Isolates Of Aeromonas Salmonicida Research Articles

Release of chromatin extracellular traps by phagocytes of Atlantic salmon, Salmo salar (Linnaeus, 1758)

Neutrophils release chromatin extracellular traps (ETs) as part of the fish innate immune response to counter the threats posed by microbial pathogens. However, relatively little attention has been paid to this phenomenon in many commercially farmed species, despite the importance of understanding host-pathogen interactions and the potential to influence ET release to reduce disease outbreaks. The aim of this present study was to investigate the release of ETs by Atlantic salmon (Salmo salar L.) immune cells. Extracellular structures resembling ETs of different morphology were observed by fluorescence microscopy in neutrophil suspensions in vitro, as these structures stained positively with Sytox Green and were digestible with DNase I. Immunofluorescence studies confirmed the ET structures to be decorated with histones H1 and H2A and neutrophil elastase, which are characteristic for ETs in mammals and other organisms. Although the ETs were released spontaneously, release in neutrophil suspensions was stimulated most significantly with 5 μg/ml calcium ionophore (CaI) for 1 h, whilst the fish pathogenic bacterium Aeromonas salmonicida (isolates 30411 and Hooke) also exerted a stimulatory effect. Microscopic observations revealed bacteria in association with ETs, and fewer bacterial colonies of A. salmonicida Hooke were recovered at 3 h after co-incubation with neutrophils that had been induced to release ETs. Interestingly, spontaneous release of ETs was inversely associated with fish mass (p < 0.05), a surrogate for age. Moreover, suspensions enriched for macrophages and stimulated with 5 μg/ml CaI released ET-like structures that occasionally led to the formation of large clumps of cells. A deeper understanding for the roles and functions of ETs within innate immunity of fish hosts, and their interaction with microbial pathogens, may open new avenues towards protecting cultured stocks against infectious diseases.

Agreement between the categorization of isolates of Aeromonas salmonicida and Yersinia ruckeri by disc diffusion and MIC tests performed at 22℃.

Standard disc diffusion and MIC test procedure were used to investigate the susceptibility of two hundred and fifty-one isolates collected from infected fish in France to florfenicol, oxolinic acid and tetracycline. The tests were performed at 22±2℃ and for the 177 Yersinia ruckeri they were read after 24-28hr incubation and for the 74 Aeromonas salmonicida isolates they were read after 44-48hr. Applying epidemiological cut-off values to the susceptibility data generated in these tests, the isolates were categorized as wild-type or non-wild-type. The agent-specific categories into each isolate were placed on the basis of the data generated by the two methods were in agreement in 98% of the determinations made. It is argued that, with respect to categorising isolates, disc diffusion and MIC methods can be considered as equally valid at this temperature and after both periods of incubation.

AsaGEI2d: a new variant of a genomic island identified in a group of Aeromonas salmonicida subsp. salmonicida isolated from France, which bears the pAsa7 plasmid.

Genomic islands (Aeromonas salmonicida genomic islands, AsaGEIs) are found worldwide in many isolates of Aeromonas salmonicida subsp. salmonicida, a fish pathogen. To date, five variants of AsaGEI (1a, 1b, 2a, 2b and 2c) have been described. Here, we investigate a sixth AsaGEI, which was identified in France between 2016 and 2019 in 20 A.salmonicida subsp. salmonicida isolates recovered from sick salmon all at the same location. This new AsaGEI shares the same insertion site in the chromosome as the other AsaGEI2s as they all have a homologous integrase gene. This new AsaGEI was thus named AsaGEI2d, and has five unique genes compared to the other AsaGEIs. The isolates carrying AsaGEI2d also bear the plasmid pAsa7, which was initially found in an isolate from Switzerland. This plasmid provides resistance to chloramphenicol thanks to a cat gene. This study reveals more about the diversity of the AsaGEIs.

Marine Plastics from Norwegian West Coast Carry Potentially Virulent Fish Pathogens and Opportunistic Human Pathogens Harboring New Variants of Antibiotic Resistance Genes.

To our best knowledge this is the first study characterizing fish pathogens isolated from marine plastics from the West coast of Norway for their potential for pathogenicity using whole genome sequencing. Marine plastic polymers identified as polyethylene, polyethylene/ethylene vinyl acetate copolymer and polypropylene, yielded a total of 37 bacterial isolates dominated by Pseudomonas spp. (70%). Six isolates representing either fish pathogens or opportunistic human pathogens were selected for whole genome sequencing (WGS). These included four isolates belonging to Aeromonas spp., one Acinetobacter beijerinckii isolate and one Morganella morganii isolate. Three Aeromonas salmonicida isolates were potentially virulent and carried virulence factors involved in attachment, type II and type VI secretion systems as well as toxins such as aerA/act, ahh1, ast, hlyA, rtxA and toxA. A. salmonicida and Acinetobacter beijerinckii carried new variants of antibiotic resistance genes (ARGs) such as β-lactamases and chloramphenicol acetyltransferase (catB), whereas Morganella morganii carried several clinically relevant ARGs. Our study shows that marine plastics carry not only potentially virulent fish pathogens but also multidrug resistant opportunistic human pathogens like M. morganii and may serve as vectors for transport of these pathogens in the marine environment.

Pharmacokinetic Data Show That Oxolinic Acid and Flumequine Are Absorbed and Excreted Rapidly From Plasma and Tissues of Lumpfish.

This study examined the uptake, tissue distribution and elimination of the antibacterial agents oxolinic acid and flumequine in lumpfish (Cyclopterus lumpus L.) by use of LC-MS/MS following a single oral administration of 25 mg/kg fish given in feed. Lumpfish are increasingly used as cleaner fish for removal of sea lice on commercially farmed salmon. The production of lumpfish is successful, but there are challenges with bacterial infections and the number of antibacterial treatments has increased in recent years. As the lumpfish is a novel species to farming, there is a need for pharmacokinetic data and establishment of protocols for efficient antibacterial treatment. The current study describes the pharmacokinetic properties of oxolinic acid and flumequine in lumpfish. Absorption of oxolinic acid was moderate and was characterized by a calculated peak plasma concentration (Cmax) of 2.12 μg/ml after 10.3 h (Tmax) and an elimination half-life (t1/2β) of 21 h. Area under curve (AUC) and AUC from 0 to 24 h (AUC0−24h) were calculated to be 60.9 and 34.0 h μg/ml, respectively. For flumequine, plasma Cmax was found to be 2.77 μg/ml after 7.7 h (Tmax) with t1/2β of 22 h. The area under the curve (AUC) and AUC from 0 to 24 h (AUC0−24) were calculated as 104.3 and 50.3 h μg/ml, respectively. Corresponding Cmax values in muscle, liver, and head-kidney for oxolinic acid were 4.01, 3.04, and, 4.68 μg/g, respectively and Tmax of 11.1, 9.2, and 10.0 h, respectively. For flumequine, Cmax values of 4.16, 4.01, and 7.48 μg/g were obtained in muscle, liver, and head kidney, respectively, with corresponding Tmax values of 10.2, 10.3, and 6.0 h. Antimicrobial susceptibility values as determined by minimum inhibitory concentration (MIC) analyses against 28 isolates of Aeromonas salmonicida isolated from diseased lumpfish ranged from 0.06 to 15 μg/ml for oxolinic acid and 0.024 to 6.25 μg/ml for flumequine. Bimodal distributions in susceptibility to both oxolinic acid and flumequine were observed. The combination of pharmacokinetic properties and MIC data make possible calculation of efficient treatment doses, which are needed to improve the welfare of lumpfish and minimize development of antibiotic resistant bacteria.

Pharmacokinetics of florfenicol in lumpfish (Cyclopterus lumpus L.) after a single oral administration

Farming of lumpfish for biological removal of sea lice from farmed Atlantic salmon has expanded rapidly in Europe and Canada over the last 5–6 years and the lumpfish has become an economically important species. There are, however, health challenges associated with bacterial diseases. In recent years, there has been an increase in antibacterial treatments prescribed for this fish species despite a lack of knowledge regarding pharmacokinetics and effect of treatment with different antibiotics.The present study examined the uptake, tissue distribution, metabolism and elimination of the antibacterial agent florfenicol in lumpfish (Cyclopterus lumpus L.) following a single oral administration of 10 mg/kg fish given in feed. Plasma, head kidney, liver and muscle from six fish were sampled at each time point and analysed by liquid chromatography/mass spectrometry (LC-MS). Absorption was moderate for this drug characterised by a calculated peak plasma concentration (Cmax) of 3.55 μg/ml obtained after 21.2 hours (Tmax) and the elimination halflife (t1/2β) relatively extended in plasma at 30 hours. Area under curve (AUC) and AUC from 0 to 24 hours (AUC0-24h) were calculated to be 248 and 61 h μg/ml, respectively. Cmax was calculated to 2.99 μg/g in muscle, 2.54 μg/g in liver and 4.70 μg/g in head kidney with corresponding Tmax of 22.1, 26.4 and 19.4 h, respectively. The main metabolite, florfenicol-amine was found in low concentrations in plasma and all tissues examined. The minimum inhibition concentrations (MIC) for florfenicol of 28 of Aeromonas salmonicida isolates from diseased lumpfish ranged from 0.39 to 1.56 μg/ml.The pharmacokinetical data presented here make an important basis for efficient antibacterial treatment for lumpfish using florfenicol and for calculation of suitable withdrawal time. Knowledge of florfenicol pharmacokinetics, combined with determination of antibiotic resistance among fish pathogenic bacteria and the effect of antibacterial agents on diseased lumpfish in vivo are important for the welfare of lumpfish and prevention of resistant bacteria.

Aeromonas salmonicida isolates: Attachment ability and sensitivity to four disinfectants

Aeromonas spp. are closely associated with food spoilage, and often enter the food chain through cross-contamination during processing from biofilms on food-contacted surfaces. The attachment abilities of two high-spoilage Aeromonas salmonicida isolates were compared. The sensitivities of these isolates in biofilm to four disinfectants, namely sodium hypochlorite, chlorine dioxide, acidic electrolyzed water (AEW) and neutral electrolyzed water (NEW), were assayed. The results indicated that isolate 1425 showed higher attachment ability as compared to isolate 1435 at initial attachment (30 min) under a short-term attachment scenario (5 h), reaching more than 5 log CFU/cm2. Under a long-term attachment scenario (7 days), isolate 1425 formed a mature biofilm after 5 days of incubation, with the highest observed cell numbers of nearly 9 log CFU/cm2. The dynamics of biofilm formation of isolate 1435 were relatively stable, with cell population ranging from 6.25 to 7 log CFU/cm2. The composition of the extracellular polymeric substances and the morphology of the mature biofilm were identified by Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) and Confocal Laser Scanning Microscopy (CLSM). The mature biofilm formed by isolate 1425 showed more resistance to tested disinfectants than that of isolate 1435. Sodium hypochlorite (200 mg/L) and AEW (40 mg/L) showed greater lethal effects than the other disinfectants. Moreover, 40 mg/L of AEW was as effective as 200 mg/L of sodium hypochlorite. These findings indicated that the biofilm formed by microorganisms associated with food spoilage and safety could not be ignored during food processing and that AEW may be a promising alternative to sodium hypochlorite for the elimination of such biofilms.

Comparative pathogenicity of Vibrio spp., Photobacterium damselae ssp. damselae and five isolates of Aeromonas salmonicida ssp. achromogenes in juvenile Atlantic halibut (Hippoglossus hippoglossus)

Juvenile Atlantic halibut (~100mg, Hippoglossus hippoglossus) were exposed to Vibrio proteolyticus, a Vibrio spp. isolate, Photobacterium damselae ssp. damselae and five different isolates of Aeromonas salmonicida ssp. achromogenes via an hour-long bath immersion to ascertain their variation in pathogenicity to this fish species. Results were analysed using Kaplan-Meier survival analysis. Analysis of the data from challenges using A.salmonicida ssp. achromogenes revealed three survival values of zero and a spread of values from 0 to 28.43. Challenges using a Vibrio spp isolate, V.proteolyticus and P.damselae resulted in Kaplan-Meier survival estimates of 31.21, 50.41 and 57.21, respectively. As all bacterial species tested could induce juvenile halibut mortalities, they must all be considered as potential pathogens. However, the degree of pathogenicity of A.salmonicida is isolate dependent.

VapA (A-layer) typing differentiates Aeromonas salmonicida subspecies and identifies a number of previously undescribed subtypes.

Sequence variation in a region of the virulence array protein gene (vapA; A-layer) was assessed in 333 ('typical' and 'atypical') isolates of the fish pathogenic bacterium Aeromonas salmonicida. Resulting similarity dendrograms revealed extensive heterogeneity, with nearly all isolates belonging to either of 14 distinct clusters or A-layer types. All acknowledged A.salmonicida subspecies (except ssp. pectinolytica, from which no vapA sequence could be obtained) were clearly separated, and notably, all isolates phenotypically identified as ssp. salmonicida formed a distinct and exclusive A-layer type. Additionally, an array of un-subspeciated atypical strains formed several equally prominent clusters, demonstrating that the concept of typical/atypical A.salmonicida is inappropriate for describing the high degree of diversity evidently occurring outside ssp. salmonicida. Most representatives assessed in this study were clinical isolates of spatiotemporally diverse origins, and were derived from a variety of hosts. We observed that from several fish species or families, isolates predominantly belonged to certain A-layer types, possibly indicating a need for host-/A-layer type-specific A.salmonicida vaccines. All in all, A-layer typing shows promise as an inexpensive and rapid means of unambiguously distinguishing clinically relevant A.salmonicida subspecies, as well as presently un-subspeciated atypical strains.

Protein expression and transcription profiles of three strains of Aeromonas salmonicida ssp. salmonicida under normal and iron-limited culture conditions.

BackgroundAeromonas salmonicida is an important fish pathogen that produces a wide and varied array of virulence factors. Here we used iron deprivation by addition of the chelator 2’2-dipyridyl to induce the expression of several such virulence factors in three isolates of Aeromonas salmonicida (one avirulent and two virulent). By using SDS-PAGE followed by mass spectrometry, we identified proteins that appeared differentially expressed under these conditions. The differential transcription of the identified gene products were subsequently measured by reverse transcription quantitative real-time PCR (RT-qPCR).ResultsOur initial screening using SDS-PAGE identified five proteins that appeared differentially expressed in virulent and avirulent isolates or, within the same isolates, between bacteria cultivated under iron-rich or iron-deprived conditions. The transcription of the genes coding for these proteins were subsequently quantified by RT-qPCR. Results of this analysis demonstrated that the gene coding for alkyl hydroperoxide reductase (AhpC), a protein involved in oxidative stress response, was transcribed at a higher rate in the virulent strain as compared to the avirulent strain. Additionally, it was observed that addition of an iron chelator to the culture medium lead to a reduction of the transcription levels of the regulatory histone-like nucleoid structuring protein (H-NS). This was consistent in all three isolates. On the other hand, the transcription levels of the virulence array protein (VapA) and the protein ATP-synthetase F (ATPF) displayed only limited changes, despite being the dominant component of a protein fraction that displayed changes during the preliminary SDS-PAGE screening. This was true regardless of the culture conditions and of the isolates considered. Finally, transcription of the enzyme enolase was upregulated in the iron-deprived broths in all isolates.ConclusionsWe identified several genes differentially expressed under culture conditions known to lead to the overexpression of virulence factors. In addition, we identified alkyl hydroperoxide as being overexpressed in the virulent isolates compared to the avirulent isolates. The results from this study will contribute to enhance our understanding of the virulence of A. salmonicida and may suggest new directions for further research.

A rapid method of improving the criteria being used to interpret disc diffusion antimicrobial susceptibility test data for bacteria associated with fish diseases

This work presents an analysis of the consequence of using data sets containing small numbers of disc diffusion inhibition zones to calculate epidemiological cut-off values. Twenty replicate data sets that contained 10, 20, 40, 60, 80 and 100 zone size measurements were obtained by randomly sampling a previously published data set that consisted of inhibition zone measurements for discs containing 30 µg florfenicol, 30 µg oxytetracycline and 2 µg oxolinic acid against 217 isolates of Aeromonas salmonicida. Application of normalised resistance interpretation allowed epidemiological cut-off values to be estimated from all of these 360 data sets. The precision associated with the replicate cut-off values was a linear function of the log of the number of data points used to generate them. The frequency with which false sensitive and false resistance errors would have resulted from the application of the cut-off values generated from small data sets using normalised resistance interpretation were compared with those that would have resulted from the application of the breakpoints that have been reported as being currently in use. This comparison demonstrated that most laboratories could improve their interpretation of the disc diffusion data they obtain for any species by performing normalised resistance interpretation of their own data for that species. This would be true even if they possessed inhibition zone data for as few as ten members of that species.

Transferable, multiple antibiotic and mercury resistance in Atlantic Canadian isolates of Aeromonas salmonicida subsp. salmonicida is associated with carriage of an IncA/C plasmid similar to the Salmonella enterica plasmid pSN254

The aim of this study was to examine the molecular basis for multiple antibiotic and mercury resistance in Canadian isolates of Aeromonas salmonicida subsp. salmonicida. Phenotypic and genotypic methods were employed to identify plasmid-associated antibiotic and mercury resistance genes and to determine the organization of those genes in multidrug-resistant (MDR) A. salmonicida isolates. The MDR phenotype was transferable via conjugation using Escherichia coli, Aeromonas hydrophila and Edwardseilla tarda as recipients. Antibiotic and mercury resistance genes were carried by a conjugative IncA/C plasmid. Three distinct antibiotic resistance cassettes were characterized; first a class I integron containing an aadA7 gene encoding for an aminoglycoside-3'-adenyltransferase, the second cassette showed 99.9% nucleotide sequence homology to a cassette previously identified in the Salmonella enterica IncA/C plasmid pSN254, containing floR, tetA, sulII and strA/strB sequences. The third cassette showed 100% nucleotide sequence similarity to a transposon-like element, containing a bla(CMY-2) beta-lactamase in association with sugE and blc sequences. This element is known to be widely distributed among clinical and food-borne Salmonella and other Enterobacteriaceae throughout Asia and the United States. Mercury resistance was linked to the presence of a mer operon that showed 100% nucleotide sequence homology to the mer operon carried by plasmid pSN254. Each MDR A. salmonicida isolate carried the same plasmid, which was related to plasmid pSN254. This is the first report of plasmid-mediated florfenicol-resistant A. salmonicida in North America. In addition, it is the first report of a plasmid-associated AmpC beta-lactamase sequence in a member of the Aeromonadaceae.

Carbohydrate analysis and serological classification of typical and atypical isolates of Aeromonas salmonicida: A rationale for the lipopolysaccharide-based classification of A. salmonicida

The cell envelope of Aeromonas salmonicida contains a lipopolysaccharide (LPS) essential for the physical integrity and functioning of bacterial cell membrane. Using a recently developed in-source fragmentation technique, we screened 39 typical and atypical isolates of A. salmonicida and established their O-chain polysaccharide structure by capillary electrophoresis–mass spectrometry (CE–MS), compositional and linkage analyses and comparison to the previously determined O-chain polysaccharide structure of A. salmonicida strain A449. These studies have demonstrated that A. salmonicida isolates fall into three distinct structural types, types A–C, based on chemical structures of their respective O-chain polysaccharide components. Subsequent immunoblotting and serological studies with salmon polyclonal antisera produced to formalin-fixed cells of A. salmonicida strains A449, N4705 and 33659 representing three structural types A–C revealed that variations in the O-chain polysaccharide structure have led to significant serological differences between strains belonging to type A and non-type A, where non-type A species include chemically separated structural types B and C. Due to the presence of common antigenic determinants shared by their respective O-chain polysaccharide components, serological cross-reactions were observed between A. salmonicida strains belonging to structural types B and C. These findings suggest the possibility of developing LPS-based classification system of A. salmonicida sub-species consisting of two serologically distinct types, type A and non-type A.

Epidemiologic cutoff values for antimicrobial agents against Aeromonas salmonicida isolates determined by frequency distributions of minimal inhibitory concentration and diameter of zone of inhibition data

"Epidemiologic cutoff values for antimicrobial agents against Aeromonas salmonicida isolates determined by frequency distributions of minimal inhibitory concentration and diameter of zone of inhibition data" published on 01 Apr 2007 by American Veterinary Medical Association.

Epidemiologic cutoff values for antimicrobial agents against Aeromonas salmonicida isolates determined by frequency distributions of minimal inhibitory concentration and diameter of zone of inhibition data

To develop epidemiologic cutoff values by use of frequency distributions for susceptibility to 4 antimicrobial agents when tested against a representative population of a major aquaculture pathogen, Aeromonas salmonicida. 217 typical and atypical A salmonicida isolates obtained from 20 states and 12 countries. Species identification of A salmonicida isolates was confirmed by detection of specific nucleotide sequences by use of a PCR assay. Minimal inhibitory concentration (MIC) and diameter of the zone of inhibition for oxytetracycline, ormetoprim-sulfadimethoxine, oxolinic acid, and florfenicol were determined for each isolate in accordance with standardized antimicrobial susceptibility testing methods that have been approved by the Clinical and Laboratory Standards Institute for bacterial isolates from aquatic animals. Susceptibility data were tabulated in a scattergram and analyzed by use of error rate bounding. Susceptibility tests for oxytetracycline, ormetoprim-sulfadimethoxine, and oxolinic acid revealed 2 distinct populations of bacteria. Isolates tested against florfenicol clustered into a single population. Oxolinic acid susceptibility data revealed higher MICs in the non-United States A salmonicida isolates. Slow-growing (atypical) A salmonicida isolates were generally more susceptible than typical isolates for all antimicrobials, except oxolinic acid. Use of frequency distributions of susceptibility results to develop epidemiologic cutoff values appears to be applicable to aquatic isolates. Frequency distributions of susceptibility results for A salmonicida revealed clear divisions between isolate susceptibilities. This type of data, considered in conjunction with pharmacokinetic and efficacy data, may be useful for developing clinical breakpoints for use in aquaculture.

Analysis of plasmid profiles of Aeromonas salmonicida isolates by pulsed field gel electrophoresis

Plasmid constitutions of Aeromonas salmonicida isolates were characterised by flat-bed and pulsed field gel electrophoresis. Resolution of plasmids by pulsed field gel electrophoresis was greater and more consistent than that achieved by flat-bed gel electrophoresis. The number of plasmids separated by pulsed field gel electrophoresis varied between A. salmonicida isolates, with five being the most common number present in the isolates used in this study. Plasmid profiles were diverse and the reproducibility of the distances migrated facilitated the use of principal components analysis for the characterisation of the isolates. Isolates were grouped according to the number of plasmids supported. Further principal components analysis of groups of isolates supporting five and seven plasmids showed a spatial separation of plasmids based upon distance migrated. Principal components analysis of plasmid profiles and antimicrobial minimum inhibitory concentrations could not be correlated suggesting that resistance to antimicrobial agents is not associated with either one plasmid or a particular plasmid constitution.

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc r) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance ( tet) gene carried [ tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS 6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn 1721, and their ability to transfer their Tc r plasmids to an Escherichia coli recipient at 15 °C. Five of the isolates, with genetically related Tc r plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7 × 10 −4 to 2.8 × 10 −6 per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 °C is the lowest temperature that has been documented in bacteria.

Protection against atypical furunculosis in Atlantic halibut, Hippoglossus hippoglossus (L.); comparison of a commercial furunculosis vaccine and an autogenous vaccine.

Atlantic halibut, Hippoglossus hippoglossus (L.), was shown to be sensitive to infection by three different isolates of Aeromonas salmonicida ssp. achromogenes in pre-challenge tests using intraperitoneal (i.p.) and intramuscular (i.m.) injections as well as bath challenges. A commercial furunculosis vaccine, Alphaject 1200, and an autogenous vaccine, AAS, based on the challenge strain, induced immune protection as shown in challenge tests 8 weeks post-immunization. The survival rate of vaccinated fish after i.p. challenge was 100%, whereas mortality of control fish was 61%. Employing i.m. challenge, relative percentage survival induced by the furunculosis vaccine and the AAS vaccine was 47 and 44, respectively. Mortality of i.m. injected controls was 68%. Vaccinated fish behaved normally following vaccination but the weight gain was significantly reduced in vaccinated fish 8 weeks post-vaccination compared with control fish receiving phosphate-buffered saline. At the same time, intra-abdominal adhesions were observed in fish injected with either of the two vaccines or adjuvant alone. Antibody response against A. salmonicida ssp. achromogenes was detected in sera from fish receiving either vaccine.

Characterization of class 1 integrons associated with R-plasmids in clinical Aeromonas salmonicida isolates from various geographical areas.

Class 1 integrons were found in 26 of 40 antibiotic-resistant isolates of the fish pathogen Aeromonas salmonicida from Northern Europe and North America. Three different dhfr genes, conferring trimethoprim resistance, and one ant(3")1a aminoglycoside resistance gene were identified as gene inserts. The gene cassettes tended to be conserved among isolates from a particular geographical area. Nineteen isolates transferred R-plasmids carrying different tet determinants to Escherichia coli in filter mating assays, and in 15 cases, the class 1 integrons were co-transferred. Transferable sulphadiazine, trimethoprim and streptomycin resistances were invariably encoded by integrons. It thus appears that integron-encoded antibiotic resistance genes contribute substantially to the horizontal spread of antimicrobial resistance within this species, being associated with conjugative plasmids.

Non‐pigment‐producing isolates of Aeromonas salmonicida subspecies salmonicida: isolation, identification, transmission and pathogenicity in Atlantic salmon, Salmo salar L.

Four non‐pigment‐producing isolates and two pigment‐producing isolates of Aeromonas salmonicida sp. salmonicida were isolated from the head‐kidney of diseased farmed Atlantic salmon, Salmo salar L. The cultural, morphological and biochemical features of the isolates were compared with those of reference strains. Injection and cohabitation experiments were performed. The only difference between the non‐pigment‐producing isolates and the pigment producing reference strains of A. salmonicida ssp. salmonicida was the inability of the former to produce pigment. In the injection experiments, the investigated non‐pigment‐producing isolate produced a significantly higher mortality compared with the mortality caused by the reference strain, whereas no difference in mortality was detected in the cohabitation experiments.