The Use of Extracellular Membrane Vesicles for Immunization against Francisellosis in Nile Tilapia (Oreochromis niloticus) and Atlantic Cod (Gadus morhua L.)

Abstract

Francisellosis in fish is caused by the facultative intracellular Gram-negative bacterial pathogens Francisella noatunensis ssp. noatunensis and Francisella orientalis. The disease is affecting both farmed and wild fish worldwide and no commercial vaccines are currently available. In this study, we tested isolated membrane vesicles (MVs) as possible vaccine candidates based on previous trials in zebrafish (Danio rerio) indicating promising vaccine efficacy. Here, the MV vaccine-candidates were tested in their natural hosts, Atlantic cod (Gadus morhua L.) and Nile tilapia (Oreochromis niloticus). Injection of MVs did not display any toxicity or other negative influence on the fish and gene expression analysis indicated an influence on the host immune response. However, unlike in other tested fish species, a protective immunity following vaccine application and immunization period could not be detected in the Atlantic cod or tilapia. Further in vivo studies are required to achieve a better understanding of the development of immunological memory in different fish species.

Keywords: Atlantic cod; fish disease; francisellosis; membrane vesicles; tilapia; vaccine.

Figure A1

Immune genes with no significant differences in gene expression levels between the different treated groups during the vaccine trial in tilapia. Gene expression levels of the investigated genes were analyzed by qPCR and are displayed as log₂ of relative gene expression. The gene expression levels were tested for significant differences in between the treatments; (p < 0.05; one-way ANOVA with multiple comparison using Tukey test; error bars indicate mean ± SEM).

Figure A2

Immune genes with no significant differences in gene expression levels between the different treated groups during the toxicity trial in Atlantic cod. Gene expression level of different investigated genes were analyzed by qPCR and are displayed as relative gene expression. The gene expression levels were tested for significant differences in between the treatments (p < 0.05; one-way ANOVA with multiple comparison using Tukey test; error bars indicate mean ± SEM).

Figure 1

Immune responses in tilapia 14 days after injection of 40 or 400 µg of Fo membrane vesicles (MVs) compared to saline-injected or untreated fish. (A) Log2 of relative mRNA abundance of a selection of immune genes performed by qPCR on isolated spleen from eight fish per group. (B) Enzyme-linked immunosorbent assay (ELISA) measuring specific IgM against Fo. Absorbance was measured at 450 nm. Labelling with different letters (a; b) indicates significant differences between groups; groups that do not share letters are significantly different. The absence of letters indicates a lack of significant differences (p < 0.05; one-way ANOVA with multiple comparisons using Tukey test; error bars indicate mean ± SEM (standard error of the mean)). Note that the scale on Y-axis can differ in (A).

Figure 2

Survival and bacterial burden of adult tilapia immunized with Fo MVs and subsequently challenged with Fo. (A) Kaplan–Meier curve of cumulative survival (%) of tilapia immunized with 40 µg Fo MVs, injected with adjuvant or a saline solution, or left untreated after a cohabitation challenge with shedders injected with 3 × 10⁵ CFU/mL of Fo. (B) Graphic represents median log₂ of Fo content in heart, muscle and kidney tissues collected during vaccine trial. The bacterial burden was detected by qPCR of genomic DNA isolated from the respective tissues. Different letters (a; b) indicate significant differences; groups that do not share letters are significantly different. The absence of letters indicates a lack of significant differences (p < 0.05; one-way ANOVA with multiple comparisons using Tukey test; error bars indicate mean ± SEM).

Figure 3

Immune gene expression levels in Atlantic cod immunized with Fnn MVs and challenged with Fnn. Sample analysis was performed by qPCR on the spleen and kidney at two time-points—30 and 61 dpc. The relative gene expression of different investigated genes displayed as log₂ of relative mRNA abundance. Results marked with different letters (a; b) indicate significant difference; groups that do not share letters are significantly different. The absence of letters indicates the lack of significant differences (p < 0.05; one-way ANOVA with multiple comparisons using Tukey test; error bars indicate mean ± SEM). Note that scales on the Y-axis may differ.

Figure 4

Detection of anti-Fnn IgM in the serum of Atlantic cod immunized with Fnn MVs assayed by ELISA in both the toxicity and vaccine trials. In the toxicity trial, the fish were immunized with either 40 or 400 µg of Fnn MVs. In the vaccine trial, the fish were immunized with either 40 µg of Fnn MVs mixed with an adjuvant or with adjuvant alone. In both trials, a saline-injected group was included in addition to an untreated group (control). In the toxicity trial, the serum was collected 14 days after Fnn MV injection. Serum samples were obtained from three fish from each respective group at given time-points during the vaccine experiment. Results marked with different letters (a; b) show the significant difference; groups that do not share letters are significantly different (p < 0.05; one-way ANOVA with multiple comparison using Tukey test; error bars indicate mean ± SEM).