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 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 establish the maximum tolerated dose of Clostridium novyi-NT spores in tumor-bearing dogs and evaluate spore germination within tumors and tumor response. Animals-6 client-owned dogs. Procedures-A standard dose-escalation study was planned, with maximum tolerated dose defined as the highest dose at which 0 or 1 of 6 dogs had dose-limiting toxicoses (DLT). Dogs received 1 dose of C novyi-NT spores IV. Toxicoses were graded and interventions performed according to specific guidelines. Grade 3 or higher toxicosis or any toxicosis combination that substantially affected patient status was considered DLT. Clinical response was measured by use of response evaluation criteria in solid tumors at 28 days. Results-The first 2 dogs had DLT. The dose was decreased. Two of the next 4 dogs had DLT; therefore, dose administration was stopped because the study endpoint had been reached. The most common toxicosis was fever (n = 6 dogs). Two dogs developed abscesses (1 within a nasal carcinoma and 1 splenic abscess) attributable to C novyi-NT infection; both required surgical intervention. Clostridium novyi-NT was cultured from 1 of 6 tumors. Five dogs were available for response assessment (4 had stable disease; 1 had progressive disease). Conclusions and Clinical Relevance-Results indicated that C novyi-NT can germinate within tumors of dogs. Toxicosis, although common and sometimes severe, was manageable with treatment. Further studies in dogs with superficial tumors may allow for continued dose escalation and provide information for use in clinical trials in veterinary and human oncology.

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 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 tear film osmolality and electrolyte composition in healthy horses. ANIMALS 15 healthy adult horses. PROCEDURES Each horse was manually restrained, and an ophthalmic examination, which included slit-lamp biomicroscopy, indirect ophthalmoscopy, and a Schirmer tear test, was performed. Tear samples were collected from both eyes with microcapillary tubes 3 times at 5-minute intervals. The tear samples for each horse were pooled, and the osmolality and electrolyte concentrations were measured. The mean (SD) was calculated for each variable to establish preliminary guidelines for tear film osmolality and electrolyte composition in healthy horses. RESULTS The mean (SD) tear film osmolality was 283.51 (9.33) mmol/kg, and the mean (SD) sodium, potassium, magnesium, and calcium concentrations were 134.75 (10), 16.3 (5.77), 3.48 (1.97), and 1.06 (0.42) mmol/L, respectively. The sodium concentration in the tear film was similar to that in serum, whereas the potassium concentration in the tear film was approximately 4.75 times that of serum. CONCLUSIONS AND CLINICAL RELEVANCE Results provided preliminary guidelines with which tear samples obtained from horses with keratopathies can be compared. Measurement of tear film osmolality in these horses was easy and noninvasive. The tear film concentration of divalent cations was greater than expected and was higher than the divalent cation concentrations in the tear films of rabbits and humans. These data may be clinically useful for the diagnosis and monitoring of hyperosmolar ocular surface disease in horses.

Objective-To determine the degree of agreement between 2 analyzers for measurement of total CO2 concentration (ctCO2) in equine plasma. Animals-6 healthy untrained horses, 6 trained Standardbreds undergoing a simulated race protocol, and 135 trained Standardbreds at a racetrack. Procedures-Jugular venous blood samples were obtained from all horses. Two analyzers (commonly used analyzer A and less expensive analyzer B) were used to measure plasma ctCO2 in each sample. Validation of both analyzers was conducted in accordance with guidelines established by the Clinical and Laboratory Standards Institute and involved characterization of linearity, total analytic error, and bias estimation. Results-Total analytic error (instrument SD) was 0.58 mmol/L (coefficient of variation, 1.6%) and 0.49 mmol/L (coefficient of variation, 1.4%) for analyzers A and B, respectively, when measuring an aqueous standard containing 36.0 mmol of CO2/L. A 1 g/L decrease in plasma protein concentration corresponded to an increase in ctCO2 measured with analyzer B of 0.065 mmol/L. A difference plot indicated that analyzer B produced values 2.7% higher than analyzer A for 103 samples from the 6 trained and exercised Standardbreds (mean plasma protein concentration, 67 g/L). Conclusions and Clinical Relevance-Analyzer B provided adequate precision and linearity for measurement of ctCO2 from 5 to 40 mmol/L and was therefore suitable for measuring ctCO2 in equine plasma, provided allowances are made for changes in plasma protein concentration.

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 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 compare results of a commercially available device for oscillometrically measured blood pressure (OBP) with invasively measured blood pressure (IBP) in awake and anesthetized dogs. ANIMALS 19 adult dogs (mean ± SD body weight, 17.8 ± 7.5 kg). PROCEDURES Blood pressures were measured in dogs while they were awake and anesthetized with isoflurane. The OBP was recorded on a thoracic limb, and IBP was simultaneously recorded from the median caudal artery. Agreement between OBP and IBP was evaluated with the Bland-Altman method. Guidelines of the American College of Veterinary Internal Medicine (ACVIM) were used for validation of the oscillometric device. RESULTS In awake dogs, mean bias of the oscillometric device was −11.12 mm Hg (95% limits of agreement [LOA], −61.14 to 38.90 mm Hg) for systolic arterial blood pressure (SAP), 9.39 mm Hg (LOA, −28.26 to 47.04 mm Hg) for diastolic arterial blood pressure (DAP), and −0.85 mm Hg (LOA, −40.54 to 38.84 mm Hg) for mean arterial blood pressure (MAP). In anesthetized dogs, mean bias was −12.27 mm Hg (LOA, −47.36 to 22.82 mm Hg) for SAP, −3.92 mm Hg (LOA, −25.28 to 17.44 mm Hg) for DAP, and −7.89 mm Hg (LOA, −32.31 to 16.53 mm Hg) for MAP. The oscillometric device did not fulfill ACVIM guidelines for the validation of such devices. CONCLUSIONS AND CLINICAL RELEVANCE Agreement between OBP and IBP results for awake and anesthetized dogs was poor. The oscillometric blood pressure device did not fulfill ACVIM guidelines for validation. Therefore, clinical use of this device cannot be recommended.