Kenneth W. Hasson

Experimental Infection of Western Hemisphere Penaeid Shrimp with Asian White Spot Syndrome Virus and Asian Yellow Head Virus

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 di...

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 Davidsons 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 Davidsons 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 Davidsons 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. Davidsons 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 Davidsons, 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.

The geographic distribution of Taura Syndrome Virus (TSV) in the Americas: determination by histopathology and in situ hybridization using TSV-specific cDNA probes

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 Davidsons 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.

Genetic variation and immunohistochemical differences among geographic isolates of Taura syndrome virus of penaeid shrimp.

Taura syndrome virus (TSV) is an important virus infecting penaeid shrimp in the western hemisphere. Genetic variation and immunohistochemical differences of 20 TSV isolates collected from the USA, Taiwan, Mexico and Nicaragua were compared. Capsid protein genes CP1 (546 bp) and CP2 (584 bp) were amplified by RT-PCR and the cDNAs were sequenced. Pairwise comparison of nucleotide sequences showed a 0-2.4% difference in CP1 and a 0-3.5% difference in CP2. Phylogenetic analyses clustered the TSV isolates into two groups: one contained USA, Taiwan and some Mexican isolates, the other contained Mexican isolates only. Immunohistochemical analysis using a TSV-specific monoclonal antibody produced positive results for the USA and Taiwan isolates but negative results for the Mexican and Nicaraguan isolates. Molecular and immunohistochemical data suggest the existence of at least two TSV strains, one of which might have evolved following contact with a new penaeid host, Penaeus stylirostris.

Chronic toxicity and histopathological studies with Benlate®, a commercial grade of benomyl, in Penaeus vannamei (Crustacea: Decapoda)

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/1 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.