Gang-Joon Heo

Nematode worm infections (Camallanus cotti, Camallanidae) in guppies (Poecilia reticulata) imported to Korea

Abstract Camallanus cotti [Jpn. J. Zool. 1(1927) 169] is a common intestinal nematode parasite of guppies. It was first reported from several kinds of freshwater fishes in Japan in 1927, and is thought to be native to continental Asia, from Japan westwards, perhaps extending as far as the Nile delta. Because of the worldwide trade in aquarium fishes, it is now found in many fishes in several continents. Guppies are one of the most popular tropical pet fishes in Korea. Here we report C. cotti infection of cultured guppies in Korea, originally imported from Indonesia, for the first time. In a tropical fish farm, mortality reached up to 30% and most guppies did not show any clinical signs except in heavily infected fish, in which worms were extruded from the anus. Prevalence of infection was 71% (59/83 fish), and there was no preference for host sex (34 of 45 female fish and 25 of 38 male fish). Although we found C. cotti in dead guppies, we could not conclude that C. cotti infection was the cause of death, because some fish died without C. cotti infection. In addition, we detected high numbers of the histophagous ciliate, Tetrahymena corlissi (Thompson, 1955) in these fish. We suspect that both C. cotti and T. corlissi infections contributed to death, but more detailed studies are required.

Prevalence of Salmonella spp. in pet turtles and their environment

Pet turtles are known as a source of Salmonella infection to humans when handled in captivity. Thirty four turtles purchased from pet shops and online markets in Korea were examined to determine whether the turtles and their environment were contaminated with Salmonella spp. Salmonella spp. were isolated from fecal samples of 17 turtles. These isolates were identified as S. enterica through 16S rRNA gene sequencing. The isolation rate of Salmonella spp. from the soil and water samples increased over time. We concluded that a high percentage of turtles being sold in pet shops were infected with Salmonella spp., and their environments tend to become contaminated over time unless they are maintained properly. These results indicate that pet turtles could be a potential risk of salmonellosis in Korea.

Metagenomics analysis of gut microbiota and immune modulation in zebrafish (Danio rerio) fed chitosan silver nanocomposites

Abstract In this study, we evaluated the effects of chitosan silver nanocomposites (CAgNCs) supplemented diet on gut microbial community, goblet cell density, gut morphometry and mRNA expression of immune related and mucin encoding genes in zebrafish. Zebrafish gut microbiota analysis results clearly showed the reduction of phylum Proteobacteria. However, they remained as the major bacterial group in gut with CAgNCs supplemented diet, while the abundance of phylum Fusobacteria and phylum Bacteroidetes were increased notably compared to the control diet fed fish. Total goblet cell density was significantly increased at 30 and 60 days in CAgNCs supplemented group (1.6–fold and 2.0‐fold, respectively) compared to the control group indicating enhanced immune function in the gut. CAgNCs supplementation has also increased villi height significantly in the zebrafish mid gut at the end of 30 (95.5 ± 3.7 &mgr;m) and 60 days (144.40 ± 4.8 &mgr;m) compared to control diet fed fish at 30 (86.90 ± 3.7 &mgr;m) and 60 days (96.2 ± 4.8 &mgr;m). Furthermore, mRNA expression of immune related genes such as TNF‐&agr; (6.2‐fold), IL‐10 (5.0‐fold), IL‐12 (9.2‐fold), IRF‐1 (5.2‐fold), Defbl1 (3‐fold), Lyz (5.1‐fold) and mucin encoding genes were significantly upregulated (above 2‐fold) compared to that of control group. The current study revealed that CAgNCs supplemented diet engenders promising effects on fish gut immunity by enhancing beneficial microbial populations, goblet cell density, villi length, and transcriptional regulation of immune related and mucin encoding genes. Graphical abstract Figure. No Caption available. HighlightsCAgNCs supplemented diet alters the zebrafish gut microbiota, increases the goblet cell density and villi height.CAgNCs supplemented diet modulates the immune responses in zebrafish.CAgNCs supplemented diet would be a better immunostimulant for aquaculture.

Two molluscan BCL-2 family members from Manila clam, Ruditapes philippinarum: molecular characterization and immune responses.

Apoptosis based immune responses are important component of host defense in mollusks. In this study, we have identified two novel molluscan BCL-2 cDNAs from Manila clam, Ruditapes philippinarum and named as RpBCL-2A and RpBCL-2B. There were four and three highly conserved BCL-2 homology (BH) regions in RpBCL-2A and RpBCL-2B, respectively suggesting these two genes could be different isoforms of anti-apoptotic BCL-2 family. Phylogenetic results revealed that Manila clam BCL-2 genes were clustered closely with invertebrate BCL-2 members. It gives evidence of their common origin and conserved features of invertebrate BCL-2 family. RpBCL-2A and 2B were expressed in tissue-specific manner showing the highest and lowest level of expression in gills and hemocytes, respectively. However there was no clear expression profile difference between two genes. After Vibrio tapetis challenge, transcriptional responses of RpBCL-2A and RpBCL-2B were induced in gills and hemocytes with high variation that could be due to effects of immune reactions of other host defense molecules.

Molecular cloning and characterization of SOCS-2 from Manila clam Ruditapes philippinarum

Suppressor of cytokine signaling (SOCS) family members are key regulators of immunological homeostasis. In this study, we have discovered the SOCS-2 member from Manila clam Ruditapes philippinarum and further analyzed its immune responses against lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C). Amino acid sequence of RpSOCS-2 consists of cytokine inducible SRC homology 2 (SH2) and SOCS box domains similar to vertebrate SOCS counterparts. It has the highest amino acid identity (41%) with Pacific oyster (Crassostrea gigas) SOCS-2 and showed close evolutional relationship with disk abalone (Haliotis discus discus) SOCS-2. Tissue specific expression results showed that RpSOCS-2 was constitutively expressed in all examined tissues with the highest level in gill tissue of un-challenged clams. RpSOCS-2 mRNA expression was up-regulated by LPS and poly I:C challenge in gills. Discovery of RpSOCS-2 homologue and expression analysis would support for understanding evolutional relationships and their role in innate immune responses in mollusks, respectively.

Multilocus sequence typing analysis of Pseudomonas aeruginosa isolated from pet Chinese stripe-necked turtles (Ocadia sinensis)

Our research sought to characterize the phylogeny of Pseudomonas aeruginosa isolated from pet Chinese stripe-necked turtles (Ocadia sinensis) to better understand its evolutionary relation to other isolates and increase understanding of a potential zoonotic pathogen transmitted through direct contact with pet turtles. Thirty-one Pseudomonas aeruginosa isolates were obtained from both immature and adult turtles sold in pet shops in Korea. To characterize the phylogenic position of Chinese stripe-necked turtle-borne P. aeruginosa relative to other strains, multilocus sequence typing (MLST) analysis was performed due to the accessibility and breadth of MLST databases. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were sequenced and the results were compared with data from the MLST database. The genes were further used for phylogenetic analysis of P. aeruginosa using concatenated gene fragments. Both rooted and unrooted phylogenetic trees were generated. Eleven distinct sequence types were present within the isolates among which seven were new. Expanding an unrooted phylogenetic tree to include P. aeruginosa MLST sequences isolated from various other geographic locations and sources revealed a divergent cluster containing the majority of isolates obtained from turtles. This suggests that P. aeruginosa strains particularly well-adapted for inhabiting turtles occupy a distinct phylogenetic position.

Chitosan nanoparticles: A positive immune response modulator as display in zebrafish larvae against Aeromonas hydrophila infection

&NA; Chitosan nanoparticles (CNPs) were synthesized by ionic gelation method and its immunomodulatory properties were investigated in zebrafish larvae. Average particle size and zeta potential were 181.2 nm and +37.2 mv, respectively. Initially, toxicity profile was tested in zebrafish embryo at 96 h post fertilization (hpf) stage using medium molecular weight chitosan (MMW‐C) and CNPs. At 5 &mgr;g/mL, the hatching rate was almost similar in both treatments, however, the survival rate was lower in MMW‐C compared to CNPs exposure, suggesting that toxicity effect of CNPs in hatched larvae was minimal at 5 &mgr;g/mL compared to MMW‐C. Quantitative real time PCR results showed that in CNPs exposed larvae at 5 days post fertilization (5 dpf) stage, immune related (il‐1&bgr;, tnf‐&agr;, il‐6, il‐10, cxcl‐18b, ccl34a.4, cxcl‐8a, lyz‐c, def&bgr;l‐1, irf‐1a, irf‐3, MxA) and stress response (hsp‐70) genes were induced. In contrast, basal or down regulated expression of antioxidant genes (gstp‐1, cat, sod‐1, prdx‐4, txndr‐1) were observed. Moreover, zebrafish larvae (at 5 dpf stage) exposed to CNPs (5 &mgr;g/mL) showed higher survival rate at 72 h post infection stage against pathogenic Aeromonas hydrophila challenge compared to controls. These results suggest that although CNPs can have toxic effects to the larvae at higher doses, CNPs exposure at 5 &mgr;g/mL could enhance the immune responses and develop the disease resistance against A. hydrophila, which could be attributed to its strong immune modulatory properties. Graphical abstract Figure. No caption available. HighlightsSynthesized CNPs at 5 &mgr;g/L exposure was non‐toxic to zebrafish at 96 hpf stage.CNPs showed immune modulatory and disease resistant against A. hydrophila in 5 dpf stage zebrafish after non‐toxic exposure.Biodegradable CNPs could be a potential immunostimulant for larval aquaculture.

Potential enterotoxicity and antimicrobial resistance pattern of Aeromonas species isolated from pet turtles and their environment

To investigate the potential enterotoxicity and antimicrobial resistance of aeromonads from pet turtles as a risk for human infection, one hundred and two Aeromonas spp. were isolated from the feces, skin and rearing environments of pet turtles and identified by biochemical and gyrB sequence analyses. Aeromonas enteropelogenes was the predominant species among the isolates (52.9%) followed by A. hydrophila (32.4%), A. dharkensis (5.9%), A. veronii (4.9%) and A. caviae (3.9%). Their potential enterotoxicities were evaluated by PCR assays for detecting genes encoding cytotoxic enterotoxin (act) and two cytotonic enterotoxins (alt and ast). 75.8% of A. hydrophila isolates exhibited the act+/alt+/ast+ genotype, whereas 94.4% of A. enteropelogenes isolates were determined to be act−/alt−/ast−. In an antimicrobial susceptibility test, most isolates were susceptible to all tested antibiotics except amoxicillin, ampicillin, cephalothin, chloramphenicol and tetracycline. Non-susceptible isolates to penicillins (ampicillin and amoxicillin) and fluoroquinolones (ciprofloxacin and norfloxacin) were frequently observed among the A. enteropelogenes isolates. Few isolates were resistant to imipenem, amikacin, ceftriaxone and cefotaxime. Collectively, these results suggest that pet turtles may pose a public health risk of infection by enterotoxigenic and antimicrobial resistant Aeromonas strains.

Prevalence of Citrobacter spp. From Pet Turtles and Their Environment

Abstract Pet turtles are considered a source of bacterial infection to humans when handled in captivity. Turtles purchased from 9 pet shops and 8 online markets in Korea were examined to determine whether the turtles and their environment were contaminated with Citrobacter spp. Biochemical tests and morphology revealed that Citrobacter spp. were isolated from 7 fecal and 76 environmental samples. Among the 7 fecal isolates, 5 bacteria were identified as Citrobacter freundii through 16S rRNA gene sequencing. The isolation rate of Citrobacter spp. from soil and water samples increased over time. Each of the isolates antibiotic resistance was characterized with a disk diffusion test. The strains showed susceptibility against amikacin, ceftriaxone, ciprofloxacin, imipenem, sulfamethoxazole/trimethoprim, and tetracycline, but were resistant to cefoxitin, cephalotin, and chloramphenicol. These results indicate that pet turtles are a potential source of Citrobacter infection in humans in Korea.

Communications: Efficacy of Norfloxacin for the Control of Edwardsiella tarda Infection in the Flounder Paralichthys olivaceus

Abstract Efficacy of norfloxacin was evaluated in laboratory and field studies for control of Edwardsiella tarda infection in the flounder Paralichthys olivaceus. In disc diffusion tests, five E. tarda strains showed high sensitivity to norfloxacin. Minimum inhibitory concentrations of norfloxacin against the five isolates were approximately 0.016–0.031 μg/mL. In laboratory studies in which flounder were challenged with E. tarda, a significant (P < 0.05) reduction in mortality was observed among fish receiving norfloxacin in feed at 100 mg/kg body weight (or more) daily for 3 d compared with mortality among nonmedicated controls. Similar results were achieved when 100 mg norfloxacin/kg body weight was used in field trials.