Human gut microbiome, diet, and mental disorders.

Diet is one of the most important external factor shaping the composition and metabolic activities of the gut microbiome. The gut microbiome plays a crucial role in host health, including immune system development, nutrients metabolism, and the synthesis of bioactive molecules. In addition, the gut microbiome has been described as critical for the development of several mental disorders. Nutritional psychiatry is an emerging field of research that may provide a link between diet, microbial function, and brain health. In this study, we have reviewed the influence of different diet types, such as Western, Mediterranean, vegetarian, and ketogenic, on the gut microbiota composition and function, and their implication in various neuropsychiatric and psychological disorders.

Fish Iridoviridae: infection, vaccination and immune response.

Each year, due to climate change, an increasing number of new pathogens are being discovered and studied, leading to an increase in the number of known diseases affecting various fish species in different regions of the world. Viruses from the family Iridoviridae, which consist of the genera Megalocytivirus, Lymphocystivirus, and Ranavirus, cause epizootic outbreaks in farmed and wild, marine, and freshwater fish species (including ornamental fish). Diseases caused by fish viruses of the family Iridoviridae have a significant economic impact, especially in the aquaculture sector. Consequently, vaccines have been developed in recent decades, and their administration methods have improved. To date, various types of vaccines are available to control and prevent Iridoviridae infections in fish populations. Notably, two vaccines, specifically targeting Red Sea bream iridoviral disease and iridoviruses (formalin-killed vaccine and AQUAVAC® IridoV, respectively), are commercially available. In addition to exploring these themes, this review examines the immune responses in fish following viral infections or vaccination procedures. In general, the evasion mechanisms observed in iridovirus infections are characterised by a systemic absence of inflammatory responses and a reduction in the expression of genes associated with the adaptive immune response. Finally, this review also explores prophylactic procedure trends in fish vaccination strategies, focusing on future advances in the field.

Feed and immersion challenges with lymphocystis disease virus (LCDV) reveals specific mechanisms for horizontal transmission and immune response in senegalese sole post-larvae.

The horizontal transmission of lymphocystis disease virus (LCDV) through contaminated water and feed (using artemia as vehicle) and the associated immune gene expression profiles in Senegalese sole post-larvae were investigated. All specimens analyzed were positive for LCDV DNA detection at 1-day post-challenge (1 dpc) with the highest viral levels in specimens infected through the immersion route. However, the percentage of LCDV-positive animals and number of viral DNA copies dropped progressively at 2 and 7 dpc. The histological analysis identified structural changes in the skin, muscle and gills of sole post-larvae LCDV-challenged by immersion. In situ hybridization confirmed a wide distribution of LCDV in the skin, gut, surrounding vessels in trunk muscle and head kidney in the immersion route, while the signals were restricted to the liver and lamina propria in the feeding treatment. Expression analysis using a set of 22 genes related to innate immune defense system demonstrated clear differences in the time-course response to LCDV as function of the infection route. Most antiviral defense genes, the proinflammatory cytokines, the complement c3, g-type lysozyme and T-cell markers cd4 and cd8a were rapidly induced in the feeding-infected post-larvae, and they were remained activated at 2 dpc. In contrast, in the immersion-infected post-larvae the induction of most defensive genes was delayed, with a low intensity at 2 dpc. All these data demonstrate that LCDV can horizontally infect Senegalese sole post-larvae through the water or feed although with different patterns of histopathological disorders, virus distribution and route-specific expression profiles.

Lymphocystis disease virus (LCDV-Sa), polyomavirus 1 (SaPyV1) and papillomavirus 1 (SaPV1) in samples of Mediterranean gilthead seabream.

Lymphocystis disease, caused by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of tumour-like lesions on the skin of affected animals associated with several environmental factors and/or with stress due to the intensive culture conditions of fish farms. In a previous study, the genomes of a new LCDV species, LCDV-Sa, were detected, together with 2 previously unknown viruses, Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1). Gilthead seabream from 17 fish farms in Spain, Italy and Turkey were sampled between 2009 and 2015 to investigate the role of the newly described SaPV1 and SaPyV1 viruses in lymphocystis disease development. Our results show that in diseased fish, either or both of the new viruses are almost invariably detected together with LCDV (98%). In asymptomatic fish, these viruses were detected in a much lower percentage (28%) and mostly in concurrence with LCDV (24%). These data confirm the suspected association among the 3 different viruses during lymphocystis disease development in gilthead seabream and warrant future studies to establish their respective contributions.

Modification of betanodavirus virulence by substitutions in the 3' terminal region of RNA2.

Betanodaviruses have bi-segmented positive-sense RNA genomes, consisting of RNAs 1 and 2. For some members of the related genus alphanodavirus, the 3' terminal 50 nucleotides (nt) of RNA2, including a predicted stem-loop structure (3'SL), are essential for replication. We investigate the possible existence and role of a similar structure in a reassortant betanodavirus strain (RGNNV/SJNNV). In this study, we developed three recombinant strains containing nucleotide changes at positions 1408 and 1412. Predictive models showed stem-loop structures involving nt 1398-1421 of the natural reassortant whereas this structure is modified in the recombinant viruses harbouring point mutations r1408 and r1408-1412, but not in r1412. Results obtained from infectivity assays showed differences between the reference strains and the mutants in both RNA1 and RNA2 synthesis. Moreover, an imbalance between the synthesis of both segments was demonstrated, mainly with the double mutant. All these results suggest an interaction between RNA1 and the 3' non-coding regions (3'NCR) of RNA2. In addition, the significant attenuation of the virulence for Senegalese sole and the delayed replication of r1408-1412 in brain tissues may point to an interaction of RNA2 with host cellular proteins.

Differential antiviral activity of European sea bass interferon-stimulated 15 protein (ISG15) against RGNNV and SJNNV betanodaviruses.

ISG15 is an antiviral protein acting intracellularly, by conjugation to viral or cellular proteins, or extracellularly, as cytokine. In this work, an in vitro system, consisting of E-11 cells over-expressing European sea bass ISG15 (Dl_ISG15_E11 cells), has been developed to evaluate the European sea bass ISG15 protein activity against RGNNV and SJNNV isolates. Regarding RGNNV, RNA2 copy number and viral titres were similar in E-11 and Dl_ISG15_E11 cells, and the cellular survival analyses demonstrated that Dl_ISG15_E11 cells were not protected from this virus. In contrast, ISG15 compromises SJNNV replication, since a reduction of the SJNNV genome synthesis has been recorded. The ISG15 anti-SJNNV activity was confirmed by viral titration and survival assays. In addition, a role of the intracellular ISG15 in modulating the transcription of endogenous genes has being recorded, with tlr3 gene being knocked out and e3 gene being up-regulated in RGNNV-inoculated Dl_ISG15_E11 cells. Sea bass ISG15 has also been detected extracellularly, and its activity has been evaluated by co-culture. The survival rate of RGNNV-inoculated E-11 cells increased from 25% to 46% when they were co-cultured with ISG15-producing cells. Similarly, the survival rate of SJNNV-inoculated E-11 cells increased from 27% to 51% in co-culture with ISG15-producing cells. To our knowledge, this is the first description of a differential antiviral activity of an ISG15 protein against two betanodavirus species, and the first evaluation of the cytokine-like activity of a fish ISG15 protein on non-immune cells.

Immuno-related gene transcription and antibody response in nodavirus (RGNNV and SJNNV)-infected European sea bass (Dicentrarchus labrax L.).

The immune response of European sea bass to RGNNV and SJNNV infections has been evaluated by quantifying the transcription of some genes involved in the IFN I system, as well as in the inflammatory and adaptive immune mechanisms. The transcription of IFN-I, ISG-12, ISG-15 and MxA genes has been analyzed in brain and head kidney, showing that RGNNV genotype induces a more intense response of the IFN I system than SJNNV in both organs. In addition, the results obtained indicate the importance of the inflammatory response in nodavirus pathogenesis, with the transcription of IL-8 and TNF-α significantly higher in brain than in head kidney, being RGNNV the strongest inductor. An important difference between the immune response induced by both genotypes refers to the IgM titre in sera, which was higher in SJNNV-inoculated fish. The acquired response is also important locally, since TR-γ transcription is higher in brain than in head kidney (especially in the RGNNV-inoculated group). To our knowledge, this is the first study addressing the sea bass anti-SJNNV immune response.

Concurrence of Iridovirus, Polyomavirus, and a Unique Member of a New Group of Fish Papillomaviruses in Lymphocystis Disease-Affected Gilthead Sea Bream.

UNLABELLED: Lymphocystis disease is a geographically widespread disease affecting more than 150 different species of marine and freshwater fish. The disease, provoked by the iridovirus lymphocystis disease virus (LCDV), is characterized by the appearance of papillomalike lesions on the skin of affected animals that usually self-resolve over time. Development of the disease is usually associated with several environmental factors and, more frequently, with stress conditions provoked by the intensive culture conditions present in fish farms. In gilthead sea bream (Sparus aurata), an economically important cultured fish species in the Mediterranean area, a distinct LCDV has been identified but not yet completely characterized. We have used direct sequencing of the virome of lymphocystis lesions from affected S. aurata fish to obtain the complete genome of a new LCDV-Sa species that is the largest vertebrate iridovirus sequenced to date. Importantly, this approach allowed us to assemble the full-length circular genome sequence of two previously unknown viruses belonging to the papillomaviruses and polyomaviruses, termed Sparus aurata papillomavirus 1 (SaPV1) and Sparus aurata polyomavirus 1 (SaPyV1), respectively. Epidemiological surveys showed that lymphocystis disease was frequently associated with the concurrent appearance of one or both of the new viruses. SaPV1 has unique characteristics, such as an intron within the L1 gene, and as the first member of the Papillomaviridae family described in fish, provides evidence for a more ancient origin of this family than previously thought. IMPORTANCE: Lymphocystis disease affects marine and freshwater fish species worldwide. It is characterized by the appearance of papillomalike lesions on the skin that contain heavily enlarged cells (lymphocysts). The causative agent is the lymphocystis disease virus (LCDV), a large icosahedral virus of the family Iridoviridae In the Mediterranean area, the gilthead sea bream (Sparus aurata), an important farmed fish, is frequently affected. Using next-generation sequencing, we have identified within S. aurata lymphocystis lesions the concurrent presence of an additional LCDV species (LCDV-Sa) as well as two novel viruses. These are members of polyomavirus and papillomavirus families, and here we report them to be frequently associated with the presence of lymphocysts in affected fish. Because papillomaviruses have not been described in fish before, these findings support a more ancient origin of this virus family than previously thought and evolutionary implications are discussed.

Photobacterium toruni sp. nov., a bacterium isolated from diseased farmed fish.

Three bacterial strains were isolated from liver and spleen of diseased farmed redbanded seabream (Pagrus auriga) in south-west Spain. Their partial 16S rRNA gene sequences clustered within those of the genus Photobacterium, showing high similarity (98.6-99.3 %) to the type strains of Photobacterium iliopiscarium, P. piscicola, P. kishitanii, P. aquimaris and P. phosphoreum. Multilocus sequence analysis using six housekeeping genes (gapA, topA, mreB, ftsZ, gyrB and 16S rRNA) confirmed the new strains as forming an independent branch with a bootstrap value of 100, likely to represent a novel species. To confirm this, we used whole genome sequencing and genomic analysis (ANIb, ANIm and in silico DNA-DNA hybridization) obtaining values well below the thresholds for species delineation. In addition, a phenotypic characterization was performed to support the description and differentiation of the novel strains from related taxa. Cells were Gram-stain-negative, motile bacilli, chemo-organotrophic and facultatively anaerobic. They fermented glucose, as well as galactose and d-mannose, without production of gas. Oxidase and catalase were positive. The predominant cellular fatty acids were C16 : 1ω7c/C16 : 1ω6c and C16  :  0. The predominant respiratory quinone (Q-8) and major polar lipids (phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol) were inferred from annotated genes in the genome of strain H01100410BT, which had a G+C content of 38.6 mol%. The results obtained demonstrate that the three strains represent a novel species, for which the name Photobacterium toruni sp. nov. is proposed. The type strain is H01100410BT (=CECT 9189T=LMG 29991T).

Target organs for lymphocystis disease virus replication in gilthead seabream (Sparus aurata).

The lymphocystis disease (LCD), the main viral pathology described in cultured gilthead seabream (Sparus aurata), is a self-limiting condition characterized by the appearance of hypertrophied fibroblasts (named lymphocysts) in the connective tissue of fish, primarily in the skin and fins. The causative agent of the disease is the Lymphocystis disease virus (LCDV), a member of the Iridoviridae family. In the present study, LCDV genome and transcripts were detected by real-time PCR in caudal fin, as well as in several internal organs, such as intestine, liver, spleen, kidney and brain, from asymptomatic, diseased and recovered gilthead seabream juveniles. These results indicate that the LCDV has a broad range tissue tropism, and can establish a systemic infection, even in subclinically infected fish. As showed by in situ hybridization, the permissive cells for LCDV infection seem to be fibroblasts, hepatocytes and cells of the mononuclear phagocyte system. Histopathological alterations associated with LCD were observed in all the organs analysed, including necrotic changes in liver and kidney, inflammatory response in the intestine submucosa or brain haemorrhage, although lymphocysts were only detected in the dermis of the caudal fin. Nevertheless, these histological changes were reverted in recovered animals.

Gene expression profiles associated with lymphocystis disease virus (LCDV) in experimentally infected Senegalese sole (Solea senegalensis).

In the present study, the pathogenesis of lymphocystis disease virus (LCDV) and the immune gene expression patterns associated with this viral infection were determined in the flatfish Senegalese sole. The results indicate that LCDV spreads rapidly from the peritoneal cavity through the bloodstream to reach target organs such as kidney, gut, liver, and skin/fin. The viral load was highest in kidney and reduced progressively thorough the experiment in spite of the viral major capsid protein gene was transcribed. The LCDV injection activated a similar set of differentially expressed transcripts in kidney and intestine although with some differences in the intensity and time-course response. This set included antiviral-related transcripts (including the mx and interferon-related factors irf1, irf2, irf3, irf7, irf8, irf9, irf10), cytokines (il1b, il6, il8, il12 and tnfa) and their receptors (il1r, il8r, il10r, il15ra, il17r), chemokines (CXC-type, CC-type and IL-8), prostaglandins (cox-2), g-type lysozymes, hepcidin, complement fractions (c2, c4-1 and c4-2) and the antigen differentiation factors cd4, cd8a, and cd8b. The expression profile observed indicated that the host triggered a systemic defensive response including inflammation able to cope with the viral challenge.

SJNNV down-regulates RGNNV replication in European sea bass by the induction of the type I interferon system.

European sea bass is highly susceptible to the betanodavirus RGNNV genotype, although the SJNNV genotype has also been detected in this fish species. The coexistence of both genotypes may affect the replication of both viruses by viral interaction or by stimulation of the host antiviral defense system in which the IFN I system plays a key role. IFN I triggers the transcription of interferon-stimulated genes, including Mx genes, whose expression has been used as a reporter of IFN I activity. The present study evaluated the effect of a primary exposure to an SJNNV isolate on a subsequent RGNNV infection and analyzed the role of the IFN I system in controlling VNNV infections in sea bass using different in vivo approaches. VNNV infection and Mx transcription were comparatively evaluated after single infections, superinfection (SJ+RG) and co-infection (poly I:C+RG). The single RGNNV infection resulted in a 24% survival rate, whereas the previous SJNNV or poly I:C inoculation increased the survival rate up to 96 and 100%, respectively. RGNNV replication in superinfection was reduced compared with RGNNV replication after a single inoculation. Mx transcription analysis shows differential induction of the IFN I system by both isolates. SJNNV was a potent Mx inducer, whereas RGNNV induced lower Mx transcription and did not interfere with the IFN I system triggered by SJNNV or poly I:C. This study demonstrates that an antiviral state exists after SJNNV and poly I:C injection, suggesting that the IFN I system plays an important role against VNNV infections in sea bass.

Genetic characterization and transcription analyses of the European sea bass (Dicentrarchus labrax) isg15 gene.

Fish interferons are cytokines involved in its resistance to viral infections by inducing the transcription of several interferon-induced genes, such as isg15. The aim of the present study was the genetic characterization of the European sea bass isg15 gene, describing the regulatory motifs found in its sequence. In addition, an in vivo analysis of transcription in response to betanodavirus (RGNNV genotype) and poly I:C has been performed. The analysis of the resulting sequences showed that sea bass isg15 gene is composed of two exons and a single 276-bp intron located at the 5'-UTR region. The full length cDNA is 1143-bp, including a 102-bp 5'-UTR region, a 474-bp ORF, and a 291-bp 3'-UTR region. Several mRNA-regulatory elements, including three unusual ATTTA instability motifs in the intron, and four ATTTA motifs along with a cytoplasmic polyadenylation element in the 3'-UTR region, have been found in this sequence. The in vivo analyses revealed a similar kinetics and level of transcription in fish brain and head kidney after poly I:C inoculation; however, the induction caused by RGNNV started earlier in brain, where the upregulation of isg15 gene transcription was high. The present study contributes to further characterize the European sea bass IFN I response against RGNNV infections.

Application of a new real-time polymerase chain reaction assay for surveillance studies of lymphocystis disease virus in farmed gilthead seabream.

BACKGROUND: Lymphocystis disease (LCD) is the main viral infection reported to affect cultured gilthead seabream (Sparus aurata) in Europe. The existence of subclinical Lymphocystis disease virus (LCDV) infection in this fish species has been recognised by using polymerase chain reaction (PCR)-based methods. Nevertheless, these methods do not provide quantitative results that can be useful in epidemiological and pathological studies. Moreover, carrier fish have been involved in viral transmission, therefore the use of specific and sensitive diagnostic methods to detect LCDV will be relevant for LCD prevention. RESULTS: We have developed a real-time PCR (qPCR) assay to detect and quantify LCDV. The assay was evaluated for viral diagnosis in surveillance studies in gilthead seabream farms, and also to identify viral reservoirs in a hatchery. The prevalence of LCDV infection in the asymptomatic gilthead seabream populations tested varied from 30 to 100 %, including data from one farm without previous records of LCD. Estimated viral load in caudal fin of subclinically infected fish was two to five orders of magnitude lower than in diseased fish. The qPCR assay allowed the detection of carrier fish in broodstock from a farm with a history of clinical LCD in juvenile fish. In addition, the quantitative detection of LCDV was achieved in all samples collected in the hatchery, including fertilized eggs, larvae and fingerlings, and also rotifer cultures and artemia metanauplii and cysts used for larval rearing. CONCLUSIONS: The qPCR assay developed in this study has proved to be a rapid, sensitive, and reliable method for LCDV diagnosis, which could be valuable to identify LCDV reservoirs or to study viral replication in gilthead seabream.

Neurodevelopmental Disorders Associated with Gut Microbiome Dysbiosis in Children.

The formation of the human gut microbiome initiates in utero, and its maturation is established during the first 2-3 years of life. Numerous factors alter the composition of the gut microbiome and its functions, including mode of delivery, early onset of breastfeeding, exposure to antibiotics and chemicals, and maternal stress, among others. The gut microbiome-brain axis refers to the interconnection of biological networks that allow bidirectional communication between the gut microbiome and the brain, involving the nervous, endocrine, and immune systems. Evidence suggests that the gut microbiome and its metabolic byproducts are actively implicated in the regulation of the early brain development. Any disturbance during this stage may adversely affect brain functions, resulting in a variety of neurodevelopmental disorders (NDDs). In the present study, we reviewed recent evidence regarding the impact of the gut microbiome on early brain development, alongside its correlation with significant NDDs, such as autism spectrum disorder, attention-deficit/hyperactivity disorder, Tourette syndrome, cerebral palsy, fetal alcohol spectrum disorders, and genetic NDDs (Rett, Down, Angelman, and Turner syndromes). Understanding changes in the gut microbiome in NDDs may provide new chances for their treatment in the future.

Influence of human gut microbiome on the healthy and the neurodegenerative aging.

The gut microbiome plays a crucial role in host health throughout the lifespan by influencing brain function during aging. The microbial diversity of the human gut microbiome decreases during the aging process and, as a consequence, several mechanisms increase, such as oxidative stress, mitochondrial dysfunction, inflammatory response, and microbial gut dysbiosis. Moreover, evidence indicates that aging and neurodegeneration are closely related; consequently, the gut microbiome may serve as a novel marker of lifespan in the elderly. In this narrative study, we investigated how the changes in the composition of the gut microbiome that occur in aging influence to various neuropathological disorders, such as mild cognitive impairment (MCI), dementia, Alzheimer's disease (AD), and Parkinson's disease (PD); and which are the possible mechanisms that govern the relationship between the gut microbiome and cognitive impairment. In addition, several studies suggest that the gut microbiome may be a potential novel target to improve hallmarks of brain aging and to promote healthy cognition; therefore, current and future therapeutic interventions have been also reviewed.

Immune response of DNA vaccinated-gilthead seabream (Sparus aurata) against LCDV-Sa infection: relevance of the inflammatory process.

Lymphocystis disease is one of the main viral pathologies affecting cultured gilthead seabream (Sparus aurata) in the Mediterranean region. Recently, we have developed a DNA vaccine based on the major capsid protein (MCP) of the Lymphocystis disease virus 3 (LCDV-Sa). The immune response triggered by either LCDV-Sa infection or vaccination have been previously studied and seem to be highly related to the modulation of the inflammatory and the IFN response. However, a comprehensive evaluation of immune-related gene expression in vaccinated fish after viral infection to identify immunogenes involved in vaccine-induced protection have not been carried out to date. The present study aimed to fulfill this objective by analyzing samples of head-kidney, spleen, intestine, and caudal fin from fish using an OpenArray® platform containing targets related to the immune response of gilthead seabream. The results obtained showed an increase of deregulated genes in the hematopoietic organs between vaccinated and non-vaccinated fish. However, in the intestine and fin, the results showed the opposite trend. The global effect of fish vaccination was a significant decrease (p<0.05) of viral replication in groups of fish previously vaccinated, and the expression of the following immune genes related to viral recognition (tlr9), humoral and cellular response (rag1 and cd48), inflammation (csf1r, elam, il1ß, and il6), antiviral response (isg15, mx1, mx2, mx3), cell-mediated cytotoxicity (nccrp1), and apoptosis (prf1). The exclusive modulation of the immune response provoked by the vaccination seems to control the progression of the infection in the experimentally challenged gilthead seabream.

Immunogene expression analysis in betanodavirus infected-Senegalese sole using an OpenArray® platform.

The transcriptomic response of Senegalese sole (Solea senegalensis) triggered by two betanodaviruses with different virulence to that fish species has been assessed using an OpenArray® platform based on TaqMan™ quantitative PCR. The transcription of 112 genes per sample has been evaluated at two sampling times in two organs (head kidney and eye/brain-pooled samples). Those genes were involved in several roles or pathways, such as viral recognition, regulation of type I (IFN-1)-dependent immune responses, JAK-STAT cascade, interferon stimulated genes, protein ubiquitination, virus responsive genes, complement system, inflammatory response, other immune system effectors, regulation of T-cell proliferation, and proteolysis and apoptosis. The highly virulent isolate, wSs160.3, a wild type reassortant containing a RGNNV-type RNA1 and a SJNNV-type RNA2 segments, induced the expression of a higher number of genes in both tested organs than the moderately virulent strain, a recombinant harbouring mutations in the protruding domain of the capsid protein. The number of differentially expressed genes was higher 2 days after the infection with the wild type isolate than at 3 days post-inoculation. The wild type isolate also elicited an exacerbated interferon 1 response, which, instead of protecting sole against the infection, increases the disease severity by the induction of apoptosis and inflammation-derived immunopathology, although inflammation seems to be modulated by the complement system. Furthermore, results derived from this study suggest a potential important role for some genes with high expression after infection with the highly virulent virus, such as rtp3, sacs and isg15. On the other hand, the infection with the mutant does not induce immune response, probably due to an altered recognition by the host, which is supported by a different viral recognition pathway, involving myd88 and tbkbp1.

Evaluation of Gilthead Seabream (Sparus aurata) Immune Response after LCDV-Sa DNA Vaccination.

Lymphocystis disease is the main viral pathology reported in gilthead seabream. Its etiological agent is Lymphocystis disease virus 3 (LCDV-Sa), genus Lymphocystivirus, family Iridoviridae. There are no effective treatments or vaccines for LCDV control, thus the main aim of this study was to develop a DNA vaccine, and to evaluate both the protection conferred against LCDV-Sa infection and the immune response in vaccinated fish. The vaccine was constructed by cloning the mcp gene (ORF LCDVSa062R) into pcDNA3.1/NT-GFP-TOPO. Two independent vaccination trials were conducted. In the first one, 5-7 g fish were intramuscularly injected with the vaccine (pcDNA-MCP) or the empty-plasmid, and the distribution and expression of the vaccine was investigated. Furthermore, vaccinated fish were challenged with LCDV-Sa in order to access the protective capacity of the vaccine. In the second trial, 70-100 g fish were vaccinated as specified, and the immune response was evaluated analyzing the expression of 23 immune-related genes and the production of specific antibodies. The results showed that the vaccine triggers an immune response characterized by the overexpression of genes relating to the inflammatory process, but not the innate antiviral immunity relating to type I IFN (interferon), and also induces the production of specific neutralizing antibodies, which could explain the protection against LCDV-Sa in vaccinated fish.

Are the defined substrate-based methods adequate to determine the microbiological quality of natural recreational waters?

Monitoring the microbiological quality of water used for recreational activities is very important to human public health. Although the sanitary quality of recreational marine waters could be evaluated by standard methods, they are time-consuming and need confirmation. For these reasons, faster and more sensitive methods, such as the defined substrate-based technology, have been developed. In the present work, we have compared the standard method of membrane filtration using Tergitol-TTC agar for total coliforms and Escherichia coli, and Slanetz and Bartley agar for enterococci, and the IDEXX defined substrate technology for these faecal pollution indicators to determine the microbiological quality of natural recreational waters. ISO 17994:2004 standard was used to compare these methods. The IDEXX for total coliforms and E. coli, Colilert, showed higher values than those obtained by the standard method. Enterolert test, for the enumeration of enterococci, showed lower values when compared with the standard method. It may be concluded that more studies to evaluate the precision and accuracy of the rapid tests are required in order to apply them for routine monitoring of marine and freshwater recreational bathing areas. The main advantages of these methods are that they are more specific, feasible and simpler than the standard methodology.