| Summary: | Chapter 1 reviews the literature about the immunological aspects of taeniid cestode infections and the existing vaccines against Taenia solium cysticercosis in pigs. One of the most promising vaccines is TSOL18, a protein that has been identified in the oncosphere of Taenia solium and expressed as a recombinant molecule in E. coli. Repeated experimental trials have shown that this vaccine is able to protect up to 100% of the immunised pigs against a challenge infection with T. solium. Antibodies raised by the vaccine are capable of killing the parasite in in vitro cultures and it is believed that antibody and complement mediated killing of invading parasites is the major protective immune mechanism induced by vaccination with TSOL18. The identification of the villages with a high risk of T. solium infection, which could subsequently be used in the vaccine trial, is reported in chapter 2. A survey was conducted in 150 households owning 1756 pigs in the rural areas of Mayo-Danay division in the far north region of Cameroon. A questionnaire survey was carried out to collect information on the pig farming system and to identify potential risk factors for T. solium cysticercosis infection in pigs. Blood samples were collected from 398 pigs with the aim of estimating the sero-prevalence of Taenia solium cysticercosis. The results showed that 90.7% of the pigs were free roaming during the dry season and that 42.7% of households keeping pigs in the rural areas had no latrine facility. Seventy six percent of the interviewed pig owners affirmed that the members of the household used open field defecation. ELISA for antigen and antibody detection showed an apparent prevalence of porcine cysticercosis of 24.6% and 32.2%, respectively. A Bayesian approach using the conditional dependence between the two diagnostic tests indicated that the true sero-prevalence of cysticercosis in Mayo-Danay was 26.6%. Binary logistic regression analysis indicated that the lack of knowledge of the taeniasis-cysticercosis complex and the absence of a pig pen in the household were associated with pig cysticercosis. Chapter 3 reports the investigations that were undertaken to characterise whether the principal antibody specificities raised by TSOL18 in pigs were against linear or conformational determinants. TSOL18 was expressed in two truncated forms representing either the amino terminal portion or the carboxy terminal portion, with the two truncations overlapping in sequence by 25 amino acids. The original protein (designated TSOL18N—) and the two truncations (TSOL18N—-1 and TSOL18N—-2) were used in inhibition ELISA to determine their ability to inhibit the binding of protective pig antibodies to TSOL18. TSOL18N— was shown to be capable of completely inhibiting the binding of pig anti-TSOL18N— antibodies to TSOL18N— in ELISA. However, neither TSOL18N—-1 nor TSOL18N—-2, either alone or combined, was capable of inhibiting any detectable amount of reactivity of pig anti-TSOL18N— antibodies with TSOL18N—. It is concluded that the dominant antibody specificities, and likely the host-protective specificities, of TSOL18 are conformational epitopes. Chapter 4 describes the development of an antibody detection test for the specific diagnosis of porcine cysticercosis. A fraction with a major band of 14 kDa was obtained from crude cyst fluid (CF) of T. solium cysticerci by 2-step chromatography. A first fraction isolated by gel filtration was purified using an anion exchange column on High Performance Liquid Chromatography (HPLC). Evaluation of the analytic sensitivity of this fraction (F3) was carried out in an antibody detection enzyme-linked immunosorbent assay (Ab-ELISA-F3) using serum samples from pigs experimentally infected with different doses of T. solium eggs. The cross-reactivity of F3 was evaluated with serum samples from pigs that were naturally or experimentally infected with Taenia hydatigena, Taenia saginata asiatica, Fasciola hepatica, Trichinella spiralis, Metastrongylus apri, Trypanosoma congolense or Sarcoptes scabiei, and with serum samples of rabbits hyper-immunised with cyst fluid of T. hydatigena or T. solium. Analysis of the specificity of the F3 showed that serum samples of pigs infected with other parasites did not recognise this antigen. Cross-reaction with T. hydatigena occurred in ELISA using CF as antigen, but the F3 antigen fraction was not recognized by the rabbit hyper-immune serum against T. hydatigena. Evaluation of the diagnostic sensitivity and specificity of the Ab-ELISA-F3 was done by a non-parametric Receiver Operating Characteristic (ROC) analysis using serum samples from Zambian and Cameroonian village pigs. The results from the ROC analysis yielded a low diagnostic value (area under ROC curve= 0.48) with the sera from the Zambian pigs while a relatively high diagnostic value was obtained with the sera from Cameroonian pigs (area under ROC curve= 0.78). In Chapter 5 the efficacy of the TSOL18 vaccine is assessed under field conditions in the Mayo-Danay district. Two hundred and forty 2-3 month old piglets belonging to 114 individual households were involved in the study. In each household one or more pairs of piglets were included, with one animal of each pair being vaccinated and the other acting as a non-vaccinated control. Vaccinated animals received two initial immunizations intramuscularly in the neck one month apart with 200μg TSOL18 plus 5mg Quil A. At the time of the second immunization both vaccinated and control animals received an oral dose of 30mg/kg oxfendazole. Vaccinated animals received a third immunization approximately 3 months after the first immunization. Antibody responses to the vaccine were assessed at different time intervals by ELISA. Necropsies were undertaken when the pigs were approximately 12 months of age. All parasites were counted in half of the body musculature and in the brain. Two hundred and twelve animals were available for necropsy at the end of the trial (110 vaccinated; 102 controls). Viable T. solium cysticerci were identified in 20 control pigs (prevalence 19.6%), including 14 animals that had estimated total body burdens of > 1000 cysticerci. No cysticerci were found in any of the vaccinated animals indicating that the vaccine provided a very high level of protection (P< 0.0001) against naturally acquired infection with T. solium in pigs. Combined application of TSOL18 vaccination and a single oxfendazole treatment in pigs is a simple and relatively sustainable procedure that has the potential to control T. solium transmission in endemic areas and, indirectly, reduce the number of new cases of neurocysticercosis in humans. In chapter 6, the similarity of the antibody responses of pigs and mice to TSOL18 antigen is highlighted. Four IgG1 monoclonal antibodies (MoAb) were produced against the conformational epitopes of TSOL18. It was shown that pig antisera inhibit the binding of these MoAbs in a competition ELISA, indicating that pig and mouse antibodies against TSOL18 vaccine react with the same conformational epitopes. For this reason, monoclonal antibodies raised in mice immunized with TSOL18 could be a valuable source of antibodies for further characterisation of the host-protective epitopes of the vaccine. A monoclonal antibody-based inhibitive enzyme-linked immunosorbent assay (mi-ELISA) was developed. Serum samples of TSOL18-vaccinated and non-vaccinated pigs were used. In all the vaccinated and protected pigs screened at necropsy, anti-TSOL18 antibodies inhibited the binding of a monoclonal antibody (Mab25D12C1) specific to the conformational epitopes of TSOL18 antigen, suggesting an immune response that correlates with protection. This result was in agreement with the results obtained in an indirect ELISA, which showed that all the vaccinated and protected pigs had developed antibodies to the TSOL18 vaccine. In chapter 7 the efficacy of the TSOL18 vaccine is compared with that of other vaccines, which are currently being tested under field conditions. Recommendations are made for implementing a vaccination programme in Cameroon. Future research activities are suggested to improve our knowledge on the duration of the immunity of the vaccine and various other aspects of the vaccine production and delivery.
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