Isolation of a highly pathogenic Vibrio pelagius strain associated with mass mortalities of turbot,Scophthalmus maximus (L.), larvae

A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain. Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected. The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being < 5 bacteria mL(-1) for larvae 1 week post-infection and 3.9 x 10(5) bacteria mL(-1) in post-larvae at day 12 post-infection. The bacterial strain was recovered in pure culture from the internal organs of infected fish. Histological lesions in post-larvae exhibited swelling and necrosis of gill secondary lamellae, sloughing of intestinal mucosa and necrosis of haematopoietic tissue in the kidney. Vibrio pelagius (Hq 222) was able to grow in sterile sea water when incubated at room temperature or at 15 degrees C. Vibrio pelagius (Hq 222) was more adherent to the turbot cell lines TV-1 and TF than Escherichia coli. In both cell lines, the number of adhered bacteria increased with incubation time.     

Existence of two O-serotypes in the fish pathogen Pseudomonas anguilliseptica

The serological characteristics of a group of 32 Pseudomonas anguilliseptica strains isolated in Spain from seabream (Sparus aurata) and turbot (Scophthalmus maximus) were compared with a total of 18 collection strains of this bacterial species with different geographical and host origin. The employment of different techniques, including slide agglutination, microagglutination and dot blot, allowed us to establish two serological groups, one comprising practically all the eel isolates, and the other including the majority of isolates from other fish species. The study of the lipopolysaccharides (LPS) and outer membrane proteins (OMP) corroborated these results, indicating that the serological differences among strains are due to the somatic antigen and not to antigenic determinants of protein nature. Therefore, a serological scheme of two "O" serotypes is proposed for P. anguilliseptica. The results obtained will be of importance for epidemiological studies as well as for the design of adequate vaccine formulations.     

Evidence for the facultative intracellular behaviour of the fish pathogen Vibrio ordalii

Vibrio ordalii is an extracellular, Gram‐negative bacterium that produces vibriosis in salmonids. While pathogenesis is not fully understood, this bacterium has numerous likely genes for adhesion, colonization, invasion factors and, as recently suggested, intracellular behaviour. Therefore, this study aimed to clarify possible intracellular behaviour for V. ordalii Vo‐LM‐18 and ATCC 33509^T in the fish‐cell lines SHK‐1 and CHSE‐214. Confocal microscopy revealed Vo‐LM‐18 and ATCC 33509^T inside cytoplasm in both fish‐cell lines at 4 hr post‐inoculation (hpi). At 8 and 16 hpi, the proportion of fish cells invaded by both strains increased. Moreover, intracellular V. ordalii were observed after 8 hpi inside mouse embryonic fibroblasts (MEF), demonstrating that entry was not due to a cellular phagocytosis process. Flow cytometry confirmed immunocytochemistry results, with both V. ordalii evidencing statistically significant differences in the number of infected cells between 8 and 16 hpi. Interestingly, V. ordalii infection did not significantly damage fish cells, as determined by LDH liberation. Viable counts at 8 hpi detected, on average for both lines, 176 ± 47 CFU/ml of culturable intracellular Vo‐LM‐18 and ATCC 33509^T cells. These in vitro findings support the facultative intracellular behaviour of V. ordalii and may be of importance for understanding pathogenicity and survival in aquatic environments.     

First isolation of Tenacibaculum maritimum from wedge sole, Dicologoglossa cuneata (Moreau)

The first isolation of Tenacibaculum maritimum from wedge sole, Dicologoglossa cuneata, is reported. The pathogen was recovered from ulcers of cultured fish, from three different outbreaks. The six isolates obtained were biochemically and serologically characterized and diagnosis was confirmed by polymerase chain reaction using specific primers and partial 16S rRNA gene sequencing. The isolates constituted a homogeneous phenotypic group; however, they belong to two of the different serotypes described within this species. A virulence evaluation of the isolates using Wedge sole fry was also performed.     

Insights into the virulence‐related genes of Edwardsiella tarda isolated from turbot in Europe: genetic homogeneity and evidence for vibrioferrin production

Edwardsiella tarda has long been known as a pathogen that causes severe economic losses in aquaculture industry. Insights gained on E. tarda pathogenesis may prove useful in the development of new methods for the treatment of infections as well as preventive measures against future outbreaks. In this report, we have established the correlation between the presence of virulence genes, related with three aspects typically involved in bacterial pathogenesis (chondroitinase activity, quorum sensing and siderophore‐mediated ferric uptake systems), in the genome of E. tarda strains isolated from turbot in Europe and their phenotypic traits. A total of 8 genes were tested by PCR for their presence in 73 E. tarda isolates. High homogeneity was observed in the presence/absence pattern of all the strains. Positive results in the amplification of virulence‐related genes were correlated with the detection of chondroitinase activity in agar plates, in vivo AHL production during fish infection and determination of type of siderophore produced by E. tarda. To the best of our knowledge, this is the first study carried out with European strains on potential virulence factors. Furthermore, we demonstrated for the first time that E. tarda produces the siderophore vibrioferrin.     

Evolution of drug resistance and minimum inhibitory concentration to enrofloxacin in <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> strains isolated in fish farms

The in vitro susceptibility of 63 isolates of Tenacibaculum maritimum from four fish farms to eight chemotherapeutic agents used for the treatment of bacterial diseases in fish were assessed. The results indicated that all strains were resistant to oxolinic acid and susceptible to amoxicillin, nitrofurantoin, florfenicol, oxytetracycline and trimethoprim-sulphamethoxazole. However, some isolates presented resistance to enrofloxacin and flumequine, ranging from 10 to 30%, and from 25 to 60%, respectively, depending on the farm sampled. These data were used in an attempt to predict whether the resistance to enrofloxacin was static or evolved during the time of sampling from 2003 to 2004. A relationship between the use of enrofloxacin and levels of resistance was detected in the studied farm, increasing significantly from no resistant isolates in 2003 to 44.8% resistant strains in 2004, the year in which this drug was commonly employed. This result was accompanied by a marked decline of about 29.2% of the inhibition zone sizes for the T. maritimum strains in comparison to the initial values (average 21.5 mm). Minimum inhibitory concentration (MIC) of enrofloxacin for 100 T. maritimum strains was determined by the microdilution method. Twenty isolates were resistant to enrofloxacin (> 256 μg ml⁻¹), while the remaining strains showed a bimodal distribution, which ranged from 0.5 to 32 μg ml⁻¹. Our interpretation of the enrofloxacin MIC data suggests that the breakpoint for T. maritimum should be 4 μg ml⁻¹. However, similar studies in other laboratories are necessary to validate this breakpoint value.     

Molecular characterization of Portuguese strains of Yersinia ruckeri isolated from fish culture systems

A total of 23 Portuguese strains of Yersinia ruckeri , the causative agent of enteric redmouth disease (ERM), were comparatively studied by means of lipopolysaccharide (LPS) and outer membrane protein (OMP) analysis, plasmid profiling and ribotyping in order to investigate the heterogeneity among isolates and the usefulness of these methods as epidemiological markers. Only two LPS profiles were observed among the isolates studied, corresponding with the serotypes O1 and O3 of Y. ruckeri . A higher heterogeneity was detected analysing the OMP, seven different patterns being observed. Although some isolates carried different small plasmids, all the serotype O1 isolates showed a plasmid band of 50 MDa and all the serotype O3 strains shared in common a extrachromosomic DNA band of 30 MDa. The analysis of the ribopatterns obtained using three different enzymes, separated the strains into 10 ribotypes, indicating genetic heterogeneity among the isolates. The heterogeneity was greater within serotype O1 isolates, six ribotypes being detected, than within serotype O3 in which only three ribotypes were found. Although OMP analysis and ribotyping can be useful for the differentiation of strains on the basis of farm and/or season of isolation, we consider that ribotyping is the best candidate for epidemiological studies because it is easier to perform and offers a slightly better discriminative power.     

Health status of two salmonid aquaculture facilities in North Portugal: characterization of the bacterial and viral pathogens causing notifiable diseases

A comparative bacteriological and virological survey was conducted in two fish farms in the North of Portugal. The fish species examined included cultured rainbow trout, Oncorhynchus mykiss (Walbaum), and brown trout, Salmo trutta L., as well as wild fish captured near both facilities. The microbial load in the internal organs of apparently healthy fish was nonitored over a year, an all the disease problems occurring during this period were investigated. Although both farms presented intermediate levels of infection(30–40% infected fish), farm B showed the poorest microbiological quality since constant but low mortalities were observed throughout the year. Flavobacterium and Psedomonas-Xanthomonas were the predominant bacterial groups, comprising around 40–50% of the isolates from each farm. In farm B, members of the Enterobacteriaceae and mortile Aeromonas also showed significant prevalence (about 20%). The only outbreak of a notifiable disease was an occurrence of furunculosis, caused by Aeromonas salmonicida subsp. salmoncida, in farm A. However, Yersinia ruckeri was isolated not only from diseased fish, but also from asymptomatic fish, usually in mixed infections with motile Aeromonas or infections with motile Aeromonas or infectious pancreatic necrosis virus (IPNV). While Y. ruckeri isolates associated with mortalities belonged to the serotype O1 (subgroup a), those isolated from asymtomatic fish corresponded to serotype O3. Two strains of IPNV (serotype Ab) were isolated in farm B, which represents the first viral agent detected in Portuguese aquaculture. Qualitative and quantitative differences in microbial load were observed between cultured and wild fish. No notifiable bacterial or viral pathogens were detected in any of the feral species studied.     

Characterization of Edwardsiella tarda strains isolated from turbot, Psetta maxima (L.)

The biochemical, serological and molecular characteristics of a group of 21 Edwardsiella tarda strains isolated from turbot, Psetta maxima, in two different areas of Europe were analysed and compared with a total of 13 strains of this bacterial species with different geographical and host origins. All the turbot isolates were biochemically identical to the E. tarda strains included as reference. The use of different techniques including microagglutination, dot blot and Western blot of lipopolysaccharides allowed us to determine that all the turbot isolates constitute an homogeneous and distinctive serological group. Genetic analysis by randomly amplified polymorphic DNA (RAPD) analysis demonstrated that although the E. tarda strains from turbot were compiled in a unique group using the primers P3 and P6, two clonal lineages could be detected when oligonucleotides P4 and P5 were employed.