Molecular Epidemiological Investigation And Genotype Analysis Of Four Intestinal Protozoa In Sika Deer In Northeast China

Cryptosporidium,E.bieneusi,G.duodenum and Blastocystis,are four important zoonotic intestinal protozoa that can infect humans,wildlife,companion animals,birds and a variety of domestic animals.Normal human infections are usually asymptomatic,but can cause severe diarrhea in children or immunocompromised people.Domestic animals or captive wild animals have been found in different regions of our country to be able to infect these parasites.However,it was unclear about the spread of these four intestinal protozoa in the sika deer in Northeast China.In order to examine the infection of the four intestinal protozoa and the zoonosis risk on skia deer in Northern China,a total of 818 feces samples were collected from 13 farms in four Province from September 2017 to December 2018 investigate the epidemiological characteristics of four intestinal protozoa.By PCR amplification,electrophoresis,sequencing,sequence splicing and phylogenetic analysis,the main results obtained were as following:1 Based on the nested PCR amplification of the 18 S r RNA of the Cryptosporidium,the infection rate of Cryptosporidium was 1.1%(9/818,95% CI 0.39-1.82),and the infection rate was in the range from 0%~1.49% in 4 regions,and the highest infection rate was found in Jilin Province 1.49%(8/538,95% CI 2.80-13.86,),However,no Cryptosporidium infection was detected in Liaoning Province and Inner Mongolia region.Among other categories,the post-weaned sika deer infection rate was 1.14%(9/787;95% CI 0.40-1.89)higher than the pre-weaned 0%(0/31);the male sika deer infection rate was 1.29%(9/699,95% CI 0.45-2.12)higher than the female sika deer 0%(0/119);the infection rate of breeding bases was 1.49%(8/465;95% CI 0.53-2.90)higher than that of single pastures 0.28%(1/353,95% CI-0.27-0.84).The infection rate of Cryptosporidium in different domesticate modes and regions was no significantly different using the statistical analysis(P >0.05).A total of one species(Cryptosporidium deer)was identified by sequence analysis.2 Based on the nested PCR amplification of the ITS gene of the E.bieneusi,the infection rate of E.bieneusi was 13.57%(111/818,95% CI 11.22-15.92),and the infection rate was in the range from 0%~17.84% in 4 regions,and the highest infection rate was found in Jilin Province 17.84%(96/538,95% CI 14.61-21.08).However,no E.bieneusi infection was detected in Inner Mongolia region.Among other categories,the post-weaned sika deer infection rate was 14.1%(111/787,95% CI 11.67-16.54)higher than the pre-weaned 0%(0/31);the male sika deer infection rate was 15.88%(111/699,95% CI 13.17-18.59)higher than the female sika deer 0%(0/119);the infection rate of breeding bases was 20.22%(94/465,95% CI 16.55-23.88)higher than that of single pastures 4.82%(17/353,95% CI 2.57-7.06).The infection rate of E.bieneusi in different domesticate modes and regions was significantly different using the statistical analysis(P <0.01).Six known genotypes(JLD-Ⅲ,JLD-Ⅸ,JLD-Ⅷ,Ebp C,BEB6 and I)and 10 novel genotypes(namely LND-Ⅰ,and JLD-ⅩⅤ to JLD-ⅩⅩⅢ)were found in sika deer in this study,and E.bieneusi genotype BEB6 were identified as the predominant species/genotypes in skia deer.genotype Ebp C clustersed in Group 1d,five Genotype(JLD-III,JLD-XIX,JLD-XVII,JLD-XXII,and JLD-XVI)in Group 1a,and other 10 gentype(JLD-IX,JLD-VIII,BEB6,I,JLND-I,JLD-XV,JLD-XXIII,JLD-XX,JLDXVII and JLD-XXI)in Group 2.The multilocus sequence typing(MLST)method was used to analyze the polymorphism and population genetic structure of E.bieneusi positive samples by selecting gene loci MS1,MS3,MS4 and MS7.The results showed that 16 positive isolates of 111 positive samples were successfully amplified at two loci simultaneously,resulting in 10 distinct multilocus genotypes(MLGs),The results show that different subtypes between the same genotype and E.bieneusi of Sika deer in Northeast China was rich in genetic diversity.3 Based on the nested PCR amplification of the BG gene,GDH gene and TPI gene of the Giardia duodenum.Three positive samples were detected at the BG gene,three positive samples were detected at the GDH gene,no positive samples were detected at the TPI gene,and one sample was detected simultaneously in BG and GDH,the total infection rate detected was 0.61%(8/818,95% CI 0.00-1.15).The infection rate was in the range from 0%~0.93% in 4 regions,and the highest infection rate was found in Jilin Province 0.93%(5/538,95% CI 0.12-1.74),however no G.duodenum infection was detected in other four region.Among other categories,the post-weaned sika deer infection rate was 0.64%(5/787,95% CI 0.00-1.19)higher than the pre-weaned 0%(0/31);the male sika deer infection rate was 0.72%(5/699,95% CI 0.00-1.34)higher than the female sika deer 0%(0/119);the infection rate of breeding bases was 1.08%(5/465,95% CI 0.13-2.02)higher than that of single pastures 0%(0/353).Sequence analysis showed that the sika deer was infected with G.duodenum Assemblage E and Assemblage A in Northeast China.and and the Assemblage E was the dominant genotype.4 Based on the conventional PCR amplification of the Small Subunit ribosome RNA(18S r RNA)of the Blastocystis,the infection rate of Blastocystis was 1.22%(10/818,95% CI 0.47-1.98).The infection rate was in the range from 0%~1.86% in 4 regions,and the highest infection rate was found in Jilin Province 1.86%,(10/538,95% CI 0.71-3.00),however,no Blastocystis infection was detected in other regions.Among other categories,the post-weaned sika deer infection rate was 1.27%(10/787,95% CI 0.49-2.05)higher than the pre-weaned 0%(0/31);the female sika deer infection rate was 3.36%(4/119,95% CI 0.10-6.64)higher than the male sika deer 0.86%(6/699,95% CI 0.17-1.54);the infection rate of breeding bases was 2.15%(10/465,95% CI 0.83-3.47)higher than that of single pastures 0%(0/353).The infection rate of Blastocystis in different gender category was significantly different using the statistical analysis(P <0.05).In this study,2 types of Blastocystis genotypes(ST10 and ST14)were identified,and the ST10 was the dominant genotype.This study used molecular biology methods to investigate the molecular epidemiological characteristics of four intestinal protozoa in Northeast China and the risk of zoonotic diseases.The research results not only provide scientific basis for the prevention and control of four intestinal protozoan diseases in sika deer,also provides basic data for the prevention and control of the spread of four intestinal protozoa between humans and animals.