Name: Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens (Video) | JoVE Journal
Uploaded: 2012-03-15T12:00:00.000+00:00
Duration: 11 min 18 s
Description: Learn micro-injection techniques for studying host-pathogen interactions in zebrafish embryos using fluorescent bacteria. This method enables live imaging of immune cell responses to Salmonella and Mycobacterium infections.

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The authors would like to thank Chao Cui, Floor de Kort, Esther Stoop and Ralph Carvalho for images in Figure 4, Ulrike Nehrdich and Davy de Witt for fish care, and other lab members for helpful discussions. This work is supported by the Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Ministry of Education, Culture and Science, the European Commission 7th framework project ZF-HEALTH (HEALTH-F4-2010-242048), the European Marie-Curie Initial Training Network FishForPharma (PITN-GA-2011-289209), and by the Australian NHMRC (637394). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.

<ol><li>Meijer, A. H., Spaink, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host-pathogen+interactions+made+transparent+with+the+zebrafish+model%5BTitle%5D)+AND+%22Curr.+Drug+Targets%22%5BJournal%5D)">Host-pathogen interactions made transparent with the zebrafish model.</a> Curr. Drug Targets. 12, 1000-1017 (2011).</li>
<li>Davis, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+visualization+of+mycobacterium-macrophage+interactions+leading+to+initiation+of+granuloma+formation+in+zebrafish+embryos%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.</a> Immunity. 17, 693-702 (2002).</li>
<li>vander Sar, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+embryos+as+a+model+host+for+the+real+time+analysis+of+Salmonella+typhimurium+infections%5BTitle%5D)+AND+%22Cell+Microbiol%22%5BJournal%5D)">Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections.</a> Cell Microbiol. 5, 601-611 (2003).</li>
<li>Zakrzewska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophage-specific+gene+functions+in+Spi1-directed+innate+immunity%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Macrophage-specific gene functions in Spi1-directed innate immunity.</a> Blood. 116, 1-11 (2010).</li>
<li>Le Guyader, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Origins+and+unconventional+behavior+of+neutrophils+in+developing+zebrafish%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Origins and unconventional behavior of neutrophils in developing zebrafish.</a> Blood. 111, 132-141 (2008).</li>
<li>Herbomel, P., Thisse, B., Thisse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ontogeny+and+behaviour+of+early+macrophages+in+the+zebrafish+embryo%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Ontogeny and behaviour of early macrophages in the zebrafish embryo.</a> Development. 126, 3735-3745 (1999).</li>
<li>Alibaud, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Mycobacterium+marinum+TesA+mutant+defective+for+major+cell+wall-associated+lipids+is+highly+attenuated+in+Dictyostelium+discoideum+and+zebrafish+embryos%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos.</a> Mol. Microbiol. 80, 919-934 (2011).</li>
<li>Carvalho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+screen+for+tuberculosis+progression%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A high-throughput screen for tuberculosis progression.</a> PLoS One. 6, 16779-16779 (2011).</li>
<li>van der Sar, A. M., Spaink, H. P., Zakrzewska, A., Bitter, W., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specificity+of+the+zebrafish+host+transcriptome+response+to+acute+and+chronic+mycobacterial+infection+and+the+role+of+innate+and+adaptive+immune+components%5BTitle%5D)+AND+%22Mol.+Immunol%22%5BJournal%5D)">Specificity of the zebrafish host transcriptome response to acute and chronic mycobacterial infection and the role of innate and adaptive immune components.</a> Mol. Immunol. 46, 2317-2332 (2009).</li>
<li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115-e1115 (2009).</li>
<li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stages+of+embryonic+development+of+the+zebrafish%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li>
<li>Westerfield, M. The Zebrafish Book. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWesterfield%2C+M+%22A+Guide+for+the+Laboratory+Use+of+Zebrafish+%28Brachydanio+rerio%29%22">A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio)</a>. , The University of Oregon Press. Eugene. (1993).</li>
<li>Gutzman, J. H., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+Brain+Ventricle+Injection%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Zebrafish Brain Ventricle Injection.</a> J. Vis. Exp. (26), e1218-e1218 (2009).</li>
<li>Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mpeg1+promoter+transgenes+direct+macrophage-lineage+expression+in+zebrafish%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.</a> Blood. 117, 49-56 (2011).</li>
<li>Stockhammer, O. W., Zakrzewska, A., Hegedus, Z., Spaink, H. P., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptome+profiling+and+functional+analyses+of+the+zebrafish+embryonic+innate+immune+response+to+Salmonella+infection%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Transcriptome profiling and functional analyses of the zebrafish embryonic innate immune response to Salmonella infection.</a> J. Immunol. 182, 5641-5653 (2009).</li>
<li>Renshaw, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+transgenic+zebrafish+model+of+neutrophilic+inflammation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108, 3976-3978 (2006).</li>
<li>Stoop, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+embryo+screen+for+mycobacterial+genes+involved+in+the+initiation+of+granuloma+formation+reveals+a+newly+identified+ESX-1+component%5BTitle%5D)+AND+%22Dis.+Model+Mech%22%5BJournal%5D)">Zebrafish embryo screen for mycobacterial genes involved in the initiation of granuloma formation reveals a newly identified ESX-1 component.</a> Dis. Model Mech. 4, 526-536 (2011).</li>
<li>Levraud, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+the+zebrafish+IFN+receptor%3A+implications+for+the+origin+of+the+vertebrate+IFN+system%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Identification of the zebrafish IFN receptor: implications for the origin of the vertebrate IFN system.</a> Journal of Immunology. 178, 4385-4394 (2007).</li>
<li>Levraud, J. P., Colucci-Guyon, E., Redd, M. J., Lutfalla, G., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+analysis+of+zebrafish+innate+immunity%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">In vivo analysis of zebrafish innate immunity.</a> Methods Mol. Biol. 415, 337-363 (2008).</li>
<li>Brothers, K. M., Newman, Z. R., Wheeler, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+imaging+of+disseminated+candidiasis+in+zebrafish+reveals+role+of+phagocyte+oxidase+in+limiting+filamentous+growth%5BTitle%5D)+AND+%22Eukaryot.+Cell%22%5BJournal%5D)">Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth.</a> Eukaryot. Cell. 10, 932-944 (2011).</li>
<li>Colucci-Guyon, E., Tinevez, J. Y., Renshaw, S. A., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategies+of+professional+phagocytes+in+vivo%3A+unlike+macrophages%2C+neutrophils+engulf+only+surface-associated+microbes%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils engulf only surface-associated microbes.</a> J. Cell Sci. 124, 3053-3059 (2011).</li>
<li>van Soest, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+static+immersion+and+intravenous+injection+systems+for+exposure+of+zebrafish+embryos+to+the+natural+pathogen+Edwardsiella+tarda%5BTitle%5D)+AND+%22BMC+Immunology%22%5BJournal%5D)">Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda.</a> BMC Immunology. 12, 58-58 (2011).</li>
<li>Adams, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+tolerance+in+replicating+mycobacteria+mediated+by+a+macrophage-induced+efflux+mechanism%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism.</a> Cell. 145, 39-53 (2011).</li>
<li>Ellett, F. L., Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Computational+quantification+of+fluorescent+leukocyte+numbers+in+zebrafish+embryos%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Computational quantification of fluorescent leukocyte numbers in zebrafish embryos.</a> Methods in Enzymology. , (2011).</li>
<li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113-e1113 (2009).</li>
<li>Mathias, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resolution+of+inflammation+by+retrograde+chemotaxis+of+neutrophils+in+transgenic+zebrafish%5BTitle%5D)+AND+%22J.+Leukoc.+Biol%22%5BJournal%5D)">Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish.</a> J. Leukoc. Biol. 80, 1281-1288 (2006).</li>
<li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish%5BTitle%5D)+AND+%22BMC+Dev.+Biol%22%5BJournal%5D)">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Dev. Biol. 7, 42-42 (2007).</li>
<li>Gray, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+intravital+imaging+of+macrophage+and+neutrophil+behaviour+during+inflammation+using+a+novel+transgenic+zebrafish%5BTitle%5D)+AND+%22Thromb.+Haemost%22%5BJournal%5D)">Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish.</a> Thromb. Haemost. 105, 811-819 (2011).</li>
</ol>

The authors have nothing to disclose.

The infection methods described in this article are frequently used to study the function of host innate immunity genes or bacterial virulence genes1. The intravenous micro-injection methods (protocols 5 and 6.1) are used most often for such studies. The caudal vein at the posterior blood island is the most convenient location for intravenous injection of 1-day-old embryos. As the caudal vein becomes more difficult to penetrate at later stages, we prefer the Duct of Cuvier as intravenous injection site for embryos at 2-3 dpf. In all cases it is critical to inject the embryos with consistent numbers of bacteria, which should be checked by plating injection inocula for cfu counting. The following aspects must be considered to achieve reproducible injections. Firstly, it is essential to have high-quality glass microcapillary injection needles. If the needle tip is too large, the injection will create a puncture hole large enough for bleeding to occur and the injected bacteria will flow out of the blood circulation. Secondly, bacteria must be injected directly into the blood circulation. If the needle tip is not inside the vein or if injection fluid spreads into the yolk sac extension, the embryo must be discarded from the experiment. Thirdly, using PVP40 as a carrier for injecting M. marinum will assist in preventing the bacteria from sinking in the glass needle. PVP40 improves the homogeneity of the suspension, resulting in more reproducible inocula throughout the duration of injections. PVP40 may also be used for S. typhimurium injections, but here it is less important because the larger size and bright fluorescence of the bacteria allows a visual control over the injection inoculum during experiments. For practising the injections we recommend the use of phenol red dye (1% v/v) or 1 &mu;m fluorescent spheres available in different colors (Invitrogen). Once the technique is mastered, it takes approximately 30 minutes to inject 50-100 embryos, including the time required for aligning the embryos on agarose plates and adjusting the bacterial injection volume.
While intravenous injections into the blood island (protocol 5) or into the duct of Cuvier (protocol 6.1) are most commonly used, the alternative routes of infection described in this video article have proved useful to study macrophage and neutrophil chemotaxis (protocols 6.2, 6.3, and 6.4), to study growth of attenuated bacterial strains (protocol 6.5), and to adapt the zebrafish infection model for high-throughput applications (protocol 6.6) 4-8. The described micro-injection methods can also be applied for injections of viruses 18, 19, fungal spores 14, 20 or protozoan parasites (Maria Forlenza, personal communication). A useful addition to the injection procedures described in this video article is a recently described procedure for subcutaneous injection of bacteria into zebrafish embryos 21. This procedure was applied to study phagocytosis of bacteria by neutrophils, which were found to efficiently engulf bacteria on tissue surfaces but, in contrast to macrophages, were virtually unable to phagocytose bacteria upon injection into the blood or into fluid-filled body cavities. For subcutaneous injections embryos are positioned similar as for the tail muscle injections described in this video article, but the needle is inserted just under the skin to inject the bacteria over a somite.
It is important to consider that micro-injection is essentially an artificial route of infection. However, early embryos are highly resistant to external exposure to bacterial pathogens. In fact, immersion assays, where embryos are bathed in a bacterial suspension, are not reproducible in our hands 22. While some embryos may become infected upon immersion, we have observed a large variation in mortality rates and in the expression of inflammatory marker genes between individual embryos in such assays. The micro-injection methods described in this article achieve reproducible infections and are particularly useful to study bacterial interactions with host innate immune cells. An efficient approach to quantify bacterial infection in individual embryos is to analyze the fluorescent images of infected embryos with custom-made, dedicated pixel quantification software 17, 23. This approach has been shown to correlate well with results of cfu counts after plating of infected embryos. Similarly, relative changes in leukocyte numbers per embryo have been quantified by digital image analysis in transgenic leukocyte fluorophore reporter lines; this approach would be applicable to quantifying the host leukopoietic response to infection24.
The function of host innate immune genes can be investigated efficiently in vivo by combining the described infection models with morpholino knockdown. Morpholinos are synthetic antisense oligonucleotides that target specific RNAs and reduce the expression of the gene products when injected into 1-cell stage zebrafish embryos as shown in other video articles in this journal 10, 25 . In morpholino knockdown studies, it is of great importance that all embryo groups to be compared are staged correctly prior to bacterial injections. This is especially important because embryos have a developing immune system that becomes increasingly competent over time.
There are several transgenic reporter lines currently available to distinguish the major innate immune subsets, macrophages and neutrophils, in zebrafish embryos. The mpx and lyz promoters have been used to generate neutrophil reporter lines 16, 26, 27, and the csf1r (fms) and mpeg1 promoters were used to produce macrophage reporters 14, 28 . While csf1r (fms) is additionally expressed in xanthophores, mpeg1 expression is exclusively in macrophages. As shown in this video article, the recently developed mpeg1 reporter lines are highly useful for imaging bacterial phagocytosis by macrophages.

<table><tbody><tr><td>Phenol Red</td><td>Sigma-Aldrich</td><td>P0290</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>228210</td><td></td></tr><tr><td>Difco Middlebrook 7H10 agar</td><td>BD Biosciences</td><td>262710</td><td></td></tr><tr><td>BBL Middlebrook OADC Enrichment</td><td>BD Biosciences</td><td>211886</td><td></td></tr><tr><td>Difco Middlebrook 7H9 Broth</td><td>BD Biosciences</td><td>271310</td><td></td></tr><tr><td>BBL Middlebrook ADC Enrichment</td><td>BD Biosciences</td><td>211887</td><td></td></tr><tr><td>Tween 80</td><td>Sigma-Aldrich</td><td>P1754</td><td></td></tr><tr><td>Polyvinylpyrrolidone (PVP40)</td><td>Calbiochem</td><td>529504</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Phenylthiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629</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>Methyl cellulose</td><td>Sigma-Aldrich</td><td>M0387</td><td></td></tr><tr><td>SeaPlaque Agarose (low melting point agarose)</td><td>Lonza Inc.</td><td>50100</td><td></td></tr><tr><td>FluoSpheres (1.0 &amp;mu;m, red fluorescent (580/605))</td><td>Invitrogen</td><td>F8821</td><td></td></tr><tr><td>FluoSpheres (1.0 &amp;mu;m, yellow-green fluorescent (505/515))</td><td>Invitrogen</td><td>F8823</td><td></td></tr></tbody></table>

infection of zebrafish embryos with intracellular bacterial pathogens

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.

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

The overall goal of this procedure is to micro inject zebrafish embryos with fluorescent bacteria for live imaging of the interaction with host immune cells. This is accomplished by first preparing and loading the injection needle with bacterial suspensions. Next, the embryos are staged and aligned on an injection plate.Then the bacterial inoculum is injected using a route to achieve either a local or a systemic bacterial infection. Finally, the embryos are mounted and the embryos are imaged. Ultimately, results can be obtained that show intracellular localization of bacterial pathogens inside fluorescent phagocytes of the transparent zebrafish embryonic host through fluorescence and confocal microscopy.This method can help answer key questions in the immunology field because the zebrafish embryo model is very powerful for life imaging of host pathogen interactions, Generally, individuals new to this method will struggle because it requires a lot of practice To reproducibly micro inject bacteria into the blood circulation of zebrafish embryos for systemic infection or into specific compartments to achieve local infections. After pulling glass micro capillary needles and beveling the tips to a 45 degree angle, stage the zebra fish embryos at 28 hours post fertilization by checking for consistent blood circulation. The start of pigmentation in the eye, a straight tail and the heart being positioned just ventrally to the eye.Once the embryos are anesthetized, use a micro loader tip to load the needle with a previously prepared bacterial inoculum. Mount the loaded needle onto a micro manipulator connected to a stand and position it under a stereo microscope. Set the injection time to 0.2 seconds and the compensation pressure to 15 Hector Pascals.Adjust the injection pressure between 700 and 900 Hector Pascals to obtain the correct injection volume for the needle used. Set the micro manipulator with the loaded needle into the correct position prior to injecting. Place the anesthetized embryos on a flat 1%agarose injecting plate and remove any excess egg water.Next, use a hair loop tool to line up the embryos for each injection. Move the plate by hand during injections to orient the embryos with their tails pointing towards the needle tip. Place the needle tip directly above the coddle vein close to the urogenital opening.Pierce the periderm with the needle tip and inject the desired dose of fluorescently labeled bacteria. We use approximately 250 colony forming units or CFUs of salmonella typhimurium and approximately 120 CFUs of mycobacterium marum. The injected bacterial suspension will follow the blood flow through the coddle vein towards the heart monitor if the injection was performed correctly by checking for an expanding volume of the vascular system directly after the pulse.Frequently check that the injection volume remains the same during the experiment. To provide a control for the consistency of the injections throughout the experiment, inject a drop of bacteria directly onto a drop of sterile PBS on bacterial growth medium. After approximately every 30th embryo injection, plate out this drop and count the bacterial colonies after incubation.To determine the CFU in the injection volume, use a fluorescent stereo microscope to observe individual fluorescent styria cells circulating in the bloodstream directly after injection and discard embryos that are not properly injected. Fluorescent aggregates of M marum bacteria should be visible by two days post infection and grow larger over time. To inject into the duct of cuvier, line up anesthetize two to three days Post fertilization embryos.As for coddle vein injection at a 45 degree angle from the dorsal side of the embryo, insert the needle into the starting point of the duct of cuvier. Just dorsal to the location where the duct starts broadening over the yolk sac for hindbrain ventricle injecting position. Anesthetize 32 hours post fertilization embryos with their dorsal side towards the needle tip.Insert the needle into the hindbrain ventricle from an anterior position without touching the neuro helium to inject into tail muscles. Position anesthetized one to two days post fertilization embryos with their tail pointing toward the needle tip and with the needle at an approximately 65 degree angle, inject into the muscle above the urogenital opening for otic vesicle injecting. Orient anesthetize two to three days post fertilization embryos with the tails pointing to towards the needle.Inject at a 65 degree angle and at low pressure to inject into the node cord using one to two days. Post fertilization embryos with the tail pointing away from the needle. Insert the needle through the tail muscle tissue into the cord.For a yolk injection of the 16 to 1000 cell stage embryos pierce the needle through the corion into the center of the yolk to image the infection. Anesthetize the infected embryos in a 1%augurous layered Petri dish covered with egg water containing trica. Using a hair loop tool aligned the embryos in the correct position for imaging under a fluorescent stereo microscope.Individual fluorescent s tym cells can be observed circulating in the bloodstream directly after injection. If a different position than the lateral view is required. Mount the embryos in 1.5%methyl cellulose and use a hair loop tool to manipulate the embryo into the required position to image the infection using an inverted confocal microscope.Place a drop of low melting point aeros on a glass bottom dish. Place the anesthetized embryo into the aero drop with limited amount of egg water and use a hair loop tool to manipulate the embryo into position. Let the aero solidify and submerge the aero drop in egg water containing trica.The sample is now ready for confocal imaging injection of salmonella typhimurium or mycobacterium marum bacteria into the blood island of embryos at one day. Post fertilization results in the rapid phagocytosis by macrophages dissemination of the relatively large and brightly fluorescent DS.Red labeled styrian bacteria can be imaged directly with stereo fluorescence and confocal imaging at two hours. Post-infection shows that many bacteria are phagocytose by fluorescent macrophages.An injection dose of 250 CFU of wild type styria will induce a strong pro-inflammatory response and is lethal within a day. In contrast, the intravenous injection of erum leads to a persistent infection where infected macrophages form tight aggregates that are considered as the initial stages of granulomas, which are the hallmark of tuberculosis. Confocal imaging of such a granuloma like aggregate in the transgenic EG one EGFP line at five days post-infection shows the intracellular growth of m cherry labeled M num bacteria.Inside the green fluorescent macrophages, bacteria can be injected into the hind brainin ventricle, which is a compartment devoid of macrophages at 32 hours post fertilization injection of 20 to 100 M cherry labeled M marum bacteria into this compartment leads to the rapid infiltration by macrophages that phagocytose the bacteria as shown here. By using the transgenic M-P-X-E-G-F-P injection of approximately 20 CFU of Styria into the otic vesicle leads to the attraction of neutrophils at three hours post infection. While this response is not observed in PBS control injections, the notochord, which appears to be resistant to infiltration by leukocytes, is a permissive compartment for the growth of erum mutants that are strongly attenuated when injected in other tissues.Following yoke injection of a dose of 2240 CFU erum bacteria spread over several days into the embryonic tissues and form granuloma like aggregates. Similar to those observed with the conventional intravenous injection method. While attempting this procedure, it's important to remember that proper staging of the zebrafish embryos is critical because the embryonic immune system becomes increasingly competent during development.Following this procedure, other methods like transcriptome profiling and immunohistochemistry can be performed in order to answer additional questions like how the embryonic innate immune system responds to pathogenic infections.

infection-of-zebrafish-embryos-with-intracellular-bacterial-pathogens

Research

JoVE Journal

Immunology and Infection

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.

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

This video describes a technique of intraperitoneal injection, a method for delivering solutions into the abdominal cavity of Adult Zebrafish.

Intraperitoneal Injection: A Method of Solution Delivery into the Abdominal Cavity of an Adult Zebrafish

Source: Zhang, X., et al. A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection. J. Vis. Exp. (2019)This video demonstrates fluorescence imaging of zebrafish embryos injected with Staphylococcus aureus, enabling quantification of infection progression and bacterial distribution to assess the impact of biomaterial microspheres on host-pathogen interactions in vivo.

 Fluorescence Imaging of Biomaterial-Associated Bacterial Infection in Zebrafish Embryos

Source: Zhang, X. et. al., A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection. J. Vis. Exp. (2019)This video demonstrates a zebrafish embryo model for studying infections linked to implanted biomaterials. Injecting Staphylococcus aureus with polystyrene microspheres enhances bacterial survival, confirming the infection-promoting role of implanted materials.

In Vivo Analysis of Implant-Associated Bacterial Infections Using Zebrafish Embryos

Source: Shi, J., et al., Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens. J. Vis. Exp. (2020)This video demonstrates the procedure for preparing an Agrobacterium tumefaciens culture by inducing virulence gene expression with acetosyringone, followed by antibiotic removal through washing and resuspension, to generate a transformation-ready suspension for gene delivery into plant tissues.

Microinjection of Bacteria into the Caudal Vein of Zebrafish Embryos

Source: Bernut, A., et al., Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos. J. Vis. Exp. (2015).This video demonstrates a protocol for isolating and quantifying Mycobacterium abscessus from an infected zebrafish embryo. The procedure involves embryo euthanasia, tissue lysis, and homogenization to release the bacteria, followed by centrifugation and resuspension in a surfactant buffer. Finally, the bacterial suspension is serially diluted, plated...

Isolation and Cultivation of Bacteria from Zebrafish Embryos to Assess Infection Load

Source: Flores, E., et al. Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae. J. Vis. Exp. (2019).This video demonstrates the method of establishing a foodborne bacterial intestinal infection model in zebrafish larvae by feeding them paramecia infected with fluorescent E. coli.

Modeling Intestinal Infection in Zebrafish Larvae Using Foodborne Bacteria

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.

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

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.

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

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.

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

There is currently a lack of in vivo models amenable to drug screening against chronic bacterial infections. Here, we describe a protocol for wound infection by a Pseudomonas aeruginosa clinical isolate to generate a persistent infection in zebrafish larvae.

Modeling Persistent Pseudomonas aeruginosa Infection in Wounded Zebrafish Larvae

Learn micro-injection techniques for studying host-pathogen interactions in zebrafish embryos using fluorescent bacteria. This method enables live imaging of immune cell responses to Salmonella and Mycobacterium infections.

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

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