Fish & Shellfish Immunology (2001) 11, 523–535
doi:10.1006/fsim.2000.0333
Available online at http://www.idealibrary.com on
Humoral immune parameters of cultured Atlantic halibut
(Hippoglossus hippoglossus L.)
SIGRUN LANGE*, BJARNHEIÐUR K. GUÐMUNDSDOTTIR AND BERGLJOT MAGNADOTTIR
Institute for Experimental Pathology, University of Iceland, Keldur,
IS-112 Reykjavík, Iceland
(Received 27 September 2000, accepted 21 December 2000)
Several humoral immune factors were studied in a group of cultured halibut
(Hippoglossus hippoglossus L.). The serum protein and IgM concentration
was comparable to levels seen in other teleost species. A strong antibody
activity against TNP-BSA was observed but not against other antigens tested.
Lysozyme and anti-protease activity was detected and showed variable heat
sensitivity. Unlike the anti-protease activity, the lysozyme activity of the sera
was not sensitive to storage at 20 C. No spontaneous haemolytic activity
was observed and the sera had no bactericidal e#ect on any of the bacterial
strains tested. Iron binding capacity of the sera was high. Individual variation
was considerable in all the factors tested.
2001 Academic Press
Key words:
anti-protease, complement, halibut (Hippoglossus hippoglossus
L.), immunoglobulin, iron, lysozyme, natural antibodies.
I. Introduction
Although vaccination is the most e$cient method of controlling persistent
pathogens associated with aquaculture, recent studies have shown that
the use of non-specific stimulants, which enhance elements of the innate
immune system, can lead to increased protection against di#erent pathogens
and improved larval survival (Austin & Austin, 1987; Ellis, 1988; Press &
Lillehaug, 1995; Dalmo et al., 2000).
The non-specific defence system provides a first line of defence against
pathogen attack and plays a vital role in preventing the establishment of
infection. The non-specific defence does not alter on repeated exposure and
consists of physical barriers, humoral and cellular factors. These defence
mechanisms can be divided into four distinct groups according to their mode
of action: microbial growth inhibitors, enzyme inhibitors, lysins, agglutinins
and precipitins (Alexander & Ingram, 1992; Dalmo et al., 1997). A high level of
non-specific antibodies is sometimes seen in fish serum, commonly showing
anti-TNP/DNP, anti-DNA or anti-Thyr activity (Vilain et al., 1984; Gonzalez
et al., 1988; Marchalonis et al., 1993). In some species of fish, persistent
pathogens or adverse conditions may induce a high level of non-specific
antibodies (Michel et al., 1990). In other species, such as cod, these natural
*Corresponding author. E-mail: [email protected]
1050–4648/01/060523+13 $35.00/0
523
2001 Academic Press
524
S. LANGE ET AL.
antibodies seem to be a natural parameter of the immune defence system
(Magnadottir et al., 1999a,b).
Halibut is a recent and promising species in commercial aquaculture.
Studies have been carried out on its cultural requirements, its susceptibility to
common fish pathogens and protection by vaccination or other measures
(Mangor-Jensen et al., 1998; Bricknell et al., 1999, 2000; Bowden et al., 2000;
Dalmo et al., 2000). Studies of its immune system have been limited and the aim
of the present work was to take a closer look at several humoral immune
parameters of cultured halibut. These include immunoglobulin concentration,
non-specific antibody activity, haemolytic activity, lysozyme activity, antiprotease activity, iron binding capacity and bactericidal activity of serum.
Furthermore, the e#ect of temperature and storage on lysozyme activity and
anti-protease activity of the sera was tested.
II. Materials and Methods
THE FISH
Halibut (Hippoglossus hippoglossus L.), mean weight 1·43 (0·24) kg and
2 years of age were obtained from the Icelandic fish farm Fiskeldi Eyjafjardar
hf in Thorlakshofn, Iceland. The fish were cultured in a tank (diameter=9 m;
water depth=1·8 m) with a flow-through system, at 7 C and with a salinity of
32 ppt. Life mass in the tank was 48 kg m 3 and the halibut were fed
commercial food pellets (LAXÁ, Iceland). The fish group under study was the
first generation in culture, descending from 40 females. At the farm the fish
had been routinely tested for health status at 3-monthly intervals by the Fish
Disease Laboratory, Keldur, and were always declared disease-free and
healthy.
SAMPLING
Blood (1–3 ml) was collected from a vessel in the gills of 31 fish. The blood
was allowed to clot at room temperature for 2 h and then overnight at 4 C.
Serum was collected after centrifuging at 750g for 10 min and stored in
aliquots at 20 C.
PROTEIN MEASUREMENTS
A protein assay kit from Pierce, U.S.A., was used for protein estimation
following the manufacturer’s instructions. The kit was based on the analytical
method described by Bradford (1976) with bovine serum albumin (BSA, Sigma,
U.S.A.) as the standard protein.
IgM ISOLATION
Pooled sera from three individuals were used for IgM isolation. IgM was
purified as described by Magnadottir (1990, 1998). The method involved
chromatography of serum on a CM A$ Gel Blue column (Bio-Rad, U.S.A.),
using the FPLC system from Pharmacia (Denmark). Unbound fractions were
HUMORAL IMMUNE PARAMETERS OF ATLANTIC HALIBUT
525
collected, proteins precipitated by ammonium sulphate and analysed by gel
filtration (Suprose 6, Pharmacia) followed by ion exchange chromatography
(MonoQ, Pharmacia). Fractions were tested for IgM by Western blotting using
polyclonal anti-halibut IgM and purity was checked by SDS-PAGE and silver
staining. To estimate the concentration of purified IgM, the optical density at
280 nm was measured and the concentration calculated with the equation:
1 mg IgM ml 1 gives an O.D. reading of 1·1 at 280 nm (Magnadottir et al.,
1999a).
POLYCLONAL ANTI-IgM (pAb) PRODUCTION
Polyclonal antibodies against halibut IgM were prepared in mouse ascitic
fluid according to the method of Overkamp et al. (1988). The pAb titre was
determined by indirect ELISA (Maloy et al., 1991) and the titre was set as the
reciprocal value of the highest dilution that gave 2–3 times the O.D. reading of
the bu#er control. The pAb preparation used in this paper had a titre of
425 000.
IMMUNOGLOBULIN (IgM) CONCENTRATION
The total immunoglobulin (IgM) concentration in 27 halibut serum samples
was measured using a competitive ELISA (Magnadottir & Gudmundsdottir,
1992). Microtrays (96 wells) were coated with halibut IgM (10 g ml 1, 100 l
well 1) in a coating bu#er (0·1 M sodium carbonate bu#er, pH 9·6, Sigma).
Residual sites were blocked with 1% gelatine in the coating bu#er (100 l
well 1) for 1 h at room temperature. Halibut serum, in two-fold serial
dilutions (10 2 to 10 5, 50 l well 1), was incubated for 1 h at 37 C with
polyclonal mouse anti-halibut IgM antibodies (50 l well 1), diluted in PBSTween (PBS made from tablets, Sigma, 0·1% Tween added) so that the
antibody was not in excess, based on the titration curve. Bound antibody was
detected with alkaline phosphatase-labelled goat anti-mouse Ig (100 l well 1,
Dako Denmark) in PBS-Tween. Between these steps the microtitre plates were
washed with PBS-Tween. The substrate was p-nitrophenyl phosphate (100 l
well 1, Sigma), the stop solution was 3N NaOH (50 l well 1) and O.D. was
read at 405 nm. For plotting of a standard graph each plate contained purified
halibut IgM dilutions, in place of the serum, and the IgM concentration v. O.D.
at 405 nm was plotted. The serum IgM concentration of each sample was
interpolated from the standard graph and the mean calculated.
NON-SPECIFIC ANTIBODY ACTIVITY
The antibody activity of 30 halibut serum samples against six antigens was
measured using an ELISA test described by Magnadottir & Gudmundsdottir
(1992). The antigens used were TNP-BSA (trinitrophenyl-bovine serum
albumin, containing 21 TNP residues per BSA molecule, prepared at our
laboratory), ssDNA (single-stranded DNA from calf thymus, Sigma),
thyroglobulin (bovine, Sigma), BSA (bovine serum albumin), LPH (Limulus
polyphemus haemocyanin, Sigma) and glycogen (from Mytilus edulis, Sigma).
526
S. LANGE ET AL.
Microtrays were coated with 100 l well 1 of the antigen diluted to 10 g ml 1
in the coating bu#er, except TNP-BSA, which was diluted to 5 g ml 1 and
ssDNA which was diluted to 20 g ml 1. Blocking of residual sites was
performed with 1% gelatine in coating bu#er for 1 h at room temperature.
Sera were tested in the dilution 10 2 (50 l well 1) and incubated overnight
at 4 C. Mouse polyclonal anti-halibut-IgM antibodies (50 l well 1, incubated
for 1 h at 37 C), followed by alkaline phosphatase-conjugated goat anti-mouse
Ig antibodies (50 l well 1, incubated for 1 h at 37 C), were used for the
detection of bound halibut antibodies. Between steps the microtrays were
washed with PBS-Tween. The substrate was p-nitrophenyl phosphate (100 l
well 1), the stop solution was 3N NaOH (50 l well 1) and O.D. was read at
405 nm.
LYSOZYME ACTIVITY
The lysozyme activity of 30 sera was measured using a method based on the
ability of lysozyme to lyse the bacterium Micrococcus lysodeikticus (Ellis,
1990b). Preliminary work involved the determination of the optimum pH
(pH 6·2) and incubation temperature (22 C). On a 96-well microtray, 100 l of
serum in four two-fold serial dilutions (1/5–1/40) in phosphate bu#er (0·05 M,
pH 6·2) was mixed with 100 l of a 0·4 mg ml 1 suspension of Micrococcus
lysodeikticus (Sigma) in phosphate bu#er. The microtray was incubated at
22 C and O.D. was read at 590 nm at 0, 15, 30 and 60 min. For a positive control,
serum was replaced by Hen Egg White lysozyme (ICN, U.S.A., serial dilutions
starting at 1·6 g ml 1) and for a negative control, bu#er replaced serum.
A unit of lysozyme activity was defined as the amount of serum causing a
decrease in the O.D. reading of 0·001 min 1.
The influence of temperature on lysozyme activity was tested by incubating
10 sera at 22, 37, 45 and 56 C for 30 min, prior to the assay. The e#ect of
storage on lysozyme activity was tested by repeating the assay on 30 sera after
storing at 20 C for 5 months.
ANTI-PROTEASE ACTIVITY
The anti-trypsin activity of 29 sera was measured according to the method
described by Ellis (1990a), with some modifications (Magnadottir et al., 1999a).
Twenty microlitres of the test serum were incubated with 20 l of standard
trypsin solution (Sigma T-7409, 1000–2000 BAEE, 5 mg ml 1) for 10 min at
22 C. Then 200 l of 0·1 M phosphate bu#er, pH 7·0, and 250 l 2% azocasein
(Sigma A-2765) were added and incubated for 1 h at 22 C. Then 500 l of 10%
trichloro acetic acid (TCA) were added and incubated further for 30 min at
22 C. The mixture was centrifuged at 4000g for 5 min and 100 l of the
supernatant was transferred to a 96-well non-absorbent microtray (Nunc,
Denmark) containing 100 l of 1 N NaOH well 1. The O.D. was read at 430 nm.
For a 100% control, bu#er replaced the serum and for a negative control,
bu#er replaced both serum and trypsin. The percentage inhibition of trypsin
activity by each serum sample was calculated by comparing it to the 100%
control sample. Heat sensitivity was measured by incubating five sera at 22,
HUMORAL IMMUNE PARAMETERS OF ATLANTIC HALIBUT
527
37, 45 and 56 C prior to the assay. The e#ect of storage on anti-protease
activity was measured by repeating the assay on 30 sera after storing at
20 C for 3 weeks and then after 5 months.
IRON BINDING CAPACITY
The total iron content (TI, g ml 1) and unsaturated iron binding capacity
(UIBC, g ml 1) of 19 halibut sera were determined using a kit (Sigma no. 565)
based on the method described by Persijn et al. (1971). The manufacturer’s
procedure was followed with the modifications that all volumes were reduced
by a factor of 10 (Langston et al., 1998). Total iron binding capacity (TIBC, g
ml 1) was calculated as the sum of TI and UIBC and the percent saturation
was calculated as (TI/TIBC)100.
HAEMOLYTIC ACTIVITY
The spontaneous haemolytic (SH) activity of 30 halibut sera (complement
activity by the alternative pathway) was measured using a method described
by Magnadottir et al. (2000) with the modification that the test bu#er was not
supplemented with EDTA. The target cells were red blood cells from sheep
(s-RBC), rainbow trout (rbt-RBC) or rabbit (r-RBC) and the reaction bu#er was
a complement fixation test bu#er containing Mg2+ and Ca2+ (prepared from
tablets, Oxoid, U.K.), with 0·1% gelatine (CFT-G). In brief, 0·5% suspensions
of s-RBC, rbt-RBC and r-RBC were prepared in CFT-G. The test was carried
out in 96-well ‘round bottom’ microtrays (Nunc) and the halibut serum (100 l
well 1, diluted 10 1 to 10 3 in CFT-G) was incubated with the di#erent RBCs
(50 l well 1) for 1 h at 4, 22 or 37 C, with occasional shaking. Then the tray
was centrifuged, the supernatant collected and the O.D. read at 405 nm. A graph
of O.D. values v. serum dilution was drawn and the SH50% calculated, i.e. the
dilution that gave 50% lysis. The test was carried out on fresh and unfrozen
sera and also on sera that had been stored at 20 C.
BACTERICIDAL ACTIVITY OF HALIBUT SERUM
Bactericidal activity of five halibut sera on six di#erent bacteria
(Aeromonas salmonicida spp. achromogenes, Aeromonas hydrophila, Yersinia
ruckerei, Escherichia coli, Bacillus subtilis and Lactobacillus sp.) was tested.
The bacterial strains were grown in tryptose broth (DIFCO, U.S.A.) and mixed
with 0·7% tryptose agar each (1 ml bacteria suspension in 9 ml tryptose agar).
Sterilized filter paper discs, soaked with halibut serum (5 l serum per disc)
were put on the Petri dish containing 1·2% tryptose agar and covered with the
mixture of bacteria suspension and 0.7% tryptose agar. The agar plates were
left at room temperature until confluent bacterial growth had formed in the
top-agar. The bactericidal e#ect would be indicated in plaques around the
filter paper discs. Commercial discs (Oxoid, U.K.) containing oxytetracyclin
and ampicillin were used as a control.
528
S. LANGE ET AL.
Table 1. Total protein (n=29) and IgM concentration (n=27) in halibut serum
Parameter
Total protein
IgM
%IgM
Mean
Standard deviation
Min–max
n
41·31 mg ml 1
2·31 mg ml 1
5·58%
9·73 mg ml 1
0·74 mg ml 1
2·07%
20–69 mg ml 1
0·8–3·8 mg ml 1
2·6–10·3%
29
27
27
2.5
Box Plot
2.25
2
405 nm
1.25
O.D.
1.75
1
1.5
0.75
0.5
0.25
0
BSA Thyroglobulin TNPBSA
ssDNA
LPH
Glycogen
Antigen
Fig. 1. The natural antibody activity of halibut serum. The results are expressed as box
plots where the median is the line within each box, the boxes indicate 25–75
percentiles, the tails 10–90 percentiles and the circles the extreme values (n=30).
III. Results
SERUM PROTEIN AND IMMUNOGLOBULIN (IgM) CONCENTRATION
The total protein content of halibut serum varied from 20 to 69 mg ml 1
(mean value about 40 mg ml 1). The immunoglobulin concentration varied
from 0·8 to 3·8 mg ml 1 which was 2·6 to 10·3% of the total protein content in
the serum (Table 1).
NON-SPECIFIC ANTIBODY ACTIVITY
High antibody activity was obtained against TNP-BSA (mean O.D.405 =1·962),
whereas the response against the other antigens was relatively low, as shown
in Fig. 1. Activity against BSA (mean O.D.405 =0·099), ssDNA (mean
O.D.405 =0·098) and glycogen (mean O.D.405 =0·135) was negligible, whereas some
activity was observed against thyroglobulin (mean O.D.405 =0·213) and LPH
(mean O.D.405 =0·567) (Fig. 1).
HUMORAL IMMUNE PARAMETERS OF ATLANTIC HALIBUT
500
529
Box
450
Lysozyme units ml
–1
400
350
300
250
200
150
100
50
0
22
37
45
Incubation temperature (°C)
56
Fig. 2. The e#ects of temperature on the lysozyme activity of halibut serum. The
median is the line within each box, the boxes indicate 25–75 percentiles, the tail
10–90 percentiles and the circles the extreme values (n=10).
LYSOZYME ACTIVITY
The mean lysozyme activity was 285·5 units ml 1 measured at room
temperature 2 days after bleeding of the fish. When sera (n=10) were
incubated at di#erent temperatures (22, 37, 45 and 56 C) before testing, a
decreasing lysozyme activity was observed with rising temperature. Thus, the
mean lysozyme activity was 372·6 units at 22 C, a slight reduction was seen at
37 C (370·6 units), a 30% reduction at 45 C (267·2 units) and a 60% reduction
at 56 C (148·0 units) (Fig. 2).
When the lysozyme assay was repeated 5 months later at room temperature
for 30 sera, the mean activity was 284·6 units ml 1.
ANTI-PROTEASE ACTIVITY
The mean anti-trypsin activity of 29 sera was 72·90% 2 days after the fish
was bled. When sera (n=5) were incubated at di#erent temperatures before
testing, a decreasing anti-trypsin activity was observed with increasing
temperature. Thus, the trypsin inhibition at 56 C was only 35% of the trypsin
inhibition at 22 C (Fig. 3).
When the test was repeated 3 weeks later, using sera stored at 20 C, the
anti-trypsin activity of the halibut sera was reduced by 36%, and 5 months
later it had reduced to 50% of the original value (Fig. 4).
IRON BINDING CAPACITY
The total iron concentration of halibut serum (n=19) ranged from 0·11 to
2·03 g ml 1, the unbound iron binding capacity and total iron binding
530
S. LANGE ET AL.
Percentage of trypsin inhibition
80
Box Plot
70
60
50
40
30
20
10
0
22
37
45
Incubation temperature (°C)
56
Fig. 3. The e#ects of temperature on the anti-protease activity of halibut serum. The
median is the line within each box, the boxes indicate 25–75 percentiles and the tail
10–90 percentiles (n=5).
capacity ranged from 2 to 11 g ml 1 and the percentage saturation was 1·35
to 27·6% (Table 2).
HAEMOLYTIC ACTIVITY
No spontaneous haemolytic activity was detected in halibut sera tested.
Neither incubation temperatures (4, 22 and 37 C), RBC type (sheep, rabbit,
rainbow trout) nor EDTA had any e#ect.
BACTERICIDAL ACTIVITY OF HALIBUT SERUM
The halibut sera showed no bactericidal activity against any of the bacteria
tested.
IV. Discussion
In the present study innate humoral parameters of cultivated halibut were
examined. The fish came from a cultivated stock of halibut, which was of the
same age (2 years) and similar size (approximately 1·5 kg) and was kept under
identical conditions in a single tank.
The total protein concentration of halibut serum was comparable to
the level seen in other flatfish like wild dab (Hutchinson & Manning, 1996)
and other teleost species like wild or cultured cod (Israelsson et al., 1991;
Magnadottir et al., 1999a,b) and free living or cultured salmonids (Olesen
& Jørgensen, 1986; Havarstein et al., 1988). The IgM concentration of
halibut serum was comparable to the IgM level seen in rainbow trout (Olesen
& Jørgensen, 1986) but lower than is generally observed in cod serum
(Magnadottir et al., 1999a,b). Considerably higher serum protein and IgM
HUMORAL IMMUNE PARAMETERS OF ATLANTIC HALIBUT
100
531
Box Plot
Percentage of trypsin inhibition
90
80
70
60
50
40
30
20
10
0
2 days
3 weeks
Time
5 months
Fig. 4. The e#ect of storage 20 C on the anti-protease activity of halibut serum. The
median is the line within each box, the boxes indicate 25–75 percentiles and the tail
10–90 percentiles (n=30).
Table 2. The total iron (TI), unsaturated iron binding capacity (UIBC), total iron
binding capacity (TIBC) and percentage iron saturation (% I satn) in halibut serum
(n=19)
Parameter
TI
UIBC
TIBC
% I satn
Mean
Standard deviation
Min–max
n
0·65 g ml 1
5·86 g ml 1
6·51 g ml 1
10·21%
0·47 g ml 1
1·51 g ml 1
1·51 g ml 1
6·96%
0·11–2·03 g ml 1
3·04–9·93 g ml 1
2·15–10·71 g ml 1
1·35–27·6%
19
19
19
19
values were seen in a previous study of halibut, which was of wild origin and
larger (4–5 kg) than in the fish used in the present study (Magnadottir, 1998).
This suggests that these parameters may be influenced by the size of the
halibut as has been observed in cod (Magnadottir et al., 1999b).
A relatively strong activity (O.D. values >1·25) against TNP-haptenated BSA
was observed, whereas the activity against the other antigens tested was low.
High anti-TNP activity has been observed in many fish species from di#erent
phylogenetic levels (Gonzalez et al., 1988; Marchalonis et al., 1993). The lack of
cross-reactivity with other antigens observed in the present study, which
indicates exclusive specificity for TNP, has been seen in other teleost species.
In chondrostean and elasmobranch species, on the other hand, cross-reactivity
with other antigens seems to be the general rule (Gonzalez et al., 1988). The
function of the anti-TNP antibodies is not known. In cod, which does not
produce significant amount of specific antibodies (Espelid et al., 1991;
Schrøder et al., 1992), there are indications that they may be activated and
532
S. LANGE ET AL.
involved in the first line of non-specific immune defence (Magnadottir et al.,
2000).
Both lysozyme and anti-protease activity were detected in halibut serum.
Lower values were obtained for the lysozyme activity of these halibut sera
than in a comparable study on wild dab (Hutchinson & Manning, 1986). The
heat sensitivity of lysozyme and anti-protease activity varied. Very slight
reduction in lysozyme activity was observed following incubation at 37 C and
about 30% reduction following incubation at 45 C. The anti-protease activity,
on the other hand, was more heat-sensitive and showed 30% reduction in
activity following incubation at 37 C and about 60% reduction following
incubation at 45 C. Similarly, the lysozyme activity of halibut serum was not
sensitive to storage at 20 C, contrary to the observation by Hutchinson &
Manning (1986) studying the lysozyme activity in dab serum. The anti-protease
activity of halibut serum was, however, sensitive to storage at 20 C,
showing about 40% reduction in activity following storage for 3 weeks at
20 C. After this period the rate of loss of activity slowed down considerably.
This is in agreement with the observations by Ellis (1987). The labile
component contributing to the anti-protease activity of fish serum is the
2-macroglobulin, whereas other components like antithrombin and antiplasmin are less heat-labile (Ellis, 1987; Alexander & Ingram, 1992; Barrett
& Starkey, 1973; Starkey & Barrett, 1982). These components probably contribute to the remaining anti-protease activity observed in the halibut serum
after prolonged storage at 20 C. Overall the anti-protease activity of fresh
halibut serum was similar to that of cod serum assayed after 6-month storage
at 20 C (Magnadottir et al., 1999a,b). This suggests a di#erent sensitivity to
storage by these two species. On the other hand, in a study by Bowden et al.
(1997), using a di#erent methodology from the present study, cultured halibut
sera had the highest value of 2-macroglobulin binding capacity in five
cultured fish species tested. Nevertheless, it may be premature to assume
that species containing lower levels of 2-macroglobulin are necessarily
any less able to defend against exogenous proteinases. In a study by Moyner
et al. (1993), no relationship could be found between 2-macroglobulin and
proteinase inhibition during a furunculosis challenge in Atlantic salmon.
The total iron content of halibut serum was relatively low or similar to the
levels observed in cod (Magnadottir et al., 1999a,b) and lower than in salmon
(Langston et al., 1998). The percentage saturation on the other hand was
higher than in cod, but not as high as in salmon (Langston et al., 1998). Again,
the individual variation in all the iron parameters measured was considerable.
The iron binding capacity of fish serum, attributed to its transferrin content,
can act as a growth inhibitor, withholding iron from certain iron-requiring
pathogens and thus delaying or hindering bacterial multiplication (Alexander
& Ingram, 1992). The relatively high iron binding capacity suggests that this
may be an e#ective parameter in the halibut serum in delaying or hindering
certain bacterial infections.
No spontaneous haemolytic activity was observed in these halibut sera.
This activity is attributed to complement activity of the alternative pathway
and is relatively high in fish compared with mammals (Sakai, 1992). The lack
of spontaneous haemolytic activity may be an inherent characteristic of
HUMORAL IMMUNE PARAMETERS OF ATLANTIC HALIBUT
533
halibut or caused by environmental factors. Another explanation could be
nutrition deficiency, similar to the depletion of serum alternative complement
pathway activity in gilthead seabream, caused by alpha-tocopherol and n-3
HUFA dietary deficiencies described by Montero et al. (1998).
Halibut serum did not have a bactericidal e#ect on any of the bacteria
tested. This was in spite of the relatively strong lysozyme and anti-protease
activity and the high iron binding capacity of the serum. This shows that other
components, e.g. complement factors, are required to kill or inhibit the growth
of these bacteria in vitro.
As mentioned above, all the parameters examined in this study showed
considerable individual variation. This was in spite of the homogeneity of the
group with respect to its origin, size and cultural conditions. Other studies
have shown that humoral immune parameters of teleosts may be influenced
by such factors as size, environmental temperature, nutritional conditions,
density and disease status of the fish (Alexander & Ingram, 1992; Magnadottir
& Gudmundsdottir, 1992; Montero et al., 1998; Magnadottir et al., 1999a,b).
Studies of this nature have not been carried out on halibut but, as mentioned
above, there were indications that some of the humoral parameters might be
influenced by the size of the fish. The group sampled in the present study was
not genetically homogeneous as about 40 females of wild origin contributed
eggs to the hatchery. Variations in immune parameters observed must therefore be primarily attributed to variations in the genetic makeup of the fish and
their inherent variable response to the environment. The considerable individual variations seen in the immune parameters examined in the present
study demonstrate the di$culties facing scientists searching for a suitable
humoral parameter to monitor fish health in aquaculture.
The authors would like to thank Birgir Kristjansson and the sta# at Fiskeldi
Eyjafjardar, Thorlakshofn, Iceland, for providing the fish and sampling facilities.
Thanks are also due to Eggert Gunnarsson, Keldur, for helping with the antibody
production.
References
Alexander, J. B. & Ingram, G. A. (1992). Noncellular nonspecific defense mechanisms of
fish. Annual Review of Fish Diseases 2, 249–279.
Austin, B. & Austin, D. A. (1987). Bacterial Fish Pathogens: Disease in Farmed and
Wild Fish, pp. 111–195. Chichester: E. Horwood Ltd.
Barrett, A. J. & Starkey, P. M. (1973). The interaction of 2-macroblobulin with
proteinases. Biochemical Journal 133, 709–724.
Bowden, T. J., Butler, R., Bricknell, I. R. & Ellis, A. E. (1997). Serum trypsin inhibitory
activity in five species of farmed fish. Fish & Shellfish Immunology 7, 377–385.
Bowden, T. J., Lester, K., MacLachlan, P. & Bricknell, I. R. (2000). Preliminary study
into the short term e#ects of adjuvants on Atlantic halibut (Hippoglossus
hippoglossus L.). Bulletin of the European Association of Fish Pathologies 20,
148–151.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein dye binding. Analytical
Biochemistry 72, 248–254.
Bricknell, I. R., Bowden, T. J., Bruno, D. W., MacLachlan, P., Johnstone, R. & Ellis,
A. E. (1999). Susceptibility of Atlantic halibut, Hippoglossus hippoglossus (L.) to
infection with typical and atypical Aeromonas salmonicida. Aquaculture 175,
1–13.
534
S. LANGE ET AL.
Bricknell, I. R., Bowden, T. J., Verner-Je#reys, D. W., Bruno, D. W., Shields, R. J. &
Ellis, A. E. (2000). Susceptibility of juvenile and sub-adult Atlantic halibut
(Hippoglossus hippoglossus L.) to infection by Vibrio anguillarum and e$cacy of
protection induced by vaccination. Fish & Shellfish Immunology 10, 319–327.
Dalmo, R. A., Ingebrigsten, K. & Bøgwald, J. (1997). Non-specific defence mechanisms
in fish, with particular reference to the reticuloendothelial system (RES).
Journal of Fish Diseases 20, 241–273.
Dalmo, R. A., Arild, A. K., Arnesen, S. M., Tobias, P. S. & Bøgwald, J. (2000). Bath
exposure of Atlantic halibut (Hippoglossus hippoglossus L.) yolk sac larvae to
bacterial lipopolysaccharide (LPS): absorption and distribution of the LPS and
e#ect on fish survival. Fish & Shellfish Immunology 10, 107–128.
Ellis, A. E. (1987). Inhibition of the Aeromonas salmonicida extracellular protease by 2
macroglobulin in the serum of rainbow trout. Microbial Pathogenesis 3, 167–177.
Ellis, A. E. (1988). General principles of fish vaccination. In Fish Vaccination (A. E.
Ellis, ed.) pp. 1–19. London: Academic Press.
Ellis, A. E. (1990a). Serum antiproteases in fish. In Techniques in Fish Immunology
(J. S. Stolen, T. C. Fletcher, D. P. Anderson, B. S. Roberson & W. B. van
Muiswinkel, eds) pp. 95–99. Fair Haven, NJ: SOS Publications.
Ellis, A. E. (1990b). Lysozyme assays. In Techniques in Fish Immunology (J. S. Stolen,
T. C. Fletcher, D. P. Anderson, B. S. Roberson & W. B. van Muiswinkel, eds)
pp. 101–103. Fair Haven, NJ: SOS Publications.
Espelid, S., Rødseth, O. M. & Jørgensen, T. Ø. (1991). Vaccination experiments and
studies of the humoral immune responses in cod, Gadus morhua L., to four
strains of monoclonal-defined Vibrio anguillarum. Journal of Fish Diseases 14,
185–197.
Gonzalez, R., Charlemagne, J., Mahana, W. & Avrameas, S. (1988). Specificity of
natural serum antibodies present in phylogenetically distinct fish species.
Immunology 63, 31–36.
Havarstein, L. S., Aasjord, P. M., Ness, S. & Endresen, C. (1988). Purification and
partial characterization of an IgM-like serum immunoglobulin from Atlantic
salmon (Salmo salar L.). Development and Comparative Immmunology 12,
773–785.
Hutchinson, T. M. & Manning, M. J. (1996). Seasonal trends in serum lysozyme activity
and total protein concentration in dab (Limanda limanda L.) sampled from Lyme
Bay, U.K. Fish & Shellfish Immunology 6, 473–482.
Israelsson, O., Petersson, A., Bengten, E., Wiersma, E. J., Andersson, J., Gezelius, G. &
Pilstrom, L. (1991). Immunoglobulin concentration in Atlantic cod, Gadus
morhua L., serum and cross-reactivity between anti-cod antibodies and
immunoglobulins from other species. Journal of Fish Biology 39, 265–278.
Langston, A. L., Bricknell, I. R. & Ellis, A. E. (1998). Iron binding capacity of peripheral
blood leucocyte lysates from Atlantic salmon (Salmo salar L). In Methodology in
Fish Diseases Research (A. C. Barnes, G. A. Davidson, M. P. Hiney & D.
McIntosh, eds) pp. 111–116. Aberdeen: Fisheries Research Services.
Magnadottir, B. (1990). Purification of immunoglobulin from the serum of Atlantic
salmon (Salmo salar L.). Icelandic Agriculture Science 4, 49–54.
Magnadottir, B. (1998). Comparison of immunoglobulin (IgM) from four fish species.
Icelandic Agriculture Science 12, 51–63.
Magnadottir, B. & Gudmundsdottir, B. K. (1992). A comparison of total and specific
immunoglobulin levels in healthy Atlantic salmon (Salmo salar L.) and in
salmon naturally infected with Aeromonas salmonicida subsp. achromogenes.
Veterinary Immunology and Immunopathology 32, 179–189.
Magnadottir, B., Jonsdottir, H., Helgason, S., Bjornsson, B., Jørgensen, T. Ø.
& Pilstrom, L. (1999a). Humoral immune parameters of Atlantic cod
(Gadus morhua L.) I: the e#ects of environmental temperature. Comparative
Biochemistry and Physiology Part B 122, 173–180.
Magnadottir, B., Jonsdottir, H., Helgason, S., Bjornsson, B., Jørgensen, T. Ø. &
Pilstrom, L. (1999b). Humoral immune parameters of Atlantic cod (Gadus
HUMORAL IMMUNE PARAMETERS OF ATLANTIC HALIBUT
535
morhua L.) II: the e#ects of size and seasonal factors. Comparative Biochemistry
and Physiology Part B 122, 181–188.
Magnadóttir, B., Jónsdóttir, H., Helgason, S., Björnsson, B., Solem, S. T. & Pilström, L.
(2001). Immune parameters of immunized cod (Gadus morhua L.). Fish &
Shellfish Immunology 11, 75–89.
Maloy, W. L., Coligan, J. E. & Paterson, Y. (1991). Indirect ELISA to determine
antipeptide antibody titer. In Current Protocols in Immunology (J. E. Coligan,
A. M. Kruisbeek, D. H. Margulies, E. M. Shevach & W. Strober, eds) Vol. 2,
Unit 9.4.
Mangor-Jensen, A., Harboe, T., Shields, R. J., Gara, B. & Naas, K. E. (1998). Atlantic
halibut, Hippoglossus hippoglossus L., larvae cultivation literature, including a
bibliography. Aquaculture Research 29, 857–886.
Marchalonis, J. J., Hohman, V. S., Thomas, C. & Schluter, S. F. (1993). Antibody
production in sharks and humans: A role for natural antibodies. Comparative
Biochemistry and Physiology Part B 17, 41–53.
Michel, C., Gonzalez, R., Bonjour, E. & Avrameas, S. (1990). A concurrent increasing of
natural antibodies and enhancement of resistance to furunculosis in rainbow
trout. Annales de Recherches Veterinaires 21, 211–218.
Montero, D., Tort, L., Izquierdo, M. S., Robaina, L. & Vergara, J. M. (1998). Depletion
of serum alternative complement pathway activity in gilthead seabream
caused by -tocopherol and n-3 HUFA dietary deficiencies. Fish Physiology and
Biochemistry 18, 399–407.
Møyner, K., Røed, K. H., Sevatdal, S. & Heum, M. (1993). Changes in nonspecific parameters in Atlantic salmon, Salmo salar L., induced by Aeromonas
salmonicida infection. Fish & Shellfish Immunology 3, 253–265.
Olesen, N. J. & Jørgensen, P. E. V. (1986). Quantification of serum immunoglobulin
in rainbow trout Salmo gairdneri under various environmental conditions.
Diseases of Aquatic Organisms 1, 183–189.
Overkamp, D., Mohammed-Ali, S., Cartledge, C. & Landon, J. (1988). Production of
polyclonal antibodies in ascitic fluid of mice: technique and applications.
Journal of Immunoassay 9, 51–68.
Persijn, J.-P., van der Slik, W. & Riethorst, A. (1971). Determination of serum iron and
latent iron-binding capacity (LIBC). Clinica Chimica Acta 35, 91–98.
Press, C. McL. & Lillehaug, A. (1995). Vaccination in European salmonid aquaculture:
a review of practices and prospects. British Veterinary Journal 151, 45–69.
Sakai, D. K. (1992). Repertoire of complement in immunological defence mechanisms of
fish. Annual Review of Fish Diseases 2, 223–247.
Schrøder, M. B., Espelid, S. & Jørgensen, T. Ø. (1992). Two serotypes of Vibrio
salmonicida isolated from diseased cod (Gadus morhua); virulence, immunological studies and vaccination experiments. Fish & Shellfish Immunology 2,
211–221.
Starkey, P. M. & Barrett, A. J. (1982). Evolution of 2-macroglobulin. The demonstration in a variety of vertebrate species of a protein resembling human
2-macroglobulin. Biochemical Journal 205, 91–95.
Vilain, C., Wetzel, M.-C., Du Pasquier, L. & Charlemagne, J. (1984). Structural and
functional analysis of spontaneous anti-nitrophenyl antibodies in three cyprinid
fish species: carp (Cyrinus carpio), goldfish (Carassius auratus) and tench (Tinca
tinca). Developmental and Comparative Immunology 8, 611–622.