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Title: Serological characterization of Trypanosoma (Nannomonas) congolense using in vitro-derived metacyclic forms
Author: Frame, Iain Alexander
ISNI:       0000 0001 3481 1125
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1989
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This thesis describes the development and application of techniques to characterize the metacyclic variable antigen type (M-VAT) repertoires of stocks of Trypanosoma congolense which were isolated from a defined geographical area. Seventeen stocks of T. congolense were isolated from domestic dogs in a 320 hectare area around Kakumbi, Chipata District, Zambia. Six of these stocks were cloned and stabi 1 ated as TREU 1881, TREU 1885, TREU 1894, TREU 1896, TREU 2034 and TREU 2037. Insect form j_n vitro cultures were initiated from the proboscides, proventriculi or midguts of tsetse flies infected with the cloned stocks. The cultures produced antigenically stable metacyclic trypanosomes in large numbers on a regular basis In vitro-derived metacyclic forms were used as reference antigens in four serological assays. Firstly, monoclonal antibodies were prepared to the metacyclic surface antigens of one stock, T. congolense TREU 1885. Nine monoclonal antibodies were produced which, individually, recognized between 6% and 18% of the metacyclic population. When pooled, the monoclonal antibodies stained 70-84% of the entire M-VAT repertoire. The monoclonal antibodies were used in an indirect fluorescence antibody test (IFAT) to determine whether M-VATs which occurred in TREU 1885 were present in the metacyclic populations of the other stocks in culture. None of the M-VATs present in the other in vitro-deri ved stocks were recognized by the monoclonal antibodies indicating that TREU 1885 M-VATs were distinct and characteristic for that stock. A cross protection assay was carried out in mice using viable, in vitro-derived trypanosomes. Groups of mice were infected with each of the six stocks, then treated 10-14 days post-infection with either diminazene aceturate or isometamidium chloride. Fourteen days post-treatment, each group was challenged with one of the six stocks. The animals were immune to challenge only against the infecting stock indicating that the six stocks cultured in vitro were antigenically distinct. In order to facilitate serological typing of the 17 stocks, an enzyme-linked immunosorbent assay (ELISA) and IFAT were developed using glutaraldehyde- and formalin-fixed intact trypanosomes respectively. Twenty-one day post-infection antisera from rabbits were used to identify M-VAT specific immune responses to the metacyclics of the cultured stocks. The ELISA, although shown to be M-VAT specific in early experiments, failed to distinguish between antigenically distinct stocks of the reference collection. A triple labelling IFAT which detected the IgG-specific immune response of the host was shown to be the most effective technique for distinguishing M-VAT repertoires using fixed trypanosomes. Using this assay, sera raised against the 17 original isolates were examined using in vitro-derived metacyclic trypanosomes from the six cloned stocks as antigens. Fluorescence was observed only between known homologous antisera and trypanosomes thus indicating that from 17 stocks, at least seven serodemes were present in this area The reasons for the wide diversity in serodeme distribution in this small area are discussed in relation to the tsetse fly and game animal populations. The value of serodeme characterization and its use in determining the ecology and natural history of T. congolense and relevance to strategies for implementing rational control measures are also discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Zoology