Role of Salmonella enterica subspecies enterica serovar Enteritidis pathogenicity island-2 in chickens

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 host’s 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

Effect of the Salmonella Pathogenicity Island 2 Type III Secretion System on Salmonella Survival in Activated Chicken Macrophage-Like HD11 Cells

In order to better identify the role of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (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 wild-type Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) strain, a SPI-2 mutant S. Typhimurium strain, a wild-type Salmonella enterica subspecies enterica serovar Enteritidis (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-uptake (PU) by the HD11 cells, up to 24 h PU, while the E. coli strain was no longer recoverable by 3 h PU. We can conclude from these observations that the SPI-2 T3SS of S. Typhimurium and S. Enteritidis 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

NOMENs Land: The Place of Eponyms in the Anatomy Classroom

The law of Non-Original Malappropriate Eponymous Nomenclature (NOMEN) states that no phenomenon is named after its discoverer (Stigler, 1980; Aresti and Ramachandran, 2012; Aronson, 2014). However, eponymous terms are rife in the anatomical and medical literature. Here the authors support the argument that eponymous terms do not have a firm place and should not be used in anatomy education

Physical activity for antenatal and postnatal depression in women attempting to quit smoking: randomised controlled trial

Background: Antenatal depression is associated with harmful consequences for both the mother and child. One intervention that might be effective is participation in regular physical activity although data on this question in pregnant smokers is currently lacking. Methods: Women were randomised to six-weekly sessions of smoking cessation behavioural-support, or to the same support plus 14 sessions combining treadmill exercise and physical activity consultations. Results: Among 784 participants (mean gestation 16-weeks), EPDS was significantly higher in the physical activity group versus usual care at end-of-pregnancy (mean group difference (95% confidence intervals (CIs)): 0.95 (0.08 to 1.83). There was no significant difference at six-months postpartum. Conclusion: A pragmatic intervention to increase physical activity in pregnant smokers did not prevent depression at end-of-pregnancy or at six-months postpartum. More effective physical activity interventions are needed in this population. Trial registration: Current Controlled Trials ISRCTN48600346. The trial was prospectively registered on 21/07/2008

List of bacterial strains used in this study.

a<p>Dr. B. Finlay, University of British Columbia, Vancouver, British Colombia.</p>b<p>Dr. C. Poppe, Laboratory for Foodborne Zoonoses, Health Canada, Guelph, Ontario.</p

Recovery of <i>Salmonella</i> from the cell monolayer fraction of HD11 cells over time.

<p>The ability of SPI-2 mutants (<i>S. </i>Typhimurium Δ<i>ssaR</i> and <i>S.</i> Enteritidis ΔSPI-2) to survive in the chicken macrophage HD11 cell line was compared to their parent wild-type strains (<i>S.</i> Typhimurium SL1344 and <i>S.</i> Enteritidis Sal18) as well as to the non-pathogenic <i>E. coli</i> strain DH5α. <b>Panel A</b> shows the recovered CFU/ml from the cell monolayer fraction at 0.5 h post-uptake (PU), prior to addition of gentamicin. <b>Panels B</b>, <b>C</b>, <b>D</b>, and <b>E</b> show the recovered CFU/ml from the cell monolayer fraction after the addition of gentamicin at 3, 6, 12, and 24 h PU respectively. *, <i>p-</i>value<0.05; **, <i>p</i>-value<0.01; ***, <i>p</i>value<0.001. Note that the scale of the Y-axis is linear, and differs between time points.</p

Hydrogen peroxide production by activated HD11 cells.

<p>Fold difference in hydrogen peroxide production in HD11 cells 12 hours after stimulation with PMA compared to that of unstimulated cells, as measured by luminescence produced by the reaction between hydrogen peroxide, luminal, and HRP.</p

HD11 chicken macrophage-like cells.

<p><b>Panel A</b> shows HD11 cells that have not been stimulated with PMA, while <b>Panel B</b> shows HD11 cells 12 hours after stimulation with PMA. Photographs were taken under 10X magnification.</p

Wild-type <i>S.</i> Typhimurium strain SL1344 in the media fraction over time.

<p>HD11 cell nuclei are stained blue, both intra- and extracellular bacteria are green, and extracellular bacteria appear red or yellow. <b>Panel A</b> shows an HD11 cell loaded with wild-type <i>S.</i> Typhimurium strain SL1344 at 0.5 h PU, just prior to addition of gentamicin. <b>Panels B</b> and <b>C</b> show whole HD11 cells containing SL1344 at 3 and 6 h PU, respectively, after addition of gentamicin. <b>Panels D</b> and <b>E</b> show fragmented HD11 cells containing SL1344 at 12 and 24 h PU, respectively, after the addition of gentamicin. Whole and fragmented cells containing SL1344 were visible at all time points in the media fraction.</p