Differential effects of exposure to parasites and bacteria on stress response in turbot Scophthalmus maximus simultaneously stressed by low water depth.

The stress response of turbot Scophthalmus maximus was evaluated in fish maintained 8 days under different water depths, normal (NWD, 30 cm depth, total water volume 40 l) or low (LWD, 5 cm depth, total water volume 10 l), in the additional presence of infection-infestation of two pathogens of this species. This was caused by intraperitoneal injection of sublethal doses of the bacterium Aeromonas salmonicida subsp. salmonicida or the parasite Philasterides dicentrarchi (Ciliophora:Scuticociliatida). The LWD conditions were stressful for fish, causing increased levels of cortisol in plasma, decreased levels of glycogen in liver and nicotinamide adenine dinucleotide phosphate (NADP) and increased activities of G6Pase and GSase. The presence of bacteria or parasites in fish under NWD resulted in increased cortisol levels in plasma whereas in liver, changes were of minor importance including decreased levels of lactate and GSase activity. The simultaneous presence of bacteria and parasites in fish under NWD resulted a sharp increase in the levels of cortisol in plasma and decreased levels of glucose. Decreased levels of glycogen and lactate and activities of GSase and glutathione reductase (GR), as well as increased activities of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and levels of nicotinamide adenine dinucleotide phosphate (NADPH) occurred in the same fish in liver. Finally, the presence of pathogens in S. maximus under stressful conditions elicited by LWD resulted in synergistic actions of both type of stressors in cortisol levels. In liver, the presence of bacteria or parasites induced a synergistic action on several variables such as decreased activities of G6Pase and GSase as well as increased levels of NADP and NADPH and increased activities of GPase, G6PDH and 6PGDH.

Effective qPCR methodology to quantify the expression of virulence genes in Aeromonas salmonicida subsp. salmonicida.

AIMS: This study aimed to select and validate different methodological strategies to quantify the expression of the virulence genes ascC and ascV by qPCR in Aeromonas salmonicida subsp. salmonicida (Aer. salmonicida). METHODS AND RESULTS: Using the geNorm, Normfinder and BestKeeper algorithms, reference genes for the qPCR were selected based on their in vitro expression stabilities in three Aer. salmonicida strains. Gene amplification efficiency was calculated by Real-time PCR Miner and LinReg PCR programmes, which have not been used previously in the analysis of bacterial gene expression. The expression of the ascC and ascV virulence genes in a virulent Aer. salmonicida strain was evaluated by three quantification models, including single (least or most stable) or three most stable reference genes, combined with constant or specific gene amplification efficiency. The most stable reference genes were gyrB, proC and rpoC, while rpoD and fabD were the least stable. Quantification models showed different expression patterns. CONCLUSIONS: The optimal strategy to quantify mRNA expression was to use a combination of the three algorithms and the quantification model including the three most stable reference genes. Real-time PCR Miner or LinReg PCR were valuable tools to estimate amplification efficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods used in this study gave more reliable expression data using qPCR than previously published methods. The quantification and expression dynamics of virulence genes will contribute to a better understanding of how Aer. salmonicida interacts with its host and the environment, and therefore to the prevention of epizootics due to this pathogen.

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.

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.

Presence of a lethal protease in the extracellular products of Vibrio splendidus-Vibrio lentus related strains.

The presence of a lethal extracellular 39-kDa protease, a virulence determinant of a Listonella pelagia strain which produces vibriosis in turbot, was determined in the extracellular products (ECP) of 33 Vibrionaceae strains. Both immunological and enzymatic techniques distinguished this specific protease from other Vibrionaceae proteins. It was detected in 15% (5/33) of the ECPs assayed belonging to strains of the Vibrio splendidus-V. lentus related group isolated in Galician aquaculture systems (NW Spain). As these strains were associated with diseased octopus and cultured turbot, were able to colonize the internal organs of fish and produced a lethal ECP for fish, they are a potential risk for the health of reared aquatic organisms.

Characterization of Vibrio strains isolated from turbot (Scophthalmus maximus) culture by phenotypic analysis, ribotyping and 16S rRNA gene sequence comparison.

AIM: The aim of the present study was to clarify the taxonomic status of Vibrio strains isolated from an aquaculture system and to compare the results of the identifications made by phenotypic and molecular methods. METHODS AND RESULTS: Fifty-one Vibrio strains isolated from a turbot (Scophthalmus maximus) aquaculture system were characterized by ribotyping and 16S rRNA gene sequencing. The strains had been identified phenotypically in a previous numerical taxonomy analysis as Vibrio anguillarum, V. mediterranei, V. splendidus, V. aestuarianus, V. ordalii, V. fischeri and V. scophthalmi. Cluster analysis of ribotype patterns showed that the strains were separated into two main groups: V. splendidus-V. lentus and V. scophthalmi groups. The use of 16S rRNA gene sequence allowed differentiation among V. splendidus biovar I and V. lentus strains. CONCLUSIONS: The molecular methods identified strains of V. splendidus biovar I, V. lentus and V. scophthalmi, showing discrepancies with phenotypic characterization. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular methods, as 16S rRNA gene sequence analysis, are necessary for the identification of phenotypically close species to avoid mis-identifications. Interestingly, this is the first report of V. lentus strains associated to turbot culture.

Vibrio lentus associated with diseased wild octopus (Octopus vulgaris).

Studies were undertaken to identify the bacteria involved in a disease of wild octopus (Octopus vulgaris). Signs of the disease include round hard lesions in the arms or head mantle, leading, in advanced cases, to the loss of skin and the exposure of the muscle beneath. Bacterial strains isolated from sterile organs have been evaluated taxonomically and by experimental infections. Different phenotypes and ribotypes of Vibrio lentus were identified. Experimental infection by bath challenge demonstrated that V. lentus was able to reproduce the skin lesions, colonize the internal organs and induce mortality in healthy octopuses. V. lentus was re-isolated from the skin lesions and gill heart of dead octopuses, as confirmed by numerical taxonomy analysis. No effects were produced in sea bream or turbot by intraperitoneal injection of the bacterial isolate.

Purification and partial characterisation of a fish lethal extracellular protease from Vibrio pelagius.

The purification, characterisation and lethal effect of an extracellular protease present in the extracellular products (ECPs) of a pathogenic Vibrio pelagius (7P) strain are described. The extracellular protease was purified by size-exclusion high-performance liquid chromatography and characterised by enzymatic assays. The lethal effect was evaluated by injection into fish. The native protease had a molecular mass of 39 kDa, was active on casein and L-leucyl-beta-naphthylamide (LNA) and its metalloprotease nature was shown by the LNA inhibition profile. Kinetic studies on the hydrolysis of casein and LNA confirmed a competitive inhibition of one substrate with respect to the other. The temperature assays showed that both aminopeptidolytic and caseinolytic activities were labile at 70 degrees C for 3 min. The N-terminal amino acid sequence of 7P protease revealed a high degree of homology with other metalloproteases of Vibrio species that are implicated in virulence. The purified 7P protease showed an LD(50) of 1.77 microgprotein/g fish for turbot. The quick lethal effect (<24h) and the macroscopic damage (external haemorrhagic areas, principally on fins and mouth, petechial haemorrhages in internal organs, but with no external or internal apparent necrotic areas) detected in the host were similar to those obtained by injection of total ECP and live cells of 7P strain. An extracellular protease with endopeptidolytic and exopeptidolytic activities, responsible for the lethal effect of ECP and clinical signs of vibriosis in turbot was purified from a pathogenic V. pelagius (7P) strain.

A 45-kDa acetylcholinesterase protoxin of Aeromonas hydrophila: purification and immunogenicity in fish.

A rabbit antiserum to the 15-kDa acetylcholinesterase toxin neutralised the lethal effect of the 15-kDa toxin of Aeromonas hydrophila when injected into trout. However, immunisation of fish with the 15-kDa toxoid failed to induce an antibody response, and a higher molecular mass form of this toxin was purified from the extracellular products with the aim of inducing an immune response in fish. The optimal conditions for production of extracellular products by A. hydrophila strain B32 were studied to increase the concentration of this protoxin. The extracellular products were fractionated by molecular exclusion chromatography to yield a purified protoxin with an estimated molecular mass of 45 kDa by SDS-PAGE and which gave a positive reaction in Western blotting with the rabbit anti-15-kDa toxin serum. Since the 45-kDa protoxin showed lower specific acetylcholinesterase activity than the active 15-kDa toxin, the behaviour of the active site was studied using specific inhibitors. This 45-kDa protoxin was 13.3-fold less toxic than the 15-kDa toxin and induced antibody production in fish.

Characterization by numerical taxonomy and ribotyping of Vibrio splendidus biovar I and Vibrio scophthalmi strains associated with turbot cultures.

Twelve Vibrio strains were examined phenotypically in 91 biochemical characters and genotypically by ribotyping. Ten were isolated from sea water and two from diseased turbot (Scophthalmus maximus). All isolates originated from one experimental system located in Ría de Vigo (Galicia, north-west Spain). Different type strains were used for comparative purposes. The taxonomic position was analysed with the NTSYST-pc and similarities among strains were calculated by the Simple Matching coefficient (SSM). rRNA gene restriction patterns were performed with the HindIII enzyme. The SSM coefficient separated the 12 Vibrio strains into two groups which included strains that showed a SSM coefficient quite similar to V. splendidus biovar 1 (ATCC 33125) and V. scophthalmi (CECT 4638). None of 91 phenotypical characters were specific in distinguishing both species. The ribotyping confirmed the taxonomic classification of strains. The pathogenicity of each strain was evaluated; 10 environmental strains were avirulent and two, isolated from diseased turbot, were virulent. Different biotypes and ribotypes were found among the avirulent isolates. This work showed ribotyping to be a valuable tool for identification and confirmed the necessity of extending the ribotype database within closely related Vibrio species in order to clarify the taxonomic position.

Numerical taxonomy of gram-negative, facultative anaerobic bacteria isolated from skin of turbot (Scophthalmus maximus) and surrounding water.

A numerical taxonomic study of 473 gram negative heterotrophic facultative anaerobic bacteria isolated from skin of turbot (Scophthalmus maximus) and its culture water was performed. The study included 53 type and reference strains belonging to the genera Vibrio, Aeromonas and Listonella. The strains were characterized using 90 tests and data were examined by Simple Matching coefficient (S(SM)) and Jaccard coefficient (S(J)). UPGMA (unweighted pair group method, arithmetic average) defined 66 phena at S(SM) values > or = 84% and 27 groups at S(SM) > or = 80%. Six phena were defined as Vibrio albensis, V. (Listonella) anguillarum, V. splendidus biotype I, V. fischeri, V. ordalii and V. scophthalmi including reference strains. Some groups clustered different phena for one species, although others as the V. anguillarum related strains and inactive Vibro group required S(SM) > or = 84% to define species. More studies are necessary to identify the Vibrio spp. strains and to confirm some species identifications.

Siderophore production by Aeromonas salmonicida subsp. salmonicida. Lack of strain specificity.

Siderophore production, presence of iron-regulated outer membrane proteins and siderophore specificity was determined among 17 isolates of Aeromonas salmonicida subsp. salmonicida obtained from Spain and Scotland. All grew in the presence of ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA) and siderophore production was detected using chrome azurol S (CAS) agar, confirming the presence of a high-affinity siderophore iron-uptake mechanism. The Arnow test confirmed that all isolates produced a catechol siderophore. Cross-feeding assays with indicator bacteria showed the absence of anguibactin, enterobactin, 2,3-dihydroxybenzoic acid (DHBA) and the hydroxamate siderophore, aerobactin, in the iron-restricted supernants of a representative isolate which cross fed 15/17 A. salmonicida isolates tested. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of the same 2 major iron-regulated outer membrane proteins (IROMPs) in all isolates when grown in iron-restricted conditions and siderophore strain specificity as assessed by cross-feeding experiments was not apparent. Thus, with respect to IROMP and siderophore production A. salmonicida appears to be a homogeneous species.

The acetylcholinesterase ichthyotoxin is a common component in the extracellular products of Vibrionaceae strains.

In previous work, it was reported that a strain of Aeromonas hydrophila (B32) produces the most potent lethal toxin with neurotoxic activity described so far for fish. In the present study, the presence and distribution of this acetylcholinesterase toxin lethal for fish were determined in extracellular products (ECP) of 42 Vibrionaceae strains using both immunological and colorimetric methods. This neurotoxin was shown to be present in the majority of the ECP from the Aeromonas and Vibrio strains tested and is responsible for the specific acetylcholinesterase activity. Also, although the Western blot and Ouchterlony techniques are valid as qualitative methods for the detection of this toxin, the Western blot procedure was 100-fold more sensitive than the Ouchterlony technique.

Surface phenotypic characteristics and virulence of Spanish isolates of Aeromonas salmonicida after passage through fish.

Eleven strains of Aeromonas salmonicida were passaged twice by intraperitoneal injection through rainbow trout and reisolated from the kidney of moribund fish. The surface characteristics and virulence of the strains changed following passage through fish. None of the in vitro tests used could effectively predict the in vivo virulence.

Effects of the acetylcholinesterase toxin of Aeromonas hydrophila on the central nervous system of fish.

The purified acetylcholinesterase (AcChE) toxin, crude extracellular products (ECP) or viable virulent Aeromonas hydrophila were injected intraperitoneally into rainbow trout in different sublethal and lethal doses. When fish showed signs of morbidity, brain tissue was excised and assayed for acetylcholinesterase activity. In all cases there was a large increase in AcChE activity (about 40-fold for purified AcChE-toxin). This was shown to be due to an accumulation of the fish's own AcChE and not the bacterial toxin. Nevertheless, the latter was detected in brain homogenates from fish in all treatment groups using a rabbit antiserum to the purified toxin to probe Western blots of brain homogenates, demonstrating that the toxin does gain access to brain tissue and is produced during in vivo infection. The results strongly suggest that this toxin plays a central role in the pathogenesis of A. hydrophila infection.

Purification and characterisation of an extracellular metalloprotease, serine protease and haemolysin of Aeromonas hydrophila strain B32: all are lethal for fish.

Three different lethal (for rainbow trout, Salmo gairdneri) extracellular toxins were purified by HPLC from the culture supernatants of Aeromonas hydrophila strain B32 which had been isolated from rainbow trout. A metalloprotease, MW 38 kDa, was stable at 56 degrees C for 10 min, had no cytotoxic activity and and LD50 of 150 ng/g fish. In narrow range isoelectric-focusing (IEF) the enzyme had 11 isomers with (pls) between 4.12 and 4.8. A serine protease (22 kDa) was stable at 56 degrees C for 10 min, possessed cytotoxic activity and had an LD50 of 150 ng/g fish. In IEF, multiple isomers possessed pls between 4.5-5.2. The haemolysin had alpha-haemolytic activity (68 kDa) multiple isomers in IEF with pl range 4.5-5.1 and an LD50 of 2 micrograms/g fish. It was stable after heating to 56 degrees C for 20 min, 60 degrees C for 10 min and possessed esterase activity on beta-naphthyl acetate. These latter properties suggest it may be a novel haemolysin distinct from alpha- and beta-haemolysin.

An extracellular acetylcholinesterase produced by Aeromonas hydrophila is a major lethal toxin for fish.

A hitherto unrecognised lethal toxin from the extracellular products (ECP) of Aeromonas hydrophila is described. The pure toxin was 300 times more toxic than the crude ECP and is the most toxic substance so far described from this bacterium, with a minimum lethal dose of 0.05 micrograms g-1 fish. The toxin had high acetylcholinesterase activity and occurred in native ECP as a monomeric 15.5 kDa polypeptide. The purified toxin had five isoelectric focusing forms ranging from pl 4.45 to 4.70. The ECP of each of six strains of A. hydrophila isolated from fish possessed acetylcholinesterase activity suggesting that the toxin is common in this species. The toxin was not a cytolysin and produced no gross pathology in injected fish. Its enzymic nature, low lethal dose, lack of tissue pathology and its apparent narcotic effect suggest that this toxin may act upon the central nervous system of the fish.

Virulence properties and enterotoxin production of Aeromonas strains isolated from fish.

The biological activities in vivo and in vitro of 59 motile Aeromonas spp. isolated from fish and water tanks were simultaneously analyzed in poikilothermic and homoiothermic systems. A total of 64.3% of the isolates tested were pathogenic for fish, and 62% of Aeromonas hydrophila and A. sobria isolates either virulent or nonvirulent for fish were enterotoxigenic. Although the majority of the strains were proteolytic and amylolytic and produced DNase, other activities, such as elastase and staphylolysis, were only present in A. hydrophila. Most of the strains (96%) produced hemolysins, and 68% had agglutinating capacity, but neither isolates pathogenic for fish nor enterotoxigenic isolates showed specificity for trout or human erythrocytes, respectively. The production of siderophores, agglutination in acriflavine, and precipitation after boiling were found not to be useful tests for screening virulent strains. Although statistical analysis revealed a significant relationship between virulence for fish and positive results for arabinose and sucrose fermentations, elastase, and hemolysis of human erythrocytes, only lysine decarboxylase showed a significant positive relationship with enterotoxigenicity. Using extracellular products from representative Aeromonas strains with different virulence markers and belonging to distinct O serogroups, we demonstrated a lack of correlation between cytotoxicity for fish and homoiothermic cell lines and pathogenicity. The extracellular products from selected pathogenic A. hydrophila strains were lethal for rainbow trout and displayed proteolytic, hemolytic, and cytotoxic activities which were simultaneously lost after heat treatment. The findings reported here indicate that it is not possible to establish a common and single mechanism involved in the invasion of Aeromonas spp. in poikilothermic and homoiothermic hosts.

Characterization of extracellular metallo- and serine-proteases of Aeromonas hydrophila strain B51.

The extracellular proteases of Aeromonas hydrophila B51 were stable on heating (56 degrees C) and on storage at 4 degrees C or -20 degrees C. Inhibitor studies showed that 72% of the total activity was inhibited by EDTA (a metalloprotease inhibitor) and 26% was inhibited by phenylmethanesulphonyl fluoride (a serine protease inhibitor). Analytical isoelectric focussing revealed the presence of 33 proteins in the crude extracellular products. Using a casein overlay technique three separate zones of proteolytic activity were detected: a zone with pI 6.5-6.8, formed of two closely focussed bands (possibly isomers of the same protease) and completely inhibited by EDTA; a single band with pI 7.0, which was inhibited by EDTA; and a diffuse zone with pI 8.3-8.5, which was only partially inhibited by EDTA. It is concluded that the serine protease activity focussed in this latter zone. These results indicate the presence of at least four, and possibly five proteases. Our results differ substantially from those reported by other workers using different isolates and it is suggested that significant differences in the character of extracellular products and extracellular proteases exist between different isolates of A. hydrophila.

Evaluation of different assay systems for identification of environmental Aeromonas strains.

Important biochemical reactions in conventional tests were compared with counterpart reactions in two multiple test systems, API-20E (Analytab Products, Plainview, N.Y.) and Aeromonas hydrophila medium, to evaluate their accuracy for the identification of motile Aeromonas spp. isolated from fish. In a total of 49 Aeromonas spp. isolates and 10 A. hydrophila reference strains, false-negative or -positive reactions were detected in the Voges-Proskauer test, indole production, gelatinase activity, production of gas, fermentation of arabinose, and lysine decarboxylase reaction. A good correlation was found, among the three identification systems, for the fermentation of mannitol and inositol as well as for the arginine dihydrolase and ornithine decarboxylase tests. The failure of A. hydrophila medium in the detection of gas indicates that this medium is not entirely suitable for defining aerogenic or anaerogenic strains. From the results of the present study, we consider that of the identification method and taxonomic scheme to be adopted for environmental Aeromonas spp. must be standardized.