Development of Monoclonal Antibody-Based Assays for Detection and Differentiation of Trypanosome Species in The Tsetse Fly (Glossina Spp.)

Abstract

Diagnosis of trypanosome infections in tsetse (Glossina spp.) is currently based on dissection and microscopic examination of different organs of the vector for the presence of infecting trypanosomes. This procedure is slow and labour intensive, and it is not accurate. The work reported in this thesis was conducted with the objective of developing a field applicable, monoclonal antibody (MoAb)-based assay for the detection of, and differentiation between trypanosome species in infected Glossina species. The development of a more accurate alternative technique to the dissection method would provide a useful tool for estimation of the trypanosomiasis risk in various localities. MoAbs had been previously generated against invariant antigens of various trypanosome subgenera, including the Trypanozoon, Nannomonas and Duttonella, and the T. congolense species. Some of these MoAbs were selected and used in the studies described in this thesis. It was, however, necessary to produce additional trypanosome species-specific MoAbs for several reasons: Firstly, there was no T. simiae specific MoAb. Secondly, the selected T. vivax specific MoAbs were less sensitive compared with some of the T. congolense and Nannomonas species-specific MoAbs, in terms of the minimum number of procyclics or epimastigotes that they could detect. Thirdly, some of the selected MoAbs could not be used in the Western immunoblot assay to identify the antigens involved. The additional trypanosome species-specific MoAbs produced included three Trypanozoon subgenus-specific MoAbs (KT39a/18.17, KT43/33.32 and KT43/27.32), two Duttonella subgenus-specific MoAbs (KD32/48.17 and KD37/19.3), two Nannomonas subgenus-specific MoAbs (KN4/13.9 and KN5/6.15) and one T. simiae specific MoAb KNS7/14.X. Characterization of the specific antigens, using the indirect immunofluorescent antibody test x x i v (IFAT), Western immunoblot analysis, proteinase-K digestion and periodate oxidation, showed that some of the new MoAbs detected trypanosome species or subgenus-specific antigens that previously had not been identified. These new antigens included a T, brucei species-specific antigen which localized in the cytoplasm of T. brucei procyclics and differed from previously identified antigens which were located at the cell membrane. Employing the nitrocellulose membrane (NC)-based dot-enzyme-linked immunosorbent assay (dot-ELISA), it was found that the selected MoAbs could identify and differentiate between in vitro propagated trypanosome species. The dot-ELISA detected in vitro propagated T. brucei, T. congolense and T. simiae procyclics, and T. vivax epimastigotes, in both single and artificially mixed preparations. The assay had a specificity more than 99.9% and a sensitivity as good as 10 trypanosomes per dot for some T. brucei, T. congolense and Nannomonas specific MoAbs. The integrity of trypanosome antigens applied onto NC membrane remained unaffected for up to 60 days of storage at 4°C under desiccated conditions or under similar conditions at room temperature (17-26°C). Each of the derived MoAbs was able to detect isolates of the respective trypanosome species obtained from different geographical areas, and none cross-reacted with T. grayi. Furthermore, each of the T. congolense specific MoAbs reacted with all three types of this parasite species (savannah, Kilifi and riverine-forest types) that were tested. The dot-ELISA developed, however, could not be applied directly to the diagnosis of trypanosome infections in tsetse because of non-specific staining of NC membrane by samples prepared from tsetse gut, due to the presence of pigments and undigested blood meals. This non-specific staining was eliminated by prior decoloration of tsetse gut samples on NC membrane, using 5% hydrogen peroxide (H2 O2 ). This finding enabled successful detection and differentiation of T. brucei, T. congolense and T. simiae parasites in the gut of experimentally infected Glossina, using a modified dot- XXV ELISA. The sensitivity of the assays was; 90.5% in detecting T. brucei infections, 85.4% in detecting T. congolense infections and 94.4% in detecting T. simiae infections. The sample from the gut of each tsetse fly could be replicated in 15 dots onto NC membrane for testing. When stored on NC membrane at room temperature (19-26°C) under desiccated conditions, the samples did not show any loss in activity over a period of 90 days. The dot-ELISA technique was also used successfully to detect T. brucei parasites in the salivary glands of experimentally infected Glossina. This application of the technique was, however, achieved following the substitution of Na2 EDTA buffer for PBS or PSG which were used in detecting T. brucei in infected tsetse gut. The assay was more than 99.9% specific and 90% sensitive in detecting T. brucei in infected tsetse salivary glands. Trypanosoma congolense and T. vivax infections were successfully detected in the mouthparts of experimentally infected tsetse flies using dotELISA. However, it was not possible to replicate tsetse proboscide samples for testing with different MoAbs due to the very low parasite numbers in this organ. The recommended strategy, therefore, was to randomly sort all tsetse flies suspected to be infected with T. congolense, T. simiae or T. vivax into three separate groups and test them with different MoAbs. In a limited field evaluation of the dot-ELISA, 2 out of 104 G. pallidipes flies identified by dissection and microscopy to carry midgut infections, were also positive by dot-ELISA. Using the dissection technique, such infections would have been diagnosed as immature T. brucei and/or T. congolense and/or T. simiae. However, by the dot-ELISA, it was possible to determine with certainty that the two flies were infected with T. congolense parasites. Furthermore, the dot-ELISA detected T. congolense antigens in the midguts of an additional 6(5.8%) of the 104 G. pallidipes and 17(4.4%) of 390 G. longipennis that were negative by the dissection method, suggesting that the dot-ELISA may be more sensitive than the dissection method. This x x v i ability of the dot-ELISA to detect more infections than the dissection method, suggests that the MoAb-based dot-ELISA is not only a more precise tool for identification and differentiation of trypanosome species in the vector, but that it may also be more sensitive than the dissection method.