Published Ahead of Print 27 December 2013. 10.1128/AEM.03580-13. 2014, 80(5):1710. DOI: Appl. Environ. Microbiol. Neely and Jeffrey H. Withey Jonathan P. Allen, Sarah Bajer, Kevin Ginsburg, Melody N. Donna L. Runft, Kristie C. Mitchell, Basel H. Abuaita, cholerae Colonization and Transmission Zebrafish as a Natural Host Model for Vibrio http://aem.asm.org/content/80/5/1710 Updated information and services can be found at: These include: REFERENCES http://aem.asm.org/content/80/5/1710#ref-list-1 at: This article cites 52 articles, 23 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on July 22, 2014 by WAYNE STATE UNIVERSITY http://aem.asm.org/ Downloaded from on July 22, 2014 by WAYNE STATE UNIVERSITY http://aem.asm.org/ Downloaded from
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Published Ahead of Print 27 December 2013. 10.1128/AEM.03580-13.
2014, 80(5):1710. DOI:Appl. Environ. Microbiol. Neely and Jeffrey H. WitheyJonathan P. Allen, Sarah Bajer, Kevin Ginsburg, Melody N. Donna L. Runft, Kristie C. Mitchell, Basel H. Abuaita, cholerae Colonization and TransmissionZebrafish as a Natural Host Model for Vibrio
http://aem.asm.org/content/80/5/1710Updated information and services can be found at:
This article cites 52 articles, 23 of which can be accessed free
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Receive: RSS Feeds, eTOCs, free email alerts (when new
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Zebrafish as a Natural Host Model for Vibrio cholerae Colonizationand Transmission
Donna L. Runft, Kristie C. Mitchell, Basel H. Abuaita,* Jonathan P. Allen,* Sarah Bajer, Kevin Ginsburg, Melody N. Neely,Jeffrey H. Withey
Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan, USA
The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is asso-ciated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novelanimal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract afterexposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithe-lium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naivefish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes wereapparent. 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. choleraecolonization, transmission, and environmental survival.
Vibrio cholerae, the cause of the severe human diarrheal diseasecholera, is also a ubiquitous inhabitant of coastal regions
around the globe. As is the case for all species within the Vibriogenus, V. cholerae is an aquatic bacterium that may be found bothfreely swimming and in association with various forms of aquaticflora and fauna (1–5). The environmental lifestyle and reservoirsof V. cholerae have only in recent years become the subject ofvigorous research and remain poorly understood.
Over 200 V. cholerae serogroups have been identified from en-vironmental sampling. However, only the O1 and O139 sero-groups are capable of causing cholera. The O1 serogroup is furthersubdivided into two biotypes, classical and El Tor (6). Classicalbiotype V. cholerae is thought to have caused the first six of theseven known cholera pandemics beginning in 1817 and producesa more severe form of cholera. El Tor V. cholerae is responsible forthe seventh pandemic, which began in 1961 and continues to thepresent day. El Tor strains are thought to be better suited forenvironmental survival, although the reasons for this are not clear.However, classical biotype strains are currently very difficult, ifnot impossible, to isolate from the environment, suggesting thatEl Tor strains have fully occupied the V. cholerae environmentalniche. O139 serogroup strains, which caused large cholera out-breaks in the 1990s, have been shown to be derived from El Torstrains (7). In recent years some hybrid strains that closely resem-ble El Tor strains but also contain genetic material from classicalstrains have been isolated from cholera patients (8–10).
To become a human pathogen, V. cholerae must be ingested incontaminated water or seafood. After ingestion, V. cholerae sensesnumerous signals resulting in production of virulence factors thatpermit colonization of the human intestine and ultimately causethe diarrhea that will transmit V. cholerae back into the environ-ment. The two major human virulence factors are cholera toxin(CT), which directly causes the characteristic secretory diarrhea incholera patients (11, 12), and the toxin-coregulated pilus (TCP),which is required for intestinal colonization (13, 14). Virulencegene expression is controlled by a complex cascade of positive andnegative transcription regulators (15). In addition to these majorvirulence factors, which are required for causing human cholera,
other virulence factors are implicated in human noncholera diar-rhea caused by V. cholerae (16–18). Unlike the two V. choleraeserogroups that cause cholera, a wide variety of serogroups cancause noncholera diarrhea in humans (19, 20).
Several mammalian animal models for V. cholerae colonizationand pathogenesis are in current usage. The most common modelsused for the study of mammalian pathogenesis are the 3- to 5-day-old “infant mouse” model (21) and the adult rabbit ligated ilealloop and removable intestinal tie-adult rabbit diarrhea (RITARD)models (22–24). These models are useful for the study of V. chol-erae virulence, but neither the mouse nor the rabbit is a naturalhost for V. cholerae. No pathogenesis is evident in infant mice, andthe pathogenesis caused by V. cholerae in adult rabbits does notstrongly resemble that of human cholera. The adult rabbit modelsalso require survival surgery and significant manipulation of theanimal. The recently rediscovered infant rabbit model (25) doesproduce a disease state somewhat similar to that of human chol-era, but again the rabbit is not a natural host of V. cholerae andsignificant manipulation is required to produce colonization inthe infant rabbit. The adult mouse has been used for V. choleraestudies, but the disease produced in adult mice does not resemblehuman cholera and is not dependent on the major virulence fac-tors 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
Received 31 October 2013 Accepted 20 December 2013
* Present address: Basel H. Abuaita, Department of Microbiology andImmunology, University of Michigan Medical School, Ann Arbor, Michigan, USA;Jonathan P. Allen, Department of Microbiology-Immunology, NorthwesternUniversity Feinberg School of Medicine, Chicago, Illinois, USA.
used. One such model is the drosophila model. V. cholerae hadpreviously been found to colonize insect egg masses, and recentwork has determined that V. cholerae will also colonize the dro-sophila 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 inde-pendent of the major virulence factors required for human chol-era, indicating that other colonization factors and toxins may beinvolved in the environmental lifestyle of V. cholerae (29, 30).Given that most V. cholerae strains in the environment are not O1or O139 serogroup pandemic strains, it follows that V. choleraewould have colonization factors for environmental niches not car-ried on the pathogenicity islands involved in human cholera.
An ideal natural model for V. cholerae would be an animalwithin which V. cholerae may be found in its natural habitat. Re-cent work published by Senderovich et al. found non-O1 V. chol-erae colonizing the intestinal tracts of 10 different wild-caught fishspecies (3). This was the first evidence that V. cholerae may usevertebrate fish as a vector both for increasing bacterial populationand potentially for transport over long distances. This study alsosuggested that V. cholerae may potentially be a commensal in fish.
In the current study, we investigated whether the well-de-scribed zebrafish, Danio rerio, could serve as a vertebrate fishmodel for V. cholerae. Zebrafish have a long and extremely suc-cessful history as model organisms for many biological processesranging from development to bacterial pathogenesis (31, 32). Be-cause the biology of zebrafish is so well understood, its potential asa model for V. cholerae opens many new pathways to understand-ing the V. cholerae environmental lifestyle. Furthermore, the nat-ural habitat of zebrafish in Asia broadly overlaps areas of choleraendemicity, strongly suggesting that there is a natural associationbetween zebrafish and V. cholerae in the wild (33). The zebrafishprovides a natural course of infection model and thus should be anexcellent method for studying the environmental lifestyle of V.cholerae, its requirements for intestinal colonization in both fishand humans, and transmission of the disease from infected touninfected hosts.
MATERIALS AND METHODSEthics statement. This study was carried out in strict accordance with therecommendations in the Guide for the Care and Use of Laboratory Ani-mals of the National Institutes of Health. All animal work was conductedaccording to the relevant guidelines of the Public Health Service, Office ofLaboratory Animal Welfare, Animal Assurance no. A3310-01, and wasapproved by the Wayne State University IACUC, protocol number A01-14-10.
Bacterial growth. V. cholerae strains (Table 1) were grown either onLB medium prior to use in animal experiments or as described in the text.Intestinal homogenates were plated on LB agar containing 100 �g/mlstreptomycin and 40 �g/ml X-Gal (5-bromo-4-chloro-3-indolyl-�-D-ga-lactopyranoside).
Zebrafish. Six- to 9-month-old ZDR wild-type zebrafish were used forthe experiments with adult zebrafish. For the larva infections, zebrafish at5 days postfertilization (dpf) were used. Zebrafish were bred and main-tained as previously reported (34). For anesthesia, zebrafish were placed in100 ml of 168 �g/ml Tricaine (ethyl-3 aminobenzoate methanesulfonatesalt; catalog no. A50040; Sigma) solution. For euthanasia of zebrafish, thedose of Tricaine was doubled, and fish remained in the solution for 25 to30 min. All animal protocols were approved by the Wayne State Univer-sity IACUC committee.
Oral gavage of zebrafish. Zebrafish were first anesthetized by placingthem in Tricaine solution. After the fish were sufficiently anesthetized (�4min), they were removed, rinsed in fresh water without anesthetic, andplaced, dorsal side up, between the open jaws of a gauze-wrapped hemo-stat on a wedge of Styrofoam to position the head at the correct angle,creating a stage for inoculation (as described in reference 34). Zebrafishwere 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�l of a washed bacterial suspension. The end of the tubing was gentlyinserted into the zebrafish esophagus, and inoculum was slowly added bydepression of the plunger of the syringe. The zebrafish were then placedinto a 400-ml beaker with a perforated lid containing 200 ml of tank water(sterilized double-distilled water [ddH2O] with 60 mg/liter of InstantOcean aquarium salts [35]). Four to six zebrafish were added to eachbeaker and placed into a glass front incubator set at 27°C for the durationof the experiment.
Inoculation of zebrafish via water. Bacterial cultures were washedonce in phosphate-buffered saline (PBS) and diluted to the correct con-centration using PBS before adding to the tank water. Bacterial concen-trations 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. Fourto six zebrafish were then placed into a 400-ml beaker with a perforated lidcontaining 200 ml of tank water (sterilized ddH2O with 60 mg/liter ofInstant Ocean aquarium salts [35]) and the bacterial inoculum. Each bea-ker was placed into a glass front incubator set at 27°C for the duration ofthe experiment.
Transmission experiments. A group of 4 zebrafish, marked on thedorsal 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 weremoved to another beaker of freshwater two times to remove external V.cholerae organisms. The infected fish were then added to a larger beaker of400 ml water containing 4 naive zebrafish. After 24 h, the fish were sacri-ficed and intestinal V. cholerae populations were enumerated as describedbelow.
Determination of V. cholerae colonization of intestine. At desig-nated time points, fish were removed from the beaker and euthanized asdescribed above. Intestines were aseptically removed, placed into 300 �l ofsterile PBS, and homogenized using a micro-tissue grinder (Kontes PelletPestle motorized tissue grinder; Fisher). Serial dilutions of the homoge-nate were made and plated onto selective media for enumeration.
Experiments using zebrafish larvae. Five-day-postfertilization ze-brafish 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 expressedgreen fluorescent protein (GFP) from the tcpA promoter. At the desig-nated time points, larvae were removed from the well with the bacteriaand washed in sterile tank water twice and then placed into a well with aeuthanizing dose of Tricaine solution. Larvae were then mounted on amicroscope slide in Tricaine inside a 1-mm-thick washer glued to theslide. A coverslip was placed on top of the washer, and the larvae wereviewed with a Zeiss Axioskop 40 Fluorescence microscope at a magnifica-tion of �100. In some instances, paramecia were also added to the wellwith the V. cholerae to facilitate uptake of the bacteria.
Histology of V. cholerae-infected zebrafish intestines. At 24 hpostinfection, adult zebrafish were removed from tank water and eutha-nized. An incision was made using a scalpel along the ventral line of each
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 �toxTJW879 E7946 �toxT; PtcpA-gfpa Strains O395 and E7946 were from a laboratory collection. STR, streptomycin.
fish, and then it was placed in Dietrich’s fixative for 24 to 48 h. Next, thezebrafish were placed in tissue cassettes and dehydrated through a series ofgraded ethanol. Following a final 1-h wash in 100% ethanol, the fish wereincubated in toluene for 1 h and placed in Clearify (American MasterTechScientific Inc.) for 12 to 18 h. The fish were then incubated in a bottle ofmolten paraffin heated in a 60°C water bath for at least 1 h, the paraffinwas changed, and the fish were incubated for another 12 to 18 h in thesame water bath. Finally, the fish were embedded in 60°C paraffin andplaced on ice to cool until the paraffin was solidified. The paraffin blockswere cut at 3 �m, placed on Superfrost Plus Gold microscope slides(Fisher Scientific), and dried in a 55°C oven for at least 24 h before stain-ing. Sections were stained with anti-V. cholerae polyclonal antibody (KPLBacTrace) and counterstained with a secondary antibody conjugated toAlexa Fluor 568 (A11011; Molecular Probes). Stained sections wereviewed with a Zeiss Axioskop 40 Fluorescence microscope at a magnifica-tion of �1,000.
RESULTSExposure of zebrafish to V. cholerae results in robust intestinalcolonization. We began our investigation of zebrafish as a V. chol-erae host model by inoculating individual fish with 106 CFU viaoral gavage, followed by enumeration of V. cholerae in the intesti-nal tract 24 h postinfection. This is the method used in the infantmouse model, and gavage has the advantage of controlling thenumber of bacteria in the inoculum. V. cholerae was specificallyselected by plating intestinal homogenates on medium containingstreptomycin, as all strains used in these experiments are strepto-mycin resistant. Unlike experiments performed with infant mice,which have little or no intestinal microbiota, zebrafish have anintact intestinal microbiota, so selection for V. cholerae is essential.To further distinguish V. cholerae from other intestinal bacteriathat are naturally streptomycin resistant, X-Gal was also added tothe plates, as V. cholerae will form blue colonies but the otherintestinal bacteria will not. The results of these experiments indi-cated that V. cholerae does indeed robustly colonize the zebrafishintestinal tract (data not shown). Fish infected by gavage typicallyhad upwards of 105 V. cholerae organisms colonizing their intes-tinal tract after 24 h. However, the anatomy of the zebrafishesophagus presented a problem with gavage that affected repro-ducibility, and many fish did not become colonized due to regur-gitation of the inoculum. Additionally, the goal of this work was toexplore a natural host model, so manipulating the fish with anes-
thesia and gavage was undesirable. The gavage experiments were,however, successful in determining that V. cholerae can colonizethe zebrafish intestinal tract in large numbers.
Our next effort was to simulate a more natural infectious routeby simply adding V. cholerae to a beaker of 200 ml water contain-ing several zebrafish. After 24 h of exposure to V. cholerae, the fishwere sacrificed and tested for intestinal colonization. Various in-fectious doses, ranging from 106 to 1010 bacteria per beaker, weretested (data not shown); the lowest infectious dose that achievedconsistent colonization levels was 108 V. cholerae bacteria per bea-ker, i.e., 5 � 105 V. cholerae cells per milliliter of water. Exposure ofzebrafish to this dosage of V. cholerae via water resulted in largenumbers of V. cholerae in the intestinal tract 24 h postinfection(Fig. 1A); approximately 104 V. cholerae cells per fish intestine wasthe typical observation, although there was a several-log rangeobserved in different fish. Increasing the infectious dose to 1010 V.cholerae cells, i.e., 5 � 107 cells per milliliter, resulted in a tighterrange of colonization among individual fish, with most fish havingbetween 104 and 106 V. cholerae cells colonizing their intestinaltracts. Both classical and El Tor biotype O1 pandemic strains wereable to colonize zebrafish intestinal tracts, although the El Torbiotype, on average, exhibited a slightly higher bacterial load (Fig.1). V. cholerae was not detected in significant numbers in the na-res, gills, scales, fins, spleen, or heart (data not shown). Theseresults suggest that the intestine is specifically targeted and is theonly site of colonization for V. cholerae in zebrafish. Furthermore,colonization of zebrafish intestine by V. cholerae does not result ininvasive infection. This is very similar to what occurs in humansand mammalian animal models for V. cholerae.
Given the high numbers of V. cholerae colonizing the intestinaltract after 24 h, we next explored earlier time points to determinehow quickly colonization occurred. As shown in Fig. 2, we as-sessed colonization in zebrafish exposed to either V. cholerae clas-sical strain O395 (�1010 CFU per 200 ml) or V. cholerae El Torstrain E7946 (�109 CFU per 200 ml) at 2 h, 6 h, and 24 h postex-posure. Both biotypes were highly colonized as early as 2 h post-exposure, indicating that V. cholerae enters the zebrafish intestinein high numbers over a very short time frame. Numbers for bothbiotypes were quite consistent between fish at 2 h and 6 h postex-posure, with greater variability observed at 24 h postexposure.
FIG 1 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 calculatedafter 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 O1serogroup classical biotype V. cholerae strain. Statistical significance indicated above the data was determined by Student’s t test.
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Histological examination of colonized zebrafish intestinaltracts revealed clumps or microcolonies of V. cholerae in closecontact with the epithelial surface. As shown in Fig. 3, individualV. cholerae curved bacilli were visible at the epithelial surface at the24 h time point after exposure of zebrafish to V. cholerae biotype ElTor in water. V. cholerae cells were visualized in sections of fixedzebrafish by fluorescence microscopy using a primary polyclonalantibody directed against V. cholerae and a secondary monoclonalantibody carrying the fluorescent tag. The contact between V.cholerae and the intestinal epithelial surface observed in zebrafishvery closely resembles the interaction between V. cholerae and theepithelial surface observed in mammalian models (36, 37).
V. cholerae biotype El Tor has a colonization advantage inzebrafish. V. cholerae biotype El Tor has apparently completelyreplaced the classical biotype in the environment and as an agentof human cholera. The two biotypes have numerous differences,including changes in virulence regulation, metabolism, sensitivityto antibiotics, and possession of accessory toxins. Differences infish colonization could provide one potential explanation for thetakeover by El Tor in the environment. To examine this possibil-ity, we compared the ability of classical and El Tor biotypes tocolonize zebrafish. While both biotypes robustly colonize ze-brafish, we consistently observed somewhat higher bacterial loadsin zebrafish infected with V. cholerae biotype El Tor, although
FIG 2 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 serialdilutions of the intestinal homogenates.
FIG 3 Fluorescence micrographs of V. cholerae colonizing the zebrafish intestinal epithelium. Fish were exposed to V. cholerae for 24 h in water and thensacrificed, fixed, and prepared for sectioning. Bacteria were visualized using a polyclonal primary antibody against V. cholerae and a secondary antibody carryinga fluorescent tag. (A) Uninfected fish; (B, C, D) infected fish. Magnification, �1,000.
there was variation from fish to fish (Fig. 1). Because we observeddifferences between levels of El Tor and classical biotype coloni-zation of the zebrafish intestine at the 24 h time point, the questionarose as to whether these differences would be maintained for along time or would vary. To answer this question, colonizationlevels at the 24, 48, and 72 h time points were compared (Fig. 4).The results of these experiments indicate a clear difference be-tween the classical and El Tor biotypes. V. cholerae classical bio-type was cleared from zebrafish intestinal tracts by 72 h postexpo-sure. However, V. cholerae biotype El Tor was retained in thezebrafish intestinal tracts at high levels even 6 days postexposure.This result suggests that the El Tor biotype has acquired genes thatallow it to colonize fish for a prolonged period. This observation isconsistent with the hypothesis that increased success of V. choleraeEl Tor within the fish reservoir potentially abetted the disappear-ance of classical V. cholerae from worldwide environmentalniches.
Zebrafish colonized by V. cholerae transmit the bacteria tonaive fish. As described above, zebrafish are rapidly colonized byV. cholerae after exposure in water. The colonized zebrafish alsoexhibit signs of pathogenesis, primarily diarrhea, which leads tofouling of the water by infected fish. A likely function in the envi-ronment for this V. cholerae-induced diarrhea would be to en-hance escape of newly replicated bacteria back into the aquaticniche. This could also potentially enable colonization of other fishthat are near the infected fish, leading to rapid population growthof V. cholerae within a school of fish.
To test the hypothesis that infected fish could transmit theinfection to naive fish, we exposed groups of zebrafish to V. chol-erae for 2 h as described above. Two fish were sacrificed at thispoint to assess their intestinal colonization levels, and we typicallysaw between 104 and 105 V. cholerae cells per fish. The remaininginfected fish were marked by fin clipping to distinguish them fromuninfected fish. The infected, clipped fish were twice washed inbeakers of clean water to remove external V. cholerae and thenadded to another, larger beaker of clean water containing a groupof naive zebrafish. The fish were kept together for 24 h and theneuthanized, and intestinal colonization by V. cholerae was as-sessed. The results indicate that every previously uninfected fishbecame colonized by V. cholerae after 24 h of exposure to infectedfish (Fig. 5).
The major human V. cholerae virulence factors are not re-quired for zebrafish colonization. Intestinal colonization in hu-mans and most mammalian animal models requires productionof TCP. Although TCP is not directly involved in adherence of V.cholerae to the epithelial surface (14), it has been hypothesized thatmicrocolony formation mediated by TCP is crucial for effectivecolonization (13, 38, 39). We investigated whether TCP or viru-lence factors that are coregulated with TCP are essential for ze-brafish colonization by using V. cholerae deficient for toxT, themajor virulence transcription activator (15), to infect zebrafish.�toxT V. cholerae does not produce TCP, CT, accessory coloniza-tion factors, or several other coregulated gene products (40–43).Our results indicate that �toxT V. cholerae colonizes zebrafish aswell as wild-type toxT V. cholerae (Fig. 6). Our finding is consistentwith the previous observation that non-O1 strains, which do notcarry the Vibrio pathogenicity island (VPI) genes required for TCP
FIG 4 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 containing108 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 intestinewas calculated after plating serial dilutions of intestinal homogenates 24 h, 48 h, 72 h, or 144 h postinfection. (A) Results from classical biotype strain O395infection. (B) Results from El Tor biotype strain E7946 infection.
FIG 5 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 twiceand placed in a fresh beaker with naive “recipient” fish for 24 h. Data shownwere collected from plating serial dilutions of intestinal homogenates 24 h afterexposure of the naive fish to the infected fish. Strains used: classical, O395; ElTor, E7946.
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production, colonize wild fish species (3). Our finding is also con-sistent with the fact that the vast majority of V. cholerae strainspresent in the environment do not possess CTX�, which carriesthe genes encoding CT (44), or the VPI, which carries the genesencoding TCP, other ToxT-regulated genes, and toxT itself (45).
Zebrafish larvae are colonized by V. cholerae. All the experi-ments described above were performed using mature adult ze-brafish. Next, we investigated whether we could observe the up-take of V. cholerae into the digestive tract of zebrafish larvae. Bytaking advantage of the transparency of zebrafish larvae and usingV. cholerae expressing GFP, we could potentially observe activeuptake and colonization of the zebrafish.
Our results indicate that zebrafish larvae are rapidly colonizedby V. cholerae. GFP-producing V. cholerae cells were clearly evi-dent in the digestive tract at 2 h postexposure in water (Fig. 7).Figure 7C shows fluorescent V. cholerae cells just past the mouthand also beginning to colonize the intestine. At 24 h postexposure,abundant fluorescence in the intestinal tract is visible in larvaeexposed to GFP-producing V. cholerae, whereas no fluorescence isvisible in unexposed larvae. These results indicate that V. choleraeenters the zebrafish larva’s digestive tract simply by exposurethrough water, leading to rapid and robust intestinal colonization.
DISCUSSION
Here we describe use of the zebrafish as a novel animal model forthe study of the human pathogen V. cholerae. This work estab-lishes both a fish model for V. cholerae and a natural host modelfor V. cholerae. The use of a natural host and natural route ofinfection should provide new opportunities to determine factorsrequired for intestinal colonization, pathogenesis, and transmis-sion that cannot be realized using current mammalian animalmodels.
Zebrafish have numerous advantages over existing V. choleraeanimal models. This new model requires no manipulation of theanimal host, whereas mammalian animal models require substan-tial manipulation for V. cholerae colonization to occur. The infantmouse model, which is probably the most frequently used animalmodel for V. cholerae, requires oral gavage to establish intestinalcolonization, and an infected mouse does not exhibit diarrheadespite the production of CT in the intestinal tract (21). No signsof pathogenesis in infant mice are produced unless inocula greaterthan 108 bacteria are used, in which case the cause of death is still
not dehydration. Infant mice also do not have a significant micro-biota. The main advantage of the infant mouse model is that TCPproduction is required for colonization, as has been observed inhumans. However, the absolute requirement for TCP makes iden-tification of other potential virulence factors, such as the still-unknown factors that allow V. cholerae to adhere directly to theepithelial 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 performwithout substantial training. The rabbit RITARD model, whileproducing an infection that is closer to the cholera disease statethan other models, has similar limitations (23, 24). The infantrabbit model, which produces a state that is the most similar to thehuman disease, requires pretreatment of the animal with antibi-otics to eliminate microbiota, anesthesia, and administration ofthe inoculum with buffers by oral gavage and is also expensive (25,46). The adult mouse model has been very useful for studyingaccessory toxins but does not produce a disease state like that ofcholera and has the usual limitations of artificial host models (26,27, 47).
Zebrafish colonization of the intestine occurs via a natural pro-cess and in the presence of the normal fish microbiota. This shouldpermit future study related to the interplay between commensalsand V. cholerae that is not possible using mammalian animal mod-els. Recent research that examined the natural microbiota of ze-brafish found that the Vibrio genus was highly represented, al-though which Vibrio species were present was not determined(48). The prolonged colonization that we observed with V. chol-erae biotype El Tor suggests that V. cholerae may even be a ze-brafish commensal. Future work will determine whether this isindeed the case. Adult zebrafish also have a fully functioning im-mune system, with both innate and adaptive arms similar to thoseof humans. This strong similarity between components of im-mune system between zebrafish and humans should facilitate ex-
FIG 6 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 themean bacterial load per fish. Total colonization per intestine was calculatedafter plating serial dilutions of intestinal homogenate 24 h postinfection.Strains used were the El Tor strain E7946 and a derivative of E7946 having acomplete in-frame toxT deletion.
FIG 7 Colonization of zebrafish larvae by V. cholerae. Larvae were exposed toV. cholerae for the indicated time and then fixed for microscopy. GFP-produc-ing V. cholerae cells were visualized by fluorescence microscopy, overlaid onlight micrographs of the zebrafish larvae. (A) Uninfected larva; (B) infectedlarva 24 h after exposure; (C) infected larva 2 h after exposure; (D) ventral viewof infected larva 2 h after exposure.
tensive studies on the immune response to V. cholerae. Futureexperiments using zebrafish mutant strains with defects in im-mune response should help us to better understand both innateand acquired immunity to V. cholerae that will likely parallel theresponse in the human gut, which has been difficult to study. Theobservation that zebrafish larvae are also colonized should facili-tate future studies on colonization during development of theadaptive immune response.
The fact that infected zebrafish can transmit V. cholerae to na-ive fish provides an opportunity to study V. cholerae transmissionin great detail. Currently, natural transmission is essentially im-possible to study in mammalian models, as all of them requireeither gavage or survival surgery to administer the bacteria. There-fore, the zebrafish is likely to provide much new information ongenetic factors important for V. cholerae transmission in futurestudies.
Our observation that V. cholerae El Tor colonizes zebrafish fora longer time than classical V. cholerae may help to explain how ElTor strains have completely replaced classical strains as the causeof human cholera worldwide. Classical strains have become ex-tremely rare in the environment and may actually be extinct, al-though there is evidence that the CT-encoding CTX� derivedfrom classical biotype strains remains (9, 49–52). In recent years,so-called hybrid biotype V. cholerae has become a significant causeof human cholera. However, the only significant difference be-tween El Tor strains and the hybrid strains is within the CTX�genome (51), suggesting that El Tor strains have simply under-gone a recombination event with a classical CTX� genome. If fishare an important reservoir and/or vector for increasing the V.cholerae population, as we believe, then even a small increase infitness gained by El Tor strains could translate to a huge popula-tion advantage over time in the environment. This could lead tocomplete filling of the environmental niches by V. cholerae El Torand subsequent extinction of classical V. cholerae.
In summary, here we describe the use of zebrafish as a novelanimal model for the study of V. cholerae colonization and trans-mission. This new model provides many advantages over existinganimal models for V. cholerae and should facilitate many newavenues of research on both the environmental lifestyle of V. chol-erae and its pathogenesis in fish and humans.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant R21AI095520from the National Institute of Allergy and Infectious Diseases.
We thank members of the Neely and Withey labs for helpful discus-sions.
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