Cryptosporidium Epidemiology Investigation On Non-human Primates And Species Identification And Genotyping Of Cryptosporidium Isolates From Some Isolates

Epidemiological investigation on non-human primates cryptosporidiosis was carried out in several areas. 5 Cryptosporidium isolates derived from non-human primates were made nested PCR-RFLP detection and analysis; genotypic analysis of the Small-Subunit rRNA (SSU rRNA) gene were carried out to identify Cryptosporidium species and genotype of non-human primates, conclude Cryptosporidium molecular evolution relationship of non-human primates and other species.Then we could found non-human primates cryptosporidiosis infection source and mechanism of transmission in order to provide theoretical foundation for prevention of non-human primates cryptosporidiosis.To study the prevalence of Cryptosporidium in non-human primates, a survey was performed in 739 fecal samples with the modified acid fast stain and the Sheater's sugar flotation technique. As a result, 52 fecal samples were positive, the average infection rate of Cryptosporidium sp. was 7.04%(52/739). The infection rate of 142 samples use the modified acid fast stain was 18.31%(26/142), The infection rate of 597 samples use the Sheater's sugar flotation technique was 4.36%(26/597). The infection rate was different in the different areas and the different seasons. These Cryptosporidium sp. was identified C.parvum according to the shape and size of oocysts.The Small-Subunit rRNA(SSU rRNA) specific fragments of 5 non-human primates Cryptosporidium isolates were amplified by nested PCR. The PCR products were connection and transformation, cloned and sequenced. The sequence length was 830-840bp. The PCR products were digested by SspI restriction enzyme to determine species and genotype. One isolates was got 2 fragments at about 450bp and 380-390bp, Based on length of restriction fragments, this isolates was close C. muris. Other 4 isolates were got 3 fragments which about 100bp, 250bp and 450bp. These four isolates were initially considered to be C. parvum or C.hominis.In order to determine species/ genotype of Cryptosporidium isolates, 5 non-human primates Cryptosporidium isolates were amplified by nested PCR, cloned and sequenced based on 18S rRNA gene. The sequence length was 830-840bp. 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 software such as Clustal X 1.81, and DNAstar 4.0. There were 2 clusters. One isolate was close to stomach parasitic group and other four isolates were close to intestinal parasitic group. Homology analysis showed that SH01 isolates have 99.3% homology with C. muris, other four isolates have 99.2%,99.8%,99.9% and 99.4% homology with C.parvum monkey genotype, respectively.Based on PCR-RFLP, genotypic and subgenotypic analysis , the species/ genotype of isolates in this experiment were reached consensus and identified as one C. muris-like and four C.parvum monkey genotype. The molecular biology evidence for non-human primates infected C. muris-like was found for the first time. This study provided a rationale for understand the Cryptosporidium relationship with non-human primates and other mammal, and provided a reference for evaluate human and non-human primates close activity security.