Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. The SPI-2 T3SS has been identified as vital for survival and replication of S. Typhimurium and S. Enteritidis in mouse macrophages, as well as full virulence in mice. In order to test the ability of SE SPI-2 mutants to survive in vivo we used a chicken isolate of SE (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type (WT) strain Sal18: Sal18 attTn7::tet and Sal18 attTn7::cat, while the other two groups received the WT strain (Sal18 attTn7::tet) and one of two mutant strains (Sal18 attTn7::cat ÄspaSÄssaU or Sal18 ÄSPI-1ÄSPI-2::cat). From this study we conclude that S. Enteritidis deficient in the SPI-1 and SPI-2 systems are out-competed by the WT strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains; a WT strain and three other strains missing either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge (PC) we observed a reduced systemic spread of the SPI-2 mutants, but by day 3 the mutants systemic distribution levels matched that of the WT strain. Based on these two studies, we conclude that the SPI-2 T3SS facilitates invasion and systemic spread of S. Enteritidis in chickens, but alternative mechanisms for these processes appear to exist. Several structural components of the T3SSs encoded by SPI-1 and SPI-2 are exposed to the hosts immune system prior to/during the infection/invasion process, making them potential vaccine candidates. Several of these candidates genes were cloned, the proteins overproduced, purified, and formulated as vaccines for use in further studies. SPI-2 T3SS proteins used for vaccine studies included the secretin, SsaC, the needle, SsaG, the filament, SseB, and a part of the translocon, SseD, as well as a number of effectors, SseI, SseL, SifA, and SifB. The first vaccine study involved vaccination of SPF chickens with SseB and SseD, followed by challenge with the WT S. Enteritidis strain Sal18. Additional studies evaluated whether hens vaccinated with SPI-2 T3SS structural or effector components could mount a significant humoral immune response (as measured by serum immunoglobulin Y [IgY] titres), whether these antibodies could be transferred to progeny (as measured by egg yolk IgY titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with the WT S. Enteritidis strain Sal8. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgY that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered SE per bird in most situations. In order to better identify the role of the SPI-2 T3SS in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a WT S. Typhimurium strain, a SPI-2 mutant S. Typhimurium strain, a WT S. Enteritidis strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-infection (PI) up to 24 h PI, while the E. coli strain was no longer recoverable by 3 h PI. We can conclude from these observations that the SPI-2 T3SS is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment as E. coli is effectively eliminated.

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. The SPI-2 T3SS has been identified as vital for survival and replication of S. Typhimurium and S. Enteritidis in mouse macrophages, as well as full virulence in mice. In order to test the ability of SE SPI-2 mutants to survive in vivo we used a chicken isolate of SE (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type (WT) strain Sal18: Sal18 attTn7::tet and Sal18 attTn7::cat, while the other two groups received the WT strain (Sal18 attTn7::tet) and one of two mutant strains (Sal18 attTn7::cat ÄspaSÄssaU or Sal18 ÄSPI-1ÄSPI-2::cat). From this study we conclude that S. Enteritidis deficient in the SPI-1 and SPI-2 systems are out-competed by the WT strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains; a WT strain and three other strains missing either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge (PC) we observed a reduced systemic spread of the SPI-2 mutants, but by day 3 the mutants systemic distribution levels matched that of the WT strain. Based on these two studies, we conclude that the SPI-2 T3SS facilitates invasion and systemic spread of S. Enteritidis in chickens, but alternative mechanisms for these processes appear to exist. Several structural components of the T3SSs encoded by SPI-1 and SPI-2 are exposed to the hosts immune system prior to/during the infection/invasion process, making them potential vaccine candidates. Several of these candidates genes were cloned, the proteins overproduced, purified, and formulated as vaccines for use in further studies. SPI-2 T3SS proteins used for vaccine studies included the secretin, SsaC, the needle, SsaG, the filament, SseB, and a part of the translocon, SseD, as well as a number of effectors, SseI, SseL, SifA, and SifB. The first vaccine study involved vaccination of SPF chickens with SseB and SseD, followed by challenge with the WT S. Enteritidis strain Sal18. Additional studies evaluated whether hens vaccinated with SPI-2 T3SS structural or effector components could mount a significant humoral immune response (as measured by serum immunoglobulin Y [IgY] titres), whether these antibodies could be transferred to progeny (as measured by egg yolk IgY titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with the WT S. Enteritidis strain Sal8. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgY that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered SE per bird in most situations. In order to better identify the role of the SPI-2 T3SS in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a WT S. Typhimurium strain, a SPI-2 mutant S. Typhimurium strain, a WT S. Enteritidis strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-infection (PI) up to 24 h PI, while the E. coli strain was no longer recoverable by 3 h PI. We can conclude from these observations that the SPI-2 T3SS is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment as E. coli is effectively eliminated.