Donald V Lightner

The University of Arizona, Tucson, Arizona, United States

Are you Donald V Lightner?

Claim your profile

Publications (204)294.41 Total impact

  • Source
    Kathy F.J. Tang, Donald V. Lightner
    [Show abstract] [Hide abstract]
    ABSTRACT: Three insecticidal toxin complex (tc)-like genes were identified in Vibrio parahaemolyticus 13-028/A3, which can cause acute hepatopancreatic necrosis disease in penaeid shrimp. The three genes are: a tcdA-like gene (7710-bp), predicted to code for a 284 kDa protein; a tcdB-like gene (4272-bp), predicted to code for a 158 kDa protein; and a tccC3-like gene (2916-bp), predicted to encode a 107 kDa protein. All 3 predicted proteins contain conserved domains that are characteristic of their respective Tcs proteins. By RT-PCR, all 3 tc-like genes were found to be expressed in this bacterium. Through genome walking and the use of PCR to join contigs surrounding these 3 genes, a genomic island (87,712-bp, named tc-GIvp) was found on chromosome II localized next to the tRNA-gly. The GC content of this island, which is not found in other Vibrio species, is 40%. The tc-GIvp is characterized to have 60 ORFs encoding regulatory or virulence factors. These include: a type 6 secretion protein VgrG, EAL domain-containing proteins, fimbriae subunits and assembly proteins, invasin-like proteins, peptidoglycan-binding proteins and Tcs proteins. The tc-GIvp also contains 21 transposase genes, suggesting that it was acquired through horizontal transfer from other organisms.This article is protected by copyright. All rights reserved.
    FEMS Microbiology Letters 10/2014; · 2.05 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Acute hepatopancreatic necrosis disease (AHPND), which has also been referred to as early mortality syndrome (EMS), initially emerged as a destructive disease of cultured shrimp species in Asia in 2009. The pathogen associated with the disease, Vibrio parahaemolyticus, subsequently spread to the Western Hemisphere and emerged in Mexico in early 2013. The spread to the Western Hemisphere is a major concern to shrimp producers in the region. To date, the only peer-reviewed published method for determining whether mortalities are due to AHPND is through histological examination. A novel PCR detection method was employed to assess samples from Mexico in order to confirm the presence of the pathogen in this country. This manuscript details the detection methods used to confirm the presence of AHPND in Mexico. Both immersion and per os challenge studies were used to expose the Penaeus vannamei to the bacteria in order to induce the disease. Histological analysis confirmed AHPND status following the challenge studies. Also provided are the details of the molecular test by PCR that was used for screening candidate V. parahaemolyticus isolates. A rapid PCR assay for detection of AHPND may help with early detection and help prevent the spread of AHPND to other countries.
    Diseases of Aquatic Organisms 08/2014; 111(1):81-86. · 1.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Timely pond-side detection of white spot syndrome virus (WSSV) plays a critical role in the implementation of bio-security measures to help minimize economic losses caused by white spot syndrome disease, an important threat to shrimp aquaculture industry worldwide. A portable device, namely POCKIT™, became available recently to complete fluorescent probe-based insulated isothermal PCR (iiPCR), and automatic data detection and interpretation within one hour. Taking advantage of this platform, the IQ Plus™ WSSV Kit with POCKIT system was established to allow simple and easy WSSV detection for on-site users. The assay was first evaluated for its analytical sensitivity and specificity performance. The 95% limit of detection (LOD) of the assay was 17 copies of WSSV genomic DNA per reaction (95% confidence interval [CI], 13 to 24 copies per reaction). The established assay has detection sensitivity similar to that of OIE-registered IQ2000™ WSSV Detection and Protection System with serial dilutions of WSSV-positive Litopenaeus vannamei DNA. No cross-reaction signals were generated from infectious hypodermal and haematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), and hepatopancreatic parvovirus (HPV) positive samples. Accuracy analysis using700 L. vannamei of known WSSV infection status shows that the established assayhassensitivity93.5% (95% CI: 90.61-95.56%) and specificity 97% (95% CI: 94.31-98.50%). Furthermore, no discrepancy was found between the two assays when 100 random L. vannamei samples were tested in parallel. Finally, excellent correlation was observed among test results of three batches of reagents with 64 samples analyzed in three different laboratories. Working in a portable device, IQ Plus™ WSSV Kit with POCKIT system allows reliable, sensitive and specific on-site detection of WSSV in L. vannamei.
    PLoS ONE 01/2014; 9(3):e90545. · 3.53 Impact Factor
  • Source
    Kathy F J Tang, Marc Le Groumellec, Donald V Lightner
    [Show abstract] [Hide abstract]
    ABSTRACT: White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the aquaculture industry around the world. During 2010 to 2012, WSSV caused severe mortalities in cultured penaeid shrimp in Saudi Arabia, Mozambique and Madagascar. To investigate the origins of these WSSV, we performed genotyping analyses at 5 loci: the 3 open reading frames (ORFs) 125, 94 and 75, each containing a variable number of tandem repeats (VNTR), and deletions in the 2 variable regions, VR14/15 and VR23/24. We categorized the WSSV genotype as {N125, N94, N75, ΔX14/15, ΔX23/24} where N is the number of repeat units in a specific ORF and ΔX is the length (base pair) of deletion within the variable region. We detected 4 WSSV genotypes, which were characterized by a full-length deletion in ORF94/95, a relatively small ORF75 and one specific deletion length in each variable region. There are 2 closely related genotypes in these 3 countries: {6125, del94, 375, Δ595014/15, Δ1097123/24} and {7125, del94, 375, Δ595014/15, Δ1097123/24}, where del is the full-length ORF deletion. In Saudi Arabia, 2 other related types of WSSV were also found: {6125, 794, 375, Δ595014/15, Δ1097123/24} and {8125, 1394, 375, Δ595014/15, Δ1097123/24}. The identical patterns of 3 loci in these 4 types indicate that they have a common lineage, and this suggests that the WSSV epidemics in these 3 countries were from a common source, possibly the environment.
    Diseases of Aquatic Organisms 09/2013; 106(1):1-6. · 1.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A new emerging disease in shrimp, first reported in 2009, was initially named early mortality syndrome (EMS). In 2011, a more descriptive name for the acute phase of the disease was proposed as acute hepatopancreatic necrosis syndrome (AHPNS). Affecting both Pacific white shrimp Penaeus vannamei and black tiger shrimp P. monodon, the disease has caused significant losses in Southeast Asian shrimp farms. AHPNS was first classified as idiopathic because no specific causative agent had been identified. However, in early 2013, the Aquaculture Pathology Laboratory at the University of Arizona was able to isolate the causative agent of AHPNS in pure culture. Immersion challenge tests were employed for infectivity studies, which induced 100% mortality with typical AHPNS pathology to experimental shrimp exposed to the pathogenic agent. Subsequent histological analyses showed that AHPNS lesions were experimentally induced in the laboratory and were identical to those found in AHPNS-infected shrimp samples collected from the endemic areas. Bacterial isolation from the experimentally infected shrimp enabled recovery of the same bacterial colony type found in field samples. In 3 separate immersion tests, using the recovered isolate from the AHPNS-positive shrimp, the same AHPNS pathology was reproduced in experimental shrimp with consistent results. Hence, AHPNS has a bacterial etiology and Koch's Postulates have been satisfied in laboratory challenge studies with the isolate, which has been identified as a member of the Vibrio harveyi clade, most closely related to V. parahemolyticus.
    Diseases of Aquatic Organisms 07/2013; 105(1):45-55. · 1.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp. The major targets of WSSV infection are tissues of ectodermal and mesodermal embryonic origin, predominantly the cuticular epithelium and subcuticular connective tissues. Recently, we discovered a WSSV variant in Penaeus indicus that heavily infects the subcuticular connective tissue, with very slight indications in the cuticular epithelium. The variant was also unusual in that WSSV accumulations were found in the interstitial spaces of both the subcuticular connective tissue and the lymphoid organ. This WSSV variant was confirmed through immunohistochemistry with an anti-WSSV VP28 monoclonal antibody, and also by in situ hybridization with a VP28 DNA probe. By in situ hybridization, shrimp with variant and typical histology were shown a deletion in ORF94, which is characteristic of a new type of WSSV found in Saudi Arabia; apparently, the loss of this ORF is not associated with the variant's reduced capability of infecting the cuticular epithelium cells.
    Journal of Invertebrate Pathology 02/2013; · 2.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The bacteria that cause necrotizing hepatopancreatitis in Penaeus vannamei adversely affect penaeid shrimp cultured in the Western Hemisphere. 16S rRNA and gyrase B gene analyses determined the taxonomic position of these bacteria. The name 'Candidatus Hepatobacter penaei' is proposed for these pathogenic bacteria, a member of the Rickettsiales order.
    Applied and Environmental Microbiology 12/2012; · 3.95 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Three Litopenaeus vannamei families, from a breeding program in Panama and with possible WSSV resistance, were challenged per os with a reference isolate of White spot syndrome virus originally obtained from China in 1995 (WSSV-CN95). These F8, F9 and F12 generation families were developed from founder stocks a decade ago and were survivors of white spot disease. Juvenile shrimp used for WSSV challenge averaged 1.5 g, and they were stocked at 50 to 96 animals per tank into nine 1000 L fiberglass tanks containing artificial seawater at 30 ppt salinity and 26 °C. Three of the 1000 L tanks were used as negative control tanks, with one tank for each family. Six 1000 L tanks were used for challenging the three families with WSSV, with two replicate tanks for each family. A positive control consisting of 20 “Kona” SPF reference line L. vannamei (average weight 1.5 g) was included and challenged with WSSV in a 90 L glass aquarium. The Kona stock was fed the same batch of WSSV infected tissue as the three Panamanian families to confirm infectivity and to provide a basis with which to compare final survival. WSSV infected minced frozen shrimp tissue was fed at a rate of 5% of average body weight one time on day 0. All tanks were equipped with air diffusers to provide sufficient aeration and an acclimated crushed oyster shell internal recirculating biological filter. Each tank was covered with a plastic sheet to contain aerosols and minimize water temperature fluctuations. The experimental tanks were checked daily and moribund animals were collected when observed and preserved in Davidson's AFA fixative. Mortalities in the three Panamian families ceased at 17 days post challenge. Two survivors from each tank were preserved for histology and five shrimp per tank were individually tested by qPCR to determine their WSSV status and viral load. Survival at termination in the negative control families was 95%, 98% and 100%. Survival in the Kona line WSSV positive control was 0% with all the Kona line shrimp dead by day 6 post infection. At termination on day 17, survival of Panamanian selected families in the WSSV challenged groups was 23%, 57% and 26% for families LP-1, LP-2 and LP-3, respectively. This is the first time in the scientific literature that significant resistance of L. vannamei against WSSV under controlled conditions is reported.
    Aquaculture 11/2012; s 368–369:36–39. · 2.01 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Prior to 2004, Colombian shrimp farming benefited from a selection program in which Penaeus vannamei stocks were developed with resistance to Taura syndrome disease (TS). However since 2004, TS reappeared as a significant disease. In 2010, an apparently new strain of TSV (designated as CO 10) was collected in Colombia. Its genome was sequenced and compared with six other fully sequenced isolates. This analysis revealed that the TSV CO 10 is closely related to the isolates from Hawaii and Venezuela. Phylogenetic analysis based on capsid protein 2 (CP2) region from 59 TSV isolates shows that the recent Colombian isolates (2006-2010) form a new cluster and differ from the previous Colombia isolates (1994-1998) by 4% in nucleotide sequence. The virulence of this CO 10 isolate was similar to a Belize TSV determined through experimental infection in P. vannamei showing 100% mortalities and similar survival curves. By RT-qPCR for TSV, the viral loads were also close in the infected shrimp from both CO 10 and Belize at the order of 1×10(10)copies per μl RNA. To develop TSV-resistant lines, the candidate shrimp should be challenged with virus strains that have been isolated most recently from the regions where they will be cultured. This study suggests that the TSV present in Colombian shrimp farms during the last 5years is a new TSV strain with high virulence.
    Journal of Invertebrate Pathology 09/2012; · 2.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) are highly pathogenic to penaeid shrimp and have caused significant economic losses in the shrimp culture industry around the world. During 2010 and 2011, both WSSV and TSV were found in Saudi Arabia, where they caused severe mortalities in cultured Indian white shrimp Penaeus indicus. Most outbreaks of shrimp viruses in production facilities can be traced to the importation of infected stocks or commodity shrimp. In an attempt to determine the origins of these viral outbreaks in Saudi Arabia, we performed variable number of tandem repeat (VNTR) analyses for WSSV isolates and a phylogenetic analysis for TSV isolates. From the WSSV genome, the VNTR in open reading frames (ORFs) 125 and 94 were investigated with PCR followed by DNA sequence analysis. The genotypes were categorized as {N125, N94} where N is the number of repeat units in a specific ORF, and the subscript indicates the ORF (i.e. ORFs 125 and 94 in this case). From 15 Saudi Arabia WSSV isolates, we detected 3 genotypes: {6125, 794}, {7125, del94}, and {8125, 1394}. The WSSV genotype of {7125, del94} appears to be a new variant with a 1522 bp deletion encompassing complete coding regions of ORF 94 and ORF 95 and the first 82 bp of ORF 93. For TSV genotyping, we used a phylogenetic analysis based on the amino acid sequence of TSV capsid protein 2 (CP2). We analyzed 8 Saudi Arabian isolates in addition to 36 isolates from other areas: SE Asia, Mexico, Venezuela and Belize. The Saudi Arabian TSV clustered into a new, distinct group. Based on these genotyping analyses, new WSSV and TSV genotypes were found in Saudi Arabia. The data suggest that they have come from wild shrimp Penaeus indicus from the Red Sea that are used for broodstock.
    Diseases of Aquatic Organisms 07/2012; 99(3):179-85. · 1.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Pacific white shrimp Penaeus vannamei that were pre-exposed to Taura syndrome virus (TSV) and then challenged with yellow head virus (YHV) acquired partial protection from yellow head disease (YHD). Experimental infections were carried out using specific-pathogen-free (SPF) shrimp which were first exposed per os to TSV; at 27, 37 and 47 d post infection they were then challenged by injection with 1 × 104 copies of YHV per shrimp (designated the TSV-YHV group). Shrimp not infected with TSV were injected with YHV as a positive control. Survival analyses comparing the TSV-YHV and YHV (positive control) groups were conducted, and significant survival rates were found for all the time groups (p < 0.001). A higher final survival was found in the TSV-YHV group (mean 55%) than in the positive control (0%) (p < 0.05). Duplex reverse transcription quantitative PCR was used to quantify both TSV and YHV. Lower YHV copy numbers were found in the TSV-YHV group than in the positive control in pleopods (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.001) and lymphoid organ (LO) samples (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.01). In situ hybridization assays were conducted, and differences in the distribution of the 2 viruses in the target tissues were found. The foci of LO were infected with TSV but were not infected with YHV. This study suggests that a viral interference effect exists between TSV and YHV, which could, in part, explain the absence of YHD in the Americas, where P. vannamei are often raised in farms where TSV is present.
    Diseases of Aquatic Organisms 04/2012; 98(3):185-92. · 1.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Seafood is a highly traded food commodity. Farmed and captured crustaceans contribute a significant proportion with annual production exceeding 10 M metric tonnes with first sale value of $40bn. The sector is dominated by farmed tropical marine shrimp, the fastest growing sector of the global aquaculture industry. It is significant in supporting rural livelihoods and alleviating poverty in producing nations within Asia and Latin America while forming an increasing contribution to aquatic food supply in more developed countries. Nations with marine borders often also support important marine fisheries for crustaceans that are regionally traded as live animals and commodity products. A general separation of net producing and net consuming nations for crustacean seafood has created a truly globalised food industry. Projections for increasing global demand for seafood in the face of level or declining fisheries requires continued expansion and intensification of aquaculture while ensuring best utilisation of captured stocks. Furthermore, continued pressure from consuming nations to ensure safe products for human consumption are being augmented by additional legislative requirements for animals (and their products) to be of low disease status. As a consequence, increasing emphasis is being placed on enforcement of regulations and better governance of the sector; currently this is a challenge in light of a fragmented industry and less stringent regulations associated with animal disease within producer nations. Current estimates predict that up to 40% of tropical shrimp production (>$3bn) is lost annually, mainly due to viral pathogens for which standard preventative measures (e.g. such as vaccination) are not feasible. In light of this problem, new approaches are urgently required to enhance yield by improving broodstock and larval sourcing, promoting best management practices by farmer outreach and supporting cutting-edge research that aims to harness the natural abilities of invertebrates to mitigate assault from pathogens (e.g. the use of RNA interference therapeutics). In terms of fisheries losses associated with disease, key issues are centred on mortality and quality degradation in the post-capture phase, largely due to poor grading and handling by fishers and the industry chain. Occurrence of disease in wild crustaceans is also widely reported, with some indications that climatic changes may be increasing susceptibility to important pathogens (e.g. the parasite Hematodinium). However, despite improvements in field and laboratory diagnostics, defining population-level effects of disease in these fisheries remains elusive. Coordination of disease specialists with fisheries scientists will be required to understand current and future impacts of existing and emergent diseases on wild stocks. Overall, the increasing demand for crustacean seafood in light of these issues signals a clear warning for the future sustainability of this global industry. The linking together of global experts in the culture, capture and trading of crustaceans with pathologists, epidemiologists, ecologists, therapeutics specialists and policy makers in the field of food security will allow these issues to be better identified and addressed.
    Journal of Invertebrate Pathology 03/2012; 110(2):141-57. · 2.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: About 3.5 million metric tons of farmed shrimp were produced globally in 2009 with an estimated value greater than USD$14.6 billion. Despite the economic importance of farmed shrimp, the global shrimp farming industry continues to be plagued by disease. There are a number of strategies a shrimp farmer can employ to mitigate crop loss from disease, including the use of Specific Pathogen Free (SPF), selectively bred shrimp and the adoption of on-farm biosecurity practices. Selective breeding for disease resistance began in the mid 1990s in response to outbreaks of Taura syndrome, caused by Taura syndrome virus (TSV), which devastated populations of farmed shrimp (Litopenaeus vannamei) throughout the Americas. Breeding programs designed to enhance TSV survival have generated valuable information about the quantitative genetics of disease resistance in shrimp and have produced shrimp families which exhibit high survival after TSV exposure. The commercial availability of these selected shrimp has benefitted the shrimp farming industry and TSV is no longer considered a major threat in many shrimp farming regions. Although selective breeding has been valuable in combating TSV, this approach has not been effective for other viral pathogens and selective breeding may not be the most effective strategy for the long-term viability of the industry. Cost-effective, on-farm biosecurity protocols can be more practical and less expensive than breeding programs designed to enhance disease resistance. Of particular importance is the use of SPF shrimp stocked in biosecure environments where physical barriers are in place to mitigate the introduction and spread of virulent pathogens.
    Journal of Invertebrate Pathology 03/2012; 110(2):247-50. · 2.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Shrimp farming in the Americas began to develop in the late 1970s into a significant industry. In its first decade of development, the technology used was simple and postlarvae (PLs) produced from wild adults and wild caught PLs were used for stocking farms. Prior to 1990, there were no World Animal Health Organization (OIE) listed diseases, but that changed rapidly commensurate with the phenomenal growth of the global shrimp farming industry. There was relatively little international trade of live or frozen commodity shrimp between Asia and the Americas in those early years, and with a few exceptions, most of the diseases known before 1980 were due to disease agents that were opportunistic or part of the shrimps' local environment. Tetrahedral baculovirosis, caused by Baculovirus penaei (BP), and necrotizing hepatopancreatitis (NHP) and its bacterial agent Hepatobacterium penaei, were among the "American" diseases that eventually became OIE listed and have not become established outside of the Americas. As the industry grew after 1980, a number of new diseases that soon became OIE listed, emerged in the Americas or were introduced from Asia. Spherical baculovirus, caused by MBV, although discovered in the Americas in imported live Penaeus monodon, was subsequently found to be common in wild and farmed Asian, Australian and African penaeids. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) was introduced from the Philippines in the mid 1970s with live P. monodon and was eventually found throughout the Americas and subsequently in much of the shrimp farming industry in the eastern hemisphere. Taura syndrome emerged in Penaeus vannamei farms in 1991-1992 in Ecuador and was transferred to SE Asia with live shrimp by 1999 where it also caused severe losses. White Spot Disease (WSD) caused by White spot syndrome virus (WSSV) emerged in East Asia in ∼1992, and spread throughout most of the Asian shrimp farming industry by 1994. By 1995, WSSV reached the eastern USA via frozen commodity products and it reached the main shrimp farming countries of the Americas located on the Pacific side of the continents by the same mechanism in 1999. As is the case in Asia, WSD is the dominant disease problem of farmed shrimp in the Americas. The most recent disease to emerge in the Americas was infectious myonecrosis caused by IMN virus. As had happened before, within 3years of its discovery, the disease had been transferred to SE Asia with live P. vannamei, and because of its impact on the industry and potential for further spread in was listed by the OIE in 2005. Despite the huge negative impact of disease on the shrimp farming industry in the Americas, the industry has continued to grow and mature into a more sustainable industry. In marked contrast to 15-20years ago when PLs produced from wild adults and wild PLs were used to stock farms in the Americas, the industry now relies on domesticated lines of broodstock that have undergone selection for desirable characteristics including disease resistance.
    Journal of Invertebrate Pathology 03/2012; 110(2):174-83. · 2.67 Impact Factor
  • D V Lightner
    [Show abstract] [Hide abstract]
    ABSTRACT: Reviewed in this paper are the steps for listing or de-listing of an aquatic animal disease, the current list of OIE listed aquatic animal diseases, and the reporting requirements for listed diseases by member countries. The current OIE listed aquatic animal diseases includes two diseases of amphibians, nine of fish, seven of mollusks, and eight of crustaceans. Of interest is the difference in importance of the listed diseases in each of the four groups of aquatic animals. In mollusks, parasitic diseases dominate the list, while in fish and crustaceans virus diseases are dominant. Whether a listed disease is due to a virus, fungus, bacterium or a parasite, the occurrence of the disease may adversely affect international trade among trading partners that have, or do not have, the listed disease. By its very nature, the international trade in terrestrial animals and aquatic animals, and their products, is influenced by national and international politics. When the occurrence of an OIE listed or emerging disease becomes an issue between trading partners, trade restrictions may be put in place and disputes are often a consequence. The World Trade Organization named the OIE as the reference body for animal health as it relates to international trade. This action recognized the 88 year history of the work by the OIE in disease control, listing of diseases, the development of the terrestrial and aquatic codes and the diagnostic manuals, and the prompt notification of members by the OIE of the occurrence of listed diseases. The intent of the WTO with this action was likely to minimize disease related trade disputes brought before the WTO.
    Journal of Invertebrate Pathology 03/2012; 110(2):184-7. · 2.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A reovirus (tentatively designated as Callinectes sapidus reovirus, CsRV) was found in the blue crabs C. sapidus collected in Chesapeake Bay in 2005. Histological examination of hepatopancreas and gill from infected crabs revealed eosinophilic to basophilic, cytoplasmic, inclusions in hemocytes and in cells of connective tissue. A cDNA library was constructed from total RNA extracted from hemolymph of infected crabs. One clone (designated as CsRV-28) with a 532-bp insert was 75% identical in nucleotide sequence (and 95% similar in translated amino acid sequence) to the quanylytransferase gene of the Scylla serrata reovirus (SsRV). The insert of CsRV-28 was labeled with digoxigenin-11-dUTP and hybridized to sections of hepatopancreas and gill of infected C. sapidus, this probe reacted to hemocytes and cells in the connective tissue. No reaction was seen in any of the tissues prepared from uninfected crabs. Thus, this in situ hybridization procedure can be used to diagnose CsRV.
    Journal of Invertebrate Pathology 09/2011; 108(3):226-8. · 2.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The Penaeus vannamei nodavirus (PvNV), which causes muscle necrosis in Penaeus vannamei from Belize, was identified in 2005. Infected shrimp show clinical signs of white, opaque lesions in the tail muscle. Under transmission electron microscopy, the infected cells exhibit increases in various organelles, including mitochondria, Golgi stacks, and rough endoplasmic reticulum. Cytoplasmic inclusions containing para-crystalline arrays of virions were visualized. The viral particle is spherical in shape and 19 to 27 nm in diameter. A cDNA library was constructed from total RNA extracted from infected shrimp. Through nucleotide sequencing from the cDNA clones and northern blot hybridization, the PvNV genome was shown to consist of 2 segments: RNA1 (3111 bp) and RNA2 (1183 bp). RNA1 contains 2 overlapped open reading frames (ORF A and B), which may encode a RNA-dependent RNA polymerase (RdRp) and a B2 protein, respectively. RNA2 contains a single ORF that may encode the viral capsid protein. Sequence analyses showed the presence of 4 RdRp characteristic motifs and 2 conserved domains (RNA-binding B2 protein and viral coat protein) in the PvNV genome. Phylogenetic analysis based on the translated amino acid sequence of the RdRp reveals that PvNV is a member of the genus Alphanodavirus and closely related to Macrobrachium rosenbergii nodavirus (MrNV). In a study investigating potential PvNV vectors, we monitored the presence of PvNV by RT-PCR in seabird feces and various aquatic organisms collected around a shrimp farm in Belize. PvNV was detected in mosquitofish, seabird feces, barnacles, and zooplankton, suggesting that PvNV can be spread via these carriers.
    Diseases of Aquatic Organisms 05/2011; 94(3):179-87. · 1.73 Impact Factor
  • Kathy F J Tang, Donald V Lightner
    [Show abstract] [Hide abstract]
    ABSTRACT: We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.
    Diseases of Aquatic Organisms 02/2011; 93(3):191-8. · 1.73 Impact Factor
  • Linda M Nunan, Donald V Lightner
    [Show abstract] [Hide abstract]
    ABSTRACT: A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by Lo et al. (1996) and outlined as the recommended PCR diagnostic assay in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.
    Journal of virological methods 01/2011; 171(1):318-21. · 2.13 Impact Factor
  • Source
    G. D. Stentiford, D. V. Lightner
    [Show abstract] [Hide abstract]
    ABSTRACT: White Spot Disease (WSD) caused by White Spot Syndrome Virus (WSSV) is listed as ‘non-exotic’ to the European Union in EC Directive 2006/88. Two other viral diseases (Taura Syndrome and Yellowhead Disease) are listed as exotic. Despite the listing of WSD as a non-exotic disease, definitive case reports have not been officially reported, or published in the peer-reviewed literature. Here we report on a series of outbreaks of WSD in three European Union (EU) Member States (Greece, Italy and Spain) and in one non-EU European country (Turkey) over the period 1995 to 2001. Samples were submitted by industry representatives over this period and were therefore not officially reported to the Competent Authorities of respective European Member States. At least one of the cases appeared to be associated with the feeding of imported shrimp carcasses from Asia to broodstock while other cases were associated with the importation of post-larvae between hatcheries and on-growing facilities from outside of Europe and further movements within Europe. These case reports demonstrate the ability for WSSV to cause disease and mortality in penaeid shrimp farmed at European ambient temperatures. Furthermore, they demonstrate potential for the introduction of WSSV to new geographic areas via the movement of live crustaceans and their products, both from outside of the EU, and between EU Member and non-member countries within the European region.
    Aquaculture. 01/2011; 319(1):302-306.

Publication Stats

3k Citations
294.41 Total Impact Points

Institutions

  • 1977–2014
    • The University of Arizona
      • Department of Veterinary Sciences and Microbiology
      Tucson, Arizona, United States
  • 2009–2010
    • Ludong University
      Shan-tang, Jiangxi Sheng, China
  • 2006–2007
    • Department of Livestock Development
      • Department of Livestock Development
      Bangkok, Bangkok, Thailand
  • 2005
    • Oceanic Institute
      Waimānalo, Hawaii, United States
  • 2000
    • Max Planck Institute for Biology of Ageing
      Köln, North Rhine-Westphalia, Germany
  • 1997
    • Université de Montpellier 1
      Montpelhièr, Languedoc-Roussillon, France
  • 1990
    • Observatoire des Sciences et des Techniques
      Lutetia Parisorum, Île-de-France, France