The Genetic Characteristics Analysis Of Anaplasma Phagocytophilum From Sheep

Anaplasma phagocytophilum is an obligate intracellular gram-negative bacteria,mainly parasitizing in neutrophils.A.phagocytophilum can cause an emerging tick-borne natural focal zoonotic disease.Not only this agent pose a serious threat to human health,but also is there a significant impact on the animal husbandry economic benefit.In addition,A.phagocytophilum strains exist genetic diversity in a variety of host animals and ticks.This study aims to analyze genetic characteristics of A.phagocytophilum sheep-borne applying molecular biological technology in China.1.In order to get a general knowledge of the prevalence of A.phagocytophilum in dairy goats in China,and provide the scientific reference for prevention and control of this disease,and promote the development of dairy goats industry,the present study was carried out to reflect the epidemiology of A.phagocytophilum in dairy goats in partial regions of China.From July 2012 to September 2014,a total of 300 blood samples and 204 milk samples were collected from Luoyang Yiyang,Luoyang Jianxi,Xinmi,Nanyang Xixia,Kaifeng,Pingdingshan and Shangqiu Ningling in Henan province,Kunming in Yunnan province,Fuping,Linyou,Jingyang and Longling in Shanxi province.All samples were tested by the nested PCR.The results showed that A.phagocytophilum were detected only in some blood samples not in milk samples,with infection rate as 18%(54/300).The A.phagocytophilum infection of dairy goats in different areas was different,with Luoyang Yiyang100%(5/5),Shanxi Linyou76.67%(46/60),Pingdingshan 12.50%(2/16)and Shanxi Gaoling 2.86%(1/35).While there was no infection in Luoyang Jianxi,Zhengzhou Xinmi,Nanyang Xixia,Kaifeng,Ningling,Kunming,Yunnan,Shanxi Jingyang and Shanxi Fuping.The A.phagocytophilum infection of dairy goats from different varieties was different.The A.phagocytophilum infection rate is the highest as 83.87%(26/31)for Hybrid dairy goat,higher as 22.83%(21/92)for Guanzhong dairy goat,next as 11.11%(7/63)for Local dairy goats and no infection for Saanen dairy goat.The A.phagocytophilum infection rate of dairy goats from different feeding ways was also different.A.phagocytophilum infection rate of dairy goats in grazing is up to 38.85%(54/139).While there is no infection in those of stabling feeding.What's more,after gene sequence analysis,the results indicated that there appeared three base differences in one of the 54 A.phagocytophilum strains in this study.Phylogenetic analysis showed that there were closer relations between the A.phagocytophilum strains and the isolate(U02521)from humans,and the homology was 99.1%~99.5%,with potential zoonotic risk.2.In order to get a general knowledge of the genetic characteristics of A.phagocytophilum from sheep,and to make it clear whether there was with potential zoonotic risk,the present study was carried out to reflect genetic characteristics of A.phagocytophilum from sheep in our country.From May 2012 to September 2014,a total of 140 positive DNA samples were collected from Luoyang,Zhengzhou,Kaifeng,Nanyang,Xinxiang,Jiyuan,Pingdingshan,Sanmenxia and Zhumadian in Henan province,Kunming in Yunnan province,Chaoyang in Liaoning province,Linqu in Shandong province,Guiyang and Qinglong in Guizhou province,Jincheng in Shanxi province,Heilongjiang in Heilongjiang province and Fuping,Linyou,Jingyang and Longling in Shanxi province.All samples were tested by the nested PCR based on the 16 S rRNA,MSP4(major outer membrane protein)and groESL(heat shock proteins)gene,and then were sequenced.Last,the phylogenetic analysis were performed using Mega5.05.The results show that there appeared five different types among 140 16 S rRNA gene sequences,respectively named genotype A-E.There were closer relations between genotype D and the isolate(U02521)from humans,and the homology was 99.1%~99.5%,with only one base difference.There were 2~7 base differences between different genotypes.There were differences in the distribution of the five genotypes between different regions.Genotype A was in all surveyed provinces,while genotype B-E presented only in some provinces.For example,genotype B only existed in Shanxi.genotype C only existed in Luoyang in Henan.Genotype D only existed in Shanxi,Heilongjiang and Luoyang in Henan.Genotype E was only found in Zhengzhou in Henan.The phylogenetic tree of MSP4 gene of A.phagocytophilum showed the 47 gene sequences were divided into three clusters,a total seven different types,respectively named genotype?a,?b,?c,?d,?a,?b and ?.There were differences in the distribution of the seven genotypes in different regions.Some regions presented seven genotypes of MSP4 gene,while some regions only presented one or two genotypes.There appeared nine sequence types in the groESL gene sequences,respectively named genotype A-I.There were differences in sequence types for different areas in China.There were seven genotypes icluding genotype A-G in total in Henan province,six genotypes including genotype A,C,D,E,H and I in Shanxi province,five genotypes including genotype A,B,D,F and G in Shanxi province,only one genotype-genotype A in Guizhou and Heilongjiang province.Shandong and Liaoning province respectively found genotype C and genotype E.In this study,PCR was applied to test blood and milk samples of dairy goats in partial areas in China.The results showed that positive of A.phagocytophilum were found in the blood samples,indicating that there was A.phagocytophilum infection of dairy goats in China.In this study,the PCR based on the 16 S rRNA,MSP4 and groESL gene were used to analyze the genetic characteristics of A.phagocytophilum from sheep in our country for the first time.As a result,five 16 S rRNA genotypes,seven MSP4 genotypes and nine groESL genotypes were found,showing that there existed diversity in A.phagocytophilum from sheep in China.In addition,there were closer relations between the A.phagocytophilum strains from sheep and the isolates(U02521)from humans based on 16 S rRNA gene sequence analysis.The homology was 99.1%~99.8%.There was only one base difference between genotype D and U02521,with potential zoonotic risk.This will lay the foundation to further develop the monitoring of zoonosis and the research of the protection of public health security.