Vibrio anguillarum Phages Protect against Vibriosis in Atlantic Salmon

Vibrio anguillarum is the causative agent of a fatal hemorrhagic septicaemia disease (vibriosis) which affects >50 species of fish, including the economically important Salmo salar (Atlantic salmon), Oncorhynchus mykiss (rainbow trout), Psetta maxima (turbot) and Sparus aurata (sea bream).  Increasing restrictions on the use of antibiotics to control bacterial diseases in aquaculture (due to concerns of negative environmental impact and bacterial resistance), have encouraged the development of new strategies.  Three V. anguillarum serotypes have been associated with vibriosis: O1, O2 and O3.    While commercial vaccines to protect against V. anguillarum vibriosis outbreaks caused by the O1 and O2 serotypes are available, O3-serotype-outbreaks cannot currently be prevented by vaccination.  Bacteriophages (or ‘phage’: viruses that infect and have the potential to kill bacteria) have been isolated for the bacterial fish pathogens Vibrio harveyi, Flavobacterium psychrophilum and Aeromonas salmonicida, and appear to show potential for controlling these bacteria.  Although one A. salmonicida phage has also been demonstrated as effective against a V. anguillarum strain, the ability of this phage to protect fish from vibriosis has not been proven.

Higuera et al. (2013) provide the first evidence that V. anguillarum phages have the ability to protect fish against experimental infection with V. anguillarum.  Phages were isolated from bivalve (clam and mussel) tissues of samples purchased in the central market of Santiago, Chile.  Chile has a growing salmon industry, where (unlike Europe) most of the pathogenic isolates of V. anguillarum are known to exhibit the O3 serotype.  The authors isolated six phages via plaque assay, using V. anguillarum strain PF4 (previously isolated from an Atlantic salmon fish farm in Chile and identified as serotype O3) and 0.22 μm filtered bivalve homogenate.  Examination of host range (performed via spot tests) showed that all isolated phages infected different strains of V. anguillarum.  Higuera et al. (2013) investigated whether one of the phages they isolated was able to prevent V. anguillarum vibriosis in S. salar via two separate ‘challenge’ experiments.

Three treatments were applied to groups of 15 individual S. salar: 1) V. anguillarum strain PF4 added directly to tank water; 2) Phage added directly to tank water immediately after PF4 addition; 3) Medium only added to tank water (control).  In the treatment where only PF4 were added, fish begun to die after one day, and 6 days later only 7% of the fish had survived.  Where phage was added directly after PF4 (at a multiplicity of infection (MOI) of one PFU per bacterial cell) 100% of the fish survived up to ten days later. The control group suffered 30% mortality ten days after media addition.  No V. anguillarum was recovered from dead individuals or tank water in the control group.  Similar results were obtained when a MOI was increased to 20.  The authors also note that phage was found to be present in the phage-treated tank seven days post infection. 

The second challenge experiment investigated whether the same phage could protect fish from vibriosis in farming conditions.  Groups of 100 S. salar were exposed to the same treatments as listed above (with the addition of a ‘phage only’ treatment) on an experimental fish farm located in Southern Chile.  Conditions differed from the previous experiment in several ways, including the exchange of 50% of the tank water every day, and the use of seawater (to adjust salinity).  Higuera et al. (2013) report that in tanks where V. anguillarum was added without phage, the fish had suffered a 40% mortality after 20 days, whereas when phage was also added (at an MOI of 10), 100% of the fish survived (phage alone had no observable affect).  The authors conclude that their observations strongly suggest that the phage they isolated can protect Atlantic salmon against infection by V. anguillarum strain PF4.  They postulate that the high survival rate of fish treated with PF4 and without the addition of phage in farming conditions (at 60%), may be explained by the fact that 30% of the water used was obtained directly from the sea, which therefore contained different populations of bacteria that might have competed against PF4, or even acted as a Probiotic.

This study provides some interesting evidence for the efficacy of phage therapy in the treatment of V. anguillarum vibriosis in fish, yet there are some nagging issues with the method employed.  It appears that no replication was included in the lab-based challenge experiment, with just one tank employed for each condition.  As 15 fish were used for each treatment, it is not obvious why the authors did not (at the least) split these into three groups of five individuals, thus allowing some statistical analysis (and therefore a nod towards taking natural variability into account).  Likewise, although 100 individuals were used in the fish-farm experiment, no account of tank number or mention of statistical analysis is made when the fish survival rates are quoted.  Further work would benefit from the inclusion of replication in experimental design, and perhaps filtration of seawater in order to investigate the theory of competitive bacteria in reducing fish infection (as oppose to other factors, e.g. fish immunity/resistance to infection). 

Another issue which the authors do not fully address is that of bacterial resistance to infection by phage.  This has been cited as a potential problem in the use of phage therapy for other applications (e.g. preventing coral disease, Efrony et al., 2006), and although the authors of this study determined that PF4 has a resistance rate of 4.3 x 10^-6 when infected with the phage isolate, they appear to somewhat gloss over the problem of resistance, by stating that these levels seem too low to cause morbidity or mortality in fish (and that resistant bacteria may be less pathogenic).  It seems likely that selection would increase the levels of PF4 resistance to the phage isolate with continued phage administration, thus a cocktail of phages (as suggested for the treatment of coral disease by Efrony et al., 2006), rather than this one isolate, may be the answer to long term protection of Atlantic salmon from V. anguillarum vibriosis in aquaculture.  A different strategy entirely may be to employ probiotic bacteria (Sorroza et al. 2012, as reviewed on this blog by Matt).  Nevertheless, this study appears to indicate that phage therapy may be one promising route of investigation in the fight against bacterial diseases such as V. anguillarum in the aquaculture industry.

Higuera, G., Bastías, R., Tsertsvadze, G., Romero, J. & Espejo, R. T. Recently discovered Vibrio anguillarum phages can protect against experimentally induced vibriosis in Atlantic salmon, Salmo salar. Aquaculture 392-395, 128–133 (2013).