Fish & Shellfish Immunology (1997) 7, 349–353

SHORT COMMUNICATION

The antibody response of snakehead, Channa striata

Bloch, to Aphanomyces invaderis

K. D. THOMPSON,1 J. H. LILLEY,1 S. CHINABUT2 AND A. ADAMS1

1
Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland,
U.K. and 2Aquatic Animal Health Research Institute, Kasetsart University
Campus, Bangkok, Thailand

(Received 13 November 1996, accepted in revised form 30 January 1997)

Key words: Epizootic Ulcerative Syndrome, fungus, snakehead fish, anti body
response, Western blot analysis.

Epizootic Ulcerative Syndrome (EUS) is a devastating disease a#ecting both wild and
farmed freshwater and brackish water fish in Southeast and South Asia. This disease
has had serious implications for food sources and local economies of the region (Lilley
et al., 1992). Ever since the emergence of EUS in the early eighties, no singular
aetiological agent has been conclusively identified as causing the disease, even though
a variety of bacteria and viruses have been associated with the condition (Frerichs
et al., 1986; Roberts et al., 1994). These infectious agents appear opportunistic in nature
and are thought to have only a secondary role in causing infection. A specific
Aphanomyces fungus, on the other hand, has been consistently isolated from fish
infected with EUS and shown to be capable of reproducing typical EUS lesions
(Roberts et al., 1994; Lilley et al., 1992). The fungus associated with EUS has recently
been named Aphanomyces invaderis by Willoughby et al., (1995).
Histological examinations of infected tissue reveal that a distinctive inflammatory
response may be produced against the fungus by the immune defences of the fish
(Roberts et al., 1994). In an attempt to halt the invasion of fungus through the tissues
of the fish, macrophages surround and envelop invading mycelium by forming granu-
lomas (Chinabut et al., 1995). However, no reports of a humoral response against
Aphanomyces in fish are available in the literature. This paper presents such an
account in snakehead, Channa striata Bloch.
The fungi used in the present study, outlined below, have been previously described
by Lilley & Roberts (1997). Spore suspensions were prepared from the fungi according
to Willoughby & Roberts (1994). Briefly, agar plugs of mycelium (4 mm) were placed in
Petri dishes containing glucose–peptone–yeast (GPY) broth and cultured for 4 days at
20) C. Suspensions of secondary zoospores were prepared by washing the mycelium
mats five times with sterile distilled water, then incubating them overnight at 20) C in
sterile filtered pond water diluted 1:2 with distilled water (Willoughby & Roberts,
1994). The zoospores were prepared by microwaving them for 10 s, after which time the
zoospores were no longer motile. Both the zoospore suspensions and homogenised
mycelium mats (used to produce the zoospores) were used to immunise the fish.
Mycelium extracts were prepared for sodium dodecyl sulphate polyacylamide gel
electrophoresis (SDS-PAGE) by adding double strength GPY to the zoospore suspen-
sions, culturing them for 2 days at 20) C then extracting the mycelium mats using a
modification of the method described by Wood (1988). Liquid nitrogen was
349
1050-4648/97/050349+05 $25.00/0/fi970084 ? 1997 Academic Press Limited
350 K. D. THOMPSON ET AL.

kDa
207
112
80

45

36
27
18

1 2 3 4 5
Fig. 1. Separation of Aphanomyces mycelium extract by SDS-PAGE. The gel (4–20%) was stained
with Coomassie blue. Lanes: (1) PA7; (2) F3SA; (3) NJM9030; (4) 3P; (5) FDL458. Approximately
20 ìg protein was applied to each lane.

used to freeze the mycelium prior to extraction and polyvinyl pyrrolidone was omitted
from the extraction procedure.
Snakehead fish (500 g), obtained from Suphanburi Province, Thailand,
were maintained at the Aquatic Animal Health Research Institute (AAHRI), Kasetsart
University Campus, Bangkok, Thailand. Three groups of fish were used in the study.
The first group (n=8) were non-vaccinated healthy control fish. The second group
(n=5) received a primary intramuscular (i.m.) injection of microwaved PA7 spores (1 ml
containing 5000 spores ml "1), followed by an intraperitoneal injection of microwaved
PA7 spores mixed with homogenised mycelium (1 ml containing 5000 spores ml "1
mixed with 1 mat of mycelium) 10 days later. The third group, consisting of non-
vaccinated healthy fish, were challenged with an i.m. injection of viable motile spores
from strain PA7 (1 ml containing 5000 spores ml "1). The first two groups of fish were
maintained at a water temperature of 27) C, while the third group was maintained at
20) C throughout the challenge period. Blood was collected from the caudal vein of fish
on day 30 of the study. Blood samples were also collected from seven snakeheads
infected with EUS during a natural outbreak of the disease in Pichit Province,
Thailand. These fish had also been sampled for fungal, viral and bacterial analysis
and both A. invaderis (Lilley & Roberts, 1997) and rhabdoviruses were recovered
(Kanchanakhan, 1996).
Sera from the fish of each group were pooled and screened by Western blot analysis.
SDS-PAGE was performed using precast acrylamide separating gels (4–20%) (Bio-Rad
Laboratories, Hemel Hampstead, U.K.). Fungal extracts (10 ìl of 2 mg protein ml "1
and 10 ìl sample bu#er) were applied to each lane of the gel, which was then subjected
to electrophoresis for 45 min at 200 V. Fungal antigens were transferred from the gel to
sheets of nitrocellulose membrane by a wet blotting system using 50 V for 60 min. The
gels were stained with 0.1% (w/v) Coomassie blue R250, while the nitrocellulose
membranes were washed with Tris-bu#ered saline with Tween-20 (TTBS) (0.05 M Tris,
0.15 M NaCl, 0.1% v/v Tween-20 pH 7.6) and blocked with 1.0% (w/v) bovine serum
albumin before applying the fish sera [1/10 dilution in phosphate-bu#ered saline (PBS)
(0.2 M, pH 7.3)]. The nitrocellulose membranes were incubated for 60 min at 20) C,
washed twice with TTBS, and incubated for 60 min at 20) C with rabbit anti-snakehead
IgM (diluted 1/100 in PBS) (courtesy of Jitkasem Chanphong, AAHRI). They were
washed twice with TTBS, then goat anti-rabbit IgG-HRP conjugate (Scottish Antibody
 ANTIBODY RESPONSE OF CHANNA STRIATA BLOCH 351

(a) kDa (b) kDa

207 207
112 112
80 80
45 45

36 36
27 27
18 18

8 8

1 2 3 4 5 1 2 3 4 5

(c) kDa (d) kDa

207 207
112 112
80 80
45 45

36 36
27 27
18 18

8 8

1 2 3 4 5 1 2 3 4 5
Fig. 2. The response of snakehead anti-Aphanomyces (strain PA7) sera against extracts of
mycelium from Aphanomyces by Western blot analysis. (a) healthy fish; (b) fish immunised with
spores and mycelium from strain PA7; (c) fish experimentally challenged with PA7; and (d) fish
naturally infected with Aphanomyces Lanes: (1) PA7; (2) F3SA; (3) NJM9030; (4) 3P; (5) FDL458.

Production Unit, Carluke, Scotland) (diluted 1/100 in TTBS) was applied to

the membranes for 1 h. The assay was developed with 3*3-diaminobenzidine tetrahydro-
chlodide (DAB) (Sigma) (6 mg) dissolved in 10 ml of substrate bu#er (20 mM Tris
hydrochloride, 500 mM sodium chloride, 30 ìl hydrogen peroxide, pH 7.5) until bands
appeared. The reaction was stopped by washing the membranes with distilled water
for 10 min.
Similarities in the SDS-PAGE protein profiles were evident between the extracts of
strains PA7, NJM9030 and 3P and these were markedly di#erent to the profiles
obtained with strains F3SA and FDL458 (Fig. 1). Aphanomyces invaderis, strain PA7
(lane 1) was isolated from snakehead fish (Channa striata Bloch) during outbreaks of
EUS in Thailand in 1995. Aphanomyces piscicida, strain NJM9030 (lane 3) had been
isolated from ayu, Plecoglossus altivelis Temminck and Schlegel, during a mycotic
granulomatosis infection in Japan (Hatai et al., 1977) and Aphanomyces sp., strain 3P
(lane 4) was obtained from grey mullet, Mugil cephalus L., infected with redspot disease
(RSD) in Australia (Fraser et al., 1992). It has been speculated that the latter two fungi
are in fact the same as the agent which is responsible for EUS in Southeast Asia
(Callinan, 1994; Hatai et al., 1994; Callinan et al., 1995; Lilley & Roberts, 1997). Thus,
352 K. D. THOMPSON ET AL.

this would explain the resemblance between their SDS-PAGE profiles. The two
remaining strains of Aphanomyces, F3SA, a saprophytic Aphanomyces sp. (lane 2)
isolated from snakehead fish and A. astaci, FDL458 (lane 5), recovered from white
clawed crayfish, Austropotamobius pallipes Lereboullet (Alderman et al., 1986), did not
result in an EUS infection in fish artificially challenged with the fungus (Lilley &
Roberts, 1997). Callinan et al., (1995) has also reported similarities between SDS-PAGE
profiles of Aphanomyces sp. isolated from fish infected with RSD and EUS.
The response of the snakehead anti-Aphanomyces sera to the di#erent Aphanomyces
mycelium extracts by Western blot analysis reflected the similarities found between the
protein profiles of the Aphanomyces strains by SDS-PAGE (Fig. 2). Sera from healthy
fish elicited a weak non-specific reaction with all mycelium extracts, particularly with
bands present at around 40 and 55 kDa in the extracts of the pathogenic strains of
Aphanomyces (PA7, NJM9030 and 3P) [Fig. 2(a), lanes 1, 3 and 4, respectively]. Sera
from fish immunised with the spores and mycelium from strain PA7 produced a
stronger reaction with these extracts than was observed with healthy fish sera,
particularly with the 40 and 55 kDa bands [Fig. 2(b)]. These sera also identified a band
at 37 kDa, unique to the mycelium extracted from saprophytic Aphanomyces strain,
F3SA (lane 2), while only an undefined region of staining between 36 and 80 kDa was
observed with the sera against A. astaci (FDL458) [Fig. 2(b), lane 5]. As well as
recognising the bands mentioned above, antisera from both the experimentally chal-
lenged fish and fish naturally infected with A. invaderis [Fig. 2(c) and (d)], identified a
low molecular weight band at around 10 kDa, which was not detected by the immu-
nised sera from group two fish immunised with heat killed fungi and which was not
apparent on the profiles of the saprophytic strain of Aphanomyces (F3SA) or with A.
astaci (FDL458). The results suggest that the 10 kDa band is not immunogenic to the
fish when it is presented as a vaccine in the form of microwaved fungal particles, but
is immunogenic when the fungus is actually growing within the fish. A possible
explanation for the di#erences in the antigenicity of this band may be due to
alterations in its structural confirmation during vaccine preparation compared to
in situ fungal growth within the fish. These findings have serious implications for the
development of vaccines against EUS.
The authors thank the Overseas Development Agency (project no. R5997) for
supporting this project and the sta# of AAHRI, Kasetsart Campus University,
Bangkok, Thailand for their assistance throughout the study. Prof. K. Hatai, Dr G. C.
Fraser and Dr D. J. Alderman for provision of fungal strains.

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