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Identification and characterisation of microorganisms in Australian wildlife ticks

Loh, Siew May (2018) Identification and characterisation of microorganisms in Australian wildlife ticks. PhD thesis, Murdoch University.

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Globally, ticks are well known vectors of a number of pathogens, including members of the genera Anaplasma, Borrelia, Ehrlichia, Babesia, and Theileria, which are associated with a range of tick-borne diseases (TBD). These bacteria and blood-borne parasites are widely studied overseas due to their significant medical and veterinary impacts. In Australia, in recent decades, research into ticks and TBD has been focused on mammalian hosts, particularly livestock and companion animals. The relationship between wildlife and their ticks, on the other hand, are less studied. Despite disease concerns associated with ticks in Australia caused primarily by the Australian paralysis tick, Ixodes holocyclus, and other introduced species such as the bush tick, Haemaphysalis longicornis, and the brown dog tick, Rhipicephalus sanguineus, the impacts of native wildlife ticks are not known. The main objective of this thesis was to identify and characterise novel microorganisms and potential tick-borne pathogens residing in ticks that parasitise Australian wildlife.

Wildlife ticks (n=1,000) were sourced across Australia from wildlife hospitals and sanctuaries, and genomic DNA (gDNA) was extracted from a subsample (n=196). Following the amplification of the bacterial 16S rRNA gene V1-V2 hypervariable region, next generation sequencing (NGS) on the Illumina MiSeq platform was used to assess the microbial composition. Results revealed the presence of a Borrelia species in Bothriocroton concolor ticks collected from echidnas (Tachyglossus aculeatus), prompting further screening of additional B. concolor ticks (n=97) using primers targeting the 16S ribosomal RNA (rRNA), flagellin (flaB), the 60 kDa heat shock protein (groEL), the glycerophosphodiester phosphodiesterase (glpQ), and the DNA gyrase (gyrB) genes. Sanger sequencing and phylogenetic analysis revealed that the Borrelia sp. identified in the B. concolor ticks was unique and distantly related to the Relapsing Fever Borrelia, with the reptile-associated (REP) Borrelia group, its sister taxa. Therefore, this novel species was described phylogenetically and named ‘Candidatus Borrelia tachyglossi’, representing the first Borrelia species to be molecularly characterised in native Australian ticks.

Next Generation Sequencing analyses of the bacterial 16S rRNA gene V1-V2 hypervariable region also revealed the presence of Ehrlichia spp. belonging to the Anaplasmataceae family, which were further characterised using Sanger sequencing and phylogenetic analysis of the 16S rRNA gene, the citrate synthase (gltA) and groEL genes to evaluate the relationship between the Ehrlichia detected in the echidna ticks to other Ehrlichia spp. Phylogenetic analyses showed that the Ehrlichia sequences derived from the echidna ticks were unique and shared the closest nucleotide identity (99.2%) with ‘Candidatus Ehrlichia ornithorhynchi’ detected in platypuses and I. ornithorhynchi ticks, clustering with ‘Candidatus Ehrlichia khabarensis’ as their closest sister species, while dissimilar to other described Ehrlichia species.

This thesis also presents the first identification of a Theileria fuliginosa-like species in Ixodes australiensis ticks collected from western grey kangaroos (Macropus fuliginosus) in Western Australia. In addition, Theileria orientalis was identified in one Haemaphysalis longicornis tick from a red fox (Vulpes vulpes) and new genotypes of Theileria and Babesia spp. were identified in ticks from bandicoots (Perameles nasuta) and brushtail possums (Trichosurus vulpecula), respectively. Collectively, phylogenetic analyses of the Babesia and Theileria spp. derived from Australian marsupials demonstrates that they are unique and different from their overseas counterparts. Currently, little is known about the pathogenicity of these newly identified bacteria and piroplasms in wildlife ticks.

In conclusion, the results presented in this thesis provide an essential foundation for future research and valuable insights into the presence of important tick-borne microorganisms and the microbial diversity in ticks associated with Australian wildlife.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Life Sciences
United Nations SDGs: Goal 15: Life on Land
Supervisor(s): Oskam, Charlotte, Irwin, Peter and Ryan, Una
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