Complex Feline Disease Mapping Using a Dense Genotyping Array.

The current feline genotyping array of 63 k single nucleotide polymorphisms has proven its utility for mapping within breeds, and its use has led to the identification of variants associated with Mendelian traits in purebred cats. However, compared to single gene disorders, association studies of complex diseases, especially with the inclusion of random bred cats with relatively low linkage disequilibrium, require a denser genotyping array and an increased sample size to provide statistically significant associations. Here, we undertook a multi-breed study of 1,122 cats, most of which were admitted and phenotyped for nine common complex feline diseases at the Cornell University Hospital for Animals. Using a proprietary 340 k single nucleotide polymorphism mapping array, we identified significant genome-wide associations with hyperthyroidism, diabetes mellitus, and eosinophilic keratoconjunctivitis. These results provide genomic locations for variant discovery and candidate gene screening for these important complex feline diseases, which are relevant not only to feline health, but also to the development of disease models for comparative studies.

One-year study evaluating efficacy of an iodine-restricted diet for the treatment of moderate-to-severe hyperthyroidism in cats.

PURPOSE: This study sought to determine the efficacy of a therapeutic iodine-restricted diet during a 12 -month interval in cats with moderately to severely increased total thyroxine (TT4) concentrations. PATIENTS AND METHODS: Eight hyperthyroid cats with serum creatinine <2.0 mg/dL and TT4 >6.0 µg/dL (reference interval 1.5-4.0 µg/dL) were enrolled. Each cat underwent an initial physical examination, complete blood count, serum chemistry panel, thyroid profile, thyroid scintigraphy, and echocardiogram. Physical examination and all blood samplings were repeated at 1, 2, 3, 6, and 12 months after enrollment. Clients were instructed to feed only the therapeutic iodine-restricted diet throughout the entire study. RESULTS: Median TT4 was 8.4 (range 6.2-24.0) µg/dL at study onset. Thyroid scintigraphy was abnormal in all cats, confirming hyperthyroidism. Six out of eight cats had normal serum TT4 after 4 weeks of feeding the therapeutic diet. The two cats that did not achieve normalization of TT4 at 4 weeks had the highest initial TT4 concentrations. Three cats were withdrawn from the study due to emergence of chronic kidney disease. CONCLUSION: An iodine-restricted diet was able to control moderate-to-severe hyperthyroidism in most (six out of eight) of the cats by 4 weeks. Cats with highest baseline TT4 took substantially longer before TT4 concentrations normalized, if at all.

Assessment of canine autologous platelet-rich plasma produced with a commercial centrifugation and platelet recovery kit.

OBJECTIVES: To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and confirm reproducibility of platelet enrichment, as well as determine the platelet activation status in the final product of a commercial platelet-rich plasma kit using canine blood. METHODS: Venous blood from 20 sedated client-owned dogs was used to prepare platelet-rich plasma (PRP) from a commercial kit. Complete blood counts were performed to determine erythrocyte, leukocyte, and platelet numbers in both whole blood (WB) and resultant PRP. The WB and PRP samples from jugular (fast collection) and cephalic (slow collection) venipuncture were also compared. P-selectin externalization was measured in WB and PRP samples from 15 of 20 dogs. RESULTS: This commercial kit produced an average percent recovery in platelets of 64.7 ± 17.4; erythrocytes of 3.7 ± 0.8, and leukocytes of 31.6 ± 10.0. Neutrophil, monocyte, and lymphocyte percent recovery was 19.6 ± 7.2, 44.89 ± 19.8, and 57.5 ± 10.6, respectively. The recovery of platelets from jugular venipuncture (59.7 ± 13.6%) was lower than from cephalic recovery (68.8 ± 19.1%). The mean percent P-Selectin externalization for WB, PRP, and PRP with thrombin was 25.5 ± 30.9, 4.5 ± 6.4, and 90.6 ± 4.4 respectively. CLINICAL SIGNIFICANCE: Cellular reproducibility of this kit was confirmed and platelets were concentrated within autologous serum. Additionally, measurements of P-selectin externalization showed that platelets are inactive in PRP unless stimulated to degranulate.

The associations between serum adiponectin, leptin, C-reactive protein, insulin, and serum long-chain omega-3 fatty acids in Labrador Retrievers.

Obesity has been associated with an increased inflammatory response and insulin resistance due to adipose tissue-derived adipokines and increases in C-reactive protein (CRP). Dogs appear to be similar to other species with the exception of adiponectin, which might not be affected by obesity status. Serum long-chain polyunsaturated fatty acid concentrations have been positively and negatively associated with serum adipokines. The aim of the study was to examine the relationship between leptin, CRP, adiponectin, and insulin to body condition score (BCS) and to the long-chain omega-3 fatty acids in serum lipoproteins, including alpha-linolenic acid, eicosapentaenoic acid (EPA), docosapentanenoic acid (DPA), and docosahexaenoic acid (DHA) as a reflection of dietary omega-3 status in the Labrador Retriever. Seventy-seven Labrador Retrievers were evaluated for BCS, percent fasting serum lipoprotein fatty acid concentrations, as well as serum leptin, adiponectin, insulin, and CRP. A multivariable general linear regression model was constructed to examine the association between the dependent variables leptin, CRP, adiponectin, and insulin and the predictor variables of BCS, age, and sex, as well as concentrations of alpha-linolenic acid, EPA, DHA, and DPA. Adiponectin concentration was positively associated with age (P<0.0008), EPA (P=0.027) and negatively associated with DHA (P=0.008). Leptin concentration was positively associated with an increased DHA (P=0.009), BCS (P<0.0001), age (P=0.02), and decreased DPA (P=0.06). Insulin concentration was only associated with BCS (P<0.0001), and no meaningful associations were found for CRP. Longer chain omega-3 fatty acids may play a role in regulating adiponectin concentrations in dogs. However, because insulin concentrations were associated only with BCSs, further examination of the role of adiponectin in canine obesity is warranted. EPA and DPA may reduce the overall inflammatory state in dogs as these omega-3 fatty acids reflect increased adiponectin (increased EPA and decreased DHA) and decreased leptin (decreased DHA and increased DPA).

Evaluation of dietary energy intake and physical activity in dogs undergoing a controlled weight-loss program.

OBJECTIVE: To quantify physical activity and dietary energy intake in dogs enrolled in a controlled weight-loss program and assess relationships between energy intake and physical activity, sex, age, body weight, and body condition score (BCS). DESIGN: Prospective clinical study. ANIMALS: 35 client-owned obese dogs (BCS > 7/9). PROCEDURES: Dogs were fed a therapeutic diet with energy intake restrictions to maintain weight loss of approximately 2%/wk. Collar-mounted pedometers were used to record the number of steps taken daily as a measure of activity. Body weight and BCS were assessed at the beginning of the weight-loss program and every 2 weeks thereafter throughout the study. Relationships between energy intake and sex, age, activity, BCS, and body weight at the end of the study were assessed via multivariable linear regression. Variables were compared among dogs stratified post hoc into inactive and active groups on the basis of mean number of steps taken (< or > 7,250 steps/d, respectively). RESULTS: Mean ± SD daily energy intake per unit of metabolic body weight (kg(0.75)) of active dogs was significantly greater than that of inactive dogs (53.6 ± 15.2 kcal/kg(0.75) vs 42.2 ± 9.7 kcal/kg(0.75), respectively) while maintaining weight-loss goals. In regression analysis, only the number of steps per day was significantly associated with energy intake. CONCLUSIONS AND CLINICAL RELEVANCE: Increased physical activity was associated with higher energy intake while maintaining weight-loss goals. Each 1,000-step interval was associated with a 1 kcal/kg(0.75) increase in energy intake.

The effects of weight loss on adipokines and markers of inflammation in dogs.

Evidence suggests that adipose tissue-derived adipokines induce mild inflammation and may play a role in insulin resistance associated with diabetes. The present study was designed to examine a series of adipokines and markers of inflammation in dogs before and after a successful weight loss. The study included fasting serum samples from twenty-five dogs before and after a weight-loss programme. Serum C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) were measured as indicators of chronic inflammation, while serum adipokines including total adiponectin, high-molecular-weight (HMW) adiponectin, resistin and leptin were also examined. Medians for CRP (before, 10.0 (interquartile range 5.4-15.0) µg/ml; after, 5.6 (interquartile range 3.8-7.0) µg/ml) and MCP-1 (before, 212 (interquartile range 157-288) ng/ml; after, 185 (interquartile range 143-215) ng/ml) decreased significantly after weight loss. Medians for resistin showed a mild, yet significant reduction (before, 67.1 (interquartile range 44.4-88.5) pg/ml; after, 60.5 (interquartile range 32.3-67.1) pg/ml), while leptin showed a dramatic decrease after weight loss (before, 18.9 (interquartile range 10.8-35.4) ng/ml; after, 6.6 (interquartile range 3.9-10.2) ng/ml). Serum total adiponectin and HMW adiponectin were unchanged on all analyses performed. These data suggest that weight loss can decrease chronic inflammation; however, the clinical implications of this decrease are not well elucidated in dogs. Surprisingly, there was no increase in total or HMW serum adiponectin after weight loss, as observed previously in human subjects. The lack of change in total and HMW adiponectin might explain why insulin resistance and type 2 diabetes are less prevalent in obese dogs when compared with humans and cats.

Cross-sectional study to investigate the association between vitamin D status and cutaneous mast cell tumours in Labrador retrievers.

Epidemiological data indicate that low serum vitamin D concentrations are associated with an increased risk of a variety of human tumours. Cutaneous mast cell tumours (MCT) occur more frequently in dogs than in any other species. Canine MCT express the vitamin D receptor, and vitamin D derivatives have in vitro and in vivo anti-tumour activity. We sought to examine the association between vitamin D serum level and MCT in Labrador retrievers, a dog breed predisposed to MCT development. To examine this association, serum 25-hydroxyvitamin D3 (25(OH)D3) concentrations were examined in eighty-seven Labrador retrievers, including thirty-three with MCT and fifty-four unaffected controls. The relationship between cases and controls and 25(OH)D3 level, age and body condition score were evaluated using univariate and multivariate analyses. Potential differences in vitamin D oral intake, calculated on the basis of a dietary questionnaire, were also evaluated between groups. Mean 25(OH)D3 concentration (104 (SD 30) nmol/l) in dogs with MCT was significantly lower than that of unaffected dogs (120 (SD 35) nmol/l; P = 0.027). The mean calculated vitamin D intake per kg body weight in Labrador retrievers with MCT was not statistically different from that of unaffected Labrador retrievers (0.38 (SD 0.25) and 0.31 (SD 0.22) µg/kg body weight, respectively; P = 0.13). These findings suggest that low levels of 25(OH)D3 might be a risk factor for MCT in Labrador retrievers. Prospective cohort studies are warranted.

Use of pedometers to measure the relationship of dog walking to body condition score in obese and non-obese dogs.

The objective of the present study was to utilise an accurate canine pedometer methodology and to assess the relationship between activity and body condition score (BCS) in dogs. Initial methodology validation used videography and pedometer step measurements to assess actual steps taken in comparison with pedometer readings for twenty large, medium and small dogs. During the validation, dogs considered to be medium or large breed showed no significant difference between pedometer readings and actual steps taken. A total of seventy-seven obese and non-obese dogs over 35 cm (14 inches) shoulder height and over 10 kg were recruited from a dog obesity clinic and a community sample to assess daily walking activity. Body condition scoring and pedometer steps were assessed on three separate weeks during a 10-week period. During the activity monitoring, daily step counts ranged from 5555 to 39 970 steps/d among the seventy-seven medium and large dogs. Dogs' BCS were inversely correlated with average daily steps (Spearman's ρ = - 0.442, P < 0.0001). The present study identified a significant inverse correlation between daily walking steps and BCS over a range from 4 to 9 out of 9 (P < 0.0001).

Potential role for peptidylarginine deiminase 2 (PAD2) in citrullination of canine mammary epithelial cell histones.

Peptidylarginine Deiminases (PADs) convert arginine residues on substrate proteins to citrulline. Previous reports have documented that PAD2 expression and activity varies across the estrous cycle in the rodent uterus and pituitary gland, however, the expression and function of PAD2 in mammary tissue has not been previously reported. To gain more insight into potential reproductive roles for PAD2, in this study we evaluated PAD2 expression and localization throughout the estrous cycle in canine mammary tissue and then identified possible PAD2 enzymatic targets. Immunohistochemical and immunofluorescence analysis found PAD2 expression is low in anestrus, limited to a distinct, yet sparse, subset of epithelial cells within ductal alveoli during estrus/early diestrus, and encompasses the entire epithelium of the mammary duct in late diestrus. At the subcellular level, PAD2 is expressed in the cytoplasm, and to a lesser extent, the nucleus of these epithelial cells. Surprisingly, stimulation of canine mammary tumor cells (CMT25) shows that EGF, but not estrogen or progesterone, upregulates PAD2 transcription and translation suggesting EGF regulation of PAD2 and possibly citrullination in vivo. To identify potential PAD2 targets, anti-pan citrulline western blots were performed and results showed that citrullination activity is limited to diestrus with histones appearing to represent major enzymatic targets. Use of site-specific anti-citrullinated histone antibodies found that the N-terminus of histone H3, but not H4, appears to be the primary target of PAD activity in mammary epithelium. This observation supports the hypothesis that PAD2 may play a regulatory role in the expression of lactation related genes via histone citrullination during diestrus.