Distribution Of Three Intestinal Protozoa In Crab-eating Macaques In Hainan And Zoonatic Potential

Cryptosporidium spp.,Enterocytozoon bieneusi,and Giardia duodenalis are the most common zoonotic parasites.They often infect humans through environmental factors,such as water-borne transmission(drinking water,recreational water,and irrigation water)and food-borne transmission(vegetables and aquatic products).Studies have increasingly shown that non-human primates(NHPs)are common hosts of these three zoonotic parasites,while most genotypes and subtypes in NHPs were also found in humans.Therefore,NHPs are one of the most important animal sources of these parasites to infect humans.Many studies have focused on non-human primates kept in zoos,but few have explored the genetic diversity and public health significance of these pathogens in intensive breeding primates.In this study,we examined the genetic diversity of the three pathogens in 1452 crab-eating macaques(Macaca fascicularis)in a commercial facility in Hainan,China.The genetic diversity of genotypes and subtypes within these species has been analyzed to assess the zoonotic risk and public health implications.Altogether,Cryptosporidium was detected using 18S rRNA-based PCR in 132(9.1%)sampled animals.Four Cryptosporidium species were identified,namely C.hominis(86),C.parvum(30),Cryptosporidium muris(15)and Cryptosporidium ubiquitum(1).The identified C.parvum,C.hominis and C.ubiquitum were further subtyped by using gp60 PCR.Among them,C.parvum belonged to subtypes in two known subtype families,namely IIoA14G1(18)and IIdA19G1(2).In contrast,C.hominis mostly belonged to two new subtype families Im and In,which are genetically related to la and Id,respectively.The C.hominis subtypes identified included ImA18(38),InA14(6),InA26(6),InA17(1)and IiA17(3).The C.ubiquitum isolates belonged to subtype family ?d.By subtype,ImA18 and IIoA14G1 were detected in animals with diarrhea whereas the remaining ones were mostly found in asymptomatic animals.Compared with C.parvum and C.muris,higher oocyst shedding intensity was observed in animals infected with C.hominis,especially those infected with the ImA18 subtype.Enterocytozoon bieneusi was detected by PCR analysis based in internal transcribed spacer(ITS)loci in 461(31.7%)fecal specimens from the animals.Nine E.bieneusi genotypes were detected in this study by DNA sequence analysis of the internal transcribed spacer of the rRNA gene,namely Type IV(236/461),Peru8(42/461),Pongo2(27/461),Peru11(12/461),D(4/461)and PigEbITS7(1/461)previously seen in NHPs as well as humans,and Macaque3(119/461),CM2(17/461)and CM3(3/461)that had been only detected in NHPs.The PCR amplification efficiencies were 81.8%(72/88),80.7%(71/88),86.4%(76/88)and 89.8%(79/88)at the MS1,MS3,MS4,and MS7 loci,respectively.Of the 88 samples,53 were successfully amplified at all four loci.Phylogenetic analyses based on sequences and allelic data showed the appearance of four major subpopulations(SPs)in the study population.The MLGs of the NHP-specific ITS genotype Macaque3 clustered in SP3 and SP4,suggesting these two subpopulations could be NHP-specific.The strong intragenic linkage disequilibrium(LD)among the five genetic loci and a positive standardized index of association(ISA)in allelic profile data indicated the presence of an overall clonal population of E.bieneusi in crab-eating macaques.The inconsistent segregation of isolates among loci suggested some occurrence of genetic recombination.G.duodenalis was detected by PCR analysis based on triosephosphate isomerase(tpi),glutamate dehydrogenase(gdh)and ?-giardin(bg)loci in 469(32.3%)fecal specimens from the animals.DNA sequence analyses of the tpi,gdh and bg loci identified all G.duodenalis specimens as having assemblage B.Altogether,eight(4 known and 4 new),seven(6 known and 1 new)and seven(4 known and 3 new)subtypes were seen at the tpi,gdh and bg loci,leading to the detection of 53 multi-locus genotypes(MLG-B-hn01 to MLG-B-hn53).Most of them were genetically related to those previously seen in common Old-World monkeys.Single-site-based genotyping tool cannot accurately assess the genetic diversity and public health significance of Giardia,so multi-site-based genotyping tool makes the typing results more reliable.In this study,the infection rate of intestinal parasites in crab-eating macaques with diarrhea symptoms was significantly higher than without diarrhea symptoms.It is speculated that mixed infection of intestinal protozoa may be the main reason for aggravating the symptoms of diarrhea in crab-eating macaques.At the same time,young monkeys are more susceptible to intestinal parasites,and most of the individuals infected with multi-worm species are young monkeys,which has important guiding significance for the feeding methods of cynomolgus monkeys.The above results are helpful for assessing the genetic diversity of Cryptosporidium,Enterocytozoon bieneusi,and Giardia to control the transmission of these three pathogens in the environment and animals.