Duodenal and colonic mucosal S100A8/A9 (calprotectin) expression is increased and correlates with the severity of select histologic lesions in dogs with chronic inflammatory enteropathy.

BACKGROUND: Calprotectin, a damage-associated molecular pattern protein of the S100/calgranulin family, is a potential marker of gastrointestinal inflammation in dogs and mainly originates from activated macrophages and granulocytes. Increased calprotectin concentrations are reported in feces and serum samples from dogs with chronic inflammatory enteropathy (CIE), but mucosal calprotectin expression has not been extensively investigated in canine CIE. Thus, we aimed to evaluate gastrointestinal mucosal concentrations of calprotectin in 62 dogs (44 dogs with CIE compared to 18 healthy Beagles) using a particle-enhanced turbidimetric immunoassay method. Additionally, we assessed the relationship of gastric, duodenal, jejunal, ileal, and colonic mucosal calprotectin levels with the clinical disease severity (canine clinical inflammatory bowel disease activity index, CIBDAI), histopathologic findings, clinical outcome, and serum albumin concentrations to further evaluate the potential of calprotectin as a biomarker for CIE. RESULTS: Mucosal calprotectin concentrations in dogs with CIE were significantly higher in the duodenum (median: 276.2 µg/g) and colon (median: 298.2 µg/g) compared to healthy controls (median: 94.3 µg/g, P = 0.0039; and median: 112.0 µg/g, P = 0.0061). Similar numerical differences in the ileum and cecum were not statistically significant, and mucosal calprotectin concentrations correlated significantly among the different gastrointestinal segments. Histologic lesion severity was linked to mucosal calprotectin concentrations for inflammatory and structural histology criteria in the duodenum and colon (all P < 0.05). Higher mucosal calprotectin levels in the duodenum and across all segments correlated with lower serum albumin concentrations (both P < 0.05); duodenal mucosal calprotectin concentrations were more than sixfold higher in hypoalbuminemic dogs (median: 1441 µg/g, n = 4) than normoalbuminemic dogs (median: 227 µg/g, n = 40). There was no significant association of mucosal calprotectin levels with CIBDAI scores or individual clinical outcomes. CONCLUSIONS: These results show that duodenal and colonic mucosal calprotectin concentrations are increased in dogs with CIE, providing further supporting evidence for the diagnostic potential of fecal calprotectin (presumably reflecting mucosal) concentrations and in dogs with CIE. Further longitudinal research is needed to assess changes in mucosal calprotectin concentrations with clinical response to treatment vs. mucosal disease remission and to determine the clinical utility of fecal calprotectin concentrations to diagnose and monitor dogs with CIE in clinical practice.

Diagnostic value of endoscopic retrograde cholangiopancreatography and therapeutic value of endoscopic sphincterotomy in dogs with suspected hepatobiliary disorders.

BACKGROUND: Conventional diagnostic methods have some limitations in diagnosing specific causes of canine hepatobiliary disorders. In the evaluation of the hepatobiliary system in dogs, ultrasonography (US) is the first imaging method of choice. Nonetheless, endoscopic retrograde cholangiopancreatography (ERCP) has also been proven to be a practicable technique for evaluating canine hepatobiliary (endoscopic retrograde cholangiography, ERC) and pancreatic duct (endoscopic retrograde pancreatography, ERP) disorders, providing additional therapeutic options by sphincterotomy (EST). To date, the efficacy and safety of diagnostic and therapeutic ERCP has not been evaluated in veterinary medicine literature. The present study sought to report complications and outcomes of dogs undergoing ERCP and EST, and to assess the usefulness of diagnostic ERCP by comparing the findings of US, ERCP and histopathological findings in liver and pancreas. RESULTS: This retrospective case series comprises data collected from 15 dogs that underwent successful ERC/ERCP. Nine dogs underwent EST following ERC. US and ERC were best in agreement when assessing the common bile duct. In case of disagreement between the modalities, the ERC findings of the ductal structures were in line with the available pathology findings more often than the US findings, whereas the opposite was noted for the gallbladder. The technical success rates were 88.2% for ERC, 66.7% for ERP, and 81.8% for EST, with no major complications during or immediately after the procedure. Immediate bile flow after EST was recorded in 7/9 dogs but only four showed coinciding clinical and laboratory improvement and four dogs were euthanized within 1-6 days after EST. CONCLUSIONS: US remains a valuable initial diagnostic imaging method for hepatobiliary disorders and allows good assessment of the gallbladder. ERC can serve as a complementary procedure for diagnostic assessment of the hepatobiliary duct disorders. However, in order to improve the outcomes of EST, careful selection of patients for the procedure would require more advanced diagnostic imaging of the hepatobiliary area.

In-frame deletion in canine PITRM1 is associated with a severe early-onset epilepsy, mitochondrial dysfunction and neurodegeneration.

We investigated the clinical, genetic, and pathological characteristics of a previously unknown severe juvenile brain disorder in several litters of Parson Russel Terriers. The disease started with epileptic seizures at 6-12 weeks of age and progressed rapidly to status epilepticus and death or euthanasia. Histopathological changes at autopsy were restricted to the brain. There was severe acute neuronal degeneration and necrosis diffusely affecting the grey matter throughout the brain with extensive intraneuronal mitochondrial crowding and accumulation of amyloid-ß (Aß). Combined homozygosity mapping and genome sequencing revealed an in-frame 6-bp deletion in the nuclear-encoded pitrilysin metallopeptidase 1 (PITRM1) encoding for a mitochondrial protease involved in mitochondrial targeting sequence processing and degradation. The 6-bp deletion results in the loss of two amino acid residues in the N-terminal part of PITRM1, potentially affecting protein folding and function. Assessment of the mitochondrial function in the affected brain tissue showed a significant deficiency in respiratory chain function. The functional consequences of the mutation were modeled in yeast and showed impaired growth in permissive conditions and an impaired respiration capacity. Loss-of-function variants in human PITRM1 result in a childhood-onset progressive amyloidotic neurological syndrome characterized by spinocerebellar ataxia with behavioral, psychiatric and cognitive abnormalities. Homozygous Pitrm1-knockout mice are embryonic lethal, while heterozygotes show a progressive, neurodegenerative phenotype characterized by impairment in motor coordination and Aß deposits. Our study describes a novel early-onset PITRM1-related neurodegenerative canine brain disorder with mitochondrial dysfunction, Aß accumulation, and lethal epilepsy. The findings highlight the essential role of PITRM1 in neuronal survival and strengthen the connection between mitochondrial dysfunction and neurodegeneration.

Altered Basal Autophagy Affects Extracellular Vesicle Release in Cells of Lagotto Romagnolo Dogs With a Variant ATG4D.

Lagotto Romagnolo breed dogs develop a progressive neurological disease with intracellular vacuolar storage when homozygous for a variant in the autophagy-related gene 4D (ATG4D). A lysosomal enzyme deficiency has not been proven in this disease, despite its overlapping morphology with lysosomal storage diseases. Instead, basal autophagy was altered in fibroblasts from affected dogs. The aim of this study was to clarify the origin of the limiting membrane of the accumulating vacuoles and determine whether altered basal autophagy affects the extracellular release of vesicles in cells from diseased dogs. When assessed by immunoelectron microscopy, the membrane of the cytoplasmic vacuoles in affected tissues contained ATG4D, markers for autolysosomes (microtubule-associated protein 1A/B light chain 3 and lysosome-associated membrane protein 2) and for recycling endosomes (transferrin receptor 2), indicating that the vacuoles are hybrid organelles between endocytic and autophagic pathways. Ultracentrifugation, nanoparticle tracking analysis, and mass spectrometry were used to analyze the vesicles released from cultured fibroblasts of affected and control dogs. The amount of extracellular vesicles (EVs) released from affected fibroblasts was significantly increased during basal conditions in comparison to controls. This difference disappeared during starvation. The basal EV proteome of affected cells was enriched with cytosolic, endoplasmic reticulum, and mitochondrial proteins. Heat shock proteins and chaperones, some of which are known substrates of basal autophagy, were identified among the proteins unique to EVs of affected cells. An increased release of extracellular vesicles may serve as a compensatory mechanism in disposal of intracellular proteins during dysfunctional basal autophagy in this spontaneous disease.

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.

ANLN truncation causes a familial fatal acute respiratory distress syndrome in Dalmatian dogs.

Acute respiratory distress syndrome (ARDS) is the leading cause of death in critical care medicine. The syndrome is typified by an exaggerated inflammatory response within the lungs. ARDS has been reported in many species, including dogs. We have previously reported a fatal familial juvenile respiratory disease accompanied by occasional unilateral renal aplasia and hydrocephalus, in Dalmatian dogs. The condition with a suggested recessive mode of inheritance resembles acute exacerbation of usual interstitial pneumonia in man. We combined SNP-based homozygosity mapping of two ARDS-affected Dalmatian dogs and whole genome sequencing of one affected dog to identify a case-specific homozygous nonsense variant, c.31C>T; p.R11* in the ANLN gene. Subsequent analysis of the variant in a total cohort of 188 Dalmatians, including seven cases, indicated complete segregation of the variant with the disease and confirmed an autosomal recessive mode of inheritance. Low carrier frequency of 1.7% was observed in a population cohort. The early nonsense variant results in a nearly complete truncation of the ANLN protein and immunohistochemical analysis of the affected lung tissue demonstrated the lack of the membranous and cytoplasmic staining of ANLN protein in the metaplastic bronchial epithelium. The ANLN gene encodes an anillin actin binding protein with a suggested regulatory role in the integrity of intercellular junctions. Our study suggests that defective ANLN results in abnormal cellular organization of the bronchiolar epithelium, which in turn predisposes to acute respiratory distress. ANLN has been previously linked to a dominant focal segmental glomerulosclerosis in human without pulmonary defects. However, the lack of similar renal manifestations in the affected Dalmatians suggest a novel ANLN-related pulmonary function and disease association.

Concentrations of medetomidine enantiomers and vatinoxan, an α2-adrenoceptor antagonist, in plasma and central nervous tissue after intravenous coadministration in dogs.

OBJECTIVE: To quantify the peripheral selectivity of vatinoxan (L-659,066, MK-467) in dogs by comparing the concentrations of vatinoxan, dexmedetomidine and levomedetomidine in plasma and central nervous system (CNS) tissue after intravenous (IV) coadministration of vatinoxan and medetomidine. STUDY DESIGN: Experimental, observational study. ANIMALS: A group of six healthy, purpose-bred Beagle dogs (four females and two males) aged 6.5 ± 0.1 years (mean ± standard deviation). METHODS: All dogs were administered a combination of medetomidine (40 µg kg-1) and vatinoxan (800 µg kg-1) as IV bolus. After 20 minutes, the dogs were euthanized with an IV overdose of pentobarbital (140 mg kg-1) and both venous plasma and CNS tissues (brain, cervical and lumbar spinal cord) were harvested. Concentrations of dexmedetomidine, levomedetomidine and vatinoxan in all samples were quantified by liquid chromatography-tandem mass spectrometry and data were analyzed with nonparametric tests with post hoc corrections where appropriate. RESULTS: All dogs became deeply sedated after the treatment. The CNS-to-plasma ratio of vatinoxan concentration was approximately 1:50, whereas the concentrations of dexmedetomidine and levomedetomidine in the CNS were three- to seven-fold of those in plasma. CONCLUSIONS AND CLINICAL RELEVANCE: With the doses studied, these results confirm the peripheral selectivity of vatinoxan in dogs, when coadministered IV with medetomidine. Thus, it is likely that vatinoxan preferentially antagonizes α2-adrenoceptors outside the CNS.

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.

A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease.

Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136) in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s) and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

Basal Autophagy Is Altered in Lagotto Romagnolo Dogs with an ATG4D Mutation.

A missense variant in the autophagy-related ATG4D-gene has been associated with a progressive degenerative neurological disease in Lagotto Romagnolo (LR) dogs. In addition to neural lesions, affected dogs show an extraneural histopathological phenotype characterized by severe cytoplasmic vacuolization, a finding not previously linked with disturbed autophagy in animals. Here we aimed at testing the hypothesis that autophagy is altered in the affected dogs, at reporting the histopathology of extraneural tissues and at excluding lysosomal storage diseases. Basal and starvation-induced autophagy were monitored by Western blotting and immunofluorescence of microtubule associated protein 1A/B light chain3 (LC3) in fibroblasts from 2 affected dogs. The extraneural findings of 9 euthanized LRs and skin biopsies from 4 living affected LRs were examined by light microscopy, electron microscopy, and immunohistochemistry (IHC), using antibodies against autophagosomal membranes (LC3), autophagic cargo (p62), and lysosomal membranes (LAMP2). Biochemical screening of urine and fibroblasts of 2 affected dogs was performed. Under basal conditions, the affected fibroblasts contained significantly more LC3-II and LC3-positive vesicles than did the controls. Morphologically, several cells, including serous secretory epithelium, endothelial cells, pericytes, plasma cells, and macrophages, contained cytoplasmic vacuoles with an ultrastructure resembling enlarged amphisomes, endosomes, or multivesicular bodies. IHC showed strong membranous LAMP2 positivity only in sweat glands. The results show that basal but not induced autophagy is altered in affected fibroblasts. The ultrastructure of affected cells is compatible with altered autophagic and endo-lysosomal vesicular traffic. The findings in this spontaneous disease provide insight into possible tissue-specific roles of basal autophagy.

Infection with Possible Novel Parapoxvirus in Horse, Finland, 2013.

A horse in Finland exhibited generalized granulomatous inflammation and severe proliferative dermatitis. After euthanization, we detected poxvirus DNA from a skin lesion sample. The virus sequence grouped with parapoxviruses, closely resembling a novel poxvirus detected in humans in the United States after horse contact. Our findings indicate horses may be a reservoir for zoonotic parapoxvirus.

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.

A SEL1L mutation links a canine progressive early-onset cerebellar ataxia to the endoplasmic reticulum-associated protein degradation (ERAD) machinery.

Inherited ataxias are characterized by degeneration of the cerebellar structures, which results in progressive motor incoordination. Hereditary ataxias occur in many species, including humans and dogs. Several mutations have been found in humans, but the genetic background has remained elusive in dogs. The Finnish Hound suffers from an early-onset progressive cerebellar ataxia. We have performed clinical, pathological, and genetic studies to describe the disease phenotype and to identify its genetic cause. Neurological examinations on ten affected dogs revealed rapidly progressing generalized cerebellar ataxia, tremors, and failure to thrive. Clinical signs were present by the age of 3 months, and cerebellar shrinkage was detectable through MRI. Pathological and histological examinations indicated cerebellum-restricted neurodegeneration. Marked loss of Purkinje cells was detected in the cerebellar cortex with secondary changes in other cortical layers. A genome-wide association study in a cohort of 31 dogs mapped the ataxia gene to a 1.5 Mb locus on canine chromosome 8 (p(raw) = 1.1x10(-7), p(genome) = 7.5x10(-4)). Sequencing of a functional candidate gene, sel-1 suppressor of lin-12-like (SEL1L), revealed a homozygous missense mutation, c.1972T>C; p.Ser658Pro, in a highly conserved protein domain. The mutation segregated fully in the recessive pedigree, and a 10% carrier frequency was indicated in a population cohort. SEL1L is a component of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery and has not been previously associated to inherited ataxias. Dysfunctional protein degradation is known to cause ER stress, and we found a significant increase in expression of nine ER stress responsive genes in the cerebellar cortex of affected dogs, supporting the pathogenicity of the mutation. Our study describes the first early-onset neurodegenerative ataxia mutation in dogs, establishes an ERAD-mediated neurodegenerative disease model, and proposes SEL1L as a new candidate gene in progressive childhood ataxias. Furthermore, our results have enabled the development of a genetic test for breeders.

The DoGA consortium expression atlas of promoters and genes in 100 canine tissues.

The dog, Canis lupus familiaris, is an important model for studying human diseases. Unlike many model organisms, the dog genome has a comparatively poor functional annotation, which hampers gene discovery for development, morphology, disease, and behavior. To fill this gap, we established a comprehensive tissue biobank for both the dog and wolf samples. The biobank consists of 5485 samples representing 132 tissues from 13 dogs, 12 dog embryos, and 24 wolves. In a subset of 100 tissues from nine dogs and 12 embryos, we characterized gene expression activity for each promoter, including alternative and novel, i.e., previously not annotated, promoter regions, using the 5' targeting RNA sequencing technology STRT2-seq. We identified over 100,000 promoter region candidates in the recent canine genome assembly, CanFam4, including over 45,000 highly reproducible sites with gene expression and respective tissue enrichment levels. We provide a promoter and gene expression atlas with interactive, open data resources, including a data coordination center and genome browser track hubs. We demonstrated the applicability of Dog Genome Annotation (DoGA) data and resources using multiple examples spanning canine embryonic development, morphology and behavior, and diseases across species.

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.

A homozygous missense variant in the alkaline phosphatase gene ALPL is associated with a severe form of canine hypophosphatasia.

Inherited skeletal disorders affect both humans and animals. In the current study, we have performed series of clinical, pathological and genetic examinations to characterize a previously unreported skeletal disease in the Karelian Bear Dog (KBD) breed. The disease was recognized in seven KBD puppies with a variable presentation of skeletal hypomineralization, growth retardation, seizures and movement difficulties. Exome sequencing of one affected dog revealed a homozygous missense variant (c.1301T > G; p.V434G) in the tissue non-specific alkaline phosphatase gene, ALPL. The identified recessive variant showed full segregation with the disease in a cohort of 509 KBDs with a carrier frequency of 0.17 and was absent from 303 dogs from control breeds. In humans, recessive and dominant ALPL mutations cause hypophosphatasia (HPP), a metabolic bone disease with highly heterogeneous clinical manifestations, ranging from lethal perinatal hypomineralization to a relatively mild dental disease. Our study reports the first naturally occurring HPP in animals, resembling the human infantile form. The canine HPP model may serve as a preclinical model while a genetic test will assist in breeding programs.

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.