Bacterial Biogeography of the Colon in Dogs With Chronic Inflammatory Enteropathy.

The intestinal microbiota is believed to play a role in the pathogenesis of inflammatory bowel disease in humans and chronic inflammatory enteropathy (CIE) in dogs. While most previous studies have described the gut microbiota using sequencing methods, it is fundamental to assess the spatial distribution of the bacteria for a better understanding of their relationship with the host. The microbiota in the colonic mucosa of 22 dogs with CIE and 11 control dogs was investigated using fluorescence in situ hybridization (FISH) with a universal eubacterial probe (EUB338) and specific probes for select bacterial groups. The number of total bacteria labeled with EUB338 probe was lower within the colonic crypts of dogs with CIE compared to controls. Helicobacter spp. and Akkermansia spp. were decreased on the colonic surface and in the crypts of dogs with CIE. Dogs with CIE had increased number of Escherichia coli/Shigella spp. on the colonic surface and within the crypts compared to control dogs. In conclusion, the bacterial microbiota in the colonic mucosa differed between dogs with and without CIE, with depletion of the crypt bacteria in dogs with CIE. The crypt bacterial species that was intimately associated with the host mucosa in control dogs was composed mainly of Helicobacter spp.

S100A12 concentrations and myeloperoxidase activities are increased in the intestinal mucosa of dogs with chronic enteropathies.

BACKGROUND: Intestinal mucosal S100A12 and myeloperoxidase (MPO) are inflammatory biomarkers in humans with inflammatory bowel disease (IBD). However, these biomarkers have not been studied in the intestinal mucosa of dogs with chronic enteropathies (CE), even though dogs with CE have increased S100A12 concentrations in feces and serum. This study investigated mucosal S100A12 concentrations and MPO activities in both dogs with CE and healthy Beagles. ELISA (S100A12 concentrations) and spectrophotometric methods (MPO activity) were used. The associations of both biomarkers with canine IBD activity index (CIBDAI), histopathologic findings, clinical outcome, and serum albumin concentrations were also investigated. We studied intestinal mucosal samples originating from different intestinal regions of 40 dogs with CE and 18 healthy Beagle dogs (duodenum, ileum, colon, and cecum). RESULTS: Compared with healthy Beagles, mucosal S100A12 concentrations in dogs with CE were significantly higher in the duodenum (p < 0.0001) and colon (p = 0.0011), but not in the ileum (p = 0.2725) and cecum (p = 0.2194). Mucosal MPO activity of dogs with CE was significantly higher in the duodenum (p < 0.0001), ileum (p = 0.0083), colon (p < 0.0001), and cecum (p = 0.0474). Mucosal S100A12 concentrations in the duodenum were significantly higher if the inflammatory infiltrate consisted mainly of neutrophils (p = 0.0439) or macrophages (p = 0.037). Mucosal S100A12 concentrations also showed a significant association with the severity of total histopathological injury and epithelial injury in the colon (p < 0.05). Mucosal MPO activity showed a significant association (p < 0.05) with the severity of total histopathological injury, epithelial injury, and eosinophil infiltration in the duodenum. There was no significant association of both biomarkers with CIBDAI or clinical outcome. CONCLUSIONS: This study showed that both mucosal S100A12 concentrations and MPO activities are significantly increased in the duodenum and colon of dogs with CE; mucosal MPO was also increased in the ileum and cecum. Future research should focus on assessing the clinical utility of S100A12 and MPO as diagnostic markers in dogs with CE.

The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro.

BACKGROUND: For a good probiotic candidate, the abilities to adhere to intestinal epithelium and to fortify barrier function are considered to be crucial for colonization and functionality of the strain. The strain Lactobacillus acidophilus LAB20 was isolated from the jejunum of a healthy dog, where it was found to be the most pre-dominant lactobacilli. In this study, the adhesion ability of LAB20 to intestinal epithelial cell (IECs) lines, IECs isolated from canine intestinal biopsies, and to canine, porcine and human intestinal mucus was investigated. Further, we studied the ability of LAB20 to fortify the epithelial cell monolayer and to reduce LPS-induced interleukin (IL-8) release from enterocytes. RESULTS: We found that LAB20 presented higher adhesion to canine colonic mucus as compared to mucus isolated from porcine colon. LAB20 showed adhesion to HT-29 and Caco-2 cell lines, and importantly also to canine IECs isolated from canine intestinal biopsies. In addition, LAB20 increased the transepithelial electrical resistance (TER) of enterocyte monolayers and thus strengthened the intestinal barrier function. The strain showed also anti-inflammatory capacity in being able to attenuate the LPS-induced IL-8 production of HT-29 cells. CONCLUSION: In conclusion, canine indigenous strain LAB20 is a potential probiotic candidate for dogs adhering to the host epithelium and showing intestinal barrier fortifying and anti-inflammatory effects.

S100A12 concentrations and myeloperoxidase activity in the intestinal mucosa of healthy dogs.

BACKGROUND: Relatively few laboratory markers have been evaluated for the detection or monitoring of intestinal inflammation in canine chronic enteropathies, including inflammatory bowel disease (IBD). Previous research found that the intestinal mucosal levels of S100A12 and myeloperoxidase (MPO), as biomarkers of gut inflammation, were elevated in human patients with IBD. To date, the S100A12 and MPO levels in intestinal mucosal samples from either healthy dogs or from dogs suffering from IBD remain unreported. Therefore, this study aimed to evaluate the mucosal S100A12 and MPO levels in four different parts of the intestine (duodenum, jejunum, ileum and colon) in 12 healthy laboratory Beagle dogs using the ELISA and spectrophotometric methods, respectively. RESULTS: Based on histological examinations, the recorded findings for all the samples were considered normal. The mucosal concentration of S100A12 in the ileum was significantly higher than in all other segments of the intestine (p < 0.05). MPO activity was significantly higher in the ileal, jejunal and duodenal than in colonic mucosal samples (p < 0.05). Moreover, its concentration was higher in the jejunum than in the duodenum. CONCLUSIONS: This study showed that S100A12 and MPO are reliably detectable in canine intestinal mucosa. The assays used appeared to be sufficient to further evaluate the role of S100A12 and MPO in the pathogenesis of canine chronic enteropathies, including IBD. These biomarkers may play a role in the initial detection of gut inflammation suggesting the need for further investigations to confirm IBD or to differentiate between IBD subtypes. Understanding the role of S100A12 and MPO in the pathogenesis of chronic intestinal inflammation in future may result in an improved understanding of canine chronic intestinal inflammation.

Can Chromoendoscopy Improve the Early Diagnosis of Gastric Carcinoma in Dogs?

Chromoendoscopy has improved the early diagnosis of gastric cancer in humans but its usefulness in dogs is unknown. This study aimed at assessing whether adding narrow band imaging (NBI) or indigo carmine (IC) chromoendoscopy (CE) can improve the diagnostic yield of standard white light endoscopy (WLE). We compared the real-time findings of canine WLE, NBI-CE, and IC-CE and corresponding histology reports with endoscopic mucosal pattern assessment templates used in human medicine. Belgian Shepherd dogs are predisposed to gastric carcinoma. Therefore, 30 dogs of this breed served as the study population. According to histology, 17/30 dogs had mucosal changes (mucous metaplasia, glandular dysplasia, and gastric carcinoma). Diagnostic yield was best when targeted biopsies were taken with WLE and NBI-CE combined (15/17 cases). WLE alone positively identified only 8/17 cases and missed a gastric carcinoma in 3/6 cases. CE assessment templates based on macroscopic mucosal patterns, broadly used in human medicine, were not readily applicable in dogs. In conclusion, the study provides evidence that using CE in dogs has the potential to improve the diagnosis of precancerous gastric mucosal pathology and early gastric carcinoma. However, current image assessment templates from human medicine need major adjustments to the patterns of canine gastric mucosa.

Gastric mucosal pathology in Belgian Shepherd dogs with and without clinical signs of gastric disease.

BACKGROUND: Gastric carcinoma (GC) is uncommon in dogs, except in predisposed breeds such as Belgian Shepherd dogs (BSD) of the Tervuren and Groenendael varieties. When GC is diagnosed in dogs it is often late in the disease, resulting in a poorer prognosis. The aim of this prospective clinical study was to investigate possible associations of gastric mucosal pathologies with clinical signs, laboratory test results and GC in BSD. An online survey gathered epidemiological data to generate potential risk factors for vomiting as the predominant gastric clinical sign, and supported patient recruitment for endoscopy. Canine Chronic Enteropathy Clinical Activity Index (CCECAI) score and signs of gastroesophageal reflux (GER) were used to allocate BSD older than five years to either Group A, with signs of gastric disease, or Group B, without signs. Findings in the clinical history, laboratory tests and gastric histopathology of endoscopic biopsies were statistically analysed in search of associations. RESULTS: The online survey included 232 responses. Logistic regression analysis recognized an association of vomiting with gagging, poor appetite and change in attitude. Recruitment for endoscopy included 16 BSD in Group A (mean age 9.1 ± 1.8 years, mean CCECAI = 3.1 ± 2.2 and signs of GER); and 11 in Group B (mean age 9.8 ± 1.4 years, CCECAI = 0, no signs of GER). Seven (25.9%) of the 27 BSD (Group A 4/16, Group B 3/11) had leukopenia. Serum C-reactive protein tended to be increased with more advanced GC (P = 0.063). Frequency of GC, mucosal atrophy, mucous metaplasia, or glandular dysplasia did not differ between groups. GC was frequently diagnosed (6/27), even without clinical signs (2/11). The odds ratio for vomiting (OR = 9.9; P = 0.016) was increased only when glandular dysplasia was present. GC was associated with mucous metaplasia (P = 0.024) and glandular dysplasia (P = 0.006), but not with mucosal atrophy (P = 1). CONCLUSIONS: GC can develop as an occult disease, associated with metaplasia and dysplasia of the gastric mucosa. Suggestive clinical signs, notably vomiting, should warrant timely endoscopy in BSD. Extensive endoscopic screening of asymptomatic dogs remains, however, unrealistic. Therefore, biomarkers of mucosal pathology preceding clinical illness are needed to support an indication for endoscopy and enable early diagnosis of GC.

Correction to: Canine breeds associated with gastric carcinoma, metaplasia and dysplasia diagnosed by histopathology of endoscopic biopsy samples.

An amendment to this paper has been published and can be accessed via the original article.

Primary hyperparathyroidism caused by bilateral parathyroid cystic carcinoma in a cat.

CASE SUMMARY: A 16-year-old neutered female Korat cat presented with chronic vomiting, mild azotaemia and mild hypercalcaemia. Physical examination revealed bilateral palpable masses on each side of the trachea. Laboratory results were consistent with primary hyperparathyroidism, diagnostic imaging findings with cystic thyroid or parathyroid masses, and fine-needle aspiration cytology with thyroid hyperplasia or adenoma. In order to confirm whether one or two of the masses were the cause of the hyperparathyroidism, cystic fluid was aspirated from both for parathyroid hormone concentration measurement. The concentration was shown to exceed that of the serum manyfold in both samples, confirming both masses to be functional and of parathyroid origin. A total parathyroidectomy and thyroidectomy were performed on the right side, and a subtotal thyroidectomy and a subtotal to total parathyroidectomy on the left, without any major postoperative complications. Histopathology was consistent with bilateral parathyroid carcinoma. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this report is the first to describe a rare case of bilateral parathyroid cystic carcinoma in a cat. It highlights the usefulness of determining parathyroid hormone concentration in the cystic fluid of a suspected neoplastic parathyroid mass preoperatively. It also demonstrates that it may be possible to remove most of the cervical parathyroid and thyroid tissue of a cat without causing any clinically relevant hypocalcaemia or iatrogenic hypothyroidism. However, serum concentrations of ionised calcium, thyroxine and creatinine should be closely monitored in the postoperative period in order to detect and control possible complications.

Canine breeds associated with gastric carcinoma, metaplasia and dysplasia diagnosed by histopathology of endoscopic biopsy samples.

BACKGROUND: Gastric carcinoma (GC) is a rather rare pathological finding in dogs, with the exception of some breeds which seem predisposed. The etiopathogenesis is largely unknown in dogs, whereas in humans GC often develops from gastric mucosal metaplasia and dysplasia. This study investigates whether dogs of certain breeds are more often subject to gastroduodenoscopy (GDS), and diagnosed with GC, mucosal metaplasia or dysplasia. A retrospective clinical database search was performed at the Veterinary Teaching Hospital at the University of Helsinki, Finland. The following inclusion criteria were applied to estimate relative risk for metaplasia/dysplasia and GC: dogs from pure breeds with at least five individuals subject to GDS with histopathology of gastric biopsies. RESULTS: Between 2006 and 2016, from a total of 54945 canine patients presented, 423 dogs underwent GDS. Inclusion criteria were met in 180 dogs of 20 different pure breeds. Eight dogs had GCs (mean age = 9.8 ± 1.7 years): Belgian Tervuren (n = 4), Collie (n = 2), Golden Retriever (n = 1) and Jack Russel Terrier (n = 1). Fourteen dogs of eight breeds had gastric mucosal metaplasia or dysplasia. A log-binomial statistical model revealed that dogs in the following breeds had a significantly higher probability to undergo GDS than the others in the study population: Australian Terrier, Belgian Tervuren, Cairn Terrier, Collie and Siberian Husky. Belgian Tervuren was found at higher risk to be diagnosed with GC [RR = 19 (5.7-63.9; P < 0.0001)], as well as mucosal metaplasia/dysplasia [RR (7.6; 2.95-19.58; P < 0.0001)], as compared to the other breeds included. Shetland Sheepdog had an increased RR (5.83; 1.75-19.45; P = 0.0041) for metaplasia. CONCLUSIONS: The results indicate a very low incidence of GC in dogs. The Belgian Tervuren, however, appears as predisposed. The histopathologic descriptions of mucosal changes such as metaplasia and dysplasia were also rare, but were more frequent in the Belgian Tervuren. Previous reports of these changes in dogs are very scarce, but they might be presumably related to GC in dogs, as they are in humans. Future research should investigate the possible role of metaplasia and dysplasia in the development of GC in dogs, especially those of predisposed breeds.

Identification of matrix metalloproteinase-2 and -9 activities within the intestinal mucosa of dogs with chronic enteropathies.

BACKGROUND: Matrix metalloproteinases (MMPs) 2 and 9 are zinc- and calcium-dependent endopeptidases involved in the breakdown and reconstitution of extracellular matrix under both physiological and pathological conditions. Mucosal MMP-2 and -9 activities have been reported to be upregulated in the intestine of humans with inflammatory bowel disease (IBD), and in animal models of IBD. However, their involvement in the pathogenesis of canine chronic enteropathies (CE) is unknown. This study investigated mucosal pro- and active MMP-2 and -9 activities in dogs with CE and healthy dogs using gelatin zymography, and also to determine the association of their activities in dogs with CE with the canine IBD activity index (CIBDAI), histopathologic findings, the clinical outcome, and hypoalbuminemia. Intestinal mucosal samples from duodenum, ileum, colon, and cecum were collected from 40 dogs with CE and 18 healthy Beagle dogs. RESULTS: In dogs with CE, the number of samples positive for mucosal pro- and active MMP-2 was significantly higher in the duodenum (P < 0.0001 and P = 0.011, respectively), ileum (P = 0.002 and P = 0.018, respectively), and colon (P < 0.0001 and P = 0.002, respectively), compared with healthy controls. Mucosal pro-MMP-9-positive samples in the duodenum and colon were significantly more frequent in dogs with CE than in healthy dogs (P = 0.0004 and P = 0.001, respectively). Despite the presence of mucosal samples positive for active MMP-9 in the intestinal segments of dogs with CE, the difference compared to healthy controls did not reach statistical significance. None of the intestinal mucosal samples in healthy dogs showed gelatinolytic activity corresponding to the control bands of active MMP-2 and -9. Mucosal active MMP-9 activities displayed a significant positive association with the severity of neutrophil infiltration in the duodenum (P = 00.040), eosinophils in the cecum (P = 00.037), and the CIBDAI score for ileum samples (P = 0.023). There was no significant association of pro- and active MMP-2 and -9 levels with the clinical outcome or hypoalbuminemia. CONCLUSIONS: This study is the first to demonstrate upregulation of mucosal pro- and active MMP-2 and pro-MMP-9 in the intestine of dogs with CE compared to healthy dogs. The results provide supporting evidence for the possible involvement of MMP-2 and -9 in the pathogenesis of canine CE.

Comparison of intestinal expression of the apical sodium-dependent bile acid transporter between dogs with and without chronic inflammatory enteropathy.

BACKGROUND: Intestinal absorption of bile acids is mediated by the apical sodium-dependent bile acid transporter (ASBT). Fecal bile acid dysmetabolism has been reported in dogs with chronic inflammatory enteropathy (CIE). OBJECTIVE: Characterization of ASBT distribution along the intestinal tract of control dogs and comparison to dogs with CIE. ANIMALS: Twenty-four dogs with CIE and 11 control dogs. METHODS: The ASBT mRNA and protein expression were assessed using RNA in situ hybridization and immunohistochemistry, respectively. The concentrations of fecal bile acids were measured by gas chromatography-mass spectrometry. The fecal microbiota dysbiosis index was assessed with a quantitative polymerase chain reaction panel. RESULTS: In control dogs, ASBT mRNA expression was observed in enterocytes in all analyzed intestinal segments, with highest expression in the ileum. The ASBT protein expression was restricted to enterocytes in the ileum, cecum, and colon. Dogs with CIE had significantly decreased expression of ASBT protein in the ileum (P = .001), which was negatively correlated with histopathological score (ρ = -0.40; Pcorr = .049). Additionally, dogs with CIE had a significantly increased percentage of primary bile acids in feces compared to controls (P = .04). The fecal dysbiosis index was significantly higher in dogs with CIE than in control dogs (P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: These findings indicate that ileal protein expression of ASBT is downregulated in dogs with CIE. This change may be linked to the inflammatory process, intestinal dysbiosis, and fecal bile acid dysmetabolism observed in these patients.

Oral tylosin administration is associated with an increase of faecal enterococci and lactic acid bacteria in dogs with tylosin-responsive diarrhoea.

The term tylosin-responsive diarrhoea (TRD) is used for canine recurrent diarrhoea cases for which no underlying cause can be found after extensive diagnostic investigations, but which show a response to the antibiotic tylosin in a few days. The objective of this prospective, one-arm longitudinal trial was to assess the effects of oral tylosin administration on the faecal levels of potentially probiotic bacteria, such as Enterococcus spp. and lactic acid bacteria (LAB), in dogs with TRD. This trial included 14 client-owned suspected TRD dogs that were on tylosin treatment and had firm faeces. Treatment was then terminated and dogs were followed up for up to 2 months to determine the recurrence of diarrhoea. Once diarrhoea started, dogs received tylosin (orally, 25 mg/kg, once daily for 7 days). At the end of the treatment period, stools were firm again in 11 dogs (TRD dogs); three dogs continued having diarrhoea and were excluded from the study. Faecal samples were collected at all three time-points for culture of LAB and enterococci. In TRD dogs, the colony counts of Enterococcus spp. (P = 0.003), LAB (P = 0.037), tylosin-resistant Enterococcus spp. (P <0.001) and LAB (P <0.001) were significantly higher when the dogs were on tylosin treatment and had normal faecal consistency compared to when they had diarrhoea following discontinuation of tylosin. In conclusion, cessation of diarrhoea in TRD dogs with tylosin treatment could be mediated by selection of a specific lactic acid population, the Enterococcus spp., due to their potential probiotic properties.

Efficacy of two low-dose oral tylosin regimens in controlling the relapse of diarrhea in dogs with tylosin-responsive diarrhea: a prospective, single-blinded, two-arm parallel, clinical field trial.

BACKGROUND: Despite its wide acceptance as a treatment for canine chronic enteropathies, the macrolide antibiotic tylosin lacks official oral dosage recommendations. Not even textbooks share consensus about the dose; daily recommendations vary from 25 to 80 mg/kg and dosing intervals from one to three times daily. RESULTS: All eight dogs responded to the 5 mg/kg dose, and six of seven dogs responded to the 15 mg/kg dose. The mean fecal consistency scores at the 25 mg/kg tylosin dosage were no significantly different from scores at the 5 mg/kg or 15 mg/kg tylosin dosages (P=0.672, P=0.345). CONCLUSIONS: Interestingly, 14/15 (93%) of the dogs responding to a dose of 25 mg/kg tylosin once daily for seven days also responded to the lower dosages at diarrhea relapse. The data indicate that a suitable dose of tylosin for treating diarrhea relapse in canine TRD could be as low as 5 mg/kg once daily for seven days.

Identification of matrix metalloproteinase-2 and -9 activities within intestinal mucosa of clinically healthy beagle dogs.

Matrix metalloproteinases (MMPs) 2 and 9 are zinc-dependent endopeptidases that contribute to the control of breakdown and reconstitution of extracellular matrix under both normal and pathological conditions. The main objective of this study was to identify the presence of MMP-2 and -9 in the mucosa of the small and large intestines of clinically healthy beagle dogs using gelatin zymography technique. Intestinal mucosa samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were taken from 12 healthy laboratory beagle dogs and examined histologically. Based on WSAVA histology standards, recorded findings of all samples were considered insignificant. Pro-MMP-2 and -9 activities were found in 17/48 (35%) and 25/48 (52%) of the samples, respectively. Among four different parts of the intestine of 12 dogs, the ileum had the highest positivity rates of 7/12 (58.3%) and 8/12 (66.7%) for pro-MMP-2 and -9 activities, respectively. However, statistical analysis showed no significant difference of pro-MMP-2 and -9 activities between the separate parts of the intestine (P>0.05). None of the intestinal samples showed gelatinolytic activity corresponding to the control bands of active MMP-2 and MMP-9. This study showed that pro-MMP-2 and -9 could be detected in the intestinal mucosa of healthy dogs using zymography, which seems to be a useful tool to evaluate the role of MMP-2 and -9 in the pathogenesis of canine chronic enteropathies, including inflammatory bowel diseases.

Effect of tylosin on dogs with suspected tylosin-responsive diarrhea: a placebo-controlled, randomized, double-blinded, prospective clinical trial.

BACKGROUND: The macrolid antibiotic tylosin has been widely used to treat canine chronic diarrhea, although its efficacy is based on anecdotal reports and experimental studies in dogs and not on strong scientific evidence. The term tylosin-responsive diarrhea (TRD) refers to diarrheal disorders responding to tylosin therapy within a few days. In TRD, the stool remains normal as long as tylosin treatment continues, but diarrhea reappears in many dogs within weeks after discontinuation. The aim of our trial was to assess the effect of tylosin on fecal consistency compared with a placebo treatment in dogs with suspected TRD and additionally to establish whether tylosin in dogs with recurrent diarrhea is as effective as empirical studies and anecdotal reports suggest. METHODS: Subjects comprised 71 client-owned dogs that, according to the owners, had previously been treated successfully with tylosin due to recurrent diarrhea of unknown etiology. At the initial examination, where there were no signs of diarrhea, the dogs were randomly assigned in a 2:1 ratio to a tylosin or placebo group. During a two-month follow-up the owners evaluated the fecal consistency according to previously published guidelines. When diarrhea recurred, either tylosin (25 mg/kg q 24 h, 7 days) or placebo treatment was initiated orally. Treatment outcome was evaluated as the mean of fecal consistency scores assigned during the last three days of the treatment period. To test for differences between the tylosin and placebo group in the proportion of responders, Pearson's Chi-squared test and Fisher's exact test were applied. RESULTS: Sixty-one dogs met the selection criteria and were followed for two months. During the follow-up 27 dogs developed diarrhea and either tylosin or placebo treatment was started. The proportion of dogs with normal fecal consistency at the end of treatment was 85% (17/20) in the tylosin group and 29% (2/7) in the placebo group (Pearson's Chi-squared test p = 0.0049 and Fisher's exact test two-sided, p = 0.0114). CONCLUSIONS: Our findings indicate that tylosin is effective in treating recurrent diarrhea in dogs. The dose of 25 mg/kg once daily appears sufficient. No changes specific to TRD were detected in the examinations.