Chapter 2 Animal Models of Tuberculosis : Zebrafish

Over the past decade the zebrafish (Danio rerio) has become an attractive new vertebrate model organism for studying mycobacterial pathogenesis. The combination of mediumthroughput screening and real-time in vivo visualization has allowed new ways to dissect host pathogenic interaction in a vertebrate host. Furthermore, genetic screens on the host and bacterial sides have elucidated new mechanisms involved in the initiation of granuloma formation and the importance of a balanced immune response for control of mycobacterial pathogens. This article will highlight the unique features of the zebrafish– Mycobacterium marinum infection model and its added value for tuberculosis research. Animal Models of Tuberculosis: Zebrafish 2 Why would one use zebrafish (Danio rerio) to study tuberculosis (TB)? Although zebrafish are vertebrates, they do not have lungs, an obvious caveat for studying a pulmonary disease. Furthermore, at present it is unclear whether Mycobacterium tuberculosis can give rise to successful infections in cold-blooded animals. Robert Koch tried to infect coldblooded animals, including a turtle, a goldfish, three eels, and five frogs. After two months, none of them showed any sign of disease, whereas most mammals were either clearly ill or showed tubercles upon autopsy (Koch, 1884). Despite these drawbacks, zebrafish have emerged as a valuable organism to study infectious diseases and especially TB (Grunwald and Eisen, 2002; Meeker and Trede, 2008; Ramakrishnan, 2013). The power of the model, real-time imaging of biological processes, was first exploited for TB by the group of Ramakrishnan (Davis et al., 2002), leading the way to study mycobacterial virulence factors and host characteristics in real time in a living vertebrate animal. In recent years, the strength of the zebrafish model has been greatly extended with the increasing availability of transgenic zebrafish lines, improved imaging techniques, and a growing list of genetic tools and large-scale mutant analysis. This article will highlight the unique features of the zebrafish–Mycobacterium marinum infection model and its added value for TB research. What is the zebrafish-M. marinum model of tuberculosis? To appreciate the zebrafish—M. marinum model of TB, it is important to discuss the basic traits and tools of both the zebrafish and its natural pathogen M. marinum. General properties of zebrafish Advantageous features of the zebrafish include their small size (adults are 3to 5-cm long), the possibility of keeping them at high population density (5 fish/L), and their ease of breeding—a single female can lay up to 300 eggs a week (Meijer and Spaink, 2011). Zebrafish embryos develop externally and are transparent during embryo and larval stages, making it possible to follow host–pathogen interaction in real time. In contrast to other animal models, the zebrafish can be studied during the first weeks of development. In this period, the embryo solely relies on the innate immune system (Figure 1) (Meeker and Trede, 2008; Novoa and Figueras, 2012; van der Sar et al., 2004b; Van Der Vaart et al., 2012), which provides the opportunity to study the contribution of innate immunity to disease in an isolated fashion. Furthermore, it allows for distinguishing between this arm of immunity and a combined innate and adaptive immune response in the context of infection, like in adult fish, which have a complex adaptive immune system akin to that of mammals (Figure 1) (Meeker and Trede, 2008; Renshaw and Trede, 2012; Traver et al., 2003; Van Der Vaart et al., 2012). The finalized whole-genome sequence of zebrafish (Howe et al., 2013) reveals that ~70% of human genes have at least one obvious zebrafish orthologue. Because of the genetic possibilities (Amsterdam and Hopkins, 2006; Blackburn et al., 2013; Lesley and Ramakrishnan, 2008; Meijer and Spaink, 2011) and the Animal Models of Tuberculosis: Zebrafish 3 Figure 1. Development of zebrafish immunity in comparison with the human immune system A Z eb ra fi sh H u m an D ev el o p m en t R ef