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

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

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

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

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

Salmonella enterica Serovar Enteritidis Pathogenicity Island 1 Is Not Essential for but Facilitates Rapid Systemic Spread in Chickens▿

Salmonella enterica subsp. enterica serovar Enteritidis is a leading cause of human food-borne illness that is mainly associated with the consumption of contaminated poultry meat and eggs. To cause infection, S. Enteritidis is known to use two type III secretion systems, which are encoded on two salmonella pathogenicity islands, SPI-1 and SPI-2, the first of which is thought to play a major role in invasion and bacterial uptake. In order to study the role of SPI-1 in the colonization of chicken, we constructed deletion mutants affecting the complete SPI-1 region (40 kb) and the invG gene. Both ΔSPI-1 and ΔinvG mutant strains were impaired in the secretion of SipD, a SPI-1 effector protein. In vitro analysis using polarized human intestinal epithelial cells (Caco-2) revealed that both mutant strains were less invasive than the wild-type strain. A similar observation was made when chicken cecal and small intestinal explants were coinfected with the wild-type and ΔSPI-1 mutant strains. Oral challenge of 1-week-old chicken with the wild-type or ΔSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection of the liver and spleen was delayed in birds that were challenged with the ΔSPI-1 strain. These data demonstrate that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of the organism in chickens