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Identification of fish source Vibrio alginolyticus and evaluation of its bacterial ghosts vaccine immune effects.

ABSTRACT: Vibrio alginolyticus (V. alginolyticus) is a common pathogen for humans and marine aquatic animals. Vibriosis of marine aquatic animals, caused by V. alginolyticus, has become more prevalent globally in recent years. Hence, a safe and effective vaccine is urgently needed for the control of this disease. Here, the strain 16-3 isolated from the large yellow croaker (Larimichthys crocea) suffered from canker was identified as V. alginolyticus based on morphological, biochemical, and 16S rDNA sequencing analysis. Then, recombinant temperature-controlled lysis plasmid pBV220-lysisE was electroporated into the strain 16-3 to generate V. alginolyticus bacterial ghosts (VaBGs) by inducing lysis gene E expression, and the safety and immune effects of VaBGs were further investigated in mice and large yellow croaker. The results showed that VaBGs were as safe as formalin-killed V. alginolyticus cells (FKC) to mice and fish. Compared with FKC and PBS groups, significant elevations of the serum agglutinating antibody titer, serum bactericidal activity, lymphocyte proliferative responses, and levels of four different cytokines (Th1 type: IL-2, TNF-?; Th2 type: IL-4 and IL-6) in serum were detected in the VaBGs group, indicating that a Th1/Th2-mediated mixed immune response was elicited by the VaBGs. More importantly, after challenged with the parent strain 16-3, VaBGs-vaccinated mice and fish showed higher protection than FKC-vaccinated mice, the relative percent of survival (RPS) being 60%, 66.7% and 40%, respectively. Taken together, this is the first demonstration that the newly constructed V. alginolyticus ghosts may be developed as a safe and effective vaccine against V. alginolyticus infection in aquaculture.

SUBMITTER: Cao J

PROVIDER: S-EPMC6011932 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Identification of fish source Vibrio alginolyticus and evaluation of its bacterial ghosts vaccine immune effects.

Cao Ji J Zhang Jiajun J Ma Lin L Li Lin L Zhang Wenchang W Li Jinnian J

MicrobiologyOpen 20180119 3

Vibrio alginolyticus (V. alginolyticus) is a common pathogen for humans and marine aquatic animals. Vibriosis of marine aquatic animals, caused by V. alginolyticus, has become more prevalent globally in recent years. Hence, a safe and effective vaccine is urgently needed for the control of this disease. Here, the strain 16-3 isolated from the large yellow croaker (Larimichthys crocea) suffered from canker was identified as V. alginolyticus based on morphological, biochemical, and 16S rDNA sequen ...[more]

PMID: 29349911

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Project description:Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis gene E of bacteriophage PhiX174. In this study, Vibrio parahaemolyticus ghosts (VPGs) were generated by chemically-induced lysis and the method is based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH), acetic acid, boric acid, citric acid, maleic acid, hydrochloric acid, and sulfuric acid. The MIC values of the respective chemicals were 3.125, 6.25, <50.0, 25.0, 6.25, 1.56, and 0.781 mg/mL. Except for boric acid, the lysis efficiency reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs appeared completely DNA-free, which was confirmed by quantitative real-time PCR. Besides, lipopolysaccharides (LPS) extracted from the NaOH-induced VPGs showed no distinctive band on SDS-PAGE gel after silver staining. On the other hand, LPS extracted from wild-type bacterial cells, as well as the organic acids-induced VPGs showed triple major bands and LPS extracted from the inorganic acids-induced VPGs showed double bands. It suggests that some surface structures in LPS of the NaOH-induced VPGs may be lost, weakened, or modified by the MIC of NaOH. Nevertheless, Limulus amoebocyte lysate assay revealed that there is no significant difference in endotoxic activity between the NaOH-induced VPGs and wild-type bacterial cells. Macrophages exposed to the NaOH-induced VPGs at 0.5 × 10⁶ CFU/mL showed cell viability of 97.9%, however, the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. Like Escherichia coli LPS, the NaOH-induced VPGs are an excellent activator of pro-inflammatory cytokines (IL-1β and iNOS), anti-inflammatory cytokine (IL-10), and dual activities (IL-6) in the stimulated macrophage cells. On the other hand, the induction of TNF-α mRNA was remarkable in the macrophages exposed with wild-type cells. Scanning electron microscopy showed the formation of trans-membrane lysis tunnel structures in the NaOH-induced VPGs. SDS-PAGE and agarose gel electrophoresis also confirmed that cytoplasmic proteins and genomic DNA released from the VPGs to culture medium through the lysis tunnel structures. Taken together, all these data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.

Dataset Information

Identification of fish source Vibrio alginolyticus and evaluation of its bacterial ghosts vaccine immune effects.

Publications

Identification of fish source Vibrio alginolyticus and evaluation of its bacterial ghosts vaccine immune effects.

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