OBJECTIVE To examine the equine foot for the presence of sensory receptors including Merkel cells and small lamellated Pacinian-like corpuscles (SLPCs). SAMPLE Forefeet obtained from 7 horses following euthanasia for reasons other than foot disease. PROCEDURES Disarticulated feet were cut into either sagittal sections or cross sections and immersed in neutral-buffered 4% formalin. Following fixation, samples were obtained from the midline of the dorsal aspect of the hoof wall and from the frog (cuneus ungulae) between the apex and central sulcus. The formalin-fixed, paraffin-embedded hoof wall and frog sections were routinely processed for peroxidase immunohistochemistry and stained with H&E, Alcian blue, and Masson trichrome stains for histologic evaluation. RESULTS Sensory myelinated nerves and specific receptors were identified within the epidermal and dermal tissues of the equine foot including the hoof wall laminae, coronet, and frog. Merkel cells were identified with specific antisera to villin, cytokeratin 20, and protein gene product 9.5 in coronet epidermis and hoof wall. These cells were interspersed among basilar keratinocytes within the frog, coronary epidermis, and secondary epidermal laminae. The SLPCs were present within the superficial dermis associated with the central ridge of the frog (ie, frog stay). Numerous S100 protein and protein gene product 9.5 immunoreactive sensory nerves in close proximity to these receptors were present throughout the dermal tissues within both the frog and hoof wall. CONCLUSIONS AND CLINICAL RELEVANCE The presence of Merkel cells and SLPCs that are known to detect tactile and vibrational stimuli, respectively, further defined the diverse range of neural elements within the equine foot.

OBJECTIVE To develop a high-resolution melting (HRM) assay to detect the g.66493737C>T polymorphism in the myostatin gene (MSTN) and determine the frequency of 3 previously defined g.66493737 genotypes (T/T, T/C, and C/C) in warmblood horses. SAMPLES Blood samples from 23 horses. PROCEDURES From each blood sample, DNA was extracted and analyzed by standard PCR methods and an HRM assay to determine the MSTN genotype. Three protocols (standard protocol, protocol in which a high-salt solution was added to the reaction mixture before the first melting cycle, and protocol in which an unlabeled probe was added to the reaction mixture before analysis) for the HRM assay were designed and compared. Genotype results determined by the HRM protocol that generated the most consistent melting curves were compared with those determined by sequencing. RESULTS The HRM protocol in which an unlabeled probe was added to the reaction mixture generated the most consistent melting curves. The genotypes of the g.66493737C>T polymorphism were determined for 22 horses (16 by HRM analysis and 20 by sequencing); 14, 7, and 1 had the T/T, T/C, and C/C genotypes, respectively. The genotype determined by HRM analysis agreed with that determined by sequencing for 14 of 16 horses. The frequency of alleles T and C was 79.5% and 20.5%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HRM analysis may be a faster and more economical alternative than PCR methods for genotyping. Genotyping results might be useful as predictors of athletic performance for horses.

OBJECTIVE To evaluate gene expression and DNA copy number in adipose tissue-derived stromal cells (ADSCs) and in ADSC-derived neurosphere-like cell clusters (ADSC-NSCs) generated from tissues of chronically paraplegic dogs. ANIMALS 14 client-owned paraplegic dogs. PROCEDURES Dorsal subcutaneous adipose tissue (< 1 cm3) was collected under general anesthesia; ADSCs were isolated and cultured. Third-passage ADSCs were cultured in neural cell induction medium to generate ADSC-NSCs. Relative gene expression of mesenchymal cell surface marker CD90 and neural progenitor marker nestin was assessed in ADSCs and ADSC-NSCs from 3 dogs by quantitative real-time PCR assay; expression of these and various neural lineage genes was evaluated for the same dogs by reverse transcription PCR assay. Percentages of cells expressing CD90, nestin, glial fibrillary acidic protein (GFAP), and tubulin β 3 class III (TUJ1) proteins were determined by flow cytometry for all dogs. The DNA copy number stability (in samples from 6 dogs) and neural cell differentiation (14 dogs) were assessed with array-comparative genomic hybridization analysis and immunocytochemical evaluation, respectively. RESULTS ADSCs and ADSC-NSCs expressed neural cell progenitor and differentiation markers; GFAP and microtubule-associated protein 2 were expressed by ADSC-NSCs but not ADSCs. Relative gene expression of CD90 and nestin was subjectively higher in ADSC-NSCs than in ADSCs. Percentages of ADSC-NSCs expressing nestin, GFAP, and TUJ1 proteins were substantially higher than those of ADSCs. Cells expressing neuronal and glial markers were generated from ADSC-NSCs and had no DNA copy number instability detectable by the methods used. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested ADSCs can potentially be a safe and clinically relevant autologous source for canine neural progenitor cells. Further research is needed to verify these findings.

OBJECTIVE To determine variance effects influencing ground reaction forces (GRFs) in a heterogeneous population of lame dogs during trotting. ANIMALS 30 client-owned dogs with thoracic limb lameness and 31 dogs with pelvic limb lameness. PROCEDURES GRFs, velocity, height at the dorsal aspect of the scapulae (ie, withers), and shoulder height were obtained. Each dog was trotted across a force platform at its preferred velocity. Variance effects for 12 velocity and associated relative velocity (V*) ranges were examined. RESULTS Individual dog, velocity, V*, and limb significantly influenced GRFs. Withers height V* ranges were associated with small variance in GRFs, but all absolute and V* ranges were associated with significant effects for all 4 limbs and both types of lameness. Significant changes in lame limb GRFs and velocity in ipsilateral trials in dogs with thoracic limb and pelvic limb lameness were evident with trial repetition. Withers height V* range of 0.55 to 0.93 captured a large proportion of trials (> 90%) in dogs with thoracic limb or pelvic limb lameness, with limited effects on peak vertical force and vertical impulse. CONCLUSIONS AND CLINICAL RELEVANCE Trial repetition caused alterations to GRFs and subject velocity that may have confounded assessment of lameness, which supported the concept that a priori selection of a velocity or V* range for force platform gait analysis should use a range that captures valid trials efficiently while minimizing GRF variance. These ranges typically would span the preferred velocity of subject dogs, such as withers height V* of 0.55 to 0.93.

OBJECTIVE To characterize clinical findings for polysaccharide storage myopathy (PSSM) in warmblood horses with type 1 PSSM (PSSM1; caused by mutation of the glycogen synthase 1 gene) and type 2 PSSM (PSSM2; unknown etiology). SAMPLE Database with 3,615 clinical muscle biopsy submissions. PROCEDURES Reported clinical signs and serum creatine kinase (CK) and aspartate aminotransferase (AST) activities were retrospectively analyzed for horses with PSSM1 (16 warmblood and 430 nonwarmblood), horses with PSSM2 (188 warmblood and 646 nonwarmblood), and warmblood horses without PSSM (278). Lameness examinations were reviewed for 9 warmblood horses with PSSM2. Muscle glycogen concentrations were evaluated for horses with PSSM1 (14 warmblood and 6 nonwarmblood), warmblood horses with PSSM2 (13), and horses without PSSM (10 warmblood and 6 nonwarmblood). RESULTS Rhabdomyolysis was more common for horses with PSSM1 (12/16 [75%] warmblood and 223/303 [74%] nonwarmblood) and nonwarmblood horses with PSSM2 (221/436 [51%]) than for warmblood horses with PSSM2 (39/147 [27%]). Gait abnormality was more common in warmblood horses with PSSM2 (97/147 [66%]) than in warmblood horses with PSSM1 (1/16 [7%]), nonwarmblood horses with PSSM2 (176/436 [40%]), and warmblood horses without PSSM (106/200 [53%]). Activities of CK and AST were similar in warmblood horses with and without PSSM2. Muscle glycogen concentrations in warmblood and nonwarmblood horses with PSSM1 were significantly higher than concentrations in warmblood horses with PSSM2. CONCLUSIONS AND CLINICIAL RELEVANCE Rhabdomyolysis and elevated muscle glycogen concentration were detected in horses with PSSM1 regardless of breed. Most warmblood horses with PSSM2 had stiffness and gait abnormalities with CK and AST activities and muscle glycogen concentrations within reference limits.

OBJECTIVE To determine pharmacokinetics and pulmonary disposition of minocycline in horses after IV and intragastric administration. ANIMALS 7 healthy adult horses. PROCEDURES For experiment 1 of the study, minocycline was administered IV (2.2 mg/kg) or intragastrically (4 mg/kg) to 6 horses by use of a randomized crossover design. Plasma samples were obtained before and 16 times within 36 hours after minocycline administration. Bronchoalveolar lavage (BAL) was performed 4 times within 24 hours after minocycline administration for collection of pulmonary epithelial lining fluid (PELF) and BAL cells. For experiment 2, minocycline was administered intragastrically (4 mg/kg, q 12 h, for 5 doses) to 6 horses. Plasma samples were obtained before and 20 times within 96 hours after minocycline administration. A BAL was performed 6 times within 72 hours after minocycline administration for collection of PELF samples and BAL cells. RESULTS Mean bioavailability of minocycline was 48% (range, 35% to 75%). At steady state, mean ± SD maximum concentration (Cmax) of minocycline in plasma was 2.3 ± 1.3 μg/mL, and terminal half-life was 11.8 ± 0.5 hours. Median time to Cmax (Tmax) was 1.3 hours (interquartile range [IQR], 1.0 to 1.5 hours). The Cmax and Tmax of minocycline in the PELF were 10.5 ± 12.8 μg/mL and 9.0 hours (IQR, 5.5 to 12.0 hours), respectively. The Cmax and Tmax for BAL cells were 0.24 ± 0.1 μg/mL and 6.0 hours (IQR, 0 to 6.0 hours), respectively. CONCLUSIONS AND CLINICAL RELEVANCE Minocycline was distributed into the PELF and BAL cells of adult horses.

OBJECTIVE To compare intraosseous pentobarbital treatment (IPT) and thoracic compression (TC) on time to circulatory arrest and an isoelectric electroencephalogram (EEG) in anesthetized passerine birds. ANIMALS 30 wild-caught adult birds (17 house sparrows [Passer domesticus] and 13 European starlings [Sturnus vulgaris]). PROCEDURES Birds were assigned to receive IPT or TC (n = 6/species/group). Birds were anesthetized, and carotid arterial pulses were monitored by Doppler methodology. Five subdermal braided-wire electrodes were used for EEG. Anesthetic depth was adjusted until a continuous EEG pattern was maintained, then euthanasia was performed. Times from initiation of euthanasia to cessation of carotid pulse and irreversible isoelectric EEG (indicators of death) were measured. Data (medians and first to third quartiles) were summarized and compared between groups within species. Necropsies were performed for all birds included in experiments and for another 6 birds euthanized under anesthesia by TC (4 sparrows and 1 starling) or IPT (1 sparrow). RESULTS Median time to isoelectric EEG did not differ significantly between treatment groups for sparrows (19.0 and 6.0 seconds for TC and IPT, respectively) or starlings (88.5 and 77.5 seconds for TC and IPT, respectively). Median times to cessation of pulse were significantly shorter for TC than for IPT in sparrows (0.0 vs 18.5 seconds) and starlings (9.5 vs 151.0 seconds). On necropsy, most (14/17) birds that underwent TC had grossly visible coelomic, pericardial, or perihepatic hemorrhage. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that TC might be an efficient euthanasia method for small birds. Digital pressure directly over the heart during TC obstructed venous return, causing rapid circulatory arrest, with rupture of the atria or vena cava in several birds. The authors propose that cardiac compression is a more accurate description than TC for this procedure.

Escherichia coli isolates from chickens with colibacillosis were assigned to phylogenetic groups based on multiplex polymerase chain reaction (PCR) and antibacterial resistance of E. coli belonging to these groups was examined. Furthermore, the gyrA gene of isolates was sequenced and a phylogenetic tree was generated. A total of 84 E. coli isolates were grouped using multiplex PCR of TSPE4.C2, chuA, yjaA, and gadA molecular markers. Four phylogenetic groups were identified with strains divided as follows: 16 in group A (19.05%), 17 in group B1 (20.24%), 23 in group B2 (27.38%), and 28 in group D (33.33%). Escherichia coli isolates belonging to phylogenetic groups B2 and D were resistant to Soltrim and Flumequine unlike the majority of E. coli isolates that belonged to groups A and B1, and which were susceptible to these antibiotics. The phylogenetic results based on gyrA gene sequences from multiplex PCR revealed that E. coli phylogenetic grouping was in accordance with the clusters obtained in the phylogenetic tree. In conclusion, the comparative sequence analysis of gyrA sequences provides a firm framework for an accurate classification of E. coli and related taxa and may constitute a pertinent phylogenetic marker for E. coli.

OBJECTIVE To determine anatomic reference points for 4 turtle species and to evaluate data on relative anatomic dimensions, signal intensities (SIs), and position of selected organs within the coelomic cavity by use of MRI. ANIMALS 3 turtle cadavers (1 red-eared slider [Trachemys scripta elegans], 1 yellow-bellied slider [Trachemys scripta scripta], and 1 Coastal plain cooter [Pseudemys concinna floridana]) and 63 live adult turtles (30 red-eared sliders, 20 yellow-bellied sliders, 5 Coastal plain cooters, and 8 hieroglyphic river cooters [Pseudemys concinna hieroglyphica]). PROCEDURES MRI and necropsy were performed on the 3 turtle cadavers. Physical examination, hematologic evaluation, and whole-body radiography were performed on the 63 live turtles. Turtles were sedated, and MRI in transverse, sagittal, and dorsal planes was used to measure organ dimensions, position within the coelomic cavity, and SIs. Body positioning after sedation was standardized with the head, neck, limbs, and tail positioned in maximum extension. RESULTS Measurements of the heart, liver, gallbladder, and kidneys in sagittal, transverse, and dorsal planes; relative position of those organs within the coelom; and SIs of the kidneys and liver were obtained with MRI and provided anatomic data for these 4 turtle species. CONCLUSIONS AND CLINICAL RELEVANCE MRI was a valuable tool for determining the position, dimensions, and SIs of selected organs. Measurement of organs in freshwater chelonians was achievable with MRI. Further studies are needed to establish reference values for anatomic structures in turtles. Results reported here may serve as guidelines and aid in clinical interpretation of MRI images for these 4 species.