2.1. Experimental fish and bacterial strain
Grass carp (n ¼ 2000) were obtained from the Xinmin Aquatic
Breeding Center (Heyang, Shaanxi, China), with the total length and
body weight of 3.0 ± 0.5 cm and 1.0 ± 0.1 g (data are presented as
mean values ± SD), respectively. Fish were acclimatized in five 300 L
aquaria with 28 C in aerated water under laboratory conditions for
twoweeks prior to the beginning of experiments and fed to apparent
satiation twice a day (10:00, 16:00) with a diet of commercial dry
pellets (Wuhan Tianlong Feed Company, Wuhan, China). Based on
the primers combination strategy of Vazquez-Juarez et al. [25], grass
carp were checked randomly to verify pathogen free status of
A. hydrophila prior to the trial. All protocols strictly adhered to the
guide for care and use of laboratory animals, and were approved by
Northwest A&F University animal protection committee.
A. hydrophila strain XS91-4-1 was preserved in our laboratory
with 20% glycerol at 80 C. During the experimental period,
A. hydrophila were cultured in Luria Bertani (LB) broth at 28 C and
grown overnight with constant shaking.