Directory UMM :Data Elmu:jurnal:A:Aquaculture:Vol184.Issue1-2.Apr2000:
Aquaculture 184 Ž2000. 31–44
www.elsevier.nlrlocateraqua-online
White spot baculovirus syndrome in the Indian
shrimp Penaeus monodon and P. indicus
P.R. Rajan
a,1
, P. Ramasamy a,1, V. Purushothaman b,
G.P. Brennan c,)
a
Department of Zoology, Life Sciences Building, UniÕersity of Madras, Chennai 600025, Madras, India
Department of Microbiology, Madras Veterinary College, TANUVAS, Chennai 600007, Madras, India
School of Biology and Biochemistry, The Queen’s UniÕersity of Belfast, 97 Lisburn Road, Belfast BT9 7BL
Northern Ireland, UK
b
c
Accepted 8 September 1999
Abstract
Sporadic occurrences of white spot baculovirus ŽWSBV. infections have been reported in
shrimp farms throughout the maritime states of India. WSBV presents as a reddish discolouration
with white spots on the exoskeleton and epidermis with muscle opacity. Onset of the disease is
extremely rapid with mass mortalities. Infected juveniles and sub-adults of Penaeus indicus and
P. monodon become lethargic surface frequently, exhibit loss of balance, with reduced feeding and
preening activities. The nuclei of WSBV-infected epithelial Žhypodermal., septal and secretory
cells of the gill filaments exhibit basophilic hypertrophied nuclei with a reduced cytoplasmic
volume. Massive tissue disintegration occurred in the ectodermal and mesodermal tissues. The
electron-dense nucleoplasm of the gill epithelial cells is mostly replaced with virions. Electron
microscopic examination revealed the presence of double-enveloped, non-occluded, rod-shaped
virions with a tube-like or branched extension and empty capsids. The numbers of mitochondria,
endoplasmic reticulum ŽER. and Golgi were also reduced, as were the numbers of secretory or
storage vesicles. WSBV is considered to be the main causative agent responsible for mass
mortalities of juveniles and sub-adults in the cultured Indian penaeid shrimp, P. monodon and P.
indicus. WSBV is highly pathogenic and readily transmitted from diseased shrimp to healthy
susceptible shrimp via, contaminated water, faeces and by scavenging on dead infected shrimp. It
may affect all stages of shrimp. The spread of the disease from cultured to natural systems and
vice versa cannot be dismissed. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: White spot baculovirus syndrome; Penaeus monodon; Endoplasmic reticulum
)
1
Corresponding author. E-mail: g.brennan@qub.ac.uk
E-mail: karthika@giasmd01.vsnl.net.in.
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 1 5 - 4
32
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
1. Introduction
Sporadic outbreaks of white spot virus-like infections have been encountered in
cultured penaeid shrimp farms throughout the maritime states of India wiping out total
stocks. Although the causative agent has not yet been clearly identified in the Indian
penaeid shrimp, it was suspected to be systemic ectodermal and mesodermal baculovirus
ŽRamasamy et al., 1995; Muralimanohar et al., 1996.. White spot syndrome was initially
reported in several species of shrimp in a number of Asian countries including India,
Indonesia, Taiwan and Thailand ŽChou et al., 1995; Wang et al., 1995; Lightner, 1996;
Chang et al., 1996; Durand et al., 1996; Wongteerasupaya et al., 1996; Lo et al.,
1996a,b.. The causative agent is thought to be a non-occluded white spot baculovirus
ŽWSBV. ŽInouye et al., 1994; Momoyama et al., 1994; Nakano et al., 1994; Takahashi
et al., 1994; Chou et al., 1995; Wang et al., 1995; Durand et al., 1996. and has been
reported in both cultured and wild Taiwan shrimp, in fresh water prawn, crabs, copepods
and insects ŽLo et al., 1996b.. WSBV is extremely virulent with rapid onset and is now
emerging as the single most important threat to the future of the shrimp industry in Asia.
The histopathology and characteristics of the WSBV appear similar to that of
Systemic mesodermal and ectodermal baculovirus ŽSEMBV. ŽWang et al., 1995;
Wongteerasupaya et al., 1995; Takahashi et al., 1996; Lo et al., 1996a,b. but differ from
Monodon baculovirus ŽMBV. and Baculovirus mid-gut necrosis ŽBMN. ŽSano et al.,
1984; Mari et al., 1993; Wongteerasupaya et al., 1995.. Although the clinical signs of
WSBV have been described in Indian shrimp ŽAnonymous, 1995; Karunasagar et al.,
1997., no systematic histopathological, experimental infection nor ultrastructural studies
of the WSBV virions have been described. Additionally, there have been conflicting
reports on the size and shape of the virus and the size of the genome ŽWongteerasupaya
et al., 1995, 1996; Lo et al., 1996a.. Some of the WSBV isolates were considered as
PmNOBII Ž Penaeus monodon, non-occluded baculovirus ŽNOB. II. ŽWongteerasupaya
et al., 1995, 1996; Takahashi et al., 1996. while other isolates were described as
PmNOBIII ŽWang et al., 1995; Karunsagar and Karunasagar, 1997.. Therefore, a need
for further critical studies is apparent to determine the size, shape and the clinical–pathological signs of WSBV in both naturally and experimentally infected Indian shrimp.
2. Materials and methods
WSBV-infected juveniles and sub-adults of P. monodon and P. indicus were
collected from a number of aquaculture farms located in Andhra Pradesh ŽGudur, Kota
and Nellore. and in Tamil Nadu ŽChidambaram, Muthupettai, Pattukottai Sirkhazi and
Tuticorin. India ŽFig. 1., between 1994 and 1999. Tissues from live, moribund WSBVinfected shrimp were prepared for light microscopy and transmission electron microscopy. The tissues selected for investigation were the gills, lymphoid organ, hepatopancreas, heart, stomach, mid-gut, hind-gut and nerve cord. For light microscopy,
tissues were fixed in Davidson’s fixative ŽBell and Lightner, 1988., dehydrated in a
graded series of alcohols and embedded in paraffin wax. Sections, 5 mm in thickness,
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
33
Fig. 1. South India map showing sampling U-sites of WSBV-infected shrimp.
were cut and mounted on glass slides, stained with Harris’s haematoxylin and eosin and
examined for pathological changes.
For transmission electron microscopy, tissues were fixed in 4% glutaraldehyde
buffered to pH 7.2 with 0.1 M sodium cacodylate–HCl containing 3% sucrose and 0.5%
sodium chloride for 24 h at 48C. Following post-fixation in 1% osmium tetroxide
ŽOsO4 ., the tissues were dehydrated in alcohols, infiltrated and embedded in resin and
polymerized for 48 h at 608C. Sections, 70–80 nm in thickness, were cut on a Reichert
Ultracut E ultramicrotome, mounted on bare 200 mesh copper grids, double stained with
uranyl acetate and lead citrate and viewed in a JEOL 100CX transmission electron
microscope.
34
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
The specimens used for viral isolation and experimental infections were obtained
from field studies in a shrimp farm in Tuticorin, Tamil Nadu, where an outbreak of
WSBV had been detected in July 1997. Live, moribund WSBV-infected shrimp were
collected, transported in dry ice and stored at y408C until required for viral isolation
and experimental infection.
2.1. Viral isolation and purification
WSBV-infected tissues of P. monodon were ground into powder in the presence of
liquid nitrogen. The virus was isolated using the method of Wang et al. Ž1995.. The viral
pellet was resuspended in TE buffer Ž10 mM Tris–HCl and 1 mM
ethylenediaminetetra-acetic acid wEDTAx., pH 8.0 and stored at y408C until required
for the confirmation of Rivers postulates.
2.2. Experimental-infection
To prove Rivers’ postulate, the following experiments were designed. Juveniles and
sub-adults of P. monodon, P. indicus, P. semisulcatus and Metapenaeus dobsoni were
collected from Muttukadu estuary, Chennai ŽMadras. and transported to the laboratory in
aerated sea water. Brooders of P. indicus were collected from the Kovalam coast, Tamil
Nadu, post-larval P. monodon were collected from a hatchery in Tamil Nadu and
transported to the laboratory. All specimens were maintained in the laboratory in filtered
seawater Ž28 to 32 ppt. at an ambient room temperature Ž308C. and fed with autoclaved
pellet feed. After an acclimatization period of 48 h, with no obvious signs of infection or
mortalities, the shrimp were experimentally infected with WSBV using a number of
different procedures and observed up to 20 days for signs of post-infection morbidity,
mortality and mode of transmission. The procedures employed are shown below.
Ž1. Healthy shrimp at different stages of growth were fed a single meal of minced
tissues of WSBV-infected P. monodon and subsequently fed with pellet feed.
Ž2. WSBV-infected tissues Ž25 mg. of P. monodon Žgnathothorax. were homogenized in 80 ml Leibovitz L 15 medium, centrifuged and the supernatant filtered through a
0.45-mm Sartorius filter and the tissue filtrate was inoculated into filtered, sea water
tanks containing different growth stages of healthy shrimp.
Ž3. Faecal pellets from WSBV-infected shrimp were collected and inoculated into
uninfected shrimp in tanks containing P. monodon, P. indicus and M. dobsoni.
Fig. 2. Sixty-day-old WSBV-infected P. monodon, collected from Chidambaram. Note the white spots Žw. on
the carapace and abdominal segments. Scale bar s 2.2 cm.
Fig. 3. Ža,b. One hundred-day-old P. monodon collected from Tuticorin, showing white spots Žw. on the
carapace Ža. and abdominal segments Žb.. Scale bar s1.7 cm.
Fig. 4. Ža,b. Isolated carapaces from WSBV-infected P. monodon illustrating the appearance of light and
heavily infected shrimp. Scale bar s1 cm.
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
35
Ž4. WSBV virions isolated and purified by ultracentrifugation Ž1,000,000 = g. were
diluted with 500 ml Leibovitz L 15 medium and 10 ml was injected into the lateral side
36
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
of the second abdominal segment of healthy P. monodon and P. indicus. Uninfected
controls were maintained in filtered seawater.
Experimentally infected shrimp, which died or became moribund during the 20 day
trial, were removed from the tanks and fixed in Davidson’s fixative for histological
studies, the surviving shrimp were stored at y408C for further studies.
3. Results
In infected P. monodon and P. indicus, WSBV presents as a reddish body colouration with characteristic white spots on the exoskeleton and epidermis with muscle
opacity ŽFigs. 2–4.. The epidermal cells of the stomach, gills, cuticular epidermis,
lymphoid organ and heart of P. monodon and P. indicus exhibited hypertrophied nuclei
during the early stages of viral infection ŽFigs. 5–9.. The cell cytoplasm was still visible
but became noticeably thinner as viral development progressed until it was fully
transparent surrounding the nucleus. In the middle stage of virus development, the
infected cells displayed hypertrophied nuclei with marginated basophilic chromatin. As
infection progressed, the nucleus became enlarged and the staining reaction changed
from acidophilic to basophilic. Eventually the nuclei disintegrated, leaving a large
vacant area. Massive tissue disintegration was observed in the gills, stomach, lymphoid
organ, heart, mid-gut, hind-gut and nervous tissues.
The hepatopancreas remained free of infection. Electron microscopic examination of
white spot-diseased shrimp revealed the occurrence of a non-occluded, rod-shaped,
double-enveloped WSBV some of which possessed a tube-like, or branched extension
and empty capsids ŽFigs. 10–15.. The virions measured 154 " 2 by 373 " 5 nm and
nucleocapsids 111 " 1.5 by 293 " 7 nm Ž n s 100.. WSBV-infected epithelial Žhypodermal. cells, septal cells and secretory cells of the gill filaments exhibited hypertrophied
nuclei with a reduced cytoplasmic volume. The numbers of mitochondria, endoplasmic
reticulum ŽER. and Golgi were also reduced, as were the numbers of secretory or
Fig. 5. Light photomicrograph of 60-day-old P. monodon collected from Sirkhazki, showing WSBV infected
gill epithelial cells. Note an hypertrophied nucleus Žhn. and considerable damage and degeneration of the gill
tissue with numerous large vesicles Žunlabelled arrows.. Scale bar s15 mm.
Fig. 6. P. monodon, 30 day old, collected from Pattukotai, showing infected Žhn. and uninfected Žun.
hypertrophied nuclei in the gill epithelial tissue. Degeneration of the gill epithelium is indicated by the
unlabelled arrows. Scale bar s10 mm.
Fig. 7. P. monodon, 100 day old, collected from Tuticorin showing WSBV-infected lymphoid tissue
exhibiting nuclear hypertrophy Žhn. in the stromal matrix cells. Scale bar s 20 mm.
Fig. 8. Enlarged light photomicrograph image showing hypertrophied nuclei Žhn. in the nerve cells of
100-day-old WSBV-infected P. monodon collected from Tuticorin. Scale bar s 20 mm.
Fig. 9. Section of spongy connective tissue from the stomach of 100-day-old P. monodon, collected from
Gudur, showing WSBV-infected cells containing hypertrophied nuclei Žhn. and the nature of the associated
degeneration. Uninfected cells Žun.. Scale bar s10 mm.
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
37
storage vesicles. The hypertrophied nuclei lacked heterochromatin and contained enveloped virions and empty capsids. The electron-dense nucleoplasm of the gill epithelial
cells was almost totally replaced with virions. The nuclear envelope was disorganized,
appeared thick and consisted of fibrous elements and numerous pores ŽFigs. 10–14..
Cytolysis and disintegration of the gill epithelial cells was observed ŽFigs. 5, 6 and 11..
38
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
39
Shrimp examined from the various locations in Andra Pradesh and Tamil Nadu
displayed similar histopathological characteristics of infection.
Studies carried out on a shrimp farm located in Tuticorin, Tamil Nadu, revealed an
outbreak of white spot disease confirmed by the appearance of dead shrimp in the
feed-check trays and at the margins of the pond, an area frequented by shrimp and from
which birds gathered moribund or dead shrimp. The gut of these animals appeared
empty. The numbers of dead shrimp increased on a daily basis and mass mortalities
were encountered. Examination of the infected stocks revealed the presence of white
spots on the exoskeleton with reddish body colouration and muscle opacity. Further
histological examination of the infected tissues showed hypertrophied nuclei, marginated
chromatin and widespread focal necrosis. The presence of virions in the infected tissues
was confirmed by transmission electron microscopy.
The results of the experimental infections of WSBV are listed in Table 1. Characteristic white spots appeared on the inner surface of the carapace and somites, which were
numerous and more prominent in P. monodon. In P. indicus, it was necessary to
remove the carapace to confirm infection. On examination, the various species and
stages of shrimp exhibited the characteristic histopathological signs of WSBV infection.
Intramuscular injection was the most rapid route of infection in P. monodon and P.
indicus. The infected shrimp become lethargic with reduced feeding and preening
activities. As the infection progressed, the shrimp stopped feeding, surfaced frequently
and exhibited a loss of balance. The cannibalistic behaviour of some shrimp feeding on
parts of WSBV-infected siblings was also observed. Organ disintegration and cell lysis
occurred as severity of infection increased. A mortality rate of 100% occurred within 1
week post-infection. The controls remained infection-free.
4. Discussion
The occurrence of WSBV has been shown to be the causative agent responsible for
mass mortalities in juveniles and sub-adults of the cultured Indian penaeid shrimp, P.
Fig. 10. Section through a WSBV-infected gill epithelial cell Žhn. from 100-day-old P. monodon. Free virions
Žv. are enclosed by a thick layer the nuclear membrane Žnl.. Mitochondria were absent. Bacterial cells Žb. are
present in the cytoplasm of the infected cell. Scale bar s 0.5 mm.
Fig. 11. Transmission electron micrograph of WSBV-infected gill epithelial cells, from a 100-day-old P.
indicus, collected from Tuticorin. Hypertrophied nuclei Žhn. contain virions Žv. and there is degeneration of
the nuclear membrane, nucleoplasm and cell membrane Žunlabelled arrows., and a thickening of the nuclear
membrane Žnl.. Scale bar s1 mm.
Fig. 12. P. indicus, 100 day old, collected from Tuticorin. WSBV-infected gill epithelial cell containing
tube-like, sometimes branched capsule envelopes Žc. and virions Žv.. Scale bar s 0.5 mm.
Fig. 13. WSBV-infected gill epithelial cell of 100-day-old P. indicus, collected from Chidambaram, showing a
cross-sectional view ŽCv. of virions in their envelope capsule Že., and nuclear capsids Žnc.. Also present are
empty capsules Žc. enclosed within the nucleus and limited by the nuclear membrane Žnl.. Virions Žv.. Scale
bar s 0.5 mm.
40
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
Fig. 14. Transmission electron micrograph of WSBV-infected gill epithelial cells of 100-day-old P. indicus,
collected from Chidambaram. The nuclear membrane has degenerated however, a thick nuclear membrane Žnl.,
encloses the virions Žv.. Mitochondria Žm.. Scale bar s 0.5 mm.
Fig. 15. Transmission electron micrograph of a portion of a WSBV-infected gill epithelial cell from a
100-day-old P. monodon, collected from Sirkhazki, showing virions, nucleo-capsids Žnc. and the capsule
envelope Žc.. Scale bar s 0.25 mm.
monodon and P. indicus and both ultrastructural and experimental evidence is presented
to support this finding. Mass mortalities caused by WSBV were reported in the cultured
shrimp, P. japonicus from Japan ŽInouye et al., 1994; Nakano et al., 1994; Takahashi et
al., 1994., Taiwan ŽChou et al., 1995. and Thailand ŽWongteerasupaya et al., 1995..
In the early stages of infection, some shrimp were observed cannibalizing dead
infected shrimp thus promoting the spread of WSBV in the population. Infection in P.
monodon was readily recognized by the presence of numerous white spots on the
carapace, while in P. indicus, it was necessary to remove the carapace to confirm
infection, usually accompanied by a reddish body colouration, ŽWang et al., 1995;
Chang et al., 1996; Wongteerasupaya et al., 1996; Lo et al., 1996b..
While WSBV infects the ecto- and meso-dermal tissues, it was absent in the
hepatopancreatic cells, suggesting it is similar to SEMBV infection ŽWongteerasupaya et
al., 1995.. To date, WSBV is the only known crustacean baculovirus that affects a wide
Table 1
Mortality of WSBV experimentally infected shrimp
Method of Species
infection
challenged
Growth
stage
Total number
of shrimp
challenged
Day Žs . Post-infection and percentage mortality
Day 1
Day 2
Percentage of
Mortality
Day 3
Day 4
Day 5
Day 6
Day 7
Control Experi- Control Experi- Control Experi- Control Experi- Control Experi- Control Experi- Control Experi- Control Experi- Control Experimental
mental
mental
mental
mental
mental
mental
mental
mental
P. monodon
P. monodon
P. monodon
P. indicus
P. indicus
P. indicus
P.
semisulcatus
M. dobsoni
Inoculation P. monodon
of WSBV- P. monodon
infected
P. indicus
tissue
P. indicus
filtrate
M. dobsoni
in to
filtered
tank water
Inoculation P. monodon
of WSBV- P. monodon
infected
P. indicus
faecal
P. indicus
pellet
M. dobsoni
in filtered
tank water
Injection
P. monodon
of 10 m l
P. indicus
purified
WSBV
a
Post-larvae
Juveniles
Sub-adults
Juveniles
Sub-adults
Broodstock
Sub-adults
50
55
65
22
50
48
15
100
115
150
67
129
49
25
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
45
2
5
13
16
20
0
0
0
0
0
0
0
0
52
23
45
38
25
573
0
0
0
0
0
0
0
0
–
55
35
28
29
23
20
0
0
0
0
0
0
0
–
17
14
16
29
–
64
0
0
0
0
0
0
0
–
2.5
2
5
2
–
16
0
0
0
0
0
0
0
–
–
–
–
–
–
–
0a
0a
0a
0a
0a
0a
0a
100
100
100
100
100
100
100
Juveniles
Juveniles
Sub-adults
Juveniles
Sub-adults
Sub-adults
20
30
25
30
30
15
55
45
35
60
45
20
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
13
6
10
7
0
0
0
0
0
0
0
40
13
46
20
36
40
0
0
0
0
0
0
49
40
23
27
29
55
0
0
0
0
0
0
11
33
26
37
29
5
0
0
0
0
0
0
–
–
–
7
–
–
0
0
0
0
0
0
–
–
–
–
–
–
0a
0a
0a
0a
0a
0a
100
100
100
100
100
100
Juveniles
Sub-adults
Juveniles
Sub-adults
Sub-adults
20
15
20
15
15
25
15
30
25
25
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8
17
7
4
16
0
0
0
0
0
44
40
47
52
48
0
0
0
0
0
40
53
43
44
32
0
0
0
0
0
8
–
3
–
4
0
0
0
0
0
–
–
–
–
–
0
0
0
0
0
–
–
–
–
–
0a
0a
0a
0a
0a
100
100
100
100
100
Sub-adults
Sub-adults
15
15
25
25
0
0
0
0
0
0
32
28
0
0
56
52
0
0
12
20
0
0
–
–
0
0
–
–
0
0
–
–
0a
0a
100
100
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
Shrimp
were fed
with
WSBVinfected
tissues of
P.
monodon
One hundred percent survival of shrimps in control even on 20th day, Ž – . none of the WSBV-infected shrimps survived.
41
42
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
range of tissues in the penaeid shrimp as well as other crustaceans, whereas MBV,
BMNV and PBV are tissue and host specific. WSBV ŽPmNOBIII. has been described
from P. monodon by Wang et al. Ž1995., Chang et al. Ž1996., Karunsagar and
Karunasagar Ž1997. and Karunasagar et al. Ž1997. while Wongteerasupaya et al. Ž1996.
reported the occurrence of WSBV ŽPmNOBII. infection in ecto- and meso-derm-derived
tissues from P. monodon as well as P. chinensis, P. indicus, P. japonicus, P.
merguiensis, P. monodon, and P. Õannamei obtained from China, Thailand, Indonesia
and India. This suggests that WSBV is a substantial pathogen for a range of penaeid,
crustacean and insect hosts with a wide geographic distribution.
Histopathological studies of WSBV-infected P. monodon and P. indicus revealed the
occurrence of basophilic hypertrophied nuclei with a reduced volume of eosinophilic
cytoplasm in the infected cells. Occlusion bodies have previously been reported in
WSBV-infected hypertrophied nuclei ŽWang et al., 1995. but were not reported here.
The extensive tissue damage observed in the infected hosts may be sufficient to reduce
the physiological and functional efficiencies of the hosts, and thus give rise to the
characteristic clinical signs of WSBV epizootics in shrimp. Histopathological observations on experimentally infected tissues reflect the findings of those observed in
naturally infected shrimp, and agrees with the findings of Chang et al. Ž1996. who
reported nuclear hypertrophy, cell lysis and tissue degeneration, in experimentally
infected P. monodon.
The hypodermal cells of the gills of WSBV-infected shrimp contained reduced
numbers of mitochondria, ER, Golgi complex and secretory or storage vesicles in the
cytoplasm. The nucleus showed hypertrophy, the nucleoplasm was less electron-dense
and a thick nuclear envelope consisted of fibrous elements, numerous pores and
enveloped virions. This is the first description of the ultrastructural pathology of
WSBV-infected Indian shrimp.
From the gross morphological, ultrastructural and histopathological information, the
viral infection was identified as white spot syndrome-associated baculovirus and classified as a member of the genus NOB of the subfamily Nudibaculovirinae of Baculoviridae and the isolate represents PmNOB ŽFrancki et al., 1991; Lightner, 1993; Wang et
al., 1995; Wongteerasupaya et al., 1995.. A tube-like projection was observed extending
from one end of the WSBV virions, the function and significance of which is unknown.
Wang et al. Ž1995. have also described a tail-like projection from the virions isolated
from P. monodon. Additionally, they have described the capsid to possess parallel
cross-striations composed of rings of subunits in a stacked series.
Experimental infection studies with WSBV have confirmed Rivers’ postulate. WSBV
isolates from diseased shrimp, successfully infected healthy penaeid shrimp. Intramuscular injection of purified virions caused 100% mortality within a few days post-infection
Ž1–5 days.. In contrast, natural routes of infection took longer to establish before
causing mortality. Infected shrimp did not survive beyond 7 days post-infection. These
studies indicate that WSBV is highly pathogenic and readily transmitted from diseased
shrimp to healthy susceptible shrimp via, contaminated water, faeces of infected shrimp
and through scavenging on dead infected shrimp, and require only a few days to invade,
multiply and exhibit the characteristic signs and pathogenicity associated with infected
hosts leading to mortality. It may affect all stages of penaeid and metapenaeid shrimp.
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
43
Chou et al. Ž1998. also showed, using shrimp infectivity tests of WSBV, that moralities
reached 100% within 4–6 days and that WSBV was readily transmitted across different
penaeid shrimp. It is still uncertain whether WSBV exists in latent forms in larvae or
brood stocks and which factors make them virulent or non-virulent. The control shrimp
remained healthy for more than 20 days.
Environmental factors may contribute to epizootics of WSBV particularly during the
monsoon season with associated changes in salinity, heavy surface run-off and turbidity
ŽAnonymous, 1995; Karunsagar and Karunasagar, 1997.. Stress is another contributing
factor leading to mass mortalities ŽLo et al., 1996b.. WSBV has a widespread occurrence over a range of hosts, some of which may act as vectors in natural and cultured
systems. Supamattaya et al. Ž1998. have shown that crustaceans, commonly found in
shrimp culture ponds, may act as viral reservoirs and aid transmission of WSBV. The
interaction between cultured penaeid shrimp and other presumed reservoir hosts, known
to inhabit shrimp farms, estuaries and coastal waters are of great interest and understanding them may help in the management of the disease ŽLo et al., 1996b.. Untreated source
water contaminated with possible vectors may be one route of entry for the virus in
culture systems. However, whether similar routes of infection exist in the Indian
situation needs to be examined and investigated at the ultrastructural and molecular
levels.
The current study is the first report describing the morphological and ultrastructural
changes in WSBV-infected Indian P. monodon and P. indicus. The release of
hatchery-reared WSBV-infected post-larvae by farmers, naturalists or even by chance,
into the environment to replenish natural stocks must be avoided because it may have a
negative feed back impact in addition to increasing the spread of the disease from
cultured systems to natural coastal systems.
References
Anonymous, 1995. SEMBV — an emerging viral threat to cultured shrimp in Asia. CP Shrimp News 3, 2–3.
Bell, T.A., Lightner, D.V., 1988. A Handbook of Normal Penaeid Shrimp Histology. World Aqua. Soc., Baton
Rouge, pp. 1–114.
Chang, P.S., Lo, C.F., Wang, Y.C., Kou, G.H., 1996. Identification of white spot syndrome associated
baculovirus ŽWSBV. target organs in the shrimp Penaeus monodon by in situ hybridization. Dis. Aquat.
Org. 27, 131–139.
Chou, H.Y., Huang, C.Y., Wang, C.H., Chiang, H.C., Lo, C.F., 1995. Pathogenicity of a baculovirus infection
causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis. Aquat. Org. 23, 165–173.
Chou, H.Y., Haung, C.Y., Lo, F.C., Kou, G.H., 1998. Studies on transmission of white spot syndrome
associated baculovirus ŽWSBV. in Penaeus monodon and P. japonicus via waterborne contact and oral
ingestion. Aquaculture 164, 263–276.
Durand, S., Lightner, D.V., Nunan, L.M., Redman, R.M., Mari, J., Bonami, J.R., 1996. Application of gene
probes as diagnostic tools for white spot baculovirus ŽWSBV. of penaeid shrimp. Dis. Aquat. Org. 27,
59–66.
Francki, R.I.B., Fauquet, C.M., Knudson, D.L., Brown, F. ŽEds.., 1991. Classification and nomenclature of
viruses. Arch. Virol., Suppl., 2, pp. 117–123.
Inouye, K., Miwa, S., Oseko, N., Nakano, H., Kimura, T., 1994. Mass mortalities of cultured kuruma shrimp
Penaeus japonicus, in Japan in 1993: electron microscopic evidence of the causative virus. Fish Pathol. 29,
149–158.
44
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
Karunsagar, I., Karunasagar, I., 1997. Strategies for management of white spot disease in shrimp. Souvenir of
the National Aquaculture week 24th–7th Feb., 1997. Aquaculture Foundation of India, Neelankarai,
Chennai, India. pp. 37–39.
Karunasagar, I., Otta, S.K., Karunasagar, I., 1997. Histopathological and bacterial study of white spot
syndrome of Penaeus monodon along the west coast of India. Aquaculture 153, 9–13.
Lightner, D.V., 1993. Diseases of cultured penaeid shrimp. In: McVey, J. ŽEd.., CRC Handbook of
Mariculture, Vol. 1, Crustacean Aquaculture, 2nd edn. CRC Press, Boca Raton. pp. 393–486.
Lightner, D.V. ŽEd.., 1996. A Handbook of Pathology and Diagnostic Procedures for Diseases of Penaeid
Shrimp. World Aquaculture Soc., Baton Rouge.
Lo, C.F., Leu, J.H., Ho, C.H., Chen, C.H., Peng, S.E., Chen, Y.T., Chou, C.M., Yeh, P.Y., Huang, C.J., Chou,
H.Y., Wang, C.H., Kou, G.H., 1996a. Detection of baculovirus associated with white spot syndrome
ŽWSBV. in penaeid shrimp using polymerase chain reaction. Dis. Aquat. Org. 25, 133–141.
Lo, C.F., Ho, C.H., Penug, S.E., Chen, C.H., Hsu, H.C., Chiu, Y.L., Chang, C.F., Liu, K.F., Su, M.S., Wang,
C.H., Kou, G.H., 1996b. White spot syndrome baculovirus ŽWSBV. detected in cultured and wild shrimp,
crabs and other arthropods. Dis. Aquat. Org. 27, 215–225.
Mari, J., Bonami, J.R., Poulos, B., Lightner, D.V., 1993. Preliminary characterization and partial cloning of
the genome of a baculovirus from Penaeus monodon ŽPm SNPVs MBV.. Dis. Aquat. Org. 16, 207–215.
Momoyama, K., Hiraoka, M., Nakano, H., Koube, H., Inouye, K., Oseka, N., 1994. Mass mortalities of
cultured kuruma shrimp, Penaeus japonicus, in Japan in 1993: histopathological studies. Fish Pathol. 29,
141–148.
Muralimanohar, B., Sundararaj, D., Selvaraj, D., Sheela, P.R.R., Chidambaram, P., Mohan, A.C., Ravishankar,
B., 1996. An outbreak of SEMBV and MBV infection in cultured Penaeus monodon in Tamil Nadu.
Indian J. Fish. 43, 403–406.
Nakano, H., Koube, H., Umezaea, S., Momoyama, K., Hiraoka, M., Inouye, K., Oseka, N., 1994. Mass
mortalities of cultured kuruma shrimp, Penaeus japonicus, in Japan in 1994: epizootiological survey and
infection trials. Fish Pathol. 29, 135–139.
Ramasamy, P., Brennan, G.P., Jayakumar, R., 1995. A record and prevalence of Monodon baculovirus from
post-larval Penaeus monodon in Madras, India. Aquaculture 130, 129–135.
Sano, T., Nishimura, T., Fukuda, H., Hayashida, T., Momoyama, K., 1984. Baculoviral mid-gut gland necrosis
ŽBMN. of Kuruma shrimp Ž Penaeus japonicus . larvae in Japanese intensive culture systems. Helgol.
Meeresunters. 37, 255–264.
Supamattaya, K., Hoffmann, R.W., Boonyaratpalin, S., Kanchanaphum, P., 1998. Experimental transmission
of white spot syndrome virus ŽWSSV. from black tiger Penaeus monodon to the sand crab Portunnus
pelagicus, mud crab Scylla serrata and krill Acetes sp. Dis. Aquat. Org. 32, 79–85.
Takahashi, Y., Itami, T., Kondom, M., Maeda, M., Fujii, R., Tomonaga, S., Supamattaya, K., Boonyaratpalin,
S., 1994. Electron microscopic evidence of bacilliform virus infection in Kuruma shrimp Ž Penaeus
japonicus .. Fish Pathol. 29, 121–125.
Takahashi, Y., Itami, T., Maeda, M., Suzuki, N., Kasornchandra, J., Supamattaya, K., Khongpradit, R.,
Boonyaratpalin, S., Kondo, M., Kawai, K., Kusuda, R., Hirono, Aoki, T., 1996. Polymerase chain reaction
ŽPCR. amplification of bacilliform virus ŽRV-PJ. DNA baculovirus ŽSEMBV. DNA in Penaeus monodon
Fabricius. J. Fish Dis. 19, 399–403.
Wang, C.H., Lo, C.F., Leu, J.H., Chou, C.M., Yeh, P.Y., Chou, H.Y., Tung, M.C., Chang, C.F., Su, M.S.,
Kou, G.H., 1995. Purification and genomic analysis of Baculovirus associated with white spot syndrome
ŽWSBV. of Penaeus monodon. Dis. Aquat. Org. 23, 239–242.
Wongteerasupaya, C., Vickers, J.E., Sriurairatana, S., Nash, G.L., Akarajamorn, A., Boonsaeng, V., Panyim,
S., Tassanakajon, A., Withyachumnarnkul, B., Flegel, T.W., 1995. A non-occluded, system baculovirus
that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn
Penaeus monodon. Dis. Aquat. Org. 21, 69–77.
Wongteerasupaya, C., Wongwisansri, S., Boonsaeng, V., Panyim, S., Pratanpipat, P., Nash, G.L., Withyachumnarnkul, B., Flegel, T.W., 1996. DNA fragment of Penaeus monodon baculovirus PmNOBII gives
positive in situ hybridization with white-spot viral infections in six penaeid shrimp species. Aquaculture
143, 23–32.
www.elsevier.nlrlocateraqua-online
White spot baculovirus syndrome in the Indian
shrimp Penaeus monodon and P. indicus
P.R. Rajan
a,1
, P. Ramasamy a,1, V. Purushothaman b,
G.P. Brennan c,)
a
Department of Zoology, Life Sciences Building, UniÕersity of Madras, Chennai 600025, Madras, India
Department of Microbiology, Madras Veterinary College, TANUVAS, Chennai 600007, Madras, India
School of Biology and Biochemistry, The Queen’s UniÕersity of Belfast, 97 Lisburn Road, Belfast BT9 7BL
Northern Ireland, UK
b
c
Accepted 8 September 1999
Abstract
Sporadic occurrences of white spot baculovirus ŽWSBV. infections have been reported in
shrimp farms throughout the maritime states of India. WSBV presents as a reddish discolouration
with white spots on the exoskeleton and epidermis with muscle opacity. Onset of the disease is
extremely rapid with mass mortalities. Infected juveniles and sub-adults of Penaeus indicus and
P. monodon become lethargic surface frequently, exhibit loss of balance, with reduced feeding and
preening activities. The nuclei of WSBV-infected epithelial Žhypodermal., septal and secretory
cells of the gill filaments exhibit basophilic hypertrophied nuclei with a reduced cytoplasmic
volume. Massive tissue disintegration occurred in the ectodermal and mesodermal tissues. The
electron-dense nucleoplasm of the gill epithelial cells is mostly replaced with virions. Electron
microscopic examination revealed the presence of double-enveloped, non-occluded, rod-shaped
virions with a tube-like or branched extension and empty capsids. The numbers of mitochondria,
endoplasmic reticulum ŽER. and Golgi were also reduced, as were the numbers of secretory or
storage vesicles. WSBV is considered to be the main causative agent responsible for mass
mortalities of juveniles and sub-adults in the cultured Indian penaeid shrimp, P. monodon and P.
indicus. WSBV is highly pathogenic and readily transmitted from diseased shrimp to healthy
susceptible shrimp via, contaminated water, faeces and by scavenging on dead infected shrimp. It
may affect all stages of shrimp. The spread of the disease from cultured to natural systems and
vice versa cannot be dismissed. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: White spot baculovirus syndrome; Penaeus monodon; Endoplasmic reticulum
)
1
Corresponding author. E-mail: g.brennan@qub.ac.uk
E-mail: karthika@giasmd01.vsnl.net.in.
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 1 5 - 4
32
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
1. Introduction
Sporadic outbreaks of white spot virus-like infections have been encountered in
cultured penaeid shrimp farms throughout the maritime states of India wiping out total
stocks. Although the causative agent has not yet been clearly identified in the Indian
penaeid shrimp, it was suspected to be systemic ectodermal and mesodermal baculovirus
ŽRamasamy et al., 1995; Muralimanohar et al., 1996.. White spot syndrome was initially
reported in several species of shrimp in a number of Asian countries including India,
Indonesia, Taiwan and Thailand ŽChou et al., 1995; Wang et al., 1995; Lightner, 1996;
Chang et al., 1996; Durand et al., 1996; Wongteerasupaya et al., 1996; Lo et al.,
1996a,b.. The causative agent is thought to be a non-occluded white spot baculovirus
ŽWSBV. ŽInouye et al., 1994; Momoyama et al., 1994; Nakano et al., 1994; Takahashi
et al., 1994; Chou et al., 1995; Wang et al., 1995; Durand et al., 1996. and has been
reported in both cultured and wild Taiwan shrimp, in fresh water prawn, crabs, copepods
and insects ŽLo et al., 1996b.. WSBV is extremely virulent with rapid onset and is now
emerging as the single most important threat to the future of the shrimp industry in Asia.
The histopathology and characteristics of the WSBV appear similar to that of
Systemic mesodermal and ectodermal baculovirus ŽSEMBV. ŽWang et al., 1995;
Wongteerasupaya et al., 1995; Takahashi et al., 1996; Lo et al., 1996a,b. but differ from
Monodon baculovirus ŽMBV. and Baculovirus mid-gut necrosis ŽBMN. ŽSano et al.,
1984; Mari et al., 1993; Wongteerasupaya et al., 1995.. Although the clinical signs of
WSBV have been described in Indian shrimp ŽAnonymous, 1995; Karunasagar et al.,
1997., no systematic histopathological, experimental infection nor ultrastructural studies
of the WSBV virions have been described. Additionally, there have been conflicting
reports on the size and shape of the virus and the size of the genome ŽWongteerasupaya
et al., 1995, 1996; Lo et al., 1996a.. Some of the WSBV isolates were considered as
PmNOBII Ž Penaeus monodon, non-occluded baculovirus ŽNOB. II. ŽWongteerasupaya
et al., 1995, 1996; Takahashi et al., 1996. while other isolates were described as
PmNOBIII ŽWang et al., 1995; Karunsagar and Karunasagar, 1997.. Therefore, a need
for further critical studies is apparent to determine the size, shape and the clinical–pathological signs of WSBV in both naturally and experimentally infected Indian shrimp.
2. Materials and methods
WSBV-infected juveniles and sub-adults of P. monodon and P. indicus were
collected from a number of aquaculture farms located in Andhra Pradesh ŽGudur, Kota
and Nellore. and in Tamil Nadu ŽChidambaram, Muthupettai, Pattukottai Sirkhazi and
Tuticorin. India ŽFig. 1., between 1994 and 1999. Tissues from live, moribund WSBVinfected shrimp were prepared for light microscopy and transmission electron microscopy. The tissues selected for investigation were the gills, lymphoid organ, hepatopancreas, heart, stomach, mid-gut, hind-gut and nerve cord. For light microscopy,
tissues were fixed in Davidson’s fixative ŽBell and Lightner, 1988., dehydrated in a
graded series of alcohols and embedded in paraffin wax. Sections, 5 mm in thickness,
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
33
Fig. 1. South India map showing sampling U-sites of WSBV-infected shrimp.
were cut and mounted on glass slides, stained with Harris’s haematoxylin and eosin and
examined for pathological changes.
For transmission electron microscopy, tissues were fixed in 4% glutaraldehyde
buffered to pH 7.2 with 0.1 M sodium cacodylate–HCl containing 3% sucrose and 0.5%
sodium chloride for 24 h at 48C. Following post-fixation in 1% osmium tetroxide
ŽOsO4 ., the tissues were dehydrated in alcohols, infiltrated and embedded in resin and
polymerized for 48 h at 608C. Sections, 70–80 nm in thickness, were cut on a Reichert
Ultracut E ultramicrotome, mounted on bare 200 mesh copper grids, double stained with
uranyl acetate and lead citrate and viewed in a JEOL 100CX transmission electron
microscope.
34
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
The specimens used for viral isolation and experimental infections were obtained
from field studies in a shrimp farm in Tuticorin, Tamil Nadu, where an outbreak of
WSBV had been detected in July 1997. Live, moribund WSBV-infected shrimp were
collected, transported in dry ice and stored at y408C until required for viral isolation
and experimental infection.
2.1. Viral isolation and purification
WSBV-infected tissues of P. monodon were ground into powder in the presence of
liquid nitrogen. The virus was isolated using the method of Wang et al. Ž1995.. The viral
pellet was resuspended in TE buffer Ž10 mM Tris–HCl and 1 mM
ethylenediaminetetra-acetic acid wEDTAx., pH 8.0 and stored at y408C until required
for the confirmation of Rivers postulates.
2.2. Experimental-infection
To prove Rivers’ postulate, the following experiments were designed. Juveniles and
sub-adults of P. monodon, P. indicus, P. semisulcatus and Metapenaeus dobsoni were
collected from Muttukadu estuary, Chennai ŽMadras. and transported to the laboratory in
aerated sea water. Brooders of P. indicus were collected from the Kovalam coast, Tamil
Nadu, post-larval P. monodon were collected from a hatchery in Tamil Nadu and
transported to the laboratory. All specimens were maintained in the laboratory in filtered
seawater Ž28 to 32 ppt. at an ambient room temperature Ž308C. and fed with autoclaved
pellet feed. After an acclimatization period of 48 h, with no obvious signs of infection or
mortalities, the shrimp were experimentally infected with WSBV using a number of
different procedures and observed up to 20 days for signs of post-infection morbidity,
mortality and mode of transmission. The procedures employed are shown below.
Ž1. Healthy shrimp at different stages of growth were fed a single meal of minced
tissues of WSBV-infected P. monodon and subsequently fed with pellet feed.
Ž2. WSBV-infected tissues Ž25 mg. of P. monodon Žgnathothorax. were homogenized in 80 ml Leibovitz L 15 medium, centrifuged and the supernatant filtered through a
0.45-mm Sartorius filter and the tissue filtrate was inoculated into filtered, sea water
tanks containing different growth stages of healthy shrimp.
Ž3. Faecal pellets from WSBV-infected shrimp were collected and inoculated into
uninfected shrimp in tanks containing P. monodon, P. indicus and M. dobsoni.
Fig. 2. Sixty-day-old WSBV-infected P. monodon, collected from Chidambaram. Note the white spots Žw. on
the carapace and abdominal segments. Scale bar s 2.2 cm.
Fig. 3. Ža,b. One hundred-day-old P. monodon collected from Tuticorin, showing white spots Žw. on the
carapace Ža. and abdominal segments Žb.. Scale bar s1.7 cm.
Fig. 4. Ža,b. Isolated carapaces from WSBV-infected P. monodon illustrating the appearance of light and
heavily infected shrimp. Scale bar s1 cm.
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
35
Ž4. WSBV virions isolated and purified by ultracentrifugation Ž1,000,000 = g. were
diluted with 500 ml Leibovitz L 15 medium and 10 ml was injected into the lateral side
36
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
of the second abdominal segment of healthy P. monodon and P. indicus. Uninfected
controls were maintained in filtered seawater.
Experimentally infected shrimp, which died or became moribund during the 20 day
trial, were removed from the tanks and fixed in Davidson’s fixative for histological
studies, the surviving shrimp were stored at y408C for further studies.
3. Results
In infected P. monodon and P. indicus, WSBV presents as a reddish body colouration with characteristic white spots on the exoskeleton and epidermis with muscle
opacity ŽFigs. 2–4.. The epidermal cells of the stomach, gills, cuticular epidermis,
lymphoid organ and heart of P. monodon and P. indicus exhibited hypertrophied nuclei
during the early stages of viral infection ŽFigs. 5–9.. The cell cytoplasm was still visible
but became noticeably thinner as viral development progressed until it was fully
transparent surrounding the nucleus. In the middle stage of virus development, the
infected cells displayed hypertrophied nuclei with marginated basophilic chromatin. As
infection progressed, the nucleus became enlarged and the staining reaction changed
from acidophilic to basophilic. Eventually the nuclei disintegrated, leaving a large
vacant area. Massive tissue disintegration was observed in the gills, stomach, lymphoid
organ, heart, mid-gut, hind-gut and nervous tissues.
The hepatopancreas remained free of infection. Electron microscopic examination of
white spot-diseased shrimp revealed the occurrence of a non-occluded, rod-shaped,
double-enveloped WSBV some of which possessed a tube-like, or branched extension
and empty capsids ŽFigs. 10–15.. The virions measured 154 " 2 by 373 " 5 nm and
nucleocapsids 111 " 1.5 by 293 " 7 nm Ž n s 100.. WSBV-infected epithelial Žhypodermal. cells, septal cells and secretory cells of the gill filaments exhibited hypertrophied
nuclei with a reduced cytoplasmic volume. The numbers of mitochondria, endoplasmic
reticulum ŽER. and Golgi were also reduced, as were the numbers of secretory or
Fig. 5. Light photomicrograph of 60-day-old P. monodon collected from Sirkhazki, showing WSBV infected
gill epithelial cells. Note an hypertrophied nucleus Žhn. and considerable damage and degeneration of the gill
tissue with numerous large vesicles Žunlabelled arrows.. Scale bar s15 mm.
Fig. 6. P. monodon, 30 day old, collected from Pattukotai, showing infected Žhn. and uninfected Žun.
hypertrophied nuclei in the gill epithelial tissue. Degeneration of the gill epithelium is indicated by the
unlabelled arrows. Scale bar s10 mm.
Fig. 7. P. monodon, 100 day old, collected from Tuticorin showing WSBV-infected lymphoid tissue
exhibiting nuclear hypertrophy Žhn. in the stromal matrix cells. Scale bar s 20 mm.
Fig. 8. Enlarged light photomicrograph image showing hypertrophied nuclei Žhn. in the nerve cells of
100-day-old WSBV-infected P. monodon collected from Tuticorin. Scale bar s 20 mm.
Fig. 9. Section of spongy connective tissue from the stomach of 100-day-old P. monodon, collected from
Gudur, showing WSBV-infected cells containing hypertrophied nuclei Žhn. and the nature of the associated
degeneration. Uninfected cells Žun.. Scale bar s10 mm.
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
37
storage vesicles. The hypertrophied nuclei lacked heterochromatin and contained enveloped virions and empty capsids. The electron-dense nucleoplasm of the gill epithelial
cells was almost totally replaced with virions. The nuclear envelope was disorganized,
appeared thick and consisted of fibrous elements and numerous pores ŽFigs. 10–14..
Cytolysis and disintegration of the gill epithelial cells was observed ŽFigs. 5, 6 and 11..
38
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
39
Shrimp examined from the various locations in Andra Pradesh and Tamil Nadu
displayed similar histopathological characteristics of infection.
Studies carried out on a shrimp farm located in Tuticorin, Tamil Nadu, revealed an
outbreak of white spot disease confirmed by the appearance of dead shrimp in the
feed-check trays and at the margins of the pond, an area frequented by shrimp and from
which birds gathered moribund or dead shrimp. The gut of these animals appeared
empty. The numbers of dead shrimp increased on a daily basis and mass mortalities
were encountered. Examination of the infected stocks revealed the presence of white
spots on the exoskeleton with reddish body colouration and muscle opacity. Further
histological examination of the infected tissues showed hypertrophied nuclei, marginated
chromatin and widespread focal necrosis. The presence of virions in the infected tissues
was confirmed by transmission electron microscopy.
The results of the experimental infections of WSBV are listed in Table 1. Characteristic white spots appeared on the inner surface of the carapace and somites, which were
numerous and more prominent in P. monodon. In P. indicus, it was necessary to
remove the carapace to confirm infection. On examination, the various species and
stages of shrimp exhibited the characteristic histopathological signs of WSBV infection.
Intramuscular injection was the most rapid route of infection in P. monodon and P.
indicus. The infected shrimp become lethargic with reduced feeding and preening
activities. As the infection progressed, the shrimp stopped feeding, surfaced frequently
and exhibited a loss of balance. The cannibalistic behaviour of some shrimp feeding on
parts of WSBV-infected siblings was also observed. Organ disintegration and cell lysis
occurred as severity of infection increased. A mortality rate of 100% occurred within 1
week post-infection. The controls remained infection-free.
4. Discussion
The occurrence of WSBV has been shown to be the causative agent responsible for
mass mortalities in juveniles and sub-adults of the cultured Indian penaeid shrimp, P.
Fig. 10. Section through a WSBV-infected gill epithelial cell Žhn. from 100-day-old P. monodon. Free virions
Žv. are enclosed by a thick layer the nuclear membrane Žnl.. Mitochondria were absent. Bacterial cells Žb. are
present in the cytoplasm of the infected cell. Scale bar s 0.5 mm.
Fig. 11. Transmission electron micrograph of WSBV-infected gill epithelial cells, from a 100-day-old P.
indicus, collected from Tuticorin. Hypertrophied nuclei Žhn. contain virions Žv. and there is degeneration of
the nuclear membrane, nucleoplasm and cell membrane Žunlabelled arrows., and a thickening of the nuclear
membrane Žnl.. Scale bar s1 mm.
Fig. 12. P. indicus, 100 day old, collected from Tuticorin. WSBV-infected gill epithelial cell containing
tube-like, sometimes branched capsule envelopes Žc. and virions Žv.. Scale bar s 0.5 mm.
Fig. 13. WSBV-infected gill epithelial cell of 100-day-old P. indicus, collected from Chidambaram, showing a
cross-sectional view ŽCv. of virions in their envelope capsule Že., and nuclear capsids Žnc.. Also present are
empty capsules Žc. enclosed within the nucleus and limited by the nuclear membrane Žnl.. Virions Žv.. Scale
bar s 0.5 mm.
40
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
Fig. 14. Transmission electron micrograph of WSBV-infected gill epithelial cells of 100-day-old P. indicus,
collected from Chidambaram. The nuclear membrane has degenerated however, a thick nuclear membrane Žnl.,
encloses the virions Žv.. Mitochondria Žm.. Scale bar s 0.5 mm.
Fig. 15. Transmission electron micrograph of a portion of a WSBV-infected gill epithelial cell from a
100-day-old P. monodon, collected from Sirkhazki, showing virions, nucleo-capsids Žnc. and the capsule
envelope Žc.. Scale bar s 0.25 mm.
monodon and P. indicus and both ultrastructural and experimental evidence is presented
to support this finding. Mass mortalities caused by WSBV were reported in the cultured
shrimp, P. japonicus from Japan ŽInouye et al., 1994; Nakano et al., 1994; Takahashi et
al., 1994., Taiwan ŽChou et al., 1995. and Thailand ŽWongteerasupaya et al., 1995..
In the early stages of infection, some shrimp were observed cannibalizing dead
infected shrimp thus promoting the spread of WSBV in the population. Infection in P.
monodon was readily recognized by the presence of numerous white spots on the
carapace, while in P. indicus, it was necessary to remove the carapace to confirm
infection, usually accompanied by a reddish body colouration, ŽWang et al., 1995;
Chang et al., 1996; Wongteerasupaya et al., 1996; Lo et al., 1996b..
While WSBV infects the ecto- and meso-dermal tissues, it was absent in the
hepatopancreatic cells, suggesting it is similar to SEMBV infection ŽWongteerasupaya et
al., 1995.. To date, WSBV is the only known crustacean baculovirus that affects a wide
Table 1
Mortality of WSBV experimentally infected shrimp
Method of Species
infection
challenged
Growth
stage
Total number
of shrimp
challenged
Day Žs . Post-infection and percentage mortality
Day 1
Day 2
Percentage of
Mortality
Day 3
Day 4
Day 5
Day 6
Day 7
Control Experi- Control Experi- Control Experi- Control Experi- Control Experi- Control Experi- Control Experi- Control Experi- Control Experimental
mental
mental
mental
mental
mental
mental
mental
mental
P. monodon
P. monodon
P. monodon
P. indicus
P. indicus
P. indicus
P.
semisulcatus
M. dobsoni
Inoculation P. monodon
of WSBV- P. monodon
infected
P. indicus
tissue
P. indicus
filtrate
M. dobsoni
in to
filtered
tank water
Inoculation P. monodon
of WSBV- P. monodon
infected
P. indicus
faecal
P. indicus
pellet
M. dobsoni
in filtered
tank water
Injection
P. monodon
of 10 m l
P. indicus
purified
WSBV
a
Post-larvae
Juveniles
Sub-adults
Juveniles
Sub-adults
Broodstock
Sub-adults
50
55
65
22
50
48
15
100
115
150
67
129
49
25
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
45
2
5
13
16
20
0
0
0
0
0
0
0
0
52
23
45
38
25
573
0
0
0
0
0
0
0
0
–
55
35
28
29
23
20
0
0
0
0
0
0
0
–
17
14
16
29
–
64
0
0
0
0
0
0
0
–
2.5
2
5
2
–
16
0
0
0
0
0
0
0
–
–
–
–
–
–
–
0a
0a
0a
0a
0a
0a
0a
100
100
100
100
100
100
100
Juveniles
Juveniles
Sub-adults
Juveniles
Sub-adults
Sub-adults
20
30
25
30
30
15
55
45
35
60
45
20
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
13
6
10
7
0
0
0
0
0
0
0
40
13
46
20
36
40
0
0
0
0
0
0
49
40
23
27
29
55
0
0
0
0
0
0
11
33
26
37
29
5
0
0
0
0
0
0
–
–
–
7
–
–
0
0
0
0
0
0
–
–
–
–
–
–
0a
0a
0a
0a
0a
0a
100
100
100
100
100
100
Juveniles
Sub-adults
Juveniles
Sub-adults
Sub-adults
20
15
20
15
15
25
15
30
25
25
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
8
17
7
4
16
0
0
0
0
0
44
40
47
52
48
0
0
0
0
0
40
53
43
44
32
0
0
0
0
0
8
–
3
–
4
0
0
0
0
0
–
–
–
–
–
0
0
0
0
0
–
–
–
–
–
0a
0a
0a
0a
0a
100
100
100
100
100
Sub-adults
Sub-adults
15
15
25
25
0
0
0
0
0
0
32
28
0
0
56
52
0
0
12
20
0
0
–
–
0
0
–
–
0
0
–
–
0a
0a
100
100
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
Shrimp
were fed
with
WSBVinfected
tissues of
P.
monodon
One hundred percent survival of shrimps in control even on 20th day, Ž – . none of the WSBV-infected shrimps survived.
41
42
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
range of tissues in the penaeid shrimp as well as other crustaceans, whereas MBV,
BMNV and PBV are tissue and host specific. WSBV ŽPmNOBIII. has been described
from P. monodon by Wang et al. Ž1995., Chang et al. Ž1996., Karunsagar and
Karunasagar Ž1997. and Karunasagar et al. Ž1997. while Wongteerasupaya et al. Ž1996.
reported the occurrence of WSBV ŽPmNOBII. infection in ecto- and meso-derm-derived
tissues from P. monodon as well as P. chinensis, P. indicus, P. japonicus, P.
merguiensis, P. monodon, and P. Õannamei obtained from China, Thailand, Indonesia
and India. This suggests that WSBV is a substantial pathogen for a range of penaeid,
crustacean and insect hosts with a wide geographic distribution.
Histopathological studies of WSBV-infected P. monodon and P. indicus revealed the
occurrence of basophilic hypertrophied nuclei with a reduced volume of eosinophilic
cytoplasm in the infected cells. Occlusion bodies have previously been reported in
WSBV-infected hypertrophied nuclei ŽWang et al., 1995. but were not reported here.
The extensive tissue damage observed in the infected hosts may be sufficient to reduce
the physiological and functional efficiencies of the hosts, and thus give rise to the
characteristic clinical signs of WSBV epizootics in shrimp. Histopathological observations on experimentally infected tissues reflect the findings of those observed in
naturally infected shrimp, and agrees with the findings of Chang et al. Ž1996. who
reported nuclear hypertrophy, cell lysis and tissue degeneration, in experimentally
infected P. monodon.
The hypodermal cells of the gills of WSBV-infected shrimp contained reduced
numbers of mitochondria, ER, Golgi complex and secretory or storage vesicles in the
cytoplasm. The nucleus showed hypertrophy, the nucleoplasm was less electron-dense
and a thick nuclear envelope consisted of fibrous elements, numerous pores and
enveloped virions. This is the first description of the ultrastructural pathology of
WSBV-infected Indian shrimp.
From the gross morphological, ultrastructural and histopathological information, the
viral infection was identified as white spot syndrome-associated baculovirus and classified as a member of the genus NOB of the subfamily Nudibaculovirinae of Baculoviridae and the isolate represents PmNOB ŽFrancki et al., 1991; Lightner, 1993; Wang et
al., 1995; Wongteerasupaya et al., 1995.. A tube-like projection was observed extending
from one end of the WSBV virions, the function and significance of which is unknown.
Wang et al. Ž1995. have also described a tail-like projection from the virions isolated
from P. monodon. Additionally, they have described the capsid to possess parallel
cross-striations composed of rings of subunits in a stacked series.
Experimental infection studies with WSBV have confirmed Rivers’ postulate. WSBV
isolates from diseased shrimp, successfully infected healthy penaeid shrimp. Intramuscular injection of purified virions caused 100% mortality within a few days post-infection
Ž1–5 days.. In contrast, natural routes of infection took longer to establish before
causing mortality. Infected shrimp did not survive beyond 7 days post-infection. These
studies indicate that WSBV is highly pathogenic and readily transmitted from diseased
shrimp to healthy susceptible shrimp via, contaminated water, faeces of infected shrimp
and through scavenging on dead infected shrimp, and require only a few days to invade,
multiply and exhibit the characteristic signs and pathogenicity associated with infected
hosts leading to mortality. It may affect all stages of penaeid and metapenaeid shrimp.
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
43
Chou et al. Ž1998. also showed, using shrimp infectivity tests of WSBV, that moralities
reached 100% within 4–6 days and that WSBV was readily transmitted across different
penaeid shrimp. It is still uncertain whether WSBV exists in latent forms in larvae or
brood stocks and which factors make them virulent or non-virulent. The control shrimp
remained healthy for more than 20 days.
Environmental factors may contribute to epizootics of WSBV particularly during the
monsoon season with associated changes in salinity, heavy surface run-off and turbidity
ŽAnonymous, 1995; Karunsagar and Karunasagar, 1997.. Stress is another contributing
factor leading to mass mortalities ŽLo et al., 1996b.. WSBV has a widespread occurrence over a range of hosts, some of which may act as vectors in natural and cultured
systems. Supamattaya et al. Ž1998. have shown that crustaceans, commonly found in
shrimp culture ponds, may act as viral reservoirs and aid transmission of WSBV. The
interaction between cultured penaeid shrimp and other presumed reservoir hosts, known
to inhabit shrimp farms, estuaries and coastal waters are of great interest and understanding them may help in the management of the disease ŽLo et al., 1996b.. Untreated source
water contaminated with possible vectors may be one route of entry for the virus in
culture systems. However, whether similar routes of infection exist in the Indian
situation needs to be examined and investigated at the ultrastructural and molecular
levels.
The current study is the first report describing the morphological and ultrastructural
changes in WSBV-infected Indian P. monodon and P. indicus. The release of
hatchery-reared WSBV-infected post-larvae by farmers, naturalists or even by chance,
into the environment to replenish natural stocks must be avoided because it may have a
negative feed back impact in addition to increasing the spread of the disease from
cultured systems to natural coastal systems.
References
Anonymous, 1995. SEMBV — an emerging viral threat to cultured shrimp in Asia. CP Shrimp News 3, 2–3.
Bell, T.A., Lightner, D.V., 1988. A Handbook of Normal Penaeid Shrimp Histology. World Aqua. Soc., Baton
Rouge, pp. 1–114.
Chang, P.S., Lo, C.F., Wang, Y.C., Kou, G.H., 1996. Identification of white spot syndrome associated
baculovirus ŽWSBV. target organs in the shrimp Penaeus monodon by in situ hybridization. Dis. Aquat.
Org. 27, 131–139.
Chou, H.Y., Huang, C.Y., Wang, C.H., Chiang, H.C., Lo, C.F., 1995. Pathogenicity of a baculovirus infection
causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis. Aquat. Org. 23, 165–173.
Chou, H.Y., Haung, C.Y., Lo, F.C., Kou, G.H., 1998. Studies on transmission of white spot syndrome
associated baculovirus ŽWSBV. in Penaeus monodon and P. japonicus via waterborne contact and oral
ingestion. Aquaculture 164, 263–276.
Durand, S., Lightner, D.V., Nunan, L.M., Redman, R.M., Mari, J., Bonami, J.R., 1996. Application of gene
probes as diagnostic tools for white spot baculovirus ŽWSBV. of penaeid shrimp. Dis. Aquat. Org. 27,
59–66.
Francki, R.I.B., Fauquet, C.M., Knudson, D.L., Brown, F. ŽEds.., 1991. Classification and nomenclature of
viruses. Arch. Virol., Suppl., 2, pp. 117–123.
Inouye, K., Miwa, S., Oseko, N., Nakano, H., Kimura, T., 1994. Mass mortalities of cultured kuruma shrimp
Penaeus japonicus, in Japan in 1993: electron microscopic evidence of the causative virus. Fish Pathol. 29,
149–158.
44
P.R. Rajan et al.r Aquaculture 184 (2000) 31–44
Karunsagar, I., Karunasagar, I., 1997. Strategies for management of white spot disease in shrimp. Souvenir of
the National Aquaculture week 24th–7th Feb., 1997. Aquaculture Foundation of India, Neelankarai,
Chennai, India. pp. 37–39.
Karunasagar, I., Otta, S.K., Karunasagar, I., 1997. Histopathological and bacterial study of white spot
syndrome of Penaeus monodon along the west coast of India. Aquaculture 153, 9–13.
Lightner, D.V., 1993. Diseases of cultured penaeid shrimp. In: McVey, J. ŽEd.., CRC Handbook of
Mariculture, Vol. 1, Crustacean Aquaculture, 2nd edn. CRC Press, Boca Raton. pp. 393–486.
Lightner, D.V. ŽEd.., 1996. A Handbook of Pathology and Diagnostic Procedures for Diseases of Penaeid
Shrimp. World Aquaculture Soc., Baton Rouge.
Lo, C.F., Leu, J.H., Ho, C.H., Chen, C.H., Peng, S.E., Chen, Y.T., Chou, C.M., Yeh, P.Y., Huang, C.J., Chou,
H.Y., Wang, C.H., Kou, G.H., 1996a. Detection of baculovirus associated with white spot syndrome
ŽWSBV. in penaeid shrimp using polymerase chain reaction. Dis. Aquat. Org. 25, 133–141.
Lo, C.F., Ho, C.H., Penug, S.E., Chen, C.H., Hsu, H.C., Chiu, Y.L., Chang, C.F., Liu, K.F., Su, M.S., Wang,
C.H., Kou, G.H., 1996b. White spot syndrome baculovirus ŽWSBV. detected in cultured and wild shrimp,
crabs and other arthropods. Dis. Aquat. Org. 27, 215–225.
Mari, J., Bonami, J.R., Poulos, B., Lightner, D.V., 1993. Preliminary characterization and partial cloning of
the genome of a baculovirus from Penaeus monodon ŽPm SNPVs MBV.. Dis. Aquat. Org. 16, 207–215.
Momoyama, K., Hiraoka, M., Nakano, H., Koube, H., Inouye, K., Oseka, N., 1994. Mass mortalities of
cultured kuruma shrimp, Penaeus japonicus, in Japan in 1993: histopathological studies. Fish Pathol. 29,
141–148.
Muralimanohar, B., Sundararaj, D., Selvaraj, D., Sheela, P.R.R., Chidambaram, P., Mohan, A.C., Ravishankar,
B., 1996. An outbreak of SEMBV and MBV infection in cultured Penaeus monodon in Tamil Nadu.
Indian J. Fish. 43, 403–406.
Nakano, H., Koube, H., Umezaea, S., Momoyama, K., Hiraoka, M., Inouye, K., Oseka, N., 1994. Mass
mortalities of cultured kuruma shrimp, Penaeus japonicus, in Japan in 1994: epizootiological survey and
infection trials. Fish Pathol. 29, 135–139.
Ramasamy, P., Brennan, G.P., Jayakumar, R., 1995. A record and prevalence of Monodon baculovirus from
post-larval Penaeus monodon in Madras, India. Aquaculture 130, 129–135.
Sano, T., Nishimura, T., Fukuda, H., Hayashida, T., Momoyama, K., 1984. Baculoviral mid-gut gland necrosis
ŽBMN. of Kuruma shrimp Ž Penaeus japonicus . larvae in Japanese intensive culture systems. Helgol.
Meeresunters. 37, 255–264.
Supamattaya, K., Hoffmann, R.W., Boonyaratpalin, S., Kanchanaphum, P., 1998. Experimental transmission
of white spot syndrome virus ŽWSSV. from black tiger Penaeus monodon to the sand crab Portunnus
pelagicus, mud crab Scylla serrata and krill Acetes sp. Dis. Aquat. Org. 32, 79–85.
Takahashi, Y., Itami, T., Kondom, M., Maeda, M., Fujii, R., Tomonaga, S., Supamattaya, K., Boonyaratpalin,
S., 1994. Electron microscopic evidence of bacilliform virus infection in Kuruma shrimp Ž Penaeus
japonicus .. Fish Pathol. 29, 121–125.
Takahashi, Y., Itami, T., Maeda, M., Suzuki, N., Kasornchandra, J., Supamattaya, K., Khongpradit, R.,
Boonyaratpalin, S., Kondo, M., Kawai, K., Kusuda, R., Hirono, Aoki, T., 1996. Polymerase chain reaction
ŽPCR. amplification of bacilliform virus ŽRV-PJ. DNA baculovirus ŽSEMBV. DNA in Penaeus monodon
Fabricius. J. Fish Dis. 19, 399–403.
Wang, C.H., Lo, C.F., Leu, J.H., Chou, C.M., Yeh, P.Y., Chou, H.Y., Tung, M.C., Chang, C.F., Su, M.S.,
Kou, G.H., 1995. Purification and genomic analysis of Baculovirus associated with white spot syndrome
ŽWSBV. of Penaeus monodon. Dis. Aquat. Org. 23, 239–242.
Wongteerasupaya, C., Vickers, J.E., Sriurairatana, S., Nash, G.L., Akarajamorn, A., Boonsaeng, V., Panyim,
S., Tassanakajon, A., Withyachumnarnkul, B., Flegel, T.W., 1995. A non-occluded, system baculovirus
that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn
Penaeus monodon. Dis. Aquat. Org. 21, 69–77.
Wongteerasupaya, C., Wongwisansri, S., Boonsaeng, V., Panyim, S., Pratanpipat, P., Nash, G.L., Withyachumnarnkul, B., Flegel, T.W., 1996. DNA fragment of Penaeus monodon baculovirus PmNOBII gives
positive in situ hybridization with white-spot viral infections in six penaeid shrimp species. Aquaculture
143, 23–32.