Development of a Taqman real-time PCR assay for rapid detection and quantification ofVibrio tapetisin extrapallial fluids of clams

Abstract

The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD) in the Manila clam Venerupis (=Ruditapes) philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

Vibrio tapetis Displays an Original Type IV Secretion System in Strains Pathogenic for Bivalve Molluscs

“…We found a conserved gene cluster that potentially encodes a Type IV secretion system in 13 of the 17 genomes (Table S4 ) and was absent in the non-virulent strains GDE, GTRI, LP2, and HH6087 (Figure 6 ). This finding is in line with recent real time PCR experiments targeting T4SS elements in these strains (Bidault et al, 2015 ). The T4SS cluster of V. tapetis strains consists of the vir operon ( virB2, virB3, virB4, virB6, virB8, virb9, virB10, virB11 ) plus genes of unknown functions.…”

“…Additionally, VirB4, a component of the Type IV secretion system (Juhas et al, 2008 ; Christie et al, 2014 ), is systematically present in the genome of pathogenic V. tapetis strains but absent from non-pathogenic strains (ie. not able to reproduce BRD significantly after in vivo pallial challenge in V. philippinarum ) (Bidault et al, 2015 ).…”

Ancient DNA analysis identifies marine mollusc shells as new metagenomic archives of the past

“…S1, Table S4) and isolated respectively from a BRD‐positive R. philipinarum clam in France in 1990 (Paillard, Maes, & Oubella, ; Borrego, Castro, & Luque, ), and from a healthy Atlantic halibut ( Hippoglossus hippoglossus ) in Scotland in 2002 (Reid, Duncan, Laidler, Hunter, & Birkbeck, ). DNA was extracted following (Bidault, Richard, Le Bris, & Paillard, ). Shotgun HTS reads were obtained by applying the protocol described above on 1 μg of DNA extract sheared using a diagenode Bioruptor ultrasonicator (four cycles of: 15 s at high intensity; 90 s off), and amplifying libraries for seven cycles before single‐end sequencing on an Illumina HiSeq2000 platform at the Danish National High‐Throughput DNA Sequencing Centre.…”

Microbial Diseases of Bivalve Mollusks: Infections, Immunology and Antimicrobial Defense

“…The rate of larval mortality can reach 100%, especially if larvae are reared in static systems at high temperature and density. Diagnosis of bacterial diseases is based on macroscopic inspection of shell valves in combination with PCR, which is the most sensitive and rapid method [ 21 ].…”