Objective—To evaluate molecular and histologic characteristics of the superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT), and suspensory ligament (SL) and assess trace-mineral concentrations in serum, liver, and hair of juvenile llamas with metacarpophalangeal and metatarsophalangeal joint hyperextension. Animals—12 juvenile llamas (6 with bilateral hyperextension of metacarpophalangeal joints, metatarsophalangeal joints, or both and 6 clinically normal control llamas). Procedures—Radiography and ultrasonography of metacarpophalangeal and metatarsophalangeal regions were performed. Llamas were euthanized, and SDFT, DDFT, and SL samples were collected for histologic evaluation of collagen and elastin content and orientation, proteoglycan content, and collagen type III immunohistochemistry. Total RNA was isolated from SL tissue, and gene expression of collagen types I and III, lysyl oxidase, and matrix metalloproteinase-13 was evaluated via real-time quantitative reverse transcriptase PCR assay. Liver, serum, and hair samples were evaluated for trace mineral content. Results—Collagen type III gene expression and proteoglycan content were significantly increased in SL samples of affected juvenile llamas, compared with those of control llamas. No difference was detected in collagen and elastin content and orientation or in gene expression of collagen type I, lysyl oxidase, or matrix metalloproteinase-13 between groups. Affected llamas had significantly increased serum molybdenum and decreased liver cobalt concentrations, compared with values for control llamas. Conclusions and Clinical Relevance—Increased collagen type III gene expression and proteoglycan content in SL samples of affected juvenile llamas provided evidence of ongoing SL matrix repair. Trace mineral differences may have been attributable to dietary imbalances in affected llamas.

Objective—To determine whether a mutation in the fibrillin 2 gene (FBN2) is associated with canine hip dysplasia (CHD) and osteoarthritis in dogs. Animals—-1,551 dogs. Procedures—Hip conformation was measured radiographically. The FBN2 was sequenced from genomic DNA of 21 Labrador Retrievers and 2 Greyhounds, and a haplotype in intron 30 of FBN2 was sequenced in 90 additional Labrador Retrievers and 143 dogs of 6 other breeds. Steady-state values of FBN2 mRNA and control genes were measured in hip joint tissues of fourteen 8-month-old Labrador Retriever–Greyhound crossbreeds. Results—The Labrador Retrievers homozygous for a 10-bp deletion haplotype in intron 30 of FBN2 had significantly worse CHD as measured via higher distraction index and extended-hip joint radiograph score and a lower Norberg angle and dorsolateral subluxation score. Among 143 dogs of 6 other breeds, those homozygous for the same deletion haplotype also had significantly worse radiographic CHD. Among the 14 crossbred dogs, as the dorsolateral subluxation score decreased, the capsular FBN2 mRNA increased significantly. Those dogs with incipient hip joint osteoarthritis had significantly increased capsular FBN2 mRNA, compared with those dogs without osteoarthritis. Dogs homozygous for the FBN2 deletion haplotype had significantly less FBN2 mRNA in their femoral head articular cartilage. Conclusions and Clinical Relevance—The FBN2 deletion haplotype was associated with CHD. Capsular gene expression of FBN2 was confounded by incipient secondary osteoarthritis in dysplastic hip joints. Genes influencing complex traits in dogs can be identified by genome-wide screening, fine mapping, and candidate gene screening.

The present study investigated whether the 14-3-3 η and γ proteins, which are potent matrix metalloprotease (MMP) stimulators, are detectable in the synovial fluid of dogs with cranial cruciate ligament rupture (CCLR). Synovial fluid samples from 7 dogs with unilateral CCLR and control samples from 4 dogs without a history of any joint inflammation or any other abnormalities underwent Western blot analysis for the 14-3-3 η, γ, and σ proteins as well as MMP-1 and MMP-3. Craniocaudal and lateral radiographic projections of the stifle joint were evaluated for the presence and severity of 13 specific radiographic markers of osteoarthritis and graded numerically. The Spearman method was used to detect any correlation between the 14-3-3-η level in the synovial fluid and the radiograph-based grade. The η isoform was present only in the samples from the dogs with CCLR. The levels of 14-3-3-γ, MMP-1, and MMP-3 were significantly higher in the samples from the dogs with CCLR than in the control samples (P < 0.05). However, there was no significant difference between the CCLR and control samples in the level of the σ isoform. The Spearman method showed a significant correlation between the 14-3-3-η level in the synovial fluid and the presence of either patellar osteophytes or lateral or medial (or both) condylar periarticular osteophytes (P < 0.05). The MMP stimulatory effect of the 14-3-3 η and γ isoforms may be the reason for the high levels of MMP-1 and MMP-3 observed. Thus, 14-3-3 proteins, especially the η isoform, may be important markers of osteoarthritis caused by CCLR.

Objective—To determine effects of various concentrations of retinoic acid (RA) or a synthetic RA receptor antagonist (LE135) on equine chondrocytes or bone marrow—derived equine mesenchymal stem cells (BMDMSCs) in monolayer cultures. Sample—Articular cartilage and BMDMSCs from 5 clinically normal horses. Procedures—Monolayers of chondrocytes cultured in standard media and of BMDMSCs cultured in chondrogenic media were treated with RA at concentrations of 0, 0.1, 1, or 10μM or LE135 at concentrations of 0, 0.1, 1, or 10μM on day 0. On days 7 and 14, samples were analyzed for DNA concentration, chondrocyte morphology or features consistent with chondrogenesis (ie, chondral morphology [scored from 0 to 4]), and gene expression of collagen type Ia (CI), collagen type II (CII), and aggrecan. Results—Chondrocytes treated with RA had more mature chondral morphology (range of median scores, 3.0 to 4.0) than did untreated controls (range of median scores, 0.5 to 0.5). Chondrocytes treated with LE135 did not sustain chondrocyte morphology. All BMDMSCs had evidence of chondral morphology or high CII:CI ratio. Retinoic acid (1 or 10μM) or LE135 (10μM) treatment decreased DNA content of BMDMSC cultures. At 0.1 and 1μM concentrations, LE135 weakly but significantly increased chondral morphology scores, compared with untreated controls, but lack of aggrecan expression and lack of increased CII:CI ratio, compared with that of controls, did not affect chondrogenesis. Conclusions and Clinical Relevance—RA promoted maturation and hypertrophy in chondrocytes but not BMDMSCs in monolayer cultures. Deficiency or blockade of RA may prevent hypertrophy and maturation of differentiated chondrocytes.

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.

A fast, sensitive, and specific reverse-transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed that involved a single tube and a 1-step reaction for detecting infectious bursal disease virus (IBDV). Four specific primers were used for amplification of the VP2 gene of IBDV. The amplified LAMP products were detected by DNA electrophoresis and by direct observation with the naked eye in the presence of SYBR Green I. The sensitivity of RT-LAMP was determined to be 0.01 fg of IBDV viral RNA. This assay for IBDV is more sensitive than the conventional RT-polymerase chain reaction assay, which has a detection limit of 1 ng. The LAMP assay was also assessed for specificity and was found to precisely discriminate between positive and negative test samples. This newly established LAMP assay, combined with RT, is a practical diagnostic tool because IBDV-infected and uninfected clinical samples collected from an experimental farm could be discriminated. Full verification of a sample’s IBDV status was obtained within 40 min of extraction of the viral RNA, which could then be directly added to the RT-LAMP reaction mixture.

Objective: To evaluate influence of electrode position on cardioversion energy (CE; energy delivered in the shock at which cardioversion was achieved) during transvenous electrical cardioversion (TVEC) in horses with atrial fibrillation. Animals: 37 horses with atrial fibrillation (41 cardioversion events). Procedures: Records were reviewed to identify horses that underwent TVEC for treatment of atrial fibrillation. Signalment and CE were recorded. Electrode positions in the right atrium and pulmonary artery were identified on intraoperative radiographs. An orthogonal coordinate space was created, and electrode y- and z-axis coordinates and shadow lengths were determined. Trigonometric modeling was used to estimate x-axis electrode positions that resulted in observed shadows. Postmortem casts of catheterized horses were used to assess electrode paths and anatomic relationships. Model assumptions were tested by use of these and a theoretical data set. Relationships between signalment, electrode position, and CE were assessed via multivariate analysis. Results: Sex and y-axis differences between electrode positions were significant predictors of CE. Population stratification based on examination of residuals improved model strength; populations differed in z-axis variables and in CE. Decreasing distance between electrodes and pulmonary artery electrode positions ventral to the right atrium were associated with increased CE. Agreement between estimated and actual x-axis coordinates was poor. Conclusions and Clinical Relevance: Optimal electrode positioning can reduce the energy requirement for successful TVEC and may eventually support application of TVEC under short-term IV anesthesia and potentially increase chances of treatment response. Further investigation into these relationships is warranted.

Objective: To assess the effect of computed tomography (CT) scan protocols (radiation amounts) and fabrication methods on biomodel accuracy and variability. Sample: Cadaveric femur of a Basset Hound. Procedures: Retroreconstructions (n = 158) were performed of 16 original scans and were visually inspected to select 17 scans to be used for biomodel fabrication. Biomodels of the 17 scans were made in triplicate by use of 3 freeform fabrication processes (stereolithography, fused deposition modeling, and 3-D printing) for 153 models. The biomodels and original bone were measured by use of a coordinate measurement machine. Results: Differences among fabrication methods accounted for 2% to 29% of the total observed variation in inaccuracy and differences among method-specific radiation configurations accounted for 4% to 44%. Biomodels underestimated bone length and width and femoral head diameter and overestimated cortical thickness. There was no evidence of a linear association between thresholding adjustments and biomodel accuracy. Higher measured radiation dose led to a decrease in absolute relative error for biomodel diameter and for 4 of 8 cortical thickness measurements. Conclusions and Clinical Relevance: The outside dimensions of biomodels have a clinically acceptable accuracy. The cortical thickness of biomodels may overestimate cortical thickness. Variability among biomodels was caused by model fabrication reproducibility and, to a lesser extent, by the radiation settings of the CT scan and differences among fabrication methods.

Objective: To determine the effectiveness of preinduction hyperbaric oxygen treatment (HBOT) in ameliorating signs of experimentally induced endotoxemia in horses. Animals: 18 healthy adult horses. Procedures: Horses were randomly assigned to 1 of 3 equal-sized treatment groups to receive normobaric ambient air and lipopolysaccharide (LPS), HBOT and LPS, or HBOT and physiologic saline (0.9% NaCl) solution. Horses were physically examined, and blood was obtained for a CBC and to determine concentration or activity of plasma tissue necrosis factor-α, blood lactate, and blood glucose before the horses were treated with HBOT and then intermittently for 6 hours after administration of LPS or physiologic saline solution. Results: All LPS-treated horses developed signs and biochemical and hematologic changes consistent with endotoxemia. Treatment with HBOT significantly ameliorated the effect of LPS on clinical endotoxemia score but did not significantly improve other abnormalities associated with endotoxemia. Conclusions and Clinical Relevance: The protective effect of HBOT was minimal, and results did not support its use as a treatment for horses prior to development of endotoxemia.

Objective: To noninvasively evaluate physiologic postprandial adaptations of the heart in snakes. Animals: 6 juvenile Paraguay anacondas (Eunectes notaeus). Procedures: The heart of each anaconda was echocardiographically evaluated after food was withheld for 28 days as well as 3 and 10 days after feeding. Physical measurements included body length, weight, and circumference at the level of the heart. Echocardiographic measurements included heart rate and 2-D total and internal ventricular area. From these measurements, total ventricular volume as well as the myocardial area as a surrogate of myocardial mass was calculated. Results: No significant changes in body length, weight, and circumference were found. Significant increases in heart rate (from 45 to 58 beats/min), total ventricular volume (from 4.63 to 5.54 mL), and myocardial area (from 0.7 to 0.81 cm2) were detected 10 days after feeding, compared with results obtained prior to feeding after food had been withheld for 28 days. No pericardial effusion was detected at any time point. Conclusions and Clinical Relevance: Echocardiographic evaluation of the heart of anacondas was performed, and feeding resulted in concentric cardiac hypertrophy. Physiologic fluctuation of cardiac dimensions should be considered when cardiac imaging is performed in snakes.