Succinate dehydrogenase mutant of Listonella anguillarum protects rainbow trout against vibriosis.

Listonella anguillarum is a Gram-negative facultative anaerobic rod causing hemorrhagic septicemia in marine and rarely in freshwater fish. Succinate dehydrogenase (SDH) plays an important role in the tricarboxylic acid (TCA) cycle by oxidizing succinate to fumarate while reducing ubiquinone to ubiquinol. Recent studies indicate that central metabolic pathways, including the TCA cycle, contribute to bacterial virulence. However, the role of SDH in L. anguillarum virulence has not been studied. Here, we report in-frame deletion of the succinate dehydrogenase iron-sulfur protein (SDHB) and its role in L. anguillarum virulence in rainbow trout. To accomplish this goal, upstream and downstream regions of the L. anguillarum sdhB gene were amplified in-frame and cloned into a suicide plasmid. The chromosomal sdhB gene of L. anguillarum was deleted by homologous recombination. Virulence and immunogenicity of the L. anguillarum ΔsdhB mutant (LaΔsdhB) were determined in rainbow trout. Results show that LaΔsdhB was highly attenuated in rainbow trout, and fish immunized with LaΔsdhB displayed high relative survival rate after exposure to wild type L. anguillarum. These findings indicate SDH is important in L. anguillarum virulence in rainbow trout, and LaΔsdhB could be used as an immersion, oral, or injection vaccine to protect rainbow trout against vibriosis.

Phenotypic and genetic diversity of coexisting Listonella anguillarum, Vibrio harveyi and Vibrio chagassi recovered from skin haemorrhages of diseased sand smelt, Atherina boyeri, in the Gulf of Trieste (NE Adriatic Sea).

AIMS: This study identified and characterized coexisting Vibrios associated with haemorrhagic skin lesion bearing sand smelt fishes (Atherina boyeri) in north-eastern Adriatic Sea. METHODS AND RESULTS: Bacteria were isolated from external skin lesions of four samples, and representative morphotypes grown on thiosulfate-citrate-bile salt-sucrose agar were isolated. In total 25 isolates, presumptively assigned to Vibrio genus, were biochemically characterized and were grouped in 10 phenotypic profiles. Phenotypes were heterogeneously distributed among the diseased sand smelt analysed; only one phenotype was recovered from all the samples. Sequencing of 16S rRNA was performed to identify representatives of all phenotypes. Phylogenetic analysis using the neighbour-joining method revealed six isolates clustered within the Vibrio harveyi group, three clustered with known Vibrio chagasii strains and three clustered with Listonella anguillarum. CONCLUSIONS: Vibrios with a broad phenotypic variability were found in the external lesions of diseased A. boyeri. In total three species of Vibrio were identified: V. harveyi showed the wider phenotypical and ribotypical heterogeneity while L. anguillarum shared similar biochemical characteristics with typical strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Previously unreported coexistence of potential pathogenic species colonizing diseased A. boyeri has ecological as well as epidemiological significance.

The genus Listonella MacDonell and Colwell 1986 is a later heterotypic synonym of the genus Vibrio Pacini 1854 (Approved Lists 1980)--a taxonomic opinion.

We analysed the taxonomic position of the genus Listonella based on phylogenetic, genomic and phenotypic data. The species of the genus Listonella were nested within the genus Vibrio according to the 16S rRNA gene sequence-based phylogenetic tree. The closest neighbour of Vibrio (Listonella) anguillarum strains LMG 4437(T) and ATCC 68554 (=strain 775) was Vibrio ordalii LMG 13544(T), with more than 99.5% 16S rRNA gene sequence similarity. Furthermore, Vibrio (Listonella) pelagius is highly related to Vibrio splendidus. According to average amino acid identity (AAI), multilocus sequence analysis (MLSA) and Karlin genome signature, the closest neighbour of L. anguillarum ATCC 68554 is V. ordalii LMG 13544(T), with 95% AAI, 98% MLSA and 5 in Karlin. V. anguillarum ATCC 68554 and Vibrio cholerae N16961 had 77% similarity in AAI, 85% in MLSA and 14 in the Karlin signature. Phenotypic analyses of previously published data for V. (L.) anguillarum and V. (L.) pelagius revealed that the genus Listonella is extremely similar to the genus Vibrio. V. ordalii and L. anguillarum strains yielded up to 67% DNA-DNA hybridization. There are only a few phenotypic features that might be used to discriminate these two species: L. anguillarum is positive for the Voges-Proskauer reaction, citrate utilization, starch hydrolysis, lipase activity and acid production from glycerol, sorbitol and trehalose, whereas V. ordalii is negative for these traits. We suggest that the genus Listonella is a later heterotypic synonym of the genus Vibrio and propose to use the names Vibrio anguillarum and Vibrio pelagius in place of Listonella anguillarum and Listonella pelagia, respectively.

[Characterization of Listonella anguillarum as the aetiological agent of vibriosis occurred in cultured ayu (Plecoglossus altivelis) in Ninghai country, China].

OBJECTIVE: Ayu (Plecoglossus altivelis) vibriosis threatens ayu aquaculture seriously caused by mass mortality due to severe infections. We characterized the vibriosis pathogen of ayu in Ninghai country. METHODS: A dominant strain was isolated and identified by a series of biochemical and physiological tests. The lethal dose 50% (LD50) was calculated by the modified Karber's method. PCR amplification and sequence analysis were used to further identify the pathogen. RESULTS: LD50 of ayu-H080701 was 1.2 x 10(4) CFU to ayu. PCR amplification showed that the bacterial universal primers for 16S rRNA gene and the specific primers for the metalloprotease (MP) gene of Listonella anguillarum worked. 16S rRNA gene analysis showed that ayu-H080701 shared 99.4%-99.5% nucleotide identical to L. anguillarum isolates, while 94.3% and 91.9% nucleotide identical to L. pelagius and Photobacterium damselae respectively. Metalloprotease analysis showed that ayu- H080701 shared 97.6%-98.8% amino acid sequence identical to L. anguillarum isolates, while lower than 75.6 % to other bacteria. Phylogenetic analysis showed that ayu-H080701 grouped constantly with L. anguillarum isolates. CONCLUSION: The biochemical, physiological tests and sequence analysis all strongly supported the identification of the pathogen causing ayu vibriosis in Ninghai country, China, as an isolate of L. anguillarum.