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Introduction of bacterial and viral pathogens from imported ornamental finfish in South Korea

Introduction of bacterial and viral pathogens from imported ornamental finfish in South Korea Background: Live fish import may lead to the unintended introduction of pathogens. We examined the monthly distribution of microbial pathogens in ornamental finfish imported into South Korea over a 6-month period. Results: Vibrio alginolyticus was detected in one lemon damsel in June and July; V. vulnificus was detected in one lemon damsel, one caerulean damsel, and one pearl-spot chromis and one ocellaris clownfish in July, April, and May, respectively; Photobacterium damselae was detected in one ocellaris clownfish and one caerulean damsel in June and July, respectively; V. anguillarum was detected in one pearl-spot chromis in February; V. harveyi was detected in one ocellaris clownfish and two mandarin fish in February and April, respectively; Yersinia ruckeri was detected in a pearlscale goldfish group in June and July and in two colored carp groups in July; and Lactococcus garvieae was detected in a lemon damsel group and a sutchi catfish group in July and May, respectively. European catfish virus, the only viral pathogen detected, was found in two sutchi catfish groups in May. Conclusion: This study is the first to identify pathogenic species and the presence or absence of pathogens (non- quarantine diseases) in imported ornamental finfish. These results demonstrate that various pathogens with the potential to harm indigenous fish populations can accompany ornamental finfish imported into South Korea. Keywords: Imported ornamental finfish, Non-quarantine diseases, Vibriosis, Bacterial pathogens, Viral pathogens Background and Singapore. As a result, the large number of The import value of live aquarium fish into South Korea imported fish may have exacerbated the problem of has increased by more than twofold from $2.011 million exotic fish pathogens being brought into South Korea. in 1997 to $4.724 million in 2010 and continues to in- Import of a variety of live fish species can lead to the crease each year (Korea Agro-Fisheries & Food Trade unintended introduction of pathogens. In Australia, for Corporation in South Korea). Most of the ornamental example, the government has stringent regulations for fish (90%) imported into South Korea are freshwater live imported ornamental species that include mandatory species (about 1000 in total) that are cultured in aquar- quarantine periods of 1–3 weeks and health certification. iums. However, marine species (approximately 500) are Despite these measures, there have been many outbreaks caught in the wild. Most of the freshwater fish are of exotic viral, bacterial, fungal, protozoan, and meta- imported from tropical Asian countries such as zoan infections among ornamental fish in Australian Malaysia, Indonesia, and China (Moravec et al. 1999), aquaculture farms. Aeromonas salmonicida is associated whereas marine fish are from Indonesia, the Philippines, with ulcers in pearlscale goldfish (Carassius auratus), and Edwardsiella ictaluri with systemic disease in rosy * Correspondence: poka96@sunmoon.ac.kr; kyj5088@hanmail.net barbs (Pethia conchonius) (Humphrey 1995). E. tarda Hee Jae Choi and Jun Wook Hur contributed equally to this work. was isolated from fighting fish (Betta splendens) Department of Pharmaceutical Engineering & Biotechnology, Sun Moon imported from Singapore and quarantined in Australia University, Asan-si, Republic of Korea Department of Aquatic Life Medical Sciences, Sun Moon University, in 1985 (Humphrey et al. 1986). Although E. tarda is a Seonmun-ro 221 Tangjeong-myeon, Asan-si, Chungnam 336-708, Republic of ubiquitous bacterium in freshwater environments, it was Korea not known to cause mortality in aquafarm species and Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 2 of 9 was found during a large survey of bacterial pathogens fish per month. Sutchi catfish (Pangasius sutchi)was infecting salmonids in Australia between 1981 and 1985 imported every month with 20 fish being sampled each (Humphrey et al. 1987). Furthermore, it was found to month. Five fish each of lemon damsel (Pomacentrus cause subdermal abscesses in wild eels (Anguilla rein- moluccensis), caerulean damsel (Pomacentrus caeruleus), hardtii) in Queensland in 1990 (Eaves et al. 1990) and and whitetail dascyllus (Dascyllus aruanus) along with mortality in farmed rainbow trout (Oncorhynchus three pearl-spot chromis (Chromis notata) were sampled mykiss) in New South Wales in 1993 (Reddacliff et al. in February and March, followed by ten fish each per 1996). Non-quarantined diseases are negligible. When month being sampled April through July. Five colored however, a new or exotic disease is introduced, there is carp (Cyprinus carpio haematopterus) were sampled in always the risk of serious pathogenic or transmissible February, and ten were sampled each month from diseases, resulting in enormous economic losses in com- March through July. Mandarin fish (Synchiropus splendi- mercial aquaculture or in wild aquatic systems. dus) were sampled over 4 months from February The role of ornamental fish in the transmission of through May, at a frequency of five fish in February and non-quarantine pathogens and the establishment of in- March and ten in April and May. fection and disease has received little attention in South Korea. The Korean government has been investigating Selection of candidate bacterial and viral pathogens from the influx of aquatic quarantine pathogens via imported imported ornamental finfish living aquatic animals (National Fishery Products Qual- Bacterial and viral pathogens in ornamental finfish were ity Management Service in South Korea). However, these selected based on those most likely to infect finfish ac- surveys had not focused on non-quarantine diseases as- cording to information from the World Organization for sociated with imported ornamental finfish until 2015. Animal Health (OIE) and survey data from the National We have addressed this in the current study by exam- Fishery Products Quality Management Service (NFQS) ining the presence of non-quarantine bacterial and viral in South Korea for species imported in 2012–2015. Spe- pathogens in imported ornamental finfish, identification cifically, we selected pathogens for which there are of the pathogens, and phylogenetic analysis of the dis- standard international methods for detection (Table 2). eases detected. Bacterial pathogens included Lactococcus garvieae, Yersi- nia ruckeri (the cause of enteric redmouth disease Methods (ERM)), and the etiological agents of vibriosis, such as Sampling of imported ornamental finfish Vibrio anguillarum, V. alginolyticus, V. harveyi, V. vulni- We examined the presence of microbial pathogens in or- ficus, V. penaeicida, and Photobacterium damselae namental finfish imported into Korea. Ornamental fin- (formerly V. damsel). Viral pathogens included European fish specimens varied in terms of the number of samples catfish virus (ECV), infectious hematopoietic necrosis and their size and weight (Table 1). Imported fish were virus (IHNV), viral nervous necrosis (VNN), and infec- randomly sampled for 6 months from February through tious spleen and kidney necrosis virus-like (ISKNV-like). July 2016. Fish sampled included ocellaris clownfish Olive flounder (Paralichthys olivaceus) artificially in- (Amphiprion ocellaris), pearlscale goldfish (Carassius fected with Vibrio anguillarum or viral hemorrhagic auratus), and Nile tilapia (Oreochromis niloticus) at ten septicemia virus (VHSV) was used as positive Table 1 Number of imported ornamental finfish sampled per month. We examined a total of 581 fish representing 10 species Water Common name (scientific name) Total samples (fish) per month (2016) Average Average length weight February March April May June July (cm) (g) Sea Colored carp (Cyprinus carpio haematopterus) 5 10 10 10 10 10 5.3 1.21 Pearlscale goldfish (Carassius auratus) 10 10 10 10 10 10 5 5.53 Sutchi catfish (Pangasius sutchi) 20 20 20 20 20 20 8 2.41 Nile tilapia (Oreochromis niloticus) 10 10 10 10 10 10 5.0 2.57 Fresh Pearl-spot chromis (Chromis notata) 3 3 10 10 10 10 4.3 1.06 Caerulean damsel (Pomacentrus caeruleus) 5 5 10 10 10 10 5.5 2.24 Lemon damsel (Pomacentrus moluccensis) 5 5 10 10 10 10 4.3 1.44 Mandarin fish (Synchiropus splendidus) 5 5 10 10 0 0 7.3 5.91 Whitetail dascyllus (Dascyllus aruanus) 5 5 10 10 10 10 3.5 1.15 Ocellaris clownfish (Amphiprion ocellaris) 10 10 10 10 10 10 4.2 1.47 Monthly sampling numbers for each species of fish from February to July 2016 are shown Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 3 of 9 Table 2 PCR primers used for the detection of bacterial and viral pathogens Pathogen Primer name Sequence Reference L. garvieae pLG1 5′-CAT AAC AAT GAG AAT CGC-3′ Baeck et al. 2006 pLG2 5′-GCA CCC TCG CGG GTT G-3′ Y. ruckeri YER8 5′-GCG AGG AGG AAG GGT TAA GTG-3′ Gibello et al. 1999 YER10 5′-GAA GGC ACC AAG GCA TCT CTG-3′ V. anguillarum Van-ami8 F 5′-ACA TCA TCC ATT TGT TAC-3′ Gonzalez et al. 2003 Van-ami417 R 5′-CCT TAT CAC TAT CCA AAT TG-3′ V. alginolyticus gyrB Fw 5′-GAG AAC CCG ACA GAA GCG AAG-3′ Zhou et al. 2007 gyrB Re 5′-CCT AGT GCG GTG ATC AGT GTT G-3′ V. harveyi VH-4F 5′-GTG ATG AAG AAG CTT ATC GCG ATT-3′ Kim et al. 2014 VH-7R 5′-CGC CTT CTT CAG TTA ACG CAG G-3′ V. vulnificus vvhA Fw 5′-TTC CAA CTT CAA ACC GAA CTA TGA-3′ Panicker et al. 2004 vvhA Re 5′-ATT CCA GTC GAT GCG AAT ACG TTG-3′ V. penaeicida Fw 5′-AA TAT TGC ACA ATG GGC GC-3′ Saulnier et al. 2000 Re 5′-G CGC TTT ACG CCC AGT AAT TCC G-3′ P. damselae PD-2F 5′-CAA GAC ATC ATC GAT GTG ATG CGT-3′ Kim et al. 2014 PD-2R 5′-GAA ACT TTA CCA TCT ACC ACT TTG-3′ European catfish virus (ECV) Fw 5′-ATGCGCTCCTCCGGGTCAAAG-3′ OIE 2011 Re 5′-CATCATGAACGGCTCGATGG-3′ Infectious hematopoietic necrosis virus (IHNV) IHNV-GF 5′-AGA GAT CCC TAC ACC AGA GAC-3′ Emmenegger et al. 2000 IHNV-GR 5′-GGT GGT GTT GTT TCC GTG CAA-3′ Viral nervous necrosis (VNN) VNN F 5′-CGT GTC AGT CAT GTG TCG CT-3′ Nishizawa et al. 1996 VNN R 5′-CGA GTC AAC ACG GGT GAA GA-3′ Infectious spleen and kidney necrosis virus–like (ISKNV-like) P1 F 5′-ATG TCT GCA ATC TCA GGT-3′ Xu et al. 2008 P2 R 5′-TTA CAG GAT AGG GAA GCC TG-3′ NP1 (NP nested) 5′-GCG TTT GAT GCG ATG GAG AC-3′ NP2 (NP nested) 5′-ACG GCA GAG ACA CGG TAG GC-3′ experimental controls for each polymerase chain reac- PCR assay. As a large number of yellow or green col- tion (PCR) assay used for disease identification. The onies of different types formed, these were classified ac- positive control for the PCR method was used to elimin- cording to those with the same morphology, and 5–10 ate uncertainty regarding the method of disease detec- different types of colonies were used for PCR diagnosis. tion in PCR assay. For detection of the other pathogens (L. garvieae, Y. ruckeri, ECV, IHNV, VNN, and ISKNV-like), the col- Sampling of imported ornamental fish and identification lected target organs of the finfish were pooled into two of microbial pathogens or four groups with five fish per group, and the genomic To detect bacterial or viral infections in imported spe- DNA extracted using a GeneAll Exgene Tissue SV kit cies, we analyzed quarantine records and procured regu- (GeneAll Biotechnology, Seoul, Korea), or the genomic lar samples from import companies listed in the NFQS. RNA derived from virus purified using a RNeasy Mini Samples were kept in their imported culture water, un- kit (Qiagen, Valencia, CA, USA). The RNA was reverse exposed to the Korean environment. The finfish were transcribed to generate cDNA using Power cDNA syn- anesthetized, their livers, spleens, and kidneys harvested thesis kit (INtRON, Korea). The isolated genomic DNA using sterilized dissection tools, and the tissues evalu- and cDNA were used as template in PCR assays to de- ated for disease identification. As the first line of screen- tect the genes of the pathogen. ing, we used thiosulphate-citrate-bile salts-sucrose (TCBS) agar plates as a selective medium for identifying PCR assay Vibrio bacteria. To identify Vibrio spp., the yellow or Pathogen detection and analysis were performed using green colonies on the TCBS medium were identified by PCR methods previously described, as referenced in Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 4 of 9 Table 2. Details on the primer sets used for the detection Results of each pathogen are shown in Table 2. PCR mixes con- Screening pathogens by TCBS agar culture and PCR tained 10 μl of 2 × ExPrime Taq™ Premix (GENET BIO, detection Korea), which included 1 unit of ExPrime Taq DNA We analyzed the monthly prevalence of bacterial and Polymerase, Tris-HCl (pH 9.0), PCR enhancer, viral pathogens based on the total finfish samples (NH4) SO , 4 mM MgCl , enzyme stabilizer, sediment, (Table 3). Two pathogens were detected in February, V. 2 4 2 loading dye, and 2.0 mM dNTPs mixture; 0.5 μM of each anguillarum in a pearl-spot chromis and V. harveyi in −1 primer of the relative primer sets; 1 μlof10 diluted an ocellaris clownfish. No pathogens were detected in total DNA template; and distilled water to a final total the imported finfish tested in March. Among the fish volume of 20 μl. The thermal cycling profiles included species imported in April, there were two cases of V. an initial 95 °C for 3 min (tissue-extracted DNA) or 10 harveyi detected in mandarin fish and one case of V. min (colony-extracted DNA), then 30 sequential amplifi- vulnificus detected in each of the pearl-spot chromis and cation cycles, followed by a final extension step of 72 °C caerulean damsel fish. In May, there was a positive find- for 7 min. The amplification cycles were ing for L. garvieae in a group of sutchi catfish, V. vulnifi- pathogenic-specific and consisted of denaturation at 95 ° cus in an ocellaris clownfish, and two cases of ECV C for 30 s, annealing at 50 °C for 1 min, and template ex- detected in separate groups of sutchi catfish. In June, tension at 72 °C for 30 s for L. garvieae, ESV, and IHNV; there was one case each of Y. ruckeri in a group of denaturation at 95 °C for 30 s, annealing at 58 °C for 1 pearlscale goldfish, P. damselae in an ocellaris clownfish, min, and template extension at 72 °C for 30 s for V. and V. alginolyticus in a lemon damsel. In July, eight anguillarum, V. penaeicida, V. vulnificus,NNV,and specimens were tested positive for genetic material from ISKNV-like; or denaturation at 95 °C for 30 s, annealing bacterial pathogens. These included three cases of vibri- at 60 °C for 1 min, and template extension at 72 °C for osis (V. alginolyticus in a lemon damsel, V. vulnificus in 30 s for Y. ruckeri, V. harveyi, V. alginolyticus,and P. both a lemon damsel and a caerulean damsel, and P. damselae. The PCR products were sequenced by Cosmo damselae in a caerulean damsel); a case of Y. ruckeri in a Genetech (Seoul, Korea) and confirmed using the basic pearlscale goldfish and two cases in separate groups of local alignment search tool (BLAST) on the National colored carp; and one case of L. garvieae in a lemon Center for Biotechnology Information (NCBI) website damsel. (https://www.ncbi.nlm.nih.gov). Phylogenetic analysis of screened pathogen gene Phylogenetic analysis sequences For the bacterial pathogens, 16S rRNA gene sequences Phylogenetic trees based on the detected genes of the were obtained for L. garvieae, P. damselae, and Y. ruck- pathogens were generated using MEGA 7 software. eri. The gyrase B subunit (gyrB) gene sequence was ob- Alignment with known sequences in the GenBank data- tained for V. alginolyticus. For the Vibrio spp., amiB base showed that the genetic sequence detected in sequences of V. anguillarum, which encode N-acetyl- lemon damsel, which encoded for only 385-aa, had a muramoyl-L-alanine amidase, RNA polymerase beta high similarity (99%) with the 16S rRNA genes of L. gar- subunit (rpoB) sequences of V. harveyi and vvhB se- vieae from Oncorhynchus mykiss (KM604702.1) in India quences of V. vulnificus, which encodes a chaperone, and Oreochromis niloticus (KM209202.1) in Indonesia. were obtained from analysis of the PCR products. For The 388-aa fragment detected in ocellaris clownfish analysis of the viral pathogen ECV, gene sequences of showed 99% similarity with GenBank sequences for the the major capsid protein (MCP) were obtained. Each se- 16S rRNA gene of P. damselae from Lates calcarifer in quence detected was aligned using the NCBI GenBank India (MF164181.1) and 96% similarity with Oplegnathus database. fasciatus of China (KF956381.1) (Fig. 1a). The genetic For phylogenetic analysis, the aligned sequences were sequence derived from the pathogen isolated from the evaluated using the MEGA 7 (Molecular Evolutionary colored carp encoded for a 496-aa fragment and shared Genetic Analysis 7) software program (Kumar et al. 99% similarity with the 16S rRNA gene of Y. ruckeri 2016). The alignments of the predicted protein or nu- from Salmo salar of Norway (CP023184.1) and grass cleotide sequences were revised by excluding regions carp of China (KF003196.1) (Fig. 1b). with uncertain alignment at the ends of the sequences. In the Vibrio spp., the 349-aa gyrB sequence isolated The phylogenetic trees were constructed using the from the lemon damsel showed relatedness to V. algino- neighbor-joining method with bootstrap analysis (1000 lyticus from Trachurus trachurus in Japan (CAR48209.1) replications), which showed that the pathogen genes de- with a 97% sequence similarity (Fig. 1c). The 551-aa tected in this study were placed into the same clade with fragment of the V. anguillarum amiB that was detected respective species in GenBank. in pearl-spot chromis had 99% similarity to the amiB of Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 5 of 9 Table 3 Pathogens detected by polymerase chain reaction in imported finfish sampled from February to July 2016 (5 fish/group) Fish sample Pathogen Number of pathogen-positive fish (or group)/total fish sampled by month February March April May June July Ocellaris clownfish V. harveyi 1 fish/10 0/10 0/10 0/10 0/10 0/10 P. damselae 0/10 0/10 0/10 0/10 1 fish/10 0/10 V. vulnificus 0/10 0/10 0/10 1 fish/10 0/10 0/10 Pearlscale goldfish Y. ruckeri 0/10 0/10 0/10 0/10 1 group/10 1 group/10 Sutchi catfish L. garvieae 0/20 0/20 0/20 1 group/20 0/20 0/20 ECV 0/20 0/20 0/20 2 groups/20 0/20 0/20 Lemon damsel L. garvieae 0/5 0/5 0/10 0/10 0/10 1 group/10 V. alginolyticus 0/5 0/5 0/10 0/10 1 fish/10 1 fish/10 V. vulnificus 0/5 0/5 0/10 0/10 0/10 1 fish/10 Pearl-spot chromis V. anguillarum 1 fish/3 0/3 0 /10 0/10 0/10 0/10 V. vulnificus 0/3 0/3 1 fish/10 0/10 0/10 0/10 Caerulean damsel V. vulnificus 0/5 0/5 1 fish/10 0/10 0/10 1 fish/10 P. damselae 0/5 0/5 0/10 0/10 0/10 1 fish/10 Colored carp Y. ruckeri 0/5 0/10 0/10 0/10 0/10 2 groups/10 Mandarin fish V. harveyi 0/5 0/5 2 fish/10 0/10 –– Whitetail dascyllus – 0/5 0/5 0/10 0/10 0/10 0/10 Nile tilapia – 0/10 0/10 0/10 0/10 0/10 0/10 V. anguillarum isolated from Lateolabrax japonicus of We detected the V. alginolyticus gyrB gene in lemon China (CP016095.1) (Fig. 1d). The 481-aa region of rpoB damsel during a 2-month period (detection rates of 10% detected in mandarin fish demonstrated 99% identity to in both June and July). The gyrB gene detected in caeru- V. harveyi rpoB from Trachinotus ovatus of China lean damsel had 97% similarity with the V. alginolyticus (CP018680.2) (Fig. 1e). The 579-aa segment of V. vulnifi- isolate from Trachurus in Japan according to sequence cus vvhB detected in caerulean damsel was 94% similar alignment and analysis, with a close association being to V. vulnificus vvhB from Mya arenaria (CP016322.1), demonstrated in the phylogenetic tree. V. alginolyticus is Mactra veneriformis (CP015513.1) of South Korea, Oreo- responsible for epizootic outbreaks and mortality in chromis of Taiwan (CP009262.1), and eastern oyster of farmed gilt-head sea bream (Sparus aurata L.) and the USA (KF255370.1) (Fig. 1f). puffer fish (Fugu vermicularis vermicularis) (Balebona et In addition to the bacterial sequences detected, the al. 1998b; Colorni et al. 1981; Noguchi et al. 1987) and viral sequences also aligned with known sequences in has caused massive losses for the aquaculture industry the GenBank database. The 395-aa fragment of the ECV in China (Xie et al. 2005). In sea bream, symptoms of in- MCP gene detected from sutchi catfish demonstrated fection include septicemia, hemorrhage, and fluid accu- 99% sequence similarity with the ECV gene of Silurus mulation in the peritoneal cavity (Balebona et al. 1998a; glanis of Spain (YP 006347612.1) (Fig. 1g). Colorni et al. 1981). Ours is the first report of V. algino- lyticus infection in lemon damsel. V. vulnificus was detected in lemon damsel in July, Discussion caerulean damsel in April and July, pearl-spot chromis The occurrence of bacterial or viral diseases in aquacul- in April, and ocellaris clownfish in May, at a 10% detec- ture is a major problem that can lead to enormous eco- tion rate in each case. Phylogenetic analysis revealed that nomic losses worldwide. Vibrio species are widely the vvhB detected in caerulean damsel shared 94% simi- distributed in marine environments and estuaries and larity with the V. vulnificus isolates from Mya arenaria are some of the most abundant pathogens in aquafarms and Mactra veneriformis of South Korea. This pathogen (Austin and Austin 1993a; Bergh et al. 2001; Hjeltnes has been shown to cause infectious diseases in Japanese and Roberts 1993; Lightner 1993). V. anguillarum, V. and European eels in Spain, Sweden, and Norway alginolyticus, V. harveyi, and V. vulnificus are among the (Biosca et al. 1991, 1997; Muroga et al. 1976). V. vulnifi- main pathogens causing vibriosis in several finfish spe- cus has been detected in marine fish collected along the cies (Austin and Zhang 2006; Balebona et al. 1998b; west coast of India at an incidence of 16.6% (Thampuran Romalde 2002), including in South Korea. and Surendran 1998) and has also been detected in Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 6 of 9 Fig. 1 Phylogenetic trees and electrophoresis detection of gene sequences from the pathogens. Phylogenetic comparison between other sequences and the present detected sequences (●, denoted by a bold circle) based on phylogenetic tree of a 16S rRNA gene sequences obtained from P. damselae in ocellaris clownfish, b 16S rRNA gene sequences obtained from Y. ruckeri in colored carp, c gyrB gene sequences obtained from V. alginolyticus in lemon damsel, d amiB gene sequences obtained from V. anguillarum in pearl-spot chromis. e rpoB gene sequences obtained from the V. harveyi in mandarin fish, f vvhB gene sequences obtained from V. vulnificus in caerulean damsel, and g MCP gene sequences obtained from ECV in sutchi catfish. h Electrophoresis detection of pathogen bands in agarose gel (1%). [Lines 1, 4, 6, 10: 100 bp ladder (GeneSTA™), Line 2: V. harveyi (454 bp), Line 3: V. vulnificus (205 bp), Line 5: V. alginolyticus (337 bp), Line 7: P. damselae (533 bp), Line 8: V. anguillarum (429 bp), Line 9: Y. ruckeri (575 bp), Line 11: L. garvieae (1100 bp), Line 12: ECV (625 bp)] Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 7 of 9 other countries such as Spain, the Netherlands, and et al. 2001; Colorni et al. 2003; Fadaeifard et al. 2012; Japan. Kang et al. 2004; Kawanishi et al. 2005; Kusuda et al. V. anguillarum was detected in approximately 1/3 of 1991; Lee et al. 2001; Prieta 1993; Ravelo et al. 2003). pearl-spot chromis imported in February. The amiB of Various countries with aquafarms have reported L. gar- V. anguillarum that was detected from pearl-spot chro- vieae infections, including Israel, Italy, Taiwan, Spain, mis had 99% similarity with the amiB of V. anguillarum Japan, Australia, Turkey, and the UK (Baya et al. 1990; derived from Lateolabrax japonicus of China. V. anguil- Eldar et al. 1994, 1995, 1996; Kitao 1993; Kusuda et al. larum was first reported in European eels (Bergman 1976; Nieto et al. 1995; Rasheed and Plumb 1984), as 1909) and is widely found in both cultured and wild fish, well as South Korea (Kang et al. 2004). including Salmo salar L. (salmon), rainbow trout (Onco- Y. ruckeri was detected in pearlscale goldfish (one of rhynchus mykiss), bream, eel, mullet, catfish, and tilapia two groups in June and July each) and colored carp (Oreochromis spp.) in salt or brackish water. Infections (both groups in July). The Y. ruckeri 16S rRNA gene de- cause fatal hemorrhagic septicemic disease (Aguirre-- rived from colored carp showed 99% similarity with the Guzman et al. 2004; Frans et al. 2011; Paillard et al. Y. ruckeri 16S rRNA gene isolated from Salmo salar of 2004; Toranzo et al. 2005). Norway and grass carp of China. The disease ERM, The V. harveyi rpoB sequence was detected in ocellaris which is caused by Y. ruckeri, can lead to mortality in clownfish (10% infection rate in February) and mandarin farmed salmonids (Austin and Austin 1993b). There fish (20% infection rate in April). The rpoB gene se- have been no reports to date of ERM in pearlscale gold- quence detected in the mandarin fish demonstrated 99% fish or colored carp; in addition, it has only been associ- identity with the gene from Trachinotus ovatus of China. ated with farmed eel in South Korea (Joh et al. 2010, This pathogen is known to affect crevalle jack (Caranx 2013). hippos), summer flounder (Paralichthys dentatus), sand- Finally, ECV was the only viral pathogen detected in bar shark (Carcharhinus plumbeus), and lemon shark the current study but was found at a rate of 10% in (Negaprion brevirostris) (Austin and Zhang 2006; sutchi catfish imported in May. The ECV MCP gene de- Kraxberger-Beatty et al. 1990; Bertone et al. 1996; Col- tected in sutchi catfish showed 99% sequence similarity well and Grimes 1984; Grimes et al. 1984; Lee et al. with that isolated from Silurus glanis of Spain. In 2002; Soffientino et al. 1999). Germany, ECV caused high morbidity and mortality in P. damselae was detected in ocellaris clownfish (10% sheatfish and catfish hosts in a 1989 outbreak, causing detection rate in June) and in caerulean damsel (10% de- 100% mortality in infected fish (Ahne et al. 1989). tection rate in July). Analysis of P. damselae using the In this study, Vibrio species, L. garvieae and P. damse- 16S rRNA gene detected in ocellaris clownfish revealed lae, were already known to exist in South Korea, but that it had 96% similarity to the P. damselae derived ECV and Y. ruckeri have not been reported in South from Oplegnathus fasciatus of China and resulted in the Korea yet. Therefore, Y. ruckeri and ECV are exotic in- relatedness demonstrated by the phylogenetic tree. This fectious diseases, and it is necessary to continuously pathogen has previously been shown to affect cultures of monitor and analyze the effects of the pathogenicity of red-banded sea bream, common sea bream, white seab- these diseases. ream, and marine rainbow trout in Denmark (Labella et We were unable to identify any individuals in the al. 2011; Pedersen et al. 2009). The mortality rates range imported samples with unusual symptoms of disease in- between 5% and 94% depending on the season, and the fection. The culture water imported with the samples species and age of the fish (Labella et al. 2011). was cloudy only for the lemon damsel and ocellaris L. garvieae was detected in lemon damsel (50% infec- clownfish. However, we did not detect pathogens in cul- tion rate in July) and sutchi catfish (25% infection rate in ture water. We did not observe any symptoms of latent May). Sequencing analysis of the 16S rRNA gene de- stage diseases in the ornamental finfish. We also tected in lemon damsel had high similarity (99%) with screened for genes of V. penaeicida, IHN, VNN, and the L. garvieae 16S rRNA gene isolated from Oncorhyn- ISKNV-like as pathogens in imported finfish, but none chus mykiss in India and Oreochromis niloticus in were detected during the sampling period of the study. Indonesia. This pathogen was first discovered in rainbow Furthermore, we did not detect genes of the selected trout raised on a Japanese fish farm in the 1950s (Hos- pathogens in whitetail dascyllus or Nile tilapia during hina et al. 1958) but has since been isolated as a the test period. disease-causing agent in rainbow trout, yellowtail, tilapia, Japanese eel (Anguilla japonica), olive flounder (Para- Conclusions lichthys olivaceus), gray mullet catfish, wild wrasse (Coris The frequency of detecting bacterial and viral pathogens aygula), black rockfish (Sebastes schlegeli), amberjack among the finfish imported into South Korea from Feb- (Seriola dumerili), and kingfish (Seriola lalandi) (Chen ruary through July 2016 gradually increased over time. Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 8 of 9 This may have been associated with an increase in water Received: 25 October 2018 Accepted: 30 January 2019 temperature. Domestic interest in imported aquatic spe- cies is increasing; exotic finfish are bred as a hobby, as References well as for consumption. However, this may lead to an Aguirre-Guzman G, Ruiz HM, Ascencio F. A review of extracellular virulence increase in imported pathogens in South Korea. In product of Vibrio species important in diseases of cultivated shrimp. Aquac Australia, a wide range of viruses, bacteria, and parasites Res. 2004;35:1395–404. Ahne W, Schlotfeldt HJ, Thomsen I. Fish viruses: isolation of an icosahedral was identified in ornamental fish during quarantine in cytoplasmic deoxyribovirus from sheatfish (Silurus glanis). Zentralbl the period from 1999 to 2004 (Chong and Whittington Veterinarmed B. 1989;36:333–6. 2005), and these pathogens had negative effects in Austin B, Austin DA. Vibriosis. In: Austin B, Austin DA, editors. Bacterial fish pathogens and disease in farmed wild fish. England: Springer; 1993a. aquatic animals. 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Mar Biotechnol. 2002;4:267–77. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Fisheries and Aquatic Sciences Springer Journals

Introduction of bacterial and viral pathogens from imported ornamental finfish in South Korea

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Copyright © 2019 by The Author(s)
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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

Background: Live fish import may lead to the unintended introduction of pathogens. We examined the monthly distribution of microbial pathogens in ornamental finfish imported into South Korea over a 6-month period. Results: Vibrio alginolyticus was detected in one lemon damsel in June and July; V. vulnificus was detected in one lemon damsel, one caerulean damsel, and one pearl-spot chromis and one ocellaris clownfish in July, April, and May, respectively; Photobacterium damselae was detected in one ocellaris clownfish and one caerulean damsel in June and July, respectively; V. anguillarum was detected in one pearl-spot chromis in February; V. harveyi was detected in one ocellaris clownfish and two mandarin fish in February and April, respectively; Yersinia ruckeri was detected in a pearlscale goldfish group in June and July and in two colored carp groups in July; and Lactococcus garvieae was detected in a lemon damsel group and a sutchi catfish group in July and May, respectively. European catfish virus, the only viral pathogen detected, was found in two sutchi catfish groups in May. Conclusion: This study is the first to identify pathogenic species and the presence or absence of pathogens (non- quarantine diseases) in imported ornamental finfish. These results demonstrate that various pathogens with the potential to harm indigenous fish populations can accompany ornamental finfish imported into South Korea. Keywords: Imported ornamental finfish, Non-quarantine diseases, Vibriosis, Bacterial pathogens, Viral pathogens Background and Singapore. As a result, the large number of The import value of live aquarium fish into South Korea imported fish may have exacerbated the problem of has increased by more than twofold from $2.011 million exotic fish pathogens being brought into South Korea. in 1997 to $4.724 million in 2010 and continues to in- Import of a variety of live fish species can lead to the crease each year (Korea Agro-Fisheries & Food Trade unintended introduction of pathogens. In Australia, for Corporation in South Korea). Most of the ornamental example, the government has stringent regulations for fish (90%) imported into South Korea are freshwater live imported ornamental species that include mandatory species (about 1000 in total) that are cultured in aquar- quarantine periods of 1–3 weeks and health certification. iums. However, marine species (approximately 500) are Despite these measures, there have been many outbreaks caught in the wild. Most of the freshwater fish are of exotic viral, bacterial, fungal, protozoan, and meta- imported from tropical Asian countries such as zoan infections among ornamental fish in Australian Malaysia, Indonesia, and China (Moravec et al. 1999), aquaculture farms. Aeromonas salmonicida is associated whereas marine fish are from Indonesia, the Philippines, with ulcers in pearlscale goldfish (Carassius auratus), and Edwardsiella ictaluri with systemic disease in rosy * Correspondence: poka96@sunmoon.ac.kr; kyj5088@hanmail.net barbs (Pethia conchonius) (Humphrey 1995). E. tarda Hee Jae Choi and Jun Wook Hur contributed equally to this work. was isolated from fighting fish (Betta splendens) Department of Pharmaceutical Engineering & Biotechnology, Sun Moon imported from Singapore and quarantined in Australia University, Asan-si, Republic of Korea Department of Aquatic Life Medical Sciences, Sun Moon University, in 1985 (Humphrey et al. 1986). Although E. tarda is a Seonmun-ro 221 Tangjeong-myeon, Asan-si, Chungnam 336-708, Republic of ubiquitous bacterium in freshwater environments, it was Korea not known to cause mortality in aquafarm species and Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 2 of 9 was found during a large survey of bacterial pathogens fish per month. Sutchi catfish (Pangasius sutchi)was infecting salmonids in Australia between 1981 and 1985 imported every month with 20 fish being sampled each (Humphrey et al. 1987). Furthermore, it was found to month. Five fish each of lemon damsel (Pomacentrus cause subdermal abscesses in wild eels (Anguilla rein- moluccensis), caerulean damsel (Pomacentrus caeruleus), hardtii) in Queensland in 1990 (Eaves et al. 1990) and and whitetail dascyllus (Dascyllus aruanus) along with mortality in farmed rainbow trout (Oncorhynchus three pearl-spot chromis (Chromis notata) were sampled mykiss) in New South Wales in 1993 (Reddacliff et al. in February and March, followed by ten fish each per 1996). Non-quarantined diseases are negligible. When month being sampled April through July. Five colored however, a new or exotic disease is introduced, there is carp (Cyprinus carpio haematopterus) were sampled in always the risk of serious pathogenic or transmissible February, and ten were sampled each month from diseases, resulting in enormous economic losses in com- March through July. Mandarin fish (Synchiropus splendi- mercial aquaculture or in wild aquatic systems. dus) were sampled over 4 months from February The role of ornamental fish in the transmission of through May, at a frequency of five fish in February and non-quarantine pathogens and the establishment of in- March and ten in April and May. fection and disease has received little attention in South Korea. The Korean government has been investigating Selection of candidate bacterial and viral pathogens from the influx of aquatic quarantine pathogens via imported imported ornamental finfish living aquatic animals (National Fishery Products Qual- Bacterial and viral pathogens in ornamental finfish were ity Management Service in South Korea). However, these selected based on those most likely to infect finfish ac- surveys had not focused on non-quarantine diseases as- cording to information from the World Organization for sociated with imported ornamental finfish until 2015. Animal Health (OIE) and survey data from the National We have addressed this in the current study by exam- Fishery Products Quality Management Service (NFQS) ining the presence of non-quarantine bacterial and viral in South Korea for species imported in 2012–2015. Spe- pathogens in imported ornamental finfish, identification cifically, we selected pathogens for which there are of the pathogens, and phylogenetic analysis of the dis- standard international methods for detection (Table 2). eases detected. Bacterial pathogens included Lactococcus garvieae, Yersi- nia ruckeri (the cause of enteric redmouth disease Methods (ERM)), and the etiological agents of vibriosis, such as Sampling of imported ornamental finfish Vibrio anguillarum, V. alginolyticus, V. harveyi, V. vulni- We examined the presence of microbial pathogens in or- ficus, V. penaeicida, and Photobacterium damselae namental finfish imported into Korea. Ornamental fin- (formerly V. damsel). Viral pathogens included European fish specimens varied in terms of the number of samples catfish virus (ECV), infectious hematopoietic necrosis and their size and weight (Table 1). Imported fish were virus (IHNV), viral nervous necrosis (VNN), and infec- randomly sampled for 6 months from February through tious spleen and kidney necrosis virus-like (ISKNV-like). July 2016. Fish sampled included ocellaris clownfish Olive flounder (Paralichthys olivaceus) artificially in- (Amphiprion ocellaris), pearlscale goldfish (Carassius fected with Vibrio anguillarum or viral hemorrhagic auratus), and Nile tilapia (Oreochromis niloticus) at ten septicemia virus (VHSV) was used as positive Table 1 Number of imported ornamental finfish sampled per month. We examined a total of 581 fish representing 10 species Water Common name (scientific name) Total samples (fish) per month (2016) Average Average length weight February March April May June July (cm) (g) Sea Colored carp (Cyprinus carpio haematopterus) 5 10 10 10 10 10 5.3 1.21 Pearlscale goldfish (Carassius auratus) 10 10 10 10 10 10 5 5.53 Sutchi catfish (Pangasius sutchi) 20 20 20 20 20 20 8 2.41 Nile tilapia (Oreochromis niloticus) 10 10 10 10 10 10 5.0 2.57 Fresh Pearl-spot chromis (Chromis notata) 3 3 10 10 10 10 4.3 1.06 Caerulean damsel (Pomacentrus caeruleus) 5 5 10 10 10 10 5.5 2.24 Lemon damsel (Pomacentrus moluccensis) 5 5 10 10 10 10 4.3 1.44 Mandarin fish (Synchiropus splendidus) 5 5 10 10 0 0 7.3 5.91 Whitetail dascyllus (Dascyllus aruanus) 5 5 10 10 10 10 3.5 1.15 Ocellaris clownfish (Amphiprion ocellaris) 10 10 10 10 10 10 4.2 1.47 Monthly sampling numbers for each species of fish from February to July 2016 are shown Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 3 of 9 Table 2 PCR primers used for the detection of bacterial and viral pathogens Pathogen Primer name Sequence Reference L. garvieae pLG1 5′-CAT AAC AAT GAG AAT CGC-3′ Baeck et al. 2006 pLG2 5′-GCA CCC TCG CGG GTT G-3′ Y. ruckeri YER8 5′-GCG AGG AGG AAG GGT TAA GTG-3′ Gibello et al. 1999 YER10 5′-GAA GGC ACC AAG GCA TCT CTG-3′ V. anguillarum Van-ami8 F 5′-ACA TCA TCC ATT TGT TAC-3′ Gonzalez et al. 2003 Van-ami417 R 5′-CCT TAT CAC TAT CCA AAT TG-3′ V. alginolyticus gyrB Fw 5′-GAG AAC CCG ACA GAA GCG AAG-3′ Zhou et al. 2007 gyrB Re 5′-CCT AGT GCG GTG ATC AGT GTT G-3′ V. harveyi VH-4F 5′-GTG ATG AAG AAG CTT ATC GCG ATT-3′ Kim et al. 2014 VH-7R 5′-CGC CTT CTT CAG TTA ACG CAG G-3′ V. vulnificus vvhA Fw 5′-TTC CAA CTT CAA ACC GAA CTA TGA-3′ Panicker et al. 2004 vvhA Re 5′-ATT CCA GTC GAT GCG AAT ACG TTG-3′ V. penaeicida Fw 5′-AA TAT TGC ACA ATG GGC GC-3′ Saulnier et al. 2000 Re 5′-G CGC TTT ACG CCC AGT AAT TCC G-3′ P. damselae PD-2F 5′-CAA GAC ATC ATC GAT GTG ATG CGT-3′ Kim et al. 2014 PD-2R 5′-GAA ACT TTA CCA TCT ACC ACT TTG-3′ European catfish virus (ECV) Fw 5′-ATGCGCTCCTCCGGGTCAAAG-3′ OIE 2011 Re 5′-CATCATGAACGGCTCGATGG-3′ Infectious hematopoietic necrosis virus (IHNV) IHNV-GF 5′-AGA GAT CCC TAC ACC AGA GAC-3′ Emmenegger et al. 2000 IHNV-GR 5′-GGT GGT GTT GTT TCC GTG CAA-3′ Viral nervous necrosis (VNN) VNN F 5′-CGT GTC AGT CAT GTG TCG CT-3′ Nishizawa et al. 1996 VNN R 5′-CGA GTC AAC ACG GGT GAA GA-3′ Infectious spleen and kidney necrosis virus–like (ISKNV-like) P1 F 5′-ATG TCT GCA ATC TCA GGT-3′ Xu et al. 2008 P2 R 5′-TTA CAG GAT AGG GAA GCC TG-3′ NP1 (NP nested) 5′-GCG TTT GAT GCG ATG GAG AC-3′ NP2 (NP nested) 5′-ACG GCA GAG ACA CGG TAG GC-3′ experimental controls for each polymerase chain reac- PCR assay. As a large number of yellow or green col- tion (PCR) assay used for disease identification. The onies of different types formed, these were classified ac- positive control for the PCR method was used to elimin- cording to those with the same morphology, and 5–10 ate uncertainty regarding the method of disease detec- different types of colonies were used for PCR diagnosis. tion in PCR assay. For detection of the other pathogens (L. garvieae, Y. ruckeri, ECV, IHNV, VNN, and ISKNV-like), the col- Sampling of imported ornamental fish and identification lected target organs of the finfish were pooled into two of microbial pathogens or four groups with five fish per group, and the genomic To detect bacterial or viral infections in imported spe- DNA extracted using a GeneAll Exgene Tissue SV kit cies, we analyzed quarantine records and procured regu- (GeneAll Biotechnology, Seoul, Korea), or the genomic lar samples from import companies listed in the NFQS. RNA derived from virus purified using a RNeasy Mini Samples were kept in their imported culture water, un- kit (Qiagen, Valencia, CA, USA). The RNA was reverse exposed to the Korean environment. The finfish were transcribed to generate cDNA using Power cDNA syn- anesthetized, their livers, spleens, and kidneys harvested thesis kit (INtRON, Korea). The isolated genomic DNA using sterilized dissection tools, and the tissues evalu- and cDNA were used as template in PCR assays to de- ated for disease identification. As the first line of screen- tect the genes of the pathogen. ing, we used thiosulphate-citrate-bile salts-sucrose (TCBS) agar plates as a selective medium for identifying PCR assay Vibrio bacteria. To identify Vibrio spp., the yellow or Pathogen detection and analysis were performed using green colonies on the TCBS medium were identified by PCR methods previously described, as referenced in Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 4 of 9 Table 2. Details on the primer sets used for the detection Results of each pathogen are shown in Table 2. PCR mixes con- Screening pathogens by TCBS agar culture and PCR tained 10 μl of 2 × ExPrime Taq™ Premix (GENET BIO, detection Korea), which included 1 unit of ExPrime Taq DNA We analyzed the monthly prevalence of bacterial and Polymerase, Tris-HCl (pH 9.0), PCR enhancer, viral pathogens based on the total finfish samples (NH4) SO , 4 mM MgCl , enzyme stabilizer, sediment, (Table 3). Two pathogens were detected in February, V. 2 4 2 loading dye, and 2.0 mM dNTPs mixture; 0.5 μM of each anguillarum in a pearl-spot chromis and V. harveyi in −1 primer of the relative primer sets; 1 μlof10 diluted an ocellaris clownfish. No pathogens were detected in total DNA template; and distilled water to a final total the imported finfish tested in March. Among the fish volume of 20 μl. The thermal cycling profiles included species imported in April, there were two cases of V. an initial 95 °C for 3 min (tissue-extracted DNA) or 10 harveyi detected in mandarin fish and one case of V. min (colony-extracted DNA), then 30 sequential amplifi- vulnificus detected in each of the pearl-spot chromis and cation cycles, followed by a final extension step of 72 °C caerulean damsel fish. In May, there was a positive find- for 7 min. The amplification cycles were ing for L. garvieae in a group of sutchi catfish, V. vulnifi- pathogenic-specific and consisted of denaturation at 95 ° cus in an ocellaris clownfish, and two cases of ECV C for 30 s, annealing at 50 °C for 1 min, and template ex- detected in separate groups of sutchi catfish. In June, tension at 72 °C for 30 s for L. garvieae, ESV, and IHNV; there was one case each of Y. ruckeri in a group of denaturation at 95 °C for 30 s, annealing at 58 °C for 1 pearlscale goldfish, P. damselae in an ocellaris clownfish, min, and template extension at 72 °C for 30 s for V. and V. alginolyticus in a lemon damsel. In July, eight anguillarum, V. penaeicida, V. vulnificus,NNV,and specimens were tested positive for genetic material from ISKNV-like; or denaturation at 95 °C for 30 s, annealing bacterial pathogens. These included three cases of vibri- at 60 °C for 1 min, and template extension at 72 °C for osis (V. alginolyticus in a lemon damsel, V. vulnificus in 30 s for Y. ruckeri, V. harveyi, V. alginolyticus,and P. both a lemon damsel and a caerulean damsel, and P. damselae. The PCR products were sequenced by Cosmo damselae in a caerulean damsel); a case of Y. ruckeri in a Genetech (Seoul, Korea) and confirmed using the basic pearlscale goldfish and two cases in separate groups of local alignment search tool (BLAST) on the National colored carp; and one case of L. garvieae in a lemon Center for Biotechnology Information (NCBI) website damsel. (https://www.ncbi.nlm.nih.gov). Phylogenetic analysis of screened pathogen gene Phylogenetic analysis sequences For the bacterial pathogens, 16S rRNA gene sequences Phylogenetic trees based on the detected genes of the were obtained for L. garvieae, P. damselae, and Y. ruck- pathogens were generated using MEGA 7 software. eri. The gyrase B subunit (gyrB) gene sequence was ob- Alignment with known sequences in the GenBank data- tained for V. alginolyticus. For the Vibrio spp., amiB base showed that the genetic sequence detected in sequences of V. anguillarum, which encode N-acetyl- lemon damsel, which encoded for only 385-aa, had a muramoyl-L-alanine amidase, RNA polymerase beta high similarity (99%) with the 16S rRNA genes of L. gar- subunit (rpoB) sequences of V. harveyi and vvhB se- vieae from Oncorhynchus mykiss (KM604702.1) in India quences of V. vulnificus, which encodes a chaperone, and Oreochromis niloticus (KM209202.1) in Indonesia. were obtained from analysis of the PCR products. For The 388-aa fragment detected in ocellaris clownfish analysis of the viral pathogen ECV, gene sequences of showed 99% similarity with GenBank sequences for the the major capsid protein (MCP) were obtained. Each se- 16S rRNA gene of P. damselae from Lates calcarifer in quence detected was aligned using the NCBI GenBank India (MF164181.1) and 96% similarity with Oplegnathus database. fasciatus of China (KF956381.1) (Fig. 1a). The genetic For phylogenetic analysis, the aligned sequences were sequence derived from the pathogen isolated from the evaluated using the MEGA 7 (Molecular Evolutionary colored carp encoded for a 496-aa fragment and shared Genetic Analysis 7) software program (Kumar et al. 99% similarity with the 16S rRNA gene of Y. ruckeri 2016). The alignments of the predicted protein or nu- from Salmo salar of Norway (CP023184.1) and grass cleotide sequences were revised by excluding regions carp of China (KF003196.1) (Fig. 1b). with uncertain alignment at the ends of the sequences. In the Vibrio spp., the 349-aa gyrB sequence isolated The phylogenetic trees were constructed using the from the lemon damsel showed relatedness to V. algino- neighbor-joining method with bootstrap analysis (1000 lyticus from Trachurus trachurus in Japan (CAR48209.1) replications), which showed that the pathogen genes de- with a 97% sequence similarity (Fig. 1c). The 551-aa tected in this study were placed into the same clade with fragment of the V. anguillarum amiB that was detected respective species in GenBank. in pearl-spot chromis had 99% similarity to the amiB of Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 5 of 9 Table 3 Pathogens detected by polymerase chain reaction in imported finfish sampled from February to July 2016 (5 fish/group) Fish sample Pathogen Number of pathogen-positive fish (or group)/total fish sampled by month February March April May June July Ocellaris clownfish V. harveyi 1 fish/10 0/10 0/10 0/10 0/10 0/10 P. damselae 0/10 0/10 0/10 0/10 1 fish/10 0/10 V. vulnificus 0/10 0/10 0/10 1 fish/10 0/10 0/10 Pearlscale goldfish Y. ruckeri 0/10 0/10 0/10 0/10 1 group/10 1 group/10 Sutchi catfish L. garvieae 0/20 0/20 0/20 1 group/20 0/20 0/20 ECV 0/20 0/20 0/20 2 groups/20 0/20 0/20 Lemon damsel L. garvieae 0/5 0/5 0/10 0/10 0/10 1 group/10 V. alginolyticus 0/5 0/5 0/10 0/10 1 fish/10 1 fish/10 V. vulnificus 0/5 0/5 0/10 0/10 0/10 1 fish/10 Pearl-spot chromis V. anguillarum 1 fish/3 0/3 0 /10 0/10 0/10 0/10 V. vulnificus 0/3 0/3 1 fish/10 0/10 0/10 0/10 Caerulean damsel V. vulnificus 0/5 0/5 1 fish/10 0/10 0/10 1 fish/10 P. damselae 0/5 0/5 0/10 0/10 0/10 1 fish/10 Colored carp Y. ruckeri 0/5 0/10 0/10 0/10 0/10 2 groups/10 Mandarin fish V. harveyi 0/5 0/5 2 fish/10 0/10 –– Whitetail dascyllus – 0/5 0/5 0/10 0/10 0/10 0/10 Nile tilapia – 0/10 0/10 0/10 0/10 0/10 0/10 V. anguillarum isolated from Lateolabrax japonicus of We detected the V. alginolyticus gyrB gene in lemon China (CP016095.1) (Fig. 1d). The 481-aa region of rpoB damsel during a 2-month period (detection rates of 10% detected in mandarin fish demonstrated 99% identity to in both June and July). The gyrB gene detected in caeru- V. harveyi rpoB from Trachinotus ovatus of China lean damsel had 97% similarity with the V. alginolyticus (CP018680.2) (Fig. 1e). The 579-aa segment of V. vulnifi- isolate from Trachurus in Japan according to sequence cus vvhB detected in caerulean damsel was 94% similar alignment and analysis, with a close association being to V. vulnificus vvhB from Mya arenaria (CP016322.1), demonstrated in the phylogenetic tree. V. alginolyticus is Mactra veneriformis (CP015513.1) of South Korea, Oreo- responsible for epizootic outbreaks and mortality in chromis of Taiwan (CP009262.1), and eastern oyster of farmed gilt-head sea bream (Sparus aurata L.) and the USA (KF255370.1) (Fig. 1f). puffer fish (Fugu vermicularis vermicularis) (Balebona et In addition to the bacterial sequences detected, the al. 1998b; Colorni et al. 1981; Noguchi et al. 1987) and viral sequences also aligned with known sequences in has caused massive losses for the aquaculture industry the GenBank database. The 395-aa fragment of the ECV in China (Xie et al. 2005). In sea bream, symptoms of in- MCP gene detected from sutchi catfish demonstrated fection include septicemia, hemorrhage, and fluid accu- 99% sequence similarity with the ECV gene of Silurus mulation in the peritoneal cavity (Balebona et al. 1998a; glanis of Spain (YP 006347612.1) (Fig. 1g). Colorni et al. 1981). Ours is the first report of V. algino- lyticus infection in lemon damsel. V. vulnificus was detected in lemon damsel in July, Discussion caerulean damsel in April and July, pearl-spot chromis The occurrence of bacterial or viral diseases in aquacul- in April, and ocellaris clownfish in May, at a 10% detec- ture is a major problem that can lead to enormous eco- tion rate in each case. Phylogenetic analysis revealed that nomic losses worldwide. Vibrio species are widely the vvhB detected in caerulean damsel shared 94% simi- distributed in marine environments and estuaries and larity with the V. vulnificus isolates from Mya arenaria are some of the most abundant pathogens in aquafarms and Mactra veneriformis of South Korea. This pathogen (Austin and Austin 1993a; Bergh et al. 2001; Hjeltnes has been shown to cause infectious diseases in Japanese and Roberts 1993; Lightner 1993). V. anguillarum, V. and European eels in Spain, Sweden, and Norway alginolyticus, V. harveyi, and V. vulnificus are among the (Biosca et al. 1991, 1997; Muroga et al. 1976). V. vulnifi- main pathogens causing vibriosis in several finfish spe- cus has been detected in marine fish collected along the cies (Austin and Zhang 2006; Balebona et al. 1998b; west coast of India at an incidence of 16.6% (Thampuran Romalde 2002), including in South Korea. and Surendran 1998) and has also been detected in Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 6 of 9 Fig. 1 Phylogenetic trees and electrophoresis detection of gene sequences from the pathogens. Phylogenetic comparison between other sequences and the present detected sequences (●, denoted by a bold circle) based on phylogenetic tree of a 16S rRNA gene sequences obtained from P. damselae in ocellaris clownfish, b 16S rRNA gene sequences obtained from Y. ruckeri in colored carp, c gyrB gene sequences obtained from V. alginolyticus in lemon damsel, d amiB gene sequences obtained from V. anguillarum in pearl-spot chromis. e rpoB gene sequences obtained from the V. harveyi in mandarin fish, f vvhB gene sequences obtained from V. vulnificus in caerulean damsel, and g MCP gene sequences obtained from ECV in sutchi catfish. h Electrophoresis detection of pathogen bands in agarose gel (1%). [Lines 1, 4, 6, 10: 100 bp ladder (GeneSTA™), Line 2: V. harveyi (454 bp), Line 3: V. vulnificus (205 bp), Line 5: V. alginolyticus (337 bp), Line 7: P. damselae (533 bp), Line 8: V. anguillarum (429 bp), Line 9: Y. ruckeri (575 bp), Line 11: L. garvieae (1100 bp), Line 12: ECV (625 bp)] Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 7 of 9 other countries such as Spain, the Netherlands, and et al. 2001; Colorni et al. 2003; Fadaeifard et al. 2012; Japan. Kang et al. 2004; Kawanishi et al. 2005; Kusuda et al. V. anguillarum was detected in approximately 1/3 of 1991; Lee et al. 2001; Prieta 1993; Ravelo et al. 2003). pearl-spot chromis imported in February. The amiB of Various countries with aquafarms have reported L. gar- V. anguillarum that was detected from pearl-spot chro- vieae infections, including Israel, Italy, Taiwan, Spain, mis had 99% similarity with the amiB of V. anguillarum Japan, Australia, Turkey, and the UK (Baya et al. 1990; derived from Lateolabrax japonicus of China. V. anguil- Eldar et al. 1994, 1995, 1996; Kitao 1993; Kusuda et al. larum was first reported in European eels (Bergman 1976; Nieto et al. 1995; Rasheed and Plumb 1984), as 1909) and is widely found in both cultured and wild fish, well as South Korea (Kang et al. 2004). including Salmo salar L. (salmon), rainbow trout (Onco- Y. ruckeri was detected in pearlscale goldfish (one of rhynchus mykiss), bream, eel, mullet, catfish, and tilapia two groups in June and July each) and colored carp (Oreochromis spp.) in salt or brackish water. Infections (both groups in July). The Y. ruckeri 16S rRNA gene de- cause fatal hemorrhagic septicemic disease (Aguirre-- rived from colored carp showed 99% similarity with the Guzman et al. 2004; Frans et al. 2011; Paillard et al. Y. ruckeri 16S rRNA gene isolated from Salmo salar of 2004; Toranzo et al. 2005). Norway and grass carp of China. The disease ERM, The V. harveyi rpoB sequence was detected in ocellaris which is caused by Y. ruckeri, can lead to mortality in clownfish (10% infection rate in February) and mandarin farmed salmonids (Austin and Austin 1993b). There fish (20% infection rate in April). The rpoB gene se- have been no reports to date of ERM in pearlscale gold- quence detected in the mandarin fish demonstrated 99% fish or colored carp; in addition, it has only been associ- identity with the gene from Trachinotus ovatus of China. ated with farmed eel in South Korea (Joh et al. 2010, This pathogen is known to affect crevalle jack (Caranx 2013). hippos), summer flounder (Paralichthys dentatus), sand- Finally, ECV was the only viral pathogen detected in bar shark (Carcharhinus plumbeus), and lemon shark the current study but was found at a rate of 10% in (Negaprion brevirostris) (Austin and Zhang 2006; sutchi catfish imported in May. The ECV MCP gene de- Kraxberger-Beatty et al. 1990; Bertone et al. 1996; Col- tected in sutchi catfish showed 99% sequence similarity well and Grimes 1984; Grimes et al. 1984; Lee et al. with that isolated from Silurus glanis of Spain. In 2002; Soffientino et al. 1999). Germany, ECV caused high morbidity and mortality in P. damselae was detected in ocellaris clownfish (10% sheatfish and catfish hosts in a 1989 outbreak, causing detection rate in June) and in caerulean damsel (10% de- 100% mortality in infected fish (Ahne et al. 1989). tection rate in July). Analysis of P. damselae using the In this study, Vibrio species, L. garvieae and P. damse- 16S rRNA gene detected in ocellaris clownfish revealed lae, were already known to exist in South Korea, but that it had 96% similarity to the P. damselae derived ECV and Y. ruckeri have not been reported in South from Oplegnathus fasciatus of China and resulted in the Korea yet. Therefore, Y. ruckeri and ECV are exotic in- relatedness demonstrated by the phylogenetic tree. This fectious diseases, and it is necessary to continuously pathogen has previously been shown to affect cultures of monitor and analyze the effects of the pathogenicity of red-banded sea bream, common sea bream, white seab- these diseases. ream, and marine rainbow trout in Denmark (Labella et We were unable to identify any individuals in the al. 2011; Pedersen et al. 2009). The mortality rates range imported samples with unusual symptoms of disease in- between 5% and 94% depending on the season, and the fection. The culture water imported with the samples species and age of the fish (Labella et al. 2011). was cloudy only for the lemon damsel and ocellaris L. garvieae was detected in lemon damsel (50% infec- clownfish. However, we did not detect pathogens in cul- tion rate in July) and sutchi catfish (25% infection rate in ture water. We did not observe any symptoms of latent May). Sequencing analysis of the 16S rRNA gene de- stage diseases in the ornamental finfish. We also tected in lemon damsel had high similarity (99%) with screened for genes of V. penaeicida, IHN, VNN, and the L. garvieae 16S rRNA gene isolated from Oncorhyn- ISKNV-like as pathogens in imported finfish, but none chus mykiss in India and Oreochromis niloticus in were detected during the sampling period of the study. Indonesia. This pathogen was first discovered in rainbow Furthermore, we did not detect genes of the selected trout raised on a Japanese fish farm in the 1950s (Hos- pathogens in whitetail dascyllus or Nile tilapia during hina et al. 1958) but has since been isolated as a the test period. disease-causing agent in rainbow trout, yellowtail, tilapia, Japanese eel (Anguilla japonica), olive flounder (Para- Conclusions lichthys olivaceus), gray mullet catfish, wild wrasse (Coris The frequency of detecting bacterial and viral pathogens aygula), black rockfish (Sebastes schlegeli), amberjack among the finfish imported into South Korea from Feb- (Seriola dumerili), and kingfish (Seriola lalandi) (Chen ruary through July 2016 gradually increased over time. Choi et al. Fisheries and Aquatic Sciences (2019) 22:5 Page 8 of 9 This may have been associated with an increase in water Received: 25 October 2018 Accepted: 30 January 2019 temperature. 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