Donald V Lightner

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• Aranguren, L. F., Aranguren, L. F., Salazar, M., Salazar, M., Tang, K., Tang, K., Caraballo, X., Caraballo, X., Lightner, D., & Lightner, D. (2013). Characterization of a new strain of Taura syndrome virus (TSV) from Colombian shrimp farms and the implication in the selection of TSV resistant lines. Journal of Invertebrate Pathology, 112(1), 68-73.
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  PMID: 23022573;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 1×1010copies 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. © 2012 Elsevier Inc.
• F., K., Groumellec, M. L., & Lightner, D. V. (2013). Novel, closely related, white spot syndrome virus (WSSV) genotypes from Madagascar, Mozambique and the Kingdom of Saudi Arabia. Diseases of Aquatic Organisms, 106(1), 1-6.
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  PMID: 24062547;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, N 94, N75, δX14/15, δX 23/24} 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, δ1097 123/24} 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. © 2013 Inter-Research.
• Lightner, D., Aranguren, L. F., Salazar, M., Tang, K., Caraballo, X., & Lightner, D. V. (2013). Characterization of a new strain of Taura syndrome virus (TSV) from Colombian shrimp farms and the implication in the selection of TSV resistant lines. Journal of invertebrate pathology, 112(1).
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  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 5 years is a new TSV strain with high virulence.
• Lightner, D., Nunan, L. M., Pantoja, C. R., Gomez-Jimenez, S., & Lightner, D. V. (2013). "Candidatus Hepatobacter penaei," an intracellular pathogenic enteric bacterium in the hepatopancreas of the marine shrimp Penaeus vannamei (Crustacea: Decapoda). Applied and environmental microbiology, 79(4).
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  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, which are members of the Rickettsiales order.
• Lightner, D., Tang, K. F., Le Groumellec, M., & Lightner, D. V. (2013). Novel, closely related, white spot syndrome virus (WSSV) genotypes from Madagascar, Mozambique and the Kingdom of Saudi Arabia. Diseases of aquatic organisms, 106(1).
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  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.
• Lightner, D., Tang, K. F., Pantoja, C. R., Redman, R. M., & Lightner, D. V. (2013). A histological variant of white spot syndrome virus (WSSV) from the Kingdom of Saudi Arabia. Journal of invertebrate pathology, 113(1).
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  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.
• Lightner, D., Tran, L., Nunan, L., Redman, R. M., Mohney, L. L., Pantoja, C. R., Fitzsimmons, K., & Lightner, D. V. (2013). Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Diseases of aquatic organisms, 105(1).
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  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.
• Nunan, L. M., Pantoja, C. R., Gomez-Jimenez, S., & Lightner, D. V. (2013). "Candidatus hepatobacter penaei," an intracellular pathogenic enteric bacterium in the hepatopancreas of the marine shrimp Penaeus vannamei (Crustacea: Decapoda). Applied and Environmental Microbiology, 79(4), 1407-1409.
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  PMID: 23241970;PMCID: PMC3568585;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, which are members of the Rickettsiales order. © 2013, American Society for Microbiology.
• Tang, K. F., Tang, K. F., Pantoja, C. R., Pantoja, C. R., Redman, R. M., Redman, R. M., Lightner, D. V., & Lightner, D. V. (2013). A histological variant of white spot syndrome virus (WSSV) from the Kingdom of Saudi Arabia. Journal of Invertebrate Pathology, 113(1), 82-85.
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  PMID: 23454062;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. © 2013 Elsevier Inc.
• Tran, L., Nunan, L., Redman, R. M., Mohney, L. L., Pantoja, C. R., Fitzsimmons, K., & Lightner, D. V. (2013). Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Diseases of Aquatic Organisms, 105(1), 45-55.
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  PMID: 23836769;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 in - duced 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. © Inter-Research 2013.
• , J., White-Noble, B., Schofield, P., Chamorro, R., & Lightner, D. (2012). Report of significant WSSV-resistance in the Pacific white shrimp, Litopenaeus vannamei, from a Panamanian breeding program. Aquaculture, 368-369, 36-39.
• Aoki, T., Bonami, J., Lightner, D., Lo, C., Stentiford, G., Leu, J., Vlak, J., Soderholl, K., Walker, P., Flegel, T., Wang, H., Xu, X., & Yang, H. (2012). Nimaviridae. Virus Taxonomy: 9th Report of the International Committee on Taxonomy of Viruses, 229-234.
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  In A.M.Q. King, M.J. Adams, E.B. Carstens and E J. Lefkowitz, editors: Virus Taxonomy. 9th Report of the International Committee on Taxonomy of Viruses, Oxford, Elsevier
• Aranguren, L. F., F., K., & Lightner, D. V. (2012). Protection from yellow head virus (YHV) infection in Penaeus vannamei pre-infected with Taura syndrome virus (TSV). Diseases of Aquatic Organisms, 98(3), 185-192.
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  PMID: 22535868;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-pathogenfree (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 TSVYHV 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. © Inter-Research 2012.
• Aranguren, L., Tang, K., & Lightner, D. (2012). Protection from yellow head virus (YHV)infection in Penaeus vannamei pre-infected withTaura syndrome virus (TSV). Diseases of Aquatic Organisms, 98, 185-192.
• Bakonyi, T., Bonning, B., Lightner, D., Chen, Y., Nakashima, N., Valles, S., & Christian, P. (2012). Dicistroviridae. Virus Taxonomy: 9th Report of the International Committee on Taxonomy of Viruses, 840-845.
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  In A.M.Q. King, M.J. Adams, E.B. Carstens and E.J. Lefkowitz, editors: Virus Taxonomy. 9th Report of the International Committee on Taxonomy of Viruses, Oxford, Elsevier
• Bakonyi, T., Bonning, B., Lightner, D., Chen, Y., Nakashima, N., Valles, S., & Christian, P. (2012). Iflaviridae. Virus Taxonomy: 9th Report of the International Committee on Taxonomy of Viruses, 846-849.
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  In Andrew M.Q. King, Michael J. Adams, Eric B. Carstens, and Elliot J. Lefkowitz, editors: Virus Taxonomy. 9th Report of the International Committee on Taxonomy of Viruses. Oxford. Elsevier
• Bonami, J., Lightner, D., Snijder, E., Cowley, J., Walker, P., de, G. R., & Flegel, T. (2012). Roniviridae. Virus Taxonomy: 9th Report of the International Committee on Taxonomy of Viruses, 829-834.
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  In A.M.Q. King, M.J. Adams, E.B. Carstens and E.J. Lefkowitz, editors: Virus Taxonomy. 9th Report of the International Committee on Taxonomy of Viruses. Oxford, Elsevier
• Cuéllar-Anjel, J., White-Noble, B., Schofield, P., Chamorro, R., & Lightner, D. V. (2012). Report of significant WSSV-resistance in the Pacific white shrimp, Litopenaeus vannamei, from a Panamanian breeding program. Aquaculture, 368-369, 36-39.
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  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. © 2012 Elsevier B.V.
• F., K., Navarro, S. A., Pantoja, C. R., Aranguren, F. L., & Lightner, D. V. (2012). New genotypes of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) from the Kingdom of Saudi Arabia. Diseases of Aquatic Organisms, 99(3), 179-185.
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  PMID: 22832716;Abstract: White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) are highly patho genic 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. © Inter-Research 2012.
• Lightner, D. (2012). Global transboundry disease politics. Journal of Invertebrate Pathology, 110, 184-187.
• Lightner, D. V., & Lightner, D. V. (2012). Global transboundry disease politics: The OIE perspective. Journal of Invertebrate Pathology, 110(2), 184-187.
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  PMID: 22434003;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. © 2012 Elsevier Inc.
• Lightner, D. V., Lightner, D. V., Redman, R. M., Redman, R. M., Pantoja, C. R., Pantoja, C. R., Tang, K. F., Tang, K. F., Noble, B. L., Noble, B. L., Schofield, P., Schofield, P., Mohney, L. L., Mohney, L. L., Nunan, L. M., Nunan, L. M., Navarro, S. A., & Navarro, S. A. (2012). Historic emergence, impact and current status of shrimp pathogens in the Americas. Journal of Invertebrate Pathology, 110(2), 174-183.
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  PMID: 22434000;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 3. years 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 15-20. years 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. © 2012 Elsevier Inc.
• Lightner, D. V., Lightner, D. V., Redman, R., Pantoja, C., Tang, K., Noble, B., Schofield, P., Mohnay, L., Nunan, L., & Navarro, S. (2012). Historic emergence, impact and current status of shrimp pathogens in the Americas. Journal of Invertebrate Pathology, 110, 174-183.
• Lightner, D. V., Stentiford, G., Neil, D., Peeler, E., Shields, J., Small, H., Flegel, T., Vlak, J., Jones, B., Morado, F., Moss, S., Lotz, J., Bartholomay, L., Behringer, D., Hauton, C., & Lightner, D. V. (2012). Disease will limit future food supply from the global crustacean fishery and aquaculture sectors. Journal of Invertebrate Pathology, 110, 141-157.
• Lightner, D., Aranguren, L. F., Tang, K. F., & Lightner, D. V. (2012). Protection from yellow head virus (YHV) infection in Penaeus vannamei pre-infected with Taura syndrome virus (TSV). Diseases of aquatic organisms, 98(3).
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  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.
• Lightner, D., Lightner, D. V., Tang, K. F., Navarro, S. A., Pantoja, C. R., & Aranguren, F. L. (2013). New genotypes of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) from the Kingdom of Saudi Arabia. Diseases of aquatic organisms, 99(3).
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  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.
• Moss, S. M., Moss, S. M., Moss, D. R., Moss, D. R., Arce, S. M., Arce, S. M., Lightner, D. V., Lightner, D. V., Lotz, J. M., & Lotz, J. M. (2012). The role of selective breeding and biosecurity in the prevention of disease in penaeid shrimp aquaculture. Journal of Invertebrate Pathology, 110(2), 247-250.
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  PMID: 22434005;Abstract: About 3.5 million metric tons of farmed shrimp were produced globally in 2009 with an estimated value greater than USD$Boyle, M.L.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. © 2012 Elsevier Inc.
• Moss, S., Moss, D., Arce, S., Lightner, D., & Lotz, J. (2012). The role of selective breeding and biosecurity in the prevention of disease in penaeid shrimp aquaculture. Journal of Invertebrate Pathology, 110, 247-250.
• Schneemann, A., Bonami, J., Lightner, D., Johnson, K., Nakai, T., & , R. (2012). Nodaviridae. Virus Taxonomy: 9th Report of the International Committee on Taxonomy of Viruses, 1061-1068.
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  In A. M.Q. King, M.J. Adams, E.B. Carstens and E.J. Lefkowitz, editors: Virus Taxonomy. 9th Report of the International Committee on Taxonomy of Viruses, Oxford, Elsevier
• Stentiford, G. D., Stentiford, G. D., Neil, D. M., Neil, D. M., Peeler, E. J., Peeler, E. J., Shields, J. D., Shields, J. D., Small, H. J., Small, H. J., Flegel, T. W., Flegel, T. W., Vlak, J. M., Vlak, J. M., Jones, B., Jones, B., Morado, F., Morado, F., Moss, S., , Moss, S., et al. (2012). Disease will limit future food supply from the global crustacean fishery and aquaculture sectors. Journal of Invertebrate Pathology, 110(2), 141-157.
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  PMID: 22434002;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. © 2012 Elsevier Inc.
• Tang, K., Navarro, S., Pantoja, C., Aranguren, F., & Lightner, D. (2012). New genotypes of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) from the Kingdom of Saudi Arabia. Diseases of Aquatic Organisms, 99, 179-185.
• F., K., & Lightner, D. V. (2011). Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon. Diseases of Aquatic Organisms, 93(3), 191-198.
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  PMID: 21516971;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 × 108 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. © Inter-Research 2011.
• Heres, A., Redman, R., & Lightner, D. (2011). Histopathology of Spiroplasma penaei systemic infection in experimentally infected Pacific white shrimp, Penaeus vannamei. The Israeli Journal of Aquaculture Bamidgeh, IIC(63), 589-596.
• Heres, A., Redman, R., & Lightner, D. V. (2011). Histopathology of Spiroplasma penaei systemic infection in experimentally infected pacific white shrimp, Penaeus vannamei. Israeli Journal of Aquaculture - Bamidgeh, 63(1).
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  Abstract: Penaeus vannamei shrimp were challenged with a suspension of a pathogenic isolate of Spiroplasma penaei prepared from a 72-h culture. The route of challenge was by intramuscular injection of the bacterial suspension into the third abdominal segment. Lesion development was evaluated in moribund shrimp collected and fixed in Davidson's fixative 96 h post challenge. The predominant host responses to infection by S. penaei observed by histological examination were the general systemic development of hemocytic nodules (often melanized) and poorly organized hemocytic infiltration. Such lesions were most prevalent in the lymphoid organ, gill filaments, heart, connective tissue, antennal gland, and skeletal muscle. The presence of S. penaei in the lesions was verified by in situ hybridization using a digoxigenin (DIG)-labeled probe specific to the spiralin gene of Spiroplasma spp. Transmission electron micrographs (TEM) showed S. penaei cells free in the cytoplasm of lymphoid organ cells. The cultures of S. penaei used for this study and infected abdominal tissue were verified by PCR using spiroplasma-specific primers that amplify a fragment from a variable region of the 16S rDNA gene sequence.
• Lightner, D. (2011). Status of shrimp diseases and advances in shrimp health management. Diseases in Asian Aquaculture, VII, 121-134.
• Lightner, D. (2011). Virus diseases of farmed shrimp in the Western Hemisphere (the Americas): a review. Journal of Invertebrate Pathology, 106, 110-130.
• Lightner, D. V., & Lightner, D. V. (2011). Virus diseases of farmed shrimp in the Western Hemisphere (the Americas): A review. Journal of Invertebrate Pathology, 106(1), 110-130.
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  PMID: 21215359;Abstract: Penaeid shrimp aquaculture is an important industry in the Americas, and the industry is based almost entirely on the culture of the Pacific White Shrimp, Litopenaeus vannamei. Western Hemisphere shrimp farmers in 14 countries in 2004 produced more than 200,000 metric tons of shrimp, generated more than $2 billion in revenue, and employed more than 500,000 people. Disease has had a major impact on shrimp aquaculture in the Americas since it became a significant commercial entity in the 1970s. Diseases due to viruses, rickettsial-like bacteria, true bacteria, protozoa, and fungi have emerged as major diseases of farmed shrimp in the region. Many of the bacterial, fungal and protozoan caused diseases are managed using improved culture practices, routine sanitation, and the use of chemotherapeutics. However, the virus diseases have been far more problematic to manage and they have been responsible for the most costly epizootics. Examples include the Taura syndrome pandemic that began in 1991-1992 when the disease emerged in Ecuador, and the subsequent White Spot Disease pandemic that followed its introduction to Central America from Asia in 1999. Because of their socioeconomic significance to shrimp farming, seven of the nine crustacean diseases listed by the World Animal Organization (OIE) are virus diseases of shrimp. Of the seven virus diseases of penaeid shrimp, five are native to the Americas or have become enzootic following their introduction. The shrimp virus diseases in the Americas are increasingly being managed by exclusion using a combination of biosecurity and the practice of culturing domesticated specific pathogen-free (SPF) stocks or specific pathogen-resistant (SPR) stocks. Despite the significant challenges posed by disease, the shrimp farming industry of the Americas has responded to the challenges posed by disease and it has developed methods to manage its diseases and mature into a sustainable industry. © 2010 Elsevier Inc.
• Lightner, D., Nunan, L. M., & Lightner, D. V. (2011). Optimized PCR assay for detection of white spot syndrome virus (WSSV). Journal of virological methods, 171(1).
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  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.
• Lightner, D., Tang, K. F., & Lightner, D. V. (2011). Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon. Diseases of aquatic organisms, 93(3).
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  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.
• Lightner, D., Tang, K. F., Messick, G. A., Pantoja, C. R., Redman, R. M., & Lightner, D. V. (2011). Histopathological characterization and in situ detection of Callinectes sapidus reovirus. Journal of invertebrate pathology, 108(3).
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  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.
• Lightner, D., Tang, K. F., Pantoja, C. R., Redman, R. M., Navarro, S. A., & Lightner, D. V. (2011). Ultrastructural and sequence characterization of Penaeus vannamei nodavirus (PvNV) from Belize. Diseases of aquatic organisms, 94(3).
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  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.
• Nunan, L. M., & Lightner, D. V. (2011). Optimized PCR assay for detection of white spot syndrome virus (WSSV). Journal of Virological Methods, 171(1), 318-321.
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  PMID: 21111001;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. © 2010 Elsevier B.V.
• Nunan, L., & Lightner, D. (2011). Optimized PCR assay for detection of white spot syndrome virus (WSSV). Journal of Virological Methods, 171, 318-321.
• Stentiford, G. D., & Lightner, D. V. (2011). Cases of White Spot Disease (WSD) in European shrimp farms. Aquaculture, 319(1-2), 302-306.
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  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. © 2011.
• Stentiford, G., & Lightner, D. (2011). Cases of White Spot Disease (WSD) in European shrimp farms. Aquaculture, 319, 302-306.
• Tang, K. F., Pantoja, C. R., Redman, R. M., Navarro, S. A., & Lightner, D. V. (2011). Ultrastructural and sequence characterization of Penaeus vannamei nodavirus (PvNV) from Belize. Diseases of Aquatic Organisms, 94(3), 179-187.
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  PMID: 21790065;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. Phylo genetic 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. © Inter-Research 2011.
• Tang, K. F., Tang, K. F., Messick, G. A., Messick, G. A., Pantoja, C. R., Pantoja, C. R., Redman, R. M., Redman, R. M., Lightner, D. V., & Lightner, D. V. (2011). Histopathological characterization and in situ detection of Callinectes sapidus reovirus. Journal of Invertebrate Pathology, 108(3), 226-228.
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  PMID: 21925184;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. © 2011 Elsevier Inc.
• Tang, K., & Lightner, D. (2011). Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon. Diseases of Aquatic Organisms, 93, 191-198.
• Tang, K., Messick, G., Pantoja, C., Redman, R., & Lightner, D. (2011). Histopathological characterization and in situ detection of Callinectes sapidus reovirus. Journal of Invertebrate Pathology, 108, 226-228.
• Tang, K., Pantoja, C., Redman, R., Navarro, S., & Lightner, D. (2011). Ultrastructural and sequence characterization of Penaeus vannamei nodavirus (PvNV) from Belize. Diseases of Aquatic Organisms, 94, 179-187.
• Aranguren, L. F., Tang, K. F., & Lightner, D. V. (2010). Quantification of the bacterial agent of necrotizing hepatopancreatitis (NHP-B) by real-time PCR and comparison of survival and NHP load of two shrimp populations. Aquaculture, 307(3-4), 187-192.
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  Abstract: A real-time quantitative PCR (qPCR) assay was developed using a TaqMan probe to detect and quantify the necrotizing hepatopancreatitis bacterium (NHP-B) in Penaeus vannamei. A pair of primers which amplify a 67bp DNA fragment and a TaqMan probe were selected from the 16S rRNA gene of NHP-B genome. A positive control plasmid DNA was used to demonstrate that the NHP qPCR assay has a detection limit of 100 copies and a log-linear range up to 108 copies. An NHP-B challenge test using two different populations, Colombia (COL) and Specific Pathogen Free KONA line (KONA), was conducted. Higher final survival and a better survival curve were found in the COL population. No differences in NHP-B bacterial load were found in hepatopancreas (HP) analyzed from the survivors from each population. Sensitivity of the qPCR test was much higher than the conventional PCR (100 copies vs 1×105 copies). © 2010 Elsevier B.V.
• Aranguren, L., Tang, K., & Lightner, D. (2010). Quantification of the bacterial agent of necrotizing hepatopancreatitis (NHP-B) by real-time PCR and comparison of survival and NHP load of two shrimp populations. Aquaculture, 307, 187-192.
• Côté, I., & Lightner, D. V. (2010). Hyperthermia does not protect Kona stock Penaeus vannamei against infection by a Taura syndrome virus isolate from Belize. Diseases of Aquatic Organisms, 88(2), 157-160.
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  PMID: 20225676;Abstract: This study evaluated the susceptibility of Penaeus vannamei, Kona stock-line, to infection by an isolate of Taura syndrome virus from Belize (TSV-BZ) under hyperthermic conditions (32°C). Shrimp exposed to the reference Hawaii-94 isolate of TSV (TSV-HI) showed resistance to infection at 32°C as demonstrated by the absence of mortality, histopathological lesions and decreased viral load by qPCR. However, at 32°C, shrimp were fully susceptible to the disease caused by TSV-BZ, exhibiting high mortality, severe histopathological lesions and increased viral load. This susceptibility of shrimp to TSV-BZ infection under hyperthermic conditions was independent of the route of infection (injection vs. per os) and the salinity of the water (11 vs. 28). TSV-BZ might be a temperature-permissible mutant of TSV. © Inter-Research 2010.
• Heres, A., Heres, A., Lightner, D. V., & Lightner, D. V. (2010). Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis. Journal of Invertebrate Pathology, 103(1), 30-35.
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  PMID: 19836398;Abstract: A pathogenic Spiroplasma penaei strain was isolated from the hemolymph of moribund Pacific white shrimp, Penaeus vannamei. The shrimp sample originated from a shrimp farm near Cartagena, Colombia, that was suffering from high mortalities in ponds with very low salinity and high temperatures. This new emerging disease in a marine crustacean in the Americas is described as a systemic infection. The multilocus phylogenetic analysis suggests that S. penaei strain has a terrestrial origin. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S rDNA and 23S-5S rDNA intergenic spacer region, were reconstructed using the distance-based Neighboring-Joining (NJ) method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborate the observations by other investigators using the 16S rDNA gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to Spiroplasma insolitum and distantly related to Spiroplasma sp. SHRIMP from China. © 2009 Elsevier Inc. All rights reserved.
• Lightner, D., & Redman, R. (2010). The global status of significant infectious diseases of farmed shrimp. Asian Fisheries Society Journal, 23(4), 383-426.
• Lightner, D., Côté, I., & Lightner, D. V. (2010). Hyperthermia does not protect Kona stock Penaeus vannamei against infection by a Taura syndrome virus isolate from Belize. Diseases of aquatic organisms, 88(2).
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  This study evaluated the susceptibility of Penaeus vannamei, Kona stock-line, to infection by an isolate of Taura syndrome virus from Belize (TSV-BZ) under hyperthermic conditions (32 degrees C). Shrimp exposed to the reference Hawaii-94 isolate of TSV (TSV-HI) showed resistance to infection at 32 degrees C as demonstrated by the absence of mortality, histopathological lesions and decreased viral load by qPCR. However, at 32 degrees C, shrimp were fully susceptible to the disease caused by TSV-BZ, exhibiting high mortality, severe histopathological lesions and increased viral load. This susceptibility of shrimp to TSV-BZ infection under hyperthermic conditions was independent of the route of infection (injection vs. per os) and the salinity of the water (11 vs. 28). TSV-BZ might be a temperature-permissible mutant of TSV.
• Lightner, D., Heres, A., & Lightner, D. V. (2010). Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis. Journal of invertebrate pathology, 103(1).
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  A pathogenic Spiroplasma penaei strain was isolated from the hemolymph of moribund Pacific white shrimp, Penaeus vannamei. The shrimp sample originated from a shrimp farm near Cartagena, Colombia, that was suffering from high mortalities in ponds with very low salinity and high temperatures. This new emerging disease in a marine crustacean in the Americas is described as a systemic infection. The multilocus phylogenetic analysis suggests that S. penaei strain has a terrestrial origin. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S rDNA and 23S-5S rDNA intergenic spacer region, were reconstructed using the distance-based Neighboring-Joining (NJ) method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborate the observations by other investigators using the 16S rDNA gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to Spiroplasma insolitum and distantly related to Spiroplasma sp. SHRIMP from China.
• Lightner, D., Muller, I. C., Andrade, T. P., Tang-Nelson, K. F., Marques, M. R., & Lightner, D. V. (2010). Genotyping of white spot syndrome virus (WSSV) geographical isolates from Brazil and comparison to other isolates from the Americas. Diseases of aquatic organisms, 88(2).
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  White spot syndrome virus (WSSV) is a viral pathogen that has caused significant economic losses in shrimp farming. Variable-number tandem repeats (VNTRs) (open reading frame [ORF] 94, 125 and 75), a large deletion (ORF 23/24) and a transposase were proposed as molecular markers for genotyping. WSSV-infected shrimp Litopenaeus vannamei were collected in 2 Brazilian regions (Santa Catarina and Bahia) from 2005 to 2008. DNA was extracted and PCR of the variable regions was performed, followed by sequencing. All Santa Catarina samples showed the same number of repeats for the minisatellites analyzed. Bahia samples showed a different pattern for the regions, indicating that there are at least 2 different WSSV genotypes in Brazil. Both Brazilian isolates have an 11453 bp deletion in ORF 23/24 when compared with WSSV-TW (Taiwan), which has the full sequence for this locus. The Brazilian WSSV isolates were compared with WSSV isolates from other countries in the Americas (USA, Panama, Honduras, Mexico and Nicaragua); the repeat number patterns for the 3 VNTR regions analyzed were different between the Brazilian isolates and the other western-hemisphere isolates. This may be due to mutations in WSSV after its introduction into the different countries. Our results also show that WSSV found in Bahia and Santa Catarina very likely originated from different sources of contamination.
• Lightner, D., Nunan, L. M., Poulos, B. T., Navarro, S., Redman, R. M., & Lightner, D. V. (2010). Milky hemolymph syndrome (MHS) in spiny lobsters, penaeid shrimp and crabs. Diseases of aquatic organisms, 91(2).
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  Black tiger shrimp Penaeus monodon, European shore crab Carcinus maenas and spiny lobster Panulirus spp. can be affected by milky hemolymph syndrome (MHS). Four rickettsia-like bacteria (RLB) isolates of MHS originating from 5 geographical areas have been identified to date. The histopathology of the disease was characterized and a multiplex PCR assay was developed for detection of the 4 bacterial isolates. The 16S rRNA gene and 16-23S rRNA intergenic spacer region (ISR) were used to examine the phylogeny of the MHS isolates. Although the pathology of this disease appears similar in the various different hosts, sequencing and examination of the phylogenetic relationships reveal 4 distinct RLB involved in the infection process.
• Muller, I. C., P., T., F., K., R., M., & Lightner, D. V. (2010). Genotyping of White spot syndrome virus (WSSV) geographical isolates from Brazil and comparison to other isolates from the Americas. Diseases of Aquatic Organisms, 88(2), 91-98.
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  PMID: 20225670;Abstract: White spot syndrome virus (WSSV) is a viral pathogen that has caused significant economic losses in shrimp farming. Variable-number tandem repeats (VNTRs) (open reading frame [ORF] 94, 125 and 75), a large deletion (ORF 23/24) and a transposase were proposed as molecular markers for genotyping. WSSV-infected shrimp Litopenaeus vannamei were collected in 2 Brazilian regions (Santa Catarina and Bahia) from 2005 to 2008. DNA was extracted and PCR of the variable regions was performed, followed by sequencing. All Santa Catarina samples showed the same number of repeats for the minisatellites analyzed. Bahia samples showed a different pattern for the regions, indicating that there are at least 2 different WSSV genotypes in Brazil. Both Brazilian isolates have an 11453 bp deletion in ORF 23/24 when compared with WSSV-TW (Taiwan), which has the full sequence for this locus. The Brazilian WSSV isolates were compared with WSSV isolates from other countries in the Americas (USA, Panama, Honduras, Mexico and Nicaragua); the repeat number patterns for the 3 VNTR regions analyzed were different between the Brazilian isolates and the other western-hemisphere isolates. This may be due to mutations in WSSV after its introduction into the different countries. Our results also show that WSSV found in Bahia and Santa Catarina very likely originated from different sources of contamination. © Inter-Research 2010.
• Nunan, L. M., Poulos, B. T., Navarro, S., Redman, R. M., & Lightner, D. V. (2010). Milky hemolymph syndrome (MHS) in spiny lobsters, penaeid shrimp and crabs. Diseases of Aquatic Organisms, 91(2), 105-112.
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  PMID: 21387989;Abstract: Black tiger shrimp Penaeus monodon, European shore crab Carcinus maenas and spiny lobster Panulirus spp. can be affected by milky hemolymph syndrome (MHS). Four rickettsia-like bacteria (RLB) isolates of MHS originating from 5 geographical areas have been identified to date. The histopathology of the disease was characterized and a multiplex PCR assay was developed for detection of the 4 bacterial isolates. The 16S rRNA gene and 16-23S rRNA intergenic spacer region (ISR) were used to examine the phylogeny of the MHS isolates. Although the pathology of this disease appears similar in the various different hosts, sequencing and examination of the phylogenetic relationships reveal 4 distinct RLB involved in the infection process. © Inter-Research 2010.
• Yan, D. C., Tang, K. F., & Lightner, D. V. (2010). A real-time PCR for the detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp. Journal of Fish Diseases, 33(6), 507-511.
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  PMID: 20367743;Abstract: Hepatopancreatic parvovirus (HPV) causes a common shrimp disease that occurs in many shrimp farming regions, especially in the Indo Pacific, and infects most of the cultured penaeid species. There are seven geographic HPV isolates known, so a method to detect different HPV types is needed. We developed a sensitive and generic real-time PCR assay for the detection of HPV. A pair of primers and TaqMan probe based on an HPV sequence obtained from samples of Fenneropenaeus chinensis from Korea were selected, and they were used to amplify a 92 bp DNA fragment. This real-time PCR was found to be specific to HPV and did not react with other shrimp viruses. A plasmid (pHPV-2) containing the target HPV sequence was constructed and used for determination of the sensitivity of this assay. The assay could detect a single copy of plasmid DNA, and it was used successfully in finding HPV in shrimp samples from the China-Yellow Sea region, Taiwan, Korea, Thailand, Madagascar, New Caledonia and Tanzania. © 2010 Blackwell Publishing Ltd.
• Andrade, T. P., & Lightner, D. V. (2009). Development of a method for the detection of infectious myonecrosis virus by reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow hybrid assay. Journal of Fish Diseases, 32(11), 911-924.
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  PMID: 19531063;Abstract: We report the development of a reverse-transcription loop-mediated isothermal amplification and nucleic acid lateral flow method (RT-LAMP-NALF) for detection of infectious myonecrosis virus (IMNV). The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The sensitivity of RT-LAMP (using two and three primer pairs) and nested RT-LAMP (using three primer pairs) was compared by real-time reverse-transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. The detection of RT-LAMP (three primer pairs) products was accomplished by using a NALF-test strip. The RT-LAMP-NALF showed equivalent sensitivity to RT-LAMP (using three primer pairs), and it was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. On the other hand, the RT-LAMP-NALF was 10 and 100 times less sensitive than nested RT-PCR and real-time RT-PCR, respectively. The simplified RNA extraction method ranged from 4.4 × 106 to 2.2 × 108 IMNV copy numbers μL-1 RNA, and it was similar with the standard RNA extraction (from 1.2 × 106 to 6.3 × 107 IMNV copy numbers μL-1 RNA). These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings. © 2009 Blackwell Publishing Ltd.
• Côté, I., Poulos, B. T., Redman, R. M., & Lightner, D. V. (2009). Development and characterization of a monoclonal antibody against Taura syndrome virus. Journal of Fish Diseases, 32(12), 989-996.
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  PMID: 19602090;Abstract: We produced a panel of monoclonal antibodies (MAbs) from the fusion of Taura syndrome virus variants from Belize (TSV-BZ) immunized BALB/cJ mouse spleen cells and non-immunoglobulin secreting SP2/0 mouse myeloma cells. One antibody, 2C4, showed strong specificity and sensitivity for TSV in dot-blot immunoassay and immunohistochemistry (IHC) analysis. The MAb reacted against native TSV-BZ, TSV variants from Sinaloa, Mexico (TSV-SI) and TSV variants from Hawaii (TSV-HI) in dot-blot immunoassay. By IHC, the antibody identified the virus in a pattern similar to the digoxigenin-labelled TSV-cDNA probe for the TSV-BZ, TSV-HI and TSV-SI variants, but not for the TSV variants from Venezuela (TSV-VE) and the TSV variants from Thailand (TSV-TH). MAb 2C4 did not react against other shrimp pathogens or with normal shrimp tissue. Western blot analysis showed a strong reaction against CP2, a region of high antigenic variability amongst TSV variants. This antibody has potential diagnostic application in detection and differentiation of certain TSV biotypes. © 2009 Blackwell Publishing Ltd.
• Lightner, D. V., Pantoja, C. R., Redman, R. M., Hasson, K. W., & Menon, J. P. (2009). Case reports of melamine-induced pathology in penaeid shrimp fed adulterated feeds. Diseases of Aquatic Organisms, 86(2), 107-112.
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  PMID: 19902839;Abstract: Shrimp (Penaeus monodon and P. vannamei) specimens were submitted to the University of Arizona's Aquaculture Pathology Laboratory (UAZAPL) and to the Texas Veterinary Medical Diagnostic Laboratory (TVMDL) in 2007 and 2008 from shrimp-rearing facilities in India and Indonesia for histological testing. These were found to present prominent golden to greenish-brown needle-and plate-like birefringent crystals within multifocal hemocytic granulomas in the antennal gland tubules and peritubular hemal sinuses. Their appearance was very similar to melamine-cyanuric acid-induced crystals previously described from cat and dog kidneys with melamine-associated renal failure (MARF). Significant chronic mortalities were reported from the affected P. vannamei farms in Indonesia, but were not observed in the affected P. monodon facility in India. Shrimp feed was suspected as the source of melamine due to the similarity of the shrimp antennal gland lesions to those present in MARF. 'Normal' and 'suspect' feed samples from the facilities in Indonesia and India were sent to regional laboratories for analysis. Melamine was detected in 2 of 4 feed samples from an affected Indonesian farm. Melamine was not detected in 'normal' feed from the Indian facility, but it was found in 2 'suspect' samples (Feeds A and B) at levels of 183.39 and 112.50 ppm, respectively. A bioassay of Feed A with P. vannamei at UAZAPL confirmed that the melamine-contaminated feed induced prominent granulomas in the antennal gland with the characteristic crystals within 10 d of the first feeding, experimentally confirming the direct relationship of melamine-adulterated feed to the unique pathology observed.
• Lightner, D., Wertheim, J. O., Tang, K. F., Navarro, S. A., & Lightner, D. V. (2009). A quick fuse and the emergence of Taura syndrome virus. Virology, 390(2).
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  Over the last two decades, Taura syndrome virus (TSV) has emerged as a major pathogen in penaeid shrimp aquaculture and has caused substantial economic loss. The disease was first discovered in Ecuador in 1991, and the virus is now globally distributed with the greatest concentration of infections in the Americas and Southeast Asia. To determine the evolutionary history of this virus, we constructed a phylogeny containing 83 TSV isolates from 16 countries sampled over a 16-year period. This phylogeny was inferred using a relaxed molecular clock in a Bayesian Markov chain Monte Carlo framework. We found phylogenetic evidence that the TSV epidemic did indeed originate in the Americas sometime around 1991 (1988-1993). We estimated the TSV nucleotide substitution rate at 2.37 x 10(-3) (1.98 x 10(-3) to 2.82 x 10(-3)) substitutions/site/year within capsid gene 2. In addition, the phylogeny was able to independently corroborate many of the suspected routes of TSV transmission around the world. Finally, we asked whether TSV emergence in new geographic locations operates under a quick fuse (i.e. rapid appearance of widespread disease). Using a relaxed molecular clock, we determined that TSV is almost always discovered within a year of entering a new region. This suggests that current monitoring programs are effective at detecting novel TSV outbreaks.
• Navarro, S. A., Tang, K. F., & Lightner, D. V. (2009). An improved Taura syndrome virus (TSV) RT-PCR using newly designed primers. Aquaculture, 293(3-4), 290-292.
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  Abstract: Taura syndrome virus (TSV)-specific primers, designated as 7171F/7511R, were designed to improve the sensitivity of RT-PCR detection. This pair of primers was shown to detect TSV isolates representative of four phylogenetic lineages: Belize, Americas, SE Asia, and Venezuela. Its detection limit was determined to be 20 copies of the TSV genome, 100 times more sensitive than the TSV primers 9992/9195 currently being used by many laboratories and recommended for surveillance and diagnostic applications by the Office International des Epizooties. Primers 7171F/7511R were found to be specific to TSV and did not to react to either Infectious hypodermal and hematopoietic necrosis virus (IHHNV), White spot syndrome virus (WSSV), Yellow head virus (YHV), or Infectious myonecrosis virus (IMNV). This new RT-PCR method was shown to detect TSV in chronically infected Penaeus vannamei that had survived up to 236 days after exposure to TSV. © 2009 Elsevier B.V. All rights reserved.
• Wertheim, J. O., Tang, K. F., Navarro, S. A., & Lightner, D. V. (2009). A quick fuse and the emergence of Taura syndrome virus. Virology, 390(2), 324-329.
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  PMID: 19524995;Abstract: Over the last two decades, Taura syndrome virus (TSV) has emerged as a major pathogen in penaeid shrimp aquaculture and has caused substantial economic loss. The disease was first discovered in Ecuador in 1991, and the virus is now globally distributed with the greatest concentration of infections in the Americas and Southeast Asia. To determine the evolutionary history of this virus, we constructed a phylogeny containing 83 TSV isolates from 16 countries sampled over a 16-year period. This phylogeny was inferred using a relaxed molecular clock in a Bayesian Markov chain Monte Carlo framework. We found phylogenetic evidence that the TSV epidemic did indeed originate in the Americas sometime around 1991 (1988-1993). We estimated the TSV nucleotide substitution rate at 2.37 × 10- 3 (1.98 × 10- 3 to 2.82 × 10- 3) substitutions/site/year within capsid gene 2. In addition, the phylogeny was able to independently corroborate many of the suspected routes of TSV transmission around the world. Finally, we asked whether TSV emergence in new geographic locations operates under a quick fuse (i.e. rapid appearance of widespread disease). Using a relaxed molecular clock, we determined that TSV is almost always discovered within a year of entering a new region. This suggests that current monitoring programs are effective at detecting novel TSV outbreaks. © 2009 Elsevier Inc. All rights reserved.
• Yan, D., Yan, D., Tang, K. F., Tang, K. F., Lightner, D. V., & Lightner, D. V. (2009). Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp. Journal of Invertebrate Pathology, 102(2), 97-100.
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  PMID: 19622362;Abstract: A real-time PCR method was developed to detect monodon baculovirus (MBV) in penaeid shrimp. A pair of MBV primers to amplify a 135 bp DNA fragment and a TaqMan probe were developed. The primers and TaqMan probe were specific for MBV and did not cross react with Hepatopancreatic parvovirus (HPV), White spot syndrome virus (WSSV), Infectious hypodermal and haematopoietic virus (IHHNV) and specific pathogen free (SPF) shrimp DNA. A plasmid (pMBV) containing the target MBV sequence was constructed and used for determination of the sensitivity of the real-time PCR. This real-time PCR assay had a detection limit of one plasmid MBV DNA copy. Most significantly, this real-time PCR method can detect MBV positive samples from different geographic locations in the University of Arizona collection, including Thailand and Indonesia collected over a 13-year period. © 2009 Elsevier Inc.
• Andrade, T. P., Redman, R. M., & Lightner, D. V. (2008). Evaluation of the preservation of shrimp samples with Davidson's AFA fixative for infectious myonecrosis virus (IMNV) in situ hybridization. Aquaculture, 278(1-4), 179-183.
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  Abstract: The potential negative effect of prolonged storage of shrimp tissues in Davidson's AFA fixative on in situ hybridization (ISH) signal was demonstrated previously for Taura syndrome virus (TSV), which has a single-stranded RNA genome. In this study we evaluated if prolonged storage of infectious myonecrosis virus (IMNV) infected shrimp in Davidson's AFA (Alcohol, Formaldehyde, Acetic acid) fixative will degrade its double-stranded RNA genome resulting in false negative ISH reactions. Twenty-one shrimp (3 g) specific-pathogen-free Litopenaeus vannamei were used in this study. Three shrimp were used as negative control and 18 shrimp were inoculated with a tissue homogenate prepared from frozen IMNV-infected L. vannamei obtained from Brazil in 2003 (positive control). Shrimp were collected at Day 12 post-injection and fixed in Davidson's AFA for five different preservation times (1, 2, 4, 7 and 10 days). After the different fixation times, the Davidson's AFA was replaced with several changes of 70% ethanol until the pH was stable. IMNV lesions were confirmed in all positive control shrimp by routine H & E histology and ISH. Myonecrosis lesions were strongly positive by ISH at all five preservation times evaluated. Hence, in the present report it was found that the length of time (up to 10 days) in Davidson's AFA did not have a deleterious effect on the ISH reaction for IMNV. © 2008 Elsevier B.V. All rights reserved.
• Côté, I., Navarro, S., Tang, K. F., Noble, B., & Lightner, D. V. (2008). Taura syndrome virus from Venezuela is a new genetic variant. Aquaculture, 284(1-4), 62-67.
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  Abstract: In early 2005, the Aquaculture Pathology Laboratory of the University of Arizona received samples of diseased shrimp, Penaeus vannamei, from a Taura syndrome (TS) outbreak in the Lake Maracaibo region of Venezuela. Histopathology and in situ hybridization (ISH) were performed and the results confirmed the presence of Taura syndrome virus (TSV). The viral isolate was sequenced and presented a 93% similarity with the TSV reference strain from Hawaii (TSV-HI94). Immunohistochemistry (IHC), dot blot immunoassay and bioassays were also performed. While processed samples reacted only faintly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassay, the Venezuelan isolate of TSV (TSV-VE05) presented mortality comparable to TSV-HI94 in P. vannamei SPF Kona stock. These data indicate that a new variant of the virus was responsible for the outbreak of TS in Venezuela. © 2008 Elsevier B.V. All rights reserved.
• Lightner, D., Nunan, L. M., Pantoja, C., & Lightner, D. V. (2008). Improvement of a PCR method for the detection of necrotizing hepatopancreatitis in shrimp. Diseases of aquatic organisms, 80(1).
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  Necrotizing hepatopancreatitis (NHP) is considered to be one of the most important bacterial diseases affecting penaeid shrimp culture and is caused by an unclassified Gram-negative, pleomorphic, intracellular Alphaproteobacterium. Due to the enteric nature of the bacteria, PCR is the one non-lethal method available for detection of the pathogen. Over a decade ago, a PCR protocol was developed for detection of NHP, which over the subsequent years was shown to occasionally generate false positive reactions. The University of Arizona Aquaculture Pathology Laboratory has developed a set of primers and PCR cycling parameters that have been tested on a variety of DNA templates, using 2 types of PCR reagent systems, which eliminated the generation of false positive amplicons.
• Lightner, D., Tang, K. F., Pantoja, C. R., & Lightner, D. V. (2008). Nucleotide sequence of a Madagascar hepatopancreatic parvovirus (HPV) and comparison of genetic variation among geographic isolates. Diseases of aquatic organisms, 80(2).
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  A segment of Madagascar hepatopancreatic parvovirus (HPV) genomic sequence (5742 nucleotides) was determined through PCR and direct sequencing. This nucleotide sequence was compared to isolates from Australia, Thailand, Korea, and Tanzania, and the mean distance was determined to be 17%. The Madagascar HPV is closest to the Tanzania isolate (12%), followed by isolates from Korea (15%), Australia (17%) and Thailand (20%). Analysis of the genomic structure revealed that this HPV sequence is comprised of one partial Left open reading frame (ORF) (349 amino acids, aa) and complete Mid (578 aa) and Right (820 aa) ORFs. The amino acid sequences of the 3 ORFs were compared among isolates. The Right ORF was found to have the highest variation with a mean distance of 24%. This was followed by the Left and Mid ORF with distances of 13 and 7%, respectively. A phylogenetic analysis based on the amino acid sequence of the Right ORF divides 7 HPV isolates into 3 well-separated groups: Korea, Thailand, and Australia. The Madagascar HPV clustered with the Korea and Tanzania isolates. In Madagascar, HPV has been detected by histological examination since the 1990s. PCR analysis of a recent (2007) sampling showed a 100% prevalence. HPV was also detected in Mozambique with a 100% prevalence. High (95%) prevalence of HPV was found in wild Penaeus merguinesis collected from New Caledonia. These results indicate that HPV displays a high degree of genetic diversity and is distributed worldwide among populations of penaeid shrimp.
• Nunan, L. M., Pantoja, C., & Lightner, D. V. (2008). Improvement of a PCR method for the detection of necrotizing hepatopancreatitis in shrimp. Diseases of Aquatic Organisms, 80(1), 69-73.
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  PMID: 18714686;Abstract: Necrotizing hepatopancreatitis (NHP) is considered to be one of the most important bacterial diseases affecting penaeid shrimp culture and is caused by an unclassified Gram-negative, pleomorphic, intracellular Alphaproteobacterium. Due to the enteric nature of the bacteria, PCR is the one non-lethal method available for detection of the pathogen. Over a decade ago, a PCR protocol was developed for detection of NHP, which over the subsequent years was shown to occasionally generate false positive reactions. The University of Arizona Aquaculture Pathology Laboratory has developed a set of primers and PCR cycling parameters that have been tested on a variety of DNA templates, using 2 types of PCR reagent systems, which eliminated the generation of false positive amplicons. © Inter-Research 2008.
• Poulos, B. T., Noble, B. W., & Lightner, D. V. (2008). Comparison of Taura syndrome virus (TSV) detection methods during chronic-phase infection in Penaeus vannamei. Diseases of Aquatic Organisms, 82(3), 179-185.
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  PMID: 19244969;Abstract: Methods to detect Taura syndrome virus (TSV) were assessed for their ability to detect the virus during chronic phase infection in the Pacific white shrimp Penaeus vannamei. In situ hybridization (ISH), immunohistochemistry (IHC) using monoclonal antibody 1A1, conventional RT-PCR and real-time quantitative (q)RT-PCR were compared using shrimp sampled over 60 wk following experimental TSV infection. Between Weeks 7 and 60, hematoxylin-eosin histology confirmed the presence of lymphoid organ spheroids (LOS) and an absence of lesions in the cuticular epithelium. ISH detected TSV in LOS over the duration of the study. IHC was generally less sensitive than ISH, and after Week 24, was often unable to confirm TSV infection. Detection of TSV by RT-PCR was highly dependent on sample source after Week 43, where viral RNA was detected in 12 of 14 hemolymph samples but only 5 of 16 pleopod samples. qRT-PCR detected TSV over the 60 wk in both hemolymph and pleopods, although RNA copy numbers in pleopods were consistently lower throughout the study. This study demonstrates that ISH and qRT-PCR are the most reliable methods for detecting TSV during late chronic phase infection. RT-PCR was also reliable if hemolymph was used as the sample source. © Inter-Research 2008.
• Tang, J., Ochoa, W. F., Sinkovits, R. S., Poulos, B. T., Ghabrial, S. A., Lightner, D. V., Baker, T. S., & Nibert, M. L. (2008). Infectious myonecrosis virus has a totivirus-like, 120-subunit capsid, but with fiber complexes at the fivefold axes. Proceedings of the National Academy of Sciences of the United States of America, 105(45), 17526-17531.
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  PMID: 18981418;PMCID: PMC2582295;Abstract: Infectious myonecrosis virus (IMNV) is an emerging pathogen of penaeid shrimp in global aquaculture. Tentatively assigned to family Totiviridae, it has a nonsegmented dsRNA genome of 7,560 bp and an isometric capsid of the 901-aa major capsid protein. We used electron cryomicroscopy and 3D image reconstruction to examine the IMNV virion at 8.0-Å resolution. Results reveal a totivirus-like, 120-subunit T = 1 capsid, 450 Å in diameter, but with fiber complexes protruding a further 80 Å at the fivefold axes. These protrusions likely mediate roles in the extracellular transmission and pathogenesis of IMNV, capabilities not shared by most other totiviruses. The IMNV structure is also notable in that the genome is centrally organized in five or six concentric shells. Within each of these shells, the densities alternate between a dodecahedral frame that connects the threefold axes vs. concentration around the fivefold axes, implying certain regularities in the RNA packing scheme. © 2008 by The National Academy of Sciences of the USA.
• Tang, K. F., Pantoja, C. R., & Lightner, D. V. (2008). Nucleotide sequence of a Madagascar hepatopancreatic parvovirus (HPV) and comparison of genetic variation among geographic isolates. Diseases of Aquatic Organisms, 80(2), 105-112.
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  PMID: 18717063;Abstract: A segment of Madagascar hepatopancreatic parvovirus (HPV) genomic sequence (5742 nucleotides) was determined through PCR and direct sequencing. This nucleotide sequence was compared to isolates from Australia, Thailand, Korea, and Tanzania, and the mean distance was determined to be 17%. The Madagascar HPV is closest to the Tanzania isolate (12%), followed by isolates from Korea (15%), Australia (17%) and Thailand (20%). Analysis of the genomic structure revealed that this HPV sequence is comprised of one partial Left open reading frame (ORF) (349 amino acids, aa) and complete Mid (578 aa) and Right (820 aa) ORFs. The amino acid sequences of the 3 ORFs were compared among isolates. The Right ORF was found to have the highest variation with a mean distance of 24%. This was followed by the Left and Mid ORF with distances of 13 and 7%, respectively. A phylogenetic analysis based on the amino acid sequence of the Right ORF divides 7 HPV isolates into 3 well-separated groups: Korea, Thailand, and Australia. The Madagascar HPV clustered with the Korea and Tanzania isolates. In Madagascar, HPV has been detected by histological examination since the 1990s. PCR analysis of a recent (2007) sampling showed a 100% prevalence. HPV was also detected in Mozambique with a 100% prevalence. High (95%) prevalence of HPV was found in wild Penaeus merguinesis collected from New Caledonia. These results indicate that HPV displays a high degree of genetic diversity and is distributed worldwide among populations of penaeid shrimp. © Inter-Research 2008.
• Andrade, T. P., Srisuvan, T., Tang, K. F., & Lightner, D. V. (2007). Real-time reverse transcription polymerase chain reaction assay using TaqMan probe for detection and quantification of Infectious myonecrosis virus (IMNV). Aquaculture, 264(1-4), 9-15.
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  Abstract: Infectious myonecrosis, caused by Infectious myonecrosis virus (IMNV), is an important emerging disease of shrimp that has affected the production of cultured Litopenaeus vannamei in Northeast Brazil. In this study we report the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe to detect this virus in shrimp. The real-time RT-PCR showed a strong linear correlation (r2 = 0.986) between threshold cycles (CT) and RNA quantities. The assay gave negative results for other viruses, including Yellow head virus (YHV), Taura syndrome virus (TSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), and White spot syndrome virus (WSSV) and the necrotizing hepatopancreatic bacterium (NHPB). This real-time RT-PCR assay can detect as few as 10 IMNV copy numbers/μl RNA, while the nested RT-PCR can detect no fewer than 1000 IMNV copy numbers/μl RNA. Specific-pathogen-free L. vannamei were used in the infectivity assay. There were one control group (Group 1) and one viral challenged group (Group 2), from which shrimp were sampled for RT-PCR and histological analysis. The RNA from dead shrimp was extracted and tested for IMNV by nested and real-time RT-PCR. The shrimp in Group 1 showed 100% survival, while those in Group 2 showed a 0% survival. The first mortality in the Group 2 was observed at Day 8 post-inoculation (p.i.); and the mortalities dramatically increased after Day 40 p.i. Histological sections from Group 2 shrimp taken at intervals throughout the study exhibited acute to chronic phase lesions of IMNV infection, and consecutive tissue sections reacted to the IMNV-specific cDNA probes by in situ hybridization. The real-time RT-PCR detected the presence of IMNV in all 30 of the challenged specimens in Group 2. In contrast, the nested RT-PCR detected the presence of IMNV in 23 of the 30 specimens. The real-time RT-PCR revealed that the 7 specimens not detected by nested RT-PCR contained relatively low IMNV copy numbers compared to the other 23 specimens. These results demonstrate that the real-time RT-PCR developed in this study is a sensitive diagnostic method for IMNV.
• Eddy, F., Powell, A., Gregory, S., Nunan, L. M., Lightner, D. V., Dyson, P. J., Rowley, A. F., & Shields, R. J. (2007). A novel bacterial disease of the European shore crab, Carcinus maenas - Molecular pathology and epidemiology. Microbiology, 153(9), 2839-2849.
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  PMID: 17768229;Abstract: Several rickettsia-like diseases have been reported in arthropods (insects and crustaceans), some of which result in significant losses of economically important species such as shrimp and crabs. This study reports on the molecular pathology of a recently emerged disease of the European shore crab, Carcinus maenas, termed milky disease - named as a result of the unusual milky appearance of the haemolymph (blood). This disease was more prevalent (>26 %) during summer months when the water temperature in a pilot crab farm was approximately 19 °C. The putative causative agent of the disease was a Gram-negative bacterium that could not be cultured on a range of agar-based growth media. Diseased crabs showed significant reductions in free blood cell numbers and total serum protein. Such animals also displayed raised levels of glucose and ammonium in blood. Ultrastructural and in situ hybridization studies revealed that the causative agent associated with milky disease multiplied in the fixed phagocytes of the hepatopancreas (digestive gland), ultimately to be released into the haemolymph, where the circulating blood cells showed little response to the presence of these agents. Attempts to induce the infection by short-term temperature stress failed, as did transmission experiments where healthy crabs were fed infected tissues from milky disease affected individuals. Sequence analysis of the 16S rRNA gene from the milky disease bacteria indicated that they are a previously undescribed species of α-proteobacteria with little phylogenetic similarity to members of the order Rickettsiales. © 2007 SGM.
• F., K., Navarro, S. A., & Lightner, D. V. (2007). PCR assay for discriminating between infectious hypodermal and hematopoietic necrosis virus (IHHNV) and virus-related sequences in the genome of Penaeus monodon. Diseases of Aquatic Organisms, 74(2), 165-170.
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  PMID: 17432046;Abstract: We developed a PCR assay that can detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) but that does not react with IHHNV-related sequences in the genome of Penaeus monodon from Africa and Australia. IHHNV is a single-stranded DNA virus that has caused severe mortality and stunted growth in penaeid shrimp. Recently, IHHNV-related sequences were found in the genome of some stocks of P. monodon from Africa and Australia. These virus-related sequences have a high degree of similarity (86 and 92 % identities in nucleotide sequence) to the viral genome, which has often generated false-positive reactions during PCR screening of these stocks. For this assay, a pair of IHHNV primers (IHHNV309F/R) was selected. The sequences of these primers match (100% of nucleotides) the target sequence in IHHNV, but mismatch 9 or 12 nucleotides of the genomic IHHNV-related sequences. This PCR assay was tested with various IHHNV isolates and with a number of samples of shrimp DNA that contained IHHNV-related sequences. This assay can reliably distinguish IHHNV DNA from shrimp DNA: it only detects IHHNV. Also, this pair of primers was included in a duplex PCR to detect IHHNV and simultaneously determine the presence of an IHHNV-related sequence. Using these primers, the PCR assay has a sensitivity equivalent to a PCR assay commonly used for detecting IHHNV in Litopenaeus vannamei, and can be used for routine detection. © Inter-Research 2007.
• F., K., Pantoja, C. R., Redman, R. M., & Lightner, D. V. (2007). Development of in situ hybridization and RT-PCR assay for the detection of a nodavirus (PvNV) that causes muscle necrosis in Penaeus vannamei. Diseases of Aquatic Organisms, 75(3), 183-190.
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  PMID: 17629112;Abstract: A nodavirus (tentatively named PvNV, Penaeus vannamei nodavirus) that causes muscle necrosis in P. vannamei was found in Belize in 2004. From 2004 to 2006, shrimp samples collected from Belize exhibited clinical signs, white, opaque lesions in the tails and histopathology similar to those of shrimps infected by infectious myonecrosis virus (IMNV). Histological examination revealed multifocal necrosis and hemocytic fibrosis in the skeletal muscle. In addition, basophilic, cytoplasmic inclusions were found in striated muscle, lymphoid organ and connective tissues. However, IMNV was not detected in these shrimps by either RT-PCR or in situ hybridization, suggesting that these lesions may be caused by another RNA virus. Thus, a cDNA library was constructed from total RNA extracted from hemolymph collected from infected shrimp. One clone (designated PvNV-4) with a 928 bp insert was sequenced and found to be similar (69% similarity when comparing the translated amino acid sequences) to the capsid protein gene of MrNV (Macrobrachium rosenbergii nodavirus). The insert of PvNV-4 was labeled with digoxigenin-11-deoxyuridine triphosphate (dUTP) and hybridized to tissue sections of P. vannamei with muscle necrosis collected in Belize and from laboratory bioassays. The samples were positive for PvNV infection. Positively reacting tissues included skeletal muscle, connective tissues, the lymphoid organ, and hemocytes in the heart and gills. In addition, we experimentally infected both P. vannamei and P. monodon with PvNV prepared from Belize samples. A nested RT-PCR assay developed from the PvNV-4 cloned sequence showed that both species are susceptible to PvNV infection. © Inter-Research 2007.
• Lightner, D. V., Riggs, A., Corbin, J. S., & Ostrowski, A. C. (2007). Taura syndrome virus in specific pathogen-free Penaeus vannamei originating from Hawaii and in P. vannamei stocks farmed in France?. Diseases of Aquatic Organisms, 74(1), 77-79.
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  PMID: 17425266;Abstract: It is the opinion of the authors of this Comment on Do et al. (2006), that those authors incorrectly interpreted their test results, which are more likely the result of mislabeling of samples or within-laboratory contamination, and that the TSV isolates found in Penaeus vannamei in Korea in 2004 and 2005 did not originate from Hawaii as claimed by the authors, but from a country (or countries) in southeast Asia. Finally, we believe that the authors did not follow proper international guidelines, extend a professional courtesy to the supplier of the disputed shrimp sample, nor take a critical approach in interpreting their own data. It is unfortunate that the authors did not follow through with additional testing, or seek a second opinion from an independent laboratory, before implicating shrimp imported from Hawaii as the source of TSV in Korea. © Inter-Research 2007.
• Lightner, D., Tang, K. F., Navarro, S. A., & Lightner, D. V. (2007). PCR assay for discriminating between infectious hypodermal and hematopoietic necrosis virus (IHHNV) and virus-related sequences in the genome of Penaeus monodon. Diseases of aquatic organisms, 74(2).
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  We developed a PCR assay that can detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) but that does not react with IHHNV-related sequences in the genome of Penaeus monodon from Africa and Australia. IHHNV is a single-stranded DNA virus that has caused severe mortality and stunted growth in penaeid shrimp. Recently, IHHNV-related sequences were found in the genome of some stocks of P. monodon from Africa and Australia. These virus-related sequences have a high degree of similarity (86 and 92% identities in nucleotide sequence) to the viral genome, which has often generated false-positive reactions during PCR screening of these stocks. For this assay, a pair of IHHNV primers (IHHNV309F/R) was selected. The sequences of these primers match (100% of nucleotides) the target sequence in IHHNV, but mismatch 9 or 12 nucleotides of the genomic IHHNV-related sequences. This PCR assay was tested with various IHHNV isolates and with a number of samples of shrimp DNA that contained IHHNV-related sequences. This assay can reliably distinguish IHHNV DNA from shrimp DNA: it only detects IHHNV. Also, this pair of primers was included in a duplex PCR to detect IHHNV and simultaneously determine the presence of an IHHNV-related sequence. Using these primers, the PCR assay has a sensitivity equivalent to a PCR assay commonly used for detecting IHHNV in Litopenaeus vannamei, and can be used for routine detection.
• Lightner, D., Tang, K. F., Pantoja, C. R., Redman, R. M., & Lightner, D. V. (2007). Development of in situ hybridization and RT-PCR assay for the detection of a nodavirus (PvNV) that causes muscle necrosis in Penaeus vannamei. Diseases of aquatic organisms, 75(3).
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  A nodavirus (tentatively named PvNV, Penaeus vannamei nodavirus) that causes muscle necrosis in P. vannamei was found in Belize in 2004. From 2004 to 2006, shrimp samples collected from Belize exhibited clinical signs, white, opaque lesions in the tails and histopathology similar to those of shrimps infected by infectious myonecrosis virus (IMNV). Histological examination revealed multifocal necrosis and hemocytic fibrosis in the skeletal muscle. In addition, basophilic, cytoplasmic inclusions were found in striated muscle, lymphoid organ and connective tissues. However, IMNV was not detected in these shrimps by either RT-PCR or in situ hybridization, suggesting that these lesions may be caused by another RNA virus. Thus, a cDNA library was constructed from total RNA extracted from hemolymph collected from infected shrimp. One clone (designated PvNV-4) with a 928 bp insert was sequenced and found to be similar (69% similarity when comparing the translated amino acid sequences) to the capsid protein gene of MrNV (Macrobrachium rosenbergii nodavirus). The insert of PvNV-4 was labeled with digoxigenin-11-deoxyuridine triphosphate (dUTP) and hybridized to tissue sections of P. vannamei with muscle necrosis collected in Belize and from laboratory bioassays. The samples were positive for PvNV infection. Positively reacting tissues included skeletal muscle, connective tissues, the lymphoid organ, and hemocytes in the heart and gills. In addition, we experimentally infected both P. vannamei and P. monodon with PvNV prepared from Belize samples. A nested RT-PCR assay developed from the PvNV-4 cloned sequence showed that both species are susceptible to PvNV infection.
• Lightner, D., Tang, K. F., Redman, R. M., Pantoja, C. R., Groumellec, M. L., Duraisamy, P., & Lightner, D. V. (2007). Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection. Journal of invertebrate pathology, 96(3).
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  An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue-green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e-124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.
• Montgomery-Brock, D., Tacon, A. G., Poulos, B., & Lightner, D. (2007). Reduced replication of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei held in warm water. Aquaculture, 265(1-4), 41-48.
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  Abstract: Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a serious pathogen, which for the last 24 years has been responsible for negatively affecting the growth and survival of some species of cultured penaeid shrimp. The Pacific white shrimp Litopenaeus vannamei is particularly affected by IHHNV as the virus can cause poor growth and physical deformities in infected populations. There are no known treatments for shrimp affected with IHHNV and management tools for preventing this disease are limited to the exclusion of the virus from cultured shrimp populations. Previous studies have determined that warm-water culture conditions inhibit the replication rate of two other important shrimp viruses, White spot syndrome virus (WSSV) and Taura syndrome virus (TSV) in L. vannamei. The purpose of this study was to compare the effect of water temperature on the replication rate of IHHNV in shrimp held in warm water (32.8 ± 1.0 °C), compared to the replication rate of IHHNV in shrimp held in cool water (24.4 ± 0.5 °C). A total of 72 specific pathogen-free (SPF) L. vannamei were used for this study. They were divided into four groups, namely 1) saline-injected shrimp held in warm water (31.1 ± 1.0 °C), 2) saline-injected shrimp held in cool water (26.3 ± 0.6 °C), 3) IHHNV-injected shrimp held in warm water (32.8 ± 1.0 °C), and 4) IHHNV-injected shrimp held in cool water (24.4 ± 0.5 °C). The concentration of IHHNV in shrimp collected from day 1 through day 17 post-exposure was measured with real time polymerase chain reaction (PCR) using the TaqMan® technique. The results from samples collected on days 6, 12, and 17 post-exposure showed a statistically significant difference (p < 0.05) between the viral load of the IHHNV-injected shrimp held in warm water versus the IHHNV-injected shrimp held in cool water. The IHHNV-injected shrimp held in the warm-water conditions averaged 1.44 × 103, 1.77 × 103, and 1.20 × 105 virus copies/50 ng of shrimp tissue DNA on days 6, 12, and 17, respectively. The IHHNV-injected shrimp in the cool-water conditions averaged 2.25 × 105, 1.8 × 106, and 4.6 × 106 virus /50 ng DNA on days 6, 12, and 17, respectively. These results clearly indicate that under these conditions water temperature had a profound effect on the replication rate of IHHNV in L. vannamei. The results support the findings of the previous studies and further point to the potential application of elevated environmental temperature as a management strategy for certain viral diseases within the shrimp farming industry.
• Nunan, L. M., Lightner, D. V., Pantoja, C. R., Stokes, N. A., & Reece, K. S. (2007). Characterization of a rediscovered haplosporidian parasite from cultured Penaeus vannamei. Diseases of Aquatic Organisms, 74(1), 67-75.
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  PMID: 17425265;Abstract: Mortalities of Penaeus vannamei, cultured in ponds in Belize, Central America, began during the last part of the grow-out cycle during the cold weather months from September 2004 through February 2005. Tissue squashes of infected hepatopancreata and histological examination of infected shrimp revealed that the mortalities might have been caused by an endoparasite. To confirm the diagnosis, DNA was extracted from ethanol preserved hepatopancreata and the small-subunit rRNA gene was sequenced. The 1838 bp sequence was novel and phylogenetic analysis placed the P. vannamei parasite within the phylum Haplosporidia as a sister taxon to a clade that includes Bonamia and Minchinia species. In situ hybridization was performed using anti-sense DNA probes that were designed to hybridize specifically with the parasite's nucleic acid. This organism presents similar characteristics to those of a haplosporidian that infected cultured P. vannamei imported from Nicaragua into Cuba, as described by Dyková et al. (1988; Fish Dis 11:15-22). © Inter-Research 2007.
• Redman, R. M., & Lightner, D. V. (2007). Opportunities for training in shrimp diseases. Developments in Biologicals, 129, 137-146.
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  PMID: 18306527;Abstract: Opportunities for formal training in shrimp diseases were not available 30 years ago. This was because the shrimp farming industry was in its infancy with few significant disease issues and even fewer shrimp disease specialists investigating the causes of production losses. In 2006, more than two million metric tons of the marine penaeid shrimp were farmed, accounting for more than half of the world's supply. With most of the world's shrimp fisheries at maximum sustainable yields, the ratio of farmed to fished shrimp appears likely to continue to increase. The remarkable growth of sustainable shrimp farming was made possible through the development of methods to diagnose and manage disease in the world's shrimp farms. This occurred as the result of the development of training opportunities in shrimp disease diagnosis and control methods and the application of that knowledge, by an ever increasing number of shrimp diseases specialists, to disease management at shrimp farms. The first type of formal training to become generally available to the industry was in the form of special short courses and workshops. The first of these, which was open to international participants, was given at the University of Arizona in 1989. Since that first course several dozen more special short courses and workshops on shrimp diseases have been given by the University of Arizona. Dozens more special courses and workshops on shrimp diseases have been given by other groups, including other universities, industry cooperatives, governments and international aid agencies, in a wide range of countries (and languages) where shrimp farming constitutes an important industry. In parallel, graduate study programs leading to post graduate degrees, with shrimp disease as the research topic, have developed while formal courses in shrimp diseases have not become widely available in veterinary or fisheries college curricula in the USA and Europe, such courses are appearing in university programs located in some of the shrimp farming countries of SE Asia. The trend towards more formal training programs in shrimp diseases and disease management is likely to continue as the industry continues to mature and become increasingly sustainable.
• Tang, K. F., Tang, K. F., Redman, R. M., Redman, R. M., Pantoja, C. R., Pantoja, C. R., Groumellec, M. L., Groumellec, M. L., Duraisamy, P., Duraisamy, P., Lightner, D. V., & Lightner, D. V. (2007). Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection. Journal of Invertebrate Pathology, 96(3), 255-260.
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  PMID: 17585932;Abstract: An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue-green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140 nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0 kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e-124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV. © 2007 Elsevier Inc. All rights reserved.
• Bonnichon, V., Lightner, D. V., & Bonami, J. (2006). Viral interference between infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus in Litopenaeus vannamei. Diseases of Aquatic Organisms, 72(2), 179-184.
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  PMID: 17140141;Abstract: White spot syndrome virus (WSSV) is highly virulent and has caused significant production losses to the shrimp culture industry over the last decade. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) also infects penaeid shrimp and, while being less important than WSSV, remains a major cause of significant production losses in Litopenaeus vannamei (also called Penaeus vannamei) and L. stylirostris (also called Penaeus stylirostris). These 2 viruses and their interactions were previously investigated in L. stylirostris. We report here laboratory challenge studies carried out to determine if viral interference between IHHNV and WSSV also occurs in L. vannamei, and it was found that experimental infection with IHHNV induced a significant delay in mortality following WSSV challenge. L. vannamei infected per os with IHHNV were challenged with WSSV at 0, 10, 20, 30, 40 and 50 d post-infection. Groups of naïve shrimp infected with WSSV alone died in 3 d whereas shrimp pre-infected with IHHNV for 30, 40 or 50 d died in 5 d. Real-time PCR analysis showed that the delay correlated to the IHHNV load and that WSSV challenge induced a decrease in IHHNV load, indicating some form of competition between the 2 viruses. © Inter-Research 2006.
• Bray, W. A., Williams, R. R., Lightner, D. V., & Lawrence, A. L. (2006). Growth, survival and histological responses of the marine shrimp, Litopenaeus vannamei, to three dosage levels of oxytetracycline. Aquaculture, 258(1-4), 97-108.
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  Abstract: Toxicity of oxytetracycline (OTC) in prepared feed for penaeid shrimp was evaluated in a 42-day trial with Litopenaeus vannamei (initial mean weight, 10.3 g ± 0.504 S.D.). Four treatments consisted of: (1) 0.0 g/kg OTC (control feed), (2) 4.5 g/kg OTC (1× treatment, maximum recommended dose), (3) 13.5 g/kg OTC (3× treatment) and (4) 22.5 g/kg OTC (5× treatment). Each treatment consisted of 11 replicate tanks, with 8 shrimp per replicate. The test period was three times the recommended OTC label dosing period (14 days). This target animal safety study was conducted under GLP (Good Laboratory Practices) conditions, as part of submissions to the U.S. Food and Drug Administration Center for Veterinary Medicine for approval of therapeutic use of OTC in penaeid shrimp feeds. Growth rate (weight gain) was found to be OTC-dose-related. There was no significant difference in mean weight gain between control and 1× OTC treatments (9.1 g and 9.2 g, respectively), but growth was strongly depressed in the 3× and 5× treatments (1.50 g and 4.02 g, respectively). There did not appear to be a dose response in mean percent survival of test shrimp (range: 93.2% to 98.9%). Abnormal soft exoskeletons (= shell, cuticle) were observed in 66.7% to 90.9% of shrimp in the 3× and 5× treatments after 42 days, compared with 0.0% and 9.1% in control and 1× treatments, respectively (from sample N = 3 per tank = N = 33 per treatment, 37.5% of individuals stocked). The high numbers of soft exoskeletons observed could be related to a deficiency of calcium/magnesium, principal shell components, due to divalent cation-chelating properties of OTC. Some dose-related histological changes were apparent in the hepatopancreas (HP) of experimental shrimp sampled after 42 days of OTC feeds. Only slight changes were apparent in the histological presentation of the HPs of the shrimp from the 0× (untreated control) and the 1× treatment levels. In contrast, shrimp sampled from the 3× and 5× OTC treatment levels showed generally reduced levels of lipid droplet storage in the HP, and some necrosis and sloughing of the HP tubule epithelium. The virtually complete absence of HP lipids and the presence of moderate atrophy of the proximal portion of the HP tubules, indicated by markedly reduced tubule epithelial cell height, were the principal characteristics of the HP of shrimp sampled from the 5× OTC group. Mitotic activity in E-cells, a measure of the regenerative capability of the HP, was constant across all experimental groups regardless of the OTC level in the experimental feed. Mean OTC consumed per shrimp in the 1× treatment was estimated to be 0.09813 g over 42 days, compared with 0.16850 g and 0.29687 g in the 3× and 5× treatments, respectively. Feeds containing OTC at higher levels (3× and 5×) were consumed at much lower rates than control and 1× treatments, indicating lower palatability and/or toxicity of extreme dosage levels. The 3× and 5× treatments consumed approximately 46% less feed than the control and 1× treatments. © 2006 Elsevier B.V. All rights reserved.
• Covarrubias, M. M., Osuna-Duarte, A., Garcia-Gasca, A., Lightner, D. V., & Mota-Urbina, J. (2006). Prevalence of necrotizing hepatopancreatitis in female broodstock of white shrimp Penaeus vannamei with unilateral eyestalk ablation and hormone injection. Journal of Aquatic Animal Health, 18(1), 19-25.
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  Abstract: The present study analyzed the relationship between unilateral eyestalk ablation and steroid hormone injection in female white shrimp Penaeus vannamei, the prevalence of necrotizing hepatopancreatitis (NHP) caused by the intracellular rickettsia-like bacterium NHP-B. Two simultaneous bioassays determined the onset and development of NHP in broodstock. Wet-mount analysis, conventional histopathology, and in situ hybridization with an NHP-B-specific DNA probe were used to assess the prevalence and degree of severity of NHP and to confirm NHP-B infection. Wet-mount and histopathological results showed that females with unilateral eyestalk ablation had a 64% prevalence of NHP at grades 2 and 4, while females receiving the hormone injection showed a 23% prevalence of NHP at grades 3 and 4; control shrimp injected with phosphate-buffered saline (PBS) displayed a 9% prevalence of NHP at grade 3, whereas nonablated controls showed no signs of disease. The rickettsia-like specific DNA probe demonstrated a positive hybridization signal in NHP-B, and the hepatopancreas, the target tissue for the bacterium, showed a strong positive signal. Significant differences (P < 0.05) were found in females with unilateral eyestalk ablation compared with nonablated controls, in hormone-injected females compared with PBS-injected controls, and in females with unilateral eyestalk ablation compared with the hormone injection group. We conclude that unilateral eyestalk ablation enhances NHP disease prevalence in broodstock reared under maturation conditions. © Copyright by the American Fisheries Society 2006.
• Lightner, D. V., Poulos, B. T., Tang-Nelson, K., Pantoja, C. R., Nunan, L. M., Navarro, S. A., Redman, R. M., & Mohney, L. L. (2006). Application of molecular diagnostic methods to penaeid shrimp diseases: Advances of the past 10 years for control of viral diseases in farmed shrimp. Developments in Biologicals, 126, 117-122.
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  PMID: 17058487;Abstract: The most important diseases of farmed penaeid shrimp have infectious aetiologies. Among these are diseases with viral, rickettsial, bacterial, fungal and parasitic aetiologies. Diagnostic methods for these pathogens include the traditional methods of gross pathology, histopathology, classical microbiology, animal bioassay, antibody-based methods, and molecular methods using DNA probes and DNA amplification. While methods using clinical chemistry and tissue culture are standard methods in veterinary and human diagnostic laboratories, the former has not been routinely applied to the diagnosis of penaeid shrimp diseases and the latter has yet to be developed, despite considerable research and development efforts that have spanned the past 40 years. No continuous shrimp cell lines, or lines from other crustaceans, have been developed. Hence, when molecular methods began to be routinely applied to the diagnosis of infectious diseases in humans and domestic animals in the mid- to late 1980s, the technology was applied to the diagnosis of certain important diseases of penaeid shrimp for which only classical diagnostic methods were previously available. A DNA hybridization assay for the parvovirus IHHNV was the first molecular test developed for a shrimp disease. This was followed within a year by the first PCR test for MBV, an important baculovirus disease of shrimp. Today, shrimp disease diagnostic laboratories routinely use molecular tests for diagnostic and surveillance purposes for most of the important penaeid shrimp diseases.
• Lightner, D., Poulos, B. T., & Lightner, D. V. (2006). Detection of infectious myonecrosis virus (IMNV) of penaeid shrimp by reverse-transcriptase polymerase chain reaction (RT-PCR). Diseases of aquatic organisms, 73(1).
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  Infectious myonecrosis virus (IMNV) infecting cultured Litopenaeus vannamei in Brazil is a double-stranded RNA virus that causes a slowly progressive disease with cumulative mortalities of up to 70%. The disease is currently diagnosed using a combination of gross signs (primarily skeletal tail muscle necrosis with white opaque discoloration), histopathology, and in situ hybridization with a digoxigenin-labeled gene probe. A rapid and sensitive method for definitive diagnosis of the disease was developed using reverse-transcriptase polymerase chain reaction (RT-PCR). Two primer sets were used to detect 328 and 139 bp amplicons in a nested RT-PCR assay. Using RNA extracted from purified virions, the first step reaction detected 100 copies of the IMNV viral genome whereas the nested step detected 10 copies. The primers were shown to be specific for IMNV and no amplicons were detected using RNA extracted from shrimp infected with other penaeid shrimp viruses (Taura syndrome virus [TSV], yellowhead virus [YHV], infectious hypodermal hematopoietic necrosis virus [IHHNV] and white spot syndrome virus [WSSV]).
• Lightner, D., Poulos, B. T., Tang, K. F., Pantoja, C. R., Bonami, J. R., & Lightner, D. V. (2006). Purification and characterization of infectious myonecrosis virus of penaeid shrimp. The Journal of general virology, 87(Pt 4).
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  The causative agent of myonecrosis affecting cultured Penaeus vannamei in Brazil was demonstrated to be a virus after purification of the agent from infected shrimp tissues. Purified viral particles were injected into specific pathogen-free P. vannamei, resulting in a disease that displayed the same characteristics as those found in the original shrimp used for purification. The virus was named infectious myonecrosis virus (IMNV). The viral particles were icosahedral in shape and 40 nm in diameter, with a buoyant density of 1.366 g ml(-1) in caesium chloride. The genome consisted of a single, double-stranded (dsRNA) molecule of 7560 bp. Sequencing of the viral genome revealed two non-overlapping open reading frames (ORFs). The 5' ORF (ORF 1, nt 136-4953) encoded a putative RNA-binding protein and a capsid protein. The coding region of the RNA-binding protein was located in the first half of ORF 1 and contained a dsRNA-binding motif in the first 60 aa. The second half of ORF 1 encoded a capsid protein, as determined by amino acid sequencing, with a molecular mass of 106 kDa. The 3' ORF (ORF 2, nt 5241-7451) encoded a putative RNA-dependent RNA polymerase (RdRp) with motifs characteristic of totiviruses. Phylogenetic analysis based on the RdRp clustered IMNV with Giardia lamblia virus, a member of the family Totiviridae. Based on these findings, IMNV may be a unique member of the Totiviridae or may represent a new dsRNA virus family that infects invertebrate hosts.
• Lightner, D., Tang, K. F., & Lightner, D. V. (2006). Infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia. Virus research, 118(1-2).
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  We found an infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequence within the shrimp genome in populations of Penaeus monodon from Africa and Australia. IHHNV is a single-stranded DNA virus that has caused severe mortality and stunted growth in penaeid shrimp. Recently, IHHNV-related sequences were found in samples of P. monodon from Madagascar and Tanzania. These sequences vary considerably (14 and 8%, respectively) from that of IHHNV found in association with viral epidemics. Laboratory bioassays were carried out with P. monodon and Litopenaeus vannamei to determine if either of these IHHNV-related sequences is infectious. We used juvenile and adult P. monodon containing the virus-related sequences from four geographic regions to generate inocula and tissues for feeding. Specific pathogen free P. monodon and L. vannamei were used as indicator shrimp. During the 2-4 week bioassays, none of the indicator shrimp showed signs of infection or disease. Results of both PCR assays and histological examination of the indicator shrimp were negative for IHHNV infection, indicating that the Africa type IHHNV-related sequences are not infectious. With the shrimp containing the Madagascar type IHHNV-related sequence (designated as type A), we performed genome walking at the 3' end of the virus-related sequence and found that this virus-related sequence is part of the P. monodon genome. A fragment of 1.9 kb flanking sequence was cloned and sequenced. Sequence analysis showed that this flanking sequence contains shrimp microsatellite DNA. Also, its translated amino acid sequence was highly similar to a retrotransposon. This result provides molecular evidence that the type A IHHNV-related sequence is shrimp DNA. This sequence was found in the P. monodon collected from Africa and Australia.
• Poulos, B. T., & Lightner, D. V. (2006). Detection of infectious myonecrosis virus (IMNV) of penaeid shrimp by reverse-transcriptase polymerase chain reaction (RT-PCR). Diseases of Aquatic Organisms, 73(1), 69-72.
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  PMID: 17240754;Abstract: Infectious myonecrosis virus (IMNV) infecting cultured Litopenaeus vannamei in Brazil is & double-stranded RNA virus that causes a slowly progressive disease with cumulative mortalities of up to 70%. The disease is currently diagnosed using a combination of gross signs (primarily skeletal tail muscle necrosis with white opaque discoloration), histopathology, and in situ hybridization with a digoxigenin-labeled gene probe. A rapid and sensitive method for definitive diagnosis of the disease was developed using reverse-transcriptase polymerase chain reaction (RT-PCR). Two primer sets were used to detect 328 and 139 bp amplicons in a nested RT-PCR assay. Using RNA extracted from purified virions, the first step reaction detected 100 copies of the IMNV viral genome whereas the nested step detected 10 copies. The primers were shown to be specific for IMNV and no amplicons were detected using RNA extracted from shrimp infected with other penaeid shrimp viruses (Taura syndrome virus [TSV], yellowhead virus [YHV], infectious hypodermal hematopoietic necrosis virus [IHHNV] and white spot syndrome virus [WSSV]). © Inter-Research 2006.
• Poulos, B. T., Tang, K. F., Pantoja, C. R., Bonami, J. R., & Lightner, D. V. (2006). Purification and characterization of infectious myonecrosis virus of penaeid shrimp. Journal of General Virology, 87(4), 987-996.
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  PMID: 16528049;Abstract: The causative agent of myonecrosis affecting cultured Penaeus vannamei in Brazil was demonstrated to be a virus after purification of the agent from infected shrimp tissues. Purled viral particles were injected into specific pathogen-free P. vannamei, resulting in a disease that displayed the same characteristics as those found in the original shrimp used for purification. The virus was named infectious myonecrosis virus (IMNV). The viral particles were icosahedral in shape and 40 nm in diameter, with a buoyant density of 1.366 g ml-1 in caesium chloride. The genome consisted of a single, double-stranded (dsRNA) molecule of 7560 bp. Sequencing of the viral genome revealed two non-overlapping open reading frames (ORFs). The 5′ ORF (ORF 1, nt 136-4953) encoded a putative RNA-binding protein and a capsid protein. The coding region of the RNA-binding protein was located in the first half of ORF 1 and contained a dsRNA-binding motif in the first 60 aa. The second half of ORF 1 encoded a capsid protein, as determined by amino acid sequencing, with a molecular mass of 106 kDa. The 3′ ORF (ORF 2, nt 5241-7451) encoded a putative RNA-dependent RNA polymerase (RdRp) with motifs characteristic of totiviruses. Phylogenetic analysis based on the RdRp clustered IMNV with Giardia lamblia virus, a member of the family Totiviridae. Based on these findings, IMNV may be a unique member of the Totiviridae or may represent a new dsRNA virus family that infects invertebrate hosts. © 2006 SGM.
• Srisuvan, T., Noble, B. L., Schofield, P. J., & Lightner, D. V. (2006). Comparison of four Taura syndrome virus (TSV) isolates in oral challenge studies with Litopenaeus vannamei unselected or selected for resistance to TSV. Diseases of Aquatic Organisms, 71(1), 1-10.
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  PMID: 16921995;Abstract: Taura syndrome virus (TSV) infection in TSV-resistant (TSR) and TSV-susceptible (Kona) Litopenaeus vannamei (also called Penaeus vannamei) was investigated using histology, in situ hybridization (ISH), conventional reverse transcription polymerase chain reaction (RT-PCR) assays, and SYBR-Green real-time RT-PCR analysis. The shrimp were challenged by feeding with minced tissues of L. vannamei infected with 4 genotypic variants of TSV (Bz01, Th04, UsHi94, and Ve05). Survival probabilities of TSR shrimp were higher than those for Kona shrimp with all 4 variants. Th04, UsHi94, and Ve05 gave no Taura syndrome lesions with TSR shrimp. In contrast, TSR shrimp challenged with Bz01 and Kona shrimp with all 4 TSV variants exhibited severe necrosis of cuticular epithelial cells and lymphoid organ spheroids, indicative of acute and chronic phases of TSV infection, respectively. TSV was not detected by RT-PCR in TSR shrimp infected with Th04, UsHi94, and Ve05, or in Kona shrimp infected with Ve05 but was detected in TSR shrimp infected with Bz01 and in Kona shrimp infected with Bz01, Th04, and UsHi94. Real-time RT-PCR revealed that mean TSV copy numbers in TSR shrimp infected with Bz01, Th04, and UsHi94 were significantly (p < 0.0005) lower than those in Kona shrimp. In contrast, mean TSV copy numbers in TSR and Kona shrimp infected with Ve05 were not significantly different (p > 0.4). The results show that TSR L. vannamei are susceptible to infection but give high survival rates following challenge by all 4 variants of TSV. © Inter-Research 2006.
• Srisuvan, T., Pantoja, C. R., Redman, R. M., & Lightner, D. V. (2006). Ultrastructure of the replication site in Taura syndrome virus (TSV)-infected cells. Diseases of Aquatic Organisms, 73(2), 89-101.
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  PMID: 17260828;Abstract: Taura syndrome virus (TSV) is a member of the family Dicistroviridae that infects Pacific white shrimp Litopenaeus vannamei (also called Penaeus vannamei), and its replication strategy is largely unknown. To identify the viral replication site within infected shrimp cells, the viral RNA was located in correlation with virus-induced membrane rearrangement. Ultrastructural changes in the infected cells, analyzed by transmission electron microscopy (TEM), included the induction and proliferation of intracellular vesicle-like membranes, while the intracytoplasmic inclusion bodies and pyknotic nuclei indicative of TSV infection were frequently seen. TSV plus-strand RNA, localized by electron microscopic in situ hybridization (EM-ISH) using TSV-specific cDNA probes, was found to be associated with the membranous structures. Moreover, TSV particles were observed in infected cells by TEM, and following EM-ISH, they were also seen in close association with the proliferating membranes. Taken together, our results suggest that the membranous vesicle-like structures carry the TSV RNA replication complex and that they are the site of nascent viral RNA synthesis. Further investigations on cellular origins and biochemical compositions of these membranous structures will elucidate the morphogenesis and propagation strategy of TSV. © Inter-Research 2006.
• Tang, K. F., & Lightner, D. V. (2006). Infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequences in the genome of the black tiger prawn Penaeus monodon from Africa and Australia. Virus Research, 118(1-2), 185-191.
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  PMID: 16473428;Abstract: We found an infectious hypodermal and hematopoietic necrosis virus (IHHNV)-related sequence within the shrimp genome in populations of Penaeus monodon from Africa and Australia. IHHNV is a singlestranded DNA virus that has caused severe mortality and stunted growth in penaeid shrimp. Recently, IHHNV-related sequences were found in samples of P. monodon from Madagascar and Tanzania. These sequences vary considerably (14 and 8%, respectively) from that of IHHNV found in association with viral epidemics. Laboratory bioassays were carried out with P. monodon and Litopenaeus vannamei to determine if either of these IHHNV-related sequences is infectious. We used juvenile and adult P. monodon containing the virus-related sequences from four geographic regions to generate inocula and tissues for feeding. Specific pathogen free P. monodon and L. vannamei were used as indicator shrimp. During the 2-4 week bioassays, none of the indicator shrimp showed signs of infection or disease. Results of both PCR assays and histological examination of the indicator shrimp were negative for IHHNV infection, indicating that the Africa type IHHNV-related sequences are not infectious. With the shrimp containing the Madagascar type IHHNV-related sequence (designated as type A), we performed genome walking at the 3′ end of the virus-related sequence and found that this virus-related sequence is part of the P. monodon genome. A fragment of 1.9 kb flanking sequence was cloned and sequenced. Sequence analysis showed that this flanking sequence contains shrimp microsatellite DNA. Also, its translated amino acid sequence was highly similar to a retrotransposon. This result provides molecular evidence that the type A IHHNV-related sequence is shrimp DNA. This sequence was found in the P. monodon collected from Africa and Australia. © 2006 Elsevier B.V. All rights reserved.
• Chayaburakul, K., Lightner, D. V., Sriurairattana, S., Nelson, K. T., & Withyachumnarnkul, B. (2005). Different responses to infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus monodon and P. vannamei. Diseases of Aquatic Organisms, 67(3), 191-200.
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  PMID: 16408834;Abstract: Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is widespread in cultured Penaeus monodon and P. vannamei in Thailand. It causes runt-deformity syndrome that is characterized by physical abnormalities and stunted growth in P. vannamei, but causes no apparent disease in P. monodon. In both species, the virus may produce Cowdry Type A inclusions in tissues of ectodermal and mesodermal origin, but these are common in P. vannamei and rare in P. monodon. The virus can be more easily detected in both species by IHHNV-specific PCR primers. By in situ hybridization (ISH) using specific IHHNV probes, fixed phagocytes associated with myocardial cells tended to show strong positive reactions in both shrimp species. Ovarian and neural tissue (neurons in the nerve ganglia and glial cells in the nerve cord) were ISH positive for IHHNV only in P. vannamei. By transmission electron microscopy, necrotic cells were found in the gills of IHHNV-infected P. vannamei, while paracrystalline arrays of virions and apoptotic cells rather than necrotic cells were found in the lymphoid organ of IHHNV-infected P. monodon. Thus, it is possible that apoptosis in P. monodon contributes to the absence of clinical disease from IHHNV. These findings reveal different responses to IHHNV infection by the 2 shrimp species. A curious feature of IHHNV infection in P. monodon was inconsistency in the comparative viral load amongst tissues of different specimens, as detected by both ISH and real-time PCR. This inconsistency in apparent tissue preference and the reasons for different cellular responses between the 2 shrimp species remain unexplained. © Inter-Research 2005.
• Erickson, H. S., Poulos, B. T., F., K., Bradley-Dunlop, D., & Lightner, D. V. (2005). Taura syndrome virus from Belize represents a unique variant. Diseases of Aquatic Organisms, 64(2), 91-98.
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  PMID: 15918471;Abstract: A Taura syndrome virus (TSV) isolate from cultured Penaeus vannamei grown in Belize, Central America was characterized and shown to be a unique isolate. Mortality rates in laboratory infections of specific pathogen-free (SPF) P. vannamei, reactivity of the virus with monoclonal antibody (MAb) 1A1 and phylogenetic analysis demonstrated that the Belize isolate (BLZ02TSV) is a new variant of TSV. The Hawaiian 1994 TSV isolate (HI94TSV, GenBank AF277675) was used as the reference isolate for these studies. Laboratory infections of SPF P. vannamei with BLZ02TSV demonstrated higher mortalities and earlier onset of mortalities compared to infections with HI94TSV Shrimp tissues infected with BLZ02TSV reacted with a TSV-specific gene probe by in situ hybridization and were positive by RT-PCR using TSV diagnostic primers, thus indicating that the isolate was TSV. However, Western blot analysis and immunohistochemistry using MAb 1A1 demonstrated that BLZ02TSV did not react with the antibody, suggestive of changes in the VP1 region of the genome that codes for the polypeptide to which MAb 1A1 binds. Phylogenetic analysis of a 1.3 kbp fragment of the TSV VP1 capsid region revealed that BLZ02TSV represents a distinct group among more than 29 isolates of TSV studied thus far. This research demonstrates that BLZ02TSV is a unique isolate of TSV and reiterates a problem related to the use of MAb 1A1 for detection of TSV in clinical specimens. © Inter-Research 2005.
• Hennig, O. L., Arce, S. M., Moss, S. M., Pantoja, C. R., & Lightner, D. V. (2005). Development of a specific pathogen free population of the Chinese fleshy prawn, Fenneropenaeus chinensis: Part II. Secondary quarantine. Aquaculture, 250(3-4), 579-585.
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  Abstract: The Oceanic Institute (OI), in collaboration with researchers from the University of Arizona (UAZ), established a population of specific pathogen free (SPF) Fenneropenaeus chinensis through Secondary Quarantine (SQ) in Kona, Hawaii. Shrimp were 0.38 g (S.D. ± 0.13 g) when they entered the nursery/growout module of the SQ facility. Juveniles from five maternal families were stocked in separate 230-L nursery tanks, as was a sixth family comprised of shrimp from two of the seven original spawns. After tagging, representative shrimp from all six families were stocked in a 50-m2 concrete raceway that was enclosed in a biosecure greenhouse. After 155 days in the growout module, mean shrimp weight was 23.4 g (S.D. ± 7.25 g) and females (29.1 ± 4.61 g) were 74% larger than males (16.7 ± 2.44 g). A Sprung Structure™ was erected to enclose the biosecure maturation and hatchery module of the SQ facility. Broodstock were subjected to an acclimation process where they were kept in seawater at 22°C, followed by a reduction in water temperature of 1°C/day until the temperature reached 17°C. At this time, unilateral eyestalk ablation was performed on female broodstock and water temperature was further reduced at the same rate until it reached 12°C. After 2 weeks, water temperature was raised to 17°C at a rate of 1°C/day to induce ovarian development. Production of an F1 generation of Fe. chinensis was successfully accomplished using artificial insemination, and representative shrimp from this generation have tested negative for all specifically listed pathogens using histology and PCR/RT-PCR. Of particular interest with Fe. chinensis is their cold tolerance. This species may represent a valuable resource for shrimp farmers in temperate and sub-tropical climates to grow a second "winter" crop, thereby increasing production and profitability for the shrimp farmer. © 2005 Elsevier B.V. All rights reserved.
• Lightner, D. V. (2005). Biosecurity in shrimp farming: Pathogen exclusion through use of SPF stock and routine surveillance. Journal of the World Aquaculture Society, 36(3), 229-248.
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  Abstract: Biosecurity, as it is being applied to shrimp aquaculture, may be defined as the practice of exclusion of specific pathogens from cultured aquatic stocks in broodstock facilities, hatcheries, and farms, or from entire regions or countries for the purpose of disease prevention. To make a biosecurity program a functional concept in shrimp aquaculture, the relevant risks should be identified and the appropriate biosecurity measures put into practice to mitigate those risks. Examples of biosecurity measures put into place for this purpose may include such basics as site selection when the intent is to locate a new shrimp culture facility in an area where certain diseases are not enzootic. Standard facility/farm operating procedures can be adapted to minimize the risks of disease introduction and spread within a facility through such concepts as pretreatment of all source water, and reduced or "zero" water exchange. Stocking shrimp culture facilities with domesticated shrimp stocks that are free of specific diseases ("Specific Pathogen Free" or SPF) and/or with stocks resistant to specific disease agents (SPR) is perhaps the most important single component of a biosecurity program. The example set by the development of domesticated SPF stocks of Litopenaeus vannamei has helped to make biosecure shrimp culture feasible. The development of these and other SPF stocks, and the diagnostic methods to develop and monitor them for specific diseases and disease causing agents, have been milestones in the development of the international shrimp farming industry in recent years, and it has contributed to the species rivaling Penaeus monodon as the dominant farmed shrimp species. The regular monitoring (surveillance) of shrimp stocks in biosecure culture facilities is a necessary component of a biosecurity plan, as is having in place a contingency plan for disease containment and eradication should a breach occur in the physical and managerial components of a biosecure facility and a targeted disease occur. © Copyright by the World Aquaculture Society 2005.
• Lightner, D., Erickson, H. S., Poulos, B. T., Tang, K. F., Bradley-Dunlop, D., & Lightner, D. V. (2005). Taura syndrome virus from Belize represents a unique variant. Diseases of aquatic organisms, 64(2).
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  A Taura syndrome virus (TSV) isolate from cultured Penaeus vannamei grown in Belize, Central America was characterized and shown to be a unique isolate. Mortality rates in laboratory infections of specific pathogen-free (SPF) P. vannamei, reactivity of the virus with monoclonal antibody (MAb) 1A1 and phylogenetic analysis demonstrated that the Belize isolate (BLZ02TSV) is a new valiant of TSV. The Hawaiian 1994 TSV isolate (HI94TSV, GenBank AF277675) was used as the reference isolate for these studies. Laboratory infections of SPF P. vannamei with BLZ02TSV demonstrated higher mortalities and earlier onset of mortalities compared to infections with HI94TSV. Shrimp tissues infected with BLZ02TSV reacted with a TSV-specific gene probe by in situ hybridization and were positive by RT-PCR using TSV diagnostic primers, thus indicating that the isolate was TSV. However, Western blot analysis and immunohistochemistry using MAb 1A1 demonstrated that BLZ02TSV did not react with the antibody, suggestive of changes in the VP1 region of the genome that codes for the polypeptide to which MAb 1A1 binds. Phylogenetic analysis of a 1.3 kbp fragment of the TSV VP1 capsid region revealed that BLZ02TSV represents a distinct group among more than 29 isolates of TSV studied thus far. This research demonstrates that BLZ02TSV is a unique isolate of TSV and reiterates a problem related to the use of MAb 1A1 for detection of TSV in clinical specimens.
• Lightner, D., Srisuvan, T., Tang, K. F., & Lightner, D. V. (2005). Experimental infection of Penaeus monodon with Taura syndrome virus (TSV). Diseases of aquatic organisms, 67(1-2).
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  Clinical signs and lesions of Taura syndrome virus (TSV) infection in Penaeus monodon have not been documented although the virus has been detected in this shrimp species by reverse transcription polymerase chain reaction (RT-PCR). This study provides the first evidence of TSV infection in P. monodon by histological and in situ hybridization (ISH) analyses. We performed experimental bioassays with groups of P. monodon using inocula of P. monodon and Litopenaeus vannamei (Th04PmTSV and Th04LvTSV, respectively), which were collected from Thailand in 2004 and found to be positive for TSV by RT-PCR. Samples of shrimp for histological and ISH analyses were collected on Days 2, 14, and 28 post-inoculation. Mortality among TSV-inoculated P. monodon appeared on Day 3, with 2 out of 10 shrimp dying. Severe necrosis of cuticular epithelial cells and lymphoid organ spheroids, indicative of acute and chronic phase lesions of TSV infection, respectively, were detected in the samples. Sequence analyses of the capsid protein 2 (CP2) gene showed that Th04PmTSV and Th04LvTSV isolates were different; however, both belonged to a phylogenetic family of Asian TSV isolates. The results of this study demonstrated that both mortality and histological lesions are associated with TSV infection in P. monodon.
• Lightner, D., Tang, K. F., & Lightner, D. V. (2005). Phylogenetic analysis of Taura syndrome virus isolates collected between 1993 and 2004 and virulence comparison between two isolates representing different genetic variants. Virus research, 112(1-2).
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  Taura syndrome virus (TSV) is highly pathogenic to Litopenaeus vannamei (Pacific white shrimp) and has caused significant economic loss in the shrimp culture industry. It was first reported from Ecuador in 1992 and has since become widely distributed throughout the Americas and southeast Asia (SE Asia). To determine the genetic relationship among various geographic isolates, we amplified and sequenced a 1.3 kb fragment of the TSV capsid protein gene 2 (CP2) from each of 34 isolates collected from cultured penaeid shrimp stocks in Ecuador, Colombia, Honduras, USA, Mexico, Belize, Thailand, China, and Indonesia. An additional six CP2 sequences obtained from GenBank were included in the analysis. The results indicated low genetic variation (0--5.6% for nucleotide sequence and 0--7.0% for deduced amino acid sequence) among these 40 isolates. A phylogenetic analysis based on the deduced CP2 amino acid sequence revealed three distinct groups: Americas, Belize, and SE Asia. The Belize and SE Asia groups were separated from each other by a 4.7% difference in amino acid sequence. The Belize and Americas groups differed by 4.4%. The Americas and SE Asia groups were the closest, separated by a difference of only 3.3%. Comparison between Belize and Hawaii TSV (reference strain for Americas group) indicated that Belize TSV was more virulent than Hawaii TSV. In bioassays, the Belize isolate caused 50% mortality by 3 days, while the Hawaii isolate caused 50% mortality over 4--6 days. Based on the phylogenetic analysis and virulence comparison, the Belize TSV isolate should be considered as a new variant.
• Nunan, L. M., Lightner, D. V., Oduori, M. A., & Gasparich, G. E. (2005). Spiroplasma penaei sp. nov., associated with mortalities in Penaeus vannamei, Pacific white shrimp. International Journal of Systematic and Evolutionary Microbiology, 55(6), 2317-2322.
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  PMID: 16280489;Abstract: A new bacterial strain, designated SHRIMPT, isolated from the haemolymph of the Pacific white shrimp, Penaeus vannamei, was serologically distinct from other spiroplasmas. Cells of this strain were helical in form and variable in length. Examination by electron microscopy revealed wall-less cells delineated by a single cytoplasmic membrane. The organisms grew well in M1D media supplemented with 2 % NaCl. Strain SHRIMPT grew at temperatures of 20-37 °C, with optimum growth occurring at 28 °C. The strain catabolized glucose and hydrolysed arginine, but did not hydrolyse urea. The G+C content of the DNA was 29 ± 1 mol%. Strain SHRIMPT (=ATCC BAA-1082T=CAIM 1252T) is designated the type strain of a novel species, Spiroplasma penaei sp. nov., which represents a new subgroup (I-9) of the group I spiroplasmas. © 2005 IUMS.
• Pantoja, C. R., Song, X., Xia, L., Gong, H., Wilkenfeld, J., Noble, B., & Lightner, D. V. (2005). Development of a specific pathogen-free (SPF) population of the Chinese fleshy prawn Fenneropenaeus chinensis - Part 1: Disease Pre-screening and Primary Quarantine. Aquaculture, 250(3-4), 573-578.
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  Abstract: A total of 36 gravid female shrimp Fenneropenaeus chinensis, captured off the coast of Qingdao, Peoples Republic of China, from April 20 through April 24, 2001, were examined by PCR for the presence of WSSV and HPV, the two most prevalent viral diseases affecting this species of shrimp in this region of China. Eight spawns, derived from 8 different females, were selected from shrimp where no WSSV or HPV had been detected. Nauplii obtained from 7 individual spawns and unhatched eggs from one other shrimp were transported to the University of Arizona, in Tucson, Arizona, USA, for larval rearing and production of postlarvae under quarantine protocols. The resultant postlarvae were kept in Primary Quarantine for approximately 2 months, while additional tests were performed to determine their specific pathogen-free (SPF) status according to criteria set by the United States Marine Shrimp Farming Program (USMSFP). By the time the shrimp had reached an average weight of approximately 0.4 g, no USMSFP specific listed pathogens had been detected in any of the 7 hatches and the shrimp were released into Secondary Quarantine at the Oceanic Institute in Kailua-Kona, Hawaii for production of a F1 generation. This F1 generation may be used as a SPF founder population for propagation and selection programs. © 2005 Elsevier B.V. All rights reserved.
• Srisuvan, T., Tang, K. F., & Lightner, D. V. (2005). Experimental infection of Penaeus monodon with Taura syndrome virus (TSV). Diseases of Aquatic Organisms, 67(1-2), 1-8.
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  PMID: 16385801;Abstract: Clinical signs and lesions of Taura syndrome virus (TSV) infection in Penaeus monodon have not been documented although the virus has been detected in this shrimp species by reverse transcription polymerase chain reaction (RT-PCR). This study provides the first evidence of TSV infection in P. monodon by histological and in situ hybridization (ISH) analyses. We performed experimental bioassays with groups of P. monodon using inocula of P. monodon and Litopenaeus vannamei (Th04PmTSV and Th04LvTSV, respectively), which were collected from Thailand in 2004 and found to be positive for TSV by RT-PCR. Samples of shrimp for histological and ISH analyses were collected on Days 2, 14, and 28 post-inoculation. Mortality among TSV-inoculated P. monodon appeared on Day 3, with 2 out of 10 shrimp dying. Severe necrosis of cuticular epithelial cells and lymphoid organ spheroids, indicative of acute and chronic phase lesions of TSV infection, respectively, were detected in the samples. Sequence analyses of the capsid protein 2 (CP2) gene showed that Th04PmTSV and Th04LvTSV isolates were different; however, both belonged to a phylogenetic family of Asian TSV isolates. The results of this study demonstrated that both mortality and histological lesions are associated with TSV infection in P. monodon. © Inter-Research 2005.
• Surachetpong, W., Poulos, B. T., Tang, K. F., & Lightner, D. V. (2005). Improvement of PCR method for the detection of monodon baculovirus (MBV) in penaeid shrimp. Aquaculture, 249(1-4), 69-75.
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  Abstract: Penaeus monodon-type baculovirus (MBV) infects and causes disease in hatchery-reared larvae and early stages of juvenile P. monodon. In this study, an improved 1-step PCR method was developed for the detection of MBV. The oligonucleotide primers, assigned as 261F and 261R, were designed from a genomic clone of MBV. Using extracted DNA from hepatopancreas or feces of ten geographic isolates of MBV infected shrimp, the PCR reaction yields a 261 bp fragment specific to the MBV sequence. Primers 261F/R did not cross-react with DNA from other shrimp viruses or with uninfected shrimp tissue. The detection limit of this primer pair is 100 copies of positive control plasmid DNA. This improved molecular method provides a diagnostic tool for identification of MBV in infected shrimp tissue collected from different geographic regions. © 2005 Elsevier B.V. All rights reserved.
• Tang, K. F., & Lightner, D. V. (2005). Phylogenetic analysis of Taura syndrome virus isolates collected between 1993 and 2004 and virulence comparison between two isolates representing different genetic variants. Virus Research, 112(1-2), 69-76.
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  PMID: 16022902;Abstract: Taura syndrome virus (TSV) is highly pathogenic to Litopenaeus vannamei (Pacific white shrimp) and has caused significant economic loss in the shrimp culture industry. It was first reported from Ecuador in 1992 and has since become widely distributed throughout the Americas and southeast Asia (SE Asia). To determine the genetic relationship among various geographic isolates, we amplified and sequenced a 1.3 kb fragment of the TSV capsid protein gene 2 (CP2) from each of 34 isolates collected from cultured penaeid shrimp stocks in Ecuador, Colombia, Honduras, USA, Mexico, Belize, Thailand, China, and Indonesia. An additional six CP2 sequences obtained from GenBank were included in the analysis. The results indicated low genetic variation (0-5.6% for nucleotide sequence and 0-7.0% for deduced amino acid sequence) among these 40 isolates. A phylogenetic analysis based on the deduced CP2 amino acid sequence revealed three distinct groups: Americas, Belize, and SE Asia. The Belize and SE Asia groups were separated from each other by a 4.7% difference in amino acid sequence. The Belize and Americas groups differed by 4.4%. The Americas and SE Asia groups were the closest, separated by a difference of only 3.3%. Comparison between Belize and Hawaii TSV (reference strain for Americas group) indicated that Belize TSV was more virulent than Hawaii TSV. In bioassays, the Belize isolate caused 50% mortality by 3 days, while the Hawaii isolate caused 50% mortality over 4-6 days. Based on the phylogenetic analysis and virulence comparison, the Belize TSV isolate should be considered as a new variant. © 2005 Elsevier B.V. All rights reserved.
• Bradley-Dunlop, D. J., Pantoja, C., & Lightner, D. V. (2004). Development of monoclonal antibodies for detection of necrotizing hepatopancreatitis in penaeid shrimp. Diseases of Aquatic Organisms, 60(3), 233-240.
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  PMID: 15521322;Abstract: Monoclonal antibodies (MAbs) were produced against necrotizing hepatopancreatitis bacteria (NHP-B) of penaeid shrimp. The MAbs tested in dot-immunoblot (D-IB) assays were capable of detecting the NHP-B in hepatopancreas samples collected from moribund juvenile Litopenaeus vannamei during an experimentally induced NHP-B infection. The MAbs were also screened by immunohistochemistry (IHC) using case submissions that were determined to be infected not only by histology, but also polymerase chain reaction (PCR) and in situ hybridization (ISH) assays using specific digoxigenin (DIG)-labeled probes on histological sections prepared from naturally infected shrimp. Two of the MAbs were chosen for development of detection methods for NHP. The MAbs were tested using IHC methods on Davidson's alcohol-formalin-acetic acid (AFA) fixed tissue sections and identified NHP-B infected cells and tissues in a pattern similar to that seen with DIG-labeled NHP-specific gene probes. None of the MAbs reacted with tissue from specific pathogen-free (SPF) shrimp or with shrimp tissues infected with a rickettsia-like bacteria, Vibrio sp., Campylobacter sp., and Spiroplasma sp. The MAbs were found to be negative against these other organisms, demonstrating that they are species specific and useful for rapid diagnostic detection of NHP-B.
• Gong, H., Jiang, D. -., Lightner, D. V., Collins, C., & Brock, D. (2004). A dietary modification approach to improve the osmoregulatory capacity of Litopenaeus vannamei cultured in the Arizona desert. Aquaculture Nutrition, 10(4), 227-236.
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  Abstract: A modified diet was formulated for Arizona inland shrimp farming and tested as a method for reducing moult-associated mortalities presumed due to trace mineral deficiencies. The experimental diet was supplemented with additional dietary magnesium, potassium, phospholipids and cholesterol to a commercial shrimp feed (Rangen 45/10, which was also used as the control diet). The modified diet was tested at Arizona Mariculture Associates (AMA), while the control diet was used at a nearby inland shrimp farm, Desert Sweet Shrimp Farm (DSSF). Both feeds were used throughout the culture season of 2001. Earthen pond-reared Litopenaeus vannamei at intermoult stages (C-D0) and ranging from 7 to 30 g were sampled at intervals for determination of haemolymph osmolality (HO). Results showed that the modified diet had not only resulted in larger size shrimp at harvest, but also improved osmoregulatory capacity (OC). HO of DSSF shrimp decreased as shrimp grew bigger, whereas HO of AMA shrimp was maintained at a stable level, or showed a slightly positive linear relationship with weight. The hyper-OC of shrimp from AMA (462 mOsm kg-1) was greater than that from DSSF (398 mOsm kg-1). Shrimp at AMA fed the experimental diet presented no mass moult-associated mortalities. To further investigate the iso-osmotic point of shrimp reared in AMA, a group of six salinity gradients were designed by mixing oceanic salts into the well water to form 5, 8.5, 11.4, 14.4, 17.8, 20.7 p.p.t. medium. HO of subadult shrimp (25 g in average) were then evaluated 48 h after they had been transferred from 5 p.p.t. pond water to the medium. Shrimp HO increased with external salinity, and a plateau formed as salinity reached at 11.4 p.p.t. and higher. The iso-osmotic point of shrimp was estimated to be 695.5 mOsm kg-1, equivalent to 26.1 p.p.t. in AMA well water.
• Lightner, D., Bradley-Dunlop, D. J., Pantoja, C., & Lightner, D. V. (2004). Development of monoclonal antibodies for detection of necrotizing hepatopancreatitis in penaeid shrimp. Diseases of aquatic organisms, 60(3).
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  Monoclonal antibodies (MAbs) were produced against necrotizing hepatopancreatitis bacteria (NHP-B) of penaeid shrimp. The MAbs tested in dot-immunoblot (D-IB) assays were capable of detecting the NHP-B in hepatopancreas samples collected from moribund juvenile Litopenaeus vannamei during an experimentally induced NHP-B infection. The MAbs were also screened by immunohistochemistry (IHC) using case submissions that were determined to be infected not only by histology, but also polymerase chain reaction (PCR) and in situ hybridization (ISH) assays using specific digoxigenin (DIG)-labeled probes on histological sections prepared from naturally infected shrimp. Two of the MAbs were chosen for development of detection methods for NHP. The MAbs were tested using IHC methods on Davidson's alcohol-formalin-acetic acid (AFA) fixed tissue sections and identified NHP-B infected cells and tissues in a pattern similar to that seen with DIG-labeled NHP-specific gene probes. None of the MAbs reacted with tissue from specific pathogen-free (SPF) shrimp or with shrimp tissues infected with a rickettsia-like bacteria, Vibrio sp., Campylobacter sp., and Spiroplasma sp. The MAbs were found to be negative against these other organisms, demonstrating that they are species specific and useful for rapid diagnostic detection of NHP-B.
• Lightner, D., Nunan, L. M., Pantoja, C. R., Salazar, M., Aranguren, F., & Lightner, D. V. (2004). Characterization and molecular methods for detection of a novel spiroplasma pathogenic to Penaeus vannamei. Diseases of aquatic organisms, 62(3).
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  Traditionally, Spiroplasma spp. have only been isolated from the surfaces of flowers and other plant parts, from the guts and hemolymph of various insects, and from vascular plant fluids (phloem sap) and insects that feed on these fluids. In this article, we report the first pathogenic spiroplasma to be discovered in shrimp and the results of its characterization through histological evaluation, in situ hybridization assays, transmission electron microscopy, 16S rRNA sequence homology, and injection infectivity studies. In addition, molecular methods are described that were developed for the detection of this microorganism, which was determined to be the causative disease agent in Colombian farm-raised Penaeus vannamei suffering from high mortalities. Using standard histological methods and in situ hybridization assays, it was confirmed that P. vannamei was infected with this pathogenic spiroplasma. Histological analysis revealed systemic inflammatory reactions in affected organs/tissues. In an attempt to identify the bacteria, frozen infected P. vannamei samples, from the initial epizootic, were used to sequence the 16S rRNA gene and develop molecular detection methods. The 16S rRNA gene was amplified by PCR and then sequenced. The sequence data were analyzed using the GenBank BLAST search and the results revealed a 98% homology with Spiroplasma citri, a pathogen of citrus trees. The 16S rRNA sequence data were evaluated for development of unique PCR primers to the putative spiroplasma. Using PCR primers developed for the spiralin gene of Spiroplasma spp., a digoxigenin-labeled probe was developed and tested. This probe was species-specific, with no positive reactions or cross-reactivity occurring with other bacterial samples tested in this format.
• Lightner, D., Tang, K. F., Wang, J., & Lightner, D. V. (2004). Quantitation of Taura syndrome virus by real-time RT-PCR with a TaqMan assay. Journal of virological methods, 115(1).
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  A real-time RT-PCR assay was developed using a TaqMan probe to detect and quantify Taura syndrome virus (TSV) in penaeid shrimp. A pair of RT-PCR primers, which amplify a 72 bp DNA fragment, and a TaqMan probe were selected from open reading frame 1 (ORF1) of the TSV genome. The primers and TaqMan probe used in this assay reacted with TSV isolates from Hawaii, Texas, Colombia, Mexico, Belize, Indonesia, and Thailand, but neither with RNA of healthy shrimp nor with an isolate of yellow head virus. A plasmid (pTSV-1) that contains the target TSV sequence was constructed and used to generate positive control RNA through in vitro transcription. The positive control RNA was used to demonstrate that the real-time RT-PCR assay has a detection limit of 100 copies and a log-linear range up to 10(8) copies of TSV RNA. This quantitative method was found to be highly reproducible, with low intra- and inter-assay variation. Coefficient of variation (CVs) values were 0.04-8.9 and 0.05-3.7%, respectively, for replicates within and among assays. This assay was used to quantify TSV in both acutely and chronically infected shrimp in a laboratory experiment. The quantities of TSV in the tissues of pleopods and gills were not significantly different, and there was no difference in TSV levels between the acutely and chronically infected groups. However, in the chronically infected shrimp, the quantities of TSV were one to two orders of magnitude higher in the lymphoid organ than in either gills or pleopods. This assay proved to be specific with high sensitivity, and it can be used to detect and quantify TSV in shrimp samples.
• Nunan, L. M., Pantoja, C. R., Salazar, M., Aranguren, F., & Lightner, D. V. (2004). Characterization and molecular methods for detection of a novel spiroplasma pathogenic to Penaeus vannamei. Diseases of Aquatic Organisms, 62(3), 255-264.
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  PMID: 15672883;Abstract: Traditionally, Spiroplasma spp. have only been isolated from the surfaces of flowers and other plant parts, from the guts and hemolymph of various insects, and from vascular plant fluids (phloem sap) and insects that feed on these fluids. In this article, we report the first pathogenic spiroplasma to be discovered in shrimp and the results of its characterization through histological evaluation, in situ hybridization assays, transmission electron microscopy, 16S rRNA sequence homology, and injection infectivity studies. In addition, molecular methods are described that were developed for the detection of this microorganism, which was determined to be the causative disease agent in Colombian farm-raised Penaeus vannamei suffering from high mortalities. Using standard histological methods and in situ hybridization assays, it was confirmed that P. vannamei was infected with this pathogenic spiroplasma. Histological analysis revealed systemic inflammatory reactions in affected organs/tissues. In an attempt to identify the bacteria, frozen infected P. vannamei samples, from the initial epizootic, were used to sequence the 16S rRNA gene and develop molecular detection methods. The 16S rRNA gene was amplified by PCR and then sequenced. The sequence data were analyzed using the GenBank BLAST search and the results revealed a 98 % homology with Spiroplasma citri, a pathogen of citrus trees. The 16S rRNA sequence data were evaluated for development of unique PCR primers to the putative spiroplasma. Using PCR primers developed for the spiralin gene of Spiroplasma spp., a digoxigenin-labeled probe was developed and tested. This probe was species-specific, with no positive reactions or cross-reactivity occurring with other bacterial samples tested in this format.
• Nunan, L. M., Tang-Nelson, K., & Lightner, D. V. (2004). Real-time RT-PCR determination of viral copy number in Penaeus vannamei experimentally infected with Taura syndrome virus. Aquaculture, 229(1-4), 1-10.
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  Abstract: This study examined the viral copy number as determined by real-time RT-PCR, in different tissue samples from Penaeus vannamei exposed to Taura syndrome virus (TSV). Two routes of exposure, injection and per os, were investigated. Six different body parts from each shrimp were assessed for viral copy numbers. Eight shrimp were analyzed per treatment. In addition, eight specific pathogen free (SPF) P. vannamei were analyzed and served as a negative control. The tissue samples examined included: whole tail muscle, tail muscle (shell removed), gills, pleopods, head (cephalothorax with the hepatopancreas included) and tail fan. The results from these experiments showed a significant level of difference between the SPF and the injection treatments. As was expected, there was also a significant difference between the negative control and the per os treatment groups. There was no significant difference between the viral copy number contained in different body parts from the injection experiment. These results contrasted to the per os results, in which there was a statistically significant difference between tail/gills, tail/head, tail/tail fan, whole tail/tail fan and pleopods/tail fan. The tail samples had the lower viral copy numbers, as did the whole tail and pleopods when compared to the tail fan. The mean viral copy number per nanogram of total RNA (x̄ cn/ng tRNA) extracted in the injection study ranged from 1.4 × 105 in the gills to 2.3 × 105 in the whole tail. In the per os experiment, the x̄ cn/ng of extracted tRNA ranged from 2.5 × 10 4 in the tail muscle to 4.3 × 105 in the head. When these values are converted to mean viral copy number per gram (x̄ cn/g) of tissue, the values increased in range. In the injection study, the x̄ cn/g of tissue ranged from 1.2 × 109 in the tail fan to 7.4 × 109 in the head. These values contrast to the x̄ cn/g of tissue in the per os study in that the lowest value of 1.7 × 108 was in the tail muscle and the highest x̄, 1.7 × 1010, was in the head. Overall, all body parts from infected shrimp, regardless of treatment type, quantitatively analyzed by real-time RT-PCR, determined the presence of TSV. © 2004 Elsevier Science B.V. All rights reserved.
• Pantoja, C. R., Navarro, S. A., Naranjo, J., Lightner, D. V., & Gerba, C. P. (2004). Nonsusceptibility of primate cells to Taura syndrome virus. Emerging Infectious Diseases, 10(12), 2106-2112.
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  PMID: 15663846;PMCID: PMC3323408;Abstract: Taura syndrome virus (TSV), a pathogen of penaeid shrimp and member of the family Dicistroviridae, was recently reported to have the ability to infect primate cells. We independently retested this hypothesis. Three lines of primate cells FRhK-4, MA-104, and BGMK, which are highly susceptible to infection by human picornaviruses, were challenged with TSV. Viral replication was assayed by real-time reverse transcription-polymerase chain reaction using cell media samples collected on days 0, 4, and 7 postchallenge. By day 7, genome copy numbers had decreased 25%-99%. No cytopathic effect was observed after 7 days. An in situ hybridization assay, with gene probes specific for detection of TSV, was negative for TSV in challenged cells. The infectivity of residual virus in the cell culture media at day 7 was confirmed by bioassay using TSV-free indicator shrimp (Litopenaeus vannamei). TSV did not infect the primate cells tested, and no evidence of zoonotic potential was found.
• Tang, K. F., Wang, J., & Lightner, D. V. (2004). Quantitation of Taura syndrome virus by real-time RT-PCR with a TaqMan assay. Journal of Virological Methods, 115(1), 109-114.
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  PMID: 14656468;Abstract: A real-time RT-PCR assay was developed using a TaqMan probe to detect and quantify Taura syndrome virus (TSV) in penaeid shrimp. A pair of RT-PCR primers, which amplify a 72bp DNA fragment, and a TaqMan probe were selected from open reading frame 1 (ORF1) of the TSV genome. The primers and TaqMan probe used in this assay reacted with TSV isolates from Hawaii, Texas, Colombia, Mexico, Belize, Indonesia, and Thailand, but neither with RNA of healthy shrimp nor with an isolate of yellow head virus. A plasmid (pTSV-1) that contains the target TSV sequence was constructed and used to generate positive control RNA through in vitro transcription. The positive control RNA was used to demonstrate that the real-time RT-PCR assay has a detection limit of 100 copies and a log-linear range up to 108 copies of TSV RNA. This quantitative method was found to be highly reproducible, with low intra- and inter-assay variation. Coefficient of variation (CVs) values were 0.04-8.9 and 0.05-3.7%, respectively, for replicates within and among assays. This assay was used to quantify TSV in both acutely and chronically infected shrimp in a laboratory experiment. The quantities of TSV in the tissues of pleopods and gills were not significantly different, and there was no difference in TSV levels between the acutely and chronically infected groups. However, in the chronically infected shrimp, the quantities of TSV were one to two orders of magnitude higher in the lymphoid organ than in either gills or pleopods. This assay proved to be specific with high sensitivity, and it can be used to detect and quantify TSV in shrimp samples. © 2003 Elsevier B.V. All rights reserved.
• Vanpatten, K. A., Nunan, L. M., & Lightner, D. V. (2004). Seabirds as potential vectors of penaeid shrimp viruses and the development of a surrogate laboratory model utilizing domestic chickens. Aquaculture, 241(1-4), 31-46.
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  Abstract: White spot syndrome virus (WSSV), Taura syndrome virus (TSV), Yellow head virus (YHV), and Infectious hypodermal and hematopoietic necrosis virus (IHHNV) have caused significant economic losses of farmed penaeid shrimp worldwide. Because the presence of large numbers of seabirds at shrimp grow-out ponds may be related to disease transmission, we hypothesized that these birds may be capable of carrying infectious viral particles in their feces from affected ponds to nearby unaffected ponds, and perhaps even to nearby farms. Because of the difficulties posed by working with wild fowl in a laboratory setting, a surrogate model, employing domestic white leghorn chickens (Gallus domesticus), was developed to compare with the detection and infectivity results from captive laughing gulls (Larus atricilla). Using standard histology, PCR/RT-PCR and infectivity challenges, this study demonstrated that IHHNV and TSV remained infectious for up to 1 day following passage through both chickens and seagulls, while no viable WSSV or YHV were found following passage through the gut of either bird species. This study demonstrated that wild seabirds may serve as mechanical vectors of TSV and IHHNV by passing infectious particles to aquatic environments in their feces, and that chickens may be used as a surrogate laboratory model for seabirds in such viral infectivity tests. © 2004 Elsevier B.V. All rights reserved.
• Durand, S. V., Redman, R. M., Mohney, L. L., Tang-Nelson, K., Bonami, J. R., & Lightner, D. V. (2003). Qualitative and quantitative studies on the relative virus load of tails and heads of shrimp acutely infected with WSSV. Aquaculture, 216(1-4), 9-18.
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  Abstract: There is currently concern and controversy in the shrimp industries of the Americas about the risk posed by the importation and reprocessing of shrimp infected with white spot syndrome virus (WSSV) and yellow head virus (YHV). To further understand the risk, more knowledge concerning the quantitative virus load of infected shrimp is needed. The present study was carried out to better define, using qualitative and quantitative methods, the relative virus load of shrimp heads and tails. For these studies, specific pathogen-free (SPF) Penaeus vannamei were infected with WSSV. Emergency harvest of these shrimp was simulated by collecting the infected shrimp at the onset of postinfection mortalities and determining the relative virus loads of the head and tails by quantitative real-time PCR and histology methods. Routine histology and in situ hybridization assay with a WSSV-specific DNA probe demonstrated qualitatively similar levels of WSSV infection in the heads and tails of experimental infected shrimp. The novel real-time PCR method demonstrated quantitatively that the head had a slightly higher WSSV load than did the tail. However, since the tail represents 58% of the total body weight, the total virus load on a per weight basis turns out to be similar in the head (49%) and tail (51%) of the same shrimp with acute phase WSSV infections. In proportion to the total tail weight, the virus load of the peeled shell represents 55% of the total viral load in tail. © 2003 Elsevier Science B.V. All rights reserved.
• F., K., Poulos, B. T., Wang, J., Redman, R. M., Shih, H., & Lightner, D. V. (2003). Geographic variations among infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolates and characteristics of their infection. Diseases of Aquatic Organisms, 53(2), 91-99.
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  PMID: 12650241;Abstract: Nucleotide sequence variations of a 2.9 kb fragment of infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolated from samples of Penaeus monodon were determined and compared with an isolate from Hawaii. The infection characteristics of these isolates were examined by histology, in situ hybridization, and laboratory challenge studies with P, vannamei. Isolates of IHHNV were obtained from samples collected from the SE Asia region (the Philippines, Thailand, and Taiwan). Isolates of putative IHHNV were obtained from African samples (Tanzania, Madagascar, and Mauritius). The Philippine isolate had a very high nucleotide sequence identity (99.8%) to Hawaii IHHNV. The Thailand isolate showed a slightly lower identity (96.2%). The putative IHHNV sequences collected from Tanzania and Madagascar showed greater divergence from Hawaii IHHNV, 8.2% difference for Tanzania and 14.1% difference for Madagascar. A phylogenetic analysis showed that the Philippine IHHNV clustered with IHHNV found in the western hemisphere. This supports the theory that the Philippines was the origin of IHHNV that was first detected in Hawaii, In the laboratory infection study, both the Philippine and Thailand IHHNV were passed into P. vannamei, and the infected shrimp did not suffer any mortalities. In another laboratory infection, P. vannamei injected with a tissue homogenate of P. monodon from Madagascar, which tested positive for IHHNV by PCR, did not demonstrate IHHNV infection, suggesting that this putative IHHNV is not infectious to P. vannamei.
• Lightner, D., Nunan, L. M., Noble, B., Le Groumellec, M., & Lightner, D. V. (2003). Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P. monodon. Diseases of aquatic organisms, 54(1).
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  A rickettsia-like bacterium (RLB), which caused severe mortalities of commercially farmed Penaeus monodon in the southwest region of Madagascar, was investigated to determine whether the organism would produce the same disease in P. vannamei. Two series of bioassays were performed to determine whether this RLB could be transmitted to P. vannamei through injection and per os exposure. The first series of challenge bioassays used frozen, RLB-infected P. monodon tissue from Madagascar as the inoculum and feed for the injection, and per os bioassays with specific pathogen free (SPF) P. vannamei. In the second series of bioassays, frozen RLB-infected P. vannamei tissue derived from the first series of injection bioassays was used as the inoculum to challenge by injection and per os SPF P. vannamei. Disease status was determined through standard histological techniques and by in situ hybridization assays with a digoxigenin-labeled probe specific for this RLB. The results indicated that P. vannamei did develop the RLB infection when injected with either RLB infected P. monodon or P. vannamei tissue homogenates. This contrasts with results from the per os exposure to the RLB in which the disease could not be reproduced.
• Lightner, D., Nunan, L. M., Poulos, B., Redman, R., Le Groumellec, M., & Lightner, D. V. (2003). Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea). Diseases of aquatic organisms, 53(1).
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  Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.
• Lightner, D., Tang, K. F., Poulos, B. T., Wang, J., Redman, R. M., Shih, H., & Lightner, D. V. (2003). Geographic variations among infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolates and characteristics of their infection. Diseases of aquatic organisms, 53(2).
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  Nucleotide sequence variations of a 2.9 kb fragment of infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolated from samples of Penaeus monodon were determined and compared with an isolate from Hawaii. The infection characteristics of these isolates were examined by histology, in situ hybridization, and laboratory challenge studies with P. vannamei. Isolates of IHHNV were obtained from samples collected from the SE Asia region (the Philippines, Thailand, and Taiwan). Isolates of putative IHHNV were obtained from African samples (Tanzania, Madagascar, and Mauritius). The Philippine isolate had a very high nucleotide sequence identity (99.8%) to Hawaii IHHNV. The Thailand isolate showed a slightly lower identity (96.2%). The putative IHHNV sequences collected from Tanzania and Madagascar showed greater divergence from Hawaii IHHNV, 8.2% difference for Tanzania and 14.1% difference for Madagascar. A phylogenetic analysis showed that the Philippine IHHNV clustered with IHHNV found in the western hemisphere. This supports the theory that the Philippines was the origin of IHHNV that was first detected in Hawaii. In the laboratory infection study, both the Philippine and Thailand IHHNV were passed into P. vannamei, and the infected shrimp did not suffer any mortalities. In another laboratory infection, P. vannamei injected with a tissue homogenate of P. monodon from Madagascar, which tested positive for IHHNV by PCR, did not demonstrate IHHNV infection, suggesting that this putative IHHNV is not infectious to P. vannamei.
• Nunan, L. M., Noble, B., Groumellec, M. L., & Lightner, D. V. (2003). Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P. monodon. Diseases of Aquatic Organisms, 54(1), 43-48.
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  PMID: 12718469;Abstract: A rickettsia-like bacterium (RLB), which caused severe mortalities of commercially farmed Penaeus monodon in the southwest region of Madagascar, was investigated to determine whether the organism would produce the same disease in P. vannamei. Two series of bioassays were performed to determine whether this RLB could be transmitted to P. vannamei through injection and per os exposure. The first series of challenge bioassays used frozen, RLB-infected P. monodon tissue from Madagascar as the inoculum and feed for the injection, and per os bioassays with specific pathogen free (SPF) P. vannamei. In the second series of bioassays, frozen RLB-infected P. vannamei tissue derived from the first series of injection bioassays was used as the inoculum to challenge by injection and per os SPF P. vannamei. Disease status was determined through standard histological techniques and by in situ hybridization assays with a digoxigenin-labeled probe specific for this RLB. The results indicated that P. vannamei did develop the RLB infection when injected with either RLB infected P. monodon or P. vannamei tissue homogenates. This contrasts with results from the per os exposure to the RLB in which the disease could not be reproduced.
• Nunan, L. M., Poulos, B., Redman, R., Groumellec, M. L., & Lightner, D. V. (2003). Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea). Diseases of Aquatic Organisms, 53(1), 15-23.
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  PMID: 12608564;Abstract: Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65°C, otherwise cross-reactivity was observed with other types of bacteria.
• Pantoja, C. R., & Lightner, D. V. (2003). Similarity between the histopathology of white spot syndrome virus and yellow head syndrome virus and its relevance to diagnosis of YHV disease in the Americas. Aquaculture, 218(1-4), 47-54.
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  Abstract: The similarity of lesions, especially of the lymphoid organ (LO), caused by severe white spot syndrome virus (WSSV) infection and those once thought to be diagnostic for yellow head virus (YHV) infection have been the cause of misdiagnosis of YHV disease at several pathology laboratories in the Americas. In order to illustrate this similarity, and to demonstrate the risk of misdiagnosis, specimens of shrimp Penaeus setiferus and P. vannamei, experimentally infected with WSSV, were examined by conventional H&E histology and by in situ hybridization (ISH) with nonradioactively labeled gene probes specific for detection of each virus. H&E analysis of shrimp with severe WSSV infection revealed the presence of severe necrosis of the lymphoid organ and of the fibrous connective tissue that were very similar to that caused by YHV infection. Five representative shrimps, displaying severe lymphoid organ necrosis, were selected and subjected to in situ hybridization with the gene probe specific for detection of YHV, and the results were negative. When the same specimens were subjected to in situ hybridization with the WSSV-specific gene probes, a strong positive reaction to the probes was observed both systemically and within the lymphoid organ. Clinical samples of 10 shrimps originating from several Latin American countries and diagnosed with severe WSSV infection by H&E histology and displaying severe lymphoid organ necrosis were also examined by in situ hybridization. No reaction to the YHV probe was observed in any of these specimens. Hence, we have concluded that severe infection by WSSV may result in systemic necrosis, and necrosis of the lymphoid organ, in particular, which is very similar to that caused by YHV. Since the presence of YHV has not been confirmed in the Americas, it is recommended that in cases when YHV infection may be suspected after conventional H&E analysis, additional evidence should be obtained by other means such as in situ hybridization with a YHV-specific probe and/or by RT-PCR. © 2003 Elsevier Science B.V. All rights reserved.
• Durand, S. V., & Lightner, D. V. (2002). Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp. Journal of Fish Diseases, 25(7), 381-389.
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  Abstract: Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested (Penaeus stylirostris, P. monodon, P. vannamei) ranged from 2.0 × 104 to 9.0 × 1010 WSSV copies μg-1 of total DNA (n=26). In whole moribund post-larvae, 4.3 × 109 WSSV copies μg-1 of DNAwere detected which is equivalent to 5.7 × 1010 WSSV copies g-1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in < 4 h).
• Erickson, H. S., Zarain-Herzberg, M., & Lightner, D. V. (2002). Detection of Taura syndrome virus (TSV) strain differences using selected diagnostic methods: Diagnostic implications in penaeid shrimp. Diseases of Aquatic Organisms, 52(1), 1-10.
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  PMID: 12517000;Abstract: Anecdotal industry reports of Taura syndrome (TS) epizootics in a Taura syndrome virus (TSV) tolerant strain of Penaeus stylirostris and collected evidence of field TS epizootics in P. stylirostris suggested that a distinct new TSV strain might have emerged since 1994. The Ecuadorian 1992 TSV genome published in GenBank is virtually identical to the Hawaiian 1994 TSV isolate (HI94TSV) used as reference throughout this investigation. Three other geographic and year isolates of TSV from naturally occurring TS epizootics of cultured penaeid shrimp were obtained from Mexico (SIN98TSV and MX99TSV from P. vannamei and SON2KTSV from P. stylirostris). Selected TSV diagnostic methods set forth by the Office International des Epizooties were utilized as the basis for isolate analysis. By Southern blot, TSV probes P15 and Q1 reacted specifically with all the diagnostic reverse transcription polymerase chain reaction (RT-PCR) fragments. Additionally, labeled RT-PCR amplicons from the TSV isolates amplified by routine diagnostic RT-PCR primers gave positive in situ hybridizations with TSV, indicating that all 4 isolates shared homology. By Western blot, immuno-dot blot, and immunohistochemistry, all TSV-purified isolates reacted with TSV polyclonal antibody (PAb). However, with TSV monoclonal antibody (MAb) 1A1 all isolates, except SIN98TSV, reacted, indicating that the difference in isolate SIN98TSV is within VP1, the target for MAb 1A1. The amino acid (AA) sequence of SIN98TSV VP1, MX99TSV VP1 and SON2KTSV VP1 has a 98% homology with the reference HI94TSV VP1. A span of 12 AAs are identified in SIN98TSV VP1 containing significant AA substitutions which may account for a conformational change of the antigenic epitope sufficient to prevent MAb 1A1 from binding. The implications of these results with respect to the antibody-based diagnosis of TSV are discussed.
• F., K., & Lightner, D. V. (2002). Low sequence variation among isolates of infectious hypodermal and hematopoietic necrosis virus (IHHNV) originating from Hawaii and the Americas. Diseases of Aquatic Organisms, 49(2), 93-97.
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  PMID: 12078987;Abstract: A 2.9 kb fragment of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) genome, which contains the coding sequence of putative non-structural and capsid proteins, was amplified and sequenced from each of 14 IHHNV isolates collected from cultured penaeid shrimp stocks in Hawaii and various sites in the Americas between 1982 and 1997. The sequence comparison indicates that the IHHNV genome is very stable, with 99.6 to 100% similarity among these 14 isolates. Only nucleotide substitutions were found. The percentage of substitution was higher in the putative capsid proteins region (1.3%) than in the putative non-structural proteins region (0.6%). Out of 25 substitutions found, 14 resulted in amino acid changes. There is no apparent association between clinical outcomes and particular amino acid substitutions. Based on genetic distances, the isolates were clustered into 3 groups that generally correspond with their geographic origins.
• F., K., Spann, K. M., Owens, L., & Lightner, D. V. (2002). In situ detection of Australian gill-associated virus with a yellow head virus gene probe. Aquaculture, 205(1-2), 1-5.
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  Abstract: A digoxigenin-labeled gene probe for yellow head virus (YHV) was used to detect gill-associated virus (GAV) in Penaeus monodon from Australia via in situ hybridization. In GAV-infected shrimp, positively reacting tissues included: lymphoid organ, gills, antennal gland, and cuticular epithelium of the stomach. This demonstrates that a YHV probe can be used as a diagnostic tool for GAV and supports previous suggestions that these two viruses are closely related. © 2002 Elsevier Science B.V. All rights reserved.
• Lightner, D., Erickson, H. S., Zarain-Herzberg, M., & Lightner, D. V. (2002). Detection of Taura syndrome virus (TSV) strain differences using selected diagnostic methods: diagnostic implications in penaeid shrimp. Diseases of aquatic organisms, 52(1).
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  Anecdotal industry reports of Taura syndrome (TS) epizootics in a Taura syndrome virus (TSV) tolerant strain of Penaeus stylirostris and collected evidence of field TS epizootics in P. stylirostris suggested that a distinct new TSV strain might have emerged since 1994. The Ecuadorian 1992 TSV genome published in GenBank is virtually identical to the Hawaiian 1994 TSV isolate (HI94TSV) used as reference throughout this investigation. Three other geographic and year isolates of TSV from naturally occurring TS epizootics of cultured penaeid shrimp were obtained from Mexico (SIN98TSV and MX99TSV from P. vannamei and SON2KTSV from P. stylirostris). Selected TSV diagnostic methods set forth by the Office International des Epizooties were utilized as the basis for isolate analysis. By Southern blot, TSV probes P15 and Q1 reacted specifically with all the diagnostic reverse transcription polymerase chain reaction (RT-PCR) fragments. Additionally, labeled RT-PCR amplicons from the TSV isolates amplified by routine diagnostic RT-PCR primers gave positive in situ hybridizations with TSV, indicating that all 4 isolates shared homology. By Western blot, immuno-dot blot, and immunohistochemistry, all TSV-purified isolates reacted with TSV polyclonal antibody (PAb). However, with TSV monoclonal antibody (MAb) 1A1 all isolates, except SIN98TSV, reacted, indicating that the difference in isolate SIN98TSV is within VP1, the target for MAb 1A1. The amino acid (AA) sequence of SIN98TSV VP1, MX99TSV VP1 and SON2KTSV VP1 has a 98% homology with the reference HI94TSV VP1. A span of 12 AAs are identified in SIN98TSV VP1 containing significant AA substitutions which may account for a conformational change of the antigenic epitope sufficient to prevent MAb 1A1 from binding. The implications of these results with respect to the antibody-based diagnosis of TSV are discussed.
• Lightner, D., Tang, K. F., & Lightner, D. V. (2002). Low sequence variation among isolates of infectious hypodermal and hematopoietic necrosis virus (IHHNV) originating from Hawaii and the Americas. Diseases of aquatic organisms, 49(2).
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  A 2.9 kb fragment of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) genome, which contains the coding sequence of putative non-structural and capsid proteins, was amplified and sequenced from each of 14 IHHNV isolates collected from cultured penaeid shrimp stocks in Hawaii and various sites in the Americas between 1982 and 1997. The sequence comparison indicates that the IHHNV genome is very stable, with 99.6 to 100% similarity among these 14 isolates. Only nucleotide substitutions were found. The percentage of substitution was higher in the putative capsid proteins region (1.3%) than in the putative non-structural proteins region (0.6%). Out of 25 substitutions found, 14 resulted in amino acid changes. There is no apparent association between clinical outcomes and particular amino acid substitutions. Based on genetic distances, the isolates were clustered into 3 groups that generally correspond with their geographic origins.
• Mari, J., Poulos, B. T., Lightner, D. V., & Bonami, J. (2002). Shrimp Taura syndrome virus: Genomic characterization and similarity with members of the genus Cricket paralysis-like viruses. Journal of General Virology, 83(4), 915-926.
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  PMID: 11907342;Abstract: The single-stranded genomic RNA of Taura syndrome virus (TSV) is 10205 nucleotides in length, excluding the 3′ poly(A) tail, and contains two large open reading frames (ORFs) that are separated by an intergenic region of 207 nucleotides. The ORFs are flanked by a 377 nucleotide 5′ untranslated region (UTR) and a 226 nucleotide 3′ UTR followed by a poly(A) tail. The predicted amino acid sequence of ORF1 revealed sequence motifs characteristic of a helicase, a protease and an RNA-dependent RNA polymerase, similar to the non-structural proteins of several plant and animal RNA viruses. In addition, a short amino acid sequence located in the N-terminal region of ORF1 presented a significant similarity with a baculovirus IAP repeat (BIR) domain of inhibitor of apoptosis proteins from double-stranded DNA viruses and from animals. The presence of this BIR-like sequence is the first reported in a single-stranded RNA virus, but its function is unknown. The N-terminal amino acid sequence of three TSV capsid proteins (55, 40 and 24 kDa) were mapped in ORF2, which is not in the same reading frame as ORF1 and possesses and AUG codon upstream of the structural genes. However, the intergenic region shows nucleotide sequence similarity with those of the genus Cricket paralysis-like viruses, suggesting a similar non-AUG-mediated translation mechanism. The structure of the TSV genome [5′ UTR-non-structural proteins-intergenic UTR-structural proteins-3′ UTR-poly(A) tail] is similar to those of small insect-infecting RNA viruses, which were recently regrouped into a new virus genus, Cricket paralysis-like viruses.
• White, B. L., Schofield, P. J., Poulos, B. T., & Lightner, D. V. (2002). A laboratory challenge method for estimating Taura syndrome virus resistance in selected lines of Pacific white shrimp Litopenaeus vannamei. Journal of the World Aquaculture Society, 33(3), 341-348.
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  Abstract: Among the strategies being developed to improved survival and harvest yields in the farming of Pacific white shrimp Litopenaeus vannamei is breeding domesticated family lines and the selection for further development of those lines that demonstrate resistance to Taura syndrome virus (TSV) challenge in the laboratory. A standardized laboratory challenge method for measuring TSV resistance by per os exposure to the virus, relative to a reference stock of L. vannamei, was developed and used to screen a total of 176 family lines provided by five different companies over a period of several months. All challenged shrimp were exposed to TSV per os by feeding minced TSV-positive shrimp carcasses at ∼ 10% of the shrimp biomass once per day for three consecutive days. Studies were carried out for a minimum of 14 d from the first day (day 0) of exposure to TSV. The survival rates obtained following TSV challenge of the selected L. vannamei families ranged from 0% to 100%, with a mean of 31%. The reference line of L. vannamei ("Kona line") gave survival rates of 0% to 37% with a mean of 13%. The results of the present study demonstrate that the use of a relatively simple laboratory challenge procedure provides a mechanism to evaluate and compare resistance to TSV among selected L. vannamei families and to predict the performance of selected stocks in farm environments where TSV is enzootic.
• Håstein, T., Hill, B. J., Berthe, F., & Lightner, D. V. (2001). Traceability of aquatic animals. OIE Revue Scientifique et Technique, 20(2), 564-583.
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  PMID: 11548527;Abstract: Effective methods of traceability are urgently required for use in research as well as in different types of aquaculture operations and to control trade in aquatic animals and products. In regard to the marking of fish, many different tagging methods have been described and the method to be used depends on the purpose and need for tagging. In contrast, for molluscs and crustaceans, only a few methods of marking such animals have been described, due to the practical difficulties. The authors first describe the different methods for tracing fish and fishery products, by means of external tags, such as Floy tags, Carlin tags and passive integrated transponder tags; chemical marking using inorganic substances such as silver nitrate or potassium nitrate, pigments, oxytetracycline, etc.; and several different types of electronic devices in which basic information such as the strain of fish, farm of origin or weight can be stored. Genetic traceability using deoxyribonucleic acid profiling is developing quite rapidly for cultured brood stocks and wild populations. This technique may be used with very high degrees of confidence to assign to or exclude animals or products from their claimed origin, paternity or strain, and may be used as evidence in court proceedings. The second section of this paper describes the traceability of live molluscs for restocking and for human consumption. In these applications, genetic markers have been demonstrated to be suitable. Mechanical tagging on a small scale for research purposes has also been used. Otherwise, the only means of tracing live molluscs are the movement documents and the labelling on boxes that certifies the origin of the commodity. The third section describes the methods available for tracing live and dead crustaceans. A large variety of physical tagging methods for decapod crustaceans is described, such as the injection of biological stains (fast green, Niagara sky blue, trypan red and blue) and external tags such as coloured streamer tags, wire tags and a variety of anchor tags. Furthermore, a number of different internal coding methods, such as the coded micro-wire tags and injected elastomer tags are discussed in detail. As is the case for fish, genetic molecular techniques are also applied in population studies of crustaceans; some of the molecular genetic methods are described. Prawns for human consumption are most frequently packed whole or as tails after the necessary sorting, washing and freezing and the only way of performing a traceback is through documents relating to movement, invoices, health certificates and labelling of the boxes. The minimum requirements for labelling would be the content of the packages, i.e. species, quantity, identification of the manufacturer (name and address), packing place, importer/exporter or vendor of the product, in addition to the loading bill number.
• 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. Journal of the World Aquaculture Society, 32(3), 330-334.
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  Abstract: In March 2000, 104 wild caught Litopenaeus vannamei broodstock, captured off the Pacific coast of Panama, were screened for the following penaeid viruses: infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV). The purpose of this study was to determine the prevalence of IHHNV and WSSV in wild shrimp in this area of the Western Hemisphere and to acquire specific pathogen free (SPF) L. vannamei for inclusion into the Oceanic Institute's genetic breeding program. The prevalence of the viruses was determined using the dot blot hybridization format, which is a commercially available molecular method for detecting these viruses. Dot blot hybridization assays can be used as an initial screening method to detect moderately to highly infected shrimp. The results from the dot blot assays indicated the prevalence of IHHNV in 28% and WSSV in 2% of the 104 hemolymph samples tested. Results from this study were used to establish the initial candidate SPF status of the animals that were assessed and to determine the prevalence of two serious pathogens of penaeid shrimp captured from the wild of the Pacific Ocean in the Central American region off the coast of Panama.
• Pantoja, C. R., & Lightner, D. V. (2001). Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level. Diseases of Aquatic Organisms, 44(2), 87-96.
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  PMID: 11324820;Abstract: A post-embedding in situ hybridization procedure was developed to detect hepatopancreatic parvovirus (HPV) of penaeid shrimp at the ultrastructural level. The procedure was optimized using sections of resin-embedded hepatopancreas from HPV-infected juvenile Penaeus monodon and postlarval P. chinensis. The hepatopancreata were fixed using various fixatives, dehydrated, and embedded in the hydrophilic resin Unicryl™. A 592 bp HPV-specific DNA probe, labeled with DIG-11-dUTP, was tested both on semi-thin and ultra-thin sections and examined by light and electron microscopy, respectively. Hybridized probe was detected by means of an anti-DIG antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal intensities were similar with all fixatives tested, but ultrastructure was best preserved with either 2 or 6 % glutaraldehyde. Post-fixation with 1% osmium tetroxide improved ultrastructure but markedly decreased hybridization signal and induced non-specific deposition of gold and silver. Under optimized conditions, this technique was used to successfully follow the development of HPV from absorption and transport through the cytoplansm to nuclear penetration, replication and release by cytolysis. The probe signal was consistently observed among necrotic cell debris within the lumen of hepatopancreatic tubules, within the microvillous border of tubule epithelial cells, within the cytoplasm, and within diagnostic HPV intranuclear inclusion bodies. The nucleolus and karyoplasm of patently infected cells (i.e., showing HPV intranuclear inclusion bodies) were almost devoid of signal. Electron-lucent structures, known as intranuclear bodies, commonly found within the virogenic stroma, showed only weak labeling. This is the first use of in situ hybridization to detect HPV nucleic acids with the electron microscope. The technique should be useful for studying the pathogenesis of HPV.
• Phromjai, J., Sukhumsirichart, W., Pantoja, C., Lightner, D. V., & Flegel, T. W. (2001). Different reactions obtained using the same DNA detection reagents for Thai and Korean hepatopancreatic parvovirus of penaeid shrimp. Diseases of Aquatic Organisms, 46(2), 153-158.
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  PMID: 11678231;Abstract: Hepatopancreatic 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.
• Poulos, B. T., Pantoja, C. R., Bradley-Dunlop, D., Aguilar, J., & Lightner, D. V. (2001). Development and application of monoclonal antibodies for the detection of white spot syndrome virus of penaeid shrimp. Diseases of Aquatic Organisms, 47(1), 13-23.
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  PMID: 11797911;Abstract: Monoclonal antibodies (MAbs) were produced against white spot syndrome virus (WSSV) of penaeid shrimp. The virus isolate used for immunization was obtained from China in 1994 and was passaged in Penaeus vannamei. The 4 hybridomas selected for characterization all produced MAbs that reacted with the 28 kD structural protein by Western blot analysis. The MAbs tested in dot-immunoblot assays were capable of detecting the virus in hemolymph samples collected from moribund shrimp during an experimentally induced WSSV infection. Two of the MAbs were chosen for development of serological detection methods for WSSV. The 2 MAbs detected WSSV infections in fresh tissue impression smears using a fluorescent antibody for final detection. A rapid immunohistochemical method using the MAbs on Davidson's fixed tissue sections identified WSSV-infected cells and tissues in a pattern similar to that seen with digoxigenin-labeled WSSV-specific gene probes. A whole mount assay of pieces of fixed tissue without paraffin embedding and sectioning was also successfully used for detecting the virus. None of the MAbs reacted with hemolymph from specific pathogen-free shrimp or from shrimp infected with infectious hypodermal and hematopoietic necrosis virus, yellow head virus or Taura syndrome virus. In Western blot analysis, the 2 MAbs did not detect any serological differences among WSSV isolates from China, Thailand, India, Texas, South Carolina or Panama. Additionally, the MAbs did not detect a serological difference between WSSV isolated from penaeid shrimp and WSSV isolated from freshwater crayfish.
• Tang, K. F., & Lightner, D. V. (2001). Detection and quantification of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp by real-time PCR. Diseases of Aquatic Organisms, 44(2), 79-85.
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  PMID: 11324819;Abstract: A real-time PCR method using a fluorogenic 5′ nuclease assay and a PE Applied Biosystems GeneAmp 5700 sequence detector was developed to detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. A pair of PCR primers to amplify an 81 bp DNA fragment and a fluorogenic probe (TaqMan probe) were selected from ORF1 (open reading frame 1) of the IHHNV genome. The primers and TaqMan probe used in this assay were shown to be specific for IHHNV and did not react with either hepatopancreatic parvovirus (HPV), white-spot syndrome virus (WSSV), or shrimp DNA. A plasmid, pIHHNV-P4, containing the target IHHNV sequence was constructed and used as a positive control. The concentration of pIHHNV-P4 was determined through spectrophotometric analysis and the plasmid was used for quantitative studies. This real-time PCR assay had a detection limit of 10 copies and a log-linear range up to 5 × 107 copies of IHHNV DNA. The assay was then used to quantify IHHNV in infected shrimp collected from 5 locations: Hawaii, Panama, Mexico, Guam, and the Philippines. The quantitative analysis showed that wild-caught, large juvenile Penaeus stylirostris collected from the Gulf of California (Mexico) in 1996 were naturally infected with IHHNV and contained up to 109 copies of IHHNV μg-1 of DNA. Similar quantities of IHHNV were detected in hatchery-raised, small juvenile P. stylirostris collected from Guam in 1995 and in farm-raised, post-larval P. monodon from the Philippines in 1996. Laboratory-infected P. stylirostris contained approximately 108 copies of IHHNV 31 d after being fed with IHHNV-infected shrimp tissue. In contrast, individuals of Super Shrimp®, a line of P. stylirostris selected for IHHNV resistance, showed no signs of infection 32 d after ingesting IHHNV-infected shrimp tissue. Laboratory-infected P. vannamei also contained approximately 108 copies of IHHNV 30 d after being fed infected shrimp tissue. A time-course study of IHHNV replication in juvenile P. vannamei showed that the doubling time in the exponential growth phase was approximately 22 h.
• Aubert, H., & Lightner, D. V. (2000). Identification of genetic populations of the pacific blue shrimp penaeus stylirostris of the gulf of California, Mexico. Marine Biology, 137(5-6), 875-885.
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  Abstract: Species are often composed of discrete breeding units (i.e. populations or stocks) which, while not reproductively isolated from other such groups, may have limited opportunities to exchange genetic material because of geographic distance, barriers to migration, or spawning asynchrony. Low levels of gene flow between stocks may result, over time, in their genetic divergence, and species that are subdivided into morphologic or genetically distinct stocks are said to be structured. The aim of our investigation was to test whether or not Penaeus stylirostris from the Gulf of California (Mexico) was structured into genetically distinct populations. Shrimp samples were collected in 1996 from six regions of the Gulf where specimens with distinct morphologic characteristics had previously been identified. Statistical analysis of 324 RAPD loci (randomly amplified polymorphic DNA), resolved through polyacrylamide gels and scored for each of 78 specimens, permitted the quantification and comparison of between-stock genetic differences. The finding that genetically discrete stocks of P. stylirostris can be found in a small portion of the geographic distribution range of the species, disagrees with the long-held perception that this resource is panmictic in nature. This new evidence is not only of interest for selective breeding programs in the shrimp aquaculture industry, but is also relevant to the management of the Mexican shrimp fishery which, at present, is perceived and managed as a single stock.
• Durand, S. V., Tang, K. F., & Lightner, D. V. (2000). Frozen commodity shrimp: Potential avenue for introduction of white spot syndrome virus and yellow head virus. Journal of Aquatic Animal Health, 12(2), 128-135.
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  Abstract: Since 1992, white spot syndrome virus (WSSV) and yellow head virus (YHV) have caused mortalities in cultured shrimp throughout Asia. By 1995, WSSV was detected in Texas and South Carolina, and the virus has also been recently reported in Central and South America (Nicaragua, Honduras, Guatemala, Panama, Colombia, Peru, and Ecuador). The importation of live infected shrimp is the principal mechanism by which exotic viruses may be introduced to new geographic regions. However, another probable mechanism is via the importation of infected commodity shrimp from regions where the pathogens are enzootic. Ten different lots of imported frozen tails of Penaeus monodon were screened for WSSV and YHV by polymerase chain reaction (PCR) and reverse transcriptase (RT) PCR analysis. In 8 of 10 samples tested, WSSV was detected, and YHV was found in 3 out of the 10 samples. Six of the 10 sample sets of frozen shrimp gave strong positive tests for WSSV or YHV by PCR, and these were selected for bioassay with specific pathogen-free P. vannamei, which were used as the indicator shrimp for infectious virus. The indicator P. vannamei were exposed to WSSV- or YHV-positive tissues either per os or by injection. Infectious WSSV resulted in 100% mortality in the indicator P. vannamei in four of nine bioassays. One of the three YHV bioassays produced mortalities in the indicator shrimp, and the other two in vitro assays demonstrated the presence of the virus in the tissues of the indicator shrimp. Infections were confirmed by histology, PCR or RT-PCR, or by in situ hybridization. The results of the study indicated that WSSV and YHV were present in the different lots of imported frozen P. monodon that were tested, and that both viruses were infectious to P. vannamei either by injection or by per os exposure.
• Nunan, L. M., 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. Marine Biotechnology, 2(4), 319-328.
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  Abstract: A rapid and reliable polymerase chain reaction (PCR) method was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in peneid shrimp. The oligonucleotide primers amplify a 1681-bp fragment of IHHNV, which encompasses the coding sequence for one of the viral coat proteins. The PCR method detects IHHNV in hemolymph and homogenized tissue obtained from the cephalothorax or pleopods of infected shrimp. The technique was also successfully applied to tissue samples preserved in 70% ethanol. The correct size fragment was amplified using IHHNV obtained from six different geographic regions in three different species of penaeid shrimp. No DNA extraction method was necessary for this technique. The use of hemolymph or pleopods provides a nondestructive screening method by which to test juveniles and adult broodstock for the presence of IHHNV.
• Pantoja, C. R., & Lightner, D. V. (2000). A non-destructive method based on the polymerase chain reaction for detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp. Diseases of Aquatic Organisms, 39(3), 177-182.
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  PMID: 10768285;Abstract: Current methods to detect hepatopancreatic parvovirus (HPV) infection of penaeid shrimp depend on invasive techniques that require dissecting the organs infected by this virus. However, sacrificing valuable stocks in order to determine their HPV status can be a drawback in the case of breeding programs. A method was developed for HPV detection by applying a polymerase chain reaction (PCR) assay to fecal samples collected from live HPV-infected shrimp Penaeus chinensis. A pair of PCR primers, 1120F/1120R, which amplify a 592 base pair (bp) region from the virus genome, was designed from previously known HPV sequence information (HPV clone HPV8). PCR amplification with these primers generated a product of the expected size directly from the crude feces of HPV-infected shrimp but not from the feces of specific pathogen-free (SPF) shrimp. The HPV origin of the amplified product was validated by means of an in situ hybridization assay where the product of the amplification, labeled with digoxigenin (DIG)-11-dUTP, showed an intense reaction within hepatopancreatic cells displaying characteristic HPV lesions on HPV-infected shrimp. No reaction to this probe was observed when reacted in situ with sections of the hepatopancreas of SPF specimens or to sections of shrimp infected by the infectious hypodermal and hematopoietic necrosis virus (IHHNV), another parvovirus of penaeid shrimp. These primers were tested for specificity against homologous and nonhomologous viruses and no product was amplified. A fragment of the expected size was obtained only when purified HPV or purified HPV8 plasmid was used as template DNA. Under optimized conditions, these primers detected as little as 1 fg of purified HPV8 plasmid DNA, equivalent to approximately 300 HPV particles. Analysis of fecal samples by PCR may prove useful for non-lethal screening of valuable shrimp of unknown HPV status. This same strategy also might be used for detection of other enteric viruses that infect penaeid shrimp.
• Tang, K. F., & Lightner, D. V. (2000). Quantification of white spot syndrome virus DNA through a competitive polymerase chain reaction. Aquaculture, 189(1-2), 11-21.
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  Abstract: A competitive polymerase chain reaction (PCR) method for quantification of white spot syndrome virus (WSSV) genome was developed. A pair of WSSV primers, designated WSSV341F/R, was selected to amplify a 341-bp DNA fragment from the WSSV genome. For a competitive internal standard, we constructed and cloned a 289-bp DNA fragment, the result of a 52-bp deletion from the 341-bp amplicon. In a competitive PCR reaction, we co-amplified the target WSSV DNA with known concentrations of the internal standard using WSSV341F/R primers. The amplicons from WSSV DNA and from internal standard DNA differed in size and could be distinguished after gel electrophoresis. The concentration of WSSV genomes was determined from its relation to the concentration of the internal standard. The log-log plot of the ratio of the amplicons (internal standard: WSSV) on the internal standard concentration was linear. Using this competitive PCR procedure, we quantified WSSV DNA in the samples of hemolymph and tissues of the cephalothorax of individual WSSV-infected shrimp. The number of WSSV genomes in both hemolymph and tissues corresponded to the severity of infection determined by histological evaluation. In addition, the changes in number of WSSV genomes in the hemolymph during the course of the infection were determined. (C) 2000 Elsevier Science B.V.
• Tang, K. F., 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(3-4), 203-210.
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  Abstract: A susceptibility study of postlarvae (PL) and juvenile Super Shrimp®, a selected line of Penaeus stylirostris, was conducted to compare their resistance to infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection to that of a specific pathogen free (SPF) population of P. vannamei. Super Shrimp® PLs were fed with IHHNV-infected shrimp tissue for 2 days and then maintained on a pelletized ration for an additional 28 days. PLs were sampled at days 0, 1, 2, 3, 4, 6, 10, 15, 20, 25 and 30. There was no apparent mortality during the experimental period. Tissue DNA extracted from the PLs was analyzed for the presence of IHHNV by PCR. Low levels of IHHNV were detected only in DNA extracts from samples at days 1, 2, and 3. No IHHNV DNA was detected from days 4 to 30. The days that the PLs were weakly IHHNV-PCR positive were during the period that they were being fed with IHHNV-tissue, and thus, the IHHNV DNA signal was suspected to be from the infected tissue used as a feed. Through both histology and in situ hybridization, we confirmed that tissues of Super Shrimp® PLs were not infected with IHHNV. PCR results of another IHHNV challenge study with juveniles of Super Shrimp® were similar to those with PLs. These results indicate that IHHNV did not replicate in the PL and juvenile Super Shrimp®. In contrast, P. vannamei juveniles, which were used as a positive control, showed a more intense IHHNV infection, as determined by PCR detection, beginning at 6 days postchallenge and increasing throughout the remainder of the study. In addition, the IHHNV-infected P. vannamei at 30 days postchallenge showed histological changes characteristic of IHHNV infection and had a positive reaction for IHHNV with in situ hybridization. Our studies show that Super Shrimp® are resistant to IHHNV infection. This is the first unequivocal demonstration of resistance to viral infection in shrimp. (C) 2000 Elsevier Science B.V.
• Wang, Q., Nunan, L. M., & Lightner, D. V. (2000). Identification of genomic variations among geographic isolates of white spot syndrome virus using restriction analysis and Southern blot hybridization. Diseases of Aquatic Organisms, 43(3), 175-181.
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  PMID: 11206732;Abstract: White spot syndrome virus (WSSV) is widely distributed in most of the Asian countries where penaeid shrimp are cultured, as well as in some regions of the USA, Six geographic isolates of WSSV - 1 each from penaeid shrimp from China, India, Thailand, and the US states of Texas and South Carolina, and 1 isolated from crayfish at the National Zoological Park in Washington, DC - were compared by combining the methods of restriction analysis and Southern blot hybridization. DNA was extracted from purified viruses and then digested with selected endonucleases: AccI, BgIII, ClaI, BamHI, EcoRI, HindII, HaeI, SacI and XhoI. The blots were detected with digoxigenin-11-dUTP-labeled WSSV genomic probes: LN4, C42 and A6. No distinctive differences among the 5 WSSV isolates from penaeid shrimp were detected; however, differences in the WSSV isolate from crayfish were observed. A 2.8 kb DNA fragment originating from the crayfish isolate and encompassing the LN4 region was subcloned into pBluescript and sequenced for comparison with the LN4 fragment from the Thailand WSSV isolate. The results indicate that some genomic components of WSSV from different geographic regions share a high degree of homology. This method can be used to distinguish between the WSSV isolate from crayfish and the WSSV isolates from penaeid shrimp.
• Wang, Q., Poulos, B. T., & Lightner, D. V. (2000). Protein analysis of geographic isolates of shrimp white spot syndrome virus. Archives of Virology, 145(2), 263-274.
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  PMID: 10752552;Abstract: Six geographic isolates of the white spot syndrome virus (WSSV) of penaeid shrimp, from China, India, Thailand, South Carolina, Texas, as well as from crayfish kept at the US National Zoo in Washington D. C, were compared by electron microscopy and sodium sulfate polyacrylamine gel electrophoresis (SDS-PAGE). Amino acid compositions of four of the major structural polypeptides of the South Carolina WSSV were analyzed, and three of the four polypeptides were partially sequenced from their NH2 termini. The morphologies of purified virions of the six geographic isolates of WSSV were indistinguishable by transmission electron microscopy. By SDS-PAGE, the protein profiles of the six isolates were very similar, but not identical. They all contained three major polypeptides with sizes of approximately 25, 23 and 19 kDa. A fourth major polypeptide at the 14.5 kDa position was observed in four of the geographic isolates. The WSSV isolated from crayfish presented a slightly different structural protein profile, particularly with regard to the protein in the 19 kDa range that appeared larger in size than those of the other isolates. The NH2 terminal amino acids of the 25, 23 and 14.5 kDa polypeptides of the South Carolina WSSV were sequenced as MDLSFTLSVVTA, MEFGNLTNLDVA, and VARGGKTKGRRG, respectively. No significant homologous sequence was found in the GenBank. These protein sequences have been submitted to the SWISS-PROT Protein Data Bank and assigned the accession numbers P82004, P82005 and P82006.
• Arcier, J., Herman, F., Lightner, D. V., Redman, R. M., Mari, J., & Bonami, J. (1999). A viral disease associated with mortalities in hatchery-reared postlarvae of the giant freshwater prawn Macrobrachium rosenbergii. Diseases of Aquatic Organisms, 38(3), 177-181.
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  Abstract: During mortality outbreaks in hatchery-reared Macrobrachium rosenbergii postlarvae (PL) in Guadeloupe Island (French West Indies) during 1997, an associated viral disease was discovered and the agent was subsequently isolated. The clinical signs presented by severely affected PL consisted essentially of an opaque whitish appearance of the abdomen. Histopathological changes in affected PL were characterized predominantly by pale to darkly basophilic, often reticulated, cytoplasmic inclusions in the connective tissue cells of most organs and tissues. The isolated virus was approximately 30 nm in diameter as observed with an electron microscope by negative staining. By its location, structure and size it could be related to different families of the small RNA cytoplasmic viruses such as the Picornaviridae or the Nodaviridae. Its characterization is in progress.
• Hasson, K. W., Lightner, D. V., Mari, J., Bonami, J., Poulos, B. T., Mohney, L. L., Redman, R. M., & Brock, J. A. (1999). The geographic distribution of Taura Syndrome Virus (TSV) in the Americas: Determination by histopathology and in situ hybridization using TSV-specific cDNA probes. Aquaculture, 171(1-2), 13-26.
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  Abstract: Representative archived Litopenaeus vannamei samples (117 total), originating from 13 different countries and collected between 1992 to 1996, were analyzed by in situ hybridization to verify the presence of Taura Syndrome Virus (TSV) within pathodiagnostic acute phase TS histological lesions. The in situ assay results showed that TSV was present in one or more representative samples analyzed from each country (76 of 117 samples or 65%), thus, confirming the original histological diagnosis of TSV infection. The false negative in situ hybridization results, obtained for 35% of the samples assayed (41 in total), were attributed to over-fixation with Davidson's AFA (acetic acid, formaldehyde, alcohol) solution and consequent acid hydrolysis of TSV genomic RNA within pathodiagnostic TSV lesions. The collective findings of this disease survey assisted in documenting the spread and current distribution of TSV over a 5-year period and definitively established the presence of TSV within TS diseased shrimp originating from Ecuador when and where the disease was first recognized in 1992. These findings further strengthen the existing evidence that TS has a viral, not a toxic, etiology and indicate that either a single TSV strain, or very similar strains of the same virus, are responsible for the TSV panzootic that has been expanding in the Americas since 1992.
• Hasson, K. W., Lightner, D. V., Mohney, L. L., Redman, R. M., & White, B. M. (1999). Role of lymphoid organ spheroids in chronic Taura syndrome virus (TSV) infections in Penaeus vannamei. Diseases of Aquatic Organisms, 38(2), 93-105.
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  Abstract: Lesion development was documented in Penaeus vannamei juveniles with experimentally induced, chronic phase Taura syndrome virus (TSV) infections, by both routine histology and in situ hybridization, during a 48 wk time course study. Histologically, the defining characteristics of TSV chronicity in P. vannamei include the absence of acute phase histological lesions, a low prevalence of ectopic spheroid development, and rapid successive lymphoid organ spheroid (LOS) formation and morphogenesis. Three distinct LOS morphotypes (Types A, B, and C) were identified by light microscopy. The earliest detectable LOS, Type A, appeared to evolve from activated LO tubule phagocytes that had sequestered TSV. The succeeding LOS, Type B, contained necrotic cells that were consistently TSV-positive by in situ hybridization for up to 32 wk following an acute phase infection. These persistent, long-term infections suggested that TSV replication occurred within Type B LOS, and this satisfied the definition of a chronic infection. The terminal Type C LOS were consistently found to be TSV-negative and characterized by cells with condensed basophilic nuclei, a reduction in overall cell size, and progressive atrophy leading to degradation without an inflammatory response. These cellular changes are characteristic of apoptotic cells, suggesting that TSV-infected LOS cells self-destruct; resulting in TSV elimination. TSV infections appear to have 3 potential outcomes: (1) the virus may continue to replicate within LOS cells unchecked, (2) it may be eliminated by LOS cells, or (3) viral replication and elimination may occur concurrently, resulting in persistent infections. Ectopic spheroids were TSV-induced and observed in sites normally occupied by tegmental glands within appendages, suggesting that they developed from either hemolymph-borne phagocytes or fixed phagocytes associated with the gland. We suggest that these cellular masses arise from migrating and/or resident phagocytes transformed in response to chronic viral infections or non-self substances too small for hemocyte encapsulation. The possibility that spheroid development represents an unexplored and significant branch of the cell-mediated immune response of penaeid shrimp is discussed.
• Hasson, K. W., Lightner, D. V., Mohney, L. L., Redman, R. M., Poulos, B. T., & White, B. M. (1999). Taura syndrome virus (TSV) lesion development and the disease cycle in the Pacific white shrimp Penaeus vannamei. Diseases of Aquatic Organisms, 36(2), 81-93.
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  Abstract: The Taura syndrome virus (TSV) disease cycle was redefined through histological and gene probe analysis of experimentally infected specific pathogen-free (SPF) Penaeus vannamei sampled at timed intervals. The cycle consists of 3 overlapping, but clinically and histologically distinct, phases: a ~7 d peracute to acute phase, a ~5 d transition phase (previously termed the chronic or recovery phase), and a definitive chronic phase. The acute phase is characterized by the rapid development of severe, multifocal to diffuse cuticular epithelial necrosis and high mortalities. Using in situ hybridization analysis, infected pre-lytic cuticular epithelial cells display very strong TSV-positive probe signals, and a total of 3 stages of acute phase necrosis are described. Surviving P. vannamei then enter the transition phase, which is distinguished histologically by multifocal melanized lesions within regions of the cuticular epithelium (resolving acute phase lesions), focal active acute phase lesions, and the onset of lymphoid organ (LO) spheroid development. Gene probe analysis of transitionally infected shrimp reveals probe-positive foci of active acute phase lesions, a diffuse probe signal within the walls of morphologically normal LO tubules and/or focal probe signals within developing LO spheroids. Shrimp surviving this stage enter the chronic phase infection after ecdysis. The defining characteristics of the chronic phase include the cessation of mortalities, the resumption of normal behavioral patterns, the complete absence of visible melanized lesions and acute phase histological lesions of the cuticular epithelium, and marked LO hypertrophy directly resulting from the rapid development of numerous LO spheroids, some of which are TSV positive by in situ hybridization analysis.
• Lightner, D. V. (1999). The penaeid shrimp viruses TSV, IHHNV, WSSV, and YHV: Current status in the Americas, available diagnostic methods, and management strategies. Journal of Applied Aquaculture, 9(2), 27-.
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  Abstract: Viral diseases have emerged during this decade as serious economic impediments to successful shrimp farming. While nearly 20 distinct viruses or groups of viruses are known to infect penaeid shrimp, only four, WSSV, YHV, IHHNV, and TSV, pose a threat to the future of penaeid shrimp culture in the Western Hemisphere. TSV and IHHNV have caused serious disease epizootics throughout the Americas and Hawaii. IHHNV was described nearly 20 years ago when it was found to be responsible for cumulative losses of cultured Penaeus stylirostris that often exceed 90%. The threat of high mortalities posed by IHHNV has historically curtailed interest in the culture of this species in favor of the more IHHNV-resistant P. vannamei. While relatively resistant, IHHNV nonetheless infects P. vannamei and causes runt deformity syndrome (RDS) in which affected shrimp display reduced growth, highly variable sizes, lower production, and sometimes reduced survival. Taura Syndrome in P. vannamei is the virtual "mirror image" of IHHN disease in P. stylirostris. Following its recognition in 1992 as a distinct disease of P. vannamei in Ecuador, Taura Syndrome and its viral agent TSV spread rapidly throughout many of the shrimp farming regions of the Americas. Cumulative mortalities due to TSV in affected juvenile P. vannamei populations have ranged from 40 to 95%. Because P. stylirostris was found to be innately TSV-resistant, genetically selected IHHNV-resistant lines of this species disease are being developed and marketed in the Americas. Some shrimp farmers are using domesticated, selected stocks of P. vannamei that show improved resistance to TSV, while others have continued to use wild stocks that are also showing increased resistance, perhaps through intense natural selection. The viruses of the Yellow Head Disease (YHV) and the White Spot Syndrome (WSSV) were first recognized in 1991-92 in Asia, and by 1996 the two diseases had spread and had caused major pandemics throughout much of the shrimp farming regions of East Asia, Southeast Asia, Indonesia, and India. In late 1995, WSSV was found in North America for the first time in P. setiferus at a shrimp farm in south Texas, WSSV has since been detected in cultured and wild shrimp, crabs, and freshwater crayfish at multiple sites in the eastern and southeastern U.S., as well as being commonly found (along with YHV) in imported frozen commodity shrimp. Because Western Hemisphere penaeids are highly susceptible to WSSV and YHV, the introduction and establishment of either of the viruses poses a significant threat to the shrimp farming industry. The available detection methods for IHHNV, TSV, WSSV, and YHV include traditional methods that employ gross signs, clinical history, histopathology, and bioassay with susceptible shrimp hosts. Molecular and serological methods have also been developed, and specific gene probes, monoclonal antibodies, or PCR methods are readily available for each of the four viruses.
• Lo, C., Hsu, H., Tsai, M., Ho, C., Peng, S., Kou, G., & Lightner, D. V. (1999). Specific genomic DNA fragment analysis of different geographical clinical samples of shrimp white spot syndrome virus. Diseases of Aquatic Organisms, 35(3), 175-185.
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  Abstract: White spot syndrome (WSS) has been found in many species of shrimp and crabs, not just in Asia but globally. The causative agent is known as white spot syndrome virus (WSSV). In order to clarify the relatedness of WSSV from various geographic regions, we compared the viral DNA of a number of clinical samples of WSSV: (1) China96-116A from Penaeus chinensis, (2) India95-314 from Penaeus monodon, (3) grocery store95-204 and grocery store96-115 from P. monodon possibly originating from Thailand, (4) crayfish97-25 from Orconectes punctimanus collected from the U.S. National Zoo, (5) Thailand95-46 from experimentally infected Penaeus vannamei, (6) South Carolina97-64 from P. vannamei, and (7) Texas95-242 and Texas96-7 from P. vannamei. These specimens were first examined by dot hybridization analysis with nucleic acid probes derived from a WSSV Taiwan isolate. Although the intensity of the hybridization signals varied, and although some specimens of India95-314, crayfish97-25, Texas95-242 and Texas96-7 failed to give a detectable hybridization signal with certain probes, the broad consistency of dot hybridization data suggests that these WSSV clinical samples from different geographical locations are closely related. Following this analysis, all the specimens were examined using 10 virus-specific polymerase chain reactions (PCR). The amplification products were subsequently digested with Cfo I, Hae III, Hpa II and Rsa I restriction endonucleases to determine if there were any DNA fragment polymorphisms in the WSSV clinical samples. The results highlighted the genetic relatedness of all the WSSV clinical samples with the possible exception of a series of Texas viral samples which could be distinguished from the other geographic samples in some of the PCR-based tests.
• Morales-Covarrubias, M., Nunan, L. M., Lightner, D. V., Mota-Urbina, J., Garza-Aguirre, M., & Chávez-Sánchez, M. (1999). Prevalence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in wild adult blue shrimp penaeus stylirostris from the Northern Gulf of California, Mexico. Journal of Aquatic Animal Health, 11(3), 296-301.
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  Abstract: Conventional histopathology and molecular methods, including dot blot hybridization with a specific DNA probe and polymerase chain reaction (PCR), were used to assess the prevalence and degree of severity of infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in wild adult blue shrimp Penaeus stylirostris captured in the Northern Gulf of California. Through histopathological analysis, a presumptive diagnosis of IHHNV was initially determined by demonstrating the presence of Cowdry type A inclusion bodies. Prevalence rate ranged from 80% to 100% in females and was 60% in males. Using the dot blot technique, the results showed that the presence of IHHNV varied in females from 86% to 89% and in males from 56% to 57%. Results were further validated using PCR to assess the prevalence of IHHNV in eggs and sperm. The PCR results showed that IHHNV prevalence in unfertilized eggs was 100% and in sperm was 60%. We concluded that wild adults of P. stylirostris are infected with IHHNV but that they do not show clinical signs of disease. Our data suggest that in the 10th year of the IHHNV epizootic in the Gulf of California, a hostpathogen relationship might have reached a putative equilibrium.
• Pantoja, C. R., Lightner, D. V., & Holtschmit, K. H. (1999). Prevalence and geographic distribution of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in wild blue shrimp Penaeus stylirostris from the Gulf of California, Mexico. Journal of Aquatic Animal Health, 11(1), 23-34.
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  Abstract: A histopathological survey of wild blue shrimp Penaeus stylirostris from the Gulf of California, Sonora, Mexico, revealed the presence of infectious hypodermal and hematopoietic necrosis virus (IHHNV). The survey performed at 39 sampling stations during August-September 1990, showed the prevalence of IHHNV infection to be 46% in the upper Gulf zone and 26% in the central-lower Gulf zone. The presence of IHHN virus was confirmed with the IHHNV-specific gene probe BA401 by means of in situ hybridization. Specimens of the Pacific brown shrimp P. californiensis (also known as yellowleg shrimp) and Pacific white shrimp P. vannamei, incidentally captured during the survey, were also analyzed and found to be infected by IHHNV. The survey demonstrated that IHHNV had widespread distribution in a region where it had not been previously detected and, presumably, was not present before 1987. These findings suggest that IHHNV has become established in wild populations of P. stylirostris, and perhaps in P. californiensis and P. vannamei, in the Gulf of California.
• Poulos, B. T., Kibler, R., Bradley-Dunlop, D., Mohney, L. L., & Lightner, D. V. (1999). Production and use of antibodies for the detection of Taura syndrome virus in penaeid shrimp. Diseases of Aquatic Organisms, 37(2), 99-106.
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  Abstract: Monoclonal (MAb) and polyclonal (PAb) antibodies were produced against the penaeid shrimp virus, Taura syndrome virus (TSV), isolated from naturally infected Penaeus vannamei from farms in Ecuador, Hawaii and Texas. The PAbs produced in both chickens and mice were capable of detecting TSV in the hemolymph of shrimp during the acute phase of infection by an immunoblot assay. The MAbs were produced using BALB/cByJ mouse spleen cells fused with non-immunoglobulin-secreting SP2/0-Ag-14 mouse myeloma cells. Three MAbs of different immunoglobulin isotypes (IgGγ 1κ, IgGγ 2bκ and IgGγ 3κ) were compared in an immunoblot assay to determine their reactivity to hemolymph from TSV-infected shrimp during the acute and chronic phases of infection and their cross-reactivity, if any, with other shrimp viruses. Western blots of purified TSV were used to compare the specificities of the 3 MAbs for the structural proteins of the virus. The MAbs were used to monitor a laboratory-induced TSV infection in juvenile P. vannamei, and they were capable of detecting the virus in the hemolymph of both acute and chronic phase samples. The results with chronic phase hemolymph samples were variable and indicated the need to develop an immunoassay in which the virus in a sample is captured by one antibody and then detected with a second antibody. The availability of MAbs with different specificities for TSV viral proteins and the ability to produce PAbs in chickens will make it possible to develop a sensitive capture assay for rapid detection of the virus in field situations.
• Tang, K. F., & Lightner, D. V. (1999). A yellow head virus gene probe: Nucleotide sequence and application for in situ hybridization. Diseases of Aquatic Organisms, 35(3), 165-173.
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  PMID: 10228874;Abstract: A portion of the genome of yellow head virus (YHV) of penaeid shrimp was cloned and the cDNA fragment (1161 bp) was designated clone 3-27. The fragment was labeled with digoxigenin and hybridized in situ to tissue sections of YHV-infected Penaeus vannamei. Positively reacting tissues included those of the lymphoid organ, cuticular epithelium, and gills. In addition, connective tissue of hepatopancreas, heart, antennal gland, hematopoietic organ, nerve tract, midgut cecum and muscle reacted to the probe. The probe was highly specific since it hybridized only to tissues from YHV-infected shrimp. It did not react to those of uninfected shrimp or shrimp infected with WSSV (white spot syndrome virus), IHHNV (infectious hypodermal and hematopoietic necrosis virus), or TSV (Taura syndrome virus). The clone was sequenced, and primers were synthesized for rapid detection of YHV in hemolymph using RT-PCR (reverse transcription-polymerase chain reaction). The strand that constituted the viral sequence in the cDNA was also determined via RT-PCR and in situ hybridization with a single-stranded RNA (ssRNA) probe.
• Wang, Q., White, B. L., Redman, R. M., & Lightner, D. V. (1999). Per os challenge of Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles with six geographic isolates of white spot syndrome virus. Aquaculture, 170(3-4), 179-194.
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  Abstract: White spot syndrome virus (WSSV) is one of the most important pathogens of penaeid shrimp. It is widely distributed in most Asian countries where penaeid shrimp are cultured, as well as in the Gulf of Mexico and SE USA. The virulence of six geographic isolates of WSSV was compared using Litopenaeus vannamei postlarvae and Farfantepenaeus duorarum juveniles. The six geographic isolates of WSSV originated from China, India, Thailand, Texas, South Carolina, as well as from crayfish maintained at the USA National Zoo. For challenge studies, virus infected tissues were given per os to L. vannamei postlarvae and Fa. duorarum juveniles. Resultant WSSV infections were confirmed by histological examination. The cumulative mortality of L. vannamei postlarvae reached 100% after challenge with each of the six geographic isolates of WSSV. However, the Texas isolate caused mortalities more rapidly than did the other shrimp isolates; the crayfish WSSV isolate was the slowest. In marked contrast, cumulative mortalities of juvenile Fa. duorarum reached only 35-60%, and varied among the geographic isolates of WSSV. Interestingly, in Fa. duorarum, the Texas WSSV isolate was also the most virulent, while the crayfish WSSV was the least virulent. The findings suggest that slight differences in virulence exist among geographic isolates of WSSV, and that susceptibility may vary with species and lifestages of the host.
• Costa, R., Mermoud, I., Mari, J., Bonami, J. R., Hasson, K., & Lightner, D. V. (1998). Investigations of Penaeus stylirostris disease (Syndrome 93) in New Caledonia, exploring a viral hypothesis. Aquaculture, 164(1-4), 311-322.
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  Abstract: Semi-intensive farming of Penaeus stylirostris (Mexican strain) is a developing industry in New Caledonia. Since 1993 crops have experienced mortality episodes (named 'Syndrome 93') which are strongly affecting the industry. A search for bacteria in the hemolymph of moribund shrimps revealed bacterial septicemia. The Vibrio spp. initially isolated were variable and usually weakly pathogenic. Therefore, another research was investigated. Histological sections of moribund shrimps revealed not only classical vibriosis lesions, but also numerous pycnotic cells and intracytoplasmic basophilic bodies, which resemble those described in the case of 'yellow head disease' and 'Taura syndrome'. These observations suggested a possible participation of viral agents in 'Syndrome 93'. To explore this viral hypothesis, five cephalothorax from moribund shrimps caught in 1994 were ground, centrifuged and filtered on a 0.22 μm membrane. Filtered aliquots were kept at -76°C, then injected into groups of presumably healthy P. stylirostris (1 g), from New Caledonia and specific pathogen-free P. stylirostris and P. vannamei from Tucson, AZ. While 100% mortality was observed in groups injected with the filtrate 6 h to 5 days after injection, no significant mortality occurred in the control groups. Basophilic bodies similar to those described above were observed in experimentally infected moribund shrimps. This demonstrates the existence of ultrafiltrable entities in diseased shrimps which can experimentally reproduce the lesions, symptoms and mortalities characteristic of 'Symdrome 93'. Specific gene probe were used (dot blot) to detect Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and showed that 94 out of 95 moribund shrimps were positive but the detected levels were very low. Purification trials using centrifugation on sucrose and cesium chloride gradients failed to detect IHHNV which is probably present in small quantities. However, some fractions revealed an abundance of calibrated particles of about 100 mm of variable shape following examination of negative staining under transmission electron microscopy. Nucleic acid extraction trials and electrophoresis of these fractions on agarose gel revealed small quantities of a monocatenary DNA molecule of about 10 Kb, probably of viral origin. Results suggest the presence of the IHHNV, and probably of a second virus in shrimp crops in New Caledonia. The etiological role of each of those entities in 'Syndrome 93' is currently being studied.
• Durand, S., Lightner, D. V., & Bonami, J. R. (1998). Differentiation of BP-type baculovirus strains using in situ hybridization. Diseases of Aquatic Organisms, 32(3), 237-239.
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  PMID: 9676249;Abstract: BP-type viruses infect wild and famed shrimp species in the Americas and can cause high mortality in larval stages. Histologically, BP disease is characterized by the presence of occlusion bodies (OBs) in the nucleus of hepatopancreatic epithelial cells. By in situ hybridization using different molecular probes, we tested shrimp infected with BP-type viruses collected from several geographical areas. In one shrimp, a case of double infection by 2 different BP-type viruses was noted. This has not been previously reported. The molecular probes recognized only the PAcific strain of BP. This specificity of the probe was confirmed by in situ hybridization tests with some infected shrimp collected from the Atlantic and Pacific coasts. The probes reacted only with infected shrimp obtained from the Pacific coast. These results suggest the existence of at least 2 different BP-type viruses and show that specific probes can be used to differentiate between them.
• Lightner, D. V., & Redman, R. M. (1998). Shrimp diseases and current diagnostic methods. Aquaculture, 164(1-4), 201-220.
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  Abstract: In less than 30 yr, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of U.S. dollars in revenue, and augmentation of the world's food supply with a high value crop. Concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. Major epizootics have plagued the world's shrimp culture industries. The most important diseases of cultured penaeid shrimp have had viral or bacterial etiologies, but a few important diseases have fungal and protozoan agents as their cause. Diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. While tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. The need for rapid, sensitive diagnostic methods led to the application of modem biotechnology to penaeid shrimp disease. The industry now has modem diagnostic genomic probes with nonradioactive labels for viral pathogens like IHHNV, HPV, TSV, WSSV, MBV, and BP. Additional genomic probes for viruses, for bacterial pathogens like NHP and certain Vibrio spp., and Microsporidia have also been developed. Highly sensitive detection methods for some pathogens that employ DNA amplification methods based on the polymerase chain reaction (PCR) now exist, and more PCR methods are being developed for additional agents. These advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease.
• Lightner, D. V., & Redman, R. M. (1998). Strategies for the control of viral diseases of shrimp in the Americas. Fish Pathology, 33(4), 165-180.
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  Abstract: Viral diseases have severely impacted many of the penaeid shrimp farming industries of the world causing significant production and economic losses. Nearly 20 distinct viruses, or groups of viruses, are known to infect penaeid shrimp. Viruses belonging to the WSSV, MBV, BMN, HPV, IHHNV, and YHV groups have been important pathogens of cultured shrimp in Asia and the Indo-Pacific regions, while TSV, IHHNV, and BP have been the principal viruses of concern in the Americas. Numerous strategies have been attempted for the control of viral diseases in penaeid shrimp aquaculture. These strategies range from the use of improved culture practices (i.e. where sources of virus contamination are reduced or eliminated, sanitation practices are improved, stocking densities are reduced, etc.) to stocking "specific pathogen-free" (SPF) or "specific pathogen resistant" (SPR) species or stocks. In the Americas many strategies have been employed in efforts to reduce production losses due to the enzootic viruses IHHNV, BP, and TSV. Improved husbandry practices have been successfully employed for the control of BP, and for nearly a decade, this virus has seldomly been reported as an economic constraint to successful shrimp culture. Until recently, the popularity and use of the relatively IHHNV resistant species Penaeus vannamei, in preference to the culture of the more IHHNV susceptible P. stylirostris, was characteristic of the shrimp farming industries of the Americas. The popularity of P. vannamei began to decline when TSV emerged as a very serious pathogen of this species in 1992 and then spread to virtually all of the shrimp growing regions of the Americas during the ensuing four years. Because P. stylirostris was found to be innately TSV resistant, two domesticated, genetically selected SPR strains of this species, which are resistant to IHHN disease, are currently being developed and marketed in the Americas. In some regions, these SPR stocks of TSV and IHHNV resistant P. stylirostris are replacing P. vannamei stocks in culture. Other shrimp farming interests are using wild or domesticated stocks of P. vannamei that show improved resistance to TSV. While resistance to TSV was used as a selection criteria for the domesticated stocks of P. vannamei, natural selection for TSV resistance appears to be occurring in wild stocks where TSV has been enzootic for several years. The same selective process for IHHNV resistance seems to be occurring in some wild stocks of P. stylirostris.
• Lightner, D. V., Hasson, K. W., White, B. L., & Redman, R. M. (1998). Experimental infection of western hemisphere penaeid shrimp with Asian white spot syndrome virus and Asian yellow head virus. Journal of Aquatic Animal Health, 10(3), 271-281.
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  Abstract: Postlarval and juvenile stages of four species of western hemisphere penaeid shrimp (Penaeus aztecus, P. duorarum, P. setiferus, and P. vannamei) were experimentally challenged with white spot syndrome virus (WSSV) and yellow head virus (YHV) isolates originating from Asia. Challenge exposures were accomplished by feeding minced tissue from WSSV- or YHV-infected shrimp tissues. The WSSV challenge of postlarval shrimp resulted in severe infections in P. setiferus and P. vannamei and less severe infections in P. aztecus and P. duorarum. The WSSV challenge of juvenile shrimp (~1 g) resulted in severe infections and 100% cumulative mortality in P. setiferus and P. vannamei, moderate infections and 27% cumulative mortality in P. aztecus, and no signs of infection and 0% cumulative mortality in P. duorarum. The YHV challenge caused serious disease and mortality in juveniles of all four species, but postlarval shrimp appeared resistant to YHV because no virus-related signs of infection, mortality, or distinctive histopathology were detected. The findings of this preliminary study suggest that Asian WSSV and YHV are potentially serious pathogens for the species of western hemisphere penaeid shrimp that were tested.
• Mari, J., Bonami, J., & Lightner, D. V. (1998). Taura syndrome of penaeid shrimp: Cloning of viral genome fragments and development of specific gene probes. Diseases of Aquatic Organisms, 33(1), 11-17.
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  PMID: 9653455;Abstract: The ssRNA genome extracted from purified Taura Syndrome Virus (TSV) was transcribed into double-stranded, blunt-ended cDNA and was used to construct cDNA libraries either in pUC 18 or in pBluescript II KS-vectors. Twelve recombinant plasmids chosen after screening of the libraries were subjected to restriction enzyme digestions for determination of size inserts and restriction maps. Two of them, pP15 and pQ1, were selected for probe construction. The inserts, 1500 and 1300 base pairs (bp) respectively, were DIG-11dUTP-labelled and the corresponding probes were named P15 and Q1. On northern blots and dot blots, using different denaturation methods, the 2 probes hybridized specifically with extracted RNA-TSV genome, TSV and infected TS shrimp homogenates. No positive hybridization was obtained with other shrimp viruses tested [Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Hepatopancreatic Parvovirus (HPV)]. The specificity of the 2 probes was confirmed by in situ hybridization on histological sections of TS diseased shrimps.
• Mohney, L. L., Poulos, B. T., Brooker, J. H., Cage, G. D., & Lightner, D. V. (1998). Isolation and identification of mycobacterium peregrinum from the pacific white shrimp penaeus vannamei. Journal of Aquatic Animal Health, 10(1), 83-88.
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  Abstract: Mycobacterium peregrinum was isolated from multifocal, melanized nodular lesions in the carapace of the cultured marine shrimp Penaeus vannamei. This is the first identification of this species of Mycobacterium in shrimp. The lesions appeared to be the result of opportunistic infections in otherwise healthy animals. The presence of M. peregrinum in cultured shrimp has a direct negative impact on the marketability of shrimp because it causes obvious black lesions. The potential for accidental infections of shrimp farm or packing plant workers from handling infected shrimp is of concern since nodular skin lesions induced by the bacterium are difficult to treat.
• Nunan, L. M., Poulos, B. T., & Lightner, D. V. (1998). Reverse transcription polymerase chain reaction (RT-PCR) used for the detection of Taura Syndrome Virus (TSV) in experimentally infected shrimp. Diseases of Aquatic Organisms, 34(2), 87-91.
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  PMID: 9828404;Abstract: Taura Syndrome Virus (TSV) has adversely affected the shrimp culture industries of the Americas. First recognized in 1992, this vital agent has spread throughout the shrimp growing regions of South and Central America to become established in North America in the short span of 5 yr. Diagnostic methods for TSV include histopathology, bioassay using susceptible Penaeus vannamei as the indicator species and in situ hybridization with TSV specific complimentary DNA (cDNA) gene probes. An additional method for detecting TSV is through the use of reverse transcription polymerase chain reaction (RT-PCR). Two oligonucleotide primers were selected using the sequence information from a cloned cDNA segment of the TSV genome. The primers, designated 9195 and 9992, used in the RT-PCR procedure amplify a 231 base pair (bp) fragment of the cDNA. Using the RT-PCR technique, TSV has been detected in the hemolymph of P. stylirostris and P. vannamei with experimentally induced TSV infections.
• Nunan, L. M., Poulos, B. T., & Lightner, D. V. (1998). The detection of White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) in imported commodity shrimp. Aquaculture, 160(1-2), 19-30.
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  Abstract: Transmission of exotic pathogens occurs through a variety of means, including migration with humans and animals, rapid transit by land, sea or air or through the shipment of infected frozen food products. White Spot Syndrome Virus (WSSV) and Yellow Head Virus (YHV) have caused mass mortalities of cultured shrimp in Asia beginning in 1992. In 1995, these viruses appeared for the first time in the Western Hemisphere causing high mortalities in farm reared shrimp in Texas, USA. The purpose of this study was to determine if WSSV and YHV are present in frozen shrimp products imported into the United States from Asia. Infectivity assays, transmission electron microscopy (TEM), and polymerase chain reaction (PCR) showed these viruses were detectable and infectious in frozen shrimp imports. Frozen shrimp were used to infect indicator shrimp (Penaeus stylirostris) which resulted in mortalities. The cause of these mortalities was determined by histology and TEM to be by YHV. PCR confirmed the presence of WSSV in the frozen, purchased products. The results from this study indicate that exotic shrimp pathogens can be transmitted via imported frozen products.
• Bonami, J., Aubert, H., Mari, J., Poulos, B. T., & Lightner, D. V. (1997). The polyhedra of the occluded baculoviruses of marine decapod crustacea: A unique structure, crystal organization, and proposed model. Journal of Structural Biology, 120(2), 134-145.
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  Abstract: The baculoviruses of marine penaeid shrimp, PmSNPV and PvSNPV (MBV type and BP type, respectively), have distinctly different occlusion bodies (OBs) from those of the insect baculoviruses. In contrast to insect baculovirus, the penaeid baculovirus OB is unenveloped and formed by large subunits (SuOBs), as observed by electron microscopy after negative staining. The polyhedrin subunits measure 17 to 23 nm in diameter and appear icosahedral, resembling full and empty vital particles. Although these SuOBs look similar in morphometrics to shrimp parvoviruses, their density, polypeptide composition, and UV spectra are more characteristic of proteins than nucleoproteins. Common to the two shrimp baculovirus OBs that were investigated is the aggregation of three icosahedral SuOBs into a triplet. The observed difference in their crystalline structure is directly related to the way in which triplets attach to each other to form the OB. In the BP- type OB, the triplets form alternating parallel rows in all three dimensions. On the other hand, in the MBV-type OB, four triplets form a hollow sphere which we call a 'rosette,' the building blocks of the MBV-type OB. We assembled models for the penaeid baculovirus OB as an alternative to those hypothesized for insect baculovirus OBs.
• Bonami, J., Hasson, K. W., Mari, J., Poulos, B. T., & Lightner, D. V. (1997). Taura syndrome of marine penaeid shrimp: Characterization of the viral agent. Journal of General Virology, 78(2), 313-319.
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  PMID: 9018052;Abstract: The causative agent of Taura syndrome (TS) was recognized in 1994 to be viral in nature and tentatively classified as belonging to either the family Picomaviridae or Nodaviridae. The work reported here has led to a more definitive classification of this new penaeid virus. Located within the cytoplasm of infected cuticular epithelial cells of penaeid shrimp, the virus is a 31 to 32 nm icosahedral particle with a buoyant density of 1.338 ± 0.001 g/ml. Three major (55, 40 and 24 kDa) and one minor (58 kDa) polypeptides constitute its proteinic capsid. Its genome contains a single molecule of ssRNA, which is polyadenylated at the 3' end and approximately 9 kb in length. Based on these characteristics, we believe that TS virus should be included in the family Picornaviridae. Ecuadorian and Hawaiian TS virus isolates were found to be identical in their biophysical, biochemical and biological characteristics, and should be considered as the same virus.
• Durand, S., Lightner, D. V., Redman, R. M., & Bonami, J. (1997). Ultrastructure and morphogenesis of White Spot Syndrome Baculovirus (WSSV). Disease of Aquatic Organisms, 29(3), 205-211.
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  Abstract: Since 1993 non-occluded baculoviruses, associated with a syndrome with high mortalities, have been reported in cultured penaeid shrimp from Asia anti the Indo-Pacific region. Infections are typically accompanied by the presence of white spots on the cuticle. Numerous names were given to the virus(es) in early reports on the disease, but the syndrome is increasingly known as White Spot Syndrome (WSS) and its viral agent(s) as White Spot Syndrome Baculovirus (WSSV). The WSS virion is a stocky rod-shaped particle with an apical envelope extension. The nucleocapsid is cylindrical with asymmetric ends, and has a superficial segmented appearance. The pattern of degradation confirms that the nucleocapsid is a cylinder formed by stacks of rings, which are in turn composed of 2 rows of regularly spaced subunits WSSV replication takes place in the nucleus and is first indicated by chromatin margination and nuclear hypertrophy. Viral morphogenesis begins by the formation of membranes de nova in the nucleoplasm and by the elaboration of segmented, empty, king tubules. These tubules break into fragments to form naked empty nucleocapsids. After that, membranes envelop the capsids leaving an open extremity. The nucleoproteins, which have a filamentous appearance, enter the capsid through this open end. When the core is completely formed, the envelope narrows at the open end and forms the apical tail of the mature virion.
• Garza, J. R., Hasson, K. W., Poulos, B. T., Redman, R. M., White, B. L., & Lightner, D. V. (1997). Demonstration of infectious taura syndrome virus in the feces of seagulls collected during an epizootic in Texas. Journal of Aquatic Animal Health, 9(2), 156-159.
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  Abstract: Laughing gulls Larus atricilla were observed feeding on diseased shrimp Penaeus vannamei during an epizootic of the Taura syndrome virus (TSV) at a south Texas farm in May 1995. Fresh fecal samples were collected from a gull roosting site on a pond levee where gulls that fed on diseased shrimp had roosted overnight. The feces samples were tested for the presence of TSV by injection bioassay. A cell-free clarified homogenate of the fecal material was injected into indicator P. vannamei, and the shrimp were observed for 9 d. Cumulative mortalities of the two replicate groups of indicator shrimp were 45% and 70%. Moribund indicator shrimp collected and preserved for histological examination exhibited lesions pathognomonic for acute-phase infections by TSV. The presence of TSV in the induced lesions was confirmed with in situ hybridization with cDNA probes specific for a portion of the TSV genome. The presence of infectious TSV in the gulls' feces supports the hypothesis that these birds are a probable transport vector of the virus within and among nearby shrimp farms.
• Hasson, K. W., Hasson, J., Aubert, H., Redman, R. M., & Lightner, D. V. (1997). A new RNA-friendly fixative for the preservation of penaeid shrimp samples for virological detection using cDNA genomic probes. Journal of Virological Methods, 66(2), 227-236.
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  PMID: 9255734;Abstract: In situ hybridization analysis of shrimp histological sections, utilizing Taura syndrome virus (TSV) specific cDNA probes, is the most sensitive diagnostic technique presently available for the detection of this penaeid shrimp viral disease. However, false negative genomic probe results are obtained frequently from samples of Pacific white shrimp, Penaeus vannamei, that have been preserved with Davidson's AFA (acetic acid, formaldehyde, alcohol) fixative and that, otherwise, demonstrate pathognomonic TSV lesions by routine histology. This problem was linked to prolonged storage of shrimp samples in Davidson's fixative, which is highly acidic (pH ~ 3.5-4). Degradation of TSV genomic RNA was hypothesized to be due to either fixative- induced acid hydrolysis and/or acidophilic endogenous ribonuclease activity. Routine H and E histology and in situ hybridization analyses were conducted on equal numbers of TSV infected P. vannamei juveniles that were preserved for four different time periods (2, 6, 10 and 14 days) with either Davidson's fixative or a new, near neutral (pH ~ 6.0 - 7.0), RNA-friendly fixative (R-F) that was developed by the authors. In situ hybridization assays were conducted with and without RNase precautions and all of the samples tested contained moderate to severe TSV lesions by routine histology. Davidson's preserved samples produced weak TSV probe signals after 2 days fixation, but did not react with the probes in those samples that were stored for > 6 days in the fixative. In contrast, TSV was detectable by gene probe in all of the time treatment samples preserved with the new R-F fixative. Equivalent in situ hybridization results were obtained when the same samples were analyzed in the absence of RNase-free conditions. These findings suggest that TSV RNA is degraded when samples are stored in an acidic fixative, such as Davidson's, for more than 2 days and that this problem can be prevented through preservation of shrimp samples with R-F fixative. The efficacy of this new fixative is demonstrated and the results show that RNase-free conditions are not necessary for conducting TSV in situ hybridization analyses.
• Lightner, D. V., Redman, R. M., Poulos, B. T., Nunan, L. M., Mari, J. L., & Hasson, K. W. (1997). Risk of spread of penaeid shrimp viruses in the Americas by the international movement of live and frozen shrimp. OIE Revue Scientifique et Technique, 16(1), 146-160.
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  PMID: 9329114;Abstract: Within the past decade, viral diseases have emerged as serious economic impediments to successful shrimp farming in many of the shrimp-farming countries of the world. In the western hemisphere, the viral agents of Taura syndrome (TS) and infectious hypodermal and haematopoietic necrosis have caused serious disease epizootics throughout the shrimp-growing regions of the Americas and Hawaii, while in Asia the viral agents of white spot syndrome (WSS) and yellow head (YH) have caused pandemics with catastrophic losses. The international transfer of live shrimp for aquaculture purposes is an obvious mechanism by which the viruses have spread within and between regions in which they have occurred. Shrimp-eating gulls, other seabirds and aquatic insects may also be factors in the spread of shrimp viruses between and within regions. Another potentially important mechanism for the international spread of these pathogens is the trade in frozen commodity shrimp, which may contain viruses exotic to the importing countries. The viral agents of WSS, YH and TS have been found, and demonstrated to be infectious, in frozen shrimp imported into the United States market. Mechanisms identified for the potential transfer of virus in imported frozen products to domestic populations of cultured or wild penaeid shrimp stocks include: the release of untreated liquid or solid wastes from shrimp importing and processing plants directly into coastal waters, improper disposal of solid waste from shrimp importing and processing plants in landfills so that the waste is accessible to gulls and other seabirds, and the use of imported shrimp as bait by sports fishermen.
• Mohney, L. L., Williams, R. R., Bell, T. A., & Lightner, D. V. (1997). Residues of oxytetracycline in cultured juvenile blue shrimp, Penaeus stylirostris (Crustacea: Decapod), fed medicated feed for 14 days. Aquaculture, 149(3-4), 193-202.
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  Abstract: Juvenile shrimp fed for 14 days on oxytetracycline (OTC) medicated feed with a potency of 1500 mg OTC kg-1 of feed reached a level of 33 μg OTC g-1 of shrimp tail muscle within 4 days, as measured by the microbial agar plate diffusion assay method for oxytetracycline in tissues, approved by the Association of Official Analytical Chemists. The concentration of OTC remained between 3.3-5.2 μg OTC g-1 of shrimp tail muscle from Day 4 through Day 14 of the feeding period. These concentrations were greater than the Minimum Inhibitory Concentration for sensitive organisms. By the fifth day following cessation of feeding medicated feed the drug content in shrimp tail muscle was less than the detectable limit for the method, or 0.4 μg of OTC g-1 shrimp tissue.
• Nunan, L. M., & Lightner, D. V. (1997). Development of a non-radioactive gene probe by PCR for detection of white spot syndrome virus (WSSV). Journal of Virological Methods, 63(1-2), 193-201.
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  PMID: 9015290;Abstract: Combining primers created from the sequence information of two baculo-like viruses of penaeid shrimp, Baculovirus penaei (BP) and Monodon baculovirus (MBV), produced a 750 bp band on a 0.8% agarose gel using White Spot Syndrome Virus (WSSV), from Penaeus monodon, as the DNA template. The PCR fragment was ligated to a plasmid vector, (pGEM-T) and transformed, creating a 3.7 Kbp clone. The DNA insert was sequenced, and the original primer pair was located. Using restriction enzymes, the insert was isolated, excised and non-radioactively labeled. This cloned labeled fragment was tested by in situ hybridization for specificity and reactivity with BP, MBV and WSSV-infected shrimp tissues. The major advantage of this novel method of gene probe development is that no DNA sequence information of the targeted infectious agent needed to be known or available. In addition, tedious viral isolation and purification was circumvented. In this study, knowledge of the possible viral strain was important in limiting the PCR primer pairs investigated. The use of arbitrary primers designed for PCR assays from two other possibly related shrimp viruses, increased the likelihood that a generated PCR product would be specific for WSSV.
• Overstreet, R. M., Overstreet, R. M., Lightner, D. V., Lightner, D. V., Hasson, K. W., Hasson, K. W., McIlwain, S., McIlwain, S., Lotz, J. M., & Lotz, J. M. (1997). Susceptibility to Taura Syndrome Virus of Some Penaeid Shrimp Species Native to the Gulf of Mexico and the Southeastern United States. Journal of Invertebrate Pathology, 69(2), 165-176.
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  Abstract: Experimental studies demonstrated thatPenaeus setiferus,but notPenaeus aztecusorPenaeus duorarum,could be killed by Taura syndrome virus (TSV). However, specimens ofP. setiferusthat survived infection and bothP. aztecusandP. duorarumat least 79 days postexposure that did not demonstrate gross signs of infection were shown to harbor virus by bioassay usingPenaeus vannamei,a highly susceptible target host. Consequently, all three of those penaeids native to the southeast United States can serve as carriers or reservoir hosts of TSV without necessarily exhibiting disease. Infections inP. setiferustook longer to cause mortality than inP. vannameiand killed a smaller percentage of that host. Also, histological lesions diagnostic of TSV infection were not always evident in sectioned tissue of infectedP. setiferus,and they generally were more conspicuous during Days 4-7 postexposure compared with lesions that also occurred at both earlier and later days in tissues ofP. vannamei.Infections could be produced by injection, ingestion, and incorporation of the infective material into dietary brine shrimp. There appeared to be a difference in susceptibility to TSV disease by different stocks ofP. setiferus,but different experiments produced conflicting evidence regarding a relationship between age and predilection to mortality. Large and small specimens of equal-aged shrimp succumbed similarly to TSV infections for bothP. vannameiandP. setiferus.The nonnative speciesP. chinensisdemonstrated a high susceptibility to experimental TSV disease. © 1997 Academic Press.
• 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(1-2), 1-8.
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  Abstract: An experimental non-lethal serodiagnostic kit was evaluated for screening candidate specific pathogen-free (SPF) Penaeus vannamei broodstock under typical field conditions. In this trial, a total of 26 adult P. vannamei was screened for the presence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) using an IHHNV dot blot field kit. The non-radioactive IHHNV in situ hybridization method was used as a standard for comparison. The results showed a significant relationship between the outcome of the dot blot kit and the outcome of the in situ method. In particular, the test results using the experimental kit were consistent with the in situ hybridization method. Based on these findings, the experimental serodiagnostic kit may provide a more rapid, cost-effective diagnostic method than traditional techniques for IHHNV detection.
• Durand, S., Lightner, D. V., Nunan, L. M., Redman, R. M., Mari, J., & Bonami, J. (1996). Application of gene probes as diagnostic tools for White Spot Baculovirus (WSBV) of penaeid shrimp. Disease of Aquatic Organisms, 27(1), 59-66.
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  Abstract: Since 1993, similar baculoviruses, which cause high mortalities in penaeid shrimp, have been reported from China, Japan, Korea, Thailand and Taiwan. All these baculovirus-caused diseases are characterized by the presence of white spots in the cuticle. To isolate the agent of the disease referred to as White Spot Syndrome (WSS) and White Spot Baculovirus (WSBV), in this paper, Penaeus vannamei and P. stylirostris were experimentally inoculated with homogenates of infected Penaeus monodon from Thailand. In transmission electron microscopy (TEM), the enveloped WSBV virions have a size of about 350 nm long and 130 nm wide. The nucleocapsids range from 300 to 420 nm in length and 70 to 95 nm in diameter and show a superficially segmented appearance. Also present are unique particles, which have not been observed before, measuring 400 nm in length and 120 nm in width. These particles are larger than most of the usual nucleocapsids and have a cross- batched superficial appearance. After nucleic acid extraction, EcoRI-digested fragments of the WSBV genome were cloned. Four of these fragments were characterized and used as non-radioactive probes lableled with DIG-11-dUTP. By in situ hybridization, the probes hybridized with material located in nuclei of all WSBV-infected tissues. Previously reported target tissues, such as connective tissue, epithelial tissue and also hemocytes, clearly showed positive hybridization with the probes. In addition, some light infection was revealed in the muscle and nervous tissue.
• Lightner, D. V. (1996). Epizootiology, distribution and the impact on international trade of two penaeid shrimp viruses in the Americas. OIE Revue Scientifique et Technique, 15(2), 579-601.
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  PMID: 8890382;Abstract: Marine penaeid shrimp are affected by appproximately twenty viruses, the majority of which were discovered as a result of their negative effects on aquaculture. In the Americas, infectious hypodermal and haematopoietic necrosis (IHHN) virus and Taura syndrome (TS) virus have had a significant negative impact on aquaculture industries and, in one instance, on a commercial fishery. Both viruses have become widely distributed as a consequence of the movement of host stocks for aquaculture. IHHN virus (IHHNV) causes catastrophic losses in cultured and wild Penaeus stylirostris. In marked contrast, P. vannamei is relatively resistant to IHHN but infection results, nonetheless, in poor culture performance. TS virus (TSV) is the 'mirror image' of IHHNV in its effect on P. stylirostris and P. vannamei. TSV causes catastrophic losses in P. vannamei, whereas P. stylirostris is highly resistant to TS. In the less than three years since the discovery of TSV in Ecuador in 1992, the virus has spread rapidly and caused massive production losses in most shrimp-growing countries in the Americas.
• Lightner, D. V., Hasson, K. W., White, B. L., & Redman, R. M. (1996). Chronic toxicity and histopathological studies with Benlate^®, a commercial grade of benomyl, in Penaeus vannamei (Crustacea: Decapoda). Aquatic Toxicology, 34(2), 105-118.
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  Abstract: Juvenile Pacific white shrimp, Penaeus vannamei (Crustacea: Decapoda) were exposed to 0, 0.01, 0.1, 1.0 mg/l of benomyl (Benlate® OD; DuPont) in a 30-day static renewal bioassay. The levels tested were below the 96 h LC50 of approximately 10 mg benomyl/l for juvenile penaeid shrimp. Exposure to benomyl at 1.0 mg/l resulted in death of some shrimp after 11 to 12 days, and approximately 80% cumulative mortality by 19 days. No overt toxicity or histopathological changes were noted at 0.01 or 0.1 mg benomyl/l after 30 days of exposure. Moribund shrimp (in the 1.0 mg benomyl/l treatments from day 11 to 19) displayed lethargy, anorexia, opaque musculature, and cuticular hyperchromatophorism. Histological examination of affected shrimp revealed distinctive pathological changes which were confined to the hepatopancreas (HP) and the associated anterior midgut caecum. The most significant lesions included a marked atrophy of the HP, intertubular hemocytic inflammation, often with melanized foci, and necrosis and desquamation of the HP epithelial cells. Moreover, all shrimp examined from the 1.0 mg/l dose level displayed HP epithelial cells that were enormously hypertrophied and presented as conspicuous giant megahepatopancreatocytic (MCy) cells. These benomyl-induced MCy cells are a unique lesion which is distinct from other reported toxic and infectious diseases of penaeid shrimp. Hence, the presence of MCy cells in degenerate HP tubules may provide pathognomonic indicators of benomyl toxicity in penaeid shrimp and perhaps other crustaceans.
• Bonami, J. -., Mari, J., Poulos, B. T., & Lightner, D. V. (1995). Characterization of hepatopancreatic parvo-like virus, a second unusual parvovirus pathogenic for penaeid shrimps. Journal of General Virology, 76(4), 813-817.
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  PMID: 9049326;Abstract: The hepatopancreatic parvo-like virus (HPV) of penaeid shrimp was extracted from infected shrimp tissues, purified and subsequently characterized. The viral particles, icosahedral in shape, are 22 nm in diameter and possess a buoyant density of 1.41 g/ml. They contain ssDNA, of approximately 5 kb in size which encodes a single polypeptide of 54 kDa. On the basis of its general characteristics this pathogenic agent belongs to the Parvoviridae family, but because of two unusual characteristics (capsid protein formed with a single polypeptide and genome structure more closely related to the autonomous parvoviruses rather than the densoviruses), it seems to constitute a novel group in the Parvoviridae family.
• Bonami, J. R., Bruce, L. D., Poulos, B. T., Mari, J., & Lightner, D. V. (1995). Partial characterization and cloning of the genome of PvSNPV (=BP-type virus) pathogenic for Penaeus vannamei. Diseases of Aquatic Organisms, 23(1), 59-66.
• Hasson, K. W., Lightner, D. V., Poulos, B. T., Redman, R. M., White, B. L., Brock, J. A., & Bonami, J. R. (1995). Taura syndrome in Penaeus vannamei: Demonstration of a viral etiology. Diseases of Aquatic Organisms, 23(2), 115-126.
• Lightner, D. V., Redman, R. M., Hasson, K. W., & Pantoja, C. R. (1995). Taura syndrome in Penaeus vannamei (Crustacea: Decapoda): Gross signs, histopathology and ultrastructure. Diseases of Aquatic Organisms, 21(1), 53-59.
• Mari, J., Lightner, D. V., Poulos, B. T., & Bonami, J. -. (1995). Partial cloning of the genome of an unusual shrimp parvovirus (HPV): Use of gene probes in disease diagnosis. Diseases of Aquatic Organisms, 22(2), 129-134.
• Park, E. D., Lightner, D. V., Milner, N., Mayersohn, M., Park, D. L., Gifford, J. M., & Bell, T. A. (1995). Exploratory bioavailability and pharmacokinetic studies of sulphadimethoxine and ormetoprim in the penaeid shrimp, Penaeus vannamei. Aquaculture, 130(2-3), 113-128.
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  Abstract: The pharmacokinetics and bioavailability of sulphadimethoxine (SDM) and ormetoprim (OMP) were examined, following simultaneous administration in penaeid shrimp. The hemolymph concentration versus time data following intra-sinus injection for both SDM (42 mg/kg) and OMP (8.6 mg/kg) were well described by a multiexponential equation suggesting multicompartmental behavior. The SDM:OMP parameter estimates of systemic clearance, steady-state volume of distribution, and disposition half-life were 215:1765 ml/kg · h, 1319:34 382 ml/kg, and 9.0:17.8 h, respectively. Hemolymph protein binding of SDM and OMP was 5.2% and 12.1%, respectively. The bioavailabilities of SDM and OMP were 30% and 32%, respectively. Peak hemolymph concentration (Cmax) and time of occurrence of that value (Tmax) for SDM following a single oral dose (210 mg/kg) were 25.0 μg/ ml at 4 h, while the corresponding values for OMP were (dose, 42 mg/kg) 0.70 μg/ml at 4 h. The percent of the available oral dose 2 h post-administration for SDM:OMP in the hemolymph, muscle, and hepatopancreas were 5.0:0.6%, 7.8:3.4%, and 2.4:24.8%, respectively. Hemolymph and muscle tissue concentrations were below detectable limits after 48 h for SDM and 24 h for OMP. The SDM:OMP drug combination has good potential as a shrimp chemotherapeutant in that they were rapidly and moderately absorbed, have relatively short half-lives and low hemolymph protein binding which translates to the bulk of absorbed drugs being available for a therapeutic response. © 1995.
• Bruce, L. D., Lightner, D. V., Redman, R. M., & Stuck, K. C. (1994). Comparison of traditional and molecular detection methods for Baculovirus penaei infections in larval Penaeus vannamei. Journal of Aquatic Animal Health, 6(4), 355-359.
• Bruce, L. D., Redman, R. M., & Lightner, D. V. (1994). Application of gene probes to determine target organs of a penaeid shrimp baculovirus using in situ hybridization. Aquaculture, 120(1-2), 45-51.
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  Abstract: Gene probes developed to the penaeid shrimp virus, Baculovirus penaei (BP), were utilized to determine the target organs infected by BP. BP-DNA fragments were nonradioactively labelled with digoxigenin and used to probe paraffin-embedded, fixed tissue sections using an in situ hybridization assay system. BP-infected tissue sections were microscopically examined for a blue-purple probe precipitate indicative of a positive reaction for BP. Particular attention was given to the reproductive tissues of mature shrimp, to determine if vertical transmission might occur. BP positive cells were located only in the hepatopancreatic tubule epithelium and in the midgut mucosa. These findings indicate that BP is a gut-infecting baculovirus that is transmitted horizontally by the per os route and not by true vertical transmission routes. © 1994.
• Lightner, D. V., & Redman, R. M. (1994). An epizootic of necrotizing hepatopancreatitis in cultured penaeid shrimp (Crustacea: Decapoda) in northwestern Peru. Aquaculture, 122(1), 9-18.
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  Abstract: In January and February of 1993 a severe epizootic of necrotizing hepatopancreatitis (NHP) began to affect a number of commercial penaeid shrimp farms in the Tumbes area of northwestern Peru and adjacent areas of Ecuador. Histological and transmission electron microscopical (TEM) examination of affected juvenile Penaeus vannamei showed massive infections of the hepatopancreas by a pleomorphic intracellular Gram-negative bacterium. The agent was named Peru NHP (PNHP) for its geographical occurrence and for the type of lesion with which it was associated. A very similar bacterium (TNHP) has been found to be associated with a serious epizootic disease in shrimp farms in Texas. Before the cause of the Peru epizootic of PNHP was determined and corrective measures were taken, nearly half of Peru's 44 active shrimp farms had been so seriously affected that they were closed by June of 1993. © 1994.
• Meyers, T. R., Lightner, D. V., & Redman, R. M. (1994). A dinoflagellate-like parasite in Alaskan spot shrimp Pandalus platyceros and pink shrimp P.borealis. Diseases of Aquatic Organisms, 18(1), 71-76.
• Mohney, L. L., Lightner, D. V., & Bell, T. A. (1994). An epizootic of vibriosis in Ecuadorian pond-reared Penaeus vannamei Boone (Crustacea: Decapoda). Journal - World Aquaculture Society, 25(1), 116-125.
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  Abstract: A massive epizootic of vibriosis began in November 1989 and continued through May 1990 in nursery and grow-out ponds in a number of shrimp farms located in the Gulf of Guayaquil, Ecuador. The level of cumulative mortalities ranged from slight in some ponds to >90% in others. Examination of affected Penaeus vannamei led to the isolation of several closely related Vibrio species. The predominant species were identified as Vibrio parahaemolyticus, V. vulnificus, and V. alginolyticus. The epizootic coincided with the second year of a severe drought in Ecuador. Discharge of fresh water by the Guayas River was reduced and salinities and relative nutrient concentrations were elevated in the Gulf of Guayaquil. These environmental conditions were ideal for the increased growth of the causative vibrios in the estuarine waters and are believed to be factors that contributed to the 1989-1990 epizootic. -from Authors
• Park, E. D., Lightner, D. V., & Park, D. L. (1994). Antimicrobials in shrimp aquaculture in the United States: regulatory status and safety concerns.. Reviews of Environmental Contamination and Toxicology, 138, 1-20.
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  PMID: 7938783;Abstract: The consumption of seafood, especially shrimp, increases yearly in the U.S. The U.S. is the second largest importer of shrimp in the world, consuming more than 11% of the total world production. Aquaculture is becoming an increasingly important source of the world's shrimp, currently accounting for approximately 30% of the world's supply. Unfortunately, in this era of international trade deficits, U.S. production of aquacultured shrimp is insignificant (< 0.1%) compared with world production. As shrimp aquaculture expands in the U.S., so does the use of intensive farming techniques. Shrimp aquaculture is like any other animal husbandry industry in that shrimp are subject to disease, especially under intensive farming methods. In penaeid shrimp, the primary diseases associated with mortalities are usually viral or bacterial. The majority of bacterial infections in penaeid shrimp are attributable to Vibrio species, with mortalities ranging from insignificant to 100%. However, the rapid growth of this industry has outpaced efforts by researchers, pharmaceutical companies, and federal regulatory agencies to provide approved therapeutants for shrimp disease management. Approval of drugs and their surveillance for compliance with regulations applicable to seafoods, including aquacultured goods, is the responsibility of the FDA. There are three general areas of concern regarding human health when chemotherapeutants are used in aquaculture: (1) residues of drugs in fish destined for human consumption; (2) development of drug resistance in human pathogenic bacteria; and (3) direct toxic effects to humans from handling of drugs. Currently, there are no antibacterials approved for shrimp aquaculture in the U.S. One of the major obstacles in the development and approval of new drugs for aquaculture is the cost of conducting the required studies. The high cost to pharmaceutical companies discourages investment in shrimp chemotherapeutant research, since the current U.S. market for such products is small. Unfortunately, the U.S. shrimp aquaculture industry will remain small without legal availability of chemotherapeutants. Oxytetracycline (OTC) and Romet-30 are two antibacterials currently approved in the U.S. for catfish and salmonid aquaculture. Shrimp aquaculture facilities outside of the U.S. routinely use these drugs, as well as others, in the treatment of bacterial disease outbreaks. Much of the work required for OTC approval by the FDA for penaeid shrimp has been completed.(ABSTRACT TRUNCATED AT 400 WORDS)
• Park, E. D., Lightner, D. V., Stamm, J. M., & Bell, T. A. (1994). Preliminary studies on the palatability, animal safety, and tissue residues of sarafloxacin-HCl in the penaeid shrimp, Penaeus vannamei. Aquaculture, 126(3-4), 231-241.
• Poulos, B. T., Lightner, D. V., Trumper, B., & Bonami, J. R. (1994). Monoclonal antibodies to a penaeid shrimp parvovirus, infectious hypodermal and hematopoietic necrosis virus (IHHNV). Journal of Aquatic Animal Health, 6(2), 149-154.
• Poulos, B. T., Mari, J., Bonami, J., Redman, R., & Lightner, D. V. (1994). Use of non-radioactively labeled DNA probes for the detection of a baculovirus from Penaeus monodon by in situ hybridization on fixed tissue. Journal of Virological Methods, 49(2), 187-193.
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  PMID: 7822460;Abstract: Clones isolated from a genomic library of the baculovirus PmSNPV (= MBV) were used to prepare DNA probes for detection of PmSNPV in shrimp tissue. Davidson's AFA fixed shrimp tissues were probed using DNA that had been labeled with the hapten, digoxigenin, by an in situ hybridization assay. The DNA probes readily distinguished PmSNPV-infected from uninfected shrimp tissue. The reactions were localized to the hepatopancreas and midgut, the sites where this virus is detected by standard histological methods. The probes did not react to shrimp infected with Baculovirus penaei (BP = PvSNPV). © 1994.
• Bruce, L. D., Redman, R. M., Lightner, D. V., & Bonami, J. R. (1993). Application of gene probes to detect a penaeid shrimp baculovirus in fixed tissue using in situ hybridization. Diseases of Aquatic Organisms, 17(3), 215-221.
• Lightner, D. V., Lightner, D. V., Redman, R. M., & Redman, R. M. (1993). A Putative Iridovirus from the Penaeid Shrimp Protrachypene precipua Burkenroad (Crustacea: Decapoda). Journal of Invertebrate Pathology, 62(1), 107-109.
• Lightner, D. V., Redman, R. M., Moore, D. W., & Park, M. A. (1993). Development and application of a simple and rapid diagnostic method to studies on hepatopancreatic parvovirus of penaeid shrimp. Aquaculture, 116(1), 15-23.
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  Abstract: Hepatopancreatic parvovirus (HPV) is one of about a dozen viruses known to infect penaeid shrimps. HPV is apparently cosmopolitan in distribution, having been reported from wild and/or cultured penaeid shrimp in Asia, Africa, Australia and North and South America. However, the importance of the virus as a pathogen remains poorly understood, possibly due in part to the relative difficulty of diagnosis of HPV infections and to the possibility that HPV infections may be masked in multiple infections with other pathogens. Until now diagnosis of HPV has been dependent upon the histological demonstration of pathognostic Feulgen positive, basophilic intranuclear inclusion bodies in hepatopancreatic epithelial cells, which has limited diagnostic capability for HPV to suitably equipped laboratories. A rapid field diagnostic method for HPV infections was developed that uses Giemsa-stained impression smears of the hepatopancreas. The impression smear method was shown to compare favorably in diagnostic sensitivity to the standard histopathological method for HPV when applied to the diagnosis of HPV infections in postlarval and juvenile Penaeus chinensis (Osbeck). © 1993.
• Mari, J., Bonami, J. -., & Lightner, D. (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. Journal of General Virology, 74(12), 2637-2643.
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  PMID: 8277269;Abstract: The infectious hypodermal and haematopoietic necrosis virus (IHHNV), pathogenic for penacid shrimp, is an icosahedral unenveloped particle, 22 nm in diameter, with an ssDNA linear genome, and proposed to be a member of the Parvoviridae. A large majority of minus-strand DNA is incorporated into the capsids compared to the plus-strand. A small amount of reannealed plus- and minus-strands (dsDNA) obtained after nucleic acid extraction was blunt-ended and cloned into the system pUC18/Escherichia coli strain DH5α. Selected clones were studied and characterized using restriction enzymes. One of them, BQ31, was used to construct different sized probes labelled with digoxigenin-11-dUTP. These probes failed to hybridize with DNA of some insect parvoviruses and with DNA of a parvo-like virus of shrimp. They reacted strongly with dilutions of homogenized IHHNV-infected shrimp tissues and, conversely, did not react with uninfected shrimp tissues. They hybridized in situ, in sections of infected animals, labelling strongly the target cells and particularly the nuclear Cowdry type A inclusion body, which is the most diagnostic characteristic of this disease.
• Williams Jr., E. H., Bunkley-Williams, L., Grizzle, J. M., Peters, E. C., Lightner, D. V., Harshbarger, J., Rosenfield, A., & Reimschuessel, R. (1993). Epidemic misuse [3]. Nature, 364(6439), 664-.
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  PMID: 8355774;
• Luedeman, R., & Lightner, D. V. (1992). Development of an in vitro primary cell culture system from the penaeid shrimp, Penaeus stylirostris and Penaeus vannamei. Aquaculture, 101(3-4), 205-211.
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  Abstract: Studies were carried out to improve the methods for producing primary cell cultures of penaeid shrimp and to make these procedures routine and practical. Supplements tested included: lobster hemolymph, shrimp muscle extract, fetal bovine serum and hybridoma quality fetal bovine serum. Grace's Insect Medium supplemented with hybridoma quality fetal bovine serum was found to provide the best results. The optimum conditions for shrimp cells in Grace's Insect Medium were determined to be an osmolality of approximately 700-750 mmol/kg, a temperature range of 25-28 °C and incubation in a normal atmosphere. Using the methods developed, monolayers of primary cultures of ovarian epithelioid cells from Penaeus stylirostris and Penaeus vannamei were routinely obtained with 80% confluence within a 2-day period. © 1992.
• Williams, R. R., Dehdashti, B., & Lightner, D. V. (1992). Performance of an aquatic multispecies system in evaluating the effects of a model microbial pest control agent on nontarget organisms. Journal of Toxicology and Environmental Health, 37(2), 247-264.
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  PMID: 1404484;Abstract: A recirculating multispecies test system was developed in conjunction with a study of the fate and persistence of a model microbial pest control agent on nontarget marine and freshwater organisms. The basic unit of the system was a 113-I glass aquarium with vertical biological filters in the center of the aquarium, such that two compartments were formed. This allowed the sequestration of predator and prey species within the same system. Organisms from six phyletic groups were subjected to a genetically altered strain of Pseudomonas putida for 15-29 d in either artificial seawater or fresh water. The system was able to maintain the animals for these periods with a minimum of maintenance. Additionally, the system design lent itself to disinfection, dismantling, and rebuilding between experiments with a minimum of labor, and has potential for longer-term studies.
• Bruce, L. D., Trumpet, B. B., & Lightner, D. V. (1991). Methods for viral isolation and DNA extraction for a penaeid shrimp baculovirus. Journal of Virological Methods, 34(3), 245-254.
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  PMID: 1660489;Abstract: Procedures for the purification of virions and nucleocapsids of Baculovirus penaei (BP) of penaeid shrimp and subsequent extraction of the viral nucleic acid are described. BP-infected hepatopancrata, from two species of shrimp from different geographical locations in the Americas, were removed and homogenized in a solution of TN buffer (0.01 M Tris-HCl, 0.10 M NaCl, pH 8.0). The homogenized mixture was strained through a 100-mesh screen to remove large pieces of tissue and centrifuged to concentrate the remaining material. The pellet was suspended in TN buffer and layered on to a handmade CsCl gradient. Fractions were collected according to the bands observed in the gradient, and the optical density at 254 nm was recorded for each fraction. The resultant data was tabulated and graphed. Additionally, each fraction was examined by transmission electron microscopy to determine relative numbers of viral particles present. Large amounts of virus consistently corresponded to a specific band in the gradient, which produced a peak when the spectrophometric data was graphed. Nucleic acid was then extracted from the purified viral particles. Removal of polysaccharides was accomplished with the addition of CTAB/NaCl. The BP DNA was visualized on an agarose gel with phage lambda DNA markers for size estimation, and a preliminary endonuclease digestion was performed using BamHi. © 1991.
• Dehdashti, B., & Lightner, D. V. (1991). Observations on the biology of Hyalella azteca (Amphipoda) in a closed system. A study of a model ecosystem in microgravity. Crustaceana, 61(3), 233-240.
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  Abstract: Viable populations were maintained in the closed ecosystem for up to 15 months, the only external input being photoradiation. One colony was given to Soviet astronauts for a 14 day flight onboard Cosmo-2044. Conditions in space had no apparent adverse effects on adults or developing embryos. -M.Dean
• Bonami, J. -., Trumper, B., Mari, J., Brehelin, M., & Lightner, D. V. (1990). Purification and characterization of the infectious hypodermal and haematopoietic necrosis virus of penaeid shrimps. Journal of General Virology, 71(11), 2657-2664.
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  PMID: 2254754;Abstract: Infectious hypodermal and haematopoietic necrosis (IHHN) is one of the most important viral diseases of cultured penaeid shrimps and is potentially a limiting factor in the development of farming projects for some species of these shrimps. Although the IHHN agent was recognized early as being viral in origin, attempts to characterize it were inconclusive because of difficulties in obtaining sufficient amounts of purified virions to permit its characterization. Recent improvements of purification procedures have allowed the physicochemical characterization of this virus. Purified IHHNV is a non-enveloped icosahedral particle averaging 22 nm in diameter, exhibiting a mean buoyant density of 1.40 g/ml in CsCl. The genome is a single molecule of ssDNA with an estimated size of 4.1 kb by molecule length measurement in transmission electron microscopy. As determined by SDS-PAGE, the particle contains four polypeptides with M(r)s of 74K, 47K, 39K and 37.5K, respectively. From its characteristics, this virus could be a member of the Parvoviridae family.
• Lightner, D. V., Redman, R. M., & Ruiz, E. (1989). Baculovirus penaei in Penaeus stylirostris (Crustacea: Decapoda) cultured in Mexico: Unique cytopathology and a new geographic record. Journal of Invertebrate Pathology, 53(1), 137-139.
• Bell, T. A., & Lightner, D. V. (1987). An outline of penaeid shrimp culture methods including infectious disease problems and priority drug treatments.. Veterinary and Human Toxicology, 29 Suppl 1, 37-43.
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  PMID: 3617440;
• Lightner, D. V., & Brock, J. A. (1987). A lymphoma-like neoplasm arising from hematopoietic tissue in the white shrimp, Penaeus vannamei Boone (Crustacea: Decapoda). Journal of Invertebrate Pathology, 49(2), 188-193.
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  PMID: 3559262;Abstract: A pond-reared adult female Pacific white shrimp (Penaeus vannamei Boone, Crustacea: Decapoda) from an experimental shrimp culture facility in Hawaii was found to possess a lymphatic neoplasm. Present in this animal were bilateral hypertrophied hematopoietic nodules present ventral and lateral to the ventral nerve cord, and smaller multiple ectopic foci of similar appearing lymphoid cells in the gills, the subcutis, and other tissues. The lesions contained numerous anaplastic and hypertrophied lymphoid cells, many of which displayed bizarre mitotic figures, including polypolar metaphase figures. Numerous multinuclear giant cells present in the lesion were presumed to have originated from cells with polypolar mitotic figures. The characteristics of the cells composing these lesions, the expansive and invasive nature of the lesions, and the presence of ectopic foci of neoplastic cells support classification of this lesion as a hematopoietic sarcoma. Focal lesions of the type that are diagnostic of infections by the penaeid shrimp virus IHHN were present in the neoplastic hematopoietic tissue and other tissues of this shrimp, suggesting the possible role of viral infection in the development of neoplastic lesions in this animal. © 1987.
• Lightner, D. V., Moore, D. W., & Redman, R. M. (1987). A tumor-like hernia of the hepatopancreas and associated tissues of the penaeid shrimp Penaeus stylirostris (Crustacea: Decapoda). Aquaculture, 65(3-4), 359-362.
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  Abstract: A sub-adult cultured blue shrimp, Penaeus stylirostris (Crustacea: Decapoda) possessed a prominent papilliform tumor-like growth that protruded from the mid-dorsal surface of its carapace. Subsequent study showed the growth not to be a true tumor, but to be a hernia of the hepatopancreas and associated organs. The unusual lesion was probably the result of a puncture wound. © 1987.
• Lightner, D. V., & Redman, R. M. (1985). A parvo-like virus disease of penaeid shrimp. Journal of Invertebrate Pathology, 45(1), 47-53.
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  Abstract: Cultured populations of four penaeid shrimp species (Crustacea, Decapoda) from four separate culture facilities in Asia were found to be adversely affected by a disease of presumed viral etiology. Individual shrimp with the disease displayed nonspecific signs, including poor growth rate, anorexia, reduced preening activity, increased surface fouling, and occasional opacity of tail musculature. These signs were accompanied by mortalities during the juvenile stages, after apparently normal development through the larval and postlarval stages. Accumulative mortality rates in epizootics in Penaeus merguiensis and P. semisulcatus reached as high as 50 to 100%, respectively, of the affected populations within 4 to 8 weeks of disease onset. The principal lesion, common to all four species, was necrosis and atrophy of the hepatopancreas, accompanied by the presence of large prominent basophilic, PAS-negative, Fuelgen-positive intranuclear inclusion bodies in affected hepatopancreatic tubule epithelial cells (hepatopancreatocytes). These inclusion bodies presumably developed from small, eosinophilic, intranuclear bodies that were also present in the affected tissues. Electron microscopy of affected hepatopancreatocytes revealed aggregations of 22- to 24-nm-diameter virus particles within the electron-dense granular inclusion body ground substance. The virus particle size and morphology, the close association of the nucleolus with the developing inclusion body, and the presence of intranuclear bodies within developing inclusion bodies are similar to cytopathological features reported for parvovirus infections in insects and vertebrates. It is suggested that this presumed virus disease of cultured penaeid shrimp be called HPV for Hepatopancreatic Parvo-like Virus disease. © 1985.
• Bell, T. A., & Lightner, D. V. (1984). IHHN virus: Infectivity and pathogenicity studies in Penaeus stylirostris and Penaeus vannamei. Aquaculture, 38(3), 185-194.
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  Abstract: Infectivity studies of infectious hypodermal and hematopoietic necrosis virus (IHHNV) were conducted on Penaeus stylirostris and P. vannamei (representing species highly susceptible and highly refractory to the disease) via intramuscular injections of the virus. Distinctive histological lesion patterns were observed between species. Six target organ systems were compared interspecifically. The gills and nerve cord/ganglia were shown to undergo significantly higher degrees of tissue damage in infected P. stylirostris compared with infected P. vannamei. Pathogenetic differences correlated well with observed differences in epizootiology between the two species. The infectivity study confirmed that IHHN disease is virus-caused. Cell-free extracts from IHHNV-infected shrimp, when injected into healthy, susceptible shrimp, produced mortalities and Cowdry type A intranuclear eosinophilic inclusion bodies presently considered pathognomonic for the disease. © 1984.
• Hobson, J. F., Carter, D. E., & Lightner, D. V. (1984). Toxicity of a phthalate ester in the diet of a penaied shrimp. Journal of Toxicology and Environmental Health, 13(4-6), 959-968.
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  PMID: 6492211;Abstract: The toxicity of di-2-ethylhexyl phthalate (DEHP), a ubiquitous environmental pollutant, was measured experimentally as a contaminant in shrimp aquaculture feeds. Diets containing 40 to 50,000 ppm DEHP were fed to Penaeus vannamei for 14 d at 4% body weight/d. DEHP concentrations in shrimp, diet, and water were measured by electron-capture gas chromatography. Whole-body residues in shrimp were 18 ppm at the highest dose, and bioconcentration factors were inversely proportional to dose. DEHP in water was ≤ 1.7 ppb for all dose levels. No increased mortality or histopathological alterations were observed at any dose. Absorption of DEHP by P. vannamei was measured in static 24- and 96-h bioassays. Diets enriched with [14C] DEHP to levels of 60, 600, and 6000 ppm were fed at 2% body weight/d. At all dose levels, 3.7% of total radioactivity was measured as body burden in shrimp and 40% as polar aspects in test water after 96 h. DEHP by oral administration was absorbed, metabolized, and excreted, and this process was linear with dose for the dose range studied.
• Hose, J. E., Lightner, D. V., Redman, R. M., & Danald, D. A. (1984). Observations on the pathogenesis of the imperfect fungus, Fusarium solani, in the California brown shrimp, Penaeus californiensis. Journal of Invertebrate Pathology, 44(3), 292-303.
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  Abstract: The pathogenesis of Fusarium solani infections was studied using a highly susceptible species, Penaeus californiensis. In two experiments, F. solani infections were established in artificially wounded and infected juvenile and adults of P. californiensis, while similar control groups were wounded but not artificially infected. In the first experiment, the progress of F. solani infections in 15-g cultured juveniles of P. californiensis was followed by gross inspection and by light and electron microscopical study of the wound area. Developing F. solani infections were produced with a success rate of 100% within 14 days postinfection, and deaths due to disease of greater than 50% of the artificially infected group had occurred by 24 days postinfection. Infections were histologically characterized by (1) expansion of lesions into the tissue surrounding the point of entry, (2) destruction of invaded tissues by host granuloma formation and possibly by fungal enzymes and toxins, and (3) hemocyte encapsulation of hyphae with eventual melanization of the encapsulating hemocytes. In the second study, wild adult P. californiensis were artificially infected with F. solani, and changes in several of their hemolymph constituents [alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT), glucose, total protein, hemocyte count, and hematopoietic tissue mitotic index] were determined as the disease developed. Significant differences occurred in these hemolymph parameters of shrimp with advanced F. solani infections compared to those of uninfected, unwounded control shrimp or those with early F. solani infections. Hemolymph from severely infected shrimp was hypoproteinemic, contained lower numbers of circulating hemocytes, and frequently failed to coagulate. © 1984.
• Lightner, D. V., & Redman, R. M. (1984). Intranucleolar cystalline bodies in the hepatopancreas of the blue shrimp, Penaeus stylirostris. Journal of Invertebrate Pathology, 43(2), 270-273.
• Lightner, D. V., Redman, R. M., & Bell, T. A. (1983). Infectious hypodermal and hematopoietic necrosis, a newly recognized virus disease of penaeid shrimp. Journal of Invertebrate Pathology, 42(1), 62-70.
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  PMID: 6886467;Abstract: Populations of the Pacific blue shrimp, Penaeus stylirostris, reared at the University of Arizona's experimental shrimp culture facility on Oahu in Hawaii from late 1980 through 1981, were severely affected by a highly acute and lethal disease of viral etiology. Also found to be susceptible to the disease were P. vannamei and P. monodon. The disease was named infectious hypodermal and hematopoietic necrosis (IHHN) disease to describe the principal lesions observed. The histopathology of acute and subacute IHHN disease in these species was dominated by the presence of conspicuous eosinophilic intranuclear-inclusion bodies of the Cowdry type A variety in ectodermally (especially the cuticular hypodermis) and mesodermally (especially the hematopoietic tissues) derived tissues that were undergoing necrosis. Electron microscopy of affected tissues demonstrated the presence of two or three types of virus-like particles with cubic morphology and diameters of 17 to 27 nm that suggest IHHN virus to be either a parvo- or picornavirus. © 1983.
• Lightner, D. V., Redman, R. M., & Bell, T. A. (1983). Observations on the geographic distribution, pathogenesis and morphology of the baculovirus from Penaeus monodon Fabricius. Aquaculture, 32(3-4), 209-233.
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  Abstract: A baculovirus disease of Penaeus monodon Fabricius named monodon baculovirus (MBV) was found in populations of postlarval P. monodon that originated from three separate sources in the Southwestern Pacific region. The pathogenesis, lesion development and morphology of the virus was the same regardless of the origin of the P. monodon population. The apparent target organs and tissues for MBV are the hepatopancreatic tubule and duct epithelium of postlarvae, juveniles and adults, and the anterior midgut epithelium of very young postlarvae. It was not determined if MBV produces patent disease in the larval stages of P. monodon, nor how it is transferred from brood stock to their offspring. MBV disease may cause serious losses in compromised populations (especially in postlarvae and young juveniles), but under conditions favorable to the host, fewer mortalities occurred and only low-grade infections of the hepatopancreas were detected. Three stages in the pathogenesis of MBV disease were noted in the hepatopancreas. Stage 1 hepatopancreatocytes had slightly hypertrophied nuclei, but contained no occlusion bodies (a highly specialized type of inclusion body) and few completed virions; stage 2 cell nuclei also had hypertrophied nuclei, but contained developing occlusion bodies and completed virions; stage 3 cell nuclei contained mature occulusion bodies and abundant numbers of completed free and occluded virions. Cell necrosis and cytolysis or sloughing (releasing virus and occlusion bodies into the gut lumen) followed stage 3 development in living hosts. The morphology of MBV is consistent with members of the genus Baculovirus subgroup A. The MBV particles are rod-shaped, singly enveloped, and replicate in the nucleus, occur free or within proteinaceous polyhedral occlusion bodies, and are assumed to contain DNA as the nucleic acid type. The nucleocapsid of MBV measured 42 ± 3 nm by 246 ± 15 nm, while the enveloped virions measured 75 ± 4 nm by 324 ± 33 nm. © 1983.
• Duhamel, R. C., Finerty, M., Lightner, D. V., & Brendel, K. (1982). Reexamination of the amino acid compositions of proteins from penaeid shrimp: failure to find uncommonly-high tryptophan levels. Comparative Biochemistry and Physiology -- Part B: Biochemistry and, 73(2), 301-304.
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  Abstract: 1. 1. A soluble fraction and an insoluble fraction were isolated from cuticle-free tail tissue of Penaeus vannamei and their amino acid compositions compared to analogous fractions previously isolated from Penaeus setiferus by Thompson & Thompson (Comp. Biochem. Physiol. 27, 127-132 (1968); 35, 471-477 (1970)) in which tryptophan contents of 119.3 and 190.4 residues/1000 were reported. 2. 2. The previously-reported tryptophan values are surprisingly high, because tryptophan is generally the least abundant amino acid in proteins. In this study, a maximum of 9.7 tryptophan residues/1000 were found. 3. 3. Extraction with 1 M NaCl followed by 0.5 M acetic acid solubilized 94% of the total protein but no hydroxyproline, indicating that shrimp collagen is uniformly insoluble, perhaps because of extensive cross-linking. © 1982.
• Lightner, D. V., Redman, R. M., Price, R. L., & Wiseman, M. O. (1982). Histopathology of aflatoxicosis in the marine shrimp Penaeus stylirostris and P. vannamei. Journal of Invertebrate Pathology, 40(2), 279-291.
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  PMID: 6815280;Abstract: The acute and subacute toxicity of aflatoxin B1 to the marine shrimp Penaeus stylirostris and P. vannamei (Order: Decapoda, Class: Crustacea) was investigated. Experimental shrimp were exposed to a range of concentrations of the toxin directly by intramascular injection (from 2 to 160 μg aflatoxin B1/g body weight), or by multiple per os dosing with the feed (from 53 to 300 μg aflatoxin B1/g feed) for up to 25 days. The histopathogenesis of aflatoxicosis in the aflatoxin-exposed animals was followed and found to be time and dose dependent in the hepatopancreas, mandibular organ, and in the hematopoietic organs. Less significant and/or inconsistent lesions were also observed in other organs and tissues, but a time-dose dependency was not noted. The principal lesions of aflatoxicosis in penaeid shrimp occur in the hepatopancreas and the mandibular organ. In the former organ, subacute and acute aflatoxicosis is expressed as necrosis of the hepatopancreatic tubule epithelium that proceeds from the proximal (older) portion of the tubules, in the center of the organ, to the peripheral (younger) tubule tips. A marked intertubular hemocytic inflammation followed by encapsulation and fibrosis of affected tubules follows in subacute aflatoxicosis, but is not as developed as in acute aflatoxicosis. The mandibular organ in aflatoxicosis displays a necrosis of the peripheral epithelial cells of the cords within the gland that progresses proximally to the central vein. Only a slight hemocytic inflammation accompanies the degenerative changes in this latter organ. © 1982.
• McKee, C., & Lightner, D. V. (1982). Effect of several algicides and surfactants on the filamentous bacterium Leucothrix mucor Oersted. Applied and Environmental Microbiology, 43(3), 715-718.
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  PMID: 16345979;PMCID: PMC241899;
• Williams, R. R., Hose, J. E., & Lightner, D. V. (1982). Toxicity and residue studies of cultured blue shrimp treated with the algicide Cutrine-Plus. Progressive Fish-Culturist, 44(4), 196-201.
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  Abstract: Laboratory bioassays on the blue shrimp (Penaeus stylirostris) were conducted with Cutrine-Plus, a chelated copper algicide registered for use in fish hatcheries but not for shrimp culture facilities. The 24- and 96-h LC50's for blue shrimp were found to be 151.3 and 19.5 mg/L copper as Cutrine-Plus, respectively. Cutrine-Plus was found to be safe (i.e., produced no statistical mortality) at a use rate of 1.0 mg/L for a 24-h static exposure. The 24-h EC50 for gill melanization was 0.64 mg/L copper. In residue studies no accumulation of copper in whole-body tissue due to the treatments could be shown.
• Wiseman, M. O., Price, R. L., Lightner, D. V., & Williams, R. R. (1982). Toxicity of aflatoxin B₁ to penaeid shrimp. Applied and Environmental Microbiology, 44(6), 1479-1481.
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  PMID: 6818903;PMCID: PMC242216;Abstract: Single intramuscular injections of aflatoxin B1 into the tail muscle of Penaeus stylirostris produced 24- and 96-h median lethal doses of 100.5 (78.3 to 129.0) and 49.5 (29.8 to 82.3) mg/kg, respectively. A toxicity curve showed no threshold at the levels tested. The mortality response in a feeding study with P. vannamei was not dose dependent, but tissue and organ damage were similar to that seen in injected animals.
• Lightner, D. V., & Redman, R. M. (1981). A baculovirus-caused disease of the penaeid shrimp, Penaeus monodon. Journal of Invertebrate Pathology, 38(2), 299-302.
• Danald, D. A., Ure, J., & Lightner, D. V. (1979). PRELIMINARY RESULTS OF OZONE DISINFECTION OF SEAWATER CONTAINING THE POTENTIAL SHRIMP PATHOGENS VIBRIO SP. AND FUSARIUM SOLANI.. Ozone: Science and Engineering, 1(4), 329-334.
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  Abstract: Studies to determine the feasibility of using ozone to disinfect seawater for use in controlled environment shrimp culture were run at the University of Arizona-University of Sonora Environmental Shrimp Culture Station at Puerto Penasco, Mexico. Ozone was injected into seawater containing known natural populations of Vibrio spp, and known introduced populations of Fusarium solani. Total residual oxidant (TRO) concentrations and total viable plate counts for these pathogens were made during and post ozonization. TRO at a concentration of 2. 6mg/1 was sufficient to kill greater than 99. 9% of the Vibrio spp. present in the seawater within a 5-min exposure period. Greater than 99. 9% of the introduced conodiospores of F. solani were killed within 5 minutes after exposure to approximately 2. 1 mg/1 TRO.
• Hunter, B., Magarelli Jr., P. C., Lightner, D. V., & Colvin, L. B. (1979). Ascorbic acid-dependent collagen formation in penaeid shrimp. Comparative Biochemistry and Physiology -- Part B: Biochemistry and, 64(4), 381-385.
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  PMID: 233793;Abstract: 1. 1. This study tested the hypothesis that black death, the ascorbic acid (AsA) related disease of penaeid shrimp, is related to collagen underhydroxylation. 2. 2. Collagen measured as hydroxyproline (HYP) in healthy Penaeus californiensis (Holmes) and P. stylirostris (Stimpson) of a wide range of masses was determined. The results revealed a logarithmic relationship between total body collagen HYP and body weight fitting the equation y = 90×1.18 where y = total collagenous HYP (μg) and x = body weight (g). 3. 3. Shrimp tissues most subject to mechanical trauma (subcutis, hindgut and gills) had the highest collagenous HYP levels and were most consistently and severely affected by an ascorbic acid (AsA) deficiency disease. 4. 4. Prolyl hydroxylase (PH) activity was demonstrated in tissues of P. californiensis and P. stylirostris by hydroxylation of [3,4-3H]proline. 5. 5. AsA was required for shrimp PH activity using a chicken embryo substrate. 6. 6. Nutritional trials revealed that dietary AsA was required for proline hydroxylation in collagen formation in P. californiensis. © 1979.
• Magarelli Jr., P. C., Hunter, B., Lightner, D. V., & Colvin, L. B. (1979). Black death: an ascorbic acid deficiency disease in penaeid shrimp. Comparative Biochemistry and Physiology -- Part A: Physiology, 63(1), 103-108.
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  Abstract: 1. 1. The physiological requirement for ascorbic acid (AsA) has been established for two species of penaeid shrimp: Penaeus californiensis (Holmes) and Penaeus stylirostris (Stimpson). 2. 2. The AsA deficiency disease syndrome, called black death disease (BD), has been characterized by melanized lesions in the loose connective tissue under the exoskeleton, on the abdomen, on the carapace, in the gills, and in the foregut and hindgut. 3. 3. Tissue levels of AsA showed a positive correlation with intake of AsA and with change in survival and negatively correlated with occurrence of BD. 4. 4. Experiments demonstrated a statistical advantage (P < 0.05) in growth and survival in P. californiensis and for growth in P. stylirostris fed diets containing AsA. © 1979.
• Fisher, W. S., Nilson, E. H., Steenbergen, J., & Lightner, D. V. (1978). Microbial diseases of cultured lobsters: A review. Aquaculture, 14(2), 115-140.
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  Abstract: Six microbial diseases of cultured American lobsters (Homarus americanus) determined to be of significant concern to commercial aquaculture are discussed in relation to present knowledge of characteristics of the causative microorganisms, lobster susceptibility, environmental influence, severity, gross and microscopic recognition, physiological changes, contagion and control. The diseases reviewed here include shell disease, Gaffkemia, microbial epibiont disease, Lagenidium disease, Haliphthoros disease, and Fusarium disease. © 1978.
• Lightner, D. V. (1978). Possible toxic effects of the marine blue-green alga, Spirulina subsalsa, on the blue shrimp, Penaeus stylirostris. Journal of Invertebrate Pathology, 32(2), 139-150.
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  PMID: 103972;Abstract: Blooms of a marine species of blue-green algae identified as Spirulina subsalsa (Cyanophyta, Oscillatoriacae) were found to be related to a particular disease syndrome in raceway-reared blue shrimp, Penaeus stylirostris. The disease was characterized by necrosis of the lining epithelium of the midgut, dorsal cecum, and hindgut gland, and a consequent hemocytic enteritis. Bacterial infections due predominately to Vibrio alginolyticus were common in affected shrimp and presumed to be a secondary condition resulting from necrosis of the gut epithelium. These bacterial infections were expressed as local abscesses near or on the gut or as fulminating septicemias. © 1978.
• Corliss, J., Lightner, D., & Zein-Eldin, Z. P. (1977). Some effects of oral doses of oxytetracycline on growth, survival and disease in Penaeus aztecus. Aquaculture, 11(4), 355-362.
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  Abstract: Two size groups of brown shrimp (Penaeus aztecus) were fed a formulated feed containing 0, 100, 1 000 or 5 000 mg of oxytetracycline/kg of feed. Growth and survival were measured after a 3-week drug consumption period. The shrimp were then inoculated with Vibrio alginolyticus, and survival was monitored during the following 24 h. At all three concentrations of oxytetracycline, small shrimp (mean initial wet weight 143.4 mg) consumed approximately one-third the amount of feed consumed by those fed the control diet with no oxytetracycline, yet growth was more rapid with diets containing 100 and 1 000 mg of oxytetracycline/kg of food than with the control diet. Larger shrimp (mean initial wet weight 458.1 mg) receiving oxytetracycline consumed about one-fourth the feed consumed by those on the oxytetracycline-free diet. Some growth inhibition was apparent in these shrimp at all oxytetracycline concentrations. Maximum drug consumption rate, based on actual feed intake, was approximately 1 300 mg oxytetracycline per kg body weight per day for small shrimp, but only 370 mg per kg body weight per day for larger shrimp. All shrimp fed 0, 100 or 1 000 mg of oxytetracycline/kg of feed died within 24 h following inoculation with a standard dose (70% light transmission at 520-540 nm) of Vibrio alginolyticus. All the small shrimp and 70% of the large shrimp fed at the 5 000-mg drug level died, but death generally took place later in the 24-h period than with those fed at the lower drug concentrations. All shrimp fed 5 000 mg oxytetracycline/kg of feed and inoculated with a 1 : 100 dilution of the standard dose of Vibrio alginolyticus survived. All small shrimp and 90% of the large shrimp survived injection of sterile saline. © 1977.
• Lightner, D. V., & Redman, R. (1977). Histochemical demonstration of melanin in cellular inflammatory processes of penaeid shrimp. Journal of Invertebrate Pathology, 30(3), 298-302.
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  Abstract: Brown-to-black pigment deposits present in association with sites of hemocytic activity in penaeid shrimp (Crustacea, Decapoda) were demonstrated to be melanin by use of histochemical techniques. The brown-black pigment was associated with cellular inflammatory disease processes of infectious and noninfectious etiologies in Penaeus californiensis, P. stylirostris, P. vannamei, and P. duorarum. © 1977.
• Solangi, M. A., & Lightner, D. V. (1976). Cellular inflammatory response of Penaeus aztecus and P. setiferus to the pathogenic fungus, Fusarium sp., isolated from the California brown shrimp, P. californiensis. Journal of Invertebrate Pathology, 27(1), 77-86.
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  Abstract: Host response of two penaeid species, Penaeus aztecus and P. setiferus, from the Gulf of Mexico to the pathogenic fungus Fusarium sp. isolated from the California brown shrimp, P. californiensis, was studied in vivo. The hemocytic response to this fungus was traced histologically in the gills. Both species showed complete resistance to infection by the fungal spores when normal or wounded shrimp were held in seawater containing the spores or when spores were injected directly into the shrimp in low concentrations. Complete melanization and encapsulation of the micro- and macroconidia were observed. Spore dosages of 3.2 × 106 or more were lethal, apparently due to mechanical blockage of the blood sinuses of the gills. © 1976.
• Fontaine, C. T., Bruss, R. G., Sanderson, I. A., & Lightner, D. V. (1975). Histopathological response to turpentine in the white shrimp, Penaeus setiferus. Journal of Invertebrate Pathology, 25(3), 321-330.
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  Abstract: Observations are presented on the inflammatory response to a highly irritative substance, turpentine, injected into the abdominal musculature of the white shrimp, Penaeus setiferus. Injections of the irritant were administered with a tuberculin syringe between the fifth and sixth segments. Penaeid shrimp were found to be highly sensitive to turpentine, even when administered in small dosages. When sterile petroleum jelly was mixed with the turpentine to reduce the dispersion rate, the shrimp's "internal defense mechanism" was able to combat effectively the effect of the irritant. Postinjection observations of the tissues at the site of injection, gill, heart, and hepatopancreas were made at 8, 16, 24, 32, 40, 48, 72, 96, 120, 168, and 240 hr, and at 15, 20, 30, 40, 50, 60, and 120 days. The induced cellular inflammatory response consisted of infiltrating hemocytes and fibrocytes resulting in the formation of fibrous capsules, brown melanized nodules, and fibrous scar tissue in all tissues examined. The gills and hepatopancreas showed considerable tissue destruction early, but were eventually cleared of the histopathological effects of the turpentine and later appeared normal. However, extensive tissue destruction was easily distinguishable in the heart and abdominal muscle even at 120 days postinjection. © 1975.
• Lightner, D. V., & Fontaine, C. T. (1975). A mycosis of the American lobster, Homarus americanus, caused by Fusarium sp. Journal of Invertebrate Pathology, 25(2), 239-245.
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  PMID: 1167883;Abstract: A mycosis in cultured American lobsters, Homarus americanus, is described. The causative agent, a pigment-producing Fusarium sp., was isolated from diseased lobsters from an experimental "lobster farm" in New York. Affected lobsters had "black spots" of various sizes on the exoskeleton and appendages and brownish discoloration of the gills. Hyphae and conidia of the Fusarium sp. were present in or on these lesions. © 1975.
• Fontaine, C. T., & Lightner, D. V. (1974). Observations on the phagocytosis and elimination of carmine particles injected into the abdominal musculature of the white shrimp, Penaeus setiferus. Journal of Invertebrate Pathology, 24(2), 141-148.
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  PMID: 4414874;Abstract: White shrimp, Penaeus setiferus, were injected in the abdominal musculature with 0.03 ml of a 1.4% carmine-saline solution and were kept at a temperature of 26°-28°C and a salinity of 23-26‰. Tissue samples were taken at hourly intervals of 1, 6, 12, 18, 24, 30, 36, 42, and 72 hr and at 4, 6, 8, 10, 12, 16, 19, and 33 days post-injection and were examined histologically to determine the sites of phagocytosis and elimination of foreign particulate matter. Within 1 hr post-injection, extracellular clumps of the carmine particles were formed in the hemolymph. These clumps had been invaded by hemocytes at 18 hr, but they persisted throughout the study. Phagocytosis of the particles was accomplished by hemocytes circulating in the hemolymph and by fixed phagocytes in the gill, heart, loose connective tissue, and blood sinusoids in the abdomen. The fate of some phagocytized carmine was climination by the migration of hemocytes through the epithelium of the gills, gut, hepatopancreas, and through the extremities of the pereiopods and the pleopods. Encapsulations or brown nodules were formed in the musculature of the pereiopods around necrotic hemocytes that had phagocytized carmine. A large blister or cyst filled with carmine was formed in the gill cover of one specimen. The obsevation of carmine particles at 33 days post-injection indicates a slow clearance rate of large amounts of abiotic particulate matter in penaeid shrimp. © 1974.
• Lightner, D. V. (1974). Case reports of ossifying fibromata in the striped mullet.. Journal of wildlife diseases, 10(4), 317-320.
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  PMID: 4215891;
• Lightner, D. V., Salser, B. R., & Wheeler, R. S. (1974). Gas-bubble disease in the brown shrimp (Penaeus aztecus). Aquaculture, 4(C), 81-84.
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  Abstract: Gas-bubble disease in larval and juvenile brown shrimp (Penaeus aztecus) is described. Stage II protozoeal, larval brown shrimp developed the disease after being placed in water warmed in a closed heater that did not allow excess gas to escape. Juvenile brown shrimp developed gas-bubble disease after being held in a pressure tank for 35 h. The pressure in the tank had been maintained at 2 585 mm Hg with compressed air. The implications of the finding that shrimp are susceptible to gas-bubble disease are discussed in relation to shrimp culture activities, particularly those that use heated water. © 1974.
• Fontaine, C. T., & Lightner, D. V. (1973). Observations on the process of wound repair in penaeid shrimp. Journal of Invertebrate Pathology, 22(1), 23-33.
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  Abstract: The Petersen disk tag is a standard mark for penaeid shrimp, and attachment of the tag involves the insertion of a stainless steel pin through the shrimp's abdomen, resulting in a relatively large puncture wound. The wound healing process first observed at 24 hr post-tagging showed a pronounced hemocytic infiltration of the wound area. Hemocytes in contact with the pin became fusiform, began adhering to one another, and formed several concentric layers around the pin. Scattered foci of bacteria or nercrotic tissue in the vicinity of the would also became encapsulated by concentric layers of fusiform hemocytes, thereby forming nodules. Melanin appeared in association with the layers of hemocytes nearest the pin and in the nodules. Hemocytic infiltration was lollowed by the appearance of fibrocytes and the deposition of collagenlike fibers along the would channel 48 hr after wounding. Involution of epidermis and consequential cuticular involution into the would channel began at 96 hr after wounding. Complete epidermal and cuticular formation along the wound occurrd by 384 hr post-tagging. © 1973.
• Lightner, D. V., & Fontaine, C. T. (1973). A new fungus disease of the white shrimp Penaeus setiferus. Journal of Invertebrate Pathology, 22(1), 94-99.
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  PMID: 4719278;Abstract: A primary mycosis of larvae of the white shrimp, Penaeus setiferus, is described. The disease first became apparent in larvae in the second protozoeal stage and disappeared as the shrimp reached the first mysis stage. Affected shrimp became immobilized by near complete tissue destruction and replacement by the expanding mycelium. The fungus was found to be Lagenidium sp. and was infective to larval brown shrimp, Penaeus aztecus. © 1973.
• Sparks, A. K., & Lightner, D. V. (1973). A tumorlike papilliform growth in the brown shrimp (Penaeus aztecus). Journal of Invertebrate Pathology, 22(2), 203-212.
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  PMID: 4784765;Abstract: A brown shrimp, Penaeus aztecus, from an experimental pond of the Texas Parks and Wildlife Department's Marine Laboratory at Palacios, Texas, possessed a papilliform tumorlike growth on the right ventrolateral aspect of the sixth abdominal segment. Gross and histological study revealed the lesion to be confluent with the carapace, epidermis, and subepidermal connective tissue. The growth, tentatively diagnosed as a benign neoplasm, consists of grossly hypertrophied and normal tissue elements. © 1973.

Proceedings Publications

• Reantaso, M. B., & Lightner, D. V. (2013, June 25-27). FAO.2013. Report of the FAO/MARD Technical Workshop on Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Syndrome (AHPNS) of Cultured Shrimp (under TCP/VIE/3304). Hanoi, Vietnam. In FAO sponsored conference in Hanoi.

Presentations

• Lightner, D. V., Redman, R. M., Pantoja, C. R., Noble, B. L., Nunan, L. M., & Tran, L. (2013, June 25-27). Documentation of an Emerging Disease (Early Mortality Syndrome) in SE Asia and Mexico. FAO and Ministry of Agriculture and Rural Development (MARD) sponsored conference. Hanoi, Vietnam: FAO and MARD, Hanoi, Vietnam.
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  I presented a ~30 minute Power Point on the EMS situation in SE Asia and Mexico.
• Lightner, D. (2012, August). Characterization, distribution and case definition of EMS/AHPNS. Asia Pacific Emergency Regional Consultation on Shrimp Early Mortality Syndrome (EMS)/Acute Hepatopancreatic Necrosis Syndrome (AHPNS). Bangkok, Thailand.
• Lightner, D. (2012, March). Documentation of a New Disease (Early Mortality Syndrome) in South China & Vietnam in 2010 and 2011. Aquaculture America 2012. Las Vegas, NV.
• Lightner, D. (2012, May). New genotype of WSSV from the Kingdom of Saudi Arabia, Mozambique and Madagascar. Antananarivo, Madagascar.
• Lightner, D. (2012, November). Development and Spread of a New Shrimp Disease (Early Mortality Syndrome) in SE Asia. Hanoi, Vietnam: Food and Agricultural Organization of the United Nations and Ministry of Agriculture and Rural Development, Vietnam.
• Lightner, D. (2012, November). Major Virus Diseases Affecting Penaeid Shrimp with an Emphasis on WSSV. St. Louis, MO, USA: Monsanto.
• Lightner, D. (2012, October). Development and Spread of a New Shrimp Disease (Early Mortality Syndrome) in SE Asia. Global Aquaculture Advocate, Global Outlook for Aquaculture Leadership (GOAL). Bangkok, Thailand.
• Lightner, D. (2012, September). Current Status of White Spot Disease (Caused by WSSV) & its Effect in Shrimp Culture. CESAIBC. Ensenada, Mexico.
• Lightner, D. V. (2012, June). 2012 Shrimp Pathology Short Course. Shrimp Pathology Short Course. Tucson, AZ.
• Lightner, D. (2011, March). IHHN disease and strain divergence of its viral agent. Aquaculture America. San Diego, CA.
• Lightner, D. V. (2011, August). Global transboundary disease politics: the OIE perspective. OECD Symposium - Disease in Aquatic Crustaceans: Problems and Solutions for Global Food Security. Halifax, Canada: OECD.
• Lightner, D. V. (2011, August). Historic emergence, impact and current status of shrimp pathogens in the Americas. OECD Symposium - Disease in Aquatic Crustaceans: Problems and Solutions for Global Food Security. Halifax, Canada: OECD.
• Lightner, D. V. (2011, July). Presentations on IMN and other significant diseases in the region. Workshop on IMNV. Las Vegas, NV: Fisheries Department and Shrimp Club of Indonesia.
• Lightner, D. V. (2011, June). 2011 Shrimp Pathology Short Course. 2011 Shrimp Pathology Short Course. Tucson, AZ.
• Lightner, D. V. (2011, June). Global health issues - crustaceans. OIE Global Conference on Aquatic Animal Health Progremmes: Their Benefits for Global Food Security. Pnamama City, Panama: OIE.
• Lightner, D. V. (2011, November). Disease management through the use of specific pathogen-free stocks, biosecurity, molecular diagnositc methods and best management practices. El Boom de la Innovacion tecnologica en la industria acuicola, V Simposio Nicovita. Tegucigalpa, Honduras.
• Lightner, D. V. (2011, October). 30 day mortality syndrome (acute hepatopancreatic degeneration syndrome, AHPDS). XIII Congreso Ecuatoriano de Acuiculture & Aquaexpo. Guayaquil, Ecuador.
• Lightner, D. V. (2011, October). Infectious Myonecrosis: Its status as an OIE-listed disease and its transfer from Brazil to Indonesia. X Simposio Internacional de Camaron de Cultivo y Exhibicion Comercial. Las Vegas, NV.
• Lightner, D. V. (2011, October). Panamanian breeding program produces shrimp families with resistance to WSSV. XIII Congreso Ecuatoriano de Acuiculture & Aquaexpo. Guayaquil, Ecuador.

Others

• Aranguren, L. F., Tang, K., & Lightner, D. V. (2013, March/April). Study: TSV Exposure May Lessen YHV Effects In White Shrimp.. Global Aquaculture Advocate.
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  Aranguren, L.F., K. Tang and D.V. Lightner. 2013. Study: TSV Exposure May Lessen YHV Effects in White Shrimp. Global Aquaculture Advocate March/April. pp 84-87.
• Tran, L., Nunan, L., Redman, R., Lightner, D. V., & Fitzsimmons, K. (2013, July/August). EMS/AHPNS: Infectious Disease Caused By Bacteria. Global Aquaculture Advocate.
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  Tran, L., L.M. Nunan, R.M. Redman, D.V. Lightner and K. Fitzsimmons. 2013. EMS/AHPNS: Infectious Disease Caused By Bacteria. Global Aquaculture Advocate July/August, pp. 16-18.
• Lightner, D. (2012, Fall). New disease in SE Asia. Panorama Acuicola Magazine.
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  Una nueva enfermedad en camaron.
• Lightner, D. V., & Tang, K. (2012, May/June). New WSSV & TSV genotypes in KSA. Global Aquaculture Advocate.
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  Pages 20-21
• Wyban, J., Noble, B., & Lightner, D. (2012, July/August). SPF line testing for IMNV and TSV resistance. Aquaculture Asia Pacific.
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  Volume 8, Issue 4
• , J., Chamorro, R., White-Noble, B., Schofield, P., & Lightner, D. (2011, Fall). White shrimp Litopenaeus vannamei significantly resistant against WSSV.
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  Volume: 16, Issue 5, pgs 46-48
• , J., Chamorro, R., White-Noble, B., Schofield, P., & Lightner, D. (2011, July/August). Testing finds resistance to WSSV in shrimp from Panamanian breeding program. Global Aquaculture Advocate.
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  Pages: 65-66
• , J., White-Noble, B., Schofield, P., Chamorro, R., & Lightner, D. (2011, November/December). Selected families of white shrimp Litopenaeus vannamei from Panama breeding program show important resistance to WSSV. AQUA Culture Asia Pacific Magazine.
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  Pages: 36-37
• Arce, S., Moss, S., & Lightner, D. (2011, May/June). Biosecurity principles for sustainable production using SPF shrimp. Global Aquaculture Advocate.
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  Pages: 14-16
• Lightner, D. (2011, Fall).
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  Volume: 16, Issue 4, pgs 8-20
• Nunan, L., & Lightner, D. (2011, January/February). One-step PCR for white spot syndrome detection. Global Aquaculture Advocat.
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  Page: 74
• Pantoja, C., Navarro, S., & Lightner, D. (2011, May/June). Ring test compare PCR results from diagnostic laboratories. Global Aquaculture Advocate.
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  Pages: 36-37