Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Poultry Science
Non Technical Summary
Understanding the relationship between the chicken gut microbiota, diet composition - including prebiotics and probiotics, and resistance to Salmonella would be of particular interest to the poultry producers and consumers. We therefore plan to study and disseminate knowledge about the best practices for reducing the incidences of Salmonella in poultry and Salmonellosis in humans - caused by the consumption of poultry products including eggs. Our integrated approach will include the best practices for optimizing the chicken's gut microbiota via modulations of the dynamic relationship between prebiotics, probiotics and vaccines.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
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. Understanding the relationship between the chicken gut microbiota, diet composition - including prebiotics and probiotics, and resistance to Salmonella would be of particular interest to the poultry producers and consumers. We therefore plan to study and disseminate knowledge about the best practices for reducing the incidences of Salmonella in poultry and Salmonellosis in humans - caused by the consumption of poultry products including eggs. Our integrated approach will include the best practices for optimizing the chicken's gut microbiota via modulations of the dynamic relationship between prebiotics, probiotics and vaccines.
Project Methods
Experiment 1: Three hundred 1-day old pullets will be randomly assigned to one of 3 treatment groups (control, prebiotic, and Salmonella vaccine treatment) of 100 birds/group. Within each group, birds will be placed into one of 2 animal isolation chambers (50 birds/isolator). The control group will be fed a standard starter diet, the prebiotic group will be fed a diet supplemented with galactooligosaccharide (GOS, 1.0% w/w) to promote the growth of endogenous Firmicutes, while the vaccine group will receive 108 cfu/bird of an attenuated ST-vaccine by oral gavage. At 4 weeks of age one isolator/treatment group will be challenged with 105 cfu/bird of wild type Salmonella Enteriditis (SE) by oral gavage. To understand how promoting the growth of endogenous bacteria, or the introduction of live attenuated Salmonella vaccine strains affect the diversity of the microbiota, and how those changes are related to colonization by SE, cecal contents will be collected from 5 birds/treatment group at 0, 1, 2, 3, 4, 5, 6, 7, and 8 weeks and analyzed individually by bTEFAP (see Methods). In addition, the cecal content and sections of liver and spleen will be collected at 0, 1, 2, 3, 4, 5, 6, 7, and 8 weeks and analyzed for the presence of SE colonization of the gut (cecal contents) and invasion into the host (liver and spleen). Finally, based on the analysis of the data collected above cecal contents containing specific species found exclusively (or significantly enriched) in animals with the lowest SE colonization levels will be isolated, cultured and characterized for use in Experiment 2. Experiment 2: Four hundred 1-day old pullets will be randomly assigned to one of 4 treatment groups (control, prebiotic, commercial probiotic, candidate probiotic) of 100 birds/group. Within each group, birds will be placed into one of 2 animal isolation chambers (50 birds/isolator). As described above, the control group will be fed a standard starter diet, the prebiotic group will be fed a diet supplemented with GOS (1.0% w/w), the third group will be fed a diet supplemented with FM-B11 (IVS-Wynco LLC, Springdale, AR), a commercially available probiotic previously demonstrated to suppress colonization by SE (See Relevant Body of Knowledge), and the fourth group will receive 108 cfu/bird of the organisms identified in Expt 1 as being associated with decreased SE colonization. Methods: Animals: 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. Approximately 100mg of sample will be resuspended in 100µl of 1X PBS with lysozyme and 200µl of ATL buffer in a 1.5ml microcentrifuge tube and incubated at 37°C for 30 minutes. Following the initial incubation, 20µl of proteinase K will be added and the mix will be incubated overnight at 56°C. Then tubes will be centrifuged at 800g for 5 minutes. The supernatant will be transferred to a Qiagen S-block and sonicated in a VWR B2500A sonicator for 30 minutes at 3 kHz and 65°C. Following the sonication step, the S-block will be transferred to the BioRobot Universal.The V1-V3 region of the bacterial 16S rDNA will be amplified using forward primer composed of the Roche Titanium Fusion Primer A (5'-CGTATCGCCTCCCTCGCGCCATCAG-3'), a 10 basepair MID barcode unique to each of the samples and the universal bacteria primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3'). The reverse primer will be composed of the Roche Titanium Primer B (5'-CTATGCGCCTTGCCAGCCCGCTCAG-3') the identical 10 basepair MID as the forward primer and the reverse bacteria primer 338R (5'-TGCTGCCTCCCGTAGGAGT-3'). Each sample will be gel purified individually using the Qiagen Gel Extraction Kit and then pooled based on equal mass. The pooled 16S rDNA amplicons will be sequenced on a Roche GS FLX plus sequencer using the Titanium sequencing reagents and protocols. Chimeric sequences will be depleted from the trimmed data set using the Black Box Chimera Check (B2C2) tool. Culturing of cecal microbiota: Culturing of samples collected from chicken cecum samples will be carried out essentially as described by Goodman et al.. Freshly collected cecal samples will be transferred into an anaerobic chamber within 30 min of their collection and placed in prereduced PBS with 0.1% cysteine (PBSC). The sample material will be suspended by vortexing for 5 min, and the suspension will be allowed to stand at room temperature for 5 min to permit large insoluble particles to settle to the bottom of the tube. Dilutions of the supernatants will be plated onto prereduced TYG supplementedBrain-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. 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. Viable counts will be determined for each bird's organs by plating 0.1 ml aliquots of each dilution in duplicates on LB and XLT-4 media containing the appropriate antibiotics. Plates will be incubated overnight at 37o C before counting. Samples showing no viable counts on the first decimal dilution (i.e., <102 CFU/g) will be enriched overnight at 37oC in Tetrathionate Broth before streaking on XLT-4 agar plates containing the appropriate antibiotics. The presence of Salmonella in the different samples (i.e., from vaccinated and challenged birds) will be confirmed by VITEK®@ REF-21 341 (Biomerieux, Inc.), and published real-time PCR protocols. IgG and IgA titer determinations: Levels of anti-Salmonella IgG and IgA will be assessed in serum, and IgA in crop washes, and intestinal washes by indirect ELISA using killed/sonicated Salmonella cells as the coating antigen as previously described and using HRP labeled anti-chicken IgG or anti-chicken IgA to detect anti-Salmonella IgG and IgA, respectively. Statistical Analysis: Significant differences between the different treatment groups for the mean viable bacterial counts of the ST vaccine(S) and the Salmonella Challenge strains (i.e., WT Virulent S. Typhimurium and WT virulent S. Enteriditidis) in samples taken from the different Organs will be determine by analysis of variance (ANOVA) with Students's test. The limit of significance will be set to p<0.05.