OBJECTIVE To investigate regional differences of relative metabolite concentrations in the brain of healthy dogs with short echo time, single voxel proton magnetic resonance spectroscopy (1H MRS) at 3.0 T. ANIMALS 10 Beagles. PROCEDURES Short echo time, single voxel 1H MRS was performed at the level of the right and left basal ganglia, right and left thalamus, right and left parietal lobes, occipital lobe, and cerebellum. Data were analyzed with an automated fitting method (linear combination model). Metabolite concentrations relative to water content were obtained, including N-acetyl aspartate, total choline, creatine, myoinositol, the sum of glutamine and glutamate (glutamine-glutamate complex), and glutathione. Metabolite ratios with creatine as the reference metabolite were calculated. Concentration differences between right and left hemispheres and sexes were evaluated with a Wilcoxon signed rank test and among various regions of the brain with an independent t test and 1-way ANOVA. RESULTS No significant differences were detected between sexes and right and left hemispheres. All metabolites, except the glutamine-glutamate complex and glutathione, had regional concentrations that differed significantly. The creatine concentration was highest in the basal ganglia and cerebellum and lowest in the parietal lobes. The N-acetyl aspartate concentration was highest in the parietal lobes and lowest in the cerebellum. Total choline concentration was highest in the basal ganglia and lowest in the occipital lobe. CONCLUSIONS AND CLINICAL RELEVANCE Metabolite concentrations differed among brain parenchymal regions in healthy dogs. This study may provide reference values for clinical and research studies involving 1H MRS performed at 3.0 T.

OBJECTIVE To evaluate the potency of vecuronium and duration of vecuronium-induced neuromuscular blockade in dogs with centronuclear myopathy (CNM). ANIMALS 6 Labrador Retrievers with autosomal-recessive CNM and 5 age- and weight-matched control dogs. PROCEDURES Dogs were anesthetized on 2 occasions (1-week interval) with propofol, dexmedetomidine, and isoflurane. Neuromuscular function was monitored with acceleromyography and train-of-four (TOF) stimulation. In an initial experiment, potency of vecuronium was evaluated by a cumulative-dose method, where 2 submaximal doses of vecuronium (10 μg/kg each) were administered IV sequentially. For the TOF's first twitch (T1), baseline twitch amplitude and maximal posttreatment depression of twitch amplitude were measured. In the second experiment, dogs received vecuronium (50 μg/kg, IV) and the time of spontaneous recovery to a TOF ratio (ie, amplitude of TOF's fourth twitch divided by amplitude of T1) ≥ 0.9 and recovery index (interval between return of T1 amplitude to 25% and 75% of baseline) were measured. RESULTS Depression of T1 after each submaximal dose of vecuronium was not different between groups. Median time to a TOF ratio ≥ 0.9 was 76.7 minutes (interquartile range [IQR; 25th to 75th percentile], 66.7 to 99.4 minutes) for dogs with CNM and 75.0 minutes (IQR, 47.8 to 96.5 minutes) for controls. Median recovery index was 18.0 minutes (IQR, 9.7 to 23.5 minutes) for dogs with CNM and 20.2 minutes (IQR, 8 to 25.1 minutes) for controls. CONCLUSIONS AND CLINICAL RELEVANCE For the study dogs, neither potency nor duration of vecuronium-induced neuromuscular blockade was altered by CNM. Vecuronium can be used to induce neuromuscular blockade in dogs with autosomal-recessive CNM.

Interferon (IFN)-γ has been shown to be associated with immunity to Marek’s disease virus (MDV). The overall objective of this study was to investigate the causal relationship between IFN-g and vaccine-conferred immunity against MDV in chickens. To this end, 3 small interfering RNAs (siRNAs) targeting chicken IFN-g, which had previously been shown to reduce IFN-γ expression in vitro, and a control siRNA were selected to generate recombinant avian adeno-associated virus (rAAAV) expressing short-hairpin small interfering RNAs (shRNAs). An MDV challenge trial was then conducted: chickens were vaccinated with herpesvirus of turkey (HVT), administered the rAAAV expressing shRNA, and then challenged with MDV. Tumors were observed in 4 out of 10 birds that were vaccinated with HVT and challenged but did not receive any rAAAV, 5 out of 9 birds that were administered the rAAAV containing IFN-γ shRNA, and 2 out of 10 birds that were administered a control enhanced green fluorescent protein siRNA. There was no significant difference in MDV genome load in the feather follicle epithelium of the birds that were cotreated with the vaccine and the rAAAV compared with the vaccinated MDV-infected birds. These results suggest that AAAV-based vectors can be used for the delivery of shRNA into chicken cells. However, administration of the rAAAV expressing shRNA targeting chicken IFN-γ did not seem to fully abrogate vaccine-induced protection.

OBJECTIVE To determine whether thioredoxin (TRX)-1 can be used as a valid biomarker for oxidative stress in dogs. ANIMALS AND SAMPLES 10 Beagles and Madin-Darby canine kidney cells. PROCEDURES Madin-Darby canine kidney cells were used to verify antigen cross-reactivity between human and canine anti–TRX-antibodies. Dogs were assigned to receive 21% or 100% O2 (5 dogs/group) via an artificial respirator during a 3-hour period of isoflurane anesthesia (starting at 0 hours). Blood and urine samples were collected before (baseline) and at 6, 12, 24, and 48 hours after commencement of inhalation anesthesia. Concentrations of TRX-1 and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in plasma and urine samples were analyzed; urine concentrations were reported as ratios against urine creatinine concentration. RESULTS Canine TRX-1 was recognized by monoclonal human anti-TRX-1 antibodies (clones of adult T-cell leukemia-derived factor [ADF]-11 and ADF21) by western blot analysis. Results of an ELISA indicated that plasma TRX-1 concentration and urine TRX-1-to-creatinine concentration ratio increased rapidly after the 3-hour period of hyperoxia with maximal peaks at 12 and 6 hours, respectively. Urine 8-OHdG-to-creatinine concentration ratio also increased significantly after hyperoxia induction. However, unlike the rapid increase in urine TRX-1-to-creatinine concentration ratio, maximal urine 8-OHdG-to-creatinine concentration ratio was attained at 48 hours after hyperoxia induction. These variables remained unchanged from baseline in the control group. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that human anti-TRX monoclonal antibodies cross-reacted with canine TRX, and plasma TRX-1 concentrations were rapidly increased in dogs following an oxidative stress challenge. Thus, TRX may be a valuable clinical biomarker for detecting oxidative stress more rapidly than 8-OHdG in dogs.

OBJECTIVE To determine effects of equipotent concentrations of fentanyl and isoflurane, compared with isoflurane alone, on cardiovascular variables in New Zealand White rabbits (Oryctolagus cuniculus). ANIMALS 6 adult female New Zealand White rabbits. PROCEDURES Rabbits were anesthetized with isoflurane, and lungs were mechanically ventilated. The minimum alveolar concentration (MAC) of isoflurane alone (baseline) and with fentanyl administered IV to achieve 3 targeted plasma concentrations was determined for each rabbit by means of an electrical stimulus. Cardiovascular variables were measured in a separate experiment at 1.3X isoflurane MAC and equipotent doses of isoflurane plus fentanyl at the same 3 targeted plasma concentrations. Blood samples were collected for measurement of blood gas variables and plasma fentanyl concentrations. Treatment effects were evaluated by repeated-measures ANOVA followed by 2-tailed paired t tests with sequentially rejective Bonferroni correction. RESULTS Mean ± SD MAC of isoflurane was 1.95 ± 0.27%. Mean measured plasma fentanyl concentrations of 4.97, 8.93, and 17.19 ng/mL reduced isoflurane MAC by 17%, 37%, and 56%, respectively. Mean measured plasma fentanyl concentrations during cardiovascular measurements were 5.49, 10.26, and 18.40 ng/mL. Compared with baseline measurements, heart rate was significantly lower at all 3 plasma fentanyl concentrations, mean arterial blood pressure and systemic vascular resistance were significantly higher at mean fentanyl concentrations of 10.26 and 18.40 ng/mL, and cardiac output was significantly higher at 18.40 ng of fentanyl/mL. CONCLUSIONS AND CLINICAL RELEVANCE Administration of fentanyl in isoflurane-anesthetized rabbits resulted in improved mean arterial blood pressure and cardiac output, compared with isoflurane alone. This balanced anesthesia technique may prove useful in the management of clinical cases in this species.

The objective of this study was to determine the effect of additional human chorionic gonadotrophin (hCG) on the ovarian response of gilts previously treated with 200 IU hCG combined with 400 IU equine chorionic gonadotrophin (eCG) (eCG/hCG). Seventy-one prepuberal gilts (105 ± 7.5 kg) were assigned to groups: i) eCG/hCG (hCG-0; n = 25); ii) eCG/hCG followed by 100 IU of hCG at 24 h (hCG-100; n = 24); iii) eCG/hCG followed by 200 IU hCG at 24 h (hCG-200; n = 10); and iv) controls (CON; n = 12). Ovulation response was assessed by ovarian dissection or real-time ultrasonography. Additional hCG did not significantly improve numbers of gilts ovulating. Numbers of corpora lutea increased with hCG, and was higher in hCG-200 (P < 0.01). Compared to hCG-0, the frequency of cysts in gilts was higher in hCG-100 (P < 0.05) and further increased in hCG-200 (P < 0.01). The number of cysts per gilt was dose-dependently increased by additional hCG. We conclude that supplemental hCG will increase the number of corpora lutea but will be associated with follicular cyst development in a dose dependent manner.

OBJECTIVE To determine bactericidal effects of enrofloxacin, florfenicol, tilmicosin, and tulathromycin on clinical isolates of Mannheimia haemolytica at various bacterial densities and drug concentrations. SAMPLE 4 unique isolates of M haemolytica recovered from clinically infected cattle. PROCEDURES Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined for each drug and isolate. Mannheimia haemolytica suspensions (10(6) to 10(9) CFUs/mL) were exposed to the determined MIC and MPC and preestablished maximum serum and tissue concentrations of each drug. Log10 reduction in viable cells (percentage of cells killed) was measured at various points. RESULTS Bacterial killing at the MIC was slow and incomplete. After 2 hours of isolate exposure to the MPC and maximum serum and tissue concentrations of the tested drugs, 91% to almost 100% cell killing was achieved with enrofloxacin, compared with 8% growth to 93% cell killing with florfenicol, 199% growth to 63% cell killing with tilmicosin, and 128% growth to 43% cell killing with tulathromycin over the range of inoculum tested. For all drugs, killing of viable organisms was evident at all bacterial densities tested; however, killing was more substantial at the MPC and maximum serum and tissue drug concentrations than at the MIC and increased with duration of drug exposure. Rank order of drugs by killing potency was enrofloxacin, florfenicol, tilmicosin, and tulathromycin. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that antimicrobial doses that equaled or exceeded the MPC provided rapid killing of M haemolytica by the tested drugs, decreasing opportunities for antimicrobial-resistant subpopulations of bacteria to develop during drug exposure.

OBJECTIVE To determine effects of increasing plasma fentanyl concentrations on the minimum alveolar concentration (MAC) of isoflurane in rabbits. ANIMALS 6 adult female New Zealand White rabbits (Oryctolagus cuniculus). PROCEDURES Rabbits were anesthetized with isoflurane in oxygen; ventilation was controlled and body temperature maintained between 38.5° and 39.5°C. Fentanyl was administered IV by use of a computer-controlled infusion system to achieve 6 target plasma concentrations. Isoflurane MAC was determined in duplicate by use of the bracketing technique with a supramaximal electrical stimulus. Blood samples were collected for measurement of plasma fentanyl concentration at each MAC determination. The MAC values were analyzed with a repeated-measures ANOVA followed by Holm-Sidak pairwise comparisons. RESULTS Mean ± SD plasma fentanyl concentrations were 0 ± 0 ng/mL (baseline), 1.2 ± 0.1 ng/mL, 2.2 ± 0.3 ng/mL, 4.4 ± 0.4 ng/mL, 9.2 ± 0.4 ng/mL, 17.5 ± 2.6 ng/mL, and 36.8 ± 2.4 ng/mL. Corresponding mean values for isoflurane MAC were 1.92 ± 0.16%, 1.80 ± 0.16%, 1.60 ± 0.23%, 1.46 ± 0.22%, 1.12 ± 0.19%, 0.89 ± 0.14%, and 0.70 ± 0.15%, respectively. Isoflurane MAC for plasma fentanyl concentrations ≥ 2.2 ng/mL differed significantly from the baseline value. In 3 rabbits, excessive spontaneous movement prevented MAC determination at the highest plasma fentanyl concentration. CONCLUSIONS AND CLINICAL RELEVANCE Fentanyl reduced isoflurane MAC by approximately 60% in New Zealand White rabbits. Further studies will be needed to investigate the cardiorespiratory effects of isoflurane and fentanyl combinations in rabbits; however, fentanyl may prove to be a useful adjunct to inhalation anesthesia in this species.

OBJECTIVE To isolate and characterize endothelial colony-forming cells (ECFCs; a subtype of endothelial progenitor cells) from peripheral blood samples of horses. SAMPLE Jugular venous blood samples from 24 adult horses. PROCEDURES Blood samples were cultured in endothelial cell growth medium. Isolated ECFCs were characterized by use of functional assays of fluorescence-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) uptake and vascular tubule formation in vitro. Expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor 2, and von Willebrand factor) and hematopoietic (CD14) cell markers was assessed through indirect immunofluorescence assay and flow cytometry. The number of passages before senescence was determined through serial evaluation of DiI-Ac-LDL uptake, vascular tubule formation, and cell doubling rates. RESULTS Samples from 3 horses produced colonies at 12 ± 2.5 days with characteristic endothelial single layer cobblestone morphology and substantial outgrowth on expansion. Equine ECFCs formed vascular tubules in vitro and had uptake of DiI-Ac-LDL (74.9 ± 14.7% positive cells). Tubule formation and DiI-Ac-LDL uptake diminished by passage 5. Equine ECFCs tested positive for von Willebrand factor, vascular endothelial growth factor receptor 2, CD34, and CD105 with an immunofluorescence assay and for CD14 and CD105 via flow cytometry. CONCLUSIONS AND CLINICAL RELEVANCE ECFCs can be isolated from peripheral blood of horses and have characteristics similar to those described for other species. These cells may have potential therapeutic use in equine diseases associated with ischemia or delayed vascularization.

OBJECTIVE To investigate the anti-inflammatory and immunomodulatory properties of tulathromycin in vitro and in experimental models of Actinobacillus pleuropneumoniae–induced pleuropneumonia and zymosan-induced pulmonary inflammation in pigs. ANIMALS Blood samples from six 8- to 30-week-old healthy male pigs for the in vitro experiment and sixty-five 3-week-old specific pathogen–free pigs. PROCEDURES Neutrophils and monocyte-derived macrophages were isolated from blood samples. Isolated cells were exposed to tulathromycin (0.02 to 2.0 mg/mL) for various durations and assessed for markers of apoptosis and efferocytosis. For in vivo experiments, pigs were inoculated intratracheally with A pleuropneumoniae, zymosan, or PBS solution (control group) with or without tulathromycin pretreatment (2.5 mg/kg, IM). Bronchoalveolar lavage fluid was collected 3 and 24 hours after inoculation and analyzed for proinflammatory mediators, leukocyte apoptosis, and efferocytosis. RESULTS In vitro, tulathromycin induced time- and concentration-dependent apoptosis in neutrophils, which enhanced their subsequent clearance by macrophages. In the lungs of both A leuropneumoniae– and zymosan-challenged pigs, tulathromycin promoted leukocyte apoptosis and efferocytosis and inhibited proinflammatory leukotriene B4 production, with a concurrent reduction in leukocyte necrosis relative to that of control pigs. Tulathromycin also attenuated the degree of lung damage and lesion progression in A pleuropneumoniae–inoculated pigs. CONCLUSIONS AND CLINICAL RELEVANCE Tulathromycin had immunomodulatory effects in leukocytes in vitro and anti-inflammatory effects in pigs in experimental models of A pleuropneumoniae infection and nonmicrobial-induced pulmonary inflammation. These data suggested that in addition to its antimicrobial properties, tulathromycin may dampen severe proinflammatory responses and drive resolution of inflammation in pigs with microbial pulmonary infections.