OBJECTIVE To determine the physiochemical properties and pharmacokinetics of 3 midazolam gel formulations following buccal administration to dogs. ANIMALS 5 healthy adult hounds. PROCEDURES In phase 1 of a 2-phase study, 2 gel formulations were developed that contained 1% midazolam in a poloxamer 407 (P1) or hydroxypropyl methylcellulose (H1) base and underwent rheological and in vitro release analyses. Each formulation was buccally administered to 5 dogs such that 0.3 mg of midazolam/kg was delivered. Each dog also received midazolam hydrochloride (0.3 mg/kg, IV). There was a 3-day interval between treatments. Blood samples were collected immediately before and at predetermined times for 8 hours after drug administration for determination of plasma midazolam concentration and pharmacokinetic analysis. During phase 2, a gel containing 2% midazolam in a hydroxypropyl methylcellulose base (H2) was developed on the basis of phase 1 results. That gel was buccally administered such that midazolam doses of 0.3 and 0.6 mg/kg were delivered. Each dog also received midazolam (0.3 mg/kg, IV). All posttreatment procedures were the same as those for phase 1. RESULTS The H1 and H2 formulations had lower viscosity, greater bioavailability, and peak plasma midazolam concentrations that were approximately 2-fold as high, compared with those for the P1 formulation. The mean peak plasma midazolam concentration for the H2 formulation was 187.0 and 106.3 ng/mL when the midazolam dose administered was 0.6 and 0.3 mg/kg, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that buccal administration of gel formulations might be a viable alternative for midazolam administration to dogs.

OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro. SAMPLE Venous blood samples from 40 adult horses. PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities. RESULTS Treatment with LPS resulted in a significant delay in apoptosis after incubation for 12 and 24 hours (neutrophils from blood samples of 40 horses). There was a significant correlation between increases in LPS dose and decreases in apoptosis after incubation for 24 hours (3 experiments, each of which involved neutrophils obtained from the same 3 horses at 3 separate times). Caspase-9 activity, but not caspase-3 or -8 activity, was significantly reduced in LPS-treated neutrophils after incubation for 12 hours (neutrophils from blood samples of 17 horses). Treatment with a TLR4 inhibitor or intrinsic and extrinsic inducers of apoptosis prevented LPS-delayed apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE LPS treatment delayed apoptosis of equine neutrophils in vitro for up to 24 hours in a dose-dependent manner by alteration of the intrinsic pathway of apoptosis and was dependent on TLR4 signaling. Increased neutrophil life span may contribute to the development of a systemic inflammatory response syndrome in endotoxemic horses.

OBJECTIVE To investigate the effects of respiratory phase, body position, beam center location, and gastric distention on radiographic assessment of liver size in dogs. ANIMALS 12 Beagles. PROCEDURES Liver length and the ratio of liver length to T11 length were determined on lateral radiographic views obtained with various techniques. Images were acquired at maximal expiration or maximal inspiration, with dogs in right or left recumbency, with the beam centered on the caudal border of the scapula or the 13th rib, and after food was withheld or with gastric distention. Effects on organs adjacent to the liver were assessed with CT. Changes of the thoracic cavity during the respiratory cycle were investigated with fluoroscopy. RESULTS Liver length was significantly greater on radiographs obtained at maximal expiration than at maximal inspiration, but there was no increase in the ratio of liver length to T11 length. Body position, beam center location, and gastric distention did not significantly affect liver size. For CT, location of the spleen and stomach and location or size of the liver did not change markedly between right and left recumbency. Fluoroscopy revealed that thoracic width was less at maximal expiration than maximal inspiration. CONCLUSIONS AND CLINICAL RELEVANCE Liver length was greater at maximal expiration than at maximal inspiration because of a smaller thoracic width. Body position, beam center location, and gastric distention did not affect liver length. The ratio of liver length to T11 length was not significantly affected by any of the factors investigated.

OBJECTIVE To investigate the effect of lipopolysaccharide (LPS) on type VII collagen– cleaving matrix metalloproteinases (MMPs) in the lamellar tissue of extracorporeally perfused equine limbs. SAMPLE 10 right forelimbs and 3 left forelimbs collected from 10 adult horses after slaughter at a licensed abattoir. PROCEDURES Extracorporeal perfusion of the isolated equine limbs was performed for 10 hours under physiologic conditions (control-perfused limbs; n = 5) and with the addition of 80 ng of LPS/L of perfusate (LPS-perfused limbs; 5). Lamellar tissue specimens were then collected from the dorsal aspect of the hooves. Additionally, corresponding control specimens were collected from the 3 nonperfused left forelimbs. Immunohistochemical analysis was performed on paraffin-embedded tissue blocks with antibodies against total (latent and active) MMP-1, MMP-2, MMP-8, and MMP-9 as well as antibody against active MMP-9. Intensity of immunohistochemical staining was scored, and stain distribution in the lamellar tissue was noted. RESULTS Staining intensity of total and active MMP-9 was significantly increased in LPS-perfused versus control-perfused limbs. No such difference was identified for MMP-1, MMP-2, and MMP-8. CONCLUSIONS AND CLINICAL RELEVANCE Of the 4 MMPs that are capable of degrading type VII collagen, MMP-9 was the only one for which production increased in the lamellar tissue of isolated equine limbs perfused with versus without a clinically relevant concentration of LPS. These results suggested that MMP-9 may be involved in initiation of pathological changes in lamellar tissue in endotoxin-induced laminitis, whereas MMP-1, MMP-2, and MMP-8 may be less relevant.

OBJECTIVE To evaluate the plasma disposition of mycophenolic acid (MPA) and its derivatives MPA glucuronide and MPA glucoside after twice-daily infusions of mycophenolate mofetil (MMF) in healthy cats for 3 days and to assess the effect of MMF administration on peripheral blood mononuclear cell (PBMC) counts and CD4+-to-CD8+ ratios. ANIMALS 5 healthy adult cats. PROCEDURES MMF was administered to each cat (10 mg/kg, IV, q 12 h for 3 days). Each dose of MMF was diluted with 5% dextrose in water and then administered over a 2-hour period with a syringe pump. Blood samples were collected for analysis. A chromatographic method was used to quantitate concentrations of MPA and its metabolites. Effects of MMF on PBMC counts and CD4+-to-CD8+ ratios were assessed by use of flow cytometry. RESULTS All cats biotransformed MMF into MPA. The MPA area under the plasma concentration–time curve from 0 to 14 hours ranged from 14.6 to 37.6 mg·h/L and from 14.4 to 22.3 mg·h/L after the first and last infusion, respectively. Total number of PBMCs was reduced in 4 of 5 cats (mean ± SD reduction, 25.9 ± 15.8% and 26.7 ± 19.3%) at 24 and 48 hours after the end of the first infusion of MMF, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Plasma disposition of MPA after twice-daily IV infusions for 3 days was variable in all cats. There were no remarkable changes in PBMC counts and CD4+-to-CD8+ ratios.

OBJECTIVE To determine reference intervals for total nucleated cell count, total protein concentration, pH, RBC count, and percentages of neutrophils, lymphocytes, and large mononuclear cells in synovial fluid samples (SFSs) obtained from the carpal and tarsal joints of healthy swine. ANIMALS 54 healthy commercial finisher pigs that had no evidence of lameness or gross joint swelling. PROCEDURES Each pig was anesthetized, and SFSs were collected from 1 carpal and 1 tarsal joint for fluid analysis, cytologic evaluation, bacterial culture, and PCR analyses for common swine joint pathogens. Each pig was euthanized after SFS collection, and synovial tissue samples were collected for histologic assessment. If necessary, postmortem SFSs were collected. RESULTS Overall, 37 of 50 tarsal and 46 of 53 carpal SFSs met inclusion criteria of sufficient volume, no gross blood contamination, and negative results of bacterial culture and PCR analyses, and were from joints with histologically normal synovial tissues. For the carpal and tarsal joints, upper reference limits were as follows: total nucleated cell count, 3,281 cells/μL and 2,368 cells/μL, respectively; total protein concentration, 3.6 g/dL and 3.6 g/dL, respectively; pH, 7.2 and 7.0, respectively; RBC count, 0.8 × 10(6) cells/μL and 0.1 × 10(6) cells/μL, respectively; and percentage of neutrophils, 46.5% and 33.7%, respectively; percentage of lymphocytes, 40.6% and 56.3%, respectively; and percentage of large mononuclear cells, 92.0% and 95.3%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results have provided reference intervals for selected variables in SFSs obtained from the carpal and the tarsal joints of healthy swine, which should be useful in diagnostic investigations of swine lameness and arthritis.

OBJECTIVE To compare the disposition of fentanyl citrate after a single IV injection in isoflurane-anesthetized red-tailed hawks (Buteo jamaicensis) and Hispaniolan Amazon parrots (Amazona ventralis). ANIMALS 6 adult red-tailed hawks and 6 adult Hispaniolan Amazon parrots. PROCEDURES Anesthesia was induced and maintained with isoflurane; intermittent positive-pressure ventilation was provided. The minimum alveolar concentration of isoflurane was determined for each bird by use of the bracketing method and a supramaximal electrical stimulus. Fentanyl (20 μg/kg) was administered IV. Arterial (red-tailed hawks) or jugular venous (Hispaniolan Amazon parrots) blood samples were obtained immediately before and 1, 2, 4, 8, 15, 30, 60, 120, 180, 240, and 480 minutes (red-tailed hawks) and 1, 5, 10, 15, 30, 60, 120, and 180 minutes (Hispaniolan Amazon parrots) after fentanyl administration. RESULTS A 3-compartment and a 2-compartment model best described fentanyl pharmacokinetics in red-tailed hawks and Hispaniolan Amazon parrots, respectively. Median apparent volume of the central compartment and volume of distribution at steady state were 222 mL/kg and 987 mL/kg, respectively, for the red-tailed hawks and 5,108 mL/kg and 13,079 mL/kg, respectively, for the Hispaniolan Amazon parrots. Median clearance and elimination half-life were 8.9 mL/min/kg and 90.22 minutes, respectively, for the red-tailed hawks and 198.8 mL/min/kg and 51.18 minutes, respectively, for the Hispaniolan Amazon parrots. CONCLUSIONS AND CLINICAL RELEVANCE Pharmacokinetic results for fentanyl in isoflurane-anesthetized red-tailed hawks and Hispaniolan Amazon parrots indicated large differences and should strongly discourage extrapolation of doses between these 2 species.

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

Salmonella is an important cause of foodborne illnesses in humans. Food-producing animals, including swine, are a major source of Salmonella in food products. This study investigated on farm Salmonella fecal shedding in pigs at different production stages - from weaning to marketing - and its association with the presence of Salmonella in tissues at slaughter. Fourteen groups from 8 commercial farrowing sources (N = 809 pigs) were monitored 5 times from birth to slaughter. Fecal and tissue samples were collected from pigs and cultured for Salmonella. A survey was conducted to collect farm management information. A multi-level mixed-effects logistic regression modelling method was used to analyze Salmonella shedding over time and the association between Salmonella shedding and the presence of Salmonella in tissue samples. Salmonella was recovered from 13% (421/3339) of fecal samples collected from 809 pigs over the course of the study. Overall, 35% (284) of pigs shed Salmonella at least once, while 12% (99) shed more than once. Salmonella shedding increased as pigs aged (P = 0.01) and increased in the summer months (P < 0.01). Salmonella was isolated from tissue samples collected from 23% (134/580) of pigs; however, the presence of Salmonella at slaughter was not associated with on farm shedding. The seasonal trend in Salmonella shedding and its association with age may be used to identify high-risk groups and implement more effective control measures accordingly. The identification of repeat shedders warrants interventions that target this source of infection on swine farms.

OBJECTIVE To assess and compare 2 injection techniques for conducting ocular anterior segment indocyanine green angiography (ASICGA) and sodium fluorescein (SF) angiography in horses. ANIMALS 3 healthy adult female horses (age range, 19 to 25 years). PROCEDURES Horses were sedated, jugular catheters were placed, and manual restraint was used to ensure proper positioning for the angiography procedure. Two injection techniques (IV and intra-arterial) were performed for each horse 1 week apart. Intravenous injections of 0.25% indocyanine green (ICG; 50 mg) and 10% SF (10 mg/kg) were administered via the jugular catheter. Intra-arterial injections of ICG (1 mg) and SF (1 mg/kg) were administered into the common carotid artery with ultrasound guidance. Angiography was performed by use of an adaptor system comprised of a modified digital single-lens reflex camera, camera adaptor, and lens. Imaging was performed at a rate of 3 images/s for 60 seconds immediately following ICG injection, then at 2, 3, 4, and 5 minutes after injection. The SF was injected 5 minutes thereafter. RESULTS ASICGA allowed visual identification of the arterial, capillary, and venous phases of angiography. Intra-arterial administration provided superior dye fluorescence, sharper contrast, and faster dye passage than IV administration. Visibility of the iris vasculature was limited with SF, and extravasation of SF was noted. No clinically important adverse events were detected. CONCLUSIONS AND CLINICAL RELEVANCE ASICGA images were obtainable with both injection techniques; however, visibility of the iris vasculature was better with intra-arterial administration of ICG. The ASICGA technique may serve as a viable ocular imaging modality for horses.