1,720,987 research outputs found
Geographic variations among infectious hypodermal and hematopoietic necrosis virus (IHHNV) isolates and characteristics of their infection
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Application of recombinant techniques for detection of the penaeid shrimp baculovirus, BP
No full textResearch was conducted to develop gene probes to Baculovirus penaei (BP) from a cloning of BP genomic DNA and to test the applications of the gene probes. Five non-homologous probes representing >20% of the total BP genome were tested in dot blot and in situ hybridization assays. Four different dot blot hybridization assay systems were evaluated. Results obtained from radioactive labeling of the probes indicate that the probes are specific for BP DNA, while the results obtained from the three non-radioactive labeling systems suggest problems with non-specific reactivity and sample preparation for these methods. The results also indicated that in situ hybridization is presently the optimum assaying system for BP gene probes. The BP gene probes were used with an in situ hybridization assay to gather initial data concerning the mode of transmission for BP
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Taura syndrome of marine penaeid shrimp: Discovery of the viral agent and disease characterization studies
No full textThese studies were begun during the summer of 1994, W2 years after the recognition of Taura Syndrome (TS), a lethal disease of farm-raised Penaeus vannamei in Ecuador of then unknown etiology. Utilizing specific pathogen free (SPF) P. vannamei test shrimp, the combined results of four initial TS bioassays suggested that the disease had an infectious instead of a toxic etiology as originally reported. The hypothesis that TS was virus-caused prompted the application of a shrimp parvovirus (IHHNV) purification protocol, which resulted in the discovery of Taura Syndrome virus (TSV), a previously unrecognized virus that was isolated from TS diseased shrimp tissues. Three serial infectivity studies were performed in which purified cell-free extracts of TSV were injected into SPF P. vannamei test shrimp and the criteria of Rivers' postulates were fulfilled, establishing that TS has a viral etiology. In situ hybridization assays for the detection of TSV resulted in frequent false negative gene probe results. This problem was due to fixative-induced acid hydrolysis of the TSV RNA genome resulting from tissue fixation with Davidson's solution (pH 3.5-4). Development and use of a neutral fixative, R-F (RNA-Friendly) fixative, was shown to prevent this problem. The pathogenesis of TSV lesions was analyzed in experimentally infected, time course sampled SPF P. vannamei. The TSV disease cycle was found to consist of three clinically and histologically distinct overlapping phases; an W7 d acute phase, an W5 d transition phase and a long term cyclic chronic phase of at least 8 months duration. TSV susceptibility studies of two endemic North American (P. setiferus and P. aztecus) and one Asian penaeid species (P. chinensis) showed that P. aztecus and P. chinensis juveniles were susceptible, whereas, P. setiferus appeared refractory to TSV infection. The geographic range of TSV within the Americas was documented based on gene probe and histological findings of archived P. vannamei samples dating from 1992 to 1996. TSV was detectable within P. vannamei submitted from Ecuador when the disease was first recognized (1992) and has since spread into 12 other countries. The causes and effects of the international controversy of a viral versus a toxic etiology for TS are discussed and a solution offered to prevent similar disputes in the future.This item was digitized from a paper original and/or a microfilm copy. If you need higher-resolution images for any content in this item, please contact us at [email protected]
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Characterization of white spot syndrome virus of penaeid shrimp: Genomic cloning and sequencing, structural protein analyzing and sequencing, genetic diversity, pathology and virulence
No full textThe purpose of this dissertation was to characterize virulence, genomic and protein composition of a newly emerged virus of penaeid shrimp: white spot syndrome virus (WSSV). A partial genomic library, covering approximately 30-50% of the genome, of WSSV isolated from crayfish Orconectes punctimanus, was constructed by digesting viral DNA with endonuclease ClaI and cloning into the system pBluescript-JM109. Three viral inserts of approximately 2.2 kb, 2.8 kb and 6.3 kb, named as QW245, CR44, and QW237 respectively, were sequenced and analyzed. Six geographic isolates of WSSV, from China, India, Thailand, South Carolina, Texas, as well as from crayfish obtained from the US National Zoo in Washington D.C. were compared by electron microscopy (TEM) and SDS-PAGE. All viral isolates contained three major polypeptides of 25, 23 and 19 kDa. A fourth major polypeptide at the 14.5 kDa position was observed in four of the viral isolates. The 19 kDa polypeptide of the crayfish WSSV appeared larger in size than that of the other isolates. Amino acid composition of four of the major structural polypeptides of the South Carolina WSSV was analyzed. The NH₂ terminal amino acids of the 25, 23 and 14.5 kDa polypeptides of the SC WSSV were sequenced as MDLSFTLSVVTA, MEFGNLTNLDVA, and VARGGKTKGRRG, respectively. The genomic composition of the six geographic isolates of WSSV were compared by combining the methods of restriction analysis using nine endonucleases AccI, BglII, ClaI, BamHI, EcoRI, HindII, HaeI, SacI, XhoI and Southern blot hybridization applying three digoxigenin-11-dUTP labeled WSSV genomic probes LN4, C42 and A6. No distinctive difference among five WSSV isolated from penaeid shrimp was detected; differences were observed in the crayfish isolate of WSSV. The virulence of the six geographic isolates of WSSV were compared by per os challenge of Litopenaeus vannamei postlarvae and juveniles, and Farfantepenaeus duorarum juveniles. The Texas WSSV caused higher and more rapid mortalities; the crayfish WSSV caused lower and less rapid mortalities. L. vannamei postlarvae and juveniles were very susceptible to WSSV infection, while Fa. duorarum juveniles showed moderate resistance.This item was digitized from a paper original and/or a microfilm copy. If you need higher-resolution images for any content in this item, please contact us at [email protected] file replaced with corrected file September 2023
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Morphologic and genetic characterization of wild populations of shrimp of the genus Penaeus within the Gulf of California, Mexico: New social, political, and management dilemmas for the Mexican shrimp fishery
No full textSpecies are often composed of discrete breeding units called populations, demes, or stocks. Each stock, while not reproductively isolated from other conspecific populations, may have limited opportunities to interbreed with others due to geographic separation. Allopatric stocks are favored, fortuitously arise, or become extinct, through differential selective pressures (including fishing and disease) acting either locally or throughout the geographic range of a species. Usually, these selection processes result in genetic and morphological dissimilarities between stocks. Whereas changes in the genotype are commonly reflected in the morphology of the individual, shifts in morphology do not always result from alterations of the genotype; morphological change can be induced by environmental factors (phenotypic plasticity). Therefore, it is essential to combine morphological with genetic analyses in studies of wild population identification. The goals of my research were to identify and characterize morphological stocks of Penaeus stylirostris and P. vannamei in the Gulf of California, and to conduct genetic analysis on the identified morphotypes of P. stylirostris to confirm their population structure. Seventy-eight variables were obtained from 417 specimens of P. stylirostris (representative of 21 fishing grounds) and 218 P. vannamei (representing 14 fishing grounds) and analyzed by principal component and canonical variate analyses. Morphometric analysis revealed three distinct regional stocks of P. vannamei and five of P. stylirostris. Genetic structure of P. stylirostris populations was tested from 78 samples (representing six fishing grounds in the Gulf) of male total genomic DNA extracted from frozen shrimp tails. The DNA samples were adjusted in distilled water to a final concentration of 10 ng/mul, and amplified with 20 random 10-mer primers (Operon) on a Perkin Elmer Thermocycler. The PCR RAPD profiles were used to identify between stock genetic differences. The novel adoption of sequencing gels to analyze RAPD profiles, proved useful in demonstrating (significant) genetic differences between specimens from all six fishing grounds. My research demonstrates the importance and applicability of combining morphological and genetic analyses in studies of wild stock identification. The relevance of my findings to the management of the Mexican shrimp fishery and the aquaculture industry are thoroughly discussed.This item was digitized from a paper original and/or a microfilm copy. If you need higher-resolution images for any content in this item, please contact us at [email protected] file replaced with corrected file April 2023
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Hepatopancreatic parvovirus of penaeid shrimp (HPV): Partial cloning and genome characterization, in situ hybridizationat the ultrastructural level, geographic diversity and non-invasive detection
No full textThe genome of a Korean isolate of Hepatopancreatic parvovirus (HPV) was partially cloned, sequenced and characterized. Random PCR amplification of viral DNA was combined with conventional cloning methods to generate three clones named HPV8 (2,136 bp insert), HPV3 (1,560 bp insert), and CP1139 (413 bp insert). DNA sequencing demonstrated overlapping regions between HPV8 and HPV3 and between HPV3 and CPII39. The combined sequence of these three clones encompass approximately 3,350 bp of the total 5,000 bp estimated for the HPV genome. A large open reading frame (1,692 bp) was found within clones HPV3/CPII39 encoding a polypeptide of 549 residues (∼60 kDa) whose amino terminus shows 100% homology with the first 12 residues sequenced from an apparently single 54 kDa (by SDS-PAGE) structural protein found in a Korean isolate of HPV. Two new gene probes EC.592 (592 bp) and EC.350 (350 bp) were developed by PCR amplification of previously constructed HPV (Korean) clones and labeled with DIG11-dUTP. These probes recognize different regions of the HPV genome. The specificity of both probes was confirmed by in situ hybridization using HPV-infected Penaeus chinensis (Korean) as a positive control and specific-pathogen free P. vannamei and IHHNV-infected P. stylirostris, as negative controls. Both probes were used in in situ hybridization to compare their reaction to HPV-type lesions detected by conventional H&E histology in 7 species of HPV-infected shrimp, and one of freshwater prawn, from 13 countries. The results of this comparison strongly suggest the existence of genomic differences among these geographic isolates. A post-embedding in situ hybridization assay at the electron microscope level was developed to detect HPV nucleic acids on HPV-infected hepatopancreata from P. chinensis and P. monodon . Hybridized probe (EC.592) was detected with an anti-DIG sheep antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal was observed within HPV-infected hepatopancreatic cells, which was specifically located within intranuclear viral inclusions, cytoplasm, microvillous border, and associated to necrotic debris within the lumen of hepatopancreatic tubules. A non-destructive method, based on the PCR, was developed to detect HPV by examination of crude fecal samples from HPV-infected shrimp.This item was digitized from a paper original and/or a microfilm copy. If you need higher-resolution images for any content in this item, please contact us at [email protected] file replaced with corrected file August 2023
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Characterization of Taura syndrome virus (TSV) isolates from Penaeid shrimp: Pathology, virulence, structural protein analysis and genetic diversity, and, Development of the aquaculture pathology diagnostic laboratory database
No full textIn the research reported here, the pathology, virulence, and strain differences of Taura syndrome virus (TSV) was studied. Initial studies on TSV pathogenesis compared the survival of juveniles of a highly Taura syndrome (TS) susceptible line of Penaeus vannamei, a line of TS resistant P. vannamei, and an innately TS resistant P. stylirostris line following TSV challenge by feeding (per os) or injection methods, in the absence of horizontal transmission via cannibalism and/or absorption from the water. Per os_TSV challenge resulted in I00% survival in P. stylirostris, but challenge by per os exposure produced significant mortality commencing on about the same post-exposure day in both SPF and SPR P. vannamei (P < 0.001), suggesting that P. stylirostris is significantly (P < 0.001) more resistant toper os TSV infection and presentation of TS disease than either SPF or SPR P. vannamei. The potential roles of the cuticular lining of the stomach and hindgut and unlined portions of the gut in TSV resistance in penaeid shrimp are discussed as factors where an innate resistance mechanism was postulated to explain the observed differences between the different species and populations of shrimp in TSV susceptibility. To investigate apparent TSV strain differences, three 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) and one TSV isolate from Belize, Central America (BLZ02TSV from P. vannamei) were analyzed and compared to the reference TSV isolate (HI94TSV) by selected TSV diagnostic and genetic analysis methods. The results show that screening of penaeid shrimp broodstock and postlarvae by MAb I Al testing will not detect all TSV isolates, possibly leading to false negative results, further spread of TSV and re-emergence of TS in regions where it has been eradicated. The putative VP1 antigenic epitope recognized by TSV MAb 1A1 is identified, with SIN98TSV and BLZ02TSV having 70.0% and 80.0% AA homology, respectively, within the 10 AA region. There are three distinct electropherotypes and 'serotypes' of TSV, with electropherotype A (TSV Etype-A) and serotype A (TSV-A) representing those TSV isolates conforming to VP1 properties of the Hawaiian 1994 TSV isolate, electropherotype B (TSV Etype-B) and serotype B (TSV-B) representing those TSV isolates conforming to the VP1 properties of the Sinaloan 1998 TSV isolate, and electropherotype C (TSV Etype-C) and TSV serotype C (TSV-C), representing those TSV isolates conforming to the VP1 properties of the Belize 2002 TSV isolate. In a parallel activity, the University of Arizona (UAZ) Aquaculture Pathology Diagnostic Laboratory (APL) Case Database (DB) and the UAZ Aquaculture Pathology Diagnostic Laboratory Client Address Book Database (AB), relational databases, were created using FileMaker Pro software, are used to keep an up to date and accurate record of all UAZAPL diagnostic and research case and client information and may be searched and sorted to find case data and/or client information of interest
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Development of in vitro primary cell cultures from the penaeid shrimp, Penaeus stylirostris and Penaeus vannamei and evaluation of a potential application.
No full textStudies have been carried out to improve the methods for producing primary cell cultures from penaeid shrimp and to make these procedures routine and practical. Using the methods developed, monolayers of primary cultures of ovarian epithelioid cells from Penaeus stylirostris and P. vannamei were routinely obtained with 60 to 80% confluences within a two day period. A variety of commercially available synthetic tissue culture media and media supplements were tested. It was found that Grace's Insect Medium supplemented with hybridoma quality fetal bovine serum provided the best results. Different culture conditions were tested and it was found that the optimum conditions for shrimp cell growth was a 25-28\sp\circC temperature range with a normal atmosphere. Efforts to infect these primary shrimp ovarian cell cultures from P. stylirostris with infectious hypodermal and hematopoietic necrosis virus (IHHNV) provided inconclusive results
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Histopathological, biological and molecular characteristics of the pathogenic Spiroplasma penaei isolated from the hemolymph of infected Pacific white shrimp, Penaeus vannamei
No full textBiological and molecular characteristics of the pathogenic mollicute, Spiroplasma penaei, isolated from the hemolymph of infected Pacific white shrimp, Penaeus vannamei, were investigated. The doubling times of a S. penaei were 6.13 h (2% NaCl) and 3.43 h (no salt) under aerobic conditions, and 6.63 h (2% NaCl) and 3.22 h (no salt) under anaerobic conditions. Small diffuse white colonies with granular centers, surrounded by small satellite colonies that appeared embedded in the agar matrix, were detected on solid M1D medium (2% Noble agar) under aerobic conditions at 28°C. The genome size of the S. penaei was 1778 Kb, as determined by pulsed-field gel electrophoresis using undigested DNA. Reduction of virulence of S. penaei was not detected in serial passage 24 and 76 isolates but passage 131 isolate was attenuated as indicated by the number of surviving shrimp and histological findings of challenged P. vannamei. Toxicity was not detected in supernatant fractions of M1D medium cultures of S. penaei isolates. The most predominant host responses to the S. penaei reference isolate and to serial passage isolates were hemocytic nodules and hemocytic infiltration observed in hematoxylin and eosin-stained histological sections. Transmission electron microscopy of the lymphoid organ of experimentally infected P. vannamei depicted S. penaei without cell wall and free in the cytoplasm of lymphoid organ cells. The lesions observed in histological sections were verified by in situ hybridization using a digoxigenin (DIG)-labeled probe specific to the spiralin gene of Spiroplasma spp. 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 and 23S-5S ISR, were constructed using the distance-based Neighboring-Joining 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 corroborates the observations by other investigators using the 16S gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to S. insolitum and distantly related to Spiroplasma sp. SHRIMP from China
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Studies on Two Genomic Variants of Taura Syndrome Virus: Infection under Hyperthermic Conditions and Detection with a Novel Monoclonal Antibody
No full textTaura syndrome (TS) is one of the most devastating diseases affecting the shrimp farming industry worldwide. The causative virus, Taura syndrome virus (TSV), has been identified. My work is centred on the development of monoclonal antibodies against TSV. I have also characterized a novel variant of the virus from Venezuela and evaluated the effect of hyperthermia on TSV infection. This work has resulted in 3 manuscripts, which constitute the core of this dissertation. The taxonomy throughout this dissertation is done according to Holthuis (1980).The first manuscript describes the production of a monoclonal antibody reacting with the Belize strain of TSV. The antibody, MAb 2C4, exhibits good sensitivity and specificity for TSV in immunohistochemistry (IHC) and dot blot immunoassay. MAb 2C4 reacted with the TSV-HI94, TSV-SI98 and TSV-BZ02 variants, but not with the TSV-VE05 and TSV-TH05 variants. This antibody adds and improves tools to those available for TSV diagnosis.Chapter three describes a relatively novel variant of TSV from Venezuela, which was characterized by our laboratory. By genetic sequencing, this new isolate exhibits a 94% similarity with TSV-HI94. IHC, dot blot immunoassay and bioassays were also performed. While processed samples reacted only weakly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassays, TSV-VE05 presented mortality comparable to TSV-HI94 in Penaeus vannamei. These data confirm the presence of TSV in Venezuela and that a new variant of the virus was responsible for the outbreak of TS.In chapter four, the behavior of TSV infection under hyperthermic conditions was examined. I compared the susceptibility of Kona stock P. vannamei to the infection by two variants of TSV under hyperthermic conditions (32oC). Shrimp, infected with TSV-HI94, were resistant to infection at high temperature. However, under the same hyperthermic conditions, the challenged shrimp were fully susceptible to the infection by TSV-BZ02. This susceptibility to TSV-BZ02 at higher temperatures was independent both of the route of infection and of the salinity of water. I conjecture that TSV-BZ02 might be a temperature permissible mutant of TSV
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