Molecular Characterization Of Enterocytozoon Bieneusi,giardia Duodenalis And Cryptosporidium Sp.in Captive Masked Palm Civets (Paguma Larvata) In Southern China

Enterocytozoon bieneusi,Giardia duodenalis and Cryptosporidium spp.are common enteric pathogens in humans and various wild and domestic animals,causing diarrhea or other gastrointestinal symptoms.Humans can be infected by these pathogens through anthroponotic or zoonotic route.Masked palm civets are known to play an important role in the transmission of some zoonotic pathogens.In recent years,masked palm civet has been widely farmed in southern China.However,the distribution and zoonotic potential of Enterocytozoon bieneusi,Giardia duodenalis and Cryptosporidium spp.in these animals remain unclear.Therefore,this preliminary study was conducted to detect and assess zoonotic potential of E.bieneusi,G.duodenalis and Cryptosporidium spp.in captive masked palm civets in southern China.In this study,we collected 889 fecal specimens from four masked palm civets farms in Hainan,Guangdong,Jiangxi and Chongqing,southern China,and analyzed for the prevalence,genotypes and subtypes of these pathogens by nested PCR based DNA sequencing.Theχ~2 test was applied to compare differences in the prevalence of these pathogens among different farms and age groups.Then,the multilocus sequence typing(MLST)was used to further explore the population structure and genetic diversity of E.bieneusi in masked palm civets.Altogether,the infection rates of E.bieneusi,G.duodenalis and Cryptosporidium spp.were 53.3%(474/889),3.8%(34/889)and 0.1%(1/889),respectively.Theχ~2 test showed infection rates of E.bieneusi on farms in Hainan and Chongqing were significantly higher than those in Guangdong and Jiangxi(P<0.01),while the G.duodenalis infection rate on the farm in Guangdong was significantly higher than those in Hainan and Jiangxi(P<0.01),suggested the transmission of E.bieneusi and G.duodenalis is different in these farms.By age,E.bieneusi infection rates in animals of<1 year(61.0%;P<0.01)and 1～2(53.5%;P<0.05)years were significantly higher than in animals of>2 years(40.9%).Significantly higher prevalence of G.duodenalis was detected in animals of 1～2 years(10.9%)than those of<1 year(2.8%,P<0.01)and>2 years(2.4%,P<0.01),suggested the age may be a risk factor in the transmission of E.bieneusi and G.duodenalis in masked palm civets.Sequence analysis revealed two known E.bieneusi genotypes(Peru8 and J)and11 novel ones(named as PL1～PL11).Among them,PL1 and PL2 were the dominant genotypes.The phylogenetic analysis showed PL4,5,9,10 and 11 were placed in zoonotic Group 1,while PL1,2,3,6,7 and 8 in Group 2.Further MLST analysis of E.bieneusi have revealed a high genetic diversity and the occurrence of some genetic recombination among isolates on the farm in Jiangxi which is the largest among the four farms and has been established for over 20 years.Additionally,the zoonotic genotypes were only found in this farm.The long history of the farm and high genotype diversity have probably facilitated the genetic exchange among E.bieneusi isolates and the emergence of zoonotic genotypes in farmed masked palm civets.Among the G.duodenalis-positive specimens,the dominant assemblage B was zoonotic.Further multilocus genotyping of assemblage B has revealed that all 13 multilocus genotypes in civets formed a cluster related to those from humans.A novel Cryptosporidium genotype named as Civet genotype I was found.The phylogenetic analysis based SSU r RNA,gp60 and hsp70 genes showed this genotype was genetically related to the Cryptosporidium bamboo rat genotype II.This first report of E.bieneusi,G.duodenalis and Cryptosporidium sp.in farmed masked palm civets in southern China extends our knowledge on the taxonomy and transmission of these pathogens in captive wild animals,which would be beneficial to the development of intervention measures against these pathogens.