Combined Dietary Administration of Chlorella fusca and Ethanol-Inactivated Vibrio proteolyticus Modulates Intestinal Microbiota and Gene Expression in Chelon labrosus.

The use of functional feeds in aquaculture is currently increasing. This study aimed to assess the combined impact of dietary green microalgae Chlorella fusca and ethanol-inactivated Vibrio proteolyticus DCF12.2 (CVP diet) on thick-lipped grey mullet (Chelon labrosus) juvenile fish. The effects on intestinal microbiota and the transcription of genes related to metabolism, stress, and the immune system were investigated after 90 days of feeding. Additionally, the fish were challenged with Aeromonas hydrophila and polyinosinic-polycytidylic acid (poly I:C) to evaluate the immune response. Microbiota analysis revealed no significant differences in alpha and beta diversity between the anterior and posterior intestinal sections of fish fed the control (CT) and CVP diets. The dominant genera varied between the groups; Pseudomonas and Brevinema were most abundant in the CVP group, whereas Brevinema, Cetobacterium, and Pseudomonas were predominant in the CT group. However, microbial functionality remained unaltered. Gene expression analysis indicated notable changes in hif3α, mhcII, abcb1, mx, and tnfα genes in different fish organs on the CVP diet. In the head kidney, gene expression variations were observed following challenges with A. hydrophila or poly I:C, with higher peak values seen in fish injected with poly I:C. Moreover, c3 mRNA levels were significantly up-regulated in the CVP group 72 h post-A. hydrophila challenge. To conclude, incorporating C. fusca with V. proteolyticus in C. labrosus diet affected the microbial species composition in the intestine while preserving its functionality. In terms of gene expression, the combined diet effectively regulated the transcription of stress and immune-related genes, suggesting potential enhancement of fish resistance against stress and infections.

Unusual Surge of Acute Hepatitis A Cases in 2016 and 2017 in Malaga, Southern Spain: Characterization and Relationship with Other Concurrent European Outbreaks.

We aimed to describe the Hepatitis A virus (HAV) cases that arose in Malaga (Spain) in 2016 and 2017 when the European Centre for Disease Prevention and Control (ECDC) reported several outbreaks among men who have sex with men (MSM). Therefore, we conducted a retrospective study gathering demographic, clinical, and immunological data from the acute HAV patients attending our hospital between March 2016 and December 2017. Additionally, VP1/P2A region was amplified from serum samples, sequenced, and genotyped. We finally performed a phylogenetic analysis, including the HAV strains from the other European outbreaks. A total of 184 HAV cases were reported, with the highest number in March 2017. The cohort mostly comprised Spaniards (81.0%), males (84.8%), and MSM (72.3%), with a median age of 33.0 years (interquartile range (IQR) = 25.0-43.0). Most patients exhibited symptoms. In addition, a successful amplification and sequencing of the VP1/P2A region was performed in 25 out of 106 serum samples (23.6%). All the sequences belonged to the genotype IA, and 20 were phylogenetically related to VRD_521_2016, first described in the United Kingdom (UK). In conclusion, HAV cases emerged in Malaga in 2016 and 2017, showing an epidemic character phylogenetically related to the predominant strain first detected in the UK. Characteristics of the cohort were similar to those from the European outbreaks.

In Vitro and In Vivo Effects of Ulvan Polysaccharides from Ulva rigida.

One of the main bioactive compounds of interest from the Ulva species is the sulfated polysaccharide ulvan, which has recently attracted attention for its anticancer properties. This study investigated the cytotoxic activity of ulvan polysaccharides obtained from Ulva rigida in the following scenarios: (i) in vitro against healthy and carcinogenic cell lines (1064sk (human fibroblasts), HACAT (immortalized human keratinocytes), U-937 (a human leukemia cell line), G-361 (a human malignant melanoma), and HCT-116 (a colon cancer cell line)) and (ii) in vivo against zebrafish embryos. Ulvan exhibited cytotoxic effects on the three human cancer cell lines tested. However, only HCT-116 demonstrated sufficient sensitivity to this ulvan to make it relevant as a potential anticancer treatment, presenting an LC50 of 0.1 mg mL-1. The in vivo assay on the zebrafish embryos showed a linear relationship between the polysaccharide concentration and growth retardation at 7.8 hpf mL mg-1, with an LC50 of about 5.2 mg mL-1 at 48 hpf. At concentrations near the LC50, toxic effects, such as pericardial edema or chorion lysis, could be found in the experimental larvae. Our in vitro study supports the potential use of polysaccharides extracted from U. rigida as candidates for treating human colon cancer. However, the in vivo assay on zebrafish indicated that the potential use of ulvan as a promising, safe compound should be limited to specific concentrations below 0.001 mg mL-1 since it revealed side effects on the embryonic growth rate and osmolar balance.

Zoonotic Parasites in Playgrounds in Southern Spain: A One Health Approach.

Zoonotic parasitic diseases are considered a global threat to public health. In this sense, canines and felines may be infected by different cosmopolitan parasites, with playgrounds serving as an important focus of infection for humans, as well as domestic or wild animals. Knowledge of the epidemiological situation of parasites in animal reservoirs integrated into the environment, identifying the spread pathways, is a key element for an effective response to this threat. Thus, the aim of this study was to assess the frequency of intestinal parasites with zoonotic potential in 120 playgrounds in the Malaga province (Spain). Samples were processed and analysed following standard parasitological procedures. Some 36.7% of playgrounds were parasite-positive with one or more zoonotic parasites. The most common parasites recovered were nematodes (60.0%), followed by protozoan species (33.3%) and cestodes (6.7%). In the parasite-positive playgrounds, Toxocara spp. (17.0 ± 3.5%) and Giardia duodenalis (17.0 ± 3.4%) were the most predominant parasites. In addition, 34.1% of playgrounds were infected with multiple parasites. Our results show a high presence of parasitic forms with zoonotic potential in playgrounds in Malaga, Spain. Due to the close contact between pets and humans in playgrounds, the potential zoonotic risk may increase if prevention and control measures are not designed.

Antioxidant, Immunomodulatory and Potential Anticancer Capacity of Polysaccharides (Glucans) from Euglena gracilis G.A. Klebs

The present study was carried out to determine the bioactivity of polysaccharides extracted from Euglena gracilis (EgPs). These were characterized by FT-IR and GC-MS. Cytotoxicity analyses (MTT) were performed on healthy human gingival fibroblast cell lines (HGF-1), obtaining an IC50 of 228.66 µg mL-1, and cell lines with anticancer activity for colon cancer (HCT-116), breast cancer (MCF-7), human leukemia (U-937, HL-60) and lung cancer (NCl-H460), showing that EgPs have anticancer activity, mainly in HTC-116 cells (IC50 = 26.1 µg mL-1). The immunological assay determined the immunomodulatory capacity of polysaccharides for the production of proinflammatory cytokines IL-6 and TNF-α in murine macrophages (RAW 264.7) and TNF-α in human monocytes (THP-1). It was observed that the EgPs had a stimulating capacity in the synthesis of these interleukins. The antioxidant capacity of polysaccharides and their biomass were analyzed using the ABTS method (18.30 ± 0.14% and (5.40 ± 0.56%, respectively, and the DPPH method for biomass (17.79 ± 0.57%). We quantitatively profiled HGF-1 proteins by liquid chromatography-tandem mass spectrometry analysis, coupled with 2-plex tandem mass tag labelling, in normal cells. In total, 1346 proteins were identified and quantified with high confidence, of which five were considered to be overexpressed. The data is available through ProteomeXchange, under identifier PXD029076.

Estudio mediante técnicas moleculares del tanatomicrobioma intestinal en la estimación del intervalo post mortem temprano empleando un modelo de ratón / Early post mortem interval estimation in a mouse model using molecular analyses of the gut thanatomicrobiome

Introducción: en la actualidad, la falta de métodos cuantitativos fiables ha llevado a distintas líneas de investigación a buscar un modelo que prediga el intervalo post mortem (IPM). El tanatomicrobioma, presente desde el momento de la muerte, parece sufrir cambios predecibles y que siguen una correlación con el IPM.Materiales y métodosse ha analizado experimentalmente el comportamiento del tanatomicrobioma en la región del intestino delgado posterior y del colon ascendente durante las primeras 24 h de descomposición en Mus musculus. Para ello, se ha llevado a cabo una aproximación molecular basada en el análisis del gen ribosomal 16S (ARNr 16S) mediante electroforesis en gel con gradiente desnaturalizante (DGGE) y, seguidamente un análisis de la alfa y beta diversidad.Resultadoslos resultados basados en el análisis de los índices de diversidad ecológica reflejaron cambios estadísticamente significativos antes de las 12 h, y un descenso de la diversidad a partir de esas 12 h postmortem, siendo este estadísticamente significativo en las 2 regiones intestinales analizadas. Por otro lado, el estudio comparativo de las comunidades microbianas mostró que cambian estructurada y diferenciablemente desde el momento de la muerte, alejándose en similitud de las mostradas en vida (IPM 0 h).Discusiónestos resultados coinciden con el descenso de la diversidad sugerido a largo plazo por distintos autores. Sin embargo, en las condiciones del estudio, se ha visto que este descenso no se inicia hasta las 12 h. Conclusión como conclusión, se han podido establecer, según los cambios en la diversidad bacteriana, fases de la dinámica bacteriana durante la descomposición que podrían ayudar a mejorar los modelos de correlación microbiana para la estimación del IPM. (AU)

Introduction: Currently, the lack of reliable quantitative methods has led different research lines to find a model that predicts the postmortem interval (PMI). The thanatomicrobiome, present from the moment of death, has been shown to change in predictable ways, allowing a correlation with PMI.Materials and methodsIn this study, the shifts of the thanatomicrobiome in the region of the posterior small intestine and the ascending colon in Mus musculus during the first 24 hrs of decomposition have been analyzed experimentally. For this purpose, a molecular approach based on the analysis of the 16S ribosomal gene (16S rRNA) and a denaturing gradient gel electrophoresis (DGGE) was adopted, followed by analyses of the ecological diversity indices Alpha and beta diversity.ResultsThe results based on the analysis of the ecological diversity indices reflected statistically significant changes before 12 hrs, and a decrease in diversity after 12 hrs postmortem, this being statistically significant in the two intestinal regions analyzed. Moreover, the comparative study of microbial communities indicated distinct and structured changes from the moment of death, with shifts in the degree of similarity from the composition detected in life (PMI 0 hrs).DiscussionThese results agree with other studies demonstrating a decrease in microbial diversity. However, under the conditions of the study, this decrease does not begin until 12 hrs after death. Conclusions: In conclusion, by examining the dynamics of bacterial diversity our study has identified phases during decomposition that could help to improve microbial correlation models for PMI estimation. (AU)

Vaccination of Gilthead Seabream After Continuous Xenoestrogen Oral Exposure Enhances the Gut Endobolome and Immune Status via GPER1.

In fish culture settings, the exogenous input of steroids is a matter of concern. Recently, we unveiled that in the gilthead seabream (Sparus aurata), the G protein-coupled estrogen receptor agonist G-1 (G1) and the endocrine disruptor 17α-ethinylestradiol (EE2) are potent modulators in polyreactive antibody production. However, the integral role of the microbiota upon immunity and antibody processing in response to the effect of EE2 remains largely unexplored. Here, juvenile seabreams continuously exposed for 84 days to oral G1 or EE2 mixed in the fish food were intraperitoneally (i.p.) immune primed on day 42 with the model antigen keyhole limpet hemocyanin (KLH). A critical panel of systemic and mucosal immune markers, serum VTG, and humoral, enzymatic, and bacteriolytic activities were recorded and correlated with gut bacterial metagenomic analysis 1 day post-priming (dpp). Besides, at 15 dpp, animals received a boost to investigate the possible generation of specific anti-KLH antibodies at the systemic and mucosal interphases by the end of the trial. On day 43, EE2 but not G1 induced a significant shift in the serum VTG level of naive fish. Simultaneously, significant changes in some immune enzymatic activities in the serum and gut mucus of the EE2-treated group were recorded. In comparison, the vaccine priming immunization resulted in an attenuated profile of most enzymatic activities in the same group. The gut genes qPCR analysis exhibited a related pattern, only emphasized by a significant shift in the EE2 group's il1b expression. The gut bacterial microbiome status underwent 16S rRNA dynamic changes in alpha diversity indices, only with the exposure to oral G1, supporting functional alterations on cellular processes, signaling, and lipid metabolism in the microbiota. By the same token, the immunization elevated the relative abundance of Fusobacteria only in the control group, while this phylum was depleted in both the treated groups. Remarkably, the immunization also promoted changes in the bacterial class Betaproteobacteria and the estrogen-associated genus Novosphingobium. Furthermore, systemic and mucosal KLH-specific immunoglobulin (Ig)M and IgT levels in the fully vaccinated fish showed only slight changes 84 days post-estrogenic oral administration. In summary, our results highlight the intrinsic relationship among estrogens, their associated receptors, and immunization in the ubiquitous fish immune regulation and the subtle but significant crosstalk with the gut endobolome.

Cultivated and Wild Juvenile Thick-Lipped Grey Mullet, Chelon labrosus: A Comparison from a Nutritional Point of View.

The thick-lipped grey mullet (Chelon labrosus) is a nominee fish species for aquaculture diversification in Spain because it is an omnivore and euryhaline species, but limited knowledge about the nutritional attributes of this species is available. This study aimed to characterize the chemical composition of wild and cultured fish. The muscle proximate composition, and fatty acid and amino acid profiles were assessed. The cultivated specimens showed a higher lipid content and lower protein and ash contents compared with the wild specimens. The predominant tissue fatty acids in both the wild and cultivated fish were palmitic acid (16:0), oleic acid (18:1n-9) and docosahexaenoic acid (DHA, 22:6n-3). A higher content of arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (20:5n-3) and DHA were detected in the muscle of wild mullets, while the fish supplied with commercial pellets showed higher quantities of monounsaturated fatty acids, and lower quantities of saturated fatty acids and polyunsaturated fatty acids (PUFAs). Regarding PUFAs, n-3 fatty acids were predominant in wild mullets, while n-6 and n-9 were more abundant in farmed fish. In terms of amino acid composition, except for histidine in wild specimens, the amino acid amounts were higher than the FAO/WHO standard. In conclusion, C. labrosus may contribute to improving the dietary intake of highly polyunsaturated n-3 fatty acids, with a benefit to human health, owing to that fact that a 100-g fillet portion of cultivated and wild C. labrosus can provide 770 mg and 1160 mg of EPA and DHA, respectively, which exceeds the 250 mg dietary daily intake recommended by the FAO/WHO.

TarSynFlow, a workflow for bacterial genome comparisons that revealed genes putatively involved in the probiotic character of Shewanella putrefaciens strain Pdp11.

Probiotic microorganisms are of great interest in clinical, livestock and aquaculture. Knowledge of the genomic basis of probiotic characteristics can be a useful tool to understand why some strains can be pathogenic while others are probiotic in the same species. An automatized workflow called TarSynFlow (Targeted Synteny Workflow) has been then developed to compare finished or draft bacterial genomes based on a set of proteins. When used to analyze the finished genome of the probiotic strain Pdp11 of Shewanella putrefaciens and genome drafts from seven known non-probiotic strains of the same species obtained in this work, 15 genes were found exclusive of Pdp11. Their presence was confirmed by PCR using Pdp11-specific primers. Functional inspection of the 15 genes allowed us to hypothesize that Pdp11 underwent genome rearrangements spurred by plasmids and mobile elements. As a result, Pdp11 presents specific proteins for gut colonization, bile salt resistance and gut pathogen adhesion inhibition, which can explain some probiotic features of Pdp11.

Accumulation and Depuration of Pathogenic and Indicator Microorganisms by the bivalve mollusc, Chamelea gallina L, Under Controlled Laboratory Conditions.

The comparative accumulation and depuration processes for several microorganisms ( Escherichia coli , Salmonella typhimurium , Vibrio parahaemolyticus , Aeromonas hydrophila , Streptococcus faecalis , Staphylococcus aureus , and MS-2 coliphage) by the striped venus, Chamelea gallina , under controlled laboratory conditions were studied. Microorganisms accumulated rapidly in bivalves during the first 6 h, with accumulation rates between 3.2 to 360.5 organisms/h depending on the type of microorganism. The relative patterns and rates of elimination of the microorganisms suggest that they are eliminated from shellfish in two different ways. One is of a mechanical nature that results in microbial elimination during the first 12 h. The other elimination mechanism depends upon the microbial species and their accumulated number. All microorganisms tested were eliminated completely by the molluscs after 3 d of depuration, except MS-2 bacteriophages. Results indicate that MS-2 coliphages may be a more reliable indicator of the microbial depuration efficiency by the shellfish under laboratory conditions than E. coli .