Nuria Castro

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.

Effectiveness of bivalent vaccines against Aeromonas hydrophila and Lactococcus garvieae infections in rainbow trout Oncorhynchus mykiss (Walbaum)

Lactococcus garvieae and Aeromonas hydrophila are bacterial pathogens affecting salmonids and other fish species and cause of heavy losses in aquaculture. Diseases caused by these bacteria can be controlled satisfactory by immunization using monovalent vaccines. In this study, the protective efficacy of two bivalent vaccines against L. garvieae and A. hydrophila was evaluated in rainbow trout (Oncorhynchus mykiss). Bivalent formulations, containing formalin-inactivated bacteria, were prepared as an aqueous bacterin and as an adjuvanted vaccine using montanide ISA-763. Protection against L. garvieae and A. hydrophila was tested at day 30 and 90 post-vaccination. High levels of protection were achieved for the aqueous and adjuvanted bivalent vaccines against L. garvieae (RPS of 100% and 95.3%) and A. hydrophila (RPS of 100% and 95.3%) at day 30 post-vaccination. Significant differences (p < 0.05) were found between the RPS at days 30 and 90 post-immunization with a decrease in the protection levels for the aqueous bivalent vaccine against L. garvieae (RPS 76.2%) and A. hydrophila (RPS 85%), but not for the adjuvanted vaccine (RPS of 90% against L. garvieae and 95% against A. hydrophila). In addition, high antibody levels were observed in the vaccinated fish at day 15 post-immunization using both vaccines. Our results demonstrate that these bivalent vaccines can effectively protect rainbow trout against L. garvieae and A. hydrophila and could offer an appropriate strategy to prevent these infections in rainbow trout farms.

Multilocus sequence analysis of the marine bacterial genus Tenacibaculum suggests parallel evolution of fish pathogenicity and endemic colonization of aquaculture systems.

ABSTRACT The genus Tenacibaculum, a member of the family Flavobacteriaceae, is an abundant component of marine bacterial ecosystems that also hosts several fish pathogens, some of which are of serious concern for marine aquaculture. Here, we applied multilocus sequence analysis (MLSA) to 114 representatives of most known species in the genus and of the worldwide diversity of the major fish pathogen Tenacibaculum maritimum. Recombination hampers precise phylogenetic reconstruction, but the data indicate intertwined environmental and pathogenic lineages, which suggests that pathogenicity evolved independently in several species. At lower phylogenetic levels recombination is also important, and the species T. maritimum constitutes a cohesive group of isolates. Importantly, the data reveal no trace of long-distance dissemination that could be linked to international fish movements. Instead, the high number of distinct genotypes suggests an endemic distribution of strains. The MLSA scheme and the data described in this study will help in monitoring Tenacibaculum infections in marine aquaculture; we show, for instance, that isolates from tenacibaculosis outbreaks in Norwegian salmon farms are related to T. dicentrarchi, a recently described species.

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.

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.

Furunculosis in Senegalese sole (Solea senegalensis) cultured in a recirculation system

IN recent years, the mariculture industry in Spain has focused on searching for new species to rear other than turbot ( Scophthalmus maximus ), gilthead sea bream ( Sparus aurata ) and sea bass ( Dicentrarchus labrax ), with the aim of increasing diversification. Senegalese sole ( Solea senegalensis ) is a flatfish belonging to the family Soleidae, which is of special interest for mariculture because of its high commercial value, its scarcity in the markets and its biological characteristics, which allow it to be ideally suited to aquaculture conditions. Although this fish species has been reared in extensive aquaculture in the marshes of the southern Atlantic coast of Spain and Portugal (Dinis and others 1999), at present some marine farms are introducing it in Galicia (north-west Spain), with promising results (Olmedo and others 2003). Until now, the most common bacterial diseases affecting cultured sole have been photobacteriosis, vibriosis and tenacibaculosis (Toranzo and others 2003). Aeromonas salmonicida subspecies salmonicida , the causal agent of so-called ‘typical’ furunculosis, was reported for the first time in 1894 in Germany, during a disease outbreak at a Bavarian brown trout ( Salmo trutta ) hatchery (Emmerich and Weibel 1894). Currently, this pathogen has a wide geographical distribution, including most European countries, and Canada and Japan, among others. In Spain, A salmonicida subspecies salmonicida was isolated for the first time in Galicia in 1987 from rainbow trout ( Oncorhynchus mykiss ) cultured in freshwater. In seawater culture, furunculosis was detected in 1989 in Atlantic salmon ( Salmo salar ) and trout; the disease most probably originated from the transport of infected yearling brown trout from central Spain (Toranzo and others 1990). The incidence of furunculosis in salmonids cultured in freshwater and seawater has increased steadily in Spain in subsequent years, constituting a significant threat to other cultured fish species. In 1992, an outbreak of furunculosis was diagnosed for …

Comparative polyphasic characterization of Streptococcus phocae strains with different host origin and description of the subspecies Streptococcus phocae subsp. salmonis subsp. nov.

A polyphasic study was undertaken to clarify the taxonomic position of Streptococcus phocae strains isolated from Atlantic salmon (Salmo salar) cage-farmed in Chile. Four salmon and three seal isolates showed minor differences in the SDS-PAGE protein analysis. Thus, a major protein band present in the salmon isolates, of approximately 22.4 kDa, was absent in the pinniped strains, regardless of the growth media employed. In addition, the pinniped strains showed protein bands with molecular masses of 71.5 and 14.2 kDa, when grown on trypticase soy agar supplemented with 1% NaCl, or 25.6 kDa, when grown on Columbia blood agar, not present in the Atlantic salmon strains. A high similarity in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS spectra of the strains was observed, although some minor peaks were absent in the fish isolates. Fatty acid methyl esters from isolates with different host origin significantly (P<0.05) differed in the content of C16:0, C17:0, C18:1ω9c, C20:4ω6,9,12,15c and summed features 3, 5 and 8. The salmon isolates formed a separate cluster in the phylogenetic analysis of housekeeping genes, separately or as concatenated sequences. Sequence divergences among salmon and seal strains were in the range of inter-subspecies differentiation for groEL (2.5%), gyrB (1.8%), recN (2.1%), rpoB (1.7%) and sodA (2.0%) genes. DNA-DNA hybridization results confirmed those of sequencing, showing reassociation values between seal and salmon strains close to the borderline of species definition. Differences in growth at low temperatures and in the haemolytic capacities were also observed between both groups of isolates. On the basis of all these results, the salmon isolates represent a novel subspecies of S. phocae, for which the name Streptococcus phocae subsp. salmonis subsp. nov. is proposed. The type strain is C-4T (=CECT 7921T=DSM 24768T). The subspecies Streptococcus phocae subsp. phocae subsp. nov. is automatically created. An emended description of S. phocae is also provided.

First description of Edwardsiella tarda in Senegalese sole, Solea senegalensis (Kaup)

Edwardsiella tarda is a Gram-negative, motile, rodshaped, member of the family Enterobacteriaceae. E. tarda has been isolated from a great variety of fish species, shellfish, reptiles, amphibians, birds, mammals and from the environment contaminated by animals or man. Although first descriptions of E. tarda hypothesized that infections occurred preferentially at warm water temperatures (Plumb 1999), subsequent studies described edwardsiellosis in water temperatures between 10 and 18 C and in cold seasons (Lui & Tsai 1980; Castro, Toranzo, Barja, Núñez & Magariños 2006). In recent years, edwardsiellosis caused by E. tarda has had a serious impact in fish culture, for example, as an important emergent pathogen in European turbot, Scopthalmus maximus (L.), culture (Castro et al. 2006; Toranzo 2007). The Senegalese sole, Solea senegalensis (Kaup), is of great interest to aquaculturists because of its relatively fast growth and good commercial prospects. It has a high market demand with a high commercial value and would permit a more diversified production from aquaculture producers. At present, pasteurellosis (Photobacterium damselae subsp. piscicida) (Magariños, Romalde, LopezRomalde, Morinigo & Toranzo 2003), vibriosis (Vibrio harveyi) (Rico, Tapia-Paniagua, Martı́nezManzanares, Balebona & Moriñigo 2008) and tenacibaculosis (Tenacibaculum maritimum) (Cepeda & Santos 2002) are considered the most important bacterial pathogens affecting sole culture. However, as happens with other fish cultures, once the development of prophylactic measures and/or suitable vaccination programmes control these diseases, previously unrecognized pathogenic agents may assume greater prominence. In this communication, we report the first case of edwardsiellosis caused by E. tarda in farmed Senegalese sole. During the spring of 2010, an episode of mortality occurred in 100 g sole reared in a marine farm in Galicia (north-western Spain), and water temperature was around 17 C and salinity 32%. Turbot were also cultured in the rearing facility using the same water recirculation system but in different tanks. Examination of dead fish on the farm revealed the presence of cutaneous lesions in the dorsal surface, tumefactions around the eyes and haemorrhages in their ventral surface. Affected sole were sent to the laboratory for microbiological analysis. Fishes were killed with tricaine methanesulphonate (MS-222), necropsied under aseptic conditions and examined for internal lesions. Sole presented abundant ascitic fluid, anaemic liver and kidney with petechial haemorrhages. To determine the causative agent of this pathology, a group of 30 affected fish were subjected to routine microbiological analysis. Internal organs (spleen, liver and kidney) as well as external lesions Journal of Fish Diseases 2012, 35, 79–82 doi:10.1111/j.1365-2761.2011.01325.x

A multiplex PCR for the simultaneous detection of Tenacibaculum maritimum and Edwardsiella tarda in aquaculture.

A specific and sensitive multiplex PCR (mPCR) method was developed as a useful tool for the simultaneous detection of two important flatfish pathogens in marine aquaculture, Tenacibaculum maritimum and Edwardsiella tarda. In fish tissues, the average detection limit for these mPCR-amplified organisms was 2 × 10 ⁵ ± 0.2 CFU/g and 4 × 10 ⁵ ± 0.3 CFU/g, respectively. These values are similar or even lower than those previously obtained using the corresponding single PCR. Moreover, mPCR did not produce any nonspecific amplification products when tested against 36 taxonomically related and unrelated strains belonging to 33 different bacterial species. Large amounts of DNA from one of the target bacterial species in the presence of low amounts from the other did not have a significant effect on the amplification sensitivity of the latter.

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.