OBJECTIVE To produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions. SAMPLE Cultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred. PROCEDURES Cell cultures were transfected by electroporation with a plasmid (pNIT) that expresses the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg), which can be controlled by a doxycycline-responsive promoter. Cells that stably integrated the TAg were selected and expanded to passage 25. For each passage, differentiation and fusion properties of the cells were determined and immunocytochemical analyses were performed to evaluate expression of TAg and other muscle-specific proteins. Optimum conditions that led to cell differentiation into myotubes were also determined. RESULTS Compared with nontransfected control cells, myogenic, desmin-positive cells expressed the TAg when incubated at 33°C and could be maintained in culture for numerous passages. Reduced expression of TAg was identified in cells incubated at 37°C or when incubated with doxycycline at 33°C. Expression of TAg was not detected when cells were incubated with doxycycline at 37°C, and when serum was withdrawn from the culture medium, those clones differentiated into a pure population of multinucleated myotubes. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that production of an immortalized clonal equine skeletal muscle cell line was possible. A clonal equine skeletal muscle cell line will be a valuable in vitro tool for use in equine physiology and disease research.

OBJECTIVE To determine changes in dimensions of feline skin samples as a result of histologic processing and to identify factors that contributed to changes in dimensions of skin samples after sample collection. SAMPLE Cadavers of 12 clinically normal cats. PROCEDURES Skin samples were obtained bilaterally from 3 locations (neck, thorax, and tibia) of each cadaver; half of the thoracic samples included underlying muscle. Length, width, and depth were measured at 5 time points (before excision, after excision, after application of ink to mark tissue margins, after fixation in neutral-buffered 10% formalin for 36 hours, and after completion of histologic processing and staining with H&E stain). Measurements obtained after sample collection were compared with measurements obtained before excision. RESULTS At the final time point, tissue samples had decreased in length (mean decrease, 32.40%) and width (mean decrease, 34.21%) and increased in depth (mean increase, 54.95%). Tissue from the tibia had the most shrinkage in length and width and that from the neck had the least shrinkage. Inclusion of underlying muscle on thoracic skin samples did not affect the degree of change in dimensions. CONCLUSIONS AND CLINICAL RELEVANCE In this study, each step during processing from excision to formalin fixation and histologic processing induced changes in tissue dimensions, which were manifested principally as shrinkage in length and width and increase in depth. Most of the changes occured during histologic processing. Inclusion of muscle did not affect thoracic skin shrinkage. Shrinkage should be a consideration when interpreting surgical margins in clinical cases.

OBJECTIVE To determine by use of an in vitro model the potential for translocating sufficient numbers of bacteria into a joint during arthrocentesis through cellulitic tissue to cause sepsis. SAMPLE Culture media containing 4 concentrations of Staphylococcus aureus and needles of 3 sizes. PROCEDURES Needles (22, 20, and 19 gauge) were inserted through Mueller-Hinton agar that contained known concentrations of S aureus (10(3),10(4),10(5), and 10(6) CFUs/mL). After a needle exited through the medium, any agar plug within the needle bore was ejected into a sterile syringe and the contaminated portion of the needle was harvested. Sterile saline (0.9% NaCl) solution was used to emulsify the agar plug and wash the contaminated portion of the needle. The resulting solution was cultured to determine the number of bacterial CFUs that could be deposited into a joint during arthrocentesis through contaminated tissue. RESULTS Needle gauge and bacterial concentration were both associated with the number of bacterial CFUs deposited after insertion through contaminated agar. Although all needle sizes were capable of bacterial translocation sufficient to cause septic arthritis, ORs for 20- and 22-gauge needles translocating > 33 CFUs of S aureus were significantly higher than the OR for a 19-gauge needle. The ORs for 20- or 22-gauge needles translocating > 33 CFUs of S aureus (the minimum population of S aureus known to cause joint sepsis) were 0.22. CONCLUSIONS AND CLINICAL RELEVANCE Results for this in vitro model indicated that caution should be used when performing arthrocentesis through cellulitic tissue.

While pancreatitis is now recognized as a common ailment in cats, the diagnosis remains challenging due to discordant results and suboptimal sensitivity of ultrasound and specific feline pancreatic lipase (Spec fPL) assay. Pancreatitis also shares similar clinical features with pancreatic carcinoma, a rare but aggressive disease with a grave prognosis. The objective of this pilot study was to compare the plasma proteomes of normal healthy cats (n = 6), cats with pancreatitis (n = 6), and cats with pancreatic carcinoma (n = 6) in order to identify potential new biomarkers of feline pancreatic disease. After plasma protein separation by 2-dimensional gel electrophoresis, protein spots were detected by Coomassie Brilliant Blue G-250 staining and identified by mass spectrometry. Alpha-1-acid glycoprotein (AGP), apolipoprotein-A1 (Apo-A1), and apolipoprotein-A1 precursor (Pre Apo-A1) appeared to be differentially expressed, which suggests the presence of a systemic acute-phase response and alteration of lipid metabolism in cats with pancreatic disease. Future studies involving greater case numbers are needed in order to assess the utility of these proteins as potential biomarkers. More sensitive proteomic techniques may also be helpful in detecting significant but low-abundance proteins.

Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD.

OBJECTIVE To determine pharmacokinetic and pharmacodynamic properties of the novel factor Xa inhibitor apixaban in clinically normal cats. ANIMALS 5 purpose-bred domestic shorthair cats. PROCEDURES A single dose of apixaban (0.2 mg/kg, PO) was administered to each cat (time 0), and blood samples were obtained at 0, 15, 30, 45, 60, 120, 240, 360, 480, and 1,440 minutes. After a 1-week washout period, another dose of apixaban (0.2 mg/kg, IV) was administered to each cat, and blood samples were obtained at 0, 5, 10, 15, 30, 45, 60, 120, 240, 360, 480, and 1,440 minutes. Apixaban concentrations in plasma were measured via liquid chromatography–tandem mass spectrometry. Pharmacodynamic effects of apixaban were determined with a commercial assay for factor × activity, which measures endogenous factor Xa activity chromogenically. RESULTS Factor Xa was inhibited as a function of time after a single dose of apixaban administered orally or IV, and a direct inverse correlation with the plasma apixaban concentration was detected. Pharmacokinetic analysis revealed moderate clearance, short half-life, and high bioavailability for apixaban. A 2-compartment model was fit to the IV pharmacokinetic data; compartmental modeling could not be used to adequately describe the oral data because of substantial interindividual variability. CONCLUSIONS AND CLINICAL RELEVANCE Results inticated that apixaban was an effective inhibitor of factor Xa in cats. Further studies will be needed to determine pharmacokinetics and pharmacodynamics after multidose administration, effects of cardiac disease on pharmacokinetics and pharmacodynamics, dosing recommendations, and efficacy of apixaban for use in the treatment and prevention of thromboembolic disease in cats.

OBJECTIVE To evaluate the use of rebound and applanation tonometry for the measurement of intraocular pressure (IOP) and to assess diurnal variations in and the effect of topical anesthesia on the IOP of healthy inland bearded dragons (Pogona vitticeps). ANIMALS 56 bearded dragons from 4 months to 11 years old. PROCEDURES For each animal following an initial ophthalmic examination, 3 IOP measurements were obtained on each eye between 9 AM and 10 AM, 1 PM and 2 PM, and 5 PM and 7 PM by use of rebound and applanation tonometry. An additional measurement was obtained by rebound tonometry for each eye in the evening following the application of a topical anesthetic to evaluate changes in the tolerance of the animals to the tonometer. Descriptive data were generated, and the effects of sex, time of day, and topical anesthesia on IOP were evaluated. RESULTS Bearded dragons did not tolerate applanation tonometry even following topical anesthesia. Median daily IOP as determined by rebound tonometry was 6.16 mm Hg (95% confidence interval, 5.61 to 6.44 mm Hg). The IOP did not differ significantly between the right and left eyes. The IOP was highest in the morning, which indicated that the IOP in this species undergoes diurnal variations. Topical anesthesia did not significantly affect IOP, but it did improve the compliance for all subjects. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that rebound tonometry, but not applanation tonometry, was appropriate for measurement of IOP in bearded dragons. These findings provided preliminary guidelines for IOP measurement and ophthalmic evaluation in bearded dragons.

OBJECTIVE To assess efficiency of gravity filtration to enhance recovery of equine bone marrow elements including stem and progenitor cells. ANIMALS 12 healthy adult horses. PROCEDURES Bone marrow aspirates were collected from the fifth sternebral body and filtered by gravitational flow to obtain bone marrow elements. Raw and harvested bone marrow and marrow effluent were evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, mesenchymal stem cell CFUs, cell viability, and differentiation capacity. Isolated cells were analyzed for CD90 and major histocompatibility complex (MHC) class I and II antigens. RESULTS Mean cell viability of harvested bone marrow was 95.9%. Total WBCs and platelets were efficiently captured on the filter (> 95%), and mean recovery in harvested bone marrow was 30%. Cytologic cell differential counts indicated that the percentage of neutrophils was significantly less and the progenitor cell population was significantly higher and concentrated 1.56-fold in harvested bone marrow, compared with results for raw bone marrow. Flow cytometry and cell culture were used to characterize harvested bone marrow cells as positive for expression of CD90 and negative for MHCI and MHCII, which indicated stem cells with a multipotent phenotype that differentiated into chondrocytes, osteocytes, adipocytes, and tenocytes. CONCLUSIONS AND CLINICAL RELEVANCE Gravitational filtration of bone marrow efficiently yielded platelets and cells and produced a progenitor-enriched, leukocyte-reduced product, compared with raw bone marrow.

Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and <6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.

The objective of this study was to determine the effects of propofol on the minimum alveolar concentration of sevoflurane needed to prevent motor movement (MACNM) in dogs subjected to a noxious stimulus using randomized crossover design. Six, healthy, adult beagles (9.2 ± 1.3 kg) were used. Dogs were anesthetized with sevoflurane on 3 occasions, at weekly intervals, and baseline MACNM (MACNM-B) was determined on each occasion. Propofol treatments were administered as loading dose (LD) and constant rate infusion (CRI) as follows: Treatment 1 (T1) was 2 mg/kg body weight (BW) and 4.5 mg/kg BW per hour; T2 was 4 mg/kg BW and 9 mg/kg BW per hour; T3 was 8 mg/kg BW and 18 mg/kg BW per hour, respectively. Treatment MACNM (MACNM-T) determination was initiated 60 min after the start of the CRI. Two venous blood samples were collected and combined at each MACNM-T determination for measurement of blood propofol concentration using high-performance liquid chromatography method (HPLC). Data were analyzed using a mixed-model ANOVA and are presented as least square means (LSM) ± standard error of means (SEM). Propofol infusions in the range of 4.5 to 18 mg/kg BW per hour resulted in mean blood concentrations between 1.3 and 4.4 μg/mL, and decreased (P < 0.05) sevoflurane MACNM in a concentration-dependent manner. The percentage decrease in MACNM was 20.5%, 43.0%, and 68.3%, with corresponding blood propofol concentrations of 1.3 ± 0.3 μg/mL, 2.5 ± 0.3 μg/mL, and 4.4 ± 0.3 μg/mL, for T1, T2, and T3, respectively. Venous blood propofol concentrations were strongly correlated (r = 0.855, P < 0.0001) with the decrease in MACNM. In dogs, propofol decreased the sevoflurane MACNM in a concentration-dependent manner.