Identification And Phylogenetic Analysis Of Cryptosporidium Of Giant Pandas

Lots of data have reported about the wild animals infected with Cryptosporidium. Up to now, at least12of24valid species of Cryptosporidium were identified:C. andersonis, C. muris, C. baileyi, C. canis, C. felis, C. galli, C. hominis, C. parvum, C. meleagridis, C. saurophilum, C. ubiquitum and C. serpentis. Among the12valid species, C. muris, C. parvum, C.canis, C.felis, C. meleagridis, C. ubiquitum and C. hominis were zoonotic species. Therefore, the research of Cryptosporidiosis of giant pandas will play a significantly important role in public health.Now, about giant panda infected Cryptosporidium hasn’t been reported. Based on that, we studied a survey of prevalence of Cryptosporidium infecting giant pandas captived in Conservation and Research Centre for the Giant Panda of BI FENG XIA, ya’an city. The morphology of the oocysts, the sequence analysis of the gene18S rRNA、 HSP70、COWP and actin, and the fragment of Restriction enzymes Sspl and Vspl were researched to identified the species and genotype.The sheather’s sugar flotation technique was used for the survey of pervalance of Cryptosporidium in giant pandas and one panda was detected positive. The fecal of the positive panda was examined for60days in order to observe the shedding of oocysts. In morphology, the isolate (oocysts) ranged in size from3.95-5.51μm (length)×3.29-4.70μm (width), with an average size of4.60×3.99μm (n=50), and the ratio was1.15. The sequences of giant panda-Cryptosporidium were amplified by nested PCR of18S rRNA, HSP70, COWP and actin genes. Sequences anlysis show that the sequences of the isolates were different from all reference sequences for each of the gene characterized, and there were no difference in the same locus.The isolates were closely related to Cryptosporidium bear genotype with11,10,56and12nucleotide differences, respectively. The homology to bear genotype at the18S rRNA locus was98.6%, which is comparable to that between C. parvum and C. hominis (99.2%), or between C. muris and C. andersoni (99.4%). At the HSP70and actin loci, the similarities were max with99.4%and98.8%, respectively. However, fragment analysis through digestion of18S rRNA secondary PCR products with Sspl and Vspl indicated that the visible bands were observably distinguishing from restriction fragment lengths of other common Cryptosporidium genotypes and species.Based on these data, the Cryptosporidium isolated from giant panda was considered as a new genotype, Cryptosporidium giant panda genotype. This was the first report of giant panda infected Cryptosporidium, the identification of new genotype significantly made up for phylogenetic relationship and host adaptation among spieces. Likewise, it provided important reference for further research about the biological characteristics of Cryptosporidium giant panda genotype. In addition, this study contributed genetic data to prevention of giant pandas Cryptosporidiosis.Moreover, the author also did some research about other intestinal protozoa along with the study of prevanlence of giant pandas infected Cryptosporidium. The results showed that the prevalence of this study was obviously lower than wild pandas.