Dylan Clements

Prevalence of radiographic signs of degenerative joint disease in a hospital population of cats.

The prevalence of radiographic signs of degenerative joint disease (including appendicular osteoarthritis) among a hospital population of 218 cats was 33·9 per cent (74 cats), and the prevalence of signs of appendicular joint osteoarthritis was 16·5 per cent (36 cats). Half of the cases of appendicular joint osteoarthritis had no apparent radiographic or historical cause, and clinical signs of lameness were recorded in only six of them, all of which had an apparent radiographic cause. The 74 cats with radiographic signs of degenerative joint disease were on average significantly older than the 144 cats in which there were no radiographic signs of the disease.

Gene expression markers of tendon fibroblasts in normal and diseased tissue compared to monolayer and three dimensional culture systems

BackgroundThere is a paucity of data regarding molecular markers that identify the phenotype of the tendon cell. This study aims to quantify gene expression markers that distinguish between tendon fibroblasts and other mesenchymal cells which may be used to investigate tenogenesis.MethodsExpression levels for 12 genes representative of musculoskeletal tissues, including the proposed tendon progenitor marker scleraxis, relative to validated reference genes, were evaluated in matched samples of equine tendon (harvested from the superficial digital flexor tendon), cartilage and bone using quantitative PCR (qPCR). Expression levels of genes associated with tendon phenotype were then evaluated in healthy, including developmental, and diseased equine tendon tissue and in tendon fibroblasts maintained in both monolayer culture and in three dimensional (3D) collagen gels.ResultsSignificantly increased expression of scleraxis was found in tendon compared with bone (P = 0.002) but not compared to cartilage. High levels of COL1A2 and scleraxis and low levels of tenascin-C were found to be most representative of adult tensional tendon phenotype. While, relative expression of scleraxis in developing mid-gestational tendon or in acute or chronically diseased tendon did not differ significantly from normal adult tendon, tenascin-C message was significantly upregulated in acutely injured equine tendon (P = 0.001). Relative scleraxis gene expression levels in tendon cell monolayer and 3D cultures were significantly lower than in normal adult tendon (P = 0.002, P = 0.02 respectively).ConclusionThe findings of this study indicate that high expression of both COL1A2 and scleraxis, and low expression of tenascin-C is representative of a tensional tendon phenotype. The in vitro culture methods used in these experiments however, may not recapitulate the phenotype of normal tensional tendon fibroblasts in tissues as evidenced by gene expression.

Gene expression in canine atopic dermatitis and correlation with clinical severity scores

BACKGROUND Canine atopic dermatitis (cAD) is a common condition in dogs that may be a naturally occurring model for human atopic dermatitis (hAD). Despite this, comparative research is limited, particularly into the genetic background of cAD. OBJECTIVES 1. Measure candidate gene expression in cAD skin using quantitative real time PCR (qPCR). 2. Correlate gene expression to clinical cAD scores (Canine Atopic Dermatitis Extent and Severity Index[CADESI]-03 and intradermal allergen test [IDT]). METHODS mRNA was extracted from biopsies of non-lesional and lesional skin from atopic dogs, and healthy skin from non-atopic dogs. Gene expression was quantified using qPCR, and compared between non-lesional atopic, lesional atopic and healthy skin. Gene expression in atopic skin was correlated with clinical severity (CADESI-03) and the number of positive reactions on an IDT. RESULTS Of the 20 quantified genes, 11 demonstrated statistically significant altered mRNA expression between atopic and healthy skin; dipeptidyl-peptidase-4 (DPP4), phosphatidylinositol-3,4,5-trisphosphate-5-phosphatase-2 (INPPL1), serine protease inhibitor kazal type-5 (SPINK5), sphingosine-1-phosphate lyase-1 (SGPL1), peroxisome proliferator-activated receptor gamma (PPARgamma), S100 calcium-binding protein A8 (S100A8), Plakophilin-2 (PKP2), Periostin (POSTN), Cullin4A, TNF-alpha and metalloproteinase inhibitor-1 (TIMP-1). Three genes correlated with CADESI-03: serum amyloid A 1 (SAA-1), S100A8, and PKP2; and four with IDT results: mast cell protease I (CMA1), SAA-1, S100A8 and SPINK5. CONCLUSION Genes with altered expression included those relevant to skin barrier formation and immune function, suggesting both are relevant in the pathogenesis of AD. Many of these genes reflect the proposed pathogenesis in hAD, supporting the use of dogs as a model for hAD. Furthermore, these genes may be considered suitable targets for future genetic and protein function studies in human and canine AD.

Kinematic analysis of the gait of 10 labrador retrievers during treadmill locomotion

The trotting gait of 10 sound, adult labrador retrievers was analysed using kinematic gait analysis on a purpose-built treadmill using video-based motion analysis software. The maximal angular displacement, minimal angular displacement, average angular displacement, and the maximal positive and negative angular velocities of the right elbow and right stifle were measured over five gait cycles at defined time points during each of five two-minute sessions. The dogs’ trotting gait was not repeatable, either for individual dogs during the first session or between sessions, or between dogs at the same time points during a session.

Gene (mRNA) expression in canine atopic dermatitis: microarray analysis

Genes potentially involved in the pathology of canine atopic dermatitis (AD) were identified using gene expression microarrays. Total RNA extracted from skin biopsies was hybridized to an Agilent Technologies custom-designed 22K canine array. The arrays were analysed using Genedata Analyst software. Data were corrected for multiple hypothesis testing and tested for significance using the National Institute on Aging array analysis tool. For comparison, data were divided into separate groups: lesional atopic (n = 16), nonlesional atopic (n = 17) and healthy controls (n = 9). Fifty-four genes were differentially expressed at a significance level of 0.05 in canine AD compared to healthy controls. Sixteen genes were differentially expressed in both nonlesional and lesional atopic skin, 26 genes only in nonlesional skin and 12 only in lesional skin. These genes were associated with innate immune and inflammatory responses, cell cycle, apoptosis, barrier formation and transcriptional regulation. The most dysregulated gene in lesional skin was S100A8, which showed an almost 23-fold increase in expression. This is a pro-inflammatory cytokine located in the epidermal differentiation complex. Microarray analysis is a novel technique in canine AD. Significant changes in gene expression were identified in atopic skin. These were relevant to skin barrier formation and the immune response, suggesting that they both participate in AD. Gene expression restricted to lesional skin may be involved in inflammatory changes, whereas those shared or restricted to nonlesional skin may reflect the atopic phenotype. Investigating gene polymorphisms in the targets identified in this study will help improve our understanding of the genetic basis of this disease.

Western blot analysis of the IgG responses of ruminants infected with Neospora caninum and with Toxoplasma gondii

The IgG antibody responses of sheep, goats and cattle inoculated subcutaneously with live Neospora caninum tachyzoites of the NC1 isolate were analysed by Western blotting. Antibodies were detected against a wide range of NC1 tachyzoite antigens (6.5 to 80 kDa). The dominant antibody responses were directed against proteins at 36.5-38, 45.5-48.5, 52-53.5, 58, 58.5, 59.5, 60.5, 62, 63.5, 64, 66.5, 67, 67.5, 68.5 and 69.5 kDa, with sera from all three species. These sera were also used to probe blots of Toxoplasma gondii antigen and, while a number of protein bands were recognized, there was no consistency within or between animal species. The IgG antibody responses of sheep, goats and cattle orally infected with T. gondii oocysts of the M3 isolate were analysed by the same methods. Antibodies were detected to a range of S48 toxoplasma tachyzoite antigens (11 to 83 kDa). The dominant antibody responses were directed against proteins at 11, 16-17, 21.5, 22.5-23.5, 26-28.5, 32-35, 49.5, 50.5, 53, 54.5, 60.5 and 61 kDa, with sera from all three species. These sera were also used to probe blots of N. caninum antigen; antibody responses to numerous antigens were detected but showed little consistency within or between animal species.

A comparison of radiographic, arthroscopic and histological measures of articular pathology in the canine elbow joint

Validation of radiographic and arthroscopic scoring of joint pathology requires their comparison with histological measures of disease from the same joint. Fragmentation of the medial coronoid process (FMCP) is a naturally occurring disease of the canine elbow joint that results in osteoarthritis, and the objectives of this study were to compare the severity of histopathological changes in the medial coronoid process (MCP) and medial articular synovial membrane with gross radiographic scoring of elbow joint osteophytosis and the arthroscopic assessment of the MCP articular cartilage surface. Radiographic scoring of osteophytosis and the arthroscopic scoring of visual cartilage pathology of the MCP correlated moderately well with the histopathological evaluation of cartilage damage on the MCP and synovial inflammation in the medial part of the joint, but not with bone pathology in the MCP. Marked cartilage pathology on the MCP was identified in joints with either no radiographic evidence of osteophytosis or with mild cartilage damage that was evident arthroscopically.

A Deletion in the Canine POMC Gene Is Associated with Weight and Appetite in Obesity-Prone Labrador Retriever Dogs

Summary Sequencing of candidate genes for obesity in Labrador retriever dogs identified a 14 bp deletion in pro-opiomelanocortin (POMC) with an allele frequency of 12%. The deletion disrupts the β-MSH and β-endorphin coding sequences and is associated with body weight (per allele effect of 0.33 SD), adiposity, and greater food motivation. Among other dog breeds, the deletion was only found in the closely related flat-coat retriever (FCR), where it is similarly associated with body weight and food motivation. The mutation is significantly more common in Labrador retrievers selected to become assistance dogs than pets. In conclusion, the deletion in POMC is a significant modifier of weight and appetite in Labrador retrievers and FCRs and may influence other behavioral traits.

Reference genes for canine skin when using quantitative real-time PCR.

Quantitative real-time PCR (qPCR) facilitates the quantification of mRNA expression. Accurate qPCR analysis of gene expression requires the normalisation of data using a reference or housekeeping gene which is expressed at a similar level in all tissues tested. GAPDH is the most well known and most widely used reference gene but many papers have demonstrated that it is not stably expressed in different tissues. The aim of this study was to measure reference gene stability in canine skin using real-time qPCR. Skin samples from healthy control dogs (n=7) and dogs with atopic dermatitis (lesional skin n=7 and non-lesional skin n=7) were used to quantify seven reference genes (IMP, CG14980, S7, HIRA, GAPDH, RPL13A and SDHA) in canine whole skin. Three different statistical programs (Bestkeeper, GeNorm and Normfinder) were used to assess the stability of the reference genes. The results confirmed that GAPDH is not a stably expressed reference gene in canine skin; this finding may influence interpretation of previous qPCR studies on canine skin using this as a reference gene. RPL13A and CG14980 were found to be the most stably expressed genes in canine whole skin and would be more suitable as reference genes in future studies.

Clinical assessments of increased sensory sensitivity in dogs with cranial cruciate ligament rupture

Dogs with chronic pain have a compromised quality of life. Repeatable and accurate sensory assessments form a means by which the hypersensitivity likely to reflect chronic pain may be quantified. These assessments can be applied to individuals to identify those that may benefit from improved analgesic relief. In this study four sensory assessments were evaluated in dogs presenting with a naturally occurring chronic painful condition (cranial cruciate ligament rupture, CCLR) and were compared with healthy control animals of similar age and weight. Inter-digital von Frey filament and thermal sensitivity tests revealed that the affected hind limb of dogs with CCLR was significantly more sensitive than the opposing limb. Static weight bearing and gait parameter scores were also reduced in the affected hind limb compared to the opposing hind limb of dogs with CCLR; no such differences were found between the hind limbs of healthy (control) dogs. The quantitative sensory tests permitted the differentiation of limbs affected by CCLR from healthy limbs. Dogs presenting with CCLR demonstrate objectively quantitative sensory sensitivities, which may require additional consideration in case management.