lactoferricin-b and Fish-Diseases

Zebrafish (Danio rerio) was used as a bioreactor to produce bovine lactoferricin (LFB), which has wide-ranging antimicrobial activity. We constructed an expression plasmid in which LFB was fused with green fluorescent protein (GFP) and driven by zebrafish beta-actin promoter. After microinjection, six transgenic founders were screened on the basis of GFP appearance. Among them, a stable ZBL-5 line was selected by the ubiquitous and strong expression of GFP. Using PCR and Western blot analysis, we confirmed that the recombinant LFB-GFP protein was produced by the F2 progeny derived from the ZBL-5 line. The bactericidal agar plate assay proved that the functional domain of LFB was released from the LFB-GFP fusion protein, resulting in strong bactericidal activity against Escherichia coli, Edwardsiella tarda and Aeromonas hydrophila. Furthermore, adult zebrafish were given one feeding of fifty 72-hpf transgenic embryos. The treated fish were then immersed in freshwater containing 1 x 10(5) CFU ml(-1)E. tarda for 7 days. The survival rate of the treated zebrafish was significantly higher than that of fish fed with fifty wild-type embryos (75 +/- 12.5% versus 4 +/- 7.2%). This line of evidence suggested that pathogen resistance can be enhanced by using transgenic embryos containing LFB-GFP as a food supplement for fish, while, at the same time, reducing the demand of chemical antibiotics.

Topics: Actins; Animals; Animals, Genetically Modified; Anti-Bacterial Agents; Bioreactors; Blotting, Western; Cattle; Dietary Supplements; Eggs; Fish Diseases; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Immunity, Innate; Lactoferrin; Polymerase Chain Reaction; Recombinant Proteins; Zebrafish

Antibiotics are commonly employed in most fish aquacultures to prevent disease. One major risk in this practice is that antibiotic-resistant pathogens may be selected. Therefore, we wanted to examine the feasibility of producing an economical, non-antibiotic alternative. The microalga Nannochloropsis oculata is an essential phytoplankton used as live feed for fish larvae. We attempted to culture N. oculata in a way that would provide an organism against bacterial pathogenic infection. To test this idea, we constructed an algae-codon-optimized bovine lactoferricin (LFB) fused with a red fluorescent protein (DsRed) driven by a heat-inducible promoter, which is a heat shock protein 70A promoter combined with a ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2' promoter from Chlamydomonas reinhardtii. After electroporation, we examined 491 microalgal clones and generated two stable transgenic lines, each expressing a stable transgene inheritance for at least 26 months. This was confirmed by the positive detection of the mRNA transcript and the protein of LFB-DsRed produced by the transgenic microalgae. To test the efficacy of the antimicrobial peptide LFB, medaka fish (Oryzias latipes) were adapted from freshwater to seawater and were fed with the transgenic algae by oral-in-tube delivery method. Bacterial infection with 1 x 10(5)Vibrio parahaemolyticus per fish was induced 6h thereafter by oral-in-tube delivery as well. For medaka fish fed with 1 x 10(8) transgenic algae per fish, the average survival rate after a 24-h period of infection was much higher than that of medaka fed with wild-type algae (85+/-7.1% versus 5+/-7.1%). This result suggests that medaka fish fed with the LFB-containing transgenic microalgae will have bactericidal defense against V. parahaemolyticus infection in its digestive tract.

Topics: Algal Proteins; Animals; Blotting, Western; Eukaryota; Fish Diseases; Gastrointestinal Tract; Lactoferrin; Organisms, Genetically Modified; Oryzias; Plasmids; Protoplasts; Recombinant Proteins; Survival Analysis; Vibrio Infections; Vibrio parahaemolyticus