Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection

Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.     

Characterization of Aeromonas salmonicida subsp. salmonicida: a comparative study of strains of different geographic origin

A total of 130 strains of the fish pathogen Aeromonas salmonicida isolated in Denmark, Norway, Scotland, Canada and the USA were examined. The strains originated from farmed salmonid fish. The biochemical, physiological and serological characteristics, antibiotic resistance patterns and cell surface-related properties were compared. Aeromonas salmonicida was found to be remarkably consistent in general cultural and biochemical characteristics. It is noteworthy that the strains were positive in the fermentation of L-arabinose and were negative in the fermentation of D-arabinose. All the strains were highly proteolytic. It was observed, however, that 5% of the strains did not digest calf and trout serum and the production of haemolysin and degradation of casein by the same strains were delayed compared with the other strains. Common to all of the rough strains were auto-aggregation and ability to bind the dyes Coomassie brilliant blue and Congo red and the majority of these strains were highly hydrophobic. The strains were tested for their susceptibility to 22 antibacterial agents. Antibiotic resistance profiles of Aer. salmonicida indicated that resistance to the quinolones and oxytetracycline was increasing and that multi-resistant strains were found in several countries. The variation found in antibiograms could have potential as epidemiological markers in certain geographic areas.     

Bottom substrate preference in wild and reared turbot Psetta maxima L.

In a field experiment, breed turbot of an average length between 7–25 cm was released into a small bay. During the following week; diffusion rate, mortality and catchability of the released fish was estimated daily, by using a normal distribution model. In this model the size of the fish was incorporated in such a way, that it was possible to retrieve information about how each of the estimated parameters depended on the fish size. In addition, the length of the prey items found in the stomach was measured and compared to the prey length available in the habitat. Turbot was found to adjust to eat the available prey fast, after 3 days approximately 100% had detectable signs of prey in their stomach.     

Molecular characterization and quantitative analysis of superoxide dismutases in virulent and avirulent strains of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A. salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB. The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O(2)(-)-catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative sodA were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of A. salmonicida subsp. salmonicida strain A449 when cultured in vitro and in vivo.     

Association of Type III secretion genes with virulence of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida possesses a number of potential virulence factors, including a recently identified plasmid-encoded Type III secretion system. A number of field isolates of A. salmonicida subsp. salmonicida were examined for the presence of Type III secretion genes. Using in vitro experiments, it was found that field isolates containing such genes are cytotoxic to fish cell lines, whereas those that lack these genes are not. Using a rainbow trout in vivo model, the virulence of a wild type A. salmonicida subsp. salmonicida strain (Strain JF2267), which possesses Type III secretion genes, was compared to that of a laboratory derivative of the same strain that has lost these genes. While Strain JF2267 was virulent towards rainbow trout, its derivative was not. The A. salmonicida subsp. salmonicida Type Strain ATCC 33658T, which also lacks Type III secretion genes, was also found to be avirulent by this challenge model. The findings from both the in vitro and in vivo experiments suggest that the presence of Type III secretion genes is associated with the virulence of this important fish pathogen.     

Aeromonas salmonicida subsp. salmonicida in the light of its type-three secretion system

Aeromonas salmonicida subsp. salmonicida is an important pathogen in salmonid aquaculture and is responsible for the typical furunculosis. The type-three secretion system (T3SS) is a major virulence system. In this work, we review structure and function of this highly sophisticated nanosyringe in A. salmonicida. Based on the literature as well as personal experimental observations, we document the genetic (re)organization, expression regulation, anatomy, putative functional origin and roles in the infectious process of this T3SS. We propose a model of pathogenesis where A. salmonicida induces a temporary immunosuppression state in fish in order to acquire free access to host tissues. Finally, we highlight putative important therapeutic and vaccine strategies to prevent furunculosis of salmonid fish.     

Evidence for a type III secretion system in Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host-range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen.     

异育银鲫源杀鲑气单胞菌杀鲑亚种的分离鉴定

【目的】分离鉴定江苏省扬州市养殖场异育银鲫患病病原。【方法】采用常规的理化特性和分子生物学的方法,对从濒死异育银鲫肝脏处分离到的菌株YZ-1进行表型生物学、分子生物学及药敏试验的系统研究。【结果】该菌株16S r RNA基因(序列长度1 446 bp,Gen Bank登录号为JX164202)与其它杀鲑气单胞菌16S r RNA基因一致性在99%-100%之间,构建发育树确定该菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.salmonicida)。人工回感可导致异育银鲫死亡。药敏试验结果显示:对头孢呋辛、复方新诺明、恩诺沙星等23种抗生素敏感;对阿米卡星、四环素、大观霉素、头孢拉定等11种抗生素中度敏感;对青霉素G、链霉素、庆大霉素、氟苯尼考、万古霉素等10种抗生素耐药。【结论】研究结果证实引起异育银鲫死亡的病原为杀鲑气单胞菌杀鲑亚种。     

Small subunit rRNA gene sequences of Aeromonas salmonicida subsp. smithia and Haemophilus piscium reveal pronounced similarities with A. salmonicida subsp. salmonicida

The small subunit ribosomal RNA (SSU rRNA) encoding genes from reference strains of Aeromonas salmonicida subsp. smithia and Haemophilus piscium were amplified by polymerase chain reaction and cloned into Escherichia coli cells. Almost the entire SSU rRNA gene sequence (1505 nucleotides) from both organisms was determined. These DNA sequences were compared with those previously described from A. salmonicida subsp. salmonicida, subsp. achromogenes and subsp. masoucida. This genetic analysis revealed that A. salmonicida subsp. smithia and H. piscium showed 99.4 and 99.6% SSU rRNA gene sequence identity, respectively, with A. salmonicida subsp. salmonicida.     

Evidence against dormacy in the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida

Results indicate that A. salmonicida does not enter an unculturable dormant state. The resuscitation of dormant cells by nutrient broth described by previous workers appears to be due to the presence of small numbers of viable, culturable cells too few to be detected by the sampling protocol employed.     

Structural studies of the core region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The core oligosaccharide structure of the in vivo derived rough phenotype of Aeromonas salmonicida subsp. salmonicida was investigated by a combination of compositional, methylation, CE-MS and one- and two-dimensional NMR analyses and established as the following: [carbohydrate: see text] where R=alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1--> or alpha-D-Galp-(1--> (approx. ratio 4:3). Comparative CE-MS analysis of A. salmonicida subsp. salmonicida core oligosaccharides from strains A449, 80204-1 and an in vivo rough isolate confirmed that the structure of the core oligosaccharide was conserved among different isolates of A. salmonicida.     

Three small,cryptic plasmids from Aeromonas salmonicida subsp. salmonicida A449

The nucleotide sequences of three small (5.2-5.6 kb) plasmids from Aeromonas salmonicida subsp. salmonicida A449 are described. Two of the plasmids (pAsa1 and pAsa3) use a ColE2-type replication mechanism while the third (pAsa2) is a ColE1-type replicon. Insertions in the Rep protein and oriV region of the ColE2-type plasmids provide subtle differences that allow them to be maintained compatibly. All three plasmids carry genes for mobilization (mobABCD), but transfer genes are absent and are presumably provided in trans. Two of the plasmids, pAsa1 and pAsa3, carry toxin-antitoxin gene pairs, most probably to ensure plasmid stability. One open reading frame (ORF), orf1, is conserved in all three plasmids, while other ORFs are plasmid-specific. A survey of A. salmonicida strains indicates that pAsa1 and pAsa2 are present in all 12 strains investigated, while pAsa3 is present in 11 and a fourth plasmid, pAsal1, is present in 7.     

Structural characterization of the lipid A region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The lipid A components of Aeromonas salmonicida subsp. salmonicida from strains A449, 80204-1 and an in vivo rough isolate were isolated by mild acid hydrolysis of the lipopolysaccharide. Structural studies carried out by a combination of fatty acid, electrospray ionization-mass spectrometry and nuclear magnetic resonance analyses confirmed that the structure of lipid A was conserved among different isolates of A. salmonicida subsp. salmonicida. All analyzed strains contained three major lipid A molecules differing in acylation patterns corresponding to tetra-, penta- and hexaacylated lipid A species and comprising 4'-monophosphorylated beta-2-amino-2-deoxy-d-glucopyranose-(1-->6)-2-amino-2-deoxy-d-glucopyranose disaccharide, where the reducing end 2-amino-2-deoxy-d-glucose was present primarily in the alpha-pyranose form. Electrospray ionization-tandem mass spectrometry fragment pattern analysis, including investigation of the inner-ring fragmentation, allowed the localization of fatty acyl residues on the disaccharide backbone of lipid A. The tetraacylated lipid A structure containing 3-(dodecanoyloxy)tetradecanoic acid at N-2',3-hydroxytetradecanoic acid at N-2 and 3-hydroxytetradecanoic acid at O-3, respectively, was found. The pentaacyl lipid A molecule had a similar fatty acid distribution pattern and, additionally, carried 3-hydroxytetradecanoic acid at O-3'. In the hexaacylated lipid A structure, 3-hydroxytetradecanoic acid at O-3' was esterified with a secondary 9-hexadecenoic acid. Interestingly, lipid A of the in vivo rough isolate contained predominantly tetra- and pentaacylated lipid A species suggesting that the presence of the hexaacyl lipid A was associated with the smooth-form lipopolysaccharide.     

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.     

Absence of population structure of turbot (Psetta maxima) in the Baltic Sea

We found low, albeit significant, genetic differentiation among turbot (Psetta maxima) in the Baltic Sea but in contrast to earlier findings we found no evidence of isolation by distance. In fact temporal variation among years in one locality exceeded spatial variation among localities. This is an unexpected result since adult turbot are sedentary and eggs are demersal at the salinities occurring in the Baltic. Our findings are most likely explained by the fact that we sampled fish that were born after/during a large influx of water to the Baltic Sea, which may have had the consequence that previously locally and relatively sedentary populations became admixed. These results suggest that populations that colonize relatively variable habitats, like the Baltic, face problems. Any adaptations to local conditions that may build up during stable periods may quickly become eroded when conditions change and/or when populations become admixed. Our results indicate that the ability of turbot to survive and reproduce at the low salinity in the Baltic is more likely due to phenotypic plasticity than a strict genetic adaptation to low salinity.     

Quantitative and qualitative evaluation of iNOS expression in turbot (Psetta maxima) infected with Enteromyxum scophthalmi

Enteromyxum scophthalmi is the causative agent of turbot enteromyxosis, an intestinal parasitisation that produces severe desquamative enteritis leading to a cachectic syndrome and eventually the death. It is well known the importance of the innate immune response against parasites in fish, with the release of antimicrobial substances such as reactive oxygen and nitrogen species, produced by the inducible nitric oxide synthase (iNOS). This enzyme is mainly found in phagocytes, but also in structural cells from the intestinal mucosa. The aim of this study was to characterize iNOS in intestine and lymphohaematopoietic organs (spleen and anterior kidney) of turbot by means of immunohistochemistry in order to assess the possible changes of this enzyme through the infection. The presence of the enzyme was evaluated in control and E. scophthalmi-infected turbot. The results showed immunoreactivity in the apical border of enterocytes and mild staining of goblet cells in both control and infected turbot although it was more evident and widespread in infected turbot compared to control. Moderate numbers of iNOS+ cells were present in the lamina propria-submucosa of fish which presented moderate and severe inflammatory infiltrates at this level. In spleen and kidney, iNOS+ cells were scattered through the parenchyma and, in severely infected fish, tended to be allocated near the vascular structures and melano-macrophage centres. The number of positive cells at the lymphohaematopoietic organs was significantly higher in infected turbot and increased as infection progressed. The increase in the expression of iNOS in the tissues of E. scophthalmi-infected turbot was more evident in individuals with severer lesions. The measurement of the levels of iNOS during turbot enteromyxosis reveals a possibly delayed response that would not able to eliminate the parasites but would exacerbate mucosal injury.     

Development of the eye in the turbot Psetta maxima (Teleosti) from hatching through metamorphosis

Development of the eyes during the larval and metamorphic stages of the turbot Psetta maxima (Teleosti) was studied using microscopy. Events during differentiation of both eyes occur simultaneously, and no differences between he migrating and no-migrating eye were observed during metamorphosis. At hatching, the eyes are rudimentary, consisting of a neuroepithelial optic cup and a small lens. During larval development, major changes occur in the lens and retina, in which cones are the only photoreceptors. The appearance of rods is delayed until metamorphosis. The outer ocular layers (sclera and choroid) arise during larval development as thin connective layers with little differentiation. These layers undergo important changes just before and during metamorphosis. These results indicate that development of the individual components of the eye occurs at different times. Those of ectodermal origin appear early, providing a simple visual organ during larval life. By metamorphosis, the eye shows adult characteristics, including two types of photoreceptors, a rich choroid vascular supply and ocular structures involved in protecting, shaping, and moving the eye. J Morphol 233:31–42, 1997. © 1997 Wiley-Liss, Inc.     

Dietary arginine degradation is a major pathway in ureagenesis in juvenile turbot (Psetta maxima)

Recent studies indicate that urea excretion is responsive to protein intake and that turbot, Psetta maxima, appear to differ from other species by their urea excretion pattern and levels. This study was undertaken to evaluate the influence of dietary nitrogen and arginine on ureagenesis and excretion in turbot. Juvenile turbot (29 g) were fed semi-purified diets containing graded levels of nitrogen (0-8% dry matter) and arginine (0-3% dry matter) for 6 weeks. Growth data showed that turbot have high dietary nitrogen (123 mg/kg metabolic body weight/day) and very low dietary arginine (9.3 mg/kg metabolic body weight/day) requirements for maintenance. Requirements for unit body protein accretion were 0.31 g and 0.15 g for nitrogen and arginine respectively. Post-prandial plasma urea levels and urea excretion rates showed that urea production was significantly (P<0.05) influenced by dietary arginine levels. While hepatic arginase (EC 3.5.3.1) activity increased significantly (P<0.05) with increasing dietary arginine levels, activities of other enzymes of the ornithine urea cycle were very low. Our data strongly suggest that the ornithine urea cycle is not active in the turbot liver and that dietary arginine degradation is a major pathway of ureagenesis in turbot.