Download Evidence of existence of infectious hypodermal and hematopoietic

Evidence of existence of infectious hypodermal and hematopoietic
necrosis virus in penaeid shrimp cultured in China
Yang, B., Song, X.-L., Huang, J*, Shi, C.-Y., Liu, L.
Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture,
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, PR China
Abstract
Infectious hypodermal and hematopoietic necrosis virus is the causative agent of a shrimp disease which causes
economic losses in global industry. A pair of primers, I2814F/I3516R, was designed from the IHHNV genomic
sequence AF218266 (GenBank) that encodes for structural protein corresponding to nucleotides 2814—3516, which
amplifies a 703 base pair (bp) region from the virus genome. PCR amplification with the primers generated a product of
the expected size from the purified IHHNV DNA of Litopenaeus vannamei and IHHNV-infected penaeid populations
but not from the IHHNV-free shrimp, white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV). The
PCR amplicon described above was labeled with digoxigenin (DIG)-11-dUTP as a probe used for dot blot hybridization
and in situ hybridization test. Under the optimized PCR conditions, the primers were detected as little as 20 fg of
purified IHHNV DNA, which contained only 8.83×103 copies of IHHNV, 1000 fold greater than using dot blot
hybridization, which provides the greatest sensitivity for detection of IHHNV. Sections of histopathology showed
eosinophilic intranuclear inclusions (Cowdry type A inclusions or CAIs) in infected tissues while In situ hybridization,
cells displayed the intense reaction with the DIG-labeled probe. PCR assay was developed to detect IHHNV in
penaeid shrimp and other crustaceans from the rearing ponds of China (March 2001-June 2004). The positive rate was
51.5% (154 out of 299 penaeid shrimp samples) and 8.3% (2 out of 24 crab samples) respectively. The survey
demonstrated that the presence of IHHNV in China.
Keywords: IHHNV; Litopenaeus vannamei; PCR; in situ hybridization; Dot blot hybridization; Histopathology
Introduction
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the major causes of diseases in cultured
penaeid shrimp. It was first found in juvenile Litopenaeus stylirostris in Hawaii (Lightner et al., 1983a). IHHNV is a
small, icosahedral, non-enveloped virus containing a single-stranded linear DNA genome of approximately 4.1kb in
length (Bonami et al., 1990; Mari et al., 1993). It is considered to be a member of the Parvoviridae family (Bonami et
al., 1990) according to its morphology and biochemical structure and has been recently closely related to Mosquito
Brevidensoviruses (Shike et al., 2000).
Infection with IHHNV does not produce any pathognomonic gross clinical signs (Lightner et al., 1983a), although
runt-deformity syndrome (RDS) has been noted in infected juvenile Litopenaeus vannamei and Penaeus monodon(Bell
and Lightner, 1984; Kalagayan et al., 1991; Primavera and Quinitio, 2000). IHHNV has ever been reported in ovarian
tissues and fertilized egg of Fenneropenaeus chinensis (Zhang and Sun, 1997). The economic losses impacting on L.
vannamei is that range between 10% and 50% by comparison with IHHNV-free crops (Lightner and Redman, 1998). In
farms, horizontal and vertical transmission may transmit the IHHNV through ingestion of infected dead individuals and
infected broodstocks (Bell and Lightner, 1984; Lotz, 1997).
Commonly recently L. vannamei becomes one of the major cultured species of aquaculture industry in China since the
white spot disease outbroke in 1993. With importing broodstocks and postlarvae of animals from the countries which
have the relative populations of the penaeid shrimp into China, it is important, first to screen IHHNV free populations.
In situ hybridization method, PCR, nested PCR and real-time PCR analysis provide the highest available detection
sensitivity for IHHNV (Lightner et al., 1994; Motte et al., 2003., Nunan et al., 2000; Tang and Lightner, 2001).
Infectious hypodermal and hematopoietic necrosis virus gene probe serodiagnostic field kit was used for screening of
candidate specific pathogen-free L. vannamei broodstock (Carr et al., 1996). However, there is no more detection
methods reported about this disease in China. The aim of this study was to evaluate the diagnostic method described
above for screening IHHNV-free animals and survey the existence of IHHNV in cultured populations of penaeid shrimp
in China, although it is being reported here for the first time.
Materials and methods
Sample preparation for PCR analysis
* Corresponding author. Mailing address: 106 Nanjing Road, Qingdao, Shandong 266071, PR China. Tel: +86 532-85823062. Fax:
+86 532-85811514, E-mail: [email protected]
Proceedings of the 11th International Symposium on Veterinary Epidemiology and Economics, 2006
Available at www.sciquest.org.nz
L. vannamei broodstock samples (10 out of 32 broodstock population) and adult samples (15 out of 75 adult population)
were collected from Hainan province of south of China in 2001. Postlarval L. vannamei, F. chinensis, juvenile L.
vannamei, adult L. vannamei, F. chinensis, Marsupenaeus japonicus, broodstock L. vannamei, L. stylirostris, Artemia,
Nereidae, Rotatoria and crab (Hemigrapsus penicillatus) were obtained from four shrimp farming ponds of northern
China between 2001 and 2004.
IHHNV-infected penaeid shrimp samples were postlarvaes of L.vannamei from north of China whose size was 0.7 cm.
The samples were alive and appeared to be clinically normal.
IHHNV virions were purified according to a protocol developed by Bonami et al (Bonami et al., 1990).
For assay of PL, whole shrimp was used. For juvenile shrimp, the head was removed. For adult and broodstock, the gills
were used. One penaeid shrimp and other crustaceans were considered as a separate sample for the PCR analysis and
was processed separately using disposable equipment to avoid cross-contamination. The total DNA was extracted from
the tissues with a high pure PCR template preparation kit (Roche Molecular Biochemicals). During the procedure of
DNA extraction, it has to be careful to exclude shrimp eyes from samples, as these are known to contain a PCR
inhibitor.
PCR amplification for IHHNV diagnosis
The PCR Primers were selected by the published IHHNV genomic sequence (accession No. AF218266, GenBank) that
encodes for structural protein corresponding to nucleotides 2814—3516. Primers were designed using Prime Premier
5.0 software (PREMIER Biosoft International). The upstream (I 2814F) and downstream (I 3516R) primer sequences
were 5'- TAA TGA AGA CGA AGA ACA CGC CGA AGG -3' and 5'- TGG GTA GAC TAG GTT TCC AAG GGA
TGG TT -3', respectively which were used to amplify a 703 bp fragment and synthesized by biosynthesis. The PCR was
carried out in PCR express (Thermo Hybaid), and in a 25 µl reaction containing 1× PCR buffer, 200 µM each of dATP,
dTTP, dGTP, and dCTP. 2.5 U of Taq DNA polymerase (Promega), 200 pg of purified IHHNV DNA from L. vannamei
template and double distilled water to adjust the final volume to 25µl. Using a “hot start” before adding polymerase that
is for 10 min at 94°C, 5 min at 55°C. The optimal conditions were tested by varying the primer concentration (9.375,
18.75, 37.5, 75 and 150 ng), MgCl2 concentration (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mM), annealing temperature (50, 55, 57,
60 and 65°C), PCR cycle number (20, 25, 30, 35 and 40 cycles), with a final extension at 72°C for 5 min, followed by a
4°C incubation until they could be retrieved from thermal cycler. A 10-µl reaction mixture of each tested sample were
analyzed using 1.2% agarose gel electrophoresis. Identification of a 703 bp band in the gel was a evidence of the
presence of IHHNV in the sample. Furthermore, for comparative purposes, the optimized PCR conditions were
evaluated.
Specificity for detection of IHHNV DNA was determined using optimized PCR conditions with different sources of
template DNA including total tissue DNA from IHHNV-free shrimp L. vannamei (250 ng), white spot syndrome virus
DNA (WSSV DNA, 375 ng), hepatopancreatic parvovirus DNA (HPV DNA, 1 µg) and the total tissue DNA (213 ng)
from IHHNV-infected shrimp L. vannamei (1 µl to 25 µl reaction of PCR). Control positive (purified IHHNV DNA)
and negative (double distilled water) were also included. For electrophoresis, 10µl of each PCR mixture was run
simultaneously and amplified fragments were observed in agarose gel (1.2%) stained with Ethidium bromide under UV
light. To determine sensitivity, purified IHHNV DNA was 10-fold serially diluted (20 pg to 0.2 fg), used as the DNA
template for PCR and the product were detected as described above.
Dot blot and in situ hybridization
The gene probe I703 for dot-blot and in situ hybridization was prepared using the “PCR Dig Labeling Mix” (Roche
molecular biochemicals ref. 1585550). The reaction mixture contained IHHNV DNA (20 pg/µl), Digoxigenin-11-dUTP
and other necessary components required by optimized PCR conditions. The labeled PCR product was then purified
with a gel extraction mini kit (Watson Biotechnologies, Inc). The labeled DNA was resuspended in TE buffer and stored
at -20°C until used.
The procedures used for dot blot analysis were as follows. DNA solutions of all the sources described above, TE buffer
and shrimp pond water were denatured by boiling for 10 min in 0.2 ml Eppendorf® tubes and transferred to ice for 5
min. A 1-µl volume of each sample was applied to a nitrocellulose membrane by using a 1.0 µl capillary tube pipet. The
membrane was air-dried and then baked at 80°C for 2 hr. DIG-labeled probe I703 prepared as described above was
diluted in hybridization buffer to a concentration of 20 ng ml-1 and incubated with membrane overnight at 65°C for
hybridization. After reacting with anti-digoxigenin antibodies conjugated with the enzyme alkaline phosphatase and the
steps of washing in Buffer I, the hybridized membrane was then maintained in development solution including the
reagents NBT (nitroblue tetrazolium) and BCIP (5-bromo-4-chloro-3-indoyl phosphate) at dark, room temperature for
1hr, and then we record the results
Proceedings of the 11th International Symposium on Veterinary Epidemiology and Economics, 2006
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For in situ hybridization procedure, the whole postlarval shrimp samples included IHHNV-infected and IHHNV-free
shrimp of L. vannamei and F. chinensis from northern China whose size were 0.7 cm. The samples were alive and fixed
in Davidson’s AFA fixative (Bell and Lightner, 1984). The embedded samples were cut to 5 µm thickness. The samples
of sections subjected to in situ hybridization with digoxigenin-labeled IHHNV-specific gene probe I703. In situ
hybridization was performed according to the protocol of Lightner (1996)(Lightner, 1996). The hybridized sections
were examined by light microscope at 100× magnification. Control slides positive and negative for IHHNV were also
included.
Southern blot hybridization
Southern blot hybridization was performed to localize the 703 bp PCR product within the genomic of purified IHHNV
DNA from the diseased L. vannamei. It can also evaluate the specificity and sensitivity of the DIG-labeled probe I703.
For this purpose, The purified IHHNV DNA which was 10-fold serially diluted (20 pg to 0.2 fg), used as the DNA
template for PCR was depurinated by soaking gel in 0.25 M HCl after the electrophoresis, neutralized in 1.5 M
NaCl/0.5 M NaOH, and neutralized in 1 M Tris (pH 7.5)/1.5 M NaCl. Then subsequently transferred to a nitrocellulose
membrane in the buffer of 10×SSC overnight. The membrane was then used for hybridization with DIG-labeled 703bp
PCR product and the procedure was the same as dot blot hybridization.
Histopathology
The same paraffin blocks to be examined by in situ hybridization were consecutively sliced and processed for
preparation of standard H&E stained tissue sections as described by Bell & Lightner (Bell and Lightner, 1988). Then
they were examined the stained sections by light microscope at 100× magnification.
Results
PCR amplification and analysis of the product
The optimum PCR conditions were carried out containing 1× PCR buffer, 200 µM each of dATP, dTTP, dGTP and
dCTP. 2.5 U of Taq DNA polymerase, 75 ng of each primer (2814F and 3516R), 20 pg of IHHNV DNA template and
double distilled water to adjust the final volume to 25 µl. Using a “hot start” before adding polymerase for 10 min at
94°C, 5 min at 55°C. The optimal conditions were as follows, 1 min at 94°C, 1 min at 57°C, 1 min at 72°C (40 cycles),
then extended at 72°C for 5 min, followed by a 4°C incubation.
Fig. 1. (A) PCR amplification of 10-fold serially diluted purified IHHNV DNA from Litopenaeus
vannamei using the primer I2814F and I3516R which yield a 703bp PCR product.
M: DL2,000 DNA ladder. Lane 1 to 6: purified IHHNV DNA at concentrations of 20, 2.0 pg and 200, 20, 2.0, 0.2 fg, blank control
respectively. The amplicon at 20 fg of IHHNV DNA is not distinctly visible in the photograph but could be seen in the actual gel. (B)
Southern blot hybridization analysis of agarose gel from (A) hybridized with DIG-labeled \ probe I703.
Under the optimized PCR conditions, a single product of the expected size 703 bp was detected. No amplification
fragment at 703 bp was present for IHHNV-free shrimp of L. vannamei, WSSV DNA and HPV DNA. The lowest
amount of the purified IHHNV DNA template that could be detected was 20 fg, which contained only 8.83×103 copies
Proceedings of the 11th International Symposium on Veterinary Epidemiology and Economics, 2006
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of IHHNV (Fig. 1). By Southern blot hybridization, the 703 bp amplicon could be detected by DIG-labeled probe I703
at 0.2 fg, 100 times lower concentration than it could be visualized in the agarose gel.
Histopathology
Sections of histopathology showed eosinophilic intranuclear inclusions (Cowdry type A inclusions or CAIs) that are
pathognomonic for IHHNV infections in connective tissues. Mayer-Bennett H&E. (Fig. 3) Severity of infection grade
was grade 2 according to the degree of target tissue and organ damage (Lightner et al, 1983b).
Dot blot and in situ hybridization
The result of dot blot hybridization demonstrated that the probe produced by PCR labeling hybridized with IHHNV
DNA and gave an intense signal. The limit of detection was 25 pg to purified IHHNV DNA from L. vannamei, 27 ng to
total tissue DNA from IHHNV-infected shrimp of L. vannamei, but could not react with total tissue DNA from
IHHNV-free shrimp of L. vannamei, WSSV DNA and HPV DNA (Fig. 2).
Fig. 2. Dot blot hybridization of IHHNV DNA and the samples using DIG-labeled probe I703 on a
nitrocellulose membrane.
A1 to A9: 2-fold serially diluted purified IHHNV DNA from Litopenaeus vannamei(2.0×102 pg to 8.0×10-1 pg, respectively ); B1 to
B9: 2-fold serially diluted total tissue DNA(2.0×102ng to 2.0×10-1ng, respectively) from IHHNV-infected shrimp of L. vannamei; C1
to C9: 2-fold serially diluted DNA(2.5×102ng to 1.0ng, respectively) from IHHNV-free L. vannamei; D1 to D9: supernatant of
IHHNV-free shrimp in TE buffer; E1: DNA extracted from white spot syndrome virus (375 ng); E2: DNA extracted from
hepatopancreatic parvovirus (1 µg); E3: TE buffer; E4:distilled water; E5:shrimp pond water; E6 to E9: Blank of control
Fig. 3. Histological detection and in situ hybridization of IHHNV-infected Litopenaeus vannamei.
H&E staining of connective tissue (A, B), pathognomonic eosinophilic Cowdry A (CAI) intranuclear inclusion body (arrow). In situ
detection with DIG-labeled probe I703 in gill (C) and epithelium (D), displaying CAIs with a strong positive reaction to IHHNV
probe in cells of gill (arrow) and in the numerous cells of epithelium. Bar = 10µm
The in situ hybridization was used to confirm IHHNV infection and the result strongly showed positive reaction in all of
the target tissues for IHHNV. In gills and epidermis, the sections showed in Fig. 3 that several IHHNV infected cell
nuclei have reacted with the DIG labeled probe I703. Viral DNA is stained dark blue to black by the detection reaction
for DIG-labeled probe & Bismarck Brown. Furthermore, the in situ hybridization analysis detections showed the
Proceedings of the 11th International Symposium on Veterinary Epidemiology and Economics, 2006
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presence of IHHNV in tissues where conventional H&E analysis did not show evidence of infection. No reaction to the
probe was observed in the sections of IHHNV-free shrimp of L. vannamei and F. chinensis.
Detection of IHHNV in samples by PCR
An epidemiological survey was performed from 2001-2004 along the Chinese coast (Table 1) with a total of 299
cultivated penaeid shrimps, 24 wild crabs and 106 other crustaceans (artemia, nereidae rotatoria) from the rearing ponds
analyzed for IHHNV detection based on 1-step PCR. In total, 154 out of 299 (51.5%) penaeid shrimp samples and 2 out
of 24 (8.3%) crab samples were positive for the target virus. In case of shrimps, IHHNV prevalence was around 59.5%
for L. vannamei. There were no F. chinensis, M. japonicus, L. stylirostris and other crustaceans (artemia, nereidae
rotatoria) found to be positive, suggesting a low prevalence. On the basis of these results, it suggests that infections
occurred mainly in L. vannamei and crab may play an important role in the horizontal transmission of IHHNV.
Table 1. Prevalence of IHHNV (as determined by PCR) in Penaeid shrimp and other crustaceans from
the rearing ponds of China (March 2001-June 2004).
Year
2001
Sampling station
South of China
North of China
2002
North of China
2003
North of China
2004
North of China
Species
L. vannamei broodstock
Total number analyzed
10
Total IHHNV positive
0
Prevalence (%)
0
L. vannamei adult
15
5
33
L. vannamei broodstock
L. stylirostris broodstock
12
10
0
0
0
0
F. chinensis adult
10
0
0
15, respectively
40
36
7, respectively
26
40
10
10, respectively
24
80
10
10
0
20
32
0
12
10
0
0
2
75
0
0
0
50
89
0
46
25
0
0
8
94
0
0
Artemia, Nereidae, Rotatoria
L. vannamei postlarvae
L. vannamei juvenile
Artemia, Nereidae, Rotatoria
L. vannamei postlarvae
L. vannamei adult
F. chinensis postlarvae
Artemia, Nereidae, Rotatoria
Crab(Hemigrapsus penicillatus)
L. vannamei postlarvae
M. japonicus adult
Artemia
Discussion
Infectious hypodermal and hematopoietic necrosis virus is one of the highly pathogenic viruses of Penaeid shrimp and
has been studied since its discovery in the early 1980s. More and more methods of diagnosis are established to
inspect the prevalence of the disease and screen the SPF individuals in cultured shrimp. The vertical transmission of
IHHNV through female L. vannamei was confirmed by analyzing embryos and larvae produced, respectively, by
IHHNV-infected and IHHNV-free females fertilized by IHHNV-free males. The embryos and larvae produced from
IHHNV-free females were free of virus whereas embryos and larvae produced from IHHNV-infected females were also
found to be infected through the nested-PCR analysis (Motte et al., 2003). It is necessary to detect the IHHNV for the
cultural system and health certification of SPF status. This especially showed great significance in the program of
shrimp culture in China. The study of this paper is the first report with the relative research about that country.
The specificity of the primers I2814F and I3516R, designed from the IHHNV genomic sequence AF218266 (GenBank)
was illustrated by absence of amplification from related IHHNV or non-related WSSV and HPV viruses. The specificity
of these primers was further validated by the results of the dot blot and in situ hybridization assay performed with the
probe prepared with the products amplified from purified IHHNV DNA of L. vannamei. At dilutions of 10-4 of the
original template of IHHNV DNA was sufficient for detection by PCR. The results showed that under the conditions
established for this assay, the I2814F and I3516R primers were capable of detecting as little as 20 fg of purified IHHNV
DNA which contained only 8.83×103 copies of IHHNV, more than 1000 times lower concentration than what they
could be visualized by using dot blot hybridization which provides the greatest sensitivity for detection of IHHNV. The
light infection of the animals would not be detected by using dot blot hybridization and so the method is not suitable for
screening the carrirs with low viral loads. Furthermore PCR is also used to detect the IHHNV of other marine animals
that carry the virus (Shi et al., 2003). Different PCR assays would offer different levels of sensitivity. The relevant
studies have been reported previously, e.g. PCR method for the detection of IHHNV (Nunan et al., 2000) detection and
quantification of IHHNV by real-time PCR (Tang and Lightner, 2001), detection and quantification of IHHNV and
Proceedings of the 11th International Symposium on Veterinary Epidemiology and Economics, 2006
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WSSV using real-time PCR and SYBR green chemistry(Dhar et al., 2001). The described assays are about a 1000-fold
greater sensitivity than we described here. However, from limit of our result, it is evident that IHHNV has already
presented in China.
In situ hybridization, as the principal diagnostic histological characteristic of IHHNV infection, CAIs were strongly
recognized by the probe I703 in nucleus of many cell types in IHHNV-infected shrimp and provided the greatest
diagnostic certainty available for this agent. Additionally, from the comparison between in situ hybridization and
histopathology, the tissue where in situ hybridization test showed obvious presence of IHHNV while no evidence of
infection by the virus could be observed by conventional H&E histology. This result suggests that in early or low-level
chronic infections, there is no CAIs formated in some tissue, and they may escape the detection of H&E analysis.
Recent work suggests that there is sufficient sequence variation among geographical isolations from diverse locations in
Asia, thus to suggest that multiple geographical strains of the virus exist (Tang and Lightner, 2002). It is similar to
Hepatopancreatic parvorirus (HPV) of shrimp. The DNA sequnce of HPV from Thai shrimp Litopenaeus monodon
(HPVmon) differs from HPV of F. chinensis (HPVchin) by approximate 30%. Phromjai (Phromjai et al., 2002) gave a
detection research on HPV between the two species. The primers designed in this paper is sensitive to IHHNV from L.
vannamei and there is no evidence to verify whether it also be suitable to IHHNV from other species of penaeid shrimp.
Over recent years, more and more disease organisms which were believed to be absent from China have been
discovered here. Previously, there had no studies on IHHNV prevalence in China. The preliminary investigations in our
laboratory indicated that almost 51.5% shrimp and 8.3% crabs which collected from the culture areas of China during
2001-2004 found to be IHHNV-infected after one-step PCR diagnosis. This, however, is an estimation of point
prevalence and there could be temporal and spatial variations. These results may get from the higher prevalence under
2-step or real-time PCR conditions. These finding suggests the presence of IHHNV in China. It may be assumed that
the commercial trades of broodstock and PL shrimp, the latent carriers or lightly infected populations, transfer and
spread IHHNV. However, more research will be needed to determine the geographic distribution, species of host and to
evaluate the damage of its prevalence.
Acknowledgements
This work was supported by the project State Major Basic Research Development Program (Grant 1999012002),
National Nature Science Foundation of China (Grant 30271020), Municipal Nature Science Foundation of Qingdao
(Grant 03-2-JZP-10) and the Scientific Activity Foundation for Homewards Talent of the Ministry of Personnel. The
authors express their appreciation to Dr. Xiao-pei Wu for her careful review for the manuscript.
References
Bell, T.A., Lightner, D.V., (1988). A Handbook of Normal Penaeid Shrimp Histology. World Aquaculture Society.
Bell, T.A., Lightner, D.V., (1984). IHHN virus: infectivity and pathogenicity studies in Penaeus styliostris and Penaeus
vannamei. Aquaculture 38, 185-194.
Bonami, J.R., Trumper, B., Mari, J., Brehélin, M., Lightner, D.V., (1990). Purification and characterization of the
infectious hypodermal and haematopoietic necrosis virus of penaeid shrimps. J. Gen. Virol. 71, 2637-2664.
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.
Dhar, A.K., Roux, M.M., Klimpel, K.R., (2001). Detection and quantification of infectious hypodermal and
hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR green
chemistry. J. Clin. Microbiol. 39(8), 2835-2845.6
Kalagayan, H., Godin, D., Kanna, R., Hagino, G., Sweeney, J., Wyban, J., (1991). IHHN virus as an etiological factors
in Runt-Deformity Syndrome (RDS) of juvenile Penaeus vannamei cultured in Hawaii. J. World Aquac. Soc. 22,
235-243.
Lightner, D.V., Redman, R.M., Bell, T.A., (1983a). Infectious hypodermal and hematopoietic necrosis, a newly
recognized virus disease of penaeid shrimp. J. Invertebr. Pathol. 42, 62-80.
Lightner, D.V., Redman, T.A., Bell, T.A., (1983b). Detection of IHHN virus in Penaeus stylirostris and P. vannamei
imported into Hawaii. Journal of the World Aquaculture Society. 14, 212-225.
Lightner, D.V., Redman, R.M., (1998). Shrimp diseases and current diagnostic methods. Aquaculture 164, 201-220.
Lightner, D.V., Poulos, B.T., Bruce, L., Redman, R.M., Nunan, L., Pantoja, C., Mari, J., Bonami, J.R., 1994.
Development and application of genomic probes for use as diagnostic and research reagents for the Penaeid shrimp
parvoviruses IHHNV and HPV and BP. USMSFP 10th Anniversary Review, GCRL, Special Publication 1, 59-85.
Lightner, D.V., (1996). A handbook of shrimp pathology and diagnostic procedures of diseases of cultured penaeid
shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA.304P.
Lotz, J.M., (1997). Special topic review: viruses, biosecurity and specific pathogen-free stocks in shrimp aquaculture.
World J. Microbiol. Biotechnol. 13, 405-413.
Proceedings of the 11th International Symposium on Veterinary Epidemiology and Economics, 2006
Available at www.sciquest.org.nz
Mari, J., Bonami, J.R., Lightner, D.V., (1993). Partial cloning of the genome of infectious hypodermal and
haematopoietic necrosis virus, an unusual parvovirus pathogenic for penaeid shrimps; diagnosis of the disease using
a specific probe. J. Gen. Virol. 74, 2637-2643.
Motte, E., Yugcha, E., Luzardo, J., Castro, F., Leclercq, G., Rodrífuez., Miranda, P., Borja, O., Serrano, J., Terreros, M.,
Montalvo, K., Narváez, A., Tenorio, N., Cede•o, V., Mialhe, E., Boulo, V., (2003). Prevention of IHHNV vertical
transmission in the white shrimp Litopenaeus vannamei. Aquaculture 219, 57-70.
Nunan, L., Poulos, B.T., Lightner, D.V., (2000). Use of polymerase chain reaction for the detection of infectious
hypodermal and hematopoietic necrosis virus in penaeid shrimp. Mar. Biotechnol. 2(4), 319-328.
Phromjai, J., Boonasaeng, V., Withyachumnarnkul, B., Flegel, T.W., (2002). Detection of hepatopancreatic parvovirus in
Thai shrimp Penaeus monodon by in situ hybridization, dot blot hybridization and PCR amplification. Dis. Aquat.
Org. 51, 227-232.
Primavera, J.H., Quinitio, E.T., (2000). Runt-deformity syndrome in cultured giant tiger prawn Penaeus monodon. J.
Crustac. Biol. 20, 796-802.
Shi, C.Y., Huang, J., Yang, B., Song, X.L., Xu, H.S., (2003). The detection of four shrimp viruses using PCR and
RT-PCR. Marine Fisheries Research 24(1), 1-5.
Shike, H., Dhar, A., Burns, J., shimizu, C., Jousset, F., Klimpe, K., Bergoin, M., (2000). infectious hypodermal and
haematopoietic necrosis virus of shrimp is related to mosquito brevidensoviruses. Virology 276, 167-177.
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(2), 79-85.
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
Zhang, J.X., Sun, X.Q., (1997). A preliminary study on the virus in the eggs of Penaeus chinensis. Oceanography of
Huanghai&Bohai Seas 15(1), 48-51.
This paper was submitted to Vet. Microbiol. in Nov, 2005.
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