Vibrio species isolated from diseased farmed sole,Solea senegalensis (Kaup), and evaluation of the potential virulence role of their extracellular products

Bacteria isolated from an outbreak with moderate mortalities in farmed sole, Solea senegalensis (Kaup), in the south of Spain were identified as Vibrio harveyi and V. parahaemolyticus. Only bacterial strains showing swarming were virulent in sole and caused mortalities in experimentally inoculated fish. However, the signs of the disease were only reproduced with V. harveyi. The intramuscular inoculation of the extracellular products (ECPs) of both species produced mortalities in inoculated fish and the appearance of surface ulcers in the case of V. harveyi. However, the inoculation of sublethal doses of ECPs to fish showed a protective effect against V. harveyi.     

哈氏弧菌胞外产物对红鳍东方鲀的致病性

采用平板玻璃纸覆盖技术提取哈氏弧菌(Vibrio harveyi)H-06091菌株的胞外产物(ECP),通过肌肉和腹腔注射2种方式研究其对红鳍东方鲀(Fugu obscurus)的致病性。采用双层纸片法对其主要酶成份进行初步研究;测定其对绵羊红细胞和红鳍东方鲀红细胞的溶血活性;将哈氏弧菌在不同温度下(15℃、20℃、25℃、30℃、35℃、40℃)培养36h和25℃下培养不同时间(12h、18h、24h、36h、48h、60h、72h)后提取其胞外产物并对其蛋白质含量进行测定;经SDS-PAGE分离粗提胞外产物中蛋白成分,并用Dot-ELISA和Western-blot方法对其成分进行分析。实验结果显示,该胞外产物对红鳍东方鲀具有致病性;具有淀粉酶和酪蛋白酶活性,不具有卵磷脂酶、脂肪酶和明胶酶活性;对红鳍东方鲀红细胞有溶血活性,对绵羊红细胞无溶血活性;胞外产物蛋白质总含量在35℃培养36h后出现高值,与温度变化无明显相关性;25℃下培养24h、36h和48h的粗提胞外产物经SDS-PAGE分离,有3条蛋白质条带最明显,分子量分别约为31kD、38kD、60kD;其中分子量约为60kD蛋白条带,经Dot-ELISA和Western-blot方法证实其为半胱氨酸蛋白酶-1。根据以上结果认为哈氏弧菌H-06091菌株胞外产物含有多种毒力因子,与该菌的致病性密切相关。     

Presence of a lethal protease in the extracellular products of Vibrio splendidus-Vibrio lentus related strains

The presence of a lethal extracellular 39-kDa protease, a virulence determinant of a Listonella pelagia strain which produces vibriosis in turbot, was determined in the extracellular products (ECP) of 33 Vibrionaceae strains. Both immunological and enzymatic techniques distinguished this specific protease from other Vibrionaceae proteins. It was detected in 15% (5/33) of the ECPs assayed belonging to strains of the Vibrio splendidus-V. lentus related group isolated in Galician aquaculture systems (NW Spain). As these strains were associated with diseased octopus and cultured turbot, were able to colonize the internal organs of fish and produced a lethal ECP for fish, they are a potential risk for the health of reared aquatic organisms.     

市售仔虾中副溶血性弧菌的检测

应用常规细菌分离法对西宁市某几个农贸市场采集的70份仔虾样品进行了副溶血性弧菌的检测,经细菌形态观察、嗜盐性试验、细胞色素氧化酶试验和生化特性测定,鉴定为副溶血性弧菌。结果检出阳性菌株4株,阳性率为5.7%(4/70),同时通过致病性试验证明4株分离菌对小白鼠有致死效应。     

养殖河蟹弧菌病病原菌分离鉴定及其胞外产物的致病性

从杭州-养蟹场的病蟹体内分离到3株细菌,经形态学检查,生化性质测定,鉴定为副溶血弧菌,人工感染健康触,48h内均发生死亡,死亡率100%,证实副溶血弧菌为河触的致病菌。药敏试验结果表明,此病原菌对链霉素,利福平,卡那霉素,复方新诺明,环丙沙星,氟哌酸,四环素,氯霉素,氟嗪酸,复达欣,菌必治,萘啶酸等药物高度敏感。分离菌株培养物经理化方法处理获得的胞外产物蛋白具有明胶酶,几丁质酶,淀粉酶,酪蛋白酶,脂酶,磷脂酶等多种酶活性及溶血活性,并对河触具有明显的致病作用。     

养殖河蟹弧菌病病原茵分离鉴定及其胞外产物的致病性

从杭州一养蟹场的病蟹体内分离到3株细菌,经形态学检查、生化性质测定,鉴定为副溶血弧菌。人工感染健康蟹,48h内均发生死亡,死亡率100％,证实副溶血弧菌为河蟹的致病菌。药敏试验结果表明,此病原菌对链霉素、利福平、卡那霉素、复方新诺明、环丙沙星、氟哌酸、四环素、氯霉素、氟嗪酸、复达欣、菌必治、萘啶酸等药物高度敏感。分离菌株培养物经理化方法处理获得的胞外产物蛋白具有明胶酶、几丁质酶、淀粉酶、酪蛋白酶、脂酶、磷脂酶等多种酶活性及溶血活性,并对河蟹具有明显的致病作用。     

Comparative assessment of Vibrio virulence in marine fish larvae

Vibrionaceae infections are a major obstacle for marine larviculture; however, little is known about virulence differences of Vibrio strains. The virulence of Vibrio strains, mostly isolated from vibriosis outbreaks in farmed fish, was tested in larval challenge trials with cod (Gadus morhua), turbot (Scophthalmus maximus) and halibut (Hippoglossus hippoglossus) using a multiwell dish assays with single‐egg/larvae cultures. The strains differed significantly in virulence as some caused a high mortality of larva reaching 100% mortality after a few days, while others had no or only marginal effects on survival. Some Vibrio strains were pathogenic in all of the larva species, while some caused disease only in one of the species. Twenty‐nine of the Vibrio anguillarum strains increased the mortality of larvae from at least one fish species; however, pathogenicity of the strains differed markedly. Other Vibrio species had no or less pronounced effects on larval mortalities. Iron uptake has been related to V. anguillarum virulence; however, the presence or absence of the plasmid pJM1 encoding anguibactin did not correlate with virulence. The genomes of V. anguillarum were compared (D. Castillo, P.W. D'Alvise, M. Middelboe & L. Gram, unpublished data) and most of the high‐virulent strains had acquired virulence genes from other pathogenic Vibrio.     

Regulation of Vibrio alginolyticus virulence by the LuxS quorum-sensing system

Quorum sensing (QS) is a bacterial intercommunication system that controls the expression of multiple genes in response to population density. The LuxS QS system regulates the expression of several virulence factors in a wide variety of pathogenic bacteria. LuxS has been characterized to be responsible for producing a type of autoinducer, AI-2, which stimulates the expression of the luciferase operon in Vibrio harveyi. Vibrio alginolyticus is established as an opportunistic pathogen of several marine animals, and its LuxS QS system remains undefined. To investigate the pathogenic role of luxS in V. alginolyticus, the luxS mutants of both the standard strain ATCC 33787 and a fish-clinical isolate MVP01, named MYJS and MYJM, respectively, were constructed. The mutation resulted in reduced lethality to Pagrus major. Intraperitoneal LD(50) of MYJS and MYJM increased by 15- and 93-fold, respectively. The two luxS mutants exhibited a lower growth rate and defective flagellar biosynthesis. They also showed a significant decrease in protease production and an increase in both extracellular polysaccharide production and biofilm development. The results suggest that the LuxS QS system plays an important role in regulating the expression of virulence factors in V. alginolyticus.     

1种致病性鳗弧菌胞外蛋白酶的分离纯化及性质

从山东省莱州海区自然发病的花鲈（Lateolobrax japanicus）体内分离到1株致病性鳗弧菌，经硫酸铵盐析、DEAE-Sepharose Fast Flow和Sephadex-G100凝胶层析等方法从其培养液中分离纯化了1种胞外蛋白酶。用SDS-PAGE电泳测得蛋白质的分子量为36.7kD，最适温度为50℃，对热不稳定，70℃15min完全失去活力；最适pH为7.0；1mmol/L的PMSF对酶活性无影响，部分金属离子如Cu^2 、Fe^2 、Fe^3 、Zn^2 对酶活力有抑制作用，而Ca^2 对酶有一定程度的激活作用，1mmol/L EDTA能完全抑制酶的活性，表明该酶是1种金属蛋白酶。     

Phytoplankton succession in intensive marine shrimp culture ponds treated with a commercial bacterial product

Succession of phytoplankton dominance was studied in shrimp culture ponds treated with commercial bacterial products. Diatoms were dominant and the cyanobacteria were absent in both treated and control ponds at the beginning of the culture period. After 34 days, the diatoms significantly decreased whereas cyanobacteria increased in both ponds. Chlorophyll a increased from a mean of 35.56 mg m^?3 in the first phase to 186.00 mg m^?3 in the final phase, and from 42.12 mg m^?3 to 242.81 mg m^?3 in the treated and control ponds respectively. Cyanobacteria were significantly higher in the control compared with the treated ponds during the final phase of the culture. Algal bioassay showed that the addition of nitrogen either alone or with silica to pond water significantly increased the specific growth rate of Chaetoceros calcitrans. The specific growth rate of Oscillatoria sp. significantly increased when a combination of nitrogen, phosphorus and carbon was added to the pond water. Addition of silica seemed to depress the growth rate of Oscillatoria sp. Nutrient enrichment should be minimized and the supply of nitrogen and silica should be adequate for promoting the growth of beneficial phytoplankton in aquaculture systems.     

Virulence properties of Moritella viscosa extracellular products

Moritella viscosa is the causative agent of winter ulcer disease of marine fish. Knowledge of its pathogenicity is limited and there are no reports comparing the virulence properties of a collection of bacterial isolates. The in vivo and in vitro virulence of the extracellular products (ECP) of 22 M. viscosa isolates was screened. Two non-virulent Canadian isolates and a Norwegian isolate with reduced virulence produced non-lethal ECP. Correlation was obtained between cytotoxin and haemolysin production of M. viscosa. Isolates from salmon produced ECP with lower cytotoxic and haemolytic activities than ECP of isolates originating from other hosts. Correlation was not found between lethality of ECPs in salmon and cytotoxic or haemolytic activities. All isolates secreted esterases and a metallopeptidase (MvP1), degraded starch and produced siderophores. Variable levels of ECP protein concentration, different enzymatic activities and siderophore production could not explain differences in virulence. The results show that virulent M. viscosa isolates secrete a lethal toxic factor of unknown nature and that cytotoxin production may reflect host adaptation. Cell-culture models may not be optimal for determining the virulence of M. viscosa, as no association between cytotoxicity and bacterial virulence was obtained. Non-virulent strains may be useful in future research on M. viscosa virulence, as construction of mutants has not been successful.     

Salinity a significant environmental factor for Vibrio harveyi virulence in Fenneropenaeus indicus

The effect of ambient salinity on the haemolymph variables of Fenneropenaeus indicus and its susceptibility to Vibrio harveyi infection under salinity stress has been studied. Adult shrimps were acclimated to 5‰ (hypo osmotic), 25‰ (iso osmotic) and 35‰ (hyper osmotic) salinity levels and the animals were injected with a mid logarithmic culture of V. harveyi at sub lethal level and haemolymph parameters were analysed. Haemolymph proteins, intracellular superoxide anion production, phenoloxidase (PO), alkaline phosphatase (ALP) and acid phosphatase (ACP) activity were found to be at elevated level both at 5‰ and 35‰ post challenge. The haematological responses showed a progressive increase (P < 0.05) up to post challenge day 5 (PCD 5) followed by a considerable decline at all salinities with the lowest being at 35‰. The alterations in the variables were higher in shrimps held at 5‰. However, the V. harveyi infection was severe in animals held at 35‰. The reduction in the parameters could be correlated with the decrease in survival rate of shrimps at 35‰ with a concurrent increase in V. harveyi at this salinity. Multiple regression analysis revealed that ACP (P < 0.001), haemocyte protein HCP (P < 0.001) and PO (P < 0.05) could explain 91% variability in the shrimp survival. These parameters may be used as effective shrimp health indicators. It is evident from the study that ambient salinity alters the haemolymph variables, modulates the virulence in V. harveyi and makes the shrimps more vulnerable to infection at higher salinity. The virulence of V. harveyi is increased at 35‰ salinity as being evidenced from the high mortality at this salinity. The study emphasizes the importance of salinity as an important environmental factor both in terms of host susceptibility and virulence of the pathogen.     

The role of rpoS in the regulation of Vibrio alginolyticus virulence and the response to diverse stresses

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu²⁺, Pb²⁺, Hg²⁺ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.     

Comparative genome sequencing to assess the genetic diversity and virulence attributes of 15 Vibrio anguillarum isolates

Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. However, the diversity and virulence mechanisms of this pathogen are still insufficiently known. In this study, we aimed to increase our understanding of V. anguillarum diversity and virulence through comparative genome analysis of 15 V. anguillarum strains, obtained from different hosts or non‐host niches and geographical regions, among which 10 and 5 strains were found to be virulent and avirulent, respectively, against sea bass larvae. First, the 15 draft genomes were annotated and screened for putative virulence factors, including genes encoding iron uptake systems, transport systems and non‐ribosomal peptide synthetases. Second, comparative genome analysis was performed, focusing on single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). Five V. anguillarum strains showed a remarkably high nucleotide identity. However, these strains comprise both virulent and avirulent strains towards sea bass larvae, suggesting that differences in virulence may be caused by subtle nucleotide variations. Clearly, the draft genome sequence of these 15 strains represents a starting point for further genetic research of this economically important fish pathogen.     

White Spot Syndrome Virus in cultured shrimp: A review

Shrimp is one of the main aquaculture species in the world. Different viruses affect them, which causes serious mortality to economically important species, such as Penaeus monodon, Litopenaeus vannamei and L. stylirostris, among others. White spot syndrome virus or WSSV is a highly lethal, stress‐dependent virus, which belongs to the family Nimaviridae, genus Whispovirus. Three WSSV virus isolates were first detected in 1992 in Thailand, Taiwan and China. Later, a fourth isolate of the virus was detected in the Americas in 1999. This virus has a large circular double‐stranded DNA genome with different sizes (292.9–307.2 kb), where the diverse isolates show differences in virulence. This virus infects a wide range of aquatic crustaceans by vertical and horizontal transmission, with different mortality results. The spread of infection between regions may be due to infected shrimp and carriers such as other crustaceans, seabirds, aquatic arthropods or other vectors. The aim of this work is to describe the current knowledge on the status, transmission, pathology, isolation, control and genomic characteristics of WSSV.     

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.     

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...     

虾池浮游微藻与养殖水环境调控的研究概况

在对虾养殖过程中,虾池中的浮游微藻群落结构和对虾的健康养殖有着密切关系,一些有益微藻能调节水体溶氧量（DO）和酸碱度（pH）,吸收氨氮（NH4-N）和亚硝氮（NO2-N）等有害物质,有效地调控养殖环境。但一些能分泌毒素的微藻也会给对虾的健康生长带来危害。数量和生物量占微藻总量比例均较高的浮游微藻优势种对整个虾池微藻群落结构的稳定起着重要作用,主导着微藻群落的功能发挥。不同养殖模式、养殖季节、养殖地域以及养殖阶段虾池中浮游微藻的优势种类分布、多样性等群落特征有差异。浮游微藻群落中优势种的变动规律和虾池中各种环境因子的动态密切相关,环境因子的变动会影响浮游微藻群落结构的变动。文章综述了对虾养殖生产实践中浮游微藻群落结构和生态调控特征的研究概况,并对在养殖中构建优良微藻藻相的方法进行了探讨。