Bacteriophage therapy for the control of Vibrio harveyi in greenlip abalone (Haliotis laevigata)

Highlights

• Bacteriophage therapy can be used for bacterial infections in abalone

• Pathogenic Vibrio harvey was successfully controlled with the use of specific bacteriophages

• Bacteriophage therapy has the potential to be a useful therapeutic tool in the abalone industry

Abstract

Bacteriophage therapy has demonstrated promising results towards the control of bacterial infections within the aquaculture industry as an alternative therapy to antibiotics. This current research describes the efficacy of bacteriophage therapy in controlling vibriosis within abalone (Haliotis laevigata). Two bacteriophages were isolated and used in in vitro assays to determine the effect of each specific phage on the growth of specific Vibrio harveyi isolates. To demonstrate efficacy, an in vivo bioassay was performed using abalone (H. laevigata) at a water temperature of 24 °C. Characterisation of the two isolated phages revealed they were from the family, Siphoviridae. The phages had different antimicrobial abilities towards different Vibrio harveyi isolates in the in vitro assays and the bioassay demonstrated improved survival of abalone treated with bacteriophage. The results of the bioassay showed that the negative control and phage control were statistically identical, with 100% survival, but they were different from the positive bacterial control, with a 0% survival. The bacteria + phage treated group showed increased accumulative survival of 70% compared to the positive control. This is the first study to demonstrate the efficacy of bacteriophage therapy treatment for abalone. The study demonstrates that if abalones are treated with bacteriophage shortly after Vibrio harveyi infection, the accumulative survival could be significantly increased.

Introduction

As most commercial land-based abalone farms are intensive systems (Handlinger et al., 2005, Stone et al., 2014), the industry is under high risk of mass mortality when the animals are exposed to infectious agents or the culture conditions are undesirable (Cardinaud et al., 2014b). Common pathogens, which cause serious infections in intensive abalone production systems includes viruses, bacteria, and parasites. (Vandepeer, 2006, Liggins et al., 2010, Mayfield et al., 2011).

Vibriosis is one of the primary bacterial diseases in the aquaculture industry. This disease is caused by a range of Vibrio species, which can lead to mass mortality in a relatively short time (Jiang et al., 2013). Vibriosis-associated mass mortalities in abalone have been reported in France (Travers et al., 2008b, Pichon et al., 2013, Cardinaud et al., 2014a, Meistertzheim et al., 2014), Australia (Handlinger et al., 2005, Travers et al., 2009b, Dang et al., 2012, Stone et al., 2014), and Japan (Travers et al., 2009b). Abalone vibriosis usually occurs when animals are immunosuppressed due to conditions such as elevated water temperature or spawning (Pichon et al., 2013, Cardinaud et al., 2014a). During the summer period, when the water temperatures are above 23 °C, there have been mass mortalities on commercial farms, referred to as summer mortality in abalones (Travers et al., 2008b, Lange et al., 2014, Stone et al., 2014). Previous studies showed that individual abalone undergoing gonad development are more susceptible to vibriosis, while immature abalone are more resistant to natural infection (Travers et al., 2009a, Meistertzheim et al., 2014). Since controlling water temperature on farms during such elevated temperatures is impractical for long periods of time, there is an industry need for effective vibriosis treatments for mature abalone.

Antibiotics have traditionally been used in the treatment of bacterial diseases in aquaculture species (Defoirdt et al., 2007, Karunasagar et al., 2007); however, the efficacy of antibiotics is decreasing due to increasing rates of antimicrobial bacterial resistance. In some countries, antibiotics have been banned as the residues pose a threat to human health and the environment (Defoirdt et al., 2007, Phumkhachorn and Rattanachaikunsopon, 2010, Rong et al., 2014). Alternative control methods are required by the seafood industry to reduce mortality and minimize the impact on human health and the environment. Consequently, bacteriophage (phage) therapy is considered one such potential alternative treatment (Karunasagar et al., 2007, Crothers-Stomps et al., 2010, Phumkhachorn and Rattanachaikunsopon, 2010, Silva et al., 2014b).

Phage therapy has been applied as a treatment for vibriosis within the seafood industry (Defoirdt et al., 2011). There are reports of phage application involving live animals such as post larvae prawns (Karunasagar et al., 2007), oysters (Pelon et al., 2005, Rong et al., 2014), fish larvae production (Silva et al., 2014b), and Atlantic salmon (Higuera et al., 2013), however, there are no published studies to determine if abalone can be effectively treated using the therapy. Phage therapy has also been used for seafood processing, for example, as surface treatment of channel catfish and raw salmon fillets (Soni and Nannapaneni, 2010, Soni et al., 2010), and raw oysters (Jun et al., 2014a). There is a single report of a wild type phage association with black abalone (Haliotis cracherodii), providing protection from the rickettsial withering syndrome (Stobart et al., 2012), which is associated with intracytoplasmic rickettsia-like organisms (Friedman et al., 2014). Friedman et al. (2014) showed that the presence of natural phages reduced mortality in abalone populations, but they did not apply phage as a therapy. Yu et al. (2013) isolated and characterized five lytic phages from an abalone farm and found there was potential to apply phage therapy for controlling V. owensii induced vibriosis in abalone. However, there are no further studies attempting to use the selected phages as treatment for abalone vibriosis.

This study aimed to determine whether bacteriophage therapy could be used to control bacterial diseases in greenlip abalone (Haliotis laevigata) using V. harveyi as an infection model. The hypotheses of this study were that phage(s) will inhibit or decrease the growth of the model bacteria in vitro, and selected phage(s) can be used to control vibriosis in abalone.