Virulence of Vibrio harveyi responsible for the "Bright-red" Syndrome in the Pacific white shrimp Litopenaeus vannamei

Vibrio harveyi (Vh) CAIM 1792 strain was isolated from Litopenaeus vannamei affected with "Bright-red" Syndrome (BRS). The strain grew in 1-10% NaCl, at 15-35°C and was resistant to ampicillin (10 μg), carbenicillin (100 μg) and oxytetracycline (30 μg). The lowest MIC was for enrofloxacine (0.5 μgml(-1)). The in vivo and in vitro toxicity of bacterial cells and the extracellular products (ECPs) of Vh CAIM 1792 grown at 1.0%, 2.0% and 4.0% NaCl were evaluated. Adherence ability, enzymatic activities and siderophore production of bacterial cell was tested. The ECPs exhibited several enzymatic activities, such as gelatinase, amylase, lipase, phospholipase and caseinase. These ECPs displayed a strong cytotoxic effect on HELA cell line at 6 and 24 h. Challenges using 10(3) CFU g(-1) caused opacity at the site of injection and over 80% shrimp mortality before 24 h p.i. (post-injection). Mortality caused by the ECPs was higher than mortalities with bacteria, especially in the first hours p.i. Bacteria were re-isolated from hemolymph samples of moribund shrimp and identified as Vh CAIM 1792 by rep-PCR. Histological analysis of shrimp L. vannamei injected with Vh CAIM 1792 revealed generalized necrosis involving skeletal muscle (MU) at the injection site, the lymphoid organ (LO), heart and connective tissues. Melanization within the MU at the site of injection was also observed as well as hemocytic nodules within the hearth and MU at 168 h p.i. LO was the target organ of BRS. Necrosis of the MU at the injection site was the main difference in comparison to other shrimp vibriosis.     

Noradrenaline modulates the immunity of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in response to pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (18.4 +/- 1.2 g) were injected individually with noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1). For the shrimp that received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1), the THC decreased by 15%, 21% and 32%, phenoloxidase activity decreased by 15%, 31% and 31%, respiratory burst decreased by 13%, 21% and 32%, and SOD activity decreased by 46%, 56% and 55%, respectively, after 2 h. The phagocytic activity and clearance efficiency of shrimp that received noradrenaline at either dose decreased significantly after 2 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency returned to normal values after 4, 4, 8, 24, 16 and 8 h, respectively, in the shrimp that received noradrenaline at either dose. In another experiment, L. vannamei which had received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1) were challenged after 1h by injection with V. alginolyticus at 1.0 x 10(5) colony-forming units (cfu)shrimp(-1) and then placed in seawater of 20 per thousand. The cumulative mortality of shrimp that received noradrenaline at either dose was significantly higher than that of shrimp that received saline after 4 h, and at the termination of the experiment (48 h after the challenge). It is therefore concluded that noradrenaline administration at 10(-6) mol shrimp(-1) or less causes immune modulation of L. vannamei.     

Effects of dopamine on the immunity of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in response to pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (20.0+/-1.5 g) were injected individually with dopamine at 10(-8), 10(-7) and 10(-6)mol shrimp(-1), respectively. For the shrimp that received dopamine at 10(-7) and 10(-6)mol shrimp(-1), the THC decreased by 25% and 39%, phenoloxidase activity decreased by 15% and 32%, respiratory burst decreased by 21% and 36%, and SOD activity decreased by 50% and 63%, respectively, after 4 h. The phagocytic activity and clearance efficiency of shrimp that received dopamine at either dose decreased significantly after 2 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency returned to normal values after 16, 8, 8, 24, 16 and 4 h, respectively, for the shrimp that received dopamine at either dose. In another experiment, L. vannamei which had received dopamine at 10(-8), 10(-7) and 10(-6)mol shrimp(-1) were challenged after 1 h by injection with V. alginolyticus at 1.0x10(5) colony-forming units (cfu) shrimp-1 and then placed in seawater of 20 per thousand. The cumulative mortality of shrimp that received dopamine at either dose was significantly higher than that of shrimp that received saline after 8 h, and of shrimp that received saline at the termination of the experiment (48 h after the challenge). It is therefore concluded that dopamine administration at 10(-6)mol shrimp-1 or less causes immune modulation of L. vannamei.     

Effect of saponin immersion on enhancement of the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The haemocyte count, phenoloxidase (PO) activity, specific alpha(2)-macroglobulin (alpha2-M) activity, respiratory burst, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, phagocytic activity, and clearance efficiency against Vibrio alginolyticus were examined when the white shrimp Litopenaeus vannamei (10.42+/-2.0g) were immersed in seawater (34 per thousand) containing different concentrations of saponin (0, 0.5, 1 and 2mgL(-1)) for 24, 48 and 72h. Hyaline cells (HC), the total haemocyte count (THC), specific alpha2-M activity, respiratory burst, SOD activity, and GPx activity directly increased with the saponin concentration, whereas PO activity was inversely related to the saponin concentration. White shrimp L. vannamei that were immersed in saponin at 1 and 2mgL(-1) showed increased phagocytic activity and clearance efficiency to V. alginolyticus over 48-72h. In another experiment, shrimp immersed in seawater containing different concentrations of saponin after 72h were challenged with V. alginolyticus at 3.2x10(5) colony-forming units (cfu)shrimp(-1), and then placed in seawater. The survival rate of shrimp immersed in seawater containing saponin at either dose was significantly higher than that of control shrimp after 12h, as well as at the termination of the experiment (5days after the challenge). It was therefore concluded that L. vannamei immersed in water containing saponin at 2mgL(-1) or less exhibited an immunomodulatory effect, as well as protection against V. alginolyticus infection.     

Effect of copper sulfate on the immune response and susceptibility to Vibrio alginolyticus in the white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei (Boone) held in 35 per thousand seawater were challenged with Vibrio alginolyticus at a dose of 3 x 10(5) colony-forming units (cfu) shrimp(-1), and then placed in water containing concentrations of Cu2+ at 0 (control), 1, 5, 10 and 20 mg l(-1). Mortality of shrimp in 5, 10 and 20 mg l(-1) Cu2+ was significantly higher than those in 1 mg l(-1) Cu2+ and the control solution after 24-96 h. In another experiment, L. vannamei which had been exposed to control, 1, 5, 10 and 20 mg l(-1) Cu2+ for 24, 48 and 96 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity and clearance efficiency to V. alginolyticus. Copper concentrations at 1 mg l(-1) or greater for 24h resulted in decreased THC, phenoloxidase activity, phagocytic activity and clearance efficiency, whereas copper concentration at 20 mg l(-1) caused significant increase in respiratory burst of L. vannamei. In conclusion, concentration of Cu2+ at 1 mg l(-1) or greater increased the susceptibility of L. vannamei to V. alginolyticus infection by a depression in immune ability. The release of superoxide anion by L. vannamei exposed to 20 mg l(-1) Cu2+ was considered to be cytotoxic to the host.     

Administration of hot-water extract of brown seaweed Sargassum duplicatum via immersion and injection enhances the immune resistance of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, and respiratory burst were examined when the white shrimp Litopenaeus vannamei (10.42+/-1.39 g) were immersed in seawater (34 per thousand) containing hot-water extract of brown alga Sargassum duplicatum at 100, 300 and 500 mg l(-1), or injected with hot-water extract of S. duplicatum at 2, 6, 10 and 20 microg g(-1). These parameters increased significantly when the shrimp were immersed in seawater containing hot-water extract at 300 and 500 mg l(-1) after 1 h, or when the shrimp were injected with hot-water extract at 10 and 20 microg g(-1) after 1 day. L. vannamei that were injected with hot-water extract at 6, 10 and 20 microg g(-1) had increased phagocytic activity and clearance efficiency to V. alginolyticus after 1-6 days. In another experiment, L. vannamei which had been immersed in seawater containing hot-water extract at 100, 300 and 500 mg l(-1), or injected with hot-water extract at 2, 6, 10 or 20 microg g(-1) were challenged with V. alginolyticus at 1 x 10(6), or 1.4 x 10(6) colony-forming units (cfu) shrimp(-1), and then placed in seawater. The survival of shrimp that received hot-water extract at either dose was significantly higher than that of control shrimp after 2 days, as well as at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei that were immersed in hot-water extract of S. duplicatum at 300 mg l(-1), or the shrimp that were injected with hot-water extract at 10 microg g(-1) or less had increased immune ability as well as resistance to V. alginolyticus infection.     

Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei

The haemogram, phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, lysozyme activity, and the mitotic index of haematopoietic tissue (HPT) were examined after the white shrimp Litopenaeus vannamei had been fed diets containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 0.5, 1.0, and 2.0 g kg(-1) for 7-35 days. Results indicated that these parameters directly increased with the amount of extract and time, but slightly decreased after 35 days. RBs, SOD activity, and GPx activity reached the highest levels after 14 days, whereas PO and lysozyme activities reached the highest levels after 28 days. In a separate experiment, white shrimp L. vannamei, which had been fed diets containing the extract for 14 days, were challenged with Vibrio alginolyticus at 2 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1), and then placed in seawater. The survival rate of shrimp fed the extract-containing diets was significantly higher than that of shrimp fed the control diet at 72-144 h post-challenge. We concluded that dietary administration of the G. tenuistipitata extract at ≤1.0 g kg(-1) could enhance the innate immunity within 14 days as evidenced by the increases in immune parameters and mitotic index of HPT in shrimp and their enhanced resistance against V. alginolyticus and WSSV infections. Shrimp fed the extract-containing diets showed a higher and continuous increase in the humoral response indicating its persistent role in innate immunity.     

The immunostimulatory effects of hot-water extract of Gelidium amansii via immersion, injection and dietary administrations on white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), phenoloxidase activity, and respiratory burst were examined when white shrimp Litopenaeus vannamei were immersed in seawater (34 per thousand) containing hot-water extract of red alga Gelidium amansii at 200, 400 and 600 mg l(-1), injected with hot-water extract at 4 and 6 microg g(-1) shrimp, and fed diets containing hot-water extract at 0, 0.5, 1.0 and 2.0 g kg(-1). These parameters increased significantly when shrimp were immersed in seawater containing hot-water extract at 400 and 600 mg l(-1) after 1h, when shrimp were injected with hot-water extract at 6 microg g(-1) shrimp after one day, and when shrimp were fed diets containing hot-water extract at 1.0 and 2.0 g kg(-1) after 14 days. Phagocytic activity and clearance efficiency were significantly higher for the shrimp that were fed diets containing hot-water extract at 1.0 and 2.0 g kg(-1) than those of shrimp that were fed diets containing hot-water extract at 0 and 0.5 g kg(-1) after 14 and 28 days. In a separate experiment, L. vannamei which had received hot-water extract via injection, or fed diets containing hot-water extract, were challenged after 3h or 28 days with V. alginolyticus at 2 x 10(6) cfu shrimp(-1) and 1 x 10(6) cfu shrimp(-1), respectively, and then placed in seawater. The survival of shrimp that were injected with hot-water extract at 6 microg g(-1) was significantly higher than that of control shrimp after 1 day, and the survival of shrimp fed diets containing hot-water extract at 0.5, 1.0 and 2.0 g kg(-1) increased significantly after 3 days as well as at the end of the experiment (6 days after the challenge), respectively. It was concluded that L. vannamei that were immersed in hot-water extract at 400 mg l(-1), injected with hot-water extract at 6 microg g(-1) shrimp, and fed hot-water extract of G. amansii at 2.0 g kg(-1) or less showed increased immune ability as well as resistance to V. alginolyticus infection.     

The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under low and high pH stress

White shrimp Litopenaeus vannamei (also known as Penaeus vannamei) held in 34 per thousand seawater at pH 8.2 were injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 8 x 10(5) colony-forming units (cfu) shrimp(-1), and then transferred to tanks at pH 6.5, 8.2 (control) and 10.1, respectively. After 24-168 h, the mortality of V. alginolyticus-injected shrimp that were transferred to pH 6.5 and pH 10.1 tanks was significantly higher than that of V. alginolyticus-injected shrimp held at pH 8.2. In another experiment, L. vannamei held at pH 8.2 following transfer to pH 6.5, 8.2 (control) and 10.1 for 6, 12, 24, 72 and 120 h were examined for immune parameters, phagocytic activity, and the clearance efficiency of shrimp against V. alginolyticus. The results indicated that the shrimp that were transferred to pH 6.5 and 10.1 showed significantly decreased phenoloxidase (PO) activity, respiratory burst, phagocytic activity, and clearance efficiency against V. alginolyticus over 6-72 h; significantly decreased superoxide dismutase (SOD) activity over 6-24h; and decreased total haemocyte count (THC) over 12-72 h. Shrimp transferred to pH 10.1 showed significantly decreased granular cell counts, and THC after 6h, and decreased SOD activity after 72 h. The immune parameters of shrimp transferred to pH 6.5 and 10.1 returned to the original values after 120 h. However, shrimp transferred to pH 6.5 still maintained lower phagocytic activity, and clearance efficiency against V. alginolyticus, and shrimp transferred to pH 10.1 still maintained lower clearance efficiency against V. alginolyticus. It was therefore concluded that low pH and high pH stress decrease the resistance of white shrimp L. vannamei against V. alginolyticus and decrease several parameters of the immune response.     

Safety and protective effect of a disinfectant (STEL water) for white spot syndrome viral infection in shrimp

The efficacy of STEL water for protection against white spot syndrome virus (WSSV) infection was evaluated using shrimp. The LC50 of residual chlorine (Cl-) in STEL water for brood-stock and 2-mo-old shrimp were 2.3 and 3.2 ppm, respectively. All 2-month-old shrimp raised in seawater containing more than 40 microl 2l(-1) of a WSSV-infected tissue homogenate died within 3 d post-exposure (dpe). Thus, a 10-fold dose of 400 microl 2 l(-1) was used in the disinfection tests. Low concentrations of STEL water effectively prevented mortality of shrimp at this challenge dose. All 2-month-old shrimp exposed to seawater with 400 microl of viral homogenate disinfected with STEL water at Cl- concentrations over 0.125 ppm for 1 and 10 min, lived until 5 dpe. With 5-mo-old shrimp, all positive control shrimps died within 3 dpe, whereas most shrimp reared in seawater disinfected with STEL water for 1 h before addition of homogenate lived until 5 dpe. Results suggested that continuous disinfection of seawater with STEL water may be effective for preventing WSSV infection in shrimp.     

The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress

The white shrimp Litopenaeus vannamei were challenged with tryptic soy broth (TSB)-grown Vibrio alginolyticus at a dose of 1 x 10(6) colony-forming units (cfu) shrimp(-1), and then placed in water containing concentrations of nitrite-N at 0 (control), 1.12, 5.15, 11.06 and 21.40 mg l(-1). Mortality of shrimp in 5.15, 11.06 and 21.40 mg l(-1) was significantly higher than those in the control solution after 48-168 h. L. vannamei that had been exposed to control, 0.98, 4.94, 9.87 and 19.99 mg l(-1) nitrite-N for 96 h were examined for THC (total haemocyte count), phenoloxidase activity, and respiratory burst (release of superoxide anion). The THC and phenoloxidase activity decreased when the shrimp were exposed to 4.94, 9.87 and 19.99 mg l(-1) nitrite-N, whereas, the respiratory burst increased significantly at 9.87 and 19.99 mg l(-1) nitrite-N after 96 h. It is therefore suggested that nitrite in water caused a depression in the immune ability of L. vannamei and an increased susceptibility to V. alginolyticus infection, together with an increase of superoxide anion production, possibly to cytotoxic levels for the host.     

The immune stimulatory effect of sodium alginate on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (9.4-11.3 g) were injected individually with sodium alginate at 10, 20 or 50 microg g(-1). No significant differences in THC, DHC and superoxide dismutase activity were observed among the shrimp injected with saline and those injected with sodium alginate at 10, 20 or 50 microg g(-1). However, L. vannamei injected with sodium alginate at 20 microg g(-1)increased its phenoloxidase activity and respiratory burst after 2 days and one day, respectively. L. vannamei injected with sodium alginate at 50 microg g(-1)maintained a higher phagocytic activity and clearance efficiency to V. alginolyticus after 4 days. In another experiment, L. vannamei which had been injected with sodium alginate, were challenged with V. alginolyticus at 2x10(5)colony-forming units (CFU) shrimp(-1)and then placed in seawater of 34 per thousand. The survival of shrimp that received sodium alginate at either dose was significantly higher than that of control shrimp at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei received sodium alginate at 10 microg g(-1)or more and increased its immune ability and resistance from V. alginolyticus infection.     

Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1h after the rLV-CHH injection and then returned to normal after 3h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.     

Time course of necrotizing hepatopancreatitis (NHP) in experimentally infected Litopenaeus vannamei and quantification of NHP-bacterium using real-time PCR

Necrotizing hepatopancreatitis (NHP), a severe bacterial disease affecting penaeid shrimp aquaculture, is caused by a gram-negative, pleomorphic, intracellular alpha-proteobacterium referred to as the NHP-bacterium (NHPB). The time course of NHP was investigated in experimentally infected juveniles of Kona stock Litopenaeus vannamei. Susceptible animals were individually isolated in 41 of aerated artificial seawater at salinity 30 +/- 1 ppt and maintained in a water bath at 30 +/- 1 degree C for 60 d. A total of 120 individuals were exposed per os to a 0.05 g piece of NHPB-infected hepatopancreas and 100 controls were exposed to uninfected tissue. At intervals of 3, 6, 9, 16, 23, 30, 37, 44, and 53 d post-exposure, 6 shrimp exposed to NHPB-infected tissue and 4 controls were randomly removed from the experiment; hepatopancreas samples were processed for histological and molecular analysis, and feces were processed for molecular diagnosis of NHPB infection. NHPB was first detected in the hepatopancreas through histology at 6 d post-exposure. All control shrimp were diagnosed as NHPB negative. NHPB infections classified as stage I (scattering of hepatopancreatic tubules with adjacent epithelial cells containing NHPB) and stage II (numerous infected tubules with occasional hemocyte infiltration) were observed from 6 to 37 d post-exposure. All animals that experienced NHPB-induced mortality from 16 to 51 d post-exposure were at stage III (numerous necrotic tubules, dense hemocyte infiltration, and presence of granulomas). NHPB is capable of infecting all hepatopancreatic cell types including embryonic, resorptive, fibrillar and blister-like cells. The percent of hepatopancreatic tubules containing NHPB in epithelial cells increased over time, representing bacteria multiplication and spread. Real-time PCR allowed for quantification of NHPB in hepatopancreas and feces. Over the course of infection, NHPB was present at 10(3) to 10(7) copies mg(-1) of hepatopancreas and 10(1) to 10(5) copies mg(-1) of feces. Lethal infections contained 10(6) to 10(7) copies mg(-1) of hepatopancreas and 10(3) to 10(6) copies mg(-1) of feces.     

Modulation of the innate immune system in white shrimp Litopenaeus vannamei following long-term low salinity exposure

Immune parameters, haemocyte lifespan, and gene expressions of lipopolysaccharide and β-glucan-binding protein (LGBP), peroxinectin (PX), integrin β, and α2-macroglobulin (α2-M) were examined in white shrimp Litopenaeus vannamei juveniles (0.48 ± 0.05 g) which had been reared at different salinity levels of 2.5‰, 5‰, 15‰, 25‰, and 35‰ for 24 weeks. All shrimp survived during the first 6 weeks. The survival rate of shrimp reared at 2.5‰ and 5‰ was much lower (30%) than that of shrimp reared at 15‰, 25‰, and 35‰ (76%~86%) after 24 weeks. Shrimp reared at 25% grew faster. Shrimp reared at 2.5‰ and 5‰ showed lower hyaline cells (HCs), granular cells (GCs), phenoloxidase activity (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme activity, but showed a longer haemocyte lifespan, and higher expressions of LGBP, PX, integrin β, and α2-M. In another experiment, shrimp which had been reared at different salinity levels for 24 weeks were challenged with Vibrio alginolyticus (6 × 10(6) cfu shrimp(-1)), and WSSV (10(3) copies shrimp(-1)) and then released to their respective seawater. At 96-144 h, cumulative mortalities of shrimp reared at 2.5‰ and 5‰ were significantly higher than those of shrimp reared at 15‰, 25‰, and 35‰. It was concluded that following long-term exposure to 2.5‰ and 5‰ seawater, white shrimp juveniles exhibited decreased resistance against a pathogen due to reductions in immune parameters. Increases in the haemocyte lifespan and gene expressions of LGBP, integrin β, PX, and α2-M indicated that shrimp had the ability to expend extra energy to modulate the innate immune system to prevent further perturbations at low salinity levels.     

The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei

White shrimp, Litopenaeus vannamei, which had been injected with chitin at 4, 6 and 8 microg g(-1) or chitosan at 2, 4 and 6 microg g(-1), were challenged with pathogen Vibrio alginolyticus at 2 x 10(6) colony-forming units (cfu) shrimp(-1) and then placed in seawater of 34 per thousand. The survival of shrimp that received chitin or chitosan at either dose was significantly higher than that of control shrimp after 1 day, and at the termination of the experiment (6 days after the challenge). In another experiment, the total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, and phagocytic activity to V. alginolyticus were measured when L. vannamei (10.4 +/- 0.7 g) were injected individually with chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1). L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased significantly its THC and respiratory burst after 2 days. L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) still maintained significantly higher phenoloxidase activity after 6 days. L. vannamei received chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased its phagocytic activity against V. alginolyticus after 1 day, respectively. It is therefore concluded that L. vannamei that received chitin at 6 microg g(-1) or chitosan at 4 microg g(-1) or less increased its immune ability and resistance to V. alginolyticus infection.     

Impact of exposure to bacteria on metabolism in the penaeid shrimp Litopenaeus vannamei

We hypothesized that aggregation of bacteria and hemocytes at the gill, which occurs as part of the shrimp's antibacterial immune defenses, would impair normal respiratory function and thereby disrupt aerobic metabolism. Changes in oxygen uptake and lactate accumulation were determined in Litopenaeus vannamei, the Pacific white shrimp, following injection with either saline (control) or a strain of the gram-negative bacterium Vibrio campbellii that is pathogenic in crustaceans. The rate of oxygen uptake was determined during the first 4 h after injection and after 24 h. Injection of bacteria decreased oxygen uptake by 27% (from 11.0 to 8.2 micromol g-1 h-1) after 4 h, while saline-injected shrimp showed no change. Decreased oxygen uptake persisted 24 h after Vibrio injection. In well-aerated water, resting whole-animal lactic acid levels increased in shrimp injected with bacteria (mean=2.59 micromol lactate g-1+/-0.39 SEM, n=8) compared to saline-injected control shrimp, but this difference did not persist at 24 h. Exposure to hypercapnic hypoxia (PCO2=1.8 kPa, PO2=6.7 kPa) also resulted in significant whole-body lactic acid differences (mean=3.99 and 1.8 micromol g-1 tissue in Vibrio and saline-injected shrimp, respectively). Our results support the hypothesis that the crustacean immune response against invading bacteria impairs normal metabolic function, resulting in depression of oxygen uptake and slightly increased anaerobic metabolism.     

The immune response of white shrimp Litopenaeus vannamei following Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei injected with saline, and injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 1.0 x 10(5) and 1.8 x 10(5) colony-forming units (cfu) shrimp(-1) were examined for hyaline cell (HC) counts, granular cell (GC) counts, total haemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after 1-168 h. Shrimp that received no injection served as the control. The shrimps which received V. alginolyticus at both doses showed significant decreases in these parameters after 6-96 h. The values for HC and SOD activity decreased earlier and then RB. The time to cause maximum depletion of haemocytes (haemocytopenia), PO activity, RB, and SOD activity were 12, 72, 48, and 24 h post-injection, respectively. The HC, GC, and RB returned to the original values earlier at 72 h, followed by SOD activity at 96 h, and then PO activity at 168 h post-infection. It was concluded that an injection of V. alginolyticus rapidly reduced the shrimp's immunity by decreasing HC, GC, SOD activity, RB, and PO activity within 3-24 h, followed by a slow recovery during 72-168 h post-injection. Furthermore, white shrimp L. vannamei which received V. alginolyticus showed a 6-9 h later response in PO activity, and a 72-96 h later recovery of PO activity, compared to the responses in RB and SOD activity indicating their roles in shrimp defence and immunity.     

The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus at different salinity levels

White shrimp Litopenaeus vannamei held in 25 per thousand seawater were injected with TSB-grown Vibrio alginolyticus (1 x 10(4) cfu shrimp(-1)), and then transferred to 5, 15, 25 (control) and 35 per thousand. Over 24-96 h, the mortality of V. alginolyticus-injected shrimp held in 5 per thousand and 15 per thousand was significantly higher than that of shrimp held in 25 per thousand and 35 per thousand, and the mortality of V. alginolyticus-injected shrimp held in 5 per thousand was the highest. Shrimp held in 25 per thousand and then transferred to 5, 15, 25 (control) and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V. alginolyticus after 12-72 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency decreased significantly for the shrimp held in 5 and 15 per thousand after 12 h. It is concluded that the shrimp transferred from 25 per thousand to low salinity levels (5 and 15 per thousand) had reduced immune ability and decreased resistance against V. alginolyticus infection.     

Dietary administration of sodium alginate enhances the immune ability of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

Haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase (SOD) activity, glutathione peroxidase (GPX) activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured in white shrimp Litopenaeus vannamei juveniles (12.3 +/- 1.2 g) which had been fed diets containing sodium alginate at 0.5, 1.0, 2.0 g kg(-1) after five months. L. vannamei fed a diet containing 2.0 g kg(-1) sodium alginate had increased phenoloxidase activity, respiratory burst and SOD activity, but decreased GPX activity significantly. L. vannamei fed a diet containing 2.0 g kg(-1) sodium alginate had increased phagocytic activity and the shrimp fed a diet containing sodium alginate at 0.5, 1.0 or 2.0 g kg(-1) had increased clearance efficiency to V. alginolyticus. In another experiment, L. vannamei, which had been fed control diet, or sodium alginate-containing diets after 5 months, were challenged with V. alginolyticus at 2 x 10(6) colony-forming units (CFU) shrimp(-1) and then placed in seawater of 15 per thousand. The survival of shrimp fed a diet containing 2.0 g kg(-1) after one day, and the survival of shrimp fed diets containing sodium alginate at 0.5 and 1.0 g kg(-1) after 2-4 days increased significantly, as compared to that of shrimp fed control diet. It is therefore concluded that administration of sodium alginate in the diet at 2.0 g kg(-1) or less could enhance the immune ability of L. vannamei and increase its resistance to V. alginolyticus infection.