Development of a colony hybridization method for the enumeration of total and potentially enteropathogenic Vibrio parahaemolyticus in shellfish

2014 ◽  
Vol 186 ◽  
pp. 22-31 ◽  
Author(s):  
Elisabetta Suffredini ◽  
Loredana Cozzi ◽  
Gianni Ciccaglioni ◽  
Luciana Croci
2004 ◽  
Vol 67 (11) ◽  
pp. 2424-2429 ◽  
Author(s):  
G. E. KAUFMAN ◽  
G. M. BLACKSTONE ◽  
M. C. L. VICKERY ◽  
A. K. BEJ ◽  
J. BOWERS ◽  
...  

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°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.


2020 ◽  
Vol 83 (11) ◽  
pp. 2014-2017
Author(s):  
JESSICA L. JONES ◽  
KERI A. LYDON ◽  
WILLIAM C. WALTON

ABSTRACT Vibrio parahaemolyticus and Vibrio vulnificus are naturally occurring human pathogenic bacteria commonly found in estuarine environments where oysters are cultured. The use of triploid oysters has increased due to their rapid growth rate and because they maintain a high quality throughout the year. Previous work suggested levels of Vibrio spp. may be lower in triploid oysters than diploid oysters. Therefore, this study aimed to determine whether there is a difference in the abundances of V. parahaemolyticus and V. vulnificus between half-sibling diploid and triploid American oysters (Crassostrea virginica). In four trials, 100 individual oysters (either iced or temperature abused) were analyzed for V. parahaemolyticus and V. vulnificus by using direct plating followed by colony hybridization. Mean levels of V. parahaemolyticus in iced and abused diploid oysters were 3.55 and 4.21 log CFU/g, respectively. Mean levels in iced and abused triploid oysters were 3.49 and 4.27 log CFU/g, respectively. Mean levels of V. vulnificus in iced and abused diploid oysters were 3.53 and 4.56 log CFU/g, respectively. Mean levels in iced and abused triploid oysters were 3.54 and 4.55 log CFU/g, respectively. The differences in Vibrio spp. abundances between diploid and triploid oysters was not significant (P > 0.05). However, the differences across treatments were significant (P < 0.05), with the exception of V. parahaemolyticus levels in trial 3 (P = 0.83). Variation between individual oysters was also observed, with 12 of 808 measurements being outside of the 95th percentile. This phenomenon of occasional statistical outliers (“hot” or “cold” oysters) has been previously described and supports the appropriateness of composite sampling to account for inherent animal variability. In summary, the data indicate that abundances of V. parahaemolyticus and V. vulnificus are not dependent on the ploidy of cultured oysters but vary with the type of handling. HIGHLIGHTS


2006 ◽  
Vol 69 (11) ◽  
pp. 2770-2772 ◽  
Author(s):  
JESSICA L. NORDSTROM ◽  
RACHEL RANGDALE ◽  
MICHAEL C. L. VICKERY ◽  
ANDREA M. B. PHILLIPS ◽  
SHELLEY L. MURRAY ◽  
...  

Reliable methods are needed to detect total and pathogenic Vibrio parahaemolyticus. One marker of V. parahaemolyticus virulence is the thermostable-related hemolysin. We developed an alkaline phosphatase–labeled DNA probe method for the specific detection and enumeration of trh-positive V. parahaemolyticus by colony hybridization. The probe was tested against a panel of 200 bacterial strains and determined to be specific for trh-positive V. parahaemolyticus. Additionally, the trh alkaline phosphatase probe colony hybridization was successfully used to detect and enumerate trh-positive V. parahaemolyticus in seafood and water samples collected from the United States and the United Kingdom.


1994 ◽  
Vol 61 (4) ◽  
pp. 529-535 ◽  
Author(s):  
Ralf Tatzel ◽  
Wolfgang Ludwig ◽  
Karl Heinz Schleifer ◽  
Peter R. Wallnöfer

SummaryA total of 529 bacterial strains have been isolated from milk and cream sampled at different sites in a dairy production plant under conditions selective for aerobic sporeforming bacteria. Identification with classical methods based on morphological, physiological and biochemical criteria showedBacillus licheniformisto be the most frequently occurringBacillussp. The investigation also revealed 62 unidentified strains. Classical identification methods were time consuming (3–7d), lacked specificity and —because of their dependence on phenotypic gene expression—sometimes produced ambiguous results. Consequently, a colony hybridization method developed for the identification ofB. licheniformisstrains and using non-radioactive labelled 23S rRNA targeted oligonucleotide probes was also used. Identification ofB. licheniformiswith borth classical and hybridization methods revealed diverging identification results for 70 strains.


2019 ◽  
Vol 157 ◽  
pp. 37-42 ◽  
Author(s):  
Karanth Padyana Anupama ◽  
Kundar Deeksha ◽  
Ariga Deeksha ◽  
Iddya Karunasagar ◽  
Indrani Karunasagar ◽  
...  

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