DNA probe for Aeromonas salmonicida.

A DNA fragment that is specific to Aeromonas salmonicida has been isolated from a genomic DNA library by differential hybridization. The specificity of this fragment as a DNA probe for A. salmonicida was shown by hybridization against reference strains and clinical isolates of A. salmonicida, related aeromonads, and species from several other bacterial genera. The sensitivity of detection by a polymerase chain reaction test, based on this fragment, was approximately two A. salmonicida cells.     

DNA probe for Aeromonas salmonicida.

A DNA fragment that is specific to Aeromonas salmonicida has been isolated from a genomic DNA library by differential hybridization. The specificity of this fragment as a DNA probe for A. salmonicida was shown by hybridization against reference strains and clinical isolates of A. salmonicida, related aeromonads, and species from several other bacterial genera. The sensitivity of detection by a polymerase chain reaction test, based on this fragment, was approximately two A. salmonicida cells.     

RAPD analysis of Aeromonas salmonicida and Aeromonas hydrophila

The randomly amplified polymorphic DNA (RAPD) technique was used to analyse the genetic differentiation of 13 strains of Aeromonas salmonicida subsp. salmonicida , and seven strains of Aer. hydrophila. Reproducible profiles of genomic DNA fingerprints were generated by polymerase chain reaction (PCR) using a single randomly designed primer. The RAPD profiles of all the non-motile aeromonads, Aer. salmonicida subsp. salmonicida were identical. However, profiles of the motile aeromonads, Aer. hydrophila differed between isolates. These findings reveal genomic homogeneity in Aer. salmonicida subsp. salmonicida and genetic variety in Aer. hydrophila strains.     

The development of random DNA probes specific for Aeromonas salmonicida

RAPD-PCR has been used to produce DNA probes for Aeromonas salmonicida . DNA hybridization studies showed that RAPD-PCR fragments of the same size did not necessarily hybridize to each other and therefore these sequences were not always homologous. However, a single RAPD-PCR fragment (designated 15e) was identified as being common to Aer. salmonicida . Subsequently, 15e was found to comprise five DNA fragments of similar size which differed in their nucleotide sequences. All five fragments were evaluated as DNA probes for the specific detection of Aer. salmonicida DNA: two hybridized specifically to DNA of all Aer. salmonicida isolates tested, including the four current subspecies and atypical isolates; one hybridized to subspecies salmonicida , achromogenes and masoucida , but not subspecies smithia ; one hybridized to subspecies salmonicida and achromogenes , but not subspecies masoucida or smithia ; and one hybridized to subspecies salmonicida , achromogenes and smithia , but not subspecies masoucida . It is believed that these fragments could be useful as non-radioactive probes for the safe and rapid diagnosis of these fish pathogens.     

The development of random DNA probes specific for Aeromonas salmonicida

RAPD-PCR has been used to produce DNA probes for Aeromonas salmonicida. DNA hybridization studies showed that RAPD-PCR fragments of the same size did not necessarily hybridize to each other and therefore these sequences were not always homologous. However, a single RAPD-PCR fragment (designated 15e) was identified as being common to Aer. salmonicida. Subsequently, 15e was found to comprise five DNA fragments of similar size which differed in their nucleotide sequences. All five fragments were evaluated as DNA probes for the specific detection of Aer. salmonicida DNA: two hybridized specifically to DNA of all Aer. salmonicida isolates tested, including the four current subspecies and atypical isolates; one hybridized to subspecies salmonicida, achromogenes and masoucida, but not subspecies smithia; one hybridized to subspecies salmonicida and achromogenes, but not subspecies masoucida or smithia; and one hybridized to subspecies salmonicida, achromogenes and smithia, but not subspecies masoucida. It is believed that these fragments could be useful as non-radioactive probes for the safe and rapid diagnosis of these fish pathogens.     

Restriction endonuclease fingerprinting analysis of Canadian isolates of Aeromonas salmonicida

Restriction endonuclease fingerprinting (REF) analysis was used to examine total cellular DNA prepared from 56 independent field isolates of the fish pathogen, Aeromonas salmonicida. DNA was digested singly with the restriction enzymes EcoRI and HindIII, and the resulting fragments separated by polyacrylamide gel electrophoresis and visualized by silver staining. The REF patterns of typical isolates of A. salmonicida subsp. salmonicida were distinct from those of A. hydrophila, A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and atypical isolates of A. salmonicida subsp. salmonicida. Differences between strains of typical A. salmonicida subsp. salmonicida could also be distinguished. Canadian isolates examined could be assigned to 1 of 12 different groups (REF groups), with the majority of the isolates belonging to REF groups 1 and 5. REF group 1 strains were isolated from British Columbia and New Brunswick while REF group 5 isolates were found in Ontario. None of the European strains examined had REF patterns identical to those of Canadian isolates. Based on REF analysis, there was little genetic heterogeneity detected among 23 isolates from two short-term studies of naturally occurring infections. Several different REF groups were seen among A. salmonicida collected over a 10-year period from coho salmon from the Credit River. Consistent with earlier biochemical and hybridization studies, the REF data suggest that A. salmonicida is a clonal pathogen. REF analysis can, however, permit the identification of subgroups, which may be useful in epidemiological studies.     

Survival of cells and DNA of Aeromonas salmonicida released into aquatic microcosms

D. DEERE, J. PORTER, R.W. PICKUP AND C. EDWARDS. 1996. The survival of the bacterial fish pathogen Aeromonas salmonicida , and persistence of its DNA, were monitored in aquatic microcosms using selective culture and most probable number PCR. Bacterial cells and naked DNA were released into natural non-sterile microcosms consisting of lake sediment overlayered with lake water. Two different types of surface sediment were used. One was sandy in character, taken from the shoreline whilst the other was a littoral loamy surface mud. Inoculated cells and naked DNA became undetectable from water overlayers within 4 weeks of release. Colony counts of Aer. salmonicida declined below detectable limits after 4 weeks in loamy sediment or 7 weeks in sandy sediment; however, naked DNA and DNA from released cells remained detectable for more than 13 weeks.     

Pulsed-field gel electrophoriesis analyis of Aeromonas salmonicida ssp. salmonicida

A total of 133 strains of Aeromonas salmonicida ssp. salmonicida, isolated from a wide variety of sources, were characterized by pulsed-field gel electrophoresis patterns. Sixteen profiles were demonstrated, with one profile being predominant in samples from all the countries and species of fish. Our results suggest a clonal distribution of this subspecies, with a predominant clone being responsible for most of the outbreaks worldwide.     

Siderophore production by Aeromonas salmonicida.

Growth under conditions of iron-restriction and the production of siderophores was examined in 21 typical and 14 atypical strains of Aeromonas salmonicida. With the exception of one atypical strain, all strains grew and multiplied in the presence of the high-affinity iron chelators ethylenediamine di(o-hydroxyphenylacetic acid), alpha, alpha'-dipyridyl or transferrin. Chrome azurol S agar was used to screen bacterial strains growing under these conditions for the production of siderophores. Siderophore production was detected only in the typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains, where they were associated with an iron-binding activity. The siderophore was extracted from iron-restricted culture supernatant of one strain by adsorption onto an XAD-7 resin; it behaved as a 2,3-diphenol-catechol in several colorimetric assays. The results indicate that although both typical and atypical strains of A. salmonicida grow and multiply under conditions of iron-restriction, they use different iron-uptake mechanisms, siderophore-mediated and siderophore-independent, respectively. In cross-feeding assays, growth of typical strains was stimulated only by homologous iron-restricted supernatant, suggesting strain differences in the siderophore produced. However, one strain produced a culture supernatant with growth-stimulating activity for other typical and also atypical strains.     

Survival of Aeromonas salmonicida in lake water

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Survival of Aeromonas salmonicida in river water

Abstract By definition, Aeromonas salmonicida is found in fish but never in surface water. However, this does not explain the reason for explosive out-breaks of furunculosis among populations of salmonid fish which have never been exposed to the disease. Evidence is presented, from laboratory-based experiments, which show that A. salmonicida survives in freshwater, beyond the period necessary for plate counts to reach zero. These cells may subsequently be re-activated by the addition of nutrient. It may be assumed, therefore, that A. salmonicida survives outside of fish, by entering a dormant phase.     

Acquisition of iron by Aeromonas salmonicida.

The ability of six typical and three atypical strains of Aeromonas salmonicida to sequester Fe3+ from the high-affinity iron chelators ethylenediaminedihydroxy-phenylacetic acid, lactoferrin, and transferrin was determined. Typical strains were readily able to sequester Fe3+ and used two different mechanisms. One mechanism was inducible and appeared to involve production of a low-molecular-weight soluble siderophore(s). Iron uptake by this mechanism was strongly inhibited by ferricyanide. One virulent strain displayed a second mechanism which was constitutive and required cell contact with Fe3+-lactoferrin or -transferrin. This strain did not produce a soluble siderophore(s) but could utilize the siderophore(s) produced by the other strain. Fe3+ uptake by this stripping mechanism was strongly inhibited by dinitrophenol. Atypical strains displayed a markedly reduced ability to sequester iron from high-affinity chelators, although one of them was able to utilize the siderophores produced by the typical strain. In all strains examined, Fe3+ limitation resulted in the increased synthesis of several high-molecular-weight outer membrane proteins.     

The Bombyx mori genome: analysis by DNA reassociation kinetics

The size and nucleotide sequence complexity of the Bombyx mori genome has been determined from the kinetics of reassociation of its DNA. Nonrepeated DNA comprises 55% of the genome, and the remainder is divided equally between sequences repeated roughly 500 and 50000 times. Non-repeated sequence DNA virtually free of repeated sequences was prepared by partial reassociation and subsequent fractionation on hydroxyapatite. The nucleotide sequence complexity of this component was determined relative to DNA from B. subtilis and E. coli. After correction for the size of the repeated sequence fraction, the DNA content of the Bombyx mori genome was calculated to be 0.53±0.02×10^?12 g. This value compares favorably with the DNA content of haploid B. mori spermatids and mature sperm determined cytophotometrically by Rasch (1973).     

异育银鲫源杀鲑气单胞菌杀鲑亚种的分离鉴定

【目的】分离鉴定江苏省扬州市养殖场异育银鲫患病病原。【方法】采用常规的理化特性和分子生物学的方法,对从濒死异育银鲫肝脏处分离到的菌株YZ-1进行表型生物学、分子生物学及药敏试验的系统研究。【结果】该菌株16S r RNA基因(序列长度1 446 bp,Gen Bank登录号为JX164202)与其它杀鲑气单胞菌16S r RNA基因一致性在99%-100%之间,构建发育树确定该菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.salmonicida)。人工回感可导致异育银鲫死亡。药敏试验结果显示:对头孢呋辛、复方新诺明、恩诺沙星等23种抗生素敏感;对阿米卡星、四环素、大观霉素、头孢拉定等11种抗生素中度敏感;对青霉素G、链霉素、庆大霉素、氟苯尼考、万古霉素等10种抗生素耐药。【结论】研究结果证实引起异育银鲫死亡的病原为杀鲑气单胞菌杀鲑亚种。     

Occurrence of a capsule in Aeromonas salmonicida

Aeromonas salmonicida grown in a medium with excess glucose as carbon source produces both capsular and exocellular polysaccharides. The capsular polysaccharide is composed of glucose, mannose, rhamnose, N-acetylmannosamine and mannuronic acid in the molar ratios of approximately 5:3:0.75:2:1. The extracellular polysaccharide is similarly constituted, but in the molar ratios of approximately 4.75:10.5:1.5:2:1. The capsular and exocellular polysaccharides did not cross-react with monoclonal antibodies against the A-layer or the O-antigen lipopolysaccharide.     

Assessing glycosidic linkage flexibility: Conformational analysis of the repeating trisaccharide unit of Aeromonas salmonicida

Summary A detailed conformational analysis was performed for the synthetic branched trisaccharide -d-Man-NAc-(14)-[-d-Glc-(13)]-l-Rha 1 which represents the repeating unit of the O-antigenic polysaccharide of Aeromonas salmonicida. The study was based on 26 experimental NOE curves from 1D transient NOE experiments, employing Gaussian-shaped inversion pulses at 600 MHz. Eight of the NOE curves were interglycosidic and thus useful for an analysis of glycosidic linkage orientations. Metropolis Monte Carlo (MMC) simulations and minimum-energy calculations with the program GEGOP were used to obtain theoretical NOE curves which were compared to the experimental ones. MMC simulations with different temperature parameters of 310, 600, 900 and 2000 K allowed identification of NOEs which are sensitive towards different conformation distributions-not only different conformations-at both glycosidic linkages in 1. A comparison of trisaccharide 1 with the constituent disaccharides -d-ManNAc-(14)-l-Rha 2 and -d-Glc-(13)-l-Rha 3 revealed effects of branching on glycosidic linkage flexibility. A quantitative evaluation was facilitated by the introduction of entropy-related flexibility parameters. Our study indicates a notable restriction of flexibility, especially at the (13) linkage in 1. Although overall flexibility in 1 is reduced as compared to the constituent disaccharides 2 and 3, it cannot be neglected altogether. In summary, combined transient NOE experiments and MMC simulations provide a simple approach to analyse glycosidic linkage flexibility.     

Structural studies of the core region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The core oligosaccharide structure of the in vivo derived rough phenotype of Aeromonas salmonicida subsp. salmonicida was investigated by a combination of compositional, methylation, CE-MS and one- and two-dimensional NMR analyses and established as the following: [carbohydrate: see text] where R=alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1--> or alpha-D-Galp-(1--> (approx. ratio 4:3). Comparative CE-MS analysis of A. salmonicida subsp. salmonicida core oligosaccharides from strains A449, 80204-1 and an in vivo rough isolate confirmed that the structure of the core oligosaccharide was conserved among different isolates of A. salmonicida.     

Molecular characterization and quantitative analysis of superoxide dismutases in virulent and avirulent strains of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A. salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB. The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O(2)(-)-catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative sodA were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of A. salmonicida subsp. salmonicida strain A449 when cultured in vitro and in vivo.