Juan J. Borrego

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.

A continuous cell line from the cultured marine fish gilt-head seabream (Sparus aurata L.)

Abstract Conspecific cell lines provide an efficient experimental system with many applications for improving the productivity of commercial fish species. A continuous cell line SAF-1 has been developed from fin tissues of an adult gilt-head seabream ( Sparus aurata ) without immortalising treatments. The cells grow in DME-F12 basal medium supplemented with 5% foetal bovine serum at the optimal temperature of 25 °C and have been sub-cultured approximately 70 times over a 1.5 year period. They show a fibroblast-like morphology, high efficiency of plating, doubling time of 2 days and high survival after being frozen. Karyotype and DNA content of the cell line reveal a mostly euploid and rather stable cell population, which is of great interest for cytogenetic studies. This cell line has been shown to be susceptible to several fish viruses and to bacterial extracellular products.

Coliphages as an indicator of faecal pollution in water. Its relationship with indicator and pathogenic microorganisms

Abstract The study was designed to test the proposal that Escherichia coli specific bacteriophages might serve as universal faecal pollution indicators in water. A highly specific, sensitive and rapid technique for the detection and quantification of these virus particles was developed. The numerical relationship between E. coli and its parasitic phages was investigated in three different aqueous ecosystems such as sea water in the vicinity of sewage outfalls, river water contaminated by domestic and industrial sewage discharges, and estuarine waters, and found to be very close. In addition, the results obtained indicate that the coliphages are good indicators of the presence of the pathogenic microorganisms studied. In nearly all the water samples tested, the results suggest that coliphages are better indicators of faecal pollution than the classical indicator systems currently employed.

Infectious pancreatic necrosis virus: biology, pathogenesis, and diagnostic methods.

Publisher Summary Infectious pancreatic necrosis virus (IPNV) is the etiological agent of an acute contagious systemic disease of several species of freshwater and marine fish, molluscs, and crustacean. IPNV is the widespread of the piscine viruses. IPNV belongs to the genus Aquabirnavirus within the family Birnaviridae. The family contains three genera: genus Aquabirnavirus (type species, IPNV and yellowtail ascites virus) of fish, genus Avibirnavirus (type species, infectiousbursal disease virus, IBDV) of birds, and genus Entomobirnavirus (type species, Drosophila X virus, DXV) of insects. Several diagnostic methods for IPNV have been reported, including the fluorescent antibody technique, the immunostaphylococcus- protein A test (ISPA), the coagglutination test, the enzyme-linked immunosorbent assay (ELISA), immunoblots and Western blots, and immunoperoxidase phosphatase cell staining (IP). More recently, molecular probes for the detection of nucleic acids using the polymerase chain reaction have been developed and applied for the diagnosis of fish viruses. Techniques involving detection and characterization of the viral genome or polypeptides are also considered these include DNA-based techniques, mainly nucleic acid hybridization, and the polymerase chain reaction.

Survey of bacterial pathologies affecting farmed gilt-head sea bream (Sparus aurata L.) in southwestern Spain from 1990 to 1996

Abstract A bacteriological survey in three fish farms with intensive culture of gilt-head sea bream (Sparus aurata L.) was conducted in southwestern Spain (provinces of Cadiz, Malaga, and Huelva) from 1990 to 1996. All the disease problems occurring during this period in the S. aurata were investigated, focusing on the isolation, characterization, antimicrobial resistance and pathogenicity of the bacterial isolates affecting the fish. The main pathogenic microorganisms isolated were Vibrio (67.8%), Pseudomonas (13.5%), Photobacterium damsela subsp. piscicida (6.7%), Cytophaga/Flexibacter-like bacteria (4.8%), Aeromonas (0.5%), and Gram positive bacteria (6.7%). Although the highest percentages of isolates corresponded to Vibrio and Pseudomonas spp., the strains of P. damsela subsp. piscicida and Cytophaga/Flexibacter-like bacteria caused epizootics with highest degree of mortalities. From the present study, on the basis of the pathogenicity test, we suggest the consideration of V. anguillarum, V. alginolyticus, V. harveyi and V. splendidus as primary pathogenic species for gilt-head sea bream.

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.

Evaluation of different bacteriophage groups as faecal indicators in contaminated natural waters in southern England

Abstract River and seawater affected by faecal discharges were analysed to evaluate the reliability of coliphages and F-specific RNA bacteriophages as indicators of the microbiological quality of waters. F-specific RNA bacteriophages showed no direct relationship with the levels of faecal pollution, and this group was never detected in samples with a low level of enteroviruses (1–10 pfu/10 l.). In contrast, coliphages were constantly detected in the same samples. The concentration of coliphages detected in the samples with 1–10 and > 10 pfu/101. of enteroviruses were similar and higher than the Economical European Community guide and imperative levels of faecal coliforms (100 and 2000 FC/100 ml, respectively). Therefore, coliphages would be considered as an optimal indicator of the microbiological quality of the natural waters.

Coliphages as an indicator of faecal pollution in water : their survival and productive infectivity in natural aquatic environments

The capability of coliphages as indicators of faecal pollution was tested, on the basis of their survival and infectivity in two natural aquatic environments (marine and river). The results obtained indicate that coliphages show a similar inactivation rate to Salmonella in all types of water studied. On the other hand, the phage productive infection of Escherichia coli cells in water and environmental conditions depends on the physiological characteristics of the host bacteria, which generally cannot grow optimally in these conditions. In conclusion, the coliphages may be considered good indicators of faecal pollution in natural waters.

Relationships between salmonella spp and indicator microorganisms in polluted natural waters

Abstract The relationships between Salmonella spp and indicator microorganisms (total coliforms, faecal coliforms, faecal streptococci, Clostridium perfringens and coliphages) were investigated in three different aquatic environments affected by wastewater discharges. The results indicated that the statistical correlations obtained depended, basically, on the source of faecal discharges. The microorganisms most closely correlated with Salmonella spp were faecal coliforms and C. perfringens , the latter also yielding the highest linear regression slope values. The detection percentages of Salmonella spp were high even at a low degree of pollution, which allowed detection of the pathogens in the absence of classical indicator microorganisms in the sample.

Occurrence of Salmonella spp in estuarine and coastal waters of Portugal.

The presence of Salmonella and its relationship with indicator organisms of fecal pollution, such as total coliforms, fecal coliforms and fecal streptococci, was studied at two marine zones in Portugal. Seventeen different Salmonella serotypes were isolated and identified, S. virchow was the most frequently isolated (21.6%). In addition, a high percentage (35.1%) was recorded for some Salmonella serotypes of clinical significance, namely S. enteritidis, S. infantis, S. typhimurium and S. virchow. In any of the samples from the two zones Salmonella was not detected in the absence of any of the indicator organisms. However, the incidence of Salmonella as a function of indicator concentration intervals established by the EEC standards was 0, 10 and 19.3% at guide values of total coliforms, fecal coliforms and fecal streptococci, respectively in the Faro samples (south of Portugal). In contrast, Salmonella incidence rates of 37.5, 36.4 and 33.3% were recorded at the corresponding guide values the Caminha samples (north of Portugal). No significant correlations (p>0.005) were obtained between Salmonella and the indicators at the sampling stations; however, total coliforms and fecal streptococci were the indicators most closely related to Salmonella in Caminha and Faro samples, respectively. Survival experiments in Escherichia coli, Enterococcus faecalis and S. typhimurium, using diffusion chambers, were performed to verify whether the lack of correlation between indicators and Salmonella was due to different inactivation rates in seawater. The results indicate that survival percentages of the three microorganisms tested were similar after 48 h of exposure to seawater.