A canine case of Ehrlichia canis infection without a history of being in an endemic area in Japan.

A mixed-breed, 8-year-old male dog developed neutropenia, thrombocytopenia, and hyperglobulinemia. Bone marrow hyperplasia and splenic plasmacytosis were cytologically observed. The dog had never been outside of Tokyo or Shizuoka Prefecture. Splenectomy was performed to confirm and remove the cause of splenic plasmacytosis. A histopathological diagnosis of splenic plasmacytoma was made; however, serum protein electrophoresis showed polyclonal gammopathy. Further screening was performed, and Ehrlichia canis infection was confirmed. The dog was treated with doxycycline for 5 weeks. After the antibiotic therapy, no relapse of neutropenia, thrombocytopenia, hyperglobulinemia, or positive polymerase chain reaction result of E. canis infection was observed for 3 years. Careful attention should be given to ehrlichiosis when exploring the cause of pancytopenia or hyperglobulinemia, regardless of the travel history.

Large granular lymphocyte lymphoma in the skin and urinary bladder of a dog.

A 10-year-old female Cavalier King Charles Spaniel presented with hematuria, pollakiuria and skin rash. Based on the histopathological and cytological examination of the skin and bladder mucosa, the dog was diagnosed with large granular lymphocytic (LGL) lymphoma of the bladder and skin. The dog responded well to the initial chemotherapy with nimustine for 3 months. Since recurrence of skin erosion and bladder wall thickening were observed, the dog was subsequently administered chemotherapy with other anticancer drugs, including chlorambucil, vincristine, doxorubicin, L-asparaginase, cytosine arabinoside, and cyclophosphamide. The dog survived for 11 months and died due to tumor-related disseminated intravascular coagulation. This is the first report of a canine case of LGL lymphoma in the skin and bladder.

Case Report: 1-Year Follow-Up of Vagus Nerve Stimulation in a Dog With Drug-Resistant Epilepsy.

A vagus nerve stimulation (VNS) system was surgically implanted to treat drug-resistant epilepsy in a 5-year-old male Shetland Sheepdog. At regular visits during a 1-year follow-up, treatment efficacy and adverse effects were assessed, and programmable stimulation parameters were adjusted to optimize stimulation intensity while avoiding adverse effects. The frequency of generalized tonic-clonic seizures was reduced by 87% after the initiation of VNS. The owner reported that the dog regained his personality, and the quality of life of both the dog and owner improved. The only adverse effect of VNS was a cough that was controlled by adjusting stimulation parameters. There were no surgical complications or other issues with the VNS device. This is the first long-term evaluation of VNS therapy in a dog, and the results obtained suggest that gradual adjustments of VNS parameters facilitate optimum VNS dosing.

Successful treatment and long-term follow up of idiopathic immune-mediated polyarthritis with mycophenolate mofetil in a cat.

CASE SUMMARY: A 7-year-old neutered male Norwegian Forest Cat was presented with decreased appetite and activity, weight loss, fever, neutrophilia and hyperglobulinaemia. A physical examination showed painful stifle joints and enlarged popliteal lymph nodes. Blood examination showed neutrophilia, hyperglobulinaemia and increased serum amyloid A. Urinalysis, thoracic and abdominal radiographs, and abdominal ultrasonography were unremarkable. Synovial fluid from the knee joints had diminished viscosity and revealed neutrophilic inflammation on the smear. There was no evidence of infection in a microbiological culture of the synovial fluid. A diagnosis of idiopathic immune-mediated polyarthritis (IMPA) was made. Prednisolone was initiated at 2 mg/kg q24h PO and tapered with additional immunosuppressants (leflunomide, ciclosporin A and methotrexate); however, prednisolone could not be discontinued. Informed consent was obtained from the owner and mycophenolate mofetil (MMF) at a dosage of 10 mg/kg q12h PO was initiated on day 798. There were no adverse effects of MMF and prednisolone was discontinued on day 1183. Clinical signs resolved and the cat's general condition remained stable with MMF alone at a dosage of 10 mg/kg q48h PO on day 1600. RELEVANCE AND NOVEL INFORMATION: There is limited information describing feline IMPA and its treatment options other than the use of prednisolone. This is the first report of the successful treatment and long-term follow-up of feline IMPA with MMF. MMF may be a safe and effective option as an additional immunosuppressant in feline IMPA.

Conversion of mesenchymal stem cells into a canine hepatocyte-like cells by Foxa1 and Hnf4a.

INTRODUCTION: Hepatocytes, which account for the majority of liver tissue, are derived from the endoderm and become hepatocytes via differentiation of hepatic progenitor cells. Induced hepatocyte-like (iHep) cells and artificial liver tissues are expected to become useful, efficient therapies for severe and refractory liver diseases and to contribute to drug discovery research. The establishment of iHep cell lines are needed to carry out liver transplants and assess liver toxicity in the rising number of dogs affected by liver disease. Recently, direct conversion of non-hepatocyte cells into iHep cells was achieved by transfecting mouse adult fibroblasts with the Forkhead box protein A1 (Foxa1) and hepatocyte nuclear factor 4 homeobox alpha (Hnf4α) genes. Here, we applied this conversion process for the differentiation of canine bone marrow stem cells (cBMSCs) into hepatocyte-like cells. METHODS: Bone marrow specimens were collected from four healthy Beagle dogs and used to culture cBMSCs in Dulbecco's Modified Eagle's Medium (DMEM). The cBMSCs displayed the following characteristic features: plastic adherence; differentiation into adipocytes, osteoblasts and chondrocytes; and a cell surface antigen profile of CD29 (+), CD44 (+), CD90 (+), CD45 (-), CD34 (-) and CD14 (-), or CD11b (-) and CD79a (-), or CD19 (-) and HLA class II(-). The cBMSCs were seeded in a collagen I-coated plate and cultured in DMEM with 10% fetal bovine serum and transfected with retroviruses expressing Foxa1 and Hnf4α the following day. Canine iHep cells were differentiated from cBMSCs in culture on day 10, and were analyzed for morphology, RNA expression, immunocytochemistry, urea production, and low-density lipoprotein (LDL) metabolism. RESULTS: The cBMSCs expressed CD29 (98.06 ± 1.14%), CD44 (99.59 ± 0.27%) and CD90 (92.78 ± 4.89%), but did not express CD14 (0.47 ± 0.29%), CD19 (0.44 ± 0.39%), CD34 (0.33 ± 0.25%), CD45 (0.46 ± 0.34%) or MHC class II (0.54 ± 0.40%). The iHep cells exhibited morphology that included circular to equilateral circular shapes, and the formation of colonies that adhered to each other 10 days after Foxa1 and Hnf4α transfection. Quantitative RT-PCR analysis showed that the expression levels of the genes encoding albumin (ALB) and cadherin (CDH) in iHep cells on day 10 were increased approximately 100- and 10,000-fold, respectively, compared with cBMSCs. Corresponding protein expression of ALB and epithelial-CDH was confirmed by immunocytochemistry. Important hepatic functions, including LDL metabolic ability and urea production, were increased in iHep cells on day 10. CONCLUSION: We successfully induced cBMSCs to differentiate into functional iHep cells. To our knowledge, this is the first report of canine liver tissue differentiation using Foxa1 and Hnf4α gene transfection. Canine iHep cells are expected to provide insights for the construction of liver models for drug discovery research and may serve as potential therapeutics for canine liver disease.

Three-dimensional spheroid culture of canine hepatocyte-like cells derived from bone marrow mesenchymal stem cells.

INTRODUCTION: Primary cultured hepatocytes are an important model for early safety evaluations of newly developed drugs. Many factors, however, hinder the wider applications of this technology, especially the difficulty to maintain these cells in long-term culture. To date, creating a stable supply of human or animal hepatocytes with proper hepatic function in vitro has not been achieved. Furthermore, frequently harvesting hepatocytes from living donors for use in culture is highly invasive and simply not feasible. We have previously reported that canine bone marrow-derived mesenchymal stem cells (cBMSCs) can be effectively converted into induced hepatocyte-like cells (iHep cells); however, these cells had reduced function in comparison to mature hepatocytes. In recent studies, spheroid formation-based three-dimensional (3D) culture has been noted to greatly increase hepatocyte function; nevertheless, no reports have described the use of this technology for culturing canine hepatocytes. Therefore, in this study, we aimed to establish a 3D spheroid culture using converted canine iHep cells to investigate their function as hepatocytes. METHODS: The iHep cells were prepared by introducing two genes, namely, the Forkhead box A1 (Foxa1) and hepatocyte nuclear factor 4 homeobox alpha (Hnf4α), into cBMSCs seeded onto an ultra-low attachment microplate to induce spheroid formation. Thereafter, the hepatic functions of these spheroids were evaluated using immunocytochemistry, as well as qualitative and quantitative PCR. RESULTS: Notably, albumin was observed in the iHep spheroids and the expression of hepatic genes, such as albumin and drug metabolism CYP genes, could also be detected. Another interesting finding was evident upon further comparing the quantified albumin gene and CYP2E1 gene expressions in the two-dimensional and three-dimensional culture systems; notably, a 100- to 200-fold increase in gene expression levels was observed in the three-dimensional spheroids when compared to those in conventional monolayers. CONCLUSIONS: Upon incorporating three-dimensional technology, we managed to achieve iHep spheroids that are closer in gene expression to living liver tissue compared to conventional monolayer cultures. Thus, we are one step closer to creating a sustainable in vitro hepatocyte model. Furthermore, we believe that this system is capable of maintaining the stable drug metabolizing capacity of canine hepatocytes in vitro, which might be useful in improving current drug assessment studies.

Distribution of regulatory T cells in inflammatory colorectal polyps of miniature dachshunds.

Inflammatory colorectal polyp (ICRP) is an emerging disease in Miniature Dachshunds (MDs). Animals with this disease exhibit multiple polyps with severe neutrophil infiltration that respond to immunosuppressive therapy. Macrophages in polypoid lesions have been described to play an important role in neutrophil infiltration in the lesion by producing IL-8. In contrast, IL-10, an anti-inflammatory cytokine, was also reported to be upregulated in polypoid lesions, but its significance in the pathogenesis of ICRP has not been clarified. Regulatory T cells (Tregs) are the main source of IL-10 production and contribute to the maintenance of intestinal homeostasis. Therefore, the objective of this research was to compare the distribution of Tregs in polypoid lesions of ICRPs and the association between the distribution and expression of pro- or anti-inflammatory cytokines. Tissue biopsy specimens of polypoid lesions were collected from 28 MDs with ICRP. Those of macroscopically non-polypoid colonic mucosa from 24 MDs with ICRPs and 21 control dogs were further included as controls. Real-time quantitative polymerase chain reaction was used to quantify gene expression of IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-17, IL-22, IFN-γ, TNF-α, TGF-ß, and forkhead box protein P3 (Foxp3) in each tissue sample. The numbers of Foxp3-positive cells (Tregs) and ionized calcium binding adapter molecule 1 (Iba-1)-positive cells (macrophages) were determined by immunohistochemistry. The gene expression of IL-1ß, IL-6, IL-8, TNF-α, IFN-γ, IL-17, IL-10, TGF-ß, and Foxp3 was significantly upregulated in polypoid lesions relative to control levels. The numbers of Foxp3-positive Tregs and Iba-1-positive macrophages were significantly increased in polypoid lesions compared to those in the non-polypoid colonic mucosa of MDs with ICRPs and control dogs. The upregulation of IL-10 was moderately correlated with the distribution of Tregs in polypoid lesions from MDs with ICRPs. In addition, the relative upregulation of IL-1ß, IL-6, and IL-8 in polypoid lesions, compared to expression in non-polypoid colonic mucosa of MDs with ICRPs, was significantly greater than that of IL-10. These results indicate that increases in Treg numbers and anti-inflammatory cytokines in polypoid lesions comprise reactive changes in response to the inflammation, which warrants further investigation.

Age-related analysis of the gut microbiome in a purebred dog colony.

Dogs are model animals that can be used to study the gut microbiome. Although the gut microbiome is assumed to be closely related to aging, information pertaining to this relationship in dogs is limited. Here, we examined the association between the canine gut microbiome and age via a bacterial 16S rRNA gene amplicon sequence analysis in a colony of 43 Japanese purebred Shiba Inu dogs. We found that microbial diversity tended to decrease with aging. A differential abundance analysis showed an association of a single specific microbe with aging. The age-related coabundance network analysis showed that two microbial network modules were positively and negatively associated with aging, respectively. These results suggest that the dog gut microbiome is likely to vary with aging.

Fecal microbiome in dogs with inflammatory bowel disease and intestinal lymphoma.

Although alteration of commensal microbiota is associated with chronic gastrointestinal (GI) diseases such as inflammatory bowel disease (IBD) in dogs, the microbiota composition in intestinal lymphoma, an important differential diagnosis of canine IBD, has not been investigated. The objective of this study was to compare the fecal microbiota in dogs with IBD, dogs with intestinal lymphoma, and healthy dogs. Eight dogs with IBD, eight dogs with intestinal lymphoma, and fifteen healthy dogs were included in the study. Fecal samples were analyzed by 16S rRNA gene next-generation sequencing. Rarefaction analysis failed to reveal any difference in bacterial diversity among healthy dogs and diseased dogs. Based on PCoA plots of unweighted UniFrac distances, the bacterial composition in dogs with intestinal lymphoma was different from those observed in dogs with IBD and healthy dogs. When compared with healthy dogs, intestinal lymphoma subjects showed significant increases in organisms belonging to the Eubacteriaceae family. The proportion of the family Paraprevotellaceae and the genus Porphyromonas was significantly higher in dogs with IBD compared to healthy dogs. These observations suggest that dysbiosis is associated with intestinal lymphoma as well as IBD in dogs.

Analysis of fecal short chain fatty acid concentration in miniature dachshunds with inflammatory colorectal polyps.

Short chain fatty acids (SCFAs) play an important role in the maintenance of colonic homeostasis, and their depletion has been reported in various gastrointestinal disorders. Inflammatory colorectal polyps (ICRPs) are a recently recognized disease specific to miniature dachshunds (MDs), and fecal dysbiosis with a reduction of SCFA-producing bacteria has been reported with this disease. Therefore, this study was performed based on the hypothesis that a reduced SCFA concentration associates with the development of ICRPs. We recruited 11 ICRP-affected MDs and 25 control MDs. Their fecal SCFA concentrations and bacterial proportions were quantified using high performance liquid chromatography and quantitative real-time PCR, respectively. The feces of ICRP-affected MDs contained lower amounts of propionic acid and lower proportions of Bifidobacterium than the feces of control MDs. Furthermore, fecal proportions of Bifidobacterium, Firmicutes and Lactobacillus exhibited significant positive correlations with fecal concentrations of total SCFAs and/or propionic acid; fecal Escherichia coli proportions correlated negatively with fecal concentrations of total SCFAs, as well as acetic, propionic and butyric acid. This result indicates an association between fecal dysbiosis and fecal SCFA concentrations; these phenomena may contribute to ICRP pathogenesis in MDs. Potential therapeutic targeting of the reduced propionic acid concentration using probiotics, prebiotics or SCFA enemas merits further study.

Fecal dysbiosis in miniature dachshunds with inflammatory colorectal polyps.

Chronic gastrointestinal disease is associated with the alteration of gastrointestinal microbiota. Inflammatory colorectal polyps (ICRPs) are commonly observed in miniature dachshunds (MDs) in Japan and are characterized by multiple polyps that are restricted in the colorectal mucosa with severe neutrophil infiltration. This study was aimed to compare the fecal microbiota of ICRP-affected MDs with that of healthy MDs. High-throughput sequencing of amplicons derived from the V3-V4 region of the 16S rRNA gene was applied using the Illumina MiSeq system. Principal coordinates analysis revealed that fecal microbiota of ICRP-affected MDs was significantly altered compared with that of healthy MDs. Proportions of Fusobacteriaceae, Helicobacteraceae, Porphyromonadaceae, and Turicibacteraceae were significantly more abundant in ICRP-affected MDs, while those of Lachnospiraceae were significantly less abundant in ICRP-affected MDs compared with healthy MDs. These results suggest that the dysbiosis is associated with ICRPs and is a potential therapeutic target, though further investigations are needed.

Regulation of p16 gene expression by histone H3 acetylation in canine lymphoid tumor cell lines.

In order to investigate whether suppression of the p16 gene is mediated by histone H3 acetylation in 4 canine lymphoid tumor cell lines, the gene's acetylation status was examined. In 2 canine lymphoid tumor cell lines with low p16 mRNA expression (GL-1 and UL-1), the acetylation level was lower than that in CL-1 cells with high p16 mRNA expression. The expression of the p16 gene in these 2 cell lines was markedly restored after culture in the presence of a histone deacetylase inhibitors trichostatin A, indicating that p16 was inactivated by hypoacetylation. Findings obtained this study will add new insights and lead to the better understanding of the disease pathogenesis and future development of epigenetic therapeutic strategies.

Efficacy of leflunomide for treatment of refractory inflammatory colorectal polyps in 15 Miniature Dachshunds.

Inflammatory colorectal polyp (ICRP), common in miniature dachshunds, presents with hematochezia, tenesmus and mucoid feces. Although an 80% response rate has been reported when treated with prednisolone and cyclosporine, effective treatment is needed for the remaining 20% of ICRP dogs. Leflunomide is an immunosuppressive drug reported as effective in several immune-mediated diseases. In the present study, we retrospectively evaluated the efficacy and adverse effects of leflunomide in 15 ICRP dogs that were refractory to treatment with prednisolone and cyclosporine. Treatment efficacy was assessed by endoscopy, clinical symptoms and rectal palpation. Adverse effects were determined by clinical symptoms and blood testing during follow-up. The leflunomide treatment response rate was 93.3%. The median dosage of leflunomide and the median response time were 3 mg/kg (range: 1.7-4.0 mg/kg) and 35 days (range: 20-119 days), respectively. Adverse effects observed included lethargy (3 dogs), anorexia (1 dog), respiratory symptoms (1 dog), leukocytopenia (2 dogs), thrombocytopenia (1 dog), anemia (1 dog) and liver enzyme elevation (8 dogs). Most of the adverse effects improved with symptomatic treatment and leflunomide discontinuation or dosage reduction. In conclusion, leflunomide treatment is effective in ICRP dogs refractory to treatment with prednisolone and cyclosporine. Because several adverse effects were observed, close monitoring is needed during leflunomide treatment follow-up.

Expression profile of circulating serum microRNAs in dogs with lymphoma.

Serum microRNAs (miRNAs) are mediators of cell-to-cell communication and alter the cellular microenvironment; they are stable for hours under certain conditions in body fluids despite the presence of RNases. Certain miRNAs have been found to be altered in the serum or plasma of humans with various cancers and may represent promising, non-invasive biomarkers for various diseases in humans and animals. The objective of this study was to determine the expression profile of circulating miRNAs in the serum of dogs with lymphoma. Serum samples were obtained from 61 dogs with lymphoma and 40 control dogs, and real-time reverse transcription-polymerase chain reaction was used for miRNA measurement. In order to select candidate genes, a comprehensive expression analysis was undertaken prior to validation of several candidate miRNAs. Of 277 miRNAs, five (let-7b, miR-223, miR-25, miR-92a, and miR-423a) were selected as candidates. The expression levels of four miRNAs (let-7b, miR-223, miR-25, miR-92a) were significantly reduced in the lymphoma group, whereas miR-423a levels were significantly increased compared to the controls. When the lymphoma cases were categorized into high- or low-grade as well as into their anatomic form, miR-25 levels were lower in the serum samples from the lymphoma group compared to those from the control group. Although the biological function of serum miRNAs still remains unclear, determining their functional roles in serum and tissues will contribute not only to the identification of potential biomarkers but also to the elucidation of the pathogenesis of canine lymphoma.

Polymorphisms of nucleotide-binding oligomerization domain 2 (NOD2) gene in miniature dachshunds with inflammatory colorectal polyps.

Inflammatory colorectal polyps (ICRPs) frequently occur in miniature dachshunds (MDs) in Japan, typically form multiple polyps with severe neutrophil infiltration. ICRPs are speculated as a novel, breed-specific canine inflammatory bowel disease. Pattern recognition receptors (PRRs) play an important role in the differentiation of pathogens from commensal bacteria and food antigens, and polymorphisms of various PRRs have been shown to be associated with human and canine IBD. We recently reported that the reactivity of nucleotide-binding oligomerization domain 2 (NOD2), toll-like receptor (TLR) 1/2, TLR2, and TLR2/6 are greater in ICRP-affected MDs than that in controls. Therefore, this study was aimed to investigate single nucleotide polymorphisms (SNPs) of PRRs associated with ICRPs in MDs. Mutational analysis of canine NOD2, TLR1, TLR2, and TLR6 genes was performed with six ICRP-affected MDs, five control MDs, and five healthy beagles. The mutational analysis identified 13 non-synonymous SNPs in NOD2, TLR1, TLR2, and TLR6 genes, of which six SNPs in NOD2 exon 3 were further analyzed in an association study using 63 ICRP-affected MDs, 82 control MDs, and 237 control dogs of various breeds. Four of the SNPs (A1532G, T1573C, C1688G, and G1880A of the NOD2 gene) were in Hardy-Weinberg equilibrium and in complete linkage disequilibrium in MDs, and their minor allele frequencies were significantly lower in ICRP-affected MDs than in control MDs (0.016 vs. 0.140, P=0.0002). The calculated inheritance model was an additive model (odds ratio=0.10, 95% confidence interval=0.02-0.45, P=0.0001), which indicates that the haplotype with minor alleles in these SNPs (A, T, C, and G in A1532G, T1573C, C1688G, and G1880A) possess a protective effect regarding the development of ICRPs. However, these SNPs were not specific for MDs, although the minor allele frequencies of these SNPs in control MDs were significantly lower than in other breed dogs. These results suggest that the identified four SNPs (A1532G, T1573C, C1688G, and G1880A in the NOD2 gene) may play a role in the pathogenesis of ICRPs in MDs. Because the majority of MDs and other breed dogs do not have the protective alleles, their absence may not be a specific cause of ICRPs in MDs but rather contribute to the development of inflammation.

Functional analysis of pattern recognition receptors in miniature dachshunds with inflammatory colorectal polyps.

Inflammatory colorectal polyps (ICRPs) frequently occur in miniature dachshunds (MDs) in Japan. MDs with ICRPs develop multiple polyps with severe neutrophil infiltration that respond to immunosuppressive therapy. Therefore, ICRPs are thought to constitute a novel, breed-specific form of canine inflammatory bowel disease (IBD). Pattern recognition receptors (PRRs) play a key role in the distinction of pathogens from commensal bacteria and food antigens. Dysfunction resulting from genetic disorders of PRRs have been linked to human and canine IBD. Therefore, we analyzed the reactivity of PRRs in MDs with ICRPs. Twenty-six MDs with ICRPs and 16 control MDs were recruited. Peripheral blood-derived monocytes were obtained from each dog and then stimulated with PRR ligands for 6 and 24 hr; subsequently, messenger RNA (mRNA) expression levels and protein secretion of IL-1ß were quantified using quantitative real-time PCR and ELISA, respectively. The levels of IL-1ß mRNA and protein secretion after stimulation with a nucleotide-binding oligomerization domain 2 (NOD2) ligand were significantly greater in monocytes from ICRP-affected MDs than in those from control MDs. In addition, IL-1ß protein secretion induced by toll-like receptor (TLR) 1/2, TLR2 and TLR2/6 stimulation was also significantly greater in ICRP-affected MDs. These results suggest that reactivity against NOD2, TLR1/2, TLR2 and TLR2/6 signals is enhanced in ICRP-affected MDs and may play a role in the pathogenesis of ICRPs in MDs. Additional studies of the genetic background of these PRRs should be performed.

Inflammatory colorectal polyps in miniature Dachshunds frequently develop ventrally in the colorectal mucosa.

This study explored the hypothesis that inflammatory colorectal polyps (ICRPs) in miniature Dachshunds are more likely to occur ventrally in the colorectum. Angle-fixed colonoscopic images were collected from 11 miniature Dachshunds with ICRPs and randomly rotated. Macroscopic severity at 12 divided angles was scored by four veterinarians blinded to the rotation angle. Mean prevalence and severity scores of ICRPs were significantly higher ventrally than dorsally (P < 0.01).

Effect of oral administration of metronidazole or prednisolone on fecal microbiota in dogs.

Gastrointestinal microbiota have been implicated in the pathogenesis of various gastrointestinal disorders in dogs, including acute diarrhea and chronic enteropathy. Metronidazole and prednisolone are commonly prescribed for the treatment of these diseases; however, their effects on gastrointestinal microbiota have not been investigated. The objective of this study was to evaluate the effects of these drugs on the gastrointestinal microbiota of dogs. Metronidazole was administered twice daily at 12.5 mg/kg to a group of five healthy dogs, and prednisolone at 1.0 mg/kg daily to a second group of five healthy dogs for 14 days. Fecal samples were collected before and after administration (day 0 and 14), and 14 and 28 days after cessation (day 28 and 42). DNA was extracted, and the bacterial diversity and composition of each sample were determined based on 16S ribosomal RNA (rRNA) gene sequences using next-generation sequencing (Illumina MiSeq). In the group administered metronidazole, bacterial diversity indices significantly decreased at day 14, and recovered after the cessation. Principal coordinates analysis and hierarchical dendrogram construction based on unweighted and weighted UniFrac distance matrices revealed that bacterial composition was also significantly altered by metronidazole at day 14 compared with the other time points. The proportions of Bacteroidaceae, Clostridiaceae, Fusobacteriaceae, Lachnospiraceae, Ruminococcaceae, Turicibacteraceae, and Veillonellaceae decreased, while Bifidobacteriaceae, Enterobacteriaceae, Enterococcaceae, and Streptococcaceae increased at day 14 and returned to their initial proportions by day 42. Conversely, no effect of prednisolone was observed on either the bacterial diversity or composition. Reducing pathogenic bacteria such as Fusobacteria and increasing beneficial bacteria such as Bifidobacterium through the administration of metronidazole may be beneficial for promoting gastrointestinal health; however, further investigations into the effects on diseased dogs are needed.

Intestinal protease-activated receptor-2 and fecal serine protease activity are increased in canine inflammatory bowel disease and may contribute to intestinal cytokine expression.

Serine proteases elicit cellular responses via protease-activated receptor-2 (PAR-2) which is known to regulate inflammation and the immune response. Although the gastrointestinal tract is exposed to large amounts of proteolytic enzymes, the role of PAR-2 in canine inflammatory bowel disease (IBD) remains unclear. The objective of this study was to investigate the effects of PAR-2 activation on inflammatory cytokine/chemokine gene expression in canine intestine and the expression of intestinal PAR-2 and fecal serine protease activity in dogs with IBD. Duodenal biopsies from healthy dogs were cultured and treated ex vivo with trypsin or PAR-2 agonist peptide, and inflammatory cytokine/chemokine gene expression in the tissues was then quantified by real-time PCR. PAR-2 mRNA and protein expression levels in the duodenal mucosa were examined by real-time PCR and immunohistochemistry, respectively. Fecal serine protease activity was determined by azocasein assay. In ex vivo-cultured duodenum, trypsin and PAR-2 agonist peptide induced significant up-regulation of mRNA expression levels of interleukin-1 ß (IL-1ß), IL-8, mucosae-associated epithelial chemokine (MEC) and fractalkine, and this up-regulation was inhibited by a serine protease inhibitor. Duodenal PAR-2 mRNA and protein expression levels were higher in dogs with IBD than in healthy control dogs. Fecal serine protease activity was significantly elevated in dogs with IBD, and the level of activity correlated positively with the clinical severity score. These results suggest that PAR-2 may contribute to the pathogenesis of canine IBD by inducing expression of inflammatory mediators in response to luminal serine proteases.

Expression profiling of pattern recognition receptors and selected cytokines in miniature dachshunds with inflammatory colorectal polyps.

Inflammatory colorectal polyps (ICRPs) are commonly seen in miniature dachshund (MD) dogs; typically, multiple polyps form with severe neutrophil infiltration. ICRP is thought to be a novel form of inflammatory bowel disease (IBD), but its etiology has not been investigated. The innate immune system is implicated in the pathogenesis of both human and canine IBD. Therefore, the aim of the current study was to evaluate the messenger RNA (mRNA) expression profiles of pattern recognition receptors (PRRs) and cytokines in ICRPs. Polyp tissues were collected by colonoscopic biopsies from 24 MDs with ICRPs. Non-polypoid colonic mucosa was collected from all MDs with ICRPs and 21 clinically healthy beagles (as the controls). The expression levels of the mRNAs encoding toll-like receptors (TLRs) 1-10; nucleotide-binding oligomerization domain (NOD)-like receptors NOD1 and NOD2; and cytokines IL-1ß, IL-6, IL-8/CXCL8, and TNF-α were evaluated by quantitative real-time RT-PCR. Three of the 10 well-known candidate reference genes were selected as housekeeper genes based on analyses from the GeNorm, NormFinder, and BestKeeper programs. Levels of TLR1, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, NOD2, and all cytokines were significantly upregulated in the polyps relative to those in the controls. There was significant decrease in the expression levels of TLR3 and NOD1 in the polyp tissues compared to the non-polypoid colonic mucosa obtained from MDs with ICRPs. All upregulated PRR mRNAs were positively correlated with all proinflammatory cytokine mRNAs. This study demonstrated the dysregulation of PRRs and proinflammatory cytokines in ICRPs of MDs, which may play an important role in the pathogenesis of this disease.