Siderophores produced by Vibrio anguillarum in vitro and in infected rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Ten strains of Vibrio anguillarum produced three different types of iron-binding compounds when cultured under different conditions. These were (1) a common phenolate siderophore produced by all strains. (2) a hydroxamate siderophore produced by three strains and (3) a second phenolate siderophore, tentatively identified as anguibactin, produced by V. anguillarum strain 775 and two other strains, all of which contained a plasmid of 45–50 Md. The relative affinities of these siderophores, determined by competition for ⁵⁵Fe was: anguibactin < hydroxamate siderophore < common phenolate siderophore. However, under these conditions, none removed iron from purified aerobactin. Experimental infection of rainbow trout. Oncorhynchus mykiss (Walbaum), showed that only the common phenolate siderophore was detected in the kidney and spleen of fish infected with strains 91079 and NCIMB6. The hydroxamate siderophore produced in vitro by strain NCIMB6 was not detected in vivo. However, in the kidney of fish infected with strain 775, both the common phenolate siderophore and anguibactin were detected, showing that a second uptake system is required by strain 775 in vivo and that the iron-uptake system based on the common phenolate siderophore is defective.     

Growth inhibition of the salmon pathogen Vibrio ordalii by a siderophore produced by Vibrio anguillarum strain VL4355

Abstract. Thirty strains of V. anguillarum were tested for the production of inhibitory substances against closely-related bacteria using the deferred antagonism test. Only one strain, Vibrio anguillarum VL4355, inhibited strains of V. ordalii and this effect was blocked by the addition of iron salts to the culture medium. Siderophore production was investigated for this strain. Results from bioassays suggested that strain VL4355 produced a siderophore related to anguibactin, the plasmid-encoded phenolate siderophore produced by V. anguillarum strain 775. However, when plasmid DNA was compared for strains 775 and VL4355 the Bam HI-generated restriction profiles were different, although hybridization experiments indicated some homology. Using the chrome-azurol sulphate assay to measure siderophore production, strain VL4355 yielded significantly higher values than other V. anguillarum strains. Amberlite XAD-2 was used to produce concentrated siderophore preparations from strains VL4355 and 775. Both preparations were inhibitory to the growth of strains of V. ordalii , but not V. anguillarum , as were solutions of the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The difference in sensitivity to iron-limiting conditions for V. ordalii and V. anguillarum , coupled with the inability of V. ordalii to utilize ferric-anguibactin, could reflect different mechanisms of iron uptake for these two organisms.     

Transmission of protease genes into a protease-deficient mutant of Aeromonas salmonicida in river sediments

Abstract. The transformation of Aeromonas salmonicida with DNA fragments from bacterial cell-free sonicates was investigated with intraspecific, interspecific band intergeneric fish pathogenic bacteria including Aeromonas salmonicida, Aeromonas hydrophila, Pseiidomonas fluorescens and Vibrio anguillarum strains as donor bacteria. A phenotypic marker for transformation was extracellular protease production since a protease-deficient mutant NTG-1 induced from pathogenic A. salmonicida strain A-7301 by mutagenesis was used as a recipient. This mutant was non-pathogenic to rainbow trout. The mutant was incubated with each sonicate at 20°C for 20 days with a nutrient-poor medium containing a trace (5 μg/ml each) of both humic acid and tryptone in the presence of clean river sand (100 g/100 ml medium) corresponding with an environment of rivers. During the incubation, the survival of mutant NTG-1 cells was observed and protease positive NTG-1 cells were isolated from each culture. The protease production of the isolates was due to the transmission of protease genes of the donor strains. The activity of proteases produced by the transformants extra-cellularly was determined. These transformants induced with the sonicates of the parent strain, intraspecific strain and with the sonicates of the interspecific A. hydrophila strain were pathogenic to rainbow trout, whereas the transformants derived with the sonicates of the intergeneric strains P. fluorescens and V. anguiUarum showed non-pathogenicity, although all the donor strains, with the exception of the P. fluorescens strain, were pathogenic. These findings are interesting since they demonstrate that trausformation in A. salmonicida occurs with considerable ease even intergenencally and interspecifically, as well as intraspecifically in river environments, and that there is a large difference in the lethal toxicity of extracellular protease produced by these bacteria.     

Siderophores and related outer membrane proteins in Vibrio spp. which are potential pathogens of fish and shellfish

Abstract. A total of eight reference strains and 43 environmental isolates of Vibrio species that are potential fish pathogens, were assayed for the production and utilization of siderophores. Chemical and biological assays indicated that all species produced phenolate compounds and only some strains of V. cholerae non-O1, V. parahaemolyticus and V. fluvialis produced hydroxamates. Bioassays indicated that all species produced compounds that stimulated the growth of the homologous and the heterologous species in low-iron media. The catechol-type siderophores produced may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants. However, the chromatographic analysis and absorption spectra of supernatants and their extracts showed some differences among catechols excreted by Vibrio species. The hydroxamate compounds produced by some strains of V. fluvialis and V. parahaemolyticus were different from the aerobactin. The synthesis of iron-regulated outer membrane proteins was also examined in representative strains of each species. The molecular size of the main induced proteins ranged between 74 and 95kDa, and were relatively species specific.     

发病鲆鲽类分离菌株的16S rRNA基因测序分析

细菌性疾病是中国海水养殖鲆鲽类的主要病害,为全面了解病原菌种类,本研究对1999~2012年从山东、江苏、河北、天津等沿海地区养殖场发病鲆鲽鱼类中分离得到的124株优势菌株进行了16S rRNA基因测序和系统发育学分析。将基因序列与GenBank核酸序列数据库进行相似度比对分析,结果显示,有83株与弧菌属(Vibriosp.)细菌相似度最高,11株与气单胞菌属(Aeromonas sp.)细菌相似度最高,4株与爱德华氏菌属(Edwardsiella sp.)细菌相似度最高,26株为其他15种属的细菌。根据系统发育学分析结果,进一步将66株菌鉴定为16个种,优势种为溶藻弧菌(V.alginolyticus)、哈氏弧菌(V.harveyi)、鳗弧菌(V.anguillarum)、杀鲑气单胞菌(A.salmonicida)和迟缓爱德华氏菌(E.tarda)。选择其中的9株鳗弧菌和4株迟缓爱德华氏菌进行人工感染实验,结果显示,其中7株鳗弧菌和3株迟缓爱德华氏菌对大菱鲆(Scophthalmus maximus)有较强的致病性。研究结果可为阐明中国海水养殖鲆鲽类的流行病发生历史、病原种类、病原监测及疾病控制提供重要参考。     

健康养殖南美白对虾肠道细菌的抗菌活性

用双层琼脂扩散法检测从健康养殖南美白对虾肠道内分离出的111株菌对8株病原菌的抗菌活性。结果表明:111株菌对白假丝酵母菌,非01群霍乱弧菌,嗜水气单胞菌指示菌无抗菌活性,48.6%的菌株对余下的5株指示菌有抗菌活性,其中气单胞菌属和发光杆菌属对荧光假单胞菌与金黄色葡萄球菌的抑制性最强。各菌属中以气单胞菌属,发光杆菌属的抗菌谱最广,弧菌属、肠杆菌科、黄单胞菌属、芽孢杆菌属、小球菌属次之。土壤杆菌属、棒状杆菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属则没有抗菌活性。     

Desalted Cod Spoilage Flora Inhibition by Citric Acid and Potassium Sorbate Combination

ABSTRACT

Desalted cod spoilage isolates were tested in culture broth for growth inhibition by citric acid and potassium sorbate. Preservative effect was also evaluated in refrigeratedcod juice and desalted cod spiked with Shewanella putrefaciens and Pseudomonas fluorescens orputida. Combination of 0.15% citric acid and 0.1% potassium sorbate inhibited growth of approximately 60% of isolates, including the former two strains. Total inhibition was observed for S. putrefaciens and partial inhibition was noticed for P. fluorescens or putida, in both treated juice and desalted cod. Preservatives delayed growth of halotolerant bacteria in cod. Citric acid plus potassium sorbate addition lowered the pH to extend desalted cod shelf life.     

采用通用引物PCR配合SSCP和RFLP技术检测鱼病病原菌

采用通用引物PCR（UPPCR）、PCR－RFLP、PCR－SSCP技术,研究快速鉴别鱼病病原菌的分子生物学诊断技术。结果发现,采用细菌16S rRNA基因保守区特异性引物,以嗜水气单胞菌、鲁克氏耶尔森菌、鳗弧菌、柱状曲挠杆菌、乙型链球菌、荧光假单胞菌等部分常见鱼病病原菌为对象,可以建立一种UPPCR技术。该技术能在保证实验条件不变的基础上,检出上述所有细菌,并还可检出大肠杆菌和双歧杆菌等非鱼病病原菌。并且认为,该法与SSCP配合即采用UPPCR－SSCP技术能较好地鉴别被检菌而用于鱼病病原菌的快速诊断。     

粘帚霉对几种水生生物有害菌的抑制作用测定

采用对峙培养法测定了粘帚霉16株菌株对嗜水气单胞菌、耶耳森菌、鳗弧菌、褐藻酸降解菌-MA10和褐藻酸降解菌-MB16的作用。试验结果表明,粘帚霉中NCH-3-1、YJS-3-2、GJ-1-1、SS-1-1、SHW-2-1、SHW-1-1、BD-2-2 7株菌株对供试的5种有害菌表现出较强的抑制作用。     

The use of API 20E for the identification of Vibrio anguillarum and V. ordalii

Abstract. Sixty-eight strains of Vibrio anguillarum , five of V. ordalii and the type strains of V. alginolyticus, V. carchariae, V. damsela and V. parahaemolyticus were compared using the API 20E gallery. Within the V. anguillarum strains, distinct groups could be separated mainly on the basis of their reaction on indole production and the fermentation of amygdalin and arabinose. Vibrio ordalii , the former V. anguillarum biotype 2, could easily be separated from V. anguillarum and from the other fish pathogenic Vibrio spp.     

斑节对虾“红体病”细菌性病原的初步研究

从患“红体病”的斑节对虾中分离到８个菌株,通过致病力试验证实其中两个菌株是引起斑节对虾“红体病”的主要病原,使对虾死亡率分别达到７０％和９０％。根据生化试验,确定这两个菌株分别是嗜水气单胞菌和副溶血弧菌。抗药性实验的结果表明,嗜水气单胞菌菌株对强力霉素较为敏感,副溶血弧菌菌株对氟哌酸、红霉素、青霉素和强力霉素较为敏感。     

In situ adherence of Vibrio spp. to cryosections of Atlantic salmon, Salmo salar L., tissue.

Pathogenic and presumed non-pathogenic bacteria isolated from fish were tested for their adhesion to cryosections from different mucosal surfaces of Atlantic salmon, Salmo salar L. Adhered bacteria were detected by immunohistochemistry. Mucus was stained and fixed with Alcian blue after incubation of bacteria. The majority of the bacteria tested, i.e. Vibrio anguillarum serotype O1 , Vibrio salmonicida , Vibrio viscosus, Flexibacter maritimus and 'gut vibrios', i.e. Vibrio iliopiscarius and intestinal isolates of V. salmonicida , all adhered to mucus on all salmon epithelial surfaces tested, including sections from the foregut, hindgut, pyloric caeca, gills and skin. In contrast, V. anguillarum serotype O2, including both serotypes O2a and O2b, did not adhere to mucus, but did adhere to all other components of the tissues. As a positive control for adhesion of bacteria on cryosections, Escherichia coli was bound to piglet ileal mucosal lining, and as a negative control for adhesion, Staphylococcus aureus was found not to bind to any of the tissues tested. The present study shows that adhesion to mucus was not restricted to pathogenic bacteria, and furthermore, that not all pathogenic bacteria studied adhered to mucus. Hence, on the basis of these findings, the present authors suggest that V. anguillarum O2 may have an invasion strategy which does not involve adhesion to mucus, and thus, differs from the other pathogenic bacteria in the present study, which all bound to salmon mucus.     

创伤弧菌、溶藻弧菌外膜蛋白特性的比较研究

对用Sarkosyl法分离的创伤孤菌、溶藻弧菌、副溶血弧菌、鳗弧菌的外膜蛋白进行了初步的比较分析.这4种弧菌外膜蛋白的SDS-PAGE和Western blotting的图谱有相似性亦有差异.SDS-PAGE显示,4种弧菌中除副溶血弧菌外均能分离到47、38 ku的外膜蛋白;创伤弧菌和溶藻弧菌存在4种共同的外膜蛋白,大...     

Serotyping of Vibrio anguillarum isolated from diseased freshwater fish in Japan

Abstract. During the period from 1965 to 1980, 263 Vibrio anguillarum strains from ayu, Plecoglossiis altivelis (Temminck & Schlegel), two from rainbow trout. Salmo gairdneri Richardson, and two from eel, Anguilla japonica (Temminck & Schlegel), were collected from fish suffering from vibriosis in various parts of Japan. On the basis of cross-agglutination and cross-absorption tests with thermostable (O) antigens, six distinct serotypes (A, B, C, D, E and F) were established among 12 selected strains of V. anguillarum . 241 strains isolated from ayu and two strains from rainbow trout belonged to serotype A, six strains from ayu and one strain from eel to serotype B, 12 strains from ayu to serotype C, three strains from ayu to serotype D, one strain from ayu to serotype E, and one strain from eel to serotype F. V. anguillarum strains belonging to serotypes D, E and F have not been detected from ayu, rainbow trout and eel since 1973; these serotypes appear to be minor types. V. anguillarum strain NCMB 6 and 1669 belong to our serotype A and V. anguillarum 813 to our serotype C.     

病原性鳗弧菌(Vibrio anguillarum)双重PCR与LAMP检测方法的建立

本研究检测了分离自发病大菱鲆、半滑舌鳎及鲤鱼的22株病原鳗弧菌(Vibrio anguillarum)毒力相关基因的携带情况,并建立了病原鳗弧菌的分子生物学检测方法。以PCR方法检测8个毒力相关基因的分布,结果显示,22株病原鳗弧菌均可扩增出6个基因(empA、vah1、vah4、flaA、rtxA和tonB)目的条带,未扩增出virA和angM基因;针对vah4和rtxA设计引物进行双重PCR扩增,同一PCR反应体系可扩增出两条目的条带,灵敏度为2.4×103 CFU/ml,对照菌无任何扩增条带;以vah4设计引物进行LAMP扩增,病原鳗弧菌可扩增出阶梯状条带,呈现阳性反应,6株对照菌无阶梯状扩增条带且呈现阴性反应,LAMP扩增灵敏度为2.4×101 CFU/ml。LAMP检测灵敏度是双重PCR的100倍,LAMP技术与PCR比较,操作简便、快速、灵敏度高且不需昂贵仪器,LAMP检测鳗弧菌的方法更适合于养殖生产实际应用。     

Interaction of the fish pathogen Aeromonas hydrophila with tilapia, Oreochromis aureus (Steindachner), phagocytes

Abstract. Interaction of Aeromonas hydrophila and tilapia, Oreochromis aureus (Steindachner), phagocytes was studied in vitro. All virulent and avirulent strains of A. hydrophila tested could multiply in non-activated and Freund's complete adjuvant activated phagocytes. Activated phagocytes increased the uptake of bacteria into cells, and the rates of intracellular replication for these bacteria were faster than in non-activated phagocytes. Among the A. hydrophila strains examined, virulent strain PPD134/91 replicated at the fastest rate inside phagocytic cells and produced cytopathic effect in the phagocytes in the shortest incubation time. Opsonized avirulent A. hydrophila were sensitive to phagocyte-mediated killing or unable to grow in phagocytes. Serum components and phagocytes may together prevent the growth of avirulent A. hydrophila in fish. The release of extracellular oxygen radicals during phagocytosis was examined using chemiluminescence assay (CL). Virulent strains induced CL responses but avirulent strains did not. This suggests that the virulent strains interacted with the phagocytes somewhat differently from the avirulent strains.     

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction

Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL⁻¹ of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g⁻¹ tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.     

The effect of iron limitation growth conditions on the cell and extracellular components of the fish pathogen Pasteurella piscicida

The effect of iron limitation, using the iron-chelating agent 2,2 dipyridyl, on the electrophoretic profiles of outer membrane proteins (OMPs) and extracellular products (ECPs) from 21 Pasteurella piscicida strains isolated from Europe and Japan was investigated. In addition, the effect of iron-limited and iron-surplus growth conditions on caseinase activity in culture supernatants of the pathogen was examined. The majority of P. piscicida strains, from Greece, Italy and France, cultured under iron-limited conditions, produced four novel OMPs (63 and three at and above 200 kDa). In contrast, iron-regulated outer membrane proteins were not induced in Japanese strains. Electrophoretic analysis of the ECPs from the pathogen grown under iron surplus and iron limitation revealed a large range of products and additional high molecular mass (MM) bands were evident under iron-limited conditions. When culture supernatants were analysed for their activity, most of the bacteria tested showed elevated activities under iron limited conditions. Finally, neither hydroxamate nor phenolate type siderophores could be detected with any of the chemical assays used.     

Proteases of the Aeromonas hydrophila complex: identification, characterization and relation to virulence in channel catfish, Ictalurus punctatus (Rafinesque)

Abstract. Traditional biochemieal techniques and a stain to detect proteases in polyacrylamide gels were used to identify and partially characterize three proteases, P1, P2 and P3, produced by Aeromonas hydrophila strain Ah 22. P1 was found to be a heat-labile serine protease with an optimum pH of 7·5, while P2 is a heat-stable metalloprotease with an optimum pH of 8·0, and P3 is a moderately heat-stable metalloprotease with peak activity beween pH 7 and 11. A comparison of 17 other strains of the A. hydrophila complex indicated that four produced P1, P2 and P3. Two strains produced just P1 and P3; one produced only P3; six produced two different serine proteases, P2a and P2b; and two produced a number of uncharacterized proteases. Virulence studies in age-0 + channel catfish indicated no correlation between either quantitative or qualitative protease production and virulence.