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Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5-7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm 2 ) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Stewart, Kopper, McKinney-Aguirre, Veerasamy, Sahoo, Freund and Gonzalez.)

We investigated the effects of season and geographic location on detection of nucleic acids of potential enteric pathogens (PEPs) or their toxins (PEP-Ts) in feces of horses ≥6-mo-old in the United States. Results of 3,343 equine diarrhea PCR panels submitted to Idexx Laboratories for horses >6-mo-old were reviewed. Submission months were grouped into 4 seasons, and states were grouped into 4 geographic regions. Logistic regression was performed to assess effects of season and region on detection rates of PEPs and PEP-Ts. Agresti-Coull CIs were determined. Detection rate of Salmonella enterica was higher in the South in summer compared to all other regions, and was also higher in the South in fall compared to the Midwest and Northeast. The Neorickettsia risticii detection rate was lower during summer in the West and higher in fall in the Midwest. Detection of Cryptosporidium spp. was lower during spring, summer, and winter in the West. Differences were not identified for detection rates of Clostridioides difficile , Clostridium perfringens , Lawsonia intracellularis , Rhodococcus equi , equine rotavirus, and equine coronavirus. Overall, our data support seasonal and regional differences in detection rates of S. enterica , N. risticii , and Cryptosporidium spp. in horses ≥6-mo-old in the United States.

In a recent issue of the Lancet, the prevalence of Inflammatory Bowel Disease (IBD) was estimated at 7 million worldwide. Overall, the burden of IBD is rising globally, with direct and indirect healthcare costs ranging between $14.6 and $31.6 billion in the U.S. alone in 2014. There is currently no cure for IBD, and up to 40% of patients do not respond to medical therapy. Although the exact determinants of the disease pathophysiology remain unknown, the prevailing hypothesis involves complex interplay among host genetics, the intestinal microenvironment (primarily bacteria and dietary constituents), and the mucosal immune system. Importantly, multiple chronic diseases leading to high morbidity and mortality in modern western societies, including type II diabetes, IBD and colorectal cancer, have epidemiologically been linked to the consumption of high-calorie, low-fiber, high monosaccharide, and high-fat diets (HFD). More specifically, data from our laboratory and others have shown that repeated consumption of HFD triggers dysbiotic changes of the gut microbiome concomitant with a state of chronic intestinal inflammation and increased intestinal permeability. However, progress in our understanding of the effect of dietary interventions on IBD pathogenesis has been hampered by a lack of relevant animal models. Additionally, current in vitro cell culture systems are unable to emulate the in vivo interplay between the gut microbiome and the intestinal epithelium in a realistic and translatable way. There remains, therefore, a critical need to develop translatable in vitro and in vivo models that faithfully recapitulate human gut-specific physiological functions to facilitate detailed mechanistic studies on the impact of dietary interventions on gut homeostasis. While the study of murine models has been pivotal in advancing genetic and cellular discoveries, these animal systems often lack key clinical signs and temporal pathological changes representative of IBD. Specifically, some limitations of the mouse model are associated with the use of genetic knockouts to induce immune deficiency and disease. This is vastly different from the natural course of IBD developing in immunologically competent hosts, as is the case in humans and dogs. Noteworthily, abundant literature suggests that canine and human IBD share common clinical and molecular features, such that preclinical studies in dogs with naturally occurring IBD present an opportunity to further our understanding on disease pathogenesis and streamline the development of new therapeutic strategies. Using a stepwise approach, in vitro mechanistic studies investigating the contribution of dietary interventions to chronic intestinal inflammation and "gut leakiness" could be performed in intestinal organoids and organoid derived monolayers. The biologic potential of organoids stems from the method's ability to harness hard-wired cellular programming such that the complexity of the disease background can be reflected more accurately. Likewise, the effect of therapeutic drug candidates could be evaluated in organoids prior to longitudinal studies in dog and human patients with IBD. In this review, we will discuss the value (and limitations) of intestinal organoids derived from a spontaneous animal disease model of IBD (i.e., the dog), and how it can heighten understanding of the interplay between dietary interventions, the gut microbiota and intestinal inflammation. We will also review how intestinal organoids could be used to streamline the preclinical development of therapeutic drug candidates for IBD patients and their best four-legged friends., Competing Interests: Authors KA, AEJ and JPM are co-founders of 3D Health Solutions, Inc. Author EG is employed by Ceva Sante Animale and acts as a consultant of 3D Health Solutions, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kopper, Iennarella-Servantez, Jergens, Sahoo, Guillot, Bourgois-Mochel, Martinez, Allenspach and Mochel.)

Background: Rates of detecting ≥1 potential enteric pathogens (PEP) or toxins (PEP-T) in feces, blood, or both of horses ≥6 months of age with enteric disease and impact of multiple detections on outcome of horses with colitis has not been reported., Objective: To determine detection rates of PEP/PEP-T in feces, blood, or both of horses with enteric disease and effect of detecting multiple agents on outcome of horses with colitis., Animals: Thirty-seven hundred fifty-three fecal samples submitted to IDEXX Laboratories and 239 fecal and blood samples submitted to Michigan State University's Veterinary Diagnostic Laboratory (MSUVDL)., Methods: Retrospective evaluation of PEP/PEP-T testing results was performed to determine rates of detection of 1 or more PEP/PEP-T. Impact of detecting multiple agents on outcome was assessed in 239 horses hospitalized for colitis., Results: One or more PEP/PEP-T was detected in 1175/3753 (31.3%) and 145/239 (60.7%) of samples submitted to IDEXX Laboratories and MSUVDL, respectively. In a hospitalized cohort, survival to discharge was lower (76%) in horses with 1 agent, compared to horses with either no (88%) or multiple (89%) agents. There was no difference (P = .78) in days of hospitalization between horses with 0 (1-17), 1 (1-33), and > 1 positive (1-20) result. There was no difference in cost of hospitalization (P = .25) between horses with 0 ($2357, $1110-15 553), 1 ($2742, $788-11 005), and >1 positive ($2560, $1091-10 895) result., Conclusions and Clinical Importance: Detection rates of PEP/PEP-T in horses with colitis vary with cohorts and tests performed. Detection of more than 1 PEP or PEP-T did not affect outcome., (© 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

The objective of this study was to determine if transferable antimicrobial resistance (AMR) genes are present in commercial animal probiotics. DNA was extracted from 50 probiotics, tested for the presence of bacterial DNA, and analyzed by polymerase chain reaction (PCR) for the presence of 8 transferrable AMR genes, including tetracycline, erythromycin, aminoglycoside, sulfonamide, and trimethoprim. Samples that were positive by PCR were confirmed by genome sequencing. Forty-seven (94%) products contained bacterial DNA. Of these, 97% contained at least 1 AMR gene, and 82% contained 2 or more. These results indicate that further evaluation of the risk for transmission of these AMR genes may be warranted., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

The nematode Trichuris muris has been shown to interact with specific enteric bacteria, but its effects on the composition of its host's microbial community are not fully understood. We hypothesized that Trichuris muris -infected mice would have altered colon microbiota as compared with uninfected mice. Colon histopathology and microbial community structure and composition were examined in mouse models of colitis (C3BirTLR4 -/- IL10 -/- and C3H/HeJ TLR4 -/- IL10 +/+ mice) with and without T. muris infection, in uninfected C3BirIL10 -/- mice with and without spontaneous colitis, and in normal C3H/ HeJ mice. T. muris -infected mice developed colon lesions that were more severe than those seen in IL10-deficient mice. Ap- proximately 80% of infected IL10 -/- mice had colon neutrophilic exudates, and some had extraintestinal worms and bacteria. The composition and structure of proximal colon microbiota were assessed by using terminal restriction fragment length polymorphism analysis targeting the bacterial 16S rRNA gene. Colon microbiota in C3BirIL10 -/- and C3H/HeJ mice differed both qualitatively and quantitatively. Trichuris infection significantly altered the relative abundance of individual operational taxonomic units [OTU] but not the composition (presence or absence of OTU) of colon microbiota in the 2 mouse genotypes. When C3BirIL10 -/- and C3H/HeJ mouse OTU were considered separately, Trichuris was found to affect the microbiota of C3BirIL10 -/- mice but not of C3H/HeJ mice. Even though 34 of the 75 (45%) C3BirIL10 -/- mice had spontaneous colitis, neither qualitative nor quantitative differences were detected in microbiota between colitic or noncolitic C3BirIL10 -/- mice or noncolitic C3H/HeJ mice. Therefore, Trichuris -infected mice developed distinct microbial communities that were influenced by host background genes; these alterations cannot be attributed solely to colonic inflammation.

Objective: Vancomycin resistant enterococci (VRE) are of significant public health concern. The identification of VRE in livestock and food has increased. The objective of this study was to determine if the transferrable vancomycin resistance genes vanA and vanB were present in probiotics marketed for use in animals that claimed to contain Enterococcus spp., Results: Of the 40 products selected, Enterococcus spp. DNA was successfully extracted from 36 products. Of these 36 products with enterococcal DNA, 2 (6%) had a PCR product consistent with vanA which was confirmed by sequencing. None of the products appeared to contain vanB.

The objective of this study was to determine whether there was an association between type of colloid administered and survival of horses with enterocolitis (N = 92). A retrospective review of medical records of horses with enterocolitis treated with plasma or hetastarch (HES) between January 1, 2005 and December 31, 2011 was performed. Data collected included signalment, outcome, physical and clinicopathologic findings, and volume and type of colloid administered. Sixty-nine horses (75%) were treated with plasma and 23 horses (25%) were treated with HES. After accounting for confounding variables, horses treated with plasma (80% survival) were more likely to survive to discharge than horses treated with HES (47% survival; P = 0.041) despite similar disease severity at admission. This study provides support that use of natural colloids may be superior to treatment with synthetic colloids in horses with enterocolitis. A prospective, multi-center trial comparing outcome of critically ill equine patients treated with natural or synthetic colloids is warranted., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Background: Obtaining commercial fluids for intravenous administration (IVF) was challenging during a recent shortage. This necessitated use of custom-made non-sterile fluids for intravenous administration (JUGs) in some hospitals. There are no studies comparing outcome of horses treated with JUG versus IVF and limited information is available about adverse effects of JUGs., Hypothesis/objectives: To evaluate death, complications, blood pH, and plasma electrolyte concentrations of horses that received JUG versus IVFs., Animals: One hundred eighty-six horses that received IVFs and 37 that received JUGs., Methods: A retrospective review of medical records was performed to identify horses that received IVFs or JUGs during hospitalization. Information including survival to discharge, complications (fever [>38.5°C], jugular vein phlebitis/thrombosis, arrhythmia, or laminitis), blood pH, and plasma electrolyte concentrations were obtained., Results: There was no difference (P = .67) in survival to discharge for horses that received JUGs (78%) compared to horses that received IVFs (87%). Horses that received JUGs were more likely to develop a jugular vein complication (3 of 37 versus 1 of 186, odds ratio 17.2 [95% CI 1.9-389.8], P = .04). Horses that received JUGs were more likely to have electrolyte abnormalities consistent with hyperchloremic metabolic acidosis., Conclusion and Clinical Importance: Veterinarians using JUGs should obtain informed client consent because of a potential increased risk of jugular vein complications. Chloride content of JUGs should be considered to limit development of hyperchloremic metabolic acidosis., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Objective: To investigate effects of body condition on permeability of intestinal mucosa in horses., Animals: 13 horses (7 obese and 6 lean) from 8 to 15 years of age., Procedures: Body condition score was assessed, and an oral sugar test (OST) was performed to evaluate glucose and insulin dynamics. Horses were allowed a 2-week diet acclimation period and were then euthanized. Tissue samples were collected from the jejunum, ileum, cecum, pelvic flexure, right dorsal colon, and rectum. Mucosal permeability was assessed by measuring transepithelial resistance and lipopolysaccharide (LPS) flux across tissue samples mounted in Ussing chambers., Results: 5 obese horses and 1 lean horse had evidence of insulin dysregulation, whereas 1 obese and 5 lean horses had no abnormalities in results of the OST. Results for the OST were not available for 1 obese horse. Mucosal transepithelial resistance did not differ in any intestinal segment between obese and lean horses. Obese horses had a significantly higher LPS flux across jejunal mucosa, compared with results for lean horses, but there were no significant differences between obese and lean horses for other intestinal segments., Conclusions and Clinical Relevance: Obese horses may have had greater paracellular mucosal permeability of jejunal mucosa to LPS, compared with that for lean horses. This finding was consistent with data for the gastrointestinal mucosa of humans and mice and supported the hypothesis that obese horses may be at higher risk from chronic exposure to increased amounts of LPS, compared with the risk for lean horses.

We used terminal restriction fragment polymorphism (T-RFLP) analysis to assess (1) stability of the fecal microbiota in dogs living in environments characterized by varying degrees of exposure to factors that might alter the microbiota and (2) changes in the microbiota associated with acute episodes of diarrhea. Results showed that the healthy canine GI tract harbors potential enteric pathogens. Dogs living in an environment providing minimal exposure to factors that might alter the microbiota had similar microbiotas; the microbiotas of dogs kept in more variable environments were more variable. Substantial changes in the microbiota occurred during diarrheic episodes, including increased levels of Clostridium perfringens, Enterococcus faecalis, and Enterococcus faecium. When diet and medications of a dog having a previously stable microbiota were changed repeatedly, the microbiota also changed repeatedly. Temporal trend analysis showed directional changes in the microbiota after perturbation, a return to the starting condition, and then fluctuating changes over time.