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J L Barja

Publications and source records attributed to J L Barja.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of Edwardsiella tarda strains isolated from turbot, Psetta maxima (L.).

The biochemical, serological and molecular characteristics of a group of 21 Edwardsiella tarda strains isolated from turbot, Psetta maxima, in two different areas of Europe were analysed and compared with a total of 13 strains of this bacterial species with different geographical and host origins. All the turbot isolates were biochemically identical to the E. tarda strains included as reference. The use of different techniques including microagglutination, dot blot and Western blot of lipopolysaccharides allowed us to determine that all the turbot isolates constitute an homogeneous and distinctive serological group. Genetic analysis by randomly amplified polymorphic DNA (RAPD) analysis demonstrated that although the E. tarda strains from turbot were compiled in a unique group using the primers P3 and P6, two clonal lineages could be detected when oligonucleotides P4 and P5 were employed.

Agglutination Tests↗
PubMed2005

Development of a rapid, sensitive and non-lethal diagnostic assay for the detection of viral haemorrhagic septicaemia virus.

A non-lethal diagnostic procedure based on polymerase chain reaction (PCR) technology was developed to detect viral haemorrhagic septicaemia virus (VHSV). Sensitivity of the assay was tested using purified viral RNA and seeded tissues. Detection limits of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay were estimated to be 10 fg of purified RNA and 0.97 x 10(3) or 10(0) TCID(50)/g of seeded tissue, depending on the experimental approach employed (viral adsorption allowed for 1 or 24h). Addition of nested PCR increased sensitivity up to 100-fold when cDNA excised from the agarose gel was used as template. Both, RT-PCR and nested RT-PCR, as well as Southern blot were applied to RNA extracted from blood of experimentally infected brown trout and the results were compared with those obtained by applying the same techniques to tissues and also with those of conventional viral isolation in cell culture. The superiority of the nested RT-PCR applied to blood samples has been clearly demonstrated in terms of sensitivity, obtaining positive results in 85% of fish tested, as against 40% obtained by RT-PCR and Southern blot, and only 5% viral isolations in cell culture. This procedure could turn into an important tool for screening of wild stocks as well as valuable individuals in commercial fish farms, since it makes to kill the fish unnecessary.

Animals↗
PubMed2005

Isolation of lymphocystis disease virus from sole, Solea senegalensis Kaup, and blackspot sea bream, Pagellus bogaraveo (Brunnich).

Two viruses were isolated from cultured sole, Solea senegalensis, and wild blackspot sea bream, Pagellus bogaraveo, and preliminarily characterized as lymphocystis disease viruses (LCDVs). Viral isolates were characterized by morphological, biochemical and biophysical properties. In addition, the susceptibility of four fish cell lines was also tested. LCDV isolates developed cytopathic effects on the SAF-1 cell line at 5 and 6 days post-infection and reached titres of 10(6) TCID50 mL(-1). The antigenic and structural protein analysis of the two new LCDV isolates showed identical profiles to that obtained for LCDV strain Leetown NFH (ATCC VR-342), used as a reference viral strain, and for an LCDV isolate collected from gilt-head sea bream, Sparus aurata, cultured in southern Spain. Molecular confirmation was performed by polymerase chain reaction. Specific primers for LCDV produced a 270-bp DNA fragment, the expected size for LCDV.

Animals↗
PubMed2004

Restriction fragment length polymorphisms and sequence analysis: an approach for genotyping infectious pancreatic necrosis virus reference strains and other aquabirnaviruses isolated from northwestern Spain.

Reference strains of infectious pancreatic necrosis virus resembling the 10 recognized serotypes and local isolates of aquabirnaviruses isolated in northwestern Spain from reservoirs (mollusks) and from asymptomatic and carrier cultured fish were genotyped by restriction fragment length polymorphism (RFLP) and nucleic acid sequence analyses. The RFLP analysis yielded seven genogroups, each of which was clearly correlated with a serotype. Sequence analysis of the three open reading frames provided quite similar results in terms of genogrouping. Based on the results of this study and in order to unify the two types of assays, we propose placing aquabirnaviruses into six genogroups, four of which can be subdivided into two genotypes based on a two-step restriction analysis. The genotyping corresponds with serotyping as follows: genogroup I includes two genotypes corresponding to serotypes A9 (genotype I.1) and A1 (genotype I.2); genogroup II corresponds to serotype A3; genogroup III includes genotypes III.1 (serotype A2) and III.2 (serotype B1); genogroups IV and V include two genotypes, each corresponding to serotypes A5, A6, A7, and A8 (genotypes IV.1, IV.2, V.1, and V.2, respectively);and genogroup VI corresponds to serotype A4. As expected, most local isolates belonged to genotype III.1 and genogroup II. However, a few local isolates corresponded to the American types of genogroup I. Finally, based on the results of this study and due to its simplicity, the two-step restriction analysis assay is proposed as a method for typing new isolates of aquabirnaviruses, and the results correspond to the results of conventional serotyping.

Animals↗
PubMed2004

Comparison of different primer sets for the RT-PCR detection of hepatitis A virus and astrovirus in mussel tissues.

In the present study, the efficiency of several primer sets for the RT-PCR detection of hepatitis A virus (HAV) and astrovirus from both crude viral extracts and experimentally infected shellfish tissues was evaluated. Differences were observed depending on the primer set employed in the sensitivity of amplification of both viral types. For HAV primers, HAV240/HAV68 yielded the higher sensitivity: showing a detection limit of 0.02-0.1 infectious particles/microL or mg of tissue (either crude extracts or seeded mussel tissues). Regarding detection of AsV, a better performance was observed with primer set A1/A2 achieving a sensitivity of 0.1-1 PFU/microL or mg of tissue. The results obtained in this work strongly indicated that selection of primer sets to be employed for the routine detection of enteric viruses was a critical point in the design of the RT-PCR protocols.

Animals↗
PubMed2002

Isolation of viral hemorrhagic septicemia virus from Greenland halibut Reinhardtius hippoglossoides caught at the Flemish Cap.

Viral hemorrhagic septicemia virus (VHSV) was isolated from apparently healthy Greenland halibut Reinhardtius hippoglossoides caught in the Flemish Cap, a deep fishing ground in the North Atlantic Ocean in international waters near Newfoundland. The identity of the virus was confirmed by electron microscopy, immunodot, seroneutralization and reverse transcriptasepolymerase chain reaction. In the serology assays, all isolates reacted in the immunodot assay with a polyclonal antiserum against the European VHSV Type Strain F1, and were neutralized by the same antiserum, although most of the strains showed low or moderate neutralization titers. None of the isolates were detected by immunofluorescence using a specific monoclonal antibody against a nucleocapsid-related protein of VHSV F1. This is the first report of VHSV isolated from wild Greenland halibut, which represents a new host species for the virus, and it is also the first evidence of VHSV in a location close to the Atlantic coast of North America. This isolation indicates that VHSV is more widely distributed than has been thought, and appears to support a marine origin of this virus.

Animals↗
PubMed2002

Prevalence of enterovirus and hepatitis A virus in bivalve molluscs from Galicia (NW Spain): inadequacy of the EU standards of microbiological quality.

A study of the presence of hepatitis A virus (HAV) and enterovirus (EV) in shellfish from the northwestern coast of Spain, one of the most important mussel producers in the world, was carried out employing dot-blot hybridization and RT-PCR techniques. In addition, bacterial contamination of the samples was evaluated by Escherichia coli (EC) counts, according to the European Union (EU) standards of shellfish microbiological quality. Shellfish samples included raft-cultured and wild mussels, as well as wild clams and cockles. Bacterial counts showed that the majority of samples (40.8%) could be classified as moderately polluted following the EU standards, and therefore should undergo depuration processes. However, differences in bacterial contamination were observed between cultured mussel and wild shellfish. Thus, percentage of clean samples (<230 EC/100 g shellfish) was clearly higher in cultured mussels (49.1%) than in wild mussels (22.8%) or clams and cockles (10.7%). HAV was detected in 27.4% and EV in 43.9% of the samples that were analyzed. Simultaneous detection of both viral types occurred in 14.1% of the samples. Statistical tests of dependence (chi-square test) showed no relationship either between viral and bacterial contamination, or between the presence of HAV and EV. Comparative analysis of hybridization and RT-PCR for viral detection yielded different results depending on the virus type that was studied, RT-PCR being effective for HAV but not for EV detection. The obtained results reinforce once again the inadequacy of bacteriological standards to assess viral contamination and suggest that although virological analysis of shellfish is possible by molecular techniques, interlaboratory standardization and validation studies are needed before the routine use in monitoring shellfish microbiological safety.

Animals↗
PubMed2001

Global market: shellfish imports as a source of reemerging food-borne hepatitis A virus infections in Spain.

A total of 16 mollusk imports from South America to Spain, including clam and scallop species, were analyzed for hepatitis A virus (HAV), due to the great concern about this type of food after an important hepatitis A outbreak in eastern Spain in September 1999. In addition, clams from the stock that had caused the outbreak were also tested. Of the 17 stocks, four were positive for the presence of HAV RNA as demonstrated by RT-PCR and Southern hybridization. Contradictory analyses confirmed the results of the primary tests in all cases. The findings obtained in this work strongly support the role of mollusk imports from endemic areas of HAV as an important vehicle of hepatitis A, and demonstrate the imperative need for sanitary control measures to prevent future outbreaks of this disease.

Blotting, Southern↗
PubMed2000

Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae.

A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon.

Animals↗
PubMed2000

Presence of phospholipase-D (dly) gene coding for damselysin production is not a pre-requisite for pathogenicity in Photobacterium damselae subsp. damselae.

The presence of the phospholipase-D (dly) gene as pre-requisite for virulence of Photobacterium damselae subsp. damselae for poikilotherm and homoiotherm animals was investigated in a total of 17 strains isolated from fish, shellfish, mammals and seawater. With this aim, we developed two PCR protocols. A simple PCR using primers flanking the almost complete dly gene, and a multiplex-PCR using two sets of primers directed to internal fragments of the dly and 16S rRNA genes. Only six of the 17 Ph. damselae subsp. damselae strains studied harboured the dly gene regardless of their haemolytic activity against sheep or rabbit erythrocytes as well as their virulence for mammals and marine fish. In fact, all strains but one were pathogenic for one or both animals, with LD(50)values ranging from 1x10(3)and 3x10(5)bacteria for turbot, and 2x10(6)and 8x10(7)cells for mice. The PCR results were corroborated in dot blot hybridization experiments employing a DNA probe directed to an internal region of the dly gene. From the data obtained in this work, we can conclude that the presence of the dly gene is not an indicative of the pathogenicity of Ph. damselae subsp. damselae and, therefore, the role of damselysin as the main virulence factor of this marine bacterium for poikilotherm and homoiotherm hosts should be re-evaluated.

Animals↗
PubMed2000

Existence of two geographically-linked clonal lineages in the bacterial fish pathogen Photobacterium damselae subsp. piscicida evidenced by random amplified polymorphic DNA analysis.

In this work, we applied the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida), an important pathogen for different marine fish. Regardless of the oligonucleotide primer employed, the 29 isolates of Ph. damselae subsp. piscicida tested were separated into two groups, the RAPD-PCR analysis differentiated the European strains from the Japanese strains. The similarity between both groups estimated on the basis of the Dice coefficient was 75-80%. These results show that European and Japanese isolates of Ph. damselae subsp. piscicida, regardless of their host fish species, belong to two different clonal lineages. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.

Animals↗
PubMed2000

Diversity of infectious pancreatic necrosis virus strains isolated from fish, shellfish, and other reservoirs in Northwestern Spain.

A comparison was done of 231 strains of birnavirus isolated from fish, shellfish, and other reservoirs in a survey study that began in 1986 in Galicia (northwestern Spain). Reference strains from all of the infectious pancreatic necrosis virus serotypes were included in the comparison, which was done by neutralization tests and agarose and polyacrylamide gel electrophoresis of the viral genome. The neutralization tests with antisera against the West Buxton, Spajarup (Sp), and Abild (Ab) strains showed that most of the Galician isolates were European types Sp and Ab; however, many isolates (30%) could not be typed. Results from agarose gels did not provided information for grouping of the strains, since all were found to have genomic segments of similar sizes. Analysis of polyacrylamide gels, however, allowed six electropherogroups (EGs) to be differentiated on the basis of genome mobility and separation among segments, and a certain relationship between EGs and serotypes was observed. A wide diversity of electropherotypes was observed among the Galician isolates, and as neutralization tests showed, most of the isolates were included in EGs corresponding to European types Ab and Sp. Only 6.5% of the isolates had the electropherotype characteristic of American strains.

Animals↗
PubMed1999

Genetic analysis of turbot pathogenic Streptococcus parauberis strains by ribotyping and random amplified polymorphic DNA.

Ribotyping and RAPD profiling of a collection of 18 Streptococcus parauberis strains isolated from diseased turbot in Galicia (NW Spain) was performed in order to analyze the possible genetic variability within this bacterial fish pathogen. In addition, the value of this technique for intraspecific classification and epidemiological studies was evaluated. Ribopatterns of DNA digested with three endonucleases and hybridized with a cDNA probe complementary to highly conserved sequences in the 16S and 23S rRNA genes showed a great homogeneity among the turbot isolates. Compared with ribotyping, RAPD appeared to be a reliable and fast technique for discriminating between isolates of S. parauberis on the basis of their farm of isolation and, therefore, represents a powerful tool for epidemiological studies of this fish pathogen.

Animals↗
PubMed1999

Arthrobacter rhombi sp. nov., isolated from Greenland halibut (Reinhardtius hippoglossoides).

Two strains of a hitherto undescribed Gram-positive coryneform bacterium isolated from Greenland halibut (Reinhardtius hippoglossoides) were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains constitute a new line within the genus Arthrobacter. The nearest relatives of the bacterium from fish were members of the Arthrobacter nicotianael Arthrobacter sulfureus group. The unknown bacterium was readily distinguished from these species by phenotypic methods. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Arthrobacter rhombi sp. nov. The type strain of Arthrobacter rhombi is CCUG 38813T.

Animals↗
PubMed1999

16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.

Animals↗
PubMed1997

Immunization with bacterial antigens: Vibrio infections.

Within the genus Vibrio, the species causing the most economically important diseases in marine culture are Vibrio anguillarum, V. ordalii, V. salmonicida and V. vulnificus biotype 2. For these bacterial fish pathogens host range, clinical importance, virulence mechanisms, the antigenic variants relevant to vaccination, the existence of genetic intraspecific diversity and the available vaccines including commercial or domestically produced will be described. Among the 10 serotypes described in V. anguillarum, only serotypes O1, O2 and O3 have been associated with mortality in a great variety of farmed and feral fish worldwide. Whereas serotype O1 is a very homogeneous group from the biochemical, serological and genetic stand-point, within serotype O2 and O3 two antigenic entities have been detected. Moreover these two serotypes present a remarkable genetic heterogeneity. However, many of the available commercial vibriosis vaccines include in their formulations only V. anguillarum serotype O1 in combination with V. ordalii (formerly V. anguillarum biotype 2). In addition no commercial vaccine provides information about the subgroup(s) used as representative of V. anguillarum O2. Recently, Vibrio species taxonomically related to V. anguillarum (VAR) have been isolated from diseased fishes. An extensive characterization of these VAR organisms allowed us to distribute them into at least seven O-serogroups. The inclusion of representative VAR strains in the vibriosis vaccines need to be discussed. V. ordalii, V. salmonicida and V. vulnificus are homogeneous species with respect to biochemical reactions, serology and degree of virulence, possess a narrow host range and seem to be restricted to some geographic areas. Although iron acquisition systems can be involved in the virulence mechanisms of these pathogens, only in V. anguillarum has it been clearly demonstrated that the ability to scavenge iron from the host is a crucial virulence determinant. The role of exotoxins and cell surface associated properties in the Vibrio infections remains to be elucidated.

Animals↗
PubMed1996

Adherence and invasive capacities of the fish pathogen Pasteurella piscicida.

Pasteurella piscicida strains were weakly or moderately adherent to cell lines, the levels of attachment being variable depending on the cells employed. All the isolates exhibited the highest binding capacity to CHSE-214 cells. Adhesive capacities were affected by heat and sugars but not by proteinase K or by treatment with antisera raised against the lipopolysaccharides of P. piscicida, implicating components of glycoprotein(s) as ligands in the adhesion process. The isolates showed a great binding capacity to intestines from the marine fish hosts gilthead sea bream, sea bass and turbot, with values ranging from 10(4) to 10(5) bacteria/g. Although the P. piscicida strains showed a weak invasiveness in the poikilothermic cell lines employed as in vitro model, the bacteria remained viable inside the infected cells at least for 2 days. The invasion process was inhibited by cytochalasin D indicating the active participation of the host cytoskeleton in the internalization of P. piscicida.

Animals↗