Molecular Characterization Of Cryptosporidium Spp. From Quail In Henan Province

The epidemiological investigation of quail cryptosporidiosis was carried out in Henan province. The 239 Cryptosporidium isolates derived from quail.Selecting 45 isolates in the total were amplified by nested-PCRRFLP detection and analysed; genotypic/genotype analysis of the 18S rRNA gene and Actin gene, determing molecular phylogenetic relationship between isolates drived from quail of this province and other species, concluding quail infection source and transmission mechanism in order to provide theoretical basis for prevention of cryptosporidiosis.To study the epidemiology of Cryptosporidium in quail of Henan Province, a total of 1818 fecal samples were collected from 47 quail farms which breed about 200,000 quails in 5 areas, with the Sheather's sugar flotation and modified acid fast stain techniques. The infection rate of Cryptosporidium in quail is 13.15%(239/1818). By morphology determined, two Cryptosporidium species were found, which are C. baileyi and C. meleagridis. The data shown that the infection rate of Cryptosporidium has no relative with quail'species, however, which is correlation with territory, season and age. It provids the detail information to prevention the cryptosporidiosis of quail and public health security.Intestinal parasitism is one of the important factors restricting the development of poultry industry, so we did not only research the cryptosporidium of quail, but also detect the other intestinal parasites. In result , Cryptosporidium, Coccidium , Nematod were detected from the samples of quail, the infection rate are 13.15%(239/1818), 81.02%(1475/1818), 11.88%(217/1818), respectively. The data shown that the Intestinal parasites has relative with territory, season and quail'age.This investigation has provid the detail information to prevention and cure the intestinal parasitosis of quail.The18S rRNA specific fragments of 45 isolates were amplified by nested PCR. The PCR products were digested by SspI restriction enzyme and VspI restriction enzyme to determine species and genotype. Fragments of 573,267bp and 621,115,104bp were got after digested with SspI and fragments of 621,115,104bp and 457,171,115,104bp were got after digested with VspI. Based on length of restriction fragments, the 45 isolates were initially considered to be C. baileyi and C. meleagridis, in which the sample 41was infected both Cryptosporidium spp.In order to determine species/ genotype of cryptosporidium isolates from quail of Henan province, specific fragments of 18S rRNA gene, were amplified by nested PCR and sequenced. Then, Blast or 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 the 40 isolates were identified as C.baileyi,in which 3 isolates has one base difference from the other. The 2 isolates were identified as C.meleagridis .To the further reseach the C.meleagridis from quail, 18SrRNA gene and Actin gene specific fragment of the 2 isolates were amplified by nested PCR, and sequenced, then, Blast or Fasta 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 DNAstar4.0. Based on the phylogenetic analysis, the two were identified as C.meleagridis.Based on PCR-RFLP, genotypic and subgenotypic analysis,the species/genotype of isolates in this experiment were reached consensus and identified as C.baileyi and C.meleagridis. it can be concluded that quail cryptosporidium is one important source to the human cryptosporidiosis .