Stimulating the immune response of Litopenaeus vannamei using the phagocytosis activating protein (PAP) gene

High mortality in the shrimp farming industry is caused by several pathogens such as white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio harveyi (V. harveyi). A PAP (Phagocytosis activating protein) gene able to activate phagocytosis of shrimp hemocytes was cloned into the eukaryotic expression vector phMGFP. In vitro expression was confirmed by transfection of PAP-phMGFP into CHO (Chinese Hamster Ovary) cells and the expression of the Green Fluorescent Protein (GFP) was observed. In order to activate the phagocytic activity of shrimp, 20, 40 and 80 μg/shrimp of this PAP-phMGFP vector were injected into Litopenaeus vannamei muscle. After challenged with WSSV, 40 μg/shrimp produced the highest relative percent survival (77.78 RPS). Analysis for the expression of the GFP gene in various tissues showed the expression mostly in the hemolymph of the immunized shrimp. The expression level of PAP and proPO (Prophenoloxidase) gene were highest at 7 days after immunization. This agreed with the efficiency of protection against WSSV that also occurred 7 days after immunization with the highest RPS of 86.61%. However there was no protection 30 days after immunization. Hemocytes of shrimp injected with PAP-phMGFP had 1.9 folds and 3 folds higher percentage phagocytosis and phagocytic index than the shrimp injected with PBS. Accordingly, copies of WSSV reduced in the PAP-phMGFP injected shrimp. In addition, PAP-phMGFP also protected shrimp against several pathogens: WSSV, YHV and V. harveyi, with RPS values of 86.61%, 63.34% and 50% respectively. This finding shows that the immune cellular defense mechanisms in shrimp against pathogens can be activated by injection of PAP-phMGFP and could indicate possible useful ways to begin to control this process.     

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.     

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.     

The immunostimulatory effect of hot-water extract of Gracilaria tenuistipitata on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were examined in the white shrimp Litopenaeus vannamei (10.3+/-1.5 g) injected individually with hot-water extract of Gracilaria tenuistipitata at 4 or 6 microg g-1. L. vannamei receiving hot-water extract of G. tenuistipitata at either dose increased significantly its THC, phenoloxidase activity, and respiratory burst after 2 days. L. vannamei received hot-water extract of G. tenuistipitata at 6 microg g-1 increased its phagocytic activity and clearance efficiency to V. alginolyticus after 1 day. In another experiment, L. vannamei which had been injected with hot-water extract of G. tenuistipitata were challenged with V. alginolyticus at 2x10(6) colony-forming units (cfu) shrimp-1 and then placed in seawater of 34 per thousand. The survival of shrimp that received hot-water extract of G. tenuistipitata at 6 microg g-1 was significantly higher than that of shrimp that received saline and the control shrimp after 3 days, and at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei receiving the hot-water extract of G. tenuistipitata at 6 microg g-1 or less increased its immune ability and resistance to V. alginolyticus infection.     

Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus

Pacific white shrimp (Litopenaeus vannamei) were injected with Taura syndrome virus (TSV) to assess shrimp immune responses and survival. TSV-infected shrimp suffered high mortality, but mock-infected and untreated shrimp experienced no mortality. Moribund shrimp were a pale, reddish colour and were lethargic and soft-shelled. Their haemolymph was clear red and coagulated poorly. In TSV-infected shrimp, the total haemocyte count (THC), hyalinocyte and granulocyte counts, and total plasma protein decreased significantly to 21%, 24%, 17% and 56% of untreated control values, respectively. Haemocyanin decreased to 67%, and clottable proteins to 80% of control values (P< 0.01). Copper and calcium ions, haemocytic transglutaminase (TGase) activity and plasma growth inhibitory activity against Vibrio harveyi also decreased significantly. Generation of intrahaemocytic superoxide anion, O(-2), in TSV-infected shrimp was significantly greater (P< 0.05) than in both control groups, no matter whether glucan stimulated or unstimulated. But the relative increase of intrahaemocytic O(-2) generation in TSV-infected shrimp response to glucan stimulation was lower in both controls. Plasma phenoloxidase (PO) activity increased significantly in TSV-infected shrimp. The plasma bacterial agglutinin titre against E. coli and V. harveyi, growth inhibition of E. coli and the concentration of magnesium ions in TSV-infected shrimp did not change significantly.In conclusion, ten of thirteen haemolymph parameters changed significantly during the host-TSV interaction. These parameters might be valuable references of shrimp health status.     

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.     

'Bright-red' syndrome in Pacific white shrimp Litopenaeus vannamei is caused by Vibrio harveyi

Since July 2005, recurrent outbreaks of vibriosis have occurred in shrimp farms in northwestern Mexico. Moribund Litopenaeus vannamei associated with mass mortalities were lethargic and displayed red discoloration spots on their abdomen, and hence were called 'bright-reds' by farmers. Shrimp submitted for diagnosis were examined using wet tissue mounts, bacteriological assays and their respective minimum inhibitory concentration (MIC), and histology. A dominant yellow bacterial colony was isolated in thiosulphate citrate bile salts-sucrose (TCBS) agar and identified by molecular methods as Vibrio harveyi strain CAIM 1792. Pathogenicity of the V. harveyi strain was demonstrated in L. vannamei. The lowest MIC against Vibrio isolates from bright-red shrimp was obtained with enrofloxacine (3.01, SD = 5.96 pg ml(-1)). Histology detected severe necrosis in lymphoid organ tubules, muscle fibers, and connective tissue, as well as melanization and hemocytic nodules associate with microcolonies of Gram-negative bacilli. Bacteria from severely affected shrimp were dispersed from the haemocoel to other tissues causing a systemic vibriosis. The data indicate that V. harveyi strain CAIM 1792 is the cause of bright-red syndrome (BRS) and represents a threat to the Mexican shrimp farming industry.     

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.     

Enhanced cellular immunity in shrimp (Litopenaeus vannamei) after 'vaccination'

It has long been viewed that invertebrates rely exclusively upon a wide variety of innate mechanisms for protection from disease and parasite invasion and lack any specific acquired immune mechanisms comparable to those of vertebrates. Recent findings, however, suggest certain invertebrates may be able to mount some form of specific immunity, termed 'specific immune priming', although the mechanism of this is not fully understood (see Textbox S1). In our initial experiments, either formalin-inactivated Vibrio harveyi or sterile saline were injected into the main body cavity (haemocoel) of juvenile shrimp (Litopenaeus vannamei). Haemocytes (blood cells) from V. harveyi-injected shrimp were collected 7 days later and incubated with a 1:1 mix of V. harveyi and an unrelated gram positive bacterium, Bacillus subtilis. Haemocytes from 'vaccinated' shrimp showed elevated levels of phagocytosis of V. harveyi, but not B. subtilis, compared with those from saline-injected (non-immunised) animals. The increased phagocytic activity was characterised by a significant increase in the percentage of phagocytic cells. When shrimp were injected with B. subtilis rather than vibrio, there was no significant increase in the phagocytic activity of haemocytes from these animals in comparison to the non-immunised (saline injected) controls. Whole haemolymph (blood) from either 'immunised' or non-immunised' shrimp was shown to display innate humoral antibacterial activity against V. harveyi that was absent against B. subtilis. However, there was no difference in the potency of antibacterial activity between V. harveyi-injected shrimp and control (saline injected) animals showing that 'vaccination' has no effect on this component of the shrimp's immune system. These results imply that the cellular immune system of shrimp, particularly phagocytosis, is capable of a degree of specificity and shows the phenomenon of 'immune priming' reported by other workers. However, in agreement with other studies, this phenomenon is not universal to all potential pathogens.     

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.     

Effects of rutin from Toona sinensis on the immune and physiological responses of white shrimp (Litopenaeus vannamei) under Vibrio alginolyticus challenge

Rutin is a bioflavonoid with strong antioxidant activity. To investigate the regulatory roles of rutin in various functions in crustaceans, we examined physiological (haemolymph glucose, lactate, and lipid) and innate non-specific immune responses (total haemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (release of superoxide anion, O(2)(-)) and superoxide dismutase (SOD) activity) to the pathogen Vibrio alginolyticus in white shrimp (Litopenaeus vannamei) that were individually injected with rutin extracted from Toona sinensis at 10, 20, or 50 microg g(-1). Results showed that PO activity and respiratory burst of L. vannamei increased obviously (P<0.05) when injected with rutin at a dose of 20 and 50 microg g(-1) after 12 and 24 h, respectively. Both the THC and SOD activities of experimental and control groups revealed no significant difference at all doses. L. vannamei injected with rutin at either dose maintained lower glucose, lactate, and lipid levels in response to V. alginolyticus challenge after 12-36, 24-48, and 24-60 h, respectively. The survival rate of L. vannamei that received rutin at either dose was significantly higher than that received saline after 48-72 h. It was, therefore, concluded that the immune ability and resistance against V. alginolyticus infection of L. vannamei receiving rutin at > or = 10 microg g(-1) increased.     

Anti-CHH antibody causes impaired hyperglycemia in Penaeus monodon

Crustacean hyperglycemic hormone (CHH) plays a major role in controlling glucose level in the haemolymph and also triggers important events during molting and reproductive cycles. In Penaeus monodon, three types of CHH, namely Pem-CHH1, Pem-CHH2 and Pem-CHH3, have been previously characterized. In this study, mouse polyclonal antibody was raised against recombinant Pem-CHH1 that was expressed in Escherichia coli. The anti-Pem-CHH1 antibody recognized all three types of Pem-CHHs but did not cross-react with either related hormone, molt-inhibiting hormone of P. monodon, or unrelated human growth hormone. The hyperglycemic activity in the extract from the eyestalk neural tissues was significantly depleted after incubating with anti-Pem-CHH antibody. Direct injection of the antibody into shrimp caused about 30-50% reduction in the haemolymph glucose level. The result demonstrates the ability of anti-Pem-CHH1 antibody to deplete the activity of CHH in vivo, and thus provides a possibility of using anti-Pem-CHH1 antibody to inhibit the hormone activity as a strategy to modulate growth and reproduction in this species.     

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.     

The immune response of the white shrimp Litopenaeus vannamei and its susceptibility to Vibrio infection in relation with the moult cycle

The white shrimp Litopenaeus vannamei (8.0-14.4 g) was examined for haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity, and clearance efficiency to the pathogen Vibrio alginolyticus in relation with moult cycle (postmoult, A, B; intermoult, C; premoult, D(0)/D(1)D(2)/D(3)). Granular cells were the highest at C and D(0)/D(1)stage, and the lowest at A stage. Hyaline cells and THC (total haemocyte count) were higher at C stage, but lower at postmoult stages. Phenoloxidase activity was the highest at C stage, and the lowest at A stage. Respiratory burst was lower at A stage. Phagocytic activity of shrimps against V. alginolyticus decreased significantly at postmoult and premoult stages. Additionally, the clearance efficiency of shrimps to V. alginolyticus was significantly lower for shrimps at A stage than those at C stage. In another experiment, L. vannamei at different moult stages were injected with tryptic soy broth (TSB)-grown V. alginolyticus (1x10(5)cfu shrimp(-1)) and then held in 34% seawater. After 10 h, the mortality of V. alginolyticus-injected shrimps was significantly higher for shrimps at postmoult stage than those at intermoult stage. Over 48-120 h, the mortality of V. alginolyticus-injected shrimps was 50.0%, 33.3% and 40.0% at postmoult, intermoult and premoult stage, respectively. It is concluded that L. vannamei showed a decrease in resistance at A stage through a reduction of its haemocyte count, phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency against V. alginolyticus.     

Hyperglycemic and osmotic effects of dopamine and recombinant hormone CHH-B1 in the Pacific white shrimp Litopenaeus vannamei

The effects of dopamine (DA) on crustacean hyperglycemic hormone (CHH) release and osmoregulation were investigated in the white shrimp Litopenaeus vannamei. Application of 2 μg of the recombinant CHH-B1_His hormone or 2?×?10^?6 mol L^?1 of DA to intact shrimp caused an increase in the hemolymph glucose levels 1 h post-injection, suggesting that DA might stimulate hyperglycemia through CHH release from the sinus glands. This assumption was supported by similar experiments using bilaterally eyestalk-ablated shrimp. Additionally, rCHH-B1_His restored the osmoregulatory capacity (OC) of shrimp under hyperosmotic conditions to basal values 2 h post-injection, whereas DA led to an OC decrease in shrimp at all sampling times. These neuroendocrine factors may be involved in the control of metabolism and osmoregulation in L. vannamei and could be important for its adaptation to different environmental conditions.     

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.     

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.     

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.