Natural cytotoxic activity in seabream (Sparus aurata L.) and its modulation by vitamin C

Growth of pathogen bacterium. Enterococcus was not affected in tryptic soy broth (TSB) medium containing ammonia-N concentration in the range of 0-5.14 mg l(-1). Giant freshwater prawn Macrobrachium rosenbergii (8-12 g) were challenged with Enterococcus which had been incubated for 24 h in TSB medium containing different concentrations of ammonia-N at 0-5.14 mg l(-1) Cumulative mortality of M. rosenbergii was higher for the bacteria incubated in TSB medium having ammonia-N at 0 and 0.26 mg l(-1), than those incubated in TSB medium having 1.28, 2.57 and 5.14 mg l(-1) ammonia-N after 24 h of challenge. However, cumulative mortality of prawn was significantly higher for the bacteria incubated in TSB medium with no ammonia added after 120 h of challenge. The prawns (8-12 g) were challenged with Enterococcus previously incubated in TSB medium for 24 h, then placed in water having concentrations of ammonia-N at control (0.06 mg l(-1)), 0.55, 1.01, 1.68 and 3.18 mg l(-1). Mortality of prawns increased directly with ammonia-N concentrations after 72 h challenge. The pranws (20-30 g) which had been exposed to control, 0.55, 1.68 and 3.18 mg (-1) ammonia-N for 7 days were examined for the total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity and respiratory burst of haemocytes. Phenoloxidase activity decreased when the prawns were exposed to ammonia-N greater than 0.55 mg l(-1). The respiratory burst increased significantly at 0.55 mg l(-1) but decreased significantly at 1.68 and 3.18mg (-1) ammonia-N. No significant difference in haemocyte count was observed among the prawns at different ammonia-N concentrations. It is suggested that ammonia in water decreases the virulence of Enterococcus, and reduces the immune resistance of M. rosenbergii.     

Trichlorfon, an organophosphorus insecticide, depresses the immune responses and resistance to Lactococcus garvieae of the giant freshwater prawn Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory bursts (release of superoxide anions), superoxide dismutase (SOD) activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (18.2+/-2.1 g) were individually reared in water containing concentrations of trichlorfon of 0, 0.2, or 0.4 mg L(-1) for a 144-h period. No significant differences in the THC were observed among prawns at the beginning and following 144 h of exposure to 0, 0.2, and 0.4 mg L(-1) trichlorfon. However, phenoloxidase activity significantly decreased when the prawns were exposed to trichlorfon at 0.2 and 0.4 mg L(-1), and no significant differences were observed between the two concentrations at any sampling time. The total production of superoxide anion by M. rosenbergii significantly increased with exposure to 0.2 and 0.4 mg L(-1) trichlorfon, and no significant differences were observed between the two concentrations during the 144-h exposure period. However, M. rosenbergii exposed to 0.2 and 0.4 mg L(-1) trichlorfon showed decreased SOD activity from 48 to 144 h. Phagocytic activity and clearance efficiency to L. garvieae significantly decreased when prawns were exposed to 0.2 and 0.4 mg L(-1) trichlorfon for 48 h. In another experiment, prawns were challenged with 5 x 10(5) colony-forming units (cfu)prawn(-1) of L. garvieae, then reared in water containing different concentrations of trichlorfon. The onset of mortality was earlier in prawns exposed to trichlorfon compared to those exposed to the zero control. The cumulative mortality of prawns directly increased with ambient trichlorfon concentrations in the range of 0-0.4 mg L(-1) after 168 h. It is concluded that exposure of M. rosenbergii to trichlorfon at 0.2 mg L(-1) or more caused cytotoxicity resulting in depression of the immune response, and increased its susceptibility to L. garvieae infection.     

Effects of intrinsic and extrinsic factors on the haemocyte profile of the prawn, Macrobrachium rosenbergii

The giant freshwater prawn Macrobrachium rosenbergii was investigated for its total haemocyte count (THC) based on season, sex, size and feeding rate. The THC, when the prawns were subjected to injections of foreign materials was also investigated. The prawns displayed the highest and lowest THC in autumn and winter respectively, with no significant difference between male and female, or among animals with a body weight range of 7-115 g. The prawns displayed the lowest THC at D3 stage, and the highest in C stage. The prawns displayed the lowest THC when they were fed at 0.1% feeding rate among feeding rates of 0.1%, 0.5%, 1.0% and 1.5% body weight x day(-1). Prawns injected with carbon powder and Enterococcus showed increased THC during the first 6 h. Prawns injected with saline and carbon powder had the lowest THC after 30 h, and recovered to the normal value after 54 h. Prawns injected with Enterococcus showed the lowest THC after 42 h, and showed delayed recovery.     

Effect of benzalkonium chloride stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii

Addition of benzalkonium chloride (BKC) at 0, 0.3, 0.6 and 1.0 mg l(-1) to tryptic soy broth (TSB) had no effect on growth of Lactococcus garvieae, a bacterial pathogen of the giant freshwater prawn Macrobrachium rosenbergii. However, injection of the cultured cells into prawns at a dose of 4 x 10(6) colony-forming units (cfu) prawn(-1) resulted in significantly higher mortality at 120 h (p < 0.05) in prawns injected with cells grown in the absence of BKC than in prawns injected with cells grown in the presence of BKC. In other experiments, prawns were injected with TSB-grown L. garvieae (4 x 10(6) and 3 x 10(5) cfu prawn(-1)) and then held in water containing BKC at 0, 0.3, 0.6 and 1.0 mg l(-1). After 120 h, mortality was significantly higher in all the BKC treatments than in the control without BKC. Prawns showed no significant differences in total hemocyte count (THC) or differential hemocyte count (DHC) amongst treatment and control groups. However, 96 h exposure to 0.3 mg l(-1) BKC or more resulted in a decrease in phenoloxidase activity and an increase in respiratory burst to levels considered to be cytoxic. In summary, exposure of L. garvieae to BKC at 0.3 mg l(-1) or more decreased its virulence to M. rosenbergii, while exposure of M. rosenbergii to BKC at 0.3 mg l(-1) or more increased its susceptibility to L. garvieae infection.     

Effect of nitrite on interaction between the giant freshwater prawn Macrobrachium rosenbergii and its pathogen Lactococcus garvieae

Addition of nitrite-N at 1.5 mg l(-1) in tryptic soy broth (TSB) significantly (p < 0.05) decreased the growth rate of the bacterial pathogen Lactococcus garvieae and significantly (p < 0.05) reduced mortality compared to zero nitrite controls when injected into giant freshwater prawns Macrobrachium rosenbergii at 5 x 10(5) colony-forming units (CFU) per prawn. In other experiments, whereby prawns were injected with TSB-grown L. garvieae (5 x 10(5) CFU prawn(-1)) and then held in water containing nitrite-N, mortality at 72 h post-injection was significantly (p < 0.05) higher for prawns held in water containing 1.68 mg l(-1) nitrite than at lower concentrations. Prawns exposed to different concentrations of nitrite-N were examined for THC (total hemocyte count), phenoloxidase activity, respiratory burst, phagocytic activity and bacterial clearance efficiency. No significant differences in THC and phenoloxidase activity were observed among treatments. With prawns exposed to nitrite-N for 168 h (7 d) at 1.59 mg l(-1), phagocytic activity and clearance efficiency decreased, while at 1.15 mg l(-1) or more, respiratory burst increased, generating the superoxide anion at levels considered cytoxic to the host. We conclude that nitrite-N at 1.68 mg l(-1) causes depression in the immune response and increased mortality in M. rosenbergii infected with L. garvieae.     

The susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae and its resistance under copper sulfate stress.

Addition of copper sulfate (0.1 to 0.4 mg l(-1)) to tryptic soy broth (TSB) had no effect on growth rate of the bacterial pathogen Lactococcus garvieae. Giant freshwater prawns Macrobrachium rosenbergii were injected with L. garvieae (4 x 10(6) colony-forming units [cfu] prawn(-1)) grown in TSB or TSB containing copper sulfate at 0.1, 0.2, 0.3 or 0.4 mg l(-1). After 48 h, the cumulative mortality was significantly (p < 0.05) higher for prawns exposed to L. garvieae grown in 0.4 mg l(-1) copper sulfate than at the lower concentrations examined. In other experiments, prawns were injected with TSB-grown L. garvieae (4 x 10(6) and 2 x 10(5) cfu prawn(-1)), then held in water containing copper sulfate. After 8 h the mortality of L. garvieae-exposed prawns held in water containing 0.4 mg l(-1) copper sulfate was significantly higher than prawns held in water containing 0.2 and 0.3 mg l(-1) copper sulfate. At the lower L. garvieae density, cumulative mortality of prawns increased directly with ambient copper sulfate concentrations in the range of 0.2 to 0.4 mg l(-1). All prawns survived a 168 h exposure to 0.1 mg l(-1) copper sulfate. Prawns exposed to different concentrations of copper sulfate were examined for hemocyte density, phenoloxidase activity and respiratory burst. No significant differences in hemocyte density were observed among treatments. In prawns following a 48 h exposure to 0.1 mg l(-1) copper sulfate, phenoloxidase activity was decreased, but respiratory burst was increased. In conclusion, copper sulfate increased the virulence of L. garvieae to M. rosenbergii and modulated its immune system. Copper sulfate at 0.1 mg l(-1) decreased susceptibility of M. rosenbergii to L garvieae infection, whereas at 0.2 mg l(-1) the susceptibility was increased. The generation of superoxide anion by M. rosenbergii exposed to copper sulfate at a concentration higher than 0.2 mg l(-1) was considered to be cytoxic.     

Dopamine depresses the immune ability and increases susceptibility to Lactococcus garvieae in the freshwater giant prawn, Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (16.2 +/- 2.1 g) were individually injected with saline, or dopamine at 0.5, 5.0, or 50.0 pmol prawn(-1). The results show that a transient period of immunosuppression occurred between 2 and 8 h after injection of dopamine for all immune parameters except circulating haemocytes and all immune parameters returned to control values within 8-16 h after receiving dopamine. Injection of dopamine also significantly increased the mortality of M. rosenbergii challenged with the pathogen L. garvieae. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility to L. garvieae in M. rosenbergii.     

Effect of potassium permanganate stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii

This work is part of a continuing series of investigations on the effect of commonly used aquaculture chemicals on the immune resistance and susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae. The methodology has been described in earlier publications of the series. Potassium permanganate at 1.0 mg l(-1) in tryptic soy broth (TSB) had no effect on the growth rate of L. garvieae. The mortality of M. rosenbergii challenged with 4 x 10(6) colony-forming units (cfu) prawn(-1) of TSB-grown L. garvieae was significantly greater than that of challenged controls. Addition of potassium permanganate at 1.0 mg l(-1) in TSB significantly increased the virulence of L. garvieae to M. rosenbergii. Exposure of M. rosenbergii to potassium permanganate prior to challenge with TSB-grown L. garvieae at 4 x 10(6) and 3 x 10(6) cfu prawn(-1) revealed that 96 h mortality was significantly lower for prawns held in water containing 0.3 mg l(-1) of the chemical than for prawns in water containing 1.0 mg l(-1) or no chemical. Potassium permanganate caused no significant changes in total hemocyte counts and differential hemocyte counts, compared to the control treatments. However, a concentration of 1.0 mg l(-1) or more for 96 h resulted in decreased phenoloxidase activity, phagocytic activity and clearance efficiency. Respiratory burst increased with exposure to 0.3 mg l(-1). In conclusion, treatment with potassium permanganate at 0.3 mg 1(-1) was effective in reducing M. rosenbergii mortality from L. garvieae infection, but higher concentrations had a negative effect, probably due to reduced prawn defenses.     

Effects of dissolved oxygen on survival and immune responses of scallop (Chlamys farreri Jones et Preston)

This experiment investigated the effects of dissolved oxygen (DO) on the survival and immune responses of scallop Chlamys farreri. The scallops (initial mean dry weight of soft tissue 1.52+/-0.10 g) were cultivated in the seawater with different DO levels (8.5, 6.5, 4.5, and 2.5mg l(-1), respectively) for 21 d. Each treatment had triplicate groups of 35 animals. During the experimental period, the scallops were fed with Spirulina maxima, and water temperature ranged from 15.2 degrees C to 17.5 degrees C, salinity from 29.5 per thousand to 32.5 per thousand and pH from 7.5 to 8.2. Survival, specific growth rate (SGR) and total haemocyte count (THC) were examined at the end of the study, and superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (ALP), were examined at 12 h, 24 h, Day 7, Day 14 and Day 21 after being exposed to the graded DO levels. The lower DO levels (2.5 and 4.5mg l(-1))resulted in lower survivals of scallops, and the survival (81.7%) at 2.5mg 1(-1)DO was significantly lower than those (100.0%) at 8.5 and 6.5mg l(-1) DO. Similarly, the SGR and THC of scallop gradually reduced with decreasing DO levels, and reached significant levels at 2.5mg l(-1) DO (P<0.05). At higher DO levels (8.5 and 6.5mg l(-1)), the SOD activity maintained rather stable during the entire sampling period. At lower DO levels (4.5 and 2.5mg l(-1)), however, the SOD activity significantly increased at 12 h, and then significantly decreased to the levels below the normal. At the two lower DO levels, ACP activities had no significant changes before Day 7, and then declined to the levels that were significantly lower than the normal. Significantly higher ALP activity was only observed at 12 h in the treatment of 2.5mg l(-1) DO, but in all other treatments and sampling times it fluctuated in a narrow range. In conclusion, less than 4.5mg l(-1) DO reduced the survival and depressed the immune responses of C. farreri.     

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.     

Dietary administration of bovine lactoferrin influences the immune ability of the giant freshwater prawn Macrobrachium rosenbergii (de Man) and its resistance against Aeromonas hydrophila infection and nitrite stress

Haemocyte count, phenoloxidase activity, agglutinin levels, total protein content, bacterial clearance efficiency, resistance to the pathogen Aeromonas hydrophila and nitrite stress were measured in the giant freshwater inter-moult sub-adult prawn Macrobrachium rosenbergii (15-20 g) which had been fed diets containing bovine lactoferrin (Lf) at 50, 100, 200mg kg(-1) feed for 7 or 14 days. M. rosenbergii fed a diet containing 100mg Lf kg(-1) diet for 7 days showed significant (P<0.05) increase in total protein levels, agglutination titres against bacteria A. hydrophila and rabbit RBC, phenoloxidase activity, bacterial clearance (as observed through reduced number of circulating bacteria) as well as survival against A. hydrophila challenge. Increased bacterial clearance was also noticed in prawns fed Lf at 50 or 200mg kg(-1) for 14 days compared to control. Feeding of Lf at 50mg kg(-1) diet for 7 or 14 days was able to enhance only PO activity and reduce percent mortality against A. hydrophila challenge compared to its control. Total haemocyte count was higher in the lowest dose of Lf feeding, i.e. 50mg kg(-1) for 7 days. However, there was no significant alteration in the differential haemocyte population with respect to graded levels of Lf feeding for 7 or 14 days. A notable reduction in mortality percent after 120 h of nitrite stress was observed in prawn fed Lf at 100mg kg(-1) diet for 14 days. On the contrary, feeding of the highest dose of Lf, i.e. 200mg kg(-1) diet for 14 days failed to stimulate most of the innate immune parameters or reduce the percent mortality against A. hydrophila challenge or nitrite stress. It is therefore concluded that administration of Lf in the diet at 100mg kg(-1) for 7 days could enhance the immune ability of M. rosenbergii and increase its resistance to A. hydrophila infection or nitrite stress.     

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.     

Effect of hypoxic stress on the immune response and the resistance to vibriosis of the shrimpPenaeus stylirostris

The immune response of shrimp exposed to severe hypoxia, 1 mg O₂ml⁻¹for 24 h, was measured in terms total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity (PO) and respiratory burst by NBT reduction. Results showed that hypoxia induced a significant decrease (P<5%) of the THC due to a significant decrease of semi-granular cells (SGC) (P<5%) and hyaline cells (HC) (P<5%). There was also a decrease in the NBT reduction. On the other hand, PO activity increased significantly (P<1%) and seemed to be related to a lower amount of plasma inhibitors regulating the prophenoloxidase system. Experimental infection with a virulentVibrio alginolyticuswas carried out by intramuscular injection of 2×10⁴colony forming units (cfu) per shrimp. The mortality of stressed shrimp was 48% while the mortality of control shrimp was 32%. This difference was significant (P<1%). The variations of immunological parameters, except for the PO activity, seem to be consistent with thein vivoresponse against the pathogenicVibrio. Coyright 1998 Academic Press     

Effect of ammonia on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Taiwan abalone Haliotis diversicolor supertexta held in 30 parts/per thousand seawater and 26 degrees C were injected with TSB-grown Vibrio parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then placed in water containing different concentrations of ammonia-N (un-ionized plus ionized ammonia) at 0.01 mg l(-1) (control), 1.12, 3.22, 5.24 and 10.18 mg l(-1). Mortality of abalone increased directly with ambient ammonia-N concentration. After 12 h, the mortality of V. parahaemolyticus-injected abalone held in 3.22 mg l(-1) ammonia-N was significantly higher than those placed in 1.12 mg l(-1) ammonia-N and the control solution. In another experiment, the abalone which had been exposed to control, 1.08, 3.16, 5.37 and 10.34 mg l(-1) ammonia-N for 24, 72 and 120 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), and phagocytic activity and clearance efficiency to V. parahaemolyticus. The abalone when exposed to 3.16 mg l(-1) ammonia-N had decreased THC after 72 h, and decreased phenoloxidase activity, phagocytic activity and clearance efficiency after 24 h. However, the abalone when exposed to 3.16 mg l(-1) ammonia-N had increased respiratory burst after 24 h. The immune parameters except superoxide anion seemed to be suppressed in a dose-dependent fashion after 24 h. It is concluded that ammonia caused a depression in immune parameters and an increase in mortality of H. diversicolor supertexta from V. parahaemolyticus infection.     

Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus

Growth of Vibrio alginolyticus was not affected by TSB medium containing ammonia-N concentration in the range of 0-20 mg l(-1). White shrimp Litopenaeus vannamei (7-12 g in the intermolt stage) were challenged with V. alginolyticus, which had been incubated for 24 h in the TSB medium containing different concentrations of ammonia-N (0, 1, 5. 10 and 20 mg l(-1)). There was no significant difference in cumulative mortality for shrimp incubated in the TSB medium containing 0, 1, 5, 10 and 20 mg l(-1)ammonia-N after 120 h of challenge. The shrimps were challenged with V. alginolyticus previously incubated in the TSB medium for 24 h, then placed in water containing concentrations of ammonia-N at 0.01 mg l(-1)(control), 1.10, 5.24, 11.10 and 21.60 mg l(-1). Mortality of shrimp in 5.24, 11.10 and 21.60 mg l(-1)was significantly higher than those in the control solution (0.01 mg l(-1)) after 48-168 h. Shrimps which had been exposed to control, 1.10, 5.24, 11.10 and 21.60 mg l(-1)ammonia-N for 7 days were examined for THC (total haemocyte count), granular cells, hyaline cells, phenoloxidase activity, release of superoxide anion, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V. alginolyticus. No significant difference in THC, hyaline cells and granular cells were observed among shrimps at different ammonia-N concentrations. Phenoloxidase activity however, decreased when the shrimps were exposed to 5.24 mg l(-1)ammonia-N and greater after 7 days. The release of superoxide anion increased significantly, whereas SOD activity decreased significantly at 21.60 mg l(-1)ammonia-N. With shrimps exposed to 11.21 and 21.22 mg l(-1)ammonia-N for 7 days, phagocytic activity and clearance efficiency to V. alginolyticus significantly decreased. It is therefore suggested that ammonia in water caused a depression in the immune response and an increase in mortality of L. vannamei from the V. alginolyticus infection.     

Effect of nitrite on immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Taiwan abalones Haliotis diversicolor supertexta held in 30% per thousand seawater and 26 degrees C were injected with tryptic soy broth (TSB)-grown Vibrioparahaemolyticus (1.6 x 10(5) CFU [colony-forming units] abalone(-1)), and then placed in water containing different concentrations of nitrite-N (nitrite as nitrogen): 0.01 mg l(-1) (control), 1.05, 3.04, 5.10 and 10.06 mg l(-1). Mortality of the abalones increased in direct parallel to ambient nitrite-N concentration. Over 12 to 48 h, the mortality of V. parahaemolyticus-injected abalones held in 3.04 mg l(-1) nitrite-N was significantly higher than that of abalones in the control solution. Abalones that had been exposed to control, 0.96, 2.95, 5.03 and 10.16 mg l(-1) nitrite-N for 24, 72 and 120 h were examined for THC (total hemocyte count), phenoloxidase activity, respiratory bursts (release of superoxide anion), phagocytic activity, and clearance efficiency of V. parahaemolyticus. The THC increased in abalone after 72 h exposure to 0.96 and 2.95 mg l(-1) nitrite-N, but decreased in abalones after 24 h exposure to 5.03 and 10.16 mg l(-1) nitrite-N. Phenoloxidase activity and respiratory bursts increased, while phagocytic activity and clearance efficiency decreased in abalones exposed to > or = 0.96 mg l(-1) nitrite-N for 24 h. It is concluded that nitrite-N in water at concentrations as low as 0.96 mg l(-1) weakens the immune response and increases mortality of H. diversicolor supertexta infected with V. parahaemolyticus.     

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.     

Clearance of white spot syndrome virus (WSSV) and immunological changes in experimentally WSSV-injected Macrobrachium rosenbergii

A time course experimental challenge of WSSV was carried out to examine the clearance of WSSV in Macrobrachium rosenbergii and the consequent immunological changes. The experimental animals were injected with WSSV and the samples of gills, pleopods, head soft tissue and hemolymph were collected at different intervals of 1, 3, 5, 10, 25, 50, 75 and 100days post infection (p.i.). WSSV infection and clearing were confirmed by single step PCR, nested PCR and bioassay. At 3days p.i., M. rosenbergii became lethargic and stopped feeding in contrast to the control prawns that behaved and fed normally. However, the WSSV-injected prawns suffered no mortality during the experimental period and recovered without any further gross signs of disease or any mortality over a period of 100days p.i. The single step PCR analysis showed positive at 1, 3 and 5days p.i. in gills, head soft tissue, pleopods and hemolymph, and all the organs showed negative at 10days p.i. onwards. The nested PCR results showed that all organs were positive for WSSV from 3days p.i. and extended up to 25days p.i. At 50days p.i, head soft tissue sample alone showed WSSV-positive while all other organs were negative by nested PCR. All the organs at 75 and 100days p.i. showed nested PCR negative for WSSV as observed in the control prawn. The hemolymph collected from experimentally infected M. rosenbergii at 1, 3 and 5days p.i. caused 100% mortality at 40h p.i., 55h p.i. and 72h p.i, respectively in Penaeus monodon whereas hemolymph collected at 10, 25, 50, 75 and 100days p.i. failed to cause mortality in shrimp. The moribund shrimp showed WSSV-positive and surviving shrimp showed negative by PCR. Immunological parameters such as proPO, O(2)(-) and clotting time in WSSV-injected M. rosenbergii were found to be significantly higher than those of the control groups, whereas THC and superoxide dismutase were significantly lower when compared to control groups.     

亚硝酸盐胁迫对罗氏沼虾血细胞及其抗氧化酶活力的影响

【背景】亚硝酸盐是虾类集约化养殖过程中最常见的毒性污染物之一,研究亚硝酸盐胁迫对罗氏沼虾血细胞的毒性以及抗氧化酶在抗胁迫防御中的作用,能够为罗氏沼虾养殖过程中的亚硝酸盐中毒防治提供理论参考。【方法】以不同浓度(0、1、5和10 mg·L~(-1))的亚硝态氮(NO_2~--N)对罗氏沼虾进行胁迫,于胁迫后的0、6、12、24和48 h取样,应用流式细胞术检测血细胞活性氧(ROS)含量和细胞凋亡率,同时测定血细胞总数(THC)和胞内抗氧化酶活力。【结果】1 mg·L~(-1)NO_2~--N在48 h内对血细胞ROS含量、凋亡率和THC均无显著影响。5 mg·L~(-1)NO_2~--N胁迫24 h,血细胞ROS含量显著上升,THC显著下降,胁迫48 h凋亡率显著提高。10 mg·L~(-1)NO_2~--N胁迫6 h,血细胞ROS含量和凋亡率均显著上升,胁迫12 h THC显著下降。血细胞的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPx)的活力均不同程度地被NO_2~--N胁迫所诱导,CAT活力主要在胁迫前期提高,而GPx活力在胁迫后期提高。【结论与意义】亚硝酸盐存在浓度和时间毒性效应,一定浓度的亚硝酸盐会诱导虾血细胞产生ROS,这些ROS的过量产生诱导了血细胞发生凋亡,继而导致THC下降,这一氧化胁迫过程可能是亚硝酸盐对罗氏沼虾产生细胞毒性的重要机制之一。抗氧化酶活力的诱导表明抗氧化酶在亚硝酸盐胁迫过程中发挥防御作用。     

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 °C, 17 °C and 25 °C respectively. Thereafter, a recovery period of 24 h at 17 °C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immunomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 °C and 24 h of air exposure at 17 °C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 °C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 °C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 °C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops.