Immune response of zebrafish (Danio rerio) against a newly isolated bacterial pathogen Aeromonas hydrophila I. Rodrı ´guez, B. Novoa, A. Figueras* Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Eduardo Cabello 6, Vigo, Spain Received 11 February 2008; revised 7 May 2008; accepted 11 May 2008 Available online 18 May 2008 KEYWORDS Zebrafish; Aeromonas; Cytokine; Inflammation; Immune response; Flow cytometry; Extracellular products Abstract A strain of Aeromonas hydrophila associated with unusual mortalities in zebrafish (Danio rerio) culture facilities was isolated, identified and characterized. In challenge exper- iments, adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection with viable bacteria and its extracellular products (ECPs) reaching very high mortalities in a few hours. The infection, by the viable bacteria or the ECPs, caused cell death in kidney, due to the cytotoxic and haemolytic activities of the bacterial ECPs. Moreover, the infection affected the release of oxygen (ROS) and nitrogen (NO) reactive free radicals. To determine if this A. hydrophila infection induces an inflammatory response, mRNA expression levels of tumour necrosis factor-a (TNFa), interleukin-1b (IL-1b), interferon-g (IFNg) and inducible nitric oxide (iNOS) were assessed by real time PCR. The expression levels of TNFa, IL-1b and IFNg were upregulated in the kidneys of infected zebrafish with viable bacteria, heat-killed bacteria and ECPs. Expression levels of iNOS were upregulated by ECPs. Mortality rate (LD 50 ) and histo- pathology were also determined. ª 2008 Elsevier Ltd. All rights reserved. Introduction Zebrafish (Danio rerio) has been extensively used to study vertebrate development [1] and recently the interest has grown in the fields of human disease, cancer and immunol- ogy [2e10]. The zebrafish with a complete (innate and adaptative) immune system has advantages over other ver- tebrate infection models, such as mice and rats, because of its small size, relatively rapid life cycle and ease of breed- ing, transparency of early life stages and rapid growth allowing a high number of genetic screens and real-time visualization. Experimental infections using zebrafish have been conducted with different bacteria [11e17] and viruses [18e21]. Aeromonas spp. are ubiquitous aquatic ecosystems bacteria [22]. In particular, Aeromonas hydrophila has been reported as an important pathogen for humans and for lower vertebrates, including amphibians, reptiles and fish [23]. These bacteria have often been isolated from humans with diarrhoea [24e26] and in systemic illness of * Corresponding author. E-mail address: pato1@iim.csic.es (A. Figueras). 1050-4648/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.05.002 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/fsi Fish & Shellfish Immunology (2008) 25, 239e249