Growth-deficient mutants of Aeromonas hydrophila PPD 134/91 were induced by transposon mini-Tn5 mutagenesis, with Escherichia coli SM10λpir (pUT::mini-Tn5Km) as the Tn5 donor. Growth-deficient mutants were distinguished by their abilities to grow on TSA plates supplemented with tetracycline and neomycin, and were extremely slow in growth on rich and minimal media. These mutants were further characterized by their growth rates, serum resistance, surface characters, production of exoenzymes, invasion into EPC cells, and degree of virulence (LD50) in blue gourami. All seven attenuated strains were slower in growth and three were protease-deficient. Three attenuated mutants (M56, M57, and M40B) were chosen for vaccination experiments. After being challenged with the wild type PPD134/91, survival abilities of these vaccinated fish were compared with unvaccinated controls. Among the three transposon mutants, M57 offered the best protection. Vaccination of blue gourami with 106 CFU of live transposon mutant M57 by intraperitoneal injection resulted in a 40% increase in survival at both LD50 and 5 x LD50 doses. Intraperitoneally injected formalin-killed PPD134/91 at 107 CFU conferred similar protection when challenged at LD50 dose, while no protection was observed at 5 x LD50 dose. This study shows that attenuated growth-deficient mutants are promising candidates for the development of live vaccines against A. hydrophila infections in fish.
- Aeromonas hydrophila
- Diseases and their control in fish
- Live vaccine