Infected Investigation On Dairy Cow Cryptosporidiosis And Preparation Of McAb Against Cryptosporidium Parvum

KM femina Mices about 15g Immunosuppressived by DEX were infected with standard Cryptosporidium paruvm oocysts.We had purified the oocysts from the fecal by cane sugar discontinuous gradient after 6 days infected,extracted the DNA from the standard Cryptosporidium parvum and standard Cryptosporidium andersoni, designed the two pairs primer according to the downloaded sequences of 18S rRNA from NCBI,after Nested PCR,the productions were undertook by agarose gel electrophoresis,obtained duplicate bands about 508bp and 514bp,recovered the PCR productions and enzyme cut by ECOT14â… and cloned and sequenced.We had succeeded to establishing the way of detection Cryptosporidium Tyzzer by Nested PCR-RFLP,and distinguished the C.p from C.an.An epidemiology of cryptosporidia in cows was surveyed with cane sugar discontinuous gradient and Nested PCR-RFLP on seven farms in shanghai area.Six farms were positive,the positive rate was 85.71%.691 samples were individually collected from these farms and 128 samples were positive,the total infectious rate of Cryptosporidium Tyzzer was 18.52%,Two type of oocysts were found,they were identified as C.parvum with infection rate of 6.22%(43/691),and C.andersoni 12.3%(85/691)respectively according to agaroseelectrophoresis,RFLP and sequence. The infectious rates of calves,beefs and cows were 26.03%(82/315),15.38% (40/260),4.79%(7/146),The infectious rates of calves is much higher than that of both beefs and cows.Different farm had different infectious rate,the infectious rates were 39.58%,26.74%,0,3.80%,19.44%,12.71%,14.63%.C.parvum in samples' homology of 18S rRNA were 99.2%compare to C.parvum publication on NCBI,and 85.2%-85.8%compare to C.andersoni.C.andersoni in samples' homology of 18S rRNA were 97.4%-98.4%compare to C.andersoni publication on NCBI,and 85.4% compare to C.parvum.The oocyts of C.parvum were purified from samples fecal of cows in shanghai area,KM femina Mices about 15g Immunosuppressived by DEX were infected.The oocysts were purified from the fecal by cane sugar discontinuous gradient after 6 days infected,800rpm,centrifuged 5min,repeated 5 times,freeze-thawed and sonicated. BALB/c mice were immunized for four times with purified oocyst antigen of C.parvum.Spleen cells of the mice were selected to fuse with SP2/0 cells by PEG1450.First the hybridized cell were cultured by HAT selective medium,and then were cultured by HT selective medium,the last were cultured by conventional culture medium.The positive wells were selected by indirect ELISA,and after 3 generations of clone by limited dilution,2 hybridized cell clones(1D5,4E12)against C.parvum were obtained.The solution titers were 1:3200 and 1:1600,and ascite titers were 1:102400 and 1:51200.The monoclonal ascites were examined with indirect Immunofluorescence assay,two were anti-oocyst.Two ascites clones were purified and passed through SDS-PAGE electrophoresis,the result shown that the heavy chains were approximately 54kDa,and light chains was about 24kDa.Molecular weights were about 94 kDa and 85 kDa.