The Epidemiology Of Mycoplasma Ovis And The Research Of The Adhesion Mechanism During Infection With Mycoplasma Suis

Eperythrozoon is an uncultivated, well-less bacterium that parasitizes the surface of host’s erythrocytes or separates from the plasma. This pathogen is generally latent infection and can reduce the body’s immune system so that the carriers are more susceptible to the secondary infection with other diseases, leading to mortality. At present, there are several methods to diagnose eperythrozoonosis, however, they are not specific for Mycoplasma ovis (M. ovis) and usually omit some infected carriers. Therefore, the establishment of a quick and effective diagnostic method is the key to the prevention and treatment of this disease. In this study, a semi-nested PCR assay was developed and was used to detect the prevalence of M. ovis in Hubei province compared with conventional PCR (Neimark et al.,2004). In order to study the invasion mechanisms of Eperythrozoon in the protein level, Far western blotting (Far WB) and pull-down assay were used to study the interaction between protein of adhesion-associated Eperythrozoon and membrane proteins of erythrocyte.1. The establishment of a semi-nested PCR assay for the detection of M. ovisAccording to the published16S rRNA gene sequence of M. ovis from GenBank (EU165509.1), primers were designed and a semi-nested PCR was developed. The sensitivity of the new developed assay was as high as10-10fg. Specificity analysis revealed it had no cross reaction with M. suis, M. wenyonii, M. ovipneumoniae, Theileria sergenti as well as Babesia orientalis.2. The epidemiology investigation of M. ovis in Hubei province of China.A total of371blood samples, collected from seven different regions in Hubei province of China, were detected for the presence of M. ovis using both semi-nested PCR and conventional PCR(Neimark et al2004).151(41%) samples were positive by the new developed semi-nested PCR, while only97(26%) were positive by conventional PCR. These results indicated that the semi-nested PCR was much more sensitive than the conventional PCR. Statistical analysis found that the positive rate of samples from the north (47%) and south (31%) of Yangtse River was significantly different (*P<0.05) whereas gender was not significantly different (P>0.05). The present study demonstrated that the semi-nested PCR is more suitable for detection of M. ovis and the investigation of epidemiology.3. The interactions between the adhesion-related proteins of Mycoplasma suis and the membrane proteins of erythrocyte In this study, the natural red blood cell membrane proteins were acquired. GAPDH and OSGEP was first measured by BCA kit and diluted to5μg/mL as a template for Far WB experiment. The results showed that GAPDH/OSGEP interacted with a variety of red blood cell membrane proteins and Band3and GPA were the most obvious ones. Considering the important role and the proportion of these two proteins, the further study will be focus on Band3and GPA.4. The clone and prokaryotic expression of Band3and GPA gene of erythrocyteAccording to the published gene sequences of Band3and GPA, two pairs of specific primers were designed and the target fragments were amplified from cDNA extracted from15-day-old pig spleen and bone marrow. The sequencing results showed that the fragment sizes of Band3and GPA were1077bp and495bp, respectively. The prokaryotic expression plasmids pGEX-6P-1-Band3, pGEX-6P-1-GPA were established and expressed in E.coli BL21. Western blot (WB) analysis showed that the two proteins were highly expressed. Optimal conditions for the expression of BL21/pGEX-6P-1-Band3, BL21/pGEX-6P-1-GPA were37℃5h and ITPG concentration was0.8mmol/L,0.6mmol/L, respectively.5. Identification of the interactions between the adhesion-related proteins of Mycoplasma suis and the membrane proteins of erythrocyteThe supernatant obtained in optimal conditions were used in His-tag pull-down assay. The results showed that Band3/GPA could interact with GAPDH/OSGEP. The parallel experiments were also established to determine the specificity of the test and confirmed Prey proteins were not binding to the resin or GST tag.In the present study, a semi-nested PCR assay was developed and was used to detect the prevalence of M. ovis in Hubei province compared with conventional PCR (Neimark et al.,2004). Besides, the natural red blood cell membrane proteins were successfully obtained. The erythrocyte membrane proteins Band3and GPA were cloned, expressed and western blot. Far WB and His-tag pull-down assay determined Band3and GPA had an interaction with GAPDH and OSGEP, respectively. This study proved the role of Band3/GPA during the infection with M. suis and provided a theoretical basis for the invasion mechanism of Eperythrozoon.