Health, religious, and commercial aspects justify the need for meat species identification. The lack of officially approved methods prompts the undertaking of research on validation of isoelectric focusing of proteins (IEF) for official purposes. Samples were prepared from pigs (Sus scrofa ferus domestica), cattle (Bos taurus), and poultry (Gallus gallus domesticus). Meat mixtures were made by blending 50%, 25%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.2% meat of other species. Samples were examined on ultrathin polyacrylamide gels with pH 3–9 gradient. The results of the study confirmed the stable and reproducible pattern of meat protein bands. The detection limit of raw meat admixtures from pigs, cattle, and poultry mostly ranged from 2% down to 0.2% (0.2% for poultry). However, the IEF method can be used to detect the addition of pig meat to bovine meat in an amount higher than 3%. At the significant mixture level (i.e at least 5% addition of meat of another species) IEF proves itself with 100% specificity, sensitivity, and accuracy. The achieved detection limits provide a basis for recommending the IEF method for routine tests in laboratories detecting the species origin of meat.

The aim of the study was to estimate the diagnostic sensitivity (DSe) and specificity (DSp) of an agar gel immunodiffusion (AGID) assay for detection of myxoma virus (MYXV) in the classical form of myxomatosis and to compare its diagnostic performance to that of molecular methods (IAC-PCR, OIE PCR, and OIE real-time PCR). A panel of MYXV-positive samples of tissue homogenates with low (1 PCR unit – PCRU) and high (3,125 PCRU) virus levels and outbreak samples were used for method comparison studies. The validation parameters of the AGID assay were assessed using statistical methods. The AGID attained DSe of 0.65 (CI₉₅%: 0.53–0.76), DSp of 1.00 (CI₉₅%: 0.40–1.00), and accuracy of 0.67 (CI₉₅%: 0.55–0.76). The assay confirmed its diagnostic usefulness primarily for testing samples containing ≥3,125 PCRU of MYXV DNA. However, in the assaying of samples containing <3,125 PCRU of the virus there was a higher probability of getting false negative results, and only molecular methods showed a 100% sensitivity for samples with low (1 PCRU) virus concentration. The overall concordance of the results between AGID and IAC-PCR was fair (ĸ = 0.40). Full concordance of the results was observed for OIE PCR and OIE real-time PCR when control reference material was analysed. Findings from this study suggest that AGID can be used with some limitations as a screening tool for detection of MYXV infections.

OBJECTIVE To determine the pharmacokinetics of florfenicol, terbinafine, and betamethasone acetate after topical application to canine auricular skin and the influence of synthetic canine cerumen on pharmacokinetics. SAMPLE Auricular skin from 6 euthanized shelter dogs (3 females and 3 neutered males with no visible signs of otitis externa). PROCEDURES Skin adjacent to the external opening of the ear canal was collected and prepared for use in a 2-compartment flow-through diffusion cell system to evaluate penetration of an otic gel containing florfenicol, terbinafine, and betamethasone acetate over a 24-hour period. Radiolabeled 14C-terbinafine hydrochloride and 3H-betamethasone acetate were added to the gel to determine dermal penetration and distribution. Florfenicol absorption was determined by use of high-performance liquid chromatography–UV detection. Additionally, the effect of synthetic canine cerumen on the pharmacokinetics of all compounds was evaluated. RESULTS During the 24-hour experiment, mean ± SD percentage absorption without the presence of synthetic canine cerumen was 0.28 ± 0.09% for 3H-betamethasone acetate, 0.06 ± 0.06% for florfenicol, and 0.06 ± 0.02% for 14C-terbinafine hydrochloride. Absorption profiles revealed no impact of synthetic canine cerumen on skin absorption for all 3 active compounds in the gel or on skin distribution of 3H-betamethasone acetate and 14C-terbinafine hydrochloride. CONCLUSIONS AND CLINICAL RELEVANCE 3H-betamethasone acetate, 14C-terbinafine hydrochloride, and florfenicol were all absorbed in vitro through healthy auricular skin specimens within the first 24 hours after topical application. Synthetic canine cerumen had no impact on dermal absorption in vitro, but it may serve as a temporary reservoir that prolongs the release of topical drugs.

OBJECTIVE To determine the pharmacokinetics of detomidine hydrochloride administered IV (as an injectable formulation) or by the oral-transmucosal (OTM) route (as a gel) and assess sedative effects of the OTM treatment in healthy dogs. ANIMALS 12 healthy adult dogs.PROCEDURES In phase 1, detomidine was administered by IV (0.5 mg/m2) or OTM (1 mg/m2) routes to 6 dogs. After a 24-hour washout period, each dog received the alternate treatment. Blood samples were collected for quantification via liquid chromatography with mass spectrometry and pharmacokinetic analysis. In phase 2, 6 dogs received dexmedetomidine IV (0.125 mg/m2) or detomidine gel by OTM administration (0.5 mg/m2), and sedation was measured by a blinded observer using 2 standardized sedation scales while dogs underwent jugular catheter placement. After a l-week washout period, each dog received the alternate treatment. RESULTS Median maximum concentration, time to maximum concentration, and bioavailability for detomidine gel following OTM administration were 7.03 ng/mL, 1.00 hour, and 34.52%, respectively; harmonic mean elimination half-life was 0.63 hours. All dogs were sedated and became laterally recumbent with phase 1 treatments. In phase 2, median global sedation score following OTM administration of detomidine gel was significantly lower (indicating a lesser degree of sedation) than that following IV dexmedetomidine treatment; however, total sedation score during jugular vein catheterization did not differ between treatments. The gel was subjectively easy to administer, and systemic absorption was sufficient for sedation. CONCLUSIONS AND CLINICAL RELEVANCE Detomidine gel administered by the OTM route provided sedation suitable for a short, minimally invasive procedure in healthy dogs.

Two agar gel immunodiffusion (AGID) kits for the serodiagnosis of bovine leukemia virus (BLV) were imported from Europe and were compared with North American kits. The BLV AGID kits from North America and from Europe differed significantly. The punches were different, as were the pattern distribution in the agar of the reference and the test sera, resulting in differences in the reading of the immunoprecipitation lines. Based on the testing of 1200 serum samples from cattle, the European kits gave a good correlation with the American kits, as indicated by their respective kappa values. However, the European kits were found to be less sensitive when evaluated against weakly positive samples from field specimens or following a dilution trial. Only 65% and 50% of the weakly positive samples detected by the American kit #1 were detected by the European kits #2 and #3, respectively. The American kit was also capable of detecting BLV antibodies in 45% of strongly positive samples diluted 1/50 in negative sera, while antibodies were detected in only 15% of the samples with the European kit #2 and in none of the samples with the European kit #3. False negatives were also detected with the European kits. Among the false negatives, the degree of expected reactions was weak (European kit #2) or of varying degrees of positivity (European kit #3). Besides the differences in format and performance, the BLV-AGID kits in Europe are evaluated with the National Standard Serum E4 while a proficiency panel composed of a quadruplicate set of 10 reference sera is used in Canada to monitor the kits. Based on the overall observations, we noted a lack of standardization between the BLV-AGID kits used in North America and in Europe.

Oral keratinocytes from dogs were cultured on either collagen gels or artificial matrices at the air-liquid interface, and the expression of keratinocyte antigens and basement membrane components was determined, using various monoclonal and polyclonal antibodies. Keratinocytes grown on collagen gels expressed pemphigus vulgaris, pemphigus foliaceous, and bullous pemphigoid antigens. Diffuse, suprabasal, and superficial keratinocyte membrane differentiation antigens identified by various monoclonal antibodies also were expressed in a pattern identical to that observed in the native tissue. Laminin and type-IV collagen were deposited at the keratinocyte-collagen interface in a patchy distribution. When synthetic matrices were used, the oral keratinocytes differentiated, but to a lesser extent than cells grown on collagen gels. Antigen expression for cells grown on synthetic matrices was similar to that for cells on collagen, except for failure of the keratinocytes on synthetic membranes to express superficial cell antigens and pemphigus foliaceous antigens.

OBJECTIVE To evaluate hemodynamic, respiratory, and sedative effects of buccally administered detomidine gel and reversal with atipamezole in dogs. ANIMALS 8 adult purpose-bred dogs. PROCEDURES Arterial and venous catheters were placed. Baseline heart rate, respiratory rate, cardiac output (determined via lithium dilution with pulse contour analysis), oxygen delivery, systemic vascular resistance, arterial blood gas values, and sedation score were obtained. Detomidine gel (2.0 mg/m2) was administered on the buccal mucosa. Cardiopulmonary data and sedation scores were obtained at predetermined times over 180 minutes. Atipamezole (0.1 mg/kg) was administered IM at 150 minutes. Reversal of sedation was timed and scored. Data were analyzed with an ANOVA. RESULTS Compared with baseline values, heart rate was lower at 45 to 150 minutes, cardiac output and oxygen delivery were lower at 30 to 150 minutes, and systemic vascular resistance was increased at 30 to 150 minutes. There were no significant changes in Paco2, Pao2, or lactate concentration at any time point, compared with baseline values, except for lactate concentration at 180 minutes. All dogs became sedated; maximum sedation was detected 75 minutes after administration of detomidine. Mean ± SD time to recovery after atipamezole administration was 7.55 ± 1.89 minutes; sedation was completely reversed in all dogs. No adverse events were detected. CONCLUSIONS AND CLINICAL RELEVANCE Buccally administered detomidine gel was associated with reliable and reversible sedation in dogs, with hemodynamic effects similar to those induced by other α2-adrenoceptor agonists. Buccally administered detomidine gel could be an alternative to injectable sedatives in healthy dogs.

8OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture. SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4). PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens from freshly harvested equine lungs and from healthy and heaves-affected horses. Immunologic techniques were used to evaluate the contractile phenotype of ASM cells in culture. RESULTS Characteristic ASM cells were successfully cultured from endobronchial tissue or biopsy specimens from both healthy and heaves-affected horses, and their contractile phenotype was maintained for up to 7 passages. Moreover, the capacity of cells at the seventh passage to contract in a collagen gel in response to methacholine was maintained. CONCLUSIONS AND CLINICAL RELEVANCE ASM cells isolated from equine endobronchial tissue and biopsy specimens were able to maintain a contractile phenotype in long-term cell cultures, suggesting they could be used for tissue engineering and in vitro studies of equine ASM cells.

OBJECTIVE To determine the prevalence of naturally occurring anti-dog erythrocyte antigen (DEA) 7 antibodies in DEA 7–negative dogs from Spain and Italy. ANIMALS 252 DEA 7–negative dogs from a population of 312 dogs that were previously tested for DEA 1, DEA 4, and DEA 7. PROCEDURES A plasma sample was obtained from each dog and evaluated for anti-DEA 7 antibodies by the use of gel column agglutination. Each plasma sample underwent major crossmatching with RBCs from DEA 7-positive dogs. Samples that resulted in agglutination were then crossmatched with RBCs from DEA 1-negative, DEA 4-positive, and DEA 7–negative dogs to confirm the presence of anti-DEA 7 antibodies. Results were then used to calculate the risk for a delayed transfusion reaction in a DEA 7–negative dog with anti-DEA 7 antibodies after a transfusion with blood that was not crossmatched or typed for DEA 7. RESULTS 96 of 252 (38.1%) plasma samples contained anti-DEA 7 antibodies. A DEA 7–negative dog with anti-DEA 7 antibodies had a 5.9% chance of developing a delayed hemolytic reaction after transfusion with blood not crossmatched or typed for DEA 7. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that canine blood used for transfusion should be crossmatched with the blood or plasma of the intended recipient prior to transfusion to minimize the likelihood that the recipient will develop a hemolytic reaction associated with anti-DEA 7 antibodies. Ideal canine blood donors should be negative for both DEA 1 and DEA 7.

The purpose of this research was to study changes in the salivary proteome of healthy pigs in stressful situations to identify any potential new salivary biomarker of stress. Three groups of animals were subjected to 3 stress models: snaring restraint followed by simulated sampling of vena cava blood; brief transport by road; and restriction of movement in a digestibility cage. Saliva was obtained from each animal before and 15 and 30 min after the induction of stress. The samples from the animals that showed the greatest increase in salivary cortisol concentration were pooled and run on 2-dimensional gels. Coomassie Brilliant Blue R-250 was used for spot detection and mass spectrometry for spot identification. Statistical analyses showed that 2 proteins had significant differences in expression before and after the induction of stress. These proteins were identified as odorant-binding protein and fragments of albumin. Further studies will be necessary to confirm the value of using these proteins as salivary biomarkers of stress in pigs.