The mitochondrial genome of Angiostrongylus mackerrasae is distinct from A. cantonensis and A. malaysiensis

Abstract

The native rat lungworm (Angiostrongylus mackerrasae) and the invasive rat lungworm (Angiostrongylus cantonensis) occur in eastern Australia. The species identity of A. mackerrasae remained unquestioned until relatively recently, when compilation of mtDNA data indicated that A. mackerrasae sensu Aghazadeh et al. (2015b) clusters within A. cantonensis based on their mitochondrial genomes (mtDNA). To re-evaluate the species identity of A. mackerrasae, we sought material that would be morphologically conspecific with A. mackerrasae. We combined morphological and molecular approaches to confirm or refute the specific status of A. mackerrasae. Nematodes conspecific with A. mackerrasae from Rattus fuscipes and Rattus rattus were collected in Queensland, Australia. Morphologically identified A. mackerrasae voucher specimens were characterized using amplification of cox1 followed by the generation of reference complete mtDNA. The morphologically distinct A. cantonensis, A. mackerrasae and A. malaysiensis are genetically distinguishable forming a monophyletic mtDNA lineage. We conclude that A. mackerrasae sensu Aghazadeh et al. (2015b) is a misidentified specimen of A. cantonensis. The availability of the mtDNA genome of A. mackerrasae enables its unequivocal genetic identification and differentiation from other Angiostrongylus species.

Further studies of neuroangiostrongyliasis (rat lungworm disease) in Australian dogs: 92 new cases (2010–2020) and results for a novel, highly sensitive qPCR assay

“…This assay was subsequently utilized by VPDS. Briefly, total DNA was obtained from L 1 A. cantonensis SYD.1 isolated using faecal samples from an infected rat (Červená et al, 2019;Valentyne et al, 2020). DNA was isolated from a single L 1 and 3 L 1 that had been stored in 80% ethanol.…”

“…Angiostrongylus qPCR was run using primers (S0947, S0948) and probe (S0949) targeting AcanR3990 (William Sears et al, 2020, submitted). This highly sensitive qPCR assay amplifies DNA of both A. cantonensis and A. mackerrasae, the 2 Angiostrongylus species endemic in rats in Australia (Červená et al, 2019;Valentyne et al, 2020). All primers and a fluorescently labelled probe (FAM/ZEN/3 ′ IBFQ) were ordered from Integrated DNA Technologies Australia (Baulkham Hills, Australia).…”

Using cerebrospinal fluid to confirm Angiostrongylus cantonensis as the cause of canine neuroangiostrongyliasis in Australia where A. cantonensis and Angiostrongylus mackerrasae co-exist

“…As a generalisation, NA is assumed to be caused by infection with A. cantonensis ( Barratt et al., 2016 ). In Australia, however, two Angiostrongylus species – the invasive emerging A. cantonensis and the native A. mackerrasae – are known to be neurotropic in their definitive rat hosts ( Bhaibulaya, 1974 ; Červená et al., 2019 ; Valentyne et al., 2020 ). A lack of methods enabling species differentiation has prevented the definitive confirmation or exclusion of A. mackerrasae as a cause of clinical disease in dogs.…”

“…Recently, the species status of A. cantonensis and A. mackerrasae was re-confirmed using complete mitochondrial (mt) DNA, where a 7.1–7.5% difference between the two was demonstrated ( Valentyne et al., 2020 ). The molecular discrimination of Angiostrongylus spp.…”

“…To do so, we utilised archived and curated CSF samples from canine NA cases from Australia ( Lee et al., 2021 ). The PCR assay targeted the highly variable cox 1 region, which is able to discriminate reference A. cantonensis DNA from that of A. mackerrasae ( Valentyne et al., 2020 ). The cox 1 DNA sequence results were further verified by the adoption of a nuclear marker enabling DNA sequence analysis of ITS2 rDNA assay, which was capable of amplifying both A. cantonensis and A. mackerrasae .…”