Pathogenicity of Aeromonas hydrophila Isolated From Diseased Largemouth Bass (Micropterus salmoides) and Development of an Inactivated Vaccine.

Abstract

Aeromonas hydrophila can cause disease in various aquatic animals, but there exist no effective alternatives to control its outbreak. In this study, diseased largemouth bass were collected from the breeding farm Lake Dahong (Chongqing, China), a strain SK-2 was isolated and identified as A. hydrophila. In order to investigate the pathogenicity of the strain and the protective efficacy of the inactivated vaccine, we firstly evaluated the virulence factors including hemolytic, proteolytic, and amylolytic activities. The results displayed that the diameters of hemolytic, proteolytic, and amylolytic circles reached 0.94 ± 0.10, 2.15 ± 0.16, and 1.51 ± 0.14 cm respectively. Median lethal dose (LD₅₀) was tested and the LD₅₀ value was calculated as 1.39 × 10⁵ CFU/g. Besides, a challenge test was conducted and two levels (LD₅₀ and 1/10 LD₅₀) were set for intraperitoneal injection. Significant immune responses were found in the serum and head kidney samples, with the higher superoxide dismutase (SOD), peroxidase (POD) and malondialdehyde (MDA) levels (p < 0.05). The results of qPCR revealed that tumour necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interferon-γ (IFN-γ) expression levels were elevated in the liver and head kidney samples, reaching a stable state at 48 h. Furthermore, the inactivated vaccine of SK-2 was prepared using formalin and 0.85% saline (CG) and the inactivated vaccine of SK-2 (AHIV) was intraperitoneally injected for 28-d immunisation. The results indicated the SOD and lysozyme (LZM) activities in the serum of the AHIV group were dramatically higher (p < 0.05) than those in the CG group, the catalase (CAT) and MDA levels were remarkably increased (p < 0.05) compared with the CG group, and these immune-related genes exhibited conspicuous upregulation (p < 0.05), suggesting the inactivated vaccine of SK-2 could bring about subdued oxidative stress in the innate immunity of largemouth bass. The wide-type SK-2 was challenged into the largemouth bass post 28-d immunisation, and the relative protection was calculated as 86.21%. These findings provide theoretical support for the bacterial disease control of largemouth bass.