Evaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis.

Intestinal mucus barrier disruption may occur with chronic inflammatory enteropathies. The lack of studies evaluating mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced goblet cell (GBC) numbers and/or mucin 2 (MUC2) expression, which are responsible for mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is undetermined if Ki-67 immunoreactivity, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to histologic severity in GC and LPC. Study objectives included comparing Ki-67 immunoreactivity, GBC population and MUC2 expression in dogs with GC, LPC and non-inflamed colon; and exploring the use of ribonucleic acid (RNAscope®) in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs over an eight-year period. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colitis (NC) (n=10) group based on final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope® ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per dog were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using image analysis software. Spearman's correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic score (WHS) and measured variables. Linear regression models were used to compare relationships between WHS with KI67%, GBC%, and MUC2%; and between GBC% and MUC2%. Median WHS was highest in dogs with GC. Median KI67% normalised to WHS was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation overall. Median MUC2% normalised to WHS in the NC group (10.02%; range, 3.05-39.09%) was two- and three-fold higher than in the GC and LPC groups respectively. With increased colonic inflammation, despite minimal changes in GBC% overall, MUC2 expression markedly declined in the LPC group (-27.4%; 95%-CI, -49.8, 5.9%) and mildly declined in the GC and NC groups. Granulomatous colitis and LPC likely involve different pathways regulating MUC2 expression. Decreased MUC2 gene expression is observed in dogs with chronic colitis compared to dogs without colonic signs. Changes in MUC2 expression appear influenced by GBC activity rather than quantity in GC and LPC.

Nodular hyperplasia of lymphoglandular complexes in dogs: A potential diagnostic pitfall for rectal masses.

Lymphoglandular complexes are components of the gut-associated lymphoid tissue that are characterized by submucosal lymphoid aggregates invested by projections of mucosal epithelium. Reports of pathology involving these structures are rare in both human and veterinary literature. Here, the authors report 2 cases of rectal masses excised from dogs following a period of tenesmus and hematochezia. In both animals, the masses were composed of lymphoid tissue closely encompassing tubuloacinar structures. Immunohistochemistry and polymerase chain reaction antigen receptor rearrangement testing demonstrated that the lymphoid population was polyclonal, comprising T and B cells arranged in loosely follicular aggregates centered on the epithelial foci. In light of these findings, a diagnosis of lymphoglandular complex nodular hyperplasia was reported. To the authors' knowledge, this is the first report of this condition in dogs.

Biomarkers of endothelial activation and inflammation in dogs with organ dysfunction secondary to sepsis.

Introduction: Alteration in endothelial function during sepsis is thought to play a key role in the progression of organ failure. We herein compared plasma concentrations of endothelial activation biomarkers vascular endothelial growth factor (VEGF), hyaluronan (HA), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF), as well as inflammatory mediator concentrations (IL-6, IL-8, IL-10, C-reactive protein and monocyte chemoattractant protein-1) in dogs with sepsis to healthy dogs. Methods: This study was a multicenter observational clinical trial conducted at two university teaching hospitals from February 2016 until July 2017. The study included 18 client-owned dogs hospitalized with sepsis and at least one distant organ dysfunction, as well as 20 healthy dogs. Plasma biomarker concentrations were measured using ELISA. Severity of illness in dogs with sepsis was calculated using the 5-variable acute physiologic and laboratory evaluation (APPLEFAST) score. Biomarker concentrations were compared between septic and healthy dogs using linear models. Results: Septic peritonitis was the most frequent source of sepsis (11/18; 61%), followed by pneumonia (4/18; 22%). Ten dogs (56%) had only 1 organ dysfunction, whereas 3 dogs (17%) had 2, 3 (17%) had 3, 1 (6%) had 4 and 1 (6%) had 5 organ dysfunctions. The median APPLEFAST score in the septic dogs was 28.5 (Q1-Q3, 24-31). Mean plasma concentrations of all endothelial and inflammatory biomarkers, except vWF, were higher in the sepsis cohort than in controls. The mean endothelial biomarker concentrations in the septic cohort ranged from ~2.7-fold higher for HA (difference in means; 118.2 ng/mL, 95% credible limit; 44.5-221.7) to ~150-fold for VEGF (difference in means; 76.6 pg./mL, 95% credible limit; 33.0-143.4), compared to the healthy cohort. Fifteen dogs with sepsis (83%) died; 7 (46%) were euthanized and 8 (53%) died during hospitalization. Conclusion: Dogs with naturally occurring sepsis and organ dysfunction had higher mean concentrations of biomarkers of endothelial activation and inflammation compared to healthy dogs, broadening our understanding of the pathophysiology of sepsis secondary to endothelial dysfunction.

Prednisolone prescribing practices for dogs in Australia.

Although prednisolone is a routinely prescribed medication in dogs, there is a lack of information regarding prednisolone prescribing practices by veterinarians. This study aims to describe characteristics of dogs receiving prednisolone, disease processes treated, doses prescribed as well as to identify factors influencing the dose rate in Australia. The VetCompass Australia database was queried to identify dogs prescribed prednisolone between 1 July 2016 to 31 July 2018 (inclusive). A random sample of 2,000 dogs from this population were selected. Dog demographic data, prednisolone dose and indication for prescription were collated. Indicated dose for the condition treated was compared to prescribed dose. Multivariable linear regression was used to identify patient-level characteristics associated with prescribed prednisolone dose. A large and small breed dog cohort, treated for the same disease process, were compared for differences in dosing. Median age of dogs was 73 (range 2 to 247) months and median body weight was 17 (range 1.56 to 90) kg. Median prescribed prednisolone dose was 0.8 mg/kg/day, with most dogs receiving an anti-inflammatory dose (0.3-1 mg/kg/day, 58%). Prednisolone prescriptions were predominantly for diseases of the integument (n = 1645, 82%) followed by unknown indication and respiratory disease. A total of 152 dogs (8%) were prescribed immunosuppressive doses of prednisolone for conditions where an anti-inflammatory dose would be recommended. Increases in bodyweight were associated with lower doses on mg/kg scale but higher doses on a mg/m2 scale (p < 0.001). Overall, prednisolone was primarily used as an anti-inflammatory in this population, with some inappropriate use of immunosuppressive doses. Increasing bodyweight was associated with a small reduction in dose in mg/kg, suggesting that clinicians are adjusting prednisolone dose rates based on dog bodyweight.

A retrospective multi-center study of treatment, outcome, and prognostic factors in 34 dogs with disseminated aspergillosis in Australia.

BACKGROUND: Disseminated aspergillosis (DA) in dogs has a guarded prognosis and there is a lack of a gold standard treatment protocol. OBJECTIVE: To retrospectively assess survival times and factors influencing survival times. ANIMALS: Dogs diagnosed with DA from January 2007 to June 2017. METHODS: Disseminated aspergillosis case data were retrieved from 13 Australian veterinary referral centers, with a diagnosis confirmed with culture or PCR. Factors influencing survival time after diagnosis were quantified using a Cox proportional hazards regression model. RESULTS: Thirty-four dogs met the study inclusion criteria. Twenty-two dogs were treated with antifungal treatment and 12 dogs received no antifungal treatment. Accounting for censoring of dogs that were either still alive on the date of data collection or were loss to follow-up, dogs treated with itraconazole alone (n = 8) had a median survival time (MST) of 63 (95% CI: 20-272) days compared to 830 (95% CI: 267-1259) days for the n = 14 dogs that received multimodal antifungal therapy ( χ 2 test statistic 8.6; df = 1; P < .01). The daily hazard of death (DHOD) for dogs with abnormally high serum creatinine concentration at the time of diagnosis was 7.4 (95% CI: 1.9-29) times that of dogs with serum creatinine within the reference interval. CONCLUSION AND CLINICAL IMPORTANCE: Serum creatinine concentration at the time of diagnosis is a useful prognostic indicator for survival after a diagnosis of DA. The MST for dogs treated with multimodal antifungal therapy is longer than itraconazole alone and warrant further investigation (P < .01).

Hierarchical modelling of immunoglobulin coated bacteria in dogs with chronic enteropathy shows reduction in coating with disease remission but marked inter-individual and treatment-response variability.

Chronic enteropathies are a common problem in dogs, but many aspects of the pathogenesis remain unknown, making the therapeutic approach challenging in some cases. Environmental factors are intimately related to the development and perpetuation of gastrointestinal disease and the gut microbiome has been identified as a contributing factor. Previous studies have identified dysbiosis and reduced bacterial diversity in the gastrointestinal microbiota of dogs with chronic enteropathies. In this case-controlled study, we use flow cytometry and 16S rRNA sequencing to characterise bacteria highly coated with IgA or IgG in faecal samples from dogs with chronic enteropathy and evaluated their correlation with disease and resolution of the clinical signs. IgA and IgG-coated faecal bacterial counts were significantly higher during active disease compared to healthy dogs and decreased with the resolution of the clinical signs. Characterisation of taxa-specific coating of the intestinal microbiota with IgA and IgG showed marked variation between dogs and disease states, and different patterns of immunoglobulin enrichment were observed in dogs with chronic enteropathy, particularly for Erysipelotrichaceae, Clostridicaceae, Enterobacteriaceae, Prevotellaceae and Bacteroidaceae, families. Although, members of these bacterial groups have been associated with strong immunogenic properties and could potentially constitute important biomarkers of disease, their significance and role need to be further investigated.

From Bench Top to Clinics: How New Tests Can Be Helpful in Diagnosis and Management of Dogs with Chronic Enteropathies.

In this article, we review different tests that have been researched in dogs with chronic enteropathy. The usefulness of these tests either to assess etiology, to differentiate between treatment response, or to monitor treatment response is discussed. The tests are divided in those that are commercially available and those that hold promises for further development.

Suspected pancreatic carcinoma needle tract seeding in a cat.

CASE SUMMARY: A 15-year-old male neutered domestic longhair cat was referred for investigation of a pancreatic nodule. Fine-needle aspiration of the nodule was performed on two occasions, 2 weeks apart, and cytology revealed pyogranulomatous inflammation and moderately dysplastic exocrine pancreatic epithelium, suspicious for neoplasia. Thoracic radiographs were unremarkable and a partial pancreatectomy was performed. On histopathology, the nodule was diagnosed as a moderately differentiated pancreatic adenocarcinoma. Two weeks after surgery, a firm subcutaneous nodule was detected on the left ventrolateral abdomen. Cytology of the nodule was suggestive of pancreatic carcinoma and needle tract seeding was suspected. With palliative treatment, the cat lived a further 136 days. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this represents the first report of suspected transabdominal needle tract seeding of pancreatic carcinoma following fine-needle aspiration in veterinary medicine. Veterinarians should consider this when discussing risks of pancreatic fine-needle aspiration with owners and should attempt to minimise the number of needle aspirations where possible.

Chronic Enteropathy In Canines: Prevalence, Impact And Management Strategies.

In this article, the studies about the prevalence of chronic enteropathy are reviewed as well as the information regarding short- and long-term prognosis for dogs treated with the three most common therapies; these include dietary modification, antibiotics, and immunosuppressants. Although the data available are limited, most studies support a good to excellent long-term response in dogs that have a successful food trial, whereas the response is poor with antibiotics or on-going treatment is required to retain remission. There is a risk of antimicrobial resistance developing with inappropriate use of antimicrobials such as in these situations. The published information highlights the need for alternative strategies to antibiotic treatment to manipulate the GI microbiome, and in the final part of this article studies on the use of probiotic for the treatment of chronic enteropathy are reviewed.

The utility of diagnostic tests for immune-mediated hemolytic anemia.

BACKGROUND: A definitive diagnosis of immune-mediated hemolytic anemia (IMHA) can be difficult to make. However, it is critical to differentiate IMHA from other causes of anemia due to the impact on prognosis and outcome for IMHA patients. Recently published American College of Veterinary Internal Medicine recommendations for the diagnosis of IMHA should be followed to concurrently confirm ongoing anemia, verify in vivo hemolysis, and detect anti-erythrocyte antibodies. The reliability of immunologic IMHA tests varies depending on which test is used and how it is performed. OBJECTIVES: Our aims were to determine which tests are currently used in veterinary medicine to diagnose IMHA and review the utility of assays that have historically been used to diagnose IMHA. METHODS: A short survey was designed to see which diagnostic tests for IMHA were currently being used by veterinary practices. The survey was distributed via list-serves to veterinarians and veterinary technologists. A literature review was performed to report the utility of diagnostic tests for the diagnosis of IMHA. RESULTS: Survey respondents indicated a variability in test protocols used to diagnose IMHA. Most respondents perform saline agglutination or Coombs' tests to detect anti-erythrocyte antibodies. Additional tests that can be used to support a diagnosis of IMHA are discussed in this review. CONCLUSIONS: A standardized diagnostic approach should be followed to differentiate IMHA from other causes of anemia. Test methodology can vary from one laboratory to another, and clinicians should be familiar with the procedures used by their laboratory.

ACVIM consensus statement on the diagnosis of immune-mediated hemolytic anemia in dogs and cats.

Immune-mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune-mediated erythrocyte destruction, and adverse consequences of long-term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence-based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for IMHA, comorbidities, and causality. Succinct evidence summary statements were written, along with screening recommendations. Statements were refined by conducting 3 iterations of Delphi review with panel and task force members. Commentary was solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted. The resulting document is intended to provide clinical guidelines for diagnosis of, and underlying disease screening for, IMHA in dogs and cats. These should be implemented with consideration of animal, owner, and geographical factors.

Effect of immunosuppressive drugs on cytokine production in canine whole blood stimulated with lipopolysaccharide or a combination of ionomycin and phorbol 12-myristate 13-acetate.

A pharmacodynamic assay has been previously developed to monitor ciclosporin treatment in dogs by assessing inhibition of cytokine transcription after whole blood stimulation with 12-myristate 13-1 acetate and ionomycin (PMA/I). In this study, whole blood stimulation with either PMA/I or lipopolysaccharide (LPS) was used to assess the effect of multiple drugs (azathioprine, ciclosporin, mycophenolate, leflunomide and prednisone) after a 7-day treatment course on production of cytokines measured with a multiplex assay in healthy dogs (n = 4 for each treatment). Interleukin-10 (IL-10), interferon gamma (IFNγ) and tumour necrosis factor alpha (TNFα) were significantly activated by PMA/I stimulation and IL-6, IL-10 and TNFα by LPS stimulation, in the absence of immunosuppressive drugs. After ciclosporin treatment, IL-10, IFNγ and TNFα production was significantly reduced after stimulation with PMA/I compared to pre-treatment. After prednisone treatment, TNFα production was significantly reduced after stimulation with PMA/I or LPS compared to pre-treatment. No significant change was observed after treatment with azathioprine, leflunomide or mycophenolate. This methodology may be useful to monitor dogs not only treated with ciclosporin, but also with prednisone or a combination of both. Further studies are needed to assess the use of this assay in a clinical setting.

Changes in duodenal CD163-positive cells in dogs with chronic enteropathy after successful treatment.

Chronic enteropathy (CE) in dogs is characterized retrospectively per treatment response as food-responsive enteropathy (FRE), antibiotic-responsive enteropathy (ARE), and immunosuppressant-responsive enteropathy (IRE) - the latter most resembling inflammatory bowel disease in people. The aim of this study was to characterize duodenal macrophages (Mϕ) in CE using immunohistochemistry; with calprotectin (CAL) as a marker of early differentiated Mϕ and CD163 expression as a marker for resident Mϕ in the duodenum before and after treatment. Prior to treatment, dogs with FRE and IRE had a lower CD163+/CAL+ ratio than control dogs (CTRL) in crypts; this increased significantly and normalized compared with CTRL after treatment. Conversely, the CD163+/CAL+ ratio in dogs with ARE was comparable to that in healthy dogs before and after treatment. In summary, these results suggest that Mϕ play a role in the pathogenesis of CE in FRE and IRE, with a decrease in resident Mϕ and an increase in early differentiated Mϕ, but not in ARE dogs. Mϕ normalize after successful treatment.

Comparison of a chlorambucil-prednisolone combination with an azathioprine-prednisolone combination for treatment of chronic enteropathy with concurrent protein-losing enteropathy in dogs: 27 cases (2007-2010).

OBJECTIVE: To compare treatment protocols for chronic enteropathy and concurrent protein-losing enteropathy that used prednisolone in conjunction with either azathioprine or chlorambucil in dogs. DESIGN: Retrospective case series. ANIMALS: 27 dogs. PROCEDURES: All dogs had hypoalbuminemia (serum albumin concentration, < 18.0 g/L) and chronic enteropathy as diagnosed via complete gastrointestinal tract investigations including intestinal biopsy. Dogs received either an azathioprine-prednisolone combination (group A; n = 13) or a chlorambucil-prednisolone combination (group C; 14). Response to treatment was assessed by evaluation of body weight gain, serum albumin concentration, and duration of primary treatment. RESULTS: No significant pretreatment differences were detected between groups for any baseline variable (signalment and weight), clinicopathologic variable (albumin, cobalamin, and folate concentrations), or histopathologic findings. After treatment, serum albumin concentration and weight gain were significantly greater in group C. Median survival time for group A dogs was 30 days (95% confidence interval, 15 to 45 days) and was not reached for group C dogs. Duration of primary treatment was positively associated with the histopathologic presence of mild lacteal dilatation and use of a chlorambucil-prednisolone combination. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that a chlorambucil-prednisolone protocol is more efficacious for treatment of chronic enteropathy and concurrent protein-losing enteropathy, compared with an azathioprine-prednisolone combination. Given these findings, a prospective randomized clinical trial is warranted.

Detection of extended-spectrum-beta-lactamase-positive Escherichia coli in bile isolates from two dogs with bacterial cholangiohepatitis.

This is the first report of Escherichia coli isolates producing CTX-M-15, the predominant type of extended-spectrum ß-lactamase (ESBL) associated with clinical disease in humans in the United Kingdom, in a United Kingdom pet dog. This report also describes the first isolation of CTX-M/Tem ESBL-positive E. coli from bile in dogs with hepatobiliary disease.

Unusual systemic signs in a dog with sterile neutrophilic-macrophagic lymphadenitis and nodular panniculitis.

A 7 mo old female English springer spaniel was presented with diarrhea, vomiting, apathy, and hyperthermia. Further examinations revealed generalized lymphadenomegaly consistent with sterile neutrophilic-macrophagic lymphadenitis and pulmonary involvement. Subcutaneous nodules developed one day after presentation. Histology was consistent with sterile idiopathic nodular panniculitis and vasculitis. No infectious organism was isolated. The dog responded to prednisolone, but relapsed during medication tapering. Cyclosporine had to be added to control the disease. No further relapse had occurred 98 wk after the first presentation. This is an unusual presentation of a systemic sterile neutrophilic-macrophagic lymphadenitis with nodular panniculitis and vasculitis associated with gastrointestinal and pulmonary signs.

Evaluation of lymphocyte apoptosis in dogs with inflammatory bowel disease.

OBJECTIVE: To determine whether lymphocyte apoptosis in intestinal mucosae is more common in healthy dogs than dogs with inflammatory bowel disease (IBD) and whether numbers of apoptotic cells increase after successful treatment of affected dogs. ANIMALS: 8 dogs with IBD (IBD dogs) and 8 healthy control dogs. PROCEDURES: Biopsy specimens of the duodenum and colon were obtained via endoscopy from dogs with IBD before and after 10 weeks of standard treatment and compared with specimens obtained from control dogs. Expression of activated caspase 3 (Casp3), caspase-cleaved fragment p85 from poly-ADP-ribose polymerase (PARP), and B-cell leukemia/lymphoma 2 (Bcl-2) was measured in the duodenal (villous tip and base) and colonic mucosae. RESULTS: Expression of Casp3 was greater in the duodenal villous tips of control dogs, compared with expression in similar tissues from dogs with IBD before or after treatment. Despite clinical improvement of dogs with IBD, expression of Casp3 did not increase after treatment. Expression of PARP did not differ between groups at any time point. Expression of Bcl-2 was greater at all 3 tissue sites in control dogs, compared with expression at the same sites in dogs with IBD. Furthermore, Bcl-2 expression in duodenal villous tips was higher in dogs with IBD after treatment but was not higher elsewhere. In control dogs, expression patterns for all 3 markers were similar between sites (villous tip > villous base > colon). CONCLUSIONS AND CLINICAL RELEVANCE: Expression of Casp3 in lymphocytes in duodenal villous tips was significantly reduced in dogs with IBD, compared with expression in healthy dogs, but no increase was detected following successful treatment of IBD. Increased expression of Bcl-2 may be a potential marker of the success of treatment.