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.Lactococcus garvieae, one of the major gram-positive cocci pathogenic for fish, is considered a serious problem in cultured marine and freshwater fish species such as yellowtail (Seriola quinqueradiata) in Japan and rainbow trout (Onchorynchus mykiss) in Europe and Australia (4,8,(10)(11)(12)(19)(20)(21). In Spain, this disease has appeared in rainbow trout farms since 1991 (9, 28) and at present is considered one of the most important risk factors in the trout industry during the summer months. Besides fish, L. garvieae has been isolated from cows and buffalo (6, 31); it has also been recovered from humans sources (13,14). In addition, it has been isolated recently from diseased freshwater prawns (Macrobrachium rosembergii) in Taiwan (5). All these facts indicate the expanding importance of L. garvieae.Despite the role of this microorganism as an infectious agent, few studies of the epidemiology of L. garvieae recovered from fish have been published until now. Epidemiological investigations depend on the availability and reliability of highly discriminatory typing systems which may differentiate between strains from different sources (23). Ribotyping (RT) and pulsed-field gel electrophoresis (PFGE) have been used for epidemiological characterization of L. garvieae and have shown high genetic variability within this species (12, 35). The typing schemes proposed by these authors are complicated and have limited epidemiological value. Moreover, the RT and PFGE techniques usually involve time-consuming steps and specific equipment (16,25). Randomly amplified polymorphic DNA (RAPD) is an accesible and sensitive method based on the use of arbitrary primers to amplify polymorphic segments of DNA (38). Although this technique has been widely used in recent years for the study of genetic diversity among isolates of a number of bacterial fish pathogens (3,18,24,27,30,33,34,37) to our knowledge no studies were reported for L. garvieae.In the p...

Tenacibaculum maritimum is the etiological agent of marine flexibacteriosis disease, with the potential to cause severe mortalities in various cultured marine fishes. The development of effective preventive measures (i.e. vaccination) requires biochemical, serological and genetic knowledge of the pathogen. With this aim, the biochemical and antigenic characteristics of T. maritimum strains isolated from sole, turbot and gilthead sea bream were analysed. Rabbit antisera were prepared against sole and turbot strains to examine the antigenic relationships between the 29 isolates and 3 reference strains. The results of the slide agglutination test, dot-blot assay and immunoblotting of lipopolysaccharides (LPS) and membrane proteins were evaluated. All bacteria studied were biochemically identical to the T. maritimum reference strains. The slide agglutination assays using O-antigens revealed cross-reaction for all strains regardless of the host species and serum employed. However, when the dot-blot assays were performed, the existence of antigenic heterogeneity was demonstrated. This heterogeneity was supported by immunoblot analysis of the LPS, which clearly revealed 2 major serological groups that were distinguishable without the use of absorbed antiserum: Serotypes O1 and O2. These 2 serotypes seem to be host-specfic. In addition, 2 sole isolates and the Japanese reference strains displayed cross-reaction with both sera in all serological assays, and are considered to constitute a minor serotype, O1/O2. Analysis of total and outer membrane proteins revealed that all strains share a considerable number of common bands that are antigenically related.

No abstract

Brown Ring Disease (BRD) is a bacterial disease caused by Vibrio tapetis which affects cultured clams and causes heavy economic losses. In this study, 28 V. tapetis strains isolated from 5 different hosts were intraspecifically characterized by 3 different polymerase chain reaction-(PCR-) based typing methods: enterobacteria repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP)-PCR and randomly amplified polymorphic DNA (RAPD)-PCR. Cluster analysis of genetic profiles obtained from these molecular techniques clearly showed the existence of 3 genetic groups strongly correlated to the host origin. The first group was formed by 23 V. tapetis strains isolated from Manila clam Ruditapes philippinarum, 1 isolated from venus clam Venerupis aurea, and 1 isolated from common cockle Cerastoderma edule, all collected from France and Spain. The second group was formed by 2 strains isolated from carpet-shell clam R. decussatus cultured in the northwest of Spain. The third group was composed of 1 strain isolated from Atlantic halibut Hippoglossus hippoglossus from the UK. We concluded that the 3 typing methods based on PCR were useful for the intraspecific typing of V. tapetis strains, and that they can potentially be used as a fast and reliable tool for epidemiological studies in the future. KEY WORDS: Vibrio tapetis · RAPD · ERIC-PCR · REP-PCR · Typing Resale or republication not permitted without written consent of the publisherDis Aquat Org 69: [175][176][177][178][179][180][181][182][183] 2006 demonstrated some genetic variations among V. tapetis strains (Castro et al. 1997, Romalde et al. 2002, Chevalier et al. 2003.Repetitive element PCR is a group of techniques that generates DNA fingerprints which can be utilized for the discrimination of bacterial species and/or strains (Versalovic et al. 1991(Versalovic et al. , 1994. These methods involve the application of oligonucleotide primers based on families of short, highly conserved extragenic repetitive sequences, including the repetitive extragenic palindromic (REP) and the enterobacterial repetitive intergenic consensus (ERIC) sequences (Stern et al. 1984, Hulton et al. 1991. REP and ERIC sequences are present in species throughout the Enterobacteriaceae family (Versalovic et al. 1991, Bachellier et al. 1999. The use of appropriate outward-facing PCR primers directed at these repeated sequences generates multiple amplification products, which reflect distance polymorphisms between adjacent DNA repeats. PCR with primers based on ERIC and REP sequences has been successfully used to differentiate bacterial strains from diverse species (de Bruijn 1992, Bennasar et al. 2002, Bruant et al. 2003, Hahm et al. 2003.The RAPD assay is based on the use of short random sequence primers, 9 to 10 bases in length, which hybridize with sufficient affinity to chromosomal DNA sequences to render a pattern of bands that is used for fingerprinting bacterial strains (Welsh & McClelland 1990, Williams et al. 1990. A number of studies have reported...

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 conditions. Genetic analysis by means of randomly amplified polymorphic DNA demonstrated the existence of variability within P. anguilliseptica. Two major genetic groups were established, which were related to the host origin of the isolates. One group comprised the majority of eel isolates while the other comprised the isolates obtained from other fish species. These results will be important in understanding the epidemiological aspects of P. anguilliseptica.