Precipitins in the serum of the atlantic salmon

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 4, pp. 641-651, 1980 0145-305X/80/040641-ii$02.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.

PRECIPITINS

IN THE SERUM OF THE ATLANTIC SALMON

J. B. Alexander, Department of Biology, University of Salford, Annexe, Salford, M6 6WZ, U.K.

Gardner

Street

ABSTRACT Sera were collected from Atlantic salmon from rivers and estuaries in Northwest England, Northern Ireland, Wales and Southwest England in the years 1966 to 1975, during an outbreak of Ulcerative Dermal Necrosis (UDN), and examined against extracts of diseased tissue (DTE) and amylopectln (Ap) by gel diffusion and single radial Immunodlffuslon (SRID). From 1966 to 1970 the percentage of salmon from fresh water containing preclpltlns to DTE increased to almost 100% and remained at that level. The majority of sera gave a preclpltln llne with Ap. In SRID with I% Ap in the agar, two preclpltln rings occurred: an outer, diffuse and an inner, dense ring. The dominant preclpltln reacting with DTE is not an Immunoglobulln but an antlbody-mlmetlc protein called ~(-preclpltln.

INTRODUCTION In 1964 ulcers were reported on the skin o£ adult Atlantic salmon (Salmo salar), returning to the rivers of southwest Ireland. Similar lesions were seen in Lancashire and Cumbrla in 1965 and later in the rest of the British Isles. These ulcers are referred to as Ulcerative Dermal Necrosis (UDN). There is a high mortality and the ulcers rapidly become colonlsed by bacteria, especially Aeromonas and Pseudomo~as, and fungi particularly Saprolegnla. No satisfactory aetlology has been proposed. The sera of Atlantic salmon contain two classes of preclpltlns which react with extracts of diseased salmon tissue [I] and o£ 641

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PRECIPITINS IN SAI/~ON

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fungi, especially Saprolegnla [2,3,4,5]. The predominant preclpitln after isolation appears to be an a(-globulln with a molecular weight of 170,000 [6]. It is not an Immunoglobulin and the name O~-preclpitln has been proposed. Recently, preclpltlns have been found in the serum of salmon and other fish which react with the components of starch, especially amylopectln [7,8]. Amylopectln is an O((I-¢4) polymer of glucose containing up to 4% @((I-96) linkages which results in a branched-chaln structure. This is the first antigen to be identified that reacts with o(preclpitin and has made possible the measurement of the concentration of this antibody-mlmetlc protein in serum. This paper reports the distribution of these preclpltins in salmon collected in the British Isles between 1966 and 1975 and the relative amounts of the preclpitins to amylopectln. MATERIALS

AND METHODS

Serum was collected from Atlantic salmon as previously described [7]. Diseased tissue from heavily infected salmon was homogenised in 0.5% NaCI, kept at 4°C for 24 h and centrifuged at 25,000 rpm for 60 mln. The supernatant was decanted and used as antigen (DTE). Amylopectln batch No 56140 (Koch-Light Lab Ltd) was used at a concentration of 5% in I% sodium azlde. Sera were examined for the presence of preclpltlns by the gel diffusion method of Ouchterlony [9J. The concentrations of preclpltlns to amylopectin were determined by single radial immunodlffuslon (SRID) with I% amylopectin incorporated in the agar instead of antiserum. A pooled salmon serum (SP14) was made from 45 male and female flsh caught at Broadralne in 1973 and was used as a refere nce.

RESULTS Preclpltlns

to DTE.

The results for fish caught at sea and in the t i d a l reaches of rivers are summarlsed in Table I. An examination of fish caught off the coast of Northern Ireland revealed 2 negatives out of 344. All sera from fish caught in the tidal reaches of rivers were positive with the exception of 16 out of 57 fish from the Foyle in 1969. A total of 989 serum samples were collected in the non-tldal reaches of rivers in Great Britain and N Ireland. Of these 869 came from the Rivers Lune, Kent, Leven, Ribble, Hodder and Wyre in Northwest England; the remalnin~ 120 were collected in Devon (44), Cornwall (19), South W a l e s ~ 1 9 ) and N Ireland (38). In NW England, samples were available from 1966 to 1975 and the majority were collected from hatcheries at Broadralne on the River Lune and Langcllffe on the Ribble usually during the months of November, December and January (Winter fish). The numbers and percentages of positive male and female Winter f i s h were analysed for each year (Table II).

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PRECIPITINS IN SALMON

643

TABLE I. Precipitlr~ to Diseased Tissue Extracts in the Sera of Salmon caught in the Sea and Tidal Reaches of Rivers. Enviromme nt Locality

Disease

Marine

Greenland

Clean

Northern Ireland

Clean

Estuary

Upper Tidal

Devon NW England

Year

Pre cipi tlms + - %+

1969 1970 1971 Total

161 94 89 344

Total Clean Diseased

Devon Foyle

Clean

Ban

Clean

Total

I(I?) 4

19b9/70 1975 Total

6

2 2

100 100 98 99

161 94 91 346

11 22 33

0 0 0

IO0 I00 I00

11 22 33

2 2 21 100 250 13 388

0 0 0 16 0 0 16

100 100 100 86 100 100 96

2 2 21 116 250 13 404

Clean Clean

NW England

Atlantic

0 0

TABLE II. Preciptims to Diseased Tissue Extracts in the Sera of Diseased Atlantic Salmon caught during the Autumn and Winter in the Nontidal Sections of the Rivers Lune, Kent, Leven, Hodder, Ribble and Wyre analysed for Year of Collection and Sex. Year 19661967 1968 1969 1970 1971 1972 1973 1974 1975 Totals *

Females

Males

+

-

%+

+

_

3 26 62 54 56 53 29 10 9 14

3 11 I I 2 1 0 0 0 0

50 70 98 98 97 98 100 100 100 100

12 26 30 53 33 67 104 102 35

16 4 0 I 3 0 I 0 0

316

19

95

462

25

Total %+

+

_

43 87 100 98 92 100 99 100 100

3 42 88 84 109 86 96 114 111 49

3 28 5 I 3 4 0 I 0 0

100 99 100 100

95

782

45

95

the time of collection believed to be the Autumn includes 5 sera for which the sex was not recorded

%+

50 60~ 95 99 97 96

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Only 8 of the Winter fish were recorded as 'clean' and all of these were positive. The remaining 34 samples from NW England were collected at different times of the year. The results from all areas were analysed for time of year and for place of collection (Tables III and IV). TABLE III. Preclpltlns to Disease Tissue Extracts in the Sera of Diseased Atlantic Salmon collected at Different Times of the Year (a) in Northwest England, (b) other Areas, (c) all areas, in the Nontidal Sections of Rivers. Time of year

+

-

%+

Spring Summer Winter

9 14 782

11 0 45

45 100 95

TOTAL

805

56

94

Spring Summer Wlnter

3 16 T7

8 2 14

27 89 85

TOTAL

96

24

80

Spring Summer Winter

12 30 859

19 2 59

39 94 94

TOTAL

901

80

92

(a) NW England.

(b) Other areas

(c) All areas

TABLE IV. Preclpltlns to Diseased Tissue Extracts in the Sera of Atlantic Salmon caught in the Non-tldal Reaches of Rivers analysed for Area of Collection. J J m m J m m J ~ m m J J m m J m J m j J m m m J J m J m m m ~ m m m ~ m m j J m m m J j m m m ~ m m m m m m m m m m

Place

+

27

-

%+

Devon CorrJ,all South Wales N Ireland NW England

17 15 37 813

17 2 4 I 56

61 89 80 97 94

TOTAL

909

80

92

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PRECIPITINS IN SALMON

645

TABLE V. The Numbers and Percentages of Atlantic Salmon collected in the Sea off the Coast of Northern Ireland with Preclpitins to Amylopectln in their Sera. Year

1969 1970 1971

Positive

%+

Strong NO

%

Weak NO %

7 24 66

21 77 96

18 7 3

53 23 4

Negative NO

64 100 100

9

%

26

0 0

FIG I. Gel diffusion of salmon sera against amylopectin (Ap) and diseased salmon tissue extracts (DTE). N.B. Salmon sera SSI, SS2, SS3 and SS4 gave strong multiple lines against DTE and a weak or no line against Ap. SS5 and SS6 gave a precipitin line with Ap and only weak lines with DTE. Precipitins to Am~lopectin Starch is a mixture of 2 polymers of glucose - amylose and amylopectln. In both glucose subunits are linked O ( ( I - ~ ) but in amylopectin up to 4% ~ ( I - ~ 6 ) linkages occur. This results in a branched structure. Both of these react with o&-precipitin but amylopectin gives stronger lines. Sera were collected from 781 salmon in NW England and challenged with amylopectin. Good precipitin lines occurred in 771, 5 showed weak but distinct reactior~ and 5 showed no reaction. No precipitln lines with DTE were observed in 28 of these sera. Some sera which gave strong reactions and produced multiple precipitin lines with DTE, gave weak or negative

646

reactions true.

PRECIPITINS

with

amylopectin

IN SALMON

(Figure

I).

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The converse was also

Sera from 137 marine fish caught at Torr Head and Carnlough were examined (Table V). Samples which were negative with amylopectln were positive with DTE, both tests being performed on the same plates. One of the two marine sera from 1971 which showed a negative reaction with DTE gave a weak line with amylopect in. An additional 351 sera were examined; 303 from the River Foyle, 10 from Devon, 26 from N Ireland and 12 from C o r ~ a l l . Seventeen of 53 fish from the Foyle in 1970 produced a strong precipitin line whilst the remainder showed weak reactions. Of the 250 collected in 1975, 91% gave a strong preclpltln line and the remainder were weak. In the Devon samples, 8 were strong and 2 weak. Whereas 6 gave no llne with DTE. All the Cor~all samples (12) were positive.

~'

i'

},

,i

FIG 2. Single Radial Immunodiffuslon of Salmon Sera. The Agar contained I% Amylopectln. Note the Inner D e ~ e Rings and the Outer Diffuse OneS.

Single Radial Immunodlffuslon. Salmon serum examined by SRID showed 2 rings of precipitation, an inner, dense one and an outer, more diffuse ring (Figure 2). The concentration of the outer, more diffuse ring differed in males and females. In general, the concentration in females was about half that in males. The concentrations fluctuated from year to year but there was a gradual increase in both sexes.

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PRECIPITINS IN SALMON

U

647

I

I

l

t

I

!

I

i

I IM

I 70

I ?!

I PI~

I P'3

I P'l,

M

TF~Rm OF COt.LECTION

FIG 3. Graph of the variations in the means of the concentrations of the inner and outer rings found in male (x) and female (o) Winter fish from Northwest England between 1967 and 1975. TABLE VI. Concentration of Outer Ring Precipitins to Amylopectin in the Serum of Atlantic Salmon as a Percentage of the Concentration in SP14. Measured by Single Radial Immunodiffuslon. 1967

1968

1969

1970

1971

1972

1973

1974

1975

WINTER FISH FROM NW ENGLAND Females Mean SE Number

27.9 7.9 15

34.9 3.4 47

32.6 5.7 33

Males Mean SE Number

67.6 11.5 12

96.9 11.3 26

54.U 104.5 10.4 7.3 28 52

65.6 6.7 41

45.2 7.0 42

65.8 35.3 45.8 15.0 7.11 8.9 10 7 14

62.1 117.2 104.2 ~9.0 103.2 4.4 5.8 4.4 4.5 5.1 14 54 102 81 34

MARINE SUMMER FISH FROM N IRELAND Mean SE Number

65.1 11.5 14

213.5 237.4 202.9 8.0 8.7 5.1 30 59 72

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TABLE VII. Concentration of the Inner ring Preclpltins to Amylopectln in the Serum of the Atlantic Salmon as a Percentage of the Concentration in SP14. Measured by Single Radial Immunodiffusion.

1967

1868

1969

1970

1971

1972

1973

1974 1975

WINTER FISH FROM NW ENGLAND Females Mean SE Number

52 13.4 15

127 10.1 48

70 106 176 190 224 294 95 6.8 6.9 8.3 17.4 34.8 55.2 15.3 33 45 42 14 10 8 14

92 13.9 12

107 14.1 26

77 121 143 200 222 377 101 6.1 6.0 21.4 6.8 6.5 22.4 7.6 28 52 14 54 102 82 34

Males Mean SE Number

MARINE SUMMER FISH FROM N IRELAND Mean SE Number

204 196 372 14.7 11.6 17.3 29 59 72

The changes in the inner ring were more pronounced. Between 1967 and 1970, the concentration of the inner ring fluctuated between 52% and 105% for female fish and 77% and 120% in males. From 1969 the values increased each year until in 1974 they reached 376% for males and 296% in females. A similar increase occurred in marine fish between 1970 and 1971. In both estuarlne and freshwater fish for 1975, there was a return to the pre-1971 value. These results were obtained by straight llne extrapolation and greatly exceed the standards and probably only place the sera in order of concentration. A plot of the sq~sre of the diameter of each ring against the dilution of pooled serum gave a straight line after 24 h incubation for the inner ring, and after 48 h for the outer. Concentrations of the rings were measured in the sera of fish collected in the winter at Broadralne and Langcliffe from 1967 to 1975, in the summer off the coast of N Ireland and in the estuary of the River Foyle. The means and standard errors of the estimates were calculated for both sexes, for each year (Tables VI and VII). They are shown graphically in Figure 3. The concentrations of the inner and outer rings in the sera were compared by one-way analysis of variance and there was 49% correlat ion.

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PRECIPITINS

IN SALMON

649

DISCUSSION The dominant preclpltln which reacts wlth DTE In the Atlantic salmon Is not an immunoglobulln but an antlbody-mimetlc protein called 0(-preclpltln [2,~]. The precipitate formed is not soluble In EDTA and Is, therefore, not C-reactlve protein. 'Clean' and diseased flsh both gave negative results. In most positive cases more than one precipltln line was formed wlth DTE. Many of the flsh which were negative to DTE were positive with amylopectln and vlce versa. This may indicate that the reactions were specific and that amylopectln and the antigens of DTE reacted with different preclpltlns. only one of the six Greenland specimens was positive. This was a low proportion compared wlth the other marine groups. These fish were caught off the coast of Greenland In 1967 by the Ministry of Agriculture, Fisheries & Food and could have originated from any of the North Atlantic countries. Perhaps variations among the fungal populations within these different areas influenced the results. However, the possibility of loss of activity due to storage during transit cannot be overlooked. Wlth 2 exceptions, all flsh caught off the coast of Northern Ireland gave preclpltln lines wlth DTE. Of these, 26% (9) of the flsh sampled In 1969 failed to react wlth amylopectin. As wlth the Greenland sera, these negative results could have been due to poor storage as the samples had been frozen and thawed at least once.

In the non-tldal reaches of rivers In NW England, the proportion of salmon wlth preclpltlns varied from year to year and between sexes. When UDN first occurred In thls area, the proportion of flsh wlth precipltlns to DTE was 50%. In females the percentage increased and by 1968, over 98% were positive. In males, the percentage positive dld not exceed 95% until 1969. Since then both have fluctuated slightly, with the highest rLumber of negative flsh In any one year being four. Throughout the year In NW England, flsh taken from non-tldal sections of rivers In the spring showed the highest proportion of negative results, followed by those collected In the winter. All Summer flsh were positlve. The flsh which failed to glve preclpltin lines were also diseased. If the preclpltln has a protective role, Its reduction In badly diseased animals could be expected. However, the sera of these flsh still contained small amounts of C~-preclpltln [6]. There are at least two explanations for an increase In the inner r i n g component revealed by the SRID plates. Firstly, it may be an induced effect. The increase began about 1969 and flsh returning to spawn at thls tlme and later would have been In the rivers as parr w h e n UDN first occurred. Those returning after 1971 could have spent the whole of their river stage In the presence of UDN. They could, therefore, have been In contact wlth the antigens of UDN and thls could have stimulated production of more precipltln. Thls behavlour resembles an

650

PRECIPITINS IN SALMON

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immune r e s p o ~ e with the induction of specific preclpitlns. Alternatively, the change may have been due to genetic selection. When the disease first occurs in a river a high percentage o£ the adult fish die. I£ the preclpitln confers an advantage on the animal, then one could expect selection for fish producing large amounts of this protein. UDN first occurred in 1965 to 1966 in NW England and N Ireland and the majority of the offspring (FI) from these animals would have returned in 1971 and 1972. In both areas the major increase in concentration coincides with this return. This may indicate that the second hypothesis is the more likely. However, it does not explain the dramatic fall in 1975, which occurred in both estn~rine and freshwater animals, and is not explained by either hypothesis. It is possible that future annual measurements may resolve this question. Although the immunological role of ~ preclpitin is not clear, it is involved in the response of salmon to disease.

I would like to thank Professor W.E. Kershaw, CMG, VRD, MD, DSc, formerly Chairman of the Department of Biology for the excellent facllltles he provided. I wish to thank Mr G.C. Shearer, MRCVS, and Professor E.L. Cooper for their helpful criticism of the manuscript. I also gratefully acknowledge the assistance and facilities given by past and present employees and riparian owners of the Northwest Regional Water Authority, the Southwest Regional Water Authority, the former Department of Agriculture (Northern Ireland), the Foyle Fisheries Commission, the Severn Regional Water Authority, and the Welsh National Water Development Authority. Thanks are also due to Mr D. Smythe formerly of Carnlough and the McNeal Brothers of Torr Head for permission to sample at their bagnets and to the late Mr A. Noble for samples from the Bann Fishery. REFERENCES

I. ALEXANDER, J.B., WILSON, J.G.M. and KERSHAW, W.E. respor~e in salmon. J. Fish Biol., R, 348, (1970).

Immune

2. HODKINSON, M. and HUNTER, A. Immune response of U.D.N.infected salmon to Saprolegnla. J. Fish Biol., 2, 305, 1970. 3. HODKINSON, M. and HUNTER, A. The effect of culture media on Saprolegnla antigens. Mycopath. Mycol. appl., 43, 347, 1971. 4. HUNTER, A. Fungi and their antibodies thesis, Unlverslty of Salford. 1976.

in

salmon.

Master's

5. WILSON, J.G.M. Immunological aspects of fungal disease in fish. In: Recent Advances in Aquatic Mycology. E.B. Gareth Jones (Ed.), L o n ~ , Elek S c l e n ~ . 1976. P.573.

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6. DAVIES, D.H. The isolation and characterization of the light 'antibody' from Atlantic salmon (Salmo salar). Doctoral thesis, University of Salford. 1975. . ALEXANDER, ~Salmo salar) thesls,

J.B. The serum proteins of the Atlantic salmon with special reference to preclpltins. Doctoral University of Salford. 1976.

8. AL-SALMON, H.A.M. The antigens of fungi associated with salmon disease. Master's thesis, University of Salford. 1976. 9. OUCHTERLONY, O. In vitro method for testing producing capacity of diphtheria bacteria. Acta. ~ath. microbiol. S cand., 25, ]86, 194~.

the

toxin-