Progress 01/15/24 to 01/14/25
Outputs
Target Audience: The target audience reached during this reporting period were academic scholars and food industry professionals. The primary means of communication was through academic conferences, posters, presentations, listserv and individual emails. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? During the reporting period, the PI and co-PIs trained graduate and undergraduate students in conducting research in poultry microbiology and safety. Specifically, the student learned to prepare phytochemical nanoemulsions followed by their characterization, culture of microorganisms in a BSL-2 setting, conducting in-vivo poultry trials, conducting inactivation studies on poultry products, processing samples for microbial enumeration, recording and statistically analyzing microbial data. In addition, students learnt microbiome analysis, bioinformatics analysis for microbiome study, and volatile fatty acid analysis using Mass-spectrometry. How have the results been disseminated to communities of interest? Results have been disseminated to communities of interest through conference presentations (poster and oral presentations), newsletters, listserv and emails. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, we plan to continue our work on the research objectives as per our time-line plan. In addition, we aim to publish the work described in this report to peer-reviewed journals.
Impacts
What was accomplished under these goals?
Specific accomplishments and progress made during the reporting period (2024-2025) is described below. Project 1:Effect of Eugenol nanoemulsion in inactivating Salmonella Enteritidis survival on broiler chicken skin during refrigerated storage. Introduction/Background: Contamination of broiler carcasses with Salmonella Enteritidis (SE) during slaughter and subsequent processing is a persistent challenge endured by the U.S. poultry industry. The pathogen colonizes the ceca in high numbers leading to poultry product contamination during processing and subsequent foodborne human infections. Despite several traditional postharvest interventions, the occurrence of SE in broiler carcasses and products suggest the current interventions are not completely effective, and hence, there is a need to innovate novel strategies to control SE contamination on poultry products. Eugenol (EG; phytocompound from clove) has been shown to exert anti-SE efficacy, however, the low water dispersibility of EG hampers its application as a carcass wash treatment. This study investigated the anti-Salmonella efficacy of Eugenol in its nanoemulsion form (EGNE) as dip treatments simulating the chill tank application during poultry processing to inactivate S. Enteritidis on broiler chicken skin. Methods: EGNE was formulated with Gum Arabic and lecithin (GAL) as emulsifiers. Chicken skins were spot-inoculated with a 5-strain cocktail of SE (107 CFU/sample), followed by dipping in water (control) or water containing Peracetic acid (PAA) 0.02%, GAL 0.5%, EG, or EGNE at 1.25% for 15 min at 4oC. Post treatment, skin samples were subjected to refrigerated storage for 14 days. Samples were enumerated for surviving SE on chicken skin and in wash water on days 0, 1, 3, 7 and 14. All experiments had triplicate samples, repeated thrice, and analyzed using two-way ANOVA (p<0.05). Results: EGNE had a particle size of ~86 nm, poly dispersity index of <0.3 and zeta-potential of ~-32.3 mV. In baseline (SE inoculated skin, not washed), ~6.79 log CFU/sample SE were recovered on day 0. SE counts decreased to ~5.23 log CFU/sample by day 14 of storage (p<0.05). Immersing SE inoculated skins in water for 15 min did not reduce SE on skin as compared to baseline and ~6.68 log CFU/sample were recovered on day 0. By day 14 of storage, ~5.69 log CFU/sample of SE were recovered on control samples (p<0.05). Washing with water containing GAL or PAA did not reduce SE on skin during storage as compared to control (p>0.05). Washing with EGNE 1.25% for 15 min was more effective than PAA for all storage times, and more effective than EG on day 1, 3, 7 and 14 (p<0.05). In comparison to control, EGNE effectively reduced SE on skin by ~1.32 log CFU as early as day 0, and improved in efficacy by day 14, reducing the pathogen by ~2.76 log CFU (p<0.05). EGNE and EG reduced SE in wash water to below detection limit (p<0.05). Conclusions: Results suggest that EGNE could potentially be used as a natural antimicrobial wash to reduce SE on poultry products. Project 2: Effect of supplementingEugenol nanoemulsion in chicken drinking water onSalmonella Enteritidis colonization in broiler chickens, effect on gut microbiome, volatile fatty acid profile and production parameters. Introduction/Background: Poultry serve as a reservoir host for Salmonella Enteritidis (SE), a major poultry-associated foodborne pathogen, and significant contributor to foodborne illnesses globally and in the United States. Consumption of contaminated poultry products is a major source of SE infection in humans. The pathogen colonizes the ceca of broiler chickens in high numbers leading to product contamination during slaughter. Currently, pre-harvest strategies are implemented on farm with minimal antimicrobial success. Eugenol (EG), a Generally Recognized as Safe status compound obtained from clove has been extensively researched for its anti-Salmonella efficacy, however, the low water solubility of EG thwarts its application as a potential drinking water supplement for broiler chickens. In this study, the efficacy of Eugenol oil in its nanoemulsion form as an in-water supplement in reducing SE colonization in broiler chickens was investigated. Methods: Eugenol nanoemulsion (EGNE) was formulated by EG oil, Deionized water (DI water), Gum Arabic and Lecithin (GAL) as emulsifying agents, followed by subjecting the mixture to sonication. A total of 160 Day-old Cornish cross broiler chickens were procured and randomly allotted to 4 groups (10 birds/treatment/timepoint; n=2). Groups included control (fresh tap water), emulsifier control (GAL), EG or EGNE 0.03%, respectively. Broiler chickens were acclimated for 6 days, and on day 7, GAL, EG, or EGNE 0.03% was supplemented in chicken drinking water till day 28. On day 14, birds were inoculated by oral gavage with a 4-strain cocktail of SE (S. Enteritidis strain 12, 21, 28, 31; ~9 log CFU/bird). Necropsies were performed on day 21 and 28. Cecal content was collected and enumerated for SE. Weekly body weight gain, feed and water intake were measured. Two independent trials were conducted. Data was analyzed using one-way ANOVA (p<0.05). Results:EGNE had a particle size of ~89 nm, PDI of <0.3 and zeta-potential of ~ -34.45 mV. For SE inoculated birds supplemented fresh tap water (control), ~3.70 and 4.09 log CFU/g SE were recovered in cecal content on day 21 respectively, for Trial 1 and Trial 2. On day 28, ~2.00 and 2.58 log CFU/g SE were recovered in cecal content respectively, for Trial 1 and Trial 2. Emulsifiers (GAL) and EG oil dose 0.03% did not reduce SE colonization in ceca on day 21 and 28 as compared to control (p>0.05). In water supplementation of EGNE 0.03% reduced SE colonization on day 21 by ~1.29 and 1.63 log CFU/g respectively for Trial 1 and 2 (p<0.05). On day 28, EGNE 0.03% supplementation reduced SE colonization by ~1.38 and 1.65 log CFU/g, respectively, as compared to control (p<0.05). No significant difference in body weight gain, feed, and water consumption or FCR were observed in any treatments as compared to control. Studies investigating the effect of phytochemical treatments on chicken cecal microbiome, and volatile fatty acid profile is currently underway. Conclusions:Supplementing EGNE in drinking water could potentially be used to control SE colonization in broiler chickens.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Allen, J., Balasubramanian, B., De Almeida, A.L., Battles, O., Connors, M., Upadhyaya, I., Upadhyay, A. Eugenol nanoemulsion in chicken drinking water reduces Salmonella Enteritidis colonization in broiler chickens. Conference of Research Workers in Animal Diseases (CRWAD), Chicago, IL, January 18-21, 2025.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2025
Citation:
Allen, J., Luo, Y., Upadhyaya, I., Upadhyay, A. Effect of Eugenol nanoemulsion in inactivating Salmonella Enteritidis survival on broiler chicken skin during refrigerated storage. Poultry Science Association Annual Meeting, Ohio, July 14-17, 2025.
- Type:
Other
Status:
Published
Year Published:
2024
Citation:
Allen, J., and Upadhyay, A. Investigating the potential of eugenol nanoemulsion in reducing Salmonella Enteritidis colonization in broiler chickens and survival on poultry products. Ph.D. Dissertation Proposal, Department of Animal Science, University of Connecticut, November 15, 2024.
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Progress 01/15/23 to 01/14/24
Outputs
Target Audience:The target audience reached during this reporting period were academic scholars and food industry professionals. The primary means of communication was through academic conferences, posters, presentations, listserv and individual emails. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the reporting period, the PI and co-PIs trained graduate and undergraduate students in conducting research in poultry microbiology and safety. Specifically, the student learned to prepare phytochemical nanoemulsions followed by their characterization, culture of microorganisms in a BSL-2 setting, conducting in-vivo poultry trials, conducting inactivation studies on poultry products, processing samples for microbial enumeration, recording and statistically analyzing microbial data. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through conference presentations (poster and oral presentations), newsletters, listserv and emails. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan to continue our work on the research objectives as per our time-line plan. In addition, we aim to publish the work described in this report to peer-reviewed journals.
Impacts
What was accomplished under these goals?
Specific accomplishments and progress made during the reporting period is described below. Project 1: Investigating the efficacy of Eugenol nanoemulsion in controlling Salmonella Enteritidis on poultry products. Salmonella Enteritidis (SE) is a major foodborne pathogen in the United States responsible for ~260,000 infections annually. Consumption of contaminated poultry products is a major source of SE infection as chickens act as reservoir host for SE wherein the pathogen colonizes the ceca leading to product contamination during slaughter. To reduce Salmonella colonization in live birds and their dissemination on poultry products, pre-harvest interventions are employed with limited antimicrobial effect. Therefore, this highlights the need to develop novel post-harvest interventions to reduce SE survival on poultry products. In this regard, Eugenol (EG) is a Generally Recognized as Safe status plant compound obtained from the buds of cloves. EG has been extensively researched for its anti-Salmonella efficacy, however, the low water solubility of EG thwarts its application as a potential carcass wash treatment. This study investigated the efficacy of Eugenol oil in its nanoemulsion form (EGNE) as dip treatments in inactivating SE on broiler chicken skin. EGNE was prepared with Gum Arabic and lecithin (GAL) as emulsifiers using ultrasonication, a high energy method. Chicken skins were spot-inoculated with a 5-strain cocktail of SE (107 CFU/sample), followed by dipping in sterile DI water (control) or water containing Peracetic acid (PAA) 0.02%, GAL 0.5%, EG, or EGNE at 0.3, 0.6, 1.25% for 15, 30, 240 or 480 min at 4oC. The efficacy of the highest dose-time combination of EGNE was tested in reducing SE on multiple skins (to mimic whole broiler carcasses chilled collectively in a chill tank at a poultry processing facility). Briefly, SE inoculated skins (10 samples per treatment) were collectively washed in a receptacle containing 400 mL sterile DI water (control), PAA 0.02%, GAL 0.5%, EGNE or EG 1.25% at 4? for 480 min. Post treatment, the surviving SE on chicken skin and in wash water was enumerated. Moreover, SE population was enumerated in wash water, followed by enrichment if undetectable. All experiments had triplicate samples, repeated thrice, and analyzed using one-way ANOVA (p<0.05). EGNE had a particle size of ~82 nm, PDI of <0.3 and zeta-potential of ~-36.70 mV. In baseline (SE inoculated skin, not washed), ~7.21 log CFU/sample SE were recovered. Washing with water or GAL did not reduce SE on skin (p>0.05). All nanoemulsion concentrations were effective, as early as 15 min, in reducing SE by ~0.81, 1.45, 1.74 log CFU/sample, respectively, as compared to control (p<0.05). EGNE 1.25% was more effective than corresponding EG treatment at all timepoints and reduced SE by ~3.26 log CFU/sample as compared to control by 480 min. No significant difference between EGNE 1.25% and PAA was observed at 15 or 30 min wash time (p>0.05). However, at 240 and 480 min wash time, EGNE 1.25% was more effective than PAA and reduced SE by additional 1.5 log CFU (p<0.05). For collective skin wash experiments, EGNE 1.25% reduced SE by ~2.85 to 3.64 log CFU/sample as compared to control, GAL, PAA and EG 1.25% (p<0.05). For wash water (480 min), ~ 4.88, 4.76 and 1.41 log CFU/mL SE was recovered in control, GAL, PAA treatments respectively. EGNE and EG 1.25% inactivated SE population in wash water undetectable, and tested enrichment negative as compared to control (p<0.05). EGNE could potentially be used as a natural antimicrobial wash to reduce SE on poultry products. Project 2:Effect of prophylactic supplementation of Eugenol nanoemulsion in reducing Salmonella Enteritidis colonization in broiler chickens. Salmonella Enteritidis (SE) is a major foodborne pathogen which causes ~20% of reported Salmonellosis infections in the United States. Consumption of contaminated poultry products is a major source of SE infection as chickens act as reservoir host for SE wherein the pathogen colonizes the ceca leading to product contamination during slaughter. To reduce Salmonella colonization in live birds thus reducing SE dissemination on poultry products, pre-harvest strategies on the farm are employed with varied degrees of efficacy. Generally Recognized as Safe status plant compounds such as Eugenol (EG) obtained from the buds of clove has been extensively researched for its anti-Salmonella efficacy, however, the low water solubility of EG thwarts its application as a potential in-water supplement for broiler chickens. This study investigated the efficacy of in-water supplementation of Eugenol nanoemulsion (EGNE) in reducing SE colonization in broiler chickens. EGNE was prepared by high-energy sonication using Gum Arabic and Lecithin (GAL) as emulsifiers. Thereafter, Cornish cross broiler chickens were procured and randomly allocated to 12 groups (20 birds/treatment; n=2). Groups included control (fresh tap water), emulsifier control (GAL), EG or EGNE 0.03, 0.06% for non-challenge (no SE) and challenged (SE infected), respectively. On day 7, EG or EGNE 0.03, 0.06% doses were supplemented in drinking water till day 28. On day 14, birds assigned to challenge group were inoculated by oral gavage with a 4-strain cocktail of SE (S. Enteritidis strain 12, 21, 28, 31; ~9 log CFU/bird). Necropsies were performed on day 21 and 28 where 120 birds were sacrificed on day 21, with remaining 120 birds sacrificed on day 28. Cecal content was collected per necropsy and enumerated for SE. Weekly body weight gain, feed and water intake were measured. Two independent trials were conducted, with pen as the experimental unit. Data was analyzed using one-way ANOVA (p<0.05). EGNE had a particle size of ~82 nm, PDI of <0.3 and zeta-potential of ~-36.70 mV. For SE inoculated birds supplemented fresh tap water (control), ~3.70 and 2.89 log CFU/g SE were recovered in cecal content on day 21 and 28 respectively. Emulsifiers (GAL) and EG oil doses (0.03, 0.06%) did not reduce SE colonization in ceca on day 21 and 28 as compared to control (p>0.05). In water supplementation of EGNE (0.03 and 0.06%) was not effective on day 21, however, on day 28 reduced SE colonization by ~2.34 and 1.36 log CFU/g in cecal content respectively, as compared to control (p<0.05). No reduction in body weight gain was observed in any treatments, except for EGNE 0.06% dose. Results suggest that EGNE could potentially be used to control SE colonization in broiler chickens.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
J. Allen., B. Balasubramanian., S.A. Baskaran., Y. Luo., A. Upadhyay. Application of Eugenol Nanoemulsion in controlling Salmonella Enteritidis on poultry products. Reciprocal Meat Conference, Oklahoma City, June 16-19, 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
J. Allen., B. Balasubramanian., S.A. Baskaran., Y. Luo., A. Upadhyay. Eugenol nanoemulsion: A natural antimicrobial for inactivating Salmonella Enteritidis on broiler chicken skin for improving product safety. International Association for Food Protection Annual Meeting, Long Beach, CA, July 14-17, 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Allen, J., Balasubramanian, B., Shah, T., Shah, C., Leticia De Almeida, A., Walunj, A., Upadhyay, A. Effect of prophylactic supplementation of eugenol nanoemulsion in reducing Salmonella Enteritidis colonization in broiler chickens. Poultry Science Association Annual Meeting, Louisville, KY, July 15-18, 2024.
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Progress 01/15/22 to 01/14/23
Outputs
Target Audience: The target audience reached during this reporting period includes academic scholars, students, industry professionals. The primary means of communication was listserv, conference presentations and individual emails. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training:Students trained to submit an abstract to a scientific conference, present their research and write a manuscript. How have the results been disseminated to communities of interest?The results have been disseminated via conference presentations, abstracts, and peer-reviewed manuscripts. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, I plan to work on objectives of the grant as per our time-line plan. In addition, I aim to publish the work described in this report in reputed food microbiology and safety journals.
Impacts
What was accomplished under these goals?
Response: During this reporting period, the PI and co-PIs trained the graduate students to work on the project. The trained graduate students presented the research performed at national and international conferences (please refer to product section). A description of the accomplishments is provided below. Project 1:A Multiomic Analysis of Chicken Serum Revealed the Modulation of Host Factors Due toCampylobacter jejuniColonization and In-Water Supplementation of Eugenol Nanoemulsion. Campylobacter jejuniis a foodborne pathogen that causes campylobacteriosis globally, affecting ~95 million people worldwide. MostC. jejuniinfections involve consuming and/or handling improperly cooked poultry meat. To better understand chicken host factors modulated byCampylobactercolonization, we explored a novel LCMS-based multiomic technology using three experimental groups: (1) negative control, (2) positive control, and (3) eugenol nanoemulsion (EGNE) treatment (supplemented with 0.125% EGNE in the water) of broiler chickens (n = 10 birds/group). Birds in groups two and three were challenged withC. jejunion day 7, and serum samples were collected from all groups on day 14. Using this multiomic analysis, we identified 1216 analytes (275 compounds, seven inorganics, 407 lipids, and 527 proteins). The colonization ofC. jejunisignificantly upregulated CREG1, creatinine, and 3-[2-(3-Hydroxyphenyl) ethyl]-5-methoxyphenol and downregulated sphingosine, SP d18:1, high mobility group protein B3, phosphatidylcholines (PC) P-20:0_16:0, PC 11:0_26:1, and PC 13:0_26:2. We found that 5-hydroxyindole-3-acetic acid significantly increased with the EGNE treatment when compared to the positive and negative controls. Additionally, the treatment increased several metabolites when compared to the negative controls. In conclusion, this study revealed several potential targets to controlCampylobacterin broiler chickens. Project 2:Effect of continuous exposure to Trans-cinnamaldehyde on induction of resistance in Salmonella Enteritidis. Antibiotic-resistant Salmonella has been associated with numerous outbreaks in the U.S. and is a significant concern in poultry production. Generally Recognized as Safe status phytochemical Trans-cinnamaldehyde (TC) has been extensively researched in the past two decades as a potential antibiotic alternative for the poultry industry. However, whether exposure to TC leads to resistance development in S. Enteritidis (SE) has not been explored. This study investigated the effect of continuous exposure of S. Enteritidis to sub-inhibitory concentration (SIC) of TC at 37°C for 20 days. In addition, the effect of SIC of TC on expression of antimicrobial genes critical for resistance development in SE was investigated. Poultry-derived isolates, S. Enteritidis 12 and 28 were cultured in presence or absence (control) of SIC (1/3 MIC of minimum inhibitory concentration) of TC, ampicillin (AMP), ciprofloxacin (CIP) and nalidixic acid (NA) in sterile TSB at 37°C for 24 h. After 24 h, the S. Enteritidis strains were sub-cultured by adding 50 μL of overnight growth strain to 10 mL fresh TSB, followed by incubation under the same conditions. The process was repeated for 20 days. The MIC of TC was estimated on days 0, 5, 10, 15 and 20 to determine MIC shifts against SE 12 and SE 28. On day 0 and 20, RNA was extracted from TC and AMP exposed samples of SE 12, followed by cDNA synthesis, and RT-qPCR analysis. All experiments had triplicate samples, repeated thrice, and analyzed using one-way ANOVA at p<0.05. Results revealed that subculturing with SIC of TC for 20 days did not result in MIC increase in SE 12 and 28 as compared to control (P>0.05). However, S. Enteritidis 12 and 28 exposed to SICs of AMP, CIP, and NA increased MIC by 5th day of passage (P<0.05). For SE 12 strain, the MIC increased by 5, 7 and 15-fold on 20th day when exposed to NA, AMP and CIP respectively, as compared to control (P<0.05). For SE 28 strain, the MIC increased by 3-fold on 20th day when exposed to all antibiotics as compared to control (P<0.05). Gene expression results revealed that TC downregulated antimicrobial resistance genes when exposed to the compound for 24h (day 0) as compared to control (P<0.05). By 20th day, TC downregulated genes that play a key role in DNA replication processes (gyrA and parE) as compared to AMP, in which these genes were significantly upregulated (P<0.05). In addition, AMP resulted in a 1-to-2-fold increase in the expression of acrA, mdtK as compared control (P<0.05). Results suggest that exposure to SIC concentration of TC does not induce resistance in S. Enteritidis. However, further investigation exploring the effect of extended exposures (>20 days), dose, or strain variability is warranted.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Wagle, B.R., Quach, A., Yeo, S., Assumpcao, A.L., Arsi, K., Donoghue, A.M. and Jesudhasan, P.R., 2023. A Multiomic Analysis of Chicken Serum Revealed the Modulation of Host Factors Due to Campylobacter jejuni Colonization and In-Water Supplementation of Eugenol Nanoemulsion. Animals, 13(4), p.559.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Wagle, B., Arsi, K., Assumpcao, A., Donoghue, A., Jesudhasan, P. 2022. Identification of biomarkers associated with Campylobacter colonization in broiler chickens. Poultry Science Vol. 101 (E-suppl.1).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Allen, J., Balasubramanian, B., Shah, T., Upadhyay, A. Effect of continuous exposure to Trans-cinnamaldehyde on induction of resistance in Salmonella Enteritidis. Poultry Science Annual Conference, Philadelphia, PA, July 15-18, 2023.
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The target audience reached during this reporting period were academic scholars and food industry professionals. The primary means of communication was listserv and individual emails. Changes/Problems:There have been supply-chain issues related to some of the products we need to accomplish our research goals. For example, essential supplies such as sterile petri dishes, sterile tips were back ordered to up to 6 months several times that delayed some of our progress. We are actively addressing theseissues by approaching multiple vendors to maintain a regular supply of materials and keep the experiments going. What opportunities for training and professional development has the project provided?During the reporting period, the PI and co-PIs hiredgraduate students and trained the students in conducting research in poultry microbiology and safety. Specifically, the student learned to prepare phytochemical nanoemulsions followed by their characterization, culture of microorganisms in a BSL-2 setting,conducting in-vivo poultry trials, processing samples for microbial enumeration, recording and statistically analyzing microbial data. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, I plan to work on objective 1 and 2 of the grant as per our time-line plan. In addition, I aim to publish the work described in this report in reputed food microbiology and safety journals.
Impacts
What was accomplished under these goals?
Response: During this reporting, the PI and co-PIs hired/supported graduate students to work on the project. Specific progress made is presented below. The projects described below contributed to objective 1 and 2 of the grant. Project 1: Effect of plant-derived compound eugenol on Campylobacter jejuni proteome and virulence critical for colonization in chickens. Campylobacter jejuni is an important foodborne pathogen that causes severe diarrhea in humans. Chickens act as the reservoir host for Campylobacter, wherein the pathogen colonizes the ceca leading to contaminated poultry products during slaughter. The potential of natural intervention strategies, including plant-derived antimicrobials have been investigated to reduce chicken cecal colonization of C. jejuni. However, the underlying molecular mechanisms of these strategies are still unknown. This study investigated the effect of eugenol (a generally recognized as safe status phytochemical obtained from clove oil), on the whole cell proteomic profile of C. jejuni. In addition, the effect of eugenol on factors critical for cecal colonization (bacterial motility, attachment to epithelial cells) in chickens were studied using 0.4% agar for a motility assay and cell culture analysis using chicken primary enterocytes isolated from broilers. C. jejuni (strain S-8, isolated from commercial broilers) was cultured in the presence or absence (control) of sub-inhibitory concentrations of eugenol (0.01%) for 24 h followed by SDS-PAGE based protein extraction. The effect of eugenol on expression of C. jejuni proteome was quantified using LC-MS/MS analysis followed by targeted proteomics and data analysis using Scaffold Proteomic software. The experiment was conducted three times and the samples were run in triplicates. Whole cell proteomic analysis identified more than 600 proteins in C. jejuni with many virulent proteins modulated by eugenol. The major groups of proteins that were identified contribute to physiological process (sensory systems, biological regulators, developmental processes) and virulent attributes (motility systems, adhesion, quorum sensing) in C. jejuni. Eugenol reduced the expression of major virulence proteins contributing to biological adhesion (PorA, CadF), motility system (MotA, MotB, FliA, FliD, FliF, fliL, fliY), energy taxis (IlvH, CetA, CetB), molecular transport (TatA, TatB, TolB) and Quorum sensing (LuxS) when compared to controls (P<0.05). Follow up motility and attachment assays revealed that eugenol was effective in reducing the motility (> 50% reduction) and attachment of C. jejuni to primary chicken enterocytes (P<0.05). Overall, these results delineate the prospective mechanism of action of eugenol on C. jejuni and the potential of using this phytochemical to control C. jejuni colonization in chickens. Project 2: Effect of plant-derived compound eugenol and nanoemulsion form on Campylobacter jejuni colonization in broiler chickens. Campylobacter jejuni is the leading cause of human foodborne illness globally and is strongly linked with the consumption of contaminated poultry products. Chickens are the reservoir host of C. jejuni, where the pathogen colonizes the ceca, thereby leading to contamination of carcass during slaughter. A reduction in cecal C. jejuni colonization would directly translate into reduced product contamination and risk of human infections. This study investigated the efficacy of in-water supplementation of a Generally Recognized as Safe compound, eugenol (EG) as nanoemulsion (NE) in reducing C. jejuni colonization in broiler chickens. In two separate trials, day-old chicks (Cobb 500) were weighed and randomly assigned to one of two groups (C. jejuni inoculated or not-inoculated). Within each group, chicks were supplemented with EG (normal or NE) in drinking water at 0, 0.0625, 0.125 or 0.25% for 14 days (n=10 birds/treatment/trial). Birds in the inoculated group were challenged with a 4-strain mixture of C. jejuni (~ 106 CFU/ml; 250 µl/bird) on day 7. Cecal samples were collected on day 14 for enumeration of surviving C. jejuni. In addition, the effect of EG on the cecal microbiome was investigated. In-water supplementation of 0.125% EG normal reduced C. jejuni counts by ~ 1.4 Log CFU/g in both trials. Similarly, the 0.125% EG NE reduced C. jejuni colonization by ~ 1.5 and 3.7 Log CFU/g in trials 1 and 2 respectively as compared to controls (P<0.05). No reduction in feed and water consumption or body weight gain was observed with the supplementation of EG at 0.125% or lower doses (P>0.05). Illumina MiSeq based microbiome analysis revealed that administration of EG and EGNE at tested concentrations modulated the cecal microbiome at genus level in both inoculated and not-inoculated groups (P<0.05) without affecting phyla (P>0.05). Results suggest that EGNE could potentially be used to control C. jejuni colonization in broiler chickens. Project 3: Effect of plant-derived compound trans-cinnamaldehyde and nanoemulsion form on Campylobacter jejuni colonization in broiler chickens. Campylobacter jejuni is a major foodborne pathogen that causes severe gastroenteritis in humans. Chickens act as the reservoir host for C. jejuni, wherein the pathogen colonizes the ceca thereby leading to contamination of the carcass during slaughter. Reducing C. jejuni cecal colonization could potentially reduce the risk of human infections. This study investigated the efficacy of in-water supplementation of Trans-cinnamaldehyde (TC; generally recognized as safe status compound from Cinnamon bark) nanoemulsion in reducing C. jejuni cecal colonization in 14-day-old broiler chickens. In addition, the effect of TC on colonization factors (motility, attachment to chicken enterocytes) was investigated using a motility bioassay and cell culture analysis. In two separate trials, day of hatch broiler chickens (Cobb 500; 10 birds/treatment/trial) were supplemented with TC (normal or nanoemulsion form) in drinking water at 0, 0.0625, 0.125, 0.25, 0.5, and 1% level for 14 days. On day 7, the birds were challenged with a four-strain cocktail of C. jejuni (~6 log CFU/bird) by oral gavage. On day 14, the birds were sacrificed and C. jejuni colonization in cecal contents were quantified by dilution and plating of cecal contents on Campylobacter Line agar. In addition, Ethidium monoazide based real-time PCR was employed to quantify bacterial viability in cecal contents. Data were analyzed using ANOVA with GraphPad ver. 6. Differences between the means were considered significantly different at P<0.05. Administration of TC nanoemulsion (Polydispersity index <0.3; size ~100-200 nm; zeta potential ~ -0.35 mV) in drinking water at 0.25% reduced C. jejuni colonization by ~1 or 2 logs CFU/mL in trial 1 or trial 2 as compared to respective controls (P<0.05). Follow up mechanistic analysis revealed that TC reduced pathogen motility and attachment to primary chicken enterocytes (P<0.05). No reduction in feed consumption, water intake or body weight gain was observed in 0.25% or lower concentration treatments as compared to controls (P>0.05). Results suggest that TC nanoemulsion could potentially be used to control C. jejuni colonization in broiler chickens.
Publications
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