저자는 도움이 토론에 대한 각각 tdTomato와 사비의 발현을 허용 pTEC27과 pTEC15의 관대 한 선물을 사러 - 클로드과 L. 라마 크리슈를 제공하는 케이 Kissa에 감사드립니다. 프랑스 국립 연구 기관의 프로젝트 (ZebraFlam ANR-10-MIDI-009과 DIMYVIR ANR-13-BSV3-007-01) 및 유럽 공동체의 일곱 번째 프레임 워크 프로그램의이 작품의 일부를 형성 (FP7-사람들-2011-ITN) 부여 계약에 따라 NO. PITN-GA-2011-289209 마리 퀴리 초기 교육 네트워크 FishForPharma합니다. 우리는 CM 듀폰에 자금을 지원하기위한 협회 그레고리 Lemarchal 및 Vaincre 라 Mucoviscidose (RF20130500835)을 감사하는 것이 좋겠다.

The authors have nothing to disclose.

제브라 피쉬는 최근 실시간 (36)의 넓은 필드와 공 촛점 이미징을 사용하여 세균 감염의 역학을 공부하는 우수한 척추 동물 모델 시스템으로 떠오르고있다. 함께 마이크로 주입 방법 (프로토콜 4)와 분산 마이코 박테리아 현탁액 (프로토콜 2.2)의 조합은 재현 전신 감염, 후속 모니터링 및 호스트 식세포와 세균의 상호 작용에 특별한 초점을 맞춘 감염의 진행의 시각화 할 수 있습니다. 엠...

미코 농양은 인간의 임상 증후군의 넓은 스펙트럼을 일으키는 새로운 병원균이다. 이러한 피부 감염뿐만 아니라 심각한 만성 폐 감염, 주로 면역 및 낭포 성 섬유증 환자 1,2,3,4에서 발생을 포함한다. M.를 농양은 인간의 원내 및 인성 감염을 담당하는 주요 빠르게 성장하는 마이코 박테리아 종으로 간주된다. 또한, 몇 가지 최근의 보고서는 가능성을 강조하는 M. 농양은 혈액 - 뇌 장벽을 통과하여 중추 신경계 (CNS) 5,6- 중요한 병변을 유도 할 수있다. 빠른 재배자, M.를 임에도 불구하고 또한 농양 전시 육아 종성 구조 내에서 년 동안 묵비권과 폐 7 caseous 병변을 생성 할 수있는 능력을 포함하여 결핵균의 그와 관련된 여러 가지 병원성 기능. 더 놀라운 낮은 센입니다엠의 sitivity 항생제에 농양, 상당한 치료 실패율 8,9로 이어지는 치료하기가 매우 어렵습니다 이러한 감염을 렌더링. 이 종의 중요한 위협은 주로 주요 공중 보건 기관 (10)에 관심과 폐 이식 (11)에 대한 금기 사항이다 항생제에 고유의 저항이다. M. 농양 표시 다른 임상 결과로 이어질 부드러운 (S) 또는 거친 (R) 식민지 형태 형. S 균주 달리, R 박테리아 로프 또는 끈형 구조 (12, 13)로 이어지는 단으로 성장하는 경향이있다. 세포 또는 동물 중 모델을 기반으로 여러 독립적 인 연구는 연구의 하이퍼 독성 표현형은 14, 15를 morphotype 밝혔다. 역학 연구, M.의 가장 심각한 경우 가입일 농양, 폐 감염이 연구와 관련이있는 것으로 나타납니다하는 유일한 변형 16 개의 변종이감염된 호스트 3 년 동안 지속하는 것으로 나타났습니다. morphotype 차이는 표면 관련 glycopeptidolipids (GPL) 12 (S)에 존재 또는 (R)에서 손실에 의존한다. 그러나, 현재 이용 가능한 셀룰러 / 동물 모델의 본질적인 한계는 M.에 의한 연구를 사용 농양 감염, R 또는 S 변종의 병태 생리 학적 사건에 대한 우리의 지식은 불분명 남아있다. 정맥 또는 에어로졸 경로를 통해 면역 능력이 쥐의 감염은 지속적 감염 및 생체 내 약물 감수성 검사 (17)에 대해 연구하는 마우스의 사용을 방해, 일시적 식민지로 연결됩니다. 따라서, 호스트 응답의 조작 의무가 동물 모델을 개발하는 것은 큰 도전이다. 이러한 맥락에서, 감염의 포유 동물 모델 비는 비용, 속도 및 윤리적 양호성 O 등 여러 가지 이점을 제공한다 (18)를 포함 초파리 melanogaster의 최근 개발되어왔다마우스 모델을 VER. 감염의 제브라 피쉬 (다니오 레 리오 (rerio)) 모델은 또한 비 침습적 영상, M.의 진행과 연대하여, 시각적으로 탐구하고있다 살아있는 동물 19 농양 감염. 중요한 개념의 증거는 엠에 대한 생체 내 항생제 평가에 대한 적합성을 입증하기 위해 설립되었다 17, 20 농양. 제브라 널리 다양한 병원균과 숙주 면역 시스템 (21) 사이의 상호 작용을 연구하기 위해 지난 수십 년 동안 사용되어왔다. 이 다른 척추 동물 모델의 증가 성공 동기 부여 및 다수의 바이러스와 세균 감염 19,22,23,24,25,26,27,28,29의 더 나은 이해를 위해 사용을 검증 주요 독특한 기회에 의존합니다. 대부분의 다른 동물 모델에 반해, 제브라 피쉬 배아는 비 침습적 형광 이미징 (30). 이는 헥타르 있도록 광학적으로 투명M. 공부를 LED에 농양은 세균의 형태 학적 가소성의 예를 나타내는 세포 외 cording의 설명과 남중, 전례없는 세부 제브라 피쉬 배아를 감염. Cording는 면역 체계의 파괴의 새로운 메커니즘 및 급성 엠의 발병을 촉진하는 핵심 메커니즘을 나타냅니다 농양 감염 (19). 이 보고서는 M.의 병태 생리 학적 특성을 해독하는 제브라 피쉬 배아를 사용하는 새로운 도구와 방법을 설명합니다 감염 농양과 간균과 선천성 면역 시스템 사이의 친밀한 상호 작용을 연구하는. 먼저, 그 자체 박테리아 접종원 배아 준비, 감염의 처리를 포함하는 마이크로 인젝션 상세한 프로토콜이 제시된다. 구체적 방법은 M.를 평가하도록 같은 호스트의 생존과 세균 부담 등의 다양한 매개 변수를 측정하여 농양 독성이되게됩니다. 특별한 초점은 방법에 기재되어 있습니다, 시공간 수준에서, 운명과 진행 감염과 엠에 호스트 면역 반응을 모니터링 비디오 현미경을 사용하여 농양. 또한, M. 동안의 기여와 대 식세포의 역할을 조사하기 위해 감염 농양, 방법 (genetically- 또는 화학적 기반 중 하나 방법을 사용) 대 식세포가 고갈 된 배아 설명되어 있습니다를 생성합니다. 마지막으로, 프로토콜은 고정 또는 생활 배아를 사용하여 대 식세포 나 호중구가 문서화와 특정 상호 작용을 시각화합니다. 이 보고서의 목적은 엠에 새로운 광명을 비춰하는 추가 연구를 자극하는 것입니다 농양 독성 메커니즘 및 제어되지 않은 급성 감염 프로세스의 확립에 cording 특히 역할.

<table><tbody><tr><td>BBL MGIT PANTA</td><td>BD Biosciences</td><td>245114</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Euromedex</td><td>04-100-811-E</td><td></td></tr><tr><td>Catalase from Bovine Liver&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C40</td><td></td></tr><tr><td>Difco Middlebrook 7H10 Agar</td><td>BD Biosciences</td><td>262710</td><td></td></tr><tr><td>Difco Middlebrook 7H9 Broth</td><td>BD Biosciences</td><td>271310</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Oleic Acid</td><td>Sigma-Aldrich</td><td>O1008</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Delta Microscopie</td><td>15710</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma-Aldrich</td><td>319244</td><td></td></tr><tr><td>Tween 80</td><td>Sigma-Aldrich</td><td>P4780</td><td></td></tr><tr><td>Agar</td><td>Gibco Life Technologie</td><td>30391-023</td><td></td></tr><tr><td>Low melting agarose</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Instant Ocean Sea Salts&amp;nbsp;</td><td>Aquarium Systems Inc</td><td></td><td></td></tr><tr><td>Borosilicate glass capillaries&amp;nbsp;</td><td>Sutter instrument Inc</td><td>BF100-78-10</td><td>1 mm O.D. X 0.78 mm I.D.</td></tr><tr><td>Micropipette puller device&amp;nbsp;</td><td>Sutter Instrument Inc</td><td></td><td>Flamming/Brown Micropipette Puller p-87</td></tr><tr><td>Microinjector</td><td>Tritech Research&amp;nbsp;</td><td></td><td>Digital microINJECTOR, MINJ-D</td></tr><tr><td>Tweezers</td><td>Sciences Tools inc</td><td></td><td>Dumont # M5S&amp;nbsp;</td></tr><tr><td>Microloader Tips</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

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.

다양한 해부학 적 부위 (32)를 주입 할 수 있지만, 꼬리 정맥 주사는 종종 생존 실험 세균 부담 결정, 식세포 작용 활성 또는 코드를 포함하여 형성 후속 분석을위한 전신 감염을 생성하는 데 사용된다. 꼬리 근육 주사 주입 (그림 3A)의 사이트에있는 대 식세포의 모집을 평가하는 데 사용됩니다. 조사 엠의 R과 S 변종의 독성을 비교하려면 농양, 형광 박테리아 현탁?...

Watch this Scientific Journal Video about Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos at JoVE.com

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.

영상 병인과의 Cording를 해독하고<em&gt; 미코 농양</em&gt; Zebrafish의 배아에서

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

deciphering-imaging-pathogenesis-cording-mycobacterium-abscessus

Research

JoVE Journal

Immunology and Infection

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

10.2K 조회수.  Université Montpellier. 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. 

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

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Zebrafish의 배아에 주입 마이크로 비드

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

연구

발생학

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

의 이미징 HIV-1 봉투 유발 Virological 시냅스와 합성 지질 Bilayers에 신호

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

면역학 및 감염병학

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

세포내 세균성 병원균과 Zebrafish 태아의 감염

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

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.

Zebrafish 애벌레의 분해해 칸디다 증의 비침습 이미징

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

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

설립과 연구에 높은 처리량 설정 최적화<em&gt; 표피 포도 구균</em&gt;와<em&gt; Mycobacterium의 박테</em&gt; 감염 신약 개발의 모델로

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Zebrafish 태아 모델 Vivo 시각화 및 Intravital 소재 분석 관련 된 황색 포도상구균 에 감염

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

생체공학

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.

진 균 marinum 감염 성인 Zebrafish에 모델링 결핵

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

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

제브라피시 배아에서 사체내 현미경 검사를 통해 진균 감염 시 대식세포 리액세포 사멸 시 시각화

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

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.

호스트 병원체 상호 작용의 분석을 위한 아스퍼질러스 포자를 가진 얼룩말 물고기 애벌레의 감염

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

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

영상 병인과의 Cording를 해독하고

요약

더 많은 비디오 탐색