ABSTRACT
Objective To analyze the biological characteristics of a mutant strain of Salmonella ty-phimurium SL1344 with sseK2-deletion (SL1344△sseK2) in order to provide reference for further study of safe and effective live vaccines. Methods The mutant strain SL1344△sseK2 with a deletion of 1047 bp in sseK2 gene was constructed through a two-step allelic exchange using recombinant suicide plasmid. Its com-plemented strain, SL1344C△sseK2, was also constructed. Biological and immunological characteristics of the mutant strain were detected. Results PCR, double-enzyme digestion and sequencing analysis showed that the mutant strain SL1344△sseK2 and the complemented strain SL1344C△sseK2 were successfully con-structed. The serotype of the mutant strain was 1,4,[5],12:i:1,2, identical to the parent strain SL1344. In addition, the mutant strain showed no significant change in biochemical characteristics or growth rate and was genetically stable in vitro. Compared with the parent strain SL1344, the virulence of SL1344△sseK2 was attenuated in BALB/ c mice. The median lethal dose of SL1344△sseK2 for 6-week-old BALB/ c mice was 3. 44×108 colony-forming units (CFU), which was 1620 times lower than that of SL1344. Oral immuniza-tion with SL1344△sseK2 protected 62. 5％ of the mice against challenge with wild Salmonella typhimurium strains on 17 d after vaccination. The levels of serum IgG antibody peaked on 14 d after immunization. No significant difference in biological characteristics was observed between the complemented and the parent strains, indicating that the mutant strain was basically complemented to the wild-type strain.Conclusions The mutant strain SL1344△sseK2 was constructed successfully and genetically stable with sig-nificantly attenuated virulence and good immunogenicity. This study suggested that sseK2 gene played an im-portant role in regulating the virulence of SL1344, which might provide reference for further study of its func-tion and for assessing its potential as a candidate live attenuated vaccine.
ABSTRACT
Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.