Studies On The Antigenic Characteristics And Phylogenetic Analysis Of 16S RRNA Gene Of E.wenyoni

Eperythrozoonosis of cattle is a contagious disease caused by E.wenyoni. For a long time, it was often neglected because of its lower prevalence, even though there are many E.wenyoni -carriers in the fields. However, recent investigations have indicated that the incidence of this disease is considerably increasing, and it has become one of the main diseases which hinder the development of cattle production. Many problems, such as its morphology, classified status, accurate diagnosis and effective prevention of this disease, need to be systematically identified and built up. In this study, the characteristics of antigen and 16S rRNA gene of E.wenyoni were investigated using samples of blood from the spontaneously infected cattle. Firstly, the morphologic characteristics and motility of E.wenyoni, its effect on hematological and immune indexes were determined in terms of the pathogenesis and paved the way for the diagnosis of this disease. Secondly, the antigen was identified tentatively with SDS-PAGE and western blotting after E.wenyoni was isolated from blood in order to develop a specific-antigen-based assay and good quality vaccine for the E wenyoni control. Finally, to decide the classified position of E.wenyoni in terms of systematic evolution, the 16S rRNA gene of E.wenyoni was amplified by PCR, and the amplified product was sequenced after clone, the phylogenetic tree was constructed and discussed.It was shown microscopically that the infected erythrocytes lost their morphology, the E.wenyoni had the characteristics of polymorpholism and refractivity. There is also a characteristics of motility in the E.wenyoni free in the plasma, when they attached to erythrocytes, it looks to stop to move but in fact they make the erythrocytes shave. The tests of hematology showed that with the increment of the infective extent, the values of RBC, HGB and HCT extremely decreased, in contrast, the indexes of WBC, osmotic fragility of erythrocyte and ESR increased obviously. The results of immune indexes showed that the formation rates of C3bRR and ICR, the percentage of T lymphocyte ANAE~+ in cattle seriously infected with E.wenyoni were decreased. This indicated that the immune response of cattle was inhibited when they were seriously infected with E.wenyoni.The result of SDS-PAGE showed that the molecular weights of E.wenyoniantigen covered 24kD~150kD. Nine domain proteins were clearly isolated, and their molecular weights were 141.34 kD, 107.27 kD, 86.03 kD, 68.05 kD, 56.21 kD, 44.30 kD, 32.99 kD, 28.89 kD, and 24.59kD, respectively. Three specific proteins were screened with western blotting, and their molecular weights were 147.31 kD, 49.03 kD and 35.72 kD, respectively.Parts of the gene sequence of the 16S rRNA in E.wenyoni were successfully cloned. The comparison of sequences showed that the determined sequence was most similar to those in GenBank (AF016546) (similarity level, 97.9%), Phylogenetic analysis indicated that the resulting sequence was more similar to those of Mycoplasma spp.(homo!ogy level, about 70%), but was farther from Anaplasma spp.( homology level, about 50%). These data indicated that E.wenyoni seems to belong to Mycoplasma, but not Ricketlsiales, Anaplasmataceae.