Phylogenetic Analysis And Multilocus Sequence Typing Of Cryptosporidium Spp.and Enterocytozoon Bieneusi In Northeast China

Cryptosporidiosis and microsporidiosis are enteric diseases in humans and other animals mainly caused by Cryptosporidium spp. and Enterocytozoon bieneusi, respectively. They can cause self-limiting diarrhea in immunocompetent hosts and persistent and life-threatening diarrhea in immunocompromised persons, especially AIDS patients.In this study, the nested PCR amplification of small subunit ribosomal RNA (SSU rRNA) gene and internal transcribed spacer (ITS) region were used respectively for the detection of Cryptosporidium spp. and E. bieneusi. Fecal samples were collected from a few farms of HeilongJiang Province. Cryptosporidium spp. were detected in samples from calves and adult cattle, identified to be Cryptosporidium andersoni (identity100%with GenBank database), which were susceptible for bovine. The average infection rate was19.44%(7/36), in which calves infection (60%) was significantly higher than the prevalence of adult cattle (9.09%)(P=0.013); E. bieneusi were only detected in fecal samples from sheep and identified as genotype BEB6, which was first reported to be found in cattle on farms of USA in2007. This was the first report of this genotype in sheep. The results showed that Cryptosporidiosis in HeilongJiang Province were mainly caused by C. andersoni, and calves showed more susceptibly than the adults.Another nested PCR amplification of internal transcribed spacer (ITS) region was used for the detection and identification of E. bieneusi genotypes in duodenal mucosal specimens from livestock and poultry with serious diarrheal. Specimens were collected from over twenty cities/areas from northeast China.3of14chicken duodenal mucosal specimens from HeilongJiang Province and Jilin Province were positive for E. bieneusi with the infection rate21.43%(3/14);55of177pig duodenal mucosal specimens from varies cities/areas in northeast China were positive for E. bieneusi with the infection rate31.07%(55/177), the infection rate of HeilongJiang Province (31.11%,42/135) was similar to Jilin Province (43.33%,13/30)(P=0.199). Samples of which come from Qiqihar, Shuangyashan, Jixi in HeilongJiang Province, Tongliao in Liaoning Province and Tianjin City (including its subordinate county-level cities, county, etc.) were negative and the rest were all positive. In this study19genotypes were detected including9newly identified, named as CS-1to CS-8and CC-1according to the international nomenclature. Each of the new genotype sequence had SNPs with others and different from every known genotype sequence. By using the multilocus sequence typing (MLST) tool described by Feng,7samples were typed at loci MS1, MS3, MS4, and MS7respectively which were positive for E. bieneusi from HeilongJiang Province, Jilin Province and Inner Mongolia. As the results of previous study, ITS genotypes of those samples including EbpC, Henan-â…£, CHN7and two DNA sequencing failure samples at the ITS locus. The results showed that each sample had same sequences or only single SNP at multiple locus with other information from all over the world. Comparing with the host-adapted ITS genotypes sequences at all four locus and we used in this study, the basic features of ITS region corresponded to microsatellite and minisatellite locus. No.299specimen identified as genotype CHN7was first reported in Changchun had a highly similar genetic distance in MS1, MS3, MS4, MS7loci with other genotypes also found in pigs; although genotype Henan-â…£ and Henan-â…¤ were first identified in human from Henan Province, Henan-â…£ had close genetic distance with other zoonotic genotypes while Henan-â…¤ was similar to host-specificity genotypes at MS1locus and other loci had no successfully amplification. This was further evidence for the results before that genotype Henan-â…£ had zoonotic potential, on the other hand inferred that genotype Henan-â…¤ was host-specificity only for human. By applying this method and integrated analysis of multiple biological label data, we can give qualitative and speculation for E. bieneusi for its infectious, genetic characteristics of sub-population and other aspects. This will lay the foundation database for the future research of E. bieneusi.