Manual of Diagnostic Tests for Aquatic Animals (2003)

  PART 4
..«  		 SECTION 4.1.
  		 CHAPTER 4.1.6.
..«  »»
  		 Summary
? - Index

CHAPTER 4.1.6.

INFECTIOUS HYPODERMAL AND
HAEMATOPOIETIC NECROSIS

SUMMARY

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is the smallest of the known penaeid shrimp viruses. The IHHN virion is a 22 nm, nonenveloped icosahedron, with a density of 1.40 g/ml in CsCl, contains linear single-stranded DNA with an estimated size of 4.1 kb, and has a capsid with four polypeptides of molecular weight 74, 47, 39, and 37.5 kD. Because of these characteristics, IHHNV has been classified as a member of the family Parvoviridae (5, 6, 14, 16, 24).
 
Following its discovery, infection by IHHNV was demonstrated to cause acute epizootics and mass mortality ((90%) only in Penaeus stylirostris, with the juvenile and subadult life stages being the most severely affected. In marked contrast, IHHNV causes the chronic disease 'runt-deformity syndrome' in P. vannamei in which reduced, irregular growth and cuticular deformities, rather than mortalities, are the principal effects (1, 2, 7, 9, 10, 12, 14, 20). Selected shrimp stocks that are not only resistant to IHHN disease, but are also refractory to infection have been developed (14, 35). Some members of populations of P. stylirostris and P. vannamei that survive IHHNV infections and/or epizootics, may carry the virus for life and pass the virus on to their progeny and other populations by vertical and horizontal transmission (1, 20, 28).
 
IHHNV has a world-wide distribution among cultured penaeid shrimp. IHHNV is commonly found in wild penaeid shrimp in the eastern Pacific from Peru to Mexico. Although IHHNV has been reported from cultured P. vannamei and P. stylirostris in most of the shrimp-culturing regions of the Western Hemisphere and in wild penaeids throughout their range along the Pacific coast of the Americas (Peru to northern Mexico), it has not been found in wild penaeid shrimp on the Atlantic coast of the Americas (1, 2, 4-31, 33, 34). IHHNV has been also reported in cultured penaeid shrimp from Pacific islands including the Hawaiian Islands, French Polynesia, Guam, and New Caledonia. In the Indo-Pacific region, the virus has been reported from cultured and wild penaeid shrimp in East Asia, South-East Asia, and the Middle East (20). An IHHN-like virus has been reported from Australia (32). However, because this agent does not react with the DNA probe BS4.5 to IHHNV, it is not closely related to the strain(s) of IHHNV that do react with the probe and that are widely distributed in East Asia and in the Americas (20). Although demonstrated in wild shrimp from the Indo-Pacific, there is a paucity of data available on the distribution of this virus in wild shrimp from this region. The geographical distribution of IHHNV in shrimp has increased significantly since its discovery in the early 1980s. Recent work suggests that there is sufficient sequence variation among geographical isolates from diverse locations in Asia to suggest that multiple geographical strains of the virus exist (37). IHHNV causes serious disease in two species of Western Hemisphere penaeid shrimp and it is considered to be a potential pathogen in several other Western and Eastern Hemisphere species of penaeid shrimp. While infection by IHHNV has been demonstrated to cause disease in P. stylirostris, P. vannamei and P. monodon, natural or experimental infections in other penaeid species have been observed without the occurrence of disease (8, 10, 17-20, 22).
 
IHHNV typically infects cells in tissues of ectodermal and mesodermal origin, and only rarely is it detected in endoderm-derived tissues, such as the epithelial mucosal cells of the midgut, midgut caeca and hepatopancreas. In P. stylirostris, IHHNV infection may result in high mortalities, while in some strains of P. stylirostris, in P. vannamei, and possibly in P. monodon, reduced growth and cuticular deformities may be the only outcome of infection (15, 19, 20, 22).
 
Molecular diagnostic tests are the surveillance methods for IHHNV in penaeid shrimp. With molecular methods unique portions of the IHHNV genome are detected by dot-blot hybridisation using specific DNA probes or by polymerase chain reaction (12, 14, 20, 24, 27, 28, 30, 32, 34-37). While PCR methods provide the greatest sensitivity for detection of IHHNV (14, 28, 30, 35-37), the use of IHHNV-specific DNA probes with in-situ hybridisation applied to paraffin sections provides the greatest diagnostic certainty available for this agent (20). A variety of other diagnostic methods can be used to provide presumptive and confirmed diagnoses of IHHNV infection. Among these are routine histology, bioassays, and methods that use monoclonal antibodies against IHHNV with indirect fluorescent, enzyme-linked immunosorbent, and Western blot assays. With standard histological methods, diagnostic intranuclear eosinophilic inclusion bodies are demonstrated in specific target tissues (8, 20).
 
Eradication methods for IHHNV can be applied to certain aquaculture situations. These methods are dependent on eradication of infected stocks, disinfection of the culture facility, the avoidance of re-introduction of the virus (from other nearby culture facilities, wild shrimp, etc.), and re-stocking with IHHNV-free postlarvae that have been produced from IHHNV-free broodstock (10, 20, 21).
 

DIAGNOSTIC PROCEDURES

The methods currently available for surveillance, detection, and diagnosis of infectious hypodermal and haematopoietic necrosis virus (IHHNV) infections 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; and R& D = the method is in development but is not yet available though commercial sources.
 

Table 1. IHHNV surveillance, detection and diagnostic methods

Method
 
 Screening
 
 Presumptive
 
 Confirmatory
 
 
 
 Larvae
 
 PLs
 
 Juveniles
 
 Adults
 
  
 
  
 
Gross signs
 
 -
 
 -
 
 -
 
 -
 
 +
 
 -
 
Direct BF/LM
 
 -
 
 -
 
 -
 
 -
 
 -
 
 -
 
Histopathology
 
 -
 
 -
 
 -
 
 -
 
 ++
 
 ++
 
Bioassay
 
 -
 
 -
 
 -
 
 -
 
 +
 
 +
 
Transmission EM
 
 -
 
 -
 
 -
 
 -
 
 +
 
 +
 
Antibody-based methods
 
 -
 
 -
 
 -
 
 -
 
 R& D
 
 R& D
 
DNA probes - dot-blot
 
 -
 
 ++
 
 ++
 
 ++
 
 ++
 
 ++
 
DNA probes - in situ
 
 -
 
 +++
 
 +++
 
 +++
 
 +++
 
 +++
 
PCR
 
 +
 
 +++
 
 +++
 
 +++
 
 +++
 
 +++
 

PLs = postlarvae; BF = bright field; LM = light microscopy; EM = electron microscopy;
PCR = polymerase chain reaction.

Sampling procedures: see Chapter I.3.
 
1.   Standard Screening Methods for IHHNV
 
     Surveillance methods for IHHNV use nonradioactive DIG-labelled gene probes and polymerase chain reaction (PCR) methods. Nonradioactive DIG-labelled DNA probes for IHHNV are now commercially available as ShrimProbeTM kits from DiagXotics (Wilton, Connecticut [CT], USA) in dot-blot and in-situ hybridisation formats. The probe is labelled with a nonradioactive label, digoxigenin-11-dUTP (DIG-11-dUTP). The protocols given below use the GeniusTM System developed by Boehringer Mannheim Biochemicals (this company now owned by Roche Diagnostic Corporation) and was adapted from the GeniusTM System User's Guide for Membrane Hybridization and from Boehringer Mannheim's Nonradioactive In Situ Hybridization Application Manual (12, 20, 27, 34).
 
     Gene probe and PCR methods provide greater diagnostic sensitivity than do more traditional methods for IHHN diagnosis that employ classic histological methods. Furthermore, these methods have the added advantage of being applicable to nonlethal testing of valuable broodstock shrimp. A haemolymph sample may be taken with a tuberculin syringe, or an appendage (a pleopod for example) may be biopsied, and used as the sample for a direct dot-blot test or PCR assay (4, 12, 20, 30, 32).
 
     Several PCR methods are available for IHHNV detection (28, 30, 35-37), and several companies market PCR test kits. PCR kits are available for IHHNV in the USA from DaigXotics (Wilton, CT, USA) or from Farming Intelligene Technology (Taipei China). However, very recent information on IHHNV suggests that there are multiple geographical strains and that some primer sets will not react with some geographical strains of the virus isolates (37). Hence, confirmation of unexpected positive and/or negative PCR results with a second primer set using another diagnostic method is advisable.
 
     1.1.   Dot-blot hybridisation procedure
 
          The dot-blot hybridisation method using a DIG-labelled DNA probe for IHHNV follows generally the methods outlined in Lightner (16) that are given below. Formulas for the required reagents are given in Section 1.1.1. below.
 
          i)   Prepare a positively charged nylon membrane (Roche Diagnostics Cat. No. 1-209-299): cut pieces to fit samples and controls and mark with soft-lead pencil making 1 cm squares for each sample. Include a positive and a negative control on each filter. Lay out on to a piece of filter paper (Whatman 3MM).
 
          ii)   If necessary, dilute samples to be assayed in TE plus 50 µg/ml salmon sperm DNA, using 1 µl sample in 9 µl buffer in 1.5 ml microcentrifuge tubes. Samples for dot-blots can be haemolymph or tissues homogenised in TN buffer.
 
          iii)   Boil samples for 10 minutes and quench on ice for 5 minutes. Briefly microfuge samples in the cold to bring down all liquid and to pellet any coagulated protein. Keep on ice until samples are dotted on to the membrane.
 
          iv)   Dot 1-3 µl of each sample on to an appropriate place on the filters. Allow to air-dry and then fix samples on to the membrane by baking at 80°C for 30 minutes or by UV cross-linking using a DNA transilluminator for 3 minutes.
 
          v)   Turn on a water bath to 68°C and prepare prehybridisation solution. For a 10 x 15 cm membrane, prepare 8 ml per membrane. Switch on a stirring hot plate set to 'low' and stir and warm for 30 minutes until the blocking agent has dissolved and the solution is cloudy. Also prepare some heat-seal bags that are slightly larger in size than the membrane. Five to six bags will be needed per membrane.
 
          vi)   Remove membranes from the oven or transilluminator and put into a heat-seal bag with 4 ml per membrane of prehybridisation solution. Seal the bags and put into a 68°C water bath for 0.5-1 hour.
 
          vii)   Boil the DIG-labelled probe for 10 minutes, quench on ice and then microfuge in the cold to bring all the liquid down. Keep on ice. Remove the prehybridisation solution from the bags. Add 2 ml of fresh prehybridisation solution to each bag and then add the correct, predetermined amount of DIG-labelled probe to each, mixing well as it is being added. Seal the bags, place back in the 68°C water bath and incubate for 8-12 hours.
 
          viii)   Wash membranes well with:
 
2 x standard saline citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) 2 x 5 minutes at room temperature
0.1 x SSC/0.1% SDS 3 x 15 minutes at 68°C
(use 4 ml/filter and seal in bags)
Buffer I 1 x 5 minutes at room temperature
Buffer II 1 x 30 minutes at room temperature
Buffer I 1 x 5 minutes at room temperature
(Buffers are prepared ahead of time).
 

 
          ix)   React the membranes in bags with anti-DIG AP conjugate (Roche Diagnostics 1-093-274) diluted 1/5000 in Buffer I. Use 3 ml per membrane, incubate for 30-45 minutes at room temperature on a shaker platform.
 
          x)   Wash membrane well with:
 
Buffer I 2 x 15 minutes at room temperature
Buffer III
 
 1 x
 
 5 minutes at room temperature
 

 
          xi)   Develop the membranes in bags using 3 ml per membrane of development solution (nitroblue tetrazolium salt [NBT]/Xphosphate in Buffer III) made up just prior to use. React in the dark at room temperature for 1-2 hours. Stop the reactions in Buffer IV and dry the membranes on 3MM filter paper.
 
          xii)   Photograph the results. (Colour fades over time.)
 
          xiii)   Store dry membranes in heat-seal bags.
 
          .   1.1.1. Reagent formulas for dot-blot method
 
          i)   TN buffer
 
20 mM Tris/HCl 3.15 g Tris/HCl
0.4 M NaCl 23.38 g NaCl
DD H2O 1000 ml

 
               pH to 7.4; autoclave to sterilise; store at 4°C.
 
          ii)   Sample dilution buffer: Buffer IV plus 50 µg/ml salmon sperm DNA. Prepare salmon (or herring) sperm DNA as follows:
 
               .   Dissolve 2.5 mg salmon sperm DNA in 5 ml DD H2O (500 µg/ml).
               .   Shear the DNA by passing the solution through an 18-20-gauge needle several times.
               .   Boil the DNA for 10-15 minutes to denature it.
               .   Filter 45 ml Buffer IV through a 0.45 µm filter.
               .   Mix 5 ml of sheared, denatured DNA with the 45 ml of filtered Buffer IV.
               .   Store at -20°C (can keep usable amount at 4°C).
 
          iii)   20 x SSC buffer:
 
3 M NaCl 175.32 g NaCl
0.3 M Na citrate 88.23 g Na citrate.2H2O
DD H2O 1000 ml
pH to 7.0; filter through 0.45 µm filter; autoclave to sterilise; store at 4°C.
 

 
          iv)   5 x SSC buffer
 
5 x SSC 250 ml 20 x SSC stock
DD H2O 750 ml
Filter through 0.45 µm filter; store at 4°C.
 

 
          v)   Prehybridisation solution (prepare 30 minutes prior to use)
 
1% blocking agent 0.1 g blocking reagent (Roche Diagnostics 1-096-176)
0.1% Sarkosyl 50 µl 20% Sarkosyl
0.02% SDS 20 µl 10% sodium dodecyl sulfate
5 x SSC buffer 10 ml 5 x SSC buffer
Heat on a 'low' setting with stirring for 30 minutes to dissolve blocking agent.
 

 
          vi)   Wash buffers
 
               2 x SSC, 0.1% SDS:
               Mix 100 ml 20 x SSC with 895 ml DD H2O; filter through 0.45 µm filter; add 5 ml 20% SDS and mix; store at room temperature.
 
               0.1 x SSC, 0.1% SDS:
               Mix 5 ml 20 x SSC with 990 ml DD H2O; filter through 0.45 µm filter; add 5 ml 20% SDS and mix; store at room temperature.
 
          vii)   Buffer I
 
100 mM Tris/HCl 12.11 g Tris Base
150 mM NaCl 8.77 g NaCl
DD H2O 1000 ml
pH to 7.5 with HCl; filter through 0.45 µm filter; store at 4°C (can make a 10 x stock, autoclave; filter the 1 x buffer).
 

 
          viii)   Buffer II
 
0.5% blocking agent 0.05g blocking reagent (Roche Diagnostics 1-096-176)
Buffer I 10 ml
Heat on a low setting with stirring for 30 minutes to dissolve blocking agent.
 

 
          ix)   Buffer III
 
100 mM Tris/HCl 12.11 g Tris Base
100 mM NaCl 5.84 g NaCl
DD H2O 1000 ml
pH to 9.5 with HCl  
Then add:  
50 mM MgCl2 10.16 g MgCl2.6H2O
Filter through 0.45 µm filter; store at 4°C.
 

 
          x)   Buffer IV ('TE')
 
10 mM Tris/HCl 1.21 g Tris base
1 mM EDTA 0.37 g ethylene diamine tetra-acetic acid.2 H2O (disodium salt)
DD H2O 1000 ml
pH to 8.0 with HCl; filter through 0.45 µm filter; store at 4°C.
 

 
          xi)   Development solution (prepare just prior to use)
 
Nitroblue tetrazolium salt 45 µl NBT (Roche Diagnostics 1-383-213)
(75 mg/ml in 70% dimethylformamide)
5-bromo-4-chloro-3-indoyl phosphate, toluidine salt (50 mg/ml in dimethyl-formamide) 35 µl X-phosphate
(Roche Diagnostics 1-383-221)
Buffer III
 
 10 ml

 
     1.2.   In-situ hybridisation procedure
 
          The in-situ hybridisation method using a DIG-labelled DNA probe for IHHNV follows generally the methods outlined in Lightner (20) and given below. Formulas for the required reagents are given in Section 1.2.1.
 
          i)   Embed tissue in paraffin and cut sections at ~4 µm thickness. Put sections on to positively charged microscope slides (do not put gelatin in water to float sections; use just water).
 
          ii)   Put slides in a slide rack, such as a Tissue-Tek rack. Heat the slides in an oven for 45 minutes at 60°C. In the staining centre, rehydrate the tissue as follows:
 
Citri Solve* 3 x 5 minutes each
Absolute alcohol 2 x 1 minutes each
95% alcohol 2 x 10 dips each
80% alcohol 2 x 10 dips each
50% alcohol 1 x 10 dips
Distilled water six rinses (do not let slides dry out)
* Citri Solve is a xylene substitute.
 

 
          iii)   Wash the slides for 5 minutes in phosphate buffered saline (PBS or TNE). Prepare fresh proteinase K at 100 µg/ml in PBS (or TNE). Place slides flat in a humid chamber, pipette on 500 µl of the proteinase K solution and incubate for 10-15 minutes at 37°C. Drain fluid on to blotting paper.
 
          iv)   Return slides to slide rack. Fix sections in 0.4% cold formaldehyde for 5 minutes at room temperature.
 
          v)   Incubate slides in 2 x SSC for 5 minutes at room temperature.
 
          vi)   With slides flat, add 0.5-1 ml prehybridisation buffer and incubate in a humid chamber for 15-30 minutes at 37°C.
 
          vii)   Boil the DIG-labelled probe for 10 minutes and quench on ice; spin briefly in the cold and keep on ice. Dilute the probe to 25 ng/ml in prehybridisation solution and cover the tissue with 250 µl of the solution. Incubate the slides for 2-4 hours at 42°C or overnight at 37°C in a humid chamber. Drain fluid on to blotting paper. During this incubation, put the wash buffers at 37°C to prewarm them.
 
          viii)   Place slides in slide rack. Wash the slides as follows:
 
2 x SSC 2 x 5-30 minutes at 37°C
1 x SSC 2 x 5 minutes at 37°C
0.5 x SSC
 
 2 x 5 minutes at 37°C

 
          ix)   Wash the slides for 5 minutes in Buffer I at room temperature. Put the slides flat in a humid chamber and block with 0.5 ml per slide of Buffer II. Incubate for 15 minutes at 37°C. Drain the fluid on to blotting paper.
 
          x)   Dilute the anti-DIG AP conjugate (Boehringer Mannheim 1-093-274) 1/1000 in Buffer II (1 µl anti-DIG AP per 1 ml buffer). Cover tissue with 500 µl of diluted conjugate and incubate in a humid chamber for 30 minutes at 37°C.
 
          xi)   Place the slides in a slide rack. Wash in Buffer I twice for 5-10 minutes each time at room temperature. Wash one time with Buffer III for 5-10 minutes.
 
          xii)   Prepare the development solution by first adding 4.5 µl NBT per 1 ml buffer III. Mix well. Then add 3.5 µl X-phosphate per ml of solution and mix well. Pipette on 500 µl per slide and incubate in a humid chamber in the dark for 2-3 hours at room temperature.
 
          xiii)   Stop the reaction by returning the slides to a slide rack and washing in Buffer IV for 15 minutes at room temperature.
 
          xiv)   Counterstain the slides by dipping for 5 minutes in 0.5% aqueous Bismarck brown Y.
 
          xv)   Dehydrate the slides in the staining centre as follows:
 
95% alcohol 3 x 10 dips each
Absolute alcohol 3 x 10 dips each
Citri Solve (or xylene) 4 x 10 dips each
Do not allow the slides to dry out - leave them in the last Citri Solve (or xylene) container until ready for cover-slips.
 

 
          xvi)   Mount with cover-slips and mounting medium (Permount).
 
          xvii)   Examine the slides under bright-field for dark-blue or black precipitate that marks sites where IHHNV DNA is present. Pathodiagnostic intranuclear Cowdry type A inclusions are well marked with probe. Also often marked are host cell nuclei without obvious inclusions, cytoplasmic inclusions, and accumulation of free virus in the tissue spaces and haemolymph.
 
          NOTE: Always run a known positive and negative control.
 
          .   1.2.1. Reagent formulas for in-situ hybridisation method
 
          i)   10 x phosphate buffered saline
 
NaCl 160 g
KH2PO4 4 g
Na2HPO4 23 g
KCl 4 g
DD H2O 1950 ml (qs to 2 litres)

 
               pH to 8.2 with NaOH; autoclave to sterilise; store at room temperature. To make 1 x PBS, dilute 100 ml 10 x PBS in 900 ml DD H2O; Filter 1 x solution through 0.45 µm filter; store at 4°C.
 
          ii)   10 x Tris/NaCl/EDTA (TNE) buffer
 
Tris base 60.57 g
NaCl 5.84 g
EDTA 3.72 g
DD H2O 900 ml (qs to 1 litre)

 
               pH to 7.4 with concentrated or 5 M HCl. To make 1 x TNE, dilute 100 ml 10 x TNE in 900 ml DD H2O; Filter 1 x solution through 0.45 µm filter; store at 4°C.
 
          iii)   Proteinase K, 100 µg/ml (prepare just prior to use)
 
PBS 10 ml 1 x PBS
Proteinase K
 
 1 mg

 
          iv)   0.4% formaldehyde
 
37% formaldehyde 5.4 ml
DD H2O 500 ml

 
               Store at 4°C; can be reused up to four times before discarding.
 
          v)   Prehybridisation buffer (50 ml final volume)
 
4 x SSC 10 ml 20 x SSC
50% formamide 25 ml 100% formamide
1 x Denhardt's 2.5 ml 20 x Denhardt's
5% dextran sulfate 10 ml 25% dextran sulfate
Warm to 60°C  

 
               Boil 2.5 ml of 10 mg/ml salmon sperm DNA and add to buffer for final concentration of 0.5 mg/ml salmon sperm DNA; store at 4°C.
 
          vi)   20 x SSC buffer
 
3M NaCl 175.32 g NaCl
0.3 M Na3C6H5O7.2H2O 88.23 g Na citrate.2H2O
DD H2O 1000 ml (qs)
pH to 7.0; autoclave; store at 4°C.

 
               To make 2 x SSC, dilute 100 ml 20 x SSC in 900 ml DD H2O; To make 1 x SSC, dilute 50 ml 20 x SSC in 950 ml DD H2O; To make 0.5 x SSC, dilute 50 ml 20 x SSC in 1950 ml DD H2O. Filter solutions through 0.45 µm filter; store at 4°C.
 
          vii)   20 x Denhardt's solution
 
BSA (Fraction V) 0.4 g bovine serum albumin
Ficoll 400 0.4 g Ficoll
PVP 360 0.4 g polyvinylpyrollidine
DD H2O 100 ml
Filter solutions through 0.45 µm filter; store at 4°C. Aliquot 2.5 ml into small tubes and store frozen.
 

 
          viii)   25% dextran sulfate
 
Dextran sulfate 25 g
DD H2O 100 ml
Mix to dissolve; store frozen in 10 ml aliquots.
 

 
          ix)   Salmon sperm DNA (10 mg/ml)
 
Salmon sperm DNA 0.25 g
DD H2O 25 ml

 
               To prepare, warm the water and slowly add the DNA with stirring until completely dissolved; boil for 10 minutes; shear the DNA by pushing through an 18-gauge needle several times; aliquot 2.5 ml into small tubes and store frozen; boil for 10 minutes just before using to facilitate mixing in the buffer.
 
          x)   10 x Buffer I
 
1 M Tris/HCl 121.1 g Tris base
1.5 M NaCl 87.7 g NaCl
DD H2O 1000 ml (qs)
pH to 7.5 with HCl. Autoclave; store at 4°C.

 
               To make 1 x Buffer I, dilute 100 ml of 10 x stock in 900 ml DD H2O. Filter through 0.45 µm filter; store at 4°C.
 
          xi)   Buffer II (blocking buffer)
 
Blocking reagent 0.25 g Blocking reagent (Roche Diagnostics 1-096-176)
Buffer I 50 ml 1x Buffer I
Store at 4°C for up to 2 weeks.
 

 
          xii)   Buffer III
 
100 mM Tris/HCl 1.21 g Tris base
100 mM NaCl 0.58 g NaCl
DD H2O 100 ml (qs)
pH to 9.5 with HCl  
Then add:  
50 mM MgCl2 1.02 g MgCl2.6H2O
Filter through 0.45 µm filter; store at 4°C.
 

 
          xiii)   10% polyvinyl alcohol (PVA)
 
Polyvinyl alcohol 10 g
DD H2O 100 ml

 
               To prepare, slowly add PVA to water while stirring on low heat. (It takes 2-3 hours for PVA to go into solution.) Dispense 10 ml per tube and store frozen at -20°C.
 
          xiv)   Development solution
 
               Mix 90 ml Buffer III with 10 ml of 10% PVA. Store at 4°C. Just prior to use, for each 1 ml of Buffer III with PVA add:
 
4.5 µl NBT 75 mg NBT/ml in 70% dimethylformamide
(Roche Diagnostics 1-383-213)
3.5 µl X-phosphate
 
 5-bromo-4-chloro-3-indoyl phosphate, toluidine salt (50 mg/ml in dimethylformamide)
(Roche Diagnostics 1-383-221)

 
          xv)   Buffer IV
 
10 mM Tris/HCl 1.21 g Tris base
1 mM EDTA 0.37 g EDTA.2H2O (disodium salt)
DD H2O 1000 ml
pH to 8.0 with HCl. Filter through 0.45 µm filter; store at 4°C.
 

 
          xvi)   0.5% Bismarck Brown Y
 
Bismarck Brown Y 2.5 g
DD H2O 500 ml

 
               Dissolve the stain in water. Filter through a Whatman No. 1 filter; store at room temperature.
 
     1.3.   Polymerase chain reaction
 
          The PCR method for IHHNV follows generally the methods outlined in Lightner (20) and is given below. Cumulative experience with the technique has led to modifications with respect to template (DNA extraction of clinical specimens), choice of primers, and volume of reaction. The primers described below were derived from the GenBank sequence AF218266 (30). The protocols for thermal cycling and the PCR reaction mix are described in Nunan et al. (31).
 
          i)   Use as template, the DNA extracted from ground tissue homogenate (TN buffer, 0.4 M NaCl, 20 mM Tris, pH 7.4) or haemolymph (collected with a small amount of 10% sodium citrate) or from tissue or haemolymph that were fixed in 95% ethanol and then dried. A control consisting of tissue or haemolymph from known negative animals should be included during the DNA extraction step. The DNA can be extracted by a variety of methods but excellent results have been obtained using kits from Roche Diagnostics (Cat. No. 1-796-828) or Qiagen (Cat. No. 51304), or reagents from Gibco Life Sciences (DNazol Cat. No. 10503-027). Spectrophotometric readings of the final DNA will indicate the purity of the DNA and the amount of total DNA extracted from the sample. Use 1-5 µl of extracted DNA per 50 µl reaction volume.
 
               Note: Homogenised tissue material or haemolymph that has not had the DNA extracted may also be used directly as template, but the concentration of virus will be lower and there may be substances present that will inhibit the PCR.
 
          ii)   The following controls should be included in every PCR assay for IHHNV: a) DNA from a known negative tissue sample; b) DNA from a known positive sample (either from tissue or haemolymph or from a plasmid clone that contains the fragment that the specific set of primers amplifies; and c) a 'no template' control.
 
          iii)   Use as primers, primers 389F and 389R, which elicit a band 389 bp in size from IHHNV-infected material, or primers 77012F and 77353R, which elicit a band 356 bp in size from IHHNV-infected material. Prepare primers at 100 ng/µl in distilled water. Keep frozen at -70°C.
 
Primer
 
 Sequence
 
 G:C ratio
 
 Temperature
 
389F
 
 5'-CGG-AAC-ACA-ACC-CGA-CTT-TA-3'
 
 50
 
 72°C
 
389R
 
 5'-GGC-CAA-GAC-CAA-AAT-ACG-AA-3'
 
 45
 
 71°C
 
77012F
 
 5'-ATC-GGT-GCA-CTA-CTC-GGA-3'
 
 50
 
 68°C
 
77353R
 
 5'-TCG-TAC-TGG-CTG-TTC-ATC-3'
 
 55
 
 63°C
 

 
               Note: The 77012/77353 primers are from the region in the genome that codes for the structural proteins, whereas the 389F/R primers are from the region that codes for nonstructural proteins. If a test result is ambiguous with one set of primers, the second set can be used for confirmation of the result from a different region of the viral genome.
 
          iv)   Use a 'hot start' method for the polymerase: if you use Applied Biosystem's AmpliTaq Gold, this involves a 5-minute step at 95°C to denature DNA prior to the primers binding and activation of the enzyme. This programme is then linked to the cycling programme (35 cycles) and an extension programme. The programme is set as follows:
 
Hot start Programme 1 5 minutes 95°C  
Linked to Programme 2 30 seconds 95°C  
    30 seconds 55°C 35 cycles
    1 minutes 72°C  
Linked to Programme 3 7 minutes 72°C  
Linked to
 
 Programme 4 4°C until off  

 
          v)   Prepare a 'master mix' consisting of water, 10 x PCR buffer, the four dNTPs, the two primers, MgCl2, AmpliTaq Gold and water (assume use of 1 µl of template; if using more, adjust water accordingly). Add mix to each tube. Use thin-walled tubes designed for PCR. Always run a positive and a negative control.
 
               'Master Mix':
 
DD H2O 32.5 µl x number of samples
10 x PCR buffer 5 µl x number of samples
10 mM dTTP 1 µl x number of samples
10 mM dATP 1 µl x number of samples
10 mM dCTP 1 µl x number of samples
10 mM dGTP 1 µl x number of samples
25 mM MgCl2 4 µl x number of samples
Forward primer (100 ng/µl) 1.5 µl x number of samples
Reverse primer (100 ng/µl) 1.5 µl x number of samples
AmpliTaq Gold
 
 0.5 µl x number of samples

 
               Vortex this solution to mix all reagents well; keep on ice.
 
               Note: The volume of the PCR reaction may be modified. Previously, the PCR reactions for IHHNV were run in 100 µl volumes, but it is not necessary to use that amount of reagents, therefore 50 µl volumes are described in this procedure. Likewise, the PCR reactions can also be run in volumes as small as 25 µl. To do this, increase or decrease the volume of the reagents accordingly.
 
          vi)   Add 49 µl Master Mix to each tube and then add 1 µl of the sample to be tested.
 
          vii)   Vortex each tube, spin quickly to bring down all liquid. If your thermal cycler does not have a heated lid to prevent condensation, then carefully overlay the top of each sample with
25-50 µl mineral oil and re-cap the tubes. Insert tubes into thermal cycler and start programme 1 ('hot start'), which is linked to cycling, extension and soak cycles.
 
          viii)   If mineral oil was used, recover samples from under the mineral oil using a pipette set at 50 µl and transfer to a fresh tube. Using the long-tipped pipette tips (designed for loading gels) results in less oil being carried over with the sample.
 
          ix)   Run 10 µl of the sample in a 1.5% agarose gel (containing 0.5 µg/ml ethidium bromide to stain the DNA). Look for the 389 bp band (if using primers 389F and 389R) or for the 356 bp band (if using primers 77012F and 77353R). Bands are not always seen, as it is necessary to have at least 10 ng DNA/µl to see DNA in a gel. A Southern transfer of the gel or a dot-blot can be run for more sensitive detection. The DNA can also be precipitated (0.3 M sodium acetate and 2.5 volumes 100% ethanol, -70°C, for 1-3 hours, centrifuge for 20 minutes) and resuspended in 1/10th volume (i.e. 4 µl) TE (10mM Tris, 1 mM EDTA, pH7.5) or water and either re-run in the gel or tested in a dot-blot.
 
2.   Diagnostic Methods for Confirmatory Tests
 
     2.1.   Gross signs
 
          .   2.1.1. IHHN disease in Penaeus stylirostris
 
               IHHNV often causes an acute disease with very high mortalities in juveniles of the species. Vertically infected larvae and early postlarvae do not become diseased, but in approximately 35-day old or older juveniles, gross signs of the disease may be observed, followed by mass mortalities (1, 2, 8, 9, 17-20, 22, 25, 26). In horizontally infected juveniles, the incubation period and severity of the disease is somewhat size and/or age dependent, with young juveniles always being the most severely affected. Infected adults seldom show signs of the disease or mortalities (1, 2).
 
               Gross signs are not IHHN specific, but juvenile P. stylirostris with acute IHHN show a marked reduction in food consumption, followed by changes in behaviour and appearance. Shrimp of this species with acute IHHN have been observed to rise slowly in culture tanks to the water surface, there to become motionless and then to roll-over and slowly sink (ventral side up) to the tank bottom. Shrimp exhibiting this behaviour may repeat the process for several hours until they become too weak to continue, or until they are attacked and cannibalised by their healthier siblings. Penaeus stylirostris at this stage of infection often have white or buff-coloured spots (which differ in appearance and location from the white spots that sometimes occur in shrimp with white spot syndrome virus infections) in the cuticular epidermis, especially at the junction of the tergal plates of the abdomen, giving such shrimp a mottled appearance. This mottling later fades in P. stylirostris. In P. stylirostris and in P. monodon with IHHN, moribund shrimp are often distinctly bluish in colour, with opaque abdominal musculature (17-20, 22).
 
          .   2.1.2. IHHN disease in Penaeus vannamei
 
               The chronic disease, runt deformity syndrome (RDS), occurs in this species as a result of IHHNV infection. The severity and prevalence of RDS in infected populations of juvenile or older P. vannamei may be related to infection during the larval or early postlarval stages (7, 8-13, 16-20, 22). RDS has also been reported in cultured stocks of P. stylirostris. Juvenile shrimp with RDS display bent or deformed rostrums, wrinkled antennal flagella, cuticular roughness, and other cuticular deformities. Populations of juvenile shrimp with RDS display a relatively wide distribution of sizes with many smaller than expected ('runted') shrimp. The coefficient of variation (CV = the standard deviation divided by the mean of different size groups within a population) for populations with RDS is typically greater than 30% and may approach 90%, while IHHNVfree (and thus RDS-free) populations of juvenile P. vannamei and P. stylirostris usually show CVs of 10-30% (7, 10, 11, 13, 16, 20, 22).
 
     2.2.   Histological method
 
          Histological demonstration of prominent intranuclear, Cowdry type A inclusion bodies provides a provisional diagnosis of IHHNV infection. These characteristic IHHN inclusion bodies are eosinophilic and often haloed (with haematoxylin and eosin stains of tissues preserved with fixatives that contain acetic acid, such as Davidson's AFA and Bouin's solution), intranuclear inclusion bodies within chromatin-marginated, hypertrophied nuclei of cells in tissues of ectodermal (epidermis, hypodermal epithelium of fore- and hindgut, nerve cord and nerve ganglia) and mesodermal origin (haematopoietic organs, antennal gland, gonads, lymphoid organ, and connective tissue). Intranuclear inclusion bodies due to IHHNV may be easily confused with developing intranuclear inclusion bodies due to WSSV infection. In-situ hybridisation assay of such sections with a specific DNA probe to IHHNV provides a definitive diagnosis of IHHNV infection (see Section 1.2.) (3, 15, 20).
 
     2.3.   Enhancement of infection
 
          The prevalence and severity of IHHNV infections may be 'enhanced' in a quarantined population by rearing shrimps in relatively crowded or stressful conditions. The 'crowding stress' factors may include high stocking densities and marginal water quality (i.e. low dissolved oxygen, elevated water temperature, or elevated ammonia or nitrite) in the holding tank water. These conditions may encourage expression of low-grade IHHNV infections and the transmission of the agent from carriers to previously uninfected hosts in the population resulting in increased prevalence and severity of infections that can be more easily detected using the available diagnostic and detection methods for IHHNV (20).
 
     2.4.   Molecular methods
 
          See Sections 1.1.-1.3.
 
          Real-time PCR methods have been developed for the detection of IHHNV. These methods offer extraordinary sensitivity that can detect a single copy of the target sequence from the IHHNV genome (14, 35). While providing extraordinary sensitivity and the promise of being the most appropriate PCR technology for pathogen detection, widespread adoption of these methods by diagnostic facilities that service the international shrimp culture and related industries is not likely to occur in the near future. The methods, which are evolving rapidly, require highly trained technicians, costly reagents, and very expensive equipment. Some very costly real-time thermal cyclers placed on the market only a few years ago are deemed to be obsolete by their manufactures and, therefore, the sale and service of these instruments have been discontinued. These instruments have been replaced by the next generation of instruments that employ the newest technology.
 

REFERENCES

1.   Bell T.A. & Lightner D.V. (1984). IHHN virus: Infectivity and pathogenicity studies in Penaeus stylirostris and Penaeus vannamei. Aquaculture, 38, 185-194.
 
2.   Bell T.A. & Lightner D.V. (1987). IHHN disease of Penaeus stylirostris: effects of shrimp size on disease expression. J. Fish Dis., 10, 165-170.
 
3.   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.
 
4.   Bell T.A., Lightner D.V. & Brock J.A. (1990). A biopsy procedure for the non-destructive determination of IHHN virus infection in Penaeus vannamei. J. Aquat. Anim. Health, 2, 151-153.
 
5.   Bonami J.R., Brehelin M., Mari J., Trumper B. & Lightner D.V. (1990). Purification and characterization of IHHN virus of penaeid shrimps. J. Gen. Virol., 71, 2657-2664.
 
6.   Bonami J.R. & Lightner D.V. (1991). Chapter 24. Unclassified Viruses of Crustacea. In: Atlas of Invertebrate Viruses, Adams J.R. & Bonami J.R., eds. CRC Press, Boca Raton, Florida, USA, 597-622.
 
7.   Bray W.A., Lawrence A.L. & Leung-Trujillo J.R. (1994). The effect of salinity on growth and survival of Penaeus vannamei, with observations on the interaction of IHHN virus and salinity. Aquaculture, 122, 133-146.
 
8.   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.
 
9.   Brock J.A., Lightner D.V. & Bell T.A. (1983). A review of four virus (BP, MBV, BMN, and IHHNV) diseases of penaeid shrimp with particular reference to clinical significance, diagnosis and control in shrimp aquaculture. Proceedings of the 71st International. Council for the Exploration of the Sea, C.M. 1983/Gen:10/1-18.
 
10.   Brock J.A. & Main K. (1994). A Guide to the Common Problems and Diseases of Cultured Penaeus vannamei. Oceanic Institute, Makapuu Point, P.O. Box 25280, Honolulu, Hawaii, USA, 241 pp.
 
11.   Browdy C.L., Holloway J.D., King C.O., Stokes A.D., Hopkins J.S. & Sandifer P.A. (1993). IHHN virus and intensive culture of Penaeus vannamei: effects of stocking density and water exchange rates. J. Crustacean Biol., 13, 87-94.
 
12.   Carr W.H., Sweeney J.N., Nunan L., Lightner D.V., Hirsch H.H. & Reddington J.J. (1996). The use of an infectious hypodermal and hematopoietic necrosis virus gene probe serodiagnostic field kit for the screening of candidate specific pathogen-free Penaeus vannamei broodstock. Aquaculture, 147, 1-8.
 
13.   Castille F.L., Samocha T.M., Lawrence A.L., He H., Frelier P. & Jaenike F. (1993). Variability in growth and survival of early postlarval shrimp (Penaeus vannamei Boone 1931). Aquaculture, 113, 65-81.
 
14.   Dhar A.K., Roux M.M. & Klimpel K.R. (2001). Detection and quantification of infectious hypodermal and hematopoeitic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR green chemistry. J. Clin. Microbiol., 39, 2835-2845.
 
15.   Kalagayan G., Godin D., Kanna R., Hagino G., Sweeney J., Wyban J. & Brock J. (1991). IHHN virus as an etiological factor in runt-deformity syndrome of juvenile Penaeus vannamei cultured in Hawaii. J. World Aquaculture Soc., 22, 235-243.
 
16.   Lightner D.V. (1983). Diseases of Cultured Penaeid Shrimp. pp. In: CRC Handbook of Mariculture. Vol. 1. Crustacean Aquaculture, McVey J.P., ed. CRC Press, Boca Raton, Florida, USA, 289-320.
 
17.   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.
 
18.   Lightner D.V. (1993). Diseases of penaeid shrimp. In: CRC Handbook of Mariculture: Crustacean Aquaculture, McVey J.P., ed. CRC Press, Boca Raton, Florida, USA.
 
19.   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.
 
20.   Lightner D.V. (1996). The penaeid shrimp viruses IHHNV and TSV: epizootiology, production impacts and role of international trade in their distribution in the Americas. Rev. sci. tech. Off. int. Epiz., 15, 579-601.
 
21.   Lightner D.V., Bell T.A., Redman R.M. & Perez L.A. (1992). A collection of case histories documenting the introduction and spread of the virus disease IHHN in penaeid shrimp culture facilities in Northwestern Mexico. ICES Marine Science Symposia, 194, 97-105.
 
22.   Lightner D.V., Mohney L.L., Williams R.R. & Redman R.M. (1987). Glycerol tolerance of IHHN virus of penaeid shrimp. J. World Aquaculture. Soc., 18, 196-197.
 
23.   Lightner D.V., Poulos B.T., Bruce L., Redman R.M., Mari J. & Bonami J.R. (1992). New developments in penaeid virology: application of biotechnology in research and disease diagnosis for shrimp viruses of concern in the Americas. 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, 233-253.
 
24.   Lightner D.V., Redman R.M. & Bell T.A. (1983). Infectious hypodermal and hematopoietic necrosis a newly recognized virus disease of penaeid shrimp. J. Invertebr. Pathol., 42, 62-70.
 
25.   Lightner D.V., Redman R.M., Bell T.A. & Brock J.A. (1983). Detection of IHHN virus in Penaeus stylirostris and P. vannamei imported into Hawaii. J. World Mariculture Soc., 14, 212-225.
 
26.   Mari J., Bonami J.R. & Lightner D.V. (1993). Partial cloning of the genome of infectious hypodermal and hematopoietic necrosis virus, an unusual parvovirus pathogenic for penaeid shrimps; diagnosis of the disease using a specific probe. J. Gen. Virol., 74, 2637-2643.
 
27.   Martinez-Cordova L.R. (1992). Cultured blue shrimp (Penaeus stylirostris) infected with infectious hypodermal and hematopoietic necrosis virus in northwestern Mexico. The Progressive Fish Culturist, 54, 265-266.
 
28.   Morales-Covarrubias M.S. & Chavez-Sanchez M.C. (1999). Histopathological studies on wild broodstock of white shrimp Penaeus vannamei in the Platanitos area, adjacent to San Blas, Nayarit, Mexico. J. World Aquaculture Soc., 30, 192-200.
 
29.   Morales-Covarrubias M.S., Nunan L.M., Lightner D.V., Mota-Urbina J.C., Garza-Aguirre M.C. & Chavez-Sanchez M.C. (1999). Prevalence of IHHNV in wild broodstock of Penaeus stylirostris from the upper Gulf of California, Mexico. J. Aquat. Anim. Health, 11, 296-301.
 
30.   Nunan L.M., Arce S.M., Staha R.J. & Lightner D.V. (2001). Prevalence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) in Litopenaeus vannamei in the Pacific Ocean off the coast of Panama. J. World Aquaculture Soc., 32, 330-334.
 
31.   Nunan L.M., Poulos B.T. & Lightner D.V. (2000). Use of polymerase chain reaction (PCR) for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. Mar. Biotechnol., 2, 319-328.
 
32.   Owens L., Anderson I.G., Kenway M., Trott L. & Benzie J.A.H. (1992). Infectious hypodermal and hematopoietic necrosis virus (IHHNV) in a hybrid penaeid prawn from tropical Australia. Dis. Aquat. Org., 14, 219-228.
 
33.   Pantoja C.R., Lightner D.V. & Holtschmit K.H. (1999). Prevalence and geographic distribution of IHHN parvovirus in wild penaeid shrimp (Crustacea: Decapoda) from the Gulf of California, Mexico. J. Aquat. Anim. Health, 11, 23-34.
 
34.   Tang K.F.J., Durand S.V., White B.L., Redman R.M., Pantoja C.R. & Lightner D.V. (2000). Postlarvae and juveniles of a selected line of Penaeus stylirostris are resistant to infectious hypodermal and hematopoietic necrosis virus infection. Aquaculture, 190, 203-210.
 
35.   Tang K.F.J. & Lightner D.V. (2001). Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR. Dis. Aquat. Org., 44, 79-85.
 
36.   Tang K.F.J. & Lightner D.V. (In press). Low sequence variation among isolates of infectious hypodermal and hematopoietic necrosis virus (IHHNV) originating from Hawaii and the Americas. Dis. Aquat. Org.,
 
37.   Tang K.F.J. & Lightner D.V. (2002). High genetic variation among isolates of infectious hypodermal and hematopoietic necrosis virus (IHHNV) collected from southeast Asia, Madagascar and east Africa. Book of Abstracts, Aquaculture America 2002. World Aquaculture Society, Baton Rouge, LA, USA. p. 328.
 

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