Prevalence and characterization of verotoxin-producing Escherichia coli (VTEC) in cattle from an Ontario abattoir
Musafiri Karama, Roger P. Johnson, Robert Holtslander, Scott A. McEwen, Carlton L. Gyles

Abstract — This study determined the prevalence of verotoxin (VT)-producing Escherichia coli (VTEC) in Ontario beef cattle at slaughter and characterized the isolates by serotype, virulence factors, virulence markers, and antimicrobial resistance. Cultures of rectal feces from 500 animals were screened for VT by an enzyme-linked immunosorbent assay (ELISA) and by polymerase chain reaction (PCR) for genes vt1, vt2, and eae. The VT-ELISA-positive samples were tested by a VT-immunoblot to isolate VTEC colonies. The prevalence rates of VTEC by VT-ELISA and PCR were 10.2% [95% confidence interval (CI), 7.8% to 13.2%] and 6.2% (95% CI, 4.4% to 8.7%), respectively. Colonies of VTEC were isolated from 27 (53%) of the 51 VT-ELISA-positive samples and belonged to 24 serotypes, which did not include O157:H7. Twelve of the serotypes have been implicated in disease in humans. Virulence profiling of the isolates by PCR revealed that 2 (8%) were eae-positive, 5 (21%) had vt1 only, and 19 (79%) had vt2, of which 3 had vt2 only, 7 had vt1 + vt2, 4 had vt2 + vt2c, 2 had vt2 + vt2c + vt2d, 2 had vt1 + vt2 + vt2c, and 1 had vt1 + vt2 + vt2c + vt2d. The distribution of selected plasmid-encoded putative virulence genes was as follows: ehxA, 63%; espP, 46%; saa, 67%; and subA, 54%. Nine of the 24 isolates were resistant to 1 or more antimicrobials. Major conclusions are that the VTEC prevalence of 10.2% was among the lower rates reported for beef cattle, a high proportion of the isolates had vt2 genes, the subA gene was reported for the 1st time in Canadian VTEC, and the absence of O157 VTEC likely reflects the use of a technique that detected all VTEC.

Pig and herd level prevalence of Toxoplasma gondii in Ontario finisher pigs in 2001, 2003, and 2004
Zvonimir Poljak, Catherine E. Dewey, Robert M. Friendship, S. Wayne Martin, Jette Christensen, Davor Ojkic, John Wu, Eva Chow

Abstract — The objective of this study was to estimate the apparent and true prevalence of exposure to Toxoplasma gondii in Ontario finisher pigs. During the study period (2001 to 2004), sera from 6048 pigs were tested with a commercial enzyme-linked immunosorbent assay (ELISA); 103 farms were included 1 to 3 times in the study. True prevalence was estimated using a Bayesian approach. Apparent prevalence at the pig level was 1.59% [95% confidence interval (CI): 0.45, 2.99] in 2001, 0.06% (95% CI: 0.00, 0.46) in 2003, and 0.26% (95% CI: 0.00, 0.82) in 2004. Apparent prevalence at the herd-level was 13.7% (95% CI: 7.5, 22.3) in 2001; 1.25% (95% CI: 0.03, 6.77) in 2003, and 3.75% (95% CI: 0.78, 10.6) in 2004. Similarly, posterior Bayesian estimates of true prevalence at the pig level were 1.7% [95% probability interval (PI): 1.2, 2.2] in 2001, 0.2% (95% PI: 0.04, 0.4) in 2003, and 0.3% (95% PI: 0.1, 0.7) in 2004. At the herd level, posterior estimates of prevalence were 11.6% (95% PI: 7.4, 16.8) in 2001, 0% (95% PI: 0.0, 2.5) in 2003, and 1.2% (95% PI: 0.0, 5.0) in 2004 when a herd cut-point = 1 was used. Exposure to T. gondii in finishing pig farms in Ontario appears to be infrequent.

Comparison of the selection of antimicrobial resistance in fecal Escherichia coli during enrofloxacin administration with a local drug delivery system or with intramuscular injections in a swine model
Romain Béraud, Louis Huneault, Dave Bernier, Francis Beaudry, Ann Letellier, Jérôme R.E. del Castillo

Abstract — This study evaluated, for the first time, the selection of antibiotic resistance in fecal Escherichia coli, a potential reservoir of genes of resistance, during the prolonged exposure to fluoroquinolones after the implantation of a local drug delivery system (LDDS) in a swine model. Fourteen pigs were randomly assigned to group IM (5 mg/kg/day of intramuscular enrofloxacin — EFX) or LD (surgical implantation of EFX-polymethyl-methacrylate peri-femoral implants). Blood samples were collected daily for determination of plasma EFX and ciprofloxacin (CFX) concentrations. Fecal samples were collected daily to determine the E. coli counts and the susceptibility patterns of its isolates as evaluated by antibiotic disk diffusion tests. In both groups, EFX administration significantly reduced the bacterial counts after 2 days. During recolonization, the bacterial counts remained lower than baseline in group IM but not significantly, and almost reached pre-treatment levels in group LD. Susceptibility to EFX, CFX, and nalidixic acid of recolonizing E. coli in LD pigs slightly decreased but remained within the limit of “susceptible” isolates. In contrast, quinolone susceptibility of recolonizing E. coli in IM pigs dropped dramatically (P < 0.0001). In addition, intramuscular exposure to fluoroquinolones significantly decreased the susceptibility of E. coli to ampicillin and trimethoprim-sulfamethoxazole (P < 0.05). In conclusion, the use of a dosing regimen that minimized the intestinal output of fluoroquinolones also minimized the selection of resistance to several classes of antibiotics. This could represent another advantage of LDDS usage compared to long-lasting systemic administration of fluoroquinolones.

PrP genotype frequencies of Quebec sheep breeds determined by real-time PCR and molecular beacons
Yvan L’Homme, Anne Leboeuf, Johanne Cameron

Abstract — The allele and genotype frequencies of the prion protein gene (PrP), known to have an impact on scrapie susceptibility, were determined by real-time PCR for 500 Quebec purebred rams. Molecular beacons were very efficient in discriminating the 5 alleles investigated. Polymorphisms at coding positions 136, 154, and 171 of the PrP gene were analyzed using 3 separate real-time PCR reactions and a total of 7 molecular beacons. A total of 4 different alleles (ARQ, ARR, AHR, and VRQ) were observed at different frequencies among the 7 breeds of sheep investigated. Results show that more than 50% of the rams in every breed carried at least one ARR allele, which is considered the most resistant to scrapie. The susceptibility ARQ allele was also present in every breed and together with the ARR allele, they were the most frequent alleles found in Quebec rams. The VRQ allele associated with the highest susceptibility to scrapie occurred in 5 of the 7 breeds, although at low frequencies. Overall, the results indicate that the frequencies of PrP alleles and genotypes in common breeds of sheep in Quebec make it feasible to reduce scrapie risk by selective breeding.

Pharmacokinetics of intravenous tramadol in dogs
Chantal J. McMillan, Alex Livingston, Chris R. Clark, Patricia M. Dowling, Susan M. Taylor, Tanya Duke, Rolf Terlinden

Abstract — The purpose of this study was to determine the pharmacokinetics of tramadol and the active metabolite mono-O-desmethyltramadol (M1) in 6 healthy male mixed breed dogs following intravenous injection of tramadol at 3 different dose levels. Verification of the metabolism to the active metabolite M1, to which most of the analgesic activity of this agent is attributed to, was a primary goal. Quantification of the parent compound and the M1 metabolite was performed using gas chromatography. Pharmacodynamic evaluations were performed at the time of patient sampling and included assessment of sedation, and evaluation for depression of heart and respiratory rates. This study confirmed that while these dogs were able to produce the active M1 metabolite following intravenous administration of tramadol, the M1 concentrations were lower than previously reported in research beagles. Adverse effects were minimal, with mild dose-related sedation in all dogs and nausea in 1 dog. Analgesia was not documented with the method of assessment used in this study. Tramadol may be useful in canine patients, but additional studies in the canine population are required to more accurately determine the effective clinical use of the drug in dogs and quantification of M1 concentrations in a wider population of patients.

Effects of medetomidine and midazolam alone or in combination on the metabolic and neurohormonal responses in healthy cats
Teppei Kanda, Yoshiaki Hikasa

Abstract — The purpose of this study was to investigate the effects of a medetomidine–midazolam combination on some neurohormonal and metabolic variables in healthy cats. Five cats were used repeatedly in each of 5 groups, which were injected intramuscularly with physiological saline solution (control), 0.5 mg/kg of midazolam, 40 µg/kg of medetomidine, 80 µg/kg of medetomidine, and 40 µg/kg of medetomidine plus 0.5 mg/kg of midazolam. Blood samples were taken 10 times over 24 h from a catheter introduced into the jugular vein. Plasma concentrations of glucose, insulin, glucagon, cortisol, nonesterified fatty acids (NEFAs), norepinephrine, and epinephrine were determined. In addition, the duration of lateral recumbency, rectal temperature, heart rate, and respiratory rate were examined. The combination of medetomidine and midazolam enhanced the duration of lateral recumbency and reduced the hyperglycemia induced by medetomidine alone. Recovery from hypoinsulinemia induced by the medetomidine–midazolam combination tended to be more rapid than when the same dose of medetomidine was used alone. The decrease in plasma norepinephrine levels induced by medetomidine alone was diminished by the addition of midazolam. Midazolam alone did not significantly change the plasma glucose, insulin, glucagon, cortisol, epinephrine, or NEFA concentration, but increased the norepinephrine concentration. This study revealed that the combination of medetomidine and midazolam produces minimal neurohormonal and metabolic changes when compared with medetomidine alone in cats.

Characterization of endothelin receptors in the peripheral lung tissues of horses unaffected and affected with recurrent airway obstruction
Sumanth Polikepahad, Masudul Haque, Joseph Francis, Rustin M. Moore, Changaram S. Venugopal

Abstract — The purpose of the study was to determine and compare the expression of endothelin (ET) receptors in the peripheral lungs of healthy horses and those affected with recurrent airway obstruction (RAO) using reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, Western blot analysis, and immunohistochemical techniques. Two groups of horses (7 healthy and 7 RAO-affected) were selected from a pool of horses destined for euthanasia. The grouping of horses was based on the history, clinical scoring, and pulmonary function testing. After euthanasia, gross postmortem evaluation of the lungs was conducted, and lung samples were collected and either stored at -80°C or fixed in zinc-formalin for 12 h. The RT-PCR was performed by using specific primers for ETA and ETB receptors, and ß-actin. To determine the relative gene expression real-time PCR was performed. To detect ET receptor protein expression, Western blotting and immunohistochemical studies were performed using polyclonal antibodies against ETA and ETB receptors and ß-actin. The ET receptor expression was determined by performing either densitometric analyses or scoring of immunostaining. Statistical analyses were performed to detect differences in receptor expression within and between the 2 groups. The results indicated that ET receptor expression, particularly ETB receptors, was significantly greater in the peripheral lungs of RAO-affected horses than in those of healthy horses. Clinical trials using ET receptor antagonists, particularly ETB antagonists might help in developing a therapeutic strategy to treat this career-ending disease.

A retrospective epidemiological study of clinical signs and familial predisposition associated with aseptic meningitis in the Norwegian population of Nova Scotia duck tolling retrievers born 1994–2003
Kristin P. Anfinsen, Mette Berendt, Flora J.H. Liste, Therese R. Haagensen, Astrid Indrebo, Frode Lingaas, Oyvind Stigen, Lis Alban

Abstract — Aseptic meningitis (AM) is a disease that causes grave clinical signs such as intensive neck pain, fever, and lethargy. The severity of this disease is reflected in the fact that affected animals require long-term, and in chronic cases, lifelong therapy with corticosteroids. A number of dogs must be euthanized because of therapeutic failure. In recent years, the Norwegian population of Nova Scotia duck tolling retrievers has experienced an increase in individuals with AM. The aim of the present study was to investigate the prevalence of AM and to pursue the suspicion of hereditary factors influencing an accumulation of AM cases in the breed. Using the Norwegian Kennel Club registery, a random sample (362 dogs) stratified by year of birth was drawn from the total population born from 1994 to 2003 (1525 individuals). The owners were contacted and questioned about clinical signs of AM in their dogs. Subsequently, the practising veterinarians and the breeders of positive responders were contacted in order to confirm a clinical diagnosis of AM and to identify possible affected family members. Pedigrees of AM positive individuals and affected relatives were investigated. The study estimated a prevalence of AM of 2.5%. For all affected dogs, it was possible to trace the pedigree of both parents of affected dogs back to a specific founder dog. The genealogical investigation strongly indicates that genetic factors are involved in the etiology of the disease.

Cardiac isoenzymes in healthy Holstein calves and calves with experimentally induced endotoxemia
Simon F. Peek, Fred S. Apple, Mary Ann Murakami, Peter M. Crump, Susan D. Semrad

Abstract — This paper describes a controlled study designed to establish normal values for cardiac troponins I and T (cTnI and cTnT) and CK-MB mass in healthy newborn Holstein calves, and to compare values for cTnI, cTnT, CK-MB and total creatine kinase (CK) with age-matched calves experiencing experimentally induced endotoxemia. Nineteen healthy Holstein bull calves, 48 to 72 h of age were used. Baseline cTnI, cTnT, CK-MB and total CK measurements were obtained from control (n = 9) and experimental (n = 10) calves. Controls then received physiological saline and experimental calves received endotoxin (O55:B5 Escherichia coli LPS) intravenously after which cardiac biomarkers and total CK were measured at 3 h, 6 h, 12 h, and 24 h post-initiation of infusion. Measured values were analyzed and compared using analysis of variance (ANOVA) by repeated measure design, with statistical significance set at P < 0.05. The cardiac biomarker cTnT was not detected in any calf at any time point, and CK-MB was only detected in 5 of 95 samples. The cTnI was significantly increased compared to baseline and controls, 3 h post lipopolysaccharide (LPS) infusion. Total CK was significantly increased in LPS administered calves at 18 and 24 h post infusion. The mean, standard deviation, and range for cTnI in healthy controls were 0.023 ng/mL (s = 0.01), and 0.01 to 0.05 ng/mL, respectively. In conclusion, LPS administration was associated with rapid and significant increases in cTnI but CK-MB and cTnT were not detected in the plasma of healthy calves. Total CK values increased significantly following LPS administration. Biochemical evidence of myocardial injury occurs within 3 h following LPS administration to neonatal Holstein calves.

Effects of the opioid remifentanil on the arrhythmogenicity of epinephrine in halothane-anesthetized dogs
Natache A. Garofalo, Francisco J. Teixeira-Neto, Denise S. Schwartz, Maria do Carmo F. Vailati, Paulo V.M. Steagall

Abstract — Opioids may exert a protective effect against ventricular arrhythmias via a vagally mediated mechanism. This study evaluated the effects of the opioid remifentanil on arrhythmogenicity of epinephrine during halothane anesthesia. Eight dogs were assigned to 2 treatments in a randomized crossover design, with 1-week intervals between treatments. Anesthesia was maintained with 1.3% end-tidal halothane in oxygen and mechanical ventilation to maintain eucapnia. A constant rate infusion of remifentanil (0.72 µg/kg/min) was administered throughout the study in the experimental treatment, while control animals received physiologic saline as placebo. The arrhythmogenic dose of epinephrine (ADE), defined as 4 premature ventricular complexes (PVCs) within 15 s, was determined by administering progressively increasing infusion rates of epinephrine (2.5, 5.0, and 10 µg/kg/min), allowing 20 min intervals between each infusion rate. In both treatments, epinephrine infusions induced bradyarrhythmias and atrioventricular conduction disturbances, which were followed by escape beats and PVCs. In the remifentanil treatment, mean ± s ADE values (11.3 ± 4.9 µg/kg) did not differ from values observed in control animals (9.9 ± 6.1 µg/kg). On the basis of the ADE model for assessing the arrhythmogenity of drugs during halothane anesthesia, the present study did not demonstrate a protective effect of remifentanil (0.72 µg/kg/min) against ventricular arrhythmias in dogs.

Detection of respiratory pathogens in air samples from acutely infected pigs
Joseph R. Hermann, Susan L. Brockmeier, Kyoung-Jin Yoon, Jeffrey J. Zimmerman

Abstract — Pathogens causing significant respiratory disease in growing pigs include Porcine reproductive and respiratory syndrome virus, Porcine circovirus 2, swine influenza virus, porcine respiratory coronavirus, Mycoplasma hyopneumoniae, and Bordetella bronchiseptica. The objective of this research was to characterize the respiratory excretion of these pathogens by acutely infected pigs. Pigs were inoculated under experimental conditions with 1 pathogen. Samples were collected from the upper respiratory tract and exhaled air. All pathogens were detected in swabs of the upper respiratory tract, but only M. hyopneumoniae and B. bronchiseptica were detected in expired air from individually sampled, acutely infected pigs. These findings suggest either that the acutely infected pigs did not aerosolize the viruses or that the quantity of virus excreted was below the detection threshold of current sampling or assay systems, or both, at the individual-pig level.

Comparative pathogenicity of early and recent isolates of avian metapneumovirus subtype C in turkeys
Binu T. Velayudhan, Sally L. Noll, Anil J. Thachil, David A. Halvorson, Daniel P. Shaw, Sagar M. Goyal, Kakambi V. Nagaraja

Abstract — The objective of the present study was to compare the pathogenicity of early and recent isolates of avian metapneumovirus subtype-C (aMPV-C) in turkeys. Two-week-old turkeys were inoculated with early and recent isolates of aMPV-C. Clinical signs were monitored. Tissues were examined for viral ribonucleic acid (RNA), lesions, and viral antigen by reverse transcription-polymerase chain reaction (RT-PCR), histopathology and immunohistochemistry, respectively. Birds infected with the recent isolate had higher clinical sign scores than those infected with the early isolate. Only the recent isolate produced a multifocal loss of cilia in the nasal turbinate of infected birds. Immunohistochemistry revealed intense staining of aMPV antigen in turbinate and trachea of birds infected with the recent isolate. The findings indicate that the recent isolate produced more severe clinical signs and lesions in turkeys compared to the early isolate. The recent isolate could be ideal for the development of a challenge model for aMPV infection in turkeys.