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. 2021 May 3;9(5):451.
doi: 10.3390/vaccines9050451.

Construction and Characterization of an Aeromonas hydrophila Multi-Gene Deletion Strain and Evaluation of Its Potential as a Live-Attenuated Vaccine in Grass Carp

Jihong Li  1   2 , Shilin Ma  1 , Zhi Li  3 , Wei Yu  1 , Peng Zhou  1 , Xiang Ye  1 , Md Sharifull Islam  3   4 , Yong-An Zhang  1   2   5 , Yang Zhou  1   5 , Jinquan Li  3
Affiliations

Construction and Characterization of an Aeromonas hydrophila Multi-Gene Deletion Strain and Evaluation of Its Potential as a Live-Attenuated Vaccine in Grass Carp

Jihong Li et al. Vaccines (Basel). .

Abstract

Aeromonas hydrophila is an important pathogen that causes motile Aeromonas septicemia (MAS) in the aquaculture industry. Aerolysin, hemolysin, serine protease and enterotoxins are considered to be the major virulence factors of A. hydrophila. In this study, we constructed a five-gene (aerA, hly, ahp, alt and ast) deletion mutant strain (named Aeromonas hydrophila five-gene deletion strain, AHFGDS) to observe the biological characteristics and detect its potential as a live-attenuated vaccine candidate. AHFGDS displayed highly attenuated and showed increased susceptibility to fish blood and skin mucus killing, while the wild-type strain ZYAH72 was highly virulent. In zebrafish (Danio rerio), AHFGDS showed a 240-fold higher 50% lethal dose (LD50) than that of the wild-type strain. Immunization with AHFGDS by intracelomic injection or immersion routes both provided grass carp (Ctenopharyngodon idella) significant protection against the challenge of the strain ZYAH72 or J-1 and protected the fish organs from serious injury. Further agglutinating antibody titer test supported that AHFGDS could elicit a host-adaptive immune response. These results suggested the potential of AHFGDS to serve as a live-attenuated vaccine to control A. hydrophila infection in aquaculture.

Keywords: Aeromonas hydrophila; grass carp; immune response; live-attenuated vaccine; virulence.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phenotype characterization of AHFGDS. (A) Confirmation of strain AHFGDS by PCR using detecting primers covering (lane 1–10) or within (lane 11–20) the in-frame deletion fragments of the genes ast, alt, ahp, hly and aerA. Lane 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, ZYAH72 with primer sets P5/P6, P13/P14, P21/P22, P29/P30, P37/P38, P7/P8, P15/P16, P23/P24, P31/P32, P39/P40, respectively. Lane 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, AHFGDS with above primer sets, respectively. Marker is shown to the left (M). (B) Growth curves in LB medium over 12 h period. (C) β-hemolytic phenotype of the strains grown on sheep’s blood agar. (D) Cytotoxicity results after 2 h of incubation with the CIK cell line. The results were presented as the mean ± SD. *** p < 0.001.
Figure 2
Figure 2
Blood (A) and skin mucus (B) killing assay of wild-type ZYAH72 and AHFGDS mutant. The experiments were performed three times independently. The data shown were obtained from a representative experiment and presented as the mean ± SD. NS: not significant, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
(A) Survival curves for zebrafish challenged with wild-type ZYAH72 and AHFGDS at doses of 3.4 × 105 CFU/fish. (B) Symptoms of zebrafish infected by wild-type ZYAH72, AHFGDS or control group.
Figure 4
Figure 4
The mRNA levels of TGF-β, IL-1β and TNF-α coding genes in grass carp trunk kidney (A), spleen (B) and head kidney (C) of AHFGDS or wild-type ZYAH72 infected group. The mRNA level of each gene was normalized to that of β-actin. The experiments were performed three times independently. The data shown were obtained from a representative experiment. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
(A) Serum agglutinating antibody titers in grass carp immunized with AHFGDS through different administration routes. (B) Immunization with AHFGDS protects grass carp against lethal challenges with wild-type ZYAH72 or J-1. *** p < 0.001.
Figure 6
Figure 6
Histopathological examinations. Samples collected from fish vaccinated with AHFGDS or PBS (control group) at 48 h post wild-type ZYHA72 infection. The tissues were stained with hematoxylin and eosin. Black arrow indicates lymphocyte recruitment, red arrow indicates goblet cells, and green arrow indicates red blood cell necrosis.
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