Molecular Epidemiology Of Cryptosporium In Scale Chicken Farms In Henan Province

Cryptosporidium spp. are important zoonotic protozoa that infect more than 240 animals, including humans. Cryptosporidiosis can cause severely harmful disease to human and animals. In this study, epidemiological investigation on chicken cryptosporidiosis was conducted by the Sheather's sugar flotation technique and the modified acid-fast staining method in Henan province. Moreover, based on cross transmission using young chicken and nested PCR amplification of 18S rRNA gene, the partial Cryptosporidium isolates obtained were used to study the pathogenicity and molecular characteristics, thus to provide a theory foundation for studying the molecular epidemiology of cryptosporidiosis.1. In order to study the prevalence of cryptosporiosis in scale chicken farms in Henan Province , a survey on Cryptosporidium was performed from August 2006 to July 2007 by the Sheather's sugar flotation technique and the modified acid-fast staining method. 2015 fecal specimens were examined from eight scale chicken farms and 37 moderate farms in ten districts. The results showed that the infection rate of laying hens was 10.57%(163/1542) and the broilers was 3.17%(15/473). of all isolates,0-30d, 31-60d and 121-180d chicken were found to be more easily infected,and the infection percentage were3.70%,24.60% and 12.87% respectively. According to the characterization of morphology, the oocysts collected were identified as Cryptosporidium baileyi. The investigation revealed the infection rate of Cryptosporidium was the highest from March to May and lowest from December to next February.2. Cross-infection tests were carried out on pathology, ultrastructure of some organs with pathologic histology technique and scanning electron microscope technique, 3-day-old chickens were inoculated Cryptosporidium oocysts from laying hens. The results showed that the prepatent period was 4 days, and the patent period lasted 18 days, and the peak period was in 7-14 after infected. Main clinical signs were be odserved in the respiratory system. The oocysts mainly resideed in larynx, trachea, bursa of Fabricius and cloaca.3. According to the published 18S rRNA gene primer, 32 isolates were amplified by nested PCR. The PCR products were digested by sspI restriction enzyme and vspI restriction enzyme respectively to determine species and genotype. After digestes with in sspI restriction enzyme , 28 isolates were separated into 573bp ,267bp, and 4 isolates were separated in 450bp,267bp,111bp. Fragments of 108bp, 251bp and 485-495bp were got after digested with sspI; with vspI restriction enzyme digested, 28 isolates were separated into 621bp,115bp,104bp etc,4 isolates were separated in 457bp,171bp,115bp etc. Based on length of restriction fragments, the isolates were initially considered to C. baileyi and C.meleagridis.4. In order to determine species/ genotype of cryptosporidium isolates from chicken of Henan province, specific fragments of 18S rRNA gene, were amplified by nested PCR and sequenced. Then, Blast 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, Phylip3.65 and DNAStar 4.0. Based on the phylogenetic analysis of 18S rRNA gene the 28 isolates were identified as C.baileyi; four isolates were in the same clade with EF641025,EF17938, which were downloaded from GenBank ,and could be identified as C.meleagridis .According to the results of the investigation of henan province and the collected dates ,we found that cryptosporidium was exited broadly in henan. Both of C. baileyi and C.meleagridis exist in chicken farmes, but C. baileyi was the predominant species according to our results. It was verified that C. baileyi oocysrs were mainly found in BF and respiratory system though the infection assay. 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.meleagridi .there were some mutation in some isolates.This paper provides some reference of Molecular Epidemiology of Cryptosporium in scale chicken farms in Henan province. Its useful in assessment of the public health significance of Cryptosporidium in henna chicken farmsBoth of C. baileyi and C.meleagridis were existed in chicken farmes, but C. baileyi was the predominant species according to our results. It was verified that C. baileyi oocysrs were mainly found in BF and respiratory system though the infection assay. 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.meleagridi .there were some mutation in some isolates.This paper provides some reference of Molecular Epidemiology of Cryptosporium in scale chicken farms in Henan province. Its useful in assessment of the public health significance of Cryptosporidium in henan chicken farms.