Fish and Shellfsh Immunology 107 (2020) 497–510 Available online 8 November 2020 1050-4648/© 2020 Elsevier Ltd. All rights reserved. Full length article Pathogenicity and immunogenicity of Edwardsiella piscicida ferric uptake regulator (fur) mutations in zebrafsh Banikalyan Swain a, * , Cole T. Powell a , Roy Curtiss III a a University of Florida, Department of Infectious Diseases & Immunology, College of Veterinary Medicine, Gainesville, FL, 32608, USA A R T I C L E INFO Keywords: E. piscicida Fur Vaccine Immunogenicity Zebrafsh ABSTRACT Edwardsiella piscicida is the etiological agent of edwardsiellosis in fsh and causes severe economic losses in global aquaculture. Vaccination would be the most effective method to prevent infectious diseases and their associated economic losses. The ferric uptake regulator (Fur) is an important transcriptional global regulator of Gram- negative bacteria. In this study, we examined the regulatory function of Fur in E. piscicida. We designed a strain that displays features of the wild-type virulent strain of E. piscicida at the time of immunization to enable strains frst to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. Regulated delayed attenuation in vivo is based on the substitution of a tightly regulated araC P araBAD cassette for the promoter of the fur gene such that expression of this gene is dependent on arabinose provided during growth. Thus, following E. piscicida mutant colonization of lymphoid tissues, the Fur protein ceases to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. We deleted the promoter, including all sequences that interact with activator or repressor proteins, for the fur gene, and substituted the improved araC P araBAD cassette to yield an E. piscicida strain with the ΔP fur170 :TT araC P araBAD fur deletion-insertion mutation (χ16012). Compared to the wild-type strain J118, χ16012 exhibited retarded growth and enhanced siderophore production in the absence of arabinose. mRNA levels of Fur-regulated genes were analyzed in iron deplete or replete con- dition in wild-type and fur mutant strains. We observed zebrafsh immunized with χ16012 showed better colonization and protection compared to the Δfur (χ16001). Studies showed that E. piscicida strain χ16012 is attenuated and induces systemic and mucosal IgM titer in zebrafsh. In addition, we found an increase in transcript levels of tnf-α, il-1β, il-8 and ifn-γ in different tissues of zebrafsh immunized with χ16012 compared to the unimmunized group. We conclude that, E. piscicida with regulated delayed attenuation could be an effective immersion vaccine for the aquaculture industry. 1. Introduction Aquaculture accounts for approximately half of all fsh production globally and continues to be one of the fastest growing agricultural in- dustries [1]. However, the industry has incurred serious economic losses due to Edwardsiella piscicida (formerly known as E. tarda), a gram-negative, facultatively anaerobic bacterium of the family Entero- bacteriaceae. E. piscicida causes edwardsiellosis in a variety of freshwater and marine fsh species important to the aquaculture industry including catfsh (Ictalurus furtatus), tilapia (Tilapia nilotica), Indian major carp (Catla catla) and turbot (Scophthalmus maximus) [2,3]. The emergence of antibiotic-resistant bacteria as a result of fsh farming practices has prompted the development of vaccines as a safe, cost-effective alternative antibiotics. The challenge in developing an effective vaccine is constructing a vector that has minimal associated virulence, but still induces maximal immunogenic responses in the host. One way to achieve this is through the use of live attenuated vaccines (LAVs) in which the virulence of the pathogen is reduced by the deletion of specifc genes encoding virulence factors. Several LAVs for E. piscicida have already been developed by deletion of genes encoding potential virulence factors including the twin-arginine translocation system TatABCD, chorismic acid stynthetase ArOc, and an uncharacterized DcrB family protein ETAE_0023 [4–6]. However, this means of attenu- ation is often associated with reduced immunogenicity due to the decreased ability of the attenuated strain to colonize and persist in in- ternal effector lymphoid tissues in the immunized individual. * Corresponding author. E-mail address: swainbanikalyan@yahoo.com (B. Swain). Contents lists available at ScienceDirect Fish and Shellfsh Immunology journal homepage: www.elsevier.com/locate/fsi https://doi.org/10.1016/j.fsi.2020.10.029 Received 18 March 2020; Received in revised form 22 October 2020; Accepted 31 October 2020