AEM Accepts, published online ahead of print on 27 December 2013 Appl. Environ. Microbiol. doi:10.1128/AEM.03580-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.

Zebrafish as a natural host model for Vibrio cholerae colonization and transmission

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Donna L. Runft, Kristie C. Mitchell, Basel H. Abuaita1, Jonathan P. Allen2, Sarah Bajer, Kevin

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Ginsburg, Melody N. Neely, and Jeffrey H. Withey*

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Department of Immunology and Microbiology, Wayne State University School of Medicine,

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Detroit, MI

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Current Address: Department of Microbiology and Immunology, University of Michigan

Medical School, Ann Arbor, MI 2

Current Address: Department of Microbiology-Immunology, Northwestern University Feinberg

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School of Medicine, Chicago, IL

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*Corresponding author. Mailing address: Department of Immunology and Microbiology, Wayne

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State University School of Medicine, 540 E. Canfield, Detroit, MI 48201.

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Telephone: 313-577-1316

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Fax: 313-577-1155

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Email: [email protected]

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Running Title: Zebrafish as a natural V. cholerae Model

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Abstract

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The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae.

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V. cholerae in the environment is associated with several varieties of aquatic life, including

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insect egg masses, shellfish and vertebrate fish. Here we describe a novel animal model for V.

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cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish

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intestinal tract after exposure in water with no manipulation of the animal required. Colonization

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occurs in close contact with the intestinal epithelium and mimics colonization observed in

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mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naïve fish, which

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then become colonized. Striking differences in colonization between classical and El Tor biotype

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V. cholerae were apparent. The zebrafish natural habitat in Asia heavily overlaps cholera

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endemic areas, suggesting that zebrafish and V. cholerae evolved in close contact with each

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other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization,

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transmission and environmental survival.

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Introduction

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Vibrio cholerae, the cause of the severe human diarrheal disease cholera, is also a

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ubiquitous inhabitant of coastal regions around the globe. As is the case for all species within the

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Vibrio genus, V. cholerae is an aquatic bacterium that may be found both freely swimming and

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in association with various forms of aquatic flora and fauna (1-5). The environmental lifestyle

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and reservoirs of V. cholerae have only in recent years become the subject of vigorous research

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and remain poorly understood.

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Over 200 V. cholerae serogroups have been identified from environmental sampling.

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However, only the O1 and O139 serogroups are capable of causing cholera. The O1 serogroup is

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further subdivided into two biotypes, classical and El Tor (6). Classical biotype V. cholerae is

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thought to have caused the first six of the seven known cholera pandemics beginning in 1817 and

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produces a more severe form of cholera. El Tor V. cholerae is responsible for the seventh

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pandemic, which began in 1961 and continues to the present day. El Tor strains are thought to be

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better suited for environmental survival, although the reasons for this are not clear. However,

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classical biotype strains are currently very difficult, if not impossible, to isolate from the

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environment, suggesting that El Tor strains have fully occupied the V. cholerae environmental

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niche. O139 serogroup strains, which caused large cholera outbreaks in the 1990s, have been

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shown to be derived from El Tor strains (7). In recent years some hybrid strains that closely

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resemble El Tor strains but also contain genetic material from classical strains have been isolated

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from cholera patients (8-10).

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To become a human pathogen, V. cholerae must be ingested in contaminated water or

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seafood. After ingestion, V. cholerae senses numerous signals that result in production of

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virulence factors that permit colonization of the human intestine and ultimately cause the

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diarrhea that will transmit V. cholerae back into the environment. The two major human

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virulence factors are cholera toxin (CT), which directly causes the characteristic secretory

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diarrhea in cholera patients (11,12), and the toxin-coregulated pilus (TCP), which is required for

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intestinal colonization (13,14). Virulence gene expression is controlled by a complex cascade of

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positive and negative transcription regulators (15). In addition to these major virulence factors

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that are required for causing human cholera, other virulence factors are implicated in human non-

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cholera diarrhea caused by V. cholerae (16-18). Unlike the two V. cholerae serogroups that cause

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cholera, a wide variety of serogroups can cause non-cholera diarrhea in humans (19,20).

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Several mammalian animal models for V. cholerae colonization and pathogenesis are in

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current usage. The most common models used for the study of mammalian pathogenesis are the

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3-5 day old “infant mouse” model (21) and the adult rabbit ligated ileal loop and RITARD

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models (22-24). These models are useful for the study of V. cholerae virulence but neither the

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mouse nor the rabbit is a natural host for V. cholerae. No pathogenesis is evident in infant mice

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and the pathogenesis caused by V. cholerae in adult rabbits does not strongly resemble human

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cholera. The adult rabbit models also require survival surgery and significant manipulation of the

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animal. The recently rediscovered infant rabbit model (25) does produce a disease state

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somewhat similar to human cholera, but again the rabbit is not a natural host of V. cholerae and

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significant manipulation is required to produce colonization in the infant rabbit. The adult mouse

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has been used for V. cholerae studies but the disease produced in adult mice does not resemble

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human cholera and is not dependent on the major virulence factors required to produce human

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cholera (26). The adult mouse is, however, a good model for studying V. cholerae accessory

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toxins (27).

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Non-mammalian V. cholerae animal models are less widely used. One such model is the

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drosophila model. V. cholerae had previously been found to colonize insect egg masses and

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recent work has determined that V. cholerae will also colonize the drosophila digestive tract and

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even kill the insect host (28). Therefore drosophila may be a more natural model for

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environmental V. cholerae. The pathogenesis observed in drosophila is largely independent of

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the major virulence factors required for human cholera, indicating that other colonization factors

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and toxins may be involved in the environmental lifestyle of V. cholerae (29,30). Given that 4

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most V. cholerae strains in the environment are not O1 or O139 serogroup pandemic strains, it

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follows that V. cholerae would have colonization factors for environmental niches not carried on

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the pathogenicity islands involved in human cholera.

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An ideal natural model for V. cholerae would be an animal within which V. cholerae may

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be found in its natural habitat. Recent work published by Senderovich et al. found non-O1 V.

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cholerae colonizing the intestinal tracts of 10 different wild-caught fish species (3). This was the

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first evidence that V. cholerae may use vertebrate fish as a vector for both increasing bacterial

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population and potentially for transport over long distances. This study also suggested that V.

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cholerae may potentially be a commensal in fish.

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In the current study, we investigated whether the well-described zebrafish, Danio rerio,

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could serve as a vertebrate fish model for V. cholerae. Zebrafish have a long and extremely

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successful history as model organisms for many biological processes ranging from development

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to bacterial pathogenesis (31,32). Because the biology of zebrafish is so well understood, its

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potential as a model for V. cholerae opens many new pathways to understanding the V. cholerae

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environmental lifestyle. Furthermore, the natural habitat of zebrafish in Asia broadly overlaps

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cholera endemic areas, strongly suggesting that there is a natural association between zebrafish

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and V. cholerae in the wild (33). The zebrafish provides a natural course of infection model and

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thus should be an excellent method for studying both the environmental lifestyle of V. cholerae,

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its requirements for intestinal colonization in both fish and humans, and transmission of the

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disease from infected to uninfected hosts.

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Materials and Methods 5

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Ethics statement. This study was carried out in strict accordance with the

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recommendations in the Guide for the Care and Use of Laboratory Animals of the National

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Institutes of Health. All animal work was conducted according to relevant guidelines of the

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Public Health Service, Office of Laboratory Animal Welfare, Animal Assurance # A3310-01,

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and was approved by the Wayne State University IACUC, protocol # A 01-14-10.

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Bacterial growth. V. cholerae strains were grown on either LB medium prior to use in

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animal experiments as described in the text. Intestinal homogenates were plated on LB agar

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containing 100 μg/ml streptomycin and 40 μg/ml X-Gal.

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Zebrafish. Six to nine month old ZDR wild type zebrafish were used for the

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experiments with adult zebrafish. For the larvae infections, 5 days post fertilization (dpf)

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zebrafish were used. Zebrafish were bred and maintained as previously reported (34). For

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anesthesia, zebrafish were placed in 100 mls of 168 ug/ml Tricaine (ethyl-3 aminobenzoate

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methanesulfonate salt, Sigma A50040) solution. For euthanasia of zebrafish, the dose of

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Tricaine was doubled and fish remain in the solution for 25 – 30 minutes. All animal protocols

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were approved by the Wayne State University IACUC committee.

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Oral gavage of zebrafish. Zebrafish were first anesthetized by placing them in Tricaine

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solution. After the fish were sufficiently anesthetized (~4 minutes), they were removed, rinsed

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in fresh water without anesthetic and placed, dorsal side up, between the open jaws of a gauze

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wrapped hemostat on a wedge of styrofoam to position the head at the correct angle, creating a

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stage for inoculation (as described in (34). Zebrafish were inoculated using polyethylene tubing

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(PE-10, Braintree Scientific) attached to a 3/10cc syringe with a ½ inch, 29 gauge needle

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containing 20ul of a washed bacterial suspension. The end of the tubing was gently inserted into

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the zebrafish esophagus and inoculum was slowly added by depression of the plunger of the

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syringe. The zebrafish were then placed into a 400 ml beaker with a perforated lid containing

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200 ml of tank water (sterilized ddH20 with 60 mg/L of Instant Ocean aquarium salts (35)).

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Four to six zebrafish were added to each beaker and placed into a glass front incubator set at

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27˚C for the duration of the experiment.

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Inoculation of zebrafish via water. Bacterial cultures were washed once in PBS and

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diluted to the correct concentration using PBS before adding to the tank water. Bacterial

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concentrations ranged from 106 to 109 per beaker (~4 x 103 to 4 x 106 cfu/ml) and inoculum was

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added to the tank water before the fish. Four to six zebrafish were then placed into a 400 ml

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beaker with a perforated lid containing 200 ml of tank water (sterilized ddH20 with 60 mg/L of

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Instant Ocean aquarium salts (35)) and the bacterial inoculum. Each beaker was placed into a

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glass front incubator set at 27˚C for the duration of the experiment.

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Transmission experiments. A group of 4 zebrafish, marked on the dorsal fin for

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idenitification, was exposed to a total of 109 to 1010 V. cholerae in 200 ml water as described

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above. After 3 hours, the fish were moved to another beaker of fresh water two times to remove

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external V. cholerae. The infected fish were then added to a larger beaker of 400 ml water

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containing 4 naïve zebrafish. After 24 hours the fish were sacrificed and intestinal V. cholerae

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were enumerated as described below.

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Determination of V. cholerae colonization of intestine. At designated time points, fish

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were removed from the beaker and euthanized as described above. Intestines were aseptically

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removed, placed into 300 ul of sterile PBS and homogenized using a micro tissue grinder

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(Kontes Pellet Pestle motorized tissue grinder, Fisher). Serial dilutions were made of the

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homogenate and plated on selective media for enumeration.

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Experiments using zebrafish larvae. Five day post-fertilization (dpf) zebrafish larvae

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were placed into 1 x 106 cfu/ml V. cholerae diluted into 1ml tank water in a 12-well plate and

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incubated for 2 to 24 hours at 27˚C. The V. cholerae strain (JW879) was carrying a plasmid that

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expressed green fluorescent protein (GFP) from the tcpA promoter. At the designated time

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points, larvae were removed from the well with the bacteria and washed in sterile tank water

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twice and then placed into a well with a euthanizing dose of Tricaine solution. Larvae were then

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mounted on a microscope slide in Tricaine inside of a 1mm thick washer glued to the slide. A

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coverslip was placed on top of the washer and the larvae were viewed with a Zeiss Axioskop 40

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Fluorescent microscope at 100X magnification. In some instances paramecium were also added

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to the well with the V. cholerae to facilitate uptake of the bacteria.

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Histology of V. cholerae-infected zebrafish intestines. At 24 hours post infection, adult

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zebrafish were removed from tank water and euthanized. An incision was made using a scalpel

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along the ventral line of each fish then it was placed in Dietrich’s fixative for 24-48 hours. Next,

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the zebrafish were placed in tissue cassettes and dehydrated through a series of graded ethanol.

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Following a final 1-h wash in 100% ethanol, the fish were incubated in toluene for 1 hour and

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placed in ClearifyTM (American MasterTech Scientific Inc) for 12-18 hours. The fish were then

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incubated in a bottle of molten paraffin heated in a of 60°C water bath for at least 1 hour, the paraffin

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was changed, and the fish were incubated for another 12-18 hours in the same water bath. Finally,

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the fish were embedded in 60°C paraffin and placed on ice to cool until the paraffin was solidified.

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The paraffin blocks were cut at 3 μm, placed on Superfrost® Plus Gold microscope slides (Fisher

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Scientific) and dried in a 55°C oven for at least 24 hours before staining. Sections were stained with 8

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α−V. cholerae polyclonal antibody (KPL BacTrace) and counterstained with secondary antibody

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conjugated to Alexa Fluor 568 (Molecular Probes, A11011). Stained sections were viewed with

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a Zeiss Axioskop 40 Fluorescent microscope at 1000X magnification.

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

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began our investigation of zebrafish as a V. cholerae host model by inoculating individual fish

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with 106 cfu via oral gavage, followed by enumeration of V. cholerae in the intestinal tract 24

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hours post-infection. This is the method used in the infant mouse model, and gavage has the

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advantage of controlling the number of bacteria in the inoculum. V. cholerae was specifically

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selected by plating intestinal homogenates on media containing streptomycin, as all strains used

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in these experiments are streptomycin resistant. Unlike experiments performed in infant mice,

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which have little or no intestinal microbiota, zebrafish have intact intestinal microbiota, so

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selection for V. cholerae is essential. To further distinguish V. cholerae from other intestinal

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bacteria that are naturally streptomycin resistant, X-Gal was also added to the plates as V.

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cholerae will form blue colonies but the other intestinal bacterial will not. The results of these

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experiments indicated that V. cholerae does indeed robustly colonize the zebrafish intestinal tract

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(data not shown). Fish infected by gavage typically had upwards of 105 V. cholerae colonizing

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their intestinal tract after 24 hours. However, the anatomy of the zebrafish esophagus presented a

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problem with gavage that affected reproducibility and many fish did not become colonized due

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to regurgitation of the inoculum. Additionally, the goal of this work was to explore a natural host

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model, so manipulating the fish with anesthesia and gavage was undesirable. The gavage

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experiments were, however, successful in determining that V. cholerae can colonize the

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zebrafish intestinal tract in large numbers.

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Our next effort was to simulate a more natural infectious route by simply adding V.

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cholerae to a beaker of 200 ml water containing several zebrafish. After 24 hours exposure to V.

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cholerae the fish were sacrificed and tested for intestinal colonization. Various infectious doses,

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ranging from 106 to 1010 bacteria per beaker, were tested (data not shown); the lowest infectious

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dose that achieved consistent colonization levels was 108 V. cholerae per beaker, i.e. 5 x 105 V.

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cholerae per milliliter of water. Exposure of zebrafish to this dosage of V. cholerae via water

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resulted in large numbers of V. cholerae in the intestinal tract 24 hours post-infection (Fig. 1A);

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approximately 104 V. cholerae per fish intestine was the typical observation, although there was

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a several log range observed in different fish. Increasing the infectious does to 1010 V. cholerae,

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i.e. 5 x 107 per milliliter, resulted in a tighter range of colonization among individual fish, with

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most fish having between 104 and 106 V. cholerae colonizing their intestinal tracts. Both classical

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and El Tor biotype O1 pandemic strains were able to colonize zebrafish intestinal tracts,

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although the El Tor biotype, on average, exhibited a slightly higher bacterial load (Fig. 1). V.

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cholerae was not detected in significant numbers in the nares, gills, scales, fins, spleen or heart

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(data not shown) These results suggest that the intestine is specifically targeted and is the only

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site of colonization for V. cholerae in zebrafish. Furthermore, colonization of zebrafish intestine

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by V. cholerae does not result in invasive infection. This is very similar to what occurs in

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humans and mammalian animal models for V. cholerae.

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Given the high numbers of V. cholerae colonizing the intestinal tract after 24 hours, we

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next explored earlier time points to determine how quickly colonization occurred. As shown in

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Fig. 2, we assessed colonization in zebrafish exposed to either classical strain O395 (~1010 CFU 10

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per 200 ml) or El Tor strain E7946 (~109 CFU per 200 ml) V. cholerae at 2 hours, 6 hours, and

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24 hours post exposure. Both biotype were highly colonized as early as 2 hours post exposure,

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indicating that V. cholerae enter the zebrafish intestine in high numbers over a very short time

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frame. Numbers for both biotypes were quite consistent between fish at 2 hours and 6 hours post

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exposure, with greater variability observed at 24 hours post exposure.

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Histological examination of colonized zebrafish intestinal tracts revealed clumps or

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microcolonies of V. cholerae in close contact with the epithelial surface. As shown in Fig. 3,

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individual V. cholerae curved bacilli were visible at the epithelial surface at the 24 hour time

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point after exposure of zebrafish to El Tor biotype V. cholerae in water. V. cholerae were

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visualized in sections of fixed zebrafish by fluorescence microscopy using a primary polyclonal

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antibody directed against V. cholerae and a secondary monoclonal antibody carrying the

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fluorescent tag. The contact between V. cholerae and the intestinal epithelial surface observed in

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zebrafish very closely resembles the interaction between V. cholerae and the epithelial surface

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observed in mammalian models (36,37).

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

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V. cholerae has apparently completely replaced classical biotype in the environment and as an

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agent of human cholera. The two biotypes have numerous differences, including changes in

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virulence regulation, metabolism, sensitivity to antibiotics, and possession of accessory toxins.

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Differences in fish colonization could provide one potential explanation for the takeover by El

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Tor in the environment. To examine this possibility, we compared the ability of classical and El

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Tor biotype V. cholerae to colonize zebrafish. While both biotypes robustly colonize zebrafish,

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we consistently observed somewhat higher bacterial loads in zebrafish infected with El Tor

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biotype V. cholerae, although there was variation from fish to fish (Fig. 1). Because we observed 11

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differences between levels of El Tor and classical biotype colonization of the zebrafish intestine

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at the 24 hr time point, the question arose as to whether these differences would be maintained

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for a long time period or would vary. To answer this question, colonization levels at the 24, 48,

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and 72 hr time points were compared (Fig. 4). The results of these experiments indicate a clear

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difference between the classical and El Tor biotypes. Classical biotype V. cholerae was cleared

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from zebrafish intestinal tracts by 72 hours post-exposure. However, El Tor biotype V. cholerae

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was retained in the zebrafish intestinal tracts at high levels even 6 days post exposure. This result

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suggests that the El Tor biotype has acquired genes that allow it to colonize fish for a prolonged

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period. This observation is consistent with the hypothesis that increased success of El Tor V.

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cholerae within the fish reservoir potentially abetted the disappearance of classical V. cholerae

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from worldwide environmental niches.

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Zebrafish colonized by V. cholerae transmit the bacteria to naïve fish. As described

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above, zebrafish are rapidly colonized by V. cholerae after exposure in water. The colonized

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zebrafish also exhibit signs of pathogenesis, primarily diarrhea, which leads to fouling of the

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water by infected fish. A likely function in the environment for this V. cholerae-induced diarrhea

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would be to enhance escape of newly replicated bacteria back into the aquatic niche. This could

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also potentially enable colonization of other fish that are near the infected fish, leading to rapid

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population growth of V. cholerae within a school of fish.

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To test the hypothesis that infected fish could transmit the infection to naïve fish, we

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exposed groups of zebrafish to V. cholerae for 2 hours as described above. Two fish were

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sacrificed at this point to assess their intestinal colonization levels and we typically saw between

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104 and 105 V. cholerae per fish. Remaining infected fish were marked by fin clipping to

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distinguish them from uninfected fish. The infected, clipped fish were twice washed in beakers 12

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of clean water to remove external V. cholerae, then added to another, larger beaker of clean

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water containing a group of naïve zebrafish. The fish were kept together for 24 hours, then

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euthanized and intestinal colonization by V. cholerae was assessed. The results indicate that

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every previously uninfected fish became colonized by V. cholerae after 24 hours exposure to

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infected fish (Fig. 5).

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

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colonization. Intestinal colonization in humans and most mammalian animal models requires

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production of TCP. Although TCP is not directly involved in adherence of V. cholerae to the

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epithelial surface (14), it has been hypothesized that micro-colony formation mediated by TCP is

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crucial for effective colonization (13,38,39). We investigated whether TCP or virulence factors

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that are co-regulated with TCP are essential for zebrafish colonization by using V. cholerae

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deleted for toxT, the major virulence transcription activator (15), to infect zebrafish. ΔtoxT V.

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cholerae does not produce TCP, CT, accessory colonization factors, or several other coregulated

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gene products (40-43). Our results indicate that ΔtoxT V. cholerae colonizes zebrafish as well as

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wild-type toxT V. cholerae (Fig. 6). Our finding is consistent with the previous observation that

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non-O1 strains, which do not carry the Vibrio pathogenicity island (VPI) genes required for TCP

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production, colonize wild fish species (3). Our finding is also consistent with the fact that the

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vast majority of V. cholerae strains present in the environment do not possess the CTXΦ, which

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carries the genes encoding CT (44), or the VPI, which carries the genes encoding TCP, other

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ToxT-regulated genes, and toxT itself (45).

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Zebrafish larvae are colonized by V. cholerae. All the experiments described above

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were performed using mature adult zebrafish. Next we investigated whether we could observe

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uptake of V. cholerae into the digestive tract of zebrafish larvae. By taking advantage of the 13

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transparency of zebrafish larvae and using V. cholerae expressing green fluorescent protein

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(GFP), we could potentially observe active uptake and colonization of the zebrafish.

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Our results indicate that zebrafish larvae are rapidly colonized by V. cholerae. GFP-

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producing V. cholerae were clearly evident in the digestive tract at 2 hours post exposure in

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water (Fig. 7). Fig. 7C shows fluorescent V. cholerae just past the mouth and also beginning to

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colonize the intestine. At 24 hours post exposure, abundant fluorescence in the intestinal tract is

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visible in larvae exposed to GFP-V. cholerae, whereas no fluorescence is visible in unexposed

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larvae. These results indicate that V. cholerae enter the zebrafish larvae digestive tract simply by

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exposure through water, leading to rapid and robust intestinal colonization.

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Discussion

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Here we describe use of the zebrafish as a novel animal model for the study of the human

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pathogen V. cholerae. This work establishes both a fish model for V. cholerae and a natural host

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model for V. cholerae. The use of a natural host and natural route of infection should provide

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new opportunities to determine factors required for intestinal colonization, pathogenesis, and

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transmission that cannot be realized using current mammalian animal models.

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Zebrafish have numerous advantages over existing V. cholerae animal models. This new

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model requires no manipulation of the animal host, whereas mammalian animal models require

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substantial manipulation for V. cholerae colonization to occur. The infant mouse model, which is

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probably the most frequently used animal model for V. cholerae, requires oral gavage to

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establish intestinal colonization and an infected mouse does not exhibit diarrhea despite the

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production of CT in the intestinal tract (21). No signs of pathogenesis in infant mice are 14

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produced unless inocula greater than 108 bacteria are used, in which case the cause of death is

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still not dehydration. Infant mice also do not have significant microbiota. The main advantage of

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the infant mouse model is that TCP production is required for colonization, as has been observed

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in humans. However, the absolute requirement for TCP makes identification of other potential

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virulence factors, such as the still unknown factors that directly adhere V. cholerae to the

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epithelial surface, difficult. The rabbit ligated ileal loop model, which is better than the mouse

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model at assessing CT production, requires survival surgery, is expensive, and is difficult to

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perform without substantial training. The rabbit RITARD model, while producing an infection

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that is closer to the cholera disease state than other models, has similar limitations (23,24). The

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infant rabbit model, which produces a state the most like human disease, requires pre-treatment

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of the animal with antibiotics to eliminate microbiota, anesthesia, administration of the inoculum

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with buffers by oral gavage, and is also expensive (25,46). The adult mouse model has been very

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useful for studying accessory toxins but does not produce a disease state like cholera and has the

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usual limitations of artificial host models (26,27,47).

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Zebrafish colonization of the intestine occurs via a natural process and in the presence of

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the normal fish microbiota. This should permit future study related to the interplay between

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commensals and V. cholerae that is not possible using mammalian animal models. Recent

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research that examined the natural microbiota of zebrafish found the Vibrio genus was highly

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represented, although which Vibrio species were present was not determined (48). The prolonged

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colonization we observed with El Tor biotype V. cholerae suggests that V. cholerae may even be

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a zebrafish commensal. Future work will determine whether this is indeed the case. Adult

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zebrafish also have a fully functioning immune system, with both innate and adaptive arms

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similar to those of humans. This strong similarity between components of immune system 15

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between zebrafish and humans should facilitate extensive studies on the immune response to V.

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cholerae. Future experiments using zebrafish mutant strains with defects in immune response

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should help us to better understand both innate and acquired immunity to V. cholerae that will

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likely parallel the response in the human gut, which has been difficult to study. The observation

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that zebrafish larvae are also colonized should facilitate future studies on colonization during

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development of the adaptive immune response.

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The fact that infected zebrafish can transmit V. cholerae to naïve fish provides an

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opportunity to study V. cholerae transmission in great detail. Currently natural transmission is

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essentially impossible to study in mammalian models, as all of them require either gavage or

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survival surgery to administer the bacteria. Therefore the zebrafish is likely to provide much new

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information on genetic factors important for V. cholerae transmission in future studies.

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Our observation that El Tor V. cholerae colonize zebrafish for a longer time period than

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classical V. cholerae may help to explain how El Tor strains have completely replaced classical

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strains as the cause of human cholera worldwide. Classical strains have become extremely rare in

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the environment and may actually be extinct, although there is evidence that the CT-encoding

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CTXΦ derived from classical biotype strains remains (9,49-52). In recent years, so-called hybrid

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biotype V. cholerae has become a significant cause of human cholera. However, the only

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significant difference between El Tor strains and the hybrid strains is within the CTXΦ genome

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(51), suggesting that El Tor strains have simply undergone a recombination event with a classical

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CTXΦ genome. If fish are an important reservoir and/or vector for increasing the V. cholerae

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population, as we believe, then even a small increase in fitness gained by El Tor strains could

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translate to a huge population advantage over time in the environment. This could lead to

16

355

complete filling of the environmental niches by El Tor V. cholerae and subsequent extinction of

356

classical V. cholerae.

357

In summary, here we describe the use of zebrafish as a novel animal model for the study

358

of V. cholerae colonization and transmission. This new model provides many advantages over

359

existing animal models for V. cholerae, and should facilitate many new avenues of research on

360

both the environmental lifestyle of V. cholerae and its pathogenesis in fish and humans.

361 362 363

Acknowledgements

364

This work was supported by Public Health Service grant R21AI095520 from the National

365

Institute of Allergy and Infectious Diseases. We thank members of the Neely and Withey labs for

366

helpful discussions.

367 368

17

369

Strain O395 E7946 JW JW

Relevant Genotype O1 Serogroup V. cholerae, classical biotype. StrepR O1 Serogroup V. cholerae, El Tor biotype. StrepR E7946 ΔtoxT PtcpA-gfp

Source Laboratory collection Laboratory collection

370 371

Table 1. Bacterial strains used in this work.

18

Reference

372

References

373 374

1.

Halpern, M., Broza, Y. B., Mittler, S., Arakawa, E., and Broza, M. (2004) Chironomid egg

375

masses as a natural reservoir of Vibrio cholerae non-O1 and non-O139 in freshwater

376

habitats.[erratum appears in Microb Ecol. 2004 Aug;48(2):285]. Microbial Ecology 47, 341-349

377

2.

from the eastern oyster, Crassostrea virginica. Appl Environ Microbiol 41, 559-560

378 379

3.

Senderovich, Y., Izhaki, I., and Halpern, M. (2010) Fish as reservoirs and vectors of Vibrio cholerae. PLoS ONE 5, e8607

380 381

Hood, M. A., Ness, G. E., and Rodrick, G. E. (1981) Isolation of Vibrio cholerae serotype O1

4.

Tamplin, M. L., Gauzens, A. L., Huq, A., Sack, D. A., and Colwell, R. R. (1990) Attachment of

382

Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters. Appl

383

Environ Microbiol 56, 1977-1980

384

5.

Rawlings, T. K., Ruiz, G. M., and Colwell, R. R. (2007) Association of Vibrio cholerae O1 El

385

Tor and O139 Bengal with the Copepods Acartia tonsa and Eurytemora affinis. Appl Environ

386

Microbiol 73, 7926-7933

387

6.

Raychoudhuri, A., Mukhopadhyay, A. K., Ramamurthy, T., Nandy, R. K., Takeda, Y., and Nair,

388

G. B. (2008) Biotyping of Vibrio cholerae O1: time to redefine the scheme. Indian J Med Res

389

128, 695-698

390

7.

Emergence and evolution of Vibrio cholerae O139. Proc Natl Acad Sci U S A 100, 1304-1309

391 392

8.

395

Safa, A., Nair, G. B., and Kong, R. Y. (2010) Evolution of new variants of Vibrio cholerae O1. Trends Microbiol 18, 46-54

393 394

Faruque, S. M., Sack, D. A., Sack, R. B., Colwell, R. R., Takeda, Y., and Nair, G. B. (2003)

9.

Safa, A., Sultana, J., Dac Cam, P., Mwansa, J. C., and Kong, R. Y. (2008) Vibrio cholerae O1 hybrid El Tor strains, Asia and Africa. Emerg Infect Dis 14, 987-988

19

396

10.

Nguyen, B. M., Lee, J. H., Cuong, N. T., Choi, S. Y., Hien, N. T., Anh, D. D., Lee, H. R.,

397

Ansaruzzaman, M., Endtz, H. P., Chun, J., Lopez, A. L., Czerkinsky, C., Clemens, J. D., and

398

Kim, D. W. (2009) Cholera outbreaks caused by an altered Vibrio cholerae O1 El Tor biotype

399

strain producing classical cholera toxin B in Vietnam in 2007 to 2008. J Clin Microbiol 47, 1568-

400

1571

401

11.

intestine. Annu Rev Med 24, 19-23

402 403

12.

Holmgren, J., and Lonnroth, I. (1975) Mechanism of action of cholera toxin. Specific inhibition of toxin-induced activation of adenylate cyclase. FEBS Lett 55, 138-142

404 405

Sharp, G. W. (1973) Action of cholera toxin on fluid and electrolyte movement in the small

13.

Thelin, K. H., and Taylor, R. K. (1996) Toxin-coregulated pilus, but not mannose-sensitive

406

hemagglutinin, is required for colonization by Vibrio cholerae O1 El Tor biotype and O139

407

strains. Infect Immun 64, 2853-2856

408

14.

Tamamoto, T., Nakashima, K., Nakasone, N., Honma, Y., Higa, N., and Yamashiro, T. (1998)

409

Adhesive property of toxin-coregulated pilus of Vibrio cholerae O1. Microbiol Immunol 42, 41-

410

45

411

15.

cholerae virulence gene expression. Infect Immun 75, 5542-5549

412 413

Matson, J. S., Withey, J. H., and DiRita, V. J. (2007) Regulatory networks controlling Vibrio

16.

Ogawa, A., Kato, J., Watanabe, H., Nair, B. G., and Takeda, T. (1990) Cloning and nucleotide

414

sequence of a heat-stable enterotoxin gene from Vibrio cholerae non-O1 isolated from a patient

415

with traveler's diarrhea. Infect Immun 58, 3325-3329

416

17.

Shin, O. S., Tam, V. C., Suzuki, M., Ritchie, J. M., Bronson, R. T., Waldor, M. K., and

417

Mekalanos, J. J. (2011) Type III secretion is essential for the rapidly fatal diarrheal disease caused

418

by non-O1, non-O139 Vibrio cholerae. MBio 2, e00106-00111

419 420

18.

Morris, J. G., Jr., Wilson, R., Davis, B. R., Wachsmuth, I. K., Riddle, C. F., Wathen, H. G., Pollard, R. A., and Blake, P. A. (1981) Non-O group 1 Vibrio cholerae gastroenteritis in the

20

421

United States: clinical, epidemiologic, and laboratory characteristics of sporadic cases. Ann Intern

422

Med 94, 656-658

423

19.

Bagchi, K., Echeverria, P., Arthur, J. D., Sethabutr, O., Serichantalergs, O., and Hoge, C. W.

424

(1993) Epidemic of diarrhea caused by Vibrio cholerae non-O1 that produced heat-stable toxin

425

among Khmers in a camp in Thailand. J Clin Microbiol 31, 1315-1317

426

20.

Hughes, J. M., Hollis, D. G., Gangarosa, E. J., and Weaver, R. E. (1978) Non-cholera vibrio

427

infections in the United States. Clinical, epidemiologic, and laboratory features. Ann Intern Med

428

88, 602-606

429

21.

Klose, K. E. (2000) The suckling mouse model of cholera. Trends Microbiol 8, 189-191

430

22.

Formal, S. B., Kundel, D., Schneider, H., Kunevn, and Sprinz, H. (1961) Studies with Vibrio cholerae in the ligated loop of the rabbit intestine. Br J Exp Pathol 42, 504-510

431 432

23.

intestinal tie-adult rabbit diarrhea model. Infect Immun 35, 952-957

433 434

24.

25.

26.

27.

Olivier, V., Salzman, N. H., and Satchell, K. J. (2007) Prolonged colonization of mice by Vibrio cholerae El Tor O1 depends on accessory toxins. Infect Immun 75, 5043-5051

441 442

Olivier, V., Queen, J., and Satchell, K. J. (2009) Successful small intestine colonization of adult mice by Vibrio cholerae requires ketamine anesthesia and accessory toxins. PLoS ONE 4, e7352

439 440

Ritchie, J. M., Rui, H., Bronson, R. T., and Waldor, M. K. (2010) Back to the future: studying cholera pathogenesis using infant rabbits. MBio 1

437 438

Spira, W. M., Sack, R. B., and Froehlich, J. L. (1981) Simple adult rabbit model for Vibrio cholerae and enterotoxigenic Escherichia coli diarrhea. Infect Immun 32, 739-747

435 436

Spira, W. M., and Sack, R. B. (1982) Kinetics of early cholera infection in the removable

28.

Blow, N. S., Salomon, R. N., Garrity, K., Reveillaud, I., Kopin, A., Jackson, F. R., and Watnick,

443

P. I. (2005) Vibrio cholerae infection of Drosophila melanogaster mimics the human disease

444

cholera. PLoS Pathog 1, e8

21

445

29.

Purdy, A. E., and Watnick, P. I. (2011) Spatially selective colonization of the arthropod intestine

446

through activation of Vibrio cholerae biofilm formation. Proc Natl Acad Sci U S A 108, 19737-

447

19742

448

30.

susceptibility to intestinal Vibrio cholerae infection. Cell Microbiol 11, 461-474

449 450

31.

32.

33.

34.

35.

Atkinson, M. J., and Bingman, C. (1997) Elemental composition of commerical seasalts. Journal of Aquariculture and Aquatic Sciences 8, 39-43

459 460

Phelps, H. A., Runft, D. L., and Neely, M. N. (2009) Adult zebrafish model of streptococcal infection. Curr Protoc Microbiol Chapter 9, Unit 9D 1

457 458

Engeszer, R. E., Patterson, L. B., Rao, A. A., and Parichy, D. M. (2007) Zebrafish in the wild: a review of natural history and new notes from the field. Zebrafish 4, 21-40

455 456

Allen, J. P., and Neely, M. N. (2010) Trolling for the ideal model host: zebrafish take the bait. Future microbiology 5, 563-569

453 454

Sullivan, C., and Kim, C. H. (2008) Zebrafish as a model for infectious disease and immune function. Fish Shellfish Immunol 25, 341-350

451 452

Berkey, C. D., Blow, N., and Watnick, P. I. (2009) Genetic analysis of Drosophila melanogaster

36.

Jones, G. W., Abrams, G. D., and Freter, R. (1976) Adhesive properties of Vibrio cholerae:

461

adhesion to isolated rabbit brush border membranes and hemagglutinating activity. Infect Immun

462

14, 232-239

463

37.

the Toxin-Coregulated Pilus in the Infant Mouse Model. J Bacteriol 193, 5260-5270

464 465

Krebs, S. J., and Taylor, R. K. (2011) Protection and Attachment of Vibrio cholerae Mediated by

38.

Kirn, T. J., Lafferty, M. J., Sandoe, C. M., and Taylor, R. K. (2000) Delineation of pilin domains

466

required for bacterial association into microcolonies and intestinal colonization by Vibrio

467

cholerae. Mol Microbiol 35, 896-910

468 469

39.

Sun, D., Lafferty, M. J., Peek, J. A., and Taylor, R. K. (1997) Domains within the Vibrio cholerae toxin coregulated pilin subunit that mediate bacterial colonization. Gene 192, 79-85

22

470

40.

Toxboxes in the Vibrio cholerae Cholera Toxin Promoter. J Bacteriol 194, 5255-5263

471 472

Dittmer, J. B., and Withey, J. H. (2012) Identification and Characterization of the Functional

41.

Withey, J. H., and DiRita, V. J. (2005) Activation of both acfA and acfD transcription by Vibrio

473

cholerae ToxT requires binding to two centrally located DNA sites in an inverted repeat

474

conformation. Mol Microbiol 56, 1062-1077

475

42.

divergently transcribed aldA and tagA genes. J Bacteriol 187, 7890-7900

476 477

43.

44.

Waldor, M. K., and Mekalanos, J. J. (1996) Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272, 1910-1914

480 481

Withey, J. H., and DiRita, V. J. (2006) The toxbox: specific DNA sequence requirements for activation of Vibrio cholerae virulence genes by ToxT. Mol Microbiol 59, 1779-1789

478 479

Withey, J. H., and DiRita, V. J. (2005) Vibrio cholerae ToxT independently activates the

45.

Karaolis, D. K., Johnson, J. A., Bailey, C. C., Boedeker, E. C., Kaper, J. B., and Reeves, P. R.

482

(1998) A Vibrio cholerae pathogenicity island associated with epidemic and pandemic strains.

483

Proc Natl Acad Sci U S A 95, 3134-3139

484

46.

tract: lessons from animal studies. Curr Top Microbiol Immunol 337, 37-59

485 486

Ritchie, J. M., and Waldor, M. K. (2009) Vibrio cholerae interactions with the gastrointestinal

47.

Nygren, E., Li, B. L., Holmgren, J., and Attridge, S. R. (2009) Establishment of an adult mouse

487

model for direct evaluation of the efficacy of vaccines against Vibrio cholerae. Infect Immun 77,

488

3475-3484

489

48.

Roeselers, G., Mittge, E. K., Stephens, W. Z., Parichy, D. M., Cavanaugh, C. M., Guillemin, K.,

490

and Rawls, J. F. (2011) Evidence for a core gut microbiota in the zebrafish. Isme Journal 5, 1595-

491

1608

492

49.

Na-Ubol, M., Srimanote, P., Chongsa-Nguan, M., Indrawattana, N., Sookrung, N., Tapchaisri, P.,

493

Yamazaki, S., Bodhidatta, L., Eampokalap, B., Kurazono, H., Hayashi, H., Nair, G. B., Takeda,

494

Y., and Chaicumpa, W. (2011) Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in

495

Thailand. Indian J Med Res 133, 387-394 23

496

50.

Halder, K., Das, B., Nair, G. B., and Bhadra, R. K. (2010) Molecular evidence favouring step-

497

wise evolution of Mozambique Vibrio cholerae O1 El Tor hybrid strain. Microbiology 156, 99-

498

107

499

51.

Lee, J. H., Choi, S. Y., Jeon, Y. S., Lee, H. R., Kim, E. J., Nguyen, B. M., Hien, N. T.,

500

Ansaruzzaman, M., Islam, M. S., Bhuiyan, N. A., Niyogi, S. K., Sarkar, B. L., Nair, G. B., Kim,

501

D. S., Lopez, A. L., Czerkinsky, C., Clemens, J. D., Chun, J., and Kim, D. W. (2009)

502

Classification of hybrid and altered Vibrio cholerae strains by CTX prophage and RS1 element

503

structure. J Microbiol 47, 783-788

504

52.

Safa, A., Bhuyian, N. A., Nusrin, S., Ansaruzzaman, M., Alam, M., Hamabata, T., Takeda, Y.,

505

Sack, D. A., and Nair, G. B. (2006) Genetic characteristics of Matlab variants of Vibrio cholerae

506

O1 that are hybrids between classical and El Tor biotypes. J Med Microbiol 55, 1563-1569

507 508 509 510

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Figure Legends

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Fig. 1. V. cholerae colonization of zebrafish intestines after exposure in water. 4-5 fish

513

were added to 200 ml water containing 108 V. cholerae (panel A) or 1010 V. cholerae (panel B).

514

Data shown here are compiled from multiple experiments. Each dot represents the data from one

515

fish. Total colonization per intestine was calculated after plating serial dilutions of intestinal

516

homogenates 24 hr post infection. Strain E7946 is an O1 serogroup El Tor biotype V. cholerae

517

strain and O395 is an O1 serogroup classical biotype V. cholerae strain. Statistical significance

518

indicated above the data was determined by student’s t test.

519

Fig. 2. V. cholerae colonization of zebrafish at earlier time points. Zebrafish were

520

exposed to either (A) 3 x 1010 V. cholerae classical strain O395 or (B) 3 x 109 V. cholerae El Tor

521

strain E7946. At the indicated time points, fish were sacrificed and intestinal V. cholerae levels

522

determined by plating of serial dilutions of the intestinal homogenates.

523

Fig. 3. Fluorescence micrograph of V. cholerae colonizing the zebrafish intestinal

524

epithelium. Fish were exposed to V. cholerae for 24 hours in water and then sacrificed, fixed and

525

prepared for sectioning. Bacteria were visualized using polyclonal primary antibody against V.

526

cholerae and secondary antibody carrying a fluorescent tag. A: Uninfected fish. B,C,D: infected

527

fish. 1000x magnification.

528

Fig. 4. Time course of classical and El Tor biotype V. cholerae colonization after

529

exposure in water. 4-5 fish were added to 200 ml water containing 108 V. cholerae. Each dot

530

represents the data from one fish and the horizontal bar indicates the mean bacterial load per fish.

531

Total colonization per intestine was calculated after plating serial dilutions of intestinal

25

532

homogenates 24 hr, 48 hr, 72 hr, or 144 hr post infection. A. Results from classical biotype strain

533

O395 infection. B. Results from El Tor biotype strain E7946 infection.

534

Fig. 5. Transmission of V. cholerae from infected fish to naïve fish. 4-5 “donor” fish

535

were exposed to V. cholerae in water for 3 hr, then washed twice and placed in a fresh beaker

536

with naïve “recipient” fish for 24 hr. Data shown are collected from plating serial dilutions of

537

intestinal homogenates 24 hr after exposure of the naïve fish to the infected fish. Strains used:

538

classical: O395; El Tor: E7946.

539

Fig. 6. Effect of toxT deletion on zebrafish colonization by El Tor V. cholerae. Each dot

540

represents the data from one fish and the horizontal bar indicates the mean bacterial load per fish.

541

Total colonization per intestine was calculated after plating serial dilutions of intestinal

542

homogenate 24 hr post infection. Strains used were the El Tor strain E7946 and a derivative of

543

E7946 having a complete in-frame toxT deletion.

544

Fig. 7. Colonization of zebrafish larvae by V. cholerae. Larvae were expose to V.

545

cholerae for the indicated time, then fixed for microscopy. GFP-producing V. cholerae were

546

visualized by fluorescence microscopy, overlaid on light micrographs of the zebrafish larvae. A.

547

Uninfected larva. B. Infected larva 24 hours after exposure. C. Infected larva 2 hours after

548

exposure. D. Ventral view of infected larva 2 hours after exposure.

26

Fig 1 Fig.

P = 0.058

P = 0.042

A.

B.

E7946 El Tor 24 HPI

O395 Classical 24 HPI

Fig 2 Fig.

A.

B.

Fig. 3 Uninfected Fig. g 2 A.

B.

C.

D.

Fig. g 4 A.

B.

Fig. g 5

Fig. g 6

E7946 WT

E7946 ΔtoxT

Fig. g 7

A

B

C

D

Zebrafish as a natural host model for Vibrio cholerae colonization and transmission.

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several vari...
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