Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish

Abstract

The zebrafish (Danio rerio) is a widely used model for developmental biology, neurobiology, toxicology, and genetic disease. Recently, the zebrafish has been recognized as a valuable model for infectious disease and immunity. In this study the pathogenesis and inflammatory cytokine response of zebrafish to experimental Edwardsiella tarda infection was characterized. In challenge experiments, zebrafish embryos were susceptible to infection by immersion. Adult fish were susceptible to challenge by intraperitoneal (ip) injection but not static immersion unless the epithelial layer was perturbed by scraping prior to exposure. To determine if E. tarda infection induces a typical acute inflammatory response, mRNA expression levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) were assessed by quantitative real-time PCR. The expression levels of IL-1β and TNFα were significantly upregulated in infected zebrafish embryos and adults. The methods developed in this study will be particularly valuable for targeted gene disruption studies of host immune components and in zebrafish genetic screens.

Introduction

Recently, zebrafish have been recognized for their value as a model for infectious disease. Genomic comparisons and the discovery of evolutionarily conserved host defense strategies, such as the interferon mediated anti-viral response [1], [2] and toll-like receptors [3], [4], suggest that studies of infectious disease in zebrafish will lead to a better understanding of non-specific immunity and resistance. Several research groups have attempted to identify pathogens that can infect and cause disease in zebrafish. In a zebrafish model for streptococcal infection published by Neely et al. [5], adult zebrafish were exposed to Streptococcus iniae and Streptococcus pyogenes by intraperitoneal (ip) and intramuscular (im) injection. Davis et al. [6] found that injection of zebrafish embryos with Mycobacterium marinum led to the aggregation of macrophages into granuloma-like structures, a potential model for tuberculosis. Adult zebrafish that were injected ip with M. marinum were shown to develop granuloma-like lesions and displayed acute or chronic infections in a dose-dependent manner [7]. Salmonella typhimurium expressing the red fluorescent protein, DsRed, has been used to monitor the progression of infection in zebrafish embryos injected with these bacteria [8].

Edwardsiella tarda was chosen for this study because it has been established as a significant pathogen in a variety of fish species and grows at temperatures suitable for zebrafish maintenance. A member of the Enterobacteriacae family, this intracellular pathogen grows at an optimum temperature range of 30–37 °C [9]. In the species most commonly associated with E. tarda infection, channel catfish (Ictalurus punctatus), Japanese eel (Anguilla japonica), and Japanese founder (Paralichthys olivaceus), the disease caused by E. tarda, edwardsiellosis, is a generalized septicemia, which is often associated with poor water quality and stress [10]. Disease signs may include small cutaneous lesions that can develop into necrotic abscesses, distended abdomen and swollen anus due to the accumulation of ascitic fluid, pigment loss, enlarged kidney, and abscesses on internal organs [10]. In addition to finfish, E. tarda has been found to infect other species including reptiles, birds, amphibians, aquatic mammals, and humans [10]. Natural infection of fish is thought to be through waterborne contact with E. tarda. Susceptible fish species may take up E. tarda through the skin, gills, or oral route [11], [12]. In this study, we examined E. tarda as a potential pathogen capable of causing disease and stimulating the inflammatory immune response in both zebrafish embryos and adults.

When a pathogen is able to penetrate the external barriers of an organism and cause infection, initially a non-specific inflammatory response is triggered. This immediate and vigorous response is mediated by the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα). These cytokines play a critical role in initiating the pro-inflammatory cytokine cascade, in recruitment and activation of macrophages, and in stimulation of the adaptive immune response. IL-1β has been isolated from several fish species including rainbow trout (Oncorhynchus mykiss) [13], carp (Cyprinus carpio) [14], sea bass (Dicentrarchus labrax) [15], sea bream (Sparus aurata) [16], spotted catshark (Scyliorhinus canicula) [17] and, most recently, zebrafish (Accession # AY340959). TNFα has been cloned and characterized in rainbow trout [18], brook trout (Salvelinus fontinalis) [19], carp [20], channel catfish [21], sea bream [22], Japanese flounder [23], and zebrafish (Accession # AY427649).

We report the successful infection of zebrafish embryos and adults with E. tarda, resulting in the induction of disease-related immune responses and pathology. Experimental infection of zebrafish with E. tarda produced infection kinetics and histopathology that are indicative of acute infection in both embryos and adults. Analyses of IL-1β and TNFα mRNA expression profiles revealed evidence of upregulation of the inflammatory response in zebrafish embryos and adults as a result of E. tarda infection. The methods and results presented in this study provide valuable tools for the further investigation of host–pathogen interactions and innate immunity in the zebrafish model.