Construction And Immunobiological Features Of Salmonella Pullorum ΔfurΔspeDE Strain

Salmonella enterica serovar Pullorum(Salmonella Pullorum) is an avian pathogenic gram-negative bacterium. Young chickens can be infected by Salmonella Pullorum with pullorum disease (PD) following high morbidity and mortality. Infected adult chickens are bacterial carriers with no clinical symptoms. In addation to horizontal transmission, Salmonella Pullorum also can transmit vertically through ovary and eggs, leading to low rate of eggs’hatchability and high rate of young chickens’death. As a result, Salmonella Pullorum has become one of harmful pathogens in poultry industry with huge economic losses in developing countries. A series of measures have been taken to control this pathogen in developed and developing countries, including vaccines, antibiotics, biosecurity, and ’test-and-cull’ policies. After many years of practice, it has been proved that vaccine may be the effective measures to control Salmonella infection in poultry. Among three kinds of vaccines being used for controlling Salmonelloses containing inactivated vaccine, subunit vaccine and live attenuated vaccine, live attenuated vaccine could be the promising vaccine for its good security, genetic stability and sufficient immunoprotection.The critical balance between safety and immunogenicity should be carefully considered to develop live attenuated vaccine. So it is difficult to select target gene(s) to construct an attenuated vaccine strain. In this study, fur and speDE were deleted to construct Salmonella Pullorum S06004△fur△speDE, and the immunobiological properties of S06004△furAspeDE were tested as live attenuated vaccine.1. Construction and identification of Salmonella Pullorum △fur△speDE strainIn this study, S06004△fur△speDE strain was constructed using λ-red homologous recombination system. The PCR and DNA sequencing indicated that fur and speDE genes were deleted in S06004△fur△speDE strain. Comparing to parent strain S06004, some biochemical characteristics of S06004△fur△speDE were changed. S06004△fur△speDE and S06004△fur grew slower than S06004, S06004△speDE was similar to S06004 in the growth rate. Analysis of genetic stability manifested that S06004△furAspeDE strain was confirmed with good genetic stability and the deleted gene could not be reversed. The LD50 in chickens showed that the virulence of S06004△fur△speDE, S06004△fur and S06004AspeDE had a significant drop, respectively, about 105 times,73.4 times and 4.95 times comparing to the virulence of S06004. All results indicated that attenuated S06004△fur△speDE is safe for chickens and could be used as a live attenuated vaccine candidate for further study.2. Preliminary analysis on immunobiological features of attenuated Salmonella Pullorum △fur△speDE strainThe immunobiological features of attenuated S06004△fur△speDE strain were further analyzed on the model of Salmonella infection to cells and chickens. Avian macrophage cell line HD-11 was used as in vitro model in adhesion, invasion and proliferation assay, S06004△fur△speDE could reduce its ability to invade HD-11 cells and to proliferate within the HD-11 cells, but its ability of adhesion was almost unchanged. Chickens were used as in vivo model to analyze the rule of clearance and immunoprotection of S06004△fur△speDE strain, 3-days-old chickens were administered orally and intramuscularly respectively, in the orally inoculated group, the bacteria of S06004AfurAspeDE were cleared in chicken liver and spleen after 10 days post infection(dpi); in the intramuscularly inoculated group, S06004AfurAspeDE were cleared in chicken liver and spleen after 3 weeks post infection(wpi). The immunoprotective efficacy assay demonstrated that S06004△fur△speDE could provide significant immunoprotection against S06004 challenge at 14 dpi, and at the same time, the pathological section showed, the degree of pathological changes in chickens liver and spleen in the group immunized with S06004A△fur△speDE following the challenge of S06004 were lighter than that of the control group not being immunized. The net weight in immunized group with the challenge of S06004 gained more than that of those not being immunized. Based on indirect ELISA, serum IgG antibodies and mucous IgA antibodies were detected, the level of serum IgG antibodies could be detected at 4 dpi and peaked at 3 wpi, the level of mucosal IgA antibodies could be elicited after 1 wpi and peaked at 3 wpi. The mRNA levels of cytokines in spleens were quantitatively analyzed by quantitative real-time PCR (qRT-PCR), the level of IFN-y was up-regulated. In summary, S06004△fur△speDE has the potential to be used as a live attenuated vaccine candidate to prevent and control Salmonella Pullorum because of its good safty and sufficient immunogenicity.