Occurrence of white spot syndrome disease (WSSD) in farmed
Penaeus indicus in Iran: Clinical, histopathological, cytological and
polymerase chain reaction (PCR) observations
M. Afsharnasab1*, S. Akbari2, B.Tamjidi3, F. Laloi4 & M. Soltani5,
1 Iranaian Fisheries Research Organization, Theran, Iran, 2Fish Health Unit,
Faculty of Veterinary Medicine, University of Shiraz, Shiraz, Iran; 3Fisheries
Research Center, Ahvaz, Iran; 4Fishereis Research Center, Sari, Iran;
5Department of Aquatic Animal Health, Faculty of Veterinary Medicine,
University of Tehran, Tehran, Iran
ABSTRACT
During June till July 2002 a rapid and high mortality occurred in cultured P.
indicus in Abadan region located in the south of Iran. Affected shrimps were
clinically lethargic showing typical white spots inside carapace with loss of
appetite. They then become reddish in body coloration and showing yellow
swollen hepatopancreases. Histological examinations of fixed tissues in
Davidson from affected shrimps resulted in observations of intranuclear
Cowdry type-A inclusion bodies in all examined tissues except for
hepatopancreas under light microscopy. In transmission electron microscopic
(TEM) examinations the nuclei of infected cells were slightly hypertrophic with
marginated chromatins along the nuclear membranes and disappearance of
nucleoli. In the late stage of disease outbreak the intranuclear Cowdry type A
inclusion bodies were seen in the profiles. The virions were elliptical to short
rods with trilaminar envelopes that measured 248±87×107±11nm and
nucleocapsids were 162±15×59±27nm. Polymeras chain reaction (PCR)
studies using commercial white spot syndrome virus kit revealed two bands of
356 bp and 403 bp of DNA fragments. This is the first report of WSSD in
farmed P. indicus in Iran.
KEY WORDS: Penaeus indicus, white spot syndrome virus, histopathology,
transmission electron microscopy, PCR
Corresponding authors: E-mail: mafsharnasab@yahoo.com
INTRODUCTION
White spot syndrome (WSS; synonymly white spot baculovirus /white spot
syndrome baculovirus disease) (Lightner 1996) is one of the most important
shrimp diseases. It is of the global occurrence and it affects most of the
commercially cultured shrimp species (Inouye et al. 1994, Chou et al. 1995,
Lightner 1996, Flegel 1997, Spann and Lester, 1997). The disease is
characterised by distinct white spots in the cuticle, lethargy with cumulative
mortalities often reaching 100% within 2 to 7 days post-infection (Chou et al.
1995, Huang et al. 1995, Wongteeraspaya et al. 1995). The causative agent
of WSS is an enveloped, non occluded, rod-shaped DNA virus known as white
spot syndrome virus (WSSV) (Wang et al.1995, Lightner 1996). A general
name referred to as “white spot syndrome (WSS)”is often used for a number
of outbreaks by viral agents e.g. systemic ectodermal and mesodermal
baculovirus (SEMBV) which gives gross signs of infection in the form of white
spots on the carapace at the late stage of infection (Wongteerasupaya et al.
1995). Similar viral infections were found in different countries implicated with
several penaeid species and the causative agents have been given different
designation, namely white spot syndrome baculovirus (Chou et al. 1995),
hypodermal and hematopoiteic necrosis baculovirus (HHNBV) (Huang et al.
1995) and penaeid rod shaped DNA virus (PRDV) (Takahashi et al. 1994).
These viruses from different locations appeared to be very similar in disease
symptoms, histopathology, morphology and genomic structure (Flegel 1997).
Therefore, they were increasingly considered as the same or of different
strains, and grouped as white spot syndrome virus (WSSV) (Kasornchandra
and Boonyaratpalin 1998). Analysis of a genomic segment of WSSV favoured
the view that this virus was either a member of a new genus (whispovirus)
within the baculoviridea or a member of an entirely new virus family
Whispoviridae (van Hulten et al. 2000).
Recent rapid development of prawn farming in Iran has raised a number of
treats to the industry including health management issues. It was expected to
increase the total production from 7000mt in 2001 to 8000mt in 2002.
However, the occurrence of a highly contagious disease with rapid mortality
in some farmed P. indicus caused a significant reduction in production to
4500mt in 2002. This paper present the results of our investigations on the
causative agents of this mortality in farmed P. indicus.
MATERIAL AND METHODS
About 100 PL20 and Juvenile live specimens of affected cultured P. indicus
were collected from Abadan region, south of Iran during June till July 2002.
The collection of samples was based on case history from the field and
general inspections including wet mount preparation in the laboratory. About
30 moribund shrimp samples were collected in cold Davidson’s fixative for 24
to 72 hours (large shrimps were first injected with the fixative and were then
kept in the fixative for longer period) at room temperature, transferred to
50% ethyl alcohol until required for routine histological process (Bell and
Lightner 1988, Lightner 1996). Sections of paraffin embedded tissues were
cut at 5 µm, stained with modified Mayer’s hematoxylin and eosin/phloxine
(H&E/Phloxine) staining (Luna 1968, Bell and Lightner 1988) and were then
examined under compound microscope.
Ultrathin sections for transmission electron microscopy (TEM) examinations
were prepared by obtaining 1-2 mm3 pieces of exoskeleton, hepatopancreas,
lymphoid organ, heart, midgut and gills of moribund shrimps and fixing with
cold 4% glutaradehyeds in 0.2 M cacodylate phosphate buffer (pH 7.4) at 4°C
overnight and postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer
for 1-2 hours. The tissues were then dehydrated in graded acetone and
embedded in the resin mixture. Approximately 1µm thick sections were
prepared from each tissue block and stained with 1% methylene blue for
approximately 1 minute at 60º C. The 50-80 nm ultrathin sections were then
prepared from the blocks showing desired degree of tissue destruction or the
presence of nuclear inclusion bodies using diamond knives on a
ultramicrotome. Mounted sections on a copper grid were then stained with
uranyl acetate and lead citrate (Hayat 1986, Adams & Bonami 1991) and
examined under CM 10 Philips transmission electron microscope (TEM).
Twenty samples including gill and hepatopancreas were also collected and
fixed in 75% alcohol for polymeras chain reaction (PCR) studies. A
commercial white spot syndrome virus kit (Single–Tube Nested DNA
Amplification Kit for detection of white spot syndrome virus, Cat. No. KWSFT
Genensis Biotechnology Sdn. Bhd, Selangor, Malaysia) was used for PCR
works. Briefly, aliquots (2µl) of DNA samples were added to 23µl of reaction
mixture for DNA amplification. Thermo cycling conditions for the PCR were:
94º C for 3 min (1 cycle), followed by 94º C for 30 sec, 60º C for 30 sec, 72º
C for 30 sec (5 cycles), followed by 94º C for 25 sec, 60º C for 25 sec, 72º C
for 30 sec (15 cycles), followed by 94º C for 25 sec, 50º C for 25 sec, 72º C
for 25 sec (6 cycles), followed by 94º C for 25 sec, 49º C for 25 sec, 72º C for
25 sec, followed by 72º C for 3 min as final extension. PCR products (10µl)
were mixed with loading solution containing bromophenol blue and were
analysed by electrophoresis on 2-2.5% agarose gel containing 1µg/ml
ethidium bromide solution. According to the kit guidance positive samples
infected with WSSV will result in the amplification of a 356-bp viral DNA band
for light infected samples while moderate to severely infected samples will
result in the amplification of both 403-bp and 356-bp bands.
RESULTS
Clinical observations
Clinically affected P. indicus were lethargic in behavior showing loss of
appetite, followed by the appearance of moribund shrimps swimming near the
surface at the edge of ponds within a few days post-outbreak. Also, pink to
reddish-brown coloration of the body surface and white spots of about 0.52mm in diameter on the cuticle especially on the inner surface of the
exoskeleton of cephalothorax and abdomen were predominant observable
gross lesions (Figure 1). The cuticle could be easily separated from the
underlying epidermis and the hepatopancreas became yellow-white with a
swollen and fragile texture (Figure 2). Cuticular deformities such as broken or
withered antennae and damaged rostrum, opaque abdominal musculature
and melanised gills were also frequently observable. Wet mount examination
of the gills showed the contamination with some epicomensal and fouling
organisms, in particular with Zoothamanium spp. A mortality of 70-100%
occurred within 3 days post-outbreak of disease in some farms showing
typical white spots sings on the shells of the affected shrimps.
Histopathological findings by light microscopy
Histologically, there were intraunclear eosinophilic Cowdry type-A inclusion
bodies observable in gills, midgut, cuticular epidermis, lymphoid organ, heart
and connective tissue (Figures 3, 4 & 5). Such inclusion bodies were not
finding in the infected hepatopancratic epithelial cells (HECs) even in
moribund specimen (Figure 6). Although HECs have no viral inclusion, the
infiltrated hemocytes within interstitial spaces were highly infected. The
hepatopancreas showed cellular vacuolation resulting in diminution of the
tubule lumen. These might be the reasons why hepatopancreas become
swollen and fragile. The early stage of infection was generalized by epithelial
hyperplaisa containing hypertrophic nuclei with marginated chromatins and
eosinophilic to basophilic intranuclear inclusion bodies. With the progress of
infection the inclusion bodies were separated by a halo from the marginal
chromatin. At the advanced stage of infection the nuclei were disintegrated,
and the halo spaces were seen in the cells. There was also focal to multifocal
necrosis together with nuclear hypertrophy and vacuolar space around the
nucleus containing the inclusions.
Transmission electron microscopy
Under TEM the nuclei of infected cells showed slight hypertrophy and
marginated chromatins along the nuclear membranes. The nucleolus and the
chromatin were fused causing the central spaces of the nucleus to become
thin and hemogenus (Figure 7). The progressive of infected cells revealed
intranuclear ovoid, elongated or elliptical virions resembling typical nonoccluded baculo-like forms within remarkable hypertrophic nuclei. The virions
which were both tangently and crossly cut measured 248 ± 87×162 ±15 nm
and the nucleocapsids of 162 ± 15 × 59±27 nm with a well development
trilaminar envelope (Figure 8). In some hypertrophic nuclei the immature viral
particles with empty capsid, nucleocapsid and circular envelope along with
fully mature virions were detected in the nucleoplasms.
Polymerase chain reaction (PCR)
As showed in Figure 9 two bands of 356-bp and 403-bp fragments were
observed after 30 cycles of PCR amplification of the viral genomic of
examined samples. As mentioned before according to the kit guidance the
positive samples infected with WSSV will result in the amplification of a 356bp viral DNA band for light infected samples, while moderate to severely
infected samples will result in the amplification of both 403-bp and 356-bp
bands. Also, the appearance of 232 bp shrimp DNA product (false negative
control) and 153 bp WSSD DNA products in the positive control confirms the
validity of the obtained result.
DISCUSSSION
The clinical observations including white spots on the carapace and
appendages and yellowish discoloration of hepatopancreas cuticular,
deformities such as broken or withered antennae and damaged rostrum,
opaque abdominal musculature and melanised gills with rapidly high mortality
up to 100% observable in this outbreak resembling WSSD as described by
other authors e.g. Momoyama et al. (1994); Takahashi et al. (1994); Chou et
al. (1995); Wang et al. (1995); Wongteerasupaya et al. (1995); Wang et
al.(1999). However, such clinical findings could be seen in other shrimp viral
infections such as IHHNV causing by a 22nm Parvovirus agent (Lightner
1996).
Widespread focal to diffuse cellular degeneration, unclear hypertrophy in most
tissues of ectodermal and mesodermal origins, necrosis in hepatopancreas,
haemocytic infiltration and hypertrophic nuclei containing eosinophilic to
basophilic inclusion bodies of affected cells have been described as some
microscopic characteristics of white spot syndrome disease by Nash &
Akarajamon (1995), Chou et al. (1995) and Wang et al. (1999). In the
present study, identical histological features were observed in both early and
late stages of the affected P. indicus except for ovarian tissues that showed
prominent necrosis and inclusion bodies.
Since 1992, the etiological agents of white spot syndrome disease in cultured
Penaeid shrimps have been described by several researchers from different
countries (e.g. Inouye et al. 1994; Takahashi et al. 1994; Huang et al.1995;
Chou et al.1995; Wang et al. 1995; Wonggteerasupaya et al. 1995; Wang
2000). Comparatively, the length and width of the observed viral capsids in P.
indicus are almost identical to other studies with a slight different length for
this viral particle that may be due to deviating in tangential section.
Although, the size of observed virus in this study is similar to Penaeus
monodon baculovirus (MBV) and baculoviral midgut gland necrosis virus
(BMN), its virion is much thicker in width and has darker or denser viral core
as well as having an appendage without inclusion bodies. However, MBV has
been only detected from the hepatopancreas and midgut (Lightner et al.
1983), while BMN differs from this virus in term of viral morphology (an
occluded baculovirus) and its restriction to hepatopancreas and midgut
tissues (Sano et al. 1981, 1984).
Moreover, except their similarity in morphology, all these agents associated
with white spot disease are different in target organ, histopathology, viral
nucleic acid content, clinical signs and epidemiological aspects. Therefore,
probably all these causative agents associated with white spot disease are the
same or of different strains.
Primer sets have been designated for PCR identification of WSSV by a number
of researchers from different countries such as Taiwan, Thailand, Japan and
India. Wongteerasupaya et al. (1995) reported an average size of about 168
kbp. for SEMBV genomic DNA fragment in the agarose gel. Similar result was
obtained by Wang et al. (1995) who have estimated the genomic DNA
above150 kbp for the viral agent associated with WSS in P. monodon. Also,
the size of viral agent responsible for WSSD in Malaysian prawn farming has
been estimated 305±30x127±11nm (Wang et al. 1999). In the present study
using commercial WSSV specific primer set (Single-Tube Nested DNA
Amplification Kit) the genomic DNA of viral agent from affected P. indicus
estimated about 356 bp and 403 bp. Therefore, because of similar clinical
observations, histopathological and cytological findings as well as PCR results
both WSSV from Malaysia and Iran are presumed to be closely related.
The causative agents of WSSD from different locations appeared to be very
similar in disease features, histopathology, morphology and genomic structure
(Flegel 1997). Therefore, they have been increasingly considered as the same
or of different strains and grouped as white spot syndrome virus (WSSV)
(Kasornchandra and Boonyaratapalin 1998). Analysis of a genome segment of
WSSV favoured the view that the observed virus in farmed P. indicus in Iran
is either a member of a new genus (whispovirus) within the baculovirus or a
member of a entirely new virus family, whispoviridae (van Hulten et al. 2000).
In this regard more information is required to firstly study the virus sequencing
and phylogenetic analysis and to secondly evaluate its pathogenicity in in vivo conditions.