Abstract
Vibrio owensii DY05 is a serious pathogen causing epizootics in the larviculture of ornate spiny lobster Panulirus ornatus. In the present study a multi-tiered probiotic screening strategy was used to identify a probiotic combination capable of protecting P. ornatus larvae (phyllosomas) from experimental V. owensii DY05 infection. From a pool of more than 500 marine bacterial isolates, 91 showed definitive in vitro antagonistic activity towards the pathogen. Antagonistic candidates were shortlisted based on phylogeny, strength of antagonistic activity, and isolate origin. Miniaturized assays used a green fluorescent protein labelled transconjugant of V. owensii DY05 to assess pathogen growth and biofilm formation in the presence of shortlisted candidates. This approach enabled rapid processing and selection of candidates to be tested in a phyllosoma infection model. When used in combination, strains Vibrio sp. PP05 and Pseudoalteromonas sp. PP107 significantly and reproducibly protected P. ornatus phyllosomas during vectored challenge with V. owensii DY05, with survival not differing significantly from unchallenged controls. The present study has shown the value of multispecies probiotic treatment and demonstrated that natural microbial communities associated with wild phyllosomas and zooplankton prey support antagonistic bacteria capable of in vivo suppression of a pathogen causing epizootics in phyllosoma culture systems.
Highlights
The ornate spiny lobster (Panulirus ornatus) is considered a prospective aquaculture species based on encouraging growout potential [1] and lucrative market value [2]
Antagonistic Bacteria The Well-diffusion Agar Assay (WDAA) confirmed 62 of 149 isolates recovered from replica plating and 29 of 356 culture collection isolates as antagonistic towards V. owensii DY05
To select antagonistic strains likely capable of interaction with phyllosoma hosts, representatives were shortlisted based on their phylogenetic identity, the strength of their in vitro antagonism in WDAA, and their environmental origin, with preferences given towards natural prey-derived isolates and bacteria associated with P. ornatus phyllosomas or their environments
Summary
The ornate spiny lobster (Panulirus ornatus) is considered a prospective aquaculture species based on encouraging growout potential [1] and lucrative market value [2]. Vibrio owensii is an emerging pathogen, with the type strain DY05 demonstrated as the etiological agent of a disease causing mass mortalities of cultured P. ornatus larvae (phyllosomas) [7,8]. We have previously shown that the planktonic form is central to vectored transmission of V. owensii DY05 [7], it was pertinent to investigate the ability of probiotic candidates to inhibit planktonic growth. Since biofilms are refuges for pathogens in aquaculture systems [15,16] and pathogen biofilms on natural tissues are inherently tolerant to conventional antimicrobial therapies [17], we wanted to investigate the ability of probiotic candidates to inhibit biofilm formation under conditions of exclusion, competition and displacement
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