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Cryptosporidiosis is caused by the obligate intracellar protozoan parasites, Cryptosporidium,which infects the epithelium of gastrointestinal and respiratory tract of animals and humans,has recently emerged as a global public health problem. Nowadays,cryptosporidiosis was considered as one of the suspicious index of acquired immunodeficiency syndrome(AIDS) and one of the six diseases leading to diarrhea worldly.However,up to the present,the epidemiology and genotypes of Cryptosporidium parasites in cows in Shanghai city,are not distinctly understood. Furthermore,there is no effective treatment available for cryptosporidiosis in human and animals,although large numbers of antiparasitic,antifungal,antibiotic,or antivirval agents were investigated.Consequently,the present study was undertaken to investigate the molecular detecting techniques,epidemiology in cows in Shanghai city,and screening of drugs against cryptosporidiosis in vivo and in vitro.1.Development of Nested PCR Assay for Detection of CryptosporidiumTo develop a sensitive and species-specific nested PCR diagnostic technique for detection and identification of Cryptosporidium oocysts,two pairs of primers were designed on the base of the 18S rRNA gene published in Genbank.The results showed that the primers were highly differential to C.parvum and C.andersoni,no amplification products were detected in the control DNA samples extracted from E.bovis,Neospora,Toxoplasma gondii and E.coli.The least dosages detected by nested PCR were five oocysts in one gram feces.The fragments of 514 bp and 509 bp were amplified by nested PCR from C.parvum and C.andersoni.In conclusion, nested PCR was suitable for the molecular epidemiology survey of Cryptosporidium.2.Development of Real-time PCR Assay for Detection and Quantitation of Cryptosporidium in cowsReal-time PCR method for detecting Cryptosporidium in cows was established with primers and probe designed according the 18S rRNA of GenBank and recombinant plasmid as DNA template.This method was validated on positive samples including C. parvum and C.andersoni.The results showed that amplification of DNAs from the C. parvum and C.andersoni were detected(SYBR Green),C.andersoni were detected (TaqMan) and others(C.bailey,Toxoplasma gondii,Neospora caninum and Escherichia coli) were not.The sensitivity of the assay was 5 copies of recombinant plasmid and 5 ooeysts per one gram of feces.In summary,a rapid,sensitive and specific real-time PCR method for the detection of Cryptosporidium from fecal samples has been developed,which could be served as a valuable tool for epidemiological survey.3.Molecular Epidemiology and Genotyping of Cryptosporidium Parasites in Cows in ShanghaiThe present study was designed to investigate the epidemiology of Cryptosporidiosis in cows in shanghai.Fecal samples were detected by nested PCR and real time PCR.The amplified fragments were cloned,sequenced and digested by EcoT14I to identify.As a result,a total of 101(16.03%) animals were detected positive by nested PCR in the five farms,and 98(15.56%) animals were positive by real time PCR(SYBR Green),58(9.2%) in C.andersoni(TaqMan).The prevalence in the spring,summer,autumn,and winter were 14.61%,10.56%,16.89%,and 23.08 %,which vary significantly among the four seasons(χ~2=9.20,P＞0.05).And there was a significant difference between the prevalence in winter and that in summer(χ~2=8.64, P＜0.01).The prevalence of the calves(17.28%) was not significantly higher than that of the grown cows(16.46%)(χ~2=0.04,P＞0.05),but significantly higher than that of the milch cows(χ~2 = 4.15,P＜0.05).Oocysts morphology analysis,RFLP analysis, phylogenetic analysis and a distances matrix generated from these sequences showed that C.andersoni and C.parvum were identified in cows in Shanghai city,and the C. parvum isolate belonged to the species C.parvum were most similar to the C.parvum "mouse" genotype.It is concluded that the prevalence of cows was high,and there are two species of Cryptosporidium in Shanghai city,the isolates of C.parvum is C. parvum "mouse" genotype.There might be a cross-transmission between cows and mice.4.The suppressive effect of Chinese Herbal Medicinal Ingredients on C.parvum infectionThe present study was designed to investigate the suppressive effect of Astrgalus Polysaccharides(APS),Oxymatrine(OM),Paromomycin(PRM),Glycyrrhizie acid (GA),Chlorogenic acid(CA),and Polydatin(PD) on C.parvum infection in vitro,and APS,OM and PRM fon C.parvum infection in vivo.The results in vitro showed that the C.parvum adhesion to MDBK cells were significantly suppressed by APS,the therapeutic effect of the OM and PRM groups was better than those of others(p＜0.05 or p＜0.01).The results in vivo showed that the C.parvum adhesion to MDBK cells were significantly suppressed by APS and OM(p＜0.05 or p＜0.01),the therapeutic effect of the OM and PRM groups was better than that of APS(p＜0.05 or p＜0.01).In conclusion,APS,OM and PRM could effectively inhibit the growth of C.parvum infection in vitro and in vivo.
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