L. F. Kubena

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.

Foodborne Salmonella ecology in the avian gastrointestinal tract

Foodborne Salmonella continues to be a major cause of salmonellosis with Salmonella Enteritidis and S. Typhimurium considered to be responsible for most of the infections. Investigation of outbreaks and sporadic cases has indicated that food vehicles such as poultry and poultry by-products including raw and uncooked eggs are among the most common sources of Salmonella infections. The dissemination and infection of the avian intestinal tract remain somewhat unclear. In vitro incubation of Salmonella with mammalian tissue culture cells has shown that invasion into epithelial cells is complex and involves several genetic loci and host factors. Several genes are required for the intestinal phase of Salmonella invasion and are located on Salmonella pathogenicity island 1 (SPI 1). Salmonella pathogenesis in the gastrointestinal (GI) tract and the effects of environmental stimuli on gene expression influence bacterial colonization and invasion. Furthermore, significant parameters of Salmonella including growth physiology, nutrient availability, pH, and energy status are considered contributing factors in the GI tract ecology. Approaches for limiting Salmonella colonization have been primarily based on the microbial ecology of the intestinal tract. In vitro studies have shown that the toxic effects of short chain fatty acids (SCFA) to some Enterobacteriaceae, including Salmonella, have resulted in a reduction in population. In addition, it has been established that native intestinal microorganisms such as Lactobacilli provide protective mechanisms against Salmonella in the ceca. A clear understanding of the key factors involved in Salmonella colonization in the avian GI tract has the potential to lead to better approach for more effective control of this foodborne pathogen.

Bactericidal effect of sodium chlorate on Escherichia coli O157:H7 and Salmonella Typhimurium DT104 in rumen contents in vitro.

Escherichia coli O157:H7 and Salmonella Typhimurium DT104 are important foodborne pathogens affecting the beef and dairy industries and strategies are sought to rid these organisms from cattle at slaughter. Both pathogens possess respiratory nitrate reductase that also reduces chlorate to the lethal chlorite ion. Because most anaerobes lack respiratory nitrate reductase, we hypothesized that chlorate may selectively kill E. coli O157:H7 and Salmonella Typhimurium DT104 but not potentially beneficial anaerobes. In support of this hypothesis, we found that concentrations of E. coli O157:H7 and Salmonella Typhimurium DT104 were reduced from approximately 1,000,000 colony forming units (CFU) to below our level of detection (< or = 10 CFU) following in vitro incubation (24 h) in buffered ruminal contents (pH 6.8) containing 5 mM added chlorate. In contrast, chlorate had little effect on the most probable number (mean +/- SD) of total culturable anaerobes (ranging from 9.9 +/- 0.72 to 10.7 +/- 0.01 log10 cells/ml). Thus, chlorate was bactericidal to E. coli O157:H7 and Salmonella Typhimurium DT104 but not to potentially beneficial bacteria. The bactericidal effect of chlorate was concentration dependent (less at 1.25 mM) and markedly affected by pH (more bactericidal at pH 6.8 than pH 5.6).

Efficacy of Zeolitic Ore Compounds on the Toxicity of Aflatoxin to Growing Broiler Chickens

Commercially available zeolitic ore compounds, when incorporated into the diets at 0.5%, were evaluated for their ability to reduce the deleterious effects of 3.5 mg aflatoxin/kg feed on growing broiler chickens from 1 day to 3 weeks of age. In a series of four experiments, the compounds used included the following: mordenite (particle size of -20 mesh; Zeomite); clinoptilolite (particle size of -20 mesh; Zeobrite); SC Zeolite (particle size of -20 mesh); and clinoptilolite (particle size of -35 mesh; Clino 1) or clinoptilolite (particle sizes of -20 plus +35 mesh; Clino 2). Results demonstrated that 0.5% Zeobrite, Clino 1, or Clino 2 added to aflatoxin-contaminated diets did not significantly (P < 0.05) diminish the toxicity of high concentrations of aflatoxin to growing broiler chicks. Zeomite mordenite ore reduced the toxicity of aflatoxin to growing chicks by 41%, as indicated by weight gains, liver weight, and serum biochemical measurements, which compares favorably with its in vitro binding capacity to aflatoxin. SC Zeolite reduced weight-gain toxicity of aflatoxin by approximately 29%.

Effect of sodium chlorate on Salmonella Typhimurium concentrations in the weaned pig gut

Salmonella cause economic losses to the swine industry due to disease and compromised food safety. Since the gut is a major reservoir for Salmonella, strategies are sought to reduce their concentration in pigs immediately before processing. Respiratory nitrate reductase activity possessed by Salmonella also catalyzes the intracellular reduction of chlorate (an analog of nitrate) to chlorite, which is lethal to the microbe. Since most gastrointestinal anaerobes lack respiratory nitrate reductase, we conducted a study to determine if chlorate may selectively kill Salmonella within the pig gut. Weaned pigs orally infected with 8 x 10(7) CFU of a novobiocin- and nalidixic acid-resistant strain of Salmonella Typhimurium were treated 8 and 16 h later via oral gavage (10 ml) with 0 or 100 mM sodium chlorate. Pigs were euthanized at 8-h intervals after receiving the last treatment. Samples collected by necropsy were cultured qualitatively and quantitatively for Salmonella and for most probable numbers of total culturable anaerobes. A significant (P < 0.05) chlorate treatment effect was observed on cecal concentrations of Salmonella, with the largest reductions occurring 16 h after receiving the last chlorate treatment. An observed treatment by time after treatment interaction suggests the chlorate effect was concentration dependent. Chlorate treatment may provide a means to reduce foodborne pathogens immediately before harvest.

Review on the role of dietary zinc in poultry nutrition, immunity, and reproduction

Zinc is an important nutrient in animal metabolism. In poultry, zinc serves not only as a nutrient but can also be used as a dietary supplement to manipulate the reproductive system of the bird. This article summarizes the general biochemistry, physiology, and nutritional aspects of zinc metabolism to provide a brief overview on what is known regarding zinc. The potential role of zinc in poultry immune response, Salmonella infection, and molting are emphasized.

Induced moulting issues and alternative dietary strategies for the egg industry in the United States

The United States (U.S.) poultry industry continues to implement induced moulting to extend egg production in commercial laying flocks. Achieving an optimal moult requires dietary manipulation to cause a complete regression of the reproductive organs and cessation of egg production. This is followed by rejuvenation and initiation of an additional egg laying cycle. Currently feed withdrawal is the primary means to initiate moult and is regarded as an optimal approach for achieving post-moult performance. However, removal of feed can lead to potential physiological stress in laying hens as well as an increased susceptibility to Salmonella enteritidis colonization and invasion. To retain the ecological benefits of induced moult will require development, testing and implementation of alternative dietary approaches that minimizes these problems and increase the egg production and egg quality benefits associated with the additional egg laying cycles. Strategies for accomplishing this are discussed.

The Influence of a Fructooligosaccharide Prebiotic Combined with Alfalfa Molt Diets on the Gastrointestinal Tract Fermentation, Salmonella Enteritidis Infection, and Intestinal Shedding in Laying Hens

Molting is a natural process, which birds undergo to rejuvenate their reproductive organs. The US poultry egg production industry has used feed withdrawal to effectively induce molt; however, susceptibility of Salmonella Enteritidis has encouraged the development of alternative methods. Previous research conducted in our laboratory showed that alfalfa is effective at molt induction and provides equivalent postmolt production numbers and quality when compared with feed withdrawal. In the attempt to further increase the efficacy of alfalfa molt diet and decrease the chicken susceptibility to Salmonella Enteritidis during molt, fructooligosaccharide (FOS) was added to a combination of 90% alfalfa and 10% layer ration in 2 levels (0.750 and 0.375%). Ovary and liver colonization by Salmonella Enteritidis in 3 and 2 of the 4 trials, respectively, were reduced (P <or= 0.05) in hens fed FOS-containing diets compared with hens subjected to feed withdrawal. Significant decreases in ce-cal Salmonella Enteritidis counts were also observed in 2 of the 4 trials. In 3 of the 4 trials, the same diets did not affect (P > 0.05) the production of cecal total volatile fatty acids when compared with hens undergoing feed withdrawal. However, in all 3 alfalfa molt diets, the concentrations of lactic acid were greater (P <or= 0.05) than hens with feed withdrawal, but no differences (P > 0.05) were observed among hens fed alfalfa combined with FOS and hens fed alfalfa/layer ration without FOS. Overall, given the similarities between hens fed 0.750% FOS (H) and 0.375% FOS (L), molt diets combined with the lower level of FOS should be sufficient.

Comparison of Two Hydrated Sodium Calcium Aluminosilicate Compounds to Experimentally Protect Growing Barrows from Aflatoxicosis

Two formulations of hydrated sodium calcium aluminosilicate (HSCAS-1 and HSCAS-3), anti-caking agents for mixed feed, were added to the diets of growing barrows and were evaluated for their potential to diminish the clinical signs of aflatoxicosis. The experimental design consisted of 8 barrows (2 replicates of 4 each/treatment) assigned to 1 of the following 6 treatment diets (total of 48): 1) 0 g of HSCAS-1 or HSCAS-3 and 0 mg of aflatoxin (AF)/kg of feed (control); 2) 5 g HSCAS-1/kg of feed; 3) 5 g HSCAS-3/kg of feed; 4) 3 mg AF/kg of feed; 5) 3 mg AF plus 5 g HSCAS-1/kg of feed; or 6) 3 mg AF plus 5 g HSCAS-3/kg of feed. Barrows were maintained in indoor concrete-floored pens, with feed and water -available for 28 days (from 8 to 12 weeks of age). Barrows were observed twice daily and weighed weekly, and blood samples were collected at day 28 for hematologic, immunologic, and serum biochemical measurements. At the termination of the study, barrows were euthanized and necropsied. Barrow body weight gains were diminished, compared to those of controls, by consumption of AF alone and both of the AF plus HSCAS diets; however, the AF plus HSCAS-1 and AF plus HSCAS-3 barrow body weight gains were significantly greater (P < 0.05) than those of the AF-alone barrows, No toxic responses or performance differences were noticed for barrows consuming either of the HSCAS-alone diets. Serum concentrations of alkaline phosphatase, gamma glutamyltransferase, calcium, cholesterol, albumin, triglycerides, and urea nitrogen were altered in barrows of the AF-alone treatment. The use of HSCAS prevented most but not all of the AF-induced changes in biochemical values. Immunologic measurements that were adversely affected by AF included mitogen-induced lymphoblastogenesis and peritoneal macrophage activity and function. The addition of HSCAS to AF-contaminated diets protected barrows from some of these toxic changes. Although immunologic measurements in the AF plus HSCAS groups were significantly different than those of the AF-alone group, values were still not equivalent to those of controls. These findings suggest that HSCAS-1 and HSCAS-3 are equal in their ability to protect against the toxicity of AF. Although these compounds may offer a novel approach to the preventive management of aflatoxicosis in livestock and poultry, HSCAS is not approved by the US Food and Drug Administration for treatment of animal diets for prevention of mycotoxicosis.

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.