CHAPTER 2.3.4.
1
2
3
SPHERICAL BACULOVIROSIS (PENAEUS MONODON-TYPE BACULOVIRUS)
4
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P. monodon from Africa (Lightner, unpublished data) further suggesting that MBV (or the species PemoNPV) is made up of more than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 body’s 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
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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 28°C in 33 parts per thousand (ppt) sea water (39–41).
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).
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2.3.3.
1
2
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).
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73
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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).
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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).
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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 spawner’s 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
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Chapter 2.3.4. – Spherical baculovirosis (Penaeus monodon-type baculovirus)
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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).
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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
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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 50–150 PL,
depending on their size/age) may be necessary to obtain sufficient sample material (extracted nucleic acid) to run a diagnostic assay.
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3.4. Best organs or tissues
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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.
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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.
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4.
Diagnostic methods
4.1. Field diagnostic methods
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140
4.1.1.
Clinical signs
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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
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151
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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.
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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 µm to nearly 20 µm.
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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).
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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).
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4.2.3.
175
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182
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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 Davidson’s 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.
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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 4–6 µm thickness are cut. Routine histological stains such as Mayer Bennett’s 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 Gram’s tissue stains. For example, Brown and Brenn’s 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).
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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
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Chapter 2.3.4. – Spherical baculovirosis (Penaeus monodon-type baculovirus)
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203
yellow-green against a pale green background under epi-fluorescence (barrier filter of 0–515 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).
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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.
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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
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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
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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.
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4.3.1.2.3.2. Dot-blot hybridisation procedure for MBV
233
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235
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237
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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).
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4.3.1.2.3.3. In-situ hybridisation procedure
6
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Chapter 2.3.4. – Spherical baculovirosis (Penaeus monodon-type baculovirus)
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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
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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’
62°C (68.9°C)
MBV1.4r
5’-TTG-GCA-TGC-ACT-CCC-TGA-GAT-3’
64°C (70.8°C)
MBV1.4NF
5’-TCC-AAT-CGC-GTC-TGC-GAT-ACT-3’
64°C (70.8°C)
MBV1.4NR
5’-CGC-TAA-TGG-GGC-ACA-AGT-CTC-3’
66°C (72.8°C)
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 µl of preheated (65°C) lysis buffer
(100 mM Tris/HCl, 100 mM ethylene diamine tetra-acetic acid [EDTA], 1% sodium dodecyl sulphate, pH 8.0) and
incubated at 65°C for 5–10 minutes.
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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 µl) is added and the mixture is incubated for 2 hours at 65°C with
occasional inversion.
273
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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 µl of the
aqueous layer is transferred to a fresh microfuge tube containing 150 µl 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 –20°C ethanol are added with 1 µl of 20 mg/litre glycogen to precipitate the DNA.
279
vii)
DNA is precipitated by incubation at –20°C overnight or by incubation at –70°C for 1 hour.
280
281
282
viii) DNA is pelleted at 12,000 g for 15 minutes at 4°C. The resulting DNA pellet is rinsed twice, first with 500 µl 80% cold
ethanol and centrifuged at 12,000 g for 10 minutes at 4°C, 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 µl dilution buffer (10 mM Tris/HCl, pH 8.0, 0.1 mM EDTA, pH
8.0) at room temperature overnight or at 37°C for 2 hours. Following spectrophotometric analysis, and prior to PCR,
the DNA is diluted to 50 ng/µl in dilution buffer.
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Chapter 2.3.4. – Spherical baculovirosis (Penaeus monodon-type baculovirus)
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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 µM of each MBV1.4F and MBV1.4R, 2.5 U of Taq, and made up to a final volume of 50 µl.
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 96°C for 5 minutes; 40 cycles of 94°C for
30 seconds, 65°C for 30 seconds, 72°C for 60 seconds; and one cycle of 72°C for 7 minutes.
295
296
vi)
The second step of the nested PCR is accomplished with 0.5 µl 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 µM 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 µl.
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 96°C for 5 minutes; 35 cycles of 94°C for
30 seconds, 60°C for 30 seconds, 72°C for 60 seconds; and one cycle of 72°C 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 µl of gelloading buffer (0.25% [w/v] bromophenol blue, 15% [w/v] Ficoll-type 400, 100 mM EDTA, pH 8.0) to 10 µl 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 µl PCR beads*
23.5 µl
Primer 261F (0.3 µM)
Primer 261R (0.3 µM)
DNA template (50–450 ng of DNA)
0.5 µl
0.5 µl
0.5 µl
*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
8
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
c
d
d
d
d
d
Bioassay
d
d
d
d
c
c
Direct LM
b
b
c
c
a
a
Histopathology
b
b
c
c
a
a
Transmission EM
d
d
d
d
d
a
Antibody-based assays
d
d
d
c
d
d
DNA Probes in situ
c
c
c
c
a
a
PCR
a
a
a
a
a
a
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
9
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
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64.
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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
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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,
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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
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6.
BROCK J.A. & LIGHTNER D.V. (1990). Diseases of crustacea. Diseases caused by microorganisms. In: Diseases of Marine Animals, Vol. III, Kinne O.,
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8.
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12.
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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
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DOUBROVSKY A., PAYNTER J.L., SAMBHI S.K., ATHERTON J.G. & LESTER R.J.G. (1988). Observations on the ultrastructure of baculovirus in Australian
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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.
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21.
JOHNSON P.T. & LIGHTNER D.V. (1988). Rod-shaped nuclear viruses of crustaceans: gut-infecting species. Dis. Aquat. Org., 5, 123–141.
400
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22.
LE BLANC B.D. & OVERSTREET R.M. (1991). Effect of dessication, pH, heat and ultraviolet irridation on viability of Baculovirus penaei. J. Invertebr.
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402
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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, 217–219.
404
24.
LEWIS D.H. (1986). An enzyme-linked immunosorbent assay (ELISA) for detecting penaeid baculovirus. J. Fish Dis., 9, 519–522.
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25.
LIGHTNER D.V. (1988). Diseases of cultured penaeid shrimp and prawns. In: Disease Diagnosis and Control in North American Marine
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27.
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28.
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29.
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417
30.
LIGHTNER D.V. & REDMAN R.M. (1981). A baculovirus-caused disease of the penaeid shrimp, Penaeus monodon. J. Invertebr. Pathol., 38, 299–302.
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32.
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38.
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45.
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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
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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
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51.
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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, 207–215.
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the International Committee on Taxonomy of Viruses. Arch. Virol. (Suppl. 10). Springer-Verlag, Vienna, Austria and New York, USA, 586 pp.
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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, 267–272.
VOGT G. (1992). Transformation of anterior midgut and heaptopancreas by monodon baculovirus (MBV) in Penaeus monodon postlarvae.
Aquaculture, 107, 239–248.
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,
123–131.
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