DETECTION AND CHARACTERIZATION OF RICKETTSIAE IN WESTERN AUSTRALIA

The aim of this study was to address the shortfall in current, in-depth knowledge of Western Australian rickettsiae investigating in particular, the role of native and feral animals as reservoir hosts. Two novel species of rickettsiae were detected in ticks collected from native and feral animals and humans, these were characterised using molecular methods. A preliminary investigation into the pathogenic potential of one of the isolates was also undertaken. To facilitate investigation, ectoparasites (principally ticks) were opportunistically collected from across the State, with an emphasis on native and feral animals and people. All ectoparasites were screened for rickettsial infection using a polymerase chain reaction incorporating Rickettsia-specific citrate synthase gene (gltA) primers. Preliminary sequencing was performed on representative PCR-positive samples from each geographical location, vertebrate host and ectoparasite in order to identify and characterize the infecting rickettsia. Isolation in cell culture and further genotypic characterization was then performed. Finally, a serosurvey and questionnaire were implemented in one of the study areas to determine whether people were being infected with a Rickettsia spp. and whether infection was associated with clinical signs. Ectoparasite collection produced three genera of ticks (Ixodes, Amblyomma and Haemaphysalis) from native animals, feral pigs and people, primarily from the southwest of Western Australia and Barrow Island in the Pilbara region. Ticks from a number of sources were shown to be infected with rickettsiae by the PCR, including feral pigs, people, bobtail lizards, kangaroos, bandicoots, burrowing bettongs, common brushtail possums and yellow-footed antechinus. Genotypic characterization of positive amplicons from ticks revealed the presence of two novel spotted fever group rickettsiae. Rickettsia gravesii sp. nov., named in honour of Dr Stephen Graves, was identified extensively throughout the southwest of the State and on Barrow Island in Ixodes, Amblyomma and Haemaphysalis spp. ticks from multiple hosts. Candidatus Rickettsia antechini was detected in Ixodes spp. only from yellow-footed antechinus in Dwellingup. In addition, a novel Bartonella spp. (Bartonella sp. strain Mu1) was also detected from Acanthopsylla jordani fleas collected from yellow-footed antechinus in Dwellingup. Rickettsia gravesii sp. nov. is most closely related to the Rickettsia massiliae subgroup of the spotted fever group and to R. rhipicephali in particular. Sequence similarities between this novel species and the subgroup were 99.7%, 98.4%, 95.8% and 97.4% based on its 16S rRNA, gltA, ompA and ompB genes respectively. Candidatus Rickettsia antechini also demonstrated a close relationship to the R. massiliae subgroup (99.4%, 94.8% and 97.1% sequence similarity based on its gltA, ompA and ompB genes respectively). The two novel Western Australian species demonstrated 98.4%, 96.3% and 96.7% sequence similarity to each other based on gltA, ompA and ompB genes respectively indicating separate species. The novel Bartonella spp. (Bartonella sp. strain Mu1) detected in fleas collected from yellow-footed antechinus in Dwellingup demonstrated greatest gltA gene sequence similarity to Bartonella strain 40 at 86.1%. Results from the serosurvey and questionnaire-based investigation into the zoonotic importance of R. gravesii sp. nov. on Barrow Island supported the results of the tick study and suggested that a tick-borne rickettsia(e) was infecting people on the island. However, a significant association between seroconversion and a history of symptoms consistent with a rickettsiosis was not found, and it is possible therefore, that R. gravesii sp. nov. produces only asymptomatic infections. Future work on rickettsiae in Western Australia will involve phenotypic characterization of the novel species, further investigation of their epidemiology and pathogenicity and an ongoing search for additional undiscovered species.