Aquaculture 392–395 (2013) 128–133

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Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Recently discovered Vibrio anguillarum phages can protect against

experimentally induced vibriosis in Atlantic salmon, Salmo salar
Gastón Higuera a, 1, Roberto Bastías a,⁎, 1, George Tsertsvadze b, Jaime Romero a, Romilio T. Espejo a
a
Laboratorio de Biotecnología, Instituto de Nutrición y Tecnología de los Alimentos (INTA), University of Chile, Santiago, Chile
b
George Eliava Institute of Bacteriophage, Microbiology and Virology, Tbilisi, Georgia

a r t i c l e i n f o a b s t r a c t

Article history: Vibrio anguillarum is a marine bacterium that can cause vibriosis in several fish species of economic impor-
Received 23 January 2013 tance. The use of bacteriophage is an alternative strategy to control vibriosis in aquaculture systems. Here,
Received in revised form 8 February 2013 we present the isolation and characterization of six phages that are able to infect the pathogenic strain of
Accepted 11 February 2013
V. anguillarum, PF4. These phages all possess a similar double stranded DNA (dsDNA) genome but, according
Available online 20 February 2013
to their restriction pattern, can be differentiated into three types. The phages exhibited a similar host range,
Keywords:
infecting both V. anguillarum and V. ordalii but not V. parahaemolyticus strains. The CHOED phage protected
Vibrio anguillarum Salmo salar against experimentally induced vibriosis with the strain PF4. The presence of the phage increased
Bacteriophage the survival of fish to 100% when it was used with a MOI of 1 and 20, versus less than 10% of survival in the
Phage absence of the phage. To our knowledge, this is the first report of the ability of V. anguillarum phages to pro-
Salmo salar tect fish against experimental infection with V. anguillarum, and our results support the development of
Vibriosis phage therapy as a valid alternative for the control of vibriosis in salmonid aquaculture.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction emergence of multi-resistant bacteria (Cabello, 2006). Although

Vibrio anguillarum (also known as Listonella anguillarum) is a ma-
rine Gram-negative bacterium that is the etiologic agent of vibriosis, a
fatal hemorrhagic septicemia disease that affects more than 50 fresh-
and salt-water fish species, including several species that are of eco-
nomic importance to the aquaculture industry, such as the Atlantic
salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), turbot
(Psetta maxima), sea bass, and sea bream (Sparus aurata) (Toranzo
et al., 2005). Moreover, some bivalve mollusks and crustaceans are
also affected by this bacteria (Paillard et al., 2004).
To date, 23 O serotypes (O1-O23) have been found; however, the
O1, O2 and, in a lesser way, the O3 serotype have been associated
with vibriosis (Frans et al., 2011; Silva-Rubio et al., 2008). Recently,
the genome of the V. anguillarum 775 strain, which is of the O1 sero-
type, was sequenced, and the analysis of this genome demonstrated
that the majority of the genes that encode essential cellular functions
and pathogenicity are located on the first of the two chromosomes
that the bacteria possess (Naka et al., 2011).
Antibiotics are still the main therapeutic tool for controlling bacte-
rial diseases in aquaculture. However, their use is becoming increas-
ingly restricted, due to their negative environmental impact and the
⁎ Corresponding author at: Institute of Marine Biology, Biotechnology, and Aquaculture,
Hellenic Centre for Marine Research, Gournes, Heraklion 71003, Crete, P.O. BOX 2214,
Greece. Tel.: +30 2810337761; fax: +30 2810 337778.
E-mail addresses: robastias@gmail.com, roberto@hcmr.gr (R. Bastías).
1
Both authors contributed equally to this work.
there are multiple commercial vaccines to protect fish against out-
breaks of vibriosis that are caused by the O1 and O2 serotypes, the
outbreaks that are caused by serotype O3 cannot be completely
prevented (Mikkelsen et al., 2007). Because of this, there is increasing
interest in the development of new strategies to control pathogenic
bacteria in aquaculture. One option is the use of bacteriophages (or
phages), which are viruses that infect bacteria and are present in all
environments. Bacteriophages have the potential to kill their host
bacteria and have therefore been used to control pathogenic bacteria
in different fields with very promising results (Hagens and Loessner,
2007; Kutateladze and Adamia, 2010; Nakai and Park, 2002). Recently,
several phages that infect various bacterial fish pathogens have been
isolated (Vibrio harveyi, Flavobacterium psychrophilum, etc.), and they
have shown potential in controlling these bacteria (Castillo et al.,
2012; Crothers-Stomps et al., 2010; Karunasagar et al., 2007; Nakai
and Park, 2002; Park and Nakai, 2003; Stenholm et al., 2008). One
phage that infects Aeromonas salmonicida and exhibits a broad host
range was isolated and was demonstrated to be effective against a
V. anguillarum strain. However, its ability to protect fish from vibriosis
has not yet been proven (Pereira et al., 2011).
Although in Europe most of the pathogenic isolates of V. anguillarum
correspond to the O1 and O2 serotypes, in Chile, where there is a grow-
ing salmon farming industry, most of the pathogenic isolates exhibit an
O3 serotype. Here, we describe seven strains of V. anguillarum and the
closely related species Vibrio ordalii, which were isolated in Chile and
have been phenotypically characterized previously (Silva-Rubio et al.,
2008), as well as the isolation and characterization of six phages that

0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaculture.2013.02.013
 G. Higuera et al. / Aquaculture 392–395 (2013) 128–133
infect V. anguillarum. Additionally, we demonstrate the ability of one of
these phages to protect S. salar from infection by this pathogenic bacte-
ria. To our knowledge, this is the first report that validates the potential
of phages to control V. anguillarum infections and, consequently, to pre-
vent vibriosis outbreaks in fish.
2. Material and methods
2.1. Strains and media
The V. anguillarum strains Va PF4, Va PF7 and Va PF8 were donated
by Chilean diagnostic companies (Veterquimica and Biovac). These
V. anguillarum strains were isolated from different fish farms in the
south of Chile and were previously identified as V. anguillarum serotype
O3 by PCR-based and serological analysis (Silva-Rubio et al., 2008). The
identity of these strains as V. anguillarum was confirmed sequencing the
16S rRNA gene (GenBank accession numbers KC579365, KC579366 and
KC579367). The strain PF4 of V. anguillarum, which was used in the
bacterial challenges, was deposited in the PCM (Polish Collection of
Microorganisms), and the collection number was B/00045. The Vibrio
parahaemolyticus strain VpKX (RIMD2210633) correspond to the pan-
demic strain O3:K6 and was obtained from the Research Institute for
Microbial Diseases, Osaka University, Osaka, Japan. Vp57.5 corresponds
to the pandemic strain O3:k6 isolate obtained in Chile. Both strains have
been widely characterized as molecular and phenotypical (Fuenzalida
et al., 2006; Garcia et al., 2013; Matsumoto et al., 2000). The strain
Vp1193 (HER1193) is the host of the phage KVP40 and both were pur-
chased from The Félix d'Hérelle Reference Center for Bacterial Viruses.
All V. parahemolyticus strains were confirmed by growth in selective me-
dium (CHROMagar™ Vibrio) and the presence of the thermolabile he-
molysin gene (tlh) (Bej et al., 1999). The strains Vo ATCC33509 and Va
ATCC19264 correspond to the type strains of V. ordalii and V. anguillarum
respectively and were purchased from the American Type Culture Collec-
tion (ATCC). The bacteria were grown in artificial sea water (23.4 g/L
NaCl, 24.7 g/L MgSO4 ×7H2O, 1.5 g/L KCl, and 1.43 g/L CaCl2 ×2H2O,
pH 6.5) that had been supplemented with 1% Bactotryptone (Gifco)
and 0.5% yeast extract, at 25 °C with reciprocal shaking. TCBS medium
(Thiosulfate Citrate Bile Salts Sucrose) was used as a selective medium
for Vibrio species.
2.2. Phage isolation and production
The phages were isolated from bivalve samples that were purchased
in the central market of Santiago, Chile (Comeau et al., 2005). Briefly,
soft tissues from clams and mussels were homogenized, centrifuged
and pelleted, and the resulting supernatants were diluted 10-fold with
artificial seawater and filtered (0.22 μm). Phages in the filtrate were
detected by plating 100 μL of the supernatant, using the standard
method for a double-layer agar plaque assay and the PF4 strain of
V. anguillarum as a host; the plates were incubated overnight at 25 °C
to observe the lysis plaques. One isolated plaque was picked from the
double-layer agar plaque assay and re-plated twice to ensure clonal
phage stocks. For phage growth, 50 mL of a V. anguillarum PF4 culture
(~10 8 cells per mL, optical density 0.2–0.3 at 600 nm) was infected at
a multiplicity of infection (MOI) of 10 and incubated overnight. The cul-
ture was centrifuged at 5000 ×g for 10 min., and the supernatant was
filtered (0.22 mm filter) and subsequently stored at 4 °C.
2.3. Phage characterization
To test the sensitivity of the phages to chloroform, aliquots of the
phage stocks were treated with chloroform (0.05% final concentration),
and the untreated stocks were titered in parallel with the treated ali-
quots. The sensitivity of the phages to cold was also tested by storing al-
iquots of each phage at −80 °C in 50% glycerol for 2 weeks, after which
129
the stored aliquots and the original phage stocks were titered in
parallel.
Examination of the host range was performed by the spot test,
using the different bacterial strains to make the lawns, and 20 μL
drops of each phage to test the sensitivity of the bacteria. The bacteria
were incubated overnight at 25 °C to observe the zone of clearing
(Kutter, 2009). The broad host range vibriophage KVP40 was includ-
ed in this assay as a reference (Miller et al., 2003).
The ratio of bacterial resistance to infection by the CHOED phage
was determined by growing serial dilutions of the PF4 strain of
V. anguillarum from an exponential culture (OD620nm = 0.5) in the
presence or absence of a large amount of bacteriophage CHOED
(MOI > 100). The frequency was obtained by calculating the quotient
of the titers of the PF4 strain grown with and without phage.
For the DNA extraction, samples were incubated with DNase
(2 mg/mL) and RNase (100 mg/mL) for 1 h at 37 °C. These samples
were subsequently treated with 500 mg/mL proteinase K for 15 min,
at 65 °C. Sodium dodecyl sulfate (SDS) was added to a final concentra-
tion of 0.5% (w/v). After incubation at 65 °C for 45 min, the solution was
extracted twice with phenol–chloroform. Finally, the DNA was precipi-
tated by the addition of a 1/10 volume of 3 M sodium acetate (pH 5.0)
and two volumes of absolute ethanol at −20 °C. After the pellet was
washed with 70% ethanol, it was dissolved in TE buffer (0.01 M Tris,
0.001 M EDTA, pH 7.5). Restriction site mapping was performed by
digesting the DNA with the restriction enzyme HindIII (Promega),
according to the manufacturer's instructions. Fragments were separat-
ed by electrophoresis on a 7.5% polyacrylamide gel for 2 h at 70 V,
using the GeneRuler 1 kb DNA ladder (Fermentas) as a marker.
Transmission electron microscopy (TEM) was used to study the nu-
cleocapsid ultrastructure of the bacteriophages. Samples were prepared
on collodium copper grids, negatively contrasted with 2% uranyl ace-
tate, and examined using an electron microscope 100SX (Jeol, Japan)
at 80 KV and an instrumental magnification of 50,000.
2.4. Bacterial challenge
All of the protocols used for the challenges were supervised by a
veterinarian and approved by the ethical committee of the INTA to
comply with the European policy on the “3 Rs”, Reduce, Refine, and
Replace, to obtain reliable scientific information by using the fewest
animals possible.
The challenges in the laboratory facilities were performed under
three tank conditions, one tank for each condition, as follows: Groups
of 15 S. salar of 8–25 g per tank (100 L) were maintained in aerated
dechlorinated freshwater with 1% NaCl at 15 °C, with a water re-
circulation system. A fresh culture of the V. anguillarum strain PF4 was
added directly to the water in the corresponding tanks (bacteria alone
and bacteria plus phage), to a final concentration of 5×105 CFU/mL,
while an equal volume of fresh medium was added to the control tank.
The CHOED phage was added directly to the water immediately after
the addition of the bacteria, at the necessary concentration to obtain
the desired multiplicity of infection (MOI).
The challenges in the fish farm facilities were performed with
groups of 100 S. salar of 20–25 g per tank (250 L). The fish were
maintained at 12–15 °C, in fresh water that was adjusted to 1% of sa-
linity with seawater and in the presence of a water recirculation sys-
tem that exchanged 50% of the water every day. The tank conditions
for the challenges were set up as the conditions for the previous chal-
lenges were, with the addition of one tank condition to test the effect
of the CHOED phage alone on the fish. In this tank, the phages were
added at the same concentration as in the tank with bacteria and
phage.
The mortality in the tanks was monitored daily for each challenge. All
of the dead fish were removed from the tanks and were examined for
the presence of pathogenic bacteria on the skin, kidneys and liver; sam-
ples from these tissues were inoculated in TCBC plates (Verner-Jeffreys
130 G. Higuera et al. / Aquaculture 392–395 (2013) 128–133

et al., 2007). The concentration of bacteria and phage in the water tanks
was monitored by inoculating the corresponding dilution in TCBS plates
to detect the bacteria, and using the double-layer agar plaque assay to
determine the phage concentration.

3. Results

3.1. Bacteriophage isolation

Nine samples of shellfish that were obtained from the central mar-
ket of Santiago were analyzed during September of 2010 to detect
phages that could infect the PF4 strain of V. anguillarum isolated
from an Atlantic Salmon (S. salar) from a fish farm near Chiloe Island
(Chile) and previously identified as a serotype O3 (Silva-Rubio et al.,
2008). Bacteriophages were detected as plaque forming units (PFU)
using the standard double-layer agar plaque assay (see Material and
Methods section). Four samples tested positive for the presence of
bacteriophages, and 6 PFU were isolated from clams and mussels
and were named according to the type of sample (Table 1). All of
the isolated phages were resistant to chloroform treatment and
were active after 2 weeks in storage at − 80 °C.

3.2. Bacteriophage characterization

Genomic DNA from the recovered phages (309, ALMED, CHOED,

ALME, CHOD and CHOB) was isolated, and its size was determined
by electrophoresis in a low-percentage agarose gel, with a high
size-range molecular DNA ladder. All of the isolates possessed a ge- Fig. 1. Restriction pattern of the bacteriophage genomes. Digestion of the genomes was
nome 47–48 kb (Table 1) in size, and all of the genomes were made performed with the enzyme HindIII. The marker is the GeneRuler 1 kb DNA ladder
(Fermentas).
of double-stranded DNA, as they were digested by the restriction en-
donuclease HindIII (Fig. 1). The restriction pattern revealed that the
phages that were named ALME, ALMED and CHOB most likely corre- V. ordalii and V. anguillarum but not the V. anguillarum ATCC19264 type
spond to a single clone or are closely related (Schnabel and Jones, strain.
2001). The same conclusion can be drawn for the phages that were
named 309 and CHOED, while phage CHOD was shown to be closely 3.3. Challenge with V. anguillarum and protection with bacteriophages
related to, but different from, this last group. The ALMED, CHOED
and CHOD phages were used to determine the morphology of the To determine whether the CHOED phage could prevent vibriosis
phage groups for each of the three restriction patterns and were de- that was caused by V. anguillarum in S. salar, the challenges were
posited in the PCM, with the collection numbers F/00073, F/00074, performed in three parallel conditions: the PF4 strain of V. anguillarum
and F/00072, respectively. The electron microscopy revealed that was added to the first tank, the CHOED phage was added to the second
the ALMED phage has an icosahedral head that is 50 nm in diameter tank, and the third tank was a control tank, containing only fresh cul-
and a long contractile tail (Fig. 2a), while the CHOED phage also has ture medium. Fig. 3 shows two representative independent challenges
an icosahedral head that is 50 nm in diameter, but no tail was ob- with different MOIs. When the bacteria were added to a final concen-
served (Fig. 2b). Phage CHOD could not be observed by electron mi- tration of 5 × 10 5 CFU/mL of water in the tank, fish began to die on day
croscopy, despite several attempts. 1 after the addition of the bacteria, and by day 6, only 7% of the fish
Every phage that was isolated infected different strains of survived. However, when the CHOED phage was added with the bac-
V. anguillarum and V. ordalii, but did not infect any of the teria at a ratio of one PFU per bacterial cell, the fish remained alive
V. parahaemolyticus strains that were tested (Table 2). All of the up to 10 days post-infection, while in the control tank approximately
phages produced zones of clearing on the susceptible strains grown 70% of the fish survived by 10 days post-addition (Fig. 3a). The mortal-
in solid medium but with different intensities. The V. anguillarum ity that was observed in the control tank is not related to the bacteria,
ATCC19264 type strain was resistant to the six isolated phages. Addition- as V. anguillarum was not recovered from the water or from the dead
ally, the broad host range vibriophage KVP40 (Miller et al., 2003) that was fish of those tanks. When the amount of phage was increased to 20
included as a reference in the assay was able to infect V. parahaemolyticus, phage units per bacterial cell, similar results were observed (Fig. 3b).
On day 1 post-infection, the concentration of Vibrio bacteria in the
tank without phage was 2.8 × 106 CFU/mL, and the concentration had
Table 1
Bacteriophages of V. anguillarum isolated during 2010. The phages were considered
decreased to 1 × 103 CFU/mL by day 3 post-infection, while in the tank
sensitive when the titer decreased by more than three orders of magnitude. with phage, the concentration of Vibrio bacteria was 2.9× 104 CFU/mL
on day 1 post-infection, and by day 3 post-infection, the bacterial con-
Name Sample Date of DNA size Chloroform Cold
centration was below the limits of our detection (b1×103 CFU/mL). By
isolation (Kb) sensitivity sensitivity
(−80 °C) day 7 post-infection, the bacteria could not be detected in any of the
tanks. The phage was present in the water; even 7 days post-infection,
309 Mussels 03/9/2010 48 No No
ALMED Clams 28/9/2010 47 No No the concentration of phage was 1×106 PFU/mL. Overall, these results
CHOED Mussels 28/9/2010 48 No No strongly suggest that the CHOED phage can prevent the infection of fish
ALME Clams 28/9/2010 47 No No with the PF4 strain of V. anguillarum.
CHOD Mussels 28/9/2010 48 No No To test whether the CHOED phage can also protect fish from vibri-
CHOB Mussels 12/9/2010 47 No No
osis in farming conditions, a new set of challenges was performed in
 G. Higuera et al. / Aquaculture 392–395 (2013) 128–133 131

Fig. 2. Transmission electron microscopy of V. anguillarum bacteriophages. ALMED (a) and CHOED (b). ALMED shows a binary symmetry, with a head and a long tail. No tail was
observed in the CHOED phage samples. The scale bar represents 100 nm.

the facilities of an experimental fish farm (Centro experimental that most isolated aquatic bacteriophages and, in particular, most of
Chinquihue) located in southern Chile (Chinquihue, 41° 27′ S/72° the vibriophages belong to the Siphoviridae, Myoviridae and Podoviridae
57′ W). The results suggested that, after 20 days of infection with families (Comeau et al., 2006; Crothers-Stomps et al., 2010; Wommack
the bacteria, only 60% of the fish remained alive in the tanks. Howev- and Colwell, 2000). It was not possible to determine the morphology of
er, when 10 PFU of phage per bacterial cell was added, 100% survival the CHOD phage, but it is probable that it is morphologically similar to
was observed 20 days after infection (Fig. 4). The CHOED phage alone the CHOED and 309 phages because of its closely related restriction pat-
had no observable effect on the fish when it was added to the control tern (Fig. 1). None of the isolated phages could infect the V. anguillarum
tanks (Fig. 4). The results demonstrate that despite the high survival ATCC19264 type strain, but they can infect the V. ordalii ATCC33509
rates observed in the tanks infected with the bacteria, it is still possi- type strain. This can be explained by the fact that V. anguillarum and
ble to observe a protective effect of the CHOED phage against infec- V. ordalii are closely related (Schiewe et al., 1981; Wiik et al., 1995) and
tion by the PF4 strain of V. anguillarum. are difficult to distinguish, and V. ordalii ATCC33509 is closely related to
some V. anguillarum Chilean strains (Fernandez and Avendano-Herrera,
4. Discussion 2009).
The results presented here strongly suggest that the CHOED phage
To our knowledge, these are the first reported bacteriophages to can protect Atlantic salmon against infection by the PF4 strain of
exhibit the potential to control V. anguillarum infection in S. salar. V. anguillarum. The phage can most likely infect the bacteria in the
Phages were not previously considered to be an alternative method water as soon as it is added to the tank, because 3 days post-infection,
for preventing the vibriosis caused by V. anguillarum, despite the the bacteria was undetectable in the water (b 10 3 CFU/mL on TCBS
abundant and diverse populations of bacteriophages in marine envi- plates); the phage, however, was still present, even 7 days post-
ronments (Wommack and Colwell, 2000) and previous reports that infection. However, because previous work has shown that phages
several phages are able to infect Vibrios (Bastias et al., 2010; can enter the internal tissues of fish after ingestion (Verner-Jeffreys et
Comeau et al., 2006; Sen and Ghosh, 2005; Shivu et al., 2007). Some al., 2007), bacterial colonization could also have begun inside the fish.
phages with potential uses in the aquaculture industry have been The challenge with bacteria alone resulted in a higher survival rate
previously isolated (Crothers-Stomps et al., 2010; Karunasagar et al., in the fish farm facilities (Fig. 4) than in the laboratory facilities
2007; Nakai and Park, 2002; Park and Nakai, 2003; Thiyagarajan et (Fig. 3). This difference could be explained by the fact that in the
al., 2011). Here we describe 6 phages that according to the restriction fish farm, nearly 30% of the water used came directly from the sea.
patterns of their genomes, can be differentiated into three groups. Therefore, this water could contain different populations of bacteria
However, all of the phage groups infect a similar host range, which or even Vibrios, which could compete or act as a probiotic against
can be explained by the fact that all of the phages were isolated using the PF4 strain of V. anguillarum that was used to infect the fish,
the PF4 strain of V. anguillarum as a host. Electron microscopy revealed decreasing its pathogenic potential (Austin et al., 1995; Kesarcodi-
that the ALMED phage has an icosahedral head with a contractile tail, Watson et al., 2008; Ninawe and Selvin, 2009). In fact, different colo-
while the CHOED phage has an icosahedral head, and no tail was ob- ny morphologies were observed when water from these tanks was
served. This finding is in agreement with previous studies suggesting examined on TCBS plates. When the same procedure was performed
in the laboratory facilities, only one type of colony was observed.
Table 2 We determined that the PF4 strain of V. anguillarum exhibits a re-
Host range of the isolated V. anguillarum bacteriophages. sistance rate of 4.3 × 10 −6 when infected with the CHOED phage. This
means that after infection with the phage, there is a possibility that a
Phage Bacterial strains
small number of resistant bacteria remain in the tanks that is below
VoATCC VpKX Vp1193 Vp57.5 VaATCC VaPF4 VaPF7 VaPF8 our detection limits. However, this small quantity of bacteria seems
33509 19264
to be too few to cause morbidity and mortality in fish. Moreover, pre-
309 +++ − − − − +++ − +++ vious reports have suggested that bacteria that are resistant to phage
ALMED ++ − − − − ++ − +
infection could be less fit or could lose their pathogenic proprieties
CHOED +++ − − − − +++ − +++
ALME +++ − − − − ++ − ++ (Capparelli et al., 2010; Filippov et al., 2011), although more analysis
CHOD +++ − − − − +++ − +++ is needed to determine if this is the case.
CHOB ++ − − − − ++ − ++ One of the remaining questions about the potential use of phage to
Kvp40 +++ +++ +++ +++ − ++ NDa NDa control bacterial disease in aquaculture relates to the method used to
a
Not determined, “+” intensity of lysis, “−“ no lysis. administer the phages into the tanks. Phages have been added in
132 G. Higuera et al. / Aquaculture 392–395 (2013) 128–133
Fig. 3. Challenge of S. salar with the PF4 strain of V. anguillarum and the bacteriophage CHOED, with a MOI of 1 (a) and 20 (b). Empty squares and filled squares represent the per-
centage of fish survival in tanks with the bacteria, with or without phage, respectively. Filled circles represent the percentage of fish survival in the control tanks.
different ways, namely through intraperitoneal injections, water baths,
or with food, and all of these methods have different results (Imbeault
et al., 2006; Karunasagar et al., 2007; Park and Nakai, 2003; Verner-
Jeffreys et al., 2007). Success in the use of phages against pathogenic
bacteria could be related to the method of its addition. It is not
completely clear how V. anguillarum infects the fish, but it is known
that the colonization of the skin plays an important role in the develop-
ment of the disease (O'Toole et al., 2004; Weber et al., 2010). For this
reason, the addition of the phages directly to the water of the tanks like-
ly helps the phage to attack the bacteria throughout all of the steps of
fish infection, which is likely not the case when the method of intraper-
itoneal injection is used. The addition of the phages to the water is also a
promising method for preventing infection during the early stages of
larval rearing, especially in species that require live feeds because
phages could exclude the pathogenic bacteria from the water without
harming the beneficial microbiota. In addition, the fact that the
CHOED phage remains in the water of the tanks for a week allows us
to consider its use as a prophylactic tool to prevent V. anguillarum
infections.
Acknowledgments
We thank Samuel Valdevenito for providing the bacterial strains
and Elena Sarropoulou from the Institute of Marine Biology, Biotech-
nology and Aquaculture from the Hellenic Centre for Marine Research
for obtaining the sequence of the 16S rRNA genes. This work was par-
tially supported by grants INNOVA 240107 and FONDECYT 1110425,
1110253.
Fig. 4. Challenge of S. salar with the PF4 strain of V. anguillarum and the bacteriophage
CHOED, with a MOI of 100 in aquaculture farm conditions (Chinquihue). Empty
squares and filled squares represent the percentage of fish survival in tanks with the
bacteria, with or without phage, respectively. Empty circles represent the percentage
of fish survival in tanks with only phage, and filled circles represent the percentage
of fish survival in control tanks.
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