Sequence analyses at mitochondrial and nuclear loci reveal a novel Theileria sp. and aid in the phylogenetic resolution of piroplasms from Australian marsupials and ticks.

Liisa A Ahlstrom,Amanda D Barbosa,Charlotte L Oskam,Peter J Irwin,Timothy J Portas,Una M Ryan,Jill Austen,J Anthony Friend

doi:10.1371/journal.pone.0225822

Abstract

The order Piroplasmida encompasses two main families: Babesiidae and Theileriidae, containing tick-borne pathogens of veterinary and medical importance worldwide. While only three genera (Babesia, Cytauxzoon and Theileria) comprising piroplasm parasites are currently recognised, phylogenetic studies at the 18S rRNA (18S) gene suggest that these organisms represent at least ten lineages, one of which comprises the relatively unique and highly diverse Theileria spp. from Australian marsupials and ticks. As an alternative to analysing 18S sequences alone, sequencing of mitochondrial genes has proven to be useful for the elucidation of evolutionary relationships amongst some groups of piroplasms. This research aimed to characterise piroplasms from Australian native mammals and ticks using multiple genetic markers (18S, cytochrome c, oxidase subunit III (cox3) and cytochrome B (cytB)) and microscopy. For this, nearly complete piroplasm-18S sequences were obtained from 32 animals belonging to six marsupial species: eastern bettong (Bettongia gaimardi), eastern quoll (Dasyurus viverrinus), eastern grey kangaroo (Macropus giganteus), swamp wallaby (Wallabia bicolor), quokka (Setonix brachyurus) and Gilbert’s potoroo (Potorous gilbertii). The organisms detected represented eight novel Theileria genotypes, which formed five sub-clades within the main marsupial clade containing previously reported Australian marsupial and tick-derived Theileria spp. A selection of both novel and previously described Australian piroplasms at the 18S were also successfully characterised, for the first time, at the cox3 and cytB loci, and corroborated the position of Australian native theilerias in a separate, well-supported clade. Analyses of the cox3 and cytB genes also aided in the taxonomic resolution within the clade of Australian Piroplasmida. Importantly, microscopy and molecular analysis at multiple loci led to the discovery of a unique piroplasm species that clustered with the Australian marsupial theilerias, for which we propose the name Theileria lupei n. sp.

Highlights

Piroplasm is a generic term used to describe apicomplexan haemoprotozoan parasites belonging to the order Piroplasmida
Six blood samples were collected from animals belonging to four marsupial species housed at Tidbinbilla Nature Reserve in the Australian Capital Territory (ACT): eastern bettong (Bettongia gaimardi), eastern quoll (Dasyurus viverrinus), eastern grey kangaroo (Macropus giganteus) and swamp wallaby (Wallabia bicolor) (Table 1)
The 33 nearly complete marsupial-derived piroplasm 18S sequences obtained in the present study represented eight distinct Theileria genotypes, which formed five sub-clades (A-E) within the main clade containing previously reported Australian marsupial/tick-derived Theileria (Fig 1A)

Summary

Introduction

Piroplasm is a generic term used to describe apicomplexan haemoprotozoan parasites belonging to the order Piroplasmida This order encompasses two main families: Babesiidae and Theileriidae, containing three main genera (Babesia, Theileria and Cytauxzoon) which are tick-borne pathogens of veterinary and medical importance worldwide [1, 2]. Classical taxonomy of piroplasms has been based predominantly on transmission dynamics, host cell type(s) infected, vertebrate host preferences, and parasite morphology [8, 9]. Theileria and Cytauxzoon species are thought to be limited to transstadial transmission in the tick and undergo extra-erythrocytic schizogony in nucleated cells, prior to the erythrocytic stage. Piroplasms belonging to the Babesia sensu stricto taxonomic group exhibit transstadial and transovarial transmission in the tick and exclusive infection of erythrocytes in the vertebrate host. Based on morphological features, the babesias are broadly grouped into two forms: small Babesia (trophozoites are 1.0 to 2.5 μm; e.g., B. gibsoni, B. rodhaini) and large Babesia (2.5 to 5.0 μm; e.g., B. bovis, B. caballi) [13]

Objectives

Methods

Results

Discussion

Conclusion