| Background and PurposeEnterocytozoon bieneusi was an important zoonotic pathogen,which was capable to infect intestine of humans and various animals.It can be transmitted by the fecal-oral route,via either direct contact or ingestion of contaminated food or water and cause persistent diarrhea and even death in immunocompromised persons.The pathogen existed around the world and was more common in tropical regions.As the only tropical island province of China,however,it was unclear that the prevalence,genotype distribution and epidemic characteristics of E.bieneusi in humans and animals in Hainan province.Up to date,there were no effective control measures of E.bieneusi.Therefore,the study on molecular epidemiology of E.bieneusi in humans and various animals in Hainan Province to determine the prevalence and molecular genetic characteristics,and to assess the risk of zoonotic transmission,which was helpful for the control of E.bieneusi in this area.MethodsFrom July 2018 and December 2019,feces samples from 1264 humans(including 352 from diarrhea outpatients,303 from diarrhea patients in children’s hospital,340 from Chinese college students,253 from kindergarten children,and 43from international students)and 1812 animals(including 193 from long-tailed macaques,173 from rhesus macaques,369 from wild rats,24 from red-bellied squirrels,93 from asiatic brush-tailed porcupines,117 from bamboo rats,188 from pigs,314 from cattle,and 341 from black goats)were collected in Hainan Province.All DNA specimens were examined by nested PCR amplification of the internal transcribed spacer(ITS)region of the r RNA gene and all the positive amplification products were sequenced,to determine the prevalence and genotype characteristics of E.bieneusi by sequence analysis,and to assess the zoonotic potential by sequence homology and phylogenetic analysis.Chi-square test(continuous correction or Fisher’s exact probability method)was used for comparison between count data groups and the probability(P)value<0.05 was considered significant.Results(1)The total positive rate of E.bieneusi in humans was 3.7%(47/1264).Among them,the highest positive rate of E.bieneusi was in diarrhea outpatients(5.8%,19/352);followed by diarrhea patients in children’s hospital(5.3%,16/303),college students(2.9%,10/340),and international students(2.3%,1/43),the lowest positive rate was in kindergarten children(0.4%,1/253)(χ~2=18.856,P<0.05).The positive rate of diarrhea patients(5.6%,35/628)was significantly higher than that in healthy people(1.9%,12/636)(χ2=11.955,P<0.05).The overall positive rate of E.bieneusi in animals was 21.0%(381/1812).Among them,the highest positive rate of E.bieneusi was in pigs(46.8%,88/188),followed by long-tailed macaques(30.6%,59/193),black goats(24.0%,82/341),wild rats(18.7%,69/369;including 7 species of wild rat),bamboo rats(15.4%,18/117),rhesus macaques(15.0%,26/173),cattle(9.9%,31/314),asiatic brush-tailed porcupines(7.5%,7/93),and the lowest positive rate(4.2%,1/24)was observed in red-bellied squirrels.(2)A total of 28 ITS genotypes were identified in humans,10 known genotypes(CHG5,CHG3,SHR1,CHG2,Type-IV,Ebp C,Ebp A,Pig EBITS7,CHG19,and CM21)and 18 novel genotypes(HNH-1 to HNH-18).A total of 46 genotypes were identified in animals,including 26 known genotypes(including Type-IV,D,Peru8,Pig EBITS7,Peru11,CM1,Ponog2,CM2,WL21,Henan-III,ESH-02,Ebp A,MJ14,S7,CS-4,CHG5,Ebp C,CHG19,SHR1,BEB4,J,I,CHG3,CHG2,CM21,and AHG1)and 20 novel genotypes(HNM-I to HNM-VIII,HNR-I to HNR-V,HNR-VII,HNP-I,HNP-III,HNP-IV,HNC-I,HNG-I,and HNG-II).(3)In the present study,a total of 64 genotypes were identified in humans and animals,all of which were classified into group 1,group 2,group 12 and group 13 on the phylogenetic tree.Among them,53 genotypes belong to group 1 or group 2,one genotype(S7)belong to group 12,and group 13 contains 8 genotypes in humans and3 genotypes in animals(SHR1 was identified in both humans and animals).Conclusions(1)This is the first study on the identification of E.bieneusi from humans and animals in Hainan Province.The overall rate of E.bieneusi in humans was 3.7%,and diarrhea associated with the infection of E.bieneusi.The prevalence of different animals varies with ranging from 4.2%to 46.8%.(2)A total of 64 genotypes were identified in humans and animals in Hainan Province,of which novel genotypes accounted for 59.4%(38/64).There were 30novel genotypes belonged to group 1(54.5%,18/33)or Group 2(60.0%,12/20)with8 genotypes identified in humans belong to Group 13,indicating the unique epidemic characteristics of E.bieneusi in Hainan Province.(3)All known genotypes identified in humans were found in the animals at the same time,indicating that the possibility of transmission from animals to humans and a higher risk of zoonotic potential of E.bieneusi in Hainan Province. |
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