Polymorphic microsatellite markers for genetic analysis of collagen genes in suspected collagenopathies in dogs

Defects in collagen proteins cause a variety of disorders in humans. It can be expected that collagen gene mutations are involved in collagenopathies in dogs. The collagen genes COL3A1, COL5A1, COL5A2, COL6A1, COL6A3, COL9A1, COL9A2, COL9A3, COL10A1 and COL11A1 were identified on the canine genome based on the homology with the human genes. Simple sequence repeats (microsatellites) were found in the chromosomal regions of these genes and investigated for polymorphism in Labrador Retrievers, Bernese Mountain dogs, Boxer dogs and German Shepherd dogs by PCR and subsequent detection of the DNA products. Nine informative microsatellite markers were identified. The markers closely situated to COL9A1, COL9A2 and COL9A3 were used to investigate the involvement of the genes in cranial cruciate ligament rupture in Boxer dogs. It was found that these genes are probably not involved in this abnormality. The markers described here will be useful for a candidate gene approach of suspected collagenopathies specific to dog breeds.     

Ascites due to pre-sinusoidal portal hypertension in dogs: a retrospective analysis of 17 cases

Accumulation of a pure transudate abdominal effusion in the absence of significant hypoalbuminaemia is uncommon in dogs and is due to pre-sinusoidal portal hypertension. Reported causes of pre-sinusoidal portal hypertension vary, but suggest a reasonable prognosis. A retrospective analysis of 17 dogs that presented to our institution with ascites due to pre-sinusoidal portal hypertension identified idiopathic hepatic fibrosis or canine chronic hepatitis as the underlying cause in the majority of cases. Twelve (70.5%) dogs were 4 years of age or younger at time of presentation. Total serum protein was higher in dogs with chronic hepatitis than it was in dogs without inflammatory disease. The prognosis was generally poor and no histological, imaging or biochemical parameters were useful as prognostic indicators. Dogs died or were euthanased due to severe clinical signs associated with the portal hypertension and/or perceived poor prognosis.     

PCR screening for candidate etiological agents of canine hepatitis

PROBLEM ASSESSED: Hepatitis, either acute or chronic, is a relatively common hepatic disease in dogs. Several forms of canine hepatitis can occur, some with a defined cause, most cases have an unknown etiology. The similarities between canine hepatitis and human viral hepatitis suggest that canine hepatitis may have a viral etiology too. OBJECTIVE: To test liver tissue of dogs with hepatitis for the presence of candidate agents based on their known association with hepatitis in other mammals. METHODS AND APPROACH: The following infectious agents were tested by PCR: Hepadnaviridae, Helicobacter spp., Leptospira spp., Borrelia spp., hepatitis A virus, hepatitis C virus and hepatitis E virus. Also canine adenovirus and parvovirus were included. Ninety-eight liver tissue samples of dogs with various histologically diagnoses forms of hepatitis were tested. Primers were designed on conserved regions in the genome of each of these agents, to increase the likelihood of detection by PCR. To further increase sensitivity, nested PCRs for all agents were designed. Finally, for each agent a nested short primer PCR (SPP) was performed. RESULTS: None of these agents were detected by nested PCR and nested SPP. However, in two acute hepatitis liver samples parvovirus was detected by nested PCR, and one of these was also detected by nested SPP. CONCLUSIONS: Hepatitis in dogs is not caused by agents with high homology to known infectious agents that cause hepatitis in other species.     

Current concepts in the treatment of canine chronic hepatitis

Chronic hepatitis is a common disorder in dogs seen by general practitioners. Several new drugs have been marketed for treating this disease. Unfortunately, there are few controlled studies that examine the efficacy of these medications for the treatment of canine chronic hepatitis. A rational therapeutic approach can be implemented based on histopathologic findings of a liver biopsy. A liver biopsy is essential for establishing a definitive diagnosis and guiding the optimal therapy. The biopsy allows characterizing the inflammatory process, quantitating hepatic copper concentrations, and determining if fibrosis is present. Copper associated hepatopathy is treated with zinc and copper chelators. Idiopathic chronic hepatitis is thought to be immune mediated. The treatment of idiopathic chronic hepatitis consists of controlling inflammation (prednisone, azathioprine), reversing fibrosis (colchicine), and protecting against oxidant damage (vitamin E, ursodeoxycholic acid, S-adenosylmethionine). The prognosis for chronic hepatitis is quite variable. Dogs with end-stage disease have a poor prognosis, while dogs diagnosed earlier can have a mean survival of years. Early diagnosis and intervention are key to the successful treatment of dogs with chronic hepatitis.     

Expression of the high mobility group A1 (HMGA1) and A2 (HMGA2) genes in canine lymphoma: analysis of 23 cases and comparison to control cases

Overexpression of high mobility group A (HMGA) genes was described as a prognostic marker in different human malignancies, but its role in canine haematopoietic malignancies was unknown so far. The objective of this study was to analyse HMGA1 and HMGA2 gene expression in lymph nodes of canine lymphoma patients. The expression of HMGA1 and HMGA2 was analysed in lymph node samples of 23 dogs with lymphoma and three control dogs using relative quantitative real‐time RT‐PCR. Relative quantity of HMGA1 was significantly higher in dogs with lymphoma compared with reference samples. HMGA2 expression did not differ between lymphoma and control dogs. With the exception of immunophenotype, comparison of disease parameters did not display any differences in HMGA1 and HMGA2 expression. The present findings indicate a role of HMGA genes in canine lymphoma. This study represents the basis for future veterinary and comparative studies dealing with their diagnostic, prognostic and therapeutic values.     

Lobular Dissecting Hepatitis in Juvenile and Young Adult Dogs

A specific form of chronic hepatitis is described in 21 dogs. The disease appears to develop in the neonatal and juvenile period. Predominant clinical signs include weight loss, ascites, and jaundice. The liver is small, with a smooth surface or a limited number of hyperplastic nodules. A characteristic feature of the hepatitis is disruption of the hepatic architecture by complete fibrotic dissection of the lobular parenchyma into individual and small groups of hepatocytes with subsequent portal hypertension. This lesion may represent a specific pattern of hepatic reaction in the immature animal, and resembles human neonatal hepatitis.     

Quantification of MDR-1 gene expression in canine tissues by real-time reverse transcription quantitative polymerase chain reaction

MDR-1 gene product mediated multidrug resistance is thought to play a major role in the outcome of chemotherapy in some canine tumors, especially malignant lymphoma. In the present study, MDR-1 RNA expression in normal lymph node and liver tissue as well as in tumor biopsies from 23 dogs with lymphomas and two dogs with liver tumors was measured by real-time RT-quantitative PCR. MDR-1 gene expression was detected in all samples analyzed. Comparably high MDR-1 RNA levels were measured in all normal liver tissues, one of the lymphomas and a cholangiocarcinoma. MDR-1 expression levels in canine lymphomas were found to vary over a wide range with most tumors expressing relative low levels. Interestingly, gastrointestinal lymphomas expressed higher MDR-1 RNA levels than multicentric lymphomas (p = 0.03). In conclusion, real-time RT-quantitative PCR appears to be a suitable method for sensitive and quantitative determination of MDR-1 gene expression in canine normal and neoplastic tissues.     

Polymerase chain reaction analysis for viruses in paraffin-embedded myocardium from dogs with dilated cardiomyopathy or myocarditis

OBJECTIVE: To perform polymerase chain reaction (PCR) analysis on paraffin-embedded myocardium from dogs with dilated cardiomyopathy (DCM) and dogs with myocarditis to screen for canine parvovirus, adenovirus types 1 and 2, and herpesvirus. SAMPLE POPULATION: Myocardial specimens from 18 dogs with an antemortem diagnosis of DCM and 9 dogs with a histopathologic diagnosis of myocarditis were evaluated. PROCEDURE: Paraffin-embedded myocardial specimens were screened for viral genome by PCR analysis. Positive-control specimens were developed from cell cultures as well as paraffin-embedded tissue specimens from dogs with clinical and histopathologic diagnoses of viral infection with canine parvovirus, adenovirus types 1 and 2, and herpesvirus. The histologic characteristics of all myocardial specimens were classified regarding extent, location, and type of inflammation and fibrosis. RESULTS: Canine adenovirus type 1 was amplified from 1 specimen from a dog with DCM. Canine parvovirus, adenovirus type 2, and herpesvirus were not amplified from any myocardial specimens. Histologic analysis of specimens from dogs with DCM revealed variable amounts of fibrosis; myocardial inflammation was observed in 1 affected dog. Histopathologic analysis of specimens from dogs with myocarditis disclosed variable degrees of inflammation and fibrosis. CONCLUSIONS AND CLINICAL RELEVANCE: Viral agents canine parvovirus, adenovirus types 1 and 2, and herpesvirus are not commonly associated with DCM or active myocarditis in dogs. Additional studies evaluating for nucleic acid from viruses that less commonly affect dogs or different types of infectious agents may be warranted to gain insight into the cause of DCM and myocarditis in dogs.     

Inherited copper toxicosis in the Bedlington terrier: a report of two clinical cases

The clinical signs, laboratory findings and pathological changes are described in two cases of inherited copper toxicosis in the Bedlington terrier. The first case presented with acute signs of depression, vomiting, anorexia, weight loss and jaundice while the second case followed a more chronic course with less severe clinical signs which included weight loss and ascites. Both dogs had elevated circulating levels of alanine aminotransferase (ALT), however other haematological and biochemical parameters, while reflecting liver involvement, varied between the two cases. Chemical analysis of the liver revealed elevated copper levels in both cases (951·7 and 1093·4 μg/g wet weight respectively; normal less than 150 μg/g). These levels, however, are less than some affected but asymptomatic Bedlington terriers. Pathologically the first case had micronodular cirrhosis, while the second had focal hepatitis with fibrosis. Both dogs showed vacuolation of the white matter in the cerebrum, cerebellum, midbrain and medulla. Attention is drawn to the similarities and differences between copper toxicosis in the Bedlington terrier and Wilson's disease in man.     

Assessment of collagen genes involved in fragmented medial coronoid process development in Labrador Retrievers as determined by affected sibling-pair analysis

OBJECTIVE: To evaluate the involvement of various collagen genes in the development of fragmented medial coronoid process (FCP) in Labrador Retrievers. SAMPLE POPULATION: 93 dogs originating from 13 litters were used in the study; FCP was diagnosed in 35 dogs, and each affected dog had at least 1 sibling that was also affected. Twelve dams and sires were included in the analysis. All dogs were purebred Labrador Retrievers except for 2 litters (offspring of a female Golden Retriever-Labrador Retriever mixed-breed dog). PROCEDURES: For each dog, DNA was isolated from blood samples. Polymorphic microsatellite markers adjacent to 14 candidate genes (ie, COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL6A3, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, and COL24A1) were analyzed by use of PCR assays; genotypes were determined via automated detection of DNA products. The level of allele sharing between pairs of affected siblings was assessed. RESULTS: Among the 93 dogs, allele sharing of the 14 collagen genes was determined as follows: COL1A1, 45%; COL1A2, 47%; COL2A1, 37%; COL3A1, 32%; COL5A1, 43%; COL5A2, 32%; COL6A3, 36%; COL9A1, 45%; COL9A2, 49%; COL9A3, 38%; COL10A1, 46%; COL11A1, 52%; COL11A2, 47%; and COL24A1, 47%. CONCLUSIONS AND CLINICAL RELEVANCE: Because siblings share 50% of their genome at random, the fact that the percentages of allele sharing among the analyzed collagen genes were not significantly > 50% indicates that these genes are not determinant candidates for FCP in Labrador Retrievers. The gene for the vitamin D receptor could also be excluded because of its proximity to COL2A1.     

Serum and bronchoalveolar lavage fluid endothelin-1 concentrations as diagnostic biomarkers of canine idiopathic pulmonary fibrosis

Background: Diagnosis of canine idiopathic pulmonary fibrosis (IPF) is challenging. Endothelin‐1 (ET1) is a biomarker of IPF in humans, but whether ET1 can detect and differentiate IPF from other canine respiratory diseases is unknown. Objective: To evaluate whether measurement of the concentration of ET1 in serum and bronchoalveolar lavage fluid (BALF) can be used to distinguish canine IPF from chronic bronchitis (CB) and eosinophilic bronchopneumopathy (EBP). Animals: Twelve dogs with IPF, 10 dogs with CB, 6 dogs with EBP, 13 privately owned healthy West Highland White Terriers (WHWT), and 9 healthy Beagle dogs. Methods: Prospective, case control study. ET1 concentration was determined by ELISA in serum and in BALF. Results: No significant difference in serum ET1 concentration was detected between healthy Beagle dogs and WHWT. Serum ET1 concentration was higher in dogs with IPF (median interquartile range; 2.32 pg/mL, 2.05–3.38) than healthy Beagle dogs (1.28, 1.07–1.53; P < .001), healthy WHWT (1.56, 1.25–1.85; P < .001), dogs with EBP (0.94 0.68–1.01; P = .001), and dogs with CB (1.54 0.74–1.82; P = .005). BALF ET1 concentration was below the detection limit in healthy WHWT and in dogs with CB, whereas it was measurable in all dogs with IPF. A cut‐off serum concentration of 1.8 pg/mL had a sensitivity of 100% and a specificity of 81.2% for detection of IPF, with an area under the receiver operating characteristic curve of 0.818. Conclusions and Clinical Importance: Serum ET1 can differentiate dogs with IPF from dogs with EBP or CB. ET1 can be detected in BALF of dogs with IPF.     

Chronic hepatitis in the English springer spaniel: clinical presentation, histological description and outcome

Medical records and liver histology of 68 English springer spaniels (ESS) with a histological diagnosis of CH were reviewed retrospectively. PCR was performed on liver tissue for canine adenovirus-1 (CAV-1), canine parvovirus, canine herpesvirus and pathogenic Leptospira species. Follow-up information was obtained to calculate survival times. Median age at presentation was three years seven months (range, seven months to eight years five months) and there were 48 female and 20 male dogs. Clinical signs were non-specific and five dogs were asymptomatic. All dogs had an increase in serum activity of one or more hepatobiliary enzymes. Histopathology demonstrated hepatocyte necrosis and apoptosis with varying amounts of fibrosis. A predominantly lymphoplasmacytic infiltrate throughout the hepatic parenchyma was found in all 68 dogs, but 45 of these dogs also had a neutrophilic component to the inflammatory infiltrate. There was no significant copper accumulation and no aetiological agent was identified by PCR. The median survival time was 189 days (range, 1 to 1211 days), 38 dogs died within three months and 12 dogs survived more than a year following diagnosis.     

Acute phase protein concentrations in dogs with nasal disease

The concentrations of C-reactive protein (CRP), serum amyloid A, haptoglobin (Hp) and α(1)-acid glycoprotein were measured in dogs with clinical signs of nasal disease and compared with those of healthy dogs in order to determine the expression of these proteins in cases of canine nasal disease. A significant difference (P<0.001) between the symptomatic group and the control group was found for both CRP and Hp. Among the animals with nasal disease, a significant intergroup difference (P<0.05) was found in the expression of Hp between dogs with aspergillosis and those with chronic rhinitis.