Multiplex PCR analysis of virulence genes and their influence on antibiotic resistance in Enterococcus spp. isolated from broiler chicken.

Enterococcus spp. are opportunistic pathogens that cause lameness in broiler chickens, resulting in serious economic losses worldwide. Virulence of Enterococcus spp. is associated with several putative virulence genes including fsr, efm, esp, cylA, cad1, ace, gelE, and asa1. In this study, multiplex polymerase chain reaction (PCR) for the simultaneous detection of these virulence genes in Enterococcus spp. was developed, and detection limits for E. faecium, E. faecalis, and E. hirae were 64.0 pg/µL, 320.0 pg/µL, and 1.6 ng/µL DNA, respectively. Among 80 Enterococcus isolates tested, efm and cad1 were detected in all 26 E. faecium samples, and only cad1 was observed in E. hirae. Additionally, the presence of virulence genes in 25 E. faecalis isolates were 100% for cad1, 88.0% for gelE, 64.0% for fsr, 44.0% for asa1, 16.0% for cylA, and 4.0% for esp. No virulence genes were found in E. gallinarum isolates. A total of 49 isolates were resistant to tigecycline and to at least 2 different classes of antibiotics. The most prevalent resistance was to ciprofloxacin (73.5%), quinupristin/dalfopristin (55.1%), and tetracycline (49.0%). No strains were resistant to vancomycin or linezolid. This is the first multiplex PCR assay to simultaneously detect eight virulence genes in Enterococcus spp., and the method provides diagnostic value for accurate, rapid, and convenient detection of virulence genes. Additionally, we report the prevalence of virulence genes and antimicrobial resistance in Enterococcus isolates from commercial broiler chickens suffering lameness.

Loop-mediated isothermal amplification assays for Enterococcussp., Escherichiacoli and Staphylococcusaureus in chicken.

Bacterial chondronecrosis with osteomyelitis (BCO) is a major cause of lameness in broiler chicken, and results in serious economic losses worldwide. Although the pathogenesis mechanism leading to lameness is not entirely understood, some strains of Enterococcussp., avian pathogenic Escherichia coli or Staphylococcus aureus have been long recognized as important causative pathogens. To prevent the progression of Enterococcussp., avian pathogenic E. coli or S. aureus infections, we developed rapid, sensitive and convenient diagnostic assays using loop-mediated isothermal amplification (LAMP). Entero-Common-LAMP assays were developed for simultaneous detection of eight Enterococcus species. To target specific microorganisms, seven Entero-Specific-LAMP assays for E. faecalis, E. faecium, E. hirae, E. gallinarum, E. avium, E. duransand E. cecorum were developed, as well as E. coli-LAMP and S. aureus-LAMP assays. Considering the prevalence and economic impact of Enterococcussp., E. coli and S. aureus, the 10 different LAMP assays which were developed have considerable potential as routine diagnostic methods in the field or in resource-limited environments.

Pathogenesis and genetic characteristics of novel reassortant low-pathogenic avian influenza H7 viruses isolated from migratory birds in the Republic of Korea in the winter of 2016-2017.

In this study, we characterized H7 subtype low-pathogenicity (LP) influenza A viruses (IAVs) isolated from wild bird habitats in the Republic of Korea from 2010 to early 2017. Through national surveillance, 104 H7 IAVs were isolated, accounting for an average of 14.9% of annual IAV isolations. In early 2017, H7 subtypes accounted for an unusually high prevalence (43.6%) of IAV detections in wild birds. Phylogenetic analysis revealed that all the viruses isolated in the winter of 2016-2017 fell within cluster II of group C, belonging to the Eurasian lineage of H7 IAVs. Notably, cluster II of group C included the H7 gene from the highly pathogenic H7N7 IAV that was detected in northeastern Italy in April of 2016. Through a gene-constellation analysis, the H7 LPIAVs that we isolated constituted ≥11 distinct genotypes. Because the viruses belonging to the genotypes G2.1 and G1 were observed most frequently, we compared the replication and transmission of representative viruses to these genotypes in specific-pathogen-free chickens. Notably, the representative G2.1 strain was capable of systemic replication and efficient transmission in chickens (as evidenced by virus isolation and histopathological examination) without any clinical signs except mortality (in one infected chicken). The efficient subclinical viral replication and shedding of the G2.1 virus in chickens may facilitate its silent spread among poultry after introduction. Given that wild birds harbor novel strains that could affect poultry, our results highlight the need for enhanced IAV surveillance in both wild birds and poultry in Eurasia.

Loop-mediated isothermal amplification assay for detection of four immunosuppressive viruses in chicken.

Loop-mediated isothermal amplification (LAMP) methods to detect chicken infectious anemia virus (CIAV), reticuloendotheliosis virus (REV), and Marek's disease virus (MDV), and a reverse transcription (RT)-LAMP assay to detect infectious bursal disease virus (IBDV), were developed. The CIAV-LAMP, REV-LAMP, MDV-LAMP, and IBDV-RT-LAMP methods were performed using four sets of six primers targeting the VP1 gene of CIAV, the gp90 gene of REV, the Meq gene of MDV, and the VP2 gene of IBDV. The results (a change in color) were observed visually. The methods showed high specificity and sensitivity. The detection limits were 50 genomic copies of CIAV, 16 genomic copies of REV, 20 genomic copies of MDV, and 250 genomic copies of IBDV. When used to test clinical samples, the results of the LAMP assays were in 100% agreement with a previously described PCR. Therefore, the LAMP assays are simple, rapid, highly sensitive, and specific methods for detecting four immune-suppressive viruses.

High prevalence of blaCTX-M group genes in Aeromonas dhakensis isolated from aquaculture fish species in South Korea.

The prevalence of resistant genes against ß-lactams in 119 Aeromonas strains was determined. A large number (99.2%) of the present fish strains were resistant to one or more ß- lactams including ceftiofur, amoxicillin-clavulanic acid, ampicillin, piperacillin and cefpodoxime. Among antibiotic resistance phenotypes, the simultaneous resistance to all ß-lactams occurred in 25.2% (n=30) of all strains, which consisted of 18 strains of A. dhakensis, 8 strains of A. caviae, 2 strains of A. hydrophila and only one strain of A. veronii. For exploring genetic background of the antibiotic resistances, multiple PCR assays were subjected to detect ß-lactamase-encoding genes, bla(TEM), bla(OXA-B) and bla(CTX-M). In the results, the bla(TEM-1) gene was harbored in all strains, whereas only 3 strains harbored bla(OXA) gene. In the case of bla(CTX-M) gene, the gene was detected in 21.0% (25 out of 119) of all strains, which countered with 80% (20 out of 25) of A. dhakensis, 8% (2 out of 25) of A. caviae and 12% (3 out of 25) of A. hydrophila. In addition, most of the bla(CTX-M) positive strains showed simultaneous resistance to all ß-lactams (18 out of 30 strains). In sequence analysis for bla(CTX-M) genes detected, they were CTX-M group 1-encoding genes including bla(CTX-M-33) from 3 eel strains of A. dhakensis. Therefore, A. dhakensis obtained from cultured fish could represent a reservoir for spreading genes encoding CTX-M group 1 enzymes and hence should be carefully monitored, especially for its potential risk to public health.

Bacterial pathogens and flora isolated from farm-cultured eels (Anguilla japonica) and their environmental waters in Korean eel farms.

The purpose of this study was to investigate bacterial pathogens and flora in both sick and clinically healthy eels, Anguilla japonica, and the environmental rearing waters of Korean eel farms. Between 2003 and 2010, a total of 621 sick eels were submitted for diagnosis, while 216 healthy eels and 87 environmental water samples were collected during a survey of 26 eel farms in Korea. Seven different bacterial species were obtained from 183 isolates, which were recovered from the internal organs of the 621 sick eels. The most frequently isolated bacterium was Edwardsiella tarda (71.0%), followed by Aeromonas hydrophila (9.3%), Citrobacter freundii (7.7%), Aeromonas veronii (6.0%), Listonella anguillarum (2.7%), Plesiomonas shigelloides (2.2%), and Pseudomonas anguilliseptica (1.1%). From the eel and water samples of the survey, a total of 472 isolates from 34 different species belonging to 15 genera of bacteria were isolated. The most prevalent genus of bacteria was Aeromonas spp. (141/472, 29.8%). Among the 34 types of bacterial species, C. freundii (20.1%) and A. hydrophila (19.9%) were the most frequently isolated. The results of this study indicate that a wide range of bacterial species, which can act as primary or opportunistic pathogens, may be recovered from clinically healthy eels and rearing waters. This study provides baseline information about bacterial pathogens and floral contamination for the control and treatment of bacteria in Korean eel farms.

Characterization of Edwardsiella tarda isolated from farm-cultured eels, Anguilla japonica, in the Republic of Korea.

We surveyed the occurrence of edwardsiellosis on eel farms and investigated the characteristics of Edwardsiella tarda isolated from farm-cultured eels in the Republic of Korea. The occurrence rate of edwardsiellosis was 72% in the investigated samples. Among the edwardsiellosis cases, 46% were found to be mixed infections, with parasites and other kinds of bacteria. Some of the biochemical characteristics of the E. tarda isolates were different from those of the previously reported E. tarda isolated from several kinds of fish from different countries, especially in terms of hydrogen sulfide and indole production. The E. tarda isolated from the eels in the Republic of Korea had the characteristics of two biogroups, the wild-type biogroup and biogroup 1. The enzymatic activity of the E. tarda showed similar patterns to previously reported E. tarda strains and ATCC strains. This is the first it has been reported that E. tarda isolated from farm-cultured eels had some different biochemical characteristics from those of previously reported E. tarda isolated from several kinds of fish.

Development of duck hepatitis A virus type 3 vaccine and its use to protect ducklings against infections.

A variant type of duck hepatitis A virus (DHAV), DHAV-3 was recently discovered in South Korea and China. Sequence analyses verified that the variant is genetically or serologically different from the DHAV-1 and DHAV-2 types. Duck hepatitis had been reported in South Korea since 1985 and an attenuated DHAV-1 vaccine had efficiently prevented epidemics of DHAV-1 until 2002. Despite the DHAV-1 based vaccine in use the novel DHAV-3 circulating in South Korea remains to be a threat to duckling farming. To develop a live attenuated vaccine against DHAV-3, a representative isolate, AP-04203, was therefore attenuated by repeated passages in SPF chicken embryos 100 times. The 100th passaged virus, AP-04203P100, did not cause clinical sign and mortality in 1-day-old ducklings as well as reversion of virulence capacity. The ducklings vaccinated with AP-04203P100 virus (10(3.0)ELD(50)/0.2ml) on 1-day-old age via the intramuscular injection were well protected from 2 days after challenge with pathogenic AP04203P1 virus via the intramuscular route. In addition, the vaccine candidate also exhibited complete protection against currently circulating pathogenic DHAV-3 isolates. In conclusion, we demonstrate that the live attenuated virus, AP-04203P100, is a promising vaccine candidate facilitating the prevention of duck hepatitis caused by DHAV-3 around East Asia including South Korea.

Comparison of antigenic proteins from Lactococcus garvieae KG- and KG+ strains that are recognized by olive flounder (Paralichthys olivaceus) antibodies.

Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae strains. In immunoproteomic analysis, there was a significant difference in the 2-DE immunoblot profiles of the L. garvieae strains using sera collected from fish surviving infection with either L. garvieae strains KG9408 or NSS9310. These sera reacted with 8 and 7 unique antigenic protein spots, respectively. Heat shock protein (HSP) 70 and DNA-directed RNA polymerase were among the specific antigens recognized by the anti-NSS9310 serum. In addition, the anti-NSS9310 and anti-KG9408 olive flounder sera reacted with 25 common antigenic protein spots of all the L. garvieae strains, which included elongation factor (EF)-Tu, arginine deiminase (AD), inosine-5'-monophosphate dehydrogenase (IMPD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphomannomutase (PMM), L-lactate dehydrogenase (L-LDH), 6-phosphofructokinase and UDP-galactose 4-epimerase (UDP-galactose). Based on the present results, the 8 antigens recognized by the anti-KG9408 serum and the 25 common antigens recognized by both sera may serve as potential markers for developing an effective vaccine against this bacterium.

Experimental infection of chickens, ducks and quails with the highly pathogenic H5N1 avian influenza virus.

Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype have spread since 2003 in poultry and wild birds in Asia, Europe and Africa. In Korea, the highly pathogenic H5N1 avian influenza outbreaks took place in 2003/2004, 2006/2007 and 2008. As the 2006/2007 isolates differ phylogenetically from the 2003/2004 isolates, we assessed the clinical responses of chickens, ducks and quails to intranasal inoculation of the 2006/2007 index case virus, A/chicken/Korea/IS/06. All the chickens and quails died on 3 days and 3-6 days post-inoculation (DPI), respectively, whilst the ducks only showed signs of mild depression. The uninoculated chickens and quails placed soon after with the inoculated flock died on 5.3 and 7.5 DPI, respectively. Both oropharyngeal and cloacal swabs were taken for all three species during various time intervals after inoculation. It was found that oropharyngeal swabs showed higher viral titers than in cloacal swabs applicable to all three avian species. The chickens and quails shed the virus until they died (up to 3 to 6 days after inoculation, respectively) whilst the ducks shed the virus on 2-4 DPI. The postmortem tissues collected from the chickens and quails on day 3 and days 4-5 and from clinically normal ducks that were euthanized on day 4 contained the virus. However, the ducks had significantly lower viral titers than the chickens or quails. Thus, the three avian species varied significantly in their clinical signs, mortality, tissue virus titers, and duration of virus shedding. Our observations suggest that duck and quail farms should be monitored particularly closely for the presence of HPAIV so that further virus transmission to other avian or mammalian hosts can be prevented.

Phenotypic characteristics of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus).

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis, respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges-Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.

Very virulent infectious bursal disease virus isolated from wild birds in Korea: epidemiological implications.

To explore the epidemiological link between infectious bursal disease virus (IBDV) in wild birds and domestic chickens in Korea, we examined 107 free-living wild birds, representing 7 species, that were found dead of apparent natural causes in Korea over the past two years for the presence of IBDV. Five birds were tested positive for IBDV by RT-PCR assay: black-billed magpie (n=1), mallard duck (n=2), bean goose (n=1) and white-fronted goose (n=1). IBDV was isolated from RT-PCR-positive tissues following chicken embryo inoculation. Sequence analysis of the VP2 gene indicated that all of the isolates from the wild birds encode amino acids A222, I242, I256, I294 and S299 of VP2, which are conserved among strains of very virulent IBDV (vvIBDV). Phylogenetic analysis revealed that the wild bird IBDV isolates are closely related to strains of vvIBDV. An IBDV isolate from a magpie showed 60% mortality in SPF chickens and severe bursal atrophy. The epidemiological implications of IBDV in free-living wild birds are discussed. To our knowledge, this is the first report of vvIBDV in free-living wild birds.

Differential diagnosis between type-specific duck hepatitis virus type 1 (DHV-1) and recent Korean DHV-1-like isolates using a multiplex polymerase chain reaction.

Duck hepatitis can be caused by four types of viruses: duck hepatitis virus (DHV) type 1 (DHV-1), DHV-1a (a variant strain of DHV-1), DHV-2 and DHV-3. In Korea, duck hepatitis has been associated with two types of DHV-1, original DHV-1 type-specific strain (DHV-1s) and the recently reported DHV-1 variant strains (DHV-1v). The pathogenicity and pathological findings of ducklings infected with the recent DHV-1v isolates, AP-04114 and AP-04203, were almost identical to those infected with members of the DHV-1s, DHV-HS and the type-specific strain DRL-62. To be able to monitor the epidemiological patterns exhibited by the two Korean types, a specific gene-based differential diagnostic method based on multiplex polymerase chain reaction was developed. The primers selected were designed to bind to and amplify conserved regions within the RNA-dependent RNA polymerase (3D) gene, the complete capsid (P1) region or the 5'-untranslated region to distinguish between the DHV-1s and DHV-1v groups. The described multiplex polymerase chain reaction method was able to selectively recognize ducklings infected with either of the two groups of Korean isolates. The method was also able to distinguish between DHVs and other avian-originated RNA viruses. The detection limit of the diagnostic method was determined to correspond to 10(3) copies viral RNA and 100 pg used as starting template. As a result, the use of this test allows rapid and early diagnosis of two different virus types affecting the commercial duckling industry.

Highly pathogenic avian influenza virus (H5N1) in domestic poultry and relationship with migratory birds, South Korea.

During the 2006-2007 winter season in South Korea, several outbreaks of highly pathogenic avian influenza virus (H5N1) were confirmed among domestic poultry and in migratory bird habitats. Phylogenetic analysis showed that all isolates were closely related and that all belong to the A/bar-headed goose/Qinghai/5/2005-like lineage rather than the A/chicken/Korea/ES/2003-like lineage.

Development of one-step reverse transcriptase-polymerase chain reaction to detect duck hepatitis virus type 1.

A one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed and optimized for the detection of duck hepatitis virus type 1 (DHV-1) using the Viral Gene-spin viral DNA/RNA extraction kit. A pair of DHV-1-specific primers was designed against the gene encoding RNA-dependent RNA polymerase (3D gene). Using RNA prepared from duckling liver samples infected with two reference and seven Korean field isolates of DHV-1, one-step RT-PCR with DHV1-specific primers amplified a 467-bp fragment. Under the same conditions, no amplification was observed for 14 other avian pathogenic viruses and bacteria. Using RNA prepared from serial dilutions of the DHV-1 with the supernatant of the uninfected duckling liver homogenate (10% w/v), the one-step RT-PCR assay was found to be sensitive to 10 50% egg lethal dose (ELD50) 0.1 ml(-1) of DHV-1. Furthermore, this method detected DHV-1 from the livers and allantoic fluid of duck embryos dying before 3 days postinoculation (PI) and of chicken embryos that were chilled at 3 days PI. Therefore, this one-step RT-PCR method is rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.

Molecular analysis of duck hepatitis virus type 1 reveals a novel lineage close to the genus Parechovirus in the family Picornaviridae.

Duck hepatitis virus type 1 (DHV-1) was previously classified as an enterovirus, based primarily on observed morphology and physicochemical properties of the virion. The complete nucleotide sequences of two strains (DRL-62 and R85952) of DHV-1 have been determined. Excluding the poly(A) tail, the genomes are 7691 and 7690 nt, respectively, and contain a single, large open reading frame encoding a polyprotein of 2249 aa. The genome of DHV-1 is organized as are those of members of the family Picornaviridae: 5' untranslated region (UTR)-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3' UTR. Analysis of the genomic and predicted polyprotein sequences revealed several unusual features, including the absence of a predicted maturation cleavage of VP0, the presence of two unrelated 2A protein motifs and a 3' UTR extended markedly compared with that of any other picornavirus. The 2A1 protein motif is related to the 2A protein type of the genus Aphthovirus and the adjacent 2A2 protein is related to the 2A protein type present in the genus Parechovirus. Phylogenetic analysis using the 3D protein sequence shows that the two DHV-1 strains are related more closely to members of the genus Parechovirus than to other picornaviruses. However, the two DHV-1 strains form a monophyletic group, clearly distinct from members of the genus Parechovirus.

Highly pathogenic avian influenza (H5N1) in the commercial domestic ducks of South Korea.

The present study reports the clinical, virological and pathological findings observed in a natural outbreak of highly pathogenic avian influenza in farmed commercial ducks. The ducks developed clinical signs, including mild respiratory distress, depression, mild diarrhoea, loss of appetite and increasing mortality (up to 12%). At necropsy, multifocal mottled necrosis was commonly found in the pancreas with splenomegaly, hepatomegaly, and swollen kidneys. Microscopically, there was necrotized pancreatitis and hepatitis, and lymphocytic meningoencephalitis and myocarditis. Influenza viral antigen was demonstrated in areas closely associated with histopathological lesion. Avian influenza virus was isolated from the caecal tonsil, faeces, and kidney of the domestic ducks. The isolated virus was identified as a highly pathogenic H5N1, with a haemagglutinin proteolytic cleavage site deduced amino acid sequence of ... QREKRKKR/GLFGAIAG ... In order to determine the pathogenicity of the isolate, eight 6-week-old specific pathogen free chickens were inoculated intravenously with the virus, and all birds died within 24 h after inoculation. This is the first report of an outbreak of highly pathogenic avian influenza with clinical signs in commercial domestic ducks in South Korea.

Characterization of highly pathogenic H5N1 avian influenza A viruses isolated from South Korea.

An unprecedented outbreak of H5N1 highly pathogenic avian influenza (HPAI) has been reported for poultry in eight different Asian countries, including South Korea, since December 2003. A phylogenetic analysis of the eight viral genes showed that the H5N1 poultry isolates from South Korea were of avian origin and contained the hemagglutinin and neuraminidase genes of the A/goose/Guangdong/1/96 (Gs/Gd) lineage. The current H5N1 strains in Asia, including the Korean isolates, share a gene constellation similar to that of the Penfold Park, Hong Kong, isolates from late 2002 and contain some molecular markers that seem to have been fixed in the Gs/Gd lineage virus since 2001. However, despite genetic similarities among recent H5N1 isolates, the topology of the phylogenetic tree clearly differentiates the Korean isolates from the Vietnamese and Thai isolates which have been reported to infect humans. A representative Korean isolate was inoculated into mice, with no mortality and no virus being isolated from the brain, although high titers of virus were observed in the lungs. The same isolate, however, caused systemic infections in chickens and quail and killed all of the birds within 2 and 4 days of intranasal inoculation, respectively. This isolate also replicated in multiple organs and tissues of ducks and caused some mortality. However, lower virus titers were observed in all corresponding tissues of ducks than in chicken and quail tissues, and the histological lesions were restricted to the respiratory tract. This study characterizes the molecular and biological properties of the H5N1 HPAI viruses from South Korea and emphasizes the need for comparative analyses of the H5N1 isolates from different countries to help elucidate the risk of a human pandemic from the strains of H5N1 HPAI currently circulating in Asia.

Parasitic infections in live freshwater tropical fishes imported to Korea.

We examined 15 species of ornamental tropical fishes originating from Southeast Asia to determine the cause of losses among 8 fish farms in Korea. A total of 351 individuals belonging to 5 different families (1 species of Characidae, 6 of Cichlidae, 3 of Cyprinidae, 1 of Heleostomatidae, and 4 of Poecilidae) were collected for the purpose of detecting metazoan and protozoan parasites. Parasites were fixed and stained using routine methods, and identified. We found 3 ciliates, 2 monogeneans, 1 nematode, and 1 copepod from 7 host species. Of these, Ichthyophthirius multifiliis was the most common parasite in our study, and together with Trichodina sp., caused mass mortality of Sumatra barb Puntius tetrazona at 1 farm. We also found Camallanus cotti and Tetrahymena corlissi from guppies Poecilia reticulata, both for the first time in Korea. Farmers consider these 2 pathogens to be the most serious ones in Korea. Gussevia asota from oscar Astronotus ocellatus, and Gyrodactylus bullatarudis from platy Xiphophorus maculatus were also found in Korea for the first time. We believe that appropriate quarantine practices for tropical ornamental fishes should be introduced because the failure to require and implement quarantines has already resulted in the accidental introduction of exotic parasites to fish farms, and because these parasites can cause further economic losses if they become established in the wild.