Generation, safety and immunogenicity of an Actinobacillus pleuropneumoniae quintuple deletion mutant SLW07 (ΔapxICΔapxIICΔorf1ΔcpxARΔarcA).

We inactivated a virulence determinant, ArcA, in an Actinobacillus pleuropneumoniae quadruple deletion mutant SLW06 (ΔapxICΔapxIICΔorf1ΔcpxAR, serovar 1), and a quintuple deletion mutant SLW07 was generated. SLW07 showed decreased adherence to and invasion of host cells, compared to its parent strain SLW06. SLW07 was more sensitive in RAW264.7 macrophage-mediated phagocytosis and clearance. SLW07 was less virulent in mice. An immunization assay indicated that both SLW07 and SLW06 preferentially stimulated T helper cell type 2 response in mice. Live vaccines induced the production of interleukin-6 and tumor necrosis factor-α by splenic lymphocytes. Furthermore, the protective immunity of SLW07 was not affected after ArcA mutation. Immunization with SLW07 could provide a complete protection following virulent A. pleuropneumoniae challenge in mice. Our results suggest that SLW07 is a promising live vaccine candidate, which is further attenuated from and shares similar protective efficacy with its quadruple deletion parent SLW06.

Construction, characterization and evaluation of the protective efficacy of the Streptococcus suis double mutant strain ΔSsPep/ΔSsPspC as a live vaccine candidate in mice.

Streptococcus suis serotype 2 (S. suis 2) causes sepsis and meningitis in piglets and humans, and results in one of the most serious bacterial diseases affecting the production of commercial pigs around the world. Due to the failure of the current inactivated vaccine to protect against the disease, development of a new attenuated live vaccine against S. suis 2 by deleting essential virulence factors is urgently needed. We have previously reported the construction and characterization of an SsPep single gene deletion mutant strain ΔSsPep based on S. suis 2. Our previous results have shown that SsPep plays a critical role in the pathogenesis of S. suis 2. In this study, a precisely defined double-deletion mutant ΔSsPep/ΔSsPspC of S. suis 2 without antibiotic-resistance markers was constructed based on ΔSsPep, and the levels of virulence of the wild-type (WT) and ΔSsPep/ΔSsPspC were compared in a mouse experimental infection model. We demonstrated that the double mutant ΔSsPep/ΔSsPspC was less virulent than the WT, and could induce a noticeable antibody response. Analysis of IgG subclasses (IgG1 and IgG2a) indicated that both Th1 and Th2 responses were induced by ΔSsPep/ΔSsPspC, although the IgG2a (Th1) response predominated over the IgG1 (Th2) response. Moreover, ΔSsPep/ΔSsPspC could confer 90% protective efficacy against challenge with a lethal dose of fully virulent S. suis 2. Taken together, these data demonstrate that ΔSsPep/ΔSsPspC can be used as an effective live vaccine and provide a novel strategy against infection of S. suis 2.

Deletion of the znuA virulence factor attenuates Actinobacillus pleuropneumoniae and confers protection against homologous or heterologous strain challenge.

The znuA gene is known to be important for growth and survival in Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida under low Zn(2+) conditions. This gene is also present in Actinobacillus pleuropneumoniae serotype 1; therefore, the aim of this study was to investigate the existence of a similar role for the znuA gene in the growth and virulence of this organism. A precisely defined ΔznuA deletion mutant of A. pleuropneumoniae was constructed based on the sequence of the wild-type SLW01 using transconjugation and counterselection. This mutation was found to be lethal in low-Zn(2+) medium. Furthermore, the ΔznuA mutant strain exhibited attenuated virulence (≥22-fold) as well as reduced mortality and morbidity in a murine (Balb/C) model of infection. The majority of the bacteria were cleared from the lungs within 2 weeks. The ΔznuA mutant strain caused no adverse effects in pigs at doses of up to 1.0×10(9) CFU/mL. The ΔznuA mutant strain induced a significant immune response and conferred 80% and 100% protection on immunised pigs against challenge with A. pleuropneumoniae strains belonging to homologous or heterologous serovars, respectively, compared to the blank controls. The data obtained in this study indicate the potential of the mutant ΔznuA strain for development as a live vaccine capable of inducing reliable cross-serovar protection following intratracheal immunisation.