1. Introduction
Vibrionaceae is a genetic and metabolic diverse family of heterotrophic bacteria which are widespread in aquatic environments around the world [
1]. Several vibrios are able to infect a wide range of aquatic animals and constitute therefore a large problem in aquaculture [
2]. One of the most important is
Vibrio anguillarum, which causes the disease vibriosis and is responsible for large-scale losses in the aquaculture industry [
3,
4]. Chemotherapy against vibriosis is associated with a major concern due to the risk of antibiotic-resistance developing in the pathogenic bacteria [
5]. Vaccines against vibrio have been successful in preventing disease [
6,
7], however, they are often not useful at the larval stage, as the immune system is not fully developed. Therefore, alternative methods for the control and treatment of
V. anguillarum infections in fish larvae and fry are needed. The use of bacteriophages (phages) has been explored in several studies as a treatment of pathogens in aquaculture [
4,
8,
9,
10,
11,
12,
13]. Pereira et al. [
4] and Mateus et al. [
11] did in vitro assays with phages infecting different bacteria responsible for the diseases vibriosis and furunculosis and showed that both single-phage suspensions and phage cocktails could inactivate the bacteria [
4,
11]. However, often regrowth of phage tolerant bacteria was observed within 24 h after phage treatment [
11,
13]. Phage addition to shrimp larvae infected with
V. harveyi caused a reduction in the pathogen load and significantly increased shrimp survival compared to untreated controls groups as well as parallel treatments with antibiotics [
8,
9]. Another study on zebrafish larvae infected with
V. anguillarum [
12] also found significantly enhanced larvae survival after phage addition. Successful phage treatment in Atlantic salmon (
Salmo salar L.) infected with
V. anguillarum strain PF4 was found for phage CHOED, resulting in complete elimination of pathogen-induced mortality when phages were added at a high multiplicity of infection [
10]. Together, the previous experimental approaches demonstrate that phage therapy can be a feasible alternative method to control specific
Vibrio pathogens in aquaculture. However, the use of phages is complicated by the fact that multiple strains of the
Vibrio pathogens with different phage susceptibility patterns may coexist in aquaculture environments [
14]. The implications of strain diversity for the efficiency of phage control may be overcome either by combining several phages which target a broad range of pathogenic hosts, or to use a broad-host-range phage which can infect multiple strains within a given species or even multiple species [
15]. The phage KVP40 represents a broad-host-range phage which infects at least eight species of
Vibrio sp. (
V. parahaemolyticus,
V. alginolyticus,
V. natriegens,
V. cholerae,
V. mimicus,
V. anguillarum,
V. splendidus, and
V. fluvialis) and one
Photobacterium sp. (
P. leignathi) [
16]. All of these species contain a 26-kDa outer membrane protein named OmpK, which is a receptor for phage KVP40 [
17].
The application of phages for controlling pathogens may be hampered by the development of phage resistance in the bacteria [
18], and several mechanisms have been described in
V. anguillarum which can eliminate or reduce bacterial sensitivity to phages and thus limit the efficiency and duration of phage control [
19].
The aim of this study was to examine the effect of phage KVP40 on the survival of turbot and cod larvae challenged with four different V. anguillarum strains. Larval mortality and abundance of bacteria and phages were quantified to determine the potential of using phage KVP40 to control V. anguillarum infections during the early larval stage. In general, phage KVP40 was able to reduce or delay the mortality of both turbot and cod larvae in all the challenge trials and reduce larval mortality imposed by the background population of pathogens.
The results demonstrated that phage KVP40 reduced the mortality imposed by the added pathogens as well as other Vibrio pathogens already present in the environment during the initial 1–4 days of the experiment, emphasizing the potential of using phages to reduce turbot and cod mortality at the larval stage.
3. Discussion
In general, the addition of phage KVP40 reduced or delayed the mortality of turbot and cod larvae challenged with
V. anguillarum, with the largest effect observed for strain 4299, where the relative turbot and cod mortality was reduced by 22–33% and 72%, respectively, by the end of the experiment. In most of the challenges, the positive effect of phage KVP40 addition on larval survival was maintained throughout the incubation period. However, incubation with strain PF430-3 showed a temporary effect of phage addition on mortality and larval mortality reached the same level as in the bacterial challenges (without phage) after 4–10 days. Since the phage was maintained in high concentrations throughout the experiment, it is likely that strain PF430-3 was protected against infection, which supports previous observations that strain PF430-3 can reduce its susceptibility to phage KVP40 by forming aggregates or biofilm, creating spatial refuges [
20].
In addition to the specific V. anguillarum pathogens, other pathogens already associated with the fish eggs prior to the experiments were present in the experiments. This allowed an assessment of the effects of phages on both the mortality caused by the V. anguillarum strains and the mortality imposed by the natural background pathogen communities. The decrease in mortality recorded for all the phage controls (without V. anguillarum) compared to the nonchallenge controls (without phage and V. anguillarum) demonstrated a strong effect of phage KVP40 on the initial bacterial pathogen communities associated with the eggs. This was supported by the observation that >85% of the isolated colonies originating from the background bacterial community were susceptible to phage KVP40.
Despite the large fraction of phage susceptible strains, the bacterial abundance increased in all the incubations over time, and only in cod challenge trial 2 did addition of phage KVP40 reduce the bacterial abundance for multiple days. This suggested that during the experiment, pathogens that were not infected by KVP40 (i.e., non-Vibrio pathogens and possibly phage-resistant V. anguillarum strains) replaced the phage susceptible strains, and thus were the main cause of mortality in the experiments. This was supported by the increased effects of phages on mortality in cod challenge trial 2, where the eggs were pretreated with 25% glutaraldehyde. These results emphasized that the growth of other pathogens than V. anguillarum was the main cause of mortality in the experiments that were not pretreated with glutaraldehyde, and that phage KVP40 was able to significantly reduce mortality imposed by the added V. anguillarum strains.
Consequently, even though the presence of a bacterial background pathogen community masked the effect of phage KVP40 on the added
V. anguillarum strains, it at the same time provided a more realistic demonstration of how the addition of phage KVP40 will affect an infected aquaculture system. These results emphasized the potential of phage KVP40 to control not only the added host strains but also a broader range of pathogens present in the rearing facilities. Similar results were obtained for the two broad-host-range KVP40-like phages
φSt2 and
φGrn1 infecting the fish pathogens
V. alginolyticus [
21]. These phages were able to reduce the natural
Vibrio load present in
Artemia live feed cultures used in fish hatcheries. The current study is, however, the first demonstration of a positive effect of phage application on larval survival by reducing the natural microbiota, rather than exclusively focusing on the effects of one added pathogen. While the composition of the background microbiota was not analyzed in the current study, previous studies have found that bacterial communities associated with cod and turbot eggs in rearing units were dominated by
Pseudomonas,
Alteromonas,
Aeromonas, and
Flavobacterium [
22], but also
Vibrio has been shown to be prevalent in these environments [
23]. In our study, the high fraction of bacteria growing on
Vibrio-selective TCBS medium combined with the high susceptibility to phage KVP40 suggested that the background bacterial community was dominated by
Vibrio or
Vibrio-related species, as the phage KVP40 has been shown to infect at least eight
Vibrio species and one
Photobacterium [
16]. This was also supported by preliminary analysis of the microbiome associated with the turbot eggs used in challenge trial 2, which showed dominance of
Vibrio species (Dittmann, unpublished results). The differences in mortality in the control treatments (nonchallenged control and KVP40 control) between different experiments may therefore reflect differences in the composition of background bacterial community, representing differences in virulence and KVP40 susceptibility. Further, higher incubation temperature of the turbot than cod eggs may also have increased bacteria-induced mortality in the turbot experiments. In one of the treatments (cod challenge trial 1 with strain 90-11-286), addition of phage KVP40 increased larval mortality (
Figure 4c). Specific secondary metabolites or toxins released during cell lysis may potentially inhibit larval growth [
24]. However, since this was not observed in any of the other treatments, it is not likely that the viral lysates affected the cod larvae. Alternatively, the viral lysates may have stimulated growth of other specific pathogens already present in the experiment, as also indicated by the enhanced bacterial growth in the phage added culture (
Figure 6c). Previous studies have shown that lysogenization of
V. harveyi with phage VHS1 increased the virulence of the bacterium against black tiger shrimp (
Penaus monodon) by the phage encoded toxin associated with hemocyte agglutination ([
25]). There has not been any indication of lysogenization of
Vibrio pathogens with phage KVP40, and the production of a KVP40-encoded toxin is therefore not a likely explanation for the observed increase in larval mortality in this experiment.
Our results support previous attempts to control pathogens in aquaculture by use of phages. A challenge trial in Atlantic salmon using
V. anguillarum strain PF4, a close relative to strain PF430-3 used in the current study [
13], showed 100% survival using the phage CHOED, independent of the original multiplicity of infection (MOI) [
10]. The efficiency of this phage on fish survival compared to the current study most likely relates to the fact that larger fish are more robust against infections by co-occurring pathogens than larvae. A delay in mortality after phage addition was also observed by Imbeault et al. [
26] and Verner-Jeffreys et al. [
27] in brook trout and Atlantic salmon, respectively, infected with
A. salmonicida using different phages. While Imbeault et al. [
26] were able to delay the onset of disease and reduce the mortality to 10%, Verner-Jeffreys et al. [
27] also demonstrated a delay in the mortality, but only observed a temporary effect of the phages in survival.
Previous in vivo challenge studies with a positive outcome of phage therapy were conducted on >5 day old larvae [
12] or fish averaging 15–25 grams [
10], while our study was conducted on eggs which hatched during the course of the challenge trials. Eggs and newly hatched larvae are more sensitive to the infection by pathogenic
V. anguillarum and other pathogens than late stages due to the inefficient protection provided by the intestinal microflora associated with their gut mucosa, which constitutes a primary barrier [
28]. Despite the general frailty of newly hatched larvae, we demonstrated a significant phage-mediated reduction in mortality of cod and turbot larvae in experimental challenge trials with
V. anguillarum pathogens in combination with the natural pathogenic bacteria associated with the incubated fish eggs. These results emphasize that phage therapy is a promising approach to reduce pathogen load and mortality in marine larviculture.