Huggins Lucas G, Koehler Anson V, Schunack Bettina, Inpankaew Tawin, Traub Rebecca J
Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Victoria 3050, Australia.
Bayer Animal Health GmbH, 51373 Leverkusen, Germany.
Pathogens. 2020 Apr 1;9(4):258. doi: 10.3390/pathogens9040258.
Bacterial canine vector-borne diseases are responsible for some of the most life-threatening conditions of dogs in the tropics and are typically poorly researched with some presenting a zoonotic risk to cohabiting people. Next-generation sequencing based methodologies have been demonstrated to accurately characterise a diverse range of vector-borne bacteria in dogs, whilst also proving to be more sensitive than conventional PCR techniques. We report two improvements to a previously developed metabarcoding tool that increased the sensitivity and diversity of vector-borne bacteria detected from canine blood. Firstly, we developed and tested a canine-specific blocking primer that prevents cross-reactivity of bacterial primer amplification on abundant canine mitochondrial sequences. Use of our blocking primer increased the number of canine vector-borne infections detected (five more and three more infections) and increased the diversity of bacterial sequences found. Secondly, the DNA extraction kit employed can have a significant effect on the bacterial community characterised. Therefore, we compared four different DNA extraction kits finding the Qiagen DNeasy Blood and Tissue Kit to be superior for detection of blood-borne bacteria, identifying nine more , two more , one more infection and more putative bacterial pathogens than the lowest performing kit.
细菌性犬媒传播疾病是热带地区一些最危及犬类生命的疾病的病因，通常研究较少，有些还对同居人群构成人畜共患病风险。基于下一代测序的方法已被证明能够准确鉴定犬类中多种媒介传播细菌的特征，同时也比传统PCR技术更灵敏。我们报告了对先前开发的元条形码工具的两项改进，这提高了从犬类血液中检测到的媒介传播细菌的灵敏度和多样性。首先，我们开发并测试了一种犬特异性阻断引物，该引物可防止细菌引物扩增与丰富的犬线粒体序列发生交叉反应。使用我们的阻断引物增加了检测到的犬媒传播感染数量（多检测到5种和3种感染），并增加了发现的细菌序列的多样性。其次，所使用的DNA提取试剂盒可能对所鉴定的细菌群落有显著影响。因此，我们比较了四种不同的DNA提取试剂盒，发现Qiagen DNeasy Blood and Tissue Kit在检测血源细菌方面表现更优，与表现最差的试剂盒相比，多鉴定出9种、2种、1种感染以及更多假定的细菌病原体。
