Moritella viscosa isolated from farmed Atlantic cod (Gadus morhua)

Transcription

1 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 109 Moritella viscosa isolated from farmed Atlantic cod (Gadus morhua) Colquhoun D.J. 1, Hovland H. 2, Hellberg H. 3, Haug T. 1 and Nilsen H. 3 National Veterinary Institute, P.O. Box 8156 Dep, 0033 Oslo, Norway 1. Cod Culture Norway, Kollsnes Naeringspark, 5337 Rong, Norway 2. National Veterinary Institute, P.O. Box 1253 Sentrum, Bergen, Norway 3. Abstract The first isolation of Moritella viscosa, a causative agent of winter ulcer in Atlantic salmon (Salmo salar), from farmed Atlantic cod (Gadus morhua) is reported. The bacteria was isolated from two post-spawning broodstock cod displaying skin lesions. The isolations occurred in Norway in April 2002, when water temperature was approximately 7 o C. Identification of the bacteria as M. viscosa was confirmed using phenotypical testing and sequencing of 16S rdna. Introduction Moritella viscosa (Benediktsdóttir et al. 2000), first described as Vibrio viscosus (Lunder et al. 2000) is routinely and extensively isolated from salmon suffering from winter ulcer in Norway. Since its first isolation from diseased sea-farmed salmon in Norway in the 1980`s (Lunder 1992), the bacteria has also been isolated regularly in Iceland (Benediktsdóttir et al. 2000) and at least twice in Scotland (Bruno et al. 1998; Birkbeck et al. 2000). Winter ulcer is associated with low water temperatures and is characterised by external lesions of varying severity in affected fish, although extensive internal pathology may also be present. Despite the introduction of commercial vaccines directed against M. viscosa, which appear to provide reasonable levels of protection against experimental challenge, winter ulcer continues to be a source of loss to the salmon farming industries of Norway and Iceland. With increasing diversification of aquaculture it may be expected that bacterial diseases, both familiar and new, will be encountered. Marine farming of Atlantic cod (Gadus morhua) is expected to become the next boom industry in Northern European aquaculture. Although cod farming is presently quite small scale, bacterial infections including various serotypes of Vibrio anguillarum have already made their presence felt. Whether M. viscosa will prove to be a pathogen of importance in cod farming remains to be seen, but from experimental challenge of cod with M. viscosa isolated from natural outbreaks in salmon (Gudmundsdóttir et al. 2001), prior to the isolation of the cod isolates described here, we know that cod are susceptible (at least experimentally) to infection with this bacterium. Previously, M. viscosa has been isolated from farmed Atlantic salmon (Salmo salar), rainbow trout (Onchorhynchus mykiss) and from cap-

2 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 110 tive wild-caught plaice (Pleuronectes platessa) (Lunder et al. 2000). To the best of our knowledge this paper describes the first isolation of M. viscosa from Atlantic cod. Case history The isolations took place on a land-based broodstock unit in the county of Hordaland, Norway in April The cod, which had been held in tanks since January 2002, were a mixture of wild-caught and F1 cultured fish, and were in somewhat poor condition following spawning. Water temperature at the time of investigation was 7 o C, but had recently been considerably lower. Four fish within a population of approximately 300 post-spawning broodstock (average size 5-6 kg) cod were observed to have skin lesions (2.5 x 2.5 cm) on the posterior dorsal surfaces. The lesions, similar to those described in salmon (Lunder 1992), penetrated the epidermis and dermis, thereby exposing the underlying muscle. Materials and methods Bacterial isolation Bacteriological samples from kidney and from surface ulcers were sown onto heart infusion agar (Difco) containing 2% NaCl and 5% bovine blood (BA), incubated at 15 o C and initially read after 48h. Incubation and observation was extended for a further five days. Physiological and biochemical characterisation Cells from cultures grown for 48h at 15 o C on BA were used in all tests. Unless otherwise noted the final NaCl content of test media was 2%. All test media with the exception of those used to determine temperature range were incubated at 15 o C. Otherwise biochemical characterisation was carried out using standard methods. The oxidation/ fermentation test was performed in a basal glucose medium (Difco). Arginine dihydrolase, lysine and ornithine decarboxylase tests (Müller 1955) were observed for seven days. API20E tests (Biomérieux) were inoculated with bacteria suspended in 50% artificial seawater. Aerobic carbohydrate assimilation was tested using API 50 CH (Biomérieux) kits in which the bacteria were suspended in a media comprising 1.5g agar, 2.0g ammonium sulphate, 0.250g CAS amino acids and 1 ml trace element solution per litre. Isolates were subjected to slide agglutination testing (Andersen and Dixon 1985) utilising polyclonal rabbit antisera raised against M. viscosa NCIMB T. Sequencing of 16S rdna The 16S rdna gene of strain 2002/09/ was amplified using PCR and primers FD1 and RP2 (Weisburg et al. 1991). DNA sequencing was performed using the BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, CA) and an ABI prism 3100 Avant capillary sequencer (Applied Biosystems). Sequence analysis was performed using the sequencher program (Gene Codes Corp, Genetics Computer Group (GCG, Oxford Molecular, London, England)) and BLAST search analysis (Altschul et al., 1997). Results Bacteriology Of the four fish sampled, mixed bacterial cultures were retrieved from all ulcer samples. No bacteria were recovered from kidney samples. Two of the ulcer cultures were dominated by grey, â-haemolytic, viscous colonies.

3 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 111 Test NVI 2002/09/ T NCIMB V iscous colonies Gram -stain G-negative rods G-negative rods M obility O xidative/fermentative + / + + / + Gas from glucose Vibbriostat (0/129) sensitive & S lightly S lightly & C atalase # ALO (Müller) - / + / - / +/ - - # G elatinase prod. Acid from: Cellobiose Mannose Trehalose H emolysis (5% bovine blood agar) + * + Growth at 4 0 C Growth at 30 0 C Table 1. Phenotypical testing & Sensitivity to 0/129 is typically less in M. viscosa compared to most other Vibrio spp. * Relatively weak â- hemolysis, extending only slightly beyond colony border. More distinct â- hemolysis, extending beyond colony border. # Production of acid from glucose and basic products from lysine in Müller media is typically weak for M. viscosa. One of these cultures was compared phenotypically with M. viscosa type strain NCIMB T. Both cod isolates reacted positively in the M. viscosa slide agglutination test. Morphological and biochemical results are summarised in tables 1, 2 and 3. 16S rdna sequencing A sequence of 1465bp was obtained from the strain under study and submitted to Genbank under accession number AY BLAST searching revealed a high degree of homology with the M. viscosa type strain. Our sequence was aligned with the two available 16S rdna M. viscosa sequences from the type strain (NCIMB T ). Over 1465bp, our sequence was found to differ from accession AJ by only one base pair and from Y17574 by three base pairs (plus one ambiguous base in Y17574).

4 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 112 Test Table 2. API20E N VI 2002/09/ Discussion and conclusions NCIMB 13584T ONPG ADH LDC ODC CIT H2S URE TDA IND VP G EL G LU MAN INO SOR RHA SAC MEL AMY ARA On examination of the presented results there would appear to be some inconsistencies regarding production of acid from glucose during O/F testing and the very weak production of acid from glucose (and basic products from lysine) in Müller dihydrolase/ decarboxylase testing, both in laboratory produced media and in AP20E kits. The reasons for this are not clear, but may be due to differences in ph of the medias involved i.e. basal O/F media = ph 7.4 and Müller media = ph 6.0. Such weak reactions in Müller media are unusual amongst the psychrophilic vibrios encountered in our diagnostic work and are considered characteristic of M. viscosa strains identified in our laboratories. That the tested bacteria did not assimilate glucose in API 50 CH tests, whilst producing acid in the basal glucose medium is not easily explained. While this may suggest a problem with the media used to inoculate the API 50 CH tests, glucose assimilation is routinely obtained on testing of other bacteria using the same medium. Importantly, identical results were achieved for both the type strain and the cod strain. Discrepancies between our API 50 CH results and those of Bruno et al. (1998) may be due to differences in inoculating media. M. viscosa has a stringent requirement for levels of NaCl not normally present in media supplied with commercial kits. For this reason we constructed a media including 2% NaCl for use in API 50 CH based assimilation tests. The inoculating media used by Bruno et al (1998) was not described. As for M. viscosa NCIMB T the isolate under study was biochemically rather unreactive. Both strains gave identical results under phenotypical testing, sufficient to differentiate M. viscosa from its closest relatives (Table 9. Lunder et al. 2000). When combined with 16S rdna, and positive agglutination with specific anti-sera we can identify the strain under study as M. viscosa with a high degree of certainty. Although there was no significant mortality or loss associated with the reported isolation, it is important that field health workers are aware of the possibility of isolation of M. viscosa from farmed cod. Although M. viscosa isolated in Norway are, phenotypically, normally very homogenous, the cod isolate described here has a somewhat increased

5 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 113 Substrate Continues next column NVI 2002/09/ NCIMB 13584T G lycerol Erythritol D-Arabinose L-Arabinose R ibose D-Xylose L-Xylose Adonitol Methyl-xyloside Galactose D-Glucose D-Fructose D-Mannose L-Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol a Methyl-D-mannoside a Methyl-D-glucoside N-Acetyl-glucosamine Amygdaline Arbutine Esculine Salicine Cellobiose M altose Lactose Melibiose Saccharose Trehalose Inuline Melezitose Substrate Table 3. API 50 CH NVI 2002/09/ NCIMB 13584T D-raffinose A midon G lycogene Xylitol ßGentiobiose D-Turanose D-Lyxose D-tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2 ceto-gluconate 5 ceto-gluconate haemolytic activity compared to the type strain. This has also been seen (personal observation) in a minority of M. viscosa strains isolated from salmon. It was, unfortunately, not possible to test the susceptibility of farmed cod to the cod isolate during the present study. Given the likely increase in cod farming in coming years, investigations into such susceptibility and vaccine protection should be performed. It is of interest that another cultured marine species, turbot, is not protected against M. viscosa isolated from salmon, following administration of vaccines designed for Atlantic salmon (Björnsdóttir et al. 2003).

6 Bull. Eur. Ass. Fish Pathol., 24(2) 2004, 114 References Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., Miller W., and Lipman D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research. 25(17): Anderson D.P. and Dixon O.W. (1985) Fish Biologics Guide: Regimens and protocols for the production and use of antisera, antigens and other reagents for fish disease serodiagnostics. U.S. Fish and Wildlife Service. Kearneysville, West Virginia, USA. Benediktsdóttir E., Verdonck L., Sproer C., Helgason S. and Swings J. (2000) Characterisation of Vibrio viscosus and Vibrio wodanis isolated at different geographical locations: a proposal for reclassification of Vibrio viscosus as Moritella viscosa comb. nov. International Journal of Systematic and Evolutionary Microbiology. 50: Birkbeck T.H., Billcliffe B., Laidler A. and Cox D.I. (2000) The relationship between Aeromonas sp. NCIMB 2263, a causative agent of skin lesions in Atlantic salmon, Vibrio marinus (Moritella marina) and Vibrio viscosus. Journal of Fish Diseases. 23: Björnsdóttir B., Gudmundsdóttir S., Magnadõttir B. and Gudmundsdóttir B.K. (2003) Vaccination of turbot (Scophthalmus maximus) against atypical furunculosis and winter ulcers. Poster presentation. EAFP International Conference, Malta. Bruno D., Griffiths J., Petrie J. and Hastings T.S. (1998) Vibrio viscosus in farmed Atlantic salmon Salmo salar in Scotland: field and experimental observations. Diseases of Aquatic Organisms. 34: Gudmundsdóttir B.K., Magnadóttir B. and Gudmundsdóttir S. (2001) Experimental infection of juvenile turbot (Scopthtalmus maximus), Halibut (Hippoglossus hippoglossus) and cod (Gadus morhua) with the bacterium Moritella viscosa. Poster presentation. EAFP International Conference, Dublin. Lunder T. (1992) Winter ulcer in Atlantic salmon: A study of pathological changes, transmissability and bacterial isolates. Dr scientarium thesis. Norwegian School of Veterinary Science. Lunder T., Sørum H., Holstad G., Steigerwalt A.G., Mowinckel P. and Brenner D.J. (2000). Phenotypic and genotypic characterisation of Vibrio viscosus sp. nov. and Vibrio wodanis sp. nov. isolated from Atlantic salmon (Salmo salar) with winter ulcer. International Journal of Systematic and Evolutionary Microbiology. 50: Müller V. (1955) Simplified tests for some amino acid decarboxylases and for the arginine dihydrolase system. Acta Pathol. Microbiol. Scand. 36: Weisburg W.G., Barns S.M., Pelletier D.A. and Lane D.J. (1991) 16S Ribosomal DNA amplification for phylogenetic study. J. Bact. 173 (2):