Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
Veterinary Pathology
Non Technical Summary
Post-weaning diarrhea (PWD) caused by enterotoxigenic Escherichia coli (ETEC) is an economically important enteric disease in weaned piglets. Currently, antibiotics are used prophylactically and therapeutically in swine industry to control ETEC in pigs. The overuse of antibiotics is directly linked to the emergence of antibiotic-resistant bacteria and poses a threat to both human and animal health. Therefore, the use of antibiotics in the industry is being tightly re-examined by regulatory agencies. In this context, identifying a safe antibiotic alternative to prevent PWD in piglets becomes a real emergency.Recent studies confirm that disruption of gut microbiome (gut-dysbiosis) induced during weaning transition is one of the major predisposing factors for PWD in pigs. Therefore, compounds that can stabilize the gut microbiome during weaning transition could potentially be used for controlling PWD. This proposal investigates the efficacy of carvacrol (CR), a food grade phytophenol, in preventing gut-dysbiosis and PWD in piglets. Besides exerting potent anti-virulence properties against major enteric pathogens, including E. coli, CR has been found to possess a protective effect against gut dysbiosis by modulating the gut microbiome abundance and diversity in animal models. We hypothesize that CR supplementation in weaning diet could protect piglets from gut dysbiosis and subsequent PWD. For testing this hypothesis, piglets will be fed with weaning diet supplemented with and without CR. On day 10, piglets will be challenged with ETEC, and the diarrhea score, bacterial load, and histologic scores will be determined post infection. The fecal microbiome of different treatment groups will be analyzed using high throughput sequencing technology.The results from this research will provide the swine industry with a readily available non-antibiotic feed supplement to control PWD in piglets. The proposed research will also provide insights on 'gut microbiome modulation' as a potential strategy to control PWD in swine operations.
Animal Health Component
90%
Research Effort Categories
Basic
10%
Applied
90%
Developmental
(N/A)
Goals / Objectives
Post-weaning diarrhea (PWD) caused by enterotoxigenic Escherichia coli (ETEC) is an economically important enteric disease in weaned piglets. Currently, antibiotics are used prophylactically and therapeutically in swine industry to prevent and control ETEC in pigs. The overuse of antibiotics is directly linked to the emergence of antibiotic-resistant bacteria and poses a threat to both human and animal health. Therefore, the use of antibiotics in the industry is being tightly re-examined by regulatory agencies. In this context, identifying a safe antibiotic alternative to prevent PWD in piglets becomes a real emergency.The results from this research will provide the swine industry with a readily available non-antibiotic feed supplement to prevent and control E. coli carriage and PWD in piglets.Objectives:To determine the effect of in-feed CR supplementation in reducing ETEC carriage and post-weaning diarrhea in pigletsSince the association between gut-dysbiosis and PWD is well established, intervention strategies that stabilize or reversing such changes in microbiome during weaning transition could be an effective approach to prevent and control PWD in piglets. CR could be a potential antibiotic alternative for combating PWD in piglets due to the following properties: 1) Protective effect of CR on animal gut microbiome; 2) Antivirulence properties against important enteric pathogens; 3) Natural food-grade compound generally regarded as safe by the FDA, and 4) Antidiarrheal properties.To the best of our knowledge, no in-depth studies have been conducted so far to determine the effect of phytophenols including CR on composition and diversity of piglet gut microbiome utilizing high throughput sequencing platforms. Moreover, no investigations have done so far to test a specific monoterpenoid phyophenol such as CR for their efficacy against PWD in pigs. It has been recently established that CR positively alters gut microbiome composition and selectively inhibits the growth of unfavorable gut bacteria including enteric pathogens in different animal models. We hypothesize that in feed CR supplementation could potentially prevent gut-dysbiosis and subsequent PWD caused by ETEC. The ultimate goal of this project is to test the efficacy of CR supplementation in prevention and control of ETEC infection and promote gut health in piglets, and thus our future projects will be directed towards the flexibility of CR administration, which includes the delivery of solubilized CR in drinking water.2. To determine the effect of CR supplementation on gut microbiome of weaned piglets using high throughput 16s rRNA sequencingSince the association between gut-dysbiosis and PWD is well established, intervention strategies that stabilize or reversing such changes in microbiome during weaning transition could be an effective approach to prevent and control PWD in piglets. Moreover, previous research conducted by the PI demonstrated a protective effect of CR against gut-dysbiosis and diarrhea in a mouse model using 16s rRNA sequencing. High throughput sequencing techniques could provide in-depth details of microbiome modulation induced by CR supplementation in normal, and ETEC infected piglets, potentially yielding useful information for swine industry to prevent and control PWD in piglets. Additionally, the results from this project will help the scientific community to develop new treatment strategies and novel compounds that stabilize piglet gut microbiome and prevent and control PWD in commercial swine operations.The results from this research will provide the swine industry with a readily available non-antibiotic feed supplement to control PWD in piglets. The proposed research will also provide insights on 'gut microbiome modulation' as a potential strategy to control PWD in swine operations.
Project Methods
Materials and methodsAnimals: Animal studies will be carried out in accordance with the protocol approved by the Institutional Animal Care and Use Committee, Iowa State University. Animals will be monitored for weight loss and diarrhea, for the entire duration of the trial.Effect of CR on postweaning enteric colibacillosis in weaned piglets: Twenty four suckling piglets weaned and weighed at 3 weeks of age, will be randomly divided into four experimental groups (n=6 per group), which will be housed separate pens in groups with ad-libitum access to starter diet and drinking water. Different treatment groups are listed in Table 1. The treatment groups (Control group (C), Carvacrol control Group (CR), E. coli control group (EC) and Carcavrol + E. coli group (CR+EC). CR and CR+EC group will be provided a starter diet supplemented with CR (1000 mg/kg feed) till the end of the study (21 days). The dosage of CR (1000 mg/kg feed) is selected based on previously published in vivo pig studies [22].CR supplementation at this concentration (1000 mg/kg feed) was found to have no harmful effect on the intestinal tissue and beneficial gut flora in vitro and in vivo [17, 20, 23]. On day 10th of the study (10-day post weaning), animals in EC, CR+EC, groups will be challenged with approximately 1010 CFU enterotoxigenic E. coli (F18 positive clinical isolate with Stx2). Control groups will be administered with phosphate buffer saline (PBS) using oral intragastric tube. Animals will be weighed and observed for diarrhea for 7 days and fecal samples will be collected at different time points (0, 3, and 7 day post-infection). The piglets will be sacrificed on day 7 post infection, and the intestinal samples will be collected for histopathology.Determination of incidence of diarrhea and diarrhea index: The number of pigs showing diarrhea will be recorded daily throughout the duration of the study. The severity of diarrhea will be evaluated using standard fecal consistency score system (Marquardt et al., 1999). Scores 0-3 represent firm normal feces (0), pasty with possible slight diarrhea (1), semi-liquid or definitely unformed feces (2) or very watery and frothy diarrhea (3). Both individual and pen fecal scores will be recorded everyday post ETEC challenge. Diarrhea index will be calculated as sum of feces score/total number of pigs.Fecal sample collection and quantification of E. coli in porcine feces: Fecal samples (swabbed directly from the rectum) will be collected each morning from all piglets from day 0, 3 and 7 day post challenge. The piglets will be euthanized by penestrating captive bolt or intravenous injection of barbuituate overdose. Jejunal, ileal and colonic contents will be collected and stored at -80o C for molecular analysis. Total DNA will be isolated from 200 mg of feces using QIAamp Fast DNA Stool Mini Kit (QIAGEN) as per manufacturer's instruction. Real-Time PCR will be performed using CybrGreen Q-PCR Kit (New England BioLabs) in a Stratagene MX5000 real-time PCR machine as per previously published protocol and oligonucleotide primers [24]. Total DNA was isolated from 200 microliters of E. coli suspension of known titers will be used as a standard, and the absolute quantification of E. coli will be performed by standard curve method [24].Histopathology: After sacrifice on 7-day post challenge, the mid-jejunal segments (5 cm in length) will be collected, opened longitudinally and rinsed thoroughly with physiological saline at 4oC before fixing in 4% paraformaldehyde for subsequent histological analysis. The samples will be sectioned at 5 μm thickness and stained with hemotoxylin and eosin. These will be acquired with 100× magnifications using an Olympus BX51 microscope (Olympus Optical Company, Tokyo, Japan). Intestinal villus height and villus crypt depth will be measured using Image-Pro Plus 6.0 image processing and analysis system (Media Cybernetics, Bethesda, MD, USA).Sequencing of the taxonomic marker and data analysis: DNA will be extracted from 0.25 g of fecal samples from all treatment groups using the MoBio PowerMag Soil 96 well kit (MoBio Laboratories, Inc). The microbiome analysis will be set up as a completely randomized design with treatments done in replicates of six. Partial bacterial 16S rRNA genes (V4) will be amplified using 30 ng of extracted DNA as template. The cleaned pool will be sequenced on MiSeq (Illumina, Inc). Forward and reverse reads from the paired-end sequencing will be first merged using the fastq.join script. Qiime 1.8 will then be used for additional data analysis. Comparisons of specific OTUs between treatment groups will be made at the phylum, order, and genus level using OTUs detected in at least 25% of samples in a given group included in the analysis. Biological effect sizes will be estimated using the linear discriminant analysis effect size (LEfSe) method. A CoVennTree (Comparative weighted Venn Tree) analysis will be performed to assess differences in the microbial population structure between groups.Statistical analysis: The results of the animal study will be expressed as means ± standard errors of the means (SEM). The differences between the experimental groups will be compared using the analysis of variance (ANOVA). Two-way ANOVA will be used to compare experimental groups across the days. The differences between two groups will be analyzed using unpaired Student's t-test. "N1" Chi-squared test will be used to compare incidence rate between two different treatments. The statistical significance level will be set at a P < 0.05. Alpha diversity (chao1) will be compared using a non-parametric two sample t-test with 999 Monte Carlo permutations. Beta diversity (Bray-Curtis dissimilarity) will be compared using a two-sided student's two-sample t-test with Bonferroni correction. The frequency of detection (group significance) of specific OTU calls within groups will be compared using a Kruskal-Wallis non-parametric analysis of variance. followed by correction for multiple comparison using the Benjamini and Hochberg False Discovery Rate (FDR) method. A FDR of 5% will be utilized to determine significance.Quality Assurance/Quality Control Plan: All clinical observations will be done by trained personnel to ensure consistency and repeatability of clinical scoring. F18 and Stx2 positive E. coli clinical isolate will be provided by ISU Veterinary Diagnostic Laboratory (VDL). All in vitro assays and experiments will be performed will be done either by Dr. Mooyottu's lab in duplicates, and replicated thrice following published protocols, by ISU DNA facility, or ISU VDL following their Quality Assurance and Quality Control policies.