Hamid Boudra

Binding of Fusarium mycotoxins by fermentative bacteria in vitro.

Aims:  Fusarium toxins can occur in conserved forages impairing farm animal performances and health. On‐farm biological decontamination methods could be an alternative to traditional physico‐chemical methods. In this work, the ability to remove Fusarium toxins by fermentative bacteria was evaluated in vitro.

Cell wall component and mycotoxin moieties involved in the binding of fumonisin B1 and B2 by lactic acid bacteria

Aims:  The ability of lactic acid bacteria (LAB) to bind fumonisins B1 and B2 (FB1, FB2) in fermented foods and feeds and in the gastrointestinal tract could contribute to decrease their bioavailability and toxic effects on farm animals and humans. The aim of this work was to identify the bacterial cell wall component(s) and the functional group(s) of FB involved in the LAB–FB interaction.

Screening of fermentative bacteria for their ability to bind and biotransform deoxynivalenol, zearalenone and fumonisins in an in vitro simulated corn silage model

Fermentative bacteria can potentially be utilized to detoxify corn silage contaminated by Fusarium toxins. The objective of the present study was to test a large number of these bacteria for their ability to bind and/or biotransform deoxynivalenol (DON), zearalenone (ZEN) and fumonisins B1 and B2 (FB1, FB2) in conditions simulating corn silage. A total of 202 strains were screened in contaminated, pH 4, corn infusion inoculated with 5 × 108 CFU ml–1. Eight Lactobacilli and three Leuconostoc biotransformed ZEN into α-zearalenol, but no biotransformation was detected for DON and fumonisins. In contrast, most strains were capable of binding Fusarium toxins. The most effective genera were Streptococcus and Enterococcus, capable of binding up to 33, 49, 24 and 62% of DON, ZEN, FB1 and FB2, respectively. The ability to bind Fusarium toxins seems to be a common property of fermentative bacteria and could help to decrease their toxicity in animals.

Fungal secondary metabolites from Monascus spp. reduce rumen methane production in vitro and in vivo1

Decreasing methanogenesis without affecting fermentation and digestion of feeds in the rumen can reduce the environmental impact of ruminant production and have a beneficial effect on feed conversion efficiency. In this work, metabolites produced by Monascus spp. molds were assayed for their antimethanogenic activity in vitro and in vivo. The capacity of 7 strains of Monascus to produce secondary metabolites was assessed in solid media. Monitored metabolites included the statins monacolin K, pravastatin, and mevastatin, and the mycotoxin citrinin. Ethanolic extracts from 5 different solid media from 2 selected strains were tested in vitro. Fermentation was not negatively affected by any treatment, but one extract decreased methane production (P < 0.05). This extract was further assayed in 3 consecutive batch incubations where a marked decrease in methane was observed in the third batch (P < 0.05). In contrast, methane produced in flasks with pure monacolin K was not different from controls (P > 0.05). Rice on which the selected Monascus sp. was grown also decreased methane production when used as substrate for in vitro incubations (P < 0.05). The effect of Monascus-fermented rice on methane production was then assayed in vivo. Six wethers were adapted to a diet containing rice grain and hay (1:1 ratio). Rice was then replaced by fermented rice and given to animals for nearly 2 wk. Animals were monitored for a further 2 wk after the treatment. Daily methane emissions decreased (P < 0.05) by 30% after 2 to 3 d into the treatment and remained low throughout the administration period. This change was associated with reduced ruminal acetate to propionate ratio and decreased numbers of methanogens as detected by quantitative PCR (P < 0.05). In contrast, no changes in the methanogenic community were observed by denaturing gradient gel electrophoresis (DGGE). Total bacteria numbers increased (P < 0.05) with changes in the DGGE profile community, whereas protozoa were not affected by the treatment. Methane emissions and the acetate to propionate ratio remained numerically less in the 2 wk posttreatment as compared with measures before treatment. Metabolites produced by Monascus appear to have an inhibitory effect on methanogens and decreased methanogenesis in vitro and in short-term in vivo without any apparent negative effect on rumen fermentation. This strategy deserves to be further explored and could be an abatement option under certain feeding situations.

Rumen microbial communities influence metabolic phenotypes in lambs

The rumen microbiota is an essential part of ruminants shaping their nutrition and health. Despite its importance, it is not fully understood how various groups of rumen microbes affect host-microbe relationships and functions. The aim of the study was to simultaneously explore the rumen microbiota and the metabolic phenotype of lambs for identifying host-microbe associations and potential biomarkers of digestive functions. Twin lambs, separated in two groups after birth were exposed to practices (isolation and gavage with rumen fluid with protozoa or protozoa-depleted) that differentially restricted the acquisition of microbes. Rumen microbiota, fermentation parameters, digestibility and growth were monitored for up to 31 weeks of age. Microbiota assembled in isolation from other ruminants lacked protozoa and had low bacterial and archaeal diversity whereas digestibility was not affected. Exposure to adult sheep microbiota increased bacterial and archaeal diversity independently of protozoa presence. For archaea, Methanomassiliicoccales displaced Methanosphaera. Notwithstanding, protozoa induced differences in functional traits such as digestibility and significantly shaped bacterial community structure, notably Ruminococcaceae and Lachnospiraceae lower up to 6 folds, Prevotellaceae lower by ~40%, and Clostridiaceae and Veillonellaceae higher up to 10 folds compared to microbiota without protozoa. An orthogonal partial least squares-discriminant analysis of urinary metabolome matched differences in microbiota structure. Discriminant metabolites were mainly involved in amino acids and protein metabolic pathways while a negative interaction was observed between methylotrophic methanogens Methanomassiliicoccales and trimethylamine N-oxide. These results stress the influence of gut microbes on animal phenotype and show the potential of metabolomics for monitoring rumen microbial functions.

Effect and stability of gliotoxin, an Aspergillus fumigatus toxin, on in vitro rumen fermentation

Aspergillus fumigatus is a toxicogenic fungus usually found in contaminated animal feeds, especially in conserved forages where it can produce several mycotoxins. Gliotoxin, one of the most important toxic metabolites produced by this fungus, has antibacterial, immunosuppressive and apoptotic effects. Ruminants due to the high proportion of forages they receive in the ration would be particularly exposed to gliotoxin. The objective of this work was (1) to assess the effect of gliotoxin on in vitro rumen fermentation and (2) to determine the effect of fermentation on gliotoxin stability. Gliotoxin did not affect rumen fermentation at concentrations found in naturally contaminated feeds. No effects were observed up to a concentration of 20 μg toxin ml−1 and an extremely high toxin concentration (80 μg ml−1) was necessary to affect dry matter degradation, gas and total volatile fatty acids production by 24, 37 and 18%, respectively (p<0.01). In addition, the toxin was unstable in the rumen environment with 90% disappearance at 6 h of incubation (p<0.05). In contrast, extracts of A. fumigatus cultures containing gliotoxin at concentrations several times lower than that used for experiments with pure toxin had a negative effect on fermentations indicating the toxicity and possible synergism of other metabolites produced by this fungus. Extracts containing 8.8 μg gliotoxin ml−1 decreased dry matter degradation, gas and volatile fatty acids production by 28, 46 and 35%, respectively (p<0.01). Identification of these toxic metabolites and assessment of the rate of passage of gliotoxin to the lower intestinal tract is necessary to evaluate the potential risk of these toxins to ruminants.

Effectiveness of modified yeast cell wall extracts to reduce aflatoxin B1 absorption in dairy ewes

This study investigated the effect of a modified yeast cell wall extract preparation (YCW) on the excretion of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) in feces, urine, and milk of dairy ewes fed an aflatoxin-contaminated diet. Sixteen ewes in mid-lactation were assigned to 4 treatment groups: control, AF (60 μg of AFB1/kg of feed), YCW (2 g/kg of feed), and AF+YCW. The trial consisted of a short-term (3-d) exposure period followed by a long-term (21-d) exposure period. At the end of each exposure period, milk, urine, and feces were collected over 72 h. The treatments did not affect feed intake, milk production, milk composition, or body weight. The presence of AFM1 was detected in all matrices, whereas AFB1 was only present in feces. Daily excretion was higher following long-term exposure and reached 26.9 μg of AFB1/d in feces, 37.2 μg of AFM1/d in feces, and 10.7 μg of AFM1/d in urine. Supplementation with YCW was effective in increasing aflatoxin excretion in feces in the long-term exposure (up to 156% increase). The effect was accompanied by a trend of decreasing urinary excretion of AFM1. In contrast, the addition of YCW to the contaminated diet did not affect the transfer of aflatoxins from feed to milk under the present experimental conditions with low-producing ewes. The transfer rates of AFM1 in milk ranged from 0.24 to 0.54%. In conclusion, feed supplementation with YCW reduced the absorption of AFB1 and increased the elimination of AFB1 and AFM1 in ewe feces. Yeast cell wall extract could be used to protect ruminants from chronic exposure to aflatoxins present in feeds.

Dietary carbohydrate composition modifies the milk N efficiency in late lactation cows fed low crude protein diets.

Nitrogen emissions from dairy cows can be readily decreased by lowering the dietary CP concentration. The main objective of this work was to test whether the milk protein yield reduction associated with low N intakes could be partially compensated for by modifying the dietary carbohydrate composition (CHO). The effects of CHO on digestion, milk N efficiency (milk N/N intake; MNE) and animal performance were studied in four Jersey cows fed 100% or 80% of the recommended protein requirements using a 4×4 Latin square design. Four iso-energetic diets were formulated to two different CHO sources (starch diets with starch content of 34.3% and NDF at 32.5%, and fiber diets with starch content of 5.5% and NDF at 49.1%) and two CP levels (Low=12.0% and Normal=16.5%). The apparent digestible organic matter intake (DOMI) and the protein supply (protein digestible in the small intestine; PDIE) were similar between starch and fiber diets. As planned, microbial N flow (MNF) to the duodenum, estimated from the urinary purine derivatives (PD) excretion, was similar between Low and Normal CP diets. However, the MNF and the efficiency of microbial synthesis (g of microbial N/kg apparently DOMI) were higher for starch v. fiber diets. Milk and milk N fractions (CP, true protein, non-protein N (NPN)) yield were higher for starch compared with fiber diets and for Normal v. Low CP diets. Fecal N excretion was similar across dietary treatments. Despite a higher milk N ouput with starch v. fiber diets, the CHO modified neither the urinary N excretion nor the milk urea-N (MUN) concentration. The milk protein yield relative to both N and PDIE intakes was improved with starch compared with fiber diets. Concentrations of β-hydroxybutyrate, urea and Glu increased and those of glucose and Ala decreased in plasma of cows fed starch v. fiber diets. On the other hand, plasma concentration of albumin, urea, insulin and His increased in cows fed Normal compared with Low CP diets. This study showed that decreasing the dietary CP proportion from 16.5% to 12.0% increases and decreases considerably the MNE and the urinary N excretion, respectively. Moreover, present results show that at similar digestible OM and PDIE intakes, diets rich in starch improves the MNE and could partially compensate for the negative effects of Low CP diets on milk protein yield.

Time ofAspergillus flavus infection and aflatoxin formation in ripening of figs

Figs in an orchard were inoculated with an aflatoxigenicAspergillus flavus strain in two ways by spore injection or by dusting at three maturation stages: firm ripe, shrivelled, and dried. Fruits were individually examined for fungal development and analyzed for aflatoxin B1 (AF B1) after 2, 4, 6, 8 and 10 days. Fruit injected at the first stage showed fungal development and AF B1 contamination within two days. The toxin level increased sharply to 1 ppm after 10 days. The mean level of AF B1 (284.75 ng/g) was significantly higher than those observed in other conditions. Figs dusted at the first stage showed only a tiny fungal growth even after 10 days. AF B1 appeared after 6 days with a low frequency (35%), mean level (7.6 ng/g) and a great variation among figs (0.22–15 ng/g). Among fruits inoculated during the shrivelled fig and dried fruit stages, no fungal growth was observed and AF B1 was detected with a lower incidence in association with low mean levels (less than 1.25 ng/g). Methods of prevention of aflatoxin contamination at the critical step, the firm ripe stage, are discussed.

Simultaneous analysis of the main markers of nitrogen status in dairy cow's urine using hydrophilic interaction chromatography and tandem mass spectrometry detection

Microbial protein synthesis and nitrogen balance status in ruminants can be evaluated by the presence of metabolites in urine. This work aims to develop and validate a simple and sensitive method for simultaneous determination of nine markers of nitrogen status in ruminants urine using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS system, operated in multiple reaction monitoring (MRM) in positive mode, was used for determining selected purine (allantoin, uric acid, xanthine, and hypoxanthine) and pyrimidine derivatives (β-aminobutyric acid and β-alanine), which are used to estimate rumen microbial protein synthesis - the main source of protein for ruminants. Creatinine, creatine and urea, three other metabolites involved in nitrogen metabolism were also measured by this method. The procedure was based on a simple dilution of urine samples in acetonitrile, followed by LC-ESI-MS/MS analysis. Chromatographic separation was tested with three different columns. A zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) column provided an optimal separation for all metabolites. Precision of the method was typically below 10%, and accuracy was above 90% with the exceptions of allantoin and urea at pH 6 and 3, and β-alanine at pH 3. Metabolites were stable after 3 months of storage at -20°C, except for xanthine, hypoxanthine and β-aminobutyric acid that lost up to 48, 50 and 39% of initial concentration after only 1month of storage in acidified urine. This LC-ESI-MS/MS method is more specific, unequivocally detecting target metabolites at lower detection limits than methods using UV detection. The method was suitable for the determination of all metabolites tested. The developed method was subsequently used to compare total and spot urine sampling obtained from dairy cows fed diets with contrasting levels of protein.