Therapeutic efficacy of superactive charcoal in rats exposed to oral lethal doses of T-2 toxin.

Superactive charcoal, a compound known to complex with many toxins, was evaluated in this study for its effectiveness in preventing death in rats given an oral lethal dose of 8 mg/kg body weight of T-2 toxin. The median effective dose of oral superactive charcoal in preventing deaths in rats was 0.175 g/kg body weight. Concurrent use of cathartics, such as sorbitol, magnesium sulfate and sodium sulfate, to facilitate removal of the superactive charcoal:T-2 toxin complex formed in vivo did not enhance the survival rates of rats. One gram per kilogram body weight oral superactive charcoal enhanced survival times and survival rates in rats given 8 mg/kg of T-2 toxin as late as 3 hr after the T-2 toxin was administered. Some benefit in survival rate may be derived from giving the superactive charcoal as late as 5 hr after the T-2 toxin.

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.

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.

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.

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.

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).

Toxicosis in dairy cattle exposed to poison hemlock (Conium maculatum) in hay: isolation of Conium alkaloids in plants, hay, and urine.

Cattle in two herds developed signs of bloating, increased salivation and lacrimation, depression, respiratory distress, ataxia, and death after ingestion of hay that contained large amounts of poison hemlock (Conium maculatum). Twenty of 30 Angus cows and calves were affected in the first herd (2 died). In the second herd, 5 of 30 Holstein heifers were affected (1 died). The Conium alkaloids, coniine and gamma-coniceine, were quantified in the hay, the plants from the responsible hayfield, and the urine of affected animals.

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.

Clinical and pathologic findings of blue-green algae (Microcystis aeruginosa) intoxication in a dog.

A healthy dog developed signs of lethargy and vomiting after ingesting water from a tide pool containing blue-green algae. Fulminant hepatic failure occurred, and the dog was euthanized 52 hours later. At necropsy, the liver was large, friable, and discolored a dark red. Histopathology showed hepatocyte dissociation, degeneration, and necrosis. The alga was identified as Microcystis aeruginosa, a known hepatotoxin. The intraperitoneal administration of lyophilized cell material from the bloom caused hepatic necrosis in mice.

Effect of time and storage temperature on cholinesterase activity in blood from normal and organophosphorus insecticide-treated horses.

Delays between time of sampling and time of testing are common; therefore, the length of time that blood can be stored at various temperatures was evaluated for effects on cholinesterase activity. Six horses were treated with 16 g of trichlorfon per os, 6 horses were treated with 15 g of dichlorvos per os, and 10 horses were untreated controls. The cholinesterase activity in whole blood from each horse was measured using an adaptation of the Ellman colorimetric method. The blood from each horse was then divided into 3 groups and stored at 5 C (refrigerated), 20 C (room temperature), or 38 C (incubated). Subsequent cholinesterase activities were measured daily and then at weekly intervals. The cholinesterase activities did not significantly increase or were measured daily and then at weekly intervals. The cholinesterase activities did not significantly increase or decrease (P > 0.05) in the blood from the untreated horses until after 1 week for any of the 3 temperature groups. The cholinesterase activities did not significantly increase or decrease (P > 0.05) in the stored blood from the trichlorfon-treated horses for 4 weeks in all 3 temperature groups. The cholinesterase activities significantly increased (P < 0.05) in the stored blood from the dichlorvos-treated horses after 1 week when the blood was refrigerated and by 24 hours when the blood was stored at room temperature or incubated. Therefore, blood from normal or organophosphate-treated horses can be used for cholinesterase evaluation for up to 1 week when stored at 5 C.

Type C botulism in turkeys: determination of the median toxic dose.

The median toxic dose (TD50) of type C botulinum in turkeys was determined using an up-and-down method for toxicologic testing. Birds were dosed intravenously with 10, 20, or 40 mouse lethal dose per kilogram body weight (MLD/kg) of type C botulism toxin and observed for 5 days. Administration of toxin resulted in acute death, posterior paresis, or no effect, depending upon the dose. The TD50 causing posterior paresis was calculated as 25.75 MLD/kg (confidence interval = 17.08-38.82 MLD/kg). Posterior paresis was identified as the principal sign associated with sublethal botulism toxicosis in turkeys. The resultant posterior paresis was similar to the clinical syndrome observed in an unsolved field investigation.

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.

Chemical study of the hepatotoxins from Microcystis aeruginosa collected in California.

Four cyclic peptide toxins were purified and quantified from the aqueous extract of algal cell material utilizing high performance liquid chromatography, thin layer chromatography, and fast atom bombardment mass spectrometry. The cyclic peptide toxins appear to be similar structurally to hepatotoxins from previously identified blooms of the blue-green alga Microcystis aeruginosa.

Estrogenic activity in forages: diagnostic use of the classical mouse uterine bioassay.

The classical mouse uterine bioassay was evaluated and adapted for routine diagnostic use in response to requests for evaluation of forages suspected of being estrogenic. Forages were extracted in acetone or 10% ethanol in acetone (v/v). Extracts were mixed with ground corn-based mouse feed. Immature female mice (n = 3/group) were fed a total of 100 g of the ground feed for 5 days. Body weights were monitored before and after the trial. After 6 days, the mice were euthanized and uterine weights were determined. Mean uterine weights were compared using 1-way analysis of variance with preselected contrasts for individual means. Selected uteruses were fixed in 10% neutral buffered formalin for histologic examination. Control feeds, diethylstilbestrol (DES), estradiol, coumestrol, feeds with no reported estrogenic properties, and a feed that caused hyperestrogenism in cattle were tested. Moderate levels of estrogenic compounds resulted in dose-responsive uterine enlargements (10-270 ppm coumestrol over 5 days). Extremely high levels of estrogen frequently resulted in feed refusal and lack of uterine enlargement (10 ppm DES, 100 ppm estradiol). Diagnostically significant estrogenic activity was recovered from the feed known to have been estrogenic in cattle. The classical mouse uterine bioassay was relatively inexpensive, quick, repeatable, and capable of detecting clinically relevant coumestrol levels in hay.

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.

Lead concentrations in blood and milk from periparturient dairy heifers seven months after an episode of acute lead toxicosis.

In September 1988, 100 of 300 yearling dairy heifers developed blindness, tachypnea, foaming at the mouth, chewing, and facial fasciculations. Twenty-five animals died. Lead toxicosis was diagnosed based on the clinical signs and the presence of excessive concentrations of lead in whole blood, liver, kidney, and rumen contents of affected animals. The source of the lead was sudan grass silage that had been contaminated by soil that contained up to 77,000 mg/kg of lead. Lead concentrations were determined approximately 7 months after the acute episode of lead toxicosis. Whole blood and milk samples were obtained from heifers and a group of control cows 2 weeks prior to (blood only), at the time of, and 2 and 4 weeks after freshening. No lead was found in any of the milk samples (detection limit = 0.055 mg/liter). Animals that had been severely affected by lead toxicosis experienced a transient increase in whole blood lead concentrations at freshening that was not high enough to be considered toxic. No similar increases in blood lead were observed for control cows or heifers that had experienced milder toxicosis. These findings suggest that at parturition lead is mobilized into the blood of cattle previously exposed to excessive lead.

The correlation between serum selenium and blood selenium in cattle.

The selenium (Se) concentration of paired blood and serum samples from cattle was determined by 2 methods: 1) atomic absorption spectroscopy using hydride generation (HG-AAS), and 2) inductively coupled argon plasma emission spectroscopy using hydride generation (ICP). Samples from 327 cattle were analyzed by HG-AAS, and samples from 344 cattle were analyzed by ICP. The data were examined by linear regression analysis, and the technique of inverse prediction was utilized to determine prediction intervals for estimating blood Se concentration from known serum Se concentration. The correlation coefficients, by simple linear regression of serum Se on blood Se, were 0.79 (r2 = 0.62) and 0.88 (r2 = 0.77) for the HG-AAS data and the ICP data, respectively. For the HG-AAS data, the inverse prediction formula for estimating blood Se when serum Se is known, at the 95% prediction interval, was [formula; see text]. For the ICP data, the inverse prediction formula for estimating blood Se when serum Se is known, at the 95% prediction interval, was [formula; see text]. The prediction intervals were quite wide, and the accuracy of estimating blood Se from a known serum Se was not useful for diagnostic purposes. The use of serum Se concentration to assess nutritional status of cattle with respect to Se does not appear to be appropriate.

Zinc toxicosis due to ingestion of a penny in a gray-headed chachalaca (Ortalis cinereiceps).

Zinc toxicosis was diagnosed in a gray-headed chachalaca (Ortalis cinereiceps) due to ingestion of a copper-plated zinc penny. Histopathological lesions were most marked in the pancreas. These lesions included apoptosis, zymogen granule depletion, and loss of normal acinar architecture. There was also severe gizzard erosion. Heavy metal analysis revealed abnormal levels of zinc and iron in the liver. Iron pigment in the liver was most concentrated in Kupffer cells. This, along with evidence of erythrophagocytosis in the spleen, suggested that extravascular hemolysis was also associated with zinc toxicosis in this case.

A syndrome in commercial turkeys in California and Oregon characterized by a rear-limb necrotizing skeletal myopathy.

A necrotizing skeletal myopathy of rear limbs was diagnosed in 17 flocks of commercial turkeys. The mean mortality attributed to the myopathy was 2.29% (range = 0.13-9.7%) over a mean period of 9.6 days (range = 6-14 days). The mean age of the birds at the time of onset was 7.4 weeks (range = 4-10.5 weeks). Clinically, birds experienced an episode of watery droppings and high-pitched crying, followed by rear-limb paresis or paralysis. Creatine kinase and aspartate aminotransferase were markedly elevated in birds with the myopathy. Grossly, a few birds had pale streaking in the muscles of the thighs and legs. Histologically, acute and subacute degeneration was present in myofibers of the legs, abdomen, thighs, back, and tail. The subacute lesion was characterized by marked sarcolemmal cell proliferation. Feed analyses ruled out selenium deficiency and the presence of mycotoxins as etiologies. Monensin was present in approved usage or only slightly elevated levels. A known potentiating antibiotic was being used concurrently with monensin in only one flock.

Recurrent transient paresis in a turkey flock.

Recurring episodes of extreme leg weakness and associated mortality were documented in a turkey flock at 8 to 15 weeks of age. Flock mortality attributed to posterior paresis was approximately 12%, or 4800 of 40,000 turkeys. Four of six open-confinement units were affected. Gross and histological examinations revealed no significant lesions. Immunology and virology were uninformative. There were no significant differences in serum chemistry between clinically affected and normal turkeys. Testing of feed, water, soil, and tissues revealed no common toxicants. Isolation and supportive care for affected turkeys, both in the laboratory and in the field, frequently resulted in full recovery. Injection of a test group of affected turkeys with Type C botulism antitoxin appeared to enhance recovery. However, repeated attempts to detect botulism toxin in serum, liver, or cecal contents using mouse bioassay procedures were unsuccessful.