Abstract
Vibrio anguillarum is a pathogenic bacterium that infects flounder resulting in significant losses in the aquaculture industry. The VAA protein previously identified in flounder is associated with a role in immune protection within these fish. In the present study, a recombinant DNA plasmid encoding the VAA gene of V. anguillarum was constructed and its potential as a DNA vaccine, to prevent the infection of V. anguillarum in flounder fish, investigated. We verified the expression of the VAA protein both in vitro in cell lines and in vivo in flounder fish. The protective effects of pcDNA3.1-VAA (pVAA) were analyzed by determination of the percentage of sIgM+, CD4-1+, CD4-2+, CD8β+ lymphocytes, and the production of VAA-specific antibodies in flounder following their immunization with the DNA vaccine. Histopathological changes in immune related tissues, bacterial load, and relative percentage survival rates of flounder post-challenge with V. anguillarum, were all investigated to assess the efficacy of the pVAA DNA vaccine candidate. Fish intramuscularly immunized with pVAA showed a significant increase in CD4-1+, CD4-2+, and CD8β+ T lymphocytes at days 9, 11, and 14 post-vaccination, reaching peak T-cell levels at days 11 or 14 post-immunization. The percentage of sIgM+ lymphocytes reached peak levels at weeks 4–5 post-immunization. Specific anti-V. anguillarum or anti-rVAA antibodies were induced in inoculated fish at days 28–35 post-immunization. The liver of vaccinated flounder exhibited only slight histopathological changes compared with a significant pathology observed in control immunized fish. Additionally, a lower bacterial burden in the liver, spleen, and kidney were observed in pVAA protected fish in response to bacterial challenge, compared with pcDNA3.1 vector control injected fish. Moreover, the pVAA vaccine confers a relative percentage survival of 50.00% following V. anguillarum infection. In summary, this is the first study indicating an initial induction of the T lymphocyte response, followed by B lymphocyte induction of specific antibodies as a result of DNA immunization of flounder. This signifies the important potential of pVAA as a DNA vaccine candidate for the control of V. anguillarum infection.
Highlights
Flounder (Paralichthys olivaceus) is a common fish species within the aquaculture industry in the North of China
The flow cytometry (FCM) results revealed that the proportion of cells that could express VAA proteins following transient transfection with pVAA was ∼11.7%, while no cells expressing VAA protein were observed in the pcDNA3.1 transfected cell population (Figure 1B)
immunofluorescence assay (IFA) analysis of HINAE cells transfected with the pVAA showed a green fluorescence specific for VAA protein expression
Summary
Flounder (Paralichthys olivaceus) is a common fish species within the aquaculture industry in the North of China. Vaccines confer safety advantages for both fish and the environment [3], while the use of chemicals and antibiotics are associated with the potential disadvantages of accumulation in the flesh of animals, the appearance of drug-resistant strains, and contamination of the aquatic environment [4, 5]. Such vaccines targeted to V. anguillarum, are not yet available for use in China and any prospects of their development are limited by a dearth of information about the adaptive immune response of fish following vaccination. The aim of this study was to construct a recombinant DNA plasmid containing the VAA gene of V. anguillarum and investigate any immune protection effects observed after vaccination
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