Baylisascaris-associated neural larval migrans in a group of juvenile Red-necked wallabies

Heindrich Snyman, John Sallaway, Patricia Bell-Rogers, Nathan Bennoit, Hugh Cai

Animal Health Laboratory, University of Guelph, Guelph, ON (Snyman, Bell-Rogers, Bennoit, Cai), Sallaway Equine Professional Corporation (Sallaway) 

AHL Newsletter, 2021;25(2):20.

In early winter, a group of 8 to10-month-old juvenile Red-necked wallabies (Macropus rufogriseus) from a captive zoo population experienced an unusual disease event.  Initially a single joey presented with sudden peracute death without any premonitory clinical abnormalities, followed shortly after by the onset of slow progressive neurological deterioration in a few juvenile cohorts.  Neurological abnormalities included ataxia, proprioceptive deficits, opisthotonus, and grand mal seizures provoked by somatosensory or visual stimulation.  An in-clinic postmortem on the first joey did not reveal any obvious gross abnormalities and a broad array of representative tissues were collected in formalin and submitted to the Animal Health Laboratory for histopathology.

Regionally within the white matter of the mesencephalon, brain stem, and cerebellum of this joey, there was prominent expansion of Virchow-Robbins spaces by dense aggregates of lymphocytes and macrophages up to ten cell layers thick (perivascular cuffing).  Scattered throughout the adjacent neuropil were multiple irregular malacic foci that were filled with glial cells, Gitter cells, lymphocytes, gemistocytic astrocytes, and rare individual neutrophils.  Given the predominantly mononuclear encephalitis, the concurrent onset of neurological symptoms in other individuals in this cohort, as well as a particular species susceptibility, acute toxoplasmosis was initially considered the most likely differential diagnosis.  However, PCR testing of formalin-fixed paraffin embedded (FFPE) tissue scrolls, as well as IHC staining of affected brain segments, tested negative for Toxoplasma gondii.

Due to progressive neurological decline, and a concern to maintain quality of life, an additional affected joey was euthanized and both fresh and formalin-fixed brain along with other tissues were submitted for further investigation.  Similar regionalized changes were present, this time within the brain stem, mesencephalon, and thalamus, however, both the perivascular and malacic inflammatory infiltrates also included small loose to moderately dense clusters of eosinophils.  In addition, there were a few small oval to oblong linear pockets filled with dense aggregates of Gitter cells that resembled acute malacic tracts.  Together with the eosinophilic inflammation, this was highly suspicious for aberrant metazoan nematode migration and indeed deeper sections of the malacic regions in the thalamic region revealed a few embedded characteristic ascarid nematode larvae and a diagnosis of neural larval migrans due to Baylisascaris sp. was made.

Further species confirmation was sought through a Baylisascaris sp. PCR test, targeting the mitochondrial Cytochrome-C oxidase subunit 2 gene (COX2).  Both fresh frozen brain as well as FFPE scrolls from the thalamic block from the second joey and FFPE scrolls from affected brain in the first joey were tested; a positive result was obtained only from the FFPE scrolls from the second joey.  Sequencing results of the PCR product were closely aligned to reported sequences for both Baylisascaris procyonis (raccoon roundworm) (305/306 base pairs, 99.67% identity) and Baylisascaris columnaris (skunk roundworm) (304/306 base pairs, 99.35% identity).  Although this close match didn’t allow for complete differentiation between the two species, the slightly higher homology and situational observation of multiple raccoon latrines on site suggest that raccoon roundworm remains the most likely etiology.  Perhaps the curious nature of these young individuals and investigation of latrine sites placed them at a higher risk for exposure than the older cohorts in their mob. 

B. procyonis is a well-known cause of neural larval migrans (cerebrospinal nemotodiasis), with infections having been recorded in many different species of birds and mammals.  The eggs can remain infective in raccoon latrine sites for many years.  The pathogenesis involves the emergence of larvae in the gastrointestinal tract which then migrate through the wall to the portal vasculature and liver, and then to the lungs where they enter systemic circulation and especially disseminate to the brain and eyes.  Most cases are encapsulated in an eosinophilic granuloma, but in some cases, the nematodes migrate throughout the brain leaving behind necrotic tracts filled with debris and eosinophilic inflammation.  In most cases the larvae are not visible in histological sections as the worms continue to migrate after the death of the animal.  Other species of Baylisascaris could also cause similar lesions, although infection with these species is far less common than infection with raccoon roundworm: B. melis (badgers), B. columnaris (skunks), B. laevis (woodchuck), B. schroederi (pandas), B. devosi (fishers and martens), and B. transfuga (bears).  Neural larval migrans from Aelurostrongylus cantonensis (rodent feces) is also a common differential.  

Negative test results with the fresh brain samples was probably due to the multifocal nature of this infection, simply indicating that no worms were present within the pieces of sampled brain.  This result, as well as the negative result obtained from FFPE scrolls from the first joey, probably also indicate minimal antigen shedding of the nematode during migration; therefore, best results with PCR testing would necessitate the physical presence of whole or fragments of nematodes in the sample.   AHL

Figure 1. Brain (thalamus) with perivascular cuffing and scattered foci of malacia and gliosis with five adjacent embedded cross and tangential sections of nematode larvae consistent with neural larval migrans of an ascarid nematode. H&E stain.

Figure 2. Larvae are ~ 30 to 50 µm wide with a 3-5 µm thick cuticle, prominent lateral chords and characteristic lateral alae, coelomyarian-polymyarian musculature, pseudocoelom, and an intestine lined by uninucleate columnar epithelial cells.  Eosinophils are present within the adjacent neuropil. H&E stain.

References

1. Church M, Terio K, Keel M. Chapter 12: Procyonidae, Viverridae, Hyenidae, Herpestidae, Eupleridae, and Prionodontidae.  In: Pathology of Wildlife and Zoo Animals.  Terio K, McAloose D and St. Leger J, eds. Elsevier, 2018:314-315.

2. Dangoudoubiyam S, et al. PCR assays for detection of Baylisascaris procyonis eggs and larvae. J Parasitol 2009;95(3):571-7.

3. Roug A, et al. Cerebral larva migrans caused by Baylisascaris spp. in a free-ranging North American porcupine (Erethizon dorsatum). J Wildl Dis 2016;52(3):763-5.