Intestinal Explant Cultures from Gilthead Seabream (Sparus aurata, L.) Allowed the Determination of Mucosal Sensitivity to Bacterial Pathogens and the Impact of a Plant Protein Diet.

The interaction between diet and intestinal health has been widely discussed, although in vivo approaches have reported limitations. The intestine explant culture system developed provides an advantage since it reduces the number of experimental fish and increases the time of incubation compared to similar methods, becoming a valuable tool in the study of the interactions between pathogenic bacteria, rearing conditions, or dietary components and fish gut immune response. The objective of this study was to determine the influence of the total substitution of fish meal by plants on the immune intestinal status of seabream using an ex vivo bacterial challenge. For this aim, two growth stages of fish were assayed (12 g): phase I (90 days), up to 68 g, and phase II (305 days), up to 250 g. Additionally, in phase II, the effects of long term and short term exposure (15 days) to a plant protein (PP) diet were determined. PP diet altered the mucosal immune homeostasis, the younger fish being more sensitive, and the intestine from fish fed short-term plant diets showed a higher immune response than with long-term feeding. Vibrio alginolyticus (V. alginolyticus) triggered the highest immune and inflammatory response, while COX-2 expression was significantly induced by Photobacterium damselae subsp. Piscicida (P. damselae subsp. Piscicida), showing a positive high correlation between the pro-inflammatory genes encoding interleukin 1ß (IL1-ß), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2).

Molecular and histopathological characterization of Photobacterium damselae in naturally and experimentally infected Nile tilapia (Oreochromis niloticus).

Mass mortality has occurred among cultured Nile tilapia, Oreochromis niloticus, on fish farms in Manzala, Dakahlia province, Egypt, in the summer season, 2019. Moribund fish were reported with deep ulcers, septicaemic lesions and sampled for bacterial isolation. In this study, most isolates were subjected to bacteriological examination, antibiotic sensitivity test, 16S rRNA gene sequencing and histopathological examination. Following isolate identification, intraperitoneal challenge of Nile tilapia with a bacterial suspension 2 × 106  CFU/ml was performed. Samples from liver, spleen and kidney were collected for histological and biochemical analysis. The results showed a high similarity (99%) to Photobacterium damselae strains using phylogenetic analysis of 16S rRNA. P. damselae exhibited resistance to amoxicillin and erythromycin, as well it was highly sensitive to chloramphenicol and doxycycline. Moreover, haemorrhage, oedema, hemosiderosis and melanomacrophage activation in the liver and head kidney of infected fish were detected by light and electron microscopy. Also, significant higher levels of CAT and SOD in the spleen and head kidney, as well as the serum levels of NO were observed in experimentally challenged O. niloticus, compared to the control fish. Our data identified P. damselae for the first time from infected Nile tilapia, describing its sensitivity to a variety of antibiotics, histopathological alterations and oxidative stress impact, and it could be useful indicators for understanding P. damselae pathogenesis, which might provide a preventive efficacy for P. damselae.

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.

Inclusion of dietary Ulva ohnoi 5% modulates Solea senegalensis immune response during Photobacterium damselae subsp. piscicida infection.

Macroalgae represent valuable sources of functional ingredients for fish diets, and the influence of supplemented aquafeeds on growth performance has been studied for some fish and seaweed species. In the present work, the potential immunomodulation exerted by U. ohnoi (5%) as dietary ingredient was investigated in Senegalese sole. After feeding with the experimental diets for 90 d, fish immune response before and after challenge with Photobacterium damselae subsp. piscicida (Phdp) was assessed. In absence of infection, systemic immune response was not modified by 5% U. ohnoi dietary inclusion for 90 d. Thus, no differences in liver and head kidney immune gene transcription or serum lysozyme, peroxidase, antiprotease and complement activities were observed based on the diet received by Senegalese sole specimens. Regarding mucosal immune parameters, no changes in gene transcription were detected in the skin and gills, whilst only tnf, cd4 and cd8 were significantly up-regulated in the intestine of fish fed with U. ohnoi, compared to the values obtained with control diet. On the contrary, when S. senegalensis specimens were challenged with Phdp, modulation of the immune response consisting in increased transcription of genes encoding complement (c1q4, c3, c9), lysozyme g (lysg), tumor necrosis factor alpha (tnfα) as well as those involved in the antioxidant response (gpx, sodmn) and iron metabolism (ferrm, hamp-1) was observed in the liver of fish fed with U. ohnoi. In parallel, decreased inflammatory cytokine and complement encoding gene transcription was displayed by the spleen of fish receiving the algal diet. Though mortality rates due to Phdp challenge were not affected by the diet received, lower pathogen loads were detected in the liver of soles receiving U. ohnoi diet. Further research to investigate the effects of higher inclusion levels of this seaweed in fish diets, feeding during short periods as wells as to assess the response against other pathogens needs to be carried out.

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.

Role of AIP56 disulphide bond and its reduction by cytosolic redox systems for efficient intoxication.

Apoptosis-inducing protein of 56 kDa (AIP56) is a major virulence factor of Photobacterium damselae subsp. piscicida, a gram-negative pathogen that infects warm water fish species worldwide and causes serious economic losses in aquacultures. AIP56 is a single-chain AB toxin composed by two domains connected by an unstructured linker peptide flanked by two cysteine residues that form a disulphide bond. The A domain comprises a zinc-metalloprotease moiety that cleaves the NF-kB p65, and the B domain is involved in binding and internalisation of the toxin into susceptible cells. Previous experiments suggested that disruption of AIP56 disulphide bond partially compromised toxicity, but conclusive evidences supporting the importance of that bond in intoxication were lacking. Here, we show that although the disulphide bond of AIP56 is dispensable for receptor recognition, endocytosis, and membrane interaction, it needs to be intact for efficient translocation of the toxin into the cytosol. We also show that the host cell thioredoxin reductase-thioredoxin system is involved in AIP56 intoxication by reducing the disulphide bond of the toxin at the cytosol. The present study contributes to a better understanding of the molecular mechanisms operating during AIP56 intoxication and reveals common features shared with other AB toxins.

Involvement of Hsp90 and cyclophilins in intoxication by AIP56, a metalloprotease toxin from Photobacterium damselae subsp. piscicida.

AIP56 (apoptosis inducing protein of 56 kDa) is a key virulence factor secreted by virulent strains of Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes septicemic infections in several warm water marine fish species. AIP56 is systemically disseminated during infection and induces massive apoptosis of host macrophages and neutrophils, playing a decisive role in the disease outcome. AIP56 is a single-chain AB-type toxin, being composed by a metalloprotease A domain located at the N-terminal region connected to a C-terminal B domain, required for internalization of the toxin into susceptible cells. After binding to a still unidentified surface receptor, AIP56 is internalised through clathrin-mediated endocytosis, reaches early endosomes and translocates into the cytosol through a mechanism requiring endosomal acidification and involving low pH-induced unfolding of the toxin. At the cytosol, the catalytic domain of AIP56 cleaves NF-κB p65, leading to the apoptotic death of the intoxicated cells. It has been reported that host cytosolic factors, including host cell chaperones such as heat shock protein 90 (Hsp90) and peptidyl-prolyl cis/trans isomerases (PPIases), namely cyclophilin A/D (Cyp) and FK506-binding proteins (FKBP) are involved in the uptake of several bacterial AB toxins with ADP-ribosylating activity, but are dispensable for the uptake of other AB toxins with different enzymatic activities, such as Bacillus anthracis lethal toxin (a metalloprotease) or the large glycosylating toxins A and B of Clostridium difficile. Based on these findings, it has been proposed that the requirement for Hsp90/PPIases is a common and specific characteristic of ADP-ribosylating toxins. In the present work, we demonstrate that Hsp90 and the PPIases cyclophilin A/D are required for efficient intoxication by the metalloprotease toxin AIP56. We further show that those host cell factors interact with AIP56 in vitro and that the interactions increase when AIP56 is unfolded. The interaction with Hsp90 was also demonstrated in intact cells, at 30 min post-treatment with AIP56, suggesting that it occurs during or shortly after translocation of the toxin from endosomes into the cytosol. Based on these findings, we propose that the participation of Hsp90 and Cyp in bacterial toxin entry may be more disseminated than initially expected, and may include toxins with different catalytic activities.

Genetics of resistance to photobacteriosis in gilthead sea bream (Sparus aurata) using 2b-RAD sequencing.

BACKGROUND: Photobacteriosis is an infectious disease developed by a Gram-negative bacterium Photobacterium damselae subsp. piscicida (Phdp), which may cause high mortalities (90-100%) in sea bream. Selection and breeding for resistance against infectious diseases is a highly valuable tool to help prevent or diminish disease outbreaks, and currently available advanced selection methods with the application of genomic information could improve the response to selection. An experimental group of sea bream juveniles was derived from a Ferme Marine de Douhet (FMD, Oléron Island, France) selected line using ~ 109 parents (~ 25 females and 84 males). This group of 1187 individuals represented 177 full-sib families with 1-49 sibs per family, which were challenged with virulent Phdp for a duration of 18 days, and mortalities were recorded within this duration. Tissue samples were collected from the parents and the recorded offspring for DNA extraction, library preparation using 2b-RAD and genotyping by sequencing. Genotypic data was used to develop a linkage map, genome wide association analysis and for the estimation of breeding values. RESULTS: The analysis of genetic variation for resistance against Phdp revealed moderate genomic heritability with estimates of ~ 0.32. A genome-wide association analysis revealed a quantitative trait locus (QTL) including 11 SNPs at linkage group 17 presenting significant association to the trait with p-value crossing genome-wide Bonferroni corrected threshold P ≤ 2.22e-06. The proportion total genetic variance explained by the single top most significant SNP was ranging from 13.28-16.14% depending on the method used to compute the variance. The accuracies of predicting breeding values obtained using genomic vs. pedigree information displayed 19-24% increase when using genomic information. CONCLUSION: The current study demonstrates that SNPs-based genotyping of a sea bream population with 2b-RAD approach is effective at capturing the genetic variation for resistance against Phdp. Prediction accuracies obtained using genomic information were significantly higher than the accuracies obtained using pedigree information which highlights the importance and potential of genomic selection in commercial breeding programs.

Molecular and phenotypic characterization of Photobacterium damselae among some marine fishes in Lake Temsah.

Photobacterium damselae species are one of the most devastating bacterial pathogens in mariculture worldwide. Some species of Photobacterium are pathogenic for marine animals and human. They are the causative agents of photobacteriosis, formerly known as pasteurellosis. A total of (202) marine fishes of three different species were represented as: seabass (Dicentrarchus labrax), seabream (Sparus aurata) and gray mullet (Mugil capitus) randomly collected from Lake Temsah at Ismailia governorate along the parallel Pelagic road to the lake in the governorate from August 2015 to July 2016. The clinical picture and gross lesions of the diseased fishes were recorded. Isolation and identification of suspected bacteria using traditional and molecular methods. Samples from affected organs were collected for studying the histopathological alterations of these pathogens. Fifty one fishes were found to be infected with Photobacterium damselae subsp. Piscicida. Seabass (Dicentrarchus labrax) was the most infected fish species (23), followed by seabream (Sparus aurata) (18) finally gray mullet (Mugil capitus) was (10). 91fishes were found to be infected with P. damselae subsp. damselae, seabass (Dicentrarchus labrax) was the most infected fish sp. (36), followed by seabream (Sparus aurata) (32), then gray mullet (Mugil capitus) (23). The results indicated that, the total prevalence of P. damselae subsp. piscicida in all examined species (25.24%), the highest seasonal prevalence was recorded in summer season (37.09%) followed by autumn (26%) then spring (20.37%) and winter (11.11%). On the other hand, the total prevalence of P. damselae subsp. damselae in all examined species (45.04%), the highest seasonal prevalence was recorded in summer season (67.74%) followed by autumn (52%) then spring (29.62%) and winter (19.44%). Molecular diagnosis with conventional PCR used to confirm the traditional isolation was applied by using specific primers of two genes (polycapsular saccharide gene and urease C gene). The histopathological studies of naturally infected marine fishes showed severe inflammatory reactions in different organs with accumulation of melanomacrophages and necrosis. The results confirm that P. damselae subspecies damsalea is the most prevalent pathogen between marine fishes, and seabass (Dicentrarchus labrax) was the highly affected marine fishes in this study.

Transcriptome changes in response to temperature in the fish pathogen Photobacterium damselae subsp. damselae: Clues to understand the emergence of disease outbreaks at increased seawater temperatures.

The marine bacterium Photobacterium damselae subsp. damselae (Pdd) is a generalist and facultative pathogen that causes disease in a wide range of marine animals including fish species of importance in aquaculture. Disease outbreaks in fish farms have been correlated with an increased water temperature during summer months. In this study, we have used RNA sequencing to analyze the transcriptome of Pdd RM-71 cultured at two different temperatures, which simulated temperature conditions experienced during free swimming lifestyle at mid latitudes in winter months (15°C) and during outbreaks in aquaculture in warm summer months (25°C). The enhanced bacterial growth of Pdd observed at 25°C in comparison to 15°C suggests that an elevated seawater temperature contributes to the build-up of a sufficient bacterial population to cause disease. In comparison to growth at 15°C, growth at 25°C resulted in the upregulation of genes involved in DNA synthesis, nutrient uptake, chemotaxis, flagellar motility, secretion systems and antimicrobial resistance. Plasmid-encoded virulence factors, which include a putative adhesin/invasin OmpU, a transferrin receptor and a serum resistance protein, were also upregulated. Transcription factor RpoS, genes involved in cold shock response, modulation of cell envelope and amino acid metabolism, as well as genes of yet unknown function were downregulated at 25°C. Notably, the gene encoding damselysin cytotoxin (Dly) was among the most highly transcribed genes at the two assayed temperatures, at levels comparable to the most highly expressed housekeeping genes. This study contributes to our understanding of the regulatory networks and biology of a generalist marine bacterial pathogen, and provides evidence that temperature regulates multiple physiological and virulence-related functions in Pdd.

Comparative pathogenicity of Vibrio spp., Photobacterium damselae ssp. damselae and five isolates of Aeromonas salmonicida ssp. achromogenes in juvenile Atlantic halibut (Hippoglossus hippoglossus).

Juvenile Atlantic halibut (~100 mg, Hippoglossus hippoglossus) were exposed to Vibrio proteolyticus, a Vibrio spp. isolate, Photobacterium damselae ssp. damselae and five different isolates of Aeromonas salmonicida ssp. achromogenes via an hour-long bath immersion to ascertain their variation in pathogenicity to this fish species. Results were analysed using Kaplan-Meier survival analysis. Analysis of the data from challenges using A. salmonicida ssp. achromogenes revealed three survival values of zero and a spread of values from 0 to 28.43. Challenges using a Vibrio spp isolate, V. proteolyticus and P. damselae resulted in Kaplan-Meier survival estimates of 31.21, 50.41 and 57.21, respectively. As all bacterial species tested could induce juvenile halibut mortalities, they must all be considered as potential pathogens. However, the degree of pathogenicity of A. salmonicida is isolate dependent.

Secreted Citrate Serves as Iron Carrier for the Marine Pathogen Photobacterium damselae subsp damselae.

Photobacterium damselae subsp damselae (Pdd) is a Vibrionaceae that has a wide pathogenic potential against many marine animals and also against humans. Some strains of this bacterium acquire iron through the siderophore vibrioferrin. However, there are virulent strains that do not produce vibrioferrin, but they still give a strong positive reaction in the CAS test for siderophore production. In an in silico search on the genome sequences of this type of strains we could not find any ORF which could be related to a siderophore system. To identify genes that could encode a siderophore-mediated iron acquisition system we used a mini-Tn10 transposon random mutagenesis approach. From more than 1,400 mutants examined, we could isolate a mutant (BP53) that showed a strong CAS reaction independently of the iron levels of the medium. In this mutant the transposon was inserted into the idh gene, which encodes an isocitrate dehydrogenase that participates in the tricarboxylic acid cycle. The mutant did not show any growth impairment in rich or minimal media, but it accumulated a noticeable amount of citrate (around 7 mM) in the culture medium, irrespective of the iron levels. The parental strain accumulated citrate, but in an iron-regulated fashion, being citrate levels 5-6 times higher under iron restricted conditions. In addition, a null mutant deficient in citrate synthase showed an impairment for growth at high concentrations of iron chelators, and showed almost no reaction in the CAS test. Chemical analysis by liquid chromatography of the iron-restricted culture supernatants resulted in a CAS-positive fraction with biological activity as siderophore. HPLC purification of that fraction yielded a pure compound which was identified as citrate from its MS and NMR spectral data. Although the production of another citrate-based compound with siderophore activity cannot be ruled out, our results suggest that Pdd secretes endogenous citrate and use it for iron scavenging from the cell environment.

Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis.

The genus Photobacterium, one of the eight genera included in the family Vibrionaceae, contains 27 species with valid names and it has received attention because of the bioluminescence and pathogenesis mechanisms that some of its species exhibit. However, the taxonomy and phylogeny of this genus are not completely elucidated; for example, P. logei and P. fischeri are now considered members of the genus Aliivibrio, and previously were included in the genus Vibrio. In addition, P. damselae subsp. piscicida was formed as a new combination for former Vibrio damsela and Pasteurella piscicida. Moreover, P. damselae subsp. damselae is an earlier heterotypic synonym of P. histaminum. To avoid these incovenences draft and complete genomic sequences of members of Photobacterium are increasingly becoming available and their use is now routine for many research laboratories to address diverse goals: species delineation with overall genomic indexes, phylogenetic analyses, comparative genomics, and phenotypic inference. The habitats and isolation source of the Photobacterium species include seawater, sea sediments, saline lake waters, and a variety of marine organisms with which the photobacteria establish different relationships, from symbiosis to pathogenic interactions. Several species of this genus contain bioluminescent strains in symbiosis with marine fish and cephalopods; in addition, other species enhance its growth at pressures above 1 atmosphere, by means of several high-pressure adaptation mechanisms and for this, they may be considered as piezophilic (former barophilic) bacteria. Until now, only P. jeanii, P. rosenbergii, P. sanctipauli, and the two subspecies of P. damselae have been reported as responsible agents of several pathologies on animal hosts, such as corals, sponges, fish and homeothermic animals. In this review we have revised and updated the taxonomy, ecology and pathogenicity of several members of this genus. [Int Microbiol 20(1): 1-10 (2017)].

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish.

Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacterized chromosomal loci encoding a phospholipase (PlpV) and a collagenase (ColP). PlpV was ubiquitous in the subspecies and exerted hemolytic activity against fish erythrocytes, which was enhanced in the presence of lecithin. ColP was restricted to a fraction of the isolates and was responsible for the collagen-degrading activity in this subspecies. Consistent with the presence of signal peptides in PlpV and ColP sequences, mutants for the type II secretion system (T2SS) genes epsL and pilD exhibited impairments in phospholipase and collagenase activities. Sea bass virulence experiments and cell culture assays demonstrated major contributions of PhlyC and PlpV to virulence and toxicity.IMPORTANCE This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, Photobacterium damselae subsp. damselae To date, studies on the genetic basis of virulence were restricted to the pPHDD1 plasmid-encoded toxins Dly and PhlyP. However, the vast majority of the recent isolates of this pathogen from fish farm outbreaks lack this plasmid. Here we demonstrate that the plasmidless strains produce two hitherto uncharacterized ubiquitous toxins encoded in chromosome I, namely, the hemolysin PhlyC and the phospholipase PlpV. We report the main roles of these two toxins in fish virulence and in cell toxicity. Our results constitute the basis for a better understanding of the virulence of a widespread marine pathogen.

Two routes of infection with Photobacterium damselae subsp. piscicida are effective in the modulation of the transcription of immune related genes in Solea senegalensis.

The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important disease outbreaks affecting cultured fish species including the flatfish Solea senegalensis. In the present work, transcription of iron metabolism related genes (TF, FERR-M, HP-1 and HAMP-1) as well as innate immune system components such as complement proteins (C3 and C7), lysozyme (LYS-G), TNF family (TNFα, TRAF-3), NCCRP-1 and heat shock protein encoding genes (HSP70, HSP90AA, HSP90AB and GP96) has been determined in the liver and kidney of S. senegalensis specimens after Phdp infection. Intraperitoneal injection (IP) and immersion (IM) routes have been used for infection. Fish developed specific antibodies in both cases, higher levels being detected in IP infected specimens. Both infection routes resulted in increased relative transcript levels of FERR-M, HP-1 and HAMP-1 genes and TF decreased relative transcription, conducting to lower iron availability for the pathogen. This response can be considered as a strategy to limit iron availability for Phdp, a pathogen capable to obtain iron from transferrin. Relative transcription of genes encoding lysozyme and complement factors C3 and C7 were also increased regardless the infection route; the liver was the main organ involved in the initial stages and the kidney in later stages of the infection. TNFα and TRAF-3 relative gene transcription increased 24h post-infection. TRAF-3 gene induction was detected 30 d post-infection, whilst no changes in TNFα were observed 72h or 30 d post-infection. NCCRP-1 changes were observed after IP infection in the liver and kidney; however, IM infection resulted only in slight changes in the kidney of infected fish. This different response observed maybe related to a lower number of invaded cells by the pathogen. Finally, changes in HSP90AB and GP96 have been detected after infection by both routes. Different late modulation has been observed in assayed genes depending on the route of infection. Thus, only LYS-G, TF, NCCRP-1, GP96 and HSP90AB gene transcription was modulated 30 d post-infection in the kidney of IM infected specimens; however, IP infected fish showed modulation in a higher number of genes both in liver and kidney tissues. The implications of these responses in resistance to infection by Phdp need to be elucidated.

Pathogenesis of Photobacterium damselae subspecies infections in sea bass and sea bream.

Photobacterium damselae is a Gram negative bacterium causes photobacteriosis, a worldwide septicemic disease in aquaculture including sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). The pathogenicity of bacterial subspecies and the disease pathological changes in natural and experimental infections have thus far yielded inconsistency of effective preventive measures. This study aimed to represent a comprehensive analysis of the potential pathogenic capacities of the two subspecies of P. damselae in cultured sea bass and sea bream in the Northwestern region of Egypt. Diseased 321 sea bass and 257 sea bream, in addition to 99 healthy sea bass fingerlings were sampled from three farms located along the Mediterranean Sea. P. damselae subspecies were isolated from diseased fish and characterized using bacteriological, molecular, and antimicrobial susceptibility methods. Healthy fish were challenged by a virulent P. damselae subsp. piscicida, monitored for disease signs and mortality, and the histopathological abnormalities and hematological disorders were carried out. Clinical signs and gross lesions in naturally infected sea bass and sea bream showed great similarities with absence of a subspecies-specific characteristic sign or lesion. The two subspecies were recovered through the entire year from individual fish sample, suggests a coexistence of two subspecies endemic infection. In diseased sea bass, 38.32% and 16.20% were positive for P. damselae subsp. piscicida and subsp. damselae, respectively. However in diseased sea bream, 44.47% and 26.46% were positive for P. damselae subsp. piscicida and subsp. damselae, respectively. High mortalities and devastating clinicopathologic abnormalities represented by sever clinical signs, hematological disorders and histological abnormalities strengthen the pathogenicity of P. damselae subspecies in the two fish species and therefore, a vaccination strategy against both subspecies should be taken into account.

First description of 'Chalky back' phenomenon in banana prawns (Fenneropenaeus merguiensis) and its possible association with Vibrio and Photobacterium species.

Here we report a newly identified 'Chalky back' phenomenon in banana prawns (Fenneropenaeus merguiensis) farmed in North Queensland, Australia. This was characterized by localized white discoloured segmentation of the cervical groove, moreover, after cooking the prawns exploded, making them unfit for commercial sale. Histological examination revealed breakdown of gut and abdominal muscle tissue in some moribund specimens. We selectively isolated Vibrio spp., which are known prawn pathogens, from healthy and Chalky back specimens. Isolated bacteria were identified, typed and tested for the presence of eight virulence genes (VGs), biofilm formation, adherence and cytotoxicity to fish cells. In all, 32 isolates were recovered and identified as Vibrio harveyi, V. owensii, V. sinaloensis-like, V. campbellii, V. shilonii, Vibrio sp. and Photobacterium damselae using 16S rRNA gene sequencing. All V. harveyi carried VGs coding for haemolysin, toxR and flagella; formed biofilm; and adhered to both cell lines. This was similar to the V. sinaloensis-like strains that were only isolated from Chalky back specimens. Our data suggest that Vibrio spp. may play a role in the pathogenesis of Chalky back. This study is the first report of Chalky back phenomenon in farmed banana prawns that needs to be closely monitored by the industry.

A Transmissible Plasmid-Borne Pathogenicity Island Confers Piscibactin Biosynthesis in the Fish Pathogen Photobacterium damselae subsp. piscicida.

The fish pathogen Photobacterium damselae subsp. piscicida produces the siderophore piscibactin. A gene cluster that resembles the Yersinia high-pathogenicity island (HPI) encodes piscibactin biosynthesis. Here, we report that this HPI-like cluster is part of a hitherto-uncharacterized 68-kb plasmid dubbed pPHDP70. This plasmid lacks homologs of genes that mediate conjugation, but we found that it could be transferred at low frequencies from P. damselae subsp. piscicida to a mollusk pathogenic Vibrio alginolyticus strain and to other Gram-negative bacteria, likely dependent on the conjugative functions of the coresident plasmid pPHDP60. Following its conjugative transfer, pPHDP70 restored the capacity of a vibrioferrin mutant of V. alginolyticus to grow under low-iron conditions, and piscibactin became detectable in its supernatant. Thus, pPHDP70 appears to harbor all the genes required for piscibactin biosynthesis and transport. P. damselae subsp. piscicida strains cured of pPHDP70 no longer produced piscibactin, had impaired growth under iron-limited conditions, and exhibited markedly decreased virulence in fish. Collectively, our findings highlight the importance of pPHDP70, with its capacity for piscibactin-mediated iron acquisition, in the virulence of P. damselae subsp. piscicida. Horizontal transmission of this plasmid-borne piscibactin synthesis gene cluster in the marine environment may facilitate the emergence of new pathogens.

Novel host-specific iron acquisition system in the zoonotic pathogen Vibrio vulnificus.

Vibrio vulnificus is a marine bacterium associated with human and fish (mainly farmed eels) diseases globally known as vibriosis. The ability to infect and overcome eel innate immunity relies on a virulence plasmid (pVvbt2) specific for biotype 2 (Bt2) strains. In the present study, we demonstrated that pVvbt2 encodes a host-specific iron acquisition system that depends on an outer membrane receptor for eel transferrin called Vep20. The inactivation of vep20 did not affect either bacterial growth in human plasma or virulence for mice, while bacterial growth in eel blood/plasma was abolished and virulence for eels was significantly impaired. Furthermore, vep20 is an iron-regulated gene overexpressed in eel blood during artificially induced vibriosis both in vitro and in vivo. Interestingly, homologues to vep20 were identified in the transferable plasmids of two fish pathogen species of broad-host range, Vibrio harveyi (pVh1) and Photobacterium damselae subsp. damselae (pPHDD1). These data suggest that Vep20 belongs to a new family of plasmid-encoded fish-specific transferrin receptors, and the acquisition of these plasmids through horizontal gene transfer is likely positively selected in the fish-farming environment. Moreover, we propose Ftbp (fish transferrin binding proteins) as a formal name for this family of proteins.

Photobacterium damselae subsp. damselae major virulence factors Dly, plasmid-encoded HlyA, and chromosome-encoded HlyA are secreted via the type II secretion system.

Photobacterium damselae subsp. damselae is a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyApl) and the chromosome-encoded HlyA (HlyAch) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn10 transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion in epsL, a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement of epsL in HlyAch secretion. In addition, mutation of epsL in a pPHDD1-harboring strain caused an almost complete abolition of hemolytic activity against sheep erythrocytes, indicating that epsL plays a major role in secretion of the plasmid-encoded HlyApl and Dly. This was further demonstrated by analysis of different combinations of hemolysin gene mutants and by strain-strain complementation assays. We also found that mutation of the putative prepilin peptidase gene pilD severely affected hemolysis, which dropped at levels inferior to those of epsL mutants. Promoter expression analyses suggested that impairment of hemolysin secretion in epsL and pilD mutants might constitute a signal that affects hemolysin and T2SS gene expression at the transcriptional level. In addition, single epsL and pilD mutations caused a drastic decrease in virulence for mice, demonstrating a major role of T2SS and pilD in P. damselae subsp. damselae virulence.