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.

Anticardiolipin and antinuclear antibodies in cancer patients--a case control study.

OBJECTIVE: To investigate patients with cancer for the frequency of IgG and IgM aCL and ANA in comparison to a group of age- and sex-matched controls. METHODS: Serum levels of IgG and IgM anticardiolipin antibodies (aCL), antinuclear antibodies (ANA) and anticytoplasmic antibodies were evaluated in 145 cancer patients, including 20 patients with thromboembolic disease (TED) and compared with age- and sex-matched controls. RESULTS: Higher levels of IgG aCL were found in patients compared to controls (p < 0.02). However, there appeared to be no difference in serum aCL levels between TED and the remaining cancer patients. No difference was found in the frequency of antinuclear and anticytoplasmic antibodies between patients and controls and the autoantibody presence in patients was usually not associated with concomitant autoimmune disease. CONCLUSION: Apart from increased levels of non-thrombogenic associated IgG aCL, there was no evidence for significantly enhanced B cell autoreactivity in this large collection of cancer patients compared to controls.

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.