Evidence for horizontal gene transfer, gene duplication and genetic variation as driving forces of the diversity of haemolytic phenotypes in Photobacterium damselae subsp. damselae

Photobacterium damselae subsp. damselae, a marine bacterium that causes infections in marine animals and in humans, produces up to three different haemolysins involved in virulence, which include the pPHDD1 plasmidencoded damselysin (Dly) and HlyApl, and the chromosome-encoded HlyAch. We screened 45 isolates from different origins, and found a correlation between their haemolytic phenotypes and the differential haemolysin gene content. All highly and medium haemolytic strains harboured pPHDD1, with amino acid substitutions in HlyApl and HlyAch being the cause of the medium haemolytic phenotypes in some pPHDD1-harbouring strains. Weakly haemolytic strains contained only hlyAch, whereas nonhaemolytic isolates, in addition to lacking pPHDD1, either lacked hlyAch or contained a hlyAch pseudogene. Sequence analysis of the genomic context of hlyAch uncovered an unexpected genetic diversity, suggesting that hlyAch is located in an unstable chromosomal region. Phylogenetic analysis suggested that hlyApl and hlyAch originated by gene duplication within P. damselae subsp. damselae following acquisition by horizontal transfer. These observations together with the differential distribution of pPHDD1 plasmid among strains suggest that horizontal gene transfer has played a main role in shaping the haemolysin gene baggage in this pathogen.This work was supported by grant EM2012/043 from the Xunta de Galicia, Spain, and by grant AGL2012-39274- C02-01 from the Ministry of Economy and Competitive- ness (MINECO) of Spain (cofunded by the FEDER Programme from the European Union)

Lymphocystis disease virus: its importance in aquaculture

Lymphocystis disease virus (LCDV) is the causative agent of a well-known fish viral disease that is characterized by hypertrophy of fibroblastic cells in the connective tissue. This viral disease affects more than 125 wild and cultured species of teleost fish from marine and freshwater environments and has a cosmopolitan geographical distribution. In aquaculture systems, the prevalence of LCDV infection is very high, likely reflecting the horizontal transmission of this virus. The incidence rate of the disease may reach 70%, causing significant economic losses for the aquaculture industry. This review provides information on the taxonomy, viral properties, epizootiology and pathogenesis, diagnostic methods and control measures of LCDV infection in fish.This review has been supported by an Excellence Project from the Andalusian Government (Junta de Andalucia) (Ref. P12-RNM 2261). E.J. Valverde was supported by a fel lowship from Ministerio de Ciencia e Innovacion, Spanish Government (Ref. BES-2011-043607

Description of New and Amended Clades of the Genus Photobacterium.

Phylogenetic relationships between species in the genus Photobacterium have been poorly studied despite pathogenic and ecological relevance of some of its members. This is the first phylogenetic study that includes new species of Photobacterium (validated or not) that have not been included in any of the previously described clades, using 16S rRNA sequences and multilocus sequence analysis (MLSA) in concatenated sequences of gyrB, gapA, topA, ftsZ and mreB housekeeping genes. Sequence analysis has been implemented using Maximum-parsimony (MP), Neighbour-joining (NJ) and Maximum likelihood (ML) treeing methods and the predicted evolutionary relationship between the Photobacterium clades was established on the basis of bootstrap values of >75% for 16S rRNA sequences and MLSA. We have grouped 22 species of the genus Photobacterium into the following 5 clades: Phosphoreum (comprises P. aquimaris, “P. carnosum,” P. iliopiscarium, P. kishitanii, P. phosphoreum, “P. piscicola” and “P. toruni”); clade Profundum (composed of P. aestuarii, P. alginatilyticum, P. frigidiphilum, P. indicum, P. jeanii, P. lipolyticum, “P. marinum,” and P. profundum); clade Damselae (two subspecies of P. damselae, damselae and piscicida); and two new clades: clade Ganghwense (includes P. aphoticum, P. aquae, P. galatheae, P. ganghwense, P. halotolerans, P. panuliri and P. proteolyticum); and clade Leiognathi (composed by P. angustum, P. leiognathi subsp. leiognathi and “P. leiognathi subsp. mandapamensis”). Two additional clades, Rosenbergii and Swingsii, were formed using a phylogenetic method based on 16S rRNA gene, although they are not confirmed by any MLSA methods. Only P. aplysiae could not be included in none of the established clade, constituting an orphan clade

Virulence properties of three new Photobacterium species affecting cultured fish.

https://v2.sherpa.ac.uk/id/publication/6895Several 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

Lymphocystis disease virus (LCDV-Sa), polyomavirus 1 (SaPyV1) and papillomavirus 1 (SaPV1) in samples of Mediterranean gilthead seabream

Lymphocystis disease, caused by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of tumour-like lesions on the skin of affected animals associated with several environmental factors and/or with stress due to the intensive culture conditions of fish farms. In a previous study, the genomes of a new LCDV species, LCDV-Sa, were detected, together with 2 previously unknown viruses, Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1). Gilthead seabream from 17 fish farms in Spain, Italy and Turkey were sampled between 2009 and 2015 to investigate the role of the newly described SaPV1 and SaPyV1 viruses in lymphocystis disease development. Our results show that in diseased fish, either or both of the new viruses are almost invariably detected together with LCDV (98%). In asymptomatic fish, these viruses were detected in a much lower percentage (28%) and mostly in concurrence with LCDV (24%). These data confirm the suspected association among the 3 different viruses during lymphocystis disease development in gilthead seabream and warrant future studies to establish their respective contributions

S- and N-doped carbon quantum dots: Surface chemistry dependent antibacterial activity.

https://v2.sherpa.ac.uk/id/publication/16741Sulfur and nitrogen-doped carbons quantum dots (S-CQDs and N-CQDs) were obtained using a simple hydrothermal treatment of S- or N-containing organic compounds/polymers. They were evaluated for their bactericidal activity against representative Gram-negative (Escherichia coli, CECT 831) and Grampositive (Bacillus subtilis subsp. subtilis 168) bacterial strains, using a qualitative estimation approach. Quantitative tests revealed greater effectiveness of N-CQDs compared to S-CQDs. The bactericidal activity of the dots was linked to their specific surface chemistry, and their sizes in the range of nanometers. In the case of the N-CQDs, amides and amines played the most important role in enhancing bactericidal function. They caused a bacterial death which was linked to the electrostatic interactions between their protonated forms and the lipids of the bacterial cell membrane. It is also possible that the ability to activate oxygen species by the CQDs surface played some role. S-CQDs showed a much lower bactericidal activity compared to that of N-CQDs. These dots (S-CQDS), containing mainly a negatively charged surface due to dissociation of sulfonic/carboxylic groups and sulfates, showed a size dependent rather than a chemistry dependent (electrostatic interactions) inhibition of the Gram-positive bacterial growth. This is the first study where the role of different heteroatoms incorporated to CQDs is examined in the context of the bactericidal activity

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

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

Artemia spp., a Susceptible Host and Vector for Lymphocystis Disease Virus.

Di erent developmental stages of Artemia spp. (metanauplii, juveniles and adults) were bath-challenged with two isolates of the Lymphocystis disease virus (LCDV), namely, LCDV SA25 (belonging to the species Lymphocystis disease virus 3) and ATCC VR-342 (an unclassified member of the genus Lymphocystivirus). Viral quantification and gene expression were analyzed by qPCR at di erent times post-inoculation (pi). In addition, infectious titres were determined at 8 dpi by integrated cell culture (ICC)-RT-PCR, an assay that detects viral mRNA in inoculated cell cultures. In LCDV-challenged Artemia, the viral load increased by 2–3 orders of magnitude (depending on developmental stage and viral isolate) during the first 8–12 dpi, with viral titres up to 2.3 102 Most Probable Number of Infectious Units (MPNIU)/mg. Viral transcripts were detected in the infected Artemia, relative expression values showed a similar temporal evolution in the di erent experimental groups. Moreover, gilthead seabream (Sparus aurata) fingerlings were challenged by feeding on LCDV-infected metanauplii. Although no Lymphocystis symptoms were observed in the fish, the number of viral DNA copies was significantly higher at the end of the experimental trial and major capsid protein (mcp) gene expression was consistently detected. The results obtained support that LCDV infects Artemia spp., establishing an asymptomatic productive infection at least under the experimental conditions tested, and that the infected metanauplii are a vector for LCDV transmission to gilthead seabream

Nervous necrosis virus (NNV) vaccination of carrier Senegalese sole (Solea senegalensis).

Viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), is one of the most threatening diseases affecting marine farmed fish, especially in early developmental stages. In addition, subclinical infections are commonly detected in both farmed and wild fish. In recent years numerous efforts have been made to achieve effective vaccines for the protection of different fish species against NNV infection. Vaccination experiments are always conducted on pathogen-free animals, but subclinically infected individuals may respond differently to immunization. In this study, we have observed a different response between experimentally-induced-NNV carrier Senegalese sole and NNV-free animals when immunized with a BEIinactivated vaccine, whereas mock-vaccinated carrier fish behaved like the NNV-free group.This research was funded by Ministerio de Ciencia, Innovación y Universidades (MICIU), the Agencia Estatal de Investigación (AEI) and FEDER, grant number RTI2018-094687-B-C21 . Dr. Sandra Souto was funded with a postdoctoral grant from Consellería de Cultura, Educación y Universidad, Xunta de Galicia (postdoctoral grant ED481D-2022/024 ). The authors are grateful to Stolt Sea Farm for kindly providing the fish, J. Franqueira for technical assistance and Native English School of Languages for the English revision

Photobacterium toruni sp. nov., a bacterium isolated from diseased farmed fish.

https://v2.sherpa.ac.uk/id/publication/14638Three bacterial strains were isolated from liver and spleen of diseased farmed redbanded seabream (Pagrus auriga) in south-west Spain. Their partial 16S rRNA gene sequences clustered within those of the genus Photobacterium, showing high similarity (98.6–99.3 %) to the type strains of Photobacterium iliopiscarium, P. piscicola, P. kishitanii, P. aquimaris and P. phosphoreum. Multilocus sequence analysis using six housekeeping genes (gapA, topA, mreB, ftsZ, gyrB and 16S rRNA) confirmed the new strains as forming an independent branch with a bootstrap value of 100, likely to represent a novel species. To confirm this, we used whole genome sequencing and genomic analysis (ANIb, ANIm and in silico DNA–DNA hybridization) obtaining values well below the thresholds for species delineation. In addition, a phenotypic characterization was performed to support the description and differentiation of the novel strains from related taxa. Cells were Gram-stainnegative, motile bacilli, chemo-organotrophic and facultatively anaerobic. They fermented glucose, as well as galactose and D-mannose, without production of gas. Oxidase and catalase were positive. The predominant cellular fatty acids were C16 : 1 !7c/C16 : 1!6c and C16 : 0. The predominant respiratory quinone (Q-8) and major polar lipids (phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol) were inferred from annotated genes in the genome of strain H01100410BT, which had a G+C content of 38.6 mol%. The results obtained demonstrate that the three strains represent a novel species, for which the name Photobacterium toruni sp. nov. is proposed. The type strain is H01100410BT (=CECT 9189T=LMG 29991T)