VetoFish - Manuel des tests de diagnostic pour les animaux aquatiques

Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 4.1.5.
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CHAPTER 4.1.5.

SPHERICAL BACULOVIROSIS
(Penaeus monodon-type baculovirus)

SUMMARY
Penaeus monodon-type baculovirus (MBV) is an occluded baculo-like virus that contains double-stranded DNA as its nucleic acid type (20, 22, 24). 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 Taxomony of Viruses (ICTV) (28), but it appears as Penaeus monodon NPV, or PemoNPV, in the Seventh Report of the ICTV (37). Although PemoNPV may be the most correct name for the virus, the term MBV will be used to designate this virus (and its closely strains) in this Aquatic Manual.

MBV is considered to be a potentially serious pathogen in the larval, postlarval, and early juvenile stages of its host penaeid shrimps and prawns. MBV has a wide geographical distribution and diverse host range, and multiple strains of the virus are likely to exist (1, 2, 5, 6, 8-21, 25, 29, 30, 34, 40). MBV infections are characterised by the presence of prominent, spherical intranuclear occlusion bodies in affected epithelial cells of the hepatopancreas and midgut, or free within lysed cell debris in the faeces (5, 20). Crowding or environmental stress may enhance the pathogenicity and increase the prevalence of MBV in its hosts (2, 13, 20, 24, 31). Transmission and infection by MBV is exclusively by the oral route in which cannibalism and faecal-oral contamination are the principal mechanisms of transmission (17, 20, 31, 32, 35).

MBV-type baculoviruses are widely distributed in cultured and wild penaeid shrimp and prawns in the Eastern Hemisphere and have been observed or reported from Australia, East Asia, South-East Asia, India, East Africa, the Middle East, from many of the Indo-Pacific countries (1, 2, 5, 6, 8-15, 18-24, 26, 27, 29, 30, 34, 40, 41). MBV-type baculoviruses have been found in cultured (but not in wild) shrimp and prawns only at sites in the Mediterranean and in West Africa (20). In most of these examples, the affected stocks were either introduced P. monodon or the facility had a recent history of P. monodon introduction. MBV has been found in introduced stocks of P. monodon in the Western Hemisphere. MBV has been observed in introduced P. monodon in the Pacific in Tahiti and Hawaii, and in the Americas in a number of shrimp-farming sites in North America, South America, and the Caribbean. Despite the simultaneous culture in a number of farms, and the consequent direct exposure of certain Western Hemisphere penaeids to MBV-infected P. monodon, MBV did not produce significant infections in native species, nor has it become established in shrimp farms or in wild stocks (20, 23).

Several surveillance methods are available for use in certification of the MBV-infection status of shrimp and prawn stocks. The simplest method is based on the microscopic demonstration of the characteristic occlusion bodies produced by the virus. With direct microscopy, characteristic occlusion bodies (clusters of refractive spheres for MBV) are demonstrated in wet-mounts of whole larvae, of excised portions of the hepatopancreas from postlarvae or older shrimp, or of faeces from large juvenile to adult shrimp. Wet-mount examination of the faeces of adult broodstock for characteristic occlusion bodies may be used as a nonlethal method to detect carriers. Histology of fixed specimens may also be used for surveillance. Routine histology provides a positive diagnosis of MBV infection when characteristic occlusion bodies are demonstrated in hypertrophied nuclei of mucosal epithelial cells of the hepatopancreas or midgut. Molecular methods for MBV are also available and provide a satisfactory method for surveillance applications. An antibody-based enzyme-linked immunosorbent assay for diagnosis of MBV has been reported (16), but the method used polyclonal antibodies and the antibodies are not commercially available. Gene probes applicable to in-situ hybridisation assays and polymerase chain reaction methods are also available for MBV (3, 7, 16, 20, 26, 28, 38, 39). A nested PCR method for MBV may provide the most sensitive method available for screening and surveillance applications (3).

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 steps to eliminate faecal contamination of spawned eggs and larvae by thoroughly washing nauplii or eggs with formalin, iodophores, and clean sea water (9).

DIAGNOSTIC PROCEDURES
Infection of the hepatopancreas by Penaeus monodon-type baculovirus (MBV) is among the most readily diagnosable 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 postlarvae stages, which are commonly moved in regional and international commerce (5, 6, 19, 20). Highly sensitive molecular methods for MBV are also available and provide alternative methods for surveillance applications, especially for nonlethal testing of broodstock (3, 7 20, 23). Hence, there are several options to choose from as surveillance methods for certification of the MBV-infection status of shrimp and prawn stocks.

The methods currently available for surveillance, detection, and diagnosis of MBV are listed in Table 1. The designations used in the Table indicate: - = the method is presently unavailable or unsuitable; ? = the method is available but untested; + = the method has application in some situations, but cost, accuracy, or other factors severely limits its application; ++ = the method is a standard method with good diagnostic sensitivity and specificity; and +++ = the method is the recommended method for reasons of availability, utility, and diagnostic specificity and sensitivity.

Sampling procedures: see Chapter I.3.

Table 1. Surveillance, detection and diagnostic methods for the nuclear polyhedrosis virus MBV

Method

Screening

Presumptive

Confirmatory

Larvae

PLs

Juveniles

Adults

Gross signs

-

-

-

-

+

-

Direct BF/LM

+++

+++

+

++

+++

+++

Histopathology

+++

+++

++

++

+++

+++

Bioassay

-

-

-

-

+

+

Transmission EM

-

-

-

-

+

+

Antibody-based methods

-

-

-

-

+

+

DNA probes - dot-blot

-

-

-

-

-

-

DNA probes - in situ

-

-

-

-

+++

+++

PCR

+++

+++

+++

+++

+++

+++

1.	Standard Screening Methods for MBV
	1.1.	Direct microscopic methods
		.	1.1.1. Wet-mount 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 preparations, MBV occlusion bodies are shown to be intranuclear and to range in diameter from less than 0.1 µm to nearly 20 µm. Staining the tissue squash with a stain such as 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, 6, 19, 20, 23).
		.	1.1.2. Faecal examination technique
			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 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 (similar in size to Baculovirus penaei [BP] occlusion bodies). 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. In fresh faeces, MBV occlusions often remain in clusters, held together by the nuclear membrane (5, 9, 19, 20 36).
			Collected faeces may also be used as the sample for nonlethal testing for MBV by polymerase chain reaction (PCR). PCR will provide greater diagnostic sensitivity for low-grade infections than will direct microscopic examination (3, 7, 16, 20, 26, 38).
	1.2.	Histological methods
		See Section 2.2.
	1.3.	Polymerase chain reaction procedure
		PCR methods have been developed for MBV. Primer sets designed from certain segments of the MBV genome are unique and provide positive test results only in samples infected with particular stains of this virus (11, 19, 27, 29, 33, 38, 39).
		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, 7, 16, 33).
		The following controls should be included in every PCR assay for MBV: a known negative tissue or 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.
		.	1.3.1. DNA extraction
			DNA extraction kits are convenient and commercially available. Otherwise, refer to Section 1.3.1. in Chapter 4.1.4. Tetrahedral baculovirosis (BP) for a suitable DNA extraction procedure.
		.	1.3.2. MBV PCR method - segment of the polyhedrin gene
			A PCR method with primer sequences was published by Lu et al. (25). Two primers, p35 and p36, were designed using insect baculovirus polyhedrin gene sequences as a reference, and they amplify a 674 base pairs (bp) segment of the MBV polyhedrin gene. The primer sequences are:
			p35:	5'-AC(CT)-TA(CT)-GTG-TCA-GAC-AAC-AAA-TA(CT)-TAC-AAA-3'
			p36:	5'-GG(CT)-GCG-TC(GT)-GG(CT)-GCA-AA(CT)-TT(CT)-TT(AT)-AC(CT)-TT(AG)-AA-3'
			i)	The reaction is carried out in a final volume of 100 µl of a reaction mixture containing 67 mM Tris/HCl, pH 8.0, 2 mM MgCl₂, 0.2 mM of each dNTP, 15 pmol of each primer, and 1 unit of Taq DNA polymerase.
			ii)	A drop of mineral oil is added to cover the reaction mixture (if the thermal cycler used does not have a heated lid), which is then amplified through 35 cycles (95°C for 1 minute, 45°C for 1 minute, and 72°C for 2 minutes) in a thermal cycler.
			iii)	The 674 bp PCR product may be visualised by electrophoresis in a 1% agarose gel or by Southern transfer and hybridisation with a labelled MBV probe prepared from the same DNA segment.
		.	1.3.3. MBV PCR method - segment of the polymerase gene
			A PCR method with primer sequences was published by Hsu et al. (16). Two primers, pA1 and pA2, were designed using a conserved sequence from the insect baculovirus polymerase gene as a reference, and they amplify a 511 bp segment of the MBV polymerase gene. The primer sequences are:
			pA1:	5'-GTG-CTG-CAA-CGA-CTG-GAC-TGT-CC-3'
			pA2:	5'-TGA-TCC-TAG-GCG-TGC-CTG-TAT-TGG G-3'
			i)	The reaction is carried out in a final volume of 100 µl of a reaction mixture containing 10 ng of viral DNA, 1.6 µM each of the two primers, 10 µl of 10 x Dynazyme buffer and five units of Dynazyme (Finnzymes, Espoo, Finland), and 20 mM mixture of all four dNTP.
			ii)	The reaction mixes were overlaid with mineral oil (if required), and then heated to 94°C for 10 minutes before starting the PCR in a thermal cycler.
			iii)	The first five cycling parameters are: denaturing for 2 minutes at 94°C, annealing for 1 minute at 42°C, extension for 1 minute at 72°C.
			iv)	This is followed by 30 cycles using the following parameters: denaturing at 94°C, annealing for 1 minute at 59°C, and extension for 1 minute at 72°C. During the last cycle, extension is 10 minutes. PCR reactions are stopped at 4°C.
			v)	The 511 bp PCR product may be visualised by electrophoresis in a 2% agarose gel.
		.	1.3.4. MBV nested PCR method
			A nested PCR method capable of detecting low concentrations of MBV (down to eight viral genome equivalents) was published by Belcher & Young (3). 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 (3). 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 %GC method (values in parentheses).
			DNA extraction
			i)	PCR inhibitors were noted by the authors of this method (3) to be present in DNA samples prepared from whole MBV-infected postlartval Penaeus monodon when using the extraction method recommended by Wang et al. (42) 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 (postlarvae, shrimp hepatopancreas, faeces) are 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 glycogen to precipate 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 12000 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 in dilution buffer.
			Nested PCR method
			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.
			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/litre ethidium bromide.
2.	Diagnostic Methods for Confirmatory Tests
	Confirmation of infection by MBV may be accomplished with any of the methods listed in Section 1 (i.e. wet-mounts of hepatopancreas tissue squashes or of faecal strands, or by PCR). The other methods available for confirmatory diagnosis of MBV include: autofluorescence with phloxine, and routine histological methods (4-6, 19, 20, 23, 36).
	2.1.	Autofluorescence method with phloxine stain
		Another method for detecting MBV occlusion bodies is based on the fluorescence of phloxine-stained occlusion bodies (5, 20, 36). 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 haematoxylin and eosin (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.
	2.2.	Histological methods
		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 all routine histological studies of shrimp (4, 20). 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 24-48 hours, and then transferred to 70% ethyl alcohol for storage. After transfer to 70% ethyl alcohol, fixed specimens may be transported 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 ref. 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 5-7 µm thickness are cut. Routine histological stains such as Mayer-Bennett's or Harris H& E, Giemsa stains, and Gram tissue-staining methods may be used for the demonstration of MBV diagnostic spherical occlusion bodies in hepatopancreatocytes, gut epithelial cells, or gut lumen (5, 6, 10, 20, 24). Typically, MBV-infected hepatopancreatic (or occasionally midgut) cells will present markedly hypertrophied nuclei with single or, more often, multiple occlusion bodies, chromatin diminution and margination. Occlusion bodies may be stained bright red with H& E stains, and intensely, but variably, with Gram's tissue stains. 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 decolouration, etc.) than the surrounding tissue, aiding in demonstrating their presence in low-grade infections (19, 20, 23).
	2.3.	Molecular methods
		Nonradioactive DIG-labelled gene probes to MBV have been developed (20, 23, 26-28, 33). Gene probe and PCR methods may provide greater diagnostic sensitivity in detecting low-grade infections than do more traditional wet-mount or histological techniques. Furthermore, PCR methods (see Section 1.3.) have the added advantage of being applicable to nonlethal testing of faecal samples collected from valuable broodstock shrimp.
		DIG-labelled DNA probes for representative strains of BP and MBV are commercially available as ShrimProbeTM kits from DiagXotics (Wilton, Connecticut, USA). The probes are labelled with a nonradioactive 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.
		.	2.3.1. 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, but 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 (20).
		.	2.3.2. *In-situ* hybridisation procedure
			The in-situ hybridisation protocol given in detail in Section 1.2. of Chapter 4.1.6. Infectious hypodermal and haematopoietic necrosis virus (IHHNV) uses the GeniusTM System developed by Boehringer Mannheim Biochemicals (now Roche Molecular Biochemicals) and was adapted from the Boehringer Mannheim's Nonradioactive In Situ Hybridization Application Manual. An additional step is required for the in-situ hybridisation test for MBV. Follow steps i-vi of Section 1.2., in-situ hybridisation procedure for IHHNV, in Chapter 4.1.6. For MBV substitute the following modifications to step vii before proceeding with steps viii-xvii as given for IHHNV:
			i)	(Modified step vii): Boil the DIG-labelled probe for 10 minutes and quench on ice; spin briefly in the cold (~4-10°C) using a refrigerated centrifuge or a chilled microcentrifuge) to bring all the liquid down to the base of the microcentrifuge tube; keep on ice. Dilute the probe to 50 ng/ml in prehybridisation solution and cover the tissue with 500 µl of the solution. Denature the double-stranded viral DNA in the tissue of the histological section by placing the slides on a 85°C heat block (or on aluminium foil that is placed over a boiling water bath) for 6-10 minutes. Quench the slides on ice for 5 minutes. Incubate the slides overnight at 42°C in a humid chamber. Drain the fluid on to blotting paper. During this incubation step, keep the wash buffers at 37°C to prewarm them.
			ii)	Proceed with steps viii-xvii as given for IHHNV.
			With DIG-labelled probes, accumulations of MBV viral DNA within infected cell nuclei, in cytoplasmic phagosomes, or in necrotic tissue debris are stained blue to a dark blue-black. Although they contain virus, occlusion bodies do not normally react with DIG-labelled DNA probes because the occlusion body protein crystalline matrix does not permit penetration of the probe (20).

1.	Anderson I.G., Shariff M., Nash G. & Nash. M. (1987). Mortalities of juvenile shrimp Penaeus monodon, associated with Penaeus monodon baculovirus, cytoplasmic reo-like virus and rickettsial and bacterial infections, from Malaysian brackish water ponds. Asian Fish. Sci., 1, 47-64.
2.	Baticados M.C.L., Pitogo, C.L., Paner M.G., de la Peza L.D., Tendencia E.A. (1991). Occurrence and pathology of Penaeus monodon baculovirus infection in hatcheries and ponds in the Philippines. Israeli J. Aquaculture - Bamidgeh, 43, 35-41.
3.	Belcher C.R. & Young P.R. (1998). Colourimetric PCR-based detection of monodon baculovirus in whole Penaeus monodon postlarvae. J. Virol. Methods, 74, 21-29.
4.	Bell T.A. & Lightner D.V. (1988). A Handbook of Normal Shrimp Histology. Special Publication No. 1. World Aquaculture Society, Baton Rouge, Louisiana, USA, 114 pp.
5.	Bondad-Reantaso M.G., McGladdery S.E., East I. & Subasinghe R.P. (eds) (2001). Asia Diagnostic Guide to Aquatic Animal Diseases. FAO Fisheries Technical Paper 402, Supplement 2. Rome, Italy, 240 pp.
6.	Brock J.A. & Lightner D.V. (1990). Diseases of crustacea. Diseases caused by microorganisms. In: Diseases of Marine Animals, Vol. III, Kinne O., ed. Biologische Anstalt Helgoland, Hamburg, Germany, 245-349.
7.	Chang P.S., Lo C.F., Kou G.H. & Chen S.N. (1993). Purification and amplification of DNA from Penaeus monodon-type baculovirus (MBV). J. Invertebr. Pathol., 62, 116-120.
8.	Chen S.N., Chang P.S. & Kou G.H. (1989). Observation on pathogenicity and epizootiology of Penaeus monodon baculovirus (MBV) in cultured shrimps in Taiwan. Fish Pathol., 24, 189-195.
9.	Chen S.N., Chang P.S. & Kou G.H. (1990). Infection route and eradication of Penaeus monodon baculovirus (MBV) in larval giant tiger prawns, Penaeus monodon. In: Diseases of Cultured Penaeid Shrimp in Asia and the United States, Fulks W. & Main K.L., eds. Oceanic Institute, Honolulu, Hawaii, USA, 177-184.
10.	Chen S.N., Chang P.S., Kou G.G. & Lightner D.V. (1989). Studies on virogenesis and cytopathology of Penaeus monodon baculovirus (MBV) in giant tiger prawn (Penaeus monodon) and the red tail prawn (Penaeus penicillatus). Fish Pathol., 24, 89-100.
11.	Chen S.N., Lo C.F., Liu S.M. & Kou G.H. (1989). The first identification of Penaeus monodon baculovirus (MBV) in cultured sand shrimp, Metapenaeus ensis. Bull. Eur. Assoc. Fish Pathol., 9, 62-64.
12.	Doubrovsky A., Paynter J.L., Sambhi S.K., Atherton J.G. & Lester R.J.G. (1988). Observations on the ultrastructure of baculovirus in Australian Penaeus monodon and Penaeus merguiensis. Aust. J. Mar. Freshwater Res., 39, 743-749.
13.	Fegan D.F., Flegel T.W., Sriurairatana S. & Waiyakruttha M. (1991). The occurrence, development and histopathology of monodon baculovirus in Southern Thailand. Aquaculture, 96, 205-217.
14.	Fukuda H., Momoyama K. & Sano T. (1988). First detection of monodon baculovirus in Japan. Nippon Suisan Gakkaishi, 54, 45-48.
15.	Hao N.V., Thuy D.T., Loan L.T.T., Phi T.T., Phuoc L.H., Duong H.H.T., Corsin F. & Chanratchakool P. (1999). Presence of the two viral pathogens WSSV and MBV in three wild shrimp species (Penaeus indicus, Metapenaeus ensis and Metapenaeus lysianassa) cultured in the mangrove forest of Ca Mau Province. Asian Fish. Sci., 12, 309-325.
16.	Hsu Y.L., Wang K.H., Yang Y.H., Tung M.C., Hu C.H., Lo C.F., Wang C.H. & Hsu T. (2000). Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies. Dis. Aquat. Org., 40, 93-99.
17.	Johnson P.T. & Lightner D.V. (1988). Rod-shaped nuclear viruses of crustaceans: gut-infecting species. Dis. Aquat. Org., 5, 123-141.
18.	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.
19.	Lightner D.V. (1988). Diseases of cultured penaeid shrimp and prawns. In: Disease Diagnosis and Control in North American Marine Aquaculture, Sindermann C.J. & Lightner D.V., eds. Elsevier, Amsterdam, The Netherlands, 8-127.
20.	Lightner, D.V. (Ed.) (1996). A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA, 304 pp.
21.	Lightner D.V., Hedrick R.P., Fryer J.L., Chen S.N., Liao I.C. & Kou G.H. (1987). A survey of cultured penaeid shrimp in Taiwan for viral and other important diseases. Fish Pathol., 22, 127-140.
22.	Lightner D.V. & Redman R.M. (1981). A baculovirus-caused disease of the penaeid shrimp, Penaeus monodon. J. Invertebr. Pathol., 38, 299-302.
23.	Lightner D.V & Redman R.M. (1998). Shrimp diseases and current diagnostic methods. Aquaculture, 164, 201-220.
24.	Lightner D.V., Redman R.M. & Bell T.A. (1983). Observations on the geographic distribution, pathogenesis and morphology of the baculovirus from Penaeus monodon Fabricius. Aquaculture, 32, 209-233.
25.	Lu C.C., Tang F.J.K. & Chen S.N. (1996). Morphogenesis of the membranous labyrinth in penaeid shrimp cells infected with Penaeus monodon baculovirus (MBV). J. Fish Dis., 19, 357-364.
26.	Lu C.C., Tang F.J.K., Kou G.H. & Chen S.N. (1993). Development of a Penaeus monodon-type baculovirus (MBV) DNA probe by polymerase chain reaction and sequence analysis. J. Fish Dis., 16, 551-559.
27.	Lu C.C., Tang F.J.K., Kou G.H. & Chen S.N. (1995). Detection of Penaeus monodon-type baculovirus (MBV) infection in Penaeus monodon Fabricius by in situ hybridization. J. Fish Dis., 18, 337-345.
28.	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.
29.	Nash G., Poernomo A. & Nash M.B. (1988). Baculovirus infection in brackishwater pond cultured Penaeus monodon Fabricius in Indonesia. Aquaculture, 73, 1-6.
30.	Natividad J.M. & Lightner D.V. (1992). Prevalence and geographic distribution of MBV and other diseases in cultured giant tiger prawns (Penaeus monodon) in the Philippines. In: Diseases of Cultured Penaeid Shrimp in Asia and the United States, Fulks W. & Main K., eds. The Oceanic Institute, Makapuu Point, Honolulu, Hawaii, USA, 139-160.
31.	Natividad J.M. & Lightner D.V. (1992). Susceptibility of the different larval and postlarval stages of black tiger prawn, Penaeus monodon Fabricius, to monodon baculovirus (MBV). In: Diseases in Asian Aquaculture I, Shariff M., Subasinghe R. & Arthur J.R., eds. Fish Health Section, Asian Fisheries Society, Manila, Philippines, 111-125.
32.	Paynter J.L., Vickers J.E. & Lester R.J.G. (1992). Experimental transmission of Penaeus monodon-type baculovirus (MBV). In: Diseases in Asian Aquaculture I, Shariff M., Subasinghe R. & Arthur J.R., eds. Fish Health Section, Asian Fisheries Society, Manila, Philippines, 97-110.
33.	Poulos B.T., Mari J., Bonami J.R., Redman R. & Lightner D.V. (1994). Use of non-radioactively labeled DNA probes for the detection of a baculovirus from Penaeus monodon by in situ hybridization on fixed tissue. J. Virol. Methods, 49, 187-194.
34.	Spann K.M. & Lester R.J.G. (1996). Baculovirus of Metapenaeus bennettae from the Moreton Bay region of Australia. Dis. Aquat. Org., 27, 53-58.
35.	Spann K.M., Lester R.J.G. & Paynter J.L. (1993). Efficiency of chlorine as a disinfectant against monodon baculovirus (MBV). Asian Fish. Sci., 6, 295-301.
36.	Thurman R.B., Lightner D.V., Bell T.A. & Hazanow S. (1990). Unique physicochemical properties of the occluded penaeid shrimp baculoviruses and their use in diagnosis of infections. J. Aquat. Anim. Health, 2, 128-131.
37.	Van Regenmortel M.H.V., Fauquet C.M., Bishop D.H.L., Carstens E.B., Estes M.K., Lemon S.M., Maniloff J., Mayo M.A., McGeoch D.J., Pringle C.R. & Wickner R.B. (2000). Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, USA, 1162 pp.
38.	Vickers J.E., Lester R.J.G., Spradbrow P.B. & Pemberton J.M. (1992). Detection of Penaeus monodon-type baculovirus (MBV) in digestive glands of postlarval prawns using polymerase chain reaction. In: Diseases in Asian Aquaculture I. Shariff M., Subasinghe R.P., & Arthur J.R., eds. Fish Health Section, Asian Fisheries Society, Manila, Philippines, 127-133.
39.	Vickers J.E., Lester R.J.G., Spradbrow P.B. & Pemberton J.M. (1994). Evidence for homology between polyhedrin genes of baculoviruses of a prawn and an insect. J. Invertebr. Pathol., 63, 207-208.
40.	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.
41.	Vogt G. (1992). Transformation of anterior midgut and heaptopancreas by monodon baculovirus (MBV) in Penaeus monodon postlarvae. Aquaculture, 107, 239-248.
42.	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.


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