Source: OKLAHOMA STATE UNIVERSITY submitted to
DIETARY MODULATION OF INNATE HOST DEFENSE OF CHICKENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0225687
Grant No.
(N/A)
Project No.
OKL02811
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2011
Project End Date
Sep 30, 2016
Grant Year
(N/A)
Project Director
Zhang, GU.
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Animal Science
Non Technical Summary
The widespread use of antibiotics at sub-therapeutic levels in the animal industry has been criticized for the contamination of food products and the environment with unwanted antibiotic residues and potential emergence and dissemination of microorganisms that are resistant to these agents. To reduce antimicrobial resistance and to maintain optimal animal health without the use of conventional antibiotics, we sought to explore the potential for dietary modulation of innate immuity in the control and prevention of important foodborne pathogens using the chicken as an animal model. Such an immune-boosting approach is expected to be broadly applicable in the control and prevention of other pathogens in all other food animal species, offering great potential of enhancing animal biosecurity and production efficiency, while minimizing the use of antibiotics and emergence of drug-resistant pathogens.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023299101033%
3113299110033%
3073299109034%
Knowledge Area
302 - Nutrient Utilization in Animals; 311 - Animal Diseases; 307 - Animal Management Systems;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1090 - Immunology; 1010 - Nutrition and metabolism; 1100 - Bacteriology;
Goals / Objectives
The overall objective is to evaluate the efficacy of dietary modulation of antimicrobial peptide (AMP) synthesis in the control of foodborne pathogens in the chicken. The central hypothesis is that strategies to enhance the production of AMPs will confer on chickens the capacity to fight off infections, enhance survival, and reduce bacterial shedding. We plan to test the hypothesis and accomplish the overall objective by pursuing the following three specific objectives: 1) Search for AMP-inducing dietary factors, 2) Investigate the efficacy of selected dietary factors in protecting animals and reducing bacterial shedding, and 3) Study the molecular mechanisms of AMP induction by selected dietary factors. It is our goal to develop a truly innovative, cost-effective strategy to augment animal immunity and disease resistance with no reliance on conventional antibiotics, which cause an increasing public health concern over the development of antibiotic resistance.
Project Methods
A range of vitamins, essential amino acids, and trace minerals, and fatty acids will be tested individually or in combination in chicken cells. The dietary factor(s) that gives strong antimcrobial peptide (AMP) gene induction in vitro will be confirmed in chickens by a series of dose-response and time-course experiments. The top three dietary factors that are among the most potent in inducing AMP gene expression will be fed to chickens following experimental infections with S. enteritidis. Animal survival and bacterial titer in the cecum and feces will be examined. The top 1-2 dietary factors that are most potent in inducing AMP gene expression in vitro and reducing bacterial shedding in vivo will be selected for further investigation of the molecular mechanisms. Promoter analysis of inducible AMP genes will be conducted. Signaling pathways involved in dietary factor-mediated AMP induction will be screened and studied for their significance. Next-generation sequencing will also be employed to profile the transcriptional response of the chicken cells to selected dietary factors.

Progress 10/01/11 to 09/30/16

Outputs
Target Audience:Livestock producers, veterinary doctors, veterinary immunologists, and animal nutritionists are all interested in the outome of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The students had the opportunities to participate in professional meetings on the university campus and at the national level as well. How have the results been disseminated to communities of interest?The research outcomes have been disseminated to the scientific community by participating in various professional conferences such as Conference on Research Workers in Animal Diseases and Arkansas Nutrtional Conference. Results have also been published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In collaboration with a Chinese group, we examined whether 1,25-dihydroxy vitamin D3 (1,25D3) could stimulate avian β-defensin (AvBD) gene expression in chickens. We used chicken embryonic intestinal epithelial cells and peripheral blood mononuclear cells to study the effect of 1,25D3 on the expression of AvBDs. We observed that 1,25D3 is able to up-regulate the expression of several AvBDs in both chicken cell types. Furthermore, AvBDs were not directly induced by 1,25D3, as cycloheximide completely blocked 1,25D3-induced expression of AvBDs. Our observations suggested that 1,25D3 is capable of inducing AvBD gene expression and is a potential antibiotic alternative for disease control and prevention in chickens.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Sunkara, L.T., A.R. Curtis, and G. Zhang. 2015. Biology, Expression, and Regulation of Host Defense Peptides: A Minireview. Advances in Animal and Veterinary Sciences 3: 9-20.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Lyu, W., A.R. Curtis, L.T. Sunkara, and G. Zhang. 2015. Transcriptional regulation of antimicrobial host defense peptides. Current Protein and Peptide Science 16: 672-679.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Robinson, K., Z. Deng, Y. Hou, and G. Zhang. 2015. Regulation of intestinal barrier function by host defense peptides. Frontiers in Veterinary Science 2: 57.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang, L., L. Lu, S. Li, G. Zhang, L. Ouyang, K. Robinson, Y. Tang, Q. Zhu, D. Li, Y. Hu, and Y. Liu. 2016. 1,25-Dihydroxyvitamin-D3 induces avian ?-defensin gene expression in chickens. PLoS ONE 11: e0154546.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Livestock producers, veterinary doctors, veterinary immunologists, and animal nutritionists will be interested in the outcomes of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The research outcomes have been disseminated to the scientific community by participating in various professional conferences such as Conference on Research Workers in Animal Diseases and Arkansas Nutrtional Conference. Results have also been published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?The possible synergy among otherdietary compounds will be evaluated in chicken cells and live animals. Additionally, we will continue to search for additional small-molecule compounds with the ability to induce host defense peptide gene expression and disease resistance in chickens

Impacts
What was accomplished under these goals? Host defense peptides (HDP) have both microbicidal and immunomodulatory properties. Specific induction of endogenous HDP synthesis has emerged as a novel approach to antimicrobial therapy. Cyclic adenosine monophosphate (cAMP) and butyrate have been implicated in HDP induction in humans. However, the role of cAMP signaling and the possible interactions between cAMP and butyrate in regulating HDP expression in other species remain unknown. Here we report that activation of cAMP signaling induces HDP gene expression in chickens as exemplified by β-defensin 9 (AvBD9). We further showed that, albeit being weak inducers, cAMP agonists synergize strongly with butyrate or butyrate analogs in AvBD9 induction in macrophages and primary jejunal explants. Additionally, oral supplementation of forskolin, an adenylyl cyclase agonist in the form of a Coleus forskohlii extract, was found to induce AvBD9 expression in the crop of chickens. Furthermore, feeding with both forskolin and butyrate showed an obvious synergy in triggering AvBD9 expression in the crop and jejunum of chickens. Surprisingly, inhibition of the MEK-ERK mitogen-activated protein kinase (MAPK) pathway augmented the butyrate-FSK synergy, whereas blocking JNK or p38 MAPK pathway significantly diminished AvBD9 induction in chicken macrophages and jejunal explants in response to butyrate and FSK individually or in combination. Collectively, these results suggest the potential for concomitant use of butyrate and cAMP signaling activators in enhancing HDP expression, innate immunity, and disease resistance in both animals and humans.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu, H., J. Zhang, S. Zhang, F. Yang, P. Thacker, G. Zhang, S. Qiao, and X. Ma. 2014. Oral administration of Lactobacillus fermentum I5007 favors intestinal development and alters the intestinal microbiota in formula-fed piglets. Journal of Agricultural and Food Chemistry 62: 860-866.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Bommineni, Y.R., G.H. Pham, L.T. Sunkara, M. Achanta, and G. Zhang. 2014. Immune regulatory activities of fowlicidin-1, a cathelicidin host defense peptide. Molecular Immunology 59: 55-63.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhang, G., and L.T. Sunkara. 2014. Avian host defense peptides: from biology to therapeutic potential. Pharmaceuticals 7: 220-247.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Sunkara, L.T., X. Zeng, W. Jiang, A.R. Curtis, and G. Zhang. 2014. Cyclic AMP synergizes with butyrate in promoting beta-defensin 9 gene expression in chickens. Molecular Immunology 57: 171-180.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Livestock producers, veterinary doctors, veterinary immunologists, and animal nutritionists will be interested in the outcomes of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? The research outcomes have been disseminated to the scientific community by participating in various professional conferences such as Conference on Research Workers in AnimalDiseases andArkansas Nutrtional Conference.Results have also been published inpeer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? The possible synergy among different dietary compounds will be evaluated in chicken cells and live animals. Additionally, we will continue to search for additional small-molecule compounds with the ability to induce host defense peptide gene expression in chickens.

Impacts
What was accomplished under these goals? Dietary modulation of the synthesis of endogenous host defense peptides (HDPs) represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections. However, HDP regulation by dietary compounds such as butyrate is species-dependent. To examine whether butyrate could induce HDP expression in pigs, we evaluated the expressions of a panel of porcine HDPs in IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and primary monocytes in response to sodium butyrate treatment by real-time PCR. We revealed that butyrate is a potent inducer of multiple, but not all, HDP genes. Porcine β-defensin 2 (pBD2), pBD3, epididymis protein 2 splicing variant C (pEP2C), and protegrins were induced markedly in response to butyrate, whereas pBD1 expression remained largely unaltered in any cell type. Additionally, a comparison of the HDP-inducing efficacy among saturated free fatty acids of different aliphatic chain lengths revealed that fatty acids containing 3-8 carbons showed an obvious induction of HDP expression in IPEC-J2 cells, with butyrate being the most potent and long-chain fatty acids having only a marginal effect. We further investigated a panel of butyrate analogs for their efficacy in HDP induction, and found glyceryl tributyrate, benzyl butyrate, and 4-phenylbutyrate to be comparable with butyrate. Identification of butyrate and several analogs with a strong capacity to induce HDP gene expression in pigs provides attractive candidates for further evaluation of their potential as novel alternatives to antibiotics in augmenting innate immunity and disease resistance of pigs.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Patil, A., A.J. Ouellette, W. Lu, and G. Zhang*. 2013. Rattusin, an intestinal ?-defensin-related peptide in rats with a unique cysteine spacing pattern and salt-insensitive antibacterial activities. Antimicrobial Agents and Chemotherapy 57: 1823-1831.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Wang, A.N., C.J. Cai, X.F. Zeng, F.R. Zhang, G. Zhang, P.A. Thacker, J.J. Wang, and S.Y. Qiao. 2013. Dietary supplementation with Lactobacillus fermentum I5007 improves the anti-oxidative activity of weanling piglets challenged with diquat. Journal of Applied Microbiology 114: 1582-1591.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Zeng, X., L.T. Sunkara, W. Jiang, M. Bible, S. Carter, X. Ma, S. Qiao, and G. Zhang. 2013. Induction of porcine host defense peptide gene expression by short-chain fatty acids and their analogs. PLoS One 8: e72922.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Jiang, W., L.T. Sunkara, X. Zeng, D. Zhuo, S.M. Myers, and G. Zhang. 2013. Differential regulation of human cathelicidin LL-37 by free fatty acids and their analogs. Peptides 50: 129-138.


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Livestock producers, veterinary doctors, veterinary immunologists, and animal nutritionists will be interested in the outcomes of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? The research results have been presented in professional meetings and published in peer-reviewed articles. What do you plan to do during the next reporting period to accomplish the goals? We will plan on testing additional compounds and their combinations for their role in HDP induction and disease resistance in chickens.

Impacts
What was accomplished under these goals? We have found that induction of chicken host defense peptides (HDPs) is largely in an inverse correlation with the aliphatic hydrocarbon chain length of free fatty acids in chicken HD11 macrophages and primary monocytes, with short-chain fatty acids (SCFAs) being the most potent, medium-chain fatty acids moderate and long-chain fatty acids marginal. Additionally, three SCFAs, namely acetate, propionate, and butyrate, exerted a strong synergy in augmenting HDP gene expression in chicken cells. Consistently, supplementation of chickens with a combination of three SCFAs in water resulted in a further reduction of Salmonella enteritidis in the cecum as compared to feeding of individual SCFAs. More importantly, free fatty acids enhanced HDP gene expression without triggering proinflammatory interleukin-1β production. Taken together, oral supplementation of SCFAs is capable of boosting host immunity and disease resistance, with potential for infectious disease control and prevention in animal agriculture without relying on antibiotics.

Publications

  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Ma, X., P. Fan, L. Li, S. Qiao, G. Zhang, and D. Li. 2012. Butyrate promotes the recovery of intestinal wound -healing through its positive effect on the tight junctions. Journal of Animal Science 90: 266-268.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Sunkara, L.T., W. Jiang, and G. Zhang. 2012. Modulation of chicken antimicrobial host defense peptide gene expression by free fatty acids. PLoS One 7: e49558.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Zhang, G., L.T. Sunkara, X. Zeng, W. Jiang, and A. Curtis. 2012. Development of Immune Boosting Dietary Supplements as Alternatives to Antibiotics. International Symposium on Alternatives to Antibiotics, Sep 25-28, 2012. Paris, France.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Jiang, W., L.T. Sunkara, and G. Zhang. 2013. Differential regulation of human cathelicidin LL-37 by free fatty acids and their analogs. 24th Annual Research Symposium, OSU, Feb 20-22, 2013. Stillwater, OK.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Sunkara L.T., W. Jiang, and G. Zhang. 2013. Role of histone acetylation, cAMP signaling, and mitogen-activated protein kinases in butyrate-induced host defense peptide gene expression in chicken HD11 macrophage cells. Annual Meeting of the American Association of Immunologists, Abstract #P1283, May 3-7, 2013, Honolulu, HI.


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: After screening of a range of dietary factors including fatty acids, vitamins, essential amino acids, and trace minerals, we found that short-chain fatty acids (SCFAs) are among the most potent agents in inducing HDP gene expression in different chicken cell lines and and in live chickens as well. Furthermore, HDP induction is largely in an inverse correlation with the aliphatic hydrocarbon chain length of free fatty acids, with SCFAs being the most potent, medium-chain fatty acids moderate, and long-chain fatty acids marginal. Additionally, three SCFAs, namely acetate, propionate, and butyrate, exerted a strong synergy in augmenting HDP gene expression in chicken cells. Consistently, supplementation of chickens with a combination of three SCFAs resulted in a further reduction of Salmonella enteritidis in the cecum as compared to feeding of individual SCFAs. More importantly, free fatty acids enhanced HDP gene expression without triggering proinflammatory interleukin-1β production. PARTICIPANTS: Dr. Guolong Zhang managed and coordinated the entire project and participated in the in vitro and in vivo experiments. Dr. Jerry Ritchey at Oklahoma State University helped with pathology of the chicken infection studies. Dr. Hyun S. Lillehoj at USDA-ARS provided the chicken HD11 macrophage cell line for in vitro analysis. Dr. Susan Lamont at Iowa State University provided Salmonella enteritidis type 13a for chicken infection studies. TARGET AUDIENCES: Livestock producers, immunologists, and nutritionists will all benefit from the advances of this research. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Oral supplementation of SCFAs alone or in combination with other dietary supplements is capable of boosting host immunity and disease resistance, with potential to be developed as a promising antibiotic-alternative feeding strategy in disease control and prevention for all food animal species. The research outcomes may offer great potential of enhancing animal biosecurity and production efficiency, while minimizing the use of antibiotics and emergence of drug-resistant pathogens.

Publications

  • Sunkara, L.T., W. Jiang, M. Achanta, and G. Zhang. 2011. Modulation of Antimicrobial Host Defense Peptide Gene Expression by Free Fatty Acids. Proceedings of the 92nd Annual CRWAD Conference, Abst. #65P, Chicago, IL.
  • Lynch, S., L.T. Sunkara, and G. Zhang. 2012. Augmentation of host defense peptide gene expression and disease resistance of chickens by intestinal microbial metabolites. 7th Annual Research Symposium of Summer Student Research Training Program, Center for Veterinary Health Sciences, OSU, Stillwater, OK.
  • Achanta, M., L.T. Sunkara, D. Gan, Y.R. Bommineni, W. Jiang, and G. Zhang. 2012. Tissue expression and developmental regulation of chicken cathelicidin antimicrobial peptides. Journal of Animal Science and Biotechnology 3: 15.
  • Sunkara, L.T., and G. Zhang. 2011. Short-Chain Fatty Acids Enhance Disease Resistance of Chickens by Inducing Antimicrobial Host Defense Peptide Gene Expression. Annual BMBGSA Graduate Research Symposium, OSU, Stillwater, OK.
  • Zhang, G., L.T. Sunkara, X. Zeng, W. Jiang, and A. Curtis. 2012. Development of Immune Boosting Dietary Supplements as Alternatives to Antibiotics. International Symposium on Alternatives to Antibiotics, Paris, France. Sep 25-28, 2012.