We examined the detailed cellular localization of the rhoptry-associated protein-1 (RAP-1) of Babesia bovis merozoites by the post-embedding method of immuno-electron microscopy (IEM) using the anti-RAP-1 monoclonal antibody (mAb) 1C1 in order to substantiate the result in our previous report by an indirect immunofluorescent antibody test and the pre-embedding method of IEM. RAP-1 slightly distributed only in cytoplasm of merozoites, especially around the nucleus, but not in the rhoptry organelle at an early stage after invasion into a red blood cell. Then, the RAP-1 was accumulated in the rhoptry organelle and was also found in the iRBC cytoplasm, which suggests that synthesis and release of RAP-1 may occur in the iRBC. Finally, the RAP-1 was found within and around merozoites after the breakdown of the iRBC. The results of the present study suggest that the RAP-1 of B. bovis merozoites functions not only in the invasion into RBC but also in the escape from iRBC.

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The resting cysts of Colpoda cucullus were resistant to not only drying, but high and low temperatures. The critical temperatures for the survival of wet cysts ranged from 40 ℃ (3-hr exposure) to 45℃ (10-min exposure), and those of the dried cysts ranged from 80 ℃ (3-hr exposure) to 100 ℃ (30-min exposure). Frozen (-30 ℃, 3 hr) and remelted wet cysts also survived. Ca²⁺-induced resting cysts excysted when the external Ca²⁺ was removed. However, the excystment-inducing effect by Ca²⁺ removal was canceled by the addition of cations such as Na⁺, K⁺, and Mg²⁺ in the surrounding medium. When the mature cysts transformed in a diluted buffer without any other salts were temporally exposed to Ca²⁺ and subsequently resuspended in the buffer without Ca²⁺, excystment was induced in a majority of cysts. The addition of chlorophyll-derived molecules in the surrounding medium induced excystment, but suppressed encystment. This suggests that the excystment-inducing and encystment-suppressing activities of cereal infusion are attributed to water-soluble porphyrins derived from chlorophyll. The encystment was induced when the vegetative cells were suspended at a high density, and the substitution of non-living particles such as polystyrene latex particles (PLP) for the living cells also showed an encystment-inducing effect. The result suggests that mechanical cell-to-cell contact induces encystment.

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To develop DNA vaccines against cryptosporidiosis, a plasmid coding an immunodominant protein of Cryptosporidium parvum sporozoite, p23 (pCX-p23) and another plasmid coding a fusion protein containing whole the p23 and the Fc portion of mouse immunogloblin G1 (pCX-p23Fc). Vaccination of BALB/c mice with the plasmid, pCX-p23 and pCX-p23Fc induced the production of antibodies against p23. Although both of splenocytes of mice immunized with the plasmids pCX-p23 and pCX-p23Fc expressed interleukin-4 and interferon-γ, after the in vitro stimulation by p23 antigen, the interferon-γ expression level of pCX-p23Fc immunized mice was much higher than that of pCX-p23 immunized mice. These results suggest a possibility of the plasmid pCX-p23Fc as a DNA vaccine candidate against cryptosporidiosis.

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Morphological changes during resting cyst formation (encystment) of Colpoda cucullus were examined. At 1 hr after encystment induction, a number of vesicles containing the electron-dense and toluidine blue (TB) positive materials were fused with large vacuoles to excrete the contents, and the vacuoles opened to the extracellular space to excrete. In this stage, the rounded precystic cells were surrounded by the first-synthesized cyst wall layer (L1 layer), and the cilia were resorbed inside the L1 layer. Thereafter (1-2 hr), a secondary layer (L2 layer) was formed and subsequently a deeply TB stained substance was diffused into the space between the plasma membrane and L2 layer. At 3-6 hr, numerous endoplasmic reticula (ER) associated with ribosomes occupied the cytoplasm, indicating that cyst wall precursors such as glycoconjugates were being synthesized. By a week after encystment induction, the cyst wall composed of three distinct layers covered with a fibrous (mucous) coat was completed. The kinetosomes were still observed in 3-day old cysts.

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