S. Núñez

Microflora associated with healthy and diseased turbot (Scophthalmus maximus) from three farms in northwest Spain

A comparative analysis of the microbiological quality of three turbot (Scophthalmus maximus) farms (A, B, and C) located in Galicia (northwest Spain) is given. The microbial load and types of bacteria in the internal organs (liver and kidney) of apparently healthy fish was monitored over a year, and all the disease problems occurring during this survey were analyzed. The percentage of healthy turbot in which positive bacterial growth was obtained was relatively high in the three ongrowing facilities. Farm A exhibited the poorest conditions of fish health with an average of 42% fish infected, while farm B showed the best microbiological quality with 27% of turbot harbouring bacteria in the internal organs. In all three farms, a wide range of bacteria was found in healthy turbot with Vibrio (V. splendidus-V. pelagius, V.fisheri-V. harveyi and Vibrio spp.) and Pseudomonas spp. being the predominant groups comprising at least 80% of the total bacterial isolates in each farm. The highest number of pathological problems (22) with the most diverse bacterial flora occurred in farm A. Vibrio spp. and Pseudomonas spp. were the most prevalent bacteria recovered from diseased turbot. Haemorrhages in palate and jaws, tail and fins, and ulcerative lesions were the most frequent external clinical sings of diseased fish recorded in the three farms. However, it was not possible to associate a particular bacterial species with a specific pathology. Routine use in farm A of oxolinic acid and nitrofurantoin may have led to the development in the Vibrio strains of resistances to both chemotherapeutants (up to 25%).

Usefulness of the API-20E system for the identification of bacterial fish pathogens.

Abstract A total of 223 isolates belonging to motile Aeromonas, A. salmonicida, Vibrio anguillarum, Pasteurella piscicida and Yersinia ruckeri species were tested by the API-20E system and the results compared with those obtained with standard biochemical tube and plate tests. Depending on the species, the API-20E yielded false negative and positive reactions for fermentation of different sugars, lysine decarboxylase (LDC), Voges Proskauer (VP), citrate and gelatinase reactions. Thirteen of 32 (41%) A. salmonicida strains and 9 of 53 (17%) Y. ruckeri isolates were correctly identified using the API index. Forty-five of 69 (65%) motile Aeromonas tested (including 34 A. hydrophila, 10 A. sobria and 1 A. caviae strains) were identified as A. hydrophila. The low discrimination profiles generated by 9 Yersinia and 2 motile Aeromonas isolates were avoided using the supplementary tests cited in the API index. In the case of V. anguillarum and P. piscicida, which are not at present included in the API-20E system, 35 of 53 strains of V. anguillarum were wrongly identified as A. hydrophila, and all the P. piscicida isolates were incorrectly identified as Pseudomonas fluorescens/Ps. putida. A large number of isolates, including some reference strains, produced uncoded profiles. From these results, we consider that the API-20E system will be a useful tool for field diagnosis of bacterial fish diseases if its database is expanded to include V. anguillarum, P. piscicida, A. sobria and A. caviae species and the new profiles generated by strains of species already considered. In addition, some reactions necessary to discriminate between strains which share the same profile number are proposed.

Development of an effective Edwardsiella tarda vaccine for cultured turbot (Scophthalmus maximus).

Since 2004 Edwardsiella tarda has become one of the most important emerging pathogens in turbot aquaculture industry in Europe causing serious economic losses. Therefore, this study aimed to design an effective vaccination strategy to prevent edwardsiellosis in this fish species. Two vaccine formulations, an adjuvanted vaccine and an aqueous bacterin, and different routes of administration, bath and intraperitoneal injection (i.p.), were tested. The effectiveness of the different immunization strategies was evaluated in terms of relative percent survival (RPS) and antibody levels. On the basis of the results obtained we recommend the i.p. administration of a non-mineral oil adjuvanted vaccine via i.p., which confers RPS values over 90% at least 6 months post-vaccination.

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.

ASSOCIATION OF A MORAXELLA SP. AND A REO-LIKE VIRUS WITH MORTALITIES OF STRIPED BASS, MORONE SAXATILIS

ABSTRACT: During winter 1987, mortalities occurred in juvenile striped bass ( Morone saxatilis ) in the discharge channel of a power plant located on the Potomac River, Maryland, USA. Affected fish showed large hemorrhagic lesions along the dorso-lateral portions of the body where scales were missing. Hemorrhages were found in the swim bladder and the liver was pale, enlarged, and mottled. Large amounts of membranous material were seen connecting the liver and body wall. The gills of all fish examined were invaded by the parasites Trichodina and Ergasilus.

Phenotypic and Genetic Characterization of Pseudomonas anguilliseptica Strains Isolated from Fish

Abstract The phenotypic and genetic characteristics of a group of European strains of Pseudomonas anguilliseptica isolated from different hosts are described and compared with those of the type strain CECT (Spanish Collection of Type Cultures) 899 originally isolated from Japanese eels Anguilla japonica in Japan. The taxonomic analysis of these isolates revealed high homogeneity of characteristics regardless of the geographic origin and host. Variable results were only obtained for citrate utilization and the hydrolysis of Tween 20 and starch. Based on comparison with conventional tests, consistent results in the miniaturized system API20NE were only achieved for the P. anguilliseptica isolates when bacterial inocula were prepared from blood agar and spectrophotometrically adjusted to an optical density of 1 (at an absorbance of 580 nm). Although P. anguilliseptica is not included in the API database, this miniaturized system can be useful for the identification of this microorganism under the above condi...

Identification of Vibrio harveyi isolated from diseased cultured wedge sole Dicologoglossa cuneata

We report the first isolation of Vibrio harveyi from wedge sole Dicologoglossa cuneata. The pathogen was recovered from ulcers and internal organs of ailing cultured fish, from 7 different outbreaks between 2004 and 2006. The 15 isolates found were phenotypically characterized using biochemical tests and BIOLOG GN plates, which revealed high phenotypic diversity. Diagnosis was confirmed with PCR using V harveyi specific primers and partial 16S and 23S rRNA gene sequencing. A virulence evaluation of the isolates was also performed using fry and juvenile wedge sole. Significant mortalities were recorded by intraperitoneal injection; however, no mortalities were recorded by bath immersion.

Recommendation of an Appropriate Medium for In Vitro Drug Susceptibility Testing of the Fish Pathogen Tenacibaculum maritimum

ABSTRACT In the present study, Anacker and Ordal agar, marine agar (MA), and Flexibacter maritimus medium (FMM) were compared with the dilute versions of Mueller-Hinton agar (DMHA) medium recommended by the National Committee for Clinical Laboratory Standards (NCCLS) for their use in disk diffusion tests with Tenacibaculum maritimum strains and to calculate the MICs of five drugs by the Etest method. Preliminary growth tests performed with 32 strains of this pathogen on each medium revealed that all strains failed to grow on DMHA, while the remaining media supported good growth of all isolates. In the susceptibility tests, which were carried out with the other three media, all strains were resistant to oxolinic acid and were highly susceptible to amoxicillin and trimethoprim-sulfamethoxazole, showing a good correspondence with the Etest values, which ranged from 0.064 to 0.75 and 0.006 to 1.5 μg/ml, respectively. Enrofloxacin and oxytetracycline produced significantly smaller inhibition zones and MICs on MA than on the other media assayed. However, fast, clear, and well-defined zones of inhibition were displayed for all strains at 24 h of incubation only on FMM by both the disk diffusion assay and Etest. In addition, FMM prepared with commercial sea salts instead of seawater was also suitable for bacterial isolation as well as for susceptibility testing. On the basis of these results, the use of FMM to determine the in vitro susceptibility of T. maritimum and its inclusion in a future revision of the NCCLS M42 report are recommended.

Characterization of Vibrio tapetis strains isolated from diseased cultured Wedge sole (Dicologoglossa cuneata Moreau)

The first isolation of Vibrio tapetis from Wedge sole (Dicologoglossa cuneata) is reported. The bacterium was recovered from ulcers of ailing cultured fish, from two different outbreaks occurred in spring 2005. The four isolates found (a200, a201, a204 and a255) were biochemically, genetically and serologically characterized and diagnosis was confirmed by PCR V. tapetis specific primers and multilocus sequencing analysis (MLSA). The isolates constituted a homogeneous phenotypic and genotypic group, being distinct to the already serological and genetic groups defined within the species. A virulence evaluation of the isolate a255 was also carried out; however this strain was unable to induce disease in fry and juvenile Wedge sole.

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.