RESUMO
Vaccination against pathogens involved in bovine respiratory disease (BRD) is a useful tool to reduce the risk of this disease however, it has been observed that the commercially available vaccines only partially prevent the infections caused by Pasteurella multocida and Mannheimia haemolytica. Therefore, it is recommended to search for new adjuvant strategies to minimise the economic impact of this respiratory syndrome. A possibility to improve the conventional vaccine response is to modulate the immune system with probiotics, since there is accumulating evidence that certain immunomodulatory strains administered around the time of vaccination can potentiate the immune response. Considering veterinary vaccines are frequently tested in murine models, we have developed an immunisation schedule in BALB/c mice that allows us to study the immune response elicited by BRD vaccine. In order to evaluate a potential strategy to enhance vaccine efficacy, the adjuvant effect of Enterococcus faecalis CECT7121 on the murine specific humoral immune response elicited by a commercial vaccine against BRD was studied. Results indicate that the intragastric administration of E. faecalis CECT7121 was able to induce an increase in the specific antibody titres against the bacterial components of the BRD vaccines (P. multocida and M. haemolytica). The quality of the humoral immune response, in terms of antibody avidity, was also improved. Regarding the cellular immune response, although the BRD vaccination induced a low specific secretion of cytokines in the spleen cell culture supernatants, E. faecalis CECT7121-treated mice showed higher interferon-γ production than immunised control mice. Our results allowed us to conclude that the administration of E. faecalis CECT7121 could be employed as an adjuvant strategy to potentiate humoral immune responses.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas Bacterianas/imunologia , Complexo Respiratório Bovino/prevenção & controle , Enterococcus faecalis , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/imunologia , Probióticos , Animais , Anticorpos Antibacterianos/sangue , Afinidade de Anticorpos/imunologia , Vacinas Bacterianas/administração & dosagem , Complexo Respiratório Bovino/imunologia , Bovinos , Citocinas/biossíntese , Feminino , Esquemas de Imunização , Camundongos , Camundongos Endogâmicos BALB C , Pasteurellaceae/imunologia , Probióticos/administração & dosagemRESUMO
AIMS: To analyse the effect of Enterococcus faecalis CECT7121 on intestinal epithelial cells (IECs) and its effects on the mucosal immune response. METHODS AND RESULTS: Enterococcus faecalis CECT7121 showed a high adhesion capacity to completely and heterogeneously differentiated human intestinal epithelial cell line (Caco-2 cells). In addition, the contact of this bacterium with Caco-2 cells did not induce inflammatory chemokines (IL-8 and CCL-20). The presence of IgA(+) and IL-6(+) cells in the small intestine, as well as the production of inflammatory cytokines (TNFα, IL-6 and IL-12) in the gut, was determined after intragastric inoculation of Ent. faecalis CECT7121 in BALB/c mice. The administration of Ent. faecalis CECT7121 increased the number of IgA(+) cells in the intestinal lamina propria without modifying the percentage of IL-6(+) cells. No differences were observed in the cytokines measured in the intestinal extracts between probiotic-treated and control mice. CONCLUSIONS: Enterococcus faecalis CECT7121 stimulates local mucosal immunity and adheres to IECs without inducing inflammatory signals. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that, apart from its already reported systemic immune activity, Ent. faecalis CECT7121 has a modulatory effect at a local level.
Assuntos
Enterococcus faecalis/fisiologia , Células Epiteliais/imunologia , Imunidade nas Mucosas/efeitos dos fármacos , Mucosa Intestinal/imunologia , Intestinos/imunologia , Probióticos/administração & dosagem , Animais , Células CACO-2 , Citocinas/biossíntese , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Humanos , Interleucina-6/imunologia , Interleucina-8/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: Allergic diseases are featured by an increased production of IgE due to an imbalance in the immune response towards a Th2 profile. In this work, the ability of Enterococcus faecalis CECT7121 to regulate this Th2-exaggerated response in a murine model of ovalbumin (OVA)-induced allergy was studied. METHODS: BALB/c mice intragastrically inoculated with E. faecalis CECT7121 before and during a subcutaneous immunization protocol with OVA were studied in comparison with an immunized control group. The allergen-specific immune response (IgE, IgG, IgG1 and IgG2a) was assessed. The proliferative activity of memory splenocytes and the levels of IL-4, IL-5, IL-13, IL-10, IL-12 and IFN-γ were also determined. RESULTS: Upon treatment with E. faecalis CECT7121 the following effects were observed: (1) a decrease in specific IgE levels, (2) an increase in anti-OVA IgG2a levels, (3) the levels of anti-OVA IgG and IgG1 remained unaltered, (4) a reduction in the proliferation rate of memory cells, (5) a decrease in the levels of the Th2 cytokines IL-4, IL-5 and IL-13, and (6) the secretion of IL-10, IL-12 and IFN-γ remained unchanged. Moreover, the incubation of human basophils with non-viable E. faecalis CECT7121 together with an allergen preparation induced the release of ß-hexosaminidase at levels that were lower than control reactions and similar i.g. the spontaneous release. CONCLUSIONS: In this model, the i.g. administration of E. faecalis CECT7121 hampers the establishment of the OVA-induced allergic immune response, suggesting that this strain could be useful for the treatment of IgE-mediated allergic diseases.
Assuntos
Alérgenos/imunologia , Antígenos de Bactérias/imunologia , Dessensibilização Imunológica , Enterococcus faecalis/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/prevenção & controle , Ovalbumina/imunologia , Animais , Citocinas/biossíntese , Feminino , Imunidade Celular , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes CutâneosRESUMO
AIMS: To study the anti-tumour effects of Enterococcus faecalis CECT7121 on LBC cells, an aggressive murine T-cell lymphoma that kills the host in 18 days when is intraperitoneally (i.p.) administrated. METHODS AND RESULTS: In vitro studies have shown that LBC cell proliferation was inhibited by Ent. faecalis CECT7121 stimulus in a dose-dependent manner, inducing apoptosis. The production of ceramide was involved in the latter effect. To undertake in vivo studies, syngeneic BALB/c mice pre-treated i.p. with Ent. faecalis CECT7121 (2.5 × 10(8 ) CFU) were challenged i.p. with LBC cells (1.0 × 10(6) cells) the day after. On day 30 post-inoculation of LBC cells, 70% of Ent. faecalis CECT7121 pre-treated mice survived, whereas no survivals were recorded in the control group. A group of surviving mice was re-challenged with LBC cells, and 89% of them survived. Upon stimulation with irradiated LBC cells, spleen cell proliferation, high IFNγ, IL-12 and IL-10 levels were observed in surviving animals. CONCLUSIONS: Enterococcus faecalis CECT7121 affected multiple factors of the tumour establishment by the following methods: down-regulating the LBC cell proliferation and inducing apoptosis in these cells; and enhancing the immune response that protects animals from lymphoma challenge and re-challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrate that Ent. faecalis CECT7121 has potential as a probiotic that could facilitate the development of novel complements to therapeutic strategies against oncological diseases.
Assuntos
Enterococcus faecalis , Linfoma de Células T/prevenção & controle , Probióticos , Animais , Linhagem Celular Tumoral , Feminino , Memória Imunológica , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Lactic acid bacteria are the most adequate microorganisms for natural preservation of food. In the present work, the strain of Enterococcus faecalis CECT7121 was employed in the manufacture of craft dry-fermented sausages and its performance as a biopreservative was analysed. This strain is devoid of the genes for haemolysin and gelatinase and does not produce biogenic amines. It is sensitive to almost all the antibiotics tested and opsonophagocytic assays showed that it is devoid of a capsule. This strain had a high LD50 (10(11)CFU ml(-1)) in mice. No statistical differences were found between control and sausages inoculated with E. faecalis CECT7121 regarding the production of lactic acid, pH variation over time, reaching a minimum pH value of 5.1, and sensory analysis in both series. Sausages inoculated with E. faecalis CECT7121 had lower viable counts of Enterobacteriaceae, Staphylococcus aureus and other Gram-positive cocci at the end of fermentation and 7 days and no viable enterobacteria and S. aureus were recovered at the end of drying. E. faecalis CECT7121 did not affect the growth of Lactobacillus spp. but it displaced the autochthonous populations of enterococci. E. faecalis CECT7121 was recovered in each time point as assessed by its inhibitory activity on Listeria monocytogenes and S. aureus. These results would indicate that the addition of E. faecalis CECT7121 during the manufacture of craft dry-fermented sausages offers an interesting alternative for biopreservation.
Assuntos
Enterococcus faecalis/genética , Aditivos Alimentares , Conservantes de Alimentos , Produtos da Carne/microbiologia , Bacteriocinas/farmacologia , Aminas Biogênicas/análise , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , Farmacorresistência Bacteriana/efeitos dos fármacos , Enterococcus faecalis/isolamento & purificação , Gelatinases/genética , Cocos Gram-Positivos/isolamento & purificação , Proteínas Hemolisinas/genética , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Lactobacillus/isolamento & purificação , Lipase/metabolismo , Probióticos/farmacologia , Controle de Qualidade , Técnica de Amplificação ao Acaso de DNA Polimórfico , Staphylococcus aureus/isolamento & purificação , beta-Lactamases/metabolismoRESUMO
AIMS: To assess the inhibitory activity on Gram-positive and Gram-negative bacteria of several species of enterococci recovered from a natural corn silage. METHODS AND RESULTS: The inhibitory activity of strains of Enterococcus faecalis (58), Enterococcus faecium (35), Enterococcus gallinarum (3) and Enterococcus casseliflavus (4) were studied employing indicator strains from various sources (clinical, food and ATCC). Enterococcus faecalis MR99, the only strain with inhibitory activity, inhibited other enterococci, Listeria spp., Staphylococcus aureus, Clostridium spp., Bacillus spp., Escherichia coli, Shigella sonnei and Shigella flexneri. The bacterium contained only one conjugative pheromone-responsive plasmid. The partially chromatography-purified MR99 enterocin (PPE) had a molecular weight of approx. 5000 Da and a pI of 6.2, was sensitive to proteolytic enzymes and could be extracted in benzene and butanol. It appeared stable to adjustment of pH 4.0, 5.0, 6.0, 7.0 and 8.0 and was resistant to heat. Inactivation was at 15 min at 121 degrees C. Enterocin MR99 was bactericidal on strains of Listeria monocytogenes, Staph. aureus, and bovine mastitis agents, it was bacteriostatic on E. coli. Although enterocins MR99 and AS48 have inhibitory activity on Gram-negative bacilli, PCR studies demonstrated a lack of relationship between them. CONCLUSIONS: The active component had a protein nature, was resistant to heat and presented a wide inhibitory spectrum. SIGNIFICANCE AND IMPACT OF THE STUDY: The biological properties of Ent. faecalis MR99 suggest that this strain merits further investigations so it can be applied in human and veterinary health programmes.
Assuntos
Bacteriocinas/farmacologia , Enterococcus/fisiologia , Microbiologia de Alimentos , Silagem/microbiologia , Zea mays/microbiologia , Bacillus/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/análise , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Clostridium/efeitos dos fármacos , Meios de Cultura , Eletroforese em Gel de Poliacrilamida/métodos , Enterococcus faecalis/fisiologia , Enterococcus faecium/fisiologia , Escherichia coli/efeitos dos fármacos , Focalização Isoelétrica/métodos , Listeria/efeitos dos fármacos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/ultraestrutura , Microscopia Eletrônica/métodos , Plasmídeos/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Shigella/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/ultraestruturaRESUMO
The structure and protective activity of tetanus antibodies elicited in rabbits after whole-cell pertussis diphtheria-tetanus vaccine (DTPw) vaccination was studied. ELISA antibody levels and toxin neutralisation activity (TNT) were measured in individual serum samples. The ratio of symmetric and asymmetric (functionally monovalent) IgG molecules was determined by concanavalin A (Con A) chromatography. This test is based on the fact that the carbohydrate group responsible for the molecular asymmetry has high affinity for the lectin Con A. Asymmetric molecule ratio was observed to increase with immunisation time, as well as differences between TNT and ELISA levels. All serum samples were overestimated by ELISA as compared to TNT assay, in line with the markedly higher proportion of asymmetric molecules which have lower toxin neutralising activity. Protective levels could not be predicted reasonably from ELISA results below 0. 222 IU/ml, because this methodology fails to discriminate between both types of antibodies and only an in vivo serum neutralisation procedure (TNT) reflects the true neutralising serum activity.