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D V Lightner

Publications and source records attributed to D V Lightner.

At least 19 recordsLinked to original sources
PubMed1999

A yellow head virus gene probe: nucleotide sequence and application for in situ hybridization.

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.

Animals↗
PubMed1998

Reverse transcription polymerase chain reaction (RT-PCR) used for the detection of Taura syndrome virus (TSV) in experimentally infected shrimp.

Taura Syndrome Virus (TSV) has adversely affected the shrimp culture industries of the Americas. First recognized in 1992, this viral 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.

Animals↗
PubMed1998

Taura syndrome of penaeid shrimp: cloning of viral genome fragments and development of specific gene probes.

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.

Animals↗
PubMed1998

Differentiation of BP-type baculovirus strains using in situ hybridization.

BP-type viruses infect wild and farmed 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.

Americas↗
PubMed1997

Development of a non-radioactive gene probe by PCR for detection of white spot syndrome virus (WSSV).

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.

Animal Diseases↗
PubMed1997

A new RNA-friendly fixative for the preservation of penaeid shrimp samples for virological detection using cDNA genomic probes.

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 approximately 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 approximately 6.0-7.0), RNA-friendly fixative (R-F) that was developed by the authors. In situ hybridization assays were conducted with and without R Nase 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.

Animals↗
PubMed1997

Taura syndrome of marine penaeid shrimp: characterization of the viral agent.

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 Picornaviridae 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.

Animals↗
PubMed1997

Risk of spread of penaeid shrimp viruses in the Americas by the international movement of live and frozen shrimp.

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.

Americas↗
PubMed1996

Epizootiology, distribution and the impact on international trade of two penaeid shrimp viruses in the Americas.

Marine penaeid shrimp are effected by approximately 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.

Animals↗
PubMed1995

Characterization of hepatopancreatic parvo-like virus, a second unusual parvovirus pathogenic for penaeid shrimps.

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.

Animals↗
PubMed1994

Antimicrobials in shrimp aquaculture in the United States: regulatory status and safety concerns.

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)

Animals↗
PubMed1994

Use of non-radioactively labeled DNA probes for the detection of a baculovirus from Penaeus monodon by in situ hybridization on fixed tissue.

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).

Animals↗
PubMed1993

Epidemic misuse.

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Animals↗
PubMed1992

Performance of an aquatic multispecies system in evaluating the effects of a model microbial pest control agent on nontarget organisms.

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 non-target 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.

Animals↗
PubMed1991

Methods for viral isolation and DNA extraction for a penaeid shrimp baculovirus.

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.

Animals↗
PubMed1990

Purification and characterization of the infectious hypodermal and haematopoietic necrosis virus of penaeid shrimps.

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 Mrs of 74K, 47K, 39K and 37.5K, respectively. From its characteristics, this virus could be a member of the Parvoviridae family.

Animals↗