White spot syndrome virus epizootic in cultured Pacific white shrimp Litopenaeus vannamei (Boone) in Taiwan

White spot syndrome virus (WSSV) has caused significant losses in shrimp farms worldwide. Between 2004 and 2006, Pacific white shrimp Litopenaeus vannamei (Boone) were collected from 220 farms in Taiwan to determine the prevalence and impact of WSSV infection on the shrimp farm industry. Polymerase chain reaction (PCR) analysis detected WSSV in shrimp from 26% of farms. Juvenile shrimp farms had the highest infection levels (38%; 19/50 farms) and brooder shrimp farms had the lowest (5%; one of 20 farms). The average extent of infection at each farm was as follows for WSSV‐positive farms: post‐larvae farms, 71%; juvenile farms, 61%; subadult farms, 62%; adult farms, 49%; and brooder farms, 40%. Characteristic white spots, hypertrophied nuclei and basophilic viral inclusion bodies were found in the epithelia of gills and tail fans, appendages, cephalothorax and hepatopancreas, and virions of WSSV were observed. Of shrimp that had WSSV lesions, 100% had lesions on the cephalothorax, 96% in gills and tail fans, 91% on appendages and 17% in the hepatopancreas. WSSV was also detected in copepoda and crustaceans from the shrimp farms. Sequence comparison using the pms146 gene fragment of WSSV showed that isolates from the farms had 99.7–100% nucleotide sequence identity with four strains in the GenBank database – China ( AF332093 ), Taiwan ( AF440570 and U50923 ) and Thailand ( AF369029 ). This is the first broad study of WSSV infection in L. vannamei in Taiwan.     

Genetic variation of white spot syndrome virus (WSSV) in Pacific white shrimp Litopenaeus vannamei (Boone 1931) culture of Thailand

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X_14/15, X_23/24) (N₇₅‐N₁₂₅‐N₉₄) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (5950_14/15, 10971_23/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (5950_14/15, 10971_23/24) (3₇₅‐6₁₂₅‐7₉₄). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.     

Development of a monoclonal antibody‐based flow‐through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon

A flow‐through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL^?1 crude WSSV protein, respectively. The FTA could be completed in 8–10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1‐step polymerase chain reaction (PCR) and in between that of the 1‐ and 2‐step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post‐larvae, WSSV was first detected 14, 16 and 18 h post‐infection (hpi) by FTA, immunodot and one‐step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one‐step PCR, respectively. The FTA was more sensitive (25/27) than one‐step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4–8 °C. The FTA is available as a rapid test kit called ‘RapiDot’ for the early detection of WSSV under field conditions.     

Purification of white spot syndrome virus by iodixanol density gradient centrifugation

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60 000  g ) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80 000  g ) through a discontinuous iodixanol gradient (phosphate‐buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1 000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures.     

Evidence for the presence of white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild Penaeus monodon (Fabricius) broodstock,in the southeast coast of India

A survey on the presence of the viruses of two economically significant diseases, white spot syndrome virus (WSSV) and monodon baculovirus (MBV) in wild‐collected Penaeus monodon broodstock, was conducted during different seasons of the year in two major coastal areas of southeast India. The broodstock were collected along the coast of Tamil Nadu and Andhra Pradesh during summer, premonsoon, monsoon and post‐monsoon seasons for three consecutive years. A total of 7905 samples were collected and subjected to MBV screening, and 6709 samples that were screened as MBV negative were diagnosed for WSSV. MBV was detected using rapid malachite green staining and WSSV by nested polymerase chain reaction. Prevalence data of the viruses were analysed using the EpiCalc 2000 program at 95% confidence interval. Samples collected from the Andhra Pradesh coast displayed a slightly higher prevalence of WSSV and MBV infection than those collected from Tamil Nadu, although this difference was not statistically significant (P > 005). In addition, it was found that the prevalence of both WSSV and MBV infections fluctuated according to season. Data on prevalence of these viruses in broodstock would be useful to develop strategies for shrimp health management along the southeast coast of India.     

Effects of acute change in salinity and moulting on the infection of white leg shrimp (Penaeus vannamei) with white spot syndrome virus upon immersion challenge

In the field, moulting and salinity drop in the water due to excessive rainfall have been mentioned to be risk factors for WSSV outbreaks. Therefore, in this study, the effect of an acute change in environmental salinity and shedding of the old cuticle shell on the susceptibility of Penaeus vannamei to WSSV was evaluated by immersion challenge. For testing the effect of abrupt salinity stress, early premoult shrimp that were acclimated to 35 g L^?1 were subjected to salinities of 50 g L^?1, 35 g L^?1, 20 g L^?1, 10 g L^?1 and 7 g L^?1 or 5 g L^?1 and simultaneously exposed to 10^5.5 SID₅₀ mL^?1 of WSSV for 5 h, after which the salinity was brought back to 35 g L^?1. Shrimp that were transferred from 35 g L^?1 to 50 g L^?1, 35 g L^?1 and 20 g L^?1 did not become infected with WSSV. Shrimp became infected with WSSV after an acute salinity drop from 35 g L^?1 to 10 g L^?1 and lower. The mortality in shrimp, subjected to a salinity change to 10 g L^?1, 7 g L^?1 and 5 g L^?1, was 6.7%, 46.7% and 53.3%, respectively (P < 0.05)_. For testing the effect of moulting, shrimp in early premoult, moulting and post‐moult were immersed in sea water containing 10^5.5 SID₅₀ mL^?1 of WSSV. The resulting mortality due to WSSV infection in shrimp inoculated during early premoult (0%), ecdysis (53.3%) and post‐moult (26.72%) demonstrated that a significant difference exists in susceptibility of shrimp during the short moulting process (P < 0.05). The findings of this study indicate that during a drop in environmental salinity lower than 10 g L^?1 and ecdysis, shrimp are at risk for a WSSV infection. These findings have important implications for WSSV control measures.     

Effects of a partitioned aquaculture system on water quality and growth of Penaeus vannamei

The effects of system partition on the water quality were studied using three shrimp ponds (Ts), and each one of the shrimp pond was connected individually with a fish pond by piping and pump. Three other isolated shrimp ponds were designated as the Cs treatment. The results showed that the implementation of the system can improve the water quality in the shrimp ponds. Particulate organic matter (POM), chemical oxygen demand (COD), nitrite nitrogen (NO₂–N) and nitrate nitrogen (NO₃–N) in the Ts were lower than those in the Cs during the experiment, and in some cases, they were significantly lower (p < 0.05). Throughout most of the experimental period, the soluble reactive phosphorus in the Ts treatment were greater than that in the Cs treatment (p < 0.05), and N/P ratio of the water column was lower in the Ts treatment than in the Cs treatment throughout most of the experimental period (p < 0.05). These results indicate that the partitioned system contributed to the stability of water quality and N/P ratio in the water. Vibrio parahaemolyticus was dominant in the third to seventh weeks in Cs, and the dominant condition was found only in seventh week in Ts. The result indicated that system may contribute to the slowing down of the breakout of V. parahaemolyticus in the shrimp pond. According to the redundancy analysis, COD, chlorophyll a and POM were mainly positively related to the dominance index of V. parahaemolyticus, and maybe POM can serve as an indicator to reflect the trend of V. parahaemolyticus.     

Effects of different carbon sources and dietary protein levels in a biofloc system on growth performance,immune response against white spot syndrome virus infection and cathepsin L gene expression of Litopenaeus vannamei

A 5‐week study was performed to evaluate the effect of spoilage date extract (SDE) as the biofloc carbon source on Litopenaeus vannamei (5.4 ± 0.3 g) performance. The two levels of dietary protein (15% and 25% crude protein) and two carbohydrate sources (molasses‐M and SDE‐P) were tested including: M15, M25, P15 and P25. The minimum (0.2 ± 0.0 mg/L) and the maximum (0.5 ± 0.0 mg/L) of total ammonia nitrogen were observed in the P15 and M25 groups respectively. The highest protein efficiency ratio (6.1 ± 0.3) and protein productive value (112.3 ± 5.8%) were found in the P15 group (p < 0.05). No significant difference was found between biofloc treatments in the expression of cathepsin L gene in hepatopancreas (p > 0.05). The number of total haemocyte count (THC), semigranular cells (SGC) and granular cells (GC) of shrimp in SDE‐based biofloc treatments was relatively higher than those in molasses‐based biofloc treatments. Following the white spot syndrome virus (WSSV) challenge, a significant decrease in THC, SGC, GC and hyaline cell values was observed in all treatments (p = 0.001). Plasma biochemical parameters were significantly influenced by dietary protein levels, biofloc carbon sources as well as WSSV challenge test. In conclusion, SDE successfully could be used as an alternative carbon source for establishing a biofloc system in L. vannamei production.     

Quantitative analysis of an experimental white spot syndrome virus (WSSV) infection in Penaeus monodon Fabricius using competitive polymerase chain reaction

White spot syndrome virus (WSSV) has been a major pathogen of cultured Penaeus monodon Fabricius in Malaysia since 1994. As quantitative study on the replication of WSSV is in its infancy, competitive polymerase chain reaction (PCR) was used for quantitative study of an experimental WSSV infection per os in growout P. monodon . Gills, abdominal integument and abdominal muscle were selected for viral quantification. Infection was detectable as early as 14 h postinfection (h p.i.) in both gills and integument, but the infection in muscle was only detected at 24 h p.i. Gill tissue had the highest viral load, followed by integument and muscle. Typical viral growth curves were obtained for all organs with distinct phases of eclipse (0–24 h p.i.), logarithmic (24–48 h p.i.) and the plateau (48–120 h p.i.). Cumulative mortality rapidly increased from 48 h p.i. and reached 100% at the end of the plateau phase at 120 h p.i. Gross signs of white spots and reddish discoloration were also obvious in moribund individuals from the plateau phase. Based on the three phases of viral growth, WSSV infection was classified into light, moderate and heavy infection stages.     

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.     

Fungus,Aspergillus niger,fermented groundnut oil cake as a fishmeal alternative in the diet of Penaeus vannamei

Commercial de‐fatted groundnut oil cake (GNC) fermented with the fungus Aspergillus niger was evaluated as a fishmeal alternative in the diet of Penaeus vannamei. A 45‐day growth trail was performed using nine iso‐nitrogenous and iso‐lipidic diets. Untreated/fermented GNC was included at the rate of 0 (control), 25, 50, 75 and 100 g/kg by replacing fishmeal (w/w). Each diet was randomly assigned to triplicate group of 20 shrimps (initial weight of 3.09 ± 0.03 g). Results revealed that shrimp fed with diets having untreated GNC up to 50 g/kg has no significant difference in growth, whereas the inclusion level was enhanced to 100 g/kg with fermented GNC with no deleterious effect. The negative linear trend was found for SGR with increasing the inclusion of both untreated and fermented GNC. The feed and protein efficiency measures, viz. feed conversion ratio, protein efficiency ratio and apparent protein utilization, were better in shrimps fed with diets having fermented GNC than those fed the respective level of untreated GNC. The broken‐line analysis indicated that the inclusion of 72.5 g/kg fermented GNC showed the best FCR. No significant difference was observed in survival (86.67%–96.67%) between the dietary treatments. There was a significant difference in ether extract of shrimp between the treatments, whereas other parameters were not affected. Haemolymph indices showed a significant difference in total protein, glucose, cholesterol and triglycerides between control and test diets. The results conclude fermented GNC, which can be better than untreated one in the diet of shrimp.     

Effect of fungal fermentation on apparent digestibility coefficient for dry matter,crude protein and amino acids of various plant protein sources in Penaeus vannamei

Four commercial solvent‐extracted oilseed meals/cakes, viz. soybean meal (SBM), groundnut oil cake (GNC), rapeseed meal (RSM) and sunflower oil cake (SFC), were fermented with fungus Aspergillus niger, and its effect on apparent digestibility coefficient (ADC) was studied in Penaeus vannamei. Reference diet and eight experimental diets containing 700 g/kg reference diet and 300 g/kg test ingredient with 5 g/kg chromic oxide were formulated. Each diet was randomly allotted to three tanks containing ten shrimps. Shrimps were adapted to the experimental diets for a week, and faeces were collected using Falcon tube from second week onwards. The ADC of all the ingredients significantly (p < .05) increased with fermentation and the increase being higher in SBM (78.46%–91.71%) for dry matter and in SFC (71.51%–87.02%) for protein. Analysis of variance showed that the ADC of both dry matter and protein significantly (p < .05) differed in treatments (p = <.001) and ingredients (p = <.001). The average ADC of ingredients was ranked as SBM > GNC > RSM > SFC. The most digestible essential amino acid (EAA) in fermented ingredients was methionine in SBM, arginine in GNC, valine in RSM and histidine in SFC. A better improvement in amino acid digestibility was observed in fermented SFC. Results indicated that P. vannamei efficiently digests fermented ingredients compared to unfermented ones.     

Optimization of fixation methods for image analysis of the hepatopancreas in whiteleg shrimp,Penaeus vannamei (Boone)

Pathology in penaeid shrimps relies on histology, which is subjective, time‐consuming and difficult to grade in a reproducible manner. Automated image analysis is faster, objective and suitable for routine screening; however, it requires standardized protocols. The first critical step is proper fixation of the target tissue. Bell & Lightner's (A Handbook of Normal Penaeid Shrimp Histology, 1988, The World Aquaculture Society, Baton Rouge) fixation protocol, widely used for routine histology of paraffin sections, is not optimized for image analysis, and no protocol for frozen sections is described in the available literature. Therefore, the aim of this study was to optimize fixation of the hepatopancreas (HP) from whiteleg shrimp (Penaeus vannamei) for both paraffin and frozen sections using a semiquantitative scoring system. For paraffin sections, four injection volumes and three injection methods were compared, for frozen sections, four freezing methods and four fixation methods. For paraffin sections, optimal fixation was achieved by increasing threefold the fixative volume recommended by Bell and Lightner, from 10% to 30% of the shrimp body weight, combined with single injection into the HP. Optimal fixation for frozen sections was achieved by freezing the cephalothorax with liquid nitrogen, followed by fixation of the section with 60% isopropanol. These optimized methods enable the future use of image analysis and improve classical histology.     

Effect of starvation and refeeding on the hepatopancreas of whiteleg shrimp Penaeus vannamei (Boone) using computer‐assisted image analysis

Under normal farming conditions, shrimp can experience starvation periods attributable to disease outbreaks or adverse environmental conditions. Starvation leads to significant morphological changes in the hepatopancreas (HP), being the main organ for absorption and storage of nutrients. In the literature, limited research has described the effect on the HP of periods of starvation followed by refeeding and none in whiteleg shrimp (Penaeus vannamei) using computer‐assisted image analysis (CAIA). This study describes the effect of starvation and starvation followed by refeeding on the HP of whiteleg shrimp using CAIA. Visiopharm^® software was used to quantify the following morphological parameters, measured as ratio to the total tissue area (TLA): total lumen area (TLA:TTA), haemocytic infiltration area in the intertubular spaces (HIA:TTA), B‐cell vacuole area (VBA:TTA), lipid droplet area within R cells (LDA:TTA) and F‐cell area (FCA:TTA). Significant changes were measured for HIA:TTA and LDA:TTA during starvation (increase in HIA:TTA associated with decrease in LDA:TTA) and starvation followed by refeeding (decrease in HIA:TTA associated with increase in LDA:TTA). In the future, HIA:TTA and LDA:TTA have the potential to be used in a pre‐emptive manner to monitor the health of the HP, facilitate early diagnosis of diseases and study the pathophysiology of the organ.     

Aspergillus niger fermented plant protein mix as a potential substitute for fishmeal in the diet of Penaeus vannamei (Boone, 1931)

A 45‐day feeding trial was conducted to evaluate the utilization of fermented plant protein mix (FPPM) as a fishmeal substitute in the diet of Penaeus vannamei. Six isonitrogenous and isolipidic diets were formulated by substituting fishmeal at 0% (control), 50%, 60%, 70%, 80% and 90% (FR‐0, FR‐50, FR‐60, FR‐70, FR‐80 and FR‐90, respectively) using FPPM. Shrimps fed with diets FR‐50, FR‐60 and FR‐70 had no significant difference in weight gain (173.22%–178.07%) with the control group (179.50%). However, the broken‐line regression indicated that 70.8% fishmeal could be substituted by FPPM. The trend of digestibility was reflected in growth performance. Survival had no significant difference among the treatments. Fishmeal substitution had significant (p < .05) variations in protease and amylase activity, but not in lipase. Except lipid, other body composition was not affected due to dietary change. A decrease (p < .05) in haemolymph total protein, cholesterol and triglycerides was observed to increase in fishmeal substitution. A higher fishmeal substitution (FR‐90) had significantly (p < .05) lower total haemocyte count and the reverse was true for phenoloxidase. Results conclude that a combination of fermented ingredients would be more effective rather using alone in shrimp feed, with a great potential in reducing the pressure on fishmeal.