Fish & Shellfish Immunology (1996) 6, 123–133

Preliminary study of the detection of antibodies tolymphocystis disease virus in flounder, Platichthys flesus

L., exposed to contaminated harbour sludge

PETER DIXON*, DICK VETHAAK†, DAVID BUCKE¶ AND MICHAEL NICHOLSON‡

*Ministry of Agriculture, Fisheries and Food, Fish Diseases Laboratory,The Nothe, Weymouth, Dorset, DT4 8UB, U.K.; †Ministry of Transport,

Public Works and Water Management, Directorate-General for Public Worksand Water Mangement, National Institute for Coastal and Marine

Management/RIKZ, Ecotoxicology Section, P.O. Box 207, 9750 AE Haren,The Netherlands; and ‡Ministry of Agriculture, Fisheries and Food,

Fisheries Laboratory, Pakefield Road, Lowestoft, Suffolk, U.K.

(Received 4 July 1995, accepted in revised form 9 October 1995)

Flounder, Platichthys flesus L., were exposed to polluted harbour sludge, or tosludge wash-o# in mesocosms for three years. Towards the end the study, apilot experiment was initiated in which flounder antibodies against lympho-cystis disease virus were measured in fish held in the mesocosms for 2·5 or 3years. Although both the sub-sample sizes and number of fish with detectableantibody were small, it was found that between the two sampling periods therewas a significant reduction in the number of antibody-positive fish in themesocosm containing polluted harbour sludge, compared to fish in the meso-cosm containing sludge wash-o#, or a control mesocosm. The significance ofthat finding is discussed.

Key words: flounder, lymphocystis disease, antibody detection, pollution,immunomodulation.

I. Introduction

For many years there has been concern that long-term exposure to pollutantsin the aquatic environment may influence the health of fish populations. Thee#ects may be direct, e.g. pulp mill e%uents causing fin erosions (Lindesjöö &Thulin, 1990) or the e#ects may be more subtle, such as predisposing the fishto infectious disease. Such latter e#ects may be mediated directly or indirectlyby modulation of the non-specific and/or specific immune defence systems offish (see recent reviews by Dunier & Siwicki, 1993; Wester et al., 1994).Demonstration that these e#ects are actually taking place in the aquatic(especially the marine) environment is extremely di$cult, although suchstudies have been done (Weeks & Warinner, 1984, 1986; Bengtsson & Larson,1986; Warinner et al., 1988; Weeks et al., 1986). For that reason, many

*Author to whom correspondence should be sent.¶Present address: DB Aquatic Pathology Service, Chasers Folly, 3b Roundhayes Close,

Weymouth, Dorset, DT4 0RN, U.K.

1231050–4648/96/020123+11 $12.00/0

investigations into the e#ects of pollutants on fish health have been conductedunder experimental conditions. In the majority of such studies, fish have beenexposed to pollutants individually or in combination for various periods oftime and the ability of fish to respond immunologically or their susceptibilityto disease have been measured (Dunier & Siwicki, 1993).In an attempt to mimic more closely what may occur in the marine

environment, fish, usually flatfish, have been held on sediments obtainedfrom areas of high and negligible pollution before measurements of immuno-competence and/or disease status have been taken (Bucke et al., 1989; Bucke& Dixon, 1992; Secombes et al., 1991). Even so, those experiments are farremoved from the conditions occurring in the marine environment. In afurther step to reproduce natural conditions, but still maintain control of testanimals, experiments have been conducted in large-scale mesocosms in TheNetherlands over a 3-year period (Vethaak, 1993, 1994). In those studies,incidence and prevalence of skin ulcers, fin rot, lymphocystis disease (LD) andliver disease in flounder (Platichthys flesus L.), amongst other data, wererecorded. During the course of the experiment, an enzyme-linked immuno-sorbent assay (ELISA) to measure flounder antibody against lymphocystisdisease virus (LDV), the cause of LD, was developed (Lorenzen & Dixon, 1991),and a pilot study was initiated to measure the antibody response of flounder inthe mesocosms to LDV. As well as providing information on previous exposureof fish to LDV, in addition to the data on current infection obtained bymacroscopic examination, the data would be used to determine whetherimmunomodulation had occurred. This is a report of the pilot study.

II. Materials and Methods

MESOCOSM EXPERIMENT

The mesocosm design has been described by Vethaak (1993). In brief, 3 largeconcrete basins, each 40 m#40 m#3 m deep were used. Mesocosm 1 con-tained clean sand and was the reference mesocosm. Mesocosm 2 also con-tained clean sand, but received e%uent water from Mesocosm 3. The lattermesocosm contained dredged spoil from Rotterdam Harbour. Mesocosms 1 and2 were each stocked with 1200 one-year old flounder and Mesocosm 3 wasstocked with 400 flounder of the same age. For most contaminants there was aclear pollution gradient, with the highest values in the directly pollutedmesocosm and the lowest values in the reference mesocosm. For example, theratio of the concentrations of the PCB congener CB-153 in the sediments ofMesocosms 1, 2 and 3 was 59: 319: 1110 ìg kg"1 organic content (OC), and thecorresponding ratio of PAHs (sum of benzo(b)fluoranthene, benzo(k)fluoran-thene and benzo(a)pyrene) was 9·0: 12·7: 29·3 mg kg"1 OC. Further data on thecontaminants can be found in Vethaak (1993).

SAMPLING

The mesocosm experiment ran from May 1990 to May 1993 and fish weresampled at 6-monthly intervals over that period. However, blood was onlycollected from the fish at the last two sampling periods, in November 1992(25 fish/mesocosm) and May 1993 (remaining fish) when the fish were aged

124 P. DIXON ET AL.

approximately 3·5 year and 4 year respectively. Length, weight, sex and grosslesions were recorded (as well as other data, to be reported in anothercommunication). Fish were then bled from the caudal vein and the bloodallowed to clot at 4) C. Serum was removed, clarified by low speed centri-fugation (1000 g for 10 min) and stored with 0·02% sodium azide at "20) C.

ANTIBODY DETECTION

The ELISA described by Lorenzen & Dixon (1991) was used. LDV purifiedfrom lymphocystis lesions collected from flounder caught in local Dutchcoastal waters was used as capture antigen, in order to lessen any chance ofthe results being a#ected by possible di#erent serotypes of the virus. The serawere initially screened at a 1:10 dilution to determine the number contain-ing detectable specific antibody. Positive sera were then titrated in 2-folddilutions from 1:20 to 1:1280.

STATISTICAL ANALYSIS

The data were analysed by creating a dichotomous variable, y, for each fish,taking the value y=1 when the antibody was detected, and y=0 when theantibody was not detected. Then y was treated as the outcome of a Bernoullitrial with probability P of the antibody being present, and 1-P of the antibodybeing absent.The factors Treatment (mesocosm), Sampling Occasion, fish Sex, and the

covariate fish Length, were related to P by a logistic equation

where i, j, k refer to the levels of Treatment, Sampling Occasion and Sex, andl is fish length. The model terms were tested by calculating the di#erences inthe deviances computed with the corresponding model terms included andexcluded. Under the null hypothesis of no e#ect, the di#erence in the deviancehas approximately a Chi-squared distribution with degrees of freedom givenby the di#erence in the number of model terms (McCullagh & Nelder, 1989).

III. Results

The antibody titres and fish data are shown in Tables 1 and 2. Table 3summarises the numbers of fish and mean antibody levels by SamplingOccasion and Treatment. The simple average titres are broken into theproportions of fish for which the antibody was detected and the correspondingaverage antibody levels. Of the 161 fish tested, only 47 (29·2%) had a detectableantibody response, which makes statistical analysis of the level of responsedi$cult. Also, from Table 3, the standard errors of the average antibody levelsare large relative to the di#erences between the groups. Therefore, the datawere analysed only in terms of presence or absence of antibody in the fish. Theprobability of fish producing antibody was modelled as a generalised linearmodel incorporating Treatment, Sampling Occasion, fish Sex and Length. The

DETECTION OF LYMPHOCYSTIS ANTIBODIES 125

Table1.Titration

ofantibodiesagainstLDVinflounderseracollectedinNovember,1992

Mesocosm1

Mesocosm2

Mesocosm3

Fish

no.

Sex

Length

(cm)

Weight

(g)

Antibody

titre1

Gross

LD2

Fish

no.

Sex

Length

(cm)

Weight

(g)

Antibody

titre1

Gross

LD2

Fish

no.

Sex

Length

(cm)

Weight

(g)

Antibody

titre1

Gross

LD2

1M

32·1

371·0

1F

27·0

190·7

1F

30·3

260·0

+2

M26·4

244·9

2F

30·8

315·0

2F

29·6

298·6

1/80

3M

31·0

303·5

3F

29·1

226·4

3M

28·1

203·6

4F

34·7

387·8

4M

23·7

138·1

1/20

4F

26·0

175·8

1/640

+5

M30·8

296·2

5M

26·0

168·5

+5

F36·2

505·2

6F

31·2

341·2

1/40

6F

30·4

278·5

6F

36·8

518·2

7M

27·1

195·3

>1/1280

7F

29·4

289·4

1/40

7M

28·6

164·3

8M

31·5

315·3

1/60

8F

28·2

260·4

1/10

8M

27·7

231·2

1/320

9F

31·7

349·7

9M

26·5

176·9

1/10

9M

31·0

323·0

+10

F34·1

403·7

10F

31·1

306·9

10F

31·1

274·3

+11

F29·0

262·0

11F

29·2

245·7

11M

29·8

265·3

1/320

12M

30·5

260·8

+12

M28·4

191·1

+12

M30·2

238·2

Noserum3

+13

F28·3

244·4

13M

27·8

218·0

1/20

13F

23·9

134·8

+14

F29·7

250·7

1/20

+14

F30·7

272·8

14M

31·6

349·8

15F

27·7

239·9

1/10

+15

F27·1

198·3

15M

30·2

299·6

16F

27·4

224·5

1/20

+16

M27·8

166·5

1/20

16F

31·8

311·5

17F

30·7

255·4

17M

36·0

245·4

1/20

17F

33·2

390·6

Noserum

18M

26·3

193·9

1/60

+18

F26·5

163·9

1/640

+18

M30·2

230·0

1/40

19M

27·5

209·6

1/20

19F

27·6

196·9

+19

F34·3

382·2

20M

28·6

225·9

1/20

20F

27·8

211·2

20F

33·2

328·4

Noserum

+21

M28·0

252·0

21M

29·5

229·5

21F

33·4

306·1

1/80

+22

M25·7

178·8

22M

26·8

194·6

1/40

22M

32·3

326·2

1/640

23F

27·5

217·4

23M

28·5

229·8

+23

F33·3

368·3

24M

24·9

205·9

24F

29·9

247·0

24F

30·8

237·6

Noserum

+25

F25·3

159·5

25F

30·7

249·3

1/10

25F

32·3

311·1

1Allseraweretested;titresofrespondersareshown;2+=macroscopicLDobserved;3serumlostintransit.

analysis of deviance of the model terms is shown in Table 4. The combinede#ects of Treatment and Sampling Occasion were just significant at the 5%level. However, the individual terms just failed to be significant at the5% level. Neither fish Sex nor Length were significant.Further examination of the results showed that whereas the marginal e#ect

of Treatment averaged over the Sampling Occasions showed only a smalle#ect, there was a large e#ect due to Treatment on the second SamplingOccasion (May 1993), but not on the first (November 1992). Table 5 showsseparate analyses of deviance for each Sampling Occasion, with no evidence ofany di#erence at November 1992, but significance at the 1% level at May 1993.

IV. Discussion

A mathematical model of LD in flounder predicted that the frequency ofimmune fish would increase with age (Lorenzen et al., 1991). That predicationwas confirmed by a serological survey of flounder from the Elbe Estuary(Lorenzen & Dixon, 1991). In the same study the model did not hold forflounder from the Eider Estuary, but that may have been because the agestructure of the sample was biased towards younger fish. If the model held forthe flounder in these mesocosms, it would be expected that there would be amaintenance or increase in the number of antibody responders in May 1993compared with November 1992, but that appeared not to be the case. Therewas a significantly lower number of antibody-positive fish in Mesocosm 3 inMay 1993 compared with November 1992. The small numbers of fish sampledand the small numbers of those which had detectable antibodies to LDVmade the results of this study di$cult to analyse. However, the resultsobtained suggest a trend towards reduction in the numbers of immune fish inmesocosms containing the highest levels of pollutants.It is premature to state categorically from these data that it was the

pollutants which caused this trend. For instance the origin of the substrate inMesocosm 3 (harbour sludge) and that in the other two mesocosms (sand) hadto be di#erent because of the nature of the study; that di#erence in thestructure of the substrates may have influenced the results. Furthermore,other factors such as infection with parasites may cause immunosuppressionin fish (Woo, 1992). The sampling periods were approximately 6 months apartand these data only reflect changes occurring over that period. It may be thatthere was a natural cycle of infection with, and re-exposure to, LD, and aconcomitant cycle of increasing and declining antibody levels. Those cyclesmay not be in phase in each of the mesocosms, and what was observed was anatural decline in antibody response in the fish in Mesocosm 3. However, ifthere was no influence from the mesocosms, the cycles would be expected to bein phase.Immunomodulation by pollutants from the harbour sludge would not be

unexpected. Other studies on fish held on polluted sediments, or on fishcaptured from polluted areas, have shown there to be a diminution of theimmune response (Weeks & Warinner, 1984; Weeks et al., 1986; Warinner et al.,1988; Bucke et al., 1989). Conversely, a recent study on dab, Limanda limandaL. held on contaminated and reference sediments showed no di#erences in

DETECTION OF LYMPHOCYSTIS ANTIBODIES 127

Table2.Titration

ofantibodiesagainstLDVinflounderseracollectedinMay

1993

Mesocosm1

Mesocosm2

Mesocosm3

Fish

no.

Sex

Length

(cm)

Antibody

titre1

Gross

LD2

Fish

no.

Sex

Length

(cm)

Antibody

titre1

Gross

LD2

Fish

no.

Sex

Length

(cm)

Antibody

titre1

Gross

LD2

1M

30·5

1/20

1M

28·2

1F

33·5

+2

M27·2

+2

M26·5

+2

F33·5

3M

30·5

1/640

3F

25·8

3M

28·7

4F

30·4

1/40

4M

25·8

4M

32·0

5F

29·4

1/20

5M

27·7

5F

28·7

6F

31·6

6M

24·5

6M

30·0

+7

F34·4

1/640

+7

F30·3

1/40

7M

28·0

8M

30·3

+8

M28·3

8M

29·5

9F

33·6

9F

29·2

9F

35·2

10M

35·6

1/20

10M

29·0

10M

31·0

11M

29·8

11F

28·6

11F

35·2

+12

M27·5

12M

26·9

+12

F31·8

13M

34·5

1/10

13F

27·7

13F

36·2

14F

32·2

1/20

+14

M28·3

14M

33·7

15F

30·0

15F

31·7

+15

M32·0

+16

F32·9

16M

29·6

+16

F33·1

17F

36·2

17M

27·4

+17

F31·6

1/80

18F

32·2

18F

26·5

18F

29·7

19M

30·7

19F

25·4

+19

F30·3

20M

24·7

1/160

+20

M27·8

20F

30·3

Table2.Continued

Mesocosm1

Mesocosm2

Mesocosm3

Fish

no.

Sex

Length

(cm)

Antibody

titre1

Gross

LD2

Fish

no.

Sex

Length

(cm)

Antibody

titre1

Gross

LD2

Fish

no.

Sex

Length

(cm)

Antibody

titre1

Gross

LD2

21M

28·7

Noserum3

21M

27·6

21F

30·2

+22

M27·3

22F

25·6

22M

31·2

23F

28·7

23M

27·2

1/160

+24

M30·2

Noserum

24F

27·7

25M

27·3

25F

25·6

1/20

26M

28·2

26M

26·1

1/40

27F

25·0

27F

26·7

28M

26·5

1/160

28M

26·7

29M

25·6

1/40

29F

29·1

1/640

+30

M25·6

30M

27·3

31M

28·0

32F

28·6

33F

30·2

34M

27·5

35F

26·2

1/80

36F

24·0

1/640

37M

21·0

38F

25·4

1/160

39F

29·0

1/160

+40

M28·8

1Allseraweretested;titresofrespondersareshown;2+=macroscopicLDobserved;3serumlostintransit.

their antibody response to Vibrio anguillarum, although there were di#er-ences in gross pathology and histopathology (Bucke & Dixon, 1992). Likewise,the titres of antibody against Aeromonas salmonicida in dab exposed tosewage sludge were not significantly di#erent from those in control fish, but asignificantly greater proportion of the sewage sludge exposed fish had lownumbers of antibody secreting cells (Secombes et al., 1992). The observedchange in the present study was in the last six months of a three-year exposureperiod. That may have been because there was a slow onset of immunosup-pression during the chronic exposure to pollutants, or because of other, as yetunknown, factors.

Table 3. Summary of antibody titre data

Sampling time Mesocosm 1 Mesocosm 2 Mesocosm 3

Numbers of fish Nov 1992 25 25 21May 1993 28 40 22

Mean titre Nov 1992 69·2 (51·2) 33·2 (25·4) 101·0 (44·3)(S.E. in brackets) May 1993 63·2 (31·8) 48·5 (22·8) 3·6 (3·6)

Proportion of responders Nov 1992 0·36 (0·10) 0·40 (0·10) 0·33 (0·11)(S.E. in brackets) May 1993 0·39 (0·09) 0·22 (0·07) 0·05 (0·05)

Mean of responder titres Nov 1992 192·2 (137·5) 83·0 (62·0) 302·9 (97·1)(S.E. in brackets) May 1993 160·9 (73·3) 215·6 (82·3) 80·0 (")

Table 4. Analysis of Deviance of fitted models

Source df. Deviance Probability

Treatment+Sampling Occasion 5 13·10 2·4Treatment adjusted for Sampling Occasion 2 4·56 10·2Sampling Occasion adjusted for Treatment 1 3·61 5·8Treatment#Sampling Occasion Interaction 2 5·14 7·6Sex 1 0·11 74·4Length 1 0·88 34·9Residual 154 181

Table 5. Separate Analysis of Deviance foreach Sampling Occasion

Source df. Deviance Probability

Nov 1992Treatment 2 0·4 89·4

May 1993Treatment 2 19 0·9

130 P. DIXON ET AL.

There was no correlation between the presence of LDV antibodies and thepresence of macroscopic LD in the flounder in the mesocosms. Presence ofantibody but absence of macroscopic LD may have been because: 1) the fishwere recovering from LD but still had circulating antibody; or 2) the fish mayhave been in the early stages of infection with the virus, or manifesting asecondary response but without visible hypertrophied cells at the time ofblood sampling. Presence of macroscopic LD but absence of specific antibodymay have been because: 1) the antibody response was below the level ofsensitivity of the assay or had not yet developed; 2) immune complexes mayhave formed and depleted the serum of detectable antibody or; 3) the fishmay have been immunosuppressed.If the flounder had been immunosuppressed because of the pollutants, the

question arises as to what consequence that would have had for the fish inrelation to LD and to other pathogens. The possibilities are that: 1) therewould have been no e#ect on the fish; or 2) the fish would have had loweredresistance to pathogens. This has been discussed in relation to LD byLorenzen (1992), who postulated that immunosuppression would lead todelayed recovery (LD being a benign infection, rather than causing highmortality), which in turn would increase the prevalence of LD in thepopulation. However, if LD did induce mortality as a result of immunosup-pression, infected fish would tend to be removed from the population andhence reduce the prevalence. At the November 1992 sampling time, 36% of thefish in Mesocosm 3 exhibited gross LD, compared to 22·7% in May 1993.Although di#erent individual fish were sampled on each occasion, the preva-lence of gross LD at the population level in Mesocosm 3 reduced between 1992and 1993. This may have been caused by the cyclic nature of the disease; botha seasonal and an annual variation of LD frequency in flounder has beenobserved previously (Reiersen & Fugelli, 1984). Hence the prevalence of bothantibody-positive flounder and LD-positive flounder declined over the twosampling periods; according to Lorenzen (1992), that would occur if immuno-suppression of the fish resulted in an increased mortality. Immunosuppressionthus far has only been considered in relation to the ability of flounder torespond to LD; it is likely that a decreased ability to respond to other, morelethal pathogens may have more serious consequences for the fish.Antibody is not the only component of the host defence system to regulate

virus infections. However there are few published reports on the cellularimmune response of fish to LD. Interestingly, Faisal (1989) reported that LDitself in the gilthead sea bream, Sparus aurata L., reduced non-specificcytotoxicity and proliferative responses of leucocytes to mitogens andlipopolysaccharides. A histopathological study showed the involvement ofnon-specific and specific components of the cellular response of flounder andplaice, Pleuronectes platessa L., to LD (Russell, 1974).The ELISA for the detection of flounder antibodies against LDV was

developed after the mesocosm experiment had begun. As serum samples werenot taken from fish at the early sampling periods, it was not possible tomeasure the antibody levels throughout the course of that experiment.Nevertheless, the results of this pilot study have suggested that it would bevaluable to include LDV antibody detection in future work to add to the

DETECTION OF LYMPHOCYSTIS ANTIBODIES 131

biological and chemical data obtained from each fish. Studies on the cellularimmune response of the fish should also be done. It is recognised that manycomplex interactions will have to be analysed. In order to facilitate analysis ofthose interactions, it is essential that the immunocompetence of the fish ismeasured throughout the study, and a greater number of fish sampled. Ideally,the fish should be tagged to enable monitoring of individual fish. However,practical problems do arise because of the nature of the mesocosms them-selves; the size of the basins, and the type of substrate makes it impossible tocatch all the fish at interim sampling times unless the basins are completelydrained. That act would add a further variable to the study. Interim sub-samples of the populations can be taken, but as the same fish may not becaught on successive sampling occasions, there would be an incomplete recordof the change (if any) in the immune status of individual fish. Nevertheless,future studies will be designed to obtain the maximum information.

This article is Crown copyright.

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132 P. DIXON ET AL.

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