R. R. Marquardt

A Strain of Enterococcus faecium (18C23) Inhibits Adhesion of Enterotoxigenic Escherichia coli K88 to Porcine Small Intestine Mucus

ABSTRACT Few studies, if any, have addressed the adhesion of enterococci to the intestinal mucosa and their interference with the adhesion of pathogens, although more than 60% of probiotic preparations in the market contain strains of enterococci. The objective of this study was to investigate if Enterococcus faecium 18C23 has the ability to inhibit the adhesion of Escherichia coli K88ac and K88MB to the small intestine mucus of piglets. Approximately 9% ofE. faecium 18C23 organisms adhered to the small intestine mucus, and the adhesion was found to be specific. Living E. faecium 18C23 culture efficiently inhibited the adhesion ofE. coli K88ac and K88MB to the piglet intestine mucus. Inhibition of the adhesion of E. coli K88ac to the small intestine mucus was found to be dose dependent. Inhibition of >90% was observed when 109 CFU or more of living E. faecium 18C23 culture per ml was added simultaneously withE. coli to immobilized mucus. The substances from both the 18C23 cells and the spent culture supernatant contributed to the inhibition of adhesion of E. coli K88 to the small intestine mucus receptors. The inhibiting effect was not solely a pH effect since considerable inhibitory action was demonstrated after neutralizing the mixture or spent culture supernatant to pH 7.0. Part of the inhibition of adhesion of E. coli K88ac by E. faecium 18C23 or its supernatant might occur through steric hindrance.

The nutritive value of barley, rye, wheat and corn for young chicks as affected by use of a Trichoderma reesei enzyme preparation

Two experiments were conducted to establish the effect on the performance of growing Leghorn chicks of different concentrations of a crude enzyme preparation when added to diets containing high amounts of barley, rye, wheat or corn. In the first experiment it was shown that the enzyme which had high xylanase and cellulase activities considerably improved (P 0.05). The wheat and barley-based diets that were supplemented with enzyme yielded weight gains that were the same as obtained with the corn diets. Enzyme supplementation also improved (P 0.05). Enzyme treatment reduced the water content of the colon in birds fed diets containing all of the different grains (P<0.05) with the effect being greatest for chicks fed barley (6%) and wheat (5%), intermediate for those fed rye (3%) and lowest for the corn-fed birds (1%). These results demonstrate that a low dietary inclusion rate of a crude enzyme preparation that has high activities of two enzymes (xylanase and cellulase) can considerably improve the nutritional value of barley and rye, probably that of wheat, but not that of corn.

Use of enzymes to improve nutrient availability in poultry feedstuffs

Enzyme supplementation of cereal-based diets can significantly improve chick performance by increasing the rate of gain, efficiency of feed utilization, the apparent metabolizable energy and digestibilities of dry matter, fat and protein, with excellent improvements being obtained with diets containing rye, oats and barley. Less dramatic results are often obtained with wheat. In addition, enzyme treatment decreases the moisture content of excreta, which, together with improved dry matter digestibility, reduces the total amount of excreta produced and therefore reduces management and environmental problems. Enzyme supplementation also improves the nutritional value of lupins, and reduces the length and size of various sections of the gastrointestinal tract and the size of the pancreas of chickens. Dose-response studies with different amounts of supplemental enzymes when added to a rye-based diet demonstrated that there was a high linear correlation (r 2 > 0.91, P < 0.05) between the concentration of enzyme when transformed into its logarithmic values and the corresponding improvements in weight gain or the feed-to-gain ratio. The log-linear model shows that for every ten-fold increase in the amount of enzyme in the diet there was a two-fold and not a ten-fold incremental improvement in chick performance. These studies suggest that there is a simple relationship between the amount of enzyme added to the diet and the resulting improvements that are obtained. Overall, enzymes when properly used can produce significant improvements in chick performance and can reduce the excretion of undigested nutrients.

Effect of dietary vicine and vitamin E supplementation on the productive performance of growing and laying chickens

1. Experiments were conducted to study the effects of dietary vicine (2, 6-diamino-4, 5 dihydroxy pyrimidine-5 (beta-D-glucopyranoside)) and supplemental vitamin E on the performance of laying hens and growing chicks, haemolysis of erythrocytes than birds fed on a control diet. 3. Vicine when fed to laying hens had a very dramatic effect. It depressed food consumption, egg weight, fertility and hatchability of eggs. Packed cell volume and erythrocyte haemoglobin levels and led to increased liver weights, liver glutathione levels, liver and plasma lipid levels, plasma lipid peroxide levels and erythrocyte haemolysis in vitro. Liver protein and plasma vitamin E:lipid levels were not altered. Vitamin E supplementation slightly increased egg weights, markedly improved fertility and hatchability of eggs and lowered liver weights and lipid levels but did not affect the other factors examined. 4. It is concluded that vicine which was isolated from faba beans (Vicia faba L.) has a marked influence on the metabolism of the laying hen and only a slight effect on growing chick. Vicine or its metabolites or both cause peroxidation of cellular components which result in abnormal lipid transport of synthesis or both, increased fragility of erythrocytes, and reduced fertility. These effects are overcome to varying extents by supplemental vitamin E.

Comparison of toxicity of different mycotoxins to several species of bacteria and yeasts: use of Bacillus brevis in a disc diffusion assay

Twelve species of bacteria and two species of yeast were tested for sensitivity against 11 different mycotoxins using a disc diffusion assay. Among the bacterial species, Bacillus brevis appeared to be the most sensitive microorganism, being sensitive to eight mycotoxins (AFB1, ochratoxin A [OA], citrinin [CT], patulin [PAT], penicillic acid [PA], cyclopiazonic acid [CPA], penitrem A [PT-A], and zearalenone [ZEE]). This microorganism was not affected by a high concentration (500 μg per disc) of any of the trichothecenes (T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and deoxynivalenol). Kluyveromyces marxianus , a species of yeast, was the only microorganism that was inhibited by all four of the trichothecenes but was not inhibited by the other mycotoxins. The area of the inhibition zone produced by some of the mycotoxins such as OA with the B. brevis assay was dramatically influenced by the pH of the medium, while the toxicity of other mycotoxins such as AFB1 was relatively pH independent. The sensitivity of the B. brevis assay also tended to decrease at agar volumes above 6 ml and as the number of microbes per plate increased. The lowest amounts of the different ochratoxins; OA, OB and OC (OA ethyl ester) that could be detected under optimal conditions were 0.5, 20, and 2 μg per disc, respectively. The lowest amounts of CPA, AFB1 CT, PAT, PA, PT-A, and ZEE that were detectable were 0.5, 1, 1, 1, 1, 1, and 10 μg per disc, respectively. These results demonstrate that B. brevis can be used as a positive indicator organism to detect the presence of several common non-trichothecene mycotoxins. The results demonstrate that, used together, B. brevis and K. marxianus can be used as indicator organisms in a bioassay approach to the detection of several of the most common mycotoxins.

Effect of T-2 toxin on in vivo lipid peroxidation and vitamin E status in mice

The effects of an acute administration of T-2 toxin on vitamin E status and the corresponding degree of lipid peroxidation, as determined by the plasma and organ content of malondialdehyde (MDA), was studied in mice. The effects of T-2 toxin administration on the body weight and weights of liver, spleen and thymus were also assessed. T-2 toxin was administered in doses ranging from 1 to 6.25 mg/kg body weight, depending on the experiment, while the dietary content of vitamin E ranged from near 0 to 5000 IU/kg. There was a significant decrease in vitamin E content of plasma after the administration of the toxin with the concentrations remaining low for periods as long as 48-72 h. MDA content of liver increased significantly after 24-48 h of toxin administration in contrast to the controls. However, MDA levels returned to the control range after 72 h. The concentrations of MDA in liver were inversely related to the vitamin E content of the diet, and were always higher for the toxin-treated animals (significant linear regression between MDA content of liver and the log10 of vitamin E content of the diet). Weights of spleen and thymus decreased after T-2 toxin administration; however, the weight of liver either increased or did not change in the different experiments. In conclusion, T-2 toxin treatment of mice increased lipid peroxidation in the liver as measured by MDA production. This process was maximal after 48 h of T-2 challenge, and decreased thereafter. Plasma alpha-tocopherol levels decreased as soon as 6 h after the toxin challenge, while MDA did not increase until there was a severe depletion of vitamin E. These changes were accompanied by decrease in weight of spleen and thymus.

INDUCTION OF FREE RADICALS IN HEPATOCYTES, MITOCHONDRIA AND MICROSOMES OF RATS BY OCHRATOXIN A AND ITS ANALOGS

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and O alpha; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe3+ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe3+. The spin adduct hyperfine splitting constants indicated the presence of alpha-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.

Age-related differences in the toxicity of ochratoxin A in female rats.

Ochratoxin A (OTA) is a mycotoxin found in food and feedstuffs of plant and animal origin. OTA exposure is related to nephropathy in humans. Age-related differences, especially in nephro- and immunotoxicity of OTA, were investigated in young adult (aged 12 weeks) and old (aged 27-30 months) female SPF Wag rats, treated by gavage with 0, 0.07, 0.34 or 1.68 mg OTA/kg body weight for 4 weeks. In both age groups, survival was significantly decreased in the highest dose group. Clinical condition, body weight, clinical chemistry parameters (ALAT, ASAT, creatinin and urea) and target organs (as identified by weight and pathology - kidney, liver, adrenals, forestomach and brain) were affected by age and dose, but often more severely in old than in young rats. OTA induced primarily nephropathy. Old rats were more sensitive to induction of tubular karyomegaly and vacuolation/necrosis. In young rats, OTA induced a dose-related thickening of the basement membrane and reduction in splenic T-cell fraction. Decreased IgG levels were seen at 0.34 mg/kg OTA (young and old rats) and 1.68 mg/kg OTA (young rats). Vacuolation of the white brain matter (cerebellar medulla and ventral parts of the brain stem) was significantly increased in young rats at 0.34 and 1.68 mg/kg OTA and in old rats at 0.07 and 0.34 mg/kg OTA. It was concluded that: (1) the profiles of OTA toxicity for both age groups are similar, with the kidney and possibly the brain being primary target organs; (2) based on clinical and pathological data old rats are more sensitive to OTA than young rats; and (3) the immune system is probably not the primary target of OTA toxicity.

Free Radical Generation as Induced by Ochratoxin A and Its Analogs in Bacteria (Bacillus brevis)

Lipid peroxidation is considered as one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC, and Oα; and four synthetic analogs, d-OA, the ethylamide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in bacteria with Bacillus brevis as a model system. The uptake of the different ochratoxins by B. brevis varied substantially depending on the molecular structures. Electron paramagnetic resonance spectroscopy using α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone as a spin trapping agent showed an enhanced free radical generation due to the addition of OA and most of the analogs. The EPR signals could be further enhanced by the addition of Ca2+, a calcium ionophore and an ATPase uncoupler, whereas they were eliminated by incubating the growing cells with vitamin E. The spin adduct hyperfine splitting constants indicate the presence of α-hydroxyethyl radicals resulting from generated hydroxyl radicals, which are trapped by α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone. The results further suggest that OA induces free radical production in this model system by enhancing the permeability of the cellular membrane to Ca2+.

Mycotoxin formation in hulless barley during granary storage at 15 and 19% moisture content

Abstract Eleven-kilogram parcels of hulless barley ( Hordeum vulgare L. cv. Condor) at 15 and 19% initial moisture content were kept in simulated storage in a Manitoba farm granary for 20 weeks (June 1996–October 1996) to determine biotic and abiotic changes and mycotoxin production. Temperature, moisture content, CO 2 levels, ergosterol content, seed germination, microfloral infection, and the presence of major mycotoxins were monitored. Ochratoxin A, citrinin and sterigmatocystin reached mean levels of 24, 38 and 411 ppb by 20 weeks in the 19% moisture content barley, but were absent in the 15% moisture content barley; no other mycotoxins were detected. Penicillium species and Aspergillus versicolor (Vuill.) Tiraboschi comprised the predominant microflora. The effect of storage time was apparent at both 15 and 19% moisture content for grain temperature, Alternaria alternata (Fr.) Keissler, Penicillium species and Aspergillus versicolor . At 19% moisture content, storage time also affected moisture content, CO 2 level, ergosterol content, seed germination, and mycotoxin production. At 19% moisture content, elevated ergosterol levels at weeks 4 and 8 appear to offer early warning of the appearance of sterigmatocystin at week 12, and of ochratoxin A and citrinin at week 20.