Neurologic Baylisascaris procyonis infection in a young dog

Abstract

A 14-week-old female Boston terrier-cross dog with intermittent gastroenteritis and an eosinophilia developed progressive neurologic disease with ataxia progressing to uncontrolled paddling. Autopsy revealed Baylisascaris procyonis larvae in 4 of 7 brain sections, with severe eosinophilic meningoencephalitis. Diagnosis was confirmed with polymerase chain reaction (PCR) and DNA sequencing tests of fresh and paraffin-embedded brain in conjunction with the compatible histologic appearance.

Case description

In the fall of 2017, a 3.5 kg 14-week-old female Boston terrier-cross dog with severe and progressive neurologic disease was euthanized and submitted to the Animal Health Laboratory (AHL) at the University of Guelph for necropsy examination to document the severity and possible cause of the neurologic disease. The puppy had 5 unaffected littermates and was fostered in 2 homes, first with its mother and littermates, and later with 2 other dogs. The dog had a history of intermittent gastroenteritis the previous 5 wk, and radiographs revealed delayed gastric emptying. A zinc sulfate suspension with centrifugation fecal examination was negative for eggs (Antech Laboratories, Mississauga, Ontario). The puppy had an eosinophilia of 8.86 × 10⁹/L [reference interval (RI): 0 to 1.2 × 10⁹/L] and a lymphocytosis of 7.08 × 10⁹/L (RI: 0.69 to 4.50 × 10⁹/L). Ataxia developed 8 d before euthanasia and was progressive. One week before death the dog developed hypermetria and intention tremors. Over the course of its illness the dog had been treated with various antibiotics, antiemetics, and anticonvulsants with some fluid therapy. Despite some initial perceived improvement, the dog subsequently became agitated with decreased mentation and uncontrolled paddling. The dog was euthanized.

An autopsy was conducted on the chilled body 42 h after euthanasia. The dog was in good body condition with no significant external or internal findings.

Routine histology with preparation of hematoxylin and eosin (H&E)-stained slides was performed on major organs (lung, brain, liver, spleen, thyroid, pancreas, kidney, adrenal, skeletal muscle, heart, and intestine). Significant lesions were only seen in brain tissue. Ten sections of brain tissue examined included medulla oblongata, cerebellum, thalamus, hippocampus, as well as anterior brain stem including basal nuclei and cerebral cortex. In all sections there was severe eosinophilic meningoencephalitis with areas of necrosis and micro-abscessation. Multinucleate giant cells, histiocytes, plasma cells, and lymphocytes were commonly seen in many areas and a similar eosinophil-rich infiltrate was present in meninges. In 4 of 7 examined sections, a total of 5 small non-degenerate larval nematodes were seen within brain parenchyma. Sometimes larvae were degenerate with a severe surrounding inflammatory cell infiltrate (Figures 1A, 1B), and sometimes they were well-preserved with no significant inflammatory response evident (Figures 1C, D, E). The sections of non-degenerate larvae had a mean minimum diameter (not including alae) of 42.4 μm (range: 28.2 to 58.5 μm; n = 5), but some of these measurements were not taken mid-body. The largest diameter measured was 58.5 μm without alae or 68 μm with alae, and size and morphological features were compatible with Baylisascaris procyonis (Figure 1D) (1).

Figure 1.

A — Baylisascaris procyonis degenerate remnants (long arrow) in the brain surrounded by inflammatory cells, including some multinucleate giant cells (short arrow). Hematoxylin and eosin (H&E) stain. B — Closer view of degenerate B. procyonis (arrow) with lateral alae visible. H&E. C, D, and E — well-preserved B. procyonis larvae with no significant inflammation associated with them. D — Arrow shows the lateral alae. H&E.

A centrifugal floatation of feces obtained from the animal at necropsy did not reveal any parasites (2).

Three samples were collected from the non-fixed frozen brain. These consisted of the anterior cerebral cortex, junction of gray and white matter in the anterior cortex, and the anterior brain stem. Scrolls (consecutive microtome shavings from the paraffin histology blocks) were also prepared from 3 of the several paraffin blocks in which the parasites were clearly seen. The fresh-frozen tissue (~25 mg) was homogenized in TriReagent (Invitrogen, Waltham, Massachusetts, USA) using 2 stainless steel beads in a Mini-Beadbeater (BioSpec Products, Bartlesville, Oklahoma, USA). Nucleic acid was extracted from 100 μL of the homogenate with a MagMAX Express-96 instrument (Life Technologies, Burlington, Ontario) using the MagMAXPathogen RNA/DNA Kit (Life Technologies) with the low cell content protocol provided by the manufacturer. In addition, the 3 scroll samples (10 μm) of formalin-fixed paraffin-embedded (FFPE) dog brain tissue from the mesencephalon had DNA extracted using a QIAamp DNA FFPE kit (Qiagen, Toronto, Ontario) following the manufacturer’s protocol. The DNA extracts were tested by polymerase chain reaction (PCR) specific for B. procyonis mitochondrial cox2 gene (3). Among different tissue samples tested, brain white matter and the FFPE samples were positive with specific PCR products (146 bp) using primer sets BpF/R. Thereafter, primers Bpcox2F/2R were used to amplify a 529 bp region of the cox2 gene (3). The PCR products were purified and sequenced using both forward and reverse primers with Applied Biosystems 3730 DNA Analyzer (Life Technologies). The sequences were assembled into a consensus sequence using a bioinformatic program (Geneious 9.1.8; Biomatters, Auckland, New Zealand). After the primer sequences were trimmed, the sequences (485 bp) were used to perform a Blast search for homology sequences in the GenBank (National Center for Biotechnology Information, NCBI). The results revealed that the cox2 gene sequence of the samples had 100% homology with described B. procyonis, 99.8% with B. columnaris, 92.8% with Baylisascaris transfuga, 92.2% with Toxacaris leonine, and < 92% with other ascarid or non-ascarid nematodes. Based on the specific PCR results and sequence analysis, the larvae in this dog’s brain were confirmed to be Baylisascaris spp., most likely B. procyonis.

Discussion

This report describes the occurrence of B. procyonis larva migrans in the brain of a 14-week-old female Boston terrier-cross dog.

Baylisascaris procyonis is the common roundworm of raccoons (Procyon lotor). It has a typical ascarid life cycle with adult female worms in the raccoon intestine depositing eggs that are shed in the raccoon feces. Infected raccoons may shed 20 000 to 26 000 eggs/g (epg) of feces, with higher shedding found in juveniles (1,4). How long this high rate of shedding occurs is uncertain. In adult raccoons 1 study found 11 of 28 adult raccoons infected, with a mean of 6454 (200 to 23 600) epg of feces compared to a mean of 29 719 (200 to 228 000) epg in 62 infected juvenile raccoons, indicating reduced egg shedding due to lower numbers of adult worms with increasing raccoon age (5). Eggs can survive for years in the environment and are resistant to common disinfectants. In optimal temperature and moisture eggs develop infected larvae in 11 to 14 d. Infection is by ingesting eggs — the most common route in younger raccoons, or ingestion of third-stage larvae that are encysted within paratenic hosts — normally small rodents. Raccoons tend to defecate in favored areas referred to as “raccoon latrines,” found most often at the base or crotch of trees, but also in barn lofts, woodpiles, and other locations where large numbers of B. procyonis eggs can be found. The feces typically contain small pieces of grain or seeds, drawing small rodents and birds to these areas to pick the grain out, thus becoming exposed to the infected eggs (1,4).

Baylisascaris procyonis is a recognized zoonotic pathogen that causes visceral larva migrans in humans, mostly commonly in young children. Severe damage can occur which is sometimes fatal and is due to brain and eye migration of the larval form (6,7). Baylisascaris procyonis infection affects a wide variety of birds and mammals including dogs (1,4,8,9). In addition, dogs can serve both as definitive or paratenic hosts (1,4).

Raccoons are recognized for their threat to urban pets and their human owners as potential carriers for rabies, especially because of the recent resurgence of raccoon rabies in southwestern Ontario. It is our opinion, however, that B. procyonis, although much more common than rabies in raccoons, is not widely recognized by the public as a canine or human health hazard. We know that B. procyonis is common in the wild raccoon population of southern Ontario based on studies done on non-captive wild raccoons from the grounds of the Metropolitan Toronto Zoo where 44% (n = 10) of examined raccoons had a heavy burden (10). A more recent study detected B. procyonis in 38% (n = 128) of raccoons submitted to the Canadian Cooperative Wildlife Health Centre from southern Ontario; the parasite being found about equally in both rural and urban habitats (11).

A source of the B. procyonis was not determined in this case. The puppy and littermates were all healthy when the litter was fostered out to new homes at 8.5 wk of age and the foster owners at the first home reported that up to that age, exposure to raccoon or skunk feces was very unlikely. Given the time frame, it seems more likely that the puppy was exposed when living at the second foster home. The puppy was reported to spend some time under a spruce tree in the back yard, and had been to a horse farm, so it may have come across a “raccoon latrine” in one of these locations and eaten infected raccoon feces. The time required for the development of clinical signs after exposure is not known in dogs but is likely 2 to 4 wk depending on the infectious dose (4). In paratenic hosts, the larvae reach 50 to 80 μm in diameter 2 to 4 wk after infection (12). In lemurs clinical signs have been reported at approximately 2 wk after exposure (13). In mice CNS disease is apparent about 10 to 20 d after infection depending on the species of mouse (14).

Major differential diagnoses of cerebral larva migrans are B. procyonis, B. columnaris, and Toxocara canis with B. procyonis being the most commonly reported. Less likely other species of Baylisascaris such as B. columnaris in skunks can also cause visceral or cerebral larval migrans experimentally in mice (4), and in an urban setting where both raccoons and skunks are present, either ascarid may be involved, although nearly all reported cases have been linked to raccoons. The nematode can be identified as Baylisascaris as opposed to Toxocara species in histologic sections by their larger mid-body diameter (about 60 μm in this case), much larger single lateral alae, a prominent centrally located intestine lined by columnar cells and prominent lateral cords (Figures 1C, D, E) (1). In comparison Toxocara larvae measure 16 to 20 μm at mid-body and have much smaller alae, with the larvae having relatively larger round excretory columns dominating the body (15,16). Testing by PCR confirmed the larvae as B. procyonis or B. columnaris; however, the minor difference in the COX2 gene sequence (100% versus 99.8%, respectively) is probably not significant. In this case the parasite seen is likely to be B. procyonis because of the high percentages of this ascarid in identified cases (4) and the recognized much higher pathogenicity of B. procyonis than B. columnaris (1). Ideally, more loci should be examined to confirm the parasite as B. procyonis which can be differentiated from B. columnaris based on the SNP in NADH dehydrogenase subunit 2 (ND2), COX1 and tRNA genes in addition to COX2 gene (17). However, COX2 gene PCR was the only successful PCR on brain tissue in this case. Other PCR with long targets (1200 to 2000 bp) did not work, possibly because we did not have the purified parasite.

Of concern is the close co-habitation of humans and dogs to raccoons and their exposure to infective raccoon feces. Young orphan raccoons are cute and may be kept and handled by children. In addition, some dogs infected by B. procyonis can serve as patent hosts, further contaminating their environment with egg-laden feces.

Fatal B. procyonis larva migrans with similar lesions has been reported in dogs (9); however, we are not aware of any reports in Canada. It should be considered in dogs, especially if an eosinophilia is present and there are non-specific progressive neurologic signs. In addition, because of the tendency for raccoons to defecate in favored places and the longevity of the B. procyonis eggs, humans should be aware of the hazard represented by these “raccoon latrines” and try to recognize them, keeping dogs and children away from these areas.