Ortholinea nupchi n. sp. (Myxosporea: Ortholineidae) from the urinary bladder of the cultured olive flounder Paralichthys olivaceus, South Korea.

A new myxosporean parasite, Ortholinea nupchi n. sp. (Myxozoa; Bivalvulida), was isolated from the urinary bladder of the olive flounder Paralichthys olivaceus cultured on Jeju Island, Korea. Mature spores were subspherical in the valvular and apical views and ellipsoidal in the sutural view. The spores measured 7.6 ± 0.5 µm in length, 6.7 ± 0.3 µm in thickness, and 7.3 ± 0.5 µm in width. Two pyriform polar capsules measured 3.2 ± 0.1 µm in length and 2.7 ± 0.1 µm in width and were located at the same level at the anterior half of the myxospores. The suture line was straight in the middle of the spores, and the surface ridges ranged between five and seven, forming an intricate pattern. The result of the 18S rDNA comparison showed ≤ 93.0% similarity with other Ortholinea species. The phylogenetic tree demonstrated that O. nupchi n. sp. was closest to O. auratae and clustered with oligochaete-infecting myxosporeans (OIM) having urinary system infection tropism. Based on the comparison of environmental and host factors in the phylogenetic groups of the OIM clade, we propose that the infection of O. nupchi n. sp. originated from marine oligochaetes.

Infection dynamics of Enteromyxum leei (Myxozoa, Myxosporea) in culture water and its effects on cultured olive flounder, Paralichthys olivaceus (Temminck & Schlegel).

Enteromyxum leei is a causative agent of enteromyxosis, with a wide range of marine fish hosts. Recently, massive morbidity and mortality were caused by E. leei infection in cultured olive flounders in Korea. To reveal a relationship between E. leei abundance in culture water and the occurrence of parasite infection in host fish, we used a quantitative PCR assay targeting the 28S rDNA of E. leei in three fish farms (two where enteromyxosis had occurred and one where it did not) from April to November 2018. The gene of E. leei was detected at levels greater than 10 cells/L in the culture water where enteromyxosis occurred from July to September. Furthermore, 2 months after the detection in the water, the parasite gene (with more than 5,000 cells per 100 mg) was detected in fish intestine samples. However, in the fish farms where enteromyxosis had not occurred, the E. leei gene was detected at <10 cells in culture water (1 L) and fish intestine samples (100 mg). The quantification method used in this research provides a baseline of the infection timeline in olive flounder to develop effective management practices.

Dicyema sphyrocephalum (Phylum Dicyemida: Dicyemidae) isolated from Korean common octopus Callistoctopus minor in Korea.

BACKGROUND: Dicyemids are parasites found in the renal sac of cephalopods. The first species of dicyemid was found from kidneys of the Korean common octopus Callistoctopus minor. OBJECTIVES: This study aimed to identify the dicyemid and investigate the effect on renal sac of host. METHODS: In this study, we compared the morphological characteristics of isolate to dicyemids (Dicyema sphyrocephalum, Dicyema clavatum, and Dicyema dolichocephalum) reported from C. minor in Japan. We compared the 18S ribosomal RNA (rDNA) and cytochrome c oxidase subunit I (COI) sequences of isolate to the sequences of D. shyrocephalum and D. clavatum. The infected octopuses renal tissues were histologically compared with the tissues of uninfected individuals. RESULTS: The morphological characteristic of this isolated species corresponds to D. sphyrocephalum. The sequences similarities of 18S rDNA and COI gene of isolate are 99.7% and 98.1% with D. sphyrocephalum. We observed morphological changes in the epithelia folds of kidney at the dicyemids attached areas. CONCLUSIONS: The present study identified the isolate as D. sphyrocephalum and this is the first report of dicyemid species from Republic of Korea. Further studies on the effects of dicyemids on growth and health status of cephalopods will be needed.

Effect of Epigallocatechin Gallate on Viability of Kudoa septempunctata.

Kudoa septempunctata have been reported as a causative agent for acute transient gastrointestinal troubles after eating raw olive flounder (Paralichthys olivaceus). It raised public health concerns and quarantine control in several countries. Quantitative evaluation on viability of K. septempunctata is crucial to develop effective chemotherapeutics against it. A cytometry using fluorescent stains was employed to assess effect of three compounds on viability of K. septempunctata. Epigallocatechin gallate reduced markedly viability of K. septempunctata at 0.5 mM or more, and damaged K. septempunctata spores by producing cracks.

Evaluation of the effect of protease inhibitors on the viability of Miamiensis avidus using the WST-1 assay.

Miamiensis avidus causes scuticociliatosis in cultured olive flounders (Paralichthys olivaceus), leading to economic losses in aquaculture in Korea. Quantitative evaluation of the viability of M. avidus is important to develop an effective vaccine or chemotherapeutic agent against it. We used a colorimetric assay based on the reduction of 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) to quantify the viability of M. avidus. Using this method, we investigated the effect of protease inhibitors on the viability of M. avidus. The assay showed a clear difference in the optical density (OD) of over 104 ciliates, and the metalloprotease inhibitors 1, 10-phenanthroline and ethylenediaminetetraacetic acid (EDTA) reduced the viability of M. avidus by more than 90% when used at concentration of 5 mM and 100 µM, respectively. However, different morphological changes in the parasite were observed when exposed to these two inhibitors. These results indicate that the WST-1 assay is a simple and reliable method to quantify the viability of M. avidus, and metalloproteases are excellent targets for the development of agents and vaccines to control M. avidus infection.

A new species Myxodavisia jejuensis n. sp. (Myxosporea: Sinuolineidae) isolated from cultured olive flounder Paralichthys olivaceus in South Korea.

A new myxosporean parasite, Myxodavisia jejuensis n. sp. (Myxozoa; Bivalvulida) is described from the urinary bladder of olive flounder Paralichthys olivaceus cultured on Jeju Island, Korea. Two long lateral appendages with whip-like extensions were attached to mature spores of triangular to semi-circular shape. The spores were measured at 13.1 ± 1.1 µm in length, 17.2 ± 1.0 µm in thickness, and 13.1 ± 1.0 µm in width. Two spherical polar capsules, with a diameter of 5.0 ± 0.4 µm, were observed on opposite sides in the middle of the spore. The suture line was straight or slightly sinuous on the middle of spores. The 18S rDNA from M. jejuensis n. sp. was used in BLAST and molecular phylogenetic analysis. The results demonstrated that M. jejuensis n. sp. was closest to Sinuolinea capsularis and that the infection site tropism was correlated with the phylogeny of marine myxosporeans. In addition, we designed specific primers to detect the 18S rDNA gene of M. jejuensis n. sp.; the results showed specific amplification in M. jejuensis n. sp. among the myxosporeans isolated from the urinary bladder of the cultured olive flounder.

Kudoa ogawai (Myxosporea: Kudoidae) Infection in Cultured Olive Flounder Paralichthys olivaceus.

Since Kudoa septempuntata was identified as a causative agent of food poisoning associated with raw olive flounder Paralichthys olivaceus, interest and concern regarding the parasite have increased. However, there have been no investigations or reports of other Kudoa species infecting the fish (except for K. paralichthys, which infects the brain) in Korea. We found cysts filled with myxospores of Kudoa species in muscles of cultured olive flounder specimens and identified these to the species level. Mature spores were quadrate, measuring 8.7±0.5 µm in length, 9.2±0.4 µm in thickness, and 12.9±0.6 µm in width. The spores containing 4 polar capsules had a length of 2.1±0.2 µm and a width of 1.8±0.3 µm. The partial 18S and 28S rDNA of isolates showed 99-100% similarities with K. ogawai. Using these morphological and molecular analyses, the species was identified as K. ogawai. This study is the first report of K. ogawai infection in cultured olive flounder in Korea.

Sinuolinea capsularis (Myxosporea: Sinuolineidae) Isolated from Urinary Bladder of Cultured Olive Flounder Paralichthys olivaceus.

Sinuolinea capsularis Davis, 1917 is myxosporean that infect the urinary system of the host fish. Insufficient morphological and molecular data of S. capsularis exits, and it is therefore difficult to make an accurate identification of the parasite. We tried a series of morphological and molecular analysis to identify an myxosporean isolated from urinary bladder of cultured olive flounder, Paralichthys olivaceus, from Jeju island in the Republic of Korea. Some of them were observed under a light microscope and SEM, and remain samples were used molecular and phylogenetic analysis. Mature spores were subspherical, measuring 13.9±0.6 µm in length and 13.8±0.8 µm in width. Two spherical polar capsules on opposite sides in the middle of the spore had a diameter range of 4.3±0.4 µm. Scanning electron microscopy revealed that spores a severely twisted the suture line. By the morphological comparison and analysis, it was identified as S. capsularis. In addition, we obtained the partial 18S rDNA of S. capsularis and first registered it in NCBI. Phylogenetic analysis showed that S. capsularis clustered with Zschokkella subclade infecting the urinary system of marine fish, and it supported the infection site tropism effect on phylogeny of marine myxosporeans as well as the origin of Sinuolinea is not monophyly.

Quantitative investigation of Enteromyxum leei (Myxozoa: Myxosporea) infection and relative condition factor in cultured olive flounder Paralichthys olivaceus (Temminck and Schlegel).

Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate-buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease.

Parvicapsula curvatura n. sp. in cultured olive flounder Paralichthys olivaceus and phylogenetic characteristics of the genus Parvicapsula.

Parvicapsula curvatura n. sp. (Myxozoa; Bivalvulida) was found in the urinary bladder of olive flounder Paralichthys olivaceus cultured in a fish farm on Jeju Island, ROK. When laterally viewed, the parasite has asymmetrical curved spores that measure 9.6-11.6 µm in length. Furthermore, it has 2 subspherical polar capsules at the apex. Based on the phenotypical traits, it is most similar to P. limandae but differs in the shape of polar capsule, locality, and host specificity (family level). BLAST analysis indicated that P. curvatura was closest to P. unicornis and P. petuniae via 18S and 28S rDNA sequences, respectively. The 18S rDNA from P. curvatura was used in molecular phylogenetic analyses of Parvicapsula spp. to examine the congruence of phylogeny with spore morphology, locality, and host specificity. The results demonstrated that the spore morphotype was correlated with the phylogeny of the genus Parvicapsula, and the parasites have speciated into an oblong and semicircular spore type.

Spatial and Temporal Changes in the Distribution of Blood Fluke Infection in Nicolea gracilibranchis (Polychaeta: Terebellidae), the Intermediate Host for Cardicola orientalis (Digenea: Aporocotylidae), at a Tuna Farming Site in Japan.

Fish blood flukes of the genus Cardicola (Digenea: Aporocotylidae) are important pathogens in tuna aquaculture. Recent advances in marine blood fluke research have led to the elucidation of the lifecycles of 3 Cardicola spp. infecting tuna; all 3 flukes utilize terebellid polychaetes as the intermediate host. In our survey, we obtained large numbers of Nicolea gracilibranchis infected by larval Cardicola orientalis at our tuna farming site. To determine the spatial and temporal changes in the distribution of N. gracilibranchis surrounding tuna culture cages and their infection by C. orientalis, we conducted monthly sampling for a period of 1 yr. Terebellids were most abundant on the floats and ropes of culture cages, but a significantly higher proportion of infected N. gracilibranchis was detected on ropes, particularly up to 4 m in depth. Cardicola orientalis infection in N. gracilibranchis was clearly seasonal, with a higher infection rate between April and July. Our findings indicate that the infected terebellids inhabit specific microhabitats, and both abiotic and biotic factors likely influence blood fluke infection in the intermediate terebellid host. This information is important to better understand the general biology of marine aporocotylids and may be useful to develop a control strategy for blood fluke infection in tuna aquaculture.

Developmental stages of fish blood flukes, Cardicola forsteri and Cardicola opisthorchis (Trematoda: Aporocotylidae), in their polychaete intermediate hosts collected at Pacific bluefin tuna culture sites in Japan.

Farming of Pacific bluefin tuna (PBT), Thunnus orientalis, is a rapidly growing industry in Japan. Aporocotylid blood flukes of the genus Cardicola comprising C. orientalis, C. opisthorchis and C. forsteri are parasites of economic importance for PBT farming. Recently, terebellid polychaetes have been identified as the intermediate hosts for all these parasites. We collected infected polychaetes, Terebella sp., the intermediate host of C. opisthorchis, from ropes and floats attached to tuna cages in Tsushima, Nagasaki Prefecture, Japan. Also, Neoamphitrite vigintipes (formerly as Amphitrite sp. sensu Shirakashi et al., 2016), the intermediate host of C. forsteri, were collected from culture cages in Kushimoto, Wakayama Prefecture, Japan. The terebellid intermediate hosts harbored the sporocysts and cercariae in their body cavity. Developmental stages of these blood flukes were molecularly identified using species specific PCR primers. In this paper, we describe the cercaria and sporocyst stages of C. opisthorchis and C. forsteri and compare their morphological characteristics among three Cardicola blood flukes infecting PBT. We also discuss phylogenetic relations of the six genera of the terebellid intermediate hosts (Artacama, Lanassa, Longicarpus, Terebella, Nicolea and Neoamphitrite) of blood flukes infecting marine fishes, based on their morphological characters.

Phylogenetic study of the genus Kudoa (Myxozoa: Multivalvulida) with a description of Kudoa rayformis sp. nov. from the trunk muscle of Pacific sierra Scomberomorus sierra.

Kudoa rayformis n. sp. (Myxozoa; Multivalvulida) was observed in the trunk muscle of Pacific sierra Scomberomorus sierra caught off the coast of Tonosi, Panama. The species formed pseudocysts in myofibers and infection was subclinical. The myxospores possessed four polar capsules and spore valves, one of which had a distinct filamentous extension. This unique morphological characteristic of the myxospore validated this as a new species of Kudoa. Genetically, K. rayformis n. sp. is closest to K. inornata, with 98% and 91% similarity in 18S and 28S rDNA, respectively, but its spore shape was clearly distinct. The 18S rDNA and concatenated sequences from K. rayformis were used in molecular phylogenetic analyses of kudoids to examine the congruence of phylogeny with infection site tropism, spore morphology and cyst/pseudocyst formation. The results demonstrated that the phenotypic traits were correlated with the phylogeny of Kudoidae, and that the biological features of K. rayformis originated from the ancient Kudoidae as exhibited by the non-specific infection site tropism and the ability to infect muscle and form pseudocysts.

Discovery of intermediate hosts for two species of blood flukes Cardicola orientalis and Cardicola forsteri (Trematoda: Aporocotylidae) infecting Pacific bluefin tuna in Japan.

Fish blood flukes (Aporocotylidae) are important pathogens of farmed finfish around the world. Among them, Cardicola spp. infecting farmed tuna are considered to be serious threats to tuna farming and have received tremendous attention. We conducted periodical samplings at a tuna farming site in Japan between January and May, 2015 to determine the life cycle of Cardicola spp. We collected over 4700 terebellid polychaetes from ropes, floats and frames of tuna culture cages and found nearly 400 infected worms. Sporocysts and cercariae found in Nicolea gracilibranchis were genetically identified as Cardicola orientalis by 28S and ITS2 ribosomal DNA sequences. This was the first discovery of the intermediate host for this parasite species. Infection prevalence and the abundance of N. gracilibranchis significantly varied between sampling points and the highest number of infected terebellids were collected from ropes. We also demonstrated morphologically and molecularly that asexual stages found in a single Amphitrite sp. (Terebellidae) and adult worms isolated from farmed juvenile tuna were Cardicola forsteri. This is the first report of C. forsteri in Pacific bluefin tuna (PBT) Thunnus orientalis in Japan. Our results demonstrated that all three species of Cardicola orientalis, C. forsteri and Cardicola opisthorchis exist in Japanese farmed PBTs and that they all use terebellid polychaetes as the intermediate hosts.

Factors affecting sporoplasm release in Kudoa septempunctata.

The myxosporean parasite Kudoa septempunctata has been isolated from cultured olive flounder (Paralichthys olivaceus) and was recently identified as a cause of food poisoning in humans. Since the sporoplasm plays an important role in causing diarrhea by invading intestinal cells, the specific factors affecting the release of sporoplasm from spores should be determined. Thus, we investigated the effect of digestive and serum enzymes, fetal bovine serum (FBS), temperature, and the role of glucose in cell culture media on the release of sporoplasm. Sporoplasm release was observed in the groups treated with FBS and media containing glucose. In addition, 1,10-phenanthroline inhibited the release of sporoplasm in the FBS medium. These results indicate that K. septempunctata uses glucose for releasing its sporoplasm and that zinc or metalloprotease is related to the release mechanism. The present study provides important information for the development of agents to prevent sporoplasm release and the consequent food poisoning caused by K. septempunctata.

Bacteriophage therapy of a Vibrio parahaemolyticus infection caused by a multiple-antibiotic-resistant O3:K6 pandemic clinical strain.

BACKGROUND: Recently isolated Vibrio parahaemolyticus strains have displayed multiple antibiotic resistance. Alternatives to conventional antibiotics are needed, especially for the multiple-antibiotic-resistant V. parahaemolyticus pandemic strain. METHODS: A bacteriophage, designated pVp-1, showed effective infectivity for multiple-antibiotic-resistant V. parahaemolyticus and V. vulnificus, including V. parahaemolyticus pandemic strains. The therapeutic potential of the phage was studied in a mouse model of experimental infection using a multiple-antibiotic-resistant V. parahaemolyticus pandemic strain. We monitored the survivability and histopathological changes, quantified the bacterial and phage titers during phage therapy, and observed the immune response induced by phage induction. RESULTS: Phage-treated mice displayed protection from a V. parahaemolyticus infection and survived lethal oral and intraperitoneal bacterial challenges. CONCLUSIONS: To the best of our knowledge, this is the first report of phage therapy in a mouse model against a multiple-antibiotic­resistant V. parahaemolyticus pandemic strain infection.

The phylogenetic study on Thelohanellus species (Myxosporea) in relation to host specificity and infection site tropism.

Thelohanellus kitauei (Myxobolidae) infects cyprinid fish. The evolution of species derived from common ancestors results in the sharing of biological features. To reveal the origin of T. kitauei biological features, the correlation between phylogeny and biological features of Myxobolidae was investigated by Bayesian inference tree and Bayesian tip association significance testing. The results demonstrated that host specificity and infection site tropism were correlated with the phylogeny of Myxobolidae, and that the biological features of T. kitauei originated from the ancient Myxobolidae as exhibited by the non-specific infection site tropism and the ability to infect cyprinids.

Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi.

Philasterides dicentrarchi is a causative agent of scuticociliatosis in olive flounder Paralichthys olivaceus, aquaculture in Korea. In this study, a cDNA encoding a cathepsin L-like cysteine protease (PdCtL) of P. dicentrarchi (synonym Miamiensis avidus) was identified. To express the PdCtL recombinant protein in a heterologous system, 10 codons were redesigned to conform to the standard eukaryotic genetic code using polymerase chain reaction (PCR)-based site-directed mutagenesis. The recombinant P. dicentrarchi procathepsin L (proPdCtL) was expressed at high levels in E. coli Rosetta (DE3) pLysS with a pPET21a vector, and successfully refolded, purified, and activated into a functional and enzymatically active form. The optimal pH for protease activity was 5. Similar to other cysteine proteases, enzyme activity was inhibited by E64 and leupeptin. Immunogenicity of recombinant PdCtL was assessed by enzyme-linked immunosorbent assay, western blot, and specific anti-recombinant PdCtL antibodies were detected. Our results suggest that the biochemical characteristics of the recombinant ciliate proPdCtL protein are similar to those of the cathepsin L-like cysteine protease, that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form, and that the recombinant PdCtL acted as a specific epitope in olive flounder.

CPRMethicillin resistant coagulase-negative staphylococci isolated from South Korean ducks exhibiting tremor.

BACKGROUND: We describe coagulase-negative staphylococci (CoNS) isolates collected from ducklings exhibiting tremor in South Korea over the period of 2010 to 2011. Screening of antimicrobial susceptibility and analysis of SCCmec elements of CoNS were also investigated. RESULTS: Staphylococcus cohnii was the most frequent staphylococcus (9 isolates) and S. sciuri (4 isolates), S. lentus (3 isolate), S. simulans (1 isolate) and S. epidermidis (1 isolate) were also detected. Among the 15 antimicrobials tested in this study, resistance against oxacillin (15 isolates, 83.3%) was most frequently observed, but only one isolate (SNUDS-1) possessed mecA. This isolate was shown to possess SCCmec type III; the type 3 ccr complex and the class A mec complex. CONCLUSIONS: Based on these results, isolate SNUDS-1 was shown to possess SCCmec type III; the type 3 ccr complex and the class A mec complex. Although the SCCmec type III is not predominant in human, MR-CoNS (Methicillin resistance Coagulase-negative staphylococci) in food animals should be monitored to prevent the dissemination of antimicrobial resistance genes and resistant pathogens to the community.

Sequence-based genotyping methods to assess the genetic diversity of Riemerella anatipestifer isolates from ducklings with tremor.

The aim of this study was to investigate sequence-based genotyping methods to distinguish 27 Riemerella anatipestifer isolates from ducklings in South Korea. The 16S rRNA sequences of the 27 R. anatipestifer isolates showed 99�100% similarities to each other and to reference sequences from Genbank (AY871822.2, AY871834.2, CP002562.1, EU715016.1, EU016548.1, EU715000.1, EU715008.1 and EU715011.1). In addition, the ompA gene sequences of 25 of the 27 R. anatipestifer isolates were 100% identical to each other, and these sequences were also 100% identical to reference sequences (CP002562.1, GQ415419.1, DQ059079, FJ765034.1, AY606207.1, AF104937.1, and FJ765033.1). Alternatively, four housekeeping genes (mdh, gdh, pgi, and rpoB) and three virulence-associated genes (prtC, hagA, and sspA) were used for a multi-locus sequence typing (MLST) and a single-locus sequence typing (SLST) among R. anatipestifer isolates. Compared to 16S rRNA and the ompA gene, seven genes showed higher genetic divergence patterns, and the isolates were separated into three distinct groups in phylogenetic trees.