Establishment Of A Specific Method For Diagnosis Of Mycoplasma Haemosuis (Eperythrozoon Suis) Infection And Characterization Of A M. Haemosuis Erythrocyte Adhesin

Porcine eperythrozoonosis (PE) is an infectious disease caused by Mycoplasma haemosuis. The agent attaches to and causes deformity and damage to the red blood cells of pigs, which is suggested as an important factor associated with the development of clinical PE.To date, M. haemosuis has not been successfully cultivated, which hinders the development of more specific diagnosis methods and the study on mechanisms of host-microbe interaction. In China, diagnosis of PE has mainly been relying on the microscopic examination in Romanowsky or Giemsa stained blood smears. The drawbacks of microscopy include low specificity and sensitivity, because the readily identifiable bacteraemia is linked with the onset of acute disease while lacking in chronic infections. Standard methods for identification of M. haemosuis are needed. Recently, The molecular diagnostic methods of M. haemosuis, such as PCR assays, have been studied based on a few gene sequences published. PCR is more specific and sensitive compared to other methods and can detect early infection. In this study, a semi-nested PCR protocol based on a specific sequence of M. haemosuis was developed and the interaction of a recombinant protein encoded by a putative gene with swine RBC was further studied.Three primers were designed based on the conserved sequences of the 16S rRNA gene of M. haemosuis and M. wenyonii. DNA samples were prepared from pigs suspected of M. haemosuis infection. Sequence verification of the amplicons shown that only 16S rRNA genes of bacteria origin can be amplified, no M. haemosuis sequences were targeted. Our results indicated that 16S rRNA sequence is not an optimal target for PCR detection of M. haemosuis. To establish an more specific PCR method, three primers were designed based on a sequence (AJ504999) which is more specific to M. haemosuis. The PCR amplification parameter were first optimized with the FailSafe TM 12 PreMixes optimal PCR buffer system. Analysis of sequences obtained with this method showing that the amplicons were all derived of M. haemosuis. 300 porcine blood samples from Chongqing, Changchun and Henan were subjected for PCR analysis. Results shown that the overall infection rate of M. haemosuis in pigs was 70.76%(212/300), with 64.41%(76/118) in clinically healthy pigs and 74.73%(136/182)in pigs with symptoms such as fever. In conclusion, the PCR method represents a powerful tool for diagnosis of M. haemosuis infection and epidemiological investigation.Molecules expressed on the cell membrance of M. haemosuis are potential pathogenic factors associated with host cell (RBC) adhesion. We study a protein fragment (MSP) encoded by the longest predicted CDS (OFR2) in AJ504999 for its invovlement in binding to porcine erythrocytes. To circumvent the M. haemosuis specific translational barrier of the codon usage, msp sequence was adapted to the codon usage of E. coli and de novo synthesised. Soluble recombinant proteins (MSP-GST, MSP1-GST and MSP2-GST) were generated in E.coli and purified from bacterial lysates using Glutathione-Sepharose 4B. Erythrocyte binding and Western blot assays with the recombinant proteins clearly shown that all three proteins bound to swine RBC in a dose dependent manner while the GST protein alone did not bind. More importantly, the recombinant proteins bind to swine erythrocytes can be inhibited by GAG. Thus we hypothesized that GAG-related molecules are utilized by M. haemosuis in their interactions with host erythrocytes. The data have paved a way for futher understanding the molecular pathogenesis related to M. haemosuis infection.