In vitro antimicrobial activity of Douro wines against clinical Helicobacter pylori strains / strains121In vitro antimicrobial activity of Douro wines against clinical helicobacter pylori strains / Actividad antimicrobiana in vitro de los vinos del Duero sobre cepas clínicas de Helicobacter pylori

Abstract Aim. In vitro antimicrobial activities of seven wines (5 reds and 2 whites) from the Douro region (Iberian Peninsule) against eleven clinical strains of Helicobacter pylori were evaluated. Methods. The disk diffusion method, using Columbia Agar supplemented with horse blood (CAB), were used to determine the antimicrobial properties of some wine components against H. pylori strains. Potential interactions of antioxidants contained in the wines and two antimicrobials (amoxicillin and metronidazole) were studied by the disk diffusion method. Results. All the tested strains showed growth in CAB supplemented with 9% of the tested wines but none of them grew in media supplemented with 45% and 67.5% of wine. Similarly, all the tested strains grew in media with the concentration of proanthocyanidins present in the different types of the studied wines. The Minimal Inhibitory Concentration (MIC) values of the wine antioxidant components tested (benzoic acid, catechin, quercetin, and resveratrol) indicate that resveratrol was the most powerful inhibitory substance against H. pylori. An effect of potentiation between amoxicillin and metronidazole and the antioxidants tested was also established. The interaction of amoxicillin and resveratrol or metronidazole and catechin increased the antimicrobial activity against H. pylori. Conclusions. The results obtained suggested a potential role of resveratrol as a chemopreventive agent for H. pylori infection.

Resumen Objetivo. Se evaluó las actividades antimicrobianas in vitro de siete vinos (5 tintos y 2 blancos) de la región del Duero (Peninsula Ibérica) frente a once cepas de Helicobacter pylori de origen clínico. Métodos. Para determinar las propiedades antimicrobianas de algunos componentes del vino sobre las cepas de H. pylori se utilizaron las técnicas de difusión en disco en placas de agar Columbia suplementado con sangre de caballo (CAB). La potential interacción entre las sustancias antioxidantes presentes en los vinos y dos antimicrobianos (amoxicilina y metronidazol) se determinó usando la técnica de difusión en disco. Resultados. Todas las cepas ensayadas mostraron crecimiento en CAB suplementado con el 9% de los vinos analizados, pero no se obtuvo crecimiento de ninguna de las cepas en medios suplementados con el 45% y el 67,5% de vino. Asimismo, todas las cepas ensayadas crecieron en medios con la concentración de proantocianidinas presentes en los diferentes tipos de vinos estudiados. Los valores de concentración mínima inhibitoria (CMI) de los componentes antioxidantes de los vinos ensayados (ácido benzoico, catequina, quercetina y resveratrol) indican que el resveratrol fue la sustancia más potente en la inhibición del crecimiento de H. pylori. También se estableció un efecto de potenciación entre amoxicilina y metronidazol y los antioxidantes ensayados. Las interacciones amoxicilina + resveratrol y metronidazol + catequina aumentaron la actividad antimicrobiana contra H. pylori. Conclusiones. Los resultados obtenidos sugieren un papel potencial del resveratrol como agente quimiopreventivo de la infección por H. pylori.

Immune Response of Senegalese Sole against Betanodavirus Mutants with Modified Virulence.

Nervous necrosis virus (NNV), genus Betanodavirus, the etiological agent of the viral encephalopathy and retinopathy (VER), presents a genome with two positive-sense single-stranded RNA segments. Striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV), together with reassortants RGNNV/SJNNV, are the betanodaviruses predominantly isolated in Southern Europe. An RGNNV/SJNNV reassortant isolated from Senegalese sole (wt160) causes high mortalities in this fish species. This virus presents differences in the sequence of the 3' non-coding region (NCR) of both segments compared to RGNNV and SJNNV reference strains. Previously, it has been reported that the reversion of two of these differences (nucleotides 1408 and 1412) in the RNA2 3'NCR to the SJNNV-type (recombinant r1408-1412) resulted in a decrease in sole mortality. In the present study, we have applied an OpenArray® to analyse the involvement of sole immune response in the virulence of several recombinants: the r1408-1412 and two recombinants, developed in the present study, harbouring mutations at positions 3073 and 3093 of RNA1 3'NCR to revert them to RGNNV-type. According to the correlation values and to the number of expressed genes, the infection with the RNA2-mutant provoked the most different immune response compared to the immune response triggered after the infection with the rest of the viruses, and the exclusive and high upregulation of genes related to the complement system. The infection with the RNA1-mutants also provoked a decrease in mortality and their replication was delayed at least 24 h compared to the wt160 replication, which could provoke the lag observed in the immune response. Furthermore, the infection with the RNA1-mutants provoked the exclusive expression of pkr and the downregulation of il17rc.

Nanostructured recombinant protein particles raise specific antibodies against the nodavirus NNV coat protein in sole.

Nervous necrosis virus (NNV) reassortant strains RGNNV/SJNNV have emerged as a potent threat to the Mediterranean marine aquaculture industry, causing viral encephalopathy and retinopathy (VER) in Senegalese sole (Solea senegalensis). In this study, a cheap and practical vaccine strategy using bacterial inclusion bodies made of the coat protein of a virulent reassortant strain of this betanodavirus was devised. The nanostructured recombinant protein nanoparticles, VNNV-CNP, were administered without adjuvant to two groups of juvenile sole, one by intraperitoneal injection and the other by oral intubation. Specific antibodies were raised in vivo against the NNV coat protein via both routes, with a substantial specific antibody expansion in the injected group 30 days post homologous prime boost. Expression levels of five adaptive immune-related genes, cd8a, cd4, igm, igt and arg2, were also quantified in intestine, spleen and head kidney. Results showed cd4 and igm were upregulated in the head kidney of injected fish, indicating activation of an adaptive systemic response, while intubated fish exhibited a mucosal response in the intestine. Neither route showed significant differential expression of cd8a. The specific antibody response elicited in vivo and the lack of any signs of toxicity over the 6-week study period in young fish (n = 100), evidences the potential of the nanoparticle as a vaccine candidate.

Surveillance of viruses in wild fish populations in areas around the Gulf of Cadiz (South Atlantic Iberian Peninsula).

This report describes a viral epidemiological study of wild fish around the Gulf of Cadiz (southwestern Iberian Peninsula) and is focused on infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), and viral nervous necrosis virus (VNNV). One fish species (Chelon labrosus) was sampled inside the gulf, at the mouth of the San Pedro River. Another 29 were sampled, in three oceanographic campaigns, at sites around the Bay of Cadiz. The fish were processed individually and subjected to isolation in cell culture and molecular diagnosis. VHSV was not isolated from any species. Thirteen IPNV-type isolates were obtained from barracuda (Sphyraena sphyraena), axillary seabream (Pagellus acarne), common two-banded seabream (Diplodus vulgaris), common pandora (P. erythrinus), Senegal seabream (D. bellottii), and surmullet (Mullus surmuletus). Six VNNV isolates were obtained from axillary seabream, common pandora, black seabream (Spondyliosoma cantharus), red mullet (Mullet barbatus), Lusitanian toadfish (Halobatrachus didactylus), and tub gurnard (Chelidonichtys lucerna). In the river mouth, viruses were detected only after reamplification, obtaining prevalence percentages of IPNV and VNNV (44.4 and 63.0%, respectively) much higher than those observed in the oceanographic campaigns (25.7 and 19.6%, respectively). The opposite results were obtained in the case of VHSV after reamplification: 11.1% in the river mouth and 43.6% in the oceanic locations. Analyzing the results with respect to the proximity of the sampling sites to the coast, an anthropogenic influence on wild fish is suggested and discussed. The type of viruses and the presence of natural reassortants are also discussed.

Distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens in nervous and non-nervous organs of European seabass(Dicentrarchus labrax) during the course of an experimental challenge.

The distribution of red-spotted grouper nervous necrosis virus (RGNNV) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile European seabass (Dicentrarchus labrax) during the course of an intramuscular infection. Histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. The specific antibody response was also studied 2 months post-challenge. Viral proteins were present throughout the experimental period in the retina (inner nuclear layer, ganglion layer, outer limiting membrane, and outer plexiform layer), brain(cerebellum and tectum opticum), and liver (hepatocytes and endothelial cells). These proteins were also observed in the renal tubular cells, white pulp of spleen, and in fibroblasts and cartilage of caudal fin. This is the first report of RGNNV proteins appearing in these organs, where the immunostaining was only detected at certain sampling times after the onset of mortality. Brain and retina of virus-exposed fish showed high levels of vacuolation, while accumulation of fat vacuoles was observed in the liver. RGNNV infection also induced a specific antibody response as measured by an ELISA. In summary, this is the first study demonstrating the presence of viral proteins in cells of caudal fin, kidney and spleen of European seabass.

Tissue distribution of Red Spotted Grouper Nervous Necrosis Virus (RGNNV) genome in experimentally infected juvenile European seabass (Dicentrarchus labrax).

The distribution of viral genome in the tissues of juvenile European seabass (Dicentrarchus labrax) during the course of a Red Spotted Grouper Nervous Necrosis Virus (RGNNV) infection has not yet been described. The present study addresses this and indicates which target organs may be involved in viral replication. This information should enable more accurate detection of virus in asymptomatic carriers, and in turn help to control the spread of the disease. The aim of this study was to examine the pattern of expression of viral genomic segments RNA1 and RNA2, using two absolute real-time PCRs (RT-qPCR), over the course of a RGNNV infection after administering the virus by intramuscular injection. In situ hybridization was also used to locate the RNA2 viral segment in different organs throughout the infection. The experimental challenge provoked an acute form of viral nervous necrosis (VNN), with a resulting cumulative mortality of 37%. The RT-qPCRs designed allowed the detection of both genomic segments in all the organs tested (nervous and non-nervous tissues) at all sampling times examined. The highest viral RNA copy number was found in eyes, although viral replication appeared to begin in the brain. Viral replication was also recorded in pooled internal organs and in caudal fin. However, the increase in the viral RNA copy number in these organs did not result in an increased viral titre, which may indicate that a productive infection does not take place in non-nervous tissues, possibly due to a failure in a viral post-replication step.

Hydrophobicity and adhesion to fish cells and mucus of Vibrio strains isolated from infected fish

The hydrophobicity of 44 Vibrio strains isolated from cultured, diseased gilt-head sea bream (Sparus aurata) was determined. Three different methods were used: (1) microbial adhesion to hydrocarbons (MATH), either with phosphate buffer or with phosphate urea magnesium sulfate (PUM) buffer, (2) aggregation in the presence of salt solutions (SAT), and (3) adhesion to nitrocellulose filters (NCF). The results show that experimental conditions exerted a significant influence on hydrophobicity. Thus, Kendall rank coefficients showed the presence of correlation only for SAT and NCF, and for SAT and the MATH assay with PUM buffer. Moreover, no relationships were observed between the bacterial hydrophobicity estimated with the methods mentioned above and the ability of the strains to adhere to fish mucus or cells. These results indicate that adhesion of pathogenic Vibrio strains to host surfaces is mediated mainly by specific receptor interactions, instead of by hydrophobic interactions (AU)

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