Due to the comparatively high number of tank water samples testing positive for F. psychrophilum observed in the first subset of samples examined, we decided to screen all 2010 tank samples. Of the 85 tank water samples collected in 2010, however, only 8 (10%) were positive (range: 43 to 3,000 cells/ml) (Table 2). Table 2 Origin and percent of samples positive to F. psychrophilum   Origin

No. of samples % Positive for F. psychrophilum % of samples quantified Cells/ml Inlet and tank 2009           Inlets Ticino fish farms 60 7% 1.6% 73 to 1.5 × 104 Tanks Ticino fish farms 60 53% 1.6% 42 to 3.5 × 104 2010           Tanks Swiss fish farms 85 10% 0% 43 to 3’000 Healthy carriers 2011, 2012 Swiss fish farms 43 EPZ015938 ic50 80% 0% 0-400 In contrast to culture or FISH, F.

psychrophilum was detected in healthy and quantified in infected fish by qPCR. F. psychrophilum densities in healthy individuals were well below the QL, in a range of 0 to 15,000 cells per spleen, whereas spleens from diseased fish contained bacterial densities over the QL, in a range of 7,000 to 7.7 × 108 cells per spleen. Positive results by qPCR were reported for all spleens originating from the 4 outbreaks; FISH allowed detecting F. psychrophilum in all outbreaks while culture showed F. psychrophilum only in 3 outbreaks. Risk factors We could not show any clear correlation between the presence of F. psychrophilum and Nutlin3a the environmental parameters measured. We observed that the F. psychrophilum densities tended to increase and to cause outbreaks after changes Ergoloid in water parameters. For instance, a change in more than one ecological parameter tended to correlate with an outbreak or at least an increase of the number of F. psychrophilum in water (Figure 4). This observation, however, cannot be supported by any statistical analysis, because too few outbreaks could be analyzed during the

study period. Figure 4 Seasonal variation example. Physicochemical parameters [primary y axis: temperature (T in °C), pH of water, oxygen concentration (mg/L); secondary y axis: conductibility (μ Siemens)] measured in a selected fish farm (Ticino, Switzerland) during 2009. Detection of the pathogen in the tank water samples started on 9 June 2009 (*), the arrows indicate a flavobacteriosis outbreak in brown trout fario. Discussion This study shows that the qPCR assay developed is very sensitive and able to detect and quantify F. psychrophilum in water samples and fish spleens with no amplification of the other 130 non-target bacterial isolates. In the water samples investigated, LOD was 20 rpoC gene copies per Akt inhibitor reaction and QL 103 cells per reaction. The quantification limit was quite high: possibly random losses happened because of DNA uptake in columns during extraction of low cell concentrations. As DNA extraction from samples containing <1000 cells/μl was probably low, the quantification by qPCR was also not reliable. In a 16S rRNA gene F.