Isolation And Characterization Of Mycoplasma Leachii Sp. Nov.

Mycoplasma Leachii is an animals contagious agent, which caused mastitis and abortion in cows and polyarthritis and pneumonia in calves. Mycoplasma Leachii, represented by the type strain PG50, is one of five members of the Mycoplasma mycoides cluster and has been isolated in sporadic and outbreak cases of polyarthritis and mastitis in Australian dairy cattle. However, the pathogen has not been reported in China.In this study, severe polyarthritis in calves at a dairy farm of China was investigated, more than 300 neonatal calves showed a serious polyarthritis symptom. Some affected calves died due to many complications, permanent disfigurement of the appendicular skeleton was still evident in the calves that survived the acute stage of the disease, and therefore these calves were also eliminated. To determine the pathogens of the disease, isolation and identification of the pathogens of the joint fluids was undertaken. Firstly, the joint fluids were collected aseptically and inoculated in Broth Medium, brain heart infusion Medium, anaerobic medium, respectively, and revealed no bacterial growth. This indicated that the disease was not caused by bacterial pathogens. Secondly, Mycoplasma detection and cultures of the samples was undertaken and revealed Mycoplasma existence, two Mycoplasma strains were isolated. After cloning and purification three times, the isolates formed the visible dew-like colonies with a needle size on 1% nutrient agar solid medium, and a typical "oil fried egg"-like colony morphology with different sizes under 40x optical microscope was observed. The typical cell morphology of Mycoplasma, such as rod, filamentous and other polymorphisms,was also observed under electron microscopy. Biochemical tests showed that the Mycoplasma isolates can fermentate glucose, but not hydrolyze arginine and urea. The expected lengths of DNA fragments were amplified by using Mycoplasma 16S rRNA gene and LppA gene-specific primers from synovial fluid samples and purified cultures. Sequence comparison of 16S rRNA gene and LppA gene between the two isolates and the referenced Mycoplasma strains suggested that the isolates were most closely related to the Mycoplasma Leachii sp. nov., with identities of 99.9% for the 16S rRNA gene and of 99.6% for the LppA gene. Lastly,the polyarthritis symptoms in calves were reproduced using intra-articular injections of the organism. The results showed that the Mycoplasma isolated here belonged to the species M. Leachii., for which the names GN407 and GN408 were designated.Clinical assessment and analysis of comprehensive laboratory investigations of affected calves and synovial fluid samples indicated that Mycoplasma Leachii was the aetiological agent of the polyarthritis in calves occurred in this farm of China. Laboratory isolated strains were named GN407 and GN408. Isolation and identification of the Mycoplasma Leachii in China was the first case.