Molecular Detection And Identification Of Enterocytozoon Bieneusi Of Tarim Red Deer (Cervus Elaphus) In Southern Xinjiang

E.bieneusi is a kind of obligate intracellular parasitic eukaryote with a wide host range and genetic diversity.Currently,more than 600 genotypes have been identified based on ITS loci in humans and many animals,among which there are more than 60 deer-borne E.bieneusi genotypes.Tarim red deer is an endemic red deer subspecies in southern Xinjiang.In this study,PCR methods was used to detect and identify E.bieneusi from Tarim red deer in southern Xinjiang,in order to provide basic data for the study of E.bieneusi in Deer.(1)Based on ITS gene,630 faecal DNA samples of Tarim red deer from ten deer farms in Southern Xinjiang were detected by PCR,and 150 samples were found to be positive for E.bieneusi,with a total infection rate of 23.8%(150/630).Eight of the ten deer farms were positive for E.bieneusi,with the highest infection rate of E.bieneusi was 83.9%(26/31)in 8 farms,the lowest infection rate of E.bieneusi was 11.7%(26/222)in 5 farms.The infection rate of E.bieneusi of Tarim red deer in different deer farms was significantly different(P<0.01).The infection rates were 29.0%(20/69)、3.6%(1/28)、0.6%(1/181)、49.2%(95/193)and 20.8%(33/159)at <3 months of age,3-6 months of age,6-12 year of age,12-24 years of age,and >24 years of age,respectively.The infection rate of E.bieneusi of Tarim red deer in different age groups was significantly different(P<0.01).A total of 15 known E.bieneusi genotypes were identified by comparative analysis of sequences from 150 positive samples.The genotypes BEB6(n=81),D(n=32),COSI(n = 10),Ebp C(n = 2)and HND-IV(n = 2),HLJD-I(n = 6),CHWD1(n = 2)were zoonotic,respectively.The genotypes JLD-I(n = 2),JLD-IX(n = 3),JLD-IV(n = 1),JLD-XIV(n = 1),JLD XI(n = 4),JLD XIII(n =2),JL-VIV(n = 1)and JLD-XVII(n = 1)were idiotype,respectively.The genetic evolution tree was constructed by adjacency method,with the genotypes D,CHWD1,JLD-I,JLD-IV and Ebp C belonged to Group 1,and JLD-VIII,JLD-XIV,JLD-1,HLJD-I,JLD-XI,JLD-XVII,JLD-XIII,COS-I,HND-IV and BEB6 genotypes belong to Group 2.The results showed that E.bieneusi infection was common in captive Tarim red deer in southern Xinjiang,and its genotypes were characterized by genetic diversity.(2)The 81 samples identified as genotype BEB6 were amplified by PCR based on minisatellite(MS4)and microsatellite(MS1,MS3,MS7)gene.20,30 and 20 positive samples were amplified at MS1,MS3 and MS7,respectively.No positive samples were detected at MS4.Through gene sequence alignment analysis,eleven gene subtypes were formed at MS1 locus,which were named Type1(n = 2),Type2(n = 3),Type3(n =2),Type4(n = 2),Type5(n = 2),Type6(n = 3),Type7(n = 1),Type8(n = 1),Type9(n = 1),Type10(n = 2)and Type11(n = 1),respectively.Three subtypes were formed at MS3 locus,which were named Type1(n = 24)、Type2(n = 1)and Type3(n =5),respectively.Six subtypes were formed in MS7,which were named Type1(n= 7)、Type2(n = 1)、Type3(n = 7)、Type4(n = 2)、Type5(n = 2)and Type6(n = 1),constituting 10 MLGs.The results showed that the E.bieneusi genotype BEB6 of Tarim red deer from different farms was characterized by diverse differentiation of gene subtypes.In conclusion,this study found that E.bieneusi infection was common in captive Tarim red deer in Southern Xinjiang.Fifteen known E.bieneusi genotypes were identified,with zoonotic genotypes BEB6 and D as the dominant infection genotypes.The genetic subtype structure of Tarim red deer genotype BEB6 was studied These results will provide basic data for the epidemiological investigation of Enteromicrosporidiosis in Animals in China.