Whole-genome sequencing and analysis of Streptococcus iniae strain isolated from the brain of tilapia (Oreochromis sp.).

Streptococcus iniae is a bacterium that can infect fish, mammals, and humans. In this study, the S. iniae-313 strain was isolated from the brain of an infected tilapia, and the analysis of its sequenced genome is reported. The data revealed that S. iniae-313 carried antibiotic-resistant genes and virulence factors.

Immune response and protection efficacy of formalin-killed vaccines against Streptococcus iniae in four-finger threadfin Eleutheronema tetradactylum.

Four-finger threadfin, Eleutheronema tetradactylum farming in southern Taiwan has been facing disease problems caused by Streptococcus iniae since 2018. The development of a vaccine against infectious S. iniae in the cultured threadfin industry is necessary. Thus, this study aimed to examine the efficacy of threadfin immunized formalin-killed cells (FKC) from S. iniae GSI-111 for 42 days post-vaccination (dpv) using two doses of FKC alone (a booster at 14 dpv) as group A, and FKC mixed with ISA763A adjuvant using a single dose as group B or double doses as group C. Immunoglobulin (Ig)-M was purified from threadfin, and rabbit anti-threadfin IgM polyclonal antibodies were used to detect antibody level in immunized fish; the vaccinated group A displayed higher levels at 3 dpv and all vaccinated treatments demonstrated high antibody levels between 14 and 42 dpv. All vaccine groups showed significantly higher values of lysozyme activity at 42 dpv compared with the control group; the vaccinated A group peaked at 14 dpv. The expression profiles of pro-inflammatory and immune-related genes, TNF-α, IL-12A, and C2 were upregulated at 3 dpv, while CD8A and chemokine receptor CXCR4 were upregulated at 42 dpv. Finally, the threadfins were challenged with S. iniae at 42 dpv. The average relative percent survival was 96% for vaccination A and B treatments, and 100% for vaccination C treatment. In summary, this study demonstrated that FKC vaccines whether formulated with an adjuvant could stimulate immune response and effective protect threadfins against S. iniae infection.

Extraction of high-molecular-weight DNA from Streptococcus spp. for nanopore sequencing in resource-limited settings.

The long-read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource-limited settings, such as field conditions or low- and middle-income countries. For this purpose, protocols for extracting high-molecular-weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram-negative bacteria. However, we found that without modification, these protocols are unsuitable for gram-positive Streptococcus spp., a major threat to fish farming and food security in low- and middle-income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high-molecular-weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish-pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high-molecular-weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low-copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource-limited settings-adapted protocol for high-molecular-weight DNA extraction could be implemented successfully for similarly recalcitrant-to-lysis gram-positive bacteria, and it represents a method of choice for MinION-based disease diagnostics in low- and middle-income countries.

Molecular structure and functional responses of IgM, IgT and IgD to Flavobacterium covae and Streptococcus iniae infection in Asian seabass (Lates calcarifer Bloch, 1790).

The Asian seabass (Lates calcarifer) faces significant disease threats, which are exacerbated by intensive farming practices and environmental changes. Therefore, understanding its immune system is crucial. The current study presents a comprehensive analysis of immune-related genes in Asian seabass peripheral blood leukocytes (PBLs) using Iso-seq technology, identifying 16 key pathways associated with 7857 immune-related genes, comprising 634 unique immune-related genes. The research marks the first comprehensive report on the entire immunoglobulin repertoire in Asian seabass, revealing specific characteristics of immunoglobulin heavy chain constant region transcripts, including IgM (Cµ, ighm), IgT (Cτ, ight), and IgD (Cδ, ighd). The study confirms the presence of membrane-bound form, ighmmb, ightmb, ighdmb of IgM, IgT and IgD and secreted form, ighmsc and ightsc of IgM and IgT, respectively, with similar structural patterns and conserved features in amino acids across immunoglobulin molecules, including cysteine residues crucial for structural integrity observed in other teleost species. In response to bacterial infections by Flavobacterium covae (formerly F. columnare genomovar II) and Streptococcus iniae, both secreted and membrane-bound forms of IgM (ighmmb and ighmsc) and IgT (ightmb and ightsc) show significant expression, indicating their roles in systemic and mucosal immunity. The expression of membrane-bound form IgD gene, ighdmb, predominantly exhibits targeted upregulation in PBLs, suggesting a regulatory role in B cell-mediated immunity. The findings underscore the dynamic and tissue-specific expression of immunoglobulin repertoires, ighmmb, ighmsc, ightmb, ightsc and ighdmb in Asian seabass, indicating a sophisticated immune response to bacterial pathogens. These findings have practical implications for fish aquaculture, and disease control strategies, serving as a valuable resource for advancing research in Asian seabass immunology.

Molecular characterization, expression profiling, and functional analysis of prohibitin 1 in red seabream, Pagrus major.

Prohibitin 1 (PHB1) is ubiquitously expressed in multiple compartments within cells and is involved in the cell cycle, cell signaling, apoptosis, transcriptional regulation, and mitochondrial biogenesis at the cellular level and in the inflammation-associated and immunological functions of B and T lymphocytes. PHB1 is an important protein that performs antioxidant regulation and immune functions inside and outside cells but has not been sufficiently studied in teleost fish. Our study aimed to elucidate the functional properties and gain new insights into the biological processes and immune system of red seabream (Pagrus major), a commercially important fish cultured in South Korea and East Asia. PHB1 mRNA was most abundantly expressed in the head kidney of healthy red seabream, and significant changes in its expression were observed after artificial infection with bacteria and viruses. On analysis, reporter gene was also significantly upregulated by polyinosinic-polycytidylic acid, lipopolysaccharides, and hydrogen peroxide. Consequent to the functional characterization of PHB1 in cells via recombinant protein preparation, the activity of leukocytes was enhanced and the reactive oxygen species-induced stress in red blood cells was reduced. The results reveal the functional characteristics of PHB1 and provide new insights into the biological processes and immune system of P. major, with beneficial implications in the study of stress responses.

A first report of Streptococcus iniae infection of the spotted sea bass (Lateolabrax maculates).

This study marks the first occasion that Streptococcus iniae has been isolated, identified, and characterized as the causative pathogen in spotted sea bass (Lateolabrax maculates). Infected fish exhibited a range of external symptoms, including scale loss, bleeding from the jaw, anus, and tail, among other signs, as well as internal manifestations such as congested liver, splenomegaly, branchial anemia, yellow fat syndrome, and intestinal edema. Notably, exophthalmia and meningoencephalitis-typical symptoms associated with previous S. iniae infections-were not observed. A predominant bacterial isolate (designated 10S01) was recovered from the pure culture of spleen of a diseased spotted sea bass in Zhuhai, China. The strain was then subjected to Gram staining, biochemical profiling, and molecular confirmation through 16S rRNA and gyrB gene, corroborating its identity as S. iniae. Pathogenicity was assessed by intraperitoneal injection challenge in spotted sea bass weighing approximately 13 g/fish, revealing a LD50 of 74 cfu/g-fish. The 10S01 strain demonstrated the ability to colonize various organs, including the spleen, liver, kidney, and brain, with a relatively higher affinity for the spleen. Furthermore, antimicrobial susceptibility testing indicated that the 10S01 strain was sensitive to 14 tested antibiotics, particularly chloramphenicol, ciprofloxacin, clarithromycin, florfenicol, ofloxacin, rifampicin, and trimethoprim/sulfamethoxazole, highlighting these as preferred treatments for S. iniae infections in spotted sea bass. These findings contribute significantly to our understanding of S. iniae pathogenesis and inform the prompt and appropriate antibiotic treatment of S. iniae infections.

Streptococcus iniae in aquaculture: a review of pathogenesis, virulence, and antibiotic resistance.

One of the main challenges in aquaculture is pathogenic bacterial control. Streptococcus iniae stands out for its ability to cause high mortality rates in populations of commercially important fish populations and its recent recognition as an emerging zoonotic pathogen. The rise in identifying over 80 strains some displaying antibiotic resistance coupled with the emerging occurrence of infections in marine mammal species and wild fish underscores the urgent need of understanding pathogenesis, virulence and drug resistance mechanisms of this bacterium. This understanding is crucial to ensure effective control strategies. In this context, the present review conducts a bibliometric analysis to examine research trends related to S. iniae, extending into the mechanisms of infection, virulence, drug resistance and control strategies, whose relevance is highlighted on vaccines and probiotics to strengthen the host immune system. Despite the advances in this field, the need for developing more efficient identification methods is evident, since they constitute the basis for accurate diagnosis and treatment.

Summer mortality syndrome bacterial pathogens in farmed Nile tilapia (Oreochromis niloticus).

Background: The high summer mortality in many fish farms, which had detrimental economic and social implications, was a serious challenge that the fish industry had to deal with. Aim: With an examination of the most effective antibiotic, the ongoing research was intended to shed light on the identification of the main bacterial pathogens associated with the summer mortality syndrome in the diseased farmed Nile tilapia. Methods: Six hundred dead Nile tilapia samples that had suffered from summer mortality were collected from several fish farms between May and October of 2022. The gathered fish displayed hemorrhagic areas on the skin, scale detachment, fin degeneration, erosions, skin ulcers, and corneal opacity with unilateral and/or bilateral exophthalmia. The most prominent internal appearance was swelling of the internal organs with sanguineous ascetic fluid. Results: There were 225 bacterial isolates found. Six species were identified through phenotypic and biochemical analysis; they were Aeromonas, Vibrio, Streptococcus, Pseudomonas, Enterococcus, and Edwardsiella spp., in descending percentage, respectively. Aeromonas spp., Vibrio spp., and Streptococcus spp. were the three most frequent isolated bacterial pathogens. The identification of Aeromonas hydrophila, Vibrio spp., and Streptococcus iniae, the three most common bacterial isolates, was confirmed by molecular analysis by polymerase chain reaction. Most of the tested strains were found to be responsive to Ciprofloxacin (CIP), Gentamicin (CN), and Chloramphenicol (C) but resistant to Amoxicillin (AMX), according to an antibiotic sensitivity test. Conclusion: The three most dangerous common bacterial infections discovered during mass-farmed tilapia summer mortality are A. hydrophil a, Vibrio sp., and S. iniae. This makes it clear that high water temperatures may raise the possibility of bacterial infections, which could cause widespread tilapia mortality and substantial financial losses. Therefore, it is crucial to maintain a beneficial fish culture, environment, and husbandry practices to enhance the tilapia-rearing environment and lessen the virulence of the disease. Isolated bacterial strains showed low levels of resistance to AMX but were vulnerable to CIP, CN, and C.

Pathogenicity of Streptococcus iniae causing mass mortalities of yellow catfish (Tachysurus fulvidraco) and its induced host immune response.

The outbreak of mass mortality occurred in Tachysurus fulvidraco farm in Hubei province of China. The pathogenic strain of Streptococcus iniae (termed 2022SI08) was isolated and identified from diseased T. fulvidraco, based on morphological, physiological, and biochemical characteristics, as well as 16S rRNA gene sequence and phylogenetic analysis. Further, the whole genome of isolate S. iniae was sequenced and predicted to contain one single circular chromosome of 1,776,777 bp with a GC content of 37.14%. The genomic sequence analysis showed that 2022SI08 was positive for 204 virulent and 127 antibiotic resistant genes. The experimental challenge demonstrated the high pathogenicity of the retrieved isolate of S. iniae, with a median lethal dosage (LD50) 9.53 × 105 CFU/g. Histopathological examination indicated that the 2022SI08 strain could induce extensive tissue cell degeneration, necrosis, hemorrhage, and inflammation in the skin, gill, fin, spleen, liver, kidney, intestine, eye, and brain. Moreover, the innate immune enzyme activities in serum such as acid phosphatase and alkaline phosphatase were increased significantly at 24 and 48 h post infection (hpi) and then decreased at 168 hpi. The transcriptional profile of immune associated gene in T. fulvidraco following bacterial infection was detected at each point of time, and the results revealed clear transcriptional activation of those genes, which proving their reacting and regulatory role during the response of the host against S. iniae infection. The results revealed that S. iniae was an etiological agent in the mass mortalities of T. fulvidraco and this research will be conducive for increasing our understanding on pathogenesis and host defensive system in S. iniae invasion.

Identification, Expression and Antimicrobial Functional Analysis of Interleukin-8 (IL-8) in Response to Streptococcus iniae and Flavobacterium covae in Asian Seabass (Lates calcarifer Bloch, 1790).

In this research, the proinflammatory cytokine interleukin-8 (IL-8) was shown to play a key role in inflammatory responses in fish. This study involved the cloning of the gene that encodes IL-8 in Asian seabass (Lates calcarifer) as well as analyses of its expression and function in this fish. The expression levels of LcIL-8 indicated that it was broadly expressed in most analyzed tissues, with the most predominant expression in the whole blood 6 to 24 h after infection with S. iniae at concentrations of 105 colony-forming units (CFU)/fish (p < 0.05). After fish were immersed in F. covae, the LcIL-8 transcript was upregulated in the gills, liver and intestine, and the highest expression level was observed in the gills. However, LcIL-8 was downregulated in all the tested tissues at 48 and 96 h after infection with the two pathogenic strains, indicating that Lc-IL8 has a short half-life during the early immune responses to pathogens. Moreover, the MIC of the rLcIL-8 protein against S. iniae was 10.42 ± 3.61 µg/mL. Furthermore, functional analyses clearly demonstrated that 10 and 100 µg of the rLcIL-8 protein efficiently enhanced the phagocytic activity of Asian seabass phagocytes in vitro (p < 0.05). Additionally, in vivo injection of S. iniae following the rLcIL-8 protein indicated that 50 and 100 µg of rLc-IL-8 were highly effective in protecting fish from this pathogen (p < 0.001). The obtained results demonstrate that rLcIL-8 possesses a biological function in the defense against bacterial infections in Asian seabass.

Cellulitis and bacteremia caused by the fish pathogen,Streptococcus iniae, in an immunocompromised patient: Case report and mini-review of zoonotic disease, lab identification, and antimicrobial susceptibility.

Streptococcus iniae is a fish pathogen that can also infect mammals including dolphins and humans. Its prevalence in farmed fish, particularly tilapia, provides potential for zoonotic infections, as documented by multiple case reports. Systematic clinical data beyond cellulitis for S. iniae infection in humans, including antimicrobial susceptibility data, are unfortunately rare. Here, we present a case of cellulitis progressing to bacteremia caused by Streptococcus iniae in a functionally immunocompromised patient based on CDK4/CDK6 inhibitor and endocrine therapy, and we discuss risk factors, identification, and antimicrobial susceptibility of this rare pathogen.

Efficacy of whole-cell-based monovalent and bivalent vaccines against Streptococcus iniae and Flavobacterium covae in fingerling Asian seabass (Lates calcarifer).

Streptococcosis and columnaris caused by Streptococcus spp. and Flavobacterium spp. have been recognized as critical problems in Asian seabass aquaculture development because they cause severe mortality. In this study, we identified various isolates of S. iniae and F. covae from diseased Asian seabass farmed in Thailand for use as candidates for vaccine development. The efficacy of the vaccines was evaluated by challenge tests and immune parameter analyses in fish that received whole-cell-based monovalent and bivalent vaccines containing S. iniae (Sin) and F. covae (Fco) delivered by top-dressed feed (TD) and intraperitoneal injection (IP). The results showed that all vaccinated groups exhibited increased antibody titers compared with control fish that peaked on day 28 after booster administration with high detection levels in the Sin-IP and Fco-IP groups. Moreover, the immune responses to the injected monovalent vaccines (Sin-IP and Fco-IP) were better than the responses in the other vaccinated groups. The hematological and innate immunological parameters were significantly increased by Sin-IP and Fco-IP, particularly lysozyme activity, nitroblue tetrazolium (NBT) activity, bactericidal activity, and white blood cell numbers, and immune-related genes, including IgM, MHC-IIα, TCRß and CD4, were significantly upregulated in the head kidney, whole blood and spleen (P < 0.05). After experimental challenge, survival in the Sin-IP and Fco-IP groups was significantly higher than that in the Sin-TD, Fco-TD, Sin + Fco-TD, and Sin + Fco-IP groups, with 80.0 % and 60.0 % survival after S. iniae and F. covae infection, respectively. In contrast, survival after bacterial challenge in the control groups was 10 % in each group. Histopathological analysis revealed that Sin-IP- and Fco-IP-vaccinated fish exhibited significantly more goblet cells in the intestines and melanomacrophage centers (MMCs) in the head kidney and spleen than those in the other groups (P < 0.05). Overall, the results of our study indicated that the monovalent vaccines Sin-IP and Fco-IP provoked better vaccine efficacy and immune responses than their orally administered counterparts, and these results are consistent with those from the immunological assays that showed significantly increased responses after immunization.

Comparative genomics analysis of Streptococcus iniae isolated from Trachinotus ovatus: novel insight into antimicrobial resistance and virulence differentiation.

BACKGROUND: Streptococcus iniae is an important fish pathogen that cause significant economic losses to the global aquaculture industry every year. Although there have some reports on the genotype of S.iniae and its relationship with virulence, no genome-scale comparative analysis has been performed so far. In our previous work, we characterized 17 isolates of S.iniae from Trachinotus ovatus and divided them into two genotypes using RAPD and rep-PCR methods. Among them, BH15-2 was classified as designated genotype A (in RAPD) and genotype 1 (in rep-PCR), while BH16-24 was classified as genotype B and genotype 2. Herein, we compared the differences in growth, drug resistance, virulence, and genome between BH15-2 and BH16-24. RESULTS: The results showed that the growth ability of BH16-24 was significantly faster than that of BH15-2 at the exponential stage. Antimicrobial tests revealed that BH15-2 was susceptible to most of the tested antibiotics except neomycin and gentamycin. In contrast, BH16-24 was resistant to 7 antibiotics including penicillin, sulfasomizole, compound sulfamethoxazole tablets, polymyxin B, spectinomycin, rifampin and ceftazidime. Intraperitoneal challenge of T.ovatus, showed that the LD50 value of BH15-2 was 4.0 × 102 CFU/g, while that of BH16-24 was 1.2 × 105 CFU/g. The genome of S.iniae BH15-2 was 2,175,659 bp with a GC content of 36.80%. Meanwhile, the genome of BH16-24 was 2,153,918 bp with a GC content of 36.83%. Comparative genome analysis indicated that compared with BH15-2, BH16-24 genome had a large-scale genomic inversion fragment, at the location from 502,513 bp to 1,788,813 bp, resulting in many of virulence and resistance genes differentially expression. In addition, there was a 46 kb length, intact phage sequence in BH15-2 genome, which was absent in BH16-24. CONCLUSION: Comparative genomic studies of BH15-2 and BH16-24 showed that the main difference is a 1.28 Mbp inversion fragment. The inversion fragment may lead to abnormal expression of drug resistant and virulence genes, which is believed to be the main reason for the multiple resistance and weakened virulence of BH16-24. Our study revealed the potential mechanisms in underlying the differences of multidrug resistance and virulence among different genotypes of S.iniae.

Whole Genomic Characterization of Streptococcus iniae Isolates from Barramundi (Lates calcarifer) and Preliminary Evidence of Cross-Protective Immunization.

Lates calcarifer, also known as Barramundi or Asian seabass, is a highly productive and fast-growing species that is well suited to large-scale aquaculture due to its attractive harvestable yields (premium fish). This fish has been envisioned as having the potential to be the "Salmon of Tropics". Cultivating Lates calcarifer in aquaculture poses challenges, as the dense populations that make such aquaculture commercially viable facilitate the rapid spread of infectious diseases, which in turn significantly impact yield. Hence, the immunization of juveniles is necessary, and the development of new immunization agents enhances the efficiency of aquaculture and improves food security. In our study, we characterize seven novel strains of the bacterial pathogen Streptococcus iniae that were collected from commercial fish farms in Singapore and Australia. We find that the capsular operon in our strains is highly conserved and identify a number of major surface antigens previously described in Streptococcus. A genome analysis indicates that the present strains are closely related but form distinct strains within the S. iniae species. We then proceed to demonstrate that inoculation with the inactivated strain P3SAB cross-protects Lates calcarifer against S. iniae infections in vivo from a variety of strains found in both Singapore and Australia.

Rapid and Sensitive Detection of Streptococcus iniae in Trachinotus ovatus Based on Multienzyme Isothermal Rapid Amplification.

Infectious diseases caused by Streptococcus iniae lead to massive death of fish, compose a serious threat to the global aquaculture industry, and constitute a risk to humans who deal with raw fish. In order to realize the early diagnosis of S. iniae, and control the outbreak and spread of disease, it is of great significance to establish fast, sensitive, and convenient detection methods for S. iniae. In the present study, two methods of real-time MIRA (multienzyme isothermal rapid amplification, MIRA) and MIRA-LFD (combining MIRA with lateral flow dipsticks (LFD)) for the simA gene of S. iniae were established, which could complete amplification at a constant temperature of 42 °C within 20 min. Real-time MIRA and MIRA-LFD assays showed high sensitivity (97 fg/µL or 7.6 × 102 CFU/mL), which were consistent with the sensitivity of real-time PCR and 10 times higher than that of PCR with strong specificity, repeatability simplicity, and rapidity for S. iniae originating from Trachinotus ovatus. In summary, real-time MIRA and MIRA-LFD provide effective ways for early diagnosis of S. iniae in aquaculture, especially for units in poor conditions.

Genome-wide association study for Streptococcus iniae in Nile tilapia (Oreochromis niloticus) identifies a significant QTL for disease resistance.

Streptococcus iniae is a problematic gram-positive bacterium negatively affecting Nile tilapia (Oreochromis niloticus), one of the main aquacultural species produced worldwide. The aim of this study was to identify the genetic architecture of survival to S. iniae and identify single nucleotide polymorphism (SNPs) linked to quantitative trait loci (QTL) related to survival to S. iniae challenge. With this purpose, Nile tilapia from the Spring Genetics breeding program were sent to a controlled S. iniae challenge test where phenotypes were scored as dead for fish that died during challenge test and survivors for the fish alive at the termination of the test. Additionally, fin-clip samples from all fish in the test were collected for DNA extraction. Out of 1904 fish in the challenge test, tissue samples of 321 fish were sent for genotyping using double digest restriction site associated DNA sequencing (ddRADseq). After quality control and filtering, 9,085 SNPs were used to perform a genome-wide association study (GWAS). A significant signal in LG8 was observed indicating association with survival to S. iniae challenge, with SNPs explaining from 12% to 26% of the genetic variance. To demonstrate the usefulness of marker assisted selection (MAS) to selectively breed fish for survival to S. iniae, offspring of breeding candidates classified as "resistant" and "susceptible" based on haplotypes of the four most significant markers were sent to a controlled S. iniae challenge test. At the end of the test, the differences in mortality between the two groups were strikingly different with a final cumulative percent mortality of less than 1% and 73% for offspring from "resistant" and "susceptible" parents, respectively. These results demonstrate that MAS for improved resistance to S. iniae is feasible.

Development and Evaluation of Recombinant B-Cell Multi-Epitopes of PDHA1 and GAPDH as Subunit Vaccines against Streptococcus iniae Infection in Flounder (Paralichthys olivaceus).

Streptococcus iniae is a severe Gram-positive pathogen that can infect a wide range of freshwater and marine fish species. In continuation of our earlier studies on the development of S. iniae vaccine candidates, pyruvate dehydrogenase E1 subunit alpha (PDHA1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were highly efficacious in protecting flounder (Paralichthys olivaceus) against S. iniae. In the present study, to investigate the potential of multi-epitope vaccination strategy to prevent flounder against S. iniae infection, the liner B-cell epitopes of PDHA1 and GAPDH proteins were predicted using a bioinformatics approach and were identified by immunoassay, and recombinant B-cell multi-epitopes of PDHA1 and GAPDH (rMEPIP and rMEPIG) containing immunodominant epitope-concentrated domains were expressed in Escherichia coli BL21 (DE3) and were used as a subunit vaccine to immunize healthy flounder, while recombinant PDHA1 (rPDHA1), GAPDH (rGAPDH) and formalin-inactivated S. iniae (FKC) served as controls. Then, the immunoprotection efficacy of rMEPIP and rMEPIG was evaluated by determining the percentages of CD4-1+, CD4-2+, CD8ß+ T lymphocytes and surface-IgM-positive (sIgM+) lymphocytes in peripheral blood leucocytes (PBLs), spleen leucocytes (SPLs) and head kidney leucocytes (HKLs), as well as total IgM, specific IgM, and relative percentage survival (RPS) post immunization, respectively. It was found that fish immunized with rPDHA1, rGAPDH, rMEPIP, rMEPIG and FKC showed significant increases in sIgM+, CD4-1+, CD4-2+, and CD8ß+ lymphocytes and production of total IgM and specific IgM against S. iniae or recombinant proteins rPDHA1 and rGAPDH, which indicated the activation of humoral and cellular immune responses after vaccination. Moreover, RPS rate of the multi-epitope vaccine rMEPIP and rMEPIG groups reached 74.07% and 77.78%, higher than that of rPDHA1 and rGAPDH (62.96% and 66.67%) and KFC (48.15%). These results demonstrated that B-cell multi-epitope protein vaccination, rMEPIP and rMEPIG, could give a better protective effect against S. iniae infection, which provided a promising strategy to design the efficient vaccine in teleost fish.

Differential Expression Genes of the Head Kidney and Spleen in Streptococcus iniae-Infected East Asian Fourfinger Threadfin Fish (Eleutheronema tetradactylum).

Streptococcus iniae is a Gram-positive bacterium and is considered a harmful aquaculture pathogen worldwide. In this study, S. iniae strains were isolated from East Asian fourfinger threadfin fish (Eleutheronema tetradactylum) reared on a farm in Taiwan. A transcriptome analysis of the head kidney and spleen was performed in the fourfinger threadfin fish 1 day after infection using the Illumina HiSeq™ 4000 platform for RNA-seq to demonstrate the host immune mechanism against S. iniae. A total of 7333 genes based on the KEGG database were obtained after the de novo assembly of transcripts and functional annotations. Differentially expressed genes (DEGs) (2-fold difference) were calculated by comparing the S. iniae infection and phosphate-buffered saline control group gene expression levels in each tissue sample. We identified 1584 and 1981 differentially expressed genes in the head kidney and spleen, respectively. Based on Venn diagrams, 769 DEGs were commonly identified in both the head kidney and spleen, and 815 and 1212 DEGs were specific to the head kidney and spleen, respectively. The head-kidney-specific DEGs were enriched in ribosome biogenesis. The spleen-specific and common DEGs were found to be significantly enriched in immune-related pathways such as phagosome, Th1, and Th2 cell differentiation; complement and coagulation cascades; hematopoietic cell lineage; antigen processing and presentation; and cytokine-cytokine receptor interactions, based on the KEGG database. These pathways contribute to immune responses against S. iniae infection. Inflammatory cytokines (IL-1ß, IL-6, IL-11, IL-12, IL-35, and TNF) and chemokines (CXCL8 and CXCL13) were upregulated in the head kidney and spleen. Neutrophil-related genes, including phagosomes, were upregulated post-infection in the spleen. Our results could offer a strategy for the treatment and prevention of S. iniae infection in fourfinger threadfin fish.

Identification and expression profiling of receptor-interacting serine/threonine-protein kinase 2 in Siberian sturgeon (Acipenser baerii).

Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) is an adaptor protein of the pattern recognition receptors NOD1 and NOD2 involved in regulating inflammatory response and resisting pathogenic microbial infection. In this study, Acipenser baerii RIPK2 (AbRIPK2) was identified. The open reading frame of AbRIPK2 was 1815 bp encoding 604 amino acids. AbRIPK2 possessed the typical N-terminal kinase domain (KD) and C-terminal caspase recruitment domain (CARD). The phylogenetic tree analysis revealed that AbRIPK2 shared a relatively high identity with bony fish. Real-time fluorescence quantitative PCR (qRT-PCR) results indicated that AbRIPK2 was highly expressed in the gill, followed by muscle, liver and heart. AbRIPK2 was significantly induced in the spleen and valvular intestine after Streptococcus iniae and Aeromonas hydrophila infection. AbRIPK2 was significantly upregulated after peptidoglycan (PGN) treatment in the splenic leukocytes. This study indicated that AbRIPK2 played a potential role in resisting the pathogenic infection of Siberian sturgeon by responding to bacteria.

Identification and characterization of high mobility group box 1 and high mobility group box 2 in Siberian sturgeon (Acipenser baerii).

High mobility group box 1 (HMGB1) and high mobility group box 2 (HMGB2) were highly conserved nonhistone chromosomal proteins involved in DNA damage repair, innate immune and inflammatory response. In this study, Acipenser baerii HMGB1 (AbHMGB1) and HMGB 2 (AbHMGB2) were identified. The open reading frame (ORF) of AbHMGB1 was 621 bp which encoded 206 amino acids, and the ORF of AbHMGB2 was 630 bp encoded 209 amino acids. AbHMGB1 and AbHMGB2 were conserved compared with bony fish by phylogenetic analyzing. qRT-PCR showed that AbHMGB1 and AbHMGB2 were expressed in all examined tissues, AbHMGB1 was expressed abundantly in muscle, followed by head kidney and brain, and AbHMGB2 was highest expressed in gill, followed by brain and muscle. After Streptococcus iniae infection and PAMPs treatment, AbHMGB1 and AbHMGB2 were induced significantly. This study indicated that AbHMGB1 and AbHMGB2 are involved in the process of pathogenic infection and provided a basis for exploring the mechanism of Acipenser baerii enteritis induced by Streptococcus iniae.