Immunization against Taenia taeniaeformis in mice: identification of oncospheral antigens in polyacrylamide gels by Western blotting and enzyme immunoassay.
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Animals↗
Biomedical subjects
M D Rickard
Publications and source records attributed to M D Rickard.
Taenia taeniaeformis in mice: passive transfer of protection with sera from infected or vaccinated mice and analysis of serum antibodies to oncospheral antigens.
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Molecular vaccines against parasites.
Prophylactic vaccines can be expected to be one of the major practical outputs of parasitology research. Various groups within Australia have pursued the vaccine objective for several years, with particular emphasis on blood-stage falciparum malaria in man, intestinal helminths of sheep and cattle, cutaneous myiasis (blowfly strike) in sheep, cysticercosis in sheep and cattle, bovine babesiosis, and cattle ticks. Other vaccine programmes are concerned with giardiasis, filariasis, toxoplasmosis, fascioliasis, coccidiosis in poultry, cutaneous leishmaniasis and schistosomiasis japonica. For many years, the only available vaccine against a parasite in Australia has been the attenuated Babesia bovis vaccine produced by the Tick Fever Research Centre of the Queensland Department of Primary Industries. Strategies for achieving molecular vaccines are generally similar within the various research groups. They involve analysis of the immunology and immunochemistry of a model or in-vitro system; development of functional monoclonal antibodies; analysis of antibody specificities in clinically and/or functionally defined polyclonal sera; screening of cDNA or genomic expression libraries; peptide synthesis; identification of an appropriate vaccination schedule involving adjuvants or new recombinant DNA-based antigen delivery systems. Outlined below are five of the major vaccine programmes.
Journal Article↗
Immunochemical analysis of Taenia taeniaeformis antigens expressed in Escherichia coli.
Previously we reported the isolation of several Escherichia coli clones expressing fragments of Taenia taeniaeformis antigens as beta-galactosidase fused proteins (Bowtell, Saint, Rickard & Mitchell, 1984). Here we describe the isolation of additional antigen-expressing clones from a larval cDNA library and the assignment of these clones to 7 antigen families. These were isolated with a polyspecific rabbit antiserum raised to the oncosphere. Since this serum was capable of reacting with a large number of antigens, it was important to develop techniques for rapidly determining the identity of the native T. taeniaeformis molecule corresponding to a cloned antigen gene. These included active immunization of rabbits with fused proteins and several techniques involving affinity purification on immobilized fused proteins. The reactivity of the antigen-positive clones with sera from humans infected with related parasites was also assessed. Finally, immunization of mice with several fused proteins failed to protect against subsequent infection, although antigens previously identified as candidate host-protective antigens (Bowtell, Mitchell, Anders, Lightowlers & Rickard, 1983) have yet to be identified in the expression library.
Animals↗
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.
Ancylostoma↗
Specific antibody responses in dogs experimentally infected with Echinococcus granulosus.
Six dogs reared helminth-free were divided into 2 groups. Four dogs were infected per os with 200,000 protoscoleces each of Echinococcus granulosus and 2 were kept as uninfected controls. All the dogs were kept together until 32 days after infection, when 1 infected dog was killed, its intestine removed and the contents examined to confirm that the infection with E. granulosus had been successful. The remaining 3 infected dogs were transferred to high security housing and their feces inspected daily to establish the time infections became patent. The infected and control dogs were bled every 5 days for 75 days from the time of infection and the sera were stored at -70 degrees C. Sera were tested by the enzyme-linked immunosorbent assay (ELISA) for antibodies to E. granulosus scolex excretory/secretory (ES) antigen, protoscolex antigen and oncosphere antigen. Antibodies to scolex ES antigen and protoscolex antigen were detected in the sera of infected dogs within 2 weeks of infection. Antibody titers rose rapidly and remained at a high level until the dogs were killed 75 days after infection. Antibodies in these sera did not cross react with antigens prepared from Taenia ovis, T. hydatigena, T. pisiformis, Ancylostoma caninum, Trichuris vulpis and Toxocara canis.
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Serum antibody response following parenteral immunization with hydatid cyst fluid in sheep infected with Echinococcus granulosus.
Production of specific serum antibodies following immunization with hydatid cyst fluid antigens was investigated in sheep with Echinococcus granulosus infection and in noninfected controls. Six of 10 infected animals responded to intramuscular injection of antigen by rapid production of antibodies detected in indirect hemagglutination assays. Similar responses did not occur in any of 10 noninfected controls. It is suggested that differences in the rate of response to immunization with cyst fluid antigens between groups of sheep could be exploited in serodiagnosis of E. granulosus infection in sheep. The results also suggest that low levels of antibody found in the serum of sheep infected with E. granulosus are not the result of immunosuppression or immunological tolerance, but are due to sequestration of antigen from the immune system of the host.
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Taenia pisiformis: protective immunization of rabbits with solubilized oncospheral antigens.
Antigens were derived from hatched and activated oncospheres of Taenia pisiformis which had been separated from embryophoric debris by centrifugation on Percoll. Crude oncospheral antigen was prepared by freeze-thawing and sonication of oncospheres at 4 C, and a supernatant of crude antigen was collected following centrifugation at 100,000g. Other antigens tested were the supernatants collected after 100,000g centrifugation of crude antigen solubilized in Triton X-100, butanol, lithium diiodosalicylic acid, KCl, sodium dodecyl sulfate, or sodium deoxycholate. When groups of rabbits were immunized with the various antigens and challenged with T. pisiformis eggs, both sodium deoxycholate- and Triton X-100-solubilized antigens stimulated a level of protection similar to the crude antigen. All other antigens failed to stimulate significant protective immunity. When sodium deoxycholate-solubilized antigen was fractionated using high-performance liquid chromatography, the major host-protective components were in the fractions with molecular weight greater than 140,000. Levels of the enzyme, glutamate dehydrogenase (EC 1.4.1.2), in the serum of rabbits challenged with T. pisiformis eggs closely reflected the degree of liver damage caused by migrating larvae, and were not markedly elevated in those rabbits effectively immunized using the crude or sodium deoxycholate-solubilized antigens.
Animals↗
Mouse strain variation and effects of oocyst dose in infection of mice with Eimeria falciformis, a coccidian parasite of the large intestine.
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Animals↗
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)
Animals↗
Examination of strategies for vaccination against parasitic infection or disease using mouse models.
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Animals↗
Immunization against Taenia taeniaeformis in mice: studies on the characterization of antigens from oncospheres.
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Animals↗
Expression of Taenia taeniaeformis antigens in Escherichia coli.
Two important features of infection of mice with larvae of Taenia taeniaeformis are the ready demonstration of host protective antibodies and the ability to immunize susceptible strains of mice against first infection using crude parasite preparations. Candidate immunogens in established larvae and the invasive oncosphere have been identified by immunoprecipitation of radiolabeled parasite proteins with host-protective antibodies. To overcome the difficulties associated with purification of these antigens from parasite material, the alternative strategy of expressing parasite proteins in Escherichia coli has been adopted. Double stranded DNA complementary to mRNA from 28 day old liver larvae was inserted into the beta-galactosidase gene of the bacteriophage lambda Amp 3. Some recombinants express a fusion protein with additional parasite-encoded epitopes located at the C-terminal end of the beta-galactosidase protein. Four clones that reacted with antibodies in an E. coli colony immunoassay were selected for detailed characterization. Analysis of lysates of the selected clones by SDS-PAGE and Western blotting revealed that each clone produced an abundant fusion protein that reacted specifically with a hyperimmune anti-oncosphere serum. Sibling analysis revealed that the four antiserum-positive clones encoded three immunologically-distinct parasite antigens. The identity of the native protein of larvae encoded by one clone (designated TA10) was an abundant antigen of Mr 70,000. This approach allows the assessment of antigens expressed in E. coli as vaccines in susceptible strains of mice by direct immunization and challenge and thus the development of a model defined-antigen vaccine against a larval cestode parasite.
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Haematological and serological data from dogs raised worm-free and monospecifically infected with helminths.
Pups free from helminth infection were obtained by treating the bitch prior to parturition, and the bitch and her pups after whelping, with oxfendazole and praziquantel. They were then housed in an area free from contamination with helminth eggs. Freedom from infection was confirmed by necropsy examinations and by serological tests using the Enzyme Linked Immunosorbent Assay (ELISA). Groups of pups were infected with Toxocara canis, Echinococcus granulosus, Taenia ovis, T. pisiformis and T. hydatigena and bled at 5-day intervals. Haematological examinations and measurement of serum proteins were performed on blood samples obtained throughout 30 days of infection. Levels of serum proteins were not significantly altered in any infection. Pups infected with T. canis showed a marked increase in numbers of circulating eosinophils during the phase of larval migration through the liver and lungs; they also showed marked antibody responses to T. canis larval ES antigen. Haematological values were not significantly altered in any other pups.
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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)
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Serological diagnosis and post-operative surveillance of human hydatid disease. I. Latex agglutination and immunoelectrophoresis using crude cyst fluid antigen.
The latex agglutination (LA) test and immunoelectrophoresis (IEP) test for the diagnosis of hydatid disease due to Echinococcus granulosus were carried out on 3600 serum samples submitted to this laboratory during the period mid-1976 to March 1981. Of the 2280 samples submitted for primary diagnosis (no known past history of hydatid disease) there were 108 cases in which infection was confirmed, 99 of them with fertile cysts. The 'Arc 5' was demonstrated by IEP in the serum of 65.7% of cases with fertile cysts, and using the additional criterion of 3 or more arcs with no 'Arc 5', a further 13.1% of cases were diagnosed apparently without 'false positive' results. Antibody was less readily detected in the serum of patients with calcified or sterile cysts. The LA test was positive in 70.7% of patients with fertile cysts, but this test gave an unacceptably high rate of 17.6% 'false positive' results. The IEP and LA tests had a combined sensitivity of 87.9% for primary diagnosis. There were 65 confirmed cases of persistent or recurrent infection, of which 64 were positive with IEP. Forty-four of 48 cases where surgical cure was effected became IEP negative within 2 yr. Among 42 patients remaining IEP-positive beyond 2 yr after surgery, recurrent disease was confirmed in 31 cases, but it was not possible to obtain confirmation in the remaining 11 patients. The LA test was unsuitable for post-operative surveillance.
Antibodies↗
Serological diagnosis and post-operative surveillance of human hydatid disease. II. The enzyme-linked immunosorbent assay (ELISA) using various antigens.
The ELISA using a urease conjugated antibody and 3 antigen preparations was examined for sensitivity and specificity in hydatid diagnosis. Four groups of sera tested were: 80 samples from patients with confirmed hydatid infection; 51 latex agglutination test (LA) +ve and immunoelectrophoresis test (IEP) -ve sera from patients with miscellaneous symptoms or disorders other than hydatid disease; 195 'normal' sera from healthy donors; 115 sera from persons infected with other parasites. Antigens tested included crude sheep hydatid cyst fluid (CSHCF), CSHCF partially purified by salt fractionation (PPSHCF) and CSHCF purified by sequential affinity chromatography depletion with rabbit anti-sheep IgG, 3EgH 29-2 anti-Echinococcus monoclonal antibody and 'normal' sheep Ig (ADSHCF). All 3 antigens showed high sensitivity in detecting antibody in serum from hydatid-infected patients, and gave excellent discrimination between these samples and the LA +ve IEP -ve sera and the 'normal' sera. Cross-reactions occurred with antibodies in the sera of patients with other parasitic infections, especially other larval cestodes and filarial parasites. Superior specificity was achieved with both CSHCF and ADSHCF, and ADSHCF reacted only with a single serum sample from an E. multilocularis patient. It was concluded that a combination of ELISA and IEP was useful for the diagnosis of hydatid disease, that ELISA at a single dilution could be useful as a screening test where other larval cestode infections were not prevalent and that ELISA was not of value for post-operative surveillance.
Antibodies↗
Taenia taeniaeformis: immunoprecipitation analysis of the protein antigens of oncospheres and larvae.
Biosynthetically or exogenously labeled proteins and immunoprecipitated protein antigens of established 28-day-old larvae of Taenia taeniaeformis were compared with proteins and antigens of infective oncospheres using single and two-dimensional gel electrophoresis. Immunoprecipitation was carried out using sera from infected mice and mouse antisera raised to larvae or oncospheres, and emphasis was placed on identifying antigens common to both oncospheres and larvae. Two major larval antigens of Mr 40,000 and 200,000, designated Tt40 and Tt200, are common to somatic larval preparations and oncospheres. Additionally, two major oncosphere antigens of Mr 55,000 and 60,000, designated Tt55 and Tt60, are also present in larval excretory and secretory (i.e., ES or exoantigen) products. Information obtained from these immunoprecipitation analyses will facilitate isolation and production of common as well as stage-specific protein antigens in the development of defined-antigen vaccines in this model system of cysticercosis.
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