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Experimental infection of European crustaceans

V Corbel, Zuprizal, Z Shi, C Huang, Sumartono, J-M
Arcier and J-R Bonami
Journal of Fish Diseases 2001, 24, 377-382
White spot syndrome virus (WSSV) is one of
the names used for the virus causing white
spot disease (WSD) in shrimp. The major
clinical sign of this disease is the presence
of white spots in the cuticle of heavily
infected shrimp. Mortalities reach 100%
within 3-8 days after infection with WSSV.
Several reports have described the
explosive epidemic character of WSD in
penaeid shrimp production ponds.
Detection of WSSV in cultured and wild
shrimp, crabs and other arthropods has
demonstrated the wide range of hosts for
Shrimps infected by WSSV
The crabs Liocarcinus depurator, L. puber, Cancer
pagurus and C. maenas, the freshwater crayfish,
Astacus leptodactylus and Orconectes limosus, the
common Mediterranean grey shrimp, Palaemon
adspersus and a lobster, Scyllarus arctus were
inoculated using a tuberculin syringe with 0.1 or 0.2
mL (depending on the size of the experimental host)
of haemolymph obtained from shrimp, Penaeus
monodon, infected with WSSV.
For per os infections, animals were fed with sliced,
previously frozen, PCR-positive, WSSV infected
shrimp during the first day of the experiment.
Thereafter, they were given normal feed.
Clinical signs of infection and mortality of the
animals were subsequently recorded daily.
Haemolymph was withdrawn from moribund
and dead animals using a syringe and
examined by direct TEM. Carcasses and
haemolymph were stored at -20 °C until
further analysis.
Collected haemolymph and puri®ed virus
suspensions were negatively stained with 2%
sodium phosphotungstate (PTA, pH 7.0) on
collodoin-carbon coated grids.
Haemolymph of animals was boiled for 5 min
at 100 °C, then centrifuged at low speed. One
lL of clarified suspension was spotted on a
positively charged nylon membrane and fixed
with UV for 3 min. Hybridization followed the
protocol of Durand et al.
Samples of healthy and infected animals were
fixed in Davidson's fixative and subsequently
paraffin embedded. Sections (4-6 μm thick)
were mounted onto positively charged
microscope slides (polylysine treated) and
were used for in situ hybridization using
digoxigenin labelled probes according to the
protocol described by Durand et al. Sections
of healthy animals were also used to test the
probe specificity.
Samples of healthy and infected tissues held at 4
°C, were homogenized and centrifuged for 10
min at 100 000 g. The supernatant was boiled for
10 min. The PCR reaction solution contained
buffer 1X (100 mm Tris-HCl, 150 mm NaCl, pH
7), MgCl2 at 2.5 mm, digoxigenin labelled
nucleotide, a mix of nucleotides (2.5 mm for
each dNTP), primers sL46 and asL46 (0.5 μm), 2
units of Taq polymerase and 1 μL of the sample
supernatant (infected or healthy gills and
pleopods) for a final volume of 50 μL. The
primers were determined from the sequence of
the L46 WSSV-genome fragment.
Clinical signs were observed after 3 days
postinjection for L. puber and L. depurator
and mortality reached 50%. Successful WSSV
infections in all of the crustaceans caused a
rapid reduction in feed intake and lethargy,
as in infected shrimp. The affected crabs
(especially L. puber) displayed pale
discolouration and appeared very weak.
Moribund L. puber had some white spots on
their legs.
All the injected decapod species in this
experiment showed between 70 and 100%
mortality by 20 days post-infection. It was
common to see a rapid increase in mortality
over 4 or 5 days, which is generally
characteristic of viral infections. However, no
white spots were observed in the cuticle of
moribund or dead animals (except for L.
puber), possibly as a result of the rapid
mortality after injection
All species
infected per os or
by injection,
except C. maenas,
enveloped virions,
and free viral
WSSV in haemolymph of infected
depurator. Enveloped virion (large
arrowhead) and nucleocapsid (small
Most of the samples tested
(L. depurator, L. puber, C.
pagurus, A. leptodactylus,
O. limosus, P. adspersus
and S. arctus) showed
positive hybridization spots
with the two probes.
Positive hybridization
reactions were also seen in
species infected per os (S.
arctus, A. leptodactylus, L.
puber, P. adspersus and C.
pagurus). No reaction was
observed in the negative
control animals or in the
gills and the haemolymph
from C. maenas.
Dot-blot of infected gill samples from Palaemon
adspersus. Positive hybridization reactions are seen for
many of
the infected dead animals, while there is no reaction with
water and the negative control (healthy gill sample): T.:
control, Tÿ: negative control, SW: sterile water, IfD:
dead animals
In infected animals, subcuticular cells, connective tissue, heart
muscle, and cells in the haemal sinuses of the hepatopancreas
gave strong hybridization reactions (Figs 3±5). No hybridization
was seen in the tissues of healthy animals.
In situ hybridization in infected gill sections from
Astacus leptodactylus. The nuclei of the epidermis
(arrows) and
connective tissue cells show strong hybridization
(black) reactions
In situ hybridization in infected gill sections of Cancer
pagurus. Nuclei of the subepithelial cells (arrows) are strongly
A positive
reaction, visualized
as a specific
fragment of 494 bp,
was observed in all
of the samples
tested, except the
tissue of a healthy
PCR diagnosis of infected gill extracts from Liocarcinus
puber. L: 100 pb ladder, 1: sterile water, 2: negative
control, 3-7:
infected samples.
The ease in obtaining experimental WSSV
infections per os or by injection in European
crustaceans shows that this agent must be
considered as a potential threat to these