Winton Cheng

Dietary administration of the probiotic, Lactobacillus plantarum, enhanced the growth, innate immune responses, and disease resistance of the grouper Epinephelus coioides.

The percent weight gain (PWG) and feed efficiency (FE) of Epinephelus coioides were calculated, and the lactobacilli and total microbiota in the posterior intestines, and non-specific immune parameters of grouper, and its susceptibility to Streptococcus sp. and an iridovirus were determined when the fish were fed diets containing Lactobacillus plantarum at 0 (control), 10(6), 10(8), or 10(10) colony-forming units (cfu) kg(-1) for 4 weeks. Results showed that grouper fed a diet containing L. plantarum at the levels of 10(6), 10(8), and 10(10) cfu kg(-1) had significantly increased PGW and FE especially at 10(8) cfu kg(-1) group which were 404.6% and 1.26, respectively. L. plantarum significantly increased in the fish posterior intestines during the L. plantarum feeding period, but decreased rapidly from the intestine within 1 week after changing to the control diet (without L. plantarum). Fish fed a diet containing L. plantarum at 10(6) and 10(8) cfu kg(-1) had significantly higher survival rates than those fed the control diet after challenge with Streptococcus sp., as well as those fed 10(8) cfu kg(-1) after challenge with an iridovirus, causing increases in the survival rates of 23.3%, 20.0%, and 36.7%, respectively, compared to the control group. The alternative complement activity (ACH(50)) level of fish fed diets containing L. plantarum after 4 weeks was significantly higher than that of fish fed the control diet, and that of the 10(8) cfu kg(-1) group was significantly higher than those of the 10(6) and 10(10) cfu kg(-1) groups, which increased by 83.4% compared to the control group. The lysozyme activity and glutathione peroxidase (GPx) activity of fish fed the L. plantarum-containing diets at 10(8) and 10(10) cfu kg(-1) significantly increased compared to those fed the 10(6) cfu kg(-1)L. plantarum diet and control diet, and had increased by 76.3% and 136.6%, and 57.1% and 113.3%, respectively, compared to those fed the control diet. The phagocytic activity (PA), phagocytic index (PI), and respiratory bursts of head kidney leucocytes of fish fed 10(6), 10(8), and 10(10) cfu kg(-1)L. plantarum diets were significantly higher than those of fish fed the control diet after 4 weeks of feeding, and increased 2.2-, 2.2-, and 2.3-fold; 1.8-, 1.8-, and 2.0-fold; and 1.4-, 1.4-, and 1.4-fold, respectively, compared to the control group. We therefore recommend dietary L. plantarum administration at 10(8) cfu kg(-1) to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus of E. coioides.

Dietary sodium alginate administration affects fingerling growth and resistance to Streptococcus sp. and iridovirus, and juvenile non-specific immune responses of the orange-spotted grouper, Epinephelus coioides

The percent weight gain (PWG) and feeding efficiency (FE) of fingerling orange-spotted grouper, Epinephelus coioides, fed diets containing sodium alginate at 1.0 and 2.0 g kg(-1) were calculated on the 2nd, 4th, 6th, and 8th weeks after feeding. Survival rates of the fingerling grouper against Streptococcus sp. and an iridovirus, and non-specific immune parameters such as alternative complement activity (ACH50), lysozyme activity, natural haemagglutination activity, respiratory bursts, superoxide dismutase (SOD) activity, and phagocytic activity of juvenile grouper were also determined when the fish were fed diets containing sodium alginate at 0.5, 1.0, or 2.0 g kg(-1). The PWG and FE of fish were better when the fish were fed diets containing sodium alginate at 1.0, and 1.0 and 2.0 g kg(-1), respectively. The PWG and FE of fish fed the 0, 1.0 and 2.0 g kg(-1) sodium alginate-containing diets after 8 weeks were 271.0%, 454.4% and 327.8%, and 0.61, 0.72 and 0.68, respectively. Fish fed a diet containing sodium alginate at the level of 2.0 g kg(-1) had a significantly higher survival rate than those fed the control diet after challenge with Streptococcus sp. and an iridovirus causing an increase of survival rate by 25.0% and 16.7%, respectively, compared to the control group. The ACH(50) level of fish fed the sodium alginate-containing diets at 2.0 g kg(-1) was significantly higher than those fed the 1.0 g kg(-1) sodium alginate diet and control diet after 12 days, and had increased to 1.9-fold, compared to those fed the control diet. The lysozyme activity, phagocytic activity, respiratory bursts, and SOD level of fish fed the sodium alginate-containing diets at 1.0 and 2.0 g kg(-1) were significantly higher than those fed the control diet after 12 days, and had increased to 1.97- and 1.68-fold, 1.35- and 1.50-fold, 1.63- and 1.81-fold, and 1.23- and 1.31-fold, respectively, compared to those fed the control diet. We therefore recommend dietary sodium alginate administration at 1.0 and 2.0 g kg(-1), respectively, to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus.

Effects of the probiotic, Bacillus subtilis E20, on the survival, development, stress tolerance, and immune status of white shrimp, Litopenaeus vannamei larvae.

In this study, the probiotic, Bacillus subtilis E20, isolated from the human health food, natto, was used for white shrimp, Litopenaeus vannamei, larvae breeding to improve the larval survival rate and development by adding probiotic to the rearing water at (control), 10(8), and 10(9) cfu L(-1) salt water once every 3 days during the 14 days of breeding experiment. Thereafter, stress tolerance and immune status of postlarvae were evaluated. Shrimp larval development was significantly accelerated after adding the probiotic to the larval rearing water at a level of 10(9) cfu L(-1). The survival rate of larvae was significantly higher in the treatment with 10(9) cfu L(-1) compared to the control and the treatment with 10(8) cfu L(-1) after all larvae had metamorphosed to postlarvae. Adding the probiotic to the shrimp larvae rearing water produced a weak inhibition of bacterial growth by an analysis of the total bacterial count and presumptive Vibrio count. For stress tests, no postlarvae died when they were reared in water in which the temperature was decreased from 30 to 2 degrees C at a rate of 0.1 degrees C min(-1). Postlarvae had significantly lower cumulate mortality in the treatments with 10(8) and 10(9) cfu L(-1) compared to the control when they were suddenly exposed to fresh water and 60 per thousand salt water. A significant decrease in the cumulative mortality of postlarvae treated with the probiotic at a level of 10(9) cfu L(-1) was recorded after the sudden transfer to 300 mg L(-1) nitrite-N compared to the control and treatment with 10(8) cfu L(-1). The analysis of immune-related gene expressions showed that the gene expression of prophenoloxidase I, prophenoloxidase II, and lysozyme of larvae were significantly increased after being reared in probiotic-containing water at the levels of 10(8) and 10(9) cfu L(-1). However, no significant difference in serine proteinase or glutathione peroxidase gene expressions was recorded in this study. It is therefore suggested that 10(9) cfu L(-1) of probiotic, B. subtilis E20 adding to rearing water for shrimp larva breeding.

The immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus at different salinity levels.

Addition of NaCl at 2.5% to 3.5% to tryptic soy broth (TSB) significantly increased the growth of Vibrio parahaemolyticus. Taiwan abalone Haliotis diversicolor supertexta held in 30 per thousand seawater were injected with V. parahaemolyticus grown in TSB containing NaCl at 0.5, 1.5, 2.5, 3.5 and 4.5% at a dose of 1.6 x 10(5)colony-forming units (cfu) abalone(-1). After 48 h, the cumulative mortality was significantly higher for the abalone challenged with V. parahaemolyticus grown in 2.5% than those grown in 0.5 and 1.5% NaCl. In other experiments, abalones held in 30 per thousand seawater were injected with TSB-grown V. parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then transferred to 20, 25, 30 and 35 per thousand seawater. All abalones held in 20 per thousand were killed in 48 h. The mortality of V. parahaemolyticus-injected abalone held in 30 per thousand was significantly lower over 24-120 h. Abalone held in 30 per thousand seawater and then transferred to 20, 25, 30 and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahemolyticus after 24 and 72 h. The THC increased directly related with salinity levels. Phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahaemolyticus decreased significantly for the abalone in 20, 25 and 35 per thousand. It is concluded that the abalone transferred from 30 per thousand to 20, 25 and 35 per thousand had reduced immune ability and decreased resistance against V. parahaemolyticus infection.

Effect of dissolved oxygen on the immune response of Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Abstract Taiwan abalone Haliotis diversicolor supertexta (13–16 g) were challenged with Vibrio parahaemolyticus at a dose of 2×10 4 colony-forming units (cfu) abalone −1 previously incubated in TSB medium for 24 h, then placed in water having concentrations of dissolved oxygen (DO) at 7.70, 5.61, 3.57 and 2.05 mg l −1 . Onset of mortality occurred after 6-h exposure to 2.05 mg l −1 DO, and after 12-h exposure to 3.57 and 5.61 mg l −1 DO. Cumulative mortality of abalone at 2.05 mg l −1 DO was significantly higher than that at 3.57 mg l −1 DO, and cumulative mortality of abalone at 3.57 and 5.61 mg l −1 DO was significantly higher than that at 7.70 mg l −1 DO after 24 h. Abalones which had been placed in water for 0 to 96 h at 7.70, 5.61, 3.57 and 2.05 mg l −1 DO were examined for the total haemocyte counts (THC), phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency. No significant differences in THC and respiratory burst of abalone were observed among four treatments after 6 h. The abalone following 24-h exposure to 2.05 mg l −1 DO decreased significantly its THC and respiratory burst by 27% and 24%, respectively. Phenoloxidase activity increased significantly by 38% and 69% when abalones were exposed to 3.57 and 2.05 mg l −1 DO after 24 h, respectively. Phagocytic activity and clearance efficiency to V. parahaemolyticus decreased significantly for the abalone following 12-h exposure to 3.57 mg l −1 DO, respectively. It is concluded that DO as low as 3.57 and 2.05 mg l −1 causes depression in immune system of H. diversicolor supertexta , and increases its susceptibility to V. parahaemolyticus infection.

Dietary administration of the probiotic, Saccharomyces cerevisiae P13, enhanced the growth, innate immune responses, and disease resistance of the grouper, Epinephelus coioides.

The percent weight gain (PWG) and feeding efficiency (FE) of Epinephelus coioides were calculated. The survival of Saccharomyces cerevisiae P13 in the posterior intestines using a specific primer pair of YMR245w-F/YMR245w-R, non-specific immune parameters of grouper, and its susceptibility to Streptococcus sp. and an iridovirus were determined when the fish were fed diets containing S. cerevisiae at 0 (control), 10(3), 10(5), or 10(7) colony-forming units (cfu) kg(-1) for 4 weeks. Results showed that grouper fed a diet containing S. cerevisiae at the levels of 10(3), 10(5), and 10(7) cfu kg(-1) had significantly increased PGW and FE especially in the 10(7) cfu kg(-1) group which were 211.6% and 1.2, respectively. S. cerevisiae was able to survive in the fish posterior intestines during the S. cerevisiae feeding period. Fish fed a diet containing S. cerevisiae at 10(7) cfu kg(-1) had significantly higher survival rates than those fed the 10(3) cfu kg(-1)S. cerevisiae diet and the control diet after challenge with Streptococcus sp. and an iridovirus, with increased survival rates of 26.6% and 36.6%, respectively, compared to the challenge control group. The phagocytic activity, respiratory burst and superoxide dismutase (SOD) level of head kidney leucocytes as well as serum lysozyme activity and serum alternative complement activity (ACH(50)) of fish fed diets containing S. cerevisiae at 10(5) and 10(7) cfu kg(-1) were significantly higher than those of fish fed the 10(3) cfu kg(-1)S. cerevisiae-contained diet and the control diets after 4 weeks of feeding, and had increased by 20% and 20%, 27.6% and 19.7%, 30.5% and 36.2%, 205.8% and 169.6%, and 90.8% and 80.3%, respectively, compared to the control group. We therefore recommend dietary S. cerevisiae administration of 10(5) and 10(7) cfu kg(-1) to E. coioides to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus especially in the 10(7) cfu kg(-1) group.

Effects of temperature, pH, salinity and ammonia on the phagocytic activity and clearance efficiency of giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae

Abstract The giant freshwater prawn Macrobrachium rosenbergii (14–17 g) was exposed to different temperatures (20, 25, 30 and 35 °C), pH values (4.22, 7.27 and 9.31), salinity levels (0‰, 5‰, 10‰ and 15‰) and different concentrations (0.02 (control), 0.55, 1.20 and 3.07 mg l −1 ) of ammonia-N (ammonia as nitrogen) for 7 days. They were then examined for phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae . M. rosenbergii at temperatures of 25 and 30 °C, pH 7.27 or salinity 5‰ and 10‰ exhibited increased phagocytic activity and clearance efficiency, whereas M. rosenbergii exposed to ammonia-N at 0.55 mg l −1 or greater exhibited decreased phagocytic activity and clearance efficiency to L. garvieae . It was therefore concluded that at a temperature of 35 °C, salinity 0‰ and 15‰, pH 9.31 or exposure of ammonia-N at 0.55 mg l −1 or greater, the immune system of M. rosenbergii depressed, which in turn resulted in increased mortality from the L. garvieae infection. Prawns at temperatures of 25 and 30 °C, pH 7.27 or salinity at 5‰ however, exhibited the greatest increased resistance to the L. garvieae infection.

Immune response of white shrimp, Litopenaeus vannamei, after a concurrent infection with white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus

In the present study, we investigated immunological changes in viral-infected white shrimp, Litopenaeus vannamei. White shrimp were infected with white spot syndrome virus (WSSV) or co-infected with WSSV and infectious hypodermal and hematopoietic necrosis virus (IHHNV) as detected by polymerase chain reaction (PCR). The complete (100%) mortality rate of shrimp was caused by viral infection due to immune parameters being suppressed including decreases in phenoloxidase activity, total hemocyte counts, differential hemocyte counts, and the gene expressions of prophenoloxidase and peroxinectin. In addition, increases in lipopolysaccharide and beta-1,3-glucan-binding protein of hemocytes and the hepatopancreas, and respiratory bursts per cell, and a decrease in superoxide dismutase were found in viral-infected shrimp, which may have been related to the defense against viral infection.

The immune response of the giant freshwater prawn Macrobrachium rosenbergii and its susceptibility to Lactococcus garvieae in relation to the moult stage

Abstract The giant freshwater prawn Macrobrachium rosenbergii (12–18 g) was examined for phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity and clearance efficiency for the pathogen Lactococcus garvieae at 28 °C. Phenoloxidase activity was higher at intermoult (C stage), whereas respiratory burst was the lowest at C stage among the moult stages of A, B, C, D 1 /D 2 and D 3 . Phagocytic activity and clearance efficiency of prawns to L. garvieae decreased significantly at A, D 1 /D 2 and D 3 stages as compared with those at C stage. In another experiment, M. rosenbergii at different moult stages were injected with tryptic soy broth (TSB)-grown L. garvieae (3×10 5 cfu prawn −1 ) and then held in water. After 6 h, the mortality of L. garvieae -injected prawns at D 3 was significantly higher than that at C and A stages. After 72 h, the mortality of L. garvieae -injected prawns was 100%, 40% and 60% at D 3 , C and A stages, respectively. It was concluded that M. rosenbergii had decreased resistance at A, D 1 /D 2 and D 3 through a reduction of its phenoloxidase activity, phagocytic activity and clearance efficiency against L. garvieae . The fact that the prawns displayed higher respiratory burst, but lower phenoloxidase activity, and the fact that the phagocytic activity and clearance efficiency of prawns to L. garvieae decreased at premoult and postmoult stages are considered to be modulation of resistance factors.

Cold shock-induced norepinephrine triggers apoptosis of haemocytes via caspase-3 in the white shrimp, Litopenaeus vannamei.

The total haemocyte count (THC), haemolymph norepinephrine (NE) level, caspase-3 mRNA expression and activity levels, and apoptotic haemocyte rate were measured when shrimp Litopenaeus vannamei (20-25 g) were transferred from 28 to 22 degrees C after 0, 2, and 7 days, and the caspase-3 mRNA expression and activity levels and the apoptotic cell rate of haemocytes, in vitro, were determined after incubation with 2 x 10(-8) M NE for 0, 30, 60, and 120 min at 27 +/- 1.0 degrees C. For shrimp transferred from 28 +/- 1.0 to 22 +/- 0.5 degrees C after 2 and 7 days, the THC decreased by 17.9% and 18.0%, but the NE concentration, caspase-3 transcription and activity levels, and apoptotic cell rate increased by 62.5% and 37.3%, 5100.0% and 446.6%, 148.6% and 152.0%, and 88.7% and 200.1%, respectively, compared to those of shrimp held at 28 +/- 0.5 degrees C which served as the control. Similar tendencies were observed for the apoptotic cell rate, and caspase-3 transcription and activity levels of haemocytes exposed to 2 x 10(-8) M NE in vitro. These results suggest that NE plays an important role in the apoptosis of haemocytes in L. vannamei under hypothermal stress, which causes depressive effects on immunological responses.