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Biomedical subjects

M D Rickard

Publications and source records attributed to M D Rickard.

At least 37 records · Page 2Linked to original sources
PubMed1991

Vaccination against Taenia taeniaeformis infection in rats using a recombinant protein and preliminary analysis of the induced antibody response.

Primary screening of a cDNA expression library of Taenia taeniaeformis oncospheres in lambda gt11 bacteriophage was carried out using rabbit anti-T, taeniaeformis oncosphere serum affinity-purified from oncosphere pellets. From approximately 1.6 x 10(5) plaques, 21 single clones that were positive with the affinity-purified antibodies were isolated. Sibling analysis revealed that 17 clones out of the 21 could be assigned to five different antigen families. Only family 1 was strongly recognized by a serum prepared in a rabbit against a partially purified host-protective oncosphere antigen fraction. The fragments of lambda DNA were inserted into a pGEX plasmid vector that encodes glutathione S-transferase (GST) of Schistosoma japonicum. Clones designated TtO-18, -49.53 (family 1), 46 (family 2), 15 (family 3), 40 (family 4) and 66 (family 5) were established as subclones in pGEX-1 plasmid vectors which produced GST fusion proteins. All GST fusion proteins were soluble and recognized by anti-GST and anti-TtO sera. Three vaccination experiments with these fusion proteins using specific-pathogen-free Wistar rats revealed that all three fusion proteins of family 1 were exclusively effective against T. taeniaeformis oncosphere challenge with approximately 95% and 91% reductions in cystic metacestode and total metacestode recoveries, respectively. Rats vaccinated with fusion proteins of family 1 produced antibodies which reacted with a 21-kDa oncosphere antigen component which appeared to be a major oncosphere stage-specific antigen.

Animals↗
PubMed1991

Cestode vaccines.

Studies over the past 20 years have clearly shown the potential for developing vaccines against larval cestode infections of man and animals. The important larval cestode infections of man (Echinococcus granulosus--hydatidosis: Taenia solium--cysticercosis) involve domesticated animals as intermediate hosts in their natural life-cycles. These animals develop strong immunity against reinfection, and immunity can be artificially induced by vaccination with oncosphere antigens. A major stumbling block in developing commercial vaccines against cestodes has been the difficulty in obtaining adequate supplies of these antigens. Recent studies with Taenia ovis, a larval cestode causing cysticercosis in sheep, have demonstrated the feasibility of developing commercial vaccines against cestodes using recombinant DNA technology. A cDNA library prepared using mRNA obtained from T. ovis oncospheres was used to isolate a clone which expressed T. ovis polypeptide antigen 45W as a fusion protein with Schistosoma japonicum glutathione S-transferase (GST-45W). GST-45W gave up to 94% protection against challenge infection when used to vaccinate sheep with saponin as adjuvant. The vaccine antigen was shown by SDS PAGE to be unstable, a major disadvantage in subsequent attempts to obtain high yields of antigen for commercial production. The fusion protein has now been stabilized by reducing the size of GST-45W cDNA through deleting 19 carboxyl terminal hydropathic acids, and the resultant fusion protein GST-45W (B/X) was highly host-protective. Another experiment showed that the 45W T. ovis polypeptide cleaved enzymatically from GST-45W was still host-protective, suggesting that GST had no influence on the immunogenicity of GST-45W fusion protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

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.

Animals↗
PubMed1990

Stage-specific immunity to Taenia taeniaeformis infection in mice. A histological study of the course of infection in mice vaccinated with either oncosphere or metacestode antigens.

The course of Taenia taeniaeformis infection in mice previously vaccinated with antigens prepared from either oncosphere (TtO) or metacestode (TtM) was followed by histological examination of livers from mice killed at various times post-infection (p.i.). Distinctly different immune responses occurred in the two groups. Very few cysts were seen at any stage of infection in TtO-vaccinated mice and most of those which were present appeared histologically similar to cysts in control mice. In TtM-vaccinated mice many cysts were present from early in infection but histologically it was apparent that most were dying from 15 days p.i. because the tegument had lost its integrity, and degranulated polymorphonuclear leucocytes were present inside the parasites. These findings support earlier suggestions that stage-specific antigens are expressed in oncospheres and metacestodes. Parasites developing normally were surrounded by a halo of alcian blue staining amorphous acellular material. This material appeared to act as a barrier to attack by host inflammatory cells, and disappearance of this layer signalled death of the parasite. The possibility that the gut acted as a barrier to delay migration of oncospheres to the liver in vaccinated mice was investigated, but no evidence for this could be found.

Animals↗
PubMed1990

Cysticercosis: cellular immune responses during primary and secondary infection.

Immune reactions to cysticercosis have been extensively studied in mice. The lack of significant lymphocyte infiltration into the livers of infected mice and the obvious role of antibodies in rejection has led to the general conclusion that cellular reactions do not play a role in protection against this disease. In contrast, the present study examining the immune response to cestode infections in a large animal model (sheep) revealed the presence of a massive and highly organized cellular infiltration in the livers after a secondary Taenia hydatigena infection. The majority of the infiltrating lymphocytes were of the CD4+ phenotype with much fewer CD8+ cells present. While most gamma delta-TCR+ cells in peripheral blood are SBU-T19+, the majority of gamma delta-TCR+ lymphocytes in the liver lesions are SBU-T19- suggesting selective migration of these cells into the lesions. In contrast to the diffuse distribution of T cells in the lesions, B cells were present as distinct aggregates. In primary T. hydatigena infections, host class I and class II MHC antigens were shown, for the first time in cestode infections, to be absorbed onto the surface of the metacestode bladderwall indicating their possible involvement in parasite survival. No immune reactions were observed close to the parasite although lymphocytes and eosinophils were infiltrating the adjacent portal tract areas. Most lymphocytes in both primary and secondary infections were positive for MHC class II antigens suggesting selective recruitment of activated cells to the site of infection. Significant changes in relative and absolute numbers of lymphocyte subpopulations were also observed in the draining hepatic lymph nodes dominated by a massive increase of B cells. In contrast, at the peak of local cellular infiltration, no changes in lymphocyte subpopulations were observed in peripheral blood showing the limited usefulness of this compartment in studying cellular changes in localized infections. The vigorous cellular response observed in the livers of sheep contrasts sharply with the lack of lymphocyte infiltration reported in mice indicating that small animal models may not be appropriate to study cellular responses to cysticercosis in large animals and man.

Animals↗
PubMed1989

Vaccination against ovine cysticercosis using a defined recombinant antigen.

Cysticercosis caused by larval tapeworms is a major public health problem and a cause of substantial economic losses in the farm-animal industries. Taenia ovis in sheep is a particularly important example. Immunity to reinfection with the larvae has a central role in regulating natural transmission of the parasites, and vaccination with antigens from the early larval oncosphere stage can induce complete protection against infection. As it is impractical to obtain enough oncospheres for a commercial vaccine against these tapeworms, an alternative approach is to use recombinant DNA methods to generate a cheap and plentiful supply of antigens. We report here the expression in Escherichia coli of complementary DNA encoding T. ovis antigens as fusion proteins with the Schistosoma japonicum glutathione S-transferase. Vaccination of sheep with these fusion proteins gave significant, although not complete, immunity against challenge infection with T. ovis eggs. Commercial development of a vaccine is being pursued.

Amino Acid Sequence↗
PubMed1989

Subunit composition and specificity of the major cyst fluid antigens of Echinococcus granulosus.

The subunit composition and specificity of the major Echinococcus granulosus cyst fluid antigens were determined by immunochemical analysis using murine monoclonal antibodies against Antigen 5 and Antigen B and human sera. Immune complexes cut out from immunoelectrophoresis gels and murine hybridomas were used as a source of specific anti-Antigen 5 and anti-Antigen B antibodies. Immunoprecipitation and Western blot analyses in sodium dodecyl sulphate-polyacrylamide gels using these reagents identified Antigen 5 to be a heterodimer composed of 24-kDa and 38-kDa subunits linked by disulphide bonding. Antigen B comprised a regularly spaced group of molecules with the smallest subunit estimated to be 8 kDa and the other components each differing in size by approximately 8 kDa, i.e., 16 kDa, 24 kDa, 32 kDa etc.; all possibly derived from the 8-kDa monomer. The relative abundance of the Antigen B subunits decreased asymptotically with increasing molecular weight. Neither the Antigen 5 nor the Antigen B subunit was specific for E. granulosus. Both antigens generated readily detectable levels of specific antibody in the sera of patients with Echinococcus multilocularis, Echinococcus vogeli or E. granulosus infection. Relatively high levels of antibody to Antigen 5 were also detected in the sera of patients infected with Taenia solium. The presence of phosphorylcholine epitope(s) on Antigen 5 was confirmed.

Animals↗
PubMed1989

Lymphocyte subpopulations of sheep in protective immunity to Taenia hydatigena.

Lymphocyte subpopulations entering the liver and surrounding the rejection sites during a 9-day period after infection of immune sheep with Taenia hydatigena were identified with the aid of monoclonal antibodies against lymphocyte cell surface markers. Viable lymphocytes were isolated from the liver tissue and stained by indirect immunofluorescence for subsequent flow cytometry analysis. Over the first 6 days after challenge infection a marked increase in the ratio of T-helper to T-suppressor/cytotoxic lymphocytes was observed. SBU-T19+ lymphocytes, a CD5+ T-cell subpopulation uniquely identified in the sheep, were present in small numbers in sheep liver both before and after infection. There was a large, continuous increase of sIg+ B-cells over the 9-day observation period after infection. Eosinophils were the predominant granulocytes in the liver of infected sheep. The exact location of the leucocyte subpopulations in respect to the rejection sites in infected liver was determined by in-situ immunoperoxidase staining of frozen liver sections. The evolution of the parasite-induced leucocyte response was characterized by the appearance of a central core of eosinophils surrounded by increasing numbers of CD4+ helper T-cells. CD8+ (suppressor/cytotoxic) and SBU-T19+ T-lymphocytes were present in much smaller numbers and by day 9 after infection were located predominantly around the periphery of the lesions. Distinct foci of tightly packed B-cells developed within the lesions and increased dramatically in size over the 9-day observation period. At this time, lesions appeared as compact aggregations of leucocytes encircled by a second band of eosinophils. Both T- and B-lymphocytes within the lesions stained positive for class II major histocompatibility antigens.

Animals↗
PubMed1989

Identification of protein components of Echinococcus granulosus protoscolex antigens for specific serodiagnosis of E. granulosus infection in dogs.

Proteins of Echinococcus granulosus protoscolex excretory/secretory or deoxycholate solubilized somatic antigens were radiolabelled with 125I and immunoprecipitated with sera from dogs naturally or experimentally infected with E. granulosus and various control dog sera. Analysis of immunoprecipitates was performed using one- and two-dimensional gel electrophoresis to identify antigenic protein components specific for E. granulosus. Using both electrophoretic techniques, a basic component of Mr 27,000 and an acidic component of Mr 94,000 were defined in both excretory/secretory and somatic protoscolex antigens, and were specifically identified by 95% and 62% of 21 sera from E. granulosus-infected dogs, respectively. An abundant component of Mr 35,000 was identified by 100% of these dogs, parts of which were E. granulosus specific. Results of this study should allow identification of specific recombinant antigens for routine serodiagnosis of E. granulosus infection in dogs.

Animals↗
PubMed1989

Pre-operative albendazole therapy for recurrent hydatid disease.

Two patients with recurrent hydatid disease had a 1-month course of albendazole before surgery. In one case with a thick-walled host capsule, the scolicidal effect was incomplete, but, in the second case, in which thin-walled cysts were present, albendazole entered the cyst and was completely effective as a scolicidal agent. For thick-walled cysts, it may be necessary to use albendazole for more than 1 month pre-operatively in order to achieve a scolicidal effect.

Abdomen↗
PubMed1988

Excretory-secretory products of helminth parasites: effects on host immune responses.

Parasitic helminths excrete or secrete (ES) a variety of molecules into their mammalian hosts. The effects of these ES products on the host's immune responses are reviewed. Investigations into the source of antigenic or immunoregulatory ES products have identified the cuticular and tegumental surfaces of some nematodes and trematodes respectively as being important sources of ES products; other ES molecules are released through specialized excretory or secretory organs. It is proposed that the active shedding of surface antigens may serve as an important source of parasite antigens available to the immune system in a form in which they can be taken up and processed by antigen-presenting dendritic cells, macrophages and certain B cells for presentation to T helper cells. The ES products of nematodes, trematodes and cestodes contribute to immune evasion strategies of the parasites through mechanisms including shedding of surface-bound ligands and cells, alteration of lymphocyte, macrophage and granulocyte functions and modulation of complement and other host inflammatory responses. Immunopathology may be induced by ES products as in the development of granulomas around entrapped schistosome eggs. In some host-parasite systems ES antigens may induce host-protective immune responses and this source of protective antigens has been utilized in the successful vaccination against helminth infections, particularly against infection with trichurid nematodes and the metacestode stage of cestode parasites. The use of ES antigens in immunodiagnosis of helminth infection is also briefly discussed.

Animals↗
PubMed1988

Studies on stage-specific immunity against Taenia taeniaeformis metacestodes in mice.

The possible existence of stage-specific immune responses to Taenia taeniaeformis infection was investigated in C3H/He mice vaccinated with antigens prepared from either the oncosphere or metacestode stages. Mice were immunized twice, 2 weeks apart, with antigen in Freund's complete adjuvant. Two weeks after the second immunization they were challenged with 250 T. taeniaeformis eggs and killed day 0, 5, 10, 15, 20, 25, 30, 45 and 60 after infection. Gross examination of the livers revealed marked differences between oncosphere (TtO) and metacestode (TtM) vaccinated mice. Very few metacestodes were found in the first group but most of those that evaded the initial host attack developed like the cysts found in the control group. In contrast, many degenerating metacestodes were found in the TtM vaccinated group. In a subsequent experiment groups of mice were vaccinated with varying doses of either TtO or TtM to determine whether the qualitative differences observed above were due to antigen dose effects. However, varying antigen doses gave the same results. These data show that vaccination with oncospheres generates an immune response capable of killing invading larvae soon after infection whereas vaccination with TtM results in larvae being killed at a later stage, suggesting that there are stage-specific, host-protective antigens.

Animals↗
PubMed1988

Analysis of antibody responses to Hymenolepis nana infection in mice by the enzyme-linked immunosorbent assay and immunoprecipitation.

Serum antibody responses in two strains of mice infected with embryonated eggs of Hymenolepis nana were analysed by the enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) using sodium deoxycholate (DOC)-solubilized antigens prepared from embryonated eggs (eggs), mouse-derived cysticercoids (cysts) and adult tapeworms with immature segments only (adults). Highly susceptible dd mice, which harbour mature tapeworms for a long period (greater than 70 days), produced high levels of antibodies to all three different stages of H. nana. BALB/c mice, almost all of which expel adult tapeworms by 30 days after infection, produced high levels of antibody against egg antigens only. The high antibody titres to cyst and adult antigens in dd mice did not lead to expulsion of the worms. However, worms are rejected early in BALB/c mice when there is little or no detectable serum antibody. The antibody responses to eggs seen in BALB/c mice which had long since shed their adult worms were probably due to ingestion of eggs from faeces of other infected mice. Antibodies to eggs were not detected in BALB/c mice which were initially inoculated with eggs (day 0) and then treated with praziquantel on day 6 after the tissue phase of infection only. The different antibody responses to egg antigens and the other two antigens (cyst and adult) in BALB/c mice suggest a difference in antigen specificity between eggs and both cysts and adults. A major antigen component with Mr 32,000 appears to be specific to the egg (or oncosphere) stage of H. nana. Antibody to this major component of eggs was absorbed only with intact eggs, but not with intact cysts nor adults with immature segments only, so that the antigen appears to be on the surface of the oncosphere.

Animals↗
PubMed1988

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)

Animals↗