Beyond the A‐layer: adsorption of lipopolysaccharides and characterization of bacteriophage‐insensitive mutants of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is a fish pathogen that causes furunculosis. Antibiotherapy used to treat furunculosis in fish has led to resistance. Virulent phages are increasingly seen as alternatives or complementary treatments against furunculosis in aquaculture environments. For phage therapy to be successful, it is essential to study the natural mechanisms of phage resistance in A. salmonicida subsp. salmonicida. Here, we generated bacteriophage‐insensitive mutants (BIMs) of A. salmonicida subsp. salmonicida, using a myophage with broad host range and characterized them. Phage plaques were different depending on whether the A‐layer surface array protein was expressed or not. The genome analysis of the BIMs helped to identify mutations in genes involved in the biogenesis of lipopolysaccharides (LPS) and on an uncharacterized gene (ASA_1998). The characterization of the LPS profile and gene complementation assays identified LPS as a phage receptor and confirmed the involvement of the uncharacterized protein ASA_1998 in phage infection. In addition, we confirmed that the presence of an A‐layer at the bacterial surface could act as protection against phages. This study brings new elements into our understanding of the phage adsorption to A. salmonicida subsp. salmonicida cells.     

Comparision of a serological potency assay for furunculosis vaccines (Aeromonas salmonicida subsp. salmonicida) to intraperitoneal challenge in Atlantic salmon (Salmo salar L.)

Batch potency testing of salmonid vaccines is mainly performed by in vivo challenge, which requires a lot of animals and causes severe pain. Due to the animal welfare concerns associated with in vivo immunization challenge tests, methods which could refine, reduce or replace (3Rs) these tests are needed.The aim of this study was to assess the use of serological assay (immunization & antibody estimation with an enzyme-linked immunosorbent assay (ELISA) for batch potency testing of oil adjuvanted, inactivated commercial furunculosis vaccines. In total ten vaccines were included in the study: two commercial multi-component vaccines and two experimental single-component furunculosis vaccines with 5% and 20% antigen content (relative to the commercial vaccine), from two manufacturers. In addition two experimental single component vaccines based on A-layer positive and A-layer negative Aeromonas salmonicida respectively were included. Challenge and blood sampling were conducted 9 weeks post vaccination.There was a correlation between antibody response against A. salmonicida as measured by ELISA and protection in i.p. challenge.This study shows that the ELISA assay can be used for testing different vaccine formulations and can potentially replace in vivo challenge tests for batch potency testing of furunculosis vaccines.     

Potential Role of Specific Antibodies as Important Vaccine Induced Protective Mechanism against Aeromonas salmonicida in Rainbow Trout

Furunculosis caused by infection with Aeromonas salmonicida subsp. salmonicida has been a known threat to aquaculture for more than a century. Efficient prophylactic approaches against this disease are essential for continued growth of salmonid aquaculture. Since the introduction of successful oil-adjuvanted vaccines in the early 1990''s, a number of studies have been published on the protective as well as adverse effects of these vaccines. Most studies focus on vaccination of salmon (Salmo salar). However, rainbow trout (Oncorhynchus mykiss) are also very susceptible to infection and are vaccinated accordingly. In this study we have examined the protection against infection with a Danish strain of A. salmonicida in both vaccinated and non-vaccinated rainbow trout. A commercial and an experimental auto-vaccine were tested. The protective effects of the vaccines were evaluated through an A. salmonicida challenge 18 weeks post vaccination. Both vaccines resulted in a significantly increased survival in the vaccinated fish during a 28 day challenge period relative to non-vaccinated fish (P = 0.01 and P = 0.001 for the commercial and experimental vaccine, respectively). Throughout the entire experiment, the presence of specific antibodies in plasma was monitored using ELISA. A significant increase in specific antibody levels was seen in fish vaccinated with both vaccines during the 18 weeks between vaccination and challenge. Within 3 days post challenge, a significant decrease in specific antibodies occurred in vaccinated fish. A positive correlation was found between mean levels of specific antibodies pre challenge and overall survival. This correlation, along with the observed depletion of antibodies during the initial phase of infection, suggests that specific antibodies play an essential role in vaccine mediated protection against A. salmonicida in rainbow trout.     

Baltic salmon, Salmo salar, from Swedish river Lule älv is more resistant to furunculosis compared to rainbow trout

Background

Furunculosis, caused by Aeromonas salmonicida, continues to be a major health problem for the growing salmonid aquaculture. Despite effective vaccination programs regular outbreaks occur at the fish farms calling for repeated antibiotic treatment. We hypothesized that a difference in natural susceptibility to this disease might exist between Baltic salmon and the widely used rainbow trout.

Study Design

A cohabitation challenge model was applied to investigate the relative susceptibility to infection with A. salmonicida in rainbow trout and Baltic salmon. The course of infection was monitored daily over a 30-day period post challenge and the results were summarized in mortality curves.

Results

A. salmonicida was recovered from mortalities during the entire test period. At day 30 the survival was 6.2% and 34.0% for rainbow trout and Baltic salmon, respectively. Significant differences in susceptibility to A. salmonicida were demonstrated between the two salmonids and hazard ratio estimation between rainbow trout and Baltic salmon showed a 3.36 higher risk of dying from the infection in the former.

Conclusion

The finding that Baltic salmon carries a high level of natural resistance to furunculosis might raise new possibilities for salmonid aquaculture in terms of minimizing disease outbreaks and the use of antibiotics.     

Stable Transfection of the Diplomonad Parasite Spironucleus salmonicida

Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3×HA [where HA is hemagglutinin], 2× Escherichia coli OmpF linker and mouse langerin fusion sequence [2×OLLAS], 3×MYC) and purification of proteins (2× Strep-Tag II–FLAG tandem-affinity purification tag or streptavidin binding peptide–glutathione S-transferase [SBP-GST]) under the control of native or constitutive promoters. Three selectable gene markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr), and neomycin phosphotransferase (nptII), were successfully applied for the generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope tagged six proteins and confirmed their expression by Western blotting. Next, we demonstrated the utility of these vectors by recording the subcellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we described the creation of an S. salmonicida double transfectant suitable for colocalization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific, as well as general, diplomonad traits, benefiting research on both Giardia and Spironucleus.     

Characterization and mechanism of anti-Aeromonas salmonicida activity of a marine probiotic strain,Bacillus velezensis V4

The bacterium Aeromonas salmonicida is the causative agent of furunculosis, a systemic, ubiquitous disease of fish in the salmon family, characterized by high mortality and morbidity. Probiotics are a promising approach for prevention of furunculosis in aquaculture. A bacterial strain with anti-A. salmonicida properties, Bacillus velezensis V4, was isolated and the mechanisms underlying these properties were investigated. Anti-A. salmonicida compounds present in cell-free supernatant of V4 were purified and structurally identified as members of the iturin, macrolactin, and difficidin groups. The compounds contributed jointly to inhibition of A. salmonicida, and the diversity of the compounds was related to the versatility of their mode of action. Addition of the compounds to A. salmonicida cell suspensions reduced cell density. Analyses by confocal microscopy and scanning electron microscopy revealed cell membrane disruption, deletion of cellular content, and cell lysis of A. salmonicida. The V4 genome was sequenced, and gene clusters involved in synthesis of anti-Aeromonas compounds were detected and identified. A possible probiotic effect on growth performance of Oncorhynchus mykiss (rainbow trout) was investigated by addition of 0, 1, and 3 % (v/w) V4. Relative to control, mortality was reduced 27.25 % in the 1 % addition group and 81.86 % in the 3 % addition group. Feed coefficient ratio was reduced 19.49 % and weight gain ratio was increased 71.22 % in the 1 % addition group. Our findings demonstrate that V4 is an effective probiotic strain in O. mykiss and has clear potential for both control of furunculosis and growth promotion of aquaculture animals.

     

Cell surface hydrophobicity and macrophage association ofAeromonas salmonicida

Hydrophobic interaction chromatography and salt aggregation were used to compare the call surface hydrophobicity of strains of the fish pathogenAeromonas salmonicida which differed in their ability to produce the surface protein array known as A-layer. Presence of this superficial protein layer is crucial to the virulence of this organism and was found to coincide with a dramatic increase in cell surface hydrophobicity. Assays with in vitro cultured macrophages from either rainbow trout or mice revealed that this hydrophobic A-layer providedA. salmonicida cells with an enhanced ability to associate with phagocytic monocytes. This enhanced association was demonstrated in the absence of opsonizing antibody and may have important implications in the virulence ofA. salmonicida for fish.     

Studies of prevalence rate of furunculosis caused by infection by Aeromonas salmonicida in salmonids of the southern part of Sakhalin Island

Ichthyological studies of spawners of salmonids in the south of Sakhalin Island were studied. Cases of furunculosis disease were revealed. The agent of the disease Aeromonas salmonicida was isolated. Its morphological, physiological-biochemical, and antagonistic properties were studied, and the virulence of the isolated strains was determined. For supporting the species status of the studied strains of A. salmonicida, a molecular-genetic analysis was performed.     

Molecular Characterization of Plasmid-Mediated Oxytetracycline Resistance in Aeromonas salmonicida

Using broth conjugation, we found that 19 of 29 (66%) oxytetracycline (OT)-resistant isolates of Aeromonas salmonicida transferred the OT resistance phenotype to Escherichia coli. The OT resistance phenotype was encoded by high-molecular-weight R-plasmids that were capable of transferring OT resistance to both environmental and clinical isolates of Aeromonas spp. The molecular basis for antibiotic resistance in OT-resistant isolates of A. salmonicida was determined. The OT resistance determinant from one plasmid (pASOT) of A. salmonicida was cloned and used in Southern blotting and hybridization experiments as a probe. The determinant was identified on a 5.4-kb EcoRI fragment on R-plasmids from the 19 OT-resistant isolates of A. salmonicida. Hybridization with plasmids encoding the five classes (classes A to E) of OT resistance determinants demonstrated that the OT resistance plasmids of the 19 A. salmonicida isolates carried the class A resistance determinant. Analysis of data generated from restriction enzyme digests showed that the OT resistance plasmids were not identical; three profiles were characterized, two of which showed a high degree of homology.Aeromonas salmonicida is the causative agent of furunculosis, an economically important disease of salmonids (5). Control of this disease in aquaculture may be by prophylaxis (i.e., vaccination) (11) or by chemotherapy with a wide variety of antimicrobial compounds (5). Initially when sulfonamides were administered as food additives, they were successful (12). Subsequently, the usefulness of oxytetracycline (OT) was reported (29), and this antibiotic is still used extensively for control of furunculosis (5, 28). However, continued and widespread use of antibiotics has led to the development of resistant strains (3, 8, 15, 23). Moreover, plasmids encoding antibiotic resistance (R-plasmids) have been isolated from A. salmonicida (2, 15, 26, 27). A second generation of 4-quinolones–fluoroquinolones, notably enrofloxacin and sarofloxacin, effectively inhibits the pathogen and offers promise for the future since plasmid-encoded resistance to these compounds has not been described (7, 14, 19). However, mutational resistance to this class of compounds can develop in A. salmonicida (8, 21, 22, 32).As noted previously (28), it is difficult to make any definite conclusions about the impact of OT usage in aquaculture because of the methodological differences described in the literature. Transferable R-plasmids encoding resistance to tetracycline in A. salmonicida were described in 1971 (2, 31); subsequently, studies indicated that the frequency of OT-resistant strains of A. salmonicida was increasing. In a survey of 444 A. salmonicida isolates collected from Scottish salmon farms during 1988 to 1991, 53% of the isolates were resistant to OT (23). Using a random subsample of these isolates, researchers determined that 27% contained R-plasmids which could be transferred to Escherichia coli K-12 by conjugation (15), although no information concerning the molecular basis for the resistance was provided. Whereas there is no doubt that the results of such studies have value, it is necessary to clarify the situation with regard to the spread of R-plasmids encoding OT resistance within A. salmonicida populations. Only through precise molecular characterization of the genes encoding OT resistance and the plasmids that carry these resistance determinants will it become clear if aquaculture is facing a real threat from the use of antibiotics. To address this issue, a collection of OT-resistant isolates was used in experiments that examined the molecular basis for OT resistance and the potential environmental impact of the R-plasmids of these isolates.     

Significance of Extracellular Protease for Growth of a Heterotrophic Bacterium, Aeromonas salmonicida

The role of protease produced by a heterotrophic bacterium during growth was investigated with Aeromonas salmonicida, the pathogen of fish furunculosis, strain A-7301 and its protease-deficient mutant NTG-1 induced by mutagenesis. Strain A-7301 produced extracellular protease in a mixed amino acid medium (composed of Gly, Ala, Val, Ile, Leu, Thr, Ser, Cys, Met, Phe, Tyr, Lys, Arg, Pro, His, Try, Asp, Asn, Glu, and Gln at equal concentrations of 0.1 g/liter). Its multiplication rate was limited by the amounts of amino acids present, whereas strain NTG-1 showed no protease production despite considerable growth similar to that of A-7301. There was no difference between A-7301 and NTG-1 in amino acid requirements for growth, and seven amino acids (Gly, Ala, Val, Thr, Cys, Met, and His) were found to be indispensable. A defined level of the mixed amino acids (0.4 to 0.5 g/liter) was needed for A-7301 to initiate a large production of protease. Neither of the strains grew well in a casein medium, to which no amino acids were added. However, when a protease fraction obtained from extracellular products of A-7301 by DEAE-cellulose column chromatography was added, NTG-1 successfully reproduced in the casein medium. These results indicate that the extracellular protease plays an important role in supplying A. salmonicida cells with available amino acids as nutrients and that higher growth is closely associated with protease production which stimulates further reproduction.     

Simultaneous Detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, Three Major Fish Pathogens, by Multiplex PCR

A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.     

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.     

Functional Tn5393-Like Transposon in the R Plasmid pRAS2 from the Fish Pathogen Aeromonas salmonicida subspecies salmonicida Isolated in Norway

Tn5393c containing strA-strB was identified as part of R plasmid pRAS2 from the fish pathogen Aeromonas salmonicida subsp. salmonicida. This is the first time an intact and active transposon in the Tn5393 family has been reported in an ecological niche other than an agricultural habitat.     

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory.