Genotype And Subtype Identification Of Cryptosporidium From Three Different Animal Species

Cryptosporidium is an enteric protozoan parasite, with medical and veterinary importance, that infects a wide range of animals worldwide, including humans. Wildlife and cattle are important reservoir host of Cryptosporidium. Researchers have found a variety of Cryptosporidium in many kinds of wild animals, and there may be a cross-infection of Cryptosporidium between certain wild animals and bovine. Therefore, it is of importance and necessity to identify and analysis of genotype/subtype of Cryptosporidium between wild animals and bovine.In this study, we used saturated sucrose flotation method and molecular identification to delect the Cryptosporidium to 465 fecal fecal samples of wild animals from black bear farm, Tibetan macaque farm, takin farm, monkeys farm, Bifengxia Wildlife Zoo, Chengdu Zoo, Chengdu Giant Panda Breeding Base, Ya'an Giant Panda Protection Center. Through 18S rRNA loci,53 positive Cryptosporidium from cattle keep from dichromate saved by our laboratory were identified. Using GP60 loci and MLST technique analyze the subtypes of Cryptosporidium. And exploring the population characteristics of C. andersoni in Sichuan by structure analysis.The results showed that that wild animals infected with Cryptosporidium was negative by saturated sucrose flotation method, but delected Cryptosporidium positive in squirrel monkey and camel by molecullar idetification. The sequencing were submitted to Genebank after correction. The sequenses of Cryptosporidium isolates from squirrel monkey SCSM01 isolate and camel CDBC01 isolate were analyzed by homology and phylogenetic analysis, isolates SCSM01 and CDBC01 were the hignest homology with C. hominis and C andersoni, respectively. SCSM01 and CDBC01 each was associated with C. hominis and C. andersoni in the same branch. So they were identified as C. hominis and C. andersoni.We successfully amplified the 18S rRNA gene loci of 48 Cryptosporidium isolates from cattle (SC01-SC48), and for obtaining accession number, the sequencing results were submitted to genebank after correction. The sequenses of Cryptosporidium isolates from cattle SC01-SC48 were analyzed by homology and phylogenetic analysis. Isolates SC01-SC07, SC08-SC15, SC16-SC29 and SC30-SC48 belonged to four Ciyptosporidium species, and each had the hignest homology with C. parvum, C. lyanae, C. andersoni and C. bovis. Isolates SC01-SC07, SC08-SC15, SC16-SC29 and SC30-SC48 were each associated with C. Parvum, C. Ryanae, C. andersoni and C. bovis in the same branch. So they were identified as C. parvum, C. ryanae, C. andersoni and C. bovis, respectively. The result showed that C. bovis was the dominant species of cattle in Sichuan area. There was only a nucleotide difference between SC16-SC19 and CDBC01 at the 18S rRNA locus, while isolates SC20-SC29 and CDBC01 had no difference at the 18S rRNA locus. There may be a possibility of cross-contamination in Ciyptosporidium between the wild animals and cattle.GP60 locus was successfully used by nested-PCR amplifition to identify the subtybes of SCSM01 and SC01-SC07. For the GP60 gene, the isolate SCSM01 was identified as IkA7G4. The GP60 genes of SC01-SC07 were identified as IIdA19G1. CDBC01 isolate was identified as A6, A5, A2, A1; SC16-SC24 isolates were identified as A4, A4, A2, A1; SC25-SC28 isolates were identified as A4, A4, A4, A1; SC19 isolate was identified as A1 A4, A4, A1 by phylogenetic analysis. We got 3 groups through population structure analysis of subtypes when analysis with other subtypes of C. andersoni. The main subtype was focused on cluster 1 in this experiment, while other areas were in cluster 2, so the subtypes of C. andersoni in Sichuan were region-specific.In summary, the present study is the first detecting Cryptosporidium infection in squirrel monkey in the Sichuan region, enriching the work of cryptosporidiosis of squirrel monkeys, remedying the blank of Cryptosporidium in squirrel monkeys. And a new subtype of C. hominis was identified, filling the subtypes database of C. hominis. We identified C. andersoni re-infection in camel, and indicating C. andersoni was the dominant species infecting camel. And we detect the same Cryptosporidium species between cattle and camel, prefiguring the situation in which the Cryptosporidium may be the cross-infection between cattle and wild animals. It is the first time to detect C. parvum in cattle in Sichuan region and identify their subtypes, providing the basis data of prevention for cattle Cryptosporidiosis. And we analysis the population structure of the subtype of the C. andersoni and ascertain they has unique characteristics of the population.