Publications by authors named "Dolores Castro"

25 Publications

  • Page 1 of 1

Persistence of Lymphocystis Disease Virus (LCDV) in Seawater.

Food Environ Virol 2020 06 21;12(2):174-179. Epub 2020 Feb 21.

Departamento de Microbiología, Universidad de Málaga, 29071, Málaga, Spain.

Lymphocystis disease virus (LCDV), the causative agent of lymphocystis disease (LCD), is a waterborne pathogen that uses the external surfaces, including the gills, as portals to gain access to fish host. However, there are no data on LCDV persistence in the aquatic environment. In this study, the persistence of LCDV in natural (raw), treated (autoclaved and filtered) and synthetic seawater held at 22 and 18 °C has been evaluated. The estimated T values for LCDV in seawater ranged from 2.7 to 242 days depending on seawater type and temperature, with the highest value recorded at 22 °C in autoclaved seawater. Microbiota and temperature seem to be the main factors affecting the persistence of LCDV in seawater. The results indicated that LCDV is more stable in treated seawater than most of the fish pathogenic viruses studied so far, supporting the relevance of this medium for the prevalence of LCD in fish farms.
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http://dx.doi.org/10.1007/s12560-020-09420-6DOI Listing
June 2020

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

Viruses 2019 06 1;11(6). Epub 2019 Jun 1.

Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29017 Málaga, Spain.

Different developmental stages of spp. (metanauplii, juveniles and adults) were bath-challenged with two isolates of the Lymphocystis disease virus (LCDV), namely, LCDV SA25 (belonging to the species ) and ATCC VR-342 (an unclassified member of the genus ). Viral quantification and gene expression were analyzed by qPCR at different 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 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 × 10 Most Probable Number of Infectious Units (MPNIU)/mg. Viral transcripts were detected in the infected , relative expression values showed a similar temporal evolution in the different experimental groups. Moreover, gilthead seabream () 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 () gene expression was consistently detected. The results obtained support that LCDV infects 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.
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http://dx.doi.org/10.3390/v11060506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6630821PMC
June 2019

Feed and immersion challenges with lymphocystis disease virus (LCDV) reveals specific mechanisms for horizontal transmission and immune response in senegalese sole post-larvae.

Fish Shellfish Immunol 2019 Jun 15;89:710-718. Epub 2019 Apr 15.

IFAPA Centro El Toruño, Junta de Andalucía, Camino Tiro Pichón s/n, 11500, El Puerto de Santa María, Cádiz, Spain. Electronic address:

The horizontal transmission of lymphocystis disease virus (LCDV) through contaminated water and feed (using artemia as vehicle) and the associated immune gene expression profiles in Senegalese sole post-larvae were investigated. All specimens analyzed were positive for LCDV DNA detection at 1-day post-challenge (1 dpc) with the highest viral levels in specimens infected through the immersion route. However, the percentage of LCDV-positive animals and number of viral DNA copies dropped progressively at 2 and 7 dpc. The histological analysis identified structural changes in the skin, muscle and gills of sole post-larvae LCDV-challenged by immersion. In situ hybridization confirmed a wide distribution of LCDV in the skin, gut, surrounding vessels in trunk muscle and head kidney in the immersion route, while the signals were restricted to the liver and lamina propria in the feeding treatment. Expression analysis using a set of 22 genes related to innate immune defense system demonstrated clear differences in the time-course response to LCDV as function of the infection route. Most antiviral defense genes, the proinflammatory cytokines, the complement c3, g-type lysozyme and T-cell markers cd4 and cd8a were rapidly induced in the feeding-infected post-larvae, and they were remained activated at 2 dpc. In contrast, in the immersion-infected post-larvae the induction of most defensive genes was delayed, with a low intensity at 2 dpc. All these data demonstrate that LCDV can horizontally infect Senegalese sole post-larvae through the water or feed although with different patterns of histopathological disorders, virus distribution and route-specific expression profiles.
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http://dx.doi.org/10.1016/j.fsi.2019.04.049DOI Listing
June 2019

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

Dis Aquat Organ 2019 Jan;132(2):151-156

Universidad de Málaga, Departamento de Microbiología, Campus Universitario Teatinos, 29071 Málaga, Spain.

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.
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http://dx.doi.org/10.3354/dao03311DOI Listing
January 2019

Transcriptomic Profiles of Senegalese Sole Infected With Nervous Necrosis Virus Reassortants Presenting Different Degree of Virulence.

Front Immunol 2018 17;9:1626. Epub 2018 Jul 17.

Departamento de Microbiología, Facultad de Ciencias, Universidad de Malaga, Malaga, Spain.

Betanodaviruses [nervous necrosis virus (NNV)] are the causative agent of the viral encephalopathy and retinopathy, a disease that affects cultured Senegalese sole (). NNV reassortants, combining genomic segments from redspotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV) genotypes, have been previously isolated from several fish species. The wild-type reassortant wSs160.03, isolated from Senegalese sole, has been proven to be more virulent to sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine) in the wSs160.03 capsid protein have allowed us to obtain a mutant reassortant (rSs160.03), which provokes a 40% mortality decrease. In this study, the RNA-Seq technology has been used to comparatively analyze Senegalese sole transcriptomes in two organs (head kidney and eye/brain) after infection with wild-type and mutant strains. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wild-type isolate (with higher virulence), whereas 393 genes were differentially expressed in animals infected with the mutant strain (37.9% decrease in the number of DEGs). To study the biological functions of detected DEGs involved in NNV infection, a gene ontology (GO) enrichment analysis was performed. Different GO profiles were obtained in the following subclasses: (i) biological process; (ii) cellular component; and (iii) molecular function, for each viral strain tested. Immune response and proteolysis have been the predominant biological process after the infection with the wild-type isolate, whereas the infection with the mutant strain induces proteolysis in head kidney and inhibition of vasculogenesis in nervous tissue. Regarding the immune response, genes coding for proteins acting as mediators of type I IFN expression () and IFN-stimulated genes (, to name a few) were upregulated in animals infected with the wild-type isolate, whereas no-differential expression of these genes was observed in samples inoculated with the mutant strain. The different transcriptomic profiles obtained could help to better understand the NNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment.
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http://dx.doi.org/10.3389/fimmu.2018.01626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056728PMC
July 2018

Photobacterium malacitanum sp. nov., and Photobacterium andalusiense sp. nov., two new bacteria isolated from diseased farmed fish in Southern Spain.

Syst Appl Microbiol 2018 Sep 24;41(5):444-451. Epub 2018 May 24.

Universidad de Málaga, Departamento de Microbiología, Facultad de Ciencias, 29071 Málaga, Spain. Electronic address:

Three strains, H01100409B, H01100413B, and H27100402H, were isolated from several internal organs of diseased redbanded seabream (Pagrus auriga) reared in Andalusia (Southern Spain). All strains were studied by phenotypic, including chemotaxonomy, and genomic characteristics. Phylogenetic analysis based on concatenated sequences of six housekeeping genes (gyrB, ftsZ, topA, mreB, gapA, and 16S rRNA) supported the inclusion of the strains within the clade Phosphoreum of the genus Photobacterium, and two of the strains (H27100402H and H01100409B) formed a tight group separated from the closest species P. aquimaris. Genomic analyses, including average nucleotide identity (ANIb and ANIm) and DNA-DNA hybridization (DDH), clearly separated strains H27100402H and H01100409B from the other species within the clade Phosphoreum with values below the thresholds for species delineation. The chemotaxonomic features (including FAME analysis and MALDI-TOF-MS) of H27100402H and H01100409B strains confirmed their differentiation from the related taxa. The results demonstrated that strain H01100413B was classified as P. aquimaris and the strains H27100402H and H01100409B represented a new species each in the genus Photobacterium, for which we propose the names Photobacterium malacitanum sp. nov., type strain H27100402H (=CECT 9190=LMG 29992), and Photobacterium andalusiense sp. nov., type strain H01100409B (=CECT 9192=LMG 29994).
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http://dx.doi.org/10.1016/j.syapm.2018.04.005DOI Listing
September 2018

Description of New and Amended Clades of the Genus Photobacterium.

Microorganisms 2018 Mar 12;6(1). Epub 2018 Mar 12.

Department of Microbiology, Faculty of Sciences, Universidad de Malaga, 29071 Malaga, Spain.

Phylogenetic relationships between species in the genus 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 (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 , , , and 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 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 into the following 5 clades: Phosphoreum (comprises , "," , , , "" and ""); clade Profundum (composed of , , , , , , "," and ); clade Damselae (two subspecies of , and ); and two new clades: clade Ganghwense (includes , , , , , and ); and clade Leiognathi (composed by , subsp. and " subsp. "). 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 could not be included in none of the established clade, constituting an orphan clade.
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http://dx.doi.org/10.3390/microorganisms6010024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874638PMC
March 2018

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

Int J Syst Evol Microbiol 2017 Nov 21;67(11):4518-4525. Epub 2017 Sep 21.

Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain.

Three 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-stain-negative, 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 : 1ω7c/C16 : 1ω6c 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 H01100410B, 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 H01100410B (=CECT 9189=LMG 29991).
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http://dx.doi.org/10.1099/ijsem.0.002325DOI Listing
November 2017

Revisiting the genus Photobacterium: taxonomy, ecology and pathogenesis.

Int Microbiol 2017 Mar;20(1):1-10

Universidad de Málaga, Andalucía Tech, Departamento de Microbiología, Campus de Teatinos, Málaga, Spain.

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)].
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http://dx.doi.org/10.2436/20.1501.01.280DOI Listing
March 2017

Gene expression profiles associated with lymphocystis disease virus (LCDV) in experimentally infected Senegalese sole (Solea senegalensis).

Fish Shellfish Immunol 2017 Jul 3;66:129-139. Epub 2017 May 3.

IFAPA Centro El Toruño, Junta de Andalucía, Camino Tiro Pichón s/n, 11500 El Puerto de Santa María, Cádiz, Spain. Electronic address:

In the present study, the pathogenesis of lymphocystis disease virus (LCDV) and the immune gene expression patterns associated with this viral infection were determined in the flatfish Senegalese sole. The results indicate that LCDV spreads rapidly from the peritoneal cavity through the bloodstream to reach target organs such as kidney, gut, liver, and skin/fin. The viral load was highest in kidney and reduced progressively thorough the experiment in spite of the viral major capsid protein gene was transcribed. The LCDV injection activated a similar set of differentially expressed transcripts in kidney and intestine although with some differences in the intensity and time-course response. This set included antiviral-related transcripts (including the mx and interferon-related factors irf1, irf2, irf3, irf7, irf8, irf9, irf10), cytokines (il1b, il6, il8, il12 and tnfa) and their receptors (il1r, il8r, il10r, il15ra, il17r), chemokines (CXC-type, CC-type and IL-8), prostaglandins (cox-2), g-type lysozymes, hepcidin, complement fractions (c2, c4-1 and c4-2) and the antigen differentiation factors cd4, cd8a, and cd8b. The expression profile observed indicated that the host triggered a systemic defensive response including inflammation able to cope with the viral challenge.
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http://dx.doi.org/10.1016/j.fsi.2017.04.028DOI Listing
July 2017

Target organs for lymphocystis disease virus replication in gilthead seabream (Sparus aurata).

Vet Res 2017 04 11;48(1):21. Epub 2017 Apr 11.

Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Campus Universitario Teatinos, Malaga, Spain.

The lymphocystis disease (LCD), the main viral pathology described in cultured gilthead seabream (Sparus aurata), is a self-limiting condition characterized by the appearance of hypertrophied fibroblasts (named lymphocysts) in the connective tissue of fish, primarily in the skin and fins. The causative agent of the disease is the Lymphocystis disease virus (LCDV), a member of the Iridoviridae family. In the present study, LCDV genome and transcripts were detected by real-time PCR in caudal fin, as well as in several internal organs, such as intestine, liver, spleen, kidney and brain, from asymptomatic, diseased and recovered gilthead seabream juveniles. These results indicate that the LCDV has a broad range tissue tropism, and can establish a systemic infection, even in subclinically infected fish. As showed by in situ hybridization, the permissive cells for LCDV infection seem to be fibroblasts, hepatocytes and cells of the mononuclear phagocyte system. Histopathological alterations associated with LCD were observed in all the organs analysed, including necrotic changes in liver and kidney, inflammatory response in the intestine submucosa or brain haemorrhage, although lymphocysts were only detected in the dermis of the caudal fin. Nevertheless, these histological changes were reverted in recovered animals.
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http://dx.doi.org/10.1186/s13567-017-0428-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5387237PMC
April 2017

Evaluation of an integrated cell culture RT-PCR assay to detect and quantify infectious lymphocystis disease virus.

J Virol Methods 2016 12 15;238:62-65. Epub 2016 Oct 15.

Universidad de Málaga, Departamento de Microbiología, 29071 Málaga, Spain. Electronic address:

The lymphocystis disease virus (LCDV), a member of the Iridoviridae family, infects a wide range of fish species including gilthead seabream (Sparus aurata L.), the most important species cultured in the Mediterranean. LCDV is difficult to propagate in cell culture and does not produce clear and consistent cytopathic effects (CPE), especially in samples collected from subclinically infected fish. An integrated cell culture reverse transcription-polymerase chain reaction (ICC-RT-PCR) assay, followed by dot-blot hybridization of the RT-PCR products, was developed to improve the detection of infectious LCDV. The sensitivity of the ICC-RT-PCR assay, which can be performed in 7 d, was at least 100-fold higher than viral diagnosis obtained by CPE development. The developed assay thus allows the determination of infectious titres in samples with low viral loads, including those from asymptomatic carrier fish, in which no CPE was recorded after a 14-d incubation period. The ICC-RT-PCR assay enables rapid, specific and sensitive detection and quantification of infectious LCDV, and may be a valuable tool in the study of aspects of LCDV infection including transmission or epizootiology.
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http://dx.doi.org/10.1016/j.jviromet.2016.09.016DOI Listing
December 2016

Rapid and Sensitive Detection of Lymphocystis Disease Virus Genotype VII by Loop-Mediated Isothermal Amplification.

Food Environ Virol 2017 03 5;9(1):114-122. Epub 2016 Oct 5.

Departamento de Microbiología, Universidad de Málaga, 29071, Málaga, Spain.

Lymphocystis disease virus (LCDV) infections have been described in gilthead seabream (Sparus aurata L.) and Senegalese sole (Solea senegalensis, Kaup), two of the most important marine fish species in the Mediterranean aquaculture. In this study, a rapid, specific, and sensitive detection method for LCDV genotype VII based on loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay, performed using an apparatus with real-time amplification monitoring, was able to specifically detect LCDV genotype VII from clinically positive samples in less than 12 min. In addition, the assay allowed the detection of LCDV in all asymptomatic carrier fish analysed, identified by qPCR, showing an analytical sensitivity of ten copies of viral DNA per reaction. The LCDV LAMP assay has proven to be a promising diagnostic method that can be used easily in fish farms to detect the presence and spread of this iridovirus.
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http://dx.doi.org/10.1007/s12560-016-9265-1DOI Listing
March 2017

Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream.

J Virol 2016 10 12;90(19):8768-79. Epub 2016 Sep 12.

Centro de Investigación en Sanidad Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Valdeolmos, Madrid, Spain

Unlabelled: Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought.

Importance: Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified within S. aurata lymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.
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http://dx.doi.org/10.1128/JVI.01369-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021401PMC
October 2016

Application of a new real-time polymerase chain reaction assay for surveillance studies of lymphocystis disease virus in farmed gilthead seabream.

BMC Vet Res 2016 Apr 6;12:71. Epub 2016 Apr 6.

Departamento de Microbiología, Universidad de Málaga, 29071, Málaga, Spain.

Background: Lymphocystis disease (LCD) is the main viral infection reported to affect cultured gilthead seabream (Sparus aurata) in Europe. The existence of subclinical Lymphocystis disease virus (LCDV) infection in this fish species has been recognised by using polymerase chain reaction (PCR)-based methods. Nevertheless, these methods do not provide quantitative results that can be useful in epidemiological and pathological studies. Moreover, carrier fish have been involved in viral transmission, therefore the use of specific and sensitive diagnostic methods to detect LCDV will be relevant for LCD prevention.

Results: We have developed a real-time PCR (qPCR) assay to detect and quantify LCDV. The assay was evaluated for viral diagnosis in surveillance studies in gilthead seabream farms, and also to identify viral reservoirs in a hatchery. The prevalence of LCDV infection in the asymptomatic gilthead seabream populations tested varied from 30 to 100 %, including data from one farm without previous records of LCD. Estimated viral load in caudal fin of subclinically infected fish was two to five orders of magnitude lower than in diseased fish. The qPCR assay allowed the detection of carrier fish in broodstock from a farm with a history of clinical LCD in juvenile fish. In addition, the quantitative detection of LCDV was achieved in all samples collected in the hatchery, including fertilized eggs, larvae and fingerlings, and also rotifer cultures and artemia metanauplii and cysts used for larval rearing.

Conclusions: The qPCR assay developed in this study has proved to be a rapid, sensitive, and reliable method for LCDV diagnosis, which could be valuable to identify LCDV reservoirs or to study viral replication in gilthead seabream.
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http://dx.doi.org/10.1186/s12917-016-0696-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822239PMC
April 2016

Tissue distribution of Red Spotted Grouper Nervous Necrosis Virus (RGNNV) genome in experimentally infected juvenile European seabass (Dicentrarchus labrax).

Vet Microbiol 2011 Dec 1;154(1-2):86-95. Epub 2011 Jul 1.

IFAPA Centro El Toruño, Junta de Andalucía. Ctra N.IV, Camino de Tiro Pichón, C.P.: 11.500, El Puerto de Santa María, Cádiz, Spain.

The distribution of viral genome in the tissues of juvenile European seabass (Dicentrarchus labrax) during the course of a Red Spotted Grouper Nervous Necrosis Virus (RGNNV) infection has not yet been described. The present study addresses this and indicates which target organs may be involved in viral replication. This information should enable more accurate detection of virus in asymptomatic carriers, and in turn help to control the spread of the disease. The aim of this study was to examine the pattern of expression of viral genomic segments RNA1 and RNA2, using two absolute real-time PCRs (RT-qPCR), over the course of a RGNNV infection after administering the virus by intramuscular injection. In situ hybridization was also used to locate the RNA2 viral segment in different organs throughout the infection. The experimental challenge provoked an acute form of viral nervous necrosis (VNN), with a resulting cumulative mortality of 37%. The RT-qPCRs designed allowed the detection of both genomic segments in all the organs tested (nervous and non-nervous tissues) at all sampling times examined. The highest viral RNA copy number was found in eyes, although viral replication appeared to begin in the brain. Viral replication was also recorded in pooled internal organs and in caudal fin. However, the increase in the viral RNA copy number in these organs did not result in an increased viral titre, which may indicate that a productive infection does not take place in non-nervous tissues, possibly due to a failure in a viral post-replication step.
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http://dx.doi.org/10.1016/j.vetmic.2011.06.029DOI Listing
December 2011

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

Dis Aquat Organ 2010 Oct;92(1):31-40

Department of Microbiology, Faculty of Sciences, University of Malaga, 29071 Malaga, Spain.

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.
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http://dx.doi.org/10.3354/dao02275DOI Listing
October 2010

Characterization of the promoter region of ftsZ from Corynebacterium glutamicum and controlled overexpression of FtsZ.

Int Microbiol 2007 Dec;10(4):271-82

Department of Molecular Biology, Area of Microbiology, Faculty of Biology, University of León, Spain.

Of the five promoters detected for the ftsZ gene in Corynebacterium glutamicum, three were located within the coding region of the upstream ftsQ gene and two within the intergenic ftsQ-ftsZ region. The most distant ftsZ promoter showed activity in Escherichia coli and controlled high-level transcriptional expression of ftsZ in C. glutamicum. Quantitative Western blotting showed that all five promoters were active during the exponential growth phase and down-regulated during stationary phase. This tightly controlled expression of ftsZ in C. glutamicum indicated that small changes in the amount of FtsZ protein strongly affect bacterial cell viability. The controlled overexpression of ftsZ in C. glutamicum, using the promoter of the gntK gene (PgntK), resulted in approximately 5-fold overproduction of FtsZ, an increase in cell diameter, and a highly variable localization of the protein as spirals or tangles throughout the cell. These results suggest that the intracellular concentration of FtsZ is critical for productive septum formation in C. glutamicum. Our observations provide insight into the mechanisms used by the coryneform group, which lacks actin homologs and many regulators of cell division, to control cell morphology.
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December 2007

Co-occurrence of viral and bacterial pathogens in disease outbreaks affecting newly cultured sparid fish.

Int Microbiol 2007 Sep;10(3):193-9

Department of Microbiology, University of Málaga, Málaga, Spain.

Several microbial disease outbreaks in farm stocks of newly cultured sparid fish species, such as common seabream, redbanded seabream, and white seabream, were recorded from 2004 to 2006. This study describes the isolation and characterization of the potential causative agents, either bacteria or viruses, of these outbreaks. The isolated bacterial strains were characterized according to traditional taxonomical analyses and sequencing of a 16S rDNA fragment. Most bacteria were identified as Vibrio spp. and Photobacterium damselae subsp. damselae. The development of cytopathic effects (CPE) on different fish cell lines, the application of specific nested-PCR tests for infectious pancreatic necrosis virus (IPNV), viral nervous necrosis virus (VNNV) and viral hemorrhagic septicemia virus (VHSV), and subsequent sequence analyses were used for virus detection and identification. VNNV, related to the striped jack neural necrosis virus (SJNNV) genotype, and VHSV, related to the genotype Ia, were the only viruses detected. VNNV was isolated from the three fish species under study in five different outbreaks, whereas VHSV was isolated from common seabream and white seabream during two of these outbreaks. IPNV was not detected in any case.
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September 2007

Molecular fingerprinting of Vibrio tapetis strains using three PCR-based methods: ERIC-PCR, REP-PCR and RAPD.

Dis Aquat Organ 2006 Apr;69(2-3):175-83

Departamento de Microbiología y Parasitología, Facultad de Biología, Universidad de Santiago de Compostela, Spain.

Brown Ring Disease (BRD) is a bacterial disease caused by Vibrio tapetis which affects cultured clams and causes heavy economic losses. In this study, 28 V. tapetis strains isolated from 5 different hosts were intraspecifically characterized by 3 different polymerase chain reaction- (PCR-) based typing methods: enterobacteria repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP)-PCR and randomly amplified polymorphic DNA (RAPD)-PCR. Cluster analysis of genetic profiles obtained from these molecular techniques clearly showed the existence of 3 genetic groups strongly correlated to the host origin. The first group was formed by 23 V. tapetis strains isolated from Manila clam Ruditapes philippinarum, 1 isolated from venus clam Venerupis aurea, and 1 isolated from common cockle Cerastoderma edule, all collected from France and Spain. The second group was formed by 2 strains isolated from carpet-shell clam R. decussatus cultured in the northwest of Spain. The third group was composed of 1 strain isolated from Atlantic halibut Hippoglossus hippoglossus from the UK. We concluded that the 3 typing methods based on PCR were useful for the intraspecific typing of V. tapetis strains, and that they can potentially be used as a fast and reliable tool for epidemiological studies in the future.
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http://dx.doi.org/10.3354/dao069175DOI Listing
April 2006

Protein and glycoprotein content of lymphocystis disease virus (LCDV).

Int Microbiol 2004 Jun;7(2):121-6

Department of Microbiology, Faculty of Sciences, University of Malaga, Spain.

The polypeptide and glycoprotein composition of eight strains of the fish-pathogenic lymphocystis disease virus (LCDV) isolated from gilt-head seabream (Sparus aurata), blackspot seabream (Pagellus bogaraveo), and sole (Solea senegalensis) were determined. The protein electrophoretic patterns of all LCDV isolates were quite similar regardless of the host fish, showing two major proteins (79.9 and 55.6 kDa) and a variable number of minor proteins. Three groups of LCDV isolates were distinguished according to the number and molecular masses of the minor proteins. Eight glycoproteins were detected inside viral particles of LCDV 2, LCDV 3 and LCDV 5 isolates, but only seven glycoproteins were found inside viral particles of LCDV 1, LCDV 4, LCDV 6, LCDV 7, and LCDV 11 isolates and the reference virus ATCC VR 342 by using five lectins. LCDV glycoproteins were mainly composed of mannose and sialic acid. These glycoproteins could be part of an external viral envelope probably derived from the host cell membrane.
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June 2004

Ectoparasitic species from Canis familiaris (Linné) in Buenos Aires province, Argentina.

Vet Parasitol 2004 Feb;120(1-2):123-9

Facultad de Ciencias Naturales y Museo, CEPAVE, UNLP, Calle 2 No. 584, 1900 La Plata, Argentina.

Several arthropods that live as ectoparasites on domestic dogs can cause severe dermatitis or act as vectors of pathogenic agents, resulting in serious diseases not only in dogs, but also in humans. We studied ectoparasites found on Canis familiaris sampled in five areas in Buenos Aires province, Argentina. The prevalence of fleas, ticks and lice was analyzed, as well as their seasonal variations through the different sites studied. The kind of infestation found in each host was determined and the intensity of natural infestation was estimated. The study was carried out from October 2001 to July 2002, with 116 dogs that lived in rural areas and did not receive control treatments. In order to remove the ectoparasites, the dogs' skin was rubbed with a piece of cotton soaked in ether. All dogs had at least one species of ectoparasites. A total number of 5193 ectoparasites were found corresponding to four species, 15.7% Ctenocephalides canis, 73% Rhipicephalus sanguineus, 1.8 Linognathus setosus and 9.4% Heterodoxus spiniger. R. sanguineus was the most abundant species, and C. canis was the only flea species found. This may be due to the dogs being exclusively rural animals. Within the zones sampled, Magdalena showed the greatest prevalence, maybe as a consequence of having the highest relative humidity in relation to the other areas. Triple infestation (ticks-fleas-lice) was observed in 56.9% of the dogs; 39.6% presented double infestation, most being ticks-fleas, and only 3.4% showed simple infestation (lice). Female hosts were the most affected. Even though there were records of ectoparasites throughout all the year, a higher intensity was observed during the spring months, most likely as a result of the increase in temperature after the winter months.
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http://dx.doi.org/10.1016/j.vetpar.2003.12.001DOI Listing
February 2004

Comparison of ribotyping, randomly amplified polymorphic DNA, and pulsed-field gel electrophoresis for molecular typing of Vibrio tapetis.

Syst Appl Microbiol 2002 Dec;25(4):544-50

Departamento de Microbiología y Parasitología, Facultad de Biologia, Universidad de Santiago, Santiago de Compostela, Spain.

Brown ring disease, caused by Vibrio tapetis, is an important pathological problem in different species of cultured clams. In order to evaluate the genetic diversity of the pathogen, twenty-seven isolates of V tapetis with different origin were screened by ribotyping (RT), pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA PCR (RAPD). Restriction with PvuII, SalI, and SmaI gave 2 RT patterns, differentiating in all cases the strain 0202RD isolated from carpet-shell clams (Ruditapes decussatus) from the other strains tested. The use of NotI generated strain specific PFGE profiles, which could be grouped in two main clusters. Cluster 1 grouped all but one strain and was subdivided into six PFGE subtypes (1a to 1f) which joined at a similarity level of 75.6%. Cluster 2 included again only the isolate 0202RD. RAPD analysis yielded the same results with three different primers, this method being able to differentiate the isolates from R. decussatus from those isolated from other clam species. Of the three techniques evaluated, PFGE was the most discriminating of the three techniques evaluated, followed in discriminating power by RAPD and RT tests. On the basis of the results obtained, we conclude that the RAPD procedure, which is more rapid and easier to perform than the other techniques, shows to be very useful to analyze large amounts of strain collections from an epidemiological monitoring stanpoint. In addition, PFGE is of great utility to evaluate the genetic diversity of strains involved in an outbreak and to study the spreading of a specific clone.
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http://dx.doi.org/10.1078/07232020260517689DOI Listing
December 2002

Ultrastructure of Proechinophthirus zumpti (Anoplura, Echinophthiriidae) by scanning electron microscopy.

Mem Inst Oswaldo Cruz 2002 Sep;97(6):813-8

Museo La Plata, Facultad de Ciencias Naturales, Buenos Aires, Argentina.

The ultrastructure of Proechinophthirus zumpti Werneck, 1955, mainly the external chorionic features of the egg, is described through electronic microscopy techniques. This species was first cited in Argentina, infesting Arctocephalus australis (Zimmermann, 1873). The morphological adaptations of adults and nymphs are described in both species of Proechinophthirus parasitic on Otariidae: P. fluctus (Ferris, 1916) and P. zumpti.
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http://dx.doi.org/10.1590/s0074-02762002000600011DOI Listing
September 2002

Accumulation and Depuration of Pathogenic and Indicator Microorganisms by the bivalve mollusc, Chamelea gallina L, Under Controlled Laboratory Conditions.

J Food Prot 1991 Aug;54(8):612-618

Department of Microbiology, Faculty of Sciences, University of Malaga, 29071 Malaga, Spain.

The comparative accumulation and depuration processes for several microorganisms ( Escherichia coli , Salmonella typhimurium , Vibrio parahaemolyticus , Aeromonas hydrophila , Streptococcus faecalis , Staphylococcus aureus , and MS-2 coliphage) by the striped venus, Chamelea gallina , under controlled laboratory conditions were studied. Microorganisms accumulated rapidly in bivalves during the first 6 h, with accumulation rates between 3.2 to 360.5 organisms/h depending on the type of microorganism. The relative patterns and rates of elimination of the microorganisms suggest that they are eliminated from shellfish in two different ways. One is of a mechanical nature that results in microbial elimination during the first 12 h. The other elimination mechanism depends upon the microbial species and their accumulated number. All microorganisms tested were eliminated completely by the molluscs after 3 d of depuration, except MS-2 bacteriophages. Results indicate that MS-2 coliphages may be a more reliable indicator of the microbial depuration efficiency by the shellfish under laboratory conditions than E. coli .
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http://dx.doi.org/10.4315/0362-028X-54.8.612DOI Listing
August 1991