Molecular Epidemiology Of Enterocytozoon Bieneusi And Cyclospora In The Bos Frontails From Nujiang Area, Yunnan Province

ObjectiveCyclospora and Enterocytozoon bieneusi are common species of human intestinal parasites,which cause pollution to human living environment.They have been violently prevalent in many parts of the world.Patients with low immunity can cause dehydration diarrhea even to death,which seriously threatens human health.In China,Bos frontalis is only distributed in the Dulongjiang and Nujiang river basins in Nujiang Prefecture,Yunnan Province.It has high economic value and valuable genetic resources,and intestinal parasites can cause devastating impact on the development of the breeding industry.People and animals in the Nujiang area have frequent exposure,and the two pathogens are infected with humans and animals.Based on the specific sensitivity and high accuracy of nested PCR,we selected the Bos frontalis in Nujiang Prefecture of Yunnan Province as the survey object to investigate the infection of Bisporus and cyclosporium.Study on the Enterocytozoon bieneusi and Cyclospora of Bos frontalis whether there is a zoonotic genotype in sporozoites provides a reference and ideal prevention and control strategy for further research on Microsporidiosis and Cyclosporosis in this region.MethodsFrom September 2017 to September 2018,1129 samples of Bos frontalis feces were collected in four locations in Fugong County,Gongshan County,and surrounding villages of Nujiang Prefecture,Yunnan Province,and the sample DNA was extracted with OMG kit..Based on the polymorphic region of the ribosomal small subunit(18S rRNA)gene and the intracellular transcribed spacer(ITS)gene of the Enterocytozoon bieneusi,the sample DNA extracted by nested PCR is amplified by gel electrophoresis and will be detected.Positive samples were sent to sequencing to determine the positive rate.The sequencing results were sequenced using NCBI.Molecular biology software such as MEGA6,SPSS19.0 and Cluster X 1.83 were used for sequence alignmentanalysis and phylogenetic tree mapping to determine the species,genotype and epidemiology.Results1.In 1129 samples of Bos frontalis feces collected in Nujiang Prefecture,19 cases of Enterocytozoon bieneusi were detected by nested PCR amplification and gel electrophoresis,and the infection rate was 1.68%(19/1129,95% CI 0.93).%~2.43%),the total infection rate in the five regions was 0(0/218,),and the infection rate in Guquan Village was 2.73%(6/220,95% CI 0.58%~4.88%).The infection rate of Zuoluo Village was 0.87%(2/231,95% CI 0~2.07%),and the infection rate of Cikai Town,Gongshan County was 1.28%(3/234,95% CI 0~2.72%),Dulongjiang Township.The infection rate was 3.54%(8/226,95% CI 1.13%~5.95%),and the infection rate in five regions(P=0.026,the difference was statistically significant).The total infection rate in the four quarters was 1.10%(3/272,95% CI 0~2.34%),and the summer infection rate was 3.26%(9/276,95% CI 1.17%~5.36%).The infection rate was 1.28%(4/312,95% CI 0.03%~2.53%),the winter infection rate was 1.12%(3/269,95% CI 0~2.38%),and the infection rate in four quarters(P=0.137,The difference was not statistically significant).Four known genes were identified in 19 positive samples: EbpC(n=6),CHN3(n=3),CHN4(n=4),and BEB4(n=4);the two new genotypes were named YNNJ1,YNNJ2.2.Of the 1129 samples collected from the feces of Bos frontalis,17 were detected,and the infection rate was 1.51%(17/1129,95% CI 0.80%~2.22%).The total infection rate in the five areas was guaranteed.The infection rate of the farm was 0(0/218,),the infection rate of Guquan Village was 0.91%(2/220,95% CI 0~2.16%),and the infection rate of Yazolo Village was 1.29%(3/232,95%).CI 0~2.74%),the infection rate of Cikai Town,Gongshan County was 1.71%(4/234,95% CI 0.05%~3.37%),and the infection rate of Dulongjiang Township was 3.54%(8/226,95% CI 1.13%).~5.95%),infection rate at five sites(P=0.036,the difference was statistically significant).The total infection rate in the four quarters was 0.74%(2/272,95% CI0~1.76%),and the summer infection rate was 2.90%(8/276,95% CI 0.92%~4.88%).The rate was 0.64%(2/312,95% CI 0~1.55%),the winter infection rate was 1.86%(5/269,95% CI 0.25%~3.47%),and the infection rate in four quarters(P=0.090,difference)Not statistically significant).ConclusionThis study was the first to analyze the zoonotic genotypes and Cyclospora species of Enterocytozoon bieneusi in Dujiang cattle in Nujiang area of ??Yunnan Province.The infection rates of Enterocytozoon bieneusi in the spring,summer,autumn and winter were 1.10%(3/272),3.26%(9/276),1.28%(4/312),and 1.12%(3/269),respectively.The infection rates of summer,autumn and winter were 0.74%(2/272),2.90%(8/276),0.64%(2/312),and 1.86%(5/269),respectively.The microtypes of Pichia Enterocytozoon bieneusi were successfully identified: EbpC,CHN3,CHN4,BEB4,YNNJ1,YNNJ2,all of which were human and zoonotic genotypes.YNNJ1 and YNNJ2 were the novel Enterocytozoon bieneusi identified in this study,enriching the The genotype of the species.The analysis found that the difference in infection rate of Enterocytozoon bieneusi was statistically significant.The location was the risk factor of Enterocytozoon bieneusi infection.The infection rate was the highest in Dulongjiang Township.There was no statistically significant difference in infection rate in different quarters.Enterocytozoon bieneusi infection was not affected.Seasonal impact.In this experiment,the cyclospores were identified as Bovine circumsporozoites.The difference in infection rate between different sites of cyclosporium was statistically significant.The location was the risk factor of Cyclospora infection.The infection rate was the highest in Dulongjiang Township;There was no statistically significant difference in infection rates,and cyclosporosis infection was not affected by the season.