Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.
Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.
Vibrio cholerae is an aquatic, Gram-negative bacterium that causes the human disease cholera as well as sporadic diarrhea1,2. V. cholerae is found in the environment in many areas of the globe, often associated with other aquatic organisms. These associating organisms include plankton, insect egg masses, shellfish, and vertebrate fish species3,4,5,6,7. Several studies have isolated V. cholerae from the intestinal tracts of fish in different geographical areas7,8,9,10. The presence of V. cholerae in fish indicates that fish may act as an environmental reservoir. Fish could also be implicated in transmitting the disease to humans and in the geographic spreading of V. cholerae strains6.
To better understand how V. cholerae interacts with fish, Danio rerio, better known as zebrafish, was developed as a model system for studying V. cholerae11. Zebrafish are native to southern Asia, including the Bay of Bengal region, which is thought to be the earliest reservoir of V. cholerae. Prior to the first cholera pandemic beginning in 1817, cholera had not been reported outside of what is now India and Bangladesh. Therefore, zebrafish and V. cholerae almost certainly associated with each other over evolutionary time scales, suggesting that zebrafish are a V. cholerae host in the natural environment12.
The zebrafish model for V. cholerae is simple to execute and can be used to study the entire pathogenic V. cholerae life cycle. Fish are exposed to V. cholerae by bathing in water that has been inoculated with a known number of V. cholerae. Within a few hours, intestinal colonization takes place, followed by the production of diarrhea. Diarrhea consists of mucin, proteins, excreted bacteria, and other intestinal contents. The degree of diarrhea can be quantified using a few simple measurements13. V. cholerae that has been excreted by infected fish can then go on to infect naïve fish, completing the infectious cycle. Therefore, the zebrafish model recapitulates the V. cholerae human disease process12,14.
The most frequently used V. cholerae animal models have historically been mice and rabbits14,15,16,17,18. These models have been instrumental in adding to our knowledge of V. cholerae pathogenesis. However, because mice and rabbits are not natural V. cholerae hosts, there are limitations to what aspects of the V. cholerae life cycle can be studied. The V. cholerae colonization of mice and rabbits typically requires the absence of intestinal microbiota or a pretreatment with antibiotics to damage the intestinal microbiota. Both models require either gavage to introduce the bacteria to the digestive tract or surgical manipulation to directly inject the bacteria into the intestines. Zebrafish have an advantage in that adult fish with an intact intestinal microbiota are readily colonized and the infectious process happens naturally, without any manipulation required.
The present work demonstrates the utilization of zebrafish as a model in V. cholerae infection. The infection, dissection, enumeration of colonizing V. cholerae, and the quantification of diarrhea caused by V. cholerae will be described12,13. This model is likely to be useful to scientists interested both in the V. cholerae disease process and in the V. cholerae environmental lifestyle.
All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Wayne State University. This method was first described in Runft et al.12
1. Determination of Intestinal Colonization Levels
NOTE: Intestinal colonization is the most useful metric in the zebrafish model as it can be used to compare the relative fitness of various V. cholerae strains or the effects of mutations or gene knockouts.
2. Measurement of Fish Diarrhea
NOTE: Four different metrics were used to assess fish diarrhea: the mucin levels in the water, the overall excreted protein levels in the water, the OD600 of the water, and the V. cholerae CFU in the water13. Diarrhea normally becomes visually evident approximately 6 h after the exposure of the zebrafish to V. cholerae as described above.
V. cholerae colonization of zebrafish intestinal tracts
To provide an example of the typical colonization levels we observe, we inoculated 5 x 106 CFU of the pandemic EL Tor V. cholerae strain N16961 in 200 mL of water in a beaker containing several zebrafish. After 6 h of infection, the fish were washed in fresh water and transferred into a beaker of 200 mL of autoclaved infection water as described in the protocol. 18 h after the transfer (or 24 h after the primary infection), the fish were euthanized, and their intestines were taken to determine the colonization levels. Approximately 105 to 106 V. cholerae cells per fish intestine were typically observed in the intestinal tract 24 h post-infection (hpi) (Figure 1) with the 5 x 106 inoculum size.
Quantification of fish diarrhea
Diarrhea was quantified using the four simple assays described above. The first assay, the CFU of excreted V. cholerae, is shown in Figure 1. Serial dilutions of the water 24 hpi were plated to count the V. cholerae CFU. Relatively high levels of excreted V. cholerae are usually observed; in this example, approximately 105 V. cholerae per mL of water were detected. Uninfected fish do not produce detectible V. cholerae (data not shown).
The second assay used to quantify diarrhea is excreted mucin. Figure 2 shows the effects of three different V. cholerae infectious doses on the mucin levels in water 24 hpi. A higher infectious dose is correlated with a higher excretion of mucin into the water. As a control, four fish were placed in an identical beaker of water, but PBS was added instead of the V. cholerae suspension. The control fish excreted very little mucin.
The third diarrheal quantification assay is the OD600 of the water 24 hpi. As shown in Figure 3, the OD600 of this water was significantly higher in the beaker containing zebrafish infected with V. cholerae than in the beaker containing uninfected zebrafish.
Finally, the total protein levels were measured in the water. Water containing the infected fish contained total protein levels nearly twice that observed in the water containing the uninfected fish (Figure 4). Collectively, these four assays illustrate the effects V. cholerae infection has on zebrafish excretion.
Figure 1: Intestinal colonization of zebrafish by V. cholerae. The fish were infected for 6 h, then washed and incubated for a total of 24 h. The left side of the figure illustrates the total CFU per intestine of four fish (black dots.) The right side of the figure indicates the V. cholerae CFU per mL measured in water 24 hpi (black squares.) The means of both data sets are shown with a large horizontal line and the standard deviation are indicated by error bars. Please click here to view a larger version of this figure.
Figure 2: Mucin assay. Three different V. cholerae infectious doses were used, along with an uninfected control, as indicated under the x-axis. The mean values of the mucin detected in the water by the modified Periodic acid Schiff (PAS) assay are indicated by the black bars above each infectious dose. The error bars indicate the standard deviation. The asterisks indicate p <0.05 as determined by Student's unpaired t-test. Please click here to view a larger version of this figure.
Figure 3: OD600 assay as a proxy for diarrhea. The OD600 of 1 mL of water from the beakers containing either V. cholerae infected or uninfected fish was measured. The black bars indicate the mean value and the error bars indicate the standard deviation. The asterisks indicate p <0.05 as determined by Student's unpaired t-test. Please click here to view a larger version of this figure.
Figure 4: Total protein levels in water. The total protein was estimated by a Bradford assay as described. The black bars indicate the mean values and the error bars indicate the standard deviation. The asterisks indicate p <0.05 as determined by Student's unpaired t-test. Please click here to view a larger version of this figure.
The zebrafish is a relatively new model for studying V. cholerae but holds much promise for the future discovery of previously unknown aspects of V. cholerae biology and pathogenesis11,12,13. The adult zebrafish model has the advantages of being both a natural V. cholerae host that contains intact, mature intestinal microbiota and an environmental model. Disadvantages of the model are that the two major human virulence factors, cholera toxin and toxin-coregulated pilus, are not required for zebrafish colonization or pathogenesis12. However, this could alternatively be viewed as another advantage that could enable the identification of novel V. cholerae colonization factors and facilitate the study of other V. cholerae toxins. This is especially relevant to the vast majority of non-O1/O139 "environmental" V. cholerae strains, most of which do not produce cholera toxin or toxin-coregulated pilus and yet can still cause disease20,21.
The problems most likely to arise using this model are related to the abundant intestinal microbiota. Plating on nonselective media will make this model essentially unusable as it will be impossible to identify the V. cholerae colonies. Even using selective or partially selective media such as LB plus streptomycin or DCLS, a background of colonies from intestinal microbiota will be present. It is essential to verify that the colonies that are being counted are bona fide V. cholerae by patching the colonies produced by the plating on less selective media onto a very selective medium such as TCBS. Alternatively, the insertion of a better selective marker into the genome of a strain of interest would greatly simplify the identification of V. cholerae from intestinal homogenates.
The advantage of the assays used to quantify zebrafish diarrhea is that they are simple and inexpensive to execute13. The disadvantage is that these assays are largely nonspecific, aside from counting excreted V. cholerae CFU directly, and it can become difficult to determine if the diarrhea is directly due to V. cholerae pathogenesis, or to some other stressor. The development of variations of these or other assays to improve their specificity will likely aid in future measurements of V. cholerae pathogenesis in zebrafish.
The authors have nothing to disclose.
Thanks to Melody Neely, Jon Allen, Basel Abuaita, and Donna Runft for their efforts in helping to develop the zebrafish model. The research reported here was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R21AI095520 and R01AI127390 (to Jeffrey H. Withey). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Instrument | |||
Shaker incubator | New Brunswick Scientific, Edison, NJ | Excella E25 | |
Incubator | NUAIRE, Plymouth, MN | Auto Flow | |
Spectrophotometer | Thermo, Waltham, MA | Geaesys 6 | |
Vortex homogenizer | Minibeadbeater24 | 112011 | |
Weighing Machine | Ohaus, Columbia, MD | Adventurer Pro | |
Heat Stirer | Corning, Corning, NY | PC-420D | |
Burner | |||
automated colony counter | REVSCI | 120417B | |
Materials | |||
400 ml glass beakers | Pyrex | ||
perforated lids | Microtip holder with holes from tip box | ||
disposable plastic spoons | Office Depot, Boca Raton, FL | D15-25-7008 | |
Fish Tank System | Aquaneering, San Diego, CA | ||
RO Water Purifier | Aqua FX | TK001 | |
Fish net | Marina | ||
fish food | Tetra fin | ||
Brine Shrimp | Red jungle brand | O.S.I. pro 80 | |
Styrofoam board | |||
Pins | |||
Scalpels | Fine Scientific tools, Foster City, CA | 10000-10 | |
Forceps | Fine Scientific tools, Foster City, CA | 11223-20 | |
Vannas scissors | Fine Scientific tools, Foster City, CA | 15000-11 | |
2 ml screw cap tubes | Fisher Scientific, Hampton, NH | 02-681-375 | |
1 mm glass beads | Bio Spec | 11079110 | |
Glass beads for spreading | Sigma, St. Louis, MO | 18406-500G | |
Petri plate | Fisher Brand, Hampton, NH | FB0875713 | |
1.5 ml centrifuge tube | Midsci, Valley Park, MO | AVSS1700 | |
50 ml centrifuge tube | Corning Falcon, Corning, NY | 352098 | |
Test tubes | Pyrex | 9820 | |
Glass Pipette | Fisher Brand, Hampton, NH | 13675K | |
Micro pipettes | Sartorius Biohit, Göttingen, Germany | m1000/m200/m20 | |
Tips | Genesee Scientific, San Diego, CA | 24-150RS/24-412 | |
Chemicals | |||
Instant Ocean salts | |||
phosphate buffered saline | VWR Life Science, Radnor, PA | K813-500ml | |
Tricaine (ethyl 3-aminobenzoate methanesulfonate salt | Sigma, St. Louis, MO | A5040 | |
5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside | Sigma, St. Louis, MO | 10651745001 | |
Schiff’s reagent | Sigma, St. Louis, MO | 84655-250 mL | |
periodic acid | Fisher Scientific, Hampton, NH | 10450-60-9 | |
Mucin from porcine stomach | Sigma, St. Louis, MO | M2378-100G | |
Bovine serum albumin | Fisher Scientific, Hampton, NH | 9046-46-8 | |
Pierce 660nm Protein Assay Reagent | Thermo, Waltham, MA | 22660 | |
LB medium | |||
Trypton | BD Biosciences, San Jose, CA | 211705 | |
Teast Extract | BD Biosciences, San Jose, CA | 212750 | |
NACL | Fisher Scientific, Hampton, NH | BP358-212 | |
Agar | BD Biosciences, San Jose, CA | 214010 | |
TCBS Agar | BD Biosciences, San Jose, CA | 265020 | |
DCLS Agar | Sigma, St. Louis, MO | 70135-500gm | |
Software | |||
Microsoft office | |||
Prism 5 |