Isolation Of Theleria Equi Te-Y From Dermacentor Niveus、Analysis Of Genetic Evolution And Establishment Of Initial Culture Method In Vitro

Equine theileriosis is a kind of equine blood protozoal disease that is transmitted through vector ticks.The etiological agent is Theileia equi（formerly Babesia equi）,the World Organisation for Animal Health（OIE）lists the disease as Class B animal epidemics that a statutory reportable epidemic and is classified as a Class II animal epidemic in China.It is a global epidemic disease that has not been any vaccine manufactured.Its diagnosis and treatment are lagging behind.The incidence and mortality are high,which seriously restricts the healthy development of the horse industry.As a disease transmission and carrier,Dermacentor niveus is widely distributed in Xinjiang autonomous region,which resulting in the frequent occurrence and prevalence of the disease,which seriously affect the income of the Ili horse industry and the local herdsmen.In this study,pathogenic morphology,molecular biology,phylogenetic taxonomy,and other methods were used,set Ili River Valley as a test point to collect blood-sucking ticks on horses identify,classify,analyze cases,separate bloodborne and tickborne strains.Establish evolutionary tree based on 18S rRNA gene sequences,then establish an initial culture methods for Theileia equi in vitro,etc.These results provide the basis for the association studies of blood protozoa and mediators in the Ili Vally and also provide the comprehensive prevention and control of the disease.1.In this study,4780 hard ticks and 546 suspected horse blood samples were collected.The tick specimens were classified by morphological observation and molecular biological method;the blood samples were tested by PCR and c ELISA method,then selected the positive Theileia equi samples.The result is,D.niveus`s coxaⅠwith outer distance shorter than inner distance,the outer distance of coxaⅡ,Ⅲare narrow,tapering in shape.Spiracular plate commalike in shape,dorsal process long and narrow and curved to dorsal site,margins of base covered with single row of cyathiform,a large number of cyathiform at the top.The first and second section of abdominal inner lateral margin with seta abundant and closely packed,the third section with seta few and scattered.From these characteristics,the specimens were confirmed as D.niveus;the ITS2 gene sequencing results are 99%homology with Iranian D.niveus（GQ144706.1）,and have morphological similarity,1050 specimens were identified as D.niveus;PCR detected D.niveus carried Theileia equi`s infection rate was 7.3%.among 546 blood samples,Theileia equi`s PCR positiveness rate was 19.7%（108/546）,c ELISA`s positiveness rate was 34.7%（190/546）.D.niveus is a dominant tick species in this region.2.Specific primers were designed based on the 18S rRNA gene sequence.Te-Y strains were isolated by PCR and the DNA of the tick borne Theileia equi was sequenced and analyzed,and compared with other strains（Mongolia JQ657703.1,Riyadh KJ801928.1,Budapest KM046922.1,etc.）,using the Mega6.0 software to map the evolutionary tree of the Theileia equi.The results showed that the homology of blood-borne and tickborne was 100%,and the homology with other different strains ranged from 96%to100%,Korea（HM229407.1）and Zhao yuan（KF559357.1）has the highest homology of 100%;the average content of the base composition A,C,T and G respectively were 27.8%,18.5%,26.7%and 26.8%.The average content of A+T is 54.5%,higher than C+G content,showed A+T bias;Te-Y strain was clustered on the evolutionary tree with Zhaoyuan strain and Korean strain with a confidence level of 95 and93,respectively.the genetic distance between other local strains was 0.000 to 0.020.3.Sampled two kinds of gas conditions,first is 37°C、2%O₂、93%N₂、5%CO₂,the second is at 37°C,5%CO₂and three kinds of gas conditions,used complete medium（including M199 medium,horse serum,double-antibody and hypoxanthine）,and regularly substitute healthy red blood cell and change the medium,then established the initial in vitro cultural method of Theileia equi.The result showed that in low infection rate condition,add oxygen would help the Theileia equi growing.Theileia equi can be constantly raise 38 days,the infestation ratio was 4.0%-5.1%,highest rate was 5.4%and can subculture 6 times in total.At the other hand,the final concentration of serum was confirmed as 40%,the ability of uninactivated serum to support the growth of Theileia equi higher than that of inactivated serum.0.2m M final concentration of hypoxanthine could control erythrocytolysis and help the pathogen raise.The initial culture could provide benefits for lateral tests.