Increased genetic diversity and prevalence of co-infection with Trypanosoma spp. in koalas (Phascolarctos cinereus) and their ticks identified using next-generation sequencing (NGS).

Amanda D Barbosa,Alexander W Gofton,Amber Gillett,Una Ryan,Telleasha Greay,Andrea Paparini,Annachiara Codello,Peter Irwin,Kristin Warren,Vyacheslav Yurchenko

doi:10.1371/journal.pone.0181279

Abstract

Infections with Trypanosoma spp. have been associated with poor health and decreased survival of koalas (Phascolarctos cinereus), particularly in the presence of concurrent pathogens such as Chlamydia and koala retrovirus. The present study describes the application of a next-generation sequencing (NGS)-based assay to characterise the prevalence and genetic diversity of trypanosome communities in koalas and two native species of ticks (Ixodes holocyclus and I. tasmani) removed from koala hosts. Among 168 koalas tested, 32.2% (95% CI: 25.2–39.8%) were positive for at least one Trypanosoma sp. Previously described Trypanosoma spp. from koalas were identified, including T. irwini (32.1%, 95% CI: 25.2–39.8%), T. gilletti (25%, 95% CI: 18.7–32.3%), T. copemani (27.4%, 95% CI: 20.8–34.8%) and T. vegrandis (10.1%, 95% CI: 6.0–15.7%). Trypanosoma noyesi was detected for the first time in koalas, although at a low prevalence (0.6% 95% CI: 0–3.3%), and a novel species (Trypanosoma sp. AB-2017) was identified at a prevalence of 4.8% (95% CI: 2.1–9.2%). Mixed infections with up to five species were present in 27.4% (95% CI: 21–35%) of the koalas, which was significantly higher than the prevalence of single infections 4.8% (95% CI: 2–9%). Overall, a considerably higher proportion (79.7%) of the Trypanosoma sequences isolated from koala blood samples were identified as T. irwini, suggesting this is the dominant species. Co-infections involving T. gilletti, T. irwini, T. copemani, T. vegrandis and Trypanosoma sp. AB-2017 were also detected in ticks, with T. gilletti and T. copemani being the dominant species within the invertebrate hosts. Direct Sanger sequencing of Trypanosoma 18S rRNA gene amplicons was also performed and results revealed that this method was only able to identify the genotypes with greater amount of reads (according to NGS) within koala samples, which highlights the advantages of NGS in detecting mixed infections. The present study provides new insights on the natural genetic diversity of Trypanosoma communities infecting koalas and constitutes a benchmark for future clinical and epidemiological studies required to quantify the contribution of trypanosome infections on koala survival rates.

Highlights

Trypanosomes are blood-borne protozoans of veterinary and medical clinical significance
Sanger sequencing of amplicons obtained from these 59 samples revealed the presence of three species: T. irwini, T. gilletti and T. copemani, at prevalences of 28.6%, 1.2% and 5.4%, respectively
A total of 23 ticks were positive by single-round PCR, specific Trypanosoma DNA sequences were only obtained for 21 of these samples by Sanger sequencing (23.1%, 95% CI: 14.9–33.1%)

Summary

Introduction

Trypanosomes are blood-borne protozoans of veterinary and medical clinical significance. Koala populations in New South Wales (NSW) and Queensland (Qld) have been listed as vulnerable under the NSW Threatened Species Conservation Act 1995 and the Qld Nature Conservation (Wildlife) Regulation 2006, respectively [10, 11]. Chlamydiosis caused by Chlamydia pecorum is the main disease contributing to koala population decline [12], acting in synergy with many other variables adversely affecting koala survival (such as concurrent infections with koala retrovirus-KoRV, habitat loss, vehicle collisions, climate change and dog attacks) [13, 14]. Preliminary data suggests that trypanosome infections may be compromising the health of wild koalas, those with clinical signs of concurrent diseases, thereby contributing to population decline events [3]

Methods

Results

Discussion

Conclusion