The ribonuclease III (RNase III) is an important enzyme system that regulates non-coding RNA (ncRNA) levels. In this study, LM-ΔrncS gene deletion strain was investigated by gene overlap extension PCR (SOE-PCR) and homologous recombination techniques. The environmental stress response, biofilm formation and virulence were determined and compared between the deletion strain LM-ΔrncS and the parental strain LM EGD-e. When compared with LM EGD-e, the adaptability of LM-ΔrncS was significantly reduced (P<0.05) under the stress of 30°C/42°C, pH 9, 5% NaCl, 3.8% ethanol and 0.1% H2O2. Biofilm formation ability of LM-ΔrncS was significantly lower (P<0.05) than that of LM EGD-e. In LM-ΔrncS, the transcription levels of ncRNA SreA and SbrA genes were significantly decreased (P<0.05). The adhesion rate and invasion rate of LM-ΔrncS in RAW264.7 cells were significantly lower (P<0.01) than those of LM EGD-e, and the survival and proliferation of LM-ΔrncS in RAW264.7 cells were also significantly decreased (P<0.05). Moreover, the transcription levels of InlA, hly, prfA and SigmaB gene were significantly lower (P<0.05) than those of LM EGD-e. LD50 of LD-ΔrncS in BALB/c mice was increased by 1.49 logarithmic orders, and the survival time of the mice was significantly prolonged when compared with LM EGD-e. In addition, the bacterial load in the liver and spleen was markedly decreased, and its pathological damage was also reduced. This study confirmed that RNase III RncS is involved in the regulation of environmental stress response, biofilm formation and virulence in LMd.

Mycoplasma ovipneumoniae is an important pathogen causing respiratory disease in sheep. At present, the immune-associated antigens of M. ovipneumoniae are still unknown, which significantly limits the development of new vaccines for M. ovipneumoniae. In order to identify and characterize the immune-associated antigen genes, genomic expression library of M. ovipneumoniae was constructed and identified, from which positive clones were recognized and screened by positive serum against M. ovipneumoniae. Sequence analysis showed that these 10 clones contained 5 different genes encoding P97-like protein, P102-like protein, Translation initiation factor (IF-1), Methionine aminopeptidase (MAP) and P56 membrane protein, respectively. Three proteins including IF-1, MAP and P97-like protein were expressed in E. coli and used to immunize lambs to verify their immunogenicity, respectively. Animal immunization test confirmed that the novel protein MAP displayed a strong immunogenicity, while the immunogenicity of P97-like protein and IF-1 were relatively weak. The identification of immunogenic protein MAP provided a potentially valuable antigen candidate for the development of serological diagnostic method and subunit vaccine against M. ovipneumoniae infection.