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

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