Phage-Mediated Control of Flavobacterium psychrophilum in Aquaculture: In vivo Experiments to Compare Delivery Methods

Abstract

Phage-based approaches have gained increasing interest as sustainable alternative strategies to antibiotic treatment or as prophylactic measures against disease outbreaks in aquaculture. The potential of three methods (oral, bath, and injection) for delivering a two-component phage mixture to rainbow trout fry for controlling Flavobacterium psychrophilum infections and reduce fish mortality was investigated using bacteriophages FpV4 and FPSV-D22. For the oral administration experiment, bacteriophages were applied on feed pellets by spraying (1.6 × 108 PFU g–1) or by irreversible immobilization (8.3 × 107 PFU g–1), using the corona discharge technology (Fixed Phage Ltd.). The fish showed normal growth for every group and no mortality was observed prior to infection as well as in control groups during the infection. Constant detection of phages in the intestine (∼103 PFU mg–1) and more sporadic occurrence in kidney, spleen, and brain was observed. When fish were exposed to F. psychrophilum, no significant effect on fish survival, nor a direct impact on the number of phages in the sampled organs, were detected. Similarly, no significant increase in fish survival was detected when phages were delivered by bath (1st and 2nd bath: ∼106 PFU ml–1; 3rd bath: ∼105 PFU ml–1). However, when phages FpV4 and FPSV-D22 (1.7 × 108 PFU fish–1) were administered by intraperitoneal injection 3 days after the bacterial challenge, the final percent survival observed in the group injected with bacteriophages FpV4 and FPSV-D22 (80.0%) was significantly higher than in the control group (56.7%). The work demonstrates the delivery of phages to fish organs by oral administration, but also suggests that higher phage dosages than the tested ones may be needed on feed pellets to offer fish an adequate protection against F. psychrophilum infections.

Interactions between Rainbow Trout Eyed Eggs and Flavobacterium spp. Using a Bath Challenge Model: Preliminary Evaluation of Bacteriophages as Pathogen Control Agents

“…F. psychrophilum 950106-1/1 is a well-characterized strain isolated in 1995 (serotype Fd, virulent) [ 10 , 29 , 30 , 31 ], while F. psychrophilum 160401-1/5N was isolated in 2016 and recently characterized (serotype Th, virulent) [ 31 ]. An additional strain, F. psychrophilum FPS-S6 (serotype Th, virulent, isolated in 2017 in Sweden), was used for the production of high titer phage FPSV-D22 solutions since it was the most efficient host for phage proliferation [ 27 , 31 ]. The strains were stored at −80 °C in tryptone yeast extract salts (TYES) medium [ 32 ] and glycerol (15–20%).…”

“…I section C) was inoculated in TYES broth (5 mL, referred as TYES-B) from a −80 °C stock, incubated for 48–72 h (15 °C; 100 rpm) and then streaked onto TYES agar (TYES-B with 1.1% agar, referred as TYES-A). Single colonies were then picked (3–4 days of incubation) and inoculated in TYES-B for 48 h [ 27 ]. For bath challenge experiments, the selected bacteria were prepared according to [ 30 ].…”

“…F. psychrophilum -targeting lytic bacteriophages FpV4 (isolated in 2005 in Denmark from water with feces samples, Podoviridae family) [ 36 , 37 ] and FPSV-D22 (isolated in 2017 in Denmark from fish tissue samples, Siphoviridae family) [ 27 , 31 ] were selected for the studies. Solutions of FpV4 and FPSV-D22 were purified (0.2 μm pore size sterile filter) and stored in SM buffer (8 mM MgSO 4 , 50 mM Tris-Cl [pH 7.5], 99 mM NaCl, 0.01% gelatin) and glycerol (15%) at −80 °C [ 31 , 36 ].…”

“…After incubation for 48–72 h, the lysed cultures were then centrifuged (5000× g , 10 min, 4 °C) and filtered with a 0.2 μm pore size sterile filter (Sterivex, Millipore; Merck KGaA, Darmstadt, Germany). For the experiments in section B and C, FpV4 and FPSV-D22 crude lysates were further purified and concentrated by PEG-precipitation (24 h-incubation at 4 °C with poly-ethylene glycol 8000 (PEG-8000) and sodium chloride at a final concentration of 10% w/v and 1 M, respectively) and subsequent 0.2 µm filtration, centrifugation (10,000× g , 30 min, 4 °C), and re-suspension in either sterile TYES-B or sterile SM buffer, as described by [ 27 , 38 ].…”

“…Previous studies on phage control of Flavobacterial pathogens in rainbow trout have focused on fry and juvenile stages [ 24 , 25 , 26 , 27 , 28 ]. Here, we report for the first time the use of bacteriophages for reducing these pathogens in connection with rainbow trout eyed eggs.…”