Download Susceptibility of farmed juvenile giant grouper

Veterinary Microbiology 177 (2015) 270–279
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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic
Susceptibility of farmed juvenile giant grouper
Epinephelus lanceolatus to a newly isolated grouper iridovirus
(genus Ranavirus)
Chao Peng a,b,1, Hongling Ma a,1, Youlu Su a, Weigeng Wen a, Juan Feng a,
Zhixun Guo a, Lihua Qiu a,*
a
Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture, The South China Sea Fisheries
Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, PR China
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 7 October 2014
Received in revised form 27 February 2015
Accepted 16 March 2015
A ranavirus was isolated from the diseased farmed groupers (Grouper iridovirus in genus
Ranavirus, GIV-R), Epinephelus hybrids (blotchy rock cod, Epinephelus fuscoguttatus
, giant grouper, Epinephelus lanceolatus <), in Sanya, Hainan, in July 2013. In this study,
susceptibility of farmed juvenile giant grouper E. lanceolatus to GIV-R was determined by
intraperitoneally injection. The cumulative mortality reached to 81% at 5 day post
infection. Histologically, severe degeneration with massive pycnotic nuclei in spleen and
kidney tissues was observed, and some small-size inclusion body-bearing cells (IBCs)
existed in spleen. Hemorrhage and infiltration of inflammatory cells were presented in gill,
liver and heart along with tissue degeneration and necrosis of varying severity. The results
of immunohistochemistry analysis showed that the strongest immunolabellings were
obtained from the kidney and spleen tissues, while intermediate intensity signals were
observed in the heart, stomach, gill and liver tissues, and the weakest signals were
obtained from the intestine and brain, but no signal was obtained in eyes. Electron
microscopy revealed that spleen of moribund fish contained many viral particles in
cytoplasm. Interestingly, in surviving fish, abnormal hypertrophic cells were observed in
both splenic corpuscle and renal corpuscle, while no hypertrophic cell was observed in the
other parts of spleen and kidney tissues. Moreover, immunolabellings only stained the
hypertrophic cells in splenic corpuscle and renal corpuscle. This indicated that splenic
corpuscle and renal corpuscle play an important role in GIV-R infection and replication.
ß 2015 Elsevier B.V. All rights reserved.
Keywords:
Epinephelus lanceolatus
Histopathology
Immunohistochemistry
Ranavirus
1. Introduction
Iridoviridae, a large icosahedral enveloped viruses
present in the cytoplasm were divided into five genus:
Iridovirus, Chloriridovirus, Lymphocystivirus, Ranavirus,
* Corresponding author. Tel.: +86 20 89108308.
E-mail address: [email protected] (L. Qiu).
1
These authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.vetmic.2015.03.017
0378-1135/ß 2015 Elsevier B.V. All rights reserved.
Megalocytivirus (Jancovich et al., 2012). Iridoviruses were
well known as causative agents of serious systemic
diseases among feral, cultured, and ornamental fish in
the last decade worldwide (Wang et al., 2007). Among
family Iridoviridae, members of genus Lymphocystivirus,
Ranaviruses and Megalocytiviruses affected finfish. Both
ranaviruses and megalocytiviruses cause severe systemic
disease, occur globally and affect a diversity of hosts.
Ranaviruses are also significant pathogens of amphibians.
In contrast, lymphocystiviruses, although widespread in
C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279
fish, rarely cause economic loss (Whittington et al., 2010).
The genus Megalocytivirus included red sea bream iridovirus
(RSIV), infectious spleen and kidney necrosis virus (ISKNV),
turbot reddish body iridovirus (TRBIV), dwarf gourami
iridovirus (DGIV), Taiwan grouper iridovirus (TGIV), Sea
bass iridovirus (SBIV) and rock bream iridovirus (RBIV),
which caused significant mortality in multiple species of
marine and freshwater fish (Inoue et al., 1992; Kurita and
Nakajima, 2012; Shuang et al., 2013). Histopathological
features of genus Megalocytiviruses were the formation of
distinctive hypertrophied cells sometimes in large numbers
throughout various organs, especially spleen (Whittington
et al., 2010). The frog virus 3 (FV3), epizootic haematopoietic
necrosis virus (EHNV), European catfish virus (ECV), largemouth bass virus (LMBV), Singapore grouper iridovirus
(SGIV) and grouper iridovirus (GIV) were classified into
genus Ranaviruses, which caused severe necrosis to internal
organs of many fishes, especially in spleen and renal
haematopoietic tissue (Ahne et al., 1989; Chao et al., 2002;
Chinchar, 2002; Langdon and Humphrey, 1987; Langdon et
al., 1988, 1986; Murali et al., 2002; Pozet et al., 1992; Plumb
et al., 1996, 1999; Qin et al., 2003). More and more evidences
showed that ranavirus have become a significant cause of
disease in ectothermic animals, and that form a virological,
commercial and ecological point of view deserve additional
study (Chinchar, 2002).
In July, 2013, acute outbreaks of disease occurred
among E. pinephelus hybrid groupers (blotchy rock cod,
Epinephelus fuscoguttatus , giant grouper, Epinephelus
lanceolatus <) in Sanya, Hainan. We isolated a pathogenic
iridovirus from diseased hybrid groupers using MFF-1
cell lines, and named here as GIV-R-SY1301. Multiple
sequence analysis identified that the whole nucleotide
sequence of MCP had four base difference between king
grouper iridovirus (KGIV) and Singapore grouper iridovirus (SGIV) (Unpublished data by Ma et al.,). During 2001
and 2009, 11 isolates of iridovirus were collected from
giant grouper (E. lanceolatus) in Tainan (Huang et al.,
2011) and iridovirus similar to GIV-R was found in giant
grouper by epidemiological investigation in Hainan,
China. Because E. lanceolatus as the male parent of the
hybrid groupers has been cultivated with other groupers
271
widely in Southeast Asia. So, the ranavirus is also a
potential threat for giant grouper aquaculture. In the
present study, infection experiments were performed to
examine the susceptibility of E. lanceolatus to GIV-R. By
means of H&E and immunohistochemistry, we discussed
the histopathological changes and distribution of GIVR in
different tissues.
2. Materials and methods
2.1. Virus
GIV-R-SY1301 was isolated from naturally diseased
Epinephelus hybrids (blotchy rock cod, E. fuscoguttatus
, giant grouper, E. lanceolatus <) (Unpulished data by
Ma et al.,). GIV-R was cultured at 25 8C in MFF-1 cells
maintained using Dulbecco’s modified Eagle’s medium
(DMEM) (Invitrogen, USA) with10% (V/V) fetal bovine
serum (FBS, Gibco), 100 IU ml1penicillin G and
100 mg ml1streptomycin (Dong et al., 2008). After
centrifugation (12,000 g, 10 min, 4 8C), viral culture
supernatants were subdivided into small quantities and
stored at 80 8C until use. Titration of viral infectivity was
performed using 96-well microplates seeded with MFF-1
cells. After 5 days of culture, the appearance of cytopathic
effect (CPE) was evaluated to determine the 50% tissue
culture infectious dose (TCID50).
2.2. Experimental design
Naive healthy E. lanceolatus (5 g, average weight) were
obtained from a local farm and maintained for acclimatization in culture base of South China Sea Fisheries
Research Institute, Lingshui county, Hainan Province,
China. Animals were fed three times a day and the
seawater was changed daily with sedimentated and sandfiltrated seawater. During the experimental period, water
salinity readings were 3.2%, temperature was between 25
and 28 8C and water was kept continuous aeration.
Fishes were distributed into two groups: test group
(n = 36) and control group (n = 30). Test group was
challenged by intraperitoneally with 105.5TCID50 fish1
Fig. 1. Clinical signs of GIV-R-infected E. lanceolatus. A. Control fish. B. GIV-R infected fish showed soft muscle (black arrow) and congestion of spleen, liver
and gill (white arrow).
C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279
of virus cell supernatant. Control group was challenged by
intraperitoneally with 100 ml fish1of DMEM medium.
Both groups were maintained under similar conditions
in a separate tank. Fish were kept daily management and
mortality was monitored daily. The tissue of brain, eye,
heart, liver, spleen, stomach, intestines, kidney and gill of
moribund fish from test group were sampled for histological and immunohistochemistry analysis. When the mortality was stable, the control fish and surviving fish of test
group were also sampled for histological and immunohistochemistry analysis.
2.3. DNA extraction and PCR detection
Total genomic DNA (gDNA) was extracted from liver
tissue using DNA extraction kit special for marine
animals (Tiangen, China) following the manufacturer’s
protocol. The primer-pair F (50 -ATGACTTGTACAACGGGT30 ) and R (50 -TTACAAGATAGGGAACCCCAT-30 ) were used
to amplify the MCP of GIV-R (Huang et al., 2011). The PCR
was performed in a total reaction volume of 25 ml
containing 18.3 ml of PCR-grade water, 2.5 ml of 10
Reaction Buffer, 1 ml of dNTP mix, 1 ml of each of the
primers (10 mM), 0.2 ml r-Taq DNA polymerase
(TOYOBO) and 1 ml of template. The PCR parameters
using the primer-pair F and R were as followings: 1 cycle
of 94 8C for 10 min, 35 cycles of 94 8C for 40 s, 55 8C for
40 s, and 72 8C for 90 s, followed by the final extension at
72 8C for 10 min.
2.4. Histopathology
Tissues were fixed in 10% phosphate-buffered formalin
for at least 24 h and dehydrated in an ethanol–xylene
series before embedding in paraffin wax. Formalin-fixed,
paraffin-wax-embedded (FFPE) 5 mm tissue sections were
dewaxed in xylene, rehydrated in an ethanol series and
stained with haematoxylin and eosin (H&E).
2.5. Immunohistochemistry
Formalin-fixed, paraffin-wax-embedded (FFPE) 5 mm
tissue sections were dewaxed and rehydrated according
to the conventional method. To unmask antigen and
inactivate endogenous peroxidase, deparaffinised and
rehydrated sections were treated with 0.01 M citrate
buffer (pH 6), and heated in a microwave oven for 15 min.
Slides were then washed with 0.01 M PBS (NaCl 0.14 M;
KCl 2.7 mM; Na2HPO412H2O 10 mM; KH2PO4 1.7 mM;
pH 7.2) three times for 5 min. Tissue sections were
blocked with 5% (w/v) bovine serum albumin (BSA) for
30 min, and incubated at 37 8C for 30 min. Subsequently,
50 ml of mouse polyclonal anti-GIV-R MCP serum (1:300,
dissolved by 0.01 M PBS, donated by Dr. Chuanfu Dong
from the Sun Yat-sen University) was added and the
slides were further incubated at 4 8C for 5 h. The tissues
were then thoroughly rinsed in PBS three times for 5 min
and incubated with a secondary antibody conjugated
universal immunoenzyme polymer using GTVisonTMIII
kit (Gene Tech, China) for 30 min at room temperature.
Tissues were thoroughly rinsed in PBS and developed
with DAB. The reaction was stopped by placing the slides
in distilled water and slides were counterstained with
hematoxylin rinsed in serial graded alcohol and xylene,
and mounted with mounting media. Tissues of healthy
fish and diluent-only sections were used as negative
controls.
2.6. Electron microscopy
Tissues were fixed in 2.5% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.3, for 24 h. The samples were
then washed in phosphate buffer and finally post-fixed
in 1% osmium tetroxide for 1 h. The fixed tissues were
dehydrated in a graded series of ethanol and embedded
in Spurr’s resin. Ultrathin sections were prepared with
an ultramicrotome (Leica Ultracut R, Leica Microsystems, Wetzlar, Germany), and subsequently doublestained with uranyl acetate and lead citrate and
observed at 80 kV with a Jeol TEM-1200EX (Akishima,
Japan).
3. Results
3.1. Clinical signs and cumulative mortality
Diseased juvenile giant grouper displayed either
lethargy or a dark coloration of the body, and sometimes
ascites. The infected fish showed pale gill with petechiae,
congestion of spleen and liver, loose and soft muscle
(Fig. 1B). The mortalities started at 2 dpi and the death
fastigium was during 2–5 dpi. The cumulative mortality
was high to 81% within 5 dpi (Fig. 2). A GIV-R specific DNA
band was detected from all infected fish by PCR (Fig. 3).
3.2. Histopathology
The histopathological features of GIV-R-SY1301
infected E. lanceolatus were the severe degeneration of
spleen and kidney tissues with massive pycnotic nuclei in
the hematopoietic tissue (Fig. 4C and D). Hemorrhage and
infiltration of inflammatory cells were found in gill, liver,
heart, spleen and associated with tissue degeneration and
necrosis of varying severity.
90%
80%
Cumulative mortality
272
test group
70%
control
60%
50%
40%
30%
20%
10%
0%
1
2
3
4
5
6
Days after injection
7
8
9
Fig. 2. Cumulative mortality of E. lanceolatus after GIV-R injection.
C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279
Fig. 3. PCR detection of infected E. lanceolatus. M. DL2000 DNA marker; B1,
B2: Blank; C1, C2, C3: negative control; S1, S2, S3: diseased giant grouper.
273
Histopathological changes in spleen were the most
remarkable. Severe degeneration occurred with some
small-size inclusion body-bearing cells (IBCs) and
massive pycnotic cell nuclei in splenic pulp. Spongiosis
and disruption of ellipsoid sheaths with degeneration of
associated cells were observed (Fig. 4C). Spleen
of surviving fish showed many abnormal hypertrophic
cells in splenic corpuscle (Fig. 4E). Severe degeneration of
glomerulus and tubular epithelium were observed in
kidney. Pycnotic cell nuclei presented in the hematopoietic tissue with karyolysis of tubular epithelium. In some
case, some amorphous materials were observed in
degraded tissues (Fig. 4D). Similarly, kidney of surviving
fish showed masses of abnormal hypertrophic cells in
renal corpuscle (Fig. 4F). Considerable inflammatory
cells infiltration around the hepatic central vein and
portal area were observed in the liver, causing hepatocyte atrophied and diminished. Central vein wall
obviously thickened and partly disintegrated. The cell
Fig. 4. E. lanceolatus. Histopathological changes in spleen, kidney and gill. (A, B) Control. (C) Spleen of diseased fish, some small-size inclusion body-bearing
cells (IBCs) (white arrows) and massive pycnotic cell nuclei in splenic pulp (black arrows) were observed. (E, F) Spleen and kidney of surviving fish, masses of
abnormal hypertrophic cells in splenic corpuscle and renal corpuscle were observed (M). (D) Kidney of diseased fish, severe degeneration of glomerulus (short
arrows) and tubular epithelium (white arrows) were noticed. Pycnotic nuclei (stars) and amorphous materials (black arrows) were observed in the
hematopoietic tissue.
274
C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279
Fig. 5. E. lanceolatus. Histopathological changes in liver, heart, stomach and intestines. (A, B, E, F) Control. (C) Liver of infected fish, Infiltration of
inflammatory cells and macrophages in portal area casued hepatocyte atrophy (white arrows); venous blood wall became thick and ruptured (black arrow).
(D) Heart of infected fish, infiltration of inflammatory cells (star), atrophy of cardiomyocytes (black arrows), and swell of myocardial fibres resulting in
reduced staining affinity (white arrows) were seen. (G) Stomach of infected fish, gastric gland atrophied and partly degraded in gastric mucosa (black
arrow). (H) Gill of diseased fish, blood vessel in gill was expanded associated with tremendous erythrocyte infiltration in the sinusoid of gill filaments (star);
Epithelial hyperplasia, desquamation (black arrow), epithelial lifting (short arrow), or telangiectasis (white arrow) were observed in the secondary lamellae.
C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279
275
Fig. 6. E. lanceolatus. Transmission electron microscopic observation of spleen. Massive virions were observed in cytoplasm (white arrows).
boundaries blurred and showed necrosis in local area
(Fig. 5C). In the heart, infiltration of inflammatory cells
caused atrophy of cardiomyocytes with swelling of
myocardial fibres, resulting in reduced staining affinity
(Fig. 5D). Blood vessel in gill was expanded and fused
with tremendous erythrocyte infiltration in the sinusoid
of gill filaments. Epithelial hyperplasia, desquamation,
epithelial lifting, fusion or telangiectasis was noticed in
the secondary lamellae with the breakdown of the pillar
cell system (Fig. 5H). In stomach, gastric gland atrophied
and partly degraded in gastric mucosa (Fig. 5G). No
histopathological change was observed in the intestines,
eye and brain. Transmission electron microscopic result
showed that many enveloped hexagonal virions measuring 180–200 nm were observed in cytoplasm (Fig. 6A
and B).
3.3. Immunohistochemistry analysis
The strongest immunolabellings were obtained from
the spleen tissues. They were accumulated in the
basophilic small-size inclusion body-bearing cells and
were widespread in splenic pulp (Fig. 7C). In the kidney,
positive reactions were mainly in the renal glomerulus
and its surrounding hemopoietic tissues. But, there was
no antigen labeling existed in renal tubular epithelial
cells (Fig. 7D). In heart, strong positive immunolabellings were widespread in cardiac muscle fibers, epicardium and chambers of the heart (Fig. 9C and E). In the
liver, a few immunolabellings were observed only in the
portal area of necrotic hepatocyte (Fig. 8B). Immunolabellings in stomach were mainly in the submucosa
where had rich blood vessels (Fig. 9D), secondly in the
mucosa (Fig. 9F), and slightly in serosa. In the intestine,
positive reactions were visualized mostly in submucosa
and serosa (Fig. 8D). Immunolabellings within the gill
were detected in the afferent artery and capillary vessel
lumen, especially in capillary vessel lumen around
cartilage tissue (Fig. 7H). Weak immunolabellings were
only found in brain outer membrane (Fig. 8F). No
immunolabelling was observed in eyes. Immunolabellings were only observed in splenic corpuscle and renal
corpuscle in surviving fish of kidney and spleen (Fig. 7E
and F).
4. Discussion
Grouper, the major species being maricultured in China
and other SE Asian countries, are high-priced and popular
seafood fish. Nevertheless, iridoviruses have caused high
mortality in many cultured grouper species in the last
decades, which suggested its a severe threat to groupers
aquaculture (Chua et al., 1994; Gibson-Kueh et al., 2004;
Mahardika et al., 2004; Qin et al., 2003). Our infection
experiments results showed that naive giant grouper was
susceptible to GIV-R infection with high mortality (80%).
The newly isolated ranavirus GIV-R could cause severe
systemic disease to juvenile giant grouper, characterized
by degeneration and necrosis of varying severity in inner
organs, especially in spleen and kidney. This is the same as
ranavirus and megalocytivirus infections which also
caused systemic infection involving multiple internal
organs with high mortalities (Williams et al., 2005). What
is different is that there were only few small-size (about
10 mm) inclusion body-bearing cells (IBCs) existed in
spleen, but none in other tissues. However, other
iridoviruses diseased fishes showed systemic formation
of prominent enlarged cells as inclusion body-bearing cells
(IBCs) in different organs, such as in large yellow croaker,
striped beakperch, angelfish, farmed turbot, mandarinfish,
African lampeye and dwarf gourami with iridovirus
infection (Chen et al., 2003; He et al., 2000; Jung and
Oh, 2000; Shi et al., 2004; Sudthongkong et al., 2002), as
well as in grouper fishes with the iridovirus infection:
brown-spotted grouper (Chua et al., 1994), Malabar
grouper (Sano et al., 2002), cultured groupers (Chou
et al., 1998), juvenile humpback grouper (Mahardika
et al., 2004) and hybrid grouper (Chao et al., 2004). The
enlarged cells were about 20 mm in diameter and were
obvious larger than GIV-R infected cells. The IBCs in some
of iridovirus infected fish included three types: The early
stage of IBCs were hypertrophied blast-like cells that
possessed a basophilic cytoplasm and a centrally located,
enlarged nucleus containing prominent nucleoli; The
mature IBCs were enlarged and usually had an entirely
basophilic cytoplasm and either a centrally or marginally
located nucleus; The ballooning, degenerated IBCs contained an inclusion body with a granular appearance
within a marginally compressed, narrow cytoplasm
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C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279
Fig. 7. E. lanceolatus. Results of immunohistochemistry in spleen, kidney and gill. The signal is observed microscopically as brown yellow staining. (A, B, G)
Control. (C, D, H) Diseased fish. Strong immunolabellings were accumulated in the basophilic small-size inclusion body-bearing cells and were widespread
in splenic pulp. In kidney, strong immunolabellings were observed in renal corpuscle and its surrounding hemopoietic tissues. In gill, immunolabellings
were detected mainly around the afferent artery and capillary vessel lumen, especially in capillary vessel lumen around cartilage tissue (black arrow). (E, F)
Surviving fish of kidney and spleen. Immunolabellings were only observed in splenic corpuscle and renal corpuscle.
C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279
277
Fig. 8. E. lanceolatus. Results of immunohistochemistry in liver, intestines and brain. The signal is observed microscopically as brown yellow staining. (A, C,
E) Control. (B, D, F) Diseased fish. In liver, only a few immunolabellings were found in portal area of necrotic hepatocyte; In intestines, immunolabellings
were detected in submucosa and serosa; In brain, only few immunolabellings were found in outer membrane of brain (black arrow).
containing a pyknotic or fragmented nucleus (Mahardika
et al., 2004; Sudthongkong et al., 2002). In GIV-R infected
juvenile giant grouper, it seemed to have only small
mature IBCs in spleen. Another prominent histopathological feature of GIV-R was the formation of massive pycnotic
cell nuclei in splenic pulp and renal hematopoietic tissue.
The histopathological changes in our study were most
similar to ‘Sleepy Grouper Disease’ infected brown-spotted
grouper (Chua et al., 1994).
Immunohistochemistry using mouse GIV-R MCP antiserum showed a widespread distribution of the virus in
tissues. The strongest immunolabellings were obtained
from the kidney and spleen tissues. The intermediate
intensity signals were observed in the heart, stomach, gill
and liver tissues. The weakest signals were obtained from
the intestine and brain. The signals were specifically
located within epithelial, endothelial, leukomonocyte and
macrophages. No signal was obtained in eyes. Immunolabellings in spleen were accumulated in the basophilic
IBCs and were widespread in splenic pulp. Many reports
have proved that iridovirus infected and proliferated in the
enlarged cells or inclusion body bearing cells by IHC and
ISH. For example, immunohistochemical and nucleic acid
signals were labeled mainly in the enlarged cells in ISKNV
infected zebrafish and SGIV infected Malabar grouper
(Huang et al., 2004; Xu et al., 2008), DNA hybridization
signals were only obtained in basophilic enlarged cells of
spleen in TGIV infected Epinephelus hybrids (Chao et al.,
2004). Moreover, many viral particles were observed in
inclusion body-bearing cells of many iridovirus infected
fishes by electron microscopy (Mahardika et al., 2004;
Sudthongkong et al., 2002). Necrosis and karyolysis of
tubular epithelium was serious and immunolabellings
were only found in renal glomerulus and its surrounding
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C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279
Fig. 9. E. lanceolatus. Results of immunohistochemistry heart and stomach. The signal is observed microscopically as brown yellow staining. (A, B) Control.
(C, D, E, F) Diseased fish. In heart, strong immunolabellings were found in cardiac muscle fibers, epicardium and chambers of the heart; In stomach,
immunolabellings were mainly in submucosa and mucosa where had rich blood vessels (black arrows).
hemopoietic tissues, but not in tubular epithelium. The
results were similar to the report that no signal was found
in tubular epithelium examined by IHC and ISH (Cano et al.,
2009). This indicated that the lesions to tubular epithelium
seem not to be directly virus-related. Immunolabellings in
heart, liver, stomach, gill and digestive tract were mainly in
the sites where it had rich blood vessels. Hence, once
primary replication has taken place, virus can reach the
bloodstream, resulting in viraemia, as a step leading to
systemic infection (Ogawa et al., 1990).
On the other hand, both splenic corpuscle and renal
corpuscle of surviving fish were completely replaced by
abnormal hypertrophic cells (Fig. 54 and F), while no
hypertrophic cell was observed in the other parts of spleen
and kidney tissues, same case was found in juvenile
humpback grouper Cromileptes altivelis infected by grouper
sleepy disease iridovirus (GSDIV). Mahardika et al. (2004)
had been described them as ‘abnormal IBCs’ who contained
deformed virions and had no granular masses associated
with viral DNA and organelles (Mahardika et al., 2004).
Interestingly, immunolabellings in spleen and kidney of
surviving fish were only found in the abnormal hypertrophic cells, which confirmed that the abnormal hypertrophic cells contained massive viral major capsid protein,
perhaps, they are deformed virions, and whether they had
viral DNA remains unknown. In surviving fish, immunolabellings were only limited in splenic corpuscle and renal
corpuscle, it maybe because splenic corpuscle and renal
corpuscle prevented GIV-R from spreading to other sites, or
splenic corpuscle and renal corpuscle were the target site
of GIV-R infection and reproduction. The specific mechanism needs to further study in the future.
C. Peng et al. / Veterinary Microbiology 177 (2015) 270–279
5. Conclusion
To sum up, GIV-R can cause fatal systemic diseases to
giant grouper. It causes acute histopathological lesions in
inner organs. Antigens of GIV-R existed in most tissues of
diseased giant grouper, but distributed mainly in hemopoietic tissue of spleen and kidney. Splenic corpuscle and
renal corpuscle seem to have special role for GIV-R
infection.
Acknowledgements
This research was supported by the key Science and
Technology Program of Hainan Province under Grant No.
ZDXM20120031 and Special Foundation of Fish Disease
Prevention and Control of Guangdong Province (2012).
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