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Vol. 27: 215-225, 1996 DISEASES OF AQUATIC ORGANISMS

Dis Aquat Org Published December 12

White spot syndrome baculovirus (WSBV)

detected in cultured and captured shrimp, crabs

and other arthropods

Chu-Fang Lol, Ching-Hui HO', Shao-En pengl, Chau-Huei Chenl, Hui-Chen ~ s u ' ,

Ya-Lin ~ h i u ' ,

Chen-Fang chang2, Kuan-Fu ~ i u ~ ,

Mao-Sen Su2, Chung-Hsiung wang3,

Guang-Hsiung

KOU'~'

'Department of Zoology, National Taiwan University, Taipei, Taiwan, ROC

'Tung Kang Marine Laboratory, Taiwan Fisheries Research Institute, Tung Kang, Ping Tung, Taiwan, ROC 'Department of Plant Pathology and Entomology, National Taiwan University, Taipei, Taiwan, ROC

ABSTRACT: Whlte spot syndrome baculovirus (WSBV) has been found across ddferent shrimp species and in different Asian countries. The detection of WSBV in shrimp with white spot syndrome has already been achieved by means of l-step polymerase chain reaction (PCR). In an attempt to establish a more sensitive assay, we evaluated the effect of 2-step amplification with nested primers on the sensitivity of WSBV diagnostic PCR. The sensitivity of the 2-step amplification was 10" to 10"imes higher than that of l-step amplification. Using both techniques, we successfully detected WSBV DNA in cultured and captured shrimp, crabs and other arthropods. Cultured Penaeus monodon (black tiger shrimp),

P.

japonicus (kuruma shrimp), P penicjllatus (red tail shrimp), and Metapenaeus ensis (sand shrimp) displaying white spot syn- drome were collected from farms at different localities. One-step amplification of the DNA extracted from these shrimps consistently yielded an expected 1447 bp PCR product. Some of the tested specimens of cul- tured Scylla serrata (mud crab) that exhibited white spot syndrome were positive in l-step WSBV diag- nostic PCR, while others were positive only in 2-step WSBV diagnostic PCR. Use of the 2-step amplifica- tion protocol also detected a WSBV-specific DNA fragment in Macrobrachium rosenbergii (the giant freshwater prawn) exhibiting white spot syndrome. We also confirmed that WSBV exists in wild-caught shrimp ( P monodon, f! japonicus,

P

semisulcatus and P penicillatus) and crabs (Charybdisferiatus. Por- tunuspelagicus and P. sanguinolentus) collected from the natural environment in coastal waters around southern Taiwan. Detection of WSBV in non-cultured arthropods collected from WSBV-affected shrimp farms revealed that copepods, the pest crab Hehce tndens, small pest Palaemonidae prawn and the larvae of an Ephydridae insect were reservoir hosts of WSBV. The relatedness between WSBV and Thailand's systemc ectodermal and mesodermal baculovirus (SEMBV) is d~scussed in this paper.

KEY WORDS: White spot syndrome

. Shrimp viral disease . PCR

. Cultured and non-cultured arthropods

INTRODUCTION

White spot syndrome baculovirus (WSBV) (Lightner

1996) has been found across different shrimp species and

in different Asian countries. The principal clinical sign of

the disease associated with this virus is the presence of

white spots in the exoskeleton and epidermis of the dis-

eased shrimp. The causative virus itself consists of an

enveloped, rod-shaped nucleocapsid. It is extremely vir-

ulent, has a wide host range and targets various tissues

'Addressee for correspondence. E-mail: ghkouOccms.ntu.edu.tw

(Chou et al. 1995, Wang et al. 1995, Chang et al. 1996,

Lo

et al. 1996). The rapid onset and lethality of this disease

are remarkable. Based on the histopathology of the

affected animals and the characteristics of the virus,

WSBV is either identical or closely related to Thailand's

systemic ectodermal and mesodermal baculovirus

(SEMBV) (Wang et al. 1995, Wongteerasupaya et al.

1995,

C.

Wongteerasupaya pers. comm.). WSBV infec-

tion and its associated mortality is emerging as one of the

most significant problems for the global shrimp industry.

The amplification of selected

DNA sequences

by

polymerase chain reaction

(PCR) is fulfilling its promise

as a powerful diagnostic tool for the identification of

0 Inter-Research 1996

(2)

Dis Aquat Org 27: 215-225, 1996

pathogens (Erlich et al. 1988, Oste 1988). In recent

years, a number of researchers have drawn attention to

the need for more sensitive diagnostic techniques using

'high-tech' approaches for crustacean viruses (Brock

1992, Lightner et al. 1992, Bruce et al. 1993, 1994,

Chang et al. 1993, Man et al. 1993, 1995, Poulos et al.

1994. Wang e t al. 1995). In the case of WSBV, we have

already developed PCR primers a n d nucleic acid

probes for diagnosis (Lo et al. 1996). The specificity of

these WSBV PCR primers has already been established

(Lo e t al. 1996), and in the present paper w e use the

more sensitive nested PCR to detect the latent or pre-

patent carrier state of WSBV infection in shrimp and

other arthropod populations collected from shrimp

farms or from the natural environment.

MATERIALS AND METHODS

Oligonucleotide primers for WSBV diagnostic PCR.

A nested primer set derived from the sequence of a

cloned WSBV

Sal1

1461 bp DNA fragment (Lo et al.

1996) including 146F1, 5'-ACT ACT AAC TTC AGC

CTA TCT AG-3'; 146R1,5'-TAA TGC GGG TGT AAT

GTT CTT ACG A-3'; 146F2, 5'-GTA ACT GCC CCT

TCC ATC TCC A-3'; 146R2, 5'-TAC GGC AGC TGC

TGC ACC TTG T-3'; 146F4, 5'-AGA AGG TTA GCG

AAT GGA CTG-3'; and 146R3, 5'-CGA AGA GTA

GTG TTA GAA GAG GA-3' was utilized for l-step or

2-step WSBV diagnostic PCR. Four of these were used

in our previous study (Lo e t al. 1996).

As described previously (Lo et al. 1996), the quality

of the DNA extracted from tested organisms was

checked with a primer set amplifying a decapod gene

before the application of WSBV diagnostic PCR. For

this purpose, 2 primers, 143F (5'-TGC CTT ATC AGC

TNT CGA TTG TAG-3', where N represents G, A, T o r

C) a n d 145R (5'-TTC AGN TTT GCA ACC ATA CTT

CCC-3'), derived from a highly conserved region of the

1 8 s rRNA sequence of decapods (Kim

&

Abele 1990,

Lo et al. 1996) were used.

One-step WSBV diagnostic PCR. Three primer pairs,

146F1/146R1, 146F2/146R2 and 146F4/146R3, were

used for l-step WSBV diagnostic PCR. The DNA

sample extracted from the muscle of 1 diseased black

tiger shrimp naturally infected with WSBV was used as

template DNA for demonstrating the specificity a n d

sensitivity of the 3 primer pairs in l-step WSBV diag-

nostic PCR. The DNA samples used for amplification

totaled 0.1 pg in a 100 p1 reaction mixture containing

10 m M Tris-HC1, pH 8.8 a t 25"C, 50 mM KC1, 1.5 mM

MgCl,, 0.1

%

Triton X-100, 200 pM of each dNTP,

100 pm01 of each primer,

2

units of DynaZymeTMII

DNA Polymerase (Finnzymes Oy, Riihitontuntie 14 B,

FIN-02200 Espoo, Finland). The amplification was per-

formed in a AG-9600 Thermal Station (Biotronics Corp.

Lowell, MA, USA) for 1 cycle of 94°C for

4

min, 55°C

for 1 min. 72°C for 2 min; 39 cycles of 94°C for

1 min,

55°C for

1 min, 72°C for 2 min, plus a final 5 min ex-

tension at 72OC after 40 cycles. Control reactions con-

taining no template DNA were run for all PCR reac-

tions. A portion (10 pl) from each of the completed PCR

reactions was mixed with 1 p1 loading buffer and sub-

jected to electrophoresis on

1 %

agarose gels contain-

ing ethidium bromide at a concentration of 0.5 pg ml-',

and visualized by ultraviolet transillurnination.

Two-step WSBV diagnostic PCR. The first PCR step

was performed as described above with the outer

primer pair 146F1 a n d 146R1. After completion of the

first step, 10 p1 of the reaction mixture was added to

90 p1 of PCR cocktail containing the inner primer pair,

146F2 and 146R2, and this was then subjected to a

second step of amplification over the same 40 cycles.

For the ana!ysis of PCR products, 10 p1 of the final reac-

tion mixture was used.

Evaluation of the effect of 2-step

PCR with nested

primers o n the sensitivity of WSBV diagnostic PCR.

The relative sensitivity of l-step PCR, 2-step PCR, 1-

step PCR with Southern hybridization and l-step PCR

with dot hybridization was investigated as follows:

First step o f amplification:

Serially 10-fold diluted

(10-' to IO-') DNA samples of deproteinized DNA ex-

tracted from the muscle of diseased black tiger shrimp

naturally infected with WSBV served as template DNA

samples in the first step of amplification with the

146F1/146R1 primer set.

Second step o f amplification:

An aliquot (10 p1) of

each PCR product yielded in the first step of amplifi-

cation served a s template DNA in the second step of

amplification.

One-step PCR with Southern hybridization:

Southern

hybridization was performed with the gel used to ana-

lyze the products yielded in the first step of amplification.

After application of alkali (1.5 M NaCl and 0.5 N NaOH)

to denature the DNA, the gel was neutralized with 1 M

Tris (pH 7.4) and 1.5 M NaCl and subsequently trans-

ferred to a Hybond-N nylon membrane (Amersham,

Little Chalfont, Buckinghamshire, UK) using a Hoefer

TE 80 vacuum transfer unit (Hoefer Scientific Instru-

ments, San Francisco, CA, USA) for 60 min. A solution

of 20x SSC ( l x SSC = 150 mM NaC1, 15 mM sodium

citrate, pH 7.0) was used as transfer buffer. After cross-

linking the DNA with the membrane by UV light, the

blot was used for hybridnation with a probe derived

from the 941 bp PCR product of a reaction mixture with

the 146F2 and 146R2 primers. This probe was non-

radioactively 1abel.ed with digoxigenin (DIG)-dUTP

(Boehringer Mannheirn, Germany) by a random priming

method. The blot was hybridized at 37°C for 16 h with

the DIG-labeled probe, after prehybridization at 37°C for

(3)

Lo et a1 : WSBV d e t e c t ~ o n In arthropods

12 h in 50 % formaniide, 5 x SSC, 1 mM EDTA, 50 mM Tris (pH 8) a n d 5 x Denhardt's reagent [0.1% Ficoll-400, 0.1 % polyvinyl pyrrolidone, 0.1 % bovine serum alburmn (BSA)]. After hybridization, detection of the DIG-labeled nucleotides in blots was accomplished using a DIG Luminescent Detection f i t (Boehnnger Mannheim). The blot was exposed to Kodak XAR-5 film at 37OC for 15 to 30 min to record the chemiluminescent signal.

One-step PCR

with dot hybridization:

An aliquot (10 p1) of each of the completed PCR reactions in the first step amplification was incubated with 16 p1 of a n alkali solution (1.5 M NaCl, 0.5 N NaOH) and 24 p1 of H 2 0 a t room temperature for 5 to 7 min. After being neutralized with 4 p1 of neutralization solution (1.5 M NaCl, 1 M Tris, pH 7.4) for 10 min, the reaction niix- tures were spotted onto Hybond-N nylon paper using a 96-well dot-blot vacuum filtration manifold appara- tus (Schleicher a n d Schuell, Inc. Keene, NH, USA). The blot was rinsed with 2 x SSC. After cross-linking the DNA with the membrane by UV light, the blot was used for hybridization with a DIG-labeled probe derived from the 941 b p PCR product. The procedure for the hybridization a n d signal detection was the same a s that for Southern hybridization.

Detection of WSBV in cultured decapods. Cultured Penaeus monodon (black tiger shrimp), l? japonicus (kuruma shrimp), l? penjcillatus (red tail shrimp), Meta- p e n a e u s ensis (sand shrimp), Macrobrachlum rosen- bergii (the giant freshwater prawn) and Scylla sen-ata (mud crab) that displayed white spot syndrome (WSS) were collected at various growth stages, from various geographic regions of Taiwan, and in different seasons. These shrimp specimens were subjected to WSBV diagnostic PCR. From each specimen, 1 pereio- pod was excised a n d placed in a small vial, rapidly frozen in liquid nitrogen a n d then stored a t -70°C until use for PCR template DNA preparation. DNA was iso- lated following the method described by Lo et al. (1996) with some modification. Briefly, a piece of pereiopod (approx. 100 mg) was placed directly in a microfuge tube containing 1.2 m1 digestion buffer (100 mM NaC1, 10 mM Tris-HC1, pH 8, 25 mM EDTA, pH 8 , 0 . 5 % N- lauryl sarcosine, 0.5 mg ml-' proteinase K) a n d crushed with a disposable stick. After incubation at 65'C for 1 h , 5 M NaCl was added to adjust the NaCl concen- tration of the DNA solution to 0.7 M. The solution was then treated with 1 % N-cetyl N,N,N-trimethylammo- nium bromide (CTAB) for 10 nlin at 65°C followed by successive extractions with 1 X volume of chloroform/ isoamyl alcohol once, 1 X volume of phenol 2 to 3 times, a n d 2 X volume of chloroform/isoamyl alcohol once.

The DNA was recovered by ethanol precipitation, dried a n d resuspended in 0.1 X Tris-ethylenediamine- tetraacetic acid (1 X TE = 10 mM Tris-base, 1 mM EDTA.2Na, pH 7.6) buffer at 65OC for 30 min, before

being stored at 4°C until use for WSBV diagnostic PCR. The PCR products recovered by ethanol precipitation were further analyzed with HaeIII, HpaII, RsaI a n d Sa3A I restriction endonucleases.

Detection of WSBV in wild-captured decapods. From July through December 1995, 66 Penaeus monodon (body weight. 59 to 128 g ) , 23 l? japonicus (35 to 100 g ) , 32 l? semisulcatus (80 to 127 g ) , a n d 27 l? penicillatus (18 to 39 g ) in the adult stages and of wild origin were captured from their natural environment in the coastal waters around southern Taiwan. On their arrival at Tung Kang Marine Laboratory, specimens were ex- amined visually and found to b e in good health with no evidence of WSS. In order to minimize the effect of stressful conditions which can trigger increased viral replication in the shrimp, excision of 1 pereiopod from each of the specimens was performed immediately, a n d the excised pereipod was then subjected to 2-step WSBV diagnostic PCR. When the results of the first step of amplification became available 4 h later, one of the specimens whose pereiopod was positive for WSBV by l - s t e p PCR was killed a n d its tissues were prepared for histological observation using light iiiicroscopy ds described previously (Chou et al. 1995). The remaining shrimp specimens w e r e maintained in 500 1 tanks for a period of further observation.

Detection of WSBV in non-cultured arthropods from shrimp farms. Specimens of copepods, pest crab Hehce tridens, small pest Palaemonidae prawn a n d pupae of a n Ephydridae insect were collected from shrimp farms in epizootic areas a n d subjected to 2-step WSBV diagnostic PCR in order to ascertain whether WSBV was present in these non-cultured arthropods.

RESULTS

Oligonucleotide primers and amplicons of WSBV diagnostic PCR

The relative positions of oligonucleotide primers cor- responding to the sequences of the WSBV Sal1 1461 b p DNA fragment for WSBV diagnostic PCR a n d their aniplicons a r e indicated in Fig. 1A. Fig. 1B shows the 1- step PCR product primed by 146F1/146R1 (lane l ) ,

146F2/146R2 (lane 2) a n d 146F4/146R3 (lane 3) with each lane using the same amount of template DNA iso- lated from 1 WSBV-infected specimen of black tiger shrimp. The primer pairs achieved higher sensitivity when they yielded shorter amplicons. In the nested protocol for 2-step PCR in this study, 146F1/146R1 w a s used for outer PCR while 146F2/146R2 was used for inner PCR. However, several other combinations of primer pairs for the nested protocol were tested a n d also yielded good results (data not shown).

(4)

Dis

Aquat Org 27: 215-225, 1996

WSBV Sail l461 bp DNA fragment

Fig. 1. Pnmers and amplicons of WSBV diagnostic PCR. (A) Location and sequence of primers for WSBV diagnostic PCR; (B) amplicons primed by 146F1/146Rl (lane l ) , 146F2/146R2 (lane 2) and 146F4/146R3 (lane 3). M: pGEM DNA size marker

Evaluation of the effect of 2-step PCR with nested

primers on the sensitivity of WSBV diagnostic PCR

The sensitivity of the 2-step PCR was 103 to 104 times

higher than that of the l-step PCR (Fig.

2). The 10 1.11

of completed PCR reaction mixture of the first step of

amplification, which served as the DNA source for the

second step of amplification, contained some 1447 bp

PCR products as well as some excess from the

146F1/

146R1 primer set. Because of this, the DNA samples

that yielded a visible band of 1447 bp fragment in the

first step of amplification usually yielded 2 visible

bands (1447 and 941 bp) in the second step of amplifi-

cation (Fig. 2B). The sensitivity of 2-step PCR was also

higher than that of l-step PCR with Southern or dot

hybridization, by a factor of about 102

to 103

(Fig. 2B, C,

D).

One-step PCR

A b p l 2 3 4 5 6 7 8 9

Two-step PCR

B b p 1 2 3 4 5 6 7 8 9

One-step PCR+Southern hybridization

C b p 1 2 3 4 5 6 7 8 9

Detection of

WSBV in

cultured decapods

WSS was found in 4 major cultured species of marine

shrimp, i.e. Penaeus monodon, P japonicus,

l? penicil-

latus and Metapenaeus ensis (Fig. 3 ) . Except for

I!

penicillatus, WSS was readily observed in diseased

cultured shrimp, with the white spots being quite

marked (Fig. 3A,

B, D ) . In diseased P penicillatus,

however, the white spots were not easily seen (Fig.

3 C )

until after the carapace had been removed. One-step

amplification of WSBV DNA from the DNA samples of

shrimp that displayed WSS consistently yielded a

1447 bp PCR product as shown in the left panel of

Fig. 4. The results confirmed the presence of a rela-

tively large amount of

WSBV in the tested specimens.

Interestingly, we found that the amount of the WSBV

in shrimp from the same pond collected at the same

One-step PCR+dot hybridization 1 2 3 4 5 6 7 8 9

E::

Fig. 2. Detection of WSBV DNA in serially 10-fold diluted DNA extracted from naturally infected black tiger shrimp by (A) l-step PCR; (B) 2-step PCR; (C) 1-step PCR with Southern hybridization; and (D) l-step PCR with dot hybridization. (A) l-step PCR using 146F1 and 146R1 primer pair, (B) 2-step (nested) PCR using 146F1 and 146R1. as a n outer primer pair, and 146F2 and 146R2 a s a n inner primer pair; (C) Southern hybridization after l-step PCR using a DIG-labeled 146F2- 146R2 probe; ( D ) dot hybridization after l-step PCR using a DIG-labeled 146F2-146R2 probe. Lanes 1 to 9 correspond to 10-' to 10-Q dilution of infected shnmp DNA. PCR products

(5)

Lo et al.. WSBV detection in arthropods

Fig. 3. Cultured marine shrimp with white spot syndrome. ( A ) Penaeus monodon, ( B ) P japonicus, (C) P penicillatus, ( D ) Meta-

penaeus ensis. Scale bars = 1 cm

time was similar; this is evidenced by

the similar intensity of their PCR prod-

uct bands as shown in the left panel of

Fig.

4,

in which 2 specimens of each

shrimp species collected from the same

pond at the same time are shown. No

1

differences were observed in the rest-

riction profiles of PCR products yielded

by l? monodon, l? japonicus,

l?

penicil-

latus,

and Metapenaeus ensis,

analyzed

with

HaeIII, HpaII, RsaI

and

Sau3AI

restriction endonucleases (Fig.

4,

right

panel), which indicates the close relat-

edness of WSBV found in the different

species of shrimp collected from various

geographic regions of Taiwan and in

different seasons.

The WSS displayed by the cultured

marine crab

Scylla serrata

was readily

seen in pereiopods rather than in the

carapace. When these crabs were sub-

jected to WSBV diagnostic PCR, some

tested specimens gave a positive result

in the first step of amplification, while

others gave a positive result only after

reamplification. Unexpectedly, all tested

specimens of the cultured freshwater

prawn Macrobrachium rosenbergii with

WSS gave WSBV-positive results only

One-step WSBV DNA PCR M Pm Pi Po Me N

Haelll Hpall Rsa l Sau3Al

M Pm

pr

Pp Me Pm Pj Pp - Me E m

3

Pp

- - - - Me Pm P) Pp _M+

Fig. 4. WSBV-specific DNA fragments amplified frolll L c l l l p l O L c ylYm 3 C L I I I p I c J p l c p a l c U I I V I ~ cultured marine shrimp Penaeus monodon (Pm), P: japonicus (Pj),

P

pen~cillatus (Pp), and Metapenaeus ensis (Me) that displayed marked white spot syndrome. Two specimens for each species of

P

monodon, P: japonicus, and l? penicillatus were collected from the same pond at the same time. Left panel: l-step WSBV PCR products. Right panel: result of the PCR products analyzed with HaeIII, HpaII, RsaI and Sau3AI

(6)

220 Dis Aquat Org 27: 215-225. 1996

A

1st Step PCR

B

C

2nd Steo PCR Hoa II Rsa l Fig. 5. WSBV-spec~f~c DNA fragments amplified from template DNA samples prepared from cultured mud crab Scylla serrata and cultured freshwater shrimp Macrobrachium rosenbergii

that displayed white spot syndrome. (A) l-step WSBV PCR

products. (B) 2-step WSBV PCR products. (C) 2-step PCR prod-

ucts amplified from Penaeus monodon (Pm), S. serrata (Ss) and

M. rosenbergii (Mr) cleaved with HpalI and RsaI restriction

endonucleases. M: pGEM DNA size marker

after the second step of amplification, in

which they yielded a

94 1

bp PCR fragment.

However, analysis of this PCR product with

Hpa I1 and Rsa

I restriction endonucleases

showed similar restriction profiles to those

of the 941 bp fragments yielded by

P mon-

odon and the mud crab S. serra1.a (Fig.

5 ) .

Detection of WSBV in wild-captured decapods

Captured

specimens

of

apparently

healthy, wdd Penaeus monodon,

P japo-

nicus,

P semisulcatus and P penicillatus

were immediately tested by PCR analysls

upon their arnval at Tung Kang Manne

Laboratory. The signs of WSS, even when

present, were not readily seen in these

examined specimens (Fig 6).

The results of PCR analysis (Table

1)

indicated the presence of WSBV in all

4

of

the species investigated. Of the 66 Penaeus

4

monodon collected

50

were positive by

'2.- 4

step WSBV diagnostic PCR and 22 of these

were positive in the first step of amplifica-

tion. The l-step WSBV PCR-positive indi-

viduals usually died within 1 to

3 d after

arrival at Tung Kang Manne Laboratory.

Small white spots were observed on cara-

I

ited the typical histopathological changes

caused by WSBV infection in tissues of the

gill, the stomach, the pleopods and the

lyrnphoid organ. Cells with characteristic

hypertrophied nuclei were readily seen in

these tissues (Fig.

7 ) .

Occasionally, crabs were found amongst

Fig. 6. (A) Penaeus monodon,

(B)

P japonicus, (C) P semlsulcatus, and (D)

the captured shrimp, specifically Charyb-

P penicillatus captured from coastal waters near southern Taiwan. Scale

dis feriatus

( l

Po*unus

pelagi-

(7)

Lo et al.: WSBV detection in arthropods 221

Table 1. Results of 2-step WSBV diagnostic PCR with adult, captured Penaeus monodon, P japonicus, P semisulcatus and

P

penicillatus. Values represent no. of shrimp positive in the first and second step PCR per no. of shrimp examined WSBV diagnostic PCR F! monodon P japonicus F! seniisulcatus P penicillatus

l -step PCR 22/66 5/23 0/32 0/27

2-step PCR 50/66 14/23 2/32 3/27

(1 specimen). All of these crabs were positive In the

second step of 2-step WSBV diagnostic PCR.

Detection of WSBV in non-cultured arthropods from

shrimp farms

Copepods, pest crab Helice tndens, small pest Palae-

monidae prawn, and pupae of an Ephydridae insect

were collected from shrimp farms in epizootic areas of

WSBV infection (Fig. 8).

Template DNA samples were

prepared from the pereiopods of

15

specimens of small

Palaemonidae pest prawns and 9 specimens of H.

tri-

d e n s pest crab. They were also prepared from 10

coarctate larvae removed from the protective case of

Ephydridae pupae and 6 pooled, pelleted samples of

copepods. These samples were subjected to 2-step

WSBV diagnostic PCR. As shown in Table 2, WSBV-

positive cases were found in copepods, pest crab, pest

prawn and insects which had been recently (within

half a day) collected from shrimp farms. Four out of

10

Ephydridae pupae, 2 out of the 6 pooled samples of

copepods,

10

out of 15 pest prawn specimens and

4

out

of

11

pest crab specimens were positive by l-step

WSBV diagnostic PCR. Thus copepods, pest crab H.

tridens

and small pest Palaemonidae prawn a s well

as an Ephydridae insect are evidently reservoir hosts

for WSBV in shrimp culture facilities.

Fiq. 7. Penaeus monodon. (A]

selected l-siep

WSBV

PCR-

I

positive

P:

rno-nodon exhibit- ing the typical histopatho- logical changes caused by

WSBV

attacks. Degenerated cells characterized by hyper- trophied nuclei (arrows) are readily seen in these tissues.

(8)

222 DIS Aquat Org 27. 215-225, 1996

DISCUSSION

The specificity of the primers used here as well as

the standardization of preparation procedures for

template

DNA

a n d its quality control have been d e -

scribed previously (Lo et al. 1996). Furthermore, in

pilot studies of WSBV tissue tropism in various deca-

pods using histopathological examination,

in situ

hybridization assay a n d transmission electron micro-

scopic observation, the preliminary data show that in

most cases, a positive PCR result coincides with WSBV

histopathology a n d the presence of WSBV positive

cells as revealed by

i n situ hybridization (data not

included in t h s manuscript). Nonetheless, although

2-step PCR is very sensitive a n d reliable, there is a

need for stringent controls to eliminate the increased

chance of false positives. Therefore, in addition to a

template-free reagent control reaction, a PCR reaction

with a template is run as a supplemeniary negative

control for all PCR reactions (Lo et al. 1996). In the pre-

sent study, a sample from a previously prepared batch

of WSBV-free shrimp abdominal muscles (100 mg in

each vial; stored at -70°C) was always included in our

DNA

extraction samples (data not shown). This pro-

vided some reassurance that positive results did not

arise from contamination during processing, but false

positives a n d physical environmental contamination

a r e complex issues that will b e d s c u s s e d more fully in

papers currently in preparation.

WSBV was previously classified by Wang et al.

(1995) as the genus

Non-Occluded [sic] Baculovirus

( N O B ) of the subfamily Nudibaculovirinae of the

family

Baculoviridae in accordance with the fifth

report of the International Committee on the Taxon-

Fig. 8. (A) Insect (family: Ephydridae), (B) copepod (subclass: Copepoda, Schmackeria dubia), (C) pest prawn (farmly: Palaemonidae, Exopalaemon orientis.), and ( D ) pest crab

(Hehce tridens) collected from shrimp farms in epizoot~c ar- eas of WSBV lnfectlon. White scale bar In (A) = 3 mm; black scale bar in (B) = 0 5 mm. Arrows indicate the pupae of an

Ephydridae insect

omy of Viruses (ICTV) (Francki et al. 1991). In the sixth

report of the ICTV, however, the subfamily

Nudi-

baculovirinae a n d the genus NOB a r e not listed (Mur-

phy et al. 1995). The 2 included genera of the family

Baculoviridae a r e Nucleopolyhedro~irus

a n d

Granulo-

virus, a n d WSBV is unlikely to belong to either. The

insect viruses Oryctes rkinoceros virus (OrV) and

Heliothis zea virus 1 (HzV-l), which were previously

classified a s nonoccluded members of the family

Baculoviridae, have now been placed

in the list of unassigned invertebrate

Table 2. Results of 2-step WSBV d~agnostlc PCR in copepods (subclass: Cope- viruses,

than

a

poda), pest crab Hellce tndens, small pest prawn (family: Palaemonidae) a n d a n

nonoccluded baculoviruses have been

insect (famlly: Ephydridae) collected from s h n m p farms In epizootic areas of

found associated with a number of

WSBV infect~on. Values represent no. of specimens positwe ln the f ~ r s t and

arthropod hosts, including lnsecta,

second step PCR per no. of specimens examined

Arachnida a n d Crustacea, very Little is

WSBV diagnostic PCR Insect Copepod Pest prawn Pest crab

-

known about their molecular biology

(Sano et al. 1981, Lester e t al. 1987,

l -step PCR 4/10 2/6 10/15 4/11 2-step PCR 8/10 4/6 12/15 5/11

Johnson 1988, Johnson

&

Lightner

1988, Burand 1991, Crawford 1994,

Wang e t al. 1995, Wongteerasupaya

(9)

Lo et al.. WSBV detection ~n arthropods 223

et al. 1995). To date, it is not clear whether WSBV and

other nonoccluded baculoviruses should in fact be

included in the Baculoviridae or not. Genome sequenc-

ing, genome organization, gene order, strategy of

replication and other genetic considerations of insect

and crustacean nonoccluded baculoviruses, which are

becoming increasingly important as viral taxonomic

criteria (Murphy et al. 1995), are in urgent need of

further study before the taxonomic status of these

viruses can be decided. Notwithstanding the uncer-

tainty in the taxonomic position of nonoccluded bac-

uloviruses, in a recently published handbook of patho-

logy and diagnostic procedures for diseases of penaeid

shrimp (Lightner 1996), the present virus is called

white spot syndrome baculovirus (WSBV). Conse-

quently, in the interests of nomenclaturial consistency,

this is the name that we have used here.

Wang e t al. (1995) demonstrated that negatively

stained preparations of WSBV virions frequently had

tail-like projections extending from one end, that the

nucleocapsid components form parallel cross-striations

which are perpendicular to the longitudinal axis of the

nucleocapsid, and that the full length of WSBV

genonlic

DNA

was longer than 150 kbp. WSBV is very

close to SEMBV from Thailand

in

terms of viral mor-

phology (size, bacilliform with a tail, banding of nuc-

leocapsid) and type and size of genome. WSBV EcoR I

and BamH I restriction profiles (authors' unpubl. data)

were also found to be similar to those published for

Thailand's SEMBV (Wongteerasupaya et al. 1995). [In

this regard, please note that Thailand's SEMBV group

has informed us that the names of the restriction

enzymes were inadvertently transposed in the caption

to Fig.

4

of their article in Diseases of Aquatic Organ-

isms (Vol. 21, p. 75)

(C.

Wongteerasupaya pers.

cornm.).] Thus, SEMBV and WSBV are at least very

similar if not identical.

In several respects including viral morphology and

genome size, WSBV is distinct from the Hz-l virus but

shares some characteristics with the Oryctes rhinoc-

eros virus, a pathogenic virus of invertebrates, which

infects coleopteran insects in the family Scarabaeidae

(Burand 1991, Crawford 1994, Murphy et al. 1995,

Wang et al. 1995, Wongteerasupaya et al. 1995). Never-

theless, WSBV a n d OrV are alike only to a certain

extent, and their very similarity raises questions. What,

if any, is the relationship between insect viruses and

crustacean viruses? Are these viral agents present in

insects and crustaceans in the natural environment?

Could interactions between insects and cultured crus-

taceans contribute to the increased number of out-

breaks of shrimp viral disease? Most immediately, is it

possible that insects or other non-cultured animals act

a s reservoir hosts of crustacean viruses or vice versa?

In the present study, we have shown PCR evidence of

WSBV in the pupae of a n Ephydridae insect. It was

also detected in copepods, another non-decapod. So

far, it is not clear whether WSBV replicates in either

insects or copepods, or whether it can cause disease in

them. The interaction between cultured shrimp and

insects or other reservoir hosts that inhabit farms, estu-

aries and coastal waters are of great interest. The

dynamics involved may be very complex but under-

standing them should help prevent viral diseases.

We found that about 40% of the pest crab Helice

tri-

dens and Palaemonidae prawn collected from epi-

zootic areas of WSBV infection were positive for WSBV

by l-step diagnostic PCR. These animals were ob-

tained from farms where mass mortality of cultured

shrimp had occurred and only very few cultured

shrimp still survived. We carefully examined a large

number of carapaces removed from Palaemonidae

specimens immediately after being collected from

these farms and all of them were exceptionally clear.

Thus, these pest species are very likely asymptomatic

carriers of WSBV in farms. However, when transferred

to the laboratory, the number of the specimens positive

by l-step diagnostic

PCR increased over time (data

not shown) and all died within a few days. White spot

syndrome was readily observed in these moribund

specimens. Thus, under stressful conditions, WSBV

was able to cause disease in these pest prawns.

In Taiwan, there are

3

different sources of salt water

for shrimp farms: oceanic surface water, beach sand-

filtered sea water and water from saline wells (Chen

1990). Underground sea water (i.e. that from saline

wells) is the most popular water source for small

shrimp culture ponds in Taiwan while large earthen

ponds tend to use oceanic surface water which is

exchanged through canals by gravity flow during high

and low tides. Beach sand-filtered sea water and

underground water systems are normally pest-free,

and the threat of pathogens is much reduced. By con-

trast, there is a high risk that tidal input can introduce

pathogens, microscopic eggs and larvae of such pests

a s Palaemonidae prawn and pest crabs. We have

shown here that these pests may be major reservoir

hosts of WSBV and it is now important to continue to

examine these species in greater detail (histology,

in situ hybridization, bioassays, etc.) to determine to

what extent they might represent a real threat to the

shrimp aquaculture industry. Furthermore, the direct

discharge of water from shrimp culture systems into

drainage canals can release WSBV carriers and WSBV

itself into coastal waters. This could have a severe

negative impact on natural decapod populations and

other susceptible animals. A number of wild shrimp

populations now also carry infectious hypodermal a n d

hematopoietic necrosis virus (IHHNV) (Lotz 1992),

possibly as

a

consequence of nearby shrimp culture

(10)

224 Dis Aquat Org 27: 215-225, 1996

activities (Fulks & M a i n 1992). It is clear t h a t w a t e r s u p p l y s y s t e m s a n d poor m a n a g e m e n t of t h e culture a n d w a s t e w a t e r c a n s p e e d t h e s p r e a d of t h e virus a n d h a v e a s e v e r e n e g a t i v e i m p a c t o n t h e n a t u r a l envi- r o n m e n t .

It w a s disappointing to l e a r n t h a t WSBV w a s p r e s e n t

i n wild populations of

Penaeus monodon,

F!

japonicus,

F!

semisulcatus,

and

F!

penicillatus.

S e v e r a l possible factors m i g h t e x p l a i n t h e h i g h p r e v a l e n c e of

WSBV

infection i n F!

monodon

w h e n c o m p a r e d to t h e o t h e r s p e c i e s s t u d i e d . First, t h e black tiger s h r i m p f r e q u e n t s w a t e r s of less t h a n 30 m i n d e p t h . Unlike m a n y of t h e o t h e r p e n a e i d s h r i m p , it d o e s n o t b u r y itself i n sand b u t s t a y s o n t h e s u r f a c e of t h e s u b s t r a t e ( C h e n 1990). C o n - s e q u e n t l y , it h a s m o r e c h a n c e of b e i n g e x p o s e d t o WSBV i n coastal w a t e r s polluted b y t h e d i s c h a r g e from affected p o n d s . Secondly, m a n y p a r t s of Asia, includ-

ing T a i w a n , h a v e b e e n conducting p r o g r a m s of releas-

ing h a t c h e r y - r e a r e d postlarvae (Fulks

& M a i n 1992) o r

30 g f e m a l e s h r i m p ( C h e n 1990) into coastal w a t e r s i n

o r d e r t o r e p l e n i s h n a t u r a l s h r i m p stocks. Before w e h a v e effective diagnostic tools for all t h e k n o w n s h r i m p viruses a n d c a n certify s h r i m p stocks, t h e practice of r e l e a s i n g f a r m e d s h r i m p into t h e n a t u r a l e n v i r o n m e n t s h o u l d b e a v o i d e d b e c a u s e it m a y h a v e a n e g a t i v e i m p a c t o n n a t u r a l stocks. Conversely, t h e w i d e s p r e a d u s e of c a p t u r e d broodstock of t h e black t i g e r s h r i m p o r k u r u m a s h r i m p to p r o d u c e fry for stocking farms m a y c o n t r i b u t e to t h e viral d i s e a s e p r o b l e m s f a c e d by t h e s h r i m p c u l t u r e industry i n T a i w a n a n d m a n y o t h e r a r e a s .

Acknowledgements. This work was supported by the Council of Agriculture under grant no. 85-AST-1.1-FAD-49(27)A, 85-AST-1.1-FAD-49(27)B and the National Science Council under grant no. NSC85-2311-B002-012-B04. We are indebted to Dr Jung-Yaw Lin. Institute of Biochemistry. College of Medicine, National Taiwan University for his constructive suggestions, and for access to data bases. We thank Dr I-Chiu Liao, Director General, Taiwan Fisheries Research Institute

(TFRI), for his constructive suggestions. We thank Dr Timothy

William Flegel of Mahidol University for numerous dscus- sions on Thailand's SEMBV. We are indebted to Mr Paul

Barlow for his helpful criticism of the manuscript.

LITERATURE CITED

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(11)

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(HPV): use of gene probes in disease diagnosis.

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(1981) Baculovirus infection of cultured kuruma shrimp, Penaeus japonicus, in Japan. Fish Path01 15:185-191 Wang CH, Lo CF. Leu JH, Chou CM, Yeh PY. Chou HY, Tung

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Manuscript first received: May 8, 1996 Revised version accepted: August 28, 1996

數據

Fig.  1. Pnmers  and  amplicons  of  WSBV  diagnostic  PCR.  (A) Location  and  sequence  of  primers  for  WSBV  diagnostic  PCR;
Fig. 3.  Cultured  marine  shrimp with  white  spot syndrome. ( A )   Penaeus monodon,  ( B )   P  japonicus,  (C)  P  penicillatus,  ( D )   Meta-
Table  1. Results  of  2-step  WSBV  diagnostic  PCR  with  adult, captured  Penaeus monodon,  P  japonicus,  P  semisulcatus  and  P  penicillatus
Fig. 8. (A) Insect  (family: Ephydridae), (B) copepod  (subclass:

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