Immune Responses and Resistance against Vibrio parahaemolyticus Induced by Probiotic Bacterium Arthrobacter XE-7 in Pacific White Shrimp, Litopenaeus vannamei

A 63-d feeding experiment was conducted to determine the effects of dietary supplementation of probiotic bacterium Arthrobacter XE-7 on immune responses and resistance against Vibrio parahaemolyticus in the Pacific white shrimp, Litopenaeus vannamei . The probiotic bacteria were administered orally at four different doses of 0, 10⁶, 10⁸, and 10¹⁰ colony-forming unit (CFU)/g feed for shrimp. On Day 50, the shrimp were challenged with Vibrio parahaemolyticus by bath. On Days 7, 21, 49, and 63, six shrimp per tank were sampled to take intestine and hemolymph. With increasing dietary supplementation of probiotic bacteria, shrimp mortality decreased from 63.16% (the control) to 55.9% (10⁶ CFU/g feed), 51.75% (10⁸ CFU/g feed), and 51.78% (10¹⁰ CFU/g feed), respectively. Vibrio counts in intestine of shrimp fed probiotic bacterium Arthrobacter XE-7 was generally lower than that in the control shrimp ( P  < 0.05). The probiotic bacteria generally increased the immune parameters in shrimp, that is, total hemocyte counts, percentage phagocytosis, respiratory burst activity, and serum phenoloxidase activity. The results showed that probiotic bacterium Arthrobacter XE-7 can be used as probiotic in shrimp feed.     

Characterization of exotoxin produced by a shellfish-pathogenic Vibrio sp.

Abstract. Broth filtrate of a Vibrio species, isolated from spontaneously occurring epizootics among cultured oyster larvae, Crassostrea virginica (Gmelin), was shown to contain an exotoxin. Bioassays demonstrated that LC₅₀ value was less than 46.6 μg of toxin/l culture of oyster embryos. This quantity was produced by 2.9 × 10⁹ colony-forming units. Purified toxin showed neither proteolytic nor amylase activity; it did, however, demonstrate bacteriostatic capability. Studies showed that the toxin was heat-labile and that exposure to heat-produced toxoid had a beneficial effect on oyster embryonic development. Data revealed that although toxin was inactivated by heating, the bacteriostatic capability of the metabolite was not lost. Molecular weight of the toxin was estimated to be 68000.     

Two pathogens of Greenshell™ mussel larvae, Perna canaliculus: Vibrio splendidus and a V. coralliilyticus/neptunius-like isolate

Bacterial pathogens of Greenshell™ mussel (GSM) larvae can cause batch losses during hatchery production. Twenty-two isolates were screened using a larval bioassay. Two strains were identified as potential pathogens. Phenotypic identification of these strains revealed two non-reactive Gram-negative, oxidase positive rods. Sequencing of the 16S rRNA gene identified Vibrio splendidus and a V. coralliilyticus/neptunius -like isolate as pathogens of GSM larvae, with an ability to cause 83% and 75% larval mortality in vitro , respectively, at a concentration of 10² CFU mL⁻¹. Histopathology indicated that the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. This is the first report on pathogens of GSM larvae.     

Haemorrhagic areas in the mouth of farmed turbot, Scophthalmus maximus (L.)

Abstract. An epizootic in juvenile turbol reared on a farm located in The Ria de Vigo, Galicia, Northwest Spain, is described. The diseased turbot did not display unusual swimming behavior; the external signs of the disease were abdominal distension and haemorrbagic areas in the mouth. Internal examination of the fist showed an accumulation of reddish fluid in the peritoneal cavity. Microbiological analysis of the diseased fish revealed the presence, in pure culture in all the organs and lesions examined, of a bacterium which was characterized biochemically as Vibrio splendidus biotype I. The virulence tests showed that the V. splendidus biotype I isolate was pathogenic for rainbow trout (LD₅₀: 2.2 × 10⁴) and also for turbot (LD₅₀: 1.2 × 10⁴). The treatment of the fish using flumequine incorporated into the feed was effective in offsetting the mortality rate.     

Identification of bacteria from the normal flora of perch, Perca fluviatilis L., and evaluation of their inhibitory potential towards Aeromonas species

Pathogenic Aeromonas sobria has been identified as a causative agent of ulcerative disease in farmed European perch, Perca fluviatilis L. To study the effect of the normal intestinal bacterial flora of perch against A. sobria, we sampled 193 bacterial isolates from the perch digestive tract. The isolates were identified by sequence analysis of the 16S rRNA gene and their inhibitory potential against A. sobria was evaluated in vitro. Nineteen of the strains isolated showed inhibition and were also tested against other aeromonad and non-aeromonad fish pathogens including Yersinia ruckeri and Vibrio anguillarum. Isolates showing inhibition were primarily Pseudomonas spp.; however, inhibitory Shewanella spp., and Delftia sp. were also identified. A Pseudomonas chlororaphis isolate showed inhibition against all fish pathogens tested.     

Phenotypic analysis in the Chilean oyster, Ostrea chilensis Philippi 1845: relationship between juvenile and adult growth rates

Abstract. The Chilean oyster, Ostrea chilensis Philippi, shows high phenotypic variability for growth rate within populations. In order to determine if the serial correlation of growth rate in oysters is positive, the correlation between shell height and live weight at different stages of the life cycle in a cohort of the Chilean oyster was estimated. Four size groups of individually tagged oysters selected on the basis of their shell heights were examined. The relationships for juvenile and adult oysters were found to be positively correlated throughout the study period. The correlation coefficients ranged from r = 0.28 to r = 0.83. For shell height the coefficient of determination for market size oysters was higher in 2-year-old (r²=0.88) than in 1-year-old oysters (r²=0.09). Shell height and live weight were more highly correlated at later stages of the life cycle. The results indicate that 2-year-old oysters have a size that is indicative of performance to market size and hence is an appropriate age to select the parents to be used in breeding programmes to improve the growth rate in O. chilensis . A high mortality was detected after the natural spawning period. A physiological stress hypothesis is proposed to explain the high summer mortality in O. chilensis in the Quempillen estuary, southern Chile.     

A Parasitological Survey of Oysters Along Salinity Gradients in Coastal Louisiana

Oysters were collected along salinity gradients within four major oyster producing watersheds in Louisiana (Lake Calcasieu, Terrebonne Bay, Barataria Bay and Lake Borgne) and examined for parasites in relation to oyster condition and reproductive state. The percentage of Infection by Perkinsus marinus, Nematopsis prytherchi, N. ostrearum, Bucephalus cuculus , prokaryotic inclusion bodies, and ciliates was recorded for each site. Average pigment cell number, oyster condition, gonadal index, and the percentage of oysters with atrophic digestive diverticula were also determined.
In general, oysters experienced higher levels of parasitism in higher salinity waters. Perkinsus marinus Occurrence was correlated with salinity, an atrophied condition of the oysters, and Nematopsis spp. occurrence. Oysters in poor condition had higher concentrations of pigment cells, greater atrophy of the digestive diverticula, and increased parasitism by N. ostrearum .     

Growth,morphometrics and nutrient content of farmed eastern oysters,Crassostrea virginica (Gmelin), in New Hampshire,USA

When harvested, oysters represent a removal from the ecosystem of nutrients such as nitrogen (N) and carbon (C). A number of factors potentially affect nutrient content, but a quantitative understanding across the geographical range of the eastern oysters is lacking. This study was designed to quantify the relationships among various metrics of farmed eastern oysters near its northern geographical range focusing on nutrient content. Hatchery‐reared oysters were deployed in polyethylene bags at six sites, and were measured on multiple occasions from 2010 to 2012. A quadratic polynomial fit to the combined datasets for shell height indicated that on average a ‘cocktail’ size oyster (63 mm shell height) would be reached after 2 year, and ‘regular’ size (76 mm) would require 3 year. There were significant differences in growth rates and oyster nutrient content among the sites; means for %N in soft tissue ranged from 6.9 to 8.6, and 0.07 to 0.18 in shell. Per cent N in soft tissue and shell were highest at two sites at the mouths of rivers with elevated dissolved inorganic N concentrations in the water. Grand means (all sites, seasons and years combined) of soft tissue N and C for regular size oysters were 7.3% and 38.5%, respectively; and for shell N and C were 0.13% and 12.0% respectively. Our study extends the range of data on nutrient content of the eastern oyster to northern New England, and indicates that oyster size, seasonality, and nutrient concentration in ambient water potentially affect %N and %C content of oysters.     

Characterization of Vibrio spp. Associated with Diseased Shrimp from Culture Ponds of Andhra Pradesh (India)

Abstract.— Surveys undertaken on diseases caused by Vibrio spp. in Penaeus monodon from culture ponds of coastal Andhra Pradesh recorded the occurrence of five types of diseases: tail necrosis, shell disease, red disease, loose shell syndrome (LSS), and white gut disease (WGD). Among these, LSS, WGD, and red disease caused mass mortalities in shrimp culture ponds. Six species of Vibrio—V. harveyi , V. parahaemolyticus, V. alginolyticus, V. anguillarum , V. vulnificus , and V. splendidus —are associated with the diseased shrimp. The number of Vibrio spp. associated with each disease ranged from two to five. Additionally, shrimp with red disease had concurrent infections with white spot syndrome virus. Vibrio harveyi in the case of LSS and WGD, V. parahaemolyticus for red disease, and V. alginolyticus for shell disease are the major etiologcal agents. Differences occur in the degree of virulence of different species of Vibrio and also different isolates of the same species. Vibrio harveyi isolated from LSS shrimp is the most virulent. In general, all the Vibrio isolates from LSS shrimp tend to be more virulent as compared to their counterparts from other diseased shrimp. It is apparent that the degree of virulence of various Vibrio isolates depends on its source and the pond environmental conditions. Most of the Vibrio isolates showed susceptibility to oxytetracycline, norfloxacin, and ciprofloxacin. The luminous V. harveyi exhibited resistance to many antibiotics and susceptibility to only three drugs. Considering the emergence of antimicrobial resistant strains of Vibrio , the need for using probiotics in place of antibiotics for disease control is stressed.     

近江牡蛎HSC70基因对溶藻弧菌感染的反应

人工注射溶藻弧菌(Vibrio alginolyticus)感染近江牡蛎(Crassostrea hongkongensis),通过实时荧光定量RT-PCR方法检测了近江牡蛎6种不同组织器官(外套膜、鳃、消化腺、闭壳肌、心脏和血细胞)中HSC70基因表达量的变化;同时采用平板活菌计数法测定了近江牡蛎不同组织器官中溶藻弧菌菌量的变化。结果显示,人工注射感染溶藻弧菌后,近江牡蛎6种组织器官中HSC70基因表达量均显著性升高(P<0.05),且随时间变化均呈现出先升高后恢复至对照水平的趋势;其中鳃组织中HSC70基因分别在第6小时和第72小时出现2次显著性高表达,且在第72小时的表达量高于第6小时(P<0.05)。在多数组织器官中,HSC70基因出现显著高表达后便急剧下降至对照水平,而在消化腺中高表达持续的时间长达18 h(第6 24小时)。在注射感染溶藻弧菌后第6小时,血细胞中HSC70基因的表达量达到峰值,接近于对照水平的15倍;而在其他组织器官中,该基因表达峰值仅为对照水平的2.5倍左右。人工注射感染溶藻弧菌后3 h,3种组织(消化腺、闭壳肌和血淋巴)中均能检测到溶藻弧菌的积累,其中闭壳肌中含菌量最高,6 h时闭壳肌中含菌量急剧下降,随后又增加并维持在一定水平直至72 h;消化腺中菌量随着时间推移而增加,到达峰值后又逐步下降;血淋巴中,溶藻弧菌含量整体上随时间不断增加,到48 h稳定在较高水平。各组织中HSC70表达量与溶藻弧菌含量之间存在一定的关联性,尤其在溶藻弧菌攻击的靶器官(消化腺)中最为明显。由此可见,病原菌感染可以诱导近江牡蛎HSC70基因高表达,并且HSC70可能参与了机体抗病原菌感染的相关作用,这为进一步研究近江牡蛎HSC70基因在抗病免疫中的作用机制奠定了基础。     

Production of Pacific oysters, Crassostrea gigas Thunberg, from wild-caught and hatchery-produced seed grown at several densities on oyster shells

Abstract. Production of Pacific oysters was studied under pilot-scale conditions in Baynes Sound, British Columbia, using common bottom culture strategies. Four seeding treatments, each with a different seed per cultch density were cultured: wild-caught seed 5mm in shell height at 10 seed per cultch piece and hatchery produced seed 1-2 mm in shell height at densities of 11, 40 and 105 per cultch piece. The cultch material for all treatments was Pacific oyster shell. All seed was reared for approximately 1 year in a seed nursery located at the 2.2-m tidal level then transferred to a 1-m tidal level grow-out plot until harvest 4 years later in May 1988, Clusters of large numbers of oysters were separated and evenly distributed within the plots when the oysters attained a shell height of 60-100 mm. During the first year, growth was slow and mortalities were relatively high. All treatments produced oysters of similar size at harvest. The proportion surviving at harvest was substantially higher for the wild oysters which were initially larger at time of planting. Within the hatchery treatments proportional survival per cultch piece was inversely related to initial density on the shell; however, total production per cultch piece was positively related to initial density. Most efficient use of seed is attained at lower densities per shell; however, most efficient use of cultch and effort to handle cultch is attained at high densities.     

Toxicity to marine bivalves of culture supernatant fluids of the bivalve-pathogenic Vibrio strain NCMB 1338 and other marine vibrios

Abstract. Culture supernatant fluids from twelve Vibrio strains were toxic to spat of the European and Pacific oysters, Ostrea edulis L., and Crassostrea gigas L. At least three factors were present in culture supernates of one of the most virulent strains (NCMB 1338). The first was a heat-labile protein of molecular weight approximately 39000 which was lethal to oyster spat. This toxin was released during the stationary phase of culture and 72-h culture supernates contained 23·0LC₅₀ml when assayed against 2–3 mm Crassostrea gigas spat for 24 h at 20°C. Under the same assay conditions, 7-mm C. gigas spat were six times less sensitive to the toxin. The second factor was a relatively heat-stable ciliostatic toxin of molecular weight < 5000 and the third a proteinase of molecular weight 30000 which degraded gill segments of Mytilus edulis L. In addition, a haemolysin of molecular weight 20000 was produced. All these factors could be involved in the pathogenesis of 'vibriosis' in oyster larvae.     

Vibrio bacteria isolated from black tiger shrimp, Penaeus monodon Fabricius

Abstract. Two hundred and five isolates of Vibrio bacteria were obtained from diseased black tiger shrimp, Penaeus monodon Fabricius, cultured in Thailand during the period from February 1988 to January 1990. Ninety-six isolates of the bacteria were identified as Vibrio parahaemolyticus. The others included 74 isolates of V. vulnificus , 18 isolates of V. alginolyticus , six isolates of V. fluvialis and 11 isolates of Vibrio spp. Comparisons of some characteristics of the bacteria studied are given.     

Isolation and use of beneficial microbiota from the digestive tract of lions‐paw scallop Nodipecten subnodosus and winged pearl oyster Pteria sterna in oyster aquaculture

We isolated microbiota from the digestive tract of Nodipecten subnodosus and Pteria sterna and determined in vitro their haemolytic activity, antagonism against Vibrio spp., bacterial hydrophobicity, production of extracellular enzymes and molecular identification. Five bacterial strains were selected: RL5 and C3 (Lactobacillus spp.) and PB1‐1, PB1‐5 and PB1‐6 (Bacillus spp.). The RL5 and C3 isolates showed antimicrobial activity against Vibrio spp. and the PB1‐1, PB1‐5 and PB1‐6 isolates showed enzymatic activity for amylase, protease, lipase and cellulose; the C3 and PB1‐5 isolates were highly hydrophobic. The selected strains of bacteria were tested in vivo as probiotics, together with a treatment of ampicillin and a control without bacteria on juvenile Kumamoto oysters Crassostrea sikamea. The strains were provided individually and as mixes of isolates. Survival, growth and biochemical composition of the juveniles were determined as in vivo indicators. Juveniles grew significantly larger and faster when treated with a specific mix of isolates (MIX‐B), compared with the control. The protein, lipid and carbohydrate concentrations were also significantly higher in oysters exposed to probiotic treatments, compared with the control and the antibiotic treatment. The selected microbiota showed probiotic proprieties for cultivating C. sikamea juveniles.     

A habitat suitability index model for suspended tray culture of the Pacific oyster, Crassostrea gigas Thunberg

Abstract. A habitat suitability index (HSI) model for suspended tray culture of the Pacific oyster, Crassostrea gigas Thunberg, was constructed from existing information in the literature on oyster-environment relationships. In the model, biophysical data are used to rate aquaculture potential of coastal areas on a scale from 0.0 to 1·0. where 1·0 represents optimal conditions for growth and survival and 0·0 represents totally unsuitable habitat conditions. The model was tested with environmental, growth (increase in shell height) and secondary production (g· 100 oysters⁻¹ day⁻¹) data collected over a 14-month period for two age-classes of oysters transplanted to 10 locations along the coast of British Columbia. Canada. Regressions of HSI values calculated from entire study environmental data against oyster growth and secondary production were highly significant for both age-classes. Environmental data subsets representing short sampling programmers (1–3 months) resulted in HSI values significantly correlated to oyster growth, provided that some sampling occurred during periods of high food availability. HSI modeling techniques have potential application in site selection and coastal management of aquaculture.     

The Effects of Stocking Density,Bag Mesh Size,and Bottom Sediment on the Growth and Survival of the Eastern Oyster,Crassostrea virginica,with Emphasis on Controlling Oyster Spat Fouling

This study was designed to determine if oyster spat fouling upon adult oysters, Crassostrea virginica, cultured in coastal Georgia could be controlled by stocking density, bag mesh size, substrate type, and tidal level (intertidally or subtidally). Oyster gowth and survival was also monitored for each treatment. Sediment type had no effect on the number of spat per oyster either in intertidal or subtidal areas. Greater growth occurred in subtidal bags placed on sandmud and sandclay substrates. Intertidal mud and sandhnud bottom treatments showed the least growth. Survival of oysters grown intertidally (64%) was better the those cultured subtidally (27%). Mesh size of the oyster growing bags -had no effect upon oysler fouling or growth (shell length) and survival of the oysters they contained. Stocking density did affect oyster fouling, with lower fouling per oyster in higher density treatments. Density had no effect on oyster growth (shell length) or survival at the levels tested. Heavy fouling occurred on all oysters in the mesh and stocking density experiments while they were suspended off-bottom, but an I l -fold decrease in fouling occurred after bags were placed on the sublidal river bottom.     

牡蛎养殖过程中副溶血弧菌与水质因子间的关系

为了研究牡蛎养殖过程中副溶血弧菌与水质因子之间的关系,2009年3月到11月从广东阳西某牡蛎养殖场采集牡蛎样品检测其副溶血弧菌的感染情况,并检测水体温度、盐度、pH和溶解氧。实验结果表明:副溶血弧菌总量的检出率为94.38%。水温和副溶血弧菌总量呈正相关,盐度和副溶血弧菌总量呈负相关,pH和溶解氧与副溶血弧菌总量的相关性不明显。对水温和盐度与副溶血弧菌总量的对数进行回归分析,水温和副溶血弧菌浓度的拟合回归方程为LgVP=0.093T-0.685(R2=0.336);盐度和副溶血弧菌浓度的拟合回归方程为LgVP=1.199+0.116S-0.004S2(R2=0.217);水温和盐度对副溶血弧菌浓度的拟合回归方程为LgVP=-1.941+0.116T+0.058S-0.001S2(R2=0.414)。研究结果可为牡蛎养殖过程中副溶血弧菌的风险分析提供参考依据。     

Seasonal Fouling Stress on the Farmed Pearl Oyster,Pinctada fucata,from Southeastern Arabian Sea

Biofouling on the periostracum of pearl oysters and on the cages has been considered as a stress factor causing mortality of the farmed stock, reducing growth rates and also affecting pearl quality. In farming experiments using the pearl oyster, Pinctada fucata, at Kollam Bay (India), biofouling was found to be a problem. This study was conducted to understand the effect of fouling on the mortality of pearl oysters kept in suspended culture, to identify the main foulers, the seasonal variation in biofouling and species successions in the community with reference to abiotic factors. The average monthly mortality rate (MR) was estimated as 0.117 ± 0.002 and the monthly variations were significantly different (P < 0.01). The total fouling (0.163 ± 0.002 g/g oyster) and biofouling weights (0.166 ± 0.007 g/g oyster) were high in December when the fouling community was composed of several species and dominated by the ascidian, Didemnum sp. December was also the period when the MRs peaked indicating that this organism was the main cause of mortality in pearl farms in Kollam Bay. A clear seasonality in the fouling community (25 species belonging to nine phyla) has been observed in the present study. On the basis of this study, monthly cleaning of oysters is advocated except during December, January, and March when the cleaning should be fortnightly.     

河口水体中的菌群分析及其脱氮能力的研究

从天津渤海湾三个河口六个站位采集水样,用Zobell2216E培养基分离共得到92株异养细菌,其中G+52株,G-40株。对其形态和生理生化性状进行分析,初步研究结果表明,他们主要分属葡萄球菌、微球菌、不动杆菌、莫拉氏菌、芽孢杆菌、弧菌、产碱杆菌、肠杆菌、假单胞菌。选择假单胞菌属、葡萄球菌属、产碱杆菌属、芽孢杆菌属,研究其在含氮量较高的所采河口水样中(无机氮含量>0.3 mg/L)的脱氮能力,于0、3、6、9、12、15 h不同时间测定水样中的亚硝酸盐氮、硝酸盐氮、氨氮含量。实验结果表明经过9 h的培养测定,水样中的无机氮含量明显降低,但9 h以后除假单胞菌的水样无机氮含量继续降低之外,其他三个菌种水样无机氮含量开始上升。确定四个菌种的最佳脱氮时间为9 h,且芽孢杆菌的脱氮能力最强。     

长牡蛎MITF基因表达及与壳色的关联

为了解长牡蛎MITF基因的表达及其与壳色的关联,验证了长牡蛎中的4个MITF基因,对长牡蛎MITF氨基酸序列进行了序列分析和多序列比对,分析了长牡蛎幼体发育各时期的转录组,采用荧光定量PCR的方法研究长牡蛎各组织及黑壳、白壳牡蛎特定部位mRNA表达情况。4个MITF基因中有3个基因可能为假基因,有表达的长牡蛎MITF基因共编码448个氨基酸,为亲水性不稳定蛋白,含有N端结构域(MITF_TFEB_C3N superfamily)和高度保守的功能性结构域HLH结构域。转录组分析发现MITF在长牡蛎个体发育的各个时期均有表达,在稚贝期达到最高。组织表达结果显示MITF在外套膜中的表达水平显著高于其他组织。MITF在黑壳牡蛎闭壳肌中的表达量显著低于白壳牡蛎,而在黑壳牡蛎外套膜中的表达量高于白壳牡蛎,但不显著。在黑壳、白壳牡蛎外套膜边缘的表达量都显著高于内侧。研究表明,长牡蛎MITF基因可能在牡蛎壳形成早期就参与了黑色素的生成调控和个体的生长发育,可调控牡蛎酪氨酸酶Tyr2基因,参与外套膜和贝壳中黑色素的形成。...