In vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus.

Rotaviruses are the leading cause and coronaviruses are the major contributors of acute gastroenteritis in the young of various mammalian and avian species. Despite numerous trials and decades of research, vaccines have limited efficacy particularly for calves. As an alternative method of controlling infection, we have investigated broad spectrum antiviral agents that are not discriminatory among various viruses. This report involves testing a variety of adsorbent agents including charcoal, clay, and clay minerals to adsorb rotavirus and coronavirus in vitro. Results revealed that all the adsorbent agents had good to excellent capability of adsorbing rotavirus and excellent capability of adsorbing coronavirus. Percent adsorptions ranged from 78.74% to 99.89% for rotavirus and 99.99% for coronavirus; while sand (negative control) was < 0.01%. A high affinity binding was present as determined by a low percent desorption (0.06-3.09%). However, the adsorbent bound virus complex retained, and may have actually enhanced, infectivity.

An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections.

Crude theaflavin was extracted from black tea and then fractionated by HPLC into five components (initial peaks (IP), TF1, TF2A, TF2B, and TF3). The crude extract and the various fractions of theaflavin were collected and tested, individually and in combination, for antirotaviral activity. The mean effective concentration (EC50) was calculated and compared. Activity varied from the most active being the uncharacterized theaflavin-like initial peaks (IP) with an EC50 of 0.125 microgram/ml to the least active being theaflavin-3 monogallate (TF2A) with an EC50 of 251.39 micrograms/ ml. The combination of TF1 + TF2A + TF2B + TF3 was more active than the sum of the activities of these four fractions individually, indicating synergism among the peaks. Only the crude extract was assayed for activity against coronavirus; the EC50 was 34.7 micrograms/ml.

Prevention of maternal and developmental toxicity in rats via dietary inclusion of common aflatoxin sorbents: potential for hidden risks.

In earlier work, we have reported that a phyllosilicate clay (HSCAS or NovaSil) can tightly and selectively bind the aflatoxins in vitro and in vivo. Since then, a variety of untested clay and zeolitic minerals have been added to poultry and livestock feeds as potential "aflatoxin binders." However, the efficacy and safety of these products have not been determined. A common zeolite that has been frequently added to animal feed is clinoptilolite. Our objectives in this study were twofold: (1) to utilize the pregnant rat as an in vivo model to compare the potential of HSCAS and clinoptilolite to prevent the developmental toxicity of aflatoxin B1 (AfB1), and (2) to determine the effect of these two sorbents on the metabolism and bioavailability of AfB1. Clay and zeolitic minerals (HSCAS or clinoptilolite) were added to the diet at a level of 0.5% (w/w) and fed to pregnant Sprague-Dawley rats throughout pregnancy (i.e., day 0 to 20). Treatment groups (HSCAS or clinoptilolite) alone and in combination with AfB1 were exposed to sorbents in the feed as well as by gavage. Untreated and AfB1 control animals were fed the basal diet without added sorbent. Between gestation days 6 and 13, animals maintained on diets containing sorbent were gavaged with corn oil in combination with an amount of the respective sorbent equivalent to 0.5% of the estimated maximum daily intake of feed. Animals receiving AfB1 were dosed orally (between days 6 and 13) with AfB1 (2 mg/kg body wt) either alone or concomitantly with a similar quantity of the respective sorbent. Evaluations of toxicity were performed on day 20. These included: maternal (mortality, body weights, feed intake, and litter weights), developmental (embryonic resorptions and fetal body weights), and histological (maternal livers and kidneys). Sorbents alone were not toxic; AfB1 alone and with clinoptilolite resulted in significant maternal and developmental toxicity. Animals treated with HSCAS (plus AfB1) were comparable to controls. Importantly, clinoptilolite (plus AfB1) resulted in severe maternal liver lesions (more severe than AfB1 alone), suggesting that this zeolite may interact with dietary components that modulate aflatoxicosis. In metabolism studies, adult male Sprague-Dawley rats, maintained on diets containing 0.5% (w/w) HSCAS or clinoptilolite, were dosed orally with 2.0 mg AfB1/kg body wt. The concentration of the major urinary metabolite (AfM1) was considerably decreased in the presence of HSCAS. These results suggest that the mechanism of protection of AfB1-induced maternal and developmental toxicities in the rat may involve adsorption and reduction of AfB1 bioavailability in vivo. Importantly, this study demonstrates the potential for significant hidden risks associated with the inclusion of nonselective aflatoxin binders in feeds. Aflatoxin sorbents should be rigorously tested individually and thoroughly characterized in vivo, paying particular attention to their effectiveness and safety in sensitive animal models and their potential for deleterious interactions.

Effects of inorganic adsorbents and cyclopiazonic acid in broiler chickens.

Previous studies with cyclopiazonic acid (CPA) have indicated that this mycotoxin strongly adsorbs onto the surface of a naturally acidic phyllosilicate clay (AC). The objective of this study was to determine whether AC (and similar adsorbents) could protect against the toxicity of CPA in vivo. Acidic phyllosilicate clay, neutral phyllosilicate clay (NC, or hydrated sodium calcium aluminosilicate), and a common zeolite (CZ, or clinoptilolite) were evaluated. One-day-old broiler chicks consumed diets containing 0 or 45 mg/kg CPA alone or in combination with 1% AC, NC, or CZ ad libitum from Day 1 to 21. Body weight, feed consumption, feed:gain, hematology, serum biochemical values, and enzyme activities were evaluated. Compared to controls, CPA alone reduced body weight at Day 21 by a total of 26% and resulted in a significantly higher feed:gain ratio. Toxicity of CPA was also expressed through increased relative weights of kidney, proventriculus, and gizzard. Also, there were some alterations in hematology, serum biochemical values, and enzyme activities. Treatment with inorganic adsorbents did not effectively diminish the growth-inhibitory effects of CPA or the increased weights of organs, although there was some protection from hematological, serum biochemical, and enzymatic changes produced by CPA. The results of this study suggest that in vitro binding of CPA to clay does not accurately forecast its efficacy in vivo; the reasons for this discrepancy are not clear, but they may be related to differences in clay binding capacity and ligand selectivity for CPA in vitro vs in vivo. Predictions about the ability of inorganic adsorbents to protect chickens from the adverse effects of mycotoxins should be approached with caution and should be confirmed in vivo, paying particular attention to the potential for nutrient interactions.

Oxidative degradation and detoxification of mycotoxins using a novel source of ozone.

Practical methods to degrade mycotoxins using ozone gas (O3) have been limited due to low O3 production capabilities of conventional systems and their associated costs. Recent advances in electrochemistry (i.e. proton-exchange membrane and electrolysis technologies) have made available a novel and continuous source of O3 gas up to 20% by weight. It is possible that the rapid delivery of high concentrations of O3 will result in mycotoxin degradation in contaminated grains--with minimal destruction of nutrients. The major objectives of this study were to investigate the degradation and detoxification of common mycotoxins in the presence of high concentrations of O3. In this study, aqueous equimolar (32 microM) solutions of aflatoxins B1 (AfB1), B2 (AfB2), G1 (AfG1), G2 (AfG2), cyclopiazonic acid (CPA), fumonisin B1 (FB1), ochratoxin A (OA), patulin, secalonic acid D (SAD) and zearalenone (ZEN) were treated with 2, 10 and/or 20 weight% O3 over a period of 5.0 min and analysed by HPLC. Results indicated that AfB1 and AfG1 were rapidly degraded using 2% O3, while AfB2 and AfG2 were more resistant to oxidation and required higher levels of O3 (20%) for rapid degradation. In other studies, patulin, CPA, OA, SAD and ZEN were degraded at 15 sec, with no by-products detectable by HPLC. Additionally, the toxicity of these compounds (measured by a mycotoxin-sensitive bioassay) was significantly decreased following treatment with O3 for 15 sec. In another study, FB1 (following reaction with O3) was rapidly degraded at 15 sec, with the formation of new products. One of these appeared to be a 3-keto derivative of FB1. Importantly, degradation of FB1 did not correlate with detoxification, since FB1 solutions treated with O3 were still positive in two bioassay systems.

Individual and combined effects of fumonisin B1 present in Fusarium moniliforme culture material and diacetoxyscirpenol or ochratoxin A in turkey poults.

The individual and combined effects of feeding diets containing 300 mg fumonisin B1 (FB1), and 4 mg diacetoxyscirpenol (DAS) or 3 mg ochratoxin A (OA) were evaluated in two experiments using female turkey poults (Nicholas Large Whites) from day of hatch to 3 wk of age. When compared with controls, body weight gains were reduced 30% (Study 1) and 24% (Study 2) by FB1, 30% by DAS, 8% by OA, 46% by the FB1 and DAS combination, and 37% by the FB1 and OA combination. The efficiency of feed utilization was adversely affected by all treatments except FB1 in Experiment 2. Relative weights of the liver were significantly increased by all treatments except the DAS treatment. Serum concentrations of cholesterol were decreased and activities of aspartate aminotransferase and lactate dehydrogenase were increased and several hematological values were altered in poults fed FB1 alone and in combination with either DAS or OA. Results indicate additive or less than additive toxicity, but not toxic synergy, when poults are fed diets containing 300 mg FB1, and 4 mg DAS or 3 mg OA/kg of diet. The likelihood of encountering FB1, DAS, or OA at these concentrations in finished feed is small. However, under field conditions, other stress factors could alter the impact of these mycotoxins on the health and performance of poultry.

Adsorption of Cholera and Heat-Labile Escherichia coli Enterotoxins by Various Adsorbents: an In Vitro Study.

A variety of common inorganic adsorbents representing aluminas, zeolites, phyllosilicate clays, silica, and carbon were compared for their abilities to adsorb cholera toxin (CT) and heat-labile (LT) Escherichia coli enterotoxin. An appropriate assay system for the enterotoxins was developed using the Y-1 mouse-adrenal-tumor cell line, End points were determined by counting the number of rounded (cytotonic) cells at the relevant dilution. The adsorption varied between 177.0 × 106 and 109.6 × 102 CYTU (cytotonic titer unit) for CT with charcoal and boehmite respectively, and between 60.7 × 104 and 180.4 × 101 CYTU for LT with charcoal and boehmite respectively. Several of the other materials adsorbed CT and LT well, particularly attapulgite and sodium bentonite. The tightness of CT and LT binding to sodium bentonite and charcoal was determined by washing the adsorbent-enterotoxin pellets. Both toxins were strongly adsorbed, with dissociation of only 46.3 × 10° CYTU (<0.01 %) of the bound CT from sodium bentonite and 18.0× 101 CYTU (0.06%) of the bound LT from charcoal. The clay and charcoal pellets were assayed for their cytotonicity. Most of the activity of the adsorbed enterotoxins was lost: 93.1 and 89.6% for CT with sodium bentonite and charcoal, respectively, and 93.8 and 85.9% for LT with sodium bentonite and charcoal, respectively. The effect of dietary protein (casein) in enterotoxin adsorption by clay was also investigated. One percent casein (when adsorbed to sodium bentonite clay) completely blocked the adsorption of CT. When this protein-clay complex was treated with enzymes present in pancreatin, the digestive effect on the casein was sufficient to permit the adsorption of 137.6 × 101 CYTU of CT, although most of the blocking effect of casein remained. Further in vitro studies are needed to model the stomach, pancreatic, and intestinal digestive systems for determining if dietary proteins can block CT adsorption by clay in vivo. These results extend and support previously published data, obtained experimentally in rabbit and rat intestinal loops and from studies of children suffering spontaneous diarrhea, on the beneficial role of clays and other inorganic adsorbents in controlling enterotoxin activity.

Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of aflatoxin M1 in turkey poults. Lack of effect by activated charcoal on aflatoxicosis.

In one experiment, the effect of inorganic sorbents on the metabolic fate of aflatoxin B1 (AFB1) was studied in turkey poults. At 5 weeks of age, female poults were surgically colostomized and 9 days later orally dosed with 0.75 mg AFB1/kg BW. Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal (AC) were tested, by concomitant administration with AFB1. Urine was collected up to 48 h post-dosing and analyzed for aflatoxin M1 (AFM1) which was the major metabolite found in all treatment groups. Hydrated sodium calcium aluminosilicate, previously proven beneficial in alleviating aflatoxicosis in farm animals, reduced urinary AFM1 output when orally dosed simultaneous with AFB1. Also, acidic HSCAS and AC significantly decreased AFM1 excretion when administered concomitantly with AFB1. A second experiment was conducted to evaluate the ability of two types of AC to modify aflatoxicosis when added to aflatoxin (AF)-contaminated (from culture material) diets of turkey poults. Although AC was able to decrease AFM1 excretion in the first experiment, no protective effects from AF toxicity were observed in the feeding study.

Selective chemisorption and detoxification of aflatoxins by phyllosilicate clay.

Practical and effective strategies for the detoxification of aflatoxins are critically needed. We have shown that a phyllosilicate clay (HSCAS): i) tightly binds aflatoxins in aqueous solutions, including milk; ii) markedly decreases the bioavailability of radiolabeled aflatoxins; iii) greatly diminishes aflatoxicosis in young animals, i.e., rats, chickens, turkeys, lambs, and pigs; and iv) reduces the level of aflatoxin M1 in the milk from lactating dairy cattle and goats. In further studies, ligands with one or more of the functional groups in common with aflatoxin were reacted with HSCAS in vitro in an attempt to elucidate the specificity and mechanism of tight binding (or chemisorption). A chemisorption index (C alpha) was developed, allowing for direct comparison of various clay and zeolitic minerals with HSCAS. Chemisorption indices were determined by HPLC analysis of extracts of the supernatants and sorbed pellets (exhaustively extracted with methanol and chloroform). The beta-dicarbonyl system of aflatoxin was found to be essential for tight binding by HSCAS. Comparison of the chemisorption indices from various classes of compounds with spectral data (DRIFTS) indicated that the molecular mechanism of aflatoxin binding may involve the chelation of metal ions in HSCAS with the beta-dicarbonyl moiety in aflatoxin. Computer modeling was utilized to provide additional information. Preliminary evidence suggests that aflatoxin B1 may react at surfaces and within the interlayers of HSCAS particles. With knowledge of the mechanism involved, it has been possible to further enhance the propensity of HSCAS for aflatoxins.

Effects of phyllosilicate clay on the metabolic profile of aflatoxin B1 in Fischer-344 rats.

The phyllosilicate clay, hydrated sodium calcium aluminosilicate (HSCAS), has been shown to prevent aflatoxicosis in farm animals by reducing the bioavailability of aflatoxin. The present study was designed to determine the effects of HSCAS on the metabolism of aflatoxin B1 (AFB1) in an aflatoxin-sensitive species. Male Fischer-344 rats were orally dosed with 1.0, 0.5, 0.25 and 0.125 mg AFB1/kg body weight alone and in combination with 0.5% HSCAS. Urine samples were collected after 6, 24, 36, and 48 h. Aflatoxin M1 (AFM1) and aflatoxin P1 (AFP1) were detected in most urine samples, with or without HSCAS. AFM1 was found to be the major metabolite. Metabolite concentrations were significantly decreased in the presence of HSCAS, and more importantly, no additional metabolites were detected. Our results suggest that the AFB1-HSCAS complex was not significantly dissociated in vivo, and support earlier findings that HSCAS tightly binds aflatoxin.

Development and evaluation of a minicolumn assay for the detection of aflatoxin M1 in milk.

A practical field method for the chemiselective immobilization and detection of aflatoxin M1 in milk has been developed and is being marketed. In this new method, aflatoxin M1 is selectively adsorbed at the interface of a layer of neutral sand and a band of magnesium silicate (Florisil) packed in a glass minicolumn. Aflatoxin M1, at > or = .5 ppb in contaminated milk, can be easily visualized as a band of bright blue fluorescence. Briefly, raw or homogenized and pasteurized milk is diluted with water (1: 1, vol/vol) and passed through a C18 cartridge. Aflatoxin M1 is then partitioned by polarity, eluted with acetone-methylene chloride, and added to the minicolumn. The minicolumn is washed and viewed under long wave UV light. The limit of detection for this assay was .2 ppb, which was similar to the .3 ppb obtained using an immunoaffinity column, followed by minicolumn detection. The assay was accurate, rapid, easy to perform, and stable.