Agradecemos William T. Eckert para narrar o vídeo. Desenvolvimento do protocolo de lã de vidro fez parte da água Wisconsin e Julgamento de Saúde para Riscos entéricos (WAHTER Study), financiado pela EPA dos EUA ESTRELA Grant R831630. Alaska amostras foram coletadas por A. Reeves, A. Ramey e Meixell B. com apoio financeiro do USGS. Qualquer utilização dos nomes de comércio, produto ou empresa é apenas para fins descritivos e não implica o endosso pelo governo dos EUA.

1. Preparação da Lã de Vidro <ol><li> Antes e depois fazendo com que cada lote de filtros, esterilizar a área de trabalho com uma solução de lixívia a 10%. </li><li> Coloque luvas e jaleco. Esterilizar um balde em autoclave a 121 psi e 15 ° C durante pelo menos 20 minutos. Coloque a lã de vidro no balde estéril. </li><li> Saturar a lã de vidro com água de osmose reversa e deixe de molho durante 15 minutos. </li><li> Escorra a água de osmose reversa do balde. </li><li> Saturar a lã de vidro com 1 M de HCl e deixe de molho durante 15 minutos. </li><li> Escorra a 1 M HCl do balde. </li><li> Lavar a lã de vidro com água de osmose reversa. </li><li> Misturar bem. </li><li> Verificar o pH usando papel de pH e repetir a água de osmose inversa enxaguamento até um pH neutro é alcançada. </li><li> Deitar fora a água do enxágüe. </li><li> Saturar a lã de vidro com 1 M de NaOH e deixe de molho durante 15 minutos. </li><li> Escorra a 1 M NaOH do balde. </li><li> REPEAT a osmose inversa enxaguamento até um pH neutro é alcançada. </li><li> Deitar fora a água do enxágüe. </li><li> Em vez de um balde, uma anilha de lã de vidro pode ser construído, que é semelhante em desenho para uma máquina de lavar pipeta de vidro (Figura 1). </li><li> Cobrir a lã de vidro completamente com fosfato estéril tamponado salino (PBS), ajustada a pH 6,8. </li><li> Use lã de vidro preparada imediatamente ou armazenar a 4 ° C. Pode ser armazenado por até duas semanas. Antes de usar, certificar-se o pH é neutro uma vez que irá aumentar com o tempo. Se o pH não é neutro, re-lavar com fosfato estéril salino tamponado (pH 6,8). </li></ol> 2. Montagem do Filtro Lã de Vidro <ol><li> Perfurar um furo 11/16 polegadas em as tampas de PVC e fios de torneira para os encaixes de nylon machos adaptador pode ser aparafusado no tampões. Esta etapa é necessária apenas para a primeira assembléia. Depois disso, os tampões podem ser utilizados para ano. Aplicar Teflonfita para os fios de nylon ferragens e parafusos para as tampas. </li><li> Embale o tubo de PVC com pequenos pedaços de lã de vidro. Use um êmbolo de metal, como uma válvula de motor de carro, para embalar apertado. Embalagem não requer muita força. Embale apertado o suficiente para que a lã de vidro permanece no lugar e os canais não fazem. No entanto, não embalar tão bem a água não pode fluir através do filtro. Quando embalada de forma adequada, uma taxa de fluxo de 4 a 5 litros por minuto deve ser atingido quando o filtro está ligado a torneiras de água com pressões entre 40-60 psi. Para o tamanho de tubo de PVC especificado neste protocolo, cerca de 85 gramas de lã de vidro lavados e embalados é usado por tubo. Tara o tubo de PVC vazio em um equilíbrio de carregamento superior e embalagem com lã de vidro lavado até que a massa do tubo aumenta 85 gramas. </li><li> Inserir tela de polipropileno no tampas de PVC com conexões para o adaptador macho de nylon ligados. </li><li> Aplicar fita de Teflon para as porções roscadas do tubo de PVC. </li><li> Parafuso com tampas de PVC para a p PVCipe e uma extremidade de etiqueta filtro o influxo ea outra extremidade a saída. Isto é importante mais tarde para a etapa de eluição. Que final é rotulado entrada não importa. </li><li> Empurrar 60 ml de fosfato estéril salino tamponado (pH = 6,8) para dentro do filtro usando um cateter de ponta de uma seringa. Excesso sairá extremidade oposta. </li><li> Enrole firmemente termina com Parafilm para evitar fugas. Os filtros podem ser armazenadas até 30 dias a 4 ° C. </li></ol> 3. Amostragem <ol><li> Esterilizar tubos utilizados para a amostragem por recirculação ou imergindo itens durante 30 minutos, 0,525% NaClO (isto é, uma solução a 10% de lixívia doméstica padrão). Segue este por drenagem da solução de hipoclorito e neutralizar com tiossulfato de sódio 0,05%, feita pela adição de 25 mL de uma solução a 2% de tiossulfato de estoque de sódio anidro para um litro de água de osmose inversa. </li><li> Para a água da amostra com um pH superior a 7,5, ajustar o pH a entre 6,5 e 7,0 com HCl. Concentração de HCl pode variar de 0,25M para 1 M. Quatro litros HCl 0,5 M é geralmente suficiente para dois 800 amostras litro, dependendo do pH do ambiente da água e capacidade de tamponamento. Injectar HCl durante a amostragem utilizando uma bomba peristáltica ou Venturi (Figura 2). Ajustar a velocidade da bomba ou Venturi abertura para atingir o pH alvo. Medir o pH na linha de saída de amostra, utilizando um medidor de pH de campo. </li><li> Utilizar um pré-filtro, se as obstruções lã de vidro de filtro a partir de água com turvação elevada. (Especificações para o pré-filtro eo seu alojamento estão na linha lista de materiais.) Devido agentes patogénicos pode ser ligado a partículas retidas pelo pré-filtro deve ser eluída juntamente com o filtro de fibra de vidro (ver abaixo). </li><li> Ajustar a taxa de fluxo para entre 2 e 4 litros / minuto. Volumes de amostra típicos são entre 200 e 1500 litros. </li><li> Desligue o filtro de lã de vidro e armazenar em um saco de plástico estéril a 4 ° C durante até 48 horas. </li></ol> 4. Eluição <ol><li> Apor o filtro de lã de vidro para um anelficar com a extremidade de influxo amostra apontando para baixo dentro de uma garrafa de polipropileno. Eluir oposta da direcção do fluxo de amostra. </li><li> Empurrar 80 mL de extracto de carne estéril de 3% em 0,05 M de glicina com um pH de 9,5 para dentro do filtro. </li><li> Aguarde 15 minutos. </li><li> Empurrar outro mL 80 de extracto de carne estéril de 3% em 0,05 M de glicina com um pH de 9,5 para dentro do filtro. </li><li> Empurre o filtro de ar completa até espuma sai da entrada do filtro. </li><li> Ajustar o pH eluato entre 7,0 e 7,5 com 1 M de HCl. </li><li> Eluato Armazenar a 4 ° C durante até 24 horas ou à temperatura de -20 ° C durante períodos de tempo mais longos. </li><li> Eluir a pré-filtro, se uma for usado. Desparafuse a parte superior da carcaça do cartucho e deitar fora toda a água residual dentro do invólucro, mantendo o pré-filtro no lugar com luvas esterilizadas. </li><li> Remover o pré-filtro do cartucho de habitação e colocá-lo em um &quot;x 6&quot; 15 saco zip-lock. </li><li> Verter 200 mL de extracto de carne estéril de 3% em 0,05 M glycine com um pH de 9,5 para dentro do saco e selar de forma segura com o zip-lock. </li><li> Inverta a pré-filtro ensacado várias vezes para assegurar a totalidade da superfície entra em contacto com o extracto de carne de vaca. </li><li> Espere 15 minutos, invertendo e massageando o pré-filtro ensacado. </li><li> Abra o zip-lock. Segure o saco em torno do pré-filtro com força para espremer como extrato de carne tanto quanto possível e puxe o pré-filtro estéreis com as mãos enluvadas. </li><li> Verter o eluato extracto de carne dentro de uma garrafa de polipropileno. </li><li> Ajustar o pH eluato entre 7,0 e 7,5 com HCl 1M. </li><li> Eluato Armazenar a 4 ° C durante até 24 horas ou à temperatura de -20 ° C durante períodos de tempo mais longos. O eluato de pré-filtro pode ser analisada separadamente para agentes patogénicos ou combinado com o eluato de filtro de lã de vidro. </li></ol> 5. Os resultados representativos <table border="1"><tbody><tr><td> Patógeno </td><td> Nível de Água de Turbidez (NTU) uma </td><td> Quantidade Seeded / L b, c, d </td><td> Recuperação% ± 1 DP </td><td> Ensaios número independente </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> Enterovírus Poliovírus-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> Chorarptosporidium 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> Unidade de Turbidez nefelometria </li><li> Enterovírus enumerados por qPCR como cópias de DNA genômico / L </li><li> C. parvum enumerados por imunofluorescência como oocistos </li><li> S. entérica enumerados por cultura como formadoras de colónias-unidades </li></ol> Tabela 1. Concentração de lã de vidro com turbidez da água e densidades diferentes patógenos. <table border="1"><tbody><tr><td> Patógeno </td><td> Localização amostra de água </td><td> Valor Seeded / L a </td><td> Reco%<html> muito </td></tr><tr><td> Avian Influenza H5N2 </td><td> Sundi Lake, Anchorage Borough </td><td> 2500 </td><td> 42,9% </td></tr><tr><td> Avian Influenza H5N2 </td><td> Minto Flats, Fairbanks North Star Borough </td><td> 2500 </td><td> 36,7% </td></tr><tr><td> Avian Influenza H5N2 </td><td> Portage Valley, Anchorage Borough </td><td> 2500 </td><td> 7,8% </td></tr><tr><td> Avian Influenza H5N2 </td><td> Potter Marsh, Anchorage Borough </td><td> 2500 </td><td> 41,5% </td></tr><tr><td> Avian Influenza H5N2 </td><td> Willow Lake, Borough Yukon-Koyukuk </td><td> 2500 </td><td> 15,5% </td></tr></tbody></table><ol class="lalpha"><li> Medido por qPCR como cópias de DNA genômico / L </li></ol> Tabela 2. Concentração de lã de vidro de vírus da gripe aviária usando água de cinco sítios no Alasca. les/ftp_upload/3930/3930fig1.jpg &quot;alt =&quot; Figura 1 &quot;/&gt; Figura 1. Diagrama de máquina de lavar lã de vidro. Isto pode ser usado em vez de um balde, poupando tempo de lavagem. O conceito é semelhante a uma anilha pipeta de vidro. <img src="/files/ftp_upload/3930/3930fig2.jpg" alt="A Figura 2" /> Figura 2. Filtração lã de vidro com injecção de ácido por meio de bomba peristáltica. Observe o &quot;T&quot; conector onde a tubulação da bomba apresenta ácido para a linha de amostra entre a torneira da água e filtro de lã de vidro. o ajuste do pH é necessária apenas se a água amostrada tem um pH&gt; 7,5. Os filtros de lã de vidro são eficazes na concentração agentes patogénicos a partir de água com uma ampla gama de níveis de turvação e densidades patógeno (Tabela 1). Para testar isto, 20 litros de água declorada foi misturada com o solo seco lodo marga (0, 1,27, ou 2,75 g / L) para atingir o nível desejado de turvação e, em seguida, semeada com agentes patogénicos em várias densidades. A águafoi passado através de um filtro de lã de vidro, os agentes patogénicos concentradas no eluato foram enumerados, e este valor foi o numerador no cálculo de recuperação por cento. A quantidade de agentes patogénicos semeadas em água, isto é, o denominador do cálculo de recuperação por cento, foi determinada por primeiro semeando os agentes patogénicos em um eluato negativo, então enumerar os agentes patogénicos. O eluato negativo foi preparado pela passagem de uma amostra de 20 litros unseeded através de um filtro e de eluição. A quantificação dos agentes patogénicos semeadas em um eluato negativo evita diferenças na enumeração patógeno que poderiam resultar das diferenças matriz criada pelo filtro de lã de vidro. A importância deste passo na hora de quantificar patógenos por qPCR é discutido em Lambertini et al. 3. Uma amostra de controlo 20 litros negativo foi concentrada através de filtração em lã de vidro para determinar havia nenhum presente patógenos nativo que pudessem confundir o cálculo de recuperação por cento. A 10 uM nominal de poro de tamanho pré-filtro wtal como utilizado quando o nível de turbidez foi ≥ 215 NTU. Poliovírus foi quantificada por tempo real qPCR utilizando a concentração secundária e procedimentos de extracção de ácidos nucleicos e os iniciadores e sonda descrita no Lambertini et al. 3. Cryptosporidium parvum foi quantificada no volume de amostra final concentrado (FCSV) criado pelo procedimento de concentração secundária para poliovírus . Oocistos foram visualizadas por imunofluorescência (MeriFluor Cryptosporidium e Giardia Detecção Kit, Life Science Meridian, Inc., Cincinnati, OH). Salmonella entérica foi quantificada no FCSV por plaqueamento em agar XLD (Remel, Lenexa, KS) e contando colónia-forming- unidades. Os filtros de lã de vidro são eficazes na concentração de vírus da influenza aviária (Tabela 2). Baixa patogenicidade do vírus da gripe aviária (H5N2) foi semeado em água a partir de vários locais do Alasca e do percentual de recuperação calculado conforme descrito Above. Concentração secundária e procedimentos de ácidos nucleicos foram realizados como para o poliovírus, o vírus foi quantificada por qPCR utilizando os primers e sondas descritas no Spackman et al 4.

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Os filtros de lã de vidro têm sido usadas por vários grupos de pesquisa 3,5,6 a concentrar-se humanos vírus entéricos de uma variedade de fontes de água, tais como água potável terminado 7, a água subterrânea 8,9, água de superfície 10, a água do mar 11, as águas residuais 12, e escoamento agrícola 13. Relatamos aqui os filtros são também eficazes na concentração de vírus de influenza aviária, bem como os agentes patogénic...

<table border="1"><tbody><tr><td> Nome do reagente ou item </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Ácido clorídrico </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> A144-500 </td></tr><tr><td height="24"> De hidróxido de sódio </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> BP359-212 </td></tr><tr><td> Tampão fosfato salino Cloreto de sódio De fosfato de potássio dibásico- De fosfato de potássio monobásico- </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> De hipoclorito de sódio ou seja, água sanitária </td><td> <a href="http://www.clorox.com" target="_blank">A Clorox Co.</a> </td><td></td></tr><tr><td> Tiossulfato de sódio, anidro </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> S 475-212 </td></tr><tr><td> Extrato de carne, desidratado </td><td> <a href="http://www.bd.com" target="_blank">Becton, Dickinson and Company</a> </td><td> 211520 </td></tr><tr><td> Glycine </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> G46-500 </td></tr><tr><td> Lã de vidro lubrificada sodocalcic Ou R-11 fibra de vidro unfaced </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> Tela de polipropileno </td><td> <a href="http://www.industrialnetting.com" target="_blank">Industrial compensação</a> </td><td> xN4510 </td></tr><tr><td> 2 &quot;x4&quot; Sch 80 PVC mamilo roscadas </td><td> <a href="http://www.grainger.com" target="_blank">Grainger</a> </td><td> 6MW35 </td></tr><tr><td> 2 &quot;Sch 40 PVC cap </td><td> <a href="http://www.grainger.com" target="_blank">Grainger</a> </td><td> 5WDW3 </td></tr><tr><td> Montagem nylon Masculino adaptador (1/2 &quot;x1 / 2&quot;) </td><td> <a href="http://www.usplastic.com" target="_blank">EUA Plastic Corp</a> </td><td> 62178 </td></tr><tr><td> Os frascos para eluato-1 litro </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> 03-313-4F </td></tr><tr><td> 60 mL seringa </td><td>&quot;Http://www.fishersci.com&quot; target = &quot;_blank&quot;&gt; Fisher Scientific </td><td> NC9661991 </td></tr><tr><td> faixas de pH </td><td> <a href="http://www.whatman.com" target="_blank">Whatman</a> </td><td> 2614 991 </td></tr><tr><td> Pré-filtro, Polypropylene, 10 cartucho polegadas, 10 mM </td><td> McMaster-Carr </td><td> 4411K75 </td></tr><tr><td> Prefilter habitação </td><td> Cole-Parmer </td><td> S-29.820-10 </td></tr></tbody></table>

glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens

O primeiro passo fundamental na avaliação de níveis de patógenos em água contaminada suspeita é a concentração. Métodos de concentração tendem a ser específicas para um determinado grupo patógeno, por exemplo Ambiental dos EUA Método Agência de Protecção 1623 para Giardia e Cryptosporidium 1, o que significa que vários métodos são necessários se o programa de amostragem é a segmentação mais do que um grupo patógeno. Outra desvantagem dos métodos actuais é o equipamento pode ser complicado e caro, por exemplo, o método VIRADEL com o filtro de cartucho para a concentração de vírus 1MDS 2. Neste artigo vamos descrever como construir filtros de lã de vidro para a concentração de agentes patogênicos. Após eluição do filtro, o concentrado é passível de um passo de concentração em segundo lugar, tal como centrifugação, seguido por detecção de agentes patogénicos e enumeração por métodos de cultura ou molecular. Os filtros têm várias vantagens. A construção é fácil e os filtros podem ser construídos para umny tamanho para atender aos requisitos específicos de amostragem. As peças de filtro são baratos, tornando possível a recolha de um grande número de amostras, sem que prejudica gravemente a um orçamento do projecto. Grandes volumes de amostra (100S a 1.000 s L) pode ser concentrada, dependendo da taxa de entupimento de turvação da amostra. Os filtros são altamente portátil e com o mínimo de equipamento, tais como uma bomba e medidor de fluxo, eles podem ser implementados no campo para a amostragem de água potável acabado, água de superfície, a água subterrânea, e do escoamento agrícola. Por último, o vidro de filtração lã é eficaz para a concentração de uma variedade de tipos de organismos patogénicos para que apenas um método é necessário. Aqui nós relatamos sobre a eficácia do filtro em concentrar enterovirus humano por via aquática, S almonella enterica, Cryptosporidium parvum e vírus da gripe aviária.

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

Os filtros de lã de vidro têm sido utilizados para concentrar os vírus pela água por um número de grupos de pesquisa em todo o mundo. Aqui, mostramos uma abordagem simples para a construção de filtros de lã de vidro e demonstrar os filtros são também eficazes na concentração waterborne virais, agentes patogénicos bacterianos e protozoários.

Vidro Filtros de lã para a concentração de vírus e transmitidas pela água Agrícolas patógenos zoonóticos

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 visualizações.  United States Geological Survey. Os filtros de lã de vidro têm sido utilizados para concentrar os vírus pela água por um número de grupos de pesquisa em todo o mundo. Aqui, mostramos uma abordagem simples para a construção de filtros de lã de vidro e demonstrar os filtros são também eficazes na concentração waterborne virais, agentes patogénicos bacterianos e protozoários.

Vídeo: 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.

Métodos para a transferência rápida e localização de Patógenos Doença de Lyme no intestino 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.  ...

Imunologia e Infecção

'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.

&quot;Bioluminescent &#39;Phage Reporter para a detecção de patógenos bacterianos 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.

Vigilância da gripe aviária com FTA Cards: Métodos de campo, de Biossegurança, e questões de transporte Resolvido

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.

Detecção de vírus infecciosos de Campo mosquitos coletados por Vero Ensaio Cultura de Células

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.

A saliva, glândula salivar, e Coleta de hemolinfa de carrapatos 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.

Estabelecer uma cultura líquida, coberta de vias respiratórias humanas epiteliais Polarized Calu-3 células para estudar a resposta Cell Host para patógenos respiratórios<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.

Triagem de alta capacidade para amplo espectro químico Inibidores de Vírus RNA

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.

A administração intranasal de recombinantes Vacinas contra Influenza em quimérico rato Modelos para estudar a imunidade mucosal

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.

Fabricação de superfícies de vidro de qualidade óptica para estudar a fusão do macrófago

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.

Teste de imunohistoquímica para a detecção de antígenos lyssavírus a partir de tecidos fixos formalina

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