Beef feedlots and impact of a prebiotic on animal health
The Animal Care Committee reviewed the study and concluded that the observational research on commercial production sites did not require authorization. As such, animal care followed the code of practice for farm animals described in the guidelines for beef cattle (National Farm Animal Care Council of Canada). The producer granted permission for the post-mortems on beef calves or finishing cattle succumbing to disease and to monitor the impact of the feed additive on animal health. A shipment of five hundred beef feeder calves (227 kg) was received by a commercial beef feedlot (BF1) in Southern Alberta in December 2008. The animals appeared normal with no evidence of illness in the first two weeks. The producer purchased hay and began feeding it to the calves with no apparent problem until the next day. Fifty beef-feeder calves were dead in the morning with the only visible symptoms melena or black, "tarry" feces that are associated with gastrointestinal hemorrhage together with frank blood in the feces. Thirty-four calves were transferred to a sick pen with 16 animals seriously affected. The producer believed that the hay was responsible and removed it. Eighteen animals recovered over 7 days, the remaining 16 calves continued to decline even after antibiotic treatments were applied. The clinical symptoms suggested a mycotoxicosis including staggering, paralysis and wasting. The producer tried an alternative approach, a prebiotic feed additive, to reverse the wasting or to assist digestion. The prebiotic, Celmanax™ (VI-COR®, Mason City, IA, USA), consists of a non-living formulation of yeast cell walls or mannan-oligosaccharide (MOS) and improves cattle performance . The producer administered a single 400 ml drench of liquid Celmanax™ to all sixteen calves. We monitored the 16 calves to determine if they improved or continued to decline.
Another beef feedlot (BF2) for finishing cattle (9000 animals) in Southern Alberta experienced sudden onset death in October, 2010. Twelve cattle died overnight and after several postmortems, no apparent cause was found. The next day, two additional cattle died with no symptoms or apparent cause. After discussions with the veterinarian, a sample was removed from the mid-region of the jejunum, where we have had success in the past in identifying hemorrhaged tissue regardless of the lack of any visible hemorrhaging through the serosa.
Pathology and STEC isolation from the jejunum of JHS cases
Six beef cattle were chosen randomly from an abattoir in Southern Alberta and samples were collected from the mid-section of the jejunum. These tissues served as negative controls. The tissue from 5 beef cattle on BF1 confirmed a JHS diagnosis based on the presence of acute hemorrhaging in the jejunum, blood in the small intestine and black, "tarry" feces. To compare pathology associated with JHS diagnosis at BF1 and BF2, tissue samples were collected and the pathology recorded as described previously . Briefly, a 30 cm piece of tissue was removed from the acute hemorrhaged region of the jejunum. The blood and tissue were separated into sample tubes before serial dilutions were plated on Sorbitol MacConkey agar (SMAC; Dalynn Biologicals, Calgary, Alberta, Canada) to identify non-sorbitol fermenting bacterial colonies and on Potato Dextrose agar (PDA; Dalynn Biologicals, Calgary, Alberta, Canada) to isolate fungi. Adherent or tissue-colonizing bacteria were collected by incubating a 2.5 cm2 tissue piece in 0.1% Triton X-100 at 4°C overnight and plated on SMAC agar to determine the presence of non-sorbitol fermenting bacterial colonies. Any suspect colonies were tested as O157 and H7 using the RIM™ E. coli O157:H7 Latex test (Fisher Scientific, Ottawa, Ontario, Canada).
To elucidate whether STEC virulence determinants were critical to the development of JHS, the blood in the hemorrhaged jejunum from BF2 and the presumptive E. coli O157:H7 isolates from the hemorrhaged regions were examined for Stx1 and Stx2 expression using the Meridian™ ImmunoCard STAT!® EHEC test (Somagen, Edmonton, Alberta, Canada).
Isolation of bacterial pathogens from the jejunum of JHS cases
Washed tissue samples (2.5 cm2) were placed in 0.1% Triton X-100 overnight and the released bacteria stored at -80°C or serial dilutions were direct plated. The digesta was also stored at -80°C or serial dilutions were direct plated. A 1 to 50 μl aliquot of a diluted sample was applied to CHROMagar™ Salmonella, CHROMagar™ E. coli, CHROMagar™ O157, CHROMagar™ Salmonella Plus, and CHROMagar™ Listeria plates (Dalynn Biologicals, Calgary, Alberta). To confirm identity, the presumptive isolates with the exception of the Listeria were subjected to a Microgen™ GN-ID A + B biochemical test (Alere™ Canada, Ottawa, Ontario). Presumptive Salmonella were also subjected to a Microgen™ Salmonella Latex Agglutination test (Alere™ Canada, Ottawa, Ontario, Canada). Presumptive Listeria was identified using a Microgen™ Listeria ID test (Alere™ Canada, Ottawa, Ontario). To detect C. perfringens type A, samples were examined for characteristic features using a compound Nikon microscope set at 1000× magnification. Digesta and tissue smears were examined for the presence of large, rectangular bacilli (rod) with or without spores (ovoid, sub-terminal). Sub-cultures on blood agar were examined for rapid spreading growth.
DNA microarray assay targeting pathogenic E. coligenes
The original samples were re-examined for multiple STECs as described previously . The microarray (MaxiVir1.0) used in this study was based on earlier published work and carries 514 oligonucleotides of 70 bases in length targeting 348 virulence or virulence-related genes and 96 antimicrobial resistance or antimicrobial resistance-related genes found in gram-negative bacteria . The microarray, designed to detect a complete set of virulence genes representative of all E. coli pathotypes, includes virulence factors such as adhesins, locus of enterocyte effacement, colicins and microcins, toxins, iron acquisition and transport systems, capsular and somatic antigens, hemolysins and hemaglutinins, as well as newly recognized or putative E. coli virulence genes. Antimicrobial resistance genes included in the microarray represent different antimicrobial families such as ß-lactams, aminoglycosides, tetracycline, phenicols, trimethoprim, sulfonamide and class I integron. The microarray also carries five positive oligonucleotide controls for E. coli derived from the sequences of genes encoding tryptophanase (tnaA), beta-glucuronidase (uidA), lactose permease (lacY), beta-galactosidase (lacZ), and glutamate decarboxylase (gad). Negative controls added to this microarray consist of oligonucleotides derived from the gene sequences for the green fluorescent protein of Aequorea victoria, the lactose permease of Citrobacter freundii, and the chlorophyll synthase from Arabidopsis thaliana.
Escherichia coliDNA labeling
Bacterial DNA was labeled using Bioprime DNA labeling system (Invitrogen Life Technologies, Burlington, ON, Canada). Fifteen μl of the supernatant containing DNA was added to a final volume of 32.5 μl containing 10 μl of a random primer solution, 0.5 μl of high-concentration DNA polymerase (Klenow fragment, 40 U/μl), 5 μl of a deoxyribonucleosidetriphosphate (dNTP) mixture (1.2 mM dATP, 1.2 mM dGTP, 1.2 mM dTTP, and 0.6 mM dCTP in 10 mM Tris [pH 8.0] and 1 mM EDTA), and 2 μl of 1 mMCy5-dCTP. Labeling reactions were performed in the dark at 37°C for 3.5 h and stopped by the addition of 5 μl Na2EDTA 0.5 M (pH 8.0). The labeled samples were then purified with a PureLink PCR purification kit (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. The amount of incorporated fluorescent Cy5 dye was then quantified by scanning the DNA sample with a NanoDrop ND-1000 spectrophotometer from 200 to 700 nm. Data were analyzed using a Web-based percent incorporation calculator http://www.pangloss.com/seidel/Protocols/percent_inc.html.
Hybridization of labeled DNA
Microarrays were prehybridized at 50°C for 1 hour under a Lifterslip (25 × 60 mm; Erie Scientific Company, Portsmouth, NH, USA) using a SlideBooster hybridization workstation (model SB800; Advalytix, Germany), with 50 μl of prewarmed (37°C) digoxigenin (DIG) Easy Hyb Buffer (Roche Diagnostics, Laval, Quebec, Canada) supplemented with 5% (vol/vol) bovine serum albumin (1 mg/ml; New England Biolabs Inc., Beverly, MA). After pre-hybridization, the lifterslip was removed by dipping the slides in 0.1X SSC (saline-sodium citrate) and were air-dried. Before hybridization, the samples were dried and resuspended in 15 μl of hybridization buffer (DIG + 0.1 ug/ul ssDNA) and denatured for five minutes at 95°C. One microgram of labeled genomic DNA was hybridized on the MaxiVir1.0 microarray under a lifterslip (18 × 18 mm). The hybridization was carried out overnight at 50°C in a SlideBooster hybridization workstation. After hybridization, lifterslips were removed by dipping the slides in a 0.1X SSC and 0.1% SDS (sodium dodecyl sulfate) solution. Post-hybridization washes were performed at 37°C: two washes with 0.1X SSC and 0.1% SDS for ten and five minutes respectively and one last wash in 0.1X SSC for five minutes. The microarrays were then air-dried.
Microarray slides were scanned at 5 μm resolution with a ScanArray Lite fluorescent microarray analysis system (Perkin-Elmer, Missasauga, Ontario, Canada). Acquisition of fluorescent spots was performed using the ScanArray Express software (Perkin-Elmer, Foster City, CA). Fluorescent spot intensities were quantified with ImaGene software version 8.0 (BioDiscovery, Inc., El Segundo, CA). All the microarrays were normalized using the same method. For each subarray, the mean value for each set of duplicate spotted oligonucleotides was divided by the correction factor taken from the negative controls spots. This value was then divided by the average of the empty spots to create a signal-to-noise ratio. Oligonucleotide spots with a signal-to-noise fluorescence ratio greater than the established threshold (3 in this case), were considered positive. These ratios were then converted into binary data where a value of 0 indicates a negative probe and a value of 1 a positive probe. A threshold of 3 was chosen because it best represented spot quantification. To verify that the results were accurate, we compared the.bmp image of a given sample and the quantified result.
Isolation and identification of mycotoxigenic fungi
Feed components were collected from BF1 and the barley silage was collected from BF2. A 10 g sub-sample was finely ground and a 5 ml volume added to a PDA plate. The plate was incubated for 1 to 7 days and individual fungal isolates transferred to new PDA plates. Tissue smears were examined for fungal hyphae and suspect tissue was placed on a PDA plate. Fusarium isolates were identified by examination of micro-morphological characters and by PCR amplification and sequencing of a fragment of the EF1-a gene and comparing the sequence with the FUSARIUM-ID database [32, 33]. Penicillium isolates were identified by microscopic examination of morphology using the guide by Pitt . Aspergillus isolates were identified by microscopic examination of morphology using the guide provided by Klich .
Extraction of cytotoxins from feed components
The methods used have been described previously . The feed samples were not visibly mouldy for BF1 or BF2. The components of the total mixed ration and hay were collected from BF1 while the barley silage was collected from BF2. To extract the mycotoxins, each sample was finely ground, a 25 ml aliquot of 50% methanol was added to a 3 g sample and placed on a shaker at 200 rpm for 3 h. The supernatant was collected in another tube, and stored at 4°C until use.
Lawn assay for cytotoxicity associated with feed extracts
The lawn assay has been described previously and was used to examine the cytotoxicity of the toxins produced by Escherichia coli O157:H7 strains  and to detect mycotoxins in feeds . The assay was performed using the feed extracts in the absence or presence of 0.1% Celmanax™. Briefly, a 1% SeaKem® agarose (Mandel Scientific, Guelph, Ontario, Canada) support gel was poured into a petri dish. Next, the lawn agarose [3 ml of 3.7% SeaPlaque® agarose (Mandel Scientific, Guelph, ON, Canada)] was mixed with 3 ml of enterocyte suspension or a bovine colonic cell line and poured over the support agarose. A 5 ul aliquot of the solvent used for the extraction process served as negative controls. Each extract (5 μl) was applied with or without 0.1% prebiotic and the plate incubated for 4 h under standard culture conditions. The lawn was stained with 0.1% trypan blue (Sigma-Aldrich) and de-stained using PBS. Plates were scored the same day and the amount of extract cytotoxicity was scored as low (Cytotoxicity Score 1), moderate (Cytotoxicity Score 2) or high (Cytotoxicity Score 3) which was visualized as a faint blue spot, a blue spot or a dark blue spot respectively. These activities were compared to two standards, ground corn containing 0.1 ppm aflatoxin that had a low Cytotoxicity Score (1) and 1 ppm aflatoxin that had a high Cytotoxicity Score (3). The assay was repeated a minimum of three times.
All data were analyzed using ANOVA followed by a posthoc Tukey's test for comparison of the means. Results were considered significant if P < 0.05 and non-significant if P > 0.05.