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Interaction between the blood fluke, Sanguinicola inermis
and humoral components of the immune response of carp,
Cyprinus carpio
M. L. ROBERTS
1
,J.W.LEWIS
2
, G. F. WIEGERTJES
3
and D. HOOLE
1
*
1
Centre for Applied Entomology and Parasitology, Huxley Building, School of Life Sciences, Keele University,
Staffordshire ST5 5BG, UK
2
School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK
3
Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338,
6700AH Wageningen, The Netherlands
(Received 22 October 2004; revised 10 February 2005; accepted 10 February 2005)
SUMMARY
The effect of Sanguinicola inermis on serum antibody and complement activity in Cyprinus carpio was assessed using an
ELISA and haemolytic assays. Possible immune evasion strategies were assessed using immunodetection of host proteins
on the surface of the parasite. Carp acclimatized to 20 or 25 xC were infected by exposure to 500 cercariae or injected
intraperitoneally with 150 cercariae, and serum monitored over a 63-day period. In cercariae-injected carp, irrespective of
time and temperature, a significant increase occurred in complement activity being greatest at 25 xC. In addition, fish
exposed to the cercariae of S. inermis and maintained at 20 xC the level of complement activity was significantly higher after
5 weeks compared to controls. At 20 xC intraperitoneal injections of parasites increased serum antibody levels which peaked
after 7 days. In contrast, at 25 xC, antibody levels were maintained over 63 days. Exposure of fish to infection did not appear
to stimulate antibody production. Immunofluorescence studies revealed ‘ host-like ’ molecules on the surface of the cer-
carial body exposed to carp serum and adult flukes obtained directly from the fish or cultured for 24 h in L15 medium. The
possible role of ‘host-like ’ molecules in immune evasion is discussed and the response at different temperatures is related to
infection dynamics.
Key words: Sanguinicola inermis, Cyprinus carpio, antibody, complement, immune evasion.
INTRODUCTION
Previous studies on the immunological interactions
between Cyprinus carpio and the blood fluke
Sanguinicola inermis have revealed that infection in-
duces an intense cellular reaction. This is manifested
in a severe pathology caused by cercariae penetrating
the skin, migrations of post-cercarial juvenile adult
stages and eggs that become entrapped in host tissue
(Lee, 1990). This response involves eosinophils,
macrophages and neutrophils which encapsulate
and destroy parasite eggs in granulomatous lesions
(Richards et al. 1994a). In addition, infection is also
associated with an alteration in the cellular compo-
sition of the immune organs in carp (Richards et al.
1994b). In vitro studies (Richards et al. 1996 a, b, c)
have also revealed that adult and cercarial stages of
S. inermis induce proliferation of carp lymphocytes
and migration of carp leucocytes, although leucocyte
attachment to the parasite stages is minimal.
Previous studies have also noted that the level and
nature of the immune response of carp is determined
by a range of parameters including the duration of
infection (Richards et al. 1994a, b) and environ-
mental conditions such as temperature (Richards
et al. 1996 a) and pollution (Schuwerack et al. 200 1,
2003; Hoole et al. 2003). The role of humoral factors
such as complement and antibody in the immu ne
response of carp to this parasite has not bee n
elucidated.
The complement cascade fulfils multiple roles
within the immune system of teleosts including non-
specific and antibody-specific virucidal, bactericidal
and parasiticidal activity (Yano, 1992). Although
there have been several investigations on the associ-
ation between complement and viral and bacterial
pathogens in fish, the re has, in contrast, been a lim-
ited number of studies on the parasiticidal activity of
teleost complement. The m ajority of investigations
have been carried out on protozoan parasites. For
example, the genus Cryptobia were killed in vivo and
in vitro by activation of the alternative pathway of the
complement system of refractory fish species (Bower
& Woo, 1977 ; Wehnert & Woo, 1980), whilst the
classical pathway of complement activation, in
association with specific antibodies, has been
* Corresponding author: Centre for Applied Entomology
and Parasitology, Huxley Building, School of Life
Sciences, Keele University, Staffordshire ST5 5BG, UK.
Tel: +44 (0) 1782 583673. Fax : +44 (0) 1782 583516.
E-mail: d.hoole@biol.keele.ac.uk
261
Parasitology (2005), 131, 261–271. f 2005 Cambridge University Press
doi:10.1017/S0031182005007651 Printed in the United Kingdom
implicated in the killing of Cryptobia salmositica in
rainbow trout (Jones & Woo, 1987) and sockeye
salmon (Oncorhynchus nerka) (Bower & Evelyn,
1988). Recent studies by Buchmann, Lindenstrom &
Sigh (1 999) have also suggested that non-specific
factors including complement could play an
important role in the host response against the
pathogenic ciliate Ichthyophthirius multifiliis and that
this response may be affected by the presence of other
parasite species e.g. the monogenean Gyrodactylus
derjavini. In contrast, there have been a limited
number of investigations of the interaction between
the fish complement system and metazoan parasites.
Studies by Whyte, Chappell & Secombes (1989,
1990) revealed that the immune protection mech-
anism in rainbow trout against the eye-fluke
Diplostomum spathaceum involved both the alterna-
tive and classical pathways of complement activation.
In studies by Buchmann (1998) it was also noted that
the lethal effect of plasma from Oncorhynchus mykiss
on the monogenean Gyrodactylus derjavi ni was
mediated through the binding of complement
factor C3 to carbohydrate-rich parasite structures. In
addition to the lytic properties of complement, bio-
logically active peptides, which play important roles
in the inflamma tory processes, are produced during
the activation of the complement cascade. Several
authors have speculated that complement may be
involved in the inflammatory response of cyprinids
against metazoan parasites perhaps associated with
leucocyte chemoattraction and opsonization (e.g.
Hoole & Arme, 1986 ; Taylor & Hoole, 1993 ;
Richards et al. 1996b, c).
There have been several studies that have also
highlighted the involvement of antibodies in the
host/parasite relationship in fish (see Hoole, 1997).
Antibodies have been implicated in the immune
response to a range of parasitic infection such as
protozoa (e.g. Cobb, Levy & Noga, 1998 ; Ardelli &
Woo, 2002; Xu & Klesius, 2003), flukes (e.g. Bortz
et al. 1984 ; Aaltonen, Valtonen & Jokinen, 1997),
nematodes (e.g. Coscia & Oreste, 1998, 2000) and
tapeworms (e.g. Kennedy & Walker, 1969; Sharp ,
Pike & Secombes, 1989). There has also been in-
tensive speculation on how humoral factors in the
immune response of fish are involved in protection.
Such mechanisms as effects on parasite fecundity
(Grayson et al. 1995), binding to glandular secretions
and surface structures (Sharp et al. 1989; Williams &
Hoole, 1995), mediation of leucocyte adherence
(Hoole & A rme, 1986; Whyt e et al. 1990), induction
of parasite migration out of the fish (Clark &
Dickerson, 1997) and mediation of complement-
induced lysis (Saeij, De Vries & Wiegertjes, 2003)
have been proposed. In contrast, the mechanisms by
which fish helminth parasites evade and/or suppress
the immune response has not been extensively con-
sidered although Hoole & Arme (1983) have specu-
lated that host proteins may be acquired on the
surface of the plerocercoid of Ligula intestinalis and
used to evade the immune response in its cyprinid
hosts.
Despite an intense cellular reaction in C. carpio to
primary infections, of S. inermis, fully mature fluk es
do occur in the heart and associated efferent vessels
and commence egg production approximately 30
days post-infection at 20 xC (Kirk & Lewis, 1993). In
contrast, recent investigations carried out by Roberts
and reported by Hoole et al. (2003) have revealed that
there is a significant decrease in flukes recovered in
challenge infections compared with primary in fec-
tions. In the UK, infection of fish with S. inermis is
seasonal (Lee, 1990) with temperature appearing to
play an important role in the development of the
parasite in the intermediate and definitive hosts as
well as affecting transmission periods and the host/
parasite interactions. In this paper the association
between Sanguinicola inermis and the complement
and antibody component of the immune response in
Cyprinus carpio has thus been investigated at differ-
ent temperatures with the aim of elucidating the role
of the humoral component of the immune response
of carp to this parasite and the mechanisms by which
it might evade these immune responses.
MATERIALS AND METHODS
Source and maintenance of carp
Sanguinicola inermis-free carp, 5–10 cm in length,
obtained from Tern Fisheries, Market Drayton, UK
were acclimatized to either 20 xCor25xC for 1
month prior to use. Fish were held in dechlorinated
tap water in a 12 h light : 12 h dark lighting regime
and fed on a commercial carp diet (Mazuri Zoo
Foods, UK).
Source of infected snails
Lymnaea peregra collected from the margins of a
lake in the West Midlands, UK during May and
September were maintained using the protocol of
Kirk & Lewis (199 2) in aerated filtered pond water at
20 xC. Snails were fed Bemax snail diet and washed
lettuce leaves every 3 days and screened for cercarial
emergence which took place between 16.00 and
22.00 h. Cercariae were collected and used to infect
or inject carp experimentally within 1 h of being
released from the intermediate host (Richards et al.
1994a).
Carp infection and collect ion of experimental sera
Groups of 7–8 carp acclimatized to 20 xCor25xC
were either exposed to or injected with cercariae of
S. inermis. Infection was only carried out at 20 xCby
exposing individual fish to 500 cercariae in 400 ml of
water at 20 xC for 1 h as described by Kirk & Lewis
(1992). Injection protocols on fish maintained at
M. L. Roberts and others 262
20 xC and 25 xC comprised a single intra-peritoneal
injection of 150 live cercariae in 100 ml of sterile
phosphate buffered saline (PBS). Controls consisted
of sham-infected fish, which were maintained in
identical conditions without exposure to the parasite,
or carp injected with PBS alone. Carp were main-
tained at thei r acclimatization temperature and blood
was collected by caudal puncture from fish on day 7
post-injection (p.ij.) or post-exposure (p.i.), and then
at weekly interv als up to 49 or 63 days p.i./p.ij. for
determination of serum levels of complement or
antibody respectively. Following overnight storage
at 4 xC and centrifugation at 1000 g for 10 min serum
was collected and stored at x80 xC.
Determination of serum complement levels
Production of sheep erythrocytes sensitized with carp
antibody. Carp anti-sheep heat-inactiva ted eryth-
rocyte se rum (hiCaSE) was produced using stand ard
protocols. Briefly, 500 ml of washed sheep erythro-
cytes suspended in PBS (2r10
8
cells/ml) were injected
intraperitoneally into each of 3 carp (200–300 g) held
at 25 x C. A booster injection was given after 32 days
and 15 days post-booster the carp were bled by
caudal puncture. Pooled serum samples were heat-
inactivated (20 min at 60 xC) and stored at x20 xC.
The protocol used for the production of sensitized
sheep erythrocytes (SE) was adapted from that
described by Yano (1992). Briefly, washed sheep
erythrocytes (1r10
9
cells/ml in Hanks Balanced
Salt Solution, HBSS) exposed to a range of dilutions
of hiCaSE (1 : 50 to 1 : 1600) and a standard concen-
tration of carp serum (1 : 75), were incubated with
mixing at 25 xC for 30 min. The cells were then
washed twice in HBSS by centrifugation at 500 g.A
concentration of 1 : 200 of hiCaSE was adopted
which produced the greatest sensitisation of the
erythrocytes as indicted by the highest level of
haemolysis. Suspensions of sensitized sheep erythro-
cytes were adjusted to 5r10
8
cells/ml, stored at 4 xC
and used within 1 day of production.
Measurement of haemolytic complement activity.
Experimental serum was diluted to 260 ml with
HBSS (1 : 98) and placed in a well of a 96-well
microplate (ICN Biomedicals). Then 40 ml of sensi-
tized sheep erythrocyte suspension was added to each
well and the plate incubated at 25 xC for 1 h with
continual shaking. Prior to incubation all reagents
were held on ice to retard the action of complement.
Following incubation, the microtitre plates were
centrifuged at 350 g for 3 min and 200 ml of the cell-
free supernatant transferred to another microplate
and analysed spectrophotometrically at 540 nm. The
percentage of haemolysis induced by the serum was
determined by comparison with the optical density
reading obtained with 100% haemolysis induced by
distilled water.
Determination of serum antibody levels against
S. inermis
An ELISA was dev eloped to investigate the presenc e
and levels of anti-S. inermis antibodies in the serum
of treated and control carp held at 20 xC and 25 xC.
S. inermis cercarial homogenate was prepared from
live cercariae collected within 6 h of shedding from
Lymnaea pe regra. Cercariae were washed 3 times in
distilled water by centrifugation at 1800 g for 5 min,
re-suspended in 0
.
5 ml of distilled water and dis-
rupted by sonication. Protein determination was
carried out using a Bio-Rad protein assay kit using
human gamma globulin as a standard. Wells of a 96-
well microtitre plate (ICN Biomedicals) were coated
overnight at 4 xC with cercarial antigens using 100 ml
of cercarial homogenate at 5 mg/ml. Vacant protein
binding sites were blocked using 200 ml of 1 % dried
milk powder. Unfortunately due the limited amount
of parasite antigen available it was necessary to pool
serum from the fish at each time-point. Then 100 ml
of this serum at a dilution range of 1 : 4 to 1 : 2560 in
PBST (PBS containing 0
.
1% Tween 20) was added
to the antigen-coated wells and incubated at 30 xC for
1 h. Bound carp IgM was detected using a mono-
clonal antibody raised against the heavy chain of carp
IgM (WCI12, Secombes, van Groningen & Egberts,
1983; Koumans-van Diepen et al. 1995) and an anti-
mouse Ig peroxidase-labelled secondary antibody
(AMIg/P, Sigma). Following preliminary experi-
ments to optimize the above, 100 ml of WCI12
monoclonal at a 1 : 300 dilution with PBST was
added to each well after the excess carp serum had
been removed and the plate washed 3 times with
PBST. After a further wash, 100 ml of AMIg/P was
added to each well, incubated for 1 h at 30 xC and,
after washing, 100 ml of OPD substrate was added
and incubated for 15 min at 30 xC. The reaction was
stopped by the addition of 50 ml of 2N sulphuric acid
per well and the absorbance read at 490 nm in an
Anthos Labtec plate reader. Controls were provided
by substitution of certain steps i.e. cercarial antigen,
dried milk powder blocking, carp serum, WCI12
monoclonal antibody and/or AMIg/P with PBS.
Association of antibody with S. inermis
Adult fluke immunofluorescence. Live flukes, re-
covered from the heart and associated efferent vessels
of carp infected 35 days previously, were divided into
2 groups. One group was washed 3 times in PBS and
fixed by the addition of 4 % paraformaldehyde in
phosphate buffered saline (PBS) for 1 h at ro om
temperature (approximately 20 xC). The second
group was incubated at 20 xC in Leibovitz (L-15)
culture medium (Sigma) for 24 h (Richards et al.
1996c) prior to being washed and fixed as above.
After washing, the vacant protein binding sites were
blocked overnight at 4 xC with 3% Bovine Serum
Carp humoral immune response to S. inermis 263
Albumin (BSA) in PBS. Flukes were then incubated
with polyclonal sheep anti-carp IgM (ACIgM) at a
dilution of 1 : 100 (PBS, 1% BSA) and washed again
before incubation with anti-sheep IgG/fluorescein
isothiocyanate (FITC) conjugate (Sigma) at a di-
lution of 1 : 40 (PBS, 1% BSA) in the dark. Controls
were provided by omission of the polyclonal antibody
or labelled secondary ant ibody.
In addition, live flukes, fixed, washed and blocked
as above were incubated with the more specific
monoclonal antibody WCI12 which interacts with
heavy chain of carp immunoglobulin (Secombes et al.
1983; Koumans-van Diepen et al. 1995) at a dilution
of 1 : 100 (PBS, 1% BSA). Flukes were then washed
and treated with anti-mouse IgG antibodies directly
conjugated to FITC (AMIg/FITC) at a dilution of
1 : 40 (PBS, 1% BSA) . Controls in which either
the monoclonal antibody WCI12 or AMIg/FITC
were omitted were also included. All parasites were
washed, mounted in Citifluor (Citifluor Ltd, City
University, London) and viewed under an ultraviolet
microscope. Washes were perfo rmed 3 times and
incubations were conducted at 30 xC for 1 h, unless
stated otherwise.
Cercarial immunofluorescence
Live cercariae, concentrated to an approximate
density of 1000 per ml by centrifugation (1800 g,
1 min) were fixed by the addition of 500 mlof4%
paraformaldehyde in PBS for 1 h at room tempera-
ture. Cercariae were allowed to precipitate on ice for
15 min, washed and then incubated in 3% BSA in
PBS. Pooled serum , which had been obtained by
caudal puncture from carp that had been exposed to
500 cercariae of S. inermis 8 days previously was
added at a dilution of 1 : 100 (PBS plus 1% BSA, 0
.
1%
Triton X-100 ) and the cercariae further incubated
and then proce ssed as for adult flukes using ACIgM.
Controls consisted of serum from uninfected fish and
the omission of the polyclonal antibody.
Statistical analysis
Data obtained were tested for normality and sub-
jected to parametric analysis using a two-way
ANOVA (General Linear Model, Minitab version
11.2). Interactions between pairs of means were
further analysed using the Tukey multivariate range
test. In the comp lement assay since the percentage
haemolysis obtained was low, CH
50
could not be
determined and an arcsine transformation was car-
ried out on the data prior to statistical analysis to
equalize variances.
RESULTS
Effect of S. inermis on complement levels
Injection of carp with cercariae. Analysis of the com-
bined data for each treatment group (Figs 1 and 2),
irrespective of the time-point, revealed that signifi-
cant differences occurred between sham-injected and
cercariae-injected fish held at 20 xC(P<0
.
001) and
25 xC(P<0
.
05). In addition, when fish held at 20 xC
were compared with those held at 25 xC there were
also significant differences between the two sham-
injected groups (P<0
.
05) and the two groups injected
with cercariae (P<0
.
05). However, further analysis
revealed that there was no significant difference at
individual time-points examined in fish injected
with cercariae or indeed, when compared with the
respective time-mat ched sham-injected controls.
Carp exposed to cercariae of S. inermis. Fish that
were either uninfected or infected by exposure to 500
cercariae and maintained at 20 xC displayed a slightly
different pattern of complement activity over time
than fish that had been injected with cercariae
(Fig. 3). The leve l of complement in fish exposed to
the parasite gradually increased to a peak at 5 weeks
p.i. and then decreased to control levels by week 7.
Despite there being no overall significant difference
between control and fish exposed to S. inermis,
results for infected fish at week 5 were significantly
higher (P<0
.
05) than activity levels for the control
group at weeks 1 and 5 and the infected group at week
2. Similarly, no significant differences were detected
between fish infected by exposure to cercariae and
those injected with cercariae.
Effect of S. inermis on antibody levels
Serum collected from carp maintained at 20 xC that
had been injected intra-peritoneally with 150 live
Weeks post-injection
0
0
10
20
30
40
50
60
70
80
90
12345678
% Haemolysis
Fig. 1. The kinetics of complement activity in the serum
of carp injected with 150 live cercariae of Sangiunicola
inermis (2) or PBS sham-injected controls (%) and
maintained at 20 xC(n=7, mean¡
S.E.).
M. L. Roberts and others 264
S. inermis had increased antibody levels compared to
PBS injected control fish (Fig. 4A). These antibody
levels were highest 7 days p.ij. (i.e. approximately
1
.
9rcontrol leve ls) and gradually declined to control
levels at 56 days p.ij. Further analysis of the antibody
levels over the range of dilutions of carp serum as-
sayed revealed that over the time period, 7–14 days
p.ij., higher levels of parasite-specific antibodies
occurred in the serum from parasite injected fish
than the respective sham-injected controls (i.e. 1
.
9r
greater at 7 days; 1
.
5r greater at 14 days than their
respective controls). In contrast, in carp m aintained
at 25 xC although there was an increase in antibody
levels at 7 days p.ij. (i.e. 1
.
9r greater than control)
this was maintained over the 63 days of the exper-
iment (Fig. 4B). When considering the percentage
increase compared to controls over the initial 7–14
day period the antibody response was greater in fish
maintained at 25 xC (i.e. 7 days 73
.
2%, 14 days
40
.
7%) compared to those kept at 20 xC (i.e. 7 days
27
.
7%, 14 days 14
.
7%). Analysis of serum obtained
from fish maintained at 25 xC and exposed to 500
cercariae revealed the absence of any detectable
antibodies against cercarial antigens over a 63 day
infection period (Fig. 4C).
Interaction between carp antibody and S. inermis
Adult fluke immunofluorescence. Molecules recog-
nized by the polyclonal ACIgM antibody were
located on the tegumental surface of flukes either
fixed immediately after remov al from their host or
cultured in L-15 medium for 24 h prior to processing
(Fig. 5A and B). Fluorescence was distributed evenly
over the surface of flukes and was particularly asso-
ciated with numerous lobular projections on the
tegument. All control treatments proved negative for
specific immunofluorescence.
Immunofluorescence was, however, not observed
associated with the te gumental surface of freshly
recovered adult flukes 35 days post-infection exposed
to the monoclonal anti-carp IgM (WCI12) or in the
control groups (Fig. 6A and B).
Cercarial immunofluorescence. Immunofluorescence,
detected by using ACIgM, was observed on cercariae
incubated with infected serum (Fig. 7) and was only
associated with the cercarial body. Specific fluor-
escence was absent on the cercarial tail, the control
treatments and on cercariae incubated with serum
obtained from uninfected carp.
DISCUSSION
The present results revealed that S. inermis,in
addition to inducing a cellular reaction in C. carpio,is
also associated with an humoral response which
incorporates both non-specific i.e. complement and
specific i.e. antibody components. Previous studies
on complement activity of fish have revealed that the
activation of complement components by infectious
agents reduces the levels in the serum, a process
termed consumption by Sakai (1992). For example,
in Oncorhynchus mykiss the spontaneous activity of
X
B
A
ABX
90
80
70
60
50
40
30
20
10
0
012345678
Weeks post-exposure
% Haemolysis
Fig. 3. The kinetics of complement activity in the serum
of control (%) and infected carp, exposed to 500 cercariae
(2) maintained at 20 xC(n=8, mean¡
S.E.). Points
sharing the same letters are significantly different
(P<0
.
05).
90
80
70
60
50
40
30
20
10
0
012345678
Weeks post-injection
% Haemolysis
Fig. 2. The kinetics of complement activity in the
serum of carp injected with 150 live cercariae (2) or PBS
sham-injected controls (%) and maintained at 25 xC
(n=7, mean¡
S.E.).
Carp humoral immune response to S. inermis 265
complement has been shown to decrease significantly
within 6 days following experimental infections with
virulent strains of Aeromonas salmonicida and Vibrio
anguillarum (Sakai, 1983). Likewise, Thomas & Woo
(1989) recorded a long-term reduction in comp-
lement activity of O. mykiss infected with Cryptobia
salmositica. Although a reduction in complement
activity has also been associated with protozoan and
metazoan infections in mammals (Leid, 1988), the
present studies have revealed that in Cyprinus carpio
experimentally infected with S. inermis the levels of
serum complement activity increased. Fish injected
with live cercariae and maintained at either 20 xCor
25 xC for 7 weeks displayed significantly higher
overall levels of haemolytic activity than sham-
injected controls, suggesting that infection induced
higher complement levels. Although complement
activity was greatest in injected fish at 25 xC, an
increase in activity compared to the respective sham
control was greatest at 20 xC. This may indicate that
the effect of the parasite on complement levels is
more pronounced at this temperature. Previous stu-
dies by Richards et al. (1996 a) have also indicted that
at 20 xC cercariae of S. inermis are able to stimulate
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
S.7.14 I.7 I.14 I.21 I.28 I. 35 I. 42 I. 49 I. 56 I. 63
Treatment/days post-injection
OD 492 nm
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
S.7 S.14 I.7 I.14 I.21 I.28 I.35 I.42 I.49 I. 56 I.63
Treatment/days post-injection
OD 492 nm
A B
I.7 I.14 I.21 I.28 I.35 I.42 I.49 I.56 I.63
Treatment/days post-exposure
OD 492 nm
0.4
0.3
0.2
0.1
0.0
Control
C
Fig. 4. Primary antibody response of carp exposed to cercariae of Sanguinicola inermis. Fish injected with live cercariae
(I) or sham injected controls (S) and maintained at 20 xC (A) and 25 xC (B). (C) Fish maintained at 20 xC and exposed
to 500 cercariae (I) compared to untreated control fish (Control). Controls from 7-day and 14-day post-injection fish
were combined (7.14).
M. L. Roberts and others 266
proliferation of pronephric lymphocytes of carp;
however, this response is absent at 10 xC. The
absence of any significant difference between exper-
imental and control fish at any time-point studied
here at either 20 xCor25xC is probably due to the
high level of intraspecific variation observed.
However, there was a trend for higher complement
levels in fish injected with cercariae. These peaked
after 3 weeks p.ij. and then fell to control values by
week 7 for fish maintained at 20 xC and week 5 for
those at 25 xC, although at the latter temperature a
further increase occurred at 7 weeks p.ij. A similar
enhanced complement activity was observed by Agu,
Farrell & Soulsby (1981) in golden hamsters
(Mesocricetus auratus) experimentally infected with
Leishmania donovani over a 9-week period. Inter-
estingly, carp experimentally infected by exposure to
cercariae of S. inermis exhib it significantly higher
numbers of splenic and pronephric macrophages,
between 5 and 9 weeks p.i., than uninfected controls
(Richards et al. 1994b). It is possible therefore that
increased levels of complement activity induced in
carp injected with S. inermis resulted from increases
10
µ
m
A
B
10
µ
m
Fig. 5. Immunofluorescent labelling of molecules
recognized by the polyclonal ACIgM on the surface of
adult stage of Sanguinicola inermis. (A) Fluke fixed
immediately after removal from Cyprinus carpio.
(B) Fluke fixed after 24 h culture in L-15 medium. Note
fluorescence localized at periphery of fluke in both
instances (arrows).
10
µ
m
A
B
10
µ
m
Fig. 6. Exposure of adult Sanguinicola inermis to
monoclonal antibody WCI12 (A) with anti-mouse
IgG/fluorescein isothiocyanate (FITC) conjugate, and
(B) without anti-mouse IgG/fluorescein isothiocyanate
(FITC). Note similar level of autofluorescence and
absence of intense fluorescence at periphery of flukes.
10
µ
m
Fig. 7. Immunofluorescent labelling of molecules
recognized by the polyclonal ACIgM in cercaria of
Sanguinicola inermis incubated in serum obtained from
Cyprinus carpio infected with 500 cercariae for 8 days.
Note fluoresence localized on periphery (arrow) of
cercarial body (c). Fluorescence absent from tail region.
Carp humoral immune response to S. inermis 267
in macrophages in the spleen and pronephros.
Recent extensive studies have been carried out by
Nakao and co-workers (Kato et al. 2003 ; Nakao et al.
2003b, c ; Nakao, Uemura & Yano, 2003) on the
structure and action of different complement com-
ponents in Cyprinus carpio and have also revealed
that complement is associated with pronephric
granulocytes and macrophages (Nakao et al. 2003a)
and peripheral lymphocytes (Nakao et al. 2004).
An increase in the complement acti vity in the
serum of fish injected with or exposed to cercariae of
S.inermis compared to their respective sham controls
could alternatively be due to a reduction in the con-
sumption of complement components by S. inermis.
This would be particularly pronounced if the pro -
duction of these components is either unaffected or
increased by exposure to the parasite. In mam-
malian/trematode systems, for example the blood
fluke Schistosoma mansoni, cercarial and schistoso-
mula stages are potent activators of the alternative
pathway (Machado et al. 1975; Santoro et al. 1979).
In contrast, mechanically transformed schistosomula
lose their ability to activate and consume comp-
lement via the alternative pathway (Marikovsky et al.
1986). Si milarly, adult schistosomes are largely re-
fractory to complement activity (Fishelson, 1989).
Whether a similar mechanism of activation/evasion
occurs within the carp/S. inermis model is unknown.
However, in experimental infections several adult
worms survive and produce eggs within the heart and
blood vessels of C. carpio (Kirk & Lewis , 1992).
Carp infected by exposure to cercariae of S. inermis
and maintained at 20 xC showed a significant increase
in complement levels in the infected group from
week 2 to a peak in week 5 after which time activity
decreased to control leve ls by week 7. A peak in
activity levels at week 5 may be related to the life-
cycle of the blood fluke, as the adult parasite, which
commences egg production approximately 30 days
p.i. at 20 xC (Kirk & Lewis, 1993). Work by Santoro
et al. (1980) demonstrated a direct correlation
between elevated complement levels in S. mansoni-
infected patients and parasite egg numbers. Richards
et al. (1994 a) recorded neutrophils infiltrating areas
around the eggs of S. inermis which had become
trapped in the pronephros of infected carp. These
authors suggested that infiltration by these cells was
possibly due to parasite-derived chemotactic factors;
however, they did not rule out the role of comp-
lement in this response. In addition to differences in
complement activity between infected and control
fish, significantly higher levels of complement were
recorded in carp maintained at 25 xC compared to
fish maintained at 20 xC. The present results there-
fore corroborate previous studies (e.g. Matsuyama
et al. 1988; Carlson, Baker & Fuller, 1995) which
suggest that, in fish, complement activity is directly
correlated with temperature and raises interesting
issues relating to the seasonal transmission and
occurrence of S. inermis in UK waters. Previous
studies have revealed that temperature may play an
important role in the interactions betwe en S. inermis
and its hosts. Adults can over-winter in carp in the
wild (Lee, 1990) and mature after 2–3 months in carp
maintained at 15–18 xC (Sommerville & Iqbal, 1991)
and in less than 1 month in fish kept at 20 xC. Studies
by Lee (1990) also showed that in the UK infection of
carp primarily took place in the late summer/ autumn
when cercarial emergence was at its peak. Any cer-
carial emergence occurring in the spring was thought
to have arisen from over-wintering infected snails.
The effect of temperature on the immune response
of carp to S. inermis is also reveal ed when considering
antibody levels associated with the injection of
parasites. Antibody levels were greater and were
maintained at higher levels for a longer peri od in carp
kept at 25 xC compared to fish maintained at 20 xC.
Within the physiological range of a particular teleost
species it is widely accepted that higher environ-
mental temperatur es enhance the magnitude and
timing of antibody production (e.g. Rijkers,
Frederix-Wolters & van Muiswinkel, 1980;
Secombes et al . 1991). It is of interest that previous
studies by Richards et al. (1996 a) have also revealed
that carp lymphocytes cultured with S. inermis
extracts were also temperature sensitive i.e. pro-
liferation of pronephric lymphocytes stimulated with
sonicated S. inermis cercariae and adult flukes was
greater at 20 xC compared to 10 xC. It would thus
appear that environmental temperature may be an
important parameter in the interaction between
S. inermis and its carp host. In contrast to the above,
there was little evidence of antibodies being pro-
duced in carp infected with cercariae of S. inermis.
The dichotomy of the response to injected vs.
exposure routes of infection has important implica-
tions regarding the route and presentation of parasite
antigens, a phenomenon that also been observed by
Hoglund & Thuvander (1990) who noted that whilst
O. mykiss infected with live cercariae of Diplostomum
spathaceum did not produce an antibody response,
fish injected with frozen cercariae, diplostomulae or
metacercariae, displayed significantly higher levels of
specific antibody than control fish. In addition, Nie &
Hoole (1999) also noted that whilst carp injected with
extracts of Bothriocephalus acheilognathi produced a
significant antibody response, antibody levels in
naturally infected fish were comparable to non-
infected controls. The inability to detect antibodies
in carp experimentally infected with S. inermis may
indicate that they are rapidly bound to the surface of
the par asite or alternatively the antibody levels are
reduced as part of an immune evasion/suppression
strategy employed by the parasite.
The use of the indirect immunofluorescence
technique has revealed that molecules recognized by
the polyclonal antibody (ACIgM) are associated with
the surface of the adult S. inermis both immediately
M. L. Roberts and others 268
after removal of the parasites from the host and after
24 h in L-15 culture medium. Previous unpublished
Western blot analysis has demonstrated that the
polyclonal antibody used during this work binds to 8
protein bands in carp serum, including the heavy
chain of IgM. The positive reaction of the cercarial
body and its absence from the tail of S. inermis when
exposed to infected carp serum and the polyclonal
antibody suggests that the cercarial body and the
subsequent adult fluke may share similar surface
molecules. However, the app arent absence of IgM on
the surface of the flukes, as indicated by the absence
of surface fluorescence on flukes exposed to the
monoclonal antibody (WCI12) which recognizes the
heavy chain of carp IgM (WCI12, Secombes et al.
1983; Koumans-van Diepen et al. 1995), possibly
suggests that the surface molecules may not be
antigenic but may have a protective role.
Various parasites, for example Schistosoma man-
soni (Sher, Hall & Vadas, 1978), Brugia pahangi
(Premaratne, Parkhouse & Denham, 1989) and
Wuchereria bancrofti (Kar et al. 1993) have the ability
to acquire or mimic host molecules that are incor-
porated into their tegument. These host molecules
are thought to disguise the parasite against immune
attack. Immune evasion strategies are not exclusively
employed by mammalian parasites and while re-
search is very limited, such avoidance techniques
have been proposed for parasites infecting fish. In a
fish/cestode model Hoole & Arme (1983) proposed
that the plerocercoid of Ligula intestinalis acquired
proteins from its host cyprinid and later Williams &
Hoole (1995) detected roach (Rutilus rutilus) mole-
cules, presumed to be antibodies, on the tegumental
surface of this cestode. It is unknown whether the
presence of ‘ host-like’ molecules on the surface of
S. inermis is utilised in any immune evasion strate-
gies. However, the presence of these molecules on the
surface of the flukes that have been cultured for 24 h
does suggest they may also be parasite derived.
Sanguinicola inermis must adopt some mechanism(s)
of evading the immune response as flukes survive for
up to 42 days in the host’s circulatory system (Lee,
1990). However, this protection appears to be limited
since the present results indicate it is not as effective
against challenge infections, particularly at 8 months
p.i. (Hoole et al. 2003).
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