K.R. Verkest

Compensation for obesity-induced insulin resistance in dogs: assessment of the effects of leptin, adiponectin, and glucagon-like peptide-1 using path analysis

The hormonal mediators of obesity-induced insulin resistance and compensatory hyperinsulinemia in dogs have not been identified. Plasma samples were obtained after a 24-h fast from 104 client-owned lean, overweight, and obese dogs. Plasma glucose and insulin concentrations were used to calculate insulin sensitivity and β-cell function with the use of the homeostasis model assessment (HOMA(insulin sensitivity) and HOMA(β-cell function), respectively). Path analysis with multivariable linear regression was used to identify whether fasting plasma leptin, adiponectin, or glucagon-like peptide-1 concentrations were associated with adiposity, insulin sensitivity, and basal insulin secretion. None of the dogs were hyperglycemic. In the final path model, adiposity was positively associated with leptin (P < 0.01) and glucagon-like peptide-1 (P = 0.04) concentrations. No significant total effect of adiposity on adiponectin in dogs (P = 0.24) was observed. If there is a direct effect of leptin on adiponectin, then our results indicate that this is a positive relationship, which at least partly counters a negative direct relationship between adiposity and adiponectin. Fasting plasma leptin concentration was directly negatively associated with fasting insulin sensitivity (P = 0.01) and positively associated with β-cell function (P < 0.01), but no direct association was observed between adiponectin concentration and either insulin sensitivity or β-cell function (P = 0.42 and 0.11, respectively). We conclude that dogs compensate effectively for obesity-induced insulin resistance. Fasting plasma leptin concentrations appear to be associated with obesity-associated changes in insulin sensitivity and compensatory hyperinsulinemia in naturally occurring obese dogs. Adiponectin does not appear to be involved in the pathophysiology of obesity-associated changes in insulin sensitivity.

Distinct adiponectin profiles might contribute to differences in susceptibility to type 2 diabetes in dogs and humans

Dogs develop obesity-associated insulin resistance but not type 2 diabetes mellitus. Low adiponectin is associated with progression to type 2 diabetes in obese humans. The aims of this study were to compare total and high molecular weight (HMW) adiponectin and the ratio of HMW to total adiponectin (S(A)) between dogs and humans and to examine whether total or HMW adiponectin or both are associated with insulin resistance in naturally occurring obese dogs. We compared adiponectin profiles between 10 lean dogs and 10 lean humans and between 6 lean dogs and 6 age- and sex-matched, client-owned obese dogs. Total adiponectin was measured with assays validated in each species. We measured S(A) with velocity centrifugation on sucrose gradients. The effect of total and HMW adiponectin concentrations on MINMOD-estimated insulin sensitivity was assessed with linear regression. Lean dogs had total and HMW adiponectin concentrations three to four times higher than lean humans (total: dogs 32 ± 5.6 mg/L, humans 10 ± 1.3 mg/L, P<0.001; HMW: dogs 25 ± 4.5 mg/L, humans 6 ± 1.3 mg/L, P<0.001) and a higher S(A) (dogs: 0.78 ± 0.05; humans: 0.54 ± 0.08, P = 0.002). Adiponectin concentrations and S(A) were not lower in obese dogs (0.76 ± 0.05 in both groups; P=1). Total adiponectin, HMW adiponectin, and S(A) were not associated with insulin sensitivity in dogs. We propose that differences in adiponectin profiles between humans and dogs might contribute to the propensity of humans but not dogs to develop type 2 diabetes. Dogs with chronic, naturally occurring obesity do not have selectively reduced HMW adiponectin, and adiponectin does not appear to be important in the development of canine obesity-associated insulin resistance.

Spontaneously obese dogs exhibit greater postprandial glucose, triglyceride, and insulin concentrations than lean dogs

Dogs do not appear to progress from obesity-induced insulin resistance to type 2 diabetes mellitus. Both postprandial hyperglycemia and postprandial hypertriglyceridemia have been proposed to cause or maintain beta cell failure and progression to type 2 diabetes mellitus in other species. Postprandial glucose, triglyceride, and insulin concentrations have not been compared in lean and obese dogs. We measured serum glucose, triglyceride, and insulin concentrations in nine naturally occurring obese and nine age- and gender-matched lean dogs. After a 24-h fast, dogs were fed half their calculated daily energy requirement of a standardized diet that provided 37% and 40% of metabolizable energy as carbohydrate and fat, respectively. Fasting and postprandial glucose and triglyceride concentrations were greater in the obese dogs (P < 0.001), although the mean insulin concentration for this group was five times greater than that of the lean group (P < 0.001). Most of the 0.6 mM (11 mg/dL) difference in mean postprandial glucose concentrations between lean and obese dogs was attributable to a subset of persistently hyperglycemic obese dogs with mean postprandial glucose concentrations 1.0 mM (18 mg/dL) greater than that in lean dogs. Persistently hyperglycemic obese dogs had lower triglyceride (P = 0.02 to 0.04) and insulin (P < 0.02) concentrations than other obese dogs. None of the dogs developed clinical signs of diabetes mellitus during follow-up for a median of 2.6 yr. We conclude that pancreatic beta cells in dogs are either not sensitive to toxicity because of mild hyperglycemia or lack another component of the pathophysiology of beta cell failure in type 2 diabetes mellitus.

Basal measures of insulin sensitivity and insulin secretion and simplified glucose tolerance tests in dogs.

There is need for simple, inexpensive measures of glucose tolerance, insulin sensitivity, and insulin secretion in dogs. The aim of this study was to estimate the closeness of correlation between fasting and dynamic measures of insulin sensitivity and insulin secretion, the precision of fasting measures, and the agreement between results of standard and simplified glucose tolerance tests in dogs. A retrospective descriptive study using 6 naturally occurring obese and 6 lean dogs was conducted. Data from frequently sampled intravenous glucose tolerance tests (FSIGTTs) in 6 obese and 6 lean client-owned dogs were used to calculate HOMA, QUICKI, fasting glucose and insulin concentrations. Fasting measures of insulin sensitivity and secretion were compared with MINMOD analysis of FSIGTTs using Pearson correlation coefficients, and they were evaluated for precision by the discriminant ratio. Simplified sampling protocols were compared with standard FSIGTTs using Lins concordance correlation coefficients, limits of agreement, and Pearson correlation coefficients. All fasting measures except fasting plasma glucose concentration were moderately correlated with MINMOD-estimated insulin sensitivity (|r| = 0.62-0.80; P < 0.03), and those that combined fasting insulin and glucose were moderately closely correlated with MINMOD-estimated insulin secretion (r = 0.60-0.79; P < 0.04). HOMA calculated using the nonlinear formulae had the closest estimated correlation (r = 0.77 and 0.74) and the best discrimination for insulin sensitivity and insulin secretion (discriminant ratio 4.4 and 3.4, respectively). Simplified sampling protocols with half as many samples collected over 3 h had close agreement with the full sampling protocol. Fasting measures and simplified intravenous glucose tolerance tests reflect insulin sensitivity and insulin secretion derived from frequently sampled glucose tolerance tests with MINMOD analysis in dogs.

Adiposity and adiponectin in dogs: investigation of causes of discrepant results between two studies

Although one study showed lower adiponectin concentrations in obese dogs, other recent studies indicate that adiponectin might not be decreased in obese dogs, raising the possibility that the physiology of adiponectin is different in dogs than in humans. The aim of this study was to investigate possible causes of the discrepancy between the two largest studies to date that assessed the association between adiposity and adiponectin concentration in dogs, including the validity of the assay, laboratory error, and the effects of breed, sex, and neuter status on the relationship between adiposity and adiponectin concentrations. Adiponectin concentrations measured with a previously validated adiponectin ELISA were compared with those estimated by Western blotting analysis of reduced and denatured plasma samples. The possibility of laboratory error and the effect of EDTA anticoagulant and aprotinin were tested. Adiponectin concentration was measured by ELISA in 20 lean dogs (10 male and 10 female, 5 neutered in each sex). There was close correlation between adiponectin concentrations measured by ELISA and those estimated by Western blotting analysis (r = 0.90; P < 0.001). There was no substantial effect of EDTA, aprotinin, or laboratory error on the results. There was confounding by neuter status of the relationship between adiposity and adiponectin concentrations, but adiponectin concentrations were not significantly lower in male than in female lean dogs (females, 36 mg/L; males, 26 mg/L; P > 0.20) and were not significantly lower in intact than in neutered lean male dogs (intact, 28 mg/L; neutered, 23 mg/L; P = 0.49). We conclude that the adiponectin ELISA previously validated for use in dogs appears to be suitable for determination of canine adiponectin concentrations and that testosterone does not appear to have a strong effect on plasma adiponectin concentrations in dogs. Obesity might decrease adiponectin concentrations in intact but not in neutered dogs.

Adiponectin profiles are affected by chronic and acute changes in carbohydrate intake in healthy cats.

Adiponectin is a key adipokine that regulates carbohydrate and lipid metabolism. It circulates in stable low (LMW) and high molecular weight (HMW) forms. The aims of this study were to characterize baseline adiponectin profiles (total, LMW and HMW multimers) in healthy cats and to assess the effects of varying dietary carbohydrate content on adiponectin profiles. Cats were maintained on a diet with moderate carbohydrate content (37% metabolisable energy [ME]) for 4 weeks and then randomly allocated to either a low carbohydrate (19% ME) or high carbohydrate (52% ME) diet for 4 weeks. Fasting and postprandial plasma adiponectin profiles were measured by ELISA and sucrose gradient/Western blot. After consuming the moderate carbohydrate diet for 4 weeks, fasting total, HMW and LMW plasma adiponectin concentrations were 5.0±0.6, 2.5±0.5 and 2.6±0.2 μg/mL, respectively. After changing to the low carbohydrate diet, fasting total adiponectin was unchanged but HMW adiponectin increased and LMW adiponectin decreased. No significant postprandial changes were observed. Cats consuming the high carbohydrate diet had increased fasting total and LMW adiponectin with no change in HMW adiponectin. In the postprandial state total adiponectin was reduced and there was a trend towards a decrease in HMW (p=0.086) but not LMW multimers. These data indicate that feline adiponectin multimer profiles are similar to those reported in other species and demonstrate that changes in plasma adiponectin occur in response to chronic and acute carbohydrate intake and these reflect differential changes in adiponectin multimers.

Association of postprandial serum triglyceride concentration and serum canine pancreatic lipase immunoreactivity in overweight and obese dogs.

BACKGROUND Hypertriglyceridemia has been proposed to contribute to the risk of developing pancreatitis in dogs. OBJECTIVES To determine associations between postprandial serum triglyceride concentrations and canine pancreatic lipase immunoreactivity (cPLI) concentrations or pancreatic disease. ANIMALS Thirty-five client-owned overweight (n = 25) or obese (n = 10) dogs weighing >10 kg. METHODS Healthy dogs were prospectively recruited for a cross-sectional study. Serum triglyceride concentrations were measured before and hourly for 12 hours after a meal. Fasting cPLI and canine trypsin-like immunoreactivity (cTLI) concentrations were assayed. Cut-off values for hypertriglyceridemia were set a priori for fasting (≥ 88, ≥ 177, ≥ 354, ≥ 885 mg/dL) and peak postprandial (≥ 133, ≥ 442, ≥ 885 mg/dL) triglyceride concentrations. The association between hypertriglyceridemia and high cPLI concentrations was assessed by exact logistic regression. Follow-up was performed 4 years later to determine the incidence of pancreatic disease. RESULTS Eight dogs had peak postprandial triglycerides >442 mg/dL and 3 dogs had fasting serum cPLI concentrations ≥ 400 μg/L. Odds of high cPLI concentrations were 16.7 times higher in dogs with peak postprandial triglyceride concentrations ≥ 442 mg/dL relative to other dogs (P < .001). Fasting triglyceride concentration was not significantly associated with cPLI concentrations. None of the dogs with high triglyceride concentrations and one of the dogs with low fasting and peak postprandial triglyceride concentrations developed clinically important pancreatic disease. CONCLUSIONS AND CLINICAL IMPORTANCE Overweight and obese dogs with peak serum postprandial triglyceride concentrations ≥ 442 mg/dL after a standard meal are more likely to have serum cPLI concentrations ≥ 400 μg/L, but did not develop clinically important pancreatic disease.

Subclinical pancreatitis is more common in overweight and obese dogs if peak postprandial triglyceridemia is >445 mg/dl

Adiponectin has been investigated widely due to its association with adiposity and the metabolic syndrome in human beings. Adiponectin circulates as low- (LMW) and high-molecular weight (HMW) multimers and the latter are the more bioactive forms. There are no reports of the relative proportion (distribution) of adiponectin multimers in feline plasma. The aim of this study was to assess the association of dietary nutrient composition, body weight gain, meal feeding, and insulin sensitivity with HMW adiponectin concentration and adiponectin multimer distribution in cats.1 EVALUATION OF FOUR DNA EXTRACTION METHODS FOR THE DETECTION OF TRITRICHOMONAS FOETUS IN FELINE STOOL SPECIMENS BY POLYMERASE CHAIN REACTION. SH Stauffer, AJ Birkenheuer, MG Levy, H Marr, JL Gookin. College of Veterinary Medicine, North Carolina State University, Raleigh, NC. Feces are increasingly recognized as practical samples for molecular diagnosis of infectious disease. Extraction of PCR-quality DNA from feces can be challenging due to co-extraction of PCR inhibitors. Accordingly, we examined the effect of four commercially-available DNA extraction methods on sensitivity of PCR for detection of Tritrichomonas foetus (TF) in naturally-infected and TF-spiked feline stool. Kits evaluated included ExtractMaster Fecal DNA Extraction Kit, Epicentre Biotechnologies (Kit A); QIAamp DNA Stool Mini Kit, Qiagen (Kit B); UltraClean Fecal DNA Kit, MoBio (Kit C); and ZR Fecal DNA Kit, Zymo Research (Kit D). In accordance with manufacturer instructions, DNA was extracted from 180mg (A,B), 50mg (C), 100 & 150mg (D) aliquots of feline feces to which was added 20ml volumes containing 0–10,000 cultured feline TF. Each kit was also used to extract DNA from the feces of each of 10 naturally infected and 10 uninfected cats. DNA was eluted in 300ml (A), 200ml (B), 50ml (C), or 100ml (D) of respective elution buffer. Endogenous PCR inhibitors in extracted DNA was examined by PCR amplification of an 876 bp gene fragment of bacterial 16S rRNA. DNA was then tested by single tube nested PCR for amplification of partial ITS1, 5.8S and ITS2 rRNA genes of TF. Kit D provided the most sensitive detection of TF DNA as expressed by both organisms per DNA extraction and organisms per PCR reaction. To account for differences in DNA concentrations between kits (i.e. fecal sample size and elution volumes), the limit of detection for each kit as expressed by the number of TF per PCR reaction was as follows: Kit B 5 250, Kit A 5 167, Kit C 5 100, Kit D (150mg fecal sample) 5 5, and Kit D (100mg fecal sample) 5 0.5. PCR performed on DNA extracted from cultured TF (no feces) or TF-spiked feces (100mg) using Kit D was positive with as few as 10 TF per extraction. Further, DNA extraction using Kit D could be completed in the shortest time of all kits tested. These studies identify the ZR Fecal DNA Kit as superior to the other kits tested for extraction of PCR-qualityDNA from feline feces. ABSTRACT #2 INVESTIGATION OF ENTEROBACTER CLOACAE INFECTIONS AT A SMALL ANIMAL VETERINARY TEACHING HOSPITAL. JS Weese. University of Guelph, Guelph, Ontario.2 INVESTIGATION OF ENTEROBACTER CLOACAE INFECTIONS AT A SMALL ANIMAL VETERINARY TEACHING HOSPITAL. JS Weese. University of Guelph, Guelph, Ontario. A wide range of pathogens can cause hospital-associated (HA) infections in small animal hospitals. Among these is Enterobacter cloacae, which is one of the most clinically relevant Enterobacter spp and a common cause of HA infection in humans. Recently, multidrug resistance has become a concern, particularly with emergence of extended-spectrum beta-lactamase and extended spectrum cephalosporinase producing strains. An infection control investigation was initiated at the Ontario Veterinary College Teaching Hospital (OVCTH) in the fall of 2007 in response to anecdotal concerns about Enterobacter cloacae infections in hospitalized animals. Enterobacter cloacae was isolated from 45/36719 animals from January 1, 2005 to October 31, 2007, for an overall incidence of 1.2/ 1000 admissions. The monthly incidence rate ranged from 0 to 4.3/ 1000 admissions. Twenty-one (47%) cases were classified as community-associated, while 17 (38%) were hospital associated. Seven (15%) were community-onset but hospital associated, with three of these associated with other veterinary hospitals. There was no increase in the incidence of overall or hospital-associated infections during the study period. The urinary tract was the most common site of infection (n511, 24%). Wound infections (excluding surgical site infections) accounted for 8 (18%) of infections, with superficial and deep surgical site infections accounting for 7 (16%) and organ/space surgical site infections accounting for another 2 cases. Urinary tract infections were most common among animals with CA infection, accounting for 8/21 (38%) cases with wound infections accounting for 4 (19%) cases. Of the 24 cases associated with the OVCTH, 17 (71%) had surgery, 15 (63%) were hospitalized in the intensive care unit, 10 (42%) had indwelling urinary catheters placed, and 20 (83%) had received antimicrobials prior to onset of infection. Risk factors for E. cloacae infection could not be determined because a noninfected control group was not evaluated. Surgical site infections accounted for 9 (38%) HA cases. Overall, only 2/11 (18%) urinary tract infections were associated with prior placement of a urinary catheter. Nine (20%) animals died or were euthanized and E. cloacae was implicated as a causative or contributing factor in 5 (56%) of those cases. Two main antimicrobial phenotype patterns were identified. One (n525) was characterized by susceptibility to fluoroquinolones, tetracycline, and trimethoprim with variable susceptibility to cefoxitin while the other (n514) was characterized by resistance to these antimicrobials. Prior administration of antimicrobials was associated with presence of the more resistant phenotype (P50.044) but there was no association between this phenotype and origin of infection (P50.74) and no increase in the prevalence of this phenotype from 2005 to 2007 (P50.97). Infections with this phenotype were not associated with nonsurvival (P50.74). There was no evidence of a, HA outbreak or increase in prevalence, yet identification of multidrug resistant E. cloacae in both CA and HA infections is concerning and requires ongoing surveillance. ABSTRACT #3 STAPHYLOCOCCUS PSEUDINTERMEDIUS: A NEWLY RECOGNIZED PATHOGEN IN DOGS AND CATS. MC Faires, D Slavic, JS Weese. Ontario Veterinary College, Animal Health Laboratory, University of Guelph, Guelph, Ontario.3 STAPHYLOCOCCUS PSEUDINTERMEDIUS: A NEWLY RECOGNIZED PATHOGEN IN DOGS AND CATS. MC Faires, D Slavic, JS Weese. Ontario Veterinary College, Animal Health Laboratory, University of Guelph, Guelph, Ontario. Staphylococcus intermedius has typically been regarded as the predominant pathogenic Staphylococcus spp in dogs and cats, and a leading cause of skin and soft tissue infections. In 2005, a novel Staphylococcus species, Staphylococcus pseudintermedius, was identified. This organism is closely related to, but distinct from, S. intermedius. Gene-sequence based methods are required to differentiate these two species; however, these techniques are rarely performed in clinical laboratories, and as a result the prevalence and characteristics of S. pseudintermedius are poorly understood. Recent evidence suggests that S. pseudintermedius may actually be the predominant Staphylococcus spp in dogs and cats but misidentified as S. intermedius by diagnostic laboratories. The objective of this study was to use sequence based methods to identify putative S. intermedius isolates from dogs and cats and to evaluate antimicrobial resistance and virulence factors among S. pseudintermedius isolates. Isolates from dogs and cats identified as S. intermedius by conventional laboratory methods were obtained from the University of Guelph Animal Health Laboratory. Isolates were collected in a serial manner without selection. DNA was extracted, sequencing of the sodA gene was performed, and isolates were identified via sequence alignment with reference staphylococcal strains through GenBank (www.ncbi.nlm.nih.gov/blast/BLAST.cgi). Antimicrobial susceptibility testing was performed and PCR was used to identify various virulence factors and antimicrobial genes. A total of 25 isolates were obtained from 21 dogs and 2 cats. Medical records were not available for 2 of the isolates. 25/25 (100%) were identified as S. pseudintermedius Severity of infection ranged from superficial dermatitis to rapidly fatal necrotizing fasciitis with the majority of isolates from otitis externa 9/23 (39.1%) and urinary tract infections 6/23 (26.1%). Antimicrobial susceptibility was as follows: amoxicillin/clavulanate 23/23 (100%), ampicillin 7/ 23 (30.4%), cephalothin 23/23 (100%), clindamycin 18/23 (78.3%), gentamicin 23/23 (100%), tetracycline 18/23 (78.3%), and trimethoprim/sulfa 19/23 (82.6%). Not all antimicrobials were tested for all isolates, based on laboratory protocols regarding antimicrobial panel and site of infection. Inducible resistance to clindamycin was detected by D-test in 1 isolate reported as clindamycin-susceptible (5.6%). Detection of virulence factors and antimicrobial resistance genes is ongoing. This study identified S. pseudintermedius as an important pathogen in dogs and cats, and suggests that S. intermedius may not be a major concern in these species. Further studies are required to evaluate clinically relevant virulence factors to assist in understanding the pathogenesis of disease caused by S. pseudintermedius. 70

Insulin sensitivity is halved and fasting insulin concentration increased four times in spontaneously obese dogs

1 SEQUENCING AND CHARACTERIZATION OF THE CANINE TELOMERASE REVERSE TRANSCRIPTASE (TERT) PROMOTER. S.N. Long, E. Gault, L. Nasir, Institute for Comparative Medicine, University of Glasgow Vet School, Glasgow, Scotland, UK. Telomeres are specialized DNA-protein complexes that cap the ends of linear chromosomes and protect them from degradation and end to end fusions. Due to the ‘‘end replication problem,’’ telomeres undergo progressive shortening with each cell division. Shortening to a critical length triggers cell growth arrest and cellular senescence pathways, thereby acting as a means for regulating cellular lifespan. The enzyme telomerase is a ribonucleoprotein complex that adds nucleotides to the 39 end of telomeres, thus maintaining telomere length and bypassing cellular senescence. Telomerase activity is absent from most adult somatic cells, with expression limited to activated lymphocytes, germ cells and stem cells. In contrast, up to 90% of cancers possess telomerase activity, suggesting that telomerase activity within cells represents the acquisition of an immortal phenotype. Telomerase is composed of an RNA component, TR, a reverse transcriptase catalytic subunit, TERT, and associated proteins. Whilst TR is present in some somatic cells, the finding that TERT is only present in those cells possessing telomerase activity and that telomerase activity can be induced in telomerase-negative cells through the addition of TERT alone suggests that telomerase activity is regulated primarily through regulation of the TERT catalytic subunit. Our group has recently sequenced and characterized the canine TERT gene and identified approximately 5Kb of the upstream regulatory region. The purpose of this study was to sequence the promoter of the canine TERT gene and to identify the core region of the promoter essential for activity. PCR amplification and cloning of selected regions of the canine TERT promoter followed by luciferase assays revealed that core promoter activity is contained within a region extending approximately 300bp upstream of the ATG codon. Transient transfections in telomerase-positive canine cell lines and telomerase negative fibroblasts showed that the promoter is only active in telomerase positive cell lines. Sequence analysis demonstrated that the 59 regulatory region is GC-rich and contains no TATA or CAAT box, similar to the human TERT promoter. Motif searches revealed the presence of multiple transcription factor binding sites common to both the human and canine TERT promoters, including a single E-box, Sp1, AP1, MZF-2 and ER/Sp1 binding sites. These findings suggest that the canine TERT gene shares similar transcriptional control to the human TERT gene. Identifying the core promoter necessary for activity will enable the development of telomerase-targeted therapies in canine cancer patients similar to those investigated in human patients. ABSTRACT #2 N-ACETYLCYSTEINE DECREASES VASCULAR ENDOTHELIAL GROWTH FACTOR PRODUCTION IN CANINE HEMANGIOSARCOMA. Douglas H. Thamm, Ann M. Mitzey, Ilene D. Kurzman, David M. Vail. The Animal Cancer Center, Colorado State University (DHT, DMV) and Department of Medical Sciences, University of Wisconsin-Madison (AMM, IDK)2 N-ACETYLCYSTEINE DECREASES VASCULAR ENDOTHELIAL GROWTH FACTOR PRODUCTION IN CANINE HEMANGIOSARCOMA. Douglas H. Thamm, Ann M. Mitzey, Ilene D. Kurzman, David M. Vail. The Animal Cancer Center, Colorado State University (DHT, DMV) and Department of Medical Sciences, University of Wisconsin-Madison (AMM, IDK) Canine hemangiosarcoma (HSA) is a common disease, with very aggressive biologic behavior and short survival times with standard treatments. Novel therapies are desperately needed. As a tumor derived from vascular endothelium, antiangiogenic therapies may be uniquely efficacious for HSA. Our group and others have demonstrated the abundant expression of vascular endothelial growth factor (VEGF) and other angiogenic growth factors, as well as the receptors for these growth factors, in canine HSA cells, suggesting the possibility of autocrine signaling through these growth factor receptors. N-acetylcysteine (NAC) is a potent free radical scavenger that possesses multiple antineoplastic activities in vitro and in murine models, including inhibition of tumor and endothelial cell migration and invasion, matrix metalloproteinase activity, and VEGF production. We hypothesized that NAC would be capable of downregulating VEGF production under both normoxic and hypoxic conditions, and that this might be accompanied by decreased cell proliferation and survival as a result of attenuation of putative autocrine signaling through the VEGF receptor. Two canine HSA cell lines were cultured under normoxic conditions, or cobalt chloride (CoCl2) was added to simulate hypoxia, and varying concentrations of NAC were added. Cell supernatants and lysates were then collected and assayed for VEGF concentration using a commercially available ELISA kit. In separate experiments, HSA cells were incubated with varying concentrations of NAC and/ or doxorubicin for 72 hours, followed by determination of relative viable cell number using a commercially available ATP-based kit. One of two cell lines tested responded to simulated hypoxia with stimulation of VEGF production, suggesting that dysregulation of the hypoxia response pathways could be implicated in the pathogenesis of the disease. NAC reduced VEGF production in a dose-dependent fashion, however a higher concentration of NAC was required to inhibit CoCl2-stimulated VEGF production. NAC inhibited HSA proliferation and enhanced chemosensitivity in a dose-dependent fashion, however these effects were seen only at suprapharmacologic doses. These results provide proof of principle that redox balance is an important mediator of VEGF production in canine HSA cells, and imply that autocrine stimulation through a VEGF-VEGF receptor loop could contribute to HSA pathogenesis. It is possible that more potent antioxidants could prove clinically useful for the treatment of canine HSA in the future. ABSTRACT #3 RISK OF OSTEOSARCOMA IN RETIRED RACING GREYHOUNDS. Cynda Crawford, Julie Rosenberger, Norma Pablo. University of Florida, Gainesville, FL.3 RISK OF OSTEOSARCOMA IN RETIRED RACING GREYHOUNDS. Cynda Crawford, Julie Rosenberger, Norma Pablo. University of Florida, Gainesville, FL. Osteosarcoma (OSA) is the most common primary bone tumor diagnosed in dogs. Previous studies have determined that appendicular OSA is the most frequent form with a site predilection for metaphyseal regions of long bones, particularly the forelimbs. Several studies have identified several risk factors for OSA, including breed, age, height, sex, and neuter status. For the past decade, increasing numbers of retired racing Greyhounds have entered the pet population. Simultaneously, increased numbers of Greyhounds have been diagnosed with OSA, but there are no reports documenting their risk for this malignancy. The purpose of this study was to determine the risk of OSA in Greyhounds compared to other breeds, and to evaluate the association of host factors with this risk. In a retrospective case-control study, the medical records of all dogs diagnosed with OSA at the Veterinary Medical Teaching Hospital (VMTH) at the University of Florida from January 1996 to December 2004 were examined. Only dogs with OSA confirmed by histopathology were included in the case group. Potential risk factors recorded for each case included breed, age, sex, neuter status, and location of the OSA lesion. Prevalence for each breed was determined as the number of cases with OSA relative to the total number of the same breed seen during the same time period. Crude odds ratio (OR) and 95% confidence limits (CL) for risk in individual breeds were calculated by comparing each breed with mixed breeds, arbitrarily selected as the reference group. The risk for OSA was estimated only for breeds with 10 or more OSA cases. Unadjusted crude OR and 95% CL were estimated for age, sex, neuter status, and location of the primary lesion for the three breeds with the highest risk for OSA. P values ,0.05 were considered significant. The total number of confirmed OSA cases diagnosed at the UF VMTH from 1996 to 2004 was 172. The Greyhound had the highest breed prevalence of OSA (6.2%), followed by the Rottweiler (6.0%), Irish Wolfhound (5.7%), Irish Setter (5.7%), and Great Dane (5.2%). Compared to mixed breed dogs, the Greyhound had the highest risk for OSA (OR 5 10.5), followed by the Rottweiler (OR 5 10.1), Great Dane (OR 5 8.8), Doberman Pinscher (OR 5 3.7), Golden Retriever (OR 5 2.6), and Labrador Retriever (OR 5 2.2). Potential risk factors for OSA in the Greyhound were compared to those for the Rottweiler and Great Dane. Dogs seven to 10 years of age had the highest risk for all three breeds. There were no sex-related differences for the Greyhound or Great Dane, but spayed female Rottweilers had higher risk than sexually intact females. In all three breeds the appendicular skeleton was more likely to be affected than the axial skeleton, and forelimbs were affected as often as hindlimbs. Greyhounds have a high risk of appendicular OSA, similar to other large and giant breed dogs. ABSTRACT #4 A PROSPECTIVE STUDY OF UNFRACTIONATED HEPARIN THERAPY4 A PROSPECTIVE STUDY OF UNFRACTIONATED HEPARIN THERAPY TO PREVENT THROMBOSIS IN CANINE IMMUNE-MEDIATED HEMOLYTIC ANEMIA. EL Breuhl1, C Scott-Moncrieff1, M Brooks2. 1Purdue University, West Lafayette, IN and 2Cornell University, Ithaca, NY. Thromboembolic (TE) events are an important cause of mortality in dogs with immune-mediated hemolytic anemia (IMHA). Unfractionated heparin (UFH) is often given empirically to canine IMHA patients, However, few clinical studies have evaluated the efficacy of UFH for preventing thrombosis in this population. The objectives of this study were to develop a dosage regimen of UFH for canine IMHA based on attainment of a p

Association of adiponectin multimers with dietary nutrient composition, body weight gain, meal feeding, and insulin sensitivity in cats

Adiponectin has been investigated widely due to its association with adiposity and the metabolic syndrome in human beings. Adiponectin circulates as low- (LMW) and high-molecular weight (HMW) multimers and the latter are the more bioactive forms. There are no reports of the relative proportion (distribution) of adiponectin multimers in feline plasma. The aim of this study was to assess the association of dietary nutrient composition, body weight gain, meal feeding, and insulin sensitivity with HMW adiponectin concentration and adiponectin multimer distribution in cats.1 EVALUATION OF FOUR DNA EXTRACTION METHODS FOR THE DETECTION OF TRITRICHOMONAS FOETUS IN FELINE STOOL SPECIMENS BY POLYMERASE CHAIN REACTION. SH Stauffer, AJ Birkenheuer, MG Levy, H Marr, JL Gookin. College of Veterinary Medicine, North Carolina State University, Raleigh, NC. Feces are increasingly recognized as practical samples for molecular diagnosis of infectious disease. Extraction of PCR-quality DNA from feces can be challenging due to co-extraction of PCR inhibitors. Accordingly, we examined the effect of four commercially-available DNA extraction methods on sensitivity of PCR for detection of Tritrichomonas foetus (TF) in naturally-infected and TF-spiked feline stool. Kits evaluated included ExtractMaster Fecal DNA Extraction Kit, Epicentre Biotechnologies (Kit A); QIAamp DNA Stool Mini Kit, Qiagen (Kit B); UltraClean Fecal DNA Kit, MoBio (Kit C); and ZR Fecal DNA Kit, Zymo Research (Kit D). In accordance with manufacturer instructions, DNA was extracted from 180mg (A,B), 50mg (C), 100 & 150mg (D) aliquots of feline feces to which was added 20ml volumes containing 0–10,000 cultured feline TF. Each kit was also used to extract DNA from the feces of each of 10 naturally infected and 10 uninfected cats. DNA was eluted in 300ml (A), 200ml (B), 50ml (C), or 100ml (D) of respective elution buffer. Endogenous PCR inhibitors in extracted DNA was examined by PCR amplification of an 876 bp gene fragment of bacterial 16S rRNA. DNA was then tested by single tube nested PCR for amplification of partial ITS1, 5.8S and ITS2 rRNA genes of TF. Kit D provided the most sensitive detection of TF DNA as expressed by both organisms per DNA extraction and organisms per PCR reaction. To account for differences in DNA concentrations between kits (i.e. fecal sample size and elution volumes), the limit of detection for each kit as expressed by the number of TF per PCR reaction was as follows: Kit B 5 250, Kit A 5 167, Kit C 5 100, Kit D (150mg fecal sample) 5 5, and Kit D (100mg fecal sample) 5 0.5. PCR performed on DNA extracted from cultured TF (no feces) or TF-spiked feces (100mg) using Kit D was positive with as few as 10 TF per extraction. Further, DNA extraction using Kit D could be completed in the shortest time of all kits tested. These studies identify the ZR Fecal DNA Kit as superior to the other kits tested for extraction of PCR-qualityDNA from feline feces. ABSTRACT #2 INVESTIGATION OF ENTEROBACTER CLOACAE INFECTIONS AT A SMALL ANIMAL VETERINARY TEACHING HOSPITAL. JS Weese. University of Guelph, Guelph, Ontario.2 INVESTIGATION OF ENTEROBACTER CLOACAE INFECTIONS AT A SMALL ANIMAL VETERINARY TEACHING HOSPITAL. JS Weese. University of Guelph, Guelph, Ontario. A wide range of pathogens can cause hospital-associated (HA) infections in small animal hospitals. Among these is Enterobacter cloacae, which is one of the most clinically relevant Enterobacter spp and a common cause of HA infection in humans. Recently, multidrug resistance has become a concern, particularly with emergence of extended-spectrum beta-lactamase and extended spectrum cephalosporinase producing strains. An infection control investigation was initiated at the Ontario Veterinary College Teaching Hospital (OVCTH) in the fall of 2007 in response to anecdotal concerns about Enterobacter cloacae infections in hospitalized animals. Enterobacter cloacae was isolated from 45/36719 animals from January 1, 2005 to October 31, 2007, for an overall incidence of 1.2/ 1000 admissions. The monthly incidence rate ranged from 0 to 4.3/ 1000 admissions. Twenty-one (47%) cases were classified as community-associated, while 17 (38%) were hospital associated. Seven (15%) were community-onset but hospital associated, with three of these associated with other veterinary hospitals. There was no increase in the incidence of overall or hospital-associated infections during the study period. The urinary tract was the most common site of infection (n511, 24%). Wound infections (excluding surgical site infections) accounted for 8 (18%) of infections, with superficial and deep surgical site infections accounting for 7 (16%) and organ/space surgical site infections accounting for another 2 cases. Urinary tract infections were most common among animals with CA infection, accounting for 8/21 (38%) cases with wound infections accounting for 4 (19%) cases. Of the 24 cases associated with the OVCTH, 17 (71%) had surgery, 15 (63%) were hospitalized in the intensive care unit, 10 (42%) had indwelling urinary catheters placed, and 20 (83%) had received antimicrobials prior to onset of infection. Risk factors for E. cloacae infection could not be determined because a noninfected control group was not evaluated. Surgical site infections accounted for 9 (38%) HA cases. Overall, only 2/11 (18%) urinary tract infections were associated with prior placement of a urinary catheter. Nine (20%) animals died or were euthanized and E. cloacae was implicated as a causative or contributing factor in 5 (56%) of those cases. Two main antimicrobial phenotype patterns were identified. One (n525) was characterized by susceptibility to fluoroquinolones, tetracycline, and trimethoprim with variable susceptibility to cefoxitin while the other (n514) was characterized by resistance to these antimicrobials. Prior administration of antimicrobials was associated with presence of the more resistant phenotype (P50.044) but there was no association between this phenotype and origin of infection (P50.74) and no increase in the prevalence of this phenotype from 2005 to 2007 (P50.97). Infections with this phenotype were not associated with nonsurvival (P50.74). There was no evidence of a, HA outbreak or increase in prevalence, yet identification of multidrug resistant E. cloacae in both CA and HA infections is concerning and requires ongoing surveillance. ABSTRACT #3 STAPHYLOCOCCUS PSEUDINTERMEDIUS: A NEWLY RECOGNIZED PATHOGEN IN DOGS AND CATS. MC Faires, D Slavic, JS Weese. Ontario Veterinary College, Animal Health Laboratory, University of Guelph, Guelph, Ontario.3 STAPHYLOCOCCUS PSEUDINTERMEDIUS: A NEWLY RECOGNIZED PATHOGEN IN DOGS AND CATS. MC Faires, D Slavic, JS Weese. Ontario Veterinary College, Animal Health Laboratory, University of Guelph, Guelph, Ontario. Staphylococcus intermedius has typically been regarded as the predominant pathogenic Staphylococcus spp in dogs and cats, and a leading cause of skin and soft tissue infections. In 2005, a novel Staphylococcus species, Staphylococcus pseudintermedius, was identified. This organism is closely related to, but distinct from, S. intermedius. Gene-sequence based methods are required to differentiate these two species; however, these techniques are rarely performed in clinical laboratories, and as a result the prevalence and characteristics of S. pseudintermedius are poorly understood. Recent evidence suggests that S. pseudintermedius may actually be the predominant Staphylococcus spp in dogs and cats but misidentified as S. intermedius by diagnostic laboratories. The objective of this study was to use sequence based methods to identify putative S. intermedius isolates from dogs and cats and to evaluate antimicrobial resistance and virulence factors among S. pseudintermedius isolates. Isolates from dogs and cats identified as S. intermedius by conventional laboratory methods were obtained from the University of Guelph Animal Health Laboratory. Isolates were collected in a serial manner without selection. DNA was extracted, sequencing of the sodA gene was performed, and isolates were identified via sequence alignment with reference staphylococcal strains through GenBank (www.ncbi.nlm.nih.gov/blast/BLAST.cgi). Antimicrobial susceptibility testing was performed and PCR was used to identify various virulence factors and antimicrobial genes. A total of 25 isolates were obtained from 21 dogs and 2 cats. Medical records were not available for 2 of the isolates. 25/25 (100%) were identified as S. pseudintermedius Severity of infection ranged from superficial dermatitis to rapidly fatal necrotizing fasciitis with the majority of isolates from otitis externa 9/23 (39.1%) and urinary tract infections 6/23 (26.1%). Antimicrobial susceptibility was as follows: amoxicillin/clavulanate 23/23 (100%), ampicillin 7/ 23 (30.4%), cephalothin 23/23 (100%), clindamycin 18/23 (78.3%), gentamicin 23/23 (100%), tetracycline 18/23 (78.3%), and trimethoprim/sulfa 19/23 (82.6%). Not all antimicrobials were tested for all isolates, based on laboratory protocols regarding antimicrobial panel and site of infection. Inducible resistance to clindamycin was detected by D-test in 1 isolate reported as clindamycin-susceptible (5.6%). Detection of virulence factors and antimicrobial resistance genes is ongoing. This study identified S. pseudintermedius as an important pathogen in dogs and cats, and suggests that S. intermedius may not be a major concern in these species. Further studies are required to evaluate clinically relevant virulence factors to assist in understanding the pathogenesis of disease caused by S. pseudintermedius. 70