title: Following the infection process of vibriosis in Manila clam (Ruditapes philippinarum) larvae through GFP-tagged pathogenic Vibrio species.
abstract: Vibriosis represents the main bottleneck for the larval production process in shellfish aquaculture. While the signs of this disease in bivalve larvae are well known, the infection process by pathogenic Vibrio spp. during episodes of vibriosis has not been elucidated. To investigate the infection process in bivalves, the pathogens of larvae as V. tubiashii subsp. europaensis, V. neptunius and V. bivalvicida were tagged with green fluorescent protein (GFP). Larvae of Manila clam (Ruditapes philippinarum) were inoculated with the GFP-labeled pathogens in different infection assays and monitored by microscopy. Manila clam larvae infected by distinct GFP-tagged Vibrio spp. in different challenges showed the same progression in the infection process, defining three infection stages. GFP-tagged Vibrio spp. were filtered by the larvae through the vellum and entered in the digestive system through the esophagus and stomach and colonized the digestive gland and particularly the intestine, where they proliferated during the first 2h of contact (Stage I), suggesting a chemotactic response. Then, GFP-tagged Vibrio spp. expanded rapidly to the surrounding organs in the body cavity from the dorsal to ventral region (Stage II; 6-8h), colonizing the larvae completely at the peak of infection (Stage III) (14-24h). Results demonstrated for the first time that the vibriosis is asymptomatic in Manila clam larvae during the early infection stages. Thus, the early colonization and the rapid proliferation of Vibrio pathogens within the body cavity supported the sudden and fatal effect of the vibriosis, since the larvae exhibited the first signs of disease when the infection process is advanced. As a first step in the elucidation of the potential mechanisms of bacterial pathogenesis in bivalve larvae the enzymatic activities of the extracellular products released from the wild type V. neptunius, V. tubiashii subsp. europaensis and V. bivalvicida were determined and their cytotoxicity was demonstrated in fish and homeothermic cell lines for the first time. That activity was lost after heat treatment.
authors.affiliation: Departamento de Microbiología y Parasitología, CIBUS-Facultad de Biología y Instituto de Acuicultura, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain. Electronic address: javier.dubert@usc.es.
authors.forename: Javier
authors.initials: J
authors.lastname: Dubert
authors.affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, United States.
authors.forename: David R
authors.initials: DR
authors.lastname: Nelson
authors.affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, United States.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Spinard
authors.affiliation: Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, United States.
authors.forename: Linda
authors.initials: L
authors.lastname: Kessner
authors.affiliation: Department of Fisheries, Animal and Veterinary Sciences, University of Rhode Island, Kingston, RI 02881, United States.
authors.forename: Marta
authors.initials: M
authors.lastname: Gomez-Chiarri
authors.affiliation: Novostrea Bretagne, Route du Vieux Passage, Banastère, 56370 Sarzeau, France.
authors.forename: Fiz
authors.initials: F
authors.lastname: da Costa
authors.affiliation: Departamento de Microbiología y Parasitología, CIBUS-Facultad de Biología y Instituto de Acuicultura, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Prado
authors.affiliation: Departamento de Microbiología y Parasitología, CIBUS-Facultad de Biología y Instituto de Acuicultura, Universidad de Santiago de Compostela, Santiago de Compostela 15782, Spain.
authors.forename: Juan L
authors.initials: JL
authors.lastname: Barja
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000382:microbiology*; D004198:Disease Susceptibility; D049452:Green Fluorescent Proteins / Q000032:analysis; D054884:Host-Pathogen Interactions; D007814:Larva / Q000382:microbiology; D014733:Vibrio / Q000235:genetics / Q000502:physiology*
chemical_list: D049452:Green Fluorescent Proteins
keywords: Green fluorescent protein (GFP); Larvae; Manila clam; Vibrio spp.; Vibriosis