Leptospira interrogans Stably Infects Zebrafish Embryos, Altering Phagocyte Behavior and Homing to Specific Tissues J. Muse Davis 1 , David A. Haake 2,3 *, Lalita Ramakrishnan 4 * 1 Immunology and Molecular Pathogenesis Graduate Program, Emory University, Atlanta, Georgia, United States of America, 2 Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of America, 3 Departments of Medicine and Urology, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America, 4 Departments of Microbiology, Medicine and Immunology, University of Washington, Seattle, Washington, United States of America Abstract Leptospirosis is an extremely widespread zoonotic infection with outcomes ranging from subclinical infection to fatal Weil’s syndrome. Despite the global impact of the disease, key aspects of its pathogenesis remain unclear. To examine in detail the earliest steps in the host response to leptospires, we used fluorescently labelled Leptospira interrogans serovar Copenhageni to infect 30 hour post fertilization zebrafish embryos by either the caudal vein or hindbrain ventricle. These embryos have functional innate immunity but have not yet developed an adaptive immune system. Furthermore, they are optically transparent, allowing direct visualization of host–pathogen interactions from the moment of infection. We observed rapid uptake of leptospires by phagocytes, followed by persistent, intracellular infection over the first 48 hours. Phagocytosis of leptospires occasionally resulted in formation of large cellular vesicles consistent with apoptotic bodies. By 24 hours, clusters of infected phagocytes were accumulating lateral to the dorsal artery, presumably in early hematopoietic tissue. Our observations suggest that phagocytosis may be a key defense mechanism in the early stages of leptospirosis, and that phagocytic cells play roles in immunopathogenesis and likely in the dissemination of leptospires to specific target tissues. Citation: Davis JM, Haake DA, Ramakrishnan L (2009) Leptospira interrogans Stably Infects Zebrafish Embryos, Altering Phagocyte Behavior and Homing to Specific Tissues. PLoS Negl Trop Dis 3(6): e463. doi:10.1371/journal.pntd.0000463 Editor: Mathieu Picardeau, Institut Pasteur, France Received January 5, 2009; Accepted May 20, 2009; Published June 23, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: J.M.D. was supported by an American Society for Engineering Education predoctoral fellowship. D.A.H. was supported by VA Medical Research Funds, and by NIH/NIAID grant AI-34431. L.R. was supported by NIH R01 AI-54503 and the Burroughs Wellcome Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dhaake@ucla.edu (DAH); lalitar@u.washington.edu (LR) Introduction Though traditionally thought of as a tropical disease, leptospi- rosis is endemic worldwide due to widespread infection of urban and sylvatic rodents and other animal reservoir hosts. In areas of the world with high levels of rodent exposure, human infection is common and frequently progresses to serious disease or death [1,2]. Although much has been learned about the biology and transmission of Leptospira species, the mechanisms of their pathogenesis and host colonization remain largely unknown. Leptospires colonize the renal tubules of reservoir hosts, from where they are shed in the urine and infect new hosts via mucosal surfaces and abraded skin. In the reservoir host, there is transient low-level hematogenous dissemination, followed by chronic infection limited to the kidney [1,3,4]. In contrast, susceptible hosts experience a heavy burden of infection in the bloodstream and multiple organs. The eventual antibody response precipitates an intense inflammatory reaction associated with hepatorenal failure. A key difference between reservoir and susceptible hosts is the ability of the TLR4 innate immune receptor to recognize leptospiral lipopolysaccharide (LPS) [5,6]. Murine peritoneal macrophages are strongly stimulated by purified leptospiral LPS, while human macrophages are unable to respond to leptospiral LPS via the TLR4 pathway [5]. Taken together, these studies suggest that early containment of infection via innate mechanisms, including recognition of leptospiral antigens and phagocytosis by macrophages, is essential for effective immune defense [7]. Previous in vitro studies have demonstrated that macrophages are capable of phagocytosing leptospires [8,9]. A variety of animal models of leptospirosis have been established, each with unique advantages and drawbacks. Guinea pigs [10] and hamsters [11,12] are the primary models of hosts susceptible to acute disease, while several animals including mice [4], rats [3], monkeys [13], dogs [14] and skunks [15] can be experimentally infected and seem variously plausible as models of reservoir hosts. It is not certain to what degree these various model hosts retain features of natural infection and colonization. The zebrafish is increasingly used as a model organism for bacterial pathogenesis, with published studies of adult infection with pathogens including mycobacteria [16], streptococci [17], and Edwarsiella [18]. The ability to conduct forward genetic screens, along with the economy of infecting large numbers of animals are key advantages to this model [19,20]. Beyond these, the zebrafish embryo allows unparalleled in vivo microscopy and tracking of host-pathogen interactions involving fluorescently labeled bacteria. Minute details of the early steps of bacterial pathogenesis have been published using zebrafish embryos infected with Mycobacterium marinum [21,22,23], Salmonella enterica [22,24] and Pseudomonas aeruginosa [25]. By 32 hours post fertilization a zebrafish embryo has a circulatory system and a www.plosntds.org 1 June 2009 | Volume 3 | Issue 6 | e463