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Bastardo A.,University of Santiago de Compostela | Bohle H.,ADL Diagnostic Chile Ltd | Ravelo C.,Estacion de Investigaciones Hidrobiologicas de Guayana | Toranzo A.E.,University of Santiago de Compostela | Romalde J.L.,University of Santiago de Compostela
Diseases of Aquatic Organisms | Year: 2011

We investigated 11 strains of Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), that had been isolated from Atlantic salmon Salmo salar L. farmed in Chile and previously vaccinated against ERM. Phylogenetic analysis of the 16S rRNA gene sequences confirmed the identification of the salmon isolates as Y. ruckeri. A comparative analysis of the biochemical characteristics was made by means of traditional and commercial miniaturised methods. All studied isolates were motile and Tween 80 positive, and were identified as biotype 1. In addition, drug susceptibility tests determined high sensitivity to sulphamethoxazole/trimethroprim, oxytetracycline, ampicillin and enrofloxacin in all isolates. Serological assays showed the presence of O1a, O1b and O2b serotypes, with a predominance of the O1b serotype in 9 strains. Analysis of the lipopolysaccharide profiles and the correspondent immunoblot confirmed these results. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the outer membrane proteins revealed that all Chilean strains had profiles with a molecular weight range between 34 and 55 kDa, with 3 distinct groups based on differences in the major bands. Genotyping analyses by enterobacterial repetitive intergenic consensus (ERIC-) and repetitive extragenic palindromic (REP-)PCR techniques clearly indicated intraspecific genetic diversity among Chilean Y. ruckeri strains. © Inter-Research 2011. Source


Bastardo A.,University of Santiago de Compostela | Sierralta V.,National Major San Marcos University | Leon J.,National Major San Marcos University | Ravelo C.,Estacion de Investigaciones Hidrobiologicas de Guayana | Romalde J.L.,University of Santiago de Compostela
Aquaculture | Year: 2011

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. © 2011 Elsevier B.V. Source


Bastardo A.,University of Santiago de Compostela | Ravelo C.,Estacion de Investigaciones Hidrobiologicas de Guayana | Romalde J.L.,University of Santiago de Compostela
Environmental Microbiology | Year: 2012

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. 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. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd. Source


Bastardo A.,University of Santiago de Compostela | Ravelo C.,Estacion de Investigaciones Hidrobiologicas de Guayana | Romalde J.L.,University of Santiago de Compostela
Applied Microbiology and Biotechnology | Year: 2012

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 interrun 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. © Springer-Verlag 2012. Source


Bastardo A.,University of Santiago de Compostela | Ravelo C.,Estacion de Investigaciones Hidrobiologicas de Guayana | Castro N.,University of Santiago de Compostela | Romalde J.L.,University of Santiago de Compostela
Fish and Shellfish Immunology | Year: 2012

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. © 2012 Elsevier Ltd. Source

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