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Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either "no reliable identification" or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.

Viral hemorrhagic septicemia virus (VHSV) is the causative agent of viral hemorrhagic septicemia in fish, a disease that affects a number of teleost fish species including olive flounder (Paralichthys olivaceus). In this study, we assessed the safety and efficacy of two recombinant attenuated VHSV strains, termed A4G-G5A and ΔNV, with the purpose to select the most suitable vaccine strain. The virus strains were passaged in two commercially available cell lines, EPC and RTG-2, and the strains were also tested for residual virulence in zebrafish (Danio rerio). The A4G-G5A strain showed an attenuated growth profile in both the EPC and RTG-2 cell lines compared to wild-type (WT) VHSV (JF-09, genotype IVa), whereas the growth profile of ΔNV was comparable to the WT strains in RTG-2 cells in contrast to EPC cells. Moreover, ΔNV had higher residual virulence compared to A4G-G5A and was highly pathogenic to zebrafish. The A4G-G5A strain was chosen as vaccine candidate and tested for efficacy in in vivo fish studies in the target species, olive flounder, using an immersion vaccine scheme. Groups of fish were immunized with 10(2.5), 10(3.5), 10(4.5), and 10(5.5) TCID50/ml of A4G-G5A giving 5-13.3 cumulative percent mortality (CPM) post immunization. Immunization was followed by a challenge experiment using VHSV-WT. The relative percent survival (RPS) in immunized groups ranged from 81.6% to 100%, correlating with vaccination dose. This study demonstrates that while strain A4G-G5A has retained some residual virulence it confers high level of protection in immunized olive flounder., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The olive flounder (Paralichthys olivaceus) is a cultivated marine species that is economically important in Korea and Japan. Several bacterial pathogens have caused severe mortalities in farmed olive flounder, especially Streptococcus parauberis. We collected 145 S. parauberis isolates from diseased olive flounders from 2003 to 2008 in Jeju Island, South Korea and characterized them by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and by serology. The serological analysis divided the isolates into serotype I (62.1%) and serotype II (36.6%) and the proteome analysis divided the isolates into cluster 1 (43.4%) and cluster 2 (56.6%). All cluster 1 isolates had serotype I, but cluster 2 consisted of serotype I (32.9%), serotype II (64.6%), and others (2.5%). Further detailed analysis of the mass spectra led to identification of several specific m/z peaks that enabled discrimination between cluster 1 and 2 and between serotype I and II within cluster 2. Our results suggest that MALDI TOF MS analysis has potential as an alternative method for the rapid and reliable identification of the fish pathogen S. parauberis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Olive flounder, Paralichthys olivaceus, is an important fish species in Asia, both for fisheries and aquaculture. As the first step for better understanding the genomic structure and functional analysis, we constructed a genetic linkage map for olive flounder based on 180 microsatellites and 31 expressed sequence tag (EST)-derived markers. Twenty-four linkage groups were identified, consistent with the 24 chromosomes of this species. The total map distance was 1,001.3 cM based on Kosambi sex-average mapping, and the average inter-locus distance was 4.7 cM. Linkage between the loci was identified by an LOD score of > or =3. This linkage map may be used to map quantitative trait loci associated with important traits of the species and may assist in breeding programs.