Impact of yellow head virus outbreaks in the whiteleg shrimp,Penaeus vannamei (Boone), in Thailand

Yellow head virus (YHV) is known as a major pathogen in the black tiger shrimp, Penaeus (Penaeus) monodon. It can also cause serious mortality in farmed whiteleg shrimp, Penaeus (Litopenaeus) vannamei. However, there is no published information on the economic and/or production impact of the disease in P. vannamei. Shrimp with gross signs of YHV disease (faded body colour and 60–70% mortality) were observed in 20 study farms rearing P. vannamei in the central part of Thailand from the end of 2007 through early 2008. The estimated economic loss for these farms according to the Thai Animal Aquaculture Association was approximately US$3 million. Detailed sequence analysis of RT‐PCR amplicons from shrimp in all the study ponds revealed the presence of YHV Type 1b (YHV‐1b) alone (characterized by a 162‐bp deletion in the ORF3 region encoding the structural gene for gp116) and the absence of YHV Type 1a (YHV‐1a), the original YHV type reported from Thailand. Despite the large 162‐bp deletion (= 54 deduced amino acids) in the gp116 structural gene, histopathology of YHV‐1b infections was identical to that of YHV‐1a infections, and electron microscopy revealed that YHV‐1b virions were morphologically indistinguishable from those previously reported for YHV‐1a. In addition, an existing commercial RT‐PCR detection kit and an immunochromatographic test strip for the detection of YHV were proven to have been valid tests for both YHV‐1b and YHV‐1a. The source of the virus for these outbreaks was unlikely to have been the post‐larvae used to stock the ponds, as they were derived from domesticated specific pathogen‐free stocks free of YHV. Thus, it is possible that they originated from an unknown, natural reservoir.     

Isolation and identification of Vibrio campbellii as a bacterial pathogen for luminous vibriosis of Litopenaeus vannamei

Outbreak of luminescent disease was reported from Litopenaeus vannamei shrimp farms in Zhangpu County, Southern China during May–July 2011. The clinical signs included fluorescent, less food consumption and high mortality. Bacteria were isolated from the infected shrimps. The pathogen, a luminescent bacterium named VH1 was identified as Vibrio campbellii based on MLSA analysis (16S rDNA, rpoD and toxR). The haemolysin (hly) gene specific in V. campbellii was detected in strain VH1. Pathogenicity test using immersion infection confirmed that strain VH1 was virulent to L. vannamei postlarvae and juveniles, and the LC₅₀ value was 1.55 × 10⁶ CFU mL^?1 and 1.7 × 10⁶ CFU mL^?1 respectively.     

Production Methods and Resource Use at Litopenaeus vannamei and Penaeus monodon Farms in India Compared with Previous Findings from Thailand and Vietnam

Shrimp farms in India had average yields (pond surface basis) of 7.86 ± 1.04 (SE) m.t./ha/yr for Litopenaeus vannamei (n = 89) and 3.88 m.t./ha/yr for Penaeus monodon (n = 11). Average feed conversion ratio was 1.48 ± 0.04 for L. vannamei and 1.24 ± 0.12 for P. monodon. Land use for production ponds, supporting area, and feed ingredients averaged 0.634 ± 0.053 ha/m.t. for L. vannamei and 0.716 ± 0.087 ha/m.t. for P. monodon. Saline water volume used at farms without water exchange was 18,522 ± 4065 m³/m.t. for L. vannamei and 9528 m³/m.t. for P. monodon. Farms exchanging water used 149,188 m³/m.t. for L. vannamei and 191,500 m³/m.t. for P. monodon. Freshwater embodied in feed was 1678 ± 508 m³/m.t. for L. vannamei and 1401 ± 137 m³/m.t. for P. monodon. Average energy use for farm construction and repair, farm operations, and embodied in feed and liming materials was 82.14 ± 9.08 GJ/m.t. for L. vannamei and 59.03 ± 24.92 GJ/m.t. for P. monodon. Wildfish used to make fishmeal included in feed was 1209 kg/m.t. for L. vannamei and 1611 kg/m.t. for P. monodon. Farm infrastructure, pond management methods, yields, and resource use in India were similar to those previously reported for shrimp farms in Thailand and Vietnam.     

Infection of IHHN virus in two species of cultured penaeoid shrimp Litopenaeus vannamei (Boone) and Litopenaeus stylirostris (Stimpson) in Ecuador during El Niño 1997–98

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) was first noted in blue shrimp Litopenaeus stylirostris (Stimpson) in mid-1981. Since that time, at least 12 species of penaeoid shrimp have been reported to be infected with IHHNV. Pacific white shrimp Litopenaeus vannamei (Boone) represents a shrimp species highly refractory to the disease, whereas L. stylirostris was highly susceptible to the disease. Since the beginning of the shrimp farming industry in Ecuador, viral diseases have been observed in L. vannamei and L. stylirostris. Of these, L. vannamei represents ≈ 80% of cultured shrimp. Histopathology, ultrastructure and in situ DNA hybridization confirm the presence and assess the prevalence of IHHNV in pond-reared shrimp, and especially in abnormally small animals of both species. Although IHHNV may be considered enzootic in cultured L. vannamei in Ecuador, we did not find high prevalence (Cowdry A bodies) in specimens of diseased pond shrimp before 1996. From that time to 1998, a higher prevalence of IHHNV has been observed in both species. The epizootic of the IHHNV disease has been related to the oceanographic and climatological conditions caused by El Niño. In addition, it has been suggested that large quantities of wild shrimp post-larvae of both species that were stocked in shrimp farms, infected as latent carriers in 1997, from which the virus could spread to a larger population of these shrimp in 1998.     

Virulence and genotypes of white spot syndrome virus infecting Pacific white shrimp Litopenaeus vannamei in north‐western Mexico

White spot syndrome virus (WSSV) has caused substantial global economic impact on aquaculture, and it has been determined that strains can vary in virulence. In this study, the effect of viral load was evaluated by infecting Litopenaeus vannamei with 10‐fold serial dilution of tissue infected with strain WSSV Mx‐H, and the virulence of four WSSV strains from north‐western Mexico was assessed along with their variable number of tandem repeat (VNTR) genotypes in ORF75, ORF94 and ORF125. The LD₅₀ of the Mx‐H strain was a dilution dose of 10^?7.5; the mortality titre was 10^9.2 LD₅₀ per gram. In shrimp injected with 10^2.5 to 10^6.5 LD₅₀, no significant virulence differences were evident. Using mortality data, the four WSSV strains grouped into three virulence levels. The Mx‐F strain (intermediate virulence) and the Mx‐C strain (high virulence) showed more genetic differences than those observed between the Mx‐G (low‐virulence) and Mx‐H (high‐virulence) strains, in ORF94 and ORF125. The application of high‐viral‐load inocula proved useful in determining the different virulence phenotypes of the WSSV strains from the Eastern Pacific.     

Comparative study of the intracellular superoxide anion production in different penaeid species through the NBT‐reduction assay

The capacity of reactive oxygen intermediates production upon haemocyte stimulation is one of the most important immunoparameter utilized to assess the health status in cultivated shrimps. In the present study, we compared oxidative stress potential, by measuring the superoxide anion production in three penaeid shrimps: two wild Atlantic species, the pink shrimp Farfantepenaeus paulensis and the white shrimp Litopenaeus schmitti and one cultivated Pacific species, the white shrimp, Litopenaeus vannamei, through the nitro‐blue‐tetrazolium‐reduction assay. We also proposed an optimized experimental protocol for this assay, that produces rapid and consistent results with low levels of basal superoxide anion (O₂^?) production by unstimulated haemocytes and high levels of this oxygen radical after cell stimulation. Among the different cell elicitors used (zymosan, laminarin, lipopolysaccharide and phorbol myristate acetate), laminarin (β‐1,3‐glucans – 2 mg mL^?1) was the most potent cell activator for the haemocytes of all three penaeids and we recommend this immunostimulant to routinely evaluate shrimp respiratory burst. In general terms, the most elevated levels of O₂^? production, after cell stimulation with microbial components, were detected in L. schmitti. Interestingly, the stimulation profile of the haemocytes of L. vannamei was more similar to F. paulensis, than to L. schmitti, which is more phylogenetically related.     

Development of a multiplex PCR system for the simultaneous detection of white spot syndrome virus and hepatopancreatic parvovirus infection

A multiplex PCR kit for simultaneous detection of white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV) was developed and field testing was conducted. A 604‐bp target sequence was selected from the vp28 gene of WSSV. A primer set was developed to amplify a 338‐bp DNA fragment at the junction of the NS2 and NS1 protein genes of HPV after alignment of eight sequences from different strains. Another internal positive control primer set produced a 139‐bp PCR fragment from the β‐actin gene by alignment of this gene from Litopenaeus vannamei, Fenneropenaeus chinensis and Penaeus monodon. The detection limits, tested using purified plasmids, for WSSV and HPV were 21.4 and 19.0 copies respectively. The optimum ratio for HPV, WSSV and β‐actin was 3:1:1, with an optimum annealing temperature of 57°C. Field test of the multiplex PCR with 170 L. vannamei individuals from 17 aquaculture farms showed 41.8% coinfection with WSSV and HPV, and 40.0% and 3.5% single infection with WSSV and HPV respectively. No virus‐free shrimp farm was found. Ten wild catch F. chinensis individuals showed 60% coinfection, and 40% were infected with HPV.     

A novel micro‐organism for removing excess ammonia‐N in seawater ponds and the effect of Cobetia amphilecti on the growth and immune parameters of Litopenaeus vannamei

To investigate the feasibility of using micro‐organisms for ammonia‐N removal, six strains were isolated from Chinese white shrimp, Fenneropenaeus chinensis, seawater culture ponds in Dongying (Shandong, China). Of these, strain DY‐01, which exhibited the highest capacity to degrade ammonia‐N, removed 61.7% of the total ammonia‐N (50 mg/L) in 8 hr. An investigation of the factors affecting the removal efficiency indicated the optimum conditions to be 30°C, pH 8.0, and a salinity level of 30 g/L; 16S rDNA gene sequencing and biochemical analysis identified strain DY‐01 as Cobetia amphilecti, which has not previously been reported to degrade ammonia‐N. This strain also boosted the growth of Pacific white shrimp, Litopenaeus vannamei (p < .05), at a concentration of 10⁷ CFU/mL, with no harmful effects on the shrimp's immune system. This study has thus identified a novel aerobic nitrifying bacterium that is potentially an excellent candidate for improving the water quality in mariculture ponds.     

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.     

Daily activity pattern of the marine shrimp Litopenaeus vannamei (Boone 1931) juveniles under laboratory conditions

Lack of information on the daily activity patterns of Litopenaeus vannamei (Boone 1931) might result in inadequate feed management strategies in shrimp farms. In order to provide information to improve feed management in shrimp farms, the daily activity of juvenile shrimp reared in glass aquaria was studied, using L. vannamei (7.57±1.01 g), equivalent to 33 shrimp m^?2. Thirty‐two shrimp were observed, half in light and half in dark phase, over 280 h in 15 min h^?1 windows. Feed was provided once a day, at 10% of tank biomass. Swimming, exploration of substrate, inactivity and cleaning were recorded through instantaneous focal sampling. During the dark phase, swimming was predominant and alternated with exploration. In the light phase, inactivity was predominant, and again alternated with exploration. Cleaning occurred evenly in both the light and the dark. Substrate exploration, which characterizes the search for food, occurred in both dark and light phases, with a most intense peak 7 h after light phase onset, indicating this as the optimal feed offer.     

A diet of fructose‐enriched Artemia improves the response of juvenile Litopenaeus vannamei shrimp to acute low‐salinity challenge

The Pacific white shrimp (Litopenaeus vannamei), as an economically important species, has been reared in low‐salinity water during the last decade. To investigate how juvenile L. vannamei shrimp fed with fructose‐enriched Artemia respond to acute low‐salinity stress, the shrimp were randomly divided into four treatment groups, three groups were fed with Artemia enriched with either 100, 200 and 300 mg L^?1 of fructose and a control group fed with Artemia with no enrichment for 10 days. The results showed that the 300 mg L^?1 fructose group demonstrated the maximum survival rate and glycogen content. Additionally, the 300 mg L^?1 fructose group showed significantly higher Na⁺/K⁺‐ATPase activity, total antioxidant capacity, and expression levels of Na⁺/K⁺‐ATPase α‐subunit, V‐H ATPase α‐subunit, hexokinase, phosphofructokinase, antioxidants (SOD, CAT, GPX) and Hsp70 mRNA when compared with the control group. Furthermore, after exposure to low salinity, the mRNA levels of phosphofructokinase, V‐H ATPase α‐subunit, GPX, p38, JNK and Rac1 stayed constant in shrimp fed with fructose‐enriched Artemia but changed significantly in the control group. Thus, a diet of fructose‐enriched Artemia can improve the osmoregulation and survival of juvenile L. vannamei shrimp exposed to low salinities.     

Resource Use Assessment of Shrimp,Litopenaeus vannamei and Penaeus monodon,Production in Thailand and Vietnam

Resource use was investigated at 34 Litopenaeus vannamei and five Penaeus monodon farms in Thailand and 30 L. vannamei and 24 P. monodon farms in Vietnam. Farms varied in water surface areas for production, reservoirs, canals, and settling basins; in pond size and depth; and in water management, stocking density, feeding rate, amendment input, aeration rate, crop duration, and crops per year. Production of L. vannamei averaged 17.3 and 10.9 m.t./ha/yr, and feed conversion ratio averaged 1.49 and 1.33 in Thailand and Vietnam, respectively. On average, production of 1 m.t. of L. vannamei required 0.58 ha land, 5,400 m³ water, 60 GJ energy, and 1218 kg wildfish in Thailand and 1.76 ha land, 15,100 m³ water, 33.7 GJ energy, and 1264 kg wildfish in Vietnam. Resource use per metric ton of shrimp declined with greater production intensity. In Thailand, P. monodon was produced at 0.2–0.4 m.t./ha/yr, with no inputs but water and postlarvae. In Vietnam, P. monodon production averaged 3.60 m.t./ha/yr. Production of 1 m.t. of P. monodon required 0.80 ha land, 36,000 m³ water, 47.8 GJ energy, and 1180 kg wildfish, and resource use per ton production declined with increasing production intensity.     

Ingestion of food pellets containing Escherichia coli overexpressing the heat‐shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection

Feeding aquatic animals with bacterial encapsulated heat‐shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK‐DnaJ‐GrpE, the prokaryotic equivalents of Hsp70‐Hsp40‐Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g^?1 pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.     

Multiple infections caused by white spot syndrome virus and Enterocytozoon hepatopenaei in pond‐reared Penaeus vannamei in India and multiplex PCR for their simultaneous detection

White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow‐out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV‐positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m‐PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m‐PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively.     

New genetic recombination in hypervariable regions of the white spot syndrome virus isolated from Litopenaeus vannamei (Boone) in northwest Mexico

The white spot syndrome virus (WSSV) is a pathogen of great concern to the worldwide shrimp culture. In comparative studies on the WSSV genome, regions such as the open reading frame (ORF) 14–15 and ORF 23–24, prone to deletions and recombination, had been useful to study the evolutive relationships among viral strains. When looking for the WSSV strains infecting Litopenaeus vannamei (Boone) in northwest Mexico, we found evidence of a genetic similarity in ORF 14–15 to a strain from India and a recombination involving ORFs 78, 79 and 80. Two genotypes were found involving the insertion of a 265 base‐pair segment of ORF 108 into ORF 78 with inversions and deletions within ORFs 78, 79 and 80. The WSSV has an Asian origin and the mutations found could be an adaptation strategy to infect L. vannamei and other crustacean species of the American continent.     

Highly efficient double‐stranded RNA transfection of penaeid shrimp using cationic liposomes

Yellow head virus (YHV) is a viral pathogen of shrimp that has caused high economic loss in the Thai shrimp farming industry, and the RNA interference mechanism has potential as a virus control strategy, as previous studies have shown that injection of virus specific double‐stranded RNA (dsRNA) can inhibit viral replication in shrimp. However, to date, complete inhibition viral replication has not been achieved. This study sought to determine whether cationic liposomes mixed with an antiviral dsRNA (YHV‐Pro‐dsRNA) were able to enhance protection against YHV. The results showed that injection of a YHV‐Pro‐dsRNA/cationic liposome complex markedly increased protection against YHV as compared with naked YHV‐Pro‐dsRNA, suggesting that cationic liposomes enhance the uptake of protective dsRNA into shrimp cells.     

Effect of partial and total replacement of fish, shrimp head, and soybean meals with red crab meal Pleuroncodes planipes (Stimpson) on growth of white shrimp Litopenaeus vannamei (Boone)

Red crab meal (RCM), as a potential protein source in diets for juvenile shrimp Litopenaeus vannamei, was evaluated over a 45‐day growth trial under laboratory conditions. Eight experimental diets were tested. The basal diet contained fish (tuna by‐product), shrimp head and soybean (solvent extracted) meals as primary protein sources. Fish or soybean meals were substituted, on an equal‐protein basis, at 33%, 66% and 100% by RCM, whereas shrimp head meal (SHM) was substituted at 100%. A commercial diet was included as a reference. Final weight ranged between 2.23 and 3.36 g and growth rates (GRs) between 0.048 and 0.073 g day⁻¹. Where 66% or 100% of the protein from fish or soybean meals was substituted by RCM, the diets produced significantly higher final weights and GRs than other diets. Regression analysis showed that final weight of shrimp depended significantly on the percentage of substitution, and that the maximum weight gain would be obtained when substituting RCM for 80.2% of fish meal and 81.2% of soybean meal. Feed conversion ratio was below 1.8 for all treatments and there was no apparent relationship with other aspects of the diet. Red crab meal served as a suitable protein source for partial or total replacement of tuna by‐product, soybean and SHMs for cultivated juvenile shrimp L. vannamei.     

Hepatopancreatic and muscular distribution of oxytetracycline antibiotics in farmed pacific white shrimp (Penaeus vannamei): a physiological‐based pharmacokinetic model approach

Oxytetracycline (OTC) pharmacokinetic models previously used to investigate Penaeus vannamei have not addressed the specific problems related to drug distribution/disposition in particular tissues. This study aimed to provide an insight into OTC kinetics in the hepatopancreas and muscle based on a physiological model approach. Adult male P. vannamei at the C‐D₀ inter‐moulting stage were randomly assigned to intra‐sinus and oral administrations. In the intra‐sinus group, shrimps were dosed via the ventral sinus at an OTC level of 10.0 μg g^?1 body weight, while in the oral one, they were force fed at a dose level of 50.2 μg g^?1. The medicated animals were sampled at various time intervals until 170 h after dosing. Haemolymph, muscle and hepatopancreas samples were taken and OTC levels were determined using the validated HPLC method. A model focused on the hepatopancreas and muscle was developed. Oxytetracycline pharmacokinetic profiles in particular tissues were fitted into the model with an R² of between 0.6568 and 0.9904. Oxytetracycline muscular distributions were essentially identical for both groups and the drug did not accumulate in muscle. The distributions in the hepatopancreas for both groups were extensive, whereas that for oral administration was approximately 2.3 times greater than that for the intra‐sinus one. It was demonstrated that hepatopancreatic OTC may undergo significant first‐pass elimination with non‐linear kinetics.