Photobacterium pectinilyticum sp. nov., a novel bacterium isolated from surface seawater of Qingdao offshore.

A Gram-staining-negative, facultative aerobic, motile strain, designated strain ZSDE20T, was isolated from the surface seawater of Qingdao offshore. Phylogenetic analysis of the 16S rRNA gene of strain ZSDE20T, affiliated it to the genus Photobacterium. It was closely related to Photobacterium lutimaris DF-42 T (98.92% 16S rRNA gene sequence similarity). Growth occurred at 4-28ºC (optimum 28ºC), pH 1.0-7.0 (optimum 7.0) and in the presence of 1-7% (w/v) NaCl (optimum 3%). The dominant fatty acids were summed feature 3 (C16:1 ω7c or/and C16:1 ω6c, 34.23%), summed feature 8 (C18:1 ω7c and C18:1 ω6c, 10.36%) and C16:0 (20.05%). The polar lipids of strain ZSDE20T comprised phosphatidylethanolamine, phosphatidylcholine, lyso-phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol dimannoside, phosphatidylinositol mannosides and two unknown lipids. The major respiratory quinone was ubiquinone-8 (Q-8). The DNA G + C content of strain ZSDE20T was 45.6 mol%. Average nucleotide identity (ANI) values between ZSDE20T and its reference species were lower than the threshold for species delineation (95-96%); in silico DNA-DNA hybridization further showed that strain ZSDE20T had less than 70% similarity to its relatives. Based on the polyphasic evidences, strain ZSDE20T is proposed as representing a novel species of the genus Photobacterium, for which the name Photobacterium pectinilyticum sp. nov. is proposed. The type strain is ZSDE20T (= MCCC 1K06283T = KCTC 82885 T).

Transcriptome profiling and differential expression analysis of the immune-related genes during the acute phase of infection with Photobacterium damselae subsp. damselae in silver pomfret (Pampus argenteus).

Silver pomfret has been widely cultured in China due to its high economic value. Photobacterium damselae subsp. damselae (PDD) is a Gram-negative bacterium that has been shown to infect many fish species. To increase knowledge of the molecular mechanisms of the host defense against PDD, we conducted transcriptome analysis of head kidney in silver pomfret at 24 h and 72 h post-infection (hpi) via Illumina sequencing. The de novo assembly resulted in the identification of 79,063 unigenes, with 59,386 (75.11%) successfully annotated in public databases (NR, NT, KO, Swiss-Prot, Pfam, GO, and KOG databases). Comparison of gene expression profiles between PBS-injected fish (sham control) and PDD-challenged fish revealed 329 and 570 differentially expressed genes (DEGs) were screened at 24 hpi and 72 hpi, respectively. The DEGs were enriched in multiple immune-related pathways such as Hepatitis C, Gastric acid secretion, CAMs and Leukocyte transendothelial migration pathways, Primary immunodeficieny, ECM-receptor interaction, PI3K-Akt signaling pathway. The data obtained in the present study offers valuable information for acute immune response of silver pomfret challenged with PDD, which will facilitate further investigations on strategies against Photobacterium spp. infection in teleosts.

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida.

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Holomycin, an Antibiotic Secondary Metabolite, Is Required for Biofilm Formation by the Native Producer Photobacterium galatheae S2753.

While the effects of antibiotics on microorganisms are widely studied, it remains less well understood how antibiotics affect the physiology of the native producing organisms. Here, using a marine bacterium, Photobacterium galatheae S2753, that produces the antibiotic holomycin, we generated a holomycin-deficient strain by in-frame deletion of hlmE, the core gene responsible for holomycin production. Mass spectrometry analysis of cell extracts confirmed that the ΔhlmE strain did not produce holomycin and that the mutant was devoid of antibacterial activity. Biofilm formation of the ΔhlmE strain was significantly reduced compared to that of wild-type S2753 and was restored in an hlmE complementary mutant. Consistent with this, exogenous holomycin, but not its dimethylated and less antibacterial derivative, S,S'-dimethyl holomycin, restored the biofilm formation of the ΔhlmE strain. Furthermore, zinc starvation was found to be essential for both holomycin production and biofilm formation of S2753, although the molecular mechanism remains elusive. Collectively, these data suggest that holomycin promotes biofilm formation of S2753 via its ene-disulfide group. Lastly, the addition of holomycin at subinhibitory concentrations also enhanced the biofilms of four other Vibrionaceae strains. P. galatheae likely gains an ecological advantage from producing holomycin as both an antibiotic and a biofilm stimulator, which facilitates nutrition acquisition and protects P. galatheae from environmental stresses. Studying the function of antibiotic compounds in the native producer will shed light on their roles in nature and could point to novel bioprospecting strategies.IMPORTANCE Despite the societal impact of antibiotics, their ecological functions remain elusive and have mostly been studied by exposing nonproducing bacteria to subinhibitory concentrations. Here, we studied the effects of the antibiotic holomycin on its native producer, Photobacterium galatheae S2753, a Vibrionaceae bacterium. Holomycin provides a distinct advantage to S2753 both as an antibiotic and by enhancing biofilm formation in the producer. Vibrionaceae species successfully thrive in global marine ecosystems, where they play critical ecological roles as free-living, symbiotic, or pathogenic bacteria. Genome mining has demonstrated that many have the potential to produce several bioactive compounds, including P. galatheae To unravel the contribution of the microbial metabolites to the development of marine microbial ecosystems, better insight into the function of these compounds in the producing organisms is needed. Our finding provides a model to pursue this and highlights the ecological importance of antibiotics to the fitness of the producing organisms.

Protective efficacy of four heat-shock proteins as recombinant vaccines against photobacteriosis in Asian seabass (Lates calcarifer).

Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of photobacteriosis in marine fish and is responsible for huge losses to marine aquaculture worldwide. Efforts have been made to develop a vaccine against this disease. Heat-shock proteins (HSPs) are a family of proteins that are ubiquitous in cellular life. Bacteria produce elevated levels of HSPs as a survival strategy when exposed to stressful environments in a host during infection. This group of proteins are also important antigens that can induce both humoral and cellular immune responses. In this study, four HSPs of Phdp, HSP90, HSP33, HSP70, and DnaJ, were selected for cloning and recombinant expression. Western blotting with rabbit anti-Phdp helped identify rHSP70 and rHSP33 as immunogenic proteins. Asian seabass (Lates calcarifer) immunised with rHSP90, rHSP33, rHSP70, and rDnaJ showed 48.28%, 62.07%, 51.72%, and 31.03% relative percent survival, respectively, after being challenged with Phdp strain AOD105021. High expression levels of immune-related genes and high antibody titres were observed in the rHSP33 group, and the sera of this group also exhibited a high level of bactericidal activity against Phdp. Collectively, our results suggest that HSP33 is a potential candidate for vaccine development against Phdp infection.

Dietary arginine and citrulline supplementation modulates the immune condition and inflammatory response of European seabass.

The present study was designed to determine the modulatory effects of arginine and citrulline dietary supplementation on the immune condition and inflammatory response of European seabass, Dicentrarchus labrax. Four diets were manufactured: a control diet (CTRL) was formulated to meet the indispensable amino acids profile established for seabass. Based on this formulation, three other diets were supplemented with l-arginine at two different levels (0.5% and 1%, ARG1 and ARG2, respectively) and l-citrulline at 0.5% (CIT). Fish were fed these diets for 2 or 4 weeks under controlled conditions. At the end of 4 weeks, fish from all dietary treatments were intraperitoneally-injected with Photobacterium damselae piscicida and sampled after 4, 24 our 48 h. Immune status was characterized by a lymphocyte time-dependent decrease regardless of dietary treatment, whereas peroxidase values dropped in time in fish fed ARG1 and ARG2 and was lower at 4 weeks in fish fed ARG1 than in fish fed CTRL. Up-regulation of several genes was more evident in ARG1-and CIT-fed fish, though pro-inflammatory cytokines were down-regulated by CIT dietary treatment. Following immune stimulation, seabass fed ARG1 showed a decrease in neutrophils and monocytes circulating numbers. On the other hand, expression of 17 selected immune and inflammatory responses genes was barely affected by dietary treatments. Based on the analyzed parameters, results suggest an active role of dietary arginine/citrulline supplementation in modulating immune defences that seem to translate into a suppressed immune repertoire, mostly at the cell response level. The observed changes due to citrulline dietary supplementation were in part similar to those caused by arginine, suggesting that citrulline might have been used by macrophages as an arginine precursor and then engaged in similar immune-impairment leading mechanisms.

Virulence properties of three new Photobacterium species affecting cultured fish.

AIMS: Several virulence factors of three new Photobacterium species: Photobacterium toruni, Photobacterium malacitanum and Photobacterium andalusiense associated with diseases of cultured redbanded seabream (Pagrus auriga) were studied. The exoenzymatic activities, adherence and cytotoxic capabilities, and iron-uptake mechanisms were determined both in bacterial extracellular products (ECP) and whole bacterial cells. The histopathology damages provoked on redbanded seabream by the ECP was also studied. METHODS AND RESULTS: The highest exoenzymatic activities of the ECP were alkaline- and acid-phosphatase, phosphohydrolase and lipase. The ECP were strongly lethal for fish at 4-96 h post-inoculation (p.i). Histological changes were evident at 96 hpi of ECP, affecting head kidney, splenic parenchyma and heart. Cytotoxicity assays, on three fish lines and one human cell line, were conducted using whole bacterial cells and their ECP. The new species tested were cytotoxic only for fish cell lines using whole bacterial cells. Bacterial adherence showed an adherence index moderate on CHSE-214 cell line. All strains showed variable haemolytic activity, and were able to grow under iron-limiting conditions, although the CAS reactivitiy was very low. However, all strains produced high amounts of extracelullar citrate that could be used as iron carrier, and use haem as iron source, except the P. toruni strains because a deletion in the genomic region encoding this ability in all Vibrionaceae members. CONCLUSIONS: The toxic activity of the bacterial ECPs was thermolabile, and not associated with their thermoresistant lipopolysaccharide content. The virulence of the strains tested could not be related to the haemolytic activity. Iron uptake could be based on the use of endogenous citrate as iron carrier and P. toruni lacks the ability to use haem as iron source. SIGNIFICANCE AND IMPACT OF THE STUDY: The study analyses for the first time the virulence properties of three new species of Photobacterium pathogenic for fish.

A novel research on isolation and characterization of Photobacterium damselae subsp. damselae from Pacific white shrimp, Penaeus vannamei, displaying black gill disease cultured in China.

In June 2019, massive mortalities of cultured Penaeus vannamei occurred in a local farm in Hainan Province, China. The diseased shrimp displayed evident black gills. Three bacterial strains 20190611001, 20190611007 and 20190611022 were isolated from hepatopancreas and gills of the diseased shrimp and identified as Photobacterium damselae subsp. damselae based on the sequence analysis of 16S rRNA and toxR genes. These three isolates showed haemolytic activities. Of them, strain 20190611022 isolated from hepatopancreas was selected and processed for pathogenic analysis. The calculated median lethal dose (LD50 ) was 9.75 ± 4.29 × 105 CFU/g (body weight) by challenging P. vannameivia reverse gavage. The diseased shrimp displayed enlarged hepatopancreatic tubules and sloughing of epithelial cells in tubular lumens. The strain 20190611022 was also characterized by the testing of API 20NE systems and antibiotic susceptibility. The results of disc diffusion test showed that strain 20190611022 was sensitive to chloramphenicol, compound sulfamethoxazole, cefoperazone, ceftriaxone, ceftazidime and cefuroxime. To our knowledge, this is the first report of isolation and characterization of Photobacterium damselae subsp. damselae from natural diseased P. vannamei. Our findings can serve as a basis for further studies of its pathogenicity and provide technological support for disease controlling in shrimp aquaculture.

Genotypic diversity, and molecular and pathogenic characterization of Photobacterium damselae subsp. piscicida isolated from different fish species in Taiwan.

Photobacteriosis, caused by Photobacterium damselae subsp. piscicida (Phdp), is a serious disease in marine fish species worldwide. To date, the epidemiological characterization of this pathogen in Taiwan remains limited. In this study, we collected 39 Phdp isolates obtained from different farmed fish for phenotypic and genotypic analysis. Phenotype bioassays using API-20E and API-20NE systems showed that the Phdp is a homogeneous group. However, genotyping using the pulsed-field gel electrophoresis (PFGE) technique revealed genetic variability among Phdp isolates when 13 and 11 different PFGE band patterns were obtained with SmaI and NotI as restriction enzymes, respectively. Phylogenetic analysis using 16S rDNA and the Fur gene clustered Taiwanese isolates and other species of P. damselae in the same clade. In contrast, the ToxR phylogenetic tree, a powerful discriminatory marker, separated the two subspecies. Furthermore, the virulence-associated genes, AIP56, P55, PDP_0080, Sod and Irp1, were detected from all isolates. Virulence testing with nine representative isolates in cobia (Rachycentron canadum) and Asian sea bass (Lates calcarifer) showed that some were highly pathogenic with 80%-100% mortality rates. This study provides epidemiological data of Phdp infections in farmed fish in Taiwan, which is necessary to develop comprehensive prevention and control strategies for the disease.

Host-microbiota interactions shed light on mortality events in the striped venus clam Chamelea gallina.

Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate- and pollution-induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA-sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over-expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down-regulation of several genes involved in immune and stress response, and the over-representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment.

The intracellular signaling pathway of octopamine upregulating immune resistance functions in Penaeus monodon.

Octopamine (OA), a biogenic monoamine, is known to mediate several immune responses. This study analyzed the effects of OA on immunological regulation in the tiger shrimp Penaeus monodon. The immune parameters including total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and phagocytic activity and clearance efficiency in response to the pathogen, Photobacterium damselae, were determined when shrimp were individually injected with saline or OA at 100 or 1000 pmol shrimp-1. In addition, the intracellular second messengers in haemocyte such as Ca2+ and adenosine 3',5'-cyclic monophosphate (cAMP) were examined in shrimp receiving saline or OA at 1 or 10 nmol shrimp-1. Results showed that all of the immune parameters significantly increased at 2-4 h in OA-injected shrimp except hyaline cells in 100 pmol shrimp-1-injected shrimp at 4 h, but phenoloxidase activity per granulocyte significantly decreased at 2-4 h. However, these had returned to saline control levels after receiving OA for 8 h except differential haemocyte count and phenoloxidase activity per granulocyte for 16 h. An injection of OA also significantly increased the survival rate of shrimp challenged with Pho. damselae. Shrimp receiving OA at 1 and 10 nmol shrimp-1 significantly increased the intracellular Ca2+ concentration ([Ca2+]i) at 30-60 min and 30 min, and cAMP concentration [cAMP]i) at 5-15 min and 15 min, respectively. However, [Ca2+]i at 50-60 min, and [cAMP]i at 30-60 min returned to saline control when the shrimp received OA at 10 nmol shrimp-1, and at 1 and 10 nmol shrimp-1, respectively. These results suggest that OA administration by injection at ≤1000 pmol shrimp-1 mediates transient upregulation of immunity together with the increased resistance of P. monodon to Pho. damselae, which are modulated through intracellular Ca2+ and cAMP second messenger pathways.

Mechanism study of cyfluthrin biodegradation by Photobacterium ganghwense with comparative metabolomics.

A high-efficiency pyrethroid-degrading bacterium, Photobacterium ganghwense strain 6046 (PGS6046), was first isolated from an offshore seawater environment. Metabolomics method was used to investigate the biotransformation pathway of PGS6046 to cyfluthrin wherein 156 metabolites were identified. The growth rates of the PGS6046 cultivated in nourishing media were much higher than those cultivated in seawater, regardless of the presence of cyfluthrin. Statistical analyses revealed that the metabolic profile of PGS6046 was associated with the culture medium, the presence of cyfluthrin, and culture time. The PGS6046 cultivated in a nourishing medium was characterized by higher levels of amino acids, a lower abundance of intermediates in the tricarboxylic acid cycle, and the presence of some fatty acids than those cultivated in seawater. The effects of cyfluthrin on PGS6046 metabolism varied based on the culture medium, whereas the cyanoalanine levels increased under both culture conditions. Culture time significantly affected the metabolism of amino acids and carbohydrates in PGS6046. The present study revealed the metabolic characteristics of PGS6046 under different culture conditions and will further facilitate the exploration of the fundamental questions regarding PGS6046 and its potential applications in environmental bioremediation.

De novo transcriptome analysis of immune response on cobia (Rachycentron canadum) infected with Photobacterium damselae subsp. piscicida revealed inhibition of complement components and involvement of MyD88-independent pathway.

Cobia, Rachycentron canadum, one of the most important aquatic species in Taiwan, has suffered heavy losses from Photobacterium damselae subsp. piscicida, which is the causal agent of photobacteriosis. In this study, the transcriptomic profiles of livers and spleens from Pdp-infected and non-infected cobia were obtained for the first time by Illumina-based paired-end sequencing method with a focus on immune-related genes. In total, 164,882 high quality unigenes were obtained in four libraries. Following Pdp infection, 7302 differentially expressed unigenes from liver and 8600 differentially expressed unigenes from spleen were identified. Twenty-seven of the differently expressed genes were further validated by RT-qPCR (average correlation coefficient 0.839, p-value <0.01). Results indicated a negative regulation of complement components and increased expression of genes involved in MyD88-independent pathway. Moreover, a remarkable finding was the increased expression of IL-10, implying an inadequacy of immune responses. This study not only characterized several putative immune pathways, but also provided a better understanding of the molecular responses to photobacteriosis in cobia.

[Mechanisms and Origin of Bacterial Biolumenescence].

The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past decades. Here we discuss the mechanisms of bacterial bioluminescence. We have the main results from structural and functional analysis of the genes of lux operons, enzymes (luciferase), and mechanisms of bioluminescence in several species of marine bacteria, which belong to three genera, Vibrio, Aliivibrio, and Photobacterium (A. fischeri, V. harveyi, P. leiognathi, and P. phosphoreum), and in terrestrial bacteria of the genus Photorhabdus (Ph. luminescens). The structure and mechanisms for the regulation of the expression of the lux operons are discussed. The fundamental characteristics of luciferase and luciferase-catalyzed reactions (stages of FMNH2 and tetradecanal oxidation, dimensional structure, as well as folding and refolding of the macromolecule) are described. We also discuss the main concepts of the origin of bacterial bioluminescence and its role in the ecology of modern marine fauna, including its involvement in the processes of detoxification of the reactive oxygen species and DNA repair, as well as the bait hypothesis.

Dietary administration of sodium alginate ameliorated stress and promoted immune resistance of grouper Epinephelus coioides under cold stress.

Grouper, Epinephelus coioides, fed a diet containing sodium alginate at 0 (control, named C) and 1.0 g kg-1 (named S) at a temperature of 28 °C for 12 days, were then further individually transferred to 28 (two groups named C-28 and S-28) or 20 °C (two groups named C-20 and S-20), and immune parameters and stress indexes were measured at the beginning and after 6, 12, 24 and 48 h of exposure. Examination of immune parameters revealed that the alternative complement activity (ACH50), lysozyme activity, phagocytic activity, superoxide dismutase, and respiratory bursts significantly increased in groupers fed the sodium alginate-containing diet for 12 days, and were higher in the S-28 than those of the C-28 and S-20 groups, which were higher than those of the C-20 group from 6 to 48 h except for ACH50 at 48 h, respiratory bursts at 48 h, and lysozymes at 6 h. For the assessment of stress indicators, cortisol, glucose, and lactate levels of serum significantly decreased in grouper fed the sodium alginate-containing diet for 12 days, and were higher in the C-20 group than those of the C-28 and S-20 groups, which were higher than those of the S-20 group at 6-48 h. In another experiment, grouper fed the test diet for 12 days at a temperature of 28 °C were challenged with Photobacterium damselae subsp. piscicida at a dose of 5 × 103 colony-forming units (cfu) (g fish)-1, and then individually transferred to 28 or 20 °C. The survival rate of challenged fish of the C-28 group was significantly lower than those of challenged fish of the C-20 and S-28 groups, which were significantly lower than that of challenged fish of the S-20 group. All challenged fish of the S-20 group survived. Survival rates over 144 h were 30.0%, 70.0%, and 56.7% for the C-28, C-20, and S-28 groups, respectively. Our results indicated that dietary sodium alginate administration downregulated stress response indicators, enhanced immune responses, and prevented impacts of physiologic stress responses, immunosuppression, and susceptibility to P. damselae subsp. piscicida in grouper subjected to cold stress. Grouper cultured at 28 °C were more susceptible to P. damselae subsp. piscicida infection.

The biology and the importance of Photobacterium species.

Photobacterium species are Gram-negative coccobacilli which are distributed in marine habitats worldwide. Some species are unique because of their capability to produce luminescence. Taxonomically, about 23 species and 2 subspecies are validated to date. Genomes from a few Photobacterium spp. have been sequenced and studied. They are considered a special group of bacteria because some species are capable of producing essential polyunsaturated fatty acids, antibacterial compounds, lipases, esterases and asparaginases. They are also used as biosensors in food and environmental monitoring and detectors of drown victim, as well as an important symbiont.

Photobacterium damselae subsp. damselae, an Emerging Fish Pathogen in the Black Sea: Evidence of a Multiclonal Origin.

UNLABELLED: Photobacterium damselae subsp. damselae is considered to be an emerging pathogen of marine fish of importance in aquaculture, with a notable increase in its geographical distribution during the last several years. In this study, we carried out for the first time to our knowledge a genetic and pathobiological characterization of 14 strains isolated from sea bass (Dicentrarchus labrax) reared in the Southeastern Black Sea, where high mortalities were observed at two aquaculture farms during the summer and autumn of 2011. Heterogeneity was evidenced among strains in phenotypical traits, such as sucrose fermentation, motility, and hemolysis. Although 11 of 14 isolates were hemolytic, we found that all of the isolates lacked the pPHDD1 virulence plasmid that encodes the phospholipase-D damselysin (Dly) and the pore-forming toxin PhlyP, two hemolysins previously reported to constitute major virulence factors for turbot. Subsequent PCR and sequencing analyses demonstrated that the 11 hemolytic isolates harbored a complete hlyAch gene, a chromosome I-borne gene that encodes HlyAch hemolysin, whereas the three nonhemolytic isolates contained hlyAch pseudogenes caused by insertion sequence elements. Virulence challenges with two representative strains revealed that, albeit less virulent than the pPHDD1-harboring strain RM-71, the plasmidless hlyAch-positive and hlyAch-negative Black Sea isolates were pathogenic for sea bass. A phylogenetic analysis based on the toxR gene sequence uncovered a greater diversity in the isolates, indicating that the presence of this pathogen in the Black Sea was not caused by the introduction and spread of a single virulent clone but by the proliferation of different clones. IMPORTANCE: The geographical distribution of marine bacterial pathogens is undergoing a worldwide increase. In particular, bacteria of the group vibrios are increasingly being isolated as the causative agents of disease in novel species of cultivated fish in areas where they had not been previously reported. Here we characterize for the first time to our knowledge a collection of isolates of the fish and human pathogen Photobacterium damselae subsp. damselae from diseased sea bass reared in the Black Sea. We uncovered great genetic diversity in the Black Sea isolates of this pathogen, suggesting a multiclonal origin. We also demonstrate for the first time that these isolates bear pathogenic potential for sea bass cultures by virulence challenges.

Camellia sinensis supplemented diet enhances the innate non-specific responses, haematological parameters and growth performance in Mugil cephalus against Photobacterium damselae.

This study evaluated the effect of dietary supplementation of Camellia sinensis leaf-extract on non-specific immune responses and disease resistance of Mugil cephalus fingerling against P. damselae. Fish were fed with 0 (unsupplemented), 50, 100 and 200 mg/kg of green tea extract (GTE) supplemented diets. Results indicated that GTE decreased mortality in M. cephalus in a dose-dependent manner after challenge with P. damselae. Haematological parameters containing RBC, Hct, Hb and WBC and growth performance (weight gain) showed remarkable changes in comparison with control group. In addition, the phagocytic (PA) and respiratory burst activity (RBA) significantly increased in M. cephalus, fed 100 and 50, 100 and 200 mg/kg GTE, respectively. Lysozyme statistically increased in GTE supplemented fish. Overall, our results indicated that incorporation of C. sinensis supplemented diet at 100 and 200 mg/kg doses significantly enhanced the immune responses in M. cephalus and that the mortality percentage could be remarkably reduced after challenging the fish against P. damselae.

In vitro cytokine profile revealed differences from dorsal and ventral skin susceptibility to pathogen-probiotic interaction in gilthead seabream.

Skin is the first barrier of defense on fish, which is crucial to protection against different stressors, including pathogens. Skin samples obtained from dorsal and ventral part of Sparus aurata specimens were incubated with Photobacterium damselae subsp. piscicida (a pathogen for this fish species), with Shewanella putrefaciens Pdp11 (a probiotic bacteria isolated from healthy gilthead seabream skin) or with both bacteria. The gene expression profile of nine cytokines (il1b, tnfa, il6, il7, il8, il15, il18, il10 and tgfb) was studied by qPCR in all the skin samples. The present findings revealed different patterns of cytokine profile in dorsal and ventral skin of gilthead seabream, which could be related to the influence and susceptibility to a possible infection.

Transcription of histones H1 and H2B is regulated by several immune stimuli in gilthead seabream and European sea bass.

Histones (H1 to H4) are the primary proteins which mediate the folding of DNA into chromatin; however, and in addition to this function, histones have been also related to antimicrobial peptides (AMPs) activity in vertebrates, in fact, mammalian H1 is mobilized as part as the anti-viral immune response. In fish, histones with AMP activity have been isolated and characterized mainly from skin and gonads. One of most threatening pathogens for wild and cultured fish species nowadays is nodavirus (NNV), which target tissues are the brain and retina, but it is also able to colonize the gonad and display vertical transmission. Taking all this into account we have identified the h1 and h2b coding sequences in European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) fish species and studied their pattern of expression under naïve conditions and NNV in vivo infection. The data obtained prompted us to study their role on the immune response of gonad and head-kidney leucocytes upon viral (NNV), bacteria (Vibrio anguillarum or Photobacterium damselae), pathogen-associated molecular patterns (PAMPs) or mitogens stimulation. The h1 and h2b genes are expressed in a wide range of tissues and their expression is modify by infection or other immune stimuli, but further studies will be needed to determine the significance of these changes. These results suggest that h1 expression is related to the immune response against NNV in the brain, while h2b transcription seems to be more important in the head-kidney. Moreover, the potential role of histones as anti-viral agents is suggested and further characterization is in progress.