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Vibrio species associated with mortalities in

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hatchery-reared turbot Colistium nudipinnis and

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brill C. guntheri in New Zealand

B.K. Diggles

a,)

_{, J. Carson}

_{, P.M. Hine}

_{, R.W. Hickman}

_,

M.J. Tait

National Institute of Water and Atmospheric Research, PO Box 14-901 Kilbirnie, Wellington, New Zealand

Department of Primary Industries, Water and EnÕironment, PO Box 46, Kings Meadows, Launceston,

Tasmania 7249, Australia

Received 7 June 1999; received in revised form 24 July 1999; accepted 31 July 1999

Abstract

Studies were conducted to determine the cause of the acute mortality of juvenile turbot

Colistium nudipinnis and brill, C. guntheri in an experimental rearing facility. Gross signs of

disease included loss of appetite, erratic swimming, distended abdomens caused by an accumula-tion of clear fluid in the stomach and intestines, and haemorrhagic lesions on the underside and bases of the fins. Histological examination of the liver and kidney showed focal areas of necrosis and extensive haemorrhaging. Other lesions included necrosis and sloughing of the mucosa of the stomach and intestine, and sparse vacuolation in the brain and spinal chord. Bacteria isolated from the liver, kidney, and spleen included Vibrio splendidus I, and V. campbellii-like variants. Examination of the liver, kidney, and brain by electron microscopy failed to detect the presence of viral particles and samples of brain were negative against anti-SJNNV rabbit serum. It appears that the mortalities were due to infection by opportunist bacteria in fish predisposed by a combination of adverse factors including an acute period of poor water quality and perhaps an inadequate diet.

Keywords: Pleuronectidae; Mariculture; Disease; Vibriosis; New Zealand

1. Introduction

Flatfish of the Order Pleuronectiformes are commercially valuable species which attract considerable mariculture interest in the northern hemisphere. Bacteria, viruses,

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and protozoa have all been implicated as pathogens in flatfish culture, often causing significant economic losses in affected facilities. Disease has been an important limiting

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factor in the mariculture of turbot Scophthalmus maximus in Europe Horne et al., 1977; Lupiani et al., 1989; Figueras et al., 1992; Fouz et al., 1992, 1995; Novoa et al., 1992, 1993; Bloch and Larsen, 1993; Bloch et al., 1991; Dykova et al., 1995; Nova et

. Ž

al., 1995; Quentel and Ogier de Baulny, 1995; Lamas et al., 1996 , halibut

Hippoglos-. Ž . Ž

sus hippoglossus in Norway Grotmol et al., 1997 , and flounder Paralichthys

. Ž .

oliÕaceus in Korea Lee et al., 1991 .

There is increasing commercial interest in the mariculture of economically important species of flatfish in New Zealand waters. To date, experimental efforts have centred on

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examining the feasibility of rearing turbot Colistium nudipinnis Waite, 1910 and brill

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C. guntheri Hutton, 1873 . An acute episode of mortalities of juveniles 2–3.9 cm TL,

weight approx. 0.5 g in a mixed culture of both species occurred during initial rearing experiments in October 1997. This paper details the results of investigations into the cause of this mortality event.

2. Materials and methods

2.1. Fish

Turbot and brill larvae hatched from gametes from wild caught broodstock were raised in 500 l cylindrico-conical fibreglass tanks in a flow-through hatchery system at NIWA’s Aquaculture Research Centre, Wellington. Initial conditions of water tempera-ture and salinity were 148C and 33‰, respectively, with seawater exchanging at a rate of

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20% per hour. From 9 days post-hatching DPH , the flow rate was increased to 30% per hour, and the temperature was raised gradually, reaching 178C by 33 DPH. Larvae

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were initially fed rotifers Brachinotus plicatus with brine shrimp Artemia salina added from 9 DPH. Metamorphosis started at 18 DPH, and the juveniles were gradually

Ž Ž . .

weaned onto crumbled commercial salmon pellets Northern Roller Mills NRM NZ by 28 DPH. Fish were maintained on a 24-h light photoperiod. During the period of

mortality which started at 57 DPH, moribund juvenile turbot and brill ns30, mean TL

3.2 cm, range 2.0–3.9 cm were euthanased by sectioning of the spinal cord and examined as follows.

2.2. Gross examination

The surface of each fish was examined for lesions and signs of physical damage, and wet smears were taken from scrapes of the gills and skin and examined for the presence of protozoan and metazoan parasites.

2.3. Bacteriology

The surface of five diseased turbot was sterilised with 70% ethanol before their abdominal cavities were opened up with a ventral incision with a sterile scalpel. Samples

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of liver, kidney, and spleen were taken from juvenile fish with a flamed wire loop and

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inoculated onto thiosulphate citrate bile salt sucrose agar TCBS, Oxoid , Tryptic Soy

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Agar TSA, Gibco and TSA with 1% NaCl TSAq1 . Replicate samples of media were incubated at 15 and 208C and examined daily for bacterial colonies for 7 days. Bacteria isolated in moderate to heavy growths were subcultured to ensure purity before

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being stored at y708C in Tryptic Soy broth with 15% vrv glycerol prior to identification. Morphological examination of bacterial colonies was made on isolates recovered from storage in Tryptic Soy broth and incubated for 24–27 h at 158C on TCBS. Biochemical characterisation was undertaken on isolates recovered from storage

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in long-term preservation medium Beuchat, 1974 and subjected to 52 phenotypic tests

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using methods described by Baumann et al. 1971 , Furniss et al. 1978 , and West and

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Colwell 1984 . Data were compared to a probabilistic data matrix for Vibrio species

ŽBryant et al., 1986 using the Bacterial Identifier program Bryant and Smith, 1991 .. Ž .

An acceptable identification was reached when the identification score equalled or exceeded 0.98.

2.4. Histopathology and electron microscopy

Twenty-five diseased fish comprising a mixture of both turbot and brill were sacrificed for histology and electron microscopy. Ten of the fish were euthanased and

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immediately fixed for histology by placing the entire fish in 10% vrv formalin in seawater filtered to 0.22 mm after opening the abdominal cavity to allow entry of the fixative. All samples were embedded in paraffin wax, processed using standard histolog-ical procedures and 6 mm sections were stained with hematoxylin and eosin or Gram

stain. Fish were embedded so sagittal sections containing organs of interest gill, liver,

kidney, brain, intestine were obtained.

The remaining 15 fish were euthanased and samples of kidney, liver, and brain were

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excised, fixed for transmission electron microscopy TEM in 2.5% gluteraldehyde in filtered seawater for 2 h, washed twice in filtered seawater, postfixed in osmium tetroxide, and embedded in Spurrs resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips 420 TEM at 80 kV.

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2.5. Fluorescent antibody technique FAT

One paraffin-embedded turbot which displayed vacuolation in the brain and spinal chord was tested for the presence of nodavirus with a FAT using anti-SJNNV rabbit

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serum Office International des Epizooties, 1995; Munday and Naki, 1997 .

3. Results

3.1. Onset and gross signs of disease

Unusual mortalities began at 57 DPH, 2 days after a period of heavy rainfall which increased the organic and silt load of the incurrent water and caused a salinity drop from 36‰ to 25‰ for 24 h. Moribund turbot and brill exhibited erratic swimming, loss of

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appetite, loss of righting reflex, and increased ventilation rate. Approximately 50% exhibited distended abdomens due to the presence of a large volume of clear fluid in the stomach and intestines, and approximately 30% had focal haemorrhagic lesions on the bases of the fins, tail, and underside. No evidence of protozoan or metazoan parasitic

infection was observed in scrapes of gills and skin. Mortalities exceeded 90%

ap-.

proximately 2000 fish over a period of 4 weeks.

3.2. Bacteriology

Moderate to heavy growths of either one of two colonial types of bacteria were

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isolated in pure culture from 100% 5r5 of spleen samples and from 80% 4r5 of liver and kidney samples. One fish had a mixed infection of both types of bacteria in the liver and kidney. Both colonial types grew on TCBS, TSA, and TSAq1 media.

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Sucrose-positive colonies yellow on TCBS were identified as two variants of Vibrio

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splendidus I see Table 1 . The two isolates differed only in their ability to lyse sheep

red blood cells and utiliseD-sorbitol as a sole carbon source; the identification score for

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both isolates was 0.98. The other colonial type, sucrose negative green on TCBS, was

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identified as two V. campbellii-like variants Table 1 which differed from each other only in their ability to use melibiose as a sole carbon source. The phenotype of the two

Table 1

Biochemical characteristics of Vibrio species isolated from turbota cs: Carbon source utilisation test; r: resistant; s: sensitive.

V. campbellii-like V. campbellii-like V. splendidus I V. splendidus I

Colour on TCBS green green yellow yellow

Indole y y q q

ONPG y y q q

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Polymyxin B 50 i.u. r r s s

Aesculin y y q q

Alginase y y q q

Haemolysis sheep RBC y y y q

Sucrose acid y y q q

Mannitol acid y y q q

Cellobiose cs y y q q

Melibiose cs q y y y

Sucrose cs y y q q

D-sorbitol cs y y y q

Gluconate cs y y q q

L-citrulline cs y y q q

D-glucosamine cs y y q q

Isolates were uniformly positive for arginine dihydrolase and oxidase production, nitrate reduction, acid production from mannose, and utilization ofD-galactose,D-glucose,D-mannose, trehalose, glycerol, a-keto-glutarate, and succinate, and sensitive to 10mgrml of the vibriostatic agent Or129 and 10mgrml ampicillin. Isolates were uniformly negative for swarming, growth in 0% NaCl, production of Møllers lysine and ornithine

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decarboxylase and urease, the Voges Proskauer reaction, growth in 0.0005% wrv potassium tellurite, acid production fromL-arabinose, arbutin, salicin and sorbitol, and utilization ofL-arabinose, lactose, melezitose, xylose, ethanol, 1-propanol,D-glucuronate, amygdalin, arbutin, L-hydroxy-proline, L-leucine, and DL -3-hy-droxybutyrate.

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isolates differed from V. campbellii in that they were arginine dihydrolase-positive and sensitive to ampicillin. The identification scores for both isolates was 0.94.

3.3. Histopathology

Significant pathological lesions were found in the liver, kidney, stomach, intestine, brain, and spinal chord; however, the gills appeared normal. The liver exhibited

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generalised vacuolation, and multiple haemorrhagic and necrotic foci Fig. 1 . There was extensive haemorrhaging in the kidney with generalised necrosis of haematopoietic

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tissue and accumulation of a hyaline substance in the lumen of kidney tubules Fig. 2 . There was necrosis and sloughing of the epithelial lining and sub-mucosal layer in the

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stomach and intestine Figs. 3 and 4 in those animals which exhibited gross signs of

abdominal swelling. Sparse vacuolation was evident in the grey matter of the brain Fig.

. Ž .

5 , and moderate vacuolation in the tissue of the spinal chord Fig. 6 . Extensive haemorrhaging was evident in the area surrounding the eye, but no vacuolation was

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found in the retina Fig. 7 .

3.4. Electron microscopy

Examination of the kidney and the liver by electron microscopy found numerous rod-shaped bacteria, 1.2 to 1.4 mm=0.5 mm in dimension with a strongly electron

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dense outer membrane and cell membrane Fig. 8 . No bacteria were evident in sections

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Fig. 1. Liver of diseased turbot. Note generalised vacuolation of hepatocytes arrowheads , haemorrhagic foci

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Fig. 2. Kidney of diseased turbot. Note greatly reduced areas of haematopoietic tissue H , large haemorrhagic

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lesions arrows and hyaline substance in lumen of some kidney tubules arrowheads . Scale bars64mm.

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Fig. 3. Intestine of a diseased turbot exhibiting a grossly swollen abdomen. Necrosis N and sloughing of

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Fig. 4. Intestine of a diseased turbot exhibiting a grossly swollen abdomen. Sloughing of the necrotic mucosal

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lining M into the lumen of the intestine. Scale bars53mm.

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Fig. 5. Cerebellum of diseased turbot. Note small number of vacuoles arrow in grey matter. Scale bars160 mm.

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Ž .

Fig. 6. Spinal cord of diseased turbot. Note several vacuoles arrows . Scale bars80mm.

of brain tissue. Extensive searches of liver, kidney, and brain tissue found no evidence of viral particles in any of these organs.

Ž . Ž .

Fig. 7. Eye of diseased turbot. Note lack of vacuolation in retina r and erythrocytes E in haemorrhage inside the orbit of eye. Lslens. Scale bars107mm.

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Fig. 8. Electron micrograph of bacteria found in the kidney of diseased turbot. Scale bars200 nm.

3.5. FAT

The paraffin-embedded brain and spinal chord of a turbot which exhibited vacuola-tion in both the brain and spinal chord was negative for FAT using anti-SJNNV rabbit

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serum T. Naki, personal communication .

4. Discussion

Vibriosis is well recognised as a significant cause of disease and mortality in the

culture of flatfish Horne et al., 1977; Egidus, 1987; Lee et al., 1991; Novoa et al., 1992;

Fouz et al., 1995; Grisez et al., 1996 . The gross symptoms displayed by fish in the present study, which included abdominal distension, haemorrhaging at the base of the fins, and focal haemorrhagic lesions on the skin, are similar to those reported by Horne

Ž . Ž .

et al. 1977 for V. anguillarum infections of S. maximus, by Lupiani et al. 1989 for a mixed V. splendidus-like bacterialrreoviral infection in S. maximus, and by Lee et al.

Ž1991 for infections of P. oli. Õaceus by various Vibrio species including V. tubiashi,

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and V. damsela sPhotobacterium damselae . The haemorrhagic necrotic lesions

observed in the kidney and liver of the fish in the present study were also similar to

Ž . Ž .

lesions described by Horne et al. 1977 and Fouz et al. 1995 in S. maximus suffering from systemic bacterial septicaemias.

A range of Vibrio species have been isolated from diseased and apparently healthy

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flatfish including V. anguillarum see Toranzo and Barja, 1990 , P. damselae see Fouz

. Ž .

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Žsee Lamas et al., 1990; Novoa et al., 1992 , and V. pelagius see Novoa et al., 1992;. Ž .

Santos et al., 1997 . The pathogenicity of these bacteria is not clear for all the species

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isolated. The pathogenicity of V. anguillarum see Grisez et al., 1996 , P. damselae

Ž_{see Fouz et al., 1992 , and V. splendidus I see Angulo et al., 1994 has been verified}. Ž .

experimentally in turbot, but for the other species of Vibrio, their role as a cause of disease is unclear. V. splendidus I isolated from turbot and brill in the present study from liver, kidney, and spleen is probably a significant cause of disease based on our histopathology evidence and the known pathogenicity of V. splendidus I in flatfish. The abdominal swelling observed in diseased turbot and the associated histopathology is

consistent with observations in larvae of gilt-head seabream, Sparus aurata L. see

Sedano et al., 1996 infected with a consortium of Vibrio species including V.

splendidus I and V. fischeri. The role of the V. campbellii-like isolates as a cause of

disease is less certain. V. campbellii forms a significant component of the normal gut

flora of hatchery-reared turbot larvae reared under intensive conditions Munro et al.,

1994 . Although this species has not been reported previously as a pathogen of finfish, a

V. campbellii-like bacterium is known to be pathogenic for larvae of Penaeus indicus,

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the white tiger shrimp Sahul Hameed et al., 1996 . It has been noted that complex

bacterial floras of Vibrio species may develop in stressed turbot Lamas et al., 1990;

Bloch et al., 1991; Novoa et al., 1992 and lead to invasion by opportunistic species of

Vibrio. Several species of opportunistic Vibrio species including V. splendidus I and V. alginolyticus are known to produce extracellular proteases, esterases, and haemolysins,

aggressins likely to assist invasion and colonisation by these bacterial species Sedano et

al., 1996 . Our culture and histopathology findings point to V. campbellii-like bacteria having some pathogenic role, but further work, including fulfilment of Koch’s postu-lates, is required to establish the significance of this species as a pathogen in juvenile farmed flatfish.

The variability between isolates of V. splendidus obtained from different fish in the present study was not considered unusual as it has been demonstrated that isolates of V.

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splendidus from fish are heterogeneous Austin et al., 1997 . It is possible, however, that

the minor phenotypic differences found between the two isolates of V. splendidus, and also the two isolates of the V. campbellii-like bacteria, may have been an artefact of low reproducibility of the phenotypic tests used, or perhaps resulted from changes that occurred during storage.

We consider that the Vibrio species isolated in the present study acted as opportunist pathogens of turbot and brill because the mortality episode followed a period of adverse water quality in the form of increased organic and silt loading together with a rapid reduction in salinity from 33‰ to 25‰. Dietary deficiency from use of crumbled salmon pellets as a weaning feed cannot be discounted as an additional stressor which may have predisposed these fish to bacterial invaders, and is suggested by the highly vacuolated pathology of the liver.

The vacuolated lesions present in the brain and spinal chord of diseased turbot and brill hinted that a nodavirus may have been present. Vacuolation of these tissues is

characteristic of nodaviral infections of other species of flatfish Bloch et al., 1991;

Nguyen et al., 1994; Grotmol et al., 1997 , and are sometimes used for presumptive

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Naki, 1997 . Despite considerable effort, however, we could not visualise viral particles in these tissues using electron microscopy. The FAT using anti-SJNNV rabbit serum provides a rapid and group-specific test for at least five nodaviruses when applied to

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paraffin-embedded tissues of clinically affected fish Munday and Naki, 1997 . As the tissues of a clinically affected turbot exhibiting vacuoles in the brain and spinal chord tested negative using the FAT test, this indicated that a nodavirus was absent in this mortality event.

5. Conclusions

These juveniles were the first New Zealand turbot and brill to be reared in a hatchery. This disease outbreak was, therefore, the first recorded in flatfish mariculture in New Zealand. A combination of factors, including poor water quality and dietary inadequa-cies, appear to have contributed to mortalities which were associated with infection by opportunistic bacterial pathogens. These mortalities demonstrate that disease prevention and control will be as important in the culture of flatfish in New Zealand as they are in other areas of the world.

Acknowledgements

The authors thank Dr. B. Munday for helpful discussion on the disease syndrome and for sending paraffin-embedded samples to T. Naki of the Hiroshima University who kindly performed the FAT testing. We also wish to acknowledge the microbiology support of Toni Wagner in the identification of the Vibrio isolates and the helpful suggestions of two anonymous reviewers on drafts of the manuscript.

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B.K. Diggles et al.rAquaculture 183 2000 1–12 7

Fig. 4. Intestine of a diseased turbot exhibiting a grossly swollen abdomen. Sloughing of the necrotic mucosal

Ž .

lining M into the lumen of the intestine. Scale bars53mm.

Ž .

Fig. 5. Cerebellum of diseased turbot. Note small number of vacuoles arrow in grey matter. Scale bars160

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( ) B.K. Diggles et al.rAquaculture 183 2000 1–12 8

Ž .

Fig. 6. Spinal cord of diseased turbot. Note several vacuoles arrows . Scale bars80mm.

of brain tissue. Extensive searches of liver, kidney, and brain tissue found no evidence

of viral particles in any of these organs.

Ž . Ž .

Fig. 7. Eye of diseased turbot. Note lack of vacuolation in retina r and erythrocytes E in haemorrhage inside the orbit of eye. Lslens. Scale bars107mm.

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( )

B.K. Diggles et al.rAquaculture 183 2000 1–12 9

Fig. 8. Electron micrograph of bacteria found in the kidney of diseased turbot. Scale bars200 nm.

3.5. FAT

The paraffin-embedded brain and spinal chord of a turbot which exhibited

vacuola-tion in both the brain and spinal chord was negative for FAT using anti-SJNNV rabbit

Ž

.

serum T. Naki, personal communication .

4. Discussion

Vibriosis is well recognised as a significant cause of disease and mortality in the

Ž

culture of flatfish Horne et al., 1977; Egidus, 1987; Lee et al., 1991; Novoa et al., 1992;

.

Fouz et al., 1995; Grisez et al., 1996 . The gross symptoms displayed by fish in the

present study, which included abdominal distension, haemorrhaging at the base of the

fins, and focal haemorrhagic lesions on the skin, are similar to those reported by Horne

Ž

.

Ž

.

et al. 1977 for V. anguillarum infections of S. maximus, by Lupiani et al. 1989 for a

mixed V. splendidus-like bacterial

r

reoviral infection in S. maximus, and by Lee et al.

Ž

1991 for infections of P. oli

.

Õ

aceus by various Vibrio species including V. tubiashi,

Ž

.

and V. damsela

s

Photobacterium damselae . The haemorrhagic necrotic lesions

observed in the kidney and liver of the fish in the present study were also similar to

Ž

.

Ž

.

lesions described by Horne et al. 1977 and Fouz et al. 1995 in S. maximus suffering

from systemic bacterial septicaemias.

A range of Vibrio species have been isolated from diseased and apparently healthy

Ž

.

Ž

flatfish including V. anguillarum see Toranzo and Barja, 1990 , P. damselae see Fouz

.

Ž

.

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( ) B.K. Diggles et al.rAquaculture 183 2000 1–12 10

Ž

see Lamas et al., 1990; Novoa et al., 1992 , and V. pelagius see Novoa et al., 1992;

.

Ž

.

Santos et al., 1997 . The pathogenicity of these bacteria is not clear for all the species

Ž

.

isolated. The pathogenicity of V. anguillarum see Grisez et al., 1996 ,

P. damselae

Ž

_{see Fouz et al., 1992 , and V. splendidus I see Angulo et al., 1994 has been verified}

.

Ž

.

experimentally in turbot, but for the other species of Vibrio, their role as a cause of

disease is unclear. V. splendidus I isolated from turbot and brill in the present study

from liver, kidney, and spleen is probably a significant cause of disease based on our

histopathology evidence and the known pathogenicity of V. splendidus I in flatfish. The

abdominal swelling observed in diseased turbot and the associated histopathology is

Ž

consistent with observations in larvae of gilt-head seabream, Sparus aurata L. see

.

Sedano et al., 1996

infected with a consortium of Vibrio species including V.

splendidus I and V. fischeri. The role of the V. campbellii-like isolates as a cause of

disease is less certain. V. campbellii forms a significant component of the normal gut

Ž

flora of hatchery-reared turbot larvae reared under intensive conditions Munro et al.,

.

1994 . Although this species has not been reported previously as a pathogen of finfish, a

V. campbellii-like bacterium is known to be pathogenic for larvae of Penaeus indicus,

Ž

.

the white tiger shrimp Sahul Hameed et al., 1996 . It has been noted that complex

Ž

bacterial floras of Vibrio species may develop in stressed turbot Lamas et al., 1990;

.

Bloch et al., 1991; Novoa et al., 1992 and lead to invasion by opportunistic species of

Vibrio. Several species of opportunistic Vibrio species including V. splendidus I and V.

alginolyticus are known to produce extracellular proteases, esterases, and haemolysins,

Ž

aggressins likely to assist invasion and colonisation by these bacterial species Sedano et

.

al., 1996 . Our culture and histopathology findings point to V. campbellii-like bacteria

having some pathogenic role, but further work, including fulfilment of Koch’s

postu-lates, is required to establish the significance of this species as a pathogen in juvenile

farmed flatfish.

The variability between isolates of V. splendidus obtained from different fish in the

present study was not considered unusual as it has been demonstrated that isolates of V.

Ž

.

splendidus from fish are heterogeneous Austin et al., 1997 . It is possible, however, that

the minor phenotypic differences found between the two isolates of V. splendidus, and

also the two isolates of the V. campbellii-like bacteria, may have been an artefact of low

reproducibility of the phenotypic tests used, or perhaps resulted from changes that

occurred during storage.

We consider that the Vibrio species isolated in the present study acted as opportunist

pathogens of turbot and brill because the mortality episode followed a period of adverse

water quality in the form of increased organic and silt loading together with a rapid

reduction in salinity from 33‰ to 25‰. Dietary deficiency from use of crumbled

salmon pellets as a weaning feed cannot be discounted as an additional stressor which

may have predisposed these fish to bacterial invaders, and is suggested by the highly

vacuolated pathology of the liver.

The vacuolated lesions present in the brain and spinal chord of diseased turbot and

brill hinted that a nodavirus may have been present. Vacuolation of these tissues is

Ž

characteristic of nodaviral infections of other species of flatfish Bloch et al., 1991;

.

Nguyen et al., 1994; Grotmol et al., 1997 , and are sometimes used for presumptive

Ž

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( )

B.K. Diggles et al.rAquaculture 183 2000 1–12 11

.

Naki, 1997 . Despite considerable effort, however, we could not visualise viral particles

in these tissues using electron microscopy. The FAT using anti-SJNNV rabbit serum

provides a rapid and group-specific test for at least five nodaviruses when applied to

Ž

.

paraffin-embedded tissues of clinically affected fish Munday and Naki, 1997 . As the

tissues of a clinically affected turbot exhibiting vacuoles in the brain and spinal chord

tested negative using the FAT test, this indicated that a nodavirus was absent in this

mortality event.

5. Conclusions

These juveniles were the first New Zealand turbot and brill to be reared in a hatchery.

This disease outbreak was, therefore, the first recorded in flatfish mariculture in New

Zealand. A combination of factors, including poor water quality and dietary

inadequa-cies, appear to have contributed to mortalities which were associated with infection by

opportunistic bacterial pathogens. These mortalities demonstrate that disease prevention

and control will be as important in the culture of flatfish in New Zealand as they are in

other areas of the world.

Acknowledgements

The authors thank Dr. B. Munday for helpful discussion on the disease syndrome and

for sending paraffin-embedded samples to T. Naki of the Hiroshima University who

kindly performed the FAT testing. We also wish to acknowledge the microbiology

support of Toni Wagner in the identification of the Vibrio isolates and the helpful

suggestions of two anonymous reviewers on drafts of the manuscript.

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