16 S rRNA gene diversity and gut microbial composition of the Indian white shrimp (Penaeus indicus).

The endemic Indian white shrimp (Penaeus indicus) is an economically important crustacean species, distributed in the Indo-West Pacific region. Knowledge of its gut microbial composition helps in dietary interventions to ensure improved health and production. Here we analyzed V3-V4 hypervariable regions of the 16 S rRNA gene to examine intestinal microbiota in wild and domesticated farmed P. indicus. The study revealed that Proteobacteria, Fusobacteria, Tenericutes, and Bacteroidetes, were the dominant phyla in both the groups although there were differences in relative abundance. The dominant genera in case of the wild group were Photobacterium (29.5 %) followed by Propionigenium (13.9 %), Hypnocyclicus (13.7 %) and Vibrio (11.1 %); while Vibrio (46.5 %), Catenococcus (14 %), Propionigenium (10.3 %) and Photobacterium (8.7 %) were dominant in the farmed group. The results of the study suggest the role of environment on the relative abundance of gut bacteria. This is the first report characterizing gut microbial diversity in P. indicus, which can be used to understand the role of gut microbiota in health, nutrition, reproduction, and growth.

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 µm; 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 ±â€¯0.2 × 107 cfu/ml) was obtained at 0.6% of <360 µm chitin substrate. The biofilm formation was stabilized by 96 h of incubation at 30 °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 ±â€¯0.13 g) improved the growth (2.66 ±â€¯0.06 g) and survival (84.44 ±â€¯1.82%) compared to control (2.15 ±â€¯0.03 g; 70.94 ±â€¯0.66%) Nursery trials showed a significant reduction in occurrence of anatomical deformities like antenna cut (12.67 ±â€¯0.66%), rostrum cut (4.66 ±â€¯0.87%), and tail rot (3.33 ±â€¯0.88%), compared to animals fed with normal diet which was 24.33 ±â€¯2.72; 14 ±â€¯1.52 and 10.66 ±â€¯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.

Rock bream iridovirus (RBIV) replication in rock bream (Oplegnathus fasciatus) exposed for different time periods to susceptible water temperatures.

Rock bream iridovirus (RBIV) is a member of the Megalocytivirus genus that causes severe mortality to rock bream. Water temperature is known to affect the immune system and susceptibility of fish to RBIV infection. In this study, we evaluated the time dependent virus replication pattern and time required to completely eliminate virus from the rock bream body against RBIV infection at different water temperature conditions. The rock bream was exposed to the virus and held at 7 (group A1), 4 (group A2) and 2 days (group A3) at 23 °C before the water temperature was reduced to 17 °C. A total of 28% mortality was observed 24-35 days post infection (dpi) in only the 7 day exposure group at 23 °C. In all 23 °C exposure groups, virus replication peaked at 20 to 22 dpi (106-107/µl). In recovery stages (30-100 dpi), the virus copy number was gradually reduced, from 106 to 101 with faster decreases in the shorter exposure period group at 23 °C. When the water temperature was increased in surviving fish from 17 to 26 °C at 70 dpi, they did not show any mortality or signs of disease and had low virus copy numbers (below 102/µl). Thus, fish need at least 50 days from peaked RBIV levels (approximately 20-25 dpi) to inhibit the virus. This indicates that maintaining the fish at low water temperature (17 °C) for 70 days is sufficient to eradicate RBIV from fish body. Thus, RBIV could be eliminated slowly from the fish body and the virus may be completely eliminated under the threshold of causing mortality.

Toxicity and dose determination of quillaja saponin, aluminum hydroxide and squalene in olive flounder (Paralichthys olivaceus).

Adjuvants are substances added to vaccines to enhance the immune response of a given antigen. Most of the adjuvants are toxic at certain doses, and toxicity varies in different species. Moreover, there are no standard dosage limits set for adjuvant use in fish vaccines. We evaluated the acute toxicity, serum enzymes (AST/ALT) indicating hepatic injury and histopathological changes due to intra-peritoneal administration of different concentrations of a panel of adjuvants including quillaja saponin, aluminum hydroxide, squalene emulsion and Freund's incomplete adjuvant (FIA) with a dose ranging study of saponin (500, 160, 50, 16 and 5µgfish(-1)), aluminum hydroxide (5000, 1600, 500, 160 and 50µgfish(-1)), squalene emulsion (20, 10 and 5%), and FIA to determine the acceptable dosage for vaccination in olive flounder (Paralichthys olivaceus) fingerlings measuring 4.66±0.41g, 8.47±0.42cm. Saponin was highly toxic with a LD50 of approximately 105µgfish(-1) (22.4mgkg(-1)) causing severe histological damage and AST level was high at dose above 16µgfish(-1) and ALT, specific for liver damage was high only at 160µgfish(-1) (11U/L) and was safe at 5µgfish(-1). Aluminum hydroxide was toxic at 5000µgfish(-1) and was acceptable at dose below 1600µgfish(-1) with moderate histology and AST/ALT levels similar with control. Squalene emulsion showed increased inflammation at 20% and 10% emulsions and the inflammatory response was mild at a concentration of 5% oil emulsion and AST/ALT levels being similar to control in 10% and 5% emulsions and elevated in 20% on both sampling days. FIA was not lethal, but induced severe inflammation at injection site and around blood vessels. In comparison to FIA, saponin found to be safe at dose of 5µgfish(-1), aluminum hydroxide below 1600µgfish(-1), and squalene at 5% emulsion and could be accepted for vaccination studies. These results provide an insight for the selection of safer dose of adjuvants for intra-peritoneal vaccination of olive flounder.

Inactivated vaccine against viral hemorrhagic septicemia (VHS) emulsified with squalene and aluminum hydroxide adjuvant provides long term protection in olive flounder (Paralichthys olivaceus).

Viral hemorrhagic septicemia (VHS) in olive flounder (Paralichthys olivaceus) remains an unsolved health problem in Korean aquaculture. Vaccination plays a significant role in modern aquaculture, and the duration of protection provided is of vital importance. Here, we have demonstrated the efficacy, duration of protection and safety of an inactivated vaccine emulsified with squalene (5%) and aluminum hydroxide (0.5%). The inactivated VHS vaccine provided a moderate protection of 37% and 47% relative percent survival (RPS) at 4 and 10 weeks post vaccination (wpv). Addition of squalene and aluminum hydroxide into inactivated VHS vaccine clearly enhanced the level of protection showing 58% and 83% RPS at 4 and 10 wpv, respectively, indicating the need for adjuvants to enhance the efficacy. The vaccinated fish showed significant protection at 3, 6, 12, 18, 24, and 40 wpv (except week 57) than non-vaccinated fish to an intraperitoneal challenge of 10(7.1)TCID50/fish at 15 °C, with RPS of 60%, 64%, 71%, 55%, 52% and 50% (45% at 57 week), respectively, covering the duration of natural outbreak. Fish challenged at 18 wpv at 6 °C showed 56% RPS and protection at a low temperature. The antibody titer was high at 3 wpv with an OD of 1.08 ± 0.13, but decreased gradually and was undetectable by 24 wpv. The vaccine formulation was safe without injection site reactions, adhesions, or pigmentation observed at 6, 12, 18, or 24 wpv. Inflammatory reactions were observed in the spleen intestine at 6 and 12 wpv but were similar as control by 24 wpv. These results confirm that this vaccine is efficient and safe for olive flounder and could offer an appropriate strategy to prevent VHS without causing side effects.