Os autores gostariam de agradecer aos membros anteriores do laboratório Goodrich-Blair: Samantha Orchard, Kimberly Cowles, Erin Herbert-Tran, Greg Richards, Megan Menard, e Youngjin Parque por suas contribuições para o desenvolvimento deste protocolo. Este trabalho foi financiado pela National Science Foundation concessão IOS-0950873 e os Institutos Nacionais de Saúde NRSA comunhão FAI084441Z.

1. Esterilização ovo do inseto e criação <ol><li> Na preparação de dietas, primeiro autoclave 15 g de agar fornecida em 900-1.000 ml de H 2 O. Imediatamente após a mistura em autoclave, com 166 g de dieta de gérmen de trigo e de mistura bem em um misturador de laboratório. Despeje em um prato (ou pratos) para esfriar, em seguida, transferir dieta para folha de alumínio, enrole firmemente, loja e a 4 ° C. </li><li> Após a chegada, esterilizar M. Sexta ovos com 250 ml de solução de lixívia a 0,6% durante 2-3 min em um suporte de filtro de vidro e um aparelho com um frasco de vácuo de papel filtro de 90 milímetros, agitando ocasionalmente. </li><li> Ligue vácuo a drenagem da solução de lixívia de lavagem e ovos 3-4 vezes com 250 ml estéril H2O destilada por lavagem. </li><li> Transferir os ovos para 90 mm de papel de filtro fresco colocado numa placa de Petri tampa e deixe secar até que já não vara juntos (cerca de 20-30 min). </li><li> Transferir os ovos para a parte inferior dos recipientes de plástico com uma dieta de insectos, colocando aproximadamente 40 ovos em cada recipiente(Figura 1A). Os ovos são separados a partir da dieta para prevenir a humidade induzida contaminação fúngica. Manter os ovos, a 26 ° C com uma luz hr 16: 8 horas fotoperíodo escuro. Ovos irá iluminar a uma cor amarelo-branco antes da eclosão. </li><li> Quando incubação estiver completa, transferir cuidadosamente cerca de 25 larvas cada uma para novos recipientes com dieta sobre a parte inferior (Figura 1B). É importante para pegar cada larva pelo chifre longo preto usando uma pinça para evitar prejudicá-los durante suas frágeis primeiras fases de desenvolvimento. Incubar durante 2 dias, como anteriormente. Embora a morte larval é incomum nesta fase, alguns mortos ou, mais provavelmente, larvas developmentally atrofiado pode ser observado. Tais insectos devem ser excluídos do estudo e sacrificados por congelamento. </li><li> Para evitar os comportamentos de canibalismo, larvas de transferência para recipientes individuais, com pequenos pedaços de comida (Figura 1C) e incubar como acima. Acompanhar o desenvolvimento do inseto, fezes substituindo alimentos e limpeza UOt de embalagens de todos os dias até que eles sofrem a muda larval terceiro (geralmente 6-9 dias após a eclosão). Esta é a 4 º instar larval, caracterizada pelo aparecimento de preto do gancho como crochets em cada proeminência proleg e aumento de listras ao longo do corpo de insectos (Figura 1D). As larvas podem variar consideravelmente de tamanho, mas normalmente pesam 0,15-,4 g ao entrar na 4 ª etapa estádio. Larvas de tamanho similar deve ser escolhido para injecção. </li></ol> 2. Preparação de bactérias para Injeção <ol><li> Crescer a estirpe bacteriana (s) a ser testado de acordo com os procedimentos padrão para a fase de crescimento que pretende testar. </li><li> Pellet ul 500 de cada estirpe bacteriana a ser testado por fiação durante 2 min numa microcentrífuga a 13.000 rpm, à temperatura ambiente. </li><li> Ressuspender as células em 1 ml de fosfato salino tamponado estéril 1x (PBS) e de pelotas de novo como acima. </li><li> Ressuspender as células em 0,5 ml de 1x PBS estéril e meacerteza a densidade óptica (DO) da suspensão. Diluir todas as suspensões para o mesmo diâmetro externo, se necessário. Meio de crescimento estéril pode também ser utilizado para a ressuspensão das células e diluição em lugar de 1x PBS, se desejado. </li></ol> 3. Injeção de 4 larvas ínstares <ol><li> Prepare seriais diluições de 10 vezes da primeira estirpe bacteriana em 6 poços de uma placa de microtitulação de 96 poços em PBS 1x estéril, utilizando-se uma ponta de pipeta nova para cada diluição (Figura 2A). O volume final da suspensão de células em cada poço deve exceder o volume necessário para a injecção (10 ul por insectos) mais 20 uL para a placa de inoculo (ver passos 3.2 e 3.7). </li><li> Ul 10 cada ponto durante 5 diluições (10 ~ 2 a 10 -6) ao longo do topo de uma placa de Petri contendo um meio de crescimento adequado. Inclinar a placa, de modo que os pontos se espalhar para o centro (Figura 2B). </li><li> Remover dieta de insetos e fezes e insetos lugar em recipientes com gelo paracerca de 5 min. </li><li> Esterilizar a agulha da seringa, por lavagem 3 vezes cada em 2 tubos de etanol 70-100%, seguido de um tubo estéril de H 2 O (Figura 2C). O volume do líquido deve ser suficiente para submergir a agulha. </li><li> Quantificar as suspensões de células diluídas sob um microscópio usando um hemocitómetro. Dependendo do inoculo desejado, extrair 10 ul do microtitulação bem apropriado (Figura 2D). </li><li> Cotonete superfície do insecto com etanol a 95%, e injectar a 10 ml de suspensão celular para o insecto a um ângulo (inferior a 45 °) para trás a uma das patas falsas abdominais. Tome cuidado para não perfurar o intestino e injetar o conteúdo logo abaixo da camada epidérmica (Figura 2E). Embora alguma perda de hemolinfa (líquido límpido, verde, azul) é normal, as partículas eo líquido amarelo que emerge do local de injecção são indicativos de punção do intestino. Se isso ocorrer, sacrificar o insecto e obter um novo larva de recolocá-lo. </li><li> Repetir o passo 3.6 para cada insecto remanescente no grupo da primeira injecção. Agulha de esterilização não é necessária entre cada insecto injectado com a mesma diluição e estirpe de bactérias a menos que a contaminação ocorre. Alternativamente, um dispensador de repetição podem ser usados ​​para uma maior consistência no volume de injecção, entre as amostras. </li><li> Após a injecção de cada insecto com a primeira linhagem de bactérias, repita a etapa 3.2, desta vez manchar as suspensões de células no meio do prato e deixá-los fluir para a parte inferior (Figura 2F). Incubar as placas a temperatura adequada e contar colônias para enumerar o inóculo. Esta etapa de revestimento segundo é usado para verificar que as amostras não foram contaminadas durante o processo de injecção, uma vez que tal resultará em números de colónias diferentes entre os primeiro e segundo revestimentos. </li><li> Repita os passos 3.1 a 3.8 para cada estirpe de bactérias no experimento. Finalmente, injetar 3-5 com insetos estéreis 1x PBS usando uma sterile agulha como grupo controle negativo. Incubar os insectos a 26 ° C com uma luz hr 16: 8 hr fotoperíodo escuro. </li><li> Monitorar sobrevivência do inseto ao longo do tempo após a injeção. Morte de insectos é caracterizada como uma falta de movimento voluntário após estimulação. Insetos freqüentemente apresentam perda de apetite, diarreia (fezes, aguado amarelo), e / ou perda de água (&quot;suor&quot;) durante a infecção antes da morte; essas características pode-se notar também. </li></ol>

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Não há conflitos de interesse declarados.

A injecção directa de M. sexta larvas com bactérias entomopatogênicos, como aqui descrito, serve como um meio simples e eficaz para analisar a virulência bacteriana. O método também é altamente adaptável para se adequar a diferentes sujeitos experimentais e / ou condições. As bactérias podem ser preparados de várias maneiras, antes da injecção. No caso de X. nematophila, células selvagens cultivadas em rico em nutrientes Luria-Bertani (LB) até meados de log-fase são tipicamente o mais...

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rearing and injection of manduca sexta larvae to assess bacterial virulence

Manduca sexta, vulgarmente conhecido como o hornworm tabaco, é considerada uma importante praga agrícola, alimentando-se de plantas solanáceas, incluindo o tabaco e tomate. A susceptibilidade de M. sexta larvas para uma variedade de espécies bacterianas entomopatogênicos 1-5, bem como a riqueza da informação disponível sobre o sistema imunitário do insecto 6-8, e a sequência do genoma pendente 9 torná-lo um organismo modelo bom para ser utilizado no estudo de interacções hospedeiro-microorganismo durante a patogênese. Além disso, M. sexta larvas são relativamente grandes e fáceis de manipular e manter no laboratório relação a outras espécies de insetos suscetíveis. O seu grande tamanho, também facilita a extracção dos tecidos / hemolinfa eficiente para a análise da resposta do hospedeiro à infecção. O método aqui apresentado descreve a injecção directa de bactérias no hemocelo (cavidade de sangue) de M. larvas sexta. Esta abordagempode ser utilizado para analisar e comparar as características de virulência em várias espécies bacterianas, estirpes ou mutantes, por simplesmente controlar o tempo até à morte de insectos após a injecção. Este método foi desenvolvido para o estudo da patogenicidade de Xenorhabdus Photorhabdus e espécies, que normalmente se associam com os vectores de nematóides como um meio de ganhar a entrada do insecto. Nematóides entomopatogênicos tipicamente infectar larvas através digestivo natural ou aberturas respiratórias, e liberar os seus conteúdos simbióticas bacterianas no inseto hemolinfa (sangue) pouco depois 10. O método de injecção aqui descrito evita a necessidade de um vector nemátodo, assim, desengatar os efeitos das bactérias e nematóides sobre o insecto. Este método permite a enumeração exacta de material infeccioso (células ou proteínas) dentro do inoculo, que não é possível utilizar outros métodos existentes para a análise de entomopathogenesis, incluindo nicking 11 e ensaios de toxicidade oral 12 <em&gt;. Além disso, os ensaios de toxicidade oral abordar a virulência de toxinas segregadas introduzidos no sistema digestivo de larvas, ao passo que o método de injecção directa elimina a virulência dos inóculos de células inteiras. A utilidade do método de injecção directa, tal como descrito aqui é analisar patogênese bacteriana por monitorização da mortalidade de insectos. No entanto, este método pode ser facilmente expandido para uso no estudo dos efeitos da infecção com o M. sexta sistema imunitário. O insecto responde à infecção por meio de ambas as respostas humoral e celular. A resposta humoral inclui o reconhecimento de bactérias associadas a padrões e subsequente produção de diversos péptidos antimicrobianos 7, a expressão de genes que codificam estes péptidos pode ser monitorizada após a infecção directa através de extração de RNA e PCR quantitativa 13. A resposta celular à infecção envolve nodulação, encapsulamento, e a fagocitose dos agentes infecciosos por hemócitos 6 </sup&gt;. Para analisar estas respostas, insectos injectados podem ser dissecados e visualizadas por microscopia de 13, 14.

Um exemplo representativo de um ensaio de mortalidade dos insectos é descrito na Figura 3. Nesta experiência, os insectos foram injectados com cerca de 50 unidades formadoras de colónias (CFU) de um ou outro tipo selvagem (ATCC19061) ou uma estirpe mutante atenuada (lrp 13) de Xenorhabdus nematophila crescido a fase mid-log (n = 6 por insectos estirpe). As larvas foram observadas para, aproximadamente, 72 horas, e a percentagem de insectos vivos injectados em cada instant...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

O método descrito aqui utiliza a injecção directa de bactérias entomopatogênicos no hemocelo de<em&gt; Manduca sexta</em&gt; Larvas de insetos.<em&gt; M. sexta</em&gt; É um inseto disponível comercialmente e bem estudado. Assim, este método representa uma abordagem simples para analisar interacções hospedeiro-bacteriana a partir da perspectiva de um ou de ambos os parceiros.

Criação e injeção de<em&gt; Manduca sexta</em&gt; As larvas para avaliar a virulência bacteriana

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

rearing-injection-manduca-sexta-larvae-to-assess-bacterial

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Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

12.6K visualizações.  University of Wisconsin-Madison. O método descrito aqui utiliza a injecção directa de bactérias entomopatogênicos no hemocelo de Manduca sexta Larvas de insetos. M. sexta É um inseto disponível comercialmente e bem estudado. Assim, este método representa uma abordagem simples para analisar interacções hospedeiro-bacteriana a partir da perspectiva de um ou de ambos os parceiros.

Vídeo: Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

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 ...

Imunologia e Infecção

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Geração e Multi-fenotípica rastreio de alto teor de<em&gt; Coxiella burnetii</em&gt; mutantes por transposões

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

Aplicando um sistema de expressão induzível para estudar interferência de fatores de virulência bacteriana com Sinalização Intracelular

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

O Madagascar silvando barata como um modelo de animais não-mamíferos alternativo para investigar a eficácia da droga, patogenia e virulência

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

Identificação de marcadores de virulência de Mycobacterium abscessus para replicação intracelular em fagócitos

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

Avaliando virulência e patogênese da infecção por Aeromonas em um modelo de Caenorhabditis elegans

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Um modelo do rato para avaliar a resposta imune inata à infecção de Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Um do elevado-throughput Shigella-ensaio bactericida específico

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Ensaio de matar Opsonophagocytic para avaliar as respostas imunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...