Species Identification Based On DNA Characteristics And Preparation Of Monoclonal Antibody Of The Cryptosporidia Isolates Derived From Cows

An epidemiological survey in cows was made on one farm which was moved from Hangzhong of Zhejiang province to Zhengzhou one month ago.624 fecal samples were collected and analyzed with the saturated sugar solution flotation and Ziehl-Neelsen staining(acid-fast stain)technique.Results showed that 14 samples were infected with oocysts of cryptosporidium.The total infection rate was 2.24% (14/624).According to their morphology and characteristics,two different oocysts were identified,one was Cryptosporidium bovis,and another was C.andersoni.The infection rate of C.bovis and C.andersoni were 0.16%and 2.08%respectively.The infection rate of calves is lower than the juvenile and mature cattle,which was different with that reported recently.Cross-transmission showed that C.andersoni has no infectivity to severe combined immunodifficiecy mice(SCID)and immunosuppressed or immunocompetent Kun-ming mice.Nested PCR-RFLP by analysis of the 18SrRNA gene was used to detect Cryptosporidium from cow feces.The fragments of 18SrRNA gene were amplified by Nested PCR from the twelve Cryptosporidium isolates respectively.RFLP restriction maps of twelve isolates formed after PCR products were digested by VspI and SspI were identital to RFLP restriction maps of C.andersoni of camel and different with C. baileyi obviously.Phylogenetic analysis showed that the Cryptosporidium species formed two clades.One clade contained C.baileyi,C.bovis,C.felis,C.canis,C. meleagridis,C.parvum human genotype,C.parvum and C.wrairi.The other clade contained C.andersoni,C.muris,C.serpentis and twelve isolates from calves.In the second group,the twelve isolates formed a single taxon with the C.andersoni.This lent the evidence to the results of PCR-RFLP,and further showed the twelve isolates were all C.andersoni.The oocyts of C.andersoni strain were purified by concentration,absolute-rest precipitation,vacuum fliteration,discontinous sucrose gradients and filtration. Purified oocyts were freze-thawed and sonicated to prepare particulate antigen and treated according to WEIR to prepare soluble antigen.Balb/c mice were immunized with two kinds of antigen.Spleen cells of the immuned mice were selected to fuse with SP2/0 cells.The positive wells were selected by indirect ELISA,and after 3 to 4 generations of clone by limited dilution,three hybridized cell clones against cryptosporidium was obtained(4F7,4C10,1E3).The solution titers ranged from 1: 100 to 1:6400,and ascites titers between 1:10000 and 1:160000.The monoclonal ascite was examined with indirect immunofluorescence assay,and 2 of 3 were anti-oocysts wall,the other one was anti-sporozoite.