Development Of PCR Assay For Molecular Epidemiologic Investigation Of Anaplasmas In Sheep And Goats

Anaplasmosis, one of rickettsiosis, is caused by species of the genus Anaplasma that parasitizedon animals’ and humans’ blood corpuscle. Diseased animals show fever, anemia, weight loss, aurigo,malnutrition and so on. Serious condition may lead to the death of animals. Anaplasmosis is a tick-bornedisease, with the global climate warming and incresing about international and domestic trade in liveanimals, the disease is widely epidemic in worldwide at present. Especially Anaplasma phagocytophilum,which can causes human granulocytic anaplasmosis (HGA), is considered as zoonotic disease thatseriously threaten to human health. This study aimed to improve a PCR diagnostic method foranaplasmosis of sheep and goats and to conduct an epidemiological investigation of anaplasmosis ofsheep and goats in some parts of China. In order to provide technical support and theoretical basis forpreventing and control the Anaplasmosis in sheep and goats.The blood specimens of a total of408sheep and goats were collected from seventeen regions ofHenan, Neimeng, Liaoning, Shanxi, Shandong and Yunnan provinces of China. Firstly, Giemsa stainingmethod was used to detect the Anaplasma in the blood samples. In Giemsa staining, three species ofAnaplasma consist of A. ovis, A. bovis and A. phagocytophilum were found with their infection rate as37.5%(153/408),8.09%(33/408) and5.40%(22/408) respectively. Then the samples were subjected forthe PCR amplification of16S rRNA and MSP4genes of Anaplasma. PCR results also confirmed thosethree species of Anaplasma like-A. ovis, A. bovis and A. phagocytophilum, with varying infection rate as30.15%(A. bovis),23.53%(A. ovis) and11.52%(A. phagocytophilum). The results revealed that thesheep of different regions were infected with different dominant Anaplasma species such as the dominantspecies was A. bovis in most parts of Henan province; A. ovis in Shanxi and Neimeng provinces; A. bovisin Guizhou, Yunnan and Liaoning provinces. The highest infection rate of A. phagocytophilum was foundin sheep and goats of Shandong. Different infection rates were also observed in different rearing methods,different seasons and different breeds of sheep. Anaplasmas infection rate (59.92%) was significantlyhigher in the sheep in grazing mode than those of pan feeding (36.75%). The infection rate (61.73%) wassignificantly higher in goats than the sheep (43.09%). Summer was the peak season for Anaplasmainfection in sheep and goats with infection rate as95%in the survey areas. This study included Han sheep,Hu sheep, the Saft sheep, Inner Mongolia cashmere goats, Nubi black goat and Longling’ yellow goats toinvestigate Anaplasmas, and this was the first initiative in our country.In this study,80strains have been selected randomly from the Anaplasmas positive samples detected by the PCR amplification based on16S rRNA/MSP4genes and then one of the22representative strainsselected after gene sequencing. Homology analysis was done with the reported strains and thephylogenetic tree was established using the ClustalX1.83and DNA Star4.0software. The results showedthat the Anaplasmas of sheep and goats in the study areas have basically no great variation and have highhomology with the previously reported Anaplasmas. The homology based on16S rRNA gene locusamong the five A. bovis strains (KF4, KM11, QL6, WR12, CY6, GY3) and the previously reported strains:A. bovis Guizhou (JN558825.1), A. bovis Zhejiang (JN558829.1), A. bovis South Kroea (KC311344.1), A.bovis South Kroea (KC311346.1) was99.8%; The homology based on16S rRNA gene locus among theten A. phagocytophilum strains, GY3, JC5, KF6, LQ1, WR1, YY7with the reported A. phagocytophilumJapan (AB196721.1) and A. phagocytophilum Hubei (JN558813.1) reached100.0%, Gongyi2and LB10with the reported A. phagocytophilum Henan JN558815.1was99.1%and99.4%. According to thenomenclature of the Anaplasma new genotype, we named the new genotypes as ApLB2012-7, andApGoYi2012-11. The homology based on MSP4gene among the six A. ovis (CY3、GY8、JC12、KF4、NM12、NM32) strains and the reported strains, the highest homology of them was up to100%(HQ456348.1Yuzhong and JC12) and the lowest was99.6%(DQ674249USA and JC12). Among thethree Anaplasmas found in this study, A. phagocytophilum can parasitize both human and animals, itsphylogenetic analysis in flock has important public health significance, espcially two new genotypes(named as ApLB2012-7and ApGoYi2012-11) of A. phagocytophilum have been identified in this studywith six (ApLB2012-7) and three nucleotide (ApGoYi2012-11) differences as compared to the reportedHenan strains.