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Publication Metadata only Yellow-head virus of Penaeus monodon is an RNA virus(1995-01-01) C. Wongteerasupaya; S. Sriurairatana; J. E. Vickers; A. Akrajamorn; V. Boonsaeng; S. Panyim; A. Tassanakajon; B. Withyachumnarnkul; T. W. Flegel; Mahidol UniversityPublication Metadata only The crisis in Asian shrimp aquaculture: Current status and future needs(1998-01-01) T. W. Flegel; V. Alday-Sanz; Mahidol University, many sources of WSSV were identified. The list now includes over 40 reservoir crustaceans and perhaps even aquatic insects. All of the commonly cultivated shrimp in Asia can be infected with the virus and for many, the infection can be deadlyPublication Metadata only A non-occluded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon(1995-01-01) C. Wongteerasupaya; J. E. Vickers; S. Sriurairatana; G. L. Nash; A. Akarajamorn; V. Boonsaeng; S. Panyim; A. Tassanakajon; B. Withyachumnarnkul; T. W. Flegel; Mahidol UniversityPublication Metadata only Different reactions obtained using the same DNA detection reagents for Thai and Korean hepatopancreatic parvovirus of penaeid shrimp(2001-09-12) J. Phromjai; W. Sukhumsirichart; C. Pantoja; D. V. Lightner; T. W. Flegel; Mahidol UniversityHepatopancreatic parvovirus (HPV) can cause stunted growth and death in penaeid shrimp including Penaeus monodon. We used PCR primers and a commercial DNA probe designed from HPV of Penaeus chinensis (HPVchin) to examine HPV-infected Thai P. monodon (HPVmon). We found that the PCR primers produced a 732 bp DNA amplicon rather than the 350 bp amplicon obtained with HPVchin template and that the DNA probe gave weak to variable in situ DNA hybridization results. In addition, hybridization to PCR products from HPVmon was weak compared with hybridization with PCR products from HPVchin. By contrast, the 732 bp amplicon hybridized strongly with HPVmon-infected cells by in situ hybridization but not with uninfected shrimp tissue or other shrimp viruses, thus confirming its origin from HPVmon. Cloning, sequencing and analysis of the 732 bp amplicon showed that 696 bp (excluding the primer sequences) contained 47% GC content and had only 78% homology to 701 aligned bases from a 3350 bp DNA fragment of HPVchin from GenBank. These results explain why the reagents based on HPVchin gave a different PCR product and weak hybridization results with HPVmon, and they show that multiple primers or degenerate primers may be necessary for general detection of HPV varieties. Together with previously published information on the estimated total genome sizes for HPVchin (approximately 4 kb) and HPVmon (approximately 6 kb), these data support the contention that HPVchin and HPVmon are different varieties or species, in spite of their similar histopathology.Publication Metadata only High variation in repetitive DNA fragment length for white spot syndrome virus (WSSV) isolates in Thailand(2003-04-24) Chainarong Wongteerasupaya; Paranee Pungchai; Boonsirm Withyachumnarnkul; Vichai Boonsaeng; Sakol Panyim; T. W. Flegel; Peter J. Walker; Faculty of Medicine, Thammasat University; Srinakharinwirot University; Mahidol University; CSIRO Livestock IndustriesWhite spot syndrome virus (WSSV) presently causes the most serious losses to shrimp farmers worldwide. Earlier reports of high DNA sequence homology among isolates from widely separated geographical regions suggested that a single virus was the cause. However, we have found surprisingly high variation in the number of 54 bp DNA repeats in ORF94 (GenBank AF369029) from 55 shrimp ponds (65 shrimp samples) experiencing WSSV outbreaks in Thailand in 2000 and 2002. These were detected by PCR amplification using primers ORF94-F and ORF94-R flanking the repeat region. Altogether, 12 different repeat groups were found (from 6 to 20 repeats) with 8 repeats being most frequent (about 32%). Extracts prepared from individual shrimp in the same outbreak pond belonged to the same repeat group while those collected at the same time from separate WSSV outbreak ponds, or from the same ponds at different times, usually belonged to different repeat groups. This suggested that different outbreaks were caused by different WSSV isolates. In contrast to the highly variable numbers of repeats, sequence variation within the repeat region was confined to either T or G at Position 36. These variations may be useful for epidemiological studies on the local and global movement of WSSV, since there is high variation in the number of repeats (good for local studies) but little sequence change (good for global studies).Publication Metadata only Characterization and PCR detection of hepatopancreatic parvovirus (HPV) from Penaeus monodon in Thailand(1999-10-11) Wasana Sukhumsirichart; Chainarong Wongteerasupaya; Vichai Boonsaeng; Sakol Panyim; Siriporn Sriurairatana; Boonsirm Withyachumnarnkul; T. W. Flegel; Mahidol University; Srinakharinwirot UniversityHepatopancreatic parvovirus (HPV) causes disease in several species of penaeid shrimp. Heavy infections may result in poor growth and reduced production for shrimp farmers. From one southern Thai shrimp pond with a high prevalence of HPV infection, 790 shrimp were sampled randomly and the hepatopancreas (HP) removed. Most HP were preserved in liquid nitrogen. However, every 10th HP (79 total) was divided into 2 parts appropriately fixed for examination by transmission electron microscopy (TEM) and light microscopy. Based on light microscopy, the prevalence of HPV infection in the pond was approximately 30% and its presence was confirmed by TEM of parallel samples. The virus was subsequently purified from hepatopancreatic homogenates of the samples preserved in liquid nitrogen. Negative staining of the purified viral preparation revealed unenveloped, icosahedral viral particles 22 to 24 nm in diameter. Agarose gel electrophoresis of nucleic acid extracts revealed the presence of 2 fragments, one very intense (5.8 kb) and the other weak (4.2 kb). The larger fragment was degraded by DNase I and S1nuclease, indicating single-stranded DNA (ssDNA) characteristic of the viral family Parvoviridae. The smaller fragment was degraded by DNase I but not by S1nuclease, indicating that it comprised double-stranded DNA. A genomic DNA library of the 5.8 kb ssDNA was constructed in pUC18 and a clone containing a 659 bp fragment specific and sensitive for HPV was selected for sequencing. Based on this sequence, an HPV-specific primer set was designed to yield a 156 bp amplicon by polymerase chain reaction (PCR) amplification. The expected 156 bp amplicon was obtained only in the presence of HPV DNA template (at as little as 1 fg purified DNA) and not with nucleic acid templates extracted from healthy shrimp tissue or other shrimp pathogens. It is hoped that this PCR assay will be useful to shrimp aquaculturists for early detection and screening of shrimp larvae, parental broodstock or other possible carriers of HPV in the shrimp cultivation system.Publication Metadata only Experimental transmission of white spot syndrome virus (WSSV) from crabs to shrimp Penaeus monodon(1998-09-11) Panan Kanchanaphum; Chainarong Wongteerasupaya; Nusra Sitidilokratana; Vichai Boonsaeng; Sakol Panyim; Anchalee Tassanakajon; Boonsirm Withyachumnarnkul; T. W. Flegel; Mahidol University; Srinakharinwirot University; Thailand National Center for Genetic Engineering and Biotechnology; Chulalongkorn UniversityWhite spot syndrome virus (WSSV) of the black tiger prawn Penaeus monodon is a recently discovered baculo-like virus disease which is currently the cause of very serious and wide-spread losses in the shrimp industry in Thailand and elsewhere in Asia. Three suspected crab carriers of this virus commonly found in shrimp-rearing areas were investigated. These were Sesarma sp., Scylla serrata and Uca pugilator. All these crabs could be infected with WSSV by injection and they sustained heavy viral infections for up to 45 d (confirmed by normal histology, specific in situ DNA hybridization and PCR amplification) without visible signs of disease or mortality. All of them also transferred the disease to P. monodon via water while physically separated in aquarium cohabitation tests. Transfer of the virus to the shrimp was monitored using in situ DNA hybridization and PCR assay at 12 h intervals after cohabitation began. With U. pugilator, WSSV could be detected in the shrimp cohabitants after 24 h using PCR amplification and after 60 h using in situ hybridization. With S. serrata, the shrimp were positive for WSSV after 36 h using PCR and after 60 h using DNA in situ hybridization. With Sesarma sp. they were positive after 48 h using PCR and 72 h using in situ hybridization. These laboratory studies demonstrated that crab carriers of WSSV may pose a real threat to cultivated shrimp. However, the studies were carried out in containers with a small volume and with relatively clean sea water as compared to shrimp cultivation ponds. Pond-based studies are now needed to determine whether factors such as pond volume, pond water quality and shrimp and crab behavior can influence the rate and success of transfer.Publication Metadata only Evidence for apoptosis correlated with mortality in the giant black tiger shrimp Penaeus monodon infected with yellow head virus(2002-03-11) Kornnika Khanobdee; Chumporn Soowannayan; T. W. Flegel; Sukathida Ubol; Boonsirm Withyachumnarnkul; Mahidol UniversityHistological, cytochemical and ultrastructural changes in giant black tiger shrimp Penaeus monodon were investigated at various time intervals after injection with yellow head virus (YHV). Hemocytes, lymphoid organs (LO) and gills were the main focus of the study. After injection with YHV, onset of mortality varied from 36 h onward. By normal hematoxylin and eosin staining, the 3 tissues showed clear and increasing prevalence of nuclear condensation, pyknosis and karyorrhexis from approximately 36 h post-injection (p.i.) until death, although pathology was evident in the LO as early as 12 h p.i. in some shrimp. By nuclear DNA staining with 4′, 6-diamidino-2-phenylindole (DAPI) and by specific labeling of 3′-OH ends of nuclear DNA using a technique called terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL), cells of the 3 tissues showed evidence of chromatin condensation and DNA fragmentation, respectively. Both are generally considered to be characteristic of apoptosis. In addition to TUNEL labeling, evidence for DNA fragmentation was supported by the appearance of ∼200 base pair DNA ladders at approximately 48 h p.i. in hemocytes of YHV-infected but not uninfected shrimp. Transmission electron microscopy (TEM) of LO tissue revealed features of apoptosis in tissues of YHV-infected shrimp only. These included marginated, condensed and fragmented chromatin without concurrent cytoplasmic damage. Histological, cytochemical, ultrastructural and biochemical data were consistent with the hypothesis that widespread and progressive apoptosis occurred in susceptible shrimp infected with YHV. Although no specific tests were carried out to determine whether this purported apoptosis was the cause of mortality, moribund shrimp had extensive deterioration of vital tissues such as the hemolymph, gills, heart and LO, suggesting that many essential bodily functions had been severely compromised. This probably resulted in the gross signs of lethargy and weakness seen, and it is reasonable to suggest that further, progressive deterioration could have led to the collapse of vital functions followed by death.Publication Metadata only Detection of yellow-head virus (YHV) of Penaeus monodon by RT-PCR amplification(1997-12-30) Chainarong Wongteerasupaya; Wansika Tongchuea; Vichai Boonsaeng; Sakol Panyim; Anchalee Tassanakajon; Boonsirm Withyachumnarnkul; T. W. Flegel; Srinakharinwirot University; Thailand National Center for Genetic Engineering and Biotechnology; Mahidol University; Chulalongkorn UniversityA nucleic acid probe was developed using cDNA prepared from ssRNA extracted from yellow-head virus (YHV), a serious pathogen of the black tiger prawn Penaeus monodon. The specificity and sensitivity of this probe was established using dot-blot hybridization with nucleic acid extracts from YHV and from shrimp, bacteria and other viruses. Based on the sequence of this cloned YHV cDNA fragment, a YHV specific primer set for reverse transcription polymerase chain reaction (RT-PCR) of a 135 base pair (bp) sub-fragment was designed for detection of YHV infections in penaeid shrimp. When applied in RT-PCR with templates derived from experimentally or naturally YHV-infected shrimp and with purified YHV or YHV nucleic acid, the expected 135 bp amplification product was obtained. By contrast, nucleic acids extracted from tissue samples of healthy shrimp and from other shrimp pathogens gave no such fragment. This confirmed the specificity of the designed YHV RNA specific primers. RT-PCR based detection demonstrated high sensitivity, in that it could detect 0.01 pg of purified YHV-RNA. In a time course study of an experimental YHV infection, the RT-PCR detection showed evidence of infection at 6 to 12 h post exposure to the virus. However, histopathology typical of YHV infection [i.e. karyorhexis and pycnosis of haemocytes in haematoxylin and eosin (H and E) stained haemolymph smears] was not visible until 42 to 48 h post exposure. The results suggested that RT-PCR might be useful to shrimp aquaculturists for early detection of YHV outbreaks or for detection of asymptomatic carriers.Publication Metadata only Lethal toxicity of Vibrio harveyi to cultivated Penaeus monodon induced by a bacteriophage(1999-02-26) Lila Ruangpan; Yaowanit Danayadol; Sataporn Direkbusarakom; Siriporn Siurairatana; T. W. Flegel; Thailand National Institute of Coastal Aquaculture; Mahidol UniversityIn Southern Thailand in 1996, intense luminescence in many shrimp rearing ponds was accompanied by massive mortality resulting in total crop loss within 3 or 4 d. Mortality was correlated with gross signs which shrimp farmers called (English translation) tea-brown gill syndrome (TBGS). Histological examination of moribund shrimp revealed massive lesions of the hepatopancreas characterized by hemocytic infiltration and the presence of bacterial cells. Bacterial isolation yielded several strains tentatively identified as Vibrio harveyi on the basis of luminescence and growth on BTB Teepol agar. Representative isolate VH1039 was selected and identified by biochemical tests as V. harveyi. When strain VH1039 and the other luminescent isolates were injected into normal shrimp (1 x 107cells shrimp-1), no significant mortality was observed in comparison with control shrimp injected without bacteria. Nor was any significant mortality observed after injection of supernatant fluids from normal or sonicated bacterial cultures of VH1039 (1 x 108cells ml-1). Transmission electron microscopy (TEM) of hepatopancreatic tissue from farmed TBGS shrimp revealed bacterial cells of Vibrio morphology together with large numbers of bacteriophage particles that had round to hexagonal heads of approximately 60 nm diameter and tails of approximately 100 nm length. These were either free, attached to cell walls of intact bacteria or in various stages of replication within bacterial cells. Gills of farmed TBGS shrimp were subsequently homogenized in lobster haemolymph buffer (LHB) and membrane filtered (0.22 μm). Compared to control shrimp injected with LHB, shrimp injected with the 1000x diluted gill filtrate (DGF) showed no significant mortality. However, when DGF was injected together with 1 x 107cells of strain VH1039, there was total mortality within 48 h. High and rapid mortality concurrent with brown gills was seen only in the mixed injection group. TEM of the artificially infected shrimp tissues did show the presence of bacterial cells, but no mature bacteriophage particles or lysed bacterial cells were found similar to those seen in farmed TBGS shrimp. In further tests, addition of DGF to cultures of VH1039 induced extreme but transitory toxicity of culture filtrates from 24 to 36 h. Treatment of DGF with a germicidal lamp (UV) for 30 min or with heat at 100°C for 15 min failed to stop shrimp mortality from mixed DGF/VH1039 injections. However, mortality was stopped if the heated DGF was treated with DNase. The results suggested that a bacteriophage may sometimes mediate the toxicity of V. harveyi in Penaeus monodon by the transfer of a toxin gene(s) or a gene(s) controlling toxin production.