Barbosa Amanda D, Gofton Alexander W, Paparini Andrea, Codello Annachiara, Greay Telleasha, Gillett Amber, Warren Kristin, Irwin Peter, Ryan Una
School of Veterinary and Life Sciences, Murdoch University, Murdoch, Perth, Western Australia.
CAPES Foundation, Ministry of Education of Brazil, Brasília, Distrito Federal, Brazil.
PLoS One. 2017 Jul 13;12(7):e0181279. doi: 10.1371/journal.pone.0181279. eCollection 2017.
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.
感染锥虫属物种与考拉（灰大袋鼠）健康状况不佳和存活率降低有关，尤其是在同时存在诸如衣原体和考拉逆转录病毒等病原体的情况下。本研究描述了基于下一代测序（NGS）的检测方法在表征考拉以及从考拉宿主身上采集的两种本地蜱虫物种（全环硬蜱和塔斯马尼亚硬蜱）体内锥虫群落的患病率和遗传多样性方面的应用。在接受检测的168只考拉中，32.2%（95%置信区间：25.2 - 39.8%）至少对一种锥虫呈阳性。鉴定出了先前描述过的来自考拉的锥虫物种，包括伊尔文锥虫（32.1%，95%置信区间：25.2 - 39.8%）、吉氏锥虫（25%，95%置信区间：18.7 - 32.3%）、科普曼尼锥虫（27.4%，95%置信区间：20.8 - 34.8%）和大锥虫（10.1%，95%置信区间：6.0 - 15.7%）。诺耶斯锥虫首次在考拉中被检测到，尽管患病率较低（0.6%，95%置信区间：0 - 3.3%），并且还鉴定出了一个新物种（AB - 2017锥虫），患病率为4.8%（95%置信区间：2.1 - 9.2%）。27.4%（95%置信区间：21 - 35%）的考拉存在多达五种物种的混合感染，这显著高于单一感染的患病率4.8%（95%置信区间：2 - 9%）。总体而言，从考拉血样中分离出的锥虫序列中，相当高比例（79.7%）被鉴定为伊尔文锥虫，表明这是优势物种。在蜱虫中也检测到了涉及吉氏锥虫、伊尔文锥虫、科普曼尼锥虫、大锥虫和AB - 2017锥虫的共感染，吉氏锥虫和科普曼尼锥虫是无脊椎动物宿主中的优势物种。还对锥虫18S rRNA基因扩增子进行了直接桑格测序，结果表明该方法仅能识别考拉样本中（根据NGS）读取量较大的基因型，这突出了NGS在检测混合感染方面的优势。本研究为感染考拉的锥虫群落的自然遗传多样性提供了新见解，并为未来量化锥虫感染对考拉存活率的贡献所需的临床和流行病学研究奠定了基准。
