Evaluation of Bacillus spp Candidates for Aflatoxin B1 Biodegradation in Broiler Chickens

Poultry health has been traditionally maintained by hygenic measures, vaccinations, and the use of antibiotics. Modern husbandry management considers the use of probiotics as a natural way to protect birds against many everyday pathogens. Different strains of Bacillus spp. have proved to have beneficial effects in poultry production. However, the most used bacterium in commercial probiotics is Lactobacillus, a vegetative cell. In contrast, Bacillus spp are bacterial spores, highly resistant to harsh conditions, which makes them preferable, in some cases, to Lactobacillus because of shelf life and storage conditions. There is published information regarding mycotoxin detoxification by bacteria. Mycotoxins are a common threat for the poultry industry and different management strategies have been implemented to avoid their negative impact in the poultry industry. Additionally, not much information is provided on the effect of mycotoxin on intestinal inflammation. In chapter one, the ability of Bacillus spp. as direct-fed microbials (DFM) to biodegrade aflatoxin B1 (AFB1) by using an in vitro digestive model simulating in vivo conditions was evaluated. The experiment was performed with three groups: a) control feed; b) control feed contaminated with 0.01% AFB1; c) control feed contaminated with 0.01% AFB1 supplemented with 109 spores/g. In vitro digestion time was insufficient to confirm biodegradation of AFB1. In chapter two, two experiments were conducted in broilers to evaluate the effect of 3 concentrations of AFB1 (2, 1.5 or 1 ppm of AFB1) on gastrointestinal leakage and liver bacterial translocation (BT). Results from these experiments suggest that AFB1 does not increase gut leakage. In chapter three, three independent experiments were conducted to evaluate the biodegradation potential of previously selected Bacillus spp. provided as DFM in broiler chickens consuming feed containing different concentrations of AFB1: a) 2 ppm AFB1; b) 1.5 and 1 ppm AFB1; c) 500 ppb and 50 ppb AFB1. Even though the individual isolates incorporated in the DFM showed some in vitro activity to biodegradate AFB1, when administered in the diets at 5 different concentrations of AFB1, no significant performance differences were observed when compared with their respective control diets

Isolation, screening and identification of Bacillus spp. as direct-fed microbial candidates for aflatoxin B1 biodegradation

Objective: To evaluate the ability of Bacillus spp. as direct-fed microbials (DFM) to biodegrade aflatoxin B1 (AFB1) by using an in vitro digestive model simulating in vivo conditions. Methods: Sixty-nine Bacillus isolates were obtained from intestines, and soil samples were screened by using a selective media method against 0.25 and 1.00 μg/mL of AFB1 in modified Czapek-Dox medium. Plates were incubated at 37 °C and observed every two days for two weeks. Physiological properties of the three Bacillus spp. candidates were characterized biochemically and by 16S rRNA sequence analyzes for identification. Tolerance to acidic pH, osmotic concentrations of NaCl, bile salts were tested, and antimicrobial sensitivity profiles were also determined. Bacillus candidates were individually sporulated by using a solid fermentation method and combined. Spores were incorporated into 1 of 3 experimental feed groups: 1) Negative control group, with unmedicated starter broiler feed without AFB1; 2) Positive control group, with negative control feed contaminated with 0.01% AFB1; 3) DFM treated group, with positive control feed supplemented with 109 spores/g. After digestion time (3:15 h), supernatants and digesta were collected for high-performance liquid chromatography fluorescence detection analysis by triplicate. Results: Three out of those sixty-nine DFM candidates showed ability to biodegrade AFB1 in vitro based on growth as well as reduction of fluorescence and area of clearance around each colony in modified Czapek-Dox medium which was clearly visible under day light after 48 h of evaluation. Analysis of 16S-DNA identified the strains as Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus subtilis. The three Bacillus strains were tolerant to acidic conditions (pH 2.0), tolerant to a high osmotic pressure (NaCl at 6.5%), and were able to tolerate 0.037% bile salts after 24 h of incubation. No significant differences (P > 0.05) were observed in the concentrations of AFB1 in neither the supernatants nor digesta samples evaluated by high-performance liquid chromatography with fluorescence detection between positive control or DFM treated groups. Conclusions: In vitro digestion time was not enough to confirm biodegradation of AFB1. Further studies to evaluate the possible biodegradation effects of the Bacillus-DFM when continuously administered in experimentally contaminated feed with AFB1, are in progress

The intestinal barrier as an emerging target in the toxicological assessment of mycotoxins

Mycotoxins, the secondary metabolites of fungal species, are the most frequently occurring natural food contaminants in human and animal diets. Risk assessment of mycotoxins focused as yet on their mutagenic, genotoxic and potential carcinogenic effects. Recently, there is an increasing awareness of the adverse effects of various mycotoxins on vulnerable structures in the intestines. In particular, an impairment of the barrier function of the epithelial lining cells and the sealing tight junction proteins has been noted, as this could result in an increased translocation of luminal antigens and pathogens and an excessive activation of the immune system. The current review aims to provide a summary of the available evidence regarding direct effects of various mycotoxins on the intestinal epithelial barrier. Available data, based on different cellular and animal studies, show that food-associated exposure to certain mycotoxins, especially trichothecenes and patulin, affects the intestinal barrier integrity and can result in an increased translocation of harmful stressors. It is therefore hypothesized that human exposure to certain mycotoxins, particularly deoxynivalenol, as the major trichothecene, may play an important role in etiology of various chronic intestinal inflammatory diseases, such as inflammatory bowel disease, and in the prevalence of food allergies, particularly in children. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00204-016-1794-8) contains supplementary material, which is available to authorized users