Advances in cholera research: from molecular biology to public health initiatives.

The aquatic bacterium Vibrio cholerae is the etiological agent of the diarrheal disease cholera, which has plagued the world for centuries. This pathogen has been the subject of studies in a vast array of fields, from molecular biology to animal models for virulence activity to epidemiological disease transmission modeling. V. cholerae genetics and the activity of virulence genes determine the pathogenic potential of different strains, as well as provide a model for genomic evolution in the natural environment. While animal models for V. cholerae infection have been used for decades, recent advances in this area provide a well-rounded picture of nearly all aspects of V. cholerae interaction with both mammalian and non-mammalian hosts, encompassing colonization dynamics, pathogenesis, immunological responses, and transmission to naïve populations. Microbiome studies have become increasingly common as access and affordability of sequencing has improved, and these studies have revealed key factors in V. cholerae communication and competition with members of the gut microbiota. Despite a wealth of knowledge surrounding V. cholerae, the pathogen remains endemic in numerous countries and causes sporadic outbreaks elsewhere. Public health initiatives aim to prevent cholera outbreaks and provide prompt, effective relief in cases where prevention is not feasible. In this review, we describe recent advancements in cholera research in these areas to provide a more complete illustration of V. cholerae evolution as a microbe and significant global health threat, as well as how researchers are working to improve understanding and minimize impact of this pathogen on vulnerable populations.

Using a zebrafish model to understand adherent-invasive Escherichia coli infection.

A zebrafish model was developed to study AIEC colonization, invasion, and inflammation. This model can also be used to study the beneficial effects of a probiotic on AIEC infection of adult zebrafish. Bacteria are grown in vitro and then fish are infected with AIEC by immersion. Subsequently, colonization and inflammation can be assessed. Exposing fish to probiotic at different time points relative to AIEC can determine beneficial effects of probiotics as prophylactics or therapeutics against AIEC. For complete details on the use and execution of this protocol, please refer to Nag et al. (2022).

An adult zebrafish model for adherent-invasive Escherichia coli indicates protection from AIEC infection by probiotic E. coli Nissle.

Adherent-invasive Escherichia coli (AIEC) is an opportunistic pathogen associated with major inflammatory bowel disease, Crohn disease, and ulcerative colitis. Unfavorable conditions push commensal AIEC to induce gut inflammation, sometimes progressing to inflammation-induced colon cancer. Recently, zebrafish have emerged as a useful model to study human intestinal pathogens. Here, a zebrafish model to study AIEC infection was developed. Bath inoculation with AIEC resulted in colonization and tissue disruption in the zebrafish intestine. Gene expression of pro-inflammatory markers including interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNFα), interferon-γ (IFNγ), and S100A-10b (akin to human calprotectin) in the zebrafish intestine was significantly induced by AIEC infection. The probiotic E. coli Nissle 1917 (EcN) was tested as a therapeutic and prophylactic against AIEC infection and reduced AIEC colonization, tissue damage, and pro-inflammatory responses in zebrafish. Furthermore, EcN diminished the propionic-acid-augmented hyperinfection of AIEC in zebrafish. Thus, this study shows the efficacy of EcN against AIEC in an AIEC-zebrafish model.

An Experimental Adult Zebrafish Model for Shigella Pathogenesis, Transmission, and Vaccine Efficacy Studies.

Shigellosis has been a menace to society for ages. The absence of an effective vaccine against Shigella, improper sanitation, and unhygienic use of food and water allow the disease to flourish. Shigella can also be transmitted via natural water bodies. In the absence of a good animal model, the actual nature of pathogenesis and transmission remains unclear. Zebrafish larvae have previously been described as a model for Shigella pathogenesis. However, larval fish lack a mature intestinal microbiota and immune system. Here, the adult zebrafish was assessed as a potential model for Shigella pathogenesis. Their well-developed innate and adaptive immune responses mimic the mammalian immune system. Shigella showed a clear dose-, time-, and temperature-dependent colonization of the adult zebrafish gut. Efficacy of a three-dose immunization regime was tested using bath immunization with heat-killed trivalent Shigella immunogen. The present study demonstrates the efficacy of an adult zebrafish model for pathogenesis, transmission, and vaccine efficacy studies. IMPORTANCE Shigellosis is a diarrheal disease that is prevalent in developing countries and especially dangerous in young children. Currently, animal models for shigellosis are unable to model some aspects of the infectious cycle. Here, we describe a new shigellosis model in adult zebrafish, an increasingly common model organism for studying bacterial pathogens. The zebrafish model can be used to study Shigella colonization, transmission, and immune responses, as well as test vaccine efficacy.

Elucidating the correlation between the number of TTTTGAT heptamer repeats and cholera toxin promoter activity in Vibrio cholerae O1 pandemic strains.

A complex virulence-regulatory cascade controls expression of the cholera toxin genes (ctxAB) in Vibrio cholerae, which eventually leads to the production and secretion of choleragen (CT), responsible for rice watery diarrhoea in infected individuals. The cholera toxin promoter (PctxAB) contains a series of heptad repeats (5'-TTTTGAT-3'), which has previously been shown to play a crucial role in transcriptional regulation of ctxAB by recruiting the transcriptional activators ToxT, ToxR and the nucleoid-associated protein H-NS along the ctx promoter. The number of these repeats differs not only between the two biotypes of V. cholerae O1 strains, but also among the strains belonging to the same biotype. In this study, we examined if regulation of PctxAB is influenced in any way by the number of these repeats. Based on our observations, we posit that ctx activation indeed depends on the number of TTTTGAT heptad repeats within PctxAB, and occupation of the distal repeats by H-NS could prevent transcriptional activation of the ctx genes in V. cholerae O1 pandemic isolates. Our results suggest that ToxT-dependent transcriptional activation may not require entire displacement of H-NS and supports a recently described revised model of ToxT and H-NS mediated PctxAB transcriptional regulation.

Neutrophil-Associated Responses to Vibrio cholerae Infection in a Natural Host Model.

Vibrio cholerae, the cause of human cholera, is an aquatic bacterium found in association with a variety of animals in the environment, including many teleost fish species. V. cholerae infection induces a proinflammatory response followed by a noninflammatory convalescent phase. Neutrophils are integral to this early immune response. However, the relationship between the neutrophil-associated protein calprotectin and V. cholerae has not been investigated, nor have the effects of limiting transition metals on V. cholerae growth. Zebrafish are useful as a natural V. cholerae model as the entire infectious cycle can be recapitulated in the presence of an intact intestinal microbiome and mature immune responses. Here, we demonstrate that zebrafish produce a significant neutrophil, interleukin 8 (IL-8), and calprotectin response following V. cholerae infection. Bacterial growth was completely inhibited by purified calprotectin protein or the chemical chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), but growth was recovered by the addition of the transition metals zinc and manganese. The expression of downstream calprotectin targets was also significantly increased in the zebrafish. These findings illuminate the role of host calprotectin in combating V. cholerae infection. Inhibition of V. cholerae growth through metal limitation may provide new approaches in the development of anti-V. cholerae therapeutics. This study also establishes a major role for calprotectin in combating infectious diseases in zebrafish.

Characterization of the Immune Response to Vibrio cholerae Infection in a Natural Host Model.

The gram-negative bacterium Vibrio cholerae causes the life-threatening diarrheal disease cholera, which is spread through the ingestion of contaminated food or water. Cholera epidemics occur largely in developing countries that lack proper infrastructure to treat sewage and provide clean water. Numerous vertebrate fish species have been found to be natural V. cholerae hosts. Based on these findings, zebrafish (Danio rerio) have been developed as a natural host model for V. cholerae. Diarrheal symptoms similar to those seen in humans are seen in zebrafish as early as 6 hours after exposure. Our understanding of basic zebrafish immunology is currently rudimentary, and no research has been done to date exploring the immune response of zebrafish to V. cholerae infection. In the present study, zebrafish were infected with either pandemic El Tor or non-pandemic, environmental V. cholerae strains and select immunological markers were assessed to determine cellular immunity and humoral immunity. Significant increases in the gene expression of two transcription factors, T-bet and GATA3, were observed in response to infection with both V. cholerae strains, as were levels of mucosal related antibodies. Additionally, the cytokine IL-13 was shown to be significantly elevated and paralleled the mucin output in zebrafish excretions, strengthening our knowledge of IL-13 induced mucin production in cholera. The data presented here further solidify the relevancy of the zebrafish model in studying V. cholerae, as well as expanding its utility in the field of cholera immunology.

H-NS and ToxT Inversely Control Cholera Toxin Production by Binding to Overlapping DNA Sequences.

Vibrio cholerae infects human hosts following ingestion of contaminated food or water, resulting in the severe diarrheal disease cholera. The watery diarrhea that is characteristic of the disease is directly caused by the production of cholera toxin (CT). A complex regulatory cascade controls the production of CT and other virulence factors. However, ultimately, a single protein, ToxT, directly binds to virulence gene promoters and activates their transcription. Previously, we identified two ToxT binding sites, or toxboxes, within the cholera toxin promoter (PctxAB). The toxboxes overlap the two promoter-proximal GATTTTT heptad repeats found within PctxAB in classical biotype V. cholerae strain O395. These heptad repeats were previously found to be located within a large DNA region bound by H-NS, a global transcriptional repressor present in Gram-negative bacteria. The current model for the control of PctxAB transcription proposes complete H-NS displacement from the DNA by ToxT, followed by direct activation by ToxT-RNA polymerase (RNAP) contacts. The goal of this study was to determine more precisely where H-NS binds to PctxAB and test the hypothesis that ToxT completely displaces H-NS from the PctxAB promoter before activating transcription. The results suggest that H-NS binds only to the region of PctxAB encompassing the heptad repeats and that ToxT displaces H-NS only from its most promoter-proximal binding sites, calling for a revision of the current model involving H-NS and ToxT at PctxAB. IMPORTANCE H-NS is a global negative regulator of transcription in Gram-negative bacteria, particularly in horizontally acquired genetic islands. Previous work in Vibrio cholerae suggested that H-NS represses the transcription of cholera toxin genes by binding to a large region upstream of its promoter and that the virulence activator ToxT derepresses transcription by removing H-NS from the promoter. Here, new data support a revised model in which ToxT displaces only H-NS bound to the most promoter-proximal DNA sites that overlap the ToxT binding sites, leaving the upstream sites occupied by H-NS. This introduces a higher-resolution mechanism for the antirepression of H-NS in the control of cholera toxin production.

Vibrio cholerae Infection Induces Strain-Specific Modulation of the Zebrafish Intestinal Microbiome.

Zebrafish (Danio rerio) is an attractive model organism to use for an array of scientific studies, including host-microbe interactions. Zebrafish contain a core (i.e., consistently detected) intestinal microbiome consisting primarily of Proteobacteria. Furthermore, this core intestinal microbiome is plastic and can be significantly altered due to external factors. Zebrafish are particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae. As an intestinal pathogen, V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. Members of the resident intestinal microbial community likely must be reduced or eliminated by V. cholerae for colonization, and subsequent disease, to occur. Many studies have explored a variety of aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms but few have researched how a V. cholerae infection changes the resident intestinal microbiome. In this study, 16S rRNA gene sequencing was used to examine how five genetically diverse V. cholerae strains alter the intestinal microbiome following an infection. We found that V. cholerae colonization induced significant changes in the zebrafish intestinal microbiome. Notably, changes in the microbial profile were significantly different from each other, based on the particular strain of V. cholerae used to infect zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiota to enable colonization and that specific microbes that are targeted depend on the V. cholerae genotype.

The Vibrio cholerae Type Six Secretion System Is Dispensable for Colonization but Affects Pathogenesis and the Structure of Zebrafish Intestinal Microbiome.

Zebrafish (Danio rerio) are an attractive model organism for a variety of scientific studies, including host-microbe interactions. The organism is particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae, an intestinal pathogen. V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. While numerous studies have explored various aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms, few, if any, have examined how a V. cholerae infection alters the resident intestinal microbiome and the role of the type six secretion system (T6SS) in that process. In this study, 16S rRNA gene sequencing was utilized to investigate how strains of V. cholerae both with and without the T6SS alter the aforementioned microbial profiles following an infection. V. cholerae infection induced significant changes in the zebrafish intestinal microbiome, and while not necessary for colonization, the T6SS was important for inducing mucin secretion, a marker for diarrhea. Additional salient differences to the microbiome were observed based on the presence or absence of the T6SS in the V. cholerae utilized for challenging the zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiome to enable colonization and that the T6SS is important for pathogenesis induced by the examined V. cholerae strains. Furthermore, the presence or absence of T6SS differentially and significantly affected the composition and structure of the intestinal microbiome, with an increased abundance of other Vibrio bacteria observed in the absence of V. cholerae T6SS.

Zebrafish Models for Pathogenic Vibrios.

Vibrio is a large and diverse genus of bacteria, of which most are nonpathogenic species found in the aquatic environment. However, a subset of the Vibrio genus includes several species that are highly pathogenic, either to humans or to aquatic animals. In recent years, Danio rerio, commonly known as the zebrafish, has emerged as a major animal model used for studying nearly every aspect of biology, including infectious diseases. Zebrafish are especially useful because the embryos are transparent, larvae are small and facilitate imaging studies, and numerous transgenic fish strains have been constructed. Zebrafish models for several pathogenic Vibrio species have been described, and indeed a fish model is highly relevant for the study of aquatic bacterial pathogens. Here, we summarize the zebrafish models that have been used to study pathogenic Vibrio species to date.

Anti-virulence activity of polyphenolic fraction isolated from Kombucha against Vibrio cholerae.

The use of traditional foods and beverages or their bioactive compounds as anti-virulence agents is a new alternative method to overcome the increased global emergence of antimicrobial resistance in enteric pathogens. In the present study, we investigated the anti-virulence activity of a polyphenolic fraction previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against Vibrio cholerae O1. The isolated fraction was mainly composed of the polyphenols catechin and isorhamnetin. The fraction, the individual polyphenols and the combination of the individual polyphenols significantly inhibited bacterial swarming motility and expression of flagellar regulatory genes motY and flaC, even at sub-inhibitory concentrations. The polyphenolic compounds also decreased bacterial protease secretion and mucin penetration in vitro. In vivo study revealed that the polyphenolic fraction significantly inhibited V. cholerae induced fluid accumulation in the rabbit ileal loop model and intestinal colonization in suckling mice model. Therefore, the anti-virulence activity of the Kombucha polyphenolic fraction involved inhibition of motility and protease secretion of V. cholerae, thus preventing bacterial penetration through the mucin layer as well as fluid accumulation and bacterial colonization in the intestinal epithelial cells. The overall results implied that Kombucha might be considered as a potential alternative source of anti-virulence polyphenols against V. cholerae. To the best of our knowledge, this is the first report on the anti-virulence activity of Kombucha, mostly attributed to its polyphenolic content.

Tilapia (Oreochromis niloticus) as a Putative Reservoir Host for Survival and Transmission of Vibrio cholerae O1 Biotype El Tor in the Aquatic Environment.

Studies have reported the occurrence of Vibrio cholerae in fish but little is known about the interaction between fish and toxigenic V. cholerae as opposed to phytoplankton, which are well-established aquatic reservoirs for V. cholerae. The present study determined the role of tilapia (Oreochromis niloticus) as a reservoir host for survival and transmission of V. cholerae in aquatic environments. Three experiments were performed with one repetition each, where O. niloticus (∼2 g) kept in beakers were inoculated with four V. cholerae strains (5 × 107 cfu/mL). Firstly, infected tilapia were kept in stagnant water and fed live brine shrimp (Artemia salina) larvae daily. Secondly, infected tilapia were kept without feeding and water was changed every 24 h. Thirdly, infected tilapia were fed and water was renewed daily. Infected tilapia and non-infected controls were sacrificed on days 1, 2, 3, 7, and 14 post-inoculation and V. cholerae were enumerated in intestinal content and water. Another experiment assessed the transmission of V. cholerae from infected to non-infected tilapia. The study revealed that El Tor biotype V. cholerae O1 and V. cholerae non-O1 colonized tilapia intestines and persisted at stable concentrations during the second week of the experiment whereas the Classical biotype was undetectable after 1 week. In stagnant water with feeding, V. cholerae counts dropped to 105 cfu/ml in water and from 107 to 104 cfu/intestine in fish after 14 days. When water was renewed, counts in water decreased from 107 to 103 cfu/ml and intestinal counts went from 106 to 102 cfu/intestine regardless of feeding. All strains were transmitted from infected to naïve fish after 24 h of cohabitation. Tilapia like other fish may play an essential role in the survival and dissemination of V. cholerae O1 in aquatic environments, e.g., the seventh pandemic strains mostly. In this study, tilapia were exposed to high concentrations of V. cholerae to ensure initial uptake and follow-up studies with lower doses resembling natural concentrations of V. cholerae in the aquatic environment are needed to confirm our findings.

Internal Versus External Pressures: Effect of Housing Systems on the Zebrafish Microbiome.

Zebrafish (Danio rerio) are an attractive model organism for scientific studies, including host-microbe interactions. The organism is particularly useful for studying aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae. Previous studies have established the presence of a core zebrafish intestinal microbiome, and tracked the development of the zebrafish intestinal microbiome from the larval stage to adulthood. An unexplored matter in this host-microbe relationship is the effect of the housing system on the zebrafish intestinal and tank water microbiomes. In this study, we used 16S rRNA gene sequencing to investigate the response of zebrafish intestinal and tank water microbiomes to a change in housing conditions. Zebrafish in the separated fish tanks showed no initial differences in the structures of their intestinal microbial profiles; the same prominent bacteria were present and abundant across tanks. Immediately after the housing switch, the zebrafish intestinal microbial profiles changed in composition and structure. Within 5 days of the housing switch, the intestinal microbiome had stabilized, and changed significantly from the prehousing switch profile. This study demonstrates that although external factors can significantly perturb and alter the zebrafish intestinal microbiome, the microbiome displays a large level of selective resilience whose primary members (namely Vibrio) persist.

Characterization of the Vibrio cholerae Phage Shock Protein Response.

The phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, including Vibrio cholerae However, most of the current knowledge about the Psp response stems from in vitro studies in Escherichia coli and Yersinia enterocolitica In fact, the Psp response in V. cholerae has remained completely uncharacterized. In this study, we demonstrate that V. cholerae does have a functional Psp response system. We found that overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas other V. cholerae secretins do not. In addition, we have identified several environmental conditions that induce this stress response. Our studies on the genetic regulation and induction of the Psp system in V. cholerae suggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While a psp null strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated in V. choleraeIMPORTANCEVibrio cholerae leads a dual life cycle, as it can exist in the aquatic environment and colonize the human small intestine. In both life cycles, V. cholerae encounters a variety of stressful conditions, including fluctuating pH and temperature and exposure to other agents that may negatively affect cell envelope homeostasis. The phage shock protein (Psp) response is required to sense and respond to such insults in other bacteria but has remained unstudied in V. cholerae Interestingly, the Psp system has protein homologs, principally, PspA, in a number of bacterial clades as well as in archaea and plants. Therefore, our findings not only fill a gap in knowledge about an unstudied extracytoplasmic stress response in V. cholerae, but also may have far-reaching implications.

Glucose Metabolism by Escherichia coli Inhibits Vibrio cholerae Intestinal Colonization of Zebrafish.

The Vibrio cholerae O1 serogroup is responsible for pandemic cholera and is divided into the classical and El Tor biotypes. Classical V. cholerae produces acid when using glucose as a carbon source, whereas El Tor V. cholerae produces the neutral product acetoin when using glucose as a carbon source. An earlier study demonstrated that Escherichia coli strains that metabolize glucose to acidic by-products drastically reduced the survival of V. cholerae strains in vitro In the present study, zebrafish were fed 1% glucose and either inoculated with single V. cholerae or E. coli strains or coinfected with both V. cholerae and E. coli A significant decrease in classical biotype colonization was observed after glucose feeding due to acid production in the zebrafish intestine. El Tor colonization was unaffected by glucose alone. However, the El Tor strain exhibited significantly lower colonization of the zebrafish when either of the acid-producing E. coli strains was coinoculated in the presence of glucose. An E. coli sugar transport mutant had no effect on V. cholerae colonization even in presence of glucose. Glucose and E. coli produced a prophylactic effect on El Tor colonization in zebrafish when E. coli was inoculated before V. cholerae infection. Thus, the probiotic feeding of E. coli inhibits V. cholerae colonization in a natural host. This suggests that a similar inhibitory effect could be seen in cholera patients, especially if a glucose-based oral rehydration solution (ORS) is administered in combination with probiotic E. coli during cholera treatment.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model.

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Danio rerio as a Native Host Model for Understanding Pathophysiology of Vibrio cholerae.

Vibrio cholerae is an aquatic bacterium that causes the human disease cholera as well as milder forms of diarrhea. V. cholerae is found in the environment in association with a variety of aquatic animals, including vertebrate fish. Here we describe the use of zebrafish (Danio rerio) as a model for the pathogenic life cycle of V. cholerae. Being that fish are natural hosts for V. cholerae, the model provides several significant advantages over existing mammalian models that are not natural hosts.

Quantifying Vibrio cholerae Enterotoxicity in a Zebrafish Infection Model.

Vibrio cholerae is the etiological agent of cholera, an acute intestinal infection in humans characterized by voluminous watery diarrhea. Cholera is spread through ingestion of contaminated food or water, primarily in developing countries that lack the proper infrastructure for proper water and sewage treatment. Vibrio cholerae is an aquatic bacterium that inhabits coastal and estuarine areas, and it is known to have several environmental reservoirs, including fish. Our laboratory has recently described the use of the zebrafish as a new animal model for the study of V. cholerae intestinal colonization, pathogenesis, and transmission. As early as 6 h after exposure to V. cholerae, zebrafish develop diarrhea. Prior work in our laboratory has shown that this is not due to the action of cholera toxin. We hypothesize that accessory toxins produced by V. cholerae are the cause of diarrhea in infected zebrafish. In order to assess the effects of accessory toxins in the zebrafish, it was necessary to develop a method of quantifying diarrheal volume as a measure of pathogenesis. Here, we have adapted cell density, protein, and mucin assays, along with enumeration of V. cholerae in the zebrafish intestinal tract and in the infection water, to achieve this goal. Combined, these assays should help us determine which toxins have the greatest diarrheagenic effect in fish and, consequently, which toxins may play a role in environmental transmission.IMPORTANCE Identification of the accessory toxins that cause diarrhea in zebrafish can help us understand more about the role of fish in the wild as aquatic reservoirs for V. cholerae It is plausible that accessory toxins can act to prolong colonization and subsequent shedding of V. cholerae back into the environment, thus perpetuating and facilitating transmission during an outbreak. It is also possible that accessory toxins help to maintain low levels of intestinal colonization in fish, giving V. cholerae an advantage when environmental conditions are not optimal for survival in the water. Studies such as this one are critical because fish could be an overlooked source of cholera transmission in the environment.