Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.
The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.
Streptococcus iniae is a major aquatic pathogen that is capable of causing systemic disease in both fish and humans1. While S. iniae is responsible for large losses in the aquaculture industry, it is also a potential zoonotic pathogen, capable of causing disease in immunocompromised human hosts with clinical pathologies similar to those caused by other streptococcal human pathogens. Given its similarities with human pathogens, it is important to study S. iniae disease pathogenesis in the context of a natural host. An adult zebrafish model of S. iniae infection revealed robust infiltration of host leukocytes to the localized site of infection as well as a rapid time to host death, a time too short to involve the adaptive immune system7. In order to gain an in-depth look into the innate immune response to S. iniae infection in vivo, it is necessary to use a model that is more amenable to non-invasive live imaging.
The larval zebrafish has a number of advantages that make it an increasingly attractive vertebrate model for studying host-pathogen interactions. Zebrafish are relatively inexpensive and easy to use and maintain compared to mammalian models. Adaptive immunity is not functionally mature until 4-6 weeks post fertilization, but larvae have a highly conserved vertebrate innate immune system with complement, Toll-like receptors, cytokines, and neutrophils and macrophages with antimicrobial capabilities including phagocytosis and respiratory burst2-6,8-11. In addition, the genetic tractability and optical transparency of the embryonic and larval stages of development allow for the generation of stable transgenic lines with fluorescently labeled immune cells making it possible to examine host-pathogen interactions in real time in vivo. The generation of these transgenic lines using a photoconvertible protein such as Dendra2 allows for the tracking of individual host cell origin and fate over the course of infection12.
When developing a zebrafish larval infection model, the chosen site of microinjection will determine whether an infection is initially localized or systemic. Systemic blood infections into the caudal vein or Duct of Cuvier are most commonly used to study microbial pathogens in zebrafish and are useful for studying interactions between host and microbial cells, cytokine responses, and differences in virulence between pathogen strains. For slower growing microorganisms, early injection into the yolk sac of an embryo at the 16-1,000 cell stage can be used to generate a systemic infection13,14, with the optimal developmental stage for microinjection of a slow-growing microorganism found to be between the 16 to 128 cell stage15. However, yolk sac injections of many microbes at later stages of host development tend to be lethal to the host due to the nutrient-rich environment for the microbe and lack of infiltrating leukocytes16-18.
A localized infection usually results in directed migration of leukocytes towards the site of infection that can be easily quantified with non-invasive imaging. This type of infection can allow for dissection of the mechanisms that mediate leukocyte migration as well as investigation of different migratory and phagocytic capabilities of various leukocyte populations. Localized infections are also useful when examining differences in virulence between bacterial strains as well as studying microbe invasion mechanisms since physical host barriers must be crossed for a localized infection to become systemic. Zebrafish are typically raised at temperatures of 25-31 °C19, but they can also be maintained at temperatures as high as 34-35 °C for studies of the invasiveness of certain human pathogens with strict temperature requirements for virulence20,21.
Many different sites have been used to generate an initially localized bacterial infection including the hindbrain ventricle22, dorsal tail muscle18, pericardial cavity23, and otic vesicle (ear)5,16,24. However, it has been found that injection of bacteria into tail muscle can cause tissue damage and inflammation independent of the bacteria, which may skew results when investigating leukocyte response13. Although less damage is associated with injection into the hindbrain and although it is initially devoid of leukocytes in young embryos, the hindbrain ventricle steadily gains more immune cells over time as microglia take up residence. The hindbrain ventricle is also a more difficult location to image. The otic vesicle is a closed hollow cavity with no direct access to the vasculature25,26. It is normally devoid of leukocytes, but leukocytes can be recruited to the otic vesicle in response to inflammatory stimuli such as infection. It is also a preferred site of microinjection of bacteria in zebrafish aged 2-3 days post fertilization (dpf) because of the ease of imaging and the visualization of the injection. Therefore, we chose the otic vesicle as our site of localized bacterial infection.
The infection method used here is useful for the study of the host immune response to an initially localized infection in 2-3 dpf embryos and larvae. The focus of an inflammatory stimulus, such as infection, in a closed cavity such as the otic vesicle allows for the study of neutrophil and macrophage chemotaxis and phagocytosis. One caveat of injecting bacteria into the otic vesicle is that the ability of neutrophils to efficiently phagocytose bacteria in fluid-filled cavities may be dependent on the particular microbe. …
The authors have nothing to disclose.
The authors would like to thank lab members for zebrafish care and maintenance. This work was supported by National Institutes of Health, National Research Service Award A155397 to E. A. Harvie and NIH R01GM074827 to Anna Huttenlocher.
1.7 ml eppendorfs | MidSci | AVSS1700 | |
14 ml falcon tube | BD Falcon | 352059 | |
27 G x 1/2 in. needle | BD Biosciences | 305109 | |
96 well plate | Corning Incorporated | 3596 | |
Agar | BD Biosciences | 214030 | |
CellTracker Red | Molecular Probes, Invitrogen | C34552 | |
CNA agar | Dot Scientific, Inc | 7126A | |
Disposable transfer pipets | Fisher Scientific | 13-711-7m | |
Dissecting Scope | Nikon | SMZ745 | |
DMSO | Sigma Aldrich | D2650 | |
Ethanol 200 proof | MDS | 2292 | |
Fine tweezers | Fine Science Tools | 11251-20 | |
Gel comb | VWR | 27372-482 | 4.2 mm width, 1.5 mm thick |
Glass bottom dishes | Custom made by drilling a 16–18 mm hole in the center of a 35-mm tissue culture dish bottom and placing a 22-mm round #1 coverslip in the hole and sealing with a thin layer of Norland Optical Adhesive 68 cured by UV light. | ||
Glycerol | Fisher Scientific | G33-4 | |
High melt agarose | Denville Scientific, Inc. | CA3510-6 | |
Hydrogen peroxide | Fisher Scientific | H325 | |
Laser Scanning Confocal Microscope | Olympus | with FV-1000 system | |
Low melt agarose | Fisher | BP165-25 | |
Magnetic stand | Tritech (Narishige) | GJ-1 | |
Microinjection system | Parker | Picospritzer III | |
Microloader pipet tips | Eppendorf | 930001007 | |
Micromanipulator | Tritech (Narishige) | M-152 | |
Micropipette puller | Sutter Instrument Company | Flaming/Brown P-97 | |
Nanodrop spectrophotmeter | Thermo Scientific | ND-1000 | |
N-Phenylthiourea (PTU) | Sigma aldrich | P7629 | |
Paraformaldheyde | Electron Microscopy Sciences | 15710 | |
Petri Dishes | Fisher Scientific | FB0875712 | 100 mm x 15 mm |
Phenol | Sigma Aldrich | P-4557 | |
Phenol Red | Ricca Chemoical Company | 572516 | |
Phosphate Buffered Saline | Fisher Scientific | BP665-1 | |
Potassium hydroxide | Sigma Aldrich | P-6310 | |
Pronase | Roche | 165921 | |
Protease peptone | Fluka Biochemika | 29185 | |
Small cell culture dish | Corning Incorporated | 430165 | 35 mm x 10 mm |
Sudan Black | Sigma Aldrich | S2380 | |
Thin wall glass capillary injection needles | World Precision Instruments, Inc. | TW100-3 | |
Todd Hewitt | Sigma Aldrich/Fluka Analytical | T1438 | |
Tricaine (ethyl 3-aminobenzoate) | Argent Chemical Laboratory/Finquel | C-FINQ-UE-100G | |
Triton X-100 | Fisher Scientific | BP151-500 | |
Tween 20 | Fisher Scientific | BP337-500 | |
Yeast extract | Fluka Biochemika | 92144 |