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Featured researches published by Channarong Rodkhum.


Veterinary Microbiology | 2014

Increasing of temperature induces pathogenicity of Streptococcus agalactiae and the up-regulation of inflammatory related genes in infected Nile tilapia (Oreochromis niloticus).

Pattanapon Kayansamruaj; Nopadon Pirarat; Ikuo Hirono; Channarong Rodkhum

Temperature strongly affects the health of aquatic poikilotherms. In Nile tilapia (Oreochromis niloticus), elevated water temperatures increase the severity of streptococcosis. Here we investigated the effects of temperature on the vulnerability and inflammatory response of Nile tilapia to Streptococcus agalactiae (Group B streptococci; GBS). At 35 and 28 °C, GBS took 4 and 7h, respectively to reach the log-phase and, when incubated with tilapia whole blood, experienced survival rates of 97% and 2%, respectively. The hemolysis activity of GBS grown at 35 °C was five times higher than that of GBS grown at 28 °C. GBS expressed cylE (β-hemolysin/cytolysin), cfb (CAMP factor) and PI-2b (pili-backbone) much more strongly at 35 °C than at 28 °C. Challenging Nile tilapia reared at 35 and 28 °C with GBS resulted in accumulated mortalities of about 85% and 45%, respectively. At 35 °C, infected tilapia exhibited tremendous inflammatory responses due to a dramatic up-regulation (30-40-fold) of inflammatory-related genes (cyclooxygenase-2, IL-1β and TNF-α) between 6 and 96 h-post infection. These results suggest that the increase of GBS pathogenicity to Nile tilapia induced by elevated temperature is associated with massive inflammatory responses, which may lead to acute mortality.


Journal of Veterinary Diagnostic Investigation | 2014

Molecular characterization and virulence gene profiling of pathogenic Streptococcus agalactiae populations from tilapia (Oreochromis sp.) farms in Thailand

Pattanapon Kayansamruaj; Nopadon Pirarat; Takayuki Katagiri; Ikuo Hirono; Channarong Rodkhum

Streptococcus spp. were recovered from diseased tilapia in Thailand during 2009–2010 (n = 33), and were also continually collected from environmental samples (sediment and water) from tilapia farms for 9 months in 2011 (n = 25). The relative percent recovery of streptococci from environmental samples was 13–67%. All streptococcal isolates were identified as S. agalactiae (group B streptococci [GBS]) by a species-specific polymerase chain reaction. In molecular characterization assays, 4 genotypic categories comprised of 1) molecular serotypes, 2) the infB allele, 3) virulence gene profiling patterns (cylE, hylB, scpB, lmb, cspA, dltA, fbsA, fbsB, bibA, gap, and pili backbone–encoded genes), and 4) randomly amplified polymorphic DNA (RAPD) fingerprinting patterns, were used to describe the genotypic diversity of the GBS isolates. There was only 1 isolate identified as molecular serotype III, while the others were serotype Ia. Most GBS serotype Ia isolates had an identical infB allele and virulence gene profiling patterns, but a large diversity was established by RAPD analysis with diversity tending to be geographically dependent. Experimental infection of Nile tilapia (Oreochromis niloticus) revealed that the GBS serotype III isolate was nonpathogenic in the fish, while all 5 serotype Ia isolates (3 fish and 2 environmental isolates) were pathogenic, with a median lethal dose of 6.25–7.56 log10 colony-forming units. In conclusion, GBS isolates from tilapia farms in Thailand showed a large genetic diversity, which was associated with the geographical origins of the bacteria.


Infection, Genetics and Evolution | 2017

Comparative genome analysis of fish pathogen Flavobacterium columnare reveals extensive sequence diversity within the species

Pattanapon Kayansamruaj; Ha Thanh Dong; Ikuo Hirono; Hidehiro Kondo; Saengchan Senapin; Channarong Rodkhum

Flavobacterium columnare is one of the deadliest fish pathogens causing devastating mortality in various freshwater fish species globally. To gain an insight into bacterial genomic contents and structures, comparative genome analyses were performed using the reference and newly sequenced genomes of F. columnare including genomovar I, II and I/II strains isolated from Thailand, Europe and the USA. Bacterial genomes varied in size from 3.09 to 3.39Mb (2714 to 3101 CDSs). The pan-genome analysis revealed open pan-genome nature of F. columnare strains, which possessed at least 4953 genes and tended to increase progressively with the addition of a new genome. Genomic islands (GIs) present in bacterial genomes were diverse, in which 65% (39 out of 60) of possible GIs were strain-specific. A CRISPR/cas investigation indicated at least two different CRISPR systems with varied spacer profiles. On the other hand, putative virulence genes, including those related to gliding motility, type IX secretion system (T9SS), outer membrane proteins (Omp), were equally distributed among F. columnare strains. The MLSA scheme categorized bacterial strains into nine different sequence types (ST 9-17). Phylogenetic analyses based on either 16S rRNA, MLSA and concatenated SNPs of core genome revealed the diversity of F. columnare strains. DNA homology analysis indicated that the estimated digital DNA-DNA hybridization (dDDH) between strains of genomovar I and II can be as low as 42.6%, while the three uniquely tilapia-originated strains from Thailand (1214, NK01 and 1215) were clearly dissimilar to other F. columnare strains as the dDDH values were only 27.7-30.4%. Collectively, this extensive diversity among bacterial strains suggested that species designation of F. columnare would potentially require re-emendation.


Journal of Fish Diseases | 2008

gyrA and parC associated with quinolone resistance in Vibrio anguillarum

Channarong Rodkhum; T Maki; Ikuo Hirono; Takashi Aoki

Keywords: gyrA, gyrB, parC, parE, quinolone resis-tance, Vibrio anguillarum.Vibrio anguillarum is a Gram-negative comma-shaped bacterium with polar flagella that is amember of the family Vibrionaceae. It is the cause ofvibriosis or haemorrhagic septicaemic disease inwild and cultured marine and freshwater fish (Actis,Tolmasky & Crosa 1999; Austin & Austin 1999).Chemotherapeutic agents, including quinolones,have been widely used for the treatment of vibriosisin cultured fish and multiple drug-resistant strainsof V. anguillarum have subsequently arisen (Aoki1992; Pedersen, Tiainen & Larsen 1995; WorldHealth Organization 1999). Drug-resistance geneson the transferable R-plasmid have been previouslycharacterized, but the R-plasmid of V. anguillarumdoes not carry any genes for quinolone resistance(Aoki, Egusa & Arai 1974). The mutations in genesand amino acids associated with quinolone resis-tance in V. anguillarum are still unknown, and itwould therefore be useful to characterize thesemutations.There are two functional domains related toquinolone resistance in Gram-negative bacteria:DNA gyrase and topoisomerase IV (Hooper2000). DNA gyrase is composed of two subunits,gyrA and gyrB, whereas topoisomerase IV comprisestwo subunits, parC and parE. Topoisomerase IVhas DNA decatenating and relaxing activities, andplays an essential role in partitioning chromosomesat the terminal stage of chromosome replication(Huang 1996; Hooper 1999, 2000). The molecularmechanisms of quinolone and fluoroquinoloneresistance have been described in several organisms(Hooper & Wolfson 1993; Hooper 2000; Chen L Okuda, Hayakawa, Nishibu-chi & Nishino 1999; Ozanne, Benveniste, Tipper& Davies 2005). To understand the mechanism ofquinolone resistance in V. anguillarum, the nucle-otide sequences of the gyrA, gyrB, parC and parEgenes were sequenced and characterized in thisstudy.Twenty-five wild V. anguillarum strains isolatedfrom cultured ayu, Plecoglossus altivelis (Temminck& Schlegel), in Japan, and V. anguillarum strainH775-3 were used (Table 2). The 25 wildstrains included 10 oxolinic acid (OA)-sensitivestrains [minimal inhibitory concentration (MIC):<0.4 lgmL


Research in Veterinary Science | 2015

The study on the candidate probiotic properties of encapsulated yeast, Saccharomyces cerevisiae JCM 7255, in Nile Tilapia (Oreochromis niloticus)

Komkiew Pinpimai; Channarong Rodkhum; Nantarika Chansue; Takayuki Katagiri; Masashi Maita; Nopadon Pirarat

Saccharomyces cerevisiae JCM 7255 was tested as a probiotic candidate in tilapia after encapsulating and freeze drying. Viability and morphology during storage and during transit through simulated gut and bile conditions were determined. Growth performance, anti-streptococcal activity and gut mucosal immune parameters were also tested. The viability of encapsulated yeasts was significantly high in simulated gastric and bile conditions and remained high after storage at room temperature for 14 days. The morphology of free S. cerevisiae revealed rough, bumpy, ruptured surface during incubation in gut and bile conditions. Agar spot anti-streptococcal activity showed inhibition of 20 out of 30 strains of Streptococcus agalactiae. Supplementation improved the intestinal structure and growth performance in tilapias. Intraepithelial lymphocytes in the proximal intestine were significantly observed. Lower cumulative mortality after the oral streptococcal challenge was also seen. The results suggest that encapsulated S. cerevisiae JCM 2755 could be a potential probiotic strain in tilapia culture.


Infection, Genetics and Evolution | 2015

Genomic comparison between pathogenic Streptococcus agalactiae isolated from Nile tilapia in Thailand and fish-derived ST7 strains

Pattanapon Kayansamruaj; Nopadon Pirarat; Hidehiro Kondo; Ikuo Hirono; Channarong Rodkhum

Streptococcus agalactiae, or Group B streptococcus (GBS), is a highly virulent pathogen in aquatic animals, causing huge mortalities worldwide. In Thailand, the serotype Ia, β-hemolytic GBS, belonging to sequence type (ST) 7 of clonal complex (CC) 7, was found to be the major cause of streptococcosis outbreaks in fish farms. In this study, we performed an in silico genomic comparison, aiming to investigate the phylogenetic relationship between the pathogenic fish strains of Thai ST7 and other ST7 from different hosts and geographical origins. In general, the genomes of Thai ST7 strains are closely related to other fish ST7s, as the core genome is shared by 92-95% of any individual fish ST7 genome. Among the fish ST7 genomes, we observed only small dissimilarities, based on the analysis of clustered regularly interspaced short palindromic repeats (CRISPRs), surface protein markers, insertions sequence (IS) elements and putative virulence genes. The phylogenetic tree based on single nucleotide polymorphisms (SNPs) of the core genome sequences clearly categorized the ST7 strains according to their geographical and host origins, with the human ST7 being genetically distant from other fish ST7 strains. A pan-genome analysis of ST7 strains detected a 48-kb gene island specifically in the Thai ST7 isolates. The orientations and predicted amino acid sequences of the genes in the island closely matched those of Tn5252, a streptococcal conjugative transposon, in GBS 2603V/R serotype V, Streptococcus pneumoniae and Streptococcus suis. Thus, it was presumed that Thai ST7 acquired this Tn5252 homologue from related streptococci. The close phylogenetic relationship between the fish ST7 strains suggests that these strains were derived from a common ancestor and have diverged in different geographical regions and in different hosts.


Journal of Fish Diseases | 2017

Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter, Scortum barcoo (McCulloch & Waite)

Pattanapon Kayansamruaj; A Rangsichol; H T Dong; Channarong Rodkhum; Masashi Maita; Takayuki Katagiri; Nopadon Pirarat

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.


Diseases of Aquatic Organisms | 2016

Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia.

Ha T. Dong; Warachin Gangnonngiw; Kornsunee Phiwsaiya; Walaiporn Charoensapsri; Vuong Viet Nguyen; Pål Nilsen; Padmaja Jayaprasad Pradeep; Boonsirm Withyachumnarnkul; Saengchan Senapin; Channarong Rodkhum

Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.


Journal of global antimicrobial resistance | 2018

Quinolone-resistant phenotype of Flavobacterium columnare isolates harbouring point mutations both in gyrA and parC but not in gyrB or parE

W. Mata; C. Putita; Ha Thanh Dong; P. Kayansamruaj; Saengchan Senapin; Channarong Rodkhum

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.


Journal of Applied Microbiology | 2018

Genome characterization of piscine ‘Scale drop and Muscle Necrosis syndrome’‐associated strain of Vibrio harveyi focusing on bacterial virulence determinants

Pattanapon Kayansamruaj; Ha Thanh Dong; Ikuo Hirono; Hidehiro Kondo; Saengchan Senapin; Channarong Rodkhum

Genomic characterization of Harveyi clade vibrio strain Y6 causing ‘Scale drop and Muscle Necrosis syndrome’ (SDMN) isolated from barramundi (Lates calcarifer) in Vietnam.

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Ha Thanh Dong

Chulalongkorn University

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Saengchan Senapin

Thailand National Science and Technology Development Agency

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Ikuo Hirono

Tokyo University of Marine Science and Technology

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Takashi Aoki

Tokyo University of Marine Science and Technology

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Hidehiro Kondo

Tokyo University of Marine Science and Technology

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