Genomic and phenotypic characterization of an atypical Aeromonas salmonicida strain isolated from a lumpfish and producing unusual granular structures

Aeromonas salmonicida strains are roughly classified into two categories, typical and atypical strains. The latter mainly regroup isolates that present unusual phenotypes or hosts, comparatively to the typical strains that belong to the salmonicida subspecies. This study focuses on an uncharacterized atypical strain, M18076‐11, isolated from lumpfish (Cyclopterus lumpus) and not part of the four recognized Aeromonas salmonicida subspecies. This isolate presents an unreported phenotype in the A. salmonicida species: the formation of large granular aggregates. Granules are formed of a heterogeneous mix of live and dead cells, with live cells composing the majority of the population. Even if no mechanism was determined to cause cellular aggregation, small globular structures at the cell surface were observed, which might affect granular formation. Pan‐genome phylogenetic analysis indicated that this strain groups alongside the masoucida subspecies. However, phenotypic tests showed that these strains have diverging phenotypes, suggesting that M18076‐11 might belong to a new subspecies. Also, a pAsal1‐like plasmid, which was only reported in strains of the subspecies salmonicida, was discovered in M18076‐11. This study sheds light on unsuspected diversity in A. salmonicida subspecies and stresses the need of thorough identification when a new strain is encountered, as unique traits might be discovered.     

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.     

Evaluation of genetic relationship among six Labeo species using randomly amplified polymorphic DNA (RAPD)

Labeo species constitute an important group of fish with intense diversity and potential for commercial aquaculture in many Southeast Asian nations including the Indian subcontinent. The present investigation involves the comparative analysis of randomly amplified polymorphic DNA (RAPD) profiles of six Labeo species viz., L. bata (bata), L. calbasu (calbasu), L. dyocheilus (dyocheilus), L. fimbriatus (fimbriatus), L. gonius (gonius) and L. rohita (rohu) at the nuclear DNA variation level. Fifteen decamer random primers were chosen from 40, which amplified a total of 449 DNA fragments ranging in size from 400 to 3000 bp. Both monomorphic and polymorphic DNA bands were identified based on their presence or absence that could be used for specieswise differentiation. Similarity coefficients were calculated to quantify the genetic variation within and between species. On an average, the highest intra‐species genetic similarity value was found in calbasu (0.93) followed by rohu and fimbriatus (0.91), bata (0.87), gonius (0.86) and dyocheilus (0.77). The interspecies genetic similarity estimates among the species of Labeo were used to deduce their phylogenetic relationships. The cluster analysis showed two main clusters, one with calbasu, rohu, fimbriatus and gonius and another with bata and dyocheilus. The study provides evidence that RAPD could be used for genetic differentiation of closely related species.     

DNA fingerprinting in Indian major carps and tilapia by Bkm 2(8) and M13 probes

DNA fingerprints were obtained in three species of commercially important freshwater fishes, Labeo rohita (Hamilton). Catla catla (Hamilton) and Oreachromis mossambicus (Peters), using Bkm 2(8) and M13 multilocus probes. Bkm 2(8) gave a higher number of bands when compared with M13. However, the number of bands obtained by each probe in O. mossambicus was similar. The higher band-sharing coefficient observed in this species may be attributed to inbreeding as it arose from a small founder population. In L rohita and C. catla, the Bkm 2(8) detected similar DNA fingerprints when two enzymes Hinfi and Taqi were used. The M13 probe also gave similar fingerprints with three restriction enzymes (Hinfi, Taqi, Alui). Comparison of the DNA fingerprints obtained by Bkm 2(8) and Ml 3 showed that these two probes detected different alleles. The overall similarity of the DNA fingerprint patterns in L. rohita and C. catla may be due to their genetic closeness as indicated by their same chromosome number, C-value and their ability to produce fertile hybrids. A similar argument also holds true for the Oreochromis species where interspecies hybridization results in fertile offspring.     

Antibody-coated gold nanoparticles immunoassay for direct detection of Aeromonas salmonicida in fish tissues

Aeromonas salmonicida is the causative agent of furunculosis, a disease that affects both salmonid and non‐salmonid fish. Detection of A. salmonicida can be labour intensive and time consuming because of the difficulties in distinguishing the bacterium from other species given the wide variety of existing biochemical profiles and the slow growth characteristics which allow other organisms to overgrow the A. salmonicida. Herein, we report the development of a specific immunoassay using gold‐conjugated polyclonal antibodies for the rapid detection of A. salmonicida in fish tissues. Monodispersible 13‐nm gold nanoparticles were coated with polyclonal antibodies specific to A. salmonicida. Reddish purple agglutination of gold particles indicated the presence of A. salmonicida in samples. Positive reactions were detected visually with the naked eye. No agglutination was observed when A. salmonicida antibody‐coated gold nanoparticles were tested with other common bacterial fish pathogens, thereby verifying the specificity of the assay. The assay could detect A. salmonicida in fish tissues down to 1 × 10⁴ CFU mL^?1, and results were obtained within 45 min. The antibody‐coated gold nanoparticles were stable for at least 2 months at 4°C. The immunoassay using antibody‐coated gold nanoparticles represents a promising tool for the rapid and specific detection of A. salmonicida in fish tissues.     

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.     

Pinpointing the role of Aeromonas salmonicida in the development of skin ulcerations in common dab (Limanda limanda)

Aeromonas salmonicida was isolated from ulcerations in common dab (Limanda limanda). An experiment was performed to pinpoint its role in ulceration development, considering the importance of the skin barrier and the pigmented and non-pigmented sides. The skin of dab was treated in three zones, one where scales and epidermis were removed, one where mucus was discarded and one non-treated zone. Fish were tagged to allow individual identification and challenged with A. salmonicida. Mortality and severity of the developing lesions were recorded for 21 days post-inoculation. Starting 12 days post-inoculation, mortality occurred gradually in challenged fish; however, no direct cause could be established. Both control fish and challenged fish developed ulcerations containing A. salmonicida. Sequencing of vapA gene revealed that isolates retrieved from both groups were distinct, suggesting the presence of A. salmonicida prior to the trial. Most ulcerations developed in zones where skin was removed, suggesting that abrasion might be a predisposing factor in ulceration development. Ulcerations were also observed at the insertion site of the tag, where exposed muscle tissue might have favoured the development of ulcerations. In conclusion, A. salmonicida seems to be involved in the development of skin ulcerations in dab, although the exact pathogenesis needs to be elucidated.     

Improvements of virulence factor phenotypic tests for Aeromonas salmonicida subsp. salmonicida,a major fish pathogen

Aeromonas salmonicida subspecies salmonicida, a fish pathogen, expresses various virulence factors such as an A-layer, lipases and proteases during the infection process. Not all strains of this bacterium express the same virulence factors. It is important to be able to evaluate which factors are present when characterizing strains. The A-layer and secreted lipases and proteases are usually detected by agar-based tests that require long incubation (24 h and more) and may provide ambiguous results. In the present study, protocols have been optimized to determine the presence of these virulence factors using liquid tests. For A-layer detection, the optimized method stains the positive bacteria with Coomassie Brilliant Blue. The lipases are detected by a colorimetric biochemical reaction triggered by the degradation of p-nitrophenyl dodecanoate into a yellow product detectable by spectrophotometry, if the result is positive. Both of these tests show results in less than an hour. Finally, the protease activity is measured by clarification of a medium containing milk during an overnight bacterial growth. These new protocols provide opportunities for quicker characterization of A. salmonicida subsp. salmonicida strains and, particularly, provide more precise results.     

Phenotypic,molecular and pathological characterization of motile aeromonads isolated from diseased fishes cultured in Uruguay

Information about motile aeromonads from aquaculture systems of the Neotropical region is scarce. The aim of this study was to characterize motile Aeromonas isolated from ornamental and consumable fishes cultured in Uruguay. Biochemical and molecular methods were used for species identification. Antimicrobial susceptibility and the presence of virulence genes were evaluated. Genetic diversity was analysed by rep‐PCR, and virulence of the most representative isolates was determined by calculating the fifty lethal dose in experimentally challenged fish (Australoheros facetus). Aeromonas hydrophila and A. veronii were the most prevalent identified species (38.2% and 32.4%, respectively), whereas A. allosacharophila, A. bestiarium, A. caviae and A. punctata were less prevalent. This study constitutes the first report of these last four species in Uruguay. All isolates were resistant to at least three antimicrobials, and 82.3% of them showed multidrug resistance. Virulence genotypes were correlated with the Aeromonas species and haemolytic activity. The genotype act+/alt+/ast+/ela+/lip+ was the most prevalent (26.5%). A correlation between virulence genotypes and Aeromonas species was found. A. punctata showed a clonal structure according to rep‐PCR analysis, whereas other species showed high genetic diversity. The number of virulence genes of the isolates was related with virulence according to the experimental challenge assays.     

The effects of soybean, linseed and marine oils on aerobic gut microbiota of Arctic charr Salvelinus alpinus L. before and after challenge with Aeromonas salmonicida ssp. salmonicida

Populations of heterotrophic bacteria present in the hindgut region of Arctic charr Salvelinus alpinus L. fed dietary soybean, linseed and marine oils before challenge with Aeromonas salmonicida ssp. salmonicida and marine oil after challenge were estimated using the dilution plate technique. There were differences in bacterial composition between the rearing groups before and after challenge, as well as interindividual variations. For example, carnobacteria were only isolated from the hindgut region of fish fed soybean oil and linseed oil before challenge, whereas Carnobacterium spp. and Carnobacterium funditum‐like species were isolated from fish fed the same oils after challenge. Three non‐motile Aeromonas spp. were isolated from infected fish fed marine oil. One of these isolates was identified as identical to A. salmonicida ssp. salmonicida used in&the challenge test by microbial fingerprinting (amplified fragment length polymorphism). Electron microscopic examinations of hindgut regions demonstrated substantial numbers of bacterial cells associated with enterocytes, but bacterial colonization of the enterocyte surface varied between different rearing groups. The potential of bacteria found associated with the hindgut region to inhibit the fish pathogens A. salmonicida, Vibrio salmonicida and Vibrio anguillarum differed between rearing groups.     

Effect of intraperitoneal injection of immunostimulatory substances on allochthonous gut microbiota of Atlantic salmon (Salmo salar L.) determined using denaturing gradient gel electrophoresis

The allochthonous microbiota in the proximal and distal intestine was investigated in three groups of Atlantic salmon (Salmo salar L.) fed a commercial diet and intraperitoneally injected with (a) phosphate‐buffered saline (control), (b) lipopolysaccharide (LPS) from the fish pathogenic bacteria, Aeromonas salmonicida ssp. salmonicida, and (c) laminaran [β‐(1,3)‐d ‐glucan]. Denaturing gradient gel electrophoresis (DGGE) of the hyper variable V3 region was used to present the microbiota in different experimental groups. Sequencing and phylogenetic analysis of excised DGGE bands suggested that an intraperitoneal injection of LPS from A. salmonicida affects the allochthonous gut bacteria of Atlantic salmon to some extent, as Aeromonas enteropelogenes, Aeromonas veroni, Psychrobacter, Lactobacillus letvazi, Lactobacillus satsumensis, Pantoea, swine manure bacterium and several uncultured bacteria were unique for this group. On the other hand, the bacterial diversity of the allochthonous microbiota did not seem to be affected by injection of β‐(1,3)‐d ‐glucan. Sequences of this experimental group were most closely related to A. enteropelogenes, uncultured Escherichia and Lactobacillus aviarius ssp. aviarius.     

Genetic diversity of Nile tilapia populations revealed by randomly amplified polymorphic DNA (RAPD)

Genetic diversity of the Nile tilapia Oreochromis niloticus collected from the river Nile (Cairo, Assuit and Qena) and two Delta lakes (Burullus and Manzalla) in Egypt was examined by the analysis of randomly amplified polymorphic DNA (RAPD). Of 25 primers examined,21 primers produced 230 RAPD bands. The percentage of polymorphic bands in Manzalla (29.4%) and Burullus (24%) populations was low compared with Assuit (30.54%), Cairo (33.5%) and Qena (44.84%) populations. The highest percentage of polymorphic bands was observed in the Qena population,suggesting a greater potential for use in breeding programs. The molecular phylogenetic tree constructed by unweighted pair‐group method of analysis shows Manzalla and Burullus populations strongly linked and separate from the Assuit and Cairo populations, with Qena population as outgroup. The data serve as a baseline analysis of the current genetic diversity found among O. niloticus populations in Egypt.     

Development and Application of a Quantitative Real‐time Polymerase Chain Reaction Assay for the Detection of Aeromonas salmonicida

A rapid, economical, specific, and sensitive quantitative real‐time polymerase chain reaction (qPCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Aeromonas salmonicida from farmed Atlantic salmon, Salmo salar, with the symptoms of furunculosis. The set of primers designed from the virulence array protein (vapA) gene was specific to A. salmonicida. Compared with the conventional PCR, qPCR had a lower detection limit of 5.6 copies of the positive plasmids. The standard curve, which showed the relationship between the copies of A. salmonicida and its quantification cycle (C_q) value, could be described as follows: log (copies of A. salmonicida) = ?0.3213 C_q + 10.721. The quantitative detection of copies of A. salmonicida in different tissues of the moribund Atlantic salmon showed that A. salmonicida could be detected in all tissues; the spleen contained the largest number of A. salmonicida and then the kidney. These results suggest that the qPCR assay reported here is a specific, sensitive, and quantitative method for detecting A. salmonicida. It can be used for the routine tests of A. salmonicida in local aquaculture enterprise and for the research of infection routes of A. salmonicida to Atlantic salmon.     

Characterisation of Aeromonas strains and species by pulsed field gel electrophoresis and principal components analysis

Aeromonas genomes were investigated by restriction digesting chromosomal DNA with the endonuclease Xba I, separation of restriction fragments by pulsed field gel electrophoresis (PFGE) and principal components analysis (PCA) of resulting separation patterns. A. salmonicida salmonicida were unique amongst the isolates investigated. Separation profiles of these isolates were similar and all characterised by a distinct absence of bands in the 250kb region. Principal components analysis represented these strains as a clearly defined homogeneous group separated by insignificant Euclidian distances. However, A. salmonicida achromogenes isolates in common with those of A. hydrophila and A. sobria were shown by principal components analysis to be more heterogeneous in nature. Fragments from these isolates were more uniform in size distribution but as demonstrated by the Euclidian distances attained through PCA potentially characteristic of each strain. Furthermore passaging of Aeromonas isolates through an appropriate host did not greatly modify fragment separation profiles, indicative of the genomic stability of test aeromonads and the potential of restriction digesting/PFGE/PCA in Aeromonas typing.     

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.     

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.     

Genetic variation inferred from RAPD fingerprinting in three species of tilapia

This study used random amplified polymorphic DNA (RAPD) fingerprinting for estimating genetic variation and species differentiation in three species of tilapia. A 16-mer random primer generated RAPD markers ranging from 250 to 2400 base pairs (bp). Genetic similarity estimates obtained by pairwise comparisons based on the method of Nei and Li (1979) indicated high genetic similarity (mean genetic similarity (± sd), 0.73 (± 0.15) for Nile tilapia; 0.78 (± 0.12) for Mozambique tilapia; and 0.87 (± 0.07) for Aureus tilapia) within each of the tilapia species. The average interspecies genetic similarities obtained among the three species were 0.59 (± 0.07) for Mozambique/Nile tilapia, 0.46 (± 0.09) for Aureus/Nile tilapia and 0.38 (± 0.07) for Aureus/Mozambique tilapia pair. DNA profiles generated in each species of tilapia were unique. A total of 13 RAPD markers differentiating the three species of tilapia were detected. Our study presented RAPD markers as a new class of useful genetic markers for assessment of genetic diversity and species differentiation in tilapia.     

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.     

Genetic variation of the razor clam Ensis siliqua (Jeffreys, 1875) along the European coast based on random amplified polymorphic DNA markers

Ensis siliqua is regarded as an increasingly valuable fishery resource with potential for commercial aquaculture in many European countries. The genetic variation of this razor clam was analysed by randomly amplified polymorphic DNA (RAPD) in six populations from Spain, Portugal and Ireland. Out of the 40 primers tested, five were chosen to assess genetic variation. A total of 61 RAPD loci were developed ranging in size from 400 to 2000 bp. The percentages of polymorphic loci, the allele effective number and the genetic diversity were comparable among populations, and demonstrated a high level of genetic variability. The values of Nei's genetic distance were small among the Spanish and Portuguese populations (0.051–0.065), and high between these and the Irish populations. Cluster and principal coordinate analyses supported these findings. A mantel test performed between geographic and genetic distance matrices showed a significant correlation (r=0.84, P<0.05), suggesting an isolation by distance process.