Biomedical subjects

J L Barja

Publications and source records attributed to J L Barja.

At least 37 records · Page 2Linked to original sources

PubMed1996

The humoral immune response of turbot to recently isolated pathogenic Enterococcus strains. Cross-reactivity with other Gram-positive bacteria.

An Enterococcus sp. causing severe mortalities among farmed turbot (Scophthalmus maximus) has recently been detected in northwest Spain. We found that specific turbot serum antibodies raised against isolate RA-99.1 of the new pathogen by intraperitoneal immunization, did not cross react in enzyme-linked immunosorbent assay (ELISA) with other enterococcal or non-enterococcal Gram-positive bacteria. In immunoblotting, antibodies raised against strain RA-99.1 recognized the same components in the homologous total soluble antigen preparation (TSA) as in TSA of two other isolates of the pathogen obtained from different farms. Anti-RA-99.1 serum also recognized some components of the TSA of several other Gram-positive bacteria. Immunogold labelling indicated that the antigens which provoke the humoral immune response to this pathogen are located mainly on the bacterial surface.

Animals↗

PubMed1996

Host range susceptibility of Enterococcus sp. strains isolated from diseased turbot: possible routes of infection.

Experiments were conducted to assess the pathogenicity of Enterococcus sp. strains isolated from diseased turbot for several fish species (turbot, salmon, trout, and seabream), as well as for mice. The intraperitoneal injection assays indicated that the tested strains showed host specificity for turbot, with a high degree of virulence (50% lethal dose of 10(4) cells per g of fish). The Spanish Enterococcus sp. isolates were nonpathogenic for the other fish species studied and for mice. The possible routes of infection were determined by bath exposure (with and without prior abrasion of the skin) and by intragastric inoculations with food and feces contaminated with the pathogen. The bath challenges indicated that the Enterococcus isolates were able to overcome the defense mechanisms present on the surface of the turbot only if the skin was abraded prior to the exposure. The antibacterial activities of components of a glycoprotein nature present in the turbot skin mucus are probably responsible in part for the resistance in noninjured fish to infection. On the other hand, we demonstrated the capacity of this pathogen to overcome adverse conditions in the stomachs of fish when associated with food or fecal material, since it is able to establish an infective state and to produce mortalities after 16 to 20 days postingestion. From all of these findings, we can conclude that horizontal transmissions through water and the fecal-oral route are the main avenues of infection of turbot streptococcosis.

Animals↗

PubMed1995

Genetic transformation of Vibrio anguillarum and Pasteurella piscicida by electroporation.

Vibrio anguillarum and Pasteurella piscicida are Gram-negative bacteria which are pathogenic for marine fish and we report here the first successful transformation of these two bacteria by electroporation. The optimal conditions for electroporation included a field strength of 12.5 kV cm-1 and a time constant of 5 ms using 0.2-cm cuvettes. With these parameters, three plasmids (pSU2718, pCML, pEV3) with molecular sizes of 2.6, 5 and 13.7 kb, respectively were successfully transformed into both pathogens. V. anguillarum isolates belonging to serotypes O1 and O2 were transformed with greatest efficiency, 2.5 x 10(3) transformants per micrograms DNA, being achieved in the serotype O2 strains using plasmid pCML. Strains of serotype O3 were not transformed. In the case of P. piscicida the maximum efficiency achieved was 9.8 x 10(2) transformants per micrograms pCML plasmid DNA. This optimized system will allow development of procedures for the genetic manipulation of these pathogens.

Electroporation↗

PubMed1994

Evidence of a dormant but infective state of the fish pathogen Pasteurella piscicida in seawater and sediment.

The stability of Pasteurella piscicida strains in seawater and sediment microcosms at different temperatures (6 and 20 degrees C) was investigated during a 1-month period. Three strains of P. piscicida showed similar survival kinetics. By a standard plate count method they survived in water and sediment for only 6 to 12 days, depending on the strain and type of microcosm. During this starvation period, the metabolic activity of the cells was reduced by more than 80%. Culturable cells of each P. piscicida strain persisted better in sediment than in water, as well as at 20 degrees C compared to 6 degrees C. However, in all the microcosms, the acridine orange direct counts remained at about 10(5) cells per ml during the experimental period, which demonstrated that P. piscicida possesses a capacity to enter a viable but not culturable state. Moreover, dormant cells were always resuscitated by the addition of fresh medium to the microcosms, since we recovered numbers of culturable cells similar to the acridine orange direct counts. These resuscitated cells exhibited the same respiration rate as that seen prior to the start of the experiments. Although the biochemical, physiological, and serological characteristics; lipopolysaccharides; membrane proteins; and plasmid content of P. piscicida strains were unaffected during the starvation conditions, the dormant cells were smaller (dwarf cells) and had increased surface hydrophobicity. The starved cells maintained their infectivity and pathogenic potential for fish, with 50% lethal doses similar to those of the original strains.

Animals↗

PubMed1994

Iron uptake by Pasteurella piscicida and its role in pathogenicity for fish.

We evaluated the iron uptake mechanisms in Pasteurella piscicida strains as well as the effect of iron overload on the virulence of these strains for fish. With this aim, the capacity of the strains to obtain iron from transferrin and heme compounds as well as their ability to overcome the inhibitory activity of fish serum was analyzed. All the P. piscicida strains grew in the presence of the iron chelator ethylene-diamine-di (O-hydroxyphenyl acetic acid) or of human transferrin, which was used by a siderophore-mediated mechanism. The chemical tests and cross-feeding assays showed that P. piscicida produced a siderophore which was neither a phenolate nor a hydroxamate. Cross-feeding assays as well as preliminary chromatographic analysis suggest that this siderophore may be chemically related to multocidin. All the P. piscicida isolates utilized hemin and hemoglobin as an iron source, since the virulence of the strains increased when the fish were preinoculated with these compounds. This effect was stronger in the avirulent strains (50% lethal dose was reduced by 4 logs when fish were pretreated with hemin or hemoglobin). Only the pathogenic P. piscicida isolates were resistant to the bactericidal action of the fresh fish serum. The nonpathogenic strains grew in fish serum only when it was heat-inactivated or when it was supplemented with ferric ammonium citrate, hemin, or hemoglobin. In all the strains, at least three iron-regulated outer membrane proteins (IROMPs) (105, 118, and 145 kDa) were increased when the strains were cultured in iron-restricted medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The detection of two antigenic groups among Renibacterium salmoninarum isolates.

The analysis of the membrane proteins and their antigenic properties in a group of 14 geographically diverse strains of Renibacterium salmoninarum revealed the existence of antigenic diversity within this species. Eleven isolates, including the type strain ATCC 33209, shared a similar protein profile with a major component of 57 kDa whereas three strains showed a common pattern with a major protein of 30 kDa. The quantitative agglutination tests and Western blotting assays seem to indicate the existence of serological heterogeneity, with two distinct groups being detected.

Animals↗

PubMed1992

Detection of a vascular permeability factor in the extracellular products of Renibacterium salmoninarum.

The presence of vascular permeability factors in the extracellular products (ECP) of 10 strains of Renibacterium salmoninarum with different geographical origin and serological characteristics are reported. All the ECP produced haemorrhagic and/or oedematous zones at the injection site with a diameter ranging from 10-30 mm. However, the ECP samples did not display toxic effect in fish at the same dose as inoculated in rabbit (180-400 micrograms protein/0.1 ml). No differences were observed in the production of this dermatotoxic factor between the two antigenic groups found in this microorganism. Whereas heating (80 and 100 degrees C/15 min) the ECP samples resulted in a complete loss of their proteolytic activity, only a decrease (but not total inactivation) of the dermatotoxic effects was detected. Therefore, although proteases could be implicated in the permeability factor, they are not totally responsible for this activity.

Animals↗

PubMed1992

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.

Animals↗

PubMed1991

Plasmid associated virulence properties of environmental isolates of Aeromonas hydrophila.

The plasmid profiles, and their association with antimicrobial resistance, of 60 strains of Aeromonas hydrophila isolated from fish, shellfish and water were investigated. Only two strains were susceptible to all the antimicrobial agents tested; the highest incidences of resistance were to tetracycline (96.7%), prystanamycin (93.3%), ampicillin (91.7%) and cephalothin (91.7%). Forty strains harboured one or more plasmids and the plasmid profile most frequently detected (15%) was the association of three small plasmids of 4.2, 3.2 and 2.8 Mda. Curing experiments indicated that the plasmid-free derivative strains simultaneously lost their resistance determinants to tobramycin, neomycin, gentamicin and kanamycin. More than 90% of the strains tested produced siderophores and displayed haemolytic activity. However, the relationship between these virulence characters and the presence of plasmids was different; in 74.5% of the strains there was siderophore production and plasmids were detectable, whereas only 60% of the strains simultaneously possessed plasmids and haemolytic activity.

Aeromonas hydrophila↗

PubMed1991

Competitive dominance of antibiotic-producing marine bacteria in mixed cultures.

Competitive relationships between antibiotic-producing marine bacteria and other non-producers were studied in seawater mixed cultures. Producer strains showed a competitive advantage against non-producers as the latter were inhibited after a short time. Inhibition was also noted in mixed cultures of two producer strains. The inhibitory effect was not observed in a mixed culture with two non-producers, which indicates that an amensalist interaction occurred between populations of antibiotic-producing and non-producing marine bacteria. The results suggest that antibiotics could play an important role in the competitive relationships between marine bacterial populations.

Anti-Bacterial Agents↗

PubMed1991

MICs and MBCs of chemotherapeutic agents against Renibacterium salmoninarum.

The efficacies of 21 chemotherapeutic agents for controlling bacterial kidney disease were evaluated. The bactericidal and/or bacteriostatic effects of these drugs were tested against 11 Renibacterium salmoninarum strains with different origins. The most effective compounds displaying both bacteriostatic and bactericidal activity for all the isolates were tetracycline and erythromycin, with MICs ranging from less than 0.62 to 10.95 micrograms/ml for tetracycline and from less than 0.62 to 5.47 micrograms/ml for erythromycin. Whereas tetracycline showed identical MICs and MBCs, erythromycin showed bactericidal effects at concentrations of 5.47 to 21.87 micrograms/ml. Similarly, cefazolin and tiamulin proved to be very effective bactericidal compounds against the majority of R. salmoninarum isolates, with MBCs for 90% of the strains tested of 21.87 and 10.95 micrograms/ml, respectively. Neither nitrofuranes, quinolones, nor sulfonamides showed inhibitory effects on the growth of the strains.

Animals↗

PubMed1991

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins.

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Plasmids and factors associated with virulence in environmental isolates of Vibrio cholerae non-O1 in Bangladesh.

Plasmid profiles and factors associated with toxigenicity in 51 strains of Vibrio cholerae non-O1 isolated from water samples collected in Bangladesh were analysed. Eleven (21.5%) strains were found to harbour at least one plasmid of 1.7-115 Mda; seven of these strains shared a 115-Mda plasmid. Six of 13 strains tested gave positive cytotoxic and enterotoxic responses. However, two non-cytotoxic strains were enterotoxigenic. Only three of the six cytotoxic and enterotoxic strains caused haemagglutination of human erythrocytes which indicated that toxin production and haemagglutinating activity were unrelated in these V. cholerae non-O1 strains. Conjugal transfer assays demonstrated that the 115-Mda plasmid harboured by some of the toxigenic V. cholerae non-O1 strains carried genes coding for antibiotic resistance and cytotoxin production but not for enterotoxin production. However, this plasmid was also carried by non-toxigenic strains. Some other strains carrying no plasmids or only small-mol.-wt plasmids, were found to be toxigenic. Therefore, toxin production is not plasmid-mediated in all V. cholerae non-O1 strains. Regardless of their pathogenic potential, V. cholerae non-O1 strains possessed the capacity to grow in conditions of iron limitation and, under these conditions, synthesis of at least two new outer-membrane proteins was induced.

Bacterial Outer Membrane Proteins↗

PubMed1990

Changes in bacterial populations during red tides caused by Mesodinium rubrum and Gymnodinium catenatum in North West Coast of Spain.

Heterotrophic bacterial communities associated with four red tides caused by Mesodinium rubrum and Gymnodinium catenatum in two Galician Rias (North West Spain) were examined. Three of these were produced by the Mesodinium rubrum and the causative organism of a toxic bloom was Gymnodinium catenatum. In early stages of all the blooms, the diversity decreased but the total marine bacterial counts increased by one or two logs. Vibrio numbers were also incremented by two logs in two blooms of M. rubrum, while in the other bloom of this organism and in the red tide caused by G. catenatum a decrease in number of these bacteria was observed. A total of 116 bacterial strains were identified at the genus level and grouped into 12 phena. During the decomposition processes of two blooms of M. rubrum a zooplanktonic-type bacterial succession was observed (Vibrio, pseudomonads and Moraxella-Acinetobacter). On the other hand, during decomposition of the other red tide of M. rubrum and the bloom of G. catenatum, a typical phytoplanktonic-type succession occurred, as Pseudomonas and Moraxella groups became dominant for all the process. These results support the conflicting taxonomical position of M. rubrum. After the blooms, the changes in the community point towards the restablishment of the normal bacterial flora of the estuary (increase in diversity and decreases of bacterial numbers). Only the Vibrio strains, isolated from the non-toxic first and second red tides, displayed cytotoxic activities. A relationship among bacterial cytotoxicity and toxic effects of blooms cannot therefore be established.

Animals↗

PubMed1990

Vibrios associated with red tides caused by Mesodinium rubrum.

Vibrios were isolated from red tides caused by Mesodinium rubrum and also throughout the year in the Ria de Pontevedra, Spain. The isolates were grouped into 14 phena by numerical toxonomy. Strains associated with red tides were restricted to four phena: phena I and II were Vibrio alginolyticus, and phena III and IV were Vibrio tubiashii and Vibrio anguillarum, respectively. V. anguillarum-like strains (phena V through XI) predominated throughout the year outside the red tide areas. Cytotoxicity assays conducted in different poikilothermic and homoiothermic cell lines showed that cytotoxin production was not necessarily associated with the species selected during the red tides.

Animals↗

PubMed1990

Fish viral infections in northwest of Spain.

During a three years survey, a total of 149 samples from 20 farms of rainbow trout, salmon and turbot were examined for the presence of virus with the purpose to study the viral infections affecting cultured fish and their incidence in the fishfarms of Northwestern Spain. Infectious pancreatic necrosis virus (IPNV) was the only viral agent isolated from salmonid fish. Fry and fingerlings of trout showed the highest infection rate (24%). This virus was not detected in broodstock or embryonated eggs, although it was isolated from ovaric and seminal fluids and from juvenile carriers. From 24 samples of salmon analyzed, IPNV was only detected in one sample of juveniles. Examination of turbot led the isolation of a new virus belonging to the reoviridae family, which affected to the ongrowing population. All of the IPNV tested belonged to serotype Sp regardless of the origin of the trout stocks. During the monitorization of imported embryonated eggs, no virus was detected from any of the samples. However, in some case, IPNV was isolated when testing the fry obtained in our laboratory from those samples of imported eggs. Our findings indicate that: i) the analysis of fingerlings increase the probability to detect viral infections allowing us an optimal control of importations, and ii) most of the viral infections of fish take place in the own fish farms. The detection of mixed viral and bacterial infections emphasize the importance of carrying out an integral microbiological analysis to determine the causal agent(s) of fish mortalities.

Animals↗

PubMed1989

Influence of the growth conditions on the hydrophobicity of Renibacterium salmoninarum evaluated by different methods.

The influence of medium and salinity on the cell surface hydrophobicity of Renibacterium salmoninarum was investigated using three different methods: bacterial adherence to hydrocarbons (BATH), salt agglutination test (SAT), and binding to nitrocellulose filters (NCF). The possible relationship among hydrophobicity, haemagglutination and adherence to cell lines was also evaluated. R. salmoninarum showed to be highly hydrophobic regardless of the growth conditions or technique employed. Nevertheless, slight differences can be detected depending on the method used. In the SAT and NCF assays very uniform values were obtained within the strains. All the R. salmoninarum isolates agglutinated in (NH4)2SO4 in a range of 0.05-0.2 M and displayed a 77-100% of adherence to nitrocellulose filters. However, more variable results were observed in the BATH method depending on the hydrocarbon, buffer and strain employed. Although all of the isolates produced haemagglutinins for homeotherm erythrocytes, the majority of them failed to agglutinate poikilothermic red blood cells and were unable to adhere to fish cell lines. Therefore, a general correlation among hydrophobicity, agglutinating capacity for fish erythrocytes and adherence to fish cells can not be established for R. salmoninarum.

Agglutination Tests↗

PubMed1989

Purification and characterization of an antibacterial substance produced by a marine Alteromonas species.

An extracellular inhibitory substance produced by the marine Alteromonas strain P-31 (NCMB 2144) was isolated and purified. The inhibitor was a macromolecule with a molecular weight of 90,000 estimated by Sephadex G-100 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitory activity was antagonized by proteinase K and beta-amylase and inactivated by heating at 80 degrees C for 30 min. The purified substance exhibited two typical absorption bands in the infrared spectrum at 1,650 and 1,075 cm-1, corresponding to peptide linkages and carbohydrate residues, respectively. These findings allowed us to characterize the antimicrobial compound as a thermolabile glycoprotein. The substance exhibited a broad inhibitory spectrum, being active against clinical and environmental isolates from related and nonrelated taxonomical bacterial groups as well as against the producer strain and other similar marine bacterial strains. The inhibitory glycoprotein did not display cytotoxicity toward mammalian and fish cell lines.

Animals↗