Progress 03/01/10 to 02/28/14
Outputs
Target Audience: Poultry Scientists, Poultry Veterinarians, Extension Professionals, Poultry Farmers, Regulatory Agencies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This researchconstituted theproject of a post-doctoral associate. The project provided opportunities for traininga post-doctoral associatein molecular microbiological techniques, feeding trials with chickens, and whole genome microarray analysis. In addition, the project provided opportunities for the post-doctoral associate and project directors for presenting the research findings at conferences. How have the results been disseminated to communities of interest? The results were disseminated through peer-reviewed manuscripts in journals and presentations at the annual meeting of poultry science association. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The followings activities were completed. 1. Determined the effect of feeding thymol and carvacrol on Campylobacter persistence in broiler chickens. Two plant-derived antimicrobials, namely thymol and carvacrol were evaluated for their ability to decrease Campylobacter in chickens. A total of 450 day-old chicks were evaluated in five separate trials. In each trial, ninety birds were divided into nine groups of 10 birds each were fed with 0%, 0.25%, 0.5%, 1% or 2% thymol or carvacrol in the feed. The birds were orally infected with a five-strain mixture of C. jejuni, and samples from the cecum of each bird were collected on day 10 for Campylobacter enumeration. 2. Determined the effect of feeding eugenol and trans-cinnamaldehyde on Campylobacter persistence in broiler chickens. In three trials each containing 90 birds, day-old chicks were fed with 0%, 0.1%, 0.2%, 0.4%, or 0.8% eugenol or trans-cinnamaldehyde in the feed for 14 days. The birds were infected with C. jejuni 72 h after the start of experiment and sacrificed on day 14 to determine Campylobacter populations. 3. Determined the effect of feeding chitosan on Campylobacter persistence in broiler chickens. The ability of chitosan, a compound naturally present in crustaceans to reduce Campylobacter counts in chickens was evaluated. The birds were fed with 0%, 0.25%, 0.5%, or 1% chitosan in the feed for 14 days. The birds were infected with C. jejuni 72 h after the start of experiment and sacrificed on day 14 to determine Campylobacter populations. 4. Determined the effect of sodium octanoate to reduce Campylobacter Counts in broiler chickens Sodium octanoate, a water soluble form of caprylic acid, a natural fatty acid present in vegetable oils was evaluated for its efficacy to decrease Campylobacter populations in chickens. The birds were infected with Campylobacter as before, and given 0%, 0.175%, 0.35%, 0.7%, 1.4%, or 2.8% sodium octanoate in water for 72 h prior to sacrifice. 5. Determined the effect of feeding beta-resorcylic on Campylobacter persistence in broiler chicks The inhibitory effect of beta-resorcylic acid, a natural compound present in plants, on Campylobacter was evaluated in broiler chicks. Beta-resorcylic was fed to chicks from day of hatch until 14 days of age at 0%, 0.25%, 0.5%, or 1% level in the feed. The birds were infected with C. jejuni 72 h after the start of experiment and sacrificed on day 14 to determine Campylobacter populations. 6. Determined the effect of trans-cinnamaldehyde and eugenol on Salmonella genome In order to determine the probable genetic mechanisms behind the antimicrobial effect of trans-cinnamaldehyde and eugenol on Salmonella, we conducted a whole-genome microarray analysis of Salmonella Enteritidis treated with trans-cinnamaldehyde and eugenol. Salmonella was treated with trans-cinnamaldehyde (0.01%) or eugenol (0.04%), and RNA was extracted to study gene expression. 7. Determined the safety of plant-derived antimicrobials as feed additives in chickens by studying the hisopathological analyses of organs collected from birds fed with trans-cinnamaldehyde and eugenol. Major findings: Although carvacrol and thymol were generally effective in significantly reducing Campylobacter populations in birds, doses higher than 0.5% decreased feed intake and body weight gain birds. The 0.5% dose of chitosan in feed consistently reduced Campylobacter populations up to 3 log CFU/g in cecal contents. Feeding beta-resorcylic acid at 0.25% level to chickens had no effect on Campylobacter populations. However, in birds receiving 0.5 or 1.0% dose of beta-resorcylic acid, a 3-4 log reduction in Campylobacter counts was observed. These data suggest chitosan and beta-resoryclic acid may be effective as feed supplements to reduce enteric Campylobacter colonization in chickens. Microarray results revealed that trans-cinnamaldehyde and eugenol significantly down-regulated several Salmonella genes critical for colonization in chickens, including those required for the invasion of intestinal cells in chickens. Feeding of trans-cinnamaldehyde did not result in any abnormal changes in the liver and cecum collected from chickens, however, eugenol caused mild congestion in liver. Overall, the results indicate the potential use of several natural compounds, especially trans-cinnamaldehyde, beta-resorcylic acid and chitosan as feed additives for reducing the colonization of Salmonella and Campylobacter in chickens, thereby improving the microbiological safety of poultry meat.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Arsi, K., A.M. Donoghue, K. Venkitanarayanan, A. Kollanoor-Johny, A.C. Fanatico, P.J. Blore and D. J. Donoghue. (2014). The Efficacy of the Natural Plant Extracts, Thymol and Carvacrol Against Campylobacter Colonization in Broiler Chickens. J. Food Safety.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Anup Kollanoor-Johny, J. Frye, S. Porwollik, M. J. Darre, A. M. Donoghue, D. Donoghue, and K. Venkitanarayanan. 2012. Global gene expression analysis of Salmonella Enteritidis phage type 8 treated with subinhibitory concentrations of plant-derived compounds, trans-cinnamaldehyde or eugenol in vitro. Presented at the 101st Annual Meeting of the Poultry Science Association, Held at Athens, Georgia, July 9 to 12, 2012.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Anup Kollanoor-Johny, A. Upadhyay, S. A. Baskaran, I. Upadhyaya, S. Mooyottu, M. J. Darre, M. I. Khan, A. M. Donoghue, D. Donoghue, and K. Venkitanarayanan. 2012. Effect of therapeutic supplementation of plant compounds, trans-cinnamaldehyde and eugenol on Salmonella Enteritidis colonization in market-age broilers. Presented at the 101st Annual Meeting of the Poultry Science Association, held at Athens, Georgia, July 9-12, 2012.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Anup Kollanoor-Johny, T. E. Mattson, S. A. Baskaran, A. M. Amalaradjou, M. J. Darre, M. I. Khan, A. M. Donoghue, D. Donoghue, and K. Venkitanarayanan. 2012. Effect of food-grade carvacrol on cecal Salmonella Enteritidis colonization and cloacal shedding in 19-day-old commercial broiler chicks.
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Progress 03/01/12 to 02/27/13
Outputs
OUTPUTS: We previously reported that two GRAS-status plant-derived compounds trans-cinnamaldehyde (TC) and eugenol (EG) significantly reduced S. Enteritidis colonization in broiler chickens in vivo both prophylactically and therapeutically. In order to elucidate probable genetic mechanisms by which TC and EG reduced SE, we conducted a whole-genome microarray analysis of PT8 (one of the four isolates used for in vivo study) treated with TC and EG in vitro. Microarray included over 5,500 PCR products representing more than 99% of the ORFs of PT4 as well as four other serotypes of Salmonella. Bacteria was grown in Luria-Bertanii (LB) broth at 37C until it reached OD600 ~ 0.5. Thereafter, subinhibitory concentrations (SIC; concentrations that did not inhibit PT8 growth) of TC (0.01%) or EG (0.04%) was added to the flask and samples were drawn before (Time 0) and after the addition of molecules (n=3/plant-molecule) for RNA extraction using hot phenol-chloroform extraction method. The hybridized array slides were processed and the signals were quantified. PARTICIPANTS: Kumar Venkitanarayanan, Anup Kollanoor-Johny, Jonathan Frye, Steffen Porwollik, Michael McClelland, Annie Donoghued, Dan Donoghuee, and Michael Darre TARGET AUDIENCES: Poultry industry, Polutry scientists, Regulatory agencies PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
TC and EG down-regulated (P<0.005) PT8 genes required for expression of flagellar motility, regulation of Pathogenicity Island 1, invasion of intestinal epithelial cells, multiple transport systems and outer membrane proteins. Moreover, several metabolic and biosynthetic pathways in the pathogen were down-regulated by the plant compounds. TC and EG up-regulated expression of heat shock genes, such as dnaK, dnaJ, ibpB and ibpA in PT8 (P<0.005). Further, the results obtained from microarray were validated using a quantitative real-time PCR. This study indicated that TC and EG exert antimicrobial effects on PT8 by multiple mechanisms, including those associated with bacterial virulence, cell membrane composition and transport.
Publications
- No publications reported this period
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Progress 03/01/11 to 02/28/12
Outputs
OUTPUTS: This study investigated the therapeutic efficacy of plant compounds, trans-cinnamaldehyde (TC) and eugenol (EG) on reducing Salmonella Enteritidis (SE) in commercial, market-age broiler chickens. Eighty four (N=84) straight run, day-old, commercial broiler chicks were randomly grouped into 6 groups of 14 birds each (n=14/group): a negative control (no SE, no TC or EG), EG control (no SE, 1% EG), TC control (no SE, 0.75% TC), a positive control (SE, no TC or EG), an EG challenge group (SE, 1% EG) and a TC challenge group (SE, 0.75% TC). Before the start of each experiment, the flock was screened for any inherent Salmonella (n=12 birds/experiment). Birds were given ad-libitum access to Salmonella-free feed and water. On d 30, birds were challenged with a four-strain mixture of SE (8 log10 CFU/bird). Two birds from each group were sacrificed after 24 h (d 31) to check for colonization of SE in the cecum. Birds were given feed supplemented with TC (0.75%) or EG (1%) for 5 days before slaughter on d 42 (n=10 birds/group) for determination of SE populations in cecum and cloaca. The experiment was repeated two times. PARTICIPANTS: Venkitanarayanan, K.; Darre, M.; Khan, M.; Donoghue, D.; Donoghue, A.; Frye, J., 1. Kollanoor-Johny, A. TARGET AUDIENCES: Poultry industry, Extension agents, Polutry scientists, Regulatory agencies. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Trans-cinnamaldehyde and EG consistently reduced SE in the samples in both experiments (P<0.05). Body weights and feed consumption did not differ among the groups (P>0.05). Histological analysis revealed no abnormal changes in the cecum or liver due to supplementation of plant compounds. The results suggest that TC and EG supplemented through feed could reduce SE colonization in market-age chickens.
Publications
- Anup Kollanoor Johny, Tyler Mattson, Sangeetha Ananda Baskaran, Mary Anne Amalaradjou, Sankhiros Babapoor, Benjamin March, Satyender Valipe, Thomas Hoagland, Michael Darre, David Schreiber, Mazhar Khan, Ann Donoghue, Dan Donoghue, and K. Venkitanarayanan. 2012. Effect of Plant-Derived Compounds, Trans-Cinnamaldehyde and Eugenol in Reducing Salmonella Enteritidis Colonization in 20-Day-Old Broiler Chickens. App. Env. Microbiol. 78: 2981-2987.
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Progress 03/01/10 to 02/28/11
Outputs
OUTPUTS: Outputs As a first step for selecting optimum concentrations of the plant antimicrobials in chicken trials, the antimicrobial efficacy of four GRAS-status, plant-derived molecules namely, trans-cinnamaldehyde (TC), eugenol (EG), carvacrol (CR) and thymol (TH) against S. Enteritidis in chicken cecal contents in vitro was studied. The plant molecules were added at different concentrations (ranging from 0.1% to 1.1%) to chicken cecal contents inoculated with ~7.0 log10 CFU/ml of S. Enteritidis. The pathogen populations in the cecal contents after 15 sec, 8 h and 24 h of incubation at 40oC were determined. Duplicate samples of treatments and control were included, and the study was replicated three times. The in vivo study investigated the efficacy of feed supplemented with TC and eugenol EG for reducing S. Enteritidis colonization in 20-day-old broiler chicks. In three trials for each molecule, day-old straight run (N=75 per trial), chicks were randomly assigned to five treatment groups (n=14 per group): a negative control (no S. Enteritidis, no TC or EG), a compound control (no S. Enteritidis, TC or EG), a positive control (challenged with S. Enteritidis, no TC or EG), a low dose group (S. Enteritidis, 0.5% TC or 0.75% EG) and a high dose group (S. Enteritidis, 0.75% TC or 1% EG). Water and feed were provided ad libitum. On day 0, five birds from each trial were randomly selected and sacrificed to confirm that birds were devoid of any inherent Salmonella. On day 8, birds were inoculated with ~ 8.0 log CFU of a 4-strain mixture of nalidixic acid resistant S. Enteritidis by crop gavage. After 24 hours, two birds from each group were sacrificed and analyzed to ensure colonization of ceca with the pathogen. Six birds from each group were euthanized on days 7 and 10 after challenge, and S. Enteritidis populations in the cecum and cloaca were determined. In addition, experiments were conducted to investigate the effect of the sub-inhibitory concentrations (SIC - concentrations that do not inhibit Salmonella growth) of TC and EG on major S. Enteritidis colonization factors by cell culture and real-time quantitative PCR (RT-qPCR). PARTICIPANTS: Kumar Venkitanarayanan, University of Connecticut; Machael Darre, University of Connecticut; Mazhar Khan, University of Connecticut; Dan Donoghue, University of Arkansas, Annie Donoghure, USDA-ARS, Arkansas TARGET AUDIENCES: Poultry producers, farmers, veterinarianan ans scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results from in vitro experiments revealed that all the molecules were highly bactericidal, with the lowest concentration of TC (0.13%) reducing S. Enteritidis populations by approximately 6.0 log10 CFU/ml after 8 h and more than 8.0 log10 CFU/ml after 24 h of incubation. Trans-cinnamaldehyde at 0.35% eliminated detectable S. Enteritidis counts by 8 h of incubation. Carvacrol and EG decreased S. Enteritidis to less than 1.0 log10 CFU/ml at 0.75 and 1.12% and 0.8% and 1.2%, respectively. Although not as effective as other molecules, TH also reduced the S. Enteritidis counts. These aforementioned molecules could potentially be used to reduce S. Enteritidis in chicken ceca. In follow up chicken trials, TC at 0.5 and 0.75% and EG at 1% reduced (P < 0.05) S. Enteritidis populations in cecum (~3 log10 CFU/g) and cloaca (~2 log10 CFU/g), respectively after 10 days of infection in challenged birds. Both plant molecules did not alter (P > 0.05) the pH and cecal microflora counts. Feed intake and body weight were not significantly different for TC supplemented groups (P > 0.05). However, EG treated birds had significantly lower (P < 0.05) body weights compared to the control. TC and EG reduced motility and invasive abilities of S. Enteritidis, and down-regulated expression of virulence genes hilA, hilD, invF, flhC and motA. The results suggest that supplementation of TC through feed could be effectively used to reduce S. Enteritidis colonization in commercial broiler chickens.
Publications
- Anup Kollanoor Johny*, Michael J. Darre, Annie M. Donoghue, Dan J. Donoghue, and Kumar Venkitanarayanan. 2010. Antibacterial effect of trans-cinnamaldehyde, eugenol, carvacrol, and eugenol on Salmonella Enteritidis and Campylobacter jejuni in chicken cecal contents in vitro. J. Appl. Poultry Res. 19:237-244.
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