Os autores agradecem ao Dr. Le Trinh e Dr. Tobin para generosamente fornecer a α-catenina: linha citrino peixes e Bill Jackman por nos permitir fazer a filmagem em seu laboratório. Os autores reconhecem as fontes de financiamento Institutos Nacionais de Saúde (Grants 5P20RR016463, 8P20GM103423 e R15AI094406) e USDA (Projeto nº ME0-H-1-00517-13). Este manuscrito é publicado como principal Agricultura e Florestas Experiment Station número de publicação 3371.

NOTA: Todos os protocolos de cuidados de peixe-zebra e experimentos foram realizados de acordo com as diretrizes do NIH sob Animal Care Institucional e Comitê de Uso (IACUC) A2012-11-03 protocolo. 1. Zebrafish Elevando a 4 dias após a fertilização <ol><li> Recolhe AB peixe-zebra, ou quaisquer outras linhas transgénicas, dentro da primeira hora após a fertilização 3, como mostrado em outro vídeo 34. </li><li> Incubar 120 ovos em um 15 centímetros placas de Petri contendo 150 ml de mídia E3 (mM NaCl 5; KCl 0,17; 0,33 mM CaCl 2; 0,33 mM MgCl2; HEPES 2 mM; pH 6,8) com azul de metileno (0,3 ug / ml definitiva concentração) em um 33 ° C incubadora com 12/12 luz / fotoperíodo escuro. </li><li> Substituir o material após 6 h com E3 + PTU (1-fenil-2-tioureia, 10 ug / ml de concentração final) e remover os ovos mortos. </li><li> Incubar durante 4 dias a 33 ° C, trocando a mídia com nova E3 + PTU após 2 dias. </li></ol> 2. Eminjeção Micro-agulha Preparação <ol><li> Puxe capilar borosilicato (OD 1,2 milímetros; ID 0,69 milímetros) em um micro-agulha usando um extrator agulha com as seguintes configurações (550 Calor; 0 Pull; 130 Velocity; 110 Time). NOTA: As configurações irão variar de filamento para filamento para se obter micro-agulhas em forma semelhante (ver Figura 1) e necessitam de ser reajustados sempre que um novo filamento está instalado. </li><li> Encha uma micro-agulha com 3 ul de 0,4 um filtrou-tampão salino de fosfato (PBS; NaCl 137 mM; KCl 2,7 mM; 10 mM de Na 2 HPO 4; 1,8 mM de KH 2 PO 4, pH 7,4) utilizando uma pipeta de 20 uL microloader ponta. </li><li> Defina-se a micro-agulha num suporte de micropipeta em um micromanipulador ligado a um sistema de injecção de pressão. Ajuste a pressão de injeção para 30 PSI e a duração do pulso de 30 ms. </li><li> Usando uma placa de Petri com água destilada, inferior a micro-agulha até 1/5 da agulha está na água. Clipe da wi micro-agulhath uma pinça fina logo abaixo do nível da água. Traga a micro-agulha para fora da água e empurrar o pedal várias vezes até um bolus aparece. </li><li> Medir o diâmetro do bolo baixando a micro-agulha em um hemocitómetro em camadas com uma gota de óleo de tipo A imersão. Ajustar a duração do impulso para obter o volume desejado (ver Tabela 1). </li><li> Ajuste da contrapressão para o bólus se mantenha estável em óleo (diminuir a pressão, se aumenta o volume do bolo e aumentar a pressão de retorno se o volume de bolus diminui). Grave a duração do pulso para cada micro-agulha. </li><li> Retire a PBS da micro-agulha, aspirando-lo usando uma ponteira microloader e expulsar a esquerda-over PBS, colocando-o de volta no injector e apertar o interruptor de pulso contínua. Reserve cada micro-agulha. </li></ol> 3. Preparação Candida <ol><li> Streak Candida albicans transformada com otimizado-códon dTomato, GFP, BFP, EOS ou FarVermelho de um lote congelado (-80 ° C estoque em glicerol a 25%) usando um passador de madeira estéril para uma levedura dextrose peptine (YPD) placa de agar (10 g de extracto / L de levedura, 20 g / L de peptona, 20 g / L de dextrose , 20 g / L de agar; autoclavado e verteu-se 25 ml em placas de Petri) e incubar durante a noite a 30 ° C. </li><li> Escolha uma colónia utilizando um pino de madeira e estéril para inocular 5 ml de meio YPD líquido (10 g de extracto / L de levedura, 20 g / L de peptona, 20 g / L de dextrose, autoclavado e assepticamente em alíquotas de 5 ml em 16 x 150 milímetros de cultura de vidro tubos). </li><li> Incubar durante a noite a 30 ° C num tambor de rolos a 60 rpm. </li><li> Colete 1 ml de C. albicans cultura e transferência para um tubo de centrífuga de 1,7 ml estéril. </li><li> Centrifugar a 5000 xg por um par de segundos. Esvazie o sobrenadante e adicionar 1 ml de PBS estéril, vortex completamente. Repita isso duas vezes. </li><li> Contar as colónias utilizando um hemocitómetro por diluição do stock 1 mL de 1: 1000 em PBS estéril. </li></ol> 4. Injetar Zebrafish ema bexiga natatória <ol><li> Aquecer um prato de injecção (2% de agarose) durante 30 min em uma incubadora de 33 ° C. </li><li> No momento desejado, remover 100 dos 150 ml de E3 + UTP a partir do prato de 15 centímetros de Petri contendo o peixe para injectar, utilizando uma pipeta de 25 ml sem aspirar qualquer peixe-zebra. </li><li> Anestesiar 4 dpf peixe-zebra por adição de 2 ml de uma mistura 4 mg / ml tamponado tricaina metano sulfonato estoque (TR) para os restantes 50 ml de meio (concentração final de 200 ug / ml) e aguardar durante 15 minutos. </li><li> Sob um microscópio de dissecação, selecione peixe com uma bexiga natatória inflado. Os peixes também podem ser rastreados na época para fenótipo mais homogêneo, como a distribuição de neutrófilos em mpo: linha GFP utilizando um microscópio de epifluorescência dissecando. </li><li> Dilui-se a C. albicans de estoque a sua concentração original (passo 3.8) e 1,5 x 10 7 unidades formadoras de colónias por ml (cfu / ml) em PBS. </li><li> Transferir 30 peixes-zebra para o prato de injecção usando um tubo de transferência de plásticotte e remover o excesso de mídia. </li><li> Usando uma ferramenta de fio de pesca (0,012 polegadas de diâmetro linha de pesca super-colado em um capilar de borosilicato), fazer 3 linhas de 10 peixes, segurando o prato injeção vertical e orientar o peixe verticalmente. </li><li> Retire o excesso de mídia. </li><li> Absolutamente vortex do tubo com 1,5 x 10 7 ufc / mL C. albicans. </li><li> Encha um micro-agulha com 3 l de C. albicans utilizando uma nova ponta microloader pipeta e definir a duração do pulso para a definição apropriada (veja o passo 2.9). </li><li> Rode o prato de injeção para fazer um ângulo de 20 ° entre o micro-agulha ea cabeça do peixe, que aponta para a parte de trás da bexiga natatória (Figura 3A). </li><li> Empurrar o micro-agulha para dentro da bexiga natatória, certificando-se o furo da agulha está no lúmen, e uma vez que pressiona o pedal. </li><li> Repita o procedimento para cada peixe e transfira para um prato de recuperação (25 ml E3 + PTU) inundando o prato com um E3d esvaziando-a no prato de recuperação. </li><li> Transferir mais 30 peixes para o prato de injecção. </li><li> Repita injeção com um novo micro-agulha cheia com cuidadosamente agitadas C. albicans. </li><li> Concluir através da injecção do controlo de PBS, se necessário, utilizando um prato de injecção diferente, para evitar a contaminação e transferir para uma cápsula de recuperação separada (passo 4.10). </li></ol> 5. Triagem do Peixe para o inóculo desejado <ol><li> Preparar 40 ml de 0,4% de baixo ponto de fusão solução de agarose (LMA) através da adição de 160 mg de agarose de baixo ponto de fusão a 40 ml de E3, fervendo por microondas até à dissolução, e de se arrefecer para 37 ° C. Adicionar 400 ul de TR (concentração final de 200 ug / ml) uma vez arrefecida. </li><li> Depois de 30 minutos de recuperação, anestesiar os peixes como descrito (passo 4.3 utilizando apenas 1 ml de TR em 25 mL E3 + PTU, concentração final de 200 ug / ml). </li><li> 30 Transferência de peixe (com um mínimo de meios quanto possível) a 2 ml de LMA em peso de um barco. </li><li> Aspirar peixes individuais com uma pipeta de transferência e a placa de peixe com um LMA em gotas de uma placa de imagiologia de 96 poços, utilizando-se apenas os 10 x 6 poços centrais. </li><li> Repita até que todos os peixes para a tela são carregados para a placa de imagem. </li><li> Deixar os meios solidificar à temperatura ambiente durante 5 min. </li><li> Coloque o peixe do seu lado, certificando-se que eles estão em contato com o fundo de vidro. </li><li> Usando o objetivo 20X em um epifluorescência / microscópio confocal invertido e o filtro apropriado, registrar o número de C. células de levedura albicans na bexiga natatória de cada peixe. </li><li> Selecione o peixe com 15-25 células de levedura na bexiga natatória (ou inóculo desejado), eutanásia peixe não selecionados (800 ug / ml de TR na E3) e retornar peixe selecionado para um prato fundo Petri com 50 ml de E3 + PTU por adicionar 3 gotas de E3 para a chapa de imagem poços selecionados e aspirar o peixe com uma pipeta de plástico. </li><li> Incubar a 33 ° C durante o tempo desejado. </li></ol> 6. Imaging o Peixe Pós-screening <ol><li> No momento desejado, remover 25 dos 50 ml de meio E3 + PTU do prato fundo Petri contendo o peixe para a imagem. </li><li> Anestesiar os peixes como descrito (passo 4.3 utilizando apenas 1 ml de TR em 25 ml E3 + PTU remanescente, concentração final de 200 ug / ml). </li><li> Placa o peixe para uma placa de imagiologia de 96 poços e posição apropriadamente (passos 5,3-5,7). </li><li> Imagem por microscopia confocal utilizando os lasers adequados e filtros de emissão. Primeiro foco nos peixes usando o objetivo 4X sob contraste de interferência diferencial (DIC) e adquirir imagens da região de interesse, utilizando o objetivo 20X com os lasers confocal em modo de digitalização com 2 velocidade ms / pixel e 1024 x 600 pixel proporção. Adquirir Z-stacks com 1 mm tamanho do passo, aplicando-se o modo de filtro de Kalman de 3 frames. </li><li> Retornar para um prato fundo de Petri com 50 ml E3 + PTU ou em individual (poços de 24 poços do prato de cultura, 3 ml E3 + PTU) e incubar a 33 ° C. </li></ol> 7. Dissecando a bexiga natatória <ol><li> Encher uma seringa de 10 ml com graxa alto vácuo. </li><li> Prepara-se uma LMA 2% (passo 5.3 com 160 mg de agarose a baixo ponto de fusão em 8 ml de E3). </li><li> No momento desejado, anestesiar o peixe (passo 5.2). </li><li> Transferir 10 de peixe para um tubo de centrífuga de 1,7 mL com 1 ml de E3 e eutanásia por adição de 200 ul de 4 mg / ml tricaina (concentração final de 800 ug / ml) para o tubo de centrifugação e incubação durante 15 min à temperatura ambiente. </li><li> Prepare uma lâmina de vidro de imagem, colocando 4 gotas de graxa de vácuo em uma praça, com 1 cm de diferença em um microlâmina (75 x 25 mm, Figura 2). </li><li> Coloque 2 gotas de 2% LMA no meio da praça. </li><li> Coloque um peixe em uma lâmina de vidro separada sob um microscópio de dissecação e verificar que o coração parou de bater, retire o excesso de água. </li><li&gt; Usando uma pinça fina dissecar a bexiga natatória, mantendo a cabeça em uma posição fixa com a pinça esquerda e puxando para baixo o intestino anterior com a pinça direita (Figura 4A). </li><li> Pegue o swimbladder pelo duto pneumático com a pinça esquerda, separando-o do aparelho digestivo, e colocá-lo no centro da LMA 2% antes de ela se solidifica. </li><li> Colocar uma tampa de deslizamento (18 x 18 mm) na parte superior da lâmina de vidro de imagem e empurrá-lo até que ela toque a graxa vácuo, bem como o LMA (Figura 2). </li><li> Imagem dentro de 10 min de esvaziamento, tal como descrito na etapa 6.4. Repita o passo 7,7-7,11 com os outros peixes. </li></ol>

The authors have nothing to disclose.

Os avanços e as limitações do modelo de doença microinjeção swimbladder O modelo apresentado aqui é uma extensão do modelo de imersão mucosa candidíase descrito no Gratacap et al (2013).; acrescenta as vantagens de um tempo de infecção controlada, uma dose de infecção altamente reprodutível, e, por conseguinte, uma maior eficiência. Nós demonstramos aqui novos métodos que permitem a documentação temporais não-invasivo de dinâmica de infecção em grande detalhe, b...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table><tbody><tr><td>1.7 ml tubes</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>Deep Petri dishes</td><td>Fisher Scientific</td><td>89107-632</td><td></td></tr><tr><td>Transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>Yeast Extract</td><td>VWR Scientific</td><td>90000-726</td><td></td></tr><tr><td>Peptone</td><td>VWR Scientific</td><td>90000-264</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Agar</td><td>VWR Scientific</td><td>90000-760</td><td></td></tr><tr><td>Fine tweezers (Dumont Dumoxel #5)</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Wooden dowels</td><td>VWR Scientific</td><td>10805-018</td><td></td></tr><tr><td>Low melt agarose</td><td>VWR Scientific</td><td>12001-722</td><td></td></tr><tr><td>Flaming Brown micropipette puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Borosilicate capillary</td><td>Sutter Instruments</td><td>BF120-69-10</td><td></td></tr><tr><td>MPPI-3 injection system</td><td>Applied Scientific Instrumentation</td><td>MPPI-3</td><td></td></tr><tr><td>Back pressure unit</td><td>Applied Scientific Instrumentation</td><td>BPU</td><td></td></tr><tr><td>Micropipette holder kit</td><td>Applied Scientific Instrumentation</td><td>MPIP</td><td></td></tr><tr><td>Foot switch</td><td>Applied Scientific Instrumentation</td><td>FSW</td><td></td></tr><tr><td>Micromanipulator</td><td>Applied Scientific Instrumentation</td><td>MM33</td><td></td></tr><tr><td>Magnetic base</td><td>Applied Scientific Instrumentation</td><td>Magnetic Base</td><td></td></tr><tr><td>Tricaine methane sulfonate</td><td>Western Chemical Inc.</td><td>MS-222</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZ61 top SZX-ILLB2-100 base</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>IX-81 with FV-1000 laser scanning confocal system</td><td></td></tr><tr><td>20X microscope objective</td><td>Olympus</td><td>UPlanSApo 20x/0.75</td><td></td></tr><tr><td>Roller drum</td><td>New Brunswick Scientific</td><td>TC-7</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Glass culture tubes (16 x 150 mm)</td><td>VWR Scientific</td><td>60825-435</td><td></td></tr><tr><td>NaCl</td><td>VWR Scientific</td><td>BDH4534-500GP</td><td></td></tr><tr><td>KCl</td><td>VWR Scientific</td><td>BDH4532-500GP</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR Scientific</td><td>BDH0246-500GP</td><td></td></tr><tr><td>HEPES (Corning)</td><td>VWR Scientific</td><td>BDH4520-500GP</td><td></td></tr><tr><td>Children clay (Play-Doh)</td><td>Hasbro</td><td></td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>C69-500</td><td></td></tr><tr><td>Methylene blue</td><td>VWR Scientific</td><td>VW6276-0</td><td></td></tr><tr><td>PTU</td><td>Sigma</td><td>P7629-10G</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Hemocytometer (Hausser scientific)</td><td>VWR Scientific</td><td>15170-172</td><td></td></tr><tr><td>Type A immersion oil</td><td>Blue Marble Products</td><td>51935</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Vortex Genie</td><td>VWR Scientific</td><td>14216-184</td><td></td></tr><tr><td>Agarose (Lonza)</td><td>VWR Scientific</td><td>12001-870</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>Fishing wire</td><td>Stren</td><td></td><td></td></tr><tr><td>96 well imaging plate (Sensoplate)</td><td>Greiner Bio-One</td><td>655892</td><td></td></tr><tr><td>High vacuum grease (Dow Corning)</td><td>VWR Scientific</td><td>59344-055</td><td></td></tr><tr><td>Microslide (25 x 75 mm)</td><td>VWR Scientific</td><td>48300-025</td><td></td></tr><tr><td>Cover slips (18 x 18 mm), No 1.5</td><td>VWR Scientific</td><td>48366-045</td><td></td></tr><tr><td>15 cm Petri dish (Olympus plastics)</td><td>Genesee Scientific</td><td>32-106</td><td></td></tr><tr><td>Glycerol (EMD chemicals)</td><td>VWR Scientific</td><td>EMGX0185-5</td><td></td></tr><tr><td>24-well culture dish (Olympus plastics)</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>Weight boats (8.9 cm)</td><td>VWR Scientific</td><td>89106-766</td><td></td></tr></tbody></table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

Defesa precoce contra patógenos da mucosa consiste de uma barreira epitelial e células imunitárias inatas. A imunocompetência de ambos, e sua intercomunicação, são de extrema importância para a proteção contra infecções. As interações de células imunitárias inatas epiteliais e com um patógeno são melhor investigada in vivo, onde o comportamento complexo se desenrola ao longo do tempo e do espaço. No entanto, os modelos existentes não permitem fácil imaging espaço-temporal da batalha com patógenos ao nível da mucosa. O modelo desenvolvido aqui cria uma infecção das mucosas por injecção directa do agente patogénico fúngico, Candida albicans, em swimbladder de peixe-zebra juvenil. A infecção resultante permite imagens de alta resolução do comportamento das células epiteliais e imune inata ao longo do desenvolvimento da doença mucosa. A versatilidade deste método permite interrogatório do hospedeiro para sondar a seqüência detalhada de eventos imunológicos que levam a phrecrutamento agocyte e examinar os papéis de determinados tipos de células e vias moleculares na proteção. Além disso, o comportamento do agente patogénico como uma função do ataque do sistema imunológico pode ser iluminados simultaneamente por fluorescência utilizando-expressando proteína C. albicans. O aumento da resolução espacial da interação patógeno-hospedeiro também é possível através da técnica de dissecção da bexiga natatória rápida descrito. O modelo de infecção da mucosa descrito aqui é simples e altamente reprodutível, tornando-se uma ferramenta valiosa para o estudo da mucosa candidíase. Este sistema pode também ser amplamente traduzível a outros agentes patogénicos das mucosas, tais como micróbios micobacterianas, bacterianas ou virais que normalmente infectam através de superfícies epiteliais.

Microinjection na bexiga natatória posterior O método experimental aqui apresentado descreve a injecção de uma dose consistente de C. células de levedura albicans na bexiga natatória de 4 dpf peixe-zebra. Trabalhos anteriores com o modelo de imersão sugere que a resposta imune a bexiga natatória C. albicans é semelhante à da mucosa de mamífero candidíase 32. Aqui demonstramos um método de infecção modificado que é mais simples, mais rápida ...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Modelagem da mucosa candidíase em larval Zebrafish por swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.1K visualizações.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Vídeo: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Não-invasivos de imagem de candidíase disseminada em larvas do peixe

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Imunologia e Infecção

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Estabelecimento de Zebrafish Larval como um modelo Animal para investigar Trypanosoma cruzi motilidade In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Biologia do Desenvolvimento

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Decifrar e Imagem Patogênese e Cording de<em&gt; Mycobacterium abscessus</em&gt; Em embriões Zebrafish

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

Desenvolvimento de um Modelo de Infecção de Zebrafish Larval para Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Usando modelos de Zebrafish de infecções por vírus gripe humana A para Screen antivirais e Caracterizar imune do hospedeiro respostas de células

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modelagem de tuberculose no Zebrafish adulto infectado de Mycobacterium marinum

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Direcionando drogas para macrófagos de zebrafish larval injetando liposós carregados de drogas

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Infecção de larvas de zebrafish com esporos de Aspergillus para análise de interações hospedeiro-patógeno

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Microinjeções ventriculares cerebrais de lipopolissacarídeo em peixe-zebra larval para avaliar a neuroinflamação e a neurotoxicidade

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Neurociência

A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Source: Thrikawala, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61165">Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions.</a>&#160;J. Vis. Exp.&#160;(2020)
The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

Uma técnica zWEDGI para visualizar larvas de peixe-zebra infectadas por patógenos fúngicos

1. Preparation of&#160;Aspergillus&#160;spores for injection

	From an&#160;Aspergillus&#160;spore suspension, calculate the volume needed to obtain 1 x ...