Granulosus Antigen Research Articles

Cystic Echinococcosis: Aspects of Immune Response, Immunopathogenesis and Immune Evasion from the Human Host

Cystic echinococcosis (CE) is a neglected infectious disease caused by the larval stage of Echinococcus granulosus. It constitutes a major public health problem in developing countries. During CE, the distinguishing feature of the host-parasite relationship is that chronic infection coexists with detectable humoral and cellular responses against the parasite. In order to establish successfully an infection, E. granulosus releases molecules that directly modulate the host immune responses favoring a strong anti-inflammatory response and perpetuating parasite survival in the host. In vitro and in vivo immunological approaches, together with molecular biology and immunoproteomic technologies provided us exciting insights into the mechanisms involved in the initiation of E. granulosus infection and the consequent induction and regulation of the immune response. Here, we review some of the recent developments and discuss how these observations helped to understand the immunology of E. granulosus infection in man. Although the last decade has clarified many aspects of host-relationship in human CE, establishing the full mechanisms that cause the disease require more studies. We need to define more clearly the events that manipulate the host immune response to protect the E. granulosus from elimination and minimizing severe pathology in the host.

Development of orf virus as a bifunctional recombinant vaccine: Surface display of Echinococcus granulosus antigen EG95 by fusion to membrane structural proteins

The parapoxvirus, orf virus (ORFV) causes superficial skin lesions in infected sheep. Unattenuated ORFV is used globally to vaccinate against orf. Recombinant poxviruses are proven delivery systems and we investigated strategies to express the immunogenic Echinococcus granulosus peptide EG95 from ORFV with the aim of developing a recombinant bivalent vaccine. EG95 is an oncosphere protein of the cestode E. granulosus, a parasite responsible for causing cystic hydatid disease in a wide range of hosts including humans and grazing animals such as sheep. Recombinant viruses were produced in which EG95 was expressed by itself or fused to ORFV envelope-associated structural proteins 10 kDa and F1L. Infection studies in sheep showed that specific antibodies were produced against ORFV and EG95 and that the antibody levels against EG95 were comparable to that of animals immunized with purified EG95 in Quil A adjuvant, an immunization regime that is known to afford protection. A single exposure to the dual vaccine has potential for protecting lambs against orf and for priming against EG95 so as to respond strongly to a later injection of EG95 protein.

Microfluidic immunosensor with gold nanoparticle platform for the determination of immunoglobulin G anti- Echinococcus granulosus antibodies

This article describes a microfluidic immunosensor, developed for the detection of IgG antibodies specific to Echinococcus granulosus in human serum samples, which represents an alternative tool that can be used for the immunodiagnosis of hydatidosis in an automated way. Our device consists of a Plexiglas system with a central channel and a gold electrode. For immobilization of the E. granulosus antigen, a gold electrode was modified with the incorporation of gold nanoparticles. Immobilized antigen was allowed to react with IgG-anti- E. granulosus antibodies in samples, and these were quantified by horseradish peroxidase (HRP) enzyme-labeled secondary antibodies specific to human IgG using catechol (Q) as enzymatic mediator. HRP in the presence of hydrogen peroxide (H 2O 2) catalyzes the oxidation of Q to o-benzoquinone (P). The electrochemical reduction back to Q was detected on the gold electrode (AuE) at −0.15 V. The current obtained was proportional to the activity of the enzyme and to the concentration of antibodies of interest. The detection limit for electrochemical detection was 0.091 ng ml −1, and the within- and between-assay coefficients of variation were below 6.7%. The proposed system presents many benefits, the more relevant are: reduced complexity and costs that are considered as the most wanted features for the clinical-immunodiagnostic field.

Serodiagnosis of sheep hydatidosis with hydatid fluid, protoscolex, and whole body of Echinococcus granulosus antigens

Diagnosis of hydatidosis is based on immunodiagnostic methods along with radiological and ultrasound examinations. A great number of immunological assays have been developed for detection of anti-hydatid cyst antibodies. The principal intermediate host of Echinococcus granulosus in most endemic regions of the world is sheep. Antibodies to various antigens are detectable in the sera of some, but not all infected sheep. The objective of the present study was to develop a specific and simple antigen-based ELISA method for diagnosis of hydatidosis in sheep with different (hydatid fluid, protoscolices, and whole body of E. granulosus) antigens. A total of 100 sera were collected from sheep with hydatidosis proven by inspection of hydatid-infested livers and lungs of the sheep slaughtered in Mashhad abattoir. Hydatid fluid and protoscolex were isolated from livers or lungs of sheep with hydatid cyst in sterile conditions. Whole body of E. granulosus was isolated from intestine of infected dogs. Sera samples were examined by ELISA with different antigens. The results of antibody detection by indirect ELISA, using different antigens, showed that the hydatid fluid was the most effective antigen of those assessed for detection of infection with hydatidosis in sheep. Findings of this study indicated that antibody detection assay is a sensitive approach for diagnosis of hydatid cyst in sheep.

The Echinococcus granulosus Antigen B Gene Family Comprises at Least 10 Unique Genes in Five Subclasses Which Are Differentially Expressed

BackgroundAntigen B (EgAgB) is a major protein produced by the metacestode cyst of Echinococcus granulosus, the causative agent of cystic hydatid disease. This protein has been shown to play an important role in modulating host immune responses, although its precise biological function still remains unknown. It is generally accepted that EgAgB is comprised of a gene family of five subfamilies which are highly polymorphic, but the actual number of genes present is unknown.Methodology/Principal FindingsBased on published sequences for the family, we designed specific primers for each subfamily and used PCR to amplify them from genomic DNA isolated from individual mature adult worms (MAW) taken from an experimentally infected dog in China and individual larval protoscoleces (PSC) excised from a single hydatid cyst taken from an Australian kangaroo. We then used real-time PCR to measure expression of each of the genes comprising the five EgAgB subfamilies in all life-cycle stages including the oncosphere (ONC).Conclusions/SignificanceBased on sequence alignment analysis, we found that the EgAgB gene family comprises at least ten unique genes. Each of the genes was identical in both larval and adult E. granulosus isolates collected from two geographical areas (different continents). DNA alignment comparisons with EgAgB sequences deposited in GenBank databases showed that each gene in the gene family is highly conserved within E. granulosus, which contradicts previous studies claiming significant variation and polymorphism in EgAgB. Quantitative PCR analysis revealed that the genes were differentially expressed in different life-cycle stages of E. granulosus with EgAgB3 expressed predominantly in all stages. These findings are fundamental for determining the expression and the biological function of antigen B.

Sensitivität und Spezifität neuer kommerziell erhältlicher Tests zum Nachweis von Echinococcus-Antikörpern

Cystic echinococcosis (CE), caused by Echinococcus granulosus, and alveolar echinococcosis (AE), caused by E. multilocularis belong to the most serious parasitic diseases. Both forms of echinococcosis occur in Austria; in addition, imported cases are diagnosed and treated regularly in Austria. Diagnosis of echinococcosis is based on clinical symptoms, imaging techniques and particularly on the detection of specific antibodies in serum specimens of patients. For decades several companies have been providing commercial Echinococcus antigens and echinococcosis tests based on different methods, i.e. complement fixation test (CFT) and electrophoretic methods (CIEP, IEP) in the past and enzyme-linked immunosorbent assays (ELISA), western blot assays (WB) and indirect haemagglutination assays (IHA) in recent years. During the last years two studies have been carried out in our laboratory in order to evaluate the sensitivity and specificity of two commercial E. granulosus antigens (the synthetic p176 antigen, arc 5 antigen) and three commercial testkits (IHA from Dade Behring, IHA from Fumouze, ELISA from Novagnost-Dade Behring). Sera of patients with histologically and/or molecular biologically confirmed cystic or alveolar echinococcosis, of patients with other parasitic infections and of healthy people were tested comparatively for specific Echinococcus antibodies. The synthetic p176 antigen proved to be a highly specific but a insensitive antigen, whereas both the indirect haemagglutination assay as well as the Novagnost-ELISA showed a much higher sensitivity but only moderate specificity. Our studies demonstrated that neither the commercial antigens nor the test kits tested should be used as a primary test in a routine laboratory for the diagnosis of cystic echinococcosis or of alveolar echinococcosis.

Antibody response against hydatid fluid, protoscolex and whole body of Echinococcus granulosus antigens in lambs

Summary In this study, the immunogenicity of three types of antigens (hydatid fluid, protoscolices and whole body of E. granulosus) was investigated in lambs by ELISA. Sixteen 4-6-month-old lambs of mixed sexes were divided into 4 groups of 4 lambs (three immunized and one control group). Twelve lambs as immunized groups received 2 mg of hydatid fluid, protoscolices and whole body of E. granulosus antigens dissolved in 1 ml of PBS per immunization for each lamb, respectively. As an adjuvant, Freund's complete adjuvant (FCA) was mixed with antigens to form a water-in-oil emulsion which was inoculated subcutaneously on day 1 of the trial. Each control lamb was inoculated with a total of 2 ml of PBS emulsified in equal volumes of FCA. Lambs were boosted on day 28 with the same preparation as described above except that FCA was replaced by Freund's incomplete adjuvant (FIA). Three weeks after the second immunization, each lamb received a challenge infection with 2000 protoscolices intraperitoneally and also 10 gravid proglotid of E. granulosus orally. Sera were collected before and after immunization and serum antibodies were tested by ELISA. The results showed that the production of antibody had a significant difference between the test groups and the control (P 0.05), whereas, the level of antibody production between the lambs immunized with hydatid fluid and whole body of E. granulosus was different significantly (P<0.05). The results of this study showed that the antigens of whole body of E. granulosus might be a good candidate for immunization and diagnosis of hydatid cyst in the intermediate hosts of E. granulosus.

Evaluation of antibody against hydatid fluid, protoscolex and adult worms of Echinococcus granulosus antigens by ELISA in mice

Echinococcosis is one of the world’s relevant zoonoses, affecting both humans and their domestic animals and caused by infection with the metacestode stage of the dog tapeworm, Echinococcus granulosus. In the present study, twenty mice were randomly divided into 4 groups of 5. Each mouse in groups 1, 2 and 3 was immunized with 100 μg of hydatid cyst fluid protein, protoscolex and adult worms of E. granulosus proteins in 100 μl of phosphate-buffered saline which was emulsified with an equal volume of Freund’s complete adjuvant respectively. In group 4, mice were just immunized with above adjuvant in PBS. Mice were boosted four weeks after the first immunization with the same preparation and with Freund’s incomplete adjuvant. Three weeks later, each mouse was challenged with 2000 protoscolices intraperitoneally. Sera were collected before and after each immunization and serum antibodies were tested by ELISA. Results showed that the level of antibody in mice which were immunized with adult worms of E. granulosus on day 28 was roughly seven times higher than before immunization and was higher than hydatid cyst fluid and protoscolex. Level of antibody in mice immunized with adult worms of E. granulosus was also higher than in hydatid cyst fluid and protoscolex groups at day 49. The results of this article indicate that antigen of adult worms of E. granulosus can be used as a candidate for production of a vaccine.

Production of Monoclonal Antibodies Against the 8 kDa Subunit ofEchinococcus granulosusAntigen B (EgAgB8/2) Using DNA Immunization

Cystic echinococcosis (CE), an endemic cosmopolitan zoonotic helminthic disease caused by the larval stage of Echinococcus granulosus, lacks reliable diagnostic tools that fulfill the criteria of high sensitivity and specificity. Antigen B (AgB), a thermostable lipoprotein that constitutes a considerable fraction of the cystic hydatid fluid (HF), is being considered as a suitable source for vaccination and immunodiagnosis of CE due to its high specificity. Genetic immunization was used to immunize BALB/c mice with the second subunit of antigen B (EgAgB8/2) for the production of monoclonal antibodies (MAb). Fusion products between the spleen cells and myeloma cells produced six MAbs of the following isotypes: IgG2a (two clones), IgG2b (three clones), and IgM (one clone). The MAbs were tested for their specificity to crude sheep hydatid fluid (CSHF) versus other antigens prepared from other helminthic parasites including Toxocara canis, Acanthocheilonema viteae, Fasciola hepatica, Schistosoma mansoni, and Taenia. Five MAbs reacted with E. granulosus antigens, one showed cross reactivity with S. mansonia antigens, and one showed a high reactivity with E. granulosus but was cross reactive with all helminthic antigens tested. Using SDS-PAGE and immunoblotting under reducing conditions, all MAbs identified the four AgB subunits with molecular weights of 8, 16, 24, and 36 kDa. Further work on the specificity and sensitivity of these MAbs as well as their use in detecting circulating parasite antigens and in antigen purification will be assessed in future studies.

Serological prevalence of echinococcosis and risk factors for infection among children in rural communities of southern Ningxia, China

To assess the usefulness of serology as an indicator of Echinococcus transmission for developing preventive measures against echinococcosis in rural communities. Cross-sectional survey in 2002 among 861 children aged 7 to 18 years in Xiji County, Ningxia, China. Before ultrasound abdominal examination, a questionnaire was used to identify socioeconomic, sanitary and hygiene risk factors for echinococcal infection; filter paper blood samples were collected from each child for specific antibody detection using EmP (Echinococcus multilocularis) and EgB (Echinococcus granulosus) antigens. Transmission of both E. multilocularis and E. granulosus occur in this area. Serological prevalence was far higher than disease prevalence in this age range. We found no gender-related differences of seroprevalence among the children, suggesting an equal chance of exposure to echinococcal egg-contaminated environments. The seroprevalence distribution was associated with changes in the ecology of wild hosts for E. multilocularis contamination, and with changes in socio-geographic features of the communities for E. granulosus contamination. Serological data obtained for children in mass surveys of echinococcosis appear to be a comprehensive and useful tool to monitor changes of transmission dynamics in humans and provide 'warning signals' to decision makers for the instigation of specific control measures against the disease.

Molecular cross-talk in host–parasite relationships: The intriguing immunomodulatory role of Echinococcus antigen B in cystic echinococcosis

Cystic echinococcosis (CE), a zoonosis caused by the development of Echinococcus granulosus tapeworm larvae in the internal organs of ungulates and humans, continues to pose a major public health burden in underdeveloped and industrialised areas worldwide. Research designed to improve parasitic disease control and find out more about parasite biology has already identified a number of E. granulosus antigenic molecules. The major E. granulosus immunomodulant antigen isolated from hydatid fluid is antigen B, a 120kDa polymeric lipoprotein consisting of various 8kDa subunits. By inhibiting elastase activity and neutrophil chemotaxis and eliciting a non-protective Th2 cell response, antigen B helps the parasite evade the human response. In this review, we briefly discuss current information on the molecular characteristics and immunomodulatory properties of E. granulosus antigen B. Besides focusing on findings that provide intriguing insights into the complex interplay between host and parasite, we suggest how this information could extend the current therapeutic options in inflammatory diseases.

Echinococcus granulosus antigen B gene family: Further studies of strain polymorphism at the genomic and transcriptional levels

Echinococcus granulosus AgB gene family is constituted by five gene loci. In a previous study, we analyzed the strain variation of EgAgB2/B4 sequences. Here, we have analyzed, by SSCP and sequencing, 250 genomic clones of EgAgB1/B3/B5 gene cluster from five E. granulosus strains. Several new EgAgB genomic variants were found. EgAgB1 and EgAgB3 genomic sequences grouped E. granulosus strains by phylogenetic tools in two clusters: one formed by G1/G2 and the other by G5, G6/G7 strains, in accordance with other molecular markers. EgAgB5 genomic and cDNA sequences were only found in G1/G2 cluster. Reverse transcription-PCR analysis using subunit specific primers revealed that all the EgAgB genes were transcribed in G1 and G7 strains with the exception of EgAgB5 transcripts that were not detected in G7 strain. Interestingly, AgB2 transcripts that were probably processed by an aberrant splicing mechanism leading to a non-functional B2 protein were found in G7 strain.

Self-assembly and structural characterization of Echinococcus granulosus antigen B recombinant subunit oligomers

Echinococcus granulosus antigen B is an oligomeric protein of 120–160 kDa composed by 8-kDa (AgB8) subunits. Here, we demonstrated that the AgB8 recombinant subunits AgB8/1, AgB8/2 and AgB8/3 are able to self-associate into high order homo-oligomers, showing similar properties to that of parasite-produced AgB, making them valuable tools to study AgB structure. Dynamic light scattering, size exclusion chromatography and cross-linking assays revealed ~ 120- to 160-kDa recombinant oligomers, with a tendency to form populations with different aggregation states. Recombinant oligomers showed helical circular dichroism spectra and thermostability similar to those of purified AgB. Cross-linking and limited proteolysis experiments indicated different degrees of stability and compactness between the recombinant oligomers, with the AgB8/3 one showing a more stable and compact structure. We have also built AgB8 subunit structural models in order to predict the surfaces possibly involved in electrostatic and hydrophobic interactions during oligomerization.

Serological diagnosis of canine alveolar echinococcosis

Canine alveolar echinococcosis (AE) is a rare liver disease caused by the larval stage (metacestode) of E. multilocularis. Its diagnosis so far is rather difficult and invasive. Imaging techniques for the diagnosis have been described recently and in this study, the serological diagnosis of canine AE was evaluated in 30 dogs with confirmed AE using ELISA based on seven different antigens ( E. multilocularis-antigens: Em2G11, EmII/3–10, protoscolex (EmP), excretory/secretory and adult integument (EmAd/I); E. granulosus antigens: hydatid fluid (EgHF) and antigen B (EgAgB)) and by the detection of circulating Em2G11-antigen. EmII/3–10 and Em2G11-based tests detected AE in 50 and 53%. The combined serological investigation for specific circulating Em2G11-antigen and specific antibodies against this antigen showed a sensitivity of 77%. This strategy is useful in animals with large parasite masses and ascites. The highest sensitivities for AE were obtained by the EmAd/I and the EmP antigens (97 and 93% sensitivity) with high specificities in the control group of 76 dogs (100 and 98.7%, respectively). Heterologous E. granulosus antigens (EgHF and EgAgB) showed low sensitivities (43 and 50%, respectively) with high numbers of unspecific reactions (>16%) in the control group. In the follow-up of four dogs, the serological reaction patterns against the antigens Em2G11, EmP and EmAd/I showed some correlations with the development of the parasite mass. Intestinal infections cannot be differentiated from AE by most antigens (except EmII/3–10) and have to be excluded by the absence of coproantigens or DNA of E. multilocularis in faecal samples.

Echinococcus multilocularis: Developmental stage-specific expression of Antigen B 8-kDa-subunits

Antigen B (AgB) initially found in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa. Four genes encoding these 8-kDa-subunits have been identified from E. granulosus. In this study we isolated five genes encoding 8-kDa-subunits of AgB from Echinococcus multilocularis. Sequence comparison of isolated cDNA clones demonstrated that one of these five clones was completely identical to EmAgB8/ 1 which had been isolated previously by our group, and three of them were 94.5, 90.8, and 91.9% homologous to E. granulosus antigen B 8-kDa subunit genes, EgAgB8/ 2, EgAgB8/ 3, and EgAgB8/ 4, respectively. The remaining clone shared 51–58% homology with the nucleotide sequences of AgB genes. Gene-specific RT-PCR and Western blot analyses revealed that these genes were expressed in a developmentally regulated manner in E. multilocularis vesicles, protoscoleces, and immature adult worms. Possible functions of different expression manners are also discussed.

Endoscopic Sphincterotomy as Non-Surgical Treatment Option for Cholangitis Caused by Hepatic Hydatid Disease

Parasitosis-associated obstruction of the biliary system is a rare cause for acute cholangitis. We report the case of a North African patient presenting with right upper abdominal pain radiating into the right shoulder. An initial abdominal ultrasound scan showed several cystic lesions in the liver which were suspicious for hydatid cysts, however no dilatation of the extra- und intrahepatic bile ducts. Positive serology for Echinicoccus granulosus antigens and antibodies confirmed the diagnosis. Within one week, obstructive jaundice and cholangitis developed. Given the working hypothesis of biliary compression by hydatid cysts, a non-invasive therapy with antibiotics and antihelmintics was initiated. Following a further rise of cholestasis parameters under treatment with ceftriaxon and albendazole, an ERCP was performed. The papilla vateri was occluded by a whitish-greenish hard structure (demonstration). The injection of contrast medium showed a wide dilatation of the common bile duct and multiple contrast-free areas (demonstration). After papillotomy, a worm-like whitish-greenish structure (demonstration) followed by black bile fluid, pus and whitish debris poured into the duodenum. An endoscopic collection of the mass failed because of direct drainage of the structure into the distal duodenum. Thus, stool examinations were performed during the following days resulting in detection of the worm-like mass. Both histological and microbiological examinations revealed that the structure represented membrane parts of the known echinoccosis (demonstration). The further post-interventional follow-up was uneventful. After a stepwise normalization of the cholestasis and inflammation parameters, the patient could be discharged under continuous therapy with albendazole. Conclusion: Beside an outside compression of the biliary system caused by echinococcal cysts, an biliary obstruction caused by echinococcal elements has to be excluded in circumstances when increased cholestasis parameters and dilated bile ducts are observed in association with positive echinococcus serology. In our case, this obstruction could be corrected by endoscopic papillotomy without further need for surgical intervention.

High polymorphism in genes encoding antigen B from human infecting strains of Echinococcus granulosus

Echinococcus granulosus antigen B (AgB) is encoded by a gene family and is involved in the evasion of the host immune response. E. granulosus exists as a number of strains (G1-G10) that differ in biological characteristics. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and transcription profile of AgB in 5 E. granulosus strains. Twenty-four genomic sequences were isolated and clustered in 3 groups related to 2 of the 5 reported AgB genes. AgB4 genes were present in almost all strains, whereas AgB2 were present as functional genes exclusively in G1/G2 cluster, and as non-functional genes in G5 and the G6/G7 cluster, suggesting inter-strain variation. The AgB transcription patterns, analysed by RT-PCR, showed that AgB2 and AgB4 genes were transcribed in G1, while only the AgB4 gene was transcribed in G7 strain. Cysts from the same strain or cluster shared more genomic and cDNA variants than cysts from different strain or cluster. The level of nucleotide and deduced amino acid sequence variation observed is higher than that reported so far for coding genes of other helminths. Neutrality was rejected for AgB2 genes. These data show the genetic polymorphism of antigen-coding genes among genetically characterized strains of E. granulosus.

Experimental alveolar echinococcosis in pigs, lesion development and serological follow up

Liver lesions were found in 6/6 pigs 7 months after oral inoculation with 5000 or 35,000 Echinococcus multilocularis eggs. However, lesion morphology differed considerably among the animals. The largest lesions (3–8 mm in diameter) were found in a single pig and smaller lesions (1.5–3 mm) in three pigs. These lesions were clearly circumscribed and had pronounced central necroses and dystrophic calcifications. In contrast, most of the smallest (usually <1.5 mm in diameter) found in two other pigs, had small compact fibrotic areas and blurred borders with obvious fibrous infiltrations into the interlobular tissues. E. multilocularis specific DNA was detected by PCR in all lesion types, but metacestode viability, as assessed by in vivo intraperitoneal inoculations in jirds, could not be demonstrated. Within 1 month post inoculation, all pigs developed specific IgG antibody responses against a battery of different antigens (metacestode, cyst fluid, and protoscoleces-derived native E. multilocularis and E. granulosus antigens, affinity purified Em2G11 antigen, antigen B, recombinant Em II/3–10 antigen). Two different reaction patterns were recorded. In the two pigs with the small lesions, pronounced reactions against all crude antigens with peaks 3–5 months p.i. and clearly elevated levels until the end of the experiment were noted. In all other pigs, antibody reactions remained low in all cases. In conclusion, we demonstrated two types of E. multilocularis metacestode development in pigs with distinct immunological response patterns.

Double-antibody sandwich ELISA using biotinylated antibodies for the detection of Echinococcus granulosus coproantigens in dogs

Here we present the diagnostic evaluation of an improved double-antibody sandwich ELISA for detecting Echinococcus granulosus antigens in dog faecal samples (coproantigens). A purified rabbit IgG fraction against protoscolex excretory–secretory products was used as primary antibody, and the same fraction conjugated with biotin as secondary antibody. In order to validate the sandwich ELISA, intra- and inter-assay precision, linearity, and recovery percentages were calculated. The diagnostic evaluation of the method was carried out by investigating faecal samples from 37 dogs naturally infected with E. granulosus, 15 Echinococcus-free dogs infected with Taenia spp., 82 dogs with non-taeniid helminths and 66 dogs free of helminth infections. An overall sensitivity of 78.4% and specificity of 93.3% were determined. Positive and negative predictive values were 72 and 95%, respectively, and the diagnostic efficiency was 90.5%. In addition, the sandwich ELISA detection limit was estimated in 5.12 ng ml −1. These results are highly satisfactory, allowing the use of this methodology in surveillance and control programs for intestinal echinococcosis in dogs.

Immunogenicity of two Echinococcus granulosus antigens EgA31 and EgTrp in mice

Here, we investigate in mice the immunogenicity of two antigens EgA31 and EgTrp which are expressed by the larval stage of Echinococcus granulosus. These recombinant proteins were used alone or as a mixture (EgA31-EgTrp) to immunize BALB/c mice. By flow cytometry, we have shown that the ratio CD4+/CD8+ of splenocytes were significantly higher in the antigen-immunized groups. The specific antibody in the sera and cytokine producing splenocytes was evaluated by enzyme-linked immunosorbent assay. EgA31, EgTrp or EgA31-EgTrp elicited high antibody titer of IgG and IgA. Among IgG isotypes, IgG1 was predominant for each antigen tested alone or combined. The production of IL-12, IFN-gamma, IL-10 and IL-6 cytokines was significantly higher in mice immunized with recombinant proteins. Our results suggest that, in BALB/c mice, a mixed Th1/Th2, response to EgA31, EgTrp and EgA31-EgTrp is obtained. The use of both antigens separately or in combination as candidate vaccine proteins is discussed.