Vibriosis caused by anguillarum and V. ordalii

Atle Lillehaug and Duncan Colquhoun

Leaflet No. 71 I December 2020

ICES IDENTIFICATION LEAFLETS FOR DISEASES AND PARASITES IN FISH AND SHELLFISH

FICHES D'IDENTIFICATION DES MALADIES ET PARASITES DES POISSONS, CRUSTACES ET MOLLUSQUES

Replaces leaflets no. 27, 29, and 50

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Contents

1 Disease name ...... 1 2 Aetiological agent ...... 1 3 Susceptible species ...... 2 4 Geographical distribution ...... 2 5 Disease aetiology ...... 2 6 Significance ...... 2 7 Clinical signs and pathology ...... 3 8 Control measures and legislation ...... 3 9 Diagnostic methods ...... 3 10 References ...... 3 11 Figures ...... 5 12 Author contact details ...... 6

Vibriosis caused by Vibrio anguillarum and V. ordalii | 1

Vibriosis caused by Vibrio anguillarum and V. ordalii

Authors: Atle Lillehaug and Duncan Colquhoun

1 Disease name

Vibriosis. While the term “vibriosis” may correctly be used to describe diseases caused by a wide variety of related Vibrio species, this leaflet describes “classic vibriosis”, i.e. infections caused by the very close relatives Vibrio anguillarum and V. ordalii.

2 Aetiological agent

Vibrio anguillarum and V. ordalii are so closely related that they may well comprise two biovariants of a single species. Indeed, V. ordalii was originally described as V. anguillarum biotype II (Schiewe and Crosa, 1981) prior to its description as an independent species (Schiewe et al., 1981). Both are members of the family within the . The previously “grey” taxonomic position of V. anguillarum has recently been resolved with the proposed return of this bacterium to the genus Vibrio from the genus Listonella (Thompson et al., 2011). Thus far, 23 different serotypes have been identified in V. anguillarum (Pedersen et al., 1999). In addition, isolates not corresponding to known serotypes are still commonly identified, mainly from environmental sources, but also occasionally from fish species newly introduced to aquaculture. The majority of V. anguillarum isolates identified in association with disease in finfish belong to serotypes O1 and O2 (including various subserotypes). However, serotype O3 has also been associated with disease in a range of fish species, including European sea bass (Dicentrarchus labrax), Asian ayu (Plecoglossus altivelis), eels (Anguilla Anguilla; Tiainen et al., 1997) and salmonids in Chile (Silva-Rubio et al., 2008). Evidence also exists for a degree of host- species specificity within certain serotypes of V. anguillarum (Myhr et al., 1991). As novel sero- /genotypes may be identified on introduction of new fish species to aquaculture (Mikkelsen et al., 2011), we may expect the serotype diversity of V. anguillarum isolated from farmed fish to increase, as aquaculture expands and diversifies. The establishment of V. ordalii as an independent species (Schiewe et al., 1981) was based on phenotypical differences and DNA/DNA hybridisation. Available whole genome sequence information from a fish pathogenic V. ordalii now indicates an extremely high relationship between these species, but identified a considerably smaller genome in V. ordalii compared to V. anguillarum (Naka et al., 2011). This suggests that V. ordalii may be undergoing genomic degradation consistent with evolutionary progression from an environmental towards an obligate pathogenic lifestyle (Naka et al., 2011). Recently, strains phenotypically consistent with V. ordalii, but phylogenetically more closely related to V. anguillarum, have been identified in farmed cod in Norway (Steinum et al., 2016) and farmed lumpsucker in both Norway and the United Kingdom (Colquhoun, unpublished data). 2 | ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish

3 Susceptible species

Many different species of farmed fish (Austin and Austin, 2007), both marine and anadromous, appear susceptible to the disease. Epizootics in wild marine fish species also occur regularly, e.g. in saithe (Pollachius virens; Egidius, 1987) and big-scale sand smelt (Atherina boyeri; Yiagnisis et al., 2007). Originally described as a pathogen of eels (Bergmann, 1909), many marine fish species and both Atlantic and Pacific salmonid species are at risk, with rainbow trout (Oncorhynchus mykiss) appearing particularly susceptible (Lillehaug et al., 2003). Vibriosis outbreaks have been reported in many diverse aquaculture species, including, but not limited to, Asian ayu, European sea bass, Gilthead sea bream (Sparus aurata), turbot (Scopththalmus maximus; Larsen et al., 1994), and Atlantic cod (Gadus morhua; Colquhoun et al., 2007).

4 Geographical distribution

Vibriosis caused by V. anguillarum has a worldwide distribution in temperate waters, having been described in Europe, Asia, the Americas, and Australasia (Munday et al., 1992; Austin and Austin, 2007; Silva-Rubio et al., 2008). V. ordalii is less frequently identified, and may have a more limited distribution, having been described in North and South America, Japan, Australasia (Schiewe and Crosa, 1981; Wards et al., 1991; Colquhoun et al., 2004), and more recently in Norway (Anonymous, 2006).

5 Disease aetiology

As with all infectious diseases, vibriosis is the result of the interplay between pathogen, host and the environment. Water temperature is undoubtedly one of the major factors related to vibriosis outbreaks, with most cases involving wild and farmed fish associated with rising and relatively high water temperatures during the summer months in temperate areas (Lillehaug et al., 2003). The general association to high water temperatures is related to increased bacterial virulence/activity, but also to increased stress and reduced immunological capability in affected fish populations. Handling of fish during, e.g. grading or, paradoxically, vaccination, may also tip the balance in favour of the pathogen, resulting in the outbreak of disease.

6 Significance

Vibriosis was previously a significant problem in farmed salmonids, but its impact in salmonid culture has been drastically reduced due to the development of effective vaccines and vaccination procedures. The disease remains problematic in the marine fish-farming industries of many countries surrounding the Northern Atlantic, Mediterranean countries, and Asia/Oceania.

Vibriosis caused by Vibrio anguillarum and V. ordalii | 3

7 Clinical signs and pathology

Vibriosis can be generally described as disseminated systemic infections, for which the macroscopically visible, and non-specific external signs may include necrotic lesions and varying degrees of cutaneous haemorrhage on both body and fins, with or without exophthalmia. Internally, extensive inflammation, haemorrhage, and often ascites may be observed. While diseases caused by V. anguillarum and V. ordalii are very similar, infections caused by V. ordalii appear less virulent (Hjeltnes, 1993; Austin and Austin, 2007).

8 Control measures and legislation

While antibiotics are commonly used to treat acute outbreaks of vibriosis (Lillehaug et al., 2003; Colquhoun et al., 2007), often with good effect, there is no doubt that vaccination represents the most effective means of controlling vibriosis in aquaculture. Vibriosis vaccines were among the first vaccines developed for aquaculture use, and generally provide high levels of protection (Colquhoun and Lillehaug, 2014). Currently available vibriosis vaccines include both products for immersion- and injection-vaccination. Although intraperitoneal vaccination is generally acknowledged to provide better and longer-lasting protection, the process is relatively expensive, and requires the vaccinated fish to be above a minimum size, dependent on fish species, but generally > 10 g. The choice of administration route is therefore based on the type and size/developmental stage of the fish, as well as the value of the fish to be vaccinated. In European marine aquaculture of salmonids, the use of adjuvant polyvalent vaccines, including V. anguillarum, renders injection as the method of choice. The Vibrio agents causing disease in aquaculture have an approximate worldwide distribution, and there are no particular regulatory concerns connected to outbreaks of vibriosis or the demonstration of V. anguillarum or V. ordalii in fish.

9 Diagnostic methods

Diagnosis of vibriosis is generally based on a combination of macroscopic and histological observations, combined with the culture of the aetiological agent as the central diagnostic element. Culture of both V. anguillarum and V. ordalii is relatively easily achieved, normally by streaking from kidney tissues on general purpose media, such as Tryptone Soya Agar (TSA), Heart Infusion Agar (HIA), or Blood agar (BA). The presence of NaCl is required for growth, but the concentration of NaCl found in most general purpose agars (i.e. 0.5%) is sufficient.

10 References

Anonymous. 2006. Health situation of farmed fish 2005. Report, National Veterinary Institute. http://multiconsult.eurest.no/eng/layout/set/print/Publications/Fish-Health-Report/The- Farmed-Fish-Health-Report-2005.html. Austin, B., and Austin, D. A. 2007. Bacterial fish pathogens: disease of farmed and wild fish. 4th ed. Springer Praxis Publishing Ltd, Chichester, UK, 2007. 552 pp. https://doi.org/10.1007/978-1-4020-6069-4 4 | ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish

Bergmann, A. M. 1909. Die rote Beulenkrankheit des Aals. Bericht aus der Koniglichen Bayerischen Versuchsstation, 2: 10–54. Colquhoun, D. J., Aase, I. L., Wallace, C., Baklien, A., and Gravningen. K. 2004. First description of from Chile. Bulletin of the European Association of Fish Pathologists, 24: 185–188. Colquhoun, D. J., Aarflot, L., and Melvold, C.F. 2007. gyrA and parC mutations and associated quinolone resistance in Vibrio anguillarum serotype O2b strains isolated from farmed At- lantic cod (Gadus morhua) in Norway. Antimicrobial Agents and Chemotherapy, 51: 2597– 2599. https://doi.org/10.1128/AAC.00315-07 Colquhoun, D., and Lillehaug, A. 2014. Vaccination against vibriosis. In Fish Vaccination, Ed. R. Gudding, A. Lillehaug, and Ø. Evensen. Wiley Blackwell, Chichester, UK. pp. 172–184. Egidius, E. 1987. Vibriosis: Pathogenicity and pathology. A review. Aquaculture, 67: 15–28. https://doi.org/10.1016/0044-8486(87)90004-4 Hjeltnes, B., and Roberts, R. J. 1993. Vibriosis. In Bacterial Diseases of Fish, Ed. V. Inglis, R. J. Roberts, and N. R. Bromage. Blackwell Scientific Publications, Oxford, UK. pp. 109–121. Larsen, J. L., Pedersen, K., and Dalsgaard, I. 1994. Vibrio anguillarum serovars associated with vibriosis in fish. Journal of Fish Diseases, 17: 259–267. https://doi.org/10.1111/j.1365- 2761.1994.tb00221.x Lillehaug, A., Lunestad, B. T., and Grave, K. 2003. Epidemiology of bacterial diseases in Nor- wegian aquaculture - a description based on antibiotic prescription data for the ten-year period 1991 to 2000. Diseases of Aquatic Organisms, 53: 115–125. https://doi.org/10.3354/dao053115 Mikkelsen, H., Lund, V., Larsen, R., and 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 & Shellfish Immunology, 30: 330–339. https://doi.org/10.1016/j.fsi.2010.11.007 Munday, B. L., Carson, J., Whittington, R., and Alexander, J. 1992. Serological responses and immunity produced in salmonids by vaccination with Australian strains of Vibrio anguilla- rum. Immunology and Cell Biology, 70: 391–395. https://doi.org/10.1038/icb.1992.51 Myhr, E., Larsen, J. L., Lillehaug, A., Gudding, R., Heum, M., and Håstein, T. 1991. Characteri- zation of Vibrio anguillarum and closely related species isolated from farmed fish in Nor- way. Applied and Environmental Microbiology, 57: 2750–2757. Naka, H., Dias, G. M., Thompson, C. C., Dubay, C., Thompson, F. L., and Crosa, J. H. 2011. Complete genome sequence of the marine fish pathogen Vibrio anguillarum harboring the pJM1 virulence plasmid and genomic comparison with other virulent strains of V. anguil- larum and V. ordalii. Infection and Immunity, 79: 2889–2900. https://doi.org/10.1128/IAI.05138-11 Pedersen, K., Grisez, L., van Houdt, R., Tiainen, T., Ollevier, F., and Larsen, J. L. 1999. Extended serotyping scheme for Vibrio anguillarum with the definition and characterization of seven provisional O-serogroups. Current Microbiology, 38: 183–189. https://doi.org/10.1007/PL00006784 Schiewe, M. H., and Crosa, J. H. 1981. Molecular characterisation of Vibrio anguillarum biotype 2. Canadian Journal of Microbiology, 27: 1011–1018. https://doi.org/10.1139/m81-158 Schiewe, M. H., Trust, T. J., and Crosa, J. H. 1981. Vibrio ordalii sp. nov.: A causative agent of vibriosis in fish. Current Microbiology, 6: 343–348. https://doi.org/10.1007/BF01567009 Vibriosis caused by Vibrio anguillarum and V. ordalii | 5

Silva-Rubio, A. A., Avendaño-Herrera, R., Jaureguiberry, B. B., Toranzo, A. E., and Magariños, B. B. 2008. First description of serotype O3 in Vibrio anguillarum strains isolated from salm- onids in Chile. Journal of Fish Diseases, 31: 235–239. https://doi.org/10.1111/j.1365- 2761.2007.00878.x Steinum, T. M., Karataş, S., Martinussen, N. T., Meirelles, P. M., Thompson, F. L., and Colquhoun, D. J. 2016. Multilocus sequence analysis of close relatives Vibrio anguillarum and Vibrio ordalii. Applied and Environmental Microbiology, 82: 5496–5504. https://doi.org/10.1128/AEM.00620-16 Thompson, F. L., Thompson, C. C., Dias, G. M., Naka, H., Dubay, C., and Crosa., J. H. 2011. The genus Listonella MacDonell and Colwell 1986 is a later heterotypic synonym of the genus Vibrio Pacini 1854 (Approved Lists 1980) – a taxonomic opinion. International Journal of Systematic and Evolutionary Microbiology, 61: 3023–3027. https://doi.org/10.1099/ijs.0.030015-0 Tiainen, T., Pedersen, K., and Larsen, J. L. 1997. Vibrio anguillarum serogroup O3 and V. anguil- larum-like serogroup O3 cross-reactive species – comparison and characterization. Journal of Applied Microbiology, 82: 211–218. https://doi.org/10.1111/j.1365-2672.1997.tb03575.x Wards, B. J., Patel, H. H., Anderson, C. D., and Delisle, G. W. 1991. Characterization by re- striction endonuclease analysis and plasmid profiling of Vibrio ordalii strains from salmon (Oncorhynchus tshawytscha and Oncorhynchus nerka) with vibriosis in New-Zealand. New Zealand Journal of Marine and Freshwater Research, 25: 345–350. https://doi.org/10.1080/00288330.1991.9516488 Yiagnisis, M., Vatsos, I. N., Kyriakou, C., and Alexis, M. 2007. First report of Vibrio anguillarum isolation from diseased big scale sand smelt, Atherina boyeri Risso 1810, in Limnos, Greece. Bulletin of the European Association of Fish Pathologists, 27: 61–69.

11 Figures

Figure 1. Juvenile farmed Atlantic cod, Gadus morhua, displaying bilateral exophthalmos and erythema of the head and fin-bases, associated with Vibrio anguillarum infection. Photo: Arthur Lyngøy, Fiskr AS. 6 | ICES Identification Leaflets for Diseases and Parasites of Fish and Shellfish

Figure 2. Vibrio anguillarum serotype O2b cultured on blood agar containing 2% NaCl, displaying relatively large (4–5 mm) colonies, which display a clear area of haemolysis under individual colonies. Photo: Norwegian Veterinary Institute.

Figure 3. A Vibrio ordalii-like strain isolated from farmed Atlantic cod, Gadus morhua, cultured on blood agar containing 2% NaCl, displaying very small (< 1 mm), non-haemolytic colonies. Photo: Norwegian Veterinary Institute.

12 Author contact details

Atle Lillehaug, DVM, PhD Duncan J. Colquhoun, PhD Senior researcher Senior researcher Norwegian Veterinary Institute Norwegian Veterinary Institute P.O. Box 750 Sentrum P.O. Box 750 Sentrum N-0106 Oslo N-0106 Oslo Norway Norway E-mail: [email protected] E-mail: [email protected]