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.     

Production of acute hepatopancreatic necrosis disease toxin is affected by addition of cell‐free supernatant prepared from AI‐2‐producing Vibrio harveyi mutant

Acute hepatopancreatic necrosis disease (AHPND) causes massive mortality in shrimp ponds within the first month poststocking. The causative agent is a specific strain of Vibrio parahaemolyticus (VP_AHPND) that has acquired the capability to produce virulent binary toxins called ToxA and ToxB. This study aims to test the effect of the addition of an autoinducer‐2‐containing cell‐free supernatant (CFS) from the mutant Vibrio harveyi (VH) on growth and toxin production of VP_AHPND. The relative AI‐2‐like activity in CFS was detected by luminescence assay. The effect of CFS (5 and 9%) on growth and toxin production of VP_AHPND was evaluated. Compared to the control culture (without CFS‐VH addition), the addition of either 5 or 9% CFS‐VH affected the growth at the initial stage of VP_AHPND. Similar growth profiles of VP_AHPND were found with the addition of CFS‐VH at both concentrations. Western blot analysis suggests that the addition of CFS‐VH affected the production of both toxins. ToxA could be detected at the early hour post‐CFS‐VH inoculation, whereas the high amount of ToxB was detected when 5% CFS‐VH was added. However, interfering with the AI‐2 function with furanone, the AI‐2 antagonist resulted in a slight delay in the production of both toxins. Results from this study will help to design a novel strategy to control AHPND in shrimp culture.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

Growth parameters of whiteleg shrimp Litopenaeus vannamei and red seaweed Gracilaria corticata in integrated culturing method under zero water exchange system

Growth parameters of whiteleg shrimp Litopenaeus vannamei and red seaweed Gracilaria corticata were measured using integrated culturing method under zero‐water exchange system in a 45‐day period. A 2 × 3 factorial design was used with two levels of shrimp stocking densities and three levels of seaweed weight densities. G. corticata was cultured on a net tied to a round polyethylene frame. Culture tanks were filled with 750‐L filtered seawater. A 40‐W compact fluorescent lamp was hung over each tank to provide adequate and sufficient light for seaweed growth. Growth parameters of shrimp and seaweed such as specific growth rate (SGR), weight gained (WG) and average daily growth (ADG) were computed based on the initial and final weight of shrimp and seaweed. The maximum and minimum SGR of L. vannamei (1.97 and 1.69%/day) were observed in treatment S₁A₃ (25 shrimp/m² and 400 g seaweed/m²) and S₂A₁ (50 shrimp/m² without seaweed) respectively. The best survival rate (94.67 ± 1.33%), WG (129.9 ± 2.9%) and feed conversion ratio (1.67 ± 0.04) were also observed in treatment S₁A₃. The SGR of G.corticata in the treatment S₁A₃ (1.97 ± 0.00%/day) was significantly higher, compared to others. Strong positive correlations were obtained between the density of G. corticata and the growth parameters of L. vannamei. The red seaweed G. corticata could boost the growth parameters, survival rate and total production of L. vannamei under zero‐water exchange system.     

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.     

Mass selection for small‐sized cysts in Artemia franciscana produced in Vinh Chau salt ponds,Vietnam

Unidirectional mass truncation selection for small‐sized cysts was carried out on brine shrimp (Artemia franciscana Kellogg 1906), Vinh Chau strain, Vietnam, through cyst sieving and then culturing the selected individuals (<5% of the population) under field and laboratory conditions over two successive generations (F₁ and F₂). The results showed that at both culture scales, the cyst size of the selected line was smaller (P < 0.05) compared with the control (non‐selected) line, illustrating that there was a response to selection. The cyst size decreased about 3% per generation and the accumulated gain on the target trait was in the range 5.82–6.07% at the end of the selection process compared with the base population. The realized heritability estimation (h²) for cyst size was 0.23 ± 0.06 and 0.74 ± 0.19 in the field trials and 0.34 and 0.51 in the laboratory test for the F₁ and F₂ generation respectively. The variations found between the respective trials may be explained as the result of environmental interaction with the selection process, but the results show that a selection program for small‐sized cysts in Artemia by mass selection is possible.     

Improvement of growth performance,water quality and disease resistance against Vibrio harveyi of postlarval whiteleg shrimp (Litopenaeus vannamei) by administration of mixed microencapsulated Bacillus probiotics

The objective of this study was to investigate the effects of mixed Bacillus on growth, water quality and disease resistance against Vibrio harveyi in whiteleg shrimp (Litopenaeus vannamei). Postlarval shrimp (PL₃₀) were fed with (a) a basal diet (the control), (b) a diet containing mixed freeze‐dried Bacillus probiotics (FB) and (c) addition of mixed microencapsulated Bacillus probiotics (MB) in culture water. Addition of FB and MB probiotics improved (p < .05) growth, feed efficiency, survival and culture water quality (ammonia and nitrite) compared to the control group although there was no difference (p > .05) between the two treated groups. Bacillus numbers in gastrointestinal tracts and culture water of FB‐ and MB‐administrated shrimp were higher (p < .05) than in the control. After a 30‐day culture, shrimp were infected with V. harveyi and monitored for 10 days. A significant reduction (p < .05) in cumulative mortality was observed in FB‐ and MB‐supplemented shrimp (43.24% and 45.05%, respectively), compared to the control (63.06%). This finding demonstrated that administration of microencapsulated probiotics was as effective as freeze‐dried probiotics for improving growth, feed efficiency, survival, Bacillus in gastrointestinal tracts, water quality (ammonia and nitrite) and conferring disease resistance to V. harveyi.     

Successful rearing of whiteleg shrimp Litopenaeus vannamei larvae fed a desiccation‐tolerant nematode to replace Artemia

The nematode Panagrolaimus sp. was tested as live feed to replace Artemia nauplii during first larval stages of whiteleg shrimp Litopenaeus vannamei. In Trial 1, shrimp larvae were fed one of four diets from Zoea 2 to Postlarva 1 (PL1): (A) Artemia nauplii, control treatment; (NC) nematodes enriched in docosahexaenoic acid (DHA) provided by the dinoflagellate Crypthecodinium cohnii; (N) non‐enriched nematodes; and (Algae) a mixture of microalgae supplemented in C. cohnii cells. In Trial 2, shrimp were fed (A), (NC) and a different treatment (NS) with nematodes enriched in polyunsaturated fatty acids (PUFAs) provided by the commercial product S.presso^®, until Postlarva 6 (PL6). Mysis 1 larvae fed nematodes of the three dietary treatments were 300 μm longer (3.2 ± 0.3 mm) than control larvae. At PL1, control shrimp were 300 μm longer (4.5 ± 0.3 mm) than those fed DHA‐enriched or PUFAs‐enriched nematodes. No differences were observed in length and survival at PL6 between control larvae and those fed DHA‐enriched nematodes (5.1 ± 0.5 mm; 33.1%–44.4%). Shrimp fed microalgae showed a delay in development at PL1. This work is the first demonstration of Panagrolaimus sp. suitability as a complete substitute for Artemia in rearing shrimp from Zoea 2 to PL6.     

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...     

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.