Construction of Mycoplasma hyopneumoniae P97 Null Mutants

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia, a world-wide problem in the pig industry. This disease is characterized by a dry, non-productive cough, labored breathing, and pneumonia. Despite years of research, vaccines are marginally effective, and none fully protect pigs in a production environment. A better understanding of the host-pathogen interactions of the M. hyopneumoniae-pig disease, which are complex and involve both host and pathogen components, is required. Among the surface proteins involved in virulence are members of two gene families called P97 and P102. These proteins are the adhesins directing attachment of the organism to the swine respiratory epithelium. P97 is the major ciliary binding adhesin and has been studied extensively. Monoclonal antibodies that block its binding to swine cilia have contributed extensively to its characterization. In this study we use recombination to construct null mutants of P97 in M. hyopneumoniae and characterize the resulting mutants in terms of loss of protein by immunoblot using monoclonal antibodies, ability to bind purified swine cilia, and adherence to PK15 cells. Various approaches to recombination with this fastidious mycoplasma were tested including intact plasmid DNA, single-stranded DNA, and linear DNA with and without a heterologous RecA protein. Our results indicate that recombination can be used to generate site-specific mutants in M. hyopneumoniae. P97 mutants are deficient in cilia binding and PK15 cell adherence, and lack the characteristic banding pattern seen in immunoblots developed with the anti-P97 monoclonal antibody.

Effects of porcine MyD88 knockdown on the expression of TLR4 pathway-related genes and proinflammatory cytokines

As a critical adapter protein in Toll-like receptor (TLR)/Interleukin (IL)-1R signalling pathway, myeloid differentiation protein 88 (MyD88) plays an important role in immune responses and host defence against pathogens. The present study was designed to provide a foundation and an important reagent for the mechanistic study of MyD88 and its role TLR/IL-1R signalling pathways in porcine immunity. Lentivirus-mediated RNAi was used to generate a porcine PK15 cell line with a silenced MyD88 gene and quantitative real-time PCR (qPCR) and Western blotting were used to detect changes in the expression of critical genes in the Toll-like receptor 4 (TLR4) signalling pathway. ELISA was used to measure the levels of seven proinflammatory cytokines–interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, macrophage inflammatory protein (MIP)-1α and MIP-1β–in cell culture supernatants after MyD88 silencing. We successfully obtained a PK15 cell line with 61% MyD88 mRNA transcript down-regulated. In PK15 cells with MyD88 silencing, the transcript levels of TLR4 and IL-1β were significantly reduced, whereas there were no significant changes in the expression levels of cluster of differentiation antigen 14 (CD14), interferon-α (IFN-α) or TNF-α. The ELISA results showed that the levels of most cytokines were not significantly changed apart from IL-8 without stimulation, which was significantly up-regulated. When cells were induced by lipopolysaccharide (LPS) (0.1 μg/ml) for 6 h, the global level of seven proinflammatory cytokines up-regulated and the level of IL-1β, TNF-α, IL-6, IL-8 and IL-12 of Blank and negative control (NC) group up-regulated more significantly than RNAi group (P<0.05), which revealed that the MyD88 silencing could reduce the TLR4 signal transduction which inhibited the release of proinflammatory cytokines and finally leaded to immunosuppression.

Chrysanthemum indicum Attenuates Cisplatin-induced Nephrotoxicity both in vivo and in vitro

Chrysanthemum indicum Linné has been used in traditional medicine to treat various inflammatory diseases in East Asia. The aim of the present study was to investigate the protective effect of C. indicum ethanol extract (CILE) against cisplatin-induced nephrotoxicity. An HPLC-photodiode array method was used for fingerprint analysis of the CILE and ten major constituents were quantitatively analyzed. The protective effect of CILE on cisplatin-induced nephrotoxicity was assessed using both in vitro (porcine kidney cell; PK15 cell) and in vivo (Sprague Dawley rat) experiments. In the in vitro study, CILE enhanced PK15 cell viability after cisplatin treatment with recovered antioxidant status. Moreover, the increased p53 expression after cisplatin treatment was decreased in the CILE pretreated cells. In the in vivo study, SD rats were treated for 28 consecutive days with CILE (0, 100, 300 and 500 mg/kg). On day 23, a single dose of cisplatin (5 mg/kg) was injected to induce nephrotoxicity. The CILE pretreated group showed recovered serum renal function index with ameliorated oxidative stress. Histopathological alterations and apoptosis in the kidney were also decreased in CILE pretreated rats. Taken together, CILE could attenuate cisplatin-induced nephrotoxicity and might be a beneficial agent for acute renal failure management.

Quantitative Estimation of Porcine Endogenous Retrovirus Release from PK15 Cells

The present study focuses on the assessment of porcine endogenous retrovirus (PERV) release from PK15 cells in a time dependent manner. The highest amount of PERV A RNA was detected in PK15 cells after 16 hours of culture. The highest amount of PERV B RNA was detected in PK15 cells after 20 hours. The highest amount of both subtypes RNAs was detected in culture medium after 32 hours of culture. The peaks of PERV reverse transcriptase (RT) activity were detected after 28 h of culture in PK15 cells and after 32 hours in the culture medium. The monitoring of PERV release from PK15 cell line may be useful for the evaluation of PERV replication.