Mosquito bloodmeals can be used to determine vertebrate diversity, host preference, and pathogen exposure in humans and wildlife.

The surveillance and detection of zoonotic pathogens in animals is essential for predicting disease transmission pathways and the risks of spillover, but challenges include the costs, ethics and technical expertise required for vertebrate trapping, serum sampling and antibody or virus screening. Surveillance using haematophagous arthropods as a sampling tool offers a unique opportunity to obtain blood samples from a wide range of vertebrate species, allowing the study of host-mosquito associations, and host exposure to pathogens. We explored vertebrate diversity and potential Ross River virus (RRV) transmission pathways by analysing blood-fed mosquitoes collected in Brisbane, Australia. Host origins were identified using barcode sequencing, and host exposure to RRV was assessed using a modified plaque reduction neutralisation test. In total, 480 blood-fed mosquitoes were collected between February 2021 and May 2022. The host origins of 346 (72%) bloodmeals were identified, with humans (73%) and cattle (9%) comprising the dominant hosts. RRV seroprevalence was high in both vertebrate species with evidence of RRV exposure in 70% (21/30) of cattle and 52% (132/253) of humans. This is a novel, non-invasive method of estimating seroprevalence in vertebrate host populations. Our results highlight the potential of blood-fed mosquitoes to provide species-specific insights into pathogen transmission dynamics.