| Summary: | Both Angiostrongylus cantonensis and Angiostrongylus mackerrasae have been identified along the east coast of Australia. A lack of A. mackerrasae genomic data until 2019, however, has precluded the unequivocal identification of the Angiostrongylus species responsible for neuroangiostrongyliasis in accidental hosts such as dog and man. The availability of a whole-genome data for A. mackerrasae, including mtDNA and ITS2 rDNA, enables discrimination of A. cantonensis from A. mackerrasae. The aim of this study was to develop diagnostic PCR assays to determine the species of Angiostrongylus based on the detection of Angiostrongylus DNA sequences in the cerebrospinal fluid (CSF) of canine patients with eosinophilic meningitis. An in silico workflow utilising available cytochrome c oxidase 1 (cox1) primers streamlined the laboratory work into empirical steps, allowing optimisation and selection of a PCR assay that met the required criteria for discrimination of A. cantonensis and A. mackerrasae DNA in low-template CSF samples. The adopted cox1 qPCR assay specifically amplified and enabled the differentiation of A. cantonensis from A. mackerrasae DNA and confirmed the presence of A. cantonensis DNA in 11/50 archived CSF samples. The DNA sequences demonstrated the presence of two distinct A. cantonensis cox1 haplotypes in dogs from eastern Australia. Species identification was further confirmed via the adoption of an ITS2 rDNA assay, providing confirmation of only A. cantonensis ITS2 rDNA in the CSF samples. To our knowledge, this is the first study to unequivocally demonstrate the antemortem presence of A. cantonensis DNA in CSF from clinically affected dogs. The study confirmed the long-held assumption that A. cantonensis is the causal agent of neuroangiostrongyliasis but refutes the dogma that there was a single introduction of A. cantonensis into Australia by the demonstration of two distinct A. cantonensis cox1 haplotypes.
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