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.

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.

Philasterides dicentrarchi, a histophagous ciliate causing scuticociliatosis in olive flounder, Philasterides dicentrarchi--histopathology investigations.

Philasterides dicentrarchi is a histophagous scuticociliate infecting Korean olive flounder farms in Jeju Island, South Korea, where it causes significant economic losses. However, the route of entry of these parasites in olive flounder is currently unknown. In the present study, we attempted experimental infection with different doses (concentrations) of P. dicentrarchi by intraperitoneal (IP) injection; maximum cumulative mortality of 95% was recorded on the administration of 2.1×10(8) ciliates/ml. In small size group (3 cm length, 24±2 gwt) the ciliates were found in connective tissues of muscle, fins, and nervous tissue while in large size group (5 cm length, 47±3 gwt), the ciliates were predominantly occurred with associated symptoms of liquefaction in the central nervous system. The major clinico-pathological manifestations were loss of scales, appearance of bleached spots that coalesced to form brownish patches, hemorrhagic, and severe dermal necrotic lesion. In addition there was degeneration of muscle fibers, necrosis of the hepatic parenchyma, and severe edema of the intestinal walls, extensive fouling, necrotic degeneration and hyperplasia in the gill bronchial epithelium. In this study, many ciliates were found in the blood vessel, brain, dorsal fins, muscle, kidney, and vertebra of infected flounder. The histopathological investigations are of significant importance in view of possible routes of entry into the host and doses of ciliates that trigger fast infection for potential utility of drugs as a strategy for the control of P. dicentrarchi in farmed olive flounder.

Effectiveness and immunomodulation of chemotherapeutants against scuticociliate Philasterides dicentrarchi in olive flounder.

Philasterides dicentrarchi is a histophagous scuticociliate causes fatal scuticociliatosis in farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of scuticociliatosis due to P. dicentrarchi infections was increased from May to July (40+/-3.1% to 79.4+/-1.7%) and it decreased from August to November (63+/-2.3% to 30+/-2.6%) in olive flounder farms at Jeju Island, South Korea during 2000-2006. The prevalence of mixed infection along with Vibrio spp. bacterial infection was 49+/-7.2% than that of other mixed infection. At present no effective control measure for P. dicentrarchi infection has been described and large production losses continue. In the present study, formalin, hydrogen peroxide and Jenoclean chemotheraputants were used for bath treatment. Among Jenoclean at a low concentration of 50ppm proved effective. The results were confirmed with in vitro motility assessments and morphological changes scoring system in P. dicentrarchi. On the other hand, similar trend was noted following hydrogen peroxide treatment at this concentration, but formalin was only moderately effective. Either hydrogen peroxide or Jenoclean are the promising compounds effective at low concentrations with short application time for P. dicentrarchi. Therefore, these substances were evaluated on day 10, 20 and 30 for their ability to enhance innate immune response and disease resistance against P. dicentrarchi in olive flounder after chemotheraputants bath treatment with 100ppm for 30min per day. All the tested immune parameters were enhanced by treatment with Jenoclean, but not formalin and hydrogen peroxide. These findings suggest that Jenoclean bath treatment can be used for ensuring the heath of cultured marine fish against internal parasites such as P. dicentrarchi.

Effectiveness of chemotherapeutants against scuticociliate Philasterides dicentrarchi, a parasite of olive flounder.

The histophagous scuticociliate Philasterides dicentrarchi causes fatal scuticociliatosis in farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of scuticociliatosis with P. dicentrarchi infections was very high from May to July (40+/-3 to 79+/-2%) in olive flounder at farms of Jeju Island, South Korea, from 2000 to 2004. The prevalence of mixed infection along with Vibrio spp. infection was higher (49+/-8%) than that of scuticociliatosis alone. To date no effective control measure for P. dicentrarchi infection has been described and large economic losses continue. In the present study 3 chemotheraputants (formalin, hydrogen peroxide and Jenoclean) were used. Among these, bath treatment with Jenoclean at a low concentration of 50ppm proved effective; the results were confirmed with in vitro motility assessments and morphological changes in P. dicentrarchi. A similar trend was noted following hydrogen peroxide treatment. However, formalin was only moderately effective at this concentration. Therefore, hydrogen peroxide and Jenoclean are the promising compounds effective at low concentrations with short application time.

Histopathological changes of Korea cultured olive flounder, Paralichthys olivaceus due to scuticociliatosis caused by histophagous scuticociliate, Philasterides dicentrarachi.

Recently, mass mortality due to histophagous scuticociliate, Philasterides dicentrarachi in olive flounder, Paralichthys olivaceus occurred in aquaculture farms of Jeju Island, South Korea. We have artificially induced infection through immersion on day 12, the density of ciliates slowly decreased in the experimental tanks; on day 28 to 2300cells/ml in 3cm group and 1800cells/ml in 5cm group. The survival rate of P. olivaceus on days 24 and 28 of infection in the 3cm group was decreased to 4 and 6, respectively; however, no mortality was recorded in the 5cm group. In the control group 100% survival in the chosen period of both groups occurred except in the 3cm group whereas on days 24 and 28 the death rate was 1 and 2, respectively. Many ciliates with ingested red blood cells were observed in the gills, skeletal muscle, skin, fins and brains of infected flounder. The parasites were also observed in the lamina propria of the digestive tract, pharynx, peritoneal fluid, cerebrospinal fluid and cornea. Histopathological and inflammatory changes were characterized by swelling and generalized erythema and muscle tissue necrosis. Affected fish showed varying levels of tissue damage including severe epidermal and dermal necrotic lesions. Loss of scales, appearance of bleached spots that coalesced to form brownish patches, haemorrhagic and severe dermal necrotic lesions were the major clinico-pathological manifestations. The parasite was also seen distributed extensively in the entire brain causing widespread nerve necrosis after a prolonged period of infection. These results appear to support the hypothesis that the P. dicentrarchi may be penetrating via the gills or the skin, and then travel via the bloodstream to other parts of the body, including the body cavity.