Detection Of Mycoplasmas In Dairy Milk And Development Of A LAMP Assay For Detection Of Mycoplasma Bovis

Mycoplasmas are the known, smallest prokaryotic microorganisms which can grow in cell-free media. They lack the typical bacterial cell wall, show polyphonic morphologies and can pass through the bacterial filter. Mycoplasmas are also known as the important pathogens for humans and animals. Some of Mycoplasma species have been already demonstrated as the causative agents of cow mastitis and have caused huge economic loss in dairy industry both in Europe and North America. But the study on mycoplasma-related cow mastitis has been seldom reported in China. There is little informationt on the prevalence of Mycoplasmas in dairy milk. The predominant Mycoplasma species and their roles in pathogenosis of cow mastitis in China are not clear yet. The aim of this study is to investigate on the prevalence of Mycoplasma species in some Chinese diary farms using molecualr detection, isolation and identificaton of Mycoplasmas. The role of the dicovered Mycoplasma species in the dairy mastitis was also discussed.It was already known that more than one Mycoplasma species were reported as the causative agents of cow mastitis and there is a possibility that certain unkown species may cause cow mastitis, so a reliable genus-specific PCR for Mycoplasmas will be helpful for preliminary screenning of milk samples.Five published Mycoplasma genus-specific PCR tests were assessed with 55 strians of mycoplasmas which belongs to 17 mycoplasma species and 16 strains of different bacteria from 13 speices respectively. The PCR methods number four showed good specificity in discrimination of mycoplasma from bacteria, and it could detect 10 pg of genome DNA and 100 ccu/mL culture of M. bovis at lowest. A total of 73 samples from cattles experimentally infected with M. bovis as well as clinical cases suspected being infected with mycoplasmas, were detected by the selected PCR no. 4. The PCR gave an average agreement rate of 93.15% with the mycoplasma isolation. These results indicated that the PCR no. 4 is reliable for rapid preliminary screening of Mycoplasmas in routine laboratory diagnositic use.Using the slected Mycoplasma genus-specific PCR, a total of 1100 milk samples from 10 dairy farms in Beijing, Inner Mongolia and Ningxia of China were detected. In 407 milk samples from dairy cows with subclinical mastitis, 11.06%(45/407) of them was positive. In 46 samples from dairy cow with clinical mastitis, 10.20%(5/49) of them showed positive. In 644 milk samples from healthy dairy cow, 8.54%(55/644) of them was positive. Further identificaiton of positive samples was carried out using specific PCR for different cow mastitis related Mycoplasams: M. bovis, M. bovigenetalium, M. california, M. alkalescens and M. bovirhinis. The results showed that 98.06%(101/103) of positive samples was M. bovis and 1.94%(2/103) of them was M. alkalescens. It indicated that Mycoplasmas were prevalent in Chinese dairy milk and M. bovis was the predominant Mycoplasma species in the samples detected.To develop a rapid, simple, sensitive and specific assay for detection of the predominat Mycoplasma species of M. bovis, a loop-mediated isothermal amplification(LAMP) assay which targets the uvrC gene of M. bovis, was established by optimazation of primers and reaction procedures. The speicificity of the LAMP was evaluated with 12 strians of mycoplasmas which belongs to 11 mycoplasma species and 4 strains of different bacteria from 4 speices. The result showed that all other organisms but M. bovis were negative. It demonstrated that the develped LAMP was specific for M. bovis. The LAMP also showed a good sensitivity with detection of 1 pg of M. bovis genome DNA. The reactions of LAMP assay just requires to be insulated at 61℃ for one hour., and the result of reaction is capable of visual inspection with adding of SYBR GreenⅠ. This LAMP assay is suitable for rapid detction of M. bovis in the dairy farms and veterniary agencies with rough experimental conditions.