CHAPTER 2.3.4.

1
2
3

SPHERICAL BACULOVIROSIS (PENAEUS MONODON-TYPE BACULOVIRUS)

1.

5
6
7
8
9

For the purpose of this chapter, spherical baculovirosis is considered to be infection with Penaeus monodon-type baculovirus. Synonyms: MBV from
P. monodon was designated PmSNPV (for singly enveloped nuclear polyhedrosis virus from P. monodon) in accordance with the guidelines for virus
nomenclature published by the International Committee on Taxonomy of Viruses (ICTV) (37), and it appears as the tentative species P. monodon NPV,
or PemoNPV, in the 7th and 8th Reports of the ICTV (15, 49). Although PemoNPV may be the most correct name for the virus, the term P. monodon
baculovirus (MBV) will be used in most instances to designate this virus in this Aquatic Manual.

10

2.

Scope

Disease information

11

2.1. Agent factors

12

2.1.1.

13
14
15
16
17
18

The aetiological agent is Penaeus monodon baculovirus (MBV) (30, 32, 36). The International Committee on Virus Taxonomy lists MBV
(spherical baculovirosis) as a tentative species named PemoNPV in the genus Nucleopolyherdovirus (15). Based on the wide
geographical and host species range of MBV, the existence of different strains of MBV is likely. Polymerase chain reaction (PCR) tests
designed for East and South-East Asian isolates of MBV have recently been shown to give false-negative test results for MBV-infected
P. monodon from Africa (Lightner, unpublished data) further suggesting that MBV (or the species PemoNPV) is made up of more than
one strain

19

2.1.2.

20
21
22
23
24

MBV produces a specialised tetrahedral occlusion body in which virions are embedded in a crystalline protein (polyhedron) matrix
(46). In insects, the occlusion bodys function is to protect the occluded virions from the environment until ingested by a suitable host.
The occlusion body of MBV is expected to serve the same function. It is assumed that virions in occlusion bodies remain infectious for
months to years in the sediments (e.g. of estuaries or culture ponds) and that they are essential to the infection cycle of MBV in
different generations occupying the same nursery areas.

25
26

Free (non-occluded) virions are more subject to degradation by sunlight (UV), desiccation, microbial degradation, etc., than are
occluded virions and they are believed to remain infectious from no more than a few hours to a few days outside a host.

27

2.1.3.

28
29

Inactivation of the related occluded baculovirus (Baculovirus penaeid BP) by disinfectants, low pH, heat and UV irradiation has been
reported (22).

30
31

Desiccation (dry-outs) for control of baculovirosis is routinely used in shrimp hatcheries to disinfect spawning tanks, larval rearing
tanks and nursery tanks (see Chapter 1.1.5 Methods for disinfection of aquaculture establishments).

32

2.1.4.

33

MBV replicates in the host cell nucleus. Host-to-host transmission is per os ( 6, 21, 22, 26, 45).

Aetiological agent, agent strains

Survival outside the host

Stability of the agent (effective inactivation methods)

Life cycle

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

34

2.2. Host factors

35

2.2.1.

36
37
38
39
40
41
42

MBV infections have been reported in one or more species of the following penaeid genera/subgenera1: Penaeus, Metapenaeus,
Fenneropenaeus and Melicertus (14, 18, 23, 26, 30, 44). Experimental water-borne and per os challenge of the Japanese tiger shrimp,
Marsupenaeus japonicus, 1-day-old postlarvae (PL) with MBV failed to produce detectable infections (17). Likewise, despite the
simultaneous culture of MBV-infected P. monodon in a number of Western Hemisphere farms, and the consequent direct exposure of
certain Western Hemisphere penaeids (specifically Penaeus (Litopenaeus) vannamei, P. (L.) stylirostris and Farfantepenaeus
californiensis) to MBV, the virus did not produce infections in these species, nor has it become established in the shrimp farms or in
wild stocks of exposed regions (26, 32).

43

2.2.2.

44

All life stages, except eggs and nauplii, are susceptible to infection by MBV.

45

2.2.3.

46
47
48
49

MBV infection is typically most severe (and most readily diagnosed with simple wet-mount methods) in the larval and early PL stages
and in adult females in spawning conditions. Diagnosis is accomplished by demonstration of prominent clusters of spherical occlusion
bodies in simple wet-mounts (stained or unstained) of whole larvae, in the hepatopancreas excised from PLs, or in faecal strands
shed from spawning adult female shrimp and collected from spawning tanks.

50

2.2.4.

51

MBV is strictly enteric infecting mucosal epithelial cells of the hepatopancreas tubules and the anterior midgut (1, 6, 13, 21, 26, 32).

52

2.2.5.

53
54
55

Persistent infection occurs commonly in penaeid hosts of MBV. Wild adult P. monodon females that are heavily infected with MBV have
been shown to excrete MBV-contaminated faeces when spawning, thereby contaminating the eggs and passing the virus to the next
generation (21, 26).

56

2.2.6.

57

None known in natural infections.

58

2.2.7.

59
60

MBV occurs with wild hosts of the penaeid shrimp/prawns listed in 2.2.1. MBV infections have not been reported from non-penaeid
host species.

Susceptible host species

Susceptible stages of the host

Species or sub-population predilection (probability of detection)

Target organs and infected tissue

Persistent infection with lifelong carriers

Vectors

Known or suspected wild aquatic animal carriers

2.3. Disease pattern

61
62

2.3.1.

63
64
65
66

Transmission of MBV is horizontal by ingestion of infected tissue (cannibalism), faeces, occlusion bodies, or virus-contaminated
detritus or water (21, 26). MBV has been experimentally transmitted in the laboratory by exposure of larval or early PL P. monodon by
water-borne or per os challenge. MBV occlusion bodies were apparent by 2 days post-challenge in hepatopancreatic cells when PL-1
were challenged at 28C in 33 parts per thousand (ppt) sea water (3941).

67

2.3.2.

68
69

Prevalence is highly variable: from <1% in wild and cultured populations up to 100% in cultured populations in larval-rearing tanks and
nursery ponds (1, 7, 9, 11, 12, 26, 39, 52).

70

2.3.3.

Transmission mechanisms

Prevalence

Geographical distribution

There are two taxonomic systems for the penaeids currently in use (19, 42). In the most recent (42), which is not accepted by OIE, the penaeid subgenera were elevated to full
genera. Hence, the Latin names listed as genera/subgenera reflect both options for the taxonomy of the penaeids (19, 42).

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

71
72
73
74
75

MBV is enzootic in wild penaeids in the following regions bordering on the Indo-Pacific: East and South-East Asia, Indian subcontinent,
Middle East, Australia, Indonesia, New Caledonia, East Africa, and Madagascar. Outside the normal geographical range of P. monodon,
MBV has not been reported in wild penaeid shrimp. However, MBV has been reported from sites where introduced P. monodon has
been cultured in the Mediterranean, West Africa, Tahiti and Hawaii, as well as several sites in North and South America and the
Caribbean (5, 26).

76

2.3.4.

77
78
79
80
81
82

The larval stages (specifically protozoea and mysis) and early PL stages are the life stages where significant mortalities may occur
and they are the most easily infected in laboratory challenge studies (11, 28, 40, 41). In enzootic regions culturing P. monodon, MBV
prevalence and infection severity may be high in juveniles and adults (from 50% to nearly 100%), but without associated mortality or
morbidity. MBV infections are apparently well tolerated by P. monodon unless they are severely stressed (7, 9, 11, 12, 26, 27, 28, 39).
Nonetheless, heavy MBV infections in farmed P. monodon may suppress the growth rate, resulting in reduced survival and reduced
overall culture performance (1, 2, 7, 16, 26, 28, 38, 40).

83
84
85
86

Economic and/or production impact of the disease: MBV has caused serious disease, sometimes with high mortality rates in
hatcheries and significant production losses in farms in the Indo-Pacific. While not usually causing high mortality rates in infected
juvenile or adult stages, MBV has been documented to cause reduced growth and lower harvest production in populations that are
persistently infected with the virus (2, 26).

87

2.3.5.

88

None documented.

89

2.4. Control and prevention

Mortality and morbidity

Environmental factors

90

2.4.1.

Vaccination

91

No effective vaccination methods for MBV have been developed.

92

2.4.2.

93

No scientifically confirmed reports.

94

2.4.3.

95

No scientifically confirmed reports.

96

2.4.4.

97

No MBV-resistant stocks of the susceptible species have been demonstrated.

98

2.4.5.

99

Not applicable to MBV.

Chemotherapy

Immunostimulation

Resistance breeding

Restocking with resistant species

100

2.4.6.

Blocking agents

101

Not reported.

102

2.4.7.

103

See section 2.4.8.

104

2.4.8.

105
106
107
108
109
110
111

Hatchery: a number of husbandry practices have been applied to the prevention of MBV infections and disease. Prescreening of

Disinfection of eggs and larvae

General husbandry practices

broodstock for MBV has been somewhat effective in detecting heavily infected carriers of the virus and thereby reducing the
transmission of the disease from parent to offspring. With non-lethal testing methods, this is accomplished by simple light
microscopic examination of faecal strands (or by PCR testing of faecal strands if PCR testing facilities are readily available).
Alternatively, spent broodstock may be killed after spawning and simple light microscopic examination of a hepatopancreas squash
can be run (or the excised hepatopancreas may be tested by PCR) to determine the spawners MBV infection status. As MBV is
transmitted from adults to their offspring by faecal contamination of the spawned eggs, prevention of infection in hatcheries may be

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

112
113

achieved by taking additional steps to eliminate faecal contamination of spawned eggs and larvae by thoroughly washing nauplii or
eggs with formalin, iodophores, and clean sea water (10).

114
115
116
117
118

Nursery and grow-out ponds: MBV infections remain common in earthen-bottom ponds in regions of the Indo-Pacific where the virus

119

is enzootic (5, 7, 26). However, the incidence and prevalence of BP infections may be reduced, or totally eliminated, with lined nursery
and grow-out ponds. This is an advantage of lined ponds because MBV occlusion bodies (which resist environmental degradation and
contain infectious MBV virions) are removed with pond bottom sludge after harvest of an infected crop, allowing the previously
contaminated pond to be cleaned, disinfected and dried before being filled and restocked with MBV-free postlarvae (if available).

3.

Sampling

120

3.1. Selection of individual specimens

121

Suitable specimens for testing for infection by MBV include all life stages except eggs.

122

3.2. Preservation of samples for submission

123
124

For unstained wet-mounts of tissue squashes (excised hepatopancreas or faeces) or whole shrimp (larvae), live or freshly iced unfixed
specimens are suitable.

125

For routine histology or molecular assays see Chapter 2.3 for guidance on preservation of samples for the intended test method.

126

3.3. Pooling of samples

127
128
129

Samples taken for molecular tests may be combined as pooled samples of no more than five specimens per pooled sample of faecal strands,
juveniles, subadults and adults. However, for eggs, larvae and PL pooling of larger numbers (e.g. ~150 or more eggs or larvae or 50150 PL,
depending on their size/age) may be necessary to obtain sufficient sample material (extracted nucleic acid) to run a diagnostic assay.

130

3.4. Best organs or tissues

131
132
133

MBV infects the mucosal epithelial cells of the hepatopancreas tubules and midgut. Hence, the best organs or tissues to
sample/test/examine contain these organs/tissues. Since MBV polyhedral occlusion bodies and free virus are shed into the faeces, fresh
faecal strands may be collected and used when non-lethal testing of valuable broodstock is necessary.

134

3.5. Samples/tissues that are not suitable

135
136
137

MBV infects only the hepatopancreas and midgut mucosal epithelial cells, therefore, pleopods, haemolymph or other tissues that do not
include hepatopancreas or midgut tissue are inappropriate samples for detection of infection by MBV virus. Fresh faecal strands when
collected from juveniles, subadults, or adults may be used in some assays as a non-lethal sampling method for MBV testing.

138

4.

Diagnostic methods
4.1. Field diagnostic methods

139
140

4.1.1.

Clinical signs

141
142
143

Protozoea, mysis and early PL stages with severe MBV infections may present a whitish midgut (due to the presence of occlusion
bodies and cell debris in the faecal material) (26). Juveniles and adults present no gross signs of diagnostic value, nor do larvae or
PLs with less severe infections.

144

4.1.2.

145

None reported.

Behavioural changes

4.2. Clinical methods

146
147

4.2.1.

148

See Section 4.1

149

4.2.2.
4

Gross pathology

Direct microscopic examination

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

150
151
152
153
154
155

Infection of the hepatopancreas by P. monodon-type baculovirus (MBV) is one of the most easy to diagnose diseases of the penaeid
shrimps and prawns. The occlusion bodies formed by the virus are very conspicuous and easily demonstrated by direct light
microscopy with fresh specimens or by routine histological methods with fixed specimens. Direct microscopic methods are most
suitable for the PL stages, which are commonly moved in regional and international trade. Highly sensitive molecular methods for
MBV are also available and provide the most sensitive methods for surveillance applications, especially for non-lethal testing of
broodstock.

156
157
158
159

Wet-mounts of fresh tissue: diagnosis of MBV infections is made by the demonstration of single or multiple generally spherical
occlusion bodies in wet mounts of squash preparations of hepatopancreas or midgut examined by phase-contrast or bright-field
microscopy. In carefully prepared unstained preparations, MBV occlusion bodies are visible as single or multiple, slightly refractive,
greenish intranuclear inclusions that range in diameter from less than 0.1 m to nearly 20 m.

160
161
162

Staining the tissue squash with 0.05% aqueous malachite green aids in demonstration of the occlusion bodies by staining them more
intensely than other similar sized spherical objects, such as normal host cell nuclei, nucleoli, secretory granules, phagolysosomes,
and lipid droplets (5, 25, 26, 32).

163
164
165
166
167
168
169
170
171

Wet mounts of faecal strands: this method may be used as a non-lethal method to screen for carriers of MBV. The method can be

172
173

Collected faeces may also be used as the sample for non-lethal testing for MBV by PCR. PCR will provide greater diagnostic sensitivity
for low-grade infections than direct microscopic examination (5, 31).

174

4.2.3.

175
176
177
178
179
180
181
182
183
184
185

Histopathology may be used to provide a definitive diagnosis of MBV infection. Since 10% buffered formalin and other fixatives provide,
at best, only fair fixation of the shrimp hepatopancreas (the principal target organ for MBV), the use of Davidsons fixative (containing
33% ethyl alcohol [95%], 20% formalin [approximately 37% formaldehyde], 11.5% glacial acetic acid and 33.5% distilled or tap
water) is highly recommended for all routine histological studies of shrimp (4, 26). To obtain the best results, dead shrimp should not
be used. Only live, moribund, or compromised shrimp should be selected for fixation and histological examination. Selected shrimp
are killed by injection of fixative directly into the hepatopancreas. The cuticle over the cephalothorax and abdomen just lateral to the
dorsal midline is opened with fine-pointed surgical scissors to enhance fixative penetration (the abdomen may be removed and
discarded), the whole shrimp (or cephalothorax less the abdomen) is immersed in fixative for from 24 to no more than 48 hours, and
then transferred to 70% ethyl alcohol for storage. After transfer to 70% ethyl alcohol, fixed specimens may be transported (via post
or courier to the diagnostic laboratory) by wrapping in cloth or a paper towel saturated with 70% ethyl alcohol and packed in leakproof plastic bags.

186
187
188
189
190
191
192
193
194
195

To begin histological processing, fixed shrimp are cut-in (see Bell & Lightner [4] for a photographic guide to this procedure) to
facilitate eventual sectioning of the hepatopancreas and midgut. After dehydration, the specimens are embedded in paraffin and
sections of 46 m thickness are cut. Routine histological stains such as Mayer Bennetts or Harris haematoxylin and eosin (H&E)
may be used for the demonstration of MBV diagnostic spherical occlusion bodies in hepatopancreatocytes, gut epithelial cells, or gut
lumen. Typically, MBV-infected hepatopancreatic (or occasionally midgut) cells will present markedly hypertrophied nuclei with single
or, more often, multiple eosinophilic occlusion bodies along with chromatin diminution and margination. Occlusion bodies may be
stained bright red with H&E stains, and intensely, but variably, with Grams tissue stains. For example, Brown and Brenns histological
Gram stain, although not specific for baculovirus occlusion bodies, tends to stain occlusions more intensely (either red or purple,
depending on section thickness, time of decolourising, etc.) than the surrounding tissue, which may aid in demonstrating their
presence in low-grade infections (5, 6, 23, 25, 26, 53).

196

4.2.4.

197
198
199
200

The autofluorescence method with phloxine stain is another method for detecting MBV occlusion bodies based on the fluorescence of
phloxine-stained occlusion bodies. Aqueous 0.001% phloxine may be added to tissue squash preparations to make wet mounts of
hepatopancreas or faeces for direct examination. Histological sections stained with routine H&E containing 0.005% phloxine are also
suitable for this procedure. MBV occlusions in wet mounts of tissue squashes, in faeces, or in histological sections fluoresce bright

applied to juvenile or older shrimp, and it is perhaps most useful as a non-lethal method for screening valuable broodstock. Faecal
samples from shrimp to be tested may be obtained by placing the shrimp in an aquarium, spawning tank, or other suitable tank for a
few hours until faecal strands are present on the tank bottom. The faecal strands are best collected using a clear plastic siphon hose
(an air line fitted with a section of plastic pipette as a tip is ideal) and placed in a beaker, cup, or other suitable container. The faecal
strands may be made into wet mounts and examined directly for occlusion bodies. MBV occlusion bodies are roughly spherical,
refractive bodies that may occur singly or in clusters. In very fresh faecal strands, they may occur in clusters held together by the
nuclear membrane. The addition of a drop of 0.05% aqueous malachite green to the wet-mount preparation aids in demonstrating the
MBV occlusion bodies by staining them more intensely green than other round objects in the faeces (5, 26).

Histology

Autofluorescence

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

201
202
203

yellow-green against a pale green background under epi-fluorescence (barrier filter of 0515 nm and a 490 nm exciter filter). Other
objects in the tissues and insect baculovirus occlusion bodies do not fluoresce with this method. Hence, the method can provide a
rapid and specific diagnosis (5, 26, 47).

204

4.2.5

205

See Section 4.3.

206

4.2.6

207
208

Polyclonal antibodies produced in rabbits for detection of tetrahedral baculovirosis (BP) polyhedrin (24) cross react with MBV using
indirect fluorescent antibody test (IFAT) methods (26), but none is available for routine diagnosis of MBV infections.

209

4.2.7

210
211

MBV infection can be confirmed by demonstration of the virus (or pathognomonic occlusion bodies with occluded virions) in sections,
or demonstration of the virus in semi-purified virus preparations prepared from the hepatopancreas (13, 16, 21, 32, 33, 36).

In-situ hybridisation

Antibody-based methods

Electron microscopy/cytopathology

4.3. Agent detection and identification methods

212

4.3.1.

213

Direct detection methods

214

4.3.1.1.

Microscopic methods

215

4.3.1.1.1. Wet mounts

216

See Section 4.2.1 above

217

4.3.1.1.2. Histological sections

218

See Section 4.2.1 above

219

4.3.1.2.

Agent isolation and identification

220

4.3.1.2.1. Cell culture/artificial media

Not applicable.

221

4.3.1.2.2. Antibody-based antigen detection methods

222

See Section 4.2.6.

223

4.3.1.2.3. Molecular techniques

224
225

4.3.1.2.3.1. Molecular methods using DNA probes to MBV

226
227
228
229
230
231

Non-radioactive DIG-labelled gene probes to MBV have been developed (29, 35, 36, 43). DIG-labelled DNA probes for MBV
are commercially available as ShrimProbe kits from DiagXotics (Lawrenceville, New Jersey, USA). The probes are
labelled with a non-radioactive label, digoxigenin-11-dUTP (DIG). These probes only work well with the in-situ hybridisation
method with histological sections because there are substances present in the hepatopancreas and faeces of shrimp that
provide both false-positive and false-negative results with samples that are blotted directly and not extracted prior to
probing.

232

4.3.1.2.3.2. Dot-blot hybridisation procedure for MBV

233
234
235
236
237
238
239
240

While specific DNA probes for MBV are available, their application to dot-blot hybridisation procedures is not
recommended for most routine diagnostic applications. Pigments present in the hepatopancreas leave a coloured spot on
the hybridisation membrane that can result in the masking of a positive test or in the false interpretation of a negative
test. Likewise, pieces of chitin (which non-specifically bind DNA probes), pigments, and other materials present in the
faecal sample may also result in false-positive or false-negative dot-blot hybridisation tests. Extraction of DNA from the
hepatopancreas or faeces prior to blotting or the use of chemiluminescent or radioactively labelled probes may
circumvent these problems and is recommended. Nonetheless, the adequacy of other test methods (i.e. direct wet mounts,
histology, or PCR) has not indicated a need for the further refinement and application of the dot-blot method (26, 32).

241

4.3.1.2.3.3. In-situ hybridisation procedure

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

242
243

The in-situ hybridisation protocol given in detail for tetrahedral baculovirosis (BP) in Section 4.3.1.2.3.1 of Chapter 2.3.6
uses the same method except that a DIG-labelled probe for MBV is used.

244

4.3.1.2.3.4. Polymerase chain reaction for MBV

245
246
247

Several PCR methods have been developed for MBV and may be suitable for certain applications (8, 34, 48, 50, 51).
However, more sensitive methods have been developed recently and have been demonstrated to detect MBV from several
geographical regions (3, 46).

248
249
250
251

Substances in the hepatopancreas and faeces of shrimp have been found to inhibit the DNA polymerase used in the PCR
assay. Therefore, DNA extraction is required before PCR can be successfully applied to the detection of this virus (3, 8,
20). DNA extraction kits are convenient and commercially available. Otherwise, refer to Section 4.3.1.2.3.2 of Chapter 2.3.6
Tetrahedral baculovirosis (BP) for a suitable DNA extraction procedure.

252
253
254

The following controls should be included in every PCR assay for MBV: a known negative tissue or negative faecal sample; a
known positive tissue or faecal sample (this can be the DNA clone from which a specific set of primers was designed); and
a no-template control.

255
256
257
258

Nested PCR method for MBV (3): this nested PCR method is capable of detecting low concentrations of MBV (down to eight

viral genome equivalents). Two external and two internal primers were designed using a DNA sequence derived from the
plasmid p4Ec196, which was constructed from a 7.4 kb EcoRI fragment of an Australian isolate of MBV. The primer
sequences are:

259
260

Primer
Sequence
Temperature
MBV1.4F
5-CGA- TTC-CAT-ATC-GGC-CGA-ATA-3
62C (68.9C)
MBV1.4r
5-TTG-GCA-TGC-ACT-CCC-TGA-GAT-3
64C (70.8C)
MBV1.4NF
5-TCC-AAT-CGC-GTC-TGC-GAT-ACT-3
64C (70.8C)
MBV1.4NR
5-CGC-TAA-TGG-GGC-ACA-AGT-CTC-3
66C (72.8C)
The melting temperatures of the primers are according to the formula 2(A+T) + 4(G+C), or according to the percentage GC
method (values in parentheses).

261

DNA extraction

262
263
264

i)

PCR inhibitors were noted by Belcher & Young (3) to be present in DNA samples prepared from whole MBV-infected
PL P. monodon when using the extraction method recommended by Wang et al. (54) for BP, which incorporates
proteinase K. However, when hot phenol was used to extract the DNA, this inhibitory effect was removed.

265
266

ii)

With the hot phenol method, the sample to be tested (PLs, shrimp hepatopancreas, faeces) is freeze-dried and
ground to a powder in liquid nitrogen with a motor and pestle.

267
268
269

iii)

Approximately 300 mg of the resulting material is added immediately to 400 l of preheated (65C) lysis buffer
(100 mM Tris/HCl, 100 mM ethylene diamine tetra-acetic acid [EDTA], 1% sodium dodecyl sulphate, pH 8.0) and
incubated at 65C for 510 minutes.

270
271
272

iv)

The resulting suspension is coarsely homogenised by spot centrifugation and homogenisation with a microfuge tube
pestle. Tris/HCl-buffered phenol, pH 8.0 (600 l) is added and the mixture is incubated for 2 hours at 65C with
occasional inversion.

273
274
275
276

v)

Following centrifugation at 12,000 g for 10 minutes at room temperature, the aqueous layer is transferred to a fresh
microfuge tube and extracted twice with an equal volume of phenol/chloroform (1/1). Then, a total of 50 l of the
aqueous layer is transferred to a fresh microfuge tube containing 150 l dilution buffer and extracted once more
with an equal volume of phenol/chloroform (1/1) followed by a straight chloroform extraction.

277
278

vi)

Ammonium acetate is added to the aqueous layer to a final concentration of 2.5 M, mixed briefly, and two volumes
of 20C ethanol are added with 1 l of 20 mg/litre glycogen to precipitate the DNA.

279

vii)

DNA is precipitated by incubation at 20C overnight or by incubation at 70C for 1 hour.

280
281
282

viii) DNA is pelleted at 12,000 g for 15 minutes at 4C. The resulting DNA pellet is rinsed twice, first with 500 l 80% cold
ethanol and centrifuged at 12,000 g for 10 minutes at 4C, followed by an identical rinse and centrifugation at room
temperature.

283
284
285

ix)

286

Nested PCR steps of Belcher & Young (3):

The final DNA pellet is dried in vacuo, resuspended in 100 l dilution buffer (10 mM Tris/HCl, pH 8.0, 0.1 mM EDTA, pH
8.0) at room temperature overnight or at 37C for 2 hours. Following spectrophotometric analysis, and prior to PCR,
the DNA is diluted to 50 ng/l in dilution buffer.

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

287
288

i)

Prior to PCR, the extracted total DNA is denatured in boiling water for 3 minutes followed by quick chilling in icewater.

289

ii)

A total of 100 ng of extracted DNA is used as a template.

290
291

iii)

Each reaction tube contains 50 mM KCl, 10 mM Tris/HCl, pH 9, 0.1% Triton X-100, 0.2 mM of each dNTP, 1.5 mM MgCl2,
0.25 M of each MBV1.4F and MBV1.4R, 2.5 U of Taq, and made up to a final volume of 50 l.

292

iv)

The reaction mixes are overlaid with mineral oil (as necessary).

293
294

v)

The conditions for the first round of amplification are: one cycle of 96C for 5 minutes; 40 cycles of 94C for
30 seconds, 65C for 30 seconds, 72C for 60 seconds; and one cycle of 72C for 7 minutes.

295
296

vi)

The second step of the nested PCR is accomplished with 0.5 l of the primary PCR reaction used as template with
the internal primers.

297
298
299

vii)

The second round of amplification reaction contains 50 mM KCl, 10 mM Tris/HCl, pH 9.0, 0.1% Triton X-100, 1.5 mM
MgCl2, 0.2 mM of each dNTP, 0.25 M of each of the primers MBV1.4NF and MBV1.4NR, and 2.5 U of Taq, and made up to
a final volume of 50 l.

300

viii) The reaction mixes are overlaid with mineral oil (as necessary).

301
302

ix)

The conditions for the second round of amplification are: one cycle of 96C for 5 minutes; 35 cycles of 94C for
30 seconds, 60C for 30 seconds, 72C for 60 seconds; and one cycle of 72C for 7 minutes.

303
304
305
306

x)

Demonstration of the PCR products (533 bp first step and 361 bp second step) is accomplished by adding 1 l of gelloading buffer (0.25% [w/v] bromophenol blue, 15% [w/v] Ficoll-type 400, 100 mM EDTA, pH 8.0) to 10 l of each
reaction mixture and electrophoresis through a 0.8% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH
8.0) containing 0.5 g/litre ethidium bromide.

307
308
309

An alternative single-step PCR method is used by the OIE Reference Laboratory at the University of Arizona because it is
less prone to contamination (46). This method uses the sample type and extraction methods as described above in a
previous section.

310
311

Primers: one forward and reverse primer pair (261F/261R) selected from clone GC7 and deposited in GenBank with

312

The sequences for these primers are:

313

261F

5-AAT-CCT-AGG-CGA-TCT-TAC-CA-3

314

261R

5-CGT-TCG-TTG-ATG-AAC-ATC-TC-3

315

DNA templates:

316

i)

Extracted from hepatopancreas (frozen or ethanol fixed);

317

ii)

Extracted from whole PLs (frozen or ethanol fixed);

318

iii)

Extracted from faeces (frozen or ethanol fixed).

319

PCR reaction mixture:

accession number AY819785 produces a 261 bp amplicon (46).

Reagent (concentration)
Distilled H2O

25 l PCR beads*
23.5 l

Primer 261F (0.3 M)

Primer 261R (0.3 M)
DNA template (50450 ng of DNA)

0.5 l
0.5 l
0.5 l

*PuReTaq Ready-To-Go PCR beads, Amersham Biosciences, Buckinghamshire, UK.

320

PCR cycling parameters:

321

Primers
261F/261R

Mix/Beads
Beads*

Time
5 minutes
30 seconds, 30 seconds,
30 seconds
7 minutes

Temp. C
95
94
60
72
72

No. cycles
1
35
1

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

4.3.1.2.4. Agent purification

322

While methods for purification of MBV polyhedra and virions are published (4, 41), none is in routine use as a diagnostic method.

323
324

4.3.2.

Serological methods

325
326

Not applicable because shrimp are invertebrate animals which do not produce specific antibodies that could be used to demonstrate
infection by or prior exposure to MBV.

327

5.

Rating of tests against purpose of use

328
329
330
331
332
333

The methods currently available for targeted surveillance and diagnosis of MBV are listed in Table 5.1. The designations used in the Table indicate: a
= the method is the recommended method for reasons of availability, utility, and diagnostic specificity and sensitivity; b = the method is a standard
method with good diagnostic sensitivity and specificity; c = the method has application in some situations, but cost, accuracy, or other factors
severely limits its application; and d = the method is presently not recommended for this purpose. These are somewhat subjective as suitability
involves issues of reliability, sensitivity, specificity and utility. Although not all of the tests listed as category a or b have undergone formal
standardisation and validation, their routine nature and the fact that they have been used widely without dubious results, makes them acceptable.

334

Table 5.1. Methods for targeted surveillance and diagnosis

Method

Targeted surveillance

Presumptive
diagnosis

Confirmatory
diagnosis

Larvae

PLs

Juveniles

Adults

Gross signs

Bioassay

Direct LM

Histopathology

Transmission EM

Antibody-based assays

DNA Probes  in situ

PCR

PLs = postlarvae; LM = light microscopy; EM = electron microscopy; PCR = polymerase chain reaction.

335

Tests recommended for targeted surveillance to declare freedom from spherical baculovirosis (infection with Penaeus
monodon-type baculovirus)

336
337

6.

338
339

As indicated in Table 5.1, PCR (see appropriate section of this chapter) is the recommended method for targeted surveillance for reasons of
availability, utility, and diagnostic specificity and sensitivity.

340
341
342

Where PCR testing is not available, direct microscopy (of wet-mounts) or histopathology, as illustrated in Table 5.1 and detailed in Section 4.2.2 and
4.2.3, may provide suitable methods for targeted surveillance, provided the appropriate samples from the appropriate life stages of the host
shrimp are used.

343

7.

Corroborative diagnostic criteria

344

7.1. Definition of suspect case

345
346
347

For larvae (especially protozoea, mysis and early PL stages) of the susceptible species, a suspect case is represented by mortality of larvae
showing white midguts. For juveniles, it is represented by poor growth or poor culture performance in populations with a prior history of
MBV infection or in regions where MBV is prevalent.

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

348

7.2. Definition of confirmed case

349
350

Any combination of a molecular (PCR or ISH) test and a morphological (microscopy or histology) test using at least two of the following four
methods (with positive results):

351
352

Microscopical demonstration of spherical occlusion bodies in wet mounts of whole larvae or excised hepatopancreata. For older PLs,
juveniles and adults, spherical occlusion bodies evident in wet-mount squashes of the hepatopancreas or faeces.

353

Histological demonstration of pathognomonic intranuclear, multiple, eosinophilic, occlusion bodies.

354
355

In-situ hybridisation positive histological signal to MBV-type lesions (i.e. hypertrophied nuclei with or without pathognomonic

356

PCR positive results for MBV.

spherical occlusion bodies.

357

8.

References

358
359
360

1.

ANDERSON I.G., SHARIFF M., NASH G. & NASH. M. (1987). Mortalities of juvenile shrimp Penaeus monodon, associated with Penaeus monodon
baculovirus, cytoplasmic reo-like virus and rickettsial and bacterial infections, from Malaysian brackish water ponds. Asian Fish. Sci., 1, 47
64.

361
362

2.

BATICADOS M.C.L., PITOGO, C.L., PANER M.G., DE LA PEZA L.D., TENDENCIA E.A. (1991). Occurrence and pathology of Penaeus monodon baculovirus
infection in hatcheries and ponds in the Philippines. Israeli J. Aquaculture Bamidgeh, 43, 3541.

363
364

3.

365
366

4.

BELL T.A. & LIGHTNER D.V. (1988). A Handbook of Normal Shrimp Histology. Special Publication No. 1. World Aquaculture Society, Baton Rouge,
Louisiana, USA, 114 pp.

367
368

5.

BONDAD-REANTASO M.G., MCGLADDERY S.E., EAST I. & SUBASINGHE R.P. (EDS) (2001). Asia Diagnostic Guide to Aquatic Animal Diseases. FAO Fisheries
Technical Paper 402, Supplement 2. FAO, Rome, Italy, 240 pp.

369
370

6.

BROCK J.A. & LIGHTNER D.V. (1990). Diseases of crustacea. Diseases caused by microorganisms. In: Diseases of Marine Animals, Vol. III, Kinne O.,
ed. Biologische Anstalt Helgoland, Hamburg, Germany, 245349.

371
372

7.

CHAYABURAKUL K., NASH G., PRATANPIPAT P., SRIURARAIRATANA S. & WITHYACHUMNARNKUL. (2004). Multiple pathogens found in growth-retarded black
tiger shrimp Penaeus monodon cultivated in Thailand. Dis. Aquat. Org., 60, 8996.

373
374

8.

375
376

9.

CHEN S.N., CHANG P.S. & KOU G.H. (1989). Observation on pathogenicity and epizootiology of Penaeus monodon baculovirus (MBV) in cultured
shrimps in Taiwan. Fish Pathol., 24, 189195.

377
378
379

10.

CHEN S.N., CHANG P.S. & KOU G.H. (1990). Infection route and eradication of Penaeus monodon baculovirus (MBV) in larval giant tiger prawns,
Penaeus monodon. In: Diseases of Cultured Penaeid Shrimp in Asia and the United States, Fulks W. & Main K.L., eds. Oceanic Institute,

380
381

11.

382
383

12.

384
385

13.

BELCHER C.R. & YOUNG P.R. (1998). Colourimetric PCR-based detection of monodon baculovirus in whole Penaeus monodon postlarvae. J. Virol.

Methods, 74, 2129.

CHANG P.S., LO C.F., KOU G.H. & CHEN S.N. (1993). Purification and amplification of DNA from Penaeus monodon-type baculovirus (MBV). J.

Invertebr. Pathol., 62, 116120.

Honolulu, Hawaii, USA, 177184.

10

CHEN S.N., CHANG P.S., KOU G.G. & LIGHTNER D.V. (1989). Studies on virogenesis and cytopathology of Penaeus monodon baculovirus (MBV) in giant
tiger prawn (Penaeus monodon) and the red tail prawn (Penaeus penicillatus). Fish Pathol., 24, 89100.
CHEN S.N., LO C.F., LIU S.M. & KOU G.H. (1989). The first identification of Penaeus monodon baculovirus (MBV) in cultured sand shrimp,

Metapenaeus ensis. Bull. Eur. Assoc. Fish Pathol., 9, 6264.

COUCH J.A. (1991). Baculoviridae. Nuclear Polyhedrosis Viruses. Part 2. Nuclear Polyhedrosis Viruses of Invertebrates Other Than Insects. In:
Atlas of Invertebrate Viruses, Adams J.R. & Bonami J.R., eds. CRC Press, Boca Raton, Florida, USA, 205226.

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

386
387

14

DOUBROVSKY A., PAYNTER J.L., SAMBHI S.K., ATHERTON J.G. & LESTER R.J.G. (1988). Observations on the ultrastructure of baculovirus in Australian

388
389

15.

FAUQUET C.M., MAYO M.A., MANILOFF J., DESSELBERGER U. & BALL L.A. (2005). Virus Taxonomy. Classification and Nomenclature of Viruses. Eighth
Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, 1259 pp.

390
391

16.

FEGAN D.F., FLEGEL T.W., SRIURAIRATANA S. & WAIYAKRUTTHA M. (1991). The occurrence, development and histopathology of monodon baculovirus in
Southern Thailand. Aquaculture, 96, 205217.

392

17.

FUKUDA H., MOMOYAMA K. & SANO T. (1988). First detection of monodon baculovirus in Japan. Nippon Suisan Gakkaishi, 54, 4548.

393
394
395

18.

HAO N.V., THUY D.T., LOAN L.T.T., PHI T.T., PHUOC L.H., DUONG H.H.T., CORSIN F. & CHANRATCHAKOOL P. (1999). Presence of the two viral pathogens WSSV
and MBV in three wild shrimp species (Penaeus indicus, Metapenaeus ensis and Metapenaeus lysianassa) cultured in the mangrove forest of
Ca Mau Province. Asian Fish. Sci., 12, 309325.

396

19.

HOLTHUIS L.B. (1980). FAO Species Catalog. Vol. 1 Shrimp and Prawns of the World. FAO Fisheries Synopsis No. 125, FAO, Rome, Italy, 271 p.

397
398

20.

HSU Y.L., WANG K.H., YANG Y.H., TUNG M.C., HU C.H., LO C.F., WANG C.H. & HSU T. (2000). Diagnosis of Penaeus monodon-type baculovirus by PCR and
by ELISA of occlusion bodies. Dis. Aquat. Org., 40, 9399.

399

21.

JOHNSON P.T. & LIGHTNER D.V. (1988). Rod-shaped nuclear viruses of crustaceans: gut-infecting species. Dis. Aquat. Org., 5, 123141.

400
401

22.

LE BLANC B.D. & OVERSTREET R.M. (1991). Effect of dessication, pH, heat and ultraviolet irridation on viability of Baculovirus penaei. J. Invertebr.
Pathol., 57, 277286.

402
403

23.

LESTER R.J.G., DOUBROVSKY A., PAYNTER J.L., SAMBHI S.K. & ATHERTON J.G. (1987). Light and electron microscope evidence of baculovirus infection in
the prawn Penaeus plebejus. Dis. Aquat. Org., 3, 217219.

404

24.

LEWIS D.H. (1986). An enzyme-linked immunosorbent assay (ELISA) for detecting penaeid baculovirus. J. Fish Dis., 9, 519522.

405
406

25.

LIGHTNER D.V. (1988). Diseases of cultured penaeid shrimp and prawns. In: Disease Diagnosis and Control in North American Marine
Aquaculture, Sindermann C.J. & Lightner D.V., eds. Elsevier, Amsterdam, The Netherlands, 8127.

407
408

26.

LIGHTNER, D.V. (ED.) (1996). A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World
Aquaculture Society, Baton Rouge, Louisiana, USA, 304 pp.

409
410
411

27.

LIGHTNER D.V., BELL T.A., REDMAN R.M., MOHNEY L.L., NATIVIDAD J.M., RUKYANI A. & POERNOMO A. (1992). A review of some major diseases of economic
significance in penaeid prawns/shrimps of the Americas and Indopacific. In: Diseases in Asian Aquaculture I, Shariff M., Subasinghe R.P., &
Arthur J.R., eds. Fish Health Section, Asian Fisheries Society, Manila, Philippines, 5780.

412
413

28.

LIGHTNER D.V., HEDRICK R.P., FRYER J.L., CHEN S.N., LIAO I.C. & KOU G.H. (1987). A survey of cultured penaeid shrimp in Taiwan for viral and other
important diseases. Fish Pathol., 22, 127140.

414
415
416

29.

LIGHTNER D.V., POULOS B.T., BRUCE L., NUNAN L., PANTOJA C., MARI J. & BONAMI J.R. (1994). Development and application of genomic probes for use as
diagnostic and research reagents for the penaeid shrimp parvoviruses IHHNV and HPV and the baculoviruses MBV and BP. USMSFP 10th
Anniversary Review, GCRL Special Publication No. 1, 5985.

417

30.

LIGHTNER D.V. & REDMAN R.M. (1981). A baculovirus-caused disease of the penaeid shrimp, Penaeus monodon. J. Invertebr. Pathol., 38, 299302.

418

31.

LIGHTNER D.V & REDMAN R.M. (1998). Shrimp diseases and current diagnostic methods. Aquaculture, 164, 201220.

419
420

32.

LIGHTNER D.V., REDMAN R.M. & BELL T.A. (1983). Observations on the geographic distribution, pathogenesis and morphology of the baculovirus
from Penaeus monodon Fabricius. Aquaculture, 32, 209233.

421
422

33.

LU C.C., TANG F.J.K. & CHEN S.N. (1996). Morphogenesis of the membranous labyrinth in penaeid shrimp cells infected with Penaeus monodon
baculovirus (MBV). J. Fish Dis., 19, 357364.

Penaeus monodon and Penaeus merguiensis. Aust. J. Mar. Freshwater Res., 39, 743749.

Manual of Diagnostic Tests for Aquatic Animals 2009

11

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

423
424

34.

LU C.C., TANG F.J.K., KOU G.H. & CHEN S.N. (1993). Development of a Penaeus monodon-type baculovirus (MBV) DNA probe by polymerase chain
reaction and sequence analysis. J. Fish Dis., 16, 551559.

425
426

35.

LU C.C., TANG F.J.K., KOU G.H. & CHEN S.N. (1995). Detection of Penaeus monodon-type baculovirus (MBV) infection in Penaeus monodon Fabricius
by in situ hybridization. J. Fish Dis., 18, 337345.

427
428

36.

429
430

37.

431
432

38.

433
434
435

39.

NATIVIDAD J.M. & LIGHTNER D.V. (1992). Prevalence and geographic distribution of MBV and other diseases in cultured giant tiger prawns
(Penaeus monodon) in the Philippines. In: Diseases of Cultured Penaeid Shrimp in Asia and the United States, Fulks W. & Main K., eds. The
Oceanic Institute, Makapuu Point, Honolulu, Hawaii, USA, 139160.

436
437
438

40.

NATIVIDAD J.M. & LIGHTNER D.V. (1992). Susceptibility of the different larval and postlarval stages of black tiger prawn, Penaeus monodon
Fabricius, to monodon baculovirus (MBV). In: Diseases in Asian Aquaculture I, Shariff M., Subasinghe R. & Arthur J.R., eds. Fish Health Section,
Asian Fisheries Society, Manila, Philippines, 111125.

439
440

41.

PAYNTER J.L., VICKERS J.E. & LESTER R.J.G. (1992). Experimental transmission of Penaeus monodon-type baculovirus (MBV). In: Diseases in Asian
Aquaculture I, Shariff M., Subasinghe R. & Arthur J.R., eds. Fish Health Section, Asian Fisheries Society, Manila, Philippines, 97110.

441
442

42.

PEREZ FARFANTE I. & KENSLEY B.F. (1997). The penaeoid and sergestoid shrimps and prawns of the world: keys and diagnosis for the families and
genera. Memoires du Museum Histoire Naturelle. 175, 1233.

443
444

43.

POULOS B.T., MARI J., BONAMI J.R., REDMAN R. & LIGHTNER D.V. (1994). Use of non-radioactively labeled DNA probes for the detection of a baculovirus
from Penaeus monodon by in situ hybridization on fixed tissue. J. Virol. Methods, 49, 187194.

445
446

44.

SPANN K.M. & LESTER R.J.G. (1996). Baculovirus of Metapenaeus bennettae from the Moreton Bay region of Australia. Dis. Aquat. Org., 27, 53
58.

447
448

45.

SPANN K.M., LESTER R.J.G. & PAYNTER J.L. (1993). Efficiency of chlorine as a disinfectant against monodon baculovirus (MBV). Asian Fish. Sci., 6,
295301.

449
450

46.

SURACHETPONG W., POULOS B.T., TANG K.F.J., LIGHTNER D.V. (2005). Improvement of PCR method for the detection of monodon baculovirus (MBV) in
penaeid shrimp. Aquaculture, 249, 6975.

451
452

47.

THURMAN R.B., LIGHTNER D.V., BELL T.A. & HAZANOW S. (1990). Unique physicochemical properties of the occluded penaeid shrimp baculoviruses and
their use in diagnosis of infections. J. Aquat. Anim. Health, 2, 128131.

453
454

48.

UMESHA R.K., UMA A., OTTA S.K., KARUNASAGAR I., & KARUNASAGAR I. (2003). Detection by PCR of hepatopancreatic parvovirus (HPV) and other viruses
in hatchery-reared Penaeus monodon postlarvae. Dis. Aquat. Org,. 57, 141146.

455
456
457

49.

VAN REGENMORTEL M.H.V., FAUQUET C.M., BISHOP D.H.L., CARSTENS E.B., ESTES M.K., LEMON S.M., MANILOFF J., MAYO M.A., MCGEOCH D.J., PRINGLE C.R. & WICKNER
R.B. (2000). Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, USA, 1162
pp.

458
459
460

50.

VICKERS J.E., LESTER R.J.G., SPRADBROW P.B. & PEMBERTON J.M. (1992). Detection of Penaeus monodon-type baculovirus (MBV) in digestive glands of
postlarval prawns using polymerase chain reaction. In: Diseases in Asian Aquaculture I, Shariff M., Subasinghe R.P., & Arthur J.R., eds. Fish
Health Section, Asian Fisheries Society, Manila, Philippines, 127133.

461
462

51.

VICKERS J.E., LESTER R.J.G., SPRADBROW P.B. & PEMBERTON J.M. (1994). Evidence for homology between polyhedrin genes of baculoviruses of a
prawn and an insect. J. Invertebr. Pathol., 63, 207208.

12

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 (PmSNPV = MBV). Dis. Aquat. Org., 16, 207215.

MURPHY F.A., FAUQUET C.M., MAYO M.A., JARVIS A.W., GHABRIAL S.A., SUMMERS M.D., MARTELLI G.P. & BISHOP D.H.L. (1995). Virus Taxonomy. Sixth Report of
the International Committee on Taxonomy of Viruses. Arch. Virol. (Suppl. 10). Springer-Verlag, Vienna, Austria and New York, USA, 586 pp.
NASH G., POERNOMO A. & NASH M.B. (1988). Baculovirus infection in brackishwater pond cultured Penaeus monodon Fabricius in Indonesia.

Aquaculture, 73, 16.

Manual of Diagnostic Tests for Aquatic Animals 2009

Chapter 2.3.4. Spherical baculovirosis (Penaeus monodon-type baculovirus)

463
464

52.

465
466

53.

467
468

54.

VIJAYAN K.K., ALAVANDI S.V., RAJENDRAN K.V. & ALAGARSWAMI K. (1995). Prevalence and histopathology of monodon baculovirus (MBV) infection in
Penaeus monodon and P. indicus in shrimp farms in the south-east coast of India. Asian Fish. Sci., 8, 267272.
VOGT G. (1992). Transformation of anterior midgut and heaptopancreas by monodon baculovirus (MBV) in Penaeus monodon postlarvae.

Aquaculture, 107, 239248.

WANG S.Y., HONG C. & LOTZ J.M. (1996). Development of a PCR procedure for the detection of Baculovirus penaei in shrimp. Dis. Aquat. Org., 25,
123131.

469
470

*
* *

471
472

NB: There is an OIE Reference Laboratory for spherical baculovirosis (Penaeus monodon-type baculovirus) (see Table at the end of this Aquatic
Manual or consult the OIE Web site for the most up-to-date list: www.oie.int)

Manual of Diagnostic Tests for Aquatic Animals 2009

13