我们感谢叙述视频威廉·T·埃克特。发展的玻璃棉协议的一部分是在威斯康星州的水和卫生审判肠溶风险（WAHTER研究）资助，由美国环保署STAR格兰特R831630。 A.穿过，A. Ramey，和B Meixell阿拉斯加样品收集来自美国地质调查局的财政支持。任何贸易，产品或公司名称的使用仅是描述目的，并不意味着美国政府认可。

1。准备玻璃棉<ol><li>使每批过滤器前后，用10％漂白粉溶液消毒工作区。 </li><li>戴上手套和礼服。至少20分钟，在121°C和15磅高压灭菌消毒桶。放置在无菌桶玻璃棉。 </li><li>玻璃棉与反渗透水饱和，让浸泡15分钟。 </li><li>排水反渗透水从桶。 </li><li> 1 M盐酸饱和玻璃棉，让浸泡15分钟。 </li><li>从桶里漏1 M盐酸。 </li><li>用反渗透水冲洗玻璃棉。 </li><li>调匀。 </li><li>使用pH试纸检查pH值和重复反渗透水冲洗直到pH值达到中性。 </li><li>倒出用水冲洗。 </li><li> 1 M氢氧化钠饱和的玻璃棉让浸泡15分钟。 </li><li>从桶里漏1 M氢氧化钠。 </li><li> ŕEPEAT反渗透冲洗直到pH值达到中性。 </li><li>倒出用水冲洗。 </li><li>玻璃棉洗衣机，而不是一个水桶，可以构建，这是类似的设计，以玻璃吸管垫圈（图1）。 </li><li>玻璃棉完全覆盖无菌磷酸盐缓冲液（PBS）的调整，以pH值6.8。 </li><li>准备玻璃棉立即或商店使用4°C。它可以存储长达两个星期。在使用之前，确保pH值是中性的，因为它会随着时间的推移上升。如果不是中性的pH值，重新冲洗，用无菌磷酸盐缓冲液（pH值6.8）。 </li></ol> 2。装配玻璃纤维过滤器<ol><li>钻一个11/16英寸成男性适配器尼龙配件可以拧成的上限，PVC帽和丝锥螺纹孔。这一步是必要的，只为第一大会。此后，上限可以使用多年。适用于聚四氟乙烯尼龙接头的螺纹和螺纹瓶盖的磁带。 </li><li>包与玻璃棉小块的PVC管。使用金​​属柱塞，像汽车发动机气门，包紧。包装并不需要很大的力量。包装不够紧，使玻璃棉停留在地方和渠道没有形成。然而，不要包得那么紧水可以通过过滤器，不流。适当的包装时，每分钟4至5升的流速应达到40-60磅之间的压力过滤器附有水龙头。在该协议中指定的PVC管大小，约85克洗净，每管使用填充玻璃棉。顶级的负载平衡和与水洗玻璃棉包空PVC管，直到皮管质量增加85克。 </li><li>男性适配器尼龙件连接，插入的PVC帽聚丙烯网状。 </li><li>铁氟龙胶带，PVC管螺纹部分。 </li><li>对PVC帽螺丝的PVC带够IPE和标签过滤年底的流入和流出的另一端。这是很重要的，后来为洗脱步骤。这到底是贴上标签流入不要紧。 </li><li>推60毫升无菌磷酸盐缓冲液（pH值= 6.8）到过滤器的使用导管倾斜注射器。多余出来的另一端。 </li><li> WRAP结束与石蜡膜紧密，以避免漏水。过滤器可存储长达30天，在4°C </li></ol> 3。抽样<ol><li>抽样采用循环或浸泡30分钟的项目，在0.525％的次氯酸钠（即，一个标准的家用漂白水的10％溶液）消毒管。按照排水次氯酸钠溶液与0.05％的硫代硫酸钠，加入25毫升2％的股票无水硫酸钠解决一公升逆渗透水和消除。 </li><li>样品pH值超过7.5的水，调节pH值介于6.5和7.0用盐酸。盐酸浓度范围从0.25M到1 M。四升0.5 M盐酸一般是两个800升的样本，根据水环境的pH值和缓冲能力，足够的。使用蠕动泵或文丘里（图2）抽样过程中，注入盐酸。调节泵的转速或丘开幕，以实现目标的pH值。样线插座，使用领域的pH计测量的pH值。 </li><li>玻璃棉过滤器堵塞，如果使用高浊度水预过滤器。 （预过滤器和其住房规格的材料清单在网上）。由于病原体可以连接到被困在预过滤的颗粒，它必须随着玻璃棉过滤洗脱（见下文）。 </li><li>调整流速以2至4升/分钟。典型的样本量为200升和1500升之间。 </li><li>在4°C断开玻璃棉过滤器在一个塑料无菌袋和存储长达48小时。 </li></ol> 4。洗脱<ol><li>贴上玻璃纤维过滤器一环站在指向样品流入年底下降到聚丙烯瓶。洗脱的样品流的方向相反。 </li><li>推80毫升无菌的3％，牛肉膏0.05 M甘氨酸与过滤器的pH值9.5。 </li><li>等待15分钟。 </li><li>进入过滤器的9.5 pH值在0.05 M甘氨酸推另一无菌的3％，牛肉膏80毫升。 </li><li>推动空气彻底的过滤器，直到泡沫过滤器入口。 </li><li> 1 M盐酸洗脱液的pH值调整到7.0和7.5之间。 </li><li>商店洗脱液在4°C的长达24小时或在-20°C，时间较长。 </li><li>洗脱预过滤器，如果使用。松开住房墨盒的顶部和房屋内的所有残留的水倒掉，而持有的预过滤器的地方用无菌手套的手。 </li><li>取出从住房墨盒的预过滤器和滑入15“×6”自封袋。 </li><li>倒入200毫升无菌的3％，牛肉膏0.05 M G甜菜碱与成袋的pH值9.5和密封拉链锁安全。 </li><li>反转袋装预过滤几次，以确保整个表面接触牛肉膏。 </li><li>等待15分钟，偶尔反相和按摩袋装预过滤器。 </li><li>打开拉链锁。手柄周围的预过滤袋紧紧地挤压出尽可能多的牛肉膏和拉带无菌手套的手，预过滤器，。 </li><li>聚丙烯瓶倒入牛肉膏洗脱液。 </li><li>洗脱液的pH值调整到7.0和7.5 1M盐酸。 </li><li>商店洗脱液在4°C的长达24小时或在-20°C，时间较长。预过滤洗脱液可以单独分析病原体或玻璃棉过滤洗脱液相结合。 </li></ol><s锅类=“pdflinebreak”&gt; 5。代表结果<table border="1"><tbody><tr><td> 病原 </td><td> 水浊度（台大） </td><td> 金额种子/ L，B，C，D </td><td> 回收率（％）±1个SD </td><td> 数独立试验 </td></tr><tr><td>肠病毒脊髓灰质炎病毒萨宾第三</td><td> 0.5 </td><td> 500 </td><td> 81％±11 </td><td> 7 </td></tr><tr><td>肠脊髓灰质炎病毒萨宾三</td><td> 0.5 </td><td> 5000 </td><td> 67％±12 </td><td> 8 </td></tr><tr><td>肠病毒脊髓灰质炎病毒萨宾第三</td><td> 215 </td><td> 500 </td><td> 59％±32 </td><td> 7 </td></tr><tr><td>肠病毒脊髓灰质炎病毒萨宾第三</td><td> 215 </td><td> 5000 </td><td> 38％±22 </td><td> 6 </td></tr><tr><td>肠病毒脊髓灰质炎病毒萨宾第三</td><td> 447 </td><td> 500 </td><td> 56％±18 </td><td> 8 </td></tr><tr><td>肠病毒脊髓灰质炎病毒萨宾第三</td><td> 447 </td><td> 5000 </td><td> 63％±37 </td><td> 8 </td></tr><tr><td> 隐孢子虫 </td><td> 0.5 </td><td> 5 </td><td> 38％±14 </td><td> 7 </td></tr><tr><td> 隐孢子虫 </td><td> 0.5 </td><td> 50 </td><td> 53％±19 </td><td> 8 </td></tr><tr><td> 哭泣ptosporidium孢子虫</td><td> 215 </td><td> 5 </td><td> 40％±16 </td><td> 7 </td></tr><tr><td> 隐孢子虫 </td><td> 215 </td><td> 50 </td><td> 30％±6 </td><td> 6 </td></tr><tr><td> 隐孢子虫 </td><td> 447 </td><td> 5 </td><td> 33％±13 </td><td> 8 </td></tr><tr><td> 隐孢子虫 </td><td> 447 </td><td> 50 </td><td> 28％±11 </td><td> 8 </td></tr><tr><td> 沙门氏菌 </td><td> 0.5 </td><td> 5 </td><td> 29％±24 </td><td> 7 </td></tr><tr><td> 沙门氏菌 </td><td> 0.5 </td><td> 500 </td><td> 56％±16 </td><td> 8 </td></tr><tr><td> 沙门氏菌 </td><td> 215 </td><td> 5 </td><td> 32％±24 </td><td> 7 </td></tr><tr><td> 沙门氏菌 </TD&gt; <td> 215 </td><td> 500 </td><td> 34％±11 </td><td> 6 </td></tr><tr><td> 沙门氏菌 </td><td> 447 </td><td> 5 </td><td> 34％±18 </td><td> 8 </td></tr><tr><td> 沙门氏菌 </td><td> 447 </td><td> 500 </td><td> 31％±24 </td><td> 8 </td></tr></tbody></table><ol class="lalpha"><li>浊度单位</li><li>作为基因组拷贝/ L，肠列举的qPCR </li><li> 小隐孢子虫的卵囊免疫荧光作为列举</li><li> 沙门氏菌列举文化为菌落形成单位</li></ol> 表1。玻璃羊毛浓度不同的水浊度和病原菌密度。 <table border="1"><tbody><tr><td> 病原 </td><td> 水样中的位置 </td><td> 金额种子/ L 一 </td><td> ％RECO<html>很</td></tr><tr><td>禽流感H5N2病毒</td><td> sundi湖，安克雷奇市镇</td><td> 2500 </td><td> 42.9％ </td></tr><tr><td>禽流感H5N2病毒</td><td>明托单位，费尔班克斯北极星自治市镇</td><td> 2500 </td><td> 36.7％ </td></tr><tr><td>禽流感H5N2病毒</td><td> Portage的山谷，安克雷奇市镇</td><td> 2500 </td><td> 7.8％ </td></tr><tr><td>禽流感H5N2病毒</td><td>波特沼泽，安克雷奇市镇</td><td> 2500 </td><td> 41.5％ </td></tr><tr><td>禽流感H5N2病毒</td><td>湖边的柳树，育空地区的Koyukuk市镇</td><td> 2500 </td><td> 15.5％ </td></tr></tbody></table><ol class="lalpha"><li>定量PCR测定基因拷贝/ L， </li></ol> 表2。玻璃禽流感病毒的羊毛含量在阿拉斯加从五个地点使用的水。 les/ftp_upload/3930/3930fig1.jpg“ALT =”图1“/&gt; 图1图玻璃棉洗衣机。这可以用来代替一个桶，节约时间的冲洗。概念是类似玻璃吸管洗衣机。 <img src="/files/ftp_upload/3930/3930fig2.jpg" alt="图2" /> 图2。酸注射液由蠕动泵玻璃毛过滤。请注意样线之间的水龙头和玻璃棉过滤泵管引入酸的“T”连接器。 pH值的调整是必要的，只有水取样的pH值&gt; 7.5。 玻璃棉过滤器有效地集中从水的浊度水平和病原体的密度（表1）广泛的病原体。为了测试这一点，20升脱氯自来水混合干淤泥壤土（0，1.27，或2.75克/升），浊度达到期望的水平，然后种植在不同密度的病原体。水传递是通过玻璃纤维过滤器，并列举了在洗脱液浓缩病原体，这个值是在％的回收率计算的分子。入水，也就是％的回收率计算的分母，接种病原菌的数量决定先播种到负洗脱液病原体，然后列举的病原体。负洗脱液准备通过一个过滤器的非种子选手20公升的样品和洗脱。量化负洗脱液接种的病原体，避免病原体列举的差异可能导致玻璃棉过滤器创建矩阵的差异。这一步的重要性，通过定量PCR定量病原体时，讨论在蓝波提尼等。3。 20公升的阴性对照样品，通过玻璃棉过滤，以确定有没有可能混淆％的回收率计算的本地病原体目前集中。 10微米的名义孔径预过滤器瓦特使用时≥215 NTU的浊度水平。 脊髓灰质炎病毒定量实时定量PCR，使用二次浓度和核酸提取程序和引物和探针描述在蓝波提尼等。3。隐孢子虫是在最后集中由脊髓灰质炎病毒的二次浓度过程中创建的样本量（FCSV）量化。卵囊免疫（MeriFluor 隐孢子虫和贾第鞭毛虫检测试剂盒，经络生命科学公司，辛辛那提，俄亥俄州）的可视化。 沙门氏菌 XLD琼脂上的电镀量化的FCSV的（Remel，列涅萨，KS）和计数菌落形成单位。 玻璃棉过滤器是在有效地集中禽流感病毒（见表2）。低致病性禽流感病毒（H5N2型）从几个地点在阿拉斯加和入水播种％的回收率计算，描述ABOVE。中学浓度为脊髓灰质炎病毒;病毒核酸程序进行了定量PCR定量使用的引物和探针Spackman 等4。

<ol>
	<li>US Environmental Protection Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+1623:+Cryptosporidium+and+Giardia+in+Water+by+Filtration/IMS/FA.">Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA.</a> EPA 815-R-05-002. , (2012).</li><li>Cashdollar, J. L., Dahling, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+method+to+re-use+electropositive+cartridge+filters+for+concentrating+viruses+from+tap+and+river+water.">Evaluation of a method to re-use electropositive cartridge filters for concentrating viruses from tap and river water.</a> J. Virol. Methods. 132, 13-17 (2006).</li><li>Lambertini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+of+enteroviruses,+adenoviruses,+and+noroviruses+from+drinking+water+by+use+of+glass+wool+filters.">Concentration of enteroviruses, adenoviruses, and noroviruses from drinking water by use of glass wool filters.</a> Appl. Environ. Microbiol. 74, 2990-2996 (2008).</li><li>Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+real-time+reverse+transcription+PCR+assay+for+Type+A+influenza+virus+and+the+avian+H5+and+H7+hemagglutinin+subtypes.">Development of a real-time reverse transcription PCR assay for Type A influenza virus and the avian H5 and H7 hemagglutinin subtypes.</a> J. Clin. Microbiol. 40, 3256-3260 (2002).</li><li>Environment Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+a+new+method+for+detection+of+viruses+in+groundwater.">Optimisation of a new method for detection of viruses in groundwater.</a> Report No. NC/99/40. , (2000).</li><li>Vilagin&#233;s, P., Sarrette, B., Husson, G., Vilaginés, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass+wool+for+virus+concentration+at+ambient+water+pH+level.">Glass wool for virus concentration at ambient water pH level.</a> Water Sci. Technol. 27, 299-306 (1993).</li><li>Vivier, J. C., Ehlers, M. M., Grabow, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+enteroviruses+in+treated+drinking+water.">Detection of enteroviruses in treated drinking water.</a> Water Res. 38, 2699-2705 (2004).</li><li>Powell, K. L., Sililo, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enteric virus detection in groundwater using a glass wool trap. In: Groundwater: Past Achievements and Future Challenges. , 813-816 (2000).</li><li>Hunt, R. J., Borchardt, M. A., Richards, K. D., Spencer, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+sewer+source+contamination+of+drinking+water+wells+using+tracers+and+human+enteric+viruses.">Assessment of sewer source contamination of drinking water wells using tracers and human enteric viruses.</a> Environ. Sci. Technol. 44, 7956-7963 (2010).</li><li>van Heerden, J., Ehlers, M. M., Heim, A., Grabow, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence,+quantification+and+typing+of+adenoviruses+detected+in+river+and+treated+drinking+water+in+South+Africa.">Prevalence, quantification and typing of adenoviruses detected in river and treated drinking water in South Africa.</a> J. Appl. Microbiol. 99, 234-242 (2005).</li><li>Vilagin&#233;s, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Round+robin+investigation+of+glass+wool+method+for+poliovirus+recovery+from+drinking+water+and+sea+water.">Round robin investigation of glass wool method for poliovirus recovery from drinking water and sea water.</a> Water Sci. Technol. 35, 445-449 (1997).</li><li>Gantzer, C., Senouci, S., Maul, A., Levi, Y., Schwartzbrod, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterovirus+genomes+in+wastewater:+concentration+on+glass+wool+and+glass+powder+and+detection+by+RT-PCR.">Enterovirus genomes in wastewater: concentration on glass wool and glass powder and detection by RT-PCR.</a> J. Virol. Methods. 65, 265-271 (1997).</li><li>Borchardt, M. A., Jokela, W. E., Spencer, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+losses+in+surface+water+runoff+from+dairy+manure+applied+to+corn+fields.">Pathogen losses in surface water runoff from dairy manure applied to corn fields.</a> , (2011).</li><li>Deboosere, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+concentration+method+for+the+detection+of+influenza+A+viruses+from+large+volumes+of+surface+water.">Development and validation of a concentration method for the detection of influenza A viruses from large volumes of surface water.</a> Appl. Environ. Microbiol. 77, 3802-3808 (2011).</li><li>Lambertini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+contamination+from+operation+and+maintenance+practices+in+small+drinking+water+distribution+systems.">Virus contamination from operation and maintenance practices in small drinking water distribution systems.</a> J. Water Health. 9, 799-812 (2011).</li></ol>

没有利益冲突的声明。

玻璃棉过滤器已使用的几个研究小组3,5,6集中精力从各种水源，如成品饮用水，地下水8,9，10地表水，海水，废水12人的肠道病毒，农业径流13。在这里，我们报告的过滤器也有效的禽流感病毒以及细菌和原生动物病原体沙门氏菌 （伤寒沙门）和隐孢子虫 ，分别在集中。 deboosere 等。最近还报道了玻璃羊毛浓度禽流感病毒14。 <p class="j...

<table border="1"><tbody><tr><td> 试剂或项目名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>盐酸</td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific则</a> </td><td> A144-500 </td></tr><tr><td height="24">氢氧化钠</td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific则</a> </td><td> BP359-212 </td></tr><tr><td> 磷酸盐缓冲液 氯化钠 磷酸钾，二元 磷酸钾，二氢</td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific则</a> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific则</a> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific则</a> </td><td><html> BP358-212 BP363-500 BP362-500 </td></tr><tr><td>家用漂白水次氯酸钠即</td><td> <a href="http://www.clorox.com" target="_blank">高乐氏（Clorox）有限公司</a> </td><td></td></tr><tr><td>硫代硫酸钠，无水</td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific则</a> </td><td>小号475-212 </td></tr><tr><td>牛肉浸膏，干燥</td><td> <a href="http://www.bd.com" target="_blank">流式细胞，Dickinson公司</a> </td><td> 211520 </td></tr><tr><td>甘氨酸</td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific则</a> </td><td> G46-500 </td></tr><tr><td>上油sodocalcic玻璃棉 或 R-11的隔热unfaced玻纤</td><td> <a href="http://www.isover.com" target="_blank">依索维尔</a> <html> REF =“htt​​p://www.jm.com”目标=“_blank”&gt;约翰曼维尔</td><td> bourre 725 QN </td></tr><tr><td>聚丙烯网状</td><td> <a href="http://www.industrialnetting.com" target="_blank">工业网</a> </td><td> xN4510 </td></tr><tr><td> 2“X4”SCH 80聚氯乙烯螺纹管接头</td><td> <a href="http://www.grainger.com" target="_blank">格兰杰</a> </td><td> 6MW35 </td></tr><tr><td> 2“SCH 40 PVC帽</td><td> <a href="http://www.grainger.com" target="_blank">格兰杰</a> </td><td> 5WDW3 </td></tr><tr><td>男适配器尼龙配件（1/2“X1 / 2”） </td><td> <a href="http://www.usplastic.com" target="_blank">美国塑料公司</a> </td><td> 62178 </td></tr><tr><td>样品洗脱液-1公升瓶</td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific则</a> </td><td> 03-313-4F </td></tr><tr><td> 60毫升的注射器</td><td>“http://www.fishersci.com”目标=“_blank”&gt;尔科技</td><td> NC9661991 </td></tr><tr><td> pH值条</td><td> <a href="http://www.whatman.com" target="_blank">瓦特曼</a> </td><td> 2614 991 </td></tr><tr><td>预过滤器，聚丙烯，10英寸的墨盒，10微米; </td><td>麦克马斯特，卡尔</td><td> 4411K75 </td></tr><tr><td>预过滤器外壳</td><td>科尔 - 帕默</td><td>的S-29820-10 </td></tr></tbody></table>

glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens

关键的第一步是在怀疑受污染的水的病原体水平评估中的浓度。浓度的方法往往是具体 ​​的特定病原体，例如，美国环境保护局方法1， 贾第鞭毛虫和隐 1623，这意味着需要多种方法，抽样方案，如果针对多个病原体组。目前的方法的另一个缺点是可以是复杂和昂贵的设备，例如，集中病毒2 1MDS滤芯过滤器VIRADEL方法。在这篇文章中，我们描述了如何构建集中水源性病原体的玻璃纤维过滤器。经过过滤洗脱，浓缩是服从第二个浓度的步骤，如离心，文化或分子生物学方法的病原体检测和枚举。该过滤器有几个优点。建设是很简单的过滤器，可以建立一个NY大小为满足特定的取样要求。过滤器零件价格便宜，从而有可能严重影响项目预算的情况下收集大量的样品。大样本量（100到千件L）可以集中根据浊度堵塞率。高度便携和过滤器是用最少的设备，如泵和流量计，他们可以在现场实施抽样成品饮用水，地表水，地下水，农业径流。最后，玻璃纤维过滤是有效集中各种病原体的类型，所以只有一个方法是必要的。在这里，我们集中水性人类肠病毒， 隐孢子虫 ，S almonella肠炎，禽流感病毒的报告过滤器的有效性。

Watch this Scientific Journal Video about Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens at JoVE.com

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

玻璃棉过滤器已用于水性病毒集中了世界各地的研究小组。在这里，我们表明兴建玻璃棉过滤器的一个简单的方法，并展示过滤器也有效地集中水性的病毒，细菌和原生动物病原体。

为集中的的水性病毒和农业人畜共患病病原体的玻璃纤维过滤器

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

glass-wool-filters-for-concentrating-waterborne-viruses-agricultural

Research

JoVE Journal

Immunology and Infection

17.9K 次观看.  United States Geological Survey. 玻璃棉过滤器已用于水性病毒集中了世界各地的研究小组。在这里，我们表明兴建玻璃棉过滤器的一个简单的方法，并展示过滤器也有效地集中水性的病毒，细菌和原生动物病原体。

视频: Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

科研

免疫与感染

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

“发光法”的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

禽流感监测与FTA卡：实地方法，生物安全，和运输问题解决了

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

制备光学质量玻璃表面研究巨噬细胞融合

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

从正式固定组织检测利沙病毒抗原的免疫化学测试

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...