Name: development of a larval zebrafish infection model for clostridioides difficile
Uploaded: 2020-02-14T13:30:00
Description: Watch this Scientific Journal Video about Development of a Larval Zebrafish Infection Model for Clostridioides difficile at JoVE.com

We are grateful to Timo Fritsch for excellent animal care. We thank the members of the K&#246;ster and Steinert labs for support and helpful discussions. We thank Dr. Dandan Han for critical reading the manuscript. We gratefully acknowledge funding by the Federal State of Lower Saxony, Nieders&#228;chsisches Vorab (VWZN2889).

All animal work described here was performed in accordance with legal regulations (EU-Directive 2010/63, license AZ 325.1.53/56.1-TUBS and license AZ 33.9-42502-04-14/1418).
1. Preparation of Low Melting Agarose, Gel Plate, and Microinjection/Microgavage Needles
<ol>
	<li>Dissolve 0.08 g of low melting agarose (Table of Materials, agarose A2576) in 10 mL of 30% Danieau&#39;s medium (0.12 mM MgSO4, 0.18 mM Ca [NO3]2), 0.21 mM KCl, 1.5 mM HEPES...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+Between+the+Gastrointestinal+Microbiome+and+Clostridium+difficile.">Interactions Between the Gastrointestinal Microbiome and Clostridium difficile.</a> Annual Review of Microbiology. 69, 445-461 (2015).</li><li>Pham, L. N., Kanther, M., Semova, I., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+generating+and+colonizing+gnotobiotic+zebrafish.">Methods for generating and colonizing gnotobiotic zebrafish.</a> Nature Protocols. 3, 1862-1875 (2008).</li><li>Ransom, E. M., Ellermeier, C. D., Weiss, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+mCherry+red+fluorescent+protein+for+studies+of+protein+localization+and+gene+expression+in+Clostridium+difficile.">Use of mCherry red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile.</a> Applied and Environmental Microbiology. 81 (5), 1652-1660 (2015).</li><li>Cocchiaro, J. L., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microgavage+of+Zebrafish+Larvae.">Microgavage of Zebrafish Larvae.</a> Journal of Visualized Experiments. (72), e4434 (2013).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+of+Clostridium+difficile-Associated+Disease.">A Mouse Model of Clostridium difficile-Associated Disease.</a> Gastroenterology. 135 (6), 1984-1992 (2008).</li><li>Hutton, M. L., Mackin, K. E., Chakravorty, A., Lyras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+models+for+the+study+of+Clostridium+difficile+disease+pathogenesis.">Small animal models for the study of Clostridium difficile disease pathogenesis.</a> FEMS Microbiology Letters. 352, 140-149 (2014).</li><li>Brugman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+to+study+intestinal+inflammation.">The zebrafish as a model to study intestinal inflammation.</a> Developmental &amp; Comparative Immunology. 64, 82-92 (2016).</li><li>Goulding, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+profiles+of+infection+and+pathology+in+hamsters+infected+with+Clostridium+difficile+strains+630+and+B1.">Distinctive profiles of infection and pathology in hamsters infected with Clostridium difficile strains 630 and B1.</a> Infection and Immunity. 77, 5478-5485 (2009).</li><li>Toh, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonizing+the+Embryonic+Zebrafish+Gut+with+Anaerobic+Bacteria+Derived+from+the+Human+Gastrointestinal+Tract.">Colonizing the Embryonic Zebrafish Gut with Anaerobic Bacteria Derived from the Human Gastrointestinal Tract.</a> Zebrafish. 10, 194-198 (2013).</li><li>Bloemberg, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+static+immersion+and+intravenous+injection+systems+for+exposure+of+zebrafish+embryos+to+the+natural+pathogen+Edwardsiella+tarda.">Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda.</a> BMC Immunology. 12, 58 (2011).</li><li>D&#237;az-Pascual, F., Ort&#237;z-Sever&#237;n, J., Varas, M. A., Allende, M. 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The presented methods modify and extend an existing approach to infect zebrafish larvae by performing both injection and microgavage10,14. It also demonstrates an approach to study anaerobic pathogens with zebrafish larvae22. In addition, the protocol facilitates the analysis of innate immune cell responses in vivo upon CDI and upon colonization of C. difficile in zebrafish. The method is reproducible and easy to conduct in a rout...

C. difficile is a gram-positive, spore-forming, anaerobic, and toxin-producing bacillus that is the leading cause of severe infections in the gastrointestinal tract1. Typical symptoms of CDI include diarrhea, abdominal pain, and fatal pseudomembranous colitis, and it can sometimes lead to death1,2. Evidence has shown that host immune responses play a critical role in both the progression and outcome of this disease3. In addition to the immune response, the indigenous gut microbiota is crucial for the onset and pathogenesis of CDI4. In the past decade, both the number of cases and the mortality rate of CDI have increased significantly due to the emergence of a hypervirulent strain of C. difficile (BI/NAP1/027)5,6. A better understanding of underlying immune mechanisms and the role of microbiota during CDI will help lead to new therapeutic developments and advances, enabling better control of this epidemic.
Several animal models, such as the hamster and mouse, have been developed to provide insight into the immune defense against C. difficile7,8. However, the role of innate immune cells is still poorly understood, particularly since innate immune cell behavior is mainly derived from histological analysis or cultured cells in vitro. Therefore, establishing a transparent zebrafish model to reveal the innate immune response to C. difficile inside of a living vertebrate organism will facilitate such studies. Zebrafish larvae have a functional innate immune system, but they lack the adaptive immune system until 4&#8211;6 weeks after fertilization9. This unique feature makes zebrafish larvae an excellent model to study the isolated response and function of innate immune cells in CDI.
This report describes new methods using zebrafish larvae to study the interactions between C. difficile and innate immune cells, such as macrophages and neutrophils. First, a localized microinjection protocol that includes C. difficile inoculum and staining is presented. Using in vivo confocal time-lapse imaging, the response of neutrophils and macrophages towards the infection site is recorded, and the phagocytosis of bacteria by neutrophils and macrophages is observed. However, it has been reported that the injection itself causes tissue damage and leads to the recruitment of leukocytes independent of the bacteria10. Therefore, a noninvasive microgavage protocol to deliver C. difficile into the intestine of zebrafish larvae is subsequently described. Previous studies have demonstrated that indigenous gastrointestinal microbiota protect a host against the colonization of C. difficile11. Therefore, gnotobiotic zebrafish larvae are also used to predispose the zebrafish that are infected 12. Afterwards, intestinal dissection is performed to recover viable C. difficile and validate the duration of their presence in zebrafish intestinal tracts.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A2576</td>
			<td>Ultra-low gelling agarose</td>
		</tr>
		<tr>
			<td>Agarose low-melting (LM)</td>
			<td>Pronadisa</td>
			<td>8050</td>
			<td>It is used in agarose plates</td>
		</tr>
		<tr>
			<td>BacLight Red Bacterial Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>B35001</td>
			<td>Fluorescent dye</td>
		</tr>
		<tr>
			<td>Brain-Heart-Infusion Broth</td>
			<td>Carl Roth GmbH</td>
			<td>X916.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Brass (wild-type)</td>
			<td></td>
			<td></td>
			<td>deficient in melanin synthesis, used to generate stable transgenic lines</td>
		</tr>
		<tr>
			<td>Calcium nitrate (Ca(NO3)2)</td>
			<td>Sigma-Aldrich</td>
			<td>C1396</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Glass</td>
			<td>Harvard Apparatus</td>
			<td>30-0019</td>
			<td>Injection needles</td>
		</tr>
		<tr>
			<td>Clostridioides difficile</td>
			<td></td>
			<td></td>
			<td>R20291,, a ribotype 027 strain, TcdA+/TcdB+/CDT+ production</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Carl Roth GmbH</td>
			<td>A994</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td>open-source platform</td>
			<td></td>
			<td>Image processing</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth GmbH</td>
			<td>6763</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal needle puller</td>
			<td>Sutter instrument Inc</td>
			<td>P-87</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>168149</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite X (LAS X)</td>
			<td>Leica</td>
			<td></td>
			<td>Image processing</td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Carl Roth GmbH</td>
			<td>P026</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro injector</td>
			<td>eppendorf</td>
			<td>5253000017</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection molds</td>
			<td>Adaptive Science Tools</td>
			<td>TU1</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Carl Roth GmbH</td>
			<td>5346</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Carl Roth GmbH</td>
			<td>9265</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurocholate</td>
			<td>Carl Roth GmbH</td>
			<td>8149</td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(lyZ: KalTA4)bz17/Tg(4xUAS-E1b:EGFP)hzm3</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which in which the lyZ promoters drive the expression of EGFP fluorescent protein in neutrophils</td>
		</tr>
		<tr>
			<td>Tg(mpeg1.1: KalTA4)bz16/Tg(4xUAS-E1b:EGFP)hzm3</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which in which the mpeg1.1 drive the expression of EGFP fluorescent protein in macrophages</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Bacto</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of a larval zebrafish infection model for clostridioides difficile

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

C. difficile is strictly anaerobic, but the chromophore of fluorescent proteins usually requires oxygen to mature. To overcome this problem, a fluorescent dye was used to stain live C. difficile cells that were actively growing (R20291, a ribotype 027 strain; Figure 1A). Using the Gal4/UAS system, stable transgenic zebrafish lines were generated for live imaging, in which the mpeg1.1 or lyZ promoters drove the expression of EGFP fluorescen...

Watch this Scientific Journal Video about Development of a Larval Zebrafish Infection Model for Clostridioides difficile at JoVE.com

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

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

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

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

This method can answer the key question about the role of innate immune system in the Clostridium infection, like if and how macrophages recognize or phagocytose this pathogen. The main advantage of this method is that it can be applied to many different anaerobic bacteria, that the infection time can be manipulated, and that oral administration represents the normal human infection much closer. After culturing, transfer one milliliter of the culture into a spectrophotometer cuvette, and measure the OD600.Transfer the required amount into a fresh 1.5-milliliter tube in order to reach a final OD of one to 1.2 in one milliliter of final volume. Wash one time with one milliliter of 1X PBS, and centrifuge at 5, 000 times g for three minutes at room temperature. Resuspend in one milliliter of 1X PBS.Add three microliters of working solution of the prepared fluorescent dye into the one-milliliter bacterial suspension. Incubate the sample for 15 minutes at room temperature in the dark. After that, wash the stained Clostridioides difficile once with 1X PBS to remove residual dye, and resuspend in one milliliter of 1X PBS to achieve an OD600 of 1.0.First, place a drop of 0.8%low-melting agarose onto the zebrafish larvae in a Petri dish to cover. Gently adjust the larvae to a lateral position. Place the Petri dish on ice for 30 to 60 seconds to allow the low-melting agarose to solidify.Add 30%Danieau's medium containing 0.02%to 0.04%tricaine to cover the agarose. To prepare injection solution, add one microliter of 0.5%phenol red in PBS solution into nine microliters of the dye-stained Clostridioides difficile inoculum. Load a calibrated microinjection needle with the injection solution using a Microloader.Mount the loaded needle into a micromanipulator, and position it under a stereo microscope. Adjust the injection pressure between 600 and 900 hectopascals. Set the injection time to 0.1 to 0.3 seconds to obtain 0.5 to 1.0 nanoliters.Set the needle in the micromanipulator at an approximately 45-degree angle, pointing toward the embedded larvae. Place the needle tip above the gastrointestinal tract, close to the urogenital pore. Pierce through the agarose, then the muscle with the needle tip.Then insert it into the intestinal lumen, and inject 0.5 to 1.0 nanoliters of Clostridioides difficile. Use a fluorescence microscope to monitor the injected larvae, and use a flexible Microloader tip to pick up the properly injected larvae for confocal imaging. In a 1.5%agarose plate with grooves, place a drop of 0.8%low-melting agarose onto the zebrafish larvae to cover.Gently adjust the larvae with heads facing upright at 45-degree angles in the groove and tails against the wall of the groove. Gently operate the needle through the agarose, then into the mouth of zebrafish larvae, through the esophagus. Once the tip of the needle is inside the anterior intestinal bulb, press the injection pedal to release 0.5 to one nanoliters of bacteria culture.Fill the lumen of the intestine. Do not let it overflow from the esophagus or cloaca. Gently withdraw the needle from the mouth of the zebrafish.Following gavage, rescue the infected zebrafish larvae from the agarose with a flexible Microloader tip by first cutting the agarose away, then by lifting the larvae. Transfer these larvae into sterile 30%Danieau's medium, and rinse twice. After anesthetizing the zebrafish larvae, add 200 to 300 microliters of 1%low-melting agarose to cover the anesthetized larvae.Place the infected region of the larvae against a glass slide as closely as possible. Let the agarose solidify on ice for 30 to 60 seconds. Submerge the agarose into 30%Danieau's medium containing 0.02%to 0.04%tricaine.Proceed to image the larvae with a confocal laser scanning microscope. After euthanizing the infected zebrafish larvae, insert a needle into the dorsal trunk of zebrafish larvae close to the head to immobilize the zebrafish. Remove the head behind the gills with a lancet.Insert a second needle into the middle of the dorsal trunk. Insert a third needle into the abdomen of the zebrafish, and pull the intestine out of the body cavity. Use a microinjection needle to transfer 10 to 15 intestines into a 1.5-milliliter tube containing 200 microliters of sterile 1X PBS.Homogenize the intestines with a pestle to disrupt the tissue and prepare homogenates. Ensure the pestle reaches the bottom of the tube to disrupt all intestines completely. Into the homogenates, add Clostridioides difficile rearing medium containing D-cycloserine and cefoxitin, with or without taurocholate, and incubate in an anaerobic chamber.In this study, both neutrophils and macrophages reach the infection sites. The example of an activated macrophage phagocytizing two bacteria shows the clearing by phagocytosis and digestion of the labeled Clostridioides difficile. The microgavage to deliver fluorescence-labeled Clostridioides difficile into the intestinal lumen of macrophage and neutrophil reporter lines at five days post-fertilization mimics the natural path of Clostridioides difficile infection.However, neutrophils and macrophages did not show obvious migration to the gastrointestinal tract for up to 12 hours after microgavage. In the meantime, fluorescence of the labeled Clostridioides difficile disappeared after around five hours post-microgavage. Intestinal samples 24 hours post-infection were dissected and showed bacterial growth.No bacteria grew in the control group. At later time points, incubated samples only grew in the medium containing TCA, suggesting that total Clostridioides difficile in the gut had formed spores. 16S rDNA PCR identified the grown bacterium as Clostridioides difficile, which produce specific PCR amplicons of predictable size around 800 base pairs.Bacteria cultures grown onto a CHROMID plate appeared as typical black colonies, which further indicated that bacteria from the zebrafish intestines were Clostridioides difficile. Working with anaerobic bacteria requires that the aerobic steps are kept short because that influences the biology of the bacteria of course. Innate immune cell in zebrafish can be manipulated.For example, they can decipher the mechanism of the innate immune cell responses to the infection. Our method indicated that zebrafish can be a tool to study anaerobic pathogen from human gut. Working with multi-resistant pathogens requires that appropriate safety measures are taken.

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Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

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

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

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