Biz Video anlatan William T. Eckert teşekkür ederim. Cam yünü protokolün geliştirilmesi US EPA YILDIZ Grant R831630 tarafından finanse Enterik Riskler (WAHTER Study) için Wisconsin Su ve Sağlık Deneme parçasıydı. Alaska örnekleri USGS mali desteği ile A. Reeves, A. Ramey ve B. Meixell toplanmıştır. Ticaret, ürün veya firma adları her türlü kullanımı açıklayıcı amaçlıdır ve ABD Hükümeti tarafından onaylandığı anlamına gelmez.

1. Cam Yünü hazırlanması <ol><li> Filtreleri her bir parti öncesi ve yaptıktan sonra,% 10 çamaşır suyu çözeltisi ile çalışma alanı sterilize edin. </li><li> Eldiven ve önlük giyin. En azından 20 dakika süreyle 121 ° C ile 15 psi ile otoklavda bir kova sterilize. Steril kova cam yünü yerleştirin. </li><li> Ters osmoz su ile cam yünü Doymuş ve 15 dakika ıslatın. </li><li> Kovadan ters osmoz su boşaltın. </li><li> 1 M HCl ile cam yünü Doymuş ve 15 dakika ıslatın. </li><li> Kovadan 1 M HCl boşaltın. </li><li> Ters osmoz su ile cam yünü durulayın. </li><li> Iyice karıştırın. </li><li> PH kağıdı ile pH kontrol edilir ve nötr pH elde edilinceye kadar ters osmoz su durulama tekrarlayın. </li><li> Durulama suyu dökün. </li><li> 1 M NaOH ile cam yünü Doymuş ve 15 dakika ıslatın. </li><li> Kovadan 1 M NaOH boşaltın. </li><li> Rnötr pH elde edilinceye kadar ters osmoz durulayın EPEAT. </li><li> Durulama suyu dökün. </li><li> Bunun yerine bir kova, bir cam yünü yıkama tasarımında cam bir pipet yıkama (Şekil 1) benzer olan, inşa edilebilir. </li><li> Steril Fosfat ile tamamen kapsayacak cam yünü salin (PBS) pH 6.8 ayarlandı Tamponlu. </li><li> 4 de hazırlanmış cam hemen yün veya mağaza kullanın ° C Bu iki haftaya kadar saklanabilir. Kullanmadan önce, zaman içinde artacak gibi pH nötr olduğundan emin olun. PH nötr değilse, steril fosfat ile yeniden durulama (pH 6.8) Tamponlu Salin. </li></ol> 2. Cam Yünü Filtre Montaj <ol><li> Erkek adaptörü naylon parçaları kapaklar vidalanan böylece PVC kapaklar ve musluk konuları içine 11/16 inç delik delin. Bu aşama, sadece birinci asamblenin için gereklidir. Bundan sonra, kapaklar yıl için kullanılabilir. Teflon Uygulanaylon parçaları konuları ve kapakları için vida bandı. </li><li> Cam yünü küçük parçalar ile PVC boru paketleyin. Sıkı paketi, bir araba motoru vanası gibi, metal bir piston kullanın. Ambalaj büyük kuvvet gerektirmez. Sıkı yeterli cam yünü yerde kalır böylece paketleyin ve kanalları oluşturmazlar. Ancak, öyle sıkı su filtresi aracılığıyla akamaz paketi yok. Uygun şekilde paketlenmiş zaman, dakika başına 4 ila 5 litre debisi filtre 40-60 psi arasında basınçları ile su muslukları takılı olduğunda ulaşılabilir olmalıdır. Bu protokolde belirtilen PVC boru boyutu için, yaklaşık 85 gram yıkanmış ve paketlenmiş cam yünü boru başına kullanılır. Dara boru kütlesi 85 gram yıkanmış cam yünü ile en iyi yükleme dengesi ve paketi üzerindeki boş bir PVC boru artırır kadar. </li><li> Bağlı erkek adaptörü naylon parçaları ile PVC kapaklar içine polipropilen yerleştirin. </li><li> PVC boru dişli kısımlarına teflon bant uygulayın. </li><li> PVC p PVC kapaklar üzerine vidalayınIPE ve etiket bir filtre ucu giriş ve diğer ucunu çıkış. Bu, elüsyon aşaması için daha önemlidir. Hangi sonu girişi önemli değil etiketlenmiştir. </li><li> Steril Fosfat 60 ml bir kateter kullanarak filtre içine (pH = 6.8) Tamponlu Salin itin şırınga uçlu. Aşırı karşı uç çıkacaktır. </li><li> Wrap sızıntı önlemek için parafilm sıkıca biter. Filtreler 4 ° C de 30 gün kadar saklanabilir </li></ol> 3. Örnekleme <ol><li> 0.525% NaClO (yani, standart çamaşır suyu% 10 solüsyonu) 30 dakika öğeleri recirculating veya daldırarak örnekleme için kullanılan boru sterilize edin. Hipoklorit çözeltisi akıtılarak bu takip ve ters-osmoz bir litre su ile% 2&#39;lik bir stok susuz sodyum tiyosülfat çözeltisi ilave edilerek 25 mL yapılan% 0.05 sodyum tiyosülfat ile nötralize. </li><li> 7.5 &#39;den daha büyük bir pH değerine sahip örneği su için, HCI ile 6.5 ila 7.0 pH ayarlanır. HCl konsantrasyonu uzanabilir 0.251 M. Dört litre M 0,5 M HCl genellikle suyun ortam pH ve tamponlama kapasitesine bağlı olarak iki adet 800 litrelik örnekleri için yeterlidir. Bir peristaltik pompa veya Venturi (Şekil 2) kullanılarak örnekleme sırasında HCl enjekte edilir. Pompa hızını veya hedef pH ulaşmak için Venturi açılış ayarlayın. Bir alan pH metre kullanılarak örnek hattı çıkışında pH ölçün. </li><li> Cam yünü filtresi takunya eğer yüksek bulanıklık su ile bir ön filtreyi kullanın. (Ön filtreyi ve konut Özellikler Malzeme listesinde online olarak bulunmaktadır.) Patojenler de (bkz. aşağıda) cam yünü filtresi ile birlikte akıtılan gereken ön filtre tarafından yakalanan parçacıkları eklenebilir çünkü. </li><li> 2 ila 4 litre / dakika akış hızı ayarlayın. Tipik örnek hacmi 200 ile 1.500 litre vardır. </li><li> 48 saat için 4 ° C&#39;de bir plastik steril bir torbaya cam yünü ve filtre deposu çıkarın. </li></ol> 4. Elüsyon <ol><li> Eke dayalı bir halkaya cam yünü filtresiÖrnek girişi sonunda bir polipropilen şişeye aşağı bakacak şekilde duruyor. Örnek akış yönüne ters elüt edilmesi. </li><li> Filtre içine 9.5 pH değerine sahip 0.05 M glisin steril% 3 sığır eti ekstraktı 80 ml itin. </li><li> 15 dakika bekleyin. </li><li> Filtre içine 9.5 pH değerine sahip 0.05 M glisin steril% 3 sığır eti ekstraktı başka 80 ml itin. </li><li> Köpük Filtre girişinden dışarı çıkana kadar hava iyice filtresi itin. </li><li> 1 M HCl ile 7.0 arasında ve 7.5 ile arındırıldı pH ayarlama. </li><li> Mağaza 4 at eluat ° C daha uzun süreler için 24 saate kadar veya -20 ° C&#39;de. </li><li> Biri kullanıldığında Ön filtreyi Zehir. Sökün konut merminin ve steril eldivenli elleriyle yerde ön filtre tutarak konut içinde kalan tüm suyu dökün. </li><li> Konut kartuştan Ön filtreyi çıkarın ve 15 &quot;x 6&quot; zip-lock çanta içine kayma. </li><li> 0,05 M g steril% 3 sığır ekstresinin 200 mL dökülürBir torbaya 9.5 pH ve zip-lock ile güvenli bir şekilde mühür ile lycine. </li><li> Tüm yüzeyi sığır özü ile temas sağlamak için ön filtresi paketlenmiş birkaç kez invert. </li><li> 15 dakika bekleyin bazen ters ve torbalı ön filtreyi masaj. </li><li> Zip-kilidi açın. Ön filtreyi etrafında tutuş torba sıkıca mümkün olduğunca fazla sığır eti ekstraktı gibi sıkmak ve steril eldivenli elleriyle Ön filtreyi çekin. </li><li> Bir polipropilen şişeye sığır eti ekstraktı eluat dökün. </li><li> 1M HCI ile 7.0 arasında ve 7.5 ile arındırıldı pH ayarlama. </li><li> Mağaza 4 at eluat ° C daha uzun süreler için 24 saate kadar veya -20 ° C&#39;de. Ön filtresi eluat ayrı patojenler için analiz veya cam yünü filtre eluat ile kombine edilebilir. </li></ol> 5.. Temsilcisi Sonuçlar <table border="1"><tbody><tr><td> Patojen </td><td> Su Seviyesi Bulanıklık (NTU) bir </td><td> Miktarı Tohumlu / L b, c, d </td><td> % Kurtarma ± 1 SS </td><td> Sayı Bağımsız Denemeler </td></tr><tr><td> Enterovirus-Poliovirüs Sabin III </td><td> 0.5 </td><td> 500 </td><td> % 81 ± 11 </td><td> 7 </td></tr><tr><td> Enterovirüs Poliovirüs Sabin III </td><td> 0.5 </td><td> 5000 </td><td> % 67 ± 12 </td><td> 8 </td></tr><tr><td> Enterovirus-Poliovirüs Sabin III </td><td> 215 </td><td> 500 </td><td> % 59 ± 32 </td><td> 7 </td></tr><tr><td> Enterovirus-Poliovirüs Sabin III </td><td> 215 </td><td> 5000 </td><td> % 38 ± 22 </td><td> 6 </td></tr><tr><td> Enterovirus-Poliovirüs Sabin III </td><td> 447 </td><td> 500 </td><td> % 56 ± 18 </td><td> 8 </td></tr><tr><td> Enterovirus-Poliovirüs Sabin III </td><td> 447 </td><td> 5000 </td><td> % 63 ± 37 </td><td> 8 </td></tr><tr><td> Cryptosporidium parvum </td><td> 0.5 </td><td> 5 </td><td> % 38 ± 14 </td><td> 7 </td></tr><tr><td> Cryptosporidium parvum </td><td> 0.5 </td><td> 50 </td><td> 53 ±% 19 </td><td> 8 </td></tr><tr><td> Ağlamakptosporidium parvum </td><td> 215 </td><td> 5 </td><td> % 40 ± 16 </td><td> 7 </td></tr><tr><td> Cryptosporidium parvum </td><td> 215 </td><td> 50 </td><td> % 30 ± 6 </td><td> 6 </td></tr><tr><td> Cryptosporidium parvum </td><td> 447 </td><td> 5 </td><td> % 33 ± 13 </td><td> 8 </td></tr><tr><td> Cryptosporidium parvum </td><td> 447 </td><td> 50 </td><td> % 28 ± 11 </td><td> 8 </td></tr><tr><td> Salmonella enterica </td><td> 0.5 </td><td> 5 </td><td> % 29 ± 24 </td><td> 7 </td></tr><tr><td> Salmonella enterica </td><td> 0.5 </td><td> 500 </td><td> % 56 ± 16 </td><td> 8 </td></tr><tr><td> Salmonella enterica </td><td> 215 </td><td> 5 </td><td> % 32 ± 24 </td><td> 7 </td></tr><tr><td> Salmonella enterica </td&gt; <td> 215 </td><td> 500 </td><td> % 34 ± 11 </td><td> 6 </td></tr><tr><td> Salmonella enterica </td><td> 447 </td><td> 5 </td><td> % 34 ± 18 </td><td> 8 </td></tr><tr><td> Salmonella enterica </td><td> 447 </td><td> 500 </td><td> % 31 ± 24 </td><td> 8 </td></tr></tbody></table><ol class="lalpha"><li> Nefelometrik Bulanıklık Ünitesi </li><li> Genomik kopya / L olarak qPCR tarafından numaralandırılan Enteroviruslar </li><li> C. parvum ookistleri olarak immünofloresan tarafından numaralandırılan </li><li> S. enterica koloni oluşturan birimler olarak kültür tarafından numaralandırılan </li></ol> Değişen su bulanık ve patojen yoğunlukları ile Tablo 1. Cam yünü konsantrasyonu. <table border="1"><tbody><tr><td> Patojen </td><td> Su Numune Yer </td><td> Tutar Tohumlu / L </td><td> % Reco<html> çok </td></tr><tr><td> Kuş Gribi H5N2 </td><td> Sundi Gölü, Anchorage Borough </td><td> 2500 </td><td> % 42.9 </td></tr><tr><td> Kuş Gribi H5N2 </td><td> Minto Flats, Fairbanks Kuzey Yıldızı Borough </td><td> 2500 </td><td> % 36.7 </td></tr><tr><td> Kuş Gribi H5N2 </td><td> Portage Vadisi, Anchorage Borough </td><td> 2500 </td><td> % 7.8 </td></tr><tr><td> Kuş Gribi H5N2 </td><td> Potter Marsh, Anchorage Borough </td><td> 2500 </td><td> % 41.5 </td></tr><tr><td> Kuş Gribi H5N2 </td><td> Willow Lake, Yukon-Koyukuk Borough </td><td> 2500 </td><td> % 15.5 </td></tr></tbody></table><ol class="lalpha"><li> Genomik kopya / L olarak qPCR göre ölçülmüş </li></ol> Avian influenza virüsünün Tablo 2. Cam yünü konsantrasyonu Alaska beş siteleri su kullanarak. les/ftp_upload/3930/3930fig1.jpg &quot;alt =&quot; Şekil 1 &quot;/&gt; Şekil 1. Cam yünü yıkama diyagramı. Bu durulama zaman tasarrufu, bunun yerine bir kovanın kullanılabilir. Kavramı bir cam pipet yıkama benzer. <img src="/files/ftp_upload/3930/3930fig2.jpg" alt="Şekil 2" /> Peristaltik pompa ile asit enjeksiyonu ile Şekil 2. Cam yünü filtrasyon. Pompa hortumunu su musluğu ve cam yünü filtre arasında örnek hattı içine asit tanıtır &quot;T&quot; konnektör unutmayın. örneklenmiş suyu pH&gt; 7.5 sahipse pH ayarlaması gerekli değildir. Cam yünü filtreler bulanıklık seviyeleri ve patojen yoğunluklarda (Tablo 1), geniş bir aralık ile su patojenler konsantre etkili olmaktadır. Bunu test etmek için, dechlorinated musluk suyu 20 litre sudaki bulanıklığın istenilen seviyeye ulaşmak için kurutulmuş silt tınlı toprak (0, 1.27 veya 2.75 g / L) ile karıştırılır ve daha sonra çeşitli yoğunluklarda patojenler ekildi. Subir cam yünü filtreden geçirildi, eluat içinde konsantre patojenler numaralandırılan, ve bu değer, yüzde geri kazanım hesaplamada pay idi. , Yüzde geri kazanım hesaplama payda olan su, içine ekilmiş patojenlerin miktarı daha sonra birinci patojenler numaralandırma negatif eluat içine patojenler tohumlama ile tespit edildi. Negatif eluat bir filtre aracılığıyla bir dereceye giremeyen 20 litrelik Örnek geçirerek ve elüt kullanılarak hazırlandı. Olumsuz bir eluat içinde ekilmiş patojenler sayısallaştırabilmek cam yünü filtre tarafından oluşturulan matris farklılıklar meydana gelebilir patojen numaralandırma farklılıklar önler. QPCR tarafından patojenler miktarının Bu adımın önemi Lambertini ark olarak tartışılmıştır. 3. Bir 20 litre negatif kontrol örneği yüzde kurtarma hesaplama yanıltabilecek verebilecek hiçbir yerli patojenler bugünkü vardı belirlemek için cam yünü filtrasyon ile konsantre edildi. A 10 mikron nominal gözenek boyutu ön filtresinin wbulanıklık seviyesi ≥ 215 NTU iken olarak kullanılır. Poliovirüs Lambertini ve diğerleri tarafından tarif. 3 ikincil konsantrasyonu ve nükleik asit ekstraksiyon prosedürleri ve primerler ve prob kullanılarak gerçek zamanlı qPCR ile ölçüldü. Cryptosporidium parvum poliovirus için ikincil konsantrasyonu prosedüre göre hazırlandı nihai konsantre örnek hacmi (FCSV) içinde ölçüldü . Ookistleri immünfloresan (MeriFluor Cryptosporidium ve Giardia Algılama Takımı, Meridian Hayat Bilgisi, Inc, Cincinnati, OH) tarafından görüntülenmiştir. Salmonella enterica XLD agara plaklamayla FCSV içinde sayılabilir (Remel, Lenexa, KS) ve sayma koloni oluşturan- birimler. Cam yünü filtreleri kuş gribi virüsleri (Tablo 2) konsantre etkilidir. Düşük patojenite kuş gribi virüsü (H5N2) Alaska çeşitli yerlere su içine ekilmiş ve abov açıklandığı gibi yüzde kurtarma hesaplandıe. İkincil konsantrasyonu ve nükleik asit prosedürler poliovirus gibi gerçekleştirildi; virüsü Spackman ve diğerleri tarafından tarif primerler ve prob kullanılarak qPCR ile ölçüldü 4.

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Çıkarlarının çatışması ilan etti.

Cam yünü filtreleri gibi bitmiş içme suyu 7, yeraltı 8,9, yüzey suyu 10, deniz suyu 11, atıksu 12 olarak su kaynaklarının çeşitli insan Enterik virüsler konsantre birçok araştırma ekipleri 3,5,6 tarafından kullanılan ve edilmiştir tarım akış 13. Burada filtreler de kuş gribi virüsü gibi bakteriyel ve parazitik patojenlerde sırasıyla Salmonella enterica (serovar Typhimurium) ve Cryptosporidium parvum, ...

<table border="1"><tbody><tr><td> Reaktif veya öğenin adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Hidroklorik asit </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> A144-500 </td></tr><tr><td height="24"> Sodyum hidroksit </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> BP359-212 </td></tr><tr><td> Fosfat Tamponlu Salin Sodyum klorür Potasyum fosfat dibazik Potasyum fosfat monobazik </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td><html> BP358-212 BP363-500 BP362-500 </td></tr><tr><td> Sodyum hipoklorit yani çamaşır suyu </td><td> <a href="http://www.clorox.com" target="_blank">Clorox Co</a> </td><td></td></tr><tr><td> Sodyum tiyosülfat, susuz </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> S 475-212 </td></tr><tr><td> Sığır özü, kurutulmuş </td><td> <a href="http://www.bd.com" target="_blank">Becton, Dickinson and Company</a> </td><td> 211520 </td></tr><tr><td> Glisin </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> G46-500 </td></tr><tr><td> Yağlı sodocalcic cam yünü Veya R-11 unfaced fiberglas izolasyon </td><td> <a href="http://www.isover.com" target="_blank">Isover</a> <html> ref = &quot;http://www.jm.com&quot; target = &quot;_blank&quot;&gt; Johns Manville </td><td> Bourre 725 QN </td></tr><tr><td> Polipropilen mesh </td><td> <a href="http://www.industrialnetting.com" target="_blank">Netleştirme Endüstriyel</a> </td><td> xN4510 </td></tr><tr><td> 2 &quot;x4&quot; Sch 80 PVC dişli boru nipel </td><td> <a href="http://www.grainger.com" target="_blank">Grainger</a> </td><td> 6MW35 </td></tr><tr><td> 2 &quot;Sch 40 PVC kap </td><td> <a href="http://www.grainger.com" target="_blank">Grainger</a> </td><td> 5WDW3 </td></tr><tr><td> Erkek adaptör naylon bağlantı (1/2 &quot;x1 / 2&quot;) </td><td> <a href="http://www.usplastic.com" target="_blank">ABD Plastik A.Ş.</a> </td><td> 62178 </td></tr><tr><td> Eluat-1 litre için örnek şişeleri </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> 03-313-4F </td></tr><tr><td> 60 ml şırınga </td><td>&quot;Http://www.fishersci.com&quot; target = &quot;_blank&quot;&gt; Fisher Scientific </td><td> NC9661991 </td></tr><tr><td> pH şeritleri </td><td> <a href="http://www.whatman.com" target="_blank">Whatman</a> </td><td> 2614 991 </td></tr><tr><td> Ön filtre, Polipropilen, 10 inç kartuş, 10 mikron </td><td> McMaster-Carr </td><td> 4411K75 </td></tr><tr><td> Ön filtre konut </td><td> Cole-Parmer </td><td> S-29820-10 </td></tr></tbody></table>

glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens

Şüpheli kontamine su patojen seviyelerini değerlendirmek için önemli bir ilk adım yoğunlaşmasıdır. Konsantrasyon yöntemleri örneğin, örnekleme programını birden fazla patojen grubunu hedef ise birden çok yöntem gereklidir anlamına gelir Giardia ve Cryptosporidium 1, ABD Çevre Koruma Ajansı Yöntem 1623 belirli bir patojen grubu için özel olma eğilimindedir. Mevcut yöntem diğer bir dezavantajı, ekipman, örneğin, virüsler 2 yoğunlaştırmak için 1MDS kartuş filtre ile VIRADEL yöntemi komplike ve pahalı olabilir. Bu yazıda su kaynaklı patojenler yoğunlaştırmak için cam yünü filtreler oluşturmak için nasıl açıklar. Filtre elüsyon sonra, konsantre kültürel veya moleküler yöntemlerle patojen tespiti ve sayımı takip gibi santrifüj gibi ikinci bir konsantrasyon adım, için uygundur. Filtreleri çeşitli avantajları var. İnşaat kolaydır ve filtreler ile inşa edilebilirözel örnekleme gereksinimlerini karşılamak için ny boyutu. Filtre parça mümkün ciddi bir proje bütçe etkilemeden örnekleri, çok sayıda toplamak için yapım ucuzdur. Büyük örnek hacimleri (100s 1,000 L) örnek bulanıklık gelen tıkanma oranına bağlı olarak konsantre edilebilir. Filtreleri son derece taşınabilir ve böyle bir pompa ve akış metre gibi az ekipman ile, onlar bitmiş içme suyu, yüzey suyu, yeraltı suyu ve tarımsal yüzey akış örnekleme alanında uygulanabilir. Son olarak, cam yünü filtrasyon sadece tek bir yöntem gerekli olduğu şekilde patojen çeşitli tiplerde konsantre için etkilidir. Burada su bazlı insan entero, S almonella enterica, Cryptosporidium parvum, ve kuş gribi virüsü konsantre filtre etkinliği üzerine rapor.

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

Cam yünü filtreler dünyada araştırma gruplarının bir dizi su bazlı virüsler kullanılır konsantre edilmiştir. Burada cam yünü filtreleri oluşturmak için basit bir yaklaşım göstermek ve filtreleri de su kaynaklı viral, bakteriyel ve parazitik patojenlerde konsantre etkilidir göstermektedir.

Konsantre Su Bazlı Virüsler ve Tarım Zoonotik Patojenler için Cam Yünü Filtreler

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 Görüntüleme.  United States Geological Survey. Cam yünü filtreler dünyada araştırma gruplarının bir dizi su bazlı virüsler kullanılır konsantre edilmiştir. Burada cam yünü filtreleri oluşturmak için basit bir yaklaşım göstermek ve filtreleri de su kaynaklı viral, bakteriyel ve parazitik patojenlerde konsantre etkilidir göstermektedir.

Video: 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.

Hızlı Aktarım ve Lyme Hastalığı Patojenler lokalizasyonu için işaretleyin Gut içinde Yöntemleri

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

İmmünoloji ve Enfeksiyon

'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.

Kategori Algılama Bir Bakteriyel Patojenler &#39;Bioluminescent&#39; Muhabir Faj

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.

Kuş FTA Kartları İnfluenza Sürveyans: Alan Yöntemleri, Biyogüvenlik ve Ulaşım Sorunları çözüldü

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.

Vero Hücre Kültür Testi ile Alan toplanan Sivrisinekler Enfeksiyon Virüs Algılama

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.

Ixodes scapularis Keneler gelen tükürük, Tükürük Bezi ve hemolimf Koleksiyonu

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.

Polarize İnsan Havayolu bir sıvı kaplı Kültürünün Oluşturulması Solunum Patojenler için Host Hücre Yanıtı Okumak Epitel Calu-3 Hücreler<em&gt; In vitro</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.

RNA Virüslerinin geniş spektrumlu Kimyasal Önleyicileri için yüksek verimli tarama

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.

Kimerik fare modellerinde Rekombinant Grip aşıların burun içinde yoluyla mukozal bağışıklık Okumak

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.

Makrofaj Fusion çalışmaya imalatı kaliteli optik cam yüzeyler

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.

Formalin-Sabit Dokulardan Lyssavirüs Antijen Tespiti için İmmünohistokiniti Testi

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