Molecular Epidemiological Investigation And Development Of RAPD-SCAR Marker Of Eperythrozoon Wenyonii In Chongqing

Eperythrozoon is an extracellular bacterial organism that attaches to red blood cells or exist in blood plasma,tissue fluid and cerebrospinal fluid of animals include goats,cattles,pigs,horses, sheep,rats,dogs,blue foxes,chicken as well as human beings,and causes severe economical loss to animal husbandry industry.In this article,we designed specific primers according to the sequence data of 16S rRNA gene of Eperythrozoon wenyonii deposited in Genbank and developed polymerase chain reactions(PCRs) method to detect Eperythrozoon wenyonii.With the method we amplified the 16S rRNA gene of Eperythrozoon wenyonii from blood samples of cattles,cows and buffalos.Then we analyzed the identity of 16S rRNA genes from different hosts with DNAStar and DNAman.The results indicated that all Eperythrozoon samples from cattle,cow and buffalo were E.wenyoni with minor genetic differences.The sequence identity between cattle and buffalo was 98.6%and 98.3%between cattle and cow,while the highest identity was 99.8%between cow and buffalo.Eight counties and six region were choosed according to different geographical positions and divergent raising models in Chongqing for epidemiological investigation of Eperythrozoonsis with specific PCR method we development.The result demonstrated that 11%of cattles were infected by Eperythrozoon in Chongqing,and the infection rate of E.wenyonii showed great difference according to geographical positions,the infection rate(25.6%) in Chenkou,Wulong and Xiushan counties which mainly locate in mountain area was much higher than that in Rongchang,Beibei and Jiangbei counties which situate in hilly areas(2%).While the difference of the infection rate also happened according breeding models,the inflection rate ranged from 22.7%to 33.3%under free raising conditions,while only 6%infection rate was observed under the large-scale farming conditions.Three 16S rRNA gene sequences of E.wenyoni,E.suis and E.vois were amplified by random amplified polymorphic DNA technique(RAPD) repectively.The result shown the livestock of the same kind,the amplified bands of the infected eperythrozoon consistent with each other,but there exists a inheredity disparity of the 16S rRNA gene sequences for the infected cattle,sheep,pigs.The most nearest genetic distance index is 0.405 of the 16S rRNA gene sequence of the infected cattle and sheep,but the family relationship is relatively distant for the comparison of the infected cattle,sheep and pig,the genetic distance index is 0.714,0.673 respectively.Currently,the major method used to classify Eperythrozoon is the genetic analysis of 16S rRNA gene,however divergence raised after the analysis of Eperythrozoon 16S rRNA used by Pospischil A and colleagues,which indicated the limitations of genetic analysis only by amplification of gene and alignment of sequence.To solve this problem,3 SCARs were designed to identify Eperythrozoon infection and to distinguish different sub-infections based on the RAPD analysis of 16S rRNA from cattles,results shown SCAR1 and SCAR3 could not amplify Eperythrozoon genes as well as discriminate sub- infections,only SCAR2 had capability to be the signature of Eperythrozoon infection and could distinguish specific 16S rRNA of tattles from that of cows and buffalos, indicating the valuable application in the genetic polymorphism analysis of E.wenyonii 16S rRNA gene based on this RAPD-SCAR method.