Carmen Ravelo

Publications (10)26.72 Total impact

• Article: Highly sensitive detection and quantification of the pathogen Yersinia ruckeri in fish tissues by using real-time PCR.

  Asmine Bastardo, Carmen Ravelo, Jesús L Romalde
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  ABSTRACT: Yersinia ruckeri is the causative agent of enteric redmouth diseases (ERM) and one of the major bacterial pathogens causing losses in salmonid aquaculture. Since recent ERM vaccine breakdowns have been described mostly attributed to emergence of Y. ruckeri biotype 2 strains, rapid, reproducible, and sensitive methods for detection are needed. In this study, a real-time polymerase chain reaction (PCR) primer/probe set based on recombination protein A (recA) gene was designed and optimized to improve the detection of Y. ruckeri. The primer/probe set proved to have a 100 % analytical specificity and a sensitivity of 1.8 ag μl(-1), equivalent to 1.7 colony-forming units (CFU) ml(-1), for purified DNA, 3.4 CFU g(-1) for seeded liver, kidney, and spleen tissues, and 0.34 CFU/100 μl(-1) for seeded blood, respectively. The assay was highly reproducible with low variation coefficient values for intra- and inter-run experiments (2.9 % and 9.5 %, respectively). Following optimization, the assay was used to detect changes in the bacterial load during experimental infection. Rainbow trout (Onchorhynchus mykiss) were exposed to two strains of Y. ruckeri (biotype 1 and biotype 2) by intraperitoneal inoculation. Internal organs (liver, kidney, spleen) and blood were biopsied from dead fish daily for 15 days to quantify copies of pathogen DNA per gram of tissue. The findings showed the efficacy of this real-time PCR assay to quantify Y. ruckeri cells in the fish tissues and also confirmed this assay as a non-lethal method for the detection of this pathogen in blood samples.
  Applied Microbiology and Biotechnology 08/2012; 96(2):511-20. · 3.42 Impact Factor
• Article: A polyphasic approach to study the intraspecific diversity of Yersinia ruckeri strains isolated from recent outbreaks in salmonid culture.

  Asmine Bastardo, Carmen Ravelo, Jesús L Romalde
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  ABSTRACT: A polyphasic analysis was carried out on Yersinia ruckeri strains isolated from recently outbreaks in vaccinated fish using a combination of different phenotypic and molecular typing methods in order to study their variability and epidemiological relationships. Eighty strains were subjected to biotyping with conventional tests and API 20E system, serotyping, outer membrane protein (OMP) and lipopolysaccharide (LPS) profiling, and genetic fingerprinting by ERIC-PCR and REP-PCR techniques. The strains showed a high diversity, as evidenced by the formation of different phenotypic groups mainly related to the serotypes, LPS and OMP profiles. The diversity among all isolates, calculated as Simpson's diversity index (Di), varied between 0.35 (REP-PCR) and 0.70 (OMP). The most discriminative values (Di value ≥0.86) were obtained from any combination of three methods including biotype, serotype, API 20E profile, LPS or OMP. With the combination of all typing methods used a Di value of 0.90 was obtained. Association between different groups to the host species was evidenced. Furthermore, it seems that strains with similar characteristics are associated with recent outbreaks occurred in vaccinated fish in certain geographical areas. Our results emphasize the usefulness of using a combination of several different typing methods for epidemiological and bacterial diversity studies.
  Veterinary Microbiology 06/2012; 160(1-2):176-82. · 3.33 Impact Factor
• Article: Multilocus sequence typing reveals high genetic diversity and epidemic population structure for the fish pathogen Yersinia ruckeri.

  Asmine Bastardo, Carmen Ravelo, Jesús L Romalde
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  ABSTRACT: Yersinia ruckeri is the causative agent of enteric redmouth in fish and one of the major bacterial pathogens causing losses in salmonid aquaculture. Previously typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. In this work, we describe a multilocus sequence typing (MLST) scheme for Y.ruckeri based on the internal fragment sequence of six housekeeping genes. This MLST scheme was applied to 103 Y.ruckeri strains from diverse geographic areas and hosts as well as environmental sources. Sequences obtained from this work were deposited and are available in a public database (http://publmst.org/yruckeri/). Thirty different sequence types (ST) were identified, 21 of which were represented by a single isolate, evidencing high genetic diversity. ST2 comprised more than one-third of the isolates and was most frequently observed among isolates from trout. Two major clonal complexes (CC) were identified by eBURST analysis showing a common evolutionary origin for 94 isolates forming 21 STs into CC1 and for 6 isolates of 6 STs in the CC2. It was also possible to associate some unique ST with isolates from recent outbreaks in vaccinated salmonid fish.
  Environmental Microbiology 03/2012; 14(8):1888-97. · 5.84 Impact Factor
• Article: Effectiveness of bivalent vaccines against Aeromonas hydrophila and Lactococcus garvieae infections in rainbow trout Oncorhynchus mykiss (Walbaum).

  Asmine Bastardo, Carmen Ravelo, Nuria Castro, José Calheiros, Jesús L Romalde
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  ABSTRACT: Lactococcus garvieae and Aeromonas hydrophila are bacterial pathogens affecting salmonids and other fish species and cause of heavy losses in aquaculture. Diseases caused by these bacteria can be controlled satisfactory by immunization using monovalent vaccines. In this study, the protective efficacy of two bivalent vaccines against L. garvieae and A. hydrophila was evaluated in rainbow trout (Oncorhynchus mykiss). Bivalent formulations, containing formalin-inactivated bacteria, were prepared as an aqueous bacterin and as an adjuvanted vaccine using montanide ISA-763. Protection against L. garvieae and A. hydrophila was tested at day 30 and 90 post-vaccination. High levels of protection were achieved for the aqueous and adjuvanted bivalent vaccines against L. garvieae (RPS of 100% and 95.3%) and A. hydrophila (RPS of 100% and 95.3%) at day 30 post-vaccination. Significant differences (p < 0.05) were found between the RPS at days 30 and 90 post-immunization with a decrease in the protection levels for the aqueous bivalent vaccine against L. garvieae (RPS 76.2%) and A. hydrophila (RPS 85%), but not for the adjuvanted vaccine (RPS of 90% against L. garvieae and 95% against A. hydrophila). In addition, high antibody levels were observed in the vaccinated fish at day 15 post-immunization using both vaccines. Our results demonstrate that these bivalent vaccines can effectively protect rainbow trout against L. garvieae and A. hydrophila and could offer an appropriate strategy to prevent these infections in rainbow trout farms.
  Fish &amp Shellfish Immunology 02/2012; 32(5):756-61. · 3.32 Impact Factor
• Source

  Available from: Jesus L Romalde

  Article: Streptococcus phocae, an emerging pathogen for salmonid culture.

  Jesús L Romalde, Carmen Ravelo, Iván Valdés, Beatriz Magariños, Eduardo de la Fuente, Carolina San Martín, Rubén Avendaño-Herrera, Alicia E Toranzo
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  ABSTRACT: This work describes the characterization of the causal agent of disease outbreaks that, from 1999, occurred repeatedly during the summer months (temperatures higher than 15 degrees C) in Atlantic salmon (Salmo salar) cage-farmed in Chile affecting both smolts and adult fish cultured in estuary and marine waters, reaching in some occasions a cumulative mortality up to 25% of the affected population. Diseased fish showed exophthalmia with accumulation of purulent and haemorrhagic fluid around eyes, and ventral petechial haemorrhages. At necropsy, haemorrhage in the abdominal fat, pericarditis, and enlarged liver, spleen and kidney are common pathological changes. Gram-stained smears revealed the presence of Gram-positive cocci, beta-hemolytic, negative for oxidase and catalase tests. Although biochemical characterization of the isolates using the miniaturized system rapid ID 32 Strep suggested their assignation to genus Gemella, sequencing and RFLP analysis of the 16S rRNA revealed that bacteria associated with the mortalities belong to Streptococcus phocae. Serological studies demonstrated that all the salmon isolates are antigenically homogeneous, which can facilitate the development of preventive measures and, although sharing some antigenical determinants, they belong to a different Lancefield group than the type strain isolated from seals. On the basis of these facts, we conclude that the species S. phocae is an emerging pathogen for salmonid culture in Chile, and it should be included as a new member of the warm water streptococcosis.
  Veterinary Microbiology 08/2008; 130(1-2):198-207. · 3.33 Impact Factor
• Article: Existence of two O-serotypes in the fish pathogen Pseudomonas anguilliseptica.

  Sonia López-Romalde, Beatriz Magariños, Carmen Ravelo, Alicia E Toranzo, Jesús L Romalde
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  ABSTRACT: The serological characteristics of a group of 32 Pseudomonas anguilliseptica strains isolated in Spain from seabream (Sparus aurata) and turbot (Scophthalmus maximus) were compared with a total of 18 collection strains of this bacterial species with different geographical and host origin. The employment of different techniques, including slide agglutination, microagglutination and dot blot, allowed us to establish two serological groups, one comprising practically all the eel isolates, and the other including the majority of isolates from other fish species. The study of the lipopolysaccharides (LPS) and outer membrane proteins (OMP) corroborated these results, indicating that the serological differences among strains are due to the somatic antigen and not to antigenic determinants of protein nature. Therefore, a serological scheme of two "O" serotypes is proposed for P. anguilliseptica. The results obtained will be of importance for epidemiological studies as well as for the design of adequate vaccine formulations.
  Veterinary Microbiology 08/2003; 94(4):325-33. · 3.33 Impact Factor
• Article: Molecular fingerprinting of fish-pathogenic Lactococcus garvieae strains by random amplified polymorphic DNA analysis.

  Carmen Ravelo, Beatriz Magariños, Sonia López-Romalde, Alicia E Toranzo, Jesús L Romalde
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  ABSTRACT: In this work, we used the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Lactococcus garvieae, an important pathogen for fish. Fifty-seven strains with different hosts and geographical origins, including Japan and several countries of the Mediterranean area such as Spain, Portugal, France, Italy, England, and Turkey, were analyzed. Two primers, oligonucleotides 5 and 6 (Pharmacia Biotech) were utilized; primer 5 was the most discriminative, since allowed us to differentiate 10 RAPD -types related to the origin of the strains. Regardless of the oligonucleotide primer employed, the 57 isolates of L. garvieae studied were separated into three genetic groups, composed of the Spanish, Portuguese, English, and Turkish strains (group A), the Italian and French strains (group B), and the Japanese strains (group C). The similarity of isolates within each group, estimated on the basis of the Dice coefficient, ranged from 75 to 100%. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.
  Journal of Clinical Microbiology 03/2003; 41(2):751-6. · 4.15 Impact Factor
• Article: Use of adjuvanted vaccines to lengthen the protection against lactococcosis in rainbow trout (Oncorhynchus mykiss)

  Carmen Ravelo, Beatriz Magariños, M. Carmen Herrero, Llorenç Costa, Alicia E. Toranzo, Jesús L. Romalde
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  ABSTRACT: In this work, we evaluated the effect of the inclusion of different adjuvants in the vaccine formulation against Lactococcus garvieae in comparison with an aqueous bacterin. The aqueous and non mineral oil adjuvanted vaccines (Montanide-ISA-763-A and Aquamun) yielded a good protection 4 weeks after the immunization (RPS of 82.6, 84.8 and 86.9, respectively). The protective rates obtained for the adjuvants Montanide-IMS-2212 (water based nanoparticles with immunostimulant) and Carbomer (high molecular weight polymer organic of polyacrylic acid) were 45.7% and 56.5%, respectively. We also evaluated the duration of protection confered by Aquamun adjuvanted-vaccine in comparison with the aqueous bacterin. Three months after vaccination, the RPS values obtained with the aqueous vaccine was 40% whereas with the Aquamun adjuvanted-vaccine the protection increased to values of 92%. In addition, a long lasting protection was observed with this adjuvanted vaccine with RPS values of 90% and 83.3% in the challenges performed at 6 and 8 months post-vaccination, respectively.
  Aquaculture. 251:153-158.
• Article: Phenotypical and genetic characterization of Yersinia ruckeri strains isolated from recent outbreaks in farmed rainbow trout Oncorhynchus mykiss (Walbaum) in Peru

  Asmine Bastardo, Veronica Sierralta, Jorge León, Carmen Ravelo, Jesús L. Romalde
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  ABSTRACT: A total of 30 strains of Yersinia ruckeri causing recent outbreaks in Peruvian trout culture systems, were studied by means of biochemical characteristics, serology, lipopolysaccharide (LPS) and outer membrane protein (OMP) analysis, and ERIC and REP PCR fingerprinting. All the Peruvian isolates were found to be fermentative, oxidase negative and positive for decarboxylation of lysine and ornithine and utilization of glucose and mannitol, allowing their presumptive identification as Y. ruckeri. Sequencing of the 16S rRNA gene confirmed that isolates were indeed Y. ruckeri (> 99.98% identity). Although most of the strains studied were motile and lipase positive corresponding to the biotype 1 of Y. ruckeri, 5 of these strains were negative from both tests, being identified as biotype 2. In addition, drug susceptibility tests determined high sensitivity to sulfamethoxazole/trimethoprim, oxytetracycline, ampicillin and enrofloxacin in all the isolates. Serologically, all the Peruvian strains studied were identified as belonging to the serotype O1 subgroup a. Analysis of the lipopolysaccharide (LPS) as well as total and outer membrane proteins (OMPs) profiles and the correspondent inmunoblotting, supported these results. Genotyping performed by means of ERIC- and REP-PCR determined major correlation of the Peruvian isolates with the type strain NCIMB 2194T regardless of the biotype.
  Aquaculture. 317:229-232.
• Article: Oral immunization using alginate microparticles as a useful strategy for booster vaccination against fish lactoccocosis

  Jesús L Romalde, Asteria Luzardo-Alvárez, Carmen Ravelo, Alicia E Toranzo, José Blanco-Méndez
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  ABSTRACT: In this work, oral vaccination with encapsulated and non-encapsulated antigens was preliminary evaluated as alternative immunization procedures against trout lacotococcosis. Fish were oral immunized with a variety of different Lactococcus garvieae vaccines including encapsulated and non-encapsulated bacterial cells. An aqueous-based bacterin administered by intraperitoneal injection (i.p.) was employed as positive control. The best protective rates by oral immunization were obtained with the alginate-encapsulated vaccine (RPS of 50%); this, however, does not warrant the use of this formulation as primary immunization method. We also evaluated the efficacy of this alginate-encapsulated vaccine as booster immunization strategy. Fish were primary i.p. vaccinated with the aqueous-based vaccine and 3 months later were boostered with the oral vaccine. Four weeks after revaccination protection reached RPS values of 87%, which indicated the value of this encapsulated vaccine to increase the duration of the protection of rainbow trout against lacotoccosis.
  Aquaculture.