Effects of a mixed additive based on Saccharomyces cerevisiae and Lactobacillus rhamnosus on broilers exposed to aflatoxin B1 by contaminated feed.

The objective of the present study was to explore the influence of dietary supplementation with a mixed additive (MA) containing a probiotic and anti-mycotoxin (Saccharomyces cerevisiae RC016 and Lactobacillus rhamnosus RC007) and its interaction on the performance and health (biochemistry and liver/intestine histopathology) of broilers fed diets contaminated with aflatoxin B1 (AFB1) at 506000±22.1ng/kg. The MA contained S. cerevisiae RC016 (1×107cells/g) and L. rhamnosus RC007 (1×108cells/g) in relation 1:1. A total of sixty-one-day-old Cobb broilers were randomly allocated into four treatment groups with three replicates of 5 birds each for a five-week-old feeding experiment. The experimental diet for each treatment (T) was formulated as follows: T1, a commercial diet (CD); T2, CD+AFB1; T3, CD+0.1% MA; T4, CD+AFB1+0.1% MA. The MA improved (p<0.01) production parameters (weight gain, conversion rate, and carcass yield) and reduced (p<0.01) the toxic effect of AFB1 on the relative weight of the livers. In addition, the macro and microscopic alterations of livers and the possible intestinal injury related to histological damage in the presence of mycotoxin were reduced. The use of probiotic MA based on S. cerevisiae RC016 and L. rhamnosus RC007 in animal feed provides greater protection against mycotoxin contamination and is safe for use as a supplement in animal feed, providing beneficial effects that improve animal health and productivity. This is of great importance at the economic level for the avian production system.

Inhibitory Effect of GRAS Essential Oils and Plant Extracts on the Growth of Aspergillus westerdijkiae and Aspergillus carbonarius Strains.

The effect of essential oils (obtained using hydrodistillation) and plant extracts (ethanolic, aqueous, and hexanic extractions) of 10 different plants cultivated in Brazil were tested using the diffusion agar method, with the objective of evaluating the inhibitory effect of the oils and extracts on the mycelial growth of Aspergillus westerdijkiae NRRL 3174 and A. carbonarius RC 2054 (UNRC). Of the 40 essential oils and plant extracts analyzed, oregano essential oil and plant extract, rosemary essential oil, and the clove ethanolic extract were the best choice to obtain the growth parameters (radial growth rates (mm day-1) and lag phase (h)) due the good results presented and the volume of oil/extract obtained. Comparing all the essential oils and plant extracts that were tested for growth parameters, the best results were obtained for the clove ethanolic extract for both strains assayed. These results demonstrated an outstanding potential use of some of these products in prevention of fungal contamination in food. However, further studies need to be conducted to determine the ability of these oils and extracts to inhibit or reduce ochratoxin A production.

Intestinal toxicity of deoxynivalenol is limited by Lactobacillus rhamnosus RC007 in pig jejunum explants.

Probiotics have been explored to stimulate gut health in weaned pigs, when they started to consume solid diet where mycotoxins could be present. The aim of this study was to evaluate the effect of Lactobacillus rhamnosus RC007 on the intestinal toxicity of deoxynivalenol (DON) in an ex vivo model. Jejunal explants, obtained from 5-week-old crossbred castrated male piglets, were kept as control, exposed for 3 h to 10 µM DON, incubated for 4 h with 109 CFU/mL L. rhamnosus, or pre-incubated 1 h with 109 L. rhamnosus and exposed to DON. Histological lesions were observed, para- and transcellular intestinal permeability was measured in Ussing chambers. The expression levels of mRNA encoding six inflammatory cytokines (CCL20, IL-10, IL-1ß, TNFα, IL-8 and IL-22) were determined by RT-PCR. The expressions of the phosphorylated MAP kinases p42/p44 and p38 were assessed by immunoblotting. Exposure to DON induced histological changes, significantly increased the expression of CCL20, IL-1ß, TNFα, IL-8, IL-22 and IL-10, increased the intestinal paracellular permeability and activated MAP kinases. Incubation with L. rhamnosus alone did not have any significant effect. By contrast, the pre-incubation with L. rhamnosus reduced all the effects of DON: the histological alterations, the pro-inflammatory response, the paracellular permeability and the phosphorylation of MAP kinases. Of note, L. rhamnosus did not adsorb DON and only slightly degrade the toxin. In conclusion, L. rhamnosus RC007 is a promising probiotic which, included as feed additive, can decrease the intestinal toxicity of DON.

Safety assessment of essential oil from Minthostachys verticillata (Griseb.) Epling (peperina): 90-days oral subchronic toxicity study in rats.

Minthostachys verticillata (Lamiaceae), popularly known as peperina is largely used in popular medicine for its digestive, carminative, antispasmodic and antirheumatic properties. There are no reports of repeated exposure toxicity to guarantee their safety. The present study investigated the chemical composition, analyzed by GC-FID, and the 90-day toxicity and genotoxicity effect of M. verticillata essential oil (Mv-EO), using Wistar rats as test animals. The rats were divided into four groups (5 rats/sex/group) and Mv-EO was administered on diet at doses of 0, 1, 4 and 7 g/kg feed. The main components of Mv-EO were pulegone (64.65%) and menthone (23.92%). There was no mortality, adverse effects on general conditions or changes in body weight, food consumption and feed conversion efficiency throughout the study in male and female rats. Subchronic administration of Mv-EO did not alter the weights, morphological and histopathological analyses of liver, kidney and intestine. Genotoxicity was tested by micronucleus and comet assays. Mv-EO up to a concentration of 7 g/kg feed for 90 days did not exert a cyto-genotoxic effect on the bone marrow and cells blood of Wistar rats. These results suggest that Mv-EO appears to be safe and could be devoid of any toxic risk.

In Vitro Activity of Neem (Azadirachta indica) Oil on Growth and Ochratoxin A Production by Aspergillus carbonarius Isolates.

Aspergillus carbonarius is a saprobic filamentous fungus, food spoiling fungus and a producer of ochratoxin A (OTA) mycotoxin. In this study, the in vitro antifungal activity of neem oil (0.12% p/p of azadirachtin) was evaluated against the growth of six strains of A. carbonarius and the production of OTA. Four different concentrations of neem oil were tested in addition to three incubation times. Only the concentration of 0.3% of neem oil inhibited more than 95% of the strain's growth (97.6% ± 0.5%), while the use of 0.5% and 1.0% of neem oil showed lower antifungal activity, 40.2% ± 3.1 and 64.7% ± 1.1, respectively. There was a complete inhibition of OTA production with 0.1% and 0.3% neem oil in the four strains isolated in the laboratory from grapes. The present study shows that neem essential oil can be further evaluated as an auxiliary method for the reduction of mycelial growth and OTA production.

Pediococcus acidolactici and Pediococcus pentosaceus isolated from a rainbow trout ecosystem have probiotic and ABF1 adsorbing/degrading abilities in vitro.

Probiotics are being used in biological control of bacterial pathogens, as an alternative to antibiotics, to improve health and production parameters in fish farming. Fish farming production is severely affected by aflatoxins (AFs), which are a significant problem in aquaculture systems. Aflatoxins exert substantial impact on production, causing disease with high mortality and a gradual decline of reared fish stock quality. Some aspects of aflatoxicosis in fish, particularly its effects on the gastrointestinal tract, have not been well documented. The aim of the present study was to evaluate probiotic properties of lactic acid bacterial (LAB) strains isolated from rainbow trout intestine and feed. Moreover, AFB1-binding and/or degrading abilities were also evaluated to assess their use in the formulation of feed additives. Growth at pH 2, the ability to co-aggregate with bacterial pathogens, inhibition of bacterial pathogens, and determination of the inhibitory mechanism were tested. Aflatoxin B1 (AFB1) adsorption and degradation ability were also tested. All strains were able to maintain viable (107 cells ml-1) at pH 2. Pediococcus acidilactici RC001 and RC008 showed the strongest antimicrobial activity, inhibiting all the pathogens tested. The strains produced antimicrobial compounds of different nature, being affected by different treatments (catalase, NaOH and heating), which indicated that they could be H2O2, organic acids or proteins. All LAB strains tested showed the ability to coaggregate pathogenic bacteria, showing inhibition percentages above 40%. Pediococcus acidilactici RC003 was the one with the highest adsorption capacity and all LAB strains were able to degrade AFB1 with percentages higher than 15%, showing significant differences with respect to the control. The ability of some of the LAB strains isolated in the present work to compete with pathogens, together with stability against bile and gastric pH, reduction of bioavailability and degradation of AFB1, may indicate the potential of LAB for use in rainbow trout culture.

Potentiation of the effect of a commercial animal feed additive mixed with different probiotic yeast strains on the adsorption of aflatoxin B1.

This study potentiates the adsorbent effect for aflatoxin B1 (AFB1) of a commercial additive (CA) of animal feed, containing inactive lysate of three Saccharomyces cerevisiae strains, active enzymes, adsorbents and a selenium-amino acid complex, when the additive was mixed separately with three S. cerevisiae strains. Levels of AFB1 of 20 and 50 ng g(-1) were used to determine the binding capacity of different concentrations of CA alone and in the presence of yeast strains, as well as toxin desorption, under gastrointestinal conditions. The viability of yeasts in the presence of CA was evaluated. The results show that the CA did not affect the viability of the yeast strains assayed. CA alone showed a low percentage adsorption. At 20 and at 50 ng g(-1), CA was highly efficient in adsorbing AFB1 when combined with RC016 and RC012 strains respectively. Desorption of AFB1 by CA alone and in combination with the yeasts increased with increasing levels of CA. The results demonstrate the improvement of CA in AFB1 adsorption once it is mixed with live yeasts.

Mycobiota and mycotoxins contamination in raw materials and finished feed intended for fattening pigs production in eastern Argentina.

The aim of the present study was to determine the mycobiota and natural levels of mycotoxins such as zearalenone, fumonisin B(1), aflatoxin B(1) and ochratoxin A present in raw materials and finished fattening pig feed. Samples were examined for total fungi and genera distribution. Zearalenone, FB(1), AFB(1) and OTA contamination were determined using high pressure liquid chromatography and thin layer chromatography. Milled maize and finished feed samples showed fungal contamination over than 1 × 10(4) CFU/g. All samples contained at least one of the main mycotoxigenic genera Aspergillus, Fusarium and Penicillium. A. flavus and F. verticillioides were the most prevalent species. Only some Aspergillus section Nigri strains from suckling pig to growing pig samples were able to produce OTA. A. flavus strains from milled maize, wheat bran, suckling pig to growing pig samples were able to produce AFB(1). All samples were positive for FB(1). Sucking pig, piglet, growing and boar feed samples showed ZEA natural contamination. AFB(1) and OTA contamination were not detected. There was a 100% correlation between FB(1) and ZEA contamination in sucking pig, piglet, growing and boar feed samples; 50% piglet samples and 67% suckling pig samples showed ZEA levels over the recommended limits. The present study has shown the occurrence of two mycotoxins, FB(1) and ZEA in feed intended for fattening pig consumption. In animal production, the simultaneous presence of toxicogenic fungi and low dietary levels of mycotoxins in field conditions can cause possible health impacts and lost performance in pigs from feeding spoiled feeds.

Fungi and mycotoxins in feed intended for sows at different reproductive stages in Argentina.

The aim of this study was to evaluate fungi and contamination levels of aflatoxin B(1), ochratoxin A, fumonisin B(1), and zearalenone in raw materials and finished feed intended for sows at different reproductive stages. Total fungi, Aspergillus, Penicillium, and Fusarium species occurrence, were examined. Aspergillus flavus, A. niger aggregate spp., and F. verticillioides were the prevalent species. Fungal counts exceeded the levels proposed as feed hygienic quality limits (1 x 10(4) colony forming units) at all reproductive stages. Aflatoxin B(1), ochratoxin A, fumonisin B(1), and zearalenone were detected by high-pressure liquid chromatography. Aflatoxin levels in 80% samples of finished sow feeds were over the permitted levels of 0.02 mug g(-1) (mean 228.2 +/- 95 mug Kg(-1)). Fumonisin B(1) was detected in all tested raw materials at levels that varied from 50.3 to 1137.64 mug Kg(-1) and finished feed samples at levels that ranged from 99.8 to 512.4 mug Kg(-1). Aflatoxin B(1), zearalenone, and ochratoxin A were not detected in raw materials. All finished feeds were negative for zearalenone contamination whereas all nonpregnant gilt samples were contaminated with low OTA levels (mean 0.259 +/- 0.123). This fact requires periodic monitoring to prevent the occurrence of mycotoxicosis in animal production, to reduce the economic losses, and to minimize hazards to human health.

Rhizobacteria and their potential to control Fusarium verticillioides: effect of maize bacterisation and inoculum density.

Fusarium verticillioides is the most important seed transmitted pathogen that infects maize. It produces fumonisins, toxins that have potential toxicity for humans and animals. Control of F. verticillioides colonisation and systemic contamination of maize has become a priority area in food safety research. The aims of this research were (1) to characterise the maize endorhizosphere and rhizoplane inhabitant bacteria and Fusarium spp., (2) to select bacterial strains with impact on F. verticillioides growth and fumonisin B1 production in vitro, (3) to examine the effects of bacterial inoculum levels on F. verticillioides root colonisation under greenhouse conditions. Arthrobacter spp. and Azotobacter spp. were the predominant genera isolated from maize endorhizosphere and rhizoplane at the first sampling period, whilst F. verticillioides strains showed the greatest counts at the same isolation period. All F. verticillioides strains were able to produce fumonisin B1 in maize cultures. Arthrobacter globiformis RC5 and Azotobacter armeniacus RC2, used alone or in a mix, demonstrated important effects on F. verticillioides growth and fumonisin B1 suppression in vitro. Only Azotobacter armeniacus RC2 significantly reduced the F. verticillioides root colonisation at 10(6) and 10(7) CFU g(-1) levels under greenhouse conditions.

Radiação gama sobre a micobiota de ração avícola e Aspergillus spp / Gamma radiation on the mycoflora of poultry feed and Aspergillus species

O objetivo deste experimento foi investigar o efeito da radiação gama sobre a micobiota natural de ingredientes e ração avícola e seu impacto sobre espécies de Aspergillus spp. Para tanto, fubá, farelo de soja e ração foram expostos a uma fonte de césio-137 nas doses 0; 3,5; 8 e 15kGy. Amostras de milho, inoculadas com suspensão de conídios de Aspergillus spp., foram irradiadas com doses de 0 a 8kGy. Observou-se redução das contagens com o aumento da dose, obtendo-se eliminação da micobiota com 8kGy. Uma maior radiossensibilidade de leveduras foi observada em comparação a fungos filamentosos, e aqueles que resistiram à dose de 3,5kGy pertenciam, em sua maioria, aos gêneros Cladosporium spp., Curvularia spp., Fusarium spp. e Aspergillus spp. As espécies A. flavus e A. parasiticus mostraram-se mais radiorresistentes que as demais avaliadas. Redução na conidiogênese e presença de estruturas de resistência foram detectadas logo após a irradiação. Constatou-se também que, após repiques em meios nutritivos, os isolados irradiados recuperaram a taxa de crescimento normal. Verificou-se ainda que o estresse metabólico induzido pela irradiação promoveu aumento na produção de aflatoxina B1 e ocratoxina A.

This experiment investigated the effects of gamma radiation on the mycoflora of natural ingredients and poultry feed, and its impact on species of Aspergillus spp. Corn meal, soybean meal, feed and maize (inoculated with spore suspension of Aspergillus spp.) were irradiated with 0, 3.5, 8 and 15kGy by exposure to a 137Cs source. Increasing radiation doses led to lower counts so elimination of mycoflora was observed at 8kGy. In addition, filamentous fungi exhibited a greater resistance to radiation than yeasts and those that withstood 3.5kGy were mostly Cladosporium spp., Curvularia spp., Fusarium spp. or Aspergillus spp. A. flavus and A. parasiticus have been found to be the most resistant among the species studied. Post-irradiation reduction in the spore production as well as presence of resistance structures have been observed. When such altered colonies were isolated and grown on nutritional media again, they recovered their normal growth pattern. In addition, a significant increase in the production of aflatoxin B1 and ochratoxin A was found, probably due to metabolic stress caused by irradiation.