Biomedical subjects

J L Romalde

Publications and source records attributed to J L Romalde.

34 records · Page 2Linked to original sources

PubMed1996

Influence of the capsular layer on the virulence of Pasteurella piscicida for fish.

The influence of the capsule of Pasteurella piscicida on the cell surface properties of this microorganism as well as on the virulence and the capacity of the strains to grow in fish sera was examined. Although all the P. piscicida strains synthetized an additional exostructure in glucose-enriched-medium, only virulent strains constitutively synthetized capsule. The cell surface of all the P. piscicida isolates showed a low hydrophobic nature. No strains pelleted in broth culture (SP-) and all of them were stable after boiling (PAB). All isolates attached to the fish cell line CHSE-214 with values of adherence ranging from 2 to 5% of the initial bacterial inoculum. The presence of induced capsular material caused changes in some cell surface characteristics such as hydrophobicity and stability after boiling. A decrease in the adherence capacity of all the P. piscicida strains was also observed. However, the capsule increased the degree of virulence for fish of the nonpathogenic strains (LD50 was reduced in about 4 log) and conferred to all the isolates resistance to serum killing. Therefore, these results indicate that the presence of capsule can play an important role in the pathogenesis of P. piscicida.

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↗

PubMed1996

New molecular methods for the detection of hepatitis A and Norwalk viruses in shellfish.

Outbreaks of viral enteric diseases after consumption of shellfish are a major health risk. Methodological problems (such as toxicity for cell cultures and low viral concentrations) and the unculturability of some strains (i.e. hepatitis A virus, Norwalk virus) have made it difficult to study those viruses in the environmental samples. Currently, the analysis of the hygienic quality of marketable shellfish is determined by the use of fecal indicator bacteria, but their reliability in determining viral pollution of shellfish is very low. Recent biotechnology developments are providing available rapid, sensitive, and specific tools for detecting food-borne viruses in shellfish and in shellfish-growing waters. In this paper, a review of these new molecular methods is carried out, discussing their advantages and possible applications.

DNA Probes↗

PubMed1995

Fatty acid profiles of "Pasteurella" piscicida: comparison with other fish pathogenic gram-negative bacteria.

The fatty acid methyl ester (FAME) profiles of "Pasteurella" piscicida were determined by gas chromatography and subjected to numerical analysis in comparison with those obtained for Vibrio anguillarum, Aeromonas salmonicida and Pasteurella species of clinical origin. The bacterial species studied shared important characteristics with respect to their FAME content: in all of them the saturated and unsaturated fatty acids of 16 carbon atoms were the predominant fatty acids. However, distinguishing features could be detected for each pathogen. Using either single linkage or complete linkage algorithms, strains were divided into four phena that corresponded to the different species, but showed a high degree of correlation among them. Although single linkage discriminated strains better within each phenum, complete linkage was more useful to establish the relationships among clusters. The results obtained support the idea that "Pasteurella" piscicida is related to members of the genera Vibrio and Aeromonas and indicate the need for exhaustive genetic studies to clarify the taxonomic position of this fish pathogen.

Animals↗

PubMed1995

Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR.

A method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues by PCR was developed. Virus was added to the stomach and hepatopancreatic tissues of oysters or hard-shell clams, and viral nucleic acids were purified by a modification of a previously described method (R.L. Atmar, T.G. Metcalf, F.H. Neill, and M.K. Estes, Appl. Environ. Microbiol. 59:631-635, 1993). The new method had the following advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PCR; and (iv) use of chloroform-butanol in place of the chlorofluorocarbon trichlorotrifluoroethane. In addition, internal standards for both Norwalk virus and hepatitis A virus were made which demonstrated when inhibitors to reverse transcription-PCR were present and allowed quantitation of the viral nucleic acids present in samples. This assay can be used to investigate shellfish-associated gastroenteritis outbreaks and to study factors involved in virus persistence in shellfish.

Animals↗

PubMed1994

Capsular polysaccharide expressed by Pasteurella piscicida grown in vitro.

Pasteurella piscicida grown in a glucose-rich medium produces a capsule that can be see under light and electron microscopy. The capsular polysaccharide was purified and characterized by chemical and HPLC analysis. The polymer has the composition glucose/mannose/N-acetylgalactosamine/galacturonic acid/acetic acid in the molar ratios of approximately 2.5:1.3:0.5:0.4:2.5. The polysaccharide was immunogenic in rabbits and did not cross-react with antibodies against the O-antigen lipopolysaccharide.

Animals↗

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

In situ detection of hepatitis A virus in cell cultures and shellfish tissues.

An in situ transcription method was developed to detect hepatitis A virus RNA in both cell cultures and shellfish tissues. Radiolabeled cDNA copies were synthesized in situ by reverse transcriptase-directed transcription after annealing with a specific primer to the viral RNA. Both tritium (3H) and 35S were useful in the in situ transcription reaction, but the use of 3H resulted in a lower background and finer detail in the localization of viral particles. Application of the method to different organs of oysters which had bioaccumulated hepatitis A virus allowed the first in situ localization of the virus, specifically in stomach and hepatopancreatic tissues.

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↗

PubMed1993

Pathological activities of Yersinia ruckeri, the enteric redmouth (ERM) bacterium.

The adherence and invasive capacities as well as the pathobiological activities exhibited by Yersinia ruckeri were examined. Although adhesive ability was dependent on the cell-line employed, all the strains showed moderate adhesion and invasiveness in the salmon cell-line CHSE-214. With regard to the extracellular products (ECP) all of them were strongly toxic for fish with LD50 ranging from 2 to 9.12 micrograms protein per g fish. In addition, all the ECP samples showed caseinase, gelatinase, amylase, lipase and phospholipase activities, hydrolysed esculin and displayed hemolytic activities for trout, salmon, sheep and human erythrocytes. Heat treatment (100 degrees C for 10 min) caused the loss of all these biological activities except the hydrolysis of gelatin. On the other hand, SDS-PAGE analysis of the LPS and protein components of the ECP revealed variations among strains depending on the serotype. The lack of lethal effects of the LPS present in the ECP was also demonstrated.

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↗

PubMed1992

Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines.

Animals↗

PubMed1991

Use of pulsed field gel electrophoresis to size the chromosome of the bacterial fish pathogen Yersinia ruckeri.

Field inversion gel electrophoresis (FIGE) and contour-clamped homogeneous field (CHEF) electrophoresis were used to analyse the chromosome of Yersinia ruckeri. The 8 base-pair recognition endonucleases, NotI and SfiI, generated less than 47 DNA fragments whose size and distribution were appropriate for pulsed field separation. Each isolate displayed a characteristic restriction pattern, with about 20% of bands in common. Depending on the strain used, the estimated genome size for this bacterial fish pathogen ranged from 4460 to 4770 kilobase pairs.

Animals↗

PubMed1991

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system.

This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri. A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogeneicity of Y. ruckeri for fish.

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↗