Molecular Epidemiological Investigation And Risk Factors Assessment Of Three Kinds Of Intestinal Protozoa In Domestic Black Bears In Heilongjiang Province

Enterocytozoon bieneusi,Blastocystis spp.and Cryptosporidium are important enteric protozoa,which distributed in a variety of human and animal species and geographic locations.The infections caused by them are generally considered opportunistic.It seriously endangers the health of the host and hinders the development of animal husbandry.Although there have been many investigations and reports on E.bieneusi,Cryptosporidium and Blastocystis spp.in China,there are few reports that black bears infect these three parasites.In order to clarify the infection,popular features,gene distribution of E.bieneusi,Blastocystis spp.and Cryptosporidium,in a black bear farm in Heilongjiang province,This study collected fecal samples from 312 black bears in Heilongjiang Province.The ITS,SSU r RNA,and 18 S SSU r RNA gene sequences were used to determine the infection status of E.bieneusi,Blastocystis spp.and Cryptosporidium.And identify the E.bieneusi and Blastocystis spp.protozoa genotypes.In this study,we investigated the prevalence and genotypes of the E.bieneusi in farmed black bear in Heilongjiang province.Results showed that the overall prevalence of E.bieneusi in farmed black bear was 4.8%(15/312;95% CI 2.4-7.2).The prevalence of female black bear was6.6%(9/136;95% CI 2.4-10.8),the prevalence of male black bear was 3.4%(6/176;95% CI0.7-6.1),females are higher than males.The prevalence of young black bear was 5.6%(10/180;95% CI 2.2-8.9),the prevalence of adult black bear was 3.8%(5/132;95% CI 0.5-7.1),young are higher than audlt.The prevalence of outdoor black bear was 10.5%(6/57;95% CI 2.3-18.7),the prevalence of indoor black bear was 3.9%(9/255;95% CI 1.2-5.8),outdoor are higher than indoor.Univariate analysis implemented in SPSS 16.0 was performed to analyze the risk factors associated with the pathogen prevalence.The OR(odds thewire)and 95% CI(the confidence interval)and P values were calculated to measure the infection risks.Founding that position(outdoor: P=0.033,P<0.05;OR:3.216)can increase risk of intestinal protozoa disease.Sequence analysis of the internal transcribed spacer(ITS)enabled identification of 6 genotypes in 15 PCR positive samples,including six novel genotypes(CHB-1,CHB-2,CHB-3,CHB-4,CHB-5 and CHB-6).The genotypes formed 1 genetic groups in phylogenetic analysis,with all the E.bieneusi genotyps distributed in zoonotic Group 1.In multilocus sequence typing(MLST)of E.biensusi sequenced at the three microsatellite loci(MS1,MS3,MS7)and one the minisatellitelocus(MS4)in faremd black bear.Finally,five novel MLGs were identified.These findings demonstrated the MLST tool is more powerful for revealing genetic diversity of E.bieneusi than ITS sequencing.We can use the MLST classification method to evaluate the possibility of zoonoticosis and its transmission methods in the E.bieneusi.In this study,we investigated the prevalence and genotypes of the Blastocystis spp.in farmed black bear in Heilongjiang province.Results showed that the overall prevalence of Blastocystis spp.in farmed black bear was 14.4%(45/312;95% CI 15.7-26.8).The prevalence of female black bear was 16.2%(22/136;95% CI 9.9-22.4),the prevalence of male black bear was13.1%(23/176;95% CI 8.0-18.1),females are higher than males.The prevalence of young black bear was 18.3%(33/180;95% CI 12.6-24.0),the prevalence of adult black bear was 9.1%(12/132;95% CI 4.1-14.1),young are higher than audlt.the prevalence of outdoor black bear was 24.6%(14/57;95% CI 13.6-37.3),the prevalence of indoor black bear was 12.2%(31/255;95% CI 8.1-16.2),outdoor are higher than indoor.univariate analysis implemented in SPSS 16.0was performed to analyze the risk factors associated with the pathogen prevalence.The OR(odds thewire)and 95% CI(the confidence interval)and P values were calculated to measure the infection risks.Founding that position(outdoor:P=0.018,P<0.05;OR:2.353)and age(young:P=0.024;P<0.05;OR:2.245)can increase risk of intestinal protozoa disease.Finally,with the SSU r RNA genes sequence analysis,Blastocystis spp.genotypes ST 1 were identified in45 the PCR positive samples.Through the phylogenetic analysis,it was found that there is a possibility of zoonotic disease in this genotype.In this study,we investigated the prevalence and genotypes of the Cryptosporidium in farmed black bear in Heilongjiang province,Results showed that the overall prevalence of Cryptosporidium in farmed black bear was 0%(0/312).To sum up,this study have determined the prevalence and genotypes in three important zoonotic parasites in farmed black bear of Heilongjiang province.And assessed factors associated with an increased infection risk.Using MLST tool sequence analyzed the E.biensusi in farmed black bear and attained the more genetic informations.Which provided basic data to prevent and control E.bieneusi,Blastocystis spp.and Cryptosporidium.