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Jesús L. Romalde - One of the best experts on this subject based on the ideXlab platform.
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Polymerase Chain Reaction Amplification of Repetitive Intergenic Consensus and Repetitive Extragenic Palindromic Sequences for Molecular Typing of Pseudomonas Anguilliseptica and Aeromonas salmonicida
Journal of Aquatic Animal Health, 2008Co-Authors: Roxana Beaz-hidalgo, Sonia López-romalde, Alicia E. Toranzo, Jesús L. RomaldeAbstract:Abstract The aim of this study was to evaluate the use of two molecular techniques, repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and repetitive intergenic consensus PCR (ERIC-PCR), as epidemiological tools with which to discriminate among genetically distinct strains within two bacterial fish pathogens, Pseudomonas Anguilliseptica and Aeromonas salmonicida. A total of 30 A. salmonicida and 52 P. Anguilliseptica were analyzed. For P. Anguilliseptica, three different major fingerprints were obtained with both techniques, which defined three genomic groups: one was composed of strains isolated from eels Anguilla spp., the second of strains from turbot Scophthalmus maximus and blackspot seabream (also known as red seabream) Pagellus bogaraveo, and the third of strains from other fish species, such as gilthead seabream (also known as gilthead bream) Sparus auratus, sea bass Dicentrarchus labrax (also known as European bass Morone labrax), and salmonids. In the case of A. salmonicida, p...
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Phenotypic, serological and genetic characterization of Pseudomonas Anguilliseptica strains isolated from cod, Gadus morhua L., in northern Europe
Journal of Fish Diseases, 2007Co-Authors: Sabela Balboa, Hugh W. Ferguson, Jesús L. RomaldeAbstract:The biochemical, serological and genetic characteristics of six strains of Pseudomonas Anguilliseptica isolated from cod, Gadus morhua, in Scotland were compared to well characterized isolates of this same bacterial species but of different origin. Biochemical and physiological analyses showed that this group of isolates was highly homogeneous, their characteristics matching previous descriptions of the pathogen. Similar results were obtained for the six cod isolates in the serological assays, all of them belonging to the serotype O1. Marked homogeneity was observed also in the genetic study, analysed by means of RAPD, ERIC-PCR and REP-PCR procedures, showing that they were similar to isolates from gilthead seabream, Sparus aurata, black spot seabream, Pagellus bogaraveo, and turbot, Psetta maxima. Virulence assays demonstrated that the cod isolates were highly pathogenic for turbot and sole, Solea senegalensis, with LD50 between 7.6 · 10 4 and 5 · 10 7 bacterial cells per fish.
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Vaccination strategies to prevent emerging diseases for Spanish aquaculture.
Developments in biologicals, 2005Co-Authors: Jesús L. Romalde, Sonia López-romalde, Beatriz Magariños, Carmen Ravelo, Ruben Avendaño-herrera, Alicia E. ToranzoAbstract: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.
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Development and validation of a PCR-based protocol for the detection of Pseudomonas Anguilliseptica
Fish Pathology, 2004Co-Authors: Jesús L. Romalde, Sonia López-romalde, Beatriz Magariños, Carmen Ravelo, Alicia E. ToranzoAbstract:A PCR-based protocol for the detection of the emerging fish pathogen Pseudomonas Anguilliseptica was developed, using the primer set Psan-F/Psan-R designed on the basis of the 16S rRNA sequence. This PCR protocol was optimized and validated by testing 50 target and 38 non-target pure cultures. The method showed a 100% specificity and the detection limit obtained was of 0.7 pgDNA/PCR tube, which equates to 20 or lower bacterial cells. A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues seeded with bacteria. In addition, the developed PCR assay was also capable of direct detection of P. Anguilliseptica from tissues obtained from experimentally infected fish and fish obtained from natural outbreaks in gilthead seabream (Sparus aurata) and turbot (Scophthalmus maximus) farms. This PCR protocol can be a useful tool for a rapid, specific and sensitive diagnosis of fish pseudomonadiasis caused by P. Anguilliseptica.
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Existence of two O-serotypes in the fish pathogen Pseudomonas Anguilliseptica.
Veterinary Microbiology, 2003Co-Authors: Sonia López-romalde, Beatriz Magariños, Carmen Ravelo, Alicia E. Toranzo, Jesús L. RomaldeAbstract: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.
Alicia E. Toranzo - One of the best experts on this subject based on the ideXlab platform.
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Polymerase Chain Reaction Amplification of Repetitive Intergenic Consensus and Repetitive Extragenic Palindromic Sequences for Molecular Typing of Pseudomonas Anguilliseptica and Aeromonas salmonicida
Journal of Aquatic Animal Health, 2008Co-Authors: Roxana Beaz-hidalgo, Sonia López-romalde, Alicia E. Toranzo, Jesús L. RomaldeAbstract:Abstract The aim of this study was to evaluate the use of two molecular techniques, repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and repetitive intergenic consensus PCR (ERIC-PCR), as epidemiological tools with which to discriminate among genetically distinct strains within two bacterial fish pathogens, Pseudomonas Anguilliseptica and Aeromonas salmonicida. A total of 30 A. salmonicida and 52 P. Anguilliseptica were analyzed. For P. Anguilliseptica, three different major fingerprints were obtained with both techniques, which defined three genomic groups: one was composed of strains isolated from eels Anguilla spp., the second of strains from turbot Scophthalmus maximus and blackspot seabream (also known as red seabream) Pagellus bogaraveo, and the third of strains from other fish species, such as gilthead seabream (also known as gilthead bream) Sparus auratus, sea bass Dicentrarchus labrax (also known as European bass Morone labrax), and salmonids. In the case of A. salmonicida, p...
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Vaccination strategies to prevent emerging diseases for Spanish aquaculture.
Developments in biologicals, 2005Co-Authors: Jesús L. Romalde, Sonia López-romalde, Beatriz Magariños, Carmen Ravelo, Ruben Avendaño-herrera, Alicia E. ToranzoAbstract: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.
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Development and validation of a PCR-based protocol for the detection of Pseudomonas Anguilliseptica
Fish Pathology, 2004Co-Authors: Jesús L. Romalde, Sonia López-romalde, Beatriz Magariños, Carmen Ravelo, Alicia E. ToranzoAbstract:A PCR-based protocol for the detection of the emerging fish pathogen Pseudomonas Anguilliseptica was developed, using the primer set Psan-F/Psan-R designed on the basis of the 16S rRNA sequence. This PCR protocol was optimized and validated by testing 50 target and 38 non-target pure cultures. The method showed a 100% specificity and the detection limit obtained was of 0.7 pgDNA/PCR tube, which equates to 20 or lower bacterial cells. A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues seeded with bacteria. In addition, the developed PCR assay was also capable of direct detection of P. Anguilliseptica from tissues obtained from experimentally infected fish and fish obtained from natural outbreaks in gilthead seabream (Sparus aurata) and turbot (Scophthalmus maximus) farms. This PCR protocol can be a useful tool for a rapid, specific and sensitive diagnosis of fish pseudomonadiasis caused by P. Anguilliseptica.
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Existence of two O-serotypes in the fish pathogen Pseudomonas Anguilliseptica.
Veterinary Microbiology, 2003Co-Authors: Sonia López-romalde, Beatriz Magariños, Carmen Ravelo, Alicia E. Toranzo, Jesús L. RomaldeAbstract: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.
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Phenotypic and Genetic Characterization of Pseudomonas Anguilliseptica Strains Isolated from Fish
Journal of Aquatic Animal Health, 2003Co-Authors: Sonia López-romalde, Beatriz Magariños, Alicia E. Toranzo, S. Núñez, Jesús L. RomaldeAbstract:Abstract The phenotypic and genetic characteristics of a group of European strains of Pseudomonas Anguilliseptica isolated from different hosts are described and compared with those of the type strain CECT (Spanish Collection of Type Cultures) 899 originally isolated from Japanese eels Anguilla japonica in Japan. The taxonomic analysis of these isolates revealed high homogeneity of characteristics regardless of the geographic origin and host. Variable results were only obtained for citrate utilization and the hydrolysis of Tween 20 and starch. Based on comparison with conventional tests, consistent results in the miniaturized system API20NE were only achieved for the P. Anguilliseptica isolates when bacterial inocula were prepared from blood agar and spectrophotometrically adjusted to an optical density of 1 (at an absorbance of 580 nm). Although P. Anguilliseptica is not included in the API database, this miniaturized system can be useful for the identification of this microorganism under the above condi...
Elena Mercadé - One of the best experts on this subject based on the ideXlab platform.
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Pseudomonas guineae sp. nov., a novel psychrotolerant bacterium from an Antarctic environment.
International Journal of Systematic and Evolutionary Microbiology, 2007Co-Authors: Núria Bozal, M Jesús Montes, Elena MercadéAbstract:Two Gram-negative, cold-adapted, aerobic bacteria, designated strains M8T and M6, were isolated from soil collected from the South Shetland Islands. The organisms were rod-shaped, catalase- and oxidase-positive and motile by means of polar flagella. These two psychrotolerant strains grew between −4 and 30 °C. 16S rRNA gene sequence analysis placed strains M8T and M6 within the genus Pseudomonas. DNA–DNA hybridization experiments between the Antarctic isolate M8T and type strains of phylogenetically related species, namely Pseudomonas peli and Pseudomonas Anguilliseptica, revealed levels of relatedness of 33 and 37 %, respectively. Strain M6 showed 99 % DNA similarity to strain M8T. Several phenotypic characteristics, together with data on cellular fatty acid composition, served to differentiate strains M8T and M6 from related pseudomonads. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that strains M8T and M6 belong to the same genospecies, representing a novel species of the genus Pseudomonas, for which the name Pseudomonas guineae sp. nov. is proposed. The type strain is M8T (=LMG 24016T=CECT 7231T).
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Pseudomonas guineae sp. nov., a novel psychrotolerant bacterium from an Antarctic environment.
International journal of systematic and evolutionary microbiology, 2007Co-Authors: Núria Bozal, M Jesús Montes, Elena MercadéAbstract:Two Gram-negative, cold-adapted, aerobic bacteria, designated strains M8T and M6, were isolated from soil collected from the South Shetland Islands. The organisms were rod-shaped, catalase- and oxidase-positive and motile by means of polar flagella. These two psychrotolerant strains grew between -4 and 30 degrees C. 16S rRNA gene sequence analysis placed strains M8T and M6 within the genus Pseudomonas. DNA-DNA hybridization experiments between the Antarctic isolate M8T and type strains of phylogenetically related species, namely Pseudomonas peli and Pseudomonas Anguilliseptica, revealed levels of relatedness of 33 and 37%, respectively. Strain M6 showed 99% DNA similarity to strain M8T. Several phenotypic characteristics, together with data on cellular fatty acid composition, served to differentiate strains M8T and M6 from related pseudomonads. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that strains M8T and M6 belong to the same genospecies, representing a novel species of the genus Pseudomonas, for which the name Pseudomonas guineae sp. nov. is proposed. The type strain is M8T (=LMG 24016T=CECT 7231T).
Sonia López-romalde - One of the best experts on this subject based on the ideXlab platform.
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Experimental Pseudomonas Anguilliseptica infection in turbot Psetta maxima (L.): a histopathological and immunohistochemical study
European Journal of Histochemistry, 2009Co-Authors: Gian Enrico Magi, Sonia López-romalde, B Magariños, J Lamas, A. E. Toranzo, J. L. RomaldeAbstract:Experimental infection with Pseudomonas Anguilliseptica was performed both by intraperitoneal (i.p.) and bath route on juvenile turbot (Psetta maxima) in order to evaluate the pathology induced. Turbot was found to be sensitive to i.p. challenge (1.7×106 CFU/fish) but no to bath exposure. The i.p. challenge induced septicaemic infection and mortality. Externally, moribund fish showed distended abdomen and pale areas at day 9. The gross pathological internal signs present were abundant ascitic fluid in the peritoneal cavity, pale and enlarged spleen, pale and friable liver, and congestive and dilated gut with yellowish exudates. On histopathological examination, bacterial invasion was common in all the tissues studied but the most prominent pathological changes were observed in gut, spleen and kidney after 7 day with features of necrosis. The immunohistochemical findings support the widespread localization of the bacteria after the i.p. injection since the P. Anguilliseptica was detected in spleen from day 1 post injection, in liver, kidney and gut from day 4, in muscle from day 7 and in brain from day 9. The difficulties in infecting healthy fish by bath challenge can be explained by the opportunistic nature of this pathogen.
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Polymerase Chain Reaction Amplification of Repetitive Intergenic Consensus and Repetitive Extragenic Palindromic Sequences for Molecular Typing of Pseudomonas Anguilliseptica and Aeromonas salmonicida
Journal of Aquatic Animal Health, 2008Co-Authors: Roxana Beaz-hidalgo, Sonia López-romalde, Alicia E. Toranzo, Jesús L. RomaldeAbstract:Abstract The aim of this study was to evaluate the use of two molecular techniques, repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and repetitive intergenic consensus PCR (ERIC-PCR), as epidemiological tools with which to discriminate among genetically distinct strains within two bacterial fish pathogens, Pseudomonas Anguilliseptica and Aeromonas salmonicida. A total of 30 A. salmonicida and 52 P. Anguilliseptica were analyzed. For P. Anguilliseptica, three different major fingerprints were obtained with both techniques, which defined three genomic groups: one was composed of strains isolated from eels Anguilla spp., the second of strains from turbot Scophthalmus maximus and blackspot seabream (also known as red seabream) Pagellus bogaraveo, and the third of strains from other fish species, such as gilthead seabream (also known as gilthead bream) Sparus auratus, sea bass Dicentrarchus labrax (also known as European bass Morone labrax), and salmonids. In the case of A. salmonicida, p...
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Vaccination strategies to prevent emerging diseases for Spanish aquaculture.
Developments in biologicals, 2005Co-Authors: Jesús L. Romalde, Sonia López-romalde, Beatriz Magariños, Carmen Ravelo, Ruben Avendaño-herrera, Alicia E. ToranzoAbstract: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.
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Development and validation of a PCR-based protocol for the detection of Pseudomonas Anguilliseptica
Fish Pathology, 2004Co-Authors: Jesús L. Romalde, Sonia López-romalde, Beatriz Magariños, Carmen Ravelo, Alicia E. ToranzoAbstract:A PCR-based protocol for the detection of the emerging fish pathogen Pseudomonas Anguilliseptica was developed, using the primer set Psan-F/Psan-R designed on the basis of the 16S rRNA sequence. This PCR protocol was optimized and validated by testing 50 target and 38 non-target pure cultures. The method showed a 100% specificity and the detection limit obtained was of 0.7 pgDNA/PCR tube, which equates to 20 or lower bacterial cells. A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues seeded with bacteria. In addition, the developed PCR assay was also capable of direct detection of P. Anguilliseptica from tissues obtained from experimentally infected fish and fish obtained from natural outbreaks in gilthead seabream (Sparus aurata) and turbot (Scophthalmus maximus) farms. This PCR protocol can be a useful tool for a rapid, specific and sensitive diagnosis of fish pseudomonadiasis caused by P. Anguilliseptica.
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Existence of two O-serotypes in the fish pathogen Pseudomonas Anguilliseptica.
Veterinary Microbiology, 2003Co-Authors: Sonia López-romalde, Beatriz Magariños, Carmen Ravelo, Alicia E. Toranzo, Jesús L. RomaldeAbstract: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.
Lucas Domínguez - One of the best experts on this subject based on the ideXlab platform.
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PCR detection and PFGE DNA macrorestriction analyses of clinical isolates of Pseudomonas Anguilliseptica from winter disease outbreaks in sea bream Sparus aurata.
Diseases of Aquatic Organisms, 2002Co-Authors: M. Mar Blanco, Alicia Gibello, Miguel A. Moreno, Lucas Domínguez, Ana I. Vela, José F. Fernández-garayzábalAbstract:A PCR-based detection system for Pseudomonas Anguilliseptica was evaluated. The primer combination PAF-PAR (forward primer PAF = 5'-GACCTCGCCATTA-3', reverse primer PA R= 5'-CTCAGCAGTTTTGAAAG-3') gave a unique and specific amplification product of 439 bp at an annealing temperature of 46°C with all the P. Anguilliseptica isolates and strains (n = 56) but no amplification products were observed with any other Pseudomonas species or phylogenetically related bacteria tested. The PCR assay had a detection limit of 170 to 200 cells per PCR tube, which was improved 8-fold when the PCR amplification product was used as a nonradioactive probe in blot- ting hybridization experiments. The PCR assay allowed the specific and reliable detection of P. Anguilliseptica within 8 h, compared with up to 10 d required for its isolation and further characteri- zation by conventional microbiological approaches. Clinical isolates of P. Anguilliseptica recovered from several winter disease (WD) outbreaks diagnosed in sea bream Sparus aurata in Spain and Por- tugal between 1996 and 2001 were characterized by pulse field-gel electrophoresis (PFGE) macrore- striction analysis. The 54 clinical isolates analyzed were included in 4 different pulsotypes. Pulso- types B and C represented 54 and 25% of the isolates, respectively, and were responsible for most of the WD outbreaks diagnosed in Spain between 1996 and 2001. The implication of asymptomatic infected carriers in the dissemination and spread of WD is discussed.
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Winter disease outbreak in sea-bream (Sparus aurata) associated with Pseudomonas Anguilliseptica infection
Aquaculture, 1997Co-Authors: Ana Doménech, José F. Fernández-garayzábal, Paul A. Lawson, Judith Garcia, M. T. Cutuli, M. Mar Blanco, Alicia Gibello, Miguel A. Moreno, M. D. Collins, Lucas DomínguezAbstract:Abstract The nature of an outbreak of ‘winter disease’ affecting both juvenile and adult sea-bream ( Sparus aurata ) in several Iberian Peninsula farms from January to April of 1996 is described. The average mortality rate was approximately 10–15%, although in some fish farms mortality reached 30%. Pure cultures of aerobic Gram-negative filamentous rods were isolated from the kidney of diseased fish, as well as from some of the liver and ascitic fluid samples. 16S rRNA gene sequence analysis identified the isolates from diseased fish as Pseudomonas Anguilliseptica . The results of biochemical and physiological tests of clinical isolates and the type strain of P. Anguilliseptica were entirely consistent with the genotypic identification. The isolation of P. Anguilliseptica exclusively from the diseased fish of all affected fish farms, together with the failure to isolate this bacterium from non-affected fish of the same farms, suggest that P. Anguilliseptica is likely to be the agent responsible for the ‘winter disease’ outbreak in the sea-bream examined. The relationship of the ‘winter disease’ outbreaks to stressful environmental conditions is discussed.
