Synbiotics can be used to reduce intestinal inflammation and mitigate dysbiosis in dogs with chronic inflammatory enteropathy (CIE). Prior research has not assessed the colonic mucosal ultrastructure of dogs with active CIE treated with synbiotics, nor has it determined a possible association between morphologic injury and signaling pathways. Twenty client-owned dogs diagnosed with CIE were randomized to receive either a hydrolyzed diet (placebo; PL) or a hydrolyzed diet supplemented with synbiotic-IgY (SYN) for 6 weeks. Endoscopic biopsies of the colon were obtained for histopathologic, ultrastructural, and molecular analyses and were compared before and after treatment. Using transmission electron microscopy (TEM), an analysis of the ultrastructural alterations in microvilli length (MVL), mitochondria (MITO), and rough endoplasmic reticulum (ER) was compared between treatment groups. To explore potential signaling pathways that might modulate MITO and ER stress, a transcriptomic analysis was also performed. The degree of mucosal ultrastructural pathology differed among individual dogs before and after treatment. Morphologic alterations in enterocytes, MVL, MITO, and ER were detected without significant differences between PL and SYN dogs prior to treatment. Notable changes in ultrastructural alterations were identified post-treatment, with SYN-treated dogs exhibiting significant improvement in MVL, MITO, and ER injury scores compared to PL-treated dogs. Transcriptomic profiling showed many pathways and key genes to be associated with MITO and ER injury. Multiple signaling pathways and their associated genes with protective effects, including fibroblast growth factor 2 (FGF2), fibroblast growth factor 7 (FGF7), fibroblast growth factor 10 (FGF10), synaptic Ras GTPase activating protein 1 (SynGAP1), RAS guanyl releasing protein 2 (RASGRP2), RAS guanyl releasing protein 3 (RASGRP3), thrombospondin 1 (THBS1), colony stimulating factor 1 (CSF1), colony stimulating factor 3 (CSF3), interleukin 21 receptor (IL21R), collagen type VI alpha 6 chain (COL6A6), ectodysplasin A receptor (EDAR), forkhead box P3 (FoxP3), follistatin (FST), gremlin 1 (GREM1), myocyte enhancer factor 2B (MEF2B), neuregulin 1 (NRG1), collagen type I alpha 1 chain (COL1A1), hepatocyte growth factor (HGF), 5-hydroxytryptamine receptor 7 (HTR7), and platelet derived growth factor receptor beta (PDGFR-β), were upregulated with SYN treatment. Differential gene expression was associated with improved MITO and ER ultrastructural integrity and a reduction in oxidative stress. Conversely, other genes, such as protein kinase cAMP-activated catalytic subunit beta (PRKACB), phospholipase A2 group XIIB (PLA2G12B), calmodulin 1 (CALM1), calmodulin 2 (CALM2), and interleukin-18 (IL18), which have harmful effects, were downregulated following SYN treatment. In dogs treated with PL, genes including PRKACB and CALM2 were upregulated, while other genes, such as FGF2, FGF10, SynGAP1, RASGRP2, RASGRP3, and IL21R, were downregulated. Dogs with CIE have colonic ultrastructural pathology at diagnosis, which improves following synbiotic treatment. Ultrastructural improvement is associated with an upregulation of protective genes and a downregulation of harmful genes that mediate their effects through multiple signaling pathways.

The effects of high-dose glucocorticoids on the gastrointestinal microbiota of healthy dogs are unknown. This study’s aim was to investigate the effects of immunosuppressive doses of prednisone on the fecal microbiota and the gastric and duodenal mucosal microbiota in healthy dogs. Twelve healthy adult dogs were enrolled into a randomized, double-blinded, placebo-controlled trial. Dogs were evaluated on days 0, 14, and 28 following treatments with either prednisone (2 mg/kg/d) or placebo. Outcome measures included (1) composition and abundance of the fecal microbiota (via high-throughput sequencing of the 16S rRNA gene and qPCR-based dysbiosis index [DI]) and (2) spatial distribution of the gastric and duodenal mucosal microbiota using fluorescence in situ hybridization (FISH). No significant difference in alpha and beta diversity or amplicon sequence variants of the fecal microbiota was observed between treatment groups. Blautia spp. concentrations via qPCR were significantly decreased between prednisone group timepoints 2 and 3. Compared to placebo group dogs, prednisone group dogs showed significantly increased gastric mucosal helicobacters and increased mucosal-associated total bacteria and Bacteroides in duodenal biopsies over the treatment period. The results indicate that immunosuppressive dosages of prednisone alter the mucosal microbiota of healthy dogs in a time-dependent manner, which may disrupt mucosal homeostasis. This report is significant, since it addresses a knowledge gap in our understanding of the effects of glucocorticoids on the gastrointestinal mucosal microbiota of healthy dogs.

This retrospective study assessed the potential of blood leukocyte ratios as diagnostic biomarkers in cats with chronic enteropathies (CE). Absolute neutrophil-to-lymphocyte (NLR), neutrophil-to-monocyte (NMR), and lymphocyte-to-monocyte (LMR) ratios were calculated from the hematological profiles of 221 cats, including healthy controls (n = 73), and those diagnosed with food-responsive enteropathy (FRE, n = 59), steroid-responsive enteropathy (SRE, n = 56), or small cell lymphoma (SCL, n = 33). SCL cats had higher NLRs than SRE (p = 0.002) and FRE (p = 0.028), and lower LMRs than SRE (p = 0.012) and FRE (p = 0.001). Healthy cats had lower NLRs compared to the FRE (p < 0.001), SRE (p < 0.001), and SCL (p < 0.001) cats and higher LMRs compared to the FRE (p < 0.001), SRE (p < 0.001), and SCL (p < 0.001) cats. Receiver operating characteristic (ROC) curve analysis demonstrated that NLR ≥ 11.6 differentiated SCL from SRE with 87.5% specificity but low sensitivity (39.4%). NMR ≥ 34.5 distinguished FRE from SRE with 52.5% sensitivity and 69.6% specificity. LMR ≥ 3.72 differentiated SRE from SCL with 67.9% sensitivity and 60.6% specificity. Although significant differences in leukocyte ratios were observed among groups, their diagnostic accuracy in differentiating CE phenotypes was suboptimal. These findings suggest that the utility of NLR, NMR, and LMR as standalone diagnostic tools is limited.

ABSTRACT Background Consumption of a high‐fat, high‐carbohydrate Western‐style diet (WD) associated with obesity and inflammation in humans has not been investigated in dogs. Aims To determine the effects of WD on inflammatory indices, microbiome, and fecal bile acids (BAs) in dogs. Animals Ten adult clinically healthy dogs. Methods A dietary trial compared the effects of two home‐prepared diets: a high‐fiber, low‐fat control diet (CD) to a diet containing the macronutrient composition of WD (low‐fiber, high fat). Dietary treatments were given sequentially for three feeding periods, each lasting 1 month. Outcome measures included molecular/microbiologic testing of colonic biopsies, histopathology, inflammatory biomarkers, and quantification of fecal BA following each feeding period. Results Cell markers of apoptosis (TUNEL‐positive cells: CD1, 0.36% ± 0.2%; WD, 0.79% ± 0.5%; CD2, 0.42% ± 0.3%; 95% CI) and inflammation (NF‐ĸB area: CD1, 8.09% ± 3.3%; WD, 11.58% ± 3.4%; CD2 7.25% ± 3.8%; 95% CI), as well as serum high‐sensitivity C‐reactive protein (CD1, 2.0 ± 0.4 ng/mL; WD, 2.76 ± 0.23 ng/mL; CD2, 2.29 ± 0.25 ng/mL; 95% CI), were increased (p < 0.05) in dogs fed WD versus CD. Other perturbations seen with WD ingestion included altered (p < 0.05) colonic mucosal bacteria (bacterial counts: CD1, 301.5 ± 188.5; WD, 769.8 ± 431.9; CD2, 542.1 ± 273.9; 95% CI) and increased (p < 0.05) fecal cholic acid (median and interquartile range/IQR: CD1, 9505 [2384–33 788] peak heights; WD, 34 131 [10 113–175 909] peak heights) and serum myeloperoxidase (CD1, 46.98 ± 16.6 ng/mL; WD, 82.93 ± 33.6 ng/mL; CD2, 63.52 ± 29.5 ng/mL; 95% CI). Conclusions and Clinical Importance WD fed to clinically healthy dogs promotes colonic dysbiosis, altered fecal BA, and low‐grade inflammation independent of obesity.

BACKGROUND:In previous studies, only a subset of dogs with chronic enteropathy (CE) had an increased dysbiosis index (DI) or altered fecal metabolites or both, suggesting differences in underlying intestinal pathophysiology between these subsets. OBJECTIVES:To compare microbial functional genes and fecal metabolites between healthy dogs with DI < 0 (HC) and dogs with CE and DI > 0 (increased DI-CE) or DI < 0 (normal DI-CE). ANIMALS:Retrospective cross-sectional study including 78 HC and 138 CE dogs. METHODS:Fecal microbiome was assessed by DNA shotgun sequencing. Dysbiosis index was quantified by qPCR. Targeted fecal metabolites, long-chain fatty acids, sterols, bile acids (BAs), and carbohydrates were measured using gas chromatography-mass spectrometry (GC-MS). RESULTS:In permutational analysis of variance (PERMANOVA), functional gene profiles showed larger shifts in increased DI-CE (median R2 [95% confidence interval (CI)] = 0.12 [0.08-0.17]) than normal DI-CE (0.02 [0.01-0.04]) compared with HC (adjusted-p < 0.02), characterized by increased counts of carbohydrate and lipid degradation genes. Similarly, increased DI-CE (PERMANOVA, median R2 [95% CI] = 0.23 [0.14-0.34]) had larger shifts in fecal metabolome than normal DI-CE (0.10 [0.04-0.20]; adjusted-p < 0.02). Increased DI-CE had lower fecal unconjugated secondary BAs percentage (95% CI; HC, 88.4%-96.4%; normal DI-CE, 79.8%-99.0%; increased DI-CE, 28.1%-64.1%) and transporter-independent carbohydrates (combined ribose, xylose, rhamnose, and arabinose) concentrations (1.6-2.6; 0.7-1.8; 0.3-1.3 ng/mg; adjusted-p < 0.01). CONCLUSIONS:Results indicate differences in fecal microbial gene profiles and metabolome in increased DI-CE versus normal DI-CE and HC, suggesting dogs with an increased DI have more severe intestinal changes in metabolic functions.

Sepsis is a potentially fatal condition characterized by organ dysfunction caused by an imbalanced immune response to infection. Although an increased inflammatory response significantly contributes to the pathogenesis of sepsis, several molecular mechanisms underlying the progression of sepsis are associated with increased cellular reactive oxygen species (ROS) generation and exhausted antioxidant pathways. This review article provides a comprehensive overview of the involvement of ROS in the pathophysiology of sepsis and the potential application of antioxidants with antimicrobial properties as an adjunct to primary therapies (fluid and antibiotic therapies) against sepsis. This article delves into the advantages and disadvantages associated with the utilization of antioxidants in the therapeutic approach to sepsis, which has been explored in a variety of animal models and clinical trials. While the application of antioxidants has been suggested as a potential therapy to suppress the immune response in cases where an intensified inflammatory reaction occurs, the use of multiple antioxidant agents can be beneficial as they can act additively or synergistically on different pathways, thereby enhancing the antioxidant defense. Furthermore, the utilization of immunoadjuvant therapy, specifically in septic patients displaying immunosuppressive tendencies, represents a promising advancement in sepsis therapy.

Chronic inflammatory enteropathies (CIEs) are an important group of diseases in dogs and involve complex pathogenetic aspects. Endoscopy and histopathology are vital for documenting the disease but are less useful for subclassifying CIEs and predicting the response to treatment. However, healing of the mucosal disease process (deep remission) and ultrastructural evaluation of the mucosa have received little attention in canine CIE. Given that canine CIE shares many similarities with inflammatory bowel diseases (IBDs) in human patients—and presents a good spontaneous disease model for human IBD—this perspective article evaluates the literature on ultrastructural lesions in canine CIE and human IBD and offers future directions for the study of ultrastructural mucosal lesions in canine CIE. Such lesions might have a higher sensitivity of detection than structural changes revealed upon light microscopy and may even precede or remain after the resolution of the clinical signs and histologic lesions.

The changing notion of “companion animals” and their increasing global status as family members underscores the dynamic interaction between gut microbiota and host health. This review provides a comprehensive understanding of the intricate microbial ecology within companion animals required to maintain overall health and prevent disease. Exploration of specific diseases and syndromes linked to gut microbiome alterations (dysbiosis), such as inflammatory bowel disease, obesity, and neurological conditions like epilepsy, are highlighted. In addition, this review provides an analysis of the various factors that impact the abundance of the gut microbiome like age, breed, habitual diet, and microbe-targeted interventions, such as probiotics. Detection methods including PCR-based algorithms, fluorescence in situ hybridisation, and 16S rRNA gene sequencing are reviewed, along with their limitations and the need for future advancements. Prospects for longitudinal investigations, functional dynamics exploration, and accurate identification of microbial signatures associated with specific health problems offer promising directions for future research. In summary, it is an attempt to provide a deeper insight into the orchestration of multiple microbial species shaping the health of companion animals and possible species-specific differences.

Objectives The aims of this study were to evaluate concentrations of symmetric dimethylarginine (SDMA) in hyperthyroid cats before and after radioiodine treatment, and to compare results with other variables used to assess kidney function in cats (creatinine, urine specific gravity [USG] and glomerular filtration rate [GFR] measured by renal scintigraphy). Methods Thirteen cats diagnosed with hyperthyroidism based on clinical signs and increased serum total thyroxine (TT4) were included in this prospective study. Study design included physical examination, complete blood count, serum chemistry, TT4, urinalysis and SDMA before treatment (T0) and at 1 month (T1) and 3 months post-treatment (T3). GFR was quantified by renal scintigraphy at T0 and T3. Results Median GFR decreased significantly from baseline (3.18 ml/kg/min; range 1.35–4.87) at T3 (2.22 ml/kg/min; range 1.81–3.42 [ P = 0.005]). While median creatinine and serum urea nitrogen increased post-treatment (creatinine: T0 = 0.8 mg/dl [range 0.4–1.1], T1 = 1.3 mg/dl [range 0.9–2]; T3 = 1.65 mg/dl [range 0.8–2.8]; P <0.001; serum urea nitrogen: T0 = 23 mg/dl [range 15–26]; T1 = 27 mg/dl [range 20–40]; T3 = 27.5 mg/dl [range 20–36]; P <0.001), SDMA and USG did not change significantly (SDMA: T0 = 11 µg/dl [range 7–15]; T1 = 12 µg/dl [range 6–16]; T3 = 10.5 µg/dl [range 8–21]; P = 0.789; USG: T0 = 1.030 [range 1.011–1.059]; T1 = 1.035 [range 1.012–1.044]; T3 = 1.030 [range 1.007–1.055]; P = 0.792). Conclusions and relevance Our data suggest that factors other than GFR may affect serum SDMA in hyperthyroid cats and that SDMA does not offer an advantage over other biomarkers traditionally used to predict changes in renal function following radioiodine therapy.

DNA shotgun sequencing is an untargeted approach for identifying changes in relative abundances, while qPCR allows reproducible quantification of specific bacteria. The canine dysbiosis index (DI) assesses the canine fecal microbiota by using a mathematical algorithm based on qPCR results. We evaluated the correlation between qPCR and shotgun sequencing using fecal samples from 296 dogs with different clinical phenotypes. While significant correlations were found between qPCR and sequencing, certain taxa were only detectable by qPCR and not by sequencing. Based on sequencing, less than 2% of bacterial species (17/1190) were consistently present in all healthy dogs (n = 76). Dogs with an abnormal DI had lower alpha-diversity compared to dogs with normal DI. Increases in the DI correctly predicted the gradual shifts in microbiota observed by sequencing: minor changes (R = 0.19, DI < 0 with any targeted taxa outside the reference interval, RI), mild-moderate changes (R = 0.24, 0 < DI < 2), and significant dysbiosis (R = 0.54, 0.73, and 0.91 for DI > 2, DI > 5, and DI > 8, respectively), compared to dogs with a normal DI (DI < 0, all targets within the RI), as higher R-values indicated larger dissimilarities. In conclusion, the qPCR-based DI is an effective indicator of overall microbiota shifts observed by shotgun sequencing in dogs.

The development of 3D organoids of the small intestine is a tremendous breakthrough in drug development and biological research. However, the development of colonic organoids (i.e., colonoids) is particularly challenging due to a lack of simple, cost-effective protocols for colonoid cultivation. Here, intestinal homogenates are described as a supplement to the culture medium for maintaining and replicating colonic stem cells. Colonoids generated by this cultivation protocol demonstrate substantial proliferation and differentiation (3 months). There is a similarity between cultured colonoids and primary colon tissue regarding structure and functionality. To evaluate the functionality of colonoids, permeability testing is performed with suspensions of 4 and 40 kDa fluorescein isothiocyanate-dextran (FITC-DEX). It is observed that neither can permeate the healthy epithelial barrier. The P-glycoprotein receptor, a vital drug efflux pump mitigating potential drug toxicity, is functionally manipulated, as evidenced by its inhibition function by verapamil and monitoring uptake of Rhodamin 123. In addition, Forskolin treatment which affects chloride transport results in organoid swelling; this confirms the functional expression of the CFTR transporter in the colonoids. This protocol to generate colonoids is promising for high-throughput drug screening, toxicity testing, and oral drug development.

Inflammatory bowel disease (IBD) is a chronic, relapsing gastrointestinal (GI) disorder characterized by intestinal inflammation. The etiology of IBD is multifactorial and results from a complex interplay between mucosal immunity, environmental factors, and host genetics. Future therapeutics for GI disorders, including IBD, that are driven by oxidative stress require a greater understanding of the cellular and molecular mechanisms mediated by reactive oxygen species (ROS). In the GI tract, oxidative stressors include infections and pro-inflammatory responses, which boost ROS generation by promoting the production of pro-inflammatory cytokines. Nuclear factor kappa B (NF-κB) and nuclear factor erythroid 2–related factor 2 (Nrf2) represent two important signaling pathways in intestinal immune cells that regulate numerous physiological processes, including anti-inflammatory and antioxidant activities. Natural antioxidant compounds exhibit ROS scavenging and increase antioxidant defense capacity to inhibit pro-oxidative enzymes, which may be useful in IBD treatment. In this review, we discuss various polyphenolic substances (such as resveratrol, curcumin, quercetin, green tea flavonoids, caffeic acid phenethyl ester, luteolin, xanthohumol, genistein, alpinetin, proanthocyanidins, anthocyanins, silymarin), phenolic compounds including thymol, alkaloids such as berberine, storage polysaccharides such as tamarind xyloglucan, and other phytochemicals represented by isothiocyanate sulforaphane and food/spices (such as ginger, flaxseed oil), as well as antioxidant hormones like melatonin that target cellular signaling pathways to reduce intestinal inflammation occurring with IBD.

A key component of efforts to identify the biological and drug-specific aspects contributing to therapeutic failure or unexpected exposure-associated toxicity is the study of drug–intestinal barrier interactions. While methods supporting such assessments are widely described for human therapeutics, relatively little information is available for similar evaluations in support of veterinary pharmaceuticals. There is, therefore, a critical need to develop novel approaches for evaluating drug–gut interactions in veterinary medicine. Three-dimensional (3D) organoids can address these difficulties in a reasonably affordable system that circumvents the need for more invasive in vivo assays in live animals. However, a first step in developing such systems is understanding organoid interactions in a 2D monolayer. Given the importance of orally administered medications for meeting the therapeutic need of companion animals, we demonstrate growth conditions under which canine-colonoid-derived intestinal epithelial cells survive, mature, and differentiate into confluent cell systems with high monolayer integrity. We further examine the applicability of this canine-colonoid-derived 2D model to assess the permeability of three structurally diverse, passively absorbed β-blockers (e.g., propranolol, metoprolol, and atenolol). Both the absorptive and secretive apparent permeability (Papp) of these drugs at two different pH conditions were evaluated in canine-colonoid-derived monolayers and compared with that of Caco-2 cells. This proof-of-concept study provides promising preliminary results with regard to the utility of canine-derived organoid monolayers for species-specific assessments of therapeutic drug passive permeability.

Chronic inflammatory enteropathy (CE) is a common cause of persistent gastrointestinal signs and intestinal inflammation in dogs. Since evidence links dysbiosis to mucosal inflammation, probiotics, prebiotics, or their combination (synbiotics) may reduce intestinal inflammation and ameliorate dysbiosis in affected dogs. This study's aim was to investigate the effects of the synbiotic-IgY supplement on clinical signs, inflammatory indices, and mucosal microbiota in dogs with CE. Dogs with CE were enrolled in a randomized prospective trial. Twenty-four client-owned dogs were fed a hydrolyzed diet and administered supplement or placebo (diet) for 6 weeks. Dogs were evaluated at diagnosis and 2- and 6-week post-treatment. Outcome measures included clinical activity, endoscopic and histologic scores, inflammatory markers (fecal calprotectin, C-reactive protein), and composition of the mucosal microbiota via FISH. Eleven supplement- and nine placebo-treated dogs completed the trial. After 6 weeks of therapy, clinical activity and endoscopic scores decreased in both groups. Compared to placebo-treated dogs, dogs administered supplement showed decreased calprotectin at 2-week post-treatment, decreased CRP at 2- and 6-week post-treatment increased mucosal Clostridia and Bacteroides and decreased Enterobacteriaceae in colonic biopsies at trial completion. Results suggest a beneficial effect of diet and supplements on host responses and mucosal microbiota in dogs with CE.

Organoids are three-dimensional structures of self-assembled cell aggregates that mimic anatomical features of in vivo organs and can serve as in vitro miniaturized organ models for drug testing. The most efficient way of studying drug toxicity and efficacy requires high-resolution imaging of a large number of organoids acquired in the least amount of time. Currently missing are suitable platforms capable of fast-paced high-content imaging of organoids. To address this knowledge gap, we present the OrganoidChip, a microfluidic imaging platform that incorporates a unique design to immobilize organoids for endpoint, fast imaging. The chip contains six parallel trapping areas, each having a staging and immobilization chamber, that receives organoids transferred from their native culture plates and anchors them, respectively. We first demonstrate that the OrganoidChip can efficiently immobilize intestinal and cardiac organoids without compromising their viability and functionality. Next, we show the capability of our device in assessing the dose-dependent responses of organoids' viability and spontaneous contraction properties to Doxorubicin treatment and obtaining results that are similar to off-chip experiments. Importantly, the chip enables organoid imaging at speeds that are an order of magnitude faster than conventional imaging platforms and prevents the acquisition of blurry images caused by organoid drifting, swimming, and fast stage movements. Taken together, the OrganoidChip is a promising microfluidic platform that can serve as a building block for a multiwell plate format that can provide high-throughput and high-resolution imaging of organoids in the future.

Over the last decade, chronic inflammatory enteropathies (CIE) in dogs have received great attention in the basic and clinical research arena. The 2010 ACVIM Consensus Statement, including guidelines for the diagnostic criteria for canine and feline CIE, was an important milestone to a more standardized approach to patients suspected of a CIE diagnosis. Great strides have been made since understanding the pathogenesis and classification of CIE in dogs, and novel diagnostic and treatment options have evolved. New concepts in the microbiome-host-interaction, metabolic pathways, crosstalk within the mucosal immune system, and extension to the gut-brain axis have emerged. Novel diagnostics have been developed, the clinical utility of which remains to be critically evaluated in the next coming years. New directions are also expected to lead to a larger spectrum of treatment options tailored to the individual patient. This review offers insights into emerging concepts and future directions proposed for further CIE research in dogs for the next decade to come.

In this study, we show that ketogenic diets (KD) in healthy dogs produce significant shifts in the serum metabolome which collectively suggest down-regulation of the glycolytic and amino-acid-dependent metabolism of many tumour types. As dogs represent valuable models for preclinical evaluation of oncologic treatment modal-ities in humans, 1 the data presented in this study will provide a resource upon which future investigations on the mechanistic effect of KDs on various tumours can be based.