Shewanella putrefaciens Pdp11 extracts protect against betanodavirus infection.

Viral nervous necrosis is caused by the nervous necrosis virus (Betanodavirus genus), a naked virus with two positive-sense RNA segments. Betanodaviruses are classified into four species, being RGNNV predominant in the Mediterranean area. RGNNV causes high mortality in several fish species, including European seabass. There are two vaccines to protect seabass against RGNNV infection. In this regard, the development of strategies to protect different fish species against different viruses, such as the use of probiotics, is a key issue for the aquaculture industry. Shewanella putrefaciens Pdp11, SpPdp11, is a fish probiotic with proven positive effects on seabream and Senegalese sole, protecting those species against bacterial pathogens; however, its antiviral activity is unknown. This study evaluates the anti-RGNNV activity of sonicated-SpPdp11 extracts in vitro and in vivo. The in vitro evaluation was performed on E11 cells following 3 assays: (i) neutralization, (ii) 6-h pre-adsorption, (iii) post-adsorption, determining the inhibition of RGNNV-induced CPEs and quantifying viral replication. The immunostimulatory activity of SpPdp11 extracts was also examined, analysing the transcription of mx, hsp70, tnfα, e3 and tlr3 in E11 cells. For the in vivo evaluation, two European seabass groups were established: (i) control group, (ii) experimental group, fed with commercial pellet supplemented with SpPdp11 extracts. Animals were fed for 30 days and subsequently challenged by intramuscular injection. SpPdp11 extracts compromised RGNNV replication in E11 cells (67.3% and 55% CPE inhibition in pre-adsorption and post-adsorption assays, respectively), and modulated the transcription of all the immune-related genes examined. The highest induction was obtained for mx gene. Regarding the in vivo results, 82% of fish fed with the SpPdp11-supplemented diet survived to RGNNV infection, whereas the survival rate of fish fed with the control diet was 64%.P18-RT-1067 (Proyecto de Excelencia, Junta de Andalucia), PID2020-113637RB-C22 (MINECO/AEI/FEDER, UE). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Inhibition of nervous necrosis virus replication by Shewanella putrefaciens Pdp11 extract

One of the most relevant infectious diseases affecting farmed fish is the viral encephalopathy and retinopathy, caused by the nervous necrosis virus (NNV). The two commercial vaccines available against this virus have been designed to protect European seabass against the infection with a particular NNV species, RGNNV. Therefore, the development of strategies to protect different fish species against different viruses is a key issue for the aquaculture industry. In this regard, there are numerous studies focused on the use of probiotics, although their antiviral activity has been poorly investigated. Shewanella putrefaciens Pdp11, SpPdp11, is a probiotic with proven positive effects on gilthead seabream and Senegalese sole, protecting those species against several bac- terial pathogens; however, its antiviral activity remains to be investigated. The current study is a step forward in the use of probiotics against viral infections, evaluating the anti-RGNNV activity of sonicated-SpPdp11 extracts both, in vitro and in vivo. According to our results, SpPdp11 extracts compromised RGNNV multiplication in E11 cells, affecting viral assembly and/or exit rather than genome replication. Furthermore, this antiviral activity may be produced by the capacity of the SpPdp11-extract to induce an immune response in treated cells. This antiRGNNV activity has been confirmed in vivo, since 82% of fish fed with the SpPdp11-supplemented diet survived an experimental-RGNNV infection, whereas the survival rate of fish fed with the control diet was 64%. These results suggest that SpPdp11-supplemented feeding can be a promising prophylactic tool against RGNNV and encourage further research on other fish species and viral pathogens.This study has been supported by the projects P18-RT-1067 (Proyecto de Excelencia, Junta de Andalucia, Regional Government) and PID2020-113637RB-C22 (MINECO/AEI/FEDER, UE) (Spanish Government). Daniel Álvarez-Torres has been granted by the project PTA2020-018984-I. Funding for open access charge: Universidad de Málaga/CBUA

Modulation of the intestinal microbiota and expression of gene of senegalese sole (Solea senegalensis, Kaup 1858) after dietary administration of oxytetracycline.

Oxitetracycline and probiotics (Pdp11) administration to sole juveniles showed important changes in the species composition of intestinal microbiota that established a relationship between these species and gene expression

Changes in intestinal microbiota and gene expression of immune system parameters caused by dietary administration of alginate and Shewanella putrefaciens Pdp11 in Senegalese sole (Solea senegalensis Kaup, 1858) specimens

Dietary administration of sodium alginate single or mixed with Pdp11 modulated the intestinal microbiota diversity of sole juveniles. Both treatments promoted a change in the species present in the gut in comparison with control group. In addition, oral administration of alginate promoted the appearance of an intestinal microbiota highly influenced by the prebiotic presence. Synbiotic administration of alginate with Pdp11 promoted a more pronounced decreasing in the similarity index and in the species richness. Both treatments modulated the expression of genes related with immune system functions

Use of in vivo-induced antigen technology to identify bacterial genes expressed during solea senegalensis infection with Photobacterium damselae subsp. piscicida

The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important outbreaks affecting several fish species including flatfish Solea senegalensis. The aim of this work was to identify in vivo-induced expressed immunogenic proteins using pooled sera from fish that have experienced photobacteriosis. In vivo induced immunogenic proteins included inosine-5'-monophosphate dehydrogenase (Impdh) and alkyl hydroperoxide reductase (AhpC), two proteins involved in peptide synthesis: serine hydroxymethyl transferase (Shmt) and alanyl-tRNA synthetase (AlaRS) and the non-ribosomal peptide synthetase involved in the synthesis of the siderophore piscibactin (Irp2)

Assessment of in vitro postbiotic capabilities of the probiotic Shewanella putrefaciens pdp11 growing under different cultivation conditions containing microalgae dietary supplements widely used in aquaculture.

Probiotics have been established as potential tools to improve host health and environmental quality in aquaculture. For example,Shewanella putrefaciens Pdp11 (SpPdp11), a strain described as a probiotic for use in aquaculture (reviewedby Cámara et al. 2020). Despite the proven health benefits of probiotics, recent evidence suggests that bacterial viability is not necessary to attain effects. Thus, postbiotics have emerged as potential opportunities in the field of functional foods. The International Scientific Association for Probiotics and Prebiotics (ISAPP) convened a panel that defined postbiotics as a “preparation of inanimate microorganisms and/or their components that confer a health benefit to the host”. The present work evaluates the potential postbiotic, as extracellular products (ECPs), of SpPdp11 grown undern different cultivation conditions. These included different culture media composed of a blend of microalgae, to observe a possible synergistic effect. The ECPs obtained were evaluated for their cytotoxicity against different fish cell lines, enzymatic and antibacterial activity, and their effect on biofilm formation by several fish pathogensproyecto PID2020-113637RB-C22 de investigación financiado por MCIN/AEI/10.13039/ 50110001103

Relación entre la microbiota intestinal y la composición de ácidos grasos tras la administración del probiótico Shewanella putrefaciens Pdp11 bioencapsulado en Artemia en ejemplares larvarios de S. senegalensis

Estudios previos confirmaron que un pulso corto (10-30 dpe) del probiótico S. putrefaciens Pdp11 (2.5x10 7 cfu mL-1) bioencapsulado en Artemia y administrado a ejemplares larvarios de S. senegalensis incrementaron significativamente los niveles de lípidos totales y de ciertos ácidos grasos de los ejemplares. Dicha dieta suplementada produjo además cambios importantes en las especies microbianas intestinales de dichos organismos. La realización de un análisis de componentes principales (ACP) confirmó el efecto de la dieta probiótica en los ejemplares, sobre su microbiota y sobre a los niveles de ácidos grasos analizados

Identificación del probiótico Shewanella putrefaciens Pdp11 mediante PCR a través del transposón único que interrumpe al gen que codifica para la proteína fenazina.

Shewanella putrefaciens Pdp11 es una cepa descrita como probiótico en peces de importancia acuícola. La secuenciación de su genoma ha permitido establecer comparaciones a nivel genómico con otras cepas patógenas pertenecientes al mismo género. Como parte del estudio del genoma de Pdp11 se han identificado la presencia de 6 transposones y su ausencia en 7 cepas de Shewanella.sp (Pérez Gómez Olivia et al., 2021). En este trabajo se plantea el uso del transposón que interrumpe la proteína PhzE, implicada en la biosíntesis de la fenazina, para la identificación especifica de SpPdp11. Así como, la puesta a punto de la PCR para determinar la sensibilidad de los cebadores en la identificación del probiótico. En los cultivos acuícola, Shewanella putrefaciens Pdp11 se administra a los peces mediante su dieta en concentraciones de 10^9 ufc/gr de pienso, este trabajo permitiría la futura identificación y cuantificación del probiótico en muestras intestinales, así como el estudio del potencial de colonización del mismo.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The treatment with the probiotic Shewanella putrefaciens Pdp11 of sepecimens of Solea senegalensis exposed to high stocking densities to enhance their resistance to disease

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet.S