Immunoprotective test and whole-genome sequencing analysis of the attenuated S02 strain of Streptococcus iniae

Abstract

Background: Streptococcus iniae is one of the most serious diseases threatening tilapia aquaculture, causing huge economic losses every year. Injectable attenuated vaccines are still the best choice for preventing streptococcal diseases affecting the tilapia.

Objective: This study evaluated the safety, stability, immunogenicity, antibody production time, and immune dose of the attenuated S02 strain of S. iniae and comprehensively analyzed the possible mechanisms of its attenuated virulence at the whole-genome level.

Results: After detoxification, the S02 strain completely loses its pathogenicity to tilapia and has good immunogenicity. The results of the backpropagation safety test showed that the S02 strain did not cause disease or death to tilapia after continuous passage for 50 generations. AfterS02 was injected, the immunoglobulin M (IgM) level in the serum was significantly higher than that in the GX005 infection group within 28 days and reached its peak at 14 days. An intraperitoneal injection of 10⁹ CFUs/mL of S02 at a dose of 0.2 mL per fish had the best relative protection rate of 92.58%. The whole-genome sequencing results showed that the S02 strain had two large 0.2 Mbp segments of inversion compared to its parent virulence strain GX005, encoding 372 genes, including the virulence genes of the GNAT family N-acetyltransferase and the hyaluronic acid lyase genes of the hysA, hylA, and hylB, which are related to virulence factors.

Conclusion: This study provides theoretical data support for the prevention and control of the S. iniae infection in tilapia. The abnormal expression of important virulence genes GNAT family N-acetyltransferase and hyaluronic acid lyase genes hysA, hylA, and hylB caused by the inversion and translocation of large fragments could be the main mechanism for their attenuated virulence. This study provided theoretical support for the prevention and treatment of S. iniae infection in tilapia and the application of S02-attenuated vaccine.

Keywords: Streptococcus iniae; attenuated vaccine; immune protection; tilapia; whole genome sequencing.

Figure 1

S02 reduces the mortality rate of tilapia caused by GX005 infection (n = 100). (A,B) The death number of tilapia immunized S02 in 15 and 30 days. (C,D) The mortality of tilapia immunized S02 in 15 and 30 days. (E) The relative percent survival rates of tilapia immunized with S02 at 15 and 30 days. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test, and expressed as the mean ± SD, ****p < 0.0001 versus the (CX005 infection) Control group.

Figure 2

S02 significantly increased the level of IgM in tilapia (n = 3). Detection of changes in IgM content in tilapia serum after intraperitoneal injection of immune S02. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test, and expressed as the mean ± SD, ****p < 0.0001 versus the GX005 infection group.

Figure 3

Different dose of S02 have varying protective effects on tilapia (n = 20). (A) The number of dead fish in tilapia immunized S02 with different doses. (B) The mortality of tilapia immunized with S02 at different doses. (C) The relative percent survival rates of tilapia immunized with S02 at different doses. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test, and expressed as the mean ± SD, *p < 0.05, ****p < 0.0001 versus the CX005 infection control group.

Figure 4

Study of the effect of S02 immunization on fish of different specifications (n = 40). (A) The number of dead fish in the tilapia of different specifications immunized with S02. (B) The mortality of tilapia of different specifications immunized with S02. (C) The relative percent survival rates of tilapia of different specifications immunized with S02. **P < 0.01, ****P < 0.0001.

Figure 5

The effect of water temperature on the immune response of S02 (n = 150). (A) The number of dead fish in the tilapia immunized with S02 at different water temperatures. (B) The mortality of tilapia immunized with S02 at different water temperatures. (C) The relative percent survival rates of tilapia immunized with S02 with different water temperatures. ****P < 0.0001.

Figure 6

Composition of gene islands (n = 6). (A) Comparison of gene islands between CX005 and S02 (B) Comparison of differential gene islands between CX005 and S02. (C) Comparison heatmap of differential gene islands between CX005 and S02. Each row represents a genomic island, with an arrow denoting a gene. The length of the arrow indicates the gene length, while its direction signifies whether the gene is encoded by the sense strand or the antisense strand. The color represents the function of the gene. The letters are the annotation information based on COG (Clusters of Orthologous Groups). F represents nucleotide transport and metabolism; H represents coenzyme transport and metabolism; J represents ribosomal structure and biogenesis; K represents transcription; L represents recombination and repair; O represents post-translational modification; Q represents secondary metabolites biosynthesis; T represents signal transduction mechanisms; X represents nobilome.

Figure 7

KEGG and COG analysis (n = 6). (A–C) Comparison of COG analysis between CX005 and S02. (D,E) Comparison of KEGG analysis between CX005 and S02.

Figure 8

Deletion of hyaluronidase gene and genome collinearity analysis (n = 6). (A) Comparison of GX005 and S02 virulence genes at level 1. (B) Comparison of GX005 and S02 virulence genes at level 2. (C) Comparison heatmap of differential virulence genes between CX005 and S02. (D) Collinearity diagram of GX005 and S02 virulence genes comparison. (E) Circle diagram of differential virulence genes between CX005 and S02.

Figure 9

Comprehensive antibiotic resistance database (n = 6). (A) Comparison of antibiotic resistance genes between CX005 and S02. (B) Comparison heatmap of antibiotic resistance gene between CX005 and S02.