Characterisation of PAMP/PRR interactions in European eel (Anguilla anguilla) macrophage-like primary cell cultures

The eel (Anguilla anguilla) has been identified as a vulnerable species with stocks dramatically declining over the past decade. In an effort to support the species from overfishing of wild stocks increased interest in eel aquaculture has been notable. In order to expand the scarce knowledge concerning the biology of this species significant research efforts are required in several fields of biology. The development of cell culture systems to study the immune response is a key step towards an increased understanding of the immune response and to develop resources to support further study in this threatened species. Macrophages are one of the most important effector cells of the innate immune system. The capacity to engulf pathogens and orchestrate the immune response relies on the existence of different surface receptors, such as scavenger receptors and toll-like receptors. We have developed and described an eel macrophage-like in vitro model and studied its functional and transcriptomic responses. Macrophagelike cells from both head kidney and purified peripheral blood leukocytes were obtained and phagocytic activity measured for different whole bacteria and yeast. Moreover, based on PAMP-PRR association the innate immune response of both head kidney and PBL derived macrophage-like cells was evaluated against different pathogen-associated molecular patterns (PAMPs). Results highlight that peptidoglycan stimulation strongly induces inflammatory mRNA expression reflected in the up-regulation of proinflammatory genes IL1b and IL18 in PBL derived cells whereas IL8 is upregulated in head kidney derived cells. Furthermore TLR2 mRNA abundance is regulated by all stimuli supporting a multifunctional role for this pathogen recognition receptor (PRR) in eel macrophage-like cells

Vibrio vulnificus mutation rate: an in vitro approach

Vibrio vulnificus is a multi-host pathogenic species currently subdivided into five phylogenetic lineages (L) plus one pathovar with the ability to infect fish due to a transmissible virulence plasmid. This plasmid (or a fragment of it) has been transmitted between lineages within the species, contributing to the evolution of V. vulnificus. This study aimed to provide an experimental approximation to the V. vulnificus mutation rate by determining spontaneous mutation rates from bacterial cultures of representants of the different lineages by whole-genome sequencing. To this purpose, synonymous SNP differences, i.e., spontaneous mutation not subjected to the evolutive forces, between initial and final culture after serial growth were evaluated and used for mutation rate calculation

Eel immune response to Vibrio vulnificus infection. Host-pathogen relationship

The European eel (Anguilla anguilla), has experienced a dangerous decline in recruitment, yield and stock over the last 30 years and this decline is likely to continue into the future. Several major threats are responsible for this situation, including overfishing of glass eels for consumption, new infections by introduced pathogens, dams and blocking of migration routes. The lack of knowledge concerning the biology of this species represents a handicap to the conservation and recovery of the population. Nevertheless,the eel farming industry is increasing, therefore it could be considered one of the major risks for wild eel stock perpetuation, which is threatened principally by the lack of knowledge of the complete reproductive cycle of the European eel and by emerging bacterial, viral and parasite diseases. Vibrio vulnificus is the aetiological agent of warm-water vibriosis, a disease that constitutes the main threat to eels under culture conditions, provoking important outbreaks and can be an opportunistic pathogen for humans. V. vulnificus uses a novel characterized virulence and survival system namedMARTX (multifunctional repeat in toxin) that it is supposed to trigger a cytokine storm when it is produced in blood during the infection. It is also involved in the colonization of the gills, to invade internal organs and causes death by septicemia. We sequenced an eel immune-enriched transcriptome with Roche 454, and the data has been used to create new molecular tools for further research. We have also designed a custom eel-specific microarray (4x44K, Agilent). Our research has been focused on eel-vibrio interaction, how the host responds to the challenge and the importance of MARTX system of the bacteria for the colonization. Different challenges were performed using wild type strain (CECT4999) and Rtx double mutant strain (CT285) to evaluate mucosal immunity in the gills, the principal portal of entry.We also studied the early immune response of circulating leukocytes and the role of the erythrocytes, as one of the target cells of the lytic activity of this bacterium. Results obtained by RT-qPCR and ISH on gills demonstrate the immunocompetent function of this tissue, based on the expression and localization of immune-relates genes such as, TLRs, cytokines, chemokines and signalling molecules. Microarray studies describe key genes expressed after vibrio infection analyzed across 12h post-challenge on gills and blood (leukocytes and erythrocytes separately), specific host response against vibrio Rtx toxin, as well as, a possible function of erythrocytes in the immune response

Wild eel microbiome reveals that skin mucus of fish could be a natural niche for aquatic mucosal pathogen evolution

BACKGROUND: Fish skin mucosal surfaces (SMS) are quite similar in composition and function to some mammalian MS and, in consequence, could constitute an adequate niche for the evolution of mucosal aquatic pathogens in natural environments. We aimed to test this hypothesis by searching for metagenomic and genomic evidences in the SMS-microbiome of a model fish species (Anguilla Anguilla or eel), from different ecosystems (four natural environments of different water salinity and one eel farm) as well as the water microbiome (W-microbiome) surrounding the host. RESULTS: Remarkably, potentially pathogenic Vibrio monopolized wild eel SMS-microbiome from natural ecosystems, Vibrio anguillarum/Vibrio vulnificus and Vibrio cholerae/Vibrio metoecus being the most abundant ones in SMS from estuary and lake, respectively. Functions encoded in the SMS-microbiome differed significantly from those in the W-microbiome and allowed us to predict that successful mucus colonizers should have specific genes for (i) attachment (mainly by forming biofilms), (ii) bacterial competence and communication, and (iii) resistance to mucosal innate immunity, predators (amoeba), and heavy metals/drugs. In addition, we found several mobile genetic elements (mainly integrative conjugative elements) as well as a series of evidences suggesting that bacteria exchange DNA in SMS. Further, we isolated and sequenced a V. metoecus strain from SMS. This isolate shares pathogenicity islands with V. cholerae O1 from intestinal infections that are absent in the rest of sequenced V. metoecus strains, all of them from water and extra-intestinal infections. CONCLUSIONS: We have obtained metagenomic and genomic evidence in favor of the hypothesis on the role of fish mucosal surfaces as a specialized habitat selecting microbes capable of colonizing and persisting on other comparable mucosal surfaces, e.g., the human intestine

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

Draft Genome Sequence of Environmental Bacterium Vibrio vulnificus CladeA-yb158

We report the genome sequence of the environmental Vibrio vulnificus biotype 1_cladeA. This draft genome of the CladeA-yb158 strain, isolated in Israel, represents this newly emerged clonal group that contains both clinical and environmental strains

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

Apolipoprotein C-III in patients with systemic lupus erythematosus

Background: Systemic lupus erythematosus (SLE) has been associated with atherosclerotic cardiovascular disease (CV) and an altered lipid profile. High levels of apolipoprotein C-III (ApoC3) are associated with elevated triglyceride levels and an increased risk of CV. In the present study, we aimed to study circulating ApoC3 in patients with SLE and describe its relationship with the manifestations of the disease. Methods: This is a cross-sectional study that included 186 patients with SLE. Disease-related data, CV comorbidity, full lipid profile, and serum levels of ApoC3 were assessed. A multivariable regression analysis was performed to study how ApoC3 was related to SLE features. Results: Classic CV risk factors were significantly and strongly associated with circulating ApoC3. After a fully multivariable analysis that included classic CV risk factors and lipid profile molecules, SLICC damage (beta coef. 0.10 [95% CI 0.02?0.19] mg/dl, 0.020) and Katz severity (beta coef. 0.11 [95% CI 0.03-0.19] mg/dl, p = 0.011) indices and SLEDAI activity score (beta coef. 0.05 [95% CI 0.05-0.08] mg/dl, p = 0.004) were all independently associated with higher levels of circulating ApoC3. Conclusion: Among SLE patients, disease activity, severity, and disease damage are independently associated with higher ApoC3 serum levels.Funding: This work was supported by a grant to I.F-A. from the Spanish Ministry of Health, Subdirección General de Evaluación y Fomento de la Investigación, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016 and by Fondo Europeo de Desarrollo Regional—FEDER—(Fondo de Investigaciones Sanitarias, PI17/00083)

pilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of human health relevance

The Gram-negative bacterium Vibrio vulnificus is a common inhabitant of estuarine environments. Globally, V. vulnificus is a significant foodborne pathogen capable of causing necrotizing wound infections and primary septicemia, and is a leading cause of seafood-related mortality. Unfortunately, molecular methods for the detection and enumeration of pathogenic V. vulnificus are hampered by the genetically diverse nature of this pathogen, the range of different biotypes capable of infecting humans and aquatic animals, and the fact that V. vulnificus contains pathogenic as well as non-pathogenic variants. Here we report an alternative approach utilizing the development of a real-time PCR assay for the detection of pathogenic V. vulnificus strains based on a polymorphism in pilF, a gene previously indicated to be associated with human pathogenicity. Compared to human serum reactivity, the real-time PCR assay successfully detected pathogenic strains in 46 out of 47 analysed V. vulnificus isolates (97.9%). The method is also rapid, sensitive, and more importantly can be reliably utilised on biotype 2 and 3 strains, unlike other current methods for V. vulnificus virulence differentiation