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.

Cell-surface properties of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chilean farms.

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture, but the factors determining its virulence have not yet been elucidated. In this work, cell-surface-related properties of the isolates responsible for outbreaks in Atlantic salmon were investigated. We also briefly examined whether pathogenicity against fish varied for V. ordalii strains with differing cell-surface properties. Hydrocarbon adhesions indicated the hydrophobic character of V. ordalii, although only 4 of 18 isolates induced haemagglutination in Atlantic salmon erythrocytes. A minority of the studied isolates (6 of 18) and the type strain ATCC 33509T produced low-grade biofilm formation on polyethylene surface after 2 h post-inoculation (hpi), but no strains were slime producers. Interestingly, V. ordalii isolates showed wide differences in hydrophobicity. Therefore, we chose 3 V. ordalii isolates (Vo-LM-03, Vo-LM-18 and Vo-LM-16) as representative of each hydrophobicity group (strongly hydrophobic, relatively hydrophobic and quasi-hydrophilic, respectively) and ATCC 33509T was used in the pathogenicity studies. All tested V. ordalii strains except the type strain resisted the killing activity of Atlantic salmon mucus and serum, and could proliferate in these components. Moreover, all V. ordalii isolates adhered to SHK-1 cells, causing damage to fish cell membrane permeability after 16 hpi. Virulence testing using rainbow trout revealed that isolate Vo-LM-18 was more virulent than isolates Vo-LM-03 and Vo-LM-16, indicating some relationship between haemagglutination and virulence, but not with hydrophobicity.

PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene.

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture. To prevent and control outbreaks, a rapid, reproducible, sensitive, and effective diagnostic method is needed. We evaluated a new conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) protocol using a primer set (VohB_Fw-VohB_Rv) designed to amplify a 112 bp fragment flanking the vohB gene (coding for hemolysin production), against 24 V. ordalii strains isolated from different fish species, the V. ordalii type strain, and 42 representative related and unrelated bacterial species. The primer set was species-specific, recognizing all V. ordalii strains evaluated, with no cross-reaction with the other bacterial species. A sensitivity of 103 copies of the vohB gene was obtained with a standard curve. When the VohB_Fw-VohB_Rv qPCR protocol was applied to Atlantic salmon seeded tissues (kidney, liver, spleen, and muscle), the detection limit ranged from 5.27 × 102 to 4.13 × 103 V. ordalii CFU ml-1, i.e. 62 to 145 copies of the vohB gene, using the previously calculated standard curve. The conventional PCR also detected V. ordalii, but the total reaction time was 1 h longer. When the qPCR protocol was applied to naturally infected cage-cultured Atlantic salmon samples, 5 of 8 fish tested positive for V. ordalii, but only one of them was diagnosed as positive by direct cultivation on agar. We conclude that the PCR protocol evaluated is fast, specific, and sensitive enough to detect V. ordalii in infected tissues and is an important tool for secure diagnosis of atypical vibriosis, and is therefore helpful for the control of the disease through the prompt detection within fish populations.

Evaluation of the selective and differential ET medium for detection of Edwardsiella tarda in aquaculture systems.

AIMS: Edwardsiella tarda is an important pathogen in aquaculture where it can cause serious losses. A rapid detection of it is vital to minimize the mortalities caused by this disease, and in this work, the effectiveness of the selective differential Edw. tarda medium (ET) was evaluated for the diagnosis of edwardsiellosis as well as for its possible use in epidemiological studies. METHODS AND RESULTS: ET medium was evaluated in parallel with the commercial Salmonella-Shigella agar (SS), which is usually employed for the selective isolation of enteric bacilli. Moreover, two general media (TSA-1 and MA) were employed as a control. The results obtained showed that ET is distinctly selective for the isolation of Edw. tarda, allowing its recovery from mixed cultures and natural samples as a unique species. In contrast, although colonies of Edw. tarda could be clearly distinguishable in SS because of the appearance of a characteristic black centre, other enteric and nonenteric bacterial species were also able to grow on this medium. CONCLUSIONS: We recommend ET agar as an useful medium for the primary isolation of Edw. tarda from aquaculture samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained support ET medium as the most appropriate to develop epidemiological studies of edwardsiellosis in aquaculture and permits an earlier diagnosis of this important disease.

Intraspecific genetic variability of Edwardsiella tarda strains from cultured turbot.

Edwardsiella tarda is an enterobacterial fish pathogen that causes mortality in various fish species worldwide. In this study, we analyzed the intraspecific variability in a collection of E. tarda strains isolated from turbot. To do this we employed 4 polymerase chain reaction (PCR)-based methods: (1) random amplified polymorphic DNA (RAPD), (2) enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), (3) repetitive extragenic palindromic-PCR (REP-PCR) and (4) BOX-PCR. E. tarda isolates from different hosts were also included for comparison. E. tarda strains from turbot showed high molecular homogeneity when RAPD (primers P3 and P6), ERIC-PCR and BOX-PCR were employed. However, with regard to the REP-PCR and RAPD (primers P4 and P5) techniques, different genetic groups could be established within these isolates using either technique. The 2 RAPD types presented an 85% similarity, while those obtained with REP-PCR showed 74% similarity. Based on the results obtained, although a high genetic homogeneity was found in turbot isolates, the RAPD test (with primers P4 and P5) and REP-PCR were capable of discrimination within these strains, and they are therefore considered the most appropriate typing methods for studies of edwardsiellosis in turbot.

Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms.

A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg microl(-1) for V anguillarum, 500 fg microl(-1) for P. salmonis, and 5 pg microl(-1) for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 x 10(5) CFU ml(-1) of V anguillarum, 1.26 x 10(4) CFU m(-1) of S. phocae, and 5.33 x 10(4) CFU ml(-1) of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 +/- 0.54 x 10(7) CFU g(-1) for V. anguillarum, 9.03 +/- 1.84 x 10(5) CFU g(-1) for S. phocae, 3.8 +/- 0.78 x 10(3) CFU mg(-1) for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of -1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simultaneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms.

Serological and molecular heterogeneity among Yersinia ruckeri strains isolated from farmed Atlantic salmon Salmo salar in Chile.

We investigated 11 strains of Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), that had been isolated from Atlantic salmon Salmo salar L. farmed in Chile and previously vaccinated against ERM. Phylogenetic analysis of the 16S rRNA gene sequences confirmed the identification of the salmon isolates as Y. ruckeri. A comparative analysis of the biochemical characteristics was made by means of traditional and commercial miniaturised methods. All studied isolates were motile and Tween 80 positive, and were identified as biotype 1. In addition, drug susceptibility tests determined high sensitivity to sulphamethoxazole/trimethroprim, oxytetracycline, ampicillin and enrofloxacin in all isolates. Serological assays showed the presence of O1a, O1b and O2b serotypes, with a predominance of the O1b serotype in 9 strains. Analysis of the lipopolysaccharide profiles and the correspondent immunoblot confirmed these results. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the outer membrane proteins revealed that all Chilean strains had profiles with a molecular weight range between 34 and 55 kDa, with 3 distinct groups based on differences in the major bands. Genotyping analyses by enterobacterial repetitive intergenic consensus (ERIC-) and repetitive extragenic palindromic (REP-)PCR techniques clearly indicated intraspecific genetic diversity among Chilean Y. ruckeri strains.

Surface properties of Streptococcus phocae strains isolated from diseased Atlantic salmon, Salmo salar L.

Streptococcus phocae is an emerging pathogen for Chilean Atlantic salmon, Salmo salar, but the factors determining its virulence are not yet elucidated. In this work, cell surface-related properties such as hydrophobicity and haemagglutination, adhesion to mucus and cell lines, capsule detection, survival and biofilm formation in skin mucus and serum resistance of the isolates responsible for outbreaks in Atlantic salmon and seals were examined. Adhesion to hydrocarbons and the results of salt aggregation tests indicated most of the S. phocae were strongly hydrophobic. All isolates exhibited a similar ability to attach to the Chinook salmon embryo (CHSE) cells line, but were not able to enter CHSE cells. Haemagglutination was not detected. Our data clearly indicate that S. phocae can resist the killing activity of mucus and serum and proliferate in them, which could be associated with the presence of a capsular layer around the cells. Pathogenicity studies using seal and fish isolates demonstrated mortality or pathological signs in fish injected only with the Atlantic salmon isolate. No mortalities or histopathological alterations were observed in fish injected with extracellular products.

Evaluation of four polymerase chain reaction primer pairs for the detection of Edwardsiella tarda in turbot.

Edwardsiella tarda is an important emergent pathogen in European aquaculture, causing several mortality events in turbot Scophthalmus maximus cultures in recent years. Here, we evaluated in parallel the specificity of 4 previously published pairs of primers, gyrBF1/gyrBR1, tardaF/ tardaR, etfA and etfD, for the detection of 53 E. tarda strains isolated from different sources, as well as 18 representatives of related and unrelated bacterial species. On the basis of the obtained results, we selected the pair of primers etfD, because it was the only one that recognized all E. tarda strains without false positive reactions. The sensitivity of this primer set showed detection limits of 2 cells per reaction tube in the case of pure cultures and 200 cells per reaction tube in mixed cultures. With regard to the sensitivity in seeded turbot tissues (kidney, liver and mucus), the detection limit was 3 x 10(2) E. tarda cells per reaction. In experimentally infected turbot, the etfD primer set was able to detect the pathogen in internal organs even 1 d post-infection, with a dose of 0.1 cells g(-1) of fish. In addition, this polymerase chain reaction protocol was useful for the detection of E. tarda in the field, and, based on the findings, we propose it as the most appropriate for accurate detection of E. tarda in routine diagnosis of edwardsiellosis in fish.

Experimental Pseudomonas anguilliseptica infection in turbot Psetta maxima (L.): a histopathological and immunohistochemical study.

Experimental infection with Pseudomonas anguilliseptica was performed both by intraperitoneal (i.p.) and bath route on juvenile turbot (Psetta maxima) in order to evaluate the pathology induced. Turbot was found to be sensitive to i.p. challenge (1.7x10(6) CFU/fish) but no to bath exposure. The i.p. challenge induced septicaemic infection and mortality. Externally, moribund fish showed distended abdomen and pale areas at day 9. The gross pathological internal signs present were abundant ascitic fluid in the peritoneal cavity, pale and enlarged spleen, pale and friable liver, and congestive and dilated gut with yellowish exudates. On histopathological examination, bacterial invasion was common in all the tissues studied but the most prominent pathological changes were observed in gut, spleen and kidney after 7 day with features of necrosis. The immunohistochemical findings support the widespread localization of the bacteria after the i.p. injection since the P. anguilliseptica was detected in spleen from day 1 post injection, in liver, kidney and gut from day 4, in muscle from day 7 and in brain from day 9. The difficulties in infecting healthy fish by bath challenge can be explained by the opportunistic nature of this pathogen.

Genetic characterization of Streptococcus phocae strains isolated from Atlantic salmon, Salmo salar L., in Chile.

Streptococcus phocae is a beta-haemolytic bacterium frequently involved in disease outbreaks in seals causing pneumonia or respiratory infection. Since 1999, this pathogen has been isolated from diseased Atlantic salmon, Salmo salar, causing serious economic losses in the salmon industry in Chile. In this study, we used different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR) and restriction of 16S-23S rDNA intergenic spacer regions to evaluate the genetic diversity in S. phocae. Thirty-four strains isolated in different years were analysed. The S. phocae type strain ATCC 51973(T) was included for comparative purposes. The results demonstrated genetic homogeneity within the S. phocae strains isolated in Chile over several years, suggesting the existence of clonal relationships among S. phocae isolated from Atlantic salmon. The type strain ATCC 51973(T) presented a different genetic pattern with the PFGE, RAPD, ERIC-PCR and REP-PCR methods. However, the fingerprint patterns of two seal isolates were distinct from those of the type strain.

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.

Vaccination strategies to prevent emerging diseases for Spanish aquaculture.

In recent years, three serious diseases have emerged in Spanish aquaculture. These are lactococcosis caused by Lactococcus garvieae, which is of economical importance in rainbow trout (Oncorhynchus mykiss); pseudomonadiasis caused by Pseudomonas anguilliseptica which affects gilthead seabream (Sparus aurata) and turbot (Scophthalmus maximus); and flexibacteriosis caused by Tenacibaculum maritimum which became a devastating problem in the emerging culture of sole (Solea spp). To obtain useful information for the design and development of new vaccines, antigenic characterisation of representative strains was performed. In this work we present the strategies adopted for the vaccine formulation (strains included, use of adjuvants) and administration (route, necessity of booster, etc.). The results from laboratory and/or field vaccination trials performed showed that for lactococcosis, protection lasting for five months was obtained with an oil-adjuvanted bacterin formulation. Unadjuvanted bacterin gave only a short duration of protection, which could, however, be prolonged by an antigen boost administered via the feed. A bacterin against Pseudomonas anguilliseptica gave protection for 12 weeks when tested in an experimental challenge trial in turbot. Besides the flexibacteriosis vaccine developed by our group for turbot, and due to the antigenic host-associated variability within T. maritimum, a new bacterin was developed against this bacterium to be used specifically in sole. This new bacterin, administered to sole by intraperitoneal injection, yielded RPS values of 94 % six weeks after immunization. In conclusion, these results suggest that vaccination constitutes a cost-effective method of controlling diseases that have emerged in the most important fish species being cultured in Spain.

Pathological activities of Yersinia ruckeri, the enteric redmouth (ERM) bacterium.

The adherence and invasive capacities as well as the pathobiological activities exhibited by Yersinia ruckeri were examined. Although adhesive ability was dependent on the cell-line employed, all the strains showed moderate adhesion and invasiveness in the salmon cell-line CHSE-214. With regard to the extracellular products (ECP) all of them were strongly toxic for fish with LD50 ranging from 2 to 9.12 micrograms protein per g fish. In addition, all the ECP samples showed caseinase, gelatinase, amylase, lipase and phospholipase activities, hydrolysed esculin and displayed hemolytic activities for trout, salmon, sheep and human erythrocytes. Heat treatment (100 degrees C for 10 min) caused the loss of all these biological activities except the hydrolysis of gelatin. On the other hand, SDS-PAGE analysis of the LPS and protein components of the ECP revealed variations among strains depending on the serotype. The lack of lethal effects of the LPS present in the ECP was also demonstrated.

O-serogrouping and surface components of Aeromonas hydrophila and Aeromonas jandaei pathogenic for eels.

The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting [SP] and precipitation after boiling [PAB], profile of lipopolysaccharides [LPSs] and outer membrane proteins [OMPs]) was investigated in strains of the pathogenic species Aeromonas hydrophila and A. jandaei isolated from eels. Virulent strains of A. hydrophila reacted mostly with O:19 antiserum, and those of A. jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinée and Jansen system). Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB+ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB- strains antigenically diverse that either exhibited a heterogeneous side chain or were side chain deficient. A major 50 kDa protein was only found in the PAB+ strains, whereas major OMPs detected in PAB- strains ranged from 33 to 45 kDa irrespective of the species. Epizootic eel isolates of A. hydrophila belong to serotype O:19 and share cell-surface features with the Aeromonas highly virulent for other hosts. In contrast, epizootic A. jandaei isolates were antigenically diverse. These findings reinforce the importance of an O-serotype as an epidemiological marker in motile Aeromonas strains pathogenic for eels.

Genetic transformation of Vibrio anguillarum and Pasteurella piscicida by electroporation.

Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cm-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 x 10(3) transformants per micrograms DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 x 10(2) transformants per micrograms pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens.

Survival of fish-virulent strains of Photobacterium damselae subsp. damselae in seawater under starvation conditions.

The survival of fish-virulent strains of Photobacterium damselae subsp. damselae in seawater microcosms, with and without sediment, was investigated. The strains survived as culturable bacteria at 14 and 22 degrees C for at least 1 year, and infectivity for fish was maintained. At 5 degrees C, cells lost culturability on solid media, but this was recovered when the temperature was increased to 22 degrees C. Finally, morphological changes in the bacterium (rod to coccus), and production of vesicles and extracellular material were observed during the time of starvation. The overall results suggest that seawater and sediment can act as reservoirs for these virulent strains.

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system.

This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri. A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogeneicity of Y. ruckeri for fish.

Comparison of outer membrane protein profiles of Vibrio vulnificus biotypes 1 and 2.

The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European eels, and 12 biotype 1 strains from clinical and environmental sources have been compared. The overall profile in both biotypes was similar, and a major protein band of molecular mass 36 kDa was detected in the majority of the strains. Differences in the minor bands allowed differentiation of strains from different origins, suggesting that outer membrane protein profiles could be useful as epidemiological markers in the species V. vulnificus. Immunoblotting with antisera to whole cells of selected strains of biotypes 1 and 2 showed a strong antigenic response to outer membrane proteins 66, 60, 48, 46 and 44 kDa; these were common to all strains examined, independent of their biotypes and origins. These results demonstrate the presence of antigenically related outer membrane proteins in both biotypes of V. vulnificus.