Salmonella Immunity: Development of a Neutrophil Phagocytosis Assay and Stress Model in Swine

Our laboratory is interested in the immunomodulation of porcine defense mechanisms against bacterial intracellular pathogens such as Salmonella typhimurium and S. choleraesuis. Past studies indicate that levels of serum tumor necrosis factor-a (TNF-a) increase after intranasal challenge with S. typhimurium but not after oral inoculation. Challenge with S. choleraesuis has no effect on serum TNF-a concentration in the blood, regardless of route. Route of inoculation with S. choleraesuis has been shown to affect levels of lymphocyte proliferation. Both oral and intranasal routes of inoculation stimulate peripheral blood B-cells while the intranasal route is more effective at stimulating peripheral blood T-cells. The inoculum dose of S. typhimurium or S. choleraesuis can also play an important role in the host immune response. TNF-a concentrations in the blood are much greater after a 106 S. typhimurium challenge than after a 104 S. typhimurium challenge. At high doses (;:o:l09 CFU) S. choleraesuis causes signs of lymphocyte suppression, which may affect the ability of the immune system to eliminate the bacteria. Pigs administered an intranasal dose of 108 CFU S. choleraesuis have similar immune responses as naturally infected animals

Pathogenesis, Transmission, and Control of Salmonellosis in Swine

The objectives of our project at the National Animal Disease Center are: 1. To identify virulence factors important in the pathogenesis of Salmonella choleraesuis and Salmonella typhimurium in swine 2. To define the epidemiology and transmission of Salmonella in swine 3. To define the porcine immune response to acute and chronic Salmonella infection focusing on mechanisms to reduce or eliminate the pathogenic organism 4. To identify methods to control Salmonella in swine

Synergism between Porcine Reproductive and Respiratory

This study was conducted to investigate the effects of exposure to porcine reproductive and respiratory syndrome virus, Salmonella choleraesuis, and stress on young swine. Five-week-old segregated early weaned pigs were randomly assigned to one of eight treatments consisting of all possible combinations of three factors: S. choleraesuis (SC) on day zero, porcine reproductive and respiratory syndrome virus (PRRSV) on day three, and dexamethasone (DEX) on days three to seven. DEX was used as a proxy for stress. Treatment differences were seen in performance parameters, levels and duration of SC shedding, level and distribution of SC in tissues, clinical disease, and mortality. The results of this study provided evidence to support field observations that clinical outbreaks of PRRS are the result of interactions among concurrent infections and stressors

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for Use in the Detection of Bovine Tuberculosis in Cattle

As a consequence of continued spillover of Mycobacterium bovis into cattle from wildlife reservoirs and increased globalization of cattle trade with associated transmission risks, new approaches such as vaccination and novel testing algorithms are seriously being considered by regulatory agencies for the control of bovine tuberculosis. Serologic tests offer opportunities for identification of M. bovis-infected animals not afforded by current diagnostic techniques. The present study describes assay development and field assessment of a new commercial enzyme-linked immunosorbent assay (ELISA) that detects antibody to M. bovis antigens MPB83 and MPB70 in infected cattle. Pertinent findings include the following: specific antibody responses were detected at 90 to 100 days after experimental M. bovis challenge, minimal cross-reactive responses were elicited by infection/sensitization with nontuberculous Mycobacterium spp., and the apparent sensitivity and specificity of the ELISA with naturally infected cattle were 63% and 98%, respectively, with sensitivity improv- ing as disease severity increased. The ELISA also detected infected animals missed by the routine tuberculin skin test, and antibody was detectable in bulk tank milk samples from M. bovis-infected dairy herds. A high-throughput ELISA could be adapted as a movement, border, or slaughter surveillance test, as well as a supplemental test to tuberculin skin testing

An Oral Vaccine Based on U-Omp19 Induces Protection against B. abortus Mucosal Challenge by Inducing an Adaptive IL-17 Immune Response in Mice

As Brucella infections occur mainly through mucosal surfaces, the development of mucosal administered vaccines could be radical for the control of brucellosis. In this work we evaluated the potential of Brucella abortus 19 kDa outer membrane protein (U-Omp19) as an edible subunit vaccine against brucellosis. We investigated the protective immune response elicited against oral B. abortus infection after vaccination of mice with leaves from transgenic plants expressing U-Omp19; or with plant-made or E. coli-made purified U-Omp19. All tested U-Omp19 formulations induced protection against Brucella when orally administered without the need of adjuvants. U-Omp19 also induced protection against a systemic challenge when parenterally administered. This built-in adjuvant ability of U-Omp19 was independent of TLR4 and could be explained at least in part by its capability to activate dendritic cells in vivo. While unadjuvanted U-Omp19 intraperitoneally administered induced a specific Th1 response, following U-Omp19 oral delivery a mixed specific Th1-Th17 response was induced. Depletion of CD4+ T cells in mice orally vaccinated with U-Omp19 resulted in a loss of the elicited protection, indicating that this cell type mediates immune protection. The role of IL-17 against Brucella infection has never been explored. In this study, we determined that if IL-17A was neutralized in vivo during the challenge period, the mucosal U-Omp19 vaccine did not confer mucosal protection. On the contrary, IL-17A neutralization during the infection did not influence at all the subsistence and growth of this bacterium in PBS-immunized mice. All together, our results indicate that an oral unadjuvanted vaccine based on U-Omp19 induces protection against a mucosal challenge with Brucella abortus by inducing an adaptive IL-17 immune response. They also indicate different and important new aspects i) IL-17 does not contribute to reduce the bacterial burden in non vaccinated mice and ii) IL-17 plays a central role in vaccine mediated anti-Brucella mucosal immunity

Targeting Sphingosine Kinase 1 in Carcinoma Cells Decreases Proliferation and Survival by Compromising PKC Activity and Cytokinesis

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy