Evaluation of a cocktail of phages for the control of presumptive Vibrio parahaemolyticus strains associated to acute hepatopancreatic necrosis disease

Shrimp culture is a well‐established and fast‐growing industry that produces economic and social benefits in many countries. However, during the last years, it was severely affected by the emergence of the Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Disease (AHPND). This disease is mainly attributed to Vibrio parahaemolyticus, and currently, there is no effective cure or treatment. In this study, the use of T2A2 and VH5e bacteriophages was evaluated to control different AHPND‐positive strains (presumptively identified as V. parahaemolyticus, VP_AHPND) under laboratory conditions. Lytic effect of T2A2 and VH5e bacteriophages against different strains isolated from AHPND outbreaks was corroborated. In addition, the effectiveness of the mixture of both phages was tested on a brine shrimp experimental infection model using three highly virulent VP_AHPND strains. It has been found that phage‐treated brine shrimp had significantly higher survival percentage compared with non‐treated groups (p < .001). Also, phage cocktail was found to be harmless to the organisms. These results suggest that the phage mixture is worth considering as a possible control measure for positive AHPND strains, although it is clear that further and more extensive testing is needed.     

Update on early mortality syndrome/acute hepatopancreatic necrosis disease by April 2018

Outbreaks of acute hepatopancreatic necrosis disease (AHPND) have caused great economic losses to many shrimp‐producing countries in Asia since its first appearance in 2009. The causative agent was reported in 2013 as specific isolates of Vibrio parahaemolyticus (VP_AHPND) that were later found to harbor a plasmid (pVA) encoding the Pir‐like binary toxin genes Pir ^vpA and Pir ^vpB. VP_AHPND isolates colonize the shrimp stomach and release the binary toxins that cause massive sloughing of tubule epithelial cells followed by shrimp mortality. More recent information indicates that pVA plasmid and variants occur in many V. parahaemolyticus serotypes and also in other Vibrio species such as Vibrio campbellii, Vibrio harveyi, and Vibrio owensii. Information on such genomic and proteomic studies of different VP_AHPND isolates from different countries are reviewed. A cohort study carried out in Thailand in 2014 indicated that AHPND outbreaks account for only a portion of the disease outbreaks reported by shrimp farmers as outbreaks of early mortality syndrome (EMS). It is recommended that a regional research network and surveillance program for newly emerging or re‐emerging pathogens be established to speed up the process of diagnosis and the implementation of coordinated control measures and to avoid a repeat of the EMS/AHPND scenario.     

Identification of potential general markers of disease resistance in Exopalaemon carinicauda via three‐round challenge selection with an acute hepatopancreatic necrosis disease‐causing strain of Vibrio parahaemolyticus

Sixteen candidate disease‐resistant parameters were selected through which to evaluate the acute hepatopancreatic necrosis disease (AHPND)‐resistant capability of Exopalaemon carinicauda after three generations of selection for AHPND‐causing strain of Vibrio parahaemolyticus (VP_AHPND) resistance in our previous study. However, these parameters required further verification. In this study, another AHPND‐resistant E. carinicauda series was obtained through a short‐term selection procedure, consisting of three virulent challenge rounds of selection (about three‐week interval for each challenge) with VP_AHPND infection. After this selection, the survival rate at 144 hr post infection (hpi) increased from 23.33% to 37.78% and the observed 48‐hr LD₅₀ of VP_AHPND to shrimp increased from 10^5.5 cfu/ml to 10^6.5 cfu/ml. Then, the immune response of this AHPND‐resistant E. carinicauda was studied using the 16 candidate AHPND‐resistant parameters selected for in our previous study. The improved VP_AHPND clearance rate in hpi, increased total haemocyte counts, haemocyanin concentration, alkaline phosphatase activity and expressions of six immune‐related genes (Tollip and ALF in haemocytes and hepatopancreas; lysozyme, crustin and cathepsin B in hepatopancreas; and LGBP in haemocytes) at 24 hpi after the three‐round challenge selection suggest that these immune parameters may be reliable markers for the evaluation of the physiological status and potential AHPND‐resistant phenotypes in E. carinicauda.     

Quantitative genetics of disease resistance in different groups of Penaeus semisulcatus from preliminary controlled challenge test with Vibrio parahaemolyticus the aetiological agent of acute hepatopancreatic necrosis disease (AHPND)

Acute hepatopancreatic necrosis disease (AHPND) has caused losses to shrimp farmers worldwide owing to Vibrio parahaemolyticus (VP_AHPND). Selective breeding for disease resistance of shrimp was used as an alternative strategy for disease control. Five subsamples of shrimp from 20 full-sib families were subjected to a cohabitation challenge test. Juveniles of P. semisulcatus (mean weight 2.5 ± 0.17 g) were tested for survival after challenge with VP_AHPND. Mortality in the challenge test was recorded daily, and the experiment was terminated after 96 days when the mortality ceased. Moderate heritabilities were estimated in resistance to VP_AHPND based on a linear model (LM) and threshold model (TM). This indicates that disease resistance is a heritable trait, and a challenge test may serve as a basis for selection for resistance to VP_AHPND in P. semisulcatus. However, further investigation is needed to confirm and quantify the additive genetic variation for resistance to VP_AHPND in P. semisulcatus.     

Polyclonal antibody‐based farmer‐friendly flow‐through test for the detection of acute hepatopancreatic necrosis disease in shrimp

Early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) is currently the most significant disease of shrimp in farms of Vietnam, Thailand, Malaysia, China and Mexico, and there is a great risk that it may spread to other shrimp farming countries. Although, an array of sophisticated detection tools for AHPND available, there is a need for a sensitive, simple and rapid detection method. In this study, a simple, sensitive, rapid and polyclonal antibody‐based farmer‐friendly flow‐through assay (FTA) test has been developed for the detection of AHPND pathogen. The recombinant Photorhabdus insect‐related (Pir) A toxin‐like protein of AHPND pathogen was used to immunize rabbits at 21‐day interval observed for highest antibody titre after third booster by ELISA. The raised rabbit antiserum was purified by affinity chromatography and characterized by Western blot. The antiserum showed no cross‐reactivity with AHPND‐free Vibrio parahaemolyticus, V. anguillarum, White Spot Virus (WSV), Aeromonashydrophila and Aphanomycesinvadans. This polyclonal rabbit antiserum was used to develop a farmer‐friendly FTA test for the detection of AHPND pathogen. This simple FTA testis is more sensitive and could detect PirA^VP toxin up to 0.121 µg/ml, compared with 0.242 µg/ml by immunodot assay. Furthermore, FTA test requires only 8–10 min for completion, compared with 3 hr by immunodot thus found to be more sensitive, specific and cost‐effective. Collectively, sensitive FTA test would help shrimp farmers to take real‐time management decisions, especially emergency harvest and finally be a better hope for the prevention of AHPND.     

Development of a rapid immunochromatographic strip test for the detection of Vibrio parahaemolyticus toxin B that cause acute hepatopancreatic necrosis disease

Here, two monoclonal antibodies (MAbs) specific to different epitopes on ToxB, a toxin produced by Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (VP_AHPND), were employed to develop a rapid strip test. One MAb was conjugated to colloidal gold to bind to ToxB at the application pad, and another MAb was used to capture colloidal gold MAb–protein complexes at the test line (T) on the nitrocellulose strip. To validate test performance, a downstream control line (C) of goat anti-mouse immunoglobulin G antibody was used to capture the free colloidal gold conjugate MAb. The sample in the application buffer could be applied directly to the application well, and the test result was obtained within 15 min. The sensitivity of the kit is approximately 6.25 µg/ml of toxin, which was equivalent to the toxin produced by approximately 10⁷ cfu/ml of bacteria. This kit is convenient and easy to use since it can be used to identify VP_AHPND directly using a single colony of bacteria grown on agar culture plates. Because of its high specificity and simplicity, as well as not being reliant on sophisticated equipment or specialized skills, this strip test could be used by farmers for surveillance for ToxB-producing bacteria.     

Anti‐PirA‐like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin‐producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA‐like (rPirA) and PirB‐like (rPirB) toxins. Whole‐egg powders having IgY specific to rPirA (anti‐PirA‐IgY) and rPirB (anti‐PirB‐IgY) and IgY from non‐immunized hen (control‐IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti‐PirA‐IgY, anti‐PirB‐IgY and control‐IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti‐PirA‐IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti‐PirA‐IgY (87%) compared with the control (12%). These results confirm that addition of anti‐PirA‐IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.     

Administration of probiotic Bacillus licheniformis induces growth,immune and antioxidant enzyme activities,gut microbiota assembly and resistance to Vibrio parahaemolyticus in Litopenaeus vannamei

This study aimed to elucidate the effects of dietary Bacillus licheniformis ATCC 11946 (BL) on the growth, immune and antioxidant activities, intestinal morphology and microbiota, and susceptibility to Vibrio parahaemolyticus (VP_AHPND) in juvenile pacific white shrimp, Litopenaeus vannamei (LV). Juvenile LV (initial weight = 0.63 ± 0.001) were fed diets containing varying BL concentrations (0 (BL0), 10⁶ (BL1), 10⁷ (BL2) and 10⁸ (BL3) CFU/g feed) for 8 weeks. Growth performance, immune and antioxidant enzyme activities, and intestinal morphology significantly improved in the probiotic‐treated groups than the untreated. Regardless of the treatment group, the two dominant phyla were Proteobacteria and Bacteroidetes, whereas the two dominant genera were Ruegeria and Vibrio. Increasing inclusion of probiotics in diets led to significant increase in beneficial bacterial genera (Ruegeria, and Pseudoalteromonas) and a significant decrease in some known opportunistic pathogens (Vibrio, Tenacibaculum, Photobacterium, Kangiella and Spongiimonas) with the BL3 group witnessing the best. A 7‐day challenge study with VP_AHPND showed significantly high protection in the probiotic‐treated groups, with the BL3, BL2 and BL1 obtaining 51%, 34% and 22% relative percentage survival, respectively. In conclusion, BL at 10⁸ CFU/g feed should be used to help in shrimp production since it attained the most significant improvement.     

致急性肝胰腺坏死病副溶血弧菌(VpAHPND)自然感染三疣梭子蟹

近年来包括急性肝胰腺坏死病(AHPND)在内的多种新发疫病的流行,使我国甲壳类养殖业遭受了严重的经济损失。为了筛查导致山东潍坊某养殖场中一虾蟹混养池塘内患病三疣梭子蟹感染的可能病原,本研究采用分子生物学检测方法,对三疣梭子蟹样品进行了白斑综合征病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)、虾血细胞虹彩病毒(SHIV)、致急性肝胰腺坏死病副溶血孤菌(Vp_(AHPND))、虾肝肠胞虫(EHP)、偷死野田村病毒(CMNV)、黄头病毒(YHV)和肝胰腺细小病毒(HPV)等8种病原的检测,并对样品进行了组织病理和原位杂交分析。分子生物学检测结果显示,患病三疣梭子蟹样品呈Vp_(AHPND)阳性,而呈现WSSV、IHHNV、SHIV、EHP、CMNV、YHV和HPV阴性。对样品进行Vp_(AHPND)套式PCR第二轮扩增产物的序列测定、比对和进化树分析,结果显示,扩增产物序列与致病副溶血弧菌质粒上pirA~(vp)毒力基因片段具有99%的同源性,该序列与已报道的多个致病副溶血弧菌PirA聚在进化树的同一主分支上。组织病理学分析显示,患病三疣梭子蟹的肝胰腺小管上皮细胞坏死,心肌纤维呈溶解样病变,鳃丝上皮柱突细胞明显坏死,胸神经节的神经细胞损伤严重,并且这些组织中还可见大量的细胞核固缩现象;原位杂交结果显示,肝胰腺、心肌、鳃组织及胸神经节中的病变部位均存在Vp_(AHPND)探针的蓝紫色杂交信号。以上表明,虾蟹混养池塘中三疣梭子蟹在自然状态下感染了Vp_(AHPND),并导致肝胰腺、心肌、鳃和胸神经节发生了严重病理损伤。本研究首次在养殖三疣梭子蟹中检测到Vp_(AHPND)感染并揭示了感染所致的病理变化,相关结果为揭示Vp_(AHPND)自然宿主种类和养殖三疣梭子蟹病害防控提供了基础信息。     

Impact of farm management on expression of early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) on penaeid shrimp farms in Thailand

Asian shrimp farming industry has experienced massive production losses due to a disease caused by toxins of Vibrio bacteria, known as early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) for the last 5 years. The disease can cause up to 100% cumulative pond mortality within a week. The objective of this study was to identify factors associated with AHPND occurrence on shrimp farms. A case–control study was carried out on shrimp farms in four provinces of Thailand. Factors related to farm characteristics, farm management, pond and water preparation, feed management, post‐larvae (PL) shrimp and stock management were evaluated. Multivariable logistic regression analysis identified factors affecting AHPND occurrence at the pond level. Chlorine treatment, reservoir availability, use of predator fish in the water preparation, culture of multiple shrimp species in one farm and increased PL stocking density contributed to an increased risk of AHPND infection, while delayed first day of feeding, polyculture and water ageing were likely to promote outbreak protection. Additionally, the source of PL was found to be associated with AHPND occurrence in shrimp ponds, which requires further study at the hatchery level. Identification of these factors will facilitate the development of effective control strategies for AHPND on shrimp farms.     

Characterization of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in southern Thailand

Vibrio parahaemolyticus was isolated from shrimp of five farms located in the Pattani and Songkhla provinces of southern Thailand. Using a PCR method targeted to the unique DNA sequences derived from the plasmid (AP2 primers) and the toxin gene (AP3 primers) of V. parahaemolyticus that caused acute hepatopancreatic necrosis disease (AHPND), a total of 33 of 108 isolates were positive. In contrast, all 63 and 66 isolates of clinical and environmental V. parahaemolyticus, respectively, obtained previously from 2008 to 2014 from the same area were negative. This implied that these strains were likely to be the cause of the outbreak of AHPND in this area. Intestinal samples proved to be a better source for the isolation of V. parahaemolyticus AHPND than the hepatopancreas. All isolates were investigated for haemolytic activity, virulence genes, serotypes, genotypes and antibiotic susceptibility. All the AHPND isolates had a unique O antigen, but small variations of the K antigens were detected from different farms. In addition, the DNA profiles of V. parahaemolyticus AHPND isolates were similar, but distinct from those clinical and environmental isolates. It is postulated that the causative agent of AHPND might have originated from one clone and then slightly different serotypes subsequently developed.     

In vitro and in vivo selection of probiotic purple nonsulphur bacteria with an ability to inhibit shrimp pathogens: acute hepatopancreatic necrosis disease‐causing Vibrio parahaemolyticus and other vibrios

Shrimp cultivation has been faced with huge losses in productivity caused by infectious shrimp pathogenic vibrios, especially Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (AHPND). Hence, purple nonsulphur bacteria (PNSB) were isolated from shrimp ponds for investigating their abilities to control shrimp pathogenic Vibrio spp. and their use as probiotics for sustainable shrimp cultivation. Based on their probiotic properties, strains S3W10 and SS15 were selected because of their strong abilities to produce amylase, gelatinase and vitamin B12. However, only three PNSB strains (SS15, TKW17 and STW181) strongly inhibited V. harveyi_KSAAHRC and V. vulnificus_KSAAHRC including V. parahaemolyticusAHPND strains by secreting antivibrio compounds. Four selected PNSB also grew in the presence of pancreatic enzymes, and they were identified as Rhodobacter sphaeroides for strains S3W10, SS15 and TKW17 and Afifella marina for strain STW181. The effects of a mixed culture were also investigated as follows: T1 (S3W10 + SS15), T2 (S3W10 + TKW17) and T3 (S3W10 + STW181) on postlarval white shrimp (Litopenaeus vannamei) for 60 days by comparison with a control. All three probiotic PNSB sets significantly improved the digestive enzyme activities and shrimp growth with their proliferation in shrimp gastrointestinal tract although the shrimp survival was not significantly different. They also significantly reduced the cumulative mortality of shrimp exposed to a virulent AHPND strain (V. parahaemolyticusSR2). This is the first to conclude that selected probiotic PNSB strains have great potential to be used for shrimp cultivation to control vibrios including AHPND strains.     

对虾养殖场底泥中副溶血性弧菌的接合型质粒多样性及其与毒力之间的关系

为了阐明引起急性肝胰腺坏死病(acute hepatopancreatic necrosis disease, AHPND)副溶血性弧菌的接合型质粒在对虾养殖环境中的遗传多样性,实验从中国5个沿海省份的虾场收集了100个底泥样品,以质粒上编码接合转移蛋白的保守基因为目标,利用PCR法检测相关质粒的存在情况,并对质粒进行测序。结果显示,100个样品中有39个样品含有质粒的接合转移蛋白片段。从100个底泥样品中分离出15株副溶血性弧菌,其中13株含有1～2个质粒。质粒序列测序结果显示,这些质粒可分为8种类型/谱型,其中7种不携带pirAB,但均含有编码接合转移的基因簇。根据分离副溶血性弧菌携带质粒的8种谱型,分别选择8株副溶血性弧菌进行凡纳滨对虾攻毒实验,发现这些菌株对凡纳滨对虾的毒性有显著差异,实验虾死亡率为15%～100%。只有pirAB阳性菌株会对实验虾产生AHPND症状,死亡率为100%。对质粒组成进行分析表明,质粒之间遗传物质交换频繁,大部分质粒的遗传组成都来自一个183 kb的超大质粒pVP2HP。综上,本实验通过探究对虾养殖场底泥中结合性质粒的多样性,增强了人们对副溶血性弧菌...     

The first record of acute hepatopancreatic necrosis disease in the Philippines

A new and emerging disease is threatening the shrimp industry, a bacterial disease which contains a highly pathogenic plasmid, creating a deadly toxin that causes high mortality in shrimps. The disease has been identified as acute hepatopancreatic necrosis disease (AHPND) or commonly known as early mortality syndrome (EMS). To help the efforts of sustaining the shrimp industry, the study focused on detecting Vibrio parahaemolyticus causing AHPND/EMS affecting Penaeus vannamei (Boone, 1931) (Pacific white shrimp) and Penaeus monodon (Fabricius 1798) (Black Tiger shrimp) in different locations in the Philippines. The presence of the disease was determined by microbiological methods and polymerase chain reaction (PCR) and was confirmed by the histopathology of the hepatopancreas of infected shrimp. Results show that the prevalence of the pathogenic strain of V. parahaemolyticus from the shrimps collected were 33% in Luzon, 21% in Visayas and 5% in Mindanao. The study presents the first record of AHPND/EMS in the country. The detection of this newly emerging disease in the shrimp industry is very crucial as it is the first step in identifying affected sites towards strategizing ways to combat the disease.     

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.     

Effects on survival and bacterial community composition of the aquaculture water and gastrointestinal tract of shrimp (Litopenaeus vannamei) exposed to probiotic treatments after an induced infection of acute hepatopancreatic necrosis disease

Acute hepatopancreatic necrosis disease (AHPND) is a devastating condition impacting marine shrimp production worldwide. The objective of this study was to evaluate the effects of four probiotic formulations on Pacific white shrimp (Litopenaeus vannamei) infected with pathogenic Vibrio parahaemolyticus causing AHPND. In addition, bacterial community composition analyses of shrimp gastrointestinal tract (GIT) and aquaculture water before and after infection were conducted by sequencing variable region 4 of the 16S rRNA gene on the Illumina MiSeq platform. Treatments included: (1) Lactobacillus casei (P1), (2) L. casei and Rhodopseudomonas palustris (P2), (3) L. casei, Saccharomyces cerevisiae and R. palustris (P3), and (4) a commercial probiotic EM^® (EM), which showed shrimp survival of 11.7%, 26.7%, 36.7% and 73.3% respectively. Treatments causing lower survival showed greater relative abundance (>60%) of family Vibrionaceae in the GIT compared to treatments with higher survival. Diversity indices from GIT samples revealed that treatments showing higher survival had higher Shannon index values (4.69 ± 0.133), compared with those of treatments with lower survival (0.17 ± 0.004). Diversity indices from water samples did not show significant differences after infection (Shannon index 4.64 ± 0.53). The results showed that probiotics could effectively mitigate AHPND while maintaining diverse microbial composition in shrimp GIT, thus maintaining sustainability in the shrimp aquaculture industry.     

Effect of using sodium bicarbonate to adjust the pH to different levels on water quality,the growth and the immune response of shrimp Litopenaeus vannamei reared in zero‐water exchange biofloc‐based culture tanks

The bioflocs technology proved to be a sustainable technique used in zero‐water exchange shrimp culture systems. However, the pH and alkalinity may decrease due to the biofloc formation process and Nitrification. A 48‐day experiment was performed to investigate the effects of different pH (7.1–7.6; 7.6–8.1) conditions on water quality, the growth and the health status of shrimp in biofloc technology (BFT) through using sodium bicarbonate to adjust pH respectively. Two pH treatments and one control were compared: T0 — control, T1 — pH 7.6 — NaHCO₃, T2 — pH 8.1 — NaHCO₃, each treatment consisted of three replicate tanks (90 L water volume) and each replicate stocked with 30 shrimp (equivalent to 333 shrimp m^?3). Significant physical, chemical and biological differences (P < 0.05) were detected among treatments. At the end of the experiment, water quality, the growth and the immune response of shrimp in control were significantly lower (P < 0.05) than the other treatments. Moreover, the T2 treatment had a better performance in these three aspects. The results indicated that it was necessary to adjust the pH and alkalinity in the BFT, and a higher pH as well as alkalinity for shrimp growth and the stability of the BFT were more favourable.     

Some Limiting Factors in Superintensive Production of Juvenile Pacific White Shrimp,Litopenaeus vannamei,in No‐water‐exchange,Biofloc‐dominated Systems

Superintensive shrimp culture in zero‐exchange, biofloc‐dominated production systems is more biosecure and sustainable than traditional shrimp farming practices. However, successful application of this technology depends upon optimizing dietary formulations, controlling Vibrio outbreaks, and managing accumulative changes in water quality and composition. A 49‐d study investigated the effect of two commercial feeds of differing protein content and an indoor limited‐exchange, biofloc‐dominated culture environment on Litopenaeus vannamei performance and tissue composition, water quality and ionic composition, and Vibrio dynamics. Juveniles (5.3 g) were stocked at 457/m³ into four 40 m³ shallow raceways containing biofloc‐dominated water and fed one of two commercial feeds with differing protein content, 35 or 40%. Shrimp performance, Vibrio populations, and changes in shrimp and culture water composition were monitored. There were no significant differences (P > 0.05) in shrimp performance (survival, weight, growth, specific growth rate, total biomass, yield, feed conversion ratio, and protein efficiency ratio) or proximate composition between feed types. The 40% protein feed resulted in higher culture water nitrate and phosphate concentrations, alkalinity consumption and bicarbonate use, and higher phytoplankton density. The presence of Vibrio, specifically Vibrio parahaemolyticus, reduced shrimp survival. This survival decrease corresponded with increased culture water Vibrio concentrations. Culture water K⁺ and Mg²⁺ increased significantly (P < 0.05), and Sr²⁺, Br^?, and Cl^? decreased significantly (P < 0.05) over time. While Cu²⁺ and Zn²⁺ did increase in shrimp tissue, no heavy metals accumulated to problematic levels in culture water or shrimp tissue. These results demonstrate the importance of monitoring Vibrio populations and ionic composition in limited‐exchange shrimp culture systems.     

Optimum dietary protein‐to‐energy ratio for juvenile whiteleg shrimp,Litopenaeus vannamei,reared in a biofloc system

An 8‐week feeding trial was conducted to evaluate the optimum dietary protein‐to‐energy (P/E) ratio in juvenile whiteleg shrimp, Litopenaeus vannamei. Six diets were formulated with two protein levels (30% and 35%) and three digestible energy levels (16, 17.5 and 19 kJ/g diet) at each protein level (₃₀P₁₆, ₃₀P_17.5, ₃₀P₁₉, ₃₅P₁₆, ₃₅P_17.5 and ₃₅P₁₉). Fifty shrimp averaging 0.97 ± 0.03g (Mean ± SD) were randomly distributed in biofloc tanks and fed one of the experimental diets. Weight gain (WG), specific growth rate (SGR) and feed efficiency (FE) of shrimp fed the ₃₅P_17.5 diet were significantly higher than those of shrimp fed ₃₀P₁₆, ₃₀P_17.5 and ₃₀P₁₉ diets (p < .05). Results for non‐specific immune responses showed that diet ₃₅P_17.5 caused higher lysozyme activity in shrimp comparing to lower protein diets. Thiobarbituric acid reactive substances (TBARS) of plasma were lower for shrimp fed on diets consisting of higher protein and energy levels than shrimp fed on the ₃₀P₁₉ diet. Analyses of digestive enzyme activities showed higher trypsin activities for shrimp fed on ₃₅P_17.5 and ₃₅P₁₉ diets comparing to ₃₀P₁₆ and ₃₀P_17.5 diets. Also, hepatopancreatic lipase activity of shrimp fed ₃₅P₁₆, ₃₅P_17.5 and ₃₅P₁₉ diets were significantly higher than those of shrimp fed the other diets. In conclusion, based on the results for growth performance, biochemical parameters, immune responses, oxidative stress and enzyme activities, 35% protein and 17.5 kJ/g digestible energy (₃₅P_17.5) could be considered as the optimum protein‐to‐energy ratio in the diet of juvenile whiteleg shrimp reared in a biofloc system.     

Progress in research on acute hepatopancreatic necrosis disease (AHPND)

Acute hepatopancreatic necrosis disease (AHPND) of shrimps is an important disease, first appeared in China in 2009. Since then, AHPND has caused serious drops in shrimp production (up to 20 % worldwide). Although AHPND [originally termed as acute hepatopancreatic necrosis syndrome (AHPNS)] first appeared in 2009, it was not until 2013 that a laboratory infection model was devised and the causative agent identified as certain strains of Vibrio parahaemolyticus. AHPND has caused mortality from 40 to 100 % which usually occurs early (within approximately 35 days) after stocking shrimp fry in shrimp ponds; therefore, it was initially referred to as early mortality syndrome (EMS). Confusingly, other pathogens and environmental factors also cause EMS and are often attributed to AHPND by shrimp farmers. Frequently, farmers do not send samples for confirmatory tests requiring detection of the unique histopathology at the acute stage of disease (massive sloughing of hepatopancreatic epithelial cells without any accompanying signs of a pathogen). The gross signs presumptive of AHPND (lethargy, slow growth, empty stomach and midgut, and a pale to white, atrophied hepatopancreas) are insufficient for confirmatory diagnosis. Recently, molecular detection of AHPND bacteria using PCR has been developed, which has sped up diagnosis and increased research on the causative agent, alternative detection methods, and possible therapies. We hope that this review of research progress on AHPND will serve as a useful introduction for researchers who are currently unfamiliar with AHPND, but have backgrounds in bacterial virulence, detection, and epidemiology, and may be encouraged to participate in the research effort to reduce AHPND’s impact on shrimp cultivation.