Signalment:  

Adult male fox squirrel (Sciurus niger)

Over the course of several days, five squirrels were found dead under a tree at a residence in Colorado.Prior to death, all of the squirrels had similar clinical signs which included hindquarter paralysis, lethargy, and heavy breathing.


Gross Description:  

Two fox squirrels were presented for postmortem examination in good body condition with minimal autolysis.No evidence of trauma was identified.The adult male squirrel had no significant gross lesions and stomach contents were within normal limits, including grainy yellow-brown ingesta.The adult female had turquoise-green granular material on the fur of the upper lip and similar material within the stomach.The colonic contents were stained a distinct turquoise-green.


Histopathologic Description:

Brain, 2 sections (cerebellum and cerebral cortex including hippocampus): Diffusely throughout both sections, the white matter is characterized by moderate to severe extracellular vacuolization.Vacuoles are formed by variably swollen myelin sheaths which occasionally coalesce into large extracellular clear spaces.Dilated myelin sheaths contain normal to minimally swollen axons.Scattered throughout the grey matter of the cerebral cortex and rarely within the hippocampus are low numbers of neurons with degenerative changes, including central chromatolysis and occasional pyknosis. Rare neurons are shrunken, angular, and hypereosinophilic with karyolysis (necrosis).Clefts within the perikaryon of multiple neuronal cell bodies are consistent with fixation artifact.


Morphologic Diagnosis:  

Cerebellum and cerebral cortex: Vacuolar myelinopathy, severe, diffuse, with mild, multifocal neuronal degeneration and necrosis.


Lab Results:  

Adipose tissue contained desmethylbromethalin.


Condition:  

Bromethalin toxicosis, squirrel


Contributor Comment:  

Bromethalin toxicosis was strongly suspected based on the history and collective gross and histologic findings.This suspicion was confirmed by the presence of desmethylbromethalin in the adipose tissue.Desmethylbromethalin is a toxic metabolite of bromethalin, a potent neurotoxin and the active ingredient in a variety of rodenticides.The mechanism of action involves un-coupling of oxidative phosphorylation, resulting in decreased ATP production and diminished Na+/K+ pump activity.9,10 In the CNS, the net result is severe, acute fluid retention and a dramatic elevation in cerebrospinal fluid pressure. Bromethalin is metabolized to desmethylbromethalin th-rough N-demethylation by hepatic mixed-function oxygenases and is excreted predominantly in the bile. The oral LD50 is 2.38-5.6 mg/kg in the dog and 0.4-0.71 mg/kg in the cat.3,9 A relative resistance to toxicity has been demonstrated in species unable to metabolize bromethalin to desmethylbromethalin (e.g. guinea pigs with an LD50 of 1000 mg/kg).10 Short of chemical confirmation, diagnosis of bromethalin toxicosis is based on likelihood of exposure and development of corresponding clinical signs, including muscle tremors, seizures, dypsnea, hyperexcitability, hind limb ataxia, and paresis to paralysis.Severity and onset (2- 14 hours post-ingestion) are dose-dependent.3 -

Bromethalin is indistinguishable from anticoagulant rodenticides in appearance and color, and gross lesions are uncommon.Diffuse white matter vacuolization is the characteristic histologic lesion, and ultrastructural studies have demonstrated intramyelinic vacuoles with separation and splitting of myelin lamellae.4,5 Luxol fast blue-periodic acid Schiff stain has demonstrated myelin displacement due to edema with no apparent net myelin loss.5- Hypertrophied astrocytes and oli-godendrocytes have also been reported.5- Vacuolization of the optic nerve occurs in most cases.4,5 Similar white matter vac-uolization is seen with triethyltin and hexachlorophene neurotoxicosis.7,8- -

Bromethalin use has increased in recent years in association with new regulations prohibiting residential use of second generation anticoagulant rodenticides.While bromethalin remains readily available for over-the-counter sales, many anticoagulant rodenticides with similar names (e.g. brodifacoum, bromadiolone) have been removed.Thus, bromethalin toxicity is gaining in importance due to increased popularity of neurotoxic rodenticides.


JPC Diagnosis:  

Cerebrum and cerebellum, white matter: Vacuolar myelinopathy, diffuse, severe.


Conference Comment:  

Conference participants discussed this lesion as being very -Ç-ÿquiet histologically, with minimal if any response to the swelling of myelin sheaths. The moderator discussed how this lesion contrasts with a demyelinating lesion, which manifests histologically as a patchy, less diffuse distribution and with at least some degree of glial response.In this example of bromethalin toxicity, there is a distinct absence of swollen axons and spheroids, and the oligodendrocytes appear quiescent.

The differential diagnosis discussed by participants for a similar histologic lesion in other species included other toxicants, such as hexachlorophene and ammonia, as well as plant toxins seen in various parts of the world, such as Stypandra spp. in Australia and Helichrysum spp. in Africa.The lesions of bromethalin in the central nervous system of these squirrels also bear resemblance to those of avian vacuolar myelinopathy, which is seen in North America secondary to a cyanobacterial toxin that grows on non-native aquatic vegetation.The condition is frequently lethal and affects various avian species, such as bald eagles and American coots in the southeastern United States.6 Another cause of similar white matter specific vacuolar change includes branched-chain alpha-ketoacid decarboxylase de-ficiency (maple syrup urine disease) in cattle.

Despite the apparent frequency with which bromethalin intoxication occurs in domestic animals and wildlife species, there are surprisingly few reports in the recent professional literature.In addition to the more acute syndrome discussed above, a paralytic syndrome is also described when concentrations below the LD50 are ingested; it includes ataxia, CNS depression, and paralysis which may develop over a period of days and worsen over a period of weeks.2- The acute syndrome has also been reported to occur when smaller doses (below the LD50) are ingested in dogs and may relate to treatment with activated charcoal, which can result in idiosyncratic hypernatremia in rare cases. Dramatic changes in sodium levels can result in CNS associated clinical signs and lesions, including cortical laminar necrosis in cases of salt intoxication; conversely, osmotic demyelination can occur in cases where there is a sudden increase in sodium in a hyponatremic animal. Histologic lesions in osmotic demyelination and the changes seen in bromethalin toxicity can have similarities, although in osmotic demyelination there is myelin and oligodendrocyte loss which does not occur with bromethalin intoxication.1

The green-tinged or turquoise coloring seen in the gross image is characteristic of dyes used in certain rodenticides, as well as in some fertilizers and pesticides,2 which can make confirmation of bromethalin intox-ication challenging.1- The highest con-centration of desmethylbromethalin is us-ually found in adipose tissue, which is the most important tissue sample for diagnostic toxicology testing in suspected cases of bromethalin intoxication.2- Bromethalin is not only lipid-soluble, but also readily crosses the blood brain barrier.1 Additionally, in cases of mild white matter vacuolation, both light microscopy and ultrastructural examination may not be able to precisely differentiate the changes of bromethalin intoxication from those of autolysis, the latter of which are especially common in wildlife species.2- Neuron-specific nuclear protein (NeuN) -and glial fibrillary acidic protein (GFAP) may be useful in distinguishing subtle changes from autolytic artifact in questionable cases.Decreased NeuN immunoreactivity can indicate neuronal loss and/or metabolic stress and increased GFAP imm-unoreactivity is indicative of reactive astrocytosis.1


References:



1. Bates MC, Roady P, Lehner AF, Buchweitz JP, et al. Atypical bromethalin intoxication in a dog: pathologic features and identification of an isomeric breakdown product. BMC Vet Res. 2015; 11(244):1-9.

2. Bautista AC, Woods LW, Filigenzi MS, Puschner B. Bromethalin poisoning in a raccoon (Procyon lotor): diagnostic considerations and relevance to nontarget wildlife. J Vet Diagn Invest. 2014; 26(1):154-157.

3. Dorman, DC, Parker, AJ, Buck, WB. Bromethalin Toxicosis in the dogs. Part I: Clinical Effects. J Am Anim Hosp Assoc. 1990; 26(6): 589-594.

4. Dorman, DC, Simon, J, Harlin, KA, Buck, WB. Diagnosis of bromethalin toxicosis in the dog. J Vet Diagn Invest. 1990; 2:123-128.

5. Dorman, DC, Zachary, JF, Buck, WB. Neuropathologic findings of bromethalin toxicosis in the cat. Vet Pathol. 1992; 29:139-144.

6. Haynie RS, Bowerman WW, Williams SK, Morrison SK. Triploid grass carp susceptibility and potential for disease transfer when used to control aquatic vegetation in reservoirs with avian vacuolar myelinopathy. J Aquat Anim Health. 2013; 25(4):252-259.

7. Nakaue, HS, Dost, FN, Buhler, DR. Studies on the toxicity of hexachlorophene in the rat. Tox and Appl Pharm. 1973; 24: 239-249.

8. OShaughnessy, DJ, Losos, GJ. Peripheral and central nervous system lesions caused by triethyl- and trimethyltin salts in rats. Tox Path. 1986; 14(2).

9. Peterson, ME. Bromethalin Topical Review. Topics in Compan An Med. 2013; 28:21-23.

10. van Lier, RB, Cherry, LD. The toxicity and mechanism of action of bromethalin: a new single-feeding rodenticide. Fundam Appl Toxicol. 1988 Nov; 11(4):664-72.


Click the slide to view.



4-1. Colon, squirrel.


4-2. Stomach, squirrel.


4-3. Cerebrum and cerebellum. squirrel:


4-4. Cerebellum, squirrel.


4-5 Cerebrum, squirrel.



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