| Cryptocaryon irritans, one of the most harmful parasites in seawater fishes breeding, could infect most sea fishes farming intensively and cause mass mortality. In this study, three strains of C. irritans isolated from three places (Changle, Ningde and Fuding) in different three fishes were identified by internal transcribed spacers (ITS) sequences. Then ITS-1 sequences and partial HSP70 sequences were analyzed for understanding the phylogenetic relationship of them. Results showed little genetic diversities among them.Cryptocaryon irritans was propagated in the laboratory using Paralichthys olivaceus as a model host, controlling water temperature between 22℃ and 24℃. The morphology in each life cycle period was detailly observed and recorded. The hatchability of tomonts and the infection ability of theronts were compared between two groups, the tomonts were stored in 5℃ for two months before hatched in 27 ℃ in one group and directly hatched in 27℃ in the other group. The serum collected from the fish infected after 2 weeks and 4 weeks was detected on clumping and immobilization to theronts. The result showed that P.olivaceus can be a model host for C. irritans. Up to 40 consecutive cycles were successively established,8 to 10 days in one cycle, providing adequate materials to our later study. Trophonts parasitized on the body surface, gills and fins. Shape of Trophonts was circular or elliptic changing with moving, coated with ciliums,100 μm-400 um in size, a little bigger than needlepoint with naked eye. Four macronucleuses could be clearly observed after Wright’s staining. The protomonts were round or oval with thin membrane, adhereing to the bottom wall after swimming slowly for 2 h-8 h in sea water. The tomonts were low penetrability with the size of 328.5 ± 39.6 urn x 401.2 ± 47.4 μm. In 24h to 48h, the protoplast of tomonts began to asymmetrically divide, finally develop to 70 to 300 theronts. Some trophont-like round protoplasts were observed orbiting in the tomont. Theronts were elliptic, coated with ciliums, with the size of 30.2 ± 6.5 μm × 66.9±6.5 μm and with four macronucleuses inside. The iatchability of tomonts dropt to 7.6% in the first group. And none trophont can be detected on the fish after exposed to 1000 theronts in this group. The immobilization titers of serum antibody to theronts could be 1:320 and 1:1280 in the two-week infected fish group and the four-week infected fish group. This indicated that humoral immunity played an important role against C.irritans infection.In this study, the membrane protein extracted from theront was used to immune Balb/C mouse. Finally 11 strains of positive hybridoma cells were screened out after cell fusion, subcloning and indirect ELISA screening. The ascites contained monoclonal antibody was got from the Balb/C mouse after injection positive hybridoma cells 5D11AG5,5H9BG3,6E11CE7 and used as the first antibody in Western-blot and immunofluorescence assay. The results show that Isotyping of the three McAb were all IgM. The titer of McAb-5D11AG5 was about 1:3200, while McAb-5H9BG3 was about 1:25600 and McAb-6E11CE7 was about 1:51200. The Western-blot assay results showed that the McAb-5D11AG5 and McAb-5H9BG3 could recognize the 35kDa protein, but the McAb-6El 1CE7 couldn’t do. This indicate that recognition epitopes of McAb-6E11CE7 were conformational epitopes. However, 3 kinds of McAb all could recognize the antigens on theront in an immunofluorescence assay. McAb-5D11AG5 and McAb-5H9BG3 could recognize on the front of theronts and McAb-6E11CE7 could recognize on the whole outer membrane all of this work would make a contribution to separate and screen functional protein of C.irritans. |
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