Abstract：
BACKGROUND: Although wild ungulate populations are heavily monitored throughout Europe, we understand little of how parasites affect population dynamics, and there is no systematic, long-term monitoring of parasite diversity and parasite loads. Such monitoring is in part hampered by a lack of time- and cost-effective assay methodologies with high sensitivity and good taxonomic resolution. DNA metabarcoding has been successfully used to characterize the parasitic nemabiome with high taxonomic resolution in a variety of wild and domestic hosts. However, in order to implement this technique in large-scale, potentially non-invasive monitoring of gastrointestinal parasitic nematodes (GIN), protocol optimization is required to maximize biodiversity detection, whilst maintaining time- and cost-effectiveness.
METHODS: Faecal samples were collected from a wild moose population and GIN communities were characterized and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2 region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and cell lysis method.
RESULTS: Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative, but the proportion of target nematode sequences recovered was correlated with the parasitologically determined parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method that included mechanical cell disruption and maximized the starting material volume.
CONCLUSIONS: DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation method that involves mechanical cell disruption and maximizes the starting material volume.
METHODS: Faecal samples were collected from a wild moose population and GIN communities were characterized and quantified using both parasitological techniques (egg and larva counting) and DNA metabarcoding of the ITS2 region of rDNA. Three different isolation methods were compared that differed in the volume of starting material and cell lysis method.
RESULTS: Similar nematode faunas were recovered from all samples using both parasitological and metabarcoding methods, and the approaches were largely congruent. However, metabarcoding assays showed better taxonomic resolution and slightly higher sensitivity than egg and larvae counts. The metabarcoding was not strictly quantitative, but the proportion of target nematode sequences recovered was correlated with the parasitologically determined parasite load. Species detection rates in the metabarcoding assays were maximized using a DNA isolation method that included mechanical cell disruption and maximized the starting material volume.
CONCLUSIONS: DNA metabarcoding is a promising technique for the non-invasive, large-scale monitoring of parasitic GINs in wild ungulate populations, owing to its high taxonomic resolution, increased assay sensitivity, and time- and cost-effectiveness. Although metabarcoding is not a strictly quantitative method, it may nonetheless be possible to create a management- and conservation-relevant index for the host parasite load from this data. To optimize the detection rates and time- and cost-effectiveness of metabarcoding assays, we recommend choosing a DNA isolation method that involves mechanical cell disruption and maximizes the starting material volume.
摘要：
背景：尽管整个欧洲都对野生有蹄类动物种群进行了大量监测，我们对寄生虫如何影响种群动态知之甚少，没有系统的，长期监测寄生虫多样性和寄生虫负荷。这种监测部分受到缺乏具有高灵敏度和良好分类分辨率的时间和成本有效的测定方法的阻碍。DNA元编码已成功用于表征寄生线虫，在各种野生和家庭宿主中具有高分类学分辨率。然而,为了大规模实施这项技术，胃肠寄生线虫（GIN）的潜在非侵入性监测，需要协议优化来最大限度地检测生物多样性，同时保持时间和成本效益。
方法：从野驼鹿种群中收集粪便样本，并使用寄生虫学技术（卵和幼虫计数）和rDNAITS2区域的DNA代谢编码对GIN群落进行表征和定量。比较了三种不同的分离方法，这些方法在起始材料的体积和细胞裂解方法上有所不同。
结果：使用寄生虫学和代谢编码法从所有样品中回收了类似的线虫动物，这些方法在很大程度上是一致的。然而,与卵和幼虫计数相比，代谢编码测定显示出更好的分类分辨率和更高的灵敏度。元编码不是严格定量的，但是回收的目标线虫序列的比例与寄生虫学确定的寄生虫载量相关。使用包括机械细胞破坏和使起始材料体积最大化的DNA分离方法来最大化元粒编码测定中的物种检测率。
结论：DNA元编码是一种有前途的非侵入性技术，大规模监测野生有蹄类动物种群中的寄生虫，由于其分类分辨率高，增加测定灵敏度，以及时间和成本效益。虽然元编码不是一种严格的定量方法，尽管如此，仍有可能从该数据为宿主寄生虫负荷创建与管理和保护相关的索引。为了优化代谢编码检测的检测率以及时间和成本效益，我们建议选择一种DNA分离方法，该方法包括机械破坏细胞并最大化起始材料体积。
方法：从野驼鹿种群中收集粪便样本，并使用寄生虫学技术（卵和幼虫计数）和rDNAITS2区域的DNA代谢编码对GIN群落进行表征和定量。比较了三种不同的分离方法，这些方法在起始材料的体积和细胞裂解方法上有所不同。
结果：使用寄生虫学和代谢编码法从所有样品中回收了类似的线虫动物，这些方法在很大程度上是一致的。然而,与卵和幼虫计数相比，代谢编码测定显示出更好的分类分辨率和更高的灵敏度。元编码不是严格定量的，但是回收的目标线虫序列的比例与寄生虫学确定的寄生虫载量相关。使用包括机械细胞破坏和使起始材料体积最大化的DNA分离方法来最大化元粒编码测定中的物种检测率。
结论：DNA元编码是一种有前途的非侵入性技术，大规模监测野生有蹄类动物种群中的寄生虫，由于其分类分辨率高，增加测定灵敏度，以及时间和成本效益。虽然元编码不是一种严格的定量方法，尽管如此，仍有可能从该数据为宿主寄生虫负荷创建与管理和保护相关的索引。为了优化代谢编码检测的检测率以及时间和成本效益，我们建议选择一种DNA分离方法，该方法包括机械破坏细胞并最大化起始材料体积。
