Investigative and Diagnostic Toxicology and Feed-Related Outbreaks.

Incidences of feed contamination are rare even though enormous amounts of animal feed are manufactured. However, there are still some cases of feed-related illness in horses. Veterinarians play a crucial role in recognizing and mitigating these events and in assessing the severity of risks. Due to these risks, proper reporting and consultation with government and state agencies are crucial. Accurate diagnosis and identification of the source of poisoning are promising when a thorough case workup is performed and agencies such as veterinary diagnostic laboratories and the US Food and Drug Administration Center for Veterinary Medicine are used effectively.

Investigation of a common canine factor VII deficiency variant in dogs with unexplained bleeding on autopsy.

The factor VII (FVII) protein is an integral component of the extrinsic coagulation pathway. Deleterious variants in the gene encoding this protein can result in factor VII deficiency (FVIID), a bleeding disorder characterized by abnormal (slowed) clotting with a wide range of severity, from asymptomatic to life-threatening. In canids, a single FVIID-associated variant, first described in Beagles, has been observed in 24 breeds and mixed-breed dogs. Because this variant is present in breeds of diverse backgrounds, we hypothesized that it could be a contributing factor to unexplained bleeding observed in some canine autopsy cases. DNA was extracted from paraffin-embedded tissue samples from 67 anticoagulant-negative autopsy cases with unexplained etiology for gross lesions of hemorrhage. Each dog was genotyped for the c.407G>A (F71) variant. Experimental controls included 3 known heterozygotes and 2 known homozygotes for the F71 variant, 2 normal dogs with known homozygous wild-type genotypes (F7WF7W), and 5 dogs with bleeding at autopsy that tested positive for anticoagulant rodenticide and were genotyped as F7WF7W. All 67 cases tested homozygous for the wild-type allele, indicating that the common FVIID variant was not responsible for the observed unexplained bleeding. Our work demonstrates the usefulness of retrospective studies utilizing veterinary diagnostic laboratory databases and tissue archives for genetic studies. In the case of FVIID, our results suggest that a singular molecular test for the F71 variant is not a high-yield addition to postmortem screening in these scenarios.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Atrazine intoxication in cattle, confirmed by gas chromatography-mass spectrometry.

Ten of 40 cows died within 48 h of gaining access to a barn in which various chemicals were stored. Some of the surviving cows exhibited drooling, muscle tremors, and agitation. Postmortem examinations of 2 cows were performed in the field, and revealed nonspecific, moderate-to-severe pulmonary congestion. Liver and rumen contents, each from a different cow, were analyzed using a qualitative, multi-residue GC-MS method validated for the detection of pesticides and other chemical analytes. Using this method, extracts from the liver and rumen content samples were compared to atrazine (neat standard) and matrix-matched, control samples fortified with atrazine. GC-MS analysis detected atrazine at 215 m/z (NIST match >97%) with a retention time of ~13 min in liver and rumen content samples from our case. Detection of atrazine in the samples from the cows in this herd, combined with the clinical history, indicate that atrazine toxicity was the likely cause of clinical signs and death observed in this herd.

Commercial and Industrial Chemical Hazards for Ruminants: An Update.

Livestock can be exposed to a wide variety of commercial or industrial chemicals that have variable toxicity. Adverse effects can be due to acute or chronic illnesses and deaths or due to contamination of meat or milk intended for human consumption. A diagnosis can be challenging in the absence of a known exposure. It is critical that a complete evaluation be conducted and appropriate samples be collected for possible analysis. Appropriate experts and regulatory agencies should be consulted as soon as possible to avoid potential contaminated animal products reaching consumers.

Chronic copper toxicosis in a crossbred heifer calf.

An 8-mo-old, crossbred, heifer calf was presented to the Heeke Animal Disease Diagnostic Laboratory with a history of ataxia and altered mentation. Grossly, the liver was diffusely yellow-orange, turgid, and exuded watery, thin blood on cut section. The cortex and medulla in both kidneys were diffusely and markedly dark brown to black. The urinary bladder was filled with dark red urine. Histologically, centrilobular hepatocellular degeneration was observed, but these sections lacked necrosis. In the kidney, numerous cortical tubules contained intraluminal bright eosinophilic fluid and red-orange granular casts that stained positive for hemoglobin with the Dunn-Thompson method. The gross and histologic lesions supported a high level of suspicion for copper toxicosis. Feed and water samples from the farm were submitted for mineral analysis. The copper concentration in the feed was 118 mg/kg, and the molybdenum concentration was 0.9 mg/kg. Chronic copper toxicosis is rarely reported in cattle. The gross lesions in our case are a departure from, although similar to, previously reported cases, including lack of histologic hepatocellular necrosis. Collectively, gross and histologic lesions were compatible with copper toxicosis in this calf, and copper concentrations in the feed samples suggest a feed-mixing error.

Investigative Diagnostic Toxicology and the Role of the Veterinarian in Pet Food-Related Outbreaks: An Update.

Although most commercial pet foods are safe, there have been a few instances in which chemical or bacterial contamination have caused outbreaks of illness in animals. Because of concerns regarding cases of contaminated commercial pet food that have been reported over the past several years, some pet owners may be choosing to feed noncommercial, home-prepared diets. When pet food contamination is suspected, pet owners often seek advice from their veterinarian regarding its health impact and subsequent diagnosis. This article addresses the role of the veterinarians in pet food contamination and highlights recommended approaches to handling pet food outbreaks or recalls.

Radiation Emergencies: Dogs and Cats.

Exposure of dogs and cats to clinically significant amounts of ionizing radiation is unlikely. However, accidental release of radiation has occurred and nuclear terrorism is possible. If an incident occurs, early reaction will be by first responders, followed by state and federal emergency personnel. It is possible that veterinarians will be called upon to assist to evaluate animals for contamination and/or exposure, perform initial lifesaving tasks, and decontaminate people's pets. Therefore, veterinary professionals should understand radiation exposure, what is happening, the possible effects on animals, and how to provide veterinary care and assistance in a radiation emergency.

Rapid detection of terbufos in stomach contents using desorption electrospray ionization mass spectrometry.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an emerging analytical technique that permits the rapid and direct analysis of biological or environmental samples under ambient conditions. Highlighting the versatility of this technique, DESI-MS has been used for the rapid detection of illicit drugs, chemical warfare agents, agricultural chemicals, and pharmaceuticals from a variety of sample matrices. In diagnostic veterinary toxicology, analyzing samples using traditional analytical instrumentation typically includes extensive sample extraction procedures, which can be time consuming and labor intensive. Therefore, efforts to expedite sample analyses are a constant goal for diagnostic toxicology laboratories. In the current report, DESI-MS was used to directly analyze stomach contents from a dog exposed to the organophosphate insecticide terbufos. The total DESI-MS analysis time required to confirm the presence of terbufos and diagnose organophosphate poisoning in this case was approximately 5 min. This highlights the potential of this analytical technique in the field of veterinary toxicology for the rapid diagnosis and detection of toxicants in biological samples.

Myocardial fibrosis associated with previous ingestion of yew (Taxus sp.) in a Holstein heifer: evidence for chronic yew toxicity in cattle.

Twenty-six 5-month-old Holstein calves were accidentally exposed to discarded branches of yew bushes (Taxus sp.). Several calves were found dead approximately 24 hr after exposure; however, a few calves died several days after exposure. One calf died 18 days after the initial exposure to Taxus sp. and was examined on the farm via necropsy. Gross lesions included ascites, and dilated and flaccid myocardial ventricles. Sections of formalin-fixed heart were submitted to the Indiana Animal Disease Diagnostic Laboratory for histopathologic examination; fresh rumen contents were submitted for toxicologic testing. Histologically, large areas of myocardium were replaced by fibrous connective tissue, suggesting previous myocardial necrosis. Taxus alkaloids were identified in the rumen contents using gas chromatography-mass spectrometry. Based on the clinical history, the gross and histologic lesions, the identification of Taxus alkaloids in the rumen contents, and lack of exposure to other known cardiotoxic agents, yew toxicity was considered the cause of death in this calf. Ingestion of taxines is known to cause acute and subacute toxicity in human beings and animals; however, a chronic clinical course and severe histologic lesions have not been previously associated with yew toxicity. Although only 1 calf was examined, this case suggests that yew toxicity can result in a prolonged clinical course in cattle and can cause histologic myocardial lesions.

Investigative diagnostic toxicology and the role of the veterinarian in pet food-related outbreaks.

Due to the potential implications of food-related illnesses in animals, recognition of pet food-related outbreaks is one of the many crucial roles of the veterinarian. This article describes the veterinarian's role in investigating and reporting food-related illnesses in cats and dogs. Recommendations regarding taking thorough case histories, appropriate sample collection, effective use of veterinary diagnostic laboratories, and recommendations for reporting such illnesses are described.

Development of a gas chromatography-mass spectrometry technique to diagnose white snakeroot (Ageratina altissima) poisoning in a cow.

An 8-year-old, crossbred beef cow was referred to the Indiana Animal Disease Diagnostic Laboratory at Purdue University for a complete necropsy in October 2009. The cow was the sixth to die in a 7-day period. Affected cows were reportedly stumbling and became weak, excitable, and recumbent. Histologically, myonecrosis was severe in the skeletal muscles and mild in the heart and tongue. According to the submitter, exposure to a poisonous plant was suspected, and a plant specimen received from this case was identified as white snakeroot (Ageratina altissima). Using the white snakeroot specimen, a gas chromatography-mass spectrometry analytical method for the detection of tremetone and dehydrotremetone (2 components of white snakeroot) was developed. Both tremetone and dehydrotremetone were detected in the plant specimen. Dehydrotremetone was recovered from the liver, while neither component was recovered in the rumen content. In the past, because of the lack of standard reference material, the diagnosis of white snakeroot poisoning was based mainly on history of exposure and the presence of the plant in the rumen. The analytical method described herein can be used to document exposure to tremetone or dehydrotremetone in cases of suspected white snakeroot poisoning when coupled with the appropriate clinical signs and lesions.

Quantitative assessment of the percent fat in domestic animal bone marrow.

Measurement of the amount of fat in femoral bone marrow can provide a quantitative assessment of the nutritional status of an individual animal. An analytical method is presented for quantitating the percent fat in bone marrow from three domestic species: bovine, canine, and equine. In this procedure, fat is extracted from bone marrow using pentane, and the percent fat recovered is determined gravimetrically. Based on analyses from adult animals (normal body condition scores), the average percentage of fat in the bone marrow was >80%. In cases in which animals have been diagnosed as emaciated or exhibit serous atrophy of fat (body scores of 1 or 2), the femoral bone marrow fat was less than 20%. In domestic animals, bone marrow fat analysis can be a useful, quantitative measure that, when used in conjunction with all other data available, can support a diagnosis of starvation or malnutrition.

Chronic microcystin exposure induces hepatocyte proliferation with increased expression of mitotic and cyclin-associated genes in P53-deficient mice.

Homozygous p53 deficient knockout mice were used to assess the role of p53 in tumor promotion by the protein phosphatase inhibitor and hepatic tumor promoter microcystin-LR (MCLR). More than 50% of human cancers bear mutations in the p53 gene, and in particular, p53 tumor suppressor gene mutations have been shown to play a major role in hepatocarcinogenesis. Trp53 homozygous (inactivated p53) and age-matched wild-type control mice were assigned to vehicle or MCLR-treated groups. MCLR or saline was administered daily for up to 28 days. RNA from the 28-day study was hybridized onto Mouse Genome GeneChip arrays. Selected RNA from 28 days and earlier time points was also processed for quantitative polymerase chain reaction (PCR). Livers from the 28-day, Trp53-deficient, MCLR group displayed greater hyperplastic and dysplastic changes morphologically and increases in Ki-67 and phosphohistone H3 (mitotic marker) immunoreactivity. Gene-expression analysis revealed significant increases in expression of cell-cycle regulation and cellular proliferation genes in the MCLR-treated, p53-deficient mutant mice compared to controls. These data suggest that regulation of the cell cycle by p53 is important in preventing the proliferative response associated with chronic, sublethal microcystin exposure, and therefore, conclude that p53 plays an important role in MCLR-induced tumor promotion.

Hepatic gene expression changes in mice associated with prolonged sublethal microcystin exposure.

Microcystin-LR (MCLR) is an acute hepatotoxicant and suspected carcinogen. Previous chronic studies have individually described hepatic morphologic changes, or alterations in the cytoskeleton, cell signaling or redox pathways. The objective of this study was to characterize chronic effects of MCLR in wild-type mice utilizing gene array analysis, morphology, and plasma chemistries. MCLR was given daily for up to 28 days. RNA from the 28-day study was hybridized onto mouse genechip arrays. RNA from 4 hours, 24 hours, 4 days, 1 day, and 28 days for selected genes was processed for quantitative-PCR. Increases in plasma hepatic enzyme activities and decreases in total protein, albumin and glucose concentrations were identified in MCLR-treated groups at 14 and 28 days. Histologically, marked hepatokaryomegaly was identified in the 14-day MCLR group with the addition of giant cells at 28 days. Major gene transcript changes were identified in the actin organization, cell cycle, apoptotic, cellular redox, cell signaling, albumin metabolism, and glucose homeostasis pathways, and the organic anion transport polypeptide system. Using toxicogenomics, we have identified key molecular pathways involved in chronic sublethal MCLR exposure in wild-type mice, genes participating in those critical pathways and related them to cellular and morphologic alterations seen in this and other studies.

Fatal ricin toxicosis in a puppy confirmed by liquid chromatography/mass spectrometry when using ricinine as a marker.

Ricin, a lectin from the castor bean plant (Ricinis communis), is considered one of the most potent plant toxins. Ingestion of masticated seeds results in high morbidity, with vomiting and watery to hemorrhagic diarrhea. The prognosis varies with the number of seeds ingested, the degree of mastication, individual susceptibility, and the delay in treatment. Low mortality restricts assessment of histologic lesions, and the literature on toxicologic analysis for ricin is limited. This report describes a fatal case of castor bean ingestion in a 12-week-old Mastiff puppy, with confirmation of ricin exposure through detection of the biomarker ricinine by liquid chromatography/mass spectrometry (LC/MS). Despite supportive therapy, the puppy died several hours after presentation for acute vomiting, diarrhea, and lethargy. At necropsy, a segment of jejunum and mesenteric lymph nodes were congested. When the owner reported the presence of castor beans in the dog's feces, selected formalin-fixed and unfixed tissues were submitted for diagnostic evaluation. Histopathologic findings included superficial necrotizing enteritis of the jejunum and occasional, random foci of coagulative necrosis in the liver. The alkaloid ricinine was detected in gastric content by using a newly developed LC/MS method. This confirmation of exposure is important in the diagnosis of ricin toxicosis, because ingestion of castor beans is not always fatal, histologic lesions are nonspecific, and the degree of mastication can influence the effective dose of ricin.

Phase I clinical trial of the use of zinc phthalocyanine tetrasulfonate as a photosensitizer for photodynamic therapy in dogs.

OBJECTIVE: To determine the threshold for acute toxicosis of parenterally administered zinc phthalocyanine tetrasulfonate (ZnPcS(4)), a candidate second-generation photosensitizer, in mice and evaluate the compound's safety in a phase I clinical trial of ZnPcS(4)-based photodynamic therapy (PDT) in pet dogs with naturally occurring tumors. ANIMALS: Male Swiss-Webster mice and client-owned dogs with naturally occurring neoplasms. PROCEDURES: For the study of acute toxicosis, mice were given graded doses of ZnPcS(4). To determine safety, a rapid-titration phase I clinical trial of ZnPcS(4)-based PDT in tumor-bearing dogs was conducted. RESULTS: In mice, administration of >or= 100 mg of ZnPcS(4)/kg resulted in renal tubular necrosis 24 hours after IP injection. In tumor-bearing dogs, ZnPcS(4) doses

Degenerative myelopathy and vitamin A deficiency in a young black-maned lion (Panthera leo).

Degenerative myelopathy and vitamin A deficiency were diagnosed in a 1-year-old, female, black-maned lion (Panthera leo). Diffuse white matter degeneration characterized by dilated myelin sheaths, Wallerian degeneration, and reactive astrocytosis was present at all levels of the spinal cord. With luxol fast blue-resyl echt violet stain, bilaterally symmetrical demyelination was observed in the fasciculus cuneatus of the cervical spinal cord and in peripheral white matter of cervical, thoracic, and lumbar segments. Additionally, the ventral gray columns and brain stem nuclei contained rare chromatolytic neurons with abnormal neurofilament accumulation. Leptomeninges of the cervical spinal cord were focally adhered to the dura and thickened by fibrosis and osseous metaplasia. Vitamin A deficiency was diagnosed based on hepatic vitamin A concentration of 1.71 microg/g dry weight. Adequate hepatic vitamin A concentration for yearling to adult domestic animals ranges between 150 and 1000 microg/g dry weight. Lesions were distinct from those previously described in young captive lions with vitamin A deficiency, which had thickened skull bones and cerebellar herniation. The pathogenesis of vitamin A-associated myelopathy in this lion may be similar to that described in adult cattle, which is believed to result from spinal cord compression secondary to elevated pressure of cerebrospinal fluid.

Species comparison of vitamin K1 2,3-epoxide reductase activity in vitro: kinetics and warfarin inhibition.

A comparative study of vitamin K(1) 2,3-epoxide reductase (VKOR) activity in vitro was conducted across species. The apparent kinetic constants K(m app), V(max), and Cl(int app) were determined in bovine, canine, equine, human, murine, ovine, porcine, and rat hepatic microsomes. In addition to these enzyme kinetic constants, the IC(50) of warfarin for VKOR was determined in human, murine, porcine, and rat hepatic microsomes. Interspecies differences were observed when comparing the K(m app) (range, 2.41-6.46 microM), V(max) (range, 19.5-85.7 nmol/mg/min), and Cl(int app) (range, 8.2-18.4 ml/mg/min) values. Comparison of the IC(50) values of warfarin, across the four species tested, revealed a significant species difference between murine microsomes (0.17 microM) and rat microsomes (0.07 microM). Overall, this study indicates that there are interspecies differences regarding the in vitro reduction of vitamin K(1) 2,3-epoxide by the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase. Significant differences between the IC(50) values of murine and rat microsomes suggest differences in the susceptibility of these species to warfarin.