Collini Margherita, Albonico Francesca, Hauffe Heidi C, Mortarino Michele
Department of Biodiversity and Molecular Ecology, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all'Adige, Trento, Italy; Dipartimento di Scienze veterinarie e sanità pubblica, Università degli Studi di Milano, Milan, Italy.
Dipartimento di Scienze veterinarie e sanità pubblica, Università degli Studi di Milano, Milan, Italy.
Vet Parasitol. 2015 Jun 15;210(3-4):194-205. doi: 10.1016/j.vetpar.2015.04.007. Epub 2015 Apr 18.
The sheep tick, Ixodes ricinus L., is an important hematophagous vector of zoonotic disease of both veterinary and public health importance in Europe. Risk models for tick-borne diseases can be improved by identifying the main hosts of this species in any given area. However, this generalist tick stays on a host for only a few days a year over its life cycle, making the study of its feeding ecology difficult. In contrast, ticks can easily be collected from vegetation when they are questing. Molecular methods have proved to be a reliable alternative to field observation, but most current methods have low sensitivity and/or low identification success (i.e. hosts are only identified to taxonomic levels higher than species). In this study we use Real-time PCR coupled with High Resolution Melting Analysis (HRMA) to identify the source of the last bloodmeal in questing tick nymphs. Twenty of the most important tick hosts were grouped taxonomically and six group-specific primer sets, targeting short mitochondrial DNA regions, were designed de novo. Firstly, we show that these primers successfully amplify target host DNA (from host tissue or engorged ticks), and that HRMA can be used to reliably identify hosts to species (or genera in the case of Sorex and Apodemus). Secondly, the new protocol was tested on field-collected questing nymphs. Bloodmeal source was identified in 65.4% of 52 individuals. In 83.3% of these, the host was identified to species or genera using HRMA alone. Moreover, the primer sets designed here can unequivocally identify mixed bloodmeals. The combination of sensitivity and identification success together with the closed-tube and single step approach that minimizes contamination, make Real-time HRMA a good alternative to current methods for bloodmeal identification.
蓖麻硬蜱（Ixodes ricinus L.）是欧洲一种重要的吸血媒介，传播对兽医和公共卫生都具有重要意义的人畜共患病。通过确定该物种在任何特定区域的主要宿主，可以改进蜱传疾病的风险模型。然而，这种多食性蜱在其生命周期中每年仅在宿主身上停留几天，这使得对其取食生态学的研究变得困难。相比之下，蜱在搜寻宿主时很容易从植被上采集到。分子方法已被证明是一种可靠的替代野外观察的方法，但目前大多数方法灵敏度低和/或鉴定成功率低（即宿主仅被鉴定到高于物种的分类水平）。在本研究中，我们使用实时聚合酶链反应（PCR）结合高分辨率熔解分析（HRMA）来鉴定搜寻到的蜱若虫最后一次血餐的来源。将20种最重要的蜱宿主进行分类分组，并从头设计了针对线粒体DNA短区域的6组特异性引物。首先，我们表明这些引物成功扩增了目标宿主DNA（来自宿主组织或饱血蜱），并且HRMA可用于可靠地将宿主鉴定到物种（对于Sorex和Apodemus属则鉴定到属）。其次，在野外采集的搜寻若虫上测试了新方案。在52只个体中，有65.4%鉴定出了血餐来源。其中83.3%仅使用HRMA就将宿主鉴定到了物种或属。此外，这里设计的引物组可以明确鉴定混合血餐。灵敏度和鉴定成功率，以及封闭管和单步方法相结合，最大限度地减少了污染，使得实时HRMA成为目前血餐鉴定方法的一个很好的替代方法。
