Construction Of Three Major Pathogenicity Islands Deleted-mutants Of Salmonella Enterica Serovar Enteritidis And Evaluation Of Their Immune Efficacy In Chickens

The Gram-negative bacteria Salmonella enterica, a facultative intracellular pathogen, mainlyinfect birds. Salmonellosis, the most frequent food-borne disease in humans, usually results fromcontaminated water and food. Virulence genes of Salmonella that correlate with pathogenesismajorly locate in chromosome and plasmid. Those virulence-associated genes and regulatorscluster in specific regions of chromosomes, termed Salmonella pathogenicity islands (SPIs).However, only limited information is available on how each of these pathogenicity islandsinfluences the ability of Salmonella to infect chickens. In this study, we therefore constructedSalmonella Enteritidis mutants with single individual SPIs (SPI-1,SPI-2,SPI-3) deletedseparately, and the mutants with incoprorated SPIs deleted, and assessed their virulence inchickens, together with the innate immune response of the hosts.The modification red homologous recombination system was used because segments or genecluster greater than40kb can be removed by this single-step procedure. The chloramphenicolresistant gene flanked by homologues of (SPI-1,SPI-2,SPI-3) was amplified by PCR and ligatedinto pET28a to construct targeting plasmid, and then transformed into S. enterica strain SM6byelectric shock. Sequentially, the plasmid pKD46a was transformed and induced by L-arabinosefor recombination. The chloramphenicol resistant gene were liminated by pCP20inducingrecombinase FLP mediated recombination events. The resulting positive mutants were identifiedby PCR and designated as SM6ΔSPI-1,SM6ΔSPI-2,SM6ΔSPI-3,SM6ΔSPI-1&2,SM6ΔSPI-1&3,SM6ΔSPI-2&3,SM6ΔSPI-1-3.The mutant strains were stable after continuous subculture in LB;the growth rate of the mutant strains is higher than that of the parental strain except the SM6ΔSPI-2&3; Scanning electron microscopy (SEM) showed that the SPIs mutants display adhesiontogether except the SM6ΔSPI-2&3; We also compared the carbon source metabolism of the SPIsmutants and SM6and found a few differences; the invasion capacity of the SPI-1mutants werereduced compared to wild type strain; the adhesion capacity of the SPI mutants was obviouslylower than the wild type strain(P<0.001).The colonization ability, pathogenicity and the protection efficacy of the mutants wereevaluated by intraperitoneal injection with the wild-type strain or the SPI mutants in7day-oldSPF chickens. The results showed that the mutants did not cause associated symptoms althoughthey can colonize in tissues, and the SM6ΔSPI-1-3efficiency of colonization is lower than theSM6in the spleen and liver. The serum antibody were determined by using indirectenzyme-linked immunosorbent assay (iELISA), the results showed that antibody levels of theSPIs groups were higher than SM6group. FCM showed that the CD4+T and CD8+T subsets have been obviously changed after immunization with the SPIs mutants. After immunization, thecytokine (IL-4and IFN-γ) were detected by ELISA kit and the results revealed that the liveattenuated vaccine have higher protective efficacy to chickens challenged with SM6. The resultsof pathological examination showed that all the mutants display fewer changes than thewild-type strain. In summary, these findings demonstrate the potential of SM6ΔSPI-1-3as aneffective live attenuated vaccine. The construction of the above SPIs mutants not only laid agood foundation to elucidate the pathogenesis of salmonella and the development of safe andeffective vaccine, but also provides a new perspective for the prevention and treatment ofsalmonellosis.