White spot syndrome virus inactivation study by using gamma irradiation

The present study was conducted to investigate the effect of gamma irradiation on white spot syndrome virus(WSSV). White spot syndrome virus is a pathogen of major economic importance in cultured penaeid shrimp industries. White spot disease can cause mortalities reaching 100% within 3–10 days of gross signs appearing. During the period of culture, immunostimulant agents and vaccines may provide potential methods to protect shrimps from opportunistic and pathogenic microrganisms. In this study, firstly, WSSV was isolated from infected shrimp and then multiplied in crayfish. WSSV was purified from the infected crayfish haemolymph by sucrose gradient and confirmed by transmission electron microscopy. In vivo virus titration was performed in shrimp, Penaeus semisulcatus. The LD50 of live virus stock was calculated 10 5.4 /mL. Shrimp post-larvae(1–2 g) were treated with gamma-irradiated(different doses) WSSV(10 0 to 10-4 dilutions) for a period of 10 days. The dose/survival curve for irradiated and un-irradiated WSSV was drawn; the optimum dose range for inactivation of WSSV and unaltered antigenicity was obtained 14–15 kGy. This preliminary information suggests that shrimp appear to benefit from treatment with gammairradiated WSSV especially at 14–15 KGy.     

Selection for growth performance of tank-reared Pacific white shrimp, Litopenaeus vannamei

Seven growth-related traits were measured to assess the selection response and genetic parameters of the growth of Pacific white shrimp, Litopenaeus vannamei, which had been domesticated in tanks for more than four generations. Phenotypic and genetic parameters were evaluated and fitted to an animal model. Realized response was measured from the difference between the mean growth rates of selected and control families. Realized heritability was determined from the ratio of the selection responses and selection differentials. The animal model heritability estimate over generations was 0.44±0.09 for body weight (BW), and ranged from 0.21±0.08 to 0.37±0.06 for size traits. Genetic correlations of phenotypic traits were more variable (0.51–0.97), although correlations among various traits were high (>0.83). Across generations, BW and size traits increased, while selection response and heritability gradually decreased. Selection responses were 12.28%-23.35% for harvest weight and 3.58%–13.53% for size traits. Heritability estimates ranged from 0.34±0.09 to 0.48±0.15 for harvest weight and 0.17±0.01–0.38±0.11 for size traits. All phenotypic and genetic parameters differed between various treatments. To conclude, the results demonstrated a potential for mass selection of growth traits in L. vannamei. A breeding scheme could use this information to integrate the effectiveness constituent traits into an index to achieve genetic progress.     

Optimal content and ratio of lysine to arginine in the diet of Pacific white shrimp, Litopenaeus vannamei

The optimal quantity of dietary lysine (Lys) and arginine (Arg), and the optimal ratio of dietary Lys to Arg for Pacific white shrimp Litopenaeus vannamei were investigated. Coated Lys and Arg were added to a basal diet (37.99% crude protein and 7.28% crude lipid) to provide graded levels of Lys and Arg. The experimental diets contained three Lys levels (2.51%, 2.11%, and 1.70% of total diet), and three Arg levels (1.41%, 1.80%, and 2.21% of total diet) and all combinations of these levels were tested. Pacific white shrimp, with a mean weight of 3.62±0.1 g, were randomly distributed in 36 fiberglass tanks with 30 shrimp per tank and reared on the experimental diets for 50 days. After the feeding trial, the growth performance, survival, feed conversion rate (FCR), body composition and protease and lipase activities in the hepatopancreases of the experimental shrimps were determined. The results show that weight gain (WG), specific growth rate (SGR), FCR, body protein, body Lys and Arg content were significantly affected by dietary Lys and Arg (P <0.05) and improved when dietary Lys and Arg levels were 2.11% ～ 2.51% and 1.80%～2.21%, respectively. Protease and lipase activities in the hepatopancreases of the shrimps appeared higher when dietary Lys and Arg quantities were 2.11% ～2.51% and 1.80%～2.21%, although the difference was not statistically significant (P >0.05). Therefore, according to our results, the optimal Lys and Arg quantities in the diet of Pacific white shrimp, L. vannamei, were considered to be 2.11%–2.51% and 1.80%–2.21%, respectively, and the optimal ratio to be 1:0.88–1:1.05.     

Impact of Vibrio parahaemolyticus and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp Litopenaeus vannamei

White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect L itopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus(WSSV+Vp), or we fi rst infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection(Vp+WSSV). The effect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of V ibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction(PCR) method. L v My D88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the different mortality rates. Our results show that(1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection;(2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed signifi cant increases in the numbers of Vibrio( P 0.05);(3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 5 to 10 7 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.     

凡纳滨对虾和斑节对虾对WSSV敏感性的比较

用6.0×104拷贝、1.2×104拷贝和6.0×103拷贝3种剂量白斑综合症病毒(WSSV)对凡纳滨对虾和斑节对虾进行人工注射感染,比较了两种对虾对WSSV敏感性的差异。结果表明,凡纳滨对虾死亡时间随病毒剂量降低而延长,斑节对虾死亡时间没有明显差异;随病毒剂量的降低,凡纳滨对虾人工注射感染后病毒复制高峰时间显著延长,斑节对虾感染后病毒复制高峰时间相同,WSSV在凡纳滨对虾体内比在斑节对虾体内复制慢。对虾携带WSSV数量最低为3.3×107拷贝.g-1,最高为4.3×108拷贝.g-1。凡纳滨对虾比斑节对虾对WSSV的抵抗性更强。     

Detection of white spot syndrome virus (WSSV) ofPenaeus chinensis by in situ hybridization

White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I-digested fragments of the WSSV genome were cloned; three of these fragments were used as non-radioactive probes labeled with DIG-11-dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV-infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp. This work was supported by SRF for ROCS, SEM and the National 863 Project (Grant 819-Q-08) and Project under major State Basic Research Development Program (Grant G1999012002).     

Impact of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect Litopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus (WSSV+Vp), or we first infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection (Vp+WSSV). The eff ect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of Vibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction (PCR) method. LvMyD88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the diff erent mortality rates. Our results show that (1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection; (2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed significant increases in the numbers of Vibrio (P<0.05); (3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10⁵ to 10⁷ /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.     

Development and evaluation of a DNA microarray assay for the simultaneous detection of nine harmful algal species in ship ballast and seaport waters

Rapid, high-throughput and reliable methods are urgently required to accurately detect and monitor harmful algae, which are responsible for algal blooms, such as red and green tides. In this study, we successfully developed a multiplex PCR-based DNA microarray method capable of detecting nine harmful algal species simultaneously, namely Alexandrium tamarense, Gyrodinium instriatum, Heterosigma akashiwo, Karenia mikimotoi, Prorocentrum donghaiense, Prorocentrum minimum, Ulva compressa, Ulva ohnoi and Ulva prolifera. This method achieved a limit of detection (LOD) of 0.5 ng of genomic DNA (orders of magnitude of the deci-nanogram range) in the tested algae cultures. Altogether, 230 field samples from ship ballast waters and seaport waters were used to evaluate the DNA microarray. The clinical sensitivity and specificity of the DNA microarray assay in detecting field samples were 96.4% and 90.9%, respectively, relative to conventional morphological methods. This indicated that this high-throughput, automatic, and specific method is well suited for the detection of algae in water samples.