Ringraziamo William T. Eckert per raccontare il video. Sviluppo del protocollo di lana di vetro faceva parte del Water Wisconsin e Trial Salute per rischi enterica (WAHTER Study), finanziato dalla US EPA STAR di Grant R831630. Alaska campioni sono stati raccolti da A. Reeves, A. Ramey, e B. Meixell con il sostegno finanziario USGS. Qualsiasi uso di nomi commerciali, prodotto o azienda è per scopi descrittivi soltanto e non implica riconoscimento alcuno da parte del governo degli Stati Uniti.

1. Preparare la lana di vetro <ol><li> Prima e dopo aver fatto ogni partita di filtri, sterilizzare l&#39;area di lavoro con una soluzione di candeggina al 10%. </li><li> Indossare guanti e camice. Sterilizzare un secchio in autoclave a 121 ° C e 15 psi per almeno 20 minuti. Posizionare la lana di vetro nel secchio sterile. </li><li> Saturare la lana di vetro con acqua ad osmosi inversa e lasciare macerare per 15 minuti. </li><li> Scolare l&#39;acqua ad osmosi inversa dal secchio. </li><li> Saturare la lana di vetro con 1 M HCl e lasciate macerare per 15 minuti. </li><li> Scolare la M 1 HCl dal secchio. </li><li> Lavare la lana di vetro con acqua ad osmosi inversa. </li><li> Mescolare accuratamente. </li><li> Controllare il pH utilizzando carta pH e ripetere l&#39;acqua osmosi inversa risciacquo fino a pH neutro si ottiene. </li><li> Versare l&#39;acqua di risciacquo. </li><li> Saturare la lana di vetro con 1 M NaOH e si lascia macerare per 15 minuti. </li><li> Scolare la M 1 NaOH dal secchio. </li><li> Ripetere l&#39;osmosi inversa risciacquo fino a pH neutro si ottiene. </li><li> Versare l&#39;acqua di risciacquo. </li><li> Invece di un secchio, una rondella lana di vetro può essere costruito, che è simile nel disegno ad una rondella pipetta di vetro (Figura 1). </li><li> Coprire la lana di vetro completamente con fosfato sterile Buffered Saline (PBS) a pH 6,8. </li><li> Utilizzare la lana di vetro preparata immediatamente o conservare a 4 ° C. Può essere conservato per un massimo di due settimane. Prima dell&#39;uso, assicurarsi che il pH è neutro in quanto aumenterà nel tempo. Se il pH non è neutro, ri-sciacquare con fosfato sterile salina tamponata (pH 6,8). </li></ol> 2. Il montaggio del filtro in lana di vetro <ol><li> Praticare un foro 11/16 di pollice nei tappi in PVC e fili di rubinetto in modo che i maschi raccordi in nylon adattatori possono essere avvitati i tappi. Questo passaggio è necessario solo per la prima assemblea. Successivamente, i tappi possono essere usati per anni. Applicare Teflonnastro per i fili di nylon e raccordi a vite per i tappi. </li><li> Riporre il tubo in PVC con piccoli pezzi di lana di vetro. Utilizzare uno stantuffo metallico, come una valvola motore di un&#39;auto, per il confezionamento stretto. Imballaggio non richiede grande forza. Confezione abbastanza stretto in modo che la lana di vetro rimane al suo posto e canali non si formano. Tuttavia, non viene imballato in modo così stretto acqua non può passare attraverso il filtro. Quando imballato opportunamente, una portata di 4 a 5 litri al minuto deve essere raggiungibile quando il filtro è fissato a rubinetti con pressioni comprese tra 40-60 psi. Per le dimensioni di tubo in PVC di cui al presente protocollo, circa 85 grammi lavato e lana di vetro imballato è usato per pipe. Tare il tubo vuoto PVC su un top-loading equilibrio e pacco con lana di vetro lavata fino a quando la massa del tubo aumenta 85 grammi. </li><li> Inserire la rete in polipropilene nei tappi in PVC con raccordi maschio in nylon adattatore collegato. </li><li> Applicare il nastro di teflon alle parti filettate del tubo in PVC. </li><li> Avvitare tappi in PVC alla p PVCipe e etichetta un&#39;estremità filtro l&#39;afflusso e l&#39;altra estremità del deflusso. Questo è importante per la successiva fase di eluizione. Che fine viene etichettato afflusso non importa. </li><li> Spingere 60 mL di soluzione salina tamponata con fosfato sterile (pH = 6.8) nel filtro utilizzando un catetere punta siringa. Eccesso uscirà all&#39;estremità opposta. </li><li> Wrap termina strettamente con Parafilm per evitare perdite. I filtri possono essere memorizzati fino a 30 giorni a 4 ° C. </li></ol> 3. Campionamento <ol><li> Sterilizzare tubi utilizzati per il campionamento da parte di ricircolo o immergendo gli elementi per 30 minuti in NaClO 0,525% (cioè, una soluzione al 10% di candeggina standard). Segui questa drenando la soluzione di ipoclorito e neutralizzare con tiosolfato di sodio 0,05%, ottenuto aggiungendo 25 mL di una soluzione al 2% di sodio tiosolfato anidro stock di un litro d&#39;acqua ad osmosi inversa. </li><li> Per esempio l&#39;acqua con un pH maggiore di 7,5, regolare il pH a tra 6,5 ​​e 7,0 con HCl. HCl concentrazione può variare da 0,25M a 1 M. quattro litri 0,5 M HCl è generalmente sufficiente per due campioni di 800 litri, a seconda del pH ambientale dell&#39;acqua e la capacità di buffering. Iniettare HCl durante il campionamento utilizzando una pompa peristaltica o Venturi (Figura 2). Regolare la velocità della pompa o l&#39;apertura Venturi per raggiungere l&#39;obiettivo di pH. Misurare il pH all&#39;uscita linea del campione usando un pH-metro campo. </li><li> Utilizzare un prefiltro se gli zoccoli in lana di vetro filtro da acqua con elevata torbidità. (Specifiche per il prefiltro e la sua sede sono in linea nell&#39;elenco Materiali.) Poiché patogeni può essere collegato a particelle intrappolate dal prefiltro deve essere eluito con il filtro di lana di vetro (vedi sotto). </li><li> Regolare il flusso tra 2 e 4 litri al minuto. Volumi di campione tipici sono tra 200 e 1.500 litri. </li><li> Scollegare il filtro di lana di vetro e riporlo in un sacchetto di plastica sterile a 4 ° C per un massimo di 48 ore. </li></ol> 4. Eluizione <ol><li> Applicare il filtro di lana di vetro ad un anellostand con l&#39;estremità rivolta verso il basso afflusso di campione in una bottiglia di polipropilene. Eluire opposta alla direzione del flusso del campione. </li><li> Premere 80 ml di estratto di carne sterile 3% in 0,05 M glicina con un pH di 9,5 nel filtro. </li><li> Attendere 15 minuti. </li><li> Premere un&#39;altra 80 mL di sterile estratto di carne 3% in 0,05 M glicina con un pH di 9,5 nel filtro. </li><li> Spingere il filtro d&#39;aria che fino a schiuma fuoriesce dal filtro in ingresso. </li><li> Regolare il pH dell&#39;eluato a tra 7,0 e 7,5 con 1 M HCl. </li><li> Eluato Conservare a 4 ° C per un massimo di 24 ore oa -20 ° C per lunghi periodi di tempo. </li><li> Eluire il prefiltro se viene utilizzato. Svitare la parte superiore della cartuccia fuori sede e versare tutta l&#39;acqua residua all&#39;interno del corpo mentre si tiene il prefiltro in posizione con le mani protette da guanti sterili. </li><li> Togliere il prefiltro dalla cartuccia dell&#39;alloggiamento e farla scorrere in un 15 &quot;x 6&quot; zip-lock bag. </li><li> Versare 200 ml di estratto di carne sterile 3% in M ​​0,05 glycine con un pH di 9,5 nel sacco e sigillare saldamente con la zip-lock. </li><li> Invertire il prefiltro insaccato diverse volte per garantire l&#39;intera superficie viene a contatto con l&#39;estratto di carne. </li><li> Attendere 15 minuti, a volte invertendo e massaggiando il prefiltro insaccato. </li><li> Aprire lo zip-lock. Afferrare il sacchetto attorno al prefiltro strettamente a spremere come estratto di carne il più possibile e tirare la prefiltro con guanti sterili,. </li><li> Versare l&#39;eluato estratto di carne in una bottiglia di polipropilene. </li><li> Regolare il pH dell&#39;eluato a tra 7,0 e 7,5 con HCl 1M. </li><li> Eluato Conservare a 4 ° C per un massimo di 24 ore oa -20 ° C per lunghi periodi di tempo. L&#39;eluato prefiltro può essere analizzato separatamente per i patogeni o in combinazione con l&#39;eluato filtro lana di vetro. </li></ol> 5. Risultati rappresentativi <table border="1"><tbody><tr><td> Patogeni </td><td> Water Level torbidità (NTU) a </td><td> Importo Seeded / L b, c, d </td><td> Recupero% ± 1 SD </td><td> Trials Numero indipendenti </td></tr><tr><td> Enterovirus-Poliovirus Sabin III </td><td> 0,5 </td><td> 500 </td><td> 81% ± 11 </td><td> 7 </td></tr><tr><td> Enterovirus-Poliovirus Sabin III </td><td> 0,5 </td><td> 5000 </td><td> 67 ± 12% </td><td> 8 </td></tr><tr><td> Enterovirus-Poliovirus Sabin III </td><td> 215 </td><td> 500 </td><td> 59% ± 32 </td><td> 7 </td></tr><tr><td> Enterovirus-Poliovirus Sabin III </td><td> 215 </td><td> 5000 </td><td> 38% ± 22 </td><td> 6 </td></tr><tr><td> Enterovirus-Poliovirus Sabin III </td><td> 447 </td><td> 500 </td><td> 56 ± 18% </td><td> 8 </td></tr><tr><td> Enterovirus-Poliovirus 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> Piangereptosporidium 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> Unità di torbidità nefelometrico </li><li> Enterovirus enumerate da qPCR come copie genomiche / L </li><li> C. parvum enumerati mediante immunofluorescenza di oocisti </li><li> S. enterica enumerati dalla cultura come formanti colonie-unità </li></ol> Tabella 1. Concentrazione di lana di vetro con la torbidità dell&#39;acqua e densità diversi agenti patogeni. <table border="1"><tbody><tr><td> Patogeni </td><td> Campione d&#39;acqua Location </td><td> Importo Seeded / L a </td><td> Reco%<html> molto </td></tr><tr><td> Influenza aviaria H5N2 </td><td> Sundi Lake, Anchorage Borough </td><td> 2500 </td><td> 42,9% </td></tr><tr><td> Influenza aviaria H5N2 </td><td> Minto Flats, Fairbanks North Star Borough </td><td> 2500 </td><td> 36,7% </td></tr><tr><td> Influenza aviaria H5N2 </td><td> Portage Valley, Anchorage Borough </td><td> 2500 </td><td> 7,8% </td></tr><tr><td> Influenza aviaria H5N2 </td><td> Potter Marsh, Anchorage Borough </td><td> 2500 </td><td> 41,5% </td></tr><tr><td> Influenza aviaria H5N2 </td><td> Willow Lake, Yukon-Koyukuk Borough </td><td> 2500 </td><td> 15,5% </td></tr></tbody></table><ol class="lalpha"><li> Misurato da qPCR come copie genomiche / L </li></ol> Tabella 2. Concentrazione in lana di vetro del virus dell&#39;influenza aviaria utilizzando l&#39;acqua da cinque siti in Alaska. les/ftp_upload/3930/3930fig1.jpg &quot;alt =&quot; Figura 1 &quot;/&gt; Figura 1. Schema di rosetta lana di vetro. Questo può essere usato al posto di un secchio, risparmiando tempo di risciacquo. Il concetto è simile ad una rondella pipetta di vetro. <img src="/files/ftp_upload/3930/3930fig2.jpg" alt="Figura 2" /> Figura 2. Lana di vetro di filtrazione con iniezione di acido da pompa peristaltica. Nota: il connettore &quot;T&quot;, dove il tubo della pompa acida introduce nella linea di campione tra il rubinetto dell&#39;acqua e la lana di vetro filtro. regolazione del pH è necessaria solo se l&#39;acqua ha un pH campionata&gt; 7,5. I filtri di lana di vetro sono efficaci agenti patogeni in concentrazione da acqua con una vasta gamma di livelli di torbidità e densità patogeni (Tabella 1). Per verificare ciò, 20 litri di acqua di rubinetto senza cloro è stato mescolato con il terreno limo argilla secca (0, 1,27 o 2,75 g / L) per raggiungere il livello desiderato di torbidità e poi seminati con agenti patogeni a varie densità. L&#39;acquaè stato passato attraverso un filtro lana di vetro, i patogeni concentrati nell&#39;eluato erano enumerate, e questo valore era il numeratore nel calcolo percentuale di recupero. La quantità di agenti patogeni seminati in acqua, che è, il denominatore del calcolo recupero percentuale, è stato determinato prima semina dei patogeni in un eluato negativo di elencare i patogeni. L&#39;eluato negativo è stato preparato facendo passare una seminata campione di 20 litro attraverso un filtro e eluendo. Quantificare gli agenti patogeni seminate in un eluato negativo evita differenze di enumerazione degli agenti patogeni che potrebbero derivare da differenze matrice creata dal filtro di lana di vetro. L&#39;importanza di questo passo, nel quantificare i patogeni di qPCR è discusso in Lambertini et al. 3. Un campione di 20 litro controllo negativo è stata concentrata mediante filtrazione in lana di vetro per determinare non ci fossero native presenti agenti patogeni che potrebbero confondere il calcolo percentuale di recupero. A 10 um dimensione nominale dei pori prefiltro wcome quando il livello torbidità era ≥ 215 NTU. Poliovirus è stato quantificato in tempo reale utilizzando la concentrazione qPCR secondario e procedure di estrazione di acidi nucleici e inneschi e le sonde descritte in Lambertini et al. 3. Cryptosporidium parvum è stato quantificato nel volume finale del campione concentrato (FCSV) creato dalla concentrazione procedura secondaria di poliovirus . Oocisti sono state visualizzate mediante immunofluorescenza (MeriFluor Cryptosporidium e Giardia Detection Kit, Meridian Life Science, Inc., Cincinnati, OH). Salmonella enterica è stato quantificato in FCSV da piastrato su agar XLD (Remel, Lenexa, KS) e conteggio delle colonie di formazione- unità. I filtri di lana di vetro sono efficaci a concentrare virus dell&#39;influenza aviaria (Tabella 2). Virus a bassa patogenicità dell&#39;influenza aviaria (H5N2) è stato seminato in acqua da diverse località in Alaska e la percentuale di recupero calcolato come descritto Above. Concentrazione secondario e procedure di acidi nucleici sono stati eseguiti come per poliovirus, il virus è stato quantificato qPCR utilizzando i primer e sonde descritte in Spackman et al 4.

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I filtri di lana di vetro sono stati utilizzati da vari gruppi di ricerca a concentrarsi 3,5,6 virus enterici umani da una varietà di fonti d&#39;acqua, come acqua potabile finito 7, 8,9 acque sotterranee, acque di superficie 10, 11 di acqua di mare, acque reflue 12, e scarichi agricoli 13. Qui riportiamo i filtri sono efficaci anche a concentrare il virus dell&#39;influenza aviaria così come i patogeni batterici e protozoi Salmonella enterica (sierotipo ...

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glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens

Il primo passo fondamentale per la valutazione dei livelli di agenti patogeni in sospetto di acqua contaminata è la concentrazione. Metodi di concentrazione tendono a essere specifica per un gruppo particolare agente patogeno, per esempio gli Stati Uniti Metodo Environmental Protection Agency 1623 per Giardia e Cryptosporidium 1, il che significa che più metodi sono necessari se il programma di campionamento di mira più di un gruppo patogeno. Un altro svantaggio dei metodi attuali è l&#39;apparecchiatura può essere complicato e costoso, per esempio il metodo VIRADEL con cartuccia filtrante per concentrare il virus 1MDS 2. In questo articolo abbiamo descritto come creare filtri in lana di vetro per concentrare gli agenti patogeni a base acquosa. Dopo eluizione filtro, il concentrato è suscettibile ad una fase di seconda concentrazione, come centrifugazione, seguita da patogeno rilevamento e conteggio con metodi culturali o molecolari. I filtri sono diversi vantaggi. La costruzione è semplice ed i filtri possono essere costruiti ad unadimensioni ny per soddisfare le esigenze specifiche di campionamento. Le parti del filtro sono poco costosi, che permettono di raccogliere un gran numero di campioni senza incidere negativamente un bilancio del progetto. Grandi volumi di campione (100s a 1.000 s L) può essere concentrata a seconda del tasso di intasamento di torbidità. I filtri sono facilmente trasportabili e con attrezzatura minima, come una pompa e misuratore di portata, possono essere attuate in campo per il campionamento dell&#39;acqua finito di bere, acque superficiali, sotterranee e scarichi agricoli. Infine, filtrazione lana di vetro è efficace per concentrare una varietà di tipi di patogeni così solo metodo è necessario. Qui riportiamo l&#39;efficacia del filtro di concentrazione acquosa enterovirus umani, S almonella enterica, Cryptosporidium parvum, e il virus dell&#39;influenza aviaria.

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

I filtri di lana di vetro sono stati utilizzati per concentrare i virus acquosa da un certo numero di gruppi di ricerca in tutto il mondo. Qui mostriamo un metodo semplice per la costruzione di filtri di lana di vetro e di dimostrare i filtri sono anche efficaci in concentrazione acquosa virali e patogeni batterici e protozoi.

Filtri di lana di vetro per la raccolta virus a base acquosa e agricole agenti patogeni zoonotici

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 Views.  United States Geological Survey. I filtri di lana di vetro sono stati utilizzati per concentrare i virus acquosa da un certo numero di gruppi di ricerca in tutto il mondo. Qui mostriamo un metodo semplice per la costruzione di filtri di lana di vetro e di dimostrare i filtri sono anche efficaci in concentrazione acquosa virali e patogeni batterici e protozoi.

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.

Metodi per il trasferimento rapido e localizzazione di agenti patogeni malattia di Lyme All&#39;interno della Gut Tick

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

Ricerca

Immunologia e infezioni

'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.

Phage &#39;Bioluminescent&#39; Reporter per il rilevamento di batteri patogeni di categoria A

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.

Sorveglianza dell&#39;influenza aviaria con FTA Carte: metodi per i campi, Biosicurezza, e problemi di trasporto Risolto

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.

Il rilevamento del virus infettivo da Campo-zanzare raccolte mediante test coltura cellulare Vero

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.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche Ixodes scapularis

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.

Creazione di un liquido coperto Cultura polarizzata Airway epiteliali umane Calu-3 celle per studio cellulare risposta dell&#39;ospite agli agenti patogeni delle vie respiratorie<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.

High-throughput screening per largo spettro chimico inibitori di RNA virus

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.

Intranasale somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

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.

Realizzazione di superfici di vetro di qualità ottica per studiare macrofago Fusion

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.

Test immunoistochimico per il rilevamento dell'antigene del lyssavirus da tessuti fissati con formalina

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