Recovery of Campylobacter from feed using different enrichment media.

Studies were conducted to investigate the recovery of Campylobacter from feed. The impact of feed moisture, water activity, pH, number of background microflora and the use of different antibiotic supplements in Campylobacter enrichment broth (CEB) on Campylobacter recovery were evaluated in five studies. Broiler starter feed was inoculated with 104 -105 cfu of Campylobacter/g and stored at 24 °C and 43% RH. Enrichment culture was conducted on the day of inoculation or 24 h post inoculation and every 48 h of storage thereafter for 14 d. Feed moisture, water activity, pH and level of background microflora were not correlated with Campylobacter recovery. The incubation of feed in CEB with no antibiotic supplement resulted in the number of background microflora increasing to 109 cfu/g and the pH of the media decreasing to pH 4-5 impacting recovery. Addition of certain antimicrobial supplements to CEB reduced background microflora growth and maintained a near neutral pH. Campylobacter was recovered up to 10 days post inoculation when using CEB containing antibiotic supplements compared to 1 day in CEB. These findings suggest that Campylobacter can be recovered from feed and the type of antimicrobial supplement utilized influences recovery by controlling extraneous microbial growth which occurs during enrichment.

Isolation of Salmonella spp. from Animal Feed.

The isolation of Salmonella from feed is challenging and adjustments need to be made in order to accurately isolate the pathogen from feed. This is due to the complex nature of the feed matrix, which is both porous and fibrous. The outlined method below contains the essential components of a successful Salmonella methodology for the analysis of feed that overcomes the limitations of currently available methods.

Optimizing buffering chemistry to maintain near neutral pH of broiler feed during pre-enrichment for Salmonella.

Salmonella is a human pathogen that can accompany live broilers to the slaughter plant, contaminating fully processed carcasses. Feed is one potential source of Salmonella to growing broilers. Monitoring feed for the presence of Salmonella is part of good agricultural practice. The first step in culturing feed for Salmonella (which may be at low numbers and sub-lethally stressed) is to add it to a pre-enrichment broth which is incubated for 24 h. During the course of pre-enrichment, extraneous bacteria metabolize carbohydrates in some feed and excrete acidic byproducts, causing the pH to drop dramatically. An acidic pre-enrichment pH can injure or kill Salmonella resulting in a failure to detect, even if it is present and available to infect chickens. The objective of this study was to test an array of buffering chemistries to prevent formation of an injurious acidic environment during pre-enrichment of feed in peptone water. Five grams of feed were added to 45 mL of peptone water buffered with carbonate, Tris pH 8, and phosphate buffering ingredients individually and in combination. Feed was subjected to a pre-enrichment at 35°C for 24 h; pH was measured at 0, 18, and 24 h. Standard phosphate buffering ingredients at concentrations up to 4 times the normal formulation were unable to fully prevent acidic conditions. Likewise, carbonate and Tris pH 8 were not fully effective. The combination of phosphate, carbonate, and Tris pH 8 was the most effective buffer tested. It is recommended that a highly buffered pre-enrichment broth be used to examine feed for the presence of Salmonella.

Salmonella in commercially manufactured feeds.

We collected 886 samples (68 feed ingredient samples, 189 dust samples, and 629 feed samples) from 3 feed mills each of which produced between 100,000 and 400,000 tons of feed a year. Samples were collected on 3 d (Monday, Wednesday, and Friday), during 2 seasons (early spring and summer), and between 0700 and 1700 h approximately once per hour. Samples were collected from 5 locations within each mill: ingredient receiving, at the mixer, at the pellet mill, from pellet coolers, and at load-out. Temperatures were taken of the samples obtained at the pellet mill immediately following collection. All samples were analyzed for Enterobacteriaceae counts (EC) and Salmonella. The data confirm that feed ingredients and dust can be a major source of Salmonella contamination in feed mills. There were no differences (P < 0.05) in the Salmonella contamination rates of samples collected in spring as compared with samples collected in summer. Salmonella contamination rates were observed to be higher in samples collected on Friday compared with samples collected on Monday or Wednesday, an effect that may be management related. Data collected at the pellet mill clearly illustrate the uneven distribution of Salmonella contamination in feed as well as the need for control of dust around the pellet mill. Feed samples (both mash and pellets) contaminated with Salmonella contained significantly higher EC than samples not contaminated with Salmonella. Thus, EC may provide some indication of the likelihood of Salmonella contamination in feed samples.

Electrical remodeling in pressure-overload cardiac hypertrophy: role of calcineurin.

BACKGROUND: Myocyte hypertrophy accompanies many forms of heart disease, but its contribution to electrical remodeling is unknown. METHODS AND RESULTS: We studied mouse hearts subjected to pressure overload by surgical thoracic aortic banding. In unbanded control hearts, action potential duration (APD) was significantly longer in subendocardial myocytes compared with subepicardial myocytes. Hypertrophy-associated APD prolongation was significantly greater in subendocardial myocytes compared with subepicardial myocytes, indicating stress-induced amplification of repolarization dispersion. To investigate the underlying basis, we performed voltage-clamp recordings on dissociated myocytes. Under control unoperated conditions, subendocardial myocytes exhibited significantly less transient outward current (I(to)) than did subepicardial cells. Hypertrophy was not associated with significant changes in I(to), sustained current, or inward rectifier current densities, but peak L-type Ca(2+) current density (I(Ca,L)) increased 26% (P<0.05). Recovery from I(Ca,L) inactivation was accelerated in hypertrophied myocytes. Inhibition of calcineurin with cyclosporin A prevented increases in heart mass and myocyte size but was associated with an intermediate APD. The hypertrophy-associated increase in I(Ca,L) and the accelerated recovery from inactivation were blocked by cyclosporin A. CONCLUSIONS: These data reveal regional variation in the electrophysiological response within the left ventricle by way of a mechanism involving upregulated Ca(2+) current and calcineurin. Furthermore, these results reveal partial uncoupling of electrophysiological and structural remodeling in hypertrophy.

Studies on oxalate oxidase from beet stems upon immobilization on concanavalin A.

Oxalate oxidase (EC 1.2.3.4) was purified from beet stems and immobilized on concanavalin A. The bound enzyme showed a high resistance of denaturation and increased the storage stability at 4 degrees C. The immobilized oxidase showed a broad optimum at pH 3.5-5, compared to the free enzyme with a sharp optimum at pH 4.5. There was a 3-fold increase in the apparent Km value on immobilization. The lectin interaction also eliminated the inhibitory effect produced on the enzyme by azide, nitrate and glycollate. The stimulatory effect on the enzyme activity by the flavins was not seen with the bound enzyme. The interaction of oxidase on concanavalin A-Sepharose 4B column and its reversal with methyl alpha-D-mannoside, indicated the presence of polysaccharides. The glycoprotein nature was further confirmed by periodic acid-sciff staining procedure of the enzyme after gel electrophoresis.

Comparative toxicity of scirpentriol and its acetylated derivatives.

Scirpentriol (STO) and its seven acetylated derivatives, 3-, 4- and 15-monoacetoxyscirpenol (MAS), 3,4-, 3,15-, and 4,15-diacetoxyscirpenol (DAS), and 3,4,15-triacetoxyscirpenol (TAS) were compared for their acute oral lethality in broiler chicks, lethality in brine shrimp, and dermal toxicity in guinea pigs. Of the eight toxins, 4,15-DAS was the most toxic in the three assays, 3-MAS was the least toxic in brine shrimp and dermal assays, and 3,4-DAS was the least toxic in the chick assay. There was a difference of about a 100-fold and 20-fold, respectively, between 4,15-DAS and 3-MAS in dermal toxicity and brine-shrimp toxicity, as well as a difference of more than 16-fold between 4,15-DAS and 3,4-DAS in chick toxicity. In general, a free hydroxy group at the 3-position was a primary determinant of toxicity. Toxicity in the scirpenol family did not follow precisely the pattern reported earlier for the T-2 toxin family of trichothecene toxins, in which a decrease in the number of acyl groups was accompanied by a decrease in toxicity. At necropsy, the predominant sign in chicks was petechial hemorrhaging, primarily in the gastrointestinal tract and in the vascular beds of the beaks and the toe nails. The 4,15-DAS and 15-MAS were about 3 times more toxic in chicks than aflatoxin. All members of the scirpenol family of trichothecene mycotoxins appeared sufficiently toxic to warrant attention whenever field outbreaks occur. Apparently, brine shrimp and dermal assays are successful predictors of chick lethality by the more toxic trichothecenes and are less suitable for predicting the activity of the less toxic trichothecenes.

Nitroreduction of 6-nitrobenzo[a]pyrene: a potential activation pathway in humans.

6-Nitrobenzo[a]pyrene, an environmental pollutant, was metabolized by human intestinal microflora to 6-nitrosobenzo[a]pyrene and 6-aminobenzo[a]pyrene. The two-electron reduction product 6-nitrosobenzo[a]pyrene exhibited strong direct-acting mutagenicity in the Salmonella typhimurium assay. These results imply that 6-nitrobenzo[a]pyrene can be hazardous to human health via a nitroreduction activation pathway and opens the possibility that other nitro-polycyclic aromatic hydrocarbons that are not direct-acting mutagens may be activated in vivo by a similar mechanism.

Metabolism of 1-, 3-, and 6-nitrobenzo[a]pyrene by intestinal microflora.

The compounds 1-, 3-, and 6-nitrobenzo[a]pyrene (nitro-BaP) are environmental pollutants and have been shown to be potent bacterial mutagens. The anaerobic metabolism of these isomeric nitro-BaPs was investigated by the incubation of rat intestinal microflora with each isomer for 48 h. Aliquots were removed at several time intervals, extracted, fractionated by high-pressure liquid chromatography (HPLC), and the radioactivity determined. Metabolites were identified by comparison of their chromatographic, ultraviolet-visible absorption, and mass spectral properties with those of authentic standards. The order of the extent of nitroreduction for these isomers was 3-nitro-BaP greater than 6-nitro-BaP greater than 1-nitro-BaP. After 48 h of exposure, 84% of the added 3-nitro-BaP was present as 3-amino-BaP, 51% of the 6-nitro-BaP was metabolized to 6-amino-BaP, and 1-nitro-BaP was reduced to 1-amino-BaP (13%) and 1-nitro-BaP (4%). The order of the extent of microbial nitroreduction for these nitro-BaP isomers is different from the predictions based on electronic and steric hindrance effects. These results suggest that intestinal microflora nitroreductases exhibit a markedly high degree of substrate specificity toward nitro-BaPs that affects the extent of nitroreduction.

Effect of dietary fat level on dose response relationships during aflatoxicosis in young chickens.

The effect of aflatoxin in poultry is greater on birds fed a low fat diet, but it is not known whether this effect is associated with a lower apparent minimum effective dose (MED), altered slope of the response curve, or both. Aflatoxin at 16 dosages ranging from 0 to 3.797 micrograms/g of feed was fed to six groups of 15 young chickens per treatment ingesting a 2 or 4% fat diet for 3 wk. The weights of the body, liver, bursa of Fabricius, and spleen and the total lipid content of the liver were measured. Mathematical models were fitted to the data and dose-response curves were predicted as continuous functions of aflatoxin concentration. Quadratic polynomials fit body weight and spleen weight whereas plateau-linear models fit liver weight and liver lipid content in both 2 and 4% fat diets. The weight of the bursa of Fabricius was fit equally well by quadratic and linear plateau models. Dietary fat had negligible effects on the apparent MED (micrograms of aflatoxin per gram of feed) for body, liver and spleen weights, which were calculated from the modeling approach to be 1.37 and 1.41, 1.68 and 1.69, and 1.49 and 1.46 on 2 and 4% fat diets, respectively. The apparent MED for liver lipid content was appreciably lower for birds fed the 2% fat diet than those fed the 4% fat diet (.88 and 1.62, respectively). Similarly, the apparent MED for the bursa was 1.48 and 1.74 for birds fed the 2 and 4% fat diets, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of dietary protein level on dose response relationships during aflatoxicosis in young chickens.

It is well known that the effect of aflatoxin is enhanced by a low protein diet, but whether this is associated with a lower apparent minimum effective dose, increased slope of response curves, or both has not been investigated previously. Aflatoxin at 12 dosages ranging from 0 to 2.34 micrograms/g of feed was fed to eight groups of 10 young chickens per treatment consuming a 10.00 or 12.75% protein diet for 3 weeks. The body weights, liver weights relative to body weights, and total lipid content of the livers were determined. Mathematical models were fitted to the data and from the appropriate equations the dose-response curves were predicted as continuous functions of aflatoxin concentration. A quadratic polynomial fit body weight data on the 12.75% protein diet whereas a plateau-linear model fit body weight data on the 10.00% protein diet. This implies that in a low protein diet aflatoxin affects only one of the factors controlling growth. Plateau-linear models fit liver relative weight and liver lipid content data on both 10.00 and 12.75% diets. For both variables the lower protein diet decreased the apparent minimum effective dose and increased the positive slope of the linear response. The apparent minimum effective doses (micrograms of aflatoxin per gram of feed) in this experimental system were calculated from the modeling approach to be 1.21 and 2.00 for body weight, 1.08 and 1.65 for liver lipids, and 1.45 and 2.34 for liver relative weight in 10.00 and 12.75% protein diets, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced production of pancreatic digestive enzymes during aflatoxicosis in egg-type chickens.

As aflatoxin causes malabsorption and its toxicity is enhanced by a low protein diet, digestive enzymes formed in the pancreas apparently are influenced by aflatoxin. This hypothesis was investigated in a 2 X 2 factorial experiment. Six groups of 10 egg-type chickens per treatment were analyzed for the absence and presence of aflatoxin (0 and 4 micrograms/g diet) and for normal (12.75%) and low (10.00%) protein in soy-dextrose diets. The specific activities of pancreatic chymotrypsin, amylase, and lipase, but not trypsin, were increased significantly (P less than .01) by aflatoxin. Lowering dietary protein had no effect by itself except to increase amylase activity. Low protein and aflatoxin interacted to lessen but not prevent the effect of aflatoxin on chymotrypsin and amylase. Calculation of total pancreatic activities revealed that aflatoxin increased trypsin, chymotrypsin, amylase, and lipase to 107, 169, 113, and 119%, respectively, of control values on the low protein diet, whereas values were 99, 175, 115, and 115%, respectively, on the normal protein diet. Neither aflatoxin nor low protein altered significantly (P less than .05) the lipid content of fecal material. Thus, aflatoxicosis in egg-type chickens is characterized by a surplus of some digestive enzymes and by normal fecal lipids in contrast to the specific deficiency of amylase and lipase and steatorrhea reported earlier in meat-type chickens. Whereas malabsorption caused by aflatoxin in broilers can be accounted for in part by impaired digestion, this mechanism apparently does not occur in egg-type chickens.

Preparation of scirpentriol and triacetoxyscirpenol in good yield from cultures of Fusarium sambucinum NRRL 13495.

Crude extracts of filtrates of cultures of Fusarium sambucinum NRRL 13495 were acetylated or hydrolyzed. After chromatography on cartridge columns of silica gel and recrystallization three times from mixtures of ethyl acetate and hexane, 3,4,15-triacetoxyscirpenol (435 +/- 10 mg/liter of filtrate; mean +/- standard error [n = 3]) and the parent alcohol scirpentriol were isolated (261 +/- 29 mg/liter of filtrate; mean +/- standard error [n = 3]) in 68 and 53% yield for a 130- and 14-fold improvement, respectively, over prior reports.

Preparation of 4,15-diacetoxyscirpenol from cultures of Fusarium sambucinum NRRL 13495.

Filtrates of Fusarium sambucinum NRRL 13495 grown in a stagnant culture for 9 days contained up to 458 +/- 60 (mean +/- standard error; n = 3) mg of 4,15-diacetoxyscirpenol per liter depending on culture conditions. Extraction with ethyl acetate, chromatography on a column of silica gel, and crystallization from mixtures of ethyl acetate and hexane provided pure material in 96% yield.

Production of zearalenone, T-2 toxin, and deoxynivalenol by Fusarium spp. isolated from plant materials grown in North Carolina.

Fusarium spp. isolated from plant materials grown in the hot, humid climate of North Carolina were tested for production of mycotoxins. Isolates of F. acuminatum, F. graminearum, F. moniliforme, F. oxysporum, and F. solani produced zearalenone while isolates of F. equiseti and F. graminearum produced T-2 toxin and deoxynivalenol, respectively. This is the first report of zearalenone production by F. solani. The toxins were identified by capillary gas chromatography-mass spectrometry. These findings suggest that there are toxigenic strains of Fusarium indigenous to the warmer regions of the USA and that fasariotoxicoses of animals in this region are not necessarily the result of importing toxic grains from the cooler, upper midwestern USA.

Initial experience with a new prosthetic angioaccess device.

Long-term vascular access has increased longevity for many patients with end-stage renal disease. Much of the hospitalization in this group of patients continues to be for maintenance of reliable vascular access. Thrombosis, infection, aneurysm, and stenosis lead to serious morbidity. The Hemasite angioaccess system has been introduced in an attempt to circumvent some of these problems. We reviewed our initial experience with 90 of these devices placed in 77 patients during the past 24 months. Thirty-five devices (39%) were placed under emergency conditions when the primary access site had failed, 34 (38%) were used as the initial access procedure, and simple patient convenience was the indication 21 times (23%). Twelve patients have died, with no deaths related to the device. Twenty-eight infections and 18 thromboses accounted for the failures. Fourteen thromboses were seen with the graftless device where collateral flow existed around it. One-year patency was 46% for all devices, 38% for 34 graftless devices placed in the upper arm, and 50% for the grafted model in the upper arm position. Overall patency is not comparable to other access methods yet patient acceptance is high. Placement in the upper arm offers the highest rate of success.

Bioconversion of alpha-[14C]zearalenol and beta-[14C]zearalenol into [14C]zearalenone by Fusarium roseum 'Gibbosum'.

Cultures of Fusarium roseium 'Gibbosum' on rice were treated with [14C]zearalenone, alpha[14C]zearalenol, or beta-[14C]zearalenol to determine whether a precursor-product relationship exists among these closely related fungal metabolites. Culture extracts were purified by silica gel column chromatography and fractionated by high-pressure liquid chromatography, and the level of radioactivity was determined. Within 7 days, the beta-[14C]zearalenol was converted to zearalenone, and no residual beta-[14C]zearalenol was detectable. Most of the alpha-[14C]zearalenol added was also converted into zearalenone with 14 days. In cultures treated with [14C]zearalenone, no radioactivity was noted in any other components.

Method for detecting production of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol by Fusarium isolates.

Three methods for detecting toxigenic fusaria in culture were compared by using known producers of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol. Moist, autoclaved rice cultures of known toxigenic isolates grown in 20-ml tubes yielded oily extracts containing compounds which interfered with qualitative and quantitative analysis for the mycotoxins. Vermiculite moistened with nutrient broth in 20-ml tubes yielded a much cleaner extract. Growing the fungi on a liquid medium required a shorter incubation period, but yields of T-2 toxin and deoxynivalenol were low and variable, and the method required greater space in the incubator. Screening of the extracts by thin-layer chromatography with colorimetric spray reagents to detect the presence of these toxins permitted reduction in the number of extracts quantified by the more lengthy gas-liquid chromatographic method. Culturing in nutrient broth on vermiculite in tubes coupled to a qualitative screen before quantitation proved to be a convenient, inexpensive, and relatively rapid method that enabled reliable screening of a large number of Fusarium isolates for toxin production as compared with prior methods.