title: Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse.
abstract: Lactococcus garvieae is the causative microbial agent of lactococcosis, an important and damaging fish disease in aquaculture. This bacterium has also been isolated from vegetables, milk, cheese, meat and sausages, from cow and buffalo as a mastitis agent, and even from humans, as an opportunistic infectious agent. In this work pathogenicity experiments were performed in rainbow trout and mouse models with strains isolated from human (L. garvieae HF) and rainbow trout (L. garvieae UNIUDO74; henceforth referred to as 074). The mean LD(50) value in rainbow trout obtained for strain 074 was 2.1 × 10(2) ± 84 per fish. High doses of the bacteria caused specific signs of disease as well as histological alterations in mice. In contrast, strain HF did not prove to be pathogenic either for rainbow trout or for mice. Based on these virulence differences, two suppressive subtractive hybridizations were carried out to identify unique genetic sequences present in L. garvieae HF (SSHI) and L. garvieae 074 (SSHII). Differential dot-blot screening of the subtracted libraries allowed the identification of 26 and 13 putative ORFs specific for L. garvieae HF and L. garvieae 074, respectively. Additionally, a PCR-based screening of 12 of the 26 HF-specific putative ORFs and the 13 074-specific ones was conducted to identify their presence/absence in 25 L. garvieae strains isolated from different origins and geographical areas. This study demonstrates the existence of genetic heterogeneity within L. garvieae isolates and provides a more complete picture of the genetic background of this bacterium.
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Reimundo
authors.affiliation: Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
authors.forename: Amable J
authors.initials: AJ
authors.lastname: Rivas
authors.affiliation: Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Osorio
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Jéssica
authors.initials: J
authors.lastname: Méndez
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Pérez-Pascual
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Roberto
authors.initials: R
authors.lastname: Navais
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Gómez
authors.affiliation: Biomar Iberia, Apdo 16, 34210 Dueñas, Palencia, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Sotelo
authors.affiliation: Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
authors.forename: Manuel L
authors.initials: ML
authors.lastname: Lemos
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Guijarro
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005393:Fish Diseases / Q000382:microbiology*; D014644:Genetic Variation; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology / Q000662:veterinary*; D006801:Humans; D017045:Lactococcus / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity*; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017686:Oncorhynchus mykiss / Q000382:microbiology*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D037521:Virulence Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D037521:Virulence Factors