Isolation And Identification Of Mycoplasma Ovipneumoniae From Shandong And Studies On Out Membrane Protein Of Standard Strain Y-98

Mycoplasma ovipneumoniae (M.O) is the pathogen to Infectious pleuropneumoniae of sheep and goats, and has no cross immunity with Mycoplsam mycoides subsp. Capri. The latter pathogen could only cause pleuropneumoniae of goats. After being firstly isolated in China in 1982, M.O has made great threats to the sheep and goats. In this study, the methods with isolation and identification of M.O were demonstrated systemically based on the M.O isolated in Shandong; the serologically investigation of M.O in several breeding farm was made; the biological properties of outer membrane protein of Y98 were studied to demonstrate the pathogenic mechanism of M.O; the PCR to diagnose M.O infection rapidly was designed and preliminary applied. The studies had made the theconical support for the M.O infection in Shandong province.The study was divided into four parts:Part 1: The isolation and identification of M.O in Shandong provinceThe samples from lamb died of pleuropneumoniae were collected, and the pathogen was isolated with Hartleys medium. Through morphological, serological, biological, and electron microscope, the isolated strain YG showed the same properties with the M.O standard strain Y98. The culture of YG was inoculated into the healthy lamb though trachea, and the lamb showed the same clinical symptoms with natural infected lamb. The results demonstrated M.O was the pathogen. The SPF embryo grew slowly and died lastly after M.O being inoculated into the yolk arc. As for the propagation way of M.O, they mainly showed the binary fission, and they also showed the budding fission. Through electron microscope, the membrane of M.O was three-level structure and had floss fimbria.Part 2: The serological investigation of M.O infection in Shandong provinceThe IHA was made to 898 sheep and goats of different age and breed from Jinan, Laiwu, Liaocheng, and Dongying. The serum would be considered to be positive if the value of it was above 26. The results showed there was M.O infection in Shandong, and sheep and goats of different age and breed showed different sensitivity to M.O. The general positive rate was 28.9%, and the positive rate of sheep was32%, which was above the positive rate of goats, 24.3%. The positive rate of one-year-old sheep and goats was 42.8%, which was higher than the ones of other ages. As for the breed, Boer Goat showed the positive rate of48.3%, Dubo Sheep showed the positive rate of 47.9%. They all were higher than the positive rate of local ones and hybrid ones, which were 22.5% and 27.6%, respectively.Part 3: Studies on the outer membrane protein of Y98The results of Electron Microscope illustrated the existence of out membrane protein and fimbria, which could play an important role in the pathogenic process. With Tritonx-100 lysis and 0.6%KI extraction, the out membrane protein of Y-98 was obtained. Through the 12%SDS-PAGE analysis, the molecular weight scope was proved to be between14.4 and 125.9KD; and though the western-blot, the major protective antigens were determined as those with molecular weight of 67.6KD,39.3KDand 28.8KD. With the protein immunizing rabbits, the results demonstrated that the protein could induce rabbit produce specific antibody of high titer, and could induce effectively the cellular immune response. The sera against membrane protein could neutralize effectively with Y-98, which inhibit the deleterious effect of Y-98 on MDCK.Part 4: The construction and preliminary application of PCR to detect M.O rapidlyAccording to the conserved sequence of Mycoplasma on GenBank, the common primers were designed to amplified sequence of M.O, and the product was about 453bp. About the specific test, the PCR using the above primers could amplified the corresponding sequence from M.OY98, YG, MS, and MG, but could not amplified the sequence from Streptococcus pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus, which were the major pathogens causing pneumoniae in sheep and goats. The template was diluted to 1pg/ml, and the PCR could also detect the DNA. The result showed the good sensitivity. When using the culture of M.OY98, YG and yolk from inoculated SPF embryo, the PCR could amplified specifically the sequence of 453bp.