Dolores Castro

Vibrio tapetis sp. nov., the Causative Agent of the Brown Ring Disease Affecting Cultured Clams

A taxonomic characterization was carried out on strains of the bacteria that cause the brown ring disease of clams. On the basis of their phenotypic and genotypic characteristics, these strains can be considered to constitute a new taxonomic unit, distinct from other Vibrio species. The guanine-plus-cytosine content of the strains ranged between 42.9 and 45.5 mol% (43.2 mol% for the proposed type strain). DNA-DNA hybridization studies showed 100% intragroup relatedness, but levels of genetic relatedness to the reference strains of different Vibrio species tested ranged between 15 and 58%. The strains have all the properties characteristic of the genus Vibrio and can be clearly differentiated from other species of this genus by their growth at 4°C and their negative responses for growth at 30°C and in 6% NaCl, arginine dehydrolase, lysine decarboxylase, ornithine decarboxylase, and Voges-Proskauer reaction. The name Vibrio tapetis is proposed for the new species; strain B1090 (CECT 4600) is the type strain.

Analysis of the mechanism of quinolone resistance in nalidixic acid-resistant clinical isolates of Salmonella serotype Typhimurium

Over a period of 2.5 years, 42 cases of gastro-enteritis caused by nalidixic acid-resistant Salmonella serotype Typhimurium occurred in Malaga. The epidemiological relationship among the strains involved was investigated by analysis of plasmid profile and of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). Despite having different plasmid profiles, all 42 nalidixic-acid resistant Typhimurium isolates had evolved from one clone as shown by analysis of chromosomal DNA by PFGE. The mechanism of quinolone resistance in these Typhimurium isolates was also investigated. Analysis of outer-membrane proteins and lipopolysaccharide from quinolone-susceptible and resistant clinical isolates tested showed no differences. All nalidixic acid-resistant isolates had MICs for ciprofloxacin of 0.25 mg/L and for nalidixic acid of 1024 mg/L. Polymerase chain reaction fragments of 285 bp, containing the quinolone resistance-determining region of the gyrA gene, and of 237 bp, containing the region of parC homologous to the quinolone resistance-determining region of the gyrA gene, were sequenced. All resistant isolates presented a change at Ser-83 to Phe in the GyrA protein, but no changes were observed in the ParC protein. These findings indicated that this mutation in gyrA plays a major role in the acquisition of nalidixic-acid resistance in clinical isolates of Typhimurium.

Significance of several bacteriophage groups as indicators of sewage pollution in marine waters

Seawater samples collected from two beaches with different levels of pollution were studied for the presence of classically and newly proposed faecal indicators such as, total and faecal coliforms, faecal streptococci, coliphages, F-specific phages and bacteriophages of Bacteroides fragilis. Total and faecal coliforms showed lower survival rates in seawater than faecal streptococci, F-specific bacteriophages and coliphages. On the other hand, total coliform concentrations were only higher than those of faecal coliforms in heavily polluted seawater, although in samples with a low level of pollution, faecal streptococci and Escherichia coli C phage counts were generally greater than those showed by faecal coliforms. The low concentration in which F-specific and B. fragilis bacteriophages were detected in marine waters compared to the E. coli bacteriophage levels, is an important shortcoming for the general use of the former microorganisms as universal indicators of faecal pollution. From the results obtained in this study, it may be concluded that faecal streptococci and E. coli C bacteriophages are the most appropriate indicators of the remote pollution in marine waters.

Vibrios isolated from the cultured manila clam (Ruditapes philippinarum): numerical taxonomy and antibacterial activities

Aims: A numerical taxonomic study of halophilic Vibrio isolated from healthy and brown ring disease (BRD) affected manila clams (Ruditapes philippinarum), harvested from the Atlantic coast of south‐western Spain, was performed. 
Methods and Results: Characterization of 123 presumptive Vibrio spp. was carried out using 94 phenotypic tests. Simple matching and Jaccard similarity coefficients were used for numerical analysis. Cluster analysis by the unweighted pair group method with arithmetic averages yielded 15 phena defined at 0·81 similarity. Large phena corresponded to Vibrio tubiashii, V. splendidus biotype I and V. harveyi (phena 1, 5 and 9, respectively). The species V.splendidus biotype II, V. natriegens, V. mediterranei and V. alginolyticus were also represented. The inhibitory effect of diffusible extracellular products of the isolates against 27 strains of V.tapetis, the aetiological agent of BRD, was also investigated. Only five V. tubiashii isolates inhibited the growth of V. tapetis strains. The antimicrobial effect was inhibited by heating and depended on the culture medium. 
Conclusions: The main Vibrio species associated with manila clams were V. tubiashii, V.spendidus and V. harveyi. The antagonistic relationship established between V. tapetis and the Vibrio spp. clam microbiota may explain the failure of isolation in plating medium of V.tapetis from BRD‐affected clams on the south Atlantic coast of Spain. 
Significance and Impact of the Study: Some of the strains isolated from manila clams correspond to agarolytic strains that constitute phenon 7 and they do not fit into any of the currently described Vibrio species.

Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species.

Aims:  The development and evaluation of a protocol based on polymerase chain reaction (PCR) and nucleic acid hybridization techniques for the specific detection of lymphocystis disease virus (LCDV) in several marine fish species.

A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius)

Aims:  To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red‐spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study.

Intraspecific characterization of Vibrio alginolyticus isolates recovered from cultured fish in Spain

Aims: Intraspecific differentiation and characterization of Vibrio alginolyticus strains isolated from cultured fish in Spain.

Viability of Salmonella spp and indicator microorganisms in seawater using membrane diffusion chambers

Diffusion chambers with polycarbonate membrane-filter side walls were used to study the comparative survival of fecal indicators (Escherichia coli and Streptococcus faecalis) and enteric pathogens (Salmonella enteritidis, S. postdam, S. typhimurium, S. london and S. infantis) in natural seawater. It was observed that the percentages of sublethal injury increased with exposure to the marine environment, and that these environmental injuries depended on the microorganism considered. A large proportion of cells lost their ability to produce colonies on the selective media, but retained this capability on a nonselective medium. All microorganisms showed low survival percentages (less than 11%) after 48 hrs of exposure to seawater, but there is not a high difference among the microbial species studied.The results obtained in the present study showed that there were no differences in the survival rates between the serotypes of Salmonella tested. Moreover, Salmonella spp exhibited a similar persistence to E. coli in the marine environment.

Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream.

ABSTRACT Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought. IMPORTANCE Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae. In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified within S. aurata lymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L.

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.