Leigh Owens

The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML.

Aims: To determine the complete nucleotide sequence of the bacteriophage VHML and establish a hypothesis for the virulence conversion caused by VHML infection of Vibrio harveyi.

A new bacteriophage, VHML, isolated from a toxin-producing strain of Vibrio harveyi in tropical Australia

Some strains of Vibrio harveyi are known to be pathogenic for fish and many invertebrates including crustaceans. Despite their importance, their modes of virulence have yet to be fully elucidated. Here, we present a previously unreported bacteriophage extracted from a toxin‐producing strain of V. harveyi isolated from moribund prawn larvae in tropical Australia. Classification into the family Myoviridae was based upon morphological characteristics (an icosahedral head, a neck/collar region and a sheathed rigid tail) and nucleic acid characteristics (double‐stranded linear DNA). We have termed the bacteriophage VHML (Vibrio Harveyi Myovirus Like). VHML is a temperate bacteriophage that has a narrow host range and shows an apparent preference for V. harveyi above other vibrios (63 Vibrio isolates tested) and other genera (10 other genera were tested). The conventional methods for phage concentration and extraction of nucleic acids from phage particles were not efficient and the alternative methods that were used are discussed.

Bacterial flora from the gut of the wild and cultured banana prawn, Penaeus merguiensis

Aims: There is growing awareness of the influence of the bacterial composition of the gut on the health and growth of the host. This study compared the bacterial flora from the digestive system of the wild and cultured prawn, Penaeus merguiensis.

Vibrio owensii sp. nov., isolated from cultured crustaceans in Australia

Two bacterial strains (DY05(T) and 47666-1) were isolated in Queensland, Australia, from diseased cultured crustaceans Panulirus ornatus and Penaeus monodon, respectively. On the basis of 16S rRNA gene sequence identity, the strains were shown to belong to the Harveyi clade of the genus Vibrio. Multilocus sequence analysis using five housekeeping genes (rpoA, pyrH, topA, ftsZ and mreB) showed that the strains form a monophyletic group with 94.4% concatenated sequence identity to the closest species. DNA-DNA hybridization experiments showed that strains DY05(T) and 47666-1 had 76% DNA similarity to each other, but <70% to their closest neighbours Vibrio harveyi LMG 4044(T) (< or =55%), Vibrio campbellii LMG 11216(T) (< or =52%) and Vibrio rotiferianus LMG 21460(T) (< or =46%). Strains DY05(T) and 47666-1 could be differentiated from their relatives on the basis of several phenotypic characteristics. The major fatty acids were C(15:0) iso 2-OH and/or C(16:1)omega7, C(16:0), C(18:1)omega7 and C(14:0). Based on the polyphasic evidence presented here, it can be concluded that strains DY05(T) and 47666-1 belong to the same novel species of the genus Vibrio, for which the name Vibrio owensii sp. nov. is proposed. The type strain is DY05(T) (=JCM 16517(T)=ACM 5300(T)).

Isolation of lytic bacteriophage against Vibrio harveyi

Aims:  The isolation of lytic bacteriophage of Vibrio harveyi with potential for phage therapy of bacterial pathogens of phyllosoma larvae from the tropical rock lobster Panulirus ornatus.

Multilocus sequence analysis provides basis for fast and reliable identification of Vibrio harveyi-related species and reveals previous misidentification of important marine pathogens ☆

Vibrio harveyi and related bacteria are important pathogens responsible for severe economic losses in the aquaculture industry worldwide. Phenotypic tests and 16S rRNA gene analysis fail to discriminate species within the V. harveyi group because these are phenotypically and genetically nearly identical. This study used multilocus sequence analysis to identify 36 V. harveyi-like isolates obtained from a wide range of sources in Australia and to re-evaluate the identity of important pathogens. Phylogenies inferred from the 16S rRNA gene and five concatenated protein-coding genes (rpoA-pyrH-topA-ftsZ-mreB) revealed four well-supported clusters identified as V. harveyi, V. campbellii, V. rotiferianus and V. owensii. 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 sp. nov. is likely a junior synonym of V. owensii. Although the MLSA topologies corroborated the 16S rRNA gene phylogeny, the latter was less informative than each of the protein-coding genes taken singularly or the concatenated dataset. A two-locus phylogeny based on topA-mreB concatenated sequences was consistent with the five-locus MLSA phylogeny. Global Bayesian phylogenies inferred from topA-mreB suggested that this gene combination provides a practical yet still accurate approach for routine identification of V. harveyi-related species.

Macrobrachium rosenbergii nodavirus disease (white tail disease) in Australia

The index case of white tail disease (WTD) is presented in adult broodstock prawns Macrobrachium rosenbergii from the Flinders River in western Queensland, Australia, in mid-2007. Histological examination revealed extensive myonecrosis with massive infiltration of myonuclei and some haemocytes. Juveniles from the same broodstock but not from 3 other families displayed white muscle lesions. Low-grade chronic mortalities approaching 100% over 1 yr occurred. Reverse transcriptase polymerase chain reactions (RT-PCR) were attempted for both M. rosenbergii nodavirus (MrNV) with 2 sets of primers and for the satellite virus, extrasmall virus (XSV). All 3 PCRs generated amplicons of the expected sizes. Basic local alignment search tool (BLAST) analyses of the 3 consensus sequences identified a 91% match with MrNV viral capsid protein gene, 96% match with MrNV RNA-directed RNA polymerase gene, and a 99% match with M. rosenbergii XSV capsid protein gene. The clinical signs, histopathological lesions and RT-PCR amplicons could be reproduced in M. rosenbergii inoculated with cell-free extracts fulfilling Rivers postulates. We conclude that this is an endemic strain of MrNV as the sequences are dissimilar to strains of MrNV circulating around Asia and the Americas. This case only poorly meets the Office International des Epizooties (OIE) case definition for WTD due to the age of the prawns involved and the nature of the inclusion bodies. Perhaps the OIE case definition needs broadening.

A newly developed ELISA showing the effect of environmental stress on levels of hsp86 in Cherax quadricarinatus and Penaeus monodon

The induction of hsps by stress in Cherax quadricarinatus and Penaeus monodon was investigated using SDS-PAGE, Western blotting and ELISA techniques. Western blotting showed the presence of an immuno-reactive protein to mouse alpha-human hsp70 IgG1 monoclonal antibody at a mass of 86 kDa (hsp86) in pleopod samples but was not sensitive enough to detect differences in response to stress. An enzyme-linked immunosorbent assay (ELISA) was developed using this antibody for the detection of hsp86 in the pleopods of C. quadricarinatus and P. monodon. Using this assay, significantly higher levels of hsp86 were detected in hyperthermally stressed C. quadricarinatus (21 to 70 g) and P. monodon (14 to 32 g) and hypoosmotically stressed P. monodon (14 to 32 g). Male C. quadricarinatus and P. monodon were thermally stressed with an increase in temperature from 24 to 33 degrees C for a period of 2 h then a recovery period of 6 h. SDS-PAGE gels of thermally stressed C. quadricarinatus and P. monodon samples revealed an increase in protein band intensity at 97 kDa (C. quadricarinatus) and 43 and 35 kDa in P. monodon. A 25 kDa mass protein was induced in C. quadricarinatus when thermally stressed. P. monodon were osmotically stressed with a decrease from 31 to 15 ppt for 2 h with a recovery of 6 h. SDS-PAGE gels revealed increased intensity of bands at 35 and 43 kDa and a 100 kDa band was induced demonstrating a wide range response of protein profile to stress in these species. SDS-PAGE gels of both species investigated also revealed an apparent reduction in band intensity of the haemocyanin subunits in stressed samples. The ELISA described here constitutes the first quantitative assay for the detection of a hsp in crustaceans and the following investigations are believed to be the first to describe the response of hsps to stress in C. quadricarinatus and P. monodon. In doing so, they provide a sound basis for future studies of the role of hsps in physiological functions in commercially cultured crustaceans.

Recombinase polymerase amplification combined with a lateral flow dipstick for discriminating between infectious Penaeus stylirostris densovirus and virus-related sequences in shrimp genome.

Penaeus stylirostris densovirus (PstDV) is an important shrimp pathogen that causes mortality in P. stylirostris and runt deformity syndrome (RDS) in Penaeus vannamei and Penaeus monodon. Recently, PstDV-related sequences were found in the genome of P. monodon and P. vannamei. This led to false positive results by PCR-based detection system. Here, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed for detecting PstDV. Under the optimal conditions, 30 min at 37°C for RPA followed by 5 min at room temperature for LFD, the protocol was 10 times more sensitive than the Saksmerphrome et als interim 3-tube nested PCR and showed no cross-reaction with other shrimp viruses. It also reduced false positive results arising from viral inserts to ∼5% compared to 76-78% by the IQ2000™ nested PCR kit and the 309F/R PCR protocol currently recommended by World Organization for Animal Health (OIE) for PstDV detection. Together with simplicity and portability, the protocol serves as an alternative tool to PCR for primarily screening PstDV, which is suitable for both laboratory and field application.

Changes to the phenotypic profile of Vibrio harveyi when infected with the Vibrio harveyi myovirus-like (VHML) bacteriophage

Aims:  To determine if infection of Vibrio harveyi with the V. harveyi myovirus‐like (VHML) bacteriophage causes a change to the phenotypic profile of this species.