Protective Efficacy of Novel Oral Biofilm Vaccines against Lactococcus garvieae Infection in Mullet, Mugil cephalus

Abstract

Lactococcus garvieae (L. garvieae) is an important pathogen that causes enormous economic losses in both marine and freshwater aquaculture. At present, antibiotics are the only option for farmers to reduce the losses caused by L. garvieae. However, the usage of antibiotics leads to environmental pollution and the production of drug-resistant strains of bacteria. Therefore, vaccination is preferred as an alternative method to prevent infectious diseases. In this study, we describe an effective approach to the production of an oral biofilm vaccine, using bacteria grown on chitosan particles to form biofilms, and thus providing an inactive pathogen that enhances the immune response in fish. We observed the formation of a biofilm on chitosan particles and administered the novel oral biofilm vaccine to fish. We analyzed the immune responses, including antibody production, phagocytic ability, albumin/globulin ratio and immune-related genes, of vaccinated and control groups of black mullet. Our results show that the phagocytic ability of the biofilm vaccine group was 84%, which is significantly higher than that of the control group, and the antibody production in this group was significantly higher compared with the other group. The mRNA expression levels of immune-related genes (TLR2, IL-1β, TNF-α) were significantly upregulated in the spleen after vaccination. In challenge experiments, the relative percent survival (RPS) was 77% in the biofilm vaccine group, 18% in the whole-cell vaccine group, and 0% in the chitosan particle group at 32 days post-vaccination. In addition, we also found that the relative percent survival (RPS) at 1 day post-vaccination was 74% in the biofilm vaccine group, 42% in the whole-cell vaccine group, and 26% in the chitosan particle group. In both long-term and short-term challenge experiments, the viability of the biofilm vaccine group was significantly higher than that of the whole-cell, chitosan particle and PBS groups. We conclude that based on its protective effect, the L. garvieae biofilm vaccine is better than the whole-cell vaccine when challenged several weeks after vaccination. In addition, the biofilm vaccine also has a greater protective effect than the whole-cell vaccine when challenged immediately after vaccination. Therefore, the biofilm vaccine might represent a novel method for the prevention and treatment of L. garvieae infection.

Keywords: IgM; Lactococcus garvieae; Mugil cephalus; oral biofilm vaccine.

Figure 1

Graphical overview of the vaccination/challenge schedule and sampling time points.

Figure 2

(A) The biofilm bacteria growth curve on chitosan particles (OD/h). L. garvieae was inoculated into BHI broth with 1% NaCl. The starting inoculum was 10⁴ CFU/mL. From the same sample, the biofilm growth curve was quantified by the DMMB method, and CFU values were plotted to obtain the bacteria growth curve. The biofilm growth abilities were evaluated by determining the optical density (OD) after applying the DMMB method (from time = 0 to time = 72 h). Data are presented as mean ± SD (n = 4). Scanning electron micrographs of (B) chitosan particle surface without L. garvieae growth and (C) biofilm growth on a chitosan particle at 48 h (arrows: L. garvieae biofilm). Magnification: 1000×; bars = 50 μm.

Figure 3

Immunohistochemical (IHC) staining of the head kidney and intestine collected after vaccination. (A,C,E) PBS control. (B,D,F) Biofilm vaccine group (mag. ×400). Arrows indicate positive signals and melanomacrophage centres.

Figure 4

(A) Phagocytic activity of whole blood with different types of vaccine. The biofilm vaccine group had significantly increased phagocytic activity compared to PBS and whole-cell vaccine groups. Data are presented as mean ± SD (n = 3 fish × 3 replicates per treatment). p-values were calculated by one-way ANOVA (p < 0.01 **, p < 0.05 *). (B) Albumin–globulin ratios of plasma with different vaccines. Data are presented as mean ± SD (n = 3 fish × 3 replicates per treatment). The p-values indicate that there were no significant differences between groups.

Figure 5

Serum IgM titres of fish vaccinated with different vaccines. The biofilm vaccine group had significantly increased IgM titres compared to the PBS and whole-cell vaccine groups. (A) Biofilm antigen coating on 96-well microplate. (B) Whole-cell antigen coating on 96-well microplate. Data are presented as mean ± SD (n = 3 fish × 3 replicates per treatment). p-values were calculated by one-way ANOVA (p < 0.01 **, p < 0.05 *).

Figure 6

Expression levels of (A) TNF-α (B) C3, (C) IL-1β and (D) TLR2 in spleen samples (n = 3 fish spleen samples × 3 replicates per treatment) after vaccination in each group. Data are presented as mean ± SD, and p-values were calculated by one-way ANOVA (p < 0.05 *, p < 0.01 **).

Figure 7

Cumulative survival rate in challenge experiment 1. The mullets were challenged by immersion with 10⁷ CFU/mL of Lactococcus garvieae. Challenge was performed on day 1 after vaccination. Fish were monitored for 10 days. Statistical significance was determined by the logrank test; there were significant differences between groups (p = 0.0023).

Figure 8

Long-term analysis of challenge experiment 2. Serum antibody levels of fish vaccinated with different vaccines. Data are presented as mean ± SD (n = 3 fish × 3 replicates per treatment). p-values were calculated by one-way ANOVA. Data are significantly different (p < 0.05 *, p < 0.01 **).

Figure 9

Long-term analysis of relative percentage survival in challenge experiment 2. Cumulative survival rate of the experimental mullet challenged by immersion with 10⁷ CFU/mL of Lactococcus garvieae. Challenge was performed on day 32 post-vaccination. Fish were monitored for 30 days. Statistical significance was determined by the log-rank test; there were significant differences between groups (p < 0.0001).