The authors would like to thank the laboratory of Dr. Carol Kim for microinjection training, Clarissa Henry for advice on speeding up embryo development and use of equipment, and Nathan Lawson for contributing fli1:EGFP fish. We thank members of the Wheeler lab and Shawn Walls for critical reading of the manuscript. We would also like to thank Mark Nilan for fish care and advice, and Ryan Phennicie and Kristin Gabor for technical advice on this project. This work was funded by a MAFES research assistantship to K. Brothers, a MAFES Hatch grant E08913-08, and a NIH NCRR award P20RR016463 to R. Wheeler.

All zebrafish care protocols and experiments were performed under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01.
1. Morpholino and Larval Injection Dishes
Experimental duration: * (10-15 minutes)
Degree of difficulty: *
<ol>
	<li>For egg injections, prepare a 2% agarose solution in sterile water and microwave. When the solution has cooled pour some of it into an extra deep Pet...

No conflicts of interest declared.

The zebrafish microinjection method presented here differs from Gutzman et al.34 in that here we demonstrate injection through the otic vesicle into the hindbrain ventricle of 36 to 48 hpf larvae. The method we describe allows for consistent injection of 10-15 yeast into the hindbrain ventricle with reduced tissue damage. This protocol produces an initially local infection that spreads throughout the body by 24 hpi (Figure 1) and results in significant lethality/morbidity5</su...

<table border="1">
<tbody>
 <tr>
 <td>Name of the 			reagent</td>
 <td>Company</td>
 <td>Catalog 			number</td>
 <td>Comments 			(optional)</td>
 </tr>
 <tr>
 <td>Spawning tanks</td>
 <td>Aquatic 			habitats</td>
 <td>&nbsp;2L</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1.7 mL tubes</td>
 <td>Axygen</td>
 <td>MCT-175-C</td>
 <td></td>
 </tr>
 <tr>
 <td>Instant Ocean</td>
 <td>Fisher 			Scientific</td>
 <td>S17957C </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Extra deep 			Petri dishes</td>
 <td>Fisher 			Scientific</td>
 <td>08-757-11Z</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Standard Petri 			dishes</td>
 <td>VWR Scientific</td>
 <td>25384-302</td>
 <td></td>
 </tr>
 <tr>
 <td>Transfer 			pipettes</td>
 <td>Fisher 			Scientific</td>
 <td>13-711-7M</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Yeast Extract</td>
 <td>VWR Scientific</td>
 <td>90000-726</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Peptone</td>
 <td>VWR Scientific</td>
 <td>90000-264</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextrose</td>
 <td>Fisher 			Scientific</td>
 <td>D16-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>VWR Scientific</td>
 <td>90000-760</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disposable 			Hemocytometer</td>
 <td>VWR Scientific</td>
 <td>82030-468</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate 			Buffered Saline</td>
 <td>VWR Scientific</td>
 <td>12001-986</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dumont Dumoxel 			Tweezers</td>
 <td>VWR Scientific</td>
 <td>100501-806</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wooden Dowels</td>
 <td>VWR Scientific</td>
 <td>10805-018</td>
 <td></td>
 </tr>
 <tr>
 <td>KimWipes</td>
 <td>VWR Scientific</td>
 <td>300053-964</td>
 <td></td>
 </tr>
 <tr>
 <td>Low Melt 			Agarose</td>
 <td>VWR Scientific</td>
 <td>12001-722</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agarose for 			injection dishes</td>
 <td>VWR Scientific</td>
 <td>12002-102</td>
 <td></td>
 </tr>
 <tr>
 <td>Flaming Brown 			Micropipette Puller</td>
 <td>Sutter 			Instruments</td>
 <td>P-97</td>
 <td></td>
 </tr>
 <tr>
 <td>Hollow glass 			rods</td>
 <td>Sutter 			Instruments</td>
 <td>BF120-69-10</td>
 <td>For glass rods 			smooth glass by heating over bunsen burner&nbsp;</td>
 </tr>
 <tr>
 <td>Pipette 			Storage Box</td>
 <td>Sutter 			Instruments</td>
 <td>BX10</td>
 <td></td>
 </tr>
 <tr>
 <td>MPPI-3 			Injection system</td>
 <td>Applied 			Scientific Instrumentation</td>
 <td>MPPI-3</td>
 <td></td>
 </tr>
 <tr>
 <td>Back Pressure 			Unit</td>
 <td>Applied 			Scientific Instrumentation</td>
 <td>BPU</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micropipette 			Holder kit</td>
 <td>Applied 			Scientific Instrumentation</td>
 <td>MPIP</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Foot Switch</td>
 <td>Applied 			Scientific Instrumentation</td>
 <td>FSW</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Applied 			Scientific Instrumentation</td>
 <td>MM33</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Magnetic Base</td>
 <td>Applied 			Scientific Instrumentation</td>
 <td>Magnetic Base</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tricaine 			methane sulfonate</td>
 <td>Western 			Chemical Inc.</td>
 <td>MS-222</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dissecting 			Scope</td>
 <td>Olympus</td>
 <td>SZ61 top 			SZX-ILLB2-100 base</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Confocal 			Microscope</td>
 <td>Olympus</td>
 <td>IX-81 with 			FV-1000 laser scanning confocal system</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TC-7 Tissue 			Culture Roller 			drum with 14 inch test tube wheel</td>
 <td>New Brunswick 			Scientific</td>
 <td>&nbsp;TC-7</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Imaging Dishes</td>
 <td>MatTek 			Corporation</td>
 <td>P24G-1.0-10-F</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pipette tips for 			loading needles</td>
 <td>Eppendorf</td>
 <td>930001007</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Plate pouring 			grids</td>
 <td>Adaptive 			Science Tools</td>
 <td>TU-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Heated Stage</td>
 <td>Bioptechs Inc.</td>
 <td>Delta T-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Flat Spatula</td>
 <td>VWR Scientific</td>
 <td>82027-486</td>
 <td></td>
 </tr>
 <tr>
 <td>Plastic Sieves</td>
 <td>Wares of 			Knutsford Online</td>
 <td>12 cm</td>
 <td></td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>VWR Scientific</td>
 <td>52858-000 </td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex Genie</td>
 <td>VWR Scientific</td>
 <td>14216-184</td>
 <td></td>
 </tr>
 <tr>
 <td>16 x 150 mm 			Culture tubes</td>
 <td>VWR Scientific</td>
 <td>60825-435</td>
 <td></td>
 </tr>
 <tr>
 <td>Nanodrop</td>
 <td>Thermo 			Scientific</td>
 <td>ND 2000</td>
 <td></td>
 </tr>
 <tr>
 <td>Phenol Red</td>
 <td>VWR Scientific</td>
 <td>97062-478 </td>
 <td></td>
 </tr>
 <tr>
 <td>HCl</td>
 <td>VWR Scientific</td>
 <td>87003-216 </td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>VWR Scientific</td>
 <td>BDH4534-500GP</td>
 <td></td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>VWR Scientific</td>
 <td>BDH4532-500GP</td>
 <td></td>
 </tr>
 <tr>
 <td>MgSO4</td>
 <td>VWR Scientific</td>
 <td>BDH0246-500GP</td>
 <td></td>
 </tr>
 <tr>
 <td>Ca(NO3)2</td>
 <td>VWR Scientific</td>
 <td>BDH0226-500GP</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>VWR Scientific</td>
 <td>BDH4520-500GP</td>
 <td></td>
 </tr>
 <tr>
 <td>Morpholinos</td>
 <td>GeneTools, LLC</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae at JoVE.com

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

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.

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

non-invasive-imaging-of-disseminated-candidiasis-in-zebrafish-larvae

Research

JoVE Journal

Immunology and Infection

14.6K visualizações.  University of Maine. O desenvolvimento rápido, tamanho pequeno e transparência de peixes-zebra são enormes vantagens para o estudo do controle imunológico inato de infecção 1-4. Aqui demonstramos técnicas para infectar larvas do peixe utilizando o agente patogénico fúngica Candida albicans Por microinjeção, a metodologia usada recentemente para implicar atividade oxidase NADPH de fagócitos no controle de dimorfismo fúngico 5.