First report of Angiostrongylus vasorum (French heartworm) in red foxes (Vulpes vulpes) on Prince Edward Island

Abstract

Objective

To identify first-stage nematode larvae (L1) recovered from a red fox scat sample and adult female worms recovered from 2 red fox lungs at necropsy, using published molecular methods to confirm a morphological diagnosis of Angiostrongylus vasorum (French heartworm).

Animal

Red fox (Vulpes vulpes).

Procedure

Nematode larvae recovered from a Baermann examination survey of wild canid scats (n = 101) conducted from January 2017 to August 2020, were identified by size and morphology and subjected to PCR and DNA sequencing of the small subunit (SSU) rRNA gene, the large subunit (LSU) rRNA gene, or the second internal transcribed spacer (ITS2). In addition, these techniques were applied to adult female worms recovered from the heart/lungs of 2 red foxes (obtained from PEI trappers and stored frozen at −20°C since December of 2018 and 2020).

Results

Size and morphology of L1 recovered by Baermann examination from a wild canid scat sample (presumed to be red fox) collected near Montague, PEI and adult female worms recovered at necropsy from 2 red fox carcasses were identified as A. vasorum. Molecular analysis confirmed the larvae and adult worms were A. vasorum.

Conclusion

These findings indicated that A. vasorum has become endemic in the red fox population on PEI.

Clinical relevance

Angiostrongylus vasorum infection is potentially fatal in dogs. Veterinarians and regional diagnostic laboratories in the Maritime provinces should consider the possibility of A. vasorum infection in dogs with clinical signs of cardiopulmonary and/or central nervous system disease or bleeding disorders.

Angiostrongylus vasorum (French heartworm) is a meta-strongyloid nematode residing in the pulmonary artery and right ventricle of domestic and wild canids; the natural definitive hosts are the red fox (Vulpes vulpes) and various other species of foxes, which serve as reservoir hosts for dogs (1). Animals acquire infection by ingestion of infected intermediate hosts (land snails, slugs, frogs) or paratenic hosts (birds, frogs) (1,2). Recent studies have indicated that direct exposure to infective third-stage larvae (L3) released free into the environment from infected gastropods may be an additional transmission route (3,4). The epidemiological significance of paratenic hosts and environmental contamination in A. vasorum transmission has not been determined. The prepatent period is usually ~38 to 57 d, but ranges of 28 to 109 d have been reported (1,5). Infection in dogs can be subclinical or result in severe cardiopulmonary or other tissue damage and is potentially fatal. Clinical signs can be highly variable and the prodromol period can be months or years (1,5). The most common clinical sign reported in dogs infected with A. vasorum is a chronic cough, which may also be accompanied by dyspnea, exercise intolerance, and weight loss (5). Infection has also been associated with bleeding disorders and signs of ocular and central nervous system disease (1). Diagnosis occurs by detecting first-stage larvae (L1) shed in the feces of infected canids using the Baermann technique (5).

Angiostrongylus vasorum is considered endemic in parts of Africa, Europe, North America, and South America (1). Until the last decade, the single North American endemic focus had been on the island of Newfoundland. Computer modeling has suggested that conditions conducive to the spread of A. vasorum within North America (red fox, terrestrial gastropods) may occur throughout the eastern half of the continent and the states and province along the western coast (6). Due to the large red fox population, climatic conditions (high rainfall), and abundant terrestrial gastropod fauna, the threat of spread from Newfoundland to the Maritime provinces of New Brunswick (NB), Nova Scotia (NS), and Prince Edward Island (PEI) has been of particular concern (1). Recent research has indicated a spread of A. vasorum within North America, with infection in red foxes reported in West Virginia, USA, and in coyotes (Canis latrans) in Nova Scotia, Canada (7,8).

During a wild canid fecal scat survey (n = 101) conducted from January 2017 to August 2020, larvae identified as A. vasorum L1 based on size and morphology were recovered by Baermann examination from a scat sample presumed to be from a red fox collected near Montague, PEI (46 km east of Charlottetown in Kings County). The mean L1 length was 392 μm (range: 384 to 400 μm), and the caudal end terminated in a sinus wave-shaped kink with a dorsal spine (Figure 1). Larvae of Uncinaria stenocephala were also recovered from the sample. Confirmation of the morphological identification by molecular methods was attempted due to the possibility that the larvae were 1 of the species of Angiostrongylus infecting rodents in North America (A. blarini, A. michiganensis, and A. schmidti) (9). Larvae of these species could have been present in the scat sample due to fox predation of rodent hosts.

Figure 1.

First stage larvae of Angiostrongylus vasorum (A) recovered from a canid scat sample collected near Montague, PEI. The caudal end (B) terminates in a sinus wave-shaped kink with a dorsal spine (inset — high magnification image of tail morphology). The mean L1 length was 392 μm (range: 384 to 400 μm).

Figure 2.

Adult female Angiostrongylus vasorum nematodes recovered from the heart/lungs of a red fox trapped in December of 2018. Size bar = 2 mm.

DNA was extracted on 3 subsamples (~10 L1) from the original mixed sample comprising 80% U. stenocephala and 20% A. vasorum, using a DNeasy Blood and Tissue kit (Qiagen, Toronto, Ontario) following the manufacturer’s instructions, except that the proteinase K digestion was 18 h at 56°C. A ribosomal DNA (rDNA) Internal Transcribed Spacer 2 (ITS2) primer set AV5F (5′-CGATGACGGTAGCAATGACA-3′) and AV4R (5′-TTTGCGTGGTTCTTTACGTG-3′) specific for A. vasorum was used (10). In addition, 8 L1 of A. vasorum were isolated using the dissecting microscope from the mixed sample that contained U. stenocephala, DNA was extracted as before, and a partial fragment of the large subunit (LSU) rRNA gene sequence (~850 to 950 bp) was amplified using primers LSU537F (5′-GATCCGTAACTTCGGGAAAAGGAT-3′) and LSU531R (5′-CTTCGCAATGATAGGAAGAGCC-3′) (11).

The A. vasorum larval partial ITS 2 regions sequence (GenBank accession number: OK146880) shared 100% identity with A. vasorum from Europe (host: dog, Canis familiaris and red fox, Vulpes vulpes; MN178647, MN104952, GU045376, EU627593, EU627594, GU045374, EU627595, EU627592). The A. vasorum larval partial LSU rRNA gene region sequence (OK143439) isolated from a mixed infection with U. stenocephala shared 99.8% identity with A. vasorum (AM039758) from the red fox (Vulpes vulpes) from Canada. ITS2 and partial LSU rRNA gene were used successfully to confirm the presence of A. vasorum co-infected with U. stenocephala in a red fox scat sample.

Subsequently, during lung flushes of adult foxes (n = 76), 2 adult foxes contained 2 and 1 adult female nematodes, respectively. The 2 red foxes were trapped near Johnstons River (12 km east of Charlottetown, in Queens County) and near Larkens Pond (171 km east of Charlottetown, in Kings County). Both foxes had been frozen at −20°C between the time they were trapped in December 2018 and December 2020, respectively and the time of necropsy. Morphology of the adult worms was consistent with A. vasorum (~20 mm in length; lacked a buccal capsule and cuticular synlophe; vaginal opening located near the posterior-end of the worm). However, definitive identification of adult worms is primarily based on the morphology of the males (9). Adult worm tissue was treated as described previously for molecular identification of larvae and the DNA sequences were 100% identical to the larval isolate sequences.

Based on these results, the authors concluded that A. vasorum has spread to the red fox population of PEI and has been there since at least 2018. Currently, no data are available on the prevalence or geographic distribution within the red fox population on PEI. There was a 20-year interval in Newfoundland from the first report of A. vasorum infection in a red fox to the first diagnosis in a dog. Within 5 y after the first diagnosis in a dog, A. vasorum infection was reported in 24% of the cases of dogs with clinical signs of cardiopulmonary disease in Newfoundland (1). Although not definitively known, it is likely that the parasite slowly spread within the Newfoundland red fox population until reaching a sufficient level leading to exposure to dogs. Clinicians in NS, PEI, and NB should consider the possibility of A. vasorum infection in dogs presented with signs of chronic respiratory disease, bleeding disorders, or neurological disease. Dogs infected with French heartworm can be diagnosed by fecal examination using the Baermann technique. Due to the erratic fecal larval shedding pattern typical of most metastrongyloids, false-negative results can occur with a single Baermann examination. Therefore, multiple examinations (ideally 3) should be performed before ruling out A. vasorum infection (1). Collection of 3 consecutive day fecal samples in such cases may be advisable (5). Fenbendazole, milbemycin oxime, and moxidectin have all been used to treat cases of infection in dogs (1), whereas milbemycin oxime and moxidectin have been used as monthly preventative treatments (12,13). CVJ