Vibriosis in aquaculture

Workshop: Vibriosis in aquaculture


Wednesday 4 September 2013, 11.15-13.00 h, Room: Sonaatti 1, Tampere Hall in Tampere, Finland

Workshop Coordinators:

Olga Haenen: Leading organizer – [email protected]

Central Veterinary Institute of Wageningen UR, NRL for Fish, Crustacean and Shellfish Diseases, P.O. Box 65, 8200 AB Lelystad, The Netherlands.

B. Fouz: co-chairperson, co-organizer – [email protected]

Dept. of Microbiology and Ecology, Faculty of Biology, University of Valencia, E-46100-Burjassot, Valencia, Spain.

I. Dalsgaard: co-chairperson, co-organizer – [email protected]

National Veterinary Institute, Technical University of Denmark , DK-1870 Frederiksberg C , Denmark.


– Schedule:

O. Haenen, B. Fouz and I. Dalsgaard: General Introduction.
A.B. García, J. Benavides, P. Campos, A. Sánchez-Mut, E. Lozano, M.C. Alcázar. J.F. Amaya and M.M. Isern* (CULMAREX, Spain): Vibriosis in West Mediterranean facilities.
B. Fouz, B. and C. Amaro (Valencia University, Spain): Warm water vibriosis: Interaction Vibrio vulnificus and eel.
H. Mikkelsen (NOFIMA, Tromsø, Norway): Vibriosis in cold water fish.
S. Zrnčić and D. Oraić (Croatian Veterinary Institute, Zagreb, Croatia): Overview of vibriosis in seabass (Dicentrarchus labrax) at Croatian farms.
M.A. Travers and T. Renault (IFREMER, La Tremblade, France): Vibriosis in marine molluscs.
R. Wardle (MSD Animal Health, UK): Using a vaccine approach to protect shrimp against vibrios.
A. Hellström, and E. Säker et al. (SVA, Uppsala, Sweden): The use of MALDI-TOF for diagnosis of Vibrio species.


– Discussion:

1. Which are the most important vibriosis problems in fish culture, crustacean culture and shellfish culture?

2. Are diagnostic methods up to date, and are the used therapies effective?

3. Are prevention measures effective, and are specific vaccines further needed?

4. Which recommendations can be made for adequate prevention and control of vibriosis in aquaculture?


– Abstracts:

– Please right-click on the title of each abstract to download the Author’s provided material –


O. Haenen*, B. Fouz, and I. Dalsgaard

Organizers of this workshop

A warm welcome to all participants of the workshop!

Aquaculture in brackish and marine water is growing worldwide. New cultured species are introduced, and types of aquaculture vary from outdoor to indoor and from flow through to recirculated water, at various temperatures. In these types of aquaculture various Vibrio species play an important role, as causative agents of fish, crustacean and shellfish diseases.

In this workshop, specialists will present the newest knowledge about vibriosis problems in fish, shellfish, and crustaceans, methods for detection and typing of Vibrio species, and vaccination. It is our intention to write a joint workshop report for the EAFP Bulletin. We further like to put your attention to the VIBRIO 2014 Conference, organized by Prof. Brian Austin from the University of Stirling, held in Edinburgh in April, 2014.


A.B. García, J. Benavides, P. Campos, A. Sánchez-Mut, E. Lozano, M.C. Alcázar. J.F. Amaya and M.M. Isern*

*Grupo Culmarex, Murcia, Spain; Email: [email protected]

Ongrowing sites in Spanish Mediterranean coast are off shore located. In some locations, official ongrowing areas are present, but it’s usual to have close sites from other companies. The main cultured species are seabream (Sparus aurata) and seabass (Dicentrarchus labrax), with little presence of meagre (Argyrosomus regius). Vibriosis affects basically seabass in ongrowing sites, with much lowest incidence in bream and meagre. Vibrio anguillarum serotipe O1 has been demonstrated to be responsible agent for acute vibriosis outbreaks in sea bass and chronic outbreaks in meagre, and it has been also isolated as commensal bacteria in sea bream. Moreover, high incidences of vibriosis can occur in hatchery and pre-ongrowing facilities, as juveniles are more sensitive to the disease. V. anguillarum serotype O2 has also been isolated as secondary pathogen in diseased sea bream. Vibriosis outbreaks in sea bass occur from 13º C to 24º C. The month mortality ranges from 1% in large fish (1kg) to 15% in medium size fish (150g) without antibiotic treatment. Early detection and quick application of antibiotics by oral treatment is basic to reduce the vibriosis impact. Spread of vibriosis is quick and real. Close cages, even fish in other facilities 2 miles far away, will synchronize outbreaks. Meagre sharing facilities in the ongrowing sites can also suffer chronic mortalities (1.5% monthly). The role of communication between farmers and the Sanitary Defense Associations (ADS) are very important to improve biosecurity and control of the disease, especially in unvaccinated fish. Diagnosis is basically done by anamnesis and necropsy observations, combined with basic microbiology (phenotypic identification + antibiogram). Agglutination tests for serotyping are very useful but not easy to find. Antibiogram test results are stable, without changes among outbreaks, cages or season. Flumequine, oxytetracyclines, sulfonamides (+trimethoprim) and florfenicol are good options for vibriosis treatment. Special issues focus in low-temperature season to reach optimal antibiotic dose due to low feed ratios premix availability, and the absence of sea bass specific premixes that allow proper withdrawal times. Vaccination as preventive measure is the best option since its effectiveness has been demonstrated around 100% during the protection period using both immersion and injection procedures. Oral formulations are strongly required by the industry but they are not enough developed. Injection procedures, protocols and formulations need to be optimized to guarantee low medium weight fish to be vaccinated by intraperitoneal (ip) injection. In offshore production, in-cage ip vaccination is not a real option. Oral formulations with good effectiveness could help reducing handling and improving welfare. Protection of the stock to minimize the infection pressure has been demonstrated to be the best way to avoid vibriosis in sea bass production and, subsequently, the use of antibiotics. Combination of the suitable and available vaccination protocols has a real benefit in cost-effect balance.


B. Fouz* and C. Amaro

University of Valencia, Valencia, Spain; Email: [email protected]

Vibrio vulnificus is a bacterial species that can be commensal and pathogenic for humans, shrimps and fish. The biotype 2 (Vv Bt 2) of the species is the etiological agent of warm-water vibriosis, an hemorrhagic septicemia that mainly affects eels but also other teleosts (tilapia, derbio, trout and seabass), especially under culture conditions. It is frequently isolated from environments located in template and tropical areas but, lately, it is being increasingly isolated from Nordic Countries due to the effects of the Global warming. Vv Bt 2 comprises of three O-serovars, one of which, serovar E, is zoonotic. Eel vibriosis in brackish water is mainly due to serovar E, showing external ulcers as the differential clinical sign. Eel vibriosis in freshwater is due to serovars A or I, showing jaw degradation as a differential clinical sign. The disease has been histopathologically and microbiologically analyzed: a) the bacterium attaches to the gills and forms biofilms, b) it enters into the blood stream and c) it arrives to the internal organs where it causes cell death by septicemia. Biotype 2 is polyphyletic and probably emerged in the fish farming environment from commensal strains by acquisition of a virulence plasmid that encodes resistance to the innate immune system of the eel and other teleosts. This plasmid is transferred between strains by forming a cointegrate with a conjugative plasmid that is widespread in the species. Our team has optimized a diagnosis procedure, effective also for carriers, which uses a multiplex PCR assay that targets specific virulence-related sequences. Vaccination is highly effective protecting eels against vibriosis caused by Vv Bt 2. Protection is correlated with high specific antibody titers mainly in plasma and secondarily in skin and intestinal mucus. If antibody levels in plasma significantly decreases, an oral booster must be performed to get fish are well-protected. The developed vaccine (Vulnivaccine) and the vaccination protocol for eels have been licensed.


– Fouz, B., A. Llorens, E. Valiente & C. Amaro. 2010. A comparative epizootiologic study of the two fish-pathogenic serovars of Vibrio vulnificus biotype 2. Journal of Fish Diseases, 33: 383-390.



Helene Mikkelsen*,

Nofima, P.O. box 6122, N-9291 Tromsø, Norway ; Email: [email protected]

In Norway, we have a large production of Atlantic salmon and some minor production of trout, Atlantic cod, halibut and Arctic char. Lately there also have been a small production of cleaner fish as wrasse and lumpfish. Vibriosis caused by several species of vibrios is still a problem in Norwegian fish farming, such as Vibrio anguillarum, V. ordalii, Aliivibrio (Vibrio) salmonicida, V. splendidus, V. logei and V. tapetis (Johansen 2013). V. anguillarum, serotypes O1, O2a, O2a-biotype II, O2b are commonly isolated: serotype O1 from trout and salmon, O2 from cod. In 2012, several outbreaks of coldwater vibriosis caused by Vibrio salmonicida in salmon farms occurred in the northern part of Norway. This disease has been under control by vaccination since 1987, and it is speculated that this re-occurrence is due to increased infection pressure or inadequate vaccine regimes. These new isolates are similar to previous one shown by 16S rRNA sequencing, immunoblot and vaccine studies. Vibriosis outbreaks in cod farming is triggered by stressors as high temperature (>14 ºC), handling and vaccination. The main cause of disease is serotype O2b, but O2a and deviating isolates of O2a-biotype 11 have also been shown. Characterizations of these isolates have been done using biochemical, serological and molecular methods. We have shown that by including O2a-biotype II in the vibriosis vaccine, cod was highly protected against classical vibriosis caused by the three sero-subgroups of V. anguillarum (Mikkelsen et al. 2007, Mikkelsen et al. 2011). In order to characterize the V. anguillarum sero-sub group O2a biotype II a new method has been developed at the Veterinary Institute. The genetic typing methods Multi-Locus Sequence Analysis by comparing partially sequences of nine housekeeping genes. Multicomponent vaccines are the main means to control vibriosis in both salmon and cod. Especially in salmon this has resulted in high production and almost no use of antibiotics. In cod farming, however, vibriosis is still a significant problem. Cod fry above 3 g responds well to dip vaccination by immersion fish in high concentration of antigen, but this is difficult to accomplish in industrial scale. Larger cod > 30g obtain high protection after injection of an oil-based multicomponent vaccine including V. anguillarum O2a and O2b, and Aeromonas salmonicida subsp. achromogenes.


Johansen, R. (2013) Fiskehelserapporten 2012, Fish health report 2012. In: (ed. by R. Johansen). Veterinærinstituttet, Oslo.

Mikkelsen, H., Lund, V., Larsen, R. & Seppola, M. (2011) Vibriosis vaccines based on various sero-subgroups of Vibrio anguillarum O2 induce specific protection in Atlantic cod (Gadus morhua L.) juveniles. Fish and Shellfish Immunology, 30, 330-339.

Mikkelsen, H., Lund, V., Martinsen, L.-C., Gravningen, K. & Schrøder, M.B. (2007) Variability among Vibrio anguillarum O2 isolates from Atlantic cod (Gadus morhua L.): Characterisation and vaccination studies. Aquaculture, 266, 16-25.



Snježana Zrnčić* and Dražen Oraić

Laboratory of fish and molluscs diseases

Department of Pathology, Croatian Veterinary Institute, Zagreb, Savska 143; Email: [email protected]

Production of sea bass (Dicentrarchus labrax) along the Croatian coast has started at the early seventies, at the same time as similar projects in France and Italy. Until now there are fifty farms out of which two are big producers and the others are small, family runned farms. During last twenty years the importance of diseases control became an inevitable factor of economic production. This presentation derives from routine monitoring of several farms in different area of Croatian coast. Although several bacterial diseases were described as limiting factor in sea bass cultivation throughout the Mediterranean area, vibriosis caused by Listonella anguillarum is still one of the most damaging bacterial diseases in the national mariculture industry. In this paper results from twenty years of vibriosis study were presented with emphasis on epizootiological data which were mainly dependant on the geographical and meteorogical condition. It is possible to conclude that environmental conditions for vibriosis outbreaks are slowly, but constantly changing. The environmental conditions and the seasonal distribution of the outbreaks, as well as clinical pathological, histological and bacteriological findings will be presented. Moreover, as the part of the practical approach to the disease control, determination of antibiotic susceptibility was performed and the development of resistance to some specific antimicrobial substances was noticed. Furthermore, some progress was attained by programmes of immunoprophylaxis by implementation of nonspecific immunomodulation and vaccination programmes.


M.A. Travers1 and T. Renault*1

Ifremer, SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins, Avenue de Mus de Loup, 17390 La Tremblade, France; Email: [email protected]

Global mollusc production is continuously increasing, and has become one of the largest aquaculture activities in the world. Molluscs are the second most important group by quantity and by value. Although mollusc culture is steadily growing in importance in the aquaculture sector, populations of molluscs may suffer from severe mortality outbreaks. Among the possible causes is the occurrence of infectious diseases due to a variety of pathogens including bacteria. Vibrio species are ubiquitous in aquatic ecosystems. They are thermo dependent bacteria and are characterized by several survival strategies to persist in seawater, including adhesion to surfaces, biofilm formation and capability to enter a dormant state. Although certain Vibrio species ameliorate growth rates in molluscs, other Vibrio species influence the health of cultured marine organisms and constitute pathogens or potential pathogens. Although in some cases bacteria are easily identified, the exact identification of several marine Vibrio species based on biochemical characteristics remains difficult. Diversity of hosts and environmental conditions may induce result in discrepancies. Phenotypic and genotypic diversity may be deduced as a capability of marine Vibrio species to adapt to variable environmental conditions. As a result, gene and genome sequencing need to be taken into account for prokaryotic systematics. Bacteria belonging to the genus Vibrio were reported in association with mortality outbreaks among bivalve larvae and were also reported to induce diseases and mortality outbreaks in adult and juvenile oysters. As examples, Vibrio aestuarianus, V. splendidus and V. harveyi have been reported to be associated with summer mortality outbreaks of Pacific cupped oysters reared in open sea tidal areas. Vibrio splendidus-related species were reported in association with mortality outbreaks of molluscs. Through epidemiological studies a high genetic diversity was observed in this group suggesting a polyphyletic nature. Diagnostic biochemical characters failed in species discrimination within this group. DNA gyrase subunit B (gyrB) gene sequencing was thus used in order to characterize V. splendidus-related isolates from Pacific cupped oysters. Although several strains clustered together, they could not be assigned to any known Vibrio species. In this context, taxonomic analysis of unidentified isolates based on a polyphasic approach including gene sequencing, fluorescent amplified-fragment length polymorphism (FAFLP) fingerprinting, DNA-DNA hybridisation and biochemical tests were successfully applied by several authors to define new species.

None of the vibriosis affecting bivalves is listed by the World Organisation for Animal Health (WOAH) (OIE) and the EU (Directive 2006/88/EC). The only bacterial disease listed by the WOAH is the infection with Xenohaliotis californiensis, identified as a member of the family Rickettsiaceae in the order Rickettsiales (OIE).


R. Wardle*

MSD Animal Health, Aquaculture, UK; Email: [email protected]

This short paper will present some of the research and development work that has been carried out by MSD Animal health and its partners around the world investigating the possibilities for harnessing the “immune system” of shrimp to help control or prevent Vibriosis caused by several species an strains of vibrio pathogens including V. parahaemolyticus. The talk will illustrate some of the challenges that confront the research work in creating and measuring an effective response in shrimp from both a laboratory and field trials perspective. The objective being to stimulate discussion on the power of immunity to help shrimp famers control these ubiquitous disease challenges.


Hellström, A.* and E. Säker et al.

SVA, Uppsala, Sweden; Email: [email protected]

In this lecture, the new protein based technique for typing bacteria a.o., MALDI TOF (MALDI = Matrix-assisted-laser-desorption/ionization, TOF=Time-of-Flight) will be highlighted: Applications in biochemistry, organic chemistry, and microbiology, advantages of this technique, principle of the method, and needed extension of the data base of the fish bacteria. Vibrio species can be typed as well with this technique, but extension of the data base for this genus is necessary. In an international Club 5 project of SVA, with VetDTU and CVI Lelystad from Oct 2013 this is looked at.