Search Results (39)

Photobacterium damselae subsp. damselae (Pdd) is an increasingly common bacterium in post-mortem diagnostics of beached marine mammals, but little is known about its precise etiological responsibility. To estimate the prevalence of Pdd in stranded cetaceans from 2017 to 2022 on the Ligurian coast (Pelagos Sanctuary), we tested tissues from 53 stranded individuals belonging to four cetacean species. DNA extracts from cetacean tissue were screened using a polymerase chain reaction (PCR) assay targeting the Pdd ureC gene. Positive samples were screened by PCR for dly, hlyA_pl and hlyA_ch hemolysin genes, which were confirmed by sequencing. Twenty-two out of 53 (41.5%) cetaceans analyzed by PCR were confirmed for Pdd DNA in at least one tissue among those analyzed. Five of these cetaceans were positive for at least one of the hemolysin genes tested. In all Pdd-positive cetaceans, other pathogens that were considered responsible for the causa mortis of the animals were also found. The results provide new information on the spread of Pdd in cetaceans and support the thesis that Pdd might be an opportunistic agent that could contribute to worsening health conditions in subjects already compromised by other pathogens. However, further studies are needed to investigate and deepen this hypothesis. Full article

In mariculture facilities, bacterial infections pose significant production challenges, with potentially catastrophic impacts on fish species. Bacterial co-infections are a widespread phenomenon in the natural marine environment, although their impact on aquatic organisms remains poorly investigated. This study aimed to detail the pathological findings associated with a natural bacterial co-infection caused by three different pathogens, namely Photobacterium damselae subsp. piscicida, Tenacibaculum maritimum and Vibrio sp., as the cause of mass mortality in European sea bass. The fish had been reared in open-net cages in Sicily and later transferred for experimental research purposes to a user establishment after immunization with an inactivated vaccine. Macroscopic, cytological and histopathological examinations were performed on 109 animals, and bacterial species were identified by the 16S rRNA gene. Overall, ulcerative skin lesions, necrotizing myositis and tail rot with occasional tail loss were associated with tenacibaculosis and vibriosis, while P. damselae subsp. piscicida mainly caused granulomatous inflammation in the spleen and head kidney. Finally, an injection site reaction due to the oil-adjuvanted vaccine administered intraperitoneally was observed in the abdominal fat. Understanding the impact of bacterial pathogens is essential to manage the health and welfare of farmed fish, and the importance of a good health monitoring program cannot be overstated to avoid outbreaks and the possible emergence of new pathogens due to the intensification of the production systems, antibiotic resistance and climate changes. The study would also highlight the importance of the quarantine period when animals supplied for research come from aquaculture farms and how the main goal in the near future should be to better define the procedures to provide completely pathogen-free animals. Full article

To explore the molecular mechanisms of the Litopenaeus vannamei response to infection by Photobacterium damselae, reveal its immune response and energetic metabolic effect, and provide a valuable genetic data source for the scientific prevention and control of Vibrio infection, transcriptomic analysis, RT-qPCR, and physiological and biochemical tests were conducted. The results showed that the expression of key genes involved in lipid and carbohydrate transport, such as apolipoprotein and TPS, was upregulated after pathogenic infection, which brought the accumulation of triacylglycerol and trehalose into the hemolymph. Additionally, the pathogenic infection selectively triggered an immune response in infected L. vannamei, activating certain immune pathways, such as the serpins and MAPK pathways. The pathogenic infection suppressed the activity of phenoloxidase (PO), and the prophenoloxidase (PPO) cascade responses were suppressed by the invasive bacteria. This paper will help us understand the energetic metabolism, immune response, and activation of the immune recognition response after pathogenic infection by P. damselae, and it lays a theoretical foundation for the biological prevention and control of P. damselae infection. Full article

Spotted sea bass (Lateolabrax maculatus) is a high-economic-value aquacultural fish widely distributed in the coastal and estuarine areas of East Asia. In August 2020, a sudden outbreak of disease accompanied by significant mortality was documented in L. maculatus reared in marine cage cultures located in Nanhuang island, Yantai, China. Two coinfected bacterial strains, namely, NH-LM1 and NH-LM2, were isolated from the diseased L. maculatus for the first time. Through phylogenetic tree analysis, biochemical characterization, and genomic investigation, the isolated bacterial strains were identified as Vibrio harveyi and Photobacterium damselae subsp. piscicida, respectively. The genomic analysis revealed that V. harveyi possesses two circular chromosomes and six plasmids, while P. damselae subsp. piscicida possesses two circular chromosomes and two plasmids. Furthermore, pathogenic genes analysis identified 587 and 484 genes in V. harveyi and P. damselae subsp. piscicida, respectively. Additionally, drug-sensitivity testing demonstrated both V. harveyi and P. damselae subsp. piscicida exhibited sensitivity to chloramphenicol, ciprofloxacin, ofloxacin, orfloxacin, minocycline, doxycycline, tetracycline, and ceftriaxone. Moreover, antibiotic resistance genes were detected in the plasmids of both strains. Extracellular product (ECP) analysis demonstrated that both V. harveyi and P. damselae subsp. piscicida can produce hemolysin and amylase, while V. harveyi additionally can produce caseinase and esterase. Furthermore, infected fish displayed severe histopathological alterations, including infiltration of lymphocytes, cellular degeneration and necrosis, and loose aggregation of cells. Artificial infection assays determined that the LD₅₀ of P. damselae subsp. piscicida was 3 × 10⁵ CFU/g, while the LD₅₀ of V. harveyi was too low to be accurately evaluated. Furthermore, the dual infection of V. harveyi and P. damselae subsp. piscicida elicits a more rapid and pronounced mortality rate compared to single challenge, thereby potentially exacerbating the severity of the disease through synergistic effects. Ultimately, our findings offer compelling evidence for the occurrence of coinfections involving V. harveyi and P. damselae subsp. piscicida in L. maculatus, thereby contributing to the advancement of diagnostic and preventative measures for the associated disease. Full article

The bioactive properties of the combination of microencapsulated cell-free supernatant (CFS) from Streptococcus thermophilus and thyme extract on food-related bacteria (Photobacterium damselae, Proteus mirabilis, Vibrio vulnificus, Staphylococcus aureus ATCC29213, Enterococcus faecalis ATCC29212, and Salmonella Paratyphi A NCTC13) were investigated. The microencapsulated CFS of S. thermophilus, in combination with ethanolic thyme extract, had a particle size in the range of 1.11 to 11.39 µm. The microencapsulated CFS of S. thermophilus had a wrinkled, spherical form. In the supernatant, especially at 2% (v/w), the thyme extract additive caused a decrease in the wrinkled form and a completely spherical structure. A total of 11 compounds were determined in the cell-free supernatant of S. thermophilus, and acetic acid (39.64%) and methyl-d3 1-dideuterio-2-propenyl ether (10.87%) were the main components. Thyme extract contained seven components, the main component being carvacrol at 67.96% and 1,2,3-propanetriol at 25.77%. Significant differences (p < 0.05) were observed in the inhibition zones of the extracts on bacteria. The inhibitory effect of thyme extract on bacteria varied between 25.00 (P. damselae) and 41.67 mm (V. vulnificus). Less antibacterial activity was shown by the microencapsulated CFS from S. thermophilus compared to their pure form. (p < 0.05). As a result, it was found that microencapsulated forms of CFS from S. thermophilus, especially those prepared in combination with 2% (v/w) thyme extract, generally showed higher bioactive effects on bacteria. Full article

The upsurge in havoc being wreaked by antibiotic-resistant bacteria has led to an urgent need for efficacious alternatives to antibiotics. This study assessed the antibacterial efficacy of two isobutyl cyanoacrylate nanoparticles (iBCA-NPs), D6O and NP30, against major bacterial pathogens of fish. In vivo tests on rainbow trout were preceded by in vitro tests of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). NP30 exhibited higher efficacy than D60, but both iBCA-NPs demonstrated dose-dependent and species-specific in vitro antibacterial properties against the bacterial isolates. Generally, Gram-negative bacteria were more resistant to the iBCA-NPs. Streptococcus iniae, Tenacibaculum maritimum, and Photobacterium damselae were particularly sensitive to both iBCA-NPs. Administered to rainbow trout at 3571.4 mg (iBCA-NP)/kg feed, the iBCA-NPs produced a relative gain rate and survival rates comparable to the control (p > 0.05). The condition factor and the hepatosomatic and viscerosomatic indices of fish were indifferentiable (p > 0.05) between the iBCA-NP groups and the control. The iBCA-NPs caused no alteration in stress, oxidative stress (superoxide dismutase, SOD), plasma complement titer, or lysozyme activity. This study presents the first report of antibacterial activity of iBCA-NPs against Gram-negative bacteria. The results of this study suggest that D60 and NP30 may contribute to reducing the amounts of antibiotics and chemotherapeutic agents used in aquaculture. Full article

The use of antibiotics in open-water aquaculture is often unavoidable when faced with pathogens with high mortality rates. In addition, seasonal pathogen surges have become more common and more intense over the years. Apart from the apparent cost of antibiotic treatment, it has been observed that, in aquaculture practice, the surviving fish often display measurable growth impairment. To understand the role of gut microbiota on the observed growth impairment, in this study, we follow the incidence of Photobacterium damselae subsp. piscicida in a seabass commercial open-water aquaculture setting in Galaxidi (Greece). Fish around 10 months of age were fed with feed containing oxytetracycline (120 mg/kg/day) for twelve days, followed by a twelve-day withdrawal period, and another eighteen days of treatment. The fish were sampled 19 days before the start of the first treatment and one month after the end of the second treatment cycle. Sequencing of the 16S rRNA gene was used to measure changes in the gut microbiome. Overall, the gut microbiota community, even a month after treatment, was highly dysbiotic and characterized by very low alpha diversity. High abundances of alkalophilic bacteria in the post-antibiotic-treated fish indicated a rise in pH that was coupled with a significant increase in gut parasites. This study’s results indicate that oxytetracycline (OTC) treatment causes persistent dysbiosis even one month after withdrawal and provides a more suitable environment for an increase in parasites. These findings highlight the need for interventions to restore a healthy and protective gut microbiome. Full article

Disease outbreaks are a common problem in aquaculture, with serious economic consequences to the sector. Some of the most important bacterial diseases affecting aquaculture are caused by Gram-negative bacteria including Vibrio spp. (vibriosis), Photobacterium damselae (photobacteriosis), Aeromonas spp. (furunculosis; haemorrhagic septicaemia) or Tenacibaculum maritimum (tenacibaculosis). Lipopolysaccharides (LPS) are important components of the outer membrane of Gram-negative bacteria and have been linked to strong immunogenic responses in terrestrial vertebrates, playing a role in disease development. To evaluate LPS effects in fish, we used a hot-phenol procedure to extract LPS from common fish pathogens. A. hydrophila, V. harveyi, T. maritimum and P. damselae purified LPS were tested at different concentrations (50, 100, 250 and 500 µg mL⁻¹) at 3 days post-fertilisation (dpf) Danio rerio larvae, for 5 days. While P. damselae LPS did not cause any mortality under all concentrations tested, A. hydrophila LPS induced 15.5% and V. harveyi LPS induced 58.3% of zebrafish larvae mortality at 500 µg mL⁻¹. LPS from T. maritimum was revealed to be the deadliest, with a zebrafish larvae mortality percentage of 80.6%. Analysis of LPS separated by gel electrophoresis revealed differences in the overall LPS structure between the bacterial species analysed that might be the basis for the different mortalities observed. Full article

Some of the characteristics of probiotics used in aquaculture are the antagonistic activity against pathogens and the activation of the immune response. Moreover, some probiotics with common antigens to pathogens can produce antibodies that react with these pathogens. One of those potential probiotics is Vibrio proteolyticus DCF12.2, a strain with the capacity to produce antibodies in soles (Solea senegalensis) that react with Photobacterium damselae subsp. piscicida and Vibrio harveyi antigens. Therefore, the objective of the work was to determine the capacity of DCF12.2 to activate the immune response in the sole, providing protection against both pathogens. For this purpose, DCF12.2 was administered to cultured soles by intraperitoneal injection, bath, and diet. The DCF12.2 strain activated the gene expression related to the fish immune response and increased the antibody production with cross-reaction to P. damselae subsp. piscicida and V. harveyi cells. Challenges with P. damselae subsp. piscicida resulted in a 30–40% relative percent survival (RPS) through DCF12.2 treatments. After challenge with V. harveyi, only intraperitoneal treatment protected the fish (67% RPS). In conclusion, the DCF12.2 strain showed great potential to be used to prevent diseases caused by both pathogens, and indicates a way to use probiotics as live vaccines. Full article

This study aims to assess the effects of different dietary n-6/n-3 long-chain polyunsaturated fatty acid ratios and CHO content in the immune response of gilthead seabream. For that purpose, gilthead sea bream juveniles (initial body weight = 47.5 g) were fed for 84 days with four isoproteic (47% crude protein) and isolipidic (18% crude lipids) diets with high (20%) or low (5%) level of gelatinized starch (HS or LS diets, respectively) and included approximately 2.4% ARA or DHA. At the end of the trial, the DHA-enriched groups presented increased red blood cell (RBC) count, hemoglobin, plasmatic nitric oxide (NO) content, and antiprotease and alternative complement activities. The ARA groups had increased thrombocyte count, and plasmatic bactericidal activity against Vibrio anguillarum was lower in the fish fed the ARA/LS diet. After the feeding trial, the fish were challenged with an intraperitoneal injection (i.p.) of killed Photobacterium damselae subsp. piscicida (Phdp) and sampled at 4 and 24 h after the challenge. At 4 h after i.p., the ARA groups presented increased plasma total immunoglobulins (Ig) and bactericidal activity against V. anguillarum. In addition, the fish fed the ARA/LS diet presented lower white blood cell (WBC) and alternative complement activity. At 24 h after i.p., the ARA groups presented increased RBC, WBC, and thrombocyte numbers, total IG, plasma peroxidase activity, and casp3 expression in the distal intestine. The HS groups presented increased plasma NO content and bactericidal activity against Phdp and decreased protease, antiprotease activity, and bactericidal activity against V. anguillarum. In conclusion, high dietary DHA levels seemed to improve the immune status of unchallenged gilthead sea bream juveniles, while high dietary ARA levels improved the fish immune response to a bacterial challenge. The energy provided by dietary starch seems to be important to promote a fast response by the fish immune system after a challenge. Full article

Cantharidin, a toxic monoterpene secreted by blister beetles, has long been used in traditional Chinese and modern medicine for its unique properties. However, despite its widespread use, its effects on fish have not been studied. The aim of this study was to evaluate the potential therapeutic applications of cantharidin in fish by examining its antioxidant, hemagglutinating, hemolytic, and cytotoxic activities at different concentrations (0, 0.625, 1.25, 2.5, 5, and 10 μg mL⁻¹) in three different cell lines. In addition, the study explored the bactericidal and bacteriostatic properties of cantharidin against various fish pathogenic bacteria. The results revealed that there were no significant differences in antioxidant, hemagglutinating, or hemolytic activities between the different concentrations of cantharidin tested. However, the study found that cantharidin exhibited dose- and time-dependent cytotoxicity in seabream (Sparus aurata) erythrocytes and in SAF-1, PLHC-1, and Hela cell lines, resulting in morphological changes indicative of apoptosis. Interestingly, the highest dose of cantharidin tested demonstrated potent bactericidal activity against four marine fish opportunistic bacteria, including Vibrio harveyi, V. anguillarum, Photobacterium damselae, and Tenacibaculum maritimum, but no statistically significant changes in bacteriostatic activity were observed against any of the bacteria tested. Overall, these results provide valuable information on the potential therapeutic applications of cantharidin in fish aquaculture. Further research is needed to fully understand the mechanisms of action and to explore possible preventive uses of cantharidin in fish. Full article

A monitoring program to follow vibriosis incidents in the Greek marine aquaculture was implemented over the past 13 years. 273 isolates, from various cases originating from eight regions and nine hosts, were collected and characterized. The main aquaculture species of the survey were the European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata). Various species of Vibrionaceae were associated with vibriosis. Vibrio harveyi had the highest prevalence and was isolated throughout the year from all hosts. During the warm months, Vibrio harveyi prevailed with frequent co-isolations of Photobacterium damselae subsp. damselae and Vibrio alginolyticus, while during spring, other Vibrio species were more abundant, such as Vibrio lentus, Vibrio cyclitrophicus, and Vibrio gigantis. Phylogenetic analysis using the mreB gene and the metabolic fingerprint of the isolates showed great variability within the species of the collection. The severity of the disease and the frequency of outbreaks make vibriosis (that is, mainly attributed to V. harveyi) an important concern for the regional aquaculture sector. Full article

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative fish pathogen with worldwide distribution and broad host specificity that causes heavy economic losses in aquaculture. Although Phdp was first identified more than 50 years ago, its pathogenicity mechanisms are not completely understood. In this work, we report that Phdp secretes large amounts of outer membrane vesicles (OMVs) when cultured in vitro and during in vivo infection. These OMVs were morphologically characterized and the most abundant vesicle-associated proteins were identified. We also demonstrate that Phdp OMVs protect Phdp cells from the bactericidal activity of fish antimicrobial peptides, suggesting that secretion of OMVs is part of the strategy used by Phdp to evade host defense mechanisms. Importantly, the vaccination of sea bass (Dicentrarchus labrax) with adjuvant-free crude OMVs induced the production of anti-Phdp antibodies and resulted in partial protection against Phdp infection. These findings reveal new aspects of Phdp biology and may provide a basis for developing new vaccines against this pathogen. Full article

The nursehound Scyliorhinus stellaris is a threatened shark species and its population in the Mediterranean Sea is declining. Programs for captive breeding and repopulation in marine protected areas (MPA) are being carried out. Unfortunately, pathogens may hinder conservation plans for this species. An impactful disease of marine animals, caused by the bacteria Photobacterium damselae, has been detected with increased frequency in recent decades in both farmed and marine animals. The aim of this work was to determine the cause of a disease outbreak in eight captive nursehounds that died after 18 months of captivity. Gross necropsy observations were indicative of a presumptive diagnosis of hemorrhagic septicemia. Histological and molecular techniques were performed, to diagnose the etiological agents that could be involved in their mortality. Phylogenetic analysis indicated the presence of P. damselae, identified as subsp. damselae by PCR-duplex, and Photobacterium swingsii in the analyzed captive nursehound Scyliorhinus stellaris. Full article

Infectious diseases caused by marine bacterial pathogens inflict increasing economic losses to fisheries and aquaculture, while also posing a growing risk to public health and affected species conservation. In this study, four wild marine fish species were collected at five fishing sites in Israel, divided into two regions—north (Acre, Haifa, Shefayim) and center-south (Tel-Aviv and Ashdod), and screened for Photobacterium damselae. An initial screening was carried out using PCR analysis with specifically designed primers on DNA extracted from livers and kidneys. P. damselae-positive samples had their 16S rRNA amplicons sequenced. Later, an attempt to specify relevant sub-species was performed, using a three-layered gene screen: Car, ureC and toxR. Of 334 fish samples, 47 (14%) were found to be P. damselae-positive, of which 20 were identified as P. damselae subsp. piscicida (Phdp), two as P. damselae subsp. damselae (Phdd) and 25 could not be identified to subspecies. Our results strengthen the view that fish residing in a polluted environment are receptive of pathogenic microorganisms. To assess how the presence of pathogens may affect population management and conservation, this research should be followed by studies aimed at: (i) quantifying levels of pollutants that may affect pathogen emergence, and (ii) creating a standard pollution-level index as a basis for setting criteria, above which authorities should take measures of precaution. Full article