Identification of antigenic Edwardsiella tarda surface proteins and their role in pathogenesis

• Published in: Fish & shellfish immunology 2013-02, Vol.34 (2), p.673-682
• Main Authors: Yu, Jong Earn, Yoo, Ah Young, Choi, Kyoung-Hwa, Cha, Jaeho, Kwak, Inseok, Kang, Ho Young
• Format: Article
• Language: English
• Subjects: Animals; Antigen; Antigens, Bacterial - genetics; Antigens, Bacterial - immunology; Aquaculture; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - immunology; DNA Primers - genetics; Edwardsiella tarda; Edwardsiella tarda - genetics; Edwardsiella tarda - immunology; Edwardsiella tarda - pathogenicity; Electrophoresis, Polyacrylamide Gel; Enterobacteriaceae Infections - immunology; Enterobacteriaceae Infections - veterinary; Enzyme-Linked Immunosorbent Assay; Fish Diseases - immunology; Fish Diseases - microbiology; Goldfish; Immune responses; Immunoblotting; Immunoglobulin M - blood; Lethal Dose 50; Pathogenesis; Peptidoglycan - genetics; Peptidoglycan - immunology; Plasmids - genetics; Pleuronectiformes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Vaccine; Virulence
• ISSN: 1050-4648; 1095-9947
• DOI: 10.1016/j.fsi.2012.11.019

Edwardsiella tarda causes an infectious fish disease called edwardsiellosis. Several outer membrane proteins (OMPs) are associated with virulence factors and are attractive as vaccine candidates. In this study, 4 immuno-reactive OMPs of E. tarda were detected using anti-sera from flounder infected with E. tarda. Using matrix-assisted laser desorption/ionization mass spectrometry analyses, 2 of the 4 OMPs were identified as OmpA and murein lipoprotein (Lpp), which are highly conserved surface proteins in gram-negative bacteria. For further characterization of these surface proteins, we generated ompA- and lpp-inactivated mutants by insertion of a kanamycin cassette in the corresponding genes, and named these mutants E. tarda CK99 and CK164, respectively. As expected, immuno-reactive OmpA and Lpp proteins were absent in E. tarda CK99 and CK164, respectively, confirming that OmpA and Lpp are antigenic surface proteins. Interestingly, the LD50 value of E. tarda CK164 in fish (2.0 × 108 colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 107 CFU/fish). The LD50 of E. tarda CK99 did not differ from that of its parental strain. After administering attenuated E. tarda CK164 to fish, we monitored the E. tarda-specific immune response profile. We observed that the E. tarda-specific serum IgM titer increased in a time-dependent manner, and was much higher than the value observed after the administration of a heat-killed E. tarda control. Moreover, fish vaccinated with E. tarda CK164 were 100% protected when challenged by CK41, a pathogenic strain. Our results suggest that E. tarda CK164 can potentially be used for developing an effective live attenuated vaccine for edwardsiellosis that can be applied in the aquaculture industry. ► We identified two surface antigens of Edwardsiella tarda as OmpA and Lpp. ► We generated ompA and lpp mutant strains E. tarda CK99 and CK164, respectively. ► The virulence of E. tarda CK164 was lower than parental strain. ► Goldfish vaccinated with E. tarda CK164 were protected against pathogenic strain. ► E. tarda CK164 can be potentially used for developing live attenuated vaccine.