Discovery of potentially novel species of the Onchocercidae (Nematoda: Filarioidea) in Burmese fighting chickens (Gallus gallus): Genetic insights into avian filariasis and co-infection with Plasmodium juxtanucleare

Burmese fighting chickens (<i>Gallus gallus</i>) raised in Nakhon Ratchasima Province, eastern Thailand, were investigated for filarioids and co-infection with other haemoparasites. Microscopy and molecular diagnostic targeting the <i>cox</i>1 gene revealed that 2 out of 12 chickens tested positive for onchocercid filarioids. One chicken carried a single infection (Onchocercidae sp. CH09), characterized by unsheathed microfilariae with a cross-striated cuticle, a blunt anterior end, a short cephalic space, and a hook-like tail tip. Another chicken exhibited a mixed filarioid infection, necessitating the subcloning of two distinct isolates (Onchocercidae sp. CH07-S1 and CH07-S2). This chicken had two distinct microfilarial forms: (i) unsheathed microfilariae resembling Onchocercidae sp. CH09 and (ii) sheathed microfilariae with a short cephalic space and paired nuclei at both extremities. Genetic analyses of <i>cox</i>1 sequences demonstrated that these forms belong to two distinct species. Isolates CH09 and CH07-S1 closely resembled Onchocercidae sp. (<i>Eufilaria</i> sp.) ROE14 (GenBank: PQ564658) derived from a chicken in eastern Thailand. They constituted a clade phylogenetically distinct from other species of <i>Eufilaria</i> and Filarioidea in passerine birds and <i>Culex</i> mosquitoes, suggesting they may represent a potentially novel onchocercid or a distant <i>Eufilaria</i> species. CH07-S2 exhibited the closest genetic affinity to Onchocercidae sp. KLS08 (GenBank: PQ564657) derived from a chicken in eastern Thailand. Interestingly, they established a distinct clade from the other reported filarial genera that parasitize avian hosts, perhaps being a novel onchocercid species or genus within galliforms. <i>Plasmodium juxtanucleare</i> was identified in 6 out of 12 chickens, including one case of co-infection with onchocercids. Our study highlights the significance of molecular approaches in revealing filarial diversity and genetic relationships, while underscoring emerging hotspots of avian filariasis in Thailand and beyond.