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

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.     

A recombinant antigen with potential for serodiagnosis of Echinococcus granulosus infection in dogs

Antibodies specific for Echinococcus granulosus were affinity purified from dog serum on transfer blots containing putative serodiagnostic antigens. These antibodies and serum pools derived from dogs with E. granulosus infections were used to screen a lambda gt11 cDNA library constructed using E. granulosus protoscolex mRNA. Nine definitive antigenic clones were isolated and characterized, of which one (c10P1) gave strong specific reactions in plaque immunoassay with sera from E. granulosus infected dogs. These clones were all subcloned into the plasmid vector pGEX-1. Antigenicity of clones was confirmed in colony immunoassay and/or immunoblot. Glutathione S-transferase (GST) fusion proteins of individual subclones were produced in Escherichia coli, purified by affinity chromatography and evaluated in ELISA using sera from dogs with infections of E. granulosus, Taenia spp. or nematodes, and helminth-free dog sera. The GST fusion protein 10P1 showed a specificity of 100% in ELISA for diagnosis of E. granulosus infection in dogs despite its relatively low sensitivity. Further investigations aim to identify additional recombinant antigens and test 10P1 expressed in alternative expression systems to increase diagnostic sensitivity of the ELISA.     

Echinococcus multilocularis: purification and characterization of glycoprotein antigens with serodiagnostic potential for canine infection

We show that a conventionally purified glycoprotein component of Echinococcus multilocularis protoscolex, designated as Emgp-89, may be useful as a serodiagnostic antigen for detecting E. multilocularis infection in dogs domesticated in endemic areas. Emgp-89 was obtained from the parasite material by a simple procedure using Con A-agarose and subsequent gel filtration chromatography. The purified fraction showed a molecular weight of >4000 kDa upon gel filtration and reacted with a series of lectins that specifically bind to mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Subsequently, serodiagnostic performance of Emgp-89 was evaluated through enzyme-linked immunosorbent assays (ELISAs) by using sera from normal, domestic dogs and dogs infected with other helminths. Emgp-89 positively reacted with all 16 serum samples from E. multilocularis-infected dogs, thus showing that this antigen is highly sensitive. On the other hand, the specificity of Emgp-89-based ELISA, determined using 41 serum samples from dogs infected with other helminths, was relatively low (83%). As an attempt to improve the specificity of Emgp-89-based ELISA, we pretreated Emgp-89 with proteinase K or sodium periodate, expecting that these treatments would enable discrimination of true positives from false positives. The ELISA value increased after treatment with sodium periodate in most false-positive samples, whereas significant decreases were observed in sera from all dogs infected with E. multilocularis. Further evaluation of this antigen should be performed using sera from dogs infected with closely-related parasites, including taeniid cestodes, which are expected to prove that this serodiagnostic system is sufficiently specific for clinical and field applications.     

Diagnosis of canine echinococcosis: comparison of coproantigen detection with necropsy in stray dogs and red foxes from northern Jordan

The sandwich enzyme linked immunosorbent assay (ELISA) was used as a diagnostic test for Echinococcus granulosus infection by detecting coproantigens in 94 stray dogs Canis familiaris and eight red foxes (Vulpes vulpes) from northern Jordan. The results were analyzed in relation to actual helminth infection as revealed by necropsy. The infection rate of dogs with E. granulosus was 13.8% with a worm load ranging between 3-> 10,000 per infected dog. In contrast, eight of 13 E. granulosus infected dogs were coproantigen positive (overall sensitivity 61.5%). The sensitivity increased to 87.5% and 100% in dogs harboring > 20 and > 100 worms/dog, respectively. The specificity of coproantigen-ELISA was 91%. The greatest cross-reactivity was found in dogs infected with Dipylidium caninum. The positive and negative predictive values for the coproantigen-ELISA test were 50% and 94.2%, respectively. Thus, a coproantigen negative dog is most probably truly negative for E. granulosus. In contrast, a coproantigen positive dog may not be truly positive for E. granulosus, except if it has a high worm burden of > 100 worms/animal.     

Echinococcus multilocularis: Two-dimensional Western blotting method for the identification and expression analysis of immunogenic proteins in infected dogs

Domesticated dogs are an important potential source of Echinococcus multilocularis infection in humans; therefore, new molecular approaches for the prevention of the parasite infection in dogs need to be developed. Here, we identified and characterized an immunogenic protein of the parasite by using a proteome-based approach. The total protein extracted from protoscoleces was subjected to two-dimensional Western blotting with sera from dogs experimentally infected with E. multilocularis. Two protein spots showed major reactivity to the sera from infected dogs. The N-terminal amino acid sequences of these spots were identical to the deduced amino acid sequence of the product of the putative hsp20 gene. RT-PCR and Western blot analyses revealed that the putative hsp20 gene and its products were expressed in almost all stages of the parasite life cycle. Furthermore, recombinant hsp20 showed specific reactivity to the sera from infected dogs, suggesting that this molecule may facilitate the development of a practical vaccine.     

Production and characterization of monoclonal antibodies against excretory/secretory products of adult Echinococcus granulosus, and their application to coproantigen detection

Two IgM murine monoclonal antibodies (MAbs), EgC1 and EgC3, were produced against the excretory/secretory (E/S) products of Echinococcus granulosus adult worms. Immunoblotting revealed that both predominantly recognized a 50 kDa antigen in the somatic extract and an 85 kDa component in the E/S products. Immunolocalization showed that both MAbs reacted with the tegument of the parasite, and additionally EgC3 reacted with parenchyma and the tegument lining the external surface of the reproductive organs. A coproantigen capture ELISA was developed using a rabbit polyclonal antibody against E/S products from adult tapeworms as catching antibodies, and each one of MAbs as detecting antibody. The assays detected seven out of eight (EgC1), and eight out of eight (EgC3) experimentally infected dogs (worm burdens ranging from 61 to 57,500), using heat-treated samples obtained at prepatent period, and none (n=8) of helminth-free samples. Time course analysis showed that, after a 12-25 days lag, coproantigen levels rose above cut off O.D. values and typically peaked around 30 days post-infection (DPI) at the end of the experiment. One dog experimentally infected with Taenia hydatigena metacestodes was slightly detected as positive at different time points after 30 DPI. Both MAbs showed a similar pattern of recognition, but T. hydatigena antigens were undetectable for a longer period, and reached lower O.D. values with EgC1. Interestingly, fecal samples from two experimentally infected dogs with Echinococcus multilocularis were not recognized by the EgC1 assay, suggesting a potential value as species-specific diagnostic tool.     

Canine echinococcosis in Kyrgyzstan: using prevalence data adjusted for measurement error to develop transmission dynamics models

Echinococcosis is a major emerging zoonosis in central Asia. A cross-sectional study of dogs in four villages in rural Kyrgyzstan was undertaken to investigate the epidemiology and transmission of Echinococcus spp. A total of 466 dogs were examined by arecoline purgation for the presence of Echinococcus granulosus and E. multilocularis. In addition, a faecal sample from each dog was examined for taeniid eggs. Any taeniid eggs found were investigated using PCR techniques (multiplex and single target PCR) to improve the diagnostic sensitivity by confirming the presence of Echinococcus spp. and to identify E. granulosus strains. A total of 83 (18%) dogs had either E. granulosus adults in purge material and/or E. granulosus eggs in their faeces as confirmed by PCR. Three genotypes of E. granulosus: G1, G4 and the G6/7 complex were shown to be present in these dogs through subsequent sequence analysis. Purge analysis combined with PCR identified 50 dogs that were infected with adult E. multilocularis and/or had E. multilocularis eggs in their faeces (11%). Bayesian techniques were employed to estimate the true prevalence, the diagnostic sensitivity and specificity of the procedures used and the transmission parameters. The sensitivity of arecoline purgation for the detection of echinococcosis in dogs was rather low, with a value of 38% (credible intervals (CIs) 27-50%) for E. granulosus and 21% (CIs 11-34%) for E. multilocularis. The specificity of arecoline purgation was assumed to be 100%. The sensitivity of coproscopy followed by PCR of the isolated eggs was calculated as 78% (CIs 57-87%) for E. granulosus and 50% (CIs 29-72%) for E. multilocularis with specificity of 93% (CIs 88-96%) and 100% (CIs 97-100%), respectively. The 93% specificity of the coprological-PCR for E. granulosus could suggest coprophagia rather than true infections. After adjusting for the sensitivity of the diagnostic procedures, the estimated true prevalence of infection of E. granulosus was 19% (CIs 15-25%) and the infection pressure in the dog population was estimated to be 0.29 infections per year (CIs 0.014-0.75). Logistic regression analysis failed to identify any significant risk factors for infections for E. granulosus. After adjusting for the sensitivity of the test procedures, the estimated true prevalence for E. multilocularis was 18% (CIs 12-30%). Dogs that were restrained had a significantly lower prevalence of E. multilocularis of 11% (CIs 6-29%) compared with 26% in free-roaming dogs (CIs 17-44%) and independently within these groups hunting dogs were more likely to be infected than non-hunting dogs.     

Determination of an antigen suitable for enzyme-linked immunosorbent assay of the antibody to Bordetella bronchiseptica in guinea pigs

To establish an enzyme-linked immunosorbent assay (ELISA) technique for the serological diagnosis of infections caused by Bordetella bronchiseptica (B. bronchiseptica) in guinea pigs, the authors recently assessed the usefulness of three antigen preparations derived from the bacterial cell components: sonication antigen (S-Ag), cell surface antigen (C-Ag) and lipopolysaccharide antigen (L-Ag). The use of S-Ag for ELISA resulted in the most sensitive detection of the antibody to B. bronchiseptica from guinea pig sera immunized with killed bacteria and sera derived from naturally infected guinea pigs. Like C-Ag, S-Ag was highly specific, showing no cross-reactivity with Pasteurella multocida. Assessment of antibody formations in animals with experimentally induced infection using the three antigen preparations revealed that the antibody to S-Ag was formed earlier than antibodies to the other two antigen preparations following growth of the bacterium in the lungs. These results indicate that ELISA with S-Ag as an antigen is a useful tool for the serological diagnosis of infection by B. bronchiseptica.     

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

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.     

Infectivity of Echinococcus granulosus protoscolices under different conditions of temperature and humidity

The aim of this study was to examine the effect of different temperatures and humidities on the infectivity of Echinococcus granulosus protoscolices. Eighteen dogs (6 groups, n = 3 each) were fed with offal mince harbouring approximately 20,000 protoscolices of E. granulosus of different viabilities. Dogs were infected with E. granulosus protoscolices of: (1) 5% viability at -10 degrees C and 50% relative humidity (RH); (2) 30% viability at 0 degrees C and 60% RH; (3) 20% viability at +10 degrees C and 65% RH; (4) 15% viability at +30 degrees C and 75% RH; (5) 11% viability at +40 degrees C and 80% RH; (6) 68% viability (control group). Dogs in each group were necropsied at 29-49 days post-infection. Mean intensities of E. granulosus recovered from dogs were 256.7 +/- 60.3 in the second group; 32.7 +/- 7.1 in the third group; 40.3 +/- 15.5 in the fourth group and 1533 +/- 513 in the control group. However, no parasites were recovered from the first and fifth groups. Results obtained in the present study show that larval stages could be infective for 1 to 4 weeks during spring, autumn or winter months when maximal temperatures are approximately 0-10 degrees C. In conclusion, cold-storage depots in slaughterhouses and abattoirs where sheep carcasses might be discarded should be kept at -20 degrees C for 2-3 days, dogs should be properly controlled and adequate control programmes must be established in areas where the disease is endemic.     

Uncinaria stenocephala: antigenic characterization of larvae and adults worms using sera from naturally infected dogs

An antigenic characterization of the larval (somatic) and adult (somatic and excretory-secretory) antigens of Uncinaria stenocephala was made, employing immunoblotting and immunoblotting-inhibition with 10 selected sera from dogs naturally infected by this hookworm. The results indicated that each one of the three parasitic extracts has different antigenic components. Sixty percent of the dog sera consistently recognised four antigens of 20, 25, 30, and 38kDa of the larval extract and the immunoblotting inhibition showed that these antigens were only observed at the larval stage. All these results indicated that these antigens can be considered as major antigens and they could be useful for the immunodiagnosis of this parasitic infection.     

Morphological characteristics of Echinococcus granulosus derived from buffalo in Iran

Cystic echinococcosis is a significant parasitic disease in Iran, where a variety of animals act as intermediate hosts. In this study, 25 isolates of Echinococcus granulosus obtained from water buffalo from various parts of Iran were characterized on the basis of the morphology of the metacestode and the adult worm. The characteristics of protoscoleces from the different studied areas were nearly similar. They showed 2 rows of alternating large and small hooks and their shapes were smooth in outline. In contrast to the protoscoleces, the adult rostellar hooks showed a rough outline. The results showed that the total length, the blade lengths of the large and small hooks and the number of hooks are almost similar to those isolated from sheep but significantly different from those isolated from camels. The growth rates of adult E. granulosus (total worm length, segmentation and maturation) of buffalo origin, at 35 and 41 days post-infection of dogs, were nearly comparable to the common sheep strain. The form of the strobila and the morphology of the reproductive system were also similar to those of sheep origin. This suggests that the common sheep strain (G1) of E. granulosus may also use buffaloes as its intermediate host.     

Antigenic properties of cystatin-binding cysteine proteinases from Neodiplostomum seoulense

The antigenic properties of cysteine proteinases binding to cystatin were analyzed in Neodiplostomum seoulense, an intestinal trematode that infects humans and rodents in the Republic of Korea. Cystatin was found to effectively capture cysteine proteinases present in the crude extract of N. seoulense. The IgG levels against cystatin-binding cysteine proteinases in sera of mice infected with N. seoulense were higher than those in sera of mice immunized with the crude extract of N. seoulense. The production of IgG antibodies against cystatin-binding cysteine proteinases increased according to the length of infection period. In immunoblots of purified cystatin-binding proteinases, 2 molecules, approximately 50 kDa and 60 kDa, reacted with N. seoulense-infected mouse sera. Of the sera from patients infected with various helminths, those of sparganum-infected patients showed the strongest affinities for cystatin-binding cysteine proteinases of N. seoulense. Cystatin-binding cysteine proteinases of N. seoulense are suggested to be putative antigens for serodiagnosis of human N. seoulense infection.     

Echinococcus granulosus infection in urban areas

In a previous survey, carried out in 100 dogs in Rome by Tassi and Widenhorn (1977), two animals, which always lived in the urban area, were found infected by Echinococcus granulosus. An urban infection was therefore hypothized. During a following experiment 6 three-month old Beagle dogs were fed for nine months with raw meat sold in the butcheries of Rome as "food for pets". Two dogs of the same breed and origin were fed for the same period with canned food and were kept as control animals. At the end of the experiment three of the six dogs were found infected by E. granulosus, while the renmant three and the two controls resulted negative. An additional similar trial was carried out in Bologna. Three dogs were fed for seven months with the same kind of raw meat sold in three butcheries of Bologna, and a fourth dog was kept as control. At the end of the seven months also one of the three dogs was found infected by E. granulosus. These results seem to confirm the hypothesis of an urban infection by E. granulosus in dogs. Various possibilities of contamination of raw meat sold in butcheries as food for animals are discussed.     

The use of excretory and secretory antigens of the scolex of Taenia ovis for the serodiagnosis of infection in dogs

The excretory and secretory antigens from the evaginated scoleces of Taenia ovis were collected for 3 days in vitro, and used in an ELISA test to detect antibodies to T. ovis in the serum of dogs. When tested on sequentially collected sera, diagnostic ELISA values could be detected in many dogs 4 wk after infection, and remained for an average of a further 4 wk after worms were removed from dogs with an anthelmintic. Using an ELISA discriminant value that eliminated all false positives from 70 uninfected laboratory dog sera and from 57 uninfected farm dog sera, 54/62 true positives were found in sera from dogs infected with various numbers of T. ovis for various intervals. Sera from dogs infected with T. hydatigena gave 11/15 false positive reactions, whereas sera from 15 dogs infected with Echinococcus granulosus or 7 dogs infected with T. pisiformis were all negative. For T. ovis the test had a high repeatability, was not greatly influenced by the number of worms carried by the dog and higher titres were correlated with long-standing infections. Approximately 1,000 scoleces could be recovered from each experimentally infected sheep. Using the ELISA test with undiluted antigen and serum diluted 1:40, approximately 10 sera could be tested in duplicate with the excretions and secretions from each T. ovis scolex.     

Metalloproteinases in the larvae of Echinococcus granulosus.

Proteolytic activity in hydatid cyst fluid, cyst membranes and protoscoleces of E. granulosus was analyzed by electrophoresis in gelatin-containing polyacrylamide gels, including characterization with a set of protease inhibitors. All contained metalloproteinases in the range 60-120 kDa, with neutral/alkaline pH optima. Major activity was observed in hydatid fluid and the membranes (five bands) with both exhibiting similar electrophoretic patterns. The samples prepared from protoscoleces shared only some of these bands.     

Echinococcus granulosus down regulates the hepatic expression of inflammatory cytokines IL-6 and TNF alpha in BALB/c mice

Hydatid disease is caused by the metacestode of Echinococcus granulosus. Different experimental models have been used to understand hydatid disease. In current studies BALB/c mice were used to evaluate the hepatic response of IL-6 and TNF alpha triggered by Echinococcus granulosus. BALB/c mice were intraperitoneally infected with protoscoleces from E. granulosus; hydatid cysts appeared on the liver eight weeks after inoculation. The RNA extracted from hepatic sections was used for RT-PCR amplification with primers for IL-6, TNF alpha, IL-10, TGF beta and G3PDH. In situ cytokine expression was assessed by FISH. Complete parasite cysts on the liver surface were observed 16 weeks after infection; controls were negative. The expression of IL-6 and TNF alpha was normal at baseline and declined progressively eight weeks after infection; in some animals such expression was abrogated 16 weeks after infection. On the other hand IL-10 and TGF beta were increased progressively. Controls expressed the cytokines normally. Present results suggest that E. granulosus induces a local immunosupression probably mediated by IL-10 and TGF beta; therefore it seems possible that such a mechanism would assist the parasite in escaping the harmful host cell-mediated response.     

Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis

This study was performed to investigate whethere there is cross-reactivity between Dirofilaria immitis and three intestinal nematodes of dogs. In ELISA, D. immitis-infected dog sera obtained at the 4th molting stage (9-11 weeks) and microfilaremic stage (25-30 weeks) were shown to be highly reactive with crude extract of T. canis. In immunoblotting, some antigenic fractions, 44, 57, 88, 100 kDa of crude extract of T. canis, were found to be positive reaction with sera of dogs infected with D. immitis. However, little or no cross-reaction were observed between sera of D. immitis-infected dogs and crude extract antigen of T. vulpis or A. caninum. These result suggest that there are partial cross reaction between sera of D. immitis-infected dogs and the antigen of T. canis.     

Detection of Echinococcus granulosus coproantigens in Australian canids with natural or experimental infection

Coproparasitological and purging methods for diagnosing canids infected with the intestinal helminth Echinococcus granulosus, an important zoonotic parasite, are unreliable. Detection of coproantigens in feces of infected dogs by enzyme-linked immunosorbent assay (ELISA) is suitable for detecting patent and prepatent infections with a high degree of sensitivity and specificity. In the present study, natural and experimental infections in domestic and wild Australian canids were investigated using a coproantigen capture ELISA. Experimental infection of dogs with E. granulosus was detected at between 14 and 22 days postinfection (PI), and optical density (OD) values remained high until termination of experiments 35 days PI. After chemotherapy, coproantigen levels in infected dogs dropped rapidly, becoming negative 2-4 days after treatment. In experimentally infected red foxes (Vulpes vulpes), the coproantigen excretion profile was different, with ELISA OD levels peaking 15-17 days PI, then falling to low or undetectable levels by 30 days PI. Coproantigens were detected in the feces of naturally infected Australian wild dogs (dingoes, dingo/domestic dog hybrids) with infection levels ranging between 2 worms and 42,600. Preliminary data on the stability of coproantigen in dog feces exposed to environmental conditions indicated that there was no change in antigenicity over 6 days. The results suggest the coproantigen ELISA could be successfully used to monitor E. granulosus prevalence rates in Australian domestic dogs, foxes, and wild dogs.     

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.