Research On Cryptosporidium Species/Genotype/Subgenotype Of Human Strain In Zhengzhou

The experiment of epidemiological investigation of human cryptosporidiosis was carried out in Zhengzhou.The two Cryptosporidium isolates derived from children were made cross-transmission experiment,nested PCR-RFLP detection and analysis;genotypic analysis of the Small-Subunit rRNA(SSU rDNA) gene,70-kDa Heat Shock Protein(HSP70)gene and actin gene as well as subgenotypic analysis with GP60 gene were carried out to indentify species/ genotype/ subgenotype,determine molecular phylogenetic relationship between isolates drived from human of this area and other species,conclude human infection source and transmission mechanism in order to provide theoretical basis for prevention of human cryptosporidiosis.In order to understand the infected condition of Cryptosporidium and its epidemiological characteristics in Zhengzhou of Henan Provice,samples of fresh stool obtained from the patients including infant patients,tumour patients and common patients who were treating in 4 hospitals of Zhengzhou were detected for oocysts of Cryptosporidium by sugar centrifugal flotation method for one year.The results revealed that the total infection rate of Cryptosporidium was 0.0003% (2/6720).The two isolates were derived from infants.This suggests that the children are more vulnerable than adults.To study biological characteristics of cryptosporidium spp,the isolates were used to conduct cross-transmission experiment: Purified isolates oocysts were used to inoculate inmmunocomprise mice,mouse feces were detected for 25 days,but no cryptosporidiurn oocyst was detected from mice feces.The Small-Subunit rRNA(SSU rRNA)specific fragments of isolates were amplified by nested PCR.The PCR products were digested by SspI restriction enzyme and VspI restriction enzyme respevtively to determine species and genotype. Fragments of 108bp,251bp and 485-495bp were got after digested with SspI and fragments of 100-110bp and 590-600bp were got after digested with VspI.Based on length of restriction fragments,the two isolates were initially considered to be C.hominis.In order to determine species/genotype of cryptosporidium isolates from human in Zhengzhou,specific fragments of three genes including 18S rDNA gene,HSP70 gene,actin gene were amplified by nested PCR,cloned 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 Clustalâ…©1.81, and DNAstar 4.0.Based on the phylogenetic analysis of 18S rDNA gene,HSP70 gene and actin gene,the two isolates were idenfied as C.hominis. As a molecular marker for subgenotyping,GP60 gene specific fragment 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 Clustalâ…©1.81,and DNAstar4.0.Based on the subgenotyping analysis,the two were identified as C.hominis Id allete,and were named C.hominis Id A31GS.Based on PCR-RFLP,genotypic and subgenotypic analysis,the species/ genotype of isolates in this experiment were reached consensus and identified as C.hominis.it can be concluded that two human cryptosporidiosis patients of this experiment may be infected cryptosporidium through direct or indirect contact with oocysts which were excreted from cryptosporidiosis patients in environment and the transmission route of human cryptosporidiosis in this area is person to person cycle. pattern.