History

A 21-year-old 0.67-kg (1.47-lb) sexually intact female Indian flying fox (Pteropus giganteus) born and housed at the Baton Rouge Zoo was brought to the zoo's veterinary hospital because of lethargy, a perceived decreased desire to forage for food, and a generally poor state of well-being.

Clinical and Gross Findings

An initial physical examination was performed when the bat was awake and subsequently when it was anesthetized with isoflurane. The bat had grade IV/IV periodontal disease and was obese. Serum biochemical analysis revealed no notable abnormalities. The bat had an excellent appetite when hand fed but would not go to its bowl to eat or drink. Saline (0.9% NaCl) solution (50 mL/kg [22.7 mL/lb], SC, q 72 h) and meloxicam (0.2 mg/kg [0.09 mg/lb], SC, once and then 0.1 mg/kg [0.045 mg/lb], PO, q 24 h) were administered. Three days after initiation of SC fluid therapy and NSAID treatment, the bat developed neurologic signs, including a continuous right-sided head tilt and failure to right itself to urinate and defecate. After 2 days of persistent neurologic signs, the animal was anesthetized with isoflurane and euthanized by means of an IV barbiturate overdose owing to poor quality of life.

The result of a direct immunofluorescence antibody test for rabies virus was negative, and the bat underwent necropsy at the Louisiana Animal Disease Diagnostic Laboratory. Gross abnormalities included focally extensive (2 × 1-cm) hemorrhage along the left lateral temporal lobe with diffuse prominent congestion of meningeal vessels, and a focal 1 × 1-cm area of malacia and hemorrhage within the base of the right ventral brainstem (Figure 1).

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Photographs of the left lateral (A) and ventral surfaces (B) of the brain of an adult female Indian flying fox (Pteropus giganteus) that initially was evaluated because of lethargy, failure to eat normally, and generally not doing well. The bat was euthanized, and necropsy was performed to determine the cause of persistent neurologic signs that were refractory to treatment. In panel A, notice the focally extensive meningeal hemorrhage along the left temporal lobe (asterisk). In panel B, there is a focal area of unilateral malacia and hemorrhage within the right side of the brainstem (asterisk). Note that lissencephaly is normal in bats, hence the absence of cerebral sulci and gyri.

Citation: Journal of the American Veterinary Medical Association 252, 5; 10.2460/javma.252.5.545

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Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→

Histopathologic Findings

At necropsy, various tissue samples were collected and fixed in neutral-buffered 10% formalin, routinely processed, paraffin-embedded, sectioned at 5-μm intervals, and stained with H&E stain. The area of meningeal hemorrhage observed over the left lateral temporal lobe (Figure 1) was characterized by leptomeningeal expansion composed predominantly of neutrophils with lesser numbers of lymphocytes and plasma cells mixed with hemorrhage and cross-sections of larval nematodes. Nematodes measured approximately 170 to 220 μm in diameter and were characterized by a smooth eosinophilic cuticle, coelomyarian-polymyarian musculature, basophilic lateral cords, and an intestinal tract lined by a few large multinucleated giant cells (Figure 2). Larval nematode reproductive organs were not observed in the sections examined. A mixture of viable and degenerate nematodes was also visible in the white matter of the left cerebral hemisphere, the lumen of the left lateral ventricle, and the stroma of the choroid plexus. Viable nematodes were commonly associated with tracks of malacia containing large numbers of gitter cells and hemorrhage. Degenerate nematodes were surrounded by lymphocytes and plasma cells, with fewer eosinophils and macrophages (including multinucleated giant cells). The unilateral focus of malacia and hemorrhage observed in the right aspect of the brainstem was characterized by dissociation and rarefaction of the neuropil, with abundant infiltration by gitter cells, mononuclear perivascular cuffing, hemorrhage, and fibrin deposition.

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Photomicrographs of sections of the cerebrum and brainstem from the Indian flying fox in Figure 1. A—Section of the left temporal cerebrum and overlying leptomeninges. The latter is expanded predominantly by neutrophils mixed with hemorrhage, with a single cross section of an intralesional larval nematode (170 μm in diameter) characterized by a smooth eosinophilic cuticle, coelomyarian-polymyarian musculature, basophilic lateral cords, and an intestinal tract lined by a few large multinucleated giant cells. H&E stain; bar = 100 μm. B—Section of the left lateral ventricle. The ventricular lumen contains a single longitudinal section of a larval nematode (1.2 mm in length) with similar histologic features and mononuclear perivascular cuffing of an adjacent blood vessel composed of lymphocytes, plasma cells, and rare eosinophils. H&E stain; bar = 250 μm. C—Section of the choroid plexus. The choroid plexus stroma is locally infiltrated by a mixture of inflammatory cells, predominantly lymphocytes, macrophages, and multinucleated giant cells. A mild amount of fibrin and hemorrhage are present within the adjacent ventricular system. H&E stain; bar = 250 μm. D—Section of the right side of the brainstem that had malacia and hemorrhage grossly. Severe regional necrosis with gitter cell infiltration, hemorrhage, and generalized mononuclear perivascular cuffing are evident histologically. H&E stain; bar = 500 μm.

Citation: Journal of the American Veterinary Medical Association 252, 5; 10.2460/javma.252.5.545

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Additional Laboratory Testing

Detection of Angiostrongylus cantonensis nucleic acid was achieved through use of a previously published quantitative PCR technique¹ and 100 ng of DNA extracted from 2 samples of approximately 100 to 200 mg of fresh-frozen brain tissue. The 2 samples were run in replicate for a total of 4 reactions. All 4 reactions yielded positive results with threshold cycle values of 27.64 and 27.59 for the sample 1 replicates and 35.72 and 37.16 for the sample 2 replicates. Replicate negative controls remained negative for A cantonensis nucleic acid.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: chronic, multifocal, granulomatous, and eosinophilic meningoencephalitis with malacia, hemorrhage, and intralesional larval nematodes consistent with A cantonensis.

Case summary: aberrant cerebral angiostrongyliasis in an Indian flying fox.

Comments

For the bat of the present report, a diagnosis of aberrant cerebral larval migrans attributable to A cantonensis was made on the basis of a combination of molecular and histologic findings. Aberrant cerebral migration of A cantonensis is considered a neglected emerging disease.² Additional infectious CNS diseases previously described in flying foxes include rhabdoviral encephalitides (ie, rabies and non-rabies lyssaviruses), tick paralysis, toxoplasmosis, listeriosis, bacterial meningoencephalitis, and amebic encephalitis caused by Balamuthia mandrillaris.^3–6

Several genera of rats are known to be definitive hosts for A cantonensis, a zoonotic metastrongylid nematode.^2,7 Whereas adult nematodes reside in the lungs of the definitive hosts and are not typically associated with clinical disease, aberrant CNS migration in dead-end hosts can result in severe clinical disease and death. To date, humans and multiple domestic, zoo, and wildlife species have been identified as dead-end hosts in North America; affected nonhuman dead-end hosts that have been reported include a white-handed gibbon, horses, dogs, a lemur, an armadillo (unpublished data), and opossums.^8–10 In Australia, dogs, horses, marsupials, birds, and flying foxes are known to have been affected.^1,11–16

Definitive and dead-end hosts become infected with A cantonensis after consuming L3 larvae carried by intermediate mollusks hosts (snails and slugs), contaminated raw vegetables containing intermediate hosts or their slime, or paratenic hosts that consume mollusks such as freshwater crustaceans, amphibians, and monitor lizards.² A single snail has been documented as harboring > 10,000 infective L3 larvae.⁸ Following consumption, the L3 larvae migrate to the host's CNS through the bloodstream; in dead-end hosts, larval development ceases and results in a potentially life-threatening reaction. In contrast, minimal host reaction occurs in the CNS of definitive hosts; further maturation of L3 larvae into young adults occurs over a 2-week period, followed by their migration to regional venous systems and, ultimately, residence within pulmonary arteries wherein they become sexually mature adults and lay eggs. The eggs hatch in terminal pulmonary arteries, develop into L1 larvae, and migrate to the pharynx where they are swallowed and excreted in host feces 6 to 8 weeks later. The L1 larvae develop to infectious L3 larvae within the aforementioned intermediate hosts. The prepatent period (infection to detection of eggs in the feces) is 6 weeks.

Traditionally considered endemic in areas of Asia and the Pacific Basin, more recent reports of angiostrongyliasis have emerged from the United States (Southeastern states and Hawaii), Brazil, and Caribbean islands.^2,8,9 Infection with A cantonensis was first documented in the contiguous United States in 1987 and was alleged to have been introduced by infected rats from ships docking in New Orleans.¹⁷ This is supported by survey data obtained by Tulane University, where 113 rats (Norway rats [Rattus norvegicus; n = 94] and black rats [Rattus rattus; 19]) trapped between 1962 and 1976 were negative for A cantonensis infection, whereas 20 of 94 (21.3%) Norway rats trapped between April 1986 and February 1987 were infected.¹⁷ Enhanced global distribution of A cantonensis outside traditionally endemic regions is attributed to increased introduction of both infected rats and mollusks to new environments through establishment of global transport networks.

In humans, aberrant cerebral migration of A cantonensis can be readily prevented by avoiding consumption of raw intermediate and paratenic hosts, in part by thoroughly washing fruits and vegetables prior to consumption. In the veterinary field, however, vertebrate animals routinely consume— whether intentionally or accidently—intermediate or paratenic hosts, which makes disease prevention more challenging. Prevention represents a cultural challenge in some parts of the world, such as Thailand, where inhabitants of the northeastern provinces are known to consume koi-koi, a traditional raw snail dish.¹⁸ Consumption of raw or partially cooked livers from monitor lizards has also been reported as a means of infection in Bangkok.¹⁹

Clinical signs of aberrant cerebral migration of A cantonensis in humans can be nonspecific and include severe headache, paresthesia, nausea, and vomiting; in severe cases, coma and death can result.²⁰ In veterinary medicine, clinical signs associated with aberrant cerebral migration by A cantonensis in dogs are well described and include a variable combination of altered mentation, hind limb weakness, hyperesthesia, upper and lower motor neuron signs in the hind limbs, and urinary incontinence.¹ In both human and veterinary medicine, a presumptive diagnosis of aberrant cerebral migration of A cantonensis is supported by detection of eosinophilia in peripheral blood or CSF samples and anti-A cantonensis immunoglobulins (IgG) in CSF samples (determined by ELISA or western blot testing).^1,20 A quantitative PCR assay has also been developed to assess for the presence of A cantonensis nucleic acids in blood.²¹ Treatment of aberrant cerebral migration of A cantonensis in human and veterinary patients includes immuno-suppressive therapy (typically glucocorticoid administration) with or without concurrent anthelmintic treatment.^1,20 Treatment with anthelminthic agents alone is not recommended because of the potential for host anaphylaxis in response to dead and dying nematodes. Administration of broad-spectrum antimicrobials to veterinary patients is justified given the increased risk of bacterial infections associated with prolonged glucocorticoid treatment.¹

Two months prior to evaluation of the case described in the present report, another adult Indian flying fox born in captivity and living in the same enclosure was determined to have aberrant cerebral migration associated with intralesional nematodes. Although a definitive diagnosis was not achieved through use of PCR assays, as in the present case, the etiologic agent was presumed to have also been A cantonensis on the basis of similar histologic features. In contrast, the other bat was found dead with no premonitory signs and had no gross CNS findings. For both bats, exposure to A cantonensis was believed to have been through ingestion of vegetables or fruits contaminated with intermediate slugs or snails. At the zoo, increased efforts to prevent future infections centered on close monitoring of food for snail and slug intermediate hosts and thorough washing of fruits and vegetables prior to feeding; since implementation of these measures, no additional cases have occurred. To the authors' knowledge, this report represents the first documented case of A cantonensis infection in an Indian flying fox within North America, highlighting the importance of routine necropsy of exotic animals that have the potential to serve as sentinels for zoonotic diseases.