A rapid screen for four corticosteroids in equine synovial fluid.

Most antidoping method development in the equine industry has been for plasma and urine, though there has been recent interest in the analysis of synovial fluid for evidence of doping by intra-articular corticosteroid injection. Published methods for corticosteroid analysis in synovial fluid are primarily singleplex methods, do not screen for all corticosteroids of interest and are not adequately sensitive. The purpose of this study is to develop a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening method for the detection of four of the most common intra-articularly administered corticosteroids--betamethasone, methylprednisolone, methylprednisolone acetate and triamcinolone acetonide. Sample preparation consisted of protein precipitation followed by a basified liquid-liquid extraction. LC-MS-MS experiments consisted of a six-min isocratic separation using a Phenomenex Polar-RP stationary phase and a mobile phase consisting of 35% acetonitrile, 5 mM ammonium acetate and 0.1% formic acid in nanopure water. The detection system used was a triple quadrupole mass analyzer with thermospray ionization, and compounds were identified using selective reaction monitoring. The method was validated to the ISO/IEC 17025 standard, and real synovial fluid samples were analyzed to demonstrate the application of the method in an antidoping context. The method was highly selective for the four corticosteroids with limits of detection of 1-3 ng/mL. The extraction efficiency was 50-101%, and the matrix effects were 14-31%. These results indicate that the method is a rapid and sensitive screen for the four corticosteroids in equine synovial fluid, fit for purpose for equine antidoping assays.

Detection of diisocyanates in nesting material associated with mortality in pigeon chicks.

Diisocyanates, commonly used in the production of polyurethane foams, paints, elastomers, varnishes, and coatings, are considered among the most hazardous inhalation toxicants. The present report describes 2 unusual cases of mortality in pigeon chicks associated with nesting material contaminated by diisocyanates. Case 1 was submitted by a racing pigeon breeder who had lost all the hatchlings (n = 125) following replacement of the nesting material with a different lot. All adult birds appeared healthy, and hatchability was not significantly affected, but hatchlings became lethargic and dyspneic after a day of hatch. At necropsy, dark wet lungs were found in the hatchlings. Case 2 was submitted by a show-roller pigeon breeder. In this case, the owner reported lower hatchability, and all hatchlings (approximately 100) died within 2 days of hatching with clinical signs similar to the first case. Necropsy did not reveal any significant findings. For both cases, nesting materials were screened for toxic compounds using gas chromatography-mass spectrometry. Toluene-2,4-diisocyanate (approximately 190-290 ppm) and 4,4'-methylene diphenyl diisocyanate (unquantified) were detected in the nesting pads. While there is very limited information on toxicosis in birds, there are reports of inhalant exposure of diisocyanates causing pulmonary edema and death in various mammalian species. Although cause-effect relationship of mortality and the nesting material was not established in the present cases, the presence of toxic compounds in the nesting materials is a cause for concern. Further investigation is needed to determine the prevalence and toxicity of diisocyanates-contaminated nesting material in avian species.

Lead excretion in milk of accidentally exposed dairy cattle.

Lead (Pb) exposure in dairy cattle is associated with economic losses due to mortality and treatment costs, but with production animals there is also risk to the human food chain. The first objective of this study was to quantify the Pb concentration in milk from Pb-exposed cattle. The second objective was to correlate blood and milk Pb concentrations from individual cows. The third objective was long-term monitoring to determine the duration of milk contamination after exposure ceased. A dairy herd of more than 100 cows was accidentally exposed to Pb-contaminated feed. Milk and blood were collected for Pb analysis. Serial collection of milk samples continued for 2.5 years. The initial concentration of Pb in bulk tank milk was 0.0999 mg l⁻¹. The highest milk Pb concentration from an individual cow was 0.4657 mg l⁻¹ and the highest blood Pb concentration was 1.216 mg l⁻¹. One milk sample collected at the end of the study (day 922) contained 0.0117 mg Pb l⁻¹ of Pb. The calculated relationship between milk (y) and blood (x) Pb concentration was ln(y) = 3.4(x) - 2.21 (R² = 0.98).

Identification of protoxins and a microbial basis for red maple (Acer rubrum) toxicosis in equines.

The leaves of Acer rubrum (red maple), especially when wilted in the fall, cause severe oxidative damage to equine erythrocytes, leading to potentially fatal methemoglobinemia and hemolytic anemia. Gallic acid and tannins from A. rubrum leaves have been implicated as the toxic compounds responsible for red maple toxicosis, but the mechanism of action and toxic principle(s) have not been elucidated to date. In order to investigate further how red maple toxicosis occurs, aqueous solutions of gallic acid, tannic acid, and ground dried A. rubrum leaves were incubated with contents of equine ileum, jejunum, cecum, colon, and liver, and then analyzed for the metabolite pyrogallol, as pyrogallol is a more potent oxidizing agent. Gallic acid was observed to be metabolized to pyrogallol maximally in equine ileum contents in the first 24 hr. Incubation of tannic acid and A. rubrum leaves, individually with ileum contents, produced gallic acid and, subsequently, pyrogallol. Ileum suspensions, when passed through a filter to exclude microbes but not enzymes, formed no pyrogallol, suggesting a microbial basis to the pathway. Bacteria isolated from ileum capable of pyrogallol formation were identified as Klebsiella pneumoniae and Enterobacter cloacae. Therefore, gallotannins and free gallic acid are present in A. rubrum leaves and can be metabolized by K. pneumoniae and E. cloacae found in the equine ileum to form pyrogallol either directly or through a gallic acid intermediate (gallotannins). Identification of these compounds and their physiological effects is necessary for the development of effective treatments for red maple toxicosis in equines.

Declines in blood lead concentrations in clinically affected and unaffected cattle accidentally exposed to lead.

Lead (Pb) poisoning remains a common cause of morbidity in dairy and beef cattle. Although Pb toxicosis is typically diagnosed in cattle with clinical signs of acute or subacute Pb poisoning, it has been hypothesized that subclinical chronic exposure of cattle to Pb, which often goes undiagnosed, poses more of a risk to the human consumer. There is not adequate information on Pb kinetics to determine when or if Pb-exposed cattle can safely enter the food chain. The objectives of the current study were to determine whether subclinical elevations in blood Pb (bPb) were present in cattle from herds where 1 or more individuals had clinical Pb poisoning and to determine the half-life (t(1/2)) of bPb in Pb-exposed cattle. Samples of blood were collected and analyzed for Pb from 126 cattle from 9 farms. Blood lead concentrations ranged from below the detection limit (2.50 µg/dl) to 423.0 µg/dl. Only 11 of the 94 cattle with detectable bPb had clinical signs such as diarrhea, blindness, bruxism, or seizures. When possible, cattle with detectable bPb had serial samples taken. The mean t(1/2) calculated from 44 serially sampled cattle was 135 days (standard deviation: 125 days, range: 3-577 days). A source of Pb on the farm was determined for all but one herd.

An unusual case of relay pentobarbital toxicosis in a dog.

Sodium pentobarbital and phenytoin are common constituents of veterinary euthanasia solutions in the United States. Relay, or secondary, barbiturate toxicosis has been reported in carnivorous animals that have fed from the carcasses of euthanized livestock. This case report presents barbiturate toxicosis in a dog. A 2-year-old female spayed Australian shepherd presented comatose 2 h after ingesting an unknown substance on the beach. The material was retrieved from the stomach by gastric lavage and visually identified as fish or other animal tissue. The dog recovered with symptomatic and supportive therapy and was released on the third day of hospitalization. Tissue found on the beach near where the dog walked and a urine sample from the dog were analyzed by gas chromatography/mass spectrometry. Both samples were positive for pentobarbital and phenytoin. The tissue was consistent with mammalian blubber based on gross and histological examination. Three weeks previously, a juvenile humpback whale had stranded on the beach where the dog had ingested the unknown substance. The whale had been euthanized with a barbiturate solution, necropsied, and removed from the beach. It was not definitively determined that the pentobarbital-containing blubber ingested by the dog was from the euthanized whale, but that was the most likely source. Although attempts were made to remove the whale's remains from the beach, practical considerations made complete removal challenging, if not impossible.

Comparison of blood lead and blood and plasma δ-aminolevulinic acid concentrations as biomarkers for lead poisoning in cattle.

Lead (Pb) concentrations in whole blood and δ-aminolevulinic acid (ALA) concentrations in plasma and whole blood from 37 cattle with suspected Pb exposure were determined in order to investigate the usefulness of ALA as a biological indicator for Pb poisoning in cattle. Cows were divided into 4 groups based on blood Pb, as follows: <30 ppb (group 1), 30-100 ppb (group 2), 100-300 ppb (group 3), and >300 ppb (group 4). The derivatization reaction for ALA was improved by a greater than 2-fold measure in whole blood and by a 10-fold measure in plasma by adding 75 and 50 µl of 0.1 N HCl, respectively. Blood Pb concentrations ranged from <25 ppb to 1,006 ppb (185.5 ± 254.9 ppb), with 17 samples containing >50 ppb Pb. Delta-aminolevulinic acid concentrations in whole blood and plasma ranged from <62.7 ppb to 96.9 ppb (77.4 ± 8.4 ppb) and from <5.0 ppb to 24.0 ppb (4.6 ± 3.8 ppb), respectively. Whole blood ALA did not correlate with blood lead concentrations in any group. Increase in plasma ALA concentration was dependent on blood Pb concentration. There was no correlation between blood Pb concentration and plasma ALA concentration in group 2 (n  =  4), but correlation coefficients were 0.736 in group 3 and 0.807 in group 4, respectively. The correlation coefficient was increased to 0.851 when groups 3 and 4 were combined. Based on these observations, in cattle, plasma ALA is a more reliable biological biomarker for Pb exposure than is blood ALA.

Comparison of two methods for blood lead analysis in cattle: graphite-furnace atomic absorption spectrometry and LeadCare(R) II system.

The current study compared the LeadCare(R) II test kit system with graphite-furnace atomic absorption spectrometry for blood lead (Pb) analysis in 56 cattle accidentally exposed to Pb in the field. Blood Pb concentrations were determined by LeadCare II within 4 hr of collection and after 72 hr of refrigeration. Blood Pb concentrations were determined by atomic absorption spectrometry, and samples that were coagulated (n = 12) were homogenized before analysis. There was strong rank correlation (R(2) = 0.96) between atomic absorption and LeadCare II (within 4 hr of collection), and a conversion formula was determined for values within the observed range (3-91 mcg/dl, although few had values >40 mcg/dl). Median and mean blood pb concentrations for atomic absorption were 7.7 and 15.9 mcg/dl, respectively; for LeadCare II, medians were 5.2 mcg/dl at 4 hr and 4.9 mcg/dl at 72 hr, and means were 12.4 and 11.7, respectively. LeadCare II results at 4 hr strongly correlated with 72 hr results (R(2) = 0.96), but results at 72 hr were lower (P < 0.01). There was no significant difference between coagulated and uncoagulated samples run by atomic absorption. Although there have been several articles that compared LeadCare with other analytical techniques, all were for the original system, not LeadCare II. The present study indicated that LeadCare II results correlated well with atomic absorption over a wide range of blood Pb concentrations and that refrigerating samples for up to 72 hr before LeadCare II analysis was acceptable for clinical purposes.

Clinicopathologic, histologic, and toxicologic findings in 70 cats inadvertently exposed to pet food contaminated with melamine and cyanuric acid.

OBJECTIVE: To document clinicopathologic, histologic, and toxicologic findings in cats inadvertently exposed to pet food contaminated with melamine and cyanuric acid. DESIGN: Case series. ANIMALS: 70 cats from a single cattery inadvertently fed contaminated food that was the subject of a March 2007 recall. PROCEDURES: Clinical signs, clinicopathologic and histopathologic findings, and results of toxicologic analyses were recorded. RESULTS: Clinical signs were identified in 43 cats and included inappetence, vomiting, polyuria, polydipsia, and lethargy. Azotemia was documented in 38 of the 68 cats for which serum biochemical analyses were performed 7 to 11 days after consumption of the contaminated food. One cat died, and 13 were euthanized. Histologic examination of kidney specimens from 13 cats revealed intratubular crystalluria, tubular necrosis with regeneration, and subcapsular perivascular inflammation characterized by perivascular fibroplasia or fibrosis and inflammation with intravascular fibrin thrombi. Toxicologic analyses revealed melamine and cyanuric acid in samples of cat food, vomitus, urine, and kidneys. CONCLUSIONS AND CLINICAL RELEVANCE: In cats unintentionally fed pet food contaminated with melamine and cyanuric acid, the most consistent clinical and pathologic abnormalities were associated with the urinary tract, specifically tubular necrosis and crystalluria.

Assessment of acute injuries, exposure to environmental toxins, and five-year health surveillance of New York Police Department working dogs following the September 11, 2001, World Trade Center terrorist attack.

OBJECTIVE: To determine deployment logistics of New York Police Department (NYPD) working dogs that assisted in relief efforts at the World Trade Center (WTC) site following the September 11, 2001, terrorist attack; establish types and rates of related acute injuries and illnesses; identify environmental toxin exposures; and determine long-term (ie, 5-year) health effects of deployment. DESIGN: Prospective cohort study. ANIMALS: 27 working dogs. PROCEDURES: Deployment logistics for the period from September 11, 2001, through May 30, 2002, were determined, and acute health disorders were identified by means of physical examination; a questionnaire; interviews with dog handlers; and toxicologic (blood and hair samples), clinicopathologic, microbiologic (nasal swab specimens submitted for Bacillus anthracis culture), and radiographic methods. Long-term health surveillance ended September 21, 2006. RESULTS: Dogs worked a total of 1,428 days (15,148 hours) at the site. Seventeen of the 27 (62.9%) dogs had health disorders during the first week. Specific conditions included fatigue (incidence rate [events/1,000 active deployment hours], 13.1), conjunctival irritation (13.1), respiratory tract problems (12.4), decreased appetite (10.8), dehydration (10), and cuts (9.3). Only minor hematologic and serum biochemical abnormalities were identified. Bacterial culture of nasal swab specimens did not yield B anthracis. Only mild and infrequent health conditions were identified during the 5-year follow-up period. None of the dogs were identified as having chronic respiratory tract disease. Six dogs died of various causes. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that acute injuries and illnesses were common among NYPD working dogs deployed to the WTC disaster site, but that longterm health complications were minimal.

Rapid forensic selected reaction monitoring liquid chromatography/mass spectrometry determination of ionophore antibiotics found at toxic levels in animal feeds.

A rapid, accurate, and selective method was developed for the forensic determination of ionophore antibiotics in animal feeds. A simple extraction procedure and liquid chromatography/tandem mass spectrometry (LC/MS/MS) in the selected reaction monitoring (SRM) mode were used for rapid identification and confirmation of monensin and lasalocid in feed samples and for quantitation of monensin. Extracts from a homogenous portion of ground feeds were prepared using liquid-solid extraction and liquid-liquid extraction techniques. Feed extracts were further purified by a simple defatting and solvent wash step and then concentrated to dryness. Feed extract residues were reconstituted in 1 mL LC mobile phase and a 2 microL aliquot injected into the SRM LC/MS system. The latter system used a C18, 100 x 2.0 mm, LC column coupled to a PE-Sciex API 2000 tandem triple quadrupole mass spectrometer equipped with a TurbolonSpray LC/MS interface. Feed samples were extracted and analyzed for the determination of monensin and lasalocid within a couple of hours. Control feed samples fortified with monensin at concentrations from 50 ppb to 5 ppm provided a linear response and calibration curve across this range with a correlation coefficient of 0.996.