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

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.

Blood Parasites in Endangered Wildlife-Trypanosomes Discovered during a Survey of Haemoprotozoa from the Tasmanian Devil

“…While the present study reported the absence of Leishmania via PCR of blood samples, it is not possible to discount occult infection and future studies utilising additional sample types would be recommended. Although co-infections of Leishmania and Trypanosoma cannot be completely ruled out in the individuals tested here, the absence of Leishmania DNA is consistent with previous studies that have utilised the same assay on other Australian mammals [ 17 , 18 , 74 ]. Therefore, reports of Australia’s only endemic Leishmania species remain confined to kangaroos and biting midges (Ceratopogonidae) in the Northern Territory [ 71 , 75 , 76 ].…”

“…For example, bone marrow, skeletal muscle, tongue, brain, and liver samples have been tested positive for Trypanosoma DNA, while blood samples (including blood smears) were negative [ 87 ]. While there is a body of work demonstrating that Australian mammals are hosts to a range of endemic species of Trypanosoma , Leishmania , Babesia , Theileria , and Hepatozoon [ 17 , 18 , 19 , 31 ], the clinical impact of these haemoprotozoa is largely unknown. This is particularly important in the context of endangered species, such as the Tasmanian devil.…”

“…Over the past two decades, the use of molecular techniques has significantly extended our understanding of haemoprotozoan parasites. As a result, it has revealed that Australian marsupials and monotremes can host a high diversity of haemoprotozoa species, most of which are unique to Australia [ 17 , 18 , 19 , 20 ]. The clinical effects of co-infections in wildlife are often difficult to evaluate.…”

Haemoprotozoan surveillance in peri-urban native and introduced wildlife from Australia

“…The nearest named species, B. lohae , exhibited 98.3–98.6% similarity. According to Barbosa (2019) genetic distance thresholds within piroplasms at the 18S rRNA locus can be as little as 0.3% between named species. Therefore, it is likely that Babesia sequences from the brush-tailed possum in the present study represent a novel species.…”

Diversity and Epidemiology of Bat Trypanosomes: A One Health Perspective

“…was based solely on direct observation of the parasite in blood smears by light microscopy [ 5 ]. However, microscopy lacks sensitivity, particularly in chronic stages of infection and species descriptions based exclusively on morphological characterisation may be confounded by overlapping morphometry among species and polymorphic life-cycle stages in vertebrate hosts [ 74 , 136 , 137 ].…”