Polyvalent Bacterial Lysate with Potential Use to Treatment and Control of Recurrent Urinary Tract Infections.

Overuse of antimicrobials has greatly contributed to the increase in the emergence of multidrug-resistant bacteria, a situation that hinders the control and treatment of infectious diseases. This is the case with urinary tract infections (UTIs), which represent a substantial percentage of worldwide public health problems, thus the need to look for alternatives for their control and treatment. Previous studies have shown the usefulness of autologous bacterial lysates as an alternative for the treatment and control of UTIs. However, a limitation is the high cost of producing individual immunogens. At the same time, an important aspect of vaccines is their immunogenic amplitude, which is the reason why they must be constituted of diverse antigenic components. In the case of UTIs, the etiology of the disease is associated with different bacteria, and even Escherichia coli, the main causal agent of the disease, is made up of several antigenic variants. In this work, we present results on the study of a bacterial lysate composed of 10 serotypes of Escherichia coli and by Klebsiella pneumoniae, Klebsiella aerogenes, Enterococcus faecalis, Proteus mirabilis, Citrobacter freundii, and Staphylococcus haemolyticus. The safety of the compound was tested on cells in culture and in an animal model, and its immunogenic capacity by analysing in vitro human and murine macrophages (cell line J774 A1). The results show that the polyvalent lysate did not cause damage to the cells in culture or alterations in the animal model used. The immunostimulatory activity assay showed that it activates the secretion of TNF-α and IL-6 in human macrophages and TNF-α in murine cells. The obtained results suggest that the polyvalent lysate evaluated can be an alternative for the treatment and control of chronic urinary tract infections, which will reduce the use of antimicrobials.

Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

Chitosan Microparticles Enhance the Intestinal Release and Immune Response of an Immune Stimulant Peptide in Oncorhynchus mykiss.

The aquaculture industry is constantly increasing its fish production to provide enough products to maintain fish consumption worldwide. However, the increased production generates susceptibility to infectious diseases that cause losses of millions of dollars to the industry. Conventional treatments are based on antibiotics and antivirals to reduce the incidence of pathogens, but they have disadvantages, such as antibiotic resistance generation, antibiotic residues in fish, and environmental damage. Instead, functional foods with active compounds, especially antimicrobial peptides that allow the generation of prophylaxis against infections, provide an interesting alternative, but protection against gastric degradation is challenging. In this study, we evaluated a new immunomodulatory recombinant peptide, CATH-FLA, which is encapsulated in chitosan microparticles to avoid gastric degradation. The microparticles were prepared using a spray drying method. The peptide release from the microparticles was evaluated at gastric and intestinal pH, both in vitro and in vivo. Finally, the biological activity of the formulation was evaluated by measuring the expression of il-1ß, il-8, ifn-γ, Ifn-α, and mx1 in the head kidney and intestinal tissues of rainbow trout (Oncorhynchus mykiss). The results showed that the chitosan microparticles protect the CATH-FLA recombinant peptide from gastric degradation, allowing its release in the intestinal portion of rainbow trout. The microparticle-protected CATH-FLA recombinant peptide increased the expression of il-1ß, il-8, ifn-γ, ifn-α, and mx1 in the head kidney and intestine and improved the antiprotease activity in rainbow trout. These results suggest that the chitosan microparticle/CATH-FLA recombinant peptide could be a potential prophylactic alternative to conventional antibiotics for the treatment of infectious diseases in aquaculture.

Nucleotides, β-glucans, ascorbic acid, α-tocoferol, and different concentrations of a vitamin-mineral premix promote growth of Nile tilapia juveniles / [Nucleotídeos, β-glucanos, ácido ascórbico, α-tocoferol e diferentes concentrações de uma pré-mistura de vitaminas e minerais promovem o crescimento de juvenis de tilápia do Nilo]

ABSTRACT Feed additives, such as β-glucans, nucleotides, ascorbic acid (vitamin C), and α-tocopherol (vitamin E) can improve fish immunity and contribute to enhanced zootechnical performance of the fish. This study aimed to evaluate the effect of different concentrations of vitamin- mineral premix with or without the inclusion of an immunostimulant boost (β-glucans, nucleotides and vitamins C and E) on the zootechnical performance, hemato-immunological parameters, histological changes, and survival of Nile tilapia juveniles. To this end, isocaloric and isoproteic diets were prepared with three different concentrations of vitamin-mineral premix (1.0, 1.5, and 2.0 kg ton-1); additionally, the effects of 4% immunostimulant boost in the diet were examined considering six diets (without the immunostimulant boost: 0.1%, 0.15%, and 0.2%, and with the immunostimulant boost: 0.1% I, 0.15% I, and 0.2% I). We used 24 experimental units, each including 15 fish. Nile tilapia juveniles (1.88 g±0.25) were fed for 50 d with supplemented diets. Further, zootechnical indexes; histological changes in the liver, spleen, and intestine; intestinal morphology; and hemato-immunological parameters were evaluated. The fish that received supplementation showed higher zootechnical values, compared to those that did not receive the supplementation of immunostimulant reinforcement. Weight gain (30.45g±3.06), daily weight gain (0.60g±0.06), final weight (32.41g±3.15) and feed conversion (0.95±0.06) were higher in tilapia fed with an inclusion concentration of 0.2%. Hematological parameters were not affected by either the pre-mix concentrations or the booster of the immunostimulant. There was an increase in the number of intestinal folds, length of the fold, total area and number of goblet cells in the group supplemented with immunostimulant reinforcement. Supplementation with the immunostimulant promoted growth and improved intestinal morphology and immunological parameters of Nile tilapia juveniles.

RESUMO Os aditivos alimentares, tais como β-glucanos, nucleotídeos, ácido ascórbico (vitamina C) e α-tocoferol (vitamina E), podem melhorar a imunidade dos peixes e contribuir para um melhor desempenho zootécnico. Este estudo teve como objetivo avaliar o efeito de diferentes concentrações de premix vitamínico mineral com ou sem a inclusão de um imunoestimulante (β-glucanos, nucleotídeos e vitaminas C e E) no desempenho zootécnico, nos parâmetros hematoimunológicos, nas alterações histológicas e na sobrevivência dos juvenis de tilápia-do-Nilo. Para esse fim, foram preparadas dietas isocalóricas e isoproteicas com três concentrações diferentes de premix vitamínico e mineral (1,0, 1,5, e 2,0kg ton-1); além disso, foram examinados os efeitos da inclusão de 4% de imunoestimulante na dieta, considerando-se seis dietas (sem o imunoestimulante: 0,1%, 0,15% e 0,2%, e com a inclusão de imunoestimulante: 0,1% I, 0,15% I e 0,2% I). Foram utilizadas 24 unidades experimentais, cada uma com 15 peixes. Os juvenis de tilápia-do-Nilo (1,88g±0,25) foram alimentados, durante 50 dias, com as dietas suplementadas. Além disso, foram avaliados índices zootécnicos; alterações histológicas no fígado, baço e intestino; morfologia intestinal; e parâmetros hematoimunológicos. Os peixes que receberam suplementação apresentaram valores zootécnicos mais elevados, em comparação aos que não receberam a suplementação de reforço imunoestimulante. O ganho de peso (30,45g±3,06), o ganho de peso diário (0,60g ± 0,06), o peso final (32,41g±3,15) e a conversão alimentar (0,95±0,06) foram maiores nas tilápias alimentadas com uma concentração de inclusão de 0,2%. Os parâmetros hematológicos não foram afetados nem pelas concentrações de premix nem pelo reforço do imunoestimulante. Foi observado o aumento no número de pregas intestinais, no comprimento da prega, na área total e no número de células caliciformes no grupo suplementado com o boost imunoestimulante. A suplementação com o imunoestimulante promoveu o crescimento e melhorou a morfologia intestinal e os parâmetros imunológicos de juvenis de tilápia-do-Nilo.

Mansoa alliacea extract improves the growth performance and innate immune response of Arapaima gigas challenged with Aeromonas hydrophila and handling stress / Extrato de Mansoa alliacea melhora o desempenho de crescimento e resposta imunológica de Arapaima gigas desafiado com Aeromonas hydrophila e estresse de manejo

This study investigated the effects of dietary supplementation with Mansoa alliacea hydroalcoholic extracts on growth, blood and immune parameters of Arapaima gigas. Fish were fed for 30 days with diets enriched with 0, 4, 8, and 12 g kg-1 of M. alliacea hydroalcoholic extract and subjected to infection with Aeromonas hydrophila and handling stress. Fish fed with 8 g kg-1 of extract showed significant increase in final weight, specific growth rate and feed efficiency when compared to the other groups. Glucose, triglycerides, total proteins, and globulins increased significantly in fish fed with 8 g kg-1 of extract, whereas albumin decreased. The number of thrombocytes increased significantly with the dietary supplementation of 8 and 12 g kg-1 of extract. After the challenge with A. hydrophila and handling stress, fish fed with 8 g kg-1 of extract had significantly higher levels of glucose, globulins, and albumins, and fish fed with 8 and 12 g kg-1 of extract showed an increment of respiratory burst. Triglyceride levels dropped significantly in fish fed with 4, 8, and 12 g kg-1 of extract, whereas the number of neutrophils increased, and total thrombocytes, leukocytes and lymphocytes were higher in fish fed with 12 g kg-1 of extract. Dietary supplementation with M. alliacea extract at 8 g kg-1 was efficient in improving the growth and innate immunity of A. gigas, being potentially useful in fish farming to control the development of A. hydrophila infections.(AU)

Investigou-se os efeitos da suplementação com extrato hidroalcólico de Mansoa alliacea sobre o crescimento e parâmetros sanguíneos e imunológicos de Arapaima gigas. Os peixes foram alimentados por 30 dias com dietas enriquecidas com 0, 4, 8 e 12 g kg-1 de extrato hidroalcoólico de M. alliacea e submetidos à infecção por Aeromonas hydrophila e estresse de manejo. Os peixes alimentados com 8 g kg-1 de extrato apresentaram aumento significativo no peso final, taxa de crescimento específico e eficiência alimentar quando comparados aos demais grupos. Glicose, triglicerídeos, proteínas totais e globulinas aumentaram significativamente nos peixes alimentados com 8 g kg-1 de extrato, enquanto a albumina diminuiu. O número de trombócitos aumentou significativamente com a suplementação dietética de 8 e 12 g kg-1 de extrato. Após infecção com A. hydrophila e estresse de manejo, os peixes alimentados com 8 g kg-1 de extrato apresentaram níveis significativamente mais altos de glicose, globulinas e albuminas, e os peixes alimentados com 8 e 12 g kg-1 de extrato apresentaram incremento de explosão respiratória. Os níveis de triglicerídeos decresceram nos peixes alimentados com 4, 8 e 12 g kg-1 de extrato, enquanto o número de neutrófilos aumentou, e o número total de trombócitos, leucócitos e linfócitos foi maior nos peixes alimentados com 12 g kg-1 de extrato. A suplementação com a 8 g kg-1 de extrato de M. alliacea foi eficiente em melhorar o crescimento e a imunidade inata de A. gigas, sendo potencialmente útil na piscicultura para controlar o desenvolvimento de infecções por A. hydrophila.(AU)

Thymomodulin enhances vaccination responses against Streptococcus agalactiae in Nile tilapia / Timomodulina melhora as respostas vacinais contra Streptococcus agalactiae em tilápia-do-Nilo

Streptococcosis caused by Streptococcus agalactiae is a major economic problem for Nile tilapia Oreochromis niloticus production worldwide. Inactivated vaccines are considered an efficient method for controlling streptococcosis. However, during the vaccination process, stress during fish handling could affect the immune response. Immunomodulators, such as thymomodulin, act on the immune system and can improve vaccination responses. Thus, the aim of this study was to assess the immunomodulatory effect of thymomodulin in O. niloticus vaccinated against S. agalactiae. For this, fish were distributed in a factorial design (2x2x6), corresponding to two levels of thymomodulin supplementation (0.0 and 0.3%), two treatments (sterile phosphate buffered saline and inactivated S. agalactiae vaccine). Initially, fish received thymomodulin supplementation for 30 days. Next, fish were vaccinated by injection intraperitoneal (i.p.) of heat inactivated S. agalactiae vaccine. 15 days after vaccination, all fish were challenged i.p. with S. agalactiae and blood samples were collected at 7, 14, 21 days post infection (DPI).Results revealed that fish supplemented with thymomodulin vaccinated or not developed better protection against S. agalactiae than vaccinated fish without thymomodulin. Furthermore, fish vaccinated and supplemented with thymomodulin presented high levels of antibodies after 14 and21 DPI, suggesting that thymomodulin can promote the immune response in Nile tilapia.

A estreptococose causada pelo Streptococcus agalactiae é um grande problema econômico para a produção de tilápia-do-nilo Oreochromis niloticus em todo o mundo. As vacinas inativadas são consideradas um método eficiente para o controle da estreptococose. No entanto, durante o processo de vacinação o estresse durante o manejo dos peixes pode afetar sua resposta imune. Imunomoduladores como a timomodulina, atuam no sistema imunológico e podem melhorar as respostas vacinais. Assim, o objetivo deste estudo foi avaliar o efeito imunomodulador da timomodulina em O. niloticus vacinado contra S. agalactiae. Para isso, os peixes foram distribuídos em esquema fatorial (2x2x6), correspondendo a dois níveis de suplementação de timodulina (0,0 e 0,3%), dois tratamentos (solução salina tamponada com fosfato estéril e vacina S. agalactiae inativada). Inicialmente, os peixes receberam suplementação de timomodulina por 30 dias. Em seguida, os peixes foram vacinados por via intraperitoneal (i.p.) da vacina de S. agalactiae inativada pelo calor. 15 dias após a vacinação, todos os peixes foram desafiados i.p. com S. agalactiae e amostras de sangue foram coletadas aos 7, 14, 21 dias após a infecção (DPI). Os resultados revelaram que os peixes suplementados com timomodulina vacinados ou não desenvolveram melhor proteção contra S. agalactiae do que os peixes vacinados sem timomodulina. Além disso, os peixes vacinados e suplementados com timomodulina apresentaram altos níveis de anticorpos após 14 e 21 DPI, sugerindo que a timomodulina pode promover a resposta imune em tilápia-do-nilo.

ß-glucan mimics tissue damage signaling and generates a trade-off between head kidney and spleen to activate acquired immunity in vaccinated tilapia (Oreochromis niloticus).

The association of vaccines with immunostimulants such as ß-glucan, promote the production of cytokines, competent immune cells and antibodies. However, differences between ß-glucan types and trials make it difficult to understand ß-glucan's mechanism of action. In this study, three trials were carried out with control and fish fed ß-glucan, the first trial occurred at 15 days; the second trial occurred at 30 days when we associated ß-glucan and vaccine; and the third trial occurred at 15 days post-challenge with Streptococcus agalactiae in tilapia (O. niloticus) in order to investigate immune-related gene expression in the head kidney and spleen using real-time qPCR. We found increases in HSP70, IL-6, IL-1ß, TNF-α, IL-10, Lys and C3 predominantly in the head kidney, except for IgM expression, which prevailed in the spleen, under vaccinated + ß-glucan action. This demonstrates the trade-off presented by the head kidney and spleen after immunostimulation in order to produce acquired immunity, as well as an increase in HSP70 expression in vaccinated + ß-glucan fish. The results suggest that ß-glucan stimulates the immune response through damage-associated molecular patterns (DAMPs) recognition. Therefore, these dynamics of the immune response promote a more robust defense against disease.

Dietary ß-glucan (MacroGard®) improves innate immune responses and disease resistance in Nile tilapia regardless of the administration period.

The effects of dietary ß-glucan on innate immune responses have been shown in a number of different vertebrate species. However, there is conflicting information about the period of administration (shorter vs. longer), and it is also unclear to what extent ß-glucan's effects can be observed post-treatment in fish. Thus, we fed Nile tilapia for 0 (control group; 45 days of control diet), 15 (30 days of control followed by 15 days of ß-glucan), 30 (15 days of control followed by 30 days of ß-glucan) or 45 days with a diet containing 0.1% of ß-glucan (MacroGard®). We evaluated the growth performance at the end of the ß-glucan feeding trial and the innate immune function immediately after the feeding trial and 7 and 14 days post-feeding trial. In addition, at day 10 post-feeding trial, we assessed the tilapia's resistance against a bacterial infection. No significant differences were observed in growth performance between the groups; however, fish fed with ß-glucan for 30 and 45 days had higher (approx. 8%) relative weight gain compared to the control. Regardless of the administration period, fish fed with ß-glucan had higher innate immune responses immediately after the feeding trial such as lysozyme activity in plasma, liver and intestine and respiratory burst compared to the control, and in general these differences were gradually reduced over the withdrawal period (up to 14 days). No differences were observed in the plasma hemolytic activity of the complement or myeloperoxidase activity in plasma or intestine. Moreover, fish from the control group had early mortalities (2 vs. 4-5 days post-infection, respectively) and a lower survival rate (60 vs. 80%, respectively) compared to fish fed with ß-glucan for 15 or 30 days, and, interestingly, fish fed for 45 days with ß-glucan had no mortality. This study indicates that regardless of the administration period (i.e., 15 up to 45 days), the ß-glucan improved the innate immune responses and the tilapia's resistance to disease, and this protection could be observed up to 10 days post-feeding trial, adding in vivo evidence that ß-glucan may contribute to a trained innate immunity. Additionally, we showed that a longer period of administration did not cause immunosuppression as previously hypothesized but promoted further growth and immune performance. These findings are relevant to the aquaculture industry and demonstrate that a longer ß-glucan feeding protocol may be considered to achieve better results.

The Influence of ß-1,3-1,6-Glucans on Rabies Vaccination Titers in Cats.

ß-glucans have been shown to stimulate the immune system in several animal species. The aim of this study was to evaluate the immune stimulation capacity of a fully formulated diet with ß-1,3-1,6-glucans in cats, by assessing the rabies antibody titer after vaccination. Thirty-five healthy cats were recruited. The cats were placed into two groups and fed a standard diet in accordance with body weight. One group had the ß-glucans incorporated into the diet; the other group served as the control group. After two weeks of dietary adjustment; the rabies vaccine (Imrab® 3 TF; Merial) was administered on days 0 and 21. Blood samples were taken on days 0, 21, and 42. Titers were determined with the rapid fluorescent foci inhibition test (RFFIT). Titers at days 21 and 42 were compared between the two groups in a linear mixed effects model. This study showed that the animals receiving the non-supplemented feed had higher post-vaccination rabies antibody titers. This indicates that, in contrast to other animal species, the ß-glucan supplemented diet did not have the expected positive effect on the rabies antibody titers in cats.

Probiotic and nutritional effects of Debaryomyces hansenii on animals.

Debaryomyces hansenii comes of age as a new potential probiotic for terrestrial and aquatic animals. Probiotic properties, including inmunostimulatory effects, gut microbiota modulation, enhanced cell proliferation and differentiation, and digestive function improvements have been related to the oral delivery of D. hansenii. Its functional compounds, such as cell wall components and polyamines, have been identified and implicated in its immunomodulatory activity. In addition, in vitro studies using immune cells have shown standpoints on the possible recognition, regulation, and effector immune mechanisms stimulated by this yeast. This review describes historic, cutting-edge research findings, implications, and perspectives on the use of D. hansenii as a promising probiotic for animals. KEY POINTS: • Debaryomyces hansenii has probiotic effects in terrestrial and aquatic animals. • Nutritional effects could be associated to probiotic D. hansenii strains. • ß-D-Glucan and polyamines from D. hansenii are associated to probiotic properties. • Adoption by the industry is expected in the next years.

Immunostimulatory and antioxidant effects of supplemental feeding with macroalga Sargassum spp. on goat kids.

Algae are a rich source of bioactive compounds and health properties that have been narrowly explored in goat production systems. The aim of this study was to determine the effect of feeding diets supplemented with Sargassum spp. on antioxidant status and immune parameters in goat kids. The diets were as follows: control (basal diet without alga), Sargassum spp. 2.5% (Ss2.5), and Sargassum spp. 5% (S5) fed over a 70-day period. A total of 11 body tissues, intestinal mucus, and blood serum were sampled at necropsy. Protein content, superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO), lysozyme, and anti-protease activities were determined, as well as immunoglobulin A (IgA) and immunoglobulin G (IgG). The results indicated that Sargassum spp. supplementation increased protein content in six tissues. Antioxidant activities (SOD and CAT) and immune-related (lysozyme, MPO, and anti-protease) activities were statistically higher (P < 0.05) in Sargassum spp. groups compared with control in several tissues, intestinal mucus, and serum. Imunoglobulin A levels in intestinal mucus were higher (P < 0.05) in Sargassum spp.-supplemented groups than the control group. In conclusion, diet supplementation of Sargassum spp. improves the antioxidant status and enhances the immune parameters in goats. Sargassum spp. dietary supplementation is proposed as strategy to strengthen antioxidant status and stimulate the immune system, which helps in the control of opportunistic pathogens in goats.

ß-Glucan enhances respiratory activity of leukocytes suppressed by stress and modulates blood glucose levels in pacu (Piaractus mesopotamicus).

We evaluated the immune response of pacu fed with a ß-glucan diet (0.5%) for 10 days. After the feeding period, fish were subjected to handling and 3 h after, inoculated with Aeromonas hydrophila. Fish were sampled before handling (baseline condition), 3, 6, and 24 h and 1 week after inoculation. A higher level of blood glucose was found in fish treated with ß-glucan in baseline conditions. Handling and bacterial inoculation increased the circulating levels of cortisol and glucose and promoted the acute inflammatory response (lymphopenia and neutrophilia). ß-Glucan prevented the decrease in the respiratory activity of leukocytes observed in the control group at 3 h sampling. ß-Glucan did not affect the complement and lysozyme, which were activated 24 h after the bacterial challenge in control fish. A reduction in the number of leukocytes was found in fish treated with ß-glucan 1 week after the challenge. We suggest two plausible hypotheses for this event: (1) it could be attributed to a depletion of the immune responses or (2) it could be due to a mobilization of the leukocytes to the spleen for antigen presenting/processing. In general, ß-glucan avoided the reduction of the activity of leukocytes after stress and the bacterial challenge and increased the baseline glucose levels. Our findings confirm the immunomodulatory action of glucan and add evidence showing that glucan can have a role in stress response.

Cost-effectiveness analysis of OM-85 vs placebo in the prevention of acute respiratory tract infections (ARTIs) in children that attend day-care centers.

BACKGROUND: Children that attend day-care centers frequently contract acute respiratory tract infections (ARTIs). ARTIs represent a burden for both children and parents. Systematic reviews on the use of immunostimulants for the prevention of juvenile recurrent ARTIs have provided moderate evidence of efficacy and safety. The aim of the study was to establish whether the immunostimulant, OM-85, was cost-effective in preventing ARTIs in children 2-6 years old that attended day-care centers or preschools in Mexico. We performed a systematic review to evaluate the efficacy of OM-85. For costs, we assumed an institutional perspective, which included the costs of care and supplies over a study period of six months, during the autumn-winter seasons. We created decision trees and constructed a model to identify pharmacoeconomic parameters. We generated 1000 estimations with the bootstrap method to calculate descriptive statistics of pharmacoeconomic parameters. We evaluated cost-effectiveness compared to treatment without immunostimulants. RESULTS: The mean (SD) incidences of ARTIs were 5.59 ± 0.29 without immunostimulants and 2.97 ± 0.32 with OM-85, during the study period. The mean (25th, 75th percentile) direct costs of ARTIs were 57.04 (37.11, 76.39) US$ (US dollars) without immunostimulants and 48.53 (37.35, 58.93) US$ with OM-85, with a mean increment of - 8.51(- 17.08, 0.75) US$, and a mean cost-effectiveness of - 17.94 (- 36.48, 1.66) US$. The direct costs plus the cost of one parent missing work to care for the child with ARTI were 125.76 (102.83, 150.16) US$, without immunostimulant and 85.21 (72.15, 98.81) US$, with OM-85. The increment was - 40.55 (- 68.29, - 13.95) US$, and the cost-effectiveness was - 86.89 (- 142.37, - 29.34) US$.Part of the cost reduction was ascribed to the reduced use of medications, particularly antibiotics. CONCLUSIONS: Our results were consistent with previous clinical studies conducted in closed institutions in Mexico. OM-85 reduced the number of ARTIs and the frequency of antibiotics use. We concluded that OM-85 was cost-effective for preventing ARTIs in children that attended day-care centers, particularly when parental absenteeism was covered by the institutions.

Fishmeal substitution for Arthrospira platensis in juvenile mullet ( Mugil liza) and its effects on growth and non-specific immune parameters / Substitución de harina de pescado por Arthrospira platensis en juveniles de lisa (Mugil liza) y sus efectos en el crecimiento y parámetros del sistema inmune no-específico / Substituição da farinha de peixe por Arthrospira platensis em juvenis de tainha (Mugil liza) e seus efeitos no crescimento e nos parâmetros do sistema imune não-especifico

Abstract Background: Cyanobacterium Athrospira platensis (Spirulina) is a potential fishmeal (FM) substitute in fish diets because of its high protein content, antioxidant and immunostimulant properties. Objective: To evaluate the effects of total and partial substitution of FM with A. platensis (0, 30, 50, 70 and 100% substitution) in juvenile mullet (Mugil liza). Methods: Juvenile mullets (n=210) were maintained in a recirculation system under optimal water parameters for the species. Mullets were fed five experimental diets for 80 days. Each diet was tested in triplicate tanks. At the end of the experimental period growth parameters were measured and samples of blood, liver and spleen were taken to evaluate the immune system. Results: Full replacement (100%) of FM resulted in growth deficits and low survival. The FM replacement induced changes in the proportion of macrophages and lymphocytes. Up to 50% FM replacement increased the expression of CD3 receptors in spleen T lymphocytes (T-Cells), whereas >50% FM replacement decreased the expression of CD3 receptors. We also found that partial FM substitution diminished the apoptotic process. Conclusions: Up to 50% FM substitution with A. platensis can improve performance of non-specific immune system of mullets.

Resumen Antecedentes: La cianobacteria Arthrospira platensis (Spirulina) puede usarse como substituto potencial de la harina de pescado (HP) por su alto contenido de proteína, sus antioxidantes y sus propiedades inmunoestimulantes. Objetivo: Analizar el efecto de la substitución parcial y total de HP por A. platensis (0, 30, 50, 70 y 100% de substitución) en juveniles de lisa (Mugil liza). Métodos: Juveniles de lisa (n=210) se mantuvieron en un sistema de recirculación con parámetros de calidad de agua en niveles óptimos para la especie. Las lisas se alimentaron con las dietas experimentales durante 80 días. Cada dieta fue evaluada en triplicado. Al final del periodo experimental se midieron los parámetros de crecimiento y se colectaron muestras de sangre, hígado y bazo para evaluación del sistema inmune. Resultados: La substitución total (100%) resultó en deficiente crecimiento y baja sobrevivencia. El remplazo de HP produjo cambios en las proporciones de macrófagos y linfocitos. La substitución de hasta un 50% HP aumentó la expresión de receptores CD3 en linfocitos T del bazo. Por otro lado, la substitución mayor a 50% HP disminuyó la expresión de receptores CD3. La substitución parcial de HP disminuyó el proceso de apoptosis. Conclusiones: Proponemos una substitución de HP del 50% por A. platensis, lo cual mejora el desempeño del sistema inmune no especifico de las lisas.

Resumo Antecedentes: A cianobactéria Athrospira platensis (Spirulina) é um potencial substituto da farinha de peixe (FP) pelo seu alto conteúdo de proteína, antioxidantes e características imune estimulantes. Objetivo: Foram avaliados os efeitos da substituição parcial e total da FP por A. platensis (0, 30, 50, 70 e 100% substituição) em juvenis de tainha (Mugil liza). Métodos: Juvenis de tainha (n=210) foram mantidos em um sistema de recirculação com os parâmetros da água sendo mantidos em níveis ótimos para a espécie. As tainhas foram alimentadas com as dietas experimentais por 80 dias, cada dieta foi testada em triplicata, ao final do período experimental foram avaliados os parâmetros de crescimento e amostras de sangue, fígado e baço foram coletadas para a avaliação do sistema imune. Resultados: A substituição total de FP resultou em redução do crescimento e baixa sobrevivência. A avaliação do sistema imune demostrou que a substituição da FP produz alterações nas proporções de macrófagos e linfócitos. Provou-se que até 50% de substituição da FP incrementa a expressão de receptores CD3. Além disso, a substituição parcial da FP diminui o processo de apoptose. Conclusão: Baseado em nossos descobrimentos, se propõe a substituição de até 50% da FP por A. platensis que melhorará o desempenho do sistema imunológico não específico das tainhas.

Growth, physiological and immune responses of Arapaima gigas (Arapaimidae) to Aeromonas hydrophila challenge and handling stress following feeding with immunostimulant supplemented diets.

The current study tested the efficacy of a dietary immunostimulant additive (Aquate Fish™®) on the growth performance, and on the physiological and immune responses of Arapaima gigas. Two trials were carried out: a feeding trial for 30 days with the experimental diets and a challenge trial for 7 days, in which fish were bacterial challenge (Aeromonas hydrophila) following by 60 s handling stress. During the feeding trial, fingerlings were fed diets supplemented with 0 (control), 6, 9 and 12 g Aquate Fish™®/kg diet. Dietary supplementation did not influence feed intake, feed conversion and condition factor, but increased the final biomass, number of erythrocytes, thrombocytes, leukocytes, lymphocytes, monocytes, hemoglobin, glucose, globulins and plasma triglycerides in fish fed at a concentration of 12 g/kg diet. After bacterial infection, mortality occurred only in fish fed control treatment, whereas respiratory burst of leukocytes, number of leukocytes and lymphocytes increased in fish that received 12 g of dietary supplementation. The results indicated that dietary supplementation with 12 g of Aquate Fish™® improved biomass and immunity performance of A. gigas fingerlings, without negatively affecting blood biochemical parameters.

Levamisole modulates the cell-mediated immunity of matrinxã, Brycon amazonicus / Levamisole modula a imunidade mediada por células de Matrinxã, Brycon amazonicus

The use of immunomodulators is an alternative to improve the immune system of fish and avoid the excessive use of antibiotics. Levamisole is a synthetic anthelmintic which promotes a potent immunostimulation on innate and acquired variables of fish. In order to access the levamisole effects on matrinxã, Brycon amazonicus, fish were fed levamisole at 500 mg kg-1 of diet for seven days. After this period fish were submitted to blood collection. Immunological variables, such as leukocytes respiratory burst, total serum protein, albumin, globulin, albumin:globulin index and hematological variables, such as hematocrit, hemoglobin, red and white blood cell (WBC) and Wintrobe indexes were evaluated. The levamisole administration increased the WBC counts indicating that the immunostimulant modulates the cell-mediated immunity. The increased WBC indicate that the fish fed levamisole were in a better body condition, and probable more resistant to disease due to potential for phagocytosis activation, degranulation of neutrophilic granules, and consequently production of antibodies and immunological memory. Besides, these findings are important for news projects regarding the production of vaccines and deeper understanding of fish cell-mediated immunity.

A utilização de imunoestimulante é uma alternativa para melhorar o sistema imune de peixes e evitar o uso excessivo de antibióticos. O levamisol é um anti-helmíntico sintético que promove uma potente estimulação imunológica em peixes, aumentando variáveis inatas e adquiridas de defesa. A fim de avaliar os efeitos do levamisol sobre matrinxã, Brycon amazonicus, os peixes foram alimentados com 500 mg kg-1 de levamisol na dieta durante sete dias. Após este período os peixes foram submetidos à colheita de sangue. Variáveis imunes, tais como a atividade respiratória de leucócitos, proteína total sérica, albumina, globulina, índice albumina:globulina e os parâmetros hematológicos, tais como hematócrito, hemograma completo, hemoglobina, número de eritrócitos e o volume corpuscular médio dos eritrócitos foram avaliados. A utilização do levamisol aumentou a contagem dos leucócitos do sangue indicando que o imunoestimulante modulou a imunidade mediada por células, entretanto, nenhuma alteração foi observada nas demais variáveis. O aumento dos leucócitos do sangue indica que os peixes alimentados com levamisol estão em uma condição corporal melhor, e em um possível surto terão mais resistência às doenças e potencial para ativação da fagocitose e degranulação de neutrófilos. Além disso, os resultados são importantes para novos projetos sobre a produção de vacinas, e sobre a compreensão mais profunda da imunidade mediada por células em peixes.

Levamisole modulates the cell-mediated immunity of matrinxã, Brycon amazonicus / Levamisole modula a imunidade mediada por células de Matrinxã, Brycon amazonicus

The use of immunomodulators is an alternative to improve the immune system of fish and avoid the excessive use of antibiotics. Levamisole is a synthetic anthelmintic which promotes a potent immunostimulation on innate and acquired variables of fish. In order to access the levamisole effects on matrinxã, Brycon amazonicus, fish were fed levamisole at 500 mg kg-1 of diet for seven days. After this period fish were submitted to blood collection. Immunological variables, such as leukocytes respiratory burst, total serum protein, albumin, globulin, albumin:globulin index and hematological variables, such as hematocrit, hemoglobin, red and white blood cell (WBC) and Wintrobe indexes were evaluated. The levamisole administration increased the WBC counts indicating that the immunostimulant modulates the cell-mediated immunity. The increased WBC indicate that the fish fed levamisole were in a better body condition, and probable more resistant to disease due to potential for phagocytosis activation, degranulation of neutrophilic granules, and consequently production of antibodies and immunological memory. Besides, these findings are important for news projects regarding the production of vaccines and deeper understanding of fish cell-mediated immunity.(AU)

A utilização de imunoestimulante é uma alternativa para melhorar o sistema imune de peixes e evitar o uso excessivo de antibióticos. O levamisol é um anti-helmíntico sintético que promove uma potente estimulação imunológica em peixes, aumentando variáveis inatas e adquiridas de defesa. A fim de avaliar os efeitos do levamisol sobre matrinxã, Brycon amazonicus, os peixes foram alimentados com 500 mg kg-1 de levamisol na dieta durante sete dias. Após este período os peixes foram submetidos à colheita de sangue. Variáveis imunes, tais como a atividade respiratória de leucócitos, proteína total sérica, albumina, globulina, índice albumina:globulina e os parâmetros hematológicos, tais como hematócrito, hemograma completo, hemoglobina, número de eritrócitos e o volume corpuscular médio dos eritrócitos foram avaliados. A utilização do levamisol aumentou a contagem dos leucócitos do sangue indicando que o imunoestimulante modulou a imunidade mediada por células, entretanto, nenhuma alteração foi observada nas demais variáveis. O aumento dos leucócitos do sangue indica que os peixes alimentados com levamisol estão em uma condição corporal melhor, e em um possível surto terão mais resistência às doenças e potencial para ativação da fagocitose e degranulação de neutrófilos. Além disso, os resultados são importantes para novos projetos sobre a produção de vacinas, e sobre a compreensão mais profunda da imunidade mediada por células em peixes.(AU)

Immunomodulation of Juvenile Pacu, Piaractus mesopotamicus, by Different ß(1-3)(1-6)-D glucan Products

Abstract Stress in intensive fish farming hamper immune function of fish and cause losses by disease outbreaks, a situation that can be minimized, but cannot be completely circumvented, by the use of immunomodulators. Addition of immunomodulators to aquafeeds has thus become a common practice. β-glucan (BG) is one of most studied and effective immunomodulators, aquaculture purposes included. Extracted from cell walls of bacteria, fungi and selected cereals, BG activity depends on the source and extraction methods. This study evaluated effects of two BG products (BG1 and BG2), extracted from Saccharomyces cerevisiae under varying extraction methods and with different immune activity, on the feeding of pacu Piaractus mesopotamicus juveniles. BG1 provided higher leukocytes respiratory activity when fed at 0.5% inclusion for 10 days and 0.1% inclusion for 15 days. Both products seems to cause negative effect on lysozyme concentration and monocytes profile when fed to pacu for 15 days at 0.5% inclusion. Although the results for BG2 did not differ from control (diet devoid of BG), the proximity with the BG1 behavior is a indicative that a commercial product with smaller BG concentration can be effective when more refined technology is used in extraction process.

Sterigmatomyces halophilus ß-glucan improves the immune response and bacterial resistance in Pacific red snapper (Lutjanus peru) peripheral blood leucocytes: In vitro study.

ß-Glucans are naturally occurring polysaccharides that are produced by bacteria, fungi and yeast. They are considered immunostimulants in fish acting on non-specific defense mechanism. Yeast-derived glucans from cell wall (Sterigmatomyces halophilus, ß-Gluc/Sh) have been used for this purpose in this study. Therefore, an in vitro assay using peripheral blood leucocytes (PBLs) from Pacific red snapper was performed to evaluate the stimulant effects of ß-Gluc/Sh and zymosan A (positive control) for 12 and 24 h and after bacterial challenge with Aeromonas hydrophila at 24 h. In addition, structural characterization of this marine yeast glucan was performed by proton nuclear magnetic resonance (NMR) revealing structures containing (1-6)-branched (1-3)-ß-D-glucan. PBLs responded positively to ß-Gluc/Sh where cell viability was higher than 80%. After challenge, ß-Gluc/Sh was able to inhibit cytotoxicity caused by A. hydrophila, highlighting that the PBLs incubated with ß-Gluc/Sh significantly increased the non-specific immune response, such as phagocytic activity, respiratory burst, nitric oxide and peroxidase activities followed by an increase in superoxide dismutase and catalase activities after 12 and 24 h post-stimulation and after challenge with the pathogen. Regarding induction of antioxidant gene expression, it was more pronounced in stimulated ß-Gluc/Sh leucocytes compared to other groups at all experimental times of the trial and after bacterial challenge. Indeed, our results clearly showed the ability of leucocytes to strongly react to ß-Gluc/Sh with an increase in cytokine gene expression, particularly the IL-1ß, IL-10 and IL-17 genes. These results confirm that S. halophilus yeast-derived ß-glucan, isolated from an extreme marine environment, is beneficial for increasing innate immune response and enhancing resistance against A. hydrophila in vitro.

Suplementação com imunoestimulante em cadelas com neoplasia mamária maligna: aspectos hematológicos e bioquímicos / Immunostimulant supplementation for bitches with malignant mammary tumor: hematological and biochemical aspects

Os alimentos funcionais têm sido empregados como adjuvantes no tratamento do câncer de mama. Neste estudo avaliaram-se as respostas hematológicas e bioquímicas clínicas à ação de um alimento funcional administrado a cadelas com diagnóstico de neoplasia mamária maligna. Após a mastectomia, 16 cadelas foram divididas em dois grupos: suplementadas (S) e não suplementadas (NS) com um composto comercial contendo Saccharomyces cerevisiae, mananoligossacarídeos e nutracêuticos. Ambos grupos receberam tratamento quimioterápico com doxorrubicina e carboplatina, alternadamente, em intervalos de 21 dias, por oito sessões, totalizando 168 dias de tratamento. As avaliações clínicas e laboratoriais foram realizadas nos momentos de aplicação do tratamento. Os resultados dos perfis hematológico (hemograma, leucograma e plaquetograma) e bioquímico sérico (ureia, creatinina, albumina, bilirrubina total e direta, alanina aminotransferase, fosfatase alcalina e gama glutamiltransferase - GGT) foram analisados pelo teste de Kruskall Wallis. No grupo S comprovou-se elevação do peso corporal e não foram observados transtornos gastrointestinais ou outros sinais de alteração clínica ao longo do tratamento. Diferentemente no grupo NS, ocorreu perda de peso e alterações clínicas, como diarreia e vômito. No quadro hematológico, constatou-se leucopenia por linfopenia no grupo de cadelas NS e preservação do valores dentro dos parâmetros considerados normais para a espécie no grupo S. Dentre todas as variáveis da bioquímica clínica, constatou-se apenas a elevação da atividade sérica da GGT nos animais do grupo NS, sem alterações no grupo S. Conclui-se que cadelas com neoplasia mamária quando suplementadas com com alimento funcional imunoestimulante apresentam melhor condição clínica, hematológica e dos níveis bioquímicos, particularmente da GGT.(AU)

Functional foods have been used as adjuvant for breast cancer treatment of bitches. The aim of the present study was to evaluate hematological and clinical biochemistry response in female dogs diagnosed with malignant mammary tumors and supplemented with functional food. After the mastectomy, 16 bitches were divided into two groups: supplemented (S) and none supplemented (NS) with a commercial product of Saccharomyces cerevisiae, mannanoligosaccharides and nutraceuticals. Chemotherapy with doxorubicin and carboplatin was performed alternately at intervals of 21 days for eight sessions during 168 days of treatment. Clinical and laboratorial assessments was made at the treatment moments. The results of the hemogram (erythrogram, leukogram and platelet count) and serum biochemistry (urea, creatinine, albumin, total and direct bilirubin, alanine aminotransferase, alkaline phosphatase and gamma glutamyltransferase - GGT) were analyzed by Kruskal Wallis test. In the S group, increase body weight was observed, but gastrointestinal disorders or other clinical disorders were not detected over the treatment. In the NS group, loss of weight and clinical disorders were observed. All hematology parameters were normal in the S group; however, leukopenia and lymphopenia were detected in the bitches of the NS group. Among all the clinical biochemistry parameters tested, only serum GGT was increased in the NS group, with no changes in the S group. In conclusion, female dogs with mammary tumor supplemented with immunostimulant functional food have better clinical condition, they demonstrate normal levels of hematological and biochemical exams, particularly GGT.(AU)