D. R. Ledoux

Detection of Melamine in Gluten, Chicken Feed, and Processed Foods Using Surface Enhanced Raman Spectroscopy and HPLC

Melamine, a nitrogen-rich chemical, was implicated in pet and human food recalls in 2007, which caused enormous economic losses to the food industry. In this study, melamine concentration in wheat gluten, chicken feed, and processed foods (that is, cake and noodle) was measured by surface enhanced Raman spectroscopy (SERS) in combination with SERS-active substrates. SERS was able to rapidly detect 0.1% melamine in wheat gluten, 0.05% in chicken feed, 0.05% in cakes, and 0.07% in noodle, respectively. A partial least squares (PLS) model was established for the quantification of melamine in foods by SERS: R= 0.90, RMSEP = 0.33. In addition, SERS results were verified by HPLC analysis based on a simplified FDA method. Compared with HPLC, the SERS method is much faster and simpler, requires minimum sample preparation, but still yields satisfactory qualitative and quantitative results. These results demonstrate that it is an applicable approach to use SERS to screen foods, eliminate presumptive negative samples of melamine contamination from the sample population, and then verify presumptive positive samples using HPLC protocols. Combining these 2 methods could provide a more rapid and cost-effective way for monitoring melamine contamination in increasingly large numbers of imported foods and feed products.

Fumonisin toxicity in broiler chicks

The effects of dietary fumonisin B1 were evaluated in young broiler chicks. The experimental design consisted of 5 treatments each with 9 randomly allotted male broiler chicks. Day-old chicks were fed diets containing 0 (feed control), 100, 200, 300, or 400 mg fumonisin B1/kg feed for 21 days. Response variables measured were chick performance, organ weights, serum biochemistry, and histologic parameters. Body weights and average daily gain dramatically decreased with increasing dietary fumonisin B1, and liver, proventriculus, and gizzard weights increased. Diarrhea, thymic cortical atrophy, multifocal hepatic necrosis, biliary hyperplasia, and rickets were present in chicks fed diets containing fumonisin B,. Serum calcium, cholesterol, and aspartate aminotransferase levels all increased at higher fumonisin dietary levels. Results indicate that fumonisin, from Fusarium moniliforme culture material, is toxic in young chicks.

Effects of turmeric (Curcuma longa) on the expression of hepatic genes associated with biotransformation, antioxidant, and immune systems in broiler chicks fed aflatoxin

The objective of the present study was to evaluate the efficacy of curcumin, an antioxidant found in turmeric (Curcuma longa) powder (TMP), to ameliorate changes in gene expression in the livers of broiler chicks fed aflatoxin B(1) (AFB(1)). Four pen replicates of 5 chicks each were assigned to each of 4 dietary treatments, which included the following: A) basal diet containing no AFB(1) or TMP (control), B) basal diet supplemented with TMP (0.5%) that supplied 74 mg/kg of curcumin, C) basal diet supplemented with 1.0 mg of AFB(1)/kg of diet, and D) basal diet supplemented with TMP that supplied 74 mg/kg of curcumin and 1.0 mg of AFB(1)/kg of diet. Aflatoxin reduced (P < 0.05) feed intake and BW gain and increased (P < 0.05) relative liver weight. Addition of TMP to the AFB(1) diet ameliorated (P < 0.05) the negative effects of AFB(1) on growth performance and liver weight. At the end of the 3-wk treatment period, livers were collected (6 per treatment) to evaluate changes in the expression of genes involved in antioxidant function [catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST)], biotransformation [epoxide hydrolase (EH), cytochrome P450 1A1 and 2H1 (CYP1A1 and CYP2H1)], and the immune system [interleukins 6 and 2 (IL-6 and IL-2)]. Changes in gene expression were determined using the quantitative real-time PCR technique. There was no statistical difference in gene expression among the 4 treatment groups for CAT and IL-2 genes. Decreased expression of SOD, GST, and EH genes due to AFB(1) was alleviated by inclusion of TMP in the diet. Increased expression of IL-6, CYP1A1 and CYP2H1 genes due to AFB(1) was also alleviated by TMP. The current study demonstrates partial protective effects of TMP on changes in expression of antioxidant, biotransformation, and immune system genes in livers of chicks fed AFB(1). Practical application of the research is supplementation of TMP in diets to prevent or reduce the effects of aflatoxin in chicks fed aflatoxin-contaminated diets.

Efficacy of Turmeric (Curcuma longa), Containing a Known Level of Curcumin, and a Hydrated Sodium Calcium Aluminosilicate to Ameliorate the Adverse Effects of Aflatoxin in Broiler Chicks

A 3-wk feeding study was conducted to evaluate the efficacy of turmeric (Curcuma longa) powder (TMP), containing a known level of curcumin, and a hydrated sodium calcium aluminosilicate (HSCAS; Improved Milbond-TX, IMTX, an adsorbent, Milwhite Inc., Houston, TX) to ameliorate the adverse effects of aflatoxin B(1) (AFB(1)) in broiler chicks. Four pen replicates of 5 chicks each were assigned to each of 7 dietary treatments, which included the basal diet not containing TMP, HSCAS, or AFB(1) (control); basal diet supplemented with 0.5% food grade TMP that contained 1.48% total curcuminoids (74 mg/kg); basal diet supplemented with 0.5% HSCAS; basal diet supplemented with 1.0 mg/kg AFB(1); basal diet supplemented with 0.5% TMP and 1.0 mg/kg AFB(1); basal diet supplemented with 0.5% HSCAS and 1.0 mg/kgAFB(1); and basal diet supplemented with 0.5% TMP, 0.5% HSCAS, and 1.0 mg/kg AFB(1). The addition of TMP to the AFB(1) diet significantly (P < 0.05) improved the weight gain of chicks, and the addition of HSCAS to the AFB(1) diet significantly (P < 0.05) improved feed intake and weight gain, and reduced relative liver weight. The addition of TMP or HSCAS and TMP with HSCAS ameliorated the adverse effects of AFB(1) on some of the serum chemistry parameters (total protein, albumin, cholesterol, calcium). Further, decreased antioxidant functions in terms of level of peroxides, superoxide dismutase activity, and total antioxidant concentration in liver homogenate due to AFB1 were also alleviated by the inclusion of TMP, HSCAS, or both. The reduction in the severity of hepatic microscopic lesions due to supplementation of the AFB(1) diet with TMP and HSCAS demonstrated the protective action of the antioxidant and adsorbent used in the present study.

Fumonisin Toxicity in Turkey Poults

The effects of dietary fumonisin B, were evaluated in young turkey poults. The experimental design consisted of 3 treatments, with 24 female turkey poults allotted randomly per treatment. Day-old poults were fed diets containing 0 mg (feed control), 100 mg, and 200 mg fumonisin B1/kg feed for 21 days. Body weight gains and efficiency of feed conversion decreased linearly with increasing dietary fumonisin. Liver, kidney, and pancreas weights increased linearly with increasing dietary fumonisin, and spleen and heart weights decreased. Serum aspartate aminotransferase levels increased with increasing dietary fumonisin, and serum cholesterol, alkaline phosphatase, mean cell volume, and mean cell hemoglobin all decreased. Biliary hyperplasia, hypertrophy of Kupffers cells, thymic cortical atrophy, and moderate widening of the proliferating and degenerating hypertrophied zones of tibial physes were present in poults fed diets containing fumonisin B1. Results indicate that fumonisin B1, from Fusarium moniliforme culture material, is toxic in young poults, and the poult appears to be more sensitive to fumonisin than the broiler chick.

Effects of aflatoxin B1 and fumonisin B1 on body weight, antibody titres and histology of broiler chicks

1. Our objective was to evaluate the toxic effects of aflatoxin B1 (AFB1) and fumonisin B1 (FB1), administered singly or in combination to broilers. 2. Feeds were prepared with concentrations equal to 0, 50 and 200 µg AFB1/kg, and/or 0, 50 and 200 mg FB1/kg, and offered to broiler chicks from 8 to 41 d of age. The experimental design was totally randomised, in a 3 × 3 factorial arrangement with 9 treatments and 12 birds per treatment. Animals were vaccinated against Newcastle disease on d 14 of life and killed at 41 d. 3. Compared with controls, all mycotoxin-treated groups at 41 d had lower body weight and weight gain, and higher relative heart weight. The relative weight of the liver increased only in birds fed diets containing 200 mg FB1, singly or in combination with AFB1. 4. At 35 d, all groups receiving mycotoxin-treated rations had reduced geometrical mean antibody titres, with birds from groups fed combinations of AFB1 and FB1/kg having even lower values, when compared to the other groups. 5. Histological changes were observed only in liver from birds fed mycotoxin-contaminated rations, and in kidneys of birds fed the diet containing 200 µg AFB1 and 200 mg FB1/kg. Main alterations included vacuolar degeneration and cell proliferation of bile ducts in the liver, and hydropic degeneration in renal tubules in the kidneys. 6. We concluded that AFB1 and FB1 in combination have primarily additive effects on body weight, liver structure and immunological response of broilers at the concentrations used.

Efficacy of Solis, NovasilPlus, and MTB-100 to reduce aflatoxin M1 levels in milk of early to mid lactation dairy cows fed aflatoxin B1

An experiment was conducted to determine the efficacy of 3 adsorbents, Solis (SO; Novus International Inc.), NovasilPlus (NOV; Engelhard Corp.), and MTB-100 (MTB; Alltech), in reducing aflatoxin (AF) M(1) concentrations in milk of dairy cows fed an AF-contaminated diet. Twelve early to mid lactation dairy cows averaging 163 d in milk were used in a 4 x 4 Latin square design with 3 replications. Cows were blocked by parity, body weight, and milk production and were provided ad libitum access to feed and water. Within each replicate, cows were randomly assigned to the 4 dietary treatments for 4 consecutive 7-d periods. Dietary treatments included AF [112 microg of AFB(1)/kg of diet dry matter (DM)]; AF + 0.56% SO; AF + 0.56% NOV; and AF + 0.56% MTB. Milk samples were collected on d 6 and 7 of each of the experimental periods. Feed intake, milk production, milk fat percentage, milk protein percentage, and linear somatic cell scores were not affected by dietary treatments and averaged 22.20 kg/d of DM, 33.87 kg/d, 3.78%, 2.95%, and 1.60, respectively, across all treatments. Transfer rates of AF from feed to milk averaged 2.65, 1.48, 1.42, and 2.52% for cows fed AF, AF + SO, AF + NOV, and AF + MTB, respectively. Daily AFM(1) excretion in milk averaged 66, 37, 35, and 63 microg/d for cows fed AF, AF + SO, AF + NOV, and AF + MTB, respectively. The addition of SO and NOV to the AF diet resulted in a significant reduction in milk AFM(1) concentrations (SO, 45%; NOV, 48%) and AFM(1) excretion (SO, 44%; NOV, 46%). In contrast, MTB was not effective in reducing milk AFM(1) concentrations (4%), AFM(1) excretion (5%), or AF transfer from feed to milk (2.52%). Results indicated that SO and NOV at 0.56% of the diet were effective in reducing milk AFM(1) concentrations in cows consuming a total mixed ration containing 112 microg of AFB(1)/kg of diet DM.

An in vitro procedure for studying enzymic dephosphorylation of phytate in maize-soyabean feeds for turkey poults

An in vitro method was developed to predict inorganic P release from maize-soyabean poultry feeds containing supplemental phytase (EC 3.1.3.8), and to quantify the effect of acid phosphatase (EC 3.1.3.2), fungal protease (EC 3.4.23.6) and Aspergillus niger cellulase (EC 3.2.1.4) on phytate dephosphorylation. Pepsin (EC 3.4.23.1) and pancreatin digestion periods were preceded by a 30 min pre-incubation at pH 5.25 to simulate digestion in the crop of poultry. Pancreatin digestion was carried out in dialysis tubing, with a ratio of about 1:25 (v/v) between the digesta and dialysing medium, to simulate gradient absorption from the duodenum. The feed:water ratio was kept within physiological limits and a constant proportion of feed weight to digestive enzymes was maintained. There was a linear response to increasing dosages of phytase up to 1000 phytase units (FTU)/kg feed, and to increasing phosphate concentration in feeds. In vivo validation was performed with growing turkeys (1-3 weeks) fed on diets containing 12 g Ca/kg and 0, 500 or 1000 FTU phytase/kg in a factorial arrangement with 0, 1, 2 or 3 g supplemental phosphate/kg (from KH2PO4). After a simple transformation (variable/in vitro P = f (in vitro P)), amounts of P hydrolysed from feed samples by in vitro digestions correlated with 3-week body-weight gain (R 0.986, P < 0.0001), toe ash (R 0.952, P < 0.0001), feed intake (R 0.994, P < 0.0001) and feed efficiency (R 0.992, P < 0.0001). The dephosphorylating ability of phytase in vitro was significantly enhanced (P < 0.05) by the addition of acid phosphatase. Fungal acid protease and Aspergillus niger cellulase also enhanced the dephosphorylation process in vitro.

Toxicological and gene expression analysis of the impact of aflatoxin B1 on hepatic function of male broiler chicks

The objective of this study was to determine the effects of dietary aflatoxin B1 (AFB1) on hepatic gene expression in male broiler chicks. Seventy-five 1-d-old male broiler chicks were assigned to 3 dietary treatments (5 replicates of 5 chicks each) from hatch to d 21. The diets contained 0, 1 and 2 mg of AFB1/kg of feed. Aflatoxin B1 reduced (P<0.05) feed intake, BW gain, serum total proteins, and serum Ca and P, but increased (P<0.01) liver weights in a dose-dependent manner. Microarray analysis was used to identify shifts in genetic expression associated with the affected physiological processes in chicks fed 0 and 2 mg of AFB1/kg of feed to identify potential targets for pharmacological/toxicological intervention. A loop design was used for microarray experiments with 3 technical and 4 biological replicates per treatment group. Ribonucleic acid was extracted from liver tissue, and its quality was determined using gel electrophoresis and spectrophotometry. High-quality RNA was purified from DNA contamination, reverse transcribed, and hybridized to an oligonucleotide microarray chip. Microarray data were analyzed using a 2-step ANOVA model and validated by quantitative real-time PCR of selected genes. Genes with false discovery rates less than 13% and fold change greater than 1.4 were considered differentially expressed. Compared with controls (0 mg of AFB1/kg), various genes associated with energy production and fatty acid metabolism (carnitine palmitoyl transferase), growth and development (insulin-like growth factor 1), antioxidant protection (glutathione S transferase), detoxification (epoxide hydrolase), coagulation (coagulation factors IX and X), and immune protection (interleukins) were downregulated, whereas genes associated with cell proliferation (ornithine decarboxylase) were upregulated in birds fed 2 mg of AFB1/kg. This study demonstrates that AFB1 exposure at a concentration of 2 mg/kg results in physiological responses associated with altered gene expression in chick livers.

Effects of Fusarium moniliforme culture material containing known levels of fumonisin B1 in ducklings.

Fusarium moniliforme culture material containing fumonisin B1 (FB1) was fed to white Pekin ducklings from 1 to 21 days of age. Four dietary treatments were prepared with 0, 100, 200, and 400 mg FB1/kg ration. Ducklings fed rations containing FB1 had a dose-dependent decrease in feed intake and weight gain. Increasing levels of FB1 in the ration were associated with increasing absolute organ weights of liver, heart, kidney, pancreas, and proventriculus. Liver sphinganine to sphingosine ratios increased significantly in ducklings fed FB1. Two of eight ducklings fed a ration containing 400 mg FB1/kg died prior to the termination of the experiment. Mild to moderate hepatocellular hyperplasia was evident in all ducklings fed FB1. Mild to moderate biliary hyperplasia was also noted in the liver sections of ducklings fed 400 mg FB1/kg in the ration. Ducklings, like other poultry, are relatively resistant to the toxic effects of FB1.