Vibrio harveyi biofilm as immunostimulant candidate for high-health pacific white shrimp, Penaeus vannamei farming

The study was to develop Vibrio harveyi biofilm-based novel microbial product and its oral delivery for high health Penaeus vannamei farming. Yield of bacterial biofilm was optimized on chitin substrate (size: < 360, 360-850 and 850-1250 mu m; concentration: 0.3, 0.6 and 0.9%) in tryptone soy broth (0.15%). The biofilm was characterized by crystal violet assay, SEM and LSCM imaging; protein profiling by SDS-PAGE and LC-ESI-MS/MS. The immune stimulatory effect of the biofilm in yard experiments was evaluated by relative quantification of immune genes using real-time PCR effect on overall improvement on health status under field trials. The highest biofilm yield (6.13 +/- 0.2 x 10(7) cfu/ml) was obtained at 0.6% of < 360 mu m chitin substrate. The biofilm formation was stabilized by 96 h of incubation at 30 degrees C. Protein profiling confirmed expression of six additional proteins (SDS-PAGE) and 11 proteins were differentially expressed (LC-ESI-MS/MS) in biofilm cells over free cells of V. harveyi. Oral administration of the biofilm for 48 h confirmed to enhance expression of antimicrobial peptides, penaeidin, crustin and lysozyme in P. vannamei. Further Oral administration of biofilm for two weeks to P. vannamei (1.8 +/- 0.13 g) improved the growth (2.66 +/- 0.06 g) and survival (84.44 +/- 1.82%) compared to control (2.15 +/- 0.03 g; 70.94 +/- 0.66%) Nursery trials showed a significant reduction in occurrence of anatomical deformities like antenna cut (12.67 +/- 0.66%), rostrum cut (4.66 +/- 0.87%), and tail rot (3.33 +/- 0.88%), compared to animals fed with normal diet which was 24.33 +/- 2.72; 14 +/- 1.52 and 10.66 +/- 1.45% respectively. In vitro and in vivo studies suggest inactivated biofilm cells of V. harveyi on chitin substrate express additional antigenic proteins and when administered orally through feed at regular intervals stimulates immune response and improve growth, survival and health status of shrimp.