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African trypanosomiasis occurs in 36 African countries following the geographic distribution of the tsetse fly. About 55 million people are at risk of acquiring infection and over 300,000 are already infected (WHO news, August 1996). Domestic animals excluded from about 70% of the land encompassing the tsetse habitat. However, many wild animals co-exist with the tsetse fly and are resistant to trypanosomiasis. Our Lab chooses Cape buffalo as a wild animal model to study the trypanoresistant mechanism. Cape buffaloes survive with low parasitemia and no sip of disease in the tsetse habitat. The superior ability of Cape buffaloes to limit the density of the trypanosome population in their bloodstream is related to the presence in their plasma or serum of material that kills all species of African trypanosome (123). The previous results from our lab had shown that the trypanocidal component in Cape buffalo serum was correlated with the presence of a 146 kDa polypeptide revealed by reducing SDS-PAGE. The amino acid sequence of the 146 polypeptide was 84–100% homologous with the sequence of human, mouse and rat xanthine:oxygen oxidoreductase (XO/XDH) suggesting that the trypanocidal protein might be XO (123). The main objective of this thesis was to explore the mechanism of Cape buffalo to control African trypanosomiasis. My work has shown that Cape buffalo serum XO generates trypanocidal H2O2 during catabolism of hypoxanthine and xanthine to uric acid. Furthermore, Cape buffalo serum contains adenosine deaminase, purine nucleoside phosphorylase, and guanine deammase, which together with xanthine oxidase catabolize adenosine, inosine, guanosine, guanine, hypoxanthine, and xanthine to uric acid yielding H 2O2. Paradoxically, Cape buffalo serum also contains catalase that catabolizes H2O2 to H2O and prevents expression of trypanocidal activity under physiological condition. However, I have shown that an infection-induced decline of blood catalase activity results in the accumulation of H2O2 in Cape buffalo blood and allows the Cape buffalo serum to express trypanocidal activity. A high xanthine oxidase activity is also found in sera from Eland, Giraffe and Greater Kudu, which might be responsible for their resistance for trypanosomiasis. Trypanosusceptible animals, which support high parasitemia and die after the infection, are unable to accumulate a trypanocidal concentration of H 2O2 in their bloodstream because of their low serum xanthine oxidase activity, sustained catalase activity and/or the presence of additional serum component(s) that protect trypanosomes from H2O2. The studies contribute to the data base of host anti-trypanosomiasis mechanisms, which may ultimately provide information to develop new measures to control African trypanosomiasis.

The IPP had previously been implicated as an important site for Ig diversification in sheep and cattle but the early site for Ig diversification in sheep remained in question. Recently, fetal spleen has been shown to be a potential site of B cell development in cattle which is phylogenetically close to sheep (Lucier et al., 1998). In this study, in order to solve the problem of when, where, and how the primary immunoglobulin repertoire of sheep is generated and diversified before the onset of diversification in IPP, various tissues of fetuses at the first trimester were examined for the expression of λ light chain genes and the degree of Vλ diversity. Thus, this study has provided significant evidence for the following conclusions on early Ig Vλ diversification in sheep: (1) Two germline Vλ genes, 5.1 and 5.3 were identified as predominant participants in Ig λ light chain gene rearrangement. (2) A new Jλ gene was found and shown to be utilized in Ig λ light chain gene rearrangement. (3) At 63 days of gestation, there is little diversity seen in the Ig λ light chain repertoire outside the spleen. However, even at this early stage, there is significant diversity of λ light chain within spleen. (4) Fetal spleen is already a reservoir of extensive Ig diversity by the end of the first trimester. (5) Spleen is an earlier site of Vλ diversity than IPP, a bursa-equivalent GALT in sheep. (6) Fetal spleen may provide a partially diversified B cell stock from which a small number of precursor B cells emigrate into the IPP and undergo subsequent clonal expansion and additional diversification within the IPP follicles in sheep. (7) This study shows that multiple sites are involved in the diversification of the Vλ repertoire in sheep.

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MPM-2, a monoclonal antibody specific for phosphorylated epitopes of mitotic proteins, has been used to identify unique 75-85 kD proteins of the mammalian sperm tail, named MPM-2 reactive sperm tail phosphoproteins (MSP). Due to the loss of MPM-2 reactivity following fertilization in bovine and rabbit zygotes, additional antibodies were produced to non-phosphorylated epitopes of MSP. One rabbit polyclonal sera (rMSPab) and three mouse monoclonal antibodies were produced. These antibodies were used to immunolabel rabbit and bull whole sperm cells and resulted in nearly identical patterns and labelled the sperm tail similar to MPM-2. Immunoelectron microscopy of whole rabbit sperm and rabbit testicular sections, showed MPM-2 reactivity primarily over the outer dense fibers and the capitulum of the sperm tail. Immunolabel with rMSPab revealed labelling of the outer dense fibers, fibrous sheath and the material surrounding the centrioles of the connecting piece. Immunoblots labelled with rMSPab indicated that reactivity is predominate at a 70-85 kD region of whole rabbit, bull, boar and mouse sperm proteins, with accessory labelled bands at 62, 64 and 55 kD. Immunoblots of extracted boar sperm proteins with MSP monoclonal antibodies displayed reactive bands at 70-85 kD as well as an additional cross reactive band at 55 kD, suggesting that the 55 kD protein is immunologically related. Zygote labelling with MSP antibodies revealed that MSP remains associated with the sperm tail throughout the first cell cycle and is centered in the sperm aster. Labelling of rabbit tissue proteins (spleen, liver, heart, lung, intestine, uterus, ovary and brain) on immunoblot with rMSPab and MPM-2 revealed that MSP is unique to mammalian sperm and all tissues contain immunologically related proteins, strongly reactive at 51-53 kD region. Injection of MSP antibodies into mature bovine oocytes, followed by sperm penetration, inhibited sperm derived microtubule aster formation. Taxol treatment of injected oocytes and zygotes suggested that MSP antibody blocked aster formation, but not by preventing microtubule nucleation. These data suggest that MSP is a structural component of the sperm tail outer dense fibers, specifically dephosphorylated following fertilization and blocking MSP with specific antibodies results in the failure of the sperm aster to develop but not by interfering with microtubule nucleation. We hypothesize that MSP is a possible intermediate filament type protein, forms a structural component of the sperm tail and MSP disassembly is a prerequisite for microtubule aster formation around the paternal centrosome.