research

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PATHMEDA was coordinated by the University of Valencia. It involved a knowledge-acquisition phase consisting in secondments in partner teams at the University of Copenhagen and University of Bergen and a second phase where the new knowledge gained was adapted and optimized to Mediterranean fish species at the University of Valencia. The second phase involved the recruitment of three Marie Curie Fellows.

The most important research achievements of PATHMEDA have been

	Morphological and molecular characterization of a new Kudoa species (Myxozoa) from grey mullets based on 18S rDNA sequences. Solid evidence for important amendments to the morphological diagnosis of the genus Kudoa were presented, which will facilitate further diagnosis of these important fish parasites.
	Development and optimization of a multiplex PCR assay for detection of Vibrio vulnificus biotype2. This procedure afforded a more simple identification of the bacterium in fish, even in subclinical carrier fish.
	Elucidating the role of the metalloprotease Vvp and the virulence plasmid pR99 of V. vulnificus serovar E in surface colonization and innate immunity of eels. These studies have determined the different roles of these two elements in infecting eels and represent a huge leap forward in understanding the infection process of ells by V. vulnificus.
	Understanding the diversity of the virulence plasmid of V. vulnificus. Plasmids are DNA molecules that are separate from the chromosomal DNA, which provide a mechanism for horizontal gene transfer within a population of microbes. The plasmid content of a wide collection of strains from the three biotypes of V. vulnificus was analyzed to identify putative conjugative and virulence plasmids. Results showed that plasmids are widespread among V. vulnificus strains, which seem to substantially contribute to genetic plasticity of the species.
	Establishing mechanisms of genetic regulation of virulence and resistance to antibiotics in V. vulnificus biotype 2 serovar E. Our findings provided a deeper insight into the virulence factors of this microbe.
	Evaluation of genotypic and phenotypic methods to distinguish clinical and environmental strains of V. vulnificus. PATHMEDA evaluated different typing methods to distinguish clinical and environmental isolates. None of the phenotypic methods was reliable enough to determine the origin of the strain of a given isolate. The genotypic techniques classified the samples into three main profiles but no single genotypic method allows at the present time distinguishing environmental from clinical strains or biogroups of V. vulnificus.
	New information of the mechanisms of quorum sensing in V. vulnificus biotype 2 serovariant E. Quorum sensing is a control mechanism of gene expression that accounts for coordinated social behaviour of individual bacteria in response to extracellular signals. The studies carried out under PATHMEDA demonstrated that the gene luxS, which codifies AI-2, is present in all strains of V. vulnificus independently of its origin, biotype or serovariant. Moreover, it was also shown that V. vulnificus also produces quorum-sensing signals elicited by acylated homoserine lactones (AHLs) in strains of biotypes 1 and 2 from the water or wounds in fish and humans.
	New insights into the phylogeny and epidemiology of V. vulnificus. The phylogeny and epidemiology of V. vulnificus was studied by Multilocus Sequence Analysis by means of the partial sequencing of four cellular genes of maintenance and three of virulence of 100 environmental and clinical stocks of different biotypes, geographic serotypes and origins. Results showed that the species does not have a clonal population structure and the species is divided in two main groups and not in three as previously thought. This finding is extremely important to understand the epidemiology of V. vulnificus in the interface between wild and cultured fish stocks.
	Isolation, detection and sequencing of the Infectious Pancreatic Necrosis Virus (IPNV) from eels. The IPNV present in wild glass-eels was isolated and sequenced. A real-time PCR assay was developed to detect the pathogen in glass-eels arriving to a local eel farm in Spain. The results demonstrate that the glass-eels are already infected upon arrival to the plant.

These research results have materialized in 13 peer-reviewed publications, 14 congress presentations and four (two master and two doctoral) theses.

PUBLICATIONS

	Roig FJ, Sanjuan E, Llorens A, Amaro C. A (2010) pilF polymorphism-based PCR to differentiate Vibrio vulnificus strains potentially dangerous to public health. Applied and Environmental Microbiology 76: 1328-1333.
	HodnelandK, GarcíaR, BalbuenaJA, ZarzaC and Fouz B (2011)Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain. Journal of Fish Diseases 34: 189-202.
	Fouz B, Llorens A, Valiente E & Amaro C. 2009. A comparative epizootiologic study of the two fish-pathogenic serovars of Vibrio vulnificus biotype 2. Journal of Fish Diseases. 33: 383-202.
	Valiente E, Bruhn JB, Nielsen KF, Larsen JL, Roig FJ, Gram L, Amaro C. 2009. Vibrio vulnificus produces quorum sensing signals of the AHL-class. FEMS Microbiology Ecology 69: 16-26.
	Sanjuan E, Oliver JD, Amaro C. 2009. Evaluation of genotypic and phenotypic methods to distinguish clinical from environmental Vibrio vulnificus strains. Applied and Environmental Microbiology 75: 1604-1613.
	Roig FJ, Llorens A, Fouz B, Amaro C. 2009. Spontaneous quinolone resistance in the zoonotic serovar of Vibrio vulnificus. Applied and Environmental Microbiology 75: 2577-2580.
	Roig FJ and Amaro C. 2009. Plasmid diversity in Vibrio vulnificus biotypes. Microbiology 155: 489-497.
	Valiente E, Padrós F, Lamas J, Llorens A and Amaro C (2008) Microbial and histopathological study of the vibriosis caused by Vibrio vulnificus serovar E in eels: The metalloprotease Vvp is not an essential lesional factor. Microbial Pathogenesis 45: 386-393.
	Valiente E, Jimenez N, Merino S, Tomas JM, Amaro C (2008) Vibrio vulnificus biotype 2 serovar E gne but not galE is essential for lipopolysaccharide biosynthesis and virulence. Infection and Immunity 76: 1628-1638.
	Valiente E, Lee CT, Lamas J, Hor L and Amaro C (2008) Role of the virulence plasmid pR99 and the metalloprotease Vvp in resistance of Vibrio vulnificus serovar E to eel innate immunity. Fish and Shellfish Immunology 24: 134-141.
	Valiente E, Lee CT, Hor L, Fouz B and Amaro C (2007) Role of the metalloprotease Vvp and the virulence plasmid pR99 of Vibrio vulnificus serovar E in surface colonization and fish virulence. Environmental Microbiology 10: 328-338.
	Holzer AS, Blasco-Costa I, Sarabeev VL, Ovcharenko MO and Balbuena JA (2006) Kudoa trifolia sp. n. - molecular phylogeny suggests a new spore morphology and unusual tissue location for a well known genus. Journal of Fish Diseases 29: 743-755.
	Sanjuán E and Amaro C (2007) Multiplex PCR assay for detection of Vibrio vulnificus biotype 2 and simultaneous discrimination of serovar E strains. Applied and Environmental Microbiology 73: 2029-2032.