Pyrosequencing survey of intestinal microbiota diversity in cultured sea bass (Dicentrarchus labrax) fed functional diets.

The routine use of chemotherapy to control bacterial diseases in aquatic populations has resulted in the development and spread of antibiotic resistance. The inclusion of immunostimulants in fish diets (functional diets) is one of the main strategies to solve this threat. This study aimed to analyse the intestinal microbiota of cultured European sea bass (Dicentrarchus labrax) fed two functional diets applying pyrosequencing of PCR-amplified 16S rRNA gene. Quality-filtered reads were assigned to family and genus taxonomic levels using the Ribosomal Database Project classifier. The autochthonous intestinal microbiota of sea bass consisted of two dominant bacterial genera: Dysgonomonas (Bacteroidetes) and Ralstonia (Betaproteobacteria), but effects of diet on this dominance were observed. In fact, the genus Dysgonomonas significantly decreased in samples from fish fed functional diets, recovering control levels at the end of the study. However, Ralstonia proportion significantly raised in samples from fish fed diet C and maintained this high level along the study period. The developed protocol could be used to study the composition of bacterial communities in the fish intestine under different nutritional and environmental conditions and its impact on infection, immune system and general fitness of fish

Evaluation of genotypic and phenotypic methods to distinguish clinical from environmental Vibrio vulnificus strains.

V. vulnificus is a heterogeneous bacterial species that comprises virulent and avirulent strains from environmental and clinical sources that have been grouped into three biotypes. To validate the typing methods proposed to distinguish clinical from environmental isolates, we performed phenotypic (API 20E, API20NE and BIOLOG tests) and genetic (ribotyping and DNA polymorphisms at several loci) studies with a large strain collection representing different biotypes, origins and host range. No phenotypic method was useful for biotyping or grouping strains with regard to origin of the isolate and only BIOLOG system was reliable to identify the strains at species level. The DNA polymorphisms divided the population into three major profiles: profile 1 were vcg type C, 16S rRNA type B and vvh type 1 and included most of biotype 1 human septicemic isolates; profile 2 were vcg type E, 16S rRNA type A and vvh type 2 and included all biotype 2 isolates together with biotype 1 isolates from fish, water and some human isolates; and the last one, profile 3, were vcg type E, 16S rRNA type AB and vvh type 2 and composed by biotype 3 strains. Ribotyping divided the species into two groups, one that included profile 1 isolates of biotype 1, and the other including isolates of all three biotypes belonging to the three profiles described. In conclusion, no genotyping system was able to distinguish neither clinical from environmental strains nor biogroups within V. vulnificus species, which suggests that new typing methodologies useful for Public Health have to be developed for this species

Biochemical and molecular characterization of three serologically different Vibrio harveyi strains isolated from farmed Dicentrarchus labrax from the Adriatic Sea

Vibrio harveyi is recognized as one of the major causes of vibriosis, a disease that threatens the long-term sustainability of aquaculture. Current research shows that the Mediterranean strains of V. harveyi are serologically heterogeneous, though research comparing the traits of different strains is scarce. This study aims to describe the biochemical, physiological and genetic characteristics of three serologically different strains of V. harveyi isolated from farmed European Sea bass (Dicentrarchus labrax) from the Adriatic Sea. A total of 32 morphological and biochemical markers were examined and, the susceptibility to 13 antimicrobials tested, and then compared the results of high-throughput sequencing and in silico analyses. This study also presents the first whole genome sequences of V. harveyi isolated from European sea bass. A large number of nonsynonymous variations were detected among sequences of the three strains. The prediction analysis of resistance genes did not correspond with the in vitro antimicrobial susceptibility tests. Six virulence genes previously unrelated to virulence of vibrios were detected in all three studied strains. The results show that differences were detected at every level of comparison among the three studied strains isolated from the same fish species originating from a small geographic area

A multiplex PCR for the detection of Vibrio vulnificus hazardous to human and/or animal health from seafood

Vibrio vulnificus is a zoonotic pathogen linked to aquaculture that is spreading due to climate change. The pathogen can be transmitted to humans and animals by ingestion of raw shellfish or seafood feed, respectively. The aim of this work was to design and test a new procedure to detect V. vulnificus hazardous to human and/or animal health in food/feed samples. For this purpose, we combined a pre-enrichment step with multiplex PCR using primers for the species and for human and animal virulence markers. In vitro assays with mixed DNA from different Vibrio species and Vibrio cultures showed that the new protocol was 100 % specific with a detection limit of 10 cfu/mL. The protocol was successfully validated in seafood using artificially contaminated live shrimp and proved useful also in pathogen isolation from animals and their ecosystem. In conclusion, this novel protocol could be applied in health risk studies associated with food/feed consumption, as well as in the routine identification and subtyping of V. vulnificus from environmental or clinical samples

The Effect of the Environmental Temperature on the Adaptation to Host in the Zoonotic Pathogen Vibrio vulnificus

Vibrio vulnificus is a zoonotic pathogen that lives in temperate, tropical and subtropical aquatic ecosystems whose geographical distribution is expanding due to global warming. The species is genetically variable and only the strains that belong to the zoonotic clonal-complex can cause vibriosis in both humans and fish (being its main host the eel). Interestingly, the severity of the vibriosis in the eel and the human depends largely on the water temperature (highly virulent at 28°C, avirulent at 20°C or below) and on the iron content in the blood, respectively. The objective of this work was to unravel the role of temperature in the adaptation to the host through a transcriptomic and phenotypic approach. To this end, we obtained the transcriptome of a zoonotic strain grown in a minimum medium (CM9) at 20, 25, 28, and 37°C, and confirmed the transcriptomic results by RT-qPCR and phenotypic tests. In addition, we compared the temperature stimulon with those previously obtained for iron and serum (from eel and human, respectively). Our results suggest that warm temperatures activate adaptive traits that would prepare the bacteria for host colonization (metabolism, motility, chemotaxis, and the protease activity) and fish septicemia (iron-uptake from transferrin and production of O -antigen of high molecular weight) in a generalized manner, while environmental iron controls the expression of a host-adapted virulent phenotype (toxins and the production of a protective envelope). Finally, our results confirm that beyond the effect of temperature on the V. vulnificus distribution in the environment, it also has an effect on the infectious capability of this pathogen that must be taken into account to predict the real risk of V. vulnificus infection caused by global warming

Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets

[Background]: The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested. [Results]: The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome. [Conclusions]: This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach.This work has been carried out with financial support from the European Union under grant projects ARRAINA (FP7-KBBE-2011-288,925) to JPS, MP and VK and ParaFishControl (H2020-634429) to ASB. Additional funding has been received from Spanish Ministry of Economy and Competitiveness (MINECO) project no. AGL2013- 48560-R to JPS and ASB, and Generalitat Valenciana (PROMETEOII/2014/085) to ASB. MCP was contracted under CSIC PIE project no. 201740E013 and MINECO FPDI-2013-15741, and IE under APOSTD/2016/037 grant by the “Generalitat Valenciana”.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Developmental immunotoxicology: What underlies the critical windows of exposure?

Endocrine disruptors in general and oestrogenic compounds in particular have been widely investigated in view of their effects on several physiological processes and, therefore, their ecotoxicologic relevance. [...

European Sea Bass (Dicentrarchus labrax) immune status and disease resistance are impaired by arginine dietary supplementation

Infectious diseases and fish feeds management are probably the major expenses in the aquaculture business. Hence, it is a priority to define sustainable strategies which simultaneously avoid therapeutic procedures and reinforce fish immunity. Currently, one preferred approach is the use of immunostimulants which can be supplemented to the fish diets. Arginine is a versatile amino acid with important mechanisms closely related to the immune response. Aiming at finding out how arginine affects the innate immune status or improve disease resistance of European seabass (Dicentrarchus labrax) against vibriosis, fish were fed two arginine-supplemented diets (1% and 2% arginine supplementation). A third diet meeting arginine requirement level for seabass served as control diet. Following 15 or 29 days of feeding, fish were sampled for blood, spleen and gut to assess cell-mediated immune parameters and immune-related gene expression. At the same time, fish from each dietary group were challenged against Vibrio anguillarum and survival was monitored. Cell-mediated immune parameters such as the extracellular superoxide and nitric oxide decreased in fish fed arginine-supplemented diets. Interleukins and immune-cell marker transcripts were down-regulated by the highest supplementation level. Disease resistance data were in accordance with a generally depressed immune status, with increased susceptibility to vibriosis in fish fed arginine supplemented diets. Altogether, these results suggest a general inhibitory effect of arginine on the immune defences and disease resistance of European seabass. Still, further research will certainly clarify arginine immunomodulation pathways thereby allowing the validation of its potential as a prophylactic strategy.European Union's Seventh Framework Programme AQUAEXCEL (Aquaculture Infrastructures for Excellence in European Fish Research) [262336]; AQUAIMPROV [NORTE-07-0124-FEDER-000038]; North Portugal Regional Operational Programme (ON. 2 - O Novo Norte) , under the National Strategic Reference Framework, through the European Regional Development Fund; North Portugal Regional Operational Programme (ON. 2 - O Novo Norte), under the National Strategic Reference Framework through the COMPETE - Operational Competitiveness Programme; Fundacao para a Ciencia e Tecnologia; Fundacao para a Ciencia e Tecnologia [SFRH/BD/89457/2012, SFRH/BPD/77210/2011]; Generalitat Valenciana through the project REVIDPAQUA [ISIC/2012/003]; [PEst-C/MAR/LA0015/2013]; [UID/Multi/04423/2013]info:eu-repo/semantics/publishedVersio

Bacterial diseases of tilapia, their zoonotic potential and risk of antimicrobial resistance

Tilapia culture is an important source of income and nutrition to many rural families. Since 2000, the production of tilapia increased and reached domestic and global markets. Major farmed species is Nile tilapia (Oreochromis niloticus), in earthen ponds and cage cultures. Intensification contributed to global tilapia disease outbreaks, with bacterial infections causing mortalities and morbidities, threatening sustainable production. At tilapia farms, high nutrient concentrations, water temperature and fish densities enhance bacterial growth including virulent bacterial clones and potential zoonotic bacteria. Global warming favours this. This review respectively provides a comprehensive overview of the most common and emerging bacterial pathogens, diseases, clinical presentations and diagnostics of tilapia, including bacteria and diseases with zoonotic potential. First, common bacterial disease outbreaks, including streptococcosis, motile Aeromonas septicaemia, francisellosis, columnaris disease and vibriosis are described. Then, information on emerging bacterial infections of concern for tilapia, like edwardsiellosis through Edwardsiella ictaluri and E. tarda, as well as Aeromonas schubertii is provided. Reports of infectious bacterial tilapia disease outbreaks from other bacteria, including Lactococcus garvieae, Aerococcus viridans, Pseudomonas spp., Mycobacterium marinum and Chlamydia spp., and others are reviewed. Furthermore, bacteria with zoonotic potential, like Streptococcus agalactiae ST283, S. iniae, Aeromonas sp., E. tarda, Vibrio vulnificus pathovar (pv) piscis and M. marinum are included in the review, to provide the most current overview of the disease risks affecting production and post-harvest stages. Additionally, the status and risks of antimicrobial resistance in bacteria from tilapia and other cultured fish through imprudent use of antibiotics, and its future at a global level are provided

Micromón València (Universitat de València)

En Julio de 2017 se creó la red SWI@Spain, auspiciada por el grupo de Docencia y Difusión de la Microbiología (DDM) de la Sociedad Española de Microbiología (SEM), para desarrollar la iniciativa internacional Small World Initiative (SWI) en la península ibérica. En la Universitat de València (UV) se constituyó entonces el grupo de Innovación Docente en Microbiología (IDM) para implementar el proyecto a nivel local. Avalados por el Servei de Formació Permanent i Innovació Educativa (SFPIE) de la UV, el grupo ha llevado a cabo diferentes iniciativas relacionadas con el objetivo fundamental del proyecto: divulgar la problemática actual relacionada con el uso inadecuado de antibióticos, el incremento de bacterias resistentes a éstos y la necesidad de encontrar nuevas moléculas con actividad antibacteriana para combatir las infecciones que provocan