Journal of Fish Diseases 1995, 18. 529-537
Antibody response to crustacean ectoparasites in
rainbow trout, Oncorhynchus mykiss (Walbaum),
immunized with Argulus foliaceus L. antigen extract
N . R U A N E , ' T. K. M C C A R T H Y ^ & P. K^lhLY^
'Department of Zoology, University College
Cork, ^Department of Zoology. University College Galway, and ^Aquaculture Development Centre, Department of
Zoology, University College Cork, Ireland
Abstract. A humoral antibody response was demonstrated by ELISA in rainbow trout immunized
intraperitoneally with extracts from the branchiuran Qclopamsite Argulus foliaceus. A similar immunization protocol produced a higher titre response in a rabbit. Both trout and rabbit identified several
antigenic components on immunoblots. ELISA and immunoblotting indicated that rainbow trout
and rabbit anti-A./o/iaceus sera identified components from the parasitic copepods Lepeophtheirus
salmonis and Caligus elongatus.
Introduction
The expansive development of finfish aquaculture has been paralleled by the increasing range of pathogens
encountered. Major pathogens of marine salmonid culture in northern Europe include the ectoparasitic caligid
copepods or sea lice Lepeophtheirus salmonis (Kroyer) and Caligus elongatus (Nordmann). Macroinvertebrate
ectoparasites do not currently pose a problem in freshwater salmonid aquaculture, although epizootics have
been reported (Mendez, Ramos, Pereira & da Silva 1990). The Branchiura are an entirely parasitic class of
Crustacea infecting the skin or gill cavities of freshwater and marinefish.The class consists of a single order, the
Arguloida, with over 130 species, the majority being marine and native to North America (Bower-Shore 1940).
In Ireland, the only argulid species definitely identified is Argulus foliaceus L., a palaearctic species found on
freshwater fish including the Atlantic salmon, Salmo salar L. and brown trout, Salmo trutta L.
Knowledge of the immune response to crustacean parasites by their fish hosts is meagre (Kularatne,
Subasinghe & Shariff 1994), Previous studies have demonstrated a humoral immune response in rainbow
trout, Oncorhynchus mykiss (Walbaum), and Atlantic salmon immunized with sea lice antigens (Grayson,
Jenkins, Wrathmell & Harris 1991; Reilly & Mulcahy 1993). Cross-reacting antigens have also been
observed between both L. salmonis and C. elongatus, and their various life stages (Reilly & Mulcahy
1993). The object of this study was to demonstrate an antibody response in rainbow trout immunized with
A. foliaceus antigen, and to determine the degree of cross-reactivity with antigens from the sea lice
C. elongatus and L. salmonis.
Materials and methods
Antigen preparation
Argulus foliaceus were collected from brown trout and pike, Esox lucius L., taken from Lough Corrib,
County Galway, Ireland. Antigen extracts were prepared by homogenizing 750 mg of A. foliaceus in 3 ml
Correspondence: Dr P. Reilly, Aquaculture Development Centre, Department of Zoology, University College Cork, Ireland
529
530
N.
Ruanecta\.
of phosphate buffered saline (PBS), pH 7 4 , in a Dounce homogenizer at 0 °C. Excess paniculate
material was removed by eentrifugation at I800g for 30 min at 4 °C. The resulting pellet was
resuspended in PBS and the eentrifugation process repeated. Supematants from each step were pooled,
and used to immunize fish and rabbit. Protein concentration of the antigen extract was estimated
using the Btadford protein-dye binding assay (Bradford 1976) and adjusted to a final protein concentration of 2 mg ml"'. Sea lice antigen extracts were prepared in a similar manner using adult female
and pre-adult stages.
Immunization
procedures
Rainbow trout (/? = 8, average weight 100 g) were immunized intraperitoneally with 1 0 0 ^ of
A. foliaceus antigen extract, emulsified in 30% v/v complete Freund's adjuvant (CFA). Fourteen days
later, fish were re-immunized using antigen plus incomplete Freund's adjuvant (IFA). A similar immunization was performed 7 days later, and at a further 7 day-interval a fmal immunization of antigen less
adjuvant was administered. Therefore, each fish received 800 jjg of antigen during the immunization
procedure. Control fish (n = 5) were similarly immunized with PBS replacing antigen. Experimental
fish were maintained in aquaria with a flow-through aerated water supply and fed on standard trout diet.
Mean water temperature during the period of immunization was 18-5 °C (max. 19-8 °C, min. 17-8 °C).
Seven days post-immunization, fish were test bled. Blood was collected from the caudal vein, allowed
to clot at room temperature and stored at 4 °C for several hours prior to serum isolation by eentrifugation (4000 g for 7 min). Serum was stored at -40 °C. Mucus samples were collected by scraping the fish
surface. Subsequently, these samples were centrifuged (4000 g for 7 min.) and the supematants used in
ELISA.
One rabbit was immunized with A. foliaceus antigen extract. The immunization protocol was as
described for the fish except that immunizations were administered at 7-day intervals. Blood was collected
7 days post-immunization. Pre-immunization serum from this rabbit was used as a control.
ELISA
Fish and rabbit ELISA systems were optimized for antigen density and serum dilution by checkerboard
titrations. Polystyrene ELISA plates (96 well) (Costar, Fastbinder, Costar Corporation, Cambridge, MA,
USA) were coated with 50 ^Jg m l ' of A. foliaceus or sea lice antigen extract in 0-2 M carbonate-bicarbonate
buffer, pH 9 6, and incubated overnight at 4 °C (100 ^ antigen extract per well = 5 ;t^ antigen per well). The
following steps were performed at room temperature (~ 17 °C). For each washing step, plates were washed
with PBS containing 0 5% Tween 20, pH 7 4, on a SLT 812 SW2 platewasher (SLT Labinstruments, Salzburg, Austria). After coating, plates were washed, blocked for one hour with 3% BSA (bovine serum albumin, Sigma, Dorset, UK) in PBS, pH 7 4, at 37 °C and washed again. Test sera (or fish mucus) diluted in PBS
1% BSA, pH 7 4, were added at 1 0 0 ^ per well. Plates were incubated for one hour, washed and then
incubated with either goat anti-trout Ig (peroxidase-labelled affinity ptirified antibody to trout immunoglobulin;
Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD, USA) or goat anti-rabbit IgG (peroxidase conjugated anti-rabbit IgG, Sigma) diluted in PBS 1% BSA, pH 7 4. After washing, plates were developed with
200 lA of o-Phenylenediamine dihydrochloride, hydrogen peroxide substrate (Sigma). Plates were incubated
in the dark for 30 min, and the reaction was stopped with 50 /il 3 M H^SO^, and scanned at OD 429 nm on a
SLT Spectra platereader (SLT Labinstruments).
Antibody response to Argulus and sea lice
53
Electrophoresis
SDS PAGE
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) was performed according
to the method of Laemmli (1970) using a Mini Protean II system (Bio-Rad, Hertfordshire, UK). A 5%
stacking gel, and 7 5% or 12 5% separating gels were found satisfactory for separation of the major
antigenie components of A. foliaceus and sea lice. Prior to electrophoresis, sample extracts (at protein
concentration 1 mg ml"') were boiled for 2-5 min in equal volumes of reducing buffer (Sigma Technical Bulletin No. MWS-877PBS) and 10 lA added to each gel lane. Molecular weight standards (Sigma)
were included in all gels. Electrophoresis was conducted at 200 V for approximately 40 min and gels
were subsequently fixed and protein bands visualized with Coomassie brilliant blue R (Sigma).
Western blotting
Following SDS-PAGE, gels were equilibrated in transfer buffer [25 mM Tris, 192 mM glycine, pH 8 3;
Towbin, Staehelin & Gordon 1979 (methanol not included)]. Separated proteins were transferred
eleetrophoretically, using a Trans-Blot SD semi-dry transfer cell (Bio-Rad), onto Immobilin PVDF transfer membrane (Millipore) for Western blotting. After transfer, blots were blocked in TBS (Trizma
buffered saline) 3% BSA, pH 7 4, at 37 ""C for one hour, and subsequently probed with trout or
rabbit antisera. When using sera from immunized trout, blots were washed in running tap water
for 5 min, incubated in primary antisera (diluted 1:3 or 1:20 in TBS 1% BSA, pH 7 4) for one
hour, washed, and incubated with goat anti-trout Ig (affinity purified antibody to trout
immunoglobulin alkaline phosphatase conjugate; Kirkegaard & Perry Laboratories Inc.) diluted 1:1000
in TBS 1% BSA. Blots were then washed in tap water for 5 min. Immunoblots were developed using nitro
blue tetrazolium [NBT (Sigma), 50 mg ml"' in 70% N, N-Dimethylformamide (DMF, Sigma)], 5-bromo-4chloro-3-indolyl phosphate [BCIP (Sigma), 50 mg ml"' in 50% DMF]. Immunoblots were flooded with
substrate (33 ^ NBT + 16 5 /il BCIP in 5 ml 100 mM Tris, pH 9 5, 100 mM NaCl, 5 mM MgCl, at 37 °C)
and the colour allowed to develop (2-10 min). Development was stopped by immersing the blot
in water. The procedure when using rabbit sera was essentially similar; the primary antisera being
rabbit anti-A. foliaceus (diluted 1:1500) followed by goat anti-rabbit IgG (alkaline phosphatase
conjugate, Sigma) (diluted 1:2000). All antibody probing procedures were conducted at room
temperature on an orbital shaker (Stuart Scientific).
Results
ELISA
Humoral antibody responses from immunized rainbow trout and jnimunized rabbit, measured by ELISA,
were significantly higher than from control animals (/ = 2-078, 22 d.f.; P < 0 05 and paired t = 11 261. 11
d.f.;P<0 001, forfish and rabbit respectively) (Figs 1 & 2). The response of the immunized trout (Fig. 1)
was almost twice that of control fish at a 1:20 dilution, and was also higher than the controls for the next
six dilutions. However, immunized fish exhibited greater variation in individual responses (individual OD
values at 1:20 dilution: 0 59,0 58, 0 46,0 40,0 27, 0-22, 0-21 and 0-21) than control fish (0-27, 0-27, 0-19,
018 and 0-17), with two control fish having OD values greater than four of the immunized fish.
532
'. Ruaneei'd\.
051Immunized
- -0--- Control
1
2
3
4
Log reciprocal serum dilution
5
Figure 1. ELISA dilution curves for
immunized and control rainbow trout
sera.
1-0
Immunized
— 0 — - Control
0-8
E
c
o
I 0.4
CO
0-2
-o-~
00
1-0
--•o--c>-
2-0
30
4-0
L o g reciprocal serunn d i l u t i o n
B-O
Figure 2. ELISA dilution curves for
immunized and control rabbit sera.
Rabbit anti-A. foliaceus serum identified C. elongatus and L. salmonis antigens, as demonstrated by
ELISA (Fig. 3). The response to C. elongatus antigen was consistently higher than the response to
L. salmonis antigen. Rainbow trout ?ini\-A. foliaceus serum from the three highest responding fish (i.e. OD
values 0-59,0-58 and 0-49) at dilutions of 1:20,1:40 and 1:80 were also shown to bind to C. elongatus and
L. salmonis antigen extracts (Fig. 4). Similar to the results obtained when using rabbit sera, there was a
greater affinity for C. elongatus antigen, compared to L. salmonis antigen, with a two-fold difference in
absorbency values at the lowest dilution.
The presence of a mucosal antibody response to A. foliaceus antigens was not detected by ELISA,
absorbence values for immunized fish being 0-15 + 0-017, compared with 0-17 ± 0 015 for control fish.
Antibody response to Argulus and sea lice
1-0
533
L salmonis
- - - 0 — C. elongatus
0-8
£
c
ft e
0-6
o
c
0-4
0-2
Figure 3. Rabbit anti-A. foliaceus
serum response to L. salmonis and
C elongatus.
0-0
10
2-0
30
40
Log reciprocal serum dilution
5-0
05
L salmonis
---o— C. elongatus
04
c
5 0-3
c
(0
0-2
01
Figure 4. Rainbow trout
anti->l. foliaceus serum response to
L. salmonis and C elongatus.
0-0
12
1-4
1-6
1-8
Log reciprocal serum dilution
2-0
SDS PAGE and Western blotting
Protein components of A. foliaceus were separated on 7 5% and 12 5% acrylamide gels with the latter
12 5% gel giving better resolution (Fig. 5). Numerous bands ranging in molecular weight from approximately
100 kDa to approximately 15 kDa were evident; however, components of over 100 kDa were not intensely
stained.
Western blots of A. foliaceus antigens probed with rabbit anti-A. foliaceus serum identified
components in the molecular range of 82 to 13 kDa (Fig. 6). The main components identified had
approximate molecular weights of 82, 76, 65, 62, 41 and 31 kDa. Several components in the range of
20 to 13 kDa stained intensely, with components of 20, 18, 17, 14, 13 and especially a component of
534
N. Ruane et al.
20-1
142
Figures. SDS-PAGE (12-5%)
profile oi A. foliaceus molecular
components, with approximately
10 pg protein per lane and stained
with Coomassie briUiant blue R. The
positions of molecular weight
standards (kDa) are indicated.
66
45
36
29
24
201
14-2
Figure 6. Western blot of a 12-5%
gel (A. foliaceus antigen,
approximately 10 ^ protein per
lane) probed with rabbit
anti-A. foliaceus serum. The
positions of molecular weight
standards (kDa) are indicated.
approximately 15 kDa being clearly identified (Fig. 6). Components were not identified when using
pre-immunized control sera.
Antibody response to Argulus and sea lice
535
Western blots of A. foliaceus antigens were also probed with sera from immunized and control rainbow
trout. When using control sera or sera from immunized fish, diluted 1:20, antigens were not visualized on
immunoblots. At a dilution of 1:3, high-responding trout did identify components of approximate
molecular weight 48, 47, 29-5, 18, 17 and 15 kDa (results of these immunoblots are not shown as
background staining prevented clear photographic distinction of components).
Sea lice antigens were also probed with rabbit anti-A. foliaceus serum (Fig. 7). Caligus elongatus
components of approximate molecular weights of 100, 82, 65, 48 and 44 kDa were identified. Components of 65, 55, 48 and 44 kDa were identified on L. salmonis immunoblots probed with rabbit antiA. foliaceus sera; however, these components were less clearly stained than C. elongatus antigens.
Components were not identified when using pre-immunized control sera. Sufficient quantities of serum
were not available to permit similar blotting for rainbow trout.
Discussion
The present study represents the first reported account of a humoral antibody response in rainbow trout
(or rabbit) to A. foliaceus antigens. The regime employed for trout immunization differed from that
used in previous studies (e.g. Grayson et al. 1991; Reilly & Mulcahy 1993). Both rainbow trout
and rabbit were immunized in a similar manner, each receiving an initial immunization and three
boosting injections. Differences in the capacity of the mammalian and teleost immune systems to
respond to similar immunization procedures were highlighted, with the rabbit producing a higher titre
response and identifying more components on immunoblots.
As demonstrated by ELISA, rainbow trout serum antibody response to immunization with
A. foliaceus antigen varied. Such variation may be expected for a variety of host dependent reasons.
The phenomena of (unimmunized) control rainbow trout having serum antibody capable of identifying Argulus antigen(s) to a greater extent than immunized fish may be indicative of cross-reactivity
29
Figure 7. Western blot of a 7-5% gel (approximately 10 fJg protein per lane) probed with rabbit anti-A. foliaceus serum.
Lanes (x2): (i) A. foliaceus antigen; (2) C elongatus antigen; (3)1. salmonis antigen. The positions of molecular weight
standards (kDa) are indicated.
536
A^. Ruane a a\.
following previous natural infections with crustacean parasites; however, such infection was not
evidenced with the fish used in this study. Previous studies have also indicated that pre-smolt
Atlantic salmon serum will reaet positively with L salmonis antigen(s), using ELISA (R Reilly,
unpublished results). Such non-specific responses to crustacean antigens may be advantageous,
especially in diadromous fish which encounter pathogens, such as sea lice, on entering a new,
seawater environment.
The results of SDS PAGE and Western blotting did not clearly identify components of molecular
weight greater than 100 kDa in A. foliaceus. This may reflect the absence of such components, or more
likely, their quantity and immunogenicity, relative to components of lesser molecular weight and/or the
inability of Coomassie blue to stain such quantities of protein. Moreover, components of molecular weight
greater than 200 kDa have been identified from sea lice (Grayson et al. 1991; Reilly & Mulcahy 1993). In
excess of 10 major components were identified on Western blots probed with rabbit anti-A. foliaceus sera.
A number of components of relatively low molecular weight from 13 to 20 kDa proved to be highly
antigenic, particularly a 15-kDa component which stained intensely. Although A. foliaceus components
were not identified on immunoblots probed with rainbow trout anti-A. foliaceus sera, diluted 1:20, several
antigenic components were identified, at a lower dilution of 1:3. In common with blots probed with rabbit
sera, components of 48, 18, 17 and 15 kDa were identified. These results indicate that these particular
components were highly antigenie in both rabbit and fish.
ELISA results of rabbit and trout anti-A. foliaceus sera indicated that both irmnunized animals were
capable of identifying sea lice antigens. Results from Western blotting also indicate that common antigens
were identified from A. foliaceus, C. elongatus and L salmonis, especially components of 65, 48 and
44 kDa. It has been demonstrated that C. elongatus and L. salmonis share many common antigens (Reilly
& Mulcahy 1993), and the present results indicate that A. foliaceus also shares cross-reacting antigens
with sea lice. The results of ELISA and Western blotting also indicate that C. elongatus and L. salmonis
antigens were not equally detected by anti-A. foliaceus sera from rainbow trout or rabbit. As both
C elongatus and L. salmonis belong to the family Caligidae, this cannot be satisfactorily explained
taxonomically. Argulus foliaceus and C. elongatus may have antigens or antigenic epitopes which have
similar quantitative expression in both species compared to L. salmonis. This assumption is suggested by
results of immunoblotting, where C. elongatus components were more intensely stained than similar
components in L. salmonis.
In common with sea lice, A. foliaceus feeds on host blood (Bower-Shore 1940), and presumably
on skin mucus. Successful vaccination strategies against mucus and blood feeding crustacean ectoparasites
of teleost fish will depend on immunization routes preferentially stimulating mucosal surfaces and on
the identification of common protective antigens. The absence of a mucosal antibody response in the
present study may be reflective of the route of antigen delivery, as Atlantic salmon orally immunized
with L. salmonis antigens produce antibody in the skin mucus (P. Reilly, unpublished data). The
demonstration of an antibody response in rainbow trout immunized with .4./o//aceM5 antigen and crossreacting antigens with sea lice offers the possibility for further investagtions on a broad-based
vaccine against crustracean ectoparasites.
References
Bower-Shore C. (1940) An investigation of the common fish louse ArgH/M^/o/iar^MS (Linn.). Parasitology 32, 361-371.
Bradford M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilising the
principle of protein-dye binding. Analytical Biochemistry 72, 248-254.
Antibody response to Argulus and sea lice
537
Grayson T.H., Jenkins P.G., Wrathmell A.B. & Harris J.E. (1991) Serum responses to the salmon louse Lepeophtheirus
salmonis Kr0yer (1838) in naturally infected salmonids and immunized rainbow trout Oncorhynchus mykiss
(Walbaum) and rabbits. Fish & Shellfish Immunology 1, 141-155.
Kularatne M., Subasinghe R.P. & Shariff M. (1994) Investigations on the lack of acquired immunity by the Javanese carp,
Puntius gonionotus (Bleeker), against the crustacean parasite, Lernaea minuta (Kuang). Fish & Shellfish Immunology
4, 107-114.
Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 222,
680-685.
Mendez J., Ramos M.A., Pereira T.G. & da Silva A.M. (1990) Rainbow trout culture failure in a small lake as a result of
massive parasitosis related to careless fish introductions. Aquaculture 89, 123-126.
Reilly P. & Mulcahy M. (1993) Humoral antibody response in Atlantic salmon {Salmo salar L.) immunized with extracts derived from the ectoparasitic caligid copepods Caligus elongatus (Nordmann 1832) and Lepeophtheirus
salmonis (Kr0yer 1838). Fish & Shellfish Immunology 3, 59-70.
Towbin H.. Staehelin T. & Gordon J. (1979) Eiectrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350-^354.