Comportamento de células do sistema imune frente ao desafio com Salmonella Enteritidis em aves tratadas e não tratadas com ácidos orgânicos

A Salmonelose é uma importante zoonose, considerada a principal causa de infecções bacterianas, sendo associada ao consumo de produtos avícolas. Como alternativa de controle, ácidos orgânicos têm sido amplamente usados. No entanto, pouco se conhece sobre o estado imunológico de aves de produção, e uma avaliação deste status é necessária para proteger frente a enfermidades e para garantir à aplicação segura de agentes terapêuticos ou imunização profilática. Este trabalho teve como objetivo verificar o comportamento do sistema imunológico das aves previamente infectadas com Salmonella Enteritidis (SE) tratadas com um composto de ácidos orgânicos em diferentes concentrações administrado via água e ração comparando com as aves infectadas e não tratadas. Foram inoculados 120 frangos de corte com 1mL de SE, via oral, na concentração de 1,0 x 108 UFC/mL, no 1º e 2º dia de idade, divididos em seis tratamentos com duas repetições, utilizando 200, 400, 500 e 1000ppm do ácido orgânico. Aos 35 dias de vida das aves, foram coletados, de todos os grupos, alíquotas de sangue de 3mL em tubo contendo EDTA para a avaliação das células imunes através de citometria de fluxo. Foram analisadas as porcentagens circulantes de células CD4+, CD8β+, MHC I+, MHC II+, TCRVβ1+, TCRVβ2+ e CD28+. Para análise microbiológica foram coletadas tonsilas cecais destas aves. Observou-se com esse estudo que os ácidos orgânicos nas dosagens 1000ppm na água e 500ppm na ração durante, dois e sete dias respectivamente antes do abate, foram eficazes na redução da infecção por SE em frangos de corte, comprovadas pelo método microbiológico e demonstradas através do comportamento das células do sistema imune. No presente estudo as aves infectadas apresentaram uma proporção menor de células T auxiliares circulantes quando comparadas às aves infectadas, mas tratadas com o AO ou com o grupo não infectado. A mesma tendência pode ser observada para as células CD28+, TCRVβ1+ e MHC IIbright+, e, com menor resolução, para CD8β+

A Bifidobacterium-based synbiotic product to reduce the transmission of C. jejuni along the poultry food chain.

With the ban of dietary antimicrobial agents, the use of probiotics, prebiotics and synbiotics has attracted a great deal of attention in order to improve intestinal health and control food-borne pathogens, which is an important concern for the production of safe meat and meat products. Recently, Campylobacter jejuni has emerged as a leading bacterial cause of food-borne gastroenteritis in humans, and epidemiological evidences indicate poultry and poultry products as the main source of human infection. This work aimed at the development of a synbiotic mixture capable of modulating the gut microbiota of broiler chickens to obtain an increase of the beneficial bacteria (i.e. bifidobacteria, lactobacilli) and a competitive reduction of C. jejuni. The prebiotic compound used in the mixture was chosen after an in vivo trial: a fructooligosaccharide and a galactooligosaccharide were separately administered to broilers mixed with normal feed at a concentration of 0.5% and 3%, respectively. Quantitative PCR on DNA extracted from fecal samples revealed a significant (pb0.05) increase of Bifidobacterium spp. in broilers treated with the galactooligosaccharide, coupled to a decrease (pb0.05) of Campylobacter spp. The galactooligosaccharide was then combined with a probiotic Bifidobacterium strain (B. longum subsp. longum PCB133), possessing in vitro antimicrobial activity against C. jejuni. The strain was microencapsulated in a lipid matrix to ensure viability into the feed and resistance to stomach transit. Finally, the synbiotic mixture was administered to broiler chickens for 14 days mixed with normal feed in order to have an intake of 109 CFU of PCB133/day. Bifidobacterium spp., Lactobacillus spp., Campylobacter spp., B. longum subsp. longum and C. jejuni were quantified in fecal samples. PCB133 was recovered in feces of all animals. C. jejuni concentration in poultry feces was significantly (pb0.05) reduced in chickens administered with the synbioticmixture. This study allowed to highlight the positive effect of the synbiotic approach for C. jejuni reduction in broiler chickens, which is of fundamental importance for the safety of poultry meat consumers

Lactic acid bacteria [LAB]: potential for control of Fusarium growth

Lactic Acid Bacteria [LAB] are worldwide known as probiotic microorganisms. One of the most important probiotic feature is their strong activity against a wide range of harmful bacteria/fungi (Gaggìa et al., 2011). Production of organic acids and production of antagonistic compounds are the main mechanisms described nowadays. This work focuses on the in vitro selection of LAB strains isolated from environmental sources against Fusarium genus. Fusarium is a well distributed genus of filamentous fungi affecting plant, animal and human health. In the agricultural field, Fusarium is one of the most important plant pathogenic genera, with a record of devastating infections in various economically important plants. In this study, in vitro antagonistic effects of 18 isolates of Lactobacillus spp. were evaluated against F. oxysporum f. sp. lactucae, agent of tracheofusariosis in Lactuca sativa, F. culmorum and F. graminearum, agents of severe diseases in cereals. Fungal inhibition was performed using spot agar test in a triplicate assay (Magnusson et al., 2003). Overnight cultures of LAB were inoculated as spot on MRS agar plates and allowed to grow at 30°C for 24 h. The plates were then overlaid with 10 ml of PDA containing 104 mould spores/ml. After 48-72 h of aerobic incubation at 25°C, the zone of inhibition was measured. All LAB strains (except one) showed antifungal activity against all Fusarium species (inhibition halo between 23.7- 65.3 mm). The highest halos were obtained against F. graminearum on which 50% of the LAB strains showed a total growth inhibition of the colony. These results are probably related to the high amount of organic acids produced by LAB strains but it can also be hypothesized the presence of antifungal compounds. In vivo application of the most successful LAB strains is foreseen towards wheat and lettuce to confirm in vitro results

PERTURBATION OF FOREST SOIL MICROBIAL COMMUNITY BY METAL NANOPARTICLES

Nanotechnology is a growing field with a major role in product and process innovations. This is an opportunity to develop new products, but it also harbors risks for the environment and health. Although it is a common knowledge that some nanoparticles (NPs) have antibacterial activity, there is little information on the influence of NPs on soil microbial community (Hänsch and Emmerling, 2010). The harmful properties of silver ions to bacteria, for example, are well investigated. Silver ions may inhibit bacterial respiratory activity and may negatively interact with the bacterial cell wall enzymes. Furthermore, Ag ions may inhibit DNA replication. Perturbations of soil microbial consortia can have significant consequences for ecosystems and soil microbial consortia are influenced by and also affect plant growth (Davidson and Janssens, 2006). The aim of this work was to assess the impact of Ag, CeO2, Fe3O4, SnO2 NPs on the soil microbial community, to evaluate if NPs can affect the soil quality. 500 mg of NPs / kg of soil were mixed to an A1 horizon of Epileptic Cambisols (sandy clay loam texture; subacid pH) deriving from a forest environment and incubated at constant temperature and controlled moisture (e.g. 25°C and 60% WHC). Aliquots of soil samples were analysed after 30, 60 and 90 days of incubation with NPs to evaluate the following parameters: viable bacterial and moulds/yeasts counts , total soil microbial biomass, estimation of soil respiration, DNA extraction and PCR-DGGE. Preliminary results of the biological assays shows an increment of the basal respiration and a decrease in the amount of carbon soil microbial biomass. The relation between these parameters determine a higher metabolic quotient (qCO2) compared to the control test, that identifies a stressful situation, most evident in the thesis with Ag-NPs. Microbial plate counts evidenced no significant differences among treated and control soil except for a 2 log decrease of viable bacterial count in the Ag-treated soil after 60 days (Ag-60). Moreover, the quantification of soil DNA extractions showed a gradual decrease of DNA concentration in Ag-treated soil during incubation. PCR-DGGE was performed to evaluate NPs impact on microbial soil community. DGGE profile of control soil was mainly conserved in CeO2, Fe3O4 and SnO2 treated samples. On the contrary, Ag-treated soil evidenced a clear different profile already after the first 30 days (Ag-30). Moreover, the profile changed again in Ag-60 and remain stable in Ag-90. It is interesting to note that some bands showed an increased intensity as the incubation with Ag NPs proceeded. This preliminary results show an important influence of the Ag NPs on soil microbial community, decreasing richness and diversity. Bands sequencing are in progress to identify the microbial populations that are positively/negatively affected by NPs treatment

MODELLI DI STUDIO DI BIOMARKERS PER LA VALUTAZIONE DEL RISCHIO DI CONTAMINAZIONE DA NANOPARTICELLE D’ARGENTO NEL SUOLO (PROGETTO INESE)

Il progetto “Impact of Nanoparticles in Environmental Sustainability and Ecotoxicity” (INESE), finanziato dall’Istituto Italiano di Tecnologia (IIT), ha studiato gli effetti delle nanoparticelle (NPs) ingegnerizzate su diversi ecosistemi (acquatici e terrestri), proponendo differenti sistemi biologici di studio (piante acquatiche, ricci di mare, organismi del suolo, pomodoro-impollinatore, ecc). In particolare l’UO dell’Università di Bologna ha focalizzato l’attenzione sullo studio dei modelli di interazione tra particelle di suolo, microorganismi (biomassa microbica), invertebrati (Lumbricus rubellus) e pianta (Solanum lycopersicum). In questa sede verranno riportati i risultati ottenuti dall’impatto delle nanoparticelle di argento (Ag-NPs) nell’ecosistema suolo. La crescita esponenziale dell’uso di materiali contenenti Ag-NPs è dovuta alla loro importante azione antimicrobica, sempre più sfruttata in diversi campi: dalla medicina (bendaggi) all’abbigliamento (magliette, calzini etc.), ai prodotti di igiene personale (spazzolini da denti), dalla sanificazione delle acque reflue con conseguente produzione di biosolidi impiegati come ammendanti agricoli, all’utilizzo in agricoltura di prodotti dedicati ("Nano-Argentum 10", Fa Nanosys, Svizzera). Studi basati su monitoraggio registrano l’aumento di Ag-NPs nei diversi comparti ambientali (aria-acqua e suolo) e dimostrano che il suolo è destinato ad esserne il maggior ricettore. Il C contenuto nella biomassa microbica del suolo (Cmic) è significativamente diminuita dopo l'esposizione ad Ag-NPs rispetto al controllo, e ciò viene anche confermato dalla diminuzione del DNA microbico estraibile e dall’analisi PCR-DGGE. Quest’ultima ha mostrato la capacità battericida dell’Ag-NPs, riducendo la comunità microbica di un suolo forestale a solo 5 ceppi di batteri resistenti. Riguardo all’impatto sugli invertebrati, il maggior contenuto di Ag rispetto al controllo trovato nei tessuti dei lombrichi è stato riconfermato dall’analisi ESEM che ha mostrato la presenza di Ag-NPs nell’intestino dei lombrichi, dopo depurazione. Inoltre l’esposizione di questi organismi ad Ag-NPs ha causato significativi cambiamenti nella composizione lipidica dei tessuti, rilevando quindi condizioni di stress. La somministrazione di Ag-NPs alle piante di pomodoro tramite l’acqua di irrigazione ha messo in evidenza come le NPs possano essere traslocate dalle radici alle foglie e infine al frutto edule. I potenziali rischi per la salute umana ed ambientale dovuti all’incontrollata diffusione delle NPs costituiscono motivi di grave preoccupazione per la comunità scientifica internazionale