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Molecular discovery and genomic characterisation of Australian Tick-bourne bacteria

Gofton, Alexander (2018) Molecular discovery and genomic characterisation of Australian Tick-bourne bacteria. PhD thesis, Murdoch University.

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Abstract

Collectively, ticks transmit the greatest variety of pathogenic microorganisms of any arthropod group, and worldwide tick-borne pathogens are emerging infectious threats to people, domestic animals, and wildlife. Bacterial pathogens in the genera Anaplasma, Ehrlichia, Neoehrlichia (Family: Anaplasmataceae), and Borrelia (Family: Spirochaetaceae) are responsible for a large proportion of tick-borne infections worldwide and contain a high diversity of species, each with their own ecological niche comprising different vertebrate and tick hosts. Although well studied around the world due to their pathogenic effects on people and animals, until recently no members of these genera had been identified as indigenous to Australia. Recent findings indicate that in fact Australia does contain a wide diversity of native Anaplasma, Ehrlichia, Neoehrlichia, and Borrelia spp., however the initial identification of these organisms was based on short DNA sequences that contained insufficient genetic information for accurate taxonomic identification. The aim of this thesis was to confirm the presence of these species in native Australian ticks and to investigate the molecular systematics, taxonomy, and genomics of newly identified Australian Anaplasma, Ehrlichia, Neoehrlichia, and Borrelia spp.

Initially, due to their medical and veterinary significance, two native Australian tick species, Ixodes holocyclus and Amblyomma triguttatum subsp. were examined for novel Anaplasma, Ehrlichia, and Neoehrlichia spp. A total of 391 questing and blood-fed I. holocyclus nymphs and adults were sampled from almost the entire enzootic range of the tick from coastal north Queensland to coastal southern New South Wales, and 609 questing Am. triguttatum subsp. nymph and adult ticks were sampled from four distinct locations in southwest Western Australia (Yanchep National Park and Bungendore Regional Park), northern Western Australia (Barrow Island), and South Australia (Innes National Park). Ticks were screened for novel Anaplasma, Ehrlichia, and Neoehrlichia spp. using genus-specific PCR and qPCR assays and broader pan-Anaplasmataceae nested PCR assays.

Two new Neoehrlichia spp. named ‘Candidatus Neoehrlichia australis’ and ‘Candidatus Neoehrlichia arcana’ were identified in 8.7% and 3.1% of I. holocyclus samples, respectively, and co-infections with both species were detected in two samples.

Phylogenetic reconstructions of 16S rRNA (1,264 bp), groEL (1,047 bp), and gltA (561 bp) gene sequences clearly indicated that these new species were highly similar to, but distinct from other (northern hemisphere) Neoehrlichia spp., and formed a highly supported monophyletic clade with other Neoehrlichia spp.

In Am. triguttatum subsp. ticks a new Ehrlichia sp. named ‘Candidatus Ehrlichia occidentalis’ was identified in samples from southwest Western Australia and South Australia, but not northern Western Australia, with prevalences ranging from 4.2% in Innes National Park, South Australia, to 15.2% in Bungendore Regional Park in southwest Western Australia. Phylogenetic analyses of 16S rRNA (1,281 bp), groEL (1,067 bp), gltA (1,033 bp), and map1 (655 bp) gene sequences indicated that ‘Can. Ehrlichia occidentalis’ is distinct from all previous described Ehrlichia spp., but is most closely related to E. ruminantium and Ehrlichia sp. Panola Mtn., and forms a monophyletic clade with these species. Additionally, novel A. bovis genotypes were identified in 4.3% of tick samples from Yanchep National Park and 1.4% of samples from Barrow Island. This was the first detection of A. bovis in Australia, and phylogenetic analysis of 16S rRNA (1,265 bp) and groEL (1,184 bp) gene sequences show that they are distinct and peripheral from all other genotypes worldwide, but cluster with geographically similar genotypes from Southeast Asia. This data suggests that A. bovis may have entered the Australian continent long after its separation from other Gondwanan landmasses, perhaps during the Miocene (20-5 mya), when Australia was much closer to the Indonesian archipelago and radiations of bats and rodents first entered Australia.

Additionally, the bacterial microbiome of the platypus tick, I. ornithorhynchi, was investigated with 16S rRNA gene metabarcoding, uncovering another new Australian Ehrlichia sp. that was distinct from ‘Candidatus Ehrlichia occidentalis’. This Ehrlichia sp. was also identified in I. ornithorhynchi ticks and platypus (Ornithorhynchus anatinus) blood samples from Queensland and Tasmania that were screened with pan-Anaplasmataceae PCR assays, and 16S rRNA (1,343 bp), groEL (1,074 bp), and gltA (1,004 bp) gene sequences were produced. Intracellular inclusions consistent with Ehrlichia spp. were also observed in blood smears from Tasmanian platypuses that were positive for the novel Ehrlichia, but not in negative samples. Phylogenetic reconstructions showed conclusively that this Ehrlichia sp. was distinct from all previously described species, however, phylogentically clustered with high support with ‘Candidatus Ehrlichia khabarensis’ that was recently identified in voles in far eastern Russia, and was named ‘Candidatus Ehrlichia ornithorhynchi’.

Following the discovery of endemic Anaplasma, Ehrlichia, and Neoehrlichia spp. in native Australian ticks, an investigation co-led by the Department of Agriculture and Food, Western Australia was conducted to confirm the presence of a novel Ehrlichia sp. that had been detected in the spleen of a Bos indicus Brahman weaner calf during a routine post mortem investigation. Initially, molecular screening for A. marginale returned a short (200 bp) E. canis-like 16S rRNA sequence, however taxonomic discrimination was not possible with the short sequence, and confirmatory testing by the Australian Animal Health Laboratory did not detect any Ehrlichia or Anaplasma spp. Therefore, spleen samples were sent to the Murdoch University Vector and Water-Borne Pathogens Research Laboratory where confirmatory testing was performed with pan-Anaplasmataceae and Ehrlichia-specific PCR assays.

Testing confirmed the presence of Ehrlichia DNA in the spleen tissue, and phylogenetic analysis of Ehrlichia 16S rRNA (1,126 bp), groEL (1,040 bp), and dsb (362 bp) gene sequences indicated with high confidence that the species was E. mineirensis, a recently described species transmitted by Rhipicephalus microplus sensu lato ticks, which has been found naturally infecting cattle and mule deer in South America and Canada, where it does not cause clinical illness in cattle under natural settings. Additionally, a 16S rRNA sequence (955 bp) was also produced which was consistent with ‘Candidatus Anaplasma boleense’, a novel Anaplasma species previously identified in ticks, mosquitoes and a variety of bovines, cervines, and other wild mammals in China and Malaysia. This is the first identification of these organisms in Australia, and it is unknown how endemic these organisms are in Australia or whether they recently entered the continent with imported cattle. The animal from which these organisms were identified did not have clinical or pathological findings consistent with anaplasmosis or ehrlichiosis, and evidence from this case and overseas suggests that E. mineirensis is not a major threat to animal health. Nevertheless, it is unknown what effects these organisms may have when co-infected with tick-borne cattle fever agents such as A. marginale, and further research is needed to understand its endemicity and potential impacts on cattle health in Australia.

Finally, in collaboration with the German National Reference Centre for Borrelia, high throughput sequencing and metagenomic assembly techniques were used to construct the genomes of the recently described Australian Borrelia sp., ‘Candidatus Borrelia tachyglossi’, and B. turcica, a closely related species associated with tortoises. These Borrelia spp. are of high interest because of their unique phylogenetic position compared to the Lyme borreliosis-causing and relapsing fever-associated clades, and their unique but poorly understood associations with ixodid vectors and reptile and monotreme hosts. The genome of ‘Candidatus Borrelia tachyglossi’ was sequenced directly from a single engorged female Bothriocroton concolor tick that was removed from an echidna (Tachyglossus aculeatus) host using Illumina technology, and after assembly the full linear chromosome (937,305 bp), as well as three incomplete linear plasmids and one complete circular plasmids was recovered. Borrelia turcica, however, was sequenced from a cultured isolate (IST7), using both Illumina and Pacific Biosciences single molecule real time technologies, to produce a complete genome containing the entire linear chromosome (957,653 bp), five complete linear plasmids, and one complete circular plasmid.

Comparative genomics revealed that B. turcica and ‘Candidatus Borrelia tachyglossi’ have distinct relapsing fever-like genomes that contain characteristics such as evolutionary conserved spirochete orthologous gene clusters, horizontally acquired purine metabolism genes, large linear plasmids (megaplasmids), and components of a multiphasic antigen switching system that are unique to relapsing fever Borrelia spp. Nevertheless, whole genome phylogenetic inference highlights the evolutionary divergence between these species and the main relapsing fever Borrelia clade, and showed that these species diverged from the relapsing fever lineage very early after the Lyme borreliosis-relapsing fever split. The genomes of B. turcica and ‘Candidatus Borrelia tachyglossi’ also showed signs of lineage specific adaptation, such as expanded suites of macronutrient (purine, glycogen, and maltose) metabolism genes, and signs of genomic decay, with numerous genes involved in post transcriptional tRNA nucleoside modification either degraded into pseudogenes or deleted from the genomes. Additionally, despite being more closely related to relapsing fever Borrelia spp., these Borrelia species shared some ancestral orthologs with the Lyme borreliosis Borrelia clade, potentially indicating that these diverse organisms share some conserved phenotypic characteristics.

Work presented in this thesis provides the first insight into the diversity of tick-borne Anaplasmataceae in Australia and begins to explore their molecular systematics and taxonomy. Furthermore, assembling and characterising the genomes of B. turcica and ‘Candidatus Borrelia tachyglossi’ has resulted in significant insights into their biology and potential phenotypes in vertebrate and ticks, and provides an important foundation for continued research into Borrelia taxonomy, biology, and genomics. The outcomes of this thesis significantly informs our view of Australian ticks and their associated microorganisms and form the genesis of a wide range of research projects investigating further the biology, ecology, and potential health impacts of emerging Anaplasmataceae and Borrelia diversity in Australia.

Item Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor(s): Irwin, Peter and Ryan, Una
URI: http://researchrepository.murdoch.edu.au/id/eprint/42855
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