副溶血弧菌(Vibrio parahaemolyticus)中溶源噬菌体与其宿主菌致病力的相关性

急性肝胰腺坏死病(Acute Hepatopancreatic Necrosis Disease,AHPND)是由副溶血弧菌(Vibrio parahaemolyticus)引起的对虾病害,本研究从患AHPND的凡纳滨对虾样品中分离得到5株副溶血弧菌,采用致AHPND的副溶血弧菌(VPAHPND)的相关质粒的引物AP2进行PCR检测,表明这5株菌中均存在AHPND相关质粒。利用丝裂霉素C进行溶源性噬菌体筛选和噬菌体诱导发现,其中2株副溶血弧菌(20130629002S01和20130726001S01)可能存在溶源性噬菌体感染;从经0.5μg/ml丝裂霉素C诱导的20130629002S01和20130726001S01中分别分离得到两种噬菌体phage1和phage2。透射电镜观测显示,phage1为有尾噬菌体,phage2为球形噬菌体。将上述5株副溶血弧菌进行卤虫无节幼体人工感染实验,结果显示,它们对卤虫无节幼体均有致病力,且各分离株的毒力表现出显著性差异;20130629002S01和20130726001S01两株带有溶源性噬菌体的副溶血弧菌的致病力显著低于无噬菌体的副溶血弧菌(20130721001S02)。本研究结果表明,5株VPAHPND分离株都含有AHPND相关的质粒,表现出显著的毒力差异,可能携带不同的溶源噬菌体,也可能不携带溶源噬菌体,溶源噬菌体与副溶血弧菌各分离株的毒力并无必然相关性。     

副溶血弧菌(Vibrio parahaemolyticus)中溶源噬菌体与其宿主菌致病力的相关性

急性肝胰腺坏死病(Acute Hepatopancreatic Necrosis Disease,AHPND)是由副溶血弧菌(Vibrio parahaemolyticus)引起的对虾病害,本研究从患AHPND的凡纳滨对虾样品中分离得到5株副溶血弧菌,采用致AHPND的副溶血弧菌(VPAHPND)的相关质粒的引物AP2进行PCR检测,表明这5株菌中均存在AHPND相关质粒.利用丝裂霉素C进行溶源性噬菌体筛选和噬菌体诱导发现,其中2株副溶血弧菌(20130629002S01和20130726001S01)可能存在溶源性噬菌体感染;从经0.5 μg/ml丝裂霉素C诱导的20130629002S01和20130726001S01中分别分离得到两种噬菌体phage1和phage2.透射电镜观测显示,phage1为有尾噬菌体,phage2为球形噬菌体.将上述5株副溶血弧菌进行卤虫无节幼体人工感染实验,结果显示,它们对卤虫无节幼体均有致病力,且各分离株的毒力表现出显著性差异;20130629002S01和20130726001S01两株带有溶源性噬菌体的副溶血弧菌的致病力显著低于无噬菌体的副溶血弧菌(20130721001S02).本研究结果表明,5株VPAHPND分离株都含有AHPND相关的质粒,表现出显著的毒力差异,可能携带不同的溶源噬菌体,也可能不携带溶源噬菌体,溶源噬菌体与副溶血弧菌各分离株的毒力并无必然相关性.     

凡纳滨对虾中发光坎氏弧菌的分离、鉴定及致病性

为研究凡纳滨对虾死亡的致病机理,实验采用TCBS培养基从濒死凡纳滨对虾肝胰腺分离到浅绿色、直径3～7 mm的发荧光菌落;革兰氏染色为阴性短杆菌;经API20E鉴定,分离株PvL-1与坎氏弧菌参考菌株CAIM249相似度为90%;fitZ序列与坎氏弧菌CP020076相似度为99.31%;gapA序列与坎氏弧菌EF596552相似度为98.89%;采用坎氏弧菌种特异性引物对PvL-1进行PCR分析,可扩增出坎氏弧菌种特异性片段,不能扩增出轮虫弧菌和哈维氏弧菌特异性片段,上述结果表明PvL-1为坎氏弧菌成员。分离菌株在10%脱纤维羊血平板呈β溶血,脱脂奶粉平板出现明显透明圈,表明存在溶血性和胞外蛋白酶活性。分析了PvL-1的急性肝胰腺坏死病、溶血素基因、菌毛基因和其他毒力基因,表明PvL-1具有AP4、TLH、vcahHly、flaC、mukF、gloB、sodB和esrB等毒力基因。PvL-1可使健康凡纳滨对虾发病死亡,濒死凡纳滨对虾与自然发病虾呈现类似症状,对凡纳滨对虾半数致死浓度(LD50)2.94×104 CFU/尾;病理组织观察发现,人工感染发病凡纳滨对虾肝胰腺小管细胞脱落、崩解、血细胞浸润等,与自然发病凡纳滨对虾病理特征相同。研究表明,本次实验分离到可发光的坎氏弧菌PvL-1,对凡纳滨对虾有较强致病性,拓展了对凡纳滨对虾发光坎氏弧菌的认知。     

引起凡纳滨对虾急性肝胰腺坏死病的副溶血弧菌MLST新序列型

针对12株引起凡纳滨对虾急性肝胰腺坏死病(AHPND)的致病性副溶血弧菌,运用多位点测序技术,分析致病菌株的遗传特征。实验选择副溶血弧菌的7个管家基因dnaE、gyrB、recA、dtdS、pntA、pyrC及tnaA,对12株AHPND致病菌扩增测序。将核酸序列上传至PubMLST数据库进行比对后获得每株菌的序列型。收集其他地区AHPND致病性副溶血弧菌的多位点序列分型(MLST)数据,采用eBURSTV3及MEGA5.0软件进行遗传进化分析。结果显示,2014年中国广东分离的AHPND致病株属于一个新序列型ST1710(42、134、99、79、141、41、51),仅与库中ST415及ST975同源性较高。目前,急性肝胰腺坏死致病株的9种ST型可归为2个克隆组和5个单体。经系统发育树进一步分析可知新序列型ST1710与单体ST975遗传关系相近。本研究首次报道中国AHPND分离株的序列型,丰富了PubMLST数据库,并为其遗传进化研究奠定了理论基础。     

2株副溶血弧菌不同盐度下致病性和毒力基因差异分析

副溶血弧菌(Vibrio parahaemolyticus)是常见的食源性致病菌和海水养殖动物致病菌,目前已知的毒力基因有tlh、tdh、trh、T3SS、pirA、pirB、toxR/S、orf8等。盐度是影响细菌基因表达的关键生态因子之一,为进一步探求副溶血弧菌的致病机理,对2株分别分离自海水和淡水养殖发病凡纳滨对虾(Litopenaeus vannamei)中的副溶血弧菌在不同盐度下的生长速率、致病性进行检测,并用q-PCR方法对菌株毒力基因携带和表达情况进行定量研究。实验结果表明:海水菌株383生长速率快于淡水菌株V9,但菌株383在生长稳定期的细菌浓度明显低于菌株V9;菌株383对凡纳滨对虾的致病性明显高于菌株V9,前者的48 h半致死浓度(LD_(50))低于后者2个数量级;2株副溶血弧菌皆携带毒力基因tlh、T3SS1和pirA/B,但未检测到tdh、trh、T3SS2、toxR/S和orf8。部分毒力基因表达量检测结果显示,菌株383和菌株V9均为vcrD1表达量最高,其次是pirA,vopD1表达量最低;4个毒力基因不仅在2菌株间的表达量差别较大,而且盐度对同一菌株不同毒力基因表达的影响也是不同的。2株副溶血弧菌的毒力与pirA和vcrD1的表达量呈正相关性。研究结果为探究环境因素与副溶血弧菌致病力的关系提供了科学依据,同时也提示,淡水养殖凡纳滨对虾要防范副溶血弧菌输入的风险。     

凡纳滨对虾和罗氏沼虾肠道微生物及抗生素抗性基因多样性分析

探究凡纳滨对虾和罗氏沼虾肠道微生物及抗生素抗性基因(antibiotic resistance genes, ARGs)种类的差异。通过高通量测序和变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)技术分析2种虾肠道微生物群落结构差异和微生物多样性,并运用PCR方法检测了2种虾肠道细菌常见38种ARGs的携带情况。结果显示,获得凡纳滨对虾和罗氏沼虾肠道细菌有效序列分别为42 795和40 713条,物种注释单元(operational taxonomic unit, OTU)数目分别为124和82,分类地位明确的细菌种类分别隶属5个门、17个属和5个门、16个属。凡纳滨对虾肠道细菌的优势类群为变形菌门,所占比例为75.45%,优势菌属为副球菌属(25.83%)和不动杆菌属(25.24%);罗氏沼虾肠道细菌的优势类群是厚壁菌门(49.74%),优势菌属为乳球菌属(49.01%)和弧菌属(29.98%)。凡纳滨对虾肠道细菌(2.19)Shannon指数高于罗氏沼虾肠道细菌(1.78),表明前者肠道细菌多样性大于后者。DGGE图谱的分析结果与高通量测序一致,2种虾肠道细菌种类差异很大。PCR结果显示,凡纳滨对虾肠道细菌携带15种ARGs,罗氏沼虾肠道细菌携带14种ARGs。本实验表明凡纳滨对虾肠道细菌的群落种类多样性、OTU丰富度、物种总数和ARGs种类均高于罗氏沼虾肠道细菌,为后续肠道微生物资源的挖掘提供了理论依据。     

凡纳滨对虾细菌性红体病病原的分子特征与耐药性

为探明引起凡纳滨对虾细菌性红体病的病原,从病虾肝胰脏分离得到10株优势菌,经回归感染实验证实其为引起此次红体病的病原菌.Vitek与16S rRNA序列分析显示,分离株均为副溶血弧菌.基于dnaE-gyrB-recA-dtdS-pntA-pyrC-tnaA的多位点序列分型(multilocus sequence typing,MLST)表明,这些菌株形成3个新序列型(ST),其中1株为ST413,7株为ST414,2株为ST415;ST413包含新等位基因型recA-166与tnaA-121,ST414则含有新等位基因型gyrB-219.MLST结果提示,这些副溶血弧菌分离株并非来自单一克隆,呈现出一定水平的分子多样性.但这些菌株均含有大流行群(PG)的分子标记toxRS与VPA1168,并具有相同的毒力基因构成(tlh+ tdhtrh-T3SS1+T3SS2-)与耐药谱,其tdh与trh的缺失并未影响细菌对凡纳滨对虾的致病力.综上所述,引起此次凡纳滨对虾红体病的10株副溶血弧菌可能为PG的不同变异株.     

凡纳滨对虾感染致急性肝胰腺坏死病副溶血弧菌(Vp_(AHPND))的定量分析

对虾急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease,AHPND)是由致AHPND副溶血弧菌(AHPND-causing Vibrio parahaemolyticus,VpAHPND)携带的pVA1-like质粒所表达的PirA~(Vp)和PirB~(Vp)毒力蛋白对对虾肝胰腺的急性毒性所致。本研究用2.19×10~5 CFU/ml VpAHPND分离株20130629002S01对凡纳滨对虾(Litopenaeus vannamei)进行浸泡感染,于感染后2~9 d采集对虾的肝胰腺、鳃、肠道、肌肉组织,采用实时荧光定量PCR方法,检测各组织中的pir AVp拷贝数。结果显示,感染后凡纳滨对虾各组织均能检测到pirA~(Vp),其中,肝胰腺在感染后第4天达到峰值,为8.71×10~4 copies/mg,而鳃、肌肉、肠道分别在第3、4、5天达到峰值,分别为9.08×10~3、2.59×10~4、5.76×10~4 copies/mg。早期感染鳃组织中先出现Vp_(AHPND)的富集,在高死亡发生期,Vp_(AHPND)数量在肝胰腺和肠道出现高峰,在死亡数量逐渐下降的后期,各组织的Vp_(AHPND)均快速下降,肠道、肝胰腺和肌肉中的Vp_(AHPND)水平趋于接近。对虾肝胰腺组织病理切片显示,同一时间有临床症状的病虾和濒死对虾相比,濒死对虾表现出更严重的AHPND病理特征,且二者的组织病理特征均随着感染时间的延长变得更为严重,但检测到的Vp_(AHPND)数量呈下降趋势。研究表明,在Vp_(AHPND)感染过程中,组织中的pirA~(Vp)基因数量不能代表对虾的发病程度,发病程度及组织病理严重的AHPND样品中Vp_(AHPND)的数量不一定处于高水平状态。     

10株副溶血弧菌多位点序列分型新序列型

2011年浙江某规模化养殖场大面积爆发凡纳滨对虾红体病。病虾未检出白斑病毒与桃拉病毒,在肝胰脏分离得到10株副溶血弧菌,对其进行基于dnaE-gyrB-recA-dtdS-pntA-pyrC-tnaA的多位点序列分型。这些菌株均为新序列型(ST)菌株,其中1株为ST413,7株为ST414,2株为ST415;ST413包含新等位基因型recA-166与tnaA-121,ST414则含有新等位基因型gyrB-219。新等位基因型与新序列型已被多位点序列分型数据库确认并收录。新序列型与已知序列型均只含有3个以下相同的等位基因。结果提示,与此次凡纳滨对虾红体病相关的副溶血弧菌分离株可能经历了较高水平的分子变异,呈现出显著的分子多样性,并非来自单一克隆。本研究代表由副溶血弧菌新序列型引起凡纳滨对虾红体病的首次报道。     

凡纳滨对虾急性肝胰腺坏死病(AHPND)病原分离鉴定及其致病性分析

本研究从患急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease,AHPND)的凡纳滨对虾(Litopenaeus vannamei)肝胰腺中分离到一株优势菌,编号为20160303005-1,通过16S rRNA和分子伴侣蛋白groEL基因序列分析,并结合生理生化特征,将该细菌鉴定为副溶血弧菌(Vibrio parahaemolyticus),其血清型为O1:KUT(K untypeable)。基因分析结果显示,该菌株携带可引起对虾AHPND的相关毒力蛋白基因pirA~(VP)和pirB~(VP),但不携带副溶血弧菌临床菌株毒力基因:耐热直接溶血毒素(Thenmostable direct hemolysin,tdh)和相对耐热直接溶血毒素(TDH-related hemolysin,trh)基因。菌株对凡纳滨对虾具有较强的致病性,浸泡感染的半数致死剂量(LD_(50))为7.96×10~3 CFU/ml。对虾急性感染后,6 h肝胰腺颜色变浅,肠胃变空;9 h肝胰腺呈浅白色,萎缩变小。9 h死亡数过半,24 h全部死亡。组织病理学分析显示,感染后对虾肝胰腺小管崩塌,上皮细胞严重脱落,呈现出典型的AHPND病理症状。药敏实验结果显示,该菌对庆大霉素、环丙沙星和头孢他啶等16种药物敏感,对阿莫西林、替卡西林和头孢噻吩等5种药物表现为耐药。上述研究可为该病原的流行病学及药物防控研究提供基本数据。     

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.     

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.     

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.     

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.     

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。     

Strain and dose infectivity of Vibrio parahaemolyticus: the causative agent of early mortality syndrome in shrimp

Diseases of shrimp have contributed to billions of dollars of economic loss in the aquaculture industry. Newly emerging strains of the bacterium Vibrio parahaemolyticus produce a condition in shrimp called early mortality syndrome or acute hepatopancreatic necrosis disease. Three different V. parahaemolyticus strains were evaluated for their respective pathogenicity on shrimp, Litopenaeus vannamei, when the bacterial strains were grown under various laboratory conditions prior to inoculating shrimp. For each trial, feed was inoculated with a known concentration of bacteria and then fed to the shrimp. The early mortality syndrome strain of V. parahaemolyticus was the most lethal resulting up to 100% mortality within 24 h after being introduced to shrimp via a single feeding. The other two strains of Vibrio, one isolated from the environment and the other from a human clinical case, resulted in 0% and 30% mortality within 96 h respectively. The concentration of the early mortality syndrome strain of V. parahaemolyticus that the shrimp were exposed to directly correlated with mortality rate, which allowed for lethal or sublethal short‐term disease challenge assays to be established. Infiltration of haemocytes was also evident in the midgut caeca of shrimp infected with the early mortality syndrome strain of V. parahaemolyticus, which has not been previously reported.     

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.     

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.     

Characterization and growth conditions of Vibrio parahaemolyticus strains with different virulence degrees that cause acute hepatopancreatic necrosis disease in Litopenaeus vannamei

The phenotypic characteristics and growth kinetics at several temperatures, salinities, and pH values of three Vibrio parahaemolyticus (Vp) strains with different virulence and one nonpathogenic strain were evaluated. Independent of the virulence of the strain, a high metabolic diversity was found, which yielded different colored phenotypes on the CHROMagar? Vibrio. All strains were resistant to ampicillin and carbenicillin, and Vp AHPND+ organisms were the most sensitive to enrofloxacin. The exponential growth of Vp strains started at 1–2 hr of incubation, although no relationship was observed between the bacterial density and degree of virulence. Moreover, the growth of the most virulent strain was independent of the nutrients in the incubation media during the initial hour postinoculation. No strain grew at 4°C in 0% NaCl and pH 4, but only Vp AHPND+ grew at 44°C. For all strains, the lag phase was proportional to the NaCl concentration, and the growth was better at pH 8–9. However, the Vp AHPND? strain displayed a greater variability, was more sensitive to extreme conditions, and showed a lag phase of 9 hr independent of the pH.