Progress 07/01/12 to 10/31/14
Outputs
Target Audience: This project will be published as a scientific journal paper within a month of writing in a peer-reviewed scientific journal. The target audience(s) are other scientists in thefields of veterinary health, agriculture, immunology, and biotechnology. In addition, undergraduate and graduate students gained valuable trainingduring this reporting period, includingoneAfrican-americanundergraduate student. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project was very fruitful in this regard because Dr. Rubinelli and several assistant graduate and undergraduate students gained valuable experience learning how to 1) evaluate potential vaccine strains in in vitro tests; 2) conduct AICUC-approved vaccine trials in live chickens with appropriate controls; and 3) evaluate the vaccine trial through direct plating of marker vaccine and marker challenge strain Salmonella from the internal organs of the test chickens and through ELISA analysis of serum and intestinal mucosa for assessment of the immunogenicity of the vaccines. As stated above, the vaccine trial on the additional mutants developed during the extension period of the SBIR phase I grant gave encouraging results, and these are presently beingpreparedfor publication. Thus, Dr. Rubinelli and the trainees will also benefit professionally from the publication of the vaccine trialresults. How have the results been disseminated to communities of interest? Amanuscript to be published in a peer-reviewedscientific journalis presently being written based on the results of the vaccine trial described above. This manuscript is nearly complete and will be finished within the next month pending one additional ELISA experiment that is now in progress on the frozen intestinal mucosa to evaluate the pathogen-specific secretory IgA levels. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We generated the double deletion mutant intended. The double deletion mutant failed to grow in minimal media, as expected as it is a methionine auxotroph, but did grow in chicken feed and in fecal broth. As a result of this outcome, we generated an additional double mutant by deleting the metD gene, a gene that encodes the high affinity methionine transporter. We then used bacteriophage transduction to combine the metR and metD mutations. The resulting double mutant, metR metD, was tested for growth in minimal medium and failed to grow in it. When this minimal medium was supplemented with L-methionine it grew but when it was suplemented with D-methionine, it failed to grow. This was the expected outcome, because the metD transporter is known to transport D-methionine, but because this transporter was deleted, the double mutant could only grow if L-methionine was supplied. The metR metD double mutant was able to survive in 5% chicken feed and 5% fecal broth albeit at a lower level than the other mutants and wild type parent strain, indicating that this double mutant, like the metR hilA double mutant, would be able to survive in the chicken production environment, and this outcome would not be advantageous for a recombinant vaccine. To investigate the virulence of the metR hilA double mutant, a cytotoxicity test was performed and indicated reduced cytotoxicity compared to the control strain. Adhesion and invasion of the metR hilA double mutant in cultured human intestinal epithelium Caco-2 cells and in cultured chicken macrophage cellswas also reduced about 10 fold in comparison to the wild type control strain, indicating that the double mutant was attenuated and in this sense would be suitableas a vaccine, if the survival in the chicken broth was also undetectable. Unfortunately,as stated above, the double mutantsurvives in 5% chicken feed. The fourth goal (to assess the poultry vaccine market and needs of customers) we performed a SWOT analysis and an internet search to find competing products. These were summarized in a table. In addition to the four objectives of the phase I project, we also developed two additional mutant strains, tested these strains for their effect on Caco-2 and cultured chicken macrophage adhesion and invasion, and conducted macrophage survival measurements. These indicated that the two additional mutants were attenuated and therefore good candidates for a vaccine trial. The vaccine trial results indicated that these additional mutants were protective against a challenge with virulent Salmonella and one of the two additional mutants also was cleared from the chickens by three weeks post-vaccination. These last results were very encouraging.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2015
Citation:
The manuscript is being completed presently and will be submitted within one month of writing, probably in February 2015, to a peer-reviewed journal.
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