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OBJECTIVES: The aim of this study was to determine mutations associated with a quinolone-resistant (QR) phenotype of Flavobacterium columnare isolates. METHODS: The susceptibility of 53 F. columnare isolates to 11 antimicrobials, including 2 quinolones, was investigated by the disk diffusion method. Oxolinic acid (OXO) was subsequently chosen for minimum inhibitory concentration (MIC) assay. Sequence analysis of four genes within the quinolone resistance-determining regions (QRDRs) of OXO-resistant F. columnare compared with susceptible isolates was subsequently performed. RESULTS: The disk diffusion assay revealed that the majority of isolates were susceptible to all tested antimicrobials. However, 14 and 8 isolates were resistant to the quinolone antibiotics OXO and nalidixic acid, respectively. No multidrug resistance was observed. The MIC assay revealed five additional isolates that were resistant to OXO (≥4µg/mL), making a total of 19 OXO-resistant isolates observed in this study. DNA sequencing identified missense mutations both in parC and gyrA but not in gyrB or parE in QR F. columnare isolates. Mutation in parC resulted in the change His87→Tyr. For gyrA, 15 isolates of Thai origin exhibited a change at residue Ser83 to either Phe, Tyr or Ala, whereas 3 Vietnamese isolates contained two mutation sites (Ser83→Phe and Asp87→Tyr). CONCLUSION: This study is the first to reveal that QR phenotype F. columnare isolates harboured missense mutations both in parC and gyrA but not in gyrB or parE of the QRDRs.

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AIMS: Genomic characterization of Harveyi clade vibrio strain Y6 causing 'Scale drop and Muscle Necrosis syndrome' (SDMN) isolated from barramundi (Lates calcarifer) in Vietnam. METHODS AND RESULTS: A bacterial genome was sequenced using Illumina MiSeq platform. Multilocus sequence analysis confirmed that the bacterium belongs to Vibrio harveyi species. Further phylogenetic analysis inferred from core genome SNPs revealed a close relationship between our bacterium and the V. harveyi isolated from groupers in Taiwan and China. blastp results indicated that V. harveyi piscine strains carried numerous adhesin, secretion system, siderophore and toxin-related genes. Genome comparison between Y6 and 32 strains of V. harveyi from different origins showed that at least 17 potential virulence genes were present exclusively in the strain Y6. Many of these (six of 17 genes) were homologous to pyoverdine siderophore, a secreted high-affinity iron chelator, clusters originally found in Pseudomonas aeruginosa. Genome of V. harveyi Y6 was incorporated by a bacteriophage VHY6φ and replication protein of the phage was most similar to CTXφ described previously in Vibrio cholerae and Vibrio fischeri. However, the cholera toxin-encoding genes, namely ctxA and ctxB, were absent from VHY6φ, while the CTXφ-enterotoxin gene (zonula occludens toxin; zot) remained intact. CONCLUSIONS: Several putative virulence genes and a phage carrying toxin gene were identified in the genomes of SDMN-associated V. harveyi Y6. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confers genomic information of the piscine pathogenic V. harveyi which recently caused widespread mortality. Such information is of importance to gain insight into bacterial molecular pathogenesis.

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Emergence of a disease with clinical signs resembling megalocytivirus infection seriously affected large-scale barramundi farms in Vietnam in 2012-2014 with estimated losses reaching $435,810 per year. An oil-based, inactivated vaccine against red sea bream iridovirus (RSIV) was applied in one farm for disease prevention without analysis of the causative agent, and the farmer reported inadequate protection. Here we describe histological and molecular analysis of the diseased fish. PCR targeting the major capsid protein (MCP) of megalocytiviruses yielded an amplicon with high sequence identity to infectious spleen and kidney necrosis virus (ISKNV) genotype II previously reported from other marine fish but not barramundi. Detection of the virus was confirmed by positive in situ hybridization results with fish tissue lesions of the kidney, liver, pancreas, and brain of the PCR-positive samples. Based on the complete sequence of the MCP gene, the isolate showed 95.2% nucleotide sequence identity and 98.7% amino acid sequence identity (6 residue differences) with the MCP of RSIV. Prediction of antigenic determinants for MCP antigens indicated that the 6 residue differences would result in a significant difference in antigenicity of the two proteins. This was confirmed by automated homology modeling in which structure superimpositioning revealed several unique epitopes in the barramundi isolate. This probably accounted for the low efficiency of the RSIV vaccine when tested by the farmer.

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In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70-90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus-specific PCR. The full-length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV-like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.

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Numerous isolates of Flavobacterium columnare were previously recovered from red tilapia, Oreochromis sp., exhibiting columnaris-like disease in Thai farms, and the phenotypic and genetic characteristics were described. The objective of this study was to determine the virulence of two morphotypes (rhizoid and non-rhizoid colonies) of F. columnare and to determine their ability to adhere to and persist in red tilapia fry. The results showed that the typical rhizoid isolate (CUVET1214) was a highly virulent isolate and caused 100% mortality within 24 h following bath challenge of red tilapia with three different doses. The non-rhizoid isolate (CUVET1201) was avirulent to red tilapia fry. Both morphotypes adhered to and persisted in tilapia similarly at 0.5 and 6 h post-challenge as determined by whole fish bacterial loads. At 24 and 48 h post-challenge, fry challenged with the rhizoid morphotype exhibited significantly higher bacterial loads than the non-rhizoid morphotype. The results suggested that an inability of the non-rhizoid morphotype to persist in tilapia fry may explain lack of virulence.

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Flavobacterium columnare is the aetiological agent of columnaris disease and severely affects various freshwater aquaculture fish species worldwide. The objectives of this study were to determine the phenotypic characteristics and genetic variability among F. columnare isolates isolated from red tilapia in Thailand. Forty-four F. columnare isolates were recovered from diseased fish in different geographical locations. The isolates exhibited homologous phenotypic characteristics but exhibited genetic diversity. One isolate was assigned to genomovar I, and the remainder were assigned to genomovar II, indicating the coexistence of these genomovars but predominance of genomovar II. Phylogenetic analysis of the 16S-23S ISR sequences revealed that a subset of the Thai isolates (n = 25) contained a smaller intergenic spacer region (ISR) (523-537 bp) and formed a unique ISR phylogenetic group. Phylogenetic analysis of the 16S rRNA gene supported the unique cluster of Thai isolates. This is the first description of the phenotypic and molecular characteristics of F. columnare isolated from red tilapia in Thailand as well as five isolates of F. columnare derived from other fish species including Nile tilapia, koi carp and striped catfish.

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The genome of Vibrio anguillarum strain H775-3 was partially determined by a random sequencing procedure. A total of 2,300 clones, 2,100 from a plasmid library and 200 from a cosmid library, were sequenced and subjected to homology search by the BLAST algorithm. The total length of the sequenced clones is 1.5 Mbp. The nucleotide sequences were classified into 17 broad functional categories. Forty putative virulence-related genes were identified, 36 of which are novel in V. anguillarum, including a repeat in toxin gene cluster, haemolysin genes, enterobactin gene, protease genes, lipopolysaccharide biosynthesis genes, capsule biosynthesis gene, flagellar genes and pilus genes.

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