PDF(Pubmed)
Abstract：
BACKGROUND: Quantifying infection intensity is a common goal in parasitological studies. We have previously shown that the amount of parasite DNA in faecal samples can be a biologically meaningful measure of infection intensity, even if it does not agree well with complementary counts of transmission stages (oocysts in the case of Coccidia). Parasite DNA can be quantified at relatively high throughput using quantitative polymerase chain reaction (qPCR), but amplification needs a high specificity and does not simultaneously distinguish between parasite species. Counting of amplified sequence variants (ASVs) from high-throughput marker gene sequencing using a relatively universal primer pair has the potential to distinguish between closely related co-infecting taxa and to uncover the community diversity, thus being both more specific and more open-ended.
METHODS: We here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasite Eimeria in experimentally infected mice. We use multiple amplicons to differentially quantify Eimeria spp. in a natural house mouse population.
RESULTS: We show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish three Eimeria species in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects on Eimeria spp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated with Eimeria spp. abundance.
CONCLUSIONS: We conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect of Eimeria infection on the body condition of mice in the natural environment.
METHODS: We here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasite Eimeria in experimentally infected mice. We use multiple amplicons to differentially quantify Eimeria spp. in a natural house mouse population.
RESULTS: We show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish three Eimeria species in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects on Eimeria spp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated with Eimeria spp. abundance.
CONCLUSIONS: We conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect of Eimeria infection on the body condition of mice in the natural environment.
摘要：
背景：定量感染强度是寄生虫学研究的共同目标。我们之前已经表明，粪便样本中寄生虫DNA的数量可以作为感染强度的生物学有意义的量度，即使它与传播阶段的互补计数（球虫的卵囊）不一致。可以使用定量聚合酶链反应（qPCR）以相对高的通量定量寄生虫DNA，但是扩增需要高特异性，并且不能同时区分寄生虫物种。使用相对通用的引物对从高通量标记基因测序中对扩增的序列变体（ASV）进行计数，有可能区分密切相关的共同感染类群并揭示群落多样性。从而更加具体和更加开放。
方法：我们在这里将qPCR与使用标准PCR和基于微流体的PCR的基于测序的扩增进行比较，以定量实验感染的小鼠中的单细胞寄生虫艾美球虫。我们使用多个扩增子来差异定量艾美球虫。在自然的家鼠种群中。
结果：我们表明基于测序的定量具有很高的准确性。结合系统发育分析和共现网络，我们根据多个标记区域和基因在自然感染的小鼠中区分了三种艾美球虫。我们调查了对艾美球虫的地理和宿主相关影响。社区组成和发现，正如预期的那样，患病率在很大程度上可以用抽样地区(农场)来解释。控制这种效果，这种新方法使我们能够发现小鼠的身体状况与艾美球虫呈负相关。丰度。
结论：我们得出结论，扩增子测序为物种区分和粪便中寄生虫的同时定量提供了未充分利用的潜力。该方法使我们能够检测到艾美球虫感染对自然环境中小鼠身体状况的负面影响。
方法：我们在这里将qPCR与使用标准PCR和基于微流体的PCR的基于测序的扩增进行比较，以定量实验感染的小鼠中的单细胞寄生虫艾美球虫。我们使用多个扩增子来差异定量艾美球虫。在自然的家鼠种群中。
结果：我们表明基于测序的定量具有很高的准确性。结合系统发育分析和共现网络，我们根据多个标记区域和基因在自然感染的小鼠中区分了三种艾美球虫。我们调查了对艾美球虫的地理和宿主相关影响。社区组成和发现，正如预期的那样，患病率在很大程度上可以用抽样地区(农场)来解释。控制这种效果，这种新方法使我们能够发现小鼠的身体状况与艾美球虫呈负相关。丰度。
结论：我们得出结论，扩增子测序为物种区分和粪便中寄生虫的同时定量提供了未充分利用的潜力。该方法使我们能够检测到艾美球虫感染对自然环境中小鼠身体状况的负面影响。
