Impact of Feeding Status on the Efficacy of Depuration for Decontaminating Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas)

Vibrio parahaemolyticus causes acute human gastroenteritis and is often linked to consumption of raw oysters. Previous investigations indicated that refrigerated seawater depuration at 12.5°C could significantly reduce V. parahaemolyticus contamination in Pacific oysters; however, further optimization is necessary to achieve the regulatory target of >3.52 log most probable number (MPN)/g reduction. The current study investigated influences of algal feeding on efficacy of depuration to reduce V. parahaemolyticus in raw oysters. A V. parahaemolyticus cocktail (10290, 10292, 10293, BE 98–2029, 027-1c1) was mixed in artificial seawater (70 L) to inoculate oysters (n = 35) at 4–5 log MPN/g. Inoculated oysters were subjected to depuration with feed (algae = 0.036 ml/gram of oyster) and without feed at 12.5°C. Oysters (n = 5) were analyzed for V. parahaemolyticus using a three-tube MPN method after 0, 1, 3, 5, and 6 days of depuration. Depuration (6 days) achieved average V. parahaemolyticus reductions of 2.75 log MPN/g and 3.03 log MPN/g in the fed and unfed systems, respectively; however, feeding status did not significantly impact the efficacy of depuration to reduce V. parahaemolyticus in Pacific oysters. Further optimization of depuration is necessary to achieve the regulatory target for V. parahaemolyticus decontamination in raw oysters.     

Selection of lactic acid bacteria as candidate probiotics for Vibrio parahaemolyticus depuration in pacific oysters (Crassostrea gigas)

The application of probiotics in food is now widespread and is widely accepted by consumers. Lactic Acid Bacteria (LAB) were isolated from traditional salted fish and then characterized by its ability to inhibit Vibrio parahaemolyticus growth using minimal inhibitory concentration tests. Five out of these strains were identified as Lactobacillus delbrueckii, Lactobacillus casei and three Lactobacillus gasseri by PCR using 16S and 23S rRNA genes. Antibiofilm activity of Lactobacillus spp. extracts were also tested in 96 polystyrene plates. A potential antibiofilm effect was demonstrated as most LAB. Although most LAB extract were able to eradicate pre‐formed biofilm, results demonstrated that five Lactobacillus spp. exhibited a stronger inhibitory effect against V. parahaemolyticus in infected oysters. Vibrio parahaemolyticus viable cells number declined from 10⁶ UFC to 10⁴ UFC after 3 days of incubation with Lactobacillus spp. Probiotic applications, in biological control of seafood associated pathogens can be an alternative solution, providing consumer with a product of good quality owing to the use of non‐toxic compounds. Based on our results, LAB could be used as a bioprotective culture in oyster's depuration to prevent V. parahaemolyticus growth.     

一株兼具氨氮去除能力和对副溶血弧菌拮抗作用的有益菌的筛选及其初步应用

采用无机氮筛选培养基,从对虾养殖水体和对虾肠道中分离到3株具有脱氮效果的异养硝化菌株(编号分别为20131023A00、20131023A01和20131023A05),它们在氨氮含量为0.12％的液体筛选培养基上(pH=7.2),28℃经24 h对氨氮的转化能力分别为(38.9±0.1)％、(43.1±0.4)％和(49.9±0.5)％;采用纸片法拮抗实验选出菌株20131023A05对副溶血弧菌(Vibrio parahaemolyticus)具有拮抗作用,在1.57×105 CFU/cm2、1.57× 104 CFU/cm2和1.57× 103 CFU/cm2的副溶血弧菌平板上产生的抑菌圈直径分别为(9.14±0.05) mm、(11.57±0.03)mm和(13.59±0.02) mm.经16S rDNA序列测定,表明该菌株与杀鱼假交替单胞菌(Pseudoalteromonas piscicida)有最近的亲缘关系.在日本囊对虾(Marsupenaeusjaponicus)养殖水体中加入该株菌至2.5×105 CFU/ml时,该菌株表现出对副溶血弧菌注射感染的保护作用,该菌株浸浴组的日本囊对虾相对存活率(RPS)为35％.在凡纳滨对虾养殖水体中每3d加入该株细菌(3.13×104 CFU/ml),每天加入70％饲料量的赤砂糖,经60 d养殖,水体中氨氮含量比对照组及其他组显著降低.菌株20131023A05兼具去除氨氮和对副溶血弧菌拮抗作用,在对虾养殖生产中有广阔的应用前景.     

凡纳滨对虾感染致急性肝胰腺坏死病副溶血弧菌(VpAHPND)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PirAVp和PirBVp毒力蛋白对对虾肝胰腺的急性毒性所致。本研究用2.19×105 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pirAVp拷贝数。结果显示,感染后凡纳滨对虾各组织均能检测到pirAVp,其中,肝胰腺在感染后第4天达到峰值, 为8.71×104 copies/mg,而鳃、肌肉、肠道分别在第3、4、5天达到峰值,分别为9.08×103、2.59×104、5.76×104 copies/mg。早期感染鳃组织中先出现VpAHPND的富集,在高死亡发生期,VpAHPND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VpAHPND均快速下降,肠道、肝胰腺和肌肉中的VpAHPND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VpAHPND数量呈下降趋势。研究表明,在VpAHPND感染过程中,组织中的pirAVp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VpAHPND的数量不一定处于高水平状态。     

双重PCR检测携带有t1和tdh基因的副溶血弧菌毒力菌株

在常规PCR技术的基础上优化条件 ,建立并完善双重PCR技术 ,并通过检测阳性对照和待检样品 ,进一步确定其检测的可行性。结果表明在常规PCR方法中为阳性的样品 ,包括阳性对照以及待检样品 ,在双重PCR方法中也呈现阳性 ,为阴性的样品在本方法中则也呈现阴性 ;能在血平板中出现溶血圈的在本法中也被印证含有tdh基因。由此证实本方法确实能对tl和tdh两种基因同时进行检测 ,成功地证明了同时检测tl和tdh两种基因的双重PCR方法的可行性。该法不但具有常规PCR的优点 ,而且还能节省耗材和时间 ,可用于检测水产食品以及临床样品中的副溶血弧菌的毒力菌株     

Effects of Ozonated Water Treatment on the Microbial Population,Quality, and Shelf Life of Shucked Oysters (Crassostrea plicatula)

An ozonated water treatment system was designed for the sanitation of shucked oysters (Crassostrea plicatula). The effects of ozone (O₃) on shelf life were examined under refrigerated conditions (4 ± 1°C). Microbiological, chemical, and color quality were also evaluated by determining total aerobic plate counts (APC), total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA), and L, a, and b values. Single and orthogonal experiment results showed that oysters treated with 9 mg/L of O₃ in aqueous solution (oyster: ozonated water = 1:6, w/v) for 10 min at 5°C significantly reduced the total APC by about 2-log—from 4.97 ± 0.11 to 2.93 ± 0.17 log CFU/g (p < 0.05). O₃ treatment did not cause significant changes in TVB-N, TBA, a, and b values, although L values were significantly affected. During refrigerated storage, O₃-treated samples expressed lower TVB-N, TBA, a, and b values and higher L values than control samples. The shelf life of O₃-treated samples was 20 to 25 days, while that for the controls was 5 to 10 days. The current study confirms that treatment with ozonated water can effectively prolong the shelf life and maintain the quality of oysters.     

云芝多糖和副溶血弧菌灭活苗对斜带石斑鱼免疫功能的影响

采用注射云芝多糖和副溶血弧菌灭活苗的方法免疫斜带石斑鱼后,测定了受免鱼的吞噬细胞的吞噬活性、杀菌功能和免疫保护率。结果表明,云芝多糖组在第3天,吞噬细胞的吞噬百分比(PP)达到峰值(90%),显著高于对照组(P0.05),然后开始下降,到第7天即与对照组相当(P0.05);30 m in和60 m in的杀菌率显著提高(P0.05)。灭活苗组和云芝多糖+灭活苗组的PP值和杀菌率显著高于对照组(P0.05),并维持在较高水平,但2组之间差异不显著(P0.05);用副溶血弧菌攻毒后,斜带石斑鱼的免疫保护率达87.6%。由此可见,灭活苗不仅能提高斜带石斑鱼的特异性免疫力,而且也能增强吞噬细胞的吞噬和杀菌活性等非特异性免疫力。     

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.     

Volatile Organic Compounds Profile Obtained from Processing Steps of Pacific Oysters (Crassostrea gigas) as Perspective for Food Industry

ABSTRACT

In the heat treatment of oysters, many aroma compounds are lost in the cooking steam and water. In this study, aroma compound profiles from oyster processing were identified by gas chromatography- mass spectrometry (GC-MS). The compounds were extracted by headspace-solid phase microextraction (HS-SPME), while the best process conditions such as temperature, extraction time, and desorption time were studied. The aroma compounds were obtained from raw oysters, from oysters after thermal treatment, from water released from raw oysters, from cooking steam, and from oyster boiling juice. All compounds were identified and semi-quantified by GC-MS. The major oyster aroma compounds were 1-octen-3-ol, hexanal, and benzaldehyde. Most of the components from oysters were lost both to the cooking steam and to the oyster boiling juice. The concentration range of volatile organic compounds transferred for oyster boiling juice between 14 and 90 mg L^?1 shows that the residues from oyster processing have great technological potential, and they can be recovered for application as aromas by the food industry.     

Characterization and pathogenicity studies of Vibrio parahaemolyticus isolated from diseased freshwater prawn, Macrobrachium rosenbergii (de Man)

Vibrio parahaemolyticus, an important pathogen of freshwater prawn, Macrobrachium rosenbergii causes high mortality. Four strains of V. parahaemolyticus were isolated from the gill, haepatopancreas, hemolymph and carapace of diseased prawn. The species were characterized based on biochemical and serological tests. On performing an antibiotic assay, these strains were found to be highly susceptible to cephalaxin, tetracycline and erythomycin. During the in vitro pathogenicity test, all the strains were found to be positive to the Congo red binding assay and to be haemolytic in nature, whereas an in vivo pathogenicity test revealed that 2 × 10⁹ CFUs mL^?1of bacteria induced disease symptoms such as black colouration on the carapace, red discolouration of the exoskeleton and loss of appendages within 6 days and 80% mortality. In histopathological studies, a prominent necrosis was seen in gill lamellae, and branchial arches were thickened at places due to hyperplasia and haemocytic infiltration. Hepatopancreatic tissue showed dilation of tubules, vacuolation of hepatocytes and marked necrosis in acinar cells. Development of an immunological technique for the detection and screening of V. parahaemolyticus infection and its treatment is highly important in freshwater prawn aquaculture.     

Dynamics of Bacterial Communities in Postharvested Eastern Oyster (Crassostrea virginica) Maintained Under Refrigeration

The selective effect that refrigeration exerts on the autochthonous microbiota of the eastern oyster was investigated using culture-independent methods. Oysters from two different locations along the Gulf Coast of Mexico were analyzed concurrently during 2-week refrigeration. Ribosomal intergenic sequence analysis (RISA) generated complex bacterial community fingerprints that provided a strong association between RISA-defined clusters and sample date. Denaturing gradient gel electrophoresis (DGGE) profiles obtained with universal primers did not reflect any association based on date or geographic origin of the samples. During the storage period, Vibrio parahaemolyticus was replaced, at least in overall abundance, with other nonpathogenic Vibrio species better adapted to cold temperatures. This study shows that refrigeration itself does not limit the growth of the bacterial community nor reduces its richness.     

对虾肝胰腺坏死病副溶血弧菌双重微流控荧光定量PCR快速检测技术的建立与应用

急性肝胰腺坏死病(AHPND)是对虾养殖过程中最常见、最严重的疾病,给对虾养殖造成重大经济损失。AHPND病原种类多、基因型复杂,现有的针对不同病原的检测技术目标导向较弱、检测成本高、时间消耗长,对虾健康养殖亟待开发AHPND的精准快速诊断技术。本研究针对AHPND病原体携带编码一种二元毒素pirA和pirB大型质粒的遗传共性,基于pirA和pirB基因设计特异性引物并建立微流控荧光定量PCR检测方法。该方法对致病基因pirA和pirB特异性强、灵敏度高,最低检测限分别为5.43×10⁰和4.31×10¹ copies/μL,样品平均检测时间为26min左右。为进一步评估该方法在实际应用中的准确性,以含有pirA和pirB毒性质粒的副溶血弧菌(Vibrio parahaemolyticus)进行人工感染实验。结果表明,感染后的凡纳滨对虾(Litopenaeus vannamei)鳃丝、肝胰腺、肠道和肌肉等组织随时间的推迟均能检测到pirA和pirB;从感染2h的结果来看,pirB比pirA检出率更高。此外,致病因子pirA和pirB比toxR...     

Impact of a native predatory whelk on cultivated oysters (Crassostrea gigas) crops in San Quintin Bay, Mexico

San Quintin Bay (Baja, California, Mexico) is extensively utilized by artisanal farmers to cultivate Pacific oysters ( Crassostrea gigas ) using a rope/rack system. A large juvenile oyster mortality event was significantly correlated with the presence of native predatory whelks, Macron trochlea . Surveys, field and lab experiments were performed to identify the predatory behaviour of the whelk, determine factors affecting oyster mortality, and quantify the impact on oyster farmers. Macron trochlea was found to be a voracious, active predator, which can consume ∼4 juvenile oysters day⁻¹. Juvenile oysters suffer significantly higher mortality in the presence of whelks. Macron trochlea has the largest impact on small oysters. Once oysters grow to >30 mm they reach a size refuge above which there is much lower mortality. At average growth rates, an oyster can escape predation after ∼3 months. But, in areas of highest whelk densities, at average predation rates, whelks could consume the standing crop of juvenile oysters in approximately 43 days. For the artisanal oyster farmers of San Quintin this represents a substantial economic loss, which was unaccounted for previously. Farmers can reduce loss to whelk predation by seeding juvenile oysters in low whelk density areas until oysters reach the size refuge.     

Total Mercury Bioaccumulation in Tissues of Carnivorous Fish (Micropogonias furnieri and Cynoscion acoupa) and Oysters (Crassostrea brasiliana) from Sepetiba Bay,Brazil

Total mercury (Hg) levels were determined by cold vapor atomic absorption spectrophotometry (CVAAS) in tissues of two species of carnivorous fishes and in the soft tissue of oysters caught in Sepetiba Bay, Brazil. Hg was distributed unevenly in the organs and tissues of the fish; the mean Hg content was highest in the kidneys (0.048 μg/g wet weight) and lowest in the gills (0.004 μg/g w.w.). Mean Hg levels were higher in the oysters (0.052 μg/g w.w.) than in the fish (0.022 μg/g w.w). The total Hg concentration did not exceed the maximum concentration for food allowed by Brazilian legislation in any of the fish and oyster samples analyzed. Moreover, the Hg concentrations observed were all within the maximum level for fish recommended by the Food and Agriculture Organization (FAO; 0.5 μg/g w.w.).     

The effect of temperature on physiological energetics of a fast‐growing selective strain and a hatchery population of the Pacific oyster (Crassostrea gigas)

Growth is an important aquaculture trait, which has been one of the most important traits for genetic improvement in aquatic species. Numerous fast‐growing domesticated aquaculture organisms are being cultured, but the physiological processes underlying growth remain largely unexplored. The Pacific oyster (Crassostrea gigas) is one of the most widely farmed bivalves. In order to improve the productivity traits, a fast‐growing selective strain of C. gigas was established. In this study, we compared physiological energetics of the fast‐growing selective strain and an unselected hatchery population of C. gigas under different conditions of temperature. Components of energy gain (clearance rates, ingestion rates, absorption efficiency and absorption rates), energy loss (metabolic rates) and resulting energy budget (scope for growth) were analysed at four temperatures (15, 20, 25 and 30°C). The results indicated that selective breeding oysters had a higher energy gain compared to unselected oysters, but the selected strain and hatchery population of oysters did not significantly differ in metabolic rates at all tested temperatures (p > 0.05). The enhanced energy gain resulted in almost two times higher scope for growth in selective breeding oysters compared with unselected oysters. Physiological characteristics of C. gigas were greatly affected by temperature and showed a fluctuation with the increase in temperatures. The selective breeding oysters outperformed those of hatchery population in growth performance by an increased energy gain along with a similar energy loss. This study provided insight into physiological energetics accounting for growth improvements in fast‐growing selective strain of C. gigas.     

培育密度对长牡蛎面盘幼虫生长影响的对比试验

长牡蛎育苗期间经常出现面盘幼虫生长参差不齐现象,影响单位水体出苗量。笔者在正常培养密度范围内6～12个 ml作对比试验:低密度6～8个 ml培养的幼虫生长速度快,个体差异小;重视亲贝促熟,加强优质新鲜饵料的供给,有良好的水环境,能够减少幼虫差异显著的现象。     

干露胁迫对长牡蛎基因组DNA甲基化的影响

为探讨干露胁迫对海产贝类基因组DNA甲基化的影响,应用荧光标记甲基化敏感扩增多态性(fluorescencelabeled methylation sensitive amplified polymorphism,F-MSAP)技术,比较了不同干露条件下(0 d、0.5 d、1 d、3 d、5 d、7 d、9 d和11 d)长牡蛎(Crassostrea gigas)基因组DNA甲基化的变化。结果表明,对照组(干露处理0 d)闭壳肌与鳃组织的总体甲基化水平分别为29.76%和29.82%;干露处理0.5 d、1 d、3 d、5 d、7 d、9 d和11 d的长牡蛎全基因组甲基化水平呈现先增高后降低的趋势,其中,闭壳肌组织的总体甲基化水平分别为36.59%、38.86%、43.02%、39.30%、51.13%、46.79%和35.06%,鳃组织总体甲基化水平分别为39.39%、42.13%、39.36%、43.54%、56.19%、38.57%和28.99%;干露处理7 d的长牡蛎甲基化水平明显高于其他时期(P0.05),11 d时甲基化水平基本恢复至初始状态。甲基化变异模式分析发现,闭壳肌与鳃组织DNA甲基化变异位点存在差异,甲基化升高位点变化程度较大(P0.05)。以上结果表明,长牡蛎通过改变DNA甲基化模式来应答干露胁迫,发生了不同程度的甲基化与去甲基化反应,DNA甲基化与长牡蛎的抗逆性状密切相关。