PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene

2014 ◽  
Vol 107 (3) ◽  
pp. 223-234 ◽  
Author(s):  
R Avendaño-Herrera ◽  
JP Maldonado ◽  
D Tapia-Cammas ◽  
CG Feijóo ◽  
F Calleja ◽  
...  
Keyword(s):  
Hemolysin Gene ◽  
Vibrio Ordalii

scholarly journals Relationships between Environmental Factors and Pathogenic Vibrios in the Northern Gulf of Mexico

2010 ◽  
Vol 76 (21) ◽  
pp. 7076-7084 ◽  
Author(s):  
C. N. Johnson ◽  
A. R. Flowers ◽  
N. F. Noriea ◽  
A. M. Zimmerman ◽  
J. C. Bowers ◽  
...  
Keyword(s):  
Environmental Factors ◽  
Water Column ◽  
Vibrio Vulnificus ◽  
Environmental Parameters ◽  
Sampling Period ◽  
Pathogenicity Factor ◽  
Hemolysin Gene ◽  
Thermostable Direct Hemolysin ◽  
Pathogenic Vibrios ◽  
Abundance And Distribution

ABSTRACT Although autochthonous vibrio densities are known to be influenced by water temperature and salinity, little is understood about other environmental factors associated with their abundance and distribution. Densities of culturable Vibrio vulnificus containing vvh (V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh (thermostable direct hemolysin gene, V. parahaemolyticus pathogenicity factor), and trh (tdh-related hemolysin gene, V. parahaemolyticus pathogenicity factor) were measured in coastal waters of Mississippi and Alabama. Over a 19-month sampling period, vibrio densities in water, oysters, and sediment varied significantly with sea surface temperature (SST). On average, tdh-to-tlh ratios were significantly higher than trh-to-tlh ratios in water and oysters but not in sediment. Although tlh densities were lower than vvh densities in water and in oysters, the opposite was true in sediment. Regression analysis indicated that SST had a significant association with vvh and tlh densities in water and oysters, while salinity was significantly related to vibrio densities in the water column. Chlorophyll a levels in the water were correlated significantly with vvh in sediment and oysters and with pathogenic V. parahaemolyticus (tdh and trh) in the water column. Furthermore, turbidity was a significant predictor of V. parahaemolyticus density in all sample types (water, oyster, and sediment), and its role in predicting the risk of V. parahaemolyticus illness may be more important than previously realized. This study identified (i) culturable vibrios in winter sediment samples, (ii) niche-based differences in the abundance of vibrios, and (iii) predictive signatures resulting from correlations between environmental parameters and vibrio densities.

scholarly journals The Fish Pathogen Vibrio ordalii Under Iron Deprivation Produces the Siderophore Piscibactin

2019 ◽  
Vol 7 (9) ◽  
pp. 313 ◽  
Author(s):  
Pamela Ruiz ◽  
Miguel Balado ◽  
Juan Carlos Fuentes-Monteverde ◽  
Alicia E. Toranzo ◽  
Jaime Rodríguez ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Iron Uptake ◽  
Peptide Mass Fingerprinting ◽  
Iron Limitation ◽  
Fish Pathogen ◽  
Fish Pathogens ◽  
Iron Deprivation ◽  
Peptide Mass ◽  
Photobacterium Damselae ◽  
Vibrio Ordalii

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using β-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in V. ordalii. Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of V. ordalii cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like Photobacterium damselae subsp. piscicida and V. anguillarum, which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.

scholarly journals Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

2004 ◽  
Vol 70 (1) ◽  
pp. 621-624 ◽  
Author(s):  
Ram Savan ◽  
Arisa Igarashi ◽  
Satoru Matsuoka ◽  
Masahiro Sakai
Keyword(s):  
Rapid Detection ◽  
Japanese Flounder ◽  
Isothermal Amplification ◽  
Edwardsiella Tarda ◽  
Fish Disease ◽  
Fish Pathogen ◽  
Loop Mediated Isothermal Amplification ◽  
Diagnostic Protocol ◽  
Amplification Method ◽  
Hemolysin Gene

ABSTRACT Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65°C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen.

scholarly journals Evidence for insertion sequence-mediated spread of the thermostable direct hemolysin gene among Vibrio species.

1991 ◽  
Vol 173 (16) ◽  
pp. 5036-5046 ◽  
Author(s):  
A Terai ◽  
K Baba ◽  
H Shirai ◽  
O Yoshida ◽  
Y Takeda ◽  
...  
Keyword(s):  
Insertion Sequence ◽  
Vibrio Species ◽  
Hemolysin Gene ◽  
Thermostable Direct Hemolysin

Localization of the Replication Region of the pMJ101 Plasmid from Vibrio ordalii

Plasmid ◽  
1994 ◽  
Vol 31 (3) ◽  
pp. 242-250 ◽  
Author(s):  
Carla Bidinost ◽  
Jorge H. Crosa ◽  
Luis A. Actis
Keyword(s):  
Replication Region ◽  
Vibrio Ordalii

scholarly journals Detection of Vibrio parahaemolyticus in Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

2006 ◽  
Vol 72 (3) ◽  
pp. 2031-2042 ◽  
Author(s):  
Linda N. Ward ◽  
Asim K. Bej
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Detection Methods ◽  
Tissue Homogenate ◽  
Oyster Tissue ◽  
Reading Frame ◽  
Initial Inoculum ◽  
Hemolysin Gene ◽  
Pure Cultures

ABSTRACT We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 104 CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 109 CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

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