Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes.
The phylogenetic relationships of all known species of the genus Aeromonas, and especially Aeromonas bestiarum and Aeromonas salmonicida, were investigated on 70 strains using the rpoD sequence, which encodes the sigma70 factor. This analysis was complemented with the sequence of gyrB, which has already proven useful for determining the phylogenetic relationships in the genus. Nucleotide sequences of rpoD and gyrB showed that both genes had similar substitution rates (< 2 %) and a similar number of variable positions (34 % for rpoD versus 32 % for gyrB). Strain groupings by analysis of rpoD, gyrB and a combination of both genes were consistent with the taxonomic organization of all Aeromonas species described to date. However, the simultaneous analysis of both clocks improved the reliability and the power to differentiate, in particular, closely related taxa. At the inter-species level, gyrB showed a better resolution for differentiating Aeromonas sp. HG11/Aeromonas encheleia and Aeromonas veronii/Aeromonas culicicola/Aeromonas allosaccharophila, while rpoD more clearly differentiated A. salmonicida from A. bestiarum. The analysis of rpoD provided initial evidence for clear phylogenetic divergence between the latter two species. (+info)
Structural studies of the capsular polysaccharide and lipopolysaccharide O-antigen of Aeromonas salmonicida strain 80204-1 produced under in vitro and in vivo growth conditions.
Aeromonas salmonicida is a pathogenic aquatic bacterium and the causal agent of furunculosis in salmon. In the course of this study, it was found that when grown in vitro on tryptic soy agar, A. salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide. A combination of 1D and 2D NMR methods, including a series of 1D analogues of 3D experiments, together with capillary electrophoresis-electrospray MS (CE-ES-MS), compositional and methylation analyses and specific modifications was used to determine the structure of these polysaccharides. Both polymers were shown to be composed of linear trisaccharide repeating units consisting of 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 3-[(N-acetyl-L-alanyl)amido]-3,6-dideoxy-D-glucose[3-[(N-acetyl-L-alanyl)amido]-3 -deoxy-D-quinovose, Qui3NAlaNAc] and 2-acetamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D-quinovose, QuiNAc) and having the following structure: [-->3)-alpha-D-GalpNAcA-(1-->3)-beta-D-QuipNAc-(1-->4)-beta-D-Quip3NAlaNAc-(1-]n, where GalNAcA is partly presented as an amide and AlaNAc represents N-acetyl-L-alanyl group. CE-ES-MS analysis of CPS and O-chain polysaccharide confirmed that 40% of GalNAcA was present in the amide form. Direct CE-ES-MS/MS analysis of in vivo cultured cells confirmed the formation of a novel polysaccharide, a structure also formed in vitro, which was previously undetectable in bacterial cells grown within implants in fish, and in which GalNAcA was fully amidated. (+info)
Genetic diversity among A-proteins of atypical strains of Aeromonas salmonicida.
The virulence array protein gene A (vapA) encoding the A-protein subunit of the surface layer of 23 typical and atypical strains of Aeromonas salmonicida from salmonids and marine fish species were sequenced, and the deduced A-protein sequences compared. The A-proteins of the typical A. salmonicida ssp. salmonicida strains were shown to be identical, while amino acid variability was revealed among A-proteins of atypical strains. The highest amino acid variability appears to be in a predicted surface exposed region and is believed to result in antigenic differences among the atypical strains of A. salmonicida. (+info)
A deterministic model for the dynamics of furunculosis in chinook salmon Oncorhynchus tshawytscha.
Studies were undertaken to determine the parameters of transmission of Aeromonas salmonicida in chinook salmon Oncorhynchus tshawytscha, and to develop a deterministic model of the dynamics of experimental furunculosis. For determination of disease transmission coefficient (beta), disease-related mortality rate (alpha) and natural mortality rate (gamma), fish in 70 tanks (approximately 42 fish tank(-1)) were each exposed to a single infectious donor fish, 7 tanks were randomly selected daily and all individuals were examined for the presence of A. salmonicida in the kidney. The proportion of susceptible (S), infected (I) and removed (R, dead) individuals were determined daily. The parameters beta, alpha, gamma, reproductive ratio (R0) and threshold density were estimated to be 0.0214 infected ind. d(-1), 0.29 infected ind. d(-1), 0.00015 ind. d(-1), 3.23 and 13.56 ind., respectively. Using these parameters, a deterministic disease model of A. salmonicida infection as a cause of furunculosis was constructed. The net rate at which new individuals became infected (the incidence rate) per unit time was proportional to S x I x beta. The model-produced data for S were significantly associated with experimental data (r2 = 0.92). In brief, a simple SIR (susceptible-infected-removed) model was successfully utilized to simulate observed data (+info)
Attenuated virulence of an Aeromonas salmonicida subsp. salmonicida type III secretion mutant in a rainbow trout model.
Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a severe systemic disease affecting salmonid fish. This bacterium contains a type III protein secretion system that is responsible for the secretion and translocation of the ADP-ribosylating toxin, AexT, into the cytosol of fish cells. This study showed that inactivation of the type III secretion system by marker-replacement mutagenesis of the gene ascV, which encodes an inner-membrane component of the type III secretion system, attenuated virulence in a rainbow trout model. The isogenic ascV deletion mutant was phagocytosed by peripheral blood leukocytes but the wild-type (wt) A. salmonicida subsp. salmonicida isolate was not. Histological examination of fish experimentally infected with the wt bacterium revealed extensive tissue necrosis and bacterial aggregates in all organs examined, including the heart, kidney and liver, indicating that the isolate established a systemic infection. Cumulative mortality of fish experimentally infected with the wt bacterium reached 88%. In contrast, no mortality was observed among fish infected with the same dose of the ascV mutant, and histological examination of fish infected with this strain revealed healthy organs. The results indicate that the type III secretion system of A. salmonicida subsp. salmonicida is required to establish systemic infection. (+info)
Standardization of a broth microdilution susceptibility testing method to determine minimum inhibitory concentrations of aquatic bacteria.
A multiple laboratory study was conducted in accordance with the standards established by the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS), for the development of quality control (QC) ranges using dilution antimicrobial susceptibility testing methods for bacterial isolates from aquatic animal species. QC ranges were established for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 when testing at 22, 28 and 35 degrees C (E. coli only) for 10 different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline and trimethoprim/sulfamethoxazole). Minimum inhibitory concentration (MIC) QC ranges were determined using dry- and frozen-form 96-well plates and cation-adjusted Mueller-Hinton broth. These QC ranges were accepted by the CLSI/NCCLS Subcommittee on Veterinary Antimicrobial Susceptibility Testing in January 2004. This broth microdilution testing method represents the first standardized method for determining MICs of bacterial isolates whose preferred growth temperatures are below 35 degrees C. Methods and QC ranges defined in this study will enable aquatic animal disease researchers to reliably compare quantitative susceptibility testing data between laboratories, and will be used to ensure both precision and inter-laboratory harmonization. (+info)
Metabolic changes in Atlantic salmon exposed to Aeromonas salmonicida detected by 1H-nuclear magnetic resonance spectroscopy of plasma.
1H-NMR (nuclear magnetic resonance)-based chemometric methods have been applied for the first time to investigate changes in the plasma metabolite profiles of Atlantic salmon Salmo salar as a result of exposure to Aeromonas salmonicida subsp. salmonicida, a Gram-negative bacterium that is the etiological agent of furunculosis. Plasma samples were obtained from salmon that survived 21 d post exposure to A. salmonicida, and from a control group maintained under similar conditions. 1D 1H-NMR spectra were acquired and principal components analysis (PCA) was used to assess differences between the spectral profiles of plasma from salmon that survived an A. salmonicida challenge, and non-infected controls. PCA enables simultaneous comparison of spectra, presenting a simplified overview of the relationship between spectral data, where spectra cluster based on metabolite profile similarities and differences; information regarding the metabolite variations can therefore be readily deciphered. The major metabolite changes responsible for the spectral differences were related to modification in the lipoprotein profile and choline-based residues, with minor changes in carbohydrates, glycerol, trimethylamine-N-oxide and betaine. These changes indicated that exposure to A. salmonicida induced a characteristic biochemical response which could be used to determine the health status of salmon. This study suggests that with further development this metabolite profiling technique may be a useful tool for diagnosis of disease states in salmon and could provide a better understanding of the host-pathogen relationship which at present is poorly understood for A. salmonicida and Atlantic salmon. (+info)
Characterisation of atypical Aeromonas salmonicida infection in Arctic charr Salvelinus alpinus and European grayling Thymallus thymallus.
Cultured stocks of Arctic charr Salvelinus alpinus and European grayling Thymallus thymallus are vulnerable to infection by achromogenic atypical Aeromonas salmonicida (AAS). In Finland, natural stocks of both fish species have to be supported by restocking, and AAS infection poses a threat to successful restocking because no preventive means are available. In this study, we analysed AAS isolates from Arctic charr and European grayling and from other sources genetically, and characterised the signs and pathology of AAS infection in Arctic charr and European grayling both under farming conditions and after experimental challenge. AAS outbreaks were recorded in 1 fish farm over an 8 yr period. Among various salmonid fishes under farming conditions, only Arctic charr and European grayling were susceptible to AAS infection. The disease caused by AAS could be reproduced in both species using the same AAS strain in an experimental challenge. The course of the disease and pathology of natural and experimental AAS infection differed between the 2 species, even though only 1 strain was used for challenge. Isolates of AAS from Arctic charr and European grayling were genetically identical within a single river water basin. However, genetic heterogeneity was observed among the isolates from different water basins. In both species, AAS caused systemic infection. The results suggest that the same AAS strain could be used to develop a vaccine to protect both Arctic charr and European grayling from AAS infection. (+info)