RESUMEN
Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bonds formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots. We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus, which was found to be upregulated at the protein level in response to an injection of the ß-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate l-γ-glutamine-p-nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilising activity in crustacean blood.
Asunto(s)
Proteínas de Artrópodos , Astacoidea , Muramidasa , Animales , Muramidasa/inmunología , Muramidasa/metabolismo , Muramidasa/química , Muramidasa/genética , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Proteínas de Artrópodos/química , Astacoidea/inmunología , Astacoidea/genética , Secuencia de Aminoácidos , Filogenia , Alineación de Secuencia/veterinaria , Inmunidad Innata , Hemocitos/inmunología , Secuencia de Bases , Coagulación Sanguínea/efectos de los fármacos , Perfilación de la Expresión Génica/veterinariaRESUMEN
Over the past two decades, therapeutic antibodies have emerged as a rapidly expanding domain within the field of biologics. In silico tools that can streamline the process of antibody discovery and optimization are critical to support a pipeline that is growing more numerous and complex every year. High-quality structural information remains critical for the antibody optimization process, but antibody-antigen complex structures are often unavailable and in silico antibody docking methods are still unreliable. In this study, DeepAb, a deep learning model for predicting antibody Fv structure directly from sequence, was used in conjunction with single-point experimental deep mutational scanning (DMS) enrichment data to design 200 potentially optimized variants of an anti-hen egg lysozyme (HEL) antibody. We sought to determine whether DeepAb-designed variants containing combinations of beneficial mutations from the DMS exhibit enhanced thermostability and whether this optimization affected their developability profile. The 200 variants were produced through a robust high-throughput method and tested for thermal and colloidal stability (Tonset, Tm, Tagg), affinity (KD) relative to the parental antibody, and for developability parameters (nonspecific binding, aggregation propensity, self-association). Of the designed clones, 91% and 94% exhibited increased thermal and colloidal stability and affinity, respectively. Of these, 10% showed a significantly increased affinity for HEL (5- to 21-fold increase) and thermostability (>2.5C increase in Tm1), with most clones retaining the favorable developability profile of the parental antibody. Additional in silico tests suggest that these methods would enrich for binding affinity even without first collecting experimental DMS measurements. These data open the possibility of in silico antibody optimization without the need to predict the antibody-antigen interface, which is notoriously difficult in the absence of crystal structures.
Asunto(s)
Afinidad de Anticuerpos , Muramidasa , Muramidasa/química , Muramidasa/inmunología , Muramidasa/genética , Estabilidad Proteica , Humanos , Antígenos/inmunología , Antígenos/química , Animales , Simulación por ComputadorRESUMEN
A substantial body of work has been carried out describing the structural features of the complex between single-domain antibodies (VHHs) and antigens, and the preeminence for epitopes located at concave surfaces of the antigen. However, the thermodynamic basis of binding is far less clear. Here, we have analysed the energetic profiles of five VHHs binding to the catalytic cleft or to a noncleft epitope of hen egg lysozyme. Various binding energetic profiles with distinctive enthalpic/entropic contributions and structural distribution of critical residues were found in the five antibodies analysed. Collectively, we suggest that from an energetic point of view the binding mechanism is influenced by the shape of the epitope. This information may be beneficial for the design of tailored epitopes for VHHs and their practical use.
Asunto(s)
Epítopos/inmunología , Muramidasa/antagonistas & inhibidores , Anticuerpos de Dominio Único/farmacología , Animales , Pollos , Cristalografía por Rayos X/métodos , Epítopos/química , Muramidasa/inmunología , Unión Proteica , Anticuerpos de Dominio Único/inmunología , Resonancia por Plasmón de Superficie/métodos , TermodinámicaRESUMEN
The waste recycling of lemon peel, as a functional feed additive in aquafeed was evaluated by estimating the effects of fermented lemon peel (FLP) supplementation in diet on growth performance, innate immune responses, and susceptibility to Photobacterium damselae of grouper, Epinephelus coioides. A basal diet was added FLP at 0%, 1%, 3%, and 5%. Four tested diets were each fed to juvenile grouper (initial weight: 15.89 ± 0.10 g, triplicate groups) in a recirculation rearing system for eight weeks. Fish fed diets with 0%-3% FLP exhibited higher (p < 0.05) final weight, weight gain, and feed efficiency than fish fed the 5% FLP-diet. After challenge test, fish fed the 3% FLP-diet appeared the lowest mortality, followed by fish fed the 1% FLP-diet, and lowest in fish fed 0% and 5% FLP-diets. Plasma lysozyme activities were higher in fish fed diets with FLP than in fish fed the FLP-free control diet before challenge test. After challenge, fish fed diets with 1% and 3% FLP showed highest lysozyme activities, followed by fish fed the diet with 5% FLP, and lowest in fish fed the control diet. Hepatic malondialdehyde content was higher in fish fed the control diet than in fish fed diets with 1%-3% FLP. Results found that diets supplemented with 1%-3% fermented lemon peel can enhance lysozyme activity and resistance to pathogen P. damselae of grouper.
Asunto(s)
Citrus , Suplementos Dietéticos , Enfermedades de los Peces/inmunología , Frutas , Infecciones por Bacterias Gramnegativas/inmunología , Muramidasa/inmunología , Perciformes , Photobacterium , Animales , Susceptibilidad a Enfermedades , Fermentación , Infecciones por Bacterias Gramnegativas/veterinaria , Hígado/inmunología , Malondialdehído/inmunología , Muramidasa/sangre , Perciformes/sangre , Perciformes/inmunología , Perciformes/microbiologíaRESUMEN
The sea urchin Lytechinus variegatus is considered a good candidate for aquaculture, but bacterial diseases are a major challenge in culture conditions. The innate immunological defenses of L. variegatus to bacterial challenges were assessed through hematology parameters, in vitro phagocytosis, lysozyme activity and total plasma protein concentrations in cell-free coelomic fluid. Adult sea urchins were inoculated with Microccocus lysodeikticus, Escherichia coli and Vibrio parahaemolyticus in the cavity coelomic. Filtrated and sterile seawater (FSW) injected and non-injected sea urchins were used as control groups. Righting time, external aspects and behavior of sea urchins were evaluated. Twenty-four hours post-inoculation, we found an increase in the population of colorless spherule cells (CLS), phagocytosis, and humoral responses in sea urchins challenged by bacterial inoculations. Righting time was not affected by the treatments and apparent external signs of disease were not observed at least during 96h post-inoculation. The immunological system of L. variegatus quickly eliminated pathogenic microorganisms. CLS and lysozyme activity cooperate in the immune defenses of L. variegatus, showing an extraordinary efficiency for adjusting the immune defenses under stress caused by microbes. We recommend that the cellular and humoral markers serve as routine tests to monitor health status in sea urchins.
Asunto(s)
Lytechinus/inmunología , Animales , Escherichia coli , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/veterinaria , Infecciones por Bacterias Grampositivas/inmunología , Infecciones por Bacterias Grampositivas/veterinaria , Inmunidad Innata , Lytechinus/citología , Lytechinus/microbiología , Micrococcus , Muramidasa/inmunología , Fagocitosis , Vibriosis/inmunología , Vibriosis/veterinaria , Vibrio parahaemolyticusRESUMEN
This study investigated the immunomodulatory effects of Sargassum polycystum extract administration in rainbow trout (Oncorhynchus mykiss). S. polycystum methanolic extract was administered orally using feeding needles to individual rainbow trout at the dose of 0 (control), 1 (S1), 3 (S3) and 5 (S5) mg/100 µl/per fish twice a day for 7 days. On 1st, 5th, 3rd and 7th day, blood and tissues were collected from the fish and changes in humoral immune responses and immune-related gene expressions were determined. The result of oxidative radical production showed no difference during early stage of the experiment and was lately decreased (P < 0.05). Lysozyme activity increased on 3rd and 7th day of the study in S5 fish group and on 5th day in S3 group compared to control (P < 0.05). Myeloperoxidase activity had an increased level on the 1st and 3rd day in S1, S5 and S5 fish groups, respectively. IL-1ß gene was significantly up-regulated in kidney and intestine in all experimental groups (except on the 1st day, in the intestine of S5 fish group) compared to control (P < 0.05). IL-8 gene expression was elevated on 1st and 3rd day in kidney of all experimental fish groups. IL-6 transcript enhanced in a dose-dependent manner on 3rd and 7th day. IL-10 and IL-12 genes were also up-regulated. Survival in all treated fish groups challenged with Aeromonas hydrophila was significantly increased compared to that of control. The highest survival rate was recorded in S5 fish group (83.65%) followed by S3 fish group (82.62%). Our results suggest that S. polycystum aqueous methanolic extract is an effective immunostimulant and provide protection against A. hydrophila infection in rainbow trout at a dose of 3-10 mg/20 g body weight/day.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Aeromonas hydrophila , Mezclas Complejas/farmacología , Enfermedades de los Peces/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Oncorhynchus mykiss/inmunología , Sargassum , Administración Oral , Animales , Citocinas/genética , Citocinas/inmunología , Enfermedades de los Peces/mortalidad , Infecciones por Bacterias Gramnegativas/mortalidad , Infecciones por Bacterias Gramnegativas/veterinaria , Muramidasa/inmunología , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/microbiología , Peroxidasa/inmunologíaRESUMEN
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.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Cíclidos/inmunología , Riñón Cefálico/efectos de los fármacos , Bazo/efectos de los fármacos , Vacunas Estreptocócicas/administración & dosificación , beta-Glucanos/administración & dosificación , Inmunidad Adaptativa , Animales , Cíclidos/genética , Cíclidos/microbiología , Citocinas/genética , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/prevención & control , Proteínas de Peces/genética , Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Riñón Cefálico/inmunología , Muramidasa/inmunología , Transducción de Señal , Bazo/inmunología , Infecciones Estreptocócicas/genética , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/prevención & control , Infecciones Estreptocócicas/veterinaria , Streptococcus agalactiaeRESUMEN
This study aims to assess and determine the oral-administration of probiotic, Lactobacillus pentosus BD6 on growth performance, immunity and disease resistance of white shrimp, Litopenaeus vannamei. Lac. pentosus BD6 effectively inhibited the growth of aquatic pathogens, which was used in the test. Shrimp were fed with the control diet (without probiotic supplement) for 60 days and the probiotic-containing diets at 107, 108, 109, and 1010 cfu kg-1, respectively. Shrimp fed with the diet containing probiotic at the doses of 109-10 cfu kg-1 showed significant increase in growth performance as well as feed efficiency than that of the control. After a challenge test with Vibrio alginolyticus, shrimp fed with a probiotic diet at a dose of 1010 cfu kg-1 showed a significantly lower mortality as compared to the control and that of shrimp fed the diet containing probiotic at the levels up to 107-8 cfu kg-1. In addition, a therapeutic potential of Lac. pentosus BD6 was discovered because the cumulative mortalities of shrimp fed with probiotic and pathogen V. parahaemolyticus simultaneously were significantly lower when compared to control shrimp. Probiotic in diet at a dose of 109-10 cfu kg-1 significantly increased PO activity of shrimp, while shrimp receiving probiotic at the doses of 108-10 cfu kg-1 showed significant increase in lysozyme activity and phagocytic activity. Shrimp fed with the diet containing probiotic at the level of 1010 cfu kg-1 also indicated higher gene expression of prophenoloxidase (proPO) I, but not proPO II, lipopolysaccharide and ß-1,3-glucan-binding protein and penaeidin 4. Analysis of the bacterial microbiota of the shrimp intestine revealed that oral administration of probiotic increased the relative abundance of beneficial bacteria and reduced the abundance of harmful pathogenic bacteria in the gut flora of shrimp. Despite no statistically significant difference, an analysis of microbial diversity recorded higher species richness, Shannon-Weaver diversity index and evenness in the probiotic group, compared to the control group. It was concluded that Lac. pentosus BD6 has great antibacterial ability against a wide range of pathogens and has therapeutic potential to reduce the mortality of shrimp infected with V. parahaemolyticus. Additionally, dietary Lac. pentosus BD6 at the level of 1010 cfu kg-1 was recommended to improve growth performance, immunity and disease resistance of shrimp against V. alginolyticus.
Asunto(s)
Lactobacillus pentosus , Penaeidae , Probióticos/administración & dosificación , Vibriosis/prevención & control , Vibrio alginolyticus , Administración Oral , Animales , Catecol Oxidasa/inmunología , Resistencia a la Enfermedad , Precursores Enzimáticos/inmunología , Microbioma Gastrointestinal , Expresión Génica , Hemocitos/inmunología , Hemolinfa/inmunología , Muramidasa/inmunología , Penaeidae/genética , Penaeidae/crecimiento & desarrollo , Penaeidae/inmunología , Penaeidae/microbiología , Fagocitosis , Vibriosis/mortalidad , Vibriosis/veterinariaRESUMEN
Fermentation strategy is well documented to improve the nutritional value of agricultural waste by-products such olive cake (OC), which, in turn, provides healthy, safe, and affordable feedstuff. This study assessed the combined impact of Aspergillus oryzae-fermented OC (AFOC) on the growth performance, intestinal morphometry, blood biochemistry, lysozyme activity, gut immune-related genes, and flesh quality of Nile tilapia. We divided 225 fish into five groups and further subdivided into three replicates (n = 15 each) and fed them five diets (Control, AFOC5, AFOC10, AFOC15, AFOC20) to determine AFOC nutritional value and its optimized incorporation level in the diet. The trial continued for 3 months. The crude protein content of OC improved by 7.77% after A. oryzae fermentation, while lipid content decreased by 14.19%. In addition, growth and feed utilization significantly improved at (10.8-11.2) % AFOC dietary level. High-density lipoprotein (HDL) significantly improved, and the serum lysozyme level was significantly higher in the AFOC10 group compared to other groups. Interestingly, gut-related inflammatory cytokines tumor necrosis factor alpha (TNF- α) and interleukin 1 beta (IL-1ß) revealed higher relative mRNA expression in the AFOC10 group compared to other groups. The histomorphometric parameters was greatly influenced by the AFOC incorporation level (10%-20%). These findings suggested that A. orzae fermentation modifies the nutritional quality of OC, as seen through its positive impact on the growth performance, local and systemic immunity, and intestinal absorptive capacity of Nile tilapia. The recommended dose for dietary AFOC was around 11.
Asunto(s)
Aspergillus oryzae , Cíclidos , Suplementos Dietéticos , Olea , Animales , Bioensayo , Cíclidos/sangre , Cíclidos/genética , Cíclidos/crecimiento & desarrollo , Cíclidos/inmunología , Citocinas/genética , Fermentación , Expresión Génica , Pruebas Hematológicas , Intestinos/anatomía & histología , Intestinos/inmunología , Lipoproteínas HDL/sangre , Muramidasa/inmunología , Valor NutritivoRESUMEN
The effect of cinnamaldehyde (CM) enriched diet on immunity and cytokine gene expression in Channa striatus against Aphanomyces invadans is reported. C. striatus was uniformly divided into eight groups (n = 25 fish each) and fed with formulated diets with 0, 5, 10, and 15 mg kg-1 CM enriched diet. In healthy and infected groups fed with 5 mg kg-1 diet the leukocytes count increased significantly after 4th week; with 10 mg kg-1 CM diet the increase manifested after 6th week, but with 15 mg kg-1 not even after 8th week. In both groups, 5 mg kg-1 CM diet resulted in a significant increase in the serum total protein, albumin, and globulin levels after 4th week, whereas with other diets this effect was observed only after 6th week. Similarly, with any enriched diet the lysozyme activity increased significantly, but with 15 mg kg-1 CM diet only after 6th week. In both groups the complement activity and lymphocyte production increased significantly when fed with 5 mg kg-1 CM diet after 4th week while with other enriched diets only after 6th week. The phagocytic activity increased significantly in both groups fed with 5 mg kg-1 CM diet after 6th week, whereas the SOD activity increased after 4th week. The IgM production increased significantly in both groups fed with 5 mg kg-1 CM diet after 2nd week, while with 5 and 10 mg kg-1 CM diet after 4th week. In both groups, the expression of CXCR3α was significant on 4th week when fed with 10 mg kg-1 CM diet, while in the healthy group fed with 15 mg kg-1 CM diet the expression manifested earlier than 4th week. However, when fed with 10 and 15 mg kg-1 CM diets the increase was observed on 6th week; whereas, the expression of MHC-I reached the maximum on 6th week with any enriched diet. The results indicate that in C. striatus the innate immunity and expression of cytokine and immune related genes were significantly modulated when fed with 5 mg kg-1 CM diet on 4th week against A. invadans.
Asunto(s)
Acroleína/análogos & derivados , Aphanomyces , Enfermedades de los Peces , Peces/genética , Peces/inmunología , Expresión Génica/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Infecciones , Acroleína/administración & dosificación , Animales , Activación de Complemento/efectos de los fármacos , Dieta , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Inmunoglobulina M/inmunología , Infecciones/genética , Infecciones/inmunología , Infecciones/veterinaria , Recuento de Leucocitos , Muramidasa/inmunología , Fagocitosis/efectos de los fármacos , Especies Reactivas de Oxígeno/inmunologíaRESUMEN
An eight-week investigation was conducted to access the potential impact of dietary watermelon rind powder (WMRP) and L. plantarum CR1T5 (LP) administered individually or in combination on immunity, disease resistance, and growth rate of Nile tilapia fingerlings cultured in a biofloc system. Three hundred twenty fish (average weight 16.57 ± 0.14 g) were distributed into 16 tanks at a rate of 20 fish per tank. The fish were fed different diets: Diet 1 (0 g kg-1 WMRP and 0 CFU g-1 L. plantarum) (control), Diet 2 (40 g kg-1 WMRP), Diet 3 (108 CFU g-1 LP), and Diet 4 (40 g kg-1 WMRP + 108 CFU g-1 LP) for eight weeks. A completely randomized design (CRD) with four replications was applied. Skin mucus, serum immunity, and growth parameters were analyzed every 4 weeks, and a challenge study against S. agalactiae was conducted at the end of the experiment. The findings showed that the inclusion of WMRP + LP, administrated individually or in a mixture, significantly (P<0.05) stimulated growth, skin mucus, and serum immune parameters of Nile tilapia fingerlings compared with the control. The highest values were detected in fish fed the combination of WMRP and LP, as opposed to individual administration of either WMRP or LP, in which no significant differences were detected. Within the challenge study, the relative percent survival (RPS) in Diet 2, Diet 3, and Diet 4 was 48.0%, 52.0%, and 68.0%, respectively. Fish fed 40 g kg-1 WMRP + LP produced significantly higher RPS and protection against S. agalactiae than the other treated groups. Current results suggest that the dual administration of WMRP and LP maybe an effective feed additive for Nile tilapia grown in an indoor biofloc system, capable of improving growth parameters and increasing resistance to S. agalactiae infection.
Asunto(s)
Citrullus , Lactobacillus plantarum , Preparaciones de Plantas/farmacología , Prebióticos , Simbióticos , Alimentación Animal , Animales , Acuicultura , Cíclidos/sangre , Cíclidos/crecimiento & desarrollo , Cíclidos/inmunología , Dieta/veterinaria , Resistencia a la Enfermedad , Recuento de Leucocitos , Micrococcus , Moco/enzimología , Moco/inmunología , Muramidasa/inmunología , Peroxidasa/inmunología , Fagocitosis , Polvos , Estallido Respiratorio , Piel/inmunología , Infecciones Estreptocócicas/prevención & control , Streptococcus agalactiaeRESUMEN
Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg-1 laurel-leaf cistus extract for 45 days. After 30 days, superoxide anion production (SAP) increased in CL0.1 and CL0.5 fish groups and at the end of the study all experimental fish groups had higher SAP compared to that of the control (P Ë 0.05). Lysozyme activity (LA) was elevated in CL0.5 and CL1 treated groups on 30th day (P < 0.05), and this increase was only observed in C0.1 fish group at the end of study compared to control (P Ë 0.05). Myeloperoxidase activity was significantly increased in CL0.5 and CL1 fish groups at the end of study. IL-1ßgene expression was significantly increased in treated fish in a dose-depended manner. Similar results were observed for transcription of IL-6 and IL-8 (P < 0.05). Anti-inflammatory cytokines, IL-10 and TGF-ß were highly up-regulated in the intestine and head kidney of CL treated fish groups compared to control (P < 0.05). At the end of experiment, significantly higher final body weight, weight gain, and specific growth rate were obtained in CL0.1 treated fish group compared to control. However, growth was negatively affected in CL1 fish group (P < 0.05). CL1 fish group had also a significantly higher FCR. Amylase activity was significantly increased in all experimental fish groups compared to control (P Ë 0.05). Trypsin activity was decreased in CL0.1 and CL1 fish groups (P Ë 0.05). WBC and RBC were significantly increased (P Ë 0.05) in CL0.5 and CL1 fish groups, whereas haemoglobin, haematocrit, mean cell, mean cell haemoglobin contents were no significantly changed among control and treatment groups. Result of challenge test with A. hydrophila exhibited that survival rate in all treatment groups was significantly higher than that of control. These findings demonstrated that laurel-leaf cistus at 0.1 g kg-1 can be a suitable candidate for growth promotion, immune system induction and infection control in fish.
Asunto(s)
Carpas , Cistus , Enfermedades de los Peces/prevención & control , Infecciones por Bacterias Gramnegativas/prevención & control , Inmunidad Innata/efectos de los fármacos , Factores Inmunológicos/farmacología , Extractos Vegetales/farmacología , Aeromonas hydrophila , Amilasas/metabolismo , Animales , Recuento de Células Sanguíneas , Carpas/sangre , Carpas/genética , Carpas/inmunología , Carpas/metabolismo , Citocinas/genética , Etanol/química , Infecciones por Bacterias Gramnegativas/veterinaria , Riñón Cefálico/citología , Riñón Cefálico/inmunología , Lipasa/metabolismo , Muramidasa/inmunología , Hojas de la Planta/química , Solventes/química , Superóxidos/inmunología , Tripsina/metabolismoRESUMEN
The gut microbiota affects the physiology and metabolism of animals and its alteration can lead to diseases such as gut dysplasia or metabolic disorders. Several reports have shown that the immune system plays an important role in shaping both bacterial community composition and abundance in Drosophila, and that immune deficit, especially during aging, negatively affects microbiota richness and diversity. However, there has been little study at the effector level to demonstrate how immune pathways regulate the microbiota. A key set of Drosophila immune effectors are the antimicrobial peptides (AMPs), which confer defense upon systemic infection. AMPs and lysozymes, a group of digestive enzymes with antimicrobial properties, are expressed in the gut and are good candidates for microbiota regulation. Here, we take advantage of the model organism Drosophila melanogaster to investigate the role of AMPs and lysozymes in regulation of gut microbiota structure and diversity. Using flies lacking AMPs and newly generated lysozyme mutants, we colonized gnotobiotic flies with a defined set of commensal bacteria and analyzed changes in microbiota composition and abundance in vertical transmission and aging contexts through 16S rRNA gene amplicon sequencing. Our study shows that AMPs and, to a lesser extent, lysozymes are necessary to regulate the total and relative abundance of bacteria in the gut microbiota. We also decouple the direct function of AMPs from the immune deficiency (IMD) signaling pathway that regulates AMPs but also many other processes, more narrowly defining the role of these effectors in the microbial dysbiosis observed in IMD-deficient flies upon aging. IMPORTANCE This study advances current knowledge in the field of host-microbe interactions by demonstrating that the two families of immune effectors, antimicrobial peptides and lysozymes, actively regulate the gut microbiota composition and abundance. Consequences of the loss of these antimicrobial peptides and lysozymes are exacerbated during aging, and their loss contributes to increased microbiota abundance and shifted composition in old flies. This work shows that immune effectors, typically associated with resistance to pathogenic infections, also help shape the beneficial gut community, consistent with the idea that host-symbiont interactions use the same "language" typically associated with pathogenesis.
Asunto(s)
Péptidos Antimicrobianos/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/microbiología , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiología , Muramidasa/metabolismo , Animales , Péptidos Antimicrobianos/genética , Péptidos Antimicrobianos/inmunología , Bacterias/clasificación , Bacterias/genética , Drosophila melanogaster/genética , Drosophila melanogaster/inmunología , Femenino , Microbioma Gastrointestinal/inmunología , Interacciones Microbiota-Huesped , Sistema Inmunológico , Muramidasa/genética , Muramidasa/inmunología , ARN Ribosómico 16S/genética , SimbiosisRESUMEN
The aim of this study was to investigate the effects of dietary bile acids (BAs) on intestinal healthy status of tongue sole in terms of immunity, antioxidant status, digestive ability, mucosal barrier-related genes expression and microbiota. Three experimental diets were prepared with BA levels at 0 mg/kg (CT), 300 mg/kg (BA1) and 900 mg/kg (BA2) in a commercial basal diet. Each diet was fed to three replicates with 120 fish (10.87 ± 0.32 g) in each tank. After an 8-week feeding trial, growth parameters were significantly enhanced in both BAs supplementary groups (P < 0.05), and compared with CT group, survival rate in BA2 group was significantly improved (P < 0.05). Intestinal lysozyme activity and contents of immunoglobulin M and complement 3 were significantly increased in both BAs supplementary groups (P < 0.05), suggesting an enhancement effect on the non-specific immune response. BAs inclusion also significantly improved intestinal antioxidant capabilities by increasing antioxidase activities and decreasing malondialdehyde levels. In addition, compared with CT group, intestinal digestive ability was substantially enhanced as indicated by the significantly increased lipase activity in BA2 group (P < 0.05) and significantly increased amylase activity in BA1 and BA2 groups (P < 0.05). Coincidentally, BAs inclusion significantly upregulated the relative expression of intestinal mucosal barrier-related genes (P < 0.05). Further, dietary BAs distinctly remodeled intestinal microbiota by decreased the abundance of some potential pathogenic bacteria. In conclusion, dietary BAs supplementation is an effective way to improve the intestinal healthy status of tongue sole.
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Ácidos y Sales Biliares/farmacología , Suplementos Dietéticos , Peces Planos , Microbioma Gastrointestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Fosfatasa Alcalina/inmunología , Amilasas/metabolismo , Animales , Complemento C3/inmunología , Dieta/veterinaria , Proteínas de Peces/metabolismo , Peces Planos/genética , Peces Planos/inmunología , Peces Planos/metabolismo , Peces Planos/microbiología , Regulación de la Expresión Génica/efectos de los fármacos , Inmunoglobulina M/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Lipasa/metabolismo , Muramidasa/inmunología , Oxidorreductasas/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas de Uniones Estrechas/genéticaRESUMEN
Vaccination is the most effective, safe, and environmentally friendly method to prevent the outbreak of Photobacterium damselae subsp. piscicida (Phdp), a dangerous pathogen in aquaculture worldwide. Here, recombinant proteins of catalase, superoxide dismutase, isocitrate dehydrogenase, fructose 1,6-bisphosphate aldolase (Fba), and a mixture of all four proteins were investigated for their immunoprotective effects against photobacteriosis in Asian sea bass (Lates calcarifer). After immunization, experimental fish showed an increase in specific antibody levels and lysozyme activities, especially the Fba group. After a lethal challenge with Phdp strain AOD105021, the Fba group achieved the highest relative percentage of survival rate (70.21%) and a significantly lower bacterial load in the spleens than other groups 3 days after infection. The results suggest that Fba is a good candidate for subunit vaccine development against photobacteriosis in fish.
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Vacunas Bacterianas/inmunología , Fructosa-Bifosfato Aldolasa/inmunología , Perciformes/inmunología , Photobacterium/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Acuicultura , Carga Bacteriana/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Vacunas Bacterianas/administración & dosificación , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/prevención & control , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/prevención & control , Infecciones por Bacterias Gramnegativas/veterinaria , Muramidasa/sangre , Muramidasa/inmunología , Perciformes/microbiología , Photobacterium/enzimología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Bazo/inmunología , Bazo/microbiología , Vacunación/veterinaria , Eficacia de las Vacunas , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunologíaRESUMEN
BACKGROUND: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses. STUDY DESIGN AND METHODS: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization. RESULTS: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG. CONCLUSION: Although storage of RBCs leads to alteration of several lysophospholipids known to be capable of binding CD1D, storage-induced RBC alloimmunization responses are not impacted by recipient CD1D deficiency.
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Antígenos CD1d/inmunología , Conservación de la Sangre , Transfusión Sanguínea , Eritrocitos/inmunología , Isoanticuerpos/biosíntesis , Isoantígenos/inmunología , Lisofosfolípidos/sangre , Reacción a la Transfusión/inmunología , Alarminas/sangre , Alarminas/inmunología , Animales , Especificidad de Anticuerpos , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Sistema del Grupo Sanguíneo Duffy/genética , Sistema del Grupo Sanguíneo Duffy/inmunología , Femenino , Inmunización , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/inmunología , Isoanticuerpos/inmunología , Lisofosfolípidos/metabolismo , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Muramidasa/inmunología , Ovalbúmina/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunologíaRESUMEN
In the present study, antioxidant activity, immune responses, and growth performance of rainbow trout (Onchorhynchus mykiss) juveniles fed with diets supplemented with dandelion (Taraxacum officinalis) and lichen (Usnea barbata) extracts were assessed. Four different concentrations of aqueous methanolic extract of the plants (0% (control), 0.1%, 0.5%, and 1% (D, dandelion; L, lichen) were added to the diets, and fish were fed for 75 days. On the 15th, 45th, and 75th day of the study, liver antioxidant enzyme activities were determined, and immune responses were determined every 15th day. The results showed that SOD activity increased in the fish group of 0.1% D on the 15th and 45th day compared to control; however, it was lower in all the lichen extract-treated groups than in control at almost all sampling times, except on the 15th day in the 0.1% L group. CAT activity showed an increased value (P < 0.05) in 0.5% L and 1% L treated fish groups on the 15th day, in fish of 1% D and 1% L groups on 45th and on 75th day in 0.1% D group. GPX activity increased on the 15th day of the study in fish of 0.1% D group, on the 45th day in 1% D and 1% L groups and on the 75th day in fish of 0.5% D, 0.1% D, and 0.5% L groups (P < 0.05). G6PDH enhanced in all treatment groups compared to control on the 15th day, except in 0.1% L and 0.5% L groups. An elevated G6PDH activity was also observed on the 75th day of the study in 0.5% D, 1% D, and 0.5% L fish groups. An increase on lipid peroxidation (LP) was observed in all L groups on the 45th day of the study. Lysozyme activity was determined to be the highest in 0.5% and 1% L on the 45th day, in 0.1% L on the 60th day and in the 0.5% L fish group on the 75th day compared to control (P < 0.05). Myeloperoxidase was found to be the highest at the end of the study in 1% L fish group compared to the control (P < 0.05). In conclusion, we suggest the use of dandelion to combat oxidative stress and to lower FCR and the use of lichen to modulate the immune response in rainbow trout. The use of such products will be economical for aquaculture and harmless for the environment.
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Suplementos Dietéticos , Oncorhynchus mykiss , Extractos Vegetales/farmacología , Taraxacum , Usnea , Animales , Dieta , Radicales Libres/sangre , Radicales Libres/metabolismo , Hígado/metabolismo , Muramidasa/sangre , Muramidasa/inmunología , Oncorhynchus mykiss/crecimiento & desarrollo , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/metabolismo , Oxidorreductasas/metabolismo , Peroxidasa/sangreRESUMEN
The degree of antigen adsorption to adjuvants in subunit vaccines may significantly influence the immune responses they induce upon vaccination. Commonly used approaches for studying how the level of adsorption affects the induction of antigen-specific immune responses include (i) using adjuvants with different abilities to adsorb antigens, and (ii) comparing different antigens selected based on their ability to adsorb to the adjuvant. A weakness of these approaches is that not only the antigen adsorption level is varied, but also other important functional factors such as adjuvant composition and/or the B/T cell epitopes, which may affect immunogenicity. Hence, we investigated how changing the adsorption capabilities of a single antigen to an adjuvant influenced the vaccine-induced immune responses. The model antigen lysozyme, which displays a positive net charge at physiological pH due to an isoelectric point (pI) of 11, was succinylated to different extents, resulting in a reduction of the pI value to 4.4-5.9, depending on the degree of succinylation. A pronounced inverse correlation was found between the pI value of the succinylated lysozyme analogues and the degree of adsorption to a cationic liposomal adjuvant consisting of dimethyldioctadecylammonium bromide (DDA) and trehalose dibehenate (TDB) (CAF®01). Furthermore, increased adsorption to this adjuvant correlated directly with the magnitude of lysozyme-specific Th1/Th17 immune responses induced by the vaccine in mice, while there was an inverse correlation with antibody induction. However, high lysozyme-specific antibody titers were induced with an increased antigen dose, even upon vaccination with a strongly adsorbed succinylated lysozyme analogue. Hence, these data illustrate that the degree of lysozyme adsorption to CAF®01 strongly affects the quality of the resulting immune responses.
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Adyuvantes Inmunológicos/química , Antígenos/inmunología , Vacunas de Subunidad/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adsorción , Animales , Antígenos/administración & dosificación , Antígenos/química , Cationes/administración & dosificación , Cationes/química , Femenino , Glucolípidos/administración & dosificación , Glucolípidos/química , Inmunogenicidad Vacunal , Liposomas , Ratones , Modelos Animales , Muramidasa/administración & dosificación , Muramidasa/química , Muramidasa/inmunología , Compuestos de Amonio Cuaternario/administración & dosificación , Compuestos de Amonio Cuaternario/química , Células TH1 , Células Th17 , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/químicaRESUMEN
The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8+ T cell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer's patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR+ PP cells play an important role in the induction of antigen-specific T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR+ LysoDCs) in PP and examine their role in CD8+ T cell immune induction. Stimulation of C5aR+ LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8+ T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity.
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Complemento C5a/inmunología , Reactividad Cruzada , Mucosa Intestinal/inmunología , Listeria monocytogenes/inmunología , Ganglios Linfáticos Agregados/inmunología , Receptor de Anafilatoxina C5a/genética , Linfocitos T Citotóxicos/inmunología , Inmunidad Adaptativa , Animales , Presentación de Antígeno , Complemento C5a/genética , Complemento C5a/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunidad Mucosa , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Listeria monocytogenes/patogenicidad , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/microbiología , Muramidasa/genética , Muramidasa/inmunología , Ganglios Linfáticos Agregados/efectos de los fármacos , Ganglios Linfáticos Agregados/microbiología , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Receptor de Anafilatoxina C5a/inmunología , Análisis de la Célula Individual , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/microbiologíaRESUMEN
The efficiency of laccase-catalyzed protein cross-linking can be impacted by substrate protein structure and competing reactions. In this study, chemical grafting of ferulic acid (FA) on protein surface was applied to modulate the cross-linking of two inflexible globular proteins, lysozyme (LZM) and ovalbumin (OVA). The extent of FA-grafting was positively correlated with protein cross-linking extent, and determined the molecular weight profile and structures of the cross-linked product. While laccase-catalyzed reactions (with or without free FA mediator) did not lead to evident cross-linking of the native proteins, oligomeric (up to 16.4%), polymeric (up to 30.6%) FA-LZMs and oligomeric FA-OVA (5.1-31.1%) were obtained upon the enzymatic treatments. The cross-linking on the grafted FA sites occurred mainly through the formation of 8-5'-noncyclic-dehydro-diferulic linkages. The effects of investigated cross-linking approach on the emulsifying, foaming properties and the immunoglobulin E (IgE) binding capacity of LZM and OVA were also evaluated in relation to the structural properties of cross-linked proteins.