Progress 01/01/15 to 05/31/16
Target Audience:Those individual scientists and companies developing improved SE vaccine for the poultry industry are the target audience. Changes/Problems:
What opportunities for training and professional development has the project provided?The project gave the student, Emily Weaver, the training she needed to be admitted into veterninary school. How have the results been disseminated to communities of interest?The findings have not been disseminated for additional analyses are needed and samples are needed to be collected. In addition, this work was conceived and developed by Professor Amin Fadl who left the university. I (Cook) served as the mentor, along with Fadl to complete Emily's MS. We have goals to complete some analyses and publish a paper in 2017. What do you plan to do during the next reporting period to accomplish the goals?
What was accomplished under these goals?
First, we successfully made a mutant SE that was deficient in the virulence factor genes gidA and mnmE. Such a mutant in mice for a different stain of salmonella was protective. When an LD 50 study of protection using pathogenic wild-type SE in vaccinated and non-vaccinated chicks was used, no mortality was observed. This means that the pathogen was not lethal as anticipated, hence to see if the vaccine worked we looked at SE invasion in tissues. The vaccinated chicks when challenged with the virulent SE showed about a 1log reduction in SE in spleen, no change in the liver, and intestine and a 1 log decrease in the ceca. The level of SE in tissues did not drop to zero, hence the vaccine was not successful in preventing colonization of SE according to the model used. Second, cytokine analysis of the data showed the mutant increased sera gamma interferon levels and increased lymphocyte blastogenesis, two important measures that the mutant activated the immune system. We also found that both the mutant and the virulent pathogen suppressed sIgA levels in the mucosa. sIgA is important to the survival of birds and suppression of sIgA may be a critical strategy in SE pathogenesis for future study. In conclusion, the mutant served as a successful vaccine in reducing tissue invasion of SE but was not effective in totally eliminating colonization. New strategies may improve the development of a better vaccine.
Weaver, E.M. 2016. gidA mnmE DKO as a potential salmonla vaccine for chickens. M.S. Thesis, University of Wisconsin-Madison.
Progress 01/01/15 to 09/30/15
What opportunities for training and professional development has the project provided?THe project is currently training a graduate student. How have the results been disseminated to communities of interest?
What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will evaluate vaccination efficacy of the gidA mnmE DKO mutant and determine maternal protective immunity in the progenies of vaccinated laying hens.
What was accomplished under these goals?
The long-term goal of this project is to develop an attenuated strain of Salmonella Enteritidis (SE) for use as a live attenuated vaccine to protect poultry against SE and other serovars of Salmonella. We have previously shown that deletion of virulence gene regulator gidA attenuated Salmonella virulence in both in vitro and in vivo models of infection. Immunization of mice with the gidA mutant strain conferred protective immunity in mice by eliciting both humoral and cell-mediated immune responses, suggesting that the gidA mutant strain is a good candidate for use in a live-attenuated vaccine. Deletion of both gidA and mnmE of Salmonella Typhimurium (STM) caused a marked attenuation and mice immunized with the gidA mnmE DKO mutant were protected from a highly lethal dose challenge of WT without showing any signs or symptoms of STM associated septic shock. We hypothesize that GidA and MnmE have a significant role in the virulence of Salmonella. In this study we will conduct a complete immunological characterization of the gidA mnmE DKO mutant of SE for potential use as live-attenuated vaccine in chickens to inhibit bacterial colonization of intestinal mucosa. Additionally, this study will determine if progenies of vaccinated hens will show maternal protective immunity during the first couple of weeks of life. Objective 1: Determine the attenuation of a gidA mnmE DKO mutant in chickens. In this study, we examined the 50% lethal dose (LD50) of the gidA mnmE mutant and wild-type SE strains in one week old chickens. One-week old SPF chicks were orally infected with different doses (3x105 - 1x1010 CFU) of the wild-type (WT) and the gidA mnmE mutant and observed for 6 weeks for signs of morbidity and mortality. Data from study indicated that the gidA mnmE double knockout mutant was highly attenuated and could be used for immunization of chickens. Objective 2: Examine the ability of immunization to block Salmonella colonization. One-week old SPF chicks were immunized with the gidA mnmE mutant strain of SE and the control birds were given sterile PBS instead. Three-weeks following immunization, birds were challenged with a high dose of the WT. Chickens were euthanized at one week post-challenge and bacterial load was determined in the ceca, small intestine, spleen and liver. Data from this study showed significantly less number of bacteria was recovered from the group of chickens immunized with the mutant, indicating that immunization of chickens with the gidA mnmE mutant inhibits Salmonella colonization of intestinal mucosa and internal organs.