Biomedical subjects

J L Romalde

Publications and source records attributed to J L Romalde.

At least 19 recordsLinked to original sources

PubMed2005

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods.

AIM: The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. METHODS AND RESULTS: Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. CONCLUSIONS: Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.

Animals↗

PubMed2005

Vaccination strategies to prevent emerging diseases for Spanish aquaculture.

In recent years, three serious diseases have emerged in Spanish aquaculture. These are lactococcosis caused by Lactococcus garvieae, which is of economical importance in rainbow trout (Oncorhynchus mykiss); pseudomonadiasis caused by Pseudomonas anguilliseptica which affects gilthead seabream (Sparus aurata) and turbot (Scophthalmus maximus); and flexibacteriosis caused by Tenacibaculum maritimum which became a devastating problem in the emerging culture of sole (Solea spp). To obtain useful information for the design and development of new vaccines, antigenic characterisation of representative strains was performed. In this work we present the strategies adopted for the vaccine formulation (strains included, use of adjuvants) and administration (route, necessity of booster, etc.). The results from laboratory and/or field vaccination trials performed showed that for lactococcosis, protection lasting for five months was obtained with an oil-adjuvanted bacterin formulation. Unadjuvanted bacterin gave only a short duration of protection, which could, however, be prolonged by an antigen boost administered via the feed. A bacterin against Pseudomonas anguilliseptica gave protection for 12 weeks when tested in an experimental challenge trial in turbot. Besides the flexibacteriosis vaccine developed by our group for turbot, and due to the antigenic host-associated variability within T. maritimum, a new bacterin was developed against this bacterium to be used specifically in sole. This new bacterin, administered to sole by intraperitoneal injection, yielded RPS values of 94 % six weeks after immunization. In conclusion, these results suggest that vaccination constitutes a cost-effective method of controlling diseases that have emerged in the most important fish species being cultured in Spain.

Adjuvants, Immunologic↗

PubMed2004

Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as determined by randomly amplified polymorphic DNA-PCR.

AIM: The aim of the present study was to evaluate the intraspecific genetic variability within Tenacibaculum maritimum strains isolated from different species of marine fish. METHODS AND RESULTS: Twenty-nine strains isolated from five different fish species and three reference strains were characterized by randomly amplified polymorphic DNA (RAPD) method. Cluster analysis of RAPD-PCR profiles showed that the strains, regardless of the oligonucleotide primer employed (P2 and P6), were separated into two main groups that strongly correlated with the host species and/or O-serotypes described for this pathogen. One group composed all strains isolated from sole (Solea senegalensis and S. solea) and gilthead seabream (Sparus aurata), and the other compiled the T. maritimum isolates from yellowtail (Seriola quinqueradiata), Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus). An important exception was observed in the RAPD patterns of the reference strains, which were included in different genetic groups depending on the primer employed. CONCLUSIONS: The results obtained demonstrated genetic variability within the T. maritimum isolated from different marine fish. Such genetic variability proved to be strongly associated with the host and/or serogroups described for this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD analysis constitutes a valuable molecular technique for epidemiological studies of T. maritimum. Interestingly, this is the first report of intraspecific differentiation and characterization of T. maritimum strains isolated from cultured fish.

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↗

PubMed2003

Lactococcus garvieae in wild Red Sea wrasse Coris aygula (Labridae).

Lactococcus garvieae infection in wild wrasse Coris aygula is reported, and the serological and molecular characteristics of the isolate are described. This is the first evidence of the presence of this pathogen in the Red Sea, and it follows the recent diagnosis of Mycobacterium marinum and Streptococcus iniae in wild fish from the same region. Whether all 3 pathogens are strains endemic to the Red Sea, or recent introductions into the region, remains to be determined, but their appearance over a period of a few years in wild fish populations in the northern Red Sea is consistent with an emerging trend affecting marine organisms on a global level in areas subjected to intense anthropogenic impacts.

Animals↗

PubMed2002

Dormancy as a survival strategy of the fish pathogen Streptococcus parauberis in the marine environment.

The fate of Streptococcus parauberis in seawater and sediment microcosms at different temperatures (6 and 22 degrees C) was investigated by comparing the survival dynamics of 2 strains of this bacterial species, isolated respectively from diseased turbot and cattle. The turbot and the bovine isolate showed similar survival kinetics, remaining culturable for approximately 1 mo in water and 6 mo in sediment. A slight influence of temperature on the stability of the cells was observed, in that the number of culturable cells was about 1 log10 unit higher at 6 than at 22 degrees C. During the starvation period, the metabolic activity of the cells, after suffering a strong reduction during the first 12 d, stabilized at levels ranging from 20 to 40% of the initial values. However, in all the microcosms, the acridine orange (AO) and 4',6-diamidino-2-phenilindole (DAPI) counts remained at about 10(5) cells ml(-1) throughout the experimental period, even when cells became undetectable by standard plate count methods. The addition of fresh medium to microcosms containing nonculturable cells induced the return to culturability of S. parauberis strains. On the basis of these results, it seems that S. parauberis has the ability to enter into a viable but nonculturable (VBNC) state. Dormant cells of the turbot isolate maintained their infectivity and pathogenic potential for fish.

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↗

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

Assessment of a magnetic bead-EIA based kit for rapid diagnosis of fish pasteurellosis.

The accuracy of the magnetic beads-EIA based BIONOR AQUAEIA-Pp kit for the rapid diagnosis of pasteurellosis was evaluated. The kit reacted with all the Photobacterium damselae subsp. piscicida strains included in this study, with a detection limit of 10(4) bacteria/ml. However, non-specific reactions were observed with isolates of Ph. damselae subsp. damselae or Ph. histaminum when the bacterial concentration was high (10(9)-10(10) bacteria/ml). Similar findings in specificity and sensitivity were observed when the kit was applied to experimentally infected fish tissues. However, since those bacterial species are not usually found in the fish species susceptible to pasteurellosis, the AQUAEIA kit appears applicable for a rapid screening of the disease. In addition, when the kit was utilized to analyze cultured populations of seabream, it allowed the detection of the pathogen, not only in individuals affected by the disease, but also in asymptomatic carrier fish. Furthermore, the positive detection of Ph. damselae subsp. piscicida in broodstock gonads, seminal, and ovaric fluids, and also in eggs indicated the possibility of vertical transmission of pasteurellosis.

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

Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling.

A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis. A total of 90.9% of V. tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands. In contrast, the V. tapetis strains showed a low degree of similarity with six reference Vibrio species tested. All V. tapetis strains harbored a large plasmid of 74.5 kb. This plasmid was not detected in any of the other Vibrio species. In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V. tapetis possessed the same cleavage pattern. The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb. The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V. tapetis strains belonged to the same ribotype when the enzyme PvuII was used.

Bacterial Typing Techniques↗

PubMed1997

Edwardsiellosis in wild striped bass from the Chesapeake Bay.

The first epizootic of edwardsiellosis, caused by Edwardsiella tarda, is described. The epizootic occurred in the Chesapeake Bay, Maryland (USA) during the summer and autumn of 1994, and affected wild adult striped bass (Morone saxatilis). Clinical signs included numerous irregular coalescing hemorrhagic ulcers on the body and fins that were distinctly malodorous. Internally, the body cavity was filled with abundant yellowish or sanguinous mucoid fluid, and the visceral organs had multiple tiny white foci. The intestines contained thick white opaque mucus. Histopathological lesions included ulcerative dermatitis, cardiac endothelial hyperplasia, and necrotic foci and granulomata in multiple organs. A bacterium isolated in pure culture was characterized taxonomically and serologically as the wild-type or classical biotype of E. tarda: In infectivity trials, it was pathogenic for striped bass, gilthead seabream (Sparus aurata), and turbot (Scophthalmus maximus) with an LD50 of about 10(5) cells; however, the isolate was non-virulent for mice (LD50 > 10(8) cells). The isolate also was resistant to the bacteriolytic activity of normal fish skin mucus.

Animals↗

PubMed1997

Immunization with bacterial antigens: pasteurellosis.

Pasteurella piscicida is the aetiological agent of pasteurellosis or pseudotuberculosis, one of the most threatening diseases of wild and cultured marine fish. This bacterium has been reported from many geographical areas including USA, Japan, and the Mediterranean countries. In this review, the biochemical, serological, and molecular characteristics of the pathogen are described. In addition, its main virulence mechanisms, such as the presence of capsule, the iron uptake system, and the phospholipase activity, as well as their putative role in the pathogenicity of P. piscicida are also discussed. Finally, a detailed survey of the strategies for controlling the disease is performed, with a special emphasis on the vaccination programmes and the most effective protective antigens to be included in the vaccine formulations.

Adjuvants, Immunologic↗

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↗