PLs = postlarvae; BF = bright field; LM = light microscopy; EM = electron microscopy; ELISA = enzyme-linked immunosorbent assay; PAb/MAb = poly/monoclonal antibodies; RT-PCR = reverse-transcription polymerase chain reaction.
| 1. | Standard Screening Methods For SMV
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| | 1.1. | Histological method
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| | | The histopathological changes associated with SMVD are nonspecific, i.e. SMVD cannot be diagnosed by histopathology. Nonspecific histopathological changes can occur in the hepatopancreas, midgut, and anterior midgut caecum. Juvenile P. monodon infected experimentally with tissue extracts from clinically diseased prawns with SMVD displayed haemocytic infiltration, necrosis, and sloughing of cells into the lumens of the midgut and hepatopancreas (2). This, however, is not always seen (figure 2 of ref. 3).
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| | 1.2. | Molecular method
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| | | A nonradioactively labelled DNA probe and PCR test (in kit form) have been developed (3, 4). Commercial availability of the kits is currently being negotiated.
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| 2. | Presumptive Diagnostic Methods for SMV
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| | 2.1. | Clinical signs
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| | | There are no specific clinical signs for SMVD. Juvenile prawns in grow-out ponds with clinical viral infections may exhibit signs such as discoloration, lethargy, fouling and anorexia.
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| | 2.2. | Molecular method
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| | | See Section 1.2.
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| | 2.3. | Bioassay method
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| | | This bioassay will confirm the presence of a pathogenic virus, but does not identify the specific virus. The presence of any pathogenic virus may be detected using a relatively simple bioassay in which healthy (specific pathogen free [SPF] if available) juvenile P. monodon are exposed to suspect prawns by either feeding them with suspect prawn tissues or by injecting them with cell-free tissue extracts prepared from suspect prawns. Fraser & Owens (2) reported that SMV could be transmitted per os by feeding infected carcasses or by injection of cell-free extracts prepared from infected carcasses. With intramuscular injection of cell-free extracts, mortalities began at 14 days post-inoculation and approached 100% by 30 days post-inoculation. Disease development in the bioassay indicator prawns following per os exposure required more time, with the first mortalities occurring at ~30 days post-inoculation and reaching 76% mortality by 50 days post-inoculation (2).
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| | | To perform the bioassay, use the generalised protocol as follows:
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| | | i) | Prepare a 1:2 or 1:3 ratio (w:v) of SMV-suspect prawn heads or whole prawns with TN buffer (see Chapter 4.1.6. Infectious hypodermal and haematopoietic necrosis virus for the composition of this buffer) or sterile 2% saline prepared with distilled water.
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| | | ii) | Homogenise the mixture using a tissue grinder or blender. Do not permit the mixture to heat up by excessive homogenisation or grinding. Tissues and resulting homogenate should be kept cool during the entire protocol by maintaining on ice.
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| | | iii) | Clarify the homogenate by centrifugation at 3000 g for 10 minutes. Decant and save the supernatant. Discard the pellet.
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| | | iv) | Centrifuge the supernatant fluid at 27,000 g (15,000 rpm) for 20-30 minutes at 4°C. Decant and save the supernatant fluid. Discard the pellet.
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| | | v) | Dilute the supernatant fluid from step iv to from 1/10 to 1/100 with sterile 2% saline. This solution may now be used as the inoculum to inject indicator prawns (or filter sterilised as described in step vi).
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| | | vi) | Filter the diluted supernatant from step v using a sterile syringe (size depends on final volume of diluted supernatant) and a sterile 0.45 µm syringe filter. Multiple filters may have to be used as they clog easily. The filtrate should be collected in a sterile test tube or beaker. The solution can now be stored frozen (-20°C for short-term [weeks] storage and -80°C for long-term [months to years] storage) or used immediately to inject indicator prawns.
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| | | vii) | Indicator prawns should be from stocks of healthy (SPF if available) P. monodon.
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| | | viii) | Inject 0.01 ml per gram of body weight using a 1-ml tuberculin syringe. Indicator prawns should be injected intramuscularly into the third tail segment. If the test prawns begin to die within minutes post-injection, the inoculum contains excessive amounts of proteinaceous material and should be further diluted prior to injecting additional indicator prawns. Sudden death occurring post-injection is referred to as 'protein shock', and is the result of systemic clotting of the prawn's haemolymph in response to the inoculum.
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| | | ix) | Cell-free extract samples may be diluted (1/10 or 1/20 in TN buffer), filter sterilised (if warranted), and injected into the indicator prawns without further preparation.
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| | | x) | If a pathogenic virus is present in the inoculum, the indicator prawns should begin to die.
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| | | xi) | The presence of SMV in the indicator prawns should be confirmed by transmission electron microscopy (and in situ hybridisation by gene probe if available).
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| 3. | Confirmatory Diagnostic Methods for SMVD
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| | Confirmation of SMV infection in prawns can be achieved by the following method.
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| | 3.1. | Transmission electron microscopy
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| | | The method is essentially that of Fraser and Owens (2). Moribund prawns are sedated in cold water and then the organs of interest are removed into Petri dishes containing 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.1. The tissues are cut into 1 mm cubes, fixed for 30 minutes at room temperature (24°C) and washed twice for 10 minutes in the 0.1 M cacodylate buffer. The samples are stored in the cacodylate buffer at 4°C. Before embedding, the samples are post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hour at 24°C, and then washed twice for 10 minutes with the same buffer. The post-fixed samples are dehydrated and embedded in Spurr's resin. The sections are stained with 2% uranyl acetate in 50% ethanol acidified with hydrochloric acid for 7 minutes, rinsed four times in distilled water and blotted dry. The sections are then stained with lead acetate for 2 minutes and the rinsing step is repeated. Aggregations of virus-like particles are found associated with the nuclear membrane in the cytoplasm of the gut cells. The virions are approximately 20-25 nm in diameter (3) and hexagonally shaped suggesting icosahedral symmetry. SMV virions are not found in tissues other than the gut.
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| | 3.2. | Molecular method
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| | | See Section 1.2.
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| 1. | Albaladejo J.D., Tapay L.M., Migo V.P., Alfafara C.G., Somga J.R., Mayo S.L., Miranda R.C., Natividad K., Magbanua F.O., Itami T., Matsumura M., Nadala E.C.B., Jr. & Loh P.C. (1998). Screening for shrimp viruses in the Philippines. In: Advances in Shrimp Biotechnology, Flegel T.W., ed. National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand, 251-254.
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| 2. | Fraser C.A. & Owens L. (1996). Spawner-isolated mortality virus from Australian Penaeus monodon. Dis. Aquat. Org., 27, 141-148.
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| 3. | Owens L., Haqshenas G., McElnea C., & Coelen R. (1998). Putative spawner-isolated mortality virus associated with mid-crop mortality syndrome in farmed Penaeus monodon from northern Australia. Dis. Aquat. Org., 34, 177-185.
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| 4. | Owens L. & McElnea C. (2000). Natural infection of the redclaw crayfish cherax quadricarinatus with presumptive spawner-isolated mortality virus. Dis. Aquat. Org., 40, 219-223.
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