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Development of Novel Salmonella Control Practices and Integrated Education Program to Reduce Salmonellosis

Hassan, Hosni; Brown-Schild, Valerie; Koci, Matthew D; Stewart, Marshall; Azcarate-Peril, M Andrea; Chilcote, Amy G
North Carolina State University
Start date
End date
Long-Term Goal: The ultimate goal of the proposed integrated studies is to reduce Salmonellosis.

Objectives: 1. Characterize the changes in the chicken's gut microbiome during Salmonella infections and identify members of the microbiota associated with decreased Salmonella colonization; 2. Develop an optimized control program that promotes resistance to colonization and the development of strong mucosal immunity virulent strains of Salmonella enterica; 3. Develop innovative K-12 curriculum material in subjects related to food safety, disease transmission and next generation genetic research to engage students in agricultural sciences and communicate their role in food safety; and 4. Develop instructional materials based on the 4H national standards to communicate and teach 4H young citizens the technology and science used to produce safe food and their responsibilities in preventing food-borne disease.

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Non-Technical Summary:
Poultry products have been frequently implicated in reported cases of salmonellosis. The goal of this proposal is to develop and communicate a safe, effective and economic Salmonella control program for protecting the safety of the food supply and the health of consumers. To achieve this goal, the proposed studies will develop a novel Salmonella control strategy that promotes the establishment of an intestinal microbiota in the chicken that prevents colonization by pathogenic bacteria and enhances the mucosal immune response to Salmonella vaccination and challenge. The proposed studies addresses the research needs as identified by the "Food Safety Modernization Act", the poultry producers, the consumers, the extension/out-reach agents, and the "Food Safety Challenge Area identified in AFRI competitive grants program - Food Safety FY 2011 - Program Area Code - A4161.

A multi-disciplinary research team working with professional educators and extension agents will carry out the proposed studies to: (1) identify constituent species of the chicken's gut microbiota which correlate with increased resistance to Salmonella; (2) develop an effective Salmonella intervention program that optimizes gut microbiota and vaccination; and (3) develop educational materials for K-12 students and 4-H groups to teach consumers about food safety, the science behind preventing food borne disease, and their role in preventing salmonellosis. Research Scientists from North Carolina State University and UNC-CH will be responsible for the laboratory aspects of the project, and will work in collaboration with K-12 science and social studies teachers, and extension services agent in the dissemination of the research based educational materials.

Vaccination and SE Challenge: Attenuated Salmonella Typhimurium strain carrying a defined deletion in the gene required for Anaerobic Regulation of redox (fnr), has been generated in Dr. Hassan's laboratory. A virulent strain of S. Enteritidis will be used to challenge the vaccinated birds. The different Salmonella strains will be grown statically under aerobic conditions in LB media at 37oC. Inocula for vaccinating the birds will be prepared by growing the cultures over-night, centrifuging the cells at 6000g, Washing the cells in PBS buffer (pH 7.4) containing 1mM Mg2+. Washed cells will be suspended in PBS-Mg2+ at a cell density equal 109 CFU/ml. Each bird will receive 108 CFU live attenuated ST delta fnr strain (STFnr) by oral gavage (o.p.) at day-one. On week 4 the vaccinated birds and the controls will be challenged (o.p.) with the virulent SE strain at a dose of 106 CFU per bird. One-day old female W-36 pullets will be housed under ABLS-2 conditions, in HEAP filtered isolation chambers. These animal isolators have approximately 24 inches of feeder space, 2 nipple drinkers, and 864 sq in of floor space all of which meet or exceed the 2010 Federation of Animal Science Societies Guidelines for the care of laying hens up to 6 weeks of age. Chicks will have free access to food and water throughout the duration of the studies. At 0, 3, 7, 14, 21, 28, 31, 35, 42, and 49 days of age) 5 birds per treatment will be weighted, serum collected, and euthanized by CO2 (following approved methods in IACUC protocol), before the tissue samples/organs (liver, spleen, cecum, and crop wash) are collected for bacteriological, microbiota, and immunological analysis. Microbiome Analysis: Isolation of total DNA from cecal digesta will be carried out using a customized isolation protocol on a Qiagen BioRobot Universal and the Qiagen Blood and Tissue Isolation kit using a customized isolation protocol at the UNC Microbiome Core Facility. Dilutions of the supernatants will be plated onto prereduced TYG supplemented 49 Brain-Heart-Infusion (BHI), and MRS media, and incubated for 7 days at 42C under an atmosphere of 75% N2, 20% CO2, and 5% H2. Colonies will be collected en masse into 10 ml of prereduced PBSC for preservation of the whole microbial community by adding prereduced glycerol containing 0.1% cysteine samples (final concentration of glycerol, 20%). Additionally, individual colonies will be picked and cultured under the above described conditions in TYGS, BHI, and/or MRS broth. Stocks will be preserved in anaerobic glass vials in a -80oC freezer until further analysis. Phylogenetic identification of isolates will performed by amplification and sequencing of the 16S rDNA gene using the universal primers 8F (5'-AGAGTTTGATC(A/C)TGGCTCAG-'3) and 1492R (5'-GGTTACCTTGTTACGACTT-'3). Bacteriological Analysis: Samples of liver, spleen, cecal content, and crop wash will be collected at the specified sampling time points to enumerate viable Salmonella CFUs. The samples will be weighted and diluted 10-fold in PBS - pH 7.4. A Masticator (i.e., stomacher from NEUTEC Group, Inc.) will be used homogenize the samples. The samples will be decimally diluted in PBS buffer.

2012/07 TO 2013/06
Target Audience: 4-H agents - K-12 teachers to select the "Food Safety Kenan Fellows" - Basic scientists in the fields of Microbiology, Immunology and nutrition. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We trained two undergraduate students on all aspects of the project. Two tecyhnical research specialists participated in the studies. We selected Nine School teachers (Kenan Fellows) who will participate in the research laboratories during the summer of 2013 and develop teaching lessons on the subject matter to take to the class and exchange with other teachers throughout the State of North Carolina and the Nation. 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? A) Research: we plan to continue the deep sequencing analysis of the fecal and tissue samples collected during Experiment #1 (Objective 1) to identify the normal chicken's gut microbiota and how feeding the prebiotic (GOS, Galacto-oligosacharides) or usingour Patentend vaccine strain affectsthe gut microflora. We will also continue our immunological studies to assess the effects of vaccination and SE challenge on the mucosal immune system as outlined in the original proposal. Wealso plan to start Experiment #2 (Objective 1) where we will isolate potential probiotic organisms from the healthy "Salmonella-resistant" birds and compare it with a commercial probiotic product as well as our GOS (prebiotic) treatment. Fecal samples (Cecum) and tissue samples (liver and spleen) will be collected for bacteriological analysis. Cecum samples will also be used for microbiome analysis by pyrosequencing. Blood samples will also be collected and serum will be analyzed for Salmonella specific IgG and and gut washes will be assayed for IgA. These data are essential for achieving our first major goal, that required to develop an optimal control program to control the spread of Salmonella and reduce Salmonellosis. B) Education and Outreach: Three teams of three "Food Safety Kenan Fellows, FSKF", including an elementary, middle and high school teachers, will spend the summer of 2013in the research laboratories of the research mentors (PD Hassan, CoPD Koci, or CoPD Azcarae-Peril).Teams will be provided hands-on opportunities to participate in the research and to observe and document the scientific process in action. Each FSKF will develop hands-on curriculum units based on their research that align with the National Science Standards for the Life Sciences and other applicable strands and support a continuum of learning in across the K-12 curriculum. The research externship will be book-ended by rigorous professional development that supports the use of evidence based instructional skills and technology to promote inquiry and facilitate collaborative learning in the classroom. Fellows will work with IntelliMedia to incorporate new technological tools into instruction and curriculum, using cutting edge technology to fuse human language technologies and techniques from artificial intelligence with gaming technologies to develop high-impact interactive learning systems (See Letter on Intent to Collaborate). Specifically, the KSKFs will collaborate with IntelliMedia to develop a new version of their ?Crystal Island? video game, with a curricular focus on Salmonella in which students will explore a virtual research camp from a first person viewpoint and manipulate virtual objects, converse with characters, and use lab equipment and other resources to solve the mystery. In addition to microbiology content, scientific problem solving will be an important aspect of Crystal Island?s curriculum. Forming questions, collecting data, testing hypotheses, and reporting findings will be integral aspects of game play. FSKFs will also work with a 4-H Advisory Committee consisting of 8-10 individuals with backgrounds from the public and private sector that possess knowledge in poultry science, youth development, education, and workforce development to outline plans for their projects. They will consider target audiences and aspects of the studies with the greatest potential relevance and consult with 4-H staff to become familiar with National 4-H standards for lesson plans and evaluation. Each FSKF will alpha test and revise the lessons in their classroom during the following school year.

IMPACT: What was accomplished under these goals?
A) Research: We have finished conducting the first experiment listed under our proposed objective #1. We purchased three hundred 1-day old Hy-Line chicks from commercial supplier. The birds were randomly assigned to one of 3 treatment groups (control, prebiotic, and FNR-vaccinated) of 100 birds/group. Within each group, birds were placed into one of 2 animal isolation chambers (50 birds/isolator). Birds in the control group were fed a standard starter diet, birds in the prebiotic group were fed a diet supplemented with galactooligosaccharide (GOS, 1.0% w/w) to promote the growth of endogenous Firmicutes, and birds in the FNR-vaccinated group received 8.6x108 cfu/bird of the attenuated ST-Fnr vaccine by oral gavage. At 4 weeks of age the birds from one isolator from each treatment group were challenged with 1.7x109 cfu/bird of wild type SE by oral gavage. To assess clearance of the Fnr-vaccine strain from vaccinated birds and SE colonization of the gut and invasion into the host Cecal and liver/spleen samples (7 birds/treatment) were collected weekly (between 0 and 8 weeks) and analyzed for the presence of the vaccine strain and the SE strain. Also, cecal contents were collected from the same birds at the specified time intervals for microbiome analysis by bTEFAB in order to understand the effects of vaccination, prebiotics (GOS) and SE infection on the gut microbiota. In addition, blood serum, intestinal washes, and cecum tissue samples were collected from 7-birds/treatment at each specified time interval, for determination of IgG, IgA and the isolation of host RNA, respectively to assess the effects of the experimental treatments on the mucosal immune response. The differences in the immune responses among the treatment groups will be used to correlated with changes in SE colonization and microbiome data to determine conditions which promote the bird?s ability to resist SE. The experiment was completed on April 23, 2013. The microbiological analyses for SE-colonization have been completed and datashowed that the prebiotic treated birds as well as the Fnr-vaccinated birds were significantly less susceptible to colonization by SE than the controls. Clearly, these data validated our hypothesis and showed that we are on the right track for defining a best system/practice for increasing the chicken?s resistance to SE colonization and reducing Salmonellosis. Currently, samples are being processed to assess the immunological responses and for microbiome sequencing/analyses. We expect to the analysis of the data by ~ October 2013.

B) Education and Outreach: The process of selecting Nine Kenan fellows has been completed. The following process was followed: In November 2012, district and school administrators in 15 districts received email notification announcing the availability of the Salmonella project to K-12 teachers in their district. Recruitment efforts continued through January 20th when the application for the program closed. After an internal and mentor review of 21 applications for the project, 15 candidates were invited to in-person interviews conducted on March 7th, 2013. As a result of the interviews, three elementary, three middle and three high school teachers were selected as Food Safety Kenan Fellows. All of the 9-Food Safety Kenan Fellows attended a Kenan Fellows Program orientation event on May 4th with their Mentors in preparation for the summer externship beginning in mid-June. 4-H Co-PDs participated in the review and selection process of the Kenan fellows and the 4-H activities will also start in Mid-June.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
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Project number
Accession number
Bacterial Pathogens
Education and Training
Meat, Poultry, Game