• Journal of fish pathology
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• v.37 no.1
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• pp.49-60
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• 2024

A viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when faced with unfavorable environmental conditions, rendering them unable to grow on nutrient agar while maintaining low metabolic activity. This study explored the impact of temperature and nutrient availability on inducing VBNC state in Edwardsiella piscicida, the most important bacterial fish pathogen, and assessed its pathogenicity at VBNC state. E. piscicida was suspended in filtered sterile seawater and exposed to three different temperatures (4, 10, and 25℃) to induce the VBNC state. Subsequently, the induced VBNC cells were subjected to resuscitation by either raising the temperature to 28℃ or inoculating them in brain heart infusion broth supplemented with 1% NaCl. A propidium monoazide (PMA)-qPCR method was also developed to selectively quantify live (VBNC or culturable) E. piscicida cells. The results showed that the bacteria entered the VBNC state after approximately 1 month at 4℃ and 25℃, and 2 months at 10℃. The VBNC E. piscicida cells were successfully revived within 3 days in a nutrient-rich environment at 28℃, highlighting the significance of temperature and nutrition in inducing and resuscitating the VBNC state. In pathogenicity tests, resuscitated E. piscicida cells exhibited high pathogenicity in olive flounder comparable to cultured bacteria, while VBNC cells showed no signs of infection, suggesting they are unlikely to resuscitate in fish. In conclusion, this study contributes to our understanding of fish pathogen ecology by investigating the characteristics of the VBNC state under varying temperature and nutrition conditions.

• Journal of Life Science
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• v.31 no.1
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• pp.28-36
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• 2021

Edwardsiella piscicida is a significant cause of hemorrhagic septicemia in fish and gastrointestinal infections in humans. Survival bacteria require specialized mechanisms to adapt to environmental fluctuations. Hence, to understand the mechanism through which E. piscicida senses and responds to environmental osmolarity changes, we determined the protein expression profile and physiological properties under various salinity conditions in this study. The OmpR protein is a part of the Env-ZOmpR two-component system that has been implicated in sensing salt stress in bacteria. However, the physiological role played by this protein in E. piscicida remains to be elucidated. Therefore, in this work, the function of the OmpR protein in response to salt stress was investigated. Phenotypic analysis revealed that, in the mutant, three of the biochemical phenotypes were different from the wild type, including, citrate utilization, hydrogen sulfide, and indole production. Introduction of the plasmid containing the entire ompR gene to the mutant strain returned it to its parental phenotype. The retarded growth rate also partially recovered. Furthermore, in our studies, OmpR was not found to be related to cell motility. Taken together, our results from the mutational analysis, the growth assay, MALDI-TOF MS, qRT-PCR, and the phenotype studies suggest that the OmpR of E. piscicida is implicated in osmoregulation, growth, expression of porins (ETAE_1826), virulence-related genes (EseC, EseD and EvpC), and certain genes of unknown function (ETAE_1540 and ETAE_2706).

• Journal of fish pathology
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• v.33 no.2
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• pp.111-118
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• 2020

The genus Edwardsiella belonging to the family Enterobacteriaceae is a member of Gram-negative rod-shaped bacteria that cause disease in diverse aquatic organisms such as fish, amphibians and reptiles as well as avians and mammals including human throughout the world. This genus had been composed of three species, E. hoshinae, E. ictaluri and E. tarda, but recent researches erected two novel species, E. anguillarum and E. piscicida that were conventionally identified as E. tarda. In this study, we isolated seven strains belonging to the genus Edwardsiella from freshwater fishes that had been reared at inland fish farms in South Korea and investigated their biochemical characteristics and molecular phylogenetic relationships. The seven strains showed typical characteristics of four Edwardsiella species, E. anguillarum, E. ictaluri, E. piscicida and E. tarda, by biochemical analyses of Gram staining, indole and hydrogen sulfide (H2S) production, and API (Analytic Profile Index) 20E test. Molecular phylogenetic analyses inferred from DNA sequence data of both 16S ribosomal RNA (rRNA) and DNA gyrase subunit B (gyrB) genes were congruent with the biochemical characteristics. As a result, both biochemical and molecular phylogenetic analyses identified four strains isolated from three Anguilla species as E. anguillarum, E. piscicida and E. tarda, two strains from Pelteobagrus fulvidraco and Silurus asotus as E. ictaluri, and one strain from Moroco oxycephalus as E. piscicida. In this study, we isolated and successfully identified recently newly erected species, E. anguillarum and E. piscicida in addition to historically notorious pathogenic species, E. ictaluri and E. tarda. In the future study, systematic and comprehensive monitoring of the four Edwardsiella species are required for studying differences in pathogenicity among freshwater fishes.

• Journal of Microbiology and Biotechnology
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• v.29 no.8
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• pp.1273-1280
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• 2019

Edwardsiella piscicida is the causative agent of edwardsiellosis, which has caused enormous economic losses worldwide. In our previous research, an attenuated live vaccine known as WED and based on the virulent strain E. piscicida EIB202 can effectively protect turbots against edwardsiellosis via intraperitoneal injection, while vaccination by immersion exhibits a weaker effect. During the development of the immersion vaccine, we surprisingly found the counts of ${\Delta}pEIB202/EIB202$ colonized on zebrafish were 100 times lower than those of EIB202. However, pEIB202 carries 53 predicted ORFs and has several copies in E. piscicida EIB202, impeding the study of its function. Thus, the replication region is located at a 1,980 bp fragment (from 18,837 to 20,816 bp), containing a transcriptional repressor and a replication protein. Moreover, the minimal replication plasmid, named pRep-q77, has low copies in both E. coli and E. piscicida, but is more stable in E. piscicida than in E. coli. This work lays a foundation for further examination of the function of the virulence plasmid pEIB202.

• Journal of fish pathology
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• v.37 no.1
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• pp.61-69
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• 2024

In this study, bacterial disease monitoring was conducted from July to November 2022 for three representative bacterial species (Streptococcus parauberis, Vibrio spp. and Edwardsiella piscicida) to investigate the occurrence of bacterial diseases in farmed olive flounder (Paralichthys olivaceus) on Jeju. Over a period of five months, 3,146 olive flounders were tested. The average length and weight of the olive flounder that were sampled for the disease were found to be 32.1 cm and 357.9 g, respectively. Bacteria were detected in 1,228 olive flounders, of which 24.6% were identified as Vibrio spp.. S. parauberis and E. piscicida were identified in 6.3% and 3.2% respectively, and 3% were unknown strains that could not be identified. Also, 41 (1.3%) olive flounders were found to have both S. parauberis and Vibrio spp. and 23 (0.7%) olive flounders were found to have both E. piscicida and Vibrio spp.. In particular, S. parauberis, Vibrio spp. (V. scophthalmi) and E. piscicida were detected simultaneously in one olive flounder. In fish infected with E. piscicida, the most obvious external signs were hernias and ascites.

• Journal of fish pathology
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• v.6 no.1
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• pp.57-64
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• 1993

The properties and DNA structures of R plasmids differ depending on the species of the fish-pathogens Aeromonas hydrophila, A. salmonicida, Edwardsiella tarda, Enterococcus seriolicida, Pasteurella piscicida and Vibrio anguillarum. However, some R plasmids with the same resistance markers in similar DNA structures were found in A. hydrophila and E. tarda, as well as in A. hydrophila and A. salmonicida. R plasmids from V. anguillarum were classified into three groups according to their DNA structures. The first group was detected before 1977, the second from 1980 to 1983, and the third from 1989 to 1991. R plasmids have been retained within P. piscieida having the same DNA structures and detected at various locations and times. E. seriolicida strains carrying the same R plasmids, which were encoded with resistance to macrolide antibiotics(MLs), lincomycin(LIM), and TC, and to MLs, LIM, and CP. were distributed in yellowtail farms in various districts. The chloramphenicol-resistance(cat) gene of the R plasmids of P. piscicida was classified as CAT type I. The cat of the R plasmids of E, tarda. A. salmonicida was classified as type II. The cat of R plasmids of V. anguillarum was classified into two types. One type detected before 1977, was classified as CAT IV and the other type, detected after 1980, was classified as CAT II. Tetracycline-resistance (tet) V. anguillarum, isolated before 1977 and after 1981, was classified as Tet B and Tet G, respectively. The class D tet gene was widely distributed in R plasmids from fish-pathogens A. hydrophila, E. tarda, P. piscicida, and also V. anguillarum isolated after 1989.

• Journal of Veterinary Clinics
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• v.19 no.1
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• pp.19-22
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• 2002

The examination with 96 bacteria isolates for antibiotics susceptibility was resulted in exploring many antibiotics resistance isolates from the diseased fishes. Vibrio sp. isolates were demonstrated over 90% resistances for Penicillin (P), Amoxicillin (Amc), Erythromycin (I), Colistin (CL). Streptomycin (S). Novobiocin (NV) and Neomycin(N), these isolates were, however, appeared over 80% susceptibilities for Norfloxacin (Nor), CE and UB. In Edwardsiella tarda case, 90 to 100% high resistance was observed for P, Doxycycline (Do), Tetracyclin (Te), Oxytetracycline (T), E, CL, Sulfamethoxasoletrimetoprim (SXT), S, Oxolinic acid (OA), W, Neomycin(N), on the contrary, 90 to 100% high susceptibilities was found for Amc, Nor, Ciprofloxacin (Cip), Orbifloxacin (ORB), Enrofloxacin (ENR), Flumeguine (UB) and NA. CL, Pefloxacin (PEF),S, Flumeguine (UB), OA, NA, NV N was exhibited 90 to 100% resistances for Streptococcus sp., on the other hand, 100% susceptible to AMC and 80% susceptible to Do, Te, ENR and UB was recognised. Lastly, Photobacterium damsela subsp. piscicida was showed 100fe susceptible to Amc and 86% susceptible to NOR, CIP ENR and UB. As a consequence, fish bacterial pathogens isolated from Kyeongnam area, especially Tongyeong-si, Geoje-si, and Goseong-gun, were showed highly resistant to a variety of antibiotics available in the field.

• Journal of fish pathology
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• v.37 no.1
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• pp.37-48
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• 2024

This study evaluated in vitro antimicrobial properties of Bacillus subtilis KCTC 1326 as an environmentally friendly alternative to antibiotics. B. subtilis KCTC 1326 was characterized on biochemical properties and antibiotics susceptibility. It exhibited antimicrobial effects against all 12 species of fish bacteria used in this experiment. Among them, the largest antibacterial zone was observed for Streptococcus parauberis (34 mm), while the smallest antibacterial zone was observed for Citrobacter freundii (8 mm). Additionally, in the co-culture inhibitory assay of B. subtilis and Edwardsiella piscicida, the growth of E. piscicida was suppressed with increasing concentrations of B. subtilis KCTC 1326, with complete inhibition observed at 107 and 108 CFU/mL of B. subtilis KCTC 1326 after 24 hours of incubation. Moreover, at 48 hours of incubation, the growth of E. piscicida was inhibited across all concentration ranges of B. subtilis KCTC 1326. Therefore, this study indicated the utilizing of B. subtilis KCTC 1326 as an antimicrobial for controlling fish bacterial diseases.

• Journal of Marine Life Science
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• v.4 no.2
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• pp.86-90
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• 2019

This study aimed to compare the disinfection efficiencies of the ultraviolet and plasma systems, the two systems designed and commercialized to disinfect water in aquaculture, by putting each in a 100 ℓ water tank and concentrating 1.0 ℓ of treated water to check the changes in the number of bacteria in the samples. Each system was operated for 6 hours to culture the typical seawater bacteria in the Marine agar, Thiosulfate citrate bile salts sucrose agar and Salmonella Shigella agar media, respectively, to check the number of bacteria in the media, and the changes in the number of Edwardsiella piscicida in the treated water were checked after the artificial inoculation of E. piscicida in the disinfected seawater. As a result, the two disinfection systems showed the almost similar levels of bacterial reduction efficiency between 99.5% and 99.9%. However, the result of this study showed that, with 100 ℓ of water treated for the same length of time using the two systems, the plasma system turned out to disinfect bacteria in a shorter period of time than the UV system. However, as the changes in the number of bacteria were checked for a short length of time (6 hours) in this study, it was judged that, considering the actual aquaculture environment in which the quality of water significantly changes with feed residues, excretions and coastal contamination, etc., and a lot of biofilms and organic matter exist, the plasma system would be more efficient than the UV system as the former is capable of continuously maintaining a certain level of efficiency than the latter that is limited in terms of efficiency depending on the level of turbidity and the existence of organic matter.

• Journal of fish pathology
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• v.35 no.2
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• pp.205-214
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• 2022

In recent years, there have been many studies investigating changes in animal behavior using video tracking technology to track motion. However, there have been very few studies and results on changes in the behavior of fish infected with a pathogen. Therefore, the present study attempted to analyze the behavior of angelfish (Pterophyllum scalare) infected with bacterial pathogens using video tracking. Two cameras were placed in front of the water tank to obtain behavior data, and tracking was performed for three days until the day of death. Data such as average speed, changes in speed, the locations of the fish in the tank, and fractal dimension were statistically analyzed based on the fish speed and location in the tank of the fish. For bacterial infection, an individual angelfish was intraperitoneally injected with approximately 10⁶ CFU ml^-1 of Aeromonas hydrophila or Edwardsiella piscicida. The experiment was carried out five times for each group. Fish infected with the bacterial pathogens showed a tendency to increase in speed and to spend more time in the upper part of the tank one or two days before death. On the day the fish died, the average speed, changes in speed, and the fractal dimension value were significantly lower than the corresponding values in the control group, and the fish also remained in the lower part of the tank. Our results indicated that behavioral changes in fish could be successfully detected earlier than death using video tracking technology, and that this method presents potential for disease monitoring in aquaculture.