Immunoprotective Efficacy Of Six Cryptosporidium Antigens

Cryptosporidium is an important global epidemic zoonotic protozoa,with a wide range of hosts.The disease is now thesecond diarrhea disease(only at late site of rotavirus).Each yearthere are 0.8 million of children who under the age of 5 died for the diarrhea caused by the parasite.Although much research has been done on the basic biological characteristics,pathogenic mechanisms,immunology,genomics,etc.,and significant progress have been gained.However,there are still many problems need to face.The disease still havn't effective therapeutic drugs and preventive vaccines.Three real-time PCR assays,JVAP18S?Csp18S and CRU18 S methods were selected to test their sensitivity,specific and repeatability,and compared with nested PCR based on Cryptosporidium 18 S rDNA.The results showed that the 3 real-time PCR assays were all effective quantitate Cryptosporidium detection methods.Compared with the other two methods,the JVP18 S was more sensitive,specific and repeatable and suit for detection of field samples,especially with few parasites.After infected with Cryptospridium tyzzeri,the mice start to produc oocysts in feces at day 3 post infection(D3PI),reached peak at D9 PI,and then decreased slowly.There were a second oocyst product peak at D21 PI,and still could be detected at D30 PI.The coding gene of C.tyzzeri surface antigen CP15 was cloned and expressed successfully in prokaryotic expression system.The expressed protein was soluble and had good immunological activity.The anti-sera were acquired by immuned the recombinant CP15 protein tomiceand indirect ELISA detection showed that the prepared antiserum could react with the soluble antigen of C.parvum oocysts.Six vaccines,including 3 subunit vaccines made by rCP15,rCpSushi and r CP41,respectively,3 nucleic acid vaccines pVAX-CpCTL10,pVAX-CpTh10 and pVAX-CP15,were selected to compared their induced situation of humoral immunity and cellular immunity,and immuno protective effect to resistant artifial C.tyzzeri infection.The results showed that the levels of serum IgG antibody,the product of IL-4,IL-17,INF-? and TNF cytokines,the numbers of spleen CD4+ and CD8+ T lymphocyte and cell proliferation of spleen cell were all significantly higher than the control groups(p<0.05),indicating that the six kinds of vaccines were all induced obviously specifichumoral and cellular immune responses.The results of artificial infection test showed that the numbers of fecal oocysts production of the immunized mice were allsignificantly reduced.The 3 highest reduction rate of oocysts production were pVAX-CpCTL10,rCP41 and rCpSushi immunized groups,respecitvely.These results indicated that the six groups of vaccines all had fineprotective effect on Cryptosporidium infection.