| Cryptosporidium spp., as one of common diarrhea pathogen, can invade in human and animal gut cell and cause cryptosporidiosis. There are more than 20 species in the genus, but Crypotpsoridium parvum is the most common and harmful species. For the immune underdeveloped or immunocompromised hosts, Cryptosporidium can cause serious damage, or even threat to its life. At present, sensitive and specific diagnosis methods, effective drugs and vaccines to prevent and control the disease are still dificient. Thus, screening antigens with well immunogenicity and immunoreactivity activity and developing effective diagnosis methods and vaccine are still an important direction of cryptosporidiosis research.Two indirect ELISA methods were developed for the detection of Cryptosporidium infection in pigs using recombinant CP15/60 and CML protein as antigen. The nested PCR was selected to compare the effectiveness. Field pig serums which collected from 3 slaughter houses were detected with the two methods. The results showed that the optimization concentration of coated antigen of two recombinant proteins were all 2 μg/m L, the optimal dilution of testing serums were all 1:100 and the rabbit anti-pig Ig G/HRP sera were all 1:800. Compared with nested PCR method, the positive and negative coincidence of r CP15/60-ELISA were 100.0% and 80.0%, respectively, but the values of r CML-ELISA were 66.7% and 60.0%, respectively. Two hundred and ninty-one pig field serum samples were detected with the two methods, the positive rate of r CP15/60-ELISA was 59.5%, the r CML-ELISA was 51.5% which indicated the former was more sesitve than the later. The detection results of infected sera of Mycoplasma suis, Toxoplasma gondii, Swine fever virus, Porcine reproductive and respiratory syndrome(PRRS) were all negative. The positive rate of field pig samples deteced with r CP15/60-ELISA was 56.89%(194/341), while with r CML-ELISA was 44.47%(157/353). These results indicated that 2 sensitive and specificity ELISA detection methods of cryptosporidiosis were developed. They were suitable for laboratory preliminary screening and epidemiologic survey of pig cryptosporidiosis.In order to detect the prevetion effect of subunit vaccine developed with r CP15/60 and r CML, and compare with other antigens, the two recombinant antigens and others 4 antigens, r Cp T, r Cp Tm, r ENO, r P23 were mixed with Freund adjunts respectively to develop subunit vaccines to immnune BALB/c mice by injecting subcutaneously. The humoral and cellular immune responses were detected. The results showed that the Ig G, TNF and IL-5 level in immuned mouse serums, CD4+ and CD8+ cell numbers of spleen of immunized mice were all improved with different degrees in 6 groups, which indicates that all 6 vaccines produced specific humoral and cellular immunity. The results of artificial challenge showed that the oocysts product in fecal of each group were all reduction. The oocysts reduction rate of r CP15/60 immunized group was 45.57% which only lower than r ENO immunized group, but higher than other groups. The oocysts reduction rate of r ENO immunized group was 34.08% which only lower than r ENO immunized group, similar with r P23 immunized group, but higher than r Cp Tm and r Cp T immunized groups. This result indicated that the subunit vaccines made with r CP15/60 and r CML could induce obviously humoral and cellular immune responses and were two important cryptosporidiosis vaccine candidate antigen. |
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