Toxicity of Photobacterium damselae subsp. damselae strains isolated from new cultured marine fish

The in vivo and in vitro toxicity of bacterial cells and their extracellular products (ECPs) from 16 strains of Photobacterium damselae subsp. damselae isolated from 7 epizootic outbreaks were evaluated. On the basis of their 50% lethal dose (LD50) values (about 1 x 10(50 CFU), these strains may be considered as moderately virulent. However, their ECPs were strongly lethal for redbanded seabream Pagrus auriga causing fish death within 2 h post-inoculation (protein concentration ranged between 2.1 and 6.41 microg g(-1) fish). The bacterial ECPs tested exhibited several enzymatic activities, such as amylase, lipase, phospholipase, alkaline phosphatase, esterase-lipase, acid phosphatase, and beta-glucosaminidase. These ECPs displayed a strong cytotoxic effect on 4 fish and 2 mammalian cell lines, although this activity disappeared when ECPs were heated at 100 degrees C. The virulence of the strains tested could not be related to the hemolytic activity or to the production of the toxin damselysin. Therefore, another unknown type of toxin could play an important role in the virulence mechanisms of this bacterial pathogen.     

Molecular intraspecific characterization of Photobacterium damselae ssp. damselae strains affecting cultured marine fish

Aims: The aim of this study was to analyse the intraspecific variability of Photobacterium damselae ssp. damselae strains isolated from different cultured marine fish species using molecular typing methods. Methods and Results: Twenty P. damselae ssp. damselae strains isolated from marine fish species were used in this study. Phenotypic characterization of the strains was carried out using standard microbiological methods. Genetic characterization was conducted using three PCR‐based methods [random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR) and repetitive extragenic palindromic‐PCR (REP‐PCR)]. Dice coefficient and the unweighted pair group method with average linkage were used for numerical analyses of banding patterns. At phenotypic level, the strains analysed showed seven different profiles, which could not be related to the host fish species, geographic area or outbreak of disease. Isolates were grouped into nine and eight clusters using the RAPD technique with primers 5 and 4, respectively. In both cases, the main cluster grouped 45% of strains. The techniques ERIC‐PCR and REP‐PCR were more discriminatory, both resulting in 14 different clusters, which grouped 15–20% of the isolates. Conclusions: In this study, the techniques tested are confirmed as good tools for molecular typing, because they allow discrimination between P. damselae ssp. damselae strains isolated within the same outbreak. In addition, ERIC‐PCR and REP‐PCR methods were more adequate for rapid typing of P. damselae ssp. damselae than RAPD, allowing the discrimination at strain level. Significance and Impact of the Study: The results, in agreement with previous studies, confirmed the high intraspecific variability among isolated P. damselae ssp. damselae strains at both phenotypic and genetic levels. This suggests the existence of different clonal lineages that coexist in the same geographic area, within a short period of time (2–3 years). The discrimination at strain level can be useful to study the traceability of infections.     

Identification and characterisation of the fur genes in Photobacterium damselae ssp. piscicida and ssp. damselae

The gene encoding the ferric uptake regulator protein (fur gene) of the fish pathogenic bacterium Photobacterium damselae ssp. piscicida Strain D121 was partially amplified using degenerate oligonucleotides. Complete sequencing of the fur gene and neighbouring DNA was accomplished by primer walking. An open reading frame of 447 bp, coding for a protein of 148 amino acids, and with high homology to previously described Fur proteins, was identified. The fur gene of P. damselae ssp. damselae ATCC 35083 was subsequently amplified by PCR with specific primers and its sequence determined, showing a 99.3% similarity to the P. damselae ssp. piscicida fur gene. The P. damselae fur gene was able to complement the fur mutation of Escherichia coli Strain H1681 in an iron-dependent fashion.     

Amplified fragment length polymorphism (AFLP) and biochemical typing of Photobacterium damselae subsp. damselae

AIMS: The aim of the present study was to characterize subspecifically Photobacterium damselae subsp. damselae strains isolated from cultured Sparus aurata and Dicentrarchus labrax by means of phenotypic and molecular typing techniques (amplified fragment length polymorphism, AFLP). METHODS AND RESULTS: Seventy-one strains of P. damselae subsp. damselae were isolated from 38 cultured fishes at different fish farms located on the Mediterranean coast near Valencia, Spain. Most fish studied were asymptomatic and some were recovered during infectious outbreaks. Phenotypic characterization revealed a considerable degree of variability within the subspecies, including some characters, such as production of urease, which are used to differentiate P. damselae subsp. damselae from P. damselae subsp. piscicida. Genetic characterization was conducted on a selection of 33 strains, including two reference strains. Dice coefficient (Sd) and the unweighted pair group method with average linkage (UPGMA) were used for numerical analysis of banding patterns. AFLP type was defined on the basis of 100% similarity in the dendrogram obtained, yielding 24 distinct AFLP profiles. At 70% similarity, 13 clusters were defined, thus confirming the great variability observed for the phenotypic traits. CONCLUSIONS: The AFLP variability shown by the isolates was high enough to discriminate between different strains which colonize the same fish. However, closely related AFLP types were usually derived from strains isolated at the same fish farm, indicating an epidemiological relationship. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has confirmed that the AFLP technique allows discrimination of individual strains within P. damselae subsp. damselae for epidemiological studies, and that this subspecies exhibits greater variability than that described for subspecies piscicida.     

Crystal structures of sialyltransferase from Photobacterium damselae

Sialyltransferase structures fall into either GT-A or GT-B glycosyltransferase fold. Some sialyltransferases from the Photobacterium genus have been shown to contain an additional N-terminal immunoglobulin (Ig)-like domain. Photobacterium damselae α2–6-sialyltransferase has been used efficiently in enzymatic and chemoenzymatic synthesis of α2–6-linked sialosides. Here we report three crystal structures of this enzyme. Two structures with and without a donor substrate analog CMP-3F(a)Neu5Ac contain an immunoglobulin (Ig)-like domain and adopt the GT-B sialyltransferase fold. The binary structure reveals a non-productive pre-Michaelis complex, which are caused by crystal lattice contacts that prevent the large conformational changes. The third structure lacks the Ig-domain. Comparison of the three structures reveals small inherent flexibility between the two Rossmann-like domains of the GT-B fold.     

Photobacterium damselae ssp. damselae associated with diseased black tiger shrimp Penaeus monodon Fabricius in India

AIM: To characterize and identify Photobacterium damselae ssp. damselae present in black gill diseased Penaeus monodon collected from east coast of India. METHODS AND RESULTS: Photobacterium damselae ssp. damselae was isolated from hepatopancreas, muscles and gills by using the thiosulfate citrate bile salts sucrose agar supplemented with 1.5% NaCl (TCBS-1) medium. A total of 32 Ph. damselae ssp. damselae isolates were studied together with two reference strains. The biochemical tests and analysis of ureC and 16S rRNA genes confirmed the phenotypic characterization of the isolates as Ph damselae ssp. damselae. Experimental infection studies revealed that the LD50 values of P. monodon and P. indicus ranged from 2x10(3) to 5x10(5) CFU per shrimp and from 4x10(2) to 2x10(4) CFU per shrimp, respectively. CONCLUSIONS: Photobacterium damselae ssp. damselae was found in the internal organs of P. monodon and it showed pathogenic to shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on the Ph. damselae ssp. damselae present in the black gill diseased P. monodon in India and therefore might serve as a basis for future studies and diagnosis purpose to shrimp culturists.     

Evidence that water transmits the disease caused by the fish pathogen Photobacterium damselae subsp. damselae

The transmission through water of the disease caused by the fish pathogen, Photobacterium damselae subsp. damselae, as well as the role of the skin mucus in the initial steps of the infection, have been studied. All tested strains resisted the bactericidal activity of the mucus and showed an ability to adhere to it, but only those virulent by the intraperitoneal route were infective through water. Moribund fishes showed the typical signs of the disease: haemorrhaged areas on the body surface and ulcerative lesions with mucus degradation. These results suggest that the pathogen can be transmitted to fish through water and use the skin as a portal of entry.     

Effect of salinity on the immune response of tiger shrimp Penaeus monodon and its susceptibility to Photobacterium damselae subsp. damselae

Addition of NaCl at 2.5% to tryptic soy broth (TSB) significantly increased the growth of Photobacterium damselae subsp. damselae. Tiger shrimp Penaeus monodon held in 25 per thousand seawater were injected with P. damsela subsp. damselae grown in TSB containing NaCl at 0.5%, 1.5%, 2.5% and 3.5% at a dose of 8.48 x 10(4)colony-forming units (cfu)shrimp(-1). Over 24-96 h, the cumulative mortality was significantly higher for the shrimp challenged with P. damselae subsp. damselae grown in 2.5% NaCl than those grown in 0.5%, 1.5% and 3.5% NaCl. In another experiment, P. monodon held in 25 per thousand were injected with TSB-grown P. damselae subsp. damselae (8.48 x 10(4)cfushrimp(-1)), and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand. After 96 h, the mortality was highest for the P. damselae subsp. damselae-injected shrimp held in 5 per thousand, and the lowest for the P. damselae subsp. damselae-injected shrimp held in 25 per thousand. In a separate experiment, P. monodon held in 25 per thousand and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand were examined for immune parameters, and phagocytic activity and clearance efficiency of P. damselae subsp. damselae after 12-96 h. The THC, hyaline cell, phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency decreased significantly for the shrimp held in 5 per thousand, 15 per thousand and 35 per thousand after 12h. It is concluded that tiger shrimp P. monodon transferred from 25 per thousand to low salinity levels (5 per thousand and 15 per thousand) and high salinity (35 per thousand) had reduced immune ability and decreased resistance against P. damselae subsp. damselae infection.     

Binding of haemin by the fish pathogen Photobacterium damselae subsp. piscicida

Whole cells of virulent (DI 21 and B 51) and avirulent (ATCC 29690 and EPOY 8803-II) strains of Photobacterium damselae subsp. piscicida, grown under iron-supplemented or iron-restricted conditions, were able to bind haemin. Iron limitation resulted in an increased binding of haemin by DI 21, B 51 and ATCC 29690 cells but did not affect the haemin-binding ability of the EPOY 8803-II cells. Proteinase K treatment of whole cells markedly reduced the binding of haemin, indicating that protein receptors located at the cell surface are involved in the binding. This was confirmed by the observation that isolated total as well as outer membrane proteins from all the strains, regardless of the iron levels of the media, were able to bind haemin, with the outer membranes showing the strongest binding. Haemin binding by membrane protein extracts was not affected by heat treatment but was almost completely abolished by Proteinase K treatment, suggesting the presence of thermostable protein receptors for haemin. The capsular polysaccharide also appears to play a minor role in binding of haemin. It was concluded that constitutive as well as inducible mechanisms of haemin binding occur in P. damselae subsp. piscicida. These mechanisms would rely mainly upon the direct interaction between the haemin molecules and surface-exposed outer membrane protein receptors.     

Toxicity of nitric oxide and peroxynitrite to Photobacterium damselae subsp. piscicida

Virulent strains of Photobacterium damselae subsp. piscicida (Pdp) were grown in media with or without glucose supplementation (to enhance polysaccharide capsule formation) and the bactericidal action of nitric oxide (NO) and peroxynitrites was evaluated in a cell-free assay. Treatment with the NO-donor S-nitroso-acetyl-penicillamine (SNAP) induced a dose-and time-dependent decrease in Pdp survival. This effect was greater for strains grown without glucose supplementation (C forms) than for their counterparts grown with glucose supplementation (C(+) forms). Addition of superoxide anion (O2(-)) generating systems (Xanthine/Xanthine oxidase, glucose/glucose oxidase) to the culture media further enhanced the bactericidal effect of NO. A similar bactericidal effect, with the same pattern of sensitivity, was observed when C+ and C forms of the bacteria were treated with 3-morpholino-sydonimide hydrochloride (SIN-1), a compound which simultaneously generates NO and O2(-). Addition of superoxide dismutase (SOD) or SOD plus catalase (CAT) did not fully reverse the toxic action of SIN-1 and the bactericidal effect was similar for both C and C(+) forms suggesting that while NO alone is sufficient to cause damage in all strains of the pathogen tested, growth in glucose supplemented medium enhanced protection to reactive oxygen intermediates rather than NO.     

Evaluation of different DNA-based fingerprinting methods for typing Photobacterium damselae ssp. piscicida

This study evaluates the effectiveness of three different molecular techniques, repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and the random amplified polymorphic DNA (RAPD-PCR) for rapid typing of Photobacterium damselae ssp. piscicida strains isolated from different species of marine fish and geographic areas. The results obtained by the three methods showed that RAPD and ERIC-PCR were more discriminative for suitable rapid typing of Ph. damselae ssp. piscicida than REP-PCR. The analysis of DNA banding patterns generated by both molecular methods (RAPD and ERIC-PCR) clearly separated the strains into two main groups that strongly correlated with their geographic origin. Moreover, the REP-PCR analysis was less reproducible than the RAPD and ERIC-PCR methods and does not allow the establishment of genetic groups. RAPD and ERIC-PCR constitute valuable tools for molecular typing of Ph. damselae ssp. piscicida strains, which can be used in epidemiological studies of photobacteriosis infections.     

Construction of a safe, stable, efficacious vaccine against Photobacterium damselae ssp. piscicida

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.     

Identification of antigens for the development of a subunit vaccine against Photobacterium damselae ssp. piscicida

Photobacterium damselae ssp. piscicida (Ph.d.p.), the causative agent of photobacteriosis, is among the most important pathogens affecting finfish aquaculture globally. With the emergence of recombinant technology, subunit vaccines have been actively pursued, but mostly for viral diseases. Bacterial subunit vaccines are more difficult to develop since the bacterial genome is more complex, with numerous candidate antigens, leading to a lengthy and laborious screening process. Immunoproteomics, using western blotting on protein analyzed with 2DE and LC-MS/MS to isolate immune-reactive proteins and acquire amino acid sequences, followed by recombinant technology to clone the candidate gene, identified eight candidate antigens from Ph.d.p., which have been cloned and expressed in Escherichia coli BL21(DE3). These proteins were purified and used as antigens in an efficacy trial. Three, rHSP60, rENOLASE, and rGAPDH proteins, elicited higher specific antibody titers and stronger protective immunity than the other five and an inactivated Ph.d.p. whole bacterial vaccine. These three antigens may be candidates for the development of a subunit vaccine against Ph.d.p.     

Occurrence of both subspecies of Photobacterium damselae in mullets collected in the river Magra (Italy)

The natural reservoirs and biological characteristics of pathogenic populations of both subspecies of Photobacterium damselae in aquatic habitants remain unclear because of difficulties in obtaining pathogenic strains from the environment. In the present study, we assessed the occurrence of Photobacterium damselae subsp. piscicida and Photobacterium damselae subsp. damselae , considered to be the causative agent of past epizootic outbreaks in mullets collected in the river Magra, Italy. Two hundred and seventy-eight mullets were collected during a period of two years (2008-2009) and analyzed using multiplex PCR. During this period, 57% of fishes were positive for Photobacterium damselae subsp. piscicida and 37% for Photobacterium damselae subsp. damselae, with an higher presence in summer months although none of PCR-positive mullets showed clinical signs of disease. Our results indicate that the two micro-organisms are widespread in the population of mullets studied, and this could be a possible cause for outbreaks in favourable environmental conditions.     

Simple and rapid detection of Photobacterium damselae ssp. piscicida by a PCR technique and plating method

AIMS: To detect Photobacterium damselae ssp. piscicida using the PCR technique and plating method. METHODS AND RESULTS: Two strains of P. damselae ssp. piscicida were isolated from cultured cobia (Rachycentron canadum) at two different fish farms in Taiwan. A pair of primers was designed to detect the capsular polysaccharide gene of P. damselae ssp. piscicida by PCR. Reference strains of different genus and different clinical strains were used for this study. The expected product (410 bp) was obtained from both P. damselae ssp. piscicida and P. damselae ssp. damselae, and they were differentiated by culturing on thiosulphate citrate bile salts-sucrose agar (TCBS-1). Photobacterium damselae ssp. damselae grew on TCBS-1 producing green colonies whereas P. damselae ssp. piscicida did not grow. CONCLUSIONS: The methods used are cost and labour effective when compared with the other methods and commercially available kits. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an integrated set of methods to identify the species P. damselae and to differentiate P. damselae ssp. piscicida from P. damselae ssp. damselae.