鼻湿巾从猪流感病毒检测和隔离
Summary
The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.
Abstract
在监测猪流感A型病毒对人类和动物的健康至关重要，因为A型流感病毒迅速演变的猪群和新品种不断涌现。猪能够被感染甲型流感病毒的使得它们的出现和维持的新的流感病毒株重要主机多样谱系。抽样猪在不同的设置，如商业猪场，农产品集贸市场和活禽交易市场是非常重要的，提供当前流行IAV株的全面视图。目前的黄金标准宰前抽样技术（鼻拭子的，即集合）是劳动密集型的，因为它需要猪的物理限制。鼻抹布擦涉及在尽量减少猪的口鼻部一块布来节制的动物。鼻腔擦拭过程简单进行，并不需要专业的兽医或动物处理人员培训。 W¯¯往往微不足道略高于鼻拭子不太敏感，病毒检测和隔离率足以使鼻湿巾抽检个别猪在低应力抽样方法都需要一个可行的选择。该诉讼协议概述了需要收集一个可行鼻的步骤从个体猪擦拭。
Introduction
A型流感病毒（IAV）引起的呼吸系统疾病在许多物种，包括家禽，猪和人类。由于分段IAV基因组快速病毒进化的重配可发生新IAV株频频出现。猪是可以作为从多个宿主物种重配IAVs的混合容器中的物质。1目前IAV三个主要亚型间的北美猪（H1N1，H1N2，H3N2）通常循环，但来自人多IAV引入有导致大量IAV多样性的亚型内。IAVs感染猪2快速发展自1998年以来，当含有来自人类，禽流感和猪的病毒基因片段一个三重配IAV 3成为普遍在美国猪群中已可见一斑。4，内部基因从三重配IAV段保持IAVs目前感染猪中非常普遍。5
“>在世界范围内，IAV是显著造成呼吸系统疾病的猪其典型的临床症状包括发烧，厌食，嗜睡，咳嗽，呼吸困难，打喷嚏，流鼻涕和体重不增。IAV可以是特别昂贵的母猪养殖场，其中生殖失败是由于IAV引起发热和弱仔猪已被记录在案，6,7在美国，IAV是常用的商业猪群和广泛的抗原和基因组多样性与持续发展之间的IAV感染猪检测阻碍了该控制病毒8-11关于大流行IAV株来自重配导致猪分别实现在2009年时包含从三重配的北美猪血统和欧亚禽样猪的血统基因片段猪谱系IAV引起了世界范围内大流行的出现公共健康问题在人类12大流行性流感病毒（A型（H1N1）pdm09）具有自重配地方性猪IAV株13,14和一些新的重配株已传回人类。15重配事件和新IAV株可能引起大流行的出现，使循环IAV病毒在猪势在必行的主动监测的频率，特别是在猪的人机界面。
猪 – 人接口是IAV的双向种间传播的重要。人到猪的商业化养猪生产产生的传输是负责大量当前存在于猪群IAV多样性。农产品展销会是在美国人民和猪的comingling最大的设置和已知位点IAV的动物传播。15-21在2012年，一个变种H3N2 IAV爆发期间，案件93％的受访出席农业博览会在未来的日子发病前15基因组分析21展猪感染IAV从展猪病毒分离株与人分离株相比，证实人畜共患传输。常常不显示疾病，21-23指示需要直接诊断测试的临床症状。
对明显的病猪采样本身并不能成功地识别IAV流行的猪，不能依赖于识别IAV新毒株出现的猪之一。主动监测为检测IAV的猪，并评估其对两个猪和公共卫生威胁新兴菌株的绝对必要的。大多数IAV侦察活动是自愿的，因此需要最低限度的破坏性的方法。为IAV感染猪三大生前样本收集程序是：鼻拭子，唾液，鼻腔擦拭。对于个别采样猪检测IAV名单合成纤维插入当前建议拭子插入鼻孔的首选方法收集鼻分泌物和上皮细胞。24,25由于猪可能尽量避免此过程中，一组训练有素的人员必须手动或使用圈套根据动物的大小抑制猪26的限制处理是费力的人员，和紧张的猪。此外，展览猪经常参与多个比赛在一个公平的这样增加的压力对比赛的动物的感受可以让车主耐监测工作。
随着IAV检测的概率感染牛群从80-100％的IAV，口服液体已经成为一种流行的替代鼻拭子在猪群体的分子检测IAV的。27,28此外，口服液体可以提供更广泛的窗口IAV检测不是遵循最初的感染鼻拭子。然而，从唾液IAV隔离一直是个问题，只有50％的病毒分离的尝试导致IAV恢复。29
代替期间在猪IAV监视鼻拭子的使用鼻腔擦拭物克服了上述限制。鼻腔擦拭物不需要使用限制圈套的，可以在不强调过程的动物或证人来执行。最少的技术培训是需要收集鼻腔擦拭，这使得非兽医专业人士，包括猪的业主，收集监测样本。鼻腔擦拭物以前相比鼻拭子对甲病毒30和对于这种非侵入性采样的方法的详细的协议下面描述的检测和流感的隔离。
Protocol
Representative Results
Discussion
收集使用聚酯嘴鼻拭子样品的猪在开展IAV监控已经证明是有益的;然而，使用鼻拭子过程的阻碍，由于所需使用圈套克制的监测工作。鼻湿巾代表目前的猪采样技术的细化，旨在样品采集过程中减少压力对人和猪。而该方法已被开发用于和验证展猪设置，它可以很容易地应用于其它情形宰前检测IAV的猪是必要的（ 即商业猪，活的动物中的市场，生物医学研究等 ）
<p class="jove_content"…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.
Materials
| BBL Brain Heart Infusion | Becton, Dickinson and Company | 211059 | |
| Penicillin G Sodium Salt | MP Biomedicals, LLC | 021194537 | 1500 u/mg |
| Streptomycin Sulfate | AMRESCO LLC | 0382 | |
| Gentamicin Solution | Mediatech, Inc. | 30-005-CR | 50 mg/ml |
| Amphotericin B Solution | Fisher | S 24 25 | 250 ug/ml |
| Kanamycin Sulfate | Teknova | K2105 | 5000 ug/ml |
| TPP Rapid Filtermax System | TPP Techno Plastic Products AG | 99150 | |
| Nalgen Diagnostic Bottles | Thermo Scientific | 342002-9025 | HDPE with white PP closure |
| Dermacea Gauze Sponge, 8 ply | Covidien | 441211 | 5.08 cm × 5.08 cm (2 in. × 2 in) |
| Nitrile Exam Gloves | Saftey Choice | 19-170-010 (A-D) |
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