Birgit Puschner

Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007.

Sixteen animals affected in 2 outbreaks of pet food-associated renal failure (2 dogs in 2004; 10 cats and 4 dogs in 2007) were evaluated for histopathologic, toxicologic, and clinicopathologic changes. All 16 animals had clinical and laboratory evidence of uremia, including anorexia, vomiting, lethargy, polyuria, azotemia, and hyperphosphatemia. Where measured, serum hepatic enzyme concentrations were normal in animals from both outbreaks. All animals died or were euthanized because of severe uremia. Distal tubular lesions were present in all 16 animals, and unique polarizable crystals with striations were present in distal tubules or collecting ducts in all animals. The proximal tubules were largely unaffected. Crystals and histologic appearance were identical in both outbreaks. A chronic pattern of histologic change, characterized by interstitial fibrosis and inflammation, was observed in some affected animals. Melamine and cyanuric acid were present in renal tissue from both outbreaks. These results indicate that the pet food-associated renal failure outbreaks in 2004 and 2007 share identical clinical, histologic, and toxicologic findings, providing compelling evidence that they share the same causation.

Assessment of Melamine and Cyanuric Acid Toxicity in Cats

The major pet food recall associated with acute renal failure in dogs and cats focused initially on melamine as the suspect toxicant. In the course of the investigation, cyanuric acid was identified in addition to melamine in the offending food. The purpose of this study was to characterize the toxicity potential of melamine, cyanuric acid, and a combination of melamine and cyanuric acid in cats. In this pilot study, melamine was added to the diet of 2 cats at 0.5% and 1%, respectively. Cyanuric acid was added to the diet of 1 cat at increasing doses of 0.2%, 0.5%, and 1% over the course of 10 days. Melamine and cyanuric acid were administered together at 0%, 0.2%, 0.5%, and 1% to 1 cat per dose group. No effect on renal function was observed in cats fed with melamine or cyanuric acid alone. Cats dosed with a combination were euthanized at 48 hours after dosing because of acute renal failure. Urine and touch impressions of kidneys from all cats dosed with the combination revealed the presence of fan-shaped, birefringent crystals. Histopathologic findings were limited to the kidneys and included crystals primarily within tubules of the distal nephron, severe renal interstitial edema, and hemorrhage at the corticomedullary junction. The kidneys contained estimated melamine concentrations of 496 to 734 mg/kg wet weight and estimated cyanuric acid concentrations of 487 to 690 mg/kg wet weight. The results demonstrate that the combination of melamine and cyanuric acid is responsible for acute renal failure in cats.

Diagnostic Determination of Melamine and Related Compounds in Kidney Tissue by Liquid Chromatography/Tandem Mass Spectrometry

In 2007, it was determined that melamine, ammeline, ammelide, and cyanuric acid (abbreviated as MARC for melamine and related contaminants) had been added to wheat gluten and rice protein that were subsequently incorporated into pet food. The consumption of food tainted by MARC compounds was implicated in numerous instances of renal failure in cats and dogs. A method for the analysis of MARC compounds in kidney tissue using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) has been developed. MARC analytes were extracted by homogenization of kidney tissue in 50/40/10 acetonitrile/water/diethylamine. The homogenate was centrifuged, and an aliquot of supernatant was diluted with acetonitrile, concentrated, and fortified with a stable isotope-labeled analogue of melamine. Analytes were detected using atmospheric pressure chemical ionization and multiple reaction monitoring. Quantitation of positive samples was performed using the internal standard method and five-point calibration curves ranging between 50 and 1000 ng/mL of each analyte. The method was validated by analysis of replicate kidney tissue samples fortified with the individual analytes and by analysis of kidney samples fortified with melamine cyanurate powder at two different concentrations. This method was successfully used for routine postmortem diagnosis of melamine toxicosis in animals. Melamine was also detected by this method in paraffin-embedded tissue from animals suspected to have died of melamine toxicosis.

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.

Diagnosis of Anatoxin-a Poisoning in Dogs from North America

Anatoxin-a, a toxin produced by several genera of blue-green algae, is considered a potent neurotoxin. Ingestion of water contaminated with the toxin results in acute neurological signs and often death. This report describes fatal cases of anatoxin-a ingestion in 6 dogs, with confirmation of anatoxin-a exposure by liquid chromatography/tandem mass spectrometry (LC-MS/MS/MS). In 1 outbreak, 3 dogs developed seizures and died within an hour after swimming in a river in California, while the other outbreak involved 3 dogs that died within 1 hour after swimming in a pond in Ontario. Anatoxin-a poisoning is rarely reported in dogs as a cause of acute neurological signs and death. However, increased occurrences of blue-green algae blooms in North America make this neurotoxin an important consideration in the diagnosis of sudden death associated with environmental water exposure. This brief communication reports on the isolation and detection of anatoxin-a from environmental water sources and the stomach contents of North American dogs dying of acute neurotoxicosis. This demonstrates the first documented cases of anatoxin-a poisoning in dogs in North America and the importance of LC-MS/MS/MS in identifying neurotoxins responsible for sudden death in cases of suspected blue-green algae toxicosis; especially those cases showing no gross or histological lesions.

Patterns of mortality in free-ranging California Condors (Gymnogyps californianus).

We document causes of death in free-ranging California Condors (Gymnogyps californianus) from the inception of the reintroduction program in 1992 through December 2009 to identify current and historic mortality factors that might interfere with establishment of self-sustaining populations in the wild. A total of 135 deaths occurred from October 1992 (the first post-release death) through December 2009, from a maximum population-at-risk of 352 birds, for a cumulative crude mortality rate of 38%. A definitive cause of death was determined for 76 of the 98 submitted cases, 70%(53/76) of which were attributed to anthropogenic causes. Trash ingestion was the most important mortality factor in nestlings (proportional mortality rate [PMR] 73%; 8/11), while lead toxicosis was the most important factor in juveniles (PMR 26%; 13/50) and adults (PMR 67%; 10/15). These results demonstrate that the leading causes of death at all California Condor release sites are anthropogenic. The mortality factors thought to be important in the decline of the historic California Condor population, particularly lead poisoning, remain the most important documented mortality factors today. Without effective mitigation, these factors can be expected to have the same effects on the sustainability of the wild populations as they have in the past.

Diagnosis of Taxus (Yew) Poisoning in a Horse

A 2-year-old bay Thoroughbred colt was found dead overnight in its stall without a known history of any illness, existing disease, or toxicant exposure. No information on the clinical signs before this animals death was reported. A full necropsy was performed the next morning and revealed a mild to moderate degree of endocardial hemorrhages in both ventricles. Microscopic examination of the heart showed an acute mild mutifocal necrosis of papillary muscles and ventricles. The stomach content contained approximately 2% Taxus alkaloids as determined by gas chromatography/mass spectrometry. In the past, diagnosis of Taxus poisoning has been mainly based on history of exposure and the presence of plant parts in the gastrointestinal tract. Pathological lesions associated with Taxus poisoning have not been published for horses. Therefore, this is the first report of cardiac lesions in a horse after lethal exposure to Taxus. On the basis of these findings, it is suggested that Taxus exposure needs to be considered in the differential diagnosis of horses that die suddenly or have cardiac lesions suggestive of Taxus exposure, even if intact plant parts are not identified in the stomach by the naked eye.

Toxicosis Caused by Melamine and Cyanuric Acid in Dogs and Cats: Uncovering the Mystery and Subsequent Global Implications

Several major pet-food and human-food safety incidents occurred worldwide between 2003 and 2008, causing illnesses and deaths in children, cats, dogs, and pigs. During the 2007 outbreak of renal failure in dogs and cats in the United States, veterinary diagnostic laboratories helped identify melamine and melamine analogues as contaminants in implicated food. In 2008, thousands of infants developed renal failure from exposure to melamine alone. Management of these outbreaks depends on the collaboration of veterinary and human laboratories and clinics, government agencies, academic institutions, and food industries, along with prompt communication and sharing of data.

Microcystins in potable surface waters: toxic effects and removal strategies

In freshwater, harmful cyanobacterial blooms threaten to increase with global climate change and eutrophication of surface waters. In addition to the burden and necessity of removal of algal material during water treatment processes, bloom‐forming cyanobacteria can produce a class of remarkably stable toxins, microcystins, difficult to remove from drinking water sources. A number of animal intoxications over the past 20 years have served as sentinels for widespread risk presented by microcystins. Cyanobacterial blooms have the potential to threaten severely both public health and the regional economy of affected communities, particularly those with limited infrastructure or resources. Our main objectives were to assess whether existing water treatment infrastructure provides sufficient protection against microcystin exposure, identify available options feasible to implement in resource‐limited communities in bloom scenarios and to identify strategies for improved solutions. Finally, interventions at the watershed level aimed at bloom prevention and risk reduction for entry into potable water sources were outlined. We evaluated primary studies, reviews and reports for treatment options for microcystins in surface waters, potable water sources and treatment plants. Because of the difficulty of removal of microcystins, prevention is ideal; once in the public water supply, the coarse removal of cyanobacterial cells combined with secondary carbon filtration of dissolved toxins currently provides the greatest potential for protection of public health. Options for point of use filtration must be optimized to provide affordable and adequate protection for affected communities. Copyright

Genome-Wide Association Analysis Identifies a Mutation in the Thiamine Transporter 2 (SLC19A3) Gene Associated with Alaskan Husky Encephalopathy

Alaskan Husky Encephalopathy (AHE) has been previously proposed as a mitochondrial encephalopathy based on neuropathological similarities with human Leigh Syndrome (LS). We studied 11 Alaskan Husky dogs with AHE, but found no abnormalities in respiratory chain enzyme activities in muscle and liver, or mutations in mitochondrial or nuclear genes that cause LS in people. A genome wide association study was performed using eight of the affected dogs and 20 related but unaffected control AHs using the Illumina canine HD array. SLC19A3 was identified as a positional candidate gene. This gene controls the uptake of thiamine in the CNS via expression of the thiamine transporter protein THTR2. Dogs have two copies of this gene located within the candidate interval (SLC19A3.2 – 43.36–43.38 Mb and SLC19A3.1 – 43.411–43.419 Mb) on chromosome 25. Expression analysis in a normal dog revealed that one of the paralogs, SLC19A3.1, was expressed in the brain and spinal cord while the other was not. Subsequent exon sequencing of SLC19A3.1 revealed a 4bp insertion and SNP in the second exon that is predicted to result in a functional protein truncation of 279 amino acids (c.624 insTTGC, c.625 C>A). All dogs with AHE were homozygous for this mutation, 15/41 healthy AH control dogs were heterozygous carriers while 26/41 normal healthy AH dogs were wild type. Furthermore, this mutation was not detected in another 187 dogs of different breeds. These results suggest that this mutation in SLC19A3.1, encoding a thiamine transporter protein, plays a critical role in the pathogenesis of AHE.