Biomedical subjects

G M Blackstone

Publications and source records attributed to G M Blackstone.

3 recordsLinked to original sources

PubMed2004

Effect of intertidal exposure on Vibrio parahaemolyticus levels in Pacific Northwest oysters.

Interest in Vibrio parahaemolyticus (Vp) increased in the United States following Vp-associated gastroenteritis outbreaks in 1997 and 1998 involving the West Coast and other areas. The present study evaluated multiple aspects of Vp ecology in the Pacific Northwest with three objectives: (i) to determine the effect of low-tide exposure on Vp levels in oysters, (ii) to determine the relationship between total and pathogenic Vp, and (iii) to examine sediments and aquatic fauna as reservoirs for pathogenic Vp. Samples were collected from intertidal reefs along Hood Canal, Wash., in August 2001. Fecal matter from marine mammals and aquatic birds as well as intestinal contents from bottom-dwelling fish were tested. Total and pathogenic Vp levels in all the samples were enumerated with colony hybridization procedures using DNA probes that targeted the thermolabile direct hemolysin (tlh) and thermostable direct hemolysin (tdh) genes, respectively. The mean Vp densities in oysters were four to eight times greater at maximum exposure than at the corresponding first exposure. While tdh-positive Vp counts were generally < or = 10 CFU/g at first exposure, counts as high as 160 CFU/g were found at maximum exposure. Vp concentrations in sediments were not significantly different from those in oysters at maximum exposure. Pathogenic (tdh positive) Vp was detected in 9 of 42 (21%) oyster samples at maximum exposure, in 5 of 19 (26%) sediment samples, but in 0 of 9 excreta samples. These results demonstrate that summer conditions permit the multiplication of Vp in oysters exposed by a receding tide.

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PubMed2004

Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix.

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

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PubMed2002

Applied technique for increasing calicivirus detection in shellfish extracts.

AIMS: Optimal detection of enteric RNA viruses in clinical, environmental, and food products using reverse transcription-PCR (RT-PCR) when inhibitory substances in extracted sample materials are present. METHODS AND RESULTS: We adapted a device for detection of RNA viruses in plant tissues and insects to detect a calicivirus strain (San Miguel sea lion virus, serotype 17) in water and oyster tissue extracts. This single, compartmentalized tube-within-a-tube (TWT) device for RT-PCR-nested PCR was compared to a conventional protocol of RT-PCR-nested PCR. In the presence of 100 mg of shellfish tissue extract equivalent, this TWT device decreases the calicivirus assay detection limit 10-fold over that of conventional RT-PCR-nested PCR while maintaining an identical detection limit of viral nucleic acid suspended in water. Both the conventional and TWT methods estimated the total particle-to-infectious particle ratio for this strain of calicivirus at approximately 40 : 1. CONCLUSIONS: We believe that the TWT device with appropriate RT-PCR primers will decrease the detection limit for other calicivirus strains and RNA viruses in shellfish tissue extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe that the TWT approach is applicable to other situations where RT and/or PCR inhibitory materials are present or nucleic acid targets of bacteria or viruses are at low levels in extracts of food products or clinical specimens.

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