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.

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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 border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Supplier</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma</td>
			<td>P0290</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>228210</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H10 agar</td>
			<td>Becton, Dickenson and Company</td>
			<td>262710</td>
		</tr>
		<tr>
			<td>BBL Middlebrook OADC Enrichment</td>
			<td>Becton, Dickenson and Company</td>
			<td>211886</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H9 Broth</td>
			<td>Becton, Dickenson and Company</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>BBL Middlebrook ADC Enrichment</td>
			<td>Becton, Dickenson and Company</td>
			<td>211887</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma-Aldrich</td>
			<td>P1754</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone (PVP40)</td>
			<td>Calbiochem, Merck KGaA</td>
			<td>529504</td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
		</tr>
		<tr>
			<td>Methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0387</td>
		</tr>
		<tr>
			<td>SeaPlaque Agarose (low melting point agarose)</td>
			<td>Lonza Inc.</td>
			<td>50100</td>
		</tr>
		<tr>
			<td>FluoSpheres (1.0 &mu;m, red fluorescent (580/605))</td>
			<td>Invitrogen</td>
			<td>F8821</td>
		</tr>
		<tr>
			<td>FluoSpheres (1.0 &mu;m, yellow-green fluorescent (505/515))</td>
			<td>Invitrogen</td>
			<td>F8823</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens at JoVE.com

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

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 ...

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Immunology and Infection

43.4K Views.  Leiden University. 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.