Screening and mass spectral confirmation of beta-lactam antibiotic residues in milk using LC-MS/MS.

Milk is typically screened for beta-lactam antibiotics by nonspecific methods. Although these methods are rapid and sensitive, they are not quantitative and can yield false positive findings. A sensitive and specific method for the quantitation and mass spectral confirmation of five beta-lactam and two cephalosporin antibiotics commonly or potentially used in the dairy industry is described using high-performance liquid chromatography with tandem mass spectrometry. The antibiotics studied were ampicillin, amoxicillin, penicillin G, penicillin V, cloxacillin, cephapirin, and ceftiofur. The antibiotics were extracted from milk with acetonitrile, followed by reversed-phase column cleanup. The extract was analyzed by liquid chromatography coupled with a mass spectrometer, using a water/methanol gradient containing 1% acetic acid on a C-18 reversed-phase column. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Quantitation was based on the most abundant product ions from fragmentation of the protonated ion for amoxicillin, cephapirin, ampicillin, and ceftiofur and on the fragmentation of the sodium adduct for penicillin G, penicillin V, and cloxacillin. The method was validated at the U.S. FDA tolerance or safe level and at 5 or 2.5 ng/mL for these compounds in bovine milk. Theoretical method detection limits in milk based on a 10:1 signal to noise ratio were 0.2 ng/mL (ampicillin), 0.4 ng/mL (ceftiofur), 0.8 ng/mL (cephapirin), 1 ng/mL (amoxicillin and penicillin G), and 2 ng/mL (cloxacillin and penicillin V) using a nominal sample size of 5 mL.

Diagnosis and approach to poisoning in the horse.

Poisoning in the horse can present a highly complex case. The practitioner, owner, toxicologist, and pathologist play important roles, and all contribute information that may be important to the case. Once all the information is available, all the evidence is collected (historical, clinical, pathologic, and analytic), and proper sampling of specimens has occurred, a complete summary of the findings can be provided to the client. Based on identification of a potential toxic source and, ultimately, the diagnosis, specific treatment of affected animals and prevention of additional cases can be initiated. Consultation with a veterinary toxicologist aids in the follow-up of a poisoning case and can help to provide a thorough background to assist in preventing reoccurrence.

Botulism in the horse.

Botulism should be considered in cases where weakness, paralysis, or intolerance to exercise might be seen in the horse. Dysphagia may also be present, although it is not a consistent finding. Potential sources include carrion in hay, moldy or otherwise rotted vegetation or forage, birds carrying material from animal burial or other similar sites, and contaminated carcasses on-site. Horses, especially foals, may also suffer from toxicoinfectious botulism, a condition where the C. botulinum might colonize and produce toxin within the gastrointestinal tract. Wounds also may harbor the organism and otherwise promote botulism. Diagnosis of botulism is often a clinical diagnosis backed up by elimination of other possible infectious, injurious, or toxic causes of weakness of the horse. Definitive diagnosis and type identification in the laboratory are difficult and usually require a suitable sample of the source material. Treatment often is unrewarding unless a case is identified early and the proper antitoxin is readily available. Prevention involves common sense approaches to feeding and care of the horse and, where possible, judicious use of vaccination in endemic areas.

Diagnostic toxicology for the food animal practitioner.

In most competent veterinary diagnostic laboratories, analytic findings are interpreted by a veterinary toxicologist to determine the significance of the finding in light of historical, clinical, and pathologic findings. A veterinary toxicologist also provides consultation about possible toxic rule-outs for a case, treatment of affected animals, and prevention of additional cases. Once all of the information is available, a complete summary of the findings can be provided to the client. When the procedures outlined herein are followed, including a systematic approach to collecting all the evidence (historical, clinical, pathologic, and analytic), using proper sampling techniques, and maintaining good communication among the clinician, client, and laboratory, the usefulness of the toxicology investigation is maximized.

Comparison of two heavy metal chelators for treatment of lead toxicosis in cockatiels.

OBJECTIVE: To compare efficacy and safety of meso-2,3-dimercaptosuccinic acid (DMSA) and Ca EDTA for treatment of experimentally induced lead toxicosis in cockatiels (Nymphicus hollandicus). ANIMALS: 137 (69 females, 68 males) healthy cockatiels between 6 months and 8 years old. PROCEDURE: Lead toxicosis was induced by placing lead shot in the gastrointestinal tract. Treatment with Ca EDTA (40 mg/kg of body weight, IM, q 12 h), DMSA (40 or 80 mg/kg, PO, q 12 h), and sodium sulfate salts (SSS; 0.5 mg/kg, PO, q 48 h) was initiated 4 days after induction of lead toxicosis. Blood lead concentrations were determined, using atomic absorption spectrophotometry. Number of birds surviving and blood lead concentrations were compared among groups. RESULTS: In Phase II of the study, administration of DMSA and Ca EDTA significantly decreased blood lead concentrations when used alone or in combination in birds with lead toxicosis. Addition of SSS did not result in further decreases in lead concentrations. Eight of 12 (66.7%) birds without lead toxicosis given 80 mg of DMSA/kg did not survive to the end of the study. Lesions related to treatment with chelating agents were not detected during necropsy. CONCLUSIONS AND CLINICAL RELEVANCE: DMSA and Ca EDTA are effective chelating agents in cockatiels. Because DMSA is administered orally, it may be easier than other chelating agents for bird owners to administer at home. However, the narrow margin of safety of DMSA indicates that this agent should be used with caution.

Type C botulism in dairy cattle from feed contaminated with a dead cat.

Four hundred twenty-seven of 441 adult Holstein dairy cattle from a 1,200-cow dairy died over a 1-week period during early spring 1998. Affected animals were from 4 late lactation pens, one of which included the bull string. Signs included weakness, recumbency, watery diarrhea, and death. Eighty animals from the 4 pens were dead approximately 8 hours after the first ill cows were noted. Affected cows would collapse on stimulation and extend all 4 limbs with moderate rigidity. Several lacked lingual tonus and had abdominal breathing patterns. The animals had been fed a load of total mixed ration that included a rotten bale of oat hay containing a dead cat. No common toxicants were identified, and pathologic examination revealed no consistent lesions. Testing of tissue from the cat carcass found in the feed sample using mouse protection bioassay identified the presence of type C botulinum toxin. Samples of feed, tissue from affected animals, cat tissue from feed, milk, and serum were also tested using an enzyme-linked immunosorbent assay (ELISA) specific for type C botulinum. Two samples of rumen contents were tested and found to be positive for botulism by ELISA, and 1 of 3 liver samples had a weak positive finding. No botulinum toxin was found in milk or sera using the ELISA.

Multiresidue screen for cardiotoxins by two-dimensional thin-layer chromatography.

A two-dimensional thin-layer chromatographic method was developed for the qualitative determination of the cardiotoxins oleandrin, gitoxin, digitoxin, gitoxigenin, and grayanotoxins I, II, and III in gastrointestinal contents (stomach, rumen, colon, and cecum contents), feces, and plant material. The cardiotoxins were extracted with dichloromethane. The extract was cleaned up by charcoal and reverse phase solid-phase extraction columns. Analysis was performed by two-dimensional thin-layer chromatography on silica gel plates and visualized by aluminum chloride followed by chloramine T spray. The method detection limits were 0.05 microg/g for oleandrin, 0.1 microg/g for gitoxin, and 0.2 microg/g for the other toxicants in gastrointestinal contents and feces and were 5 times higher in plant material. Four replicate fortifications of bovine rumen contents, bovine feces, and alfalfa at these levels were all well recovered. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.

Selenium elimination in pigs after an outbreak of selenium toxicosis.

In May 1996, 150 grower pigs in 5 California counties were exposed to selenium-contaminated feed distributed by a single feed company. Feed samples from 20 herds had a mean selenium concentration of 121.7 ppm dry weight (range, 22.1-531 ppm). In San Luis Obispo County, 52 pigs in 24 herds were exposed to the feed, and 8 pigs died with signs of paralysis. Bilateral symmetrical poliomyelomalacia involving the ventral horns of the cervical and lumbar intumescence was evident on histologic examination of spinal cord from affected pigs. Of 44 surviving exposed pigs, 33 (75%) exhibited signs of selenosis, including anorexia, alopecia, and hoof lesions. Thirty-nine of 44 pigs (88.6%) had elevated (>1 ppm) blood selenium concentrations. Surviving exposed pigs were changed to a standard commercial ration containing approximately 0.5 ppm (dry weight) selenium. Blood selenium concentrations were determined weekly for 46 days following removal of the contaminated feed and were compared with values of 20 control pigs fed a standard commercial ration. Mean (+/-SD) blood selenium concentrations of exposed pigs were 3.2 +/- 2.6 ppm at the initial sampling and 0.4 +/- 0.1 ppm after 46 days. Mean blood selenium concentrations of < or = 0.3 ppm for control pigs at all samplings were significantly lower (P < 0.001) than concentrations for exposed pigs. Muscle and liver samples of 22 of the 44 exposed pigs were collected at slaughter approximately 72 days after withdrawal of the selenium-contaminated feed. Muscle samples had a mean selenium concentration of 0.36 ppm (wet weight). Liver samples had a mean selenium concentration of 1.26 ppm (wet weight). One liver sample had a selenium value in the toxic range for pigs (3.3 ppm wet weight; reference range, 0.4-1.2 ppm). A 1-compartment pharmacokinetic model of selenium elimination in exposed pigs was generated, and the geometric mean blood selenium elimination half-life was estimated to be 12 days. The 60-day withdrawal time recommended by the Food Animal Residue Avoidance Database was considered sufficient to allow safe human consumption of tissues from exposed pigs.

Normal and toxic zinc concentrations in serum/plasma and liver of psittacines with respect to genus differences.

Determination of zinc concentration in serum/plasma and tissue of caged and aviary birds is commonly requested by practitioners because of an increased awareness of zinc toxicity. However, interpretation of zinc levels is often based on normal zinc concentrations established for poultry. Also, it is likely that intergenus differences exist in normal zinc concentrations of pet birds. In an attempt to determine normal and toxic concentration ranges, zinc concentrations in liver (n = 276) and serum/plasma (n = 260) collected from psittacines between 1990 and 1998 were analyzed. Zinc concentrations were determined by inductively coupled argon plasma emission spectroscopy analysis. The results were categorized by genus and, when available, by history. Birds that were diagnosed with zinc toxicosis (on the basis of history, clinical examination, pathology, and laboratory findings) were exempt and not included in establishing normal ranges. The results indicate that important differences occur with respect to genera. For example, cockatoos and Eclectus parrots have higher normal zinc concentrations in serum or plasma than other psittacines. In addition, analysis of all the submitted cases suggests that potentially toxic zinc concentrations in livers of psittacines can be well below the range considered toxic in chickens (> 200 ppm).

Blue-green algae toxicosis in cattle.

Twenty-four of 175 heifers died after ingesting water from a stock pond containing blue-green algae (genus Microcystis) in southern Colorado. Affected cattle were found dead or had signs of nervousness, and were recumbent, weak, anorectic, and hypersensitive to noise when first examined. All cattle died within 3 days after the onset of signs. At necropsy, the rumen contained blue-green algae, and the liver was larger than normal, friable, and dark red. The most important histologic lesion was hepatocyte degeneration and necrosis. Intraperitoneal administration of lyophilized cell material from the bloom caused hepatic necrosis and death in mice, and water from the pond had clumps of cells surrounded by a clear calyx, consistent with the appearance of organisms of the genus Microcystis. Samples of pond water were examined by means of high-pressure liquid chromatography; microcystin-LR, one of the hepatotoxins produced by Microcystis spp, was found. Chromatography may be useful in the diagnosis of blue-green algae toxicosis.

Comparison of disease in calves dosed orally with oak or commercial tannic acid.

Commercial tannic acid has been used as a substitute for leaves and acorns in studies of oak toxicosis in some species. The toxicity of a commercial tannic acid given orally to calves was determined, and the clinical signs, laboratory findings, and pyrogallol production were compared with those found in calves dosed orally with oak leaves. The oak-fed calves developed the clinical signs and lesions characteristic of renal failure. Proteinuria developed by 48 hours in 1 calf and by 72 hours in the other calf. Both calves developed hematuria on day 4 and glucosuria on day 5. The blood urea nitrogen and creatinine values increased markedly on day 6. Pyrogallol was detected in the serum only at 3 and 6 hours after the calves began ingesting the oak leaves. Pyrogallol was detected in urine from 1 calf until 60 hours and in the other calf until 48 hours after the beginning of oak intake. The 2 calves that were dosed with tannic acid at the same level as found in the leaves fed to the other calves did not develop clinical signs, abnormal laboratory findings, or pyrogallol production. Calves given high levels of tannic acid at doses of 4.4-5.5 g/kg developed methemoglobinemia rather than renal disease. Therefore, commercial tannic acid given orally cannot be used as a substitute for oak in studies of toxicosis in cattle.

Sweet clover poisoning in dairy cattle in California.

Eight of 600 Holstein heifers and cows died after ingestion of sweet clover silage (Melilotus sp) that contained excessive concentrations of dicumarol caused by mold infestation. The cattle developed subcutaneous hemorrhages and bled from the vagina, became weak, were unable to move, and died. To the best of our knowledge, this is the first report of sweet clover poisoning in cattle from California and is discussed in light of previous findings in the Midwest and Canada. Sweet clover poisoning is caused by dicumarol, a fungal metabolite produced from substrates in sweet clover, and is a common livestock problem in the Northern Plains and Canada. Sweet clover poisoning should be considered in livestock animals with clinical evidence of hemostatic dysfunction, prolonged coagulation times, subcutaneous hemorrhages, and hemorrhagic abortions. Definite diagnosis of moldy sweet clover poisoning can be accomplished by analysis of serum and feed samples for dicumarol concentrations.

Comparison of nicotinic receptor binding and biotransformation of coniine in the rat and chick.

Coniine, an alkaloid from Conium maculatum (poison hemlock), is a known teratogen in many domestic species with maternal ingestion resulting in arthrogryposis of the offspring. We have previously shown that rats are not susceptible and rabbits only weakly susceptible to coniine-induced arthrogryposis. However, the chick embryo does provide a reproducible laboratory animal model of coniine-induced teratogenesis. The reason for this cross-species variation is unknown. The purpose of this study was to evaluate coniine binding to nicotinic receptors and to measure coniine metabolism in vitro between susceptible and non-susceptible species. Using the chick model, neither the peripheral nicotinic receptor antagonist d-tubocurarine chloride nor the central nicotinic receptor antagonist trimethaphan camsylate blocked the teratogenesis or lethality of 1.5% coniine (50 microliters/egg). Trimethaphan camsylate enhanced coniine-induced lethality in a dose-dependent manner. Neither nicotinic receptor blocker prevented nicotine sulfate-induced malformations but d-tubocurarine chloride did block lethality in a dose-dependent manner. Competition by coniine for [125I]-alpha-bungarotoxin to nicotinic receptors isolated from adult rat diaphragm and chick thigh muscle and competition by coniine for [3H]-cytisine to receptors from rat and chick brain were used to assess coniine binding to nicotinic receptors. The IC50 for coniine in rat diaphragm was 314 microM while that for chick leg muscle was 70 microM. For neuronal nicotinic receptors, the IC50s of coniine for maternal rat brain, fetal rat brain, and chick brain were 1100 microM, 820 microM, and 270 microM, respectively. There were no differences in coniine biotransformation in vitro by microsomes from rat or chick livers. Differences in apparent affinity of coniine for nicotinic receptors or differences in the quantity of the nicotinic receptor between the rat and chick may explain, in part, the differences in susceptibility of coniine-induced teratogenesis between these two species.

Primary photosensitization related to ingestion of alfalfa silage by cattle.

A herd of 650 Holstein cows was examined for skin disease. Approximately 400 of the lactating adults were affected, but heifers, calves, and nonlactating cows were clinically normal. The condition was characteristic of primary photosensitization. Milk production of the affected cows was normal. Affected cows did not appear to be ill, and none of the cows was icteric. Three of 7 cows had high serum gamma-glutamyltransferase activities, but in the other 4 cows, activity was within the reference range. Serum activities of other hepatic enzymes were within reference ranges in the 7 cows that were examined. Hepatic biopsy specimens from 3 cows were normal. Specimens from 4 other cows had changes that ranged from minimal to mild, chronic, lymphoplasmacytic periportal hepatitis to acute, random, necrotizing hepatitis. Development of photosensitivity was related to ingestion of alfalfa silage. Acetone extracts of the alfalfa silage, but not of other feedstuffs, were found to inhibit growth of Candida albicans under ultraviolet light. Cows experimentally fed a diet composed exclusively of the alfalfa silage developed skin lesions after 6 days, but did not have detectable serum concentrations of phylloerythrin.

Diagnosis of oleander poisoning in livestock.

Since mid-1989, 37 cases of oleander poisoning in livestock have been diagnosed at the California Veterinary Diagnostic Laboratory System. The most frequent source for oleander exposure was plant clippings. Sudden death was the most common presenting complaint. Other signs reported included diarrhea, pulmonary edema, tachycardia, cardiac arrhythmias, colic, and lethargy. In the past, a presumptive diagnosis of oleander poisoning could be based only on matching clinical signs with evidence of consumption of oleander. A new 2 dimensional Thin-layer chromatography analysis of ingesta for oleandrin and an awareness of lesions in heart muscle have greatly improved the ability to diagnose oleander toxicosis.

Diagnostic and forensic toxicology.

In most competent veterinary diagnostic laboratories, analytical findings are interpreted by the veterinary toxicologist to determine the significance of the finding in view of historic, clinical, and pathologic findings. A veterinary toxicologist also will provide consultation about possible toxic rule-outs for a case, treatment of affected animals, and prevention of additional cases. Once all of the information is available, a complete summary of the findings can be provided to the client. When the procedures outlined are followed, including a systematic approach to collecting all the evidence (historic, clinical, pathologic, and analytic), proper sampling techniques, and good communication between the clinician and the client and laboratory, the usefulness of the toxicology investigation will be maximized.

A rapid multiresidue screen for organophosphorus, organochlorine, and N-methyl carbamate insecticides in plant and animal tissues.

A multiresidue screen for the quantitative determination of 43 organophosphorus, 17 organochlorine, and 11 N-methyl carbamate insecticides in 10 g of plant or animal tissues is described. The insecticides are extracted with 5% ethanol in ethyl acetate (v/v). Samples with high lipid content are cleaned up by automated gel permeation chromatography with a 30% ethyl acetate in hexane (v/v) eluant and in-line silica gel minicolumns. Highly pigmented samples are cleaned up with class-specific solid-phase extraction columns. The concentrated extracts are analyzed by selective detection with gas chromatography or liquid chromatography. Recovery of 71 insecticides ranged from 77 to 113%. Analysis of fortified bovine liver (n = 5) resulted in an average recovery of 96 +/- 4% at the 0.5 to 0.05 micrograms/g level. Analysis of fortified alfalfa hay (n = 5) resulted in a mean recovery of 94 +/- 4% at the 0.06 to 0.5 micrograms/g level, and analysis of fortified fresh tomatoes (n = 5) resulted in an average recovery of 97 +/- 3% at the 0.06 to 0.5 micrograms/g level. Method detection limits ranged from 0.02 to 0.5 micrograms/g for the compounds studied with a nominal 10 g sample.