Spatial distribution of ochratoxin A in vineyard and sampling design to assess must contamination.

The aim of this work was to study the spatial variability of ochratoxin A (OTA) in vineyards and to define a reliable sampling protocol for bunches in order to assess OTA content before harvesting. In 2002, two vineyards with 'Negroamaro' and 'Sangiovese' grape varieties were chosen in Southern Italy. The same sampling design was applied to both vineyards. Ten plants were collected from the X-shaped path of the whole vineyard (plants 1 through 10) and 10 plants from the two central cross-perpendicular lines (plants 11 through 20). Bunches harvested from plants 1 through 10 were numbered progressively, weighed, and crushed separately, and bunches obtained from plants 11 through 20 were crushed plant by plant. Juices obtained were analyzed for OTA content by high-performance liquid chromatography. Then, a simulated approach for sampling was applied, following randomized and systematic designs. High and random variability was observed in OTA content both among bunches and among plants in the two vineyards, independent of contamination level. Simulated sampling design markedly influenced the assessment of must contamination. The best results were obtained when sampling involved one bunch per plant, in a predefined position, from at least 10 plants. Estimated means, obtained with different sampling designs, did not differ significantly from calculated mean OTA content. If the true contamination in a vineyard is 2 microg kg(-1), the limit fixed in Europe for OTA content in must and wine, the range of OTA content assessed sampling one bunch in the central position of 10 plants should lie between 2.9 and 1.4. The accuracy could be considered acceptable

Occurrence of ochratoxin A-producing fungi in grapes grown in Italy

A study was carried out to investigate fungi present on grapes grown in Italy. Aspergillus and Penicillium spp. isolates were identified and studied in vitro, and their ability to produce ochratoxin A (OA) was investigated. The survey involved nine vineyards, three located in northern Italy and six located in southern Italy. In 1999 and 2000, bunches of grapes at different growth stages were collected from all nine vineyards, and berry samples were placed in moist chambers and incubated. The resultant fungal colonies were then transferred to petri dishes containing Czapek yeast agar and incubated at 25 degrees C for 7 days; the fungal isolates were identified and then cultivated in liquid Czapek yeast medium and evaluated for their ability to produce OA. During the survey, 508 isolates were collected, with 477 belonging to Aspergillus spp. and 31 belonging to Penicillium spp. Among the aspergilli, species of the Fumigati, Circumdati, and Nigri sections were identified, with species of the Nigri section (464 isolates) largely predominating; for species of the Nigri section, 108 isolates were uniseriate, 270 were biseriate, and 86 were identified as Aspergillus carbonarius. Black aspergilli isolated over the 2 years of the study showed a very similar pattern. On average, the biseriates represented about 60% of the isolates collected in both years and were followed by uniseriates (21%) and A. carbonarius (19%). The most toxigenic strains proved to be those of A. carbonarius; about 60% of these isolates were OA producers and produced the highest levels of OA. A. carbonarius was more frequent in the south, but in both areas the percentages of OA-producing isolates remained the same

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

Monitoring of the declining trend of Polychlorobifenyls concentration in milk of contaminated dairy cows

Six pregnant heifers, coming from a herd with a history of high concentration of polychlorobifenyls (PCB) into the milk, were fed with a very low-PCB diet starting from the 6th month of pregnancy. After parturition cows were milked for at least 190 d with a maximum of 270 d. Diet was made of: corn silage (31.7% of DMI), dehydrated alfalfa (13.3% of DMI), grass hay (3.7% of DMI) and concentrate (51.3% of DMI). The average DMI was 23.12 kg/d. Milk production was recorded and samples of milk and blood were taken and analyzed for PCB (18, 28, 31, 52, 44, 101, 149, 118, 153, 138, 180 and 194 congeners) content using a GC-MS technique. The average milk yield (kg/d) of the 6 cows was 26.0, 22.5, 23.2, 24.5, 28.9, 29.3. The maximum PCB concentration of 100 ng/g of fat allowed by the Italian law was reached after 144-209 d of lactation. One animal after 204 days had a PCB concentration of 102 ng/g fat. If log10 of PCB concentration (ppb) in milk fat is regressed against days in milking (DIM) the following significant equation was obtained: log10 PCB (ng/g of milk fat)= 2.796 -0.00474 DIM; r2 0.72; P<0.01 The initial level of contamination is not the main factor affecting the time required in order for PCB to return to below the legal threshold of 100 ng/g fat, while daily milk yield significantly affects PCB excretion. Body condition and energy balance could be important factors affecting PCB excretion. In the presence of high contamination, about 6 months of lactation are required in order to obtain milk with a sufficiently low PCB content. Among the seven congeners considered by Italian legislation, PCB 101, PCB 118, PCB 138, PCB 153 constitute almost all the PCBs found in tissues and milk, with a much smaller presence of PCB 180, and the numbers of the congeners PCB 28 and PCB 52 are almost insignificant, probably because they accumulate little in the body. The congeners PCB 138 and PCB 153 are those most frequently found. For PCB 138 the liver is the principal organ of accumulation, while for PCB 153 we found equal accumulations in the liver, kidneys and tail, but a low presence in milk. The PCBs 118 and 138 are those most abundant in milks

Mycotoxin occurrence in maize produced in Northern Italy over the years 2009–2011: focus on the role of crop related factors

The occurrence of mycotoxins associated with Fusarium spp. and Aspergillus flavus in Northern Italy, and the role of cropping systems, were investigated on 140 field samples collected over the years 2009–2011. Samples were analysed for fumonisins B1 and B2 (FBs), aflatoxins B1, B2, G1 and G2 (AFs), deoxynivalenol (DON) and zearalenone (ZEN) using validated analytical methods. Information on: maize hybrid, preceding crop, tillage applied, mineral nutrition, pest and disease control, severity of European Corn Borer (ECB) attack, sowing and harvesting dates, kernel moisture at harvesting and longitude of the sampled province, were also collected. During this period there were distinct differences in FBs and AFs concentrations between years and geographic origins, and very low contamination with DON and ZEN was always found. The incidence of AFs exceeded 75% across all samples, and was almost 100% for FBs. The meteorological trends were quite different in the 3 years surveyed. 2009 was the coldest in June and the warmest in August, 2010 the most humid, and in 2011 cold weather occurred during flowering and dry conditions during ripening. The run of a logistic equation with the backward stepwise approach selected three parameters, (seeding week, ECB severity and longitude of sampling province) to predict AFB1 contamination and four parameters (year, sowing week, ECB severity and longitude of sampling province) to predict FB contamination. The internal validation gave good results, with 76% correct predictions. The probability of harvesting maize with more than 5 µg kg-1 of AFB1 varied between 86 and 5%, and the probability of harvesting maize with more than 4,000 µg kg-1 of FBs varied between 81 and 2%, respectively, for conducive and non-conducive environments. Therefore, considerable variability was found even if a limited area and only 3 years were considered

Activated Carbons: In Vitro Affinity for Aflatoxin B1 and Relation of Adsorption Ability to Physicochemical Parameters

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Reduction of Carryover of Aflatoxin from Cow Feed to Milk by Addition of Activated Carbons.

According to a double-reversal experimental design on 12 late-lactation Friesian cows the effect of two activated carbons (ACs) (CAC1 and CAC2) and a hydrated sodium calcium aluminosilicate (HSCAS) on carryover of aflatoxin B1 (AFB1) from feed to aflatoxin M1 (AFM1) in milk was determined. Cows were fed a basal diet containing AFB1 naturally contaminated corn meal and copra, During week 1 cows were fed diets containing AFB1 alone (11.28 μg of AFB1/kg of feed); in week 2 the diets contained AFB1 plus 2.0% sorbent; and in week 3 the diets again contained AFB1 alone (13.43 μg of AFB1/kg of feed). ACs reduced the analytical content of AFB1 in the pelleted feed by from 40.6% to 73.6%, whereas reduction by HSCAS was 59.2%, The AFM1 concentrations in milk in weeks 1 and 3 were higher than that in week 2, Decreases in the AFM1 excreted in the milk by addition to feed of 2% of the sorbents ranged from 22% to 45%. CAC1 and HSCAS were significantly different from each other in reducing the AFM1concentration in milk (45.3% versus 32.5%); these reductions were significantly higher than that of CAC2 (22.0%). Carryover reduction by addition of CAC1 (50%) was significantly higher than that of HSCAS (36%). Addition of 2% CAC2 did not allow pelleting of feed because of the caking action of this carbon, The lower performance of CAC2 could be related to the unsuccessful pelleting. The addition of ACs did not influence feed intake, milk production, milk composition, or body weight. Our results suggest that ACs, high-affinity sorbents for AFB1 in vitro, are efficacious in reducing AFB1 carryover from cow feed to milk. Further in vivo investigations should establish lower amounts of ACs which can be efficacious

Influence of water activity and anti-fungal compounds on development and competitiveness of Fusarium verticillioides

This investigated the roles of water activity (aw) and fungicides on the competitiveness of two Fusarium verticillioides strains against other spoilage fungi commonly present in maize (F. proliferatum, Aspergillus niger, A. flavus, A. ochraceus and Penicillium verrucosum). Fungal strains were inoculated on artificial media containing maize flour. The effects were determined of three aw levels (0.99, 0.98 and 0.95) and three fungicides (tebuconazole, procloraz and prothioconazole) on fungal interactions, the Index of Dominance (ID) of isolates and fumonisin B1+B2 (FBs) production. The two strains of F. verticillioides showed similar behaviour in conditions where water was freely available (0.99 aw); at 0.98 and 0.95 aw both F. verticilliodes strains had the lowest total ID scores (8–6 and 10–12, respectively). They showed the same ability to compete against other fungi having the highest ID scores against P. verrucosum and A. ochraceus and the lowest against A. niger and A. flavus. The lowest water activity gave (0.95 aw) was the most conducive for fumonisin production with significant differences to 0.98 and 0.99 aw. In a co-inoculation experiment, only FBs production from P. verrucosum was greater in the presence of the F. verticilliodes strains other fungi. The use of fungicides reduced Indices of Dominancy (ID) for both F. verticilliodes strains. A significant reduction in F. verticilloides growth was observed when combining water stress and fungicide treatments. This information provides increased understanding of the colonisation patterns of F. verticillioides in relation to other mycobiota and to both environmental and chemical stresses, and has implications in relation to future climate change scenarios

Penicillium populations in dry-cured ham manufacturing plants.

Seven ham manufacturing plants were sampled for 1 year to assess the mycoflora present in the air and on hams, with special attention given to potential mycotoxin producers. Temperature and relative humidity were recorded in the ripening rooms. Maturing rooms held hams from 2 to 3 through 6 to 7 ripening months, and aging rooms held hams for the following 6 to 7 months, until the 14-month ripening point, when they were ready for the market. Mean temperatures and relative humidities registered during the study were 14.9 degrees C and 62.4%, respectively, in maturing rooms and 16.3 degrees C and 57.6% in aging rooms. Aspergilli and penicillia, potential mycotoxin producers, were isolated in all the plants from the air and the ham. Aspergilli represented 5% of the isolates, while penicillia were largely dominant, with Penicillium nalgiovense being the most represented species (around 60% of the penicillia), followed by Penicillium nordicum, with 10 and 26% of the penicillia isolated, respectively, from the air or the ham. Ochratoxin A production ability, checked in vitro at 250C, was observed in 50% of the P. nordicum isolates obtained both from the air and the ham. Air and ham surface contamination by penicillia was greater in the ripening rooms, where higher temperatures were registered. A certain correlation was also observed between air and ham surface contamination. On the basis of this study, P. nordicum, the ochratoxin A producer that is notable on proteinaceous substrates, is normally present in ham manufacturing plants in Italy, even though not a dominant species. Further studies are necessary to clarify and ensure if dry-curing conditions minimize the potential risk of ochratoxin A formation in the product

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