Taxology Of Eperythrozoon 16S RDNA And Molecular Epidemiology Of Eperythrozoon On Chiken In Chongqing

Eperythrozoon,parasitized on surface of akaryocyte or liberated in blood plasma,tissue and cerebrospinal fluid,often infected animals(such as goats,cattle,pigs,horses,sheep,mice,dogs, foxes and chicken)and even human.There are lots of divergence in the classification of Eperythrozoon,which was considered as a kind of protozoon for its protozoon-like feature initially, then classified it into Rickettsiales and recently was suggested to classify into Mycoplasmatales. Through molecular biology technologies,scientists confirmed that homology of 16S rDNA between Eperythrozoon and Anaplasmataceae was low and a relatively high homology between Eperythrozoon and Mycoplasmatales.,Anaplasmatacea,Eperythrozoon Haemobartonella were classified into Mycoplasmataceae,Mycoplasma again in《Bergey's Manual of Systematic Bacteriology》published in 2005.After completion of Human Genome Sequencing Plan,age of postgenome forthcame with broad utilization of bioinformatics in the field of life science. Bioinformatic analysis based on 16S rDNA would be competent for microbe genealogical classification as an supplement of phenotype classification.In our study,many 16S rDNA sequences of representative strains of Eperythrozoon from NCBI were chose to search novel molecular markers for the exploration of genealogical classification and molecular diagnosis.Eperythrozoon 16S rDNA sequences(up to may 2008)of animals including pigs,cattle,sheep, dogs and mice and other relative spices(such as Haemobartonella and pathogenic Mycoplasma,Rickettsiales)were downloaded from GeneBank in NCBI and analyzed in homology.The results indicated that Eperythrozoon was not only more close to Haemobartonella than Rickettsia colombiensis but also one of the three apposite lineages in partial common pathogenic mycoplasma. 16S rDNA of three M.suis strains and three M.wenyonii strains were analyzed by multiple sequence alignment.It manifested that there was a kind of segments with considerable differences(about 40 bp)in hypervariable regionlocated V9 of 16S rDNA.After further secondary structure analysis,we found markedly interspecific conformational differences between those two species as well as insignificant intraspecific differences among M.suis and significant intraspecific differences among M.wenyonii.Generally, tendency of interspecific differences of secondary structure in hypervariable regions was inconsistent with that of primary structure for 16S rDNA of Mycoplasma suis, Mycoplasma wenyonii.In other word,sequences with identical secondary structure might be either identical or different at primary structure level.The differences between secondary structures may be not expressed in primary strctures.After RFLP analysis for 16S rDNA of Eperythrozoon and Haemobartonella,eight restriction enzymes,namely AluⅠ,DdeⅠ,HaeⅢ,HhaⅠ,HinfⅠ,HpaⅡ(MspⅠ),RsaⅠand XbalⅠ,were choosed to analyze specific-class taxonomically unit according to band types. Consequently,candidate molecular markers(over 100bp)distinguishing 16S rDNA of Eperythrozoon and Haemobartonella were screened by simulated restriction enzyme digestion.Designed genus specific primers according to 16S rDNA of Eperythrozoon from NCBI,two bands about 700 bp were amplified respectively from positive blood of hog and chicken by microscopic examination,and were determined as target strips by cloning and sequencing.Thus genus specific PCR method for Eperythrozoon detection was established through optimizing conditions such as Mg2~+ concentration,anneal temperature and number of cycles.The results indicated that this method had better specificity and sensitivity with specific tests and sensibility tests,and could be used in molecular diagnosis and molecular epidemiology for Eperythrozoon.According to differences of geography and terrain in Chongqing,breed styles of each county, and also breed information of each district or county in 2006,thirteen counties(Rongchang,Dazu, Yongchuan,Jiangjin,Hechuan,Bishan,Changshou and typical mountain-breed area including Wulong,Wanzhou,Yunyang,Chengkou,Qianjiang,and Xiushan.)were chose to collected blood samples and 2442 samples were obtained.Positive rates of 2442 samples tested by blood squash, Giemsa stain,universal primers and specific primers were 9.986%,3.398%,0.555%and 0, respectively.The results suggested that there was no real case of Eperythrozoon in Chongqing and PCR method was competent to diagnose chiken infected by Eperythrozoon instead of microscopic examination which had high false positive rate.