Mycobacterium marinum Infection of Adult Zebrafish Causes Caseating Granulomatous Tuberculosis and Is Moderated by Adaptive Immunity

Department of Microbiology, Box 357242, University of Washington, Seattle, WA 98195, USA.
Infection and Immunity (Impact Factor: 3.73). 12/2006; 74(11):6108-17. DOI: 10.1128/IAI.00887-06
Source: PubMed


The zebrafish, a genetically tractable model vertebrate, is naturally susceptible to tuberculosis caused by Mycobacterium marinum, a close genetic relative of the causative agent of human tuberculosis, Mycobacterium tuberculosis. We previously developed a zebrafish embryo-M. marinum infection model to study host-pathogen interactions in the context of innate immunity. Here, we have constructed a flowthrough
fish facility for the large-scale longitudinal study of M. marinum-induced tuberculosis in adult zebrafish where both innate and adaptive immunity are operant. We find that zebrafish are exquisitely
susceptible to M. marinum strain M. Intraperitoneal injection of five organisms produces persistent granulomatous tuberculosis, while the injection
of ∼9,000 organisms leads to acute, fulminant disease. Bacterial burden, extent of disease, pathology, and host mortality
progress in a time- and dose-dependent fashion. Zebrafish tuberculous granulomas undergo caseous necrosis, similar to human
tuberculous granulomas. In contrast to mammalian tuberculous granulomas, zebrafish lesions contain few lymphocytes, calling
into question the role of adaptive immunity in fish tuberculosis. However, like rag1 mutant mice infected with M. tuberculosis, we find that rag1 mutant zebrafish are hypersusceptible to M. marinum infection, demonstrating that the control of fish tuberculosis is dependent on adaptive immunity. We confirm the previous
finding that M. marinum ΔRD1 mutants are attenuated in adult zebrafish and extend this finding to show that ΔRD1 predominantly produces nonnecrotizing,
loose macrophage aggregates. This observation suggests that the macrophage aggregation defect associated with ΔRD1 attenuation
in zebrafish embryos is ongoing during adult infection.

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Available from: Lynn Connolly, Aug 04, 2014
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    • "The utility of goldfish for studying mycobacterial infection was demonstrated as early as 1926 by Joseph D. Aronson, who first identified M. marinum as a pathogen of fish in the Philadelphia aquarium (Aronson, 1926). Since then, several fish species, including zebrafish, goldfish, and medaka have been used as an in vivo model system to elucidate the pathology caused by mycobacteria (Broussard and Ennis, 2007; Hodgkinson et al., 2012; Swaim et al., 2006; Talaat et al., 1999, 1998), as well as in vitro, using primary kidney macrophages to assess their anti-mycobacterial responses (Grayfer et al., 2011; Hodgkinson et al., 2012). Recently, our group isolated and characterized goldfish primary kidney neutrophils that remained viable in culture for several days and were also functionally similar to mammalian neutrophils in that they were highly motile, exhibited degranulation and produced a robust respiratory burst response, (Katzenback and Belosevic, 2009). "
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    ABSTRACT: The lack of a reliable mammalian neutrophil in vitro culture system has restricted our ability to examine their precise roles in mycobacterial infections. Previously, we developed the procedures for the isolation and culture of primary kidney-derived neutrophil-like cells from goldfish that are functionally and morphologically similar to mammalian neutrophils. The cultured primary goldfish neutrophils exhibited prolonged viability and functional effector responses. In this study, we demonstrate that when exposed to live or heat-killed Mycobacterium marinum, goldfish neutrophils increased their mRNA levels for several pro-inflammatory cytokines (il-1β1, il-1β2, tnfα-1, tnfα-2) and the cytokine receptors (ifngr1-1, tnfr1, tnfr2). These neutrophils also exhibited chemotaxis toward live mycobacteria, internalized the bacilli, and produced reactive oxygen intermediates (ROI) in response to pathogen exposure. The survival of intracellular mycobacteria was significantly reduced in activated neutrophils, indicating a robust killing response by these teleost granulocytes. We suggest that this goldfish primary neutrophil in vitro model system will provide important information regarding neutrophil-mediated host defense mechanisms against mycobacteria in teleosts as well as in higher vertebrates. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Jul 2015 · Developmental & Comparative Immunology
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    • "During the last decade, the zebrafish (Danio rerio) has been positioned as a powerful model for immunity research not only for other fish species, but also for mammals [15] [16] [17] [18] [19]. "
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    ABSTRACT: Flavobacterium psychrophilum is a Gram-negative bacterium, responsible for the bacterial cold-water disease and the rainbow trout fry syndrome in freshwater salmonid fish. At present, there is only one commercial vaccine in Chile, made with two Chilean F. psychrophilum isolates and another licensed in Europe. The present study analyzed neutrophil migration, as a marker of innate immune activation, in zebrafish (Danio rerio) in response to different F. psychrophilum bath vaccines, which is the first step in evaluating vaccine effectiveness and efficiency in fish. Results indicated that bacterins of the LM-02-Fp isolate were more immunogenic than those from the LM-13-Fp isolate. However, no differences were observed between the same bacteria inactivated by either formaldehyde or heat. Importantly, the same vaccine formulation without an adjuvant only triggered a mild neutrophil migration compared to the complete vaccine. Observations also found that, after a year of storage at 4°C, the activation of the innate immune system by the different vaccines was considerably decreased. Finally, new vaccine formulations prepared with heat and formaldehyde inactivated LM-02-Fp were significantly more efficient than the available commercial vaccine in regard to stimulating the innate immune system.
    Full-text · Article · Feb 2015 · Journal of Immunology Research
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    • "This study is, from our point of view, the most important evidence of how the zebrafish model can be used to validate and to re-postulate host–pathogen interactions during mycobacterial infection. On the other hand, Swaim et al. (2006) reported that lymphocytes play the same critical role in controlling mycobacterial infection in fishes and mammals by the use of a defective zebrafish mutant in the rag1 gene. They also demonstrated that bacteria defective in RD1 region are also attenuated in zebrafish. "
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    ABSTRACT: Biological disease models can be difficult and costly to develop and use on a routine basis. Particularly, in vivo lung infection models performed to study lung pathologies use to be laborious, demand a great time and commonly are associated with ethical issues. When infections in experimental animals are used, they need to be refined, defined, and validated for their intended purpose. Therefore, alternative and easy to handle models of experimental infections are still needed to test the virulence of bacterial lung pathogens. Because non-mammalian models have less ethical and cost constraints as a subjects for experimentation, in some cases would be appropriated to include these models as valuable tools to explore host-pathogen interactions. Numerous scientific data have been argued to the more extensive use of several kinds of alternative models, such as, the vertebrate zebrafish (Danio rerio), and non-vertebrate insects and nematodes (e.g., Caenorhabditis elegans) in the study of diverse infectious agents that affect humans. Here, we review the use of these vertebrate and non-vertebrate models in the study of bacterial agents, which are considered the principal causes of lung injury. Curiously none of these animals have a respiratory system as in air-breathing vertebrates, where respiration takes place in lungs. Despite this fact, with the present review we sought to provide elements in favor of the use of these alternative animal models of infection to reveal the molecular signatures of host-pathogen interactions.
    Full-text · Article · Feb 2015 · Frontiers in Microbiology
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