Thioclava litoralis sp. nov., a novel species of alphaproteobacterium, isolated from surface seawater.

A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29T, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth of strain FTW29T was observed at 15-42 ℃ (optimum, 28-30 ℃), pH 4.0-9.0 (optimum, pH 5.5-7.5) and in the presence of 0.5-10% NaCl (optimum, 3.0% NaCl). Strain FTW29T showed 95.0-96.8% 16 S rRNA gene sequence similarity to various type strains of the genera Thioclava, Sinirhodobacter, Rhodobacter, Haematobacter and Frigidibacter of the family Paracoccaceae, and its most closely related strains were Thioclava pacifica DSM 10,166T (96.8%) and Thioclava marina 11.10-0-13T (96.7%). The phylogenomic tree constructed on the bac120 gene set showed that strain FTW29T formed a clade with the genus Thioclava, with a bootstrap value of 100%. The evolutionary distance values between FTW29T and type strains of the genus Thioclava were 0.17-0.19, which are below the recommended standard (0.21-0.23) for defining a novel genus in the family Paracoccaceae. In strain FTW29T, the major fatty acids identified were summed feature 8 (C18:1ω7c) and C16:0, and the predominant respiratory quinones were ubiquinone-10 and ubiquinone-9. The composition of polar lipids in strain FTW29T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid, two unidentified glycolipids and an unidentified lipid. The genome of strain FTW29T comprised one circle chromosome and six plasmids, with a G + C content of 61.4%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain FTW29T and seven type strains of the genus Thioclava were 76.6-78.4%, 53.2-56.4% and 19.3-20.4%, respectively. Altogether, the phenotypic, phylogenetic and chemotaxonomic evidence illustrated in this study suggested that strain FTW29T represents a novel species of the genus Thioclava, with the proposed name Thioclava litoralis sp. nov. The type strain is FTW29T (= KCTC 82,841T = MCCC 1K08523T).

Spartinivicinus poritis sp. nov., a red pigment-producing bacterium isolated from a scleractinian coral Porites lutea.

A Gram-stain-negative, red pigment-producing, aerobic, and rod-shaped bacterial strain (A2-2T) was isolated from a bleached scleractinian coral (Porites lutea). Strain A2-2T grew with 1.0-7.0 % (w/v) NaCl (optimum, 3.0 %), at pH 6.0-11.0 (optimum, pH 8.0), and at 18-41 °C (optimum, 35 °C). Results of phylogenetic analysis based on 16S rRNA gene sequences suggested that strain A2-2T fell within the genus Spartinivicinus and was closely related to Spartinivicinus ruber S2-4-1HT (98.1 % sequence similarity) and Spartinivicinus marinus SM1973T (98.0 % sequence similarity). The predominant cellular fatty acids of strain A2-2T were C16 : 0 (31.0 %), summed feature 3 (29.0 %), summed feature 8 (11.7 %), C12 : 0 3-OH (6.4 %), and C10 : 0 3-OH (5.5 %), while the major respiratory quinone was Q-9. The polar lipids mainly comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified phospholipid. The genome size of strain A2-2T was 6.8 Mb, with a G+C content of 40.2 mol%. The DNA-DNA hybridization value was 24.2 % between A2-2T and S. ruber S2-4-1HT and 36.9 % between A2-2T and S. marinus SM1973T, while the average nucleotide identity values were 80.1 and 88.8 %, respectively. Based on these findings, strain A2-2T could be recognized to represent a novel species of the genus Spartinivicinus, for which the name Spartinivicinus poritis sp. nov. is proposed. The type strain is A2-2T (=MCCC 1K08228T=KCTC 8323T).

Microbulbifer bruguierae sp. nov., isolated from sediment of mangrove plant Bruguiera sexangula, and comparative genomic analyses of the genus Microbulbifer.

A Gram-negative, non-motile, strictly aerobic, rod-shaped bacterium, designated as H12T, was isolated from the sediments of mangrove plant Bruguiera sexangula taken from Dapeng district, Shenzhen, PR China. The pairwise 16S rRNA gene sequence analysis showed that strain H12T shared high identity levels with species of the genus Microbulbifer, with the highest similarity level of 98.5 % to M. pacificus SPO729T, followed by 98.1 % to M. donghaiensis CN85T. Phylogenetic analysis using core-genome sequences showed that strain H12T formed a cluster with type species of M. pacificus SPO729T and M. harenosus HB161719T. The complete genome of strain H12T was 4 481 396 bp in size and its DNA G+C content was 56.7 mol%. The average nucleotide identity and digital DNA-DNA hybridization values among strain H12T and type species of genus Microbulbifer were below the cut-off levels of 95-96 and 70 %, respectively. The predominant cellular fatty acids of strain H12T were iso-C15 : 0 (22.5 %) and C18 : 1 ω7c (13.9 %). Ubiquinone-8 was detected as the major respiratory quinone. The polar lipids of strain H12T comprised one phosphatidylglycerol, one phosphatidylethanolamine, one unidentified aminoglycophospholipid, one unidentified glycophospholipid, three unidentified glycolipids, two unidentified aminolipids, and one unidentified lipid. Based on polyphasic evidence, strain H12T represents a novel species of the genus Microbulbifer, for which the name Microbulbifer bruguierae sp. nov. is proposed. The type strain is H12T (=KCTC 92859T=MCCC 1K08451T). Comparative genomic analyses of strain H12T with strains of the genus Microbulbifer reveal its potential in degradation of pectin.

Identification and functional characterization of interleukin-22 (IL-22) in orange-spotted grouper (Epinephelus coioides).

In mammals, IL-22 is considered as a critical cytokine regulating of immunity and homeostasis at barrier surfaces. Although IL-22 have been functional characterization in different species of fish, the studies about distinct responses of IL-22 in different organs/tissues/cell types is rather limited. Here, we identified and cloned IL-22 gene (named as Ec-IL-22) from grouper (Epinephelus coioides). Ec-IL-22 gene was detected in all orangs/tissues examined, and was induced in intestine, gill, spleen, head kidney, and primary head kidney/intestine leukocytes following the stimulation of LPS and poly (I:C), as well as Vibrio harveyi and Singapore grouper iridovirus infection (SGIV). In addition, the stimulation of DSS could induce the expression of Ec-IL-22 in intestine and primary leukocytes from intestine. Importantly, the treatment of recombinant Ec-IL-22 induced the mRNA level of proinflammatory cytokines in primary intestine/head kidney leukocytes. The present results improve the understanding of expression patterns and functional characteristics of fish IL-22 in different organs/tissues/cell types.

Response signatures of intestinal microbiota and gene transcription of the pearl gentian grouper to Vibrio harveyi infection.

Vibrio harveyi causes high mortality and severely limits grouper culture. The gut microbiota is an important biological barrier against pathogen invasion. In this study, we investigated dynamic changes in the intestinal microbial community, gene transcription and immune responses signatures of pearl gentian grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀) at 0, 3 and 7 days (referred to as d0, d3 and d7 groups, respectively) after infection with V. harveyi. The results demonstrated that the d7 treatment reduced the gut microbial diversity and increased the proportion of Proteobacteria and Cyanobacteria. Notably, several putative pathogenic genera (Sphingomonas and Bacteroides) proliferated, while putative probiotic genera (Rhodococcus and Lactobacillus) reduced, and these changes in intestinal bacteria might be correlated to the alterations of host immune-related molecules. The d3 and d7 treatments also altered the histomorphology and gene transcription profiles mainly associated with immune function in intestine, such as 'MAPK signaling pathway', 'Apoptosis' and 'Toll-like receptor (TLR) signaling pathway'. Furthermore, d3 group induced a homeostatic dysregulation of the antioxidant system, cytokines and TLR signaling, with a tendency to gradually return to a normal state in d7 group, along with the apoptosis process. The pathogenic infection suppressed the expression of JNK pathway and enhanced the ERK pathway. In conclusion, the dysbiosis of the intestinal bacterial communities caused by the immune changes that occurred during V. harveyi infection disrupted the intestine health in the pearl gentian grouper. These results provided a comprehensive understandings of the immune defense mechanisms in fish and valuable references to develop disease control strategies in grouper aquaculture.

A CD6 homolog of Nile tilapia (Oreochromis niloticus) conserved binding bacteria involved in the regulation of Streptococcus agalactiae induced inflammation.

As a lymphocyte-specific surface receptor belonging to the cysteine-rich superfamily of scavenger receptors, CD6 acts as a pattern recognition receptor for microbial components and is involved in the regulation of inflammatory responses. However, the characteristics and functions of CD6 molecules in lower vertebrates represented by teleost fish are unknown. In this study, a CD6 homolog (designated OnCD6) was characterized from Nile tilapia (Oreochromis niloticus), and establishing its role as a PRRs that participates in immune recognition. OnCD6 contains an open reading frame of 1872 bp that encodes a peptide of 623 amino acids, and contains two conserved SR domain. Multiple sequence alignment revealed that OnCD6 shares a relatively high level of identity with those of other species. Transcriptional expression analysis revealed that OnCD6 was constitutively expressed in immunes tissues such as head kidney and thymus. The expression level of OnCD6 in mainly immune tissues were found significantly upregulated after the injection of Streptococcus agalactiae (S. agalactiae). Moreover, OnCD6 protein was located in the head kidney and brain, mainly over the plasma membrane of lymphocytes in these immune tissues. In vitro experiments showed that CD6 extracellular protein bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of bacterial surface conserved components LPS and LTA etc. In vivo experiments demonstrated that overexpression OnCD6 before S. agalactiae challenge significantly improved tilapia survival, and this was concomitant with reduced bacterial load and pro-inflammatory cytokines (IL-1ß and TNF-α). Taken together, our results illustrated the function of CD6 molecular pattern recognition receptors (PRRs) is conserved and plays an important role in antibacterial infection.

Functional properties of ATPIF1 in the orange-spotted grouper (Epinephelus coioides) in response to viral infection.

ATP synthase inhibitory factor 1 (ATPIF1) can activate mitochondrial autophagic pathway and mediates immune response by regulating ATP synthase activity. However, the role of fish ATPIF1 on viral infection is still unknown. In this study, we identified an ATPIF1 homolog (Ec-ATPIF1) from orange-spotted grouper (Epinephelus coioides). Ec-ATPIF1 is mainly expressed in the kidney and liver. The expression of Ec-ATPIF1 was significantly up-regulated after RGNNV stimulation in vitro. Further experiments showed that overexpression of Ec-ATPIF1 inhibited the expression of viral genes (CP and RdRp) and intracellular ATP synthesis. Ec-ATPIF1 overexpression also promoted the expression of mitophagy related genes (PINK1, Parkin, BNIP3, NIX, FUNDC1, LC3), inflammation-related factors (IL-1ß, IL-6, IL-8, IL-10, TNF-α, TLR2) and interferon pathway factors (IRF1, IRF3, IRF7, MX1, ISG15, ISG56, MDA5, TRIF). While the knockdown of Ec-ATPIF1 exhibited the opposite effects on the expression of viral genes and immune-related factors above. These data suggest that Ec-ATPIF1 can impact viral infection by regulating mitophagy, ATP synthesis, the expression of inflammatory factors and interferon pathway factors. These findings will be beneficial to better explore the immune regulatory mechanisms of fish respond to viral infection.

Characterization and function analysis of cathepsin C in Marsupenaeusjaponicus.

Cathepsin C is a cysteine protease widely found in invertebrates and vertebrates, and has the important physiological role participating in proteolysis in vivo and activating various functional proteases in immune/inflammatory cells in the animals. In order to study the role of cathepsin C in the disease resistance of shrimp, we cloned cathepsin C gene (MjcathC) from Marsupenaeus japonicus, analyzed its expression patterns in various tissues, performed MjcathC-knockdown, and finally challenged experimental shrimps with Vibrio alginolyticus and WSSV. The results have shown the full length of MjcathC is 1782 bp, containing an open reading frame of 1350 bp encoding 449 amino acids. Homology analysis revealed that the predicted amino acid sequence of MjcathC shared respectively 88.42 %, 87.36 % and 87.58 % similarity with Penaeus monodon, Fenneropenaeus penicillatus and Litopenaeus vannamei. The expression levels of MjcathC in various tissues of healthy M. japonicus are the highest in the liver, followed by the gills and heart, and the lowest in the stomach. The expression levels of MjcathC were significantly up-regulated in all examined tissues of shrimp challenged with WSSV or V. alginolyticus. After knockdown-MjcathC using RNAi technology in M. japonicus, the expression levels of lectin and heat shock protein 70 in MjcathC-knockdown shrimp were significantly down-regulated, and the mortality of MjcathC-knockdown shrimp challenged by WSSV and V. alginolyticus significantly increased. Knockdown of the MjcathC reduced the resistance of M. japonicus to WSSV and V. alginolyticus. The above results have indicated that cathepsin C may play an important role in the antibacterial and antiviral innate immunity of M. japonicus.

Characterization and functional analysis of SOCS9 from orange-spotted grouper (Epinephelus coioides) during virus infection.

The suppressor of cytokine signaling (SOCS) proteins family have twelve members including eight known mammalian SOCS members (CISH, SOCS1-7) and four new discovery members (SOCS3b, SOCS5b, SOCS8 and SOCS9) that is regarded as a classic feedback inhibitor of cytokine signaling. Although the function of the mammalian SOCS proteins have been well studied, little is known about the roles of SOCS in fish during viral infection. In this study, the molecular characteristics of SOCS9 from orange-spotted grouper (Epinephelus coioides, EcSOCS9) is investigated. The EcSOCS9 protein encoded 543 amino acids with typical SH2 (389-475aa) and SOCS_box (491-527aa), sharing high identities with reported fish SOCS9. EcSOCS9 was expressed in all detected tissues and highly expressed in kidney. After red-spotted grouper nervous necrosis virus (RGNNV) infection, the expression of EcSOCS9 was significantly induced in vitro. Furthermore, EcSOCS9 overexpression enhanced RGNNV replication, promoted virus-induced mitophagy that evidenced by the increased level of LC3-Ⅱ, BCL2, PGAM5 and decreased level of BNIP3 and FUNDC1. Besides, EcSOCS9 overexpression suppressed the expression levels of ATP6, CYB, ND4, ATP level and induced ROS level. The expression levels of interferon (IFN) related factors (IRF1, IRF3, IRF7, P53), inflammatory factors (IL1-ß, IL8, TLR2, TNF-α) and IFN-3, ISRE, NF-κB, AP1 activities were also reduced by overexpressing EcSOCS9. These date suggests that EcSOCS9 impacts RGNNV infection through modulating mitophagy, regulating the expression levels of IFN- related and inflammatory factors, which will expand our understanding of fish immune responses during viral infection.

Long- and short-term dietary ß-glucan improves intestinal health and disease resistance in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀).

ß-Glucans are immunostimulants and are widely used in aquaculture industry. The present study was conducted to evaluate the effects of different periods of ß-glucan management on growth performance, intestinal health, and disease resistance in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). A commercial feed was used as control diet (CD), and the ß-glucan diet (ßD) was based on CD and further supplemented with 0.1% ß-glucan. Grouper in control and long-term ß-glucan diet (LGD) groups were fed with CD and ßD for 8 weeks, respectively. Groupers in short-term ß-glucan diet (SGD) group were fed with CD for the first 4 weeks and ßD for the last 4 weeks. We found that LGD and SGD had no effect on growth performance but reduced the mortalities of grouper after challenging with Vibrio harveyi. In addition, both LGD and SGD increased intestinal morphology, enhanced antioxidant capacity, enhanced immunity, inhibited apoptosis, altered the transcriptional profile, and activated mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathway in the intestine of grouper. Furthermore, the effect of LGD on most of the above parameters was comparable to that of SGD. In conclusion, LGD and SGD did not affect growth rate parameters but enhanced the intestinal health and disease resistance of pearl gentian grouper.

Molecular characterization and functional analysis of CD209E from Nile Tilapia (Oreochromis Niloticus) involved in immune response to bacterial infection.

CD209 plays significant roles in pathogen recognition, innate and adaptive immunity, and cell-cell interactions. In the present study, a CD209 antigen-like protein E from Nile tilapia (Oreochromis niloticus) (designated as OnCD209E) was identified and characterized. OnCD209E contains an open reading frame (ORF) of 771 bp encoding a 257 amino acid protein, as well as the carbohydrate recognition domain (CRD). Multiple sequence analysis exhibits that the amino acid sequence of OnCD209E was relatively high homologous to that of partial fish, especially the highly conserved CRD, in which four conserved disulfide-bonded cysteine residues, WIGL conserved motif and two Ca2+/carbohydrate-binding sites (EPD and WFD motifs) were founded. Quantitative real-time PCR and Western Blot revealed that OnCD209E mRNA/protein is generally expressed in all tissues examined, but with wealth in head kidney and spleen tissues. The mRNA expression of OnCD209E was significantly increased in brain, head kidney, intestine, liver, and spleen tissues in response to the stimulation with polyinosinic-polycytidylic acid, Streptococcus agalactiae and Aeromonas hydrophila in vitro. Recombinant OnCD209E protein exhibited detectable bacterial binding and agglutination activity against different bacteria as well as inhibited the proliferation of tested bacteria. Subcellular localization analysis revealed that OnCD209E was mostly localized in the cell membrane. Moreover, overexpression of OnCD209E could activate nuclear factor-kappa B reporter genes in HEK-293T cells. Collectively, these results demonstrated that CD209E may potentially involve in immune response of Nile tilapia against bacterial infection.

TLR4 involved in immune response against Vibrio Parahaemolyticus by MyD88-dependent pathway in Crassostrea hongkongensis.

Vibrio parahaemolyticus (V. parahaemolyticus) is a salt-loving gram-negative bacterium, and is the leading cause of mortality in cultured shellfish in recent years. Toll-like Receptor 4 (TLR4) is a classical pattern recognition receptor (PRRs) that recognizes pathogen-associated molecular patterns (PAMPs) of pathogenic microorganism and activates the immune response. However, the function and signal pathway of TLR4 in oyster are still unknown. In this study, a new TLR4 gene was identified from the Crassostrea hongkongensis (C. hongkongensis). The ChTLR4 contained an open reading frame of 2643 bp, encoding 880 amino acids with seven leucine-rich repeat (LRR) domains and a Toll/IL-1R (TIR) domain. The ChTLR4 shared the highest sequence identity (83.0%) with TLR4 of Crassostrea gigas. Tissue expression analysis revealed that ChTLR4 showed the highest constitutive expression in the gill and hepatopancreas, and was significantly upregulated in immune tissues post V. parahaemolyticus infection, especially in gill and hemocytes. Moreover, TLR4 silencing significantly inhibited the immune-enzyme activities, including SOD, CAT, ACP, AKP in gill and LZM in hemolymph supernatant, and increased MDA content in hemolymph supernatant. Meanwhile, the antimicrobial activities of the hemolymph supernatant were also significantly inhibited by TLR4 silencing. These data demonstrated that the ChTLR4 involved in innate immune response of C. hongkongensis against V. parahaemolyticus challenge. Finally, qRT-PCR analysis showed that ChTLR4 silencing clearly inhibited the expression of genes in TLR4-MyD88 pathway, indicating that MyD88-dependent pathway played a crucial role in ChTLR4-mediated immune response against V. parahaemolyticus.

Characterization of CD166 from Nile tilapia (Oreochromis niloticus) displays a broad pathogen recognition spectrum and involved the immune response to microbial aggression.

CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules, and its mediated adhesion plays a crucial role in different physiological and pathological phenomena, especially related to leukocyte extravasation, immune synapse stability, T cell activation and proliferation. In this study, CD166 was identified from Nile tilapia (Oreochromis niloticus, OnCD166). OnCD166 contains an open reading frame of 1671 bp that encodes a peptide of 556 amino acids, and contains five consecutive extracellular immunoglobulin domains. It's tissue distribution and expression patterns after S. agalactiae challenge were also investigated. OnCD166 is widely distributed in various tissues of healthy tilapia. After Streptococcus agalactiae challenge, OnCD166 expressions were significantly up-regulated in all tested immune tissues. Meanwhile, the recombinant OnCD166 (rOnCD166E) protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Moreover, rOnCD166E could promote phagocytosis of macrophages. Taken together, our results illustrated that OnCD166 might as a receptor involved in the immune recognition and phagocytosis against invading pathogen, which play important roles in the immune responses of Nile tilapia against bacterial pathogens.

Creb2 involved in innate immunity by activating PpMitf-mediated melanogenesis in Pteria penguin.

cAMP response element binding protein 2 (CREB2) acts as an intracellular transcriptional factor and regulates many physiological processes, including melanogenesis and melanocyte differentiation. In our previous research, the Creb2 gene has been characterized from Pteria penguin (P. penguin), but its role and regulatory mechanism in P. penguin are still unclear. In this study, first, the function of PpCreb2 in melanogenesis and innate immunity were identified. PpCreb2 silencing significantly decreased the tyrosinase activity and melanin content, indicating PpCreb2 played an important role in melanogenesis. Meanwhile, PpCreb2 silencing visibly suppressed the antibacterial activity of hemolymph supernatant, indicating that PpCreb2 was involved in innate immunity of P. penguin. Second, the PpCreb2 was confirmed to perform immune function by regulating the melanogenesis. The decreased melanin oxidation product due to PpCreb2 silencing triggered the declining of antibacterial activity of hemolymph supernatant, which then could be rescued by adding exogenous melanin oxidation products. Third, the regulation pathway of PpCreb2 involved in innate immunity was analyzed. The promoter sequence analysis of PpMitf discovered 5 conserved cAMP response element (CRE), which were specifically recognized by basic Leucine zipper domain (bZIP) of upstream activation transcription factor. The luciferase activities analysis showed that PpCreb2 could activate the CRE in PpMitf promoter via highly conserved bZIP domain and regulate the expression of PpMitf, which further regulated the PpTyr expression. Therefore, the results collectively demonstrated that PpCreb2 participated in innate immunity by activating PpMitf-mediated melanogenesis in P. penguin.

Dextran Sulfate Sodium Salt (DSS) induced enteritis in Orange-spotted grouper, Epinephelus coioides.

The enteritis is a common disease in fish farming, but the pathogenesis is still not fully understood. The aim of the present study was to investigate the inducement of Dextran Sulfate Sodium Salt (DSS) intestinal inflammation on Orange-spotted grouper (Epinephelus coioides). The fish were challenged with 200 µl 3% DSS via oral irrigation and feeding, an appropriate dose based on the disease activity index of inflammation. The results indicated that the inflammatory responses induced by DSS were closely associated with the expression of pro-inflammatory cytokines including interleukin 1ß (IL-1ß), IL-8, IL16, IL-10 and tumor necrosis factor α (TNF-α), as well as NF-κB and myeloperoxidase (MPO) activity. At day5 after DSS treatment, the highest levels of all parameters were observed. Also, the severe intestinal lesions (intestinal villus fusion and shedding), strong inflammatory cell infiltration and microvillus effacement were seen through histological examination and SEM (scanning electronic microscopy) analysis. During the subsequent 18 days of the experimental period, the injured intestinal villi were gradually recovery. These data is beneficial to further investigate the pathogenesis of enteritis in farmed fish, which is helpful for the control of enteritis in aquaculture.

Description and genomic characterization of Gallaecimonas kandeliae sp. nov., isolated from the sediments of mangrove plant Kandelia obovate.

The genus Gallaecimonas, proposed by Rodríguez-Blanco et al. (Int J Syst Evol Microbiol 60:504-509, 2010), is mainly isolated from marine environments. So far, only three species have been identified and characterized in this genus. In this study, a new Gallaecimonas strain named Q10T was isolated from the sediments of mangrove plant Kandelia obovate taken from Dapeng district, Shenzhen, China. Strain Q10T was a Gram-stain-negative, non-motile, strictly aerobic, rod-shaped bacterium, and grew with 0-8.0% (w/v) NaCl, at 10-45 °C and at pH 5.5-8.5. Phylogenetic analysis indicated that strain Q10T and the three Gallaecimonas species formed a clade in the tree, with 16S rRNA gene sequence similarities ranging from 96.0 to 97.0%. The major respiratory quinone is Q8. The polar lipids comprised aminolipid, aminophospholipid, diphosphatidylglycerol, glycolipid, phosphatidylethanolamine, phosphatidylglycerol, glycophospholipid and phospholipid. The predominant fatty acids are C16:0, C17:1ω8c, summed feature 3 (C16:1ω7c/C16:1ω6c), and iso-C16:0. The complete genome of strain Q10T is 3,836,841 bp with a G+C content of 62.6 mol%. The orthologous proteins analysis revealed 55 unique proteins in strain Q10T related to important biological processes, especially three frataxins related to iron-sulfur cluster assembly, which may play a pivotal role in environmental adaptability of this species. Based on polyphasic taxonomic data, strain Q10T is considered to represent a novel species within the genus Gallaecimonas, for which the name Gallaecimonas kandelia sp. nov. is proposed. The type strain is Q10T (=KCTC 92860T=MCCC 1K08421T). These results contribute to a better understanding of general features and taxonomy of the genus Gallaecimonas.

Comparative genomic analysis provides insights into taxonomy and temperature adaption of Aeromonas salmonicida.

Aeromonas salmonicida has long been known as psychrophiles since it is mainly isolated from cold water fish, and recent reports have revealed the existence of mesophilic strains isolated from warm sources. However, the genetic differences between mesophilic and psychrophilic strains remain unclear due to few complete genomes of mesophilic strain are available. In this study, six A. salmonicida (2 mesophilic and 4 psychrophilic) were genome-sequenced, and comparative analyses of 25 A. salmonicida complete genomes were conducted. The ANI values and phylogenetic analysis revealed that 25 strains formed three independent clades, which were referred as typical psychrophilic, atypical psychrophilic and mesophilic groups. Comparative genomic analysis showed that two chromosomal gene clusters, related to lateral flagella and outer membrane proteins (A-layer and T2SS proteins), and insertion sequences (ISAs4, ISAs7 and ISAs29) were unique to the psychrophilic groups, while the complete MSH type IV pili were unique to the mesophilic group, all of which may be considered as lifestyle-related factors. The results of this study not only provide new insights into the classification, lifestyle adaption and pathogenic mechanism of different strains of A. salmonicida, but also contributes to the prevention and control of disease caused by psychrophilic and mesophilic A. salmonicida.

Pathogenicity of Plesiomonas shigelloides and Citrobacter freundii isolated from the endangered Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon (Acipenser sinensis) is an indigenous species of China and is listed as a critically endangered species. Recently, second filial generations of Chinese sturgeon in the Yangtze River Fisheries Research Institute suffered from a severe disease. In this study, two kinds of pathogenic bacteria were isolated from diseased sturgeon and identified as Plesiomonas shigelloides and Citrobacter freundii, based on 16S rDNA gene sequence alignment analysis. Antimicrobial susceptibility testing showed that P. shigelloides was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to tocefatriaxone, piperacillin, cefoperazone, cefazolin, and ciprofloxacin. C. freundii was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to chloramphenicol, cefuroxime, norfloxacin, ciprofloxacin, and ceftazidime. The median lethal dose (LD50) values of P. shigelloides and C. freundii were 4.50 × 103 colony forming units (CFU)/g and 3.20 × 103 CFU/g, respectively. Clinical symptoms of challenged sturgeons were the same as those of naturally infected sturgeons. Histopathological examination disclosed severe damage in the viscera of P. shigelloides and C. freundii-infected sturgeons. This is the first report suggesting that P. shigelloides infection is associated with mortality of Chinese sturgeon. The results of this study revealed the pathogenesis and severe pathogenicity of P. shigelloides and C. freundii in cultured Chinese sturgeon, and offer insights into the prevention and treatment of bacterial infection caused by P. shigelloides and C. freundii in cultured sturgeons.

Construction of an attenuated glutamyl endopeptidase deletion strain of Nocardia seriolae.

The glutamyl endopeptidase homolog of Nocardia seriolae (GluNS) has been proved to be a potential virulence factor in our previous study. Present investigation was carried out to construct an attenuated N. seriolae strain by deletion with GluNS gene and evaluate its protective immunity in head snakehead. A deletion strain (NS-ΔGluNS) was established by knockout of gene GluNS from wild strain N. seriolae ZJ0503 via homologous recombination. The LD50 of NS-ΔGluNS in 3.41 × 106 cfu/mL was significantly increased than that of wild strain in 4.75 × 105 cfu/mL, indicating that the virulence of N. seriolae has been attenuated with the knockout of GluNS. Meanwhile, applying NS-ΔGluNS as an attenuated live vaccine to immune hybrid snakehead, the non-specific immunity parameters (serum LYZ, POD, ACP, and AKP activities), specific antibody (IgM) titers production and immune-related genes (MHCIα, CD4, and IL-8) expression were up-regulated in different tissues, which indicated that they were able to trigger humoral and cell-mediated immune responses. Furthermore, the protective efficacy in hybrid snakehead after vaccination with NS-ΔGluNS shown 73.53% relative percentage survival (RPS). Taken together, the attenuated NS-ΔGluNS was obtained successfully and it could elicit strong immune response and supply protective efficacy to hybrid snakehead against N. seriolae wild strain.

Molecular characterization and expression analysis of interleukin-15 (IL-15) genes in orange-spotted grouper (Epinephelus coioides) in response to Vibrio harveyi challenge.

As a member of the γc family, interleukin 15 plays an important function in the immune response. In this study, we cloned an IL15 from Epinephelus coioides (named Ec-IL15). The open reading frame of Ec-IL15 is 528 bp, encoding 175 amino acids. Sequence alignment analysis showed that EcIL-15 has a conserved Pfam: IL15 domain and four cysteine residues. Subcellular localization studies have shown that Ec-IL15 is distributed in whole cells. In healthy groupers, Ec-IL15 was expressed in all 11 tissues tested and the highest in liver. After ConA, PHA, LPS and poly I:C stimulation, Ec-IL15 expression of HKLs was significantly upregulated. After V. harveyi infection, the expression of Ec-IL15 in 9 tissues was significantly upregulated and peaked within 48 h. In addition, recombinant Ec-IL15 protein can not only stimulate HKLs proliferation and cytokine expression, but also has the potential as an immune enhancer.