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.

Recent studies showed that attenuated Salmonella-derived vaccines induce both humoral and cell-mediated immunity responses [16][17][18]. Particularly, S. enterica serovar Choleraesuis strain C500 is highly immunogenic and safe, and has been widely used in China for over 40 years to prevent piglet paratyphoid [19,20]. In the present work, we combined two M. hyopneumoniae antigens, P97R1 and NrdF (P97R1N), into S. enterica serovar Choleraesuis C501 [21]. Therefore, a recombinant attenuated S. enterica vaccine was evaluated by oral and intramuscular (IM) routes in a mouse model. The immunogenicity of S. enterica strain C501 expressing P97R1N protein was also investigated. Materials and Methods Bacterial strainsM. hyopneumoniae HNYY wild type strain isolated from tissues on primary porcine lung (Laboratory stock). Attenuated S. enterica serovar Choleraesuis strain C501 was constructed with the help of our colleagues [21]. Escherichia coli X7213 and JM105 were obtained from the Agricultural Microbiology Laboratory, Huazhong Agricultural University, China. Growth conditions and isolation of genomic DNA of M. hyopneumoniaeM. hyopneumoniae was grown in Friis modified mycoplasma broth (FMMB) as described previously [22]. The culture was grown at 37°C at 5% CO 2 until acidity was observed. Genomic DNA was extracted using a Quantum Prep AquaPure Genomic DNA Kit (Bio-Rad). AbstractThe immunogenicity of the P97 adhesin repeat region R1 (P97R1) and the ribonucleotide reductase R2 subunit (NrdF) antigens of Mycoplasma hyopneumoniae were evaluated as vaccines in mice. Mice were immunized orally or injected intramuscularly (IM) with attenuated Salmonella enterica serovar Choleraesuis strain C501 harboring a prokaryotic expression vector encoding P97R1 and NrdF (pYA97R1N). Local and systemic immune responses were analyzed in vaccinated mice. The results showed that P97R1N-specific serum IgG and IgA antibodies were detected in serum and lung samples. P97R1-specific serum IgG and IgA antibodies were also detected in serum, but in lung sample only P97R1-specific IgG response was induced. ELISA assays demonstrated that IFN-Û and IL-4 production was induced in cultural supernatants from splenocytes stimulated with specific antigens in vitro. M. hyopneumoniae whole cell-specific antibody was induced in vaccinated mice and a good immunogenic effect by this vaccine was indicated. Importantly, animals vaccinated by IM had higher levels of P97R1N-specific IgG and IgA than those vaccinated by oral route. Furthermore, mice immunized with the C501 vaccine elicited significantly higher levels of P97R1N-specific IgA and IgG in lungs and serum (P<0.05), and IFN-Û and IL-4, than those who received the live M.hyopneumoniae vaccine. Taken together, these results suggest that IM vaccination with the C501 vaccine might provide an effective method of inducing an immune response to M. hyopneumoniae.