Furunculosis In Fish Research Articles

Role of cold shock proteins B and D in Aeromonas salmonicida subsp. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus).

Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens affecting plant, insect, and mammalian hosts. However, their significance in bacterial pathogens of teleost fish remains unexplored. Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is a psychrotrophic pathogen and the causative agent of furunculosis in marine and freshwater fish. Four csp genes (cspB, cspD, cspA, and cspC) have been identified in the genome of A. salmonicida J223 (wild type). Here, we evaluated the role of DNA binding proteins, CspB and CspD, in A. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus). A. salmonicida ΔcspB, ΔcspD, and the double ΔcspBΔcspD mutants were constructed and characterized. A. salmonicida ΔcspB and ΔcspBΔcspD mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. A. salmonicida ΔcspD showed a slower growth at 28°C, biofilm formation, lower survival in low temperatures and freezing conditions (-20°C, 0°C, and 4°C), deficient in lipopolysaccharide synthesis, and low virulence in lumpfish. Additionally, ΔcspBΔcspD mutants showed less survival in the presence of bile compared to the wild type. Transcriptome analysis revealed that 200, 37, and 921 genes were differentially expressed in ΔcspB, ΔcspD, and ΔcspBΔcspD, respectively. In ΔcspB and ΔcspBΔcspD virulence genes in the chromosome and virulence plasmid were downregulated. Our analysis indicates that CspB and CspD mostly act as a transcriptional activator, influencing cell division (e.g., treB), virulence factors (e.g., aexT), and ultimately virulence.

Inactivated Aeromonas salmonicida impairs adaptive immunity in lumpfish (Cyclopterus lumpus).

Aeromonas salmonicida, a widely distributed aquatic pathogen causing furunculosis in fish, exhibits varied virulence, posing challenges in infectious disease and immunity studies, notably in vaccine efficacy assessment. Lumpfish (Cyclopterus lumpus) has become a valuable model for marine pathogenesis studies. This study evaluated several antigen preparations against A. salmonicida J223, a hypervirulent strain of teleost fish, including lumpfish. The potential immune protective effect of A. salmonicida bacterins in the presence and absence of the A-layer and extracellular products was tested in lumpfish. Also, we evaluated the impact of A. salmonicida outer membrane proteins (OMPs) and iron-regulated outer membrane proteins (IROMPs) on lumpfish immunity. The immunized lumpfish were intraperitoneally (i.p.) challenged with 104 A. salmonicida cells/dose at 8 weeks-post immunization (wpi). Immunized and non-immunized fish died within 2 weeks post-challenge. Our analyses showed that immunization with A. salmonicida J223 bacterins and antigen preparations did not increase IgM titres. In addition, adaptive immunity biomarker genes (e.g., igm, mhc-ii and cd4) were down-regulated. These findings suggest that A. salmonicida J223 antigen preparations hinder lumpfish immunity. Notably, many fish vaccines are bacterin-based, often lacking efficacy evaluation. This study offers crucial insights for finfish vaccine approval and regulations.

Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

MQM1, a bacteriophage infecting strains of Aeromonas salmonicida subspecies salmonicida carrying Prophage 3

Aeromonas salmonicida subsp. salmonicida is a Gam-negative bacterium responsible for furunculosis in fish. Because this aquatic bacterial pathogen has a rich reservoir of antibiotic-resistant genes, it is essential to investigate antibacterial alternatives, including the use of phages. Yet, we have previously demonstrated the inefficiency of a phage cocktail designed against A. salmonicida subsp. salmonicida strains due to a phage resistance phenotype associated to a prophage, namely Prophage 3. To bypass this resistance, one of the solutions is to isolate novel phages capable of infecting Prophage 3-bearing strains. Here we report on the isolation and characterization of the new virulent phage vB_AsaP_MQM1 (or MQM1), which is highly specific to A. salmonicida subsp. salmonicida strains. Phage MQM1 inhibited the growth of 01-B516, a strain carrying Prophage 3, including when combined to the previous phage cocktail. MQM1 infected 26 out of the 30 (87%) Prophage 3-bearing strains tested. Its linear dsDNA genome contains 63,343 bp, with a GC content of 50.2%. MQM1 genome can encode 88 proteins and 8 tRNAs, while no integrase or transposase-encoding genes were found. This podophage has an icosahedral capsid and a non-contractile short tail. We suggest that MQM1 may be a good addition to future phage cocktails against furunculosis to resolve the Prophage 3-resistance issue.

The Sigma Factor AsbI Is Required for the Expression of Acinetobactin Siderophore Transport Genes in Aeromonas salmonicida.

Aeromonas salmonicida subsp. salmonicida (A. salmonicida), a Gram-negative bacterium causing furunculosis in fish, produces the siderophores acinetobactin and amonabactins in order to extract iron from its hosts. While the synthesis and transport of both systems is well understood, the regulation pathways and conditions necessary for the production of each one of these siderophores are not clear. The acinetobactin gene cluster carries a gene (asbI) encoding a putative sigma factor belonging to group 4 σ factors, or, the ExtraCytoplasmic Function (ECF) group. By generating a null asbI mutant, we demonstrate that AsbI is a key regulator that controls acinetobactin acquisition in A. salmonicida, since it directly regulates the expression of the outer membrane transporter gene and other genes necessary for Fe-acinetobactin transport. Furthermore, AsbI regulatory functions are interconnected with other iron-dependent regulators, such as the Fur protein, as well as with other sigma factors in a complex regulatory network.

On-site detection of fish furunculosis by combining DNAzyme and carboxyl-functionalized graphene.

Furunculosis, which is caused by Aeromonas salmonicida, can induce septicemia, leading to the rapid death of fishes belonging to Salmonidae, Cyprinidae, and Fuscheridae, and lamprey. Targeting A. salmonicida, five DNAzyme sequences with the highest enrichment rates were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The enrichment rates were 34.78, 23.60, 8.91, 2.89, and 2.34%, respectively. The DNAzyme with the highest activity, named D-AS-2, showed specificity and sensitivity. D-AS-2 was combined with carboxyl-functionalized graphene to construct a biosensor, which showed good fluorescence response to scabies lesion samples. The diagnostic procedure was completed in <2min and can be used for the on-site diagnosis of fish diseases. A low-cost, rapid, simple, and highly specific biosensor for the diagnosis of furunculosis was established based on DNAzyme and carboxyl-functionalized graphene.

Furunculosis in fish

This datasheet on furunculosis in fish covers Identity, Overview, Associated Diseases, Pests or Pathogens, Distribution, Hosts/Species Affected, Diagnosis, Pathology, Epidemiology, Impacts, Prevention/Control, Further Information.

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes.

Aeromonas salmonicida subsp. salmonicida (hereafter A.salmonicida) is the aetiological agent of furunculosis in marine and freshwater fish. Once A.salmonicida invade the fish host through skin, gut or gills, it spreads and colonizes the head kidney, liver, spleen and brain. A.salmonicida infects leucocytes and exhibits an extracellular phase in the blood of the host; however, it is unknown whether A.salmonicida have an intraerythrocytic phase. Here, we evaluate whether A.salmonicida infects Atlantic salmon (Salmo salar) erythrocytes in vitro and in vivo. A.salmonicida did not kill primary S.salar erythrocytes, even in the presence of high bacterial loads, but A.salmonicida invaded the S.salar erythrocytes in the absence of evident haemolysis. Naïve Atlantic salmon smolts intraperitoneally infected with A.salmonicida showed bacteraemia 5days post-infection and the presence of intraerythrocytic A.salmonicida. Our results reveal a novel intraerythrocytic phase during A.salmonicida infection.

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.

Phenotype of Aeromonas salmonicida sp. salmonicida cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants and its virulence in rainbow trout (Oncorhynchus mykiss).

Precise deletion of genes related to virulence can be used as a strategy to produce attenuated bacterial vaccines. Here, we study the deletion of the cyclic-3',5'-adenosine monophosphate (cAMP) receptor protein (Crp) in Aeromonas salmonicida, the aetiologic agent of furunculosis in marine and freshwater fish. The Crp protein is a conserved global regulator, controlling physiology processes, like sugar utilization. Deletion of the crp gene has been utilized in live attenuated vaccines for mammals, birds and warm water fish. Here, we characterized the crp gene and reported the effect of a crp deletion in A.salmonicida virulent and non-virulent isolates. We found that A.salmonicida Δcrp was not able to utilize maltose and other sugars, and its generation time was similar to the wild type. A.salmonicida ∆crp showed a higher ability of cell invasion compared to the wild type. Fish challenges showed that A.salmonicida ∆crp is ~6 times attenuated in Oncorhynchus mykiss and conferred protective immunity against the intraperitoneal challenge with A.salmonicida wild type. We concluded that deletion of A.salmonicida crp influences sugar utilization, cell invasion and virulence. Deletion of crp in A.salmonicida could be considered as part of an effective strategy to develop immersion live attenuated vaccines against furunculosis.

Antimicrobial effect of the Biotronic® Top3 supplement and efficacy in protecting rainbow trout (Oncorhynchus mykiss) from infection by Aeromonas salmonicida subsp. salmonicida

Demand for more environmentally friendly practices have led to the adoption of several feed supplements by the fish farming industry. In the present study, we investigated a commercially available formula that includes a mixture of three compounds: organic acids, a phytochemical and Biomin® Permeabilizing Complex. This mixture demonstrated antimicrobial properties in vitro and was able to inhibit growth of multiple species of aquatic bacterial pathogens, including Aeromonas salmonicida.Bacterial challenge was performed using A. salmonicida and three exposure routes: intra-peritoneal injection, immersion, and cohabitation. Mortality rates following infection by injection were significantly decreased in the fish that had received the supplemented feed. Fish infected through the other routes did not show a significant difference in mortality.In term of farming performance, while the fish that had received the feed supplement showed an improvement in weight gain and final weight, these changes were not found to be statistically significant. Similarly, no significant difference was observed in the feed conversion ratio. The results of this study suggest that this feed supplement may be effective at protecting rainbow trout from fish furunculosis.

せっそう病（非定型を含む）

Aeromonas salmonicida subsp. salmonicida was originally described agent of furunculosis in salmonid fish at the end of 19th century, while atypical A. salmonicida has been isolated from a wide range of cultivated and wild fish species, non-salmonids as well as salmonids, inhabiting fresh water, brackish water and marine environments. Although A. salmonicida subsp. salmonicida is homogeneous, atypical strains are more heterogeneous with respect to biochemical characteristics and growth conditions. Based on the nucleotide sequences of 16S rDNA, Japanese isolates of atypical A. salmoicida from different fish species can be divided into four groups. This review describes the outlines and the topics of the diseases.

An investigation of the bactericidal activity of selected essential oils to Aeromonas spp.

Diseases of fishes caused by Aeromonas spp. are common, have broad host ranges and may cause high mortality. Treatments of captive-reared populations using antimicrobials are limited with concerns for bacterial resistance development and environmental dissemination. This study was done to determine whether selected plant-derived essential oils were bactericidal to Aeromonas spp. Initially, twelve essential oils were evaluated using a disk diffusion assay to an isolate of A. salmonicida subsp. salmonicida, cause of fish furunculosis. The greatest zones of inhibition were obtained with oils of cinnamon Cinnamomum cassia, oregano Origanum vulgare, lemongrass Cymbopogon citratus and thyme Thymus vulgaris. Minimum bactericidal concentrations (MBC’s) were determined for these four oils, Allimed® (garlic extract, Allium sativum) and colloidal silver to sixty-nine isolates representing nine Aeromonas spp. The lowest mean MBCs (0.02–0.04%) were obtained with three different sources of cinnamon oil. MBCs for three sources of oregano and lemongrass oils ranged from 0.14% to 0.30% and 0.10% to 0.65%, respectively, and for two thyme oils were 2.11% and 2.22%. The highest concentration (5%) of Allimed® tested resulted in MBCs to twelve isolates. A concentration of silver greater than 15mg/L would be required to determine MBCs for all but one isolate.

Non-ribosomal peptide synthetase AsbD is expressed in iron-limited conditions in the bacterial pathogen Aeromonas salmonicida

Iron is an essential nutrient for most living bacteria. One of the main mechanisms which allow bacterial pathogens to obtain iron is siderophore-mediated iron acquisition. Aeromonas salmonicida subsp. salmonicida, the aetiological agent of furunculosis in fish produces a catecholate siderophore under iron-limiting conditions. A. salmonicida siderophore biosynthesis genes include genes that encode non-ribosomal peptide synthetases. In this study using a siderophore biosynthesis gene cluster previously described, the asbD gene which encodes a non-ribosomal peptide synthetase was studied. Total protein of null mutant and wild-type strains, in iron-repleted and iron-depleted conditions, was extracted and loaded onto polyacrylamide gel. Only the wild-type strain in iron-depleted conditions expressed a 160-KDa protein. These results confirmed that asbD is an iron-regulated gene, and AsbD protein is expressed only in iron-limited conditions.

The impact of warming on the appearance of furunculosis in fish of the James Bay region, Quebec, Canada

A regional climate change impact assessment was conducted which investigated the timing of the first appearance of furunculosis caused by the bacterium, Aeromonassalmonicida, in fish of the Ouje-Bougoumou region of northern Quebec, Canada. Historical data were used to assess whether there was a temporal relationship between increased temperatures (observed climate data) and the appearance of furunculosis (traditional environmental knowledge was used to provide context). To project future impacts of climate change, climate models and lake models were used. Regression analysis revealed a significant, positive temporal trend in mean air temperature. The temperature range conducive to A. salmoncida survival coincided with the first appearance of furunculosis. In addition, it is predicted that lake temperatures will remain suitable for the presence of A. salmonicida into the future; it is likely that the disease will persist throughout the twenty-first century. Climate change appears to be a factor explaining the onset of furunculosis; however, other factors/stressors cannot be discounted, such as, the effects past mining activities near the lakes of the Ouje-Bougoumou region have had on the body burden of contaminants in the fish (and the potential effects on the fish immune system).

Effect of Lactococcus lactis CLFP 100 and Leuconostoc mesenteroides CLFP 196 on Aeromonas salmonicida Infection in Brown Trout (Salmo trutta)

Aeromonas salmonicida is the etiological agent of furunculosis in salmonid fish. This pathogen is important from an epizootic perspective because fish surviving an outbreak can remain lifelong asymptomatic carriers, serving as reservoirs of infection. As a result, the early detection and the control of infection are essential to prevent the spread of new furunculosis outbreaks. We have thus analyzed the effect of probiotic administration on the incidence of A. salmonicida in brown trout (Salmo trutta), that were subjected to temperature stress. Treatment with probiotic strains (Lactococcus lactis CLFP 100 and Leuconostoc mesenteroides CLFP 196) resulted in a higher survival rate after challenge, activation of phagocytic cells in the head kidney, and a lower rate of pathogen proliferation in the intestine as determined by real-time PCR.

Identification of iron regulated genes in the fish pathogen Aeromonas salmonicida subsp. salmonicida: Genetic diversity and evidence of conserved iron uptake systems

In this study we applied the Fur titration assay (FURTA) to isolate iron regulated genes in Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in fish. We have identified genes for siderophore biosynthesis and for ferri-siderophore transport, some of which have been previously described in this species. A gene for a hitherto uncharacterized functional hemin receptor HutE was identified, but its inactivation did not affect significantly the use of hemin as sole iron source, suggesting that redundant genes encoding hemin receptors exist in the A. salmonicida genome. Additional FURTA positive clones contained genes encoding a Flavoprotein, a transcriptional regulator protein, a DNA binding protein Fis, as well as genes encoding putative DapD and Flp/Fap-pilin proteins. A screening of gene distribution demonstrated that all the analyzed strains shared genes for siderophore biosynthesis and transport and for heme utilization, indicating that these two systems of iron acquisition are a conserved trait in this subspecies, whereas other genes are not common to all the isolates. Thus, the Fur regulon of A. salmonicida subsp. salmonicida includes both conserved and differentially occurring genes, suggesting that the genetic diversity within this fish pathogen might be higher than expected.

Identification of heme uptake genes in the fish pathogen Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in fish, can use heme as the sole iron source. We applied the Fur Titration Assay to isolate a cluster including six genes hutAZXBCD that showed similarity to heme uptake genes of other Gram-negative bacteria, and three genes orf123 of unknown function. The spatial organization of these nine genes, arranged in five transcriptional units, was similar to that of a homologous cluster in A. hydrophila. When a TonB system was provided, this cluster allowed Escherichia coli 101ESD (an ent mutant, unable to synthesize enterobactin) to utilize hemin and hemoglobin as iron sources. Mutation of hutB, a gene that encodes a predicted periplasmic hemin-binding protein, caused a drastic defect in the ability of A. salmonicida to grow with hemin as unique source of iron. Interestingly, a mutant for hutA gene (encoding the outer membrane hemin receptor) showed initially a reduced ability to grow with hemin as sole iron source, but after 24 h it achieved growth levels similar to parental strain. Thus mutation of hutA could not abolish the growth with hemin as iron source, suggesting that redundant outer membrane heme transport functions might be encoded in the A. salmonicida genome.

Identification of Siderophore Biosynthesis Genes Essential for Growth of Aeromonas salmonicida under Iron Limitation Conditions

Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, produces a catechol-type siderophore under iron-limiting conditions. In this study, the Fur titration assay (FURTA) was used to identify a cluster of six genes, asbG, asbF, asbD, asbC, asbB, and asbI, encoding proteins similar to components of the siderophore biosynthetic machinery in other bacteria. Reverse transcriptase PCR analyses showed that this cluster consists of four iron-regulated transcriptional units. Mutants with deletions in either asbD (encoding a multidomain nonribosomal peptide synthetase), asbG (encoding a histidine decarboxylase), or asbC (encoding a predicted histamine monooxygenase) did not grow under iron-limiting conditions and did not produce siderophores. Growth of the DeltaasbG strain under iron starvation conditions was restored by addition of histamine, suggesting that the siderophore in this species could contain a histamine-derived moiety. None of the mutants could grow in the presence of transferrin, indicating that A. salmonicida uses the catechol-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein. All 18 A. salmonicida strains analyzed by DNA probe hybridization were positive in tests for the presence of the asbD gene, and all of them promoted the growth of asbD, asbG, and asbC mutants, suggesting that this siderophore-mediated iron uptake system is conserved among A. salmonicida isolates. This study provides the first description of siderophore biosynthesis genes in this fish pathogen, and the results demonstrate that the asbD, asbG, and asbC genes are necessary for the production of a catecholate siderophore that is essential for the growth of A. salmonicida under iron limitation conditions.

Characterization of an ADP-ribosyltransferase toxin (AexT) from Aeromonas salmonicida subsp. salmonicida.

An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.