NB: This disease is no longer listed by the OIE.

CHAPTER 2.2.9.

SPHERICAL BACULOVIROSIS
(Penaeus monodon-type baculovirus)

1.

Scope

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) (Murphy et al. 1995), and it appears as the tentative
species P. monodon NPV, or PemoNPV, in the 7th and 8th Reports of the ICTV (Fauquet et al., 2005; Van
Regenmortel et al., 2000). 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.

2.

Disease information
2.1. Agent factors
2.1.1.

Aetiological agent, agent strains

Aetiological agent: P. monodon baculovirus (MBV) as described by Lightner & Redman (1981),
Lightner et al. (1983), Mari et al. (1993).
The International Committee on Virus Taxonomy lists MBV (Spherical Baculovirosis) as a tentative
species named PemoNPV in the genus Nucleopolyherdovirus (Fauquet et al., 2005).
Agent strains: 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 SouthEast 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infected P. monodon from Africa (Lightner, unpublished data) further suggesting that MBV (or the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.

2.1.2.

Survival outside the host

No data.

2.1.3.

Stability of the agent (effective inactivation methods)

No data.

2.1.4.

Life cycle

Not applicable.

2.2. Host factors

2.2.1.

Susceptible host species

MBV infections have been reported in one or more species of the following penaeid genera or
subgenera (the latter are between brackets): Penaeus (Penaeus), Penaeus (Metapenaeus),
Penaeus (Fenneropenaeus) and Penaeus (Melicertus) (Doubrovsky et al., 1988; Hao et al., 1999;
Lester et al., 1987; Lightner, 1996; Lightner & Redman 1981; Spann & Lester 1996). Experimental
water-borne and per os challenge of the Japanese tiger shrimp, Penaeus (Marsupenaeus) japonicus,
1-day-old postlarvae (PL) with MBV failed to produce detectable infections (Fukuda et al., 1988).
Likewise, despite the simultaneous culture of MBV-infected P. monodon in a number of farms in
various countries in the Americas (Ecuador, Brazil, Puerto Rico, and the States of Texas, South
Carolina and Hawaii of the United States of America), and the consequent direct exposure of certain
penaeids from this region (i.e. specifically Penaeus vannamei, P. stylirostris and P. 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 (Lightner, 1996; Lightner et al., 1983).

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2.2.2.

Susceptible stages of the host

Susceptible stages of the host: all life stages, except eggs and nauplii, are susceptible to infection by
MBV.

2.2.3.

Species or subpopulation predilection (probability of detection)

No data.

2.2.4.

Target organs and infected tissue

MBV is strictly enteric infecting mucosal epithelial cells of the hepatopancreas tubules and the anterior
midgut (Anderson et al., 1987; Brock & Lightner 1990; Couch, 1991; Johnson & Lightner, 1988;
Lightner, 1996; Lightner et al., 1983).

2.2.5.

Persistent infection with lifelong carriers

Persistent infection and lifelong carriers: 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 (Johnson & Lightner 1988; Lightner, 1996).

2.2.6.

Vectors

No vectors are known in natural infections.

2.2.7.

Known or suspected wild aquatic animal carriers

No data.

2.3. Disease pattern

2.3.1.

Transmission mechanisms

Transmission of MBV is horizontal by ingestion of infected tissue (cannibalism), faeces, occlusion

bodies, or virus-contaminated detritus or water (Johnson & Lightner, 1988; Lightner, 1996). 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 ppt sea water (Natividad & Lightner,
1992a; 1992b; Paynter et al., 1992).

2.3.2.

Prevalence

Prevalence of MBV is highly variable, from <1% in wild and cultured populations up to 100% in cultured
populations in larval-rearing tanks and nursery ponds (Anderson et al., 1987; Chayaburakul et al.,
2004; Chen et al., 1989b; 1989c; 1990; Lightner, 1996; Natividad & Lightner 1992a; Vijayan et al.,
1995).

2.3.3.

Geographical distribution

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, but only in the introduced P. monodon stocks (BondadReantaso et al., 2001; Lightner, 1996).

2.3.4.

Mortality and morbidity

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
(Chen et al 1989b; Natividad & Lightner 1992a; Paynter et al., 1992). In enzootic regions culturing
P. monodon, MBV prevalence and infection severity may be high (from 50% to nearly 100%) in
juveniles and adults, but without associated mortality or morbidity. MBV infections are apparently well
tolerated by P. monodon unless they are severely stressed (Chayaburakul et al., 2004; Chen et al.,
1989a; 1989b; 1989c, Lightner, 1996; Lightner et al., 1992; Natividad & Lightner 1992b). Nonetheless,
heavy MBV infections in farmed P. monodon may suppress growth rate, result in reduced survival and

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reduce overall culture performance (Anderson et al., 1987; Baticados et al., 1991; Chayaburakul et al.,
2004; Fegan et al., 1991; Lightner, 1996; Nash et al., 1988; Natividad & Lightner 1992b).

2.3.5.

Environmental factors

No data.

2.4. Control and prevention

2.4.1.

Vaccination

No effective vaccination methods for MBV have been developed.

2.4.2.

Chemotherapy

No scientifically confirmed reports of effective chemotherapy treatments.

2.4.3.

Immunostimulation

No scientifically confirmed reports of effective immunostimulation treatments.

2.4.4.

Resistance breeding

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

2.4.5.

Restocking with resistant species

Not applicable to MBV.

2.4.6.

Blocking agents

Blocking agents have not been reported.

2.4.7.

Disinfection of eggs and larvae

Because MBV is transmitted from adults to their offspring by faecal contamination of the spawned
eggs, prevention of infection in hatcheries may be 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 (Chen et al., 1990).

2.4.8.

General husbandry practices

Hatchery: a number of husbandry practices have been applied to the prevention of MBV infections and
disease. Prescreening of 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 nonlethal 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. Because MBV is transmitted from adults to their
offspring by faecal contamination of the spawned eggs, prevention of infection in hatcheries may be
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 (Chen et al., 1990).
Nursery and grow-out ponds: MBV infections remain common in earthen-bottom ponds in regions of
the Indo-Pacific where the virus is enzootic (Bondad-Reantaso et al., 2001; Chayaburakul et al., 2004;
Lightner, 1996), but incidence and prevalence of MBV infections may be reduced in lined nursery and
grow-out pond.

3.

Sampling
3.1. Selection of individual specimens
Suitable specimens for testing for infection by MBV using molecular methods (e.g. PCR, in-situ hybridisation,
etc.) include postlarvae (PL), juveniles and adults. While MBV may infect all life stages, infection severity,
and hence virus load, may be below detection limits in spawned eggs and in the larval stages, so these life
stages may not be suitable samples for MBV detection or certification for MBV disease freedom.

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3.2. Preservation of samples for submission

For routine histology or molecular assays, and guidance on preservation of samples for the intended test
method see Chapter 2.2.0.

3.3. Pooling of samples

Samples taken for molecular tests may be combined as pooled samples representing no more than five
specimens per pooled sample of juveniles, subadults and adults. However, for eggs, larvae and postlarvae
(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. See also Chapter 2.2.0.

3.4. Best organs or tissues

MBV is an enteric virus and can be detected in the hepatopanceas.
Faecal samples can be collected when non-lethal testing is required (e.g. for non-lethal testing of valuable
broodstock).

3.5. Samples/tissues that are not suitable

MBV is an enteric virus (e.g. the hepatopancreas, the midgut, or its caeca) and does not replicate
systemically.

4.

Diagnostic methods
4.1. Field diagnostic methods
4.1.1.

Clinical signs

See Section 4.2 for a description of gross clinical signs presented by shrimp infected with MBV.
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 nonlethal testing of broodstock.

Direct microscopic examination

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. 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 (Bondad-Reantaso et al., 2001; Lightner,
1988; 1996; Lightner et al., 1983).

Wet mounts of faecal strands: this method may be used as a nonlethal method to screen
for carriers of MBV. The method can be applied to juvenile or older shrimp, and it is perhaps
most useful as a nonlethal 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 tanks 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 airline 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

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staining them more intensely green than other round objects in faeces (Bondad-Reantaso et
al., 2001; Lightner, 1996).

4.1.2.

Collected faeces may also be used as the sample for nonlethal testing for MBV by PCR.
PCR will provide greater diagnostic sensitivity for low-grade infections than will direct
microscopic examination (Bondad-Reantaso et al., 2001; Lightner & Redman 1998).

Behavioural changes

Other than lethargy in severely affected PLs, no behavioural changes in infected hosts have been
reported.

4.2. Clinical methods

4.2.1.

Gross pathology

Protozoea, mysis and early PL stages with severe BP infections may present a whitish midgut (due to
the presence of occlusion bodies and cell debris in the faecal material) (Lightner, 1996). Juveniles and
adults present no gross signs of diagnostic value, nor do larvae with less severe infections.

4.2.2.

Clinical chemistry

Not applicable.

4.2.3.

Microscopic pathology

See Section 4.2.6.

4.2.4.

Wet mounts

See Section 4.1.1.

4.2.5.

Smears

Not applicable.

4.2.6.

Fixed sections

Histopathology: histology may be used to provide a definitive diagnosis of MBV infection. Because 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 (Bell & Lightner, 1988;
Lightner, 1996). 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 leak-proof plastic bags.
To begin histological processing, fixed shrimp are cut-in (see Bell & Lightner, 1998, 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 aide in demonstrating their presence in low-grade
infections (Bondad-Reantaso et al., 2001; Brock & Lightner 1990; Lester et al., 1987; Lightner 1988;
1996; Vogt, 1992).

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Autofluorescence method with phloxine stain: another method for detecting MBV occlusion bodies is
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 yellow-green against a pale green background under epifluorescence (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 (Bondad-Reantaso et al., 2001; Lightner, 1996; Thurman et al., 1990).
In-situ hybridisation: (see Section 4.3.1.2.3 below).
Antibody-based methods: polyclonal antibodies produced in rabbits for detection of Tetrahedral
Baculovirosis (BP) polyhedrin (Lewis, 1986) cross react with MBV using indirect fluorescent antibody
test (IFAT) methods (Lightner, 1996), but none is available for routine diagnosis of MBV infections.

4.2.6.

Electron microscopy/cytopathology

Electron microscopy: 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
preparation prepared from the hepatopancreas (Couch 1991; Fegan et al., 1991; Johnson & Lightner
1988; Lightner et al., 1983; Lu et al., 1996; Mari et al., 1993).

4.3. Agent detection and identification methods

4.3.1.

Direct detection methods

4.3.1.1. Microscopic methods

4.3.1.1.1. Wet mounts
See Section 4.1.1.
4.3.1.1.2. Smears
See Section 4.2.5.
4.3.1.1.3. Fixed sections
See Section 4.2.6.
4.3.1.2. Agent isolation and identification
4.3.1.2.1. Cell culture/artificial media
None reported to date.
4.3.1.2.2. Antibody-based antigen detection methods
See Section 4.2.6.
4.3.1.2.3. Molecular techniques
Molecular methods using DNA probes to MBV: non-radioactive DIG-labelled gene probes to MBV
have been developed (Lightner et al., 1994; Lu et al., 1995; Mari et al., 1993; Spann et al., 1993).
DIG-labelled DNA probes for MBV are commercially available as ShrimProbeTM 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.
Dot-blot hybridisation procedure for MBV: 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, bits of chitin (which nonspecifically 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

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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
(Lightner, 1996; Lightner et al., 1983).
In-situ hybridisation procedure: the in-situ hybridisation protocol given in detail for Tetrahedral
Baculovirosis (BP) in Section 4.3.1.2.3 of Chapter 2.2.10 uses the same method except that a DIGlabelled probe for MBV is used.
Polymerase chain reaction for MBV: Several PCR methods have been developed for MBV and
may be suitable for certain applications (Chang et al., 1993; Lu et al., 1993; Umesha et al., 2003;
Vickers et al., 1992; 1994). However, more sensitive methods have been recently developed and
demonstrated to detect MBV from several geographical regions (Belcher & Young 1998;
Surachetpong et al., 2005).
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 (Belcher & Young 1998; Chang et al., 1993; Hsu
et al., 2000). DNA extraction kits are convenient and commercially available.
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.
Nested PCR method for MBV (Belcher & Young 1998): 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:

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 per cent GC method (values in parentheses).
DNA extraction

224

i)

PCR inhibitors were noted by Belcher & Young (1998) to be present in DNA samples
prepared from whole MBV-infected PL Penaeus monodon when using the extraction method
recommended by Wang et al. (1996) for BP, which incorporates proteinase K. However, when
hot phenol was used to extract the DNA, this inhibitory effect was removed.

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.

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 sulfate, pH 8.0) and incubated at 65C for 510 minutes.

iv)

The resulting suspensison 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.

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

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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.
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.

vii)

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

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 12000 g for 10 minutes at 4C, followed
by an identical rinse and centrifugation at room temperature.
ix)

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.

Nested PCR steps of Belcher & Young (1998):

i)

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

ii)

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

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.

iv)

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

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.

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.

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.

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

x)

Demonstration of the PCR products (533 bp first step and 361 bp second step) is
accomplished by adding 1 l of gel-loading 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.

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 (Surachetpong et al., 2005). This method uses
the sample type and extraction methods as described earlier in this section.
Primers: one forward and reverse primer pair (261F/261R) selected from clone GC7 and deposited
in GenBank with accession number AY819785) produces a 261 bp amplicon (Surachetpong et al.,
2005).
The sequences for these primers are:
261F
261R

5-AAT-CCT-AGG-CGA-TCT-TAC-CA-3
5-CGT-TCG-TTG-ATG-AAC-ATC-TC-3

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DNA templates:
i)

Extracted from hepatopancreas (frozen or ethanol fixed);

ii)

Extracted from whole PLs (frozen or ethanol fixed);

iii)

Extracted from faeces (frozen or ethanol fixed).

PCR reaction mixture:

Reagent (concentration)

25 l PCR beads*

Distilled H2O

23.5 l

Primer 261F (0.3 M)

0.5 l

Primer 261R (0.3 M)

0.5 l

DNA template (50450 ng of DNA)

0.5 l

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

PCR cycling parameters:

Primers

Mix/Beads

Time

Temp. C

No. cycles

261F/261R

Beads*

5 minutes

95

30 seconds
30 seconds
30 seconds

94,
60,
72

35

7 minutes

72

4.3.1.2.4. Agent purification

None.

4.3.2.

Serological methods

None applicable.

5.

Rating of tests against purpose of use

The methods currently available for surveillance, detection, 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 and/or not available 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 standardization and validation,
their routine nature and the fact that they have been used widely without dubious results, makes them acceptable.

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Chapter 2.2.9. Spherical baculovirosis (Penaeus monodon-type baculovirus)

Table 5.1. Spherical baculovirosis (Penaeus monodon-type baculovirus) surveillance,

detection and diagnostic methods
Targeted surveillance
Method

Presumptive
diagnosis

Confirmatory
diagnosis

Larvae

PLs

Juveniles

Adults

Gross signs

Bioassay

Direct LM

Histopathology

Transmission EM

Antibody-based assays

DNA probes in situ

PCR

Sequence

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

6.

Test(s) recommended for targeted surveillance to declare freedom from infection with
Spherical baculovirosis (Penaeus monodon-type baculovirus)

Two years of history of negative test results for MBV using:

PCR performed on samples of the appropriate type and sample size;

Wet mount and/or histological results in which no spherical occlusion bodies are observed in samples of the
appropriate type and sample size.

7.

Corroborative diagnostic criteria

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

7.2. Definition of confirmed case

Any combination of at least two of the following three methods (with positive results):

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 and/or in histological sections of the hepatopancreas or faeces.

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

PCR positive results for MBV.

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Chapter 2.2.9. Spherical baculovirosis (Penaeus monodon-type baculovirus)

8.

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*
* *
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:
http://www.oie.int/en/our-scientific-expertise/reference-laboratories/list-of-laboratories/ ).
Please contact the OIE Reference Laboratories for any further information on Spherical baculovirosis
(Penaeus monodon-type baculovirus)

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