WORLD AQUACULTURE 29 aquaculture production (Harvey 2006) and Edwardsiella ictaluri is one of the most important pathogens in this industry (Shoemaker et al. 1999 ). The most highly up-regulated group of catfish genes following E. ictaluri infection are involved in iron homeostasis, including intelectin, haptoglobin, haemopexin, ceruloplasmin, transferrin and ferritin (Peatman et al. 2007, 2008, Takano et al. 2008, Liu et al. 2011a, b). This indicates there is a competition for iron between the catfish host and the bacterial pathogen E. ictaluri. Recently we characterized the iron acquisition system of E. ictaluri and evaluated its influence on pathogenesis and vaccine design for catfish (Santander and Curtiss 2012) (Santander et al. in press). We observed that E. ictaluri, grown in the absence of iron, up-regulate an outer membrane protein (Fig. 1). Also, this outer membrane protein is up-regulated in E. ictaluri Dfur mutant (Fig. 1). This outer membrane protein was identified as a hemehemoglobin TonB dependent receptor protein or HemR. The hemR gene of E. ictaluri is located in an operon that possesses the genes required for transport of heme-hemoglobin through the bacterial membrane (Santander et al. in press). E. ictaluri has a heme-hemoglobin acquisition system, regulated in a Fur–iron dependent fashion in the absence of siderophores (Fig. 1). The absence of siderophores in E. ictaluri is consistent with previous observations (Thune et al. 1999) and with the lack of known genes related to synthesis of siderophores (Williams et al. 2012). Although the absence of siderophores is not common in bacterial pathogens, the systemic nature of E. ictaluri pathogenesis correlates with the absence of siderophores. E. ictaluri Δfur and Live Attenuated Vaccine Development The ideal live attenuated bacterial vaccine should be sensitive to antibiotics, attenuated and immunogenic. Previously we adapted the suicide vector sacBR technology to precisely delete genes from the chromosome of E. ictaluri, without leaving antibiotic resistant genes or genetic scars in the chromosome (Santander et al. 2010). With the same technology, we deleted the fur gene of E. ictaluri (Fig. 1). The mutant E. ictaluri Dfur is attenuated in zebrafish (Danio rerio) and catfish hosts (Fig. 2). Synthesis of iron uptake outer membrane proteins (IROMPs), like HemR, is up-regulated inside the host after invasion. Bacterial IROMPs are not exposed to the fish immune system until a systemic infection is established. We hypothesized that synthesis of IROMPs by the E. ictaluri Dfur mutant would trigger a protective immune response in the immunized fish. After immunization with the E. ictaluri Dfur mutant, fish were protected from infection in an immersion challenge with wildtype E. ictaluri four weeks after immunization (Fig. 3). Implications for Vaccine Design The ideal vaccine for finfish aquaculture should be a “needle free” immersion-oral type that allows oral boost immunization at low cost. Well-designed, live-attenuated bacterial vaccines achieve this goal using mutant bacteria that colonize deep lymphoid tissues, triggering a potent immune response. Design of a live attenuated bacterial vaccine must consider attenuation and immune protection. For instance, the currently licensed spontaneous rifampicin-resistant (Rifr) E. ictaluri vaccine colonizes deep lymphoid tissues of fish but confers only a modest level of immune protection (Klesius and Shoemaker 1999, Williams and Lawrence 2005, Shoemaker et al. 1999). The E. ictaluri Rifr vaccine strain was selected by 33 serial passages through progressively increasing concentrations of rifampicin (Klesius and Shoemaker 2000), accumulating a set of unknown genetic alterations forced by in vitro selection. These alterations include rough lipopolysaccharide, alteration of outer membrane proteins, and changes in fatty acid content and catabolic pathways (Arias et al. 2003). This precludes easy comprehension of attenuation mechanisms and immunogenicity of vaccine in the fish host. Live attenuated vaccines for other fish diseases, such as F. psychophilum, are being developed using this selection method (LaFrentz et al. 2008). An E. ictaluri novobiocin-resistant vaccine strain has been proposed as a strategy against E. ictaluri (Pridgeon and Klesius 2010). Results thus far indicate that this general approach can result in the development of effective live attenuated bacterial vaccines for fish. Notes 1 Javier Santander, The Biodesign Institute, Center for Infectious Diseases and Vaccinology. Arizona State University. PO Box 875401. 1001 S. McAllister Avenue. Tempe, AZ 85287-5401. Tel 480-727-9047. Fax 480727-0466. Tempe, Arizona 85287. E-mail: jasantanderm@asu.edu Acknowledgments This work was supported by USDA grant CRIS-ARZR- 2009-01801, Comisión Nacional de Investigación Científica y Tecnológica (CONICYT), Gestión Propia Fellowship, Chile. I thank Ignacia Diaz for manuscript editing. (CONTINUED ON PAGE 30) FIGURE 3. Catfish immersion challenged with E. ictaluri wild type six weeks after immersion or oral vaccination with E. ictaluri Dfur-35 (107 CFU/dose) (Santander et al. in press).
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