Study On Population Structure And Investigation On Occurrence Of Important Zoonotic Intestinal Protozoan Parasites In Goats From Partial Areas Of Shaanxi Province

Protozoan parasites inhabiting intestinal tract of humans and animals are increasingly becoming one of research focuses in the field of global parasitology and public health because of their species varieties,wide distribution and serious damage.Among them,zoonotic parasites could be transmitted between humans and animals through various routes including water-and food-borne,causing severe gastrointestinal diseases,leading to significant damages to human health and animal production.Cryptosporidium,G.lamblia and Blastocystis have been recognized as important zoonotic intestinal protozoa.At present,numerous studies were focused on morphology,transmission ways and biological characteristics of these three parasites,but there are few reports about the epidemiology,genotyping and population structure in goats.Here,the prevalence,species and genotypes/assemblages/subtypes of Cryptosporidium,G.lamblia and Blastocystis were studied in goats from partial areas in Shaanxi province to evaluate the population structures of these zoonotic intestinal protozoa in goats from Shaanxi province.(1)Prevalence of Cryptosporidium,G.lamblia and Blastocystis in goatsTo explore the infection status of Cryptosporidium,G.lamblia and Blastocystis in goats in Shaanxi province,874 goat fecal samples were collected from Yuyang district of Yulin city,Baota district of Yan'an city,Fuping county of Weinan city,Qianyang county of Baoji city,Luonan county of Shangluo city and Yangling district in Shaanxi province to examine prevalence by using molecular methods and morphological characters.Microscopic observation showed that the size of the isolated Cryptosporidium oocysts was about 5 ?m,and oocysts were oval and contained an obvious oocyst residue and four naked sporozoites,while most G.lamblia existed in the form of nearly oval cysts with two nuclei,with the size of 15 ?m.Using molecular methods based on species-specific loci of 18 S rRNA,gp60,tpi,bg,gdh,the prevalence of Blastocystis was 58.0%(458/789),which was significantly higher than that of Cryptosporidium(11.8%)and G.lamblia(9.8%)(86/874),and it was identified as the dominant species.Mix infections were detected in 105 samples,accounting for 16.4%(105/639)of the total number of positive samples.Statistical analysis showed that the infection rates of three intestinal protozoa are significantly affected by goat breeds,age groups and geographical origins.The highest prevalences of Cryptosporidium(27.6%,53/192)and G.lamblia(14.6%,28/192)were found in pre-weaned goats(0-2 months),while the highest infection rate(66.5%,127/191)of the Blastocystis was detected in goats aged of 7 to 11 months old.(2)Species/genotypes of Cryptosporidium in goatsCryptosporidium species of 874 fecal samples of goats were identified based on the sequences of SSU rRNA gene fragment.Four species were found,including C.xiaoi,C.parvum,C.baileyi and C.ubiquitum.Of 101 Cryptosporidium positive samples,the numbers of C.xiaoi,C.parvum,C.baileyi and C.ubiquitum were 88,7(,5,and 1,respectively,with C.xiaoi as the predominant species.Two zoonotic species(C.parvum and C.ubiquitum)would pose potential to transmit Cryptosporidium to humans.Subtyping of C.parvum using the gp60 gene indicated four subtype IIdA19G1 and three subtype IIdA20G1.(3)Assemblages and multilocus genotypes(MLGs)of G.duodenalis in goatsAssemblages and MLGs of G.lamblia in 874 goat feces in Shaanxi province were identified based on loci of tpi,bg and gdh.Based on tpi-specific primers,a total of 65 assemblage E and six assemblage A positive samples were identified,belonging to seven assemblage E subtypes and three assemblage A subtypes.The bg-specific primers successfully amplified 49 assemblage E,four assemblage A and three assemblage B samples,forming five assemblage E subtypes,three assemblage A subtypes and three assemblage B subtypes.16 G.lamblia-positive samples were amplified at the locus gdh,and sequence analysis indicated 12 of assemblage E and two of assemblage A samples,forming four assemblage E subtypes and one assemblage A subtype.Multilocus sequence typing based on loci tpi,bg and gdh genes showed that 14 G.lamblia-positive isolates of assemblage E were successfully amplified at all three gene loci,respectively forming two,four,five assemblage subtypes and seven different MLGs.These MLGs were all observed in white cashmere goats of Yulin city and Guanzhong dairy goats of Fuping county.Among them,four,one,and four different MLGs were respectively detected in goats aged 2 months,3-6 months and 7-11 months.(4)Subtypes of Blastocystis in goatsSubtypes of 458 Blastocystis positive samples from different origins and production categories were identified based on the partial 18 S rRNA gene fragment.Six known(ST1,3,4,5,10,14)and one novel subtypes(Sd26)were identified,with the ST10 as the predominant subtype in goats from this area,accounting for 63.8%(292/458)of total subtypes.Among them,four subtypes,namely ST1,ST3,ST4 and ST5,were zoonotic subtypes.Collectively,investigation on infection status of intestinal protozoan parasites in different goat breeds from different areas of Shaanxi province clearly illustrated distribution of species,genotypes,subtypes and population structure of Cryptosporidium,G.lamblia and Blastocystis in goats in Shaanxi province,which provided valuable basic data for correlation research.