Antonio Gallo

Carryover of aflatoxin from feed to milk in dairy cows with low or high somatic cell counts

Aflatoxin M1 (AFM1) residues in milk are regulated in many parts of the world and can cost dairy farmers significantly due to lost milk sales. Additionally, due to the carcinogenicity of this compound contaminated milk can be a major public health concern. Thirty-four lactating dairy cows were utilised to investigate the relationship between somatic cell counts (SCC), milk yield and conversion of dietary aflatoxin B1 (AFB1) into milk AFM1 (carryover (CO)). The AFM1 in milk increased as soon as the first milking after animal ingestion with a pattern of increment up to the observed plateau (between 7th and 12th days of AFB1 ingestion). There was a significant (P < 0.01) effect of the milk yield whereas no effect could be attributed to the SCC levels or to the milk yield × SCC interaction. Similarly, the main effect of milk yield was also observed (P < 0.01) on the total amount of AFM1 excreted during the ingestion period. Although the plasma concentration of gamma-glutamyl transferase was significantly affected by aflatoxin administration, levels of this liver enzyme were within the normal range for lactating dairy cows. The current data suggest that milk yield is the major factor affecting the total excretion of AFM1 and that SCC as an indicator of mammary gland permeability was not related to an increase in AFM1 CO.

Review on Mycotoxin Issues in Ruminants: Occurrence in Forages, Effects of Mycotoxin Ingestion on Health Status and Animal Performance and Practical Strategies to Counteract Their Negative Effects

Ruminant diets include cereals, protein feeds, their by-products as well as hay and grass, grass/legume, whole-crop maize, small grain or sorghum silages. Furthermore, ruminants are annually or seasonally fed with grazed forage in many parts of the World. All these forages could be contaminated by several exometabolites of mycotoxigenic fungi that increase and diversify the risk of mycotoxin exposure in ruminants compared to swine and poultry that have less varied diets. Evidence suggests the greatest exposure for ruminants to some regulated mycotoxins (aflatoxins, trichothecenes, ochratoxin A, fumonisins and zearalenone) and to many other secondary metabolites produced by different species of Alternaria spp. (e.g., AAL toxins, alternariols, tenuazonic acid or 4Z-infectopyrone), Aspergillus flavus (e.g., kojic acid, cyclopiazonic acid or β-nitropropionic acid), Aspergillus fuminatus (e.g., gliotoxin, agroclavine, festuclavines or fumagillin), Penicillium roqueforti and P. paneum (e.g., mycophenolic acid, roquefortines, PR toxin or marcfortines) or Monascus ruber (citrinin and monacolins) could be mainly related to forage contamination. This review includes the knowledge of mycotoxin occurrence reported in the last 15 years, with special emphasis on mycotoxins detected in forages, and animal toxicological issues due to their ingestion. Strategies for preventing the problem of mycotoxin feed contamination under farm conditions are discussed.

Cooking quality and starch digestibility of gluten free pasta using new bean flour

The use of rice/leguminous blend may be nutritionally convenient in gluten free product manufacturing. Gluten free spaghetti was prepared with rice flour and different concentrations of bean flour (included at levels of 0%, 20% and 40%, w/w) derived from a new developed white-seeded low phytic acid and lectin free (ws+lpa+lf) bean cultivar. Protein, ash and dietary fibre contents increased linearly (P<0.05) while total starch decreased quadratically (P<0.05) with the inclusion of ws+lpa+lf bean flour. The colour of spaghetti was influenced (P<0.05) by ws+lpa+lf bean inclusion. With respect to 0% spaghetti, the inclusion of ws+lpa+lf bean increased linearly (P<0.05) the optimal cooking time and the water absorption capacity, without affecting cooking loss and texture properties. The ws+lpa+lf bean inclusion increases quadratically (P<0.05) the resistant starch content, while decreasing quadratically (P<0.05) the in vitro glycemic index. The partial replacement of rice flour with bean flour can favourably be used in gluten free spaghetti formulation.

Vaccination of Lactating Dairy Cows for the Prevention of Aflatoxin B1 Carry Over in Milk

The potential of anaflatoxin B1 (AnAFB1) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB1-KLH) in controlling the carry over of the aflatoxin B1 (AFB1) metabolite aflatoxin M1 (AFM1) in cow milk is reported. AFB1 is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB1 is AFB1 chemically modified as AFB1-1(O-carboxymethyl) oxime. In comparison to AFB1, AnAFB1 has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB1-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB1 IgG antibodies (Abs) which were cross reactive with AFB1, AFG1, and AFG2. The elicited anti-AFB1 Abs were able to hinder the secretion of AFM1 into the milk of cows continuously fed with AFB1. Vaccination of lactating animals with conjugated AnAFB1 may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.

Aflatoxins absorption in the gastro-intestinal tract and in the vaginal mucosa in lactating dairy cows

Abstract The objective of the experiment was to monitor plasma levels of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and M1 (AFM1) in lactating dairy cows fed a single oral bolus with aflatoxin naturally contaminated corn meal (Trial 1). The possible aflatoxins (AFs) absorption through mucous membranes was also investigated using the vaginal mucosa (Trial 2). In trial 1, seven lactating Holstein dairy cows were given a single oral bolus of a naturally contaminated corn meal assuring an intake of 4.89 mg AFB1, 1.01 mg AFB2, 10.63 mg AFG1 and 0.89 mg AFG2. Blood samples were collected at 0 and 5, 10, 15, 20, 25, 30 minutes after treatment. In trial 2 an aflatoxin dosage similar to that of trial 1 was provided through vaginal implant to eight lactating Holstein dairy cows. Blood samples were collected at 0 and 15, 30, 60, 180, 360 minutes after treatment. Individual milk samples of six milkings, one before and five after treatment, were also collected. Plasma and milk samples were analysed by HPLC for AFB1, AFB2, AFG1, AFG2 and AFM1 contents. In trial 1 AFB1 in plasma peaked (33.6 ng/L) as soon as 20 minutes after treatment. The plasma AFM1 was already detectable at 5 minutes (10.4 ng/L) and peaked at 25 minutes (136.3 ng/L). In trial 2 only AFB1 and AFM1 were detectable in plasma, starting from the first sampling time (15 minutes), with values of 10.7 and 0.5 ng/L, respectively. The AFB1 peaked at 30 minutes (23.9 ng/L). The AFB1 excreted in milk as AFM1 had the highest concentration (203.0 ng/L) in the first milking after treatment and decreased close to the starting values after 36 hours from treatment. The prompt appearance of studied aflatoxins, and their metabolites, in plasma suggests absorption might also take place in mouth or oesophageal mucous membranes, before the rumen compartment. Results support the hypothesis that the cytochrome P450 oxidative system, which is present in these tissues and in leukocytes, could be involved in the conversion of the AFB1 in AFM1. The absorption of AFB1 through the vaginal mucosa confirms the passive diffusion as a probable mechanism for AFB1 absorption.

Plasma glucose response and glycemic indices in pigs fed diets differing in in vitro hydrolysis indices

Different dietary starch sources can have a great impact in determining starch digestion potential, thus influencing the postprandial blood glucose response. Our objectives were to define: (i) the incremental plasma glucose response in pigs fed diets containing various sources of starch differing in in vitro digestion patterns, (ii) the in vivo glycemic index (GI) values for the same diets, (iii) the possible relationship between in vitro and in vivo data. Diets, formulated with 70% of starch from five heterogeneous sources, were characterized in depth by using two distinct in vitro evaluations. The first one was based on the Englyst-assay for nutritional classification of starch fractions, whereas the second one was based on a time-course multi-enzymatic assay up to 180 min from which the hydrolysis indices (HIs) were calculated and used as a link between the physicochemical properties of starch from diets and the in vivo responses. For the in vivo study, five jugular-catheterized pigs (35.3 ± 1.1 kg body weight) were fed one of the five diets for 6-day periods in a 5 × 5 Latin square design. On day 5, blood was collected for 8 h postprandially for evaluating glucose appearance. On day 6, blood was collected for 3 h postprandially for the estimation of the GI. Starchy diets differed for rapidly digestible starch (from 8.6% to 79.8% of total starch (TS)) and resistant starch contents (from 72.5% to 4.5% of TS). Wide between-diets variations were recorded for all the kinetic parameters and for the HI calculated from the in vitro digestion curves (P < 0.05). On the basis of the obtained HI, diets contained starch with a very low to a very high in vitro digestion potential (ranging from 26.7% to 100.0%; P < 0.05). The glucose response differed among diets (P < 0.05), with marked differences between 15 and 120 min postprandial. Overall, the ranking of incremental glucose appearance among diets agreed with their in vitro HI classification: high HI diets increased plasma glucose response more (P < 0.05) than low HI diets. Lastly, different in vivo GIs were measured (ranging from 30.9% to 100.0%; P < 0.05). The relationship between HI and GI showed a high coefficient of determination (R2 = 0.95; root mean square error (RMSE) = 15.8; P < 0.05). In conclusion, diets formulated with starches with a wide range in HI potential can strongly affect the postprandial glucose response in pigs.

Technical note: Precision and accuracy of in vitro digestion of neutral detergent fiber and predicted net energy of lactation content of fibrous feeds

The objective of this study was to test the precision and agreement with in situ data (accuracy) of neutral detergent fiber degradability (NDFD) obtained with the rotating jar in vitro system (Daisy(II) incubator, Ankom Technology, Fairport, NY). Moreover, the precision of the chemical assays requested by the National Research Council (2001) for feed energy calculations and the estimated net energy of lactation contents were evaluated. Precision was measured as standard deviation (SD) of reproducibility (S(R)) and repeatability (S(r)) (between- and within-laboratory variability, respectively), which were expressed as coefficients of variation (SD/mean × 100, S(R) and S(r), respectively). Ten fibrous feed samples (alfalfa dehydrated, alfalfa hay, corn cob, corn silage, distillers grains, meadow hay, ryegrass hay, soy hulls, wheat bran, and wheat straw) were analyzed by 5 laboratories. Analyses of dry matter (DM), ash, crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) had satisfactory S(r), from 0.4 to 2.9%, and S(R), from 0.7 to 6.2%, with the exception of ether extract (EE) and CP bound to NDF or ADF. Extending the fermentation time from 30 to 48 h increased the NDFD values (from 42 to 54% on average across all tested feeds) and improved the NDFD precision, in terms of both S(r) (12 and 7% for 30 and 48 h, respectively) and S(R) (17 and 10% for 30 and 48 h, respectively). The net energy for lactation (NE(L)) predicted from 48-h incubation NDFD data approximated well the tabulated National Research Council (2001) values for several feeds, and the improvement in NDFD precision given by longer incubations (48 vs. 30 h) also improved precision of the NE(L) estimates from 11 to 8%. Data obtained from the rotating jar in vitro technique compared well with in situ data. In conclusion, the adoption of a 48-h period of incubation improves repeatability and reproducibility of NDFD and accuracy and reproducibility of the associated calculated NE(L). Because the in vitro rotating jar technique is a simple apparatus, further improvement would probably be obtained by reducing the laboratory differences in rumen collection procedures and type of animal donors, which, however, reflect practical conditions.

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 AFB1-contaminated (LC) feedstuff (15.33 and 7.57 µg 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 Dunnetts 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 AFB1 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.

In vitro models to evaluate the capacity of different sequestering agents to adsorb aflatoxins

Eight potential aflatoxin-sequestering agents (SAs) were tested for their ability to adsorb aflatoxin B1 (AfB1) and aflatoxin G1 (AfG1) in vitro. They belong to main SA classes: silicate minerals (calcium, magnesium and sodium bentonites, kaolinite, zeolite and clinoptinolite), activated carbon and yeast cell wall-derived. The AfB1 and AfG1 used in present work were extracted from a contaminated corn meal (82.21 mg/kg of AfB1 and 97.20 mg/kg of AfG1). Three single-concentration adsorption tests, consisting of a simply-water (W), a gastro-intestinal simulating monogastric model (MM) and a ruminant model (RM) were used. The methods differed for dilution media, incubation steps and pH condition in which they were conducted. In particular, one step (2h at 39°C) at pH 7 for W; two steps (4h at 39°C) at pH 2 and 7 for MM; and a pre-incubation in rumen fluid (pH 7 for 2h at 39°C) + two steps (4h at 39°C) at pH 2 and 7 for RM, characterized each method. The AfB1:SA ratio (g/g) and dilution factor (ng of incubated AfB1:mL of volume) were chosen (1:500,000 and 4.1, respectively) to reflect field conditions. The AfB1 and AfG1 recovered in controls were 92.3% and 104.9% in W and 89.5% and 101.5% in MM; while in RM were 65.2% and 81.9%; respectively. This supported the idea of intrinsic rumen fluid factors could be involved in sequestering of aflatoxins. In the present study, three SAs (activated carbon, Mg bentonite and Na bentonite) were very efficient to sequester the available AfB1, with a sequestering activity of over 99.0% with each method. The Ca bentonite and clinoptinolite were able to bind available AfB1 in MM and RM methods, while they appeared inefficient (available AfB1 sequestered less than 80%) when W was used. The adsorption ability of zeolite was confirmed only with the W method. Ineffective or limited sequestering activity were obtained with kaolinite and yeast cell wall-derived products with each method. The AfB1 and AfG1 sequestering efficiencies observed in the present work resulted very similar showing strong and positive correlation (P<0.001) within methods (r=0.79, r=0.96 and r=0.99, respectively for W, MM and RM methods). The two simulated gastrointestinal methods (MM and RM, respectively) gave similar results and could be considered useful for in vitro pre-screening of potential sequestering agents. However, the major practical and analytical implications related to rumen fluid method suggested that MM method should be used.

In vitro production of short-chain fatty acids from resistant starch by pig faecal inoculum

The need to improve the knowledge of fermentation processes within the digestive tract in pigs is growing, particularly for ingredients that may act as potential prebiotic sources, such as resistant starch (RS). A study (based on enzymatic digestion followed by in vitro fermentation) was conducted to investigate whether various sources of RS, obtained from eight native starches characterized by inherent heterogeneous starch chemistry and structure, can influence short-chain fatty acid (SCFA) concentrations and relative production kinetics. Total and individual SCFA productions were evaluated over time and up to 72 h of incubation. The in vitro hydrolysis of native starches allowed a classification from very high [≥ 650 g/kg dry matter (DM)] to low (<50 g/kg DM) RS amount. The total SCFA production was similar between ingredients, whereas acetate and butyrate molar ratios in the SCFA profile differed (from 0.48 to 0.56 and from 0.17 to 0.25, respectively; P < 0.05). Differences in fermentation kinetic parameters for total and individual SCFA productions were observed (P < 0.05). Considering the total SCFA production after 72 h of incubation, the time at which half of the maximum production has been reached (T 1/2), the maximum rate of production (R max) and its time of occurrence (T max) differed between ingredients (P < 0.05), with values ranging from 6.1 to 11.9 h, from 0.459 to 1.300 mmol/g DM incubated per hour and from 5.1 to 9.8 h, respectively. Overall, a similar trend was observed considering individual SCFA productions. In particular, T 1/2 ranged from 6.4 to 12.5 h, from 5.5 to 12.5 h and from 6.7 to 11.3 h for acetate, propionate and butyrate, respectively (P < 0.05). For R max, differences were obtained for propionate and butyrate productions (P < 0.05), whereas no difference was recorded for acetate. In summary, our findings indicated that both quantitative and qualitative production of SCFA and related kinetics were influenced by fermentation of RS obtained from native starches characterized by heterogeneous starch characteristics. Current findings are based on an in vitro approach, and thus require further in vivo validations.