The protective effect of inactivated Flavobacterium columnare vaccine in grass carp (Ctenopharyngodon idellus)

Abstract

Flavobacterium columnare, which causes columnaris disease, is responsible for significant mortality in grass carp. Vaccination is a safe and effective measure to combat this disease, and this study aimed to investigate the immune protective effects of different treatments using an inactivated F. columnare vaccine. The vaccine was prepared by inactivating the bacteria with 0.05% formaldehyde at 4°C for 24 hours. The experiments involving grass carp were divided into two parts. In Experiment 1, the immune effects of two isolates, JX-01 (genomovar I) and MU-04 (genomovar II), were compared, along with the impact of white oil adjuvant and the number of immunizations. The results showed that when the white oil adjuvant was used as a booster, the relative percent survival (RPS) of the JW2 group and MW2 group after 8 weeks of the first immunization was 34% and 61%, respectively. In Experiment 2, only the MU-04 (genomovar II) isolate was used as an antigen, with the white oil adjuvant as a booster. The effects of different doses (CFU=10⁸, 10⁷, and 10⁶ bacteria/mL) on immune responses were compared, and the RPS values in the MW6, MW7, and MW8 groups after 4 weeks of the first immunization were found to be 38%, 57%, and 71%, respectively. Furthermore, in the cross-antigen protection experiment, the MW2 group exhibited an RPS of 55% against the JX-01 isolate, which was significantly higher than the control group (33%). These findings suggest that an inactivated vaccine comprising an appropriate antigen isolate when administered with a white oil adjuvant as a booster, can provide effective protection in grass carp.

Keywords: Flavobacterium columnare; adjuvant; grass carp; relative percent survival; vaccine.

Figure 1

Growth profiles of (A) JX-01 and (B) MU-04. JX-01 and MU-04 were cultured at 28°C in Shieh medium. Aliquots of cell culture were taken at various time points and measured for cell density at OD450. Plate counts were carried out for each bacterial strain to calculate the number of colony-forming units per milliliter (CFU/mL).

Figure 2

Western blot analysis of grass carp IgM. SDS-PAGE of grass carp IgM (A). Lane M: Protein marker. Lane 1: Natural grass carp serum (diluted 1:20). Western blot of grass carp IgM using pre-immune rabbit serum (B) or rabbit anti-grass carp rIgM serum (C) as primary antibody. Lane M: Protein marker. Lane 1: Natural grass carp serum.

Figure 3

Vaccine trial schedule in Experiment 1. Percentage survival after injection challenge with 2 × 10⁸ CFU/mL F. columnare JX-01 or MU-04 strain of the non-vaccinated and different immunized groups (J/M: JX-01 or MU-04 stain; W/P: White oil adjuvant or PBS; 1/2: single or booster immunization). (A, C, E) Challenge results at different weeks for a single immunization group; (B, D, F) Challenge results at different weeks for a booster immunization group (Immunized group/Challenge stain). Statistical analysis was based on a comparison between the immune group and the control group for the same challenge strain. The symbol * indicates 0.01<p<0.05, ** indicates 0.001<p<0.01, **** indicates p<0.0001. “ns” stands for not significant (p>0.05).

Figure 4

Vaccine trial schedule in Experiment 3. The cross challenge was used between immunized groups to test immune protection between cross antigens (Immunized group/Challenge stain). Two vaccine groups with better immune protection in Experiment 1 were used 8 weeks after the second immunization. Percentage survival in non-vaccinated and differently immunized groups after challenge with 2 × 10⁸ CFU/mL F. columnare JX-01 or MU-04 strains. Statistical analysis was based on a comparison between the immune group and the control group for the same challenge strain. The symbol * indicates 0.01<p<0.05, ** indicates 0.001<p<0.01, *** indicates 0.0001<p<0.001, **** indicates p<0.0001.

Figure 5

Vaccine trial schedule in Experiment 2. Isolate MU-04 is the parent strain of the vaccine. Percentage survival after challenge with 2 × 10⁸ CFU/mL F. columnare MU-04 strain was administered in different antigen doses (CFU =10⁶, 10⁷, and 10⁸) to the immunized groups at 2 (A), 4 (B), 6 (C), 8 (D), and 10 (E) weeks (WO: White oil group). Statistical analysis was based on a comparison between the immune group and the control group for the same challenge strain. The symbol * indicates 0.01<p<0.05, *** indicates 0.0001<p<0.001, **** indicates p<0.0001. “ns” stands for not significant (p>0.05).

Figure 6

Corresponding Experiment 1: change in anti-F. columnare IgM antibody titer in the serum of grass carp at different weeks after the initial immunization. Data are presented as Mean ± SD. The symbol * indicates p<0.05, ** indicates 0.001<p<0.01, and *** indicates p<0.001. “ns” stands for not significant (p>0.05). (A) Different immunization program groups of JX-01 strain. (B) Different immunization program groups of MU-04 strain. (J/M: JX-01 or MU-04 stain; W/P: White oil adjuvant or PBS; 1/2: once or twice immuned).

Figure 7

Corresponding Experiment 3: cross-antibody titer in the serum of the JW2 group and the MW2 group against JX-01 (A) or MU-04 antigen (B). Differences in antibody titers were analyzed using a series of non-parametric tests (Kruskal-Wallis test) followed by a Dunnet’s post hoc test to determine which groups differ significantly from each group. Data are presented as Mean ± SEM. Different letters indicate a significant difference between the two groups (p<0.05).

Figure 8

Corresponding Experiment 2: change in anti-F. columnare IgM antibody titer in the serum of grass carp at 2, 4, 6, 8, and 10 weeks after initial immunization, with different antigen doses (CFU = 10⁶, 10⁷, and 10⁸; WO: White oil group). Data are presented as Mean ± SD. The symbol * indicates p<0.05, ** indicates 0.001<p<0.01, and *** indicates p<0.001. “ns” stands for not significant (p>0.05).