Photobacterium Damselae Subsp Research Articles

Identification and characterization of toll-like receptor genes in silver pomfret (Pampus argenteus) and their involvement in the host immune response to Photobacterium damselae subsp. Damselae and Nocardia seriolae infection

Toll-like receptors (TLRs) are vital pattern recognition receptors that play a critical role in the innate immune response against pathogenic attack. Among the bacteria commonly found in the culture process of silver pomfret, Photobacterium damselae subsp. Damselae (PDD, gram-negative) and Nocardia seriolae (NS, gram-positive), can cause large-scale mortality in this fish species. However, there is currently no research on the role of TLRs in mediating the immune response of silver pomfret to these two bacterial infections. Therefore, in this study, we identified nine PaTLRs family members, including several fish-specific TLRs (TLR14 and TLR21). Phylogenetic analysis revealed that these PaTLRs genes could be classified into five subfamilies, namely TLR1, TLR3, TLR5, TLR7, and TLR11, indicating their evolutionary conservation. To further explore the interactions of TLR genes with immune-related mediators, protein and protein interaction network (PPI) results were generated to explain the association of TLR genes with TNF receptor-associated factor 6 (TRAF6) and other relevant genes in the MyD88-dependent pathway and NF-κb signaling pathway. Subsequently, RT-qPCR was conducted to verify the expression patterns of the nine TLR genes in the gills, skin, kidney, liver, and spleen of healthy fish, with most of the TLRs showing high expression levels in the spleen. Following infection with PDD and NS, these PaTLRs exhibited different expression patterns in the spleen, with PaTLR2, PaTLR3, PaTLR5, PaTLR7, PaTLR9, and PaTLR14 being significantly up-regulated. Furthermore, when spleen cells were treated with bacterial compositions, the majority of PaTLRs expression was up-regulated in response to Lipopolysaccharide (LPS) and lipophosphorylcholic acid (LTA) treatment, except for PaTLR21. Finally, changes in the expression levels of TLR-interacting genes were also observed under the stimulation of bacteria and bacterial compositions. The results of this study provide a preliminary reference for further understanding the mechanism of the innate immune response of the TLR gene family in silver pomfret and offer theoretical support for addressing the disease problems encountered during large-scale fish breeding.

Antibiotic resistance of emerging pathogenic bacteria of hybrid grouper farming in Indonesia

Abstract. Istiqomah I, Isnansetyo A, Murwantoko, Handayani DP, Lestari YN, Taslihan A, Permana IGN, Wijayanti E. 2023. Antibiotic resistance of emerging pathogenic bacteria of hybrid grouper farming in Indonesia. Biodiversitas 24: 2493-2501. A hybrid grouper is among the main coastal farming species in Asian countries, yet vibriosis is still a significant issue. Therefore, a knowledge of pathogenic strains responsible for recent cases is essential for developing appropriate control measures. The purpose of the study was to describe emerging bacterial pathogens associated with hemorrhagic disorder, skin wounds, and mortality in cantang hybrid grouper farms in Indonesia. A total of 30 bacterial strains were recovered from the kidney of diseased grouper on TCBS agar. Based on the ability to induce clinical signs and mortality of grouper juveniles owing to intraperitoneal bacterial infection at 106 CFU/fish, the bacteria were divided into three groups: pathogenic (n=11), low-pathogenic (n=2), and non-pathogenic (n=17). The pathogenic strains were putatively identified as Photobacterium damselae subsp. Damselae (n=8), Vibrio alginolyticus (n=1), Vibrio harveyi (n=2), and Vibrio azureus (n=2) based on the biochemical characteristics and 16S rRNA and DNA gyrase B subunit gene sequences. The pathogenic bacteria were sensitive to erythromycin, gentamycin, oxytetracycline, and kanamycin. However, it still has various resistant patterns to ampicillin, fosfomycin, chloramphenicol, streptomycin, rifampicin, and enrofloxacin. This study demonstrated the emergence of pathogenic P. damselae subsp. damselae, Vibrio alginolyticus, V. harveyi, and V. azureus in grouper aquaculture in Indonesia with multiple antibiotic resistance, which needed to further studies.

The Evolution of a Specialized, Highly Virulent Fish Pathogen through Gene Loss and Acquisition of Host-Specific Survival Mechanisms.

ABSTRACTPhotobacterium damselae comprises two subspecies, P. damselae subsp. damselae and P. damselae subsp. piscicida, that contrast remarkably despite their taxonomic relationship. The former is opportunistic and free-living but can cause disease in compromised individuals from a broad diversity of taxa, while the latter is a highly specialized, primary fish pathogen. Here, we employ new closed curated genome assemblies from Australia to estimate the global phylogenetic structure of the species P. damselae. We identify genes responsible for the shift from an opportunist to a host-adapted fish pathogen, potentially via an arthropod vector as fish-to-fish transmission was not achieved in repeated cohabitation challenges despite high virulence for Seriola lalandi. Acquisition of ShdA adhesin and of thiol peroxidase may have allowed the environmental, generalist ancestor to colonize zooplankton and to occasionally enter in fish host sentinel cells. As dependence on the host has increased, P. damselae has lost nonessential genes, such as those related to nitrite and sulfite reduction, urea degradation, a type 6 secretion system (T6SS) and several toxin-antitoxin (TA) systems. Similar to the evolution of Yersinia pestis, the loss of urease may be the crucial event that allowed the pathogen to stably colonize zooplankton vectors. Acquisition of host-specific genes, such as those required to form a sialic acid capsule, was likely necessary for the emergent P. damselae subsp. piscicida to become a highly specialized, facultative intracellular fish pathogen. Processes that have shaped P. damselae subsp. piscicida from subsp. damselae are similar to those underlying evolution of Yersinia pestis from Y. pseudotuberculosis.IMPORTANCE Photobacterium damselae subsp. damselae is a ubiquitous marine bacterium and opportunistic pathogen of compromised hosts of diverse taxa. In contrast, its sister subspecies P. damselae subsp. piscicida (Pdp) is highly virulent in fish. Pdp has evolved from a single subclade of Pdd through gene loss and acquisition. We show that fish-to-fish transmission does not occur in repeated infection models in the primary host, Seriola lalandi, and present genomic evidence for vector-borne transmission, potentially via zooplankton. The broad genomic changes from generalist Pdd to specialist Pdp parallel those of the environmental opportunist Yersinia pseudotuberculosis to vector-borne plague bacterium Y. pestis and demonstrate that evolutionary processes in bacterial pathogens are universal between the terrestrial and marine biosphere.

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 1β (IL1-β), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2).

Molecular techniques for the detection of bacterial zoonotic pathogens in fish and humans

Various diseases could be transmitted from aquatic species, especially fish, to humans, which is considered to be a major human health concern across the world. In recent decades, molecular techniques have been applied to examine and identify various bacterial species in fish farms. Molecular techniques are rapid, accurate, sensitive, cost-effective, and have the ability to identify specific pathogens without the need for conventional methods. Furthermore, molecular techniques are particularly useful for the detection of multiple species or in case of low template. This review study aimed to describe various molecular methods, including multiplex-polymerase chain reaction, high-resolution melting real-time, restriction fragment length polymorphism, random amplification of polymorphic DNA, nucleic acid sequence-based amplification, rolling circle amplification, fluorescence in-situ hybridization, microarray, and matrix-assisted laser desorption/ionization. In addition, we evaluated the biosensors used for the detection of zoonotic bacteria, such as Vibrio vulnificus, Vibrio cholerae, Listeria monocytogenes, Streptococcus iniae, Lactococcus garvieae, Aeromonas hydrophila, Edwardsiella tarda, Mycobacterium spp., Photobacterium damselae subsp. Damselae, and Pseudomonas fluorescens, which are transmitted from fish to humans. These bacteria are of great importance in the aquaculture industry and in terms of human health.

Photobacterium damselae subsp. damselae, a generalist pathogen with unique virulence factors and high genetic diversity.

Photobacterium damselae subsp. damselae causes vibriosis in a variety of marine animals, including fish species of importance in aquaculture. It also may cause wound infections in humans that can progress into a fatal outcome. Two major virulence factors are encoded within the large conjugative plasmid pPHDD1: the phospholipase-D damselysin (Dly) and the pore-forming toxin Phobalysin P (PhlyP). The two toxins exert hemolytic and cytolytic activity in a synergistic manner. Albeit PhlyP has close homologues in many Vibrio species, it has unique features that differentiate it from related toxins. Dly phospholipase constitutes a singular trait of P. damselae subsp. damselae among the Vibrionaceae, although related toxins are found in members of the Aeromonadaceae Fish farm outbreaks can also be caused by plasmidless strains. Such observation led to the characterization of two ubiquitous, chromosome-encoded toxins with lesser cytolytic activity: the pore forming-toxin Phobalysin C (PhlyC) and the phospholipase-hemolysin PlpV. Special attention deserves the high genetic diversity of this pathogen, with a number of strain-specific features including the cell envelope polysaccharide synthesis clusters. Fish outbreaks are likely caused by multiclonal populations which contain both plasmidless and pPHDD1-harbouring isolates, and not by well-adapted clonal complexes. Still, among such a genetic heterogeneity, it is feasible to identify conserved weak points in the biology of this bacterium: the two-component regulatory system RstAB (CarSR) was found to be necessary for maximal production of virulence factors and its inactivation severely impaired virulence.

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.

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.

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.

Effect of Different Culture Conditions on the (Twitching) Displacement of Photobacterium Damselae subsp. Piscicida

Effect of Different Culture Conditions on the (Twitching) Displacement of Photobacterium Damselae subsp. Piscicida

Occurrence of Photobacterium damselae Subsp. Piscicida in Sea-Cage Farmed Meagre (Argyrosomus regius) in Tenerife, Canary Islands, Spain

During 2008, the bacterium Photobacterium damselae subsp. piscicida was identified in meagre (Argyrosomus regius) culture during three disease outbreaks which occurred in Tenerife, Canary Islands, with mortalities reaching 50%. Typical disease signs were observed, including the characteristic spleen nodules, and bacterial identity was confirmed by biochemical and serological tests. This is the first reported isolation of Photobacterium damselae subspecies piscicida associated with mortalities in meagre.

Effects of dietary Gracilaria sp. and Alaria sp. supplementation on growth performance, metabolic rates and health in meagre (Argyrosomus regius) subjected to pathogen infection

Effects of dietary seaweed supplementation on basal physiology and health biomarkers were assessed in meagre (Argyrosomus regius) subjected to bacterial infection, using Photobacterium damselae subsp. Piscicida (Phdp) as the etiologic agent. Three test diets were prepared by supplementing a basal control formulation (44 % protein, 16 % lipid, 22 kJ g−1 energy) with 0 % seaweed (control), 5 % Gracilaria sp. or 5 % Alaria sp. During the growth trial, 180 fish (39.70 ± 0.33 g) were daily fed for 69 days with the experimental diets. After the growth trial, 60 fish from each dietary treatment were divided into two groups, infected and non-infected. The infected group was injected intraperitoneally with a saline solution (HBSS) with 2.91 x 103 CFU Phdp g−1 fish, whereas the non-infected group was injected with HBSS without Phdp. Dietary seaweed supplementation did not affect fish growth performance. Standard and routine metabolic rates, and aerobic metabolic scope did not vary significantly among dietary treatments. Conversely, maximum metabolic rate was significantly higher in fish fed Alaria sp. diet when compared to control group. Non-infected fish had higher hematocrit levels than the infected group, regardless of diet. Lactate levels were significantly higher in fish fed Alaria sp. diet when compared to control, with no interaction between diet and infection. Lipid peroxidation was significantly higher in fish fed control diet than supplemented diets. Infected groups had lower antioxidant enzymes activities when compared to non-infected. An interaction between infection and diet was found for glutathione peroxidase and reduced glutathione activities. The current study suggests that dietary seaweed supplementation modulates metabolic rates and biomarker responses in meagre, which may confer advantages in coping with biotic stressors.

Immunization of sea bream (Sparus aurata) juveniles against Photobacterium damselae subsp. piscicida by short bath: Effect on some pro-inflammatory molecules and the Mx gene expression.

Cytokines are a family of proteins derived from macrophages, lymphocytes, granulocytes, mast cells and epithelial cells and can be divided into interferons (IFNs), Interleukins (ILs) and Tumor Necrosis factors (TNFs) among others. The presence of cytokines in a wide number of fish species has been proved and several molecules types have been already cloned and sequenced. In this work some proinflamatory molecules and Mx gene were detected in the liver of vaccinated sea bream juveniles with an average body weight of 5 g. The method of immunization was by short bath and three different bacterins against the marine pathogen Photobacterium damselae subsp. piscicida were designed and used to immunize fish. Five genes encoding for five different molecules were analyzed by real time PCR: IL-1β, IL Ir-2, Cox-2, Mx and TNFα. Gene expression was quantified along four days after fish immunization and results were compared among groups. Results show that the heat-inactivated vaccine stimulates the up-regulation of IL-1β, IL Ir-2, Cox-2 and TNFα genes whereas the UV-light inactivated vaccine was the unique vaccine which stimulates the expression of Mx gene. The present is a novel study that shows by the first time the effect of the inactivation process of vaccines on the expression levels of genes involved in the defense against Photobacterium damselae subsp piscicida.

A Study on Probiotic Bacteria Isolated from Common Dentex (Dentex dentex) Larvae and their antagonistic effect on Photobacterium damselae subsp. Damselae

Bacterial species that can be considered as probiotic are gradually gaining attention and contributing to the success of aquaculture. The aim of this study was to determine the indigenous dominant probiotic bacterial species of common dentex (Dentex dentex) larvae and their effectivness against the putative fish pathogen Photobacterium damselae subsp. damselae at different feeding stages. In 2009-2010 samples of larvae and tank water were collected at 5 different occasions. After inoculations of samples, bacterial isolates were identified with standard biochemical methods. The antagonistic effects of the identified probiotic bacteria against P. damselae subsp. damselae isolated from different mariculture fishes were determined by using disc diffusion method. Bacillus cereus, B. macquariensis, and other Bacillus sp., Micrococcus luteus, Flavobacterium flevense and Flavobacterium sp. were identified as candidate probiotic species and their antagonistic effect against different P. damselae isolates was identified. The results of this study showed that B. cereus and M. luteus are more effective against Photobacterium damselae subsp. damselae. As a result, the identification of the candidate probiotic species and the widespread use of probiotics may improve the success of common dentex culture in the future.

Dexamethasone modulates expression of genes involved in the innate immune system, growth and stress and increases susceptibility to bacterial disease in Senegalese sole (Solea senegalensis Kaup, 1858)

Cortisol, the main glucocorticoid in fish, undertakes pleiotropic biological effects in response to stressors to maintain homeostasis. It can exert several actions on the immune system, growth and cellular metabolism, establishing a fine-tune regulation stress response and cross-talk interactions with other regulatory pathways. In this study, we investigated a causal relationship between high levels of glucocorticoids and susceptibility to pathogens and modification of gene expression profiles in Senegalese sole. For this purpose, we carried out two experiments using post-metamorphic individuals (21 days after hatching) that were exposed to dexamethasone (DXM), a potent glucocorticoid, in order to mimic cortisol effects. We quantified transcript levels of a wide set of genes involved in innate immune system (g-type lysozyme and hepcidin (hamp1)), HPI axis (crf, crfbp, pomcα, pomcβ, gr1 and gr2), HPT axis (tgb), cellular stress defense system (hsp70 and hsp90aa), GH/IGF axis (igf-I and igf-Ir) and the neuropeptide trh. Short-term exposure to 0.1, 1 and 10 ppm DXM provoked a reduction of pomcβ transcripts and an increase of crfbp mRNAs in a dose-dependent manner at 48 and 72 h after treatment. Moreover, g-type lysozyme transcript levels decreased significantly at 72 h whereas hamp1 mRNA levels increased at 48 h after exposure. Long-term DXM treatment (10 ppm DXM) affected negatively weight of soles (∼20% lower than controls). Moreover, reduced mRNA levels were observed for pomcβ after 1 week and igf-I and hamp1 after 2 weeks. In contrast, crfbp and crf increased mRNA levels after 2 weeks. hsp70 exhibited a dual response increasing transcript levels at 1 week after treatment and reducing thereafter. No significant changes in gene expression were observed at any time during this study for tgb, trh, hsp90aa, pomcα, gr1 and gr2. Finally, a challenge experiment using the pathogen Photobacterium damselae subsp piscicida confirmed earlier and higher mortalities in DXM-treated animals. Taken together, these data indicate that a prolonged exposure to DXM increases the susceptibility to pathogens and reduces growth. Moreover, DXM can trigger a wide cellular response modulating the expression of genes involved in the innate immune system, HPI and GH/IGF axes as well as cellular stress defense. These results are highly valuable to evaluate responses associated to aquaculture stressful conditions and discriminate specific glucocorticoid-mediated effects.

Influence of dietary administration of a probiotic strain Shewanella putrefaciens on senegalese sole (Solea senegalensis, Kaup 1858) growth, body composition and resistance to Photobacterium damselae subsp piscicida

Probiotics are currently being supplemented to cultured fish due to their benefits for fish performance. Herein, we tested the health protection and nutritional effects of probiotic Shewanella putrefaciens (Pdp11 strain), both fresh and lyophilized cells, on Senegalese sole (Solea senegalensis) juveniles. Pdp11 was incorporated into fish feed at concentration of 109 cells g−1, and then provided to juvenile sole for 2 months. Growth rates were significantly higher in fish fed the fresh probiotic compared with fish fed the control diet. Growth of fish receiving lyophilized bacteria was not improved when compared with controls. Body compositions (protein, total lipids and fatty acids profile) were similar for each dietary treatment. At the end of the study, fish were challenged by intraperitoneal inoculation with a pathogenic strain of Photobacterium damselae subsp. piscicida. Relative percentages of survival (RPS) ranged between 25% and 43.8% for fish receiving both probiotic supplemented diets. Both fresh and lyophilized Pdp11 cells conferred protection against P. damselae subsp. piscicida. However, only fresh Pdp11 enhanced Senegal sole performance. These findings should be taken into account for industry purposes in which lyophilized Pdp11 might be easier to manage.

The immune response of tiger shrimp Penaeus monodon and its susceptibility to Photobacterium damselae subsp . damselae under temperature stress

Tiger shrimp Penaeus monodon held in 25‰ seawater and 26 °C seawater were injected with Photobacterium damselae subsp . damselae grown in TSB at a dose of 8.48 × 10 4 colony-forming units (cfu) shrimp − 1 , and then reared onward at water temperatures of 22, 26 (control), 30 and 34 °C. Over 24–96 h, the cumulative mortalities of P. damselae subsp . damselae-injected shrimp held in 22 and 34 °C were significantly higher than those for injected-shrimp held in 26 and 30 °C. In a separate experiment, shrimp held in 25‰ seawater and 26 °C, and then cultured onward at 22, 26 (control), 30 and 34 °C were examined for immune parameters, phagocytic activity and clearance efficiency of P. damselae subsp . damselae at 12–96 h. THC (total hemocyte count), DHC (differential hemocyte count), phenoloxidase (PO) activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in shrimp decreased significantly after 24–96 h transfer to 22 and 34 °C. It was concluded that transfer of tiger shrimp P. monodon from 26 °C to 22 and 34 °C reduced their resistance against P. damselae subsp . damselae infection.

Variation in 16S-23S rRNA Intergenic Spacer Regions in Photobacterium damselae : a Mosaic-Like Structure

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.

Cloning and nucleotide sequence analysis of the chloramphenicol resistance gene on conjugative R plasmids from the fish pathogen Photobacterium damselae subsp. piscicida.

Transferable resistance to various drugs was investigated in Photobacterium damselae subsp. piscicida from Japan. Drug resistances were transferred via plasmids of 100, 50, and 40 kb. Resistance to chloramphenicol (Cmr) was transferred on plasmids of all 3 sizes. The Cmr gene (cat) was cloned from the 50 kb plasmids pPDP8511 and pPDP9106 transferred from P. damselae subsp. piscicida strains isolated in different years and places in Japan. Subcloning localized the cat to within 1.5 kb HindIII-HincII (or PstI) fragments. Nucleotide sequences of the coding and flanking region of the cat were determined as 1607 bp (HindIII-HincII fragment) in pPDP8511 and 1568 bp (HindIII-PstI fragment) in pPDP9106, which corresponded with the sequence from nucleotides 40 to 1607 in pPDP8511. The nucleotide sequences identified an open reading frame (ORF) encoding 213 amino acid residues with a calculated molecular mass of about 24.8 kDa, a size consistent with the molecular mass of known cat gene products, and the ORF had maximum homology (99.5%) with a Type II CAT variant from Haemophilus influenzae.