Immune gene expression in gilthead seabream (Sparus aurata) after Lymphocystis disease virus (LCDV-Sa) challenge resulting in asymptomatic infection.

AIMS: To determine the immune gene expression response of gilthead seabream (Sparus aurata) that is experimentally infected with the lymphocystivirus LCDV-Sa. METHODS AND RESULTS: Viral DNA and transcripts were detected by qPCR in all samples from fish injected with LCDV-Sa, demonstrating that the virus establish a systemic and asymptomatic infection. The expression of 23 immune-related genes was also analysed by RT-qPCR in the head kidney (HK) and intestine at several times post-infection (dpi). In HK, the expression of five type I interferon (IFN)-related genes (ifn, irf3, mx2, mx3 and isg15), il10 and ck10 was upregulated at 1-3 dpi, while genes related to the inflammation process (tnfα, il1ß, il6, casp1) were not differentially expressed or even downregulated. The expression profile in the intestine was different regarding type I INF-related genes. An upregulated c3 and ighm expression was observed in both HK and intestine at 3-8 dpi. Finally, the transcription of nccrp1 and mhcIIα was induced in HK, whereas tcrß expression was downregulated in both organs. CONCLUSIONS: LCDV-Sa seems to trigger an immune response in gilthead seabream characterized by a partial activation of type I IFN system and a lack of systemic inflammatory response which may be related to viral persistence. SIGNIFICANCE AND IMPACT OF THE STUDY: The immune response observed in gilthead seabream infected by LCDV-Sa could be implicated in the establishment of an asymptomatic persistent infection.

Virulence properties of three new Photobacterium species affecting cultured fish.

AIMS: Several virulence factors of three new Photobacterium species: Photobacterium toruni, Photobacterium malacitanum and Photobacterium andalusiense associated with diseases of cultured redbanded seabream (Pagrus auriga) were studied. The exoenzymatic activities, adherence and cytotoxic capabilities, and iron-uptake mechanisms were determined both in bacterial extracellular products (ECP) and whole bacterial cells. The histopathology damages provoked on redbanded seabream by the ECP was also studied. METHODS AND RESULTS: The highest exoenzymatic activities of the ECP were alkaline- and acid-phosphatase, phosphohydrolase and lipase. The ECP were strongly lethal for fish at 4-96 h post-inoculation (p.i). Histological changes were evident at 96 hpi of ECP, affecting head kidney, splenic parenchyma and heart. Cytotoxicity assays, on three fish lines and one human cell line, were conducted using whole bacterial cells and their ECP. The new species tested were cytotoxic only for fish cell lines using whole bacterial cells. Bacterial adherence showed an adherence index moderate on CHSE-214 cell line. All strains showed variable haemolytic activity, and were able to grow under iron-limiting conditions, although the CAS reactivitiy was very low. However, all strains produced high amounts of extracelullar citrate that could be used as iron carrier, and use haem as iron source, except the P. toruni strains because a deletion in the genomic region encoding this ability in all Vibrionaceae members. CONCLUSIONS: The toxic activity of the bacterial ECPs was thermolabile, and not associated with their thermoresistant lipopolysaccharide content. The virulence of the strains tested could not be related to the haemolytic activity. Iron uptake could be based on the use of endogenous citrate as iron carrier and P. toruni lacks the ability to use haem as iron source. SIGNIFICANCE AND IMPACT OF THE STUDY: The study analyses for the first time the virulence properties of three new species of Photobacterium pathogenic for fish.

The theoretical reliability of PCR-based fish viral diagnostic methods is critically affected when they are applied to fish populations with low prevalence and virus loads.

AIMS: The reliability of polymerase chain reaction (PCR) techniques is an important issue in viral diagnosis, and it is even crucial when they must be applied for detection of viruses in asymptomatic carriers. The problems will arise when the aim is to study wild fish populations, where the viral loads and prevalence values are extremely low. We have evaluated several PCR procedures employed by two laboratories for monitoring fish captured in several oceanographic campaigns performed in the Gulf of Cádiz. METHODS AND RESULTS: To evaluate the reliability of different diagnostics test used, we have re-analysed fish samples that had been previously subjected to diagnosis for a surveillance of viruses performed in 2010-2011 in wild fish populations. The following parameters were employed: the clinical sensitivity (Ss), the clinical specificity (Sp), the predictive positive value, the predictive negative value, and the positive and negative likelihood ratio (LR+ and LR- ). For viral nervous necrosis virus, a RT-PCR procedure supplemented by nested PCR showed the highest values (100%) for all the parameters. For viral haemorrhagic septicaemia virus, the highest values were provided by RT-PCR supplemented by dot-blot hybridization. In the case of infectious pancreatic necrosis virus, none of the procedures yielded 100% for any parameter. CONCLUSIONS: The results obtained for viral prevalence indicate: (i) that the conservation of the samples at -80°C did not affect to the capacity of detection of the virus in the tissues, and (ii) that the reproducibility of the diagnosis can be affected by factors including the staff experience and/or the materials employed. Finally, the use of a combination of procedures in advised to ensure the maximum reliability of the diagnosis when it is applied to asymptomatic fish populations. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes a strategy of combining diagnostic tests for the surveillance and monitoring of wild fish populations to reduce underestimation of the prevalence of viruses this type of populations.

Transmission of lymphocystis disease virus to cultured gilthead seabream, Sparus aurata L., larvae.

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR-hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF-1 cells. Whole-mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV-positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR-hybridization) or in larvae (PCR-hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR-hybridization, ISH and IHC. After feeding on LCDV-positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.

Cellular and molecular immune responses of the sea bass (Dicentrarchus labrax) experimentally infected with betanodavirus.

Naïve sea bass juveniles (38.4 + or - 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-beta, TCRbeta, CD4, CD8alpha, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable "in vitro" increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-beta and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.

Molecular intraspecific characterization of Photobacterium damselae ssp. damselae strains affecting cultured marine fish.

AIMS: The aim of this study was to analyse the intraspecific variability of Photobacterium damselae ssp. damselae strains isolated from different cultured marine fish species using molecular typing methods. METHODS AND RESULTS: Twenty P. damselae ssp. damselae strains isolated from marine fish species were used in this study. Phenotypic characterization of the strains was carried out using standard microbiological methods. Genetic characterization was conducted using three PCR-based methods [random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and repetitive extragenic palindromic-PCR (REP-PCR)]. Dice coefficient and the unweighted pair group method with average linkage were used for numerical analyses of banding patterns. At phenotypic level, the strains analysed showed seven different profiles, which could not be related to the host fish species, geographic area or outbreak of disease. Isolates were grouped into nine and eight clusters using the RAPD technique with primers 5 and 4, respectively. In both cases, the main cluster grouped 45% of strains. The techniques ERIC-PCR and REP-PCR were more discriminatory, both resulting in 14 different clusters, which grouped 15-20% of the isolates. CONCLUSIONS: In this study, the techniques tested are confirmed as good tools for molecular typing, because they allow discrimination between P. damselae ssp. damselae strains isolated within the same outbreak. In addition, ERIC-PCR and REP-PCR methods were more adequate for rapid typing of P. damselae ssp. damselae than RAPD, allowing the discrimination at strain level. SIGNIFICANCE AND IMPACT OF THE STUDY: The results, in agreement with previous studies, confirmed the high intraspecific variability among isolated P. damselae ssp. damselae strains at both phenotypic and genetic levels. This suggests the existence of different clonal lineages that coexist in the same geographic area, within a short period of time (2-3 years). The discrimination at strain level can be useful to study the traceability of infections.

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. METHODS AND RESULTS: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). CONCLUSIONS: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.

Detection of infectious pancreatic necrosis virus (IPNV) from asymptomatic redbanded seabream, Pagrus auriga Valenciennes, and common seabream, Pagrus pagrus (L.), using a non-destructive procedure.

A non-destructive procedure based on nested RT-PCR and dot-blot hybridization has been developed for the detection of asymptomatic IPNV-carrier fish. The pair of primers designed for RT-PCR amplified a 599-bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT-PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191-bp probe was designed for hybridization studies used in combination with the nested RT-PCR. The application of the nested RT-PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV-carriers, respectively. The combination of nested RT-PCR and dot-blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain.

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.

Microbiological quality of reclaimed water used for golf courses' irrigation.

Microbial quality of reclaimed water used for irrigation in two golf courses located in the southern Iberian Peninsula (Spain and Portugal) was evaluated. Bacterial indicators for faecal pollution (total and faecal coliforms, Escherichia coli and enterococci) were tested by membrane filtration using appropriate selective media. In addition, somatic E. coli bacteriophages, enteric viruses (entero-, hepatitis A and rota-) and Legionella pneumophila were also analysed. The results obtained showed that all wastewater treatment processes reduced adequately the number of indicator microorganisms although a significant correlation between pathogenic and indicator microorganisms tested was not found. L. pneumophila was detected by PCR but not confirmed by culture. Survival experiments of pathogenic microorganisms in aerosols and irrigated turf are conducted to determine the health hazards for the golf practice and to propose a microbial standard for wastewater used for irrigation of golf courses.

Histochemical study of lymphocystis disease in skin of gilthead seabream, Sparus aurata L.

A battery of horseradish peroxidase-conjugated lectins (Con A, WGA and DBA), as well as conventional histochemical techniques (PAS, saponification, Alcian Blue pH 0.1, 1, 2.5, chlorhydric hydrolisis, sialidase, Bromophenol blue, Tioglycollate reduction and Ferric-ferricyanide-FeIII) were used to study the content and distribution of carbohydrates, proteins and glycoconjugate sugar residues on the skin and on the lymphocystis-infected cells of gilthead seabream, Sparus aurata. Variable amounts of glycoproteins containing sialic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, mannose and/or glucose residues were observed in the cuticle and mucous cells of the corporal skin, tails and fins. Germinative and epithelial cells of the epidermis contained glycogen, proteins, carboxylated groups, as well as glycoproteins with mannose and/or glucose and N-acetyl-D-galactosamine residues. Hyaline capsule of the mature lymphocystis-infected cells was strongly stained with PAS, Alcian Blue (pH 0.5 and 2.5) and weakly positive with Alcian Blue (pH 1). Con A reacted with the granular cytoplasm, specially around hyaline capsule, and with the basophilic intracytoplasmic inclusions developed in mature lymphocystis-infected cells of Sparus aurata skin. These sugar residues (mannose and/or glucose), as well as N-acetyl-D-glucosamine and/or sialic acid and N-acetyl-D-galactosamine were not detected in the hyaline capsule of the lymphocystis disease.

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.

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.

Comparative evaluation of five serological methods and RT-PCR assay for the detection of IPNV in fish.

In the present study, six diagnostic methods for the detection of infectious pancreatic necrosis virus (IPNV) (indirect immunofluorescence, flow cytometry, immunoperoxidase, immunodot blot, immunostaphylococcus-protein A, and RT-PCR) have been comparatively evaluated using the seroneutralization as the reference assay, and 83 Spanish isolates and 3 reference strains. The most reliable methods were flow cytometry and RT-PCR which could detect virus at titers of 1x10(2) and 1x10(3) TCID50/ml, respectively. At a multiplicity of infection of 50, both assays allowed the earliest detection of IPNV at 4 h post-inoculation. Indirect immunofluorescence and immunoperoxidase assays required at least 6 h post-inoculation to detect viral antigens. The immunodot blot assay possesses low sensitivity and the immunostaphylococcus-protein A test cannot be applied for routine examination of IPNV. Positive reactions were obtained in 100% of the samples tested by seroneutralization and RT-PCR, 90.4% by the flow cytometry, 80.7% by the indirect immunofluorescence assay, 67.5% by the immunoperoxidase, 62.6% by the immunodot blot, and only 27.7% by immunostaphylococcus-protein A test. Therefore, RT-PCR and flow cytometry were the most appropriate and sensitive methods for the routine detection of IPNV from affected fish.

Development of an in situ hybridisation procedure for the detection of sole aquabirnavirus in infected fish cell cultures.

An in situ hybridisation (ISH) technique has been developed to detect sole aquabirnavirus in infected fish cell lines bluegill fibroblast (BF-2), EPC, and chinook salmon embryo cells (CHSE-214). A 613 bp cDNA probe for viral RNA coding for a fragment of VP2 protein was generated by reverse transcription polymerase chain reaction (RT-PCR) using infectious pancreatic necrosis virus (IPNV) specific DNA primers. Infected cells were strongly labelled, and no non-specific reaction was observed in non-infected cells used as negative controls. The specificity of the probe was examined by testing it against a range of IPNV serotypes such as Ab, Sp and VR-299. The ISH technique was compared with the immunofluorescence procedure to determine the sensitivity of detection of sole aquabirnavirus in BF-2 cells. The probe used in the ISH technique detected weak positivity at 8h post-inoculation (p.i.) in the cytoplasm of infected BF-2 cells inoculated with 10(3) TCID50/ml, whilst the labelling appears at 24h p.i. when the immunofluorescence technique was applied. At all other time intervals the results were equivalent.

Distribution of virus-like Particles in an Oligotrophic Marine Environment (Alboran Sea, Western Mediterranean).

Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude. In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment. Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM). The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses. In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20. VLP (virus-like particle) counts were not significantly correlated (p > 0.001) with chlorophyll a concentration or the abundance of cyanobacteria. However, there was a positive and significant correlation with bacterial abundance (p <0.001). THE ANALYSIS OF SIZE AND MORPHOLOGY OF VIRAL PARTICLES BY TEM AND THE CORRELATION OBTAINED BETWEEN THE NUMBERS OF VLP AND BACTERIA SUGGEST THAT THE MAJORITY OF THE VIRAL PARTICLES IN THE ALBORAN SEA ARE BACTERIOPHAGES. NONE OF THE INDIRECT EVIDENCE SUGGESTED THAT EUKARYOTIC ALGAE OR CYANOBACTERIA WERE IMPORTANT HOST ORGANISMS IN THESE WATERS.

Neurotoxic effect on two fish species and a PC12 cell line of the supernate of Vibrio alginolyticus and Vibrio anguillarum.

The biological effects of supernates obtained from different strains of Vibrio alginolyticus and Vibrio anguillarum isolated from diseased fish have been studied by inoculation on two fish species, eel and rainbow trout, and two fish cell lines. These supernates possess neuroexcitatory properties, and so, when they are injected into both fish species, they trigger convulsions, wriggling, contortive swimming and respiratory arrest coupled with increased respiratory reflex. Furthermore, after the application of the supernates on cultures of noradrenergic pheochromocytoma PC12 cells, an increase of acetylcholine, released from the cells was obtained. The amount of released acetylcholine depends on the source of assayed supernates and on the dose applied to the cells. On the basis of the results obtained with PC12 cells, we suggest that the supernates from pathogenic Vibrio strains injected into fish may elicit an increased release of acetylcholine in the motor endplate of some muscles related to locomotion and ventilation of the inoculated fish.

Antigenic properties and experimental transmission to several fish species of a marine birnavirus isolated from sole (Solea senegalensis).

A cross-neutralization test was used to study the antigenic relationship of an aquabirnavirus isolated from sole (Solea senegalensis), named solevirus, and several infectious pancreatic necrosis virus (IPNV) strains. Solevirus was antigenically similar to IPNV strain Sp. Transmission of the solevirus to other fish species has been determined by inoculation to freshwater and marine fish species (two salmonids and gilt-head seabream). A higher pathogenicity was obtained for the marine fish species, although solevirus caused an asymptomatic infection in all species tested, as demonstrated by the detection of viral RNA and of viral antigens in fish leucocytes, respectively, using polymerase chain reaction (PCR) and flow cytometry (FC).