The Construction Of Salmonella Dublin ΔphoQ-ΔrpoS Double-deletion Mutant And Its Preliminary Application

Salmonella dublin (S. dublin) is one of the main pathogens causing intractable diarrhea in calf paratyphoid, which has a great damage to the cattle industry due to the massive infection and higher incidence in calves. In addition, the infected cattle, S. dublin contaminated beef, incomplete sterilization of milk and its products from S. dublin infected cattle are an important source of infection to cause food poisoning. So S. dublin has a great threat on animal and public health.An effective method to prevent calf paratyphoid is vaccination. Currently, there was one live vaccine attenuated from virulent strains by continuouisly passaging in media with acetate in our country. However, this attenuated vaccine using chemical mutagenesis remains the weaknesses such as unclear genetic background, risk of back mutation and residual virulence.This study is to construct a new attenuated vaccine against S. dublin by gene deletion in order to obtain a vaccine with higher efficacy, more safety, clear genetic background, and with clear markers to differentiate the vaccination and natural infection of virulence strain. The main contents are described as follows:1. Construction of AphoQ single mutant and AphoQ-ArpoS double mutant of S.dublinBased on the original sequences of phoQ and rpoS gene clusters in the GenBank, S.dublin (CICC2149) was selected as the prototype, and the AphoQ single mutant and AphoQ-ArpoS double mutant were constructed by using Red recombinantion system, and then identified successfully by PCR.2. Study on biological characteristics of AphoQ single mutant and AphoQ-ArpoS double mutant of S.dublinThe study has confirmed that the growth rate and biochemical characteristic of mutants were consistent with those of the parent strain by growth curve and biochemical test. The virulence of mutants significantly reduced than the parent strain in Balb/c mice, the LD50 results show that the virulence of AphoQ single mutant decreased 20 times than the parent strain, and AphoQ-ArpoS double mutant was 26 times. Research on cellular adhesive and invasive ability found that mutants were significantly less adhesive and invasive for HEp-2 cells than the parent strain (P＜0.01).3. Study on immunogenicity of AphoQ single mutant and AphoQ-ArpoS double mutant in miceResearch of AphoQ single mutant and AphoQ-ArpoS double mutant on immune response by intraperitoneal injection in Balb/c mice has shown that the levels of serum antibody and cellular immunity in AphoQ and AphoQ-ArpoS immunity groups are significantly different from those of control group (P＜0.01). Mice immunized with AphoQ-ArpoS double mutant generated significantly higher IFN-y concentration in serum at 1 week after inoculation than that with AphoQ single mutant (P<0.05); however, there were no significant differences in the IgG titres and TNF-a concentration between the mice immunized with AphoQ single mutant and AphoQ-ArpoS double mutant (P>0.05). In addition, the CD4+T-cell population in the mice at 2 week after immunization with AphoQ-ArpoS double mutant was higher than that with PBS by flow-cytometric analysis (P<0.05). Therefor, AphoQ-ArpoS double mutant induced much stronger Th1-type cellular immune response than △phoQ single mutant. The results of dendritic cell infection in vitro have showed that the secretion of TNF-a in the supernatant of DCs culture are very high for all infection, and furthermore, the parent strain stimulated much higher than the deletion mutants. The secretion of IL-10 changed similarily to the section of TNF-a but lagged behind TNF-a. It may suggest that IL-10 should inhibit the inflammatory response caused by TNF-a over-expression.Protection against homologous lethal infection in mice indicated that AphoQ single mutant immunization group could provide 83.3% protection against challenge of lOLD5o of the parent strain, while AphoQ-△rpoS double mutant immunization group 100% protection with lOLD50 and 83.3% with 50LD50 of the parent strain. However, both mutants couldn’t obtain protection against challenge of 100LD50and 1000LD50 of the parent strain.Protection against heterologous lethal infection indicated that the immunization with AphoQ-ArpoS mutant produced 100% protection against challenge of lOLD50 (equal to 5 X 106CFU)of S. typhimurium and 33.3% protection against 10LD50 (equal to 5 X 109 CFU) of S. enteritidis respectively. It suggests that the AphoQ-ArpoS double mutant was able also to induce protection against heterologous challenge. The partial protection to S. enteritidis might be related to the large dose of challenge.In conclusion, immunization of S. dublin AphoQ-ArpoS double mutant would be a good candidate as a novel marker vaccine.