Nós sinceramente agradecer aos membros do laboratório de Pal para a assistência com a preparação desta demonstração. Este estudo foi suportado por concessões PHS AI076684 e AI080615 do NIH / NIAID.

1. Microinjeção de carrapatos Ixodes scapularis ninfal 1. Agulhas preparar <ol><li> Fabricar agulhas microinjeção várias aquecendo e puxando um milímetro tubos capilares de vidro (Instrumentos de Precisão World) em um dispositivo de vidro extrator micropipeta (Narishige). Remova cuidadosamente os tubos frágeis capilares. </li><li> Loja puxado agulhas (com a ponta voltada para cima) em fita adesiva em uma placa de Petri. </li></ol> 2. Preparando B. burgdorferi <ol><li> Crescer B. burgdorferi em BSK cultura de mídia 7 até a uma concentração de cerca de 10 7 células por ml. Espiroquetas são contados em um microscópio de campo escuro pelo uso de uma Petroff-Hausser contagem câmara (Hausser científica). </li><li> Pelota B. burgdorferi por centrifugação a 3000 xg por 10 minutos em temperatura ambiente. </li><li> Remover o sobrenadante e completamente ressuspender o sedimento em BSK media suavemente passando de 200 mL MICROTIP estéril para uma concentração final de 10 9 células por mL. Uma vez que as células são ressuspendidas em alta densidade celular, e pode se acumular, a suspensão celular deve ser usado para microinjeção imediatamente. </li></ol> 3. Carrapatos preparar <ol><li> Lugar claro, fita adesiva dupla face em uma lâmina de vidro. </li><li> Trabalhando em uma esteira, retire cuidadosamente os carrapatos ninfal do recipiente com uma escova pequena. </li><li> Colocar o número necessário de carrapatos a ser injetado na fita adesiva, lado ventral para cima, de frente para a mesma direção. </li></ol> 4. Carrapatos injetando <ol><li> Carga de 5 mL da B. burgdorferi cultura para dentro da agulha capilar utilizando puxado a 20 mL microloader pipeta ponta (Eppendorf). Inspecionar a agulha para bolhas de ar aprisionadas e recarregar a agulha com a cultura bacteriana, se necessário. </li><li> Ver o carrapato sob o microscópio binocular de dissecação e foco na área do carrapato abertura anal. Toque na ponta da agulha capilar, a fim de quebrar o tubo onde o diâmetro é ligeiramente menor do que a de abertura anal do carrapato, formando a agulha microinjeção. Esta etapa é fundamental como qualquer tentativa de injetar carrapatos com uma ponta de agulha não adequada provoca a morte de lesões e possíveis do carrapato injetado. </li><li> Coloque os carrapatos imobilizado sob o microscópio binocular de dissecação e concentrar-se na abertura anal, que é coberta por duas placas móveis anal. Utilizando uma pinça fina, tocar suavemente e aplicar uma pressão muito leve para qualquer área perto da abertura anal. Isto permitirá que a separação das placas anal e abertura de poros anal que se conecta ao intestino. Insira cuidadosamente a ponta da agulha um pouco para a abertura anal através da abertura forçada das placas anal. Inserção da agulha deve ser mantida a um mínimo como a ponta de vidro podem danificar o intestino posterior semitransparente que se conecta ao reto. Usando um microinjetor equipado com pedal automático (Eppendorf), injetar B. burgdorferi solução usando os seguintes parâmetros: 1.000 hectopascais (hPa) pressão de injeção, tempo de injeção 0,2 segundos e 8 de compensação de pressão hPa. Cada carrapato recebe uma única injeção. </li><li> Após a microinjeção, os carrapatos devem se comportar semelhante ao estado pré-injeção. Por exemplo, o carrapato deve rastejar em resposta a estímulos. </li><li> Nós geralmente permitem que os carrapatos se recuperar por 48 horas em uma câmara ambiental fixado em 24 ° C com 16 horas / 8 horas luz / escuridão regime de fotoperíodo e umidade de 95%. Se uma câmara não estiver disponível, carrapatos injetada pode ser armazenado em temperatura ambiente em condições de umidade dentro de um recipiente hermético, como uma câmara de dessecador regular. Se necessário, o tempo de recuperação dos carrapatos também poderia ser reduzido para algumas horas antes utilizado para a alimentação em camundongos. </li></ol> 2. B. burgdorferi localização por Microscopia Confocal de imunofluorescência 1. Carrapatos dissecar <ol><li> Coloque uma gota de solução fisiológica tamponada (PBS) em uma lâmina de vidro limpa para dissecção e outro em um slide poli-L-lisina revestido (Sigma) para microscopia. Prepare outra lâmina de vidro com fita dupla face adesiva. </li><li> Remover o carrapato do recipiente e coloque em fita dupla face adesiva, utilizando uma escova pequena. </li><li> Ver e foco do carrapato no microscópio de dissecação. Coloque uma lâmina afiada sobre o carrapato entre os pares de primeiro e segundo de pernas. Pressione firmemente o corte do carrapato em duas partes permitindo o acesso ao abdômen. Submergir imediatamente, o abdome em uma gota de PBS. </li><li> Usando uma pinça muito fina, pegue o exoesqueleto dorsal e ventral em torno do local do corte. Cuidadosamente puxe o escudo dorsal para cima e longe do carrapato expondo os divertículos intestinais acastanhadas. Tenha cuidado para manter os carrapatos dissecando sob a PBS em todos os momentos. </li><li> A semi-translúcido bun glândula salivarspindles estão localizados em ambos os lados da região anterior do intestino, e pode ser removido neste momento, se desejar. Segurando o exoesqueleto restantes no lugar com uma pinça, puxe cuidadosamente o intestino para fora do abdômen. </li><li> Limpar delicadamente o intestino, removendo quaisquer tecidos preso (por exemplo, traquéia). </li><li> Usando a ponta de uma pinça fina, transferir rapidamente o intestino carrapato em uma gota de PBS numa lâmina de vidro poli-L-lisina. Separar os intestinos em pedaços menores usando lâminas finas ou pressionando suavemente com a ponta de uma pinça fina. Aspirar cuidadosamente PBS excesso de tecidos ao redor do intestino. </li><li> Permitir que os tecidos do intestino para o ar seco à temperatura ambiente. </li><li> Corrigir o intestino carrapato por submersão em acetona por 10 minutos. Deixar secar ao ar em temperatura ambiente. Lâminas podem ser armazenados nesta etapa por meses a -20 ° C em um recipiente hermético. </li></ol> 2. Coloração <ol><li> Usando lenço de papel, remova o excesso de umidade em torno do tecido e desenhar um círculo ao redor do intestino carrapato secas por um pap pen-ou qualquer outro dispositivo de revestimento hidrofóbica barreira. Isso ajudará a manter a soluções de coloração durante as etapas de incubação subseqüentes. </li><li> Cobrir o intestino carrapato com uma ou algumas gotas de tampão de bloqueio (0,05% Tween-20, soro de cabra 5% em PBS) por 30 minutos em temperatura ambiente. O soro utilizado para o bloqueio depende da fonte de animal hospedeiro para o anticorpo. Não permita que o slide para secar a partir deste ponto em diante. </li><li> Lentamente, aspirar o tampão de bloqueio. Incubar o intestino com primário adequado e / ou soluções anticorpo secundário. Usamos isotiocianato de fluoresceína (FITC) - rotulado anti-B. burgdorferi anticorpos (Kirkegaard &amp; Perry Laboratories) na diluição de 1:100 em tampão de bloqueio por 1 hora à temperatura ambiente. Cubra o recipiente com papel alumínio para limitar a exposição à luz. </li><li> Remover a solução de anticorpos através de ligeira aspiração. Incubar o intestino com uma tinta fluorescente para os tecidos rótulo carrapato, tais como 4 &#39;,6-diamidino-2-phenylindole (DAPI) ou iodeto de propídio. Normalmente usamos 20 mg / mL de iodeto de propídio (Sigma) em PBS por 5 min. à temperatura ambiente. </li><li> Lavar 3 vezes com 0,05% Tween-20 em PBS. </li><li> Montar o slide em glicerol tamponado contendo um reagente Antifade como Slowfade (Invitrogen) e cuidadosamente cobrir com uma lamela de vidro. Lâminas podem ser armazenados nesta etapa por alguns meses a 4 ° C dentro de um recipiente hermético. Imagem e localizar B. burgdorferi sob um microscópio confocal. </li></ol> 3. Resultados representante Posição de um carrapato ninfal de microinjeção e uma imagem representando B. burgdorferi localização no intestino carrapato é apresentado na Figura 1. <img src="/files/ftp_upload/2544/2544fig1.jpg" alt="Figura 1" /> Figura 1. Microinjeção e localização de B. burgdorferi no intestino do carrapato. (A) vista ventral de uma ninfa I. carrapato scapularis posicionado para microinjeção. Anal abertura (seta) com agulha inserida microinjeção (seta) é mostrado sob a ampliação na inserção. A pinça fina é usado para aplicar pressão suave para o corpo, o que permitiu a separação das placas anal e abertura do poro anal para microinjeção. A agulha é preenchido com uma solução de Coomassie azul brilhante para melhorar a visibilidade. (B) Os resultados representativos das imagens de imunofluorescência confocal de B. burgdorferi dentro do intestino do carrapato. Região anterior de um divertículo do intestino é mostrado. Núcleos intestino e espiroquetas (seta) são rotulados com iodeto de propídio (cor vermelha) ou FITC-conjugado anti-B. burgdorferi (cor verde), respectivamente. Bar = 20 mM.

<ol>
	<li>Steere, A. C., Coburn, J., Glickstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+Lyme+disease.">The emergence of Lyme disease.</a> J Clin Invest. 113, 1093-1101 (2004).</li><li>Barthold, S. W., Diego, C., Philipp, M. T., Samuels, D. S., Radolf, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Borrelia, Molecular Biology, Host Interaction and Pathogenesis. , 353-405 (2010).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OspC+facilitates+Borrelia+burgdorferi+invasion+of+Ixodes+scapularis+salivary+glands.">OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands.</a> J Clin Invest. 113, 220-230 (2004).</li><li>Yang, X. F., Pal, U., Alani, S. M., Fikrig, E., Norgard, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+OspA/B+in+the+life+cycle+of+the+Lyme+disease+spirochete.">Essential role for OspA/B in the life cycle of the Lyme disease spirochete.</a> J Exp Med. 199, 641-648 (2004).</li><li>Zhang, X., Yang, X., Kumar, M., Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BB0323+function+is+essential+for+Borrelia+burgdorferi+virulence+and+persistence+through+tick-rodent+transmission+cycle.">BB0323 function is essential for Borrelia burgdorferi virulence and persistence through tick-rodent transmission cycle.</a> J Infect Dis. 200, 1318-1330 (2009).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TROSPA,+an+Ixodes+scapularis+receptor+for+Borrelia+burgdorferi.">TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi.</a> Cell. 119, 457-468 (2004).</li><li>Barbour, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+Lyme+disease+spirochetes.">Isolation and cultivation of Lyme disease spirochetes.</a> Yale J Biol Med. 57, 521-525 (1984).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Ixodes+scapularis+anticoagulation+by+using+RNA+interference.">Disruption of Ixodes scapularis anticoagulation by using RNA interference.</a> Proc Natl Acad Sci U S A. 101, 1141-1146 (2004).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tick+antioxidant+facilitates+the+Lyme+disease+agent's+successful+migration+from+the+mammalian+host+to+the+arthropod+vector.">A tick antioxidant facilitates the Lyme disease agent's successful migration from the mammalian host to the arthropod vector.</a> Cell Host Microbe. 2, 7-18 (2007).</li><li>Broadwater, A. H., Sonenshine, D. E., Hynes, W. L., Ceraul, S., DeSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass+capillary+tube+feeding:+a+method+for+infecting+nymphal+Ixodes+scapularis+(Acari:+Ixodidae)+with+the+Lyme+disease+spirochete+Borrelia+burgdorferi.">Glass capillary tube feeding: a method for infecting nymphal Ixodes scapularis (Acari: Ixodidae) with the Lyme disease spirochete Borrelia burgdorferi.</a> J Med Entomol. 39, 285-292 (2002).</li><li>Policastro, P. F., Schwan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+infection+of+Ixodes+scapularis+larvae+(Acari:+Ixodidae)+by+immersion+in+low+passage+cultures+of+Borrelia+burgdorferi.">Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi.</a> J Med Entomol. 40, 364-370 (2003).</li><li>Fuente, d. e. l. a., Kocan, J., M, K., Almazan, C., Blouin, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+for+the+study+and+genetic+manipulation+of+ticks.">RNA interference for the study and genetic manipulation of ticks.</a> Trends Parasitol. 23, 427-433 (2007).</li></ol>

Não há conflitos de interesse declarados.

Aqui demonstramos um procedimento baseado microinjeção de infecção rápida e eficaz de carrapatos Ixodes ninfal com o agente patogénico B. burgdorferi. Descrevemos também um procedimento confocal de imunofluorescência para a detecção de B. burgdorferi no intestino carrapato in situ. Embora a nossa demonstração envolve gut ninfal, procedimentos semelhantes são também aplicáveis ​​para outros estágios de desenvolvimento dos carrapatos, como larva ou adulto 8,9. No e...

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<td>Glass capillary tubes</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
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<td>Vertical glass puller</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>PC-10</td>
<td>&nbsp;</td>
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<td>Petroff-Hausser counting chamber</td>
<td>&nbsp;</td>
<td>Hausser scientific</td>
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<td>&nbsp;</td>
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<td>Microloader pipette tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>930001007</td>
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<td>Femtojet microinjector</td>
<td>&nbsp;</td>
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<td>Foot control FemtJet</td>
<td>&nbsp;</td>
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<td>Phosphate buffered saline</td>
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methods for rapid transfer and localization of lyme disease pathogens within the tick gut

A doença de Lyme é causada por infecção com o patógeno espiroqueta Borrelia burgdorferi, que é mantido na natureza por um ciclo de infecção do carrapato roedor-1. Um modelo murino carrapatos 2 foi desenvolvido para estudar a doença de Lyme no laboratório. Enquanto carrapatos ingênuo pode ser infectado com B. burgdorferi, alimentando-os em camundongos infectados, o processo de muda leva várias semanas ou meses para ser concluído. Portanto, o desenvolvimento de técnicas de assinalar mais rápida e eficiente a infecção, como um procedimento de microinjeção de base, é uma importante ferramenta para o estudo da doença de Lyme 3,4. O procedimento requer apenas algumas horas para gerar os carrapatos infectados e permite o controle sobre a entrega de quantidades iguais de espiroquetas em uma coorte de carrapatos. Isto é particularmente importante como a geração de B. carrapatos infectados por burgdorferi o processo de alimentação natural, utilizando camundongos não consegue garantir a taxa de infecção de 100% e, potencialmente, resulta em variação de carga de patógenos entre os carrapatos alimentados. Além disso, a microinjeção pode ser usado para infectar carrapatos com B. burgdorferi isolados em casos em que uma cepa atenuada é incapaz de estabelecer a infecção em camundongos e, portanto, não pode ser adquirida naturalmente por carrapatos 5. Esta técnica também pode ser usado para fornecer uma variedade de outros materiais biológicos em carrapatos, por exemplo, anticorpos específicos ou dupla stranded RNA 6. Neste artigo, vamos demonstrar a microinjeção de carrapatos ninfal com in vitro-grown B. burgdorferi. Iremos também descrever um método para a localização de patógenos a doença de Lyme no intestino tick usando microscopia confocal de imunofluorescência.

Watch this Scientific Journal Video about Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut at JoVE.com

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

Estudos de pesquisa a doença de Lyme muitas vezes exigem a geração de carrapatos infectados com o patógeno Borrelia burgdorferi, um processo que normalmente leva várias semanas. Aqui nós demonstramos uma microinjeção baseado procedimento de infecção do carrapato que pode ser realizado em poucas horas. Nós também demonstramos um método de imunofluorescência para na localização in situ de B. burgdorferi dentro de carrapatos.

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.  ...

methods-for-rapid-transfer-localization-lyme-disease-pathogens-within

Research

JoVE Journal

Immunology and Infection

11.5K visualizações.  University of Maryland. Estudos de pesquisa a doença de Lyme muitas vezes exigem a geração de carrapatos infectados com o patógeno Borrelia burgdorferi, um processo que normalmente leva várias semanas. Aqui nós demonstramos uma microinjeção baseado procedimento de infecção do carrapato que pode ser realizado em poucas horas. Nós também demonstramos um método de imunofluorescência para na localização in situ de B. burgdorferi dentro de carrapatos.

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

'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.

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. ...

Imunologia e Infecção

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 ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Alimentação dos Carrapatos em Animais de Transmissão e Xenodiagnóstico em Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Resgate de Vírus da Doença de Newcastle recombinante de cDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Métodos de estudo de lipídios Alterações nos neutrófilos ea subsequente formação de neutrófilos Extracelular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Metodologia para o Estudo da transferência horizontal de genes em<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Extração rápida, segura e simples Manual cabeceira de ácidos nucleicos para a detecção de vírus em amostras de sangue total

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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. ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

In Vivo Aumento da Indução de CélulaS T Reguladoras de Gut-Homing

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...