Immune Responses and Protective Efficacy of a Formalin-Killed Francisella Noatunensis Subsp. Orientalis Vaccine Evaluated through Intraperitoneal and Immersion Challenge Methods in Oreochromis Niloticus

Abstract

Francisella noatunensis subsp. orientalis (Fno), an intracellular bacterium, causes systemic granulomatous diseases, resulting in high mortality and huge economic losses in Taiwanese tilapia farming. In this study, we tested the efficacy of a formalin-killed Fno vaccine in cultured tilapia. Fno was isolated from diseased tilapia, inactivated with formalin, and mixed with the mineral oil base adjuvant (Montanide^TM ISA 763 AVG). A total of 300 tilapia were divided into two groups. The experimental group was intraperitoneally injected with 0.1 mL of vaccine, which was substituted with phosphate-buffered saline (PBS) in the control group. A booster was administered at 2 weeks post-immunization. Tilapia were challenged at 6 weeks post primary immunization by intraperitoneal (IP) injection and immersion methods. Mortality was recorded at 21 and 60 days. The results revealed that the vaccine induced a greater antibody titer and led to 71% and 76% of relative percent survival (RPS) after the IP and immersion challenge. The transcripts of proinflammatory cytokines and immune-related genes, including interleukin-1β (IL-1β), tumor necrosis factor alpha (TNFα), C-X-C motif chemokine ligand 8 (CXCL8), and interleukin-17C (IL-17C), were significantly upregulated after vaccination. Additionally, vaccinated fish had lower bacterial loads in the blood and lower granuloma intensities in the kidney, spleen, liver, and gill than control fish. The results in this study demonstrate that the inactivated Fno vaccine could be an essential resource in Taiwanese tilapia farming.

Keywords: Francisella noatunensis susbsp. orientalis; Oreochromis niloticus; formalin-killed vaccine; immune response.

Figure 1

Specific IgM antibody titers were determined by the ELISA assay from 10 fish at 2 weeks and 6 weeks after primary immunization. Asterisks (***) indicate significant differences, with p < 0.001, from the control at each time point.

Figure 2

Comparative expression profiles of IL-1β, TNFα, CXCL8, and IL-17C in the spleen (A) and kidney (B) from control and vaccinated fish (n = 6) at 24, 48, and 72 h after primary immunization. The expression of target genes was normalized to that of the RPL23 gene as a reference gene. The graph shows the relative expression levels in the vaccinated and control groups. Asterisks (*), (**), and (***) indicate significant differences with p < 0.05, p < 0.01, and p < 0.001, respectively, from the control at each time point.

Figure 3

The averages of the cumulative mortalities of tilapia challenged with the Fno vaccine strain via the intraperitoneal (IP) injection method (A) and immersion method (B) at 6 weeks post primary immunization.

Figure 4

Comparison of blood bacterial concentrations between control and vaccinated fish after 24 h, 48 h, 72 h, 120 h, and 168 h post-infection (n = 3) (A). The differences among groups were tested using a one-way ANOVA with Tukey’s post hoc test (B). NS = Not significant.

Figure 5

The intensities of granulomas in the spleen (A), kidney (B), liver (C), and gill (D) from dead and surviving fish of the control and vaccine groups after the challenge with Fno by the IP and immersion methods. The level of intensity of a granuloma or white nodule was categorized as the following: level (-) no white nodules, (+) 1–5 nodules, (++) 6–10 nodules, and (+++) more than 10 nodules [23]. The intensities of the granuloma scores were calculated as percentages of occurrence among 30 fish per group.