Multiplex PCR for differentiating Ornithodoros tick vectors in South Africa: Proof of concept

African swine fever (ASF) is a severe viral haemorrhagic disease affecting domestic pigs, maintained by ASF virus within an ancient sylvatic cycle involving Ornithodoros ticks that inhabit warthog burrows. This study aimed to develop and optimise a multiplex polymerase chain reaction (PCR) assay to differentiate three Ornithodoros species in South Africa - O. moubata, O. phacochoerus and O. waterbergensis, to enhance ASF vector identification and management. The final multiplex PCR effectively distinguished between species with specific amplicon sizes: 180 base pairs (bp) for O. moubata, 640 bp for O. phacochoerus and 1000 bp for O. waterbergensis.Contribution: This refined multiplex PCR method demonstrates proof of concept as a reliable and rapid tool for distinguishing Ornithodoros species and correlating them with their geographical origins. This assay, based on amplification size, provides crucial information about the distribution of these species, which could enhance ASF control efforts.