Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
Veterinary Diagnostic and Production Animal Medicine
Non Technical Summary
In field conditions, piglets are highly susceptible to Clostridiodes difficile (formerly Clostridium difficile) infection or CDI in a very small window, which is roughly the first week of birth. The natural resistance to CDI develops quickly along with the development of the gut microbiome, which is closely related to microbial bile acid metabolism in humans and animals. The bile acid profile, its series, and structural and functional composition during this crucial window are essential to understand the difference in CDI susceptibility between humans and pigs. So, our working hypothesis for our first aim is that the bile acid profile during the first week of life in pigs is significantly different from that of the late postnatal period (after 1 week) and that of human infants. Understanding this compositional and functional difference will potentially aid in formulating bile-acid based preventive and therapeutic strategies against CDI in piglets. To test this hypothesis, our approach will be to procure pregnant sows, keep the delivered piglets with sow until weaning period in similar field husbandry conditions, and determine bile acid profile at different periods of postnatal life by sacrificing piglets on and after the crucial susceptibility window mentioned above. Published data on human infant bile acid profiles will also be used for comparison and to determine the functional and compositional differences.Gut bile acid profile is crucial in the pathogenesis of CDI. The C. difficile spore germinant activity of primary bile acids, direct antitoxigenic nature of human and rodent primary and secondary bile acids, and strong anti-C. difficile properties of secondary bile acids are important in the host susceptibility to CDI. Therefore, early colonization piglet with C. hiranonis a bile acid converting bacteria could protect them from CDI by converting primary bile acids to secondary bile acids. Additionally, direct supplementation of secondary bile acids could potentially protect the piglets from this disease. Therefore, our working hypothesis for our second specific aim is that supplementation of secondary bile acids and colonization of bile acid converting bacteria - C. hiranonis could protect the neonatal piglets from CDI. To test this aim, our approach will be to provide a mixture of secondary bile acids or C. hiranonis after a few hours after delivery, challenge the piglets with C. difficile on 2nd day after birth, (which is the time when C. difficile naturally colonize the piglet gut) and to study the clinical signs and C. difficile induced gut pathology compared to the controls.
Animal Health Component
0%
Research Effort Categories
Basic
85%
Applied
0%
Developmental
15%
Goals / Objectives
Clostridiodes difficile infection (CDI) is a significant enteric disease that causes toxin-mediated diarrhea in pigs and humans. Pigs have notable similarities in the gastrointestinal physiology, anatomy and even share the similar strains of C. difficile. However, there is a striking difference between the susceptibility of CDI in human infants and piglets. CDI in pigs manifests as a neonatal disease, affecting piglets at their first week after birth; whereas human infants are extremely resistant to CDI. A definitive reason for this paradox is currently unknown. At this point, in the absence of other documented immuno-physiologic dissimilarities, differences in the bile acid profile at the early neonatal period could be a potential reason for differences in CDI susceptibility in piglets and human infants. Therefore, understanding these differences in the first week of piglet life could potentially provide a reasonable explanation of this paradox, based on which effective preventive strategies can be developed. Thus, we hypothesize that difference in the neonatal piglet bile acid profile is responsible for the swine-human CDI susceptibility paradox and altering the bile acid composition during the early neonatal period could protect piglets from CDI. To test these hypotheses, we propose: a) to determine the early neonatal bile acid profile of piglets to find out how it differs from the late postnatal period and human infant; 2) to determine the efficacy of early oral bile acid supplementation and bile acid converting bacterium C. hiranonis in preventing piglet CDI. To achieve this goal, we will determine the bile acid profile at different periods of postnatal life, and provide a mixture of secondary bile acids or bile acid converting bacterium- C. hiranonis in neonatal piglet to study the clinical signs and C. difficile induced gut pathology compared to the controls. The results could help the swine industry to develop novel non-antibiotic strategies to prevent CDI in piglets.
Project Methods
Specific aim 1: Determine the early neonatal bile acid profile of piglets to find out how it differs from that of late postnatal period and human infantsAnimal experiment: Six pregnant, second to fourth parity, cross-bred sows from a commercial herd negative for porcine reproductive and respiratory syndrome virus (PRRSv) will be purchased and delivered to Iowa State University (ISU) approximately one week prior to the expected farrowing date. Sows will farrow and anticipate having at least 60 piglets born alive. All 60 piglets in this study will be processed at birth with navels clamped, cut, and sprayed with 5% iodine solution (Durvet, Missouri, USA). Piglets will also be given their iron injection shortly after birth. The piglets will be housed with sows for the duration of the study. One piglet from each sow will be randomly sacrificed on days 1, 2, 3, 4, 5, 7, 10, 14, and 21 and small intestinal contents (jejunum, ileum and colon) and fresh intestinal tissues will be collected. Based on litter size, extra piglets will be assigned to be randomly sacrificed at the later periods to ensure we have at least six piglets per each time period.Measurement of bile acids by liquid chromatography-mass spectrometry. Bile acid profile of jejunal, ileal and colonic samples collected at different time points will be performed as per published protocol at PennChop microbiome lab, Philadelphia, PA using LC-ESI-MS/MS technique. Fecal levels of conjugated and unconjugated cholic acid, hyocholic acid, chenodeoxy cholic acid, deoxycholic acid, deoxycholic acid, lithocholic acid, and other intermediary bile acids will be determined as per protocol.Metagenomic analysis and determination of bile acid converting bacterial population in piglet gut: Intestinal contents at different time points will be used for microbiome analysis and identification of bile acid converting bacterial population at different time points of early and late postnatal period to determine the succession of microbial communities in relation to the bile acid profile of pre-weaning piglets. Since 16S rRNA amplicon-based analysis is not sensitive enough to determine species level microbiome changes, we will use shotgun metagenome sequencing as a higher resolution method. Since the cost of shotgun sequencing is high, ileal samples at 3, 7, and 14 days will be used for this analysis. Shotgun sequencing will be done on an Illumina genome sequencer following our previously published protocols. For shotgun metagenome data analysis, raw fastq sequences will be quality controlled using Fastqc and host reads will be removed using metaWRAP pipeline. Filtered reads will be analyzed for taxonomy using Kaiju against the proGenomes database using default parameters. The percentage abundance of each taxon was plotted using Explicit v2.10.5. In addition, the raw counts obtained for each taxon will be used for calculation of alpha and beta diversity using the phyloseq package in R. For identification of differentially abundant taxa in treatment compared to control, the raw counts of abundance of each taxon was then Log10(x+1) transformed and will be fed to DESEq2 package in R. Bacterial taxa which significantly altered from the control will be further filtered with the criteria of at least log2foldchange (Log2FC) of ≥ 2 and padj value > 0.05.Specific aim 2: Determine the efficacy of early oral bile acid supplementation and bile acid converting bacteria in preventing piglet CDI.Animal experiment and inoculations: C. difficile infection studies will be performed as per the protocol published by Arruda et all 2014 under the co-supervision of the PI. Six pregnant, second to fourth parity, cross-bred sows from a commercial herd negative for PRRSv will be purchased and delivered to ISU approximately one week prior to the expected farrowing date. On day 114 of gestation, terminal caesarian surgeries will be performed on sows and the neonatal piglets will be manually provided 10 mL of pooled colostrum and an iron injection. We anticipate at least 60 piglets will be used for the study. Piglets will be fed with milk replacer (Esbilac; Pet-Ag, Hampshire, Illinois, USA) three times daily by oral-gastric lavage using an 8 French catheter (Sovereign; Tyco/Healthcare, Mansfield, Massachusetts, USA). Briefly, the study contains 6 groups of piglets as follows: GROUP 1 -- negative control (n = 6); GROUP 2 -- Secondary bile acid mixture only (lithocholic acid and deoxycholic acid, 100 mg/kg each) (n = 6); GROUP 3 --C. hiranonis only (n = 12); GROUP 4 -- positive control (challenged with a toxigenic C. difficile strain) (n = 12); GROUP 5 -- secondary bile acid mixture and challenged with the toxigenic C. difficile strain (n = 12); and GROUP 6 -- C. hiranonis and challenged with the toxigenic C. difficile strain (n = 12). Treatments (C. hiranonis or secondary bile acid mixture) will be administrated intragastrically, approximately 4 h after birth. Piglets from GROUPS 3 and 6 received 2 × 106 C. hiranonis spores. Piglets in GROUPS 4, 5, and 6 will be challenged with 2 × 106 heat-shocked toxigenic C. difficile spores at 2nd day of birth. Piglets will be euthanized 72 h after challenge.Histopathology, quantification of toxin production and bacterial count: The intestinal segments 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 hematoxylin and eosin. These will be acquired with 100× magnifications using an Olympus BX51 microscope (Olympus Optical Company, Tokyo, Japan). 1g of loop contents will be diluted in 5 ml PBS, centrifuged at 3000g for 15 minutes and the supernatant will be collected for C. difficile toxin A/B ELISA using Techlab Tox A/B kit (Techlab, Blacksburg, VA) as per the manufacturer's instructions. Toxin quantification will be done using the standard curve method previously developed in Dr. Mooyottu's lab. Bacterial quantification will be performed using qPCR (copy number-standard curve method) with specific primers targeting C. difficile toxin B gene. DNA extracted for microbiome analysis will be used as the template for qPCR.Measurement of bile acids by liquid chromatography-mass spectrometry. Bile acid profile at postnatal day 5 will be performed as described in Aim 1.Data 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). The differences between the two groups will be analyzed using unpaired Student's t-test. "N1" Chi-squared test will be used to compare the incidence rate between two different treatments. The statistical significance level will be set at a P < 0.05.