Mapping the development of a Dinophysis bloom in a shellfish aquaculture area using a novel molecular qPCR assay.

Diarrhetic shellfish toxins produced by certain species of the marine dinoflagellate Dinophysis can accumulate in shellfish in high concentrations, representing a significant food safety issue worldwide. This risk is routinely managed by monitoring programs in shellfish producing areas, however the methods used to detect these harmful marine microbes are not usually automated nor conducted onsite, and are often expensive and require specialized expertise. Here we designed a quantitative real-time polymerase chain reaction (qPCR) assay based on the ITS-5.8S ribosomal region of Dinophysis spp. and evaluated its specificity, efficiency, and sensitivity to detect species belonging to this genus. We designed and tested twenty sets of primers pairs using three species of Dinophysis - D. caudata, D. fortii and D. acuminata. We optimized a qPCR assay using the primer pair that sufficiently amplified each of the target species (Dacu_11F/Dacu_11R), and tested this assay for cross-reactivity with other dinoflagellates and diatoms in the laboratory (11 species) and in silico 8 species (15 strains) of Dinophysis, 3 species of Ornithocercus and 2 species of Phalacroma. The qPCR assay returned efficiencies of 92.4% for D. caudata, 91.3% for D fortii, and 91.5% for D. acuminata, while showing no cross-reactivity with other phytoplankton taxa. Finally, we applied this assay to a D. acuminata bloom which occurred in an oyster producing estuary in south eastern Australia, and compared cell numbers inferred by qPCR to those determined by microscopy counts (max abund. ∼6.3 × 103 and 5.3 × 103 cells L-1 respectively). Novel molecular tools such as qPCR have the potential to be used on-farm, be automated, and provide an early warning for the management of harmful algal blooms.

Vibrio ordalii antimicrobial susceptibility testing--modified culture conditions required and laboratory-specific epidemiological cut-off values.

The present study aimed to determine oxytetracycline (OTC), florfenicol (FLO) and oxolinic acid (OXO) MICs and zone diameters for 24 Chilean Vibrio ordalii isolates using the methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals and the methods for antimicrobial disk susceptibility testing of bacteria isolated from aquatic animals guidelines published by the Clinical Laboratory Standards Institute (CLSI). The results were then used in a normalized resistance interpretation (NRI) analysis to establish tentative laboratory-specific epidemiological cut-off (ECOFF) values. MIC results were similar at the two tested temperatures (22 °C and 18 °C). At 18 °C, the NRI analysis of OTC, FLO and OXO MIC data calculated laboratory-specific ECOFF values and non-wild-type (NWT) rates to be ≤4 mg/l (24%), ≤16 mg/l (4%) and ≤8 mg/l (25%), respectively. Tests performed with all V. ordalii isolates following the officially recommended incubation temperature (22 °C) revealed difficulties in measuring inhibition zone diameters. When disk diffusion tests were performed using Mueller-Hinton agar with 1% NaCl (MHA-1) at 18 °C the inhibition zone diameter distributions showed the formation of WT populations which could be defined using NRI analysis. For OTC the laboratory-specific ECOFF value was ≥38 mm with NWT rate of 16.7%. For FLO and OXO, the laboratory-specific ECOFF values were ≥38 and ≥40, respectively, generating NWT rates of 25 and 46%, respectively. Although the CLSI suggests testing Vibrio spp. on MHA-1 at 22 °C, we found measurements of the 24 isolates were better defined and normally distributed at 18 °C. This is the first study determining the MIC and disk diffusion test of V. ordalii isolated from diseased salmonids, where laboratory-specific ECOFF values could be established. Also resistance to OTC, FLO and OXO among some Chilean isolates was demonstrated.