Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by many plant species. Due to their toxicity PAs can pose a risk to human and animal health. To detect the toxic compounds in feed materials a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed. PAs were extracted with sulphuric acid and purified with cation exchange cartridges. A newly developed solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC-MS analysis. The developed method was validated according to SANTE/11945/2015 guidelines. The recovery was from 84.1% to 112.9%, the repeatability ranged from 3.0% to 13.6%, and the reproducibility was from 4.8% to 18.9%. A sensitive and selective method for determination of PAs in feed materials has been developed and validated. All evaluated validation parameters were in accordance with EU Reference Laboratories document no. SANTE/11945/2015. Almost 41% of the analysed feed samples were positive for the presence of at least one PA.

OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation. SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds. PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44+, CD105+, and major histocompatability complex class II (MHCII; CD44+-CD105+-MHCII+ cells and CD44+-CD105+-MHCII− cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media. RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44+-CD105+-MHCII− CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates. CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.

Objective: To determine the tissue depletion profile of tulathromycin and determine an appropriate slaughter withdrawal interval in meat goats after multiple SC injections of the drug. Animals: 16 healthy Boer goats. Procedures: All goats were administered tulathromycin (2.5 mg/kg, SC) twice, with a 7-day interval between doses. Blood samples were collected throughout the study, and goats were euthanized at 2, 5, 10, and 20 days after the second tulathromycin dose. Lung, liver, kidney, fat, and muscle tissues were collected. Concentrations of tulathromycin in plasma and the hydrolytic tulathromycin fragment CP-60,300 in tissue samples were determined with ultrahigh-pressure liquid chromatography–tandem mass spectrometry. Results: The plasma profile of tulathromycin was biphasic. Absorption was very rapid, with maximum drug concentrations (1.00 ± 0.42 μg/mL and 2.09 ± 1.77 μg/mL following the first and second doses, respectively) detected within approximately 1 hour after injection. Plasma terminal elimination half-life of tulathromycin was 61.4 ± 14.1 hours after the second dose. Half-lives in tissue ranged from 2.4 days for muscle to 9.0 days for lung tissue; kidney tissue was used to determine the withdrawal interval for tulathromycin in goats because it is considered an edible tissue. Conclusions and Clinical Relevance: On the basis of the tissue tolerance limit in cattle of 5 ppm (μg/g), the calculated withdrawal interval for tulathromycin would be 19 days following SC administration in goats. On the basis of the more stringent guidelines recommended by the FDA, the calculated meat withdrawal interval following tulathromycin administration in goats was 34 days.

Objective—To determine whether selection for the homozygous A136R171 genotype that confers resistance to classic scrapie infection negatively affects production traits in sheep. Animals—996 commercial lambs obtained from 2 flocks at separate locations across 3 consecutive years. Procedures—Genotyping at codon 136 and 171 was performed by use of commercially available testing or a single-nucleotide polymorphism assay. Carcass data were collected without knowledge of genotype approximately 24 hours after slaughter by an experienced grader. The model to analyze associations between prion protein (PRNP) genotype and production traits was based on genotype, breed, or both as fixed effects and days on feed as a covariate. Results—Average daily gain was significantly associated with only combined codons 136 and 171. In flock 1, weaning average daily gain was significantly greater in AA136 sheep than heterozygotes; the difference between QR171 and RR171 sheep, compared with QQ171 sheep, were not significant although QR171 and RR171 sheep had higher values. However, in flock 2, average daily gain was significantly greater in AV136 sheep than AA136 sheep and in QR171 sheep than QQ171 sheep. Conclusions and Clinical Relevance—Findings suggest there is an advantage for average daily gain in lambs with an arginine allele at codon 171, but there were no other genotype effects on production traits. Thus, selection for the resistant arginine allele at codon 171 to comply with USDA scrapie eradication guidelines should not be detrimental to lamb production in commercial flocks. Effects of codon 136 on average daily gain were ambiguous.

Objective-To evaluate postexposure prophylaxis (PEP) in dogs experimentally infected with rabies. Procedure-29 Beagles. Procedure-Dogs were sedated and inoculated in the right masseter muscle with a salivary gland homogenate from a naturally infected rabid dog (day 0). Six hours later, 5 dogs were treated by administration of 2 murine anti-rabies glycoprotein monoclonal antibodies (mAb) and commercial vaccine; 5 received mAb alone; 5 received purified, heat-treated, equine rabies immune globulin (PHT-ERIG) and vaccine; 5 received PHT-ERIG alone; 4 received vaccine alone; and 5 control dogs were not treated. The mAb or PHTERIG was administered at the site of rabies virus inoculation. Additional vaccine doses for groups mAb plus vaccine, PHT-ERIG plus vaccine, and vaccine alone were administered IM in the right hind limb on days 3, 7, 14, and 35. Results-All control dogs and dogs that received only vaccine developed rabies. In the PHT-ERIG and vaccine group, 2 of 5 dogs were protected, whereas none were protected with PHT-ERIG alone. Use of mAb alone resulted in protection in 4 of 5 dogs. Administration of mAb in combination with vaccine provided protection in all 5 dogs. Conclusions and Clinical Relevance-Current national guidelines recommend euthanasia or a 6- month quarantine for unvaccinated animals exposed to rabies. Findings from this study document that vaccine alone following severe exposure was unable to provide protection from rabies. However, vaccine combined with mAb resulted in protection in all treated dogs, revealing the potential use of mAb in PEP against rabies in naïve dogs.

OBJECTIVE To evaluate coagulation factors in units of leukoreduced (LR) and nonleukoreduced (non-LR) canine fresh-frozen plasma (cFFP). ANIMALS 8 healthy research dogs. PROCEDURES In a crossover study, dogs were randomly assigned to 1 of 2 groups from which blood was collected and either did or did not undergo leukoreduction. After a recovery period of ≥ 28 days, the dogs were switched between protocols. After each collection, blood samples were centrifuged, and cFFP was stored frozen for later comparative analysis of coagulation factors, antithrombin, and protein C activities (reported as comparative percentages of the corresponding activities determined in a canine pooled plasma standard); prothrombin and activated partial thromboplastin times; and fibrinogen concentration. RESULTS There were no significant differences detected between results for LR cFFP, compared with those for non-LR cFFP. CONCLUSIONS AND CLINICAL RELEVANCE Although there was variation among residual activities of coagulation factors in LR and non-LR cFFP, the variations and differences were considered unlikely to impact the efficacy of LR cFFP transfused for coagulation factor replacement in dogs. However, owing to the small sample size and high variability of results in the present study, additional research with a larger sample size is required for definitive conclusions on the effects of leukoreduction on coagulation factors in cFFP and to develop treatment guidelines for LR cFFP use in dogs with congenital and acquired coagulopathies.

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.

OBJECTIVE To compare blood pressure measured noninvasively with an oscillometric device that involved use of a novel conical cuff and a traditional cylindrical blood pressure cuff. ANIMALS 17 adult hound-type dogs. PROCEDURES Dogs were anesthetized, and a 20-gauge, 1.5-inch catheter was inserted in the median sacral artery. The catheter was attached to a pressure transducer via fluid-filled noncompliant tubing, and direct blood pressure was recorded with a multifunction monitor. A specially fabricated conical cuff was placed on the antebrachium. Four sets of direct and indirect blood pressure measurements were simultaneously collected every 2 minutes. Four sets of measurements were then obtained by use of a cylindrical cuff. RESULTS The cylindrical cuff met American College of Veterinary Internal Medicine consensus guidelines for validation of indirect blood pressure measurements for mean arterial blood pressure (MAP), systolic arterial blood pressure (SAP), and diastolic arterial blood pressure (DAP). The conical cuff met the consensus guidelines for difference of paired measurements, SD, and percentages of measurements within 10 and 20 mm Hg of the value for the reference method, but it failed a correlation analysis. In addition, although bias for the conical cuff was less than that for the cylindrical cuff for SAP, MAP, and DAP measurements, the limits of agreement for the conical cuff were wider than those for the cylindrical cuff for SAP and MAP measurements. CONCLUSIONS AND CLINICAL RELEVANCE On the basis of results of this study, use of a conical cuff for oscillometric blood pressure measurement cannot be recommended.

OBJECTIVE To identify variations in glucose values concurrently obtained by use of a continuous glucose monitoring system (CGMS) at the same site, reliability of results for each site, lag time for each site, and influence of site thickness on CGMS accuracy. ANIMALS 8 random-source research dogs. PROCEDURES In experiment 1, 8 CGMS sensors were implanted bilaterally at 1 site (4 sensors/side) in 4 dogs. In experiment 2, 2 CGMS sensors were implanted bilaterally at each of 4 sites (1 sensor/side) in 8 dogs; 4 of those 8 dogs then were subjected to a glycemic clamp technique. The CGMS results were compared among sensors and with criterion-referenced results during periods of euglycemia for all 8 dogs and during hyperglycemia and hypoglycemia for 4 dogs during the glycemic clamp procedure. RESULTS Differences (median, −7 mg/dL; interquartile range [IQR], −18.75 to 3 mg/dL) between CGMS and criterion-referenced glucose concentrations differed significantly among dogs and sites; during euglycemia, they were not different from the expected normal variation between multiple sensors concurrently implanted at the same site. Differences (median, −35 mg/dL; IQR, −74 to −15 mg/dL) between CGMS and criterion-referenced concentrations were greater during changes in glucose concentrations. Thoracic sensors were most accurate but had the shortest mean functional life. CONCLUSIONS AND CLINICAL RELEVANCE Significant differences were detected between CGMS and criterion-referenced glucose concentrations. Overall clinical utility of CGMS was acceptable at all sites, with most of the values from all sensors, sites, and dogs meeting guidelines for point-of-care glucometers.

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.