Manual of Diagnostic Tests for Aquatic Animals (2003)
CHAPTER 4.1.3.
YELLOWHEAD DISEASE
SUMMARY
Yellowhead disease (YHD) of penaeid shrimp is characterised by high and rapid mortality, typically accompanied by the gross signs of yellowing of the cephalothorax and general bleaching of body colour. Histologically, moribund shrimp show systemic necrosis of cells of ectodermal and mesodermal origin, with the formation of intensely basophilic cytoplasmic inclusions (by haematoxylin and eosin staining) that arise from phagocytosed nuclei and viral inclusions. However, gross signs of infection and histopathology are not entirely reliable indicators of YHD and the use of molecular methods for confirmatory diagnosis required.
In initial Thai reports, gross signs of disease and mortality occurred within 2-4 days following an interval of exceptionally high feeding activity that ended in abrupt cessation of feeding (4, 17). Many moribund shrimp congregated near the surface at pond edges. Moribund shrimp exhibited a bleached overall appearance and a yellowish discoloration of the cephalothorax caused by the underlying yellow hepatopancreas (HP), which was exceptionally soft when compared with the brown HP of normal shrimp. In many cases, total crop loss was experienced within a few days of the appearance of shrimp showing gross signs of YHD. Yellowhead virus (YHV) can infect cultured shrimp from late postlarval stages onwards, but massive mortality is usually encountered from early to late juvenile stages in rearing ponds. Several carrier crustaceans are known or suspected (8, 39). While cessation of feeding, congregation at pond edges and a generally bleached appearance are always seen in YHD outbreaks, these disease features are not particularly distinctive for YHD. The other more pathognomonic gross signs are not always seen and thus they are not reliable, even for preliminary diagnosis of YHD.
YHV is a positive-sense, single-stranded RNA (ssRNA) virus (27, 36, 43), originally thought to be related to rhabdoviruses but now known to be related to coronaviruses and arteriviruses (32, 39). YHV has now been formally classified in the new genus Okavirus and new family Roniviridae within the viral order Nidovirales (24, 25). The status of work on YHV has been reviewed (8, 10, 12, 14, 39). Transmission electron microscopy (TEM) of YHV-infected ectodermal and mesodermal tissues shows enveloped bacilliform virions. They range from approximately 150 nm to 200 nm in length and from 40 nm to 50 nm in diameter and are located within vesicles in the cytoplasm of infected cells and in intercellular spaces (3, 4, 27). The virions arise from long, filamentous nucleocapsids (approximately 15 nm x 130-800 nm), which accumulate in the cytoplasm and obtain an envelope by budding at the endoplasmic reticulum into intracellular vesicles. Negatively stained YHV virions show regular arrays of short spikes on the viral envelope. YHV is potentially lethal to most of the commercially cultivated penaeid shrimp species.
A virus with a similar ultrastructure to that of YHV has been reported from Australia (33, 35). Gill-associated virus (GAV) was first described in moribund Penaeus monodon during an outbreak of a yellowhead-like disease (33). Previously, a yellowhead-related virus called lymphoid organ virus (LOV) was described in healthy Australian P. monodon. However, it is now evident that these are reports of the same virus, which may occur as a chronic, asymptomatic infection or an acute, fatal infection. Chronic GAV infection occurs in wild healthy P. monodon with high prevalence at some sites in eastern Australia (39) and is transmitted vertically during spawning (6). Acute GAV infection causes high mortality, but the gross signs and patterns of tissue tropism may differ from those of YHV (33). Comparison of DNA sequences of reverse-transcription polymerase chain reaction (RT-PCR) products from YHV and GAV indicates that they are closely related viral genotypes (5, 32, 39). Recently, GAV was classified as the type species of the genus Okavirus (24, 25). A third yellowhead genotype also occurs commonly in healthy P. monodon in Thailand and Vietnam, but this virus has not yet been recovered from shrimp displaying signs of YHD. The third genotype is more closely related in nucleotide sequence to GAV than to YHV. A fourth, more distantly related genotype has also recently been detected in healthy P. monodon from India.
YHD was first described as an epizootic from Thai shrimp farms (17), and subsequent outbreaks have been reported mostly from cultivated shrimp in Asia (1, 3, 4, 26, 39, 41). YHV has also been reported in farmed shrimp from the Americas (16, 18). However, these cases were not confirmed and presumptive histological diagnoses appear to have been due to severe infections with white spot virus, which can cause similar histopathology in the lymphoid organ (30). Natural YHV infections have been reported in P. monodon, Marsupenaeus japonicus, Fenneropenaeus merguiensis, Litopenaeus setiferus, Metapenaeus ensis, Palaemon styliferus and Euphasia superba (8). Laboratory trials have shown that YHV can cause high mortality in other penaeid shrimp species including Litopenaeus vannamei, L. stylirostrus, Farfantepenaeus aztecus and F. duorarum (15, 19, 20). GAV has been reported only from Australia where P. monodon appears to be the only natural host (31, 33, 35, 38). Experimental GAV infection and mortalities have also been reported in Penaeus esculentus, M. japonnicus, F. merguiensis and L. vannamei, but there is evidence of species- and age-related variation in susceptibility to disease (34, 37).
Histopathology of YHD can be used for presumptive diagnosis during outbreaks (4, 14). In moribund shrimp collected near the surface at pond edges, extensive necrosis can be found in tissues of ectodermal and mesodermal origin. The areas of necrosis are characterised by the presence of large numbers of intensely basophilic, mostly spherical, cytoplasmic inclusions ranging from approximately 2 µm in diameter or smaller. These inclusions can be seen in rapidly stained squash mounts of gills or subcuticular tissue (10), and in tissue sections. In the latter, the lymphoid organ is usually enlarged and extensively necrotised; however the subcuticular tissue of the stomach and gill tissue are the best tissues to visualise the characteristic inclusions. Grossly normal shrimp should also be collected from the same ponds where moribund shrimp are collected, for the preparation of haemolymph smears. In YHD outbreaks some of these smears will show moderate to large numbers of haemocytes with pycnotic and karyorrhectic nuclei, in the absence of bacteraemia (28). This is an early sign of impending morbidity, but it is usually absent or difficult to see in moribund shrimp because of the loss of haemocytes. Together, the results from tissues of moribund shrimp and from haemolymph smears can be used for presumptive diagnosis of a YHD outbreak. However, as severe infection with white spot virus can cause necrosis and similar histopathology in the lymphoid organ (30), confirmatory diagnosis using molecular tests is recommended. Presumptive diagnosis of GAV disease is based on gross signs including swimming near the surface and at the pond edges, cessation of feeding, a reddening of body and appendages and pink to yellow coloration of the gills (33). Lymphoid organ spheroids are commonly observed in healthy P. monodon chronically infected with GAV and lymphoid organ necrosis often accompanies GAV disease (33, 35). However, spheroid formation and degeneration of lymphoid organ tissue also occur during infection with other shrimp viruses (14, 30).
Confirmation of YHD requires more detailed analysis by RT-PCR (36, 43), Western-blot analysis (18, 20), in-situ nucleic acid hybridisation (37) and TEM (3, 4, 27). The only test currently available that reliably distinguishes YHV from GAV and other closely related viruses is RT-PCR. However, as chronic infections with YHV-related viruses appear to be common in healthy shrimp in parts of Asia, reliable disease diagnosis requires consideration of all available evidence. Dual and multiple infections with several shrimp viruses are not uncommon (9, 40) so detection of a virus in a diseased shrimp does not, in itself, establish a causal relationship. For both YHV and GAV, the use of in-situ hybridisation probes to demonstrate invasive necrotic infection of the lymphoid organ is the most reliable method of confirmatory disease diagnosis presently available (37).
Detection of YHV in carrier shrimp or other crustaceans cannot be reliably accomplished by histological methods, and more sophisticated techniques are required. As YHV readily infects species of shrimp such as L. vannamei, which are geographically separated from the YHV endemic area (15, 19, 20), bioassay in uninfected or specific pathogen free shrimp from controlled breeding programmes can be used (7, 29, 37). No continuous cell lines are presently available for the study of shrimp viruses, although YHV has been cultivated in primary cell lines from the lymphoid organ of L. vannamei (23). These cultures can be used to measure YHV titres and tissue tropism (22). However, the need to continually prepare cultures from different shrimp or from shrimp of undefined or uncertain history currently presents problems for test standardisation. Because of these limitations, it is recommended that detection of YHV for surveillance or certification of broodstock or fry for stocking, should be carried out by RT-PCR.
DIAGNOSTIC PROCEDURES
Methods for detection of yellowhead virus (YHV) and gill-associated virus (GAV) may be applied for confirmation of disease outbreaks, certification of broodstock and postlarvae used to stock rearing ponds, and for disease surveillance. For presumptive diagnosis of disease outbreaks, it is sufficient to carry out histological analysis by light microscopy of haematoxylin and eosin (H& E)-stained tissues from moribund shrimp, combined with light microscopy of stained haemolymph smears from grossly normal shrimp in the same pond. However, for definitive disease diagnosis the reverse-transcription polymerase chain reaction (RT-PCR) assay and in-situ nucleic acid hybridisation are recommended. Definitive diagnosis can also be accomplished by Western-blot analysis and transmission electron microscopy (TEM), but methods other than in-situ hybridisation and RT-PCR using suitable discriminatory primers should be used cautiously due to the high prevalence of yellow head-related viruses in healthy wild and cultured shrimp in some regions of Asia and Australia. Detection of YHV, GAV or a related virus in diseased shrimp is not, in itself, sufficient to establish a causal association. Certification of YHV or GAV infection status of broodstock and fry should be conducted using the RT-PCR assay that is sufficiently sensitive and specific to detect low-level chronic infections.
The methods currently available for surveillance, detection, and diagnosis of YHV 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. These are somewhat subjective as suitability involves issues of reliability, sensitivity, and utility.
Table 1. YHV surveillance, detection and diagnostic methods
Method
|
Screening
|
Presumptive
|
Confirmatory
|
|
Larvae
|
PLs
|
Juveniles
|
Adults
|
|
|
Gross signs
|
-
|
-
|
-
|
-
|
+
|
-
|
Histopathology
|
-
|
-
|
-
|
-
|
++
|
-
|
Transmission EM
|
-
|
-
|
+
|
+
|
+++
|
+
|
Western blot
|
?
|
?
|
+
|
+
|
+++
|
+
|
In-situ hybridisation
|
?
|
?
|
+
|
+
|
+++
|
+++
|
RT-PCR
|
+
|
+++
|
+++
|
+++
|
+++
|
+++
|
|
PLs = postlarvae; EM = electron microscopy; RT-PCR = reverse-transcription polymerase chain reaction.
Note that EM, Western blot or in-situ hybridisation cannot distinguish among YHV, GAV and yellowhead-related viruses. The viruses can be distinguished by RT-PCR using suitable discriminatory primers.
| 1. | Standard Screening Methods for YHV and GAV
|
| | 1.1. | Reverse-transcription polymerase chain reaction assay of haemolymph
|
| | | The protocol described here is adapted from Wongteerasupaya et al. (42). An alternate assay for YHV is reported by Tang & Lightner (36). A nested RT-PCR method, which is more sensitive and will detect and discriminate both YHV and GAV is described below in Section 1.2.
|
| | | Collect fresh shrimp haemolymph (50 µl), mix immediately with 500 µl of TrizolT reagent (Bethesda Research Laboratories, Gaithersberg, Maryland, United States of America [USA]) and extract RNA according to the product manual. Resuspend RNA (2 µl) in 20 µl of PCR buffer (10 mM Tris/HCl, pH 8.3, 50 mM KCl) containing 2.5 U of M-MLV (Moloney murine leukaemia virus) reverse transcriptase, 1.0 U of ribonuclease inhibitor, 0.75 µM of antisense primer (144R, below), 1 mM each of dATP, dTTP, dCTP, and dGTP, and 5 mM of MgCl2, and incubate at 42°C for 15 minutes to synthesise cDNA. Incubate the mixture at 100°C for 5 minutes to inactivate the reverse transcriptase and allow the mixture to cool to 5°C. Add the PCR mixture (10 mM Tris/HCl, pH 8.3, 50 mM KCl) containing 2.5 U of Taq DNA polymerase (Perkin Elmer Cetus), 2 mM MgCl2 and 0.75 µM of sense primer (10F, below) to give a final volume of 100 µl. Overlay the tubes with 100 µl of mineral oil and carry out PCR amplification for 40 cycles at 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, and finishing at 72°C for 10 minutes. Include a negative control containing diethyl pyrocarbonate (DEPC)-treated distilled water instead of RNA extract, and a positive control containing YHV RNA. Detect the characteristic YHV amplicon of 135 bp by electrophoresis of 20 µl aliquots through 2% agarose gels. The sensitivity of the assay is approximately 0.01 pg of purified YHV RNA (~ 103 genomes). The sequences of the PCR primers are:
|
| | | 10F: | 5'-CCG-CTA-ATT-TCA-AAA-ACT-ACG-3'
|
| | | 144R: | 5'-AAG-GTG-TTA-TGT-CGA-GGA-AGT-3'
|
| | 1.2. | Reverse-transcription polymerase chain reaction assay for differential detection of YHV and GAV
|
| | | The RT-PCR method of Wongteerasupaya et al. (42) will not detect GAV due to sequence differences from YHV at the PCR primer sites (38). The following method (provided by J.A. Cowley & P.J. Walker) is a nested RT-PCR procedure that allows detection of both YHV and GAV in the first amplification and discrimination of the genotypes in the nested amplification. The third yellowhead genotype, which occurs commonly in healthy P. monodon in some parts of Asia, will usually react as GAV in this test. The fourth genotype, which has been detected in healthy P. monodon from India, does not react at all in this test or the test described by Wongteerasupaya et al. (42).
|
| | | Fresh lymphoid organ, gill tissue or haemolymph provide a good source of RNA. Lymphoid organ and gill tissue preserved in 95% analytical-grade ethanol or stored frozen at -70°C are also suitable for total RNA preparation. Approximately 10-20 mg lymphoid organ or gill tissue or 50 µl haemolymph is disrupted in 500 µl TRIzolT reagent (Gibco-BRL) and total RNA is extracted according to the product manual. RNA is resuspended in 25 µl DEPC-treated water, heated at 56°C for 15 minutes, kept on ice and used immediately or stored at -70°C until required. Ideally, a 1/200 dilution (i.e. 2.5 µl in 500 µl DEPC-treated water) should be prepared, and absorbances A260 nm and A280 nm (UV spectrophotometer required) should be determined to quantify and check the quality of the RNA. For each set of RNA samples to be tested, DEPC-treated water and extracts known to contain either GAV or YHV RNA should be included as negative and positive controls, respectively. For cDNA synthesis, 0.1-1.0 µg RNA (1.0 µg for optimum sensitivity) and 0.7 µl of 50 pmol/µl primer GY5 are adjusted to 6 µl with DEPC-treated water, incubated at 70°C for 10 minutes and chilled on ice. cDNA is synthesised by adding 2 µl Superscript II buffer x 5 (250 mM Tris/HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 1 µl 100 mM DTT and 0.5 µl 10 mM dNTP stock mixture (i.e. 10 mM dATP, 10 mM dTTP, 10 mM dCTP, 10 mM dGTP), preheating to 42°C for 2 minutes prior to adding 0.5 µl of 200 U/µl Superscript II reverse transcriptase (Gibco-BRL) and incubating at 42°C for 1 hour. The reaction is then heated at 70°C for 10 minutes, placed on ice and spun briefly in a microcentrifuge to collect the contents of the tube. In the first PCR step, 1 µl cDNA is amplified in a 50 µl reaction containing 1 x Taq buffer (10 mM Tris/HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 1.5 mM MgCl2, 35 pmol each primer GY1 and GY4, 200 µM each of dATP, dTTP, dCTP and dGTP and 2.5 U Taq polymerase (Promega) using a 0.5 ml thin-walled tube. The reaction mixture is overlaid with 50 µl liquid paraffin and heated at 85°C for 2-3 minutes 'hot start' prior to adding cDNA. DNA is amplified using 35 cycles of 95°C for 30 seconds, 66°C for 30 seconds, and 72°C for 45 seconds, followed by final extension at 72°C for 7 minutes. In the second step, multiplex differential PCR, 0.5-5.0 µl (5.0 µl for optimum sensitivity) of the first PCR product is amplified using the reaction conditions above except that 35 pmol of each primer GY2, Y3 and G6 are used in place of GY1 and GY4, and DNA is amplified using 35 cycles of 95°C for 30 seconds, 66°C for 30 seconds, and 72°C for 30 seconds, followed by final extension at 72°C for 7 minutes. The PCR products (10 µl) are resolved in 2% agarose/TAE (Tris-acetate-EDTA [ethylene diamine tetra-acetic acid]) gels containing 0.5 µg/ml ethidium bromide alongside a suitable DNA ladder, detected using a UV transilluminator, and the gel is photographed as a record.
|
| | | A 794 bp DNA is amplified from either GAV or YHV in the first PCR step and can be visualised by agarose gel electrophoresis if virus RNA levels are sufficiently high. In the differential second PCR step, either a 406 bp DNA is amplified from GAV or a 277 bp DNA is amplified from YHV. The presence of both 406 bp and 277 bp DNA products will indicate a dual infection with GAV and YHV. The detection sensitivity of the second-step PCR is ~1000-fold greater than the first-step PCR and, depending on the level of infection, GAV or YHV RNA can be detected to a limit of 10 fg lymphoid organ total RNA.
|
| | | The sequences of RT-PCR primers generic for GAV and YHV (GY) or specific for GAV (G) or YHV (Y) are as follows:
|
| | | GY1 | 5'-GAC-ATC-ACT-CCA-GAC-AAC-ATC-TG-3'
|
| | | GY2 | 5'-CAT-CTG-TCC-AGA-AGG-CGT-CTA-TGA-3'
|
| | | GY4 | 5'-GTG-AAG-TCC-ATG-TGT-GTG-AGA-CG-3'
|
| | | GY5 | 5'-GAG-CTG-GAA-TTC-AGT-GAG-AGA-ACA-3'
|
| | | Y3 | 5'-ACG-CTC-TGT-GAC-AAG-CAT-GAA-GTT-3'
|
| | | G6 | 5'-GTA-GTA-GAG-ACG-AGT-GAC-ACC-TAT-3'
|
| | 1.3. | Reverse-transcription polymerase chain reaction assay of postlarvae
|
| | | From a nursery or hatchery tank containing 100,000 postlarvae or more, sample approximately 1000 postlarvae from each of five different points. Pool the samples in a basin, gently swirl the water in the basin and then select an assay sample from living postlarvae that collect at the centre of the basin. Choose the sample number according to the assumed or target prevalence (see Chapter I.3. Section 3.). Homogenise the sample in an appropriate volume of TrizolT reagent and extract RNA according to the product manual. For subsequent steps, follow the RT-PCR protocol for haemolymph given in Sections 1.1. or 1.2 above, using this extract as the template.
|
| | 1.4. | Western-blot assay
|
| | | Prepare reagents and carry out assays according to the protocols of Lu et al. (21) and Loh et al. (18). This includes purification of YHV virions from laboratory-infected shrimp, generation of immunoglobulins (Igs) in New Zealand white rabbits, purification of the IgG using recombinant bacterial protein-G columns and removal of cross-reacting normal shrimp antigens by adsorption on to acetone-dried, ground shrimp muscle tissue and haemolymph. For assay, remove 0.1 ml of haemolymph from live shrimp specimens and dilute in an equal volume of citrate buffer for immediate use or storage at -80°C until used. For Western blotting, use 200 µl sample, clarify at 8000 g for 5 minutes and then pellet the supernatant at 140,000 g for 5 minutes. Resuspend pellets in 100 µl 2 x loading buffer (2.5 ml 0.5 mM Tris/HCl, pH 6.8, 4 ml 10% sodium dodecyl sulfate [SDS], 2 ml glycerol, 1 µl beta-mercaptoethanol, and 0.5 ml deionised distilled water) and heat at 95°C for 5 minutes. Load a 10 µl subsample on to 5% SDS/polyacrylamide gel, and conduct electrophoresis at 200 V. Blot the gel on to a nitrocellulose membrane (pore size, 0.1 mm) in blotting buffer (3.03 g Tris base, 14.4 g glycine, and 200 ml methanol per litre) at 100 V for 1 hour. Rinse the membrane with phosphate buffered saline (PBS), pH 7.4, soak in 5% skim milk (in PBS) for 1 hour, and rinse with PBS for 5 minutes. Next, treat the membrane with a 1/1000 dilution of the primary antibody (IgG) for 1 hour, rinse three times with PBS for 5 minutes, and then treat with a 1/2500 dilution of goat anti-rabbit IgG-horseradish-peroxidase conjugate for 1 hour. Rinse again three times with PBS for 5 minutes and then treat with the substrate, 3,3',5,5'-tetramethylbenzidine, until a bluish/purple colour develops. Stop the reaction by soaking the membrane in distilled water. All incubations should be carried out at 25°C ( 2°C. Use a purified viral preparation as a positive control and identify 2-4 major protein bands characteristic of YHV at 116, 64 and 20 kDa. The sensitivity is 0.4 ng of YHV protein (~ 106 YHV virions).
|
| 2. | Presumptive Diagnostic Methods for YHV and GAV
|
| | Presumptive diagnosis of YHV can be achieved by the following three methods.
|
| | 2.1. | Rapid staining of squash mount preparations
|
| | | Collect moribund shrimp from a suspected outbreak of YHD and fix the whole shrimp or gill filaments in Davidson's fixative (14) overnight. After fixation, wash some gill filaments thoroughly with tap water to remove the fixative, and stain with Meyer's H& E (14). After staining and dehydration, when the tissue is in xylene, place a gill filament on a microscope slide in a drop of xylene and, using a fine pair of needles (a stereo microscope is helpful), break off several secondary filaments and then replace the main filament in xylene where it can be stored indefinitely as a permanent reference in a sealed vial. Being careful not to let the xylene dry, tease apart the secondary filaments on a microscope slide and remove any large fragments or particles that would thicken the mount unnecessarily. Finally, add a drop of mounting fluid and a cover-slip. Use light pressure to flatten the mount as much as possible. This procedure may also be used with thin layers of subcuticular tissue. With YHD outbreaks, examination with a x40 objective of the light microscope will reveal the presence of moderate to large numbers of deeply basophilic, evenly stained, spherical, cytoplasmic inclusions approximately 2 µm in diameter or smaller (10). This evidence should be used together with the results from haemolymph smears (see below) in making a presumptive diagnosis of a YHD outbreak. As for the fixed tissues and the filaments in xylene, these whole-mount slides can be kept as a permanent record.
|
| | | In the event that rapid results are required, the fixation step can be shortened to only 2 hours by changing the acetic acid portion of the Davidson's fixative formula to 50% concentrated HCl. For best results, this fixative should not be stored for more than a few days before use. After fixation, wash thoroughly to remove the fixative and check that the pH has returned to near neutral before staining. Do not fix for longer periods or above 25°C as this may result in excessive tissue damage that will make interpretation difficult or impossible.
|
| | 2.2. | Staining of haemolymph smears
|
| | | Haemolymph smears from moribund shrimp infected with YHV are not useful because haemocytes are usually depleted in advanced stages of YHD. Sample haemolymph from grossly normal shrimp from a suspect pond where moribund shrimp have also been collected. Draw the haemolymph into a syringe containing twice the haemolymph amount of 25% formalin or of Davidson's fixative in which the acetic acid of the formula has been replaced by either water or formalin. Mix thoroughly, ignore clots in the syringe, place a drop on a microscope slide, smear and then air-dry before staining with H& E or other standard blood smear stains. Dehydrate, add mounting fluid and add a cover-slip. Examine with the x40 lens of a light microscope. In YHD outbreaks, some of the smears will show moderate to large numbers of haemocytes with karyorhectic or pycnotic nuclei (28). It is important that the slides with these nuclei show no evidence of concomitant bacterial infection, as bacterial infections may cause similar changes in haemocytes. The results from haemolymph smears should be used together with the results from rapidly stained whole mounts (see above) or stained tissue sections when making a presumptive diagnosis of a YHD outbreak.
|
| | 2.3. | Histopathology of tissue sections
|
| | | Fix moribund shrimp from a suspected YHV outbreak in Davidson's fixative and process for preparation of standard H& E-stained tissue sections (2, 14). Examine the sections by light microscopy for the presence of moderate to large numbers of deeply basophilic, evenly stained, spherical, cytoplasmic inclusions approximately 2 µm in diameter or smaller in tissues of ectodermal and mesodermal origin (4). Tissues of the lymphoid organ, stomach subcuticulum and gills are particularly useful.
|
| 3. | Confirmatory Diagnostic Methods for YHV and GAV
|
| | Confirmatory diagnosis of YHV can be achieved by a combination of the following methods. Note that in-situ nucleic acid hybridisation, Western-blot assay or TEM may not distinguish YHV from GAV or other related viruses. As low-level chronic infections are common in some regions, detection of the presence of virus is not, in itself, evidence of aetiology. A combination in-situ nucleic acid hybridisation and discriminatory RT-PCR assay is required for definitive diagnosis.
|
| | 3.1. | Reverse-transcription polymerase chain reaction
|
| | | See Sections 1.1 and 1.2.
|
| | 3.2. | Western-blot assay
|
| | | See Section 1.4.
|
| | 3.3. | In-situ nucleic acid hybridisation
|
| | | Follow the protocol according to Tang & Lightner (36) as briefly described here. The method is suitable for detection of YHV or GAV (37). To preserve the viral RNA, fix live shrimp with neutral-buffered, modified Davidson's fixative without acetic acid (RF-fixative; 13). Process the fixed shrimp using standard histological methods and prepare 4 µm thick sections on Superfrost Plus slides (Fisher Scientific, Pennsylvania, USA). Prior to hybridisation, incubate sections at 65°C for 45 minutes, remove paraffin with Hemo de (Fisher Scientific, Pennsylvania, USA), and rehydrate through an ethanol series to water. Digest sections with proteinase K (100 µg/ml, in 50 mM Tris/HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA) for 15 minutes at 37°C, followed by post-fixation in formaldehyde (0.4%) for 5 minutes. Rinse in 2 x SSC (standard saline citrate), then prehybridise with 500 µl prehybridisation solution (4 x SSC, 50% formamide, 1 x Denhardt's, 0.25 mg/ml yeast RNA, 0.5 mg/ml sheared salmon sperm DNA, 5% dextran sulfate) at 42°C for 30 minutes. For hybridisation, overlay the sections with 250 µl hybridisation solution containing a digoxigenin-labelled probe (20-40 ng/ml) at 42°C overnight. The next day, wash the sections as follows: 2 x SSC once for 30 minutes at room temperature; 1 x SSC twice for 5 minutes at 37°C; 0.5x SSC twice for 5 minutes at 37°C. Incubate the sections with sheep anti-digoxigenin-alkaline phosphatase conjugate (Boehringer Mannheim, Germany) at 37°C for 30 minutes. Wash with 0.1 M Tris/HCl, pH 7.5, 0.15 M NaCl twice for 10 minutes at room temperature and rinse with 0.1 M Tris/HCl, pH 9.5, 0.1 M NaCl. Incubate with nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate in the dark for 1-2 hours for colour development. Counterstain with Bismarck Brown Y (0.5%), dehydrate through a series of ethanol and Hemo de, add Permount (Fisher Scientific, Pennsylvania, USA) and cover with a cover-slip. YHV-infected cells give a blue to purple-black colour against the brown counterstain. Include positive controls of YHV-infected tissue and negative controls of uninfected shrimp tissue. The diagnostic probe can be prepared by PCR labelling using the following primers:
|
| | | YHV1051F: | 5'-ACA-TCT-GTC-CAG-AAG-GCG-TC-3'
|
| | | YHV1051R: | 5'-GGG-GGT-GTA-GAG-GGA-GAG-AG-3'
|
| | 3.4. | Transmission electron microscopy
|
| | | For TEM, the most suitable tissues of moribund shrimp suspected of YHV infection are those of the lymphoid organ and gills. For screening or surveillance of grossly normal shrimp, the most suitable tissue is from the lymphoid organ. The reagents described are from ref. 14. In brief: stun live shrimp by immersion in iced water until just immobilised or kill by injection of fixative. Quickly dissect and remove small portions of target tissue (no larger than a few mm in diameter) and fix in at least 10 volumes of 6% glutaraldehyde held at 4°C and buffered with sodium cacodylate (Na[CH3]2AsO2.3H2O) solution (8.6 g Na cacodylate, 10 g NaCl, distilled water to make 100 ml, adjusted to pH 7 with 0.2 N HCl) or phosphate solution (0.6 g NaH2PO4.H2O, 1.5 g Na2HPO4, 1 g NaCl, 0.5 g sucrose, distilled water to make 100 ml, adjusted to pH 7 with 0.2 N HCl). Fix for at least 24 hours prior to processing. For long-term storage in fixative at 4°C, reduce glutaraldehyde to 0.5-1.0%. Processing involves post-fixation with 1% osmium tetroxide, dehydration, embedding, sectioning and staining with uranyl acetate and lead citrate according to standard TEM methods.
|
| | 3.5. | Bioassay
|
| | | The bioassay procedure is based on that described by Spann et al. (33) but similar procedures have been described by several other authors (19, 29, 41). Bioassay should be conducted in susceptible shrimp (see Summary) that have been certified as specific pathogen free and have been obtained from a biosecure breeding facility. Alternatively, susceptible wild or farmed shrimp to be used for bioassay should be screened by nested RT-PCR on haemolymph samples to confirm the absence of pre-existing chronic infections with YHV, GAV or related viruses. Shrimp should be maintained throughout the procedure under optimal conditions for survival of the species in laboratory culture.
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| | | Collect moribund shrimp from a disease outbreak or shrimp suspected of being carriers of infection and maintain at 4°C or on ice. Remove and discard the tail and appendages. If necessary, the whole shrimp or the retained cephalothorax may be snap-frozen and stored at
-80°C or in liquid nitrogen until required. Thaw stored samples rapidly in a 37°C water bath within two snap-seal plastic bags and then maintain at 4°C or on ice during all procedures. Remove the carapace and calciferous mouth-parts. Suspend the remaining tissues in six volumes of TN buffer (0.02 M Tris/HCl, pH 7.4, 0.4 M NaCl) and homogenise in a tissue grinder to form a smooth suspension. Clarify the homogenate at 1300 g for 20 minutes at 4°C. Remove the supernatant fluid below the lipid layer and pass through a 0.45 µm filter. Maintain the filtrate at 4°C for immediate use or snap-freeze and store in aliquots at -80°C or in liquid nitrogen. Thaw the filtrate rapidly at 37°C and maintain on ice prior to use.
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| | | Inject at least twelve juvenile (1-5 g) shrimp of a known susceptible species (P. monodon, P. esculentus, M. japonicus, F. merguiensis, L. vannamei, L. stylirostris), with 5 µl of filtrate per gram body weight into the second abdominal segment using a 26-gauge needle. Inject two equivalent groups of at least twelve shrimp with TN buffer and a filtered tissue extract prepared from uninfected shrimp. One additional group of at least twelve shrimp should be injected last with a known and calibrated positive control inoculum from shrimp infected with YHV or GAV (as required). Maintain each group of shrimp in a separate covered tank with a separate water supply for the duration of the bioassay. Ensure no inadvertent transfer of water between tanks by good laboratory practice. Observe the shrimp and record mortalities for at least 21 days or until the test and positive control groups reach 100% mortality. Collect at least one moribund shrimp from each of the four groups for examination by histology, TEM, in-situ nucleic acid hybridisation, and PCR or Western-blot analysis to confirm the presence of YHV or GAV (as required) in the sample (refer Sections above for test procedures).
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| | | Note that shrimp to be tested that are suspected of being carriers of low level chronic infections may produce an inoculum containing a very low dose of virus. In bioassay, such an inoculum may not necessarily cause mortalities, gross signs of disease or histology characteristic of a lethal infection. In this event, molecular tests or TEM must be applied to the bioassay shrimp.
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