Progress 01/29/20 to 09/30/20
Outputs
Target Audience:The target audience of the outcome of this project is the swine industry, biomedical researchers, animal and health scientists Changes/Problems:No major changes or problems reported during this period. What opportunities for training and professional development has the project provided?This project is part of a graduate student Ph.D. thesis. How have the results been disseminated to communities of interest?The manuscript on the developed modelis under preparation and will be submitted for in a peer-reviewed biomedical journal. What do you plan to do during the next reporting period to accomplish the goals?D. Plan for the next year Aim 2: Effect of C. hiranonis colonization on C. difficile pathogenesis in swine in situ ileal loop model: Results from the above experiments will provide us with the minimum time-period required for establishing reconstituted microflora within the surgically created ileal loops. To investigate the effect of C. hiranonis BAI-17 colonization on C. difficile pathogenesis in swine ileal loops, the surgical procedure described above will be repeated in 6 pigs (Figure 2, steps 1 to 4). A reconstituted microbiome mix (prepared from adult pig ileal content with and without C. hiranonis BAI-17) will be inoculated into the ileal loops of each pig. After 10-30 days of colonization (based on the results from Experiment 1), a second, small paramedian (or flank) laparotomy incision will be made for inoculating 107 CFU/ml C. difficile mixed with 0.1% taurocholic acid into the ileal loops (Figure 2, step 5) under anesthesia. The incision site will be closed and post-operative care will be provided as per standard protocol. After 2 days, animals will be euthanized to collect ileal tissues and contents for histopathologic, molecular and microbiologic analysis. We will perform surgery on a total of six animals, and on any given day, we will only perform a maximum of three surgeries (n=3/day). We will get 1 pairwise comparison (N=1) from each pig (Normal microbiome loop and Microbiome + C. hiranonis loop with and without C. difficile) which will provide total 6 repeats of (N=6) pairwise treatment comparisons from a total of six pigs. Figure 2: Surgical pig ileal loop model (steps 1-4). Following laparotomy, ileum is exteriorized and resected at two places separated by about 6- 8 inches (labelled A and B, step 1). The cut ends are anastomosed (step 2) and middle segment (labeled C) was rinsed with warm PBS, incubated with antibiotic cocktails and incised at three spots (step 3) to create four blind-ended compartments (C1-C4, step 4) and administer different treatments as indicated. Surgical pig ileal loop model of C. difficile infection (steps 1-5). After establishing the reconstituted microbiome in the pig ileal loop model, a second laparotomy will be performed to access the previously constructed compartments (C1-C4) to inoculate C. difficile spores into ileal loops with reconstituted microbiome with and without C. hiranonis (step 5). Microbiome analysis: DNA will be extracted from 0.25 g of ileal loop contents 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. Histopathology, quantification of toxin production and bacterial count: The ileal loops 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). 1 g 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. LC-ESI-MS/MS detection of each bile acid in fecal specimens or reference standards will be performed with a Shimadzu UFLC coupled to a BetaSil C18 HPLC column (50 mm × 2.1 mm × 3 μm; Thermo Fisher Scientific) and an AB Sciex API 4000 QTrap mass spectrometer (AB Sciex) running in negative-ion electrospray ionization mode (ESI) using a Turbo V ESI ion source. Authentic bile acid standards primary and secondary will be used to prepare 1 μM samples in 80% methanol and HPLC will be conducted using previously published protocol. Expected results, anticipated problems, alternative approaches, and quality control: We expect to have stable ileal loops in 100% of the animals operated. Establishment of a stable microbiome within each ileal loops is expected by 20 days post-surgery without any complications. We may observe adhesions in a small percentage of animals making a second laparotomy for inducing infection difficult. Alternatively, we may perform endoscopic surgery for inoculating C. difficile spores after the establishment of stable reconstituted flora (details in Figure 2). We expect moderate to severe C. difficile toxin-induced mucosal inflammation two days post-challenge (on the day of euthanasia) in respective ileal loops. If our hypothesis is correct, we will be able to demonstrate a reduced viable C. difficile load, lower C. difficile toxin production and a less severe (or absent) ileal inflammation along with a high concentration of secondary bile acids within the loops colonized with C. hiranonis, compared to controls. Plans for extramural grant submissions: a) USDA-NIH Dual Purpose grant program (Research in Biomedicine and Agriculture Using Agriculturally Important Domestic Animal Species- R-01): This seed grant proposal is specifically designed to yield background information for USDA-NIH 'dual-purpose dual benefit grant program RO1', which explores opportunities to use large animals in biomedical, agricultural and biological systems research. Although this funding opportunity ended last year, a strategic planning workshop (attended by PI) was held at NIH, Bethesda, MD to develop specific recommendations for future research over the next 5-10 years to facilitate interagency grants programs between the NIH, NSF, and USDA. The redesigned program emphasizes high priority areas such as gut microbiome - host interactions and utilizing swine as a better model that benefits both human and agricultural animals. b) NIH R-21 and R-15 research proposals: The results obtained from this project will be used as preliminary data for various NIH grant proposals, which include but are not limited to R-21 and R-15 funding programs. tive. c) National Pork Board (Pork checkoff) Research Grant: This project comes under important NPB subject areas such as Public Health (Antibiotic alternatives) and Infectious Diseases (C. difficile). Grant submitted (with the model developed from this project): NIAID R-21: "Investigating the role of p-cresol on the pathogenesis of C. difficile infection on a novel swine ileal loop model" PIs: Mooyottu, S and Charavaryamath, C. $410,000; not funded. Resubmission due: November 2020.
Impacts
What was accomplished under these goals?
Progress to Date At the initiation of this project we had proposed a central hypothesis that pig gut loop model serves as a relevant model to test the efficacy of bile acid converting bacterium C. hiranonis BAI-17 in controlling CDI pathogenesis. We had proposed to test our central hypothesis using the following two specific aims: 1) To develop a swine in situ ileal loop model for gut microbiome-host interface, and determine the colonization pattern and stability of specific reconstituted microbial communities within the loops 2) To investigate the efficacy of C. hiranonis in controlling CDI pathogenesis using newly developed swine in situ ileal loop model for gut microbiome-host interface Aim 1: In the first year of the study we had focused on the first specific aim which is to develop a swine in situ surgical ileal loop model for creating a suitable gut microbiome- host interface. Towards this objective, we have developed a novel surgical in situ swine ileal loop model. We created four blind ended, isolated ileal loops with intact mesenteric attachments including the draining lymph nodes within the peritoneal cavity of 1-3-month-old pigs, using aseptic surgical techniques after getting approval from the IACUC committee. Briefly, a midline celiotomy was performed under inhalant isoflurane anesthesia in 3-4-month-old pigs. An approximate 10-12-inch segment of the ileum was identified and exteriorized from the abdomen.Doyen intestinal was applied to each end followed by sharp resection of ileal segment, with preservation of mesenteric vasculature of the resected segment. The oral and aboral ends of the ileum were anastomosed in a hand-sewn fashion using 3-0 absorbable suture in a Lembert pattern.The resected segment of the ileum was packed off from the remainder of the abdomen to prevent contamination of the peritoneum and abdominal cavity during further manipulation. The ileal segment was rinsed with warm phosphate buffer saline (PBS). Further, the entire ileal segment was filled with an antibiotic cocktail containing metronidazole and enrofloxacin for 25 minutes, while the segment will be kept moist with sterile gauzes moistened with PBS. Following removal of the antibiotic cocktail by rinsing with PBS, the segment was further subdivided into four compartments (loops) (C1-C4) using simple interrupted ligatures using silk suture. Each compartment was injected with 1-2 ml of a Homogenous Human Fecal Microbiome Mixture (HFM) with and without 104 CFU/ml Clostridium hiranonis BAI-17. The ends of the ileal segment were over sewn in a Parker-Kerr suture pattern using 3-0 absorbable suture. The linea alba was closed in a simple continuous pattern using #2 absorbable suture.The overlying skin was closed in a Ford interlocking pattern using #1 non-absorbable braided nylon (or skin staples if available).Postoperative care was provided as per standard protocol. We performed the surgery on a total of five animals to date. All animals survived the surgery and recuperated without any incident. All animals started taking feed on the day of the surgery itself and was producing normal feces by the third day post-surgery. The first animal which underwent surgery was considered as a preliminary and exploratory study of the novel surgical protocol and hence was euthanized five days post-surgery to ascertain the progress of the development and characteristics of the gut microbiome in the unique environment of a closed loop. During the next batch of surgeries two of the animals were euthanized on the seventh day post-surgery to ascertain the actual study parameters and collect relevant tissue samples. The last two animals which underwent surgery is currently recuperating without incidents and will be euthanized on the seventh day post-surgery as per approved project guidelines. During necropsy, the ileal anastomoses sites were found to be patent, healing or fully healed, properly functional and intact (Figure 2). Tissue sections of the ileal loops, normal ileum, anastomoses sites were collected in 10% Neutral Buffered Formalin for histopathology to ascertain the microscopic changes and colonization characteristics that might have manifested in the unique closed ileal loop. Frozen samples of all tissues were also collected for molecular chemistry analyses to determine the recruitment of inflammatory/ pro inflammatory mechanisms in the cellular level. The luminal microbiome content of all loops and normal ileum was collected for metagenomics and 16s rRNA sequencing to analyze the identity and heterogenicity of the microbial communities. All results are pending to date. The results of the study so far indicate that the novel surgical in situ swine ileal loop model is a successful technique that can be adopted to study various aspects related to gut microbiology, inflammation, infection and pathology.
Publications
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