Abstract

The global ornamental fish trade enables translocation of exotic aquatic pathogens. In many countries, health certification and visual inspection of imported fish are key components of biosecurity to prevent the introduction of aquatic diseases. However, infected fish do not always exhibit clinical or behavioural signs of disease, and alternatives to visual inspection must be validated. This study examined the use of environmental DNA (eDNA) to detect sub-clinical parasite infections at border control. We simulated the export process of live ornamental fish in which non-infected fish, infected fish, treated fish, and non-infected fish held in contaminated water were packaged and delivered in 48 h. Quantitative PCR (qPCR) was used to detect eDNA of an ectoparasitic monogenean, Neobenedenia girellae, infecting barramundi, Lates calcarifer. The qPCR assay did not reliably detect parasite eDNA under 2 copies/µL from fish with sub-clinical infections (mean parasite intensity = 6.80 ± 4.78 S.D.), suggesting parasite infection loads tested in this study may be too low for reliable detection within the timeframe used to export live ornamental fish. Quantitative PCR tests detected parasite eDNA in 50% of infected fish and 70% of non-infected fish in contaminated transport water. This indicated a high plausibility of false negative detections because of low eDNA concentrations in transport water and false positive detections of DNA from dead parasites in the water. Environmental DNA screening methods for border control biosecurity must overcome limitations posed by low eDNA concentrations in the water, limited timeframes for sample processing, and the essential differentiation between live parasite infections and dead, non-viable parasites.

中文翻译：

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可以将环境DNA用于水族鱼类贸易中的水生生物安全吗？

摘要

全球观赏鱼贸易使外来水生病原体易位。在许多国家，进口鱼的健康证明和肉眼检查是防止水生疾病传入的生物安全的关键组成部分。但是，受感染的鱼并不总是表现出疾病的临床或行为迹象，因此必须验证视觉检查的替代方法。这项研究检查了在边界控制处使用环境DNA（eDNA）检测亚临床寄生虫感染的情况。我们模拟了活观赏鱼的出口过程，其中包装并在48小时内将未感染鱼，感染鱼，经处理鱼和未感染鱼包装在污染水中。定量PCR（qPCR）用于检测单寄生外生单生细菌新鞭毛虫的eDNA，感染澳洲肺鱼，晚钙。qPCR分析不能可靠地检测亚临床感染鱼的2拷贝/ µL以下寄生虫eDNA（平均寄生虫强度= 6.80±4.78 SD），这表明本研究中测试的寄生虫感染量可能太低，无法在一定时间内可靠检测用于出口活观赏鱼。定量PCR测试在受污染的运输水中检测到50％受感染鱼和70％非感染鱼中的寄生虫eDNA。这表明由于运输水中的eDNA浓度低和来自水中死虫的DNA的假阳性检测，假阴性检测的可能性很高。用于边界控制生物安全性的环境DNA筛选方法必须克服水中eDNA浓度低，样品处理时间有限，