Identification And Phylogenetic Analysis Of Cryptosporidium Of Bactrian Camel And Bamboo Rat

Cryptosporidium is a kind of obligate intracellular protozoa parasite, it causes significant harm to host by making them severe diarrhea. This parasite’s host range is extensive, commonly found in humans and more than 260 species of vertebrate animals. Researches had shown that wildlife is considered an important reservoir for Cryptosporidium spp., So far, several species of Cryptosporidium have been found in wild animals. In this study, we try to isolate Cryptosporidium spp. from 1370 simples of feces from wild animals from Bifengxia Wildlife Zoo, Chengdu Zoo, Guiyang Zoo, two black bear farmings and a bamboo rat farming from Ya’an, as well as giant panda from Chengdu and Ya’an; Combineg morphological and molecular methods, to identify the Cryptosporidium species, and using MLST and gp60 loci to identify their subtypes; And taking homology analysis by the phylogenetic tree based the sequences of each gene locus to research their eggs types and genotyping position.The results show that this study isolated a Cryptosporidium spp. from bactrian camel and three Cryptosporidium spp. from bamboo rats. The morphology of Cryptosporidium oocysts isolated from bactrian camel was ellipsoidal and lacked sporangium, the color was lavender. The oocysts of the isolate (SCBC02) measured 6.07-8.21μm(length)× 4.75-6.20μm (width), with an average size of 7.03x5.50μm. Three gene loci were chosed to identify the species of Cryptosporidium, and they were 18S rRNA, COWP and CpA135. Combined the morphology of Cryptosporidium oocyst and results of molecular identification, we found this isolate was Cryptosporidium andersoni. This isolate was subtyped by multilocus sequence typing technique. Amplification of the minisatellite targets (MS1, MS2, MS3 and MS 16) was achieved by nested PCR. MLST subtype of the bactrian camel isolate was identified to be A4, A4, A4 and A1 at four loci respectively. The result in this study classified as the most common subtype found in cattles in China. Immunosuppression Kun-ming, Sprague-Dawley mice and hamsters were inoculated with C.andersoni oocysts, and the results of cross-infection experiment were negative.Three isolates of Cryptosporidium from bamboo rats were morphology similar, the shape was ellipsoidal and lacked sporangium. We analyzed the sequences of 18S rRNA, COWP, CpA135, HSP70 and actin gene and constructed evolutionary trees to identify the species of them. By homology analysis, we found that the difference between YA01, YA02 YA03 of 18S rRNA locus was little, all of them were 100% homologywith Cryptosporidium parvum; In COWP site, the three isolates were 0.3% difference, and all of them were shown the highest homology with C.parvum; In HSP70 locus, there is no difference between YA01 and YA03, but these two shown 0.2% differences with YA02; In actin sites the differences between YA01, YA02 and YA03 with reference sequence homology C.parvum were 99.7%,99.2%,99.4% respectively; In CpA135 site, YA01 and YA02 were same, and their homology with YA03 was 99.3%. Phylogenetic trees observed that three Cryptosporidium spp. were associated with C.parvum in the same branch. With morphological and molecular methodtyping, we identified three isolates were Cryptosporidium parvum. three subtypes of Cryptosporidium spp. were identified and named ⅡpA9, ⅡpA9, ⅡoA13G1, what is more, YA01 (ⅡpA9), YA02 (IIpA9) were new subtypes using gp60 site.In general, this is the first time isolated Cryptosporidium spp. in bactrian camel in Sichuan, the researchs we did about it enriched the data of bactrian camel zoonosis. Furthermore, we isolated and identified three bamboo rat-derived Cryptosporidium spp., it filled in blanks of researches on parasitic diseases of bamboo rats, and take a foundation for zoonotic parasitic disease prevention and control.