key: cord-023095-4dannjjm
authors: nan
title: Research Abstract Program of the 2011 ACVIM Forum Denver, Colorado, June 15–18, 2011
date: 2011-05-03
journal: J Vet Intern Med
DOI: 10.1111/j.1939-1676.2011.0726.x
sha: 
doc_id: 23095
cord_uid: 4dannjjm

nan

Clinics ÃÃ Also see Infectious Disease abstracts ID-1 -ID-14 (Thursday, June 16, 2:15 pm -6:15 pm) ÃÃ Also see Pharmacology abstracts P-1 -P-5 (Thursday, June 16, 5:00 pm -6:15 pm) ÃÃ Also see Gasteroenterology abstracts GI-1 - June 17, 8 Hypertrophic cardiomyopathy (HCM) is the most commonly observed myocardial disease in cats. Beta-blockers and calcium channel inhibitors are frequently administered drugs to cats with preclinical HCM despite the fact that neither drug category has been proven to slow disease progression or improve survival. Ivabradine (Procorolan s , Servier, France) is a novel negative chronotropic agent used in the treatment of ischemic heart disease in people. Little is known about its efficacy and safety in cats. The purpose of this study was to determine the short-term effects of ivabradine on heart rate (HR), blood pressure, left ventricular (LV) systolic and diastolic function, left atrial (LA) performance, and clinical tolerance in healthy cats after repeated oral doses.

Ten healthy laboratory cats were involved in the present study. Physical examination, systolic blood pressure measurement, and transthoracic echocardiography were performed in all cats at baseline and after oral administration (4 weeks each) of ivabradine (0.3 mg/kg, q12 h) and atenolol (6.25 mg/cat, q12h; 1.0-1.7 mg/kg) in a prospective, double-blind, randomized, active-control, fully crossed study. A-priori non-inferiority margins for the effects of ivabradine compared to atenolol were set at 50% (f 5 0.5) based on predicted clinical relevance, observer measurement variability, and in agreement with FDA guidelines. Variables were compared by use of 2-way repeated measures ANOVA.

Ivabradine was clinically well tolerated with no adverse events observed. HR (ivabradine, Po0.001; atenolol, Po0.001; ivabradine vs. atenolol, P 5 0.721) and rate-pressure product (ivabradine, P o 0.001; atenolol, P 5 0.001; ivabradine vs. atenolol, P 5 0.847) were not different between treatments. At the dosages used, ivabradine demonstrated more favorable effects than atenolol on echocardiographic indices of left ventricular (LV) systolic and diastolic function and left atrial performance.

Ivabradine is non-inferior to atenolol with regard to effects on HR, rate-pressure product, LV function, LA performance, and clinical tolerance. Clinical studies in cats with HCM are needed to validate these findings and further assess safety. The aim of this study was to compare outcome from CPA in dogs following initial administration of either epinephrine or vasopressin during cardiopulmonary resuscitation (CPR).

Dogs having CPA in the ER or ICU of a university hospital were randomized to receive either IV epinephrine (0.01-0.02 mg/kg) or vasopressin (0.5-1.0 U/kg) in a blinded fashion immediately following establishment of IV access and again three minutes later. A standardized CPR protocol was followed. Other vasopressors were not permitted during the six minute study period, at the end of the study period additional CPR interventions were at the discretion of the managing clinician. The primary end point was return of spontaneous circulation (ROSC) within the study period; secondary end points included ROSC at any point, survival to 20 minutes, and survival to one hour.

Sixty dogs completed the study, 31 received epinephrine and 29 received vasopressin. ROSC within six minutes was 53% (13 vasopressin, 19 epinephrine p 5 0.2), ROSC at any time was 60% (15 vasopressin, 21 epinephrine p 5 0.2). Survival to 20 minutes was 32% (6 vasopressin, 13 epinephrine p 5 0.077), survival to one hour was 18% (2 vasopressin, 9 epinephrine p 5 0.027). Five dogs survived to 24 hours, one survived to hospital discharge. Of animals dying after ROSC, 13/35 were euthanized and 22/35 rearrested.

No advantage of routine substitution of vasopressin for epinephrine was seen for ROSC, a small survival advantage at one hour was seen in the group receiving epinephrine. The study also demon-strated that prospective clinical CPR research in animals is both possible and practical. Three dogs were evaluated in 3 phases. Phase-1: single-dose DiltXR at approximately 6 mg/kg PO. Phase-2: same dose q12h for 4.5 days. Phase-3: after a 14 day wash-out the single-dose protocol was repeated using cut tablets to assess affect on extended release properties. Blood pressure (BP), 6-lead ECG, echocardiogram, and 24h-ambulatory ECG were performed at baseline, and conclusion of Phase-2. Blood samples and BP was obtained 0, 1, 2, 4, 8, 12, and 24h after final dosing.

Peak median plasma diltiazem concentrations (mcg/ml) measured by HPLC for each phase were 0.0979, 0.016, and 0.07 respectively. Diltiazem concentrations were below the limit of detection in the majority of samples in phase-2. Median diltiazem concentration reached purported therapeutic concentrations (0.05-0.2 mcg/ml) by 2 h post-pill in phase-1 and 1h post-pill in phase-3. Therapeutic concentrations were maintained for 24 h in Phase-1, but only 2h in phase-3. Median BP (mmHg) was 142.7 at baseline and 126.0 at peak concentration in phase-1. Median heart rate (bpm) was 127.1 at baseline. 24h-ambulatory ECG analysis revealed the median hourly heart rate was 101.5 at baseline and 102 during phase-1. Median heart rate at peak concentration in phase-1 was 127.5.

Lack of detectable plasma diltiazem during phase-2 may be due to up-regulation of drug metabolism via P-glycoprotein (ABCB1-1) mutations. Ongoing data collection and analysis will include mutation testing.

Adiponectin (ADPN) is a cytokine produced by fat cells which has been shown to be correlated with adverse cardiac conditions in humans. In the heart, adiponectin activates several pro-survival reactions, including the AMPK pathway and COX2 receptors, which protect the heart following ischemic injury. Recent studies have shown that higher levels of ADPN influence cardiac remodeling signaling, inhibiting protein synthesis and suppressing pathological cardiac growth. In humans, ADPN plasma levels rise with decreased activity of the sympathetic nervous system and b-adrenergic agonists inhibit ADPN at the level of gene expression. In contrast C-reactive protein (CRP), a marker of systemic inflammation is elevated in humans with congestive heart failure (CHF) and correlates to the severity of disease. First, we hypothesized that dogs with CHF would have reduced ADPN and elevated CRP compared to normal dogs and that cytokine concentrations would predict severity of CHF. Second, we hypothesized that ADPN receptor-1 (R1) and ADPN protein would be elevated in the myocardium of CHF dogs reflecting a compensatory process.

We collected serum from 32 dogs (18 healthy and 18 CHF). Circulating adiponectin and CRP levels were quantified using a Mouse/Rat Adiponectin ELISA and a Canine CRP ELISA. We found lower mean CRP concentrations in normal dogs (3.5 AE 2.3 mg/ ml) than dogs with CHF (17.9 AE 17.1 mg/ml), however, the results were not statistically significant due to the large variability seen among the CHF dogs (p 5 0.07). We found greater mean ADPN concentrations in normal dogs (11.46 AE 3.3 mg/ml) than CHF dogs (9.2 AE 3.2 mg/ml) (p 5 0.05). In general, the greater the severity of the heart failure, the lower the level of serum ADPN. When the 2 tests The purpose of this study was to determine if there are any clinically important differences between the approaches (including devices) used in non invasive transvascular (interventional) closure of patent ductus arteriosus (PDA) in dogs in our institution. Initial and follow up records from all dogs (n 5 112) that underwent attempted transvascular PDA occlusion from January 2006-December 2009 were examined. Dogs were placed into 4 groups depending on the device and route of vascular access (transvenous or transarterial). Group 1: Amplatz s Canine Ductal Occluder (ACDO) (transarterial) -36 dogs; Group 2: Gianturco s or MReye Flipper s Detachable Embolization (Flipper) coil (transarterial) -38 dogs; Group 3: Amplatzer s Vascular Plug (AVP) (transarterial) -23 dogs; Group 4: Flipper coil (transvenous) -15 dogs. Statistical comparisons were made using the Kruskal-Wallis test with Mann-Whitney tests to compare pairs of groups when significance was detected. P o 0.05 was considered significant.

There was no significant difference in ages between the 4 groups. There was a significant difference in body weight between groups with dogs receiving a coil either transarterially or transvenously (groups 2 and 4) being significantly smaller than dogs receiving an ACDO or AVP. This was by design since the ACDO and AVP cannot be used in small dogs. Overall, the success rate of the total procedure (including vascular access and satisfactory PDA occlusion) was high (92%) with success rates being comparable between groups (87-97%). There was a significant difference in complication rate between groups (p o 0.0001) with the ACDO group having a markedly lower complication rate than the remaining groups (3% for ACDO versus 24-33% for the other groups). Total fluoroscopy time ranged from 3-78 minutes (median 8 minutes). Fluoroscopy time for the transvenous method was significantly longer (median 13 minutes; range 10-78 minutes) than in the remaining groups (median 6 minutes; range 3-33 minutes) (p o 0.0001). Number of dogs with residual flow immediately following the procedure and 24 hrs later was significantly less in the ACDO group than in the remaining groups (2 dogs from group 2 and 3 from group 3 had moderate persistent flow while 1 dog from group 2 and 1 from group 4 had severe persistent flow 24 hours after the procedure).

The ACDO appears superior in ease of use, complication rate, completeness of occlusion and fluoroscopy time than other devices. The remaining limiting factor with this device is patient size. Until a smaller ACDO device is marketed, coils remain the only choice for interventional closure in very small dogs ( o 2.5kg).

Previously presented at the University of California Davis, House Officers Seminar Day.

Subvalvular aortic stenosis (SAS) is one of the most commonly reported canine congenital heart defects and is inherited in Newfoundland dogs and human beings. The golden retriever and Rottweiler are breeds over-represented in dogs with subvalvular aortic stenosis; however, a genetic cause of this disease in these breeds has not been described. We performed genome wide association analysis in both normal and SAS affected Rottweilers and golden retrievers to identify chromosomal regions of interest that could implicate a causative mutation by high density single nucleotide polymorphism (SNP) array. 48 (24 unaffected/24 affected) adult golden retrievers and 48 (20 unaffected/28 affected) adult Rottweilers were included in this study. Criteria for affected included a subcostal continuous-wave Doppler aortic velocity ! 2.5 m/s and presence of a left basilar systolic ejection murmur; criteria for unaffected included a Doppler aortic velocity 1.8 m/s. DNA samples were obtained from anticoagulated blood. Genotypes were obtained using high density (173,662) SNP arrays, and genome wide association with SAS was evaluated for each breed. Significance cut-off was set at p 5 5 Â 10 À5 , and all SNPs meeting this criterion were plotted within each breed and compared across breeds using PLINK.

Affected golden retriever data implicate the most significant region of genetic variation on chromosome 21 at location 27384260 (p 5 1.15 Â 10 À5 ; odds ratio 7.58) with 11 other significant surrounding SNPs . Affected Rottweiler data also implicate the most significant region of genetic variation on chromosome 21 at location 27895300 (p 5 8.98 Â 10 À6 ; odds ratio 23.39) with 3 other significant surrounding SNPs . Other regions of statistical significance were on chromosomes 4 and 7 in the golden retriever and 11 and 38 in the Rottweiler.

Genome wide association with subvalvular aortic stenosis in the golden retriever and Rottweiler implicate overlapping chromosomal regions of interest for causative mutations on chromosome 21. The different secondary chromosomal regions of interest (CHR 4, 7 in golden retrievers and 11, 38 in Rottweilers) supports the known familial nature of this disease within different breeds and may suggest the presence of multiple mutations or breed specific disease modifiers. These data highlight the need for candidate gene evaluation on chromosome 21 in golden retrievers and Rottweilers with SAS.

Heart valves share developmental signaling pathways with bone and cartilage. Degenerative aortic valve disease in humans is characterized by valve stenosis and calcification. Recent evidence suggests that degenerative aortic valves are undergoing pathologic processes that mimic osteogenesis. Degenerative mitral valves in dogs and humans are characterized by valve regurgitation, and rarely undergo calcification. We tested the hypothesis that canine and human degenerative mitral valves might be undergoing pathologic processes that mimic chondrogenesis.

To test this hypothesis, expression of bone morphogenic protein 2 (BMP2), a chondrogenic growth factor; Sox 9, a chondrogenic transcription factor; aggrecan, a proteoglycan abundant in cartilage; and type II collagen were evaluated utilizing immunohistochemistry. Normal canine mitral valves, different stages of canine degenerative mitral valves (early, intermediate, and late), and late-stage human degenerative mitral valves were studied.

Canine and human degenerative mitral valves showed focal areas that co-expressed all four markers of chondrogenic signaling and phenotype. Valve interstitial cells and surrounding extracellular matrix in these focal areas adopted a morphologic appearance reminiscent of cartilage. Focal chondrogenesis was present in all stages of canine degenerative mitral valves, but not normal canine mitral valves. Focal areas of chondrogenesis did not coincide with nodular areas of glycosaminoglycan accumulation on the leaflet edge, but rather seemed to occur at points of chordae attachment to leaflets.

In conclusion, canine and human degenerative mitral valves undergo pathologic processes that mimic chondrogenesis. This finding suggests that mitral valve degeneration may be recapitulating developmental signaling pathways shared by heart valves and cartilage. The triggering events for chondrogenesis in mitral valves remain unknown; as does the reason why aortic and mitral valves appear to be undergoing different pathologic processes. The fact that humans exhibit degeneration of both the aortic and mitral valve, and that dogs commonly exhibit only the latter could eventually provide insight into both processes.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiomyopathy characterized by right ventricular fibrofatty infiltration and ventricular ectopy of left bundle branch block morphology (VPC) . A deletion in the striatin gene has been associated with ARVC in at least some boxer families. Syncope and sudden death (SD) occur in some affected dogs, although many affected dogs survive for years.

The objective of this study was to define clinical characteristics of ARVC in boxers that experienced SD, and compare them to those of a contemporaneous group of ARVC boxers that had not died suddenly (NSD). Data for both groups were collected from adult boxers enrolled in a long term prospective study of ARVC in which echocardiograms and 24 hour ambulatory ECG (AECG) are evaluated annually. AECG are quantitated for VPC numbers and arrhythmia grade (1-4). ARVC diagnosis requires at least 100 VPCs/ 24 hours in the absence of other disease.

Forty three adult boxers that entered the study had died suddenly at the time of analysis (SD defined as the absence of observed clinical signs within 24 hours prior to an unexpected and unexplained death). Striatin genotype was available for 28 of the 43 SD dogs (17 heterozygotes, 11 homozygotes); 19 were female (9 intact) and 24 were male (14 intact). SD occurred at a mean age of 8 years (range, 1-12); 27 SD dogs (63%) had no prior history of syncope. Twelve SD dogs (28%) were on antiarrhythmics at the time of death (metop-p0oooooooooooprolol (1), sotalol (4), amiodarone (1), procainamide (2), mexiletine & atenolol (3), atenolol (1)). Eleven SD dogs (25%) had decreased myocardial systolic function defined by a shortening fraction (%FS) o 25% (range 11-45, mean 5 27) on the most recent echocardiogram prior to SD. Median VPCs/24 hours on annual AECG was 7,700 (range 106-91,000) with a median arrhythmia grade of 4 (range 2-4).

Twenty one contemporaneously entered ARVC boxers that had survived to at least the median age of the SD group with NSD were available for comparison; 15/21 were genotyped (10 heterozygous, 1 homozygous, 4 negative), 13 were female (3 intact) and 8 male (2 intact). Twelve NSD dogs (57%) had no prior history of syncope. Median NSD group age was 10 years (range, 8-13); 11/21 (52%) were on an antiarrhythmics (sotalol (9), mexiletine & sotalol (1), mexiletine & atenolol (1)). One NSD dog had decreased %FS (NSD group %FS range 20-42, mean 5 32). The NSD median number of VPCs was 5,342 (range 622-62,622), median arrhythmia grade was 4 (range 2-4).

Striatin genotype was significantly associated with SD. No significant differences were found between groups with respect to VPC numbers or arrhythmia grade. Shortening fraction was significantly lower in the SD group (p o 0.01). SD in ARVC appears to be associated with the presence of the striatin mutation and reduced % FS, it does not appear to be associated with number of VPCS or arrhythmia grade. Coughing in the small breed dog may be related to cardiac causes associated with myxomatous mitral valve degeneration (MMVD) including pulmonary edema and compression of the mainstem bronchus by a severely enlarged left atrium, or due to respiratory causes such as tracheal and/or bronchial collapse or chronic bronchitis. The purpose of this study was to evaluate the association between left atrial enlargement and large airway collapse in dogs with MMVD and chronic cough. We hypothesized that airway collapse was independent of degree of left atrial enlargement.

Twelve dogs with MMVD and a chronic cough in the absence of congestive heart failure were prospectively evaluated with thoracic and cervical radiography, echocardiography, fluoroscopy, bronchoscopy and bronchoalveolar lavage (BAL). Group 1 dogs (n 5 8) had moderate to severe left atrial enlargement based on an echocardiographically calculated left atrial:aortic surface area [LA:Ao(a)] 4 6.

Group 2 dogs (n 5 4) had no to mild left atrial enlargement [LA:Ao(a) 6]. The site and severity of airway collapse was graded on bronchoscopy and BAL cytology was assessed for evidence of inflammation or infection. The occurrence of bronchoscopic abnormalities was compared between groups using Fisher's Exact Test. P o 0.05 was considered significant.

Age and body weight did not differ between groups. Left atrial size was interpreted radiographically as moderately to severely enlarged in 7 of 8 dogs in group 1 and as moderately enlarged in 2 of 4 dogs in group 2. Fluoroscopy revealed variable degrees of airway collapse during normal respiration and induced cough in both groups. Radiography and fluoroscopy were not accurate in identifying site and degree of collapse in either group when compared to bronchoscopy. Cervical tracheal collapse was identified during bronchoscopy in both group 1 (2 of 8) and group 2 (3 of 4) dogs but was subjectively less severe in group 1 dogs. Bronchial collapse 4 50% was evident at multiple sites in both groups of dogs with no difference between groups. All dogs had suppurative and/or lymphocytic inflammation on airway cytology. Infection was not present in either group of dogs, although non-specific light bacterial growth was detected in 6 of 8 group 1 dogs and 1 of 4 group 2 dogs (p 5 0.22).

Preliminary results failed to identify an association between left atrial enlargement and airway collapse in dogs with MMVD but did suggest that airway inflammation is common in affected dogs. Further studies are needed to identify factors contributing to airway collapse in dogs with and without MMVD.

Atenolol is often used empirically in cats with asymptomatic HCM, even though clinical and experimental evidence of efficacy is lacking. Cardiac biomarkers play a critical role in the early detection of subclinical cardiac disease, in the prediction of long-term prognosis, and in monitoring the response to therapy in humans. We hypothesized that serum concentrations of the biomarkers, Nterminal pro-brain natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI), would improve following the chronic administration of atenolol PO to asymptomatic cats with HCM.

Six Maine Coon or Maine Coon cross cats with severe HCM from the research colony at UCDavis were administered atenolol (12.5 mg PO twice a day) for 30 days. No cat had severe left ventricular dynamic outflow tract obstruction due to systolic anterior motion of the mitral valve. The concentrations of NT-proBNP and cTnI were assayed prior to drug administration and on the last day of drug administration. There was no statistically significant difference identified in NT-proBNP [median before: 394 pmol/L (range: 71-1500 pmol/L), median after: 439 pmol/L (range: 24-1500 pmol/L); p 5 0.63] or cTnI [median before: 0.24 ng/ml (range:0.10-0.97 ng/ml), median after: 0.28 ng/ml (range: 0.09-1.0 ng/ml); p 5 0.69] concentrations before and after drug administration using the Wilcoxon matched pairs test.

The cTnI finding suggests that atenolol does not reduce chronic myocyte death in cats with HCM. The lack of improvement in NT-proBNP suggests that atenolol does not improve myocardial wall stress in cats with HCM. A clinical trial is warranted to confirm or refute the findings from this study. Therefore, leptin-gene expression was investigated in blood samples of dogs with congestive heart failure (CHF; n 5 8) in comparison to dogs presented for cardiac screening (n 5 8) without abnormalities. Additionally myocardial samples (interventricular septum, right and left atrium and ventricle) of 10 dogs with no cardiac abnormalities (controls), seven dogs with acquired and three with congenital cardiac diseases were investigated using quantitative RT-PCR.

Leptin blood levels were significantly higher in dogs with CHF in comparison to dogs without diseases (p 5 0.013). There was an association with gender with higher myocardial leptin levels in female dogs with cardiac diseases (p 5 0.001). Differences between cardiac regions were present (p o 0.05) and cardiac diseases resulted in an increase in atrial leptin levels in both sexes (p 5 0.006). Interestingly, a significant reduction of myocardial leptin was present in dogs with congenital cardiac diseases (p 5 0.016), whereas acquired cardiac diseases resulted in an increase in leptin (p 5 0.035) in comparison to controls.

These results suggest that the heart might be a target of leptin action in the dog and myocardial leptin production might play a role in regulating cardiac function in an auto-and paracrine manner.

Predicting risk of CHF in asymptomatic dogs with mitral valve disease (MVD) is challenging. We examined ability of NT-proBNP to identify asymptomatic dogs at high risk for CHF. Dogs with ISACHC-1b (LA:Ao 4 1.6) were prospectively recruited; dogs with current or previous CHF or diuretic therapy were excluded. Physical examination, radiography, echocardiography, and NT-proBNP were performed at 2-9 mo intervals for 66 dogs (median follow-up 5 276 d, range, 17-1334 d). Thirty-one patients reached a study endpoint of radiographic pulmonary edema; 35 remained asymptomatic. Parameters from the visit immediately previous to onset of CHF (Future-CHF) or prior to the most recent visit without CHF (Remain-Asympt) were analyzed. Median NT-proBNP of Future-CHF (3001pmol/Lpmol/L, IQR 2255-3001) was significantly different from Remain-Asympt (1600 pmol/L, 984-2863; P 5 0.0004). Median time to CHF of Future-CHF was 86d (IQR, . Groups also differed in median LA:Ao (Future-CHF 2.20 [2.00-2.50]; Remain-Asympt 1.96 [1.85-2.13], P 5 0.0014); VHS (Future- ]; Remain-Asympt 11.3 [10.75-12.0], P 5 0.003); and LVIDd:Ao ]; Remain-Asympt 2.33 [2.15-2.64], P 5 0.0001). ROC analysis to predict if CHF would occur prior to the next visit yielded AUC 5 0.753 (95%CI, 0.632-0.851). Sensitivity was 90.3% or 77.4% and specificity 48.6% or 68.6% for NT-proBNP 41490 pmol/L or 42150 pmol/L, respectively. Mean increase in NT-proBNP between penultimate visit and two visits prior to endpoint: Future-CHF 5 1440.1 pmol/L vs. Remain-Asympt 5 110.5 pmol/L. Within 6 mo, 4.5%, 10.7%, 17.4%, and 36.7% of dogs with NT-proBNP o 1000, , and 43000 pmol/L developed CHF. NT-proBNP and heart size helped assess risk of CHF in asymptomatic MVD. Increasing the assay's upper limit of detection would likely improve utility of NT-proBNP. PIIINP is a serum biomarker of collagen biosynthesis and is described as a marker of myocardial fibrosis in human patients. We hypothesised that PIIINP concentrations would vary according to the degree of remodelling demonstrable in dogs with naturallyoccurring myxomatous mitral valve disease (MMVD).

Serum PIIINP concentrations (mg/ml) were measured in dogs with MMVD and healthy controls using a validated commerciallyavailable radioimmunoassay. Results are reported as (Mean AE SD). Non-normally distributed variables were logarithmically transformed. Comparisons of continuous variables were made between groups using t-tests and one-way ANOVAs with Tukey's post-hoc comparisons. Univariable analyses were used to evaluate associations between PIIINP and clinical characteristics (age, breed [cavalier King Charles spaniel (CKCS) yes/ no], sex, weight, heart rate [measured from ECG], treatment with ACEi [yes/ no], treatment with diuretics [yes/ no] and echocardiographic measurements [LA/Ao, LVEDD/ LVFWd, LVEDDN, LVESDN]). Multivariable analysis was initially performed with all dogs included and then repeated excluding all dogs receiving therapy. Dogs with MMVD were divided into those with no cardiomegaly (NC) (LA/Ao o 1.5 and LVEDDN o 1.8), those with cardiomegaly (LA/Ao ! 1.5 and/ or LVEDDN ! 1.8) but no clinical signs (C) and those dogs with cardiomegaly requiring treatment for congestive heart failure (CHF).

One hundred and fifty-four dogs with MMVD and 23 control dogs were studied. There was no difference in age (P 5 0.870) or weight (P 5 0.606) between the MMVD and control groups.

There was a significant difference in serum PIIINP (P 5 0.034) between normal (11.2 AE 3.66), NC (11.6 AE 4.57), C (10.1 AE 3.44) and CHF (9.4 AE 3.06) groups. Post-hoc comparisons demonstrated a difference between NC and CHF groups (P 5 0.038). There was no difference in serum PIIINP between genders (P 5 0.228). In the univariable analysis CKCS (yes/ no) (P 5 0.016) was positively associated with serum PIIINP. Age (P o 0.0001), Log (LA/Ao) (P 5 0.002) and LVEDDN (P 5 0.002) were negatively associated with serum PIIINP. In the multivariable model including all dogs, LVEDDN (P o 0.0001, B 5 À4.04 (95%CI 5 À6.11 to À1.97)), age (P 5 0.006, B 5 À0.28 (95%CI 5 À0.47 to À0.08)) and CKCS (yes/ no) (P 5 0.003, B 5 2.01 (95%CI 5 0.67 to 3.34)) were independently associated with serum PIIINP. In the multivariable model including only dogs not receiving therapy (n 5 141), LVEDDN (P 5 0.006, B 5 À4.30 (95%CI 5 À7.32 to À1.29)), age (P 5 0.011, B 5 À0.31 (95%CI 5 À0.55 to À0.07)) and CKCS (yes/ no) (P 5 0.034, B 5 1.75 (95%CI 5 0.13 to 3.37)) were independently associated with serum PIIINP. In conclusion, serum PIIINP decreases with age and with increasing LVEDDN. CKCS have higher serum PIIINP measurements independent of age and LVEDDN, which may reflect a difference in collagen turnover in this breed. Left atrial (LA) chamber dilation and congestive heart failure (CHF) are common consequences of cardiac conditions in cats. In some cases CHF is manifest as right-sided CHF (R-CHF) or pleural effusion, in other cases CHF manifests as left-sided CHF (L-CHF) or pulmonary edema. It is not always readily apparent as to which cats will develop what form of CHF. A general impression has been that LA enlargement is associated with the average burden of elevated filling pressures, but little attention has been paid to the function of the LA chamber itself. Since CHF is classically preceded by abnormal atrial chamber dilation and alterations in atrial chamber function, we want to understand how these changes may help us manage or predict CHF in the cat.

We hypothesized that cats manifesting R-CHF have LA failure with the LA acting primarily as a conduit, resulting in greater pulmonary hypertension, whereas L-CHF cats maintain some booster pump and reservoir function. We measured LA maximum and minimum areas from right parasternal long axis four-chamber views on 2D echo, and LA M-mode dimensions at maximum, minimum, and beginning of atrial contraction. LA area change, fractional shortening, active emptying fraction, and expansion index were calculated from these measurements. Right ventricular internal diastolic diameter was also measured on M-mode views. Preliminary data revealed that maximum left atrial size is not significantly different between R-CHF and L-CHF cats on 2D or M-mode measurements due to high variability. However, total left atrial fractional shortening is significantly reduced in R-CHF cats (11.32% AE 4.9) compared to L-CHF cats (19.7% AE 5.1)(p 5 0.001), and R-CHF cats have reduced left atrial active emptying fraction (6.12% AE 3.5) as compared to L-CHF cats (13.7% AE 3)(p o 0.001). Left atrial expansion ability is poorer in R-CHF cats (13.11% AE 6.7) than in L-CHF cats (25.0% AE 8)(p 5 0.002). These findings may suggest that atrial stiffness and poorer atrial function is associated with a greater degree of pulmonary venous and thus secondary pulmonary arterial hypertension resulting in pleural effusion (R-CHF). Right ventricular diameter on M-mode was increased in R-CHF cats (4.8 mm AE 2.1) when compared to L-CHF cats (2.89 mm AE 0.8)(p 5 0.002) and normal cats (2.74 mm AE 0.8)(p 5 0.002), which may also be evidence for a greater degree of pulmonary arterial hypertension in these cats. Episodic weakness and syncope are common in Boxer dogs. Reported causes include rapid ventricular tachycardia (VT) and exertion-excitement triggered neurally-mediated bradycardia (NMB) .The purpose of this retrospective study is to describe the features of presumed NMB in Boxers.

To be included in the study, each dog must have been overtly healthy with a history of exertion-excitement triggered syncope or presyncope; had a normal echocardiogram (EC); had absence of VT and fewer than 500 ventricular premature complexes (VPC) on an initial and subsequent 24 hour Holter recordings; and been alive and overtly healthy for at least six months following the initial evaluation.

A total of 27 Boxers were identified. Sixteen were male and 11 were female. Most (90%) dogs were either less than 4 or more than 7 years of age. Most dogs had multiple, but infrequent, episodes and heart rhythm was documented at the time of an episode in only 8 (30%) and only once (bradycardia) on the first Holter recording. Owners were instructed to attempt to precipitate episodes. Bradycardia related episodes were subsequently recorded in 5: during the 2nd (1), 3rd (2) or 4th (1) day of 120 hour Holter recordings and during the 5th day of an event recording (1). Collapse and bradycardia were documented during auscultation in 2 additional dogs.

The heart rate during syncope was never documented in 19 (70%) dogs. A presumptive diagnosis of NMB was based on the absence of initial and follow-up of EC abnormalities and the presence of no or few VPC during extended ECG monitoring. Multiple Holter recordings (48-168 hours) were performed in 8 of 19 (42%) dogs and event monitoring of 5 days (1) and 7 days (1) was performed in 2 additional dogs.

In conclusion, documentation of the heart rhythm during episodes of collapse was difficult, accomplished in only 30% and was unlikely during the first Holter recording. In Boxers with suspected NMB, extended ECG monitoring and implantable loop recorders may be best for HR documentation. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease with high prevalence in the boxer dog population, and is associated with sustained monomorphic ventricular tachycardia, sudden cardiac death, and replacement of myocardium with fatty or fibro-fatty tissue. Though several genes have been linked to the disease both in humans and in boxers, the etiology of ARVC is still unclear. Several mechanisms for the development of ARVC have been suggested, including dysfunction of the canonical Wnt pathway, which results in an ARVC phenotype in the mouse. The canonical Wnt pathway has been linked to many cellular functions, including growth and differentiation of adipocytes. With the recent discovery that the gene encoding striatin, a protein involved in Wnt signaling, may be involved in the development of boxer ARVC, we hypothesized that changes in the Wnt pathway may also play a role in the etiology. Here, we show changes in the localization and decreased amount of proteins affiliated with the canonical Wnt pathway.

Afflicted boxers were identified by 24-hour Holter monitoring and histopathological examination of the heart. Samples from the right ventricle RV) of 15 ARVC afflicted boxers, and 5 unafflicted dogs (1 beagle, 2 mongrels, and 2 German shepherds) were collected, fixed in 10% formalin, processed, treated with antibodies recognizingcatenin, striatin, and calnexin, and examined using confocal microscopy. Western blots were performed on 3 unafflicted RV samples, and 2 ARVC afflicted RV samples. Frozen tissue samples were homogenized in Laemmli buffer, and 10 mg of protein was loaded into each well of a 8-16% gradient gel. -catenin, an integral modulator of the Wnt pathway, and striatin were colocalized with the endoplasmic reticulum (ER) marker, calnexin. In the unafflicted animals, -catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern, with no detectable localization in the ER. In contrast, in the 15 ARVC boxers, bothcatenin and striatin were colocalized with calnexin in an ER pattern. In the afflicted samples, -catenin and striatin were not visualized to the intercalated disc and intracellular space, respectively. Western blots of striatin and -catenin revealed no changes in the amount of protein. Interestingly, a Western blot for the Wnt protein revealed a decrease in the amount of protein in ARVC samples, compared to unafflicted samples. Our preliminary data suggest that disturbances of the canonical Wnt pathway may play an etiological role in the development of ARVC in the boxer dog.

There are numerous benefits of omega-3 fatty acid supplementation in human heart disease, including reduction in arrhythmias, decreased incidence of sudden death, and improved survival in heart failure. Antithrombotic effects of omega-3 fatty acids have been demonstrated in people, which may have particular benefit in cats given their risk of thromboembolic complications with cardiac disease. Benefits also have been found in canine heart disease, and reduced serum concentrations of the omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found in dogs with congestive heart failure (CHF) secondary to DCM. To the authors' knowledge, no studies to date have investigated fatty acid concentrations in cats with cardiomyopathy. The purpose of this study was to measure serum fatty acid concentrations in normal cats and cats with cardiomyopathy.

Serum fatty acid concentrations were measured in normal cats and cats with cardiomyopathy using gas chromatography. Cats with cardiomyopathy and at least mild left atrial (LA) enlargement (LA to aortic ratio 4 1.5 on two-dimensional echocardiography from a right parasternal short axis view) were candidates for study. Normal cats had a normal history, physical examination, echocardiogram, packed cell volume, total solids and platelet count. Cats with evidence of other systemic disease or those receiving anticoagulants were excluded from the study. Normally distributed and skewed data were compared between the cardiomyopathy and control groups with independent t tests or Mann Whitney U tests, respectively. Statistical significance was set at P o 0.01.

Thirty cats with cardiomyopathy (23 neutered males and 7 neutered females) and 27 healthy controls (13 neutered males and 14 neutered females) were enrolled. Median age was 8 yr (range, 2-16 yr) in the cardiomyopathy group and 5 yr (range, 1-10 yr) in the control group (P 5 .009). Cats in the cardiomyopathy group were classified in the International Small Animal Cardiac Health Council stage 1b (n 5 10), 2 (n 5 8), 3a (n 5 3) and 3b (n 5 9). Compared to control cats, cardiomyopathic cats had higher concentrations of palmitic acid (P 5 .002) and DHA (P o .001), and lower concentrations of linoleic acid (P 5 .005). Among cats with cardiomyopathy, there was no significant correlation between any serum fatty acid concentration and left atrial size or age. These findings warrant further investigation into the role of fatty acids in cats with cardiac disease. Platelet mapping is an application of thromboelastography that relies on the generation of at least three tracings: MA THROMBIN (maximum platelet activity),MA FIBRIN (fibrin activity only), an-dMA AA or ADP (platelet activity not inhibited by arachidonic acid or ADP receptor antagonists, respectively). Using these three tracings, the % inhibition of platelets can be calculated. The purpose of this study was to evaluate the Platelet Mapping assay in normal cats and assess platelet inhibition in cats receiving clopidogrel.

Employee-owned cats with normal history, physical exam, echocardiogram, thromboelastography, packed cell volume, total solids and platelet count were eligible. Clopidogrel (18.75 mg PO Q24h) was administered for 10 days with Platelet Mapping performed on days 0 and 10. Platelet Mapping values were compared using a paired t test, with significance set at P o 0.05.

Seven cats (4 FS, 3 CM, aged 2-10 years) were enrolled. Compared to day 0, MA ADP (P o .001) and MA FIBRIN (P o .001) were lower on day 10. The latter unexpected result prompted measurement of fibrinogen concentrations at day 0 and 10 in the last 5 of these 7 cats. Fibrinogen was not different from day 0 to 10 in these 5 cats.

These results suggest that Platelet Mapping may be a simple, outpatient clinical tool to measure antiplatelet activity in cats receiving clopidogrel. This clopidogrel dose resulted in significant platelet inhibition as measured by MA ADP in all cats. Further studies correlating antiplatelet effects measured by Platelet Mapping with clinical outcomes in cats with cardiomyopathy are warranted. This study investigated the hemodynamic effects of application of an ITD in a canine model of cardiopulmonary arrest. 8 laboratory beagles which were part of a separate terminal study were anesthetized and instrumented for continuous measurement and recording of right atrial pressure, arterial pressure and carotid blood flow. Following euthanasia, CPR was performed for one minute, then a pause of one minute followed by a second one minute period at a compression rate of 100-120/minute, ventilation with 100% oxygen was delivered at eight breaths/min and 15 ml/kg tidal volume. CPR was performed with the ITD in place (ITD-CPR) and without the ITD (S-CPR) for one period each in a randomized fashion with the rescuer blinded to its application. Baseline, S-CPR and ITD-CPR data were assessed for normality, a Kruskal-Wallis one-way ANOVA on ranks was used (baseline v CPR). When appropriate a pairwise multiple comparison procedure (Dunn's method) was used. Percentage of baseline S-CPR v ITD-CPR was assessed using the Student t-test.

The right atrial diastolic pressure was significantly more negative with the ITD attached than without (p 5 0.02), the coronary perfusion pressure and carotid blood flow were significantly higher during CPR with the ITD than standard CPR (p 5 0.015, p 5 0.017). No significant differences in diastolic, mean or systolic arterial pressure or end tidal CO 2 were seen.

Application of the ITD resulted in significantly improved hemodynamics during CPR in dogs. Clinical evaluation of the device is warranted to examine whether this translates into improved success in resuscitation and survival. Left ventricular (LV) systolic dysfunction is a common problem in dogs and can be due to a variety of etiologies. One potential etiology for systolic dysfunction is persistent or paroxysmal tachyarrhythmias, leading to tachycardia-induced cardiomyopathy (TICM). In humans, TICM carries a relatively good prognosis in that remodeling may be reversible with normalization of heart rate. Differentiating between primary and secondary tachyarrhythmias in dogs with systolic dysfunction is critical for prognostic purposes as primary tachyarrhythmias may be associated with a better outcome. The goal of our study was to describe a population of dogs with TICM and to determine if treatment of arrhythmias was associated with reversible cardiac remodeling as indicated by standard echocardiographic parameters.

Medical records of dogs referred to the cardiology service of KSU VMTH from 2008 to 2010 were reviewed. TICM was defined as the presence of severe tachyarrhythmias that were reversible with treatment, systolic dysfunction or ventricular enlargement that improved with treatment of the arrhythmia, or dogs with severe tachyarrhythmias and systolic dysfunction of breeds with that are atypical for idiopathic dilated cardiomyopathy. Exclusion criteria were dogs with congenital heart disease, severe mitral regurgitation, and endocarditis. Transthoracic echocardiography, thoracic radiographs and electrocardiography (ECG) were performed in all dogs. Ventricular enlargement and systolic dysfunction were defined according to standard echocardiographic parameters. Arrhythmias were confirmed with a Holter monitor in 7 dogs.

A total of 12 dogs were included in the study. Mean age was 7 years (range 2-12 years) with 8 males (4 intact, 4 castrated) and 4 females (3 spayed, 1 intact). Four dogs had pulmonary venous congestion or pulmonary edema and two dogs had ascites. At initial presentation, the meanAESD values were as follows: heart rate 219AE75 bpm, M-mode fractional shortening (FS) 17.29AE9.05%, ejection fraction (EF) using the area-length method 30.69AE13.45%, and left atrial to aortic root ratio (LA/Ao) 1.92AE0.37. Initial meanAESD M-mode derived LV internal dimensions corrected for body weight were as follows: diastolic 1.92AE0.37 and systolic 1.49AE0.38. At least one of the following tachyarrhythmias were identified in each dog: atrial fibrillation (4), supraventricular tachycardia (5), junctional tachycardia (2), and ventricular arrhythmias (3). Ten dogs were available for follow up. Seven dogs improved in at least one of the following parameters: resolution of tachyarrhythmia (3), improved systolic function (3) Antidiuretic hormone (ADH) has been shown to be elevated in humans with congestive heart failure (CHF). Recently, antidiuretic hormone antagonists were successful during investigational treatment of refractory congestive heart failure in humans. ADH levels have been only modestly investigated in dogs with cardiac disease, primarily due to the technical difficulty in measuring ADH levels via radioimmunoassay. Based on the homologous structure of canine and human ADH, we aimed first to determine the feasibility of measuring ADH in dog plasma using a human ELISA kit, and secondly to investigate the level of ADH in dogs with congestive heart failure due to acquired cardiac disease.

ELISA assay kit validation was performed using six healthy dogs with normal clinical and echocardiographic examinations. Pooled canine plasma was spiked with synthetic ADH and intra-assay precision, dilutional parallelism, and linearity were assessed. To address the second aim of the study, samples were collected from normal dogs and dogs with heart failure due to one of two types of acquired cardiac disease: chronic degenerative valve disease (CDVD) or dilated cardiomyopathy (DCM). Patients underwent clinical, radiographic, and echocardiographic examination to confirm diagnosis, assess severity of disease, and determine presence of pulmonary edema. Whole blood was collected into EDTA tubes containing protease inhibitors, cold centrifuged, and plasma was stored at À801 until analysis. Following ether extraction, plasma ADH in each sample was measured in duplicate using a human ELISA kit. Statistical analysis included a D-Agostino and Pearson test for normality; group results were compared using a nonparametric Mann-Whitney test.

ADH was measured in canine plasma using the human ELISA kit with acceptable intra-assay precision, linearity, and dilutional parallelism. Intra-assay coefficient of variation was 11%. Twenty-four dogs were recruited for the second phase of the study. Six normal dogs and twelve dogs with radiographic evidence of pulmonary edema due to either CDVD or DCM were selected for participation. The remaining six dogs were excluded due to lack of definitive radiographic evidence of congestive heart failure. Median ADH values were 3.67 AE .38 pg/mL for the normal group (n 5 6) and 6.155 AE .94 pg/mL for the heart failure group (n 5 12). Median ADH values for the two groups were statistically different (p 5 0.0057).

Our preliminary results indicate that measuring canine ADH using a human ELISA kit is feasible and provides results with an acceptable coefficient of variation. We also showed that dogs with congestive heart failure due to CDVD and DCM have elevated ADH levels in comparison to normal dogs. Our findings motivate further investigation to assess the degree of plasma ADH level elevation and the possible use of ADH antagonists as an adjunct treatment for refractory congestive heart failure in dogs. Aortic thromboembolism (ATE) occurs in both cats and dogs. Whereas ATE in cats is strongly associated with structural heart disease and typically has an acute catastrophic presentation; the pathogenesis and presentation of ATE in dogs is less well known or understood. Further, an effective antithrombotic strategy for ATE in dogs has not been reported.

Medical records of dogs diagnosed with ATE between 2003 and 2010 were examined retrospectively. Diagnosis of ATE was based on ultrasonography, Doppler flow studies, and diminished or absent femoral pulses. Dogs were treated with various acute and chronic antithrombotic therapies. The severity of ambulatory dysfunction was graded as none, mild, moderate, severe, or non-ambulatory at presentation and after therapy. A cohort of dogs in this study received a standardized protocol of chronic warfarin therapy with or without antiplatelet drugs. Target international normalized ratio for warfarin therapy was 2 to 3.

Twenty-six dogs were diagnosed with ATE. All had an apparent mural aortic thrombus caudal to the renal arteries with most having evidence of embolization to the iliac and femoral arteries. None had structural heart disease at diagnosis. Twenty dogs (77%) were still ambulatory at diagnosis. The median duration of ambulatory dysfunction prior to presentation was 7.8 weeks (range 1 day -52 weeks). A majority of dogs (58%) had no concurrent conditions at diagnosis. Nine dogs (33%) had protein-losing nephropathy. Four dogs (15%) were hypothyroid.

Fourteen dogs were treated with a standard warfarin protocol for a median period of 22.9 months (range 0.5-53 months). Eight dogs were treated concurrently with aspirin, 2 dogs were treated concurrently with clopidogrel, and 4 dogs were treated with warfarin only. Ambulatory function improved between 1 and 3 grades in dogs treated with chronic warfarin. The median period until clinical improvement was 13.9 days (range 2-49 days). Two dogs treated with chronic warfarin therapy had documented resolution of ATE, and 5 dogs had complete resolution of ambulatory dysfunction. None of the 14 dogs treated with chronic warfarin became nonambulatory, died, or underwent euthanasia because of ATE. The median period of freedom from an adverse event was 24.2 months. No serious hemorrhagic events were reported.

Four dogs were treated with tPA. Three of these had an unfavorable outcome. Two dogs were ambulatory before tPA and become non-ambulatory after treatment. Two dogs underwent surgical thrombectomy. One had a favorable outcome until ATE recurred 14 months after surgery.

In conclusion, the pathogenesis of ATE in dogs is not associated with structural heart disease or left atrial thrombus formation. The presentation tends to be for chronic ambulatory dysfunction. Most dogs are still ambulatory at presentation. Warfarin, with or without concurrent antiplatelet therapy, is an effective antithrombotic treatment strategy for dogs with ATE.

information is known. Through previous work, investigators have encountered Norfolk Terriers (NT) with echocardiographically apparent DMVD in the absence of a heart murmur. In order to more fully understand DMVD in this breed of dog, we sought to characterize findings from the physical and echocardiographic exam, biochemical, biomarker, and nutritional profile, and select environmental variables from a cohort of apparently healthy NT.

Overtly healthy NT ! 6yrs old were recruited by 3 different veterinary hospitals and underwent historical, physical, ECG, and 2D/color-flow Doppler echocardiographic exam. Anterior mitral valve length, maximal thickness, area, and prolapse were measured from 2D images. Presence of DMVD was defined as thickened, prolapsing, or flail mitral valve leaflets in the presence of color flow Doppler evidence of mitral regurgitation. Blood samples were obtained for serum biochemistry and serotonin, plasma NT-proBNP, amino acid profile, C-reactive protein, and cardiac troponin-I.

Forty-eight dogs were entered into the study (median age, 8yrs IQR [7-10]; gender, 29F, 19M; median BCS, ). Of the 48 dogs, 23 (48%) had murmurs, 2 (4%) had mid-systolic clicks, 11 (23%) had ECG p-pulmonale, and 41 (85%) were deemed to have echocardiographic evidence of DMVD, including 18 NT without a murmur. Seven (15%), 28 (58%), and 13 (27%) dogs were classified as ISACHC 0, 1a, and 1b, respectively. Mean indexed echocardiographic mitral leaflet length (P o 0.0001), thickness (P 5 0.019), prolapse (P 5 0.0005), and LA:AoD (P 5 0.01) were significantly different between ISACHC 1a/b vs 0. Between ISACHC 1a/1b and 0, there were no differences in serum amino acids, C-reactive protein, troponin, diet, or environmental factors; however 6 different amino acids (Ala, Gly, Phe, Pro, Trp, Tyr) were significantly higher in ISACHC 1b vs. 1a. Median serum serotonin was increased in dogs with 1a/b vs. 0 (P 5 0.025). Dogs whose diets contained some canned food (P 5 0.12) and dogs residing in suburban environments (P 5 0.03) had higher serotonin concentrations. NT-proBNP tended (P 5 0.07) to be higher in ISACHC 1a/1b vs. 0. DMVD appears to be relatively common in NT and echocardiographic changes consistent with mild DMVD can be seen in dogs without a heart murmur. The results of this study establish a foundation of useful information upon which additional prospective studies can be developed. Left ventricle (LV) evaluation is one of the most important contributions of echocardiography in the assessment of cardiac function. However, LV analysis can be made from images obtained by different modes and views of the heart. The aim of this study was to compare LV measurements, shortening fraction (SF) and ejection fraction (EF) obtained from four methods: M mode in short-axis and in long-axis, bidimensional mode in short-axis and in long-axis views of the heart.

Forty normal adult German Shepherds were selected. Echocardiographic study of LV of each animal was performed by the four methods described above. ANCOVA test was used to examine the effects of axis, mode, weight and gender over LV measurements.

Isolated effect of the axis was observed for LV end-diastolic diameter (LVEDD), with greater values obtained from short-axis views. There was isolated effect of mode over EF and SF, with greater measurements derived from bidimensional mode methods. Weight correlated with all linear LV measures at least in one method, but not with EF and SF. Weight had positive effect over LV endsystolic diameter and LV end-diastole posterior wall thickness in all methods, except from M mode in short axis in the last one. Gender had isolated effect over LVEDD, males showing greater values than females in bidimensional mode in short and long axis. The combined effect of axis, gender and weight was identified in interventricular septal end-diastolic thickness.

We concluded that normal reference values obtained by different echocardiographic modes and planes should not be used interchangeably.

ABSTRACT C-29  ASSESSMENT OF LEFT VENTRICULAR DIASTOLIC FUNC-TION BY COLOR TISSUE DOPPLER IMAGING ECHO-CARDIOGRAPHY IN MAINE COON CATS TESTED FOR  MYPBC-AP31 MUTATION Hypertrophic cardiomyopathy (HCM), characterized by increased cardiac mass and diastolic dysfunction, is the most common feline heart disease. Myocardial analysis by color tissue Doppler imaging (TDI) is more sensitive than conventional echocardiography. This study evaluated diastolic dysfunction in various stages of feline HCM.

Maine Coon cats (n 5 57) were screened for the MyBPC-A31P mutation and examined with both echocardiography and TDI. Then, were phenotypically classified in: normal (n 5 45), suspects for HCM (n 5 7) and HCM group (n 5 5); and genotypically classified in: negative (n 5 28), heterozygous (n 5 26) and homozygous group (n 5 3). Myocardial velocities, measured in the basal and mild ventricular segment of the interventricular septal wall (IVS), left ventricular free wall (LVW) and in radial segment of left ventricular wall, was compared among different groups.

A significant decreased (P 5 0,01) longitudinal Em/Am at the basal segment of IVS was observed in HCM cats compared with suspects and normal cats. A significant increased (P 5 0,01) longitudinal E/Em at the basal segment of IVS was observed in HCM cats compared with suspects and normal cats. And a significant decreased (P 5 0,02) longitudinal Sm at the basal segment of the LVW was observed in heterozygous cats compared with negative cats, both without hypertrophy. There was a significant positive correlation between summated early and late diastolic velocities (EmAm) and heart rate (P o 0,001); and a positive correlation between Sm and Em velocities and heart rate (P o 0,01).

The MYBPC-A31P mutation is not consistently associated with ventricular hypertrophy and negatives cats can also develop HCM. The TDI alone is not able to identify cats with mutation before myocardial hypertrophy.

Diastolic dysfunction occurs in many cats with hypertrophic cardiomyopathy (HCM) but less is known about systolic function in various stages of HCM. Myocardial strain analysis by tissue Doppler imaging is a noninvasive echocardiographic method to assess systolic function. This study evaluated systolic function in various stages of feline HCM.

Maine Coon cats (n 5 57) were screened for the MyBPC-A31P mutation an examined with both echocardiography and strain. Then, were phenotypically classified in: normal (n 5 45), suspects for HCM (n 5 7) and HCM group (n 5 5); and genotypically classified in: negative (n 5 28), heterozygous (n 5 26) and homozygous group (n 5 3). Peak myocardial strain, measured in the basal and mildventricular segment of the interventricular septal wall (IVS), left ventricular free wall (LVW), left ventricular anterior wall (LVAW), left ventricular posterior wall (LVPW) and radial segment of left ventricular wall, was compared among different groups.

Whereas conventional echocardiography demonstrated an apparently normal contractile state based on fractional shortening, myocardial strain (at mildventricular segment of IVS) in HCM cats was significantly decreased compared with normal group (P 5 0,01). Myocardial strain (at basal segment of LVAW) also was significantly decreased in heterozygous cats compared with negative group (P 5 0,00); and was significantly decreased in heterozygous cats compared with negative group, both without ventricular hypertrophy (P 5 0,019). There was a significant negative correlation between strain values and wall thickness (P o 0,05).

This method allows detection of abnormal systolic deformation in Maine Coons cats with HCM mutation despite apparently normal systolic function. The abnormal systolic deformation already can be present in heterozygous cats without hypertrophy and increased with progressive ventricular hypertrophy. Recently, multiple advanced resting electrocardiographic (ECG) techniques have been applied in humans for detection of cardiac autonomic and repolarisation function. This has improved the diagnostic and/or prognostic value of short-time ECG in detection of common human cardiac diseases even before onset of symptoms or changes in the standard ECG. Therefore, this study investigates, if advanced ECG can predict the severity of mitral regurgitation (MR) in dogs with myxomatous mitral valve disease (MMVD) and thereby improve the diagnostic value of ECG.

The study included 77 privately owned Cavalier King Charles Spaniels (CKCSs) (age 6.0 AE 2.7 years; 30 males and 47 females). All dogs were examined by echocardiography and a short-time (3-5 min) high-fidelity 12-lead ECG, with the dog in a resting position and in sinus rhythm. Dogs were divided into 5 groups according to the degree of MR estimated as the percentage of the left atrium area using color Doppler mapping (0%; 0% o jet 15%; 15% o jet 50%; jet 4 50%; jet 5 100% and with clinical signs of congestive heart failure). ECG recordings were evaluated via custom software programs to calculate 76 different parameters, including heart rate variability (HRV), QT variability (QTV), T-wave complexity, wave morphology and 3-D ECG. One-way ANOVA determined 21 ECG parameters, which were significantly different (P o 0.05) between the 5 dog groups. Principal component factor analysis identified a 5factor model with 83.2% explained variability. QRS dipolar voltage and two repolarization indices of QTV increased significantly with MR severity, whereas total power of the frequency spectrum of RR interval and the standard deviation of QTV decreased significantly with MR severity. For the 5 selected parameters the prediction of MR jet value was tested by multiple linear regression. A correlation coefficient (R) of 0.65 indicated that the prediction value was significant (P o 0.01). If age was included in the multiple linear regression the prediction value was further increased (R 5 0.80).

Our results indicate that for a cut-off criteria of MR ! 50% jet the five selected ECG parameters could predict the severity of MR caused by MMVD in CKCSs with sinus rhythm with sensitivity 65% (78% with age inclusion) and specificity 98% (92% with age inclusion) (P o 0.05). NT-proBNP concentration is increased in canine patients with heart disease. Relatively little is known about the stimuli for release of NT-proBNP in dogs. Physical activity independent of cardiac disease and the stress of being in the hospital could influence NT-proBNP release and affect diagnosis and management of patients. We hypothesized that NT-proBNP concentration in healthy dogs would not exceed the normal reference value (900 pmol/L) following a period of exercise. The goal of this study was to examine whether physical activity could elevate plasma NT-proBNP and cause false positive results in healthy dogs.

The study population included healthy dogs 41 yr of age without heart murmur or known systemic disease, and normal 2D/color flow echocardiographic exam. Plasma samples for NT-proBNP were obtained before, immediately after, and 1 hour after a standardized 5-minute submaximal exercise regimen. The study included 14 dogs with a median age of 5.3yrs and included 6 females and 8 males. There was no statistical difference in median plasma NT-proBNP concentration across the three time points (baseline median, 617 [IQR, immediately post, ; P 5 0.11). The average coefficient of variation of NT-proBNP concentration across the exercise regimen was 23.0 AE 18.9%. In 1 of 14 dogs (7.1%), NT-proBNP increased from 790 to 1054 pmol/L immediately after exercise. The results of this study demonstrate that submaximal exercise does not significantly change median NT-proBNP concentration and the incidence of false positive results is low. Further studies should investigate effects of exercise on NT-proBNP concentrations in dogs with heart disease. Obesity is an increasing problem in veterinary medicine. Obese human patients are shown to present lower levels of natriuretic peptides, regardless of an increased volume and pressure load, what raises the possibility that the natriuretic response is impaired in these individuals. Considering the controversial findings in obese humans, and the lack of studies reported in dogs, this study proposed the evaluation of Nt-proANP concentration in obese dogs.

Nt-proANP concentration was determined prospectively in 39 obese dogs (27 females; 12 males; 6-108 months) and in 23 non-obese dogs (controls; 13 females; 10 males; 12-108 months) from a veterinary hospital population. Obesity was determined by body condition score [2 (4/9); 21 (5/9); 1 (6/9); 3 (7/9); 19 (8/9); 18 (9/9)].

Dogs were excluded if they had any primary cardiac disease, renal insufficiency, endocrine disease, or if they were receiving diuretics, vasodilators, antiepileptic drugs or corticosteroids. Commercial kits were used (Vetsign s Canine Cardio Screen Nt-pro-ANP VC3167 -Guildhay/Biomedica). Mann Whitney test was used for group comparison. Results are presented as median; interval; P25 and P75).

Nt-proANP was significantly lower in obese dogs [413.56fmol/mL (0.0-1287.14); P25 5 228.673; P75 5 595.205] than in controls [647.98fmol/mL (34.99-1596.82 ); P25 5 490.785; P75 5 957.055]; (P 5 0.004).

Results were similar to what has been found in obese humans. Lower levels of natriuretic peptides are also seen in obese heart failure patients.

This study provides important information regarding Nt-proANP concentration in obese dogs, which should be better explored characterizing the behavior of natriuretic peptides after weight loss, and also in obese dogs with primary heart disease. Left-to-right shunting patent ductus arteriosus (PDA) is one of the most common canine congenital cardiovascular defects. Human studies have shown that BNP and NT-proBNP concentrations are elevated in patients with PDA, and can be used to detect hemodynamically significant PDA. The purpose of this study was to measure NT-proBNP concentrations in dogs with PDA, and to assess whether additional indicators of hemodynamics correlate with NTproBNP. We hypothesized that NT-proBNP will serve as a simple non-invasive marker of hemodynamic significance in dogs with PDA prior to and following transcatheter ductal occlusion. NT-proBNP was measured in 30 client-owned dogs with echocardiographically normal hearts. Ten dogs with PDA were initially evaluated with thoracic radiographs, transthoracic and transesophageal echocardiography, pulmonary capillary wedge pressure (PCWP) and NT-proBNP. NT-proBNP and echocardiography were repeated at 1 day and 3 months following ductal occlusion. PCWP was repeated at 3 months.

Baseline NT-proBNP was significantly higher in PDA dogs compared to control (1877 AE 2081 pmol/L (mean AE SD), 651 AE 301; p o 0.0025). At 1 day and 3 months following ductal occlusion, NT-proBNP was 1347 AE 1256 and 466 AE 380, respectively. The following decreased significantly from baseline: PCWP (11.8 AE 4.7 to 6.4 AE 3.2 mmHg; p 5 0.018), and indexed left ventricular internal dimensions in diastole (2.26 AE 0.47 to 1.67 AE 0.19; p 5 0.006) but not significantly in systole (1.40 AE 0.31 to 1.20 AE 0.19; p 5 0.13). NT-proBNP is elevated in dogs with PDA and transductal closure is associated with a reduction in NT-proBNP, PCWP and left ventricular size. Cardiac biomarkers, particularly NT-proBNP, are becoming more commonly used in dogs and cats as part of a diagnostic work up. Multiple studies already have documented the correlation of this peptide with cardiac disease status and potential clinical implications. In a portion of these reports the manner in which samples were handled was placement of whole blood into an EDTA tube, followed by centrifugation and decanting of the supernatant that was ultimately stored at À201C or À801C prior to shipment, either with or without protease inhibition.

Our objective was to compare the NT-proBNP concentrations in feline plasma collected using the previously reported methods to the California Animal Hospital (CAH) collection method using tubes containing a protease inhibitor. This study compared NT-proBNP concentrations using the protease inhibitor tubes vs. EDTA tubes from 18 privately owned feline patients, with confirmed cardiac disease, and 6 control feline patients. For all study participants, we performed a full history and physical examination, a hematology and chemistry panel, thoracic radiographs, ECG, and echocardiogram.

In each study participant, at least 4 ml's of whole blood was drawn from a peripheral vein, and transferred to a plastic EDTA tube. The sample was centrifuged within 1 hour after collection. 1 ml of plasma was then transferred to a tube containing a protease inhibitor, which was stored at 41C until being shipped within 24 hours of collection. The remaining plasma was placed into 2 separate microtubes, which did not contain a protease inhibitor. One microtube was then stored and shipped as previous studies have reported (À201C, Styrofoam container, shipment within 24 hours), and the second microtube was frozen at À801C. All samples were shipped, received and analyzed within 24 hours of collection.

Results of this study showed that no difference was found between the 2 frozen sample methods (663 pmol/L and 650 pmol/L p 5 0.40). It was determined that both frozen methods had lower NT-proBNP levels (655 and 646 pmol/L) when compared to plasma samples shipped in protease inhibitor tubes (756 and 794 pmol/L). The findings of this trial demonstrate that the NT-proBNP levels are significantly different between samples placed in EDTA tubes vs. contain protease inhibitor (p 5 0.008 and p 5 0.0006). Utilizing protease inhibitor tubes allows more accurate measurement of plasma NT-proBNP. As for its relevance for future research and publications, authors should take care to investigate the manner in which blood samples were handled and the conclusion/results of these studies should be taken in light of the methodologies used in collecting, storing, shipping and analyzing the samples.

Degenerative mitral valve disease (DMVD) is one of the most common heart disease and is present approximately 75% of the canine heart disease. Although the high prevalence exists in small dogs, the underlying molecular mechanism of its pathophysiology is rarely known. DMVD is functionally and pathologically similar in humans and dogs, thus, there will be a common pathogenesis in human and dogs with naturally occurring DMVD. Serotonin and serotonin-related mechanisms have been implicated as a cause of valvular disease in human and animals, including spontaneous DMVD in dogs. Increased circulating 5HT concentration as a potential source of heightened 5HT signaling is demonstrated in small dogs with DMVD. The aim of this study was to investigate whether serum 5HT concentrations were associated with severity of naturally occurring DMVD in small dogs and to investigate potential associations of dog characteristics on serum 5HT concentrations in our study population. Forty-eight dogs were included in this study and were classified into control and DMVD groups according to the results of physical and echocardiographic examinations. Based on the LA:Ao ratio, dogs with DMVD were classified as follow: Control (LA:Ao ratio 1.5 and no MR), mild (LA:Ao ratio 1.5 and MR), moderate (1.5 o LA:Ao ratio 1.8 and MR), Severe (LA:Ao ratio 41.8 and MR).

Serum serotonin concentrations were measured by ELISA. An overall significant difference (P o .05) was found among 4 groups and 5HT concentrations (control, 72.38 ng/ml [51.34-95.11 DMVD, ). Significantly higher 5HT concentrations were observed in dogs with moderate (P o .05) and severe (P o .05) DMVD, compared with concentration in control group. Additionally, 5HT concentration in dogs with moderate DMVD were significantly higher (P o .05) than concentration in dogs with mild DMVD. Also, dogs with severe DMVD had significantly higher 5HT concentration than dogs with mild (P o .05) and moderate (P o .05) DMVD. There was no significant association of age, platelet, and LVIDd, on serum 5HT concentration, however, weak correlation between serum 5HT increased significantly and LA:Ao ratio (R 2 5 .211, P o .05) was observed. The results of this study indicate that serum 5HT concentrations were higher with increasing severity of spontaneous DMVD, which may be the potential cause to advance the progression of DMVD. Further studies should be performed to reveal the role of 5HT in inducing and accelerating spontaneous DMVD and to investigate if lowering serum 5HT concentration could alter the progression of DMVD. The objective of this prospective study was to evaluate the utility of cardiac troponin I (cTnI) in differentiating between underlying etiologies of pericardial effusion in the canine patient. Patients were prospectively recruited at time of diagnosis of novel pericardial effusion. Serum samples were collected prior to pericardiocentesis. Patients were evaluated by echocardiography and classified with the diagnosis of hemangiosarcoma (HSA), heart base tumor (HBT), or unknown etiology at the initial evaluation based on established characteristic echocardiographic findings. Idiopathic pericardial effusion (IPE) was defined by histopathology, echonegative for a mass lesion with no recurrence of pericardial effusion 46 months, or symptom free 412 months from time of enrollment. Patients were excluded from analysis if a diagnosis could not be established based on above criteria or concurrent moderate azotemia (Creatinine 43.0 mg/dL) was present at time of sample collection. Serum samples were frozen and analyzed in batches within 60 days of collection by a cTnI assay with a 0.2ng/mL lower limit sensitivity.

Sixty-three patients were recruited over a one year period with 15 patients excluded due to lack of diagnosis (13) or azotemia (2). Median cTnI levels of dogs with HSA (n 5 24), HBT (n 5 19), and IPE (n 5 5) were 14.8 ng/dL (interquartile range (IQR) o 0.2-11.3), 0.23 ng/dL (IQR o 0.2-0.29), and o 0.2 ng/dL (IQR o 0.2-o 0.2) respectively. Concentrations of cTnI differed significantly between dogs with HSA and HBT (p 5 0.001) and IPE (p 5 0.0029). There was no difference between cTnI concentrations between HBT and IPE dogs (p 5 0.911). Receiver operating curve analysis to determine the optimal cutoff for differentiation of dogs affected with HSA and both HBT and IPE revealed a significant (p 5 o 0.001) area under the curve (0.79). A cut-off point of cTnI of 40.78 yielded a sensitivity of 67% (95% CI, 45-84%) and specificity of 95% (95% CI, 79-99%). Utilizing a higher cut-off point of 43.0 yielded a lower sensitivity of 50% (95% CI, 29-71%), but a higher specificity of 100% (95% CI, 86-100%) which may have more clinical utility given the disparity in prognoses of the etiologies compared.

In conclusion, this study supports the diagnostic utility of cTnI concentrations to delineate between patients with HSA and other etiologies of pericardial effusion, but does not reliably differentiate between dogs with IPE and other neoplastic etiologies. The pathogenesis of degenerative mitral valve disease (DMVD) in dogs remains to be fully elucidated. The high sheer stress caused by mitral regurgitation damages the endothelial surface of the valve, and a previous study demonstrated increased transcription of intercellular adhesion molecule-1 (ICAM-1) and E-selectin in affected mitral leaflet tissue. We hypothesized that this may be responsible for platelet recruitment and adhesion, and initiation of a proliferative cascade, resulting in further myxomatous changes. The goal of this study was to compare plasma levels of ICAM-1 and E-selectin in healthy dogs and those with DMVD.

The study population included dogs with echocardiographic evidence of DMVD and healthy control dogs 41 year old with no heart murmur or known systemic diseases. DMVD dogs underwent 2D/color-flow Doppler echocardiographic exam. Blood samples were obtained for plasma ICAM-1 and E-selectin analysis using commercially available ELISA kits.

The study included 34 dogs, of which 20 had DMVD and 14 were normal. The DMVD group had a median age of 9.5yrs ) and included 6 females and 14 males. Two (10%), 13 (65%), 2 (10%) and 3 (15%) dogs were classified as ISACHC 1a, 1b, 2 and 3a, respectively. Of the control dogs, median age was 4.5yrs [2-6.5], with 5 females and 9 males. There was no statistical difference in plasma E-selectin between control dogs (median 2.71 [2.05-7.66]) and those with DMVD (2.46 [1.54-3.55]); P 5 0.35. Plasma ICAM-1 concentrations were higher in DMVD dogs (1.58 [1.20-10.58 ]) than controls (median 1.31 [1.11-1.65], but this difference did not reach statistical significance (P 5 0.22). Linear regression analysis showed no significant correlation between ICAM-1 or E-selectin and serum serotonin level, NT-proBNP or echocardiographic measures of DMVD severity (LA:Ao, LVIDd:Ao, LVIDs:Ao). The results of this study demonstrate no significant difference in circulating adhesion molecules ICAM-1 and E-selectin in dogs with DMVD as compared with healthy controls. Further studies investigating adhesion molecules within the mitral valve tissue itself are likely needed if ICAM-1 and E-selectin play a role in the pathophysiology of DMVD.

The rate of glucose utilization in the heart is greater than in other tissues, and impaired glucose uptake may play a major role in the pathogenesis of heart failure (HF). Glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (GLUTs), which includes GLUT-4, the major cardiac isoform, and GLUT-12, a recently discovered isoform, the role of which is unknown in the heart. In addition, despite the wellknown regional differences in myocardial structure and function, potential regional patterns in glucose transport have not been investigated. Thus, we hypothesized that GLUT-4 and -12 protein and gene expression would be chamber specific in healthy dogs and during chronic HF. Using a canine model of tachypacing induced chronic HF, GLUT protein and messenger RNA in both ventricles and atria were investigated by immunoblotting and real time PCR.

In control dogs, GLUT-4, but not GLUT-12, protein expression were significantly higher in the atria compared to the ventricles, with the highest content in the right atrium (RA, P o 0.001). GLUT-4 and -12 mRNA were homogeneously expressed in all the cardiac chambers. During chronic HF, GLUT -4 and -12 expression was highest in the left ventricle (LV, by 2.5 and 4.2 fold, respectively, P o 0.01), with a concomitant increase in GLUT-4 and -12 mRNA (P o 0.001). GLUT -4, but not GLUT-12, was decreased in RA during chronic HF (P 5 0.001).

Our data suggest that GLUT-4 protein was differentially expressed across the cardiac chambers in the healthy heart. During chronic HF, LV was the primary site dependent on both GLUT4and GLUT12-mediated glucose transport, which was transcriptionally regulated. In addition, the paradoxical decrease in GLUT4 content in RA may induce perturbations in atrial energy production during chronic HF. Some obese dogs are suspected to have cardiac disease because they have enlargement of the heart on thoracic radiograph. It has been reported in cats that the fat increases the cardiac silhouette, while echocardiograms revealed normal cardiac dimensions. The purpose of this study was to determine whether obesity overestimates cardiac dimension in radiographs compared to echocardiographic findings in dogs.

Twenty three obese dogs and 20 controls were included based on a 1-9 body condition scoring (BCS). Computerized radiography was obtained and VHS measurement was performed as previously described. Echocardiographic measurements were interpreted based on reference values according to lean body weight regression equations. Results for echo and VHS measurements were classified in scores as normal, mild, moderate or severe increase. Student's t test was used for comparison of VHS between groups. Mann-Whitney Rank Sum test was used to assess echocardiographic scores between groups. Spearman Rank Order Correlation was used to assess relationships between any pairs of variables between echo and VHS scores, echo vs BCS and VHS vs BCS.

Groups were similar regarding age [obese (69AE24); control (62AE27); P 5 0.368], breeds and gender distribution. Obese dogs had significantly higher VHS and echo scores compared with controls [VHS: (10.58AE0.69) vs (9.77AE0.54); P o 0.001; Echo score: range (1-4) vs (1-2); P 4 0.05]. There were no relationships between any pair of variables analyzed.

These results show that there are changes both in echo and radiographic appearance of the heart in obese dogs, but VHS overestimates cardiac silhouette compared to echo, probably related to pericardial fat accumulation. Heart rate variability (HRV) is an indirect measurement of the autonomic modulation of heart rate (HR). Reduced HRV measured from short-time electrocardiography is seen in dogs with heart failure (HF) secondary to myxomatous mitral valve disease (MMVD). However, HRV is suggested to increase with disease severity at early stages of MMVD. The aims of this study were 1) to associate HR and HRV with severity of MMVD in Cavalier King Charles Spaniels (CKCS) and 2) to compare HR and HRV between CKCS and other dog breeds in a group of dogs in HF secondary to MMVD.

One-hundred dogs were examined by echocardiography and 24hour electrocardiography. The dogs were divided into five groups: 1) CKCS with no/minimal mitral regurgitation (MR) (MR jet 15% of the left atrial area using color Doppler mapping) and no murmur, 2) CKCS with mild MR (20% o jet 50%), 3) CKCS with moderate/ severe MR (jet450%) and no clinical signs of HF, 4) CKCS in HF (HF defined as left atrium to aortic root ratio (LA/Ao) 41.5, clinical signs of HF and furosemide responsiveness) and 5) non-CKCS in HF. Dogs in HF were allowed HF therapy. Both HR and HRV were analyzed over a 24-hour period, while HRV were also analysed over a 6-hour nightly period. Analyses of variance were performed with HR or HRV as response variables and the explanatory variables dog group and echocardiographic indices of MMVD were included separately. All P-values were Bonferroni corrected.

Minimum-and mean HR were significantly higher in CKCS with moderate/severe MR and in HF compared to CKCS with no/ minimal and mild MR (all P o 0.001). Seven out of 26 HRV variables were significantly decreased in CKCS with moderate/ severe MR and in HF compared to CKCS with no/minimal and mild MR (all P o 0.02). Another 10 HRV variables showed the same groupwise differences (all P o 0.02), except that the difference between CKCS with mild MR and CKCS with moderate/severe MR did not reach statistical significance. Mminimum HR, mean HR and the HRV variables (7 and 10) differing between dog groups, also consistently decreased with increasing MR, LA/Ao and the proximal isovelocity surface area in CKCS. Non-CKCS in HF had a lower minimum HR compared to CKCS in HF (P 5 0.03) and a higher triangular index measured in both periods (all P o 0.04).

In conclusion, HR increased and most HRV variables decreased with increasing severity of MMVD in CKCS, even prior to the development of HF. Other breeds in HF secondary to MMVD had lower minimum HR, but higher triangular index compared to CKCS in HF. Although the cells in the specialized conduction system in the heart are capable of initiating their own impulse, the rate in which those impulses are generated can be influenced by autonomic nervous system. Different types of respiratory patterns can stimulate autonomic nervous system in different manners. Thus, non-sedated rabbits were studied during forced respiration aiming to evaluate the influence of this breathing pattern on heart rate.

Twenty male, one-year-old healthy New Zealand rabbits were enrolled in the study. Animals were set in right lateral recumbency and maintained that way by physical contention. Chemical sedation was not used. Partial nasal obstruction by digital compression was applied to those rabbits for five seconds, eliciting a forced inhaling and exhaling against semi closed nostrils. Heart rate was obtained by measurement of two consecutives RR intervals in the computerized electrocardiography, recorded continuously prior and during the maneuver.

Heart rate before the intervention was 251 AE 34 bpm (mean AE standard deviation). All rabbits submitted to the maneuver showed a dramatic reduction in this parameter. After nasal partial obstruction, heart rate was 142 AE 32 bpm. Data was submitted to statistical analysis by paired Student's t test and a significant difference between the heart rate before and after the maneuver was observed (p o 0.0001).

Although the exactly mechanism involved in this response was not elucidated, the presented data support the applicability of this maneuver as an efficient method for non-pharmacological heart rate reduction in rabbits. Obesity can affect cardiac function due to effects on cardiac rhythm, ventricular volume and blood pressure. The purpose of this study was to determine the effects of obesity and overweight on noninvasive systemic blood pressure and Doppler echocardiographic parameters in cats without others causes of cardiac hypertrophy.

The study groups comprised fifteen obese cats with mean body score index (BSI) of 8,8, seven overweight cats (BSI 5 6,3) and seven cats with ideal BSI (4,9). The blood pressure was measured by Doppler method and the Doppler echocardiography was performed in conscious animals. The statistical analysis was performed by analysis of variance followed by Tukey's test and Pearson's correlation. The blood pressure values of the obese cats were superior (159,12 AE 11,22 mmHg, p o 0,0001) than in overweight (134,45 AE 13,81 mmHg) and normal cats (136,90 AE 13,17 mmHg) and 57% of the obese cats had blood pressure higher than 160 mmHg. There were observed differences on the ratio of early (E) and late (A) left ventricular filling velocity (p 5 0,008) of obese animals (E/A 5 1,07 AE 0,39) compared to overweight (1,68 AE 0,37) and normal cats (1,43 AE 0,24). Seven obese cats (50%) had inversion of E/A compatible with diastolic dysfunction and there were negative correlation (r 5 À0,453, p 5 0,026) between the E/A ratio and blood pressure values. Other differences observed were increases in left ventricular septum in diastole (p 5 0,002) and in free wall in diastole (p 5 0,023) and systole (p 5 0,042) of the obese animals compared to overweight and normal cats.

These results demonstrate the possibility of cardiovascular effects related to obesity in cats, such as systemic arterial hypertension and secondary diastolic dysfunction. Diuretic therapy reduces preload, and relieves congestion secondary to cardiac dysfunction. Torsemide (torasemide) is a loop diuretic with longer duration of action, less diuretic resistance, and adjunctive aldosterone antagonism as compared to furosemide. We hypothesized that torsemide was no less effective than furosemide at diuresis, control of clinical signs, and maintenance of quality of life in dogs with congestive heart failure. A double-blinded, randomized, crossover clinical trial was performed in 7 dogs with stable heart failure receiving BID furosemide and adjunctive medications. Dogs were randomized to their current furosemide dose or torsemide (calculated as 1/10 of the daily furosemide dose divided into BID dosing). Crossover occurred at day 7 and the study ended on day 14. Clinical, laboratory, radiographic, and owner-perceived quality of life variables were evaluated on days 0, 7 and 14.

No dog developed recurrent heart failure during the study. Average furosemide dose on day 0 was 5.13 mg/kg/day (range, 2.8-9.6). Following torsemide treatment, blood urea nitrogen (P 5 0.0028), albumin (P 5 0.0287), and albumin:globulin ratio (P 5 0.0012) were significantly increased, and urine specific gravity (P 5 0.0062) and chloride (P 5 0.0051) were significantly decreased as compared to baseline and/or furosemide dosing (one-way ANOVA with Bonferroni correction). No differences in QOL were found.

Results indicate that torsemide is equivalent to furosemide at controlling clinical heart failure in dogs, and might in fact, achieve greater diuresis vs. furosemide. Larger clinical trials evaluating furosemide resistance and/or torsemide as a first-line loop diuretic for congestive heart failure in dogs with heart failure are warranted. The purpose of this study was to investigate the feasibility of speckle tracking echocardiography (STE) in healthy cats and to determine whether or not it can detect myocardial dysfunction in cats with diseased heart.

Radial and circumferential strain and strain rate were measured by STE using left ventricle short-axis view in clinically healthy cats. Eighteen cats with HCM whose LV thickness at end-diastole with 6 mm or more were evaluated with STE analysis, and compared with healthy cats. Index of left ventricular synchrony (Trs-SD) was also assessed in cats with HCM, and compared to healthy subjects. STE resulted in technically adequate images in 100% of the cats. Fusions of early and late diastolic (E and A) wave in strain rate were seen in 3 of 16 cats. Percent errors in analysis with or without simultaneous ECG monitoring were 5.3-14.7% in all parameters. Inter-and intraobserver variability of STE parameters in healthy cats was minimal (4.1-15.6%) except for the systolic circumferential strain rate. Sedation using buprenorphine and acepromazine did not affect any STE parameter. E wave in radial and circumferential strain rate of HCM cats was significantly decreased compared with healthy cats. No significant difference was seen in Trs-SD. STE analysis was considered clinically feasible to assess cardiac function in cats, and could detect myocardial dysfunction in cats with HCM. Further study is warranted to investigate to assess whether or not STE can differentiate the etiology of left ventricular concentric hypertrophy since it is clinically important. Carvedilol, a 3rd generation non-selective beta-blocker with ancillary alpha 1 -blocking and antioxidant properties may have therapeutic implications for multiple diseases in cats; however, pharmacokinetics and bioavailability of commercially prepared oral carvedilol has not been determined. HPLC for carvedilol measurement in feline plasma was validated and standardization curves created. The pharmacokinetics (PK) of carvedilol was evaluated in 5 apparently healthy male neutered adult cats (average weight of 5 kg) following single dose intravenous (IV) of 0.5 mg/kg and single dose oral administration of 1.6 to 2.0 mg/kg. Concentrations of the active parent compound, carvedilol, were detected in plasma using HPLC analysis. Lower limit of quantification was 5 ng/ml.

The mean peak concentration after IV administration of carvedilol was 8639 ng/mL (range, 901 to 8648), elimination half-life was 2.9 hours (range, 2.0 to 5.4), and clearance was 0.35 L/hr/kg. The volume of distribution was 1.18 L/hr. After a single oral administration of carvedilol, the time to peak plasma concentration was 60 minutes (range, 30 to 90 minutes) and the mean residual time was 4.8 hours. The half life was 4.44 hours. Maximal concentration 294 ng/ mL and the mean bioavailability was 15.7% with a median of 9.97% (range, 4.7% to 46%).

These data demonstrate a low bioavailability of oral carvedilol and a wide variation in cats. All cats tolerated the oral dose of carvedilol with no major adverse effects. Also, a mean residual time of 4.8 hours would suggest that a more frequent dosing schedule may be required to maintain therapeutic plasma levels. Pharmacodynamic studies investigating beta-adrenergic blockade duration may provide a more accurate dosing interval of carvedilol.

ABSTRACT C-49 EFFECTS OF SILDENAFIL CITRATE ON DOGS WITH EI-SENMENGER'S SYNDROME. K Nakamura, M Yamasaki, H Ohta, M Takiguchi. Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.

Sildenafil has shown to be effective for dogs with pulmonary hypertension; however, its efficacy for dogs with Eisenmenger's syndrome (ES) and secondary erythrocytosis has not yet been determined. The objective of this study is to determine the effect of Sildenafil for dogs with Eisenmeger's syndrome and secondary erythrocytosis.

This was a prospective, single arm, open-label study. Five clinical dogs with ES and secondary erythrocytosis were included. New York Heart Association (NYHA) functional class, PCV, and pulmonary artery acceleration time to ejection time ratio (PA AT : ET) were evaluated before and after sildenafil therapy (0.5 mg/kg, bid). NYHA functional class was significantly improved after 1 (median 2; range 1-2, P 5 0.031) and 3 months (median 2; range 1-2, P 5 0.031) of sildenafil therapy, compared with the baseline (median 3, range 2-3). PCV was significantly decreased after 1 month (62.4 AE 4.7%, P 5 0.015) and 3 months (59.9 AE 3.6%, P 5 0.01) of therapy, compared with the baseline (68.0 AE 5.6%). AT : ET was significantly increased after 1 month of therapy (0.39 AE 0.06, P 5 0.013) from the baseline (0.28 AE 0.04).

Sildenafil resolved the clinical signs and secondary erythrocytosis in dogs with ES. Sildenafil therapy could be the treatment of choice for dogs with ES. Sepsis is the number one cause of mortality in neonatal foals. The role of the RAAS and HPAA in systemic inflammation and response to stress is well documented in critically ill human neonates, but limited information exists in foals. We hypothesized that in septic foals the RAAS and HPAA will be activated by systemic inflammation and hypoperfusion and the degree of activation will be associated with severity of sepsis and mortality.

Blood samples were collected on admission from 60 septic (sepsis score 4 12), 102 sick non-septic (SNS), and 18 healthy foals of o 7 days of age. Blood concentrations of corticotropin-releasing hormone (CRH), adrenocorticotropin (ACTH), cortisol, aldosterone, angiotensin-II (ANG-II), arginine vasopressin (AVP) and plasma renin activity were determined by radioimmunoassays.

ACTH, cortisol, aldosterone, ANG-II and AVP concentrations were higher while CRH was lower in septic and SNS foals compared to healthy foals (P o 0.05). Septic non-survivor foals had higher concentrations of aldosterone, cortisol, ACTH and AVP and lower concentrations of ANG-II and CRH than survivors. AVP was associated with ANG-II in septic, and with ACTH in septic and SNS foals (P o 0.05). There was no difference in renin activity and ANG-II concentrations among foal groups. Septic foals had a higher ACTH:aldosterone ratio than healthy foals (P o 0.001).

This study shows that in response to sepsis there is RAAS and HPAA activation in critically ill foals. We propose that in sick foals AVP is more important than CRH in regulating ACTH secretion. The increased ACTH:aldosterone ratio further supports relative adrenal insufficiency in septic foals. This prospective, cohort study aimed to characterize alterations in coagulation and blood-derived inflammatory biomarkers in adult horses that developed diarrhea during hospitalization. Physical and hematological parameters were evaluated at times 0 (onset of diarrhea), 6, 12, 24 and 48h, then every 48 h until resolution of diarrhea or death. Each hematological analysis included a complete blood count (CBC), thromboelastography (TEG), partial-thromboplastin-time (PTT), prothrombin-time (PT), plasma concentrations of lactate, tumor necrosis factor alpha (TNF-a), interleukin (IL)-1, IL-6, IL-10 and NT-proC-type-natriuretic peptide (pCNP). Horses were categorized into three groups based on the duration of diarrhea and evidence of systemic inflammation. Group 0: Diarrhea o 6 h without systemic inflammation (SI); Group 1 -Diarrhea ! 6 h without SI; Group 2-Diarrhea with SI. Assessment of vital parameters and CBC established a diagnosis of SI as previously described (Levy, 2003) . Descriptive and univariate outcome analyses were based on data normality.

19 horses were enrolled, of which 16 (84.2%) survived to discharge. The mean age was 13.61/-5.3 years. Eight horses (42.1%) were categorized as group-0, 2 (10.5%) as group-1 and 9 (47.4%) as group-2. Two horses developed thrombophlebitis. Based on the results of TEG, 6/19 (31.6%) were normocoagulable, 7/19(36.8%) were hypocoagulable and 6/19 (31.6%) were hypercoagulable, at one or more time points. Of these, 7/9 (77.8%) group-2 horses were coagulopathic. Additionally, group-2 horses had a significantly lower MA than group-0 horses at baseline (43.6 AE 16.7 vs. 61.9 AE 7.2) and 6h (45.9 AE 15.9 vs. 65.5 AE 5.8). Biomarker analyses are pending.

In conclusion, SI was associated with coagulation disorders in horses with hospital acquired diarrhea. Clostridium difficile and Clostridium perfringens are commonly associated with colitis and diarrhea in equines but asymptomatic carriers exist. Reported carrier rates of toxigenic C. difficile and C. perfringens strains in feces range between 0-25% and 0-30% respectively. Toxigenic C. difficile has also been isolated from the small intestine of diseased foals and is implicated as etiologic agent of duodenitis/proximal jejunitis in adult horses however scarce information is available on prevalence in gastrointestinal compartments other than feces in healthy horses, and it is unclear whether fecal samples are good predictors of the status of proximal intestinal sites. The objectives of this study were to investigate the presence of C. difficile and C. perfringens in various intestinal compartments of healthy adult horses and to molecularly characterize isolates.

Intestinal contents were collected from the stomach, duodenum jejunum, ileum, cecum, right dorsal and left ventral colon, small colon and rectum of 10 euthanized horses free of apparent gastrointestinal disease. Enrichment culture was performed for C. difficile and C. perfringens and C. difficile isolates were further characterized via toxin gene detection and ribotyping.

C. difficile was isolated from 9/90 (10%) samples from 5/10 (50%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in three horses. Isolates were recovered from duodenum (n 5 1), right dorsal colon (n 5 3), small colon (n 5 1) and rectum (n 5 4). In one horse, the rectal sample was negative but C. difficile was isolated from a proximal site, all other horses were positive on the rectal sample if a more proximal compartment was positive. In three horses multiple compartments were positive however different strains were always present within the same horse (n 5 2). All isolates possessed genes encoding toxins A and B. Five isolates were ribotype 078 and also possessed genes encoding the binary toxin. The other isolates were ribotype 001 and were negative for the genes encoding the binary toxin. Despite using a method with a detection level as low as 9 cfu/g of feces, no C. perfringens was recovered.

Rectal samples were a good predictor of overall C. difficile carrier status (4/5 horses), however rectal samples were not always representative for the ribotype carried in more proximal compartments. The presence of variable strains within the same horse suggests transient passage of the bacterium through the gastrointestinal system rather than actual colonization although further study testing multiple colonies per site is needed. The predominance of ribotype 078 is consistent with recent emergence of this strain in this region, as earlier studies found other strains (027, 001) to be more prevalent and a variety of ribotypes were typically recovered from horses. Interestingly ribotype 078 has recently emerged as a hypervirulent strain in humans in our area. Clostridium difficile, Clostridium perfringens and Salmonella are important enteric pathogens in horses, however some healthy animals also harbour these pathogens. Point prevalence studies have reported these carriage rates, but there are no data regarding longitudinal prevalence of these enteric bacteria, information that would be useful to better understand the epidemiology of these pathogens. Additionally, antimicrobial resistance is a pressing concern. Commensal E.coli is often used as an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data from horses on farms. The objectives of this study were to longitudinally investigate the above enteric pathogens over the course of one year, molecularly characterize obtained isolates and determine the antibiotic susceptibility profile for E. coli.

Fecal samples were collected from 25 adult horses from five farms on a monthly basis over the course of one year. Selective cultures were performed for C. difficile, C. perfringens, Salmonella and E. coli. C. difficile isolates were characterized via toxin gene PCR and ribotyping. Broth microdilution was performed to assess antimicrobial susceptibility profiles of E. coli.

Clostridium difficile was isolated from 15/275 (5.45%) samples from 11/25 (44%) horses. Four horses were positive on more than one occasion, three were positive in two consecutive months. Different ribotypes were found in two of the latter horses. Most isolates were ribotype 078 (n 5 6) with ribotype 001 (n 5 5) and ribotype C (n 5 4) also identified. Ribotypes 078 and C possessed genes encoding toxins A, B and binary toxin, while ribotype 001 only possessed toxin A and B genes. Despite a detection threshold of 9 cfu/g feces, C. perfringens was not detected in any samples, nor was Salmonella. E coli was isolated from 117/225 (52%) samples. Resistance to !1 antimicrobial was present in only 19/117 (16.9%) isolates. Multidrug resistance (! 3 antibiotics) was present in 5/117 (4%). Most commonly, isolates were resistant to sulfisoxazole (17/ 117) and trimethoprim sulfamethoxazole (16/117).

The overall detection rate for toxigenic C. difficile in fecal samples of healthy horses was 5.4% which is consistent with previous studies. The cumulative prevalence of 44% was striking but only one horse shed the same strain for more than one month, indicating C. difficile shedding is a transient and dynamic state. The predominant isolation of ribotype 078 is consistent with the suspicion that this strain has emerged and become widely disseminated in this region in recent years. The low prevalence of C. perfringens and Salmonella is in agreement with some other studies. The low prevalence of antibiotic resistance in commensal E. coli was encouraging and suggests that healthy horses on pleasure horse farms are not likely a major reservoir of resistance in enteric bacteria. Type 1 Polysaccharide Storage Myopathy (PSSM) in horses is associated with a dominant missense mutation (R309H) in the skeletal muscle glycogen synthase gene (GYS1). Since disease severity varies between affected horses, we hypothesised that some clinical variability could be accounted for by the underlying genotype. 107 Belgian / Percheron horses were genotyped using a validated restriction fragment length polymorphism assay enabling grouping of horses as homozygotes (HH), heterozygotes(HR) or normal (RR). Subsequently, semimembranosis muscle samples were biopsied from each of six matched sedentary horses from each group; one sample was formalin-fixed and one fresh frozen. Sections were stained using haematoxylin and eosin, periodic acid schiff 1/diastase. Anti-dystrophin, nNOS and myosin heavy chain immunohistochemistry was performed to examine sarcolemmal intregrity, There were significant differences in resting CK activity (P 5 0.023) (median HH 5 364U/l interquartile range(IR) 332-764; HR 5 301U/l IR222-377; RR 5 260U/l IR216-320) and AST activity (P o 0.001) (AST mean HH 5 502U/l SD116; HR 5 357U/l SD92; RR 5 311U/l SD64) and muscle pathology between the 3 groups, with severity increasing RR o HR o HH. There were significantly more type 2a (P 5 0.04) and fewer type 2x fibres (P 5 0.02) in homozygotes (2a 55% SD 10.2; 2x 32% SD 18) compared with the other groups (HR 2a 38% SD 10.9, 2x 44% SD 10.8; RR: 2a 36.5% SD 12.4 2x 57% SD 11.5). More type 2a fibres contained polysaccharide inclusions in homozygotes (30% SD 11.1) than in heterozygotes (10.6% SD 6.9) (P o 0.001). Both dystrophin and nNOS expression was normally localised to the sarcolemma in pathologically normal and vacuolated fibres from mutant horses. In conclusion, sedentary homozygotes have more severe skeletal muscle pathology and higher resting plasma CK and AST activities than heterozygotes, and PSSM1 is associated with a fibre type shift towards type 2a. Although subsarcolemmal vacuolation likely disrupts the contractile apparatus's attachment to the sarcolemma, the latter's integrity appeared intact. The recumbent horse presents a logistic, diagnostic, and therapeutic challenge to the equine practitioner. There is currently very little data available on the prognosis and outcome of horses that are recumbent. Therefore, the purpose of this study was to investigate the outcome of hospitalized horses that had been recumbent in the field or in the hospital and the factors affecting their survival.

Records of horses admitted to the School of Veterinary Medicine, University of California Davis from January of 1995 to December of 2010 with a history of recumbency or horses that became recumbent while hospitalized were evaluated. A horse was defined as recumbent if it was unable to stand on its own. The medical record was examined for the following criteria: history pertaining to the current illness including treatment by the rDVM, breed, age, weight, date of presentation, physical and neurological examination findings, CBC and biochemical profile results, initial drugs administered on arrival, time spent recumbent, time spent in a sling, diagnosis, and hospitalization costs. Statistical analysis correlating factors associated with survival was performed using logistic regression.

Overall there were 112 non survivors and 49 survivors. Factors that favored survival included early initiation of treatment in the field by the rDVM, horses that tolerated a sling and spent more time in a sling, increased duration and costs of hospitalization, horses that were recumbent post anesthesia, and those recumbent due to disease of the musculoskeletal system. Factors that increased likelihood of non survival included horses that were ataxic on presentation, horses with increased BUN, horses that spent more time recumbent, those that did not tolerate a sling, and horses diagnosed with botulism and spinal cord disease.

In conclusion, this retrospective study demonstrated that both the cause of recumbency and the ability of horses to tolerate a sling had a direct effect on survival.

ABSTRACT E-7 PLASMA PEAK AND TROUGH GENTAMICIN CONCENTRA-TIONS IN HOSPITALIZED HORSES RECEIVING ONCE DAILY GENTAMICIN. JR Read 1 , PA Wilkins 2 , RD Nolen-Walston 1 . 1 University of Pennsylvania, New Bolton Center, Kennett Square, PA. 2 University of Illinois, Champaign-Urbana, IL.

Gentamicin is often used to provide gram negative antimicrobial coverage in horses at 6.6 mg/kg IV every 24 hours. Therapeutic drug monitoring in our hospital suggests larger doses are required in many clinical cases to achieve the desired concentration (8-10Â minimum inhibitory concentration) for common bacterial isolates (peak target range 32-40 mg/ml). The aim of this study was to determine the correlation between gentamicin dose and plasma concentration in hospitalized horses receiving gentamicin treatment in order to identify an optimum dose range for this population. Review of records (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) identified 71 horses ! 3 months old receiving once-daily gentamicin with peak and trough assays performed (N 5 107 sets). Spearman Rank Correlation coefficient analysis revealed a weak (R 5 0.289) but statistically significant correlation (P 5 0.003) between gentamicin dose and peak plasma concentration. Horses receiving 7.7-9.7 and 4 9.7 mg/kg gentamicin (Groups 2 and 3) had higher median peaks (31 mg/ml) than horses receiving 5.6-7.6 mg/kg (Group 1; 25.7 mg/ml). Higher doses were more likely to result in peaks 4 32 mg/ml (42 and 41%, Groups 2 and 3 respectively) than horses receiving 5.6-7.6 mg/kg (Group 1; 16%). All 22 hour post-gentamicin administration trough values were o 2 mg/ml. No correlation was found between dose and change in plasma creatinine during treatment, nor dose and trough level. These data suggest that gentamicin dosage in horses should be individually determined by therapeutic monitoring. Additionally, these data support an initial dose of 7.7-9.7 mg/kg IV every 24 hours in order to achieve desired peak concentration and an appropriately low trough concentration. Heaves is a common respiratory inflammatory disease, characterized by a pulmonary neutrophilia. This disease is also characterized by an activation of circulating neutrophils after antigen challenge but their specific role in heaves is not well understood. Also, there are anecdotal studies concerning heaves-affected horse to be more susceptible to infection. However, to our knowledge, the antibacterial host defense role mediated by circulating neutrophils was not investigated in heaves-affected horses. The objective of this study was to compare phagocytosis activity and bacterial killing by circulating blood neutrophils of heaves-affected and control horses.

Peripheral neutrophils were isolated from heaves-affected (n 5 6) and control (n 5 6) horses using a density gradient technique. The killing capacity was assessed by incubating neutrophils with Streptococcus equi spp equi and spp zooepidemicus. After 1 h of bacterianeutrophil coculture, total viable bacterial cells were measured by quantitative plating. The phagocytosis was evaluated by flow cytometry using fluorescent beads and GFP-transformed Streptococcus suis suilysin-negative mutant strain.

Circulating neutrophils from heaves-affected horses showed a significant decrease in their killing capacity toward S. zooepidemicus (p 5 0.046). A reduced, although not significant (p 5 0.1), killing capacity of S. equi by these neutrophils was also observed. The phagocytosis activity was not different between groups.

This impairment of blood neutrophil bactericidal activity in heaves-affected horses could contribute to an increase susceptibility to infection. Obesity is a common disorder of the horse, with current prevalence estimated at 30%. In people, obesity is associated with dyslipidemia, insulin resistance, mitochondrial dysfunction and downregulation of lipid and glucose metabolic pathways. In the horse, obesity is similarly associated with insulin resistance and alterations in lipid profiles; however, metabolic regulatory gene expression profiles have not been fully characterized. We hypothesized that obese horses have decreased expression of metabolic regulatory genes and decreased mitochondrial content in skeletal muscle compared with non-obese horses.

Sixteen light breed horses, 2-27 years of age were included. Body condition score (n 5 16) and neck circumference (n 5 9) were recorded. Post-mortem biopsy samples of the semi-membranosus muscle were obtained. DNA and RNA were isolated. Relative expression of the metabolic genes, peroxisome proliferator activated receptor g (PPARg), PPARg coactivator-1a (PGC-1a), fatty acid translocase (FAT) and estrogen related receptor a (ERRa) was determined by quantitative polymerase chain reaction (qPCR). Mitochondrial content was assessed by determining mitochondrial DNA/nuclear DNA ratio by qPCR, using NADH-dehydrogenase subunit 2 and cytochrome oxidase subunit 2 as mitochondrial genes and beta actin as the nuclear reference gene. Non-normal data was log transformed for analysis and a Pearson coefficient of correlation was calculated for relative gene expression, body condition score and neck circumference. A value of p o 0.05 was considered significant. Body condition score was strongly correlated with neck circumference (n 5 9, r 5 0.72, p 5 0.03). Relative expression of ERRa and GLUT-4 increased with body condition score (ERRa: n 5 16, r 5 0.66, p 5 0.005; GLUT-4: n 5 16, r 5 0.50, p 5 0.05). Copy number of the mitochondrial genes (NADH-DH and COX-2) was not related to body condition score or metabolic gene expression. Expression of GLUT-4, ERRa, PPARg, and FAT were strongly correlated to each other, but not PGC-1a. There was a strong trend towards correlation between PPARg and PGC-1a in horses with body condition score 4 6 (n 5 6, r 5 0.77, p 5 0.07).

In this study, there was no change in mitochondrial content in obese horses. Assessment of mitochondrial function in obese horses and horses with EMS is under way. The strong correlation between PPARg and PGC-1a observed only in horses with high body condition scores suggests this pathway is activated with obesity. The role of PPARg and PGC-1a in equine obesity should be further investigated to determine their potential as therapeutic targets. Upregulation of ERRa and GLUT-4 in horses with increasing body condition score is unexpected, and may indicate a compensatory response to dysfunction of a downstream pathway. Further studies to better define the role of metabolic regulatory gene expression in obese horses and those with EMS are ongoing.

Previously presented at the 7th Annual Harold Hamm Diabetes Center Research Retreat, Oklahoma City, OK. Inflammatory bowel disease is a cause of weight loss, decreased performance, and colic in horses. This condition is difficult to diagnose and clinicians rely upon absorption tests to document malabsorption. The purpose of this study was to compare glucose and xylose absorption tests in normal horses and determine the repeatability of these procedures.

Eight horses received 500 mg/kg dextrose or D-xylose powder mixed as a 10% solution in water or water alone via nasogastric intubation on three different occasions within the same week for three consecutive weeks (9 tests/horse). A crossover design was employed and the order of treatments was randomized. Blood samples were collected at time 5 0, 30, 60, 90, 120, 150, and 180 min. Data were analyzed by repeated measures ANOVA and t-tests.

Results showed that the xylose response over time differed significantly from the glucose response over time (test  time; P o 0.001). Mean time to maximum concentration differed (P o 0.001) between tests (glucose 90 min; xylose 60 min). Within-horse area under the curve, maximum concentration, and time to maximum concentration values for dextrose and xylose did not differ significantly when tests were repeated. Results indicate that glucose and xylose absorption tests are repeatable within the same horse, but plotted curves differ between tests, with peak concentrations occurring at a later time point for the glucose absorption test. We conclude that both tests provide repeatable measures of intestinal absorption, but glucose and xylose appear to differ in their rates of absorption and clearance. The purpose of this study was to examine the records of a population of Thoroughbreds with cervical vertebral malformation (CVM) and to determine which factors have an effect on these horses achieving athletic function.

This was a retrospective case study of 119 Thoroughbreds with CVM treated medically from 2002 to 2010. Forty-one were euthanized after diagnosis, while the remaining 78 were discharged for treatment. Racing records were reviewed to determine which horses raced after treatment. Horses were separated into groups based on whether or not they raced. Medical records were reviewed, and results of neurologic examination, radiographic and laboratory findings, treatments, and outcome were assessed and compared between groups.

Twenty-one of 78 horses treated medically (27%) improved enough to race. Median neurologic grade between groups was significantly different (p o 0.0001), with a hind limb grade of 2.0 (range 1-3) for the raced group and 2.5 (range 0.5-4) for the unraced. Intravertebral sagittal ratios measured from standing lateral cervical radiographs were equivocal between groups. Radiographs of all horses were examined for kyphosis, dorsal over-riding arch, caudal epiphysitis, degenerative joint disease, cystic bone lesions, and cranial stenosis of the vertebral canal. Horses with kyphosis (p 5 0.0178), degenerative joint disease (p 5 0.0497), or cranial stenosis (p 5 0.0357) at any site were less likely to return to racing.

Racing prognosis for horses with CVM treated conservatively is equivalent to that of those treated surgically as reported by Rush Moore et al (JAVMA, 1993) . Radiographic changes and neurologic grade may help serve as indicators for whether a horse will respond to conservative therapy. Since pain assessment is vital for management of colic, a valid, reliable and feasible tool for assessing the severity of acute abdominal pain in horses is urgently needed. Our aim was to construct and validate a behavior-based pain scale by methodology utilized in construction of pain scales in non-verbal humans. The project consisted of four stages. Firstly, behaviors to include in a scale were empirically identified. Thirty equine clinicians noted behaviors in each of 23 random film clips of horses with colic using a checklist. Nine behaviors (e.g. rolling, pawing, and flank watching) demonstrated good inter-observer agreement without bias (multi-rater kappas: 0.5-0.95). Secondly, the clinical judgment of experts was utilized to identify and to weight behaviors. Six expert clinicians independently expressed opinions as to which of 46 behaviors to include and the severity of pain they indicate. Two contending scales (Equine Acute Abdominal Pain Scales (EAAPS) 1 & 2) were constructed based on both the empirical and the judgmental approaches. Each included 12 identical behaviors with a 1-5 point score range; EAAPS-2 with gradations to some of the behaviors and EAAPS-1 without. In the third stage, blood cortisol and lactate levels and heart rate were shown to only approximate pain since they correlate poorly with degree of pain as assessed by visual analog scale (VAS) in 32 horses with colic and 8 controls (Spearman rho; lactate 0.359; cortisol 0.214; heart rate 0.261). Finally, reliability and validity of the pain scales were evaluated including constructs of pain; face validity, convergent and discriminate validity and extreme groups. Thirty of 40 films of horses with colic were randomly presented to 44 expert equine clinicians internationally who were randomly allocated into three groups to score pain; one group by both VAS and numerical rating scale (NRS)), and two groups, each by one of the two EAAPS scales. Inter-observer reliability of both EAAPS scales was excellent (Intraclass Correlation 0.8). Intra-observer reliability based on scores given for identical films demonstrated; 87% and 56% agreement, kappa 0.9 and 0.35, and Spearman's rho 5 0.97 and 0.58 for EAAPS-1 and 2, respectively. Both scales varied by 1 score between observations. Face validity; each group reported their scale to be valid (67% & 81%). Convergent validity; the scales compared favorably with VAS/NRS scores (Spearman's rho: 0.84-0.89). Discriminate validity; correlation to heart rate, lactate and cortisol levels was predictably low (rho 5 0.2-0.5). Extreme group validity; colic horses scored significantly higher than control horses; scores of 0.6-0.7 in controls versus 2.6-2.7 in cases. In conclusion, methodology established in human medicine but novel in veterinary medicine was used to construct and validate two clinically feasible equine abdominal pain severity scales that showed excellent reliability and validity. Further refinement of the EAAPS scale is advised prior to introduction into clinical practice. Aortic valve prolapse (AVP) is a common echocardiographic finding in horses, but when compared with mitral valve prolapse in dogs, little is known about the natural progression of this condition. Previously published data has shown that echocardiographic identification of AVP in horses is reliable, diagnostic criteria have been established and that development occurs with training. The aims of this study were to evaluate the different RNA and protein expressions of smooth muscle actin (SMA), transforming growth factor-b 1 (TGF), nitric oxide synthase (NOS) and the concentrations of elastin and collagen in normal, prolapsing and diseased cusps to evaluate what structural changes may predispose them to prolapse. Valve cusps were harvested and processed from a group of 176 horses at a commercial abattoir following disease classification using echocardiography. Horses were aged 13.7 AE 6.7years, weighing 518 AE 51 Kg and with a median body condition score of 4/9. Cusps were collected in RNAlater s and stored at À801C prior to processing. cDNA was produced from half a valve using a standard protocoland qRT-PCR performed to assess relative RNA expression of SMA, TGFb 1 , endothelial (eNOS) and inducible NOS (iNOS) and compared with the housekeeping gene 18S. A quarter of cusp was processed using an adapted commercial protocol to evaluate protein expression of SMA, TGF b 1 , eNOS and compared to vimentin. Specific antibody binding was assessed with western blotting and protein expression evaluated using dot blots. The remaining quarter cusp was used to measure soluble collagen and elastin concentrations using commercial assays 3 . Statistical analyses included one way ANOVA with post-hoc Bonferoni, paired Student's T-test, linear and logistic regression. There was no effect of gender or age on any of the measurements. Valves from animals with AVP had lower expression of SMA and Elastin compared to normal and diseased valves, increased expression of TGFb 1 and eNOS, whereas iNOS expression was greater than normal valves (Table 1) . Collagen content of valves from horses with AVP was increased compared to normal but lower than horses with valve disease. Prolapsing cusps appear to be a different phenotype from diseased cusps. Further studies will help to elucidate the significance of these findings in vivo. A clear association between heart rate (HR) and body mass has been observed across a wide range of mammalian species. Furthermore, it is well known that electrocardiographic (ECG) time intervals vary with heart rate and body mass. Within the equine species, small breeds are generally thought to have higher heart rates than large breeds. However, despite the large differences in size among different equine breeds, there is little information about normal heart rates and normal ECG time intervals in horses and ponies of different body size. Similarly, the relationship between HR and body mass in dogs of various breeds and sizes is still under debate. The goal of this study was to investigate the relationship between heart rate and ECG time intervals to body mass in apparently healthy horses and ponies and to calculate normal ranges for different weight groups.

250 adult horses and ponies at an age of 5.5 (1-30) y [median (range)] and a body weight of 479 (46-1120) kg were included in the study. All animals were considered clinically healthy based on history and physical examination. A standard base-apex ECG was recorded at a speed of 25 (n 5 4) or 50 mm/s (n 5 246) using a multiparameter monitor (Datascope Passport). During the procedure, the horses were unsedated, standing quiet in a box stall. Mean HR over 15 sec was determined for each recording. The following ECG time intervals were measured in triplicate and averaged for further analyses: PQ interval, QRS duration, QT interval, and difference between QT and QRS (QT-QRS duration). The relationship between HR, ECG time intervals, and body mass was assessed using linear regression analyses. Normal ranges (2.5% to 97.5% percentile) were calculated for 5 different weight groups. The level of significance was p 5 0.05.

Heart rate was inversely related to body mass (p o 0.0001, R 2 5 0.122). The PQ interval (p o 0.0001, R 2 5 0.413), QRS duration (p o 0.0001, R 2 5 0.147), QT interval (p o 0.0001, R 2 5 0.089), and QT-QRS duration (p o 0.0001, R 2 5 0.028) were directly related to body mass. Normal ranges for HR, PQ, QRS, and QT within the different weight groups were 36-64 bpm, 107-230 ms, 60-127 ms, 360-510 ms (o200 kg); 28-68 bpm, 162-319 ms, 74-155 ms, 362-610 ms (200-399 kg); 28-60 bpm, 211-423 ms, 89-147 ms, 390-581 ms (400-599 kg); 28-54 bpm, 220-380 ms, 87-150 ms, 367-587 ms (600-799 kg); and 24-52 bpm, 240-463 ms, 87-140 ms, 437-533 ms (4 799 kg).

We conclude that in healthy horses there is a significant but weak relationship between body mass and HR and between body mass and ECG time intervals, respectively. This study therefore supports the hypothesis that within the equine species, small breeds have faster heart rates and shorter ECG time intervals than large breeds. Therefore, body mass has to be considered when comparing HR and ECG time intervals to normal ranges in horses. Horses with pituitary pars intermedia dysfunction (PPID) often have elevated plasma ACTH concentrations. However, PPIDaffected horses rarely have resting serum cortisol levels above the reference range or adrenal hyperplasia. We hypothesized that this apparent dissociation between plasma ACTH levels and adrenal response in horses with PPID is due to the secretion of ACTH that is less biologically active than that from normal horses.

To test our hypothesis, a bioassay to evaluate ACTH activity was developed. Adrenocortical explants were harvested aseptically from normal horses at euthanasia and stimulated with plasma from healthy (n 5 9) and PPID-affected horses (n 5 11). The assay was performed three times with explants obtained from different horses. Cortisol secreted by the explants and plasma ACTH levels were measured with commercially available radioimmunoassays. Cortisol secretion stimulated by each sample was standardized to the respective explant protein concentration. Cortisol data was normalized for ACTH concentration in each plasma sample and expressed as a cortisol:protein:ACTH ratio. Ratios from horses with PPID and normal horses were compared by unpaired t-test.

Horses with PPID had significantly lower cortisol:protein:ACTH ratios compared to normal horses. (Assay 1: 0.06 AE 0.04 vs. 0.33 AE 0.11, P o 0.001; Assay 2: 0.06 AE 0.04 vs. 0.30 AE 0.10, P o 0.001; Assay 3: 0.07 AE 0.06 vs. 0.20 AE 0.13, P o 0.01). These results suggest that plasma ACTH from PPID horses is less biologically active than plasma ACTH from normal horses. Our findings give further insight into the pathophysiology of PPID and may aid in the development of novel diagnostic testing protocols. An online survey was conducted to determine perceived needs of potential employers of new ACVIM-LAIM diplomates. The survey was designed as the first step in determining what is needed for success in the various sectors of practice employing ACVIM-LAIM diplomates.

Demographic and background data were collected using questions and drop-down menus on the first page. The survey evaluated 189 skills or concepts in 26 areas of veterinary practice. Participants answered 4 questions about each skill or concept using drop-down ranked lists. Those participants that had completed an ACVIM-LAIM training program were asked 3 additional questions about whether they were taught the skill or concept during their own residency. Data were collated and descriptive statistics calculated. The mean scores or frequencies of use for each skills or concepts were ranked to determine which of the skills or concepts were most important for an entry-level diplomate.

Eighty-eight individuals participated in the survey with 86 respondents being ACVIM diplomates, 1 respondent was not board-certified and 1 respondent was an ACT diplomate. Nineteen respondents were diplomates of ACVIM and an additional specialty. Eighty-three respondents had completed an ACVIM residency. The majority of respondents were in academia (65%) with 30% being in private practice. Equine specialists prevailed (53%) followed by mixed large animal (28%) and then food animal only specialists (13%). The distribution of years post-residency was slightly skewed toward younger diplomates, but overall there was a good distribution of diplomates across years of experience. Most respondents stated that they did not make hiring decisions in their practice. Competency in disciplines other than internal medicine was expected with ultrasonography and radiology being the most desirable followed by theriogenology and lameness. Surgical skills, both equine abdominal (10%) and food animal general surgery (11%) were considered important by some respondents. Thirty-six per cent of respondents thought that a new diplomate should expect to make o $50,000 per annum, while only 13% of respondents thought that a new diplomate should expect to make ! $90,000 per annum. Not all respondents answered questions on all skills or concepts. The mean number of skills or concepts evaluated was 86 (SD 5 75) with only 18 respondents answering all 189. All skills or concepts evaluated were found to be at least somewhat important, were estimated to be used at least occasionally, were recommended for inclusion in training programs as core or elective, and some level of knowledge was expected. At least some of the respondents were taught each of the skills or concepts during their residency, practiced the skill or concept at least occasionally during their residency, and some degree of competency was expected at the time of completion of their residency. These data will provide a framework for designing future LAIM residency programs.

ABSTRACT This study evaluated pharmacokinetics and clinical safety of an oral paste formulation of a commercially available COX1-sparing NSAID in clinically healthy pony foals in a randomized controlled clinical trial. Values for complete blood count, serum chemistry profile, urinalysis, pharmacokinetic assay, and gastric endoscopy were evaluated in eighteen Shetland pony foals treated with firocoxib (0.1 mg/kg, PO, q 24 h) or placebo for 14 days. Foals were divided into 3 treatment groups. Group 1 and 2 foals received firocoxib while a 3rd group was administered an oral placebo. Gastric endoscopy was performed on group 1 and 3 foals prior to treatment and on days 7 and 14 to monitor for the presence of gastric ulcers. Group 2 and 3 foals had blood and urine samples taken sequentially for pharmacokinetic analysis, CBC, serum chemistry evaluation, and urinalysis. Physical examinations were performed prior to treatment and daily for 17 days. Data were analyzed using anova and paired t-tests (P o 0.05). None of the foals presented adverse clinical effects. There were no significant changes in CBC, biochemical profiles within groups, or differences between groups. Pretreatment gastric endoscopy scores were not significantly different from evaluations at 7 and 14 days. Firocoxib was quickly absorbed with an observed maximum concentration at 2 hr, the first sampling interval, for the majority of animals. Firocoxib plasma concentrations decreased in a log-linear manner after reaching the maximum concentration and steady state concentrations were achieved by the 7th dose. Based on the sampling times after the final and 14th dose, an average half life of 1.3 days was estimated. Administration of firocoxib did not cause any adverse effects on gastrointestinal, or hematological or serum biochemical variables, appears to have been well tolerated, and follows a predictable pharmacokinetic pattern in 4-6 week old foals. Equine herpesvirus 1 (EHV-1) is highly prevalent in most horse populations. Horses are routinely vaccinated against EHV-1, and neutralizing antibodies have helped to prevent disease. However, the USDA has recently classified EHV-1 myeloencephalopathy (EHM) as an emerging disease, in response to the apparent increase in incidence, morbidity, and mortality of EHM that suggests a change in virulence of the virus. It has been reported that cellular immune mechanisms, in particular cytotoxic T-cells (CTLs), are important in controlling EHV-1 viremia. Interferon-alpha (IFN-a) has a key function in innate immune regulation by inducing the differentiation and maturation of CTLs. Here, we investigated the influence of abortogenic (RacL11, NY03) and neuropathogenic (Ab4) EHV-1 virus strains on IFN-a, IL-4 and IL-10 secretion in equine PBMC. Equine PBMC were infected with RacL11, NY03 or Ab4 EHV-1 strains or kept in medium for 24 hours. IFN-a, IL-10 and IL-4 secretion was detected in the supernatants by a fluorescent bead-based cytokine assay. The production of IFN-a increased with increasing viral doses and similarly for all three EHV-1 strains. The production of the antiinflammatory cytokine IL-10 was significantly decreased after Ab4 infection compared to RacL11 and NY03 strains at viral infection doses of MOI 0.3-1. At high doses (MOI 3), IL-10 production was suppressed by all three EHV-1 strains. The results suggested that abortogenic and neuropathogenic EHV-1 strains equally induce antiviral IFN-a production in equine PBMC. They also illustrated the differences in the ability of EHV-1 strains to modulate anti-inflammatory IL-10. Neuropathogenic Ab4 strain had an increased potential to down-regulate IL-10 production suggesting specific viral mechanisms that interfere with the control of inflammation in the host. The variations in innate IL-10 secretion might influence the development of protective immunity and might offer an explanation why neuropathogenic Ab4 induces more severe disease, including myeloencephalopathy, than abortogenic EHV-1 strains.

Previously presented at a conference of research workers in animal disease. Rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. Treatment protocols are long, expensive and do not always produce good results. Rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. Recent studies are based on the use of the virulence associated protein A (vapA) which has been described as an important inducer of immunity against R. equi.

The present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of S. enterica typhimurium expressing VapA antigen (test group) or S. enterica typhimurium without the vapA gene (control group), previous to and following experimental challenge. Two experimental phases were established according to the immunization route: intranasal or oral vaccination up to 12 hrs following birth and at 14 days of age. The experimental and control groups were challenged on day 28 with a virulent stain of R. equi. Clinical examination, CBC and image complementary exams were used to evaluate the development of clinical signs. Immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation assays, isolation of R. equi and cytological profiles of TBW.

Clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group (2/3) and was due to R. equi pneumonia. The test group produced a more intense IgGb response when compared to controls however no statistical difference was observed. Lymphoproliferation and Th1 cytokine expression were higher in the test group. In contrast, controls produced an IL-4 response. Local IgA was significantly higher in animals immunized with Salmonella carrying vapA. Immunization protocols produced no severe toxic effects.

The vaccination of neonatal foals with S. enterica typhimurium expressing VapA was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental R.equi infection. This study was conducted to test the hypothesis that the 32 kD protein, Myristolated Alanine-Rich C-Kinase Substrate (MARCKS), is involved in equine neutrophil migration and adhesion.

In other species, MARCKS phosphorylation and dephosphorylation causes the protein to cycle between the cell membrane and cytosol, respectively. To investigate MARCKS phosphorylation in horses, neutrophils were isolated from whole blood and stimulated with platelet activating factor (PAF), leukotriene B 4 (LTB 4 ) or phorbol myristate acetate (PMA). Western blot was performed using specific phospho-MARCKS and total MARCKS primary antibodies. These results determined MARCKS phosphorylation is maximal 30 seconds following stimulation and that dephosphorylation occurs within 3 minutes.

To investigate the requirement for MARCKS in equine neutrophil chemotaxis, isolated neutrophils were pre-treated with MANS (a cell permeant peptide identical to the N-terminal 24 amino acids of MARCKS), RNS (a control peptide) or vehicle control (VC) prior to a migration assay toward known neutrophil chemoattractants (LTB 4 or PAF). Pre-treatment of equine neutrophils with MANS significantly inhibited migration while RNS pre-treatment had no effect.

To investigate MARCKS requirement in equine neutrophil adhesion, MANS, RNS or VC treated cells were stimulated to adhere to Immulon 2 plates coated with 5% FBS. Pre-treatment of equine neutrophils with MANS significantly inhibited adhesion while RNS pre-treatment had no effect.

Inhibition of MARCKS using a cell permeant peptide identical to the protein's N-terminus significantly inhibited equine neutrophil adhesion and migration. These results indicate that MARCKS is an important regulator of equine neutrophil chemotaxis and represents a potential target for anti-inflammatory therapy. Amongst other tests, a thorough neurologic examination of horses may include walking with the head elevated and during blindfolding, in order to help differentiate normal from abnormal and to help with neuroanatomically localising any lesion(s) i.e. in the ataxic horse. Consensus amongst equine neurologists suggests that gait abnormalities associated with these specific tests are often exacerbated in horses with underlying proprioceptive deficits however the effect of these tests on temporal gait characteristics in normal horses has not previously been assessed quantitatively. We hypothesized that head elevation or blindfolding, in comparison with walking in a straight line would result in a compensatory decrease in lateral (left front-on to left hind-on and right front-on to right hind-on) and diagonal coupling intervals (left front-on to right hind-on and right front-on to left hind-on) in normal horses.

Four Thoroughbreds without any history or clinical signs suggestive of neurological disease (age range 3 to 5 years) were included in the study. Retroreflective markers were applied to the withers, to the sacrum and to left and right tuber coxae; for each limb, lateral fetlock markers and dorsal and lateral hoof wall markers were used. A minimum of 3 trials each with 2-4 walk strides for each task were analysed as horses walked across an 8-force-plate runway i surrounded by a 12-camera kinematic system. ii Force-plate data were processed with semi-automated custom written Matlab iii scripts. Data were analysed with a mixed model using the statistical software R. There was a significant fixed effect of normal walk on a straight line and head elevation on left and right lateral coupling intervals (p o 0.0001) and of the left and right diagonal coupling intervals (p o 0.0001). There was no significant effect of blindfolding on neither lateral nor diagonal coupling intervals. The random effect of horse had no influence on the coupling intervals. The decrease of the lateral coupling intervals indicates a tendency towards a pacing gait during head elevation. We conclude that there is a significant change in temporal gait characteristics of non-neurologic horses when the head is elevated but not during blindfolding compared to normal walking. Current results suggest that pacing and increased variation in foot-placement during head elevation should be interpreted with caution however further work is required to determine whether the change differs between horses with neurological disease and non-neurologic disease. Hereditary Equine Regional Dermal Asthenia (HERDA) is an autosomal recessive connective tissue disorder associated with a mutation in cyclophillin B that leads to impaired collagen folding, aberrant wound repair, and corneal abnormalities. It affects young Quarter Horses, Appaloosa, and Paints. HERDA shows similarities to the human hereditary connective tissue syndrome Ehlers Danlos (EDS). Many EDS patients suffer from joint pain and osteoarthritis (OA) as adults. The similarity between EDS and HERDA raises the question whether horses suffering from HERDA develop OA. In OA, excess production of inflammatory mediators such as prostaglandin E2 (PGE 2 ) activate enzymes that degrade cartilage as well as impede wound healing. The present study examined articular cartilage from yearling horses afflicted with HERDA. We hypothesized that chondrocytes from these horses are continually activated to produce inflammatory mediators. To test this hypothesis, articular cartilage from carpal and tarsal joints of HERDA horses were evaluated using histology. PGE 2 production by chondrocyte cultures was measured by ELISA and analyzed by one-way ANOVA, Tukey post-hoc test, p o 0.05 significance. We also determined the antiinflammatory effects of an avocado/soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS) mixture (ingredients found in Cosequin s ASU) and phenylbutazone (PBZ) on chondrocytes. Cosequin s ASU and PBZ are used alone or in combination for the management of OA. Chondrocyte cultures were incubated for 24 hrs with control media alone, a clinically relevant concentration of PBZ (4 mg/ml), or the combination of ASU (NMX1000 s , 8.3 mg/ml) 1 GLU (FCHG49 s , 11mg/ml) 1 CS (TRH122 s , 20 mg/ml).

Articular cartilage from joints of five HERDA-afflicted horses showed gross and histologic evidence of osteoarthritic lesions. Chondrocyte cultures from cartilage of horses suffering from HERDA spontaneously produced greater PGE 2 than chondrocytes from normal horses (41000-fold). PBZ significantly decreased PGE 2 production by $90% (p o 0.001). The combination of ASU1-GLU1CS also significantly reduced PGE 2 production by $60% (p o 0.001). The present study supports anecdotal findings that horses suffering from HERDA are likely to develop OA. The inhibition of PGE 2 synthesis by ASU1GLU1CS suggests that this combination may be beneficial for the management of OA in HERDA.

Research supported by Nutramax Laboratories, Inc. Equine Inflammatory Airway Disease (IAD) is a common condition often treated empirically with corticosteroids. Gene expression analysis in the bronchoalveolar lavage fluid (BALF) may help understand the effects of corticosteroids in IAD. The first part of the study aimed at identifying reference genes in the BALF of IAD horses treated with corticosteroids. The second part of the study investigated the effects of dexamethasone and fluticasone propionate treatments on the mRNA expression of IL-1b, IL-4, IL-8 and IL-17.

The expression stability of seven candidate reference genes was determined in BALF taken pre-and post-treatment with dexamethasone and fluticasone propionate in horses with IAD. Primers' efficiencies were calculated using LinRegPCR. NormFinder, GeNorm and qBasePlus softwares were used to rank the genes according to their stability. GAPDH was the most stably expressed gene whereas B2M was the least stable gene. In addition, GeNorm analysis revealed that the number of genes required for optimal normalization was four (GAPDH, SDHA, HPRT, RPL32).

In the second part of the study the mRNA expression of IL-1b, IL-4, IL-8 and IL-17 was measured in BALF samples from seven IAD horses treated in a randomized cross-over design with dexamethasone (0.05 mg/kg SID, 15 days) or inhaled fluticasone propionate (3000 mcg BID with Aerohippus, 15 days). The BALF samples were taken at baseline and after each treatment period. There was no significant effect of the corticosteroids treatment on the mRNA expression of IL-1b, IL-4 and IL-8 in the BALF. The mRNA expression of IL-17 was suppressed by dexamethasone and fluticasone propionate treatments. Pneumonia is observed in horses after long distance transportation in association with confinement of horses' head position leading to a reduction in tracheal mucociliary clearance time (TMCT). We hypothesize that clenbuterol, a beta-2 agonist shown to increase TMCT in the horse, will ameliorate the affect of a fixed head position on large airway contamination and inflammation in a long-distance shipping model. Six adult horses were enrolled in a cross-over design prospective study. Horses were housed with their heads in a fixed position for 48 hours to simulate long distance transport, and treated with clenbuterol (0.8 ug/kg PO q12 h) or a placebo starting 12 hours before simulated shipping. TMCT was measured using a charcoal clearance technique. Data were collected at baseline and 48 hours, and included TMCT, tracheal wash cytology and quantitative culture, rectal temperature, CBC, fibrinogen, and serum TNFa, IL-10 and IL-2 levels. There was a 3-week washout between study arms, and each horse served as its own control. The data was analyzed using regression analysis and Wilcoxon rank-sum tests. No statistically significant difference was seen between treatment and placebo groups for any of the variables investigated. TMCT did not differ after treatment (1.71 AE 0.64 cm/min) versus placebo (1.55 AE 0.82 cm/ min; P 4 0.10), and intratracheal bacterial counts were similar for treatment (105 Â 10 3 AE 42 Â 103 cfu; P 4 0.10) and placebo (98 Â 10 3 AE 41 Â 103 cfu) groups. A reduction of tracheal b hemolytic Streptococcus. spp. after clenbuterol versus placebo was also nonsignificant (0% versus 33%; P 4 0.10). In conclusion, treatment with clenbuterol does not appear to combat the deleterious effects of this long-term shipping model. Breathing cold air during strenuous exercise is associated with airway inflammation. Under these conditions, warming and humidification of inspired air occurs in the lower respiratory tract resulting in mucosal cooling, desiccation, and hyperosmolarity. The purpose of this research was to test the hypothesis that airway hypertonicity causes inflammatory cell migration and alterations in cytokine expression associated with exercise induced airway inflammation.

Horses (n 5 9) were examined in a randomized crossover design after exposure to hypertonic aerosols (5 minute nebulization with solutions of either isotonic or hypertonic mannitol). Airway leukocytes were harvested 5 and 24 hours post aerosol challenge via bronchoaveolar lavage, and were used to determine total and differential nucleated cell count and expression of cytokinespecific mRNA.

Hypertonic aerosol challenge resulted in an increase in total number of cells 5 hr after challenge, characterized by increased macrophage (p 5 0.04) and neutrophil (p 5 0.03) concentrations, but there was no effect on airway leukocyte concentrations 24 hours after nebulization. No significant changes in the relative quantity of mRNA for airway cytokines were noted at either time point.

These data demonstrate that transient airway hypertonicity can cause airway leukocyte influx and may be responsible for the airway inflammation commonly found in athletes that exercise in cold conditions. However, our data do not support the hypothesis that hypertonicity is the sole initiating cause of changes in cytokine expression secondary to cold weather exercise. It is likely that factors such as airway temperature, shear stress or epithelial damage also play a role in this phenomenon. We studied the importance of abdominal sonograms in neonatal foals suffering from gastrointestinal conditions. We hypothesized that there would be a subgroup of neonates with sonographically detectable pneumatosis intestinalis (PI) as a reflection of a necrotizing component of the disease.

Records of foals 7 days of age hospitalized between 2005 and 2009 with signs of gastrointestinal disease were evaluated (N 5 89). The association of sonographic, clinical, pathological and clinicopathological signs with outcome and severity of disease was determined.

Pneumatosis intestinalis was imaged in 19 foals. Twenty-seven foals were classified as having necrotizing gastrointestinal disease based on the presence of gastrointestinal signs (colic, diarrhea, gastric reflux or abdominal distension) and PI detected sonographically (19), surgical (2) or pathological (6) evidence of gastrointestinal necrosis. There was a difference between overall survival rate (58%) and survival rate in foals with necrotizing disease (33%, P 5 0.005) or foals with PI detected sonographically (37%, P 5 0.02). Pneumatosis intestinalis was the only sonographic finding associated with outcome. Sonographic abnormalities in peritoneal fluid, stomach, duodenum, jejunum, cecum, umbilicus or the presence of meconium were associated (P o 0.05) with surrogates of severity of disease (hospitalization cost or days of hospitalization). Hypoproteinemia was associated with PI (P 5 0.02). The presence of blood in the feces, reflux and abdominal distension was associated with necrotizing gastrointestinal disease (P o 0.05).

Abdominal sonograms have prognostic value in neonatal gastrointestinal disease. Pneumatosis intestinalis was a common sonographic sign that worsened the prognosis. The therapeutic implications of detecting a necrotizing component of the gastrointestinal disease deserve further study. The interaction of insulin and the microvascular endothelial insulin receptor (IRc) plays an important role in the normal and insulin resistant (IR) individual. While endothelial IRc signaling is normally vasodilatory, this effect is well-documented to reverse in the IR individual, resulting in vasoconstriction. Although vascular dysfunction has been reported in sepsis-associated equine laminitis, the role of the laminar microvasculature in endocrinopathic laminitis remains poorly characterized. The purpose of this study was to characterize the pattern of IRc expression in digital laminae in ponies subjected to a dietary carbohydrate challenge that mimicked abrupt exposure to pasture rich in nonstructural carbohydrates (NSC). Mixed-breed ponies (body weight 270.9 1/-74.4 kg) received a diet of hay chop (NSC $6% on a DM basis) for 4 weeks prior to initiation of the experimental feeding protocol. Following conditioning, ponies either remained on the control diet (n 5 11) or received the same diet supplemented with sweet feed and oligofructose (total diet $42% NSC; n 5 11) for a period of 7 days. Serum insulin concentrations were measured prior to and after completion of the feeding protocol. At the end of the feeding protocol, sections of numerous tissues, including dorsal digital laminae, were collected immediately following euthanasia. The samples were formalin-fixed for 48 hours, transferred to 70% ethanol, and paraffin-embedded. Laminar sections were stained immunohistochemically for IRc using a commercially-available antibody (Abcam); the number of IRc (1) cells was quantified in 40x light microscopy fields (n 5 10) for each section. The total number of IRc (1) cells was greater in the laminae of challenged ponies than control ponies (p 5 0.0096), and there was a significant correlation between the change in serum basal insulin concentration and number of laminar IRc (1) endothelial cells (r 5 0.74; p o 0.05). While the number of IRc (1) endothelial cells was significantly greater in the dermal laminae of challenged ponies (p 5 0.0095), there was no difference in the number of interstitial IRc (1) cells (p 5 0.82). No epithelial IRc (1) cells were observed in any laminar section, and IRc (1) cells were conspicuously absent from the deep dermal tissue (including vessels). Up-regulation of IRc expression in the laminar vasculature occurs acutely in response to dietary carbohydrate challenge and accompanies hyperinsulinemia in ponies. The dramatic increase in endothelial IRc expression in the laminar microvasculature in nutritionally challenged ponies, with no apparent epithelial IRc present, suggests that hyperinsulinemia associated with exposure to increased dietary NSC may induce laminar injury by causing a similar vasoconstriction in IR equids as described in the microvasculature of IR humans. Glucose transport from the blood stream into cells, the limiting step in whole-body glucose utilization, is regulated by a family of glucose transporter (GLUT) proteins in insulin-sensitive (i.e., muscle and adipose) tissues. We previously demonstrated that GLUT4, the major isoform, is a key factor in the pathogenesis of equine insulin resistance (IR). While it has been recently demonstrated that GLUT12 (a newly discovered isoform) increases insulin-stimulated glucose transport in human muscle, its role in other tissues, particularly in the setting of IR, is not well characterized in any species. In addition, AS160 has recently emerged as a key downstream signaling molecule regulating translocation of GLUT to the cell surface, the rate-limiting step in glucose uptake. We hypothesized that GLUT12 content would be differentially expressed across tissues and that IR would induce alteration in glucose transport by affecting active cell surface GLUT12.

Biopsies of skeletal muscle, and subcutaneous and visceral adipose tissue were collected from light-breed horses, characterized as either insulin sensitive or compensated IR, based on the results of an insulin-modified frequently-sampled intravenous glucose tolerance test (n 5 5/group). We specifically quantified active cell-surface GLUT12 in these biopsies, using an innovative exofacial bismannose photolabeled assay, which has not been previously applied to GLUT12. Total GLUT12 protein expression was measured by Western blots, as well as total and phosphorylated (indicating activation of) AS160.

GLUT12 was expressed in all the depots with a significant regional effect. Total GLUT12 protein content was increased (P o 0.05) in visceral (omental and mesenteric) compared to subcutaneous (nuchal ligament and tailhead) adipose tissue and skeletal muscle of healthy horses. IR did not induce alterations in active cell-surface and total GLUT12 content nor in total and phosphorylated AS160 in any of the tissues evaluated.

Our data suggests that GLUT12 is abundant in visceral adipose tissue and is therefore likely to play a substantial role in the regulation of glucose transport. However, neither GLUT12 translocation nor AS160 activation are impaired in insulin-sensitive tissues of IR horses. It is concluded that, in contrast with GLUT4, GLUT12 does not appear to contribute to glucose transport alterations during naturally-occurring equine IR. Insulin resistance (IR), characterized by exaggerated glycemic or insulinemic responses to glucose challenge, is a key metabolic disturbance in horses that develop obesity-associated laminitis. In addition to obesity, diet and age have been demonstrated to affect tissue sensitivity to insulin in other species but these factors have received limited investigation in horses. We hypothesized that there would be greater glycemic and insulinemic responses to a sweet feed meal in aged horses, as compared to adult horses, as well as in horses adapted to a forage-only diet. Three diets, grass hay only, grass hay plus sweet feed (starch and sugar-rich, SS), and grass hay plus a fat and fiber (FF) feed, were fed to 17 mares, 8 adult (5-12 yr) and 9 aged (4 19 yr), for a 6-week adaptation period in a randomized design. Glycemic and insulinemic responses to a standardized meal of sweet feed (4 g/kg BW offered for 1 hour) were determined for 6 hours from the onset of feeding. Peak glucose and insulin concentrations and areas under the glucose or insulin vs. time curves (AUC-G, mg/ dl/360 min, and AUC-I, mU/ml/360 min) were determined and data were analyzed by one-and two-factor repeated measures ANOVA. There were no differences between age groups in glycemic responses to any of the diets. However, in aged horses peak glucose concentration (p o 0.03) and AUC-G (p o 0.01) were greater after adaptation to the forage-only diet, as compared to the other two diets. In contrast, aged horses had a greater peak insulin concentration (p o 0.05) and AUC-I (p o 0.03) than adult horses on all diets but no differences in peak insulin concentration or AUC-I was found between diets within age groups.

As hypothesized, the insulin response, but not the glycemic response, to a sweet feed meal was greater in aged horses, regardless of background diet. Further, the glycemic response was greatest after adaptation to a forage-only diet, but this finding was only significant in aged horses. morbidity, mortality, and economic loss to the equine industry. In obese humans and rodent models of nutritional obesity, systemic insulin resistance and hyperinsulinemia are followed temporally in a majority of individuals by decreased glucose tolerance, pancreatic bcell failure, and type II diabetes mellitus. In stark contrast to humans, obese horses and ponies chronically remain in what is termed a ''prediabetic'' state in human IR, characterized by hyperinsulinemic euglycemia. Few data exist describing the biology of the equine endocrine pancreas in the chronically IR animal that may both: 1) explain this unique equine endocrine physiology and 2) characterize the animal at-risk for hyperinsulinemia-associated laminitis. The purpose of the study reported here was to characterize the morphology and physiology of the equine endocrine pancreas in response to a dietary carbohydrate challenge. Twenty-two mixedbreed ponies (body weight 266.6 AE 170.5 kg) were conditioned to a diet of chopped hay (NSC $6% on DM basis) for 4 weeks; following conditioning, ponies either remained on the control diet (n 5 11), or received the same hay supplemented with sweet feed and oligofructose (total diet $42% NSC; n 5 11) for 7 days. Serum insulin concentrations were measured prior to and after completion of the feeding protocol. At the end of the feeding protocol, sections of numerous tissues, including pancreas, were collected immediately following euthanasia. The samples were formalin-fixed for 48 hours, transferred to 70% ethanol, and paraffin-embedded. Immunohistochemistry was performed on pancreas sections using a commerciallyavailable anti-insulin antibody (Abcam), and measurements of islet surface area and b-cell surface area were performed (n 5 10 islets per tissue section) using a commercially-available computer software program (Image J). There was a trend for greater total islet surface area in pancreatic tissue from ponies fed the high NSC diet when compared to the ponies on the hay diet (p 5 0.068); however, no difference was noted in b-cell surface area between diet treatments (p 5 0.12). The change in serum insulin concentration was significantly greater in the high NSC-fed ponies than in controls (403.8 1/-317.1 mIU/L vs. 1.00 1/À 4.03 mIU/L; p 5 0.002); however, this variable was not correlated with total islet surface area (r 5 0.32; p 5 0.17) or b-cell surface area (r 5 0.25; p 5 0.3). Due to the relatively modest changes in pancreatic islet surface area that accompany marked increases in serum insulin concentrations in ponies fed a high NSC diet, it is important to assess both b-cell function and insulin clearance mechanisms in future studies to delineate the mechanism(s) of hyperinsulinemia in this model. Humans that suffer from obesity show exaggerated inflammatory responses and this may be relevant to the association between increased adiposity and laminitis in horses with Equine Metabolic Syndrome (EMS). This study was performed to test the hypothesis that inflammatory responses to endotoxemia differ between healthy horses and those affected by EMS.

Six healthy adult mares and 6 horses with EMS received an intravenous infusion of lipopolysaccharide (LPS; 20 ng/kg in 60 mL sterile saline) or saline alone. A crossover design was employed with a 7-day washout period. Physical examinations were performed hourly for 9h and whole blood was collected at 30, 60, 90, 120, 180, and 240 min for assessment of inflammatory cytokine gene expression. A liver biopsy was performed between 240 and 360 min postinfusion. Data were analyzed using mixed model ANOVA.

Mean rectal temperature, heart rate, and respiratory rate increased following LPS infusion (treatment  time; P o 0.001), with higher heart (group  treatment; P 5 0.087) and respiratory rates (group; P 5 0.017) detected in EMS horses. Lipopolysaccharide infusion significantly increased whole blood gene expression of tumor necrosis factor a (TNFa), interleukin (IL)-1b (P o 0.001), IL-6 (P o 0.001), IL-8 (P o 0.001), and IL-10 (P 5 0.002), and hepatic gene expression of IL-6 (P o 0.001), IL-8 (P o 0.001), and IL-10 (P 5 0.016). Inflammatory gene expression did not differ significantly between groups, so our hypothesis was not supported. Heart rates tended to be higher when LPS was administered to horses with EMS. Elevated serum concentration of cardiac troponin I (cTnI) is a biomarker for myocardial damage in horses. Preferred times to test blood for cTnI levels following athletic performance or other events that may cause myocardial injury are not yet established and would be affected by time of release from the myocytes, location of release within the myocytes, duration of release and half-life of cTnI in the horse. This information would be necessary to more accurately and reliably test horses for myocardial injury. The aim of this study was to determine the elimination half-life (T1/2) of equine cTnI. To establish the T1/2 of equine cTnI in horses, cTnI was recombinantly expressed in E.coli. Two healthy ponies received intravenous injections of recombinant equine cTnI and plasma cTnI concentrations were measured with a point-of-care cTnI analyzer at multiple time points after injection. Standard pharmacokinetic analysis was performed to establish the elimination half-life of cTnI. For comparative purposes, data were subjected to pharmacokinetic models describing a single versus biphasic elimination profile. Elimination of recombinant equine cTnI following intravenous administration exhibits a short half-life. Establishing the T1/2 of troponin provides critical information in understanding the clinical application of this cardiac biomarker in clinical practice. This study describes a true biological cTnI T1/2, which has not been documented in any species thus far. Stall-side assessment of this cardiac biomarker in horses should enhance the ability of clinicians to detect myocardial damage and aid in the management and treatment of horses with cardiac disease. The objective of the study was to evaluate the between-pony, within-pony, between-analyser and within-analyser variation of flow-mediated vasodilation (FMD) measurement in healthy ponies, to investigate the hypothesis that FMD occurs in healthy ponies.

Six healthy, native breed, unrelated pony mares of varying weight (236-406 kg), body condition score (3/9-7/9) and age (14-25 years) were used. The median artery was occluded for 5 minutes. Twodimensional (2D) ultrasonographic images of the artery were recorded for 30 seconds prior to and for 2 minutes after occlusion. The peak luminal diameter was compared to baseline diameter to calculate the relative percentage increase in luminal size (FMD). Images were obtained from six ponies on one occasion and from one pony on six occasions. Analysis of images was performed by two independent analysers and by one analyser twice.

The mean (SD) FMD in 6 ponies was 12.57% (4.28%) and in 1 pony (6 occasions) was 7.30% (2.11%). Coefficients of variation were 34.09% and 28.84% respectively. Agreement between analysers was fair (ICC 5 0.47) and within analyser was poor (ICC 5 0.30).

FMD is used to assess endothelial function in humans and has recently been assessed for its use in canine subjects. FMD occurs and measurement is feasible in ponies. FMD could be used to assess endothelial function, in the context of laminitis or other cardiovascular diseases. Current state-of-the-art technique for measuring blood pressure (BP) in the horse is invasive and involves cannulation of the facial artery. Indirect techniques, such as oscillometry, have proven useful in the anaesthetised horse, but have not become routine in the standing horse. Monitoring BP can be indicated for the diagnosis and treatment of the hypotensive patient (ie. caused by endotoxemia, hypovolemia, Systemic Inflammatory Response Syndrome and cardiac failure) or the hypertensive patient (ie. due to equine metabolic syndrome or pain). The objective of this study was therefore to a) describe the methodology for application of oscillometric BP using a cuff applied to the tail in the standing horse and b) and to determine accuracy and precision of this method applied to the normotensive standing horse.

The oscillometric method is simple to apply in a clinical setting. A pneumatic cuff is snugly applied to the unclipped tail-base with the cuff bladder centered over the middle coccygeal artery. The tail circumference must match the manufacturers description of the cuffs diameter range. The oscillometric apparatus inflates the cuff and obtain systolic, diastolic and mean arterial BP (SAP, DAP and MAP). At least 2 consecutive measurements must be obtained. A correction of 0.7 mmHg/cm vertical distance between cuff and heart level is added to the measurement to correct for hydrostatic pressure difference.

For determination of accuracy and precision of indirect SAP, DAP and MAP, eight healthy horses (age 3 to 16 years), was equipped with an intra-arterial catheter ii in the facial artery and a commercial tail-cuff oscillometric apparatus. i Measurements were recorded every 2 minutes for 20 minutes. The data were analysed with the statistical software R using a mixed model with repeated measurements and a Bland-Altman analysis corrected for repeated measurements.

Oscillometric BP was accurate and precise for MAP (mean bias, lower confidence level, upper confidence level, variation in difference, all mmHg) (À0.3, À18.5, 19.1, 33.2, respectively) in the conscious horse but not for SAP (À1.5, À19.3, 16.3, 38.2, respectively) and DAP (0.05, 15.9, 16.0, 49, respectively) . There was no significant contribution to the statistical model of either horse or measurement number.

All horses tolerated the tail-cuff well and the method was simple to apply. Only MAP could be measured with acceptable accuracy and precision in the normotensive standing horse using the described oscillometric method. Reference intervals for thyroid hormone (TH) concentrations have not been established for donkeys. Therefore, clinicians must use reference ranges from horses, potentially leading to misdiagnosis of thyroid diseases. We hypothesized that TH concentrations are different between donkeys and horses. The purposes of this study were: a) to compare TH concentrations between donkeys and horses and, b) to determine whether the age may influence TH concentrations. Thirty-eight healthy donkeys (8.5 AE 0.8 years), mixed breeds, and 20 healthy Andalusian horses (6.4 AE 0.5 years) were used. Donkeys were divided into three groups: o 5 years (n 5 13), 5-10 years (n 5 12), and 411 years (n 5 13). Serum concentrations of total triiodothyronine (tT3), free triiodothyronine (fT3), total thyroxine (tT4), free thyroxine (fT4), reverse triiodothyronine (rT3) and thyroid-stimulating hormone (TSH) were quantified by radioimmunoassay. All blood samples were collected the same day. Neither horses nor donkeys had received any treatment for 30 days before sampling and both farms had similar production conditions. Total T3, fT3, fT4 and tT4 concentrations were higher (P o 0.01) in donkeys than horses. In contrast, no statistical differences were found for rT3 and TSH concentrations. Young donkeys ( o 5 years) had higher fT4, tT4 and rT3 concentrations compared to other donkey groups (P o 0.05). Old donkeys (411 years) had lower tT3 and fT3 concentrations than both younger donkeys groups (P o 0.05). This study shows that there are differences in TH concentrations between donkeys and horses, raising awareness on the possibility of misdiagnosis of thyroid gland dysfunction when using values from horses, being necessary to determine exclusive reference intervals for donkeys. Ovariectomy is associated with alterations of responses to many hormones, not just those associated with reproductive function. In humans and rats, ovariectomy leads to insulin resistance, increased adiposity and altered fat mobilization. The effects of ovariectomy on energy metabolism have not been reported in horses. Ovariectomized mares have been shown to respond normally to an ACTH stimulation test, but the response to suppression of the hypothalamo-pituitary-adrenal axis has not been previously described. The aim of this study was to evaluate the effect of ovariectomy on insulin response in mares and to determine if mares exhibit alterations in response to dexamethasone administration after ovariectomy.

Six healthy mares underwent an intravenous glucose tolerance test (IVGTT), an insulin sensitivity test (IST) and a dexamethasone suppression test (DST) before and 5 weeks after bilateral ovariectomy. Body weight, cortisol values at baseline, 15 and 24 hours after dexamethasone injection and ACTH values at baseline, 15 and 24 hours after dexamethasone injection, basal insulin/glucose ratio, time to reach a 60% decrease in blood glucose in the IST, time to reach baseline glucose concentration in the IVGTT and area under the curves plotting blood glucose and time to injection of glucose or insulin were compared before and after ovariectomy using a paired t-test or an ANOVA for repeated measures. Significance level was P o 0.05.

Average body weight was decreased after surgery (6kg ). The injection of dexamethasone resulted in a serum cortisol concentration of less than 1 mg/dL in all mares before ovariectomy, whereas after ovariectomy, dexamethasone injection resulted in a serum cortisol concentration of less than 1 mg/dL in 5 out of 6 mares. In all cases, ACTH concentration was within the reference range (9-35 pg/mL) before and after ovariectomy. However, ACTH concentrations at t 0 and at t 15 were significantly higher after ovariectomy. Each mare had a normal IVGTT, both before and after ovariectomy. Additionally, no significant differences were observed in basal blood glucose (84 AE 12 mg/dL before and 85 AE 3 mg/dL after) or in the time to reach glucose baseline (108 AE 66 min before and 99 AE 51 min after). Serum basal insulin concentration and insulin/glucose ratio was not significantly different before or after ovariectomy (22.0 AE 7.9 mIU/mL and 17.5 AE 8.9 mIU/mL and 0.26 AE 0.09 and 0.20 AE 0.10, respectively), nor was the average time to reach a 60% decrease in blood glucose after insulin injection (30 AE 7 min and 25 AE 9 min, respectively).

These findings suggest that, as reported in other species, the shortterm effect of ovariectomy may modify dexamethasone response in mares and that, contrary to other species, it may not modify insulin response. Equine Gastric Ulcer Syndrome (EGUS) is a common medical problem in horses. The high prevalence of gastric ulcers, vague clinical signs and negative effect on performance make it a significant clinical and economic problem within the horse industry. Current pharmaceutical treatments are expensive and alter the acidic environment of the stomach. Berries and pulp from the seabuckthorn plant (Hippophae rhamnoides) are a rich source of vitamins, trace minerals, amino acids, antioxidants, and other bioactive substances and have been used successfully to treat stomach ulcers in man and rats. The purpose of this study was to evaluate the efficacy of a commercially sold, liquid extract of seabuckthorn berries (SeaBuck TM SBT Gastro-Plus) for treatment and prevention of gastric ulcers in horses.

Eight Thoroughbred and Thoroughbred-cross horses (3-10 years of age, 5 geldings & 3 mares, 380-600 kg) were used in a blinded two-period cross-over study. Treatments consisted of control (untreated) and treatment (SeaBuck TM SBT Gastro-Plus) twice daily mixed with the grain meal. Horses were treated for 5 weeks followed by a 1 week alternating feed-deprivation period to induce or worsen existing ulcers. Gastroscopies were performed on all horses on day 0, week 5, and week 6 (at the end of the alternating feed-deprivation period). Gastric juice was aspirated and pH was measured. During gastroscopy, gastric ulcer scores were assigned to each stomach based on lesion number and severity. Horses acted as their own controls, and between each treatment period the horses had a 2-week washout period. Data was analyzed by ANOVA for repeated measures via the GLM procedure (SAS Inst. Inc., Cary, NC). When significant differences (P o 0.05) were observed, a post-hoc Tukey's test was used to determine differences.

Non-glandular gastric ulcer scores significantly increased in all control and SBT-treated horses from Week 5 to Week 6, after the feed-deprivation phase of the study. There was no significant difference in the non-glandular gastric number (P 5 0.84) and nonglandular gastric severity (P 5 0.51) scores in SBT-treated horses compared to non-treated controls. Glandular ulcer number (P 5 0.02) and glandular ulcer severity (P 5 0.02) was significantly lower in the SBT-treated horses compared to the control horses. There was no significant difference in the pH (P 5 0.06) in SBT-treated horses compared to non-treated controls. Thus, SeaBuck TM SBT Gastro-Plus, mixed in the feed twice daily, may be efficacious in controlling the severity of glandular ulcers in horses during stress, without increasing stomach pH. The availability of rapid and accurate quantitative fibrinogen measurements may be useful for evaluation of hospitalized equine patients. The Abaxis VSPro analyzer was evaluated for precision using two levels of human fibrinogen controls (300 mg/dL and 150 mg/dL), four different VSPro machines, and two different lots of cartridges, assessed over 5 subsequent days. The coefficients of variation of the assay ranged from 4% (300 mg/dL) to 8% (150 mg/ dL). We subsequently evaluated the Abaxis VSPro fibrinogen assay compared to fibrinogen concentration measured using the Beckman Coulter ACL-1000 in 50 equine samples of varying fibrinogen concentrations obtained from horses with gastrointestinal disease. All samples were measured in citrated plasma. Fibrinogen samples measured on the ACL-1000 ranged from 226 to 959 mg/dL (median 501 mg/dL). VSPro samples were run in duplicate, and the mean compared to the ACL values. Pearson correlation coefficient analysis generated an r value of 0.949 (P o 0.001). Duplicate measurements on the VSPro were strongly correlated to each other with an r value of 0.9690 (P o 0.001). Bland-Altman analysis of these samples for the VSPro compared to the ACL-1000 noted a bias of À84 AE 57 mg/dL The results of this study indicate that the VSPro benchtop fibrinogen analyzer provides accurate and precise fibrinogen data compared to the ACL-1000 reference analyzer. The immune response of foals to R. equi is incompletely understood and believed to be responsible for clinical disease caused by this pulmonary pathogen. In a recent study foals receiving a large inoculum exhibited Th2 skewing with pneumonia and a small inoculum exhibited Th1 skewing without clinical disease. We hypothesized that cytokine/chemokine production by pulmonary alveolar macrophages, in vitro, would increase with the infective dose and that the magnitude of the response would differ between foals and adults. Alveolar macrophages were obtained by bronchoalevolar lavage from 7 healthy mares and their 5-week-old foals. Macrophage cultures were infected with R. equi (337011 or 33701-) at a multiplicity of infection (MOI) of 1 or 100. Total RNA was harvested 4 and 24 hours post-infection, reverse transcribed and used as template for quantita-tive PCR. The DDCt method was used to calculate relative gene transcripts for IL-6, IL-12p40, TNFa and CXCL10.

Cellular infections at MOI 100 resulted in significantly higher expression of IL-6, IL-12p40 and TNFa mRNA transcripts compared to MOI 1. However, the dose-effect was reversed for CXCL10 with significantly lower expression at the higher MOI. There was no difference in magnitude of cytokine/chemokine responses by the alveolar macrophages between adults and foals.

Dose-dependent responses of alveolar macrophages may represent a novel mechanism by which R. equi could modulate immune responses and therefore disease. Significant down-regulation of CXCL10 mRNA transcripts associated with a higher dose is of particular interest as this chemokine plays a role in development of protective Th1 responses. The intent of this study was to develop likelihood ratios (LRs) for infection attributable to Corynebacterium pseudotuberculosis in horses based on synergistic hemolysis inhibition (SHI) test titers. Medical records for horses presented to the UC Davis Veterinary Teaching Hospital with serum submitted for SHI titer determination were evaluated and 171 cases met study inclusion criteria. These cases were grouped based on evidence of internal and/or external infection attributable to C. pseudotuberculosis and likelihood ratios with 95% confidence intervals determined.

Results showed increasing LRs indicating increasing odds for any form of active disease as titer increased with all cases considered. LRs for internal infection were 4 1 for titers ! 1280 overall and for titers 4 160 with external abscess cases excluded. No difference from 1 (and therefore no significant change in pre-test to post-test odds) was seen in any LRs for internal disease when only cases with external disease were examined (external and internal disease vs. external only). Overall, the SHI test results showed usefulness in determining internal C. pseudotuberculosis infection in horses with no evidence of external abscessation. Overall, however, higher titers were more indicative of active external or internal disease than internal disease specifically in contrast to previous reports. The SHI test was unable to distinguish internal infection when external abscesses were present. Salmonella enterica is a zoonotic pathogen that has tremendous impact on many different animal production and management systems. Rapid detection of S. enterica in fecal samples may facilitate effective infection control practices. Current detection methods require 24-48 hours (Polymerase Chain Reaction or PCR) or 48-72 hours (enriched aerobic culture) to obtain results. Alternatives have been developed, lateral flow antigen detection systems (LFADS), which are currently marketed for Salmonella detection related to food safety microbiology. The objective of this study was to evaluate two commercially available rapid Salmonella detection systems in equine feces.

Fecal samples collected from repeatedly culture-negative horses were inoculated with known concentrations of Salmonella enterica serotype Typhimurium (five uninoculated control samples, and 5 samples of each 10-fold dilution [1.9 Â 10 0 -1.9 Â 10 4 cfu/gram of feces]). All samples were aerobically cultured using a standard enrichment technique. In a blinded fashion, samples were tested using two different LFADS as well as plated on agar media for confirmatory testing.

At 24 hours of incubation, using bacterial culture as the reference method, Test 1 was correctly identify 70% of samples ( Bacterial contamination of stalls with Salmonella sp. is a serious problem in equine hospitals. Salmonella sp. exposure to horses in the facility can result in nosocomial infections which results in temporary facility closure, until the organism is eradicated. Hospital closure can result in loss of revenue, damage to reputation and interference with patient care. The purpose of this study was to evaluate three stall cleaning methods on eradication of Salmonella sp. at an equine veterinary teaching hospital (VTH).

Horses admitted to the VTH were assigned to Salmonella sp.negative stalls within areas of the VTH during the study period (September 2009 -January 2010 . When the horses were discharged stalls were randomly assigned to one of three cleaning methods (pressure-washing only [PW] , pressure washing and hand scrubbing [PWS] , or hand scrubbing only [S]) in a single period, non cross-over design. All stalls were stripped of bedding and surfaces sprayed with tap water and cleaned with a disinfectant quaternary-ammonia solution (Super HDQ Neutral, Spartan Chemical Co., Inc, Maumee, OH). The pressure-washing system (PSC Cleaning Systems, Inc., Toronto, Canada) used, provided a pressure of 3000 psi and a temperature range of 1851-2151F. Following cleaning, each stall was allowed to air dry and within 48 hours, stall surfaces were sampled using three 4 00 Â 4 00 sponges moistened with sterile saline. The person collecting the samples was masked to the method of cleaning. Sponges were submitted to the Louisiana Animal Disease Diagnostic Laboratory (LADDL) for culture of Salmonella sp. A Chi-Squared analysis was used to determine significant differences (limit p o 0.05) between cleaning methods and Salmonella sp. isolation. During the study period, 112 stalls (PW [n 5 29]; PWS [n 5 50]; S [n 5 33] were included. All stalls had negative environmental salmonella sp. cultures prior to beginning the study. For PW cleaned stalls, 6/23 (20.7%) were Salmonella sp.-positive, for PWS cleaned stalls, 12/38 (24%) were Salmonella sp.-positive, and for S cleaned stalls, 4/29 (12.1%) were Salmonella sp.-positive. Although, there were fewer Salmonella sp.-positive stalls (12.1%) in the handscrubbed stalls, cleaning method did not significantly (p 5 0.4057) affect the isolation of Salmonella sp. from the stall environment.

In conclusion, power washing alone, power washing and hand scrubbing, and hand scrubbing alone, using a quaternary-ammonia solution did not significantly affect environmental isolation of Salmonella sp. from stalls surfaces in the VTH during this study. The objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) and Rhodococcus equi isolates. A single dose of gamithromycin (6 mg/kg of body weight) was administered intramuscularly. Concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (PELF), bronchoalveolar lavage (BAL) cells, and blood neutrophils were determined using HPLC with tandem mass spectrometry detection. The minimum inhibitory concentration of gamithromycin required to inhibit growth of 90% of R. equi and S. zooepidemicus isolates (MIC 90 ) was determined. Additionally, the activity of gamithromycin against intracellular R. equi was measured.

Mean peak gamithromycin concentrations were significantly higher in blood neutrophils (8.35 AE 1.77 g/mL) and BAL cells (8.91 AE 1.65 g/mL) compared to PELF (2.15 AE 2.78 g/mL) and plasma (0.33 AE 0.12 g/mL). Mean terminal half-lives in neutrophils (78.6 h), BAL cells (70.3 h), and PELF (63.6 h) were significantly longer than that of plasma (39.1 h). The MIC 90 of S. zooepidemicus isolates was 0.125 g/mL. The MIC 90 of gamithromycin for macrolide-resistant R. equi isolates (128 g/mL) was significantly higher than that of macrolide-susceptible isolates (1.0g/ mL). The activity of gamithromycin against intracellular R. equi was similar to that of azithromycin and erythromycin. Intramuscular administration of gamithromycin at a dosage of 6 mg/kg would maintain PELF concentrations above the MIC 90 for S. zooepidemicus and phagocytic cell concentrations above the MIC 90 for R. equi for approximately 7 days. Eight Western stock yearling horses were infected with EHV-1 (Ab4) by nasopharyngeal instillation. Venous blood samples for collection of plasma were collected in Na-citrate tubes on the day prior to infection (D -1) and on D4 through D11. In addition, clinical data, nasal swabs and peripheral blood mononuclear cells (PBMC) for detection of viremia were collected on the day before infection (D -1) and on D1 through D14 post-infection. D-dimer concentrations were determined in citrated plasma samples using a latex agglutination test (Minutex D-dimer, biopool, Ireland). Viral load in PBMC was determined using quantitative PCR.

All horses showed bi-phasic fevers typical for EHV-1 infections. One horse developed acute EHM on D10 and was euthanized after samples were collected. In all horses D-dimers were undetectable on D -1 and on D4, 5 and 11. In contrast, all horses had increased Ddimer concentrations for 3 to 5 consecutive days starting on day 6 post-infection. D-dimer concentrations in 2 horses increased to 1000 ug/ml and one of these horses was the horse with acute EHM.

Interestingly, mean increased D-dimer concentrations showed timely overlap with the mean fever curve and, delayed by 1 day, with the mean viremia curve. Because plasma samples for D-dimer measurements were not collected during the first 3 days post-infection, which are typically associated with a primary fever, conclusion on the association of D-Dimers with fever of viremia await analysis of a second study currently conducted in our laboratory.

In conclusion our data indicates that during EHV-1 infection with neuropathogenic strains activation of the coagulation cascade and production of cross-linked fibrin is wide-spread; not limited to horses with clinical signs of EHM, and can be expected between days 6 and 10 post-infection. Lawsonia intracellularis is an emerging pathogen in horses and the causative agent in Equine Proliferative Enteropathy (EPE). The goal of this study was to evaluate the exposure of pre-weanling foals and broodmares to Lawsonia intracelluaris on several farms in Louisiana with a history of EPE and compare the results to several farms with no known clinical cases of EPE in foals. An additional goal of the study was to identify whether a relationship exists between Lawsonia intracelluaris and other gastrointestinal pathogens in foals. Whole blood and fecal samples were collected from 66 mares and 68 foals from four breeding farms in Louisiana. Farms A and B had no known clinical cases of EPE, while Farms C and D had previous know cases of EPE in 2009. Serum samples were examined for the presence of antibodies against Lawsonia intracellularis using an immunoperoxidase monolayer assay (IPMA). DNA was extracted from fecal samples using a commercial DNA kit and molecular detection of Lawsonia intracelluaris was assayed using real-time PCR. Fecal ova were determined using quantitative sucrose floatation. The presence of fecal Clostridium difficile toxin was measured using a commercial enzyme linked immunosorbent assay (ELISA). Three of the 4 farms examined had foals and mares with exposure to L. intracellularis as evidenced by serum antibodies against the organism. Of the total population sampled, 6 foals (8.8%) and 14 mares (21.2%) had evidence of antibodies to L. intracellularis based on serology. Three foals (4.4%) tested positive for L. intracellularis organism by fecal PCR, and all of these foals were located on farm C. Of these, one of the foals was seronegative, while the other two were seropositive. Farm C also had the highest percentage of mares (28.6%) serologically positive for L. intracellularis, while Farm A had the highest percentage of foals (14.3%) with antibody titers against L intracellaris. Farm C also had the only pairs (n 5 3) of serologically positive mares with seropositive foals. While Farm A and B had seropositive mares and/or foals, none of the foals were positive for L. intracellularis fecal shedding by PCR. All serum and fecal samples were negative for evidence of L. intracellaris on Farm D. Ten foals (14%) had fecal egg counts greater than 200 egg per gram and 2 foals (3%) were positive for C. difficile toxin. This study demonstrated evidence of natural exposure to L intracellularis on farms both with and without a history of EPE in Louisiana. Further, this study failed to establish a relationship between L intracellularis and other gastrointestinal pathogens. The objective of this study was to examine the clinical, hematological, biochemical, and outcome data from equids infected with Anaplasma phagocytophilum presented to a primary care field setting in southeastern Pennsylvania. Computerized medical records from 19 febrile equids with confirmed Anaplasma phagocytophilum infection were reviewed. Confirmation of Anaplasma phagocytophilum was defined by the presence of granular inclusion bodies seen within leukocytes or eosinophils on microscopic blood smear evaluation and/or a positive polymerase chain reaction (PCR) for Anaplasma phagocytophilum.

18 horses and 1 donkey presented with a mean fever of 104.41F and mean fever duration of 39 hours. The mean age at presentation was 9.6 years and the mean pack cell volume was 30.8%. 15/19 cases were diagnosed in the months of May to December. Equids ages 5 to 15 years had significantly lower platelet counts. 16/18 cases were positive on blood smear for inclusion bodies and 6/6 cases were positive for Anaplasma phagocytophilum on PCR. Treatments included intravenous oxytetracycline, oral doxycycline, or both. Mean treatment duration was 4.8 days and mean treatment cost was $621. 17/19 cases were normothermic within 48 hours. The treatment used in the two remaining cases was changed from oral doxycycline to intravenous oxtetracycline and was successful.

This is the first case series of equine granulocytic anaplasmosis in the mid-Atlantic states. All cases were examined and treated in the field. In order to make a definitive diagnosis, some cases required PCR. Treatment failures were documented with the use of oral doxycycline alone. 100% of the cases survived. A high incidence of clinical and possibly genetic abnormalities has been reported amongst Friesian horses including dwarfism, hydrocephalus, dissecting aortic aneurism and esophageal dysfunction.

The purpose of the current study was to develop a new electromyography (EMG) method to assess neurophysiological function of the esophagus especially for Friesian horses.

Five Friesian horses with esophageal dysfunction were included (ranging in age from 0.5-24 years and comprising 4 mares and a stallion) and two Friesian control horses (a 10-and 12-year-old gelding). All five horses with esophageal dysfunction had a history of recurrent esophageal obstruction and were examined histopathologically post-mortem. Barium contrast radiography was used as the gold standard to distinguish the diseased from the control horses. An endoscopically-guided percutaneous needle EMG procedure (Viking Quest r ; software version 11.0) was performed just caudal to the larynx and just cranial to the thoracic inlet (to monitor striated and smooth muscle, respectively) to visualize esophageal motility.

Esophageal contractility in both control horses was predominantly reflected by interference patterns associated with longer duration and lower amplitude in smooth muscle compared to striated muscle. Mean (AE SD) values were 35.1 AE 19.4 ms and 167.7 AE 96.7 mV (n 5 19 readings) and 10.8 AE 14.3 ms and 305.8 AE 233.7 mV (n 5 24 readings), respectively. In diseased horses, aperistalsis in smooth muscle was the most remarkable finding suggesting a loss of inhibitory neurogenic input resulting in aperistalsis and thus esophageal dysfunction. Preliminary findings suggest that endoscopically-guided percutaneous needle EMG might become a valuable method in elucidating the pathophysiology of dysfunction of esophageal motility especially in Friesian horses. Lymphoma affects horses of all ages. Unlike in humans, no etiologic agent has been discovered. A 9 year old Thoroughbred/Warmblood cross mare presented with signs of upper and lower respiratory disease and was subsequently diagnosed with lymphoma and equine multinodular pulmonary fibrosis (EMPF) and was positive for equine herpes virus 5 (EHV-5) in both the pulmonary tissue and the lymph nodes.

Retrospective polymerase chain reaction (PCR) testing of six lymphoma cases found that 5 of 6 of the cases were positive on PCR for EHV-5 (83.3%, p 5 0.0045, RR 5.55). Electron microscopy was performed on one sample and herpes virus particles were identified. Of the samples in which immunohistochemistry was performed (3 of 6), only T-cell rich B-cell lymphoma was identified. Samples of mesenteric or submandibular lymph nodes from 20 clinically healthy horses were submitted for EHV-5 PCR analysis; 15% were positive.

Gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as Kaposi's sarcoma and Burkitt's lymphoma. Equine herpesvirus 5, also a gamma herpesvirus, is found in association with equine lymphoma; although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia remains unknown. Pathologic events reported to occur in the digital laminae in early stages of sepsis-related equine laminitis include leukocyte extravasa-tion into the laminar interstitium, pro-inflammatory cytokine expression, and epithelial stress. While these events have been documented early in the disease process at both a developmental stage and at the onset of Obel Grade 1 (OG1) lameness in the carbohydrate overload (CHO) model of laminitis, the later events occurring at the onset of Obel Grade 3 lameness(OG3, time point at which structural failure of the laminae usually occurs) have not been determined. We hypothesized that the inflammatory events described above are sustained through OG 3 lameness, likely playing an injurious role culminating in laminar failure. Our objectives were to determine pro-inflammatory gene expression, leukocyte extravasation, and epithelial stress at OG 3 induced using the CHO model. Archived laminar tissue samples (snap frozen and paraffin embedded sections) were used from a previous CHO study at Louisiana State University (Control group [n 5 6, water], CHO group [n 5 7, corn starch]. Calprotectin (CP) immunohistochemistry (IHC) was used to assess both laminar myeloid leukocyte numbers and epithelial stress; RT-qPCR was used to assess inflammatory gene expression. Minimal inflammatory changes were present at OG3 compared to published values at OG1 stage in the CHO lameness model including decreased mRNA concentrations of cytokines (i.e. 20-fold increase in IL-6 at OG3 vs. 4 2000-fold increase at OG1, no increase in IL-1b at OG3 vs. 11-fold increase at OG1), chemokines (no change in MCP-1at OG3 vs. 4 30 fold increase at OG1, 8-fold increase in IL-8 at OG3 vs. 95fold increase at OG1) and adhesion molecules (no change in E-selectin at OG3 vs. 10-fold increase at OG1). Laminar leukocyte emigration was also decreased at the onset of OG 3 lameness compared to previously reported leukocyte infiltration at OG 1. Interestingly COX-2, underwent a greater increase at OG3 (approx. 50-fold) compared to that reported at OG1 lameness (35-fold). Finally, epithelial stress at OG3 evidenced by CP IHC did not follow the uniform widespread distribution reported at OG1 lameness, but instead was present in focal areas in which secondary epidermal laminae on either side of a common primary dermal vascular supply demonstrated increased CP signal. Overall, laminar inflammation appears to be subsiding at OG3 lameness, with epithelial stress possibly more dependent on vascular dysregulation instead of inflammatory events. The sustained increase in COX-2, central to the induced production of vasoactive prostanoids in disease processes, may play a role in vascular dysregulation. This study was conducted to characterize clinical, laboratory and postmortem findings associated with oleander toxicosis in equids and to determine factors predictive of survival in these cases.

Retrospective analysis of medical records from our veterinary medical teaching hospital from January 1, 1995 to July 15, 2010 was completed. Records of equids demonstrating detectable oleandrin in serum, plasma, urine or gastrointestinal fluid samples or detectable serum digoxin in the absence of pharmaceutical cardiac glycoside administration were included. Descriptive statistics were used to evaluate the history, physical examination, and laboratory and postmortem data of affected individuals. Logistic regression analysis was used to detect physical examination and laboratory factors significantly associated with survival.

Thirty equids met inclusion criteria of the study. Three of 30 subjects were dead on arrival or died immediately upon arrival (10%). Of the remaining 27 equids, 85% presented with gastrointestinal abnormalities, 70% were azotemic and 48% had cardiac arrhythmias. Mortality was 50% for all subjects and 44% for those treated. The predominant cause for non-survival was cardiac dysfunction. Factors significantly associated with survival included relatively decreased hematocrit and serum glucose, relatively increased serum chloride, absence of cardiac arrhythmias, and increased duration of hospitalization.

Equids with oleander toxicosis frequently present with gastrointestinal upset and may develop cardiac and renal disturbances. Patients with cardiac arrhythmias and relatively increased hematocrit and serum glucose and decreased serum chloride are significantly less likely to survive. Oleander intoxication is a differential diagnosis for colic in endemic areas, particularly with concurrent azotemia or cardiac dysrhythmia. The quantitative physicochemical approach emphasizes the importance of strong ions (Na, K, Cl, lactate), pCO 2 , and the plasma protein concentrations in determining plasma pH. Serum concentrations of strong ions, proteins, and total CO 2 are reported on modern biochemical profiles. We hypothesized that the results of serum biochemical analysis can be used for acid-base interpretation in horses. The objective was to determine whether blood pH, anion gap, and strong ion gap could be quantitatively estimated and clinically used based on the results of serum or plasma biochemical analysis.

100 horses (70 adults and 30 foals) presented to the isolation unit of our Veterinary Teaching Hospital for suspected infectious diseases were prospectively enrolled. A venous serum sample was analyzed using a Hitachi 911 or Copas 6000 C501 automated machine. Measured parameters included strong ion difference (SID 5 {Na1K}-{Cl1lac-tate}), total protein concentration (TP), and total CO 2 (tCO 2 ), with lactate being measured by blood gas analyzer. A second venous blood sample was collected into a Na-heparin blood gas syringe and analyzed for pH (pH m ), pCO 2 and concentrations of Na, K, Cl, and lactate using a Radiometer 800 Flex blood gas analyzer; SID was calculated from the measured values, and total solids (TS) were estimated using refractometry. Serum/ plasma pH (pH calc ) was calculated using Stewart's 8 factor equation from the results of serum or plasma biochemical analysis, assuming pCO 2 5 40 mmHg for serum and pCO 2 accurate for plasma. Anion gap (AG) was calculated as: AG 5 (Na1K)-(Cl1tCO 2 ). Strong ion gap (SIG) was calculated as: SIG 5 0.21x[total protein, g/L]/ (1110 {pKa-pH} )-AG. Linear regression analysis was used to compare pH calc to pH m, as well as AG and SIG to blood lactate concentrations.

Measured pH ranged from 7.03 to 7.48 (7.37 AE 0.07). Measured SID from serum biochemistry (SID SB ) ranged from 22.3 to 51.4 mEq/L (36.2 AE 4.7 mEq/L) and SID from blood gas analyzer (SID BG ) from 14.3 to 46.6 mEq/L (33.3 AE 5.4 mEq/L; R 2 5 0.59; SID BG 5 0.919 Â SID SB ). SID SB and SID BG showed small variability in measurements. TP ranged from 18 to 88 g/L (51.0 AE 13.9 g/L) and TS from 20-84 (55.2 AE 13.8 g/L; R 2 5 0.77; TS 5 1.071 Â TP). Using SID SB and tCO 2 values with constant pCO 2 , pH calc was poorly associated with pH m (R 2 5 0.27; pH calc 5 0.48 1 3.89). In contrast, using SID BG with accurate pCO 2 , pH calc was closely associated with pH m (R 2 5 0.54; pHcalc 5 0.98 1 0.15) and the equation was not different from the line of identity. Anion gap and SIG (mEq/L) calculated were significantly linearly correlated with lactate concentrations (mmol/L); AG 5 0.93 Â [lactate] 1 8.3 (R 2 5 0.39), and SIG 5 À0.91 Â [lactate] 1 0.5 (R 2 5 0.41).

We conclude that pH calc using SID SB , tCO 2 and constant pCO 2 values is not accurate. However, variability of measured biochemical parameters between machines was small, permitting use of serum biochemistry for clinical metabolic acid-base abnormalities interpretations of patients. These results reemphasize the importance of strong electrolytes and proteins in acid-base balance. Metalloproteinases (MMPs) are critically important in remodeling processes and in wound healing. However, excessive activation of MMPs by pro-inflammatory mediators including cytokines, prostaglandin E2, and nitric oxide lead to tissue breakdown. This is observed in osteoarthritis (OA) which is characterized by erosive lesions in articular cartilage. In Hereditary Equine Regional Dermal Asthenia (HERDA), afflicted horses exhibit collagen abnormalities and can have associated chronic inflammation and aberrant wound repair. HERDA affects horses with Quarter Horse bloodlines and is similar to the human hereditary connective tissue syndrome Ehlers Danlos (EDS). Many adult EDS patients suffer from joint pain and OA. We hypothesized that chondrocytes from articular cartilage of HERDA horses have increased activity of MMPs. To test this hypothesis, chondrocytes were retrieved from articular cartilage of homozygous HERDA carpal and hock joints. Chondrocytes from normal horses were also obtained for comparison. Chondrocytes were seeded at 5 x 10 6 /ml into 6-well plates and incubated at 371C, 5% CO 2 for up to seven days. Activity of secreted MMPs was determined by zymography using equal amounts of proteins for loading. Secreted MMPs were analyzed by Western blot.

Zymography showed that normal chondrocytes secreted two major bands with gelatinolytic activity observed at 92 and 72 kDa suggestive of the latent form of MMP-2 and MMP-9, respectively. Less intense bands of gelatinolytic activity were observed at about 82 and 62 kDa suggestive of the active form of MMP-2 and MMP-9. Another band of activity was also seen at 240 kDa which is suggestive of a dimer of MMP-9 that has been reported when MMPs are in excess of tissue inhibitors of metalloproteinases (TIMPs). Chondrocyte cultures from homozygous HERDA cartilage showed a similar profile but with decreased activity by 90% at 92 kDa and 10-50% increased activity at 72 kDA compared to normal chondrocytes. Western blot analysis detected MMP-2 and MMP-9 immunoreactivity in chondrocyte culture media of HERDA-afflicted and normal horses. The present study demonstrates for the first time that horses suffering from HERDA have increased MMP activity which may predispose them to the development of lesions in articular cartilage.

Research supported by Nutramax Laboratories, Inc. Equine Polysaccharide Storage Myopathy (PSSM) type 1 is a dominantly inherited glycogenosis caused by a mutation in the gene coding for skeletal muscle glycogen synthase type 1 (GYS-1). The disease has been reported to affect the Haflinger breed but so far its prevalence is unknown. Aim of this preliminary study was to estimate the occurrence of the GYS-1 mutation in Austrian Haflingers and establish which of the seven Haflinger sire lines appear mostly affected.

GYS-1 genotyping of 50 randomly chosen Haflingers was performed with a validated restriction fragment length polymorphism assay. Resting and post-exercise muscle enzyme activities (creatine kinase (CK), aspartate aminotransferase (AST), lacate dehydrogenase (LDH)) and blood lactate concentrations were compared between horses with and without the mutation.

Among the 50 horses 9 were heterozygous (HR) carrier of the mutation. No homozygotes (HH) were identified. All horses with the GYS-1 mutation were descendents of the A-or W-sire lines. The estimated HR prevalence was 18% (95% CI: 8.6-32.4%). CK activity after exercise (p 5 0.022) was significantly higher in HR horses compared with horses not carrying the mutation (RR). AST activity was significantly higher in the HR group at rest and after exercise (p o 0.001). There was no statistically significant difference in resting CK, resting and post exercise LDH activity or blood lactate between HR and RR.

Results suggest that the prevalence of HR in the Austrian Haflinger population is higher than in the overall Quarter Horse population and might be as high as 30%, similar to some Draft horse breeds. Further research is needed to establish the prevalence within the different breeding lines. Hereditary equine regional dermal asthenia (HERDA) is an autosomal recessive connective tissue disorder affecting Quarter Horse lineages. 1 Although a mutation in the gene encoding cyclophilin B has been genetically linked to HERDA, its causal association with the disease is not yet documented. 2 Previously, we demonstrated reductions in ultimate tensile strength (UTS), modulus of elasticity, and energy to failure (toughness) of skin from many corporal regions of HERDA animals. 3 Given the presumed relationship between HER-DA and abnormal collagen structure, and the predominance of Type I collagen in skin, we hypothesized that altered biomechanical properties would be detected in tendons which are rich in Type I collagen. To evaluate this hypothesis we compared the UTS, modulus of elasticity, and energy to failure of forelimb deep digital flexor tendons (DFT) from six HERDA horses to six age-matched controls. Isolated DFT was secured and pulled to failure on an Instron s 1011 Universal Testing Instrument using purpose-built cryogenic clamps. Analysis of Variance was executed using SAS 9.2 PROC GLIMMIX program (SAS Institute, 2009). P-values 0.05 were identified as significant. UTS and modulus of elasticity were significantly lower in HERDA DFT when compared with controls (P o 0.0001); energy to failure did not differ between groups. These findings document abnormal biomechanics in HERDA tendon, leading us to postulate that lower UTS and modulus of elasticity associated with the HERDA defect could convey a competitive advantage in the athletic disciplines in which this defect has segregated. (References on request). A proprietary herbal biocontamination product (BIOS) approved for cosmetic use in France, inhibits proliferation of medically relevant bacteria, mold, and viruses. These properties make BIOS potentially useful as a topical wound medication, prompting us to compare BIOS to silver sulfadiazine (SSD) in a distal extremity wound healing model in horses. 1 Using general anesthesia, two 6.25 cm 2 wounds were aseptically created on the dorsomedial aspect of all limbs. For the duration of the study, two contralateral limbs were randomly chosen to be bandaged; the other two limbs were un-bandaged -with one limb of each group being treated with 10% BIOS and the other with SSD. For each limb the most proximal wound served as an untreated control. Every 48 hours wounds were evaluated, digitally photographed, and perimeter and area determined using morphometric software (ImageJ, NIH). Analysis of variance did not identify significant differences between SSD or BIOS treatment for wound perimeter (p 5 0.76) or area (p 5 0.95). At individual time points the effect of bandaging was significant when area was evaluated (p 5 0.019) and trended toward significance for perimeter (p 5 0.084) comparisons, substantiating published reports that bandaging modifies wound healing. Difference in perimeter and area between control and treatment were highly significant (p o 0.0001), substantiating the importance of topical treatment. Over the study duration, effects of bandaging (p o 0.0001) and topical treatment (perimeter p o 0.0001; area p 5 0.0016) continued to be highly significant. BIOS performance in the equine distal extremity wound model was equivalent to SSD. Both bandaging and topical treatment significantly impacted wound healing. This effect was compounded when both variables were evaluated over time. Radiolabeled leukocytes are the only scintigraphic method currently available for identifying sites of infection and/or inflammation in horses; however the clinical applicability of this technique is limited by expense and poor efficacy. This pilot study compares the accumulation of 99m Tc-labeled IgG, PEG-liposomes and leukocytes in an equine muscle abscess model.

Three mixed breed adult horses had 2 Â 10 6 CFU S. equi zooepidemicus inoculated into the right semitendonosis to create an abscess. PEG-liposomes were prepared via the film hydration method and labeled using 200 mCi 99m Tc-hexamethyl-propylene-amine-oxime ( 99m Tc-HMPAO). Autologous leukocytes were obtained from 120 ml whole blood and labelled using 200 mCi 99m Tc-HMPAO. Commercial equine polyclonal IgG was conjugated with the chelator hydrazinonicotinamide (HYNIC) and labelled with 200 mCi 99m Tc. Radiopharmaceutical administration was initiated 24 hours after inoculation. Horses 1 and 2 received 5 mg 99m Tc-IgG, 2.7 mmol/kg 99m Tc-liposomes and 99m Tc-leukocytes, with a 48 hour interval between each radiopharmaceutical. Horse 3 received only 99m Tc-leukocytes. Scintigraphic examinations were performed at 8 and 21 hours post injection (p.i.) with each radiopharmaceutical. After the final study, horses were euthanized and tissue samples collected. The percentage of injected dose per kilogram of tissue (%ID/kg) was calculated for the region of the abscess, normal muscle and multiple organs.

Scintigraphic examinations demonstrated increased radiopharmaceutical in the region of the abscess with all three techniques at both time-points. At 8 hours p.i. abscess-to-background ratio was highest using 99m Tc-IgG (3.7 AE 0.2). At 21 hours p.i. abscess to background ratio was highest using 99m Tc-liposomes (5.9 AE 2). Tissue biodistribution data revealed abscess to muscle ratios of 36 ( 99m Tc-IgG), 24 ( 99m Tc-liposomes), and 4.1 ( 99m Tc-leukocytes).

This preliminary data demonstrates that 99m Tc-liposomes, 99m Tc-IgG and 99m Tc-leukocytes exhibit long circulating characteristics and accumulate at inflammatory/infectious foci after intravenous injection in horses. 99m Tc-IgG and 99m Tc-liposomes appear to be superior to 99m Tc-labelled leukocytes in this model. Due to its low cost and ease of preparation, 99m Tc-IgG has great potential for clinical use where identification of infectious or inflammatory foci is necessary. Digital hypothermia is used clinically to decrease the incidence of sepsis-related equine laminitis, a disease causing structural failure of digital laminae resulting in crippling lameness. Due to the fact that hypothermia was recently reported to effectively decrease laminar expression of inflammatory molecules including pro-inflammatory cytokines, chemokines and COX-2 in equine laminitis, our laboratory is investigating the effect of hypothermia on central upstream signaling cascades which may induce expression of these diverse inflammatory molecules. The p38 MAPK pathway has recently been reported to be a central component of inflammatory signaling in multiple diseases including human sepsis, and is currently being assessed as a therapeutic target. We thus hypothesized that 1) p38 MAPK is upregulated and activated in affected laminae in equine laminitis and 2) digital hypothermia inhibits inflammatory mediator expression by blocking p38 MAPK phosphorylation (indicator of p38 MAPK activation). Western hybridizations using both a total p38 MAPK and a phospho-p38 MAPK antibody were performed on archived pooled laminar samples from black walnut extract (BWE) model (10 control, 2 developmental (DEV) groups [1.5H & 3H post BWE administration] and the onset of Obel Grade 1 lameness (OG1) [n 5 5 each]) and carbohydrate overload (CHO) models (CON [n 5 8], DEV [n 5 6], OG1[n 5 6]) of laminitis, and individual laminar samples from two groups of horses from a digital hypothermia (DH) study. In the DH study, one forelimb of each horse was kept at approximately 41C in ice water and the other at ambient temperature following administration of 10 g/kg oligofructose (OF). Dorsal laminae were harvested for snap freezing at either 24 hours after OF administration (DEV, n 5 7) or at the onset of lameness (OG1, n 5 6) using protein extracted from treated and untreated digital laminae of each horse. Increased laminar concentrations of phospho-p38 MAPK were present in the developmental periods (1.5H and 3H) in the BWE model, and in both the DEV and OG1 periods in the CHO laminitis models. However, digital hypothermia had no effect on laminar phospho-p38 MAPK concentrations. Thus, p38 MAPK is activated in affected laminae in multiple models of laminitis, but does not appear to be the central signaling cascade through which hypothermia works to block the expression of inflammatory molecules. Therefore, p38 MAPK is not likely to be a viable therapeutic target as a sole source for blocking the multiple inflammatory signaling mechanisms inhibited by local hypothermia.

ABSTRACT E-60 DOES CEFQUINOME PENETRATE THE BLOOD BRAIN BARRIER IN THE NORMAL HORSE? Hollis AR 1 Duggan VE 2 and Corley KTT 3 . 1 Scott Dunn's Equine Clinic, Berkshire, UK; 2 University College Dublin, Dublin, Ireland; 3 Anglesey Lodge Equine Hospital, The Curragh, Ireland.

Meningitis is a rare but serious condition that occurs in both foals and adult horses. There is currently a restricted choice of antimicrobials that are both safe to use in horses and penetrate the blood brain barrier. Cefquinome is a fourth generation cephalosporin that has activity against Streptococcus, the most commonly reported causative organism in adult horse meningitis. Therefore, if cefquinome were to achieve therapeutic concentrations in cerebrospinal fluid following routine administration, this would be an exciting advance for the treatment of meningitis in the horse. 5 mature, healthy horses were used on 2 separate occasions, seven days apart, in a crossover design. Each horse was administered either cefquinome (1 mg/kg) or saline (equivalent volume). Cerebrospinal fluid was collected via atlanto-occipital puncture under general anaesthesia 1 and 4 hours after administration of cefquinome or saline placebo. Blood samples were collected prior to, and 1 and 4 hours after administration of cefquinome or placebo. All samples were analysed for the presence of cefquinome by a laboratory masked to treatments administered.

Cefquinome was detectable in the cerebrospinal fluid in all horses 4 hours after intravenous administration, and in 2 horses 1 hour after administration.

Cefquinome penetrates the blood-brain barrier and it is therefore a potential treatment for equine meningitis. Further investigation of the pharmacokinetics and pharmacodynamics of cefquinome in the cerebrospinal fluid is warranted to establish the optimum intravenous dose. The purpose of this study was to determine if enrofloxacin alters the pharmacokinetics of firocoxib in the horse. Firocoxib is a coxibclass nonsteroidal anti-inflammatory drug (NSAID) approved for use in horses to control pain and inflammation associated with osteoarthritis. Dosages of firocoxib are species dependent, with the recommended dose for horses being 0.1 mg/kg as an oral paste every 24 h. The main elimination pathway of firocoxib is hepatic; however the effects of concurrent administration of drugs that may inhibit its metabolism have not been evaluated.

Enrofloxacin is a synthetic antibacterial agent from the flouroquinolone group developed for veterinary use. It is primarily used for gastrointestinal, urogenital, skin and respiratory tract infections in various animals. A well acknowledged problem associated with flouroquinolone usage is their effect on the metabolism of other drugs. Co-administration of multiple drugs can result in unpredictable therapeutic outcomes. Often it is either diminished therapeutic efficacy or increased toxicity of one or more of the administered drugs. Various pharmacokinetic interactions between antimicrobials and NSAIDs have been described.

Six healthy, adult mares were administered 0.1 mg/kg of firocoxib orally. Samples were collected by direct venipuncture of the jugular vein at 0 (control), 15, 30, and 60 min, 2, 4, 8, 12, 16, 24, 48, 72 , and 96 h after administration. After a 20 day washout period the six horses were pretreated 3 days with enrofloxacin 5 mg/kg intravenously every 24 h then on the fourth day given 0.1 mg/kg of firocoxib orally. Samples were collected at 0 (control), 15, 30, and 60 min, 2, 4, 8, 12, 16, 24, 48, 72 , and 96 h after administration. All samples were stored at À801C until analysis using a validated HPLC method. The t1/2, C max , T max , AUC 0-24 and AUC 0-f after firocoxib administration were 30. 66 Angiotensin converting enzyme (ACE) inhibitors improve survival and quality of life in humans and small animals with cardiovascular and renal disease. There is limited information regarding their effects in horses. The purpose of this study was to determine the pharmacokinetics of quinapril and its effects on ACE inhibition in horses.

Six healthy horses were administered quinapril at 120 mg IV, 120 mg PO or 240 mg PO in a 3-way crossover design. Blood was collected at predetermined times for measurement of quinapril and quinaprilat concentrations using high pressure liquid chromatography, as well as ACE concentrations using a radioenzymatic assay. Normally distributed data were analyzed with one way repeated measures analysis of variance (RM-ANOVA) and non-normally distributed data were analyzed using Friedman RM_ANOVA on ranks. Significance was set at P o 0.05.

No adverse effects were observed during the study period. Plasma quinapril concentrations were low and rapidly declined after IV administration. Quinaprilat concentrations were below the limit of quantification (0.1 mg/mL). ACE activity was significantly decreased from baseline at 0.5 and 1 hour after IV dosing and at all timepoints after oral dosing. Maximum % ACE inhibition was 72, 53 and 47% with the IV, high and low oral doses, respectively.

These results suggest that, despite low plasma concentrations, quinapril has sufficient oral absorption and results in inhibition of ACE in healthy horses. Controlled studies in clinically affected horses are indicated. This study determined the pharmacokinetic profile of firocoxib in healthy neonatal foals. Foals are more sensitive to the side effects of NSAID, primarily due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. Firocoxib, a novel, second generation NSAID, is reported to have reduced side effects due to COX-2 selectivity. The pharmacokinetic profile of firocoxib in neonates has not been established. We hypothesized that firocoxib given PO to neonatal foals would achieve therapeutic concentrations in plasma.

Seven healthy foals of mixed gender were administered 0.1 mg/kg firocoxib PO q24h for nine consecutive days, commencing at 36h old. Blood was collected for firocoxib analysis at 0 (dose #1 only), 0.25, 0.5, 1, 2, 4, 8, 16 and 24h after doses #1, 5 and 9. For all other doses (2, 3, 4, 6, 7 and 8) blood was collected immediately prior to the next dose (24h trough). Elimination samples were collected after dose #9. Plasma was stored at À801C until analysis. Physical examinations were performed on foals daily and body weight obtained every two days during the sampling period.

Analysis of plasma samples by liquid chromatography-mass spectrometry revealed firocoxib was rapidly absorbed. After the initial dose, a maximum plasma concentration was reached in 30 min, minimal accumulation after repeat dosing occurred and steady state was obtained after approximately four doses. After the final dose, plasma drug concentration decreased in a linear manner with an estimated terminal t1/2 of 11h. Seventy-two hours after the final dose, firocoxib was not detectable (o 2ng/mL). Erythrocytosis is reportedly a rare finding associated with hepatocellular carcinoma in horses. The purpose of this study was to determine the relative frequency of erythrocytosis and the clinicopathologic abnormalities and hepatic histopathology associated with erythrocytosis in horses with liver disease.

Ninety-seven horses aged !1 year with clinicopathologic or clinical signs of liver disease, a complete blood count (CBC), and hepatic histopathology were included. Information on CBC, biochemical variables, and hepatic histopathology was collected from records. Data from horses with erythrocytosis (packed cell volume 4 45%) were compared to those without using the Mann-Whitney rank sum test with significance set at p o 0.05. There were no differences between groups in white blood cell count, gamma-glutamyl transferase, sorbitol dehydrogenase, aspartate aminotransferase, and alkaline phosphatase activities, total protein, albumin, globulin, blood urea nitrogen, or glucose concentrations. Fibrosis (64%), biliary hyperplasia (56%), inflammatory infiltrate (50%), megalocytosis (25%), degeneration (25%), necrosis (25%), cholestasis (25%), anisocytosis and anisokaryosis (11%), and lipidosis (3%) were observed in livers of horses with erythrocytosis. Neoplasia (3%) was observed rarely.

This study reports a high frequency of erythrocytosis in horses with liver disease. Erythrocytosis is associated with higher total bilirubin and serum bile acids concentrations. Common histopathologic changes include fibrosis, biliary hyperplasia, and inflammatory infiltrate. Hepatic neoplasia was rare. This study was performed to determine if horses diagnosed with equine proliferative enteropathy (EPE) from Lawsonia intracellularis (LI) infection had long term effects from disease based on their sale price as yearlings and race earnings.

A retrospective review of medical records of Thoroughbred horses that were treated for Lawsonia intracellularis infection between January 1, 2002 and January 31, 2008 at Hagyard Equine Medical Institute in Lexington, Kentucky was performed. Three criteria were used for inclusion in this study. First, each horse had presumptively been diagnosed with LI based on physical examination findings such as ventral edema, diarrhea, lethargy, or poor body condition. Second, horses had hypoalbuminemia of less than 2.5 mg/dL (normal reference range: 3.4-4.5 mg/dL). Third, each horse had a positive fecal polymerase chain reaction (PCR) for LI, a positive serum immunoperoxidase monolayer assay (IPMA), or both. An IPMA titer greater than or equal to 60 was considered positive for disease. 116 horses met the initial criteria.

36 of the 116 horses sold at public auction as yearlings. The sale price of these horses was compared to the average sale price of all yearlings by the same sire as the affected horse (control group). 30 of the 116 horses raced in the United States. Their monetary earnings from racing were compared to the average monetary earnings of all progeny by the same sire as the affected horse (control group). Earnings of horses that were between 3 and 7 years of age (23/30 horses) at the conclusion of the study were compared to the lifetime average earnings of the stallion's progeny. Earnings from horses that were two years of age (7/30) at the end of the study were compared to the two year old average earnings of the stallion's progeny. Monetary earnings from all races prior to December 31, 2008 were included in the study. 12 horses both sold at public auction and raced. As well as being included in the total number of horses that sold and raced, their sale records and monetary earnings were compared to the averages from their respective sire as a separate group.

This retrospective study indicated that yearling horses previously infected with LI do not sell for as much at public auction as their herdmates, but their monetary earnings from racing are not significantly different from other horses. These results should assist practitioners in guiding owners in determing if treatment of horses with EPE is appropriate and it may aid in reassuring owners that despite the poor condition of the horse during and shortly after the course of disease, horse may still have future athletic potential.

This abstract was presented at the AAEP in December 2009. Bronchopneumonia caused by Streptococccus equi subsp. zooepidemicus (S. zooepidemicus) is one of the most important causes of morbidity in weanling foals. Ceftiofur crystalline free acid (CCFA) is a long acting third-generation cephalosporin antimicrobial recently approved for the treatment of bronchopneumonia associated with S. zooepidemicus in adult horses. The objective of the present study was to determine the disposition of CCFA in plasma and pulmonary epithelial lining fluid (PELF) of weanling foals. Six healthy 4-to 5month-old weanling foals were administered a single intramuscular injection of CCFA at a dose of 6.6 mg/kg of body weight. Concentrations of desfuroylceftiofur acetamide (DCA) and related metabolites were measured by use of ultra-high performance liquid chromatography and tandem mass spectrometry.

Following IM administration, median time to maximum plasma and PELF concentrations was 24 h (12-48 h) . Mean (AE SD) peak DCA concentration in plasma (1.44 AE 0.46 mg/mL) was significantly higher than that in PELF (0.46 AE 0.03 mg/mL). Terminal half-life of DCA in plasma (74.8 AE 20.9 h) was not significantly different from that of PELF (58.5 AE 11.4 h). Time above the therapeutic target of 0.2 mg/mL was significantly longer in plasma (185 AE 20 h) than in PELF (107 AE 31 h).

Based on the results of the present study, intramuscular administration of CCFA at a dose of 6.6 mg/kg would be appropriate for the treatment of bronchopneumonia caused by S. zooepidemicus and other susceptible pathogens in weanling foals. FGF-23 is secreted by osteocytes and osteoblasts in response to hyperphosphatemia. FGF-23 enhances phosphaturia and is postulated to have a central role in the development of secondary renal hyperparathyroidism. Hyperthyroid cats have elevated plasma phosphate and parathyroid hormone concentrations, which may in part be associated with underlying chronic kidney disease (CKD). The aim of this study was to determine if plasma FGF-23 concentrations were associated with the presence of underlying CKD in hyperthyroid cats, and to investigate the changes in plasma FGF-23 concentrations that occur following treatment of HTH.

Hyperthyroid cats were recruited from two London-based first opinion practices between 1999 and 2009. Cats that were azotemic at diagnosis were excluded. HTH was treated with anti-thyroid medication alone or in combination with thyroidectomy. Cats were included in the study if they had a plasma total thyroxine concentration o 40 nmol/l documented for a six month period following commencement of treatment. Cats were classified as having azotemic CKD if they developed renal azotemia within six months of establishment of euthyroidism. Otherwise cats were deemed to have normal renal function. Stored EDTA plasma samples were assayed for FGF-23 using a recently validated ELISA. The Mann-Whitney U test and the Wilcoxon signed rank test were used to compare between the groups and assess the response to treatment respectively. Results are reported as median [25 th , 75 th percentiles]. Correlations were made using Spearman's correlation coefficient.

Thirty one cats with HTH (14 azotemic and 17 non-azotemic) were included in the study. Plasma phosphate concentrations decreased following treatment in cats that did not develop azotemia (4.84 [3.91, 5.64] mg/dl vs. 3.91 [3.38, 4.37] mg/dl; n 5 13, P 5 0.01) whereas plasma phosphate concentrations did not change significantly following treatment in cats that did develop azotemia (4.28 [3.81, 5.64] mg/ dl vs. 4.22 [3.10, 5.33] mg/dl; n 5 14, P 5 0.158). Plasma FGF-23 concentrations were significantly higher in cats that developed azotemia than cats that did not at both pre treatment (211.7 [176.4, 356 .3] pg/ml vs. 148.3 [118.8, 274 .9] pg/ml; P 5 0.039) and post treatment (514.0 [250.2, 800.0] pg/ml vs. 195.1 [160.7, 287 .3] pg/ml; P 5 0.001) timepoints. Plasma FGF-23 concentrations increased following treatment in both azotemic (P 5 0.004) and non-azotemic groups (P 5 0.025). Plasma FGF-23 concentrations and plasma phosphate concentrations were not correlated at baseline (r s 5 0.189, P 5 0.335) or following treatment (r s 5 0.136, P 5 0.472).

Plasma FGF-23 concentrations were higher in pre-azotemic cats than non-azotemic cats and increased following treatment of HTH. The reason that FGF-23 concentrations increased following treatment, particularly in the face of decreasing plasma phosphate concentrations in cats that remain non-azotemic, is unclear but may be related to the decline in glomerular filtration rate. Hyperthyroidism is a disorder resulting from the excessive production and secretion of T4 and T3 by the thyroid gland. Although the disorder and its pathological lesions have been well studied and described the cause remains illusive.

Whole blood and solid tissue samples from non-diseased, severe disease and mild disease cats based on T4 levels and thyroid histology were used in this study. Whole blood samples from 29 non-disease cats, 28 severe disease cats and 17 mild disease cats as well as solid thyroid tissue samples from 30 non-disease cats, 31 severe disease cats and 27 mild disease cats were collected and processed. The resulting total RNA samples were used for GeneChip analysis using our custom feline gene chip designed by Affymetrix. Data analysis was performed using The Partek s GS software for Gene Expression data. The Robust Multichip Average algorithm was used for background adjustment, normalization, and probe-level summarization of the raw data. ANOVA analysis was performed to find significant differentially expressed genes with a minimal False Discovery Rate control of 0.1 and a fold change of 1.3 in each direction.

During the mild disease state, pathways associated with DNA damage and apoptosis are most prominent. At later stages when the histopathological disease is more severe in addition to the aforementioned pathways others associated with TGF-beta signaling, cell adhesion and extracellular matrix remodeling take more prominence.

The analysis of this unique data set generated from the use of our proprietary GeneChip revealed molecular mechanisms that are associated with the transition from non-disease, to mild disease to severe disease, in the thyroid tissue as well as the blood. These mechanisms could provide insights into the causes of the disease and identify potential new therapeutic and diagnostic targets. Although it is well established that concurrent chronic kidney disease (CKD) develops in about 30% of hyperthyroid cats, no one has reported the use of the IRIS staging system for CKD before and after treatment of these hyperthyroid cats. The purpose of this study was to compare the effects of treatment in hyperthyroid cats with known stage 1 and 2 CKD in order to determine the effects of restoring euthyroidism or inducing hypothyroidism has on the IRIS stage in these cats. We evaluated 36 hyperthyroid cats (median age, 14 years) in this study. One day prior to treatment, serum T 4 concentration, serum chemistry analysis, complete urinalysis, and urine protein-to-creatinine ratio (UPC) were measured. All cats were again evaluated with the same parameters again 3 months after treatment with 131 I. Prior to treatment, 26 (72%) of the 36 cats had no evidence of azotemia (serum creatinine o 1.6 mg/dl), whereas 10 cats (28%) had stage 2 CKD (serum creatinine, 1.6-2.8 mg/dl). In the 36 cats, IRIS staging revealed proteinuria in 33 cats (92%), 21 with borderline proteinuria (UPC, 0.2-0.4) and 12 with overt proteinuria (UPC 4 0.4). Hyperthyroidism was cured in all 36 cats (median post-T4, 1.3 mg/dl). All cats had a good response to treatment; there were no signs of CKD except for polyuria and polydipsia in some cats. A significant (P o 0.001) increase in median values for both serum urea nitrogen (26 mg/dl to 31 mg/dl)) and creatinine (1.1 to 1.7 mg/dl) occurred after treatment. Nine of the 26 cats (34.6%) classified as nonazotemic or IRIS stage 1 prior to 131 I progressed to stage 2 CKD after 131 I. All 10 cats with stage 2 CKD before treatment remained azotemic after 131 I, with 5 cats remaining in stage 2 CKD, and 4 cats progressing to stage 3 CKD (serum creatinine, 3.1-3.7 mg/dl). There was a significant inverse relationship (P 5 0.002) between pretreatment urine specific gravity (USG) and post-treatment serum creatinine in the 36 cats. Of the 19 cats with post-treatment serum creatinine values 4 1.5 mg/dl (stage 2 to 3 CKD), 15 (79%) had pretreatment USG of o 1.040. In contrast, in the 17 cats with post-treatment serum creatinine values o 1.5 mg/dl, only 3 (18%) had pretreatment USG of o 1.040. A significant (P o 0.001) decrease in median UPC from 0.3 to 0.1 occurred after treatment, but there was no relationship between degree of proteinuria and IRIS stage in these cats. Two cats developed iatrogenic hypothyroidism after 131 I, diagnosed by finding low serum T 4 and high cTSH concentrations. Both hypothyroid cats had progressed from stage 1 before treatment to stage 2 and 3 CKD, respectively, after 131 I; after thyroxine replacement, serum creatinine decreased to near pretreatment concentrations in both cats. Conclusions: 1) IRIS stage 2 CKD is common in untreated hyperthyroid cats. 2) Progression to next higher IRIS stage is common after treatment, but most cats with remain relatively asymptomatic for CKD. 3) USG may be helpful in predicting which cat's IRIS stage will progress after 131 I. 4) Iatrogenic hypothyroidism worsens azotemia, an effect that appears reversible with replacement therapy. Home blood glucose monitoring (HBGM) of diabetic pets is likely to result in superior glycaemic control, minimizing episodes and impact of dangerous hypoglycaemia and reducing costs. Nevertheless, it has proven difficult to objectively establish a clear benefit of HBGM using biological parameters (clinical signs, blood glucose, fructosamine). The current study aimed to assess the impact of HBGM on owner perceived quality of life (QoL) aspects of diabetes mellitus (DM) treatment, using the recently validated psychometric tool DIAQoL-pet.

Owners of insulin treated diabetic cats were recruited to complete the 29-item tool, evaluating areas affecting the cat's and owner's QoL, including: worry about pet's DM, hypoglycaemia, costs, owner's desire for autonomous control over the pet's DM, etc. Item-weighted-impact-scores (IWIS), reflecting frequency and importance ratings of each item, were calculated, as well as averageweighted-impact-scores (AWIS; average IWIS of all items), as an overall measure of diabetes dependent QoL. Frequencies, IWIS and AWIS were compared between owners practising HBGM and those who did not using Mann Whitney U test (significance p o 0.05).

Two hundred and eleven owners of insulin treated diabetic cats completed the DIAQoL-pet; 161 owners practised HBGM, whereas the remaining 50 did not practise any form of home monitoring (including urine glucose). IWIS for 'excessive drinking' and 'owner wanting more control' were significantly different between the HBGM-group (mean1/-standard deviation: À2.011/À2.4 and À5.071/À4.8) and the non-HBGM-group (À3.361/À3.8 and À1.861/À3.2). There was no significant difference between the groups with regards to the IWIS for other items, including 'worry about hypoglycaemia' or 'worry about pet's DM'. Polydipsia was reported significantly more frequently in the non-HBGM-group and this was the reason for the difference between groups in this item's IWIS as it was considered of equal importance. Frequency and IWIS of reported occurrence of hypoglycaemia signs were not significantly different. AWIS for both groups was not significantly different (HBGM: À1.851/À1.3; non-HBGM: À1.871/À1.1).

The current study suggests that HBGM is predominantly practised by owners who desire more autonomous control over their cat's DM. The frequency of polydipsia was lower in the HBGM-group perhaps suggesting superior control. However, HBGM did not detectably affect the impact of the majority of QoL-items, nor the frequency of hypoglycaemic episodes. Overall diabetes dependent QoL of diabetic cat and owner, as measured per DIAQoL-pet, was unaffected by HBGM. These data argue for the use of HBGM in selected pet-owner combinations rather than as part of a practice's standard DM management protocol, although further studies are indicated. Insulin resistance is associated with impaired activation of the insulin signaling pathway in peripheral tissues such as skeletal muscle, visceral and subcutaneous (SC) adipose tissue. High plasma glucose, fatty acid and endotoxin levels are three major causes of insulin resistance in feline and human obesity and in type 2 diabetes mellitus. However, the mechanisms by which these factors influence insulin action are still unclear. Therefore, our aim was to investigate the tissue-specific expression of crucial mediators of insulin action such as the insulin-receptor substrate 1 (IRS1), the serine/threonine protein kinase B (PKB/Akt) and of the principal insulin-dependent glucose transporter protein (GLUT4) in feline models of hyperglycemia, hyperlipidemia and subacute endotoxemia.

Healthy cats were infused through the jugular vein with glucose (n 5 5), lipids (n 5 6) or lipopolysaccharide (LPS; n 5 5) for 10 days to clamp their blood concentrations at the approximate level found in untreated feline diabetes (glucose: 25-30 mmol/l; triglycerides: 3-7 mmol/l) or to induce a systemic low-grade inflammation (LPS; rectal temperature: 39.2-40.51C), respectively. Healthy control cats were infused with saline (n 5 10). On day 10, specimens were collected from skeletal muscles, visceral and SC fat and processed for IRS1 mRNA expression, total and phosphorylated PKB/Akt and GLUT4 protein expression.

Gene transcripts of IRS1 were not different between the groups. Compared to controls, skeletal muscle PKB/Akt phosphorylation was 91% lower in cats infused with glucose (P o 0.01); lipid-infused cats showed a trend for a decrease in PKB/Akt phosphorylation (31% lower than saline) and had decreased GLUT4 expression (P o 0.05) in muscle. Total (p o 0.01) and phosphorylated (P o 0.05) PKB/Akt protein expression were decreased in the SC adipose tissue of LPS-infused cats compared to controls. In these cats, phosphorylation of PKB/Akt protein was also decreased in visceral fat (p o 0.01).

Sustained hyperglycemia and, to a lesser extent, hyperlipidemia impaired insulin signaling and glucose transport pathways primarily in skeletal muscle; endotoxemia reduced insulin sensitivity mainly in adipose tissues. Thus, the development of insulin resistance in response to hyperglycemia, hyperlipidemia or endotoxemia might be affected by tissue-specific mechanisms in cats. Separately used, Single Photon Emission Computed Tomography (SPECT) and Computed Tomography (CT) both lack sensitivity and are additionally hampered by a poor anatomical location capacity and a lack of specificity, respectively. These drawbacks suggest an interest in the fusion of images obtained by the 2 techniques. The aim of this study is to test SPECT/CT fusion performance in dogs with insulinoma.

Inclusion criteria were: 1/ a biological diagnosis of insulinoma; 2/ an examination by high resolution CT scan and 111 In-pentetreotide SPECT followed by SPECT/CT fusion; 3/ a surgical or post mortem examination completed by histopathological analysis. SPECT examination showing abnormal foci and CT scan showing pancreas, lymph nodes (LN) or liver abnormalities were considered positive. In case of double positivity, presence (IMP1) or absence (IMP-) of superimposition of abnormal images was noted.

Ten dogs were included. In 2/10 dogs, superimposition of abnormalities couldn't be tested. CT scan detected 3 abnormal images [2 pancreatic nodules (PN), 1 enlarged LN (ELN)] while SPECT failed to show any abnormal uptake. Both dogs became euglycemic after removal of PN and LN designed by CT scan. In 6/ 10, all abnormal images were classified as IMP1 [6 PN, 1 ELN and 1 diffuse hepatic infiltration (DHI)]. Surgery performed on 5/6 resulted in euglycemia in 4; 1 dog remained hypoglycemic after partial removal of 1 PN. PN localization and DHI were confirmed after necropsy in the 6 th dog. In 2/10 dogs IMP1 and IMP-images were both recorded. In 1 dog, a DHI was classified as IMP1 but PN localization was IMP-: localized in the left lobe by CT scan and in the corpus by SPECT, the latest localization being confirmed after necropsy. In the other dog PN localization was IMP1 but a diffuse SPECT signal superimposing to the liver considered as normal on CT scan was noted. Hepatic biopsy confirmed SPECT results.

This study confirms an imperfect sensitivity of both CT scan and SPECT. It confirms that CT scan can be associated with unspecific abnormal images. Subject to a confirmation on a larger cohort of dogs, it indicates that IMP1 images provide specific detection and accurate localization of canine insulinomas' primary lesions and metastasis. The majority of dogs with primary hypoadrenocorticism (PH) reveal clinical and laboratory abnormalities of gluco-and mineralocorticoid deficiency. In some of them sodium and potassium levels are normal, a phenomenon currently called atypical Addison's. It has been postulated that in those cases adrenal destruction is confined to the zona fasciculata/reticularis, resulting in isolated glucocorticoid deficiency. However, there are no histological studies confirming a normal zona glomerulosa and in most reported cases diagnosis was based solely on low post-ACTH cortisol levels. The aim of the study was to evaluate aldosterone (ALDO) levels in dogs with PH with and without electrolyte abnormalities.

Seventy dogs with newly diagnosed PH were included. ALDO concentrations (RIA, Coat-A-Count s , Siemens) were measured before and 60 min after administration of 250 mg synthetic ACTH (Synacthen s , Novartis) IV. Results were compared to those of 19 healthy dogs and 17 dogs with diseases mimicking PH. To confirm that peak concentrations were not missed ALDO was additionally measured 15, 30 and 45 min after ACTH in 19 dogs (5 with PH, 14 with PH mimicking diseases). Results were analysed by means of non-parametric statistical methods (p o 0.05).

Post-ACTH ALDO was significantly lower in dogs with PH (0-253 pg/ml, median 0 pg/ml) than in healthy dogs (46-602 pg/ml, median 187 pg/ml) and in dogs with PH mimicking diseases (0-639 pg/ml, median 155 pg/ml). Low post-ACTH ALDO was found in 67/70 dogs with PH, in 64/67 of them levels were below the detection limit of the assay. Normal sodium and potassium levels were found in 5/67 dogs (7%), 6/67 dogs (9%) had hyponatremia and normal potassium, 56/67 dogs (84%) had hyponatremia and hyperkalemia. Electrolyte abnormalities ranged from mild to severe. There was no correlation between post-ACTH ALDO and sodium and a weak correlation between post-ACTH ALDO and potassium (r 5 À0.28). ALDO concentrations were not different 30, 45 and 60 min after ACTH.

The results demonstrate that ALDO levels are low in most dogs with PH independent of the degree of electrolyte abnormalities. This implicates that all three zones of the adrenal cortex are compromised and that there are mechanisms which allow maintenance of a normal electrolyte balance without ALDO. Definitive diagnosis of canine hypoadrenocorticism (HA) is based on inadequate cortisol secretion following adrenocorticotropic hormone (ACTH) administration. An abnormal serum sodium to potassium (Na:K) ratio can be used to determine whether an ACTH stimulation test is warranted. The aim of this study was to examine the utility of combining the Na:K ratio with white blood cell counts to determine whether an ACTH stimulation test is warranted.

A retrospective review of medical records of dogs examined between 2005 and 2009 was performed. 53 dogs diagnosed with HA and 110 control dogs, in which a diagnosis of HA was excluded during the study period, were included. Inclusion criteria for all 163 dogs were hospitalization with intravenous fluid therapy, a complete blood count, and serum Na and K measurements at the time of initial examination. Dogs were included in the HA group if they also had pre and post ACTH stimulation serum cortisol concentrations 1.0 mg/dl. Dogs were included in the control group if they had resting or post ACTH stimulation serum cortisol concentration 4 2.0 mg/dl. Exclusion criteria were recent administration of glucocorticoids, prior treatment of hyperadrenocorticism, or serum cortisol concentration 4 1.0 mg/dl but 2.0 mg/dl. Continuous variables were compared between groups using the Mann-Whitney U test. Receiver operating characteristic (ROC) curves were produced to assess the sensitivity and specificity of detecting HA with various cutoffs for each variable. Data is presented with 95% confidence intervals (CI) and statistical significance was defined as p o 0.05.

The Na:K ratio, neutrophil count and neutrophil:lymphocyte ratio were significantly lower in dogs with HA than in dogs without HA (p o 0.01 for each). Lymphocyte and eosinophil counts were significantly higher in dogs with HA compared to dogs without HA (p o 0.001 for each). The areas under the curve by ROC analysis were largest for Na:K ratio (0.87, CI:0.81-0.92) and lymphocyte count (0.85, CI:0.78-0.90). A Na:K ratio 39.6 was 100% sensitive (CI:93-100%) but only 15% specific (CI:9-23%) for detecting HA. A lymphocyte count ! 0.77 x10 3 cells/mL was 100% sensitive (CI:93-100%) and 35% specific (CI:27-45%). Conversely a Na:K ratio 20.1 was 51% sensitive (CI:37-65%) but 100% specific (CI:97-100%) and a lymphocyte count ! 6.1 Â 10 3 cells/mL was 9% sensitive (CI:3-20%) but 100% specific (CI:97-100%). A Na:K ratio 35.5 was 96% sensitive (CI:87-100%) and 35% specific (CI:26-44%) for detection of HA and a lymphocyte count ! 0.79 Â 10 3 cells/mL was 98% sensitive (CI:90-100%) and 36% specific (CI:27-46%) for detection of HA. A combination of this Na:K ratio ( 35.5) and lymphocyte count (! 0.79 Â 10 3 cells/mL) was 96% sensitive (CI:87-100%) and 55% specific (CI:47-66%) for detection of HA.

These results indicate that the combination of lymphocyte count and Na:K ratio results in a better screening test for HA than the use of the Na:K ratio alone. Pheochromocytoma is a malignant, catecholamine-producing, adrenomedullary tumor. Clinical signs resulting from excessive catecholamine secretion are typically non-specific, making differentiation from other adrenal tumors a challenge. Elevated plasma concentrations of the catecholamine breakdown products metanephrine (MN) and normetanephrine (NMN) are used to identify pheochromocytoma in humans. This study tested the hypothesis that plasma metanephrine concentrations are greater in dogs with pheochromocytoma than in dogs with other adrenal neoplasms, healthy dogs and dogs with non-adrenal illness.

EDTA plasma was collected from healthy dogs and unwell, hospitalized dogs with non-adrenal illness, pheochromocytoma and cortical tumors between April 2007 and October 2010. Samples were stored at À801C before measurement of free MN and NMN concentrations using high pressure liquid chromatography at the Central Laboratory for Clinical Chemistry at the University of Groningen (33 samples) or the Mayo Clinic, Rochester, Minnesota (7 samples). Kruskal-Wallis tests followed by Dunn's multiple comparison analysis were used to compare results between groups. Significance was set at P o 0.05. Results are reported as median [range] .

Eight dogs with pheochromocytoma, 5 healthy dogs, 15 dogs with non-adrenal illness and 12 dogs with cortical tumors were sampled. Pheochromocytoma was diagnosed histologically (7 dogs) or cytologically (1 dog). Cortical tumors were diagnosed histologically (9 dogs) or by response to trilostane treatment after obtaining consistent endocrine test results (3 dogs Occult hyperadrenocorticism (HAC) has been theorized to exist in which excess adrenal sex hormone secretion induces the clinical signs and laboratory changes associated with classic HAC. However, the ability of sex hormones to cause such alterations has never been closely evaluated. If sex hormones can cause a syndrome similar to classic HAC, they should be able to induce expression of classic glucocorticoid-induced genes. The purpose of the study was to determine if in vitro expression of the gene for corticosteroid-induced ALP (CIALP) could be induced by clinically relevant concentrations of cortisol and sex hormones believed to cause occult HAC.

Canine hepatocytes were purchased from a commercial source (CellzDirect or InVitro) in 6-well plates. Upon arrival (3-4 plates per shipment), the cells were allowed to recover in general media per supplier recommendations. After 4 hrs, media was changed to William's E media (-L-glutamine) containing concentrations of cortisol or sex hormones that have been documented in the literature in dogs with HAC or with purported occult HAC. Each plate was treated with a different hormone (cortisol, 17-hydroxyprogesterone [17OHP], progesterone, estradiol or androstenedione), and each well contained a different concentration (starting with no hormone added as a negative control) to evaluate a dose response. Media was changed daily. After 5 days of hormone exposure, RNA was extracted. Reverse transcription was performed and the product used for quantitative PCR for CIALP and beta-actin (Roche Lightcycler) using a gene-specific fluorescent probe for detection. Standard curves were created for each gene. All samples and standards were run in duplicate. Using the Lightcycler software (vers 4.0), CIALP expression was normalized to that of beta-actin. Fold change in expression was determined relative to the negative control. Each sex hormone was used to treat 3 plates; one plate in each shipment was treated with cortisol as a positive control.

For cortisol, a dose response was seen in expression of the CIALP gene. Compared to no cortisol, 10, 50, 100, 150, 250 and 500 nmol cortisol increased expression 2.8, 4.6, 7.1. 9.3, 9.9 and 9.8 fold, respectively. A 2-fold increase is considered significant (J.Vandesompele et al Genome Biol 2002). Expression of CIALP was not significantly induced in response to any concentration of 17OHP (10 nM maximum), progesterone (5 nM maximum), estradiol (max 400 pM maximum) or androstenedione (100 nM maximum).

We conclude that in vitro these sex hormones do not induce expression of the CIALP gene which is classically induced by cortisol in vivo; indeed, elevated serum CIALP activity is a hallmark of classic HAC. Thus, the ability of the sex hormones to induce the gene in vivo must be questioned and evaluated. Measurement of sex hormones has been advocated as an adjunct means for diagnosing typical hyperadrenocorticism (HAC), i.e. disease due to excess cortisol secretion, as well as for diagnosis of atypical HAC, i.e. disease due to excess adrenal sex hormone secretion. However, measurements in either setting have not been widely studied. Therefore, our objectives were: 1. To determine the sensitivity of 17-hydroxy-progesterone (17OHP) and estradiol concentrations pre-and post-ACTH for diagnosis of typical HAC. 2. To determine the specificity of 17OHP and estradiol concentrations preand post-ACTH for diagnosis of occult HAC.

Dogs that had PDH (n 5 12), dogs that were suspected to have HAC but proven not to (had non-adrenal illness [NAI, n 5 89]) or dogs that were healthy (n 5 20, used to establish reference ranges [RR]) were enrolled. ACTH stimulation tests were performed (5 mcg/kg cosyntropin IV); blood samples were drawn pre and 60min post; 17OHP and estradiol were measured by previously validated radioimmunoassays. A Kruskal-Wallis Rank Sum test was used to compare values between the groups. Significance was set at p o 0.05.

For basal and ACTH-stimulated 17OHP concentrations, the RR were determined to be 0.03-0.6 ng/mL (mean AE 2 s.d.; range 0.03-0.55) and 0.3-2.2 ng/mL (range 0.31-1.95), respectively. In PDH dogs, 3 and 7 had basal and post-ACTH 17OHP concentrations above the RR, respectively; in the NAI group, 23 and 25 dogs had concentrations above the RR, respectively. Thus, the sensitivity of basal and post-ACTH 17OHP measurement for diagnosis of HAC is 25% and 58%, respectively. Specificity of diagnosis is 72% and 74%, respectively. Post-ACTH 17OHP concentration was significantly different between groups.

For basal and stimulated estradiol concentrations, the RR were determined to be 81-180 pg/mL (range 89-195) and 71-170 pg/mL (range 74-151), respectively. For both basal and stimulated estradiol, 4 PDH dogs (n 5 9) had concentrations above the RR; for those with NAI (n 5 80), 14 and 15 had concentrations above the RR, respectively. Thus, the sensitivity of estradiol measurement for diagnosis of HAC is 44% for both pre-and post-ACTH. Specificity of estradiol for diagnosis for HAC is 83% and 81% for pre-and post-ACTH, respectively. Overall, 23 dogs with NAI had at least one elevated estradiol concentration (total specificity 70%). Post-ACTH estradiol concentration was not significantly different between groups.

We conclude that use of 17OHP and estradiol concentrations for diagnosis of HAC can be problematic. Sensitivity and specificity are relatively low, potentially leading to misdiagnoses. Diabetes mellitus is one of the most common feline endocrinopathies and is considered to have a similar pathophysiological basis to human type 2 diabetes. Several studies have identified risk factors for development of diabetes mellitus in cats, which include age, obesity, inappropriate diet and physical inactivity. However, to date, no specific genetic risk factors have been identified. Genome-wide association studies in humans have identified several genes that predispose to obesity and/or diabetes mellitus, one of which is the melanocortin receptor 4 (MC4R) gene. The aim of the current study was to identify polymorphisms (SNPs) in the feline MC4R gene and to use these to perform a case:control study to determine whether these candidate gene SNPs were associated with diabetes mellitus in cats.

Genomic DNA from 10 cats (6 domestic short hair [DSH], 4 Burmese) was initially analysed by PCR and direct sequencing using felMC4R-specific primers, which identified a missense mutation (MC4R:c.92 C 4 T) in the region encoding the extracellular domain of the receptor protein in DSH cats only. One hundred and nineteen DSH cats were subsequently recruited into the case:control study. Fifty nine cats were obese diabetic (29 male, 30 female), mean age 11.8 years (range 6-18y); mean weight 6.68 kg (range 5.15-10 kg). Sixty lean cats were used as controls (30 male, 30 female), mean age 13.81 years (range 9-19y), mean weight 3.99 kg (range 2.56-5.68 kg). The T to C base change alters a restriction site in the sequence recognized by the enzyme BstOI, such that DNA from cats with the mutant (C) allele can be cut, whereas that from the wild-type (T) allele cannot. Primers were designed that flanked the mutation to allow PCR amplification of this region of MC4R from genomic DNA obtained from EDTA blood. The PCR products were purified and subject to restriction fragment length polymorphism (RFLP) analysis. BstOI digestion products were then analysed by agarose gel electrophoresis. Of the 59 diabetic cats, 32 (54%) were homozygous for the mutation (CC), compared to 21 (35%) of 60 control cats. Statistical analysis (two tailed Fisher's square test) revealed that this difference between groups was statistically significant (p 5 0.0431).

In conclusion, this pilot study has identified a missense mutation in the coding sequence of MC4R. This could be an important predisposing factor for development of diabetes and/or obesity in DSH cats. Polymorphisms in a similar region of human MC4R predispose to obesity, which in turn is a major risk factor for Type 2 diabetes. Hyperadrenocorticism (HAC) is one of the most common endocrine disorders of dogs. The two most effective medical treatments are trilostane (Vetoryl s ) and mitotane (Lysodren s ). Previous studies evaluating the effect of treatment on aldosterone secretion measured the hormone at 60 min post-ACTH administration. However, the optimal sampling time would be at the time of maximal secretion, which occurs 30 minutes after the 5 mg/kg dose commonly used for the test (Carlson et al, JVIM, 2010). Thus, the true effect of either medication on aldosterone secretory capacity is unknown. Our objectives were: 1) to assess and compare the effect of treatment with trilostane and mitotane in dogs with pituitarydependent HAC (PDH) on aldosterone secretory reserve at 30 min post-ACTH stimulation and 2) to determine if changes in aldosterone concentration at that time correlate with changes in serum sodium and potassium concentrations.

Forty-six dogs being treated for PDH with either mitotane (n 5 30) or trilostane (n 5 16) have been enrolled. The dogs could be treated for any length of time. All had ACTH stimulation tests performed (5 mcg/kg cosyntropin IV); blood samples were drawn before and at 30 and 60 min post-ACTH for monitoring of cortisol and aldosterone concentration using previously validated radioimmunoassays. Ten historical normal controls were also included. Serum sodium and potassium concentrations were measured in the basal samples. A Kruskal-Wallis Rank Sum test was used to compare values between normal dogs and those treated with mitotane or trilostane. Linear regression analysis was used to determine if a correlation existed between electrolyte and aldosterone concentrations or between cortisol and aldosterone concentrations. Significance was set at the p o 0.05 level.

ACTH-stimulated aldosterone concentrations in mitotane-treated but not trilostane-treated dogs were significantly lower than that in normal dogs at both the 30 and 60 min time points. No difference was detected between aldosterone concentrations at 30 and 60 min after ACTH injection in either treatment group. A positive correlation existed between the 60-min cortisol and 30-min aldosterone concentrations in the trilostane-treated group (R 5 0.813), i.e. the peak post-ACTH concentration for each hormone, but not in dogs treated with mitotane. Basal serum sodium and potassium concentrations were not correlated with the basal aldosterone concentration in either treatment group.

In conclusion, treatment with mitotane resulted in decreased aldosterone secretory reserve, but this did not correlate with hyperkalemia or hyponatremia. Measurement of aldosterone concentrations is not predictive of electrolyte concentrations.

Previously presented at the Auburn University Phi Zeta Research Emphasis Day, November 10, 2010. Antioxidant depletion is documented in humans with hyperthyroidism, and is reversible with treatment. In addition, antioxidant depletion has been shown to increase the risk of methimazole toxicity in rats. The primary aim of this study was to determine whether deficiencies in glutathione (GSH), ascorbate (AA), or vitamin E, along with increases in urinary 8-isoprostanes, were present in hyperthyroid cats, and were reversible after radioiodine treatment. A secondary aim was to determine whether antioxidant abnormalities were associated with a prior history of methimazole toxicity.

Ongoing prospective, controlled, observational study. Otherwise healthy client-owned hyperthyroid cats presenting for radioiodine therapy (n 5 26 to date) and healthy age-matched controls (n 5 32 to date) were recruited. All cats were screened with CBC, biochemical panel, urinalysis, and T4, as well as red blood cell (RBC) GSH, plasma AA, plasma vitamin E, and urinary 8-isoprostanes. Hyperthyroid cats were re-evaluated 2 months after radioiodine treatment.

Unlike in humans, median blood antioxidants were not significantly different in hyperthyroid cats (GSH 1.5 mM; AA 11.4 mM, and vitamin E, 19 g/ml) compared to controls (GSH 1.4 mM; AA 12.5 mM, and vitamin E, 17 g/ml). Results for urinary isoprostanes are pending, and associations with methimazole toxicity will be investigated after full recruitment. RBC GSH concentrations did increase significantly (to 1.6 mM; P 5 0.019) after radioiodine treatment. However, this modest change is unlikely to be clinically significant. Preliminary data do not indicate clinically significant blood GSH, ascorbate, or vitamin E deficiencies in hyperthyroid cats. With appropriate insulin therapy and a low carbohydrate diet, up to 90% of newly diagnosed diabetic cats are eventually able to maintain euglycemia without insulin administration, and these cats are considered to have achieved remission. There are currently no published data reporting the glucose tolerance status of cats classified as being in remission, and it is unknown whether these cats are truly in diabetic remission, or should be classified as non-insulin dependent diabetics, or having impaired glucose tolerance, and/or impaired fasting blood glucose. The aim of this study was to determine fasting blood glucose concentrations and glucose tolerance status of cats in remission.

The study was a prospective study in a feline-only clinic. For inclusion, diabetic cats had to have achieved remission through insulin therapy, and insulin withheld for a minimum of two weeks.

Five diabetic cats in remission and five matched non-diabetic cats were enrolled in the study. Blood samples were obtained via the ear vein but where the cat's temperament precluded this, from the jugular.Glucose concentration was measured using a meter calibrated for feline blood (Abbott AlphaTRAK). A simplified glucose tolerance test was performed after food was withheld for 24 hours. A 22G catheter was placed in a cephalic vein three hours before the GTT was commenced, to minimize the effects of stress on blood glucose concentration. Blood glucose concentration was measured at time 0 and then a 1 g/kg dose of glucose was administered slowly via the intravenous catheter. Further blood glucose measurements were made at 2 hours and then hourly until glucose had returned to o 117 mg/dL (o 6.5 mmol/L).

In the control group, all cats had a fasting blood glucose below 117 mg/dL, and following glucose administration, glucose had returned to o 117 mg/dL by 3 hours. Fasting blood glucose in the remission group was o 126 mg/dL (7 mmol/L) in all cats except one, which had fasting blood glucose of 135 mg/dL (7.5 mmol/L). Following glucose administration, all five cats in remission had blood glucose above 117 mg/dL (6.5 mmol/L) at three hours, four were o 117 mg/dL at four hours, and one returned to o 117 mg/dL at five hours. The cat with impaired fasting glucose subsequently became diabetic after steroid administration.

The results of this study show that these cats, while no longer diabetic, have mildly impaired glucose tolerance compared to nondiabetic cats, and a minority have impaired fasting glucose. The objective of this study was to determine the role of iodine restriction in the nutritional management of cats with naturally occurring hyperthyroidism. Five domestic shorthair cats ranging in age from 8-17 years were confirmed to have hyperthyroidism based on persistently increased serum total thyroxine concentrations (TT4), palpable thyroid nodule and weight loss. Serum TT4 concentrations ranged from 55-146 nmol/l (reference range 10-55 nmol/l). The cats were then fed a low iodine containing food (0.47 ppm iodine DMB, as measured by epiboron neutron atomic activation). Serum TT4 concentrations were measured every 3 weeks. Biochemistry parameters were also evaluated at weeks 0, 6 and 9.

At 9 weeks, serum TT4 concentrations had decreased in all cats with 4 of 5 cats (80%) being euthyroid (mean 48 nmol/l; range 41-54 nmol/l). The remaining hyperthyroid cat had an initial serum TT4 of 146 nmol/l, which decreased to 83 nmol/l after being fed the iodine-restricted food. Mean decrease in TT4 for all 5 cats was 26 nmol/l (range 8-63 nmol/l). Renal parameters remained stable in all 5 cats.

These 5 cats along with 4 additional newly diagnosed hyperthyroid cats were transitioned to a similar food that contained less iodine (0.28 ppm DMB). Baseline serum TT4 concentrations in the 4 new cats ranged from 55-73 nmol/l. Serum TT4 and other biochemical parameters were monitored every 3 weeks for 9 weeks, and then every 4 weeks for an additional 8 weeks.

With the 0.28 ppm iodine food the four new cats became euthyroid with a mean TT4 concentration of 41 nmol/ (range 29-50nmol/l). The 4 euthyroid cats from the earlier feeding study had a further decrease in TT4 concentration (mean TT4 5 39 nmol/l, range 38-54nmol/l). The single non-euthyroid cat from the first study had a serum TT4 concentration of 61 nmol/l, a decrease from the baseline concentration of 83 nmol/l. The average decrease in serum TT4 for all 9 cats was 20 nmol/l (range 2-35 nmol/l).

Finally, 8 of the 9 cats were fed a third iodine-restricted food (0.17 ppm DMB) along with one other newly diagnosed hyperthyroid cat (79 nmol/l serum TT4) and evaluated every 4 weeks. All 9 cats in this evaluation were euthyroid (mean TT4 33 nmol/l; range 23-50 nmol/ l). This result included the cat whose serum TT4 remained in the hyperthyroid range in the first two evaluations. The average decrease in TT4 was 13 nmol/l (range 0-43 nmol/l). Biochemical features of renal function remained stable and no other biochemical abnormalities were observed. In summary, the results of these three feeding studies demonstrate that feline hyperthyroidism can be managed effectively with dietary iodine restriction. We have shown previously that restriction of dietary iodine (I) is a safe and effective method for decreasing serum thyroxine concentrations (TT4) in cats with hyperthyroidism. The objective of this study was to determine the maximum level of iodine in a nutritionally balanced feline mature adult food required to maintain normal serum TT4 concentrations in hyperthyroid cats currently being controlled on a food containing 0.15 ppm I (DMB) as measured by epiboron neutron atomic activation. All cats were previously diagnosed at least 14 months prior to the start of the study and their TT4 concentrations were maintained in the normal range by dietary iodine restriction for a minimum of 10 months (range 10 months-3 years). Serum TT4 concentrations ranged from 9-42 nmol/l (reference range 10-55 nmol/l) at the beginning of the study.

The cats were divided into two groups each containing 9 cats. Groups were similar in age and gender distribution (mean age 5 13.8 years, range 12-18 years). One group (Group A) was placed on a food that was formulated for mature adult cats containing 0.39 ppm I (DMB). The other group (Group B) was placed on a similar food that differed only in that it contained 0.47 ppm I (DMB). Blood was collected from all cats every three weeks and analyzed for serum TT4 concentration. Biochemistry parameters were also evaluated at weeks 0, 6 and 9.

All Group A cats exhibited increases in serum TT4 concentration (mean increase of 25 nmol/l above baseline, range 5-48 nmol/l). Seven of the cats remained in the euthyroid range (mean serum TT4 5 36 nmol/l, range-27-54 nmol/l). Two cats exceeded the upper limit of the reference range (59 and 76 nmol/l respectively). The cats in Group B also exhibited increases in serum TT4 concentration but to a greater degree than the cats in Group A (mean increase 39 nmol/l, range 20-60nmol/l). Four cats remained in the euthyroid range (mean serum TT4 5 41, range 29-49 nmol/l). The five remaining cats all exceeded the upper limit of the reference range (mean serum TT4 5 76 nmol/l, range-59-99 nmol/l).

All cats returned to a euthyroid state within 1 month of being returned to a diet containing 0.17 ppm I (DMB). It was determined that serum TT4 concentrations are not ideally controlled in the normal range in hyperthyroid cats fed a food containing ! 0.39 ppm I (DMB). Hyperthyroidism is a common disease in old cats. Excessive production of thyroid hormones is the hallmark of the disease. Three main treatments for feline hyperthyroidism include radioactive iodine, thyroidectomy, and antithyroid drugs such as methimazole. Previously we have shown that limiting dietary iodine to or below 0.27 ppm induces euthyroidism in cats with hyperthyroidism compared with a similar diet containing 0.42 ppm iodine. The objective of this study was to test whether dietary iodine at 0.32 ppm would induce euthyroidism in cats with naturally occurring hyperthyroidism.

Fourteen cats with hyperthyroidism confirmed by serum TT 4 and FT 4 measurements were stratified into two groups based on gender and age. One group (control: 4 males and 3 females, age ranged from 11 to 15 years) was given a positive control dry cat food (0.17 ppm iodine) while the other group (test: 3 males and 4 females, age ranged from 12 to 17 years) was fed a commercial dry cat food (1.9 ppm iodine) for at least 6 weeks before the study. Afterwards (week 0), the control cats continued to receive the same food while cats in the test group were given a test food (0.32 ppm iodine) for additional 12 weeks. All cats had free access to their food and deionized water during the study. Blood samples were collected during weeks 0, 3, 6, and 12 of the study.

The control cats maintained euthyroidism during the study. The test food significantly reduced serum TT 4 (72 AE 12, 43 AE 9 Ã , 42 AE 9 Ã , 40 AE 6 Ã nmol/L in weeks 0, 3, 6 and 12, respectively; Ã : p o 0.05 compared with week 0, Dunnett's t test). It also significantly reduced FT 4 at the end of the study (17 AE 2 vs. 23AE 2 pmol, week 12 vs. week 0; Dunnett's t test, p o 0.05). Serum FT 4 was within the reference range (10-55 pmol/L) in cats in both groups. Serum TT 3 , FT 3 , and TSH were not affected by the test food and were within the reference ranges (TT 3 : 0.6-1.4 nmol/L, FT 3 : 1.5-6 pmol/L, and TSH: 0-21 mU/L) in cats of both groups during the study.

This study demonstrates that dietary iodine at or below 0.32 ppm provides an effective and inexpensive therapy for cats with naturally occurring hyperthyroidism. Radioactive iodine ( 131 I) is a widely used treatment for feline hyperthyroidism. Prior to 131 I administration, many cats receive methimazole therapy. It has been suggested that recent withdrawal of methimazole prior to 131 I may increase the risk of hypothyroidism, inhibit the response to therapy, or have no effect. To further address this question, a retrospective medical records search was performed to identify hyperthyroid cats that received 131 I therapy after methimazole treatment. Inclusion criteria included documentation of the time interval between discontinuation of methimazole and 131 I administration, and measurement of thyroxine (T 4 ) at 7-14 days after 131 I. Cats were divided into 2 groups: those receiving 131 I within 1 day of stopping methimazole, and those receiving 131 I treatment 5 or more days after stopping methimazole.

Sixty cats met the inclusion criteria. Forty received 131 I within 1 day of stopping methimazole. Of those, 20 (50%) had a low T4 (o 1.2 mcg/dl), 17 (42.5%) had a normal T4 (1.2-4.8 mcg/dl), and 3 (7.5%) had an elevated T4 (4 4.8 mcg/dl) at 7-14 days after 131 I therapy. Fourteen cats received 131 I 5 or more days after stopping methimazole: 8 (57%) had a low T 4 , 5 (36%) had a normal T 4 , and 1 (7%) had an elevated T 4 at 7-14 days after 131 I therapy. The results were compared with a Fisher's exact test and there was no difference between the groups (p 5 0.76). These findings indicate that stopping methimazole therapy within 1 day of 131 I therapy does not inhibit the response to therapy. Pharmacokinetic studies evaluating synthetic insulin analogs such as glargine necessitate the ability to measure the blood concentrations of glargine without cross-reactivity to endogenous insulin. Although the cross-reactivity between endogenous human insulin assays and synthetic analogs is often known for commerciallyavailable assays, the degree of cross-reactivity of human insulin assays with feline insulin is not. The purpose of this study was to evaluate the cross-reactivity of feline insulin with a commerciallyavailable human insulin ELISA with known cross reactivity to several synthetic analogs.

Pre-and post-prandial blood samples were collected from four healthy cats immediately prior to and approximately 15 minutes following a meal, for a total of 8 samples. Dextrose was added to the meals given to two of the cats. Blood samples were immediately centrifuged and the serum was collected, aliquoted, and stored at À201C until analysis. Serum insulin levels were determined in parallel with commercially-available feline insulin and human insulin ELISAs. The ELISAs were run in duplicate and according to the manufacturer's instructions.

Concentrations of serum insulin measured by the feline insulin ELISA ranged from 12.7 ng/L to 4 700 ng/L. Despite the wide range of concentrations of feline insulin, all 8 samples evaluated with the human insulin ELISA yielded absorbance readings equal to or lower than the absorbance of the negative control, indicating no crossreactivity between the evaluated human insulin assay and feline insulin. Since this assay is reported to cross-react significantly with glargine, it is a great candidate for determination of serum glargine concentrations in cats. The aim of this prospective, controlled study was to compare the efficacy of two trilostane protocols for treatment of canine pituitary-dependent hyperadrenocorticism (PDH). Among the 28 client-owned dogs diagnosed with PDH, only the dogs weighing o 5 kg were selected (n 5 16). Group A (n 5 9; low-dose treatment group) and group B (n 5 7; high-dose treatment group) received 0.8 AE 0.3 mg of trilostane/kg orally every 12 hours and 30 mg of trilostane/ body orally every 24 hours, respectively. All of the dogs were reassessed at 2, 4, 12, and 24 weeks after the initiation of treatment.

The improvement in post-ACTH stimulation serum cortisol concentration, as well as clinical signs in group A, required more time than group B; however, 2 of 7 dogs in group B had clinical signs and abnormal laboratory findings consistent with hypoadrenocorticism after treatment for 20 weeks. Twenty-four weeks later, all of the dogs of both groups improved the abnormal clinical findings. The present study suggests that twice daily, low-dose administration of trilostane is effective in the management of canine PDH and may be safe without the potential adverse effects of once daily, high-dose treatment. However, because this study involved only a small number of dogs, a population-based control study will be needed to clarify the efficacy of low-compared to high-dose trilostane treatment. Cobalamin is essential for a variety of metabolic processes in many tissues and organs, and has effects on cell growth and peripheral and central nervous system function. Chronic distal small intestinal disease in humans, cats, and dogs has been shown to cause cobalamin deficiency. An immunoassay for the measurement of serum cobalamin concentration in these species is being used in routine practice for the diagnosis of cobalamin deficiency. In pigs, the role of cobalamin has not yet been extensively investigated. Thus, the aim of this study was to analytically validate an immunoassay, labeled for use in humans, for the measurement of cobalamin in porcine serum samples and secondly to determine serum cobalamin concentrations in weaned pigs.

For the analytical validation of the assay, serum cobalamin concentrations were measured using the commercially available IMMULITE s 2000 cobalamin immunoassay (Siemens Healthcare Diagnostics Ltd., Deerfield, IL, USA) in 30 surplus porcine serum samples from a variety of studies. Validation of the assay consisted of determination of dilutional parallelism, spiking recovery, and intra-and inter-assay variability. Additional surplus serum samples from 27 piglets from four litters at a Texas A&M University farm were obtained. Each piglet had been bled twice, the first at weaning (21 days of age) and the second one 12 days later. To investigate results in comparison between age groups, serum cobalamin concentrations were compared using a Wilcoxon matched pairs test. Significance was set at p o 0.05.

Observed to expected ratios (O/E) for serial dilutions ranged from 87.3 to 124.9% (mean AE SD: 104.2 AE 14.5%) for four different serum samples at dilutions of 1:1, 1:2, and 1:4, and from 96.8 to 118.3% (mean AE SD: 107.6 AE 15.2%) for one serum sample at dilutions of 1:2, 1:4, and 1:8. O/E for spiking recovery ranged from 87.4 to 116.7% (mean AE SD: 102.0 AE 7.5%) for five different porcine serum samples that had been spiked with each other in a 1:1 dilution. Intraassay coefficients of variation (%CV) for five different serum samples were 4.3, 5.7, 4.3, 3.7, and 6.1%. Inter-assay %CVs for five different serum samples were 4.9, 7.2, 9.6, 3.5, and 7.6%. Serum cobalamin concentration was significantly lower in piglets post weaning (median: 242 ng/L) compared to those at the time of weaning (median: 324 ng/L; p 5 0.009).

The IMMULITE s 2000 cobalamin immunoassay labeled for use in humans is linear, accurate, precise, and reproducible for measurement of serum cobalamin concentrations in pigs. This study also showed that piglets that differ in age by only 12 days have significantly different serum cobalamin concentrations. Further investigations of cobalamin concentrations in both sows and piglets at different stages of weaning are warranted. Primigravid dairy heifers can be infected with mastitis pathogens during the periparturient period. The prevalence of intramammary infection (IMI) ranges from 30-75% of quarters pre-partum and 12-45% at parturition. Some pre-partum infections self-cure before parturition, however a number of these IMIs persist into early lactation. These IMIs may impact milk production and quality and may serve as a reservoir for contagious pathogens. No study has specifically investigated the risk of an IMI persisting from the prepartum period into early lactation. The objectives of this study were to describe the prevalence of mastitis pathogens in heifers on a grazing dairy before and after parturition and calculate the relative risk (RR) and attributable fraction of population (AFP) for the association between a post-partum and pre-partum IMI.

Two-hundred-ninety-four heifers were systematically assigned to 1 of 3 groups: G1) pre-partum secretions from all mammary quarters (n 5 98), G2) no pre-partum secretions collected (n 5 98) and G3) pre-partum secretions from two diagonal quarters (n 5 98). Group assignments were designed to assess whether pre-partum sampling increased the likelihood of IMI at calving. Mammary quarter secretions were collected for bacterial culture approximately 2 weeks prior to expected calving date. Quarter milk samples were collected for bacterial culture once weekly during the 1 st 3-weeks of lactation. Bacterial isolates were classified as staphylococci, non-agalactiae streptococci and Gram-negatives. Mammary quarter samples yielding 2 different bacteria were classified as mixed infections and those yielding ! 3 bacterial types were classified as contaminated. Bacterial isolates were speciated using gene sequencing methods and strain-typed using pulse-field-gel-electrophorysis to evaluate the relatedness of bacteria isolated from pre-and post-partum samples from the same mammary quarter. Relative risk and AFP were calculated using 2 Â 2 tables.

Forty-five percent of mammary quarters had a pre-partum IMI. During the 1st 3 weeks of lactation the mean prevalence of IMI was 23.3% of quarters. Staphylococci were most frequently isolated bacteria from pre-partum secretions and milk with S. chromogenes and S. aureus being the most common species. Using data from 228 mammary quarters, the RR and AFP for the association between a post-partum and pre-partum IMI were 11 and 77%, 43 and 86%, and 12 and 68% for all staphylococci, S. aureus only and CNS only IMIs, respectively. Mammary quarters sampled pre-partum were no more likely to have a post-partum IMI than those not sampled (Chisquare, P ! 0.27). These data demonstrate that pre-partum IMIs persist into early lactation and that pre-partum secretion cultures may be a useful, not only in predicting IMI at calving, but also in assessing risk of introducing new contagious mastitis pathogens, e.g., S. aureus, into the lactating herd. Despite concerns about antimicrobial resistance and Clostridium difficile in food animals, there has been little study of the prevalence or mechanisms of resistance. This study evaluated the impact of tetracycline treatment on C. difficile shedding in veal calves and the impact on resistance.

Calves arriving on 1 veal farm received oral oxytetracycline for 5 days as per farm protocols. Calves were sampled at arrival and 6 days later. Selective culture for C. difficile was performed. Isolates were ribotyped, and tested for tetracycline susceptibility and the presence of tetracycline resistance genes. Multivariable logistic regression models were used to determine the relationship between tetracycline resistance and the presence of tetracycline resistance genes.

Clostridium difficile was isolated from 32% (56/174) and 51% (88/ 172) calves, at the first and second samples, respectively. The percentage of tetracycline resistant isolates increased from 79% to 93%. Isolates from the second sample were 3 times more likely to be tetracycline resistant (p 5 0.016) and 5 times more likely to possess tet(M) (p 5 0.004). tet(M) was detected in 13% (7/53) and 43% (39/ 91), tet(O) in 23% (12/53) and 19% (17/91) and tet(W) in 2% (1/53) and 1% (1/91) of isolates from first and second samples, respectively. tet(L), tet(K) and tet(S) were not detected. 54 resistant isolates were not carrying any of the genes investigated.

Routine tetracycline use may have had an impact on both the prevalence of C. difficile, as well as the strain distribution and resistance patterns. This is the first report of presence of tet ( The objectives of this study were to 1) estimate the prevalence of antimicrobial resistance in the study population and 2) to investigate the associations between exposures to antimicrobial drugs and antimicrobial resistance in fecal non-type specific E. coli (NTSEC) recovered from individual feedlot cattle. Two-stage random sampling was used to identify cattle for enrollment at 4 western Canadian feedlots. A fecal sample was collected per rectum from each individual at arrival and in the middle of the feeding period when cattle were rehandled as part of standard feedlot protocol. From samples collected at this second time point, a total of 2,133 NTSEC isolates were tested for susceptibility to antimicrobial drugs by disk diffusion. Parenteral and in-feed exposures to antimicrobial drugs were recorded for each individual enrolled in the study. The least square means estimates and 95% confidence intervals for the prevalence of resistance at each time point were modeled using Poisson regression. Multivariable logistic regression was used to investigate associations between antimicrobial resistance and exposure to antimicrobial drugs. Regression models were adjusted for clustering of observations among individuals and pens.

The most common resistances identified in arrival samples were sulfisoxazole (7.5%; 95%CI: 6.1-9.2), streptomycin (7.7%; 95%CI: 6.3-9.5) and tetracycline (20.0%; 95%CI: 17.7-22.6). At the second sampling point, resistance prevalence was 25.6% (95%CI: 23.5-28.0) for sulfisoxazole, 25.0% (95%CI: 22.8-27.3) for streptomycin, and 72.7% (95%CI: 70.5-75.1) for tetracycline. Logistic regression modeling identified weak associations of exposures to tetracycline and macrolide classes of drugs with antimicrobial resistance at the second time point.

ABSTRACT FA-5 PREMATURE/DYSMATURE SYNDROME IN CRIA: A RET-ROSPECTIVE STUDY OF 63 CASES (1999) (2000) (2001) (2002) (2003) (2004) (2005) . C. Gerspach, D. Anderson. The Ohio State University, Columbus OH.

Prematurity is widely acknowledged as risk factor for subsequent morbidity and mortality in llama and alpaca cria. A review of medical records for premature cria alive at the time of admission to the Veterinary Teaching Hospital between 1999 and 2005 was performed to determine risk factors of prematurity and to report the outcome and related conditions or diseases in affected cria. Medical records for 63 premature or dysmature cria were included in this study. Of these cria, 51 were alpaca and 12 llama, 36 were female and 27 were male. Reasons for referral were prematurity, failure of passive immunity, dyspnoea, weakness and failure to gain weight. Cria were presented at a mean age of 1.4 days and were premature by a mean estimated time of 19.5 days. Overall survival rate was 82.5%, with all llama cria surviving. A multivariate logistic regression model was used to identify risk factors associated with not surviving. Cria receiving camelid colostrum had a significant better outcome than cria receiving no colostrum or colostrum from different species. Dyspnea and tachypnea was associated with a poor outcome. All cria that were able to nurse, without assistance prior to referral, survived. Clinical pathology parameters most commonly associated with death were hyperphosphatemia and acidosis. Enrofloxacin is approved for the treatment of swine respiratory disease, however there are no published studies describing the pharmacokinetics of enrofloxacin at the approved dose and route in pigs (7.5 mg/kg subcutaneously). Furthermore no studies have assessed the unbound concentrations of enrofloxacin at its site of action, the extracellular tissue fluid. Therefore the objective of this study was to use an in-vivo ultrafiltration method to measure the active fraction of enrofloxacin, and the metabolite ciprofloxacin, at 3 tissue sites relevant to pigs, and to compare these concentrations with plasma concentrations collected at similar time points.

Six healthy pigs were used in this study. Pigs were recently weaned and weighed an average 16.3 kg. On the day before the experiment, pigs were anesthetized for the placement of jugular vein sampling catheters and interstitial fluid collection probes. Three ultrafiltration probes were placed in each pig in a subcutaneous site near the right shoulder, an intramuscular site along the epaxial muscles, and in the pleural space of the chest cavity. Each pig received an injection of enrofloxacin (Baytril 100, Bayer Animal Health) at a dose of 7.5 mg/ kg subcutaneously behind the left ear. Plasma and interstitial fluid samples were collected at pre-determined time points, and enrofloxacin and ciprofloxacin concentrations were measured using HPLC with fluorescence detection. Protein binding was determined with a microcentrifugation system. Pharmacokinetic data was analyzed using a one compartment model.

The analysis of plasma and ISF showed that only a small fraction of ciprofloxacin was produced in these pigs, therefore ciprofloxacin concentrations were not used in pharmacokinetic measurements. The plasma half-life (t 1/2 ), volume of distribution, clearance, and peak concentration (C max ) for enrofloxacin was 25.9 hr (AE 6.2), 6.29 L/kg (AE 1.23), 0.168 L/kg/hr (AE 0.076), and 1.07 mg/mL (AE 0.28), respectively. The concentrations from each of three tissues were not different in each pig. When pharmacokinetic values from all tissues were combined for the ISF, the t 1/2 was 23.6 hr (AE 4.1) and the C max was 1.26 mg/mL (AE 0.10). The enrofloxacin plasma protein binding was 31.1% (AE 3.28) and 37.13% (AE 16.54) at a high and low concentration, respectively.

This study has demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasma. The half-life of enrofloxacin is longer in tissues and plasma than has been reported in previous studies. The high tissue concentrations and long half-life produce an AUC/MIC ratio sufficient for the pathogens that cause respiratory infections in pigs. Ceftiofur crystalline free acid (CCFA), a long-acting ceftiofur formulation labeled for use in cattle, pigs, and horses for treatment of respiratory disease has been used for treatment of ovine respiratory infections in clinical practice. Pharmacokinetic data, however, do not exist for CCFA administered subcutaneously in sheep. The present pharmacokinetic study evaluated the single dose subcutaneous administration of CCFA in sheep (n 5 9) at 6.6 mg/kg body weight. Concentrations of ceftiofur free acid equivalents (CFAE) in plasma were measured by high performance liquid chromatography for 14 days following drug administration. Pharmacokinetics of subcutaneous CCFA in sheep were best described using a single compartment model with the following average (AE SD) parameters: area under the concentration time curve 0! 1 (206.6 hÃug/ml AE 24.8), observed maximum plasma concentration (2.4 ug/ ml AE 0.5), and observed time of maximum plasma concentration (23.1 h AE 10.1). No significant adverse drug reactions were observed. Adequate CFAE plasma concentrations were attained to effectively treat respiratory tract pathogens associated with pneumonia in sheep. The purpose of this study was to assess, using thoracic ultrasonography, the prevalence of lung lesions in pre-weaned dairy calves. Subsequent aims were to describe ultrasonographic changes within the lung, clinical respiratory score, and treatment of respiratory disease.

A longitudinal study was performed using female dairy calves from 6 commercial dairy farms in New York State. Calves were enrolled based on age. Thoracic ultrasound and clinical respiratory scoring were performed on each calf at 2 time points. A standard 5 mHz linear ultrasound probe was utilized to evaluate intercostal spaces 1 through 11 of each hemi-thorax with the calf in lateral recumbency (US1) or standing (US2). Lesion appearance, size, and location were recorded. Respiratory score (RS) was assigned based on a previously published protocol incorporating fever, nasal discharge, cough, ocular discharge and ear droop, with a higher numerical score corresponding to more severe disease. Abnormal lung on ultrasound was defined as one or more areas of !1 cm width or depth of non-aerated lung. Farm records were evaluated to identify treated calves. Calves were treated for respiratory disease at the farm manager's discretion, not based upon ultrasound findings or RS. Non-parametric methods were used to evaluate the data.

Ninety-one calves were enrolled into the study, with 6 lost to follow-up. An average of 4 minutes was spent performing the RS and ultrasound on each calf. The median ages at first (US1) and second (US2) examination were 13 (interquartile range 12-15) and 46 (interquartile range 44-47) days, respectively. The majority of calves had a low RS (o 5) and only 3.2% of calves had a RS high enough to warrant treatment based on previous recommendations (RS!5). The prevalence of calves that had abnormal lungs on ultrasound but a low RS (o 5) was 5.5% (US1) and 16.5% (US2). The prevalence of calves that had abnormal lungs on ultrasound and a high RS (! 5) was 0% (US1) and 3.5% (US2). Of the calves that had abnormal lungs on ultrasound but a low RS, 13% were treated with antimicrobials within 7 days of examination. None of the calves with high RS and abnormal lungs on ultrasound were treated with antibiotics within 7 days of examination.

This study demonstrates a high prevalence of abnormal lungs, as detected by thoracic ultrasonography, without significant clinical signs in pre-weaned dairy calves. The relatively low treatment rate in these calves may suggest an area of opportunity for improvement in calf health, welfare, and herd longevity. Further studies and follow up are needed to elucidate the significance of these findings and whether or not treatment is indicated. Literature regarding diseases causing lameness in beef cattle is limited. This retrospective study was undertaken to examine beef cattle presented for lameness. Medical records of beef cattle having a lameness examination done during the period 2007 to 2010 were reviewed and descriptive statistics generated. Lameness was classified based on clinical diagnosis. The medical records of 270 beef cattle were reviewed of which 63.2% were male and 36.8% were female. Beef cattle presented for lameness most often during the summer months (34%) and least during autumn (18%). Causes of lameness were categorized as infectious (44.6%) or non-infectious (55.4%) and infectious lameness subcategorized as either a primary disorder or a secondary infection. All cases of a primary infectious disorder were interdigital phlegmon. Secondary infections diseases included sole abscess (25.9%), septic arthritis (11.1%), tenosynovitis (2.8%), and pedal osteitis (1.3%). Non-infectious lameness included proximal limb lameness (19.6%), foot trauma (14.6%), hoof horn cracks (9.5%), hoof defects (2.5%), interdigital fibromas (1.9%), overgrown hooves (1.9%), sole bruise (1.3%), subclinical laminitis (1.3%), white line disease (0.9%), osteoarthritis (0.9%), heel erosion (0.3%), sole ulcers (0.3%), and sole hemorrhage (0.3%). The most frequently affected claw was the lateral digit of the hind limb (36.4%), followed by the medial digit of the front limb (27.1%), lateral digit of the front limb (23.6%), and the medial digit of the hind limb (12.9%). The findings of this study suggest significant differences in the frequency of disease causing lameness in beef cattle compared to published reports for dairy cattle. In people, endoscopic ultrasound (EUS) has become the technique of choice for assessing pancreatic disease and EUS-guided fineneedle aspiration (EUS FNA) has proven a useful and safe modality for characterizing pancreatic lesions. Reported complications include infections, bleeding and acute pancreatitis. In dogs, laparoscopic-assisted pancreatic biopsy has been suggested to be a safe procedure, however EUS and EUS FNA have not been evaluated in dogs so far. Thus the aim of the present study was to assess the practicability and safety of EUS examination of the abdominal cavity as well as pancreatic EUS FNA in healthy dogs. This study was approved by the Cantonal Committee for the Authorization of Animal Experimentation, Zurich, Switzerland. The study population consisted of 14 healthy beagle dogs with a median bodyweight of 13.4 kg (10.9-15.7). EUS was performed with an Olympus GF-UC140P-echoendoscope and FNA were performed using 19 G needles (Cook EchoTipUltra). After completion of the EUS-examination of the abdominal cavity from the stomach (liver, gallbladder, bile ducts, kidneys, adrenals, pancreas), the scope was advanced into the duodenum and EUS FNA of the pancreas was performed. FNA tissue acquisition was made applying negative pressure and 6 to 8 needle passes were made. All dogs received 30 mg/kg metimazole IM after EUS FNA and were re-checked ultrasonographically 20 minutes post EUS FNA. Postoperative activity was assessed using a standardized scoring system. A CBC, serum biochemistry, urinalysis and spec cPL s were measured before, as well as 24 and 48 h after EUS FNA.

The EUS examination was complete in 13/14 dogs, the pancreas could not be visualized in 1 dog. The pancreas was hypo-(4/13) to isoechoic (9/13) to the surrounding mesenterium in all cases. In 3/13 dogs parts of the pancreas presented hyperechoic. The mean measured thickness was 0.88 cm. The pancreas was aspirated in 12 dogs using a transgastric approach (3) or transduodenal approach (9). Duodenal transmural puncture was not accomplished in 1 dog where a re-sterilized needle was used. A minimal amount of peripancreatic fluid was observed in 1/12 dogs after EUS FNA. All dogs recovered uneventfully and required no further analgesia. All laboratory results including the spec cPL s measurements were within reference ranges on all three time points. Cytologically, conglomerates of exocrine pancreatic cells were seen in 8/12 cases, duodenal villous epithelial cells were seen in 11/12 cases. In 1 dog the aspirated pancreatic material was sufficient for a histological assessment. The 8 aspirates with exocrine pancreatic cells on cytology were obtained by transgastric (4) and transduodenal (4) aspirations. In conclusion, (1) EUS examination of the abdomen is feasible in medium-sized dogs, (2) the healthy canine pancreas can be difficult to visualize completely, and (3) EUS-guided pancreatic FNA using a 19 G needle is a safe procedure in healthy dogs. Studies evaluating its use in dogs with pancreatic disease are warranted to assess its clinical utility. Miniature Schnauzers have a high prevalence of idiopathic hyperlipidemia, which is characterized by an increased serum triglyceride (TG) concentration, with or without an increased serum cholesterol (CHOL) concentration. A common initial therapeutic approach for the management of hyperlipidemia is the use of a low-fat diet. Also, it is believed that low-fat diets may be beneficial in the treatment of pancreatitis in dogs. However, the efficacy of this approach has not been evaluated for either condition. The aim of the present study was to evaluate the effect of a commercially available low-fat diet on serum concentrations of TG, CHOL, and canine pancreatic lipase immunoreactivity (cPLI; measured as Spec cPL s ) in apparently healthy Miniature Schnauzers with hypertriglyceridemia.

Blood samples were collected from 15 apparently healthy Miniature Schnauzers with hypertriglyceridemia (serum triglyceride concentrations 4 108 mg/dL). Common causes of secondary hyperlipidemia were excluded based on historical information, physical examination findings, and the measurement of serum glucose, total T4, and free T4 (by ED) concentrations. The owners of the dogs were asked to switch their dog to the study diet (Royal Canin Gastrointestinal Low Fat s ; fat content: 18.6 g/1,000 Kcal) and have a second blood sample collected 8 weeks after their dog had been on the new diet. All blood samples were collected after food had been withheld for 15 hours. Serum TG, CHOL, and Spec cPL concentrations were measured both before and after the diet change. Results were compared between the two time-points using the Wilcoxon signed rank and Fisher's exact tests.

Serum TG concentrations were significantly higher before (median: 432 mg/dL) than after the diet change (median: 178 mg/dL; p 5 0.003). The proportion of dogs with hypertriglyceridemia was significantly higher before (15/15) than after the diet change (10/15; p 5 0.042). Also, the proportion of dogs with serum TG 4 500 mg/dL was significantly higher before (6/15) than after the diet change (0/15; p 5 0.016). Serum CHOL concentrations were significantly higher before (median: 296 mg/dL) than after the diet change (median: 258 mg/dL; p 5 0.004). The proportion of dogs with hypercholesterolemia was significantly higher before (8/15) than after the diet change (0/15; p 5 0.006). Finally, the difference in serum Spec cPL concentrations before (median: 88 mg/L) and after the diet change (median: 56 mg/L) approached but did not reach significance (p 5 0.052). Also, the proportion of dogs with high serum Spec cPL concentrations before (4/15) and after the diet change (0/15) was different, but this difference was not significant (p 5 0.099).

In summary, a commercially available low-fat diet was effective in reducing serum TG and CHOL concentrations in Miniature Schnauzers with hypertriglyceridemia. Toll-like receptor 5 (TLR5) is an extracellular pattern recognition receptor which recognizes flagellin present in motile bacteria. We have previously demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (SNPs) in the TLR5 gene (G22A, C100T and T1844C) and inflammatory bowel disease (IBD) in German shepherd dogs (GSDs). Recently, we have confirmed that two of these TLR5 SNPs (C100T and T1844C) are significantly associated with IBD in other canine breeds. To further substantiate the role of TLR5 in canine IBD functional analysis of these polymorphisms would be needed. Therefore the aim of this study was to determine the functional significance of the TLR5 SNPs by transfecting wild-type and mutant receptors in to human embryonic kidney cells (HEK) and carrying out nuclear factorkappa B (NF-kB) luciferase assay and IL-8 ELISA.

The TLR5 gene containing the risk haplotype for IBD (ACC) and wild-type haplotype (GTT) as determined by the case-control analysis in GSDs with IBD were cloned into plasmids expressing yellow-fluorescent protein (YFP). These were then stably transfected into HEK cells. NF-kB activity was measured by transiently transfecting the cells with NF-kB firefly and HSV-thymidine kinase promoter (pRL-TK) renilla plasmids. The cells were then stimulated with various ligands (0.1 mg/ml flagellin, 0.01 mg/ml flagellin, 1 mg/ml LPS, 1 mg/ml PAM3CSK and media control). Firefly and renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega, UK) according to the manufacturer's recommendations. The supernatants were harvested and used in an IL-8 ELISA (R&D Systems). Human TLR5 transfected HEK cells (Invivogen) served as positive controls in all experiments.

Independent T-test was used to determine the significance of relative luciferase activity and IL-8 concentration between wild-type and mutated TLR5 cells. Although there was no significant difference between the wild-type and mutated receptor when they were stimulated with 0.01 mg/ml of flagellin (p 5 0.14), there was a significant increase when the cells with mutated TLR5 were stimulated with 0.1 mg/ml of flagellin compared to the cells expressing wild-type TLR5 (p 5 0.027). Similarly, there was a significant increase in IL-8 concentration in the supernatants in the cells with the mutated TLR5 receptor when stimulated with 0.1 mg/ml flagellin compared to the wild-type (p 5 0.025-one-tailed, 0.05-two-tailed) but not with 0.01 mg/ml flagellin (p 5 0.26).

We show for the first time that polymorphisms associated with IBD are functionally hyper-responsive to flagellin compared to the wild-type receptor. This suggests that TLR5 may play a role in canine IBD and that blocking the hyper-responsive receptor found in susceptible dogs with IBD may alleviate the inappropriate inflammation seen in this disease. However, further in-vivo functional analysis of TLR5, especially at the intestinal mucosal level would be needed to confirm these findings and predict the usefulness of any future therapeutic interventions. TLR5 has been shown to play a role in the inappropriate inflammation seen in human inflammatory bowel disease (IBD). Similarly, we have recently demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (SNPs) in the canine TLR5 gene (G22A, C100T and T1844C) and inflammatory bowel disease (IBD) in German shepherd dogs (GSDs).

Therefore the aim of this study was to determine the functional significance of the TLR5 SNPs in the breed of GSDs.

The TLR5 gene containing the risk haplotype for IBD (ACC) and wild-type haplotype (GTT) were stably transfected into HEK cells. NF-kB activity was measured by transiently transfecting the cells with NF-kB firefly and HSV-thymidine kinase promoter (pRL-TK) renilla plasmids. The cells were stimulated with various TLR ligands (0.1 mg/ ml flagellin, 0.01 mg/ml flagellin, 1 mg/ml LPS, 1 mg/ml PAM3CSK and media control). Firefly and renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega, UK). The supernatants were harvested and used in an IL-8 ELISA (R&D Systems). Peripheral whole blood from dogs carrying the wild type and mutant TLR5 genes was cultured and stimulated with TLR ligands as above. Canine TNF-alpha was measured in the supernatant by commercially available ELISA (R&D Systems).

T-test was used to determine differences of relative luciferase activity, IL-8 concentration and TNF-alpha concentration between wild-type and mutated TLR5 cells. There was a significant increase in NF-kB activity when the cells with mutated TLR5 were stimulated with 0.1 mg/ml of flagellin compared to the cells expressing wild-type TLR5 (p 5 0.027), which correlated with IL-8 expression in the supernatant (p 5 0.26). Similarly, in the whole blood assay the TLR5 risk haplotype for IBD in GSDs (ACC) was significantly hyperresponsive to flagellin at a concentration of 0.1 mg/ml compared to the TLR5 wild-type haplotype (GTT) (p 5 0.001).

We show for the first time that polymorphisms associated with canine IBD in GSDs are functionally hyper-responsive to flagellin compared to the wild-type receptor. Blocking the hyper-responsive receptor found in susceptible dogs with IBD may alleviate the inappropriate inflammation seen in this disease. Proton pump inhibitors (PPI) are widely used in human and also veterinary medicine. Side-effects of PPI treatment reported in people are atrophic gastritis, gastric and esophageal cancer, and rebound hyperacidity following cessation of treatment, which has been speculated to be due to a sustained increased in circulating gastrin concentration. Moreover, long-term PPI treatment has been associated with an increased risk for osteoporosis in people. Little is known about the effect of PPI treatment on serum gastrin concentration or calcium metabolism in dogs.

Eight healthy adult research dogs (4 males and 4 females) were enrolled into the study. The dogs received an average dose of 1.1 mg/ kg of omeprazole orally twice daily for 15 days. Blood samples were collected prior to initiating the treatment and every 3 days during the 15 days of treatment and during the 15 days after discontinuation of treatment for determination of serum gastrin, ionized calcium, PTH, and 25 OH vitamin D3. Gastric fluid was collected via gastroscopy after an overnight fast for measurement of gastric pH prior to, during, and after the omeprazole treatment period. Normally distributed data were compared with a repeated measures ANOVA and post hoc Dunnett's test. Data that were not normally distributed were compared with a Friedman's test and a post-hoc Dunn's test.

Gastric fluid pH was significantly higher (p o 0.01) at the end of the treatment period (median: 7.4; range: 4.2-8.1) when compared to pretreatment values (median: 1.7; range: 1.0-6.8). Serum gastrin concentrations increased significantly from a median baseline of 10.0 ng/L (range: 10.0-27.0) to a maximum median of 379.5 ng/L (range: 49.9-566.0) at day 9 of treatment (p o 0.01). Serum gastrin remained significantly increased above baseline values from day 6 to day 15 of the treatment, but was not different from pre-treatment values 3 days after the end of the treatment. Omeprazole treatment had no effect on ionized calcium or PTH for the duration of the study. Marginal, but significant changes of 25 OH vitamin D3 were observed at day 15 (end of the treatment period -increased by 13.8%) and day 21 (6 days after the end of the treatment -decreased by 14.7%).

This study shows that treatment with omeprazole for 2 weeks results in a profound and sustained increase in serum gastrin concentration in dogs. This effect is rapidly reversible after cessation of the treatment. No effect on calcium metabolism was observed. However, this study documents only the effect of short-term treatment and it is possible that the effects of long-term administration are different. Omeprazole treatment has been associated with small intestinal bacterial overgrowth and a higher risk for infectious enteropathies in humans. Using a semi-quantitative sequencing approach, we have previously shown that omeprazole treatment may lead to alterations in both duodenal and gastric bacterial populations in healthy dogs (ACVIM 2010). However, a sequencing approach can only estimate relative proportions of genomic bacterial targets. Therefore, significant changes in the total number of bacteria could not be evaluated. The aim of this study was to quantify gastric and duodenal bacterial populations in dogs undergoing omeprazole treatment.

Eight 9 month-old healthy research dogs (4 males and 4 females) were enrolled. The dogs received an average dose of 1.1 mg/kg of omeprazole orally twice a day for 15 days. Endoscopic gastric and duodenal biopsies were harvested 30 and 15 days before starting omeprazole treatment, on the last day of treatment (day 15), and 15 days after the end of treatment (day 30). All biopsies were fixed in 10% formalin for 24 hours, processed, and embedded in paraffin blocks. Fluorescent in situ hybridization was used to quantify mucosa-associated bacteria using fluorescently-labeled probes targeting the 16S ribosomal RNA. Statistical analysis aimed to compare changes in Helicobacter spp. in gastric biopsies and total bacteria in both gastric and duodenal biopsies using the GLIMMIX and NPAR1WAY procedures in SAS s 9.2.

Bacteria were counted in 1,174 and 989 microscopic fields (63Â) obtained from 155 and 132 gastric and duodenal biopsies, respectively. In the stomach, omeprazole treatment led to a decrease in Helicobacter spp. (log of average counts AE standard error: 1.98 AE 0.14 at day 15) when compared to the counts 30 (2.43 AE 0.13, p0.0001) and 15 (2.40 AE 0.13, p0.0001) days before treatment. After completion of omeprazole treatment, Helicobacter spp. increased and returned to baseline counts (2.45 AE 0.13 at day 30, p0.0001 vs day 15). Also, in the stomach, non-Helicobacter spp. bacteria were observed more often during omeprazole treatment (median: 3, range: 0-20) than on days 30 (median: 0, range: 0-3) and 15 (median: 1, range: 0-6) before and 15 days after (median: 0, range: 0-2) omeprazole treatment; however, statistical comparison across time points did not reach significance. In the duodenum, while the median number of bacteria for all time points was zero, non-parametric comparison of median scores (number of points above median) revealed significantly higher numbers of bacteria during omeprazole treatment (p 5 0.0063).

Our results suggest that omeprazole treatment for 2 weeks leads to a lower abundance of Helicobacter spp. organisms in the stomach of healthy dogs. Also, this transient decrease in Helicobacter spp. was accompanied by a higher abundance of other bacteria in both the stomach and the proximal duodenum. The SmartPill pH.P s capsule (The SmartPill Corporation) is a wireless motility capsule that measures pH, pressure, and temperature as it passes through the gastrointestinal (GI) tract. Analysis of this data allows the calculation of gastric emptying time (GET), small and large bowel transit time (SLBTT), and total GI transit time (TGTT). This study evaluated the variability associated with repeated measurement of GI transit times and the effect of oral administration of ranitidine (Zantac s ) on GI transit times in dogs using this system. It was hypothesized that ranitidine would reduce GI transit times.

Six privately owned healthy adult dogs weighing between 25.0 kg and 41.0 kg were used. On 3 occasions each dog was fed a standard meal followed by oral administration of a capsule. Data were recorded until the capsule had passed in the dog's feces. On a 4th occasion each dog was given 75 mg of ranitidine PO q12 hrs starting 48 hrs prior to testing. The dogs were then fed the test meal and the capsule was administered as above. Ranitidine was given until the capsule had passed in the dog's feces. Proprietary SmartPill software was used to calculate GET, SLBTT, TGTT, and the median gastric pH (MGpH). Mean intra-individual and inter-individual coefficients of variation (CV%) were calculated for GET, SLBTT, and TTT for the first 3 time points. Transit times and gastric pH recorded at all 4 time points were compared using a repeated measures ANOVA. Where significant differences were identified, post-hoc testing was performed using a Bonferroni's multiple comparisons test. Significance was set at p o 0.05.

A sharp rise in pH indicating exit of the capsule from the stomach was identified in each experiment. Mean (AE SD) GET, SLBTT, and TGTT without ranitidine were 775 AE 144, 1563 AE 614, and 2338 AE 577 min, respectively. Mean GET, SLBTT, and TGTT during treatment with ranitidine were 719 AE 11, 1442 AE 885, and 2155 AE 897 min, respectively. Mean intra-individual CV% before ranitidine for GET, SLBTT, and TGTT were 12.9, 29.7, and 17.8%, respectively. Mean inter-individual CV% before treatment with ranitidine for GET, SLBTT, and TTT were 16.1, 40.5, and 24.7%, respectively. No significant differences in GET, SLBTT, or TGTT were found at any of the 4 time points. The mean MGpH during treatment with ranitidine (pH 2.85) was significantly higher than at all other time points (overall mean pH for the 3 time points: 1.73; p o 0.05).

The SmartPill system is an easy to use, ambulatory, non-invasive, non-radioactive method for assessing GI transit times in medium to large breed dogs. Measurements of GI transit times, especially SLBTT, were subject to considerable intra-individual and interindividual variation. No significant effect of oral ranitidine on GI motility was identified in this group of dogs. However, as expected, oral ranitidine caused a significant increase in gastric pH. The intestinal microbiota has been implicated in the pathogenesis of various gastrointestinal disorders in both humans and dogs. Recent metagenomic data suggest that specific bacterial groups, including bacteria within the Clostridium clusters IV and XIVa (i.e., Faecalibacterium spp., Ruminococcaceae, and Lachnospiraceae) and Bifidobacterium spp. are decreased, while Proteobacteria are increased in dogs with clinical signs of gastrointestinal disease. The objective of this study was to establish quantitative polymerase chain reaction (qPCR) assays for these specific bacterial groups and evaluate their abundance in healthy dogs and dogs with clinical signs of gastrointestinal disease.

Fecal samples were collected from 21 healthy dogs (14 females and 7 males) and 20 dogs with clinical signs of gastrointestinal disease (11 females and 9 males). Novel quantitative PCR assays were established for Faecalibacterium spp., Ruminococcaceae, and Lachnospiraceae by aligning respective group specific sequences against canine specific sequences obtained from 16S rRNA gene clone libraries and sequences available from the Ribosomal Database Project. Primers for Bifidobacterium spp. and Proteobacteria were selected from previously published studies. The specificity of the qPCR assays was confirmed by sequencing of obtained qPCR amplicons. The bacterial DNA abundance in fecal samples was compared between healthy dogs and dogs with clinical signs of gastrointestinal disease using a Mann-Whitney U test. Significance was set at p o 0.05.

A significantly lower abundance of Faecalibacterium spp. (p o 0.01) and Ruminococcaceae (p 5 0.02) was observed in dogs with clinical signs of gastrointestinal disease when compared to healthy dogs. Proteobacteria were more abundant in dogs with clinical signs of gastrointestinal disease, but this difference did not reach statistical significance (p 5 0.07). There was no significant difference in the abundance of Lachnospiraceae (p 5 0.23) and Bifidobacterium spp. (p 5 0.77) between both groups.

In conclusion, we established novel qPCR assays for Faecalibacterium spp., Ruminococcaceae, and Lachnospiraceae. We observed significant decreases in the abundance of Faecalibacterium spp. and Ruminococcaceae in dogs with clinical signs of gastrointestinal disease. These bacterial groups are considered major short-chain fatty acid producers and studies are warranted to determine if a decrease in these bacterial groups is associated with decreases in short chain fatty acid production. Further studies are also needed to determine if these bacterial shifts are associated with specific gastrointestinal disorders. The pathogenesis of chronic enteropathies (CE) in dogs likely involves complex interaction between the mucosal immune system and the intestinal microbiota. While the application of bacterial 16S rDNA sequence-based analysis has shown an association between altered microbial composition and duodenal inflammation in dogs, relatively little is known about alterations in non-invasive mucosal and luminal bacteria seen with diseases involving the ileum and colon. The present study sought to evaluate the relationship of enteric bacteria to type and severity of mucosal inflammation affecting the ileum and colon of dogs with CE.

Eleven client-owned dogs with CE involving both the small and large intestines were prospectively enrolled. CE was diagnosed on the basis of a history of chronic gastrointestinal signs, exclusion of identifiable underlying disorders, and histopathologic evidence of intestinal inflammation. Mucosal bacteria were detected in formalinfixed ileal and colonic tissue sections with fluorescence in situ hybridization (FISH) using 16S rDNA-targeted probes directed against all bacteria, Enterobacteriaceae, E. coli, Eubacterium rectale-Clostridium coccoides group, Bacteroides/Prevotella, and Helicobacter spp. Sections were examined by epifluorescence microscopy and the number of bacteria and their spatial distribution (luminal, superficial mucus, epithelial adherent, within mucosa) was determined in ten 60x fields of each section. Microbial composition in CE dogs was compared to the ileal/colonic microbiota of 7 healthy control (HC) dogs using a mixed effect ANOVA model. P values o 0.05 were considered significant.

The final diagnoses for dogs with CE included IBD (n 5 8) and lymphosarcoma (n 5 3). When compared to HC dogs, dogs with CE showed regional (ileum versus colon) imbalances in microbiota composition characterized by selective enrichment of mucosa-associated populations. Evaluation of colonic biopsies in dogs with CE showed that the total number of bacteria (P o 0.04), Clostridium (P o 0.005), Enterobacteriaceae (P o 0.04) and E. coli (P o 0.03) were increased in the adherent mucus regions of dogs with IBD as compared to HC dogs. Total bacteria (P o 0.05) and E. coli (P o 0.02) were also more numerous in dogs with LSA versus HC and IBD dogs (P o 0.04 for E. coli). Ileal biopsies from CE dogs similarly showed variable dysbiosis with increased total bacteria (P o 0.05) but decreased Helicobacter spp (P o 0.001) and Bacteroides (P o 0.02) observed within inflamed intestines as compared to HC tissues. The spatial distribution of these bacteria was also appreciably different from HC dogs, with higher numbers of bacteria generally found within the adherent mucus compartment as compared to other ileal regions.

Our data demonstrate that dogs with CE affecting the ileum and colon have altered microbiota composition that may be a cause or consequence of mucosal inflammation. Recognition of these microbiota imbalances may provide new opportunities for therapeutic intervention. Trichomonads have been rarely reported in the feces of dogs and their pathogenicity remains uncertain. Although Pentatrichomonas hominis (Ph) is considered to be a commensal that may overgrow in dogs with other causes of diarrhea, little is known regarding the history, clinical presentation or prevalence of concurrent GI infections in dogs with trichomonosis. The aim of this study was to determine whether dogs with diarrhea and trichomonosis could be distinguished from dogs having diarrhea without trichomonosis on the basis of clinical signs or presence of concurrent enteric infections.

Fecal samples from 39 dogs were submitted to NCSU from 2007-2010 for Trichomonas spp. PCR testing. DNA was extracted using a ZR Fecal DNA mini-prep kit and absence of PCR inhibitors verified by amplification of bacterial 16S rDNA. PCR for Ph and Tritrichomonas foetus (Tf) was performed as well as real-time PCR assays for 9 possible concurrent enteric infectious agents. Obtainable medical records were reviewed. All submitted fecal samples were submitted from dogs with diarrhea that was variably described as soft, mucoid, hemorrhagic, or watery.

Mean age of the dogs was 2.33 years (median 1.9; range: 2.5-120 months) and represented a total of 24 breeds. Ph, Tf, or concurrent Ph and Tf were diagnosed in 18, 1, and 1 dogs respectively (Group A). The remaining 19 dogs were negative for Ph and Tf by PCR No dogs were identified as infected with canine distemper virus or parvovirus. Five samples from each group had insufficient quantity or quality of DNA for concurrent infectious disease testing.

In this large study of canine trichomonosis, no differences in age, clinical signs, or prevalence and identity of concurrent enteric infection between diarrheic dogs with or without Ph were identified. Thus, these findings do not appear to support a primary pathogenic role for Ph as a causative agent of diarrhea in dogs. Gastrointestinal motility disorders are a common clinical problem in domestic animals. Many of the G.I. motility disorders have been treated previously with 5-HT 4 agonists although limited availability of drugs in this classification have stimulated interest in the use of new (and old) drug therapies. The dopaminergic antagonists are a group of drugs with well-known anti-emetic effects at central dopamine D 2 receptors, and putative gastrointestinal prokinetic effects at peripheral D receptors. Domperidone has been shown, for example, to reverse gastric relaxation induced by dopamine infusion in the dog. Similar studies have not been reported in the cat or rabbit, two species at risk for distal gastrointestinal motility disorders. Our aim was to study the effects, mechanisms, and sites of action of domperidone in feline colonic and rabbit gastrointestinal smooth muscle contraction.

Portions of stomach (fundus and antrum), intestine (duodenum and ileum), cecum (rabbits only), and colon (ascending and descending) were obtained from healthy cats and rabbits from 9-12 months of age. Longitudinal and circular smooth muscle strips from each site were suspended in physiologic (HEPES) buffer solution, attached to isometric force transducers, and set to optimal muscle length (L o ) using acetylcholine (ACh; 10 À4 M). Muscle strips were treated with domperidone (D; 10 À8 to 10 À4 M) in the presence or absence of ACh (10 À8 to 10 À4 M), and maximal force output (P max ) was normalized for cross-sectional area (N 5 Â 10 4 Newtons/m 2 ). Domperidone (D) had a minor direct effect of inducing feline and rabbit gastric, cecal, and colonic smooth muscle contraction. Direct effects were similar whether in the longitudinal or circular muscle orientation. The direct effect of domperidone was dose-dependent and maximal (feline colon P max 5 0.15-0.22 N; rabbit colon P max 5 0.10-0.19 N) at a dose of 10 À4 M. Domperidone had a much greater indirect effect in augmenting cholinergic (ACh; 10 À4 M) contractions in feline and rabbit gastric, cecal, and colonic smooth muscle. Domperidone-augmented cholinergic contractions were 157-200% (feline colon P max 5 1.54 AE 0.24 N ACh only; feline colon P max 5 2.03 AE 0.23N ACh 1 D) of baseline cholinergic contractions. Domperidone contractions were of a similar magnitude to those induced by cisapride. Domperidone effects were similar in mucosaintact and mucosa-dissected preparations. Domperidone contractions were unaffected by prazosin (a 1 receptor antagonist), yohimbine (a 2 receptor antagonist), or terbutaline (b 2 receptor agonist), but were somewhat attenuated by dopamine (D 2 receptor agonist) and a non-specific cholinergic antagonist (atropine).

In vitro studies show for the first time that domperidone has minor direct and major indirect effects in augmenting cholinergic contractions of feline and rabbit gastrointestinal (stomach, cecum and colon) smooth muscle. As recognition of acute and chronic pain in dogs has increased, so too has the use of non-steroidal anti-inflammatory drugs (NSAIDs) often in conjunction with tramadol. In people and rats, co-administration increases the risk of perforation and gastric injury over NSAIDs alone. Using an ex vivo model of acid injury in canine gastric mucosa, we examined the effects of indomethacin and tramadol on gastric permeability and concentrations of gastroprotective prostaglandin E 2 (PGE 2 ).

Mucosa from the gastric antrum was harvested from 5 shelter dogs immediately after euthanasia, and mounted on Ussing chambers. The tissues were equilibrated for 30-minutes prior to addition of acidic Ringer's solution (pH, 1.2). After 45-minutes of injury, the acid was replaced with neutral Ringer's and the tissues were treated with indomethacin, tramadol or both. Tissues were maintained for 210minutes total, during which time permeability was assessed electrically. Prostanoid concentrations were quantified using a commercially available ELISA. Western blots were performed for COX-1 and À2.

Recovery of gastric barrier function after acid injury was inhibited by co-administration of tramadol and indomethacin ( Figure 1 ) but not by tramadol or indomethacin alone (data not shown). Prostaglandin E 2 increased with acid injury. The increase in PGE 2 was inhibited by co-administration of indomethacin and tramadol (in pg/ml: acid injury 681.25 AE 324.88, indo 1 tramadol 149.11 AE 35.24). There was no significant effect of treatment on COX-1 or À2 expression.

Co-administration of tramadol with a non-selective NSAID inhibits the return of gastric mucosal barrier function after acid injury in canine tissue, suggesting that caution is required in prescribing concurrent use of these drugs in dogs at risk for gastric ulcers. These drugs may exert this effect by decreasing levels of gastroprotective prostanoids. Further study is needed to understand the mechanism of this drug interaction. An increased intestinal permeability (IP) has been suggested to be both cause and consequence of gastrointestinal (GI) disease, such as inflammatory bowel and celiac disease, in people. A novel tight junction regulator, larazotide acetate (Alba Therapeutics, Baltimore, MD) has been shown to significantly decrease IP in rats and in humans with celiac disease. The purpose of this study was to determine if larazotide acetate reduces IP in Soft Coated Wheaten Terriers (SCWT) and Norwegian Lundehunds (NL) with chronic GI disease and ameliorates clinical signs.

Four NL (2 females, 2 males; median age: 2.5 yrs, range: 1.5-4.5 yrs) and 9 SCWT (7 females, 2 males; median age: 5.0 yrs, range: 1.5-9.0 yrs) were enrolled based on presence of clinical signs of GI disease and hypoalbuminemia, increased fecal alpha 1proteinase inhibitor (a 1 -PI) concentrations, and/or hypocobalaminemia. SCWT with protein-losing nephropathy were excluded. Dogs were fed q12 hrs and received 0.5 mg (4 NL and 2 SCWT) or 2.0 mg (7 SCWT) of larazotide acetate PO before each meal for 90 days. Prior to start of treatment (day 1) and at the end (day 90), IP was evaluated by calculating the lactulose/rhamnose (L/R)-ratio in serum samples obtained at 30, 60, 90, and 120 min after oral dosing. Also, 3 consecutive fecal samples each were collected prior to day 1 and day 90 for N-methylhistamine (NMH) measurement. Pre-and post-treatment data were compared using a Wilcoxon signed rank test. The 0.5 mg vs. 2.0 mg dose groups were compared using a Mann-Whitney U test. Statistical significance was set at p o 0.05. L/R-ratios (medians) for the 60 min sampling time point were significantly lower on day 90 (0.046) than on day 1 (0.080; p 5 0.018). Dogs treated with 2.0 mg q12 hrs had significantly lower 60 min L/R ratios on day 90 than dogs treated with 0.5 mg (0.033 vs. 0.094; p 5 0.014). No difference was found between breeds. Fecal NMH concentrations were not different between time points, treatment groups, or breeds. Fecal a 1 -PI concentrations were available for 11 of the 13 dogs and were significantly higher on day 90 compared to day 1 (p 5 0.033). No differences were found between pre-and post-treatment serum albumin or cobalamin concentrations. Weight gain was seen in all 4 NL. Resolution of diarrhea, vomiting, hyporexia, as well as an increased activity was seen in 1 SCWT. Another SCWT had resolution of diarrhea and a decrease in pruritus. No changes in clinical signs were reported in the remaining 7 SCWT.

This study indicates that larazotide acetate might be able to reduce IP in dogs. This effect may be dose-dependent. However, not all dogs showed an improvement in clinical signs, suggesting that factors other than increased IP might have been responsible for the clinical signs in these dogs. Breed-related effects cannot be ruled out, and further studies are warranted to determine the efficacy of larazotide acetate in dogs of other breeds with GI disease. to analyze different biochemical markers, calculate clinical activity scores, and assess survival in dogs with PLE and compare them with those in dogs with food-responsive diarrhea (FRD) without protein loss. 29 dogs with PLE and 18 dogs with FRD, referred to the University of Bern, CH, were enrolled. Selection criteria included a history of chronic diarrhea (4 3 weeks), exclusion of identifiable underlying causes, and histopathologic evidence of intestinal inflammation, but not neoplasia. Underlying disorders were excluded based on CBC, chemistry profile, urinalysis, fecal analysis, trypsinlike immunoreactivity, cobalamin, folate, and transabdominal ultrasound. Also, canine pancreatic lipase immunoreactivity (spec cPL s ), C-reactive protein (CRP), calprotectin and alpha 1 -proteinase inhibitor (a 1 -PI) were measured in serum from 18 dogs and compared with 18 dogs with FRD without PLE. All dogs were scored using the canine IBD (CIBDAI) and the canine chronic enteropathy (CCE) clinical activity index (CCECAI).

Total protein, albumin (5-28.1 g/l), and total calcium (1.21-2.32 mmol/l) were decreased in all 29 dogs. Cobalamin was decreased in all but 3 dogs ( o 100-490 ng/l). Spec cPL was mildly increased in 3/18 dogs with PLE and normal in 15/18 PLE and all FRD dogs. CRP was normal in 5/18 dogs with PLE (16/18 FRD), mildly increased in 7/18 (1/18 FRD), and moderately increased in 6/18 PLE dogs (1/18 FRD). Calprotectin was slightly higher in dogs with PLE, but all PLE and FRD dogs yielded values in the normal range. Serum a 1 -PI was significantly lower in dogs with PLE than in those with FRD (p o 0.001), with 13/18 PLE dogs below the reference range (1/18 FRD). CIBDAI ranged from 4 to 16 and CCECAI from 6 to 19. At the end of the study, 12/29 dogs were still alive with survival times between 26 and 2544 days. 17/29 dogs died with a median survival of 96 days (range 2-874 days). Dogs with mildly increased CRP died earlier than dogs with a normal or moderately increased CRP (p 5 0.011), whereas albumin, calcium, Spec cPL, calprotectin, CIBDAI, and CCECAI had no significant impact on outcome and survival.

In conclusion, dogs with PLE have a significantly lower a1-PI in the serum than dogs with FRD. Furthermore, most dogs with PLE have an increased CRP and a decreased cobalamin. A mild increase in CRP appears to be a poor prognostic factor. While hypoalbuminemia is a common finding associated with chronic enteropathies, its impact on survival in this population is poorly defined. The aim of this study was to compare dogs with chronic enteropathies on the basis of their serum albumin concentration at the time of presentation. We hypothesized that dogs with a protein losing enteropathy (PLE) have a significantly shorter survival time compared to dogs with chronic enteropathies which are not hypoalbuminemic (controls).

Information obtained from the medical records included signalment, duration and characteristics of clinical signs, physical examination findings, clinicopathologic data and survival time.

One hundred seventeen cases fit the inclusion criteria; 68 in the PLE group and 49 controls. There was no statistical significance between groups for age (P 5 0.12), weight (P 5 0.17), weight loss (P 5 0.59) and body condition score (P 5 0.072). Compared to control dogs, PLE dogs had decreased serum concentrations of cobalamin (P 5 0.002), total calcium (P o 0.0001), globulin (P o 0.0001), cholesterol (P o 0.0001) and ionized calcium (P o 0.001). Survival analysis revealed a significantly decreased survival time for PLE dogs (P 5 0.0008); median survival was 701 days for PLE dogs and 4 3,500 days for controls. While the PLE group did not survive as long, survival was not directly associated with severity of hypoalbuminemia; patients with albumin concentration o 1.3 g/dL survived longer than those with mild hypoalbuminemia (1.6-1.9 g/dL).

This study supports the observation that chronic enteropathy patients have decreased survival time when presented with hypoalbuminemia; however this study suggests the severity of hypoalbuminemia is not a reliable indicator of survival. Cobalamin (vitamin B 12 ) deficiency in the Chinese Shar Pei (Shar Pei) is suspected to be hereditary. Inherited causes of cobalamin deficiency have been reported in humans and may affect absorption, transport, or cellular processing of cobalamin. Based on human and veterinary studies, an increased serum methylmalonic acid (MMA) concentration has been suggested to reflect cobalamin deficiency at the cellular level. In this context, it has been shown in humans that MMA concentrations are higher in patients with genetic disorders affecting intracellular processing than in patients with genetic defects affecting gastrointestinal processing and extracellular transport of cobalamin. Therefore, the aim of this study was to evaluate serum MMA concentrations in Shar Peis and dogs of six other breeds with cobalamin deficiency.

From 2008 In conclusion, serum cobalamin deficient Shar Peis had a 10 times higher median serum MMA concentration compared to cobalamin deficient dogs of six other dog breeds. Further studies are needed to investigate the intracellular processing of cobalamin in Shar Peis with cobalamin deficiency. Chinese Shar Peis (Shar Peis) have a high prevalence of cobalamin deficiency. Two other conditions reported frequently in this breed are Shar Pei fever and cutaneous mucinosis. Shar Pei fever is an autoimmune disorder causing periodic flare-ups and is associated with increased serum concentrations of C-reactive protein (CRP), a nonspecific marker of inflammation. Cutaneous mucinosis is characterized by excessive deposition of mucin in the dermis. Also, hyaluronic acid (HA), the main component of mucin, was shown to be significantly higher in serum from Shar Peis with cutaneous mucinosis than in healthy controls. To date, a possible association between Shar Pei fever and/or cutaneous mucinosis on one side and cobalamin deficiency on the other has not been investigated in Shar Peis. Thus, the aim of this study was to compare serum concentrations of HA (an indicator of cutaneous mucinosis) and inflammatory markers (CRP, calprotectin, and S100A12), assumed to be increased in episodes of Shar Pei fever, in Shar Peis with and without cobalamin deficiency.

Serum samples from 40 Shar Peis, collected from 2008 to 2010, were analyzed. Serum HA and CRP (reference interval (RI): 0.0-7.6 mg/L) were quantified by using commercial ELISA kits (Echelon Biosciences, Salt Lake City, UT, USA and Tridelta, Maynooth, Ireland; respectively). Serum calgranulin concentrations were measured using an in-house ELISA (calprotectin; RI: 0.9-11.9 mg/L) and RIA (S100A12; RI: 33.0-233.0 mg/L), respectively. Mann-Whitney U tests were used to compare serum HA, CRP, calprotectin, and S100A12 concentrations between Shar Peis with and without cobalamin deficiency. Significance was set at p o 0.05.

Fourteen Shar Peis were severely cobalamin deficient, defined by an undetectable serum cobalamin concentration ( o 150 ng/L). In the remaining 26 dogs, serum cobalamin concentrations were within the reference interval (251-908 ng/L). Serum concentrations of HA, CRP, calprotectin, and S100A12 were not significantly different between cobalamin deficient Shar Peis (medians: 649.9 ng/ml, 4. . Fifty percent of cobalamin deficient Shar Peis had serum calprotectin concentrations above the upper limit of the reference interval, and 43% had serum S100A12 concentrations above the suggested upper reference limit.

In this study, serum concentrations of HA, CRP, and the calgranulins did not differ between cobalamin deficient Shar Peis and Shar Peis with a normal serum cobalamin concentration. This finding leads us to speculate that increased HA and/or inflammatory markers are not associated with cobalamin deficiency in Shar Peis. Further studies are needed to investigate serum cobalamin concentrations in patients with Shar Pei fever or cutaneous mucinosis. Cobalamin deficiency (CD) has been associated with gastrointestinal and pancreatic disease in dogs. Hereditary CD has been demonstrated in Giant Schnauzers and single case reports have suggested congenital CD in the Border Collie (BC) breed. Clinicopathologic findings of CD vary and can be unspecific as cobalamin acts as a Co-Factor for a multitude of enzymatic reactions. The two most important reactions concern the conversion of methylmalonyl-CoA to succinyl-CoA and the re-methylation of homocysteine (Hcy). These two metabolites increase when cobalamin is lacking and act as markers for cobalamin availability on a cellular level. Preliminary data from dogs suggested that measurement of methylmalonic acid (MMA) may be a better diagnostic test for CD than serum cobalamin concentration. Therefore the goals of the study were (1) to establish reference values for serum cobalamin, urine MMA and plasma Hcy in healthy pet dogs, (2) to screen a larger BC population from Switzerland for CD, and (3) to perform genomic analyses on BC with CD.

For determination of reference values 35 healthy pet dogs were used. Serum cobalamin was measured using an automated chemiluminescence assay (Immulite 2000), urine MMA was determined using gas chromatography and expressed as a ratio to urine creatinine and plasma Hcy was measured using High Pressure Liquid Chromatography and fluorimetric detection. To calculate reference ranges the 10th and 90th percentile were used. Data were analyzed using non-parametric tests.

Reference ranges for cobalamin, Hcy, and MMA were: cobalamin 279.2-972.8 ng/L; urine MMA 2-4.65 mmol/mol creatinine; and plasma Hcy 4.73-18.34 mmol/L. The screened BC population comprised 113 purebred dogs and 4 BC (median 11.5 months; range 8-41) suffering from congenital CD could be identified. Clinical signs differed and consisted of tiredness (4), stunted growth (4), anemia (3), dysphagia (2) and persistent fever (1). Median (ranges) results for healthy BC and BC with CD were: for cobalamin 592 (142-1855) and 72.00 (30-139) ng/L; for urine MMA 2 (2-360) and 4148 (1800-6665) mmol/mol creatinine; for Hcy 8.5 (2.8-22.4) and 41.00 (40-86.6) mmol/L. Strikingly, healthy BC with cobalamin concentrations well within the reference range had significantly higher urine MMA concentrations compared to control dogs. Under the assumption that the four affected BC are inbred to a single founder animal, first results of genotyping on the 170 k illumina canine_HD SNP chip suggest that mutations in the CUBN and AMN gene can be excluded to cause the observed CD in these dogs. We conclude that CD is a rare familial disease in BC with variable clinical signs. To define the genomic region responsible for CD further genetic analysis is in progress. It remains to be determined why some BC have high urine MMA concentrations despite a serum cobalamin concentration within the reference range. Calprotectin is a protein complex that plays an important role in the innate immune response. Preliminary data suggest that canine calprotectin (cCP) is a useful marker for the detection of inflammation in dogs. Recently, a radioimmunoassay for the measurement of cCP has been developed and analytically validated, but this test requires the use of a radioactive tracer. Therefore, the aim of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (ELISA) for the quantification of cCP in serum and fecal specimens from dogs.

Canine calprotectin (cCP) was purified, antiserum against purified cCP was raised in rabbits, monospecific antibodies were purified by affinity chromatography, and a sandwich-ELISA was developed. Purified antibodies were used for capturing and, after coupling with horseradish peroxidase (HRP), for reporting. A HRP substrate was used for color development. The assay was analytically validated by determination of analytical sensitivity and specificity, dilutional parallelism, spiking recovery, and intra-and inter-assay variability. Control intervals for serum and fecal cCP were established from 110 and 52 healthy pet dogs, respectively, using the central 95 th percentile.

Sensitivity of the assay for serum samples assayed in a 1:400 dilution and for fecal extracts assayed in a 1:4,000 dilution was 0.3 mg/L and 3.2 mg/g, respectively. Over a wide range of the assay, there was no cross-reactivity with cS100A12, the closest structural analogue of cCP available. Observed to expected ratios (O/E) for serial dilutions ranged from 83.2-118.5% (mean AE standard deviation [SD]: 101.3 AE 10.0%) for four different serum samples, and from 81.7-129.1% (mean AE SD: 101.8 AE 14.0%) for five different fecal extracts. O/E for spiking recovery ranged from 87.8-130.4% (mean AE SD: 100.6 AE 6.5%) for four different serum samples and 6 different spiking concentrations, and from 95.9-152.0% (mean AE SD: 104.6 AE 11.6%) for 4 different fecal extracts and 6 different spiking concentrations. Intra-assay coefficients of variation (CV) for 4 different serum samples were 7.8, 5.0, 7.4, and 12.7%, and 10.0, 6.1, 6.2, and 7.0% for 4 different fecal extracts. Inter-assay CV for 4 different serum samples were 17. 2, 8.1, 9.9, and 12.6%, and 12.3, 8.3, 7.2, and 9 .6% for 4 different fecal extracts. The control intervals for serum and fecal cCP were established as 0.9-11.9 mg/L and 3.2-65.4 mg/g, respectively.

We conclude that this new ELISA for the measurement of cCP is analytically sensitive, linear, accurate, precise, and reproducible, and does not cross-react with canine S100A12. Further studies evaluating the clinical usefulness of measuring serum and/or fecal cCP are currently under way. The syndrome of hemorrhagic gastroenteritis (HGE) is characterized by a peracute onset of hemorrhagic diarrhea, vomiting, depression, and anorexia, and can be associated with a high mortality if untreated. The etiology of HGE is unknown, but it is speculated that an abnormal response to bacterial endotoxins, bacteria, or dietary components may play a role. HGE is characterized by an increased vascular/mucosal permeability, thought to represent a type I-hypersensitivity reaction, whereas inflammation and necrosis appear to be rare. However, markers of gastrointestinal (GI) inflammation and changes in the intestinal microbiota have not been studied extensively in dogs with HGE. Therefore, the aim of this study was to evaluate fecal canine calprotectin (CP) and S100A12 (A12), a 1 -proteinase inhibitor (a 1 -PI, a marker of GI protein loss), and bacterial groups that have previously been shown to be decreased (i.e., Faecalibacterium spp., Ruminococcaceae, Bifidobacterium spp.) or increased (i.e., Proteobacteria) in fecal samples from dogs with HGE.

Fecal samples from 3 consecutive days were collected from 7 dogs with HGE. Fecal CP, A12, and a 1 -PI concentrations were measured by in-house immunoassays. Bacterial DNA was extracted from each fecal sample and was analyzed for Faecalibacterium spp., Proteobacteria, Rumino-coccaceae, and Bifidobacterium spp. using quantitative PCR assays. Concentrations of fecal CP, A12, and a 1 -PI, and the abundance of bacterial DNA were compared using a Friedman test with Dunn's post-hoc tests. Significance was set at p o 0.05.

At the time of diagnosis (day 1), fecal CP, A12, and a 1 -PI were above the suggested reference intervals in 6, 6, and 5 of the 7 dogs, respectively. Until day 3, this number decreased to 2, 1, and 4, respectively. Decreases in concentrations were significant between days 2 and 3 for A12 (p 5 0.016), and between days 1 and 3 for a 1 -PI (p 5 0.012), but not for CP despite a trend (p 5 0.085). No differences in the abundance of Faecalibacterium spp. (p 5 0.085), Bifidobacterium spp. (p 5 0.192), or Proteobacteria (p 5 0.305) were observed. However, the abundance of Rumino-coccaceae was significantly lower on day 3 when compared to day 2 (p 5 0.008).

In this study, fecal markers of inflammation and GI protein loss were increased in dogs with HGE. Although the number of patients was small, following initiation of treatment, two of the markers decreased significantly. These results suggest a loss of protein into the GI tract at the onset of HGE. The lack of significant increases of Faecalibacterium spp., Bifidobacterium spp., and Ruminococcaceae, and decreases in Proteobacteria may suggest GI dysbiosis. Further longitudinal studies are needed and are currently under way to evaluate GI dysbiosis in canine HGE patients. The most recent antiemetic approved for use in dogs is maropitant citrate (Cerenia s , Pfizer Animal Health). Maropitant is a selective NK1 receptor antagonist that acts by blocking the binding of substance-P within the emetic center and chemoreceptor trigger zone.

Label dosage recommendations for maropitant citrate are 1 mg/ kg SC or 2 mg/kg orally once daily for up to 5 consecutive days (acute emesis) and 8 mg/kg orally once daily for up to 2 consecutive days (motion sickness). The study objective was to determine when steady-state is reached and the pharmacokinetics of maropitant administered at label oral dosages once daily for 14 consecutive days.

Two groups of eight healthy beagles were administered maropitant citrate at 2 or 8 mg/kg orally once daily for 14 days. Concentrations of maropitant and its metabolite were measured in plasma using a LC-MS/MS assay. Pharmacokinetic parameters were estimated using non-compartmental pharmacokinetic techniques and a modeling approach was used to estimate steady-state.

The accumulation ratio for maropitant was 2.46 (AUC0-24) and 2.03 (Cmax) for the 2 mg/kg dose; and 4.81 (AUC0-24) and 2.77 (Cmax) for the 8 mg/kg dose after 14 days. The model estimate for the number of doses required to reach 90% of steady-state was 4.30 for 2 mg/kg and 8.09 for 8 mg/kg. Three dogs experienced a single episode of vomiting.

Dosing maropitant citrate beyond the label duration was well tolerated by healthy dogs. Steady-state was reached after approximately 4 doses for daily 2 mg/kg and 8 doses for daily 8 mg/kg oral dosing.

Previously presented at the Veterinary Cancer Society, November 2010. Cobalamin (vitamin B 12 ) is involved in a variety of metabolic processes. Altered serum cobalamin concentrations have been observed in dogs with gastrointestinal disorders, such as exocrine pancreatic insufficiency (EPI) or severe and longstanding ileal disease. This study was conducted to identify breeds with a higher proportion of a decreased serum cobalamin concentration that were submitted to the Gastrointestinal Laboratory. The study was also aimed at investigating serum trypsin-like immunoreactivity (TLI) concentrations that were diagnostic for EPI in the dogs with a decreased serum cobalamin concentration. Except for CSP, breeds identified here, have not previously been identified to have a higher rate of a decreased serum cobalamin concentration. Also, a possible association between an undetectable serum cobalamin and a decreased serum TLI in AI needs to be further investigated. Calprotectin (CP) is a widely used marker for the diagnosis and monitoring of gastrointestinal (GI) inflammation in humans. Studies in humans usually report fecal CP concentrations based on a single stool sample although considerable day-to-day variability of fecal CP was found in patients with GI disease and in healthy controls. Intra-individual variation of canine CP (cCP) was also substantial in a small number of healthy dogs but has not been determined in dogs with chronic GI disease. Thus, the aim of this study was to compare the day-to-day variation of fecal cCP in dogs with chronic GI disease before and during treatment to that in healthy dogs. We hypothesized that fecal cCP would be less variable in patients with chronic GI disease than in healthy controls, and thus collection of a single fecal sample would be sufficient.

Fecal samples from 3 consecutive days were prospectively collected from 15 dogs (group A; median age: 6.1 years) referred for diagnostic work-up of chronic signs of GI disease, from 8 dogs (group B; median age: 5.6 years) with stable GI disease while being treated, and from 44 healthy adult dogs (group C; mean age: 5.4 years). Fecal samples were extracted and cCP was measured by an in-house immunoassay. Mean cCP, standard deviation, coefficient of variation (CV), and difference between maximum and minimum cCP for the 3-day sample collection period were calculated for each dog and were compared among groups using a Kruskal-Wallis test.

Fecal cCP ranged from 2.9-102.7 mg/g (median: 17.6 mg/g) in dogs with GI disease (group A), from 2.9-265.1 mg/g (median: 18.4 mg/g) in dogs of group B, and from 2.9-93.1 mg/g (median: 7.9 mg/g) in healthy controls (group C). CVs were 0-121.3% in group A (median: 20.0%), 2.0-89.4% in group B (median: 47.4%), and 0-145.2% in group C (median: 40.4%), respectively. Patients in group A appeared to have less variable fecal cCP than dogs in group B and C, but this difference was not significant (p 5 0.326). The difference between maximum and minimum cCP for the 3-day sample collection ranged from 0-31.1 mg/g in group A (median: 7.2 mg/g), from 0.1-240.0 mg/g in group B (median: 12.5 mg/g), and from 0-57.4 mg/g in group C (median: 14.8 mg/g), and were not significantly different between any of the groups (p 5 0.530).

In this study, considerable day-to-day variation of fecal cCP was found in dogs with chronic GI disease (regardless of treatment) and was comparable to that in healthy dogs. Results of this study suggest that for evaluating fecal cCP in dogs with clinical signs of GI disease, three consecutive fecal samples rather than a single fecal sample should be analyzed. Because we did not intend to evaluate the clinical usefulness of fecal cCP as a marker of GI disease in dogs, disease severity, quality, and location differed among dogs in groups A and B. The diagnostic utility of fecal cCP in dogs with GI disease is currently being investigated. It has been suggested that diagnosis of Clostridium perfringens related enteropathy should be based on the detection of the C. perfringens enterotoxin gene (cpe-gene) by PCR and/or C. perfringens enterotoxin (CPE) by ELISA in feces. However, the prevalence of the cpe-gene and CPE in dogs and especially cats with gastrointestinal disease has not yet been reported. Also, there is limited information about the stability of CPE in fecal samples at various storage conditions. The aim of this study was to evaluate the prevalence of the cpe-gene and CPE and the stability of CPE in fecal samples from dogs and cats.

To evaluate the prevalence of the cpe-gene, a total of 481 fecal samples from dogs and cats with clinical signs of gastrointestinal disease (273 dogs and 208 cats) and 109 fecal samples from those without such signs (80 dogs and 29 cats) were examined using PCR. To evaluate the prevalence of CPE, a total of 90 fecal samples from dogs and cats with clinical signs of gastrointestinal disease (31 dogs and 59 cats) and 11 dogs without such signs were evaluated using a commercially available ELISA kit (TechLab, Blacksburg, VA). The results were analyzed using a Fisher's exact test. Significance was set at p o 0.05. To evaluate the stability of CPE, 8 fecal samples from dogs and 2 from cats with clinical signs of gastrointestinal disease that were positive for CPE were examined. Also, 5 CPE negative samples from dogs were evaluated as negative controls. Each sample was subdivided into 8 aliquots and evaluated on day 0; on days 2, 5, and 10 after being stored at room temperature (RT) or 41C; and on day 10 after being stored at À201C.

The prevalence of the cpe-gene was not significantly different between dogs with signs of gastrointestinal disease (99/273; 36.3%) and dogs without (27/80; 33.8%; p 5 0.79). Also, the prevalence of the cpe-gene in cats with signs of gastrointestinal disease (80/208; 38.5%) was not significantly different compared to cats without (6/ 29; 20.7%; p 5 0.06). PCR and ELISA results were available for 90 samples. Of the 65 PCR positive samples, only 6 (9.2%) were ELISA positive. Of the 35 PCR negative samples, only 1 (2.9%) was ELISA positive. The prevalence of CPE was not significantly different between dogs with clinical signs of gastrointestinal disease (3/31; 9.7%) and those without (1/11; 9.0%; p 5 1.0). The prevalence of CPE in cats with signs of gastrointestinal disease was 4/59 (6.8%), but no samples from cats without such signs were available. When evaluating the stability of CPE, results for all aliquots were consistent with the initial result, except for one sample (on day 5, stored at RT, which was initially CPE positive).

These results indicate that only a small proportion of samples that are PCR positive for the cpe-gene are also positive for CPE. Studies are warranted to further compare the prevalence of CPE among animals with gastrointestinal disease and those without. Furthermore, the results indicate that CPE is relatively stable in fecal samples at various storage temperatures. Clostridium perfringens has been implicated as a cause of diarrhea in dogs. The main study objective was to compare two culture methods for the identification of C. perfringens. A secondary objective was to evaluate C. perfringens toxin genes a, b, b 2 , e, ı and cpe from canine isolates using a multiplex PCR and determine their prevalence in a group of normal and diarrheic dogs.

Fecal samples were collected from clinically normal (ND, n 5 105) and diarrheic dogs (DD, n 5 54) at a primary care veterinary facility. Isolation of C. perfringens was performed using direct inoculation of feces onto 5% sheep blood agar (SBA) as well as enrichment of stool in BHI broth followed by inoculation onto SBA. Isolates were tested by multiplex PCR for the presence of a, b, b 2 , e, ı and cpe genes.

C. perfringens was isolated from 84% (88/105) of ND fecal samples using direct culture and 87.6% (92/105) with BHI enrichment (p 5 0.79). In the DD, corresponding isolation rates were 90.7% and 93.8% (p 5 0.45). All isolates possessed a toxin gene. b, b 2, e, ı and cpe toxin genes were identified in 4.4%, 1.1%, 3.3%, 1.1% and 14.4% of ND isolates, respectively. In the DD group, b and b 2 were identified in 5%, e and ı were not identified and the cpe gene in 16.9% of isolates.

BHI enrichment did not significantly increase the yield of C. perfringens compared to SBA but increased time and cost involved. C. perfringens (p 5 0.64) and C. perfringens toxin genes were present in equal proportions in ND and DD groups (p ! 0.15). Culture of C. perfringens and PCR for toxin genes are of limited diagnostic utility due to the high prevalence of C.perfringens in normal dogs and the lack of apparent difference in toxin gene distribution between normal and diarrheic dogs. Endoscopic biopsies are a relatively convenient, non-invasive test for feline infiltrative intestinal disorders. Commonly, only the duodenum is examined due to cost, risks and time required to prepare the colon using lavage solutions, cathartics and/or enemas. The purpose of this study was to evaluate the consistency between endoscopic biopsies of the duodenum and ileum in cats.

Endoscopic biopsies from 70 cats which had duodenal and ileal tissue specimens were evaluated retrospectively. All slides were randomized and reviewed by a single pathologist (JM) for quality, number of biopsies, and diagnosis according to WSAVA standards. No information regarding history, clinical signs, endoscopic findings, or previous histological diagnosis was made available to the pathologist. Statistical comparison of the diagnosis of SC-LSA and IBD by intestinal location was conducted using Fisher's exact test (p o 0.05 significant).

18 of 70 cats (25.7%) were diagnosed with SC-LSA in the duodenum and/or ileum. Of these 18 cats, 7 (38.9%) were diagnosed with only duodenal SC-LSA, 8 (44.4%) were diagnosed with only ileal SC-LSA, and 3 (16.7%) had SC-LSA in both duodenum and ileum. In 8 cats with only ileal SC-LSA, 3 had severe IBD in duodenal biopsies, possibly consistent with early SC-LSA. 5 of these 8 had duodenal biopsies without evidence of SC-LSA.

Our results suggest there is a population of cats in which diagnosis of SC-LSA may only be found by evaluating ileal biopsies. Clinicians should consider performing both upper and lower GI endoscopic biopsies in cats with suspected infiltrative small bowel disease. Periodontitis is one of the most common diseases in cats and is mainly due to the presence of plaque and calculus. In this study, we investigated putative correlations between dental tartar and gingivitis and also between gingivitis and subgingival bacteria in cats.

Twelve cats (median age: 5 years; range: 1-10 years; 6 DSH and 6 Persians; 8 females and 4 males) were enrolled. Dental tartar was obtained during scaling for a dental prophylactic procedure. All cats were negative for FeLV and FIV infection as assessed by a commercial ELISA test (SNAP s FIV/FeLV Combo Test). Severity of gingivitis (scores: 0-3; 0 5 normal, 1 5 mild, 2 5 moderate, and 3 5 severe) and dental tartar (scores: 0-3) were scored in each cat. Endodontic paper points were applied for collecting a bacterial sample from the subgingival area and transferred to thioglycollate transporting media for bacterial culture. The relationship between gingivitis and tartar thickness scores was analyzed by spearman correlation. A student's t-test was used to compare the mean differences (gingivitis and tartar thickness scores) between upper and lower teeth. The association between severity of gingivitis and bacterial type was tested by chi square test.

The spearman correlation coefficient for the average gingivitis score and the average tartar thickness score was 0.91 (p o 0.05). Interestingly, the average tartar thickness scores from the upper jaw were significantly higher than those from the lower jaw (p o 0.05). The highest scores were found for the molar teeth in all cats. Bacterial culture revealed 28.9% anaerobic bacteria species (i.e., Bacteroides spp., Peptostreptococcus anaerobius, and Eubacterium aerofaciens) and 71.1% aerobic bacteria species (i.e., Pasteurella multocida, Streptococcus spp., Enterococcus spp., Staphylococcus spp., Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa). Anaerobic bacteria were found mostly in cats with higher gingivitis scores (2-3; chi square: p o 0.05), while Pasteurella multocida was found mostly in cats with lower gingivitis scores (0-1; chi square: p o 0.05). Antimicrobial sensitivity testing indicated that all of the anaerobic bacteria were sensitive to clindamycin, chloramphenicol, metronidazole, cefoxitin, or tetracycline, 90% were sensitive to erythromycin, and 80% were sensitive to penicillin. The most abundant aerobic bacterial species, Pasteurella multocida, was sensitive to cefoxitin in all cases in which it had been cultured.

These results suggest that anaerobic bacteria may be associated in the pathogenesis of severe gingivitis. These data warrant further studies of the prophylactic use of antibiotics in cats undergoing dental prophylactic procedures. Inflammatory bowel disease is the most common cause of vomiting and diarrhea in dogs. Although it can occur in any canine breed, certain breeds are more susceptible. We have previously shown that polymorphisms in the TLR4 and TLR5 gene are significantly associated with inflammatory bowel disease (IBD) in the German shepherd dog (GSD), a breed at risk of developing this disease. It would be useful to determine if these polymorphisms are significant in other canine breeds as this may allow the development of novel diagnostics and therapeutics to be applied to all canine breeds with IBD. Therefore the aim of this study was to investigate whether polymorphisms in canine TLR 4 and TLR 5 genes are associated with IBD in other non-GSD canine breeds.

Four non-synonymous SNPs in the TLR4 gene; T23C, G1039A, A1572T and G1807A and three non-synonymous SNPs in the TLR5 gene; G22A, C100T and T1844C previously identified in a mutational analysis in GSDs with IBD were evaluated in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 85 unrelated dogs with IBD consisting of 38 different non-GSD breeds from the UK were compared to a breed-matched control group consisting of 162 unrelated dogs from patients treated for noninflammatory disease at the Royal Veterinary College, London, UK.

As in the GSD IBD population the two TLR5 SNPs; C100T and T1844C were found to be significantly protective for IBD in other breeds included in this study (p 5 0.023 and p 5 0.0195 respectively).

This study confirms the protective effects of the two TLR5 SNPs (C100T and T1884C) in other canine breeds with IBD. This highlights the importance of TLR5 in the pathogenesis of canine IBD and may represent common pathological pathways of IBD in different canine breeds due to the high degree of haplotype sharing seen among breeds. This may allow for the future expansion of novel diagnostics and therapeutics to be applied to all canine breeds with IBD. Further functional studies looking at the role of TLR5 in the pathogenesis of canine IBD are needed to confirm these findings. Toll-like receptor 5 (TLR5) is an extracellular pattern recognition receptor belonging to the innate immune system. We have recently shown that three non-synonymous single nucleotide polymorphisms (SNPs) in the TLR5 gene (G22A, C100T and T1844C) are significantly associated with inflammatory bowel disease (IBD) in German shepherd dogs. In addition, we confirmed that two of these TLR5 SNPs (C100T and T1844C) were significantly associated with IBD in a population consisting of 38 different dog breeds. In order to determine if other novel SNPs exist in the TLR5 gene in addition to the ones identified in the GSD population, mutational analysis was carried out in seven Boxer dogs with IBD.

Polymerase chain reaction was carried out to amplify the TLR5 coding region in the seven dogs with IBD. Sequencing was carried out using sequence based typing with the ABI Prism Sequencing Kit (Applied Biosystems, UK) and analyzed using an ABI3100 automated sequencer (PE Applied Biosystems). Sequencing information from seven Boxer dogs with IBD from the UK were compared to the reference sequence published on the Ensemble webserver (www. ensembl.org/Canis_familiaris).

In addition to the three SNPs identified previously in the TLR5 gene, a novel non-synonymous SNP; T443C was identified in the Boxer dog population with IBD. This SNP has never been reported before and was present as the homozygote genotype in three dogs with IBD and in one dog as the heterozygote genotype. Using the Simple modular architecture research tool (SMART) web server (http:// smart.embl.de/) we were able to map the T443C SNP to the leucine rich repeat domain of the TLR5 protein. The leucine rich repeat domain is involved with ligand binding and therefore a change in the amino acid in this region may affect function, especially as the T443C SNP results in a change in the amino acid from non-polar to polar.

Our study further confirms the role of TLR5 in the pathogenesis of canine IBD. Our results suggest that in addition to shared risk polymorphisms amongst breeds, individual breeds may harbor unique SNPs arising after breed formation which may further affect their susceptibility to this disease. However, a case-control study would be needed in the Boxer dog to confirm the significance of the TLR5 T443C SNP and further functional data would be needed to elucidate the exact role of this polymorphism in canine IBD. An automated power driver device (OnControl, Vidacare) has recently become available for bone marrow aspiration (BMA) and bone marrow biopsy (BMB) in humans. The purpose of our study was to compare this automated technique to the traditional manual technique for bone marrow sampling in cats.

Twelve healthy research cats were anesthetized using a standardized protocol on 2 different occasions, 2 days apart, to have BMAs and BMBs performed by the same operator. On day 1, half of the cats were randomized to have a BMA performed at both the proximal humerus and the iliac crest, and a BMB performed at the iliac wing, using the OnControl device (15-gauge needle for BMA; 11-gauge needle for BMB). The other half of the cats had the same procedures performed using a manual technique (15-gauge Illinois needle for BMA; 11-gauge Jamshidi needle for BMB). On day 3, each cat had BMA performed at the opposite humerus and iliac crest, and a BMB performed at the proximal humerus using the opposite technique from day 1. For each procedure, the operator was given a maximum of 3 attempts to successfully collect a sample. The rate of success, as well as the number of attempts were recorded. The ''ease of use'' of the device was rated by the operator on a 5-point scale after each procedure. Using previously determined criteria, the macroscopic and microscopic qualities of the BMA and BMB samples were assessed by a board-certified pathologist, blinded to the technique used. The level of pain experienced by each cat was evaluated 6, 12, 18, 24, 36 and 48 hours following each set of procedures, using a previously validated pain scoring system. Two sample t-tests were used to compare the automated technique to the manual technique and to compare the humerus to the iliac crest site for BMAs and the humerus to the iliac wing site for BMBs.

For all procedures, at all sites, the ''ease of use'' was better for the automated technique than for the manual technique (P o 0.05). The duration of the procedure and the number of attempts to collect a sample were significantly lower with the automated technique for BMA at the proximal humerus (P o 0.05). There was no significant difference in the level of pain at any time point following each set of procedures with either technique. Performing BMA at the proximal humerus was associated with a higher rate of success (P o 0.05), a lower number of attempts (P o 0.05), a shorter duration of the procedure (P o 0.05), a higher-rated ''ease of use'' of the technique (P o 0.05), and a better quality sample (P o 0.05) when compared to sampling from the iliac crest,

In conclusion, we found the automated bone marrow sampling technique suitable for use in adult cats. This technique was easier to use than the manual technique for both BMA and BMB, and reduced the duration of the procedure and the number of attempts for successful BMA at the proximal humerus. Performing BMA at the proximal humerus was faster, easier and allowed collection of better quality samples than at the iliac crest, independently of the technique used. The fractious nature of some feline patients sometimes makes sedation or general anesthesia necessary for routine procedures such as blood collection for hematologic analyses. It has been anecdotally reported that sedation or general anesthesia could induce variations in hematologic parameters in cats, making it important for the clinician to be able to anticipate potential changes on hematologic parameters that could result from chemical restraint.

This study evaluated the effects of a standardizecd anesthetic protocol using ketamine (10 mg/kg, IV), midazolam (0.5 mg/kg, IV) and buprenorphine (10 mg/kg, IM) on the hematologic parameters of 12 healthy adult research cats. Each cat had blood samples collected before and after induction of anesthesia on 2 different occasions, 2 days apart. In total, 24 pairs of complete blood counts were obtained. Analyses were performed at a certified veterinary laboratory. Paired sample t-tests were used to determine whether there were any statistical differences between hematologic parameters before and after induction of general anesthesia, for each cat, on 2 different occasions.

Compared to preanesthetic values there was a significant decrease in red blood cell count, hemoglobin concentration, hematocrit, lymphocyte count and plasma total protein concentration after induction of anesthesia. There was no significant difference in the segmented or band neutrophil, eosinophil, basophil, monocyte and platelet counts between the samples taken before and after induction of anesthesia. On average, there was a 23.7% decrease in the red blood cell count (9.06 Â 10 12 /L to 6.91 Â 10 12 /L) (P o 0.0001), a 23% decrease in hemoglobin concentration (133.88 g/L to 103.08 g/ L) (P o 0.0001), a 24.4% decrease in the hematocrit (0.41 L/L to 0.31 L/L) (P o 0.0001), a 25.3% decrease in the lymphocyte count (3.68 Â 10 9 /L to 2.77 Â 10 9 /L) (P 5 0.0023), and a 12.1% decrease in the plasma total protein concentration (84.79 g/L to 74.54 g/L) (P o 0.0001) when samples taken before and after induction of anesthesia were compared.

If only the hematocrit was considered as a marker of anemia, 29% of the samples from these 12 healthy cats, taken while they were under general anesthesia, would have been misinterpreted as belonging to anemic patients (hematocrit o 0.285 L/L), using the reference interval established in our laboratory. None of the cats would have been considered anemic before induction of general anesthesia. In practice, the decrease in lymphocyte count following anesthesia is unlikely to be of clinical relevance, as all the samples except 2 had a lymphocyte count that was within the reference interval for cats established by our laboratory.

This study suggests that complete blood counts performed on blood taken under general anesthesia with this combination of anesthetic drugs in cats should be interpreted cautiously in order not to make a false diagnosis of anemia. The mechanism responsible for the decrease in circulating red blood cell mass following anesthesia induction in cats is unknown and requires further investigation. Rivaroxaban is an oral inhibitor of activated coagulation factor X (Xa). It is expected to have similar coagulation effects as low molecular weight heparin, without the need for injection, making it an attractive alternative for long-term anticoagulant therapy in cats.

Citrated blood obtained from five healthy adult cats was exposed in vitro to varying concentrations of rivaroxaban, followed by coagulation testing. The rivaroxaban was extracted from commercially available tablets (Xarelto s ) and dissolved in DMSO prior to addition to the blood. Tests performed included kaolin-activated thrombelastography (TEG), prothrombin time (PT), dilute PT (dPT), activated partial thromboplastin time (aPTT), and anti-factor Xa (aXa) activity.

Dose-dependent prolongations were seen in all coagulation parameters. Similar to human data, therapeutic aXa levels (between 0.5-1.0 aXa units) were achieved at in vitro concentrations between 160 and 220 mg/L. At 220 mg/L, dPT measurements were clinically prolonged in all cats (29.2 AE 4 sec vs. 18.5 AE 0.8 sec, P 5 0.148), while aPTT values were only mildly prolonged from baseline (21.9 AE 5 sec vs. 15.6 AE 2 sec, P 5 0.07). Significant prolongations were seen in dPT at 500 (60.4 AE 42 ec, P 5 0.005). TEG R time did not prolong from baseline values until concentrations of 2000 mg/L were reached (16.0 AE 9 min compared to 3.1 AE 0.7 min, P 5 0.006).

Rivaroxaban has similar coagulation effects in the cat as in other species and may play a role in feline thromboprophylaxis. Kaolinactivated TEG does not appear to be sensitive to low concentrations of rivaroxaban in the cat. Anticoagulated blood is required for platelet function studies. Sodium citrate, a calcium chelater, is the most commonly used anticoagulant to measure coagulation parameters including platelet aggregation but it may have a negative effect on platelet responsiveness. Dogs are generally considered moderate responders to collagen on platelet aggregation and are notorious for being poor or inconsistent responders to ADP-induced platelet aggregation using citrated whole blood. Hirudin, a selective thrombin inhibitor, can also be used as an anticoagulant for coagulation assays and is the anticoagulant of choice for certain assays including the Multiplate s platelet function analyzer.

Ten adult healthy dogs were used to compare whole blood platelet aggregation between citrated and hirudinated blood samples. Venous blood was collected atraumatically from the external jugular vein directly into tubes containing 3.2% trisodium citrate or hirudin. Whole blood platelet aggregation was performed (Whole-Blood Lumi-aggregometer, Chrono-log Corporation, Havertown, PA, USA) with collagen (5 mg/ml) and ADP (10 mM) as agonists. Maximal platelet aggregation (Ohms) was recorded.

There was a significant increase in collagen-induced platelet aggregation from the hirudinated samples compared to the citrated samples (31.2 AE 6.1 vs. 17.2 AE 8.6 O, p o 0.0005). There was also a significant increase in ADP-induced platelet aggregation from the hirudinated samples compared to the citrated samples (15.6 AE 6.0 vs. 2.6 AE 2.6 O, p 5 0.0001).

The results of this study show a significant difference in platelet responsiveness between citrated and hirudinated whole blood using the Chrono-log impedance aggregometer. While both collagen and ADP-induced platelet aggregation was attenuated from citrated blood samples, this was most notable for ADPinduced aggregation where almost all samples had no objective measurable platelet aggregation. It is suggested from this data that future whole blood platelet aggregation studies performed on the Chrono-log impedance aggregometer should use hirudinated blood samples although new reference limits would need to be established. Low-molecular-weight heparin (LMWH) is now used to prevent thrombotic complications in dogs. A functional assay such as the Calibrated Automated Thrombogram (CAT) may provide a new approach for monitoring LMWH therapy.

We hypothesized that CAT would detect decreased endogenous thrombin potential (ETP) in healthy dogs receiving LMWH (Fragmin s ).

Twenty-four healthy adult Beagles were included in this study and divided equally in four groups.

One dose of 50 U/kg, 100 U/kg or 150 U/kg of LMWH were given subcutaneously to healthy dogs and compared to a control group. Platelet poor plasma (PPP) was collected over a 24 hour period.

Using a repeated-measure linear model, effect of LMWH on ETP was time and dose dependent with a significant interaction (p o 0.0001). Compared to control dogs, significant differences were obtained for group 50 U/kg at T60 (p 5 0.037), for group 100 U/kg at T15 (p 5 0.013) and between T30-T240 minutes (p o 0.0001) respectively, and for group 150 U/ at T15 (p 5 0.011), between T30-T300 minutes (p o 0.0001) respectively and at T360 (p 5 0.004 The CAT assay can be employed to measure the effects of LMWH at different doses in healthy dogs, resulting in significant time and dose-dependent decreases in ETP and warrants further investigation as a tool for monitoring LMWH therapy in dogs. The purpose of this study was to determine the effects of prednisone and prednisone plus ultralow-dose aspirin on coagulation parameters in healthy dogs, with an emphasis on thromboelastography (TEG).

This was a prospective, randomized, blinded study utilizing fourteen dogs determined to be healthy based on normal physical examination, complete blood count, biochemistry, urinalysis, and fecal floatation. Dogs were evenly divided into either prednisone plus aspirin (PA) or prednisone plus placebo (PP) groups. Baseline values for TEG parameters (R, K, angle, MA, Ly30, Ly60, G, CI) were measured twice two days apart, and thrombin-antithrombin complexes (TAT), and traditional coagulation parameters (prothrombin time, activated partial thromboplastin time, d-dimer, antithrombin (AT), fibrinogen) were measured once. Each dog received 2 mg/kg/ day of prednisone, and either 0.5 mg/kg/day of aspirin (PA group) or placebo (PP group) for 14 days. A complete blood count, biochemistry profile, TEG, TAT, and traditional coagulation parameters were then repeated on each dog. Day to day variation was calculated for the TEG parameters using the two baseline measurements. The change from baseline between and within each group were compared using t-tests, or Wilcoxon 2 sample test where appropriate, for TEG, TAT, traditional coagulation parameters, and hematocrit.

Day to day variation in TEG was acceptable ( 10%) for MA, G, and angle, unacceptable (4 10%) for R, K, Ly30 and Ly60, and not meaningful for CI. Within both groups, MA, G, CI and fibrinogen significantly increased from baseline (p o 0.05). Within both groups, Ly30 and AT significantly decreased from baseline (p o 0.05). For the PP group, Ly60 significantly decreased from baseline (p 5 0.03), and approached significance for the PA group (p 5 0.0504). All other within group changes from baseline were not statistically significant (p-values 4 0.05). For all parameters, there was no difference between groups for change from baseline (p values 4 0.05).

Day to day variation in some TEG parameters is high and may preclude their clinical utility. Prednisone causes hypercoagulability in healthy dogs based on increased G, MA, and CI. The addition of ultra-low dose aspirin to prednisone has no effect on the parameters measured in this study. 'Aspirin resistance' has been identified in people and dogs that develop thrombi despite low dose aspirin therapy. Variability in platelet cyclooxygenase (COX) isoform expression is one proposed mechanism for aspirin resistance in people. Two isoforms (COX-1 and COX-2) have been identified in canine platelets. High (antiinflammatory) dose aspirin inhibits platelet function and alters expression of both COX isoforms in most dogs. This study evaluated the effects of low dose aspirin on platelet function and COX expression in normal dogs.

Twenty-five healthy client-owned dogs were evaluated before and at two time points (Days 3 and 10) during aspirin therapy (1 mg/kg PO SID). Platelet response to aspirin (Siemens PFA-100 s ; collagen/ epinephrine cartridges), was stratified into one of three groups [aspirin responders (9 dogs), non-responders (8 dogs), or inconsistent responders (8 dogs)].

Flow cytometry identified platelet COX-1 and COX-2 expression. An ELISA was used to measure urine 11-dehydro-thromboxane B 2 (11-dTXB 2 ). There were no significant differences between groups for COX-1, COX-2 or 11-dTXB 2 at any time point. When all dogs were considered as a single group, there was a significant increase (p o 0.0001Ã) in COX-1 and COX-2 mean fluorescent intensity (MFI) from baseline to Day 10, 70.1% AE 38.0 (mean AE SD) and 70.8% AE 71.2, respectively. There was a significant decrease in mean urine 11-dTXB 2 :creatinine on Day 3 and 10 by 23.4% (p 5 0.0044 Ã ) and 45% (p o 0.0001 Ã ).

As with our previous high dose studies, COX-1 expression was increased with aspirin exposure. However, there was a significant increase in COX-2 expression with low dose aspirin in contrast to the decrease seen at higher doses. Our study suggests that levels of platelet COX-1 and COX-2 expression do not influence aspirin response in dogs. Although thromboxane levels decreased in most (23 of 25) dogs on low dose aspirin, platelet function was consistently affected in only 36% of dogs, suggesting that differences in response to thromboxane may play a role in the variable affects of low dose aspirin on canine platelet function. Delayed postoperative bleeding is common in retired racing Greyhounds (RRGs), despite normal results of routine hemostasis assays. The excessive postoperative bleeding in the RRGs is not due to primary or secondary hemostatic defects, and may be due to enhanced fibrinolysis or to a clot maintenance dysfunction. Providing a method to prevent or minimize the severity of postoperative bleeding in RRGs will not only have major economic impact for owners, but also will markedly decrease the associated complications of minor or major surgeries in the breed.

Epsilon aminocaproic acid (EACA) is a potent inhibitor of fibrinolysis that also supports clot maintenance due to unknown mechanisms.

The objective of this double-blinded, prospective, randomized study was to evaluate the effects of EACA versus placebo on the prevalence of bleeding in RRGs, and to investigate its mechanism of action by using thromboelastography (TEG).

We compared the effects of EACA and placebo in 100 RRGs that underwent elective ovariohysterectomy or orchiectomy at the Veterinary Medical Center, The Ohio State University during 2 years. The main endpoint was bleeding (prevalence and severity); minor endpoints included most TEG parameters.

Thirty percent (15/50) of the RRGs in the placebo group had delayed postoperative bleeding starting 36 to 48 hours after surgery, compared to 10% (5/50) in the EACA group (P 5 0.0124).

On the TEG parameters, the R time (clot formation time) was significantly different between treatment groups (P 5 0.0321). The postoperative administration of EACA significantly decreased the prevalence of postoperative bleeding in RRGs. Thromboembolism associated with protein losing nephropathy (PLN) has been long recognized as a serious and unpredictable complication in dogs, however its prevalence remains unknown. In humans, surrogate indicators are frequently used to assess thromboembolic risk. This study aimed to investigate the prevalence of hypercoagulability in PLN dogs based on thromboelastography (TEG), and to determine whether hypercoagulability in these patients could be predicted by clinical assessments that identify systemic hypertension (systolic blood pressure 4 160 mmHg), hypoalbuminemia (serum albumin o 2.7 mg/dl), antithrombin activity (o 70%), and degree of proteinuria (urine protein:creatinine ratio [UPC] ! 2). Between March 2009-September 2010, twenty-seven dogs were identified with PLN at the Animal Medical Center. The prevalence of hypercoagulability based on a TEG G-value 4 9.6 was 83.3%. There was no statistically significant relationship, either categorically or continuously, in univariate as well as multivariate analyses of all variables. Univariate logistic regression (odds ratio; lower and upper confidence limit; P value) for hypertension was À1.18; 0.201, 6.99; 0.851; for albumin -1.37; 0.228, 8.26; 0.728; and for antithrombin activity -0.73; 0.12, 4.39; 0.728. Thus, in this patient population, in the absence of TEG, prediction of hypercoagulability using abnormalities in commonly measured clinicopathologic variables was not helpful. However, given the documented high prevalence of hypercoagulability in patients with PLN, early institution of prophylactic anti-platelet or anticoagulant therapies should be considered. Thromboelastography (TEG) is a test of global hemostasis. Due to the effects of extrinsic factors on whole blood coagulation, sample collection method (SCM) may influence results. The purpose was to determine if SCM influenced TEG using kaolin-activated citrated whole blood (WB).

Healthy dogs with normal platelet counts were prospectively enrolled. Three WB samples were obtained from each dog at least 48 hours apart from alternating jugular veins in a randomized order of three methods: 1) vacutainer s into citrated tube (VAC), 2) citrated syringe with transfer into plain tube (CIT), or 3) plain syringe with transfer into citrated tube (PLAIN). Draw time was recorded in seconds. Kaolin-activated TEG was performed, with measurement of reaction time (R), clot formation time (K), maximum amplitude (MA), and alpha angle.

Eleven dogs were enrolled. There were no significant differences in TEG indices between VAC samples and either CIT or PLAIN samples. CIT samples had a significantly higher K value (p 5 0.004) and a lower alpha angle (p 5 0.004) compared to PLAIN samples. Draw times ranged from 2-10 seconds. A longer draw time was significantly correlated (p 5 0.046; R 5 À0.35) with a shorter R time. Higher platelet count was significantly correlated (p 5 0.001; R 5 0.575) with a higher MA.

SCM did not have a significant effect on TEG parameters when comparing VAC samples to either CIT or PLAIN samples. Minimizing sample collection time and trauma during venipuncture may be important in minimizing hypercoagulable changes in TEG indices. Liquid plasma (LP) is defined as either plasma collected and refrigerated immediately after collection or fresh frozen plasma (FFP) that is thawed and stored refrigerated until use. Stability studies in people have shown that adequate clotting factor activity is preserved for at least 14 days. LP is transfused in human Level I trauma centers to critically ill people requiring rapid infusion of clotting factors as the time required to defrost FFP is considered prohibitive. The use of LP has not been described in veterinary critical care. The purposes of this study were to 1) determine the length of time required in a water bath for a unit of canine FFP to thaw and 2) describe the use of LP in a busy university emergency room (ER).

For part 1: Six units (250 ml) of canine FFP were individually thawed in a 371C water bath. The duration of time (in minutes) to thaw was recorded.

For part 2: The transfusion log was reviewed for dogs receiving LP in the last 6 months. The indications and outcome were recorded.

The mean time AE SD thaw time was 34.7 AE 1.38 minutes. Ten units of LP were transfused to 7 critically ill or injured dogs during the study time. Indications for LP transfusion included hypovolemic shock due to intra-abdominal hemorrhage in 6 dogs (2 traumatic, 4 non-traumatic) and rapid correction of hemorrhage following parenteral tissue plasminogen activator administration in 1 dog. LP volume transfused ranged from 11.1 to 50.5 ml/kg. No transfusion reactions were identified. Effect on coagulation was not consistently evaluated.

Time required to thaw a unit of FFP is greater than 30 minutes which could be detrimental in a bleeding, coagulopathic dog. LP was transfused without incident to critically ill and injured dogs and represents a potential new addition to the armamentarium of treatments in a veterinary ER setting. Further investigation of canine LP is warranted including evaluation of in vitro factor stability and in vivo efficacy in correcting coagulopathy. Immune mediated thrombocytopenia (IMT) is associated with increased morbidity and mortality. Large prospective research studies in dog platelet antibodies and clinical utilization of platelet immunoglobulin assays are limited. Potential explanations include limited availability and low specificity due to nonspecific binding. The focus of this study is to evaluate optimized direct and indirect platelet surface associated immunoglobulin (pSAIG) and staining with anti-CD61 antibodies (CD61ab) for the utilization in classifying thrombocytopenic dogs.

One hundred clinically ill and 30 apparently healthy dogs were prospectively evaluated. Data collected included a history of hemorrhage, physical examination evidence of bleeding, complete blood count, and measurement of pSAIG and CD61ab. The pSAIG assay utilized polyvalent antibodies with correction for non-specific binding by subtraction of background fluorescence with control antiserum. Thrombocytopenia was defined as o 164,000/mL and all dogs were clinically classified into 1 of 4 groups (g): g1 IMT, n 5 45, g2 Thrombocytopenia from non-immune mediated diseases, n 5 37, g3 Ill with normal platelet counts, n 5 18, g4 Healthy dogs, n 5 30.

Median platelet counts, by groups, were g1, 20,000; g2, 69,000; g3, 324,500; and g4, 212,000/mL. For the direct and indirect pSAIGs in dogs with ITP (g1), more dogs (n 5 6 and n 5 9) with clinical evidence of bleeding had antibodies compared to those who were not bleeding (n 5 3 and n 5 6). Considering only direct pSAIG the sensitivity and specificity was 13% and 95%, respectively for the diagnosis of IMT. For indirect pSAIG the sensitivity and specificity was 27% and 97%, respectively, for the diagnosis of IMT. When considering both direct and indirect pSAIG together, the sensitivity was 33% with a specificity of 93%. In g1 interference from high control antiserum background staining was noted in 26.7% of dogs and resulted in a negative direct pSAIG classification. Minimal background interference was noted in g2, g3, or g4. The percentage of platelets stained with CD61ab was significantly less in g1 (median 38, p 5 0.0001) vs. g2 (median 74, p 5 0.007) vs. g3 (median 95.1, p 5 0.857) and g4 (median 95.6).

These findings indicate the optimized platelet surface associated immunoglobulin assay has a high specificity, however poor sensitivity, for the diagnosis of IMT. The decreased CD61 staining in g1 (IMT group) may reflect decreased surface gpIIIa expression, blocking by anti-gpIIIa antibodies or other proteins, clearance by macrophages, or increased non-platelet debris and has potential applications in the diagnosis and treatment of IMT. Greyhounds have lower serum concentrations of a-globulin than other breeds, explained by negligible levels of haptoglobin (HP) measured using different methods (colorimetric, immunoturbidimetric and protein electrophoresis). The purpose of the present study was to characterize the HP gene in Greyhounds.

We isolated DNA and RNA from blood samples of AKC-registered and retired racing Greyhounds (AKCG, RRG), and a German Shepherd Dog (GSD). We sequenced the HP exons and splice sites, and conducted array comparative genomic hybridization to identify associated DNA structural variation (custom 1M Agilent oligonucleotide array). Additionally, we tested for the presence of one or multiple haplotypes spanning HP in Greyhounds using a high density SNP array (180k Illumina HD). Sequencing results of HP in both DNA and cDNA revealed three synonymous SNPs in the racing Greyhound. We did not identify structural variation overlapping or near the HP gene. Notably, we detected that the RRG and AKCG do not appear to share a specific haplotype spanning HP.

Despite having low or undetectable serum concentrations of HP, we did find that RRG HP mRNA is expressed and lacks amino acid variation. This suggests that the clinical absence of the HP is attributable to post-transcriptional HP effects or to an unknown physiological interaction. Finally, given the existence of distinct RRG and AKCG haplotypes spanning HP, it is important to characterize serum levels of HP in AKCG in follow on studies. We reported that hemoglobin in retired racing Greyhounds (RRG) has higher oxygen carrying properties and affinity than other breeds. Surprisingly, very little is known about canine hemoglobin genetics. The purpose of this study was to characterize genetics of canine beta globins.

Using computational BLAST analysis of the dog genome, we identified five beta globin genes in a single locus: two human HBElike followed by three HBB-like genes. We isolated DNA and RNA from blood of RRGs, AKC registered Greyhounds (AKCG), and German Shepherd Dog (GSD). All beta globin exons and splice sites were sequenced, and the beta globin locus was examined by array comparative genomic hybridization (custom 1M Agilent array). Additionally, we determined the number of common haplotypes that span this locus in RRGs and AKCGs using high density SNP array (180k Illumina HD).

Expression and sequence analysis of cDNA showed all five beta globin genes are actively expressed in adults. CanHBB1 and 2 were created by relatively recent segmental duplication and have identical protein sequence. CanHBB1/2 are abundantly expressed in adults; CanHBB3 is expressed at greatly reduced levels. Sequencing results revealed one rare non-synonymous single nucleotide polymorphism (SNP) in HBE1 of RRGs, but no variation that could explain their abnormal hemoglobin. We did not detect structural variation overlapping or near the beta globin locus. Notably, RRG and AKCG do not share haplotypes spanning the beta globin locus. This is the first characterization of canine hemoglobin genetics, and the first report of canine embryonic hemoglobins and their expression in adults. Sampling of the bone marrow in the dog from the costochondral (CC) junction can be performed with minimal to no sedation and readily available equipment but is not in widespread use in the United States. The aim of this study was to compare the number of attempts needed to successfully obtain a sample, the time needed for the procedure, and the sample quality between aspirates obtained from the CC junction and more traditional sites (humerus or femur) in healthy dogs when performed by novice and seasoned practitioners.

Samples were obtained from healthy anesthetized laboratory reared adult dogs after undergoing terminal endoscopic surgery. Paired samples from separate dogs were obtained by each practitioner using either a 22 gauge needle and 6 cc syringe at the CC junction or an 18 gauge Rosenthal needle and 12 cc syringe from either the proximal humerus or femur (clinician preference). Three small animal veterinary interns, one experienced technician and one boarded internist were monitored for number of attempts to success and length of time needed for success of each procedure. Slides were prepared by a single investigator and read by a blinded clinical pathologist. Data were compared using the paired t-test for normally distributed data and Wilcoxen signed rank test for non-Gaussian distributions.

Five pairs of samples from three dogs were evaluated. Two dogs had two pairs drawn from opposite limbs and ribs. Mean number of attempts to success for traditional sampling sites (1.4 1/À0.54) and time to success (8.6 minutes 1/À5.2) did not differ significantly from attempts (2.01/-0.70, p 5 0.25) or time (2.7 1/-0.98, 0.06) needed when aspirating from the CC junction. Subjectively, samples were of similar quality with regards to cellularity and number of particles present when compared within practitioners. Myeloid: erythroid ratio and percentage of lymphocytes were also not significantly different between sites (M:E ratio p 5 0.37, lymphocyte % p 5 0.07) and were within normal limits.

While there were no significant differences between the two sites in terms of number of attempts or time to success, it should be noted that the ''seasoned'' practitioners had never performed an aspirate at the CC site and had an increased number of attempts compared to the traditional sites. If the number of attempts needed for success decreases with experience, it is likely the time required would decrease as well. Both subjectively and objectively, there were no significant differences in quality or cell populations between the two sampling sites in healthy dogs. Based on this data, bone marrow aspiration from the CC junction appears to be equivalent to more traditional sampling sites in healthy dogs. Larger studies in clinically ill dogs should be performed before routinely using the site in the clinical setting. Recent research on iron homeostasis has elucidated the tightly controlled intestinal uptake of iron. Hepcidin, the major hormone limiting iron absorption and release from macrophages, is downregulated by matriptase-2, a transmembrane serine protease (Tmprss6) produced by the liver. While iron deficiency is commonly caused by chronic blood loss anemia and rarely dietary deficiency or intestinal disorders in dogs and other species, we report here the clinical to molecular investigations of a dog with iron-refractory iron deficiency anemia (IRIDA) caused by a matriptase-2 deficiency homologous to a recently described autosomal recessive disorder in humans.

The proband, a spayed female Cocker Spaniel without any clinical signs except for recent occasional idiopathic seizures, exhibited a lifelong history of microcytosis and hypochromasia but not anemia. There was no evidence of any blood loss and the dog was receiving an appropriate meat-based diet. Mean values of complete blood cell counts, performed from 0.5-5 years of age, were: hematocrit 41% (normal reference range 39-56); RBC count 9.6 Â 10 6 /mL (5.8-8.5 x 10 6 ); MCV 43 fL (62-72); MCHC 31 g/dL (33-36). Serum iron parameters revealed severe iron deficiency with serum iron 34 mg/dL (88-238); Total Iron Binding Capacity 378 mg/dL (246-450); serum iron saturation o 9% (15-50%), and ferritin 410 ng/dL (80-800). Prolonged courses of oral ferrous sulfate supplementation and several short courses of intramuscular (Dextran) injections and intravenous iron infusions did not result in improvement of any red cell or serum iron parameters. However, this dog was never anemic and the partial seizures could not be directly related to the iron deficiency status. No family members were available for further studies.

Genomic DNA was extracted from the proband's EDTA blood and the exons of the TMPRSS6 gene were amplified with flanking primers and then sequenced. In comparison to the normal canine TMPRSS6 sequence and that of a sequenced control dog we found a homozygous missense muation, R723H, toward the C-terminal end of the protein in the proband's gene.

In conclusion, the severe microcytosis and hypochromasia, low serum iron parameters and lack of a response to oral and parenteral iron therapy led to the diagnosis of IRIDA. The missense mutation in the matriptase-2 at position 723 from an arginine, which is conserved across all species currently deposited in the Genbank, to a histidine is likely the disease-causing mutation. This is the first report of an IRIDA in the dog with features very similar to those observed in humans. Dogs with naturally-occurring IRIDA may be helpful in developing and assessing novel therapies. Accidental ingestion of copper-coated zinc pennies minted after 1982 is the most common causes of zinc toxicity anemia in the dog. Zinc toxicity anemia may also be seen with ingestion of zinc from other sources as ingestion of metallic foreign material other than pennies, medicines containing zinc, and zinc supplements. The purpose of this study was to determine if there is a weight below which dogs are more susceptible to zinc toxicity anemia secondary to metallic foreign body ingestion. Records of dogs presented to the Internal Medicine service at the Veterinary Medical Center of Long Island for metallic foreign body ingestion were reviewed for signalment, weight, presenting PCV, and type of metallic foreign body ingested. Eighteen dogs met the inclusion criteria and were compared. Of the 18 dogs, there were 15 cases of coin ingestion (83%), with 11 (73%) involving ingestion of 1 or more pennies. The other 3 cases involved ingestion of a metallic object (1), decorative garland (1), and BB pellets (1).Of the 14 dogs exposed to zinc, 13 (93%) were less than 27 pounds (12.3 kgs). Of those 13 cases 11 (85%) had ingested one or more pennies. Eleven out of the 14 (79%) zinc exposure dogs were anemic at presentation. The average weight of the 11 dogs was 17.5 pounds (8 kg). This study showed that dogs less than 27 pounds appear to be more susceptible to developing anemia secondary to zinc toxicosis, with the majority of cases due to ingestion of pennies minted after 1982. Zinc toxicity anemia secondary to penny ingestion is more commonly seen in small dogs. We suspect larger dogs are able to pass pennies through the pyloric sphincter and thus not develop clinical signs. Although thrombocytopenia is common in hospitalized dogs, canine cryopreserved platelet concentrate (PC) is used infrequently. Data suggest in vitro efficacy of PC and when administered to research dogs, but efficacy is unknown in clinical patients. Study objectives were to determine clinical characteristics of dogs receiving PC as well as safety and efficacy of PC in thrombocytopenic dogs.

Medical records were evaluated retrospectively to identify dogs that received PC. Information evaluated included patient characteristics, platelet count, acute transfusion reactions, and survival. Twenty six dogs met study criteria. Dogs receiving PC ranged in age from 1-13 years (mean 7.8 years) and 17/26 (65.4%) were spayed or intact females. Hemorrhage was reported in 20/26 dogs (76.9%) prior to PC transfusion. Platelet counts prior to transfusion ranged from 0 to 77 Â 10 3 /ul (mean 29.9 1/À43.9 Â 10 3 /ul). Change in platelet count was measured in 23 dogs and the mean change was17.1 1/À 45.5 Â 10 3 /ul. Dose of PC administered ranged from 4.8 to 40 ml/kg with a mean of 14.5 1/À 8.8 ml/kg. No acute adverse reactions were reported. There was no correlation between transfusion dosage and platelet count change post transfusion. Survival to discharge occurred in 15/26 (57.7%) of dogs. The only variable correlated with survival was age with survivors being younger than non-survivors (6.4 years-old AE 3.9 vs. 10.1 years-old AE 3.1.; p 5 0.02).

Efficacy of cryopreserved PC transfusions for improving clinical outcome in dogs with thrombocytopenia is yet to be determined; however, PC is well tolerated in clinical patients. Fresh frozen plasma (FFP) is used to treat coagulopathies in dogs. Current transfusion guidelines recommend that FFP be administered within 4 hours of thawing to avoid decreasing clotting factor function and bacterial contamination. The purpose of this study was to assess clotting factor activity and bacterial contamination of FFP that had been thawed and refrigerated for 5 days.

Blood was collected from 10 client-owned healthy dogs with no known history of coagulopathy or of administration of drugs affecting coagulation. Plasma was separated from whole blood and frozen (À201C) within 30 minutes of collection. Thawed plasma was maintained at 41C (1/À21C). Aerobic and anaerobic bacterial culture, prothrombin time (PT), activated partial thromboplastin time (PTT), and factor II, VII, IX, and X analyses were tested at time of whole blood collection, FFP thaw, 24 hours post-thaw, 72 hours post-thaw, and 120 hours post-thaw.

There were no statistically significant differences in PT and PTT at any of the measured time points. Statistically significant differences occurred between initial measurements of factors II, VII, IX, and X and subsequent time points, but there was no difference in activity levels of the factors once FFP was thawed. One bacterial colony was grown from each of two samples from post-thaw plasma.

Thawed plasma protocols do not significantly decrease the function of factors II, VII, IX, and X or prolong PT and PTT. Bacterial contamination of the plasma supply seems unlikely, but strict aseptic technique should be used when obtaining plasma for patient use. Erythrocyte pyruvate kinase (PK) deficiency is the first and most common erythroenzymopathy described in dogs, cats, and humans. The PK enzyme plays a crucial role in the erythrocyte energy metabolism and its absence causes severe hemolytic anemia, often misdiagnosed as autoimmune hemolytic anemia. The disease is inherited as an autosomal recessive trait and affected dogs also develop osteosclerosis. In dogs, the enzymatic diagnosis is complicated by the anomalous expression of malfunctioning M 2 -PK expression, but breed-specific R-PK mutation tests have been reported for Basenjis, West Highland White Terriers (WHWT), and Beagles. We report here on a survey of canine PK deficiency studied at the PennGen Laboratory.

A biased group of samples were received for screening from dog breeds with known mutations as well as from dogs with chronic, prednisone-and antibiotic-resistant hemolytic anemia and their relatives. EDTA blood samples and/or cheek swabs as well as medical record information were received and genomic DNA and/ or enzyme activity testing were performed.

Among the 237 WHWTs 7% and 37% were found to be homozygous deficient dogs or carriers, respectively, with a mutant allele frequency of 0.26. The average age at the time of diagnosis was 1.5 years ranging from 2 months to 5 years of age; some samples came from Europe and South America. Of the 67 Beagles studied, 36% were affected and 3% were carriers (mutant allele frequency 0.37). The average age at the time of diagnosis was 2 years ranging from 7 months to 9 years. Surprisingly, very few samples from Basenjis were received for screening, and none showed the mutant allele. While PK-deficient Basenjis lived o 5 years, WHWT and Beagles often show milder signs and can reach 9 years of age.

Several dogs from other breeds were also examined because of chronic regenerative anemia and none had any of the known mutations seen in the other breeds. However, based upon PK enzyme activity studies, Chihuahua, Dachshund, Miniature Poodle, Spitz, Eskimo Toy, and Labrador Retriever dogs were found to be affected; they also had osteosclerosis and at least one Labrador Retriever developed severe hemochromatosis (hepatic iron 37,300 ppm; normal o 1,200 ppm, analyzed on a dry weight basis). Moreover, sequencing of the R-PK cDNA from a PK-deficient Labrador Retriever revealed a new nonsense mutation in exon 6.

In conclusion, PK deficiency appears to be a common cause for hemolytic anemia in certain breeds, and mutation testing makes screening simple. PK deficiency should also be considered in dogs of other breeds which may require the more cumbersome enzyme testing. Studies to identify new mutations will confirm and simplify the diagnosis. Supported in part by NIH grant RR02512. Immune-mediated hemolytic anemia (IMHA) is a common hematological condition observed in dogs. The diagnosis is based on clinical history, presenting signs and hematological evidence of IMHA such as regenerative anemia, leucocytosis and presence of spherocytes. The definitive diagnostic procedure is the Coomb's test (direct antiglobulin test, DAT) which is known to be highly specific but lacks diagnostic sensitivity. Direct flow cytometric assay (FCA) for IgG, IgM or C3 coated red blood cells (RBCs) detection might be more sensitive and thus could be introduced as an alternative diagnostic tool.

To investigate the usefulness of FCA for IMHA diagnosis, evaluation of IgG, IgM or C3 coated RBCs was performed from 15 dogs presented at the Veterinary Hospital at USP that fulfilled clinical and hematological criteria for IMHA. Thirty eight healthy dogs were included as controls.

DAT was performed with polyvalent and monovalent anti-dog sera with twofold serial dilutions of each one, incubated with 2% RBCs suspension at 371C and 41C. For FCA, 2% RBCs from anemic and healthy dogs were incubated with FITC anti-dog IgG, anti-dog IgM and anti-dog C3 and submitted to flow cytometry evaluation. Specific software and Mann Whitney U test were used for data analysis.

Five dogs showed positive results for DAT with polyvalent Coombs reagent at 41C (titer 64 to 4096) and 371C (titer 64 to 2048) but only three of them had agglutination titer for anti-IgG at 4 0 C (1024 to 8192) and 371C (512 to 4096). No positive results were observed for anti-IgM and anti-C3 DAT. By FC, percentage of IgG, IgM and C3 coated RBCs in normal and anemic dogs were, respectively, 1,18% and 17,11% (p o 0,001); 1,15% and 21,29% (p o 0,001); 0,66% and 6,99% (p o 0,001). IgG coated RBCs percentage were higher in dogs showing DAT positive results (min. 33,94%; max. 99,96%; median 54,25%).

Direct flow cytometric erythrocyte immunofluorescence assay is more sensitive than DAT for detection of antibodies coated RBC in anemic dogs and may provide quantitative data useful for laboratorial diagnosis of IMzHA. Bone marrow aspirates from cats are typically obtained from the ilium, humerus or femur, but may be difficult to obtain and/or of poor quality. In this study the feasibility, safety, and nature of sternal aspiration in cats was investigated. Under general anesthesia, bone marrow aspirates were obtained in a randomized order by a single investigator from the sternum and ilium of 10 healthy cats weighing 3.4-8.4 kg, with body condition scores of 5-9 (on a scale of 1-9). For sternal aspirates, cats were positioned in sternal recumbency and a 1-inch, 22-23 ga hypodermic needle attached to a 12cc syringe was inserted into the cranial manubrium and directed caudally along the long axis of the sternum. Aspirates were also obtained from the right iliac crest using an 18 ga Illinois needle attached to a 12cc syringe. Difficulty of site localization, needle insertion and advancement, and specimen aspiration, were scored from 1 (easiest) to 5 (hardest). Bone marrow smears were prepared by one investigator and reviewed by a pathologist blinded to aspiration site and cat. Sample quality was scored from 0 (no marrow particles) to 5 (excellent) based on the number of wellsmeared marrow particles on the slide. Particle cellularity was scored from 1 (4 75% fat) to 3 (o 25% fat). Post-procedure, cats were treated with tramadol (4-5 mg/kg, PO, q12h) for 3 days, and assessed for post-biopsy pain (Colorado State University Feline Acute Pain Scale, range 0 [no pain] -4 [maximum]) and site swelling (range 0 [none] -3 [marked]). Data were analyzed by ANCOVA accounting for effects of weight and body condition score.

Pneumothorax was not identified. It was significantly easier to perform sternal than iliac aspiration, but the quality of the sample was significantly better for iliac than for sternal aspirates. Because of limitations due to sample quality, bone marrow morphology in sternal samples could not be compared to iliac samples in all cats. For samples that could be compared, cellularity was identical for sternal and iliac samples from 1 cat but underestimated in the sternal sample from another cat. Myeloid:erythroid ratios and lymphocyte numbers were the same for sternal and iliac samples in 2 and 3 cats, respectively. Megakaryocyte numbers were the same in one sample, less in sternal samples compared to iliac samples from 2 cats, and overestimated in the sternal sample from 1 cat. Bone marrow cell morphology was normal in all acceptable samples. It was concluded that sternal aspiration of bone marrow using a 22-23 ga hypodermic needle is 1) easier to perform than iliac aspiration; 2) safe; but 3) provides samples of lower quality than iliac aspiration in cats. The diameter of 11-13ga Jamshidi-type needles makes bone marrow core biopsy difficult in cats. In this study, biopsies of the left humeral head were taken under anesthesia using a 1-inch, 15ga needle (EZ-IO s Intraosseous Infusion System, Vidacare) from 10 healthy cats weighing 3.4-8.4 kg with body condition scores of 5-9 (on a scale of 1-9). Humeral biopsies were compared to biopsies taken from the left iliac crest using a 2-inch, 13ga Jamshidi needle. Biopsies were performed in randomized order by one investigator. Biopsy was repeated to a maximum of 3 attempts until a specimen ! 5 mm long was obtained. Difficulty of site localization, needle insertion and needle advancement were scored from 1 (easiest) to 5 (hardest). Specimens were wrapped in tissue paper and placed in Davidson's fixative for 15 min and then transferred to formalin. Biopsy sections were reviewed by a pathologist blinded to biopsy site and cat. Biopsy length on the slide was measured, and biopsy quality was scored from 0 (no hematopoietic tissue) to 5 (! 5 intertrabecular spaces free of artifact). Post-procedure, cats were treated with tramadol (4-5 mg/kg, PO, q12h) for 3 days, and assessed for postbiopsy pain (Colorado State University Feline Acute Pain Scale, range 0 [no pain] -4 [maximum]) and swelling of biopsy sites (range 0 [none] -3 [marked]). Data were analyzed by ANCOVA accounting for effects of weight and body condition score.

There were no significant differences between 15ga and 13ga biopsies except for post-biopsy swelling, and there were no significant effects of body weight and body condition.

Six (60%) of 15 ga and 5 (50%) of 13ga biopsies were considered acceptable specimens for assessment of bone marrow architecture and morphology; all intact spaces in these biopsies had normal hematopoiesis and cell morphology. Comparison of acceptable 15 ga to 13 ga biopsy specimens from 4 cats showed no significant differences for cell density and lymphocytes/plasma cells, while cellularity, assessed as high in 2 of the 13 ga biopsies, was assessed as medium in corresponding 15ga biopsies; and megakaryocytes, assessed as 4-9/low-power field in one 13ga biopsy, were assessed as 3/low-power field in the 15 ga biopsy. Myeloid:erythroid ratios were greater in 15 ga biopsies compared to 13 ga biopsies in 2 cats, and less in the 15 ga biopsy in one cat. Discordant results between biopsies were not related to differences in quality.

In conclusion, 15 ga bone marrow biopsy of the humerus was as likely to yield a specimen of acceptable quality as was 13 ga biopsy of the ilium, and resulted in less post-biopsy swelling. Reports on canine acute liver failure (ALF) include individual or small case series of animals with a specific diagnosis. The aim of this study was to describe the clinical course, outcome and etiology of ALF in dogs presenting to a referral hospital. Medical records (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) were reviewed for a diagnosis of ALF (elevated serum bilirubin or icterus with concurrent coagulopathy or hepatic encephalopathy (HE)). Fifty cases were identified representing 22 breeds: 7 Labradors retrievers, 6 Golden retrievers, 3 German shepherds, and 3 Cocker spaniels. Median age was 6 years (1 m to Humerus, 15 ga 5.1 AE 0.9 5.6 AE 0.6 2.1 AE 0.5 6.0 AE 0.7 0 0 Ã (2.2-9.3) (3-9) (0-4) (3-10) Ilium, 13 ga 6.1 AE 0.6 9.0 AE 0.7 1.9 AE 0.6 7.5 AE 0.8 0.2 AE 0.1 0.9 AE 0.1 Ã (4.5-9.5) (6-12) (0-5) (5-12) (0-1) (0-1) 13 yrs). Presenting signs included anorexia (31/50), vomiting (26/50), polydipsia (9/50) and neurologic signs (6/50 Granulomatous hepatitis (GH) is a histopathological diagnosis characterized by focal aggregations of activated macrophages mixed with other inflammatory cells that is usually part of a systemic disease process (WSAVA). Published case reports describe many potential infectious causes, but only one retrospective study involving nine dogs with GH has detailed clinically relevant findings. The aims of this study were to describe the clinical and clinicopathologic findings in dogs with a histopathological diagnosis of GH, and to identify infectious agents using differential staining techniques, PCR, and fluorescence in-situ hybridization (FISH) in archival paraffin-embedded tissues from dogs with GH.

Medical records of dogs with a histopathological diagnosis of GH (n 5 22) were reviewed and signalment, historical toxin exposure or evidence of other systemic diseases, clinical signs, physical exam findings, clinicopathologic test results, imaging findings, concurrent diagnoses, treatments administered, and case outcome, when available, were extracted and summarized. Twelve archival formalin-fixed, paraffin-embedded hepatic tissue samples were available for special staining and molecular diagnostic testing. Two of these samples had sufficient tissue for only PCR. The mean age of dogs with GH was 7 years (median 6.5 years; range 2 to 17 years) and included 12 males and 10 females representing 14 different breeds. Common presenting complaints included inappetance or anorexia (n 5 12), weight loss (n 5 11), lethargy (n 5 9), fever (n 5 8), and vomiting (n 5 8). High mixed liver enzyme activity (14/22) was the most common clinicopathologic abnormality. Leukemia was diagnosed in one dog and copper-associated hepatopathy in 6 dogs. No infectious agents were identified using differential staining techniques. Bartonella species DNA was not PCR amplified from the extracted archival tissue. Furthermore, no bacteria were identified by means of FISH using a universal eubacterial probe. These data suggest a possible role for copper accumulation in the genesis of GH in dogs and support further evaluation of dogs with GH for evidence of copper-associated hepatopathy. Future studies should include detailed environmental histories, the collection of adequate sample volumes for quantification of hepatic copper content and the examination of frozen tissues using novel molecular diagnostic platforms. Hepatocyte copper and iron accumulation contribute to cell loss, inflammation, and fibrosis. The purpose of this study was to compare copper and iron accumulation in feline liver samples with different disease processes.

Liver biopsies (n 5 104) submitted between July 1, 2007 and June 30, 2009 were evaluated using WSAVA guidelines and categorized as non-hepatic/normal, congenital, inflammatory/infectious, neoplastic, and other. Copper (by rubeanic acid) and iron (by Prussian blue) accumulation were graded by increasing amounts (0-3) and location (centrilobular 5 CL, midzonal 5 MZ, periportal 5 PP, random 5 R). Associations between metal scores and diagnosis category were assessed using the Kruskal-Wallis test.

Histologic diagnoses were non-hepatic/normal (n 5 12), congenital (n 5 6), neoplastic (n 5 16), infectious/inflammatory (n 5 39), and other (n 5 31). Ninety-two samples were negative for copper; remaining samples were graded 1 (n 5 5), 2 (n 5 6), and 3 (n 5 1). Histologic diagnoses (pattern) for positive samples were congenital (1CL), infectious/inflammatory (7: 2CL, 1MZ, 2PP, 2R), neoplastic (2PP), and other (2CL). Iron staining was negative in 18 samples; remaining samples were graded 1 (n 5 38), 2 (n 5 40), and 3 (n 5 8). Distribution was primarily CL (n 5 38) or R (n 5 33), though MZ (n 5 13) and PP (n 5 2) distribution occurred. There were no significant differences by Kruskal-Wallis analysis for amount or location of hepatocellular iron or copper for the different disease categories.

In this study, copper accumulation was rare, had variable distribution and occurred primarily in samples with inflammatory/ infectious disease. In contrast, iron accumulation was common and did not correlate with disease category. Further prospective evaluation of copper and iron accumulation in feline liver disease and association with outcome may be warranted. Chronic hepatitis (CH) in dogs is a progressive condition without clearly defined treatment. Glucocorticoids are commonly used to stop progressive inflammation and fibrosis but are associated with significant side effects including a steroid hepatopathy that complicates enzyme monitoring. Cyclosporine is proposed as an alternative therapy, but there are no published reports of its use for canine CH.

Patient records at the CSU Veterinary Teaching Hospital were searched for histologically confirmed cases of CH treated with cyclosporine. Data were compiled on cyclosporine dosing, concurrent medications, clinical course and biochemical parameters.

13 patients over a 50-month period were identified. Serum alanine aminotransferase (ALT) decreased by an average of 71% in 12 dogs. The ALT normalized completely in 6 of 10 dogs treated for 4 60 days. In 5 of 6 dogs on 4 9 mg/kg/day, the ALT also normalized. Five of the 6 patients that exhibited clinical signs prior to treatment showed measurable improvement (weight gain, fewer gastrointestinal signs). Eight patients had hyperbilirubinemia or ascites prior to treatment; these resolved in 7. Post-treatment histopathology, available in one patient, revealed decreased severity of CH. Five patients exhibited adverse effects including gastrointestinal signs (3), gingival hyperplasia (1), and papillomatosis (1). Cyclosporine was discontinued in 2 dogs with gastrointestinal signs.

Cyclosporine was an effective therapy for many cases of CH and should be considered for patients who are refractory to or cannot tolerate glucocorticoids. Prospective clinical trials with histological documentation are needed to better define cyclosporine's effectiveness in CH. Insertion of the Veress needle and establishment of pneumoperitoneum is associated with 22 to 57% of all laparoscopic complications in humans. The purpose of this study was to determine the accuracy of interpretation of tissue impedance measurements for Veress needle location.

Two laparoscopists, blinded to impedance measurements, placed reusable Veress needles in 20 cadaverous dogs euthanized for reasons unrelated to the study. Placement order was randomized. A third individual evaluated impedance measurements using a handheld device (SensorMed, Knoxville TN) to determine correct versus incorrect needle placement. Veress needle locations were marked using contrasting colors of India Ink; tissues were dissected to determine ink locations. Impedance measurement interpretation identified 29/33 correct and 7/ 7 incorrect placements, respectively. Sensitivity, specificity, accuracy, and precision for correct Veress needle placement are listed below. Agreement was moderate (Kappa 0.50, p 5 0.01) for placements by Operator 1 and very high (Kappa 5 0.88, p o 0.01) for placements by Operator 2. Results for tissue impedance measurement interpretation are superior to published data for currently available tests. Impedance measurements accurately detected all incorrect needle placements. Comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether it increases operator detection of inappropriate Veress needle placement and decreases installment phase complication rates. Delayed detection of intestinal perforation during Veress needle insertion is associated with high mortality. The purpose of this study was to evaluate the accuracy of tissue impedance measurement interpretation for Veress needle location.

Two laparoscopists, blinded to impedance measurements, placed reusable Veress needles in 24 cadaverous cats. Placement order was randomized. A third individual evaluated impedance measurements (SensorMed, Knoxville, TN) to determine placement location. Needle locations were marked using India Ink; tissues were dissected to determine ink locations.

Impedance measurement interpretation identified 36/38 correct and 2/10 incorrect placements. All 8 undetected incorrect placements were located within the retroperitoneal fat pad. Sensitivity, specificity, accuracy, and precision for correct Veress needle placement are listed below. Correlation was absent (Kappa À0.15, p 5 0.34) for placements by Operator 1 and substantial (Kappa 0.78, p o 0.01) for Operator 2. There was no association between correct or incorrect placement and operator on Chi-squared analysis. Failure of impedance measurements to identify placement in the retroperitoneal fat pad resulted in poor accuracy and discordant Kappa statistics. Small cat size limited the number of appropriate placement sites, perhaps resulting in excessively dorsal placement. Comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether impedance measurements increase detection of inappropriate Veress needle placements or decrease installment phase complication rates. Best clinicopathologic tests detecting portosystemic shunting (PSS) in dogs remains controversial. This retrospective study examined performance of single random "fasting" and paired serum bile acids (SBA; pre-and 2-hr post-feeding) in a large population of non-icteric dogs with confirmed PSS (abdominal ultrasound, colorectal scintigraphy, radiographic or spiral-CT portography, laparotomy, or necropsy). SBA were measured by enzymatic colorimetric method with normal o 25 mmol/L. Dogs meeting inclusion criteria (n 5 568) included 467 portosystemic vascular anomalies (PSVA; 368 extrahepatic [E-PSVA], 99 intrahepatic [I-PSVA]), and 101 acquired PSS (APSS). Signalment and laboratory parameters were recorded. Non-parametric statistical analyses were used, two-tailed P o 0.05 applied with Bonferroni corrections.

Median age and weight of 74 breeds were 1.2 (0.2-12) yrs and 6.6 (0.2-48) kg, with equal gender distribution. Random "fasting" SBA detected 90% PSVA and 81% APSS, whereas post-feeding SBA detected 100% PSVA and 98% APSS. Low Protein-C (o 70% activity) occurred in 96% PSVA and 74% APSS. Low MCV and creatinine occurred in 82% and 94% of PSVA dogs, respectively; other tests were less helpful. In APSS, post-feeding SBA was superior. Compared to APSS, PSVA had significantly (P 0.002) lower MCV, cholesterol, BUN, creatinine, glucose, and Protein-C. Compared to E-PSVA, I-PSVA had significantly (P 0.009) lower post-feeding SBA, MCV, albumin, urine specific gravity, and Protein-C but higher cholesterol and glucose.

Post-feeding SBA reflect physiologically provoked bile acid challenge and should be the preferred SBA test in non-icteric dogs for PSS detection. Protein-C assists in identifying PSVA but its utility in APSS may be complicated by concurrent coagulopathies and inflammation. This study compared outcomes of treatment with adjunctive nonsteroidal anti-inflammatory drugs (NSAIDs) or anti-inflammatory glucocorticoids in dogs with severe pulmonary blastomycosis. Medical records were reviewed for dogs diagnosed with blastomycosis at the University of Illinois Veterinary Teaching Hospital between 1992 and 2007. Dogs with a presenting PaO 2 of 80 mmHg, and clinical or radiographic signs of respiratory blastomycosis were included. All dogs were treated with either itraconazole, fluconazole, amphotericin B, or a combination of these. Group 1 (G1) dogs were treated with NSAIDs and Group 2 (G2) dogs were treated with glucocorticoids as anti-inflammatory adjunctive therapy. The following comparisons were made: days of oxygen supplementation, days in hospital, survival to discharge, and long term patient survival. Mann-Whitney U tests and Chi-squared tests were performed on continuous and categorical data, respectively. P o 0.05 was considered significant.

Sixty-eight dogs fit the inclusion criteria. G1 consisted of 31 dogs and G2 consisted of 37 dogs. The two groups were found to be similar in weight, age, and sex distribution. There was no significant difference between the two groups with regard to duration of oxygen supplementation, duration of hospitalization, survival to discharge, and patient survival.

There does not appear to be a difference between the clinical course or patient outcomes between groups of dogs with severe pulmonary blastomycosis treated with NSAIDs or anti-inflammatory glucocorticoids. Further studies need to be performed to fully evaluate the impact these adjunct treatments have on prevention of ARDs and additional respiratory complications. Diagnosis of feline Histoplasma capsulatum infection traditionally relies upon identification of organisms in circulating monocytes or affected organs. In recent years, an antigen assay (AA) was developed for the diagnosis of disseminated histoplasmosis in human patients, but there is little information describing this test in cats. The goal of this study was to determine the sensitivity and specificity of H. capsulatum AA in cats with clinical disorders suggestive of histoplasmosis.

Urine and serum H. capsulatum AA results for feline patients from 3 veterinary hospitals were evaluated. Medical records were reviewed for confirmatory evidence of histoplasmosis (based on cytological or histopathological findings) or an appropriately supported alternate diagnosis.

AA results were available for 78 cats; initial testing was performed on 72 urine samples, 6 serum samples, and 1 unspecified sample. Of these cats, 17/78 had a definitive diagnosis of histoplasmosis based on organism identification, and 10 had a definitive alternate diagnosis (e.g., neoplasia, other infection) based on necropsy findings (n 5 5) or other clinical data (n 5 5). An additional 16 cats had a clinical alternate diagnosis with no cytological or histopathological evidence of histoplasmosis in the affected body system(s). The remaining cats had unverified histoplasmosis (n 5 9) or an open diagnosis (n 5 26). Of the 17 cats with confirmed histoplasmosis, 16 were positive on initial urine AA. One cat (with rectal involvement) was negative, indicating a test sensitivity of 94%. One cat was positive on urine AA but negative on serum AA. All of the 26 cats with definitive or clinical alternate diagnoses had negative results on the AA, suggesting an excellent specificity (100%). However, this result should be interpreted with caution, as the possibility of primary or concurrent histoplasmosis was only definitively excluded in the 5 patients who underwent necropsy examination.

These findings suggest that the AA for H. capsulatum is a reliable diagnostic tool in this species. A positive result appears to reliably support the presence of infection, but a small percentage of infected cats may be negative on AA. In addition, tests performed on urine may be more sensitive that those performed on serum. The purpose of this study was to evaluate the sensitivity and specificity of an Aspergillus galactomannan antigen enzyme immunoassay (GA-EIA) for the diagnosis of canine systemic aspergillosis. Serum and urine samples were collected from sick dogs at 2 hospitals (UCD and TAMU). Group 1 dogs were diagnosed with systemic aspergillosis using culture (sterile site) or microscopy and culture (non-sterile site). Group 2 dogs had clinical findings suggestive of aspergillosis but an alternate diagnosis was established. Group 3 dogs were not suspected to have aspergillosis. Samples were tested using the GA-EIA and results expressed as a galactomannan index (GMI). GMIs 4 0.5 were considered positive. Comparisons were performed using the Mann-Whitney test. There were 10 dogs in Group 1, 22 in Group 2, and 23 in Group 3. Serum was collected from all dogs, and urine from 6, 9, and 10 dogs, respectively. Serum GMIs did not differ from urine GMIs across groups. Serum GMIs of Group 1 dogs were higher than those of Group 2 and Group 3 dogs (p o 0.0001). Results from dogs in Group 2 did not differ from those in Group 3 (p 5 0.43). Two dogs in Group 1 tested negative, but had localized pulmonary infections. One dog in Group 2, which had paecilomycosis, tested positive. Two dogs in Group 3 tested positive. One was being treated with Plasmalyte. The other had a cutaneous opportunistic mycosis. These data support the utility of this assay to aid in the diagnosis of systemic aspergillosis in dogs. Anaplasma phagocytophilum, an Ixodes tick transmitted rickettsial bacterium has a wide mammalian host range that is not commonly reported in cats. Clinical signs in humans, dogs and cats are often vague and include lethargy, anorexia and malaise.

The purpose of this retrospective study was to describe the clinical signs, laboratory data and response to treatment in cats that tested positive for A. phagocytophilum on a commercially available PCR of peripheral blood (FastPanel TM ). This study describes and reports the appearance of intracellular morulae in feline neutrophils contributing to the diagnosis of A. phagocytophilum.

The A. phagocytophilum real-time PCR (RT PCR) assay consists of four multiplexed primer systems designed to detect a total of three distinct genes. Amplicons were confirmed as A. phagocytophilum by DNA sequencing. Clinicopathologic data was obtained by review of medical records and interview of primary veterinarians. Complete blood counts were available from 13/15 cats and 10/13 blood smears were reviewed.

The cats included in this study were all positive for A. phagocytophilum by real-time PCR. The cats ranged from 4 months to 13 years of age with an average age of 3.7 years. Fifteen of 15 cats had a history of tick exposure and lived in the northeastern region of the US, an Ixodes endemic area. All cats presented with lethargy, 13/15 were anorexic and 14/15 had a fever (temperature 4 103 o F). Other clinical findings included hepatomegaly, splenomegaly, ataxia and ocular changes of conjunctivitis and elevation of the nictitating membrane. Hematologic findings included leukopenia (1/13), neutropenia (1/13) and lymphopenia (4/13). Thrombocytopenia was not noted in any case. Morulae were seen within neutrophils in 2/13 cases. All cases in this report responded to treatment with doxycycline. This is the first report of the identification of morulae within neutrophils via peripheral blood smear review in cats confirmed by RT PCR to be infected with Anaplasma phagocytophilum in North America.

Infection with Anaplasma phagocytophilum should be considered in a clinically ill cat with tick exposure, living in an Ixodes endemic area that presents to a veterinarian for lethargy, anorexia and fever. The spectrum of disease manifestations and the accompanying clinicopathological abnormalities indicative of bartonellosis in dogs have not been thoroughly characterized. The objective of this unmatched case-control study was to compare signalment, clinical and pathologic findings in clinically-ill dogs suspected of a tick-borne disease that were negative for Bartonella sp. DNA (controls) as were the dogs diagnosed with bartonellosis by PCR amplification, DNA sequencing and the BAPGM (Bartonella alpha Proteobacteria growth medium) enrichment culture approach. Both groups were tested under the same laboratory conditions and in the same time frame. Medical records were reviewed for information regarding signalment, medical history, physical examination findings, clinicopathological abnormalities, microbiological data and treatment.

The study population consisted of 47 Bartonella-infected dogs and 93 non-infected dogs. Healthy dogs with no historical illnesses, such as blood donors, were excluded. The following species were amplified: B. henselae (n 5 28, 59.6%), B. vinsonii subsp. berkhoffii (n 5 20, 42.6%), B. koehlerae (n 5 3, 6.4%), B. volans-like (n 5 3, 6.4%), B. bovis (n 5 1, 2.1%). Nineteen (40.4%) Bartonella-infected dogs were febrile and lethargic and ten (21.3%) had neurological signs. Laboratory abnormalities for both groups are summarized below (number of affected dogs provided in parenthesis): Multivariate logistic regression using confounding factors was performed to establish potential associations between specific variables and Bartonella sp. infection. There were no differences in signalment, age, sex, body weight and duration of clinical signs between the two groups. Compared to the control population, infection with the genus Bartonella was associated with a diagnosis of endocarditis (P 5 0.0196, OR 5 7.95, 95%CI 5 1.39-51.11) and hypoglobulinemia (P 5 0.0057, OR 5 5.30, 95% CI 5 1.62-18.55). Controls were more likely to have joint effusion (P 5 0.0059, OR 5 5.89, 95% CI 5 1.62-27.22) and azotemia (P 5 0.0353, OR 5 2.93, 95%CI 5 1.07-8.86) than were the Bartonella sp. infected dogs.

Bartonella was detected in dogs with signs such as fever, anemia, thrombocytopenia, hyperglobulinemia and proteinuria that are typically associated with tick-borne diseases. When endocarditis or hypoglobulinemia are detected, testing for Bartonella should be prioritized. Likewise, the detection of Bartonella should prompt further testing for endocarditis, if not already investigated. 

Surveillance studies in other species depend on detection of antibodies to the highly conserved influenza A nucleoprotein (NP); however, no such antibody detection assay is approved for canine use in the U.S. The purpose of this study was to determine the diagnostic accuracy of a commercial blocking ELISA used for avian species in detecting influenza A NP antibody in dogs. Since the blocking ELISA is not a species-specific or viral subtype-specific format, we hypothesized that it would detect NP antibodies in dogs infected by influenza A virus.

Serum samples from uninfected dogs (n 5 204) and dogs naturally infected with canine influenza H3N8 (n 5 150) were tested using the IDEXX FlockChek blocking ELISA for influenza A NP antibody according to manufacturer instructions. The sample/negative control (S/N) absorbance ratios for infected dogs ranged from 0.12 to 0.67 compared to 0.53 to 1.40 for uninfected dogs. A receiver operating characteristic (ROC) curve analysis determined optimum diagnostic sensitivity (99.3%) and specificity (99.0%) at a S/N cutoff ratio of 0.647. Using this cutoff ratio, the overall diagnostic accuracy was 99.2%. Coefficients of variation for intra-assay (4.7%) and inter-assay (6.1%) testing demonstrated good repeatability with canine sera. The excellent diagnostic accuracy of the commercial blocking ELISA makes it a suitable tool for large-scale surveillance of influenza A virus exposure in dogs. Upper respiratory disease (URD) can affect a majority of cats in shelters and is one of the leading reasons for euthanasia of otherwise adoptable cats. The purpose of this study was to determine prevalence and risk factors for upper respiratory pathogens in four different models for managing unowned cats: short-term animal shelters (SHEL), long-term sanctuaries (SANC), home-based foster care (FOST), and trap-neuter-return (TNR) programs.

Conjunctival and oropharyngeal swabs were collected from 543 cats, half of which had clinical signs of URD, and tested for feline herpesvirus (FHV), feline calicivirus (FCV), Chlamydiophila felis, Bordetella bronchiseptica (Bb) and Mycoplasma felis by real-time PCR. Management model, vaccination, sex, age, body condition, and clinical signs were evaluated as risk factors for infection.

A majority of cats in all management models carried one or more organisms capable of causing URD. In many cases, prevalence was similar in cats with or without clinical signs. Unlike diseases that can be controlled by segregation of symptomatic animals, the lack of strong correlation between the presence of pathogens with the presence of clinical signs suggests that feline URD control should be managed by vaccination before or at the time of intake ,biosecurity protocols that presume all cats may be shedding pathogens, and minimizing stressful conditions that contribute to disease susceptibility. Depending on geographical location, sex, age and environment, 2-40% of cats worldwide are infected with the feline immunodeficiency virus (FIV). Knowledge of the FIV status of cats is important to limit the spread of disease and to institute appropriate health management. However, like all lentiviruses, FIV is highly variable in nucleotide sequence, and viral load in cats is variable during different disease stages. Detection of antibodies is the most widely employed diagnostic approach, but does not distinguish FIV-infected from FIV-vaccinated cats. In this study, samples from 30 FIV-seronegative cats, 30 FIVseropositive cats, and 30 FIV-historically seronegative but vaccinated cats, were analyzed by a commercial quantitative PCR (qPCR) assay and virus isolation. Replicate blood samples were coded, and then submitted for 1) qPCR (Idexx); and 2) mononuclear cell isolation with 7-day culture and viral p24 antigen detection by ELISA. For the p24 antigen ELISA, cutoff absorbance values were established from analysis of 10 FIV-negative samples. FIV infection status was pre-determined based on 2 antibody-ELISA results and vaccination history.

Results indicated that qPCR had a sensitivity of 76% for samples from FIV-seropositive cats, and a specificity of 100% and 94.1% for samples from FIV-seronegative and FIV-vaccinated cats, respectively. At a cutoff value of 3 standard deviations above the mean absorbance for p24 antigen ELISA, results from FIV-negative samples yielded a sensitivity of 69.2% for samples from FIV-seropositive cats, and a specificity of 77.1% and 64.7% for samples from FIV-seronegative and FIV-vaccinated cats, respectively. Conclusions from this study are 1) the commercial FIV qPCR assay has high specificity but limited sensitivity for diagnosis of FIV infection; 2) 7-day virus culture has limited sensitivity and specificity. Hence, detection of antibodies remains the most reliable test for diagnosis of FIV infection, but qPCR may be suitable to rule out infection. Oral disease is an important clinical problem in feline medicine and includes common painful conditions such as oropharyngeal inflammation (formerly known as gingivostomatitis) and tooth resorptive lesions. A number of infectious agents have been associated with Private veterinary clinics in the U.S. were recruited to test feline patients presenting with oral disease. Presenting cases included cats with plaque, calculus, gingivitis, stomatitis, periodontal disease, tooth resorptive lesions and other oral diseases as defined by the practitioner. All cats were tested using a commercially available point-of-care ELISA test (IDEXX Snap Combo). Confirmatory tests were not performed as part of the study. Seroprevalence was calculated as the percentage of positive tests in the study population for each virus.

A total of 11,262 cats were tested. Seroprevalence for FeLV was 6.8% and for FIV was 7.1%. Of these, 119 cats (1.0%) were infected with both viruses. Seroprevalence was higher in cats with inflammatory oral disease than in cats characterized with other types of oral disease. Of 7,805 cats with gingivitis, seroprevalence for FeLV was 7.4% and for FIV was 7.9%, with 1.1% of cats co-infected. Of 1,953 cats with stomatitis, seroprevalence for FeLV was 12.2% and for FIV was 13.9%, with 2.2% of cats co-infected.

The seroprevalence for FeLV and FIV reported in this population of cats with oral disease was higher than in a recent large study where samples from U.S. cats not specifically selected for oral disease were tested (FeLV 2.3%, FIV 2.5%). Results of this study indicate that further investigation of the role of retroviruses in cats with oropharyngeal inflammation is warranted. Reliable tests and preventive vaccines and medications for feline retroviral and heartworm (HW) infections are available, but compliance with protocols to reduce transmission is unknown. No largescale longitudinal studies evaluating prevalence over time have been reported. The purpose of this study was to determine the prevalence and risk factors for infection compared with a similar study completed for the first time 5 years previously.

Veterinary clinics and animal shelters in the US and Canada submitted results of testing using a point-of-care ELISA for FeLV antigen, FIV antibody, and HW antigen (IDEXX SNAP Triple) and risk factor information for cats tested during March-September 2010. Bivariable and multivariable analyses were used to evaluate risk factors for infections. A total of 62,301 cats were tested. Only 16% of owned cats were prescribed HW preventive. Risk of retroviral infections was increased by outdoor access, adulthood, and male gender. The most important risk factor associated with all 3 infections was clinical disease; in particular, respiratory and oral diseases and abscesses or bite wounds. Multivariate analysis revealed differences among geodivisions and across infection types.

Feline retroviral and heartworm infections are easily prevented, but difficult to treat. Despite availability of effective management protocols, compliance remains inadequate to reduce the prevalence of these infections. Improved use of preventive care and testing to identify and segregate contagious cats, particularly those at high-risk, is required to reduce the morbidity of these preventable infections. Infectious disease outbreaks are common in animal shelters and are frequently managed by depopulation when risk-assessment tools are not available. During a canine distemper virus (CDV) and parvovirus (CPV) outbreak in sheltered dogs, we used a CDV/CPV point-of-care antibody titer ELISA, a CDV quantitative RT-PCR test, and a CPV fecal antigen test as risk assessment tools to guide release of exposed dogs from quarantine and euthanasia of diseased dogs.

Serum samples (for antibody titers) and swabs of the conjunctiva and upper respiratory tract (for CDV PCR) were collected from 111 asymptomatic dogs starting on Day 4 of the outbreak. Dogs with positive CDV PCR tests were retested every 2 weeks until euthanized for progressive disease or released following recovery from infection. Dogs with clinical signs of parvoviral infection were tested using a CPV fecal antigen test.

For dogs ! 4 months old, protective antibody titers correlated with resistance to clinical disease, but 10% of dogs shed CDV. Lack of protective CDV antibody titers correlated with susceptibility to clinical infection, but most dogs recovered.

Risk assessment and outcome in 60 dogs !4 months of age Feline herpesvirus 1 (FHV-1) is a common ocular and respiratory pathogen of cats that can have clinical illness exacerbated by stress. Cyclosporine (CsA) is commonly used for the treatment of a number of inflammatory diseases in cats and can induce immune suppression. A small number of cats administered CsA to block renal transplant rejection have developed clinical signs of upper respiratory tract disease that may have been from activated FHV-1.

In this study, young adult cats experimentally inoculated with FHV-1 several months previously were divided into three groups and administered methylprednisolone acetate (8 cats, 5 mg/kg, IM, Day 0 and Day 21), CsA (8 cats, 7.0 mg/kg, PO, daily for 42 days), or a placebo (7 cats, corn syrup; 0.075 mL/kg, PO, daily for 42 days). Each cat was assigned a daily individual clinical score by a trained, masked observer using a standardized score sheet during the initial pre-treatment time period (Day -14 to Day 0) and throughout the 42 day treatment period. Each individual clinical score (conjunctivitis, blepharospasm, ocular discharge, sneezing, nasal discharge, nasal congestion, and body temperature scores), the total clinical score (sum of all parameters), the total ocular score (sum of conjunctivitis, blepharospasm, ocular discharge), and total respiratory score (sum of ocular discharge, sneezing, nasal discharge, nasal congestion) were analyzed using SAS PROC GLIMMIX with 'Treatment', 'Time', and the two-way interaction 'Treatment by Time' all as fixed effects. Statistical significance was defined as p o 0.05.

On Day 42 of the study, all of the CsA treated cats had detectable concentrations of CsA in serum (Mean 5 406.1 ng/ml; standard deviation 5 291.8 ng/ml; Median 5 388.5 ng/ml). When group mean values for clinical signs were compared over time as described, significant differences in individual clinical score measurements, in total score, total ocular score, or total respiratory score were not detected over time among any of the treatment groups.

While clinical signs of activated FHV-1 occurred in some cats administered methylprednisolone or CsA, disease was mild and self-limited in most cats and there were no significant CsA sideeffects. These results suggest that the CsA protocol described here is unlikely to reactivate latent FHV-1 infection and cause significant clinical illness. The purpose of this study was to determine the prevalence and risk factors for enteropathogens in four different models for managing unowned cats: short-term shelter, long-term sanctuary, home-based foster care, and trap-neuter-return (TNR) programs.

Fecal samples were collected from 482 cats, half with diarrhea (D) and half with normal feces (N), and tested for a panel of feline and zoonotic enteropathogens by polymerase chain reaction, antigen, and fecal flotation. Risk factors for infection evaluated include management practices, fecal consistency, and signalment.

A majority of cats had at least one enteropathogen of feline or zoonotic importance, regardless of management model or preventive healthcare protocol. For most enteropathogens, the presence or absence of diarrhea did not correlate with infection, the exceptions being T. foetus in sanctuary cats and FCoV in foster cats. Prevalence of specific enteropathogens varied between management models, reflecting differences in preventive healthcare and housing conditions. Management protocols for unowned cats were inadequate for elimination of infections present at the time of intake and for prevention of transmission of enteropathogens among shelter cats. Improved compliance with effective vaccination, deworming, sanitation, and housing protocols is needed to reduce zoonotic and feline health risks. Several allergic diseases of cats, including atopy and gingivostomatitis, can be resistant to glucocorticoids but responsive to cyclosporine. Toxoplasma gondii infection occurs in approximately 30% of cats and the effect cyclosporine therapy has on the T. gondii oocyst shedding period is unknown. The objective of this study was to determine whether administration of cyclosporine before or after T. gondii infection influences the oocyst shedding period.

The young adult cats were T. gondii seronegative when administered 1,000 T. gondii tissue cysts orally on day 42. Group 1 cats (n 5 10) were never administered cyclosporine; Group 2 cats (n 5 10) were administered cyclosporine (7.5 mg/kg, PO) daily on Days 84-126; and Group 3 cats (n 5 10) were administered cyclosporine (7.5 mg/kg, PO) daily from days 0-126. Available feces from individual cages were collected daily and fecal flotation by sugar centrifugation was performed for 84 days after T. gondii inoculation.

Group 3 shed oocysts for a significantly shorter period than Groups 1 or 2 and had a significantly lower oocyst shedding scores than Groups 1 and 2 on days 5-11 after T. gondii inoculation. Group 2 cats had completed the oocyst shedding period prior to being administered cyclosporine and repeat oocyst shedding was not detected during administration of the drug.

Administration of cyclosporine prior to T. gondii infection lessened oocyst shedding which is likely from the anti-T. gondii effects of the drug. Administration of cyclosporine using this protocol is unlikely to induce repeat T. gondii oocyst shedding in client-owned cats. Ã 5 group with diarrhea significantly different than group with normal feces P o 0.05 known about its metabolic pathways or mechanism of pathogenicity and whole genome sequencing of feline hemoplasmas has not yet been reported. The aim of this study was to completely sequence the genome of M. haemofelis to further characterise this important pathogen.

Mycoplasma haemofelis genomic DNA was purified and subjected to whole shotgun Roche 454 sequencing. Gaps were closed using targeted PCR and amplicon sequencing. Ribosomal genes and potential open reading frames (ORFs) were predicted in silico. Putative ORFs were annotated and orthologous groups identified.

Analysis showed a circular genome of 1.15 Mbp with a GC content of 38.9%. Thirty-one transfer RNAs (tRNAs) were identified, accounting for all amino acids, including a tryptophan tRNA for the opal codon (UGA). Of the 1,545 putative proteins identified, 328 (21.2%) matched to proteins from other bacterial species. In common with the pneumoniae group of mycoplasmas, the closest phylogenetic relatives of the hemoplasmas, genes involved in carbohydrate metabolism were limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source for M. haemofelis. The majority of the pentose phosphate pathway genes present in other cultivatable mycoplasmas appear to be incomplete or absent in M. haemofelis, suggesting an alternative mechanism for sourcing purine and pyramidine bases such as scavenging from the host. A gene encoding a glyceraldehyde-3-phosphate dehydrogenase homolog of the immunogenic MSG1 protein of Mycoplasma suis was present. Of the uncharacterized hypothetical proteins, 1,115 were arranged in series of orthologous repeats, or comprised fragments there-of, encoding putative proteins of approximately 200 amino acids. The predicted motifs of the majority of these putative proteins were consistent with these proteins being presented on the cell surface; an N' terminal signal peptide or transmembrane region followed by a non-cytoplasmic tail.

These data have provided valuable information as to why this pathogen remains highly fastidious; it lacks some of the metabolic pathways found in cultivatable mycoplasmas. We have also identified a homolog of a known M. suis immunogenic protein, and identified a potential mechanism for host immune system evasion by way of highly repetitive, putatively surface-expressed hypothetical proteins with variable sequences. Canine leptospirosis has been recognized as a re-emerging disease in the U.S. over the past 20 years, and several serosurveys of the prevalence of leptospiral antibodies in dogs have been published during that time. The role of cats in the epidemiology of leptospirosis has received little attention. Serosurveys of cats for exposure to or infection with leptospires have been published from other geographic areas, but none for cats in the U.S. in the past four decades. The New England states have been found to have a high incidence of canine leptospirosis. The purpose of this pilot study was to determine the prevalence of leptospiral antibodies in a population of feral cats in central Massachusetts.

Blood was collected from 63 sexually intact feral cats presented to a spay and neuter program. Microagglutination titers to Leptospira serovars autumnalis, hardjo, bratislava, icterohaemorrhagiae, canicola, pomona, and grippotyphosa were determined. Three of 63 cats (4.8%) had a positive titer to one or more serovars, with autumnalis being the most common. These results are consistent with previously published prevalence rates in feral cats. Further studies are required to determine the role of leptosporosis in clinical disease in the domestic cat. Since years the Rivalta's test is routinely used in several European countries as a tool to diagnose feline infectious peritonitis (FIP) in cats with effusion. It is inexpensive and easy to perform in private practice. There is, however, only little information about mode of action or its diagnostic value. The objectives of this study were to evaluate sensitivity, specificity, positive (PPV) and negative predic-tive values of the Rivalta's test to diagnosis of FIP and to examine if there is a correlation with any effusion or blood parameters.

Medical records of 782 cats with effusion in which the Rivalta's test was performed between 1999 and 2010 were reviewed concerning diagnosis, blood and effusion parameters, and survival time. Effusion and blood parameters were compared between Rivalta-positive and -negative effusions using the Mann Whitney U test.

Prevalence of FIP in cats with effusion was 31.7%. The Rivalta's test showed a sensitivity of 91.3%, a specificity of 66.6%, a PPV of 55.9%, and a NPV of 94.3% for the diagnosis of FIP. The PPV improved, when cats with lymphoma or bacterial infection were excluded (PPV 69.2%) and also, when only cats younger than 2 years (PPV 87.5%) or 1 year (PPV 90.8%) of age were included. The most important significantly different parameters between Rivalta-positive and -negative effusions were specific gravity as well as cholesterol, triglyceride, and glucose concentration in the effusion.

The Rivalta's test in general is a useful tool to diagnose FIP, but its sensitivity and specificity are not as high as previously assumed. If the Rivalta's test, however, is performed in young cats or if certain diseases have been ruled-out, its diagnostic value is high. Effusion total protein is not highly correlated with test outcome. Therefore, it is still unclear, which components in the effusion of cats with FIP lead to a positive Rivalta's test. Canine parvovirus (CPV) and canine distemper virus (CDV) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. Our study aimed to determine the antibody protection status of dogs at the time of admission into an animal shelter (pre-vaccination) and over the following 2 weeks after vaccination.

Serum samples were obtained from 57 incoming shelter dogs aged 4 months and older with no known history of vaccination. Immediately following serum collection, the dogs were vaccinated against CPV and CDV using a modified live vaccine (MLV). CPV and CDV antibody protection status was determined using Synbiotics TiterCHEK. Dogs with unprotective serum antibody levels against CPV and/or CDV were retested at 6-8 days post-vaccination and again at 13-15 days post-vaccination, if antibody levels were still unprotective against CPV and /or CDV. At the conclusion of the study, stored duplicate sera were submitted for batch 'gold standard' testing to determine canine distemper virus serum neutralization and canine parvovirus hemagglutination inhibition antibody titers.

Based on the Synbiotics TiterCHEK results, 43/57 dogs (75.4%) were protected against CPV and 21/57 (36.8%) were protected against CDV at intake. Older incoming dogs were more likely to be protected against CPV (P o 0.0001) and CDV (P 5 0.0174). Dogs that were spayed/neutered were more likely to be protected against CPV on intake than intact animals, although this result was not statistically significant (P 5 0.0627). The number of dogs with protective titers against CPV/CDV was increased at 6-8 days post-MLV (CPV -48/56, 85.7%; CDV -31/52, 59.6%) and further increased at 13-15 days post-MLV (CPV -54/54, 100.0%; CDV -47/48, 97.9%).

We conclude that incoming shelter dogs often do not have protective antibody titers against CPV and CDV, but older shelter dogs are more likely to be protected against CPV. Based on this population, we further conclude that a large percentage of dogs develop protective antibody titers to CPV and CDV within 1 to 2 weeks when vaccinated with a MLV. Mycoplasma spp. are common inhabitants of the feline oral cavity and so likely contaminate many cat bite abscesses. Mycoplasma spp. are cell-wall deficient and so do not respond to beta-lactam class antibiotics, the class most commonly use for the treatment of cat bite abscesses. The objectives of this study was to determine whether Mycoplasma spp. are common contaminants of cat bite abscesses and are associated with beta-lactam resistant clinical disease.

Privately owned cats with clinical evidence of an acute abscess suspected to be from a cat bite were included in the study. Participants were given a free aerobic and anaerobic culture as well as Mycoplasma spp. culture and polymerase chain reaction using Mycoplasma genus specific primers. Mycoplasma spp. amplicons were sequenced to determine the species. All cats were initially treated with appropriate wound management, were administered an antibiotic in the beta lactam class (amoxicillin-clavulanate or cefovicin), and were rechecked in person or by phone 7 days after beginning treatment.

Of the 26 cats entered into the study to date, Mycoplasma spp. were amplified from 4 cats (15.4%). Of the 2 positive samples with adequate DNA for sequencing, one was consistent with M. felis and the other was consistent with M. equigenitalium. Of the 26 cats, 25 responded by Day 7 to the initial treatment, including 3 of the 4 Mycoplasma spp. positive cats. The cat that failed initial treatment was positive for M. equigenitalium on both Day 0 and Day 7 and ultimately responded to administration of a fluoroquinolone.

The results suggest that while Mycoplasma spp. commonly contaminate cat bite abscesses, routine wound management and antibiotic therapy is adequate for control. However, as Mycoplasma spp. infections do not respond to beta lactam class antibiotic therapy, these organisms should be on the differential list for cats with abscesses that fail treatment with this antibiotic class. Molecular diagnostic assays are frequently used in clinical practice to aid in the diagnosis of suspected infectious respiratory diseases in dogs. However, most currently available assays cannot distinguish strains of the organisms used in vaccines from naturally occurring strains. Our prior studies demonstrated that previously immune adult dogs are unlikely to shed nucleic acids of vaccine strains of adenovirus 2, parainfluenza, or Bordetella bronchiseptica. However, whether this is true for puppies is unknown.

Puppies (n 5 8) at a breeding facility were moved into area without other dogs at 6 weeks of age. Swabs of the nasal and pharyngeal mucosa were collected prior to vaccination and on days 0, 1, 2, 3, 4, 5, 6, 7, 10, 14, 17, 21, 24, and 28 after vaccination with an intranasal adenovirus 2, parainfluenza, and B. bronchiseptica vaccine (IntraTrac 3, Schering Plough). The swabs were shipped on cold packs by overnight express for DNA/RNA extraction and assay in the FastPanel TM PCR Canine Respiratory Disease Profile at Antech Diagnostics.

All puppies were negative for the infectious agents prior to vaccination. After vaccination, positive assay results for parainfluenza and B. bronchiseptica were first detected on Day 1 and on Day 2 for adenovirus 2. By Day 3, DNA or RNA of the agents were amplified from all puppies from both sample sites and most samples were positive for all 3 agents through Day 10. By day 24, only one dog was still positive for B. bronchiseptica.

The results indicate that intranasal administration of adenovirus 2, parainfluenza, or Bordetella bronchiseptica vaccines commonly leads to positive molecular diagnostic assay results for a short time period after primary vaccination. These findings should be considered when assessing the results of these assays in client-owned puppies with respiratory disease. Antimicrobial resistance in Escherichia coli is an increasing concern in both human and veterinary hospitals' patients. The choice drug for treatment in dogs is enrofloxacin, a second generation fluoroquinolone (FQ) whose activity reflects, in part, ciprofloxacin. Among the difficulties in effective E. coli treatment is rapid detection of FQ resistance. The purpose of this study was to determine the specificity and sensitivity of a FRET based assay for the rapid detection of urinary tract infections caused by FQ associated multi-drug resistant E.coli. 306 clinical E. coli isolated from canine urine and 120 clinical veterinary urine samples being examined for E. coli were subjected to susceptibility testing for 14 drugs representing 6 drug classes. Pure isolates were designated NDR (no drug resistance), SDR (single drug resistance) and MDR (multi-drug resistant) (n 5 101 MDR, 116 SDR and 89 NDR). Minimum inhibitory concentration (MIC) for enrofloxacin ranged from 0.03 mg/ml to 512 mg/ml, with high MIC generally associated with MDR. Extracted DNA from culture and from urine were subjected to FRET-PCR targeting single nucleotide polymorphisms in gyrA. The resulting product was sequenced to detect other polymorphisms. Further, to determine the level of detection, microbial free canine urine was inoculated with 10 6 to 10 1 CFU/ml of 7 isolates characterized by variable susceptibility to enrofloxacin (MIC Enro 5 0.03, 0.06, 0.15, 1, 64, 128, 256 mg/ml). Of 306 pure isolates, 50 were confirmed positive for enrofloxacin resistance (MIC Enro 4 4 mg/ml), 43 of which were positively identified by the FRET-PCR assay giving a sensitivity of 86.00%. Only 1 isolate that was resistant was not detected (specificity of 96.66%). However, of the isolates expressing high level resistance (MIC 4 8 X breakpoint [64 mcg/ml]), and MDR (n 5 34), sensitivity 5 97.06%. Of the 120 urine samples 27 contained E. coli 7 of which determined to be FQ-resistant by the assay. Colony dilutions of E. coli confirmed the assay able to detect enrofloxacin resistance at as low as 101 CFU/ml. The relationship between CFUs and the peak of the -(d/dt) fluorescence of the melting curve was R2 5 0.988. These results conclude that the assay is capable of detecting not only the presence of Escherichia coli in clinical samples, but also detecting severity of fluroquinolone resistance and infection. The fluoroquinolones (FQs) are a key class of synthetic antimicrobial agents with an established history in both humans and companion animals of efficacy for treatment of urinary tract infections (UTIs) caused by E. coli, and fluoroquinolones are common therapy. Among the commonly used FQs in dogs and cats are the 2nd generation drugs, enrofloxacin, marbofloxacin, orbifloxacin (all veterinary approved) and the human drug ciprofloxacin; no 3rd and 4th generation FQ is routinely used. The purpose of this study was to assess the in vitro activity of different generation FQs toward E.coli uropathogens whose phenotype ranges from no resistance to multidrug resistance. A total number of 51 canine uropathogenic canine or feline E.coli isolates had been subjected to susceptibility testing to 6 drugs classes (15 drugs) and phenotyped as to resistance: none (NDR, n 5 12), single (SDR, n 5 15), or multiple, MDR (resistance to 2-6 drug classes; n 5 24). MDR included isolates susceptible (ENR S -MDR, n 5 12) or resistant (ENR R -MDR) to enrofloxacin. The minimum inhibition concentrations (MICs) for 11 quinolones (1-1st generation, 3-2nd generation, 4-3rd generation and 3-4th generation) were determined for these isolates using broth microdilution methods according to CLSI guidelines (E. coli ATCC s 25922 served as a negative control). MIC statistics were generated for each drug among phenotypes. The results showed that companion animal E. coli expressing NDR or SDR are largely susceptible to 2nd to 4th generation FQs. However, isolates expressing resistance to 1st or 2nd generation quinolone also express high level resistance based on the MIC 90 to 3rd and 4th generation FQs. The overall potency (MIC) for the 11 drugs for isolates not expressing ENR resistance (that is, NDR, SDR and ENR S -MDR) is GAT Canine leproid granuloma (CLG) was first reported in Brazil in 1990. Over the past 20 years, 37 cases of CLG were diagnosed in Sa˜o Paulo, Brazil, and clinical and epidemiological findings were similar to those reported in Australia. All dogs presented with one or more, uni or bilateral, ulcerated or not, papular, nodular or tumoral lesions, mainly observed in the dorsal surface of the ear, site usually more affected. In general, the lesions are painless and confined to the subcutis and skin, and it does not involve regional lymph nodes, nerves or internal organs, and systemic clinical signs frequently are absent. Short-coated breeds show a marked predisposition for this disease.

The definitive diagnosis of CLG was obtained by histological examination of skin biopsies that were stained with acid fast (Ziehl-Neelsen) and DiffQuik s .

Thirty one (83.7%) of the dogs were purebred; in this study the breed pattern comprised 19 (61.3%) Boxers, 2 (6.5%) German Shepherd and Labrador Retriever, 1 (3.2%) Dobermann, 1 (3.2%) Brazilian Terrier, 1 (3.2%) Golden Retriever, 1 (3.2%) Bulldog, 1 (2.8%) American Pitbull, 1 (3.2%) Mastiff, 1 (3.2%) Fila Brasileiro and 1 (3.2%) Cocker spaniel, 6 (16.3%) were of unknown breed. Nineteen (51.4%) of the thirty seven dogs were males. Twenty (54.1%) dogs were 4-6 years old. In most cases, dogs presented with unilateral or bilateral ear lesions, but rarely thoracic, foot and caudal lesions. The animals were successfully treated by use of rifampicin orally (''the brazilian protocol'') or enrofloxacin orally and topical rifamicin. Anaplasma phagocytophilum is being recognized more frequently in dogs in endemic areas. Currently, most suspected cases are evaluated for A. phagocytophilum antibodies by immunofluorescence assay (IFA) or ELISA. Since A. phagocytophilum is an acute disease, detection by antibody measurement may be negative on initial evaluation. It is possible that A. phagocytophilum DNA can be amplified from blood or synovial fluid prior to seroconversion.

Wild caught Ixodes scapularis adult ticks from Rhode Island were allowed to feed on 18 young adult (2-3 years), mixed sex beagles for up to 7 days. Blood (weekly for 12 weeks), serum (weekly for 12 weeks), and synovial fluid (radiocarpal joint; alternating arthrocentesis weekly for 6 weeks) were collected prior to tick attachment and then weekly after tick attachment. Joint fluid cytology was performed and total DNA was extracted from blood and synovial fluid and assayed in a proprietary real time PCR assay (FastPanel TM ) that amplifies the DNA of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeensis, and E. ewingii. Serum was assayed for A. phagocytophilum antibodies by IFA. Time to first positive results for serology and PCR were compared by paired Student's t test.

None of the beagles developed clinical evidence of disease, and no major changes in synovial fluid cytology were detected over time. Of the 18 beagles, 15 were positive for A. phagocytophilum DNA in blood or synovial fluid or IFA antibodies in at least one sample after tick attachment. Antibody titers appeared in 14 of 15 dogs from weeks 2 to 12 (median to 1 st positive 5 3 weeks AE 1). Titer magnitude ranged from 1:40 to 1:10,240. Anaplasma phagocytophilum DNA was amplified from the blood of 14 of 15 dogs with positive test results ranging from 1 to 12 weeks (median to 1 st positive 5 2 weeks AE 1.5). Anaplasma phagocytophilum DNA was amplified from synovial fluid from 8 of 15 dogs between weeks 1 to 4 (median to 1 st positive 5 4 weeks AE 1). Of the 8 dogs, 7 were PCR positive for only one week and 1 dog was PCR positive for two consecutive weeks. Of the 15 dogs, 13 were positive for A. phagocytophilum in both blood and joints by DNA analysis. Anaplasma phagocytophilum DNA was amplified from blood more quickly than seroconversion was detected by IFA antibody titer (t 5 À3.4, P o 0.01) or DNA was amplified from synovial fluid (t 5 5.4, P o 0.001).

Anaplasma phagocytophilum DNA can be amplified from the blood prior to development of detectable antibody titers by IFA. Amplification of A. phagocytophilum DNA from synovial fluid does not occur in all dogs, appears to be transient in most dogs, and a negative test result does not preclude a diagnosis of A. phagocytophilum infection. Canine granulocytic anaplasmosis and granulocytic ehrlichiosis are tick-transmitted infections caused by Anaplasma phagocytophilum (Aph) and Ehrlichia ewingii (Eew), respectively. Both organisms induce an acute clinical disease, frequently accompanied by fever, polyarthropathy and thrombocytopenia. However, Aph and Eew have different tick vectors, i.e. Ixodes scapularis and Ambylomma americanum, respectively, with different, but overlapping geographic distributions. In addition, infection outcome may be affected by other regional ticktransmitted pathogens, such as Borrelia burgdorferi (MN) or Ehrlichia chaffeensis (AR). Therefore, we compared serology and PCR results derived from dogs examined at two private practices located in highly endemic areas for either Aph or Eew. Serum collected between April-December, 2005 from Minnesota dogs (n 5 420) was tested by SNAP s 4Dx s and whole blood was tested by Aph PCR. Serum collected from Arkansas dogs (n 5 708) for 1 year beginning in August 2009 was tested using microtiter plate ELISAs for antibodies to Eew, E. canis, and E. chaffeensis (Ech) while whole blood was tested by Ehrlichia PCR. Comparisons were evaluated using Chi Square (Ã) and Binomial (w) tests with an alpha of 5%. The above results indicated that dogs are frequently exposed to both Aph and Bb in MN, whereas AR dogs are often exposed to Eew, but less frequently to Ech. Antibodies to E. canis peptides were found infrequently in both MN and AR with only 10 seroreactive dogs detected in both locations. Active Eew infection, as determined by PCR, was four times more frequent in AR pet dog seroreactors as compared to active Aph infections among Aph seroreactors. Although both organisms induce acute disease, the number of Aph and Eew PCR positive dogs that were also seropositive was relatively high suggesting that both organisms induce persistent infections or that dogs are frequently re-infected, despite the presence of a measurable humoral immune response. Additional studies are needed to determine regional infection profiles in other areas that are endemic for these pathogens. Anaplasma phagocytophilum and Ehrlichia canis are two of the most common vector borne disease agents that infect dogs and cats. While PCR assays that amplify the DNA of these agents from blood are currently available, there is minimal information concerning the performance of these assays in different commercial laboratories that utilize different techniques. The purpose of this study was to compare the E. canis and A. phagocytophilum results of two different laboratories on the same samples collected from client-owned animals.

Veterinarians in 3 states (AZ, MD, CT) were recruited to participate in the study based on high prevalence rates for E. canis or A. phagocytophilum infection. Blood in EDTA was collected from dogs or cats with fever, thrombocytopenia, or clinical evidence of polyarthritis and an equal volume of the same blood sample was simultaneously shipped on cold packs by overnight express to Colorado State and to Antech Diagnostics. Standard operating procedures at each laboratory were followed for total DNA extraction and amplification of GAPDH as the DNA control. At Colorado State University, a previously published PCR assay that amplifies the DNA of Ehrlichia spp., Anaplasma spp., Neorickettsia spp., and Wolbachia was performed on each sample with positive amplicons sequenced to determine the species. At Antech Diagnostics, a proprietary real time PCR assay (FastPanel TM ) that amplifies the DNA of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeensis, and E. ewingii was performed.

In the study to date, samples from 35 animals (30 dogs and 5 cats) have been assayed at both laboratories. DNA of A. phagocytophilum (2 cats and 2 dogs) and E. canis (1 dog) were amplified at both laboratories with a percentage agreement between laboratories of 100%.

The results to date suggest that the assay results of the two laboratories for A. phagocytophilum and E. canis are comparable. Ehrlichiosis and Bartonellosis are zoonotic diseases caused by extremely small, obligate intracellular bacteria that require a mammalian reservoir and a blood sucking arthropod vector. Human ehrlichiosis is present in Peru, with a seroprevalence as high as 23% in the highlands. Bartonella species in humans were also identified in Peru since 1905 (B. bacilliformis). Recently, a new species (B. rochalimae) was isolated from an American woman who became febrile after travelling to Peru. Dogs can become infected with the same Ehrlichia species, and the majority of Bartonella species that affect human beings. The role of dogs as reservoirs for human infections has not been clearly established, but exposure and/or infection in dogs has been used to monitor human exposure to tick-borne disease (TBD), since they share the same environment. The objective of this study was to determine the serological and molecular prevalence of Anaplasmosis, Ehrlichiosis and Bartonellosis in rural dogs in the highlands of Peru.

A total of 122 healthy adult dogs were enrolled in this study from four communities in the central highlands of Peru: Ondores, Pachacayo, San Juan de Pachayo, and Canchayllo. EDTA-blood samples were collected from 108 dogs, whereas serum samples were available from 110 dogs. Serum samples were tested for Ehrlichia canis, Anaplasma, Borrelia burgdorferi and Dirofilaria immitis infections using a qualitative dot-ELISA (Snap s 4Dx). The EDTA-blood samples were screened by conventional PCR for the groEL gene of the genus Anaplasma and Ehrlichia, and for the intergenic transcribed spacer of the genus Bartonella. Speciation was conducted by nucleotide sequencing.

Bartonella genus DNA was detected from seven of the 108 dogs (6.5%) and Ehrlichia canis DNA was detected and sequenced from one dog (0.9%). Four of the Bartonella positive samples were identified by DNA sequencing as B. rochalimae (GenBank accession numbers HQ185696 and HQ185695). The other three Bartonella positive samples were identified as B. vinsonii subspecies berkhoffii, the causative agent of endocarditis in dogs and humans. No dog was infected with Anaplasma species by DNA amplification, but one dog was seroreactive for this genus (0.9%). No specific antibodies against Ehrlichia canis and Borrelia burgdorferi and no antigens of Dirofilaria immitis were detected.

This study expands the current knowledge about TBD in Peru and describes for the first time the infection of B. rochalimae in dogs in Peru. The results suggest that dogs may play an important role in the epidemiology of this infection in humans, since they can be asymptomatic but bacteremic. Bartonella spp. DNA is commonly amplified from the blood of cats exposed to Ctenocephalides felis. In previous work, it was shown that cats administered imidacloprid and experimentally exposed to B. henselae infected cats and C. felis did not become PCR positive for B. henselae whereas untreated cats all developed infection. The purpose of this study was to determine if administration of imidacloprid to clientowned cats likely to be exposed to Bartonella spp. and C. felis in the field lessens prevalence of Bartonella spp. infection.

Veterinary students in Tennessee and Florida that owned cats that spent at least 20 days per month outside and that were willing to apply imidacloprid to their cats monthly for six months were recruited for the study. Blood for Bartonella spp. PCR assay was collected from the cats seven months after starting imidacloprid administration and assayed at Colorado State University. To serve as a control group that was unlikely to have been administered flea control products in the previous 6 months, blood was collected from feral cats during TNR programs in each of the two cities and assayed for Bartonella spp. DNA. The Bartonella spp. DNA prevalence rates between the groups were compared by chi square analysis with significance defined as P o 0.05.

The overall prevalence rates for Bartonella spp. DNA in the blood of veterinary student cats (7.4%) and the feral cats (39.1%) were significantly different (P o 0.0001). The distribution of results is shown in Table 1 .

The results suggest that Florida feral cats were more commonly exposed to C. felis than Tennessee feral cats. While the cats in the groups were not exactly matched, the student cats were allowed outdoors for approximately 20 days per month and lived in the same cities as the feral cats, so C. felis exposure rates were likely similar. As previously shown in experimentally-exposed cats, the use of imidacloprid monthly may influence transmission rates of Bartonella spp. amongst naturally-exposed cats. In an endemic area for leishmaniosis and filariosis, coinfection can occur and immunomodulation produced by Wolbachia might influence the clinical signs and progression of both diseases. The aims of the present study were 1) to determine the prevalence of Wolbachia in dogs infected with Dirofilaria immitis (Di) and other filarial nematodes, 2) to evaluate the level of coinfection of leishmaniosis and filariosis by molecular assays and 3) to evaluate any associations between Leishmania infantum (Li) infection, filariosis with or without Wolbachia and clinical presentation and outcome. Statistical differences between groups were tested for significance by the Fisher exact test using SPSS v.14.0 software (significance: p-value o 0.05).

One-hundred and eighteen owned dogs from Southeastern Spain presenting for clinical evaluation were included in the study. Criteria The results of this study highlight the increased sensitivity of PCR for diagnosis of filariosis, confirm the presence of Wolbachia in dogs from the Mediterranean basin, show the increased severity of HWD when Li-Filaria coinfection is present and suggest that Wolbachia could play a protective role for leishmaniosis. Wolbachia antigens can stimulate a Th1-type immune response, as has been previously described. However other factors (as treatment with doxycicline) might be responsible for the lower prevalence of Wolbachia among filaremic dogs infected with Li and further studies must be done to clarify this interaction. The purpose of the present study was investigate the occurrence of leishmaniasis in 200 cats in the municipality of Arac¸atuba, Sa˜o Paulo, Brazil, an endemic area for canine visceral leishmaniasis. Animals were evaluated by direct parasitological examination of lymphoid organs and serology for visceral leishmaniosis by immunosorbent assay (ELISA) and indirect immunofluorescence (IFAT). Thirteen (6.5%) out of 200 cats studied were diagnosed with visceral leishmaniasis; eight (4%) by parasitological diagnosis through cytological examination of lymphoid organs, six (3%) were considered positive by ELISA and one (0.5%) by IFAT. Only two (15.38%) out of the thirteen infected cats had clinical signs, characterized by the presence of crusty lesions on the dorsal cervical region and hepatosplenomegaly. Regarding age five cats (38.5%) had between six months and two years, being the others older than 2 years (61.5%). Only one cat (7.7%) was positive for the three employed methods. PCR confirmed Leishmania sp infection in nine (69.2%) cats, of which six were diagnosed previously by cytological examination, two by ELISA and one by the three techniques employed. Since its first description in 1912 feline leishmaniosis has been reported in several countries. The purpose of this study was to assess the prevalence of Leishmania chagasi infection in cats showing dermatologic lesions from an endemic area for visceral leishmaniasis in Brazil. Animals were evaluated by direct parasitological examination of lymphoid organs, immunohistochemical technique for detection of amastigotes in lesioned skin and serology for visceral leishmaniosis by immunosorbent assay (ELISA) and indirect immunofluorescence (IFAT). Twenty seven (49.1%) out of the 55 cats studied were diagnosed with visceral leishmaniosis. Twelve (44.4%) were positive by parasitological diagnosis; amastigote forms of Leishmania sp were identified in lymphoid organs from 10/55 (18.2%) infected cats, and immunohistochemical technique allowed the identification of nine (16.4%) positive animals. The seroprevalence of leishmaniosis was 25.4% (14/55) by ELISA and 10.9% (6/55) by IFAT. FIV specific antibodies were found in 6/55 cats (10.9%), of which 5/6 (83.3%) had leishmaniosis. Real time PCR confirmed Leishmania chagasi infection in three cats. Based on the evidence of the high occurrence of leishmaniosis in cats in this study, this disease should be included in the differential diagnosis of skin diseases of felines living in endemic areas. Blastomyces dermatiditis is a dimorphic fungus that commonly affects large-breed hunting dogs. A recent advancement in diagnosis has come with the advent of a urine antigen screening test that has both high sensitivity and moderately high specificity. Therapy for the disease involves use of antifungal agents, usually itraconazole, and length of treatment is based chiefly on resolution of clinical and radiologic signs. With the new urine antigen test, however, a noninvasive route of monitoring treatment progress is available and could be an adjunct device utilized to determine treatment efficacy and may even reveal a need for prolonged treatment. Therefore, the purpose of this study was to determine if monitoring the Blastomyces urine antigen test and comparing to pulmonary radiographic signs would elucidate the necessity for prolonged antifungal therapy, even after resolution of radiologic signs.

To this end, a retrospective case review was performed that identified a series of client-owned animals with naturally occurring blastomycosis. The inclusion criteria were radiographic pulmonary parenchymal signs consistent with fungal disease and urine antigenconfirmed blastomycosis with repeated testing of both radiographs and urine antigen quantification as monitoring parameters until negative results achieved in each. Ideally, intervals between testing dates would be between two and five months. Radiographs were considered negative if all radiographic changes had resolved or if repeated radiographs separated by at least one month were considered static after documented improvement had occurred from original diagnostic radiographs (suspected scarring). Urine antigen testing was considered negative if concentrations were less than 1.0 enzyme immunoassay units, a reference interval set by the testing laboratory.

Preliminary data analysis reveals resolution of radiographic signs of blastomycosis occurred earlier in many of the cases presented than did attaining a negative urine antigen concentration. Ceasing treatment 1 month after radiographic resolution of signs as has been recommended in the past might have resulted in premature discontinuation of therapy in many of the cases.

Monitoring of urine antigen concentrations may be of additional clinical use for determining when cessation of treatment should occur in cases of blastomycosis. Persistent elevation of urine antigen concentrations after radiographic resolution of infection may account for apparent recrudescence of blastomycosis after suspected clinical resolution. Giardia spp. and Cryptosporidium spp. are both known to cause infections in dogs and humans in the United States. Nevertheless, prevalence rates for dual infection in dogs had not been widely reported.

In this study, fecal samples from dogs housed in a northern Colorado animal shelter (n 5 121), dogs owned by veterinary students in northern Colorado (n 5 132), and dogs from the Pine Ridge reservation in South Dakota (n 5 84) were collected. Each sample was assayed with a commercially available fluorescent antibody assay that detects Giardia spp. cysts and Cryptosporidium spp. oocysts. Those samples that were positive for Giardia spp. or Cryptosporidium spp. with adequate DNA available for sequencing were genotyped by the glutamate dehydrogenase [gdh] and by the heat shock protein-70 [HSP-70] genes, respectively. Overall, 45 (13.3%) of the dogs had current evidence of a protozoal infection ( Table 1 ). The dogs from Pine Ridge reservation had the highest prevalence rates for Giardia infection and also for dual infections.

From the student dogs, sequencing was successful for the three Giardia isolates (assemblage D from 2 dogs; assemblage C from one dog) and one Cryptosporidium isolate (C. canis). From the reservation dogs, sequencing was successful for nine Giardia isolates (assemblage D from 4 dogs; assemblage C from 5 dogs) and one Cryptosporidium isolate (C. canis).

Cryptosporidium and Giardia co-infections are commonly detected in dogs; in this study dual infections were more common than Cryptosporidium infections alone. Further studies will be required to determine the clinical importance of this finding. Although the Giardia and Cryptosporidium isolates that were sequenced were the dog specific assemblages/genotypes, more samples should be analyzed in order to assess the potential for zoonotic transmission of either parasite. The current study was conducted to determine the prevalence of intestinal parasites in dogs visiting the Veterinary Teaching Hospital, Chiang Mai University, Northern Thailand. Fecal samples (n 5 301) were collected and submitted by owners between August 2009 to February 2010. Demographic and geographic data were recorded. Intestinal parasitic infection was diagnosed by both microscopic examination after zinc sulfate centrifugation flotation and commercially available IFA for Giardia spp. and Cryptosporidium spp. Polymerase chain reaction and DNA sequencing were performed on all Giardia and Cryptosporidium positive samples to provide genotyic information.

Overall prevalence of intestinal parasitic infection in dogs in Chiang Mai was 38.9%. The most prevalent parasite was Giardia spp. (24.9%) followed by Ancylostoma spp. (12.0%), Cryptosporidium spp. (7.6%), Cystoisospora spp. (6.0%), Toxocara canis (2.7%), Trichuris vulpis (2.0%), coccidian-like (1.7%), Toxascaris leonina (0.7%), and Strongyloides spp. (0.7%). The prevalence of having at least one parasite in dogs o 1 year, 1-7 years, and 4 7 years were 49.3%, 39.8%, and 27.4%, respectively. Of these infected dogs, 59.0%, 34.2%, 5.1%, and 1.7% were infected with one, two, three, and four organisms, respectively. Available DNA sequences from Giardia spp. positive samples were shown to be dog specific. Only one adequate DNA sequence was available for Cryptosporidium spp., which was shown to be C. canis.

The findings suggested that intestinal parasitic infection was common in dogs in Chiang Mai, Thailand. Dogs could be potential source for zoonotic intestinal parasitic infection since dogs in this area are allowed for free roaming. Regular deworming program is indicated to prevent not only transmission among dogs but also to human. A retrospective study was conducted on 135 parasite positive fecal specimens consisting of 90 canine, 29 feline, 14 equine and 2 from other host species, comparing recovery of eggs, protozoan cysts and coccidian oocysts using 2 standardized methods of parasite concentration: the formalin/ethyl acetate (F/EA) sedimentation concentration and the commercial Fecalyzer (flotation) kit procedures.

Specimens were processed by each technique either according to manufacturer's instructions or according to standard laboratory procedures. Formalin/ethyl acetate concentrations used at a ratio of 10 ml normal saline to 4 ml ethyl acetate for extraction of lipophilic material from pelleted stool samples, previously fixed in sodium acetate/acetic/acid/formalin (SAF) solution. Flotations with the Fecalyzer kit were performed with concentrated zinc sulfate solution (s. g. 1.22) .

The range of parasites recovered from these specimens included flagellate cysts (40 total), coccidian oocysts (28 total), ova and larvae of nematodes (80 total), and ova of trematodes (12 total) , and cestodes (16 total).

Recovery rates by Fecalyzer flotation were good for protozoan cysts, coccidian oocysts and nematode eggs and larvae, but very poor for cestode and trematode eggs. Formalin/ethyl acetate concentration showed excellent recovery of all parasites and consistently outperformed Fecalyzer in recovery rates. Recoveries by F/EA concentrations were higher by 27.5% for Giardia, by 10.7% for coccidia and by 10.0% for nematode eggs and larvae. With the exception of coccidian oocysts, based on Z-test analyses, recovery rates were significantly higher, at a confidence level of at least 95%, for all parasites, using formalin/ethyl acetate sedimentation concentration.

Although CAPC recommends the use of flotation with centrifugation methods for standard fecal ova and parasite examination for veterinary patients, sedimentation concentration methods are widely and effectively used in human diagnostic parasitology laboratories. These results provide good evidence for the use of F/EA concentration as a preferred method to flotation procedures for stool ova and parasite examinations in veterinary laboratories. Cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases. Cyclosporine inhibits calcineurin-dependent pathways of T-cell activation and the resultant cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. Little work has been done comparing the effects of these agents on cytokine production in dogs. Our study assessed these effects by measuring T-cell cytokine production using flow cytometry, and cytokine gene expression using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in activated canine T-cells treated with cyclosporine and dexamethasone.

For flow cytometric assays, peripheral blood mononuclear cells were separated using density gradients and cultured for 12 hours in the presence of cyclosporine (5, 25, or 100 ng/mL), dexamethasone (10 À7 , 10 À6 , 10 À5 M), or cyclosporine plus dexamethasone. For qRT-PCR, whole blood was cultured for 5 hours with the same drugs at the same concentrations, and RNA was then extracted from leukocytes. Expression of cytokines IL-2 and IFN-g was analyzed in PMA/ionomycinactivated T-cells by flow cytometry, and gene expression for IL-2 and IFN-g in activated T-cell populations was assessed via qRT-PCR.

Flow cytometry and qRT-PCR both demonstrated inhibition of IL-2 and IFN-g that was generally dose-dependent in response to both cyclosporine and dexamethasone. Flow cytometry results from the average of samples collected from 3 different dogs are shown in Figure A . Similar results were achieved using qRT-PCR ( Figure B ).

Suppression of IL-2 and IFN-g in activated T-cells has potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine T-cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients. Idiopathic eosinophilic diseases are described in several breeds, but are over represented in Rottweilers. The immunopathogenesis of idiopathic eosinophilic disorders is poorly characterised. Studies in people highlight the importance of cytokines, particularly interleukin-5 (IL-5), in mediating eosinophil maturation, differentiation, egress from the bone marrow, migration and polyclonal expansion. Eotaxin-2 and eotaxin-3 also appear important for induction of chemotaxis and release of reactive oxygen species from eosinophils. The aim of the current study was to establish whether definable differences in specific cytokines associated with mediation of eosinophil production and survival are present between healthy Rottweilers, non-Rottweilers and Rottweilers with non-parasitic eosinophilia. Secondly, by evaluating cytokine profiles the study aimed to improve understanding of the pathophysiology of eosinophilia therefore assisting development of potential molecular treatment options.

Quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR) assays were used to quantify messenger RNA (mRNA) encoding cytokines IL-4, IL-5, IL-10, IL-23p19, IL-12p35, IL-12p40, IL-18, interferon gamma (IFN-g) and chemokines eotaxin-2 and eotaxin-3 from peripheral blood mononuclear cell (PMBC) samples obtained from healthy non-Rottweiler dogs with normal eosinophil counts (n 5 5) and Rottweilers with normal (n 5 6), mildly increased (n 5 7) and high (n 5 3) eosinophil counts. Quantification of serum IFN-g was also performed using a commercially available canine-specific ELISA.

All samples were positive for housekeeping genes and all cytokines could be quantified with the exception of eotaxin-2 and -3. Results were normalised using three stably expressed housekeeper genes (RPL13A, SDHA and YWAZ) and a relative copy number was calculated for each sample with the sample with the fewest copies given a value of 1. No significant differences were found between groups but there was a tendency for IFN-g mRNA expression to be lower in the Rottweilers with moderate to severe eosinophilia versus control dogs (p 5 0.062). This trend was not seen in the concentration of serum IFN-g quantified by ELISA as there were no significant differences between normal and diseased animals.

In conclusion, there were no significant differences in cytokine mRNA profiles between normal dogs and Rottweilers with varying degrees of eosinophilia. Additional studies including larger numbers of affected dogs are warranted before any accurate conclusions can be made. The presence of large amount of antibody on erythrocyte membrane can accelerate red blood cell (RBC) removal process by the mononuclear phagocyte system. An antigenic stimulus such as the one promoted by vaccines, for example, can induce hypersensitivity reactions and may accelerate RBC destruction. The study objective was to evaluate the erythrocytic membrane potential in inducing lymphocyte proliferative response of recently immunized dogs.

Healthy adult dogs (n 5 17) were immunized with multiple antigens (commercial vaccine with eight antigens: distemper virus, parvovirus, coronavirus, parainfluenza virus, adenovirus, infectious hepatitis virus and leptospire; and anti-rabies). Blood samples from each animal were collected into EDTA tubes in two moments: PRE (immediately before vaccination) and POS (28 to 35 days after vaccination).

Mononuclear cells were separated by gradient, marked with CFSE-FITC and cultured. The stimuli for lymphocyte proliferation used were autologous erythrocytic membrane (AEM) and Concanavalin A (ConA). AEM was obtained by hypotonic lysis and tested in two concentrations (M1: 0.1ug/100uL; M2: 0.2 ug/100uL).

The proliferation assay was evaluated by flow cytometry and analyzed with specific software. The proliferation index (PI) was calculated dividing the fluorescence intensity of the basal sample by the stimulated one. Statistical analysis was performed using paired t-test for parametric samples and Wilcoxon test for non-parametric samples (a 5 0.05).

The For the tested concentrations, autologous erythrocytic membrane does not constitute a stimulus for lymphocyte proliferation in vitro, either before or after vaccination procedure. Additionally, there was no evidence of self-reagent lymphocytes to erythrocyte membrane after vaccination. E. coli is a common cause of canine urinary tract infection. Current treatment emphasizes eradication of established infection rather than infection prevention but increased antibiotic resistance necessitates strategies to prevent infection. Proanthocyanidins found in cranberry juice inhibit E. coli attachment to human uroepithelial cells, impairing bacterial adherence and colonization. We hypothesized that purified cranberry extract (CE) inhibits bacterial adhesion to canine uroepithelial cells.

Five healthy female dogs received an oral CE supplement (Vetoquinol; 100 mg CE/tablet) according to body weight for 30 days. Voided urine collected from each dog before (PRE) and after (30-DAY) completion of the protocol was membrane filtered (22 mm) and stored frozen (-20C). Bacterial adhesion was determined using an in vitro assay. Briefly, urine samples were incubated with an uropathogenic E. coli strain that had been subcultured to promote fimbriae expression. Urine samples containing E. coli were next incubated in 96-well plates containing methanol-fixed Madin-Darby Canine Kidney (MDCK) cells for 1-hr (35C) to permit bacterial attachment. After incubation, plates were washed to remove nonadherent bacteria and fresh media added. Plates were incubated (35C) for 4-hr to grow attached bacteria to detection level. Bacterial concentration in each well was determined using a spectrophotometer (650 nm). Results were analyzed using the Chi-square test.

CE significantly reduced bacterial adhesion by 30% (n 5 5; p 0.5) in 30-DAY urine samples compared with PRE samples. The results show that CE supplementation can reduce adhesion of uropathogenic E. coli to canine uroepithelium and suggests one mechanism by which CE might improve urinary tract health. The purpose of this study was to determine prevalence of 4 urovirulence factors (UVFs) and antimicrobial resistance in canine uropathogenic E. coli (UPEC) and to evaluate associations between UVFs and antimicrobial resistance.

Two hundred and twenty-one UPEC isolates from samples collected from 184 different canine patients submitted to The University of Tennessee Microbiology Laboratory in 2007 were evaluated. A multiplex PCR assay was used to detect cnf, hlyD, sfa/foc, and papGIII in DNA lysate. In vitro susceptibility was evaluated and if the isolate was resistant to any antimicrobial in a class, it was considered resistant to that class.

Of the 221 samples, the number of UVF expressed per isolate was: 0 5 127/221 (57%), 1 5 27/221 (12%), 2 5 4/221 (2%), 3-22/221 (10%), and 4 5 41/221 (19%). Expression of UVF was sfa (33%), hly (24%), cnf (24%), and pap (19%). Presence of 4 UVFs was associated with less resistance (p o 0.0001). The combination of hly, cnf, and sfa was associated with less resistance (p o 0.0001). When sfa was present alone, resistance was less (p o 0.0001). Average resistance to antimicrobial class by number of UVFexpressed was: 0 UVF 5 4.1 AE 3.3 classes, 1 UVF 5 1.2 AE 1.1 classes, 2 UVF 5 0.0 AE 0.0 classes, 3 UVF 5 0.8 AE 1.6 classes, and 4 UVF 5 0.5 AE 1.2 classes.

Urovirulence factors were present in a moderate number of UPEC and correlated negatively with resistance. Neither individual nor combinations of UVFs were associated with increased resistance. Obesity is associated with several comorbidities in dogs including pancreatitis, osteoarthritis, oral disease, neoplasia, and lower urinary tract disease. Investigator observations led to the hypothesis that morbidly obese dogs are more likely to have asymptomatic bacterial urinary tract infections (ABUTI) than overweight and moderately obese dogs. Therefore, a pilot study was conducted to screen for ABUTI in obese dogs. Urinalysis with urine culture and dual energy x-ray absorptiometry (DXA) were performed on fortythree dogs with body fat (BF) percentages ranging from 36 to 56%. Following DXA, subjects were categorized as obese (O)(BF 5 35-44%, n 5 17) and morbidly obese (MO)(BF 4 45%, n 5 26). No dogs had owner-reported symptoms indicative of UTI. The prevalence of ABUTI in O dogs was 6% (n 5 1) and 31% (n 5 8) in MO dogs. The dog in the O group with ABUTI was close to being MO with a BF equaling 44.3%. Of the nine dogs with positive cultures, 4 were neutered males and 5 were spayed females. The prevalence ratio of ABUTI in MO dogs was 7.5, indicating dogs with 45% or greater BF are 7.5 times more likely to have the condition then dogs o 45% BF. The results of this pilot study coincide with other surveillance data describing an increased prevalence of lower urinary tract disease in obese dogs. In conclusion, dogs with body fat percentages greater than 45% are at risk for ABUTI, and veterinarians should consider screening all morbidly obese patients for urinary tract infections. Calcium carbonate (CaC) is recommended to decrease phosphate intake in chronic kidney disease. However, its effect is poorly documented in dogs. Our objectives were to assess within-day, postprandial and CaC effects on phosphatemia variations in healthy dogs.

Phosphatemia was measured every 2 hours for 24 hours in eight adult healthy Beagle dogs in i) fasted condition and ii) a 2 Â 2 crossover design. One group received CaC mixed with maintenance diet (0.8% phosphorus), while the second group received the diet alone. After a 1-week wash-out period, groups were switched. A general linear model was used to test the period, sequence, treatment, dog and time effects on phosphatemia and the area under the phosphatemia versus time curve (AUC 0-24 ).

A significant (p o 0.001) circadian variation existed in fasted dogs. The maximum difference (mean: À1.9 mg/dL; 95% C.I.: À2.4 mg/dL; À1.4 mg/dL) was observed between 8 a.m. and midnight. The AUC 0-24 with CaC (5936 AE 533 mg.min/dL) was mildly but significantly lower (p 5 0.027) than without CaC (6239 AE 631 mg.min/dL). However, it was similar to the AUC 0-24 in fasted conditions. Feeding, with and without CaC, has minor effect on phosphatemia. However, circadian variation of fasted phosphatemia might affect its interpretation. GFR measurement permits diagnosis of kidney injury prior to development of azotemia, and is the gold standard for kidney function assessment. Accurate and rapid (o 60 min) GFR measurement has been performed in rats by simultaneous transcutaneous assay of two intravascular fluorescently-labeled markers. A recently developed analyzer assays fluorescence via a fiberoptic cable introduced through a peripheral catheter, and thus should also allow rapid GFR determination in larger species. The purpose of this study was to determine correlation and agreement between fluorescent ratiometry (FR) and iohexol plasma clearance (IPC) in dogs over a range of GFRs.

Acute kidney injury (AKI) was induced in 5 female hound-type dogs (10 mg/kg gentamicin IV q8h), and FR and IPC GFR were simulta-neously determined on days 0, 3, 6 and 9. A 9-sample, 5-hr protocol was used for IPC; FR was determined following bolus injection of a dextran conjugate mixture (2-sulfohexamine rhodamine-carboxymethyl 150 kD dextran, 5-aminofluorescein-carboxymethyl 5 kD dextran) with fluorescence measured over 60 min. GFR was calculated using 2-compartment model concentration-vs.-time curves for both techniques. Correlation was determined via Spearman's rho; agreement was analyzed via Bland-Altman plots.

IPC GFR and serum creatinine confirmed progressive AKI in all dogs. Correlation between FR and IPC was 0.91 (p o 0.001). Bland-Altman plots confirmed good agreement between techniques with slight underestimation of GFR by FR across most observed values.

These results suggest FR is suitable for GFR determination in dogs with AKI. Importantly, the portable analyzer allowed for point-of-care GFR determination in o 60 min using a peripheral vein.

Previously presented at the American Society of Nephrology Renal Week (related but not identical abstract). Dogs with protein-losing nephropathy (PLN) are at risk of thromboembolic disease, but the mechanism of hypercoagulability and the population of dogs at risk are unknown. The purpose of this study was to characterize thromboelastography (TEG) in dogs with PLN. Twenty-eight client-owned dogs with PLN (urine protein:creatinine ratio (UPC) 4 2.0) and 8 control dogs were enrolled. TEG parameters, antithrombin activity, serum biochemical profiles, and UPC were measured. TEG analyses were run in duplicate with kaolin activation; reaction time (R), clot formation time (K), maximal amplitude (MA), and G (global clot strength) were analyzed. A Wilcoxon Sum Rank Test was used to evaluate differences between groups.

Twelve PLN dogs (42.8%) were azotemic. Nineteen PLN dogs (67.8%) were hypoalbuminemic [serum albumin (SALB) o 3.0 g/dl]; 11 had SALB o 2.5 g/dl.

Dogs with PLN had higher K (p o 0.01), MA (p o 0.005) and G (p o 0.005) than controls. R was similar between the two groups. PLN dogs with SALB o 2.5 g/dl had higher G (p o 0.05) values than dogs with SALB 4 2.5 g/dl; however, even PLN dogs with normal SALB (4 3.0 g/dl) had significantly higher G values than controls (p o 0.005). No significant relationship between UPC and G, SALB and G, antithrombin and G, or SALB and antithrombin was noted using linear regression analysis.

These results indicate that antithrombin, SALB, and UPC cannot be used alone to predict hypercoagulability as assessed by TEG in dogs with PLN. A comprehensive evaluation of the coagulation system in individual patients may be necessary to predict the point at which to initiate anti-thrombotic therapy. Cystinuria is a hereditary renal tubular reabsorption defect of cystine, ornithine, lysine and arginine (collectively, COLA). The low solubility of cystine in acidic urine predisposes to the formation of uroliths. Type I cystinuria in Newfoundland and Labrador Retriever dogs is an autosomal recessive trait caused by mutations in the SLC3A1 gene, whereas in other breeds, the cause of cystinuria has not yet been determined. We report here on the clinical, biochemical and molecular features of cystinuria in Irish Terriers.

Urine and EDTA blood were collected from 222 Irish Terriers from Europe and Australia. A nitroprusside screening test was used to identify increased cystine in urine. Urinary amino acid concentrations were determined by high-pressure liquid chromatography. Cystinuric dogs were defined as having cystine calculi, a positive nitroprusside result, urinary cystine (4 179 mmol/g creatinine) and/ or a COLA concentration of 4 700 mmol/g creatinine.

All 83 females tested nitroprusside negative and had normal urinary cystine (o 150 mmol/g creatinine) and COLA (o 500 mmol/g creatinine) concentrations. The 10 intact males that formed calculi as adults exhibited cystine concentrations ranging from 323-1580 and COLA from 1029-4302 mmol/g creatinine. An additional 41 males had similarly high COLA values with cystine levels from 0-1580 mmol/g creatinine. Among the affecteds tested, 75% were nitroprusside positive. The negative nitroprusside results and/or low urinary cystine levels of affecteds may be due to precipitation of cystine in acidic urine. Sequencing the coding regions of the SLC3A1 and SLC7A9 genes from EDTA blood identified no mutations. The mode of inheritance remains undetermined. However, castration appears to lower the urinary cystine and COLA concentrations and to prevent cystine calculi formation, while diet changes have lesser effects.

In conclusion, non-type I cystinuria in Irish Terriers (and several other breeds like Mastiffs and Scottish Deerhounds) is a unique form characterized by increased aminoaciduria only in males, with lower cystine and COLA excretion and fewer and later urolith formation compared to type I cystinuria. Castrating cystinuric Irish Terriers lowers their cystine and COLA excretion and thus their risk for calculi formation. Cats and dogs that are diagnosed with acute kidney injury (AKI) and resultant uremia that is not responsive to standard medical therapy are likely to benefit from renal replacement therapies, such as intermittent hemodialysis (IHD). The purpose of this study was to evaluate the long-term outcome of patients with AKI treated with IHD, and to establish whether renal function, as determined by serum or plasma creatinine concentrations, is associated with longterm survival. Medical records of 20 cats and 35 dogs that were diagnosed with AKI, treated with IHD, and survived longer than 30 days following the last IHD treatment were retrospectively analyzed. Standard methods of survival analysis using Kaplan-Meier product limit curves and the log-rank test were performed. For all-cause mortality, the median survival time was 1823 days (95% confidence interval: 841, 4667) for cats and 1049 days (95% confidence interval: 893, 1931) for dogs. When only renal-related causes of death were taken into account, the median survival time was not reached for cats or dogs. Survival time for all-cause mortality was inversely associated with the lowest creatinine concentration within the 30 to 90 day period following the last IHD treatment (p o 0.0011 for cats, p o 0.0104 for dogs). This study demonstrates that veterinary patients that are diagnosed with AKI, treated with IHD, and survive greater than 30 days after the last IHD treatment have a good longterm prognosis and frequently die from causes that are unrelated to renal impairment. Renal fine-needle aspiration (R-FNA) is oftentimes attempted during evaluation of dogs and cats with renomegaly, mass lesions, or suspected infiltrative processes. Diagnostic utility of FNA is dependent upon the organ being sampled; additionally, in some organs, certain diagnostic imaging findings are associated with improved concordance of FNA with final diagnosis. Objectives of this study were to evaluate the diagnostic utility of R-FNA and determine whether concordance with final diagnosis is associated with specific clinicopathologic or diagnostic imaging findings. We hypothesized that R-FNA is most useful in patients with diagnostic imaging results suggestive of renal neoplasia (i.e. masses or suspected infiltrative processes). Dogs and cats that had undergone R-FNA from Jan 1, 1998 to Dec 31, 2008 were identified by database search. Patient signalment, serum creatinine and blood urea nitrogen concentration, urine specific gravity, dipstick protein, R-FNA result, and final diagnosis were recorded. Patients were excluded if abdominal radiographs or sonographic images were not available for review, or if diagnostic test results were insufficient for determination of final diagnosis. A single coauthor blinded to final diagnoses interpreted all abdominal images using a pre-set list of descriptors and grading criteria. Radiographic kidney shape, margin distortion, and ventrodorsal kidney-to-L2 ratio were evaluated. Sonographic kidney margin distortion, cortical echogenicity, and corticomedullary junction distinction were described, and presence of nodules or masses, peri-renal effusion, or a peripheral sonolucent rim was noted. Concordance of R-FNA and final diagnosis was determined, and the chi-squared or Fisher's exact test were used to determine association of concordance with the above variables; P o 0.05 was considered significant. 37 dogs and 41 cats (78 animals) met all inclusion criteria. R-FNA results were concordant with the final diagnosis in 43 (55.1%) patients, discordant in 12 (15.4%) patients, and inadequate for cytologic interpretation in 23 (29.5%) patients. Neoplasia or FIP were the final diagnoses in 19 of 43 (44.2%) and 3 of 43 (7.0%) patients with concordant results, respectively. Renal lymphoma (P 5 0.196), renal carcinoma (P 5 0.364), and renal neoplasia in general (P 5 0.451) were not associated with a higher likelihood of R-FNA and final diagnosis concordance. There was no association noted between likelihood of R-FNA and final diagnosis concordance when patients were stratified by species, serum creatinine or blood urea nitrogen concentration, urine specific gravity, dipstick proteinuria, or any diagnostic imaging variables.

This study failed to identify concurrent clinicopathologic or diagnostic imaging findings that enhanced the diagnostic utility of R-FNA. Future studies should use standardized criteria to prospectively identify patients in which R-FNA will be performed, evaluate additional variables that may be associated with increased R-FNA diagnostic utility, and directly compare the utility of R-FNA with that of other diagnostic techniques. Feline lower urinary tract disease (FLUTD) is a disease with increasing prevalence in private practices and veterinary teaching hospitals. Although several underlying causes can cause the obstructive form in male cats, the idiopathic form (feline interstitial cystitis) often is diagnosed as underlying reason in cats o 10 years. The goal of this retrospective study was to identify possible predisposing factors in order to optimize the therapy of these patients.

As a study group, 40 cats hospitalized with obstructive FLUTD at the Veterinary University of Vienna were examined during a 2 year period (2008) (2009) (2010) . As a control group 40 cats presented for other reasons were randomly chosen during the same time period. The data were examined concerning the signalment and history. Furthermore, the long-term outcome was evaluated with a questionnaire. Based on assumptions a student's t-test or a chi-square test was used.

There were no significant differences in age and breed. The body weight was significant higher in the FLUTD group than in the control group (p o 0.01). We could observe a significant risk for the disease of a weight of 4 5 kg (p o 0.01). There were significant less cat toilets in the FLUTD group compared to the control group (p o 0.05). Furthermore we could observe that in the households of FLUTD cats there was significant less than one toilet per cat (p o 0.01) and more cats diseased on FLUTD lived strictly indoor than outdoor (p 5 0.05).There were no significant differences at the time of hospitalization in age, breed, number of cats per household or season of the year between the two groups.

In summary, we could observe that cats over 5 kg body weight kept indoor with less than one toilet per cat have a significant higher possibility to be affected by obstructive FLUTD. Further studies with an extensive history of animal husbandry are needed to identify risks predispoing cats to this frequent and cost-intensive disease. Although purine uroliths (ammonium urate, sodium urate, xanthine, uric acid, etc.) represent the third most common stone type in cats, purine uroliths have the highest rate of recurrence (13% in 22 months). In dogs, mutation of the urate transporter (SLC2A9) and portovascular anomalies are common risk factors. However the underlying cause(s) for purine urolith formation in cats is unknown. The purpose of this study was to test the hypothesis that hyperuricosuria without alterations in liver function is common in cats with urate uroliths.

Urine concentrations of purine metabolites were measured by high-performance liquid chromatography in 5 cats with ammonium uroliths (cases), 5 clinically healthy, breed and gender matched cats (negative controls), and 2 cats with naturally occurring xanthine uroliths (positive controls). Prior to urine collection, all cats were fed a standard maintenance food (protein 5 7 g/100kcal) for 4 weeks. Urinary xanthine, uric acid, and allantoin concentrations and concentration to creatinine ratios were calculated and compared between groups. Also, serum pre-and post-prandial bile acid concentrations were measured.

When compared to control cats, urinary uric acid concentration was significantly higher in case cats (P 5 0.002). Xanthine was not detected in the urine of cases or negative controls. A significant difference in fasted and post-prandial serum bile acid concentrations was not detected in cases or controls (P 5 0.197, 0.212).Hyperuricosuria without increased concentrations of urinary xanthine or allantoin appears to be a risk factor for ammonium urate urolith formation in cats. An association between portovascular shunts and purine urolithiasis was not observed in this population of cats. Studies indicate that proteinuria is predictive, on a population basis, of those cats at risk of developing azotemia. SELDI-TOF-MS is a sensitive, high-throughput, proteomic technique utilising chromatographic surfaces to facilitate separation and detection of proteins and peptides within biological fluids such as urine. Individual low molecular weight (LMW) urinary proteins have been considered as potential biomarkers for renal damage but provide only a limited representation of the urinary proteome; SELDI-TOF-MS may provide a more global assessment.

Normotensive, non-azotemic geriatric cats (4 9 years) were recruited prospectively from two first-opinion clinics for routine health screening. At entry cats received a full physical examination, plasma biochemistry, evaluation of total T4 concentration and urinalysis including urine protein to creatinine ratio. Re-examination was offered at 6 and 12 months. Cats were divided into two groups based on clinical status at the 12 month re-examination (azotemic; creatinine concentration ! 2.0 mg/dL and non-azotemic). Optimisation studies were performed to facilitate the automated preparation (Biomek 3000) of CM10 (weak cation exchange) arrays for SELDI-TOF-MS analysis (Ciphergen Enterprise 4000) of urine samples from cats at entry to the study. Results are reported as median [25 th , 75 th percentile]. Mann Whitney U-test and Wilcoxon signed rank test were used to compare variables between groups and between timepoints, respectively. Ciphergen Express (3.06) software was used to analyse spectral data and a Mann Whitney U-test was used to identify clusters which differed significantly between groups (p o 0.05) at entry to the study.

Twenty non-azotemic cats were recruited, of which 10 cats developed azotemia by 12 months. No significant differences in age, body weight, biochemical or urinalysis variables were identified between groups at entry to the study. As might be expected creatinine increased significantly (1.73 mg/dL [1.58, 1.77], 2.17 [2.00, 3.23], p 5 0.002) between study entry and 12 months in the cats that developed azotaemia and there was a commensurate increase in phosphate concentration (3.81 mg/dL [3.60, 4.22], 4.65[3.94, 6 .48], p 5 0.019). Creatinine and phosphorus did not change significantly over time in the cats that did not develop azotaemia. Seven clusters with m/z values of 2822, 10 033, 10 151, 10 234, 11 635, 11 700 were found to differ significantly between groups at entry to the study.

The low protein concentration of feline urine makes the use of proteomic techniques challenging. However, this pilot study indicates that SELDI-TOF-MS can be utilised to examine the feline urinary proteome and that differences in low molecular weight protein patterns may be useful to differentiate those cats which are at risk of the development of azotemia. Further work is necessary to identify these proteins/peptides. Fibroblastic Growth Factor 23 (FGF-23) is a phosphotonin with an important physiological role in the regulation of phosphorous and vitamin D metabolism, and may therefore play a part in the development of renal secondary hyperparathyroidism. Previous studies in cats have shown parathyroid hormone (PTH) to be elevated prior to the development of azotemia. The study objectives were to explore the hypothesis that FGF-23 is a mediator of the development of renal secondary hyperparathyroidism in the nonazotemic stages of feline CKD.

Healthy, non-azotemic (plasma creatinine concentrations (Cr) o 2.0 mg/dl) geriatric cats were recruited into the study prospectively and followed for 12 months. At the study end point cats were categorised into the following 3 groups: group 1 (N 5 15)-Cr 1.58 mg/dl, group 2 (N 5 33)-Cr !1.58 mg/dl but did not meet the criteria for group 3 and group 3 (N 5 14)-Cr 4 2.0 mg/dl in association with reduced urine concentrating ability (USG o 1.035) or demonstration of persistent azotemia (Cr 4 2.0 mg/dl). Plasma samples were subjected to routine biochemical analysis, intact PTH, calcitriol and intact FGF-23 assay. Variables were compared between the 3 groups at the baseline time point. GFR was measured in an additional group of 19 cats (11 non-azotemic, 4 IRIS stage II, 4 IRIS stage III) using a corrected slope-intercept iohexol clearance method. Relationships were explored using linear regression analysis and determining the coefficient of determination (R 2 ). Results are presented as median [range] .

At the baseline time point FGF-23 concentrations were significantly higher in group 2 (208.1[51.4-814.6 ], P 5 0.001) and group 3 (237.6[127.4-908.1], P 5 0.001) compared to group 1 (126.2[69.4-505.2] ). Weak positive relationships were identified between FGF-23 and PTH (R 2 5 0.126, P 5 0.005, N 5 62) and FGF-23 and Cr (R 2 5 0.077, P 5 0.029, N 5 62). However, the positive relationships between FGF-23 and phosphate (R 2 5 0.016, P 5 0.323, N 5 62) and FGF-23 and calcitriol (R 2 5 0.085, P 5 0.212, N 5 20) were not significant. The additional group of cats in which GFR measurement was performed there was an inverse relationship between FGF-23 and GFR (R 2 5 0.208, P 5 0.040).

In conclusion, FGF-23 was elevated in cats prior to the development of azotemia. The role of FGF-23 in the development of feline renal secondary hyperparathyroidism remains to be determined and should be explored through interventional studies. However, considering the relationship between FGF-23 and GFR, it cannot be excluded that the phosphotonin is simply a marker of reduced filtration. Chronic kidney disease (CKD) is common in geriatric cats and hypoxia might contribute to the progression of this disease. The aim of this study was to evaluate urinary vascular endothelial growth factor (VEGF) as a marker of renal hypoxia.

Cats were recruited through geriatric clinics held at two first opinion London practices. VEGF was measured in stored samples using a canine ELISA kit validated for use on feline urine and indexed to creatinine concentration to yield a VEGF to creatinine ratio (VCR). Two studies were undertaken -firstly a cross-sectional analysis of clinical variables associated with VCR in cats with CKD. Diagnosis of CKD was based on concurrent findings of plasma creatinine ! 2 mg/dl and USG 1.035, with persistence of azotemia for ! 2 weeks. Only patients receiving no medical therapies were included. Normotensive and (pre-treatment) hypertensive cats were included, but borderline cases (mean systolic blood pressure 160-170 mmHg on the date of sampling) were not. Hyperthyroid cats were also excluded from this cross-sectional study. Associations between VCR and clinical data were initially assessed using the Spearman's coefficient and Mann Whitney Test. Linear regression was then used for multivariate analysis.

The second study used samples from a trial in which hypertensive cats that had been treated with amlodipine for at least 3 months were entered into a randomised cross-over study where they received placebo or benazepril (0.5 to 1 mg/kg daily) for 12 weeks in turn. VCR on placebo was compared with that on benazepril using the Wilcoxon signed ranks test. Cats with well controlled hyperthyroidism were included in this intervention study.

Results are reported as median [25th, 75th percentile]. VCR was higher (49.5[33.3, 74.1] vs. 36.1[25.6, 42 .1] fg/g, p 5 0.010) in untreated hypertensives (n 5 30) than normotensives (n 5 63). VCR was correlated with PCV (r 5 À0.236, p 5 0.024, n 5 92), UPC (r 5 0.444, p o 0.001, n 5 93), plasma phosphate (r 5 0.286, p 5 0.005, n 5 93), and USG (r 5 À0.284, p 5 0.006, n 5 93), but not plasma creatinine concentration. In the best multivariate model, PCV was associated with VCR independently of UPC (r 2 5 0.435, n 5 92). VCR was significantly reduced by Benazepril therapy (65.3 [43.1, 92 .7] fg/g) compared with placebo (76.0[48.0, 116.7] fg/g; p 5 0.031, n 5 17) with a reduction seen in 76% of cases.

These results suggest urinary VEGF excretion is associated with proteinuria in cats with CKD and might be a marker of renal hypoxia induced by low PCV. ACE inhibitor therapy might reduce urinary VEGF excretion because angiotensin II causes constriction on efferent arterioles resulting in tubular hypoxia. FGF-23 is a phosphaturic hormone. FGF-23 concentrations increase with declining renal function in humans. The objectives of this study were to validate a method for FGF-23 quantification in feline plasma and to assess the association between FGF-23 concentration and plasma creatinine or phosphate concentration in cats with chronic kidney disease (CKD).

Non-azotemic and azotemic (plasma creatinine concentration (Cr) 4 2.0 mg/dl) geriatric (4 9yrs) cats were recruited into the cross-sectional study from two London first opinion practices. Cats were excluded from the study if they were fed a phosphate restricted diet, or had evidence of concurrent disease. The cats were categorized, using a modified IRIS staging system, into the following four groups: group 1 (Cr 1.6 mg/dl), group 2 (Cr 2.0-2.8 mg/dl), group 3 (Cr 2.9-5.0 mg/dl), group 4 (Cr 4 5.0 mg/dl). Groups 2 and 3 were further subdivided based on the IRIS targets for plasma phosphate concentration (PO 4 ): group 2a (PO 4 4.5 mg/dl), group 2b (PO 4 4 4.5 mg/dl), group 3a (PO 4 5 mg/dl), group 3b (PO 4 4 5 mg/dl). FGF-23 concentrations were measured in feline EDTA plasma using a human intact FGF-23 ELISA, validated by intraand inter-assay variability and assessment of dilutional parallelism. Comparisons between groups were made using the Kruskal-Wallis test and Mann-Whitney U test, with statistical significance defined as P o 0.05. Bonferroni correction was applied where appropriate (statistical significance then determined as P o 0.008). Results are reported as median [25th, 75th percentiles]. FGF-23 concentrations ! 800pg/ml (upper limit of quantification) were assigned the value of 800pg/ml. Intra-and inter-assay variability of FGF-23 measurements were o 10.0% and dilutional parallelism between feline samples and the calibration curve were demonstrated. Plasma FGF-23 concentrations increased with increasing creatinine concentrations (group 1: 158 [115, 274] , n 5 20, group 2: 354 [239, 473] , n 5 20, group 3: 800 [425, 800], n 5 23, group 4: 800 [800, 800], n 5 14). FGF-23 measurements were significantly different between all groups (P 5 0.005 to o 0.001) except between groups 2 and 3 (P 5 0.01). FGF-23 concentrations were significantly higher in cats with higher plasma phosphate concentrations (group 2a: 329 [237, 423] , n 5 16 vs. group 2b: 576 [374, 793], n 5 4; P 5 0.047) and (group 3a: 432 [167, 800] , n 5 10 vs. group 3b: 800 [625, 800], n 5 13; P 5 0.028).

In conclusion, FGF-23 concentrations were higher in cats with more severe CKD or higher plasma phosphate concentrations as would be predicted from its known biological actions. Further work is warranted to explore the role of FGF-23 in the development of renal secondary hyperparathyroidism by measuring parathyroid hormone (PTH) and calcitriol in cats at different stages of CKD. Progressive non-cardiogenic edema and lung dysfunction are common complications of acute kidney injury (AKI) in people. Pulmonary abnormalities have not been systematically reviewed in dogs with renal azotemia, but anecdotal reports of dogs with AKI and concurrent non-cardiogenic pulmonary edema are suggestive of uremic pneumonopathy (UP), a centrally-distributed pulmonary edema syndrome associated with kidney disease in people. We therefore hypothesized that pulmonary-associated clinical signs or thoracic radiograph abnormalities are more common in dogs with renal azotemia than in non-azotemic dogs, and that this association is more likely in dogs with AKI than dogs with chronic renal failure (CRF). Our study objectives were 1) to describe thoracic radiograph and lung histopathologic abnormalities in dogs with renal azotemia, 2) to compare the occurrence of these findings in dogs with AKI, CRF, or non-systemic illness, and 3) to determine if these abnormalities are associated with shorter survival times.

Records of dogs with renal azotemia evaluated from 1/1/2000 to 8/20/2010 were reviewed; dogs which could be classified as having AKI or CRF and which had complete thoracic radiograph studies available for review were included. Dogs with primary intracranial disease and normal serum creatinine and a complete thoracic radiograph study were selected as controls. Signalment, weight, presence of pulmonary-related clinical signs, azotemia duration and severity at time of radiography, and leptospirosis antibody titer were noted. Alveolar, bronchial, interstitial, or nodular lesions were described using a 4-point scale, and lung tissue collected at time of necropsy was reviewed; both the radiologist and pathologist were blinded to final diagnoses. Significance was P o 0.05 for all analyses.

The final study population included 54 AKI, 50 CRF, and 63 control dogs. CRF dogs were older (P o 0.001) than AKI and control dogs. Pulmonary-related clinical signs were more commonly diagnosed at first evaluation in AKI dogs (29/53 dogs, 54.7%) than in CRF (13/50, 26.0%; P 5 0.003) or control dogs (9/63, 14.3%; P o 0.001). Presence of an alveolar pattern was the only radiographic finding which differed amongst groups (more common in AKI [n 5 8, 14.8%, P 5 0.047] and CRF [n 5 8, 16%, P 5 0.028] dogs than in control dogs [n 5 2, 3.2%]). There was no association between presence of an alveolar pattern and any other variable. Alveolar mineralization was the most common lesion in AKI dogs (5/8 dogs; 62.5%), with concurrent alveolar space concretions or mineralization of vessels or bronchioles noted in 1 dog each. Necropsies had not been performed in any of the CRF dogs, but mineralization was not seen in lung tissues from any control dogs (n 5 9). Neither pulmonary-associated clinical signs nor alveolar pattern were associated with median number of days from discharge until death in dogs with AKI (P 5 0.220 and 0.468, respectively) or CRF (P 5 0.280 and 0.253, respectively).

In this group of dogs, presence and type of radiographic pulmonary abnormalities were associated with renal azotemia but not with median time until death. The association between and clinical relevance of alveolar mineralization in AKI dogs were not determined, but both the radiographic and histopathologic abnormalities reported here differ from UP in people. Chronic kidney disease (CKD) is a common cause of morbidity and mortality in cats. The purpose of this study was to investigate the effects of Chinese rhubarb (Rheum officinale) supplementation on the progression of feline CKD.

Cats with stable IRIS stage II or III CKD and without comorbidity were included in the study. Cats were divided into 3 treatment groups and administered rhubarb extract (Group 1, Rubenal s , Vetoquinol, 75 mg tablet PO q 12 h), benazepril as a positive control (Group 2, 0.5 mg/kg PO q 24 h), or both (Group 3). Cats were fed a commercial renal specific diet and enteric phosphate binder as appropriate. Body weight, laboratory data, and blood pressure were recorded every 3 months for up to 34 months.

Variables between groups at enrollment and within groups over visits were compared with ANOVA and repeated measures ANO-VA, respectively. A treatment by visit interaction term was included in all repeated measures models. Significance was set at p 0.05.

Except for body weight there was no significant differences between treatment groups at enrollment. There was no significant change in body weight, hematocrit (hct), UPC, or creatinine over time as compared to baseline within any group. There was no significant difference between groups over time in regards to change in weight, hct, UPC, or creatinine. The treatment by time interaction was non-significant in all models.

Although there was no benefit associated with combination treatment, the results for rhubarb treatment alone were not different from benazepril treatment. Azodyl, an encapsulated, enteric-coated probiotic/prebiotic nutraceutical, is marketed for reduction of azotemia (BUN & Creatinine) in dogs and cats. Cat owners often sprinkle contents onto cat food to facilitate administration. However, exposure to air and stomach acid are thought to inactivate the lyophilized bacteria within the product. Therefore, we examined the ability of foodsprinkled Azodyl to reduce azotemia in cats with CKD.

10 cats with CKD were enrolled in the study and randomized receive Azodyl or placebo. Owners were provided with 3-4 capsules of Azodyl prior to enrollment to ensure compliance with administration. 2 baseline blood samples were obtained 1 month apart, and then 1 & 2 months after beginning therapy. Clinicians and owners were masked as to medication assignment. We hypothesized that a 30% decrease in BUN and/or creat in the Azodyl group would be significant, and set a 5 0.2. In order to maximize the probability of detecting a difference, we determined the % change as being the difference between the maximal baseline analyte concentration and minimal therapeutic concentration. We compared the % change between groups by Mann-Whitney U Test.

BUN and creatinine did not differ between groups. Based on these results, Azodyl, applied by sprinkling onto food fails to reduce azotemia in cats with CKD. Whether intact capsule administration reduces azotemia in cats with CKD remains unknown. Lower urinary tract disease (LUTD) occurs commonly in cats, and idiopathic cystitis (FIC) and urolithiasis account for over 80% of cases in cats less than 10 years of age. Although several strategies have been recommended, a common recommendation is to induce dilute urine resulting in more frequent urination and to dilute calculogenic constituents. In addition to conventional therapy using modified diets, traditional Chinese and Western herbs have been recommended, although only one, chorieto, has published data. We evaluated 3 commonly used herbal treatments recommended for use in cats with LUTD including (1) San Ren Tang, (2) Wei Ling Tang, and (3) Alisma. We hypothesized that these 3 Chinese herbal preparations would induce increased urine volume and decreased urine saturation for calcium oxalate and struvite.

Six healthy, spayed female, adult cats were evaluated in a placebocontrolled, randomized, cross-over design study. Cats were randomized to 1 of 4 treatments including placebo (P), San Ren Tang (SRT), Wei Ling Tang (WLT), or Alisma (A). Treatment was for 2 weeks each with a 1 week washout period between treatments. At end of each treatment period, a 24-hour urine sample was collected using modified litter boxes. Urine volume and biochemistries were measured, and urine saturation for struvite and calcium oxalate was estimated using EQUIL 1.5b. Analysis of Variance (ANOVA) was used to analyze data statistically if distributed normally and Kruskal-Wallis was used to analyze data statistically if data were not distributed normally. A p o 0.05 was considered significant.

Body weights were not different between treatments. No differences were found in 24-hour urinary analyte excretions, 24-hour urine volume, urine pH, or 24-hour urinary saturation for calcium oxalate or struvite between treatments (Table) . Urolithiasis is a multifactorial disease, frequent and recurrent in dogs in the worldwide, in which breed, sex, age, diet, some anatomical abnormalities, urinary tract infection, urine pH and some geographical and hereditary features in the populations studied have been implicated as risk factors. The effective long-term management of urolithiasis depends on identification and control of the pathophysiological mechanisms involved, which, in turn, depend on accurate knowledge of the mineral composition of the uroliths.

The aim of this study was to determine for first occasion the main epidemiological data of canine urolithiasis in Mexico. This study was developed with 491 dogs with urolithiasis from 25 of the 33 states of the country.

Chemical composition of the uroliths was determined by stereoscopic microscopy, infrared spectroscopy, scanning electron microscopy and X-ray microanalysis.

Urolithiasis affected nearly the same number of males and females; with ages ranging from two months to 15 years with a median age of 5 years. Adult animals were the most affected. Breeds more affected were Schnauzer miniature, Poodle, Dalmatian, Yorkshire terrier, Scottish terrier, Chihuahua and Bichon frisee´.

Uroliths were found in the lower urinary tract in 97.74% of the cases. Mineral composition of the uroliths was: Struvite 49.69%, followed by calcium oxalate 25.46%, purines 7.13%, silicate 6.72%, others 0.20%, mixed 8.15% and compound uroliths 2.44%. Struvite uroliths affected females in most cases, whereas calcium oxalate, purines and silicate uroliths, were mainly observed in males.

Our results are similar to studies developed in other countries and continents, though we found a higher frequency of uroliths containing silicate, either pure, mixed or compounds uroliths (10.79%); in Mexico City the frequency reached 15%. This high frequency may be due to high consumption of silicate in home-made food or in the groundwater derived from aquifers.

Acknowledgments: This work has been partially supported by a project of Waltham Foundation in Mexico and the Consejo Nacional de Ciencia y Tecnologı´a (CONACyT) of Mexico. Voiding urohydropropulsion is a non-invasive method for removing small urocystoliths from the dog, most commonly used in females due to the relatively wider and shorter urethra. This procedure is typically performed under general anesthesia to allow complete relaxation of the urethra, however, anesthesia results in longer procedure times and difficult endotracheal tube stabilization due to the vertical positioning of animals, especially in larger dogs.

The aim of this study was to devise a novel injectable sedation protocol for urohydropropulsion when cystoscopy was not concurrently required. An intravenous catheter was placed, and a combination of medetomidine (10 to 15 mg/kg IV) and hydromorphone (0.025 to 0.05 mg/kg IV) was administered, with the addition of ketamine (2 mg/ kg IV) in fractious animals; atipamezole (double volume of medetomidine, administered IM) was used as a reversal agent upon procedure completion. This protocol was considered in cardiovascularly healthy, non-diabetic dogs without evidence of urinary obstruction. Monitoring equipment included electrocardiography, blood pressure measurement, and pulse oximetry, and supplemental flowby oxygen was provided.

Two dogs received the proposed sedation protocol in order to perform urohydropropulsion. Dog one was a 3 year old female spayed Shih Tzu cross, and dog 2 was a 2 year old female spayed Standard Poodle. Ultrasonography revealed a moderate number of urocystoliths present in both dogs, measuring up to 1 mm in dog 1 and 2.3 mm in dog 2. Urohydropropulsion was performed and resulted in retrieval of 15 urocystoliths in dog 1, and approximately 20 urocystoliths in dog 2. Repeat ultrasonography revealed no uroliths present after urohydropropulsion in both dogs. The time from administration of sedation to administration of reversal agent was 6 minutes for dog 1, and 8.5 minutes for dog 2. Records were obtained from 3 dogs that had traditional general anesthetic protocols for urohydropropulsion with cystoscopy for confirmation of urocystolith removal, performed within the last 2 years, and the average anesthetic time was 64 minutes.

Subsequent to the use of medetomine-based sedation protocols for the above dogs, cystoscopy was performed in a 9 year old neutered male Golden Retriever with prostatomegaly. Medetomidine (15 ug/kg IV) and butorphanol (0.2 mg/kg IV) were administered; atipamezole (double volume of medetomidine, administered IM) was used as a reversal agent upon procedure completion. This sedation allowed adequate immobilization for cystoscopy of the urethra and urinary bladder, and endoscopic biopsying of the prostatic urethra and urinary bladder. The time from administration of sedation to administration of reversal agent was 15 minutes for this dog.

In conclusion, a novel sedative protocol for urohydropropulsion is proposed which allows for an appropriate level of sedation along with a short procedure time and rapid recovery. This sedation protocol may also be useful for certain cystoscopic procedures. analysis may be delayed for a variety of reasons, including the need for sample batching within the laboratory or shipping to an outsourced location. Therefore, it is important to know how storage of the sample may affect enzyme activity. We hypothesized that urinary NAG and GGT activity would be affected differently in samples stored by refrigeration vs. freezing.

Thirty-four canine urine samples submitted to the Clinical Pathology Laboratory at Kansas State University were included. Samples were collected from clinical patients with a variety of medical/surgical disorders and were selected based on the day of the week and a minimum volume of 10 ml. A complete urinalysis was performed on each sample; however there were no exclusion criteria based on urinalysis results. NAG and GGT activity in the urine supernatant was assessed by colorimetric assay. Aliquots of each supernatant were refrigerated for 5 days and frozen at À201C for 5 and 30 days at which time enzyme activity was re-assessed.

Compared to baseline values, enzyme activity for both NAG and GGT were stable after 5 days of refrigeration, however there were significant (P o 0.01) declines in GGT and NAG activity when urine supernatants were frozen for 5 and 30 days. Treatment for canine urinary tract infections (UTI) typically consists of 7-14 days of antimicrobial drugs in primary care veterinary practice. Compliance with this drug regimen can be difficult for some clients. Enrofloxacin is a veterinary approved fluoroquinolone antimicrobial and is useful for treatment of canine UTI. Fluoroquinolones are often used in human medicine to treat uncomplicated UTIs in women and can be prescribed for as little as 3 days. The primary objective of this study was to determine if dogs with naturally occurring uncomplicated UTI have equivalent microbiologic cure with a high dose short duration protocol of enrofloxacin, compared to a standard antimicrobial protocol.

Client-owned adult dogs with naturally occurring, uncomplicated UTI were prospectively enrolled in a multi-center clinical trial and assigned to 1 of 2 groups in a randomized blinded manner. Group 1 received treatment with 18-20 mg/kg oral enrofloxacin once daily for 3 consecutive days. Group 2 dogs were treated with 13.75-25 mg/kg oral amoxicillin-clavulante twice daily for 14 days. Both groups had urinalyses and urine cultures submitted on day 0, 10, and 21.

At the time of this interim analysis, thirty-six dogs have completed the trial. Bacteriological cure was achieved in 15 dogs (83%) treated with enrofloxacin and 14 dogs (78%) treated with amoxicillinclavulante, respectively.

These data suggest that the high-dose, short-duration enrofloxacin protocol was equally effective to the standard protocol in treating uncomplicated canine UTI in the sample patient population. and may represent a viable alternative therapeutic regimen for similar patients. Azotemia is frequent in dogs with DMVD (Nicolle et al; JVIM 2007; 21:943-949) and could result from renal hemodynamic alterations. Renal resistive index (RI) allows assessment of renal vascular resistance. The aim of this prospective study was to assess RI in dogs with different DMVD stages.

Fifty-five dogs with DVMD were used (ISACHC class 1 (n 5 28), 2 (n 5 19), and 3 (n 5 8)). Physical examination, renal ultrasonography and echo-Doppler examinations were performed in awake dogs by trained observers. Plasma creatinine, urea and NT-proBNP were measured. Statistical analyses were performed using a general linear model.

Whereas RI of renal and arcuate arteries were unaffected by ISACHC class, left interlobar RI increased (P o 0.001) from 0.62 AE 0.05 (mean AE SD) in class 1 to 0.76 AE 0.08 in class 3. Left interlobar RI was also higher (P o 0.001) in azotemic (0.74 AE 0.008) than in non azotemic (0.62 AE 0.005) dogs. Similar findings were observed for right interlobar RI. A positive effect of NT-proBNP (P 5 0.002), urea (P o 0.001), creatinine (P 5 0.002), urea-to-creatinine ratio (P o 0.001), left atrium-to-aorta ratio (P o 0.001), regurgitation fraction (P 5 0.011), systolic pulmonary arterial pressure (P o 0.001) and shortening fraction (P 5 0.028) on RI was also observed.

In conclusion, interlobar RI increases with the severity of DMVD and azotemia. A cause-effect relationship remains however to be established. Antibodies against alpha-enolase are associated with immunemediated nephritis in people. It was previously shown that vaccinated cats commonly develop antibodies against alpha-enolase. The purpose of this study was to assess for associations between alphaenolase antibodies and azotemia in privately-owned cats.

Clinically stable privately owned cats ! 10 years of age, with and without azotemia (creatinine 4 2 mg/dl), and with an available vaccine history for ! 5 years were recruited for the study. Sera were assayed for creatinine concentrations and alpha-enolase antibodies by use of previously validated techniques. Results from cats with and without azotemia were compared by Student's 2-tailed t test or Fisher's exact test with significance defined as p o 0.05.

Median ages were 15 years (range: 10-18) and 12 years (range: 10-15) for cats with (n 5 35) and without azotemia (n 5 27), respectively. There was no significant difference in vaccine events (number, type, or route of administration) between groups. Azotemic cats (34.3%) were more likely than normal cats (12.5%) to be positive for antibodies against alpha-enolase (p 5 0.016). In addition, alpha-enolase antibody concentrations were greater (p 5 0.041) in azotemic cats (mean % ELISA 5 62.5%) than cats with normal creatinine concentrations (mean %ELISA 5 47.2%).

Results of this study suggest that antibodies against alpha-enolase in cats may be associated with renal disease. Additional prospective evaluation in a larger number of cats is indicated. AKI is used in human medicine as a predictor of mortality based on the AKIN (Acute Kidney Injury Network) scoring system which utilizes relative increases in creatinine to determine stage. With this scheme, mortality has been shown to increase as the stage of kidney injury (indicated by AKIN score) increases. Accordingly, we hypothesized that this system would improve predicting prognosis in dogs and cats. We retrospectively evaluated 1088 dogs and 856 cats (2008) (2009) ) that had ! 2 creatinine measurements within 7 days, and whose first creatinine was o 1.6 mg/dl. Patients were categorized as: Level 0 (no AKI); Level 1 (second creatinine value o 1.6 mg/dL, but creatinine increased ! 0.3 mg/dL); or Level 3 (second creatinine 4 1.6 mg/dL with a creatinine increase ! 0.3 mg/dL). Thirty and 90 day survival for each level was compared to level 0. Adjusted odds ratio (OR) in dogs for 30 day survival was 1.3 for Level 1 (CI 95%, 0.8-2.2) and 3.2 (CI 95%, 1.8-5.5) for Level 2; OR for 90 day survival was 1.3 for Level 1 (CI 95%, 0.8-2.2) and 3.7 (CI 95%, 2.1-6.5) for Level 2. For cats, OR at 30 days was 1.5 (CI 95%, 0.5-4.6) for Level 1 and 3.1 (CI 95%, 1.5-6.7) for Level 2; OR for 90 day survival was 0.9 (CI 95%, 0.3-2.8) for Level 1 and 4.1 (CI 95%, 1.8-9.3) for Level 2. Thus, detecting increasing stage of AKI helps predict mortality in dogs and cats.

ABSTRACT N/U-27 FELINE URATE UROLITHIASIS: 143 CASES (2000 -2008 . J Dear 1 , R Shiraki 2 , A Ruby 2 , J Westropp 3 . 1 William R Pritchard Veterinary Medical Teaching Hospital, University of California, Davis, CA, 2 Gerald V. Ling Urinary Stone Analysis Laboratory, University of California, Davis, CA and 3 the Department of Veterinary Medicine and Epidemiology, University of California, Davis, CA.

Feline urate urolithiasis accounts for 10% of the feline stones our laboratory analyzes each year; little information is known about this disease, particularly the incidence of those cats with hepatopathies.

The objective of the study was to characterize the signalment, clinicopathologic data, and diagnostic imaging of cats with this disease as well as the salts of uric acid present.

A retrospective analysis of feline urate uroliths submitted to the Stone Lab between January 2000-December 2008 were included. From these data, primary veterinarians were solicited to submit records. Furthermore, all records from cats with urate uroliths from the VMTH were analyzed separately.

143 records were received from the primary care veterinarians. Sixteen cases were identified from the VMTH. Median values for the CBC and chemistry panels available were within the reference ranges provided, with only a few outliers present. Of the 78 cats with radiographic reports, 70 (90%) had visible evidence of uroliths. Two external cases had confirmed PSS; five cases from the VMTH had a PSS. Cats with urate uroliths and PSS were younger than cats without a documented hepatopathy (2 years vs. 7 years). The Siamese breed was overrepresented. All stones were ammonium hydrogen urate.

The pathogensis of urate uroliths in cats is poorly understood. Most cats were not completely evaluated for PSS, however, there were few clinicopathologic parameters which indicated hepatopathies were present. Further studies are warranted to evaluate genetics and purine metabolism in cats with urate uroliths to help tailor proper management and breeding strategies. 3-Indoxyl and p-cresyl sulfate (IS, and CS, respectively), small protein-bound molecules derived from gastrointestinal protein metabolism, are among the most important uremic solutes affecting morbidity and mortality in human chronic kidney disease (CKD). In the blood stream, these compounds are predominantly bound to protein, but their debilitating effects on prognosis and quality of life in CKD appear to be driven by the free fraction. The objectives of the present study were to assess the normal, physiological levels of IS and CS in healthy cats and to evaluate the correlation of the respective free and protein-bound levels.

Blood samples were taken from 105 clinically healthy adult cats enrolled at five participating veterinary practices in Germany. After centrifugation, the serum was deep frozen until transport on dry ice to the analytical laboratory. Serum creatinine and urea levels were quantified by VetTest s (IDEXX Laboratories, Inc.). Total and free IS and CS, respectively, were quantified by turbulent flow chromatography coupled with a tandem mass spectrometry detector. Statistical analysis of the results comprised i) a descriptive report of the median with upper and lower bounds of the 95% confidence interval for reference values of IS and CS, ii) a calculation of various Pearson correlation coefficients r, also tested with reference to the null hypothesis of no relationship, and iii) Wilcoxon-Mann-Whitney Utest for an estimation of the effect of hemolysis on serum IS or CS levels. Six animals with serum creatinine or urea levels outside the reference range were excluded from the calculation of reference values.

Median levels of IS in cat serum were 1.19 mg/L with upper and lower bound 95% confidence intervals at 1.46 and 0.99 mg/L, respectively. The corresponding median levels of CS were 2.11 mg/L (median) and 2.46 vs 1.57 mg/L (upper vs lower bound levels, respectively). These values showed a low, non-significant correlation with serum creatinine or urea levels. However, IS and CS serum levels were moderately correlated (total levels r 5 0.4808, p o 0001). Their respective free levels constituted about 5% of the total serum levels (r ! 0.8977, p o 0.001). Non-hemolytic samples tended to yield lower values than hemolytic samples. Due to the low number of hemolytic samples (N 5 14) , the group difference could, however, not be statistically confirmed.

The results indicate that it is sufficient to determine total levels of either IS or CS in serum while studying the effects of therapeutic or dietetic interventions on the evolution of these parameters in feline CKD. Reference values are provided for orientation towards clinically relevant changes. Disrupted urothelial differentiation has been implicated in the pathogenesis of feline idiopathic cystitis (FIC). Studies of cultured human urothelium have shown that abnormalities in urothelial differentiation and repair may be mediated by persistent 15-hydroxy-prostaglandin dehydrogenase (PGDH) activity and subsequent metabolism of cytoprotective prostaglandins. The goal of this study was to confirm persistent PGDH expression in FIC bladders compared to desmoplakin I1II expression, a marker of urothelial differentiation. Urinary bladder biopsy specimens were obtained by cystotomy from 9 symptomatic cats with chronic FIC. Cats with a history of another major disease, previous cystotomy, or recent treatment with corticosteroids, NSAIDs, antihistamines, antidepressants, or glycosaminoglycans were excluded. Urinary bladder tissue specimens were also obtained from 10 untreated clinically normal specific-pathogen-free cats. Tissue specimens were fixed in buffered 10% formalin and embedded in paraffin. Tissue sections were deparaffinized and subjected to citrate buffer microwave antigen retrieval. Tissues were stained for PGDH using a rabbit anti-PGDH antibody, an isotype negative control or goat anti-desmoplakin I1II and developed using the avidin-biotin peroxidase complex method. All FIC (9/9) and normal (10/10) cat bladder samples showed similar staining of urothelial cytoplasm for PGDH. However, desmoplakin I1II staining, found on the luminal cell surface in 4/4 normal tissues, was disrupted in 6/6 FIC bladder samples. Desmoplakin I1II staining confirmed altered urothelial differentiation in FIC cats. However, PGDH expression remained intact in FIC samples.

We hypothesize that PGDH expression in FIC may contribute to its pathophysiology due to breakdown of prostaglandins essential for urothelial healing. Additional studies will explore this hypothesis. The University of Tennessee College Of Veterinary Medicine's Picture Archiving and Communication System was searched over a 9 month period for cats that had undergone both abdominal radiographs and ultrasound during the same visit. One hundred and three cats were identified (age range o 1 to 18yrs; median 11yrs). Kidney size was determined based on radiographic and ultrasound findings. Of the included cats, 41.8% had two normal sized kidneys, 18.4% had one small and one normal, 15.5% had one large and one normal, 11.7% had two small, 8.7% had two large, and 3.9% had one small and one large kidney. The presence of mineralization, uroliths and hydronephrosis was also noted. Medical records were reviewed for clinical chemistry data and historical information concerning previous urinary disease. No significant differences were found between kidney size and renal function, kidney size and the presence of uroliths, renal mineralization and function or the presence of uroliths and function. The presence of uroliths was significantly associated with hydronephrosis. Of the 30 cats with at least one large kidney, 9 (30%) had hydronephrosis. Of the 23 cats with current or previously diagnosed uroliths, urinary tract infections or other uropathies, 10 (43.5%) had at least one small kidney. Small kidneys were commonly found in older cats, however, this correlation was not statistically significant. Based on these findings, small kidneys are more likely to be the result of urinary disease as opposed to being either congenital or due to aging. This study aimed to evaluate IFE, which has been advocated for treatment of lipid-soluble drug intoxication, in the treatment of clinically-occurring canine ivermectin toxicosis.

One Australian Shepherd and two Miniature Australian Shepherds were included. All three dogs were homozygous for the MDR-1 gene mutation. Two dogs roamed on horse ranches where ivermectin-based deworming products had recently been used. Ivermectin was administered to the third dog (165 mg/kg PO). All three dogs exhibited tremors, ptyalism, and CNS depression, which progressed over several hours to stupor in two dogs, and to a comatose state requiring mechanical ventilation in the remaining dog. A 20% formulation of IFE (Liposyn II, Hospira) was administered as a bolus (1.5 ml/kg) followed by a slow IV infusion (7.5-15 ml/kg over 30 minutes). No change was observed in the neurologic status of any patient. Lipemia visible upon blood sampling persisted for 36 hours in one dog. No other adverse effects were noted. Serum ivermectin levels confirmed ivermectin exposure in each case.

In this study, IFE administration did not result in clinical benefit in cases of ivermectin toxicosis. Brain ivermectin concentrations in MDR1 mutant/mutant genotype dogs may be too high to be overcome by IFE. Additionally, these dogs may lack P-glycoprotein-mediated biliary clearance mechanisms needed for optimal IFE function. Further investigation is needed to determine the utility and optimal dosing of IFE in canine toxicoses, to characterize its safety, and to determine how MDR-1 status may alter the efficacy of IFE in treatment of canine ivermectin intoxication. Rufinamide is a recently approved antiepileptic drug used for the treatment of seizure disorders in human patients. Rufinamide is administered at a dose of 45 mg/kg divided twice daily to achieve therapeutic concentrations of 15 mg/ml. The objective of this study was to determine the pharmacokinetic properties and short-term adverse effects of single-dose oral rufinamide in healthy dogs in preparation for a possible clinical trial evaluating the efficacy of rufinamide in the treatment of canine epilepsy.

Six healthy adult dogs were included. The pharmacokinetics of rufinamide were calculated following administration of a single mean oral dose of 20.0 mg/kg (range 18.6-20.8 mg/kg), extrapolated from the dose used in human patients. Dogs were monitored by repeat physical examinations, electrocardiograms and blood pressure assessments during the course of the study. Plasma rufinamide concentrations were determined using high-performance liquid chromatography. Pharmacokinetic data were analyzed using WinNonlin version 1.0.

No adverse effects were observed. The mean terminal half-life was 9.86 1/À 4.77 hours. The mean maximum plasma concentration was 19.561/À5.82 mg/ml and the mean time to maximum plasma concentration was 9.33 1/À 4.68 hours. Mean clearance was 1.448 1/À 0.703 L/hr. AUC inf was 410.72 1/À 175.88 mgÃh/ml.

Results of this study suggest that rufinamide given orally at 20 mg/ kg twice daily in healthy dogs should result in a plasma concentration and half-life sufficient to achieve the therapeutic level extrapolated from humans without short-term adverse effects. Further investigation into the efficacy and long-term safety of rufinamide in the treatment of canine epilepsy is warranted. The aims of this study were to investigate the ABG for (i) the prevalence of skull abnormalities; (ii) the prevalence of SM; (iii) an association between lateral ventricular size, cerebellar size and SM; and (iv) associations between SM, skull abnormalities, CSF pleocytosis and clinical signs.

Seventy-six ABGs, recruited as part of a larger epidemiological and genetic study, underwent brain and spinal MRI evaluation (3.0T General Electric Signa HDx, Milwaukee, WI). All dogs were evaluated neurologically, recording deficits and the presence of spinal pain. Sequences acquired included T2W, T1W pre-and postcontrast, and T2W FLAIR, sagittal and transverse. Cervical spinal cord central canal (CC) and or syrinx size and its percent area of spinal cord was measured using OsiriX s . The presence of Chari-like malformation (CM) was assessed by recording the presence of caudal cerebellar deviation and/or foramenal vermal herniation. Lateral ventricle and cerebellar volume was expressed as a percent of the cerebrum and intracranial volume2qa respectively. Forty-five dogs underwent atlanto-occipital cerebrospinal fluid tap at the time of MRI and the white blood cell (WBC) count was recorded. Student's t-tests were used to compare the measured variables between groups with and without skull abnormalities, spinal pain and neurological signs.

The mean age of the 30 males (24 intact) and the 46 females (34 intact) was 50.4 months (range 8-135; median 44 months). Neurological deficits and neck pain were noted in 21 (27%) and 15 (19.7%) of dogs respectively; 5 dogs (6.57%) exhibited both. Cerebellar deviation and vermal herniation were present in 37 (48.68%) and 46 (60.52%) dogs respectively; twenty-three dogs (30.26%) had both. Mean height of the CC was 2.3 mm (0-7.2 mm). Forty (52.63%) CCs were greater than 2 mm in height; the mean length of these lesions was 2.03 vertebrae (0.5-7). Mean CSF WBC count was 4.97/ml (0-39). Syrinx height and extent were significantly higher in dogs with neurological signs (size p 5 0.01; extent p 5 0.0004). There were no significant differences in syrinx sizes and extent in dogs with or without skull abnormalities or spinal pain. There were no associations of syrinx height or extent with CSF WBC count or age of dog. Intact females had a significantly lower syrinx extent than intact males (p 5 0.009). There were no significant differences in presence of spinal pain or neurological signs between dogs with or without skull abnormalities. There was a significant negative association of ventricular percentage and cerebellar percentage (p o 0.0001). There was a significant association of ventricular percentage with syrinx percentage (p 5 0.0015) and height (p 5 0.0007).

This study suggests that SM and CM are prevalent in ABGs. Syrinx size and extent are associated with neurological signs and ventriculomegaly is associated with both small cerebellar size and large syrinx size. However, SM may not be associated with CM as defined by cerebellar herniation and deviation and is not associated with CSF inflammation. The power tissue resection device (PTRD) is a hand-piece comprised of an outer cannula with motor driven vacuum-assisted inner cutting blade. This device was designed and is marketed for human neurosurgical brain/spinal cord tumor resection. The purpose of this study is to describe the use of the PTRD for intervertebral disc fenestration and to compare the effectiveness of manual fenestration to that of the PTRD.

Fifteen cadaveric lumbar spines were randomly placed into three study groups: group 1 was the control group on which no fenestrations were performed, group 2 was the manual fenestration group and group 3 was the PTRD fenestration group. The effectiveness of fenestration via both manual and PTRD was assessed by calculating the ratio of remaining nuclear weight post fenestration to total nuclear volume. Discs with lower ratios were more effectively fenestrated.

Results showed a smaller ratio of post fenestration remaining nuclear weight to nuclear volume following fenestration with the PTRD (0.23 AE 0.09) as compared to manual fenestration (0.30 AE 0.10). These results did not show statistical significance. When fenestrated samples were compared to control samples (0.39 AE 0.07), there was a statistically significant reduction in ratios.

In conclusion, the PTRD is easy to use and is as effective as the manual technique for canine intervertebral disc fenestration.

According to the human WHO classification gliomatosis cerebri (GC) is a rare astrocytic tumor affecting at least three lobes of the brain with extensive infiltration, but relative preservation of brain architecture. GC has not been reported to occur as a hereditary disease, neither in man nor in animals. Here, we report the temporally clustered occurrence of GC in a family of Bearded collies.

A 7 years old female Bearded collie with forebrain signs was presented. Differentials included inflammatory/ infectious, metabolic/ toxic, and neoplastic diseases. Within a time period of 12 months, 3 offspring of this bitch were presented with similar clinical signs. Two dogs were full siblings (2 males). The remaining female dog originated from a match with a different male dog.

MRI was performed in all 4 dogs and revealed a diffuse and extensive intra-axial lesion with moderate mass effect and midline shift. The ill defined lesion showed mainly a white matter distribution with hyperintense signal in T2-w and FLAIR images and iso-to hypointense signal in T1-w images without contrast enhancement. The lesion was bilateral in all cases, continued along the white matter extending partially into the gray matter with contact to the brain surface.

Neuropathology revealed a diffuse and extensive infiltration of the brain and spinal cord by a neoplastic glial cell population involving white and gray matter of both hemispheres, thalamus, brainstem and cerebellum in all 4 dogs. Based on the cell morphology and immunoexpression of glial fibrillary astrocytic protein by neoplastic cells diagnosis of GC was made. This is the first report of familial occurrence of GC, which is likely the result of a germ-line mutation. Several human hereditary cancer syndromes are associated with CNS tumors including amongst others the Li-Fraumeni cancer family syndrome (p53 mutation), neurofibromatosis (type 1 and 2) (neurofibromin, merlin mutation), and tuberous sclerosis (hamartin, tuberin mutation). Furthermore, familial clustering of human gliomas unassociated to the known inherited cancer syndromes has been described. In the dog, hereditary CNS tumors are not known.

The exact mode of inheritance and putative gene mutations of GC in this Bearded collie family are currently under investigation. Preliminary results are consistent with a monogenic autosomal dominant mode of inheritance, although a recessive inheritance cannot be completely ruled out at this time. Mutations in the TP53 gene were not found following amplification and sequencing of exons 5-8 in 2 affected dogs.

Previously presented at the ECVN annual meeting in Cambridge, UK.

The GM 2 gangliosidoses are characterized by a deficiency of bhexosaminidase. There are two isoforms: Hex A composed of an a and b subunit encoded by HEXA and HEXB genes respectively and Hex B with two b subunits. Hex A requires an activator encoded by GM2A. Two Japanese chin dogs with confirmed GM 2 gangliosidosis showed elevated total hexosaminidase and normal hexosaminidase A activity, a pattern associated with the AB variant in humans and consistent with prior reports in the breed. This study was performed to identify the mutation responsible using resequencing with an Applied Biosystems 3730xl DNA analyzer as previously described (Awano 2009).

Mutations in GM2A cause the AB variant in humans, but resequencing GM2A revealed no mutation that could account for the disease. Resequencing HEXA and HEXB revealed a c.967G 4 A mutation in HEXA which was homozygous in both affected dogs. Sixty-five normal Japanese Chin dogs were screened for the mutant allele; 60 were homozygous for the ancestral allele and 5 heterozygous.

This mutation predicts a p.323E 4 K substitution affecting one of two primary active-site amino acids that participate in the hydrolysis of GM 2 ganglioside. Substitution of a lysine residue at this site is likely to eliminate subunit A enzymatic activity. The apparently normal levels of hexosaminidase A activity in affected dog samples may be a result of b subunit overexpression. Human Hex B possesses low levels activity against the artificial substrate used to assess Hex A activity, but specificity of activity of the canine enzyme is not known.

Previously presented at the American Society for Neurochemistry: additional data in this abstract. Phenytoin (PHT) is the intravenous drug of choice in humans for seizure emergencies following benzodiazapines. IV fosphenytoin (FOS) is a PHT pro-drug which causes less administration related adverse events. While the short half-life of PHT is not suitable for chronic oral therapy in dogs, IV FOS has not been studied.

Two dogs received 15 mg/kg phenytoin equivalent (PE) and two dogs received 25 mg/kg PE of fosphenytoin intravenously at a rate of 50 mg PE/min. Blood for plasma levels were drawn at 10 time-points over 12 hours; total and unbound drug levels were measured by HPLC. Vital signs including EKG, blood pressure, and neurological examination were monitored.

The half-life of metabolism of FOS to PHT was $10 min, with 4 80% of FOS metabolized to PHT by 30 minutes. Eighty to 84% of PHT was protein-bound during the first 15 minutes after dosing, compared to 90-95% in humans. The elimination half-life for total PHT ranged from 2.8-3.5 hours and for unbound PHT ranged from 1.9-5.4 hours. Dogs receiving 15 mg/kg PE intravenously achieved unbound PHT plasma maximum concentrations of 2.2-2.4ug/ml at 5 minutes, consistent with human loading dose levels. Adverse events observed in some dogs included vomiting, mild ataxia, and short lived tremors, the severity of which appeared dose dependent. All dogs were clinically normal within 30 minutes of all doses.

A 15 mg/kg PE dose of IV FOS appears adequate for production of PHT levels predicted to be effective for the treatment of canine seizure emergencies. Further studies in clinical canine patients are warranted. Acquired myasthenia gravis (MG) is caused by antibodymediated inactivation of the acetylcholine receptor on the neuromuscular endplate causing focal, regional or generalized muscle weakness. Many medical treatments have been reported; however, responses to therapy and outcomes are unpredictable and death often results from aspiration pneumonia.

Therapeutic apheresis is an extracorporeal procedure that separates blood into its components for removal or specific alteration prior to return to the patient. Therapeutic plasma exchange (TPE) is an apheresis treatment in which plasma (containing pathologic antibodies) is removed and exchanged with donor plasma. TPE is used routinely to treat MG in human patients with severe disease or disease unresponsive to conventional therapy.

We report the successful use of TPE to treat 2 large breed dogs with confirmed MG (aceytlcholine receptor antibody concentration: 3.05 and 3.32 nM/L, respectively; normal concentration: o 0.6 nM/ L) that was severe and not adequately responsive to traditional therapies. Both dogs were non-ambulatory, recumbent, and demonstrated megaesophagus and aspiration pneumonia.

Three TPE treatments (1 plasma exchange each) were performed over 5 and 7 days, respectively, in each dog without complication. Both dogs became ambulatory within 3 days of starting TPE treatment with subsequent resolution of regurgitation and megaesophagus. Pyridostigmine was continued during TPE sessions and discontinued in both dogs within 3-6 months. Both dogs remain asymptomatic and have had no recurrence of MG during 16 and 4 months of follow-up, respectively.

TPE is a viable treatment option for dogs with MG that have severe disease, life-threatening complications or that remain unresponsive to traditional therapies. TPE may alleviate clinical signs more rapidly, and improve long-term outcomes when compared to historical experiences in patients with comparable disease. Clinical findings, clinicopathologic data, imaging features, and treatment of canine spinal meningiomas have been described in the veterinary literature, but histological characteristics and tumor grading have less commonly been reported. The aims of this retrospective case series were to describe the clinical, imaging, and histologic features of seven canine spinal meningiomas including a cervical spinal cystic meningioma that had imaging and intraoperative features of a subarachnoid cyst.

Medical records from dogs with a histopathological diagnosis of spinal cord meningioma presented to the Veterinary Teaching Hospital between 2006 and 2010 were reviewed. Signalment, presenting clinical signs, physical and neurologic examination, clinicopathologic data, surgery reports and available images were reviewed. All meningiomas were histologically classified and graded following the international WHO human classification for CNS tumors. Seven dogs were included, 4 males and 3 females. Median age at presentation was 8.7 years (range, 3.5-11.4 years), and median weight was 35 kg (range, 8-45 kg) . Median time between onset of clinical signs and diagnosis was 108 days (range, 45 days -1 year). Cerebrospinal fluid (CSF) analysis was performed in 4 dogs, showing increased protein concentration in 2 cases, and being normal in the other 2. Spinal radiographs revealed vertebral canal widening in one case. Myelography (4/7) showed intradural/extramedullary lesions in three cases, one of them consistent with a CSF-filled subarachnoid cavity, and an extradural lesion in one case. Magnetic resonance imaging (MRI) was performed in all cases and revealed mild to marked hyperintensity on T2W and precontrast T1W images and homogeneous contrast enhancing (CE) intradural/extramedullary masses (4 cervical and 2 thoracic) in six cases, with one of these showing an additional intramedullary CE pattern. A dural tail was identified in two dogs. One dog had a fluid-filled subarachnoid enlargement located dorsally to the spinal cord. This lesion was hyperintense on T2W, hypointense on T1W and FLAIR images, and did not enhance. It was diagnosed as a spinal subarachnoid cyst, but the histopathological study of the surgically resected mass revealed a grade I cystic meningioma. Five other cases underwent cytoreductive surgery, two transitional meningiomas (grade I) that survived 3 (alive at the time of writing) and 7 months; and three anaplastic meningiomas (grade III) that survived 10-16.6 months before neurological deterioration and euthanasia. Another anaplastic meningioma was euthanized right after diagnosis.

There are few reports grading canine spinal meningiomas, with most being grade I or II. Of the few grade III tumors reported, only one had been treated surgically and was euthanized 90 days later because of neurological deterioration. We report four grade III (anaplastic) meningiomas, three of which surgically treated and with longer survival times. Finally, cystic meningioma should be considered in the differential diagnosis of cases with imaging features consistent with arachnoid cyst because of their similar appearance, making histopathological analysis essential for a definitive diagnosis. Head trauma is a common veterinary emergency, but few prognostic indicators have been studied in dogs, making it challenging for clinicians to counsel clients about the odds of recovery. A recent meta-analysis showed that higher plasma glucose, lower plasma pH and lower hemoglobin at admission were associated with increased risk of death in human head trauma. The goal of this retrospective study was to investigate the association between admission point of care blood gas parameters and survival to discharge in dogs with head trauma.

Fifty one dogs presenting to the Cornell University Hospital for Animals with head trauma from 2007 to 2010 that had a blood gas analysis done within 1 hour of presentation were eligible for inclusion. Parameters assessed included glucose, base excess (BE), anion gap (AG), pH, hemoglobin, and sodium.

Biochemical data were found to be normally distributed using the Kolmogorov-Smirnov test. T-tests or Welch tests were used to compare parameters between survivors (S,n 5 42) and non-survivors (NS, n 5 9). Of glucose, BE, AG, pH, hemoglobin, and sodium, only mean glucose (S 5 131 mg/dl, NS 5 171.4 mg/dl, p 5 0.029) was significantly different between groups, although there was a trend for a difference in mean BE (S 5 À3.6, NS 5 À8,5, p 5 0.055). Logistic regression analysis showed that of the parameters, only BE was independently associated with outcome (odds ratio 0.79, 95% CI 5 0.63-0.98, p 5 0.036).

These results suggest that two easily measured biochemical parameters (glucose and BE) may yield useful prognostic information in dogs with head trauma, but further studies are needed to further elucidate these findings. Type I intervertebral disc disease (IVDD) commonly affects chondrodystrophic dogs. Neurological recovery and outcome following surgical decompression may be unpredictable due to suspected ischemic neuronal injury. Hyperlactatemia has been associated with spinal cord injury in humans and experimental animals. The purpose of the study was 1) to determine the relationship between serum and CSF lactate levels and 2) to compare lactate levels with neurological outcome following decompressive surgery in dogs with IVDD.

Healthy, chondrodystrophic dogs diagnosed with IVDD localized to the T3-L3 spinal cord were included. Serum lactate levels were obtained at: anaesthetic induction, skin incision, muscle dissection, and extubation. In patients with hyperlacatemia at extubation, additional samples were obtained. CSF was analyzed for lactate concentration. Neurological status was recorded at presentation and multiple times during the recovery period. 31 dogs were included in the study (3-12 years old). 22/31 dogs had normal lactate levels throughout the study. 9/31 dogs had serum hyperlactatemia prior to anaesthetic induction; 6/9 dogs returned to normal during anaesthesia and 3/9 dogs had continued hyperlactatemia until the end of the observation period. Neurological status of the dogs varied similarly between all groups. In 12/14 dogs where CSF lactate levels were measured, initial serum levels were lower than CSF lactate levels; in 5/14 dogs where CSF and serum were collected simultaneously, serum lactate concentration was consistently lower than CSF lactate.

No association between presenting neurological status or neurological outcome and serum or CSF lactate concentration was made. Neither serum nor CSF lactate concentration is useful for predicting neurological outcome in dogs with IVDD. Chiari-like Malformation (CM) has been associated with syringomyelia (SM) in Cavalier King Charles Spaniel (CKCS) and is postulated to result from a mismatch between the volume of the caudal cranial fossa and the brain parenchyma contained within. The objective of this study was to assess the role of cerebellar volume in caudal cranial fossa overcrowding and syringomyelia.

Three dimensional models were created using T2-weighted transverse magnetic resonance images in the commercial software package Mimics s . Volumes of cerebellar parenchyma were analyzed as percentages of caudal cranial fossa volume (cerebellar caudal cranial fossa percentage) and total brain parenchyma volume (cerebellar brain percentage). Data was assessed for normality and the appropriate statistical test was used to compare means/medians between groups.

Forty-five small breed dogs (SB), 58 CKCS and 31 Labradors (LD) were compared. As SM is thought to be a late onset disease process, two subgroups were formed for comparison: 21 CKCS younger than 2 years with SM (group 1) and 13 CKCS older than 5 years without SM (group 2).

CKCS had a larger cerebellar caudal cranial fossa percentage than the other groups .76%] vs. SB 47.81% [40.36-62.91%] and LD 41.32% [32.59-52.95%]; p o 0.001). The cerebellar brain percentage was also larger in CKCS compared to the other groups (CKCS 8.90% [6.62-11.46%] vs. SB 7.37% [5.25-11.34%] and LD 7.23% [6.36-9.54%]; p o 0.001). Group 1 had a significantly larger cerebellar caudal cranial fossa percentage than group 2 (53.71% AE 1.27 vs. 49.31% AE 2.35, p 5 0.001) and a significantly larger cerebellar brain percentage (9.45% AE 0.43 vs. 8.58% AE 0.55, p 5 0.021).

Our findings show that the CKCS has a relatively larger cerebellum than small breed dogs and Labradors and there is an association between increased cerebellar volume and SM in CKCS. Chiari-like malformation (CM) is nearly omnipresent in the Cavalier King Charles Spaniel (CKCS) breed. The mis-match of the caudal cranial fossa and the parenchyma within is thought to lead to syringomyelia (SM). There is currently a lack of information if the morphological changes seen in CKCS with CM are progressive or non-progressive. In this retrospective study we used established measurements of cerebral volumes, foramen magnum height and cerebellar herniation length to assess if there is a significant difference between subsequent magnetic resonance (MR) imaging of the brain of the same dog.

Electronic patient records were reviewed for CKCS with CM which had two separate MRI scans, which were a minimum of 3 months apart. CKCS with diseases affecting measurements were excluded. For the volumetric measurements three-dimensional models were created using T2-weighted transverse MR images in the medical imaging software (Mimics v12.0, Materialise n.v, 2008) . Volumes of the caudal cranial fossa parenchyma were analyzed as percentages of caudal cranial fossa volume and caudal cranial fossa volume was analyzed as a percentage of total cranial cavity volume. The volume of the ventricular system was recorded as a percentage of total parenchymal volume. Data was assessed for normality and the appropriate statistical test was used to compare means/medians.

Twelve CKCS were included with a median scan interval of 9.5 months (3-83 months). The size of the foramen magnum increased significantly between the first and second scan (1.52 AE 0.08cm vs. 1.59 AE 0.09cm; p 5 0.03), as did the length of cerebellar herniation (0.17 AE 0.05cm vs. 0.22 AE 0.09cm; p 5 0.02) and the caudal cranial fossa percentage (13.44% [9.9-15.28%] vs. 13.96% [9.9-15.48%]; p 5 0.02). There was no significant difference noted between the two time points in any of the other volumetric measurements ( This work could suggest that overcrowding of the caudal cranial fossa in conjunction with the movements of cerebrospinal fluid and cerebellar tissue secondary to pulse pressures created during the cardiac cycle causes pressures on the occipital bone. This leads to a resorption of the bone and therefore an increase in caudal cranial fossa and foramen magnum size allowing cerebellar herniation length to increase. The cord dorsum potential (CDP) is a stationary potential arising in dorsal horn interneurons after stimulation of sensory nerves. CDPs have been recorded in normal anesthetized dogs previously, and normal latency values have been determined for tibial and radial nerves. This study was undertaken to determine whether CDPs could be reliably recorded from the caudal nerves in normal dogs, thus allowing electrophysiological assessment of the cauda equina, and whether neuromuscular blockade improved recording quality.

Ten adult dogs weighing from 23.2 to 32.0 kg were anesthetized and cord dorsum recordings were compared before and after administration of atracurium. Recording needles were placed onto the dorsal lamina at intervertebral sites from L7/S1 to L2/3. Stimulations were made on the lateral aspect of the caudal vertebrae approximately 5-8 cm from the tail base. Recordings from 500 stimulations were averaged.

CDPs were recorded successfully in all dogs. Onset latency varied from 2.2 to 4.7 ms. The CDP was largest when recorded closest to the site of entry of the stimulated nerve into the cord, as determined by post-mortem examination immediately after testing in 6 dogs. Administration of atracurium did decrease muscle artifact, and in some cases helped isolate the origin of the CDP.

These data show that CDPs can be readily assessed from the caudal nerves of anesthetized dogs, with or without atracurium. Cord dorsum potentials from caudal nerves may add important information about the integrity of the cauda equina in dogs with suspected degenerative lumbosacral stenosis. Canine intracranial glial tumors and many human brain tumors express heat shock proteins (HSPs) associated with their degree of malignancy. The up-regulation of HSPs during tumor cell growth helps keep tumor proteins stable and therefore makes them a reasonable target for therapy. Ki67 expression and EC have been strong indicators of cell proliferation and dedifferentiation, respectively.The aims of this study were to determine (i) if canine meningiomas express HSP 27 and/or HSP 72; (ii) whether the expression of the HSPs was associated with Ki67 and/or E-cadherin (EC) expression; and (iii) whether peritumoral edema was associated with HSP, Ki67 and/or EC expression.

Forty-one formalin-fixed, paraffin-embedded canine intracranial meningiomas underwent immunohistochemical staining using anti-HSP 27, or 72 antibodies. These tumor samples were also immunohistochemically stained for Ki67 and EC expression. Canine mammary carcinoma and squamous cell carcinoma tissues served as the control samples, as both have previously been shown to express HSPs. Skin was used as control for Ki67 and EC. Four non-overlapping high power fields of each stained sample were selected and cell staining was analyzed using a semi-quantitative method for HSPs and Ki67; a qualitative assessment was used for EC. All analyses were performed using SAS V 9.2 (Cary, NC). Descriptive statistics of staining percentages were calculated for all tumors tested. Simple Pearson's correlation was used to test for correlations of EC area with HSP areas and Ki-67 percent positive cells and of EC intensity with HSP intensities and Ki-67 percent positive cells. All hypothesis tests were 2sided and the significance level was a 5 0.05. Thirteen meningiomas had MR images quantitatively evaluated for peritumoral edema using T2FLAIR sequences. The edema index (EI) was evaluated for an association with HSP 27, HSP 72, EC and Ki67 expression.

HSP 27 was expressed in 36% (mean 8.7% of cells; range 0-58%), HSP 72 in 52% (mean 5.2% of cells; range 0-26%) and EC in 68% of meningiomas. There was no association demonstrated between either HSP expression variable and EC or Ki-67 expression. There was also no association between the EC expression variables and Ki-67. However, there was a significant negative association between HSP 72 extent (p 5 0.03) and area (p 5 0.04) with EI.

In conclusion, HSP 27 and 72 expression was demonstrated in canine intracranial meningiomas but was not associated with Ki-67 or EC expression. This study suggests that HSPs may not have a significant role in the maintenance of canine meningiomas and so do not represent a novel treatment target for this group of tumors unlike canine glial cell tumors. However, HSP 72 may be involved in the pathogenesis of peritumoral edema in meningiomas and warrants further investigation. An extended release (XR) formulation of levetiracetam, a second generation antiepileptic drug, was recently approved for human use on a once daily basis. Although levetiracetam is clinically effective for seizure control in dogs, it requires a three times daily administration. The potential benefits of the XR formulation include reduced daily dosing leading to improved compliance and relatively constant plasma concentrations. The aim of this study was to compare the pharmacokinetics of levetiracetam XR tablets with immediate release (IR) tablets following single dosing in dogs.

Five clinically and neurologically normal mixed breed dogs were used in a cross-over design. All dogs (mean body weight 25.9kg; range 23.2-30.5) had normal hematology, serum chemistry and urinalyses. Following a 12 hour fast, each dog was administered oral IR levetiracetam (500 mg; mean dose 19.3 mg/kg; range 16.4-21.6). Heparinized blood for drug analysis was taken from each dog prior to administration and 0.25, 0.5, 0.75, 1, 2, 4 and 8 hours after. Blood was immediately centrifuged and supernatant plasma was stored at À801C until analysis. After a 4 day wash-out period, each dog was administered 500 mg oral XR levetiracetam and blood samples were taken at identical timings. Plasma samples were thawed at room temperature before preparation by solid phase extraction for HPLC analysis. Reverse phase chromatographic separation was performed. Levetiracetam and an internal standard were detected using ultraviolet spectroscopy at 205 nm. Concentrations of levetiracetam were determined by peak area comparison to the internal standard. Mean data were fit to a one compartment pharmacokinetic model with first order elimination and absorption and included a lag-phase for XR formulation.

No adverse clinical effects were noted in any of the dogs. The AUC associated with XR was 230 hr ug/mL, a 5.14 fold increase over that with IR (44.8 hr ug/mL). The absorption half-life was 3.2 hr with XR and 0.41 hr with IR, a 7.75 fold difference. The elimination halflife was 3.13 hr with XR and 2.19hr with IR, a 1.43 fold difference. The Tmax associated with XR 5.01 hr and 1.22 hr with IR, a 4.21 fold difference. The Cmax associated with XR was 18.5 mg/mL and 9.62 mg/mL with IR, a 1.92 fold difference. The plasma concentration of IR levetiracetam was not detectable at 8hr after administration whereas it was greater than 10 mg/mL at 8hr after XR administration.

Based on the AUC data, there is an approximately 5 fold increase in bioavailability of the XR compared to the IR formulation. The Cmax was approximately 2 times greater following XR administration and a high plasma level in excess of the suggested canine therapeutic concentration (5 mg/mL) for at least 8 hours. Although specific dosing recommendations cannot be made from this data, the favorable pharmacokinetics of XR over IR suggests that single, daily administration could be efficacious. Thoracic and lumbar vertebrae are frequently affected by fractures and or luxations in dogs following trauma. Surgical repair is part of the emergency treatment described for this disorder but does not guarantee improvement of the associated clinical signs. Multiple surgical repair techniques have been described but have not been compared in terms of their success and the factors associated with a positive outcome. The aims of this study were to retrospectively evaluate the effect of 3 different types of vertebral repair, injury type and injury location on outcome in dogs with thoracolumbar (TL) and lumbosacral (LS) spinal trauma.

Medical records were searched for dogs with radiographic evidence of a TL or LS vertebral fracture and or luxation (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) ; signalment, body weight and duration of disease were recorded. Dogs were retrospectively scored neurologically (0-5; normal to plegic with absent pain perception) on admission and at re-evaluation following surgery. Lesion location was classed as T3-L3 and L4-S3; dogs were evaluated as one group and as two separate groups with respect to outcome. A subset of lesions were classed as cord compression or not based on advanced imaging. Three repair techniques were evaluated (i) pins and polymethylmethacrylate (PMMA); (ii) screws and PMMA; and (iii) spinal stapling. Regression analysis was applied to test for an association between the type of surgery and a successful outcome (non-painful and ambulatory). Simple bivariate analyses were performed to investigate for variables predictive of a successful outcome.

Fifty-nine dogs were included. Twenty-eight dogs were classed as T3-L3 and 31 were L4-S3. There were 55 dogs with fractures and 43 with luxations; 23 dogs had both. Thirty-one of 35 dogs evaluated had spinal cord compression. Ten dogs were repaired with IM pins and PMMA, 18 dogs with screws and PMMA and 31 dogs with spinal stapling. Overall, there was a 78.7% success rate; there was no significant difference in outcome between the anatomic sites (p 5 0.5). All dogs initially graded as 1-3 pre-operation were classed as a successful outcome after at least one week following surgery; 79% of dogs initially graded as 4 (plegic with pain perception) were classed as successful recovery. One dog (12.5%) initially as graded as 5 (plegic with no pain perception) had a successful outcome. A low admission score was statistically predictive of a successful outcome (p o 0.0001). Surgery type was not associated with a successful recovery (p 5 0.13). Signalment, body weight, location of injury, injury type (fracture, luxation or both), presence of compression, and duration of disease did not predict outcome.

From this study, the successful recovery of dogs following surgical fixation is high and is only dependent on the neurological score at the time of admission. The choice of surgical technique does not seem to influence outcome although a prospective study comparing two surgery types is warranted to further investigate this issue the results of which can be confounded by surgeon experience and variable follow-up. Cranial thoracic intervertebral disc disease (IVDD) is extremely rare due to the presence of the intercapital ligament, although anecdotic data suggest German shepherd dogs (GSD) can share some predisposition for this disorder. The objective of the study was to retrospectively evaluate through MRI if cranial thoracic IVDD is significantly more common in GSD compare to other large breed dogs.

A search was done through database of the Ontario Veterinary College. Any GSD were a spinal MRI including T1-T10 spine was performed was recruited. A group of large-breed non-GSD was used as a control. In the midsaggital T2WI plane, three variables were assessed and graded for each intervertebral disc space T1-T9: spinal cord compression (SCC), disc degeneration (DD), and herniation. Wilcoxon sign rank test was used to assess if scores were different between groups. Exact conditional logistic regression was used to determine whether any intervertebral disc space was a risk factor. 22 GSD and 46 large breed non-GSDs were recruited. The GSD group had significantly higher scores than the non-GSD for SCC, and herniation. Regarding the individual intervertebral discs, in the GSD group T2-3, T3-4, T4-5 discs had significantly an increased risk for SCC, and T3-4 for herniation.

The results of this study show that GSD have a higher risk of cranial thoracic disc IVDD than other large breed dogs. That risk was higher in discs T2-T3, T3-4, and T4-5, particularly in T3-4. Genetic and/or conformational factors, such as weakness of the intercapital ligament, may predispose GSD to this lesion. Diskospondylitis is a common disease of the canine spine; however, few reports of MR imaging findings in dogs are available. The purpose of this study was to describe the signalment, clinical and MR imaging features in affected dogs.

Twenty-three dogs with a diagnosis of diskospondylitis based on clinical signs, MR imaging, and urine, blood, CSF and/or intervertebral disk cultures were included. Large breed dogs (4 25kg) accounted for 21of the cases. The mean age was 6.8 years with males and females equally represented. Most dogs (15/23) were ambulatory with varying degrees of pain and paresis.

MR imaging characteristics of 27 sites were reviewed. On T2W images, vertebral endplates were of mixed signal intensity (16/27) while the vertebral body was hypointense (11/27). The intervertebral disk space was hyperintense on T2W (16/27) and STIR (14/15) images and mixed signal intensity (7/13) on T1W images. Paravertebral soft tissue hyperintensities were noted on 10/27 T2W and 12/16 STIR images. Contrast enhancement occurred at 12/19 endplates and 15/19 intervertebral disk spaces. Only 4/18 vertebral bodies and 7/18 parvertebral soft tissues contrast enhanced. Intramedullary spinal cord T2W hyperintensity was noted at 10/23 sites.

Spinal cord or cauda equina compression occurred at 22/27 sites. Based on the Spearman correlation coefficient, a significant direct correlation was found between the degree of spinal cord or cauda equina compression and the patient's neurologic status (P 5 0.0011). The incidence and severity of spinal cord compression in canine diskospondylitis may have prognostic value and may have been previously underestimated using other imaging modalities. Hemilaminectomy and pediculectomy are both well described and commonly utilized techniques to access the spinal canal. These procedures are most often performed to approach a compressive lesion, such as intervertebral disc disease and neoplasia, the goal being adequate visualization of the spinal canal and access to the offending lesion. A proposed benefit of pediculectomy is preservation of the articular facets and thus better maintaining stability of the vertebral column, but at the cost of reduced access to the spinal canal. The purpose of this study was to describe standardized anatomical limits of each technique and report any observed differences that could be considered during presurgical planning.

Ten canine cadavers had both procedures performed on opposite sides to access the T11-12, T13-L1, and L2-3 spinal canal. Measurements were obtained after performing a computed tomography study of the spine and recorded from the transverse slice most representative of the defect. The surgical technique, vertebral site, and side of vertebral column were compared with the mean spinal canal and defect height using a covariate model. Dorsal and ventral remnant lamina heights were also compared.

The height of the defect relative to the spinal canal was 76-87% with hemilaminectomy and 58-75% with pediculectomy. The observed difference in defect height of 12-18% (p o 0.0001) and varied with spinal canal height. Dorsal remnant lamina height was 0.9-2.4% of spinal canal height with hemilaminectomy and 7-19% with pediculectomy. Ventral remnant lamina height ranged from 1-2% and 0.6-2.3%, respectively, though the difference was not statistically significant.

While a larger defect is expected with a hemilaminectomy procedure, our results demonstrate that this difference increases with increasing spinal canal height. Interestingly, the proportion of exposed spinal canal decreases with increasing canal height for both procedures. The difference in defect height between techniques was due to greater removal of the dorsal spinal canal, possibly making the hemilaminectomy technique better suited for more dorsal lesions, while no statistically difference in access to the ventral canal is observed. No effect of vertebral site was detected. Of note was the involvement of articular facets in half of the pediculectomy defects, involving an average of 22% of the articular facet height. This result questions the suggested benefit for the vertebral stability, but further biomechanical studies would be required. Low level laser therapy (LLLT) is a treatment used in human and veterinary medicine for a variety of clinical syndromes. Some uses in human medicine include acute pain associated with osteoarthritis, rheumatoid arthritis, tendonitis, TMJ disorders, chronic joint disorders, and wound healing. Research is currently on-going to determine the adequate wavelengths to promote effective treatment results with LLLT in these conditions. It is purported that LLLT acts via the mitochondria to increase cellular metabolism promoting wound healing and a decrease in pain and inflammation. In this study, we hypothesized that dogs treated with LLLT in conjunction with hemilaminectomy would display quicker recovery times regardless of the presence or absence of deep pain sensation.

Seventeen dogs (9 Dachshunds, 2 Chihuahuas, 2 French Bulldogs, 2 Lhasa Ahpsos, and 1 each of a Pembroke Welsch Corgi, and a Miniature Poodle) were selected and divided into two groups. The dogs ranged in age from 2 to 11 years old, weighed between 7 and 33 pounds, and underwent hemilaminectamies after acute onset of paraplegia secondary to intervertebral disc disease (surgically confirmed). One group received laser treatments on days 1 through 4 of hospitalization. The second group did not receive LLLT, but followed the same peri-operative medication protocol. The laser used in this study was an Erchonia laser model PL5000 (635nm). The hertz setting was similar for each patient using the previously established protocol for intervertebral disc disease (IVDD) with pulse rate ranging from 9Hz to 1151 Hz. All dogs received advanced imaging pre-operatively with myelogram or MRI.

Results of the study revealed that treatment with LLLT of 635nm wavelength did not shorten or improve recovery times for dogs with acute onset paraplegia secondary to IVDD after hemilaminectomy procedures. Dogs that showed recovery to ambulation at the two week recheck were consistently dogs that were deep pain positive on presentation. A lengthened recovery time or no recovery was seen in the majority of those dogs with absent deep pain on presentation as has been revealed historically in past studies. LLLT did not appear to have an effect on this result. However, there are few data describing normal glucose uptake of the canine brain for comparison with suspected or confirmed disease. Thus the purpose of this study was to assess the normal distribution of FDG uptake of canine brain structures using a high-resolution research tomography-PET and 7 T-magnetic resonance imaging (MRI) fusion system.

FDG-PET and T2-weighted MR imaging of the brain were performed on 4 healthy laboratory beagle dogs. Acquired PET and MR images were automatically co-registered by the image analysis software. On MR images, regions of interest (ROI) were manually drawn over 48 intracranial structures, including 6 gross structures (whole brain, telencephalon, diencephalon, mesencephalon, dorsal metencephalon, ventral metencephalon and myelencephalon). A standard uptake value (SUV) and relative SUV ratio (rSUV 5 SUV of ROI/SUV of whole brain) were calculated for each ROI. 7 T-MR images compensated the low anatomical resolution of PET QJ;by proving good spatial and contrast resolution for the identification of the clinically relevant brain anatomy. Among gross structures, mesencephalon and ventral metencephalon had the highest (SUV: 4.17 AE 0.23; rSUV: 1.12 AE 0.03) and the lowest (SUV: 3.34 AE 0.35; rSUV: 0.90 AE 0.06) FDG uptake respectively. When SUVs were calculated on 42 detailed regions, rostral colliculus and corpus callosum had the highest (SUV: 6.00 AE 0.16; rSUV: 1.62 AE 0.05) and the lowest (SUV: 2.81 AE 0.12; rSUV: 0.76 AE 0.06) value respectively. These data acquired from normal dog brain will be used in clinical neurology to investigate various intracranial diseases such as inflammation, neoplasm and behavioral disorders. Degenerative lumbosacral stenosis (DLSS) is a multifactorial condition affecting predominantly large breed dogs. The combination of stenosis and compressive neuropathy cause lumbar pain, lameness and neurologic dysfunction. Previous reports describe urinary and fecal incontinence in severely affected dogs. The objectives of this retrospective case series were to describe the clinical signs associated with dysuria and eventual diagnosis of DLSS in dogs, and to describe factors associated with regained micturition following prompt diagnosis and treatment.

Medical records from the University of Georgia and the University of Missouri between 1995 and 2009 of 11 dogs were reviewed. Inclusion required observation of dysuria, urine retention, absence of structural lower urinary tract disease and concurrent presumptive diagnosis of DLSS. Dysuria was defined as inability to initiate or sustain a urine stream. Urine residual volume was evaluated postvoiding. Dysuria was further evaluated using urethral contrast studies, urodynamic testing (urethral profilometry (4) and cystometry (4)), ultrasonography (5), and urine culture (8). Presumptive diagnosis of DLSS was based on imaging using plain radiography and epidurography (8), computed tomography (1) or magnetic resonance imaging (2).

Breeds represented included the German Shepherd Dog (n 5 3), Golden Retriever (n 5 2), Burnese Mountain Dog (n 5 2), and 1 each Labrador Retriever, Weimaraner, Rottweiler and mixed-breed. All dogs were male. 8 were intact at onset of clinical signs. Median body weight was 38.5 kg (range 29.5-46) and median age was 5 years (range 2-10). Median duration of clinical signs prior to admission was 2 months (range 0.25-12). Other pertinent presenting clinical signs included dyschezia (2), fecal incontinence (4), general proprioceptive ataxia (2), weakness (2), and difficulty rising (1). Physical examination findings included pelvic limb muscle atrophy (2) and prostatomegaly (1). Abnormal neurologic examination findings included postural reaction deficits (6), hyporeflexia (4), decreased tail tone (3) and lumbosacral hyperesthesia (6). Neurologic examination was normal in 3 dogs.

Dorsal laminectomy was performed and diagnosis confirmed in 9 dogs; recovery was monitored for a median of 5.5 months (range 0.25-9). Three of the 9 dogs (33%) regained normal micturition within 0.25-1.5 months of surgery. Though not statistically significant, dogs that regained micturition tended to have a shorter duration of clinical signs (median 0.25 months, range 0.25-2) versus dogs that remained dysuric (median 5 months, range 2-12). Two of the 3 dogs that regained micturition were neutered at the onset of clinical signs, but only 1of 6 dogs that remained dysuric was neutered. Signs improved in all dogs with postural reaction deficits and decreased tail tone. Hyperesthesia resolved in 5 of 6 dogs (83%) and fecal continence returned in 2 of 4 dogs (50%).

These findings suggest that following prompt diagnosis and surgical decompression, normal micturition could be regained in DLSS affected dogs presenting with signs of dysuria. Glycogen storage disease type Ia (GSDIa; von Gierke disease) is an inherited metabolic disorder resulting from a deficiency of glucose 6-phosphatase-a (G6Pase). Previous reports indicate that clinical manifestations of GSDIa occur only in individuals with homozygous expression of a p.I121L mutation. Heterozygote dogs (Het) have been previously reported to exhibit an overall normal outward phenotype. The purpose of this report is to briefly describe some differences that have been observed between Het and homozygous wild type (WT) dogs.

A colony of dogs at the University of Florida contains a mix of affected, WT, and Het individuals. In the course of studies designed to determine the effectiveness of gene therapy for correction of GSDIa in dogs, both WT and Het dogs have been utilized as controls. Available information about body weights, clinical pathology tests, fasting studies, and liver biopsies was retrieved from records for both WT and Het dogs and compared.

Although birth weights are similar, Het dogs have a slower average rate of weight gain than WT dogs and this difference is especially prominent during the first few months of life (Figure 1) . In contrast to affected dogs, both WT and Het dogs are able to maintain normal blood glucose concentrations for up to 10-12 hours of fasting, however, after longer fasts of 12-17 hours, Het dogs have lower glucose and higher lactate concentrations (Table 2 ). In addition, liver biopsy samples from Het dogs had greater apparent levels of glycogen suggested by PAS staining than did samples from WT dogs, and this correlated with the results of proton magnetic resonance spectroscopy which demonstrated 2.9 times greater glycogen content in a liver biopsy sample from a Het dog compared to a sample from a WT dog.

Together, these findings suggest that the level of G6Pase activity in heterozygote dogs does not provide a completely normal physiological, biochemical, or histological phenotype as previously reported. The glucokinase gene (GCK) encodes an enzyme involved in cellular glucose-sensing mechanisms in pancreatic beta cells and hepatocytes. GCK mRNA is present in feline pancreas but the gene is not expressed in feline liver. Hepatic GCK expression is abundant in omnivores so its absence may reflect an evolutionary adaptation of strict carnivores, like feline species. We hypothesized speciesspecific features in the GCK hepatic promoter may underlie the gene expression pattern observed in cats.

The putative feline GCK (fGCK) promoter region was located using bioinformatic software to identify homology with human GCK (hGCK). Genomic DNA from a DSH cat was subjected to direct sequencing using a series of PCR reactions with speciesspecific primers. DNA clones thus obtained were aligned to generate the feline sequence.

Direct sequencing yielded 8.9 kb of genomic DNA sequence with high homology with sequences (ACBE01359231, ACBE01359221) archived in the feline genome project. The feline sequence had six regions homologous with non-coding regions of hGCK; four of these conserved regions are upstream of the putative fGCK start. A 0.8 kb segment immediately upstream of feline hepatic exon 1 is not present in hGCK. The 0.8 kb insert is the reverse complement of a conserved sequence located downstream of exon 1 in feline and human sequences. In conclusion, the putative hepatic promoter of fGCK shares extensive homology with the hGCK promoter but contains a 0.8 kb insert not found in hGCK. Functional studies are needed to confirm the role of the unique insert in regulation of fGCK gene expression. Deuterium oxide (D 2 O) dilution has been proposed for quantifying body water content, but remains difficult to perform routinely. The objective of this study was to assess if the volume of distribution (Vd) of creatinine could be proposed as an alternative in dogs for such a measurement.

Creatinine and D 2 O Vd were measured before (C) and after induction (O) of obesity (by giving an hypercaloric diet (6100 kcal/ kg) for 6 months) in six healthy adult Beagle dogs. Creatinine (40 mg/kg) and D 2 O (200 mg/kg) were simultaneously injected by bolus iv. Blood was collected before administration and then at 5, 10, 30, 60, 120, 180, 240, 360, and 480 min post-injection (creatinine), and 10, 30, 60, 90, 120, 150, 180 and 240 min (D 2 O) . Plasma concentrations of both markers were determined. Vd was calculated using pharmacokinetic equations.

The body weight increased from 10.7 AE 0.6 (C) (mean AE SD) to 15.8 AE 1.0 kg (O). D 2 O Vd decreased from 636 AE 18 (C) to 468 AE 19 (O) mL/kg. Similarly, creatinine Vd decreased from 624 AE 31 (C) to 420 AE 25 (O) mL/kg. The individual difference between creatinine and D 2 O Vd (expressed in % of D 2 O Vd) ranged from À9.5 to 1.0% (C) and from À17.3 to À7.1% (O).

In conclusion, creatinine Vd provides a good estimate of D 2 O Vd in both normal and obese conditions. A 16 wk double blinded study was conducted comparing the affect of two foods on mobility in dogs. All work was approved by an IACUC. 53 healthy beagle dogs (7-15 years old, mixed gender) were used. 33 affected (A) and 20 non-affected (NA) dogs were identified based on orthopedic examination and radiography as having or not having evidence of naturally occurring joint pathology (presence of osteophytes, dysplasia, effusion, pain on manipulation etc) in one or more joints. A and NA dogs were evenly distributed between two locations.

Foods had nutrient profiles adequate for maintenance according to the 2009 AAFCO Official Publication. The Test food contained greater amounts of methionine, manganese, carnitine, vit. E,, vit. C, alpha linolenic acid (ALA), and eicosapentaenoic (EPA) acid: the food provided 388 mg n3 fatty acids and 847 mg n6 fatty acids per 100kcal. All dogs were fed the Control food for 4 wks followed by a 12 wk feeding period where 17 A and 10 NA dogs consumed the Test food and 16 A and 10 NA dogs the Control. Blood and urine were collected at weeks 0, 4, 8 and 12 and analyzed for serum fatty acids and urine thromboxane:creatinine ratios were determined.

Evaluators in this study were different than those making the original diagnosis and so were blinded as to treatment and diagnosis. Orthopedic exams were performed by two veterinary surgeons at each site on weeks 0, 4, 8 and 12. The same two evaluators examined the same dogs throughout. The data was evaluated for the difference between A and NA dogs and between foods with age, gender and location as covariates. Body weight, disease status, age and gender were blocked. Analysis included ANOVA repeated measures mixed procedure (SAS version 9.0) to determine treatment effects over time.Serum EPA was greater and arachidonic acid lower at weeks 4, 8 and 12 in the Test food fed dogs (p o 0.05). Urine thromboxane:creatinine ratios were decreased in the A dogs fed the Test food compared to the A dogs fed the Control food at 12 wks (p o 0.05). Lameness score was significantly improved (p o 0.05) within and between groups of dogs fed the Test food. A significantly greater proportion of A dogs fed the Test food had improvement in total 

Het (n511) Blood glucose (mg/dL) 78 (AE17) 64 (AE13) Blood lactate (mmol/L) 1.1 (AE0.5) 1.7 (AE1.1) joint score, lameness, functional disability and overall assessment score at 12 wks compared to A dogs fed the Control food. 69% of A dogs had an improved Overall Assessment score on the Test food after 4 wks and at 12 wks compared to 40% at 4 wks and 27% at 12 wks of A dogs consuming the Control food. This study shows that a food with moderate amounts of added linolenic acid and EPA can have a positive impact on systemic inflammation and mobility in 4-12 weeks.

A similar abstract will be presented at the Orthopedic Research Society meeting in January 2011 to an audience largely of orthopedic researchers interested in human orthopedics. Fat is an important dietary component, serving both as a source of energy and as a supplier of essential fatty acids (FA). Medium-chain triglycerides (MCT) contain intermediate length FA that do not rely on L-carnitine for transport across the inner mitochondrial membrane, bypassing this rate-limiting step in FA oxidation. Longchain (n-3) polyunsaturated fatty acids (PUFA) from fish oil (FO), and in particular eicosanoids derived from eicosapentaenoic acid (EPA), may protect against excessive inflammatory reactions, which may be exacerbated by eicosanoids derived from (n-6) arachidonic acid (AA). This study investigated the effects of adding MCT:FO and L-carnitine to a control diet (Prescription Diet s k/d s ) on lean body mass, and serum FA and metabolites. Forty healthy Beagles (3.1 to 14.8 y) were fed one of three foods (n 5 13 to 14 dogs each) for 6 mo. The study protocol was reviewed and approved by IACUC, Hill's Pet Nutrition, Inc. All foods were complete, balanced, and sufficient for maintenance of adult dogs; and had similar concentrations of moisture, protein, and fat (approx. 7.4%, 14.0%, 18.1%, respectively).

Composition of serum FA was determined by gas chromatography of FA methyl esters.

Metabolomic profiles of serum samples were determined from extracted supernatants that were split and run on GC/MS and LC/ MS/MS platforms, for identification and relative quantification of small metabolites. Body composition was determined by dual energy x-ray absorptiometry.

Serum concentrations of lauric and myristic FA increased; EPA and DHA increased in a dose-dependent manner; and AA decreased in dogs fed treatment food 3 (proc-mixed procedure in SAS; all P 0.05) when compared to dogs fed treatment foods 1 or 2. Serum concentrations of acetylcarnitine and succinylcarnitine increased, indicating Lcarnitine incorporation, in dogs fed treatment foods 2 and 3. Thus, a diet enriched with MCT:FO significantly altered serum FA composition, enriching (n-3) PUFA and lowering AA concentrations. There was no change in lean body mass for any of the diets compared to baseline values, and no difference between treatments, showing that all three treatment foods met protein requirements. Ten owned dogs, obese for more than 12 months (body condition score [BCS] of 9; fat mass [FM] 5 45.7 AE 1.51%) were studied. These dogs had their weight reduced by 20% (BCS 5 8; FM 5 33.5 AE 1.92%; P o 0.001) being designated weight reduced (WR) group and then were fed to maintain constant body weight during 150 days (BCS 5 8.2; P 5 0.6), designated maintenance (MAIN) group. A control (CT) group of 10 beagles was also included (BCS 5 4.5; FM 5 18.3 AE 1.38%; P o 0.01). In all groups the glucose postprandial response test was performed after 12 hours of fasting. Blood samples were taken prefeeding and after 5, 10, 15, 30, 45, 60, 120, 180, 240, 300 and 360 minutes of the consumption of cooked rice enough to the ingestion of 6g of starch/kg body weight. TNF-a and IL-6 were dosed in MILLIPLEX TM MAP panel, insulin and leptin by radioimmunoassay. Statistical analysis included paired or non-paired T-tests and Wilcoxon (P o 0.05). The regimen normalized meal glucose response, the area under the curve (AUC) of glucose for WR was lower than for obese (P o 0.05) and similar to MAIN and CT (P 4 0.05). Insulin secretion did not normalize immediately, as obese and WR exhibited similar AUC of insulin and higher values than for CT (P o 0.05). MAIN, however, presented similar AUC of insulin than CT, with lower values than obese and WR (P o 0.01), suggesting that dogs require some time to adapt their metabolism. Leptin, TNF-a, and IL-6 presented significant reductions after weight loss (P o 0.05), without differences between WR, MAIN and CT (P 4 0.05), suggesting an improvement of the pro-inflammatory state consequent to obesity. Studying food base excess (BE) modification, methionine intoxication was described. In a basal kibble dog diet (BE 5 305 mEq/kg; 1.98 g/kg of S) two dosages of ammonium sulphate and Methionine was added, resulting in diets with BE of 135 mEq/kg (4.26 g/kg of S) and À51 mEq/kg (6.65 g/kg of S), or BE of 142 mEq/kg (4.68 g/kg of S) and 4 mEq/kg (7.49 g/kg of S), respectively. A 2 Â 2 factorial plus a control diet design, resulting in five treatments, and 32 adult health beagle dogs were used, in a completed randomized design with six dogs per diet. A 5-d adaptation phase followed 3-d of total urine collection (in bottles with 100 mg of thymol). Urine were pooled by dog and analyzed for density, volume and pH. Food macroelements were determined by standards methods (AOAC, 1995) and used for BE calculation. Dog's acid-basic status was studied by blood gas analysis of venous blood, at 8:00h (pre feeding) and 6 hours after meal. A dose-dependent reduction of urinary pH was verified for both compounds (P o 0.01). Blood bicarbonate (r 5 0.98; P o 0.01), and blood base excess (r 5 0.75; P o 0.01) were highly correlated with food BE. Acidemia and reduced blood BE were verified in diets with BE close to zero (higher dose of both compounds, or 4 6.6 g/kg of S), resulting in daily or each other day vomiting episodes in the dogs. Ataxia, seizures, and vomiting were previously describe in dogs fed 47 g/kg of methionine, but our results suggest that a much lower value (27.7 g/kg) was toxic and that the safe upper limit should be between this value and 15.7 g/kg (the lower evaluated dose). In people with chronic kidney disease and heart failure, obesity is associated with longer survival times. This association, called the "obesity paradox," also has been recognized in dogs and cats with heart failure. Excess weight appears to modulate the serious deleterious effects of muscle loss in these diseases. The purpose of this study was to determine the effects of body condition and body weight changes in dogs with naturally-occurring chronic kidney disease (CKD). Dogs diagnosed with ! IRIS stage II CKD between 2008 and 2009 at Iowa State University and Tufts Cummings School of Veterinary Medicine were eligible for the study. Dogs o 1 year of age and those with acute renal failure or suspected congenital renal diseases were excluded. Medical records were reviewed using a standardized data form, and data were collected for initial body weight and body condition score (BCS, 1-9 scale), clinicopathologic values, changes in body weight and BCS, comorbidities, and treatments. Dogs were classified as underweight (BCS 5 1-3), moderate weight (BCS 5 4-6), or overweight (BCS 5 7-9). A change in body weight was defined as 4 0.2 kg. Survival times were determined for all dogs that were discharged from the hospital and lived 4 1 day. Associations between survival and BCS or body weight changes were analyzed using Cox proportional hazards models.

One hundred two dogs were enrolled in the study. At the time of diagnosis, 21 dogs were classified as IRIS Stage II, 57 dogs were Stage III and 24 dogs were Stage IV. Median body weight at baseline was 18.7 kg (range, 2.4-50.1 kg). For dogs with body condition scores recorded (n 5 74), 13 were underweight (18%), 51 were moderate (69%), and 10 were overweight (14%). For dogs that had at least two body weights recorded over the course of their disease, 22 gained weight, 46 lost weight, and 10 had no change in weight. Changes in body weight were not associated with survival; however, BCS at the time of diagnosis was significantly associated with survival. Dogs classified as underweight had a significantly shorter survival time compared to both moderate (P o 0.001) and overweight dogs (P o 0.001).

These results suggest that body condition is an important consideration in dogs with acquired chronic kidney disease. Further studies are warranted to evaluate the relationship between obesity and longer survival in dogs with CKD. Protein restriction is the cornerstone of dietary management of kidney disease. The National Research Council recommends 20% crude protein and the American Association of Feed Control Officials (AAFCO) recommends a minimum of 26% crude protein for maintenance for healthy adult cats. Protein requirement is unknown for adult cats with kidney disease. Most commercially produced cat foods for adult maintenance contains 30% or more crude protein on a dry matter basis. A typical therapeutic food for cats with kidney disease contains about 28% crude protein. The objective of the present study was to investigate whether dietary crude protein at 28.5% would be adequate for the maintenance for adult cats with impaired kidney function.

Seven adult cats, 3 female and 4 male, with age ranging from 5 to 13.7 years old (mean: 8.1 years) were used in the study. All cats had elevated serum creatinine concentration (4 1.6 mg/dl, range: 1.61-2.10 mg/dl) and reduced glomerula filtration rate (mean: 32% reduction; range: 12-60% reduction) during the study. They did not have other systematic diseases, e.g., hyperthyroidism, at the beginning of the study. Cats were fed an expanded dry food made with ingredients commonly used in commercial dry cat foods. The food contained 28.5% crude protein (chemical analysis) and 4366 kcal/kg (calculated) on a dry matter basis, or 65.3 g protein/1000 kcal. Each essential amino acid in the food was at least 130% of that recommended by AAFCO. Other nutrients in the food also exceeded AAFCO's recommendations for maintenance for adult cats. Cats were fed the food for 30 weeks. Lean body mass (dual x-ray absorptionmetry; Hologic, Hologic, Inc, MA) and serum albumin concentration were measured periodically to monitor protein status of cats.

The average lean body mass (mean AE SD) was 3.63 AE 0.82 kg, 3.72 AE 0.85 kg, 3.74 AE 0.84 kg, and 3.75 AE 0.89 kg in weeks 3, 11, 17, and 30 of the study, respectively. Paired t-test did not detect statistical difference (p 4 0.05) when comparing the lean body mass in weeks 3 versus weeks 11, 17, and 30, respectively. Serum albumin concentration were within the normal reference range during the study (mean AE SD: 3.04 AE 0.35%, 2.80 AE 0.35%, 3.04 AE 0.32%, and 3.07 AE 0.40% in weeks 3, 11, 17, and 30, respectively) . These data show that 28.5% dietary crude protein in a dry food with 4366 kcal/kg on a dry matter basis, or 65.3 g protein/1000 kcal, is adequate for maintenance for cats with impaired kidney function. In humans, several disease conditions exist that involve abnormal patterns of polyunsaturated fatty acids and similar abnormalities may be present in companion animals. Indeed there have been reports of decreased plasma arachidonic acid and reduced delta-6 desaturase activities in dogs with atopy and other skin disorders. The present study investigated serum fatty acid profiles in dogs and cats presented to the Texas A&M University Veterinary Teaching Hospital, Clinical Pathology Laboratory over the past one year period. Results were compared with normative data generated among dogs and cats from earlier feeding studies. Sera used were residual samples submitted to the laboratory for other diagnostic procedures and stored frozen for no more than 2 months after collection. The samples were grouped according to presenting disorders involving liver, kidney, digestive, and cardiac diseases. Total lipids were extracted using chloroform:methanol (2:1 v/v) and fatty acid methyl esters were prepared for capillary gas chromatography. Relative percentage distribution of individual serum fatty acids for each animal were then compared with average normative serum phospholipid fatty acid values (dogs, n 5 44; cats, n 5 29) by calculating the ratio of the value in the diseased individual to the normal mean value and used as an index of normalcy. Normalcy ratios were then plotted on a logarithmic scale with normal at 1.0. The ratio was then compared to changes greater than 3, 2 and 1 standard deviations of the normal mean values. In this way a graphical presentation of resultant values was obtained. Although the animals had been fed various commercial diets and some home-prepared foods, a number of noteworthy patterns emerged from this analysis. Dogs showed increased linoleic acid, decreased arachidonic acid, increased total monounsaturated and decreased saturated fatty acids at P o 0.001; oleic acid was increased at p o 0.01. Remarkably, these findings were similar for all canine disease categories evaluated (n 5 30, heart; n 5 17, kidney; n 5 28, liver, and n 5 28 digestive disorders). In cats, a slight decrease in arachidonic acid and large decrease in 22:0 was observed but only in heart disorders. By contrast, modest elevations of arachidonate were observed in kidney, liver, and digestive disease groups but at p o 0.01. Sample sizes of the feline sera were considerably smaller (range of 4-13 per group). A limitation of this analysis is that variability of normal data may exist depending on diet fed making comparisons less reliable However, these preliminary data suggest that metabolic diseases of dogs may depress plasma arachidonic acid independent of diet fed suggesting either reduced conversion from linoleic acid or increased utilization of arachidonate for eicosanoid production during times of metabolic stress. Conversely, in cats, increases in arachidonic acid may be associated with diet arachidonate or other mechanisms. Additional studies to verify these findings are warranted. The objective of this study was to determine whether or not Lalanyl-L-glutamine (Ala-Gln) supplementation in dogs with parvoviral enteritis improves the survival rate and ameliorates clinical signs without side effects. This randomized, double-blinded, placebo-controlled clinical trial included 39 client-owned dogs. The dogs were randomly assigned into two groups and administered Ala-Gln solution (Dipeptiven; 0.4 g/kg) or an equivalent volume of placebo orally twice a day. All of the dogs (Ala-Gln group [n 5 20] and placebo group [n 5 19]) received standard treatment while hospitalized and were monitored daily according to a clinical scoring system and diagnostic evaluation for 11 days. Among the 39 dogs, 17 (Ala-Gln-treated group [n 5 8] and placebo group [n 5 9]) were vaccinated and 22 (Ala-Gln-treated group [n 5 12] and placebo group [n 5 10]) were not vaccinated. The population consisted of 29 purebreds and 10 mixed breed dogs, with a mean age of 12.2 AE 2.2 weeks. The survival data were compared statistically by means of a log-rank test for the Kaplan-Meier survival curves.

The clinical scores of Ala-Gln-treated dogs improved significantly relative to the placebo group. There was a significant difference between the two groups in the survival distribution (P 5 0.038); specifically, 3 of the Ala-Gln-treated dogs (15.0%) died, whereas 8 of the dogs in the placebo group (42.1%) died. No side effects were associated with the administration of Ala-Gln. These results suggest that the oral administration of Ala-Gln is effective in improving clinical signs and survival rate in dogs with parvoviral enteritis. Bleeding disorders, thrombocytopenia and alterations in platelet function have been documented in humans receiving lipid-containing parenteral nutrition formulations. Despite a lack of evidence in the veterinary literature, it is believed that parenteral lipids are contraindicated in critical illness when the development of bleeding disorders is likely. The objective of this study was to determine if there is an in vitro effect on platelet function and thromboelastography (TEG) in normal dogs with varying concentrations of a 20% soybean oil emulsion (Intralipid s ).

Twelve clinically healthy dogs were used for this study. Whole blood platelet aggregation, using ADP and collagen agonists, was measured using multiple electrode aggregometry in hirudinated blood with final lipid concentrations of 0, 1, 10, and 30 mg/ml. The TEG parameters R, K, a-angle, and maximum amplitude (MA) were evaluated from citrated whole blood with equivalent final lipid concentrations as platelet aggregation.

There was no significant difference between groups with collageninduced platelet aggregation. There was a significant increase in the area under the curve (AUC) with ADP-induced aggregation at a lipid concentration of 30 mg/ml (p 5 0.027). The MA was significantly reduced at both the 10 mg/ml (p o 0.001) and 30 mg/ml (p o 0.001) lipid concentration. There was no statistical difference between groups evaluating the other TEG parameters.

While platelet aggregation appeared enhanced at the highest concentration evaluated, this concentration is not clinically relevant. The reduction in MA seems discordant but both fibrinogen and platelets contribute to the MA. Therefore the higher lipid concentrations may be interfering with fibrinogen kinetics or fibrinogenplatelet interaction. In vivo studies are indicated to determine if any of these changes are clinically significant. Rosiglitazone is a peroxisome proliferator-activated receptor gamma (PPARg) agonist and an FDA-approved anti-diabetic agent in humans that has been investigated for its ability to reduce tumor cell growth. Specifically, the combination of rosiglitazone and carboplatin has demonstrated enhanced tumor control.

The purpose of this study was to determine the peak plasma concentrations and side effect profile of rosiglitazone after oral administration in dogs with spontaneously occurring cancer. All dogs received carboplatin intravenously concurrently with oral rosiglitazone. Ten cancer-bearing dogs with normal pre-treatment hepatic and renal function were enrolled. Complete pre-treatment hematological and biochemical parameters were available in ten dogs and post-treatment parameters in nine dogs.

Peak plasma concentrations varied with dose and ranged from 150.3-959.4 ng/mL and occurred between 30 minutes and 4 hours post administration and rapidly declined after the peak. The dose limiting toxicity was hepatic at a dose of 9 mg/m 2 . There was one Grade III, two Grade I ALT, and one Grade III AST elevations noted. No changes in total bilirubin, alkaline phosphatase, or GGT values were noted. Blood glucose values remained within normal limits. Mild, self-limiting gastrointestinal and hematologic toxicities were observed when rosiglitazone was administered in combination with carboplatin.

Based on this study, the recommended dose of rosiglitazone in cancer-bearing dogs with normal hepatic function is 6 mg/m 2 orally once daily. Side effects of the combination appear similar to side effects noted with carboplatin alone. Further study is needed to determine efficacy of this combination and if more frequent dosing is required to maintain plasma concentrations. Carboplatin has shown little activity as a single agent for the treatment of canine transitional cell carcinoma (TCC). However, gemcitabine has shown synergism with carboplatin in human cell lines. The purpose of this study was to evaluate the activity of gemcitabine against canine TCC cell lines alone or in combination with carboplatin. We hypothesized that gemcitabine in combination with carboplatin would have synergistic effects in vitro. The results of this study could provide a rationale for treatment of canine TCC with the combination of these drugs.

TCC cell lines TCC-Kiss, TCC-Knapp-JS, TCC-AXA, TCC-HXC, and TCC-SH were treated with gemcitabine, carboplatin, or the combination. Cell proliferation was assessed using CyQUANT assay, cell cycle was evaluated using propidium iodide staining, and apoptosis was assessed by measuring caspase-3/7 activation. Synergy was quantified by combination index analysis using Compusyn software.

Treatment of canine TCC cell lines with carboplatin or gemcitabine decreased cell proliferation, induced cell cycle arrest, and apoptosis. When TCC cell lines were treated with gemcitabine and carboplatin in combination at a therapeutically relevant concentration (gemcitabine o 100uM, carboplatin o 250uM), a significant decrease in cell proliferation was observed compared to gemcitabine or carboplatin alone, and the drug combination was synergistic in 3 of 5 cell lines, and additive in the remaining 2 lines.

Gemcitabine exhibits biologic activity against canine TCC cell lines and carboplatin combined with gemcitabine exhibits synergistic activity at biologically relevant concentrations. Our results support further evaluation of these drugs in dogs with TCC to determine the clinical efficacy of this combination. Metronomic chemotherapy has been shown in murine models and humans to improve tumor control by inhibiting tumor angiogenesis and suppressing regulatory T cells (Treg). Treg are a subset of T lymphocytes demonstrated to be increased in humans and dogs with cancer and are thought to suppress cellular immune responses against tumors. The purpose of this study was to determine whether metronomic cyclophosphamide therapy depletes Treg and/or exhibits antiangiogenic activity in dogs with soft tissue sarcoma.

Client owned dogs with histologically confirmed grade I or II soft tissue sarcoma were administered cyclophosphamide at 12.5 mg/m 2 or 15 mg/m 2 orally once daily for 28 days. Whole blood and tumor biopsies were obtained on days 0, 14, and 28. Flow cytometric analysis of blood was performed to assess changes in T lymphocyte subsets, including CD4 1 and CD8 1 cells as well as CD4 1 FoxP3 1 Treg. Tumor microvessel density (MVD) was assessed by performing immunohistochemistry for CD146.

Five dogs were enrolled in the 12.5 mg/m 2 /day dose cohort and six dogs were enrolled in the 15.0 mg/m 2 /day dose cohort. In patients that received cyclophosphamide at 12.5 mg/m 2 /day, the mean number of Treg decreased from day 0 to 28 but there was no change in the mean percentage of Treg or MVD. For patients that received 15.0 mg/m 2 /day, both the mean number and percent of Treg as well as MVD decreased over the 28 day time period.

Cyclophosphamide at 15.0 mg/m 2 /day or greater selectively depletes Treg and inhibits angiogenesis in dogs with soft tissue sarcoma. Arsenic trioxide (ATO) is used to treat leukemias, multiple myeloma, and relapsed lymphoid malignancies in humans; its use has not been explored in veterinary oncology. Prior therapy with glucocorticoids decreases likelihood and duration of remission for dogs with lymphoma treated with chemotherapy. We hypothesized that ATO will re-sensitize glucocorticoid-resistant canine lymphoma cells to glucocorticoid-induced apoptotic death.

The OSW canine lymphoma cell line was cultured with 200 uM dexamethasone. Remaining viable dividing cells were considered resistant. Resistant cells were exposed to 0.01 uM and 0.02 uM of ATO without dexamethasone, after which cells were washed and re-exposed to 200 uM dexamethasone. After 24, 48 and 72 hours of dexamethasone exposure, cells were counted using Trypan blue stain. Apoptosis was assessed by TUNEL assays on cytospin preparations collected at 24, 48, and 72 hours from ATO-exposed and control groups. Statistical analysis was performed using 1 way ANOVA and Tukey's test.

The proportion of dead cells increased over time in both 0.01 uM and 0.02 uM ATO exposed groups. The proportion of dead cells was greater for 0.01 uM ATO (p o 0.001) and 0.02 uM (p o 0.001) groups compared to control. Apoptosis increased with increasing ATO concentration and duration of dexamethasone exposure compared to control.

These results support the effectiveness of ATO at re-sensitizing glucocorticoid-resistant canine lymphoma cells to apoptotic death following re-exposure to glucocorticoids. Ongoing gene expression studies aim to elucidate this mechanism. Additional studies to determine if this effect is seen with other chemotherapeutic agents are warranted. Lymphoma is the most common hematopoietic tumor of dogs. Protein disturbances may be associated with this disease including monoclonal gammopathies in a low percentage of cases. Serum protein electrophoresis (SPE) is routinely used to aid diagnosis of various canine diseases including lymphoma when total protein concentration is elevated. The purpose of this study was to compare SPE changes in lymphoma patients without elevated total proteins with a population of healthy dogs.

Agarose gel electrophoresis was performed on residual serum from 17 healthy control dogs and 21 untreated dogs with multicentric lymphoma (stage III -V) after measuring total protein (TP) using the biuret method. Densitometric traces of the protein bands were obtained using computer software (Totallab 100) and the albumin, alpha-1, alpha-2, beta-2 and gamma globulin subfractions were identified by visual inspection. The total protein concentration, the number of subfractions and the relative and absolute protein subfraction concentrations were then compared statistically between the two populations.

In lymphoma dogs, TP, absolute albumin, beta-2 and gamma globulin concentrations and both relative and absolute concentrations of the alpha-1 globulins were significantly lower however relative and absolute alpha-2 globulin concentrations were significantly elevated. No monoclonal gammopathies were identified in any of the dogs and not every patient with lymphoma had the above changes in their electrophoretogram.

This study has demonstrated that significant changes occur in the albumin and globulin fractions of canine lymphoma patients despite no obvious increase in TP. Further investigation is required to identify the proteins responsible for these changes. It is well known that immunophenotype has a prognostic value for the outcome of canine lymphoma, with T-cell lymphomas having a worse prognosis than B-cell lymphomas. The recent advent of flowcytometric techniques allowed easy detection of many different markers on lymphoma cells and therefore, not only distinguish between T and B cells, but also estimate possible aberration on immunophenotype. In human oncology, although some controversy persists, it seems that non-Hodgkins lymphoma and acute leukemia carrying aberrations have a worse prognosis.

The aim of this study was to evaluate the role of immunophenotype aberration in canine high-grade lymphoma considering outcome and time span to achieve complete response under chemotherapy. Samples of bone marrow, blood and lymph node suspensions from twentythree dogs were evaluated with flow-cytometry.

Eleven dogs had aberrant expression of neoplastic lymphocytes and twelve were non-aberrant. The most common aberrations found were: positivity to CD34, biphenotypes, double expression of Tantigens (CD41, CD81), diminished expression of CD45. All dogs were treated with a CHOP-based protocol. There was a significant difference for the time to achieve response to chemotherapy (partial or complete). 12/12 non aberrant lymphomas went into CR or PR after the first treatment (L-Asparaginase), while aberrant lympho-mas needed more than 2 treatment to reach CR or PR. There was a trend for a prolonged disease free interval with non-aberrant versus aberrant, although it was not statistically significant.

Aberration of immunophenotype may be a prognostic factor for canine lymphomas, but further studies with larger groups are needed. Class II major histocompatibility expression is a significant and independent predictor of prognosis in human B cell lymphoma. Low class II MHC is consistently associated with poorer outcome. The mechanism underlying this relationship is not clear, but one hypothesis is that high class II MHC allows for better antigen presentation and tumor-specific immune responses. In the this study, we investigated whether that class II MHC expression in canine B cell lymphoma was associated with remission and survival times. A total of 160 patients were categorized by level of class II MHC,expression of CD34 and cell size for on neoplastic B cells. Multivariable Cox-proportional hazard analysis was used investigate this research question using a randomly selected subset of the data, and the predictive ability of this model was validated on the remaining 1/3 of patient data. Results suggested that low class II MHC expression was associated with decreased times to relapse and death as is seen in human B cell lymphoma, and that large neoplastic cells were associated with decreased survival time. CD34 expression was not associated with patient outcomes. These findings have implications for the use of dogs to model human lymphomas, for the study of tumor vaccines, and for prediction of mortality in dogs with B cell lymphoma with a high level of specificity. One of the reasons for the failure of canine lymphoma treatment is related to the resistance of tumor cells against chemotherapy drugs. The major form of this resistance is provide by multidrug resistance ABC transporters. ABC transporters proteins comprise a large superfamily of transmembrane proteins, ATP-dependent, that extrude a large variety of drugs from the cells. Multidrug resistance phenotype in cancer cells is associated with overexpression of these transmembrane proteins. ABCG2, also known as BCRP, is a 655 residue half-transporter protein that protect hematopoetic stem cells against toxic compounds. The aim of this study was to investigate the expression of BCRP (ABCG2) in canine multicentric lymphoma. Samples were collected by fine needle aspiration of an enlarged lymph nodes, from 25 dogs with multicentric lymphoma (stage III to V) at diagnosis, and 8 normal lymph nodes (control). Dogs that were previously treated with prednisone or chemotherapy were excluded from the study. Quantitative RT-PCR was used to measure the mRNA expression level of BCRP and FLNB expression as a endogenous reference canine gene. A widely range expression value for ABCG2 expression was found for canine multicentric lymphoma. High gene expression was observed in 52% (13/25) canine lymphoma, but 48% of dogs had a lower expression when compared with normal lymph node. Gene expression was not associated with clinical staging, complete or partial remission, relapse and survival time. In conclusion, ABCG2 was expressed in canine lymph node and canine multicentric lymphoma at the diagnosis, and it was not correlated with clinical response. Osteosarcoma (OSA), the most frequent primary malignant bone tumor of dogs, is both locally aggressive and highly metastatic. Prognostic factors for canine OSA include tumor location, distant metastatic disease, and serum alkaline phosphatase (ALP) concentration. An increased serum ALP concentration is associated with poor prognosis; however the mechanisms underlying this phenomenon are currently unclear. During normal bone development ALP may be used as a marker for osteoblasts. Additionally, ALP is a downstream target of activated canonical Wnt/b-catenin signaling. Therefore, we hypothesized that increased serum ALP would be associated with increased expression of b-catenin in canine OSA. The goals of this study were: (1) characterize and compare cellular ALP expression in OSA tissue from patients with normal and high serum ALP; and (2) assess b-catenin expression in those same patient populations.

We used frozen OSA samples collected from patients with either high ALP (n 5 3) or normal ALP (n 5 3). Total RNA was isolated from the frozen tissue, converted to cDNA, and analyzed using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) with either target gene ALP (aim 1), or target gene b-catenin (aim 2). Additionally, b-catenin expression was analyzed by Western blot. QPCR data for bcatenin and ALP expression were normalized to 18S, and relative expression was calculated by the DDCt method.

The relative expression of cellular ALP was higher in high serum ALP samples compared to normal serum ALP samples: 44.74 AE 22.04 (mean relative expression AE standard deviation; p o 0.001). Further, the relative expression of b-catenin was also increased; b-catenin expression of high serum ALP samples relative to low serum ALP samples was 24.57 AE 11.41 (p o 0.001), which is also seen by Western blot.

This study begins to clarify the mechanism behind high serum ALP in canine OSA, and suggests the Wnt signaling pathway may be active in this population of patients. Further work will focus on elucidating the role active Wnt signaling plays in the biology of OSA. In the future, serum ALP status of OSA patients may help identify patients that would benefit from therapies targeting this pathway. Accurate assessment of abdominal lymph node status is of vital importance for appropriate treatment planning and determining prognosis in dogs with apocrine gland adenocarcinoma of the anal sac (AGAAS). Pretreatment knowledge of lymph node status is helpful for determining prognosis and planning the optimal extent of lymphadenectomy. In addition, pretreatment knowledge of lymph node status may help in selecting patients who might benefit from adjuvant chemotherapy and radiation therapy. Abdominal ultrasound is currently the most commonly employed test to screen for abdominal lymphadenopathy in dogs with AGAAS. Imaging studies in people indicate that magnetic resonance imaging ( To determine and compare the plasma concentration of cyclophosphamide and its metabolite 4-OHCP, within the plasma of lymphoma bearing dogs being treated with either oral or intravenous cyclophosphamide.

In this prospective study, patients were randomly assigned to either receive oral or intravenous cyclophosphamide, at a dose of 250 mg/m2. Based on a priori power calculation eight patients per treatment group were enrolled. Plasma was obtained at times 0, 15, 30, 60 minutes, and then at 2, 4, 6, 8, 24 hours post administration for evaluation of 4-OHCP concentrations by liquid chromatography-dual mass spectrometry (LC/MS/MS).

Average values were obtained for both cyclophosphamide and 4-OHCP concentrations within the plasma of both groups. The following values were obtained, half life (HL), time to maximum concentration (Tmax), maximum concentration (Cmax), and area under the curve (AUC). The Mann-Whitney statistical test was used to compare the groups. The AUC for cyclophosphamide was statistically significant (P o 0.05) when compared between the two groups. The AUC for 4-OHCP was not statistically significant between the groups. The difference between Cmax for cyclophosphamide and 4-OHCP was statistically significantly (P o 0.05) between the groups.

Although the AUC for cyclophosphamide was statistically significant between the two groups, the AUC for the active metabolite 4-OHCP was not different when administered intravenously or orally. Thus drug exposure to the active metabolite of cyclophosphamide is the same when administered intravenously or orally.

Previously The percentage of successful intraosseous (IO) catheter insertions, insertion times, and ''ease of use'' scores using the EZ-IO G3 power driver by a wide spectrum of novice participants in feline cadavers were evaluated. Novice users' mean IO catheter insertion time using the EZ-IO G3 driver was also compared to the mean IV catheter insertion time in normovolemic feline and canine patients presented to the Western College of Veterinary Medicine (WCVM) Small Animal Hospital.

Novice users included 40 WCVM personnel (8 technicians, 11 veterinary students, 5 interns, 8 residents, 8 clinicians). After watching a 5-minute EZ-IO G3 training video, each participant inserted 3 IO catheters using the EZ-IO G3 driver. Site (proximal humerus or trochanteric fossa of the femur) and side of cat (right or left) were randomized for each attempt for each participant. A 15 gauge x 15 mm long needle and a 15 gauge x 25 mm needle were used for IO catheter insertion in the humerus and femur, respectively. Participants then graded the ''ease of use'' of the EZ-IO G3 device on a Visual Analog Scale (VAS) that was converted to a 5-point scale. Twenty-six IV catheter insertions in normovolemic feline and canine patients performed by WCVM Small Animal Hospital personnel (6 technicians, 16 veterinary students, 1 intern, 1 resident, 2 clinicians) were then timed and compared to the mean IO catheter insertion time in feline cadavers by study participants using the EZ-IO G3 device.

The IO catheter was inserted correctly on every attempt by 70% (28/40) of participants. No difference was found between participant groups for mean IO catheter placement confirmation percentage (p 5 0.72). Percentage of IO catheter ''slippage off the bone'' at the time of placement did not vary across participant groups (p 5 0.12).

Mean IO catheter insertion times were all less than 10 seconds and did not differ significantly as a function of attempt number (p 5 0.93) or as a function of participant group (p 5 0.78). Participants rated the EZ-IO G3's ''ease of use'' favorably and subjective scores did not differ across participant groups with varying levels of clinical experience (p 4 0.2). Compared to the mean insertion time for IV catheterization (188 sec), mean IO catheter insertion by participants using the EZ-IO G3 (8 sec) was significantly faster (p 5 0.001).

Regardless of their level of clinical experience, participants rated the EZ-IO G3 device favorably in terms of its ''ease of use'' and their willingness to use the device in the future. Regardless of their level of clinical experience, study participants successfully placed IO catheters using the EZ-IO G3 device and did so significantly faster than the reported IV catheter insertion time in normovolemic feline and canine patients in the WCVM Small Animal Hospital. Intraosseous catheterization using the EZ-IO G3 has the potential to provide very rapid vascular access and is a skill that can be easily learned.

Previously presented at the Western College of Veterinary Medicine Undergraduate Poster Competition. Multicavitary effusion is a common cause of presentation for dogs to emergency medical centers. The goal of this study was to identify common underlying causes of multicavitary effusion as well as determine their relative importance. A retrospective analysis of 43 cases of multicavitary effusion admitted to the ICU of a tertiary referral center (Ontario Veterinary College) from 2007 to 2010 was performed. Twenty-three different breeds, with Golden and Labrador retrievers (25.6% and 14%, respectively) being most commonly seen, were included in the study. Ages ranged from 2 to 14 years with a median age of 9 years and a mean of 8.1 years. 69.8% of cases were males (30/43 cases). Most common presenting signs included lethargy (48.8%), anorexia (32.6%), vomiting (21%) and dyspnea (21%). Cavitary effusion was detected by either ultrasonography (pericardial, pleural or abdominal) or radiographs (pleural). Bicavitary effusion was present in 28 cases (65.1%) whereas 15 cases (34.9%) had tricavitary effusion. Neoplasia was found to be the most common underlying cause overall (51.2%), with hemangiosarcoma being the leading type (40.9% of neoplasia cases), followed by congestive heart failure (9.3%), gastrointestinal lymphangectasia (4.7%), peritonitis/pancreatitis (4.7%), cirrhotic liver disease (2.3%) and acute renal failure (2.3%). In 11 cases (25.6%), no underlying cause could be found. Of these, 4 (9.3% of all cases) were diagnosed as having idiopathic pericardial effusion. Taken together, these findings suggest a strong association between multicavitary effusion and diseases carrying a guarded prognosis in dogs. Infection control practices in veterinary clinics and hospitals are becoming increasingly important, with rising client expectations, growing concern about the spread of antimicrobial-resistant pathogens, and the potential for zoonotic transmission of disease. Surgical patients are at increased risk of developing infections, and can serve as sources of these pathogens for other animals and people with whom they have contact within and outside the clinic. Taking all reasonable precautions to reduce the risk of surgical site infections, beginning with preoperative preparation of the surgeon and patient, is therefore an important part of any infection control program. While guidelines are available for preoperative preparation procedures, there has been no objective investigation of compliance with these guidelines in veterinary practices. The objectives of this pilot study were to describe a range of preoperative hand scrub and surgical site preparation practices in veterinary clinics, and to determine if there were any areas that consistently require improvement.

Observation of preparation practices was performed in each of ten clinics over 9-14 days using 2-3 small wireless surveillance cameras. Data was coded for 148 surgical patients, and 31 surgeons performing a total of 190 hand scrubs. Patient hair removal was most commonly performed after induction of the animal (129/140, 92%) and using clippers (114/137, 83%) . Steps in surgical site aseptic preparation ranged from 1-4. Contact time with soap ranged from 18-369s (mean 82s, median 53s), and with alcohol from 3-220s (mean 41s, median 30.5s). Application of alcohol or antiseptic using a ''cleanest to dirtiest'' pattern was infrequent (29/66 (44%) and 11/83 (13%), respectively). Potential contamination of the surgical site occurred most frequently when the animal was moved to the surgery table after initial preparation (40/58, 69%). Preoperative alcohol hand rub was used in 2/10 facilities, but soap and water hand scrub was still more commonly used even at these clinics. Proximal-to-distal scrubbing was noted in 95/142 (67%) of soap and water scrubs. Contact time during surgeon hand preparation ranged from 7-529s (mean 144s, median 124s) for soap and water and from 4-123s (mean 34s, median 25s) for alcohol-based hand rub. Approximately 75% of the variation in contact time was due to inter-surgeon variation. No significant changes in practices were identified over the course of the observation period.

Some preoperative preparation practices were fairly consistent between clinics in this study, while others varied considerably. Contact times with preparatory solutions were often far shorter than recommended, and there was a high frequency of non-sterile contact with the surgical site during movement of patients to the surgical suite. The camera system used to perform this study did not have a significant time-dependent effect on the behavior of participants, and could be useful for performing similar field-based observational studies in the future. This prospective randomized study compared the percentage of successful intraosseous (IO) catheter insertions, insertion times, and ''ease of use'' scores using the EZ-IO G3 power driver to manual IO catheterization in feline cadavers. The IO catheter insertion time in cadavers using the EZ-IO G3 device was also compared to IV catheter insertion time in normovolemic feline and canine patients.

After a purposely limited training period, a preclinical veterinary student was timed and video-recorded as she performed 20 IO catheter placements in feline cadavers (10 IO insertions by placing an Illinois needle manually and 10 IO insertions using the EZ-IO G3). Order of technique (manual or EZ-IO G3), site of IO placement (proximal humerus or trochanteric fossa of the femur), and side of cat (right or left) were randomized for each attempt. When using the EZ-IO G3, a 15 gauge x 15 mm long needle and a 15 gauge x 25 mm needle were used for IO catheter insertion in the humerus and femur, respectively. After each attempt, the student graded the ''ease of use'' of each technique on a visual analog scale (VAS) that was converted to a 5-point scale. Twenty-six IV catheter insertions in normovolemic feline and canine patients performed by Western College of Veterinary Medicine (WCVM) Small Animal Hospital personnel (6 technicians, 16 veterinary students, 1 intern, 1 resident, 2 clinicians) were then timed and compared to the student's mean IO catheter insertion time using the EZ-IO G3.Median IO catheter insertion times for the 2 techniques were significantly different (manual IO technique 24 sec; EZ-IO G3 5 sec) (p o 0.001); the manual method took 48 seconds longer (95% confidence interval of 28 to 67 seconds) than the EZ-IO G3 method. Insertion time was more variable for the manual technique than for the EZ-IO G3. Percentage of catheter ''slippage off the bone'' and extravasation around the inserted catheter were significantly higher for placement of the manual IO catheter compared with placement of the EZ-IO G3 catheter (p o 0.001). Student's subjective ratings were more favorable and more consistent for the EZ-IO G3 technique compared to the manual technique for IO catheter insertion. Compared to the mean insertion time for IV catheterization in the WCVM Small Animal Hospital, IO catheter insertion by the student using the EZ-IO G3 was significantly faster (IV catheter 188 sec; EZ-IO G3 IO catheter 7 sec) (p o 0.001).

Intraosseous catheter insertion using the EZ-IO G3 can be said to be significantly faster, less traumatic, more user-friendly, and as effective as IO catheter placement using the manual technique. Vascular access via IO catheter insertion using the EZ-IO G3 device may be suggested to be faster than IV catheter insertion.

Previously presented at the Western College of Veterinary Medicine Undergraduate Poster Competition. Computed tomography (CT) has been widely investigated and applied as a means for non-invasive quantitative bone mineral determination in human medicine. The aim of this study was to assess age-related changes and anatomic variation in bone mineral density (BMD) using quantitative CT in normal cats. Seventeen normal cats were included in this study and divided into the following 3 age groups: o 1 year (n 5 4); 2-5 years (n 5 10); and 4 6 years (n 5 3). A computed tomographic scan of each vertebra from the 12 th thoracic to the 7 th lumbar spine, and the pelvis, was performed with a bone-density phantom (50, 100, and 150 mg/cm 3 , calcium hydroxyapatite, CIRS phantom s ). On the central transverse section, the elliptical region of interest (ROI) was drawn to measure the mean Hounsfield unit value. Those values were converted to equivalent BMD by use of the bone-density phantom and linear regression analysis (r 2 4 0.95).

The mean BMD value of the thoracic vertebrae (651.3 AE 100.4 mg/cm 3 ) was significantly higher than of the lumbar vertebrae (520 AE 119.5 mg/cm 3 ). The maximum BMD occurred at the T12, T13, and L1 levels in all age groups. There was a statistically significant difference in the mean BMD value among the 3 age groups at the T12 (P o 0.001), T13 (P o 0.001), and L4 levels (P 5 0.013), respectively. In addition, there was no significant difference between the mean BMD value of the left and right iliac bodies (485.1 AE 140.4 mg/cm 3 and 482.1 AE 148.2 mg/cm 3 , respectively). The present study suggests that age-related changes and anatomic variation in BMD values should be considered when assessing BMD using quantitative CT in cats with bone disorders. Dynamic contrast-enhanced computed tomography (DCE-CT) is a rapid and widely available method of cerebral perfusion imaging. However, there is no established reference value of cerebral blood flow (CBF) measured by DCE-CT according to a dog's age. The purpose of this study was to identify the correlation between regional CBF and aging in clinically normal dogs using DCE-CT. Fourteen dogs with no evidence of hemodynamic disorders and central nervous system dysfunction were included in this study. Dogs were assigned to the following 3 age groups: o 1 year (group 1); 3-6 years (group 2); and o 10 years (group 3). DCE-CT scans were performed at the level of the third ventricle and mesencephalic aqueduct. CBF in the gray and white matter was calculated using STROKETOOL-CT s software.

The overall mean AE standard deviation quantitative estimate for regional CBF in clinically normal dogs was 67.8 AE 12.9 ml/min/ 100 g, 44.7 AE 4.1 ml/min/100 g, and 31.0 AE 3.4 ml/min/100 g in groups 1, 2, and 3, respectively. There was no significant regional CBF difference between the right and left sides of the brain in each group. Also, a statistically significant difference in the regional CBF was observed between groups 2 and 3 (P o 0.001). Thus, aging affects the regional CBF in normal dogs and the values should be considered assessing the results of DCE-CT. According to several clinical behavior guidelines, ''toileting'' type inappropriate urination (i.e. large amounts of urine deposited in horizontal surfaces) can arise in cats suffering from a medical problem (typically lower urinary tract disease). By contrast, ''spraying'' type behaviour (i.e. possibly smaller amounts of urine deposited on vertical areas) is more typically associated with anxiety brought about by a threat to local resources, arising from either a change in the physical environment or threat to these resources from another cat. However, there is some evidence that ''sprayers'' may also be presented with a medical problem, which might be linked to the disease (e.g. painful voiding associated with crystalluria may lead to a standing posture being adopted and small amounts eliminated at a given time). This might be associated with an apprehensive state or simply a co-morbid state.

As part of a larger research project aimed at investigating behavioral and physical aspects of cats presented with inappropriate urination, owners of 14 ''spraying'' and 18 ''toileting'' cats with appropriate control subjects from the same households were recruited throughout local media coverage and the internet. The case-control dyads were brought by the owners to the veterinary hospital of the University of Sa˜o Paulo, at the same time, for a medical work-up (i.e. physical examination, complete blood count, biochemical profile, urinalysis, urine culture and abdominal ultrasound).

No significant differences between the ''sprayers'' and ''toileters'' regarding the occurrence of medical problems were found. Both groups had a similar proportion of cats affected by medical illnesses (sprayers: 35.7%, toileters: 44.4%; Chi 2 , p 5 0.618), directly or indirectly relating to the urinary system (e.g. diabetes, chronic kidney disease). In both groups, control cats also had a relatively high occurrence of medical concerns (21.4% and 27.8%, respectively for each control group). These results emphasize the importance of careful medical evaluation of cats presented for a urinary housesoiling problem. The relatively high prevalence of medical concerns among apparently healthy cats in multi-cat households may have arisen, at least in part, as a result of an inability/failure of owners to monitor individuals, thus allowing some early signs to pass unnoticed. The way in which medical and behavioral elements are linked (if at all) remain unknown but deserve further investigation. Considered as a semi-social species, domestic cats appear to be highly sensitive to the effects of social stress, especially when living in high density populations. Cats are capable of adapting to live ingroup; nonetheless, they do not appreciate living in close proximity with others as result of an environment lacking of great opportunities of escaping and hiding.

This study aimed at testing the following hypotheses: (A) owners' perceived quality of life affects cats' global levels of stress; (B) cats' global levels of stress are influenced by cats' personality; (C) cats' living style (single housing versus large group housing) does affect stress levels in cats. To our knowledge, this is the first study investigating stress levels of domestic owned cats, under natural conditions, throughout measurement of faecal glucocorticoids metabolites concentration, and taking into consideration cat personality, cat living style and owner's subjective life quality.

In this study, adrenocortical activity, as a valuable physiological indicator of emotional stress, was evaluated throughout the measurement of faecal glucocorticoids metabolites in fourteen single and sixteen in-group housed cats. Cat personality as well as owners life quality was evaluated by self reported questionnaires given to the owners to answer.

Significant differences in mean glucocorticoids metabolites concentrations (mGCM) between the two populations (i.e. single versus in-group cats) were not detected (Random effect model, p 5 0.178). However, when mGCM were taken as a function of cat personality, there were differences regarding single catstimid cats showed higher levels in comparison to easy-going (Random effect model, p 5 0.018) and bossy (Random effect model, p 5 0.020) cats. As to owner subjective life quality, a direct association between the scores given by the owners to the social dimension and mGCM was found for single cats only (i.e. the better the owner felt itself social wise the higher the mGCM of the cat; Random effect model, p 5 0.002).

Social stratification may compensate the stress resulting from spatial restriction in large in-group living cats. Other underexplored factors such as feline personality and owner life style seem to play an equally important role in domestic cats' day to day levels of stress, especially in the cats kept as single pets. In dogs, RAAS activation is a major feature of congestive heart failure (CHF). Benazepril (Fortekor s ) is a potent ACE inhibitor with well-documented effectiveness in canine CHF. Although ACE activity (ACE A ) has been used in preclinical studies as a surrogate marker of efficacy, some authors have reported a poor correlation between plasma ACE A and changes in angiotensin II (AII) or aldosterone (AL). The purpose of this study was to investigate the effect of benazepril on canine plasma renin activity/concentration (PRA/PRC), angiotensin I (AI), AII, AL, and fractional excretion of potassium (UfeK), sodium (UfeNa) and aldosterone (UfeAL). Sixteen beagle dogs were fed a low-sodium diet and dosed with placebo or benazepril tablets (10 mg PO, q24h) for 5 days. Blood and urine samples were collected on day 1 (D1) and day 5 (D5) over 24-hour periods. Data were analyzed by repeated measures ANOVA of baseline corrected values, and ANOVA of AUC 24hours . Compared with placebo, benazepril induced a significant increase in PRA and AI at D1 (p-value [PRA] :0.001, p-value [AI] :0.002) and D5 (p-value [PRA] :0.001, p-value [AI] o 0.001). No differences in PRC were noticed. Based on AUC 24hours, AII levels were 34% lower in the benazepril group at D5 (p-value [AII] :0.01). UfeAL and AL decreased by up to 27% and 24% at D1 and D5, respectively, though differences did not reach statistical significance. Benazepril markedly influences RAAS dynamics in dogs. Decreased exposure to AII and AL are likely to be the key events required to counteract pathological remodeling of the heart in CHF. This study compared two intravenous anesthetic agents, alfaxalone (ALF) (Alfaxan s , Jurox Pty. Ltd.) and propofol (PPF) (Rapinovet s , Schering Plough Animal Health) and their effects on spontaneous ventilation after induction of anesthesia in dogs at various doses.

This randomized, crossover, dose-escalation study used six dogs in weight and gender-matched pairs (3M-3F). For each drug, each dog was dosed incrementally at 1, 2, 5, 10 and 20 times the labeled anesthetic induction dose rate (ALF 2 mg/kg, PPF 6.5 mg/kg) or until a dose was reached that rendered the dog apneic. A minimum of three days was allowed between doses. For each dose administration, the entire calculated dose was delivered constantly over 1 min.

The primary variable was apnea, defined as an absence of spontaneous ventilation for 1 minute. Apneic dogs were manually ventilated with oxygen until they resumed adequate spontaneous ventilation. Once the apneic dose was determined for an individual dog for one drug, the dog began incremental doses with the alternate drug. For each anesthetic episode times were recorded from completion of induction dose to; removal of endotracheal tube, dog lifting head, dog attaining sternal recumbency and dog standing. Pulse rate, respiratory rate, SpO 2 and ETCO 2 were each measured every 5 min. Within-dog comparisons were made using the paired Student's t-test.

For both ALF and PPF all 6 dogs respired voluntarily at the labeled (1 X) dose. For PPF at 2 and 5 X doses, 4 and 0 dogs respired voluntarily respectively. For ALF at 2, 5 and 10 X doses, all 6, 4 and 1 dog respired voluntarily respectively. For all six dogs to become apneic required 5 X dose of PPF and 20 X dose of ALF. The mean no observable adverse effect dose (NOAEL) expressed as a multiple of the labeled dose was higher for ALF (4.8 X) than for PPF (1.7 X) (p 5 0.035). There were no significant differences between times to extubation, head lift or attaining sternal recumbency after ALF and PPF at 1, 2 and 5 X doses. At the 2 X dose, dogs took longer to stand after ALF (29.0 AE 7.0 min) than PPF (25.0 AE 8.3 min).

We concluded that based on anaesthetic duration, the manufacturer's labeled dose rates of 2 mg/kg for ALF and 6.5 mg/kg for PPF were equivalent. However, based on the dose escalation, the number of dogs becoming apneic at each dose-multiple is consistent with PPF having a narrower safety margin, i.e., PPF caused more respiratory depression than ALF. Parenteral levetiracetam (LEV) has been shown to rapidly attain therapeutic levels in dogs when given IV or IM, and has been used offlabel for the treatment of seizure emergencies. The purpose of this study was to determine the safety and pharmacokinetics of subcutaneously administered levetiracetam in healthy dogs. Potential application of these results would be use of SQ LEV instead of or in addition to rectal diazepam for the treatment of cluster seizures at home.

LEV was administered SQ between the shoulder blades to 4 healthy, purpose-bred hound dogs, at a dose of 60 mg/kg (undiluted). Blood samples were collected at 15, 120 and 420 minutes after LEV administration via jugular venipuncture. Plasma LEV concentrations were measured by high pressure liquid chromatography.

None of the dogs became sedated, nor was there pain evident on palpation of the injection site. Mean (standard deviation) LEV concentration was 65.2 (29.5), 114.5 (10.5) and 84.9 (20.6) mg/ml at 15, 120 and 420 minutes, respectively.

Administration of SQ LEV was well tolerated, and exceeded the suggested therapeutic range (5-45 mg/ml) within 15 minutes of administration, and remained above the range for at least 7 hours. These data indicate that SQ LEV administration may be an alternative for the at-home treatment of cluster seizures in dogs, and prospective studies in epileptic dogs are warranted. The purpose of this study was to assess the effects of CYP inhibitors (ketoconazole, chloramphenicol, fluoxetine, trimethoprim, cimetidine, and medetomidine) in varying combinations on the bioavailability of oral methadone in healthy Greyhound dogs. The IACUC approved this study.

CYP inhibitors were administered PO for 48 hours prior to methadone administration. Methadone hydrochloride was administered PO at a targeted dose of 1 mg/kg. Blood was obtained for the determination of methadone plasma concentrations by mass spectrometry. The area under the curve (AUC) of methadone for each treatment group was compared statistically to the AUC of methadone administered without inhibitors using the Mann-Whitney Rank Sum Test.

Significant increases (P o 0.001) in the methadone AUC occurred in all treatment groups which included chloramphenicol, including chloramphenicol as the only inhibitor. The magnitude of increase was at least 50 fold. Mean concentrations of methadone exceeded 10 ng/mL for at least 10 hours in all groups administered concurrent chloramphenicol. No significant increases in the AUC occurred in any of the groups which did not include chloramphenicol.

In conclusion, chloramphenicol significantly inhibits the metabolism of methadone in Greyhound dogs. As a result, the oral bioavailability of methadone is significantly increased and plasma concentrations are achieved that are reported to be effective in humans for 10-36 hours after a single oral administration. Doxycycline hyclate is used frequently in small animals, horses and exotic animals for treatment of a wide variety of infections. Because doxycycline hyclate tablets may not be suitable for oral administration in some animals, particularly horses and cats, it has been compounded into liquid suspensions. The commercially available doxycycline calcium 10 mg/ml oral suspension, Vibramycin s (Pfizer) is not suitable for use in animals due to its low concentration and flavoring that animals find unpalatable. Because of the known inherent instability of doxycline in aqueous vehicles under storage, this study was conducted to determine the potency of two formulations stored in dark and light conditions. A high pressure liquid chromatography (HPLC) assay with UV absorption at 350 nm was developed for analyzing doxycycline in formulations, in comparison to a reference standard from the United States Pharmacopeia (USP). Doxycycline hyclate 100 mg tablets were first tested for potency. The tablets were then crushed and mixed with a pharmaceutical vehicle to make two concentrations: 33.3 mg/mL and 166.7 mg/mL. The vehicle used was a 50:50 mixture of a Vehicle for Oral Solution (Ora-Sweet, USP-NF) and Vehicle for Oral Suspension (Ora-Plus, USP-NF). The suspensions were prepared in replicates of 3. Each replicate was divided, with one aliquot stored at room temperature in lighted conditions, and the other aliquot stored at room temperature in the dark. Doxycycline was extracted from the formulations and measured by HPLC at Day 0, 1, 4, 7, 14, 21, and 28 . Each replicate was tested and the potency reported as the percent doxycycline relative to the USP reference standard. On Day 0, 1, 4, and 7, the potency of each formulation was within 90-110% of the reference standard (range 93.4-109%). This value is within the accepted range cited in USP o 795 4 on Pharmaceutical Compounding-Non-Sterile Preparations. However, starting at Day 14, the potency declined dramatically and remained low for the tests performed on Day 21 and 28. The potency on Day 14, 21, and 28 was below 20% of the reference standard (range 14-18%). There was also a noticeable change in the quality of the formulation starting on Day 14, and a change in the color of the formulation to a dark brown. These results indicate that when doxycycline hyclate tablets are compounded as a suspension in an aqueous vehicle as described in this study, at 33.3 and 166.7 mg/mL under the storage conditions used in this study, potency of the formulation cannot be assured beyond 7 days. We recommend a beyond-use-day (BUD) of 7 days for formulations prepared and stored at room temperature in light or dark conditions. Therapeutic options for multidrug resistance (MDR) Escherichia coli urinary tract infections (UTI) are limited. Fosfomycin (FOS) tromethamine is an oral, broad-spectrum, cell-wall active, bactericidal drug approved for treatment of uncomplicated UTI in humans. The purpose of this study was to determine time dependency of FOS and the disposition of FOS tromethamine in dogs. Using a randomized, double crossover design, 12 client-owned dogs received FOS sodium IV (40 mg/kg) and FOS tromethamine (PO, 80 mg/kg) either with (n 5 6) or without food (n 5 6). Serum and urine were collected for 24 hr; FOS was quantitated with a bioassay (ATCC E. coli 25922, serum or ATCC Proteus vulgaris 13315, urine). In-vitro killing curves (cell counts through 24 hours) were performed at 0 (control),0.5,1,2,8,16 and 32 x MIC for MDR E. coli canine FOS susceptible (E-test s ) uropathogens. Killing curves indicated FOS to be time dependent. After IV administration, clearance (mlÃkg/hr), volume of distribution (L/kg), elimination half-life (HL; hr) and mean residence time (MRT, hr) were (mean AE sd): 210 AE 104, 0.23 AE 0.15, 0.36 AE 0.19, 1.14 AE 0.35 and 1.7 AE 0.4, respectively. For PO, C max , HL and MRT were 66 AE 21, 2.5 AE 1.09 and 5.1 AE 1.7, respectively. Serum FOS exceeded the MIC 90 reported for multidrug resistant (MDR) E. coli (1.5 mg/ml) for 7hr (IV; 2.5 mg/ml) and 12hr (PO, 9 mg/ml Diminazene is an aromatic diamidine, anti-protozoal drug that has shown promise in a small number of cases of cytauxzoonosis. In a noncontrolled case series, 5 of 6 cats with clinical cytauxzoonosis given 3 mg/ kg of diminazene aceturate survived infection. Dosage frequency was two intramuscular injections given one week apart. Commercial formulations contain the diminazene diaceturate salt. The active base is diminazene with the salt consisting of two aceturate molecules. Currently there is no data available on the pharmacokinetics of either diminazene compound in cats. The objective of this study was to determine the pharmacokinetics of diminazene diaceturate in healthy cats.

Four purpose bred cats with normal physical examination, CBC, chemistry and urinalysis were used. A powdered commercial drug formulation (Veriben s , Ceva Sanet Animale) was freshly reconstituted with sterile water to a concentration of 7 mg/mL prior to administration and sterile filtered solution. Heparinized blood samples were collected just before (hour 0) or 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72, 120 , and 168 hours after intramuscular administration of 3 mg/kg (1.68 mg/kg of diminazene base) diminazene diaceturate. The plasma was separated by centrifugation within 30 minutes of collection and frozen (À801C) until analysis. Concentrations of diminazene were measured by HPLC analysis using UV absorption and ion-pairing conditions. The pharmacokinetic profile was analyzed using a simple one-compartment model. In these cats, diminazene had a mean terminal half life (T 1/2 ) of 1.70 (1/-0.29) hrs and mean peak plasma concentration (C MAX ) 0.51 (1/À 0.11) mg/mL. The mean residence time (MRT) of diminazene was 2.45 hrs (1/À0.42). Systemic clearance (CL/F) was 1.38 (1/À 0.26) L/kg/hr. The volume of distribution per fraction absorbed (VD/F) was 3.36 (1/À 0.72) L/kg.

A single intramuscular dose of diminazene diaceturate was well tolerated by all 4 cats. Without knowing the concentration required to inhibit or kill Cytauxzoon felis, it is not yet possible to make suggestions regarding optimum dosing schedules for this drug. Additional toxicology data and studies to assess clinical efficacy for the treatment of cytauxzoonosis are indicated before routine clinical use can be considered. Meloxicam has been shown to accumulate in areas of inflammation in both the rat and human. The objective of this study was to investigate the concentration of meloxicam in synovial fluid of inflamed joints versus that of non-inflamed joints in dogs.

Eight male dogs were treated with 0.2 mg/kg of meloxicam on Day One and 0.1 mg/kg of meloxicam on Day Two. All treatments were administered orally. On Day Three reversible acute synovitis was induced in one stifle by aseptic, intra-articular administration of 1 ml sodium urate crystal suspension (10 mg/ml). In four dogs synovitis was induced in the L stifle and in four dogs the same procedure was used in the R stifle. In each dog the stifle without induction of synovitis served as the ''normal'' joint sample. A synovial fluid sample was collected from both the R and L stifle of each dog. Sample collection occurred eight hours after administration of sodium urate and twenty four hours after the last administration of meloxicam.

Synovial meloxicam concentration was analysed using High Performance Liquid Chromatography-Mass Spectrometry (HPLC/ MS-MS). The concentration of meloxicam in the inflamed versus non-inflamed joint in each dog was compared using the paired t-test. The results indicate that meloxicam preferentially accumulates in inflamed joints in the dog as meloxicam concentrations are statistically significantly higher in inflamed joints than in non-inflamed joints. No national surveillance system exists for monitoring emergent resistance in companion animals. However, E. coli resistance is an increasing therapeutic and public health concern in these in dogs and cats. The purpose of this study was to describe current resistance patterns of canine and feline pathogenic E. coli throughout the United States and identify risk factors of antimicrobial resistance.

Isolates (n 5 1512) of clinical E. coli collected from dogs or cats from May 2008 through May 2010 located in 6 different regions. Susceptibility was determined to 15 drugs (6 drug classes) by broth microdilution methods. Pharmacodyamaic statistics were described regionally. Phenotypes were determined and type of resistance was based on the number of drug classes to which resistance was expressed: none (NDR), single (SDR) and multi (MDR).

The majority of isolates were from urinary tract (71.5%) and dogs (75.7%). The proportion of resistance type for each drug was: NDR (17.5%), SDR (56.4%) and MDR (26.12%). The proportion of MDR was greatest in the Southwest (25.29%) and least in the Northwest (9.19%) (P o 0.05). For all regions, the proportion of resistance was: cephalothin (CPH, 65.2%) 4 amoxicillin-clavulanic acid (AMX, 59.4%), ampicillin (AMP, 52.9%), tricarcillin-clavulanic acid (TCX, 21.8%), doxycyline (DXY, 15.9%) 4 cefoxitin (CFX, 15.2%), cefpodoxime (CPX, 13.7%), chloramphenicol (CHP, 13.2%), enrofloxacin (ENR, 13.0%) , ciprofloxacin (CIF,12.3%), trimethoprim-sulfamethoxazole (TMX, 10.7%), ceftazidime(CFZ, 10.0%), gentamicin (GTM, 10.0%), cefotaxime (CFT, 9.2%) 4 meropenem (1.1%) (P o 0.05). The MIC 90 exceeded the resistant breakpoint for AMP, AMX, CPX, CPH, CIF, CFX, DXY and ENR whereas MIC 50 did not surpass the susceptible breakpoint. Beta-lactams (96.37%) was the most and aminoglycosides the least (0.12%) SDR. The drug class most frequently involved in MDR was beta-lactams (97.72%) and least, GEN (30.13%).

Resistance differs regionally, being greatest in the Southwest. CPH is the most and meropenam is the drug least associated with resistance; these patterns are consistent with current drugs used by veterinarians. The fluoroquinolones (FQs) are common choices for treatment of E. coli urinary tract infections (UTIs) in animals and humans. 2nd generation drugs approved in animals include enrofloxacin (ENR), marbofloxacin (MAR), orbifloxacin (ORB); human drugs include ciprofloxacin (CIP). 3rd and 4th generation FQ for humans include moxifloxacin (MOX), gatifloxacin (GAT) and ofloxacin, (OFL]), its Lisoform levofloxacin (LEV). For animals, pradofloxacin (PRA) is approved for use in Europe. The purpose of this study was to assess the in vitro activity of 1st (Naladixic acid [NAL] through 4th generation FQs (n 5 11) toward dog or cat E.coli uropathogens (n 5 51). Isolates were subjected to susceptibility testing to 6 drugs classes (15 drugs). Isolate phenotypes included no (NDR; n 5 12), single (SDR; n 5 15) or multidrug (to more than 2 drug classes; MDR; n 5 24) resistance (including ENR resistant [ENR R -MDR; n 5 12] or ENR susceptible (ENR S -MDR, n 5 12). The minimum inhibition concentrations (MICs) were determined for each isolates using broth microdilution (E. coli ATCC s 25922 served as a negative control). MIC statistics were generated for each drug among phenotypes. The overall potency (MIC 90 ) for all ENR susceptible isolates (NDR, SDR and ENR S -MDR) was GAT 4 PRA, MOX, MAR, LEV, CIP 4 SAR, ORB, OFL 4 ENR 4 NAL. Each E. coli isolate expressing NDR or SDR was susceptible to all FQ. However, isolates expressing resistance to 1st or 2nd generation FQ were also resistance to later generation drugs. Glucocorticoids (GC) are standard therapy for allergic asthma but do not reverse the underlying type I hypersensitivity. Allergenspecific immunotherapy (ASIT), a process of ''desensitization'', is potentially curative but requires identification of offending allergens. The purpose of this study was to determine if oral or inhaled GC administered at routinely used dosages would interfere with allergen identification. We hypothesized that oral but not inhaled GC would interfere with accurate identification of allergen-specific IgE using skin and serum testing in experimentally asthmatic cats.

Asthma was induced in eighteen cats using Bermuda grass allergen (BGA). Cats (n 5 6/group) were randomized to receive oral GC (10 mg prednisolone q 24 hr PO), inhaled GC (600 ug budesonide q 24hr) or placebo (gelatin capsule q 24hr PO) for one month. Intradermal skin testing (IDST) and BGA-specific IgE amounts were measured prior to, during (weeks one and four) and every two weeks after treatment until both tests were positive. A paired T test was used to compare serum IgE among groups pre-and post-treatment (P o 0.05 significant).

IDST reactivity was eliminated in 4/6 cats on oral GC, 3/6 on inhaled GC, and 1/6 placebo-treated cats. Within two weeks after stopping treatment, IDST was again positive in all cats. Contrary to our hypothesis, serum IgE reactivity to BGA was not significantly diminished by any treatment.

In conclusion, a two week withdrawal from GCs is adequate for IDST identification of allergen but no withdrawal is required prior to serum IgE testing to identify the sensitizing allergens.

Previously In people, increasing severity of asthma is associated with low serum concentrations of 25-hydroxyvitamin D (25-OH-D). 25-OH-D is thought to ameliorate lower airway inflammation primarily by decreasing the production of pro-inflammatory mediators, and by increasing the production of the anti-inflammatory cytokine IL-10. In people, serum 25-OH-D concentration is associated with sunlight exposure as well as dietary intake. Cats do not rely on sunlight for vitamin D synthesis; all vitamin D comes from dietary intake. Cats have a naturally occurring lower airway disease syndrome (LAD) that shares many features with human asthma. The goal of this study was to evaluate serum 25-OH-D concentrations in cats with LAD.

Cats with naturally developing LAD were enrolled. Criteria for a diagnosis of LAD included a history of cough, wheeze or respiratory distress, radiographic evidence of a bronchial pattern and hyperinflation, negative heartworm antigen and antibody test, and a resolution of clinical signs in response to glucocorticoids. Dietary history was obtained. 25-OH-D concentrations were determined on serum samples by a commercial laboratory.

Twelve cats with LAD were enrolled. All cats ate commercial cat food. The median 25-OH-D concentration was 112 nmol/l with a range of 65-176 nmol/l which is within the reported reference range of 65-170 nmol/l.

In contrast to human asthma, lower airway disease in cats is not associated with low serum concentrations of 25-OH-D. Interstitial lung diseases (ILD) are uncommon in dogs, with the most commonly recognized ILD idiopathic pulmonary fibrosis (''Westie fibrosis''). In human medicine, ILD represent a large umbrella of pulmonary diseases, with IPF only a subset. Other, more treatable, ILDs are also identified, and may respond to either the removal of a stimulus (hypersensitivity) or steroid therapy. The goal of this report is to describe the clinical course, including outcome, computed tomography and histopathology of dogs affected with an ILD.

The computed tomography (CT) log was reviewed for dogs that underwent thoracic CT scanning for evaluation of respiratory signs, and had changes consistent with ILD as the primary abnormality, including the presence of diffuse disease in all lobes, and at least 2 of the following: reticulation, ground glass opacity, consolidation, or traction bronchiectasis. Survival time from CT date was calculated. The presence of moderate pulmonary hypertension [pHTN] (4 50 mmHg) as estimated by tricuspid regurgitant jet, was also reported and survival times were compared with a Mann-Whitney rank sum with P o 0.05 considered significant.

Thirteen dogs were identified. Terriers and Chihuahuas were the most commonly affected breeds. Two dogs were adolescents, the remaining dogs ranged from 7-16 years, with a median of 11 years. Histopathology results (n 5 5), including moderate to severe interstitial fibrosis (3) alveolar proteinosis with fibrosis (1), and interstitial eosinophilic pneumonia (1). One had suspected cryptogenic organizing pneumonia and had a good response to glucocorticoids. Eight dogs died of respiratory failure, with a median post CT survival time of 42 days (range 2-365), two dogs died of non-pulmonary disease, 2 dogs had severe lower respiratory infections as puppies with persistent respiratory signs, and both are still alive at 4 2 years since diagnosis, 1 terrier is alive at 19 months and 1 was lost to follow up. 5 dogs had PHTN, with a median survival of 60 days (42-590), while the 5 dogs without had a survival of 180 days (range 21-730), [p 5 0.3].

Interstitial lung disease in dogs is not just idiopathic pulmonary fibrosis. Following respiratory infection, young dogs may develop an ILD with a relatively indolent course and rare ILD is steroid responsive. CT is useful to identify ILD but further research correlated with echocardiography and histopathology is advised to use it to prognosticate. Idiopathic pulmonary fibrosis (IPF) is an interstitial pulmonary disease, mainly described in West Highland white terriers (WHWT). Identification of molecular pathways important in the pathogenesis of IPF would improve our understanding of this disease and may help identify therapeutic targets. The aim of the present study was to investigate gene expression in lungs of WHWT with IPF using oligonucleotide microarray.

Total RNA was extracted from post-mortem pulmonary samples from five WHWT with IPF and five control dogs (CTRL) without pulmonary disease. The RNA was pooled from each group (IPF and CTRL) and analysed using the canine specific Affymetrix microarray technology. Genes with a minimum of a two-fold difference in expression between the two groups were selected for further analysis. The most significant biological functions for these genes were identified using Ingenuity Pathways Analysis.

More than 1000 genes were identified as having greater than twofold difference in expression. The significant biological functions associated with these genes were related to cellular movement, cellular proliferation and apoptosis. Most notable among these were genes encoding the leukocyte chemotactic proteins: CCL2 (fold change 14.93), CCL17 (12.74) and IL8 (14.32); the proteins involved in fibroblast migration; and the matrix metalloproteinases (MMPs) involved in matrix degradation: MMP7 (À17.55), MMP9 (-3.42), MMP1 (À2.76).

This study has identified genes which may be important in pathogenesis of IPF, e.g. proteins involved in leukocytes chemotaxis, fibroblast recruitment and activation, regulation of apoptosis, and extracellular-matrix turn-over. However, real-time quantitative RT-PCR studies are needed to confirm these results before any definitive conclusions can be drawn. Idiopathic pulmonary fibrosis (IPF) is an interstitial disease, mainly described in West Highland white terriers (WHWT). Defini-tive diagnosis ultimately relies on lung histopathology. Identification of specific biomarkers would be very helpful. Expression microarray is a powerful screening tool to study local gene expression in a disease state. The aim of the present study was to measure gene expression profiles in lungs of WHWT with IPF to identify potential blood or bronchoalveolar lavage fluid (BALF) biomarkers.

Total RNA was extracted from post-mortem pulmonary samples from five WHWT with histopathologically confirmed IPF and five control dogs (CTRL) without pulmonary disease. The RNA was pooled from each group (IPF and CTRL) and analysed using the canine specific Affymetrix microarray technology. IPA-Biomarkers Analysis (Ingenuity System) was used to filter and prioritize biomarkers candidates using the three following criteria: a minimum of a two-fold difference in expression between IPF and CTRL; expression of the gene in lung tissue; possible detection of the protein in blood or in BALF.

Fifty-four molecules met all the criteria. Based on difference in expression, promising proteins included CCL7 (fold change 17.8), a3-Actinin (15.7), CCL2 (14.9), Serum Amyloid A1 (14.4), IL8 (14.3), PLUNC (À25.1), MMP7 (À17.6). Some are well-known biomarkers of IPF in humans either for diagnosis (MMP7, IL8) or prognosis (CCL2).

These results provide novel potential biomarkers of canine IPF. Measurement of these proteins in blood and BALF of healthy dogs, dogs with IPF and with other respiratory diseases is needed to assess their use as biomarkers of canine IPF. Heliox is a mixture of helium and oxygen that has been used therapeutically in human medicine for treatment of airway obstruction. Helium's low density and other physical properties have been shown to reduce the work of breathing by limiting turbulence. The purpose of this study was, therefore, to evaluate respiratory parameters in response to inhaled heliox in dogs with meso-and brachycephalic conformation.

Eleven healthy dogs were recruited, five were mesocephalic and six were brachycephalic. Flow-volume loops were collected using commercial software (Buxcor) while breathing 70:30 Helium: oxygen (Heliox) and 70:30 Nitrogen:oxygen (Nitrox) in a randomized order via a low dead-space face mask. Due to the intrinsic gas properties, gas flow rates and volumes were corrected in-vitro by a conversion factor for the effect of helium on the pneumotachograph. Respiratory rate, tidal volume (ml), minute ventilation (L), inspiratory time (Ti), expiratory time (Te), peak inspiratory flow (PIF) and peak expiratory flow (PEF) were recorded while breathing heliox or nitrox. Values were compared using a paired sample t-test, with P o 0.05 considered significant.

All dogs cooperated with testing. There was no significant difference in respiratory rate, tidal volume, minute ventilation, inspiratory or expiratory times, or peak inspiratory flow. Peak expiratory flow was significantly higher (P 5 0.01) while breathing heliox than when breathing nitrox in brachycephalics but not in mesocephalics (P 5 0.22).

Heliox is well-tolerated in healthy dogs and results in an increased expiratory flow rate in brachycephalic dogs. Further investigation of heliox is warranted in dogs with airway obstruction. of this prospective multicentric study is to assess the effects that surgical correction has on the severity of clinical signs and levels of acute phase proteins (C-reactive protein [CRP] , Haptoglobin [Hp]) and cardiac troponin I (cTnI). Thirty three brachycephalic dogs with BOAS were included and evaluated before and, approximately two months, after surgical correction. The most common components of BOAS found were elongated soft palate (33/33; 100%), stenotic nares (31/33; 94%) and everted laryngeal saccules (13/33; 39.4%). Staphylectomy was performed by means of two different surgical techniques: laser (n 5 12) or electrical scalpel (n 5 21). There were significant differences between dogs depending on the surgical technique used, with a higher reduction of respiratory signs (p o 0.002) and a better postsurgical improvement (p o 0.015) with the use of laser. The levels of CRP, Hp and cTnI were categorized into normal or elevated. Before surgical treatment three (9.1%), six (18.2%) and thirteen (39.4%) dogs had elevated values of CRP, Hp and cTnI, respectively. Two months after surgical correction, five (15.1%), eleven (33.3%) and fourteen (42.4%) dogs had elevated values of CRP, Hp and cTnI, respectively. There were no statistical differences between values of CRP and cTnI before and after surgical correction but the levels of Hp increased significantly after surgical treatment (p o 0.008), probably due to postsurgical treatment with corticosteroids. As previously suggested by others, there was a statistically significant reduction of respiratory and gastrointestinal signs in dogs with BOAS submitted to surgical correction (p o 0,001).

According to the results obtained in the present study, the determination of CRP, Hp and cTnI before and two months after surgical treatment do not have a prognostic value in dogs with BOAS. Even though, near half of the dogs studied had elevated levels of cTnI (39.4%) that persisted after surgical treatment (42.4%), suggesting some degree of myocardial damage is present. Further studies are needed considering the influence of breed and age. To the authors' knowledge, this is the first description of CRP, Hp and cTnI determination in dogs with BOAS. Overweight and obesity are common conditions that lead to alterations in respiratory mechanics, airway resistance, pattern of breathing and gas exchange in humans. The objective of the present study was to investigate if there are significant differences on respiratory parameters and arterial gas analysis of obese and overweight cats, in conscious state and under general anesthesia.

Twenty nine adult cats were arranged in three groups: obese (n 5 15), overweight (n 5 7) and with ideal body score index (BSI) (n 5 7). Mean of BSI in the groups were: 8,8 (obese), 6,3 (overweight) and 4,9 (ideal BSI). Cats did not had respiratory, cardiac or others systemic diseases.

The respiratory parameters were evaluated with a ventilometer equipment coupled to facemasks in conscious cats and directly to the endotracheal tube in anesthetized cats under spontaneous respiration. The anesthesia was performed with propofol (3 AE 1,2 ml/kg) and the cats were maintained in the same anesthetic plan. The three groups were compared by analysis of variance followed by Tukey's test and conscious and anesthetized cats were compared by Student's T test, with a 5% significance level.

There were not observed differences on the respiratory parameters evaluated on ventilometry (tidal volume, expiratory and inspiratory times and peak pressures, respiratory rate and partial pressure of end tidal CO 2 (PETCO 2 )) and on arterial gas parameters (PaO 2 e PaCO 2 ) in the three groups. The PaO 2 of cats with ideal BSI was 88,1 AE 13,5 mmHg, although was not significantly different (p 5 0,06) from overweight (72,0 AE 11,9 mmHg) and obese cats (72,6 AE 14,6 mmHg). Comparison of anesthetized to conscious cats, it was detected decreases in tidal volume, expiratory and inspiratory times and peak pressures and increase in PETCO 2 in respiratory rate in the anesthetized cats. Only PETCO 2 , inspiratory time and respiratory rate in overweight cats did not differ in anesthetized cats.

These results suggest that obesity and overweight did not result in impairment of respiratory function in cats and propofol induced respiratory depression. Osteosarcoma (OSA) is the most common bone tumor in dogs, however, little is known regarding the mechanisms underlying malignant transformation in these tumors. Breeds such as Rottweilers and Greyhounds are at higher risk for developing OSA, suggesting that heritable factors play a role in this disease. MiRNAs have tumor/tissue specific roles in regulating gene expression and dysregulated miRNA expression is found frequently in cancer. We hypothesize that canine OSA is characterized by a unique miRNA expression profile(s) with dysregulation of some miRNAs being associated with specific breeds.

MiRNA expression profiling of primary OSA tumors from 6 Greyhounds and 6 Rottweilers was performed using the Nanostring Technologies nCounter miRNA Expression Assay Kit, interrogating the miRNA expression profile of 651 human miRNAs, 168 of whose mature sequences are 100% conserved between human and dog.

17 miRNAs were differentially expressed in Greyhound versus Rottweiler tumors (p o 0.05), suggesting that breed-specific dysregulation of miRNAs may contribute to the development and progression of spontaneous OSA.

Hierarchical clustering revealed distinct miRNA expression signatures in Greyhound OSA tumors as compared to Rottweilers. Based on these preliminary results, we are evaluating a larger cohort of OSA tumor samples including Greyhounds, Rottweilers, Golden Retrievers, and a mixed population of other breeds. Statistical analysis will be performed to determine the association of miRNA transcript levels with specific breeds and overall outcome. Characterization of miRNA expression in canine OSA will facilitate our understanding the biology of this disease and has the potential to identify targets for therapeutic intervention.

Originally Combination therapies using drugs with documented single-agent activity and lack of overlapping toxicities could potentially improve outcome. The hypothesis intended to be tested is that Palladia s can be safely administered concurrently with a standard weekly protocol of vinblastine (VBL), at dosages known to have activity against mast cell tumors.

Dogs with histologically confirmed measurable mast cell tumors were evaluated for eligibility to enter a standard phase I dose-finding trial (313 cohort), at a starting dose of 2.3 mg/m2 IV VBL (weekly for a total of 4 treatments) and 2.25 mg/kg PO Palladia s EOD, concurrently. Dose escalation of Palladia s was scheduled in 0.25 mg/kg increments until MTD was established or FDA label dose completed (3.25 mg/kg). Safety evaluation was performed weekly throughout the 4 week study period. Dose-limiting toxicities were described following established VCOG-CTCAE(v1.0) criteria. While antitumor response is not a primary endpoint of phase I trials, activity was documented prior to VBL treatments 2-4, and monthly thereafter, based on RECIST criteria.

Nine dogs have been enrolled; cohort 3 is filled and approaching completion of the evaluation period. Hematologic dose limiting toxicity led to 2 de-escalations of VBL. The current safe combination appears to include VBL at 1.6 mg/m2 every other week and Palladia s at 2.25 mg/kg EOD. Response was seen in all but one dog.

Without head to head trials comparing efficacy of bi-weekly VBL combined with Palladia s and VBL alone, choice of therapy should remain at the clinician's discretion.

Originally Prostate specific membrane antigen (PSMA) is a transmembrane protein expressed by tumor-associated neovasculature, but not normal blood vessels. Based upon its selective expression in endothelial cells associated with cancer, PSMA may serve as a conserved angiogenic target shared by macroscopic solid tumors of various histologies.

To investigate the feasibility of targeting a homogenous population of PSMA-expressing endothelial cells as a novel anticancer strategy, we have investigated PSMA expression in several canine hemangiosarcoma (cHSA) cell lines, and subsequently developed self-assembling nanoparticles containing diagnostic (near infrared dyes) and therapeutic (doxorubicin) cargo which selectively bind to PSMA by means of the A10 aptamer, a commercially-available oligonucleotide.

The expression of PSMA by cHSA cells was confirmed transcriptionally and translationally by real time PCR and immunohistochemistry, respectively. Selective binding and endocytosis of A10 decorated nanoparticles was studied by fluorescent microscopy. The ability of A10 decorated nanoparticles encapsulating doxorubicin to exert in vitro cytotoxic effects in cHSA cells was assessed by colony forming assays. Using a cHSA xenograft murine tumor model, clinically-relevant anticancer effects of A10 decorated nanoparticles encapsulating doxorubicin were tested.

All cHSA cell lines expressed PSMA mRNA and protein. A10 decorated nanoparticles were selectively endocytosed by PSMA-expressing cells, and when these nanoparticles encapsulated doxorubicin, significant cytotoxic effects were exerted in vitro. Finally, A10 decorated nanoparticles encapsulating doxorubicin significantly reduced the size of macroscopic cHSA tumor burdens in transplanted mice.

Diagnostic and therapeutic nanoparticles can be targeted to PSMA-expressing endothelial cells, and cHSA provides a comparative model for the future study of nanoparticle therapeutics. Canine transitional cell carcinoma (TCC) is the most common tumor of the urinary tract, and is similar to human invasive TCC in histopathologic characteristics, molecular features, sites of metastasis, and response to medical therapy. Prevalence is increasing, and novel therapies and strategies are needed to effectively treat this aggressive form of cancer in both species.

Personalized medicine techniques intend to improve treatment outcome by using patient tumor profiling to identify potential and individualized therapeutic targets. A genomic algorithm has been developed termed ''coexpression extrapolation'', or COXEN, that aims to use expression microarray data to predict drug activity in patient TCC samples. The utility of this predictive methodology has been established in other types of cancer in vitro, however its clinical utility has not yet been determined.

Validation studies of COXEN in 10 canine TCC cell lines were conducted. The goal was to determine the value of COXEN in predicting baseline sensitivity of canine TCC to 5 chemotherapy agents (gemcitabine, mitoxantrone, carboplatin, vinblastine and cisplatin) that would then be used in a proposed clinical trial. Additionally, expression data from 25 canine treatment-naı¨ve primary tumor samples were generated on an Affymetrix array platform (Canine Genome v 2.0). Both the expression data and TCC cell line data (antiproliferative effects, 50% growth inhibition or GC 50 ) were used to establish a canine specific predictive COXEN algorithm. COXEN scores for canine TCC cell-line drug activity were then analyzed. Scores predicted the activity of cisplatin, gemcitibine, and mitoxantrone in all 10 cell lines, and of carboplatin in 6 cell lines. Because all of the cell lines were sensitive to vinblastine (GI 50 o0.05 mM), the COXEN score was not predictive of its potency. Interestingly, COXEN fails to predict vinblastine response in human TCC cell line data as well. In concurrent work, comparative genomic studies to define and compare the gene expression signatures of TCC in dogs and humans provides further evidence that canine TCC is a valuable genomic model of the human disease. Current studies involve testing the chemo-predictivity of this derived canine COXEN algorithm in additional canine TCC cell lines.

Canine TCC offers an excellent model for in vitro and in vivo studies of the COXEN approach. This preclinical work will be used to guide the feasibility of future COXEN clinical trials in dogs and humans with TCC. A small molecule complex (Aminoact) isolated from bovine milk is a natural peptide mixture with multi-kinase inhibitory effects against epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor-1 (IGFR-1). Ingestion of Aminoact in people with cancer results in lower serum TNF-alpha, an increase in antioxidant superoxide dismutase (SOD) enzyme activity, and subjects' blood serum causes apopsotis in cancer cell lines. This study was designed to first assess safety and secondly the efficacy of three dosage levels of AX-3 in sustaining progression free survival (PFS) for dogs with refractory advanced and/or metastatic cancer.

The prospective, open label study included dogs of different breeds with naturally occurring histologically confirmed malignancies. The first 13 dogs received Aminoact at 1g/m 2 ; the second group of 7 dogs subsequently received the same dosage 1 350 mg of Aminoact; and the third group of dogs subsequently received 2g/ m 2 . Each dog was treated orally daily for six weeks along with 550 mg betaine HCl, that aids in peptide absorption. All patients were evaluated for toxicity using the VCOG-CTCAE and efficacy using the RECIST criteria via assessment of clinical parameters, blood work and client questionnaires.

No toxicity other than mild, transient (grade I) nausea was noted, nor were there any changes in hemograms or biochemical profiles in any patient. Dogs with tumors that were confirmed as responders (4 50% reduction in size) include pulmonary adenocarcinoma, mast cell tumor, trichoepithelioma and soft tissue sarcoma. It appears in limited studies that the response rate may be more durable at higher dosages.

The response to Aminoact is dose dependent and only transient mild toxicity was observed, which suggest the maximum effective dosage has not been reached. Further clinical studies will be valuable in determining the effective dosage and response duration. Treating cancer in dogs with Aminoact offers a unique opportunity as a model for human cancer biology and translational cancer therapeutics. Stereotactic radiation therapy (SRT) combines patient immobilization, image guidance, and intensity modulated delivery to achieve ablative radiation doses within the tumor, while preferentially sparing surrounding normal tissues. The purpose of this study was to evaluate the efficacy of SRT as a means of achieving local tumor control for canine nasal tumors.

Retrospective analysis was performed on dogs with a nasal tumor confirmed by histopathology and computed tomography, no previous surgical or radiation therapy, at least six months of follow-up, and completion of three fractions of SRT at CSU.SRT was administered via the Varian Trilogy linear accelerator once daily for three consecutive days. The Varian Eclipse treatment planwas reviewed to determine the planned target volume (PTV) and dose to 95% of the PTV. Kaplan-Meir survival analysis was performed for disease free interval (DFI) and overall survival (OS).

Sixteen patients with nasal tumors (8 adenocarcinoma/carcinomas, 2 squamous cell carcinomas, 3 chondrosarcomas, 2 osteosarcomas, and 1 undifferentiated sarcoma) were treated with SRT. A median dose of 29.1Gy was administered to 95% PTV with a median PTV of 75.3cc. SRT was well tolerated by the normal tissues with minimal, manageable side effects. To date, the median DFI is 270 days, while the median OS is 367 days.

Based upon the initial clinical experience, stereotactic radiation therapy is an emerging modality in the management of canine nasal tumors. Canine Leptospirosis can vary from subclinical infection to illness that ranges from mild to severe, including death, depending on the susceptibility of the dog, virulence of the organism, and route and degree of infection. The objective of this study was to evaluate the ability of a canine Leptospira bacterin to prevent infection and disease following challenge with virulent Leptospira canicola, L. pomona, L. grippotyphosa, or L. icterohaemorrhagiae. Groups of 8week-old beagles were vaccinated (day 0) and boosted (day 21) with placebo (n 5 10) or the 4-way bacterin (n ! 20) and subsequently challenged with each serovar. The results demonstrated that blood and various tissue samples from placebo-recipients became reliably infected, and the dogs developed typical clinical signs of Leptospirosis including loss of appetite, ocular congestion, depression, dehydration, jaundice, hematuria, melena, vomiting, petechiae, and death. In addition, placebo-recipients developed kidney and liver dysfunction. In contrast, some vaccine-recipients became infected, but the organisms were cleared quickly from the blood. Vaccinated dogs failed to develop severe clinical disease requiring medical intervention, and no animals died (p ! 0.001). A few of the vaccinated dogs developed clinical abnormalities, but the clinical signs remained mild and were self-limiting (p o 0.0001 for each serovar). Administration of the bacterin also prevented thrombocytopenia ( Ciprofloxacin, a synthetic fluoroquinolone antimicrobic, is not FDA-approved for veterinary use. However, due to recent availability of less expensive generic formulations, extra-label use of ciprofloxacin by veterinarians appears more common. Although ciprofloxacin crystalluria and uroliths have been reported in humans, we are unaware of any published reports in dogs. This is surprising since mean urine ciprofloxacin concentration (0.36 mg/ml) in dogs following a modest IV dose (13 mg/kg) was 3 times higher than the solubility of ciprofloxacin in water (0.12 mg/ml). To identify the occurrence of ciprofloxacin uroliths in dogs, records from the Minnesota Urolith Center were reviewed. Between January 2001 and December 2009, ciprofloxacin was identified in uroliths from 58 dogs; uroliths were composed of 100% ciprofloxacin in 10, mixed uroliths containing ciprofloxacin were identified in 6, a shell of ciprofloxacin was observed in 21, and ciprofloxacin surface crystals were identified in 21.

Based on an experimental study in which 83% of human volunteers consuming 1000 mg of ciprofloxacin with NaHCO3 exhibited ciprofloxacin crystalluria (urine pH 47.3), while no volunteers consuming 1000 mg of ciprofloxacin and NH4Cl to acidify urine formed crystals; we postulated that ciprofloxacin uroliths could be dissolved in acidic urine. To test this hypothesis, canine uroliths composed of 100% ciprofloxacin from a single source (6-yr-old male, English bulldog receiving 24 mg/kg of ciprofloxacin PO, q12 hr to manage superficial pyoderma; turbulent flow chromatography/tandem mass spectrometry detected 517 mg of ciprofloxacin/g of urolith) were incubated in urine at selected pH's and monitored for dissolution. Urine obtained from multiple dogs not receiving fluoroquinolones, was pooled and divided into 5 aliquots. Aliquots were adjusted with HCl or NaOH to a pH of 3, 5, 6, 7, or 8. Aliquots were capped and preserved by refrigeration; pH was monitored and readjusted weekly. Ten uroliths of approximately equal weight were randomly assigned to individual flasks containing 10 mls of urine. Flasks were constantly agitated and maintained at 381C. Every 24 hours, urine was discarded and replaced with 10 mls of urine of identical pH until stone dissolution was complete.

Ciprofloxacin urolith dissolution times at each urine pH are reported below.

Ciprofloxacin uroliths are a newly recognized disease and a potential adverse effect of ciprofloxacin administration in dogs. In vitro dissolution of ciprofloxacin uroliths was achieved in canine urine, supporting the premise that in vivo dissolution is possible. Urolith dissolution times were shortest at lower and higher pH's, which is consistent with the pKa (6.0 and 8.8) of this amphiprotic antimicrobic (more soluble at pH below the acidic pKa and above the alkaline pKa). Foods designed to promote struvite urolith dissolution may be designed for short term feeding facilitating rapid dissolution or may be formulated with a more moderate target urine pH to allow for dissolution and then life-long maintenance feeding minimizing recurrence. The purpose of this study was to compare the efficacy and rate of dissolution of a maintenance food with a struvite dissolution food.

Sixteen client-owned adult cats (13 FS, 3 MC) with naturally occurring struvite urocystoliths (mineral composition based on history, radiographs, urinalysis, urine culture and physical examination) were randomized to either a dry maintenance food (Test) or a dry food known to dissolve struvite uroliths (Control). The clinical care team and owner were blinded to treatment assignment. The Test food was formulated to provide 0.06% Mg (DM), 0.65% P, 35% protein, and a calculated target urine pH value (UpH) of 6.2-6.4. The Control food was formulated to provide 0.06% Mg (DM), 0.77% P, 35% protein, and a targeted urine pH of 5.9-6.1. Owners were advised to feed the assigned diet exclusively in an amount to maintain body condition. After diet assignment radiographs were performed at eight weekly intervals until there was no evidence of uroliths or until there was evidence that the uroliths were the same size or larger. A physical examination, complete blood count, serum chemistry profile, urinalysis and urine culture were repeated at the conclusion of the study. Statistical analysis was by ANOVA. All uroliths dissolved in all cats and both foods were palatable. Radiographs of cats fed the Control food indicated the uroliths dissolved in a significantly shorter time (mean AE Std Dev of 1.6 AE 0.7 weeks) compared to cats consuming the Test food (mean 3.9 AE 2 weeks; Po0.05).). Cats in the Control group finished the study at 1 (n 5 4), 2 (n 5 3) and 3 weeks. Cats in the Test group finished the study 1, 2, 3 (n 5 2), 4, 5, 6, and 7 weeks.

All The Minnesota Urolith Center occasionally receives uroliths for analysis that are immersed in formalin. Results of quantitative analysis of these uroliths revealed that some submitted in formalin consisted of newberyite (magnesium hydrogen phosphate trihydrate). Because newberyite is uncommonly found in uroliths formed by cats and dogs, we hypothesized that this mineral was an in vitro artifact caused by exposure of struvite (magnesium ammonium phosphate hexahydrate) to formalin. The purpose of this study was to determine if formalin alters the mineral composition of uroliths.

Urolith submissions containing stones of either 100% struvite (n 5 5 dogs and 5 cats), 100% calcium oxalate (n 5 5 dogs and 5 cats), 100% calcium phosphate apatite (n 5 5 dogs and 5 cats), 100% cystine (n 5 5 dogs and 5 cats), 100% ammonium urate (n 5 5 dogs and 5 cats), and 100% silica (n 5 5 dogs) preserved by only air drying were tested. One urolith from each submission was quantitatively analyzed by polarized light microscopy or infrared spectroscopy. A subsequent urolith from the same submission was immersed in 1 ml of 10% buffered formalin for 48 hours at room temperature. Uroliths were then air dried for 30 minutes and the analysis was repeated.

After exposure to formalin, portions of all 10 struvite uroliths were transformed into newberyite. Three (1 dog and 2 cats) of 10 ammonium urate uroliths were completely dissolved. Newberyite was not detected in any of the remaining uroliths. Likewise quantitative mineral analysis of non-struvite uroliths remained unchanged.

To avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis. We previously reported that transfusion to normal dogs of autologous erythrocyte concentrates (pRBCs) that had been stored for 21 days causes a profound inflammatory response (2X increase in leucocyte count and fibrinogen, 60X increase in C-Reactive Protein). We speculated that inflammation was due to cytokines produced during the storage period, and hypothesized that transfusion of fresh (F) pRBCs would elicit less inflammation than would stored (S) pRBCs. A whole blood unit was collected from healthy dogs (n 5 10) for pRBCs on day 0, then again on day 32. On day 35 dogs received an autologous transfusion of pRBCs stored for either 35 days (S, n 5 5) or 3 days (F, n 5 5). CBCs and in-tem thromboelastometry (CT:coagulation time, CFT:clot formation time, a:alpha, MCF:maximum clot firmness) were evaluated on blood samples collected at 0 (pre) and 5,9,24,48, and 72 hours after transfusion.

Fresh pRBCs did not elicit any change in leucocytes, platelets, or thromboelastometry. Stored pRBCs elicited a degenerative left shift (5 hr) followed by a regenerative left shift (9-48 hr), thrombocytopenia (36% decrease at 5 hr), and marked hypocoagulability characterized by prolonged CT (5,9,24 hr) and CFT (5,9 hr), and decreased a (5,9 hr) and MCF (5,9, 24 hr).

Data are mean(SD). a: p o 0.05 between groups F and S by t test. b: p o 0.05 compared to ''0'' by RM ANOVA.

Transfusion of autologous stored pRBCs elicits a greater inflammatory response than fresh pRBCs, and results in hypocoagulability on thromboelastometry. Clopidogrel is a potent antiplatelet drug that is gaining popularity in veterinary medicine for antithrombotic therapy. The parent molecule is an inactive prodrug that must be converted by hepatic isozymes to an active metabolite. The majority of the parent molecule is directed to the formation of inactive metabolites with only an extremely small proportion of parent molecule directed to the formation of the active metabolite. There are multiple hepatic isozymes responsible for the formation of the active metabolite. A non-specific hepatic isozyme inducer such as rifampin could increase the formation of the active metabolite of clopidogrel thereby increasing the pharmacodynamic response which may allow a reduced drug dose to achieve a clinical effect. We have previously presented data supporting the increased pharmacodynamic response of clopdiogrel after rifampin therapy. The goal of this study was to demonstrate an increased pharmacokinetic response of clopidogrel after rifampin induction of hepatic isozymes.

Six healthy, purpose-bred dogs were used for this study. The pharmacokinetics of clopidogrel were determined by measuring the parent molecule, primary inactive metabolite and active metabolite through LC/MS/MS. The pharmacodynamics of clopidogrel were determined by measuring collagen-induced whole blood aggregation. Blood samples were collected prior to clopidogrel administration (baseline), after 7 days of 2 mg/kg clopidogrel PO q 24 hrs, and after 7 days of 2 mg/kg clopidogrel PO q 24 hrs 1 10 mg/ kg PO q 12 hrs rifampin.

Given the absence of a known standard for the active metabolite, only a semi-quantitative assessment of active metabolite concentration can be made. There was no identifiable active metabolite peak noted at baseline or after clopidogrel treatment. However, with clopidogrel and rifampin combined administration there was an active metabolite peak identified in all dogs with a mean area of 41.7 AE 23.5. The development of the active metabolite peak was associated with an increase in the pharmacodyamic response of clopidogrel in the dogs. This is the first study in any species to document the increased formation of the active metabolite of clopidogrel in response to a strong, non-specific hepatic isozyme inducer. This increased pharmacokinetic response was associated with an increased pharmacodynamic response of clopidogrel. This data provides supportive evidence to develop therapeutic protocols to improve the pharmacodynamic response to clopidogrel in dogs that may reduce dosing requirements or correct subtherapeutic pharmacodynamic response. Critical illness-related corticosteroid insufficiency (CIRCI) has been identified in humans, foals, dogs and cats with lower-thanexpected circulating cortisol concentrations, and/or by a blunted cortisol response to ACTH stimulation. Our purpose was to determine if CIRCI exists in critically ill horses.

Endogenous plasma ACTH and serum cortisol concentrations, and cortisol at T 5 0 and T 5 30 min after 0.1 mg/kg cosyntropin, were measured by radioimmunoassay from horses with colic or systemic illness on admission, and days 2, 4 and 6 of hospitilization. Horses were divided into mild, moderate, or severe illness groups based on clinicopathologic data. Inappropriately low cortisol was defined as endogenous cortisol o mean-1SD achieved after administration of 0.1 mg/kg cosyntropin to normal horses ( o 269 nmol/L). Inadequate delta cortisol was defined as o mean delta cortisol in normal horses after 0.1 mg/kg cosyntropin ( o 159 nmol/L). Cortisol, ACTH and delta cortisol were compared using ANOVA between groups, with P o 0.05 considered significant. Fifty-eight horses classified as having mild (11), moderate (30) and severe (17) disease at admission had survival rates of 100%, 97% and 35% respectively. Admission ACTH and cortisol concentrations were highest in severely ill horses (93 AE 198pg/mL, 361 AE 137 nmol/L) compared to moderate (31 AE 36, 279 AE 137) and mildly ill horses (18.0 AE 12.0, 237 AE 133). Admission cortisol concentrations were higher overall in severely ill horses (P 5 0.016), but were low in 24% (4/17). Admission delta cortisol was low in 85% (11/13) of severely ill horses, and was associated with marked adrenal hemorrhage in non-survivors.

Severely ill horses have high cortisol and ACTH, but low cortisol and delta cortisol may indicate CIRCI secondary to adrenal hemorrhage. Equine pituitary pars intermedia dysfunction (PPID) is a common endocrinopathy of aged horses that results from neurodegeneration of the dopaminergic periventricular neurons that innervate the intermediate lobe of the pituitary. Factors that initiate spontaneous dopaminergic neurodegenerative disease remain elusive, however accumulation of misfolded a-synuclein protein and dysfunctional protein clearance have been implicated. Misfolded protein accumulation occurs due to increased protein production or decreased clearance of damaged macromolecules through the process of autophagy. While have previously demonstrated that horses with PPID have increased asynuclein in the periventricular neurons compared to controls, it remains unknown whether the protein accumulates due to increased production or decreased clearance. We hypothesized that autophagy is decreased in the pituitary neurointermediate lobe from horses with PPID compared to controls.

Neurointermediate lobe pituitary tissue was from collected from horses with PPID (n 5 12) and healthy horses (n 5 37, 2-35 years). Realtime PCR was used to determine the relative expression of autophagy genes (mTOR, Beclin1, ATG12, ATG7, ATG5, PINK, LAMP2) and a-synuclein Relative gene expression from horses with PPID were compared to healthy horses by t-test following log transformation. A Pearson coefficient of correlation was calculated comparing a-synuclein expression with autophagy gene expression.

The expression of a-synuclein, autophagy-related genes (ATG12, Beclin, Lamp2), and mTOR was greater in horses with PPID than in healthy horses. Age was not correlated to a-synuclein or autophagy gene expression. There was a significant positive correlation between expression of a-synuclein and Beclin1, ATG12, ATG7, ATG5, and PINK, but not mTOR expression. Accumulation of a-synuclein protein in horses with PPID may result from increased a-synuclein expression. Autophagy genes are upregulated in horses with PPID, suggesting a compensatory response, although these findings need to be confirmed by demonstrating an increased functional response. asynuclein expression was positively correlated to expression of autophagy genes except mTOR, suggesting a-synuclein may stimulate autophagy in an mTOR independent manner.

ACVIM FORUM SESSION 86A EFFICACY OF DELAYED ANTIVIRAL THERAPY AGAINST EHV-1 CHALLENGE. LK Maxwell 1 , LL Gilliam 1 , N Pusterla 2 , R Carmichael 1 , RW Eberle 1 , JW Ritchey 1 , TC Holbrook 1 , T Gull 1 , GB Rezabek 1 , D McFarlane 1 , CG MacAllister 1 . 1 Oklahoma State University, Stillwater, OK. 2 University of California, Davis, CA.

Equine herpes virus type-1 (EHV-1) outbreaks are often not recognized until exposed horses are at immediate risk for developing equine herpes myeloencephalopathy (EHM). The objective of this study was to determine whether delayed therapy with the antiviral drugs valacyclovir or ganciclovir could protect those horses most at risk for EHM. Eighteen aged ( 4 20 years) mares were randomized to treatment: no therapy (control), oral valacyclovir therapy, or intravenous ganciclovir therapy. Drug administration was initiated at the onset of the second febrile phase, between days 4-6 after EHV-1 inoculation (PI), and continued for one week. Neurological examinations were performed prior to the study and for three weeks PI. One horse was excluded from the study for failure to become febrile. Body temperature was significantly lower in the ganciclovirtherapy horses as compared to control horses on days 6-8 PI (P o 0.05), whereas valacyclovir-therapy horses did not differ from control horses. Viremia in whole blood, as determined by PCR, was also lower in the ganciclovir-therapy horses on days 7-10 PI and on day 7 PI in the valacyclovir-therapy horses (P o 0.05). Although antiviral drug administration did not reduce the risk of ataxia (P 5 0.06) or nasal shedding, ganciclovir therapy did decrease the severity of ataxia (P o 0.05) as compared to valacyclovir-therapy and control horses, where 0/6, 2/5, and 4/6 horses, respectively, developed at least a two grade change in ataxia. In summary, ganciclovir administration provided better protection against EHM than did valacyclovir when therapy was initiated just prior to the onset of neurological disease. Equine vaccination is amongst the most important method of prophylaxis against equine influenza virus (EIV), a pathogen in which continuous antigenic drift can lead to vaccine failure. A 6month duration of immunity (DOI) challenge infection study was conducted using commercial inactivated vaccines containing different strains of A/equine/2/influenza virus's, including Innovator TM , containing Kentucky/97 (Pfizer Animal Health, New York, NY), and Calvenza, containing a combination of Ohio/2003, Kentucky/ 95, and Newmarket93 (Boehringer Ingelheim Vetmedica, St. Joseph, MS) . The challenge virus strain was Colorado/07, the most contemporary challenge strain currently in use.

The study design was a blinded, randomized challenge trial. Three groups of 10 yearling ponies, with no history or serological evidence of EIV infection were established. Each group received one of three treatments: vaccination with Innovator TM ; vaccination with Calvenza TM ; or injection with a saline placebo. Each treatment was administered 3 times, at intervals of 1 month between the first two treatments, and 3 months between the second and third treatments. All ponies were challenged by nasal nebulization of 5X10 7 EID 50 influenza virus A/eq/2/Colorado/07 6 months after the third treatment. Clinical signs of disease, including rectal temperature, nasal discharge, anorexia, coughing, and depression, were recorded daily for 2 days prior to challenge infection, and 14 days post-challenge. Nasal shedding of EIV was measured on the same days, using a realtime PCR test procedure. EIV-specific antibody responses were measured by ELISA. Differences between groups were analyzed by non-parametric repeated measures ANOVA, and differences were declared significant when P o 0.05.

All control group ponies demonstrated clinical signs of disease consistent with EIV infection post-challenge infection, including pyrexia, nasal discharge, inappetance and partial anorexia. These signs were significantly lower in both vaccine groups; mean body temperature was elevated ( 4101.51F) for 8 days in controls, but only 2 days in vaccine groups. Nasal shedding of EIV was detected in all ponies in all groups: over the duration of the study the Calvenza group shed significantly less virus than Innovator and Control. Over time antibody titers were significantly higher in the Calvenza than the Innovator group, and both were significantly greater than controls.

This study demonstrated that both current commercial inactivated EIV vaccines have a duration of clinical protection of at least 6 months after a highly pathogenic challenge with a recent EIV isolate. Both antibody responses and virological protection differed between the vaccines. Formulation difference between the vaccines, including the EIV antigens employed, may have contributed to this performance difference. Degenerative myelopathy (DM) may be homologous to a form of amyotrophic lateral sclerosis in humans which has excitotoxic and immunologic pathogeneses described. The aims of this study were to determine (i) presence or absence of abnormalities in concentrations of CSF amino acid (AA) neurotransmitters (glutamate, glycine and gÀaminobutyric acid (GABA)) and cytokines in dogs with DM and if present (ii) investigate associations with disease severity.

Twenty-two dogs histopathologically confirmed for DM and 21 dogs with suspected DM based on thorough diagnostic investigations and 42 clinically normal age-matched control dogs were included in the study. The neurological severity of the DM dogs was graded (1-4) using an established scale. CSF was evaluated for presence of glutamate, glycine and GABA by high performance liquid chromatography and for GM-CSF, IFN-g, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IP-10, KC (keratinocyte chemoattractant), MCP-1 (monocyte chemotactic protein-1) and TNF-a using a commercially available, canine multiplex immunoassay (Millipore, Billerica, MA). All data analyses were performed using SAS V 9.2 (Cary, NC). Analyte levels were compared between DM confirmed, DM suspected and control dogs by an analysis of variance (ANOVA). Spearman correlation was used to test for correlations of analyte levels and neurological grades. All hypothesis tests were 2-sided with a 5 0.05.

There were no significant differences between individual CSF analytes in DM confirmed and DM suspected dogs. Glutamate levels were not significantly different between DM affected (mean 5 0.09 mg/ ml; range 5 0.01-0.29; SD 5 0.069) and control dogs (mean 5 0.07 mg/ ml; range 5 0.01-0.29; SD 5 0.056). Control dogs (mean 5 0.86 mg/ml; range 5 0.08-1.16; SD 5 0.213) had significantly higher levels of GABA (p o 0.0001) than DM dogs (mean 5 0.12 mg/ml; range 5 0.07-0.79; SD 5 0.12). Control dogs (mean 5 2.14 mg/ml; range 5 0.01-3.83; SD 5 0.76) also had significantly higher glycine concentrations (p o 0.0001) than DM dogs (mean 5 0.45 mg/ml; range 5 0.01-0.98; SD 5 0.24). DM-affected dogs also had significantly higher levels of IL-2 (p 5 0.03), KC (p o 0.0001) and MCP-1 (p 5 0.005) than control dogs. Neurotransmitter levels were not significantly associated with neurological grade. KC levels were significantly higher in the least affected dogs (p 5 0.0015). There were no associations with disease severity and analyte concentrations.

DM affected dogs have an imbalance of CSF AA concentrations creating a relatively excitotoxic environment. Reports in human ALS confirm an imbalance between CSF excitatory and inhibitory AAs suggesting a pathogenic role for excitotoxicity in ALS. It also appears that DM affected dogs have increases in CSF cytokines and chemokines suggestive of an immunologic component to the pathogenesis as is similar to ALS. Further prospective analysis of DM is warranted to evaluate the role of treatment on CSF variables. The pathogenesis of neuropathic pain (NP) and syringomyelia (SM) in association with Chiari-like malformation (CLM) in dogs has focused on the anatomical anomalies and secondary cerebrospinal fluid (CSF) flow abnormalities. Neuropathic pain in humans has been associated with abnormalities of neurotransmitters such as glutamate and serotonin as well as immunologic mechanisms. The aim of this study was to investigate the CSF neurotransmitter and cytokine levels in Brussels Griffon dogs (BGs) with CLM, SM and NP.

As part of an MRI study investigating the prevalence of SM in BGs, atlanto-occipital CSF was acquired from 46 dogs and stored at -80C until analysis. All dogs underwent a neurologic exam prior to MRI; Osirix s software was used to measure SM and the presence of cerebellar herniation and deviation were recorded. Deproteinized CSF samples were analysed for presence of serotonin (ng/ml), glutamate, glycine and GABA (mg/ml) by high performance liquid chromatography. All CSF samples were evaluated simultaneously for GM-CSF, IFN-g, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IP-10, KC, MCP-1 and TNF-a. A commercially available, canine multiplex immunoassay (Millipore, Billerica, MA) was used for the cytokine analysis (pg/ml). Student's t-tests were used to compare the means of neurotransmitter and cytokine values between groups with and without skull abnormalities or spinal pain. Simple Pearson's correlation was used to test for correlations of neurotransmitter and cytokine values with syrinx dimensions and correlations of neurotransmitter with cytokine values. All hypothesis tests were 2-sided and the significance level was a 5 0.05.

NP was detected in 8 dogs (17%); SM was present 24 dogs (52%); and CM was detected in 24 dogs (52%). IFN-g levels were significantly lower in dogs with NP than without (p 5 0.036). There were significant positive correlations between syrinx size and IL-8 (p 5 0.017), KC (p 5 0.025) and MCP-1 (p 5 0.003). There were significant negative correlations between IFN-g and syrinx height (p 5 0.025) and extent (p 5 0.042). There was a significant negative correlation between IL-2 and syrinx height (p 5 0.042). Neurotransmitter levels were not associated with skull abnormalities or spinal pain, but there was a positive correlation of glycine with IL-2 (p 5 0.004) and MCP-1 with glutamate (p 5 0.0147) and serotonin (p 5 0.0059).

The size of the syrinx in BGs with SM is associated with several cytokine elevations but only a decrease of IFN-g was associated with NP. Based on this study it does not appear that excitotoxicity plays a role in either SM development or NP. Further work is justified on the role of the immune system in CM, SM and NP. Current knowledge about the conservative management of disk associated cervical spondylomyelopathy (DA-CSM) is rather limited and mainly based on retrospectively retrieved data. The goals of this study were to prospectively evaluate the evolution of clinical signs in dogs treated conservatively for DA-CSM. Additionally, several potential prognostic parameters and the correlation of initial clinical signs with magnetic resonance imaging (MRI) and transcranial magnetic stimulation (TMS) were investigated.

Twenty-one dogs were included. After neurological evaluation, neurological status was graded from 0 (5 normal) to 6 (5 tetraplegia). All animals underwent low-field MRI and TMS with measurement of onset latencies and peak-to-peak amplitudes from the extensor carpi radialis and cranial tibial muscles. From the MR images, the following dimensions were calculated: remaining spinal cord area; compression ratio; vertebral occupying ratio of the spinal cord; canal height to body height ratio (CBR); canal height to body length ratio (CBLR); and the canal compromise ratio. Intraparenchymal intensity (ISI) changes were graded from 0 to 3. All dogs were reevaluated by the same person after 1, 3, 6, 12, and 24 months.

Eight of 21 dogs (38%) experienced a positive clinical evolution with improvement of clinical signs or stabilization of mild clinical signs. All dogs with a negative clinical evolution 1 month after diagnosis experienced a further progression of clinical signs resulting in a poor outcome. The opposite was true for all dogs with a positive clinical evolution after 1 month. Outcome was further significantly associated by the remaining spinal cord area and the vertebral canal compromise ratio. Prognosis was not significantly affected by clinical presentation or TMS. Progression of clinical signs, in unsuccessfully treated dogs, was generally characterized by a rapid and dramatic deterioration of neurological status. There were no significant correlations between clinical presentation, MRI and TMS. Two dogs underwent necropsy and histopathological examination. This revealed in both cases chronic Wallerian degeneration and segmental myelomalacia.

The results of this study suggest that conservative treatment of DA-CSM is associated with a rather guarded prognosis. Clinical evolution 1 month after diagnosis and selected MRI parameters can be considered as prognostic indicators. The lack of correlation between clinical presentation and outcome, medical imaging and electrophysiological evaluation is disturbing and warrants further investigation. A MRI-guided stereotactic brain biopsy system has not been clinically evaluated in dogs. The purpose of this study was to determine the ability of the Brainsight TM system to obtain histologically diagnostic samples and access the impact of this procedure on neurologic status for 72 hours after the biopsy.

Five dogs with MRI definable lesions in the brain have been enrolled. Breeds included a Pitbull mix, Pembroke Welsh Corgi, French Bulldog, Border Terrier and West Highland White Terrier. Age ranged from 5-11 years. Weight ranged from 8.5-18.8 kg.Dogs presented with seizures (n 5 5), ambulatory paresis(n 5 4), unilateral blindness(n 5 2) and head tilt(n 5 1). One dog had a normal neurologic exam. Lesions chosen for biopsy were in the olfactory and/or frontal lobes (n 5 3), parietal lobe(n 5 1), and pyriform lobe(n 5 1). Lesions were between 14-22 mm in diameter. All lesions were well-circumscribed and contrast enhancing except for one. Histologic diagnosis of meningioma(n 5 3) and granulomatous meningoencephalitis(n 5 1) were made. The poorly-circumscribed, non-contrast enhancing frontal mass yielded non-specific necrosis. Following biopsy, three dogs returned to pre-biopsy neurologic status within 12 hours. The French Bulldog took 48 hours to return to previous neurologic status due to brachycephalic syndrome that required oxygen support. One dog had acute respiratory arrest 16 hours post-biopsy. Necropsy is pending.

These results suggest that this MRI-guided biopsy system can provide an accurate histologic diagnosis of brain lesions. Biopsies of poorly-circumscribed and non-contrast enhancing brain lesions may be less diagnostic. Further evaluation is on-going to determine the true diagnostic yield and complication rate of this procedure. Concurrent malformations of the craniocervical junction are commonly identified in humans with Chiari type I malformation.

Recent evidence suggests such craniocervical junction abnormalities (CJAs) also occur in dogs suspected of having Chiari-like malformation (CLM). The purpose of this study was to objectively describe morphometric features of the craniocervical junction region of dogs with suspected CLM and to investigate for associations between these features and the occurrence of other malformations in this region.

Magnetic resonance (MR) and computed tomographic (CT) images from 274 dogs with CLM were evaluated. Three regions of neural tissue compression were assessed: cerebellar compression (CC); ventral compression at the C1/C2 articulation, termed ''medullary kinking'' (MK); and dorsal compression (DC) at the C1/C2 articulation. A compression index (CI) was calculated for all abnormal regions for each dog. Multiple logistic regression analysis was performed (p o 0.05) to ascertain whether CI values for the different regions of compression were associated with the incidence of other craniocervical junction abnormalities.

68% of dogs had MK and 38% of dogs had DC. 28% of dogs also had evidence of atlanto-occipital overlapping (AOO Medical infrared imaging (MII) is a non-invasive diagnostic imaging technique that measures skin surface temperature and generates thermal pattern maps based on predetermined color scales. Because skin temperature, dependent on regional perfusion, is under direct control of the sympathetic nervous system, MII provides information about the function of the autonomic nervous system. Because of recent advances in technology and lack of sedation needed to image patients, MII has potential use as a screening test for a variety of conditions that may result in autonomic dysregulation like Chiari-like malformation in dogs (CLM). The purposes of this study were to establish a MII protocol for dogs suspected of having CLM, to identify thermal imaging patterns for various regions of interest (ROI), to evaluate changes in thermal patterns and compare the results to those of MRI findings, considered the standard for diagnosing CLM in dogs. One hundred and five Cavalier King Charles Spaniel dogs with clinical signs attributable to CLM and confirmed CLM with MRI were evaluated with a complete blood count and chemistry profile, examination by a board certified surgeon/neurologist, multidetector CT scan of the craniocervical junction, whole body MRI and MII. The protocol for thermal imaging included cranial and caudal views of the body, full lateral right and left body views, dorsal views of the head and body, and right and left lateral views of the head. Thermal patterns were assessed with custom image recognition software. After each dog was imaged awake, general anesthesia was administered and the dogs re-imaged using the same protocol. MRI findings in dogs with severe or moderate cerebellar compression and cerebellar herniation were compared with MII results. The top of head and front of head ROI were 89.2% and 97.3% successful in identifying dogs with CLM. Based on these preliminary findings, MII may be a viable screening tool to detect CLM in dogs. Medical infrared imaging (MII) is an imaging technique that measures skin surface temperature derived from cutaneous perfusion and generates thermal pattern maps based on color scales. MII has been used as a test for a variety of conditions that cause autonomic dysregulation resulting in altered cutaneous perfusion. Acute thoracolumbar intervertebral disk disease (TLIVDD) is common in dogs. The purpose of this study was to: 1) determine the success of MII in identifying dogs with TLIVDD, 2) compare the MII localization with MRI results and surgical findings 3) determine if the MII pattern returns to that of normal dogs following decompression surgery. 72 small breed chondodystrophic dogs with TLIVDD confirmed with MRI and 14 dogs with no TLIVDD were evaluated with MRI and MII. Regions correlating with the intervertebral disk spaces were analyzed for average temperatures and thermographic patterns. Thermal patterns were assessed with computer recognition pattern analysis (CRPA) software. 21 dogs were re-evaluated 8 weeks after surgery using the same protocol. When analyzing temperature averages over a region, no significant difference was found between control and affected dogs. CRPA was 90% successful in differentiating normal from affected dogs. CRPA was 97% successful in identifying the intervertebral disk space when compared with MRI and surgical findings. Based on these findings, MII may be a viable screening tool to detect TLIVDD in dogs. Microglia physiologically shows regional topographical differences in immunophenotype and function within the central nervous system indicating the endowment for a prompt response to pathological stimuli such as trauma. Spinal cord injuries (SCI) consist of a primary injury encompassing the mechanical impact and the ''secondary wave'' of injury occurring minutes to weeks later and comprising various consecutive effects such as increased production of free radicals, excessive release of excitatory neurotransmitters and inflammatory reactions. Activated microglia has the potential to perform some of these reactions, their contribution to the secondary wave is therefore controversially discussed. It has to be considered a double-edged sword as both, beneficial and deleterious effects have been attributed to these cells.

The purpose of the presented study was to assess microglial involvement, particularly in the ''secondary wave'' following SCI. Microglia from 15 dogs with SCI was isolated and characterized ex vivo in terms of morphology, immunophenotype, and function by flow cytometry. The results were compared to region-specific findings obtained from healthy control dogs (n 5 30).

The histopathological exam confirmed the diagnosis of SCI in the cervical (n 5 5) and thoracolumbar (n 5 10) spinal cord, and revealed a significant activation of microglia/ macrophages and upregulation of myelinophagia in dogs with SCI 5 days or longer prior to euthanasia. Microglial ex vivo examination showed significantly increased expressions of B7-1, B7-2, MHC II, CD1c, ICAM-1, CD14, CD44, and CD45, and significantly enhanced phagocytosis and generation of reactive oxygen species (ROS) in SCI compared to healthy controls.

Microglial cells seem to be highly activated following SCI with an immunophenotype indicating their active role in co-stimulation of T cells, in leukocyte adhesion and aggregation, and in lipid and glycolipid presentation. Microglial phagocytosis might play a pivotal role in removal of injured or damaged cells and initialize subsequent healing processes. However, as ROS can be directly neurotoxic an enhanced microglial generation might lead to bystander damage of the traumatized spinal cord and might therefore add to the deleterious effects of the secondary wave. Modulating the microglial response in SCI might be a valuable novel therapeutic strategy alleviating further damage to the spinal cord. Thymidine kinase (TK) is a soluble biomarker present in S-phase of a salvage pathway for DNA synthesis, and can be measured in serum. TK activity correlates with stage, prognosis, and relapse in canine and human lymphoma. We previously reported the results of a pilot study evaluating TK activity in archived canine osteosarcoma, transitional cell carcinoma, and hemangiosarcoma (HSA) sera, and found elevated TK activity in 80% of canine HSA sera evaluated. The purpose of this study was to prospectively evaluate serum TK activity in a large number of dogs presenting to emergency clinics with hemoabdomen and a splenic mass, to determine if TK activity could be used as a noninvasive means to distinguish HSA versus benign conditions in this population.

Dogs presenting with hemoabdomen and a splenic mass identified on ultrasound examination were studied. Serum was collected prior to anesthesia, euthanasia or surgical intervention and frozen until batch analysis. Tissue from all patients was evaluated histologically by a single pathologist. Sera from age-matched normal dogs comprised a control population. An ELISA using azidothymidine as a TK1 substrate was used. Comparisons between groups were made using 2-tailed student T-tests, and receiver-operator characteristic (ROC) curves were generated.

Sixty-two patients and 39 normal controls were studied. There were 35 dogs with HSA, 10 dogs with other splenic neoplasia, and 17 dogs with benign diseases. Using a training set of 24 normal dogs, a cutoff of 6.55 U/L was established from the ROC curve. TK activity was significantly higher (p o 0.0001) in dogs with HSA than in the validation set of 15 normal dogs (mean1/ÀSD 5 17.711/À4.5 and 2.011/À0.6 respectively), but not between dogs with HSA and benign splenic disease (mean1/ÀSD 5 7.021/À3.7, p 5 0.13). Using a cutoff of 6.55 U/L, TK activity demonstrated a sensitivity of 0.54, specificity of 0.76, positive predictive value of 0.83 and negative predictive value of 0.45 for distinguishing HSA versus benign splenic disease. When interval thresholds of o 1.55 and 47.95 U/L were used together, diagnostic utility was markedly increased for distinguishing both HSA versus normal and HSA versus benign disease.

In conclusion, serum TK evaluation may assist in detection of canine HSA, and may also discriminate between benign disease and HSA in dogs with hemoabdomen and a splenic mass. T cell chronic lymphocytic leukemia (CLL) is a heterogeneous disease that affects a number of dog breeds. CLL patients have variable disease outcomes. The objectives of this study were to use gene expression profiling of CD8 T cell leukemias with variable outcomes in order to identify markers that can be used in routine diagnostic tests to distinguish good from poor prognosis disease, and to identify potential targets for novel therapy.

Gene expression profiling of 12 CD8 T cell leukemias (7 good, 5 poor prognosis) was conducted. Samples from 6 normal dogs were also profiled. Several differentially expressed genes were found including CD9, CD94, and CD 25. These were selected for further study using flow cytometry to determine expression of protein on the cell surface. Seventy nine cases of CD81 T cell leukemia were screened for CD 9 expression. Forty seven had associated outcome information. Based on analysis to date, CD9 expression as assessed by flow cytometry does not appear to provide prognostic information.

A monoclonal antibody to CD25 was recently made available. To date 33 patients with CD8 T cell leukemia have been profiled. CD25 is variably expressed on T cell leukemias compared to normal CD8 T cells.

CD25 is the receptor for interleukin 2. Cyclosporin, a commonly used immunosuppressive drug, inhibits IL-2 production, and has been used to treat a subset of T cell leukemias in people. Thus, the finding that CD25 is up regulated on T cell leukemias compared with normal T cells suggests a possible new therapeutic avenue. Recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of dogs. There is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (IBD). Similarly, compositional changes of the intestinal bacterial ecosystem have been associated with IBD in humans. The aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders using a next generation sequencing technique.

Fecal samples were obtained from healthy dogs (n 5 31), dogs with acute uncomplicated diarrhea (n 5 7), dogs with acute hemorrhagic diarrhea (AHD; n 5 13), and dogs with active (n 5 8) and therapeutically controlled IBD (n 5 10). The bacterial composition was analyzed by massive parallel 16S rRNA gene 454-pyrosequencing. Differences between groups were analyzed using Mann-Whitney U tests and Kruskal-Wallis tests followed by Dunn's multiple comparison tests. Statistical significance was set at p o 0.05.

Significant differences in the proportions of several bacterial groups were identified between healthy and diseased dogs. Dogs with gastrointestinal disease had significantly higher proportions of Proteobacteria (p o 0.01). Proportions of Firmicutes were lower in diseased dogs, but this difference did not reach significance (p 5 0.06). Within the Firmicutes the most notable findings were decreases in bacterial groups belonging to Clostridium clusters IV and XIVa (i.e., Ruminococcus, Dorea, and Faecalibacterium spp.; p o 0.01 for all). Dogs with AHD had the most profound changes of the microbiota, followed by dogs with acute uncomplicated diarrhea, and dogs with active IBD. Faecalibacterium spp. was the bacterial group most prominently depleted in dogs with active IBD, but was not significantly different between healthy dogs and dogs with therapeutically controlled IBD (p 5 0.66).

Results of this study revealed bacterial dysbiosis in fecal samples of dogs with various GI disorders. Bacterial changes were more profound in dogs with severe disease, but were not identified in dogs with therapeutically controlled IBD, suggesting that the microbiota is stable in non-active disease. The bacterial groups identified are considered to be important short chain fatty acid producers and may serve as candidates for the diagnosis or therapeutic monitoring of GI disease. Future studies are necessary to determine if these microbial changes correlate with functional changes in the intestinal microbiota. Ciprofloxacin oral tablets, available in a generic formulation for people, are widely used for treatment in dogs. Oral absorption data for ciprofloxacin in dogs has been variable, and too limited to guide accurate dosing. Subsequently, published doses for dogs in veterinary formularies have varied from 5 to 25 mg/kg. This study was undertaken to explore the factors that may affect oral absorption of generic ciprofloxacin in dogs, and to derive a pharmacokinetic-based dose for treating susceptible bacteria. Six healthy adult beagle dogs were used for the study (11.2 kg mean weight). After placing jugular vein catheters for collecting blood samples, these dogs were administered either a single oral dose of ciprofloxacin (250 mg tablet; mean dose 23 mg/kg), or an intravenous (IV) dose (10 mg/kg; 2 mg/mL solution). A randomized crossover design was used with a washout time between treatments. Blood was collected for plasma drug analysis for 24 hours. Ciprofloxacin concentration in plasma was analyzed using high pressure liquid chromatography (HPLC) and pharmacokinetics analyzed using a computer program. Oral absorption was also evaluated via deconvolution analysis. The oral dose was well-tolerated, but the IV dose produced transient vomiting and depression in some dogs. After the oral dose, the peak plasma concentration (C MAX ) was 4.4 mg/ mL (CV 55.9%), terminal half-life (t1/2) 2.6 hr (CV 10.8%), AUC 22.5 mg Á hr/mL (CV 62.3%), and systemic absorption (F) 63.7% (CV 41.6%). After the IV dose, the t1/2 was 3.7 hr (CV 52.3%), systemic clearance 0.587 L/kg/hr (CV 33.9%), and volume of distribution 2.39 L/kg (CV 23.7%). After examining the pharmacokinetic results from the oral dose, it was apparent that oral ciprofloxacin was absorbed well in some dogs (approximately 80%), but poorly in others (approximately 30%). To explore the factors that may have affected oral absorption, two high absorbers and two low absorbers were administered an additional oral dose as a 10 mg/mL solution (250 mg total dose) via gastric tube. After administration of the oral solution, the plasma concentrations were more uniform and consistent among dogs. Absorption of the oral solution of ciprofloxacin was 71.0% (CV 7%) with a t1/2 of 3.1 (CV 18.6%) hr and C MAX of 4.67 mg/mL (CV 17.6%). Therefore, it appears that inconsistent oral absorption of ciprofloxacin in some dogs may be formulation-dependent, and affected by tablet dissolution in the canine small intestine. Doses were calculated using the data for oral tablets in these dogs. The pharmacokinetic-pharmacodynamic (PK-PD) target was an AUC/ MIC ratio of 100. Because of the wide range in oral absorption of tablets, a dose to reach the PK-PD target ranged from Canine distemper (CD) is a highly contagious, acute or subacute systemic viral disease of dogs and other carnivores which can be controlled efficiently by the use of modified live-virus (MLV) vaccines. However, MLV strains do cross-react with molecular diagnostic tests and cause significant confusion for clinicians.

The purpose of this study was to use quantitative real-time PCR viral load information to differentiate between vaccine virus used in MLV vaccines and wildtype infections in dogs.

A real-time PCR test for CD virus (CDV) based on the P gene for phosphoprotein was used to determine viral loads in vaccinated and wildtype infected animals. A total of 158 respiratory mucosal swab samples from MLV vaccinated and asymptomatic dogs were obtained within the first 3 weeks after MLV vaccination. Based on the viral load in vaccinated animals, a cutoff value was established for the differentiation of dogs with clinical signs of respiratory distress and presumably infected with a wildtype strain of CDV. Two hundred clinical cases with known clinical and vaccination histories were analyzed to validate the cutoff value.

The CDV real-time PCR proved to be of high analytical and diagnostic sensitivity: A standard curve was established using known numbers of CDV molecules to allow absolute quantitative CDV viral load data. The limit of detection was in the single molecule range while the limit of quantitation was established at around 10 molecules per PCR reaction. A comparison to IFA showed real-time PCR to be 30% more sensitive. The CDV viral load in vaccinated animals averaged 26,738 viral particles per swab. A cutoff value of 107,903 viral particles was calculated by adding 3 standard deviations to the average value. This cutoff value correctly detected 95.2% of the vaccinated samples. Acutely infected dogs with CDV compatible clinical signs have high viral loads normally several logs higher than the cutoff value. In dogs with clinical distress, recent CDV MLV vaccination but viral loads below the cutoff value, other infectious agents were detected by using a panel of real-time PCR tests. Testing additional infectious agents in clinical settings is important in order to explain clinical signs when viral loads below cutoff values indicate that CDV is not the cause of clinical signs.

In conclusion, quantitative real-time PCR is a sensitive, rapid and reliable test regardless of recent vaccination. The use of a cutoff value will be of significant help to discriminate between vaccine interference and wildtype infection in clinical settings. Feline ureteral obstructions are a common urinary dilemma and traditional therapy is associated with substantial morbidity/mortality. Feline nephrostomy tubes are reported as being effective when pelvic drainage is required. The biggest limitation is externalized drainage, requiring careful management to prevent infection/dislodgement. The development of an indwelling ureteral bypass using a combination locking-loop nephrostomy/cystostomy tube was modified from humans, resulting in permanent indwelling drainage, reduced complications, and improved quality of life. The objective is to describe the technical and clinical outcome using a novel device called a subcutaneous ureteral bypass (SUB) in cats with ureteral obstructions.

Fifteen cats (16 kidneys) had a SUB placed for: ureterolithiasis (9), ureteral stricture (1/À stones) (6), and ureteral stent rejection (2). The median pre-and post-procedure creatinine was 6 mg/dL (range: 2.8-13) and 2.5 mg/dL (range: 2.4-9), respectively. The median pelvis diameter pre and post-procedure were 15 (range: 7-28) and 5 mm (range 3.4-6), respectively. Six French tubes were placed in 14, and 5 Fr. in 2. The bypass remained indwelling for a median of 4240 days (range 64-4450). There were 3 major complications resulting in nephrostomy tube dislodgement (2) and port leakage (1) 5 days after surgery. One patient with severe coagulopathy developed a clot which resolved with tPA infusion through the port. No SUB got occluded/obstructed long-term.

Overall, the use of a SUB for cats with ureteral obstructions can be considered a functional option when other therapies have failed or are contraindicated, but shtime. Oxidative stress is considered central to the pathogenesis of many systemic diseases. In humans, biomarkers of oxidative stress, antioxidant depletion and lipid peroxidation, have been correlated with disease severity and associated with poor clinical outcomes. Therapeutic antioxidant supplementation with NAC in glutathione (GSH)-deficient patients has shown clinical benefits, including repletion of intracellular GSH levels. We have shown that clinically ill dogs are GSH-deficient, and that GSH deficiency correlates with mortality, but it is not clear whether there are direct benefits of antioxidant intervention in these patients. The purpose of this randomized, investigator-blinded, placebo-controlled, prospective study was to evaluate the effect of NAC to normalize blood antioxidants (RBC reduced GSH (RBC GSH), plasma cysteine (CYS), serum vitamin E (vit E), and whole blood selenium (Se)), reduce lipid peroxidation (urine isoprostane/creatinine ratio (U I/ C)), and improve illness scores (SPI2) and outcome (survival to discharge) in clinically ill dogs.

Clinically ill client-owned dogs, admitted to the UW Veterinary Medical Teaching Hospital that did not receive blood transfusions, TPN, vitamins, or antioxidants were eligible for the study. Dogs enrolled in the study were randomized to receive IV infusions q. 6 h. of either NAC (1 Â 140 mg/kg and 6 Â 70 mg/kg) or equal volumes of 5% dextrose (placebo) over 48 hours. At the time of enrollment, and 2 hours following the final 48 hour infusion, blood and urine were collected to quantify RBC GSH, CYS, vit E, and Se concentrations; U I/C ratios; and calculate SPI2 scores. RBC GSH and CYS concentrations were quantified by HPLC. Commercially available HPLC, atomic absorption spectroscopy, and EIA were used to quantify vit E, Se, and U I/C ratios, respectively. Nonparametric statistical analyses were used, with results reported as medians and P o 0.05 considered significant.

Sixty-one ill dogs were randomized to either NAC (n 5 30) or placebo (n 5 31). Overall this group of ill dogs had significantly decreased RBC GSH (1.50 vs. 1.91 mM; P 5 0.0013), vit E (27 vs. 56 mg/mL; P 5 0.0002), and Se (0.37 vs. 0.55 mg/mL; P 5 0.0308) levels and elevated U I/C ratios (969 vs. 398 pg/mg; P 5 0.0005) in comparison to healthy control dogs. Dogs in the placebo group showed a significant further decrease in RBC GSH over the next 48 hours (1.48 to 1.42; P 5 0.035). NAC supplementation significantly increased plasma CYS levels (9.1 to 15.1 mM; P o 0.0001), and prevented a further decline in RBC GSH (1.55 to 1.58 mM; P 5 0.174). However, serum vit E (30 vs. 28 mg/mL), Se (0.40 vs. 0.35 mg/ mL), U I/C ratios (946 vs. 806 pg/mg), SPI2 scores (0.74 vs. 0.75), and outcome (81% vs. 76%) were not significantly different between the NAC and placebo groups after treatment.

The results of this study further support that clinically ill dogs experience oxidative stress, and suggest that antioxidant supplementation with NAC within the first 48 hours of hospitalization prevents further RBC GSH depletion. Further studies are necessary to investigate whether longer duration or combined antioxidant supplementation normalizes the redox state and impacts long-term outcome. Diabetes Mellitus in cats is very similar to Type II Diabetes in Humans, preceded by a period of insulin resistance. Evaluating insulin resistance in a cat is a time consuming, expensive, and difficult procedure. There is a need for a simple biomarker based test predictive of insulin resistance. There is a biomarker based assay predicative of insulin resistance in humans. The purpose of this study was to evaluate the utility of this assay in overweight cats and show improvement in insulin sensitivity following weight loss and weight maintenance.

The insulin resistance assay is based on the quantitative analysis of 5 metabolites (2-hydroxybuterate, creatine, palmitate, decanoylcarnitine, and oleoyl-LPC). A proprietary algorithm (Metabolon, Inc, Durham NC) was used to generate a predictive RD (Rate of Disposal) value (normal range in cats 6.5-10.5). Individuals with an RD value less than 6 will have a greater than 50% chance of being insulin resistant and an RD value less than 3 will have a greater than 90% chance of being insulin resistant.

Initial studies demonstrated that the RD values indicating insulin resistance in cats correlated with age, obesity and severity of diabetes as determined by histopathology and blood glucose levels. In a feeding study of 40 cats (4 39% vs. o 25% body fat) RD values improved from 6.19 AE 1.23 to 6.8411.38 (p 5 0.029). During weight maintenance, 19% body fat for 4 months, further improvement was observed (RD, 8.30 1 1.08 (p 5 9.2E-12)). These results demonstrate that long term weight maintenance following weight loss is critical for increasing insulin sensitivity in cats. The use of monoclonal antibodies and antibody fragments to directly target tumor antigens and neutralize their growth factors has shown promising results in human clinical trials. However, these targeted approaches have not been possible in dogs since specific tumor antigens have not been identified, monoclonal antibodies of canine origin are not available and the efficacy of xenogeneic antibodies in the dog is limited by neutralizing antibody responses. To overcome these obstacles, we have generated canine antibody phage display libraries from canine splenocytes. These libraries consist of single chain variable fragments (scFv) comprised of canine variable heavy (VH) and variable light (VL) immunoglobulin chains displayed on the surface of bacteriophage (Fig. 1) . The antigen specificity within these libraries is diverse and recapitulates the antigen-experienced immunoglobulin repertoire of the dog. We can now use simple panning techniques to isolate scFv of canine origin that bind to either known targets or unknown targets which can then be identified using standard molecular techniques.

Canine HSA is a highly aggressive malignancy of vascular endothelial cells that affects large breed dogs. Although there are no confirmed immunological targets for HSA, serum levels of Vascular Endothelial Growth Factor (VEGF) are elevated in these patients and, as in many human cancers, VEGF may represent an important therapeutic target for neutralization. We used simple panning techniques to screen canine scFv libraries generated from the spleens of dogs with HSA against canine VEGF and successfully isolated 3 scFv clones that bind and neutralize canine VEGF in vitro. These scFvs are now being taken into a murine model of canine HSA to determine whether they can inhibit tumor growth and metastases. In addition, we have panned the same antigen-experienced scFv phage display libraries against allogeneic primary canine HSA cells of low passage number to isolate canine-derived antibody fragments that can target malignant endothelial cell surface molecules. Early results demonstrate enrichment of scFv phage libraries for malignant endothelial cell binders. These scFv can be readily linked to chemotherapeutic agents or other toxins and used to deliver high doses directly to the malignant cell. This novel approach aims to reduce side effects of systemic chemotherapy and augment therapeutic response. Calcitriol, (vitamin D 3 ), has antineoplastic activity and acts synergistically to potentiate the antitumor activity of a diverse array of chemotherapeutics. CCNU, vinblastine, corticosteroids, and tyrosine kinase inhibitors, are used to treat canine mast cell tumors (MCT). Vitamin D receptor is expressed in the majority of canine MCTs, suggesting a role for calcitriol in the management of dogs with these tumors. The purpose of our study was to examine the in vitro effects of calcitriol in combination with CCNU, vinblastine, imatinib, or toceranib on canine mastocytoma C2 cells. Also, we evaluated the antitumor activity of DN101, a highly concentrated oral formulation of calcitriol, as single-agent treatment in dogs with naturally occurring MCTs.

C2 cells were incubated with serial dilutions of calcitriol (0.1-25 nM). Twenty-four hours later, cells were then treated with vehicle control or serial dilutions of CCNU (2.5-20 uM), vinblastine (1.25-10 nM), imatinib (1.67-12.5 nM), or toceranib (3.13-25 nM). Cell viability was assessed with an MTT assay after 48 hours and data was used to derive a combination index (CI: values o 1, 1, 41 indicate synergism, additivity, antagonism, respectively). In the phase II clinical trial, dogs were eligible if they had at least 1 measurable, histologically confirmed, MCT. Calcitriol was administered orally. RECIST criteria were used to assess tumor response.

Calcitriol, CCNU, vinblastine, imatinib, and toceranib each suppressed C2 cell viability in a dose-dependent manner. CI values o 1 were obtained for calcitriol (0.1-6.25 nM) combined with CCNU (5 and 10uM), vinblastine (2.5 and 5 nM), imatinib (1.67-12.5 nM) and toceranib (0.1-12.5 nM). Due to the occurrence of toxicity (vomiting, anorexia, hypercalcemia), the phase II trial was terminated early; only 10 of 20 planned patients were treated. One dog with a metastatic muzzle MCT had a complete response that lasted 89 days. Three dogs achieved partial response lasting from 74-90 days.

In summary, our in vitro data demonstrate that calcitriol combined with CCNU, vinblastine, imatinib or toceranib has synergistic effects on C2 mastocytoma cells. Antitumor responses were observed in dogs with spontaneously occurring MCTs treated orally with single-agent calcitriol, but the frequency of adverse effects was high. Together these results suggest calcitriol combination therapies might have significant clinical utility in the treatment of canine MCTs but refinement of the calcitriol-dosing regimen must be done. Cyclosporine is a potent immunosuppressive agent used to treat many canine inflammatory and immune-mediated diseases. Cyclosporine has gained popularity as an immunosuppressive agent because of a favorable toxicity profile compared to many other immunosuppressive agents. Optimal dosing regimens for cyclosporine in the dog remain unclear, primarily because standard methods that monitor effectiveness of immunosuppression have not been established. Pharmacokinetic testing is currently used during treatment with oral cyclosporine to adjust doses based on measurement of blood drug levels. Individual patients, however, often demonstrate marked variations in blood drug levels while on similar oral doses of cyclosporine, and can also demonstrate different clinical responses even at comparable drug levels, making correlation of blood cyclosporine levels and degree of disease control extremely difficult. Pharmacodynamic testing offers an alternative method for regulating cyclosporine dosing by objectively measuring the effects of cyclosporine on T-cells, the drug's main cellular target in the body. Our ACVIM Foundation-funded research has focused on developing and evaluating a comprehensive panel of biomarkers of immunosuppression that can be utilized for pharmacodynamic monitoring during treatment with cyclosporine and other immunosuppressive agents that affect T-cell function.

We have completed several studies using flow cytometry to evaluate activated T-cell expression of surface molecules (CD25 & CD95) and cytokines (IL-2, IFN-g & IL-4) as potential biomarkers. Our first study was an in vitro study evaluating expression of surface molecules and cytokines in canine T-cells exposed to varying concentrations of cyclosporine. This study established consistent drug-associated suppression of the cytokines IL-2, IFN-g and IL-4. Our second study was an in vivo study in normal dogs evaluating the effects of two doses of oral cyclosporine, a high dose considered to be reliably immunosuppressive (starting dose 10 mg/kg bid, titrated upwards as needed to attain trough drug blood levels of at least 600 ng/mL) and a lower dose used to treat atopy (5 mg/kg sid), on T-cell expression of these three cytokines. Significant suppression of IL-2 and IFN-g expression was seen at the high cyclosporine dose, while at the lower dose only IFN-g expression was suppressed. Because Tcell expression of IL-4 was not significantly suppressed at the high cyclosporine dose, IL-4 was not evaluated at the lower drug dose.

Because of specialized sample handling requirements, flow cytometry is not as practitioner friendly as other assays (such as PCR) for routine use in pharmacodynamic testing. We have therefore conducted an in vitro study comparing the effects of cyclosporine on activated T-cell expression of IL-2 and IFN-g using flow cytometry and qRT-PCR, and demonstrated dose dependent and comparable suppression of IL-2 and IFN-g using either methodology. We are currently evaluating, using qRT-PCR, the effects of oral cyclosporine on T-cell expression of IL-2 and IFN-g in normal dogs prior to moving on to pharmacodynamic trials in our clinic patients.

EFFECT OF HYPOTHYROIDISM ON REPRODUCTION IN BITCHES. DL Panciera 1 , BJ Purswell 2 , KA Kolster 2 , SR Werre 3 . Departments of 1 Small Animal Clinical Sciences, 2 Large Animal Clinical Sciences, and 3 Laboratory for Study Design and Data Analysis, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA.

Numerous reproductive abnormalities, including irregular interestrous period, anestrus, and infertility have been attributed to hypothyroidism. We previously documented reduced fertility and lower birth weight and increased periparturient mortality in pups born to bitches with experimentally-induced hypothyroidism for a median duration of 56 weeks. The purpose of this study was to evaluate reproductive function in these same bitches after hypothyroidism was treated with a replacement dose of levothyroxine.

Twelve multiparous bitches were studied. Hypothyroidism was induced in 6 dogs by administration of 1 mCi/kg 131 I. Hypothyroidism was confirmed by finding serum T4 concentrations before and 4 hours after IV administration of human recombinant TSH that were o 5 nmol/L. Levothyroxine (0.02 mg/kd q 24 h) was administered to all hypothyroid bitches. Six bitches served as euthyroid, untreated controls. Dogs were evaluated daily for signs of estrus and were bred by 1 of 2 males when serum progesterone was !5 ng/ml. Interestrous interval, gestation length, strength and duration of contractions during whelping, time between pups, number of live pups and stillbirths, viability of pups at birth, weight of pups, and periparturient mortality were recorded. The Student's t-test and ANOVA were used to compare differences between control and hypothyroid bitches for continuous, normally distributed data. The Wilcoxon rank sum test was used to analyze data between groups that was not normally distributed.

The mean duration of hypothyroidism prior to levothyroxine administration was 102 AE 8.1 weeks. Breeding took place after levothyroxine treatment for 46 AE 12.6 weeks in the hypothyroid group. All 6 dogs in the hypothyroid group and 5/6 control dogs were pregnant, while 4/8 hypothyroid and all 6 control bitches became pregnant prior to levothyroxine administration. No difference in interestrus interval or gestation length was noted between groups. During whelping, no difference in strength of contractions, contraction duration, interval between pups, or viability scores of pups was found between groups. Litter size, birth weight and peirparturient mortality were similar between groups.

Levothyroxine administration reverses the detrimental effects of hypothyroidism on fertility and neonatal health. Racing sled dogs have a high prevalence of exercise-induced gastric erosions/ulcers, with reports ranging from 50-67% of dogs running at least 100 miles in a day or less. Omeprazole reduces the severity of, but does not completely prevent, gastritis under racing conditions, and can be difficult to administer under these conditions. Famotidine can be administered in food, but has only demonstrated efficacy under less intense training conditions. The purpose of these studies was to evaluate different acid suppression strategies under racing conditions for the prevention of exercise-induced gastritis.

Experiment #1 was a randomized placebo-controlled study using 36 sled dogs (3-8 years) competing in a 330 mile race over 50-60 h. Treatment groups were famotidine (approx 1 mg/kg qd) or no treatment, beginning 2 days prior to the start of the race and proceeding until gastroscopy was performed 24h after the race. Experiment #2 was a randomized positive-control study using 52 sled dogs (2-8 years) running a mock race of 300 miles in 50h. Dogs were divided into omeprazole (approx 1 mg/kg qd, administered 30 min prior to a meal) or famotidine (approx 2 mg/kg bid) groups beginning 2 days prior to the exercise challenge and continuing for 24h after completion. Gastroscopy was performed immediately prior to the start of dosing and 24h after completion of the exercise. In all cases, mucosal appearance during gastroscopy was blindly scored using previously described scoring system.

Famotidine (1 mg/kg qd) reduced the prevalence of clinicallyrelevant, exercise-induced gastric lesions compared to no treatment (7/16 vs 11/16, p 5 0.031). Compared to famotidine at 2 mg/kg bid, omeprazole significantly decreased the severity (0.4 vs 1.2, p 5 0.0002) and prevalence (2/23 vs 7/21, p 5 0.049) of gastric lesions.

Although famotidine provides some benefit in the prevention of exercise-induced gastric lesions, neither the recommended dose nor the higher dose were considered acceptable in the prevention of exerciseinduced gastritis as between 33-44% of the dogs receiving famotidine had clinically significant lesions. A previous study examining omeprazole under racing conditions, but without careful administration on an empty stomach, resulted in a 22% prevalence of clinically significant gastric lesions. However, the bioavailability of omeprazole is reduced in the presence of food, and when the daily administration of the drug is carefully scheduled to coincide with an empty stomach, the resulting prevalence of clinically significant lesions induced by racing-intensity exercise is reduced to just over 10%. The conclusions of these studies are that omeprazole is superior to famotidine in preventing gastritis in racing sled dogs during competition. Routine administration of omeprazole is recommended to prevent stress-associated gastric disease in exercising and racing Alaskan sled dogs. Mares may be an important source of environmental contamination with Rhodococcus equi on breeding farms. Attempts to reduce fecal shedding of R equi by the mare and the effects of the mare's fecal R equi concentration on airborne concentrations in the foaling stall have not been previously reported. Twenty-one Arabian mares were treated daily with either oral gallium nitrate or placebo in a randomized double-blind study. Fecal samples were collected at day 320 of gestation (time 1), the week before foaling (time 2), and the week after foaling (time 3). Airborne concentration of R equi were measured in the stall within 6 hours post foaling using a microbial air sampling system into which standard (100-mm) culture plates with a media selective for R. equi have been loaded.

Concentration of total R equi were determined by morphological characteristics. The concentration of virulent R equi was determined using a modified colony immunoblot method. Concentrations of total and virulent R equi were compared among mares to examine effects of treatment, time, and treatment by time interaction.

There were significant (P o 0.05) effects of treatment that depended on time of sample collection. At sample times 1 and 2 there were no significant differences between groups in the fecal concentration of virulent R equi. At time 3 concentrations of virulent R equi were significantly lower among mares in the treatment group (P o 0.05) compared to control. Effects of time depended significantly on groups: for the control group, there were no significant effects of time. For the treatment group, concentrations tended to decrease over time, and concentrations at time 3 were significantly (P o 0.05) lower than those at time 1. No other differences among times for concentrations in the treatment group were statistically significant. There were no significant effects of treatment, sample time, or their interaction on the concentration of total R equi between groups; however, the pattern for these data was similar to that observed for the virulent isolates. No significant differences were determined between treatment groups for airborne concentrations of virulent or total R equi.

Treatment of mares with oral gallium nitrate significantly reduced the fecal concentrations of virulent R equi over time, but had no impact on the airborne concentration of R equi shortly after foaling. The purpose of this study was to evaluate the protein profile of bronchoalveolar lavage fluid (BALF) in horses affected with recurrent airway obstruction (RAO) and in control horses using proteomics and Western blot techniques. RAO-affected (n 5 5) and control horses (n 5 6) were subjected to an experimental exposure trial; when the RAO-affected horses showed clinical signs of disease, BALF was collected from all horses. BALF was also collected from client-owned RAO-affected horses (n 5 15) with naturally-occurring clinical signs of disease and client-owned control horses (n 5 12) from the same environments. The BALF from the experimental exposure trial horses was subjected to trypsin digestion and proteomics analysis with mass spectrometry (MS). Peaks detected with MS were identified using tandem MS analysis and database searches. Western blot was used to confirm the identity and expression levels of two proteins identified using proteomics techniques in the BALF of all horses. Data from MS experiments were analyzed with the Student's t-test to compare peak intensity between RAO-affected and control horses. Western blot band density data was analyzed with the Kruskal-Wallis ANOVA for comparison between groups of horses. Significance level was set at P o 0.05.

With MS proteomic analysis of the BALF from the experimental exposure trial horses, 2049 total peaks (peptides) were identified. Of these peaks, 100 were differentially expressed between the RAO-affected (24 over-expressed) and control horses (76 over-expressed). Identifications were made for 250 BALF proteins. Transferrin and secretoglobin were chosen for validation with Western blot. Proteomics indicated that secretoglobin was not differentially expressed between the experimental exposure trial group; this was confirmed with Western blot analysis. Western blot also showed that clientowned RAO-affected horses had lower secretoglobin expression than client-owned control horses and control horses before experimental exposure. According to the proteomics data, transferrin was over-expressed in control horses after experimental exposure compared to RAO-affected horses. While the Western blot analysis did not show a statistically significant difference in this comparison, transferrin was significantly over-expressed in control horses before experimental exposure compared to client-owned RAO-affected horses. In addition, both secretoglobin and transferrin band densities on Western blot were negatively correlated with airway obstruction and neutrophilic pulmonary inflammation.

This study demonstrates that proteomics techniques can be used in the investigation of equine BALF proteins. The proteins identified as differentially expressed between RAO-affected and control horses in this study including, but not limited to, secretoglobin and transferrin should undergo further evaluation for their use as biomarkers of RAO, and as potential targets of new therapeutic agents for RAO. Cardiotoxic effects of rattlesnake venom in the horse are not well defined. The first aim of this study was to document cardiac damage in naturally envenomated horses. Twenty horses with clinical diagnosis of snake bite were included. A snake venom ELISA was utilized to confirm envenomation when possible. Serum and plasma were collected at selected intervals. Plasma was assayed for cardiac troponin I (cTnI) using a flurometric assay (Stratus CS s , Dade Behring). Holter monitors (Zymed s , Philips) were placed at admission, 1 week and 1 month post presentation. Echocardiography was performed on available horses 5-7 months after envenomation.

The second aim of this study was to investigate potential mechanisms of the cardiac damage. Serum samples were assayed for TNFalpha using a commercial assay (Endogen). Antibody titers to Crotalus atrox venom were measured at admission, 1 week and 1 month after natural envenomation and compared to titers in vaccinated horses (Crotalus Atrox Toxoid, Red Rock Biologics).

A significant number of horses showed elevations in cTnI (p o 0.05) at one or more time point indicating myocardial damage. Holter readings revealed the presence of arrhythmias or persistent tachycardia in 19 horses. Five of twenty horses were available for echocardiography; no abnormalities were noted. Horses with increased cTnI tended to have greater TNFalpha concentrations compared with horses without increased cTnI. Peak venom titers in bitten horses were significantly higher than peak titers in vaccinated horses (p o 0.05).

Rattlesnake envenomation was associated with evidence of cardiac damage in a significant proportion of bitten horses. Further studies are needed to determine the cause as well as mechanisms to treat and/or prevent its occurrence. Little is known about the gastric mucosal flora in healthy horses and its role in gastric disease has not been critically examined. Our laboratory previously reported that a diverse microbial flora with a predominance of Streptococcus spp. and Lactobacillus spp. exists in healthy horses using Fluorescence in situ hybridization (FISH). The present study sought to further characterize the gastric mucosal flora of healthy horses using massive parallel 16SrRNA bacterial tag encoded FLX-titanium amplicon pyrosequencing (bTEFAP). Biopsies of the squamous, glandular, antral and any ulcerated mucosa were obtained from 4 healthy horses via gastroscopy after a 12-hour fast and 2 horses immediately post-mortem. DNA was extracted from the mucosal biopsies and bTEFAP and data processing was performed. Hierarchical cluster analysis based on relative abundance data on the genus level were performed to look for trends in bacterial diversity among the individual horses.

Pyrosequencing yielded between 4,500 and 13,000 reads per horse with 9238, 16587, 16587 reads in the antrum, squamous and glandular regions, respectively. The microbiome segregated into two distinct clusters: Cluster 1 comprised of 2 horses that were stabled, fed hay and sampled at post-mortem and Cluster 2 consisted of 4 horses that were pastured on grass, fed hay and biopsied gastroscopically after a 12-hour fast. Samples from different antomic regions clustered by horse rather than region. Despite being very similar at the higher taxonomic level (phyla) differences in the distribution of bacteria were seen at the genus and species level. The dominant bacteria in Cluster 1 horses were Firmicutes (483% reads/sample) consisting of mainly Streptococcus spp., Lactobacillus jensenii, L. fornicalis and Sarcina maxima. Cluster 2 had more diversity with a predominance of Proteobacteria, Bacteroidetes and Firmicutes and 51 genera identified such as Streptococcus spp., Moraxella spp., Actinobacillus spp., and others. Though the relative abundance of the individual taxonomic groups was significantly different between individual horses, no significant differences in the overall diversity could be found (as assesed by Shanon Weaver, ACE and Choa I diversity indices). Helicobacter spp. sequences were not identified in any sample (out of 58,891 reads). The ulcerated mucosa from Horse 3 (group 1) had lower diversity and higher numbers of bacteria predominated by Lactobacillus equigenerosi.

This data shows that the equine gastric mucosa harbors an abundant and diverse microbiome which is unique to each individual and differs by sampling method, fasting prior to sampling and diet. Seasonal pasture myopathy (SPM; atypical myopathy [AM] in Europe), typified by nonexertional rhabdomyolysis, occurs in pastured horses during autumn or spring. Clinical signs rapidly progress from muscular weakness to recumbency and frequently death. Extensive myonecrosis and intramyofiber lipid storage occur in highly oxidative respiratory and postural muscles. Recently, a defect of lipid metabolism called MADD has been identified in European horses with AM. This report documents the first cases of equine MADD in the United States.

Six Midwestern US horses suspected of having SPM in the spring or fall of 2009 were evaluated for MADD by urine organic acids, plasma acylcarnitines and/or muscle carnitine and histopathology. Five horses had clinical signs and clinicopathologic data consistent with severe rhabdomyolysis. One horse was found dead on pasture after 2 days of rear limb stiffness and inappetance. Urinary organic acid profiles revealed markedly elevated ethylmalonic and methylsuccinic acids, butyrylglycine, isovalerylglycine, and hexanoylglycine, consistent with equine MADD. Plasma acylcarnitine profiles from 2 horses had marked elevations of short chain acylcarnitines, while the third horse and only survivor had minor elevations of short chain acylcarnitines. Affected muscle showed extensive degeneration with intramyofiber lipid accumulation, a marked decrease in free carnitine, and high levels of carnitine esters.

SPM appears to be a highly fatal emerging disease of pastured horses in the US characterized by weakness, colic-like signs and myoglobinuria. The disease is associated with a defect in muscular lipid metabolism that can be diagnosed by performing lipid staining of muscle samples and urine organic acid profiles. Candidatus Mycoplasma haemolamae (CMhl) is a common red blood cell parasite of new world camelids. The high degree of parasitemia that develops in an infected splenectomized animal allows for the efficient collection of parasitic DNA. This DNA can then be used in the development of genetically-derived tools such as PCR and in-situ hybridization. Thus, one splenectomized animal can replace many immunologically intact animals within a research setting. The purpose of this study was to track the natural progression of CMhl parasitemia and associated clinical signs in a splenectomized alpaca.

An intact, 9-month-old, 39.1 kg male alpaca was used in this study. He had tested positive via PCR for CMhl on three different occasions, although no organisms were seen on peripheral blood smears. The alpaca was placed under general anesthesia and a ventral midline incision was made. The spleen was located, the vessels ligated, and the organ removed. Buprenorphine and flunixin meglumine were given for 2 and 4 days after surgery respectively. Body weight, attitude, rectal temperature, blood glucose, and PCV were recorded daily. In addition, a peripheral blood smear was examined daily and the percent of red blood cells that were infected with Mycoplasma organisms was determined.

The alpaca was not parasitemic prior to surgery. One percent of the RBC's contained mycoplasma on days 2 and 3 after splenectomy. Parasitic bloom developed on day 4 with 85% of the red blood cells infected, and over 70% containing 3 or more organisms. The alpaca was treated with 20 mg/kg Oxytetracycline I.V. on day 4. On postoperative day 5 no parasites were seen in the peripheral blood. The peripheral blood remained free of parasites for 11 days. On the morning of the 12 th day, 3% of the peripheral red blood cells contained Mycoplasma. By late that afternoon, 75% of the observed RBC's contained 3-4 organisms. The alpaca again received oxytetracycline. There were no more parasites observed from that time until the alpaca was euthanized 5 days later.

The alpaca lost 3.7 kg between days À1 and 12 after surgery. His weight fluctuated between 34.9 and 35.4 kg for the remainder of the study period. Blood glucose ranged between 87 and 163 mg/dL There was no major change in PCV (range 21-29%), a finding that was expected as the spleen was not available to remove infected red blood cells. Body temperature ranged between 38.1 and 39 degrees Celsius except for days 4 and 16 when more than 70% of red blood cells contained parasites. On those days rectal temperature reached 39.4 and 39.1 degrees respectively.

This study confirmed that a non-parasitemic, yet PCR positive alpaca did indeed harbor CMhl. The time from splenectomy to parasitic bloom was shorter, and the length of oxytetracycline suppression longer than has been observed in other species. Gastro-intestinal (GI) disease frequently results in increased wall thickness in many species. Identification of changes in GI wall thickness using ultrasound has proved to be a useful diagnostic tool and is widely used in human patients, small animals and horses. Although GI motility has been evaluated in cattle, normal reference ranges for wall thickness has not been reported in ruminants. The aims of this study were to report normal values for wall thickness of various GI structures and to assess the repeatability of this technique in adult dairy cows, sheep and goats. Eight healthy adult Holstein Friesian (HF) cattle (656 AE 11 Kg), eight Jersey (J) cattle (458 AE 56 kg), thirteen adult sheep (79 AE 11 Kg) and eleven adult goats (43.5 AE 8 Kg) were recruited and examined on three consecutive days. Ultrasonographic images were optimised for the structure of interest. Structures were identified based upon appearance and anatomical position. A minimum of three cineloops were obtained of the abdominal organs per intercostal space (ICS) and three along the ventral midline in each ICS; images were analysed offline. Data were analysed using ANOVA and post-hoc Bonferoni, Student's Ttest and intra-class correlation coefficients. Each structure was measured per ICS per species; if no differences were noted for structures in different ICS, then measurements were pooled. No differences were noted between HF and J cattle so data were pooled. Data are displayed in table 1. Good repeatability (ICC40.91) was obtained for all measurements and no differences were noted between animals of the same species or between days. These measurements for assessment of normal GI thickness are repeatable and may allow valuable additional information to be gained from ruminants with GI disease. Ocular infections with the infectious bovine keratoconjunctivitis (IBK) agent Moraxella bovis (M. bovis) are associated with significant economic loss in the cattle industry.Although antibiotic therapy is the treatment of choice for IBK, treatment failures are common and current vaccines are not optimally effective mainly due to antigenic variation. As a result, our laboratory has been actively investigating the therapeutic potential of Bdellovibrio bacteriovorus 109J (B. bacteriovorus); a predatory bacterium capable of attacking and inducing lysis of Gram-negative bacteria, as a new treatment for IBK.

We have previously shown that B. bacteriovorus can reduce the number of M. bovis attached to bovine epithelial cells in an in vitro model of IBK and that B. bacteriovorus can be trained to kill M. bovis as effectively as E. coli using serial passages. In this study, we hypothesized that B. bacteriovorus can remain viable in bovine tears without its prey for up to 24 hours. This hypothesis was addressed by incubating inocula of active B. bacteriovorus in its preferred media peptone yeast extract (PYE) and comparing B. bacteriovorus viability in bovine tears or phosphate buffered saline (PBS) at time 0, 2, 4, 12, and 24 hours. Using a plaque assay to quantify the mean amount of plaque forming units (PFUs) of B. bacteriovorus exposed to each treatment, it was determined that viability of B. bacteriovorus over time was comparable between treatment groups.

Overall, the results supported that B. bacteriovorus can remain viable in tears for up to 24 hours in the absence of prey bacteria. Further studies are needed to determine the therapeutic potential of B. bacteriovorus in an in vivo model of IBK. Correction of the measured ionized calcium concentration (cCa 21 ) to a pH 5 7.40 is routinely applied in experimental studies in order to assist in the interpretation of measured values relative to a reference range. The equation most commonly used for pH correction in bovine plasma is: cCa 21 pH 5 7.40 5 cCa 21 Â10 (-0.24Â{7.40 -pH}) . The validity of this equation for bovine plasma is unknown. Accordingly, our first objective was to characterize the in vitro relationship between cCa 21 and pH for bovine plasma. Feeding rations with a low dietary cation-anion difference (DCAD) during late gestation mitigates periparturient hypocalcemia in dairy cows, particularly when chloride containing acidodgenic salts are fed. The mechanism for this beneficial effect remains unclear. Our second objective was to determine whether hyperchloremia displaces calcium from binding sites to albumin, thereby increasing cCa 21 .

The in vitro relationship between plasma log(cCa 21 ) and pH in was investigated using lithium heparin anticoagulated blood from 10 healthy Holstein-Friesian calves. Plasma was harvested and tonometered with CO 2 at 371C over a pH range of 7.10-7.70. Plasma chloride concentration (cCl -) was altered by equivolume dilution of plasma with 3 electrolyte solutions of varying cCl -(97 AE 1, 110 AE 1, and 123 AE 1 mEq/L; mean AE SD).

The slope of the linear regression equation relating log(cCa 21 ) to pH for 112 tonometered plasma samples from the 10 calves was -0.24 AE 0.05 at normal values for cCa 21 (2.63 AE 0.09 mEq/L), albumin concentration (30.9 AE 2.0 g/L), and cCl -(99.1 AE 2.2 mEq/L). The experimentally-determined value for the slope for bovine plasma was identical to that determined previously for human plasma. The formula for correcting cCa 21 in bovine plasma for change in pH from 7.40 is therefore: cCa 21 pH 5 7.40 5 cCa 21 Â 10 (À 0.24 Â {7.40-pH}) . This equation is only valid at normal concentrations of albumin and chloride in plasma. Equivolume dilution of plasma by electrolyte solutions of varying cClindicated that cCa 21 pH 5 7.40 increased by 0.007 mEq for every 1 mEq/L increase in cCl -. In other words, plasma cCa 21 at a given pH increases directly in response to an increase in plasma cCl -, presumably because the additional chloride displaces calcium that is electrostatically bound to albumin. Furthermore, the increase in cCa 21 is independent to the change in plasma pH induced by an increase in cCland decrease in plasma strong ion difference.

Our finding that hyperchloremia directly increases plasma cCa 21 provides an additional mechanism by which ingestion of high chloride (acidogenic) rations prevents the clinical signs of periparturient paresis. Our finding is consistent with the results of other studies that indicate acidogenic salts that contain chloride as the predominant anion (ie, NH 4 Cl, CaCl 2 ) are more effective in increasing cCa 21 than equimolar quantities of acidogenic salts such as MgSO 4 . Coagulase negative staphylococci (CNS) are among the most common bacteria isolated from the bovine mammary gland. Historically, these bacteria were lumped together as minor mastitis pathogens. Modern molecular techniques have allowed accurate speciation and fingerprinting of the CNS species. These methodologies have recently been applied to the study of CNS in bovine mastitis. The aim of the studies presented here was to evaluate the role of individual CNS species on milk somatic cell count (SCC) and duration of intramammary infection (IMI).

In the first study, mammary quarter foremilk samples were aseptically collected from all lactating cattle ($180 head) at the University of Missouri dairy research center once monthly for 17 months for bacterial culture and milk SCC. Staphylococcal isolates were speciated by sequencing the rpoB gene and strain-typed using pulsed-field gel electrophoresis (PFGE). Using species and fingerprint data along with published definitions for staphylococcal IMI, 91 CNS IMIs were identified. Overall, 11 species of CNS were identified with Staphylococcus chromogenes, S. cohnii, S. epidermidis, and S. simulans being most prevalent. Duration of IMI and SCC data were analyzed using regression models accounting for repeated measures. Mean milk SCC and duration of IMI were found to differ between CNS species (P o 0.05). Although most IMIs were of short duration (1 month), Staphylococcus capitis and S. chromogenes IMIs had longer mean durations of infection than 3 or more of the other species isolated. Mean SCCs were under 350,000 cells/ml in most cases. However, Staphylococcus simulans and S. xylosus IMIs were more inflammatory (mean 4 600,000 cells/ml) and had a higher mean SCC than S. cohnii, S. epidermidis, and S. haemolyticus.

To examine the relationship between CNS IMI and milk SCC in a larger population of cattle, CNS isolates from the Canadian Bovine Mastitis Research Network (CBMRN) culture collection were obtained for speciation. Speciation and fingerprinting were performed as above. Isolates were from subclinical IMI from before and after the dry period and from subclinical IMI during lactation. Data associated with each isolate were obtained from the CBMRN database. Nine-hundred-thirty-eight isolates from 696 mammary quarters in 89 herds were successfully speciated. Twenty-two different species of CNS were identified. Staphylococcus chromogenes was the most frequent species identified accounting for 40% of the infections. Three species, S. chromogenes, S. xylosus, and S. simulans accounted for 475% of all infections. Data were analyzed using a linear hierarchical repeated measures mixed model. Differences in mean SCC were found between some CNS species and culture negative control quarters and also between different species of CNS (P o 0.05). Overall, our data demonstrate potential differences in pathogenicity between strains of CNS that cause bovine mastitis. Passive transfer of maternally derived antibodies via ingestion of good-quality colostrum within the first 24 hours of life is crucial for the health and future productivity of dairy calves. However, infectious diseases can be transmitted via colostrum feeding, which may require use of a colostrum replacement product or pasteurization to decrease disease transmission. While pasteurization of colostrum is effective for sterilization, heating during pasteurization can alter the viscosity of colostrum, destroys important nutritional biomolecules, and has been shown to decrease colostral IgG concentrations. The purpose of this study was to investigate the effect of high pressure processing (HPP) on the viscosity, IgG concentration, and bacterial contamination of bovine colostrum. First milking colostrum samples were collected from 18 cows from 3 different farms, and 50 ml aliquots of each sample were pooled for analysis. Pooled colostrum was processed in triplicate using an isostatic press at 400 MPa (60,000 psig) for 0, 5, 15, 30, and 45 minutes. Samples were tested for the effects of HPP on the viscosity, bacterial load (CFU/ml), and IgG concentration.

There was a significant decrease (p o 0.05) in bacterial load at each time point when compared to time 0. No significant difference in IgG concentration was found between any time points. Subjectively, the colostrum viscosity appeared to increase with the processing time, though the rheologic assessment has not been completed at this time.

HPP appears to be an effective method to decrease bacterial contamination of colostrum while maintaining appropriate IgG concentrations. Minimizing the processing time or pressure may be necessary to maintain an acceptable viscosity of the colostrum. Based on these results, additional studies are justified in order to determine the optimum combination of processing time and pressure and the effect of HPP on specific bovine pathogens. The heme-associated iron-binding apoprotein lactoferrin (LF) is known for its, anti-inflammatory, anti-parasitic, antimicrobial and bactericidal effects. Lactoferrin demonstrates ubiquity throughout mammalian host biological fluids: Saliva; tears; mammary secretions, as well as at mucosal surfaces. It is also released from immune cells under pathogenic stimulation. The purpose of this study which has been approved by Western University's Institutional Animal Care and Use Committee is to further characterize the mechanisms through which LF modulates inflammation in the face of bacterial endotoxin. It was hypothesized that LF would inhibit p38 phosphorylation. Numerous studies speak to the ability of LF to alter leukocyte function, inhibit cytokine production, and bind lipopolysaccharide (LPS); mechanisms through which it is believed to achieve its anti-inflammatory effects. Recently, investigators demonstrated its ability to interact with host DNA while others describe regulation of granulocyte adhesion and motility; elucidating its roles in the apoptotic signaling.

In earlier studies, Dawes ME, et al. demonstrated lactoferrin's ability to limit the expression of inducible cyclooxygenase-2 and the gelatinase, matrix metalloproteinase -9 by LPS-induced macrophages. The generation of these inflammatory mediators is modulated by pro-inflammatory cytokines such as interleukin-1 b (IL-1 b) and tumor necrosis factor-alpha (TNF-a), the production of both being dependent on signaling through the p38 mitogenactivated protein kinase (MAPK) pathway.

Peripheral mononuclear cells (5 Â 10 6 )isolated from buffy coat cells of healthy neonatal to 4-month old Holstein calves were cultured in the presence and absence of LF (200 ng/ml), LPS (1 mg/ml), anisomycin (25 mg/ml), a known p38 activator -the positive control, and 50 mM of SB203580, a known p38 inhibitor -the negative control. Sample lysates obtained post culture was subjected to immunoprecipitation and kinase reactions. Reactions were terminated under reducing conditions and evaluated using western immunoblotting. Phosphorylation of activated transcription factor-2 (ATF-2) by phosphorylated p38 served as the marker of investigation. Immunologically reactive ATF-2 expression by LPS and anisomycin-treated cells was compatible with a prominent band at 40 kD. Evidence of LF-induced inhibition of LPS-induced p-38 activation was observed in lanes representative of co-cultures of LF 1 LPS; LF 1 anisomycin; and anisomycin 1 SB203580, which was demonstrated by decreased immunological reactivity at 40kD. The findings here, suggest that LF interferes with LPS-induced p-38 activation of transcription factor ATF-2, in vitro. This serves as additional proof of its potential use in attenuating the systemic effects of LPS. Six (6) clinically normal, purpose-bred cats of similar age and body condition were imaged with [ 18 F] fluorodeoxyglucose ([ 18 F]FDG) and [ 18 F]FTHA by using dynamic cardiac-gated fused PET/CT for kinetic assessment of myocardial glucose and fatty acid uptake and metabolism, respectively. Kinetic tracer uptake within the myocardium was achieved by initiating image data acquisition simultaneously with tracer injection. PET data were acquired over a 1 hour period with the heart in the center of the scanner field of view.

Regions of interest were drawn in the left ventricular wall and thoracic aorta for the purpose of measuring the kinetics of tracer redistribution. Serial blood samples were also taken during PET imaging for comparison with image data. The equilibrium biodistribution of both tracers was documented 1 hour post-injection in a whole body PET/CT image. Standard echocardiographic examination of cardiac structures was also performed.

Both radiotracers remained in the plasma fraction; however, [ 18 F]FTHA was cleared from the more rapidly than [ 18 F]FDG (t 1/2 $ 2 and $ 20 min, respectively). The tracers were readily visualized within the feline myocardium in dynamic PET images and analysis of the blood pool clearance from the kinetic image data agreed with blood sampling data. Myocardial uptake of each tracer was best described by a double exponential analysis and was rapid but variable among animals (range 1-30 Bq/cc/min), although blood glucose levels were similar in all cats during image acquisition. Physiologic [ 18 F]FDG was observed in the brain, salivary tissue, gastrointestinal tract, renal pelves and urinary bladder, with [ 18 F]FTHA seen in the myocardium, liver and renal cortex. All cats were normotensive with normal echocardiographic parameters.

This study demonstrates the utility of kinetic imaging using The left ventricle (LV) shape has been suggested to change from elliptical to more globular in response to chronic volume overload. Real-time three-dimensional echocardiography (RT3DE) offers new modalities for LV assessment. The aim of the study was to investigate left ventricular changes in shape and volume occurring in response to different severities of naturally acquired myxomatous mitral valve disease (MMVD) in dogs using RT3DE.

Privately owned dogs were classified by standard echocardiography into: Healthy (20), mild (20), moderate (8) and severe MMVD (17). A LV cast was obtained using semi-automated endocardial border tracking from RT3DE dataset, from which global and regional (automatically acquired basal, mid, and apical segments based on LV long-axis dimension) end-diastolic (EDV) and endsystolic volumes (ESV), LV Long-axis dimension and RT3DE Sphericity index, were derived. Global and regional EDV and ESV increased significantly with increasing MMVD severity, assessed by MMVD group-wise comparisons and linear regression analyses using left atrial to aortic root ratio, and LV end-diastolic and end-systolic dimensions. All three segments contributed to the overall increased global volumes, but the mid EDV segment was strongest associated with increasing LV end diastolic dimension (P 5 0.048). Furthermore, LV Long axis distance and LV Sphericity index increased with increasing MMVD severity. The basal and apical EDV segments were strongest associated with Sphericity index (P o 0.0001).

In conclusion, this RT3DE study showed that increased LV EDV, primarily in the mid segment, leads to rounding of LV apical and basal segments in response to increasing MMVD severity in dogs. 84 dogs from shelters in Florida with naturally acquired DI infection were euthanized and necropsied. All adult DI in each dog were sexed using morphological features. Total worm burdens and numbers of males and females were recorded. No other information was available for any dog. All data, raw and transformed, were examined visually and descriptively. Raw numerical data were further examined by a paired T-test; log-odds transformed data were examined by logistic regression. We also conducted a binomial distribution goodness of fit analysis assuming a null hypothesis of a M:F 5 1.0.

Worm intensities ranged from 1 to 143 DI per dog. Eight dogs had unisex infections: 7/8 had all-female infections. Dogs with lowintensity dual-sex infections were more likely to have greater numbers of female DI. Overall, sex ratios were equal (paired t-test, P 5 0.7). However, logistic regression demonstrated that the probability of being female is strongly affected by the total worm intensity, with lower intensities increasing the probability of having a predominance of female worms.

Our data show that DI sex ratios in naturally-infected dogs equal 1 when examining the entire dog population, but deviate to favor female worms at low worm intensities. These data could impact adulticide treatment strategies. The reasons for sex ratio distortion in DI are unknown. We evaluated cardiac reverse remodeling after mitral valve repair under cardiopulmonary bypass (CPB) for mitral regurgitation in small breed dogs. Fifty dogs (body weight 1.8-9.3 kg, age 5-14 years) with mitral regurgitation were treated between August 2006 and November 2009. The cardiac murmur was grade 4/6-6/6. The preoperative chest X-rays showed cardiac enlargement (vertebral heart scale (VHS) 11.0-13.1). Echocardiography showed severe mitral regurgitation and left atrium enlargement (LA/Ao 2.0-4.2). After inducing anesthesia, a thoracotomy was performed in the fifth intercostal space. CPB was started by using a CPB circuit connected to carotid artery and jugular vein catheters. After inducing cardiac arrest, the left atrium was sectioned and chordae tendineae rupture confirmed. The chordae tendineae were replaced with expanded polytetrafluoroethylene. A mitral annulus plasty was also done, and the left atrium was closed. After de-clamping for restarting the heart, the chest was closed. Heart rate decreased from 118-164 bpm to 75-138 bpm. The grade of cardiac murmur was reduced to 0/6-3/6 three months postoperatively, and the heart shadow was reduced (VHS 9.8-11.5) in the chest x-rays. Echocardiography confirmed the marked reduction in mitral regurgitation and the left atrial dimensions (LA/Ao 1.2-2.2). Mitral valve repair reduced enlarged cardiac size by reduction of regurgitant rate. Pulmonary arterial hypertension (PAH) is a well recognized condition in dogs leading to considerable morbidity and mortality. The majority of therapeutics has focused on endothelial dysfunction causing reduced production of vasodilators, such as nitric oxide and prostacyclin, coupled with overproduction of vasoconstrictors, such as endothelin-1. More recently, it has been shown that the mitochondria play an important role in the development of PAH as oxygen sensors and regulators of cellular proliferation. In PAH, pulmonary artery smooth muscle cells undergo a metabolic shift from oxidative phosphorylation in the mitochondria to glycolysis in the cytoplasm as the major energy source and this leads to suppression of apoptosis and increased proliferation. Dichloroacetate (DCA) inhibits pyruvate dehydrogenase kinase to activate pyruvate dehydrogenase which catalyzes the rate limiting step for entry of pyruvate into the Krebs cycle, thus increasing mitochondrial respiration. In three different rat models of PAH, DCA has been shown prevent and reverse PAH by normalizing molecular pathology, stimulating apoptosis of pulmonary artery smooth muscle cells, and reducing pulmonary artery hypertrophy. DCA has known toxic effects, including reversible hepatotoxicity and peripheral neuropathy, and has not been studied in any species with naturally occurring PAH. The objective of this open label pilot study is to evaluate the therapeutic and toxic effects of DCA in naturally occurring canine PAH.

Three dogs with PAH diagnosed by Doppler echocardiography and no correctable underlying cause are enrolled in the study. Dogs are orally administered 25 mg/kg of DCA divided daily for 2 weeks, and then 12.5 mg/kg of DCA divided daily for the remainder of the study. At baseline, 2, 4, 8, and 12 weeks, an echocardiogram, CBC, serum chemistry profile, urinalysis, NT-proBNP, blood uric acid, blood lactate, noninvasive blood pressure, nerve conduction study, and trough DCA level (12 hr post-dose) are obtained. The measured echocardiographic parameters include peak and mean tricuspid regurgitant flow velocity and pressure gradient, peak and enddiastolic pulmonary regurgitant flow velocity and pressure gradient, pulmonary valve flow velocity acceleration time and ejection time, pulmonary valve flow velocity time integral, right ventricular myocardial performance index, tricuspid annular plane systolic excursion, and systolic tricuspid annular tissue velocity. Variables are inspected for normalcy and equality of variances and a two-sided paired t-test is used to compare the variables before and after treatment at each evaluation time.

The basis for the role of the mitochondria in PAH and the results of this pilot study will be presented to determine if DCA warrants further study as a therapy for dogs with PAH. study produced the strongest associations between the NCL phenotype and CFA2 markers. All 19 NCL-affected Tibetan Terriers were homozygous for the same haplotype which extended for 22 consecutive SNPs spanning 0.95 Mb. None of the 20 annotated genes within this target region had previously been associated with human or rodent NCL. We used DNA from NCL-affected Tibetan Terriers to resequence the coding regions and intron-exon borders of several genes harbored within the target region and found a single base pair deletion, c.1623delG, in exon 16 of positional candidate ATP13A2. This deletion produces a frame shift and a predicted premature termination codon. We genotyped all 454 Tibetan Terrier DNA samples in our collection and found all 45 NCL-affected Tibetan Terriers to be homozygous for the c.1623delG allele. Eleven additional c.1623delG homozygotes were either less than 5 years old, or lost to follow up. There were no known cases of NCL in the remaining 398 Tibetan Terriers which were either heterozygous (n 5 149) or homozygous for the ancestral allele (n 5 249). ATP13A2 is a member of group of ion transport genes and has been associated with lysosomes. Mutations in human ATP13A2 cause Kufor-Rakeb syndrome (KRS), a rare neurodegenerative disorder with clinical features that include parkinsonism plus spasticity, supranuclear upgaze paresis, and dementia. Post-mortem findings in KRS have not been reported. We conclude that NCL in Tibetan terriers is caused by a mutation in ATP13A2. Our results suggest that KRS may be a form of adult onset NCL in humans. Niemann-Pick type C (NPC) disease is a progressive neurological disorder characterized by dementia and ataxia, hepatic and pulmonary disease, and death typically within the first or second decade. Despite the identification of causative mutations, the pathogenesis is not clear and therapies to successfully treat NPC disease have been ineffective to date. The recent use of intravenously administered 2-hydroxypropylbeta-cyclodextrin (HPBCD), an FDA-designated orphan drug (May 2010), in a small number of children with NPC disease is based on favorable treatment outcome data in subcutaneously treated mouse and cat models. To rigorously evaluate the mechanistic, pharmacologic, and toxicity issues associated with HPBCD therapy in NPC disease, we have utilized the spontaneous feline NPC model harboring a missense mutation in NPC1 (pC955S), orthologous to the most common mutation in juvenile-onset patients. The feline NPC model has clinical, neuropathological and biochemical abnormalities similar to those present in juvenile-onset patients making this model homologous to the most common disease form seen in human patients. We identified that intrathecal administration of HPBCD ameliorated all clinical aspects of neurological disease at least up to 24 weeks of age (an age when untreated cats die) but had no effect on hepatic disease. We identified that while subcutaneous therapy with HPBCD at all doses ameliorated liver disease, only 8000 mg/kg substantially affected neurological disease but also resulted in early death due to pulmonary toxicity. Finally, we identified a dose-related toxic effect of HPBCD on hearing function that had not been described in any other species. Leukodystrophies are disorders of myelin synthesis and maintenance that affect CNS myelin. They are subdivided as leukodystrophies, hypomyelinating disorders and spongy degenerations. Although infrequently seen, several forms have been described in various dog breeds. We present a novel form of complex leukodystrophy consisting of hypomyelination and spongy degeneration that presents primarily with hind end tremors in Border terrier puppies. Three Border terriers from two different litters (and lineages) are described here that presented with a history of shaking movements. The youngest dog was a 3-week old male. It was the only dog affected in the litter. The other two dogs were 6-week old female littermates. There were two unaffected males in the same litter. Physical examination revealed no abnormalities. On neurological examination, the affected dogs displayed severe hind end tremors, with a characteristic swinging side-to-side movement (best described as ''rumpshaker''). The tremors also involved the head and thoracic limbs but to a lesser degree, and disappeared when the dogs were asleep or at rest. Severe cerebellar ataxia was observed when the dogs ambulated. Proprioceptive positioning was delayed in the pelvic limbs of all 3 dogs. Spinal reflexes and nociception appeared normal. Necropsy was performed in all 3 puppies. No macroscopic changes were observed. Histologic evaluation of the CNS revealed spongy degeneration and hypomyelination in all funiculi of the cervical and thoracic spinal cord. White matter of the frontal, temporal and parietal cortices had mild multifocal spongy degeneration and hypomyelination, whereas white matter of the cerebellum, medulla and pons showed severe diffuse spongy degeneration and hypomyelination with gliosis. The combination of reduced myelin formation combined with spongiform white matter changes in the absence of microglial responses suggest a complex pathogenesis affecting both oligodendrocytes' capacity to synthesize myelin and the stability of the myelin that was formed. The number of oligodendrocytes and axons appeared subjectively normal indicating a primarily hypomyelinating process.

The clinical and pathological features of this disease have not been described in any other canine leukodystrophy. The primary and most striking clinical feature is the presence of severe tremors in the hind end, causing the ''rumpshaker'' pheynotype. Genetic studies are underway to determine if the disease is inherited and the inheritance mode. A syndrome of Border collie collapse (BCC) appears to be common in dogs used for working stock. This syndrome has also been called malignant hyperthermia, heat intolerance, exerciseinduced collapse and ''wobbles''. A presumptive diagnosis of BCC can only be made by eliminating other causes of exercise intolerance and weakness. The purpose of this study was to describe the clinical features of collapse in affected dogs and determine if there were characteristic clinical or laboratory features at rest or after exercise that could aid in diagnosis.

Seven adult Border collies with a history of collapse during sheep herding (affected) and 5 adult Border collies regularly used for sheep herding but showing no signs of exercise intolerance (normal) were evaluated before and after participating in a videotaped 10 minute exercise protocol consisting ofa series of continuous short outruns and fetches of three sheep in an outdoor pen. Exercise was halted at 10 minutes or earlier if there were signs of gait or mentation abnormalities. Pre-exercise evaluation included physical examination, orthopedic and neurological exam. Pre and immediate post exercise rectal temperature, pulse and respiration, patellar reflexes, ECG, CBC, serum biochemistry profile, cortisol, arterial blood gas and plasma lactate and pyruvate concentrations were measured. Clinical parameters (gait, temperature, reflexes) and lactate and pyruvate concentrations were evaluated at intervals up to 120 minutes after exercise. Additional testing in affected dogs included measurement of acetylcholine receptor antibodies (AChRab) and DNA testing for dynamin-associated exercise induced collapse (dEIC) and the ryanodine receptor mutation associated with canine malignant hyperthermia(MH). One week after exercise affected dogs had thoracic radiographs and echocardiography performed and were anesthetized for EMG and muscle biopsies.

There were no significant differences in temperature, pulse, respiration, or any laboratory parameter at any time point between normal and affected dogs. No arrhythmias were detected. Affected dogs were negative for the DNA mutations tested and for AChR ab. Thoracic radiographs, echocardiograms, EMGs and muscle biopsies were normal.

The 5 normal dogs had no alterations in mentation or gait during or after exercise. Three of the affected dogs had exercise halted early (6 min-9 min) because of altered gait or mentation. All 7 of the affected dogs were abnormal in the 15 minutes following exercise. Abnormalities seen in all dogs included disorientation, dull mentation, swaying, falling to the side, exaggerated lifting of limbs each step, choppy gait, delayed limb protraction, scuffing of rear and/or forelegs, and crossing legs when turning. All dogs returned to normal by 30 minutes.

BCC appears to be an episodic nervous system disorder that can be triggered by exercise. Genetic testing excluded dEIC and the described canine MH mutation. Common causes of exercise intolerance were eliminated, but the cause of collapse in BCC was not determined and no clinical or biochemical marker to aid diagnosis was established. Equine Cushing's disease (ECD) is common in older horses. The purpose of this study was to determine the frequency of diagnosis, identify prognostic factors and assess owner satisfaction with treatment. The study was a retrospective cohort design evaluating equine accessions reported to the Veterinary Medical Data Base (VMDB) and the Ohio State University from 1993-2004. Proportional accessions, annual incidence and demographic characteristics of horses with ECD were compared with all accessions in the VMDB. Medical records for a subset of horses were extracted and owners contacted to obtain long-term follow up information.

Two hundred seventeen new cases of ECD were reported to the VMDB. Incidence increased from 0.25/1,000 in 1993 to 3.72/1,000 in 2002. Eighty-one percent of horses were ! 15 years of age. Average delay from onset of signs to diagnosis was 180 days (range 1 to 1,824 days). Hirsutism (84%) and laminitis (50%) were the most common clinical signs. Improvement in one or more signs 2 months after diagnosis was reported by 9/22 (41%) of horse owners. None of the clinical or laboratory data were associated with survival and, 50% of horses were alive, 4.6 years after diagnosis. 17/20 (85%) of horses were euthanatized and 13/17 (76%) were euthanatized due to conditions associated with ECD. Twenty-eight of 29 (97%) of horse owners said they would treat a second horse for ECD.

ECD is becoming a more frequent diagnosis. Fifty percent of horses survived 4.5 years after diagnosis and owners were satisfied with the horse's quality of life.

Supported by Centers of Excellence in Livestock Diseases and Human Health, College of Veterinary Medicine, University of Tennessee. The role of the hypothalamic-pituitary-adrenal (HPA) axis in sepsis has been a subject of a great deal of research. The role that the somatogenic axis plays in sepsis is less well understood and how these two axes interact during critical illness is not clear. The purpose of this study was to assess inter-relationships of adrenocorticotropin (ACTH), cortisol, and insulin-like growth factor-I (IGF-I), in septic and non-septic term foals.

Blood samples were obtained from term septic foals less than 7 days of age (n 5 20) admitted to Texas A&M University Veterinary Medical Teaching Hospital or Mid-Atlantic Equine Hospital. The foals were classified as septic by a sepsis score ! 11 and/ or a positive blood culture. Non-septic term foals less than 7 days of age (n 5 8) and having a sepsis score o 11 and a negative blood culture, were obtained from Texas A&M University Veterinary Medical Teaching Hospital and Mid-Atlantic Equine Hospital. Plasma and serum were processed from whole blood collected by jugular venipuncture upon admission, at 24 hours post admission and at 5 days post admission or at the time of discharge.

Plasma concentrations of ACTH, and serum concentrations of cortisol and IGF-I were determined by specific RIAs. Data were analyzed using linear mixed-effects modeling with foal modeled as a random effect and day of admission modeled as an ordered categorical variable; post-hoc testing of pair-wise comparisons was made using the method of Sidak. Significance was set at P o 0.05, and analyses were performed using S-PLUS software (TIBCO, Inc., Seattle, WA).

Plasma concentrations of ACTH were not significantly different between septic and non-septic foals whereas septic foals had greater serum cortisol (37 AE 8 ng/mL vs 25 AE 7 ng/mL) but lower serum IGF-I (116 AE 14 ng/mL vs 152 AE 15 ng/mL) relative to non-septic foals pooled overall sampling times. The positive association of the peripheral blood concentrations of ACTH and cortisol depended on disease status of the foals. Specifically, cortisol and ACTH were positively correlated for the septic foals (P 5 0.027) but not significantly correlated in the non-septic foals. Peripheral concentrations of ACTH and IGF-I were not significantly correlated whether data were pooled overall or stratified by sepsis status. However, peripheral concentrations of cortisol and IGF-I were negatively associated (P 5 0.019); disease status did not influence this association, although it appeared to be a stronger association for the septic than the non-septic foals. The negative correlation between serum concentrations of the adrenal axis steroid cortisol and the somatogenic axis peptide IGF-I may reflect interactions of these homeorhetic hormones. Further studies of these and other metabolic hormones in a greater number of foals are warranted to better understand how these factors contribute to survival or non-survival of critically ill foals. Botulism is a potentially fatal paralytic disorder which definitive diagnosis is difficult. The purpose of this study was to investigate if repetitive stimulation of the common peroneal nerve will aid in the diagnosis of suspected botulism in foals.

Four healthy foals were used for its comparison with 3 foals with suspected botulism. Controls were anesthetized and affected foals were sedated to avoid risks of anesthesia. The common peroneal nerve was chosen for its superficial location and easy access. Stimulating electrodes were placed along the common peroneal nerve. For recording, the active and reference electrodes were positioned over the midpoint and distal end of the extensor digitorum longus muscle, respectively. Repeated supramaximal stimulation of the nerve was performed utilizing a range of frequencies (1 to 50 Hz). Amplitude, area under the curve and percentages of decrement or increment for each M wave over subsequent potentials for each set of stimuli were analyzed.

Baseline M waves were decreased in affected foals compared to controls. A decremental response was seen at all frequencies in control foals. Decremental responses were also observed in affected foals at low frequencies. However, an incremental response in amplitude and area under the curve was seen in all affected foals at 50 Hz.

Reduced baseline M waves with incremental responses at high rates are supportive of a presynaptic neuromuscular disorder which botulism was the most likely cause in these foals. Repetitive nerve stimulation is a safe, simple, fast, and non-invasive technique that can aid in the diagnosis of suspected botulism in foals.

This study examined the frequency with which dogs are exposed to E. chaffeensis and E. ewingii relative to E. canis, which is transmitted by the more ubiquitously distributed brown dog tick (Rhipicephalus sanguineus). A total of 6,512 canine serum samples, ranging from 182 to 614 from each of the 14 participating institutions, collected at random from clinical accessions, diagnostic laboratories and/or shelters were evaluated. All serum samples were tested by three microtiter plate ELISAs using species-specific peptides for antibodies to E. canis, E. chaffeensis and E. ewingii. Zip code information for sample origin was provided by the collaborator and was used to assess seroprevalence by region. Comparisons were evaluated using the Chi-square test. Seroreactivity for at least 2 of 3 Ehrlichia spp was found in samples from every institution

Both Mississippi and Oklahoma had greater than a 7%

Samples from Ohio had the lowest aggregate seroprevalence (1.0%) with only 4 dogs E. canis seropositive, one E. ewingii seropositive and no E. chaffeensis seroreactors. The geospatial pattern of E. chaffeensis and E. ewingii seropositive samples was similar to that previously reported based on modeling seroreactivity to E. chaffeensis in white-tailed deer as well as the distribution of human monocytic ehrlichiosis (HME) cases reported by the CDC. This study provides the first large scale regional documentation of canine exposure to these three Ehrlichia spp., highlighting where infections most commonly occur and thus identifying areas where heightened awareness about these emerging vector

Urinary incontinence (UI) occurs in approximately 20% of spayed female dogs. The most common cause is urethral sphincter mechanism incompetency (USMI). Pharmacological agents are effective, however, not all dogs respond, and dogs may become refractory to treatment over time. Urethral bulking, where a compound is injected submucosally in the urethra, has been used in women and in female dogs with urinary incontinence. New synthetic compounds have been used in human medicine; the most promising is Polydimethylsiloxane (PDMS), which has been shown to be more effective than glutaraldehyde cross-linked collagen. The purpose of this descriptive clinical trial is to evaluate the safety and effectiveness of PDMS urethral bulking agent (PDMS UBA) in client-owned, spayed female dogs with naturally-occurring UI due to USMI.Twenty-two, spayed female dogs were included. Dogs had a median age of 6 years (2 to 11 years). Eighteen dog breeds were represented, and dogs weighed a median of 29.9 kg (7.3 to 62.7 kg). Average length of time of UI was 2.5 AE 2.3 years; 18/22 dogs had been treated medically, of which 2/18 were continent, 14/18 were improved, and 2/18 had no improvement. Dogs were deemed healthy based on results of physical examination, complete blood cell counts, plasma biochemical analysis, and urinalysis; urine cultures were negative.Dogs were anesthetized, positioned in dorsal recumbency, and cystoscopy performed using a 2.7 mm, 0-or 30-degree, 18 cm rigid cystoscope. Urethral bulking was performed with PDMS UBA. On average, 2.5 AE 0.9 ml were injected in 3 to 5 locations approximately 1 to 1.5 cm distal to the trigone submucosally in the proximal urethra. Good coaptation was achieved in all dogs. The procedure took on average 15.9 AE 4.3 minutes. One dog experienced urethral obstruction after the procedure; a Foley catheter was inserted for approximately 12 hours and removed at which time she urinated normally and was continent. Three dogs experienced an acute allergic reaction characterized by blepharedema and urticaria treated successfully with diphenhydramine. Dogs were discharged on day of procedure except for the one dog that experienced urethral obstruction. All dogs were treated with Meloxicam (0.1 mg/kg PO q24h for 3 days).Owners were contacted on day after discharge and 21/22 dogs were continent; 1/22 dogs was improved. Dogs were re-evaluated 1 week after discharge and 21/22 dogs were continent and 1/22 dogs Polyneuropathy in large breed dogs is a relatively common clinical problem for which the genetic basis is generally unknown. The first cases of polyneuropathy in the Leonberger breed (Leonberger polyneuropathy or LPN) were identified in 1999 by one of the authors (GDS) and a report published in 2003 (MuscleNerve 27:471-477) . In this report a spontaneous, distal and symmetrical polyneuropathy with onset between 1 to 9 years of age was described and characterized clinically, electrophysiologically, histologically and morphometrically. There were striking similarities between LPN and the Charcot-Marie-Tooth group of human inherited sensory and motor polyneuropathies, which have many known genetic mutations.A genome-wide case-control association study for LPN was performed with 53 cases and 42 controls on high-density 170K canine SNP arrays and revealed a significantly associated region on CFA 16 (P raw 5 2.36  10 À10 P genome 5 9.99  10 À5 ). A clear association of an approximately 1 Mb CFA16 haplotype with cases (P 5 1.71  10 À8 ) was observed, particularly with those cases that were affected more severely and at a younger age (P 5 2.55  10 À11 ). A positional candidate gene, ARHGEF10, which has previously been associated with peripheral nerve abnormalities in humans, was sequenced, revealing a deletion that results in a frame shift and premature stop codon. Of all Leonbergers with young onset LPN (before 4 years), 48.5% (32 of 66) have two copies of this deletion, and, of all young onset Leonbergers that are nerve biopsy positive for LPN, 59.4% (19 of 32) have two copies of this deletion. Importantly, nearly all dogs carrying two copies of the deletion (32 of 34 or 94.1%) are affected with LPN by the age of 4 years.The Leonberger breed was generated from crossing several breeds, including the St. Bernard, and a polyneuropathy clinically and histologically similar to LPN occurs in this breed. To determine if the ARHGEF10 mutation was associated with polyneuropathy in the St. Bernard, DNA was extracted from archived frozen muscle biopsy specimens from clinical cases (n 5 3). The identical ARHGEF10 Startle disease or hyperekplexia is caused by defects in mammalian glycinergic neurotransmission resulting in an exaggerated startle reflex and extensor hypertonia triggered by noise or touch. In humans and animals, startle disease is typically caused by mutations in one of three genes (GLRA1, GLRB, and SLC6A5) encoding postsynaptic glycine receptor subunits (a1 and b) or a presynaptic glycine transporter (GlyT2). A litter of seven Irish Wolfhounds was recently identified in which two puppies developed muscle stiffness and tremor beginning at 5-7 days of age post-partum. Signs were dramatic when the puppies were handled and resolved when the puppies were relaxed or sleeping. Both puppies were euthanized due to ongoing stiffness, tremor and breathing difficulties. Necropsies were performed, but no microscopic pathological abnormalities were identified in the peripheral or central nervous system.Based on the clinical signs, exons from the three candidate genes were amplified from genomic DNA isolated using PCR and directly sequenced. No deleterious polymorphisms were identified in either GLRA1 or GLRB. However, difficulties were experienced in amplifying SLC6A5 exons 2 and 3 from affected animals, although control samples were positive, suggesting that the PCR primer designs and conditions were not at fault. Further PCRs revealed that the reason for this anomaly was the presence of a homozygous 4.2 kb deletion encompassing exons 2 and 3 of the GlyT2 gene in both affected animals. This deletion is predicted to result in the loss of part of the large cytoplasmic N-terminus that is vital for trafficking of GlyT2 to synaptic sites, and a loss of all subsequent transmembrane domains via a frameshift. This genetic lesion was confirmed by defining the deletion breakpoint, Southern blotting and Multiplex Ligationdependent Probe Amplification (MLPA). This analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, suggesting recessive inheritance of this disorder. Wider testing of related animals has identified a total of 18 carriers of the SLC6A5 deletion and enabled the identification of non-carrier animals to guide future breeding strategies. Insulin resistance (IR), obesity, and type 2 diabetes affect glucagon-like peptide 1 (GLP-1) concentrations in humans and rodents, but this incretin hormone has not been examined in horses. We therefore hypothesized that GLP-1 concentrations would change in horses as obesity and IR were induced or exacerbated by overfeeding. Six horses previously diagnosed with Equine Metabolic Syndrome were provided with twice the amount of digestible energy required for maintenance as sweet feed and hay for 8 weeks. Intravenous and oral glucose tolerance tests (OGTTs) were performed at 0 and 8 weeks. Effects of time and period (0 and 8 weeks) were assessed by repeated measures ANOVA.Mean body weight increased from 438 AE 61 kg (range, 381 to 533 kg) to 464 AE 61 kg (range, 394 to 550 kg) over 8 weeks, with individual horse weight gain varying from 2 to 10%. Mean body condition score increased (P 5 0.006) from 6 AE 2 (range, 4 to 8.5) to 8 AE 1 (range, 7 to 9). Three horses developed mild laminitis. Glucagon-like peptide 1 concentrations increased over time during OGTTs (P 5 0.023), but the period  time effect was not significant (P 5 0.141). Area under the GLP-1 curve remained unaffected by weight gain, whereas area under the insulin curve increased (P 5 0.003) over time, indicating a reduction in insulin sensitivity. Obesity and IR were induced or exacerbated when horses previously diagnosed with EMS were overfed, but GLP-1 concentrations did not change as a result. Hypertonic saline solution (7.2%) (HSS) is an intravenous fluid used for the emergency treatment of intravascular volume deficits. The use of this fluid in horses with severe dehydration is controversial. The purpose of this study was to compare the use of HSS and isotonic saline solution (0.9%) (ISS) for the emergency treatment of endurance horses.Endurance horses eliminated from competition and requiring intravenous fluid therapy were eligible for enrollment in the study. Twenty-two horses were randomly assigned to receive 4 ml/kg of either HSS or ISS along with 5 L lactate Ringer's solution (LRS). Following this bolus, all horses were treated with an additional 10L of LRS. Blood and urine samples were collected before, during and after treatment. Data was compared using two-way ANOVA with repeated measures.As compared to ISS, HSS horses showed a greater decrease in PCV (p 5 0.04), total protein (p 5 0.01), albumin (p 5 0.01), and globulin (p 5 0.02). HSS horses showed a greater increase in sodium and chloride (p o 0.001) as compared to ISS horses. Horses receiving HSS had a shorter time to urination (p 5 0.03) and lower specific gravity (p o 0.001) than those receiving ISS.Results of this study indicate that HSS may provide faster restoration of intravascular volume deficits than ISS in endurance horses receiving emergency medical treatment. More profound electrolyte changes should be expected with HSS however. b 2 -adrenergic receptor agonists have been shown to increase erythrocyte carbonic anhydrase activity, which may stimulate the Jacobs-Stewart cycle and increase pulmonary circulation transvascular fluid fluxes during exercise. Increase in pulmonary transvascular fluid fluxes (J V-A ) and consequent increase in the pulmonary interstitial fluid would be detrimental for alveolar O 2 exchange during the fast erythrocyte transition time across the pulmonary capillaries. Therefore, we hypothesised that treatment with inhaled b 2 -adrenergic receptor agonist will increase J V-A and the alveolar-arterial PO 2 difference (AaDO 2 ) during exercise.Six STB horses were exercised on a high-speed treadmill at 80% VO 2 peak until fatigue. Horses were randomly assigned to treatment with salbutamol (Sal: 500mcg) or placebo (Control: Con) inhalation via Aeromask à 60 min prior to exercise, with cross over treatment used at the repeated exercise test (8 days later). Arterial and mixedvenous blood, as well as CO 2 elimination and O 2 uptake, were sampled simultaneously at rest, during exercise at 60 sec intervals until fatigue, and into recovery. Blood gases were analyzed. AaDO 2 was calculated using the inspired PO 2 (149 mmHg), and blood partial pressure of O 2 and CO 2 . Blood volume (%) changes across the lung were calculated from changes in hemoglobin and hematocrit values in venous and arterial blood. Cardiac output (Q) was calculated using the Fick equation. J V-A was calculated using Q and blood volume changes across the lung. Variables were analyzed using two-way repeated-measures ANOVA (P o 0.05).The duration of exercise to fatigue was 4.3 AE 0.3 min and 4.4 AE 0.4 min in both Con and Sal, respectively. At rest Sal had no effect on J V-A , oxygen consumption (VO 2 ), blood oxygen saturation (SO 2 ) or AaDO 2 (P40.05). At the onset of exercise J V-A increased in Con and Sal (P o 0.0001) and at fatigue reached 10.0 AE 2.4 L/min and 10.0 AE 1.6 L/min, respectively. Treatment with Sal had no effect on J V-A during exercise (P 5 0.9). At the onset of exercise SO 2 and VO 2 increased in Con and Sal (P o 0.0001). Treatment with Sal had no effect on SO 2 or VO 2 during exercise (P40.05). AaDO 2 increased during exercise in Con and Sal (P o 0.0001) and at fatigue reached 19.4 AE 2.3 mmHg and 18.1 AE 2.4 mmHg, respectively. Treatment with Sal had no effect on AaDO 2 during exercise (P 5 0.3).Inhaled b 2 -adrenergic receptor agonist salbutamol at the dose of 500mcg given 60 min before exercise did not affect the duration of exercise to fatigue, J V-A , VO 2 , SO 2 or AaDO 2 . Therefore, it had no detrimental effect on alveolar-capillary diffusion distance and the ventilation/perfusion mismatch in exercising horses. Inflammatory Airway Disease (IAD) and recurrent airway obstruction (RAO) represent two classes of equine lung inflammatory diseases that may share some similar immunologic mechanisms. There is evidence that Th2 cytokines and IL-17 play some role in RAO. IAD is a common condition in horses, but its pathophysiology is still not understood. The aim of the present study was therefore to determine the mRNA expression of Th1, Th2 and Th17 inflammatory cytokines, to understand the immunological mechanisms of IAD.The mRNA expression of ten inflammatory cytokines and chemokines was measured in the bronchoalveolar fluid (BALF) of seventeen horses with IAD and compared with ten control horses. The horses were selected based on 1-their clinical signs, 2-the inflammatory cells count in the BALF, 3-their physical examination and 4-their medical history. The mRNA expression of IL-5, IL-1b, IL-6, IL-8 and IL-10 was significantly up-regulated in BALF from horses with IAD.Furthermore, the BALF samples were subdivided in two groups based on the differential cells count 1-BALF with increased mast cells (IAD-Mast) and 2-BALF with increased neutrophils (IAD-Neutro). IL-4 was significantly down-regulated in the IAD-Neutro group compared to the IAD-Mast group. IL-17, IL-5 and IL-8 were significantly up-regulated in the IAD-Neutro group compared to the IAD-Mast group.The present study shows that IAD in horses is characterized by a Th2 and a Th17 mRNA inflammatory expression profile and that different immunological mechanisms are involved in mast cells or neutrophils accumulation in the BALF of horses with IAD. b 2 -adrenoreceptor (b 2 -AR) agonists are a class of medications that promote smooth muscle relaxation and bronchodilation in horses and humans with airway disease. Activated human peripheral blood lymphocytes (PBLs) also respond to b 2 -AR agonist stimulation by attenuating the production of cytokines associated with the pathogenesis of asthma and Recurrent Airway Obstruction (RAO). The aim of this study was to develop an in vitro technique for measuring the response of equine PBLs to stimulation with Salbutamol, a b 2 -AR agonist. This method was then used to compare the response of PBLs from RAO-affected and non-affected horses to b 2 -AR agonist stimulation. PBLs from 4 RAO and 4 nonaffected horses were cultured (4x10 6 /ml) in RPMI complete media with Concanavalin A (ConA, 2ug/10 6 cells) for 0, 1, or 2 days then stimulated with Salbutamol (30 minutes). Using flow cytometric techniques, response was measured by detecting protein kinase A phosphorylation of vasodilator stimulated phosphoprotein (VASP). Results were verified by western blot analysis. Activated PBLs were then incubated with ConA for one day were pre-incubated with b 2 or b-adrenoreceptor antagonist (ICI 118,551, Sigma s ; Atenolol, Sigma s ) for 15 minutes, followed by 30 minutes Salbutamol (500 nM) stimulation. Results were analyzed by ANOVA or ANCOVA and differences were considered significant when p o 0.05.Response to b-antagonist was only observed in activated PBLs (pre-cultured with Con A) and was greater in cells from RAO horses as compared to cells from non-affected horses. The addition of b-antagonist attenuated the response of PBLs to Salbutamol while the addition of a b 1 -antagonist had no effect. These findings indicate that activated PBLs from RAO-affected horses have a greater response to Salbutamol as compared to PBLs from non-affected horses, and this response is mediated mainly through the b 2 -AR.Human b 2 -AR are known be polymorphic and this polymorphism results in a variable response to b 2 -agonist binding that affects long term outcome in human asthmatics. Further studies are required to determine if the difference in response of PBLs from RAO affected as compared to non-affected horses is due to genetic polymorphism in the equine b 2 -AR, and whether this difference is associated with a propensity for horses to develop equine RAO.