Location: Food and Feed Safety Research
Project Number: 3091-32000-035-09-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2017
End Date: Aug 31, 2020
Evaluate the effects of epizootic bacteria, Salmonella, and Campylobacter on non-coding small RNAs derived from extracellular vesicles (EVS) in the peripheral circulation system by small RNA sequencing. Isolation and characterization of extracellular vesicles from peripheral blood by transmission electron microscopy, immunoblotting, and Nanosight particle tracking system. Determine differential expression of small RNAs and microRNAs in extracellular vesicles from infected birds. Determine the effects of Salmonella and Campylobacter on soluble factors produced by intestinal organoids. Develop in vitro models such as 3-D intestinal crypt-derived enteroids and enteric neuronal and glial cells from myenteric plexus to study host-pathogen interaction. Evaluate the effects of conditioned media from enteroids infected with Salmonella and Campylobacter on enteric neurons.
Experimental birds, bacteria challenge, and serum collection: Day-old chickens will be randomly distributed into two experimental groups: Non-infected control (20 birds) and infected (20 birds). Four days post-hatch, all birds will be orally challenged with Salmonella and Campylobacter. At 1 and 4 d post-challenge, chickens will be euthanized and necropsied. Peripheral blood (PBS) will be collected and allowed to clot for 1 h at room temperature, and will be centrifuged at 2,000×g for 10 min. Isolation of EVs: Serum will be diluted with an equal volume of PBS. EV pellets will be isolated by centrifugation at 100,000 × g for 90 min. Transmission electron microscopy (TEM), Immunoblot Analysis, and Nanoparticle Tracking System (NTS): EVs will be loaded on copper-coated electron microscopy grids, contrasted in 1% uranyl acetate, and observed using a TEM. Immunoblot will be tested if isolated EVs are positive for exosomal protein markers, HSP 70, Tsg101, and CD63. The concentrations of EVs will be characterized by NTS. Construction of small RNA libraries, Hi-Sequencing and Data Analysis: Isolated small RNA will be constructed in the small RNA library. Briefly, input RNAs will be ligated by 3' and 5' adapters, reverse-transcribed, and PCR amplified. The pooled libraries will be loaded onto two lanes of an 8-lane flowcell for cluster formation and sequenced. Raw sequence counts will be processed in a bioinformatics pipeline including a quality control filter, trimming of adapters, consolidation of reads across samples, and alignment of reads against reference databases. Each sequence read that aligns to the mature miRNA database will be normalized and tabulated in terms of its expression in each sample, its overall sum, its mean expression in each group, the ratio across groups, and measures of statistical significance using a Kruskal-Wallis test. Gene pathway will be analyzed by commercial software. Isolation of intestinal crypt-derived enteroids and organoid culture: For ex vivo analyses, small intestinal crypts from day-old chickens will be isolated and cultured to mature organoids described by Fan et al (2016). Preparation of isolated enteric neuronal and glial cells from myenteric plexus: The longitudinal and circular muscle layers of small intestine will be dissected. The diced tissues will be incubated in collagenase and dispase dissolved in HBSS with Ca2+ and Mg2+ for 30 min at 37°C. Cell suspension will be collected and cultured in DMEM/F-12 medium supplemented with N2, bFGF, EGF, penicillin/streptomycin, and L-glutamine. Infection of intestinal enteroids: Isolated intestinal enteroids will be challenged with Salmonella and Campylobacter or left uninfected. Conditioned media (CM) will be collected. The effects of CM from infected intestinal enteroids on enteric neurons: CM from non-infected and infected enteroids will be added to enteric neurons and glial cells. Kinome and RNA seq will be performed to determine the effects of the soluble factors produced from intestinal enteroids infected with Salmonella and Campylobacter on enteric neurons and glial cells.