Phylogenetic Analysis And Molecular Epidemiology Of Anaplasma Phagocytophilum

Anaplasma phagocytophilum,a gram-negative obligate intracellular parasite,has become an important tick borne pathogen threatening public health in recent years.Various kinds of animals including human beings,wild animals such as white-tailed deer and sika deer,domestic animals such as dogs,horses,cattle and sheep/goats,rodents such as white-footed mouse and wood mouse,insectivores such as shrew and hedgehog can be reservoirs of the pathogen.AP infection in humans results in human granulocytic anaplasmosis(HGA),while in animals tick borne fever(TBF),which bring great loss to animal industry as well as threaten human health.However,the epidemiology of AP in animals and the great diversity among AP strains in China have not been well studied.The aim of the present study was to investigate the epidemiology of AP in different animals,ticks in partial areas of China and the genetic diversity of the AP strains obtained in this study.1.The epidemiology of APIn order to investigate the epidemiology of AP,during 2013 to 2016,a total of 2104 blood samples originating from nine province/city/autonomous region including Henan,Shaanxi,Yunnan,Guizhou were collected from sheep,goats,cattle,dogs and African giraffe.The samples contained 1516 sheep/goats blood,120 sheep/goats milk,69 cattle blood,240 dogs blood,1 African giraffe and 158 hard ticks.Giemsa staining of blood smears and transparentized tick body were observed under light microscope to identify the existence of pathogens and for species identification,respectively.While nested PCR specially amplify 16S rRNA gene fragment of AP and Anaplasma bovis and msp4 gene fragment of Anaplasma ovis.The results showed that the total positive rate of Anaplasma was 38.4%(788/2053),while AP was found in 3.0%(61/2053)of the animals.Among 1516 sheep/goats blood samples,the prevalence of Anaplasma and AP was 45.0%and 3.4%(52/1516),respectively.The infection rate of Anaplasma and AP in sheep and goats was also different,with Anaplasma in 40.6%(262/646)of sheep,AP in 5.6%(36/646)sheep,while Anaplasma in 51.3%(424/870)of goats,AP in 1.8%(16/870)of goats.In the sheep/goats samples collected continuously in four years from Zhaobao xiang in Henan province,the Anaplasma and AP prevalence was quite high(78.2%and 47.3%),which could be related that the sampling site is a mountainous area and the animal are always grazing in field.12.5%(15/120)of the milk samples was found to be positive for Anaplasma,and 2 of them was AP positive.Among the 69 cattle blood samples,22 was positive for Anaplasma,1 was found to be infected by AP,several samples was infected by as many as four pathogens.The results of Giemsa staining,PCR analysis and ticks carrying pathogens collected from cattle suggested that the cattle in this farm may be suffering from Anaplasmosis/Theileriosis.Three stray dogs and 1 dog raised in a sheep farm were positive for AP in 240 dog blood samples.85 Haemaphysalis longicornis,16 Rhipicephalus microplus and 6 Haemaphysalis flava were identified using morphological and molecular analysis and one Haemaphysalis longicornis was positive for AP.AP was detected in an African giraffe for the first time and the mixed infection of AP,A.bovis and Theleria sp.was found in it.The above results suggested that Anaplasma was ubiquitous in animals in the investigated areas and AP was found in several animal species,which pointed out that the impact of AP on animal industry should be attached importance.On the other hand,workers and travelers in the forest areas should pay attention to ticks.2.Genetic diversity of APTo understand the genetic diversity of AP,nested PCRs were conducted to amplify the 16S rRNA、msp4、groESL and gltA genes using the obtained 220 AP positive samples.The sequences were corrected and assembled by Vector NTI Advance10 software,and then Blast on NCBI.The nucleotide sequences of every gene and the deduced amino acid sequences of coding genes were used to construct phylogenetic trees using Mega5.05 software.31 16S rRNA gene sequences,118 gltA gene sequences,162 groEL gene sequences and 52 msp4 gene sequences were obtained in this study.Among the 12 different 16S rRNA gene sequences,KX505292,identified as Ehrlichia chaffeensis,was in a same clade with an American human isolate(CP007479).KX505293 and KX505302 were identical to a China human Anaplasma isolate(KM206273).In the phylogenetic trees constructed using 8 gltA nucleotide sequences and 7 deduced amino acid sequences,the isolates in this study were arranged in two clades and cluster I contained most of the obtained isolates and one zoonotic isolate KJ700628.10 groEL nucleotide sequences and 3 deduced amino acid sequences obtained in the present study were quite different with existing sequences,with highest similarity of 90.3%with two Anaplasma spp.isolates JN055360 and JQ685509162.In the phylogenetic trees of 13 msp4 nucleotide sequences and 3 deduced amino acid sequences,the obtained sequences separated into two clades.Genetic variance analysis of AP using 16S rRNA and gltA gene showed that partial AP isolates may have zoonotic risk to some extent.Phylogenetic analysis based on groEL gene indicted that the isolates in this study are distantly related with existing isolates.While the msp4 gene sequences of the obtained isolates variate greatly.