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.

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.

Characterization of the fecal virome in dogs with chronic enteropathy.

The fecal virome has been investigated in humans and various animal species using next generation sequencing. However, limited information is available about the fecal virome of dogs with chronic enteropathy (CE). We aimed to characterize the canine fecal virome of dogs with CE and compare it with the virome of previously analyzed healthy dogs.A total of 16 adult dogs; 8 healthy dogs (data from a parallel study) and 8 dogs with CE had fecal samples assessed by viral shotgun sequencing. Fecal samples were subjected to enrichment of viral nucleic acids prior to sequencing and metagenomic analyses. Characterization of the complete genome of a canine kobuvirus was performed by Sanger sequencing. An additional 21 healthy dogs and 14 dogs with CE were further analyzed for the prevalence of canine kobuvirus.Three fecal samples from dogs with CE contained in total 3 eukaryotic viral families. In contrast, 4/8 fecal samples previously identified from healthy dogs, contained 5 eukaryotic viral families with 2 families exclusive to this group. Bacteriophages were identified in all fecal samples from CE and healthy dogs. Canine kobuvirus was identified in one dog with CE, by shotgun sequencing, and the complete genome was then characterized. This kobuvirus was classified within canine kobuvirus group, being similar to strains from Korea and China. The larger prevalence study did not detect additional samples positive for canine kobuvirus. The fecal virome of dogs with CE differs in number and type of viral families from healthy dogs. The first Australian canine kobuvirus sequence was identified and characterized from a dog with CE.

Emergence of nasal chondrosarcoma in a dog with nasal polyposis.

Whilst the malignant transformation of nasal polyps or secondary development of nasal neoplasia after chronic inflammation is likely to be relatively rare, this potential complication should be considered, and the clinician should be vigilant for evidence of malignant transformation.

Characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics.

The virome has been increasingly investigated in numerous animal species and in different sites of the body, facilitating the identification and discovery of a variety of viruses. In spite of this, the faecal virome of healthy dogs has not been investigated. In this study we describe the faecal virome of healthy dogs and dogs with acute diarrhoea in Australia, using a shotgun metagenomic approach. Viral sequences from a range of different virus families, including both RNA and DNA families, and known pathogens implicated in enteric disease were documented. Twelve viral families were identified, of which four were bacteriophages. Eight eukaryotic viral families were detected: Astroviridae, Coronaviridae, Reoviridae, Picornaviridae, Caliciviridae, Parvoviridae, Adenoviridae and Papillomaviridae. Families Astroviridae, Picornaviridae and Caliciviridae were found only in dogs with acute diarrhoea, with Astroviridae being the most common family identified in this group. Due to its prevalence, characterisation the complete genome of a canine astrovirus was performed. These studies indicate that metagenomic analyses are useful for the investigation of viral populations in the faeces of dogs. Further studies to elucidate the epidemiological and biological relevance of these findings are warranted.

Central diabetes insipidus in a cat with central nervous system B cell lymphoma.

A 6-year-old male neutered cat presented with blindness, lethargy, polydipsia, hyposthenuria and severe hypernatraemia. Central diabetes insipidus was demonstrated by means of a low measured anti-diuretic hormone (ADH) concentration in the face of hypernatraemia, and clinical response to supplementation with desmopressin. Magnetic resonance imaging of the brain showed a discrete mass in the region of the hypothalamus. The cat was euthanased and post-mortem histological examination demonstrated B cell lymphoma involving the brain, optic nerves, urinary bladder wall and diaphragm. To the authors' knowledge, this case report is the first to describe central diabetes insipidus caused by central nervous system lymphoma in the cat.

Granulomatous colitis of boxer dogs.

Granulomatous colitis (GC) is a rare, breed-specific inflammatory bowel disease of young Boxer dogs. GC has been refractory to treatment and associated with high mortality rates, but culture-independent molecular analysis has transformed therapy and prognosis by uncovering a correlation between GC and Escherichia coli invasion within colonic mucosal macrophages. GC-associated invasive E coli are similar to a newly identified E coli pathotype, "adherent and invasive E coli," that are increasingly associated with Crohn's disease in humans. Successful treatment of GC requires antimicrobials that are effective against E coli and penetrate intracellularly. Enrofloxacin is widely regarded as the antibiotic of choice.

Clinical resolution of a nasal granuloma caused by Trichosporon loubieri.

The current report describes the diagnosis of a nasopharyngeal granuloma due to a fungal infection by Trichosporon loubieri. This is the first report of successful treatment of nasal granuloma formation caused by Trichosporon species infection in a cat.

Observed occurrence of Tritrichomonas foetus and other enteric parasites in Australian cattery and shelter cats.

Cattery-housed pedigree cats, located mostly within the USA, have the highest reported prevalence of Tritrichomonas foetus (T foetus) to date. This prospective, multi-institutional, cross sectional study examines the occurrence of T foetus and other enteric parasites in cattery-housed and shelter cats within Australia, where T foetus has only recently been identified. Faecal specimens were collected from 134 cats, including 82 cattery-housed pedigree cats and 52 shelter cats. Faecal examinations performed for most cats included concentration techniques, Snap Giardia test, culture in InPouch medium, and polymerase chain reaction (PCR) amplification of T foetus ribosomal ribonucleic acid (rRNA) genes using species-specific primers. Observed occurrence of T foetus, Giardia species, Isospora species and Toxascaris leonina for cattery-housed cats (and catteries) were 0%, 7.4 (13.8)%, 10.9 (22.6)% and 1.6 (3.2)%, respectively. Observed occurrence of T foetus, Giardia species, Isospora species and hookworms for shelter cats were 0%, 11.5%, 9.8% and 4.9%, respectively. These results suggest the prevalence of T foetus in cattery-housed cats is currently much lower in Australia than in the USA, while Isospora and Giardia species infections are common.

Development of a clinical severity index for dogs with acute pancreatitis.

OBJECTIVE: To establish a clinical severity index that correlates severity of body system abnormalities with outcome in dogs with acute pancreatitis (AP) and determine the usefulness of serum C-reactive protein (C-RP) concentration as an objective measure of AP severity. DESIGN: Retrospective cohort study. ANIMALS: 61 client-owned dogs with ultrasonographically or histologically confirmed AP. PROCEDURES: Medical records of AP-affected dogs were reviewed, and signalment, physical examination findings, clinicopathologic data, and outcome (death or discharge from the hospital) were evaluated. The correlation of specific abnormalities in endocrine, hepatic, renal, hematopoietic, cardiovascular, and respiratory systems; local pancreatic complications; and intestinal integrity were evaluated, and a clinical severity index was developed for AP in dogs. The severity index score was compared with outcome and, for 12 dogs, with serum C-RP concentration. RESULTS: The clinical severity index had a good correlation with outcome and interval from hospital admission until end point (days until outcome), but there was no difference in days until outcome between survivors and nonsurvivors. All 12 dogs evaluated had high serum C-RP concentration, but this variable was not related to outcome; however, within a 2-day period after onset of clinical signs, serum C-RP concentration in survivors and nonsurvivors differed significantly. CONCLUSIONS AND CLINICAL RELEVANCE: Among AP-affected dogs, the clinical severity index may be useful for treatment comparisons and prediction of intensive management requirements. Serum C-RP concentration was best related to AP severity within a 2-day period after onset of clinical signs, but daily measurement may be more useful for monitoring progress.