Studies on the pathogenesis of streptococcal infection in cultured yellowtails Seriola spp.: effect of the cell free culture on experimental streptococcal infection

Abstract. A previous paper has revealed that experimental streptococcal infection was associated with a rapid growth of the intestinal bacteria, suggesting association of the condition with an exotoxin. The present study was undertaken to see the effect of the exotoxin on the streptococcal infection by administering cell free culture before the bacterial challenge. Cell free culture of Streptococcus sp. strain YT-3 was inoculated intramuscularly at a dosage of 0–5 ml per 100 g body weight 1 h prior to bacterial challenge. Three fish were killed at intervals after challenge for viable counting of bacteria in the viscera and blood. Intramuscular inoculation with either low virulent or virulent bacteria alone at dosages of 10⁶ to 10⁷ cells did not produce the disease in the fish, with almost complete clearance of the bacteria from the viscera and blood within 120 h. When the exotoxin was inoculated intramuscularly prior to the bacterial challenge, however, either low virulent or virulent bacteria at 10⁶ to 10⁷ cells could produce fatal infection with prominent streptococcal clinical signs.     

Lancefield group C Streptococcus dysgalactiae infection responsible for fish mortalities in Japan

A Lancefield serological group C Streptococcus sp. was isolated from cultured amberjack, Seriola dumerili Risso, and yellowtail, Seriola quinqueradiata Temminck and Schlegel, immunized with Lactococcus garvieae commercial vaccines in Japan. The isolated bacteria were Gram-positive cocci, auto-aggregating in saline, morphologically long chains in growth medium, catalase negative and alpha-haemolytic on blood agar. An almost complete gene sequence of the 16S rDNA of two isolates was determined and compared with that of bacterial strains in the database. The isolates were identified as Streptococcus dysgalactiae based on the results of the 16S rDNA sequence, the bacteriological properties and the Lancefield serological grouping. Oligonucleotide primers specifically designed for the 16S-23S rDNA intergenic spacer region of S. dysgalactiae amplified a gene from all the fish isolates, as well as the type strains alpha-haemolytic S. dysgalactiae subsp. dysgalactiae ATCC430738 and beta-haemolytic S. dysgalactiae subsp. equisimilis ATCC35666, but not those of S. equi ATCC33398, Lactococcus garvieae ATCC43921 and L. garvieae KG9408. The severe necrotic lesions of the caudal peduncle seen in experimentally infected fish were similar to those seen in naturally infected fish.     

Studies on the pathogenesis of streptococcal infection in cultured yellowtails Seriola spp.: the fate of Streptococcus sp. bacteria after inoculation

Abstract. The causative agent of streptococcal infection of yellowtails has been isolated but no information is presently available with respect to the mechanism of infection. The present study deals with the growth of the bacteria in the fish body at various periods after either per-oral or per-cutaneous challenge with Streptococcus sp. YT-3 strain at different passage levels. After percutaneous challenge with bacteria of high virulence, the kidneys retained the bacteria with the relatively high count of 10⁷ cells per gram of tissue while in other organs, although high concentrations of 10⁵-10⁶ cells were detected in 10 min, this was followed by a progressive decrease up to 24 h post-inoculation with a subsequent rapid increase during the later stages of the disease process. The highest rate of growth was obtained in the intestine, where 10⁷ cells were detected at 72 h after inoculation. After oral challenge, the bacteria were detected at high levels from organs and blood within 10 min but they were completely removed from all organs except the intestine within 24 h.     

Repeatable immersion infection with <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis> reproducing clinical signs and moderate mortality

Pseudotuberculosis is a bacterial septicaemia caused by Photobacterium damselae subsp. piscicida in several marine fish species. Yellowtail Seriola quinqueradiata is the most sensitive fish species to this disease. The internal organs of naturally infected yellowtail exhibit whitish spots, tubercle-like tissue structures, consisting of bacterial accumulations. There have been many trials for experimental infection, however adequate method of infection that reproduces moderate mortality and primary clinical signs has not yet established. Present investigation evaluated an immersion infection method by using logarithmic culture-phase bacteria resulting in higher mortality than that using stationary culture-phase bacteria. Typical white spots on the spleen and kidney were also observed constantly in dead fish. Transmission electron microscopy and fluorescent antibody microscopy showed bacterial clusters not only in the spleen and kidney but also in the blood channels in the secondary gill filaments. These results were confirmed repeatedly by plural experiments. The use of logarithmic-phase bacteria in immersion infection is an appropriate technique to reproduce moderate mortality and primary clinical signs, which will be a reliable infection method also for the challenge test of pseudotuberculosis vaccine.     

Encephalomyelitis associated with microsporidian infection in farmed greater amberjack, Seriola dumerili (Risso)

An outbreak of a disease characterized by a peculiar spiral movement in farmed greater amberjack, Seriola dumerili (Risso), occurred in Kagoshima Prefecture, Japan, in May 2008, immediately after importing the fish from China. Although neither bacteria nor viruses were detected in routine diagnostic tests, histopathological observations of the affected fish revealed severe inflammation in the tegmentum of the brain including the medulla oblongata and the anterior part of the spinal cord. In addition, a microsporidian parasite was observed in the nerve cell bodies or axons in the inflamed tissues. We identified a microsporidian small subunit rRNA gene (SSU rDNA) from the lesion, and the sequence showed 96.1% identity with that of Spraguea lophii. Subsequent in situ hybridization using probes presumably specific to the SSU rRNA confirmed that the parasite observed in histopathology harboured the identified SSU rRNA. Apparently degenerated microsporidian cells or spores were also frequently observed in tissue sections. Thus, the disease was most probably caused by the infection of a hitherto unknown microsporidian parasite that has a genetic affinity to the genus Spraguea, in the central nervous system of the amberjack.     

Seasonal and annual cycles in marine small‐scale fisheries (Ilhéus – Brazil)

Fish stock assessment is a crucial tool for resource management. Small‐scale fisheries are often multispecies and multigear. This study focuses on the search for patterns in the Ilhéus small‐scale fishery using landing data from 2000 to 2006 and climatic data. Ocyurus chrysurus and Ephinephelus spp. were the most commonly landed fish. The number of fishing trips was higher in the winter, when winds were weaker and temperatures lower. When the temperature increased, catches of Seriola dumerili were higher. Coryphaena hippurus and some rays were more abundant in the summer in the presence of stronger winds, intense rainfall and higher air temperature. Large catches of Lutjanus spp. and Scomberomorus spp. were obtained in the autumn. There is an annual seasonality in the fishery, with a cyclical landing pattern regardless of the years considered. These results are important in view of climate change, and specific strategies for better fisheries management are recommended.     

Extracellular protease production of bacteriolytic bacteria isolated from marine environments

ABSTRACT:   Fourteen bacterial strains isolated from marine environments exhibited antagonistic action against a wide range of bacteria including Vibrio spp. A double layer agar method was used for preliminary screening to determine the relative degree of growth inhibition or bacteriolysis exhibited by the isolates. Most of the antagonistic isolates were found to be Gram-negative, motile rods and were oxidase positive, and oxidative in the oxidation and fermentation test, suggesting that they are belong to the genera Pseudomonas . The antagonistic isolates lyzed the dead cells of marine Gram-negative bacteria in both plate and liquid methods. Bacteriolytic and casein hydrolytic activities were observed in the culture supernatant of the isolates. Anion exchange column chromatography (Toyopearl DEAE-650 M) was used to purify the extracellular protease produced by an antagonistic strain A1-J25a. The active fractions of protease collected from the eluted solution also exhibited bacteriolytic activity.     

Experimental infection of eggs and yolk sac larvae of halibut, Hippoglossus hippoglossus L.

Abstract Scanning electron micrographs of halibut eggs with an epiflora dominated by Flexibacter sp., showed ulcerations colonized by large numbers of bacteria. The chorion was dissolved in most ulcerations and the zona radiata was severely damaged. Infection experiments showed that exposure to these bacteria caused high mortalities at the late egg stage, hatching and the early yolk sac stage. Eggs exposed to three different Vibrio spp. showed different mortality patterns, with low mortality at hatching, followed by a continuous high mortality throughout the yolk sac stage. Mortality in the uninfected control group was low throughout the experiment. Transmission electron microscopy of the Vibrio -infected larvae showed bacteria present in the gill, heart and frontal yolk sac regions.     

基于16SrDNA分析的浒苔外生细菌多样性研究

为研究浙江近海浒苔Ulva spp.(Enteromorpha spp.)外生细菌多样性,采用传统的形态学和16S rDNA测序分析的方法,从舟山朱家尖、宁海国华电厂和奉化南沙3个地区分离到可培养的浒苔外生细菌及其周围海水细菌65株。根据细菌菌落特征和革兰氏染色结果等将分离到的细菌分为26种表型。16S rDNA序列测序比对发现:菌株与不动杆菌属(Acinetobacter)、交替单胞菌属(Alteromonas)、弧菌属(Vibrio)、假交替单胞菌属(Pseudoalteromonas)、微小杆菌属(Exiguobacterium)、芽孢杆菌属(Bacillus)、赤细菌属(Erythrobacter)、微球菌属(Micrococcus)、假单胞菌属(Pseudomonas)、深海杆菌属(Idiomarina)、Phaeobacter、Roseivirgaj和Silicibacter等23个属相应菌株具有较高同源性。对不同地区浒苔外生细菌进行了Shannon-Wiener多样性指数分析。研究表明:(1)宁海国华电厂的浒苔外生细菌多样性最丰富,多样性指数为93.98%;(2)浒苔外生细菌与其生活地区密切相关,其群落组成具有地域差异,其优势类群也不尽相同,但均归属于变形菌门。     

微囊藻水华叠加对太湖沉积物中细菌垂直分布的影响

为研究微囊藻水华爆发对沉积物中细菌的影响,加强对富营养化湖泊细菌生态分布和环境调控原理的认识,选取太湖竺山湾沉积物柱状样,经添加高浓度微囊藻培养后,采用聚合酶链式反应-变性梯度凝胶电泳（polymerase chain reaction-denaturing gradient gel electrophoresis;PCR-DGGE）方法,分析细菌在沉积物中的垂直分布特征。结果显示,微囊藻水华叠加对于沉积物剖面表层细菌群落结构影响很小,对沉积物中细菌垂直分布的影响不大,仅在7～9cm深度之间有一定影响。     

Conchiolin infection and surface coating Vibrio: shell fragility, growth depression and mortalities in cultured oysters and clams, Crassostrea virginica, Ostrea edulis and Mercenaria mercenaria

Abstract. A disease of cultured juvenile European oyster, Ostrea edulis , and American oysters, Crassostrea virginica , was characterized by infection of conchiolinous ligament and periostracum by surface-coating Vibrio spp. bacteria. A similar bacterium was isolated from diseased cultured hard clams, Mercenaria mercenaria , held in a facility about 300 km distance from the oyster hatcheries. The oysters demonstrated fragile, undercalcified peripheral shell and apparently excessive chalky deposits. Histological and scanning electron microscopical examination revealed that the oysters' eroded ligaments and perforated peripheral periostracum were infected with uniform rod-shaped bacteria. The infection appeared to interfere with normal proteinaceous and calcareous shell deposition and possibly with normal ligament function. Histological examination of the digestive system organs suggested that normal digestive processes were also interrupted. Two predominant bacteria were isolated from oyster samples and one predominant bacterium was isolated from clam samples. One of the isolates from oyster culture facilities was found almost exclusively associated with oyster shell surfaces and culture tank surfaces, this isolate was similar to the one taken from clam shell surfaces. The other predominant bacterium was found in water column samples in oyster culture facilities at levels from about 10⁵ to 10⁷ per ml. This isolate had a DNA base ratio (moles % G + C) of 43.6; the two surface-associated isolates had base ratios of 43.6 and 44.5%. These Gram-negative, oxidase positive, fermentive rods have been identified as a Vibrio spp. group. Specific antibody was produced which differentiated these bacteria from each other and from other similar isolates. Practical procedures for controlling the bacteria in shellfish culture systems are described.     

Production of diatom–bacteria biofilm isolated from Seriola lalandi cultures for aquaculture application

Controlled incorporation of selected microalgae and bacteria in aquaculture systems can be beneficial because they can act as microbiological control. That is why the characteristics of biofilm generated naturally in Seriola lalandi culture cages were analysed, their potential benefit to the growth of larvae was studied, and their controlled use for improving the larval viability and as a vector to improve incorporation of previously studied probiotic bacteria was tested. According to biodiversity results, these biofilms are composed of a diatom–bacteria mix showing a decrease in biodiversity in laboratory culture conditions being dominated by Navicula phyllepta and bacteria of the family Rhodobacteraceae. This can be produced on mesh substrates incorporated in bioreactors with rapid growth rate and adhesiveness. Preliminary results from the addition of substrates with this specific biofilm in larvae culture systems showed that it is consumed by the larvae without negative effects, while positive effects on the viability of larvae in combination with probiotics were observed. Considering preliminary results, the addition of these specific substrates with diatom–bacteria biofilms could be a good improvement for aquaculture systems and together with the use of probiotics can contribute to maintaining a stable and controlled system improving the viability of the larval fish culture in its early stages.     

DNA条形码在鰤属鱼类物种鉴定和系统进化分析中的适用性

为了探讨线粒体细胞色素C氧化酶亚基Ⅰ、Ⅱ(COⅠ、COⅡ)和16SrRNA基因在鰤属鱼类物种鉴定和群体划分中的适用性,在黄条鰤(Seriola lalandi)、高体鰤(Seriola dumerili)、五条鰤(Seriola quinqueradiata)等鰤属鱼类中克隆了 3种基因,并进行序列比对与系统进化分析....     

真空包装淡腌青鱼品质和细菌类型及数量

在零售市场和生产企业抽取4批真空包装淡腌青鱼,对其感官、化学和细菌学品质特性进行分析,并对细菌类型和数量进行研究。结果显示,产品栅栏因子及强度存在差异,盐分含量6.68%~12.06%,平均值(9.36±2.2)%;水分活度值0.75~0.91,平均值0.82±0.07;pH值6.00~6.30,平均值6.20±0.13。栅栏因子种类和强度的不同决定产品品质的差异,感官评价均可接受,过氧化值均值为19.65±8.75 meq/kg,挥发性盐基氮均值为24.50±15.30 mg/100 g,菌落总数均值为4.45±1.15 cfu/g,大肠菌群均低于30 MPN/100 g。从4批淡腌青鱼样品分离到234株细菌,73.9%是革兰氏阳性细菌,其中36.3%是葡萄球菌,31.6%是链球菌,并出现少量微球菌和棒状杆菌;由需氧革兰氏阴性杆菌不动杆菌、摩氏杆菌和鞘氨醇单胞菌组成较小的细菌菌群约占总细菌群落的18.4%,显示水分活度、盐分含量和pH值等栅栏因子与产品细菌类型和数量呈一定相关性。     

软包装即食醉鱼制品细菌学品质安全分析

对上海市场常见品牌的软包装即食醉鱼制品感官、理化、细菌学品质状况进行定性和定量研究,并对潜在病原菌安全性进行评价。结果表明,产品水分含量、水分活度、pH和盐分含量分别为43.50%~56.97%,0.93~0.97,6.07~6.73,2.41%~6.63%,差别较大;细菌菌落总数、耐热菌落总数分别是1.8×102~4.2×105cfu/g,38~3 800 cfu/g;厌氧菌落总数和金黄色葡萄球菌均低于10 cfu/g。由细菌总数计数平板分离256株细菌,鉴定残存主要细菌菌群为芽孢杆菌(34.0%)、葡萄球菌(27.3%)、玫瑰小球菌(14.0%),并出现少量的棒状杆菌(5.5%),表明热杀菌强度不足。产品中残存有一定数量的蜡样芽孢杆菌,对其潜在危害有待进一步研究。     

应用RFLP和DGGE技术分析工厂化养殖凡纳滨对虾肠道微生物群落特征

本研究应用PCR-RFLP(Polymerase Chain Reaction-Restriction Fragment Length Polymorphism)和PCR-DGGE(Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis)技术,对工厂化养殖的凡纳滨对虾肠道菌群进行多样性分析。RFLP结果显示,8月样品中不同的克隆子为5个,其中以不可培养细菌(Unculturable bacteria)为主要优势菌,其次为鲁杰氏菌属菌种Ruegeria spp.和Rhodobacterales spp.。依据微生物多样性的覆盖率分析结果表明,所构建16S rDNA克隆文库的覆盖率为97.5%。10月样品中克隆子为8个,其中以不可培养细菌、芽孢杆菌属Bacillus spp.和弧菌属Vibrio spp.为主要优势菌属,其次为Photobacterium spp.和Neptunomonas spp.;所构建16S rDNA克隆文库的覆盖率为90.8%。应用DGGE分析8月和10月对虾肠道样品,菌群以不可培养细菌和弧菌属为主,其次为Streptomyces spp.、Ruegeria spp.、Enterococcus spp.和Photobacterium damselae。     

Structural characterization of carangid fish myoglobins

The primary structures of myoglobin (Mb) from the following five carangid species were determined: yellowtail Seriola quinqueradiata, greater amberjack Seriola dumerili, yellowtail kingfish Seriola lalandi, Japanese horse mackerel Trachurus japonicus, and silver trevally Pseudocaranx dentex. The sequences were of varying composition both in the coding and in the noncoding regions, but all contained the open reading frame of 444 nucleotides encoding 147 amino acids. Amino acid sequence identities of carangid Mbs were in the range of 81-99%. The similarity of the heme pocket and associated heme-binding residues of carangid Mbs were evidence of the conservative nature of Mbs. Similar to the other teleost Mbs, carangid Mbs did not contain a D helix and had mostly conserved A and E helices as well as E-F and G-H inter-helical segments. Hydropathy profiles of carangid Mbs showed species-specific variations where silver trevally Mb exhibited generally higher hydrophobicity. Phylogenetic analysis based on the primary structures was in agreement with conventional morphological taxonomy, establishing close proximity of carangid Mbs with those of cichlid and scombroid, the other members of the Perciformes order.     

水产品中4种常见致病菌多重PCR检测方法的建立及评价

根据金黄色葡萄球菌的耐热核酸酶基因nuc、沙门氏菌的侵袭蛋白基因invA、志贺氏菌的侵染性质粒抗原H基因ipaH和副溶血性弧菌的毒力表达调控基因toxR设计引物,建立一种快速检测水产品中上述4种常见食源性致病菌的多重PCR方法。结果显示,沙门氏菌、金黄色葡萄球菌、志贺氏菌及副溶血性弧菌的多重PCR扩增片段产物大小分别为549、426、348和243 bp,该检测方法具有良好的特异性和灵敏度。设计优化了可用于水产样品中4种目标致病菌的共增菌培养基,该方法应用于35份实际水产样品的检测,并与国标方法对比验证,4种致病菌的整体符合率达94%以上,两种检测方法无显著性差异(P>0.05)。可见,该多重PCR方法可应用于水产品中沙门氏菌、金黄色葡萄球菌、副溶血性弧菌及志贺氏菌4种食源性致病菌的快速检测和分子流行病学调查。     

蟹糊贮藏中的品质变化及变质菌分析

对蟹糊贮藏中的品质和细菌相变化进行分析。结果显示,水分活度0.901产品的货架期为水分活度0.861产品的81%,产品10℃贮藏的货架期为5℃贮藏的42%,表明栅栏因子强度对产品的质量和贮藏性十分重要。蟹糊产品初始菌相较单一,主要是棒状杆菌、玫瑰色库克菌、葡萄球菌和马红球菌。贮藏中菌相组成更趋单一,至感官拒绝时,葡萄球菌比例均超过50%;特别是水分活度高的产品,无论是5℃还是10℃贮藏,至感官拒绝时,葡萄球菌比例均超过三分之二,因此可以认定葡萄球菌是蟹糊产品的优势变质菌。虽然没有检出沙门氏菌和金黄色葡萄球菌的生长,但金黄色葡萄球菌是蟹糊中的潜在病原菌。     

Nocardiosis in tank-reared Atlantic salmon, Salmo salar L.

Nocardiosis is an infection caused by aerobic Gram-positive, branching, filamentous rods of the genus Nocardia . The organism is resident in both soil and plants ( Austin & Austin 1993 ; Frerichs 1993 ), and is closely related to Mycobacterium spp. Nocardiosis caused by Nocardia asteroides and N. seriolae (previously N. kampachi ) has been reported in several finfish species, both freshwater and marine. The first reported incidence was recorded by Valdez & Conroy (1963) in neon tetras, Hyphessobrycon innesi (Myers), and subsequently in other fish species, some of commercial significance including rainbow trout, Oncorhynchus mykiss (Walbaum) ( Snieszko, Bullock, Dunbar & Pettijohn 1964 ), brook trout, Salvelinus fontinalis (Mitchill), ( Campbell & MacKelvie 1968 ) and yellowtail, Seriola quinqueradiata (Temminck & Schlegel; Kubota, Kariya, Nakamura & Kira 1968 ). Despite two of these incidences occurring in salmonids, infection by Nocardia spp. in Atlantic salmon, Salmo salar L., has not previously been reported.