Prevalence And Phylogenetic Analysis Of Piroplasma From Dogs In Zhengzhou Area

Piroplasmosis,also known as babesiosis or theileriosis,is a kind of tick-borne protozoan diseases caused by piroplasma protozoan parasites,which intracellular parasitize in the erythrocyte of variety animal and human hosts,including babesiosis and theileriosis.The clincial features of piroplasmosis contain anemia,fever,jaundice,weight loss,depression,and some other un-specific symptoms.The piroplasmosis is a worldwidely distributed include China.As well as,wide host range were documented.It can both infect human and wild or domesticate mammals,which leading to great loss in the breeding industry.At the same time,the zoonotic transmission having become an important threatening concerning the public health.This study conducted the infection and distribution investigation of piroplasma(including Babesia spp.and Theileria spp.)in dogs in Henan Province,China.It wish provides a scientific references for the prevention and control of piroplasmosis in clinical.1.During the period from April 2013 to July 2017,a total of 393 fresh anticoagulant blood samples from various dogs were collected from 10 different locations in Zhengzhou,Henan Province.The blood whole genomic DNA was extracted from the collected samples.Followed by,the DNA samples were amplified by conventional PCR and nested PCR based on its 18 S r RNA gene,and the positive amplified products were sequenced identified.The results demonstrated that the total infection rate of piroplasma in dogs was 5.34%(21/393).Positive samples were detected in three kinds of piroplasma pathogens namely,B.gibsoni(3.82%,15/393),T.luwenshuni(1.01%,4/393)and T.annulata(0.51%,2/393).There were different infection distributions of canine piroplasma among the different sampling sites,ranging from 0% to 100%.The canine B.gibsoni infection rate in large dogs(7.55%,8/106)was significantly higher than that of the medium-sized dogs(1.87%,2/107)and small dogs(2.48%,5/180),the infection rate of Rottweiler and German shepherd dog even reached 100%(1/1)and 45.45%(5/11),respectively.However,there was no significant difference in the infection rate of the family dogs(5.64%,19/337)and the stray dogs(3.57%,2/56)in different feeding patterns.There was also no significant difference in the infection rate of the male dogs(5.43%,10/184)and the female dogs(7.19%,11/153).2.The obtained 18 S r RNA gene sequences of 21 canines-derived piroplasma were compared and calibrated by using Seq Man and Clustal X 2.11 software.The calibrated sequences of Babesia spp.and Theileria spp.were carry out phylogenetic analysis,and construct genetic evolution phylogenetic tree,based on Neighbor-Joining method using MEGA6.06 software.The phylogenetic analysis indicated that the canines-derived Babesia spp.identified in present study all clustered into the branch of the previously reported B.gibsoni.This study has identified two kinds of B.gibsoni genotypes,one was DXY7 and YD11,which was 99% homology with Asian type(LC012808)in Japan and North Carolina type(AF271081)in the United States;the other one was W4 and YN21,which was 99% homology with Wuhan in China(KP666166)and United States(EU084677)B.gibsoni isolates.The phylogenetic analysis of canines-derived Theileria spp.showed that there were two genotypes of the T.annulata,namely YN16 and YN2.Among them,the YN16 99% homology with the India isolates(KT367868),and the YN2 was 99% homology with the China isolates(EU083800).There were two kinds of T.luwenshuni genotypes,one is genotype LL16,DXY28,WT10,and the other one is DF84.Among them,LL16,DXY28,WT10 formed a separate branch;whereas,DF84 99% homology with the Henan Songxian T.luwenshuni isolates(JX469518).In conclusion,the present study reported the infection and distribution of canine piroplasma in Zhengzhou area of China,and analyzed its phylogenetic evolution based on the molecular phylogeny.The study found that canine Babesia spp.all identified as B.gibsoni,indicated that the potential dominant species in dogs in Zhengzhou area.And,this is the first report of T.luwenshuni and T.annulata infection in dogs in China.The results provide a certain data support for the analysis of the epidemiology and genetic evolution of piroplasmosis in dogs in China.