Diverse Onchocercidae from Malaysian cats and Indonesian macaques: Morphological and molecular analysis of individual microfilariae using mitochondrial genomes, 28S rRNA, and Wolbachia endosymbiont sequences

During an investigation of animals as reservoirs for the filarial parasite Brugia malayi, three molecularly undescribed filarial species were co-detected. Individual microfilariae (Mf) were isolated and analyzed from blood samples of crab-eating macaques (Macaca fascicularis) from Belitung, Indonesia, and from pet dogs and cats in Sabah, Malaysia. Among 163 macaques, 33 (20.2%) were positive for large Mf (mean length 498.9 µm) similar to Dirofilaria ('Belitung I'). One macaque was infected with small Mf (mean length 150.4 µm) ('Belitung II'), with a high density of 17,150 Mf/mL. In two cats co-infected with B. malayi, Mf of a Dirofilaria species ('Sabah') with an average length of 299.1 µm were detected. Morphometric analysis of Mf showed distinct differences between these three species and other Mf described in the area. Whole genome amplification and genome sequencing of 24 individual Mf enabled phylogenetic analysis of mitochondrial genomes, and analysis of specific mitochondrial and nuclear barcode regions. The three Mf groups formed distinct clusters and could not be identified by comparison with available reference sequence. Cluster 'Belitung I' from macaques formed a sister group to other characterized Dirofilaria. Cluster 'Belitung II' included bird filariae and primate filariae of the genus Mansonella as close relatives. The cluster 'Sabah' formed a monophyletic group with the zoonotic species Dirofilaria asiatica and Dirofilaria sp. 'Thailand'. DNA of Wolbachia endobacteria was detected in Mf of 'Belitung I' and 'Sabah', but not in 'Belitung II'. These findings highlight the limited understanding of filarial diversity in macaques and cats in Asia and underscore the need for a more comprehensive approach that combines morphological and molecular data to identify and assess the pathogenicity and zoonotic potential of these parasites.