PREHARVEST FOOD SAFETY: DETERMINING THE POTENTIAL OF SALMONELLA SEROTYPE HEIDELBERG TO SYSTEMICALLY CONTAMINATE MEAT AFTER ORAL CHALLENGE OF BROILER CHICKENS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1004609
• Project No.: MIN-16-102
• Project Start Date: Dec 22, 2014
• Project End Date: Sep 30, 2018

Project Director
Kollanoor Johny, AN, .

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Animal Science

Non Technical Summary
Foodborne bacterial infections are a major cause of human enteric diseases. Salmonella Heidelberg has been causing foodborne outbreaks involving poultry products. Chickens serve as carrier-hosts for S. Heidelberg, and fecal contamination of meat during processing could present significant food safety hazard. Although the role of surface-contaminated meats in outbreaks is understood concerns about systemic transfer of S. Heidelberg to meat has not been scientifically evaluated. This proposal seeks to determine if S. Heidelberg could contaminate meat via systemic route. If found valid, strategies to control systemic transfer of S. Heidelberg to meat could be adopted improving poultry products safety.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3299	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

salmonella heidelberg

systemic infection

meat

broilers

products safety

Goals / Objectives
Major GoalTo determine the potential of Salmonella Heidelberg to systemically contaminate meat after experimental oral challenge of broiler chickens.Purpose, service, achievement, or milestone this project will provide: Foodborne bacterial infections are a major cause of human enteric diseases in the United States. Salmonella Heidelberg has been causing serious foodborne outbreaks involving poultry products. Broiler chickens serve as carrier-hosts for S. Heidelberg, and fecal contamination of meat during processing could present significant food safety hazard. Although the role of surface-contaminated meats in outbreaks is understood, concerns about systemic transfer of S. Heidelberg to meat has not been scientifically evaluated. Purpose: This proposal seeks to determine if S. Heidelberg could contaminate meat via systemic route after oral challenge of broiler chickens. Scientific achievement/milestone: The results of the study could form scientific basis to devise on-farm strategies to control systemic transfer of S. Heidelberg to meat to improve poultry products safety. A scientific manuscript highlighting the results will be published. Educational milestone: A graduate student will be trained in the process that would ultimately result in student advancement and progress of knowledge.

Project Methods
a) S. Heidelberg inoculum preparation:The ATCC and broiler meat-associated outbreak strains will be induced for resistance to nalidixic acid (NA - 50µg/ml), separately. Each strain will be grown in 1L of tryptic soy broth (TSB) containing NA for 24 hours at 37oC with shaking. The culture will be sedimented by centrifugation, and the resultant pellet reconstituted in phosphate buffered saline (PBS; pH 7.2) and diluted appropriately for use as inoculum (Kollanoor Johny et al., 2008, 2010a, 2012a, b, c).b) Ethics Statement:The project is approved by the IACUC (1403-31368A) for experiment with the ATCC strain. A modification involving experiments with the outbreak strain is awaiting approval.c) Experimental groups and S. Heidelberg challenge:Experimental groups explainedGroups-Birds-Challenge-ReasonGroup 1- 20 birds- No (100 CFU/bird) - Negative controlGroup 2- 20 birds - Yes (101 CFU/bird) - Treatment Dose 1Group 3- 20 birds - Yes (102 CFU/bird) - Treatment Dose 2Group 4- 20 birds - Yes (103 CFU/bird) - Treatment Dose 3Group 5- 20 birds - Yes (104 CFU/bird) - Treatment Dose 4Group 6- 20 birds - Yes (105 CFU/bird) - Treatment Dose 5Group 7- 20 birds - Yes (106 CFU/bird) - Treatment Dose 6Group 8- 20 birds - Yes (107 CFU/bird) - Treatment Dose 7Group 9- 20 birds - Yes (108 CFU/bird) - Treatment Dose 8Group 10- 20 birds - Yes (109 CFU/bird) - Treatment Dose 9200 birds/experiment X 3 replicates X 2 age groups (3- and 6-weeks) X 2 bacterial strains (ATCC and outbreak) = 2400 birdsCFU - colony forming units, a measure of bacterial numberFor the 3-week trials, 200 day-old broiler chicks (Ross X Ross) will be purchased and housed at the University of Minnesota BL2 research facility. All birds will be provided access ad libitum to Salmonella-free water and feed. Ten birds from the incoming flock will be randomly selected and screened for Salmonella using Sero-Quick Group kit. Birds will be weighed, wing banded, and grouped randomly into 10 groups of 20 birds per inoculum level as given in Table 1.On day 7, all birds in the inoculated groups will receive S. Heidelberg ranging from 101 to 109 CFU/bird by crop gavage (directly to the crop) (Kollanoor-Johny et al., 2009, 2012a, b, c). The control birds (10o CFU) will receive sterile PBS without bacteria (Kollanoor Johny et al., 2009). The birds will be maintained for 21 days when they will be euthanized for sample collection. For the 6-week trials, similar protocol will be followed except that the birds will be challenged with S. Heidelberg on day 25, and maintained until the end of experiment (day 42). Separate experiments will be carried out for the ATCC and outbreak strains.d) Sample analysis and measurements:d. 1. Microbiological analysis: After 24 hours of challenge, 6 birds from each group will be euthanized to ensure colonization of the pathogen in the cecum and internal organs, liver and spleen. For this, 2-5 g of samples will be collected in PBS at 4oC during necropsy and homogenized. The homogenized mixture will be diluted ten-fold in PBS, and 0.1 ml portions plated on XLD-NA plates. The plates will be incubated at 37oC for 48 hours until characteristic colonies appear. Representative colonies from XLD-NA plates will be confirmed using the Salmonella rapid detection kit. Moreover, colonies will be confirmed as S. Heidelberg using the proprietary Taqman® PCR procedure after enrichment (Life Technologies). In addition, plasma Salmonella titer (from wing vein peripheral blood) will be determined (Nandre et al., 2011) using the Chicken IgG ELISA quantitation set (Bethyl Laboratories, TX). Similarly, on day 21 (last day of the 3-week trial) the remaining birds will be slaughtered and samples of cecum, liver, spleen, blood and meat (~5 g total from breasts, thighs and drumstick) will be collected aseptically for bacteriological analysis and PCR analysis as described above. Each muscle sample will be cut into small pieces using sterile scalpel blades to resemble a finished ground product before dilution or enrichment. Furthermore, if no colonies were detected by plating, samples will be tested for surviving cells by enrichment for 48 hours at 37oC in 100 ml selenite cysteine broth (SCB) (Fernandez et al., 2002; Filho et al., 2000) containing NA, followed by streaking on XLD-NA plates. Confirmation of colonies will be done using PCR as described above. For the 6-week experiments, similar protocol will be followed except that after 5 days of challenge, i.e. on day 30, 6 birds from each group will be euthanized to ensure colonization by the pathogen in the cecum, liver and spleen. Later, on day 42, remaining birds will be euthanized for end point analysis.d. 2. Histopathology and immunohistochemistry: Liver and muscle tissues (breast, thighs and drumsticks) from 6 birds in each group from both 3- and 6-week trials will be fixed in 10% neutral-buffered formalin for 24 hours. All tissues will be externally wiped with 100% ethanol to remove any live or attached bacteria on the surface. Tissues will be processed by standard methods to obtain 4µm sections, and stained with hematoxylin and eosin (HE). Further, the sections will be used for immunohistochemistry analysis using anti-Salmonella LPS antibody by avidin-biotin complex method in Dr. Porter's laboratory as described by Gonzalez-Escobedo et al. (2013). Salmonella LPS staining could determine if infection has proceeded into these tissues, especially in the muscles.d. 3. Growth measurements: The average feed consumption and body weights of birds will be determined for all groups in all experiments. Birds will be weighed individually at the beginning and at weekly intervals. The average feed consumption per bird will be calculated as done previously (Kollanoor Johny et al., 2012a, b, c).e. Power, Model and Data Analysis:Based on the power analysis, a total of 2400 broilers are required to complete the experiments. The broiler experiments will follow a completely randomized design factorial treatment structure. Pen will be the experimental unit and the experiments will be repeated three times. The data will be analyzed using the PROC-MIXED procedure of the statistical analysis software (version 9.3, SAS Institute Inc., Cary, NC). Differences among the least square means will be detected using Fisher's least significant different test. A P value of 0.05 will be considered statistically significant. The data on body weight and feed consumption will be analyzed similarly. For histopathology, inflammation will be scored from 0-4, with 0 being normal, 1 being focal infiltrates with no necrosis, 2 being multifocal infiltrates with necrosis, 3 being widespread inflammation with extensive necrosis, and 4 being widespread infiltrates with coalescing areas of necrosis (Gonzalez-Escobedo et al., 2013). A presence or absence of S. Heidelberg by visual analysis after H&E staining will be also determined. A Student's t test will be used to detect the differences of mean histopathology scores at P<0.05.f. Efforts:The results from this study will be published in Journal of Poultry Science that has wide range of readers, including the target population. Moreover, a graduate student will be also directly benefited from the project. The presentation made at the professional meetings will deliver scientific information to the peers, industry, government officials and others who are interested. The results will be used for procuring other extensive research/integrated funds from the USDA.g.Evaluation/Milestones:A graduate student will successfully finish and defend a research thesis under the PD's guidance. A peer-reviewed publication will be produced. A presentation based on the results will be made in a professional meeting. The results will be used for preparing an integrated USDA grant proposal determining the potential of viable strategies to control systemic transfer ofSalmonellato meat.

Progress 12/22/14 to 09/30/18

Outputs
Target Audience: Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students completed their programs successfully. Their presentations acknowledged NIFA funds. Two undergraduate students are being trained in research projects as well. All my graduate students presented at national conferences, including Poultry Science annual meeting, Institute of Food Technologists annual meeting, International Association for Food Protection annual meeting, and the Minnesota Nutrition Conference. How have the results been disseminated to communities of interest?Talks by the PI: A. Kollanoor Johny. 2018. Current update on the use of essential oils in poultry. 13th Symposium on Processing and Safety of Poultry Products. MexicanNational Association of Poultry Specialists. February 15 - 16, 2018. (International). A. Kollanoor Johny. 2018. Effects of combination of antibiotic alternatives on multidrug-resistantSalmonellaHeidelberg in commercial turkeys. 4-minute lightning talk. PSA Annual meeting. July 2018. (National) Talks by the graduate students: Divek V. T. Nair, 2018. Effect of a dairy probiotic, Propionibacterium freudenreichii, on Salmonella enterica Heidelberg colonization and internal organ dissemination in commercial turkeys. 2018 Midwest ASAS Annual Meeting. Grace Dewi. 2018. Bacitracin methylene disalicylate reduces multidrug-resistant Salmonella enterica serovar Heidelberg in the cecum of commercial broiler chickens. Poultry Science Association 107th Annual Meeting. Webinar presentation by the PI: Anup Kollanoor Johny. 2017. Essential oils: What are they and why use them with poultry? Organized by eXtension.org. November 10. What do you plan to do during the next reporting period to accomplish the goals?Thisproject is ended. A new project has been initiated.

Impacts
What was accomplished under these goals? Three research abstracts from two major research projects were benefitted from the USDA Hatch Funds that were presented at scientific meetings by the graduate students. The two scientific abstracts are given below. Effect of bacitracin methylene disalicylate against multidrug resistant Salmonella enterica serovar Heidelberg in commercial broiler chickens - 2018 Minnesota Nutrition Conference (Poster presentation). Bacitracin methylene disalicylate (BMD) is a polypeptide antibiotic approved for use in poultry to improve feed efficiency, and to prevent coccidiosis-causing protozoan, Eimeria, and necrotic enteritis-causing Gram-positive bacteria, Clostridium perfringens. Broilers are also reservoir hosts to a major Gram-negative food-borne pathogen, Salmonella Heidelberg (SH), with high potential to cause invasive infections in humans. Our objective of the current study was to determine the efficacy of BMD against multidrug-resistant SH in broiler chickens. A total of six challenge studies were conducted. In all studies, BMD supplementation significantly reduced MDR SH in the cecum compared to the Salmonella controls. An average SH reduction of 4.5- and 3.0- log10 CFU/g of cecal contents (P<0.05) was observed in the 3- and 5-week-old broiler chickens (7 days after challenge), respectively. However, no significant reduction was observed in 7-wk-old birds. The results showed that BMD was highly effective on the MDR SH in the cecum of young chickens, indicating its potential to contribute to the preharvest safety of broilers. Effect of a dairy probiotic, Propionibacterium freudenreichii, on Salmonella enterica Heidelberg colonization and internal organ dissemination in commercial turkeys. 2018 Midwest ASAS Annual Meeting. Salmonella Heidelberg is a major non-typhoidal Salmonella serotype that causes foodborne illness in humans. Turkeys could harbor the pathogen in their ceca without showing obvious clinical signs, potentially resulting in contamination of carcasses during processing. Since humans could contract salmonellosis by consuming S. Heidelberg contaminated turkey products, targeted control measures minimizing the colonization of the pathogen in turkeys are required. The current study investigated the efficacy of a dairy-origin probiotic bacterium, Propionibacterium freudenreichii (PF) against S. Heidelberg using in vitro and in vivo experiments. The in vitro results revealed that PF was effective in reducing S. Heidelberg motility, multiplication, adhesion, and invasion to avian epithelial cells (P<0.05). PF possessed a high survival rate in low pH and in the presence of bile salts. PF did not possess hemolytic activity and showed susceptibility to the common antibiotics, ensuring the safety for use in turkeys. Additionally, the cell-free extracts of PF possessed antimicrobial activity against pathogens, including S. Heidelberg (P<0.05). In the in vivo experiments, the reduction in the S. Heidelberg populations ranged from 1.0- to 2.7- log10 CFU/g of the cecum in different age groups (P<0.05). In addition, PF supplementation significantly reduced S. Heidelberg invasion of liver and spleen of turkeys (P<0.05). PF colonized in high numbers in the cecum, indicating its high adherence potential. In conclusion, PF could be used as an effective probiotic in turkeys to prevent S. Heidelberg colonization and dissemination to internal organs.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Jijo Vazhakkattu Thomas, Divek V. T. Nair, Sally Noll, Timothy J. Johnson, Carol Cardona, and Anup Kollanoor Johny. (Expected 2019). Effect of turkey-derived beneficial bacteria Lactobacillus salivarius and Lactobacillus ingluviei on a multidrug-resistant Salmonella Heidelberg strain in turkey poults. Journal of Food Protection. doi:10.4315/0362-028X.JFP-18-286
• Type: Book Chapters Status: Accepted Year Published: 2018 Citation: Divek V. T. Nair and Anup Kollanoor Johny. 2018. Salmonella in poultry meat production. In Food Safety in Poultry Meat Production, Steven Ricke, Siddhartha Thakur, and Kumar Venkitanarayanan, Editors. Springer
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Grace Dewi, D.V.T. Nair, C. Peichel, S. Manjankattil, J. Langlie, and A. Kollanoor Johny. 2018. Bacitracin methylene disalicylate reduces multidrug-resistant Salmonella enterica serovar Heidelberg in the cecum of commercial broiler chickens. Poultry Science Association 107th Annual Meeting. San Antonio, TX. Poult. Sci. 97(E-Suppl. 1): 68.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Divek V. T. Nair, Jijo Vazhakkattu Thomas, Grace Dewi, Anup Kollanoor Johny. 2018. Effect of a dairy probiotic, Propionibacterium freudenreichii, on Salmonella enterica Heidelberg colonization and internal organ dissemination in commercial turkeys. 2018 Midwest ASAS Annual Meeting.
• Type: Other Status: Other Year Published: 2018 Citation: Dewi, G. Investigating the potential of lemongrass essential oil against multidrug-resistant Salmonella Heidelberg in broiler chickens. MS thesis presentation. Department of Animal Science, University of Minnesota.
• Type: Other Status: Other Year Published: 2018 Citation: Nair, DVT. Determining the efficacy of a dairy-origin probiotic, Propionibacterium freudenreichii subspecies freudenreichii against multidrug-resistant Salmonella Heidelberg in turkeys. PhD thesis presentation. Department of Animal Science, University of Minnesota.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Poultry Industry Nutritionists, Extension Agents, Poultry Scientists Changes/Problems:We includedyoung turkeys and adult turkey hens in the project in order to avoid a potential repetition of the study in broiler chickens being conducted at the Auburn University. The PI recognized this issue during the 2017 Poultry Science Annual meeting where similar abstracts presented were noticed. The issue of MDR Salmonella Heidelberg raises concernin broilers and turkeys, as this serotype of Salmonella is an emerging problem for the entire poultry industry due to the pathogen's high infection and resistance potential in humans. Although inclusion of turkeys into the research model required additional funds for research, the PIcould get institutional support from the University of Minnesota and could successfully complete turkey studies. The study has been published recently in a high impact factor journal (IF - 4.076), Frontiers in Microbiology, with due credit given to USDA NIFA Hatch funds in the Acknowledgment section. The abstract titles from the Auburn University researchers aregiven below. Differences in recovery of Salmonella Heidelberg when chicks are inoculated on days 0 and 14 - PSA Annual Meeting 2017 Environmental influences of Salmonella Heidelberg colonization in commercial broilers - IPPE Scientific Session 2017 What opportunities for training and professional development has the project provided?I have a Ph.D.student and 2 MS students currently being trained in the projects. An MSstudent has completed his program. I am also training 6 undergraduate students. How have the results been disseminated to communities of interest?Talks by PD: Kollanoor Johny. A. 2017. Use of multiple antibiotic alternatives to control multidrug-resistant Salmonella Heidelberg in turkey poults. Research Update. Minnesota Nutrition Conference, Mankato, MN, September 21 - Research update from the University of Minnesota presented in the non-ruminant nutrition section. People in attendance included industry people, researchers, extension agents. There were around 50 people in attendance. Kollanoor Johny, A. 2017. Investigating multiple antimicrobial interventions against Salmonella Heidelberg in turkey poults. IFT Annual Meeting, Las Vegas, June 28 - Symposium talk. Invited presentation to a group of industry people, researchers, academicians, teachers. There were around 50 people in attendance. Talks by graduate students: Divek V. T. Nair, J. V. Thomas, G. Dewi, T. Johnson, S. Noll, C. Cardona & A. Kollanoor Johny. 2017. Effects of multiple alternatives-to-antibiotic interventions on multidrug-resistant Salmonella Heidelberg in turkey poults (PSA annual meeting 2017, Orlando, Florida). Grace Dewi, Divek V. T. Nair & A. Kollanoor Johny. 2017. Antibacterial effect of food-grade lemongrass essential oil on Salmonella Heidelberg in vitro (PSA annual meeting 2017, Orlando, Florida). What do you plan to do during the next reporting period to accomplish the goals? Complete a few more poultry studies and complete analysis and reporting. The doctoral and MS students will defend during this project period. Two presentations are pending by the graduate students.

Impacts
What was accomplished under these goals? A manuscript was published from this project directly in a high impact factor journal (Frontiers in Microbiology IF - 4.076). The abstract is given below. Effect of Various Inoculum Levels of Multidrug-ResistantSalmonellaenterica SerovarHeidelberg (2011 Ground Turkey Outbreak Isolate) on Cecal Colonization, Dissemination to Internal Organs, and Deposition in Skeletal Muscles of Commercial Turkeys after Experimental Oral Challenge - Published in Frontiers in Microbiology 2018 Salmonellaentericaserovar Heidelberg (S. Heidelberg) is a major foodborne pathogen colonizing poultry. The pathogen is associated with a significant number of foodborne outbreaks through contaminated poultry meat, including turkeys. In this study, we determined the effect of different levels of S. Heidelberg inoculation in young and adult turkeys on the pathogen populations in cecum, liver, spleen, thigh, breast and drumstick muscles. The results indicated that, in turkey poults, the recovery of S.Heidelberg was highest in the cecum followed by spleen, liver, thigh, drumstick, and breast. The cecal colonization, dissemination to internal organs, and tissue deposition of MDRS.Heidelberg were high in poults. The pathogen recovery from the cecum of adult turkey hens ranged from 37.5 to 62.5%. The results signify the importance of controlling MDRS. Heidelberg in turkeys at the farm level to improve the safety of turkey products. Two research abstracts from major research projects were also benefitted from the USDA Hatch funds that were presented at scientific meetings by the graduate students. Effects of multiple alternatives-to-antibiotic interventions on multidrug-resistant Salmonella Heidelberg in turkey poults - PSA Annual Meeting 2017 Salmonella enterica serovar Heidelberg (SH) is one of the common Salmonella associated with poultry and is frequently isolated from turkeys. Colonization of SH in the cecum could result in the contamination of turkey carcasses after a faulty evisceration step. Reducing SH colonization in the turkey cecum could improve the microbiological safety of turkey products. We determined the efficacy of multiple alternative-to-antibiotics (A2A) interventions [probiotics, prebiotics, and vaccination] individually or in combination against SH colonization in commercial hybrid turkey poults. Results indicated that the A2A treatments resulted in significant reduction of SH in the cecum, liver, and spleen of turkey poults individually or in combination. Whereas the Prebiotic group resulted in a consistent reduction of SH in the cecum across experiments when supplemented individually, its combination with vaccination and/or Probiotic resulted in an improved reduction of SH. The results of the study indicated that combining A2A interventions could be an effective strategy to reduce SH colonization in turkeys, thereby potentially improving the microbiological safety or products. Acknowledgment - MTRPC 2015-01 & USDA NIFA Hatch (MIN-16-101). Antibacterial effect of food-grade lemongrass essential oil on Salmonella Heidelberg in vitro - PSA Annual Meeting 2017 Salmonella Heidelberg (SH), a major serovar of Salmonella linked to multiple multistate foodborne outbreaks, is frequently isolated from poultry products. The pathogen has high colonization potential in poultry that may result in contamination of the products during processing, posing a significant hazard to consumers. Since humans may contract the infection by consuming contaminated poultry, control measures to reduce or eliminate SH in poultry are required. In this regard, essential oils (EO) or their ingredients have been investigated to control several foodborne pathogens, including Salmonella. In the current study, we evaluated the efficacy of Lemongrass essential oil (LGEO), a GRAS-status EO extracted from Cymbopogon spp. against SH in vitro. Results of the study indicated the potential use of LGEO in poultry diets to improve the microbiological safety of products. Follow up in vivo studies are necessary to validate the efficacy of LGEO in poultry (USDA NIFA Hatch # MN-16-102).

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Nair DVT, Vazhakkattu Thomas J, Noll S, Porter R Jr. and Kollanoor Johny A (2018) Effect of Various Inoculum Levels of Multidrug-Resistant Salmonella enterica Serovar Heidelberg (2011 Ground Turkey Outbreak Isolate) on Cecal Colonization, Dissemination to Internal Organs, and Deposition in Skeletal Muscles of Commercial Turkeys after Experimental Oral Challenge. Front. Microbiol. 8:2680. doi: 10.3389/fmicb.2017.0268010.3389/fmicb.2017.02328
• Type: Journal Articles Status: Published Year Published: 2017 Citation: eidelberg (2011 Ground Turkey Outbreak Isolate) on to Turkey Skin. Front. Microbiol. 8:2328. doi: 10.3389/fmicb.2017.02328
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Grace Dewi, Divek V. T. Nair & A. Kollanoor Johny. 2017. Antibacterial effect of food-grade lemongrass essential oil on Salmonella Heidelberg in vitro. Poultry Science 96 (E-Suppl. 1) page 21
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Divek V. T. Nair, J. V. Thomas, G. Dewi, T. Johnson, S. Noll, C. Cardona & A. Kollanoor Johny. 2017. Effects of multiple alternatives-to-antibiotic interventions on multidrug-resistant Salmonella Heidelberg in turkey poults. Poultry Science 96 (E-Suppl. 1) page 24

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Poultry Industry Nutritionists, Extension Agents, Poultry Scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A Ph.D. student and two MS students are being trained in conducting poultry experiments under BSL2 protocols. Two undergraduate students are also being trained on this project. How have the results been disseminated to communities of interest?Talks by the PD: 1. The title of the presentation - "Effect of inoculum level on cecal colonization and organ invasion by multidrug-resistant Salmonella Heidelberg in turkey poults after experimental oral challenge". This research was presented at the Poultry Science Association annual meeting in New Orleans in 2016 to an audience of about 60 individuals that included academicians, industry professionals, students, and poultry veterinarians. 2. The title of the presentation - "Developing an 'Alternatives-to-Antibiotics' Intervention Model Against multidrug resistant Salmonella in Turkeys". This research talk was presented at the Graduate Seminar Series in the Department of Animal Science, University of Minnesota. The PD talked about how the USDA NIFA Hatch project aided in the development of the interventional approach. Around 30 graduate students and departmental faculty attended the seminar. What do you plan to do during the next reporting period to accomplish the goals?Histopathology and immunohistochemistry of the samples will be completed during the next reporting period.We will be preparing the manuscript for publication. Based on the results from this study, interventional poultry modelswill be developed.

Impacts
What was accomplished under these goals? The objective of the current study was to determine cecal colonization, organ invasion and potential skeletal muscle deposition of multidrug-resistant (MDR) S. Heidelberg (Source: 2011 ground turkey outbreak) after the experimental oral challenge of turkey poults. Two, 21-day turkey poult experiments were conducted. In each experiment, sixty (N=60 poults/experiment), day-old, straight-run, commercial turkey poults were randomly assigned to 5 challenge groups of 12 poults each. On day 1, the incoming flock was screened for any inherent Salmonella. Salmonella-free water and feed were provided ad libitum. On day 7, groups 1, 2, 3, 4 and 5 received a one-time oral inoculum dose of 10^0, 10^2, 10^4, 10^6, and 10^8 CFU/ml of S. Heidelberg in phosphate buffered saline (pH=7.2), respectively, via oral gavage. On day 9, two poults from each group (n=2 poults/group) were sacrificed for cecal samples to ensure S. Heidelberg colonization. On day 21, the remaining poults (n=10/group) from each group were euthanized, and samples were collected to determine S. Heidelberg colonization in the cecum, invasion of liver and spleen, and potential deposition in the thigh, drumstick, and breast muscles. Histopathology and immunohistochemistry were also conducted on the samples. Results indicate that the MDR S. Heidelberg outbreak isolate showed high colonization potential in the poult ceca with as low as ~4.5 log10CFU/g in the 10^2 inoculum group to as high as ~5.9 log10CFU/g in the 10^8 group, in both experiments. Spleen had more S. Heidelberg than liver (P<0.05). In addition to cecum, liver, and spleen, some of the muscle samples were tested positive for S. Heidelberg, as revealed by the microbiological analysis. Two adult turkey experiments were also conducted in continuation of the turkey poult projects.In each experiment, twenty, 11-weeks old commercial turkey poults were randomly assigned to 5 challenge groups of 4 poults each. On day 7, groups 1, 2, 3, 4 and 5 received a one-time oral inoculum dose of 0, 10^2, 10^4, 10^6, and 10^8 CFU/ml of S. Heidelberg in phosphate buffered saline (pH=7.2), respectively, via oral gavage. From day 8 onwards, fecal samples from all pens were collected to determine thepresence of S. Heidelberg. On day 18, all birds were euthanized, and samples were collected to determine S. Heidelberg colonization in the cecum, invasion of liver and spleen, and skeletal muscles. MDR S. Heidelberg outbreak isolate showed colonization potential in the adult turkey cecum with 62.5% samples positive in the highest challenge group, whereas 50% of the turkeys tested positive in the lowest inoculum group. MDR S. Heidelberg populations in the treatment groups were as low as ~0.8 log10CFU/g in the 10^2 inoculum group to as high as ~3.8 log10CFU/g in the 10^8 group. High inoculum groups (10^6, 10^8) had positive samples in the spleen. Salmonella was not isolated from the skeletal muscles except a sample in 10^2 CFU group (by enrichment). In addition, all experimental isolators tested positive for S. Heidelberg throughout the study.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: D. V. T. Nair and A. Kollanoor-Johny. 2016. Effect of Propionibacterium freudenreichii on Salmonella multiplication, motility, and association with avian epithelial cells. Poult. Sci. 00: 1-11. http://ps.oxfordjournals.org/content/early/2016/10/18/ps.pew367
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Divek V. T. Nair, Jijo V. Thomas, Tim Johnson, and A. Kollanoor Johny. 2016. Effect of Propionibacterium freudenreichii on multidrug-resistant Salmonella Heidelberg in turkey poults. Minnesota Nutrition Conference, Prior Lake, Minnesota (poster presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: A. Kollanoor Johny, Divek V. T. Nair, and J. V. Thomas. 2016. Effect of inoculum level on cecal colonization and organ invasion by multidrug-resistant Salmonella Heidelberg in turkey poults after experimental oral challenge. PSA Annual Meeting New Orleans. Poult. Sci. 95(E-Suppl. 1): 26-27.

Progress 12/22/14 to 09/30/15

Outputs
Target Audience:Poultry Industry Nutritionists, Extension Agents, Poultry Scientists Changes/Problems:As a new faculty in the department (joined in Dec 2013), I had to work for a few months (until September 2014) to set up my BSL2 pathogenic microbiology laboratory up and running with approvals from the IACUC, Institutional Biosafety Committee (IBC) and Department of Environmental Health and Safety (DEHS). I had to select and standardize the bacterial strain for inoculation and attachment on epithelial cells using tube culture and cell culture models, respectively. Moreover, although I had obtained approvals for animal studies from IACUC, I had to wait until IBC approved my first infectious agent (Salmonella Heidelberg) protocol in poultry. What opportunities for training and professional development has the project provided?I have a PhD student being trained in conducting poultry experiments under BSL2 protocols. I am also training five undergraduate students on this project. How have the results been disseminated to communities of interest?Talks by PD: Use of Probiotics and Prebiotics in poultry. Minnesota Nutrition Conference, Prior Lake, Minnesota, September 17, 2015. Attendies included industry nutritionists. I presented an overview of the currently used probiotics and prebiotics in poultry industry and stressed on mechanisms of action of these interventions by including some of my own data. Alternative Antimicrobial Agents in Poultry Production. Multistate Poultry Feeding and Nutrition Conference, Indianapolis, Indiana, May 19-21, 2015. Attendies included poultry scientists and industry nutritionists. My talk focused on using essential oils on reducing antibiotic resistant bacterial organisms in poultry production. Potential Role of Essential Oils in Organic Poultry Production. Midwest Poultry Federation Convention, Saint Paul, Minnesota, August 16-18, 2015. Attendies included organic producers, industry nutritionists, poultry scientists. In this presentation, I detailed on my research on essential oils as an intervention strategy to control infectious diseases in organic poultry. Talks by Graduate Student: Antimicrobial potential of Propionibacterium freudenreichii against Salmonella Heidelberg and Salmonella Enteritidis. 2015 PSA Annual Meeting. Attendies included poultry scientists. Graduate student presented his research data in the Food Microbiology section. What do you plan to do during the next reporting period to accomplish the goals?We are planning to complete the evaluation of data from our current project and continue experiments in adult birds.

Impacts
What was accomplished under these goals? We have completed two replicates of the study with 120 turkey poults to determine if Salmonella Heidelberg could systemically contaminate poultry (poult) meat. We will evalute our results soon. We also investigated the potential of natural interventions against Salmonella Heidelberg using an in vitro model to expand our current study to device on-farm strategies to control S. Heidelberg in poultry, as indicated in our scientific milestone section. In this study, we investigated the efficacy of a GRAS status probiotic bacterium, Propionibacterium freudenreichii N3523 (hereafter PF) against Salmonella Heidelberg in co-culture and sterile turkey cecal contents. Efficacy of PF against Salmonella Heidelberg was determined separately in co-culture medium [1:1 ratio of de Man Rogosa Sharpe (MRS for PF growth) and Tryptic Soy Broth (TSB for Salmonella Heidelberg growth)] using micro and macro broth dilution assays at 37oC. SH or SE at 105 CFU/ml was tested against different levels of PF (105 - 109 CFU/ml) for obtaining effective co-culture combinations. Then, the most effective inoculation level of PF (109 CFU/ml) was tested against Salmonella Heidelberg at 105 CFU/ml in sterile cecal contents at 37oC. Appropriate controls were included. The efficacy of PF was tested against Salmonella Heidelberg in duplicate, and the experiment was repeated three times (n=6). PF completely inactivated (~8.0 log10CFU/ml) Salmonella Heidelberg after 24 hours of incubation in co-culture and resulted in 3.5 log10CFU/ml reduction in sterile turkey cecal contents, compared to the controls after 24 hours of incubation at 37oC (P<0.05).

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: 1. Divek V. T. Nair, and Kollanoor Johny, A. 2015. Use of Probiotics and Prebiotics in Poultry. Proceedings of the Minnesota Nutrition Conference, Prior Lake, Minnesota, September 16-17, Pp 200-209. 2. Divek V. T. Nair, J. Hagen, C. Peichel, S. Hallberg, A. Bakken and A. Kollanoor Johny. 2015. Antimicrobial potential of Propionibacterium freudenreichii against Salmonella Heidelberg and Salmonella Enteritidis. Poultry Science Association Annual Meeting. Poult. Sci. 94: (E-Suppl. 1). p 14.