E. Martinez-Manzanares

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.

Bacteria recovered from diseased cultured gilthead sea bream (Sparus aurata L.) in southwestern Spain

A bacteriological study of 25 outbreaks affecting cultured gilthead sea bream (Sparus aurata L.) in southwestern Spain from 1997 to 2000 has been carried out. Each year, the highest number of outbreaks occurred during winter and affected fish with sizes ranging between 2 and 25 g. The most frequently isolated microorganisms were identified as Vibrio (69.90%). Other microorganisms belonging to Pseudomonas spp., Photobacterium damselae ssp. piscicida, Cytophaga/Flexibacterlike and Aeromonas spp. were isolated with a frequency lower than 10%. The outbreaks with the highest mortalities of cultured gilthead sea bream were due to P. damselae ssp. piscicida. An increase in the number of resistances to different antimicrobials has been observed in this study with respect to previous studies carried out in the same area. This increase was very high in the case of some species of Vibrio, Pseudomonas spp. and P. damselae ssp. piscicida. D 2003 Elsevier Science B.V. All rights reserved.

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.

Plasmid associated virulence properties of environmental isolates of Aeromonas hydrophila.

The plasmid profiles, and their association with antimicrobial resistance, of 60 strains of Aeromonas hydrophila isolated from fish, shellfish and water were investigated. Only two strains were susceptible to all the antimicrobial agents tested; the highest incidences of resistance were to tetracycline (96.7%), prystanamycin (93.3%), ampicillin (91.7%) and cephalothin (91.7%). Forty strains harboured one or more plasmids and the plasmid profile most frequently detected (15%) was the association of three small plasmids of 4.2, 3.2 and 2.8 Mda. Curing experiments indicated that the plasmid-free derivative strains simultaneously lost their resistance determinants to tobramycin, neomycin, gentamicin and kanamycin. More than 90% of the strains tested produced siderophores and displayed haemolytic activity. However, the relationship between these virulence characters and the presence of plasmids was different; in 74.5% of the strains there was siderophore production and plasmids were detectable, whereas only 60% of the strains simultaneously possessed plasmids and haemolytic activity.

Antigenic characterization of the etiological agent of the brown ring disease affecting manila clams

Summary Vibrio P1 is considered as the causative agent of Brown Ring Disease, an epizootic disease of cultured clams in Europe. Although its pathogenic implication has been clearly established, its taxonomical inclusion and virulence mechanisms are not still completely elucidated. In the present work, we have studied the outer membrane protein (OMP) and lipopolysaccharide (LPS) patterns and other antigenic characteristics of Vibrio P1 strains, and we have compared to those both reference strains of Vibrio and Vibrio strains isolated from diseased clams, by means of SDS-PAGE, immunoblotting, Western blot, slide agglutination and dot-blot techniques. The present results strongly suggest that Vibrio P1 constitutes a homogeneous group on the basis of its antigenic characteristics. Although this microorganism shares common epitopes with several Vibrio species, antigenic heterogeneity with regard to both LPS-structure and OMP-pattern is presented.

Development of immunological techniques for the detection of the potential causative agent of the brown ring disease

Abstract Current methods for the detection of the potential causative agent of the brown ring disease (BRD) in clams ( Vibrio P1) involve the isolation of the microorganism from the affected clams, and further biochemical identification. However, by using this methodology, the positive isolation of P1 strains from Spanish affected clams has not been yet successful. In the present study, three immunological techniques (slide agglutination test, immunofluorescence technique, and indirect dot-blot immunoenzymatic assay) have been developed and performed for the specific detection and identification of P1 strains compared to other Vibrio strains which routinely are isolated from clams affected by BRD. The dot-blot immunoenzymatic technique with P1 antiserum improves both the specificity and practicability of immune identification of Vibrio P1, and only a very weak cross-reactivity was recorded with some V. pelagius-V. splendidus strains isolated from diseased clams. Cross-reactions only occur at high antiserum concentrations and can be overcome by an adequate dilution of the antiserum.

Characterization of Vibrio harveyi strains recovered from diseased farmed Senegalese sole (Solea senegalensis)

Aim:  To characterize 16 Vibrio harveyi strains isolated from different epizootic outbreaks affecting farmed Senegalese sole.

Influence of the faecal pollution of marine sediments on the microbial content of shellfish

Abstract To determine the influence of faecally-contaminated sediments on the microbial content of shellfish, several indicator and pathogenic microorganisms were monitored over a period of 19 months in the Guadalhorce estuary (Malaga, South of Spain). All the microorganisms studied were found to be present in greater numbers in sediments and shellfish than in the overlying water. The occurrence of the pathogens studied in shellfish does not appear to be correlated with the levels of any particular indicator group in sediments, except for sulphite-reducing clostridia and faecal streptococci, which showed a significant relationship with the presence of Salmonella and Aeromonas hydrophila in shellfish, respectively. The results obtained in the present study suggest that an evaluation of the presence of indicator and pathogenic microorganisms in sediment may provide additional insight into long-term water quality conditions, but they do not improve the indication of pathogen presence in shellfish compared with the study of the seawater or the direct analysis of shellfish.

Immune response of gilt-head seabream (Sparus aurata) to antigens from Photobacterium damselae subsp. piscicida

Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida) is the causative agent of pseudotuberculosis (also referred to as pasteurellosis), which is one of the most important diseases affecting the culture of gilt-head seabream (Sparus aurata) in the Mediterranean countries [1]. Several studies have reported the protection against Ph. damselae subsp. piscicida by different designs of vaccines in cultured fish [2–5]. The ability of fish vaccines to induce a protective immunity is for the most part based on experimental challenge studies and/or field experiments, but the laboratory research focused on determining which antigens are the most immunogenic in fish is very scarce [6], despite this objective it is critical to identify the individual components involved in inducing protection. This kind of data could be very useful in order to develop subunit vaccines. In this way, different studies showed that vaccines consisting of immunogenic fractions can induce higher protection than inactivated whole cell bacteria in fish [7]. Measures of the antibody response have been used to help describe the impact of physiological changes, environmental conditions and vaccination [8,9]. Antibodies in the circulation constitute an important part of the resistance against pathogenic microorganisms [10]. The objective of this study is to identify the immunodominant antigens of Ph. damselae subsp. piscicida by evaluating the antigenic specificity of the humoral response in gilt-head seabream vaccinated with a bacterin in oil adjuvant, and the potential mitogenic effect of different bacterial antigens on the leucocytes of this fish. Gilt-head seabream of 100 g body weight were maintained in 100 l tanks of seawater at 20 (C with aeration. Twenty of these fish were immunised by intraperitoneal injection (i.p.) with 10 formalin-killed cells of Ph. damselae subsp. piscicida Pp8 suspended in 0.1 ml of phosphate-buffered saline (PBS) and emulsified in Freund’s incomplete adjuvant (FIA). This strain, virulent for this fish (LD50 for gilt-head seabream was 1.8 10 cfu g ), was isolated in our laboratory from diseased gilt-head seabream and was grown on Brain Heart Infusion Agar (OXOID) supplemented with 2% (w/v) NaCl (BHIAS) at 22 (C.

Relationship between indicators of fecal pollution in shellfish-growing water and the occurrence of human pathogenic microorganisms in shellfish

The suitability and efficiency of fecal indicators of pollution to determine the quality of shellfish-growing waters were studied in the estuary of Guadalhorce river (Málaga, Spain). A direct relationship between the concentration of indicator microorganisms in shellfish-growing waters and that of pathogens in shellfish ( Cardium edule and Chamelea gallina ) has not been established. However, in this study the log-normal distribution of the concentration of sulfite-reducer Clostridium spores, fecal streptococci, and coliphages showed a significant relationship with the detection of Salmonella , Vibrio parahaemolyticus , and Aeromonas hydrophila , respectively.