Tim Reuter

A biosecure composting system for disposal of cattle carcasses and manure following infectious disease outbreak.

During outbreaks of infectious animal diseases, composting may be an effective method of disposing of mortalities and potentially contaminated manure. Duplicate biosecure structures containing 16 cattle (Bos taurus) mortalities (343 kg average weight) were constructed with carcasses placed on a 40-cm straw layer and overlaid with 160 cm of feedlot manure. At a depth of 80 cm (P80), compost heated rapidly, exceeding 55 degrees C after 8 d and maintained temperatures of 55 to 65 degrees C for > 35 d. Temperatures at 160 cm (P160) failed to exceed 55 degrees C, but remained above 40 degrees C for >4 mo. To investigate rates of microbial inactivation, Escherichia coli O157:H7, Campylobacter jejuni, and Newcastle disease virus (NDV) were inoculated in manure (E. coli O157:H7 and C. jejuni approximately 10(8) CFU g(-1); NDV, approximately 10(6) EID(50) g(-1)), embedded at P80 and P160 and retrieved at intervals during composting. Escherichia coli O157:H7 and NDV were undetectable after 7 d at both depths. The C. jejuni DNA was detected up to 84 d at P80 and >147 d at P160. To estimate degradation of recalcitrant substrates, bovine brain, hoof, and rib bones were also embedded at P80 and P160 and retrieved at intervals. Residues of soft tissues remained in carcasses after opening at 147 d and bovine tissue decomposition ranked as brain > hoof > bone. More than 90% dry matter (DM) of brain disappeared after 7 d and 80% DM of hoof decomposed after 56 d. High degradation of cattle carcasses, rapid suppression of E. coli O157:H7 and NDV and reduction in viable cell densities of >6 logs for C. jejuni demonstrates that the biosecure composting system can dispose of cattle carcasses and manure in an infectious disease outbreak.

Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics

BackgroundEnvironmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA.ResultsThe concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments.ConclusionThe abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.

Investigations on genetically modified maize (Bt-maize) in pig nutrition: Chemical composition and nutritional evaluation

The objective of the present study was to determine the composition and the nutritional value of parental and transgenic maize seeds fed to pigs. The parental maize line was genetically modified to incorporate a gene from Bacillus thuringiensis (Bt) expressing a toxin against the European corn borer (Ostrinia nubilalis). Both (parental and transgenic) maize lines were analyzed for crude nutrients, starch, sugar, non-starch polysaccharides (NSP), amino acids, fatty acids, as well as for selected minerals. Furthermore, four complete diets were mixed and were analyzed for the same nutrients and some selected ingredients. The diets contained 70% maize to attain a high effect level. To evaluate the feeding value of one variety of genetically modified maize (transgenic) compared to the feeding value of the unmodified maize (parental) line, a balance study with twelve pigs was designed. Three collecting periods were used for each maize line each with six animals. The collected faeces were analyzed for crude nutrients. All measured parameters were virtually the same ( e.g. crude protein 11.59% vs. 11.06% in DM), especially the digestibility of crude protein (85.8 ± 2.3% vs. 86.1 ± 1.8%), the amount of nitrogen-free-extract (92.8 ± 0.6% vs. 93.2 ± 0.6%) and the metabolizable energy (15.7 ± 0.2% vs. 15.8 ± 0.2% MJ/kg DM) for both maize lines. Compared to the parental line, the chemical composition and digestibility of crude nutrients and energy content were not significantly affected by the genetic modification of maize. Therefore, from the view of a nutritional assessment, the genetically modified maize can be regarded as substantially equivalent to the parental maize line.

Further development of sample preparation and detection methods for O157 and the top 6 non-O157 STEC serogroups in cattle feces.

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens responsible for outbreaks of human infections worldwide. Ruminant livestock harbor STEC in their intestinal tract, and through fecal contamination possess the potential to compromise the safety of food and water. As a human health safety risk, STEC detection methods on beef carcasses and trim are needed as mandated by the USDA-FSIS. In order to monitor STEC prior to harvest and human consumption, our goal was to evaluate and/or improve detection of seven STEC serogroups in cattle feces. In comparison to traditional approaches, sample processing methods in bovine feces were evaluated using a multi-factorial Latin square design which involved freezing or freeze drying feces. Autoclaved versus non-autoclaved feces were spiked with O26:H11 or O157:H7 serotypes in various dilutions and enriched for up to 6h. Each hour, enriched aliquots were compared using traditional culture methods and quantitative polymerase chain reaction (qPCR). Furthermore, a 7-serogroup multiplex PCR (mPCR) was developed to detect O26, O45, O103, O111, O121, O145 and O157 serogroups simultaneously. The diagnostic sensitivity of our mPCR assay following 6h enrichment was superior (10CFU/g across all serogroups) compared to a previously established PCR assay (10CFU/g for O26, and O103; ≥10(4)CFU/g for all other serogroups). Obtaining viable isolates appeared to be limited by the efficiency of current immunomagnetic separation (IMS) methods, which ranged from 20 to 100% effectiveness at retrieving colonies depending on serogroup. After IMS, 70 putative STEC isolates were screened for Shiga toxin and attachment genes by mPCR. Sixty-five isolates contained one or both Shiga toxin genes.

Longitudinal Characterization of Resistant Escherichia coli in Fecal Deposits from Cattle Fed Subtherapeutic Levels of Antimicrobials

ABSTRACT Model fecal deposits from cattle fed or not fed antimicrobial growth promoters were examined over 175 days in the field for growth and persistence of total Escherichia coli and numbers and proportions of ampicillin-resistant (Ampr) and tetracycline-resistant (Tetr) E. coli. In addition, genotypic diversity and the frequency of genetic determinants encoded by Ampr and TetrE. coli were investigated. Cattle were fed diets containing chlortetracycline (44 ppm; A44 treatment group), chlortetracycline plus sulfamethazine (both at 44 ppm; AS700 treatment group), or no antibiotics (control). Fecal deposits were sampled 12 times over 175 days. Numbers of TetrE. coli in A44 and AS700 deposits were higher (P < 0.001) than those of controls and represented up to 35.6% and 20.2% of total E. coli, respectively. A time-by-treatment interaction (P < 0.001) was observed for the numbers of Tetr and AmprE. coli. Except for AmprE. coli in control deposits, all E. coli numbers increased (P < 0.001) in deposits up to day 56. Even after 175 days, high TetrE. coli numbers were detected in A44 and AS700 deposits [5.9 log10 CFU (g dry matter)−1 and 5.4 log10 CFU (g dry matter)−1, respectively]. E. coli genotypes, as determined by pulsed-field gel electrophoresis, were diverse and were influenced by the antimicrobial growth promoter and the sampling time. Of the determinants screened, blaTEM1, tetA, tetB, tetC, sul1, and sul2 were frequently detected. Occurrence of determinants was influenced by the feeding of antimicrobials. Fecal deposits remain a source of resistant E. coli even after a considerable period of environmental exposure.

Anaerobic digestion of specified risk materials with cattle manure for biogas production.

Biogas production from anaerobic digestion (AD) of specified risk materials (SRM) co-digested with cattle manure was assessed in a 3 x 2 factorial design. SRM replaced manure at 0 (control), 10% or 25% (w/w) as the substrate fed to six 2-L biodigesters maintained at 37 degrees C or 55 degrees C. Digesters were fed substrate (30 g L(-1) total volatile solids) at 6-d intervals for 90 d, with a retention time of 30 d. Keratin (<20mg) was added to each digester to model the degradation of beta-sheet rich proteins. Methane production was measured daily, and effluent was collected at feeding to monitor SRM degradation using real-time PCR analysis of bovine-specific DNA fragments. Compared with control, methane production increased by 83% or 161% (P<0.05) with 10% or 25% SRM at 37 degrees C, and by 45% and 87%, respectively, at 55 degrees C (P<0.05). Bovine DNA degradation over 6d was higher (P<0.05) at 37 degrees C as compared to 55 degrees C. Dry matter degradation of keratin at 37 degrees C decreased with increasing SRM concentration (P<0.05), whereas at 55 degrees C no difference between treatments was observed (P>0.05). Inclusion of SRM increases the production of methane during the anaerobic digestion of manure and may offer a means of deriving economic value from the disposal of SRM.

Investigations on Genetically Modified Maize (Bt-Maize) in Pig Nutrition: Fattening Performance and Slaughtering Results

A grower finisher performance trial with forty-eight pigs was designed to compare the growth performance of pigs fed diets containing either genetically modified (GM) Bt-maize (NX6262) or its parental maize (Prelude) line. During the experiment, the pigs were fed with a grower and a finisher diet both containing 70% maize investigated in a previously study which showed that they contained similar ME values and digestibility of crude nutrients. The pigs with an initial live weight of 23.9 ± 3.0kg were allotted to single boxes. During a 91 days growing period the pigs of both groups recorded equal performance in daily weight gain ( j W) 815 ± 93 vs. 804 ± 64g/d depending on equal amounts of feed intake 1.95 ± 0.15 vs . 1.94 ± 0.15kg/d (parental vs. transgenic). The results confirm equal performance among growing-finishing pigs fed parental or genetically modified maize containing diets. For slaughtering the pigs were divided into 4 groups with a different duration of the finishing period. After slaughtering the carcass characteristic were registered.

Conventional and real-time polymerase chain reaction assessment of the fate of transgenic DNA in sheep fed Roundup Ready® rapeseed meal

Conventional and real-time PCR were used to detect transgenic DNA in digesta, faeces and blood collected from six ruminally and duodenally cannulated sheep fed forage-based (F) or concentrate-based (C) diets containing 15% Roundup Ready (RR) rapeseed meal (n 3). The sheep were adapted for 14 d to F or C diets containing non-GM rapeseed, then fed the RR diets for 11 d. On day 12, they were switched back to non-GM diets for a further 11 d. Ruminal and duodenal fluids (RF, DF) and faecal samples were collected at 3 or 4 h intervals over the 4 d immediately following the last feeding of GM diets. DNA was isolated from whole RF and DF, from the cell-free supernatant fraction, and from culture fermentation liquid. Blood was collected on days 1, 5 and 9 of feeding the RR rapeseed meal. The 1363 bp 5-enolpyruvylshikimate-3-phosphate synthase transgene (epsps) was quantifiable in whole RF and DF for up to 13 h, and a 108 bp epsps fragment for up to 29 h. Transgenic DNA was not detectable in faeces or blood, or in microbial DNA. Diet type (F v. C) did not affect (P>0.05) the quantity of transgenic DNA in digesta. More (P<0.05) transgenic DNA was detected in RF than in DF, but there was an interaction (P<0.05) between sample type and collection time. In supernatant fractions from RF and DF, three different fragments of transgenic DNA ranging in size from 62 to 420 bp were not amplifiable.

Purification of polymerase chain reaction (PCR)-amplifiable DNA from compost piles containing bovine mortalities

Livestock production systems utilize composting as a method of disposal of livestock mortalities, but there is limited information on the rate and extent of carcass decomposition. Detection of specific DNA fragments by PCR offers a method for investigating the degradation of carcasses and other biological materials during composting. However, the purity of extracted DNA is critical for successful PCR analysis. We applied a method to purify DNA from compost samples and have tested the method by analyzing bovine and plant DNA targets after 0, 4, and 12 month of composting. The concentration of organic matter from composted material posed a particular challenge in obtaining pure DNA for molecular analysis. Initially extracted DNA from composted piles at day 147 was discoloured, and PCR inhibitors prevented amplification of target plant or bovine gene fragments. Bovine serum albumin improved detection by PCR (25-50 microl final volume) through the removal of inhibitors, but only when concentrations of humic acids in extracted DNA were 1.0 ng microl(-1) or less. Optimal purification of DNA from compost was achieved by chromatography using Sepharose 4B columns. The described DNA purification protocol enabled molecular monitoring of otherwise cryptic bovine and plant target genes throughout the composting process. The assay could likely be used to obtain PCR-amplifiable DNA that could be used for the detection of microbial pathogens in compost.

Prion protein detection via direct immuno-quantitative real-time PCR

We describe a simple and robust assay for the quantitative detection of prions using immuno-quantitative real-time PCR (iQ-RT-PCR) made possible by a direct conjugate of a prion-specific antibody (ICSM35) and a synthetic 99-bp DNA tail. The DNA tail was engineered to include a single ScrFI restriction site, which enabled subsequent quantification of restricted DNA tails using real-time PCR. The assay was tested with scrapie prions bound to polyvinylidene difluoride membranes and to 96-well plates coated with a capturing antibody from a commercially available immuno-based assay (TeSeE). The iQ-RT-PCR assay had a detection limit corresponding to 2.32x10(2) prion epitopes, which represented a 1000-fold increase in detection sensitivity over the commercial assay. Detection of prions from diluted scrapie-positive brain homogenate bound to membranes was linear over a range of 1.06x10(4) to 3.24x10(2) epitopes (R(2)=0.92). Given its sensitivity and versatility, the present assay has potential to enable rapid and reliable detection of agents causing transmissible spongiform encephalopathies.