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.

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.

Dietary effect of Rubus coreanus ethanolic extract on immune gene expression in white leg shrimp, Penaeus vannamei.

The objective of this study was to evaluate the effect of dietary supplementation of a Rubus coreanus ethanolic extract on immunostimulatory response in white leg shrimp Penaeus vannamei. Shrimps with an average initial weight of 0.5 ± 0.04 g were collected and acclimatized for 10 days. Four experimental diets including a control diet, a probiotic diet and 0.25 and 0.5% of R. coreanus ethanolic extract (RcEE) diets were used to feed the shrimps. After 8 weeks of culture, shrimp fed with probiotic and 0.25% RcEE diet had showed significant enhancement in the growth while shrimp fed with 0.5% RcEE diet showed significantly increased expression of immune genes and antioxidant enzymes activities. One week of challenge experiments for all the four diets fed shrimps showed decreased cumulative mortality in the 0.5% RcEE diets fed shrimps, when compared with the probiotic and 0.25% RcEE diet fed shrimp groups. The results indicates that R. coreanus ethanolic extract could be used as a herbal immunostimulant for shrimps to increase its immunity and disease resistance against the bacterial pathogen, Vibrio alginolyticus.

Hyunsoonleella jejuensis gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from seawater.

A novel marine, Gram-staining-negative, yellow-pigmented, rod-shaped bacterial strain, designated CNU004(T), was isolated from a seawater sample collected on the coastline of Jeju Island, South Korea. The strain was strictly aerobic, non-flagellated, non-gliding and oxidase- and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CNU004(T) belongs to a distinct lineage in the family Flavobacteriaceae. Strain CNU004(T) exhibited levels of 16S rRNA gene sequence similarity of 93.8-93.9 % to its nearest phylogenetic neighbours, members of the genera Gaetbulibacter, Yeosuana and Algibacter. The new isolate required sea salts or artificial seawater for growth. The optimum ranges of temperature and pH for growth were 30-35 degrees C and pH 7.0-8.0. The DNA G+C content of strain CNU004(T) was 37.7 mol%. The major fatty acids were iso-C(15 : 0), iso-C(15 : 1) G, iso-C(17 : 0) 3-OH and iso-C(15 : 0) 3-OH. Menaquinone-6 was the major respiratory quinone. Zeaxanthin was the major carotenoid pigment produced, and flexirubin-type pigments were not produced. Strain CNU004(T) was able to degrade starch and agar. Based on its phenotypic and genotypic characteristics and on the phylogenetic evidence presented, strain CNU004(T) is considered to represent a novel species of a new genus in the family Flavobacteriaceae, for which the name Hyunsoonleella jejuensis gen. nov., sp. nov. is proposed. The type strain of Hyunsoonleella jejuensis sp. nov. is CNU004(T) (=KCTC 22242(T) =DSM 21035(T)).

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

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

Antibiotic susceptibility and resistance of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus).

The rates of antibiotic susceptibility and resistance were investigated in Streptococcus iniae and Streptococcus parauberis isolates obtained from diseased olive flounders (Paralichthys olivaceus) collected from fish farms in Jeju Island, Korea. Isolates of S. iniae (n=65) were susceptible to cefotaxime, erythromycin, ofloxacin, penicillin, tetracycline and vancomycin, as demonstrated by the minimum inhibitory concentration (MIC) test. Isolates of S. parauberis (n=86) were highly resistant to erythromycin (58% of the 86 isolates tested) and tetracycline (63% of the 86 isolates tested). Fifty-four isolates of tetracycline-resistant S. parauberis contained the tet(M/O/S) genes, of which 39 and 12 isolates contained the tet(M) and tet(S) genes, respectively, whereas 3 isolates contained both the tet(M) and tet(S) genes. Among the erythromycin-resistant isolates of S. parauberis (n=50) only 14 contained the erm(B) gene. These results suggest that the tet(S) and erm(B) genes of S. parauberis are involved in the acquisition of high-level resistance to erythromycin and tetracycline. Our findings reveal a high rate of antibiotic resistance among strains of S. parauberis and emphasize the need to develop an appropriate vaccine to reduce the use of antibiotics.