Spot urine collection: A valid alternative to total urine collection for metabolomic studies in dairy cattle.

Urine is a highly suitable biological matrix for metabolomics studies. Total collection for 24-h periods is the gold standard as it ensures the presence of all metabolites excreted throughout the day. However, in animal studies, it presents limitations related to animal welfare and also due to alterations of the metabolome originating from the use of acid for preventing microbial growth or microbial contamination. In this study, we investigated whether spot urine collection is a practical alternative to total collection for metabolomic studies in lactating cows. For this purpose, we collected urine samples from 4 lactating Holstein cows fed 4 diets in a 4 × 4 Latin square design. Urine was collected for 24 h using a collecting device (i.e., total collection) or collected once per day 4 h after the morning feeding (i.e., spot urine collection). Dietary treatments differed by the amount of nitrogen content (high vs. low) and by the nature of the energy (starch vs. fiber). Urine metabolome was analyzed by 2 untargeted complementary methods, nuclear magnetic resonance and hydrophilic-interaction liquid chromatography (HILIC) coupled to a time-of-flight mass spectrometer, and by 1 targeted method, HILIC-tandem mass spectrometry. Although sampling technique had an effect on the abundance of metabolites detected, spot urine samples were equally capable of showing differences in urine metabolome than samples from total collection. When considering nitrogen levels in the diet, the robustness and precision for discriminating high- and low-nitrogen diets was equally achieved with both sampling techniques. A total of 22 discriminant metabolites associated with the N level of diets were identified from untargeted HILIC coupled to a time-of-flight mass spectrometer (n = 9) and nuclear magnetic resonance (n = 11), and 2 from targeted HILIC-tandem mass spectrometry. Alternatively, starch or fiber in the diet induced less changes in the metabolome that were not clearly discriminated independently of the sampling technique. We concluded that spot urine collection can successfully reveal differences in the urine metabolome elicited by dietary N levels and be used as a substitute of total urinary 24-h collection for metabolomic studies.

Bioavailability of aflatoxin B1 and ochratoxin A, but not fumonisin B1 or deoxynivalenol, is increased in starch-induced low ruminal pH in nonlactating dairy cows.

High-production dairy and beef systems require diets rich in starch. This practice may induce ruminal acidosis and also increase exposure to mycotoxins because starches in starch-rich diets are the main vehicles of mycotoxin contamination. The aim of this study was to investigate the effects of low ruminal pH on the bioavailability of 4 major mycotoxins [i.e., aflatoxin B1 (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), and fumonisin B1 (FB1)]. Eight nonlactating dairy cows fitted with rumen cannulas were used in a double crossover experiment. The trial was divided into 4 periods with 2 periods per crossover. Cows were divided into 2 groups receiving a low (15% dry matter basis) and high-starch diet (30.8%) with and without live yeast supplementation (1×1010 cfu per cow) in the first and second crossover, respectively. At the end of each period, cows received a single dose of mycotoxin-contaminated feed containing 0.05, 0.2, 0.24, and 0.56mg of AFB1, OTA, DON, and FB1 per kg of feed, respectively. The fecal and urinary excretion of mycotoxins and their metabolites was monitored for up to 48h postdosing. As expected, ruminal pH decreased in cows fed the high-starch diet. The high-starch diet increased the bioavailability of OTA and AFB1. Urinary excretion of OTA 24h after mycotoxin administration increased 3-fold in the high-starch diet, correlated with lower fecal excretion. Similarly, a decrease in fecal excretion of AFB1 was accompanied by an increase in urinary excretion of its major metabolite, aflatoxin M1, 48h after mycotoxin administration. In contrast to AFB1 and OTA, the bioavailability of DON and FB1 remained unchanged. Yeast supplementation had no effect on the excretion balance of these 2 mycotoxins. In conclusion, these results show that high-starch diets increased the bioavailability of OTA and AFB1, most probably through the lowering effect on ruminal pH. This greater bioavailability potentially increases the toxic effects of these mycotoxins.

Presence of aflatoxin M1 in raw, reconstituted, and powdered milk samples collected in Algeria.

Aflatoxins are potent toxic metabolites produced by Aspergillus spp. Aflatoxin M1 (AFM1) is a metabolite of aflatoxin B1 that can be present in milk, and it is a public health concern. There is scarce information on the incidence of aflatoxin M1 contamination in milk consumed in Algeria. The presence of AFM1 was investigated in raw milk samples collected between February and October 2011 from 11 dairy farms representative of Algerian production conditions and that were located around Constantine city. Reconstituted and powdered milk samples were purchased from local supermarkets. The analysis was performed by liquid chromatography-fluorescence detection after immunoaffinity purification. AFM1 was detected in 5 out of 47 samples (11 %) at levels ranging from 9 to 103 ng/L, with one sample exceeding the limit of 50 ng/L set by European regulations. Traces of AFM1 (less than 8 ng/L) were also found in 11 other samples. The incidence of AFM1 contamination was higher in imported powdered milk (29 %) than in raw milk (5 %). Although the concentration of AFM1 in contaminated samples was low, the relatively considerable prevalence found in this exploratory study justifies more detailed and continuous monitoring to reduce consumers' exposure to AFM1.

Short communication: Toxicokinetics of ochratoxin A in dairy ewes and carryover to milk following a single or long-term ingestion of contaminated feed.

Ruminal microbes have the capacity to inactivate ochratoxins, rendering ruminants less sensitive to this fungal contaminant found in cereal feeds. However, ochratoxin A has been reported in milk surveys. The objective of this study was to assess the toxicokinetics, excretion, and transmission into milk of ochratoxin A using doses similar to those of naturally occurring field contaminations. Six Lacaune dairy ewes in late lactation were separated into 2 groups that received a single dose of contaminated wheat containing 5 or 30 µg of ochratoxin A/kg of body weight. After administration, toxicokinetics and excretion were monitored for 48 h. Subsequently, ewes were administered the corresponding toxin dose daily for 24 d followed by a second toxicokinetics and excretion monitoring period for this long-term exposure. The doses used did not affect production or health of ewes. After a single dose, ochratoxin A and its main metabolite, ochratoxin α, were found in blood 1h postexposure. The maximum blood concentrations of ochratoxin A and α, respectively, were dose dependent and were observed, on average, 6 and 8h after exposure. Long-term exposure increased the maximum concentration of ochratoxin A detected in blood, whereas ochratoxin α was not affected. In contrast, the time to reach the maximum concentration was reduced to 3h for both molecules. Ochratoxins, essentially ochratoxin α, were mainly excreted in feces. Ochratoxin A and α were detected in milk at concentrations that were dose dependent but with a low carryover rate (<0.02%). Chronic administration did not increase the concentration of toxin in milk. Even though ochratoxin A can escape ruminal degradation and traces were found in milk of experimentally exposed ewes, the low carryover of ochratoxin A in milk minimizes the risk to consumers.

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.

Early life dietary intervention in dairy calves results in a long-term reduction in methane emissions.

Recent evidence suggests that changes in microbial colonization of the rumen prior to weaning may imprint the rumen microbiome and impact phenotypes later in life. We investigated how dietary manipulation from birth influences growth, methane production, and gastrointestinal microbial ecology. At birth, 18 female Holstein and Montbéliarde calves were randomly assigned to either treatment or control (CONT). Treatment was 3-nitrooxypropanol (3-NOP), an investigational anti-methanogenic compound that was administered daily from birth until three weeks post-weaning (week 14). Samples of rumen fluid and faecal content were collected at weeks 1, 4, 11, 14, 23, and 60 of life. Calves were tested for methane emissions using the GreenFeed system during the post-weaning period (week 11-23 and week 56-60 of life). Calf physiological parameters (BW, ADG and individual VFA) were similar across groups throughout the trial. Treated calves showed a persistent reduction in methane emissions (g CH4/d) throughout the post-weaning period up to at least 1 year of life, despite treatment ceasing three weeks post-weaning. Similarly, despite variability in the abundance of individual taxa across weeks, the rumen bacterial, archaeal and fungal structure differed between CONT and 3-NOP calves across all weeks, as visualised using sparse-PLS-DA. Similar separation was also observed in the faecal bacterial community. Interestingly, despite modest modifications to the abundance of rumen microbes, the reductive effect of 3-NOP on methane production persisted following cessation of the treatment period, perhaps indicating a differentiation of the ruminal microbial ecosystem or a host response triggered by the treatment in the early development phase.

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. METHODS AND RESULTS: The effect of physicochemical, enzymatic and genetic treatments of bacteria and the removal/inactivation of the functional groups of FB on toxin binding were evaluated. Treatments affecting the bacterial wall polysaccharides, lipids and proteins increased binding, while those degrading peptidoglycan (PG) partially decreased it. In addition, purified PG from Gram-positive bacteria bound FB in a manner analogue to that of intact LAB. For FB, tricarballylic acid (TCA) chains play a significant role in binding as hydrolysed FB had less affinity for LAB. CONCLUSIONS: Peptidoglycan and TCA are important components of LAB and FB, respectively, involved in the binding interaction. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria binding efficiency seems related to the peptide moiety structure of the PG. This information can be used to select probiotics with increased FB binding efficiency.

Aflatoxin M1 and ochratoxin A in raw bulk milk from French dairy herds.

Mycotoxins in milk are a public health concern and have to be regularly monitored. A survey on the presence of aflatoxin M1 (AFM1) and ochratoxin A (OTA) in raw bulk milk was conducted in 2003 in the northwest of France, the main French milk-producing basin. Randomly selected farms (n = 132) were characterized by a diet based on corn silage and containing a large proportion of on-farm produced cereals, feeding sources that are frequently contaminated by mycotoxins. Farms were surveyed twice in winter and in summer. At each sampling time, a trained surveyor completed a questionnaire recording farm management procedures and production traits. The AFM1 was found in 3 out of 264 samples but at levels (26 ng/L or less) that are below the European legislation limit of 50 ng/L. Traces of AFM1 (less than 8 ng/L) were also found in 6 other samples. The OTA was detected in 3 samples also at low levels, 5 to 8 ng/L. Farms that tested positive to the presence of mycotoxins, 12 in total including 6 farms that had traces of AFM1, differed from negative farms by a more extensive use of total mixed rations, 58 vs. 27%. In addition, the positive farms tended to have lower milk yields. Although the incidence of milk contamination with AFM1 and OTA at the farm level was low during the period studied, production and management data from the surveyed farms suggest a link between feeding management practices and mycotoxin contamination.

Development and validation of a HPLC method for the quantitation of ochratoxins in plasma and raw milk.

A HPLC method with improved sensitivity for the determination of ochratoxins (OT) A, B and alpha in plasma and milk was developed. Plasma analysis involved a simple liquid-liquid extraction with chloroform; while for milk, an additional immunoaffinity clean-up step was necessary. The method showed a good linearity (r(2)>0.999). The limit of quantification (LOQ) of OTA was 5 and 200 ng/l for milk and plasma, respectively. Average recovery was 89% in both matrices, except for OTalpha in milk that was only 18% due to poor immunoaffinity binding. OT remained stable in -20 degrees C stored samples; OTA concentration in plasma and milk did not change after 8 and 18 months of storage, respectively. The developed method has been applied to contaminated plasma and milk samples obtained from dairy ewes fed with ochratoxin-contaminated feed.

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 ß-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.

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

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.

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 ruminant's 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 (ß-aminobutyric acid and ß-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 ß-alanine at pH 3. Metabolites were stable after 3 months of storage at -20°C, except for xanthine, hypoxanthine and ß-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.

Modification of aflatoxin B1 and ochratoxin A toxicokinetics in rats administered a yeast cell wall preparation.

The cell wall of Saccharomyces cerevisiae can bind mycotoxins in vitro, but there is scarce information on whether this property decreases the absorption of mycotoxins in vivo. The effect of a yeast cell wall preparation (YCW) on toxicokinetics and balance excretion (urine and faeces) of aflatoxin B(1) (AFB1) and ochratoxin A (OTA) was tested in rats after oral administration of each toxin. The (3)H-labelled mycotoxins were used at low doses. Co-administration of YCW with AFB1 decreased the extent, but not the rate, of absorption. Concurrently, radioactivity excreted in faeces increased by up to 55% when compared with controls, whilst the excretion in urine decreased (p < 0.05). The effect of YCW on OTA was less marked, although it increased radioactivity excretion in faeces (up to 16%; p < 0.05) it did not result in changes in urine and toxicokinetic parameters. The in vivo effect is in agreement with the reported in vitro binding ability for these toxins (AFB1 > OTA). In conclusion, these results indicate that YCW could be used to protect monogastric animals against exposure to low dietary levels of selected mycotoxins.

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 B(1) and B(2) (FB(1), FB(2)) in conditions simulating corn silage. A total of 202 strains were screened in contaminated, pH 4, corn infusion inoculated with 5 x 10(8) CFU ml(-1). Eight Lactobacilli and three Leuconostoc biotransformed ZEN into alpha-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, FB(1) and FB(2), 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.

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. METHODS AND RESULTS: Twenty-nine strains of lactic (LAB) and propionic acid bacteria (PAB) were tested for their ability to remove deoxynivalenol (DON) and fumonisins B1 and B2 (FB1, FB2) from an acid, pH 4, medium. Mycotoxin removal was widespread for LAB, but differences among strains were large. Removal was up to 55% for DON, 82% for FB1 and 100% for FB2. Selected strains were also capable of removing up to 88% zearalenone. The PAB strains were less efficient than the LAB. Binding, not biodegradation appeared to be the mode of action, as no toxin derivatives were observed and removal was not impaired in nonviable bacteria. Binding was not affected by pH, except for fumonisins that decreased to nearly 0% at neutral pH. CONCLUSIONS: Selected fermentative bacteria are able to bind main Fusarium mycotoxins. SIGNIFICANCE AND IMPACT OF THE STUDY: The binding ability of selected strains could be used to decrease the bioavailability of toxins in contaminated silages.

Thermostability of Ochratoxin A in wheat under two moisture conditions.

The decomposition of ochratoxin A (OTA) was examined, under different temperature and moisture conditions. The calculated half-lives, corresponding to 50% values, were 707, 201, 12, and 6 min, respectively, at 100, 150, 200, and 250 degrees C for dry wheat and 145, 60, and 19 min, respectively, at 100, 150, and 200 degrees C for wheat heated under wet conditions. The presence of water (50%) increased the decomposition of OTA at 100 and 150 degrees C; the opposite was observed at 200 degrees C. Complete destruction of OTA within the limits of this study (100 to 250 degrees C) was not obtained.

Thermostability of Fumonisin B(1), a Mycotoxin from Fusarium moniliforme, in Corn.

Fumonisin B(1) (FB(1)) is a mycotoxin from Fusarium moniliforme that is frequently associated with corn. Thermal treatments are used in many processes concerning this cereal and its derivatives. The thermostability of this toxin in dry contaminated corn, resulting from F. moniliforme culture, was studied in different time-temperature combinations. FB(1) was quantified by instrumentalized thin-layer chromatography after a two-step sequential development and postchromatographic derivatization by p-anisaldehyde. The identity of FB(1) in extracts, before and after heat treatments, was confirmed by high-pressure liquid chromatography. For each temperature, the natural logarithm of the ratio of resulting FB(1) on initial content (In C/C(0)) is linearly correlated to exposure time. The calculated half-lives (L(50)), corresponding to the 50% value, were 10 min, 38 min, 175 min, and 8 h at 150, 125, 100, and 75 degrees C, respectively. There is a linear relationship between calculated L(50)s on a logarithmic scale and temperature. Therefore FB(1) is not significantly destroyed by the main drying processes of corn or thermal treatments used for its derivatives. Other associated means are required for detoxification.

Time of Aspergillus flavus infection and aflatoxin formation in ripening of figs.

Figs in an orchard were inoculated with an aflatoxigenic Aspergillus 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.

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 microg toxin ml(-1) and an extremely high toxin concentration (80 microg 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 microg 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.

Pharmacokinetics, metabolism and excretion of megazol, a new potent trypanocidal drug in animals.

The pharmacokinetics of megazol (CAS 19622-55-0) was investigated after intraperitoneal and oral administration of the drug (80 mg/kg) to mice. The plasma levels were significantly higher after oral administration of drug than after intraperitoneal route (33.8 micrograms/ml compared with 19.0 micrograms/ml for Cmax, 158714 micrograms.h/l compared with 96057 micrograms.h/l for AUC). When suramin (CAS 145-63-1) was administered 24 h before oral administration of megazol, megazol absorption was accelerated (2 h compared with 4 h for Tmax) but the amount absorbed was lower (19.9 micrograms/ml compared with 33.8 micrograms/ml for Cmax and 95547 micrograms.h/l vs 158714 micrograms.h/l for AUC). In the infected mice previously treated with suramin, all estimated pharmacokinetic parameters of plasma megazol were significantly modified, in particularly an increase in the apparent volume of distribution (5.6 l/kg compared with 0.9 l/kg) with a prolongation of the elimination half-life (3 h compared with 0.7 h) of megazol. Excretion of the total radioactivity of megazol was also evaluated after oral administration of 3H-megazol to rats. Total radioactivity was eliminated predominantly via the urinary route (80%) vs. 10.5% in the faeces, 9.5% remaining in the body 8 days after dosing. When unlabelled megazol was orally administered to rats with absence or presence of suramin, megazol recovered in urine and faeces 72 h dosing was: 55.7%/2% vs 20.6%/1.6%, respectively. In the urine, unchanged megazol was present as characterized by LC-MS/MS as well as 4 unknown metabolites. This study indicates that suramin significantly affects the pharmacokinetics of megazol and its elimination.