S

    1983-09-01

    The tegumental surfaces of adult male and female of Schistosoma spindale were studied by scanning electron microscopy. In general, the body surface of the male appears to be fairly uniform from anterior end to posterior end. It is characterized by the presence of transverse ridges and papillae of various types. These papillae are distributed fairly regularly over the whole body surface of the worm. The tegument lining the gynecophoral canal of the male worm is covered with numerous spines interspersed with papillae, some without cilia and some with crater-like holes in the centres and apical cilia. The tegument of the female worm is covered with smooth and perforated ridges and sensory bulbs with apical nodules.

  8. Lipid and protein composition of the surface tegument from larvae of Taenia taeniaeformis.

    Science.gov (United States)

    Mills, G L; Coley, S C; Williams, J F

    1984-04-01

    A tegumental fraction from fully developed larvae of Taenia taeniaeformis was recovered by low speed centrifugation following incubation of the parasites in a 0.1% solution of digitonin. Scanning electron microscopy of the parasite carcass revealed no surface microtrichs, and transmission electron microscopy indicated that the subtegumental layer was undamaged. The tegumental fraction, judging from the distribution of 3H-Concanavalin A, was enriched for surface components, exhibited low succinic dehydrogenase activity, and an electron microscopic examination of the pellet showed a slightly expanded but intact distal tegumental layer. The fraction, which made up 3.0% of the dry weight of the parasite, consisted of 52% protein and 32% lipid. Thirty-three proteins, ranging in Mr from 9,000 to 276,000 daltons, were detected after sodium dodecyl sulfate solubilization and polyacrylamide gel electrophoresis. Seven of these proteins were glycoproteins. Cholesterol, phosphatidylethanolamine, phosphatidylserine, and glycosphingolipids were the major lipids.

  9. Echinococcus multilocularis phosphoglucose isomerase (EmPGI): a glycolytic enzyme involved in metacestode growth and parasite-host cell interactions.

    Science.gov (United States)

    Stadelmann, Britta; Spiliotis, Markus; Müller, Joachim; Scholl, Sabrina; Müller, Norbert; Gottstein, Bruno; Hemphill, Andrew

    2010-11-01

    In Echinococcus multilocularis metacestodes, the surface-associated and highly glycosylated laminated layer, and molecules associated with this structure, is believed to be involved in modulating the host-parasite interface. We report on the molecular and functional characterisation of E. multilocularis phosphoglucose isomerase (EmPGI), which is a component of this laminated layer. The EmPGI amino acid sequence is virtually identical to that of its homologue in Echinococcus granulosus, and shares 64% identity and 86% similarity with human PGI. Mammalian PGI is a multi-functional protein which, besides its glycolytic function, can also act as a cytokine, growth factor and inducer of angiogenesis, and plays a role in tumour growth, development and metastasis formation. Recombinant EmPGI (recEmPGI) is also functionally active as a glycolytic enzyme and was found to be present, besides the laminated layer, in vesicle fluid and in germinal layer cell extracts. EmPGI is released from metacestodes and induces a humoral immune response in experimentally infected mice, and vaccination of mice with recEmPGI renders these mice more resistant towards secondary challenge infection, indicating that EmPGI plays an important role in parasite development and/or in modulating the host-parasite relationship. We show that recEmPGI stimulates the growth of isolated E. multilocularis germinal layer cells in vitro and selectively stimulates the proliferation of bovine adrenal cortex endothelial cells but not of human fibroblasts and rat hepatocytes. Thus, besides its role in glycolysis, EmPGI could also act as a factor that stimulates parasite growth and potentially induces the formation of novel blood vessels around the developing metacestode in vivo.

  10. Partial molecular characterization of Sm8, a tegumental antigen of Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Frederico GC Abath

    2002-10-01

    Full Text Available Sm8 is a major tegumental antigen of Schistosoma mansoni. The partial cDNA was isolated and analyzed. Sequence analysis revealed transmembrane compatible hydrophobic domains and a putative leucine zipper pattern. The mRNA and the protein are predominantly expressed in adult worms.

  11. Scanning electron microscopic study of the tegumental surface of adult Schistosoma sinensium.

    Science.gov (United States)

    Kruatrachue, M; Upatham, E S; Sahaphong, S; Tongthong, T; Khunborivan, V

    1983-12-01

    The SEM study of tegumental surface of adult Schistosoma sinensium reveals that the male tegument lacks tubercles or bosses; instead it is corrugated with small pits or perforated ridges. On the dorsal surface, spines are present whose number and size progressively increase towards the posterior end of the body. In addition, there are three types of papillae interspersed among the ridges and spines. The first type of papillae has crater-like holes surrounded by a circular doughnut-shaped elevation; some are ciliated and others are non-ciliated. They are generally found on the dorsal and ventral surfaces. The second is sensory papillae which are hemispherical in shape bearing apical cilia. They are found to be concentrated around the oral sucker and on the posterior end of the worm. The third is fungiform papillae without cilia which are found on the posterior end. There are short spines present on the tegument lining the gynecophoral canal of the male worm. The tegument of the female S. sinensium is corrugated with ridges on the ventral surface. Small spines are present on the anterior portion of the dorsal surface. They become larger and increased in number towards the posterior end of the worm. The three types of papillae are present but they are much fewer and less developed than those in the male worm.

  12. Functional analysis of schistosomes EF-hand domain-containing tegument proteins

    Institute of Scientific and Technical Information of China (English)

    YU FuDong; KANG Bin; LI YuanYuan; LI YiXue

    2007-01-01

    Schistosomes cause schistosomiasis disease which severely threatens human health. Little is known about the functions of EF-hand domain containing schistosomes tegument proteins other than as antigens. More possible functions of these tegument proteins were investigated with in silico analyses including protein-protein functional interaction, site-specific variation and glycosylation modification. The analysis results suggested that schistosomes could actively modulate host immune responses for its own favor through functional interactions with host proteins with immunomodulatory function, and passively regulate host immune responses through sequence variation under positive selection and glycosylating the recognition sites of host immune attack. In addition, the analysis of the C-terminal domain of these tegument proteins indicated that they could assist schistosomes in escaping host immune attacks through inhibiting chemotaxis and non-complement fixing antibody (IgG4) responses. In summary, our results suggested that these tegument antigen proteins could assist schistosomes in escaping and modulating host immune responses for self-protection during the process of host-para- site interaction.

  13. 利用细胞色素氧化酶亚单位Ⅰ分析青海藏区细粒棘球绦虫系统发育学%Phylogeny of Qinghai Plateau Echinococcus g ranulosus isolates inferred by cytochrome oxidase Ⅰ gene sequences

    Institute of Scientific and Technical Information of China (English)

    曹得萍; 樊海宁; 毋德芳; 赵海龙; 白海燕

    2014-01-01

    目的:囊型包虫病是一种广泛流行且危害严重的人兽共患寄生虫病。本文旨在分析流行于青南地区细粒棘球绦虫系统发育学及基因多态性。方法本文利用线粒体 DNA细胞色素氧化酶亚单位Ⅰ(COX Ⅰ)分子对收集到的流行于青海省的59例包虫病样本进行测序。总计71条序列(其中12条来自GenBank)碱基通过Clustal X软件比对,构建贝叶斯进化树。结果流行于青海省包虫病样本大部分与细粒棘球绦虫G1型聚在一枝,还有三个样本与多房棘球绦虫(AB018440)聚在一枝。虽大部分棘球绦虫基因型属于G1型,但各自的基因型各不相同,具有丰富的多样性。结论流行于青海省细粒棘球绦虫系统发育学比我们想象复杂。%Cystic echinococcosis is one of the most widespread and severe zoonotic helminthic diseases .To understand the phylogeny and genetic polymorphism Echinococcus granulosus (E .granulosus) prevailed in south region of Qinghai Prov-ince ,partial fragment of cytochrome oxidase Ⅰ (COX Ⅰ ) gene sequences were used to infer the phylogenetic relationship of 59 collected samples of E .granulosus in Qinghai Province .Total 72 sequences (13 sequences from GenBank) were aligned using CLUSTAL X ,and then ,Bayesian analyses were performed in Mrbayes-3 .1 .2 .The results revealed that Echinococcus spp .isolates did not form a monophyletic group .The most samples clustered with E . granulosus strain (G1) (AB297617) , but showed high genetic polymorphism .Another three samples clustered with E .multilocularis (AB018440) ,while they showed complex phylogenetic relationships among them ,further indicating that Echinococcus spp .isolates from Qinghai Prov-ince may has a more complex evolutionary history than expected .

  14. Tegumental ultrastructure of adult Quinqueserialis quinqueserialis (Trematoda: Notocotylidae): an intestinal parasite of muskrat (Ondatra zibethicus).

    Science.gov (United States)

    Naem, Soraya; Smythe, Ashleigh B

    2015-07-01

    Ten adult Quinqueserialis quinqueserialis specimens were removed from the intestine of a naturally infected muskrat, and scanning electron microscopy was used to study the morphological characteristics of the trematodes. The mature trematode, which was easy to recognize by the monostome holdfast organ, with no anterior cone, measured 2200-2500 μm in length by 900-1050 μm in width. The body was elongated and tapering at the anterior end, but the posterior end was rounded, and in some specimens was slightly truncated. The mouth opening lay at the anterior end and was surrounded by the oral sucker, which was round, small to medium in size, and subterminal. The tegument of the rim and inside of the oral sucker was smooth and had two types of papillae, domed and rosette papillae. Around the oral sucker, tegument was covered with sharp, pointed spines. The common genital pore was located on the median line of the body, posterior to the oral sucker. The cirrus had smooth tegument at the base and was armed with numerous conical spines throughout its length. The ventral surface was concave and provided with five distinct longitudinal rows of ventral papillae, which extended from the anterior to the posterior end of the body. Each row consisted of 15 to 20 papillae, making 81 to 88 papillae in all. These papillae were variable in size. In most specimens, the papillae were simple knob-like structures, but in some cases, they appeared to be bi- or trifurcate. The tegument at the base of each ventral papilla showed minute spiny pattern, but it was smooth or folded on top and had small rosette and ciliated papillae. Tegument at the edges of the worm was smooth in the mid-parts, spiny on lateral parts, and included rosette papillae. The dorsal surface of the worm was smooth and slightly convex, and the tegument was provided with two large domed papillae in one third of the anterior end of the dorsal part, few thick spines in the mid-part, and excretory pore at the level just

  15. Tegumental alterations of adult Schistosoma japonicum harbored in mice treated with a single oral dose of mefloquine.

    Science.gov (United States)

    Xiao, Shu-hua; Xue, Jian; Shen, Bing-gui

    2010-02-01

    To observe the effect of mefloquine on the tegument of adult Schistosoma japonicum harbored in mice. Twelve mice were each infected with 60-80 S. japonicum cercariae. At 35 days post-infection, 10 mice were treated orally with mefloquine at a single dose of 400 mg/kg. Two mice were sacrificed at 8 h, 24 h, 3 days, 7 days, and 14 days post-treatment respectively, and schistosomes were collected by the perfusion technique, fixed and examined under a scanning electron microscope. Schistosomes obtained from the remaining 2 untreated mice served as control. 8 h post-treatment, male and female schistosomes showed focal swelling of the worm body accompanied by extensive swelling, tough junction and fusion of tegumental ridges. Meanwhile, some of the sensory structures showed enlargement and part of them collapsed. 24 h after mefloquine administration, head portion of some male and female worms revealed high swelling accompanied by severe damage to oral sucker. 3 days post-treatment, focal swelling of worm body along the whole worm was universal. In some male and female worms, the damaged tegument fused together to form a large mass protruding from the tegumental surface. In addition, focal or extensive peeling of tegumental ridges was seen or collapse of enlarged sensory structure resulted in formation of hole-like appearance. 7 days post administration, focal swelling of worm body and damage to tegument induced by mefloquine were similar to those aforementioned, but focal peeling, collapse of enlarged sensory structures, and deformation of oral sucker in male and female worms were universal. 14 days post-treatment, individual male worm survived the treatment revealed normal appearance of tegumental ridges in head portion, although light focal swelling of worm body was still observed. Mefloquine causes focal swelling of worm body, extensive and severe damage to the tegument in adult S. japonicum.

  16. Echinococcus species from red foxes, corsac foxes, and wolves in Mongolia.

    Science.gov (United States)

    Ito, Akira; Chuluunbaatar, Gantigmaa; Yanagida, Tetsuya; Davaasuren, Anu; Sumiya, Battulga; Asakawa, Mitsuhiko; Ki, Toshiaki; Nakaya, Kazuhiro; Davaajav, Abmed; Dorjsuren, Temuulen; Nakao, Minoru; Sako, Yasuhito

    2013-11-01

    The small intestines of 420 wild canids (111 corsac foxes, 191 red foxes and 118 wolves) from Mongolia, were examined for adult worms of the genus Echinococcus. The Mongolian genotype of Echinococcus multilocularis was found in fifteen red foxes and four wolves, whereas two genotypes (G6/7 and G10) of Echinococcus canadensis were found in two and three wolves, respectively. No adult Echinococcus worms were found in the corsac foxes examined. The genotypes of E. multilocularis and E. canadensis are discussed in terms of host specificity and distribution in Mongolia. The importance of wolves in the completion of the life cycle of Echinococcus spp. is also discussed.

  17. A Multiple Functional Protein: the Herpes Simplex Virus Type 1 Tegument Protein VP22

    Institute of Scientific and Technical Information of China (English)

    Mei-li LI; Hong GUO; Qiong DING; Chun-fu ZHENG

    2009-01-01

    The herpes simplex virus type 1 (HSV-1) VP22, is one of the most abundant HSV-1 tegument proteins with an average stoichiometry of 2 400 copies per virion and conserved among alphaherpesvirinae. Many functions are attributed to VP22, including nuclear localization, chromatin binding, microtubule binding, induction of microtubule reorganization, intercellular transport, interaction with cellular proteins, such as template activating factor I (TAF-I) and nonmuscle myosin II A (NMIIA), and viral proteins including tegument protein VP16, pUS9 and pUL46, glycoprotein E (gE) and gD. Recently, many novel functions performed by the HSV-1 VP22 protein have been shown, including promotion of protein synthesis at late times in infection, accumulation of a subset of viral mRNAs at early times in infection and possible transcriptional regulation function.

  18. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host*

    Science.gov (United States)

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M.; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q.; Kilcoyne, Michelle; Hokke, Cornelis H.; Dalton, John P.; O'Neill, Sandra M.

    2016-01-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. PMID:27466253

  19. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host.

    Science.gov (United States)

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q; Kilcoyne, Michelle; Hokke, Cornelis H; Dalton, John P; O'Neill, Sandra M

    2016-10-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Carbohydrate Detection and Lectin Isolation from Tegumental Tissue of Fasciola hepatica

    Directory of Open Access Journals (Sweden)

    MB Molaei Rad

    2010-02-01

    Full Text Available "nBackground: Fascioliasis is a chronic hepatic disease and may be resulted from mechani­cal/molecular parasite adhesion to host liver tissue. The aim of this study was to detect surface car­bohydrate and lectin, carbohydrate-binding protein isolation that might be responsible of this molecular binding."nMethods: The present experimental work was conducted in the Department of Medical Parasitol­ogy and Mycology, School of Public Health, Tehran University of Medical Sciences, Te­hran, Iran.  Fasciola hepatica parasites were collected from abattoir (Saman, Tehran, Iran and surface mannose-carbohydrate was detected by fluorescein isothiocyanate (FITC conju­gated lectin (Lentil. Lectin of tegumental tissue from F. hepatica was isolated by affinity chroma­tography and detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE."nResults: Mannose carbohydrate was observed on the surface of tegumental tissue from para­site under fluorescence microscope. Carbohydrate-binding protein or lectin with MW of 50 kDa also was isolated from homogenized tegument of helminth."nConclusion: These results are important for understanding of molecular pathogenesis of F. hepat­ica at the chronic phase of fascioliasis

  1. EBV tegument protein BNRF1 disrupts DAXX-ATRX to activate viral early gene transcription.

    Directory of Open Access Journals (Sweden)

    Kevin Tsai

    2011-11-01

    Full Text Available Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs, suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus.

  2. Identification of the Nucleocapsid, Tegument, and Envelope Proteins of the Shrimp White Spot Syndrome Virus Virion

    Science.gov (United States)

    Tsai, Jyh-Ming; Wang, Han-Ching; Leu, Jiann-Horng; Wang, Andrew H.-J.; Zhuang, Ying; Walker, Peter J.; Kou, Guang-Hsiung; Lo, Chu-Fang

    2006-01-01

    The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion. PMID:16501111

  3. Estructura del quiste hidatídico producido por Echinococcus oligarthrus en el hospedero intermediario Proechimys c.f. guairae (rata espinosa en Casanare, Colombia

    Directory of Open Access Journals (Sweden)

    Gerzain Rodríguez

    2000-09-01

    Full Text Available La equinococosis es un parasitismo grave producido en el hombre y en los animales por larvas de tenias del género Echinococcus que se desarrollan en el higado, el pulmón, el bazo y en otras visceras y tejidos. De las cuatro tenias dei género Echinococcus, tres se presentan en Suramerica: E granulosus, E. vogeli y E. oligarihrus. En Colombia se han informado casos en humanos y en animales cuyo agente etiológico más frecuente es E. vogeli. En este trabajo presentamos la estructura macroscópica, microscópica y de microscopia electrónica de barrido de quistes hidatídicos múltiples y de sus protoescólices encontrados en un Proechimys c.f guairae o rata espinosa, capturado en la vereda La Plata del municipio de Pore, Casanare, durante la búsqueda de reservorios del virus de la encefalitis equina venezolana. En la autopsia del animal se encontraron quistes múltiples en el higado, el bazo y el pulmón, la gran mayoría fértiles. Están constituidos por una pared franjeada que origina cápsulas proligeras dentro de las cuales están los protoescólices. El protoescólex es el escólex inmaduro del parásito adulto formado por cuatro ventosas y un rostelo con ganchos, cuyo tamaño y morfologia corresponden con las medidas de E. oligaiihrus. Ilustramos las caracteristicas morfológicas de las larvas, el desarrollo de los protoescólices y revisamos las caracteristicas de la equinococosis en Colombia.

  4. Detection of taeniid (Taenia spp., Echinococcus spp.) eggs contaminating vegetables and fruits sold in European markets and the risk for metacestode infections in captive primates.

    Science.gov (United States)

    Federer, Karin; Armua-Fernandez, Maria Teresa; Gori, Francesca; Hoby, Stefan; Wenker, Christian; Deplazes, Peter

    2016-12-01

    Due to frequent cases of alveolar echinococcosis (AE) in captive primates in Europe, 141 samples of food, which consisting of vegetables and fruits, were investigated for contamination with egg-DNA of taeniids. Each sample consisted of at least 40 heads of lettuce as well as various vegetables and fruits. The samples were purchased at different times of the year: either from September to November (autumn), originating from greenhouses or fields in the Basel region in the North of Switzerland, or in April and May (spring) when fruit and vegetables are sourced from throughout Europe from various wholesalers. Each sample was washed, and the washing water sieved through mesh apertures of 50 μm and 21 μm, respectively. The debris, including taeniid eggs, collected on the 21 μm sieve were investigated by a multiplex PCR-analysis followed by direct sequencing. In 17 (18%) of the 95 samples collected in autumn, taeniid-DNA was detected (Taenia hydatigena in four, Taenia ovis in three, Taenia polyacantha in two and Hydatigera (Taenia) taeniaeformis in five cases). Similarly, in 13 (28%) of the 46 samples collected during spring taeniid-DNA was detected (Echinococcus granulosus s.l. in two, Taenia crassiceps in one, T. hydatigena in two, Taenia multiceps/Taenia serialis in two, Taenia saginata in one and H. taeniaeformis in five cases). Although DNA of Echinococcus multilocularis was not found specifically in this study, the detection of other fox taeniids reveals that vegetables and fruit fed to the primates at the Zoo Basel at different times of the year and from different origin are contaminated with carnivore's faeces and therefore act as a potential source of AE infections.

  5. Detection of taeniid (Taenia spp., Echinococcus spp. eggs contaminating vegetables and fruits sold in European markets and the risk for metacestode infections in captive primates

    Directory of Open Access Journals (Sweden)

    Karin Federer

    2016-12-01

    Full Text Available Due to frequent cases of alveolar echinococcosis (AE in captive primates in Europe, 141 samples of food, which consisting of vegetables and fruits, were investigated for contamination with egg-DNA of taeniids. Each sample consisted of at least 40 heads of lettuce as well as various vegetables and fruits. The samples were purchased at different times of the year: either from September to November (autumn, originating from greenhouses or fields in the Basel region in the North of Switzerland, or in April and May (spring when fruit and vegetables are sourced from throughout Europe from various wholesalers. Each sample was washed, and the washing water sieved through mesh apertures of 50 μm and 21 μm, respectively. The debris, including taeniid eggs, collected on the 21 μm sieve were investigated by a multiplex PCR-analysis followed by direct sequencing. In 17 (18% of the 95 samples collected in autumn, taeniid-DNA was detected (Taenia hydatigena in four, Taenia ovis in three, Taenia polyacantha in two and Hydatigera (Taenia taeniaeformis in five cases. Similarly, in 13 (28% of the 46 samples collected during spring taeniid-DNA was detected (Echinococcus granulosus s.l. in two, Taenia crassiceps in one, T. hydatigena in two, Taenia multiceps/Taenia serialis in two, Taenia saginata in one and H. taeniaeformis in five cases. Although DNA of Echinococcus multilocularis was not found specifically in this study, the detection of other fox taeniids reveals that vegetables and fruit fed to the primates at the Zoo Basel at different times of the year and from different origin are contaminated with carnivore's faeces and therefore act as a potential source of AE infections.

  6. In vivo activity of albendazole in combination with thymol against Echinococcus multilocularis.

    Science.gov (United States)

    Albani, Clara María; Pensel, Patricia Eugenia; Elissondo, Natalia; Gambino, Guillermo; Elissondo, María Celina

    2015-09-15

    Human alveolar echinococcosis (AE) is caused by the fox tapeworm Echinococcus multilocularis and is usually lethal if left untreated. The current strategy for treating human AE is surgical resection of the parasite mass complemented by chemotherapy with benzimidazole compounds. However, reliable chemotherapeutic alternatives have not yet been developed stimulating the research of new treatment strategies such as the use of medicinal plants. The aim of the current study was to investigate the efficacy of the combination albendazole (ABZ)+thymol on mice infected with E. multilocularis metacestodes. For this purpose, mice infected with parasite material were treated daily for 20 days with ABZ (5 mg/kg), thymol (40 mg/kg) or ABZ (5 mg/kg)+thymol (40 mg/kg) or left untreated as controls. After mice were euthanized, cysts were removed from the peritoneal cavity and the treatment efficacy was evaluated by the mean cysts weight, viability of protoscoleces and ultrastructural changes of cysts and protoscoleces. The application of thymol or the combination of ABZ+thymol resulted in a significant reduction of the cysts weight compared to untreated mice. We also found that although ABZ and thymol had a scolicidal effect, the combination of the two compounds had a considerably stronger effect showing a reduction in the protoscoleces viability of 62%. These results were also corroborated by optical microscopy, SEM and TEM. Protoscoleces recovered from ABZ or thymol treated mice showed alterations as contraction of the soma region, rostellar disorganization and presence of blebs in the tegument. However both drugs when combined lead to a total loss of the typical morphology of protoscoleces. All cysts removed from control mice appeared intact and no change in ultrastructure was detected. In contrast, cysts developed in mice treated with ABZ revealed changes in the germinal layer as reduction in cell number, while the treatment with thymol or the ABZ+thymol combination

  7. Predictors of Echinococcus multilocularis prevalence in definitive and intermediate hosts

    DEFF Research Database (Denmark)

    Takeuchi-Storm, Nao; Woolsey, Ian David; Jensen, Per Moestrup

    2015-01-01

    Echinococcus multilocularis (EM) is a pathogenic and potentially fatal cestode causing human alveolar echinococcosis (AE). A meta-analysis was conducted using a generalized estimation equation approach (GEE) to assess the effect of taxonomic, environmental and diagnostic variables on EM prevalence...

  8. Loop-mediated isothermal amplification for rapid detection of dogs infected with Echinococcus species based on copro-DNAⅡ%环介导等温扩增技术(LAMP)检测棘球绦虫感染犬粪DNA的研究Ⅱ

    Institute of Scientific and Technical Information of China (English)

    陈璐; 吾拉木·马木提; 张德亭; 金一帮

    2014-01-01

    目的:为有效地控制预防流行区犬科动物的棘球绦虫感染情况,建立一种快速检测棘球绦虫感染犬的粪便DNA检测方法-环介导等温扩增技术(loop-mediated isothermal amplification .LAMP)。方法针对细粒棘球绦虫线粒体DNA ND2基因6个位点特异性的设计4条LAMP引物,利用LAMP法和普通PCR方法同时检测棘球绦虫、肥胖带绦虫以及犬肠道内的其它寄生虫DNA来验证LAMP方法的特异性;将转入ND2基因片段的质粒作为标准阳性对照,并做梯度稀释后同时用 LAMP和 PCR方法进行检测比较两者的敏感性。此外,将采集的46份犬粪便标本提取DNA ,分别运用LAMP和犬尸体剖检进行感染犬的初步检测评估。结果 E .g mtDNA ND2基因的LAMP引物能够鉴别多房棘球绦虫和细粒棘球绦虫这两个相近的种属,并不与其它待检寄生虫发生交叉反应,且最低检测限度为4×101拷贝(灵敏度比普通PCR高出103倍)。在初步对46份犬粪便DNA检测结果中 ND2 LAMP引物显示出较好的灵敏度和特异度,χ2检验结果该方法与犬尸体剖检方法的诊断效果无统计学差异。结论本研究初步建立了LAM P技术检测细粒棘球绦虫感染犬的新方法,在各项特异度、灵敏度和样本检测中展现出较好的效果。该方法不需要昂贵的仪器设备和繁琐的电泳分析过程,有望成为流行区和基层兽医站细粒棘球绦虫感染犬检测的新方法。%To control and prevent the Echinococcus in the place ,we established a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Echinococcus species specific DNA from dog faeces .Four primers which recognizing 6 dis-tinct regions on the NADH dehydrogenase subunit 2 (ND2) gene of Echinococcus granulosus were designed and used for LAMP assay .The specificity of LAMP assay was evaluated using DNA extracted from Echinococcus granulosus , Taenia saginata , and other dog

  9. Morphology observation of new Echinococcus shiquicus in southern Qinghai plateau%青海省青南高原棘球绦虫新种-石渠棘球绦虫的形态学观察

    Institute of Scientific and Technical Information of China (English)

    曾诚

    2013-01-01

    OBJECTIVE To observe pathogen morphology of new type of Echinococcus shiquicus attested by cytb gene analysis in southern Qinghai plateau, then analyze pathogen morphology characteristics comparing with other Echinococcus. METHODS Collected Echinococcus shiquicus from Vulpes ferrilata intestine, then observed its morphology characteristics after carmine staining. Echinococcus shiquicus from Ochotona curzoniae was conducted on pathological characteristics detection after PAS staining with paraffin-embedded sections. Cytb gene-sequence was analyzed in adult and pathological tissues. RESULTS Body of adult Echinococcus shiquicus which was founded in southern Qinghai plateau was short about 1.2 -1.8mm, typical developments of adult Echinococcus shiquicus were comprised of scolex, mature proglottid and gravid proglottid.There was a small number of proglottid. Rostellum hook easily dropped off, the length of large hoop was 20.0-24.0μm, the length of small hoop was 16.0-18.0μm; body size of adult Echinococcus shiquicus was the smallest in all Echinococcus known now, head hoop number about 18-36; location of gonopore was very special, opened at mature proglottid body side about 1/3-1/4, the number of eggs in uterine of gravid proglottid was small, no-branch gravid proglottid uterine, liking bag. Characteristics pathological change under microscope: single atrial type, diameter about 10mm. There was brood pouch, which adhere to germinal layer and with lots of protoscolex; no ascus, stratum corneum was rather thick. Main pathological change was chronic proliferative inflammation. CONCLUSION As a new type Echinococcus which found in wild animal in southern Qinghai plateau, Echinococcus shiquicus enlarges research scope on Echinococcus pathogen. There are three kinds of Echinococcus in southern Qinghai plateau, such as Echinococcus shiquicus, E granulosus and E.multiocularis. By morphology observation of Echinococcus shiquicus in southern Qinghai plateau, it provides basic

  10. Impact of experimental duel infections with Schistosoma mansoni and Echinoccocus granulosus on hepatic histopathology.

    Science.gov (United States)

    Elwakil, Hala S; Ali, Nehad M; Talaat, Roba M; Osman, Wesam M

    2007-12-01

    Experimental duel infection with S. mansoni and E. granulosus was induced in mice to determine their effect on serum nitric oxide (NO) level and accordingly on the sequences of histopathological lesions affecting the liver. The results showed that serum NO level was significantly increased (pdeath of hydatid cyst in mice (GI) compared to E. granulosus (GV). So, the duel infection with the two parasites affected serum NO level and hepatic histopathology, by ameliorative or deteriorative effects, according to duration of infection with either.

  11. Ultrastructural characterization of serum-induced changes in the tegument of Taenia taeniaeformis.

    Science.gov (United States)

    Conder, G A; Marchiondo, A A; Williams, J F; Andersen, F L

    1983-10-01

    The objective of this study was to characterize complement-dependent damage to the tegument of isolated metacestodes of Taenia taeniaeformis caused by exposure to immune or normal rat serum (IRS and NRS, respectively). Metacestodes of T. taeniaeformis (34- and 69-day-old) from rats were incubated for 1 hr in 0.85% physiological saline solution (PSS), IRS, NRS, heat-inactivated at 56 C for 1 hr (delta) IRS, or delta NRS and then fixed for 2 hr in 3% glutaraldehyde. The larvae were then prepared for freeze-etching, thin sectioning, and SEM by standard techniques. Freeze-etch replicas of PSS-, delta IRS-, and delta NRS-treated larvae showed no damage, whereas those of IRS- and NRS-treated metacestodes exhibited vesiculation in the extracellular matrices, segmentation or "beading" of the microthrix tip, significant reductions in the number of intramembranous particles (IMP) in the P face of the membrane of the microthrix base, and changes in the pattern of IMP distribution in the P face of the base. Similar results were obtained from larvae prepared for thin sectioning and SEM. Additionally, thin-sectioned preparations demonstrated that in some cases the entire tegument was stripped away in IRS- and NRS-treated metacestodes. Our results have provided supportive evidence that complement-mediated lysis of larvae of T. taeniaeformis is not enhanced by the presence of antibody in serum, and we also characterized ultrastructurally the types of tegumental damage that may contribute to lysis. In addition, a possible defense mechanism used by the parasite to counter immunological attack by host phagocytic cells is proposed.

  12. In vitro and in vivo effects of 2-methoxyestradiol, either alone or combined with albendazole, against Echinococcus metacestodes.

    Science.gov (United States)

    Spicher, Martin; Naguleswaran, Arunasalam; Ortega-Mora, Luis M; Müller, Joachim; Gottstein, Bruno; Hemphill, Andrew

    2008-08-01

    The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a mainly hepatic disease characterized by continuous asexual proliferation of metacestodes by exogenous budding, resulting in the tumor-like, infiltrative growth of the parasite lesion. Current chemotherapeutical treatment of AE relies on the use of benzimidazoles (albendazole, mebendazole), but these drugs act parasitostatic rather than parasitocidal, and in case of side effects such as liver toxicity, patients are left without valuable alternatives. 2-ME2 is a natural metabolite of estradiol, with a documented anti-angiogenic and broad spectrum anti-tumour activity. Treatments of in vitro cultured E. multilocularis metacestodes with 2-ME2 (2-10 microM) showed that the drug has an adverse effect on parasite viability. First, 2-ME in vitro treatment downscaled the transcription of the 14-3-3-pro-tumorogenic zeta-isoform in E. multilocularis metacestodes. Second, scanning and transmission electron microscopy showed that the germinal layer of E. multilocularis metacestodes was dramatically damaged following 2-ME2-treatment, and the effect was dose-dependent. Similar results were obtained with E. granulosus metacestodes. Bioassays were performed in mice injected with 2-ME2-treated and albendazole-treated metacestodes, or parasites-treated with both 2-ME and albendazole in combination. These assays indicated that, despite inducing considerable damage in vitro, neither of the drugs was capable of exerting a true parasiticidal effect, but best results were achieved with a combination of both compounds. In vivo treatment in E. multilocularis-infected mice for a period of 6 weeks showed that a combined 2-ME2/albendazole based treatment lead to a reduction in parasite weight, but the results did not show statistical difference from the application of albendazole alone.

  13. Human hydatidosis granulosus in greater Cairo, Egypt: with general review.

    Science.gov (United States)

    Ibrahim, Badawyia B; Haridy, Fouad M; Hegazi, Mamdoud M; Morsy, Tosson A

    2007-08-01

    In this study, human cases (41) of proved hydatidosis granulosus were obtained from Al Kasr Al Aini University Hospitals from 2000-2006. They were 22 females with ages from 8 to 70 years and 19 males with ages from 5 to 75 years. The highly infected organ was the liver. Infection in other organs as lung, spleen, brain, eye, pelvic and bones of upper and lower limbs were reported. Most of the hydatidosis patients were from governorates of Giza (24 cases), Qalyobia (7 cases), Cairo (5 cases), Bani-Swef (3 cases), Sharkia (1 case) and Demiatta (1 case). Biopsies of infected liver and lung were processed for the histopathological studies and photographed. The results were discussed on the light of work done before Egypt.

  14. Functional analysis of transcriptional regulation of herpes simplex virus type 1 tegument protein VP22

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The herpes simplex virus type 1 (HSV-1) tegument proteins have important functions in the viral repli- cation process. In order to investigate the role of the HSV-1 tegument protein VP22 in viral replication, its transcriptional regulation of viral promoters was investigated using the chloramphenicol acetyl- transferase (CAT) assay. The results indicate that VP22 exerts a dose-dependent transcriptional in- hibitory effect on the HSV-1 α4, TK, and gC gene promoters. VP22 had the capacity to repress tran- scriptional activation of promoters via different viral transcription regulatory factors such as VP16 and ICP0, as evidenced by the specific repression of the TK and gC gene promoters by ICP0. In addition, VP22 was capable of inhibiting the promotion of ICP0 transcriptional activation in the presence of HAT PCAF, which is even more remarkable than the VP22 repression of ICP0 transcriptional activation. Fi- nally, the transcriptional inhibitory effect of VP22 on other viral promoters was demonstrated by the analysis of β-galactosidase activities in internal controls.

  15. Proteomic analysis of tegument-exposed proteins of female and male Schistosoma japonicum worms.

    Science.gov (United States)

    Zhang, Min; Hong, Yang; Han, Yanhui; Han, Hongxiao; Peng, Jinbiao; Qiu, Chunhui; Yang, Jianmei; Lu, Ke; Fu, Zhiqiang; Lin, Jiaojiao

    2013-11-01

    The interplay between sexes is a prerequisite for female growth, reproductive maturation, and egg production, and the basis of schistosome pathopoiesis and propagation. The tegument is in direct contact with the host environment and its surface membranes are particularly crucial for schistosome survival in the definitive host. In this study, a streptavidin-biotin affinity purification technique combined with LC-MS/MS was used to analyze putative tegument-exposed proteins in female and male adult Schistosoma japonicum worms. In total, 179 proteins were identified in females and 300 in males, including 119 proteins common to both sexes, and 60 female biased and 181 male biased proteins. Some (e.g., serpin and CD36-like class B scavenger receptor) were involved in host-schistosome interactions, while some (e.g., gynecophoral canal protein) were important in the interplay between sexes. Gene Ontology analysis revealed that proteins involved in protein glycosylation and lysosome were highly expressed in females, while proteins involved in intracellular signal transduction, regulation of actin filament polymerization, and proteasome core complex were highly expressed in males. These results might elucidate physiological differences between the sexes. Our study provides new insights into schistosome growth and sexual maturity in the final host and permits the screening of vaccine candidates or drug targets for schistosomiasis.

  16. Functional analysis of transcriptional regulation of herpes simplex virus type 1 tegument protein VP22

    Institute of Scientific and Technical Information of China (English)

    YU Xian; LI WeiZhong; LIU LongDing; CHE YanChun; CUN Wei; WU Wen Juan; HE ChunYan; SHAO CongWen; LI QiHan

    2008-01-01

    The herpes simplex virus type 1 (HSV-1) tegument proteins have important functions in the viral repli-cation process. In order to investigate the role of the HSV-1 tegument protein VP22 in viral replication, its transcriptional regulation of viral promoters was investigated using the chloramphenicol acetyl-transferase (CAT) assay. The results indicate that VP22 exerts a dose-dependent transcriptional in-hibitory effect on the HSV-1 α4, TK, and gC gene promoters. VP22 had the capacity to repress tran-scriptional activation of promoters via different viral transcription regulatory factors such as VP16 and ICP0, as evidenced by the specific repression of the TK and gC gene promoters by ICP0. In addition, VP22 was capable of inhibiting the promotion of ICP0 transcriptional activation in the presence of HAT PCAF, which is even more remarkable than the VP22 repression of ICP0 transcriptional activation. Fi-nally, the transcriptional inhibitory effect of VP22 on other viral promoters was demonstrated by the analysis of β-galactosidase activities in internal controls.

  17. 21 CFR 520.1872 - Praziquantel, pyrantel pamoate, and febantel tablets.

    Science.gov (United States)

    2010-04-01

    ... tapeworms (Dipylidium caninum, Taenia pisiformis, Echinococcus granulosus); hookworms (Ancylostoma caninum... for the removal and control of tapeworm Echinococcus multilocularis in dogs. (iii) Limitations. Do...

  18. 77 FR 47511 - New Animal Drugs; Cephalexin; Fentanyl; Milbemycin Oxime and Praziquantel

    Science.gov (United States)

    2012-08-09

    ... (Ancylostoma caninum), adult whipworm (Trichuris vulpis), and adult tapeworm (Taenia pisiformis, Echinococcus... tapeworm (Taenia pisiformis, Echinococcus multilocularis, and E. granulosus) infections in dogs and...

  19. Schistosoma mansoni Tegument (Smteg Induces IL-10 and Modulates Experimental Airway Inflammation.

    Directory of Open Access Journals (Sweden)

    Fábio Vitarelli Marinho

    Full Text Available Previous studies have demonstrated that S. mansoni infection and inoculation of the parasite eggs and antigens are able to modulate airways inflammation induced by OVA in mice. This modulation was associated to an enhanced production of interleukin-10 and to an increased number of regulatory T cells. The S. mansoni schistosomulum is the first stage to come into contact with the host immune system and its tegument represents the host-parasite interface. The schistosomula tegument (Smteg has never been studied in the context of modulation of inflammatory disorders, although immune evasion mechanisms take place in this phase of infection to guarantee the persistence of the parasite in the host.The aim of this study was to evaluate the Smteg ability to modulate inflammation in an experimental airway inflammation model induced by OVA and to characterize the immune factors involved in this modulation. To achieve the objective, BALB/c mice were sensitized with ovalbumin (OVA and then challenged with OVA aerosol after Smteg intraperitoneal inoculation. Protein extravasation and inflammatory cells were assessed in bronchoalveolar lavage and IgE levels were measured in serum. Additionally, lungs were excised for histopathological analyses, cytokine measurement and characterization of the cell populations. Inoculation with Smteg led to a reduction in the protein levels in bronchoalveolar lavage (BAL and eosinophils in both BAL and lung tissue. In the lung tissue there was a reduction in inflammatory cells and collagen deposition as well as in IL-5, IL-13, IL-25 and CCL11 levels. Additionally, a decrease in specific anti-OVA IgE levels was observed. The reduction observed in these inflammatory parameters was associated with increased levels of IL-10 in lung tissues. Furthermore, Smteg/asthma mice showed high percentage of CD11b+F4/80+IL-10+ and CD11c+CD11b+IL-10+ cells in lungs.Taken together, these findings demonstrate that S. mansoni schistosomula

  20. Schistosoma mansoni Tegument (Smteg) Induces IL-10 and Modulates Experimental Airway Inflammation

    Science.gov (United States)

    2016-01-01

    Background Previous studies have demonstrated that S. mansoni infection and inoculation of the parasite eggs and antigens are able to modulate airways inflammation induced by OVA in mice. This modulation was associated to an enhanced production of interleukin-10 and to an increased number of regulatory T cells. The S. mansoni schistosomulum is the first stage to come into contact with the host immune system and its tegument represents the host-parasite interface. The schistosomula tegument (Smteg) has never been studied in the context of modulation of inflammatory disorders, although immune evasion mechanisms take place in this phase of infection to guarantee the persistence of the parasite in the host. Methodology and Principal Findings The aim of this study was to evaluate the Smteg ability to modulate inflammation in an experimental airway inflammation model induced by OVA and to characterize the immune factors involved in this modulation. To achieve the objective, BALB/c mice were sensitized with ovalbumin (OVA) and then challenged with OVA aerosol after Smteg intraperitoneal inoculation. Protein extravasation and inflammatory cells were assessed in bronchoalveolar lavage and IgE levels were measured in serum. Additionally, lungs were excised for histopathological analyses, cytokine measurement and characterization of the cell populations. Inoculation with Smteg led to a reduction in the protein levels in bronchoalveolar lavage (BAL) and eosinophils in both BAL and lung tissue. In the lung tissue there was a reduction in inflammatory cells and collagen deposition as well as in IL-5, IL-13, IL-25 and CCL11 levels. Additionally, a decrease in specific anti-OVA IgE levels was observed. The reduction observed in these inflammatory parameters was associated with increased levels of IL-10 in lung tissues. Furthermore, Smteg/asthma mice showed high percentage of CD11b+F4/80+IL-10+ and CD11c+CD11b+IL-10+ cells in lungs. Conclusion Taken together, these findings

  1. The prevalance of Echinococcus multilocularis in foxes in Limburg 2002-2003

    NARCIS (Netherlands)

    Giessen JWB van der; Vries A de; Chu ML; Stortelder V; Mulder JL; Lezenne Coulander C de; Teunis P; MGB

    2004-01-01

    This report describes a survey carried out between January 2002 and March 2003 to determine the prevalence of Echinococcus multilocularis in the red fox (Vulpes vulpes) in the province of Limburg, the Netherlands. Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a very

  2. A preliminary study to understand the effect of Fasciola hepatica tegument on naïve macrophages and humoral responses in an ovine model.

    Science.gov (United States)

    Haçarız, O; Sayers, G; Mulcahy, G

    2011-02-15

    Fasciola hepatica, the liver fluke, is a highly evolved endo-parasite that uses various mechanisms to evade the host immune system. The immunosuppressive capabilities of the parasite's excretory/secretory products have been well demonstrated by previous independent studies. However, the role of the parasite's tegument in the immune responses remains to be investigated. In this study, the effect of the tegument and other fractions of adult F. hepatica (excretory/secretory, liver fluke homogenate and liver fluke homogenate without tegument) in the activation of naïve macrophages in vitro was investigated using an ovine model. In addition, an immunoproteomic approach was used to investigate the characteristics of humoral antibody responses developed in sheep against the tegument fraction. The results indicated significantly increased arginase expression in macrophages incubated with the tegument and excretory/secretory fractions. Two dimensional gel electrophoresis of the tegument demonstrated approximately 100 protein spots, with only four of these spots were highly reactive with the positive serum as determined by 2-DE immunoblotting. These results give a preliminary indication that the liver fluke tegument may play role in avoiding hosts' protective immune responses against itself.

  3. 检测动物棘球蚴的3种PCR方法的比较%Comparison of 3 PCR methods for detection of Echinococcus spp.in animals

    Institute of Scientific and Technical Information of China (English)

    周正斌; 朱淮民

    2011-01-01

    目的 比较多重PCR(muhiplex PCR)、PCR-限制性片段长度多态性(PCR-RFLP)、半巢式PCR等3种检测棘球蚴的PCR方法,优化可区分多房棘球绦虫、细粒棘球绦虫羊株(G1型)和骆驼株(G6型)、石渠棘球绦虫和带科绦虫的方法.方法 根据细粒棘球绦虫、多房棘球绦虫、石渠棘球绦虫、带科绦虫线粒体基因序列,用DNAStar软件设计半巢式PCR通用引物,建立半巢式PCR法,并与文献报道的多重PCR法、PCR-RFLP平行扩增3份巨颈绦虫囊尾蚴、48份多房棘球蚴、8份细粒棘球蚴样本DNA,比较3种检测方法的特异性和灵敏度,并以细粒棘球蚴组织DNA为模板测定3种方法的灵敏度.结果 半巢式PCR法灵敏度最高,59份样本全部扩增出561 bp条带,但需要测序才能确定虫种;多重PCR法能扩增出3份巨颈绦虫囊尾蚴(带绦虫)样本(3/3,100%),5份(5/8,62.5%)细粒棘球蚴样本,23份(23/48,47.9%)多房棘球蚴样本,其余28份样本为假阴性;PCR-RFLP扩增细粒棘球蚴、多房棘球蚴和巨颈绦虫囊尾蚴(带科绦虫)的阳性率与多重PCR相同,且PCR-RFLP不能区分细粒棘球绦虫G1型和带科绦虫.统计学分析显示半巢式PCR法的棘球蚴检出率高于PCR-RFLP法和多重PCR法.半巢式PCR法灵敏度分别是PCR-RFLP法和多重PCR法的1 000倍和100倍,最低检出细粒棘球蚴DNA浓度为253 pg/lμ.结论 根据不同研究目的,可组合选用多重PCR法、PCR-RFLP法、半巢式PCR法区分细粒棘球绦虫G1型、G6型,多房棘球绦虫,石渠棘球绦虫和带绦虫.%Objective To compare 3 methods(hemi-nested PCR,multiplex PCR and PCR-RFLP),and optimize the method for discrimination of Echinococcus spp.in animals.Methods Hemi-nested PCR primers were designed according to the mitochondria DNA sequence of the Genus Echinococcus with DNAStar software.Fifty-nine samples included E.multilocularis,E.granulosus and Taenia taeniaeformis were collected from Shiqu County.All the samples were

  4. Neglected zoonotic helminths: Hymenolepis nana, Echinococcus canadensis and Ancylostoma ceylanicum.

    Science.gov (United States)

    Thompson, R C A

    2015-05-01

    The majority of helminth parasites that are considered by WHO to be the cause of 'neglected diseases' are zoonotic. In terms of their impact on human health, the role of animal reservoirs and polyparasitism are both emerging issues in understanding the epidemiology of a number of these zoonoses. As such, Hymenolepis (Rodentolepis) nana, Echinococcus canadensis and Ancylostoma ceylanicum all qualify for consideration. They have been neglected and there is increasing evidence that all three parasite infections deserve more attention in terms of their impact on public health as well as their control.

  5. The impact of globalisation on the distribution of Echinococcus multilocularis.

    Science.gov (United States)

    Davidson, Rebecca K; Romig, Thomas; Jenkins, Emily; Tryland, Morten; Robertson, Lucy J

    2012-06-01

    In the past three decades, Echinococcus multilocularis, the cause of human alveolar echinococcosis, has been reported in several new countries both in definitive hosts (canids) as well as in people. Unless treated, infection with this cestode in people is fatal. In previously endemic countries throughout the Northern Hemisphere, geographic ranges and human and animal prevalence levels seem to be increasing. Anthropogenic influences, including increased globalisation of animals and animal products, and altered human/animal interfaces are thought to play a vital role in the global emergence of this pathogenic cestode. Molecular epidemiological techniques are a useful tool for detecting and tracing introductions, and differentiating these from range expansions.

  6. 细粒棘球绦虫原头蚴复合染色方法的探讨%Exploration on compound staining of Echinococcus granulosus protoscolices

    Institute of Scientific and Technical Information of China (English)

    李伟

    2006-01-01

    目的改进染色方法,制作结构清晰、颜色对比鲜明的细粒棘球绦虫原头蚴标本. 方法原头蚴经4%孔雀绿36 ℃初染48 h后,分别用石碳酸复红、稀释石碳酸复红、沙黄、伊红,醋酸洋红,苏木精洋红,盐酸洋红染液复染. 结果经4%孔雀绿初染的原头蚴标本分别用稀释石碳酸复红或沙黄染液复染5 s、伊红染液复染30 s、洋红染液复染5 min均能获得满意效果.染色后原头蚴头钩呈亮绿色,吸盘和其余实质部分着淡红色或浅紫红色,层次分明,立体感强.结论用孔雀绿初染,用红色染液复染制作原头蚴标本,染色效果好,操作简单,值得推广.

  7. 细粒棘球绦虫疫苗研究进展%Development in the research of Echinococcus granulosus vaccine

    Institute of Scientific and Technical Information of China (English)

    李文桂; 陈雅棠

    2003-01-01

    细粒棘球绦虫疫苗是防治囊型包虫病的一种重要途径,它包括死疫苗、分子疫苗、合成肽疫苗、DNA疫苗和重组伤寒沙门氏杆菌疫苗等,文章综述了这几种疫苗的研究进展.

  8. 胆汁对体外细粒棘球蚴原头节的作用%Effects of bile on Echinococcus granulosus protoscoleces in vitro

    Institute of Scientific and Technical Information of China (English)

    孙冯; 吕海龙; 王成华; 张晶; 雷颖; 彭心宇; 姜玉峰

    2014-01-01

    目的 探究不同浓度的胆汁在体外对细粒棘球蚴原头节生长的影响.方法 体外培养绵羊细粒棘球蚴原头节,将不同浓度的胆汁作用于原头节.0.1%伊红染色在倒置显微镜下检测原头节的活力.实验重复3次.结果 不同浓度的胆汁对原头节均有杀伤作用.浓度为100%和80%的胆汁对细粒棘球蚴原头节杀伤作用最明显,其中100%的胆汁在作用于原头节4d后死亡率就可达到100%.各浓度的胆汁与对照组比较差异有统计学意义(P<0.01).结论 胆汁对体外细粒棘球蚴原头节有抑制作用,而关于胆汁在体内的疗效有待于进一步研究.

  9. A Comparison between the Effects of Albendazole and Meben¬ dazole on the Enzymatic Activity of Excretory / Secretory Prod-ucts of Echinococcus granulosus Protoscoleces in Vitro

    Directory of Open Access Journals (Sweden)

    Seyed Jafar ADNANI SADATI

    2016-02-01

    Full Text Available Background: Hydatid cysts are formed in human body can be treated clinically by surgery or drugs such as albendazole (ABZ and mebendazole (MBZ. The purpose of this study was comparing the effects of ABZ and MBZ on glutathione-S-transferase, alkaline phosphatase and protease enzymes activities in protoscoleces of hydatid cyst. Methods: The culture supernatants containing the parasite Excretory / Secretory (E/S products were collected every 12 h for 72 h. The E/S products of treated samples with 1µg/ml ABZ and MBZ and the control one were collected and after centrifugation then protein concentrations were measured according to Bradford method. GST, ALP and protease activities of E/S products were assessed photometrically.Results: The mean of GST specific activity level in treated protoscoleces with ABZ and MBZ and in control group were obtained 69.44, 132.83 and 225.47U/mg/protein/ml respectively. The mean ALP activity level in treated protoscoleces with ABZ and MBZ and in control group were detected 19.22, 22.27 and 27.85 U/mg/protein/ml respectively. The protease activity level in treated protoscoleces with ABZ and MBZ were not detected. While the mean of protease activity level in control group was 7.61U/mg/proteins. Statistical analysis showed the significant difference between protein concentrations, the specific activities of GST, ALP and protease enzymes in treated protoscoleces in comparison with control group (P<0.05. Also, the significant difference were seen between specific activities of GST and ALP enzymes in treated protoscoleces with ABZ in comparison with treated group with MBZ (P<0.05.Conclusion: ABZ is more effective on the enzymes activities (GST and ALP as compared with MBZ. Keywords: Hydatid cyst protoscoleces, Albendazole, Mebandazole, Protease, Glutathione S-Transferase, Alkaline phosphatase

  10. 细粒棘球绦虫抗氧化蛋白TPx的免疫定位%Immunolocalization of the antioxidant protein TPx of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    侯秋莲; 王慧; 张壮志; 曹文尧; 张富春; 张文宝

    2007-01-01

    目的:研究抗重组细粒棘球绦虫抗氧化蛋白TPx(rEgTPx)多克隆抗体对天然EgTPx的结合活性,探讨EgTPx在原头蚴内的分布.方法:采集自然感染细粒棘球蚴的绵羊肝脏,在无菌条件下收集包囊内的原头蚴,经消化处理后制备石蜡切片.利用rEgTPx多克隆抗体,以间接免疫荧光法确定抗氧化蛋白EgTPx在原头蚴内的分布.结果:rEgTPx多克隆抗体能够特异性地结合天然EgTPx抗原表位,EgTPx广泛分布在原头蚴的体表皮层、皮下层和钙颗粒细胞内.结论:确定了EgTPx蛋白在细粒棘球绦虫原头蚴内的分布,为研究EgTPx在原头蚴发育的生物功能奠定基础.

  11. Ultrastructural changes in the tegument and gut of adult Fasciola hepatica following in vivo treatment with artesunate.

    Science.gov (United States)

    O'Neill, J F; Johnston, R C; Halferty, L; Brennan, G P; Fairweather, I

    2015-07-01

    An in vivo study in the laboratory rat model has been carried out to monitor changes to the tegument and gut of adult Fasciola hepatica following treatment with artesunate. Rats infected with the triclabendazole-resistant Oberon isolate were dosed orally with artesunate at a concentration of 200 mg/kg and flukes recovered 24, 48, 72 and 96 h post-treatment (pt). The flukes were processed for scanning and transmission electron microscope examination. Changes to the external surface were limited to swelling and blebbing of the interspinal tegument. There was one exception, a specimen recovered 72 h pt, which had completely lost the syncytium over the posterior region of the fluke. Internal changes to the tegumental syncytium and cell bodies were more severe and were apparent from 48 h pt onwards. Increased numbers of secretory bodies were present in the apical region of the syncytium, the basal infolds were swollen and sloughing of the apical plasma membrane was seen at 96 h pt. In the cell bodies, there was swelling and vesiculation of the cisternae of the granular endoplasmic reticulum (ger), swelling of the mitochondria and a decrease in secretory body production. Changes to the gastrodermal cells were evident from 24 h onwards. They comprised swelling and vesiculation of the ger cisternae, swelling and lysis of the mitochondria and accumulation of autophagic vacuoles and lipid droplets. The nuclei of the cells were karyopyknotic by 96 h pt. The gut was consistently more severely affected than the tegument at all time points pt, pointing to an oral route of uptake for artesunate. This study has provided information on the primary subcellular targets for drug action in the fluke.

  12. 细粒棘球蚴和多房棘球蚴的随机扩增多态性DNA分析%Analysis of Echinococcus granulosus and Echinococcus multilocularis by Randomly Amplified Polymorphic DNA

    Institute of Scientific and Technical Information of China (English)

    李文姝; 史大中

    2001-01-01

    利用RAPD技术,对细粒棘球蚴和多房棘球蚴两种包虫幼虫的基因组DNA进行扩增比较,对7种随机引物进行筛选,结果表明,在本实验条件下,其中3种引物OPB16、OPB11和OPB10的扩增产物可明显地将棘球蚴和泡球蚴在基因水平上区分开来.

  13. Cloning and Sequence Analysis of the CABPs Gene from Echinococcus granulosus%Echinococcus granulosus钙结合蛋白基因的克隆和序列分析

    Institute of Scientific and Technical Information of China (English)

    郭中敏; 徐劲; 陆家海; 单志新; 陈慧红; 余新炳

    2002-01-01

    目的获得细粒棘球蚴Calcium-binding protein(CaBPs)基因序列资料,进行序列分析,为包虫病免疫预防研究奠定基础.方法从包囊内获取细粒棘球蚴原头蚴(protoscolices),提取RNA,逆转录成cDNA,用特异引物扩增CaBPs基因;并将其克隆到T载体后进行序列测定和分析.然后登录Gene/Bank数据库.结果以cDNA为模板获得3个核苷酸长度不同的基因,分别是CaBP1为914bp,CaBP2为1095bp,CaBP3为1039bp.同源性比较结果表明:来自新疆羊源CaBPs基因序列和巴西(Brazil)羊源基因CaBPs同源性为90%以上;从cDNA获得的3个CaBPs基因序列的比较分析说明钙结合蛋白可能存在一个家族,有不同的成员组成.结论 CaBPs在E.granulosus虫体发育过程中具有十分重要的功能,到目前为止其机理尚不完全明了,CaBPs基因的获得为进一步研究其结构和功能,以及对包虫病的防治具有重要意义.

  14. [Indigenous microflora associated with the tegument of Triaenophorus nodulosus (Cestoda) and the intestine of its pike host].

    Science.gov (United States)

    Korneva, Zh V; Plotnikov, A O

    2006-01-01

    Investigations of the indigenous microflora associated with the mucous intestines of fish and its cestode parasites have been for the first time carried out using the methods of transmission and scanning electron microscopy. New data on the bacterial biodiversity in the cestode and its fish host are obtained. Nanobacteria and spirochaetes are for the first time revealed in a fish host together with the previously known bacteria forming the intestinal microflora of fish. Spirochaetes were shown to be associated with the intestines of a pike host only, while nanobacteria cover abundantly the surface of the apical parts of the intestinal microvilli and the apical parts of the microtriches in the cestode tegument. The similarity of the bacterial floras associated with the apical surface of the parasite tegument and the intestine of the host should be noted. At the same time, deeper bacterial communities represented by obligate symbionts are specific. Thus, there is a normal indigenous microflora in cestodes, associated with the tegumental surface. This symbiotic microflora has specific morphological features and provides the balance of relations in the parasite-host system.

  15. Fasciola hepatica tegumental antigens indirectly induce an M2 macrophage-like phenotype in vivo.

    Science.gov (United States)

    Adams, P N; Aldridge, A; Vukman, K V; Donnelly, S; O'Neill, S M

    2014-10-01

    The M2 subset of macrophages has a critical role to play in host tissue repair, tissue fibrosis and modulation of adaptive immunity during helminth infection. Infection with the helminth, Fasciola hepatica, is associated with M2 macrophages in its mammalian host, and this response is mimicked by its excretory-secretory products (FhES). The tegumental coat of F. hepatica (FhTeg) is another major source of immune-modulatory molecules; we have previously shown that FhTeg can modulate the activity of both dendritic cells and mast cells inhibiting their ability to prime a Th1 immune response. Here, we report that FhTeg does not induce Th2 immune responses but can induce M2-like phenotype in vivo that modulates cytokine production from CD4(+) cells in response to anti-CD3 stimulation. FhTeg induces a RELMα expressing macrophage population in vitro, while in vivo, the expression of Arg1 and Ym-1/2 but not RELMα in FhTeg-stimulated macrophages was STAT6 dependent. To support this finding, FhTeg induces RELMα expression in vivo prior to the induction of IL-13. FhTeg can induce IL-13-producing peritoneal macrophages following intraperitoneal injection This study highlights the important role of FhTeg as an immune-modulatory source during F. hepatica infection and sheds further light on helminth-macrophage interactions.

  16. Gamma-Ray Treatment of Echinococcus Protoscoleces prior to Implantation in Mice Reduces Echinococcosis

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    Qing Yuan

    2016-01-01

    Full Text Available Echinococcosis is a serious parasitic disease caused by Echinococcus tapeworms. Protoscoleces are sometimes released during surgical treatment for hydatid cysts, causing the recurrence of echinococcosis. Protoscoleces may be susceptible to radiation therapy. In this study Echinococcus protoscoleces were cultured in vitro and then divided into four different γ-ray irradiation dose groups (10 Gy, 20 Gy, 40 Gy, and 80 Gy and a blank group. The protoscoleces were then implanted into the abdominal cavity of mice. Four months later, we observed that the incidence and weight of cysts declined with the increase of irradiation dose. γ-ray irradiation can suppress the generation of Echinococcus originated from protoscolex, the reason of which is due to the damaging to the structure of Echinococcus. Irradiation may prevent echinococcosis recurrence after surgical removal of hydatid cysts.

  17. Echinococcus multilocularis--adaptation of a worm egg isolation procedure coupled with a multiplex PCR assay to carry out large-scale screening of red foxes (Vulpes vulpes) in Norway.

    Science.gov (United States)

    Davidson, Rebecca K; Oines, Oivind; Madslien, Knut; Mathis, Alexander

    2009-02-01

    Echinococcus multilocularis, causing alveolar echinococcosis in humans, is a highly pathogenic emerging zoonotic disease in central Europe. The gold standard for the identification of this parasite in the main host, the red fox, namely identification of the adult parasite in the intestine at necropsy, is very laborious. Copro-enzyme-linked immunosorbent assay (ELISA) with confirmatory polymerase chain reaction (PCR) has been suggested as an acceptable alternative, but no commercial copro-ELISA tests are currently available and an in-house test is therefore required. Published methods for taeniid egg isolation and a multiplex PCR assay for simultaneous identification of E. multilocularis, E. granulosus and other cestodes were adapted to be carried out on pooled faecal samples from red foxes in Norway. None of the 483 fox faecal samples screened were PCR-positive for E. multilocularis, indicating an apparent prevalence of between 0% and 1.5%. The advantages and disadvantages of using the adapted method are discussed as well as the results pertaining to taeniid and non-taeniid cestodes as identified by multiplex PCR.

  18. Alveolar Echinococcus species from Vulpes corsac in Hulunbeier, Inner Mongolia, China, and differential development of the metacestodes in experimental rodents.

    Science.gov (United States)

    Tang, Chong-Ti; Wang, Yan-Hai; Peng, Wen-Feng; Tang, Liang; Chen, Dong

    2006-08-01

    Adults of alveolar Echinococcus species with different uterine structures were collected from Vulpes corsac in the Hulunbeier Pasture of Northeastern China in 2001. They were Echinococcus multilocularis Leuckart, 1863 (type No. 3, similar to E. m. multilocularis), with vaselike uterus; Echinococcus cf. sibiricensis Rausch et Schiller, 1954 (type No. 1), with pyriform uterus; and Echinococcus sp. (type No. 2) with spherical uterus at segment top. The metacestode development in rodents also differed among those 3 parasites. In the case of E. multilocularis (type No. 3), many germinal cells grew on the inner surface of early cysts, most of which metastasized into host tissue to form brood vesicles or from the germinal cell layer on the inner surface of the vesicle wall. Cells also had an appearance of proliferating by means of alveolar buds from alveolar tissue that developed outward to form new alveolar foci. In Echinococcus cf. sibiricensis (type No. 1), the formation of alveolar vesicles was due to the metastasizing of germinal tissue into host tissue; protoscoleces grew in the center of alveolar vesicles. In type No. 2 (Echinococcus sp.), the formation of the alveolar vesicle was by multiplication of germinal cell layers on the inner surface of alveolar cysts; protoscoleces grew from the germinal cell layer and mesh in the vesicles. On the basis of uterine structure and on differences in development of metacestodes in experimental rodents, we propose that the 3 types of Echinococcus represent 3 independent species: E. multilocularis, Echinococcus sibiricensis, and Echinococcus sp. (type No. 2-as yet under study).

  19. The Speciesand Bioepiderniology of AlveOlar Echinococcus in Xinbaerhu West County, Eastern Inner Mongolia%内蒙古东部新巴尔虎右旗泡状肝包虫病原种类及流行学调查

    Institute of Scientific and Technical Information of China (English)

    唐崇惕; 舒利民; 唐亮; 钱玉春; 崔贵文; 康育民; 吕洪昌

    2001-01-01

    Spring (April) 12. 61%(15/119)~23.68%(9/38) and in Autumn (Sept.) 17. 60% (22/125)~6. 25% (1/16) were higher than that in Summer (July) 5.85% (20/342)~0% (0/34). 36 Vulpes corsac, 6 Vulpes vulpes and 9 Canis lupus were captured from grasslands of Xinbaerhu West County and were examined. Adults of Echinococcus granulosus were found from 3 (33. 3%) of 9 Cabis lupus, Adults of Echinococcus sibiricensis were found from 2 (5.56%) of 36 Vulpes corsac, one of the 2 positive corsac foxes was mixed infected with the adults of Echinococcus multilocularis (2.76 % ). Under experimental condition, 203 white mice (Kunmin strain) were orally infected with both gravid proglottids of E. sibircensis and E. multilocularis respectively and mixedly, which were collected from Vulpes corsac captured from Xinbaerhu West County. The alveolar cysts of E. sibiricensis were found from lungs of mice; many germinal cells masses existed in undiferrentiated embryonal cysts cavities of parasite, they metastasized off cysts into lung tissue of host to form brood capsules and protoscolice which manifested in 9th month postinfection; host reaction strong. While the alveolar cysts of E. multilocularis developed in livers of white mice; germinal cells layer which attached on inner surface of cysts wall proliferated by budding pattern to form new vesicles; the protoscolice which grew from germinal cells layer manifested in 5th and 6th months postinfection; host reaction slight. The larval stages of the two species Echinococcus in experimentally infected white mice were similar to those in naturally infected Microtus brandti.

  20. Genetic Variation of Echinococcus canadensis (G7) in Mexico

    Science.gov (United States)

    Rodriguez-Prado, Ulises; Jimenez-Gonzalez, Diego Emiliano; Avila, Guillermina; Gonzalez, Armando E.; Martinez-Flores, Williams Arony; Mondragon de la Peña, Carmen; Hernandez-Castro, Rigoberto; Romero-Valdovinos, Mirza; Flisser, Ana; Martinez-Hernandez, Fernando; Maravilla, Pablo; Martinez-Maya, Jose Juan

    2014-01-01

    We evaluated the genetic variation of Echinococcus G7 strain in larval and adult stages using a fragment of the mitochondrial cox1 gen. Viscera of pigs, bovines, and sheep and fecal samples of dogs were inspected for cystic and canine echinococcosis, respectively; only pigs had hydatid cysts. Bayesian inferences grouped the sequences in an E. canadensis G7 cluster, suggesting that, in Mexico, this strain might be mainly present. Additionally, the population genetic and network analysis showed that E. canadensis in Mexico is very diverse and has probably been introduced several times from different sources. Finally, a scarce genetic differentiation between G6 (camel strain) and G7 (pig strain) populations was identified. PMID:25266350

  1. First Report of Echinococcus equinus in a Donkey in Turkey.

    Science.gov (United States)

    Simsek, Sami; Roinioti, Erifylli; Eroksuz, Hatice

    2015-12-01

    A 2-year-old female donkey (Equus asinus) was euthanized in the Pathology Department of Firat University, Elazig, Turkey. Necropsy disclosed the presence of 7 hydatid cysts distributed throughout the lung parenchyma. One of those cysts represented the parasite material of the present study and was molecularly identified through sequencing of a fragment of cytochrome c oxidase subunit 1 (CO1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (NADH1) gene, as Echinococcus equinus. The generated CO1 sequence supports the presence of the dominant haplotype as has been described in Europe and Africa. The NADH1 sequence was found similar to sequences reported in equids in Egypt and the United Kingdom. The molecular identification of E. equinus in a donkey is being reported for the first time in Turkey.

  2. Topography and ultrastructure of the tegument of Bucephalus anguillae (Digenea: Bucephalidae), a parasite of the European eel Anguilla anguilla (Osteichthyen: Anguillidae).

    Science.gov (United States)

    Filippi, Jean-José; Quilichini, Yann; Foata, Joséphine; Marchand, Bernard

    2010-12-01

    The tegumental ultrastructure of the intestinal fluke Bucephalus anguillae was studied with the use of scanning and transmission electron microscopy. The surface of the tegument is covered by transverse ridges from which protrude numerous closely packed, digitated, and claw-shaped spines. Cobblestone-like units of the tegument were observed on the crescent-shaped formation of the rhynchus and at the posterior part of the body. Three types of sensory structures were examined, i.e., 2 uniciliated receptors and 1 without cilia. As anterior-posterior differences were observed, particular attention was given to spines and sensory receptors. Spine insertion zones and average cilia length are variable between anterior and posterior tegument areas. Ultrastructural study revealed that the tegument of B. anguillae has a typical syncytial organization with a distal cytoplasm lying over a basal matrix and cytons below. Cytoplasmic bridges allowed transit of secretory vesicles and granules. Diagrams of spines and sensory receptors were made to help in understanding the nature of these structures.

  3. Molecular Analysis of South African Ovine Herpesvirus 2 Strains Based on Selected Glycoprotein and Tegument Genes.

    Directory of Open Access Journals (Sweden)

    Fulufhelo Amanda Doboro

    Full Text Available Ovine herpesvirus 2 (OvHV-2, is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF, a generally fatal disease of cattle and other captive wild ruminants. Information on the OvHV-2 strains circulating in South Africa (SA and other African countries with regard to genetic structure and diversity, and pattern of distribution is not available. This study aimed to characterize the OvHV-2 strains circulating in SA using selected genes encoding glycoproteins and tegument proteins. To establish the genetic diversity of OvHV-2 strains, four genes, Ov 7, Ov 8 ex2, ORF 27 and ORF 73 were selected for analysis by PCR and DNA sequencing. Nucleotide and amino acid multiple sequence analyses revealed two genotypes for ORF 27 and ORF 73, and three genotypes for Ov 7 and Ov 8 ex2, randomly distributed throughout the regions. Ov 7 and ORF 27 nucleotide sequence analysis revealed variations that distinguished SA genotypes from those of reference OvHV-2 strains. Epitope mapping analysis showed that mutations identified from the investigated genes are not likely to affect the functions of the gene products, particularly those responsible for antibody binding activities associated with B-cell epitopes. Knowledge of the extent of genetic diversity existing among OvHV-2 strains has provided an understanding on the distribution patterns of OvHV-2 strains or genotypes across the regions of South Africa. This can facilitate the management of SA-MCF in SA, in terms of introduction of control measures or safe practices to monitor and control OvHV-2 infection. The products encoded by the Ov 7, Ov 8 ex2 and ORF 27 genes are recommended for evaluation of their coded proteins as possible antigens in the development of an OvHV-2 specific serodiagnostic assay.

  4. Antagonism of host antiviral responses by Kaposi's sarcoma-associated herpesvirus tegument protein ORF45.

    Directory of Open Access Journals (Sweden)

    Fan Xiu Zhu

    Full Text Available Virus infection of a cell generally evokes an immune response by the host to defeat the intruder in its effort. Many viruses have developed an array of strategies to evade or antagonize host antiviral responses. Kaposi's sarcoma-associated herpesvirus (KSHV is demonstrated in this report to be able to prevent activation of host antiviral defense mechanisms upon infection. Cells infected with wild-type KSHV were permissive for superinfection with vesicular stomatitis virus (VSV, suggesting that KSHV virions fail to induce host antiviral responses. We previously showed that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu et al., Proc. Natl. Acad. Sci. USA. 99:5573-5578, 2002. Here, using an ORF45-null recombinant virus, we demonstrate a profound role of ORF45 in inhibiting host antiviral responses. Infection of cells with an ORF45-null mutant recombinant KSHV (BAC-stop45 triggered an immune response that resisted VSV super-infection, concomitantly associated with appreciable increases in transcription of type I IFN and downstream anti-viral effector genes. Gain-of-function analysis showed that ectopic expression of ORF45 in human fibroblast cells by a lentivirus vector decreased the antiviral responses of the cells. shRNA-mediated silencing of IRF-7, that predominantly regulates both the early and late phase induction of type I IFNs, clearly indicated its critical contribution to the innate antiviral responses generated against incoming KSHV particles. Thus ORF45 through its targeting of the crucial IRF-7 regulated type I IFN antiviral responses significantly contributes to the KSHV survival immediately following a primary infection allowing for progression onto subsequent stages in its life-cycle.

  5. Molecular cloning and characterization of glutamine synthetase, a tegumental protein from Schistosoma japonicum.

    Science.gov (United States)

    Qiu, Chunhui; Hong, Yang; Cao, Yan; Wang, Fei; Fu, Zhiqiang; Shi, Yaojun; Wei, Meimei; Liu, Shengfa; Lin, Jiaojiao

    2012-12-01

    Glutamine synthetase catalyzes the synthesis of glutamine, providing nitrogen for the production of purines, pyrimidines, amino acids, and other compounds required in many pivotal cellular events. Herein, a full-length cDNA encoding Schistosoma japonicum glutamine synthetase (SjGS) was isolated from 21-day schistosomes. The entire open reading frame of SjGS contains a 1,095-bp coding region corresponding to 364 amino acids with a calculated molecular weight of 40.7 kDa. NCBIP blast shows that the putative amino acid of SjGS contains a classic β-grasp domain and a catalytic domain of glutamine synthetase. The relative mRNA expression of SjGS was evaluated in 7-, 13-, 21-, 28-, 35-, and 42-day worms of S. japonicum in the final host and higher expression at day 21, and 42 worms were observed. This protein was also detected in worm extracts using Western blot. Immunofluorescence studies indicated that the SjGS protein was mainly distributed on tegument and parenchyma in 28-day adult worms. The recombinant glutamine synthetase with a molecular weight of 45 kDa was expressed in Escherichia coli and purified in its active form. The enzyme activity of the recombinant protein was 3.30 ± 0.67 U.μg-1. The enzyme activity was highly stable over a wide range of pH (6-9) and temperature (25-40 °C) under physiological conditions. The transcription of SjGS was upregulated in praziquantel-treated worms at 2-, 4-, and 24-h posttreatment compared with the untreated control. As a first step towards the clarification of the role of glutamine synthetase in schistosome species, we have cloned and characterized cDNAs encoding SjGS in S. japonicum, and the data presented suggest that SjGS is an important molecule in the development of the schistosome.

  6. Loop-mediated isothermal amplificationfor rapid detection of dogs inefcted with Echinococcus species on copro-DNA%环介导等温扩增技术(LAMP)检测棘球绦虫感染犬粪DNA的研究

    Institute of Scientific and Technical Information of China (English)

    陈璐; 吾拉木·马木提; 陈洁; 古力帕丽·麦曼提依明

    2011-01-01

    目的 建立快速检测棘球绦虫感染犬粪便的DNA检测方法一环介导等温扩增技术(loop-mediated isothermal amplification.LAMP).方法 针对细粒棘球绦虫线粒体DNA Cox1基因6个位点特异性的设计4条LAMP引物,利用LAMP法和普通PCR方法同时检测棘球绦虫、肥胖带绦虫以及犬肠道内包括犬蛔虫和泡状带绦虫的DNA来验证LAMP方法的特异性;将转入Cox1基因片段的质粒作为标准阳性对照,并做系列10倍稀释同时用LAMP和PCR方法进行检测来比较两者的敏感性.此外,运用LAMP法检测13份犬粪DNA样本.LAMP扩增结果通过肉眼、电泳和酶切来鉴定.结果 以棘球绦虫DNA为模板的反应体系出现LAMP扩增反应产物,其它寄生虫和肥胖带绦虫没有出现LAMP扩增反应产物.LAMP检测Cox1基因的敏感性比普通PCR高出10倍,用于8只人工感染的犬检测结果均为阳性,而5只健康犬为阴性.结论 初步建立一种特异、敏感检测棘球绦虫感染犬的快速检测方法,不需要PCR仪、凝胶成像仪等昂贵的设备和电泳观察步骤,适于包虫病流行区和基层动物医院棘球绦虫感染犬的检测.%To control and prevent Echinococcus in areas where the disease was epidemic, we established a loop-mediated isothermal amplification (LAMP) assay for rapid detection of its DNA from dog faeces. The 4 primers which recognizing 6 distinct regions on the cytochrome c oxidase subunit 1 (Coxl) gene of Echinococcus granulosus were designed and used for LAMP assay. The specificity of LAMP assay was evaluated by using DNA extracted from E. granulosus and Taenia saginata and other dog intesinal parasites, such as Toxocara cants, Taenia Hydatigena. In addition, the sensitivity of LAMP assay was compared with that of conventional PCR using recombinant plasmid carrying E. granulosus Coxl gene fragment as standard template DNA after 10-fold serial dilution. Furthermore,copro-DNA samples from 13 dogs were also detected

  7. Enzymatic Shaving of the Tegument Surface of Live Schistosomes for Proteomic Analysis: A Rational Approach to Select Vaccine Candidates

    Science.gov (United States)

    Castro-Borges, William; Dowle, Adam; Curwen, Rachel S.; Thomas-Oates, Jane; Wilson, R. Alan

    2011-01-01

    Background The membrane-associated and membrane-spanning constituents of the Schistosoma mansoni tegument surface, the parasite's principal interface with the host bloodstream, have recently been characterized using proteomic techniques. Biotinylation of live worms using membrane-impermeant probes revealed that only a small subset of the proteins was accessible to the reagents. Their position within the multilayered architecture of the surface has not been ascertained. Methodology/Principal Findings An enzymatic shaving approach on live worms has now been used to release the most accessible components, for analysis by MS/MS. Treatment with trypsin, or phosphatidylinositol-specific phospholipase C (PiPLC), only minimally impaired membrane integrity. PiPLC-enriched proteins were distinguished from those released in parasite vomitus or by handling damage, using isobaric tagging. Trypsin released five membrane proteins, Sm200, Sm25 and three annexins, plus host CD44 and the complement factors C3 and C4. Nutrient transporters and ion channels were absent from the trypsin fraction, suggesting a deeper location in the surface complex; surprisingly, two BAR-domain containing proteins were released. Seven parasite and two host proteins were enriched by PiPLC treatment, the vaccine candidate Sm29 being the most prominent along with two orthologues of human CD59, potentially inhibitors of complement fixation. The enzymes carbonic anhydrase and APD-ribosyl cyclase were also enriched, plus Sm200 and alkaline phosphatase. Host GPI-anchored proteins CD48 and CD90, suggest ‘surface painting’ during worm peregrination in the portal system. Conclusions/Significance Our findings suggest that the membranocalyx secreted over the tegument surface is not the inert barrier previously proposed, some tegument proteins being externally accessible to enzymes and thus potentially located within it. Furthermore, the detection of C3 and C4 indicates that the complement cascade is initiated

  8. Ultrastructural and lectin-histochemical differences between the scolex/strobila and bladder teguments of the Taenia taeniaeformis strobilocercus.

    Science.gov (United States)

    Olson, E J; Oaks, J A; Osmundson, G D; Hildreth, M B

    2000-02-01

    The strobilocercus stage of the cat tapeworm Taenia taeniaeformis is surrounded by a single syncytial sheet of cytoplasm called the tegument. The outer membrane of the tegument covers both the scolex/strobila (S/S) and the bladder portions of the strobilocercus, but only the S/S region is resistant to intestinal digestion. It has been suggested that the glycocalyx, the surface-exposed glycoconjugates of the outer membrane, may serve to insulate underlying surface membrane components from digestion. In this study, we used lectin binding to test the hypothesis that the glycocalyx of the S/S is different from that of the bladder and that this may serve as the resistance mechanism of the S/S to digestion. Biotin-labeled lectins and an avidin-glucose oxidase detection system were applied to whole strobilocerci and to 1-microm epon-araldite plastic-embedded sections. Lectins bound to either both regions of the strobilocerci, to the S/S regions only, or did not bind at all. The restriction of some glycoconjugates to the glycocalyx of the S/S region only is consistent with our hypothesis.

  9. Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA Caracterização e otimização do líquido vesicular de Echinococcus granulosus bovino para utilização no imunodiagnóstico da hidatidose por ELISA

    Directory of Open Access Journals (Sweden)

    Oscar IRABUENA

    2000-10-01

    Full Text Available The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF obtained from fertile (FC and non-fertile cysts (NFC. Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC and NFC (PNFC, respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates.O objetivo do presente trabalho foi testar a composição química do líquido hidático bovino (BHCF obtido de cistos hidáticos férteis (FC e não férteis (NFC. Oito lotes de FC e 5 de NFC foram preparados e testados quanto à composição química, proteínas totais, proteínas derivadas do hospedeiro, conteúdo de carbohidratos e lipídeos. Não foram observadas diferenças entre os dois primeiros parâmetros sendo que o conteúdo de carbohidratos e lipídeos foi maior nos lotes FC do que nos NFC. Por SDS-PAGE foram observadas bandas de 38 e 116 kD somente nos BHCF do FC. Foram preparados dois «pools» de BHCF, um de FC (PFC e outro de NFC (PNFC. Os padrões de reconhecimento dos antígenos foram analisados por imunoblot. As condições físico-químicas para adsorção dos antígenos na superfície das placas de poliestireno (ELISA plates foram otimizadas. O valor de diagnóstico de ambos tipos de BHCF bem como a importância diagnóstica da oxidação das moléculas de carbohidratos com periodato foram analisadas por ELISA usando 42 amostras de soro de pacientes com hidatidose, 41 de pacientes com outras doenças e 15 de doadores aparentemente saudáveis. A reatividade de todos soros contra antígenos nativos foi analisada com e sem fosforilcolina livre. A melhor eficiência diagnóstica foi observada usando BHCF de PFC tratado com periodato usando tampão glicina com forte força iônica para sensibilizar as placas de ELISA.

  10. Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

    Science.gov (United States)

    Ivanova, Lyudmila; Buch, Anna; Döhner, Katinka; Pohlmann, Anja; Binz, Anne; Prank, Ute; Sandbaumhüter, Malte

    2016-01-01

    ABSTRACT Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs 1766WD1767 and 1862WE1863 are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17+)Lox-pUL36-WD/AA-WE/AA and HSV-1(17+)Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. IMPORTANCE Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several

  11. Correlation between definitive hosts of Echinococcus and echinococcosis in children in Qinghai plateau, China, 1990-2010%1990-2010年青海高原终宿主动物棘球绦虫感染与儿童棘球蚴病的相关研究

    Institute of Scientific and Technical Information of China (English)

    蔡辉霞; 王虎; 韩秀敏; 马霄

    2012-01-01

    The purpose of this study is to analyze the correlation between definitive hosts of Echinococcus and the infection of echinococcosis in children in different zones of Qinghai plateau, namely Qinghai southern plateau, Qilian mountain-He-huang valley and Qaidam Basin. Prevalence of echinococcosis in definitive hosts was identified by morphological observation on autopsy. The children with echinococcosis infection were detected by B-Ultrasound and KLISA. Prevalence of dogs infected with Echinococcus granulosus were 38. 71% (300/775), 49. 6% (124/250), and 9. 76% (4/41) in Qinghai southern plateau, Qilian mountain area and Qaidam Basin, respectively. While only in Qinghai southern plateau, dogs were found to be infected with Echinococcus mullilocularis with a prevalence of 16. 04% (98/611). Prevalence of the foxes infected with Echinococcus muhilocularis were 22. 89% (38/166) and 30. 77% (12/39) in Qinghai southern plateau and the region of Qilian mountain-He-huang valley. In these two places, wolves were found to be infected with Echinococcus granulosus, however, no Echinococcus mullilocularis was found. Prevalence rate of children aged between 5 -16 years with echinococcosis was 2. 32% (269/11 618), 0. 6% (50/8 275), 0. 12% (1/837), while sera positive rate of those children was 9. 49% (707/7 453), 3. 83% (289/7 544), and 3. 66% (28/765) in Qinghai southern plateau, Qilian mountain-Hehuang valley and Qaidam Basin, respectively. The prevalence and sera positive rates of Qinghai southern plateau were the highest in Qinghai plateau, and the significant difference was found between Qinghai southern plateau and Qilian mountain-Hehuang valley. The results suggest that children with echinococcosis are correlated with the infection degree in dogs infected with Echinococcus. The dogs infected with Echinococcus muhilocularis have significant influence on the transmission of children infected with alveolar echinococcosis in Qinghai southern plateau.%目的 分析1990-2010年

  12. Fluorescently Tagged pUL47 of Marek's Disease Virus Reveals Differential Tissue Expression of the Tegument Protein In Vivo

    Science.gov (United States)

    Arndt, Sina; Kaufer, Benedikt B.; Osterrieder, Nikolaus

    2012-01-01

    Marek's disease virus (MDV), a lymphotropic alphaherpesvirus, causes Marek's disease (MD) in chickens. MD is characterized by neurological signs, chronic wasting, and T cell lymphomas that predominate in the visceral organs. MDV replicates in a highly cell-associated manner in vitro and in vivo, with infectious virus particles being released only from feather follicle epithelial (FFE) cells in the skin. Virus produced and shed from FFE cells allows transmission of MDV from infected to naïve chickens, but the mechanisms or roles of differential virus gene expression have remained elusive. Here, we generated recombinant MDV in which we fused enhanced green fluorescent protein (EGFP) to the C terminus of the tegument protein pUL47 (vUL47-EGFP) or pUL49 (vUL49-EGFP). While vUL49-EGFP was highly attenuated in vitro and in vivo, vUL47-EGFP showed unaltered pathogenic potential and stable production of pUL47-EGFP, which facilitated direct analysis of pUL47 expression in cells and tissues. Our studies revealed that pUL47-EGFP is expressed at low levels and localizes to the nucleus during lytic replication in vitro and in lymphocytes in the spleen in vivo, while it is undetectable in tumors. In contrast, pUL47-EGFP is highly abundant and localizes predominantly in the cytoplasm in FFE cells in the skin, where MDV is shed into the environment. We concluded that differential expression and localization of MDV pUL47-EGFP tegument protein is potentially important for the unique cell-associated nature of MDV in vitro and in lymphocytes in vivo, as well as production of free virus in FFE cells. PMID:22190714

  13. Significant increase of Echinococcus multilocularis prevalencein foxes, but no increased predicted risk for humans

    NARCIS (Netherlands)

    Maas, M.; Dam-Deisz, W.D.C.; Roon, van A.M.; Takumi, K.; Giessen, van der J.W.B.

    2014-01-01

    The emergence of the zoonotic tapeworm Echinococcus multilocularis, causative agent ofalveolar echinococcosis (AE), poses a public health risk. A previously designed risk mapmodel predicted a spread of E. multilocularis and increasing numbers of alveolar echinococ-cosis patients in the province of L

  14. Spatial spreading of Echinococcus multilocularis in Red foxes (Vulpes vulpes) across nation borders in Western Europe.

    NARCIS (Netherlands)

    Vervaeke, Muriel; Giessen, Joke van der; Brochier, Bernard; Losson, Bernard; Jordaens, Kurt; Verhagen, Ron; Lezenne Coulander, Cor de; Teunis, Peter F M

    2006-01-01

    The occurrence of the fox tapeworm Echinococcus multilocularis in Red foxes was studied in Belgium and a neighbouring region in The Netherlands. A total number of 1202 foxes were analysed (1018 in Belgium and 184 in The Netherlands) of which 179 were infected with E. multilocularis (164 in Belgium a

  15. The presence of Echinococcus multilocularis in the red fox (vulpes vulpes) in the Netherlands

    NARCIS (Netherlands)

    van der Giessen JWB; Rombout Y; Limper L; van der Veen; Moolenbeek C; Franchimont H; Homan W; MGB

    1998-01-01

    Er zijn onderzoekingen gedaan naar het voorkomen van Echinococcus multilocularis bij vossen in Nederland van 1996 tot 1998. Deze parasiet is de oorzaak van alveolaire echinococcose, een ernstige parasitaire zoonose. Hiervoor zijn eerst 272 vossen onderzocht in het grensgebied met Duitsland en Belg

  16. Genetic structuring and differentiation of Echinococcus multilocularis in Slovakia assessed by sequencing and isoenzyme studies

    DEFF Research Database (Denmark)

    Snabel, V.; Miterpakova, M.; D'Amelio, S.

    2006-01-01

    Nucleotide sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and isoenzyme analysis were used to survey the genetic variability in Echinococcus multilocularis populations from Slovakia. A sample of 12 isolates acquired from 10 different districts from red foxes exhibited...

  17. A review of the current status of Echinococcus and hydatid disease,with notes on some informative achievements in China%我国棘球绦虫及棘球蚴病研究进展

    Institute of Scientific and Technical Information of China (English)

    林宇光; 卢明科; 洪凌仙

    2012-01-01

    There are seven Echinococcus pathogens of hydatid disease reported from the world, and five species are found in China. Among them, E. shiquicus, collected from eastern Tibetan plateau in Sichuan Province of China, is described as a new species by Xiao et al (2005). Its adult worm is the smallest one in Echinococcus spp. The majority of them contain a single immature proglottid and a single gravid proglottid, lacking mature proglottid. The Tibetan fox, Vulpes ferrilata , is confirmed as the final host of E. shiquicus, and the plateau pika, Ochotona curzoniae, serves as the intermediate host. In the host liver, the metacestode develops into unilocular hydatid cyst, with no daughter cyst. Domestic dog and human are not found to be infected with either adult or metacestode so far. E. granulosus is recognized with two forms on the basis of differences in host-specify. The Northern Form of E. granulosus distributes in the holarctic zone of tundra and boreal forest or Taiga. Its natural cycle is perpetuated by the predator-prey relationship existing between wolf and large deer. The Domestic Form (European Form) of E. granulosus reveals nearly cosmopolitan distribution, and its life cycle involves both domestic dog and ungulates as final and intermediate host respectively. The latter form appears to be the most important pathogen of hydatid disease in the world. In China, the unilocular dydatid disease pathogen belongs to the Domestic Form, but there probably exist complex infection with the Northern Form in some pasture lands at Northwest China. E. multilocularis or multilocular (alveolar) hydatid cyst is recognized as an important pathogen of zoonoses in the world. This cestode-pathogen distributes throughout the holarctic zone of tundra, involving Europe, Siberia, Northern Japan, subarctic islands and North America. The typical life cycle involves foxes and rodents. According to the published data from local hospitals or institutes of parasitic disease in China, during

  18. High level expression and identification of recombinant ferritin of Echinococcus groanolosus%细粒棘球蚴铁蛋白基因的克隆重组高效表达及免疫学初步研究

    Institute of Scientific and Technical Information of China (English)

    王娅娜; 丁淑琴; 王健; 张焱; 王洁; 王淑静; 赵巍

    2006-01-01

    目的构建细粒棘球蚴(Echinococcus granulosus Eg)铁蛋白(ferritin)基因的原核表达重组质粒并表达、纯化该重组蛋白,初步研究其免疫反应.方法将细粒棘球蚴铁蛋白基因亚克隆于表达载体pGEX-6p-1,转化重组体到大肠杆菌Bl21中,在异丙基-β-D-硫代半乳糖苷(IPTG)诱导下表达;用SDS-PAGE和Western-blot对表达产物进行初步鉴定,用切胶纯化的融合蛋白免疫BALB/c小鼠,通过Western-blot对该蛋白的免疫学特性进行初步研究.结果重组铁蛋白与GST以融合表达的形式在细菌中高效表达,表达产物为不可溶性的包涵体,蛋白分子量约为42 kD.融合蛋白Egferritin/GST能被细粒棘球蚴免疫的家兔血清和特异性小鼠抗血清所识别,同时特异性小鼠抗血清可识别天然抗原囊液、原头蚴可溶性蛋白中约19 kD的蛋白条带.结论成功地表达细粒棘球蚴重组铁蛋白,并且该蛋白有一定的免疫原性及抗原性.

  19. Reproduction and embryonic development in two species of squaliform sharks, Centrophorus granulosus and Etmopterus princeps: Evidence of matrotrophy?

    Science.gov (United States)

    Cotton, Charles F.; Dean Grubbs, R.; Dyb, Jan E.; Fossen, Inge; Musick, John A.

    2015-05-01

    Modes of reproduction and embryonic development vary greatly among the elasmobranchs, and prior studies have suggested that the energetic toll of embryogenesis in lecithotrophic species depletes embryonic organic matter by 20% or more. Matrotrophic species experience a lesser reduction or an increase in organic matter during embryogenesis. To investigate the maternal-embryonic nutritional relationship, we measured changes in organic matter from fertilization to near-parturition in embryos of Centrophorus granulosus and Etmopterus princeps. Embryos of C. granulosus experienced a reduction of 19.5% in organic matter, while E. princeps embryos experienced a reduction of 7.7% in organic matter over the course of embryonic development, suggesting some level of matrotrophy occurs, particularly for the latter species. Uterine villi were present in both species and developed concurrently with the embryos, increasing in length and thickness while becoming progressively vascularized. Embryos of C. granulosus were dissected to track the partitioning of water, organic matter, and inorganic matter to the liver, external yolk sac, internal yolk sac, digestive tract, and evicerated body throughout development. Mating was aseasonal for both species and spatially-mediated segregation by sex and maturity stage was observed. Ovarian cycles were concurrent for C. granulosus and consecutive for E. princeps. Size at maturity for C. granulosus was determined to be 111 cm TL for males and 143 cm TL for females, with an average fecundity of 5.3 embryos (range=4-7). Size at maturity for E. princeps was determined to be 56.5 cm TL for males and 61 cm TL for females north of the Azores and 54 cm TL for males and 69 cm TL for females near the Charlie Gibbs Fracture Zone. Average fecundity was 11.2 embryos (range=7-18) for this species. This is the first reporting of reproductive parameters for these two species, and the information provided will be valuable for informing stock assessment

  20. Impact of overgrazing on the transmission of Echinococcus multilocularis in Tibetan pastoral communities of Sichuan Province, China

    Institute of Scientific and Technical Information of China (English)

    WANG Qian; XIAO Yong-fu; Dominique A Vuitton; Peter M Schantz; Francis Raoul; Christine Budke; Maiza Campos-Ponce; Philip S Craig; Patrick Giraudoux

    2007-01-01

    Background Overgrazing was assumed to increase the population density of small mammals that are the intermediate hosts of Echinococcus multilocularis, the pathogen of alveolar echinococcosis in the Qinghai Tibet Plateau. This research tested the hypothesis that overgrazing might promote Echinococcus multilocularis transmission through increasing populations of small mammal, intermediate hosts in Tibetan pastoral communities.Methods Grazing practices, small mammal indices and dog Echinococcus rnultilocularis infection data were collected to analyze the relation between overgrazing and Echinococcus rnultilocularis transmission using nonpararnetric tests and multiple stepwise logistic regression.Results In the investigated area, raising livestock was a key industry. The communal pastures existed and the available forage was deficient for grazing. Open (common) pastures were overgrazed and had higher burrow density of small mammals compared with neighboring fenced (private) pastures; this high overgrazing pressure on the open pastures measured by neighboring fenced area led to higher burrow density of small mammals in open pastures. The median burrow density of small mammals in open pastures was independently associated with nearby canine Echinococcus multilocularis infection (P=0.003, OR=1.048).Conclusion Overgrazing may promote the transmission of Echinococcus multilocularis through increasing the population density of small mammals.

  1. A novel ex vivo immunoproteomic approach characterising Fasciola hepatica tegumental antigens identified using immune antibody from resistant sheep.

    Science.gov (United States)

    Cameron, Timothy C; Cooke, Ira; Faou, Pierre; Toet, Hayley; Piedrafita, David; Young, Neil; Rathinasamy, Vignesh; Beddoe, Travis; Anderson, Glenn; Dempster, Robert; Spithill, Terry W

    2017-08-01

    A more thorough understanding of the immunological interactions between Fasciola spp. and their hosts is required if we are to develop new immunotherapies to control fasciolosis. Deeper knowledge of the antigens that are the target of the acquired immune responses of definitive hosts against both Fasciola hepatica and Fasciola gigantica will potentially identify candidate vaccine antigens. Indonesian Thin Tail sheep express a high level of acquired immunity to infection by F. gigantica within 4weeks of infection and antibodies in Indonesian Thin Tail sera can promote antibody-dependent cell-mediated cytotoxicity against the surface tegument of juvenile F. gigantica in vitro. Given the high protein sequence similarity between F. hepatica and F. gigantica, we hypothesised that antibody from F. gigantica-infected sheep could be used to identify the orthologous proteins in the tegument of F. hepatica. Purified IgG from the sera of F. gigantica-infected Indonesian Thin Tail sheep collected pre-infection and 4weeks p.i. were incubated with live adult F. hepatica ex vivo and the immunosloughate (immunoprecipitate) formed was isolated and analysed via liquid chromatography-electrospray ionisation-tandem mass spectrometry to identify proteins involved in the immune response. A total of 38 proteins were identified at a significantly higher abundance in the immunosloughate using week 4 IgG, including eight predicted membrane proteins, 20 secreted proteins, nine proteins predicted to be associated with either the lysosomes, the cytoplasm or the cytoskeleton and one protein with an unknown cellular localization. Three of the membrane proteins are transporters including a multidrug resistance protein, an amino acid permease and a glucose transporter. Interestingly, a total of 21 of the 38 proteins matched with proteins recently reported to be associated with the proposed small exosome-like extracellular vesicles of adult F. hepatica, suggesting that the Indonesian Thin Tail week

  2. The Schistosoma mansoni tegumental-allergen-like (TAL protein family: influence of developmental expression on human IgE responses.

    Directory of Open Access Journals (Sweden)

    Colin M Fitzsimmons

    Full Text Available BACKGROUND: A human IgE response to Sm22.6 (a dominant IgE target in Schistosoma mansoni is associated with the development of partial immunity. Located inside the tegument, the molecule belongs to a family of proteins from parasitic platyhelminths, the Tegument-Allergen-Like proteins (TALs. In addition to containing dynein-light-chain domains, these TALs also contain EF-hand domains similar to those found in numerous EF-hand allergens. METHODOLOGY/PRINCIPAL FINDINGS: S. mansoni genome searches revealed 13 members (SmTAL1-13 within the species. Recent microarray data demonstrated they have a wide range of life-cycle transcriptional profiles. We expressed SmTAL1 (Sm22.6, SmTAL2, 3, 4, 5 and 13 as recombinant proteins and measured IgE and IgG4 in 200 infected males (7-60 years from a schistosomiasis endemic region in Uganda. For SmTAL1 and 3 (transcribed in schistosomula through adult-worms and adult-worms, respectively and SmTAL5 (transcribed in cercariae through adult-worms, detectable IgE responses were rare in 7-9 year olds, but increased with age. At all ages, IgE to SmTAL2 (expressed constitutively, was rare while anti-SmTAL2 IgG4 was common. Levels of IgE and IgG4 to SmTAL4 and 13 (transcribed predominantly in the cercariae/skin stage were all low. CONCLUSIONS: We have not measured SmTAL protein abundance or exposure in live parasites, but the antibody data suggests to us that, in endemic areas, there is priming and boosting of IgE to adult-worm SmTALs by occasional death of long-lived worms, desensitization to egg SmTALs through continuous exposure to dying eggs and low immunogenicity of larval SmTALs due to immunosuppression in the skin by the parasite. Of these, it is the gradual increase in IgE to the worm antigens that parallels age-dependent immunity seen in endemic areas.

  3. Schistosome tegumental ecto-apyrase (SmATPDase1 degrades exogenous pro-inflammatory and pro-thrombotic nucleotides

    Directory of Open Access Journals (Sweden)

    Akram A. Da’dara

    2014-03-01

    Full Text Available Schistosomes are parasitic worms that can survive in the hostile environment of the human bloodstream where they appear refractory to both immune elimination and thrombus formation. We hypothesize that parasite migration in the bloodstream can stress the vascular endothelium causing this tissue to release chemicals alerting responsive host cells to the stress. Such chemicals are called damage associated molecular patterns (DAMPs and among the most potent is the proinflammatory mediator, adenosine triphosphate (ATP. Furthermore, the ATP derivative ADP is a pro-thrombotic molecule that acts as a strong activator of platelets. Schistosomes are reported to possess at their host interactive tegumental surface a series of enzymes that could, like their homologs in mammals, degrade extracellular ATP and ADP. These are alkaline phosphatase (SmAP, phosphodiesterase (SmNPP-5 and ATP diphosphohydrolase (SmATPDase1. In this work we employ RNAi to knock down expression of the genes encoding these enzymes in the intravascular life stages of the parasite. We then compare the abilities of these parasites to degrade exogenously added ATP and ADP. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade these nucleotides. Suppression of SmAP or SmNPP-5 does not appreciably affect the worms’ ability to catabolize ATP or ADP. These findings are confirmed by the functional characterization of the enzymatically active, full-length recombinant SmATPDase1 expressed in CHO-S cells. The enzyme is a true apyrase; SmATPDase1 degrades ATP and ADP in a cation dependent manner. Optimal activity is seen at alkaline pH. The Km of SmATPDase1 for ATP is 0.4 ± 0.02 mM and for ADP, 0.252 ± 0.02 mM. The results confirm the role of tegumental SmATPDase1 in the degradation of the exogenous pro-inflammatory and pro-thrombotic nucleotides ATP and ADP by live intravascular stages of the parasite. By degrading host inflammatory signals

  4. WSV399, a viral tegument protein, interacts with the shrimp protein PmVRP15 to facilitate viral trafficking and assembly.

    Science.gov (United States)

    Jaree, Phattarunda; Senapin, Saengchan; Hirono, Ikuo; Lo, Chu-Fang; Tassanakajon, Anchalee; Somboonwiwat, Kunlaya

    2016-06-01

    Viral responsive protein 15 (PmVRP15) has been identified as a highly up-regulated gene in the hemocyte of white spot syndrome virus (WSSV)-infected shrimp Penaeus monodon. However, the function of PmVRP15 in host-viral interaction was still unclear. To elucidate PmVRP15 function, the interacting partner of PmVRP15 from WSSV was screened by yeast two-hybrid assay and then confirmed by co-immunoprecipitation (Co-IP). Only WSV399 protein was identified as a PmVRP15 binding protein; however, the function of WSV399 has not been characterized. Localization of WSV399 on the WSSV virion was revealed by immunoblotting analysis (in vitro) and immunoelectron microscopy (in vivo). The results showed that WSV399 is a structural protein of the WSSV virion and is particularly located on the tegument. Gene silencing of wsv399 in WSSV-infected shrimp reduced the percentage of cumulative mortality by 74%, although the expression level of a viral replication marker gene, vp28, was not changed suggesting that WSV399 might not involved in viral replication but viral assembly. Because it has already been known that tegument proteins function in capsid transport during viral trafficking and assembly, interaction between PmVRP15 on hemocyte nuclear membrane and the WSV399 viral tegument protein suggests that PmVRP15 might be required for trafficking and assembly of WSSV during infection.

  5. Genetic uniformity of Echinococcus multilocularis collected from different intermediate host species in Hokkaido, Japan.

    Science.gov (United States)

    Okamoto, Munehiro; Oku, Yuzaburo; Kurosawa, Tsutomu; Kamiya, Masao

    2007-02-01

    DNA from several isolates of Taenia taeniaeformis and Echinococcus multilocularis were digested with restriction enzymes and hybridized with digoxigenated oligonucleotide probe (CAC)5. Within the six wild isolates of Taenia taeniaeformis from Norway rats in Hokkaido, although several bands were common among isolates, fingerprinting patterns were specific to each isolate. In the case of E. multilocularis, regardless of hosts from which each isolate has been isolated, the five isolates collected from Hokkaido, showed the same fingerprinting pattern. These results indicate that there was very little genetic difference among these isolates. Although the fingerprinting pattern of E. multilocularis from St. Lawrence Is. was similar to that of the Hokkaido isolates, some bands were different from those in the Hokkaido isolates. Echinococcus multilocularis in Hokkaido seems to be closely-related genetically to that from St. Lawrence Is.

  6. Fasciola hepatica tegumental coat antigen suppresses MAPK signalling in dendritic cells and up-regulates the expression of SOCS3.

    Science.gov (United States)

    Vukman, K V; Adams, P N; O'Neill, S M

    2013-07-01

    Fasciola hepatica tegumental coat antigen (FhTeg) suppresses dendritic cell maturation and function by inhibiting IL-6, tumour necrosis factor (TNF)-α, IL-10 and IL-12 production and CD80, CD86 and CD40 cell surface marker expression in TLR4-stimulated dendritic cells. Fasciola hepatica also impairs dendritic cell function by inhibiting its phagocytic capacity and its ability to prime T cells. We have shown previously that activation of mast cells with bacterial ligands is also inhibited by FhTeg. Fasciola hepatica suppresses LPS-induced NF-κB and MAPK pathway (ERK) activation in these cells. Previously, we demonstrated that FhTeg induces expression of suppressor of cytokine signalling (SOCS)3, a negative regulator of the TLR pathway in mast cells. In this study, we show the same inhibitory effect of FhTeg on the activation of the other members of the MAPKs pathway (ERK, p38, JNK) in dendritic cells and demonstrate an enhanced expression of SOCS3, but not SOCS1, SOCS5 or PIAS3 in this process. These studies enhance our understanding of the immunomodulatory effect of helminth molecules on the TLR pathway.

  7. Effect of Praziquantel on the Tegument and Digestive Epithelium Ultrastructure of Brachylaima sp. Metacercariae Parasitizing the Edible Land Snail Cornu aspersum.

    Science.gov (United States)

    Gállego, Laia; Gracenea, Mercedes

    2016-10-01

    The edible land snail Cornu aspersum (Pulmonata: Stylommatophora) acts as second intermediate host in the cycle of Brachylaima sp. trematode, harboring free metacercariae in its kidney. The ingestion of undercooked infected snails by humans allows metacercariae to develop to adult stage in the intestine, causing brachylaimiasis. Praziquantel (PZQ) is the drug of choice to treat trematodiasis and it is effective against Brachylaima sp. metacercariae. The objective of this work was to assess, by transmission electron microscopy (TEM), the ultrastructural changes produced on the tegument and gastrodermis of the Brachylaima metacercariae recovered from C. aspersum treated with PZQ in comparison with untreated ones. Snails naturally infected by Brachylaima sp. metacercariae were treated by PZQ both individually and in groups. Metacercariae recovered from treated and control snails were processed for TEM. The tegument of untreated metacercariae was covered by a regular and thick glycocalyx. The syncytial epithelium contained abundant T2 secretory bodies appearing as membrane-bound biconcave disk-vesicles with high electron-dense and uniform content. The T2 secretory bodies located along the external area of the syncytium were mainly arranged at right angles to the apical plasma membrane. In treated metacercariae, the content of the T2 secretory bodies appeared altered, degenerating from high to low electron density, losing its uniform appearance and forming high electron-dense accumulations scattered around the periphery of the vesicle and separated by low electron-dense spaces. The presence of clusters was detectable in the central area. The characteristic arrangement of the T2 secretory bodies observed in untreated metacercariae was lost in treated ones. Vesicles near the apical area of the tegument no longer maintained their arrangement perpendicular to the apical plasma membrane. The characteristic arrangement of T2 secretory bodies and mitochondria was lost. The T2

  8. Basic and applied problems in developmental biology and immunobiology of cestode infections: Hymenolepis, Taenia and Echinococcus.

    Science.gov (United States)

    Ito, A

    2015-02-01

    Differentiation and development of parasites, including longevity in host animals, are thought to be governed by host-parasite interactions. In this review, several topics on the developmental biology of cestode infections are discussed from immunobiological perspective with a focus on Hymenolepis, Taenia and