Survey Of Dairy Cryptosporidiosis And Molecular Phylogenetic Relationship Of Cryptosporidium Isolates In Henan Province

The epidemiological survey of water buffaloes ,beef catlle and dairy cattle Cryptosporidiosis were carried out in Henan Province. Some Cryptosporidium isolates derived from water buffaloes ,beef catlle and dairy cattle were identified by microscope, nested PCR-RFLP detection and nested PCR-RFLP analysis ;genotypic analysis of the Small-Subunit rRNA (SSU rRNA) gene were carried out to indentify species/ genotype, determine molecular phylogenetic relationship between zoonotic cryptosporidium species/ genotypes, and to deduce animals infection source and transmission route.To investigate the prevalence of Cryptosporidium in water buffaloes, 257 fecal samples ,which were collected from water buffaloes in Jigong Mountain of Henan Province ,were examined by saturated sucrose solution flotation technique and the modified acid-fast staining method. The results showed that the total infection rate was 16.73%. Cryptosporidium parvum and C.andersoni were discovered by microsc- ope. C. parvum is the only zoonotic species/genotype,and the infection of C. parvum is 0.78%.Two C.bovis isolates were founded in water buffaloes more than 12 moths. The results of the present study confirm that these water buffaloes should be consider- ed as a potential zoonotic reservoir for drinking water source.In order to understand the infected status of Cryptosporidium and its epidemiological characteristics in beef catlle of Henan Provice, samples of fresh stool obtained from beef catlle were detected for oocysts of Cryptosporidium by the saturated sucrose solution and the modified acid-fast staining method. The results revealed that the total infection rate of Cryptosporidium was 4.32%(9/208). C.andersoni were only founded in beef catlle by microscope. To elucidate the genetic diversity, the partial sequences of the cryptosporidium 18S ribosomal RNA gene were targeted by nested PCR. Nine isolates from beef catlle were identificated species/ genotype/ subgenotype by nested PCR-RFLP analysis and genotypic analysis of the Small-Subunit rRNA (SSU rRNA) gene .The results show that nine isolates from beef catlle were C.andersoni .To further understand the prevalence of cryptosporidiosis among dairy frams in Henan Province,a total of 582 fecal samples were collected from less than 12-month-old calves in nine dairy farms from Zhengzhou, Kaifeng, Jiyuan and Hebi districts, the results showed the total infection rate was 26.12(152/582) by the Sheather's sucrose flotation technique and the acid-fast staining method. Thereinto, the infection rate of pre-weaned(5 days to 2 months) calves was 30.91%(51/165), and 24.22%(101/417) in post-weaned (3–12 months)calves respectively. Two species of Cryptosporidium were discovered, namely, Cryptosporidium parvum and C.anderson.. C.parvum, the only zoonotic species/genotype, constituted 50.98% of the parasite infections in pre-weaned calves ,but only 9.90%of the Cryptosporidium infections in post-weaned calves. The result of present study showed that C.parvum was of high infection rate in Henan, to verify Cryptosporidium in dairy is of important role in public health, and suggested that the type of confinement for pre-weaned dairy calves had significant influence to infection rate of two species of Cryptosporidium.The Small-Subunit rRNA(SSU rRNA) gene specific fragments of isolates from were amplifie dairy catlle by nested PCR. The PCR products were sequenced and digested by Ssp I restriction enzyme and Vsp I restriction enzyme respevtively to determine species and genotype. The amplified fragments length of two isolates were 837bp. Two of neneteen isolates'fragments of 110bp ,265bp and 420-430bp were got after digested with Ssp I ,the others fragments of 380-400 bp and 440-460 bp were got after digested with Ssp I . Two of neneteen isolates'fragments of 100-110bp and 600-630 bp were got after digested with Vsp I , two isolates'fragments of 110-180bp ,440-470 bp were got fter digested with Vsp I and the others fragments of 110-120bp,720-740bp were got after digested with Vsp I .Based on length of restriction fragments, two isolates were considered to be C.bovis , two isolates were considered to be named Cryptosporidium cervine genotype and the others were considered C.andersoni.In order to determine species/ genotype of cryptosporidium isolates from dairy catlle of Henan Province, specific fragments of 18S rRNA gene was amplified by nested PCR, and sequenced. Then, Both Blast and Fasta methods was used to search homological sequences in NCBI, DDBJ and EMBL, after that, homological sequences were alignmented. Phylogenetic tree and homological analysis were made by some biological softwares such as Clustal X 1.81, and DNAstar 4.0. Based on the phylogenetic analysis of 18S rRNA gene, two isolates were idenfied as C.bovis , two isolates were idenfied as Cryptosporidium cervine genotype and the others were idenfied as C.andersoni.This is the first identification of a genotype of Cryptosporidium cervine genotype oocyst in the feces of dairy cattle . Cryptosporidium cervine genotype can infecton mammalian including human .The results of this experiment lend an important data for further studying the biological characteristics of Cryptosporidium cervine genotype.