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 (1^st and 2^nd bath: ∼10⁶ PFU ml^-1; 3^rd 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.

Keywords: Flavobacterium psychrophilum; bacteriophages; phage-therapy; rainbow trout fry (Oncorhynchus mykiss); rainbow trout fry syndrome (RTFS).

FIGURE 1

Experimental fish trials to test phage delivery by feed (A), by bath (B), and by intraperitoneal injection (C). (A) Eight hundred and ninety-five rainbow trout fry were divided randomly in 12 aquaria (55 fish ± 4) which are represented by colored circles. Fish were fed at 2% of their body weight with either control feed (in gray), phage-immobilized (in blue), or phage-sprayed (in red) feed. Bacteriophage drawings indicate FpV4 and FPSV-D22, which were administered to fish by feed. For each group, three of the four aquaria were challenged with F. psychrophilum (1 × 10⁴ CFU fish^–1; indicated by a syringe with yellow content because of the coloration of the bacteria). Fish in the fourth aquarium were injected with sterile TYES-B as control for the infection. Two of the bacterial challenged aquaria per group were used to follow mortality. The remaining aquaria were dedicated to sampling (five fish at sampling point). (B) Hundred and twenty-five rainbow trout fry were divided in four aquaria (circles; 31 fish ± 1 per aquarium) and challenged with F. psychrophilum (1 × 10⁵ CFU fish^–1; syringe with yellow content). Fish in two aquaria were exposed to three rounds of FpV4 and FPSV-D22 phages bath (blue circles; indicated by bacteriophage drawings). Mortality was followed w = week. (C) Hundred and twenty rainbow trout (7 g), divided in six aquaria (circles, 20 fish ± 0 per aquarium), were IP challenged with F. psychrophilum (1.7 × 10⁷ CFU fish^–1; syringe with yellow content). Three days later, fish in three aquaria were exposed to phages FpV4 and FPSV-D22 by IP injection (blue circles; indicated by bacteriophage drawings). This aquaria were used to follow mortality. One additional aquarium was included for few sampling where fish were only exposed to the two component phage mix (light blue circle; 15 fish). Created with BioRender.com (the figure was exported under a paid subscription).

FIGURE 2

Experiment A. Qualitative detection of bacteriophages in fish organs (spleen, kidney, intestine, and brain) over time in fish fed with phage-immobilized (PI), phage-sprayed (PS), and control (C) feed. Blue and red colors indicate the presence of phages in organs of fish fed with phage-immobilized and phage-sprayed feed, respectively, challenged with F. psychrophilum (full color, no pattern) or not (pattern, striped). Absence of phages in the tested organ is indicated by white/blank. Positive detection = presence of one or more plaques in at least one of the technical triplicates. At each sampling point, five fish were sampled in each group except 1 day before the infection where samples were collected only from groups not supposed to be challenged with F. psychrophilum. dpi, days post infection.

FIGURE 3

Experiment A. Frequency of detection of bacteriophages and of isolation of F. psychrophilum in kidney (A), spleen (B), and brain (C) of sampled fish in the different groups over time. No phages were detected in internal organs of fish fed with control feed. Five fish were sampled at each sampling point per group. n.d., not determined. For each organ of each feed group, the total percentage of phages isolation over time is calculated with and without F. psychrophilum.

FIGURE 4

Experiment A. Quantification of bacteriophages FpV4 and FPSV-D22 in intestine (A,B), kidney (C,D), spleen (E,F), and brain (G,H) of fish fed with phage-immobilized (A,C,E,G) and phage-sprayed (B,D,F,H) feed. Values represent the average of five biological replicates per time point and error bars the standard deviation. Concentration of phages in the organs of dead fish (dead because of F. psychrophilum infection) is included in the graphs for the corresponding organ and feed type (each symbol in yellow represents a single fish). Simple linear regression lines were calculated from the log-transformed PFU over time and 95% confidence bands are also presented.

FIGURE 5

Percent survival observed in rainbow trout fry exposed to F. psychrophilum and bacteriophages in the three experiments. In (A), survival of fish fed with phage-immobilized feed (blue), phage-sprayed feed (red) feed and control feed (gray) are displayed (Experiment A). No mortality was observed in the control aquaria (data not shown). In (B), survival of fish bathed in phage solution (blue) and in control bath (gray) in Experiment B. In (C), bacteriophages were delivered with IP injections 3 days after the bacterial challenge (blue) while phage control fish were injected with sterile SM buffer (gray) (Experiment C). In all three experiments, moribund and dead fish were positive to F. psychrophilum. Final percent survival are presented for each curve in the figures. * = the curves are significantly different. 95% confidence interval is presented for each curve.