Aquaculture

ELSEVIER Aquaculture 156 (1997) 317-326

Winter disease outbreak in sea-bream (5”arus aurata) associated with Pseudomonas

anguilliseptica infection

A. Domknech a, J.F. Fernindez-Garayzgbal a, P. Lawson b, J.A. Garcia a, M.T. Cutuli a, M. Blanc0 a, A. Gibe110 a,

M.A. Moreno a, M.D. Collins b, L. Dominguez a, * ’ Departamento de Patologia Animal I (Sanidad Animall, Facultad de Veterinaria, Unirersidad Complutense,

28040 Madrid, Spain

h Department of Microbiology, BBRSC Institute of Food Research, Reading Laboratory, Reading RG6 6BZ.

UK

Received 7 October 1996; revised 1 March 1997; accepted 20 March 1997

Abstract

The nature of an outbreak of ‘winter disease’ affecting both juvenile and adult sea-bream (Sparus aurutu) in several Iberian Peninsula farms from January to April of 1996 is described. The average mortality rate was approximately lo-15%, although in some fish farms mortality reached 30%. Pure cultures of aerobic Gram-negative filamentous rods were isolated from the kidney of diseased fish, as well as from some of the liver and ascitic fluid samples. 16s rRNA gene sequence analysis identified the isolates from diseased fish as Pseudomonas anguillisepticu.

The results of biochemical and physiological tests of clinical isolates and the type strain of P.

anguillisepticu were entirely consistent with the genotypic identification. The isolation of P.

unguillisepricu exclusively from the diseased fish of all affected fish farms, together with the failure to isolate this bacterium from non-affected fish of the same farms, suggest that P.

anguillisepficu is likely to be the agent responsible for the ‘winter disease’ outbreak in the

^ Corresponding author. Tel: 34 I 3943721; fax: 34 1 3943908: e-mail: Domenech@eucmax.sim.ucm.es

00448486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOO44-8486(97)00069-O

318 A. Dominnech et al./Aquaculture 156 (19971317-326

sea-bream examined. The relationship of the ‘winter disease’ outbreaks to stressful environmental conditions is discussed. 0 1997 Elsevier Science B.V.

Kqwordst Winter disease; Pseudomonas anguilliseptica; Sea-bream

1. Introduction

Bacterial diseases are probably the major cause of economic losses affecting fish farms. In addition to established bacterial fish pathogens, such as species of Vibrio, Aeromonas or Renibacterium, responsible for severe outbreaks of disease in cultured fish (Austin and Austin, 1993), there has been in recent years an increase in the number of outbreaks associated with several emergent fish pathogenic bacterial species. This increase is favoured by the intensive systems used in aquaculture (Meyer, 1991). Opportunistic pathogens, including both Gram-positive and Gram-negative bacteria, are usually widespread in the aquatic environment or form part of the normal flora of healthy individuals, and initiate disease only when there is a breakdown in the normal environmental conditions (Roberts, 1989).

Pseudomonas anguilliseptica is an emergent, opportunistic fish pathogen that has gained in clinical significance for being responsible for mortality outbreaks in various fish species. Disease is characterised by a high mortality, and related to a decrease in the water temperatures below 11-12°C (Wiklund and Bylund, 1990; Wiklund and LonnstrBm, 1994; Berthe et al., 1995; Kusuda et al., 1995). Indeed, temperature is considered to be the major factor influencing epizootics. P. anguilliseptica was first isolated in Japanese eels ( Anguilla juponica) suffering from haemorrhagic septicemia in Japan (Wakabayashi and Egusa, 1972; Kuo and Kou, 1978). Subsequently, P. anguil-

liseptica has been isolated in several European countries affecting, in addition to eel, numerous other fish species, such as black sea-bream (Acanthopagru.s schlegeli),

salmonid fish, gilthead sea-bream (Sparus aurata), sea-bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) (Nakajima et al., 1983; Wiklund and Bylund, 1990; Berthe et al., 1995). Although the lesions observed in these species were generally less severe and extensive than those described in eels, all the available information point out the seriousness of the outbreaks in terms of high mortality, and therefore, economic losses.

The gilthead sea-bream (S. aurutu) is the major marine-fish species farmed in Spain and Portugal, and one of the most important in the Mediterranean coast. In recent years, a mortality syndrome has been recorded during winter months in Spain and Portugal, which has been responsible for severe commercial losses. This syndrome, related mainly to decreases in water temperatures to levels much lower than those recommended for optimal culture conditions (Bamabe, 1990), has been designated as ‘winter disease’ because of its unknown aetiology and non-specific symptoms and lesions (Bovo et al., 1995; Doimi, 1996). This article describes the nature of an outbreak of ‘winter disease’ affecting sea-bream in Spain and Portugal and the phenotypic and molecular genetic characteristics of the isolated organisms, P. anguilliseptica. The likely role of P. anguilliseptica as the aetiological agent of ‘winter disease’ in sea-bream and its relationship to environmental stress are discussed.

A. DomCnech et al./Aquaculture 156 (1997) 317-326 319

2. Materials and methods

2. I. Disease history

During the winter of 1995-96 several outbreaks of ‘winter disease’ occurred in eight different gilthead sea-bream farms located in the regions of Cataluna and Andalucia, Spain, and in the regions of Beira Litoral, Estremadura and Algarve, Portugal (Table 1). The average mortality rate was about IO-15%, although in some fish farms mortality reached 30%. These outbreaks occurred in juvenile and adult sea-bream, and a non- specific symptomatology was observed. The outbreaks occurred in January, when the water temperature was below 12°C and persisted until April, when the water tempera- ture increased to 18-20°C. Diseased and healthy fish from affected fish farms were transferred to the laboratory for bacteriological study.

2.2. Isolation and cultivation

An average of 20 fish from each of the different farms were examined during the study. Samples of liver, kidney, spleen, intestine, and ascitic fluid of affected and apparently healthy fish were inoculated onto Columbia Blood Agar (CA) (Bio MCrieux), Tryptone Soy Agar (TSA) (Bio MCrieux), Mac Conkey Agar (Bio Mtrieux) and TCBS Agar (Difco). All media were incubated at 22°C for 24 h up to 7 days.

2.3. Biochemical and physiological characterisation

Biochemical identification was performed using the commercial API 20E system (Bio Merieux) at 22°C and the readings were made at 24 h, 48 h, and 7 days. Enzymatic

Table I Representative bacteria1 strains of the clinical isolates obtained from diseased sea-bream in Spain” and

Portugaih during the winter of 1996 included in this study

Laboratory Reference Number Source Geographical source Fish farm Period of isolation

1123/2 Ascitic fluid Andalucia’ A February

1123/5 Kidney Andalucia A February

Il23/9 Kidney Andalucia A February

ll23/ll Kidney Andalucia A February

1137 Kidney Catalufia” B February

II38 Kidney Cataluna B February

II39 Kidney Cataluiia B February

1140 Ascitic fluid Cataluiia B February

1141 Liver Cataluna B February 1168 Kidney Algaweb C March

1169 Kidney Algarve D March 1179 Kidney Estremadura’ E March 1180 Kidney Estremadura F March

I I87 Kidney Estremadura G March 1188 Kidney Beira Litoralh H March

I203 Kidney Cataluha B April

1204 Kidney Cataluiia B April

320 A. Dome’nech et al. /Aquaculture 156 (1997) 317-326

profiles were determined in API ZYM system (Bio Mtrieux) incubated overnight at 22°C. In addition to tests included in the API 20E and API ZYM strips, isolates were tested for growth at different temperatures (4”, lo”, 25”, 30”, 37”) and NaCl concentra- tions (O%, l%, 3%) in peptone broth. The isolates were also analysed for glucose oxidation/fermentation; production of cytochrome oxidase, catalase, and H 2 S (Kliger’s medium); reduction of nitrate (1% nitrate broth); utilisation of sodium citrate (Simmon’s medium); and degradation of aesculin (Difco). All additional biochemical tests were performed in commercial media incubated at 22°C for 24 h, 48 h and 7 days. Motility was studied at 22°C and 15°C on motility test medium (Difco). The bacterial isolates included in this study are listed in Table 1. The type strain P. anguilliseptica NCIMB 1949T was also analysed in all of the physiological and biochemical studies.

2.4. Determination of 16s rRNA gene sequence

Chromosomal DNA was prepared by the method of Lawson et al. (1989). The almost complete 16s rRNA gene was amplified by PCR with universal primers pA and pH and sequenced directly using the Sequenase version 2.0 sequencing kit (USB) as described previously (Hutson et al., 1993). Data base searches, EMBL Data Library and RDP (Maidak et al., 1994) with the FASTA program (Pearson and Lipman, 1988) from the GCG package (Devereux et al., 1984) were used to screen for the nearest neighbours of the isolates. The newly determined sequences of the 16s rRNA genes of strains P.

anguilliseptica NCIMB 1949T and isolate 1123/5 have been deposited in the EMBL Data Library under accession numbers X99 540 and X99 542, respectively.

2.5. Susceptibility of antimicrobial agents

The method used was diffusion on Mueller-Hinton Agar (Bio MCrieux) using antibiotic discs (Difco). The antimicrobials tested were oxolinic acid, oxytetracycline, nitrofurantoine, chloramphenicol, flumequine and enrofloxacin.

3. Results

3. I. Clinical signs

The external appearance of the diseased sea-bream was normal, except for a frequently observed abdominal distension. Internally, the most significant clinical signs were the presence of ascitic fluid in the peritoneal cavity. The liver was pale and friable and, in a few cases, petechial haemorrhages were observed. The kidney was haemor- rhagic and the intestine congestive with fibrinous yellowish exudate. Neither external nor intestinal parasites were found.

3.2. Bacteriology

In all the affected fish farms, pure cultures of a single organism were isolated from all kidneys and from some of the liver and ascitic fluid samples of diseased fish (Table

A. Dome’nech et al./Aquaculture 156 (1997) 317-326 321

Table 2

Phenotypical and biochemical characteristics of Spanish and Portuguese isolates compared with those of the P.

anguilliseptica type strain NCIMB 1949T

Spanish isolates

(n= 11) Portuguese isolates

(n=6)

P. anguilliseptica

NCIMB 1949T

Characteristics

Gram stain

Cell morphology

Haemolysis

Cytochrome oxidase

Catalase

O/F test

Motility

Growth at 4”-30°C

Growth at 37°C

Growth at 0%3% NaCl

Growth on Mac Conkey agar

Growth on TCBS agar

Biochemical test.?

- _ - Filamentous Filamentous Bacilli-Filamentous _a _ -

+ + + + + + _ _ -

_ _ -

+ + + _ _ -

+ + +

+ + + - _ -

Citrate utilisation’ + (9) + f

Nitrate reduction _ _ -

B-galactosidase (ONPG) _ _ -

H 2 S production _ _ -

Indole production - _ -

Acid from carbohydrates” _ _ -

Urea _ _ -

Gelatin degradation _ _ +

Aesculin degradation _ _ -

Arginine dehydrolase _ - (5) +

Lysine decarboxylase _ _ -

Omithine decarboxylase _ _ -

+ : Positive reaction,

- : Negative reaction.

(n): n tested showing positive or negative reaction.

“Five isolates from the first outbreak in fish farm B located in Catalufia were haemolytic.

“All tests, except aesculin degradation, are included in the API 20E strip. Nitrate reduction, H,S production and citrate utilisation were also performed in commercial media.

‘Clinical isolates utilised citrate only in commercial Simmon’s medium.

dGlucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin, and arabinose.

1). The isolates exhibited slow growth on TSA and CA, but failed to grow on TCBS Agar. It was necessary to incubate the primary isolation plates for at least one week to observe round, grayish, convex, entire colonies of about I mm in diameter. Moreover, their growth capability decreased rapidly with further subcultures. In the few cases in which mixed bacterial cultures were obtained, the organism was recovered by sub-cul- ture on TSA and CA. The organism was not isolated from any of the apparently healthy fish studied from the same farms.

The isolates consisted of aerobic Gram-negative filamentous rods, cytochrome oxi-

322 A. Dome’nech et al. / Aquaculture 156 (19971317-326

Table 3

APY ZYM profiles of the Spanish and Portuguese isolates compared with those of P. anguilliseptica type

strain NCIMB 194gT

Hydrolysis of the following substrates Spanish isolates Portuguese isolates P. unguillisrptica

(n= 11) (n=6) NCIMB I 949T

2.Naphthyl-phosphate (phosphatase alkaline)

2.Naphthyl-hutyrate

2-Naphthyl-caprylate

2.Naphthyl-myristate

L-leucyl-2.naphthylamide

L-valyl-2-naphthylamide

L-cystyl-2-naphthylamide

N-henzoyl-DL-arginine-2-naphthylamide

N-glutaryl-phenylalanine-2-naphthylamide

2.Naphthyl-phosphate (phosphatase acid)

Naphthol-AS-BI-phosphate

h-Br-2-Naphthyl-cumgalactopyranoside

2.Naphthyl-Ppgalactopyranoside

Naphthol-AS-BI-Ppglucorinide

2-Naphthyl-currglucopyranoside

6-Br-2-naphthyl-/3bglucopyranoside

I -Naphthyl-N-acetyl-/3 txglucosaminide

6.Br-2-Naphthyl-ocn-mannopyranoside

2.Naphthyl-cut_-fucopyranoside

_ + (9)

+ (9) _

+ (9)

_ + (7)

_

_ + (5)

_ + + _

API reaction scores 0 and I were considered negative, scores 2 to 5 were considered positive.

(n): n tested showing positive reaction.

dase and catalase positive. All isolates were non-haemolytic in CA, except those isolated during the first outbreak studied in a fish farm located in CatalGa. The isolates did not hydrolyse gelatin and they were arginine dihydrolase negative (except one Portuguese strain), and utilised citrate (except two Spanish isolates). The physiological and bio- chemical characteristics of the isolates are included in Table 2. The enzymatic profiles using the API ZYM system are shown in Table 3. The profiles of the Spanish and Portuguese isolates were very similar, except for small differences in the 2-naphthyl butyrate, 2-naphthyl caprylate, and L-leucyl-2-naphthylamide test in which two Spanish strains were negative for these substrates. Slight differences were also observed for the 2-naphthyl-phosphate (phosphate acid).

16s rRNA gene sequence analysis showed the isolates from the diseased fish were genotypically homogeneous (displaying 100% sequence similarity). Comparative se- quence analysis revealed the isolates were members of the genus Pseudomonas. Since data on the fish pathogen P. anguilliseptica was not available from the EMBL Sequence Library, the 16s rRNA gene sequence of the type strain NCIMB 1949T was determined. Pairwise analysis revealed the organisms from the diseased fish corresponded with P. anguilliseptica (> 99.7% sequence similarity). In view of their close genotypic similar- ity, the phenotypic properties of P. anguilliseptica NCIMB 1949T were compared with the fish isolates (Tables 2 and 3). The results of biochemical and physiological tests were entirely consistent with the genotypic identification of the isolates as P. anguil-

liseptica.

A. Dominech et al./Aquaculture 156 (1997) 317-326 323

Antimicrobial susceptibility tests showed that all of the fish isolates and the reference strain of P. anguilliseptica were susceptible in vitro to all the tested agents.

4. Discussion

‘Winter disease’ is a syndrome characterised by severe mortality associated with stressful conditions, such as low water temperatures, but for which the aetiological agent has not been unequivocally identified. Microbiological investigations of different out- breaks have detected the presence of several agents, including Gram-negative bacteria ( Aermnonas hydrophila, enterobacteria) and viruses (Bovo et al., 1995; Doimi, 1996) any of which could be implicated with, or responsible for, the disease. In this study, organisms definitively identified as P. anguilliseptica were isolated in pure culture from the kidney and from liver and ascitic fluids (in most cases, also in pure culture) of exclusively diseased fish and not from healthy fish of the same fish farms. This finding, together with the fact that this bacterium was isolated at different times in the eight fish farms of different geographical locations far from each other, and that it has been reported as pathogenic for sea-bream (Berthe et al., 19951, suggest that P. anguillisep-

tica is likely to be the agent responsible for the ‘winter disease’ outbreak in the sea-bream examined. Recently, Bovo et al. (1995) reported the isolation of a very metabolically unreactive Gram-negative rod from two cases of ‘winter disease’ in sea-bream in Italy. Although these authors were not able to identify this bacterium, its physiological and biochemical characteristics are consistent with those of our clinical isolates, suggesting that their outbreak of ‘winter disease’ was also caused by P.

anguilliseptica. We observed only minor discrepancies among the physiological, biochemical and

enzymatic profiles of our isolates and the type strain of P. anguilliseptica (Tables 2 and 3). These inconsistencies are similar to those described elsewhere for strains of P. anguilliseptica isolated from different fish species and geographical locality (Wiklund and Bylund, 1990; Liinnstriim et al., 1994; Kusuda et al., 1995; Berthe et al., 1995). In addition, phenotypic differences may occur because some traits, such as gelatin hydroly- sis, are readily lost with subcultures. The poor growth of P. anguilliseptica in the culture media, together with the difficulty of performing standard biochemical tests (slow-growing nature and very unreactive metabolism of the organism) makes diagnosis difficult. In this respect, the sequencing of the 16s rRNA gene proved to be crucial in the recognition of P. anguilliseptica, which was subsequently confirmed by more detailed phenotypic testing. The value of 16s rRNA analysis in the diagnosis of unusual bacterial fish pathogens has been noted earlier (Domenech et al., 1993, 1996).

Only two antimicrobial agents (oxolinic acid and oxytetracycline) were used (admin- istered orally by feed) in some of the affected fish farms. The efficacy of these antibiotics was difficult to evaluate, because they were effective only when the water temperature was above 17-18°C. At lower temperatures, food intake is drastically reduced, thereby precluding discrimination between mortality reduction due to antibi- otics or water temperatures. It is pertinent to note that in some fish farms in which no antibiotic treatment was used, the mortality also decreased when the water temperature

324 A. Domknech et al. / Aquaculture 156 (1997) 317-326

reached values of 18°C or higher; which point out the self-limiting character of this infection when the water reaches these temperatures. Experimental studies have shown the rapid intraorganic multiplication of P. anguillisepticu and the subsequent develop- ment of disease in intramuscularly inoculated eels at 12°C while at 28°C there was a rapid clearance of this bacterium from tissues of infected eels (Nakai et al., 1985). This phenomenon, attributed mainly to the inactiveness of P. anguilliseptica at such higher temperatures (Muroga et al., 19771, may explain the temperature-dependence of the infection by P. anguillisepticu (Muroga and Nakajima, 1981; Nakai et al., 1985; Wiklund and Bylund, 1990; Berthe et al., 199.5).

The association of P. anguillisepticu with disease in several fish species from different European countries has increased in recent years. This may be related to the intensive farming conditions of susceptible fish species, favouring the occurrence of clinical manifestations. This is consistent with the failure to detect, in the course of this study, ‘winter disease’ outbreaks in farms in which sea-bream were maintained under less intensive farming conditions. Under stressed situations, the immune system is partially depressed, making fish more susceptible to infections by ubiquitous or oppor- tunistic pathogenic bacteria, such as P. unguillisepticu (Carson and Schmidtke, 1993; Tort et al., 1996). ‘Winter disease’ in sea-bream has been associated mainly with low water temperatures, although other stressful conditions, such as variations in salinity, nutritional deficiencies, or high fish densities, can also play a role as predisposing disease factors (Bovo et al., 1995). We observed an increase in the mortality in those farms where a decrease in the water temperature and salinity were simultaneously observed. These results reinforce the view that ‘winter disease’ is probably a multifacto- rial process favoured by different undesirable environmental conditions acting in con- cert.

5. Conclusion

Since the initial description of P. unguilliseptica in eels (Wakabayashi and Egusa, 1972; Kuo and Kou, 1978), this organism has been subsequently reported in several different fish species. In this investigation, P. anguilliseptica was isolated in pure culture from different internal organs of diseased sea-bream from all the affected fish farms studied, this indicates that the bacterium is quite probably the agent responsible for the ‘winter disease’ outbreak. The high mortality rates involving P. unguillisepticu reported to date, including the present study, suggests that this emergent bacterium may pose a potentially serious economic problem for European aquaculture in the near future.

Acknowledgements

The authors are grateful to A. Rodriguez, A. Tiana, and L. Cardoso Menezes, veterinarian technicians, who detected the outbreaks and provided the diseased sea-bream, and to the technical directors of the fish farms for their cooperation during this study. This work was in part supported by Dibaq-Diproteg S.A. (Spain), and the BBSRC (UK).

A. Dome’nech et al. / Aquaculture 156 (1997) 317-326 325

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