Studies Of Population Structure And Molecular Epidemiologic Investigation Of Four Intestinal Protozoa In Cattle In Some Areas Of Jiangxi Province

Giardia lamblia(G.lamblia),Cryptosporidium,Enterocytozoon bieneusi(E.bieneusi)and Blastocystis are four common zoonotic opportunistic intestinal protozoa distributed all over the world.Its cysts,spores or oocysts are widely existed in the environment.Giardiasis,cryptosporidiosis,microsporidiosis and blastocystisasis caused by these parasites can cause diarrhea in cattle,affect production performance,and hinder the development of animal husbandry,which is a threat to the health of people and animals.Jiangxi province is a major cattle-raising province in China,but there is no report about the infection of these four intestinal protozoa in cattle in Jiangxi province.Therefore,a total of556 fecal samples were collected from the four cities(Nanchang,Gao’an,Xinyu and Ji’an)of Jiangxi province,and molecular epidemiological investigation and genotyping of these four protozoa were carried out.In this study,the prevalence and genotype of G.lamblia were identified by nested PCR amplification of the bg,gdh and tpi gene loci,and Multi-locus Genotyping(MLGs)was used to further explore the population structure of G.lamblia.Cryptosporidium was identified by nested PCR at SSU r RNA locus,then using the Multi-locus sequence typing tool(MLST)based on four microsatellite/small satellite loci(MS1,MS2,MS3 and MS16)for subtypes detection of Cryptosporidium.E.bieneusi were examined by nested PCR amplification of the internal transcribed spacer(ITS)and MLST was used for ITS positive samples based on three microsatellite loci(MS1,MS3 and MS7)and a small satellite loci(MS4)to detect MLGs.The presence and genotype of Blastocystis were detected by using the PCR amplification based on the SSU r RNA gene.The difference in prevalence among regions,species and ages of cattle were analyzed,and the phylogenetic tree was established by Neighboor-Joining method to further gain potential public health risks for genotypes detected in the research area.The results found that the prevalence of G.lamblia,Cryptosporidium,E.bieneusi and Blastocystis in cattle in Jiangxi province were 9.2%(52/556,95% CI: 6.93-11.77),12.8%(71/556,95% CI: 9.82-15.27),5.4%(30/556,95% CI: 3.52-7.27),and 54.9%(305/556,95% CI: 50.72-58.99),respectively.The infection rate of Blastocystis is significantly higher than other three protozoa,which considered to be the dominant species of intestinal protozoa in cattle in Jiangxi province.Sequencing analysis showed that 52 G.lamblia-positive samples were detected,including two Assemblages(Assemblage A and E)and 6 new gene subtypes,forming 15 MLGs and one mixed infection.A total of 3 species of Cryptosporidium were identified in 71 Cryptosporidium positive samples,including Cryptosporidium bovis(C.bovis,1/71),Cryptosporidium ryanae(C.ryanae,7/71)and Cryptosporidium andersoni(C.andersoni,63/71).C.andersoni was predominant species in cattle in Jiangxi province.MLST analysis showed that all C.andersoni-positive samples were amplified at the four microsatellite/minisatellite loci(MS1,MS2,MS3,and MS16),forming 3(A2,A4,A5),1(A4),1(A4)and 1(A1)haplotypes,and three MLST subtypes of C.andersoni were identified(A4,A4,A4,A1;A5,A4,A4,A1;A2,A4,A4,A1).A total of eleven E.bieneusi genotypes were identified in this study,including six known genotypes(D,n = 10;I,n = 5;J,n = 4;IV,n = 4;N,n = 1;BEB4,n = 1)and five novel genotypes(JX-I to JX-V).Based on the analysis of the gene population structure,genotype D is the dominant genotype of E.bieneusi in cattle in Jiangxi province.Four known genotypes of Blastocystis(ST1,ST5,ST10,ST14)were identified.Among them,ST10 was the dominant genotype of Blastocystis.The result revealed that a local public health safety risk in research area.In summary,this study performed molecular epidemiological investigations of four intestinal protozoa in cattle in some areas of Jiangxi province,and found the cattle in Jiangxi Province were infected with G.lamblia,Cryptosporidium,E.bieneusi and Blastocystis.In four intestinal protozoa,the infection rate of Blastocystis was the highest than others.Sequencing analysis revealed the prevalence,genotype,genotype distribution and population structure of the four important intestinal protozoa in cattle.Furthermore,this study analyzed the causes of the prevalence of intestinal protozoa and clarified the dynamics and characteristics of the spread of bovine intestinal protozoa in the province,which not only provided data for the prevention and control of bovine intestinal protozoa,but also contributed to the prevention and control of public health in Jiangxi province.