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.     

First description of atypical furunculosis in freshwater farmed Atlantic salmon,Salmo salar L., in Chile

We report the first isolation, identification and characterization of a group of Chilean strains of atypical Aeromonas salmonicida isolated from freshwater farmed Atlantic salmon, Salmo salar. Affected fish showed superficial ulcers and pale liver with or without petechial haemorrhages. Outbreaks of the disease occurred in two farms in the south of Chile about 2200 km apart. Five strains were isolated in pure culture and identified by serological assays and immunofluorescence tests as belonging to Aeromonas salmonicida. Although the bacterial isolates were phenotypically homogeneous, minor differences with the reference strain A. salmonicida subsp. salmonicida ATCC 33658 were noted. Three specific primer sets and partial 16S rRNA gene sequencing allowed the identification of the Chilean isolates as atypical A. salmonicida, with A. salmonicida subsp. achromogenes and A. salmonicida subsp. masoucida as their closest relatives (100% sequence similarity). Molecular typing indicated that the atypical isolates belong to two genetic groups that were associated with the geographical origin.     

Positive correlation between Aeromonas salmonicida vaccine antigen concentration and protection in vaccinated rainbow trout Oncorhynchus mykiss evaluated by a tail fin infection model

Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin‐killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin‐killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710‐1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60‐s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.     

Exploring the efficacy of vaccine techniques in juvenile sablefish,Anoplopoma fimbria

Wild sablefish, Anoplopoma fimbria, are a valuable commercial species whose populations are declining. Fortunately, sablefish are excellent species for commercial aquaculture. Sablefish raised under high‐density conditions in the marine environment require the use of efficacious vaccines to control disease. Sablefish impacted by disease in net pens may have poor flesh quality and high mortality during grow‐out. As a result, disease can cause financial hardship for sablefish aquaculture operators. The efficacy of a multivalent vaccine preparation for sablefish, administered either by intraperitoneal (i.p.) injection or by immersion, against atypical and typical Aeromonas salmonicida, the causative agents of atypical and typical furunculosis, respectively, was examined. A. salmonicida can affect sablefish at any age and size. Consequently, an efficacious vaccine that can be appropriately and optimally administered to all life stages is desirable. Sablefish vaccinated by immersion at ~1.5 or ~4.5 g with a whole‐cell multivalent vaccine were not protected against either typical or atypical A. salmonicida. Factors that may have contributed to the ineffectiveness of the immersion vaccine are discussed. By contrast, the relative per cent survival (RPS) or potency of the whole‐cell multivalent vaccine injected i.p. in juvenile sablefish at ~50 g against typical and atypical A. salmonicida was 94.3% and 81.7% respectively. The high RPS values indicated that the vaccine successfully initiated an immune response in sablefish upon a second encounter with the pathogen.     

Systemic infection in European perch with thermoadapted virulent Aeromonas salmonicida (Perca fluviatilis)

In non‐salmonid fish, Aeromonas salmonicidacan cause local infections with severe skin ulcerations, known as atypical furunculosis. In this study, we present a systemic infection by a virulent A. salmonicidain European perch (Perca fluviatilis).This infection was diagnosed in a Swiss warm water recirculation aquaculture system. The isolate of A.  salmonicida encodes a type three secretion system (TTSS) most likely located on a plasmid similar to pAsa5/pASvirA, which is known to specify one of the main virulence attributes of the species A. salmonicida. However, the genes specifying the TTSS of the perch isolate show a higher temperature tolerance than strains isolated from cold‐water fish. The function of the TTSS in virulence was verified in a cytotoxicity test using bluegill fry and epithelioma papulosum cyprinid cells.     

Bioassay‐guided isolation and identification of active compounds from Macleaya microcarpa (Maxim) Fedde against fish pathogenic bacteria

Four alkaloids (Sanguinarine, 6‐Methoxyl‐dihydro‐chelerythrine, Cryptopine and β‐Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using bioassay‐guided isolation method, and the inhibitory activity of ethanolic extract, various fractions and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method and the microdilution assay method respectively. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram‐negative strains than other three alkaloids, with MIC values of 12.5 mg L^?1 for A. hydrophila and 50 mg L^?1 to other pathogenic bacteria. Followed by 6‐methoxyl‐dihydro‐chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg L^?1 for A. hydrophila, 100 mg L^?1 for V. harveyi, and 125 mg L^?1 for both V. anguillarum and A. salmonicida. Cryptopine and β‐allocryptopine revealed similar inhibitory activity with MIC values of 100 mg L^?1 for A. hydrophila and 200 mg L^?1 for other three bacterial species. These finding provided evidence that extract, as well as isolated compounds from M. microcarpa might be potential sources novel antibacterial agents for the treatment of fish infectious diseases.     

Analysis of DNA relationships among Aeromonas species by RAPD (randomly amplified polymorphic DNA) typing

Genetic differences between a collection of aeromonads were studied in two laboratories by analysis of randomly amplified polymorphic DNA (RAPD). A single randomly designed primer, generated reproducible profiles of genomic DNA in both laboratories for Aeromonas salmonicida subspecies salmonicida, although the profiles differed between laboratories. Analysis of atypical strains of A. salmonicida and isolates of the A. hydrophila group produced scattered profiles in both laboratories. The uniform fingerprints produced for A. salmonicida subspecies salmonicida indicate genomic homogeneity. The scattered RAPD profiles of the motile aeromonads demonstrate the genomic diversity of this group. A group of unspeciated motile aeromonads gave uniform fingerprints, suggesting the possibility of a genomically homogeneous species. Although the RAPD technique is susceptible to the effects of minor technical variations, this study has demonstrated that where there is DNA similarity, it can be recognized, and where there is diversity, differentiation can be made. RAPD promises to be useful in epidemiological studies for rapid identification of bacteria where a source of reference DNA is available and may be useful in preliminary investigations of relatedness within groups.     

Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non‐salmonid fish

Michigan's fisheries rely primarily upon the hatchery propagation of salmonid fish for release in public waters. One limitation on the success of these efforts is the presence of bacterial pathogens, including Aeromonas salmonicida, the causative agent of furunculosis. This study was undertaken to determine the prevalence of A. salmonicida in Michigan fish, as well as to determine whether biochemical or gene sequence variability exists among Michigan isolates. A total of 2202 wild, feral and hatchery‐propagated fish from Michigan were examined for the presence of A. salmonicida. The examined fish included Chinook salmon, Oncorhynchus tshawytscha (Walbaum), coho salmon, O. kisutcha (Walbaum), steelhead trout, O. mykiss (Walbaum), Atlantic salmon, Salmo salar L., brook trout, Salvelinus fontinalis (Mitchill), and yellow perch, Perca flavescens (Mitchill). Among these, 234 fish yielded a brown pigment‐producing bacterium that was presumptively identified as A. salmonicida. Further phenotypic and phylogenetic analyses identified representative isolates as Aeromonas salmonicida subsp. salmonicida and revealed some genetic and biochemical variability. Logistic regression analyses showed that infection prevalence varied according to fish species/strain, year and gender, whereby Chinook salmon and females had the highest infection prevalence. Moreover, this pathogen was found in six fish species from eight sites, demonstrating its widespread nature within Michigan.     

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.     

Atypical Aeromonas salmonicida infection in naturally and experimentally infected cod, Gadus morhua L.

Cod, Gadus morhua L., of wild origin, were reared at different temperatures for 12 months. During this period, moribund and newly dead fish were examined and samples collected for bacteriology and histopathology. Atypical Aeromonas salmonicida was isolated from 10 individuals reared at or above 7 °C. The isolates were homogeneous with respect to biochemical and antibiogram characters and similar to the ssp. achromogenes National Collection of Industrial and Marine Bacteria, UK, type strain 1110 and reference strains that have been isolated from salmonids and haddock in Iceland. Histopathological analysis of the naturally infected cod showed typical ulceration associated with atypical A. salmonicida infection and also widespread granulomatous formations. One‐year‐old cod of farmed origin, kept at 9 °C, received intraperitoneal or intramuscular injection with different doses of atypical A. salmonicida, isolated from the above wild cod. Mortalities were monitored for 28 days and the LD₅₀ calculated. The route of bacterial injection influenced the mortality rate and LD₅₀ value and affected, to some extent, the pathological changes observed and humoral immune parameters. Pathological changes, including haemorrhage, early stages of granuloma formation and necrotic changes, were seen in several organs. Infection appeared to induce non‐specific antibody activity against trinitrophenyl (TNP)‐haptenated protein and may have activated the complement system. Specific antibody response against atypical A. salmonicida was not detected.     

The ability of carnobacteria isolated from fish intestine to inhibit growth of fish pathogenic bacteria: a screening study

In the present study a microtitre plate assay was used to evaluate antagonistic activity of 157 intestinal bacteria belonging to Carnobacterium isolated from Arctic charr (Salvelinus alpinus L.), Atlantic salmon (Salmo salar L.) and wolf fish (Anarhichas lupus L.) against fish pathogenic bacteria. One hundred and forty‐nine strains isolated from Arctic charr fed; (a) different lipid levels and (b) different fatty acids were screened for their ability to inhibit growth of the fish pathogen Aeromonas salmonicida ssp. salmonicida strain AL 2020 (the causative agent of furunculosis). Carnobacterium maltaromaticum and Carnobacterium mobile isolated from fish fed a low‐lipid diet inhibited growth of the pathogen, while none of the Carnobacterium divergens isolated from fish fed the high‐lipid diet had this ability. When Arctic charr was fed different fatty acids, was the frequency of antibacterial ability of C. maltaromaticum highest in strains isolated from fish fed 4%α‐linolenic acid (18:3 n‐3) and lowest in strains isolated from fish fed 4% linoleic acid (18:2 n‐6). Extracellular growth inhibitory compounds harvested in exponential and stationary growth phase from eight carnobacteria strains isolated from three fish species were tested for their ability to inhibit growth of six fish pathogens [A. salmonicida, Vibrio splendidus strain VS11, Vibrio salmonicida strain LFI 315, Vibrio anguillarum strain LFI 317, Moritella (Vibrio) viscosa strain LFI 5000 and C. maltaromaticum (piscicola) CCUG 34645]. The highest antibacterial activity was found when cellular extracts of the producer isolate were harvested in stationary growth phase. Scanning electron microscopy (SEM) investigations of A. salmonicida showed that cell morphology was affected by the inhibitory substance produce by strain 8M851, a Carnobacterium inhibens‐like bacteria.     

Studies on the antibody response and side effects after intramuscular and intraperitoneal injection of Atlantic lumpfish (Cyclopterus lumpus L.) with different oil‐based vaccines

Atlantic lumpfish (Cyclopterus lumpus L.) is used as a biological delousing agent for sea lice (Lepeophtheirus salmonis K.) infestations in Norwegian aquaculture. Here, we present a study on the antibody response and vaccine side effects after intramuscular and intraperitoneal injection of lumpfish with two vaccines. Both vaccines contained bacterial antigens from atypical Aeromonas salmonicida A‐layer types V and VI, Vibrio anguillarum serotype O1 and Moritella viscosa sp., but one vaccine contained a vegetable oil‐based adjuvant, while the other contained a mineral oil‐based adjuvant. Intramuscular injection of the mineral oil‐based vaccine caused a high acute mortality of fish within 48 hr after immunization. Intraperitoneal injection of the mineral oil‐based vaccine resulted in a lower severity of intra‐abdominal side effects than the vegetable oil‐based vaccine. Intramuscular injection of the mineral oil‐based vaccine resulted in a significantly higher antibody response against A. salmonicida when compared to controls and the vegetable oil‐based vaccine group. The antibody response was poor against V. anguillarum and M. viscosa for all groups. Our results indicate that intramuscular injection of oil‐based vaccines might be feasible for providing immunological protection for Atlantic lumpfish against bacterial diseases, especially atypical A. salmonicida, but more work is required to identity optimal adjuvants.     

Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real‐time bioluminescence imaging

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual‐labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild‐type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24‐h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.     

Detection and quantification of Aeromonas salmonicida in fish tissue by real‐time PCR

Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real‐time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real‐time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real‐time PCR compared to 30% by a culture approach). Also, no real‐time PCR‐negative samples were found positive by culturing. A. salmonicida was detected by real‐time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real‐time PCR showed the presence of the bacterium in all examined organs (1–482 genomic units mg^?1). With a limit of detection of 40 target copies (1–2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real‐time PCR assay provides a sensitive tool for the detection of A. salmonicida.     

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 5 days post‐infection and the presence of intraerythrocytic A. salmonicida. Our results reveal a novel intraerythrocytic phase during A. salmonicida infection.     

Observations on the role of the caseinase enzyme in the pathogenesis of furunculosis

Abstract. The role of the caseinase extracellular enzyme in the pathogenesis of furunculosis was investigated. Natural and mutant strains of Aeromonas salmonicida which were unable, or limited, in their ability to produce this enzyme were studied. The virulence of the mutant strains of A. salmonicida was not significantly reduced by the lack of extracellular caseinase production. Furthermore, there was no difference in the toxicity of extracellular products produced by these mutant strains and the wild-type A. salmonicida strain studied. It is reasonable to assume from these results that the caseinase enzyme has no major role in the pathogenesis of A. salmonicida infections.     

Aeromonas salmonicida infection levels in pre‐ and post‐stocked cleaner fish assessed by culture and an amended qPCR assay

Due to increasing resistance to chemical therapeutants, the use of ‘cleaner fish’ (primarily wrasse, Labridae, species) has become popular in European salmon farming for biocontrol of the salmon louse, Lepeophtheirus salmonis (Krøyer). While being efficient de‐licers, cleaner fish mortality levels in salmon cages are commonly high, and systemic bacterial infections constitute a major problem. Atypical furunculosis, caused by Aeromonas salmonicida A‐layer types V and VI, is among the most common diagnoses reached in clinical investigations. A previously described real‐time PCR (qPCR), targeting the A. salmonicida A‐layer gene (vapA), was modified and validated for specific and sensitive detection of all presently recognized A‐layer types of this bacterium. Before stocking and during episodes of increased mortality in salmon cages, cleaner fish (primarily wild‐caught wrasse) were sampled and screened for A. salmonicida by qPCR and culture. Culture indicated that systemic bacterial infections are mainly contracted after salmon farm stocking, and qPCR revealed A. salmonicida prevalences of approximately 4% and 68% in pre‐ and post‐stocked cleaner fish, respectively. This underpins A. salmonicida's relevance as a contributing factor to cleaner fish mortality and emphasizes the need for implementation of preventive measures (e.g. vaccination) if current levels of cleaner fish use are to be continued or expanded.     

Susceptibility of corkwing wrasse Symphodus melops, goldsinny wrasse Ctenolabrus rupestis, and Atlantic salmon Salmo salar smolt, to experimental challenge with Vibrio tapetis and Vibrio splendidus isolated from corkwing wrasse

The virulence of two Vibrio strains, previously isolated from diseased corkwing wrasse Symphodus melops and identified as V. tapetis and V. splendidus, to corkwing and goldsinny wrasse Ctenolabrus rupestris and to Atlantic salmon Salmo salar, was studied under laboratory conditions. Both bacteria were shown to be opportunistically pathogenic to corkwing wrasse, causing significantly higher mortality in the challenged groups than in the controls. Bacterial cultivation of kidney samples and re-isolation of V. tapetis and V. splendidus from most mortalities confirmed the two strains as the probable cause of mortality in the challenged groups. The control group also suffered relatively high mortality, but no specific pathogens that were suspected to be the main cause of death were isolated, other than a mixture of Vibrio spp. and, in the case of one individual, atypical Aeromonas salmonicida. Following injection challenge with both bacterial strains, no mortality was recorded in Atlantic salmon. In bath challenge trials with goldsinny wrasse, only slight mortality was observed in the challenged groups and the unchallenged control group. Bacterial examination showed that atypical Aeromonas salmonicida was the probable cause of death in both bath challenged and control groups of goldsinny wrasse, and no indication of infection by any Vibrio sp. was found.     

First isolation of Vibrio tapetis and an atypical strain of Aeromonas salmonicida from skin ulcerations in common dab (Limanda limanda) in the North Sea

Skin ulcerations rank amongst the most prevalent lesions affecting wild common dab (Limanda limanda) with an increase in prevalence of up to 3.5% in the Belgian part of the North Sea. A complex aetiology of these ulcerations is suspected, and many questions remain on the exact factors contributing to these lesions. To construct the aetiological spectrum of skin ulcerations in flatfish, a one‐day monitoring campaign was undertaken in the North Sea. Fifteen fish presented with one or more ulcerations on the pigmented and/or non‐pigmented side. Pathological features revealed various stages of ulcerations with loss of epidermal and dermal tissue, inflammatory infiltrates and degeneration of the myofibers bordering the ulceration, albeit in varying degrees. Upon bacteriological examination, pure cultures of Vibrio tapetis were retrieved in high numbers from five fish and of Aeromonas salmonicida in one fish. The V. tapetis isolates showed cross‐reactivity with the sera against the representative strain of serotype O2 originating form a carpet‐shell clam (Ruditapes descussatus). Moreover, the A. salmonicida isolates displayed a previously undescribed vapA gene sequence (A‐layer type) with possible specificity towards common dab. Further research is necessary to pinpoint the exact role of these agents in the development of skin ulcerations in common dab.     

The protective effect of humic‐rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.)

When challenged with atypical Aeromonas salmonicida subsp. salmonicida, exposure of the common carp (Cyprinus carpio L.) to different humic‐rich compounds resulted in a significant reduction in infection rates. Specifically, in fish exposed to (i) humic‐rich water and sludge from a recirculating system, (ii) a synthetic humic acid, and (iii) a Leonardite‐derived humic‐rich extract, infection rates were reduced to 14.9%, 17.0% and 18.8%, respectively, as compared to a 46.8% infection rate in the control treatment. An additional set of experiments was performed to examine the effect of humic‐rich components on the growth of the bacterial pathogen. Liquid culture medium supplemented with either humic‐rich water from the recirculating system, the synthetic humic acid or the Leonardite humic‐rich extract resulted in a growth reduction of 41.1%, 45.2% and 61.6%, respectively, as compared to the growth of the Aeromonas strain in medium devoid of humic substances. Finally, in a third set of experiments it was found that while the innate immune system of the carps was not affected by their exposure to humic‐rich substances, their acquired immune system was affected. Fish, immunized against bovine serum albumin, displayed elevated antibody titres as compared to immunized carps which were not exposed to the various sources of humic substances.