A mycotoxin-deactivating feed additive counteracts the adverse effects of regular levels of Fusarium mycotoxins in dairy cows.

Little is known about the effects of commonly found levels of Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cattle. We investigated the effects of regular contamination levels, meaning contamination levels that can be commonly detected in dairy feeds, of deoxynivalenol (DON) and fumonisins (FB) in total mixed ration (TMR) on the performance, diet digestibility, milk quality, and plasma liver enzymes in dairy cows. This trial examined 12 lactating Holstein dairy cows using a 3-period × 3-treatment Latin square design. The experimental period was 21 d of mycotoxin exposure followed by 14 d of washout. During treatment periods, cows received one of 3 diets: (1) CTR (control) diet of TMR contaminated with 340.5 µg of DON/kg of dry matter (DM) and 127.9 µg FB/kg of DM; (2) MTX diet of TMR contaminated with Fusarium mycotoxins at levels higher than CTR but below US and European Union guidelines (i.e., 733.0 µg of DON/kg of DM and 994.4 µg of FB/kg of DM); or (3) MDP diet, which was MTX diet supplemented with a mycotoxin deactivator product (i.e., 897.3 µg of DON/kg of DM and 1,247.1 µg of FB/kg of DM; Mycofix, 35 g/animal per day). During washout, all animals were fed the same CTR diet. Body weight, body condition score, DM intake, dietary nutrient digestibility, milk production, milk composition and rennet coagulation properties, somatic cell count, blood serum chemistry, hematology, serum immunoglobulin concentrations, and expression of multiple genes in circulating leucocytes were measured. Milk production was significantly greater in the CTR group (37.73 kg/d) than in the MTX (36.39 kg/d) and the MDP (36.55 kg/d) groups. Curd firmness and curd firming time were negatively affected by the MTX diet compared with the other 2 diets. Furthermore, DM and neutral detergent fiber digestibility were lower after the MTX diet than after the CTR diet (67.3 vs. 71.0% and 42.8 vs. 52.3%). The MDP diet had the highest digestibility coefficients for DM (72.4%) and neutral detergent fiber (53.6%) compared with the other 2 diets. The activities of plasma liver transaminases were higher after the MTX diet than after the CTR and MDP diets. Compared with the CTR diet, the MTX diet led to slightly lower expression of genes related to immune and inflammatory functions, indicating that Fusarium mycotoxins had an immunosuppressive effect. Our results indicated that feed contaminated with regular levels of Fusarium mycotoxins adversely affected the performance, milk quality, diet digestibility, metabolic variables, and immunity of dairy cows, and that supplementation with mycotoxin deactivator product counteracted most of these negative effects.

Targeted healthy compounds in small and large-scale brewed beers.

The determination of targeted healthy compounds in the most popular small and large-scale brewed beer sold in Italy was carried out. Nitrogen compounds, fermentable sugars, total phenolic content and antioxidant capacity, ß-glucans, pyridoxine, folates and silicon were quantified. Further, amine content was determined since raw materials and brewing technology can affect their level. Significantly higher values for total phenolic content, antioxidant activity, nitrogen, folate and putrescine content were found for small scale beers. However, the statistical results were affected by the different beer styles in the small scale and large scale brewed beer groups, since the content of these components can vary between beer styles. Positive Pearson correlation was found between total phenolic content and EBC colour. Wide variations in pyridoxine, ß-glucans and fermentable sugars levels were observed both for small and large scale beers, while average silicon content of two groups was similar.

Melamine in eggs, plasma and tissues of hens fed contaminated diets.

A study was conducted to evaluate the excretion pattern of melamine from feed into eggs, plasma, kidney, liver and muscle of laying hens. In particular, 90 laying hens were randomly allocated to three dietary treatments and fed diets contaminated with melamine at a level of 2.5, 25 and 250 mg of melamine/kg of diet for T1, T2 and T3 groups, respectively. The diets were offered in six replicate boxes (five hens each) for 13 days. Eggs were collected from each group for melamine quantification on days 0, 1, 3, 6, 9 and 13. At the end of the experimental period, one hen per box was randomly selected and slaughtered to collect plasma, liver, kidney and muscle samples. During the experiment, feeding diets with increasing levels of melamine had no effect (P > 0.05) on weight gain, feed intake, egg production, egg weight and mortality of laying hens. The melamine in eggs increased from day 1 after melamine ingestion and reached a plateau between days 6 and 13 of melamine ingestion. At steady-state condition, the melamine egg concentrations increased (P < 0.01) with treatments, being 0.026, 0.352 and 4.631 mg/kg for T1, T2 and T3, respectively. Similarly, the carryover of melamine from feed to egg increased (P < 0.05) with the levels of melamine in the diets, varying from 0.50 to 0.70 and 0.84 for T1, T2 and T3, respectively. The melamine was detected in plasma of all tested groups, increasing (P < 0.01) with levels of melamine in the diets (0.030, 0.266 and 4.102 mg/l in T1, T2 and T3, respectively). Melamine was not detected in kidney, liver and muscle of hens fed T1. Except for kidney sampled in the T3, no melamine concentration higher than 2.5 mg/kg, representing the maximum allowable limit set by the US Food and Drug Administration and European Union for food and feeds, was measured. The melamine resulted higher in plasma and kidneys than in the liver and muscle both in T2 and T3. The results confirmed the presence of an excretion pattern of melamine from feed to eggs and tissues in laying hens.

Enzyme-assisted extraction for the HPLC determination of ochratoxin A in pork and dry-cured ham.

The extraction of ochratoxin A from meat products is generally carried out using chlorinated organic solvents, such as chloroform or methyl chloride, acidified with hydrochloric or o-phosphoric acid. In this study, an innovative method was developed to extract ochratoxin A from pork and dry-cured ham samples. The method was based on an enzyme-assisted extraction with pancreatin in phosphate buffer pH 7.5. Pancreatin hydrolyses the proteins, so that ochratoxin A, kept in the ionised form, is easily extracted by the aqueous solution. After purification through an immunoaffinity column, ochratoxin A is determined by HPLC with fluorescence detection. The average recovery values were higher than 90.0% and the relative standard deviations were below 5.5%. The limits of detection and of quantification were 0.06 and 0.12 µg kg(-1), respectively. A comparison between the new enzyme-assisted extraction and an established chloroform method was carried out on six naturally contaminated samples of pork and on 40 samples of dry-cured ham. Significantly higher (p<0.001) values of ochratoxin A were obtained on dry-cured ham samples by the enzyme-assisted method.

Oxidative stress parameters in silver catfish (Rhamdia quelen) juveniles infected with Ichthyophthirius multifiliis and maintained at different levels of water pH.

The aim of this study was to determine oxidative stress parameters in the liver, gill and muscle of silver catfish juveniles infected with Ichthyophthirius multifiliis and maintained at pH 5.0 or 7.0 for three days. Juveniles were infected by adding one I. multifiliis-infected juvenile and water containing theronts to tanks. After the appearance of white spots on the skin, infected juveniles exposed to pH 5.0 and 7.0 showed significantly higher thiobarbituric acid reactive substances (TBARS) levels in the liver and gills compared to uninfected juveniles. Liver of infected juveniles exposed to pH 7.0 showed higher catalase (CAT) and lower glutathione-S-transferase (GST) activities, but those maintained at pH 5.0 showed significantly higher GST activity than uninfected juveniles. The gills of infected juveniles showed significantly higher CAT (day two) and GST activity at both pH 5.0 and 7.0 compared to uninfected juveniles. Muscle of infected juveniles showed significantly lower CAT and GST activity and TBARS levels (at day three) when maintained at both pH 5.0 and 7.0 compared to uninfected juveniles. In conclusion, I. multifiliis infection induces liver and gill damage via lipid peroxidation products in silver catfish, but higher antioxidant enzyme activity could indicate a greater degree of protection against this parasite.

The presence of ochratoxin A in cord serum and in human milk and its correspondence with maternal dietary habits.

BACKGROUND: Ochratoxin A (OTA) is a mycotoxin present in food that can be found in human blood and milk. PURPOSE: The link between the nutritional habits of pregnant women both of Italian and foreign nationality resident in Italy and the presence of ochratoxin A in cord blood and in maternal milk was investigated. METHODS: The study involved 130 pregnant women. Food consumption during pregnancy was evaluated by means of the EPIC questionnaire; OTA content was determined in cord serum and maternal milk by HPLC. RESULTS: The mean daily dietary intake of OTA was 1.02 ± 1.20 and 0.87 ± 0.78 ng/kg of bodyweight for Italian and non-Italian women, respectively, but this difference was not statistically significant. The incidence of positive milk samples was 73.0 and 85.0% among the Italian and non-Italian mothers, respectively. Pork meat, soft drinks, sweets and red wine showed a significant relationship with OTA level in serum. As far as milk is concerned, a positive relationship resulted for pork meat, sweets, soft drinks and seed oils. A positive relationship between serum OTA level and the ratio serum/milk OTA was found. The intake of OTA had no effect on the cord blood creatinine level. CONCLUSIONS: This study confirms that OTA is widely present in human milk and therefore could pose a risk for the newborn.

Transfer of melamine from feed to milk and from milk to cheese and whey in lactating dairy cows fed single oral doses.

A study was conducted to evaluate the excretion pattern, after a single oral dose, of melamine from feed into milk, and the subsequent transfer to cheese and whey. The transfer of cyanuric acid was also investigated. Twenty-four lactating Holstein cows were randomly allocated to 4 treatments and received single doses of melamine as follows: 0.05, 0.50, 5.00, and 50.00 g/cow for groups D1, D2, D3, and D4, respectively. Individual milk samples were collected for melamine and cyanuric acid analyses on d 1, 2, 3, 4, 5, and 7. Milk collected individually from the second milking after melamine ingestion was used to make cheese on a laboratory scale. Melamine and cyanuric acid were extracted using a solid-phase extraction cartridge, and analyses were carried out by liquid chromatography-mass spectrometry/mass spectrometry. Maximal melamine concentrations occurred between 6 and 18 h after treatment and increased with log dose (linear and quadratic), ranging from 0.019 to 35.105 mg/kg. More than 60% of the melamine that was transferred to the milk was observed within 30 h after melamine ingestion. Melamine was not detected (limit of detection was 0.002 mg/kg) in milk 5 d after treatment in group D1, and 7 d after treatment in groups D2, D3, and D4. Blood urea nitrogen was not influenced by melamine ingestion. During cheese making, melamine was transferred mainly to the whey fraction. Cyanuric acid was not detected in any of the samples (milk, cheese, or whey). The excretion pattern of melamine in milk and whey may represent a health concern when cows ingest more than 0.50 g of melamine/d. However, only at intake levels of 5 and 50 g/d did cheese exceed the limits as set forth by the European Union. The results confirmed that melamine contamination of milk and milk products may be related not only to direct contamination, but also to adulteration of animal feeds.

Transfer of aflatoxin B1 and fumonisin B1 from naturally contaminated raw materials to beer during an industrial brewing process.

The aim of this research was to determine the fate of aflatoxins (AFs) and fumonisins (FBs) naturally occurring in raw materials (maize grit and malted barley) during four industrial brewing processes. The aflatoxin B(1) (AFB(1)) level in raw materials varied from 0.31 to 14.85 microg kg(-1), while the fumonisin B(1) (FB(1)) level (only in maize grit) varied from 1146 to 3194 microg kg(-1). The concentration in finished beer ranged from 0.0015 to 0.022 microg l(-1) for AFB(1) and from 37 to 89 microg l(-1) for FB(1); the other aflatoxins and fumonisin B(2) were not found in beer samples. The average percentage of toxins recovered in finished beer, referring to the amounts contained in raw materials, were 1.5% +/- 0.8% for AFB(1) and 50.7% +/- 4.7% for FB(1). These results were mainly due to the different solubility of the two mycotoxins during the mashing process. If raw materials comply with the limits fixed by European Commission Regulations, the contribution of a moderate daily consumption of beer to AFB(1) and FB(1) intake does not contribute significantly to the exposure of the consumer.

Effect of the inclusion of adsorbents on aflatoxin B1 quantification in animal feedstuffs.

The extraction efficiency of aflatoxin B1 (AFB1) in cattle feed containing nine adsorbents (ADSs) was investigated using two organic/aqueous solvents composed of methanol/water (80/20 v/v; MeOH) and acetone/water (85/15 v/v; AC). Samples were obtained including a highly AFB1-contaminated (HC) and a low-level AFB(1)-contaminated (LC) feedstuff (15.33 and 7.57 microg kg(-1), respectively), nine ADSs (four clay minerals; one yeast cell wall-based product; one activated carbon and three commercial ADS products) at two different levels of inclusion (10 and 20 g kg(-1)). After solvent extraction and immunoaffinity column clean-up, all samples were analysed for AFB1 by high-performance liquid chromatography (HPLC) with fluorescence detection. For each contamination level (HC and LC), the data obtained were analysed using a factorial arrangement in a completely randomized design. Means were compared with the correspondent controls using the Dunnett's test. No statistical difference was found in AFB1 levels of feedstuffs not containing ADSs when extracted with AC or MeOH, even if numerically higher values were obtained with AC. A dose-dependent effect (p < 0.01) of ADSs inclusion was observed on AFB1 recoveries that were lower when the higher ADS level (20 g kg(-1)) was included in the HC and LC feedstuffs. Higher AFB(1) recoveries were obtained using AC compared with MeOH, both in HC (75.0% versus 12.0%, respectively) and in LC (84.0% versus 22.8%, respectively) ADSs containing feedstuffs. However, when the activated carbon and the sodium bentonite were included in feeds, lower AFB1 concentrations with respect to control values (p < 0.001 and <0.05, respectively) were obtained also using AC. The data obtained in this study indicate that routine use of the MeOH solvent for AFB1 analysis of unknown feedstuffs, can produce misleading results if they contain an ADS.

Distribution of aflatoxins and fumonisins in dry-milled maize fractions.

The aim was to evaluate the distribution of aflatoxins and fumonisins in fractions derived from the dry-milling of contaminated maize. Two maize lots with different contamination levels were processed and sampled: the first (maize 1) had aflatoxin B(1) (AFB(1)) and fumonisin B(1) (FB(1)) levels of 3.6 and 5379 microg kg(-1), respectively; the second (maize 2) had corresponding levels of 91.1 and 8841 microg kg(-1), respectively. The cleaning step reduced AFB(1) and FB(1) levels by 8 and 11% in maize 1 and by 57 and 34% in maize 2. The subsequent removal of bran and germ led to a further decrease in contamination levels in the products destined for human consumption. In the latter, AFB(1) was uniformly distributed, while FB(1) was concentrated in the finer size fractions. Contamination of raw maize 1 (3.6 microg kg(-1)) was below the European Union AFB(1) limit of 5 microg kg(-1) for unprocessed maize, but among the final products only coarse flour (1.7 microg kg(-1)) was within the European Union limit of 2 microg kg(-1), while grits and fine flour showed higher levels (2.7 and 2.5 microg kg(-1), respectively). As regards cleaned maize, a different distribution of the two toxins was observed in the kernels: AFB(1) contamination was more superficial and concentrated in germ, while FB(1) contamination affected the inner layers of the kernels.

Studies on Aspergillus section Flavi isolated from maize in northern Italy.

In 2003, for the first time in Italy, significant problems arose with colonization and contamination of maize destined for animal feed with Aspergillus section Flavi and aflatoxins (AFs). This resulted in milk and derived products being contaminated with AFM(1) at levels above the legislative limit. There was little knowledge and experience of this problem in Italy. The objectives of this research were thus to study the populations of Aspergillus section Flavi in six northern Italian regions and obtain information on the relative role of the key species, ability to produce sclerotia, production of the main toxic secondary metabolites, aflatoxins and cyclopiazonic acid, and tolerance of key environmental parameters. A total of 70 strains were isolated and they included the toxigenic species A. flavus and A. parasiticus. A. flavus was dominant in the populations studied, representing 93% of the strains. Seventy percent of strains of Aspergillus section Flavi produced AFs, with 50% of strains also producing cyclopiazonic acid. Sixty-two percent of A. flavus strains and 80% of A. parasiticus were able to produce sclerotia at 30 degrees C. Using 5/2 agar, only 1 strain developed S sclerotia and 19 L sclerotia. With regard to ecological studies, growth of Aspergillus section Flavi was optimal at between 25 and 30 degrees C, while AFB(1) production was optimal at 25 degrees C. Regarding water availability (water activity, a(w)), 0.99 a(w) was optimal for both growth and AFs production, while the only aflatoxin produced in the driest condition tested (0.83 a(w)) was AFB(1). This information will be very useful in identifying regions at risk in northern Italy by linking climatic regional information to levels of fungal contamination present and potential for aflatoxin production in maize destined for animal feed. This would be beneficial as part of a prevention strategy for minimising AFs in this product.

Black aspergilli and ochratoxin A in grapes in Italy.

The objective of this research was to investigate the presence of black aspergilli in grapes grown in Italy and to study the effect of environmental and cultural factors able to influence fungal incidence and ochratoxin A (OTA) presence. In this 3-year study, black aspergilli were frequently associated with grape berries; they were present in bunches starting from setting, colonising most berries at early veraison. Aspergillus carbonarius was never dominant at the different growth stages, or in different geographic areas and years, but it was confirmed as the key fungus because of the high percentage of strong OTA producer isolates in the population. The number of OTA producer strains, isolated in each vineyard at the different growth stages, was generally very limited and they were never statistically correlated to OTA content in bunches. The effect of geographic area on fungal flora was confirmed by statistical analysis, even though a major role was played by meteorological conditions, both on fungal colonisation and OTA content in bunches. Discriminant analysis gave promising perspectives for predicting OTA presence in vineyards in the future, based on summation of degree-day and rain in the period between 21st of August and 10th of September.

Occurrence of mycotoxins and ergosterol in maize harvested over 5 years in Northern Italy.

Maize samples collected from storage bins and feed mills in Northern Italy between 1995 and 1999 were surveyed for the occurrence of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON) and fumonisin (FB1); further, ergosterol was analysed as a fungal growth marker. The incidence and mean content of AFB1 were generally low; nevertheless, a remarkable contamination was found in two samples (109 and 158 microg kg(-1)), while five others exceeded 20 microg kg(-1). DON and ZEA mean levels were significantly higher in 1996 (2716 and 453 microg kg(-1)) with respect to the other years, when mean contents ranged from 7 to 30% and from 3 to 17%, respectively, expressed in per cent of 1996 contents. FB1 was present in all samples and was by far the most remarkable mycotoxin in Northern Italian maize, with the exception of samples from 1996. The average level was 3064 microg kg(-1), 69.6% of samples resulted over 1000 microg kg(-1) and 16.9% over 5000 microg kg(-1). Significant correlations were found between ergosterol and the major mycotoxin(s) in each year (FB1 in 1995 and 1997-99; ZEA + DON in 1996). Consequently, ergosterol seems to be a good index of the toxicological quality of maize. Climatic conditions influenced the growth of different fungal species. In 1996, the first 20 days of October were extremely rainy; these weather conditions delayed the harvest until the first week of November and favoured the growth of DON and ZEA producing fungi and the synthesis of mycotoxins. On the contrary, the temperate and dry climate of the other years supported the growth of FB1-producing fungi.

Occurrence of ochratoxin A in Italian wines.

A total of 96 red wines and 15 white dessert wines produced mostly in the years 1995-97 in 19 Italian regions were analysed for ochratoxin A (OTA). The amount of OTA ranged from < 1 to 3856 ng/l the median (mean) was found to be 90 (419) ng/l for the red wines and 8 (736) ng/l for the white dessert wines. Our survey shows that the geographic region of origin has a strong influence on OTA contamination, both for red and for dessert wines: in fact, wines produced in southern Italy were markedly more contaminated. The overall median (mean) OTA concentration in the red wines produced in the four Italian areas (northwest, northeast, centre and south) was 2 (11), 90 (81), 134 (295) and 1264 (1233) ng/l. The same trend was observed for the white dessert wines: OTA concentrations of over 1000 ng/l were found in four out of five samples from southern Italy (1185, 2454, 3477, 3856 ng/l), while central and northern samples showed very low contamination. The contribution of wine to mean daily OTA intake can be considered negligible in the case of people drinking wine manufactured in northern and central Italy; this is not true if a medium drinker constantly consumes red wine produced in southern Italy in this case wine alone could supply the diet with an amount of OTA equal to or even above the tolerable daily intake of 5 ng/kg body weight recommended by the Scientific Committee on Food of the European Commission.

Activated carbons: in vitro affinity for ochratoxin A and deoxynivalenol and relation of adsorption ability to physicochemical parameters.

In vitro affinity tests were conducted to test the effectiveness of 19 activated carbons (ACs), hydrates sodium calcium aluminosilicate (HSCAS) and sepiolite (S) in binding ochratoxin A (OA) and deoxynivalenol (DON) from solution. Relationships between adsorption ability and physicochemical parameters of ACs (surface area, iodine number, methylene blue index) were tested. When 5 ml of a 4-micrograms/ml aqueous solution of OA was treated with 2 mg of AC, the ACs adsorbed 0.80 to 99.86% of the OA. HSCAS and S were not effective in binding OA. In two saturation tests carried out with increased amounts of OA (5 ml of 10-and 50-micrograms/ml aqueous solutions of OA, respectively) three ACs also showed high adsorption ability (adsorbing 92.23 to 96.57% of the OA). When 5 ml of a 4-micrograms/ml aqueous solution of DON was treated with 10 mg of AC, ACs adsored 1.83 to 98.93% of the DON. HSCAS and S were not effective in binding DON. An overall relation of adsorption ability to the physicochemical parameters of ACs was observed. The methylene blue index was more reliable than iodine number and surface area in predicting ability of ACs to adsorb OA and DON. Based on the data observed on the xxxxx eh present study as well as on aflatoxin B1 and fumonisin B1 from previous studies, it is concluded that ACs have high in vitro affinity for chemically different mycotoxins, and can be considered as potential multi-mycotoxin-sequestering agents. However, the ability to bind the main mycotoxins singly or in combination should be confirmed by in vivo investigations. Moreover, information on the amounts of AC to be added to feeds, and on the possible long-term effect on absorption of essential nutrients are needed.

Aflatoxin M1 occurrence in samples of Grana Padano cheese.

A total of 223 samples of Grana Padano cheese manufactured in 4 years (1991-94) by dairies in 11 provinces of the Po valley were checked for aflatoxin M1. Grated cheese was extracted with chloroform and the defatted extract was purified by an immunoaffinity column; aflatoxin M1 was determined by HPLC using a fluorescence detector. From the analysis of the data it has emerged that only one sample exceeded the maximum tolerated level in cheese in some European countries (250 ng/kg). Most samples (91%) were in the range 5-100 ng/kg and only 15 (6.7%) in the range 100-250 ng/kg. Notwithstanding a diffuse microcontamination, the situation regarding the AFM1 levels can be considered fairly satisfactory. Mean contamination levels of 1992 and 1994 were significantly higher (P < 0.05) than those of 1993 and 1991. No significant difference was observed among provinces or dairies of origin.