In hemoprotozoa, gene transfer technology provides an important tool to aid in the functional study of parasite genes. In this study, a transfer vector containing the enhanced green fluorescent protein (EGFP) gene laid between the Toxoplasma gondii GRA1 promoter region and the GRA2 polyA signal region was constructed and transfected into the in vitro culture of Babesia bovis by the electroporation method. On the first and second days post-transfection, clear positive fluorescences were detected in some parasite bodies, indicating the successful expression of the EGFP gene controlled by the GRA-1 promoter in B. bovis. However, the positive parasites were not ubiquitous and finally disappeared until the forth day post-transfection. This is the first time that the expression of a foreign gene has been reported in the Babesia parasite.

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To develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant vaccina virus expressing an immunodominant surface protein p23 of Cryptosporidium parvum (C. parvum) sporozoites. Antibody against p23 recognized the p23 expressed in RK13 cells infected with the recombinant vaccinia virus as an approximately 23 kDa specific band in Western blotting analysis. The immunization of Balb/c mice with the recombinant induced the production of immunoglobulin G1 (IgG1). However, the level of immunoglobulin G2a (IgG2a) production was very low. These results indicate that the p23 expressed by the recombinant vacinia virus induced predominantly a Th2 response in Balb/c mice. In the case of C57BL mice, delayed type hyper sensitivity was observed. However, the induction of antibody production against p23 was not detected.

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The role of nitric oxide in immune defense against primary infection with Babesia microti was investigated in the present study. Babesia microti infected C57B1 mice reach peak of parasitemia around day 14 PI. with approximately 45% PRBC and a moderate fall in hematocrit. C57B1 mice treated with the iNOS- inhibitor aminoguanidine reached a 55% PRBC peak parasitemia and the hematocrit dropped to approximately 29% PBCV. C57B1 iNOS-/- mice reached a peak parasitemia of more than 80% PRBC within 10 days Pl. and their hematocrit dropped to about 20% PBCV. The clearance of parasites from the peripheral blood and recovery of the hematocrit in aminoguanidine-treated C57B1 mice and iNOS-/-mice takes much longer than in the C57B1 wildtype control mice. During the course of infection there were marked differences in cytokine expression patterns of IFN-γ and TNF-α between C57B1 wildtype and C57B1 iNOS-/- mice. C57B1 wildtype mice treated with anti-IFN-γ mAB followed a different course of infection and survived the infection. On the other hand iNOS-/-mice treated with anti-IFN-γ mAB failed to control the rapid rise in parasitemia and died. Surviving animals of the iNOS-/- and anti-IFN-γ mAB-treated iNOS-/- groups subsequently developed sever kidney damage. These results show that NO is involved in early control of primary infection with Babesia microti, but also suggest that for the control of the acute stage of infection the effect of IFN-γ is not exclusively mediated through the induction of NO.

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