Development and Validation of Novel PCR Assays for the Diagnosis of Bovine Stephanofilariasis and Detection of Stephanofilaria sp. Nematodes in Vector Flies

Background: Stephanofilaria spp. nematodes are associated with cutaneous lesions in cattle and other livestock mammalian wildlife species. In Australia, Haematobia irritans exigua, commonly known as buffalo fly (BF) transmits a well-described but presently unnamed species of Stephanofilaria, which has been speculatively implicated the aetiology BF lesions. The sensitivity current techniques for detecting skin vector is low, there no genomic sequence any member genus currently available databases. Methods: To develop molecular assays detection Australian sp., biopsies were collected from freshly slaughtered typical near medial canthus. Adult microfilariae isolated using saline recovery technique. morphologically identified sp. by scanning electron microscopy. DNA was extracted internal transcribed spacer 2 (ITS2) region rDNA, cytochrome c oxidase subunit 1 (cox1) mtDNA amplified sequenced. specific polymerase chain reaction (PCR) qPCR (SYBR Green® TaqMan™) developed optimised novel ITS2 obtained. specificity each assay confirmed testing against nematode Onchocerca gibsoni Dirofilaria immitis, well host (bovine) DNA. Results: Scanning microscopy anterior posterior ends A phylogenetic analysis cox1 demonstrated that this most closely related to Thelazia callipaeda, parasitic common cause thelaziasis (or eyeworm infestation) humans, dogs, cats. Both conventional specifically Conventional PCR, TaqMan™, SYBR shown detect ng, pg, 100 fg DNA, respectively. detected single microfilaria. Conclusion: Molecular diagnostic study showed high availability an accurate sensitive PCR will assist determining its role pathogenesis lesions, understanding epidemiological dynamics. This may also have application use studies particularly S. stilesi, require confirmation.