Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Abstract

In natural infections by Trypanosoma cruzi, the vector-derived metacyclic stage is the primary agent of infection. Laboratory efforts to obtain large numbers of metacyclic trypomastigotes have been successful. However, many questions have been raised about the virulence of culture-derived metacyclics. This study was designed to identify some factors which influence metacyclogenesis and to analyze T. cruzi extracts with antisera and monoclonal antibodies developed from culture forms. Selective sub-cultivation of epimastigotes of the Tulahuen strain of T. cruzi in a liquid metacyclic culture (LMC) medium did not increase the percentages of metacyclic stages. When epimastigotes were cultured in LMC media containing proline or hydroxyproline, there was no marked improvement in metacyclic production by this strain. However, the percentages of metacyclic stages in cultures incubated in 5% CO$\sb2$ and air were two-fold greater than similar cultures in an air atmosphere. Trypanosomes cultured in LMC media containing hydroxyproline entered the death phase earlier than cultures containing proline whether they were incubated in an air atmosphere or 5% CO$\sb2$ and air. Incubation of cultures of the Tulahuen strain in a CO$\sb2$ environment increased metacyclic differentiation by as much as 60% above experimental controls. The addition of 5% CO$\sb2$ had a greater effect on metacyclic differentiation than did the addition of proline or hydroxyproline. Nineteen stable hybridomas developed against epitopes of irradiated metacyclic trypanosomes produced monoclonal antibodies (MAbs) against T. cruzi epimastigote extracts. Sixteen were of the IgM subclass, and one each of IgG$\sb1$, IgG$\sb{\rm 2a}$ and IgG$\sb{\rm 2b}$. Although all were positive by ELISA, only one (5C2.LA) produced a precipitin band, two (5A4.LA, and 5C2.LA) were positive against T. cruzi epimastigote extracts reduced with 2-mercaptoethanol, and five (1G1.LA, 5A4.LA, 5C2.LA, 5F2.LA, and 8D12.LA) produced bands by Western blot analyses of native extracts of T. cruzi epimastigotes. Sera obtained from T. cruzi-infected BALB/c and C3H/He mice reacted differently against native extracts on Western blots. The reactions varied in relation to the duration of infection of the mice. These results indicate that (1) metacyclogenesis may be influenced by the components of the culture media and the parasite strain being used, (2) the results of serological or immunological studies of Chagas' disease may be influenced by culture-derived parasites, and host strains and, (3) the humoral immune response of murine hosts may change over the course of a chronic infection.

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