Nick Bexfield

A Viral Discovery Methodology for Clinical Biopsy Samples Utilising Massively Parallel Next Generation Sequencing

Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples.

Glomerular filtration rate estimated by 3-sample plasma clearance of iohexol in 118 healthy dogs.

BACKGROUND Glomerular filtration rate (GFR) decreases in the aging human kidney, but limited data exist in dogs. HYPOTHESIS There is an effect of age and body size on estimated GFR in healthy dogs. ANIMALS One hundred and eighteen healthy dogs of various breeds, ages, and body weights presenting to 3 referral centers. METHODS GFR was estimated in clinically healthy dogs between 1 and 14 years of age. GFR was estimated from the plasma clearance of iohexol, by a compartmental model and an empirical correction formula, normalized to body weight in kilograms or liters of extracellular fluid volume (ECFV). For data analysis, dogs were divided into body weight quartiles 1.8-12.4, 13.2-25.5, 25.7-31.6, and 32.0-70.3 kg. RESULTS In the complete data set, there was no trend toward lower estimated GFR/kg or GFR/ECFV with increasing age. GFR decreased with age in dogs in the smallest weight quartile only. A significant negative linear relationship was detected between body weight and estimated GFR/kg and GFR/ECFV. Reference ranges in different weight quartiles were 1.54-4.25, 1.29-3.50, 0.95-3.36, and 1.12-3.39 mL/min/kg, respectively. Standardization to ECFV rather than kilogram body weight did not produce substantial changes in the relationships between GFR estimates and age or weight. CONCLUSIONS AND CLINICAL IMPORTANCE Interpretation of GFR results for early diagnosis of renal failure should take into account the weight and the age of the patient for small dogs.

The Cardiac Biomarker NT-proBNP Is Increased in Dogs with Azotemia

BACKGROUND Amino-terminal probrain natriuretic peptide (NT-proBNP) has been proposed as a useful biomarker for heart disease in dogs. In humans, decreased glomerular filtration rate (GFR) increases NT-proBNP. OBJECTIVE To investigate whether decreased GFR as indicated by plasma creatinine concentration is associated with increased NT-proBNP in dogs without heart disease. ANIMALS Four groups of dogs: healthy (n= 39), azotemic (n= 36), heart disease (n= 37), and congestive heart failure (CHF) (n= 7) presented to 2 teaching hospitals. METHODS Prospective observational cohort study. Plasma creatinine concentration and NT-proBNP were measured in every dog. Nonparametric tests were used to compare the differences among groups. The median and actual results for each group were compared with the manufacturers recommended and previously published suggestions for cut-off values for diagnosis of heart disease. RESULTS Median (range) plasma creatinine concentration was 1.47 (1.06-1.70), 4.36 (1.74-15.6), 1.22 (0.69-1.91), and 1.45 (0.63-1.64) mg/dL and median (range) NT-proBNP was 118 (2-673), 556 (37-1,819), 929 (212-5,658), and 3,144 (432-5,500) pmol/L for the healthy, azotemic, heart disease, and CHF groups, respectively. Pair-wise comparison indicated a significant difference among all groups for NT-proBNP (P< or = .049). Plasma creatinine concentration was significantly higher in the azotemic group compared with other groups (P < .001) but there was no significant among other groups. Application of 3 recommended cut-off values led to misclassification of dogs with azotemia as having heart disease. CONCLUSIONS Azotemia results in NT-proBNP being increased to concentrations reported as diagnostic of heart disease or heart failure in dogs. Care should be employed when interpreting the results of NT-proBNP in patients with known or possible increased plasma creatinine concentration.

Esophageal dysmotility in young dogs.

BACKGROUND Abnormal esophageal motility can exist without megaesophagus, although its prevalence in dogs is unknown and its cause has not been elucidated. HYPOTHESIS Esophageal dysmotility without overt megaesophagus exists in both symptomatic and asymptomatic young dogs, and motility can improve with age. ANIMALS Dogs examined at the Department of Veterinary Medicine, University of Cambridge for regurgitation, but without evidence of megaesophagus on radiographs, and a further group of asymptomatic dogs. METHODS Dogs underwent an initial and a repeat videofluoroscopic swallowing study. Images were reviewed and 5 criteria of esophageal motility evaluated. RESULTS Eight affected dogs were identified (median age 9 months), and terrier dogs predominated (6 terriers of 3 breeds). Esophageal motility was reduced either globally (n = 3) or segmentally (n = 5). Repeat examination at a median of 3 months revealed that regurgitation had reduced or resolved in the majority of terrier dogs, and 4 of 6 cases demonstrated an improvement in esophageal motility. Videofluoroscopic evaluation of young (median age 11 months) asymptomatic dogs of various breeds (n = 22) revealed evidence of esophageal dysmotility in 4 of 5 control terrier dogs. Repeat evaluation demonstrated an improvement in esophageal motility in 3 dogs. CONCLUSION AND CLINICAL IMPORTANCE Esophageal dysmotility without overt megaesophagus occurs in young terrier dogs, and affected animals can be symptomatic or asymptomatic. Further, an improvement in esophageal motility occurs with time in some dogs, and might represent a syndrome of delayed esophageal maturation.

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.

Breed, age and gender distribution of dogs with chronic hepatitis in the United Kingdom

Standardised histological criteria are now available for the diagnosis of canine chronic hepatitis (CH). CH is common in dogs, but no studies have reported breed, age and gender distributions in the United Kingdom (UK). The objective of this study was to determine which breeds had an increased risk for developing CH in the UK and to report the age and gender distribution for those breeds. The databases of six veterinary histopathology laboratories were searched for cases with a histological diagnosis of CH according to standardised criteria. The breed, age and gender of dogs was recorded and compared to a control population to calculate the odds ratio and 95% confidence intervals for developing CH. A total of 551 cases of CH were identified, consisting of 61 breeds. Nineteen breeds were represented by five or more cases. Breeds with an increased risk for developing CH included the American cocker spaniel, Cairn terrier, Dalmatian, Dobermann pinscher, English cocker spaniel, English springer spaniel, Great Dane, Labrador retriever and Samoyed. The median age at diagnosis for all breeds with CH was 8 years (range 7 months to 16 years). Dalmatians, Dobermann pinschers and English springer spaniels with CH were significantly younger than Cairn terriers, English cocker spaniels and Labrador retrievers with CH. Females were over-represented when all cases were examined together. In conclusion, several breeds in the UK have an increased risk of CH, some of which have not been previously reported.

Canine hepacivirus is not associated with chronic liver disease in dogs

Canine hepacivirus (CHV) has recently been identified in liver and respiratory tract samples from dogs, and comparative phylogenetic analysis has confirmed it to be the closest genetic relative of hepatitis C virus (HCV) described to date. CHV offers great potential as a model system for HCV, but only if the underlying processes of infection and pathogenesis are similar for both viruses. However, it is not yet clear if CHV is hepatotrophic. Canine chronic hepatitis (CH) is a common and usually idiopathic disease that shares similar histological features to that of HCV infection of humans. To date, no study has attempted to determine whether CHV is involved in the aetiology of liver disease in dogs. We employed two nested PCR assays, using primers targeting regions of the helicase domain of CHV NS3, to identify viral nucleic acids in liver samples from 100 dogs with CH of unknown cause in the UK. We also used a sensitive luciferase immunoprecipitation system (LIPS) assay to screen serum samples from these dogs for the presence of anti‐CHV antibodies. Surprisingly, there was no evidence of exposure to, or a carrier state of, CHV in this large cohort, suggesting that the virus is not associated with CH in UK dogs. Future work, including transmission studies, is required to understand the pathogenesis of CHV in canids before it can be proposed as a surrogate model for HCV‐induced liver disease in man.

How to... Place an oesophagostomy tube.

Clinical nutrition is a crucial but often neglected part of patient management. Nick Bexfield and Penny Watson explain when and how to place and use an oesophagostomy tube.

Treatment of canine liver disease 1. Drugs and dietary management

THE treatment of canine liver disease involves a combination of drug therapy and dietary support. This article, the first of two discussing the management of liver disease in dogs, describes the indications and actions of some of the commonly used drugs and dietary treatments for the condition. An article in the next issue of In Practice will consider how to manage the clinical signs of hepatic disorders and outline the clinical approach to some specific liver diseases in dogs.

Diagnosis of canine liver disease

LIVER disease is relatively common in dogs, but its diagnosis presents a challenge. The livers large reserve capacity means that many dogs show no clinical signs until late in the disease process and signs are often non‐specific. Findings on blood tests and diagnostic imaging are also relatively non‐specific and a biopsy is often required for definitive diagnosis. This article outlines a logical approach to the dog with suspected liver disease, describing the rational work‐up from first suspicion of disease to final biopsy‐confirmed diagnosis.