Salvador Arijo

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.

Microbial colonization of different support materials used to enhance the methanogenic process.

Macrobial colonization of the different support materials used to enhance methane production in anaerobic digestors is rapid and occurs in the first 24 h of sludge incubation. Scanning electron microscopy studies reveal a predominant presence of filamentous methanogenic forms, closely resemblingMethanosaeta (Methanothrix), which are located on the outer layer and in the bacterial framework of the biofilm. These findings are consistent with the results obtained from microbial counts using both the most probable number and epifluorescence microscopic techniques, which show an increase in the numbers of aceticlastic methanogens compared to other microbial groups involved, such as sulphate-reducing bacteria, the numbers of which are similar to those obtained under the initial conditions. Moreover, a sharp increase in the bacterial counts is observed by using the epifluorescence microscopic technique applied to homogenized samples, probably due to the count of bacteria released from the support materials.

Subcellular components of Vibrio harveyi and probiotics induce immune responses in rainbow trout, Oncorhynchus mykiss (Walbaum), against V. harveyi

Bacterial subcellular components and probiotics were successful for the stimulation of immunity and the prevention of Vibrio harveyi infections in rainbow trout, Oncorhynchus mykiss (Walbaum). Rainbow trout were immunized with whole inactivated cells of V. harveyi to obtain polyclonal antibodies against specific antigens. Western blotting showed a unique reactive band (approximately 93 kDa) between serum and bacterial proteins from outer membrane proteins (OMP) and extracellular products (ECP). Probiotics were selected according to their capability to inhibit V. harveyi. Two of these bacteria, i.e. A3-47 and A3-51, showed cross-reactivity with V. harveyi antiserum. Their OMPs and ECPs were reactive with V. harveyi antiserum in bands of approximately 93 kDa for A3-51 and higher for A3-47. In vivo tests determined that fish fed with A3-51 produced cross-reactive antibodies against V. harveyi and also, the survival of these fish infected with V. harveyi was high, being similar to the level achieved with vaccinated fish. Thus, the probiotics, when administered as live preparations, were capable of producing cross-reactive antibody against specific bacterial pathogens.

Use of the probiotic Shewanella putrefaciens Pdp11 on the culture of Senegalese sole (Solea senegalensis, Kaup 1858) and gilthead seabream (Sparus aurata L.)

The application of probiotics on aquatic animals is increasing for a better fish welfare status as well as an environment-friendly activity which are actual demands of modern aquaculture industry. A bacterium from skin mucus of healthy gilthead seabream (Sparus aurata L.) has been isolated and identified as Shewanella putrefaciens Pdp11. Different studies have been done to know its application as probiotic in the Senegalese sole and gilthead seabream farming. This article reviews the studies carried out with this probiotic microorganism focusing on the current knowledge of its in vitro and in vivo mechanisms of action. The results suggested that the probiotic S. putrefaciens Pdp11, due to its beneficial effects, could be used in the aquaculture activity of both species.

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.

Identification of Vibrio harveyi proteins involved in the specific immune response of Senegalese sole (Solea senegalensis, Kaup)

Senegalese sole cultures are frequently affected by Vibrio harveyi disease outbreaks. Vaccines in aquaculture are one of the most successful methods of preventing fish pathologies; however, these vaccines are usually composed of inactivated whole cells containing a wide pool of antigens, and some do not induce any protection against pathogens. Thus, the aim of this study was to identify immunogenic proteins of V. harveyi involved in the specific antibody production by Senegalese sole. S. senegalensis specimens were immunized, by intraperitoneal injection, with V. harveyi bacterin supplemented with inactivated extracellular polymeric substances (ECP) and Freund incomplete adjuvant to obtain polyclonal antiserum. One month later, specimens were re-inoculated with the same antigens. Sera from immunized fish were collected two months post first immunization. Strong specific immune response to V. harveyi antigens was detected by ELISA using bacterin (limit dilutions of sera were 1:64000), ECP (1:4000) and outer membrane proteins (OMP) (1:4000) as antigens. Presence of immunogenic proteins in V. harveyi ECP and OMP were determined by 2D-PAGE. For Western Blot analysis some gels were transferred onto nitrocellulose membranes and incubated with sera from S. senegalensis specimens immunized against V. harveyi. 2D-PAGE and Western Blot showed at least five reactive proteins in the ECP and two in the OMP fraction. The spots that clearly reacted with the sole antiserum were excised from stained gel, and analyzed by mass spectrometry (MALDI/TOFTOF). A database search was then performed, using MASCOT as the search method. According to the results, the five ECP spots were identified as Maltoporine, protein homologous to Metal dependent phosphohydrolase, two porins isoforms of V. harveyi and a protein homologous to the cell division protein FtsH. Reactive proteins in the OMP fraction were identified as the protein 3-hydroxyisobutyrate dehydrogenase and a protein homologous to acid phosphatase.