Download Lymphocystis disease in cultured false clown anemonefish

Aquaculture 315 (2011) 414–416
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Aquaculture
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Lymphocystis disease in cultured false clown anemonefish (Amphiprion ocellaris)
Nopadon Pirarat a,⁎, Watanyoo Pratakpiriya a, Krisaya Jongnimitpaiboon a, Kasemsri Sajjawiriyakul a,
Channarong Rodkhum b, Nantarika Chansue c
a
b
c
Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand
Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand
Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, 10330, Thailand
a r t i c l e
i n f o
Article history:
Received 29 November 2010
Received in revised form 19 December 2010
Accepted 5 January 2011
Available online 15 January 2011
Keywords:
Cultured false clown anemonefish
Lymphocystis disease
Histopathology
Transmission electron microscopy
a b s t r a c t
False clown anemonefish (Amphiprion ocellaris) is one of the most famous marine ornamental fish which have
a highly economic value in Thailand. The affected fish showed external lesions of irregular white nodules in
various sizes on skin, fins and mouth. Histopathological finding revealed clusters of lymphocystis
hypertrophied cell with thick smooth hyaline capsule. The infected cell showed an enlarged nucleus with
basophilic marginated chromatin and basophilic intracytoplasmic inclusion bodies, mainly in the peripheral
area of the cell. The typical cells were found in skin, fins, operculum, gill, spleen, and kidney. Transmission
electron microscopy revealed an icosahedral viral particle measured 200 nm in diameter with dense
nucleocapsid and a well-defined electron-lucent envelope with surface-associated fibrils. This is the first
report of lymphocystis disease in cultured false clown anemonefish in Thailand.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Anemonefish is one of the ornamental marine fish which are
mostly admired because of their various colors and beauty. Anemonefish are in the family; Pomacentridae, subfamily; Amphiprioninae and
native to warm waters of the Indian and Pacific oceans. Clownfish live
in small groups inhabiting a single anemone. Twenty eight species are
recognized, one in the genus Premnas, while the remaining are in the
genus Amphiprion. However, there are only 7–8 species in Thailand.
Five species of anemonefish; Amphiprion ocellaris, Amphiprion akallopisos, Amphiprion clarkia, Amphiprion sebae and Amphiprion ephippium,
are found in Andaman sea while the species Amphiprion polymnus and
Amphiprion perideraion are found in the gulf of Thailand.
The false clown anemonefish (A. ocellaris) are tropical marine fish
frequently found in Andaman sea. They inhabit coral reefs and sheltered
lagoons up to a depth of 15 m. False clown anemonefish have orange to
reddish-brown color with three white bands on the head, body, and
caudal peduncle. The white bands are outlined in black. The outer margin
of fins is white and the inner one is black. Clownfish or anemonefish
culture is increasingly popular and rapidly widespread in Thailand. An
expansion of anemonefish culture resulted in an increase of smuggling
anemonefish fishing from the nature. The population of anemonefish
became rapidly decreased and almost reached a crisis point. In order to
response to an uprising demand for anemonefish of domestic and
oversea market, an organization of government and private sector is now
⁎ Corresponding author.
E-mail address: [email protected] (N. Pirarat).
0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2011.01.014
focusing on the production of anemonefish larva population with high
quantity and survival rate. However, the fish culture is difficult to avoid
diseases because it only focuses on a high production with no
consideration of a management. Consequently, the cultured area is
very crowded and lacks appropriate management. This leads to stress
and epidemic of disease easily especially infectious diseases.
Lymphocystis disease (LCD), one of the common infectious diseases
which affect marine fish cultures in Thailand, was discovered in 1874
(Zhang et al., 2004). Distribution has been reported worldwide such as
in Spain (Alonso et al., 2005), France (LeDeuff and Renault, 1993), Korea,
Japan (Hossain et al., 2008) and China (Sheng et al., 2007). In Thailand,
the disease was reported by Wachirachaipaisal and Limsuwan in 1985.
Over 125 fish species were reported including 25 marine species,
belonging to 42 families (Cano et al., 2006; Sheng et al., 2007; Cano et al.,
2009). The causative agent of LCD is lymphocystis disease virus (LCDV)
which is a large virus in the genus Lymphocystivirus of the family
Iridoviridae. LCDV is an icosahedral symmetry virus, approximately
200–300 nm in diameter, and contains single linear double stranded
DNA which is circularly permuted and terminally redundant (Darai
et al., 1983; Tidona and Darai, 1997; Kitamura et al., 2006). LCD is
characterized by the external appearance of nodules, either singly or in
groups, on skin, fins, or tail of the affected fish (Walker and Hill, 1980;
Alonso et al., 2005). Although, LCD is not a fatal disease, the external
appearance might cause a significant economic loss. The principle mode
of transmission of LCD is horizontally via direct contact (Cano et al.,
2006) and external trauma (Alonso et al., 2005). Other factors such as
water contamination (Overstreet, 1988) and stress condition caused
by high population density, nutrition deficiencies, decreased dissolved
oxygen, suboptimal water quality, or human manipulation may increase
N. Pirarat et al. / Aquaculture 315 (2011) 414–416
415
Fig. 1. The false clown anemonefish, with an average body length about 1 inch, showed typical lesions of lymphocystis disease. Multiple white irregular surface nodules appeared on
skin surface, fins and mouth. The diameters of nodules were approximately 1–2 mm (Fig. 1.A). Histopathologic micrographs of the skin surface revealed hypertrophic lymphocystis
cells contained thick smooth hyaline capsules (HC) and basophilic intracytoplasmic inclusion bodies (IB) (Fig. 1.B). Magnification of lymphocystis cell (LC) at the operculum,
showing an irregular nucleus (N), marginated chromatin, unevenly stained cytoplasm, inclusion body at the cell periphery, and thick smooth hyaline capsule (HC) of the cell
membrane (Fig. 1.C). In trunk kidney, the illustrating presented two lymphocystis cells (LC) at the margin of the tubular part of kidney. Each lymphocystis cell was surrounded by a
thick smooth hyaline capsule (HC) and contained basophilic intracytoplasmic inclusion bodies and irregular nuclei with marginated chromatin (Fig. 1.D). Transmission electron
micrographs of ultra-thin sections of skin nodules revealed an electron dense icosahedral viral particles measure 200 nm in diameter (Fig. 1.E). The 200 nm-viral particles composed
of three concentric layers: outer envelope, icosahedral protein shell (1) (comprised of the major capsid protein), inner membrane (2) and central DNA core (3). This virion contained
outer fibril-like protusions (F) (Fig. 1.F).
the appearance of LCD symptoms (Paperna et al., 1982; Mellergaard and
Nielsen, 1995; Alonso et al., 2005; Cano et al., 2006).The recent study
reported that Artemia sp. might act as a reservoir host of this disease
(Cano et al., 2009). To our knowledge, LCDV infection has never been
reported in cultured false clown anemonefish. The purposes of this
study were to report the occurrence of LCDV in false clown anemonefish
and to determine the distribution of LCDV in fish organs.
2. Materials and methods
2.1. Fish samples
Eight naturally infected cultured false clown anemonefish
(A. ocellaris), with an average body length about 1 inch, were
collected from a cultured farm in central of Thailand. The morbidity
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N. Pirarat et al. / Aquaculture 315 (2011) 414–416
rate of this farm was 20%. Fish were transported to the laboratory
using a plastic bag method and then euthanized using clove oil before
complete examination. The fish showed typical external signs of LCDV
nodules in different sizes mainly on the body skin surface, fins, tail,
and mouth. Gross pathology, histopathology, and transmission
electron microscope (TEM) were used as diagnostic tools.
2.2. Pathological study
All of the fish samples were examined for gross pathology including
the location, distribution, shape, size, color, consistency and special
features of typical external lesions. Fish samples with lymphocystis
nodules were selected and fixed with 10% buffered formalin solution at
room temperature for 24 h. The fixed samples were rinsed off with tap
water and then were cut into small pieces approximately 0.5 cm in
transverse section. Next, all of the specimens were decalcified with
formic acid sodium citrate solution for 24 h and immersed in formalin
solution at room temperature for 24 h. After that, tissues were
dehydrated in ethanol and placed into xylene in order to remove
ethanol from the tissues. Furthermore, they were transferred to an
embedding mold of fresh paraffin. All paraffin blocks were then
secured to the microtome and sectioned (4–6 μm in thickness). Finally,
the flatten sections were mounted onto slides and stained with
hematoxylin and eosin (H&E) for histopathological study.
2.3. Electron microscopy
The nodular lesions of skin samples were cut into 6–8 small
squares, approximately 0.5–1 mm. and then fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) overnight at 4 °C, post-fixed
in 1% osmium tetroxide with ferricyanide in PBS at 4 °C for 1 h. The
specimens were then dehydrated in an ascending ethanol series and
finally embedded in Epon epoxy resin following standard procedures.
Ultrathin sections were double-stained with lead citrate and uranyl
acetate for observing and photographing with a transmission electron
microscope (TEM).
3. Results
3.1. Macroscopic finding
The fish samples showed multifocal to diffuse white, firm, papillomalike nodules scattered on the skin, fins, eyes and mouth (Fig. 1A). The
diameters of nodules were varied in size, approximately 1–2 mm.
3.2. Microscopic finding
Many clusters of lymphocystis cells were observed in the connective
tissues beneath the epidermis (Fig. 1B) at fins, inner layer of operculum,
and gills. Numerous hypertrophied cells with basophilic intracytoplasmic inclusion bodies were in the connective tissues of dermis and
between the scales. The lymphocystis cells were also detectable in
skeletal muscle, gill lamellae and visceral organs including spleen, head
and trunk kidney. The lymphocystis-granulomatous formations,
resulted from chronic inflammatory response, appeared in the
parenchyma of the spleen and kidney as well (Fig. 1D).
The lymphocystis hypertrophied cell, was surrounded by a thick
smooth hyaline capsule. The nucleus of lymphocystis cell was
enlarged, irregular and contained basophilic marginated chromatin.
Abundant basophilic intracytoplasmic inclusion bodies, mainly in the
peripheral area of cell, were characterized in hypertrophied cell
(Fig. 1C). The cytoplasm was stained unevenly in color by hematox-
ylin and eosin. The senile lymphocystis cells became an irregular and
broken hyaline capsule. Their nuclei disappeared and their cytoplasms
were released partly or totally.
3.3. Electron microscope
The presence of viral particles in nodules of affected skin was
observed by transmission electron micrographs. These dispersed
particles were in the cytoplasm of hypertrophied cells. Large
icosahedral viral particles, 200 nm in diameter, revealed an electron
dense nucleocapsid and well-defined electron-lucent envelopes with
surface-associated fibrils (Fig. 1E and F).
4. Discussion
This is the first report demonstrating the presence of LCDV in
cultured false clown anemonefish. The macroscopic finding was
similar to the common characteristics of LCDV infection in fish such as
infected Sarcocentruma rubrum (Wachirachaipaisal and Limsuwan,
1985) and Chanda ranga (Williams et al., 1996) which were reported
in Thailand. Histopathology and electron microscopy confirmed the
typical lymphocystis cells containing iridovirus-like particles.
Acknowledgement
This work was in part supported by a grant from the Faculty of
Veterinary Science, Chulalongkorn University, academic year 2009.
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