Mycoplasma ovipneumoniae induces caspase-8-dependent extrinsic apoptosis and p53- and ROS-dependent intrinsic apoptosis in murine alveolar macrophages.

Mycoplasma ovipneumoniae (MO) is a principle causative agent of chronic respiratory disease in ruminants, including sheep, goats, and deer, posing a great threat to the ruminant industry worldwide. However, the pathogenesis of MO infection still remains not well understood and needs further clarification. Here we report a time-dependent apoptosis in cultured murine alveolar macrophage (MH-S) cell lines in response to MO infection in vitro. Mechanistically, MO infection activated apoptosis in MH-S cells through caspase-8-dependent extrinsic pathway and through tumor protein 53 (p53)- and reactive oxygen species (ROS)-dependent intrinsic mitochondrial pathways. Moreover, MO infection promoted both transcription and translation of proinflammatory cytokine genes including interleukin-1ß (IL-1ß), IL-18, and tumor necrosis factor-α (TNF-α), in a caspase-8-, p53-, and ROS-dependent manner, implying a potential link between MO-induced inflammation and apoptotic cell death. Collectively, our results suggest that MO infection induces the activation of extrinsic and intrinsic apoptotic pathways in cultured MH-S cells, which is related to upregulated expression of proinflammatory cytokines. Our findings will contribute to the elucidation of pathogenesis in MO infection and provide valuable reference for the development of new strategies for controlling MO infection.

[Development and Application of Reverse Transcription Loop-mediated Isothermal Amplification Method for Detection of Porcine Rotavirus of Swine].

We wished to establish a method for rapid and sensitive detection of reverse transcription loop-mediated isothermal amplification(RT-LAMP)for the rapid and sensitive detection of porcine rotavirus (PoRV). According to the published PoRV VP7 sequences in GenBank,6specific primers were designed. According to the concentrations of foward and reverse primers, Bst DNA polymerase, Mg(2+), and dNTP, reaction conditions were optimized. Results revealed the concentration ratio of foward and reverse primers to be 200 nmol/L:2, 400 nmol (1:12), Bst DNA polymerase concentration to be 0.64U/µL,Mg2+concentration to be 2.5mmol/L, and dNTP concentration to be 1.0mmol/L in 1hat 60℃.The amplification effect achieved a "ladder" effect, with amplified bands being shown only for PoRV. RT-LAMP was specific and did not elicit a cross reaction with porcine epidemic diarrhea virus, transmissible gastroenteritis virus of pigs, or classical swine fever virus. The sensitivity of RT-LAMP was 1.0×10(2) copies/µL. After the reaction, inspection by the naked eye revealed positive amplification products to appears as cloudy-white precipitates, and addition of SYBR Green I showed a color change. These data demonstrate that RT-LAMP is suitable for the rapid and sensitive detection of PoRV.