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Cryptocaryon irritans is a ciliated obligate parasite of marine fish, causing white spot disease in tropical and sub-tropical regions. Recent studies have shown that fish can mount immune responses against C. irritans and acquire protection. The present study compared the protective immunity in grouper (Epinephelus coioides) against C. irritans induced by theronts, trophonts and tomonts via intraperitoneal (i.p.) injection and tentatively identified an immobilization antigen. Specific antibody titres of immunized fish serum were determined by enzyme-linked immunosorbent assay and immobilization assays at weeks 0, 1, 2, 4, 6 and 8 post-immunization, and immunized grouper were challenged with theronts to detect the survival percentage of fish. Fish immunized with theronts produced higher levels of serum antibody against C. irritans than fish immunized with trophonts or tomonts. A significantly higher level of immune protection was achieved in fish immunized with theronts but not in fish immunized with tomonts or trophonts. Western blot analysis using polyclonal immobilization antibodies of rabbit immunized with theront revealed an immunoreactive band of the protective (immobilization) antigens of C. irritans, the size of which is approximately 34 kDa. The sera of mice immunized with the protein could immobilize theronts of C. irritans. These results demonstrated that the theront stage of C. irritans elicited stronger protective immunity than the trophont and tomont stages in grouper. The tentative identification of protective antigen provides the solid foundation for the development of a defined vaccine against C. irritans.

Cryptocaryon irritans is one of the most important ectoparasites of marine fish. To identify the potential role of immune-related genes in antiparasitic immune responses in fish, we monitored the expression change of IL-8, COX-2, C-type lectin and transferrin in local and systemic immune organs of orange-spotted grouper post-C. irritans infection. IL-8 expression was up-regulated during the course of infection in the skin, while COX-2 and transferrin expression was up-regulated in the gill. COX-2 expression was significantly down-regulated in the spleen (0·7-5% of its control) and head kidney (0·5-4% of its control) post-primary infection. Transferrin expression was also down-regulated in the spleen and head kidney from 6 h to 5 days post-primary infection. However, C-type lectin expression was up-regulated in all tested organs post-infection, with the exception of day 7 in the spleen post-primary infection where the expression level was slightly down-regulated (44% of its control). These results suggest that these four immune-related genes play an important role in grouper anti-C. irritans infection and that local immune organs as the active organs contribute more than systemic immune organs to this course.

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Immobilization antigens (i-antigens) are surface membrane proteins that are widely recognized to be the ideal candidates as vaccines antigens for immunization against Cryptocaryon irritans. In this study, we cloned a putative i-antigen gene from C. irritans, which was expressed in all three stages of the C. irritans life-cycle, and localized primarily to the cell surface. The recombinant GDCI3 i-antigen was expressed and purified using the free-living ciliate, Tetrahymena thermophila as an expression system. The purified recombinant protein was recognized by rabbit anti-C. irritans antiserum and was capable of eliciting immobilizing antibodies in rabbits and fish suggesting that the antigen itself was correctly folded. Following immunization and parasite challenge, groupers vaccinated with, recombinant GDCI3 i-antigen had a 25% cumulative percent survival rate compared to 8.3% for controls. Both non-specific and parasite-specific IgMs were generated in fish following immunization, with the levels of both increasing following challenge. Parasite-specific IgM in mucus could only be elicited after challenge of the GDCI3 i-antigen vaccinated groupers. To our knowledge, this is the first report using the Tetrahymena expression system to generate C. irritans i-antigens and investigate their use for fish vaccination.

Cryptocaryon irritans (Brown, 1951) is a ciliated protozoan that was first described by Sikama, 1 who reported that it infected more than 45 fish species reared in marine aquaria. It was later renamed Ichthyophthirius marinus based on its cellular morphology, which is very similar to that of I. multifiliis, a parasitic ciliate of freshwater fish. 2 However, in 1951, Brown 3 re-described this parasite, which was found in imported fish, and named it C. irritans. This name is still in use today.