Haemoprotozoan surveillance in peri-urban native and introduced wildlife from Australia.

Vector-borne haemoprotozoans comprise a diverse group of eukaryote single-celled organisms transmitted by haematophagous (blood-feeding) invertebrates. They can cause debilitating diseases that impact wildlife, livestock, companion animals and humans. Recent research has shown that Australian wildlife host a diverse range of haemoprotozoan species; however, to date this work has primarily been confined to a few host species or isolated populations in rural habitats. There has been little investigation into the presence of these blood parasites in wildlife inhabiting urban and peri-urban areas. In this study, blood and tissue samples and ticks were collected from wildlife in New South Wales and Western Australia. Extracted DNA samples were screened with pan-specific molecular assays to determine the presence of haemoprotozoans using amplicon metabarcoding and Sanger sequencing approaches. In addition, light microscopy was performed on blood films. Eight haemoprotozoans were identified in the present study, which included species of , , and . Blood samples were collected from 134 animals; 70 black rats (), 18 common brush-tailed possums (), two bush rats (), 22 chuditch (), 20 long-nosed bandicoots (), one quenda () and one swamp rat (). Molecular screening of DNA extracted from blood samples identified 52.2% (95% CI: 43.8-60.5%) of individuals were positive for at least one haemoprotozoan species, with 19.4% (95% CI: 13.4-26.7%) positive for more than one species. The present study provides the first sequences of cf. from black rats and long-nosed bandicoots. was identified from brush-tailed possums. Two genotypes were identified from black rats and bush rats. Black rats showed the highest haemoprotozoan diversity, with five species identified. No known human pathogens that have been described in the northern hemisphere were identified in the present study, and future work is required to understand the zoonotic potential of these microbes in Australia. This work represents the first large-scale body of research using molecular tools to investigate haemoprotozoans in animals at the urban-wildland interface. Further research is needed to investigate potential consequences of infection in wildlife, particularly effects of pathogen spillover from invasive black rats to native wildlife.