Attenuated Actinobacillus pleuropneumoniae as a bacterial vector for expression of Mycoplasma hyopneumoniae P36 gene.

BACKGROUND: Actinobacillus pleuropneumoniae and Mycoplasma hyopneumoniae are causative agents of porcine pneumonia. Over the last few years, attenuated A. pleuropneumoniae live vaccines have been shown to provide protection against A. pleuropneumoniae infection. We postulated that attenuated A. pleuropneumoniae could additionally be used as a vaccine vector for protection against M. hyopneumoniae. METHODS: A mutant strain of A. pleuropneumoniae, SLW36, was constructed by replacing the urease structural gene of mutant strain SLW03 of A. pleuropneumoniae with the L-lactate dehydrogenase gene (p36) of M. hyopneumoniae by transconjugation and counter selection. The urease function and the growth kinetics of SLW36 were measured. Protein expression of P36 was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and western blotting. The attenuated virulence and immunity of SLW36 were analyzed in a mouse model. RESULTS: The mutant strain SLW36 was urease negative and four-fold less virulent than the parental strain SLW03. There were no differences in expression levels of p36 at different culture time-points and the foreign gene was stable after in vitro passage. Immunoglobulin G responses against p36 antigen and M. hyopneumoniae whole-cell antigen were detected. CONCLUSIONS: The mutant strain SLW36 can induce antibody against p36 and M. hyopneumoniae. The mutant strain SLW36 has the potential to be used as a live vaccine for protection against A. pleuropneumoniae and M. hyopneumoniae. Studies in pigs are needed to confirm protective levels of antibodies and to check for rare side-effects of the vaccine.