Molecular identification of Vibrio harveyi-related bacteria and Vibrio owensii sp. nov., pathogenic to larvae of the ornate spiny lobster Panulirus ornatus

Vibrio harveyi and related bacteria are important pathogens responsible for severe economic losses in the aquaculture industry worldwide. The ornate spiny lobster, Panulirus ornatus, is a potential valuable candidate as an aquaculture species but V. harveyi-related disease outbreaks during the extended larval life cycle are major constraints for the development of a breeding program for the aquaculture of this species at a commercial level. Bacterial identification methods such as phenotypic tests and 16S ribosomal RNA gene analysis fail to discriminate species within the V. harveyi group because these are phenotypically and genetically nearly identical. Multilocus sequence analysis (MLSA) was used to identify 36 V. harveyi-like isolates from the larval rearing system of P. ornatus and to re-evaluate the identity of other important Australian pathogens. Strains DY05 and 47666-1, isolated in northern Queensland from dying larvae of P. ornatus and Penaeus monodon prawns, clustered together and apart from currently recognised species. Biochemical tests, DNA-DNA hybridization, MLSA and fatty acid analyses confirmed that the two strains represent a new species of the V. harveyi group, described and validated as V. owensii (type strain DY05ᵀ = JCM 16517ᵀ = ACM 5300ᵀ = DSM =23055ᵀ).\ud \ud The phylogenies inferred from the 16S rRNA gene and five concatenated protein coding loci (rpoA, pyrH, topA, ftsZ and mreB) from the 36 isolates revealed four well supported clusters identified as V. harveyi, V. campbellii, V. rotiferianus and V. owensii. Although the topological patterns corroborated 16S rRNA gene phylogeny, the latter was less informative than each of the protein-coding genes taken singularly or the concatenated dataset. Results revealed that important V. campbellii and V. owensii prawn pathogens were previously misidentified as V. harveyi, and also that the recently described V. communis is likely a junior synonym of V. owensii. A two-locus phylogeny based on topA-mreB concatenated sequences was consistent with full the five-gene MLSA phylogeny. Global Bayesian phylogenies inferred from topA-mreB revealed more cases of potential V. owensii misidentifications in global databases such as the fully sequenced 1DA3 strain, initially described as V. harveyi. The topA-mreB combined analysis provides a practical yet still accurate approach for routine identification of V. harveyi-related species. A multiplex polymerase chain reaction (PCR) assay was designed to specifically detect and discriminate the highly similar species of the V. harveyi group (V. harveyi, V. campbellii, V. rotiferianus and V. owensii), as relevant pathogens of marine aquaculture animals. Four sets of specific primers were designed targeting three protein-coding loci, topA, the ftsZ and mreB, for DNA amplification in the four species. The single tube PCR reaction contained a mix of four specific and compatible primer sets, DNA from one, two, three or all the four target vibrio species and common PCR reagents. The designed PCR protocol allows single-step, simultaneous detection and discriminative identification of V. harveyi-like isolates based on the amplification of different size and specific DNA regions in each of the bacterial species. Any combination of DNA templates in the multiplex PCR mix results in a two-, three- or four-band pattern visualised in agarose gels. In cases of bacterial isolation from decapod crustacea, a qualitative assessment is included in the protocol to evaluate the DNA extraction method. This consists of the addition of previously designed primers for specific amplification of the18S rRNA gene in decapod crustacean. The multiplex PCR offers rapid and accurate identification of V. harveyi-like clinical and environmental isolates and reliable detection of potential pathogenic strains in clinical samples.\ud \ud A real-time PCR assay was also designed for detection and quantification of V. owensii species. The method used the SYTO9 technology for rapid and discriminative quantification of V. owensii by the amplification of a198-bp segment of the topA gene by specific primers. The detection limit was 20 fg of purified genomic DNA of V. owensii. Different dissociation temperatures were able to differentiate the lobster pathogen DY05ᵀ (83.2ºC) from the prawn pathogen 47666-1 (83.9ºC) due to a single nucleotide difference in the PCR products of these strains. The use of SYTO9 made the real-time assay more reproducible and cost-effective than SYBR or TaqMan technologies, respectively. The design of this real-time assay will allow detection and quantification of V. owensii pathogens, providing the aquaculture industry with a singleday reliable decision tool depending on the level of infection. As a research tool, it will allow the study of V. owensii dynamics in aquaculture rearing systems and in natural habitats.\ud \ud Early stage P. ornatus lobster larvae were experimentally challenged with V. harveyi-related isolates by Artemia-vector oral challenge or by immersion. For V. owensii DY05ᵀ, oral challenge caused 90% cumulative mortality after 48 h, while immersion (~10⁶ cfu ml⁻¹) caused lower (45%) and more gradual mortalities over eight days. Cell counts by serial dilutions suggested that high density of DY05ᵀ bacteria (~10⁶-10⁷ cfu ml⁻¹) maintained in either the animal (by oral challenge) or the water (by immersion), were responsible for mortalities. The results suggested that Artemia delivered the pathogen directly into the larval gut where they rapidly colonised the digestive system and caused sudden mortalities. For treatments with V. owensii 47666-1 and a control V. harveyi strain (RR36), very low cell densities were found within the larvae, still healthy by the end of the experiment, suggesting that cells were not able to colonise the animal tissues. High levels of extracellular products (ECPs) from high density DY05ᵀ cultures were also highly toxic to larvae of P. ornatus and caused similar symptoms as immersion treatments with live cells. Heat and digestion treatments indicated that heat-stable proteinaceous molecules secreted by DY05ᵀ are involved in its virulence to P. ornatus larvae.\ud \ud Protein analysis by sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDSPAGE) of whole-cell proteins revealed identical profiles for strains V. owensii 47666-1 and V. harveyi RR36 but differences between profiles of 47666-1 and DY05ᵀ. Comparison of SDS-PAGE band profiles between ECPs of the two strains resulted in the finding of 35 kDa OmpA_C-like protein DY05ᵀ following OFFGEL protein fractionation, electrophoresis separation and subsequent liquid chromatography mass spectrometry (LC-MS/MS). The highest identity of this protein was with the outer membrane OmpA_C-like protein of V. harveyi 1DA3, a strain that has been reclassified in this study, as a potential V. owensii strain. This protein, which shows high homology with other previously characterised OmpA-like proteins in V. harveyi, V. alginolyticus, V. proteolyticus and V. cholerae, is expressed on the surface of bacterial pathogens and is involved in the delivery of virulence factors to eukaryotic cells via outer membrane vesicles. OmpA could be involved in the potent colonisation ability of P. ornatus larvae by DY05ᵀ, which would allow proliferation and subsequent production of toxic ECPs, lethal to the animals. Future studies would further characterise toxins and OmpA-like proteins produced by DY05ᵀ in order to understand their function and regulation during infection