| | | i) | The RT-PCR assay is done in solution, using 1.0 µl of shrimp haemolymph as the RNA template, or 10 µl of total RNA extracted from tissue or haemolymph (concentration of RNA = 1-100 ng/ml).
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| | | | Note: 10% sodium citrate is commonly used as an anticoagulant for shrimp haemolymph samples and, without dilution, it may inhibit reverse transcription or PCR. Hence, diluting crude haemolymph 1/10 in water is recommended if inhibition by sodium citrate is suspected.
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| | | ii) | The following controls should be included in every RT-PCR assay for TSV: a) known TSV-negative tissue sample; b) a known TSV-positive sample (tissue or purified virus); and C) a 'no-template' control.
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| | | iii) | The GeneAmp®, EZ rTth RNA PCR kit (Applied Bioscience, Norwalk, CT) is used for all amplification reactions described here.
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| | | iv) | The optimised RT-PCR conditions (final concentrations in 50 µl total volume) for detection of TSV in Penaeus vannamei are: primers (0.46 µM each), dNTPs (300 µM each), rTth DNA polymerase (2.5 U/50 µl), manganese acetate (2.5 mM), in 5 x EZ buffer (25 mM Bicine, 57.5 mM potassium acetate, 40% [w/v] glycerol, pH 8.2).
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| | | v) | The optimised RT-PCR conditions for detection of TSV from infected P. stylirostris are the same as for P. vannamei, except that the amount of the rTth DNA polymerase should be increased to 5.0 U/50 µl.
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| | | vi) | If the thermal cycler does not have a heated lid, then light mineral oil (50 µl) is overlaid on the top of the 50 µl reaction mixtures to prevent condensation or evaporation during thermal cycling.
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| | | vii) | The RNA template and all the reagents are combined and reverse transcription is allowed to proceed at 60°C for 30 minutes, followed by 94°C for 2 minutes.
|
| | | | Note: The reaction conditions described here were optimised using an automatic DNA Thermal Cycler 480 (Perkin Elmer Cetus, Norwalk, CT). The conditions should be optimised for each thermal cycler using known positive controls.
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| | | viii) | At the completion of reverse transcription, the samples are amplified for 35 cycles under the following conditions: denaturation at 94°C for 45 seconds, and then annealing/ extension at 60°C for 45 seconds. A final extension step for 7 minutes at 60°C follows the last cycle and the process is terminated in a 4°C soak file.
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| | | ix) | Following the termination of RT-PCR, the amplified cDNA solutions are drawn off from beneath the mineral oil and placed into clean 0.5 ml microfuge tubes.
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| | | x) | A 10 µl sample of the amplified product can then be added to the well of a 2.0% agarose gel, stained with ethidium bromide (0.5 g/ml), and electrophoresed in 0.5 x TBE (Tris, boric acid, ethylene diamine tetra-acetic acid [EDTA]).
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| | | xi) | A 100 bp DNA ladder (Gibco/BRL, Gaithersburg, Maryland, USA) is used as a marker.
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| | | xii) | Details of the composition of the reagents and buffers used here may be found in Chapter 4.1.6. Infectious hypodermal and haematopoietic necrosis (IHHN).
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| 2. | Diagnostic Methods for Confirmatory Tests
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| | 2.1. | Gross signs
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| | | TS is best known as a disease of nursery- or growout-phase P. vannamei, which occurs within ~14-40 days of stocking of postlarvae into growout ponds or tanks. Hence, shrimp with TS are typically small juveniles of from ~0.05 g to <5 g. Larger shrimp may also be affected, especially if they are not exposed to the virus until they are larger juveniles or adults (3, 16, 17, 22).
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| | | TS disease has three distinct phases, acute, transition, and chronic, which are grossly distinguishable (11, 16). Gross signs presented by shrimp in the acute and especially the transition phases of TS are unique and can provide a provisional diagnosis of the disease (9, 11, 15, 16). Gross signs displayed by moribund P. vannamei with acute-phase TS include expansion of the red chromatophores giving the affected shrimp a general, overall pale reddish coloration and making the tail fan and pleopods distinctly red; hence 'red tail' disease was one of the names given by farmers when the disease first appeared in Ecuador (20). In such shrimp, close inspection of the cuticular epithelium in thin appendages (such as the edges of the uropods or pleopods) with a x10 hand lens reveals signs of focal epithelial necrosis. Shrimp showing these gross signs of acute TS typically have soft shells, an empty gut and are often in the late D stages of the molt cycle. Acutely affected shrimp usually die during ecdysis. If the affected shrimp are larger than ~1 g, moribund shrimp may be visible to sea birds at the pond edges and surface. Thus, during the peak of severe epizootics, hundreds of sea birds (gulls, terns, cormorants, etc.) may be observed feeding on affected shrimp (3-5, 8, 16, 20.).
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| | | Although only present for a few days during TS epizootics, the gross signs presented by shrimp in the transition phase can provide a tentative diagnosis of TSV infection. During the transition phase (which may be occurring while many shrimp in the affected populations are still in the acute phase and daily mortalities are high), fair to moderate numbers of shrimp in affected ponds show random, multifocal, irregularly shaped melanised cuticular lesions. These melanised spots are haemocyte accumulations indicating resolving TS lesions in the cuticular epithelium. Such shrimp may or may not have soft cuticles and red-chromatophore expansion, and may be behaving and feeding normally. After successfully molting, shrimp in the transition phase move into the chronic phase of TS in which the virus maintains a persistent infection of the lymphoid organ, perhaps for life. Such TSV carriers may pass the virus horizontally to other susceptible shrimp or possibly vertically to its progeny. Although vertical transmission is suspected, the actual mechanism of transmission of TSV from parent broodstock to their progeny has not been demonstrated (11, 15, 16, 20).
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| | | TSV typically infects cells in tissues of ectodermal and mesodermal origin. TSV infections in P. vannamei and P. stylirostris have three phases: acute, transition and chronic. The cuticular epithelium is the most severely affected tissue in the acute phase, while only the lymphoid organ seems to be infected by the virus during the chronic phase of the disease. In P. vannamei, acute-phase TSV infection may result in high mortalities, while most strains of P. stylirostris can be infected, but are resistant to the development of TS disease. Survivors of TSV acute infections pass through a brief transition phase and enter a long-term chronic phase in which the affected host may remain persistently infected for life. Such TSV carriers may pass the virus horizontally to other susceptible shrimp (2, 3, 9, 12, 13, 16, 17).
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| | 2.2. | Histological method
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| | | Diagnosis of TS in the acute phase of the disease is dependent on the histological demonstration (in haematoxylin-and-eosin-stained preparations), of multifocal areas of necrosis in the cuticular epithelium of the general body surface, all appendages, gills, hindgut, oesophagus and stomach. Occasionally affected are cells of the subcuticular connective tissues and adjacent striated muscle fibres basal to affected cuticular epithelium. Rarely, the antennal gland tubule epithelium is affected. Prominent in the multifocal cuticular lesions are conspicuous foci of affected cells that display an increased eosinophilia of the cytoplasm and pyknotic or karyorrhectic nuclei. Cytoplasmic remnants of necrotic cells are often extremely abundant in these TS acute-phase lesions and these are generally spherical bodies (1-20 µm diameter) that range in staining from eosinophilic to pale basophilic. These structures, along with pyknotic and karyorrhectic nuclei, give acute-phase TS lesions a characteristic 'peppered' or 'buckshot-riddled' appearance, which is considered to be pathognomonic for the disease. Pyknotic or karyorrhectic nuclei give a positive (for DNA) Feulgen reaction, which distinguishes them from the less basophilic to eosinophilic cytoplasmic inclusions that do not contain DNA. The absence of haemocytic infiltration or other signs of a significant host-inflammatory response distinguishes the acute phase of TS from the transitional phase of the disease (3-5, 9-12, 15, 16 20).
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| | | In the transitional phase of TS, typical acute-phase cuticular lesions decline in abundance and severity and are replaced by conspicuous infiltration and accumulations of haemocytes at the same sites. The masses of haemocytes may become melanised giving rise to the black spots that characterise the transition phase of the disease. Such lesions may show erosion of the cuticle, surface colonisation and invasion of the affected cuticle and exposed surface haemocytes by Vibrio spp. (11, 16).
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| | | Shrimp in the chronic phase of TS display no gross signs of infection, and histologically the only sign of infection is the presence of prominent LOS, which are spherical accumulations of cells that lack the central vessel of normal lymphoid organ tubules (11, 16, 17).
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| | 2.3. | Bioassay method
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| | | Further confirmation of TSV infection may be accomplished by bioassay of TSV-suspect animals with specific pathogen free (SPF) juvenile P. vannamei serving as the indicator for the virus (3, 5, 8, 11, 12, 16, 22, 27). Oral or injection protocols may be used. The oral method is relatively simple to perform and is accomplished by feeding chopped carcasses of suspect shrimp to SPF juvenile P. vannamei in small tanks. The use of a negative control tank of indicator shrimp, which receives only a normal feed, is required. When the carcass feeding (per os) protocol is used to bioassay for TSV, TS-positive indicator shrimp (by gross signs and histopathology) are typically apparent within 3-4 days of initial exposure, and significant mortalities occur by 3-8 days after initial exposure. The negative control shrimp must remain negative for gross or histological signs of TS disease and unusual mortalities (11, 12, 16).
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| | | With the injection bioassay protocol, a variety of sample types may be tested for TSV. Whole shrimp are used if they were collected during a TSV epizootic. Heads only are used if shrimp display gross transition-phase lesions (multifocal melanisation spots on the cuticle) or no clinical signs of infection (chronic phase) as the virus, if present, will be concentrated in the lymphoid organ (11, 16). For nonlethal testing of broodstock, haemolymph samples may be taken and used to expose the indicator shrimp (16).
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| | | To perform the bioassay:
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| | | i) | Prepare a 1:2 or 1:3 ratio of TSV-suspect shrimp heads or whole shrimp with TN buffer (see Chapter 4.1.6. IHHNV 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 fluid. 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 1/10 to 1/100 with sterile 2% saline. This solution may now be used as the inoculum to inject indicator shrimp (or filtered sterilised as described in step vi).
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| | | vi) | Filter the diluted supernatant fluid 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. Filtrate should be collected in a sterile test tube or beaker. The solution can now be stored frozen (recommend -20°C for short-term [weeks] storage and -80°C for long-term [months to years] storage) or used immediately to inject indicator shrimp.
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| | | vii) | Indicator shrimp should be from TSV susceptible stocks of SPF P. vannamei (such as the 'Kona stock'), which are commercially available from a number of sources in the Americas, and not from selected lines of known TSV-resistant stocks.
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| | | viii) | Inject 0.01 ml per gram of body weight using a 1 ml tuberculin syringe. Indicator shrimp should be injected intramuscularly into the third tail segment. If the test shrimp 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 shrimp. Sudden death occurring post-injection is referred to as 'protein shock', and is the result of systemic clotting of the shrimp's haemolymph in response to the inoculum.
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| | | ix) | Haemolymph samples may be diluted (1/10 or 1/20 in TN buffer), filter sterilised (if necessary), and injected into the indicator shrimp without further preparation.
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| | | x) | If TSV was present in the inoculum, the indicator shrimp should begin to die within 24-48 hours post-injection. Lower doses of virus may take longer to establish a lethal infection and shrimp should be monitored for at least 7 days post-injection.
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| | | xi) | The presence of TSV in the indicator shrimp should be confirmed by histological analysis (and in-situ hybridisation by gene probe if available) of Davidson's fixed moribund shrimp.
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| | | As a variation to the bioassay technique, a 'sentinel shrimp' system may be used. For example, TSV-sensitive stocks of small juvenile SPF P. vannamei may be held in net-pens in tanks, or in the same water system, with other shrimp of unknown TSV status to bioassay for the presence of infectious agents such as TSV.
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| | 2.4. | Antibody-based methods
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| | | MAb for detection of TSV may be used to assay samples of haemolymph, tissue homogenates, or Davidson's AFA fixed tissue sections from shrimp (28). Currently, TSV-1A1-MAb (a mouse immunoglobulin isotype IgG(1k) is available in kit form from DiagXotics (Wilton, CT, USA). TSV-1A1-Mab may be used to distinguish an apparently new strain of TSV from other strains (7). During the 1999-2000 shrimp farming seasons, a new stain of TSV emerged in Mexico that caused high mortalities (as high as 90%) in stocks of Pacific blue shrimp, P. stylirostris, that were previously resistant to TS (17, 33). What may be a second newly emerged strain of TSV appeared in 2001 in Central America where it caused similarly high mortalities in selected stocks of P. vannamei that had been selected because of demonstrated resistance to TSV (7). By not reacting with TSV-1A1-MAb, while giving positive tests for TSV with available molecular tests (i.e. RT-PCR and in-situ hybridisation), the new Mexican strain of TSV may be distinguished from the reference (Hawaiian 1994) isolate of TSV (2, 12, 25), and from other (but otherwise indistinguishable) geographical and temporal isolates of the virus that do react with TSV-1A1-MAb (7).
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| | | . | Dot-blot immunoassay method
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| | | i) | For the dot-blot immunoassay method, 1 µl of test antigen (purified virus, infected shrimp haemolymph or SPF shrimp haemolymph) is dotted on to the surface of MA-HA-N45 assay plates (Millipore, South San Francisco, California [CA], USA).
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| | | ii) | After air drying, the wells are blocked for 1 hour at room temperature with 200 µl of a buffer containing phosphate buffered saline and 0.05% Tween 20 (PBST) mixed with 10% normal goat serum (Life Technologies, Gibco BRL) and 2% Hammersten casein (Amersham Life Sciences, Arlington Heights, Illinois, USA).
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| | | iii) | The wells are washed three times with PBST and then reacted with 100 µl primary antibody (MAb or mouse polyclonal antibodies) for 30 minutes at room temperature.
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| | | iv) | Alkaline-phosphatase-labelled goat anti-mouse IgG, g chain specific, secondary antibody (Zymed, South San Francisco, CA) diluted 1/1000 in PBST plus 10% normal goat serum is used for detection (30 minutes at room temperature).
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| | | v) | After washing three times with PBST, once with PBS and once with distilled water, the reactions are visualised by development for 15 minutes at room temperature with nitroblue tetrazolium and bromo-chloro-indoyl phosphate (Roche Diagnostics, Corp.) in Tris-NaCl (100 mM each) buffer containing 50 mM MgCl2, pH 9.5.
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| | | vi) | Reactions are stopped with distilled water.
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| | | vii) | The reactions are graded using a scale from 0 to +4, with the highest intensity reaction being equivalent to the reaction generated using the MAb against the reference control consisting of semipurified TSV. A negative reaction is one in which no coloured spot is visible in the well.
|
| | | . | Other antibody-based methods
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| | | The TSV-1A1-MAb may be applicable to other antibody-based test formats (i.e. indirect fluorescent antibody or immunohistochemistry (IHC) tests with tissue smears, frozen sections, or deparaffinised fixed tissues). TSV-1A1-MAb is applicable for use in an IHC format using Davidson's AFA fixed tissue sections (7), and a kit for this procedure is available commercially (DiagXotics, Wilton, CT, USA). Application to other antibody-based test formats (i.e. indirect fluorescent antibody test with tissue smears, frozen sections, or deparaffinised fixed tissues) is possible, but still experimental at this time.
|
| | | Recent evidence indicates that there are serological differences among some geographical isolates of TSV that may cause a false-negative reaction when using TSV-1A1-MAb, which is the only TSV antibody currently available from commercial sources (DiagXotics, Wilton CT, USA). Therefore, it is recommended that unexpected results from MAb-based tests for TSV should be interpreted in the context of clinical signs, case history, and in conjunction with other test results (e.g., histology or in-situ hybridisation with a TSV-specific DNA probe).
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