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

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. [...

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

Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla

Vibriosis due to Vibrio vulnificus serovar E (biotype 2) is one of the main causes of mortality in European eels cultured in Europe. The main objective of this study was to develop a vaccine and a vaccination procedure against this pathogen. With this aim, we tested several vaccine formulations (inactivated whole-cells with and without toxoids‹inactivated extracellular products‹from capsulated and uncapsulated strains, attenuated live vaccines and purified lipopolysaccharide [LPS]) on eels maintained under controlled laboratory conditions using different delivery routes (injection and immersion). To study the immune response we estimated antibody titers and bactericidal/bacteriostatic activity in mucus and serum. To evaluate protection, we calculated the relative percent survival (RPS) after intraperitoneal (i.p.) injection and bath challenge of the pathogen. The overall results indicate that: (1) capsular antigens seem to be essential for protective immunization; (2) vaccines confer the highest protection when administered by i.p. injection; (3) booster is needed to achieve good protection by immersion; (4) enriching the vaccine with toxoids enhances protection to optimal levels (RPS values around 70 to 100%, depending on the delivery route); and (5) the protective effect in serum and mucus depends on the route of administration and seems to be related to the production of specific antibodies.Fouz Rodriguez, Belen, [email protected] ; Sanjuan Caro, Eva, [email protected] ; Amaro Gonzalez, Carmen, [email protected]

Field testing of a vaccine against eel diseases caused by Vibrio vulnificus

The field results of a vaccination programme against Vibrio vulnificus serovar E (biotype 2) in a Spanish eel farm are reported. A total of 9.5 million glass eels were vaccinated from January 1998 to March 2000 by prolonged immersion followed by 2 subsequent reimmunisations after 12 to 14 and 24 to 28 d, respectively. The acquired protection and the immune response against serovar E were estimated over a period of 6 mo after vaccination. A similar vaccination schedule was conducted with elvers in a Danish eel farm. In this case, the acquired protection and the immune response against serovar E and the new eel-pathogenic serovars, recently described in Denmark, were evaluated over a short term. The overall results show that the vaccine against V. vulnificus serovar E induces a satisfactory protective immunity during the main growth period of eels (around 6 mo) with a relative percentage survival of 62 to 86% and protects them against the new eel-pathogenic serovars. Vaccination of eels by immersion seems to be the best strategy to prevent diseases caused by V. vulnificus.Fouz Rodriguez, Belen, [email protected] ; Amaro Gonzalez, Carmen, [email protected]

Gut microbiota changes in rainbow trout, Oncorhynchus mykiss (Walbaum), during organic acid feed supplementation and Yersinia ruckeri infection

Gut microbiota (GM) composition of vertebrates are considered to influence health parameters including immunological parameters in the host but knowledge of these issues in fish are sparse. We have performed a 45 day experimental study elucidating effects of dietary supplementation of organic acids (Mera Cid) and immunostimulants on the GM composition of rainbow trout Oncorhynchus mykiss (Walbaum) and investigated its association with blood parameters and disease resistance (against enteric red mouth disease (ERM) caused the bacterium Yersinia ruckeri). A total of 640 fish received either 1) basic fish feed without organic acids or immunostimulants, 2) fish feed with immunostimulants (β6glucans, MOS, nucleotides, lactic acid bacteria, vitamin C and E) but no organic acids, 3) fish feed with immunostimulants and organic acids and 4) fish feed without immunostimulants but with organic acids. Ha lf of the fish (16 groups) were then Y. ruckeri challenged on day 21 and 28 of feeding. Haemoglobin, lysozyme and gut microbiota composition (16S rRNA gene based denaturing gradient gel electrophoresis DGGE) were determined at days 14, 30 and 45. Gut microbiota analyses showed that challenge of fish with Y. ruckeri and feeding with Mera Cid affected the intestinal content

Vibriosis in aquaculture. 16th EAFP Conference, Tampere, Finland, 4th September 2013

Aquaculture in brackish and marine water is growing worldwide. New cultured species are introduced, and types of aquaculture vary from outdoor to indoor and from flow through to recirculated water, at various temperatures. In these types of aquaculture various Vibrio species play an important role, as causative agents of fish, crustacean and shellfish diseases