| In this study, epidemiology survey on Cryptosporidium spp. from ostriches and duck in Henan province was conducted, 14 and 96 Cryptosporidium isolates were respectively obtained from ostriches and duck. Based on nested-PCR and corresponding RFLP analysis techniques, the genes of 18S rRNA, HSP70 and actin were used for genotyping. Thus, to identify the Cryptosporidium species, genotypes and subtypes of Cryptosporidium isolates from ostriches in Henan, and so to determine the phylogenetic relationship with other Cryptosporidiium species. Moreover, to infer the infection resource and transmission dynamics of cryptosporidiosis in ostriches and the results may provide a theory foundation for the prevention and treatment of cryptosporidiosis in ostriches. At the same time, in order to study the genetic difference of host-adapted Cryptosporidium to avian, Cryptosporidium isolates from different avian hosts were also included in this study. And the multiloci analyses showed that host-adaptation is a common phenomenon in genus Cryptosporidium, the sequences of different avian which come from different geographic arear are not significant deviation.1. To systematically understand the prevalence of Cryptosporidium from ostriches in Henan province, between August 2006 and August 2007, 829 fecal samples collected from six farms in Henan were examined using the Sheather's sugar flotation technique and the modified acid-fast stain method. Results showed that the average infection rate was 1.7% (14/829). Thereinto, only one farm located in Zhengzhou city was found Cryptosporidium infection and the general infection rate was 2.8% (14/506), no Cryptosporidium ifection was discovered in other three farms. Moreover, the 14 positive isolates were all come from 20～40 days old ostriches, which indicated that young ostriches were more susceptible to Cryptosporidium. The mean length and width of oocysts were 5.56 and 5.48μm respectively (4.2～6.0μm×4.5～5.0μm), with a mean shape index of 1.24 (n=100). Based on the measure of oocysts, the Cryptosporidium isolates were primarily identified as C. baileyi.2. From Octov\ber 2006 to May 2007, 564 fecal samples obtained from eight duck farms in Henan province were checked using the Sheather's sugar flotation technique and the modified acid-fast stain method. Results showed that the Cryptosporidium infection rate was 16.31% (92/564). Thereinto, four farms were found Cryptosporidium infection and the infection rate were respectively 100.00%,73.33%, 51.79% and 41.46%. 92 positive samples were all from lower than 50-day-old duck. Therefore, the young duck is more susceptible to Cryptosporidium. And there are evident difference between the infection rate and the age group of duck. The mean length and width of oocysts obtained were 5.82 and 4.40μm, respectively, with a mean shape index of 1.32 (n=100). Basen on the measurement data, the Cryptosporidium isolates from duck were primarily identified as C. baileyi.3. Nine Cryptosporidium isolates from ostriches were amplified the specific fragment of 18S rRNA gene, the length of fragment was about 820bp by sequencing analysis. At the same time, PCR products were digested by SspI and VspI restriction enzyme to determine the Cryptosporidium species and genotype. after digestion by SspI restriction enzyme, nine isolates produced identical fragments of 260bp and 570bp, while two fragments of 100-110bp and 620bp were also obtained when the PCR products were digested by VspI restriction enzyme. According to the profiles obtained, the nine Cryptosporidium isolates were identified as C. baileyi.4. In order to determine Cryptosporidium species/genotypes from the aspect of phylogenetic relationship, the specific fragments of 18S rRNA, HSP70 and actin genes were amplified from the nine Cryptosporidium isolates by nested-PCR. After sequencing, sequence similarity search was conducted by Blast and Fasta provided in the three nucleotide acid databases, i.e., NCBI, EMBL and DDBJ. Then, sequence alignment analyses, phylogenetic trees and similarity analyses were respectively performed by using the softwares of Clustal X1.81, Phylip3.67 and DNAstar4.0. Thus, based on the three gene luci, to approach and analyze the genetic difference of Cryptosporidium host-adaptation in different avian hosts.Based on PCR-RFLP analyses and multiloci phylogenetic analyses of 18S rRNA, HSP70 and actin genes, the Cryptosporidium isolates from ostriches were identified as C. baileyi. Thus, this study provides molecular biology background and essential theory foundation for the study of ostriches cryptosporidiosis in the future. In conjuction with analyes of the Cryptosporidium isolates from different avian hosts, i.e., pigeon from Henan, chicken from Beijing and Ningxia, Quain and U-chicken from Henan, the multiloci phylogenetic analyses showed that host-adaption is the general phenomenon in genus Cryptosporidium. No strict host specificity was found in C. baileyi. Moreover, the co-evaluation between Cryptosporidium spp. and hosts is also the typical characterization in the genus Cryptosporidium, i.e., with a closer phylogenetic relationship among near hosts. |
