Various "housekeeping" genes are often used as endogenous controls in gene expression experiments. We have cloned from swine, three genes commonly used as endogenous controls in other species and have characterized their relative levels of expression in various porcine tissues and their response to various cell activators. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were readily detected by northern hybridization in various tissues and in alveolar macrophages. The expression of hypoxanthine phosphoribosyltransferase (HPRT) was detected only by northern hybridization of poly-A+ enriched RNA and by reverse transcriptase-polymerase chain reaction (RT-PCR), making it more suitable for highly sensitive detection methods. Expression of GAPDH varied less among tissues than did beta-actin, making it more useful control for comparisons of gene expression between tissues with northern hybridizations. Various treatments of cultured alveolar macrophages differentially affected levels of beta-actin and GAPDH, while HPRT expression was unchanged in alveolar macrophages or spleen cells similarly treated. Therefore, while HPRT can be used as the endogenous control with sensitive detection methods such as RT-PCR, less sensitive detection methods require a more abundant gene such as GAPDH.
eThe African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (
The immune response of swine to vaccination with a live, attenuated PRRSV was assessed in the presence and absence of cytokine adjuvants or cholera toxin (CT) to address the hypothesis that adjuvant danger signals, that is, inflammatory cytokines and bacterial extoxin, stimulate a more robust immune response. Animals received four injections of recombinant porcine IL-1 and IL-6, IL-12 alone, or CT alone within 1 week of intramuscular administration of a vaccine strain of PRRSV, Ingelvac MLV. Serological and cell-mediated responses were monitored for 42 days after vaccination and for a further 10 days after challenge with the virulent VR2332 strain of PRRSV. First, the principal observation was an enhancing effect of IL-12 on the interferon gamma response to PRRSV, with a more rapid and heightened PRRSV-specific interferon gamma ELISPOT response in peripheral blood mononuclear cells. The more rapid and robust development of a cell-mediated immune response, as determined by this assay, suggests that IL-12 may influence the induction of antigen-specific T cell responses. Second, animals that received CT adjuvant displayed a more robust antibody response to GP5, the major envelope glycoprotein. Anti-GP5 titers peaked at 21 days after vaccination at more than twice the level of any other treatment, for which the peak response was at 28 days. Third, there was no evidence of PRRSV immunosuppression of immunity to unrelated antigens, including circovirus. CT is a potent mucosal adjuvant, particularly for antibody responses. It acts in part through the production of IL-1 in macrophages, but its effect was not replaced by combination treatment with IL-1 and IL-6. In sum, the results suggest that cytokine adjuvants, particularly IL-12, and CT have the potential to enhance immune responses to live viral vaccines.
An Actinobacillus pleuropneumoniae infection model in swine was established to study the expression of inflammatory cytokines during acute respiratory disease. Lavage fluid, lavage cells consisting primarily of alveolar macrophages, and lung tissue were analyzed for the presence of various cytokines at 2, 4, 8, and 24 h following endotracheal inoculation of A. pleuropneumoniae. Interleukin-1 beta (IL-1) and IL-8 mRNA levels were elevated within 2 h in lavage cells of animals inoculated with A. pleuropneumonia but not in cells from controls treated with saline-bovine serum albumin, based on Northern (RNA blot) analysis. Tumor necrosis factor (TNF) mRNA was present at low levels in all animals, and the level was not increased at any time point. In situ hybridization was more sensitive than Northern blotting and revealed elevations of all three cytokines in lavage cells within 2 to 4 h of A. pleuropneumoniae inoculation. IL-6 was detected in lavage cells by in situ hybridization but not by Northern blotting. In lung tissue obtained 18 to 24 h after A. pleuropneumoniae instillation, all cytokine mRNAs, including that of IL-6, were detected by Northern blot analysis. The levels of bioactive IL-1 and IL-6 in lavage fluids increased approximately 1,000-fold following A. pleuropneumoniae inoculation, but TNF bioactivity was not detected. Morphological localization of cytokine mRNAs by in situ hybridization indicated markedly increased levels of TNF, IL-1, and IL-8 mRNAs at the periphery of focal lung lesions. These findings indicate that inflammatory cytokines, particularly IL-1 and IL-8, are associated with the development of pleuropneumonia and may contribute to disease severity.
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