Development Of Diagnostic Methods Of Mycoplasma Wenyonii And Myocoplasma Suis And The Research Of Adhesion Mechanism

Eperythrozoon is a group of uncultivable bacteria which parasite the surface of erythrocytes of human or animals, or free in the blood plasma. Eperythrozoon can cause infections which are life threatening by acute haemolytic anaemia, or mild chronic anaemia manifested by illthrift, infertility and immune suppression. The infection of eperythrozoon can bring serious economic losses. Various of diagnostic methods for Eperythrozoon have been established, however, each method has its advantages and disadvantages. Therefore, it is in dare need of establishing a more sensitive, effective and accurate detection method. Additionally, the adhesion mechanism of Eperythrozoon is still unknown. Thus, the identification and function research of adhesion protein for eperythroon is particularly important for the clarification of the pathogenesis of this diseases.In this study, a loop-mediated isothermal amplification (LAMP) method was established for the detection of Mycoplasma wenyonii, and was used to investigate the prevalence of M. wenyonii infection in the cattle in some parts of the country, and also in the arthropods in the cowshed. Subsequently, an indirect-ELISA method based on MSG1protein was established for the detection of Mycoplasma suis, and was conducted for the preliminary clinical application. Additionally, five potential adhesion proteins of M. suis were selected from the published genome of M. suis. The adhesions of the five selected proteins to porcine red blood cells were evaluated in this study.(1) Development of loop-mediated isothermal amplification (LAMP) assay for the detection of M. wenyoniiA pair of specific primers was designed based on the16S rRNA gene of M. wenyonii, and the conventional PCR method was developed subsequently. Simutaneously, a set of specific primers was designed based on the16S rRNA gene of M. wenyonii, and the LAMP method was established. The result of sensitivity assay showed that LAMP assay was ten times more sensitive than PCR assay. A total of330blood samples of cattle from all over the country were subjected to LAMP and PCR detection,71samples were positive by the LAMP, while only62were positive by PCR. And all the PCR-positive samples were LAMP-positive. The products of9LAMP-positive but PCR-negative samples were cloned and sequenced, all sequences were verified to be M. wenyonii sequences. Additionally,17ticks,24lice,25mosquitoes and22flies were collected from a cowshed, and7ticks,13lice,16mosquitoes and22flies were tested positive by LAMP and PCR. This result indicated that these kinds of arthropods may serve as potential transmission vectors for M. wenyonii.(2) Phylogenetic analysis of M. wenyonii based on16S rRNA geneEight16S rRNA gene sequeces of M. wenyonii isolated from HN, JS, HB, SC, AH were subjected to amplification, cloning and sequencing. According to the eight sequences and the other sequences of haemotrophic mycoplasmas downloaded from Genbank, phylogenetic tree was established by using MEGA software. The tree showed that all eight sequences were located in the clade of M. wenyonii, and there is no significant difference between isolates from different areas. M. wenyonii was more closely related to Mycoplasma ovis, as compared to Mycoplasma suis. And all hemoplasma species were located within a single clade and were most closely related to Mycoplasma pneumonia group.(3) Development of indirect ELISA assay for the detection of M. suisAccording to the MSG1gene sequence published on Genbank, a pair of PCR primers was designed to amplify the main epitope of MSG1gene. After cloning and sequencing, the target gene was inserted to pET28a vector, and was expressed by prokaryotic expression system subsequently. The expression conditions for MSG1were optimized by regulating the IPTG induction time and induction concentration. An indirect ELISA assay was established for the detection of M. suis, based on the extracted recombinant MSG1protein. This method has good specificity and repeatability. The result of preliminary clinical application showed that the adult pigs had a higher prevalence of M. suis than the piglets.(4) Identification of M. suis adhesion proteinsFive putative adhesion proteins of M. suis were selected from the published genome of M. suis. The adhesions of the five selected proteins to porcine red blood cells were evaluated by adhesion ELISA and blood smears adhesion assay. Site-directed mutations were conducted to optimize the triplet codon, in order to ensure the prokaryotic expression of the five proteins. Polyclonal antibodies were prepared, purified, and labeled by biotin. The result of adhesion ELISA showed that GAPDH protein had the strongest ability to adhere to the pig red blood cell membrane, followed by OSGEP protein. Inhibition of binding assay indicated that the binding can be specifically blocked by polyclonal antibodies. The subcellular localization assay showed the surface expression of the five proteins in E. coli transformants. The result of blood smears adhesion assay was consistent with the adhesion ELISA assay. E. coli transformants gain the ability to bind to erythrocytes by expression of GAPDH and OSGEP on their surface. In summary, the established LAMP assay and indirect ELISA assay provided powerful tools to monitor the prevalence of M. wenyonii and M. suis, and also help to evaluate the potential risk factors for these diseases. The identification of M. suis adhesion proteins laid the foundation for the research of adhesion and pathogenic mechanism.