Progress 09/01/11 to 08/31/16
Outputs
Target Audience:The target audience includes scientists working in industry and academia. We have published in peer-reviewed, open-access journals and given oral presentations to interested parties in industry and academia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The grant resulted in the publication of several peer-reviewed articles and a book chapter. Graduate students and post-doctoral fellows were involved in this work. How have the results been disseminated to communities of interest?Our results have been disseminated via journal publications and oral presentations (examples are provided below). Presentations: Campylobacter jejuni exploits host cell processes. Society for General Microbiology. Convention Centre Dublin, Ireland: March 26-29, 2012. Campylobacter jejuni exploits host cell processes: Identification of C. jejuni vaccine targets. Intervet Inc. d/b/a Merck Animal Health, United Kingdom, March 30, 2012. Prevention of Human Disease by Vaccination of Food-Animals, AAAS Pacific Division, Emerging and Re-Emerging Infectious Diseases, Boise Convention Center, Boise, Idaho, June 24 - 27, 2012. Prevention of Human Disease by Vaccination of Food-Animals, Gilead Sciences, Washington State University, Lewis Alumni Centre presentation, Pullman, WA, June 11, 2012. Prevention of Human Disease by Vaccination of Food-Animals, School of Molecular Biosciences Fall 2012 Retreat, Coure d'Alene, ID, August 16, 2012. What do you plan to do during the next reporting period to accomplish the goals?This is the final report.
Impacts
What was accomplished under these goals?
We were able to address each of the three specific aims of our proposal. We found that L. acidophilus NCFM, L. crispatus JCM 5810, L. gallinarum ATCC 33199 and L. helveticus CNRZ32 inhibited the growth of C. jejuni in vitro, likely through the production of lactic acid. In addition, all four isolates examined resulted in a reduction in the median colonization of chickens by C. jejuni. L. crispatus produced the most lactic acid and resulted in the greatest reduction in C. jejuni colonization of chickens. We concluded that the ability of Lactobacillus to reduce C. jejuni colonization in chickens is based on its ability to produce lactic acid and control the metabolic activity of mixed bacterial cultures. This project sets the stage for using Lactobacillus as a probiotic in the industry. Also of interest is that we published the genome sequence for Lactobacillus crispatus JCM5810. We also demonstrated that vaccination of chickens with the CadF, FlaA, and FlpA peptides reduced the number of C. jejuni in the ceca of chickens compared to the control group (non-vaccinated C. jejuni-challenged group). A trifecta peptide containing CadF-FlaA-FlpA was determined to result in the greatest degree of protection. These findings indicated that it is possible to reduce the total number of C. jejuni in the intestines of poultry by vaccination with C. jejuni surface-exposed colonization proteins (SECPs). Other work revealed that the passive immunization with hyperimmunized egg yolk powder (HEYP) resulted in no differences in the cecal colonization of C. jejuni when compared to nontreated control chickens. This finding indicated that the treatment of chickens with HEYP, at the dose and the regimens used in the study, is not efficacious in reducing C. jejuni colonization of chickens.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Neal-McKinney JM, Lu X, Duong T, Larson CL, Call DR, et al. (2012) Production of organic acids by probiotic lactobacilli can be used to reduce pathogen load in poultry. PLoS ONE 7(9): e43928. doi:10.1371/journal.pone.0043928
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Wooten J, Liu X, Miller MJ. 2016. Draft genome sequence of Lactobacillus crispatus JCM5810, which can reduce Campylobacter jejuni colonization in chicken intestine.Genome Announc 4(2):e00255-16. doi:10.1128/genomeA.00255-16.
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Human Emerging and Re-emerging Infections: Bacterial and Mycotic Infections Gourley CR, Konkel ME. Volume II ed. Singh SK, editor. Hoboken, New Jersey: John Wiley & Sons, Inc.; 2016. Chapter 28, Campylobacter jejuni: Molecular Mechanisms and Animal Models of Colonization and Disease; p.535-554.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Neal-McKinney JM, Samuelson DR, Eucker TP, Nissen MS, Crespo R, et al. (2014) Reducing Campylobacter jejuni Colonization of Poultry via Vaccination. PLoS ONE 9(12):
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Paul NC, Al-Adwani S, Crespo R, Shah DH. Poult Sci. Evaluation of passive immunotherapeutic efficacy of hyperimmunized egg yolk powder against intestinal colonization of Campylobacter jejuni in chickens. 2014 Nov;93(11):2779-87. doi: 10.3382/ps.2014-04234. Epub 2014 Sep 11.PMID:25214556
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Salma R. Al-Adwani, Rocio Crespo, and Devendra H. Shah. Production and evaluation of chicken egg-yolk-derived antibodies against Campylobacter jejuni colonization-associated proteins. Foodborne Pathogens and Disease. June 2013, 10(7): 624-631. doi:10.1089/fpd.2012.1313.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Larson CL, Samuelson DR, Eucker TP, O'Loughlin JL, Konkel ME. The fibronectin-binding motif within FlpA facilitates Campylobacter jejuni adherence to host cell and activation of host cell signaling. Emerg Microbes Infect. 2013 Oct;2(10):e65. doi: 10.1038/emi.2013.65. Epub 2013 Oct 9. PMID:26038437
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Progress 09/01/12 to 08/31/13
Outputs
Target Audience: The target audience includes scientists working in industry and academia. We have published two articles in a peer-reviewed journals and given oral presentations to interested parties in academia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Dr. Konkel has been working in collaboration with Drs. Jeremy Lessmann, Mike Miller, and Devendra Shah. Our work includes contributions from undergraduate students (Mayumi Holly, Ross Overacker), graduate students (Salma R. Al-Adwani, Tyson P Eucker, and Derrick R Samuelson), post-doctoral fellows (Chris Gourley, Charles L. Larson, Jason Neal-McKinney, and Jason L O’Loughlin), and faculty investigators (Rocio Crespo, Michael Konkel, Mike Miller, Devendra Shah). How have the results been disseminated to communities of interest? We have published two articles in a peer-reviewed journals and given oral presentations to interested parties. What do you plan to do during the next reporting period to accomplish the goals? The goal of future work is to: 1) compare the efficacy of vaccination with multiple purified C. jejuni CAPs in reducing the load of C. jejuni in the digestive tract of chickens; and 2) generate Lactobacillus recombinant strains that express a variety of C. jejuni protein fragments. Our central hypothesis is that reducing the total number of C. jejuni in the intestines of poultry will reduce the risk of humans contracting campylobacteriosis.
Impacts
What was accomplished under these goals?
Campylobacter jejuni is responsible for a significant proportion of human morbidity and mortality in the United States and worldwide. Most cases of campylobacteriosis result from consumption of foods contaminated with chicken products. The identification of novel and existing approaches that can be used either in isolation or in combination will provide enhanced protection of chickens from C. jejuni colonization, thus providing a safer food product for consumers. The objective of Aim 1 is to identify and characterize a Lactobacillus species with probiotic activity. We reported previously that L. acidophilus NCFM, L. crispatus JCM 5810, L. gallinarum ATCC 33199 and L. helveticus CNRZ32 inhibited the growth of C. jejuni in vitro, likely through the production of lactic acid (Neal-McKinney et al., 2012, PLoS One. 2012;7(9):e43928). We have performed additional experiments this past year to further define the mechanism of the probiotic activity. The objective of Aim 2 is to evaluate the efficacy of short-term oral administration of a ‘crude’ lyophilized egg-yolk powder (LEYP) containing anti-C. jejuni CAP-specific IgY to significantly reduce or eliminate C. jejuni loads in the intestines of broiler chickens. We proposed that anti-C. jejuni CAP specific antibodies against nine C. jejuni CAPs namely: CadF, PorA, FlpA, JlpA, FlaA, Cj1349c, FlaB, and CmeC will be produced and tested for their efficacy in reducing C. jejuni loads in a chicken model. Recombinant C. jejuni CAPs were expressed in Escherichia coli and were purified by affinity chromatography. Specific-pathogen-free laying hens were hyperimmunized with each recombinant CAP to induce production of α-CAP-specific IgY. Egg yolks were collected from hens and were lyophilized to obtain egg-yolk powder (EYP) with or without α-C. jejuni CAP-specific IgY. IgY was purified from EYP, and the antibody response in serum and egg yolk was tested by indirect enzyme-linked immunosorbent assay. The α-C. jejuni CAP-specific IgY levels were significantly (P < 0.05) higher in both serum and EYP obtained from immunized hens as compared with the nonimmunized hens. Each α-C. jejuni CAP-specific IgY was found to react against the C. jejuni cells and recombinant CAPs, as detected by immunofluorescence microscopy and immunoblot assays, respectively. We also showed that α-CadF, α-MOMP, and α-CmeC IgY significantly reduced the adherence of C. jejuni to chicken hepatocellular carcinoma (LMH) cells. These findings suggest that α-C. jejuni CAP-specific IgY may be useful as a passive immunotherapeutic to reduce C. jejuni colonization in chickens. The goal of Aim 3 is to determine the efficacy of a Lactobacillus vaccine strain in reducing and preventing C. jejuni colonization. We hypothesize that a Lactobacillus strain displaying one or more peptide fragments of the C. jejuni CadF, FlpA, and FlaA proteins can be used as an oral vaccine to reduce C. jejuni colonization of chickens. Experiments are ongoing to generate Lactobacillus recombinant (vaccine) strains. In addition, we have published a paper that further defines the C. jejuni adhesin protein called FlpA. This protein is an excellent vaccine candidate. (Larson et al., 2013, Emerg. Microbes Infect. 2, e65, 2013).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Al-Adwani, S.R., Crespo, R., and Shah, D.H. 2013. Production and evaluation of chicken egg-yolk-derived antibodies against Campylobacter jejuni colonization-associated proteins. Foodborne Pathog Dis. 2013 Jul;10(7):624-31. doi: 10.1089/fpd.2012.1313.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Larson, C.L., Eucker, T.P., Samuelson, D., O'Loughlin, J.L., and M.E. Konkel. Larson, C.L., Eucker, T.P., Samuelson, D., O'Loughlin, J.L., and M.E. Konkel. 2013. The fibronectin-binding motif within FlpA facilitates Campylobacter jejuni adherence to host cell and activation of host cell signaling. Emerg. Microbes Infect. 2, e65, 2013.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: The goal of these studies are to develop effective solutions to control Campylobacter jejuni contamination on the farm (production level), thereby reducing the contamination of poultry products at the retail level. The absence of C. jejuni in chickens has no negative impact for the bird or poultry producer. Our central hypothesis is that reducing the total number of C. jejuni in the intestines of poultry will reduce the risk of humans contracting campylobacteriosis. We have focused our efforts on Aim 1 of the proposal (Identify and characterize a Lactobacillus species with probiotic activity). We evaluated the ability of four Lactobacillus strains (Lactobacillus acidophilus NCFM, Lactobacillus crispatus JCM 5810, Lactobacillus gallinarum ATCC 33199 and Lactobacillus helveticus CNRZ32) to inhibit the growth of C. jejuni in vitro and to competitively exclude C. jejuni colonization of commercial broiler chickens in vivo. Our results have been disseminated via journal publication (PLoS One) and oral presentations (below). Presentations: Society for General Microbiology. Campylobacter jejuni exploits host cell processes. Convention Centre Dublin, Ireland: March 26-29, 2012. Campylobacter jejuni exploits host cell processes: Identification of C. jejuni vaccine targets. Intervet Inc. d/b/a Merck Animal Health, United Kingdom, March 30, 2012. Prevention of Human Disease by Vaccination of Food-Animals, AAAS Pacific Division, Emerging and Re-Emerging Infectious Diseases, Boise Convention Center, Boise, Idaho, June 24 - 27, 2012. Gilead Sciences, Washington State University, Lewis Alumni Centre presentation, Pullman, WA, June 11, 2012. School of Molecular Biosciences Fall 2012 Retreat, Prevention of Human Disease by Vaccination of Food-Animals, Coure d'Alene, ID, August 16, 2012. PARTICIPANTS: We have been working in collaboration with Drs. Mike Miller, Jeremy Lessmann, and Devendra Shah. Our paper, published in PLoS One, included contributions from two graduate students (Neal-McKinney and Larson), two post-doctoral fellows (Lu and Duong), and three faculty investigators (Call, Shah, and Konkel). Dr. M.E. Konkel directed the team. TARGET AUDIENCES: The target audience includes scientists working in industry and academia. We have published one article in a peer-reviewed, open-access journal (PLoS One) and given oral presentations to interested parties in industry and academia. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcomes/Impacts Campylobacter jejuni is responsible for a significant proportion of human morbidity and mortality in the United States and worldwide. Most cases of campylobacteriosis result from consumption of foods contaminated with undercooked chicken products. The identification of novel and existing approaches that can be used either in isolation or in combination will provide enhanced protection of chickens from C. jejuni colonization, thus providing a safer food product for consumers. We have found that L. acidophilus NCFM, L. crispatus JCM 5810, L. gallinarum ATCC 33199 and L. helveticus CNRZ32 inhibited the growth of C. jejuni in vitro, likely through the production of lactic acid. In addition, all four isolates examined resulted in a reduction in the median colonization of chickens by C. jejuni. L. crispatus produced the most lactic acid and resulted in the greatest reduction in C. jejuni colonization of chickens. We concluded that the ability of Lactobacillus to reduce C. jejuni colonization in chickens is based on its ability to produce lactic acid and control the metabolic activity of mixed bacterial cultures. This project sets the stage for using Lactobacillus as a probiotic in the industry.
Publications
- Neal-McKinney, J., Lu, X., Duong, T., Larson, C.L., Call, D.R., Shah, D.H., and M.E. Konkel. Production of organic acids by probiotic lactobacilli can be used to reduce pathogen load in poultry. PLoS One. 2012;7(9):e43928. Epub 2012 Sep 4. PMID: 22962594 http://www.ncbi.nlm.nih.gov/pubmed/22962594
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