Comparative transcriptomics and histopathological analysis of crucian carp infection by atypical Aeromonas salmonicida.

Aeromonas salmonicida is a ubiquitous fish pathogen known to cause furunculosis. With the emergence of new subtypes and the expansion of the host range, it has threatened the health of a variety of marine and freshwater fish, particularly the non-salmonids, manifesting differently from the classical furunculosis. Although there have been reports of infection by atypical strains on the crucian carp, the pathogenesis and tissue pathology remain unclear. In this study, transcriptomics and histopathology were used to analyze the immune response and lesions of crucian carp infected with A. salmonicida. Comparative analysis showed 6579 differentially expressed genes (DEGs) (3428 down-regulated and 3151 up-regulated) were identified on day 5 post-infection (5 dpi). Further annotation and analysis revealed that the DEGs were enriched in enzyme regulator activity, response to oxidative stress, iron ion homeostasis and other functions, and mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), toll-like receptor (TLR), and nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) etc., and immune-related signaling pathways. Meanwhile, the four C-type lysozyme genes found in all DEGs were significantly up-regulated after infection. In addition, there was severe bleeding on the body of the infected fish. Also, the intestine, liver, spleen, and kidney showed varying degrees of inflammatory damage, especially the goblet cell hyperplasia of intestinal mucosa epithelium and degeneration and necrosis of renal tubular epithelium cells. Additionally, with the increase in pathogen concentration, the cumulative mortality increased, the severity of lesions in the hindgut and head-kidney tissues increased. The relative expression levels of four immune-related genes (TNF-α, IL-1ß, IL-11, C-lysozyme) were also significantly upregulated, compared with the control (P < 0.05). In conclusion, this study provides a scientific basis for further study on the immune response, pathological diagnosis, and prevention of crucian carp infection caused by atypical A. salmonicida.

Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping.

A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15-23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts.

Study of mesophilic Aeromonas salmonicida A527 strain sheds light on the species' lifestyles and taxonomic dilemma.

The Gram-negative bacterium Aeromonas salmonicida contains five subspecies: salmonicida, smithia, achromogenes, masoucida and pectinolytica. Pectinolytica is a mesophilic subspecies with the ability to thrive at a wide range of temperatures, including 37°C, while the four other subspecies are psychrophilic, restricted to lower temperatures. The psychrophilic subspecies are known to infect a wide range of fishes. However, there is no evidence of pathogenicity for the mesophilic subspecies pectinolytica. Study of the differences between the mesophilic and psychrophilic subspecies is hampered by the lack of completely sequenced and closed genomes from the mesophilic subspecies. A previous study reported that insertion sequences, which can induce genomic rearrangements at temperatures around 25°C, could be one of the determinants explaining the differences in lifestyle (mesophilic or psychrophilic) between the subspecies. In this study, the genome of mesophilic strain A527 of A. salmonicida was sequenced, closed and analyzed to investigate the mesophilic-psychrophilic discrepancy. This reference genome supports the hypothesis that insertion sequences are major determinants of the lifestyle differences between the A. salmonicida subspecies. Moreover, the phylogenetic analysis performed to position strain A527 within the taxonomy raises an issue regarding the intraspecies structure of A. salmonicida.

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.

AsaGEI2b: a new variant of a genomic island identified in the Aeromonas salmonicida subsp. salmonicida JF3224 strain isolated from a wild fish in Switzerland.

Aeromonas salmonicida subsp. salmonicida is the causal agent of furunculosis in salmonids. We recently identified a group of genomic islands (AsaGEI) in this bacterium. AsaGEI2a, one of these genomic islands, has almost exclusively been identified in isolates from North America. To date, Aeromonas salmonicida subsp. salmonicida JF3224, a strain isolated from a wild brown trout (Salmo trutta) caught in Switzerland, was the only European isolate that appeared to bear AsaGEI2a. We analyzed the genome of JF3224 and showed that the genomic island in JF3224 is a new variant of AsaGEI, which we have called AsaGEI2b. While AsaGEI2b shares the same integrase gene and insertion site as AsaGEI2a, it is very different in terms of many other features. Additional genomic investigations combined with PCR genotyping revealed that JF3224 is sensitive to growth at 25°C, leading to insertion sequence-dependent rearrangement of the locus on the pAsa5 plasmid that encodes a type three secretion system, which is essential for the virulence of the bacterium. The analysis of the JF3224 genome confirmed that AsaGEIs are accurate indicators of the geographic origins of A. salmonicida subsp. salmonicida isolates and is another example of the susceptibility of the pAsa5 plasmid to DNA rearrangements.

Infection of sea lamprey with an unusual strain of Aeromonas salmonicida.

The invasion of the Laurentian Great Lakes by the fish-parasitic sea lamprey has led to catastrophic consequences, including the potential introduction of fish pathogens. Aeromonas salmonicida is a bacterial fish pathogen that causes devastating losses worldwide. Currently, there are five accepted subspecies of Aeromonas salmonicida: A. salmonicida subsp. salmonicida, masoucida, smithia, achromogenes, and pectinolytica. We discuss the discovery of an isolate of A. salmonicida that is pathogenic to rainbow trout (Oncorhynchus mykiss) and exhibits unique phenotypic and molecular characteristics. We examined 181 adult sea lamprey (Petromyzon marinus) from the Humber River (Lake Ontario watershed) and 162 adult sea lamprey from Duffins Creek (Lake Ontario watershed) during the spring seasons of 2005-11. Among those, 4/343 (1.2%) sea lamprey were culture positive for A. salmonicida, whereby biochemical and molecular studies identified three of the isolates as A. salmonicida subsp. salmonicida. The remaining isolate (As-SL1) recovered from Humber River sea lamprey was phenotypically more similar to A. salmonicida subsp. salmonicida than to the four other A. salmonicida subspecies. However, unlike A. salmonicida subsp. salmonicida, As-SL1 was sucrose positive, produced an acid-over-acid reaction on triple-sugar iron medium and did not amplify with A. salmonicida subsp. salmonicida specific primers. Phylogenetic analysis based on partial stretches of the 16S rRNA and DNA gyrase subunit B genes further confirmed that the As-SL1 isolate was not A. salmonicida subsp. masoucida, smithia, achromogenes, or pectinolytica. Based on our analyses, the As-SL1 isolate is either an unusual strain of A. salmonicida subsp. salmonicida or a novel A. salmonicida subspecies. The four A. salmonicida isolates that were recovered from sea lamprey were pathogenic to rainbow trout in experimental challenge studies. Our study also underscores the potential role of sea lamprey in the ecology of infectious fish diseases.

Innate and adaptive immune responses of Arctic charr (Salvelinus alpinus, L.) during infection with Aeromonas salmonicida subsp. achromogenes and the effect of the AsaP1 toxin.

Aeromonas salmonicida subsp. achromogenes, the causative agent of atypical furunculosis in many fish species, secretes the toxic metalloendopeptidase AsaP1. This study aimed to analyze innate and adaptive immune parameters induced in Arctic charr (Salvelinus alpinus, L.) infected with wild type (wt) A. salmonicida subsp. achromogenes and its isogenic asaP1 deletion mutant (AsaP1-deficient). Head-kidney, liver and spleen were obtained from i.p. infected charr (wt, AsaP1-deficient), during a time schedule of 7 d post infection. Reverse transcription quantitative real-time PCR (RT-qPCR) was applied to study the expression of immune parameters: pro-inflammatory cytokines IL-1ß and TNF-α; anti-inflammatory cytokine IL-10; chemokines CXCL-8 (IL-8) and CC-chemokine; the cytokines IFN-γ and IL-4/13A as tracers for Th1 and Th2 immune responses, respectively; and the cell markers CD8α and CD83. In addition, lymphoid organs were histopathologically examined at days 3 and 7 post infection, including B (IgM) and T (CD3ε) cell staining. The detected immune responses were initially driven by innate mechanisms represented by the up-regulation of pro-inflammatory cytokines and chemokines and later on by adaptive Th2 related responses cumulating in B-cell recruitment as shown by regulation of immune parameters in spleen and head-kidney, with significant differences between mutant and wt infected fish. Histological sections revealed IgM-positive cells around ellipsoid arterioles in spleen, while CD3ε positive cells were found in clusters scattered all over the section. However, histopathological differences were only detected between infected and non-infected fish, but not between AsaP1-deficient mutant and wt infected fish. This work represents the first study on innate and adaptive immune responses of Arctic charr induced by a bacterial infection.

The improved PCR of the fstA (ferric siderophore receptor) gene differentiates the fish pathogen Aeromonas salmonicida from other Aeromonas species.

The members of the genus Aeromonas are autochthonous of aquatic ecosystems and several species have been associated to septicaemia, ulcerative and haemorrhagic diseases in fish, causing significant mortality in both wild and farmed, freshwater and marine fish species. The species Aeromonas salmonicida is generally recognized as the most important fish pathogen responsible for epidemic outbreaks of furunculosis in salmonids, also being able to produce infections in other cultured fish such as turbot, halibut, sea bream or goldfish. New species, i.e. Aeromonas aquariorum, Aeromonas tecta and Aeromonas piscicola, have recently been discovered and isolated from diseased fish. The species A. piscicola and Aeromonas bestiarum are practically impossible to differentiate phenotypically and genetically (when using the 16S rRNA gene) from each other and from A. salmonicida. In the present study, two previously described PCR protocols, based on the fstA and gyrB genes, for the specific detection of A. salmonicida were re-evaluated with the type strains of all Aeromonas species and with a set of A. piscicola and A. bestiarum strains. Contrary to what had been published previously it was demonstrated that the gyrB-PCR is not specific for A. salmonicida because of cross-reactions with other Aeromonas species. However, in agreement with previous results, A. salmonicida was detected on the basis of the fstA-PCR, for which an improved protocol was proposed.

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.

Clustering subspecies of Aeromonas salmonicida using IS630 typing.

BACKGROUND: The insertion element IS630 found in Aeromonas salmonicida belongs to the IS630-Tc1-mariner superfamily of transposons. It is present in multiple copies and represents approximately half of the IS present in the genome of A. salmonicida subsp. salmonicida A449. RESULTS: By using High Copy Number IS630 Restriction Fragment Length Polymorphism (HCN-IS630-RFLP), strains of various subspecies of Aeromonas salmonicida showed conserved or clustering patterns, thus allowing their differentiation from each other. Fingerprints of A. salmonicida subsp. salmonicida showed the highest homogeneity while 'atypical' A. salmonicida strains were more heterogeneous. IS630 typing also differentiated A. salmonicida from other Aeromonas species. The copy number of IS630 in Aeromonas salmonicida ranges from 8 to 35 and is much lower in other Aeromonas species. CONCLUSIONS: HCN-IS630-RFLP is a powerful tool for subtyping of A. salmonicida. The high stability of IS630 insertions in A. salmonicida subsp. salmonicida indicates that it might have played a role in pathoadaptation of A. salmonicida which has reached an optimal configuration in the highly virulent and specific fish pathogen A. salmonicida subsp. salmonicida.

Development and validation of a real-time PCR assay for the detection of Aeromonas salmonicida.

A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10(4) ± 1 × 10(4) CFU g(-1) (without enrichment) and 40 ± 10 CFU g(-1) (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.

Virulence factors of Aeromonas salmonicida subsp. salmonicida strains associated with infections in turbot Psetta maxima.

Virulence factors for Aeromonas salmonicida subsp. salmonicida (ASS) strains isolated from cultured turbot Psetta maxima L. are unknown with regard to this host. The presence of virulence genes associated with different stages of ASS infection in salmonids (vapA, tapA, fla, ascV, ascC, aexT, satA and aspA) was analysed using a polymerase chain reaction (PCR) technique in ASS strains isolated from turbot. Other ASS strains isolated from salmonids and environmental A. salmonicida (AS) strains were included for comparison. The presence of the genes was evaluated with respect to ASS virulence in turbot based on intraperitoneal and bath challenges. The genetic profile, including all of the genes studied, that was linked to virulent behaviour after intraperitoneal challenge was significantly more frequent in strains isolated from turbot than in those from salmonids or the environment. The data prove that it is not possible to predict the virulence of ASS in turbot based only on the presence of all genes tested. Moreover, the combined PCR results of vapA, aexT, ascV and ascC were useful for separating most of the ASS from environmental A. salmonicida strains. An association between virulence or genetic profile and the geographical or facility origin of the strains was not found.

An aerolysin-like enterotoxin from Vibrio splendidus may be involved in intestinal tract damage and mortalities in turbot, Scophthalmus maximus (L.), and cod, Gadus morhua L., larvae.

Vibrio splendidus is a pathogen that can cause major losses during the early stages of larval turbot rearing when live feed (rotifers or Artemia) is used. As haemolytic bacteria have often been associated with larval rearing losses, we studied the role of the V. splendidus haemolysin in infection of larvae. From a bank of over 10,000 transposon mutants of V. splendidus, two different types of haemolysin-negative mutants were obtained. Both had lost virulence for larval fish, and immunohistochemistry showed that the transposon mutant studied colonized the turbot larval intestinal tract at a similar level to the wild-type organism but did not cause damage or signs of enteritis found with the wild-type organism. One transposon insertion site was located within a gene with high homology to aerolysin, the cytolytic toxin produced by several Aeromonas spp. The haemolysin, which we have termed vibrioaerolysin, had properties similar to aerolysin and osmotic protection studies showed that it formed pores in the membranes of erythrocytes of similar diameter to those of aerolysin. The Tn10 insertion site of the second transposon mutant was in an adjacent ToxR-like gene, suggesting that this might control expression of the vibrioaerolysin. The gastroenteritis caused by Aeromonas spp. in humans is considered to be due to production of aerolysin causing cyclic AMP-dependent chloride secretion in cells of the gastrointestinal tract. Damage to the intestinal tract of marine fish larvae could occur in a similar way, and it is possible that several Vibrio spp. found in the developing bacterial flora of the larval fish gut can secrete aerolysin-like toxins leading to death of larvae in the early rearing stages. Routine bacteriological screening on blood agar plates of live feed is recommended with measures to reduce the concentrations of haemolytic bacteria in rearing systems.

Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea.

The antibiotic resistance of 16 Aeromonas (A.) salmonicida strains isolated from diseased fish and environmental samples in Korea from 2006 to 2009 were investigated in this study. Tetracycline or quinolone resistance was observed in eight and 16 of the isolates, respectively, based on the measured minimal inhibitory concentrations. Among the tetracycline-resistant strains, seven of the isolates harbored tetA gene and one isolate harbored tetE gene. Additionally, quinolone-resistance determining regions (QRDRs) consisting of the gyrA and parC genes were amplified and sequenced. Among the quinolone-resistant A. salmonicida strains, 15 harbored point mutations in the gyrA codon 83 which were responsible for the corresponding amino acid substitutions of Ser(83)→Arg(83) or Ser(83)→Asn(83). We detected no point mutations in other QRDRs, such as gyrA codons 87 and 92, and parC codons 80 and 84. Genetic similarity was assessed via pulsed-field gel electrophoresis, and the results indicated high clonality among the Korean antibiotic-resistant strains of A. salmonicida.

Atypical Aeromonas salmonicida infection in the black rockfish, Sebastes schlegeli Hilgendorf, in Korea.

Cultured black rockfish, Sebastes schlegeli, suffered mass mortalities during winter 2008 and spring 2009 in Korea, showing clinical signs of ulcer lesions and haemorrhages over their body surface. The aetiological agent was identified as Aeromonas salmonicida (strains RFAS-1, -2 and -3), which is a non-pigmented, slow-growing bacterium. Phenotypes of RFAS strains showed variation, while 16S rRNA, gyrB, rpoD, dnaJ and recA gene sequences of all the strains were affiliated to A. salmonicida. In particular, vapA gene sequences of the strains were most closely related to one of the five subspecies of A. salmonicida subsp. masoucida (=KCCM 40239(T) ). LD(50) values of RFAS-1 for intraperitoneal and intramuscular injection were 1.5 × 10(5.25) and 1.5 × 10(6.4) cfu/rockfish, respectively. However, A. salmonicida strains KCCM 40239(T) and SAS-1, which originate from masou and chum salmon, respectively, were not pathogenic to black rockfish. RFAS strains, possessing A-layer protein on their surface, exhibited ß-haemolytic activity against rockfish erythrocytes and capability to survive in rockfish serum, which seem to be associated with virulence.

The reference strain Aeromonas hydrophicla CIP 57.50 should be reclassified as Aeromonas salmonicida CIP 57.50.

The use of reference strains is a critical element for the quality control of different assays, from the development of molecular methods to the evaluation of antimicrobial activities. Most of the strains used in these assays are not type strains and some of them are cited erroneously because of subsequent reclassifications and descriptions of novel species. In this study, we propose that the reference strain Aeromonas hydrophila CIP 57.50 be reclassified as Aeromonas salmonicida CIP 57.50 based on phenotypic characterization and sequence analyses of the cpn60, dnaJ, gyrB and rpoD genes.

Outbreaks of an ulcerative and haemorrhagic disease in Arctic char Salvelinus alpinus caused by Aeromonas salmonicida subsp. smithia.

Arctic char Salvelinus alpinus farmed in different places in Austria and free of the viral diseases viral haemorrhagic septcaemia (VHS), infectious haematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) experienced disease and mortality. Diseased fish showed skin ulceration and pathological signs of sepsis. Aeromonas sp. was isolated as pure culture from the kidney of freshly euthanized diseased fish. Three independent isolates from outbreaks that occurred on 2 of the affected farms were analyzed phylogenetically by DNA sequence analysis of the rrs and gyrB genes and phenotypically with biochemical reactions. All 3 isolates were identified as Aeromonas salmonicida subsp. smithia. Analysis of virulence genes in these isolates revealed the presence of a Type III secretion system as well as several related virulence effector genes including aexT, encoding the Aeromonas exotoxin AexT, aopP and aopH. These genes are characteristic for virulent strains of typical and atypical subspecies of A. salmonicida.

Polymerase chain reaction amplification of repetitive intergenic consensus and repetitive extragenic palindromic sequences for molecular typing of Pseudomonas anguilliseptica and Aeromonas salmonicida.

The aim of this study was to evaluate the use of two molecular techniques, repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and repetitive intergenic consensus PCR (ERIC-PCR), as epidemiological tools with which to discriminate among genetically distinct strains within two bacterial fish pathogens, Pseudomonas anguilliseptica and Aeromonas salmonicida. A total of 30 A. salmonicida and 52 P. anguilliseptica were analyzed. For P. anguilliseptica, three different major fingerprints were obtained with both techniques, which defined three genomic groups: one was composed of strains isolated from eels Anguilla spp., the second of strains from turbot Scophthalmus maximus and blackspot seabream (also known as red seabream) Pagellus bogaraveo, and the third of strains from other fish species, such as gilthead seabream (also known as gilthead bream) Sparus auratus, sea bass Dicentrarchus labrax (also known as European bass Morone labrax), and salmonids. In the case ofA. salmonicida, promising results were obtained with both techniques for subspecies differentiation. Thus, two genomic profiles were obtained by ERIC-PCR. The first profile consisted of A. salmonicida subsp. salmonicida strains isolated from the different hosts. The second profile was composed of two A. salmonicida subsp. masoucida and one A. salmonicida subsp. achromogenes. Using REP-PCR, three genotypes were obtained within this pathogen that were related to the diverse subspecies analyzed. In summary, both methodologies are useful for typing distinct strains associated with different host species and therefore are helpful in epidemiological studies of P. anguilliseptica. In contrast, in the case of A. salmonicida, more studies are needed to determine their utility in discriminating the subspecies salmonicida from the other two subspecies.

Alternative host model to evaluate Aeromonas virulence.

Bacterial virulence can only be assessed by confronting bacteria with a host. Here, we present a new simple assay to evaluate Aeromonas virulence, making use of Dictyostelium amoebae as an alternative host model. This assay can be modulated to assess virulence of very different Aeromonas species.