Mucosal and systemic immune effects of Bacillus subtilis in rainbow trout (Oncorhynchus mykiss).

Bacillus spp. are well known for their probiotic properties. Hence, the long-term feeding of Bacillus spp. strains to different fish species has been proved to confer beneficial effects regarding growth or pathogen resistance, among others. However, whether these strains could function as mucosal adjuvants, up-regulating immune responses after a single administration, has not yet been investigated in fish. Thus, in the current work, we have performed a series of experiments in rainbow trout (Oncorhynchus mykiss) aimed at establishing the potential of two Bacillus subtilis spore-forming strains, designated as ABP1 and ABP2, as oral adjuvants/immunostimulants. As an initial step, we evaluated their transcriptional effects on the rainbow trout intestinal epithelial cell line RTgutGC, and in gut tissue explants incubated ex vivo with the two strains. Their capacity to adhere to RTgutGC cells was also evaluated by flow cytometry. Although both strains had the capacity to modulate the transcription of several genes related to innate and adaptive immune responses, it was the ABP1 strain that led to stronger transcriptional effects, also exerting a higher binding capacity to intestinal epithelial cells. Consequently, we selected this strain to establish its effects on splenic B cells upon in vitro exposure as well as to determine the transcriptional effects exerted in the spleen, kidney, and gut after a single oral administration of the bacteria. Our results showed that B. subtilis ABP1 had the capacity to modulate the proliferation, IgM secreting capacity and MHC II surface expression of splenic B cells. Finally, we confirmed that this strain also induced the transcription of genes involved in inflammation, antimicrobial genes, and genes involved in T cell responses upon a single oral administration. Our results provide valuable information regarding how B. subtilis modulates the immune response of rainbow trout, pointing to the usefulness of the ABP1 strain to design novel oral vaccination strategies for aquaculture.

Comprehensive transcriptome profiling and functional analysis of the meagre (Argyrosomus regius) immune system.

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection.

Tenacibaculosis induction in the Senegalese sole (Solea senegalensis) and studies of Tenacibaculum maritimum survival against host mucus and plasma.

Tenacibaculum maritimum, the aetiological agent for marine tenacibaculosis, is one of the most significant pathogens that threaten Senegalese sole, Solea senegalensis (Kaup), aquaculture. Because no immersion challenge with T. maritimum has been reported previously for this flatfish species, this study aimed to optimize bacterial yields as well as to establish a challenge model for tenacibaculosis induction. Several approaches were performed to optimize bacterial culture conditions, including treatment with non-ionic surfactants, detergents, cellulase hydrolysis and strong shaking. A prolonged bath challenge was performed for 24 h under two different temperatures, 16 and 23 °C. Moreover, mucus and plasma bactericidal activities against T. maritimum were also assessed. Culturing bacteria with strong shaking and continuous shaking provided suitable culture conditions to obtain higher bacterial yields without aggregation and fluctuation, contrary to most other treatments that showed a huge amount of bacterial aggregates. A prolonged bath method for 24 h, without skin or gill scarification, was considered suitable for disease induction with high mortality rates. Moreover, data regarding mucus and plasma bactericidal activities suggested that there is a lack of host innate immune response against T. maritimum or that this particular pathogen presents evading strategies against Senegalese sole.

Evaluation of the Potential of Marine Algae Extracts as a Source of Functional Ingredients Using Zebrafish as Animal Model for Aquaculture.

Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry.

Oxidative status and intestinal health of gilthead sea bream (Sparus aurata) juveniles fed diets with different ARA/EPA/DHA ratios.

The present work assessed the effects of dietary ratios of essential fatty acids, arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), on liver and intestine oxidative status, intestinal histomorphology and gut microbiota of gilthead sea bream. Four isoproteic and isolipidic plant-based diets were formulated containing a vegetable oil blend as the main lipid source. Diets were supplemented with ARA/EPA/DHA levels (%DM) equivalent to: 2%:0.2%:0.1% (Diet A); 1.0%:0.4%:0.4% (Diet B); 0%:0.6%:0.6% (Diet C); 0%:0.3%:1.5% (Diet D) and tested in triplicate groups for 56 days. Lipid peroxidation was higher in fish fed diets C and D while no differences were reported between diets regarding total, oxidized, and reduced glutathione, and oxidative stress index. Glutathione reductase was higher in fish fed diet A than diets C and D. No histological alterations were observed in the distal intestine. Lower microbiota diversity was observed in intestinal mucosa of fish fed diet C than A, while diets C and D enabled the proliferation of health-promoting bacteria from Bacteroidetes phylum (Asinibacterium sp.) and the absence of pathogenic species like Edwardsiella tarda. Overall, results suggest that a balance between dietary ARA/EPA + DHA promotes gilthead sea bream juveniles' health however higher dietary content of n-3 LC-PUFA might limited the presence of microbial pathogens in intestinal mucosa.

Clinical significance of anticardiolipin antibodies in juvenile idiopathic arthritis.

OBJECTIVE: Anticardiolipin antibodies (aCL) have been demonstrated in a large spectrum of autoimmune diseases. However, its occurrence in childhood, in particular in juvenile idiopathic arthritis (JIA), is not well established. The present study addressed the frequency and clinical significance of aCL in a group of JIA patients. METHODS: aCL (IgG and IgM isotypes), antinuclear antibodies (ANA), and rheumatoid factor (RF) were determined in 86 children with JIA (33 systemic, 31 polyarticular and 22 oligoarticular onset type). Thirty-two juvenile systemic erythematosus lupus patients (JSLE) and 52 healthy children formed the control groups. The disease activity and functional status of the JIA patients were scored to study their possible associations with the presence of aCL. RESULTS: Serum aCL levels above the normal range were detected in 28/86 JIA patients (32.5%), 12/32 JSLE patients (37.5%), and 3/52 healthy children (6%). Positive aCL levels were slightly or moderately elevated (usually below 30 GPL and 20 MPL). The presence of aCL was not associated with the presence of ANA or RF. Associations between aCL and clinical parameters, such as disease onset, duration, activity or severity could not be established. No JIA patient had vascular thrombosis, thrombocytopenia or "livedo reticularis". CONCLUSION: aCL occurred in low titers in JIA children, in a similar frequency to that observed in JSLE. No association with JIA clinical parameters or the clinical features classically linked to the antiphospholipid antibody syndrome were observed.

[Rheumatic diseases in adolescence] / Doenças reumáticas na adolescência.

OBJECTIVE: To present an updated review concerning the most prevalent diseases with musculoskeletal signs and symptoms that make adolescents seek medical care, giving special emphasis to rheumatic diseases. Our aim is to offer physicians and health care providers the possibility of distinct differential diagnoses, thus allowing them to establish a therapeutic approach and, if necessary, refer the patient to a specialist METHODS: Review of literature using Medline database, data obtained at our department, and the authors personal experience. RESULTS: Musculoskeletal pain is characteristic of several diseases and usually urges adolescents to seek medical care. Rheumatic diseases, especially rheumatic fever, account for nearly fifty percent of the cases. In adolescents, it is also important that the aspects regarding the diagnosis and treatment of idiopathic juvenile arthritis, arthritis associated with enthesitis, systemic lupus erythematosus, and vasculitis be considered. Fibromyalgia, reflex sympathetic dystrophy, growing pains, hypermobility syndrome, and psychogenic rheumatism are noninflammatory conditions that frequently mimic rheumatic diseases. CONCLUSIONS: Inflammatory and noninflammatory conditions, and diseases of different etiology (infectious, neoplastic, and orthopedic) are frequently associated with musculoskeletal pain. It is important that health professionals diagnose these diseases as early as possible so that prompt action can be taken and prognosis can be improved.

Antiperinuclear factor and antibodies to the stratum corneum of rat esophagus in juvenile idiopathic arthritis.

We analyzed the frequency and clinical correlates of antiperinuclear factor (APF) and antibodies to the stratum corneum of rat esophagus in 86 children with juvenile idiopathic arthritis (JIA), 32 children with juvenile systemic lupus erythematosus, and 52 healthy children. Forty-two patients with JIA (49%) were positive for APF. No association was observed between APF and current age, sex, JIA subtype, age at disease onset, or disease duration. APF was found in one patient with juvenile systemic lupus erythematosus and in no healthy child. Antibodies to the stratum corneum of rat esophagus were detected in 3 patients with polyarticular JIA. APF may be a valuable tool in the differential diagnosis of JIA.

Significado clínico dos anticorpos antinucleares na artrite reumatóide juvenil / Clinical significance of antinuclear antibodies in juvenile rheumatoid arthritis

Os anticorpos antinucleares (AAN) têm sido demonstrados em freqüência extremamente variável na artrite reumatóide juvenil (ARJ), dependendo do substrato empregado e da seleçäo dos pacientes. Além disso, a associaçÝo entre estes anticorpos e a atividade ou a gravidade da doença näo está bem estabelecida. Objetivo: Determinar a freqüência dos AAN e possível associaçöes com diferentes parâmetros clínicos da ARJ, como duraçäo, atividade e gravidade da doença. Material e métodos: 86 pacientes com ARJ foram estudados quanto à presença de AAN (pesquisados pelo método de imunofluorescência indireta em células HEp-2); 32 pacientes com lúpus eritematoso sistêmico juvenil e 52 crianças saudáveis constituíram os grupos-controles. A atividade da doença foi definida segundo uma escala de dois pontos, enquanto a gravidade foi avaliada pelo grau de capacidade funcional. Resultados: AAN foram encontrados em 36 (42 por cento) pacientes com ARJ, sendo mais prevalentes no tipo de início oligoarticular do que no sistêmico (p<0,05). Uma associaçäo com a duraçäo, atividade ou a gravidade da doença näo foi evidenciada (p>0,05), embora uma tendência tenha sido observada em pacientes do tipo de início poliarticular, em atividade de doença (P=0,054). O padräo pontilhado fino foi o mais freqëntemente observado (64 por cento), conquanto outros padröes, como o centrossômico e o do corpo intermediário, também tivessem sido notados. Conclusäo: Anticorpos antinucleares foram encontrados em menor freqüência que aquela descrita na maioria dos trabalhos da literatura para a ARJ, nÝo apresentando associaçäo com o sexo, duraçäo, atividade ou gravidade de doença. Embora o padräo de fluorescência mais freqüente tenha sido o pontilhado, a presença de padröes inusitados de fluorescência nuclear sugere que AAN na ARJ possam apresentar especificidades ainda näo definidas, que poderäo futuramente ser melhor caracterizadas