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.

Evaluation of recA sequencing for the classification of Aeromonas strains at the genotype level.

AIMS: To evaluate the usefulness of partial recA sequences for the identification of Aeromonas strains at the genotype level. METHODS AND RESULTS: A partial recA sequence was obtained from 21 type or reference strains and 33 Aeromonas isolates, collected in the South of Switzerland from human, animal and aquatic environments. The 272 bp long recA fragments showed a mean interspecies divergence of 7.8% and allowed the classification of strains at genotype level. However, some discrepancies could be observed with other gene sequence based analyses in the classification of some strains. CONCLUSIONS: The 272 bp long recA fragment is a good molecular marker to infer taxonomy of members of the genus Aeromonas, even if the primers we chose for the amplification did not allow its direct sequencing. SIGNIFICANCE AND IMPACT OF THE STUDY: In the genus Aeromonas, nucleotide sequences of some protein-encoding genes have already been evaluated as molecular markers to be used in taxonomical and epidemiological researches. This study suggests the usefulness of a recA fragment as a further sequence to investigate for these purposes.

Molecular identification of Bacillus thuringiensis var. israelensis to trace its fate after application as a biological insecticide in wetland ecosystems.

AIMS: To determine the fate of viable Bacillus thuringiensis var. israelensis (Bti) spores dispersed in the environment, using a universally applicable molecular detection methodology. METHODS AND RESULTS: Soil samples were spread on growth medium, after a temperature selection of the spores. A PCR amplification of the cry4Aa and cry4Ba insecticidal genes was applied on the colonies. Ribotyping was performed subsequently. This combined molecular method proved to be very specific for Bti, which was easily differentiated from the other B. thuringiensis serovars. A site regularly treated with Vectobac-G was chosen within the 'Bolle di Magadino' natural reserve, and monitored throughout 1 year for the detection of Bti spores. The results showed that the numbers were relatively high after insecticidal applications (1.4 x 10(5) CFU g(-1)), and decreased approx. 10-fold after 220 days. A successive treatment induced a new increase. CONCLUSIONS: The results show that yearly repeated use of Vectobac-G does not seem to have a major ecological impact on the 'Bolle di Magadino' natural reserve. Bti spores followed a trend leading to their eventual disappearance from the ecosystem, despite the seasonal application of this biological insecticide for more than a decade. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular identification of Bti cells through the PCR analysis of the delta-endotoxins genes coupled to ribotyping, is an innovative method, that has enabled the identification of this organism into wetland environments.

Molecular typing of Yersinia frederiksenii strains by means of 16s rDNA and gyrB genes sequence analyses.

The phenospecies Yersinia frederiksenii is known to comprise three genospecies, indistinguishable on the basis of phenotypic characteristics. In previous works, 13 strains, identified biochemically as Y. frederiksenii, were characterized using Multilocus enzyme electrophoresis and Ribotyping. In order to elucidate the phylogenetic position of these strains we performed their molecular typing by means of 16S rDNA and gyrB sequences analyses. Results demonstrated that gyrB is a better phylogenetic marker than 16S rDNA. The classification achieved by gyrB sequences analyses was in agreement with results obtained with more laborious methods. Moreover, a good phylogenetic identification could be reached also with partial gyrB sequences of only 350 bp.

Persistence of viral pathogens and bacteriophages during sewage treatment: lack of correlation with indicator bacteria.

The effects of different sewage treatments on the viral contamination in rivers which receive water from treatment plants without a final sand filtration step were investigated. They were all heavily contaminated with bacteriophages and human enteric viruses (detected by single step reverse transcription amplification followed by a nested polymerase chain reaction). Bacteriophages, but not faecal indicator organisms, were correlated with viral contamination.

Intestinal secretory immunoglobulin A (sIgA) response to Aeromonas exoproteins in patients with naturally acquired Aeromonas diarrhea.

The secretory immunoglobulin A (sIgA) response at the intestinal mucosa is one of the primary defense mechanisms protecting against enteric infections and may therefore be used as an indicator of bacterial enteropathogenicity. In order to understand the role played by Aeromonas strains as gastrointestinal infectious agents, the sIgA response in fecal specimens obtained from patients with naturally acquired Aeromonas diarrhea was examined. Our results demonstrated a specific sIgA response which was directed against the exoproteins produced by Aeromonas strains. The specific Aeromonas sIgA reacted with extracellular products showing molecular masses similar to those of the Aeromonas hemolytic toxins such as aerolysin and AHH1. Some reactions were directed against other proteins that are known to be important factors in the pathogenicity of Aeromonas. The specific responses highlighted are in support of the view that one should consider at least certain biotypes of Aeromonas enteropathogenic.

Epidemiological relationships between Aeromonas strains isolated from symptomatic children and household environments as determined by ribotyping.

Ribotyping was used to study the epidemiology of Aeromonas associated gastro-enteritis in young children. Ribotyping patterns of 29 Aeromonas strains (16 Aeromonas caviae, 8 Aeromonas hydrophila, 3 Aeromonas eucrenophila, 1 Aeromonas veronii, and 1 Aeromonas encheleia) isolated from primary stool cultures of sick children were compared using the GelCompare software with patterns of 104 strains (39 Aeromonas eucrenophila, 29 Aeromonas caviae, 11 Aeromonas encheleia, 10 Aeromonas hydrophila, 6 Aeromonas bestiarum, 3 Aeromonas veronii, 3 Aeromonas popoffii and 3 Aeromonas media) isolated from their household environment in order to investigate the route of transmission of these bacteria. Fifteen strains (approximately 47%) isolated from stool cultures of patients showed the same riboprofile as strains found in contacts or environment. In particular, three strains isolated from patients shared the same riboprofile with strains found in their domestic environment. The wide diffusion of potentially pathogenic Aeromonas strains in our household samples, and the high rate of asymptomatic carriers among family members, suggested that predisposing factors of the host could make children prone to an Aeromonas-related intestinal disease.

In situ analysis of sulfate-reducing bacteria related to Desulfocapsa thiozymogenes in the chemocline of meromictic Lake Cadagno (Switzerland).

Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) retrieved two clusters of sequences resembling sulfate-reducing bacteria within the family Desulfovibrionaceae. In situ hybridization showed that, similar to sulfate-reducing bacteria of the family Desulfobacteriaceae, bacteria of one cluster with similarity values to the closest cultured relatives of between 92.6 and 93.1% resembled free cells or cells loosely attached to other cells or debris. Bacteria of the second cluster closely related to Desulfocapsa thiozymogenes DSM7269 with similarity values between 97. 9 and 98.4% were generally associated with aggregates of different small-celled phototrophic sulfur bacteria, suggesting a potential interaction between the two groups of bacteria.

Signature region within the 16S rDNA sequences of Aeromonas popoffii.

To identify a group of eight Aeromonas strains of our collection showing ribotyping patterns similar to those described for the species Aeromonas popoffii, 16S rRNA gene sequence analysis was performed. Results were in agreement with the DNA binding values, and allowed the identification of a 'signature region' differentiating the A. popoffii strains from all other members of the genus Aeromonas.

In situ analysis of phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno (Switzerland).

Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) revealed the presence of a diverse number of phototrophic sulfur bacteria. Sequences resembled those of rRNA of type strains Chromatium okenii DSM169 and Amoebobacter purpureus DSM4197, as well as those of four bacteria forming a tight cluster with A. purpureus DSM4197 and Lamprocystis roseopersicina DSM229. In situ hybridization with fluorescent (Cy3 labeled) oligonucleotide probes indicated that all large-celled phototrophic sulfur bacteria in the chemocline of Lake Cadagno were represented by C. okenii DSM169, while small-celled phototrophic sulfur bacteria consisted of four major populations with different distribution profiles in the chemocline indicating different ecophysiological adaptations.

Multilocus genetic relationships between clinical and environmental Aeromonas strains.

Allelic variations in the chromosomal genome of 120 isolates of motile Aeromonas (A. hydrophila, A. sobria and A. caviae) and eight reference strains with one Plesiomonas shigelloides were assessed by analysis of electrophoretically demonstrable polymorphism in 16 genes encoding metabolic enzymes. The strains were collected from humans (n = 59) and from the aquatic environment (n = 61) in canton Tessin, Switzerland. Clustering of the electrophoretic types (ET) from a matrix of pairwise genetic distances, based on the 16 enzyme loci, confirmed the genetic distinctness of the three species. Furthermore, A. hydrophila and A. sobria were divided in three and two groups respectively. For each species clinical strains were well differentiated from those collected in the environment.