Bacteriophage therapy for the control of Vibrio harveyi in greenlip abalone (Haliotis laevigata)
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.
Section snippets
Vibrio harveyi strains
All isolates were from the School of Animal and Veterinary Sciences culture collection at the University of Adelaide, Roseworthy campus. The isolate used in the abalone bioassay was collected from oysters in Shark Bay, West Australia.
The Vibrio strains, which were used to amplify bacteriophages, were propagated using bacteriological peptone, yeast, sea salt (PYSS) agar plates (5 g bacterial peptone, 1 g yeast extract, 33 g synthetic sea salt, and 15 g agar per liter), incubated at 28 °C overnight
Prophage detection in bacterial collection
Optical density curves from the MMC assay indicated that there were no prophages in the Vibrio harveyi strains used in the subsequent experiments, as there was no changes in the growth curves for all bacterial strains (results not shown) compared to the controls. No prophage induction was indicated by optical measurements of MMC treated cultures.
Bacteriophage identification
Two bacteriophages in total were isolated and named vB_VhaS-a and vB_VhaS-tm following recommended bacteriophage nomenclature. vB_VhaS-a was from water
Discussion
This study aimed to establish an alternative treatment for vibriosis as a replacement to antibiotics in abalone production systems. A single phage, vB_VhaS-tm, was identified as most efficient in reducing the density of Vibrio harveyi isolate MO10, which was then selected as the trial phage for the bioassay. In the abalone challenge model, the phage vB_VhaS-tm resulted in 70% accumulative survival of phage treated group compared to 0% accumulative survival of the positive (bacterial) control
Acknowledgements
The original experimental isolates were gratefully provided by Dr. Nicky Buller from Animal Health Laboratories, Department of Agriculture and Food Western Australia. The abalone feed was kindly provided by Aquafeeds Australia PTY LTD.
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