Introduction
As the shrimp aquaculture industry is growing exponentially to meet increasing demands, it needs to overcome certain challenges. One of the challenges that come with intensification of aquaculture is the emergence of pathogens. Acute Hepatopancreatic Necrosis Disease (AHPND) is a bacterial infection caused predominantly by Vibrio (V.) parahaemolyticus. As the use of antimicrobials to fight infections is strongly discouraged due to the emergence of antimicrobial resistance bacteria, the search for alternatives is essential. Transferrins are a family of natural antimicrobial and immunomodulating glycoproteins, which have been shown to be effective against multiple pathogenic bacteria. Ovotransferrin (ovoTF) is an easily accessible transferrin, extracted from egg white. The aim of this study is to investigate the in vitro antimicrobial activity ovoTF towards AHPND-causing V. parahaemolyticus.
Methodology
Three AHPND-causing V. parahaemolyticus strains with different origins were used for these experiments. Different concentrations, ranging from 0.001 mg/ml to 10 mg/ml, of ovoTF were tested for their effects on growth curves, and concentrations ranging from 0.125 mg/ml to 1 mg/ml were tested for their effects on swimming and swarming motility, surface hydrophobicity and biofilm formation, and caseinase, lipase and phospholipase secretion. Live/dead flow cytometry was used to assess the ability of ovoTF to kill the bacterial cells. Furthermore, as ovoTF is able to act as a serine protease, its effect on the PirAB toxins produced by V. parahaemolyticus was investigated.
Results
Our results showed that ovoTF was able to delay growth of the bacteria significantly at a concentration of 0.1 mg/ml, and even inhibit growth at higher concentrations. Furthermore, biofilm formation by the bacteria is inhibited, which can be partly explained by the observed lower cell surface hydrophobicity after addition of the ovoTF. Swimming motility was inhibited, while swarming motility was induced. Secretion of lipase was inhibited by higher concentrations of ovoTF, while caseinase production was induced. On phospholipase secretion, the transferrin did not seem to have a significant effect. Results of live/dead flow cytometry and the degradation assay are to be included.
Clearly, ovotransferrin is putting the bacteria under pressure in vitro and from the growth assays, it can be concluded that it has a strong antibacterial effect on the AHPND-related V. parahaemolyticus. Together, these in vitro results indicate that ovoTF is a promising anti-microbial protein, and should be further investigated in vivo for its use as a natural compound for controlling AHPND in farmed shrimp.