Cytotoxicity in Vero cells and cytokines analyses in Balb/c mice as safety assessments of the probiotic mixture Saccharomyces cerevisiae RC016 and Lactobacillus rhamnosus RC007 for use as a feed additive.

The objective was to carry out cytotoxicity assays in Vero cells and cytokines analyses in Balb/c mice as safety assessments to evaluate the probiotic mixture (M) Saccharomyces cerevisiae RC016 (Sc) and Lactobacillus rhamnosus RC007 (Lr) for use as feed additive. Vero cells (104 cells per well) were exposed to Sc (2·08 × 107 , 2·08 × 106 ; 2·08 × 105 cells per ml), Lr (8·33 × 107 ; 8·33 × 106 ; 8·33 × 105 cells per ml) and their M (1 : 1). Sc concentrations did not affect the Vero cells viability; in contrast, they were lower when exposed to Lr (P Ë‚ 0·0001). Vero cells showed increasing viability with M decreasing concentrations (91% viability with M2). Control BALB/c mice received only phosphate buffer saline and the others received the M. The IL-10, IL-6 and TNFα concentrations from intestinal fluid were analysed and no significant differences were observed among treatments. The same occurred with the ratio between IL-10/TNF-α. Beneficial effects of probiotics are associated with the regulation of the excessive inflammatory response; it is desirable they can modulate the cytokines production only under pathological conditions. Here, M administration to healthy mice did not induce negative side effects and expands the knowledge about beneficial effects of using probiotic microorganisms in mixture for feed additives development.

Saccharomyces cerevisiae as a probiotic agent and a possible aflatoxin B1 adsorbent in simulated fish intestinal tract conditions / Saccharomyces cerevisiae como agente probiótico e possível adsorvente de aflatoxina B1 em condições simuladas do trato intestinal de peixes

The aim of this study was to evaluate in vitro the probiotic potential and absorption of Saccharomyces cerevisiae for the aflatoxin B1 in simulated fish intestinal tract conditions. Three yeast strains were used, two from brewery: S. cerevisiae RC1 and S. cerevisiae RC3 and one from a fish farming environment: S. cerevisiae A8L2. The selected yeasts were subjected to the following in vitro tests: homologous inhibition, self-aggregation, co-aggregation, antibacterial activity, gastrointestinal conditions tolerance and adsorption of AFB1. All S. cerevisiae strains showed good capability of self-aggregation and co-aggregation with pathogenic bacteria. All yeast strains were able to survive the gastrointestinal conditions. In acidic conditions, the factors (strain vs. time) had interaction (P=0.0317), resulting in significant variation among the strains tested in the time periods analyzed. It was observed that there was also interaction (P=0.0062) in intestinal conditions, with an increased number of cells in the 12-hour period for all strains tested. In the adsorption test, the A8L2 strain was statistically more effective (P<0.005) for both AFB1 concentrations evaluated in this study (10 and 25ng/mL). Thus, it was observed that the strains of S. cerevisiae have potential probiotic and adsorbent of AFB1.(AU)

Objetivou-se, com esta pesquisa, avaliar in vitro o potencial probiótico e adsorvente de Saccharomyces cerevisiae para aflatoxina B1 em condições simuladas do trato intestinal de peixes. Foram utilizadas três cepas de leveduras, sendo duas provenientes de cervejaria: S. cerevisiae RC1 e S. cerevisiae RC3, e uma de ambiente de piscicultura: S. cerevisiae A8L2. As leveduras selecionadas foram submetidas aos seguintes testes in vitro: inibição homóloga, autoagregação, coagregação, atividade antibacteriana, viabilidade às condições gastrointestinais e adsorção de AFB1. Todas as estirpes de S. cerevisiae mostraram boa capacidade de autoagregação e coagregação com bactérias patogênicas. Todas as estirpes de levedura foram capazes de sobreviver às condições gastrointestinais. Em condições ácidas, os fatores (cepa x tempo) tiveram interação (P=0,0317), resultando em variações significativas entre as cepas testadas nos períodos de tempo analisados. Observou-se que também houve interação (P=0,0062) em condições intestinais, havendo um aumento do número de células no período de 12h para todas as cepas avaliadas. No ensaio de adsorção, a estirpe A8L2 foi a mais eficaz estatisticamente (P<0,005), para as duas concentrações de AFB1 avaliadas neste estudo (10 e 25ng. mL-1). Dessa forma, conclui-se que as cepas de Saccharomyces cerevisiae possuem potencial probiótico e adsorvente de AFB1.(AU)

Aflatoxin-degrading Bacillus sp. strains degrade zearalenone and produce proteases, amylases and cellulases of agro-industrial interest.

Zearalenone, an oestogenic mycotoxin produced by Fusarium sp., occurs naturally in agricultural commodities. Economic losses and health concerns associated to mycotoxins has attracted research interest towards exploring novel approaches to detoxify mycotoxin-contaminated food and feed. The aim of the present work was to study the ability of 11 aflatoxin-degrading Bacillus strains to degrade ZEA. In addition, a qualitative assessment of protease, amylase and cellulase activity of the studied Bacillus strains was made. All strains were able to degrade 58-96.9% ZEA after 72 h. Toxicity towards Artemia salina was significantly reduced (P < 0.0001). Degradation extracts fluorescence decreased 50% indicating a probable cleavage of the lactone ring. Strains RC1A, RC3A and RC6A showed a remarkable enzymatic activity, showing potential to be used as feed additives.

Probiotic bacteria and yeasts adsorb aflatoxin M1 in milk and degrade it to less toxic AFM1-metabolites.

The presence of contaminants such as aflatoxins (AFs) in dairy products constitutes a serious risk to the health of consumers, especially children who are most sensitive to the adverse effects of AFs. The presence of Aflatoxin M1 (AFM1) in milk is a public health problem since dairy products are massively consumed worldwide. The aim of the present work was to select microorganisms capable of reducing AFM1 entry into the food chain through adsorption/degradation strategies. Moreover, the toxicity of AFM1 degradation products was evaluated. All tested strains had the capacity to adsorb 19%-61% AFM1 in milk. These strains also had the ability to degrade AFM1 into metabolites less toxic than the original toxin. Moreover, this is the first study to report harmless and probiotic Pediococcus pentosaceus and Kluveromyces marxianus have the ability to adsorb and degrade AFM1 to less toxic metabolites in milk.

Beneficial effects of Saccharomyces cerevisiae RC016 in weaned piglets: in vivo and ex vivo analysis.

Probiotics represents an alternative to replace antibiotics as growth promoters in animal feed and are able to control enteric bacterial diseases and to improve gut immunity. Saccharomyces cerevisiae RC016 showed previously inhibition/coagregation of pathogens) and mycotoxins adsorbent ability (aflatoxin B1, ochratoxin A and zearalenone). The aim of this work was to evaluate beneficial properties of S. cerevisiae RC016 in a non-inflammatory in vivo model in weaned piglets and in an intestinal inflammation ex vivo model induced by the mycotoxin deoxynivalenol (DON). Secretory immunoglobulin A (s-IgA) levels, intestinal cytokines, goblet cells and production parameters were evaluated in a pig model. For the in vivo assays, twelve pigs were weaned at 21 days and assigned to two groups: Control (n=6) and Yeast (n=6). Animals received yeast strain for three weeks. After 22 days the small intestine was recovered for determination of goblet cells and s-IgA. For the ex vivo assay, jejunal explants were obtained from 5 weeks old crossbred piglets and treated as follow: (1) control; (2) treated for 3 h with 10 µM DON used as an inflammatory stressor; (3) incubated with 107 cfu/ml yeast strain; (4) pre-incubated 1 h with 107 cfu/ml yeast strain and then treated for 3 h with 10 µM DON. CCL20, interleukin (IL)-1ß, IL-8 and IL-22 gene expression was determined by qPCR. Oral administration of S. cerevisiae RC016 increased s-IgA, the number of goblet cells in small intestine and all the growth parameters measured. In the ex vivo model, the cytokine profile studied showed a potential anti-inflammatory effect of the administration of the yeast. In conclusion, S. cerevisiae RC016 is a promising candidate for feed additives formulation to improve animal growth and gut immune system. This yeast strain could be able to improve the gut health through counteracting the weaning-associated intestinal inflammation in piglets.

Aflatoxin B1 adsorption/desorption dynamics in the presence of Lactobacillus rhamnosus RC007 in a gastrointestinal tract-simulated model.

AIMS: (i) To determine the aflatoxin B1 (AFB1 ) adsorption and desorption dynamics in the presence of Lactobacillus rhamnosus RC007 under simulated transit of AFB1 at each gastrointestinal tract (GIT-saliva, stomach and intestine) stage consecutively and then, separately, (ii) to study the ability of L. rhamnosus RC007 to biotransform AFB1 as a strategy that complements the adsorption process. METHODS AND RESULTS: The AFB1 adsorption and desorption assay simulating the GIT passage of AFB1 (93·89 ng g-1 ) in the presence of L. rhamnosus RC007 (108 CFU per ml) was conducted. Moreover, lactic acid production was determined. Results demonstrated that predominant environmental conditions in salivary solution induced a low AFB1 adsorption, while the transit through the gastric solution and intestinal solution allowed high percentages of adsorption and did not generate significant AFB1 desorption. CONCLUSIONS: The AFB1 adsorption and desorption dynamics in the presence of L. rhamnosus RC007 was favoured by gastric and intestinal environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of the adsorption dynamics of AFB1 with a micro-organism of interest will allow predicting its behaviour at each stage of the GIT.

Presence of aiiA homologue genes encoding for N-Acyl homoserine lactone-degrading enzyme in aflatoxin B1-decontaminating Bacillus strains with potential use as feed additives.

Microbial degradation of aflatoxins (AFs) is an alternative to the use of mycotoxin binders. The lactone ring is a possible target for microbial enzymes and its cleavage reduces AFs toxicity. The aim of this study was to isolate and identify Bacillus strains able to degrade AFB1 to less toxic metabolites and to identify aiiA genes encoding for N-acyl-homoserine lactone (AHL) lactonase to possibly correlate detoxification with the production of this enzyme. Eleven soilborne Bacillus strains were isolated and identified by MALDI-TOF MS. Ten cultures and eight cell free culture supernatants (CFCS) were able to significantly (P < 0.05) degrade 27.78-79.78% AFB1. Cell lysates were also able to degrade AFB1 (P < 0.05). Exposure to 70 and 80 °C did not affect enzyme activity. Aflatoxin B1 toxicity towards Artemia salina was reduced after degradation by each of the Bacillus strains. B. subtilis RC1B, B. cereus RC1C and B. mojavensis RC3B, amplified a fragment of 753 pb corresponding to the aiiA gene encoding for AHL lactonase. AFB1 degradation by the strains tested was due to the extracellular and intracellular enzymes. If demonstrated to be safe, these could be used to detoxify AFB1 in contaminated food or feed.

The production of yeast cell wall using an agroindustrial waste influences the wall thickness and is implicated on the aflatoxin B1 adsorption process.

The objectives of this study were: to evaluate the use of dry distillery grain soluble extract - DDGse to produce yeast biomass and to obtain cell wall (CW), to use the CW as an aflatoxin B1 (AFB1) adsorbent, to study the variation in the composition and thickness of the CW under the influence of DDGse to evaluate their implication on the adsorption process using transmission electron microscopy (TEM) and fourier-transform infrared spectroscopy (FITR). The production of biomass and CW were variable. The CW thickness values showed that S. boulardii strain grown in yeast extract peptone dextrose (YPD) or DDGse medium, with no significant differences observed. The thickness of the CW for S. cerevisiae (RC012 and VM014) were increased when the cells were grown in DDGse medium, the thickness was almost double compared to the values obtained in YPD medium. The spectra IR of each CW in the two culture media shown regions corresponding to polysaccharides, proteins and lipids. Cells grown in DDGse medium adsorbed more AFB1 than those grown in YPD. The CW adsorbed more AFB1 than the same amount of whole cell. Future studies should be done to determine the type of carbohydrates and the relationship between chitin - beta glucans responsible for mycotoxin adsorption.

Use of yeast (Pichia kudriavzevii) as a novel feed additive to ameliorate the effects of aflatoxin B1 on broiler chicken performance.

The aim of this study was to evaluate the efficacy of autochthonous Pichia kudriavzevii as a novel bioadsorbent for aflatoxin B1 (AFB1). The selection of this yeast was based on the AFB1 adsorption capacity previously demonstrated in vitro (Magnoli et al. 2016). One-day-old Cobb broilers (n = 160) were randomly assigned to four dietary treatments (T1: basal diet (B); T2: B + 0.1% yeast; T3: B + AFB1, 100 µg/kg; T4: B + 0.1% yeast + AFB1, 100 µg/kg). Performance parameters (average daily weight gain body, average daily consumption, feed conversion ratio, carcass weight, and dead weight), biochemical parameters (albumin, globulin, and albumin/globulin), liver pathological changes, and AFB1 residual levels in the liver and excreta were evaluated. Significant differences (P < 0.05) in performance parameters were observed among treatments and controls: T3 group showed the lowest average daily body weight gain value while in T4 group, the value of this parameter increased significantly (P < 0.05). T3 and T4 groups showed the lowest and highest values for average daily feed consumption, respectively. The feed conversion ratio (FC) showed no significant differences among treatments. T3 group showed the lowest dead weight and carcass weight compared with T1 group. The biochemical parameters showed no significant differences among treatments. T3 group showed macroscopic and microscopic liver changes compared to the control. Aflatoxin B1 levels (µg/g) were detected in broiler livers and showed significant differences among treatments (P < 0.05). In conclusion, native P. kudriavzevii incorporation (0.1%) in broiler diets containing AFB1 was shown to be effective in ameliorating the adverse effects of AFB1 on production.

Modelling the effect of pH and water activity in the growth of Aspergillus fumigatus isolated from corn silage.

AIMS: The aim of this work was to use mathematical kinetic modelling to assess the combined effects of aW, pH, O2 availability and temperature on the growth rate and time to growth of Aspergillus fumigatus strains isolated from corn silage. METHODS AND RESULTS: A full factorial design was used in which two factors were assayed: pH and aW . The aW levels assayed were 0·80, 0·85, 0·90, 0·92, 0·94, 0·96, 0·98 and 0·99. The levels of pH assayed were 3·5, 4, 4·5, 5, 6, 7, 7·5 and 8. The assay was performed at normal oxygen tension at 25 and 37°C, and at reduced oxygen tension at 25°C. Two strains of A. fumigatus isolated from corn silage were used. Kinetic models were built to predict growth of the strain under the assayed conditions. The cardinal models gave a good quality fit for radial growth rate data. The results indicate that the environmental conditions which take place during silage production, while limiting the growth of most micro-organisms, would not be able to control A. fumigatus. Moreover, pH levels in silage, far from limiting its growth, are also close to its optimum. Carbon dioxide at 5% in the environment did not significantly affect its growth. CONCLUSIONS: A need for a further and controlled acidification of the silage exists, as no growth of A. fumigatus was observed at pH 3·5, as long as the organoleptic characteristics of the silage are not much compromised. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus fumigatus is one of the major opportunistic pathogens able to cause illness such as allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis to rural workers. Exposure of animals to A. fumigatus spores can result in infections, particularly in those organs exposed to external invasion, such as the airways, mammary gland and uterus at birth.

Gut-borne Saccharomyces cerevisiae, a promising candidate for the formulation of feed additives, modulates immune system and gut microbiota.

The aim was to evaluate the effect of Saccharomyces cerevisiae RC016 on immune parameters and gut microbiota in healthy mice. Animals received S. cerevisiae RC016 for 10 days. Microbial translocation to liver and changes in some bacterial populations in caecum were determined. Immune stimulation was assessed at gut level (measure ofimmunoglobulin A (IgA)+ cells and luminal cytokine profile) and by evaluating the activity of peritoneal macrophages. Oral administration of S. cerevisiae RC016 did not induce microbial translocation to liver. Mice that received yeast increased the number of IgA+ cells in their intestines, the phagocytic activity of peritoneal macrophages and decreased tumour necrosis factor alpha (TNF-α) levels in the small intestine with increases of interleukin-10/TNF-αratio. Administration of S. cerevisiae RC016 caused the decline of a logarithmic unit for Enterobacteriaceae counts compared to the control. The immune and gut microbiota modulation observed demonstrates that S. cerevisiae RC016 is a promising candidate for the formulation of feed additives to improve animal productivity. The beneficial in vivo effects observed for the potential probiotic S. cerevisiae RC016 with previously reported mycotoxin-bindingproperties, demonstrated that this strain could be suitable to be included in a novel product to improve animalproductivity, with both probiotic and mycotoxin-binding properties. However, studies in the specific host will be necessary to confirm this potential.

Novel yeast isolated from broilers' feedstuff, gut and faeces as aflatoxin B1 adsorbents.

AIMS: To isolate and characterize native yeast strains from broilers' environment as feedstuff, faeces and gut, and to evaluate their binding capacity for aflatoxin B1 (AFB1 ). METHODS AND RESULTS: A total of nine yeast strains were isolated: three from feedstuff identified as Pichia kudriavzevii (2) and Clavispora lusitaniae (1), two from gut identified as Candida tropicalis and four from faeces identified as Cl. lusitaniae (3) and Cyberlindnera fabianii (1). AFB1 binding percentages varied among yeast strains and with AFB1 concentrations. To carry out adsorption studies, one strain from each genus and each origin was selected as follows: Cl. lusitaniae and P. kudriavzevii from feedstuff, Cl. lusitaniae and Cy. fabianii from faeces and Ca. tropicalis from gut. The most appropriate concentrations for cells and toxin were 107 cells per ml and 100 ng ml-1 of AFB1 respectively. All the tested yeast strains showed similar adsorption capacities independently of the origin. The adsorption isotherm studies in all yeasts assayed showed behaviour of L type or Langmuir and a varied affinity for the toxin. The stability of the AFB1 -yeast complex demonstrated the irreversibility of the binding process. CONCLUSION: Yeast strains tested in this study constitute potential AFB1 adsorbents and they possess the advantage to be native from the avian environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study makes a contribution to using native yeasts from broilers' environment for controlling chronic aflatoxicosis in avian production.

Lactobacillus rhamnosus RC007 intended for feed additive: immune-stimulatory properties and ameliorating effects on TNBS-induced colitis.

Lactobacillus rhamnosus RC007 is a potential probiotic bacterium that can exert beneficial effects as supplement for animal feed, by improving the immune status in healthy host, and by providing therapeutic benefits to infected/inflamed animals. The aim of the present work was to evaluate in vivo the beneficial properties of L. rhamnosus RC007, intended for animal feed, when administered to healthy and trinitro-benzene-sulfonic-acid (TNBS) colitis induced BALB/c mice. The administration of L. rhamnosus RC007 to healthy mice during 10 days increased the phagocytic activity of peritoneal macrophages and the number of immunoglobulin A+ cells in the lamina proper of the small intestine. Significant increases of monocyte chemotactic protein 1, interleukin (IL)-10 and tumour necrosis factor alpha (TNF-α) concentrations, and in the ratio between anti- and pro-inflammatory cytokines (IL-10/TNF-α) were observed in intestinal fluids after administration of bacteria. In the inflammation model, less body weight loss, macroscopic and histological damages in the large intestine were accompanied by increased IL-10/TNF-α ratio in the intestinal fluids of mice from the L. rhamnosus-TNBS group when compared to the TNBS group. In a healthy host, the oral administration of L. rhamnosus RC007 kept the gut immune system stimulated allowing a faster response to noxious stimulus. Mice that received L. rhamnosus RC007 also decreased the severity of the intestinal inflammation.

Effect of breast feeding time on physiological, immunological and microbial parameters of weaned piglets in an intensive breeding farm.

The aim of this work was to study the long-lasting consequences of different weaning age on physiological, immunological and microbiological parameters of weaned piglets. Piglets were weaned at 14 days (14W) or 21 days (21W). Blood samples were taken for IgG and cortisol determination on preweaning day and at 4; 20 and 40 post-weaning days. Three animals of each group were sacrificed. Small intestines for morphometric studies and secretory-IgA determination in fluid were taken. The cecum was obtained for enterobacteria, lactobacilli and total anaerobes enumeration. A significant decrease in piglet's plasma IgG concentrations was observed immediately after weaning and no differences were found between 14W and 21W. An increase in intestinal S-IgA was observed according to piglet's age. This increase was significantly higher in piglets 14W compared to piglets 21W. Animals from 14W group showed a decrease in villus length and in the number of goblet cells and intraepithelial lymphocytes. Other parameters were not affected by the weaning age. A short-term increase in cortisol was observed after weaning in both experimental groups. Enterobacteria decreased significantly after weaning in both groups, reaching values of weaning after 40 days. Lactobacilli counts decreased in both groups after weaning; however their counts were always higher than those obtained for enterobacteria. No differences were observed between 14W and 21W with regards to counts of anaerobes. The shortening of breast feeding time would favor an early synthesis of intestinal S-IgA after weaning. The changes observed in the microbiota could decrease postweaning enteric infections. However, early weaning induced negative effects on the cells of gut innate immunity and villi atrophy. This work provides knowledge about advantages and disadvantages at different weaning and long-lasting consequences on pig health. It is critical that swine producers become aware of the biological impacts of weaning age, so as to be able to decide the appropriate management strategies according to their facilities and rearing environment.

Ecophysiology of environmental Aspergillus fumigatus and comparison with clinical strains on gliotoxin production and elastase activity.

UNLABELLED: The aim of this manuscript was to study the influence of water activity (aW ) and pH in the ecophysiological behaviour of Aspergillus fumigatus strains at human body temperature. In addition, gliotoxin production and enzymatic ability among environmental (n = 2) and clinical (n = 5) strains were compared. Ecophysiological study of environmental strains was performed on agar silage incubated at 37°C, studying the interaction at eight aW levels (0·8, 0·85, 0·9, 0·92, 0·94, 0·96, 0·98 and 0·99) and eight pH levels (3·5, 4, 4·5, 5, 6, 7, 7·5 and 8). Considering the influence of the assumed lung conditions on growth of A. fumigatus (aW 0·98/0·99 and pH of 7/7·5), the optimal condition for the development of A. fumigatus RC031 was at aW 0·99 at pH 7. At aW 0·98/0·99 and pH of 7/7·5, the highest growth rate and the lowest lag phase was reported, whereas there were no significant differences at aW 0·98/0·99 and pH 7/7·5 interactions on growth of A. fumigatus RC032. Gliotoxin production of A. fumigatus strains was evaluated. The gliotoxin production was similar in clinical and environmental strains. Elastin activity was studied in solid medium, highest elastase activity index was found for clinical strain A. fumigatus RC0676, followed by the environmental strain A. fumigatus RC031. Opportunistic environmental strains can be considered as pathogenic in some cases when rural workers are exposed constantly to handling silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus fumigatus is one of the main opportunist pathogen agents causing invasive aspergillosis. Rural workers present a constant exposition to A. fumigatus spores caused by feed-borne manipulation. In this study, environmental A. fumigatus strains were able to grow and produce gliotoxin onto the studied conditions including the lung ones. Environmental and clinical strains were physiologically similar and could be an important putative infection source in rural workers.

Diversity of black Aspergilli isolated from raisins in Argentina: Polyphasic approach to species identification and development of SCAR markers for Aspergillus ibericus.

Aspergillus section Nigri is a heterogeneous fungal group including some ochratoxin A producer species that usually contaminate raisins. The section contains the Series Carbonaria which includes the toxigenic species Aspergillus carbonarius and nontoxigenic Aspergillus ibericus that are phenotypically undistinguishable. The aim of this study was to examine the diversity of black aspergilli isolated from raisins and to develop a specific genetic marker to distinguish A. ibericus from A. carbonarius. The species most frequently found in raisins in this study were Aspergillus tubingensis (35.4%) and A. carbonarius (32.3%), followed by Aspergillus luchuensis (10.7%), Aspergillus japonicus (7.7%), Aspergillus niger (6.2%), Aspergillus welwitschiae (4.6%) and A. ibericus (3.1%). Based on inter-simple sequence repeat (ISSR) fingerprinting profiles of major Aspergillus section Nigri members, a sequence-characterized amplified region (SCAR) marker was identified. Primers were designed based on the conserved regions of the SCAR marker and were utilized in a PCR for simultaneous identification of A. carbonarius and A. ibericus. The detection level of the SCAR-PCR was found to be 0.01 ng of purified DNA. The present SCAR-PCR is rapid and less cumbersome than conventional identification techniques and could be a supplementary strategy and a reliable tool for high-throughput sample analysis.

Gliotoxin production by Aspergillus fumigatus strains from animal environment. Micro-analytical sample treatment combined with a LC-MS/MS method for gliotoxin determination.

In this study, gliotoxin production by Aspergillus fumigatus strains from animal environment is studied. Moreover, a rapid, easy and environment-friendly micro-analytical sample treatment procedure coupled with LC-MS/MS was applied for the determination of gliotoxin from A. fumigatus cultures. The ability of gliotoxin production by 143 strains was assayed in yeast extract sucrose agar, and 1 ml of chloroform was used for toxin extraction without further clean-up. Mean recoveries at two spiking levels (2500 and 7000 ng/g; n = 6) were 100.3 ± 6.6 % relative SD (RSD) and 92.4 ± 3.8 % RSD. Repeatability and within-laboratory reproducibility for different concentration levels of gliotoxin (25 to 1000 ng/ml; n = 12) ranged from 0.3 to 5.4 % RSD and from 3.9 to 12.7 % RSD, respectively. The detection limit of the analytical method was 3.5 ng/g. The ability for gliotoxin production by A. fumigatus revealed that 61.5 % of the strains were able to produce the toxin at levels ranging from LOQ to 3430.5 ng/g. However, all the tested samples had similar percentages of producing strains (81.8 to 86.6 %). The micro-analytical sample treatment coupled with LC-MS/MS detection is a precise and useful methodology for determining gliotoxin from fungal extracts of A. fumigatus and allows working both fast and safely and also reducing the effect on the environment. This toxin plays a critical role in the pathobiology of A. fumigatus, and its presence in animal environments could affect animal health and productivity; in addition, there are risks of contamination for rural workers during handling and storage of animal feedstuffs.

Physiological behaviour of gliotoxigenic Aspergillus fumigatus sensu stricto isolated from maize silage under simulated environmental conditions.

Environmental conditions play a key role in fungal development. During the silage production process, humidity, oxygen availability and pH vary among lactic-fermentation phases and among different silage sections. The aim of this work was to study the physiological behaviour of gliotoxicogenic Aspergillus fumigatus strains isolated from maize silage under simulated natural physicochemical conditions - different water activities (a(W)), temperatures (Tº), pH and oxygen pressure - on the growth parameters (growth rate and lag phase) and gliotoxin production. The silage was made with the harvested whole maize plant that was chopped and used for trench-type silo fabrication. Water activity and pH of the silage samples were determined. Total fungal counts were performed on Dichloran Rose Bengal Chloramphenicol agar and Dichloran 18% Glycerol agar. The morphological identification of A. fumigatus was performed with different culture media and at different growth temperature to observe microscopic and macroscopic characteristics. Gliotoxin production by A. fumigatus was determined by HPLC. All strains isolated were morphologically identified as A. fumigatus. Two A. fumigatus strains isolated from the silage samples were selected for the ecophysiological study (A. fumigatus sensu stricto RC031 and RC032). The results of this investigation showed that the fungus grows in the simulated natural physicochemical conditions of corn silage and produces gliotoxin. The study of the physiological behaviour of gliotoxigenic A. fumigatus under simulated environmental conditions allowed its behaviour to be predicted in silage and this will in future enable appropriate control strategies to be developed to prevent the spread of this fungus and toxin production that leads to impairment and reduced quality of silage.

Comparison of toxicogenic and immunosuppressive capacity of Aspergillus fumigatus strains isolated from clinical and corn silage samples.

AIMS: To compare clinical and environmental Aspergillus fumigatus strains on their toxicogenic and immunosuppressive capacity. METHODS AND RESULTS: A total of 51 strains of A. fumigatus isolated from clinical and corn silage samples were assayed. All A. fumigatus strains were assayed for gliotoxin production, therefore strains with different gliotoxin capacities and isolated from different sources were selected and assayed for their effects on bovine macrophages and lymphocytes. Spore diffusates (SDs) obtained from all A. fumigatus strains were able to inhibit macrophage phagocytosys, regardless of their gliotoxin production capacity. However, most but not all strains were able to inhibit bactericidal activity. SDs from all A. fumigatus strains reduced lymphocytes viability. The heat treatment was not always able to inhibit the negative effect on immune cells. CONCLUSIONS: There was no difference between clinical and environmental isolates in their toxicogenic and immunosuppressive capacity. Gliotoxin would not be responsible for the immunosuppressive activity observed by the assayed A. fumigatus strains. However, gliotoxin could be present in the SD, together with some other substances. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained suggest that any environmental strain of A. fumigatus is a putative infectious strain. Prevention measures should be applied to control environmental Aspergillus conidia.