CHAPTER 2.2.9.
SPHERICAL BACULOVIROSIS
(PENAEUS MONODON-TYPE BACULOVIRUS)
1.
Scope1
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 and 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 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from Africa (Lightner, unpublished data) further suggesting that MBV (or the species PemoNPV) is made up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.
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
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NB: Version adopted by the World Assembly of Delegates of the OIE in May 2012. This disease is no longer listed by the OIE.
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Chapter 2.2.9. - Spherical baculovirosis (Penaeus monodon-type baculovirus)
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).
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 28°C in 33 ppt sea water (Natividad & Lightner, 1992a; Natividad & Lightner,
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; Chen et al., 1989c; Chen et al., 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 (Bondad-Reantaso et al., 2001; Lightner, 1996).
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Chapter 2.2.9. - Spherical baculovirosis (Penaeus monodon-type baculovirus)
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; Chen et al., 1989b; Chen et al., 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 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
2.4.8.1. 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 spawner's 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
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contamination of spawned eggs and larvae by thoroughly washing nauplii or eggs with formalin, iodophores,
and clean sea water (Chen et al., 1990).
2.4.8.2. 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.
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 50–150 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 hepatopancreas.
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.
4.1.1.1. Direct microscopic examination
4.1.1.1.1. 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
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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. 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; Lightner, 1996; Lightner et al., 1983).
4.1.1.1.2. 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 staining them more intensely green than other round objects in faeces (Bondad-Reantaso et
al., 2001; Lightner, 1996).
4.1.1.1.3. Collected faeces
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).
4.1.2. 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
4.2.6.1. 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 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
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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, 1988, 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 aide in
demonstrating their presence in low-grade infections (Bondad-Reantaso et al., 2001; Brock & Lightner,
1990; Lester et al., 1987; Lightner, 1988; Lightner, 1996; Vogt, 1992).
4.2.6.2. 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 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 (Bondad-Reantaso et al., 2001; Lightner, 1996; Thurman et al.,
1990).
4.2.6.3. in situ hybridisation
See Section 4.3.1.2.3 below.
4.2.6.4. 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.
4.2.7. Electron microscopy/cytopathology
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
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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
4.3.1.2.3.1. 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.
4.3.1.2.3.2. 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 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).
4.3.1.2.3.3. 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 DIG-labelled probe for MBV is used.
4.3.1.2.3.4. 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; Vickers et al., 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
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Chapter 2.2.9. - Spherical baculovirosis (Penaeus monodon-type baculovirus)
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’
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 per cent GC method (values in parentheses).
4.3.1.2.3.4.1. DNA extraction
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 µl of preheated
(65°C) lysis buffer (100 mM Tris/HCl, 100 mM ethylene diamine tetra-acetic acid [EDTA], 1%
sodium dodecyl sulfate, pH 8.0) and incubated at 65°C for 5–10 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 µl) is added and the mixture
is incubated for 2 hours at 65°C 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 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.
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–1 glycogen to precipitate the
DNA.
vii)
DNA is precipitated by incubation at –20°C overnight or by incubation at –70°C for 1 hour.
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.
ix)
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–1 in dilution buffer.
4.3.1.2.3.4.2. Nested PCR steps of Belcher & Young, 1998
8
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 µM of each MBV1.4F and MBV1.4R, 2.5 U of Taq, and made
up to a final volume of 50 µl.
iv)
The reaction mixes are overlaid with mineral oil (as necessary).
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.
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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.
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.
viii) The reaction mixes are overlaid with mineral oil (as necessary).
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.
x)
Demonstration of the PCR products (533 bp first step and 361 bp second step) is accomplished
by adding 1 µl of gel-loading 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 lltre–1
ethidium bromide.
4.3.1.2.3.4.3. Alternative single-step PCR method
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
5’-AAT-CCT-AGG-CGA-TCT-TAC-CA-3’
261R
5’-CGT-TCG-TTG-ATG-AAC-ATC-TC-3’
4.3.1.2.3.4.3.1. 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).
4.3.1.2.3.4.3.2. PCR reaction mixture
1.
Reagent (concentration)
25 µl PCR beads1
Distilled H2O
23.5 µl
Primer 261F (0.3 µM)
0.5 µl
Primer 261R (0.3 µM)
0.5 µl
DNA template (50‒450 ng of DNA)
0.5 µl
PuReTaq™ Ready-To-Go PCR beads™, Amersham Biosciences, Buckinghamshire, UK
4.3.1.2.3.4.3.3. PCR cycling parameters
Primers
261F/261R
Mix/Beads
Beads1
Time
Temp. °C
No. cycles
5 minutes
95
1
30 seconds
30 seconds
30 seconds
94
60
72
35
7 minutes
72
1
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Chapter 2.2.9. - Spherical baculovirosis (Penaeus monodon-type baculovirus)
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.
Table 5.1. Spherical baculovirosis (Penaeus monodon-type baculovirus)
surveillance, detection and diagnostic methods
Targeted surveillance
Larvae
PLs
Juveniles
Adults
Presumptive
diagnosis
Confirmatory
diagnosis
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
In situ DNA probes
c
c
c
c
a
a
PCR
a
a
a
a
a
a
Sequence
d
d
d
d
d
a
Method
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.
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Chapter 2.2.9. - Spherical baculovirosis (Penaeus monodon-type baculovirus)
7.2. Definition of confirmed case
Any combination of at least two of the following three methods (with positive results):
8.
•
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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*
* *
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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2016 © OIE - Manual of Diagnostic Tests for Aquatic Animals - 16/08/2016