Evaluation of a serological test system for the diagnosis of natural Echinococcus granulosus infection in dogs using E. granulosus protoscolex and oncosphere antigens

Serum antibody responses in feral or domesticated dogs naturally infected with Echinococcus granulosus or/and other common helminths were examined in an enzyme-linked immunosorbent assay (ELISA) using antigens prepared from E. granulosus protoscoleces or oncospheres. The ELISA using the protoscolex antigen was optimised with serums from experimental dogs monospecifically infected with E. granulosus or other helminth parasites, and helminth-free dogs. Anti-protoscolex antibody was detected in 16 of 22 (72.7%) serums from feral dogs with E. granulosus burdens ranging from 300 to 302,600 worms per dog. Seven serums from feral dogs which did not harbour E. granulosus at autopsy but which originated from an endemic hydatid region were tested using protoscolex antigen, and 1 serum gave a positive reaction. One hundred and two serums from dogs known never to have been infected with E. granulosus all gave negative reactions to protoscolex antigen. The sensitivity of the ELISA test proved to be superior to that which has been achieved by arecoline purging as a method of diagnosis for E. granulosus infection in dogs. For use of the assay in hydatid control or eradication campaigns, its sensitivity can be increased by choosing a lower absorbance discrimination value above which serums are regarded as having positive reactions. However, this does introduce positive reactions of some serums from dogs infected with helminths other than E. granulosus. In further development of the assay, use of defined recombinant antigens may improve both sensitivity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Echinococcus granulosus: Antigenic proteins in oncospheres and on the surface of protoscoleces identified by serum antibodies from infected dogs

Proteins present in oncospheres and on the surface of living protoscoleces of Echinococcus granulosus were radioiodinated by the lodogen technique and immunoprecipitated with sera from dogs with E granulosus infection and several categories of control sera. Analysis of immunoprecipitates was performed using sodium dodecyl-sulphate polyacrylamide gel electrophoresis to identify antigenic protein components specific for E granulosus. Sera from dogs with E granulosus infection identified antigenic proteins of around Mr 37,000, 30,000 or 22,000 in oncospheres, and proteins of around Mr 70,000, 43,000, 36,000, 27,000 (triplet), 20,000 or 14,000 on the surface of protoscoleces. These antigens appear to be both species- and stage-specific and may be useful for serological discrimination between 'current' and 'recent past' prepatent and patent E granulosus infections in dogs.     

用抗虫体表膜抗原抗体测定犬细粒棘球绦虫粪抗原

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...     

Specific antibody responses to Taenia hydatigena, Taenia pisiformis and Echinococcus granulosus infection in dogs

Groups of dogs reared free of both nematodes and cestodes were infected with Taenia hydatigena, Taenia pisiformis or Echinococcus granulosus. After infections with the Taenia spp became patent, dogs were purged to remove the worms. They were later reinfected and the second infections again removed by purging after patency. A group of 3 uninfected worm free dogs was kept as age-matched controls. The dogs were bled at intervals of 5 days and their serums tested for antibodies using the enzyme-linked immunosorbent assay (ELISA) with excretory/secretory (ES) antigens collected during in vitro incubation of evaginated scoleces (scolex ES antigen) and oncosphere antigens. Antibodies to scolex ES antigen were detected by 3 weeks after infection with each cestode species whereas antibodies to oncosphere antigen were not detected until about one week after eggs were found in the faeces of the infected dogs. Antibody responses to both oncosphere and scolex ES antigens decreased rapidly following removal of the worms by purging. Uninfected control dogs were invariably negative to both oncospheral and scolex ES antigens. There were cross-reactions between the serums from dogs infected with T. pisiformis and T. hydatigena when tested with scolex ES antigens, but oncospheral antigens showed a high degree of species specificity. Scolex ES antigens from E. granulosus were compared with those prepared from T. hydatigena and T. pisiformis for their ability to discriminate between antibodies in serums collected from dogs 31 and 32 days after infection with 100,000 protoscoleces of E. granulosus or dogs infected with Taenia spp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening for Echinococcus granulosus in dogs: Comparison between arecoline purgation, coproELISA and coproPCR with necropsy in pre-patent infections

Echinococcus granulosus is an important zoonotic infection of dogs. The purpose of the present study assessed the performance of two laboratory diagnostic methods with arecoline purgation and necropsy in infected dogs. In total 65 dogs were successfully experimentally infected with protoscoleces of E. granulosus from ovine infection. At 14-34 days post-infection groups of dogs were purged with arecoline hydrobromide and then necropsied. Faecal samples were tested at weekly intervals by coproantigen ELISA and coproPCR. The necropsy infection rate with E. granulosus was 89.2 per cent. Only 43 per cent of dogs were successfully purged after one arecoline dose; this percentage increased to 76.9% for two doses of arecoline purgation. E. granulosus coproantigen was detected by coproELISA in 82.8% of faeces. The positive and negative predictive values for coproantigen ELISA were 96 and 44.4% respectively. E. granulosus DNA was detected in pre-patent faecal samples by coproPCR in 25.9% of dogs. These results indicate that coproELISA is more sensitive than arecoline purgation for the detection of pre-patent E. granulosus infection in dogs. CoproPCR detected E. granulosus DNA in dog faeces by 21 days post-infection before egg production.     

犬细粒棘球绦虫粪抗原夹心ELISA检测方法的建立

为建立特异性和敏感性高的检验犬细粒棘球绦虫感染的方法。用细粒棘球绦虫(简称,Eg)成虫抗原分别免疫兔和绵羊,收集高免血清,纯化的高免抗体。依据抗体夹心ELISA工作原理,以兔抗体包被,检测感染Eg、不同犬带科绦虫的实验犬和空白犬粪样,绵羊抗体扑捉抗原,HRP标记兔抗绵羊IgG(1∶8 000)催化显色,用酶标仪测定OD 405nm吸光度,用以确定其特异性和敏感性。试验结果表明,敏感性为82.69%(43/52),特异性为85.88%(140/163);粪抗原在感染细粒棘球绦虫16d后可检出,最低抗原浓度为9.7ng/mL即犬感染5条成虫时可检测出阳性。该检测方法具有较好的特异性和灵敏性,为进一步研制检测细粒棘球绦虫虫体抗原ELISA检测试剂盒奠定了基础。     

Efficacy of divided doses of fospirate against immature Echinococcus granulosus infections in dogs.

Seventy dogs were used to evaluate efficacy of divided doses of fospirate against immature Echinococcus granulosus infections in dogs. Dose rates of 10 to 80 mg/kg given on 1 or 2 occasions resulted in the clearance of 70.6 to 94.5% of expected worm numbers. At least 3 treatments may be required before dogs can be free from E granulosus. Vomiting, which occurred in dogs given doses of 40 mg or more/kg, seemed to interfere with anthelmintic efficacy in some dogs.     

Comparative immunoelectrophoretic analysis of Echinococcus granulosus, Taenia hydatigena and Taenia pisiformis cyst fluid antigens by hyperimmune rabbit sera.

Cyst fluid antigens of Echinococcus granulosus, Taenia hydatigena and T pisiformis were examined by electrophoresis using homologous and heterologous hyperimmune rabbit sera to these antigens. While arc 5 forming antibodies were identified in sera from rabbits immunised with E granulosus and T hydatigena cyst fluids, antibodies responsible for forming precipitating antigen B band were detected in rabbit antisera to E granulosus, T hydatigena and T pisiformis antigens. T hydatigena cyst fluid appears to contain antigen similar to E granulosus antigen 5 and probably antigen B while T pisiformis cyst fluid has mainly an antigen close to hydatid antigen B.     

Use of two polysaccharide antigens in ELISA for the detection of antibodies to Echinococcus granulosus in sheep sera

Polysaccharide antigens were obtained from either the secretions produced during in vitro cultivation of Echinococcus granulosus protoscoleces or from mouse hydatid cyst membranes by phenol extraction. When either of these antigens was used in an enzyme-linked immunosorbent assay antibody activities were detected in sera from sheep infected 27 or more weeks earlier with at least 100 E granulosus eggs. These antibody responses were significantly higher (P less than 0.05) than those of sheep infected with Taenia hydatigena or T ovis and tested with the E granulosus antigens. Very high cross-reacting antibody responses in sera from sheep recently infected with T hydatigena were only detected with the protoscoleces secretions antigen. Neither antigen was sufficiently sensitive or specific for serodiagnostic use. However, when sera were first tested with one antigen and then with the other, and only sera that were positive in both tests were regarded as positive, the overall sensitivity and specificity of this two antigen method increased to about 80 per cent.     

Specificity of scolex and oncosphere antigens for the serological diagnosis of taeniid cestode infections in dogs

Groups of dogs raised free of helminths were monospecifically infected with the common nematodes Toxocara canis, Ancylostoma caninum and Trichuris vulpis. Serums from these dogs, and a group of dogs of unknown history but infected with Dirofilaria immitis and Dipylidium caninum, had levels of antibody to their homologous nematode antigens readily detectable by ELISA. No cross-reactions were apparent when these serums were tested by ELISA using oncosphere antigens of Taenia hydatigena, T. pisiformis and T. ovis, scolex excretory/secretory antigens of T. hydatigena, T. pisiformis and Echinococcus granulosus or protoscolex antigen of E. granulosus.     

Serological diagnosis of Echinococcus granulosus infection in sheep using cyst fluid antigen processed by antibody affinity chromatography

Serum antibody responses in sheep naturally or experimentally infected with Echinococcus granulosus and/or other larval cestodes were examined using an enzyme-linked immunosorbent assay (ELISA) with various antigens prepared from sheep hydatid cyst fluid ( SHCF ). Serum donors included: sheep experimentally infected with E. granulosus and their age-matched non-infected controls; sheep experimentally infected with other helminth parasites; sheep naturally infected with E. granulosus both from Tasmania and the Australian mainland; sheep from Tasmania naturally infected with larval cestodes other than E. granulosus; and naturally reared sheep completely free from infection with larval cestodes. Attempts were made to eliminate serological reactions which were not specific for E. granulosus by using a series of antibody affinity chromatography steps to deplete crude SHCF antigen; these included adsorption with a monoclonal antibody, 3EgH 29-2, removal of host IgG using rabbit anti-sheep IgG antibody, and removal of antigens which bound non-specifically to normal sheep immunoglobulin. The final affinity-depleted antigen product was designated AD SHCF . Specific serological reactivity in infected sheep was very low. Affinity depletion of SHCF using 3EgH 29-2 did not appear to increase the specificity of serological diagnosis of E. granulosus infection when experimentally infected sheep were compared with their non-infected controls provided the latter were age-matched with experimental animals. The other affinity adsorption steps significantly reduced non-specific background binding to antigen by normal sheep serum. Despite this reduction in background in the ELISA, only low levels of antibody could be detected in naturally-infected sheep.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening of dogs for Echinococcus granulosus coproantigen in a low endemic situation in Cyprus

In the framework of an echinococcosis surveillance and control programme in Cyprus, a commercial enzyme-linked immunosorbent assay (ELISA) (CHEKIT ECHINOTEST) designed for the detection of Echinococcus granulosus and E. multilocularis coproantigens was used in 1997-2000 for the investigation of large numbers of dogs. Most of the animals originated from areas where approximately 0.2% of the dogs had been found to be infected with E. granulosus in previous (1993-1996) arecoline surveys. The sensitivity of the coproantigen test was 83%, as determined in 35 dogs naturally infected with this cestode species. The specificity was 98% in 97 randomly selected dogs from Cyprus, but it was reduced to 80% in a group of 50 dogs, infected with Taenia spp. A total of 6551 dogs (mainly of rural origin) was examined, including three large groups (N: 2928, 1761 and 1800) from the Government Control Area (GCA) in southern Cyprus and a small group (N: 62) from the Non-Government Control Area (NonGCA) in the northern part of the island. Among the dogs from the GCA, 184 (2.8%) tested positive for coproantigen; coproantigen prevalences were 2.6, 4.9 and 1.1% in these three groups, and of 62 dogs from the NonGCA 8.1% were positive. The calculated true prevalences of E. granulosus in the dog population of the GCA ranged between 0.0 and 3.58%. The predictive values of the test, based on a 0.2% prevalence, was >99.9% for negative results, but very low (7%) for coproantigen-positive results. However, the relatively small number of coproantigen-positive dogs can be treated with praziquantel or the results can further be confirmed by arecoline purging.     

Comparative antigenic characterisation of Echinococcus granulosus and Taenia hydatigena cyst fluids by immunoelectrophoresis.

Hydatid cyst fluid from Echinococcus granulosus (HCF) and cyst fluid from Taenia hydatigena (TCF) cysts were compared in reciprocal immunoelectrophoresis (IEP) tests using homologous and heterologous antisera which were free of antibodies to host serum contaminants. The antigens for the E granulosus arc 5 were demonstrated in TCF. Antibody activity to these and other antigens common to HCF and TCF was removed from homologous antisera by absorptions with the heterologous antigenic preparation. Antigens not shared by the two metacestodes fluids were then demonstrated by IEP tests. These findings are discussed in terms of their significance to phylogenetic and immunodiagnostic studies of these parasites in their immediate hosts.     

Detection of Echinococcus granulosus coproantigens in faeces from naturally infected rural domestic dogs in south eastern Australia

OBJECTIVE: To investigate the occurrence of Echinococcus granulosus in rural domestic dogs in farming areas around Yass, New South Wales, and Mansfield and Whitfield, Victoria. DESIGN: Faeces were collected per-rectally from farm dogs voluntarily presented by their owners in four farming districts in New South Wales and two in Victoria. PROCEDURE: Faeces were collected in the field, an extract prepared from each sample and E granulosus coproantigens detected in an ELISA. Farmers were also questioned about their dog feeding and worming practices. RESULTS: Echinococcus granulosus coproantigens were detected in 99 of 344 dogs (29%) from 95 farms in south eastern New South Wales and 38 of 217 dogs (17.5%) from 43 farms in Victoria. Cross-reactions between E granulosus coproantigen trapping antibody and coproantigens in faeces from dogs monospecifically infected with other species of intestinal helminthes (Taenia ovis, T hydatigena, T pisiformis, Spirometra ericacei, Dipylidium caninum, hookworm, Toxocara canis, Trichuris vulpis) were not evident. Dietary and worming data revealed many owners fed raw meat and occasionally offal from domestic livestock and wildlife to their dogs and few owners wormed their dogs frequently enough to preclude the chance of patent E granulosus being present in their dogs. CONCLUSION: Echinococcus granulosus occurs commonly in rural dogs in south eastern Australia and an education program promoting the public health importance of responsible management of rural dogs is urgently needed.     

Echinococcus granulosus in wildlife in and around the Kosciuszko National Park, south-eastern Australia

OBJECTIVE: To investigate the distribution of Echinococcus granulosus in wild dogs and foxes and hydatidosis in wildlife coexisting with foxes and wild dogs in and around Kosciuszko National Park. DESIGN: Prospective and ad hoc surveys by necropsy of definitive and intermediate hosts. PROCEDURE: Wild dogs and foxes were trapped at one location in the Kosciuszko National Park and at 7 locations around the periphery of the Park. Feral pigs, macropodid marsupials, wombats, and feral goats were collected at some of the same locations. The animals were humanely killed, their small intestines removed in the field, the contents collected, preserved and examined microscopically. All internal organs of intermediate hosts were examined for hydatid cysts. Unidentified lesions were examined histologically. RESULTS: Echinococcus granulosus tapeworms were found in wild dogs from all locations. Prevalence ranged up to 100% with worm burdens up to 300,000 worms. Prevalence in foxes ranged up to 50% in animals recovered from 5 locations. The worm burdens were usually less than 50 E. granulosus per fox. Hydatid cysts were found in all macropodid species. Prevalence (69%) and cyst fertility (100%) were highest in swamp wallabies (Wallabia bicolour). Prevalence of cysts in feral pigs ranged up to 49%. Less than 22% of the cysts were fertile. No cysts were found in any of the wombats or feral goats. CONCLUSION: Echinococcus granulosus occurs commonly in wildlife in and around the Kosciuszko National Park. High numbers of fertile cysts in swamp wallabies, a favoured dietary item for wild dogs in this region, suggests swamp wallabies are pivotal in maintaining transmission. Physical contact with wild dogs and foxes or accidental contact with wild canid faeces is a public health risk.     

Echinococcus granulosus in farm dogs and dingoes in south eastern Queensland

Twenty-nine farms with a prevalence of greater than 20% of hydatidosis in cattle were visited in south eastern Queensland between August and December 1982. All farms carried beef cattle but none sheep. Twenty-four had dingoes and wallabies but only 8 had feral pigs. On 17 farms either macropods were killed for dog food or dogs were suspected of hunting macropods or scavenging their carcases. Purge samples were collected from 45 dogs from 23 of the 29 farms visited. The Australian sylvatic strain of Echinococcus granulosus was found in low numbers in purged intestinal content from one dog from each of 2 farms. Also, 50 intestinal tracts from dingoes from southern Queensland were examined between October 1981 and November 1983. The sylvatic strain of E. granulosus was found in 36 dingoes, the Australian mainland domestic strain in 4, and a further 5 dingoes were infected but the strain was not identified. This work indicates that domestic dogs are probably not important definitive hosts for E. granulosus in south eastern Queensland but could be an occasional source of infection for man. Dingoes are the major definitive host and worm numbers can be very high. Small foci of the domestic strain of E. granulosus may be maintained in a cycle involving dingoes, macropods and possibly feral pigs in cattle raising areas of coastal Queensland.     

A preliminary study on the use of enzyme-linked immunosorbent assay (ELISA) for detection of Trichinella spiralis infections in dogs

An enzyme-linked immunosorbent assay (ELISA) was developed to detect Trichinella spiralis infections in dogs using rabbit anti-canine IgG-horseradish peroxidase prepared according to the improved periodate method and an antigen purified from T. spiralis larvae by Sephadex G-200 chromatography. Sixty-six canine sera were tested for trichinosis by the ELISA and it showed a detection rate which was significantly higher than that by trichinoscopy. This antigen of T. spiralis appeared not to cross-react with the sera of dogs infected with Ancylostoma caninum or Taenia spp. A comparison of ortho-phenylenediamine and 5-amino-2-hydroxybenzoic acid as substrates in the ELISA did not reveal a significant difference. Pieces of filter paper saturated with a defined quantity of whole blood can be substituted for serum as a source material for the test. The relationship between worm burden and the absorbance value in ELISA is discussed.     

Evaluation of three enzyme-linked immunosorbent assays (ELISAs) for the detection of serum antibodies in sheep infected with Echinococcus granulosus

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.     

Modelling the transmission of Echinococcus granulosus in dogs in the northwest and in the southwest of Morocco

Echinococcus granulosus (E. granulosus) infection was studied in 151 dogs in two regions of Morocco: 68 dogs in the northwest (Loukkos) and 83 dogs in the southwest (Tiznit). The mean prevalence rates of echinococcosis in dogs were 58.82% (46.23-70.63%) in Loukkos and 55.42% (44.10-66.34%) in Tiznit and the mean abundances of E. granulosus per dog were 75 (59-93) and 547 (504-595), respectively. The mean abundance of E. granulosus in dogs was fitted to a negative binomial distribution by the maximum likelihood techniques to define parameters. E. granulosus was aggregated in dogs in the two regions. The prevalence of infection and the abundance of E. granulosus in dogs were fitted to mathematical models in order to determine if the parasite population is partly regulated by definitive host immunity. The best fit was obtained with the models assuming the presence of immunity. The mean time of exposure to infection was similar in the two regions and ranged from 8 months to about 2 years. The infection pressures (number of E. granulosus) obtained per dog each year were 65 (8-294) in Loukkos and 476 (316-886) in Tiznit. The proportion of dogs susceptible to infection was still high along the life of the dogs in Loukkos, while it was not different from zero in old dogs of Tiznit.     

Detection of antiplatelet immunoglobulin in thrombocytopenic dogs

Indirect enzyme-linked immunosorbent assays (ELISA) were used to detect platelet-associated immunoglobulin in sera from dogs with immune-mediated thrombocytopenia (IMT) and/or other autoimmune syndromes. One ELISA, utilizing whole platelets as the antigen substrate, readily detected antibody associated with platelets, either as specific, antiplatelet antibody or as immune complexes. This assay apparently lacked specificity because of the position reactions with sera from dogs with miscellaneous autoimmune disorders and no concurrent thrombocytopenia. Although the second ELISA, utilizing immunoaffinity purified platelet antigens was not influenced as much by immune complexes, absorbance values apparently were slightly increased. However, a small number of dogs with non-thrombocytopenic autoimmune disease tested positive. Immunoadsorption and Western immuno-blotting techniques demonstrated a complex pattern of specificities for antiplatelet antibodies. Clinical significance of these findings is discussed.