Detection and characterization of Bartonella species in Western Australia

In this study, the prevalence and genetic diversity of Bartonella species in various arthropod vectors from both wild and domestic animals in Australia were investigated using nested-polymerase chain reaction (PCR) assays and multilocus sequence analysis (MLSA). Previous studies on Bartonella species in Australia have been confined to mammalian hosts, including humans, cats, native rodents and eastern grey kangaroos. However, little is known about the status of bartonellae in arthropod vectors, which is essential in understanding the transmission dynamics of the organisms. To facilitate the investigation, ectoparasites (ticks and fleas) were collected from both wild and domestic animals from various locations in Australia. All ectoparasites were screened for Bartonella species using newly designed nested-PCRs targeting the gltA gene (citrate synthase) and the ribosomal internal transcribed spacer (ITS) region, developed as part of the present study. Multilocus sequence analysis of the 16S ribosomal RNA (rRNA), citrate synthase (gltA),cell division protein (ftsZ) and RNA polymerasebeta-subunit (rpoB) genes and the ribosomal ITS region was applied to identify and confirm the status of all Bartonella species identified in this study. Multilocus sequence analysis of the cytochrome oxidase subunit I (COI) and 18S ribosomal RNA (rRNA) genes of flea vectors harbouring a diversity of Bartonella species were analysed to characterize the extent of genetic diversity in the flea vectors and to elucidate vector-parasite associations. A phylogenetic analysis of the 5 concatenated loci identified 3 novel Bartonella species in flea vectors from marsupials in Western Australia. Candidatus Bartonella antechini was detected in fleas (Acanthopsylla jordani) from mardos (Antechinus flavipes - also called the yellow-footed antechinus). Candidatus Bartonella woyliei was detected in fleas (Pygiopsylla hilli), from brush-tailed bettongs (Bettongia penicillata– also called woylies), and Candidatus Bartonella bandicootii was detected in Pygiopsylla tunneyi fleas from western barred bandicoots (Perameles bougainville). Furthermore, a potential novel species, Bartonella sp. strain WC2 was detected in ticks (Ixodes australiensis) from woylies based on the criterion of a genetic similarity of less than 96% of the gltA locus compared with other validated Bartonella species. In the present study, the grouping of marsupial-derived Bartonella species confirmed the existence of a marsupial cluster of Bartonella species in Australia, which appears to have evolved separately to Bartonella species in other mammals. The detection of the known zoonotic Bartonella species, B. henselae and B. clarridgeiae in red foxes and their fleas (Ctenocephalides felis), indicated that red foxes could be an important reservoir of Bartonella infections for other animals and humans in the same geographical locality. Bartonella henselae and B. clarridgeiae DNA were also detected from fleas collected from pet cats in the same area. The genetic association of these zoonotic Bartonella species detected in wildlife and pet animals has demonstrated and confirmed the distribution of zoonotic Bartonella species in fleas from both wild and domestic animals in this region and a possible ecological association between the animal species. The genetic clustering of Bartonella species and flea vectors with their Australian fauna hosts suggests co-evolution of hosts, fleas and Bartonella species in Australia. In conclusion, the close association between Australian fauna, Australian fleas and Bartonella species suggests adaptation by Bartonella species to a specific ecological niche, comprised of specific mammalian hosts and specific flea vectors in particular environments.