Calvani Nichola Eliza Davies, George Sarah Deanna, Windsor Peter Andrew, Bush Russell David, Šlapeta Jan
Laboratory of Veterinary Parasitology, Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, New South Wales, Australia; Mekong Livestock Research Group, Sydney School of Veterinary Science, Faculty of Science, The University of Sydney, New South Wales, Australia.
Elanco Australasia Pty Limited, Yarrandoo R&D Centre, 245 Western Road, Kemps Creek, New South Wales, Australia.
Vet Parasitol. 2018 Feb 15;251:85-89. doi: 10.1016/j.vetpar.2018.01.004. Epub 2018 Jan 3.
Fasciolosis due to infection with Fasciola hepatica, Fasciola gigantica or their hybrids is a significant global cause of livestock production loss. Infection is commonly diagnosed by a labour-intensive sedimentation and faecal egg count (FEC), which has limited throughput and is only applicable after completion of the 8-12 week pre-patent period (PPP). A commercially-available ELISA for the detection of coprological antigen (coproELISA) enables detection prior to the completion of the PPP and is suitable for diagnosis of larger sample sizes, although the sensitivity reported under experimental infection settings can be difficult to replicate in the field, particularly in cattle. A recently-published real-time PCR workflow for the sensitive detection of Fasciola spp. DNA in faecal samples provides increased sample throughput, although the point at which this technique is first able to diagnose infection remains unknown. Other tools for the molecular diagnosis of fasciolosis, such as conventional PCR and loop-mediated isothermal amplification (LAMP), have been shown to detect F. hepatica DNA as early as 1 week post infection (WPI). In this study, faecal samples were collected weekly from 10 experimentally-infected Merino lambs and subjected to diagnosis via traditional sedimentation, coproELISA and real-time PCR. Samples were first considered positive at 6-8 WPI by coproELISA, real-time PCR and sedimentation, respectively. At 9 WPI 100% of samples were positive by all three methods. To evaluate the capacity of the real-time PCR approach to detect infection prior to completion of the PPP, two methods of sample preparation were compared at 2 WPI: (i) 150 mg raw faecal samples and (ii) 3 g faecal starting volume prior to sedimentation and pelleting. Neither method of sample preparation yielded positive results at 2 WPI suggesting that DNA amplification by real-time PCR is associated with faecal egg load.
由肝片吸虫、巨片吸虫或它们的杂交种感染引起的片形吸虫病是全球牲畜生产损失的一个重要原因。感染通常通过劳动强度大的沉淀法和粪便虫卵计数（FEC）来诊断，这种方法通量有限，并且仅适用于8 - 12周的潜隐期（PPP）结束后。一种用于检测粪便抗原的商业可用酶联免疫吸附测定（粪便酶联免疫吸附测定）能够在潜隐期结束前进行检测，并且适用于更大样本量的诊断，尽管在实验感染环境下报告的灵敏度在实际应用中可能难以复制，尤其是在牛身上。最近发表的一种用于粪便样本中片形吸虫属DNA灵敏检测的实时PCR工作流程提高了样本通量，尽管该技术首次能够诊断感染的时间点仍不清楚。其他用于片形吸虫病分子诊断的工具，如传统PCR和环介导等温扩增（LAMP），已被证明早在感染后1周（WPI）就能检测到肝片吸虫DNA。在本研究中，每周从10只实验感染的美利奴羊采集粪便样本，并通过传统沉淀法、粪便酶联免疫吸附测定和实时PCR进行诊断。粪便酶联免疫吸附测定、实时PCR和沉淀法分别在6 - 8周龄时首次将样本判定为阳性。在9周龄时，所有三种方法检测的样本100%呈阳性。为了评估实时PCR方法在潜隐期结束前检测感染的能力，在2周龄时比较了两种样本制备方法：（i）150毫克原始粪便样本和（ii）沉淀和造粒前3克粪便起始体积。两种样本制备方法在2周龄时均未产生阳性结果，这表明实时PCR的DNA扩增与粪便虫卵负荷有关。
