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Lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.)

JOURNAL OF APPLIED MICROBIOLOGY 2000年第2期89卷 317-322页

作者： Ringo, E Bendiksen, HR Wesmajervi, MS Olsen, RE Jansen, PA Mikkelsen, H Norwegian Sch Vet Sci Dept Arctic Vet Med NO-9292 Tromso Norway Univ Tromso Dept Med Microbiol Tromso Norway Finnmark Coll Dept Fisheries Alta Norway Finnmark Res Ctr Hammerfest Norway Norwegian Inst Fisheries & Aquaculture Ltd Tromso Norway

The present study reports the effect of excessive handling stress and starvation on the lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.). A relatively low population level (approximately 2 x 10(3) bacteria per gram wet tissue) of viable adherent heterotrophic bacteria was associated with the digestive tract (foregut, midgut and hindgut). Of the 752 bacterial isolates isolated from diet, water and the digestive tract, 201 isolates belonged to the carnobacteria. Of these isolates, one from the diet, one from the rearing water and 80 from the gastrointestinal tract, were further identified on the basis of 16S rDNA sequence analysis. All these isolates were identified as being Carnobacterium piscicola-like. Daily repeated stress and starvation of the fish over 11 d had no influence on the total culturable bacterial numbers or population level of C. piscicola associated with the digestive tract. C. piscicola-like isolates colonizing the various intestinal regions (foregut, midgut and hindgut) were also screened for their ability to produce growth inhibitory compounds active against the fish pathogen Aeromonas salmonicida. Of the 199 C. piscicola isolates tested, 139 inhibited growth of the pathogen.

关键词：气单胞菌属/生长和发育气单胞菌属/分离和提纯抗生作用集落计数微生物 DNA 细菌/分析 DNA 细菌/遗传学 DNA 核糖体/分析 DNA 核糖体/遗传学消化系统/微生物学脂肪酸类/分析鱼疾病/微生物学革兰氏阴性菌感染/微生物学革兰氏阴性菌感染/兽医学乳杆菌科/化学乳杆菌科/生长和发育乳杆菌科/分离和提纯表型 RNA 核糖体 16S/遗传学盐湖鲑鱼/微生物学盐湖鲑鱼/生理学动物

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Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第2期88卷 299-307页

作者： Wiklund, T Madsen, L Bruun, MS Dalsgaard, I Danish Inst Fisheries res Fish Dis Lab DK-1870 Frederiksberg C Denmark Royal Vet & Agr Univ Dept Vet Microbiol Lab Fish Dis DK-1870 Frederiksberg Denmark

Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex(R) 100 chelating resin. The primers, which were tested against strains isolated from diseased fish, healthy fish, fish farm environments and reference strains, proved to be specific for Fl. psychrophilum. The obtained detection limit of Fl. psychrophilum seeded into rainbow trout brain tissue was 0.4 cfu in the PCR tube, corresponding to 17 cfu mg(-1) brain tissue. The PCR-assay proved to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non-sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR-assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml(-1) water. The PCR-assay detected Fl. psychrophilum in water samples taken from a rainbow trout farm, but Fl. psychrophilum could not be isolated using inoculation on selective agar. The method presented here has the potential to detect low levels of Fl. psychrophilum in fish tissue and in water samples, and the technique can be a useful tool for understanding the epidemiology of Fl. psychrophilum.

关键词：脑/微生物学 DNA引物 DNA 细菌/分析鱼疾病/微生物学黄杆菌属/遗传学黄杆菌属/分离和提纯革兰氏阴性菌感染/微生物学革兰氏阴性菌感染/兽医学裂喉大麻哈鱼/微生物学聚合酶链反应/方法盐湖鲑鱼/微生物学鲑/微生物学鲑科/微生物学种特异性水微生物学动物

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Interaction between Aeromonas veronii and epithelial cells of spotted sand bass (Paralabrax maculatofasciatus) in culture

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第5期88卷 897-906页

作者： Guzman-Murillo, MA Merino-Contreras, ML Ascencio, F Biol Res Ctr Dept Marine Pathol La Paz 23000 Baja Calif Sur Mexico

An in vitro fish model to study the interaction between Aeromonas veronii and skin, gill and intestinal epithelial cells was developed using primary cultures of mucosal cells (isolated from healthy organisms). Primary cultures were exposed to Aeromonas veronii strain A186 isolated from a patient with severe gastrointestinal disease. Microbial adherence was assessed by a spectrophotometric evaluation of an enzyme-linked, biotin-streptavidin Aer. veronii cell-adhesion assay to confluent monolayers of epithelial cells on 96-well tissue culture plates. The three primary-culture cells are susceptible to Aer. veronii attachment, with the greatest binding affinity found in gills, and to a lesser extent, in skin and intestine epithelial. cells. Aer. veronii adherence was dependent on bacterial load and incubation time. The effect of glycoconjugates on Aer. veronii adhesion was investigated by pre-incubating Aer. veronii cells with monosaccharides, sialic acid-rich glycoproteins and sulphated polysaccharides. In addition, the participation of a 48-kDa Aer. veronii lectin (MCBP - mucosal constituents binding protein), with affinity for mucosal constituents, was evaluated as a putative adhesion factor of Aer. veronii to the mucosal epithelial cells of spotted sand bass by pre-incubating bacterial cells with rabbit polyclonal antibodies to Aer. veronii MCBP. Our study shows that primary-culture fish mucosal cells provide a suitable model for the study of the interactions between Aer. veronii and epithelial cells of the fish mucosa, and to study putative virulence factors of fish pathogens.

关键词：气单胞菌属/致病力气单胞菌属/生理学抗体/免疫学细菌黏附/药物作用鲈鱼细胞培养的酶联免疫吸附测定上皮细胞/微生物学鱼疾病/微生物学鱼类糖轭合物/药理学革兰氏阴性菌感染/兽医学动力学乳铁蛋白/药理学外源凝集素类/免疫学外源凝集素类/代谢时间因素动物

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Evidence that water transmits the disease caused by the fish pathogen Photobacterium damselae subsp damselae

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第3期88卷 531-535页

作者： Fouz, B Toranzo, AE Milán, M Amaro, C Univ Valencia Fac Biol Dept Microbiol E-46100 Burjassot Spain Univ Santiago de Compostela Dept Microbiol & Parasitol Burjassot Spain

The transmission through water of the disease caused by the fish pathogen, Photobacterium damselae subsp. damselae, as well as the role of the skin mucus in the initial steps of the infection, have been studied. All tested strains resisted the bactericidal activity of the mucus and showed an ability to adhere to it, but only those virulent by the intraperitoneal route were infective through water. Moribund fishes showed the typical signs of the disease: haemorrhaged areas on the body surface and ulcerative lesions with mucus degradation. These results suggest that the pathogen can be transmitted to fish through water and use the skin as a portal of entry.

关键词：细菌黏附鳗鱼疾病/微生物学鱼疾病/传播鲽形目革兰氏阴性菌感染/微生物学革兰氏阴性菌感染/传播革兰氏阴性菌感染/兽医学黏液/化学黏液/微生物学发光细菌属/致病力海水/微生物学皮肤/代谢皮肤/微生物学温度毒力水微生物学动物人类

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Difficulties in experimental infection studies with Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) using immersion, oral and anal challenges

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RESEARCH IN VETERINARY SCIENCE 2000年第2期69卷 165-169页

作者： Decostere, A Lammens, M Haesebrouck, F State Univ Ghent Dept Pathol Bacteriol & Poultry Dis B-9820 Merelbeke Belgium

This study was carried out in order to try to establish an efficacious and reliable experimental infection model for Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome, using contact, oral and anal challenges. Ten F psychrophilum strains of different origin were included. The influence of water temperature, scarification, water quality, stress and growth conditions of the pathogen on the experimental infection was assessed. For each challenge protocol, all strains failed to reproduce disease signs or mortality in rainbow trout (Oncorhynchus mykiss L.) fry. Histological and bacteriological examination of the skin, gills and internal organs of the fish 3 weeks following inoculation were found to be negative. Different hypotheses to explain the inability of the challenge models to reproduce the disease experimentally are discussed. (C) 2000 Harcourt Publishers Ltd.

关键词：疾病模型动物鱼疾病/微生物学黄杆菌属革兰氏阴性菌感染/兽医学口腔大麻哈鱼裂喉直肠应激/兽医学温度动物

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A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.)

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第7期65卷 3042-3047页

作者： Cook, M Lynch, WH Univ New Brunswick Dept Biol Fredericton NB E3B 6E1 Canada

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells, Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spanning to reduce vertical pathogen transmission.

关键词：抗菌药/药理学培养基 DNA 细菌/分析红霉素/药理学鱼疾病/微生物学革兰氏阳性菌感染/微生物学革兰氏阳性菌感染/兽医学肾/微生物学微球菌科/药物作用微球菌科/遗传学微球菌科/生长和发育微球菌科/分离和提纯卵巢/微生物学聚合酶链反应/方法 RNA 细菌/分析 RNA 信使/分析逆转录聚合酶链反应利福平/药理学盐湖鲑鱼/微生物学敏感性与特异性动物女(雌)性

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Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第9期65卷 3969-3975页

作者： Secades, P Guijarro, JA Univ Oviedo Fac Med Dept Biol Func Area MicrobiolIUBA E-33006 Oviedo Spain

A novel protease, hydrolyzing azocasein, was identified, purified, and characterized from the culture supernatant of the fish pathogen Yersinia ruckeri. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity,vas detected in peptone but not in Casamino Acid medium. Its synthesis appeared to be under catabolite repression and ammonium control. The protease was purified in a simple two-step procedure involving ammonium sulfate precipitation and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein indicated an estimated molecular mass of 47 kDa. The protease had characteristics of a cold-adapted protein, i.e., it was more active in the range of 25 to 42 degrees C and had an optimum activity at 37 degrees C. The activation energy for the hydrolysis of azocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42 degrees C. The enzyme had an optimum pH of around 8. Characterization of the protease showed that it required certain cations such as Mg2+ or Ca2+ for maximal activity and was inhibited by EDTA, 1,10-phenanthroline, and EGTA but not by phenylnethylsulfonyl fluoride. Two N-methyI-N-nitro-N-nitrosoguanidine mutants were isolated and analyzed;one did not show caseinolytic activity and lacked the 47-kDa protein, while the other was hyperproteolytic and produced increased amounts of the 47-kDa protein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal anti-47-kDa-protease serum, and zymogram analyses showed that protease activity was present in 8 of 14 strains tested and that two Y. ruckeri groups could be established based on the presence or absence of the 47-kDa protease.

关键词：酪蛋白类/代谢培养基内肽酶类/分离和提纯内肽酶类/代谢鱼疾病/微生物学鱼类免疫印迹法蛋白酶抑制药/药理学温度耶尔森菌属/酶学耶尔森菌属/生长和发育耶尔森菌感染/微生物学耶尔森菌感染/兽医学动物

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16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY 1999年第7期65卷 2942-2946页

作者： Osorio, CR Collins, MD Toranzo, AE Barja, JL Romalde, JL Univ Santiago De Compostela Fac Biol Dept Microbiol & Parasitol Santiago De Compostela 15706 Spain Univ Santiago De Compostela Fac Biol Inst Acuicultura Santiago De Compostela 15706 Spain Univ Reading Dept Food Sci & Technol Reading RG6 6AP Berks England

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data;thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.

关键词：鲈鱼 DNA引物 DNA 细菌/分析 DNA 细菌/遗传学 DNA 核糖体/分析 DNA 核糖体/遗传学鱼疾病/微生物学鱼类/微生物学基因 rRNA 革兰氏阴性菌感染/微生物学革兰氏阴性菌感染/兽医学分子序列数据发光细菌属/遗传学发光细菌属/生长和发育发光细菌属/分离和提纯聚合酶链反应/方法 RNA 核糖体 16S/遗传学敏感性与特异性序列分析 DNA 动物人类

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Systemic zygomycosis in farmed Tilapia fish

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JOURNAL OF COMPARATIVE PATHOLOGY 1999年第3期121卷 301-306页

作者： Wolf, JC Smith, SA Virginia Polytech Inst & State Univ Virginia Maryland Reg Coll Vet Med Dept Biomed Sci & Pathobiol Blacksburg VA 24061 USA

Decreased feed intake and persistent low-level mortality in a production tank of hybrid tilapia (Oreochromis niloticus x Oreochromis mossambicus x Oreochromis aureus) prompted the submission of three affected fish for diagnosis. Consistent macrosopical findings included multifocal dermal haemorrhage, excess abdominal fluid and an enlarged friable liver. On microscopical examination, broad non-septate fungal hyphae and chlamydospores were identified within numerous internal organs, often within and adjacent to blood vessels. The fungal hyphae were readily seen by silver staining (GMS) and the chlamydospores were stained deep magenta by the periodic acid-Schiff reaction. In addition to several species of Gram-negative bacteria, moderate growths of woolly white fungal colonies were obtained from the posterior part of the kidney in two of the three tilapia. These colonies were identified as a Rhizomucor sp. on the basis of the morphological characteristics of the sporulating fungi in culture. This represents the first reported episode of zygomycosis in fish. (C) 1999 Harcourt Publishers Ltd.

关键词：鱼疾病/微生物学鱼疾病/死亡率鱼疾病/病理学肾/微生物学肾/病理学肝/病理学毛霉菌病/微生物学毛霉菌病/死亡率毛霉菌病/病理学毛霉菌病/兽医学紫癜/诊断紫癜/微生物学紫癜/兽医学根毛霉菌属/分离和提纯根毛霉菌属/致病力罗非鱼属/微生物学接合菌病/微生物学接合菌病/死亡率接合菌病/病理学接合菌病/兽医学动物

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The morphology of Ichthyophonus sp in their mugilid hosts (Pisces: Teleostei) and following cultivation in vitro.: A light and electron microscopy study

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PARASITOLOGY RESEARCH 1999年第7期85卷 562-575页

作者： Franco-Sierra, A Alvarez-Pellitero, P CSIC Inst Acuicultura Torre Sal E-12595 Ribera De Cabanes Castellon Spain

The morphology of Ichthyophonus sp., a parasite of Mugil capito and Liza saliens, was investigated by light and transmission electron microscopy. The most frequent stage found in the fish hosts was the multinucleate spore, though germinating stages, hyphae, and endospores were also found. Different development patterns were observed in the media assayed for in vitro culture. Optimal growth and development were obtained in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum at pH 7. Ultrastructural features of multinucleate spores, both in the fish host and in culture, were a fibrillar thick wall and an electron-lucent matrix, with large glycogen granules, some electron-dense bodies, large vacuoles, lipid inclusions, and endoplasmic reticulum mainly appearing among the nuclei. Mitochondria with scarce tubulovesicular cristae were observed in the different stages, mainly near the wall and the germinating sites. Condensed heterochromatin was rarely seen. A nucleus-associated organelle (NAO) was frequently observed, and dictyosome cisternae and vesicles appeared in its vicinity.

关键词：培养基鱼疾病/微生物学氢离子浓度显微镜检查电子鲈形目/寄生虫学孢子真菌/超微结构接合菌亚纲/生长和发育接合菌亚纲/分离和提纯接合菌亚纲/超微结构动物牛

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