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. 2014 Mar;80(5):1710–1717. doi: 10.1128/AEM.03580-13

Zebrafish as a Natural Host Model for Vibrio cholerae Colonization and Transmission

Donna L Runft 1, Kristie C Mitchell 1, Basel H Abuaita 1,*, Jonathan P Allen 1,*, Sarah Bajer 1, Kevin Ginsburg 1, Melody N Neely 1, Jeffrey H Withey 1,✉
PMCID: PMC3957598  PMID: 24375135

Abstract

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival.

INTRODUCTION

Vibrio cholerae, the cause of the severe human diarrheal disease cholera, is also a ubiquitous inhabitant of coastal regions around the globe. As is the case for all species within the Vibrio genus, V. cholerae is an aquatic bacterium that may be found both freely swimming and in association with various forms of aquatic flora and fauna (1–5). The environmental lifestyle and reservoirs of V. cholerae have only in recent years become the subject of vigorous research and remain poorly understood.

Over 200 V. cholerae serogroups have been identified from environmental sampling. However, only the O1 and O139 serogroups are capable of causing cholera. The O1 serogroup is further subdivided into two biotypes, classical and El Tor (6). Classical biotype V. cholerae is thought to have caused the first six of the seven known cholera pandemics beginning in 1817 and produces a more severe form of cholera. El Tor V. cholerae is responsible for the seventh pandemic, which began in 1961 and continues to the present day. El Tor strains are thought to be better suited for environmental survival, although the reasons for this are not clear. However, classical biotype strains are currently very difficult, if not impossible, to isolate from the environment, suggesting that El Tor strains have fully occupied the V. cholerae environmental niche. O139 serogroup strains, which caused large cholera outbreaks in the 1990s, have been shown to be derived from El Tor strains (7). In recent years some hybrid strains that closely resemble El Tor strains but also contain genetic material from classical strains have been isolated from cholera patients (8–10).

To become a human pathogen, V. cholerae must be ingested in contaminated water or seafood. After ingestion, V. cholerae senses numerous signals resulting in production of virulence factors that permit colonization of the human intestine and ultimately cause the diarrhea that will transmit V. cholerae back into the environment. The two major human virulence factors are cholera toxin (CT), which directly causes the characteristic secretory diarrhea in cholera patients (11, 12), and the toxin-coregulated pilus (TCP), which is required for intestinal colonization (13, 14). Virulence gene expression is controlled by a complex cascade of positive and negative transcription regulators (15). In addition to these major virulence factors, which are required for causing human cholera, other virulence factors are implicated in human noncholera diarrhea caused by V. cholerae (16–18). Unlike the two V. cholerae serogroups that cause cholera, a wide variety of serogroups can cause noncholera diarrhea in humans (19, 20).

Several mammalian animal models for V. cholerae colonization and pathogenesis are in current usage. The most common models used for the study of mammalian pathogenesis are the 3- to 5-day-old “infant mouse” model (21) and the adult rabbit ligated ileal loop and removable intestinal tie-adult rabbit diarrhea (RITARD) models (22–24). These models are useful for the study of V. cholerae virulence, but neither the mouse nor the rabbit is a natural host for V. cholerae. No pathogenesis is evident in infant mice, and the pathogenesis caused by V. cholerae in adult rabbits does not strongly resemble that of human cholera. The adult rabbit models also require survival surgery and significant manipulation of the animal. The recently rediscovered infant rabbit model (25) does produce a disease state somewhat similar to that of human cholera, but again the rabbit is not a natural host of V. cholerae and significant manipulation is required to produce colonization in the infant rabbit. The adult mouse has been used for V. cholerae studies, but the disease produced in adult mice does not resemble human cholera and is not dependent on the major virulence factors required to produce human cholera (26). The adult mouse is, however, a good model for studying V. cholerae accessory toxins (27).

Nonmammalian V. cholerae animal models are less widely used. One such model is the drosophila model. V. cholerae had previously been found to colonize insect egg masses, and recent work has determined that V. cholerae will also colonize the drosophila digestive tract and even kill the insect host (28). Therefore, drosophila may be a more natural model for environmental V. cholerae. The pathogenesis observed in drosophila is largely independent of the major virulence factors required for human cholera, indicating that other colonization factors and toxins may be involved in the environmental lifestyle of V. cholerae (29, 30). Given that most V. cholerae strains in the environment are not O1 or O139 serogroup pandemic strains, it follows that V. cholerae would have colonization factors for environmental niches not carried on the pathogenicity islands involved in human cholera.

An ideal natural model for V. cholerae would be an animal within which V. cholerae may be found in its natural habitat. Recent work published by Senderovich et al. found non-O1 V. cholerae colonizing the intestinal tracts of 10 different wild-caught fish species (3). This was the first evidence that V. cholerae may use vertebrate fish as a vector both for increasing bacterial population and potentially for transport over long distances. This study also suggested that V. cholerae may potentially be a commensal in fish.

In the current study, we investigated whether the well-described zebrafish, Danio rerio, could serve as a vertebrate fish model for V. cholerae. Zebrafish have a long and extremely successful history as model organisms for many biological processes ranging from development to bacterial pathogenesis (31, 32). Because the biology of zebrafish is so well understood, its potential as a model for V. cholerae opens many new pathways to understanding the V. cholerae environmental lifestyle. Furthermore, the natural habitat of zebrafish in Asia broadly overlaps areas of cholera endemicity, strongly suggesting that there is a natural association between zebrafish and V. cholerae in the wild (33). The zebrafish provides a natural course of infection model and thus should be an excellent method for studying the environmental lifestyle of V. cholerae, its requirements for intestinal colonization in both fish and humans, and transmission of the disease from infected to uninfected hosts.

MATERIALS AND METHODS

Ethics statement.

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal work was conducted according to the relevant guidelines of the Public Health Service, Office of Laboratory Animal Welfare, Animal Assurance no. A3310-01, and was approved by the Wayne State University IACUC, protocol number A 01-14-10.

Bacterial growth.

V. cholerae strains (Table 1) were grown either on LB medium prior to use in animal experiments or as described in the text. Intestinal homogenates were plated on LB agar containing 100 μg/ml streptomycin and 40 μg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside).

TABLE 1.

Bacterial strains used in this worka

V. cholerae strain Relevant genotype or description
O395 O1 serogroup, classical biotype; STRr
E7946 O1 serogroup, El Tor biotype; STRr
JW612 E7946 ΔtoxT
JW879 E7946 ΔtoxT; PtcpA-gfp
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a

Strains O395 and E7946 were from a laboratory collection. STR, streptomycin.

Zebrafish.

Six- to 9-month-old ZDR wild-type zebrafish were used for the experiments with adult zebrafish. For the larva infections, zebrafish at 5 days postfertilization (dpf) were used. Zebrafish were bred and maintained as previously reported (34). For anesthesia, zebrafish were placed in 100 ml of 168 μg/ml Tricaine (ethyl-3 aminobenzoate methanesulfonate salt; catalog no. A50040; Sigma) solution. For euthanasia of zebrafish, the dose of Tricaine was doubled, and fish remained in the solution for 25 to 30 min. All animal protocols were approved by the Wayne State University IACUC committee.

Oral gavage of zebrafish.

Zebrafish were first anesthetized by placing them in Tricaine solution. After the fish were sufficiently anesthetized (∼4 min), they were removed, rinsed in fresh water without anesthetic, and placed, dorsal side up, between the open jaws of a gauze-wrapped hemostat on a wedge of Styrofoam to position the head at the correct angle, creating a stage for inoculation (as described in reference 34). Zebrafish were inoculated using polyethylene tubing (PE-10; Braintree Scientific) attached to a 0.3-ml syringe with a 0.5-in., 29-gauge needle containing 20 μl of a washed bacterial suspension. The end of the tubing was gently inserted into the zebrafish esophagus, and inoculum was slowly added by depression of the plunger of the syringe. The zebrafish were then placed into a 400-ml beaker with a perforated lid containing 200 ml of tank water (sterilized double-distilled water [ddH2O] with 60 mg/liter of Instant Ocean aquarium salts [35]). Four to six zebrafish were added to each beaker and placed into a glass front incubator set at 27°C for the duration of the experiment.

Inoculation of zebrafish via water.

Bacterial cultures were washed once in phosphate-buffered saline (PBS) and diluted to the correct concentration using PBS before adding to the tank water. Bacterial concentrations ranged from 106 to 1010 CFU per beaker (∼4 × 103 to 4 × 107 CFU/ml), and inoculum was added to the tank water before the fish. Four to six zebrafish were then placed into a 400-ml beaker with a perforated lid containing 200 ml of tank water (sterilized ddH2O with 60 mg/liter of Instant Ocean aquarium salts [35]) and the bacterial inoculum. Each beaker was placed into a glass front incubator set at 27°C for the duration of the experiment.

Transmission experiments.

A group of 4 zebrafish, marked on the dorsal fin for identification, were exposed to a total of 109 to 1010 V. cholerae cells in 200 ml water as described above. After 3 h, the fish were moved to another beaker of freshwater two times to remove external V. cholerae organisms. The infected fish were then added to a larger beaker of 400 ml water containing 4 naive zebrafish. After 24 h, the fish were sacrificed and intestinal V. cholerae populations were enumerated as described below.

Determination of V. cholerae colonization of intestine.

At designated time points, fish were removed from the beaker and euthanized as described above. Intestines were aseptically removed, placed into 300 μl of sterile PBS, and homogenized using a micro-tissue grinder (Kontes Pellet Pestle motorized tissue grinder; Fisher). Serial dilutions of the homogenate were made and plated onto selective media for enumeration.

Experiments using zebrafish larvae.

Five-day-postfertilization zebrafish larvae were placed into 1 ml tank water containing 1 × 106 CFU/ml V. cholerae in a 12-well plate and incubated for 2 to 24 h at 27°C. The V. cholerae strain (JW879) was carrying a plasmid that expressed green fluorescent protein (GFP) from the tcpA promoter. At the designated time points, larvae were removed from the well with the bacteria and washed in sterile tank water twice and then placed into a well with a euthanizing dose of Tricaine solution. Larvae were then mounted on a microscope slide in Tricaine inside a 1-mm-thick washer glued to the slide. A coverslip was placed on top of the washer, and the larvae were viewed with a Zeiss Axioskop 40 Fluorescence microscope at a magnification of ×100. In some instances, paramecia were also added to the well with the V. cholerae to facilitate uptake of the bacteria.

Histology of V. cholerae-infected zebrafish intestines.

At 24 h postinfection, adult zebrafish were removed from tank water and euthanized. An incision was made using a scalpel along the ventral line of each fish, and then it was placed in Dietrich's fixative for 24 to 48 h. Next, the zebrafish were placed in tissue cassettes and dehydrated through a series of graded ethanol. Following a final 1-h wash in 100% ethanol, the fish were incubated in toluene for 1 h and placed in Clearify (American MasterTech Scientific Inc.) for 12 to 18 h. The fish were then incubated in a bottle of molten paraffin heated in a 60°C water bath for at least 1 h, the paraffin was changed, and the fish were incubated for another 12 to 18 h in the same water bath. Finally, the fish were embedded in 60°C paraffin and placed on ice to cool until the paraffin was solidified. The paraffin blocks were cut at 3 μm, placed on Superfrost Plus Gold microscope slides (Fisher Scientific), and dried in a 55°C oven for at least 24 h before staining. Sections were stained with anti-V. cholerae polyclonal antibody (KPL BacTrace) and counterstained with a secondary antibody conjugated to Alexa Fluor 568 (A11011; Molecular Probes). Stained sections were viewed with a Zeiss Axioskop 40 Fluorescence microscope at a magnification of ×1,000.

RESULTS

Exposure of zebrafish to V. cholerae results in robust intestinal colonization.

We began our investigation of zebrafish as a V. cholerae host model by inoculating individual fish with 106 CFU via oral gavage, followed by enumeration of V. cholerae in the intestinal tract 24 h postinfection. This is the method used in the infant mouse model, and gavage has the advantage of controlling the number of bacteria in the inoculum. V. cholerae was specifically selected by plating intestinal homogenates on medium containing streptomycin, as all strains used in these experiments are streptomycin resistant. Unlike experiments performed with infant mice, which have little or no intestinal microbiota, zebrafish have an intact intestinal microbiota, so selection for V. cholerae is essential. To further distinguish V. cholerae from other intestinal bacteria that are naturally streptomycin resistant, X-Gal was also added to the plates, as V. cholerae will form blue colonies but the other intestinal bacteria will not. The results of these experiments indicated that V. cholerae does indeed robustly colonize the zebrafish intestinal tract (data not shown). Fish infected by gavage typically had upwards of 105 V. cholerae organisms colonizing their intestinal tract after 24 h. However, the anatomy of the zebrafish esophagus presented a problem with gavage that affected reproducibility, and many fish did not become colonized due to regurgitation of the inoculum. Additionally, the goal of this work was to explore a natural host model, so manipulating the fish with anesthesia and gavage was undesirable. The gavage experiments were, however, successful in determining that V. cholerae can colonize the zebrafish intestinal tract in large numbers.

Our next effort was to simulate a more natural infectious route by simply adding V. cholerae to a beaker of 200 ml water containing several zebrafish. After 24 h of exposure to V. cholerae, the fish were sacrificed and tested for intestinal colonization. Various infectious doses, ranging from 106 to 1010 bacteria per beaker, were tested (data not shown); the lowest infectious dose that achieved consistent colonization levels was 108 V. cholerae bacteria per beaker, i.e., 5 × 105 V. cholerae cells per milliliter of water. Exposure of zebrafish to this dosage of V. cholerae via water resulted in large numbers of V. cholerae in the intestinal tract 24 h postinfection (Fig. 1A); approximately 104 V. cholerae cells per fish intestine was the typical observation, although there was a several-log range observed in different fish. Increasing the infectious dose to 1010 V. cholerae cells, i.e., 5 × 107 cells per milliliter, resulted in a tighter range of colonization among individual fish, with most fish having between 104 and 106 V. cholerae cells colonizing their intestinal tracts. Both classical and El Tor biotype O1 pandemic strains were able to colonize zebrafish intestinal tracts, although the El Tor biotype, on average, exhibited a slightly higher bacterial load (Fig. 1). V. cholerae was not detected in significant numbers in the nares, gills, scales, fins, spleen, or heart (data not shown). These results suggest that the intestine is specifically targeted and is the only site of colonization for V. cholerae in zebrafish. Furthermore, colonization of zebrafish intestine by V. cholerae does not result in invasive infection. This is very similar to what occurs in humans and mammalian animal models for V. cholerae.

FIG 1.

FIG 1

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V. cholerae colonization of zebrafish intestines after exposure in water. Four or five fish were added to 200 ml water containing 108 (A) or 1010 (B) V. cholerae cells. Data shown are compiled from multiple experiments. Each dot represents the data from one fish. Total colonization per intestine was calculated after plating serial dilutions of intestinal homogenates 24 h postinfection. Strain E7946 is an O1 serogroup El Tor biotype V. cholerae strain and O395 is an O1 serogroup classical biotype V. cholerae strain. Statistical significance indicated above the data was determined by Student's t test.

Given the high numbers of V. cholerae colonizing the intestinal tract after 24 h, we next explored earlier time points to determine how quickly colonization occurred. As shown in Fig. 2, we assessed colonization in zebrafish exposed to either V. cholerae classical strain O395 (∼1010 CFU per 200 ml) or V. cholerae El Tor strain E7946 (∼109 CFU per 200 ml) at 2 h, 6 h, and 24 h postexposure. Both biotypes were highly colonized as early as 2 h postexposure, indicating that V. cholerae enters the zebrafish intestine in high numbers over a very short time frame. Numbers for both biotypes were quite consistent between fish at 2 h and 6 h postexposure, with greater variability observed at 24 h postexposure.

FIG 2.

FIG 2

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V. cholerae colonization of zebrafish at earlier time points. Zebrafish were exposed to either 3 × 1010 V. cholerae classical strain O395 (A) or 3 × 109 V. cholerae El Tor strain E7946 (B) cells. At the indicated time points, fish were sacrificed and intestinal V. cholerae levels were determined by plating of serial dilutions of the intestinal homogenates.

Histological examination of colonized zebrafish intestinal tracts revealed clumps or microcolonies of V. cholerae in close contact with the epithelial surface. As shown in Fig. 3, individual V. cholerae curved bacilli were visible at the epithelial surface at the 24 h time point after exposure of zebrafish to V. cholerae biotype El Tor in water. V. cholerae cells were visualized in sections of fixed zebrafish by fluorescence microscopy using a primary polyclonal antibody directed against V. cholerae and a secondary monoclonal antibody carrying the fluorescent tag. The contact between V. cholerae and the intestinal epithelial surface observed in zebrafish very closely resembles the interaction between V. cholerae and the epithelial surface observed in mammalian models (36, 37).

FIG 3.

FIG 3

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Fluorescence micrographs of V. cholerae colonizing the zebrafish intestinal epithelium. Fish were exposed to V. cholerae for 24 h in water and then sacrificed, fixed, and prepared for sectioning. Bacteria were visualized using a polyclonal primary antibody against V. cholerae and a secondary antibody carrying a fluorescent tag. (A) Uninfected fish; (B, C, D) infected fish. Magnification, ×1,000.

V. cholerae biotype El Tor has a colonization advantage in zebrafish.

V. cholerae biotype El Tor has apparently completely replaced the classical biotype in the environment and as an agent of human cholera. The two biotypes have numerous differences, including changes in virulence regulation, metabolism, sensitivity to antibiotics, and possession of accessory toxins. Differences in fish colonization could provide one potential explanation for the takeover by El Tor in the environment. To examine this possibility, we compared the ability of classical and El Tor biotypes to colonize zebrafish. While both biotypes robustly colonize zebrafish, we consistently observed somewhat higher bacterial loads in zebrafish infected with V. cholerae biotype El Tor, although there was variation from fish to fish (Fig. 1). Because we observed differences between levels of El Tor and classical biotype colonization of the zebrafish intestine at the 24 h time point, the question arose as to whether these differences would be maintained for a long time or would vary. To answer this question, colonization levels at the 24, 48, and 72 h time points were compared (Fig. 4). The results of these experiments indicate a clear difference between the classical and El Tor biotypes. V. cholerae classical biotype was cleared from zebrafish intestinal tracts by 72 h postexposure. However, V. cholerae biotype El Tor was retained in the zebrafish intestinal tracts at high levels even 6 days postexposure. This result suggests that the El Tor biotype has acquired genes that allow it to colonize fish for a prolonged period. This observation is consistent with the hypothesis that increased success of V. cholerae El Tor within the fish reservoir potentially abetted the disappearance of classical V. cholerae from worldwide environmental niches.

FIG 4.

FIG 4

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Time course of colonization by V. cholerae classical and El Tor biotypes after exposure in water. Four or five fish were added to 200 ml water containing 108 V. cholerae cells. Each dot represents the data from one fish, and the horizontal bar indicates the mean bacterial load per fish. Total colonization per intestine was calculated after plating serial dilutions of intestinal homogenates 24 h, 48 h, 72 h, or 144 h postinfection. (A) Results from classical biotype strain O395 infection. (B) Results from El Tor biotype strain E7946 infection.

Zebrafish colonized by V. cholerae transmit the bacteria to naive fish.

As described above, zebrafish are rapidly colonized by V. cholerae after exposure in water. The colonized zebrafish also exhibit signs of pathogenesis, primarily diarrhea, which leads to fouling of the water by infected fish. A likely function in the environment for this V. cholerae-induced diarrhea would be to enhance escape of newly replicated bacteria back into the aquatic niche. This could also potentially enable colonization of other fish that are near the infected fish, leading to rapid population growth of V. cholerae within a school of fish.

To test the hypothesis that infected fish could transmit the infection to naive fish, we exposed groups of zebrafish to V. cholerae for 2 h as described above. Two fish were sacrificed at this point to assess their intestinal colonization levels, and we typically saw between 104 and 105 V. cholerae cells per fish. The remaining infected fish were marked by fin clipping to distinguish them from uninfected fish. The infected, clipped fish were twice washed in beakers of clean water to remove external V. cholerae and then added to another, larger beaker of clean water containing a group of naive zebrafish. The fish were kept together for 24 h and then euthanized, and intestinal colonization by V. cholerae was assessed. The results indicate that every previously uninfected fish became colonized by V. cholerae after 24 h of exposure to infected fish (Fig. 5).

FIG 5.

FIG 5

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Transmission of V. cholerae from infected fish to naive fish. Four or five “donor” fish were exposed to V. cholerae in water for 3 h and then washed twice and placed in a fresh beaker with naive “recipient” fish for 24 h. Data shown were collected from plating serial dilutions of intestinal homogenates 24 h after exposure of the naive fish to the infected fish. Strains used: classical, O395; El Tor, E7946.

The major human V. cholerae virulence factors are not required for zebrafish colonization.

Intestinal colonization in humans and most mammalian animal models requires production of TCP. Although TCP is not directly involved in adherence of V. cholerae to the epithelial surface (14), it has been hypothesized that microcolony formation mediated by TCP is crucial for effective colonization (13, 38, 39). We investigated whether TCP or virulence factors that are coregulated with TCP are essential for zebrafish colonization by using V. cholerae deficient for toxT, the major virulence transcription activator (15), to infect zebrafish. ΔtoxT V. cholerae does not produce TCP, CT, accessory colonization factors, or several other coregulated gene products (40–43). Our results indicate that ΔtoxT V. cholerae colonizes zebrafish as well as wild-type toxT V. cholerae (Fig. 6). Our finding is consistent with the previous observation that non-O1 strains, which do not carry the Vibrio pathogenicity island (VPI) genes required for TCP production, colonize wild fish species (3). Our finding is also consistent with the fact that the vast majority of V. cholerae strains present in the environment do not possess CTXΦ, which carries the genes encoding CT (44), or the VPI, which carries the genes encoding TCP, other ToxT-regulated genes, and toxT itself (45).

FIG 6.

FIG 6

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Effect of toxT deletion on zebrafish colonization by El Tor V. cholerae. Each dot represents the data from one fish, and the horizontal bar indicates the mean bacterial load per fish. Total colonization per intestine was calculated after plating serial dilutions of intestinal homogenate 24 h postinfection. Strains used were the El Tor strain E7946 and a derivative of E7946 having a complete in-frame toxT deletion.

Zebrafish larvae are colonized by V. cholerae.

All the experiments described above were performed using mature adult zebrafish. Next, we investigated whether we could observe the uptake of V. cholerae into the digestive tract of zebrafish larvae. By taking advantage of the transparency of zebrafish larvae and using V. cholerae expressing GFP, we could potentially observe active uptake and colonization of the zebrafish.

Our results indicate that zebrafish larvae are rapidly colonized by V. cholerae. GFP-producing V. cholerae cells were clearly evident in the digestive tract at 2 h postexposure in water (Fig. 7). Figure 7C shows fluorescent V. cholerae cells just past the mouth and also beginning to colonize the intestine. At 24 h postexposure, abundant fluorescence in the intestinal tract is visible in larvae exposed to GFP-producing V. cholerae, whereas no fluorescence is visible in unexposed larvae. These results indicate that V. cholerae enters the zebrafish larva's digestive tract simply by exposure through water, leading to rapid and robust intestinal colonization.

FIG 7.

FIG 7

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Colonization of zebrafish larvae by V. cholerae. Larvae were exposed to V. cholerae for the indicated time and then fixed for microscopy. GFP-producing V. cholerae cells were visualized by fluorescence microscopy, overlaid on light micrographs of the zebrafish larvae. (A) Uninfected larva; (B) infected larva 24 h after exposure; (C) infected larva 2 h after exposure; (D) ventral view of infected larva 2 h after exposure.

DISCUSSION

Here we describe use of the zebrafish as a novel animal model for the study of the human pathogen V. cholerae. This work establishes both a fish model for V. cholerae and a natural host model for V. cholerae. The use of a natural host and natural route of infection should provide new opportunities to determine factors required for intestinal colonization, pathogenesis, and transmission that cannot be realized using current mammalian animal models.

Zebrafish have numerous advantages over existing V. cholerae animal models. This new model requires no manipulation of the animal host, whereas mammalian animal models require substantial manipulation for V. cholerae colonization to occur. The infant mouse model, which is probably the most frequently used animal model for V. cholerae, requires oral gavage to establish intestinal colonization, and an infected mouse does not exhibit diarrhea despite the production of CT in the intestinal tract (21). No signs of pathogenesis in infant mice are produced unless inocula greater than 108 bacteria are used, in which case the cause of death is still not dehydration. Infant mice also do not have a significant microbiota. The main advantage of the infant mouse model is that TCP production is required for colonization, as has been observed in humans. However, the absolute requirement for TCP makes identification of other potential virulence factors, such as the still-unknown factors that allow V. cholerae to adhere directly to the epithelial surface, difficult. The rabbit ligated ileal loop model, which is better than the mouse model at assessing CT production, requires survival surgery, is expensive, and is difficult to perform without substantial training. The rabbit RITARD model, while producing an infection that is closer to the cholera disease state than other models, has similar limitations (23, 24). The infant rabbit model, which produces a state that is the most similar to the human disease, requires pretreatment of the animal with antibiotics to eliminate microbiota, anesthesia, and administration of the inoculum with buffers by oral gavage and is also expensive (25, 46). The adult mouse model has been very useful for studying accessory toxins but does not produce a disease state like that of cholera and has the usual limitations of artificial host models (26, 27, 47).

Zebrafish colonization of the intestine occurs via a natural process and in the presence of the normal fish microbiota. This should permit future study related to the interplay between commensals and V. cholerae that is not possible using mammalian animal models. Recent research that examined the natural microbiota of zebrafish found that the Vibrio genus was highly represented, although which Vibrio species were present was not determined (48). The prolonged colonization that we observed with V. cholerae biotype El Tor suggests that V. cholerae may even be a zebrafish commensal. Future work will determine whether this is indeed the case. Adult zebrafish also have a fully functioning immune system, with both innate and adaptive arms similar to those of humans. This strong similarity between components of immune system between zebrafish and humans should facilitate extensive studies on the immune response to V. cholerae. Future experiments using zebrafish mutant strains with defects in immune response should help us to better understand both innate and acquired immunity to V. cholerae that will likely parallel the response in the human gut, which has been difficult to study. The observation that zebrafish larvae are also colonized should facilitate future studies on colonization during development of the adaptive immune response.

The fact that infected zebrafish can transmit V. cholerae to naive fish provides an opportunity to study V. cholerae transmission in great detail. Currently, natural transmission is essentially impossible to study in mammalian models, as all of them require either gavage or survival surgery to administer the bacteria. Therefore, the zebrafish is likely to provide much new information on genetic factors important for V. cholerae transmission in future studies.

Our observation that V. cholerae El Tor colonizes zebrafish for a longer time than classical V. cholerae may help to explain how El Tor strains have completely replaced classical strains as the cause of human cholera worldwide. Classical strains have become extremely rare in the environment and may actually be extinct, although there is evidence that the CT-encoding CTXΦ derived from classical biotype strains remains (9, 49–52). In recent years, so-called hybrid biotype V. cholerae has become a significant cause of human cholera. However, the only significant difference between El Tor strains and the hybrid strains is within the CTXΦ genome (51), suggesting that El Tor strains have simply undergone a recombination event with a classical CTXΦ genome. If fish are an important reservoir and/or vector for increasing the V. cholerae population, as we believe, then even a small increase in fitness gained by El Tor strains could translate to a huge population advantage over time in the environment. This could lead to complete filling of the environmental niches by V. cholerae El Tor and subsequent extinction of classical V. cholerae.

In summary, here we describe the use of zebrafish as a novel animal model for the study of V. cholerae colonization and transmission. This new model provides many advantages over existing animal models for V. cholerae and should facilitate many new avenues of research on both the environmental lifestyle of V. cholerae and its pathogenesis in fish and humans.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant R21AI095520 from the National Institute of Allergy and Infectious Diseases.

We thank members of the Neely and Withey labs for helpful discussions.

Footnotes

Published ahead of print 27 December 2013

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