ZEBRAFISH Volume 2, Number 2, 2005 © Mary Ann Liebert, Inc.
Review Paper Evolution of the Zebrafish Model: From Development to Immunity and Infectious Disease HILARY A. PHELPS and MELODY N. NEELY
ABSTRACT The successful zebrafish developmental model has now expanded to being used as a model for the analysis of host–pathogen interactions during infectious disease. Numerous pathogens have been demonstrated to infect zebrafish and new mechanisms of virulence, as well as host defense have been uncovered using this new model. In this review we summarize the literature on how the zebrafish infectious disease model is being used to decipher virulence mechanisms used by various pathogens and the host defense mechanisms initiated to combat infection.
One of the most informative methods used to study host–pathogen interactions is through animal model systems in which the responses of both the host and the pathogen can be analyzed. The availability of complete or partial genomes of numerous organisms has highlighted the evolutionary conservation of defense mechanisms used against microbial predators. New nonvertebrate model systems developed to study human disease have proven quite informative. Recently developed model host systems have been successful using plants (Arabidopsis thaliana) and nematodes (Caenorhabditis elegans) for the study of pathogenesis. Arabidopsis and C. elegans have both been used to identify numerous virulence genes in the human pathogen Pseudomonas aeruginosa.1–4 When these mutants were subsequently analyzed in a murine model of infection, a high percentage were avirulent or reduced in virulence, supporting the utility of using a nonvertebrate model for the study of human infectious disease.5,6 These nonverte-
INTRODUCTION
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to stop the progression of infectious disease will come from having a complete understanding of the virulence mechanisms used by pathogens during an infection. However, a microbial infection is made up of a complicated series of dynamic interactions between the pathogen and its host, with each interaction influencing the progression and ultimate outcome of the disease process. As the host reacts to the presence of the pathogen by initiating defense mechanisms, the microbe in turn must respond to survive the attack. A fully responsive host is necessary to analyze the changing host environment, as well as accurately study how a pathogen responds to that changing environment. Therefore, in vivo identification of virulence regulation and specific host–pathogen interactions is fundamental to the study of pathogenesis and one of the major challenges for microbial pathogenesis research. HE BEST STRATEGIES
Immunology and Microbiology Department, Wayne State School of Medicine, Detroit, Michigan.
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brate and plant models were a distinct advantage over larger mammalian systems because of the ability to easily genetically manipulate the host.1–3,7 Another model using the soil ameba Dictyostelium discoideum has been developed to study the intracellular survival mechanisms of Legionella species8 as well as the virulence mechanisms of the opportunistic human pathogen P. aeruginosa.9 Additionally, the nonvertebrate host model, Drosophila melanogaster, was used to identify the Toll family of receptors, one of the origins of host defense that is shared across a diverse range of species.10–12 The wealth of information available on Drosophila has been exploited to analyze infectious disease caused by both Mycobacterium marinum13 and Listeria monocytogenes.14
ZEBRAFISH AS AN INFECTION MODEL Recently, a new vertebrate animal model for the study of host–pathogen interactions during infection was developed using the zebrafish (Danio rerio).15,16 Successful infection of zebrafish has been demonstrated using a variety of pathogens including the zoonotic fish pathogens Mycobacterium marinum,15,17–20 Edwardsiella tarda,21,22 Salmonella arizonae,15 Vibrio anguillarum,23 and Streptococcus iniae (Fig. 1).16,24 Furthermore, infection of zebrafish has been successfully demonstrated with pathogens that are not known to infect fish such as Salmonella typhimurium,25 Bacillus subtillis,26 Escherichia coli,26 multiple species of Listeria,27 Streptococcus agalactiae (M.N. Neely and D.R. Runft, unpublished data) and Streptococcus pyogenes (Fig. 1).16,24,28 Snakehead rhabdovirus, a virus that infects warm-water fish, was also used for zebrafish infection to analyze upregulation of multiple host proteins involved in the immune response.21,29 Two additional viruses, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), were analyzed in the zebrafish for potential use as vectors for gene transfer experiments in hematopoietic cells.30 Also adding to our knowledge of infection processes in zebrafish, a novel truncated form of fibronectin (FN2) expressed on zebrafish cells was identi-
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fied by Zhao et al.,31 and was subsequently found to cause cells to be more susceptible to IHNV infection, possibly by mediating virus attachment.32 Even though several other animal models are already established, there are multiple advantages to using the zebrafish as an infectious disease model, some of which include low cost, easy maintenance, requirement of minimal laboratory space, and easy handling. The adult zebrafish size is particularly useful for monitoring disease progression because a single longitudinal section of the whole animal can be mounted on one slide for histologic analysis, instead of only a small tissue sample, as is the case with most mammalian models. For the study of vertebrate development, the growth of zebrafish embryos ex vivo has allowed analysis of mutations that would be fatal during fetal development and therefore not easily analyzed in the mouse model system. As an infectious disease model this attribute is also advantageous, because infections can be done in a stepwise fashion as immune defenses arise in the developing embryo to answer specific questions about the development and function of defense mechanisms. Another significant advantage of using the zebrafish embryos is their transparency for the first 3 weeks of life. This allows the observation of fluorescently labeled bacteria in real time inside the developing embryo. This scenario was used quite successfully to examine early host responses to infection with M. marinum.15,20 Using this technique, Davis et al.15 was able to observe two previously unrecognized macrophage-mediated mechanisms that may play a role in bacterial dissemination during infection. One mechanism was the direct transfer of bacteria between two macrophages by psuedopodial membrane tethers. A second mechanism was the phagocytosis of dead infected macrophages by uninfected macrophages, which could lead to dissemination of bacteria within the granuloma.15 Similarly, van der Sar et al.25 used DS-Red labeled S. typhimurium to analyze infection in real time, taking advantage of the transparency of the embryo to visualize infection in the same embryo over time. Injection of a very low dose (approximately 50 cells) of the pathogen into the caudal vein was lethal for the embryos in
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FIG. 1. Appearance of zebrafish 40 hours postintramuscular injection. Arrows point to site of injection. (A) Streptococcus iniae infection at 103 cfu; (B) S. pyogenes infection at 105 cfu; (C) mock infected with media alone.
30–48 hours postinfection (hpi), while strains with mutations in lipopolysaccharide synthesis genes (LPS) were attenuated for virulence. One important finding from this study was that at later time points of infection, only approximately 20–35% of Salmonella were still intracellular with the majority being extracellular in microcolonies,25 shedding light on a longstanding query in Salmonella pathogenesis as to the primary site of bacterial multiplication in vivo. An unusual phenomenon was also observed when the avirulent mutant was injected into the yolk sac, as opposed to the caudal vein.25 The avirulent mutant was able to proliferate in the yolk sac for 2 days and was then able to disseminate into the embryo. After dissemination of the bacteria, the embryo was killed rapidly. Therefore, if allowed to initially proliferate in the absence of macrophages, the avirulent mutant can kill. The authors suggest that injection into the yolk sac could be used as an in vivo growth control for mutants.25 One of the most important advantages of the zebrafish infectious disease model is the similarity of the zebrafish immune system to that of humans and other vertebrates (see below).33 This is where the zebrafish model surpasses some of the other newly developed models for
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host–pathogen analysis. While C. elegans and D. melanogastor do have many aspects of the innate immune system, they lack the components of the adaptive immune system. The zebrafish, on the other hand, has both innate and adaptive immune functions, allowing study of diseases that involve both systems. One infectious disease study capitalized on the delayed development of the adaptive immune system by infecting zebrafish embryos with M. marinum at early time points after fertilization. At the time of infection, only the innate immune system had developed, which permitted the researchers to study the contribution of innate immunity alone to granuloma formation.20 The chronically infected embryos were then allowed to develop into adults to study the contribution of the adaptive system at later stages of disease.20 The zebrafish model has also been used to analyze bacterial colonization and disease progression of pathogens that are significant problems in both wild and farmed fish populations. V. anguillarum is a gram-negative water-borne pathogen that causes hemorrhagic septicaemia and vibriosis in multiple fish species.34 Using green fluorescent protein (GFP)-labeled V. anguillarum and exploiting the transparency of zebrafish larvae, O’Toole et al.23 were able to visualize colonization of the fish in real time. This experiment allowed them to address the question of the mode and site of pathogen entry by observing the fluorescently labeled bacterium in the mouth and gastrointestinal tract of the transparent larvae after 2 hours of exposure and later in the intestine after 6 hours of exposure. Motility was also demonstrated to be a requirement for colonization of the skin of the fish, as a nonmotile mutant successfully colonized the intestine but could not colonize the skin of the zebrafish.23 Another pathogen that causes major problems in aquaculture is S. iniae, a gram-positive zoonotic pathogen of both fish and humans. In fish populations, it can cause systemic disease in more than two dozen different species in both fresh and salt water environments, potentially resulting in bacteremia, panopthalmitis and meningitis.35,36 Using adult zebrafish, Neely et al.16 were able to replicate a systemic infection, showing dissemination of bacteria
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from the site of infection to all major organs including the brain, within 22–26 hours. Histologic examination illustrated that infection pathologies closely mimicked that observed in fish infected in aquaculture.16 Examination of the entire infected fish on a single slide revealed intracellular streptococci in hepatocytes of the liver, a phenomenon involved in the pathogenesis of another Gram-positive systemic pathogen, Listeria monocytogenes,37–39 but not previously shown for a streptococcal systemic pathogen.16 Although infection of zebrafish by bacterial pathogens has been successfully demonstrated, can we take what we learn from these analyses and apply it to human infectious diseases? There is great diversity in the way in which pathogens cause disease and, just as many pathogens have developed different ways in which to survive in a host, many hosts respond to pathogens differently. Although mice have been the host model of choice for many years, and have provided us with much valuable information on virulence mechanisms, not all pathogens cause the same type of disease in mice that they do in a human infection. In these situations mice are not providing the analytical tools needed to dissect specific virulence mechanisms. Several studies have demonstrated that not only do the same clinical features of disease occur in experimental infections of mouse and zebrafish, but also in some cases, the zebrafish actually proves to be a better model for mimicking human disease characteristics. One example of such a case is infection of zebrafish by M. marinum. M. marinum is the closest genetic relative of M. tuberculosis,40 the causative agent of human tuberculosis, and is used as a model to study fundamental Mycobacterium pathogenesis. In zebrafish infections, M. marinum causes the formation of organized caseating granulomas much like those found from tuberculosis in humans caused by M. tuberculosis infection.20 However, the mouse model of tuberculosis (TB) follows a different progression than in humans, causing progressively coalescing, noncaseating lesions20,41 illustrating the advantage of using the zebrafish for analyses of Mycobacterium granuloma formation.
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Similarly, S. iniae causes a systemic infection leading to multiple organ dissemination as well as invasion of the brain and cerebral spinal fluid much like its close genetic relative, the human pathogen, S. agalactiae.16,24 Animal models for the analysis of meningitis are often very artificial, requiring direct injection into the cerebral spinal fluid or blood stream for access to the brain instead of following a natural route of infection. However, S. iniae can be isolated from the zebrafish brain in less than 24 hours from an intramuscular injection into the dorsal muscle or from short-term exposure to the pathogen after dermal abrasion.16,24 This model allows the analysis of host–pathogen interactions required for invasion of the brain from a more natural route of infection. One of the advantages of using natural fish pathogens in the zebrafish model, such as M. marinum and S. iniae, is that over time the host and bacterium have coevolved, with the pathogen adapting to life within their natural hosts, much like human-specific pathogens have adapted to the human host niche. Diseases caused by the fish pathogen often share the same clinical hallmarks of those found in humans from the human-specific pathogen counterpart. Human-specific pathogens, such as S. pyogenes, have also been used in the zebrafish infection model resulting in a fatal infection.16,24,28 While S. iniae causes a systemic infection with a large inflammatory response, S. pyogenes causes a localized necrotic infection resulting in large areas of necrosis but relatively little to no inflammation (Fig. 2).16 The surprising absence of inflammatory cells in the midst of large aggregates of bacteria has also been reported for a necrotizing myositis infection caused by S. pyogenes in an experimental baboon model.42 Moreover, a recent report of necrotizing fasciitis in human patients caused by an M14 strain of S. pyogenes, the same M type used for the zebrafish infections, found a marked absence of neutrophil infiltration in the necrotic tissue in the presence of large amounts of bacteria.43 These results demonstrate that the infection of zebrafish by S. pyogenes can be used to analyze critical steps in invasive streptococcal infections. Although not all human pathogens will cause a similar disease in the zebrafish, the above examples highlight the
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FIG. 2. Histopathologic examination of zebrafish dorsal muscle tissue after injection of streptococci. (A) and (B), 40 hours postintramuscular infection by Streptococcus iniae at a dose of 103 cfu. (C) and (D) 40 hours postintramuscular infection by S. pyogenes at a dose of 105 cfu. (A) Arrowheads outline a large region of necrosis and square indicates region of magnification in (B) (40 magnification); (B) numerous host inflammatory cells as well as cocci can be visualized in a highly necrotic region (1000 magnification); (C) arrowheads demarcate areas of necrosis and square shows region of magnification in D (40 magnification); (D) Arrow points to a large aggregate of streptococci dissecting along a facial plane (520 magnification).
utility of using the zebrafish model to study infection mechanisms relevant to human disease. Now that many bacterial pathogens have been successfully demonstrated to infect the zebrafish, the truly exciting research will be that which uses the model to ask specific questions about virulence mechanisms. Several studies have used M. marinum in the zebrafish model to address specific questions relating to Mycobacterial pathogenesis. Two different studies looked at a similar locus, called RD1 for “region of difference 1,” which is approximately 10-kb region conserved in the genome of virulent M. tuberculosis but missing from the BCG vaccine strains that are attenuated in the mouse
model of TB.44,45 Volkman et al.20 used zebrafish embryos to look at M. marinum mutants that have the entire RD1 locus deleted (RD1). While wild-type formed aggregates within 3–5 days postinfection (dpi), the RD1 mutant bacteria formed only small, transient aggregates and at a much later time point. By injecting fluorescently labeled Mycobacteria directly into the hindbrain ventricle, a site devoid of macrophages, they demonstrated that the RD1 mutant recruited macrophages as efficiently as wild type and that subsequent migration of infected macrophages out of the ventricle was also not impaired. The next group of experiments very cleverly exploited the optical
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transparency of the embryos and the ability to fluorescently label the Mycobacterium with different fluorophores. Using red-labeled wildtype bacteria and green-labeled RD1 mutant bacteria, they were able to do superinfection experiments to determine the influence of each strain on specific steps of infection. Using this technique they were able to determine that the RD1 infected macrophages actually could be recruited into aggregates if at least a few wildtype infected macrophages were present to initiate a chemotactic gradient to stimulate aggregate formation. Additionally, the RD1 locus was determined to be important for promoting cell death within the aggregates, which in turn leads to intercellular bacterial spread and increased bacterial load. By observing Mycobacterial infection of zebrafish embryos in real time the authors were able to discover that granuloma formation is actually a process that is promoted by the pathogen and leads to increased virulence.20 Also using zebrafish embryos, Gao et al.17 studied a locus adjacent to RD1, called RD1ext. Their data demonstrated that like the RD1 mutants, when genes in the RD1ext region were disrupted, the resulting mutants were severely attenuated for dissemination and may also be important for growth in vivo.17 The M. marinum–zebrafish model was also used to determine whether the origin of the clinical isolate directly related to its virulence profile.19 Results from this infection study revealed that the human isolates, from “fish tank granuloma” skin lesions, caused an acute disease in the zebrafish while the isolates from infected fish caused a chronic disease. Using DNA fingerprinting by AFLP, the researchers examined 17 different M. marinum strains isolated from various sources. The strains were genetically grouped into two AFLP clusters, with human isolates mainly sorted into one cluster and the animal isolates grouped into a separate cluster. The authors propose that even though humans become infected with M. marinum through handling fish, human isolates differ genetically from those that only infect fish.19 Further studies using the zebrafish model will allow the determination of virulence factors required for survival and persistence in either fish or humans or both.19
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Given the ease of use and inexpensive cost and maintenance of the zebrafish, another great advantage is using this model for large-scale virulence screens. A recent report documented the use of the zebrafish model in a large-scale screen of streptococcal mutants.46 Signaturetagged mutagenesis was used to produce a large library of specifically tagged mutants of the virulent S. iniae pathogen. In this assay a mixed inoculum of 12 mutants was used to infect individual zebrafish. Screening in this manner greatly increased the number of mutants that could be screened through fewer animals. Isolation and analysis of the hearts and brains of infected adult zebrafish 24 hours after infection identified mutants that could not cause a systemic infection.46 Further analysis of individual mutants proved the technique to be quite sensitive, as virulence attenuation of the mutants compared to wild type ranged from only 2 fold to over 1000 fold as measured by competitive assay.46
ZEBRAFISH AS A HOST DEFENSE MODEL The success of the zebrafish as an effective animal model for the study of vertebrate genomics and development has led to the investigation of the zebrafish immune system. As mentioned earlier, a significant advantage of this model, in terms of relating research to human disease, is that the zebrafish has a fully developed immune system with both innate and adaptive immune responses. This feature of the model provides a means of examining not only the complicated host response but also a pathogen’s ability to counter the innate and adaptive immune defenses. Understanding the zebrafish immune response allows for a full appreciation of the intricate and dynamic interplay between the host and pathogen. The advances that have been made in the study of teleost immunity demonstrate that the zebrafish is an appropriate and highly valuable model for the study of infectious disease. Together immunologic and infectious disease studies provide information that is relevant to the understanding of host–pathogen interactions in the zebrafish that directly relates to human disease.
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Innate immunity is present in all multicellular organisms in some form and provides a defense against invasion of a variety of pathogens. The mechanisms of recognition of these pathogens seem to be evolutionarily conserved from invertebrates to humans. Specific structures on pathogens called pathogen-associated molecular patterns (PAMPs) are recognized by receptors on innate immune cells.47–49 These receptors are referred to as pattern recognition molecules.50 The primary function of the innate immune system is to eliminate invading pathogens; however, in vertebrates possessing adaptive immune systems, the innate immune system has an additional purpose of serving as a trigger for the initiation of the adaptive arm of the immune system. The most significant consequences of this initiation are the upregulation of proinflammatory cytokines and modulation of an antigen-specific immune response. Such a response is crucial to combat an invading pathogen unsuccessfully cleared by the early innate defenses. While the adaptive immune response is a vital part of the host immune system, the early events in the innate immune response are equally significant and deserve attention. The antigen presenting cells and effector cells of the innate immune system are the first factors to come into contact with and recognize an invading pathogen. Although professional antigen presenting cells, or dendritic cells, have not yet been identified in zebrafish, dendritic cells have been suggested to be present in rainbow trout.51 Zebrafish and other teleosts do have antigen-presenting cells, including monocytes and tissue macrophages, which are very similar to mammalian monocytes and macrophages in both appearance and function. The major histocompatibility complex (MHC) molecules are responsible for antigen processing and presentation to lymphocytes. The MHC class I, II, and III gene loci have been identified and characterized in zebrafish.52–54 In histologic sections of adult zebrafish kidneys and spleens macrophages are easily identified.55,56 Distinguishing characteristics of these cells include their large size and their vacuolated, agranular cytoplasm.56,57 While the bone marrow is the source of blood cells in mammals, myelopoiesis occurs in the kidney of the adult zebrafish,
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the primary adult hematopoietic organ in zebrafish.58,59 Zebrafish macrophages are capable of engulfing foreign material, cellular debris and invading microorganisms (Fig. 3). A number of studies have shown engulfed remnants of apoptotic erythrocytes in phagosomes in the process of being degraded.26,60 Macrophages can be observed in embryos as early as 15 hours postfertilization (hpf)56 and Lieschke et al.56 also established that embryonic macrophages were capable of removing carbon particles from the circulation by phagocytosis within 1 hour after microinjection of embryos at 2 dpf. After phagocytosis, even early embryonic macrophages are capable of killing pathogens, where large phagosomes containing degraded material can be observed.26 Adult and embryonic zebrafish macrophages and granulocytes have been shown to be reactive oxygen speciesproducing cells, which contribute to their ability to kill phagocytosed pathogens.61 Herbomel et al.26 examined macrophage activity of 30 hpf embryos challenged with either E. coli or B. subtilis using videomicroscopy. E. coli was easily cleared from circulation completely within 3 hours, with the degradation of the bacteria observed over the next few hours. Low doses of B. subtilis were eliminated in a short amount of time as seen with E. coli infections. However, when embryos were challenged with higher doses of B. subtilis, at a concentration equal to the E. coli experiments, the macrophages became highly vacuolated and activated, but were not able to clear the bacteria. Individual phagosomes rapidly fused, forming single large phagosomes that contained intact bacteria. The bacteria remained intact for a long period of time suggesting the macrophages were having difficulty eliminating the gram-positive bacteria. By 5 hours postinjection, only two of seven embryos had cleared the circulation of bacteria. These two embryos were eventually able to kill and degrade the bacteria and survive, while the other five embryos died as a consequence of being overrun with B. subtilis. In an additional experiment, Herbomel et al.26 illustrated that macrophages are not only able to clear bacteria from the circulation, but these cells will also migrate to a site of infection in an area not nor-
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mally occupied by embryonic macrophages. Similar to mammalian macrophages, the entire population of zebrafish macrophages exhibited an activated state during an infection despite the fact that only a portion of the macrophage population migrated to the site of infection. The interaction between embryonic macrophages and the bacterium M. marinum has also been examined.15 M. marinum causes a chronic infection of macrophages resulting in granuloma formation, mimicking some aspects of the pathology seen in mammalian tuberculosis. Davis et al.15 demonstrated that embryonic zebrafish macrophages could be used to study M. marinum and granuloma formation, clarifying a problem observed in the mouse model of tuberculosis, in which aggregates of macrophages are contaminated with CD4-positive T cells at early stages of granuloma formation. The T-cell contamination of the aggregates makes it difficult to differentiate the roles of individual host factors and cells that mediate granuloma formation. Because mature lymphocytes are not present until 4 weeks postfertilization in zebrafish embryos,62 the researchers were able to determine how early innate responses contribute to the infection and the initiation of granuloma formation. Granulocytes are another cell type that an invading pathogen will encounter early in the infectious process. The zebrafish granulocytes reside in the kidney and circulation.55,56 Two separate granulocyte lineages have been identified in zebrafish to date, heterophils (or neutrophils), which appear to be functionally orthologous with mammalian neutrophils,55,56 and a unique granulocyte. This unique granulocyte seems to exhibit both basophil and eosinophil characteristics,55 and is differentiated by a granular cytoplasm with a small nonsegmented, peripherally located nucleus (Fig. 3).55,56 Basophils have been identified in other teleost species,63 but in zebrafish a distinct basophil lineage has not yet been identified. Zebrafish eosinophils/basophil granulocytes are not well characterized, and because there are significant morphologic differences between this zebrafish granulocyte and mammalian granulocytes, further analysis examining gene expression patterns and functional studies are
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necessary to determine the physiologic role this cell plays in innate immunity. In mammals, granulocytes have a significant role in acute inflammation. Lieschke et al.56 demonstrated that zebrafish heterophil/neutrophil granulocytes also play a role in acute inflammation. Using 2 and 6 dpf embryos, myeloperoxidase activity was assayed before and after the tip of an embryo’s tail was sectioned. Myeloperoxidase is a lysosomal enzyme present in the mammalian neutrophil primary granules and eosinophils. This enzyme is part of the primary defense system of the cell. Myeloperoxidase activity was not evident until 8 hours after trauma. Heterophil/neutrophil granulocytes were visible by electron microscopy in close proximity to the trauma site, and immature heterophils were found within vessels between muscle fibers. Under normal circumstances, these granulocytes are not found in the muscle tissue, which suggests that the cells sensed trauma and migrated through tissues and toward the site of inflammation. Taken together, the morphologic and functional similarity of zebrafish heterophil/neutrophil granulocytes and mammalian neutrophils, emphasizes the usefulness of the zebrafish as a model for the study of human disease. Cytotoxic cells are a third effector cell type of the innate immune system that plays an important role in the clearance of invading microorganisms. Research suggests that zebrafish and other bony fish possess two cytotoxic cell lineages that are involved in innate immunity, which include natural killer (NK) cells64–66 and nonspecific cytotoxic cells (NCC).64,67,68 Both cell types appear to be NK-like and have cytotoxic function.64,66,69 In bony fish, NCCs have been referred to as the bony fish equivalent of NK cells. However, there is evidence suggesting that NCC do not represent the NK lineage. Shen et al.64 has developed multiple cytotoxic catfish cell lines that are similar to human NK cells and have been shown to be distinct from NCC. NCCs are able to recognize protozoan parasites and are morphologically small and agranular; more closely resembling monocytes rather than NK cells, which are large and granular.64–66 A great deal of functional analysis of
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FIG. 3. Collection of images from cytospin preparations of homogenized zebrafish spleens 1 to 3 hours postintramuscular Streptococcus iniae injection. Black arrows point to representative heterophils/neutrophils. White arrows identify basophil/eosinophils. Black arrowheads point to macrophages, some of which contain intracellular S. iniae. White arrowheads identify red blood cells.
these cell lineages is still necessary to fully understand the function of both cell types. Although, there are no true NK receptor orthologs in zebrafish,65 there are related structures recently discovered in bony fish, the novel-immune-type receptors (NITRs).70–72 The function of NITRs has not been definitively shown in fish, but it has been shown that NITRs can stimulate the same signaling pathways as NK receptors when transfected into human NK cells.72 In addition, NITR genes are expressed in many of the catfish NK-like cell lines.70 Taken together, these studies suggest that NITRs may serve as functional orthologs to mammalian NK receptors.73 In addition to cellular components of innate immunity, bony fish also possess effector molecules that are highly important to the function of the innate immune system. Teleost species have a conserved, functional complement system that is similar to higher vertebrates. As with mammals, fish complement can be activated by three pathways, the classical pathway, the lectin pathway and the alternative pathway.74,75 Complement has a critical role in host
defense against invading pathogens, participating in producing opsonizing molecules, inducing inflammatory responses and forming the membrane attack complex.50 A number of the complement proteins have been identified in various bony fish species, and many of these factors have homologies to their equivalent mammalian proteins (reviewed in Holland and Lambris74). Specifically in zebrafish, complement proteins such as factor B, C2, C3, and C4 have been identified.76–78 Although C5 has not yet been examined in zebrafish, this factor has been identified in both rainbow trout and carp.79,80 C5a is a small peptide that is a very important mediator of inflammation and phagocyte recruitment.50 Boshra et al.81 has demonstrated that C5a in rainbow trout is capable of inducing leukocyte migration and contributes to inflammation. This study and others suggest that C5a has a conserved function in both humans and teleost species. There is additional evidence that zebrafish C5a is very similar to human C5a. In an adult zebrafish model for necrotizing fasciitis, a C5a peptidase insertional mutant of Streptococcus pyogenes, a
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strict human pathogen, was found to be completely attenuated (M.N. Neely and D.R. Runft, unpublished data). The data suggest that because S. pyogenes C5a peptidase is necessary for survival in the zebrafish that the zebrafish C5a complement protein can be cleaved by the peptidase, as it is in humans. However, further analysis is necessary to determine whether S. pyogenes C5a peptidase is acting directly on the zebrafish C5a complement factor. Cytokines and chemokines are important signaling molecules that link the immune system. These molecules act in a paracrine manner to affect the behavior of cells from both arms of the immune system. Altmann et al.82 was the first to clone and characterize a type I interferon (IFN) from zebrafish. Interleukin 1 (IL-1) and tumor necrosis factor- (TNF-), have also been identified and examined in the zebrafish.22 These cytokines have important roles in fighting against invading pathogens. IFN has specific antiviral activity, while TNF- and IL-1 contribute to protection against a broader range of pathogens that include viruses, bacteria, and parasitic infections in mammals. IFN synthesis can be stimulated by either viral infection or a synthetic double-stranded (ds) RNA molecule. Upon binding to the appropriate receptor, IFN induces a number of antiviral proteins that are responsible for the degradation or prevention of synthesis of viral RNA.83 These proteins include 2-5-oligoadenylate synthetase, protein kinase p68, and Mx. Altmann et al.82 examined IFN activity by using a construct of the Mx promoter linked to a reporter gene. Expression of zebrafish IFN could be stimulated by synthetic dsRNA, as demonstrated by the upregulation of the IFNinducible Mx promoter. IFN-like activity had previously been studied by looking at the induction of the Mx protein by IFN in salmonid cells.84 These functional investigations serve to illustrate that type I IFN function is conserved between mammals and fish. In an additional experiment conducted by Altmann et al.82 zebrafish hepatocytes were transfected with zebrafish IFN, and then challenged with snakehead rhabdovirus (SHRV). IFN transfected cells showed a reduction in plaque formation as compared to control cells. This study was further supported by an experiment conducted
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by Phelan et al.29 in which reverse transcription-polymerase chain reaction (RT-PCR) analysis of adult and embryo zebrafish infected with SHRV showed upregulation of both IFN and Mx mRNA. The antiviral activity of zebrafish IFN again demonstrates that the cytokine serves a similar function in fish and mammals. TNF- has an important role in initiating an inflammatory response once a pathogen has penetrated the host. IL-1 also participates in the initiation of the nonspecific inflammatory response. Together these proteins work to start a cytokine cascade that ultimately results in inflammatory cell recruitment to the site of infection, activation of these cells, and finally the response prompts activation of the adaptive immune response. Using quantitative real-time PCR analysis, IL-1 and TNF- mRNA expression levels were found to be upregulated in embryos and adult zebrafish infected with E. tarda.22 In a recent publication, Yazawa et al.85 used a construct of the Japanese flounder TNF promoter linked with GFP to examine TNF induction. The construct was introduced into zebrafish embryos by means of microinjection. When these embryos were exposed to lipopolysaccharide (LPS) GFP expression was observed, demonstrating the functionality of the zebrafish regulation of the TNF promoter in response to LPS. These experiments suggest that zebrafish IL-1 and TNF- function to stimulate the immune response as it does in mammals. The toll-like family of receptors (TLRs) is an ancient pattern recognition family, responsible for the detection of conserved markers on the surface of pathogens. These conserved structures distinguish foreign pathogens from the host, and as mentioned above, are called PAMPs.47–49 Upon TLR binding of PAMPs, adapter molecules are recruited to the TLR complex. These adapter proteins share a common Toll/interleukin-1 receptor (TIR) domain with TLRs. Signaling through adapter proteins and subsequently through downstream signaling molecules leads to the activation of mitogen-activated protein (MAP) kinase family members and translocation of NFB to the nucleus, and ultimately results in the transcription of proinflammatory cytokines.86–88
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In total, 24 putative variants of TLRs have been described in zebrafish.89,90 The zebrafish carries one or more copies of orthologs of at least 10 of the human TLR genes. Only one copy of each of four TIR-containing adapter proteins has been identified in zebrafish,89,90 including MyD88, MAL (TIRAP), TRIF (TICAM), and SARM. There is one additional adapter protein gene in humans that has not yet been described in zebrafish, TRAM (TICAM-2). Using RT-PCR analysis Meijer et al.90 confirmed expression of all the predicted TLR genes in adult zebrafish, except for the genes TLR19 and TLR20b; both genes appear to be fish-specific TLR genes and may be expressed only in a developmental stage. This investigation also confirmed the expression of IL-1 and IL-18 receptors and the four predicted TIR domain adaptor genes. To further examine TLR expression in adult zebrafish, the response to M. marinum infection was analyzed. RT-PCR analysis demonstrated that the homologues of human TLR1 (TLR1 and TLR18 in the zebrafish), TLR2, TLR5, and TLR9 genes were expressed at higher levels in the infected fish compared with the controls. While the increased expression of TLR1 and TLR2 are part of a typical host response to Mycobacterium infection, the expression of TLR5 and TLR9 are less easily explained. TLR9 is induced by unmethylated CpG dinucleotide motifs that are commonly found in viral and bacterial genomes. Zebrafish TLR9 may have been stimulated by detection of CpG motifs of M. marinum. In mammals, TLR5 responds to bacterial flagellin, but M. marinum does not produce flagella. The zebrafish TLR5 may have ligand specificities that differ from mammals, but additional function analysis is necessary to determine the role of TLR9 and TLR5 in pathogen recognition in zebrafish. The zebrafish TLR20 and TLR22 also showed increased expression, perhaps providing a fish-specific response to bacterial infection. Expression levels of the IL-1 and IL-18 receptor genes were unaffected by the infection. The zebrafish homologue of MAL showed increased expression in the infected fish, while the expression levels of the other putative TIR domain adapter genes were not responsive to Mycobacterium infection. Despite some differences, it appears that the func-
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tion of TLRs of mammals and teleosts are likely to be highly conserved. In a recently published study, Meijer et al.91 went on to examine the response of adult zebrafish to M. marinum infection by microarray analysis, with a particular interest in looking at the host immune response. Infected fish were sacrificed after 8 weeks postinfection, at which time the infected zebrafish were exhibiting signs of fish tuberculosis. Overall, the data suggested that both an innate and an adaptive immune response had been initiated, with increased expression of a number of complement components, immunoglobulin heavy and light chains and T-cell–specific genes. A number of the upregulated genes included factors involved in the inflammatory response, many of which have been shown to contribute to the response to mycobacterial infections in other mammalian systems. Conversely, there were also several immune related genes that did not show a change in expression. The expression profile may indicate that there is a specific immune response induced by M. marinum. Some of the genes not induced include important signaling molecules of the TLR cell-signaling pathway, MyD88, ECSIT, TRAF6, and TAB1. Interestingly, the signaling molecule IRAK-4 was induced in this study. IRAK-4 associates with TRAF6, leading to activation of NFB, in response to bacterial pathogens (see below). Additional analysis is necessary to determine if IRAK-4 is associating with a different signaling molecule other than TRAF6 in response to mycobacterial infection. Furthermore, a number of significant immune-related genes were not present on any of the three microarray genechips employed for this investigation. Some of the missing genes included the adapter proteins MAL, TRIF, SARM, and all of the zebrafish TLRs except TLR21. Taken together, this study adds a great deal to the understanding of host–pathogen interactions between M. marinum and the zebrafish, but due to the lack of representation of some immunityassociated genes on the microarray chips, as well as some inherent problems with microarrays in general, there are some important questions yet to be addressed. In a second study that supports the idea that zebrafish TLRs function and signal in a con-
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served manner as compared to mammalian TLRs, an orthologue of mammalian TLR3 and two important TLR signaling molecules IRAK4 and TRAF6 were identified and characterized.21 In humans, the adapter proteins contain a death domain that can associate with the death domain of IL-1R–associated kinase (IRAK). Subsequent autophosphorylation of IRAK enables its interaction with tumor necrosis factor-associated factor-6 (TRAF-6), ultimately leading to the activation of nuclear factor B (NFB) and MAP kinase.87 In the interest of examining the mechanisms of the zebrafish TLR signaling pathways, adult zebrafish and embryos were infected with either SHRV or E. tarda. TLR3 is induced during antiviral responses in mammals, resulting in proinflammatory cytokine production by signaling through TRAF6.92 In fact, TRAF6 is involved in all TLR signaling pathways.93 However, IRAK4 has a more specific role in the signaling pathway of TLR4 in response to bacterial pathogens. By looking at TLR3, IRAK-4, and TRAF6, the researchers were able to investigate separate pathways involved in recognizing bacterial and viral pathogens. An NFB-luciferase reporter plasmid was constructed to measure the activation of NFB in zebrafish liver cells. Overexpression of zebrafish IRAK4, TRAF6, or TLR3 was able to stimulate NFB activation in zebrafish liver cells, confirming the involvement of these factors in the activation of the transcription factor NFB, ultimately resulting in transcription of immunomodulatory genes. In addition, messenger RNA expression profiles of each gene in zebrafish embryos and adults were examined by quantitative real-time PCR after infection with SHRV or E. tarda. As anticipated, subsequent exposure to SHRV resulted in upregulation of only TLR3 and TRAF6 mRNA transcripts. However, infection with E. tarda resulted in a surprising increase in mRNA expression of TLR3, as well as the predicted upregulation of IRAK-4 and TRAF6 mRNA transcripts. The TLR3 results with E. tarda may again indicate an additional recognition role of zebrafish TLR3 not found in mammals. A number of studies have provided evidence that TLRs serve as an important link between innate immunity and adaptive immunity in
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mammals.48,94–96 The TLRs recognize a specific invading pathogen, and are capable of inducing the correct cell signaling that results in upregulation of costimulatory molecules and inflammatory cytokines to drive an appropriate adaptive immune response.86 Functional studies to examine adaptive immune responses in zebrafish, specifically looking at activation through TLR signaling, are feasible because teleosts have a developed adaptive immune system,97,98 which includes features such as immunoglobulin (Ig) genes expressed by B cells and T-cell receptor (TCR) genes expressed by T cells. For both mammals and zebrafish it has been shown that Ig and TCR genes are encoded by multiple gene segments distributed in arrays along the chromosome.99–101 As B and T lymphocytes develop, an intricate system of gene segments encoding multiple variable (V), diversity (D), and joining (J) regions of Ig or TCR are joined together by somatic rearrangement, also referred to as V(D)J recombination, to form a continuous functional gene encoding an antigen receptor. Both B immunoglobulin and TCR genes in the zebrafish go through V(D)J recombination.101 The recombination activating genes, rag1 and rag2, catalyze the somatic rearrangement of B immunoglobulin genes and TCR genes,102–104 and both genes have been identified in zebrafish.60 These genes have served as markers to follow development of organs containing lymphocytes.62,105,106 Wienholds et al.107 was able to generate an inactivated rag1 mutant in the zebrafish. This investigation showed that a homozygous rag1 mutant demonstrated loss of function at this locus that resulted in a complete block of V(D)J joining. Despite this severe immunodeficiency syndrome, these mutants were able to survive into adulthood and were fertile. The significance of this study is not only in the functional analysis of the zebrafish rag1 gene, but it also demonstrates a technique to generate loss of function mutations. Such mutations in immune genes are highly useful for understanding the function of the gene and how a particular immune factor impacts the progression of infectious disease. Overall, T cell distribution and function seem to be conserved in comparison with mammals.108 T cells are found in the kidney, phar-
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ynx, intestinal tract, nose, and spleen and under the skin.105,109 Functional and morphologic maturity of the zebrafish immune system occurs at 4–6 weeks postfertilization.62 Teleost show graft rejection105,110 and produce antibodies after immunization.62,111 The first immunoglobulins can be detected at approximately 4 weeks postfertilization.62 Similar to mammals, the major lymphoid organs in the zebrafish include the thymus and gut-associated lymphoid tissues, but the zebrafish anatomy differs in that the fish do not have lymph nodes or bone marrow.112 Although the adaptive immune systems of zebrafish and humans are very similar there are some significant differences worth noting. For research examining a pathogen at a stage in which adaptive immune defenses are engaged, it is important to be aware of these differences. While teleost have been shown to have an antibody response to immunization,62,111 illustrating a functional humoral response, the immunoglobulin heavy chain classes encoded on the zebrafish genome differ somewhat from both humans and mice. Previously it was thought that the zebrafish immunoglobulin heavy chain locus only encoded two heavy chain classes, immunoglobulin and .113 However, recently Danilova et al.99 examined the complete immunoglobulin heavy chain locus and identified a third heavy chain class, immunoglobulin . Furthermore, unlike mammals, class switching does not occur in zebrafish B-lymphocytes.113 An important difference between teleost and mammals is that while both possess activation-induced cytidine deaminase (AID) homologues, which contribute to receptor diversity caused by somatic hypermutation, class-switch recombination does not occur in zebrafish.114,115 The possibility exists that mammalian AID serves a dual function that does not exist in teleost species.
CONCLUSION Even though the use of zebrafish as an infectious disease model is still in the early stages, great strides have already been made. A number of pathogens have been shown to successfully infect and cause disease in the ze-
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brafish host. However, we have only scratched the surface given the vast array of pathogens that could potentially be studied in the zebrafish. The type of questions that can be addressed regarding infectious disease is also just beginning to be explored. For instance, not every pathogen must cause a fatal infection to provide important information on host– pathogen interactions. The zebrafish would be an ideal host in which to study commensal relationships. Such an investigation has recently been shown with the development of the gnotobiotic zebrafish model to determine the influence of indigenous microflora on development.116 A great number of advances have been made in the study of the immune system of zebrafish as well. Future studies will continue to provide important information on the specific immune defenses stimulated during infectious disease. In turn, infectious disease investigations will be able to examine the specific mechanisms employed by a pathogen to counter those host defenses. Given the availability of two stages of development, the transparent embryo with only innate immunity that develops into the adult with both adaptive and innate immune functions, the types of questions that can be investigated are limitless. However, because the zebrafish model is relatively new to the laboratory, there is still a need for reagents and tools to conduct these studies, as well as the continued discovery of important immunity factors. For instance, the identification of additional cell surface markers, like CD3, CD4, and CD8, would aid in the distinction between lymphocyte cell types in both immunological and infectious disease studies using zebrafish. Cytokines play a main role in stimulating and linking host immune responses. The presence of key cytokines like IFN-, IL-10, and IL-12 in other bony fish suggests that these molecules may be present in zebrafish as well.117–120 Defining whether these cytokines and others are expressed in zebrafish and if these molecules serve the same important functions in zebrafish as seen in mammals will further our knowledge about conserved vertebrate immunity. The identification of dendritic cells in the zebrafish, as has been done in the rainbow trout,51 with specific cell markers will also aid
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in determining early innate immune functions. The considerable knowledge already available from the zebrafish development community as well as the number of tools recently developed gives this new model a generous head start. As the model develops, new reagents and cell-specific markers will greatly aid in elucidating specific host–pathogen interactions.
REFERENCES 1. Mahajan-Miklos S, Tan M-W, Rahme LG, Ausubel FM. Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model. Cell 1999;96:47–56. 2. Rahme LG, Stevens EJ, Wolfort SF, Shao J, Tompkins RG, Ausubel FM. Common virulence factors for bacterial pathogenicity in plants and animals. Science 1995;30:1899–1902. 3. Rahme LG, Tan MW, Le L, et al. Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc Natl Acad Sci USA 1997;94:13245– 13250. 4. Tan MW, Rahme LG, Sternberg JA, Tompkins RG, Ausubel FM. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc Natl Acad Sci USA 1999;96: 2408–2413. 5. Mahajan-Miklos S, Rahme LG, Ausubel FM. Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts. Mol Microbiol 2000;37:981–988. 6. Tan M-W. Cross-species infections and their analysis. Annu Rev Microbiol 2002;56:539–565. 7. Darby C, Cosma CL, Thomas JH, Manoil C. Lethal paralysis of Caenothabditis elegans by Psuedomonas aeruginosa. Proc Natl Acad Sci USA 1999;96: 15202–15207. 8. Hagele S, Kohler R, Merkert H, Schleicher M, Hacker J, Steinert M. Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella. Cell Microbiol 2000;2:165–171. 9. Pukatzki S, Kessin RH, Mekalanos JJ. The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum. Proc Natl Acad Sci USA 2002;99:3159–3164. 10. Kopp EB, Medzhitov R. The toll-receptor family and control of innate immunity. Curr Opin Immunol 1999;11:13–18. 11. Dushay MS, Eldon ED. Drosophila immune responses as models for human immunity. Am J Hum Genet 1998;62:10–14. 12. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF. A family of human receptors structurally related to drosophila Toll. Proc Natl Acad Sci USA 1998;95:588–593.
13. Dionne MS, Ghori N, Schneider DS. Drosophila melanogaster is a genetically tractable model host for Mycobacterium marinum. Infect Immun 2003;71: 3540–3550. 14. Mansfield BE, Dionne MS, Schneider DS, Freitag NE. Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster. Cell Microbiol 2003;5:901–911. 15. Davis JM, Clay H, Lewis JL, Ghori N, Herbomel P, Ramakrishnan L. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity 2002;17:693–702. 16. Neely M, Pfeifer J, Caparon MG. Streptococcus-zebrafish model of bacterial pathogenesis. Infect Immun 2002;70:3904–3914. 17. Gao LY, Guo S, McLaughlin B, Morisaki H, Engel JN, Brown EJ. A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion. Mol Microbiol 2004;53:1677–1693. 18. Prouty MG, Correa NE, Barker LP, Jagadeeswaran P, Klose KE. Zebrafish-Mycobacterium marinum model for mycobacterial pathogenesis. FEMS Microbiol Lett 2003;225:177–182. 19. van der Sar AM, Abdallah AM, Sparrius M, Reinders E, Vandenbroucke-Grauls CM, Bitter W. Mycobacterium marinum strains can be divided into two distinct types based on genetic diversity and virulence. Infect Immun 2004;72: 6306–6312. 20. Volkman HE, Clay H, Beery D, Chang JC, Sherman DR, Ramakrishnan L. Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant. PLoS Biol 2004;2:e367. 21. Phelan PE, Mellon MT, Kim CH. Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio). Mol Immunol 2005;42: 1057–1071. 22. Pressley ME, Phelan PE, 3rd, Witten PE, Mellon MT, Kim CH. Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish. Dev Comp Immunol 2005;29:501–513. 23. O’Toole R, Von Hofsten J, Rosqvist R, Olsson PE, Wolf-Watz H. Visualisation of zebrafish infection by GFP-labelled Vibrio anguillarum. Microb Pathog 2004;37:41–46. 24. Miller JD, Neely MN. Zebrafish as a model host for streptococcal pathogenesis. Acta Tropica 2004;91: 53–68. 25. van der Sar AM, Musters RJ, van Eeden FJ, Appelmelk BJ, Vandenbroucke-Grauls CM, Bitter W. Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections. Cell Microbiol 2003;5:601–611. 26. Herbomel P, Thisse B, Thisse C. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 1999;126:3735–3745. 27. Menudier A, Rougier FP, Bosgiraud C. Comparative virulence between different strains of Listeria in ze-
101
EVOLUTION OF THE ZEBRAFISH MODEL
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
brafish (Brachydanio rerio) and mice. Pathol Biol (Paris) 1996;44:783–789. Brenot A, King KY, Janowiak B, Griffith O, Caparon MG. Contribution of glutathione peroxidase to the virulence of Streptococcus pyogenes. Infect Immun 2004;72:408–413. Phelan PE, Pressley ME, Witten PE, Mellon MT, Blake S, Kim CH. Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio). J Virol 2005;79:1842–1852. LaPatra SE, Barone L, Jones GR, Zon LI. Effects of infectious hematopoietic necrosis virus and infections pancreatic necrosis virus infection on hematopoietic precursors of the zebrafish. Blood Cells Mol Dis 2000;26:445–452. Zhao Q, Liu X, Collodi P. Identification and characterization of a novel fibronectin in zebrafish. Exp Cell Res 2001;268:211–219. Liu X, Collodi P. Novel form of fibronectin from zebrafish mediates infectious hematopoietic necrosis virus infection. J Virol 2002;76:492–498. Trede NS, Langenau DM, Traver D, Look AT, Zon LI. The use of zebrafish to understand immunity. Immunity 2004;20:367–379. Austin B, Austin D, Bacterial Fish Pathogens: Disease in Farmed and Wild Fish, 2nd ed. Ellis Horwood, Chichester, 1993. Eldar A, Bejerano Y, Bercovier H. Streptococcus shiloi and Streptococcus difficile: two new streptococcal species causing a meningoencephalitis in fish. Curr Microbiol 1994;28:139–143. Eldar A, Perl S, Frelier PF, Bercovier H. Red Drum Sciaenops ocellatus mortalities associated with Streptococcus iniae infection. Dis Aquat Org 1999;36:121– 127. Gregory SH, Liu C-C. CD8 T-cell-mediated response to Listeria monocytogenes taken up in the liver and replicating within hepatocytes. Immunol Rev 2000;174:112–122. Gregory SH, Sagnimeni AJ, Wing EJ. Effector function of hepatocytes and Kupffer cells in the resolution of systemic bacterial infections. J Leukoc Biol 1992;51:421–424. Rogers HW, Callery MP, Deck B, Unanue ER. Listeria monocytogenes Induces apoptosis of infected hepatocytes. J Immunol 1996;156:679–684. Rogall T, Wolters J, Flohr T, Bottger EC. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J Syst Bacteriol 1990;40:323–330. Pozos TC, Ramakrishnan L. New models for the study of Mycobacterium-host interactions. Curr Opin Immunol 2004;16:499–505. Taylor FBJ, Bryant AE, Blick KE, et al. Staging of the baboon response to group A streptococci anministered intramuscularly: a descriptive study of the clinical symptoms and clinical chemical response patterns. Clin Infect Dis 1999;29:167–177. Hidalgo-Grass C, Dan-Goor M, Maly A, et al. Effect of a bacterial pheromone peptide on host chemokine
44. 45.
46.
47.
48.
49. 50.
51.
52.
53.
54.
55. 56.
57.
58.
59. 60.
61.
degradation in group A streptococcal necrotising soft-tissue infections. Lancet 2004;363:696–703. Behr MA, Small PM. A historical and molecular phylogeny of BCG strains. Vaccine 1999;17:915–922. Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 1996;178:1274–1282. Miller JD, Neely MN. Large-scale screen highlights the importance of capsule for virulence in the zoonotic pathogen, Streptococcus iniae. Infect Immun 2005;73:921–934. Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000;406:782–787. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001;2:675–680. Janeway CA Jr., Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20:197–216. Janeway CA Jr., Travers P, Walport M, Shlomchik MJ, Immunobiology: The Immune System in Health and Disease. 5th ed. Garland Publishing, New York, 2001. Ohta Y, Landis E, Boulay T, Phillips RB, Collet B, Secombes CJ, et al. Homologs of CD83 from elasmobranch and teleost fish. J Immunol 2004;173: 4553–4560. Bingulac-Popovic J, Figueroa F, Sato A, et al. Mapping of mhc class I and class II regions to different linkage groups in the zebrafish, Danio rerio. Immunogenetics 1997;46:129–134. Michalova V, Murray BW, Sultmann H, Klein J. A contig map of the Mhc class I genomic region in the zebrafish reveals ancient synteny. J Immunol 2000;164:5296–5305. Sultmann H, Sato A, Murray BW, et al. Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping. J Immunol 2000;165:6984–6993. Bennett CM, Kanki JP, Rhodes J, et al. Myelopoiesis in the zebrafish, Danio rerio. Blood 2001;98:643–651. Lieschke GJ, Oates AC, Crowhurst MO, Ward AC, Layton JE. Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish. Blood 2001;98:3087–3096. Crowhurst MO, Layton JE, Lieschke GJ. Developmental biology of zebrafish myeloid cells. Int J Dev Biol 2002;46:483–492. Willett CE, Cortes A, Zuasti A, Zapata AG. Early hematopoiesis and developing organs in the zebrafish. Dev Dyn 1999;214:323–336. Zon LI. Developmental biology of hematopoiesis. Blood 1995;86:2876–2891. Willett CE, Cherry JJ, Steiner LA. Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish. Immunogenetics 1997;45:394–404. Hermann AC, Millard PJ, Blake SL, Kim CH. Development of a respiratory burst assay using ze-
102
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73. 74. 75.
76.
77.
PHELPS AND NEELY brafish kidneys and embryos. J Immunol Methods 2004;292:119–129. Lam SH, Chua HL, Gong Z, Lam TJ, Sin YM. Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profiling, in situ hybridization and immunological study. Dev Comp Immunol 2004;28:9–28. Bielek E, Bigaj J, Chadzinska M, Plytycz B. Depletion of head kidney neutrophils and cells with basophilic granules during peritoneal inflammation in the goldfish, Carassius auratus. Folia Biol (Krakow) 1999;47:33–42. Shen L, Stuge TB, Bengten E, et al. Identification and characterization of clonal NK-like cells from channel catfish (Ictalurus punctatus). Dev Comp Immunol 2004;28:139–152. Traver D, Herbomel P, Patton EE, et al. The zebrafish as a model organism to study development of the immune system. Adv Immunol 2003;81:253–330. Yoder JA. Investigating the morphology, function and genetics of cytotoxic cells in bony fish. Comp Biochem Physiol C Toxicol Pharmacol 2004;138: 271–280. Evans DL, Carlson RL, Graves SS, Hogan KT. Nonspecific cytotoxic cells in fish (Ictalurus punctatus). IV. Target cell binding and recycling capacity. Dev Comp Immunol 1984;8:823–833. Greenlee AR, Brown RA, Ristow SS. Nonspecific cytotoxic cells of rainbow trout (Oncorhynchus mykiss) kill YAC-1 targets by both necrotic and apoptic mechanisms. Dev Comp Immunol 1991;15:153–164. Kurata O, Okamoto N, Ikeda Y. Neutrophilic granulocytes in carp, Cyprinus carpio, possess a spontaneous cytotoxic activity. Dev Comp Immunol 1995;19:315–325. Hawke NA, Yoder JA, Haire RN, et al. Extraordinary variation in a diversified family of immune-type receptor genes. Proc Natl Acad Sci USA 2001;98: 13832–13837. Strong SJ, Mueller MG, Litman RT, et al. A novel multigene family encodes diversified variable regions. Proc Natl Acad Sci USA 1999;96:15080–15085. Yoder JA, Mueller MG, Wei S, et al. Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster. Proc Nat Acad Sci 2001;98:6771–6776. Litman GW, Hawke NA, Yoder JA. Novel immunetype receptor genes. Immunol Rev 2001;181:250–259. Holland MC, Lambris JD. The complement system in teleosts. Fish Shellfish Immunol 2002;12:399–420. Sunyer JO, Lambris JD. Evolution and diversity of the complement system of poikilothermic vertebrates. Immunol Rev 1998;166:39–57. Gongora R, Figueroa F, Klein J. Independent duplications of Bf and C3 complement genes in the zebrafish. Scand J Immunol 1998;48:651–658. Samonte IE, Sato A, Mayer WE, Shintani S, Klein J. Linkage relationships of genes coding for alpha2macroglobulin, C3 and C4 in the zebrafish: implica-
78.
79.
80.
81.
82.
83.
84.
85.
86.
87. 88.
89.
90.
91.
92.
tions for the evolution of the complement and Mhc systems. Scand J Immunol 2002;56:344–352. Seeger A, Mayer WE, Klein J. A complement factor B-like cDNA clone from the zebrafish (Brachydanio rerio). Mol Immunol 1996;33:511–520. Boshra H, Li J, Peters R, Hansen J, Matlapudi A, Sunyer JO. Cloning, expression, cellular distribution, and role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in a nonmammalian species. J Immunol 2004;172:4381– 4390. Kato Y, Nakao M, Mutsuro J, Zarkadis IK, Yano T. The complement component C5 of the common carp (Cyprinus carpio): cDNA cloning of two distinct isotypes that differ in a functional site. Immunogenetics 2003;54:807–815. Boshra H, Peters R, Li J, Sunyer JO. Production of recombinant C5a from rainbow trout (Oncorhynchus mykiss): role in leucocyte chemotaxis and respiratory burst. Fish Shellfish Immunol 2004;17: 293–303. Altmann SM, Mellon MT, Distel DL, Kim CH. Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. J Virol 2003;77: 1992–2002. Sen GC, Lengyel P. The interferon system. A bird’s eye view of its biochemistry. J Biol Chem 1992;267:5017–5020. Nygaard R, Husgard S, Sommer AI, Leong JA, Robertsen B. Induction of Mx protein by interferon and double-stranded RNA in salmonid cells. Fish Shellfish Immunol 2000;10:435–450. Yazawa R, Hirono I, Ohira T, Aoki T. Induction of Japanese flounder TNF promoter activity by lipopolysaccharide in zebrafish embryo. Mar Biotechnol (NY) 2005. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2003;85:85–95. Barton GM, Medzhitov R. Toll-like receptor signaling pathways. Science 2003;300:1524–1525. O’Neill LA. The role of MyD88-like adapters in Tolllike receptor signal transduction. Biochem Soc Trans 2003;31:643–647. Jault C, Pichon L, Chluba J. Toll-like receptor gene family and TIR-domain adapters in Danio rerio. Mol Immunol 2004;40:759–771. Meijer AH, Gabby Krens SF, Medina Rodriguez IA, He S, Bitter W, Ewa Snaar-Jagalska B, et al. Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish. Mol Immunol 2004;40:773–783. Meijer AH, Verbeek FJ, Salas-Vidal E, CorredorAdamez M, Bussman J, van der Sar AM, et al. Transcriptome profiling of adult zebrafish at the late stage of chronic tuberculosis due to Mycobacterium marinum infection. Mol Immunol 2005;42:1185–1203. Jiang Z, Zamanian-Daryoush M, Nie H, Silva AM, Williams BR, Li X. Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFkappa B
EVOLUTION OF THE ZEBRAFISH MODEL
93.
94.
95.
96.
97.
98. 99.
100.
101.
102.
103.
104.
105.
106.
107.
and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing the signaling components TLR3-TRAF6TAK1-TAB2-PKR. J Biol Chem 2003;278:16713– 16719. Suzuki N, Suzuki S, Yeh WC. IRAK-4 as the central TIR signaling mediator in innate immunity. Trends Immunol 2002;23:503–506. Kaisho T, Akira S. Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice. Trends Immunol 2001;22:78–83. Pasare C, Medzhitov R. Toll pathway-dependent blockade of CD4CD25 T cell-mediated suppression by dendritic cells. Science 2003;299:1033–1036. Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2001;2:947–950. Partula S, de Guerra A, Fellah JS, Charlemagne J. Structure and diversity of the T cell antigen receptor beta-chain in a teleost fish. J Immunol 1995;155: 699–706. Warr GW. The immunoglobulin genes of fish. Dev Comp Immunol 1995;19:1–12. Danilova N, Bussmann J, Jekosch K, Steiner LA. The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z. Nat Immunol 2005;6:295–302. Danilova N, Steiner LA. B cells develop in the zebrafish pancreas. Proc Natl Acad Sci USA 2002;99: 13711–13716. Haire RN, Rast JP, Litman RT, Litman GW. Characterization of three isotypes of immunoglobulin light chains and T-cell antigen receptor alpha in zebrafish. Immunogenetics 2000;51:915–923. Oettinger MA, Schatz DG, Gorka C, Baltimore D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 1990;248: 1517–1523. Schatz DG, Oettinger MA, Baltimore D. The V(D)J recombination activating gene, RAG-1. Cell 1989;59: 1035–1048. van Gent DC, McBlane JF, Ramsden DA, Sadofsky MJ, Hesse JE, Gellert M. Initiation of V(D)J recombination in a cell-free system. Cell 1995;81:925–934. Langenau DM, Ferrando AA, Traver D, et al. In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish. Proc Natl Acad Sci USA 2004;101:7369–7374. Willett CE, Zapata AG, Hopkins N, Steiner LA. Expression of zebrafish rag genes during early development identifies the thymus. Dev Biol 1997;182: 331–341. Wienholds E, Schulte-Merker S, Walderich B, Plasterk RH. Target-selected inactivation of the zebrafish rag1 gene. Science 2002;297:99–102.
103 108. Langenau DM, Zon LI. The zebrafish: a new model of T-cell and thymic development. Nat Rev Immunol 2005;5:307–317. 109. Danilova N, Hohman VS, Sacher F, Ota T, Willett CE, Steiner LA. T cells and the thymus in developing zebrafish. Dev Comp Immunol 2004;28:755–767. 110. Traver D, Winzeler A, Stern HM, et al. Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation. Blood 2004;104:1298–1305. 111. Anderson ED, Mourich DV, Fahrenkrug SC, LaPatra S, Shepherd J, Leong JA. Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus. Mol Mar Biol Biotechnol 1996;5:114–122. 112. Boehm T, Bleul CC, Schorpp M. Genetic dissection of thymus development in mouse and zebrafish. Immunol Rev 2003;195:15–27. 113. Cannon JP, Haire RN, Rast JP, Litman GW. The phylogenetic origins of the antigen-binding receptors and somatic diversification mechanisms. Immunol Rev 2004;200:12–22. 114. Stavnezer J, Amemiya CT. Evolution of isotype switching. Semin Immunol 2004;16:257–275. 115. Zhao Y, Pan-Hammarstrom Q, Zhao Z, Hammarstrom L. Identification of the activation-induced cytidine deaminase gene from zebrafish: an evolutionary analysis. Dev Comp Immunol 2005;29:61–71. 116. Rawls JF, Samuel BS, Gordon JI. Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota. Proc Natl Acad Sci USA 2004;101:4596–4601. 117. Inoue Y, Kamota S, Ito K, et al. Molecular cloning and expression analysis of rainbow trout (Oncorhynchus mykiss) interleukin-10 cDNAs. Fish Shellfish Immunol 2005;18:335–344. 118. Savan R, Igawa D, Sakai M. Cloning, characterization and expression analysis of interleukin-10 from the common carp, Cyprinus carpio L. Eur J Biochem 2003;270:4647–4654. 119. Yoshiura Y, Kiryu I, Fujiwara A, et al. Identification and characterization of Fugu orthologues of mammalian interleukin-12 subunits. Immunogenetics 2003;55:296–306. 120. Zou J, Yoshiura Y, Dijkstra JM, Sakai M, Ototake M, Secombes C. Identification of an interferon gamma homologue in Fugu, Takifugu rubripes. Fish Shellfish Immunol 2004;17:403–409.
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