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Streptococcus parauberis, a gram-positive bacterium, causes significant mortality in cultured fish. In Korea, S. parauberis exhibits diverse serotypes (Ia, Ib/c, and II) from diseased fish including olive flounder (Paralichthys olivaceus), each with different pathogenicity. Therefore, the development of a reliable detection and serotyping assay is essential for effective disease surveillance. From the biochemical traits, although S. parauberis can be categorized into two subgroups associated with serotypes I and II, based on pyrrolidonyl arylamidase (PYR) activity, biochemical characterization is limited in differentiating between subserotypes. Furthermore, even though PCR-based serotyping assays (multiplex PCR; mPCR) have been reported, we aimed to develop an isothermal amplification-based assay for rapid and simultaneous detection and serotyping without the need for thermal cycling equipment. Based on the consensus sequence of the capsular polysaccharide (cps) locus, multiplex recombinase polymerase amplification (mRPA) primers were designed for the simultaneous detection and serotyping of S. parauberis. The optimized primer concentrations for mRPA were 280 nM for the detection of S. parauberis, 240 nM for serotypes Ia and II, and 200 nM for serotype Ib, with optimal amplification conditions determined to be 30 min at 42 °C. In the specificity test, no cross-reactivity with other fish pathogens was observed. In addition, the sensitivity of mRPA was 0.02 ng for detecting S. parauberis and serotypes Ia and Ib/c, while it was 0.2 ng for serotyping type II. Using DNA from cultured bacteria, the novel mRPA showed a detection rate consistent with that of mPCR. Moreover, using DNA from artificially infected tissues, the detection rate by mRPA was 96.7% (29/30), which was different from the results of mPCR (80%, 24/30), which exhibited an 80% detection rate. Therefore, the novel mRPA is an effective and rapid diagnostic tool for the simultaneous detection and serotyping of S. parauberis without requiring thermal cycling equipment as well as a specific bacterial culture step. [ABSTRACT FROM AUTHOR]

This mini review deals with some controversial non-starter lactic acid bacteria (NSLAB) species known to be both human and animal pathogens but also health-promoting and probiotic. The focus is on Lactococcus garvieae, two Streptococcus species (S. uberis and S. parauberis), four Weissella species (W. hellenica, W. confusa, W. paramesenteroides, and W. cibaria), and Mammalicoccus sciuri, which worldwide, are often found within the microbiotas of different kinds of cheese, mainly traditional artisanal cheeses made from raw milk and/or relying on environmental bacteria for their ripening. Based on literature data, the virulence and health-promoting effects of these bacteria are examined, and some of the mechanisms of these actions are reviewed. Additionally, their possible roles in cheese ripening are also discussed. The analysis of the literature data available so far showed that, in general, the pathogenic and the beneficial strains, despite belonging to the same species, show somewhat different genetic constitutions. Yet, when the safety of a given strain is assessed, genomic analysis on its own is not enough, and a polyphasic approach including additional physiological and functional tests is needed. [ABSTRACT FROM AUTHOR]

ABSTRACT Streptococcus parauberis is the dominant etiological agent of streptococcosis, the most devastating bacterial disease in the olive flounder farming industry in South Korea. In this study, the distribution of serotypes, antimicrobial susceptibility, and presence of antimicrobial resistance genes (ARGs) in S. parauberis isolates obtained between 1999 and 2021 was thoroughly investigated to gain insight into the dynamics of their presence and the relationship between serotypes and antimicrobial resistance. Disk diffusion testing of 103 isolates against 10 antimicrobial agents was performed, and epidemiological cut-off values generated through normalized resistance interpretation analysis were used to classify wild-type (WT) and non-wild-type (NWT) populations. Principal component analysis and hierarchical clustering were implemented to achieve an understanding on the relationship between serotypes and antimicrobial resistance patterns. PCR-based serotyping showed that serotype Ia (67.1%) was the most prevalent in South Korea, followed by serotypes Ib/Ic (25.2%) and II (7.7%). The highest proportion of isolates was assigned to NWT against amoxicillin (80.6%), followed by oxytetracycline (77.7%) and erythromycin (48.5%). The time-scale data showed that recently obtained serotypes Ib/Ic and II isolates tended to be categorized as NWT populations resistant to more antibiotics, possibly due to microbial adaptation to antibiotic pressure. ARGs responsible for resistance to oxytetracycline and erythromycin were found only in NWT populations in serotype Ia [tet(S) and erm(B), respectively], and serotype II [tet(M) and mef(J)-msr(I), respectively]. We also found that the mef-msr gene pair in S. parauberis serotype II might be involved in low-level resistance to erythromycin. IMPORTANCE This study presents serotype distribution and antimicrobial susceptibility data along with the antimicrobial resistance genes (ARGs) of Streptococcus parauberis, which is an important bacterial fish pathogen worldwide. In particular, almost all oxytetracycline and erythromycin non-wild-type (NWT) populations harbored tet(S) or tet(M), and erm(B) or mef(J)-msr(I), respectively. Interestingly, these ARGs were distributed in a highly serotype-dependent manner, resulting in a clear correlation between the antibiogram and serotype distribution. Moreover, recent isolates belonging to serotypes Ib/Ic and II tended to be more frequently categorized as NWT against antimicrobials, including amoxicillin and cefalexin compared to old isolates, while a dramatic decrease in erythromycin and clindamycin NWT frequencies was observed in recent serotype Ia isolates, which lacked erm(B). These variations might be attributed to shifts in the antibiotics employed in South Korean aquaculture over time. The overall findings would provide important background knowledge for understanding the epidemiology of S. parauberis infection in aquaculture.

Aims Streptococcus parauberis is responsible for the development of streptococcosis in marine fish. The aim of the current study was to determine the antimicrobial susceptibility of aquatic Strep. parauberis strains, thus establishing laboratory-specific epidemiological cut-off (COWT) values to distinguish wild-type (WT) and nonwild-type (NWT) strains. Methods and results Using 220 Strep. parauberis isolates obtained from diseased Paralichthys olivaceus, Platichthys stellatus , and Sebastes schlegelii over 6 years from seven different locations in Korea, we established COWT values for eight common antimicrobial agents using the standard broth microdilution method. The COWT values calculated using MIC distribution with the NRI and ECOFFinder methods were the same or within one dilution step for the eight antimicrobials tested. Nine NWT isolates with decreased susceptibility to at least two antimicrobials and one of these isolates exhibited decreased susceptibility to six antimicrobial agents were identified using COWT values based on NRI. Conclusions Interpretive criteria for Strep. parauberis have not yet been established, and the findings of this study provide putative COWT values for eight antimicrobial agents frequently used in aquaculture in Korea. [ABSTRACT FROM AUTHOR]

Streptococcus parauberis , a gram-positive cocci, causes bacterial disease in farmed fish. The recent increase in S. parauberis infection in aquatic farms in South Korea has justified the importance of vaccine development for the prevention of this disease. In this study, we evaluated the effect of subunit vaccines prepared from recombinant M-like protein (SimA) and fibrinogen-binding protein (FBP) candidates with an aluminum hydroxide adjuvant against S. parauberis infection in olive flounder Paralichthys olivaceus. For the in vivo experiment, fish (average length, 7.18 cm; average weight, 3.5 g) were injected intraperitoneally with: phosphate buffer saline (PBS, group 1), PBS/aluminum hydroxide (group 2), FBP/aluminum hydroxide (group 3), SimA/aluminum hydroxide (group 4), and SimA/FBP/aluminum hydroxide (group 5). After 3 weeks, the fish in each group were boosted using PBS (group 1 and 2), FBP (group 3), SimA (group 4), and SimA/FBP (group 5) without adjuvant. We found that the relative percent survival of fish after S. parauberis exposure in group 2, 3, 4, and 5 was 6.25%, 18.75%, 50%, and 12.5%, respectively, whereas the mortality in groups 1 was 80%, respectively. We performed Western blot, ELISA, and quantitative real time RT-PCR (qRT-PCR) after vaccination to investigate the further efficacy of the vaccine. Western blot and ELISA of vaccinated fish serum confirmed the production of specific antibodies against SimA and FBP. Furthermore, results of qRT-PCR showed that recombinant protein SimA induced a remarkably specific-antibody response compared with that in FBP or control and increased the expression of various immune response-related genes including interleukin-8 (IL-8), toll-like receptor 2 (TLR2), tumor necrosis factor-α (TNF-α), CD4-1, and MHC II. Thus, these results indicate that SimA is a potent vaccine candidate for protection against S. parauberis infection. • SimA based vaccine against Streptococcus parauberis in fish prevents infection. • Aluminum hydroxide serves as an effective adjuvant. • SimA/adjuvant-vaccinated fish showed extended survival rate. • In addition expressed immune response-related genes and elevated antibody titer. [ABSTRACT FROM AUTHOR]

Bacterial extracellular vesicles (BEVs) are nanoscale membrane-bound structures involved in intercellular communication and transport of bioactive molecules. In this study, we described the proteomic insight and anti-inflammatory activity of Streptococcus parauberis BEVs (Sp EVs). Proteomics analysis of Sp EVs identified 6209 distinct peptides and 1039 proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated enrichment in pathways related to the biosynthesis of aminoacyl tRNA, amino acids, and secondary metabolites. Based on the predicted protein-protein interactions, we discovered key immunological proteins such as IL12A, IL12B, IL8, CD28, and NF-κB between Sp EVs and human proteins. Functionally, Sp EVs exhibit strong anti-inflammatory activity in LPS-stimulated Raw 264.7 cells by reducing the production of key inflammatory mediators. These include nitric oxide (NO), reactive oxygen species (ROS), inflammatory cytokines such as TNFα and IL6, as well as inflammation-related proteins like inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). qRT-PCR and immunoblotting results clearly indicate that Sp EVs modulate the NF-κB and MAPK pathways to induce anti-inflammatory activity. Furthermore, in vivo experiments with zebrafish larvae demonstrated that Sp EVs treatment reduced the NO and ROS production with minimal cell mortality. Finally, we validated the anti-inflammatory activity of Sp EVs in vivo by systematically assessing the inhibition of NO production, reduction in ROS generation, prevention of cell death, and modulation of NF-κB and MAPK signaling pathways. In conclusion, Sp EVs contain rich in unique proteins that play crucial roles in mediating anti-inflammatory effects. • Streptococcus parauberis extracellular vesicles (Sp EVs) are rich in unique proteins. • Sp EVs show strong anti-inflammatory activity in Raw 264.7 cells. • Sp EVs modulate the NFκB and MAPK pathways and reduce inflammatory cytokine. • In vivo study in zebrafish larvae confirmed the anti-inflammatory activity of Sp EVs. [ABSTRACT FROM AUTHOR]

The present study was conducted to assess the protective efficacy of a trivalent oral vaccine containing chitosan–PLGA encapsulated inactivated viral haemorrhagic septicemia virus (VHSV), Streptococcus parauberis serotype I and Miamiensis avidus antigens, followed by its oral (incorporated in feed) administration to olive flounder (Paralichthys olivaceus) fingerlings for a period of 15-consecutive days. After 35 days of initial vaccination, three separate challenge studies were conducted at the optimal temperature of the targeted pathogens using an intraperitoneal injection route. RPS analysis revealed moderate protection in the immunized group against all the three pathogens viz., VHSV (53.30% RPS), S. parauberis serotype-I (33.30% RPS), and M. avidus (66.75% RPS), as compared to the respective non-vaccinated challenge (NVC) control group. In addition, the immunized fish demonstrated significantly (p < 0.05) higher specific antibody titres in serum and significant (p < 0.05) upregulation in the transcript levels of immune genes of Igs (IgM, IgT, pIgR), TLRs (TLR 2, TLR 7), cytokines (IL-1β, IL-8) and complement pathway (C3) in the mucosal and systemic tissues than those of NVC control fish, suggesting orchestration of pathogen-specific host immune responses thereby favouring its combativeness against the three pathogens. The expression dynamics of IFN-γ, Mx, caspase 3 genes post VHSV challenge; IFN-γ, TLR 2, caspase 1 genes post S. parauberis serotype I challenge and CD-8α, IL-10, TNF-α genes post M. avidus challenge further substantiates the efficacy of the vaccine in stimulating antiviral, antibacterial and antiparasitic immune responses in the host resulting in their better survival. The findings from the present study reflect that the formulated trivalent oral vaccine incorporating VHSV, S. parauberis serotype I and M. avidus antigens can be a promising prophylactic strategy to prevent the associated disease outbreaks in olive flounder. • An oral vaccine (Chi/PNPs-IV + Sp + Sc) against VHSV, S. parauberis serotype I and M. avidus was formulated for olive flounder. • Encapsulation of the inactivated antigens in chitosan coated PLGA nanoparticles resulted in efficient vaccine delivery. • Vaccinated fish showed significantly (p < 0.05) higher serum antibody responses and immune gene transcripts both in systemic and mucosal tissues. • RPS of 53.30%, 33.30% and 66.75% against VHSV, S. parauberis serotype I, and M. avidus , respectively, highlights the protective efficacy of the formulated vaccine. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNAs that participate in various biological and cellular processes by regulating target gene expression. miRNAs are also known to play vital roles in the pathogenesis of various diseases, including infections, as well as the disease progression and defense responses. In this study, we examined the expression levels of pol-miR-140-3p and its target gene, kinesin family member 5A (KIF5A), in association with the Streptococcus parauberis (S. parauberis) infection, a major bacterial pathogen that causes streptococcosis in olive flounder (Paralichthys olivaceus). KIF5A is a heavy chain isoform of kinesin-1, which is known to be brain-specific, and this study is the first examination of KIF5A expression related to the regulation of miRNA in olive flounder (named PoKIF5A). There were significant differences in expression levels between infected and healthy olive flounder as the expression of pol-miR-140-3p in the infected fish was lower than that in the control, while the expression of PoKIF5A was higher in the infected fish than in the healthy controls. These contradictory results suggest that downregulated pol-miR-140-3p induces the expression of PoKIF5A against S. parauberis infection in olive flounder. • The expression levels of pol-miR-140-3p and PoKIF5A were changed in olive flounder brain during S. parauberis infection. • The conserved domains of KIF5A were analyzed using the CDD program in NCBI. • Dual-luciferase assay and qRT-PCR results showed that the expression of PoKIF5A was downregulated by pol-miR-140-3p. [ABSTRACT FROM AUTHOR]

Septin is an evolutionarily conserved family of GTP-binding proteins. Septins are known to be involved in a variety of cellular processes, including cell division, chromosome separation, cell polarity, motility, membrane dynamics, exocytosis, apoptosis, phagocytosis, DNA damage responses, and other immune responses. In this study, the sequences of the septin gene family of starry flounder were obtained using NGS sequencing, and the integrity of the sequences was verified through cloning and sequencing. At first, the amino acid sequence was annotated using the cDNA sequence, and then, the gene sequence was verified through multiple sequence alignment and phylogenetic analyses using the related conserved sequences. The septin gene family was classified into three subgroups based on the phylogenetic analysis. High conservation within the domain and homology between the genes reported in different species were confirmed. The expression level of septin gene family mRNA in each tissue of healthy starry flounder was evaluated to confirm the tissue- and gene-specific expression levels. Additionally, as a result of the analysis of mRNA expression after simulated pathogen infection, significant expression changes and characteristics were confirmed upon infection with bacteria (Streptococcus parauberis PH0710) and virus (VHSV). Based on the current results and that of previous studies, to confirm the immunological function, Septin 2, 3, and 8 were produced as recombinant proteins based on the amino acid sequences, and their role in phagocytosis was further investigated. The results of this study indicate that septin gene family plays a complex and crucial role in the host immune response to pathogens of starry flounder. • We identified the septin gene family from starry flounder (Platichthys stellatus). • The expression level of septin gene family mRNA for each tissue in a healthy starry flounder was detected in all tissues used in the experiment, and tissue- and gene-specific expression levels could be confirmed. • Septin gene family mRNA expression levels were significantly regulated by S. parauberis and VHSV. • Based on the amino acid sequence, septin 2, 3, and 8 were produced as recombinant proteins, and phagocytosis was confirmed in rSeptin 2. [ABSTRACT FROM AUTHOR]

Sargassum horneri (SH) is a brown macroalgal species commonly found along the coast of Japan, China, and Korea. SH possesses valuable bioactive compounds that can be developed as functional food ingredients as well as pharmaceutical agents for both humans and animals. In this study, SH was tested for its potential prebiotic effect. Several solvent-assisted extracts of SH were tested on the growth of three species of probiotics (LAB) (Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus brevis) and fish pathogen bacteria (Streptococcus iniae, Streptococcus parauberis, Edwersiella tarda) both in vitro and in vivo. According to the in vitro results, Celluclast extract (SHC) and crude polysaccharide extract (SHCPs) of SH showed outstanding growth enhancing activity in all LAB species and excellent antibacterial activity against pathogenic bacteria dose-dependently. Both SHC and SHCPs induced the production of secondary metabolites from LAB. The secondary metabolites successfully reduced pathogenic bacterial growth. Furthermore, in vivo experiments revealed that co-treatment with LAB and SHC/SHCPs diminished the mortality of Streptococcus parauberis infected zebrafish by modulating iNOS, COX-2 expressions. Similarly, SH act as an anti-inflammatory agent against S. parauberis infection by hindering NF-κB pathway activation. Conclusively, the results achieved from the study suggest that S. horneri has the potential to be used as a prebiotic dietary supplement and possesses a protective effect against S. parauberis infections in the aquaculture industry.

The objectives of this study were to investigate antimicrobial resistance of Streptococcus uberis and Streptococcus parauberis isolated from cows with bovine clinical mastitis in China and to examine the distribution of resistance- and virulence-related gene patterns. Antimicrobial susceptibility was determined by the E-test. Genes encoding antimicrobial resistance and invasiveness factors were examined by PCR. A total of 27 strains were obtained from 326 mastitis milk samples. Streptococcus parauberis isolates (n=11) showed high resistance to erythromycin (90.9%), followed by tetracycline (45.5%), chloramphenicol (36.4%) and clindamycin (27.3%). Streptococcus uberis isolates (n=16) were highly resistant to tetracycline (81.3%) and clindamycin (62.5%). Both species were susceptible to ampicillin. The most prevalent resistance gene in S. uberis was tetM (80.0%), followed by blaZ (62.5%) and ermB (62.5%). However, tetM, blaZ, and ermB genes were only found in 27.3, 45.5, and 27.3%, respectively, of S. parauberis. In addition, all of the isolates carried at least one selected virulence-related gene. The most prevalent virulence-associated gene pattern in the current study was sua+pauA/skc+gapC+hasC detected in 22.2% of the strains. One S. uberis strain carried 7 virulence-associated genes and belonged to the sua+pauA/skc+gapC+cfu+hasA+hasB+hasC pattern. More than 59.3% of analysed strains carried 4 to 7 virulence-related genes. Our findings demonstrated that S. parauberis and S. uberis isolated from clinical bovine mastitis cases in China exhibited diverse molecular ecology, and that the strains were highly resistant to antibiotics commonly used in the dairy cow industry. The data obtained in the current study contribute to a better understanding of the pathogenesis of bacteria in mastitis caused by these pathogens, and the findings are relevant to the development of multivalent vaccines and targeted prevention procedures.

Streptococcus parauberis is the main causative bacterial pathogen for streptococcosis in marine fish, causing a significant economic loss to the mariculture industry. In this study, we aimed to investigate the serotypes and antimicrobial susceptibilities of 83 S. parauberis isolates from diseased farmed fish (Paralichthys olivaceus, Platichthys stellatus and Sebastes schlegelii) in Korea. This study categorized the S. parauberis isolates based on PCR serotyping into serotype I (subtypes Ia and Ib/Ic) and serotype II. Serotype Ia was identified to be significantly more prevalent than serotypes Ib/Ic and II. An antimicrobial susceptibility analysis showed that all of the isolates were susceptible to amoxicillin/clavulanic acid. Overall, the isolates studied showed resistance to penicillin and ampicillin (1.2%), tetracycline (68.7%), oxytetracycline (72.3%), doxycycline (53.0%) and erythromycin (37.3%). Interestingly, we found that serotypes Ia and II showed higher degrees of tetracycline resistance than serotype Ib/Ic. The macrolide resistance gene erm(B) was present in 75.8% of the Ia isolates, whereas the mef(A) gene was not detected in any of the isolates. The tetracycline resistance genes tet(S) and tet(M) were detected in 95.2% and 60% of Ia and II isolates respectively. Moreover, β-lactam antibiotic-resistant Ia isolates contained a set of 11 mutations in PBP1A and PBP2X. Our study provides a valuable reference for surveillance of antimicrobial resistance and underscores the strict management of antibiotic use to prevent the emergence of resistant of S. parauberis serotypes. [ABSTRACT FROM AUTHOR]

Olive flounder (Paralichthys olivaceus) is the most valuable aquaculture species in Korea, corresponding to ~60% of its total production. However, infectious diseases often break out among farmed flounders, causing high mortality and substantial economic losses. Although some deleterious pathogens, such as Vibrio spp. and Streptococcus iniae , have been eradicated or contained over the years through vaccination and proper health management, the current disease status of Korean flounder shows that the viral hemorrhagic septicemia virus (VHSV), Streptococcus parauberis , and Miamiensis avidus are causing serious disease problem in recent years. Furthermore, these three pathogens have differing optimal temperature and can attack young fingerlings and mature fish throughout the year-round culture cycle. In this context, we developed a chitosan-poly(lactide-co-glycolide) (PLGA)-encapsulated trivalent vaccine containing formalin-killed VHSV, S. parauberis serotype-I, and M. avidus and administered it to olive flounder fingerlings by immersion route using a prime-boost strategy. At 35 days post-initial vaccination, three separate challenge experiments were conducted via intraperitoneal injection with the three targeted pathogens at their respective optimal temperature. The relative percentages of survival were 66.63%, 53.3%, and 66.75% in the group immunized against VHSV, S. parauberis serotype-I, and M. avidus , respectively, compared to the non-vaccinated challenge (NVC) control group. The immunized fish also demonstrated significantly (p < 0.05) higher specific antibody titers in serum and higher transcript levels of Ig genes in the mucosal and systemic tissues than those of NVC control fish. Furthermore, the study showed significant (p < 0.05) upregulation of various immune genes in the vaccinated fish, suggesting induction of strong protective immune response, ultimately leading to improved survival against the three pathogens. Thus, the formulated mucosal vaccine can be an effective prophylactic measure against VHS, streptococcosis, and scuticociliatosis diseases in olive flounder. [ABSTRACT FROM AUTHOR]

Olive flounder (Paralichthys olivaceus) is the most valuable aquaculture species in Korea, corresponding to ~60% of its total production. However, infectious diseases often break out among farmed flounders, causing high mortality and substantial economic losses. Although some deleterious pathogens, such as Vibrio spp. and Streptococcus iniae, have been eradicated or contained over the years through vaccination and proper health management, the current disease status of Korean flounder shows that the viral hemorrhagic septicemia virus (VHSV), Streptococcus parauberis, and Miamiensis avidus are causing serious disease problem in recent years. Furthermore, these three pathogens have differing optimal temperature and can attack young fingerlings and mature fish throughout the year-round culture cycle. In this context, we developed a chitosan-poly(lactide-co-glycolide) (PLGA)-encapsulated trivalent vaccine containing formalin-killed VHSV, S. parauberis serotype-I, and M. avidus and administered it to olive flounder fingerlings by immersion route using a prime-boost strategy. At 35 days post-initial vaccination, three separate challenge experiments were conducted via intraperitoneal injection with the three targeted pathogens at their respective optimal temperature. The relative percentages of survival were 66.63%, 53.3%, and 66.75% in the group immunized against VHSV, S. parauberis serotype-I, and M. avidus, respectively, compared to the non-vaccinated challenge (NVC) control group. The immunized fish also demonstrated significantly (p < 0.05) higher specific antibody titers in serum and higher transcript levels of Ig genes in the mucosal and systemic tissues than those of NVC control fish. Furthermore, the study showed significant (p < 0.05) upregulation of various immune genes in the vaccinated fish, suggesting induction of strong protective immune response, ultimately leading to improved survival against the three pathogens. Thus, the formulated mucosal vaccine can be an effective prophylactic measure against VHS, streptococcosis, and scuticociliatosis diseases in olive flounder.

Eosinophils are granular leukocytes that are evolutionarily preserved in the innate immune system of some invertebrates and vertebrates, and these cells can directly remove invading microorganisms and secrete various cytokines, and are also involved in homeostasis. These eosinophils are made up of specific granular proteins that can be differentiated from other cells, and eosinophil peroxidase (EPX) is a peroxidase released only from eosinophils that plays an important role in maintaining the main function and homeostasis of eosinophils. We obtained the sequence information of EPX for the first time from the starry flounder (Platichthys stellatus), and predicted it by amino acid sequencing to confirm sequence alignment and phylogenetic characteristics with other species. Based on analysis of the expression characteristics of PsEPX mRNA in healthy P. stellatus , it was expressed at the highest level in peripheral blood lymphocytes (PBLs) and was also expressed at a relatively high level in the head kidney and intestine, which are immune-related tissues. After artificial infection with Streptococcus parauberis and viral haemorrhagic septicaemia virus, which are the causes of major pathogenic diseases, the expression level of PsEPX was significantly regulated, which showed specific characteristics of pathogens or tissues. These results suggest that PsEPX is an important component of the immune system of P. stellatus and is considered a basic research case for the study of the immunological function of eosinophils in fish. • We identified the eosinophil peroxidase (EPX) gene from starry flounder (Platichthys stellatus). • PsEPX mRNA was highly expressed in the PBLs, head kidney and intestine of healthy starry flounder. • PsEPX mRNA expression levels were significantly regulated by S. parauberis and VHSV. [ABSTRACT FROM AUTHOR]

Background: Rock bream iridovirus (RBIV) is one of the most dangerous pathogens that causes the highest mortality in the aquaculture of rock bream (Oplegnathus fasciatus). Even though RBIV infection leads to huge economic loss, proteome studies on RBIV-infected rock bream have not been conducted to provide information about the differential protein expression pattern by the host protection system. Objective: The purpose of this study was to investigate the protein expression patterns in spleens of rock bream olive after infection by RBIV or mixed infection by RBIV and bacteria. Methods: Depending on the infection intensity and sampling time point, fish were divided into five groups: uninfected healthy fish at week 0 as the control (0C), heavily infected fish at week 0 (0H), heavily mixed RBIV and bacterial infected fish at week 0 (0MH), uninfected healthy fish at week 3 (3C), and lightly infected fish at week 3 (3L). Proteins were extracted from the spleens of infected rock bream. We used 2-DE analysis with LC–MS/MS to investigate proteome changes in infected rock bream. Results: The results of the LC–MS/MS analyses showed different protein expression profiles after infection. Proteins related to oxygen transport and energy generation, such as hemoglobin, beta-globin, and ATP synthase, were mostly expressed in the infected spleen. Whereas proteins involved in structure and cell movement, such as tubulin, myosin, actin binding proteins, and intermediate filament proteins, were down-regulated in the infected spleens. The protein expression profiles between infection by RBIV and mixed infection by RBIV and bacteria showed similar patterns. Conclusions: Our results indicated that infection by RBIV or mixed infection by RBIV and bacteria triggered energy generation and oxygen-transport, but cell migration and constructional changes in the spleen were extremely decreased. [ABSTRACT FROM AUTHOR]

Background: Olive flounder (Paralichthys olivaceus) is one of the major cultured fish species in Asia including Korea. However, the mass mortality of olive flounder caused by various pathogens leads to huge economic loss. The pathogens that lead to fish mortality include parasites, bacteria, and viruses that can cause various kinds of diseases. Objective: The purpose of this study was to investigate the protein expression patterns in the gills and spleens of olive flounder after artificial infection. We hypothesized that proteomics levels in gills and spleen may be differentially expressed depending on infectious agents. Methods: To investigate the expression pattern of proteins in gills and spleens, olive flounders were experimentally infected with VHSV (virus), S. parauberis (bacteria), or M. avidus (pathogenic ciliate). Proteins were extracted from the gills and spleens of infected olive flounder. We used 2-DE analysis with LC–MS/MS to investigate proteome changes in infected olive flounders. Results: The results of the LC–MS/MS analyses showed different protein expression profiles depending on pathogenic sources and target organs. Proteins related to cytoskeletal structure like keratin, calmodulin and actin were mostly expressed in the infected gills. Proteins involved in the metabolism pathway like glycolysis were expressed mainly in the spleens. The protein profiles of S. parauberis and VHSV infection groups had many similarities, but the profile of the M. avidus infection group was greatly different in the gill and spleen. Conclusion: Our results indicate that measures according to the characteristics of each pathogen are necessary for disease prevention and treatment of farmed fish. [ABSTRACT FROM AUTHOR]

Streptococcus parauberis is a pathogenic gram-positive bacterium that causes streptococcosis infection in fish. Since S. parauberis is becoming resistant to multiple antibiotics, the development of alternatives, such as antimicrobial peptides, has gained great attention. Octominin, derived from the defense protein of Octopus minor , showed a significant antimicrobial activity against multidrug resistance S. parauberis, with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 50 and 100 μg/mL, respectively. Furthermore, time-kill kinetics, agar diffusion, and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octominin against S. parauberis. Field emission scanning electron microscopy analysis showed morphological and ultra-structural changes in S. parauberis upon Octominin treatment. Moreover, Octominin treatment demonstrated changes in membrane permeability, induced reactive oxygen species (ROS), and its binding ability to genomic DNA, suggesting its strong bactericidal activity with multiple modes of action. We confirmed the inhibition of biofilm formation and the eradication of existing biofilms in a concentration-dependent manner. Additionally, Octominin on S. parauberis at transcriptional level exhibited downregulation of membrane formation (pgsA and cds1), DNA repairing (recF), biofilm formation (pgaB and epsF) genes, while upregulation of ROS detoxification (sodA) and DNA protecting (ahpF) related genes. An in vivo study confirmed a significantly (P < 0.05) higher relative percentage survival in Octominin-treated larval zebrafish exposed to S. parauberis (93.3%) compared to the control group (20.0%). Collectively, our results confirm that Octominin could be a potential antibacterial and anti-biofilm agent against S. parauberis. Image 1 • Octominin displayed strong antibacterial activity against Streptococcus parauberis. • Octominin involved inhibition and eradication of biofilm in S. parauberis. • S. parauberis exposed larval zebrafish are protected by Octominin treatment. [ABSTRACT FROM AUTHOR]

Streptococcus parauberis is an etiological agent that causes streptococcosis in fish, exerting a significant impact on aquaculture production. The available injectable vaccine against S. parauberis is commonly used in olive flounder species (Paralichthys olivaceus) in Korea. However, an oral alternative that overcomes the disadvantages of the injectable vaccine has long been desired in the aquaculture industry. Alginate-based microparticles have been widely used as delivery systems for oral administration in aquaculture. In the present study, we produced alginate microparticles encapsulating formalin-inactivated S. parauberis via ionic gelation, which remained stable under acidic conditions (pH 2 and 4) and at low temperatures (4 °C and 25 °C). Mixing the alginate–Gelzan complex with a commercial feed for ease of application induced a specific antibody response and exerted significant protective effects (relative survival rate: 50.0%) in small olive flounder fingerlings. Less pronounced protective effects were also observed in large fingerlings (relative survival rate: 37.5%), suggesting that protection against S. parauberis may be dependent on the size of the fish. Thus, our results indicate that alginate microparticles encapsulating an S. parauberis antigen can be used for initial vaccination in olive flounder at the fingerling stage. [ABSTRACT FROM AUTHOR]

The membrane attack complex/perforin (MACPF) superfamily consists of multifunctional proteins that form pores on the membrane surface of microorganisms to induce their death and have various immune-related functions. PFN2 is a perforin-like protein with an MACPF domain, and humans with deficient PFN2 levels have increased susceptibility to bacterial infection, which can lead to fatal consequences for some patients. Therefore, in this study, we confirmed the antimicrobial function of PFN2 in starry flounder (Platichthys stellatus). The molecular properties were confirmed based on the verified amino acid sequence of PsPFN2. In addition, the expression characteristics of tissue-specific and pathogen-specific PsPFN2 mRNA were also confirmed. The recombinant protein was produced using Escherichia coli , and the antimicrobial activity was then confirmed. The coding sequence of PFN2 (PsPFN2) in P. stellatus consists of 710 residues. The MACPF domain was conserved throughout evolution, as shown by multiple sequence alignment and phylogenetic analysis. PsPFN2 mRNA is abundantly distributed in immune-related organs such as the spleen and gills of healthy starry flounder, and significant expression changes were confirmed after artificial infection by bacteria or viruses. We cloned the MACPF domain region of PFN2 to produce a recombinant protein (rPFN2) and confirmed its antibacterial effect against a wide range of bacterial species and the parasite (Miamiensis avidus). • Perforin-2 (PFN2) from starry flounder (Platichthys stellatus) was identified. • PsPFN2 was highly expressed in the spleen, gills and PBLs of healthy starry flounder. • PsPFN2 mRNA expression levels were significantly regulated by S. parauberis and VHSV. • We confirmed the antimicrobial activities of rPsPFN2 against bacteria and M. avidus. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) constitute a group of small non-coding RNAs (~22 nucleotides) and one of their main functions is to regulate the immune responses. Gram-positive bacterium, Streptococcus parauberis is the main causative agent of "Streptococcosis" in wide range of fish species. In this study, we performed high throughput sequencing analysis to identify the miRNA profile against S. parauberis infection in the spleen of zebrafish (Danio rerio). Overall, 349 known and 151 novel miRNAs were discovered. Among them, 12 known miRNAs (dre-miR-34b, dre-miR-135a, dre-miR-200b-5p, dre-miR-146b, dre-miR-31, dre-miR-17a-3p, dre-miR-222a-3p, dre-miR-731, dre-miR-301b-3p and dre-miR-30a-3p) and 9 novel miRNAs were differentially expressed (DE) in the spleen of S. parauberis challenged zebrafish. The identified 12 DE miRNAs were predicted to regulate 721 target genes. We confirmed the miRNA expression results by validating selected known and novel DE miRNAs using qRT-PCR. Gene Ontology (GO), Kyoto Encyclopedia of Genes (KEGG) pathway analysis and miRNA-mRNA interactions implies that specific target genes of DE miRNAs are associated with immune responses. The enriched pathways included Toll-like receptor (TLR), C-type lectin, NOD-like receptor, and RIG-I-like receptor signaling pathways, etc. Especially, dre-miR-200b-5p, dre-miR-146b, dre-miR-731, dre-miR-222a-3p, and dre-miR-34b were able to target potential immune-related genes such as il10 , irak1 , traf6 , hspa8 and ikbke upon S. parauberis challenge. Thus, overall results could lay a foundation to understand the underlying immune regulatory role of miRNAs in response to pathogenic S. parauberis infection in teleosts. [ABSTRACT FROM AUTHOR]

Prosaposin (PSAP) is a precursor of saposin (SAP), which is present in lysosomal and secreted proteins. PSAP is a member of the SAP -like protein families, which comprise multifunctional proteins. In particular, their antimicrobial activity has been reported. We identified PSAP -like (PsPSAPL) sequences from starry flounder and analysed their expression and antimicrobial activity based on cDNA and amino acid sequences. PsPSAPL showed conservation of three saposin B type domains at high levels, and PsPSAPL mRNA was relatively abundantly distributed in the brain and gills of healthy starry founders. PsPSAPL mRNA showed significant expression changes in response to viral haemorrhagic septicaemia virus and Streptococcus parauberis. Synthetic peptides (PsPSAPL-1 and -2), prepared based on amino acid sequences, were used to confirm as well as analyse the antimicrobial activity against bacteria and parasites. Consequently, PsPSAPL-1 and -2 were found to significantly inhibit the growth of various bacteria and kill the Miamiensis avidus. In addition, bacterial biofilm formation was significantly inhibited. Safety was also confirmed by analysing cell haemolysis. These results indicate the immunological function of PsPSAP and the potential antimicrobial activity of the AMPs PsPSAPL-1 and -2. • The Prosaposin-like (PSAPL) was identified by NGS analysis from Platichthys stellatus. • PsPSAPL showed high homology and sequence conservation with other species PSAPL. • PsPSAPL mRNA showed significant expression change due to pathogen infection. • The antimicrobial function and in vivo safety of PsPSAPL peptides were confirmed. [ABSTRACT FROM AUTHOR]

This paper describes the predicted structure for the cps loci involved in capsule biosynthesis for Streptococcus parauberis serotypes III, IV, and V. Based on the specific serotype regions I, II, and III, a multiplex PCR protocol (mPCR) was designed to differentiate the main serotypes causing fish diseases. A real-time PCR method (qPCR) is also described to identify S. parauberis of serotype III in bacterial cultures and fish tissues. In silico and in vitro analyses revealed that both methods have a 100% specificity. The mPCR assay was optimized for the detection of S. parauberis strains of subtypes Ia (amplicon size 213 bp), subtypes Ib and Ic (both amplicon size 303 bp), serotype II (amplicon size 403 bp), and serotype III (amplicon size 130 bp) from bacterial cultures. The qPCR assay was optimized for the identification and quantification of S. parauberis serotype III strains in bacterial cultures and fish tissues. This assay achieved a sensitivity of 2.67 × 102 gene copies (equivalent to 3.8 × 10−9 ng/μl) using pure bacterial cultures of S. parauberis serotype III and 1.76 × 102 gene copies in fish tissues experimentally and naturally infected with S. parauberis of the serotype III. The specificity and sensitivity of the protocols described in this study suggest that these methods could be used for diagnostic and/or epidemiological purposes in clinical diagnostic laboratories. Key Points: • Structure of loci cps for S. parauberis of serotypes III, IV and V was described. • mPCR to differentiate S. parauberis serotypes causing disease in fish was optimized. • qPCR assay to quantify strains of S. parauberis serotype III in fish tissues. [ABSTRACT FROM AUTHOR]

Bacterial extracellular vesicles (BEVs) are nanosized structures that play a role in intercellular communication and transport of bioactive molecules. Streptococcus parauberis is a Gram-positive pathogenic bacterium that causes "Streptococcosis" in fish. In this study, we isolated S. parauberis -derived extracellular vesicles (Sp EVs), and then physicochemical and immunomodulatory properties were determined to elucidate their biological functions. Initially, the biogenesis of Sp EVs was detected using field emission scanning electron microscopy, which revealed that secretory phase Sp EVs attached to the outer surface of S. parauberis. Sp EVs had an average particle diameter and zeta potential of 168.3 ± 6.5 nm and −17.96 ± 2.11 mV, respectively. Field emission transmission electron microscopy analysis confirmed the presence of round or oval-shaped Sp EVs with clear membrane margins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed three sharp protein bands when Sp EVs were stained with Coomassie blue. In vitro toxicity of Sp EVs was assayed using the murine macrophage RAW 264.7 cells and we observed no significant (p < 0.05) viability reduction up to 50 μg/mL qRT-PCR results revealed that Sp EVs-treated (5 and 10 μg/mL) RAW 264.7 cells significantly (p < 0.05) induced the mRNA of proinflammatory (Il1β, Il6, and Tnfα) and anti-inflammatory (Il10) cytokines in a concentration-dependent manner. In vivo immunomodulatory effects of Sp EVs were investigated by injecting Sp EVs (5 and 10 μg/fish) into adult zebrafish. Transcriptional analysis based on qRT-PCR indicates significant (p < 0.05) upregulation of proinflammatory (il1β, il6 , and tnfα) and anti-inflammatory (il10) genes in a concentration-dependent manner in zebrafish kidney. Further, protein expression results in zebrafish spleen tissue confirmed the immunomodulatory activity of Sp EVs. In conclusion, Sp EVs display the characteristics of BEVs and immunomodulatory activities, suggesting their potential application as vaccine candidate. [Display omitted] • Streptococcus parauberis extracellular vesicles (Sp EVs) are nano-sized lipid membrane particles. • Sp EVs are internalized into Raw264.7 and Fathead minnow (FHM) cells. • Sp EVs are relatively nontoxic to FHM cells and larval zebrafish. • Sp EVs show immunomodulatory responses in FHM cells and zebrafish. [ABSTRACT FROM AUTHOR]

Multidrug-resistant Streptococcus parauberis causes high fish mortality in aquaculture, necessitating an urgent need for innovative control strategies. This study aimed to develop an immunizing agent against S. parauberis using exosomes isolated from the plasma of olive flounders infected experimentally with S. parauberis (Sp-Exo). Initially, we tested the in vitro immunomodulatory effect of Sp-Exo in murine macrophage RAW264.7 cells and compared it to that of exosomes isolated from naïve fish (PBS-Exo-treated). Notably, Sp-Exo treatment significantly (p < 0.05) upregulated pro-and anti-inflammatory cytokines (Il1β , Tnfα, and Il10), antimicrobial peptide, defensin isoforms (Def-rs2 and Def-ps1), and antiviral (Ifnβ1 and Isg15) genes. In vivo studies in larval and adult zebrafish revealed similar patterns of immunomodulation. Furthermore, larval and adult zebrafish exhibited significantly (p < 0.05) enhanced resistance to S. parauberis infection following treatment with Sp-Exo compared to that with PBS-Exo. Proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) approach revealed the presence of 77 upregulated and 94 downregulated differentially expressed proteins (DEPs) in Sp-Exo, with 22 and 37 significantly (p < 0.05) upregulated and downregulated DEPs, respectively. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Search Tool for the Retrieval of Interacting Genes/Proteins analyses revealed that these genes are associated with key pathways, such as innate immune responses, complement system, acute phase responses, phospholipid efflux, and chylomicron remodeling. In conclusion, Sp-Exo demonstrated superior immunomodulatory activity and significant resistance against S. parauberis infection relative to that on treatment with PBS-Exo. Proteomic analysis further verified that most DEPs in Sp-Exo were associated with immune induction or modulation. These findings highlight the potential of Sp-Exo as a promising vaccine candidate against S. parauberis and other bacterial infections in olive flounder. [Display omitted] • Streptococcus parauberis challenged plasma-derived exosomes (Sp-Exo) of olive flounder demonstrated immunomodulatory activity in Raw264.7 cells. • Sp-Exo showed greater immunomodulation in larval and adult zebrafish. • Sp-Exo has disease resistance capacity in larval and adult zebrafish against S. parauberis. • iTRAQ proteomic approach revealed immunologically important proteins in Sp-Exo. [ABSTRACT FROM AUTHOR]

Bacterial infections in fish farms increase mass mortality and rapid detection of infection can help prevent its widespread. Lactate is an important biomarker for early diagnosis of bacterial infections in farmed olive flounder (Paralichthys olivaceus). To determine the lactate levels, we designed a disposable amperometric biosensor based on Prussian blue nanozyme and lactate oxidase (LOX) entrapped in copolymer-reduced graphene oxide (P-rGO) on screen-printed carbon electrodes. Because LOX is inherently unstable, P-rGO nanosheets were utilized as a base matrix to immobilize it. After optimization in terms of enzyme loading, operating potential, and pH, the biosensor displayed maximum current responses within 5 s at the applied potential of –0.1 V vs. internal Ag/AgCl. The biosensor had Langmuir-type response in the lactate concentration range from 10 µM to 1.6 mM, a dynamic linear response range of 10–100 µM, a sensitivity of 15.9 µA mM−1 cm−2, and a lower detection limit of 3.1 µM (S/N = 3). Additionally, the biosensor featured high reproducibility, good selectivity, and stability till four weeks. Its practical applicability was tested in olive flounder infected by Streptococcus parauberis against the uninfected control. The results were satisfactory compared to those of a standard colorimetric assay kit, validating our method.

Olive flounder (Paralichthys olivaceus) is a valuable maricultured fish in Asian countries, especially Korea, Japan, and China. Streptococcosis caused by Streptococcus parauberis has resulted in significant economic loss of olive flounder aquaculture in these countries. Since the conventional formalin-killed cell vaccine (FKC vaccine) is reportedly effective only for short period, the main objective of this study was to develop new vaccine of protecting olive flounders against streptococcosis for longer period. To assess the protective effect of poly(d , l -lactide- co -glycolic acid) (PLGA) microparticle-encapsulated formalin-killed cell vaccine (PLGA vaccine), it was compared to the FKC vaccine. A bacterial agglutination test was performed using the serum of olive flounders after intraperitoneal administration of PLGA vaccine, FKC vaccine, or sterile phosphate-buffered saline (PBS). The PLGA vaccinated group showed higher agglutination titers than the FKC vaccinated group. Additionally, the percentage survival of the olive flounders was monitored after an S. parauberis challenge 10 weeks post-vaccination. In the first bioassay (challenge with 4.0 × 104 CFU/fish), percentage survival 15 days post-challenge (dpc) were 100% for the PLGA vaccine, 80% for the FKC vaccine, and 65% for PBS. In the second bioassay (4.0 × 105 CFU/fish), percentage survival 15 dpc were 100% for the PLGA vaccine, 55% for the FKC vaccine, and 0% for PBS. The results of this study showed that PLGA vaccination of olive flounder led to greater protection against streptococcosis, compared with conventional FKC vaccination. • Streptococcosis causes significant losses in olive flounder aquaculture in Asian countries including Korea • Formalin-killed vaccines confer short term protection and new vaccines with longer duration of protection are needed • A poly(d , l -lactide-co-glycolic acid) (PLGA) microparticle-encapsulated formalin-killed cell vaccine was tested • The new vaccine led to higher survival rate than the conventional vaccine. [ABSTRACT FROM AUTHOR]

In this study, the starry flounder L1 cell adhesion molecule (L1CAM) sequence was obtained using next-generation sequencing, and the integrity of the sequence was verified by cloning and sequencing. First, the amino acid sequence was predicted using the cDNA sequence, and the gene was then identified through multiple sequence alignment analysis with related sequences and phylogenetic analysis. Thus, homogeneity was confirmed. The expression level of PsL1CAM (Platichthys stellatus L1CAM) mRNA in healthy starry flounder was detected in all tissues used in the experiment, and tissue- and gene-specific expression levels were confirmed. In addition, as a result of mRNA expression analysis after artificial infection with viral hemorrhagic septicemia virus (VHSV) and Streptococcus parauberis PH0710, significant expression changes and characteristics were confirmed following infection with VHSV and S. parauberis PH0710. After artificial infection with VHSV, the expression level of PsL1CAM mRNA was significantly upregulated in almost all major tissues of the starry flounder, whereas it was significantly downregulated in mucosal-associated lymphoid tissues, such as the gills and intestine. Infection with S. parauberis PH0710 significantly upregulated the expression of PsL1CAM mRNA in almost all major tissues of the starry flounder, whereas it was significantly downregulated in the heart after infection. Our results indicate that PsL1CAM may be involved in the host immune response to starry flounders. • We identified the L1CAM from starry flounder (Platichthys stellatus). • The expression level of L1CAM mRNA for each tissue in a healthy starry flounder was detected in all tissues used in the experiment, and tissue- and gene-specific expression levels could be confirmed. • PsL1CAM mRNA expression levels were significantly regulated by S. parauberis PH0710 and VHSV. [ABSTRACT FROM AUTHOR]

Abstract Streptococcus parauberis is the major infectious agent of streptococcosis in the olive flounder (Paralichthys olivaceus), causing serious economic damage. In this study, we identified potential vaccine candidates against S. parauberis by reverse vaccinology. In total, the 2 out of 21 proteins were identified as vaccine candidates from two available S. parauberis genomes. The membrane-anchored protein SEC10/PgrA and the metal ABC transporter substrate-binding lipoprotein mtsA were potent antigenic proteins based on western blotting with mouse-derived antiserum against whole bacteria of S. parauberis serotypes I and II. In particular, metal ABC transporter substrate-binding lipoprotein (mtsA) showed similar protective immunity to that of whole-cell bacterins against S. parauberis in a zebrafish model. These results suggest that mtsA may be considered as a novel candidate in the development of vaccines against S. parauberis. Highlights • We identified 41 vaccine candidates against Streptococcus parauberis. • SEC10/PgrA and mtsA were potent antigenic proteins. • mtsA showed high protective immunity in a zebrafish model. [ABSTRACT FROM AUTHOR]

Abstract: Unusual persistent natural mortality occurred in a floating in‐pond raceway system intensively stocked with channel and hybrid catfish beginning in early November 2016 up until March 2017. The temperature during the period of outbreak ranged from 7.2 to 23.7°C. Gross examination of freshly dead and moribund fish revealed pale gills, slight abdominal distension and swollen inflamed vents. Comprehensive necropsy of 20 fish demonstrated vast amounts of bloody ascitic fluid in the coelomic cavity, visceral congestion, splenomegaly and pale friable livers but macroscopically normal kidneys, suggesting systemic bacterial infection. Bacterial cultures were initiated from skin, gills and major internal organs. Following incubation, a mixture of three bacterial colony phenotypes was observed on agar plates. Presumptive biochemical characterization of the isolates followed by 16S‐rRNA sequence analysis resulted in the identification of Aeromonas veronii, Streptococcus parauberis and Shewanella putrefaciens. Channel catfish juveniles were experimentally infected with the recovered isolates to fulfil Koch's postulates. Moreover, an antibiogram was used to evaluate the susceptibility of the isolates to antimicrobial drugs approved for use in aquaculture. Aquaflor was used successfully for treatment. Here, we report bacterial coinfection lead by A. veronii and the first identification of S. parauberis and S. putrefaciens from cultured catfish in North America. [ABSTRACT FROM AUTHOR]

The article presents a study that examined streptococcus parauberis noticed in a cultured freshwater ornamental fish, the ram cichlid Mikrogeophagus ramirezi. Mortalities had been reported after a routine sorting procedure at a commercial fish farm culturing the ram cichlid in Southern Israel in January 2014. It adds that basic factors leading to bacterial virulence were comparatively examined in the S. parauberis RC isolate and the most common causative agents of streptococcosis in fish.

Although Streptococcus parauberis is the major bacterial pathogen affecting olive flounder, the translocation and dissemination of this pathogen in infected fish are not well understood. Therefore, we conducted real-time PCR and histopathologic examination to monitor the intensity of infection in multiple organs of the olive flounder after challenge with S. parauberis through subcutaneous injection. The bacterial burden in the fish kidney, when sampled at 0, 3, and 7 dpc, was 0, 6.2 ± 4.5 × 10, and 6.7 ± 5.5 × 10 CFU/100 mg of tissue, respectively, indicating that the infection progressed rapidly over time. Of the ten different tissues sampled, the heart and the brain were the major target organs of S. parauberis based on highest copy number as detected by our modified real-time PCR method. Histopathologic examination also showed that S. parauberis caused severe inflammation accompanied by leucocyte infiltration, connective tissue expansion, and a loss of cardiomyocytes in the brain and heart of fish sampled at dpc 7. However, the number of S. parauberis-positive fish at 3 dpc was much higher in the spleen (6/8 fish) than in the remaining organs, suggesting that the spleen is targeted in the early stages of infection relative to the heart (2/8 fish) or brain (3/8 fish). This study provides essential information for studies to find treatments for the effective elimination of S. parauberis in target organs (i.e., the brain and heart) of olive flounder. [ABSTRACT FROM AUTHOR]

Two bacterial etiological agents of the disease, Streptococcus iniae and Streptococcus parauberis has been associated with fish mortalities and heavy economic loss in all over the world. Bacterial identification based on 16S rRNA sequencing is very fast, accurate and reliable in comparison to other traditional phenotype methodologies. In this study, we investigate the usefulness of this method for diagnosis and identification of Streptococcus species. We have selected 61 phylogeographic strains of Streptococcus (34 strains of S. iniae and 27 strains of S. parauberis) and designed the universal primer against the identified most hypervariable region of the 16S rRNA gene. Our universal primer able to identify any geographical strains and offers a useful and fast alternative in a clinical laboratory under routine conditions. Based on our studies, we have developed an algorithm for appropriate control of S. iniae and S. parauberis disease. We suggested the phenotype observation along with universal primer combination to detect any kind of infection or carriers at early stages. [ABSTRACT FROM AUTHOR]

The use of probiotics is a strategy employed to improve host health status and to prevent infectious diseases. The current study was aimed at investigating the diversity of lactic acid bacteria (LAB) and Bacillus species in the gastrointestinal tracts of wild marine fishes, as well as the beneficial effects of Lactococcus lactis WFLU12 as a host-derived probiotics in olive flounder. In marine fishes, wild olive flounder and rock bream were shown to be good sources of LAB and Bacillus isolation, respectively. Some isolates, including the strain WFLU12, have shown stronger inhibitory activity against various aquatic bacterial pathogens and more tolerance to low pH and bile acids compared to some strains isolated from sources other than marine. Lc. lactis WFLU12 was found to confer to olive flounder protection against streptococcosis caused by Streptococcus parauberis through competitive exclusion and increased innate immune responses. Interestingly, the natural infection rate in the probiotic fed group (33% = 10/30) was significantly lower than that in the control group (60% = 18/30). None of the nisin Z and colicin V-producing probiotic-fed fish were naturally infected by S. parauberis during the feeding period. In addition, more importantly, this promising probiotic strain significantly promoted fish growth along with better feed conversion and specific growth rate. This study demonstrates that the use of host-derived probiotics can offer a significant advantage in terms of optimum survival and function in the gastrointestinal tract of the intended host. Statement of relevance In this study, host-derived probiotic strain outperforms elimination of pathogen through competitive exclusion in the gastrointestinal tract and increased innate immune responses. More importantly, this promising probiotic strain significantly promoted fish growth along with better feed conversion. This study will provide insight into how optimal probiotics should be selected and developed. It might facilitate the replacement of commercial fish probiotic products originated from terrestrial sources with host-derived probiotics in the near future. [ABSTRACT FROM AUTHOR]

Exosomes are a group of extracellular vesicles carrying membrane proteins, lipids, RNAs, and, cytosolic proteins, which play key role in intercellular communication and homeostasis. This study describes the isolation, physicochemical, morphological and molecular characterization, toxicity, wound healing, and regeneration properties of plasma derived exosomes from naive (phosphate-buffered saline [PBS]-injected; PBS-Exo) and Streptococcus parauberis -challenged (Sp-Exo) olive flounder (Paralichthys olivaceus). The average diameters of PBS-Exo and Sp-Exo were 120.5 ± 6.1 and 113.1 ± 9.3 nm, respectively, and they presented unique cup shape morphologies. Both exosomes exhibited classical tetraspanin surface markers (CD81, CD9, and CD63) and were enriched with acetylcholinesterase. High-throughput miRNA profiling revealed differentially expressed miRNAs (log 2 fold change ≥1; P < 0.05), including 14 known and 22 novel miRNAs, in Sp-Exo. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the target genes of the miRNAs contribute towards various physiological and immunological functions, including wound healing and fin regeneration. Sp-Exo exhibited a rapid wound healing (cell migration) capacity in human fibroblast cells, and its mRNA and protein expression patterns corroborated its activity. Higher larval fin regeneration was more prevalent in Sp-Exo than in PBS-Exo, which further confirmed its functional significance. Our study provides the first basic physiochemical, morphometric, molecular (miRNA profiling), and wound healing evidences of Sp-Exo in olive flounder and highlights important miRNA cargoes in exosomes that may be potential therapeutic candidates in wound healing. [Display omitted] • Plasma-derived exosomes are isolated from Streptococcus parauberis challenged Olive flounder (Sp-Exo). • Sp-Exo exosomes exhibited characteristic morphology, physicochemical properties and tetraspanin surface markers. • miRNA profiling revealed differentially expressed cargo miRNAs in Sp-Exo. • Sp-Exo can internalize in to fish cells and are relatively non-toxic. • Sp-Exo enhances the cell migration, wound healing and regeneration. [ABSTRACT FROM AUTHOR]

A new bacteriophage, Str-PAP-1, which infects Streptococcus parauberis , was isolated and characterized. Str-PAP-1, classified as a member of the family Siphoviridae , has a double-stranded DNA genome with 35.3% GC content and 36,595 base pairs containing putative open reading frames. A BLAST search revealed that bacteriophage Str-PAP-1 bears no similarity to any previously reported bacteriophages. In a susceptibility test, 35 of the 55 (63.6%) S. parauberis field isolates tested, which were collected from streptococcosis-afflicted olive flounder ( Paralichthys olivaceus ) from the year 2010 to 2013 in Jeju-do province, Korea, were susceptible to the bacteriophage Str-PAP-1. This suggests that the bacteriophage Str-PAP-1 can be used to treat or prevent streptococcosis caused by S. parauberis . In addition, the effects of dietary supplementation with bacteriophage Str-PAP-1 at varying doses (T1: 1 × 10 5 pfu/g, T2: 1.5 × 10 5 pfu/g and T3: 2 × 10 5 pfu/g) on fish growth performance, the incidence of S. parauberis infections, and fish mortality, were evaluated. Feeding fish with the bacteriophage Str-PAP-1 improved growth (length: 9.3%, 5.4%, and 9.7%; weight: 27.1%, 21.2%, and 27.5% by T1, T2, and T3 doses, respectively), and reduced both the detection rate of S. parauberis and mortality (relative percent survival (RPS): 52.3%, 51.6%, and 53.1%, respectively) in all test groups. Thus, bacteriophage Str-PAP-1 is an environmentally friendly agent that can prevent and treat streptococcosis caused by S. parauberis . Statement of relevance S. parauberis is a major pathogenic bacterium that causes streptococcosis in farmed fish and often leads to mass mortality of farmed fish. In particular, streptococcosis caused by S. parauberis has continually increased in fish farming in Korea. Although various antibiotics have been used for the treatment of streptococcosis caused by S. parauberis , the efficacy of antibiotic-based treatment has continually decreased due to increased antibiotic resistance. The development of a new in-feed antibacterial agent that is safe, effective, and environmentally friendly is necessary. This study reports a bacteriophage that has potential as an in-feed alternative to antibiotics. [ABSTRACT FROM AUTHOR]

Long interspersed nuclear elements (LINEs) are widely distributed in the vertebrate genome, and can be either beneficial or detrimental to the host genome. Here we identified three members of LINE RT elements in the olive flounder genome. They showed high amino acid sequence identity (89-99 %), and the sequences of LINE reverse transcriptase (RT) in olive flounder are closely related to those of coral grouper, European seabass, and three-spined stickleback. Real-time reverse transcription-polymerase chain reaction analysis indicated that expression of the OF (Olive flounder)-LINE Chr3-1 RT increased more in spleen than in other tissues after treatment with the pathogen Streptococcus parauberis. These data may form the basis for further studies on the function of retroelements in infected olive flounder. [ABSTRACT FROM AUTHOR]

Enolase (ENO) is one of the surface-exposed proteins of Streptococcus iniae , which previously had been identified as a plasminogen-binding protein. In this study, ENO was evaluated to induce cross-protective immunity against S. iniae and Streptococcus parauberis which are major pathogens causing streptococcosis in fish. Immunoblot analysis shows that S. iniae recombinant ENO (rENO) produced in Escherichia coli was cross-reactive with antisera against S. iniae , and S. parauberis serotype I and II. In the challenge experiment of streptococcal infection after vaccination in zebrafish, rENO elicited a similar protection with a whole cell bacterin against S. iniae and S. parauberis , which suggests its feasibility as an efficient vaccine against streptococcosis. [ABSTRACT FROM AUTHOR]

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples. [ABSTRACT FROM AUTHOR]

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 . [ABSTRACT FROM AUTHOR]

Streptococcus parauberis is recognized as an alpha-hemolytic gram-positive bacterial pathogen associated with bovine udder mastitis. Recently, S. parauberis was identified as the major pathogen of olive flounder ( Paralichthys olivaceus) streptococcosis in Northeast Asia, a disease that causes severe economic losses by triggering massive olive flounder mortality. In this study, the complete genome sequence of S. parauberis KCTC11980 was determined and analyzed. S. parauberis KCTC11980 harbors a single chromosome of 2,179,251 bp containing 2281 predicted coding sequences and featuring an average G+C content of 35.65 %. Dot-plot analysis of the entire genomic sequence and phylogenetic analysis of the 16S rRNA gene revealed a close evolutionary relationship between S. parauberis KCTC11980 and S. uberis. Our findings could serve as a basis for understanding the pathogen-host relationship and developing new prophylactic and therapeutic strategies against fish streptococcosis. This study also enhances our understanding of the physiology and evolution of a key representative of the Streptococcaceae family. [ABSTRACT FROM AUTHOR]

We identified two lysozyme cDNA isoforms from starry flounder Platichthys stellate. The c-type and g-type starry flounder lysozyme cDNAs encoded polypeptides of 143 and 188 amino acids, respectively. Based on bioinformatic sequence characterizations, both isoforms possessed catalytic and other conserved residues essential for their functionality. Tissue distribution analysis revealed that c-type and g-type lysozyme transcripts were ubiquitously found in all tissue types examined. In particular, the c-type lysozyme mRNA was highly predominant in the spleen. On the other hand, g-type lysozyme transcripts were markedly detectable in posterior intestine, followed by kidney and spleen. In response to Streptococcus parauberis challenge, the splenic c-type, but not g-type, lysozyme mRNA level was significantly induced, indicating that the spleen is a major lysozyme producer in response to bacterial infection-mediated stress condition. At protein levels, the enhanced lysozyme activities in the skin mucus and serum were observed as early as 4 h post injection, suggesting that the lysozyme could be involved in the acute phase responses against bacterial infection. Taken together, our data indicate that S. parauberis infection elicits a lysozyme-associated systemic immune response in starry flounder, possibly in a tissue-specific and/or isoform-specific manner. [ABSTRACT FROM AUTHOR]

In recent years, Streptococcus parauberis infection has been an emerging problem in aquaculture in South Korea because of its more frequent isolation than other streptococcal bacteria including Streptococcus iniae . To develop effective treatment and prophylaxis methods against this emerging disease by S. parauberis , it is necessary to understand the underlying pathogenic mechanisms. To uncover the pathogenicity, the mutant strain of S. parauberis with a deleted phosphoglucomutase (PGM) gene which has been known to be an important virulence factor in bacterial pathogens was generated to investigate the relationship between virulence and gene function using an allelic exchange mutagenesis method. Allelic exchange mutagenesis of the phosphoglucomutase gene resulted in phenotype changes including decreased extracellular capsules, reduced buoyancy, increased hydrophobicity and reduced growth. Moreover, the S. parauberis mutant was more sensitive to innate immune clearance mechanisms including serum, mucus and phagocyte killing and could not induce mortality in olive flounder. These phenotype changes and the attenuated virulence of the pathogen to fish could be due to the reduction in capsule production by mutation of the PGM gene. The results provide evidences that phosphoglucomutase expression contributes to S. parauberis virulence in fish by affecting bacterial survival against the host's humoral and cellular defense mechanisms. [ABSTRACT FROM AUTHOR]