A useful approach for evaluating antitussive drugs in humans is to determine the sensitivity of the cough reflex to a standard challenge. The purpose of this study was to determine if methods used to induce coughing in humans would be effective when used on awake, untrained, healthy dogs for future application in therapeutic trials involving dogs with spontaneous disease. Methods tested were: mechanically stimulating the trachea by digital compression as well as by vibration from an electric shaver, neck massager, and palm sander (11 dogs), and administering nebulized irritant (3000 micromolar capsaicin), acidic (1 M citric acid), and hypotonic (deionized water) solutions using face masks (4 dogs). The threshold for success was defined as induction of at least 2 moderate or strong coughs in at least 75% of the dogs. None of the methods tested was successful. Digital compression induced soft (n = 2) or moderate (n = 1) coughing in 3 of 11 dogs tested. Nebulization of citric acid induced 1 soft cough in 1 of 4 dogs. It was concluded that coughing cannot be successfully induced in awake, healthy dogs using methods that are successful in humans. Other strategies must be developed so that cough sensitivity can be objectively and non-invasively measured in dogs for clinical research purposes.

Objective—To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies. Sample—Human, canine, and equine liver microsomes and human single CYP3A4 and CYP2C9 and their canine orthologs. Procedures—Chemical inhibitors selective for human CYP enzymes and anti-CYP antibodies were incubated with racemic ketamine and liver microsomes or specific CYPs. Ketamine N-demethylation to norketamine was determined via enantioselective capillary electrophoresis. Results—The general CYP inhibitor 1-aminobenzotriazole almost completely blocked ketamine metabolism in human and canine liver microsomes but not in equine microsomes. Chemical inhibition of norketamine formation was dependent on inhibitor concentration in most circumstances. For all 3 species, inhibitors of CYP3A4, CYP2A6, CYP2C19, CYP2B6, and CYP2C9 diminished N-demethylation of ketamine. Anti-CYP3A4, anti-CYP2C9, and anti-CYP2B6 antibodies also inhibited ketamine N-demethylation. Chemical inhibition was strongest with inhibitors of CYP2A6 and CYP2C19 in canine and equine microsomes and with the CYP3A4 inhibitor in human microsomes. No significant contribution of CYP2D6 to ketamine biotransformation was observed. Although the human CYP2C9 inhibitor blocked ketamine N-demethylation completely in the canine ortholog CYP2C21, a strong inhibition was also obtained by the chemical inhibitors of CYP2C19 and CYP2B6. Ketamine N-demethylation was stereoselective in single human CYP3A4 and canine CYP2C21 enzymes. Conclusions and Clinical Relevance—Human-specific inhibitors of CYP2A6, CYP2C19, CYP3A4, CYP2B6, and CYP2C9 diminished ketamine N-demethylation in dogs and horses. To address drug-drug interactions in these animal species, investigations with single CYPs are needed.

Objective—To determine whether canine tumor cell lines express functional tissue factor and shed tissue factor-containing microparticles. Sample—Cell lines derived from tumors of the canine mammary gland (CMT12 and CMT25), pancreas (P404), lung (BACA), prostate gland (Ace-1), bone (HMPOS, D-17, and OS2.4), and soft tissue (A72); from normal canine renal epithelium (MDCK); and from a malignant human mammary tumor (MDA-MB-231). Procedures—Tissue factor mRNA and antigen expression were evaluated in cells by use of canine-specific primers in a reverse transcriptase PCR assay and a rabbit polyclonal anti-human tissue factor antibody in flow cytometric and immunofluorescent microscopic assays, respectively. Tissue factor procoagulant activity on cell surfaces, in whole cell lysates, and in microparticle pellets was measured by use of an activated factor X-dependent chromogenic assay. Results—Canine tissue factor mRNA was identified in all canine tumor cells. All canine tumor cells expressed intracellular tissue factor; however, the HMPOS and D-17 osteosarcoma cells lacked surface tissue factor expression and activity. The highest tissue factor expression and activity were observed in canine mammary tumor cells and pulmonary carcinoma cells (BACA). These 3 tumors also shed tissue factor-bearing microparticles into tissue culture supernatants. Conclusions and Clinical Relevance—Tissue factor was constitutively highly expressed in canine tumor cell lines, particularly those derived from epithelial tumors. Because tumor-associated tissue factor can promote tumor growth and metastasis in human patients, high tissue factor expression could affect the in vivo biological behavior of these tumors in dogs.

Objective: To determine the effect of Hct on blood glucose readings of dogs obtained by use of 2 point-of-care (POC) blood glucometers and a laboratory analyzer. Animals: 184 dogs, including 139 Greyhounds. Procedures: Venous blood samples collected from 184 dogs with a range of Hcts (measured in EDTA-anticoagulated blood) were immediately analyzed with a handheld glucometer specifically developed for veterinary use and a glucometer developed for use in humans. The remainder of each blood sample was placed in fluoride oxalate tubes, and plasma glucose concentration was measured with a laboratory analyzer. Agreement between results for the POC glucometers and laboratory analyzer and effect of Hct on glucometer accuracy was assessed via regression analysis. Results: Significant differences were detected between results of the glucometers and the reference laboratory analyzer. The Hct affected the correlation between results for the glucometers and the laboratory analyzer. Deviations of the glucometers from the reference interval varied with Hct. The glucometer for veterinary use more closely correlated with the glucose concentration when Hct was within or above its reference interval. The glucometer for use in humans more closely approximated laboratory reference glucose concentrations in anemic dogs. Conclusions and Clinical Relevance: Hct had a relevant impact on the correlation between whole blood and plasma glucose concentrations in dogs. Significant variations between results obtained with the 2 glucometers could be critical when interpreting blood glucose measurements or selecting a POC glucometer for an intensive care setting and precise glycemic control in critically ill dogs.

Objective—To identify a causative mutation for dilated cardiomyopathy (DCM) in Doberman Pinschers by sequencing the coding regions of 10 cardiac genes known to be associated with familial DCM in humans. Animals—5 Doberman Pinschers with DCM and congestive heart failure and 5 control mixed-breed dogs that were euthanized or died. Procedures—RNA was extracted from frozen ventricular myocardial samples from each dog, and first-strand cDNA was synthesized via reverse transcription, followed by PCR amplification with gene-specific primers. Ten cardiac genes were analyzed: cardiac actin, α-actinin, α-tropomyosin, β-myosin heavy chain, metavinculin, muscle LIM protein, myosinbinding protein C, tafazzin, titin-cap (telethonin), and troponin T. Sequences for DCM-affected and control dogs and the published canine genome were compared. Results—None of the coding sequences yielded a common causative mutation among all Doberman Pinscher samples. However, 3 variants were identified in the α-actinin gene in the DCM-affected Doberman Pinschers. One of these variants, identified in 2 of the 5 Doberman Pinschers, resulted in an amino acid change in the rod-forming triple coiled-coil domain. Conclusions and Clinical Relevance—Mutations in the coding regions of several genes associated with DCM in humans did not appear to consistently account for DCM in Doberman Pinschers. However, an α-actinin variant was detected in some Doberman Pinschers that may contribute to the development of DCM given its potential effect on the structure of this protein. Investigation of additional candidate gene coding and noncoding regions and further evaluation of the role of α-actinin in development of DCM in Doberman Pinschers are warranted.

Objective—To assess whether the Pfirrmann system for grading lumbar intervertebral disk (IVD) degeneration in humans can also be used in dogs. Animals—202 dogs. Procedures—Magnetic resonance imaging was used to obtain images of vertebral segments from dogs, which were reviewed separately by 3 observers who graded the extent of degeneration in each visible IVD by use of the Pfirrmann classification system used for grading lumbar IVD degeneration in humans. Grading was validated against 2 factors associated with the extent of disk degeneration: type of dog (chondrodystrophic or nonchondrodystrophic breeds) and age. Results—Interobserver and intraobserver agreement for Pfirrmann grading of IVD degeneration were good (κ scores, 0.81 to 0.93). An increase in the extent of disk degeneration was positively correlated with increases in age and with chondrodystrophic breed. Conclusions and Clinical Relevance—The Pfirrmann system was reliably used to grade IVD degeneration in dogs of various breeds and ages. An increase in the extent of IVD degeneration was positively correlated with increases in age and with chondrodystrophic-type dogs.

Objective—To determine the pharmacokinetics of tramadol and its metabolites O-desmethyltramadol (ODT) and N-desmethyltramadol (NDT) in adult horses. Animals—12 mixed-breed horses. Procedures—Horses received tramadol IV (5 mg/kg, over 3 minutes) and orally (10 mg/kg) with a 6-day washout period in a randomized crossover design. Serum samples were collected over 48 hours. Serum tramadol, ODT, and NDT concentrations were measured via high-performance liquid chromatography and analyzed via noncompartmental analysis. Results—Maximum mean ± SEM serum concentrations after IV administration for tramadol, ODT, and NDT were 5,027 ± 638 ng/mL, 0 ng/mL, and 73.7 ± 12.9 ng/mL, respectively. For tramadol, half-life, volume of distribution, area under the curve, and total body clearance after IV administration were 2.55 ± 0.88 hours, 4.02 ± 1.35 L/kg, 2,701 ± 275 h•ng/mL, and 30.1 ± 2.56 mL/min/kg, respectively. Maximal serum concentrations after oral administration for tramadol, ODT, and NDT were 238 ± 41.3 ng/mL, 86.8 ± 17.8 ng/mL, and 159 ± 20.4 ng/mL, respectively. After oral administration, half-life for tramadol, ODT, and NDT was 2.14 ± 0.50 hours, 1.01 ± 0.15 hours, and 2.62 ± 0.49 hours, respectively. Bioavailability of tramadol was 9.50 ± 1.28%. After oral administration, concentrations achieved minimum therapeutic ranges for humans for tramadol (> 100 ng/mL) and ODT (> 10 ng/mL) for 2.2 ± 0.46 hours and 2.04 ± 0.30 hours, respectively. Conclusions and Clinical Relevance—Duration of analgesia after oral administration of tramadol might be < 3 hours in horses, with ODT and the parent compound contributing equally.

Escherichia coli O157:H7 remains a threat to humans via cattle-derived fecal contamination of food and water. Preharvest intervention strategies represent a means of reducing the pathogen burden before harvest. In this study, the efficacy of a commercially produced type III secreted protein (TTSP) vaccine was evaluated with the use of a commingled experimental calf infection model (30 placebo-treated animals and 30 vaccinates). The calves were vaccinated on days 0, 21, and 42 and infected with 109 colony-forming units (CFU) of E. coli O157 by oral–gastric intubation on day 56. Fecal shedding was monitored daily for 14 d. Serologic assessment revealed a robust immune response to vaccination; the serum titers of antibodies against EspA, Tir, and total TTSPs were significantly higher in the vaccinates than in the placebo-treated animals on days 21, 42, 56, and 70. Significantly less (P = 0.011) of the challenge organism was shed by the vaccinates than by the placebo-treated animals on days 3 to 10. Peak shedding occurred in both groups on days 3 to 6; during this period the vaccinates showed a mean log reduction of 1.4 (P = 0.002) and a mitigated fraction of 51%. The number of animals shedding was significantly lower among the vaccinates compared with the placebo group on days 3 to 6 (P ≤ 0.05), with a mean prevented fraction of 21%. No differences in the duration of shedding were observed. Owing to the low challenge shedding in both groups on days 11 to 14 (mean CFU/g < 10; median = 0), no significant differences were observed. These data indicate that TTSP vaccination had protective effects through significant reductions in the number of animals shedding and the number of challenge organisms shed per animal and provides evidence that TTSP vaccination is an effective preharvest intervention strategy against E. coli O157.

Objective: To determine whether therapeutic concentrations of levetiracetam can be achieved in cats and to establish reasonable IV and oral dosing intervals that would not be associated with adverse effects in cats. Animals: 10 healthy purpose-bred cats. Procedures: In a randomized crossover study, levetiracetam (20 mg/kg) was administered orally and IV to each cat. Blood samples were collected 0, 10, 20, and 40 minutes and 1, 1.5, 2, 3, 4, 6, 9, 12, and 24 hours after administration. Plasma levetiracetam concentrations were determined via high-performance liquid chromatography. Results: Mean ± SD peak concentration was 25.54 ± 7.97 μg/mL. The mean y-intercept for IV administration was 37.52 ± 6.79 μg/mL. Half-life (harmonic mean ± pseudo-SD) was 2.95 ± 0.95 hours and 2.86 ± 0.65 hours for oral and IV administration, respectively. Mean volume of distribution at steady state was 0.52 ± 0.09 L/kg, and mean clearance was 2.0 ± 0.60 mL/kg/min. Mean oral bioavailability was 102 ± 39%. Plasma drug concentrations were maintained in the therapeutic range reported for humans (5 to 45 μg/mL) for at least 9 hours after administration in 7 of 10 cats. Only mild, transient hypersalivation was evident in some cats after oral administration. Conclusions and Clinical Relevance: Levetiracetam (20 mg/kg) administered orally or IV to cats every 8 hours should achieve and maintain concentrations within the therapeutic range for humans. Levetiracetam administration has favorable pharmacokinetics for clinical use, was apparently tolerated well, and may be a reasonable alternative antiepileptic drug in cats.

Objective: To evaluate the in vitro susceptibility of various field isolates of Mycobacterium avium subsp paratuberculosis (MAP) to gallium nitrate. Sample: 10 isolates of MAP, including 4 isolated from cattle, 2 isolated from bison, 1 isolated from an alpaca, and 3 isolated from humans. Procedures: The in vitro susceptibility to gallium nitrate was tested by use of broth culture with detection of MAP growth by means of a nonradiometric automated detection method. For each MAP isolate, a series of 7 dilutions of gallium nitrate (concentrations ranging from 200 to 1,000μM) were tested. Gallium nitrate was considered to have caused 90% and 99% inhibition of the MAP growth when the time to detection for culture of the MAP stock solution and a specific concentration of gallium nitrate was delayed and was similar to that obtained for culture of the MAP stock solution (without the addition of gallium nitrate) diluted 1:10 and 1:100, respectively. Results: Gallium nitrate inhibited MAP growth in all 10 isolates. The susceptibility to gallium nitrate was variable among isolates, and all isolates of MAP were inhibited in a dose-dependent manner. Overall, the concentration that resulted in 90% inhibition ranged from < 200μM for the most susceptible isolates to 743μM for the least susceptible isolates. Conclusions and Clinical Relevance: Gallium nitrate had activity against all 10 isolates of MAP tested in vitro and could potentially be used as a prophylactic agent to aid in the control of MAP infections during the neonatal period.