Development Of A Specific PCR Assay For Mycoplasma Haemobovine Infection And Cloning Of 16S RRNA Gene Of Eperythrozoon Wenyonii And Mycoplasma Haemobovine

Eperythrozoon and Haemobartonella belong to Rickettsiales, Anaplas-mataceae, and mainly parasitize in the surface of red blood cell of variousanimal or drift away in the blood plasma, thus they are also calledhaemotrophic bacteria. These pathogens can cause the membrane structureof the red blood cell to change with severe cell damage. The symptoms ofthe infected animals include clinical anemia, choloplania and febricity, andso on. Since the report of Eperythrozoon infecting human in 1970's, therehave been much studies regarding this kind of blood nutritional hazardousfungi.There had been doubt regarding the taxonomical status ofEperythrozoon and Haemobartonella, and these microorganisms wereclassified as Eperythrozoon and Haemobartonella, belonging to Rickettsiales,Anaplasmataceae, parallel to Anaplasma. The present study used the conserved primers 27F and 1492R of procaryotic organism to amplify the16S rRNA gene of etiological agents which were suspected as Eperythrozoonand Haemobartonella. Sequencing revealed that two rRNA gene fragmentsfor Mycoplasma haemobovine (EF424082和EF460765) were obtained,1434bp and 1435bp in length, with more than 99% similarity between thetwo sequences. The sequence similarity with that of H.felis(AF178677) andH.canis(AY529641)was 94.5% and 94.7%, respectively. Therefore, weconsider that it represents Mycoplasma haemobovine in the blood of cattle.Moreover, the present study also obtained a 16S rDNA sequence of E.wenyoni-CGXD(EF221880) from dairy cow whose length was 1453bp. ThisDNA fragment had 99.8% similarity with the 16S rDNA sequence of E.wenyoni-CGX(AY769937) published by this laboratory, and had 97.7%similarity with the 16S rDNA sequence of E. wenyoni-CHUB(AY946266).These results demonstrated that Eperythrozoon and Haemobartonellashould be merged as a single genus, supporting the proposal of Neimarkand Messick that this kind of blood nutritional fungi be incorporated intoMycoplas-mamatales, Mycoplasma.Based on the 16S rRNA gene sequence of Mycoplasma haemobovineobtained in the present study, a pair of specific primers was designed aspecific PCR assay was established for the detection of Mycoplasmahaemobovine. This assay was highly specific and it did not amplify the DNAof H. felis, E. wenyoni, E. suis, Trypanosoma evansi, and Toxoplasma gondii. This technique was also quite sensitive, which can detect Bartonella DNA of36pg/μL. Application of the assay for the detection of M. haemobovine inthe clinical blood samples proved that this method is useful as a diagnostictool for the diagnosis of M. haemobovine infection and for molecularepidemiological investigation, and control of the disease it causes.