Coliform and Escherichia coli Contamination on External and Internal Surfaces of Beef Carcasses with and without Tissue Adhesion Excision.

ABSTRACT: Following removal of hides and viscera during beef processing, carcasses are inspected for tissue adhesions that can affect meat quality or harbor bacteria. Carcasses with pleural or abdominal adhesions may be diverted from the production line for manual excision and then returned to the line. No published data indicate whether adhesion excision is associated with bacterial contamination. Therefore, our objective was to determine the presence and concentration of generic Escherichia coli and non-E. coli coliforms from the internal and external surfaces of carcasses that were, or were not, diverted for adhesion excision. During 9 processing days over a 4-month period in a large commercial beef processing facility, 1,738 carcass sponge samples from 2,730 cm2 areas on both the internal and the external surfaces of carcasses with and without tissue adhesions were collected. Coliforms and E. coli were cultured and enumerated using Petrifilm procedures, and data were analyzed with mixed models. Coliforms were present at higher concentrations than E. coli, and prevalence and mean log concentrations of both coliforms and E. coli were significantly higher for samples from the external than from the internal surfaces of carcasses. However, differences in prevalence and concentration of coliforms between external and internal surfaces varied significantly based on whether carcasses had adhesions excised. The difference was greatest for coliforms present on the external (2.06 log CFU/100 cm2) versus the internal (0.93 log CFU/100 cm2) carcass surfaces without adhesions, whereas the difference in concentrations from the external (1.80 log CFU/100 cm2) and the internal (1.31 log CFU/100 cm2) surfaces of carcasses with adhesions was not as large. These results indicate that surveillance of carcass bacteria may be affected by whether the external versus the internal surfaces are sampled and whether carcasses are diverted for excision of adhesions.

Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coli.

Strains of Shiga toxin-producing Escherichia coli (STEC) are a serious threat to the health, with approximately half of the STEC related food-borne illnesses attributable to contaminated beef. We developed an assay that was able to screen samples for several important STEC associated serogroups (O26, O45, O103, O104, O111, O121, O145, O157) and three major virulence factors (eae, stx 1 , stx 2) in a rapid and multiplexed format using the Multiplex oligonucleotide ligation-PCR (MOL-PCR) assay chemistry. This assay detected unique STEC DNA signatures and is meant to be used on samples from various sources related to beef production, providing a multiplex and high-throughput complement to the multiplex PCR assays currently in use. Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a nucleic acid-based assay chemistry that relies on flow cytometry/image cytometry and multiplex microsphere arrays for the detection of nucleic acid-based signatures present in target agents. The STEC MOL-PCR assay provided greater than 90% analytical specificity across all sequence markers designed when tested against panels of DNA samples that represent different STEC serogroups and toxin gene profiles. This paper describes the development of the 11-plex assay and the results of its validation. This highly multiplexed, but more importantly dynamic and adaptable screening assay allows inclusion of additional signatures as they are identified in relation to public health. As the impact of STEC associated illness on public health is explored additional information on classification will be needed on single samples; thus, this assay can serve as the backbone for a complex screening system.

Immunogenicity and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice.

Fusobacterium necrophorum, a gram-negative, anaerobic and rod-shaped bacterium, is generally an opportunistic pathogen and causes a wide variety of necrotic infections in animals and humans. Leukotoxin, a secreted protein, is a major virulence factor. The gene encoding the leukotoxin (lktA) in F. necrophorum has been cloned, sequenced and expressed in Escherichia coli. Because of low expression levels, problems associated with purifying full-length recombinant protein, and of the physical instability of the protein, five overlapping leukotoxin gene truncations were constructed. The recombinant polypeptides (BSBSE, SX, GAS, SH, and FINAL) were expressed in E. coli and purified by nickel-affinity chromatography. The objectives were to investigate the effectiveness of the purified truncated polypeptides to induce protective immunity in mice challenged with F. necrophorum. The polypeptides, individually or in combination, and inactivated native leukotoxin or culture supernatant of F. necrophorum were homogenized with an adjuvant and injected into mice on days 0 and 21. Blood samples were collected to measure serum anti-leukotoxin antibody titers on days 0, 21 and 42 and on day 42, mice were experimentally challenged with F. necrophorum. All polypeptides were immunogenic, with GAS polypeptide eliciting the least antibody response. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection. Protection was better than the full-length native leukotoxin or inactivated supernatant.The study demonstrated that the leukotoxin of F. necrophorum carries epitopes that induce protective immunity against experimental fusobacterial infection, thus providing further evidence to the importance of leukotoxin as a major virulence factor.

Technical note: effect of removal of microbial cells by centrifugation on peptide and alpha-amino nitrogen concentrations in ruminal fluid.

We evaluated the effect of centrifuging rumen fluid prior to analysis on concentrations of alpha-amino N (AAN) and peptides. Rumen fluid was collected from steers fed grain-based diets at either various times after feeding or after dosing the rumens with solubilized casein. Fluid was either directly processed for peptide analysis by acidifying 10 ml of rumen fluid with 0.5 ml of 70% (wt/wt) perchloric acid, or first centrifuged at 500 x g for 20 min to remove protozoa and then at 30,000 x g for 15 min to remove bacterial cells prior to further processing. By removing microbial cells, intracellular AAN and peptides were not included in subsequent analyses. Concentrations of AAN were determinedusing an automated trinitrobenzene sulfonic acid assay, and peptides were determined as the increase in AAN following acid hydrolysis of the samples. When casein was not dosed, removal of microbial cells prior to analysis decreased concentrations of both AAN andpeptides, and the decrease was greater for AAN (2.2 mM)than for peptides (1.2 mM). Dosing with casein led to much higher concentrations of ruminal peptides and AAN. After casein dosing, decreases in AAN and peptidecon-centrations due to prior centrifugation (2.1 mM and 1.0 mM for AAN a nd pept ides, respectively) were similar to the decreases observed before the casein dosing. Results suggest that the contribution of intracellular AAN and peptides to the concentrations in ruminal fluid are relatively constant across broad ranges of dietary protein supply for cattle fed corn-based diets.

Cloning, sequencing, and expression of the leukotoxin gene from Fusobacterium necrophorum.

Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the whole lktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin from F. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktA hybridizing bands between isolates of the two subspecies of F. necrophorum.

Evaluation of a 5'-nuclease (TaqMan) assay for the detection of virulent strains of Yersinia enterocolitica in raw meat and tofu samples.

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5'-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with "gold standard" methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5'-nuclease assay was able to identify the organism in 100% of the repetitions when 10(2) CFU/ml or more organisms were present in pure cultures and 10(3) CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 10(5) CFU/ml or more organisms were present in pure culture and 10(6) CFU/g or more organisms were present in inoculated ground pork. The 5'-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5'-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5'-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.

Dynamics of ruminal ciliated protozoa in feedlot cattle.

Fluctuations in ciliated protozoan concentrations were monitored in 40 individually fed crossbred heifers that were stepped up to an 85% concentrate diet either slowly (12 days) or rapidly (3 days), with or without monensin (30 ppm). Ruminal fluid was withdrawn from all animals by stomach tube at the start of the study, after each group reached full feed, and at 14-day intervals thereafter throughout the finishing period until termination (day 119). Neither monensin nor speed of step-up affected (P greater than 0.10) total protozoan concentrations, ruminal pH, or lactic acid concentrations. Average protozoan concentrations peaked on day 5, progressively declined until day 56, and then increased (P less than 0.05), suggesting an adaptation to ruminal conditions. Concentrations of Isotricha spp. were higher (P less than 0.05) on the final two sampling dates than at any other time. After day 28, Entodinium, Isotricha, and Polyplastron were the only surviving genera. Protozoa were not detected in 11 heifers on day 42 and day 56, but only two animals were defaunated on day 119, indicating either exogenous or endogenous refaunation. Average protozoan concentrations were not different (P greater than 0.25) between ruminal samples collected by stomach tube the day before slaughter (2.8 x 10(5)/g) and digesta samples collected the next day (1.6 x 10(5)/g). In feedlot cattle, defaunation apparently is transitory and individual animals harbor a dynamic protozoan population that fluctuates in response to changing ruminal conditions.

Ruminal ciliated protozoa in cattle fed finishing diets with or without supplemental fat.

Ruminal samples were collected at slaughter from 364 unfasted steers fed different finishing diets to obtain information on numbers and species distribution of ciliated protozoa in feedlot cattle. Total numbers of protozoa averaged 1.59 X 10(5)/g of ruminal contents. A total of 47 steers (12.9%) were defaunated, but 4.1% of the steers possessed numbers of protozoa greater than 10(6)/g. Entodinium species did not always dominate the protozoan populations; 41 faunated steers (11.2%) were devoid of entodinia, and 79 additional steers (21.7%) possessed populations dominated (greater than 50%) by other genera. Isotricha was the most commonly occurring genus supplanting Entodinium, but Polyplastron and Epidinium were frequently present in high concentrations. Tallow and soybean soapstock supplementation reduced (P less than .05) numbers of protozoa in steers consuming wheat diets. However, yellow grease supplementation did not affect numbers of protozoa in steers fed either sorghum or corn diets. Average ruminal pH was 6.20 on the wheat diet, 6.05 on the corn diet, and 5.69 and 6.23 for the two sorghum diets, respectively. We found no correlation between ruminal pH and numbers of protozoa on any diet. The presence of relatively high protozoan concentrations and few defaunated animals in feedlot cattle necessitates reevaluation of the role that ciliated protozoa play in ruminal metabolism of animals fed processed, high-concentrate diets.

Ruminal ciliated protozoa in bison.

Ruminal contents from 79 slaughtered bison and 2 ruminally cannulated bison were collected to obtain information on total numbers and species distribution of ciliated protozoa. The bison originated from numerous herds throughout the Great Plains and were grouped into three dietary categories: (i) only forage; (ii) forage with moderate levels of supplementation; and (iii) feedlot concentrate-silage diet. Total ciliate counts were highest in bison receiving grain supplementation (210.1 x 10(4)/g) and lowest in bison consuming only forage (27.1 x 10(4)/g). All protozoan species found in bison have been reported in domestic livestock, although Ophryoscolex sp., a relatively common protozoan in cattle, was detected at low concentrations in only eight bison. The uncommon holotrich Microcetus lappus was present in five bison in concentrations reaching 8.4% of the total ciliate population. Charonina ventriculi, another infrequently observed species, was present in 18 bison, with the highest concentrations in forage-fed animals. Thirty bison possessed a type B protozoan population, characterized by Epidinium sp., Eudiplodinium maggii, and Eudiplodinium bovis. Thirty-eight bison possessed a mixed A-B population, characterized by Polyplastron sp. coexisting with low numbers of Eudiplodinium maggii or Epidinium sp. or both. Thirteen bison possessed populations lacking any remnant type B ciliate species. At least 29 of the bison possessing Polyplastron sp. were known to have been in contact with cattle, whereas all bison isolated from cattle had type B populations. The reduction of type B populations in bison becomes increasingly likely as bison production expands into areas inhabited by domestic livestock.

Comparisons of ruminal fermentation characteristics and microbial populations in bison and cattle.

Ruminal microbial populations, fermentation characteristics, digestibility, and liquid flow rates in two ruminally cannulated bison and two ruminally cannulated Hereford steers fed a prairie hay diet were compared. No significant differences in anaerobic bacterial counts, volatile fatty acid concentrations, or ruminal pHs were evident between bison and cattle. Also, no significant differences in neutral detergent fiber digestibility, indigestible fiber retention time, or intake were detected between bison and cattle, although cattle had higher levels (P less than 0.08) of ruminal dry matter and indigestible fiber than bison. Bison had a smaller (P = .02) ruminoreticular volume, faster liquid dilution rates, and faster liquid turnover times than cattle. The average ruminal ammonia nitrogen concentration was higher (P = 0.02) in bison (1.17 mg/dl) than in cattle (0.79 mg/dl). Total ciliate protozoal counts and cell volume were greater (P = 0.07) in bison (32.8 x 10(4)/g and 407.1 x 10(-4) ml/g, respectively) than in cattle (15.7 x 10(4)/g and 162.2 x 10(-4) ml/g, respectively). Bison harbored higher (P less than 0.02) numbers of Dasytricha spp., Eudiplodinium maggii, Eudiplodinium bursa, and Epidinium spp. than cattle and possessed a type B protozoan population. The cattle possessed a mixed type A-type B population that was characterized by Ophryoscolex spp. and Polyplastron spp. in association with low concentrations of Epidinium spp. and Eudiplodinium maggii.

Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives.

Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics.

Effect of lasalocid, monensin and thiopeptin on rumen protozoa.

The effects of lasalocid, monensin and thiopeptin on the total number and the generic composition of rumen protozoa were determined in vivo and in vitro. Feeding lasalocid or monensin to cattle on either high grain or high roughage diets reduced total protozoal counts. Addition of lasalocid or monensin (6 to 48 micrograms ml-1) to the in vitro rumen fermentation resulted in marked reduction in protozoal numbers. The inhibition was dose dependent. Thiopeptin had no effect on rumen protozoa either in vivo or in vitro. Among the protozoal types, holotrichs (Dasytricha, Isotricha and Charonina) were unaffected by either lasalocid or monensin. Among the entodiniomorphs, Entodinium, Diplodinium and Ophryoscolex were more sensitive than the other types. Ophryoscolex purkynei was more sensitive to monensin than to lasalocid. Protozoal inhibition by lasalocid and monensin was transient because prolonged antibiotic feeding resulted in the selection of a resistant population in the rumen of cattle.

Relation of rumen ATP concentration to bacterial and protozoal numbers.

Cultures of Streptococcus bovis and mixed populations of rumen bacteria were used to investigate the concentration of ATP and rumen bacterial numbers at various stages of growth. ATP, extracted with Tris buffer, was analyzed using the firefly luciferin-luciferase bioluminescent reaction. ATP concentrations of S. bovis and mixed cultures of rumen bacteria significantly correlated with live cell counts during the log phase of growth but not during the stationary phase. The average cellular ATP concentration of rumen bacteria was calculated to be 0.3 fg of ATP per cell. Studies done with in vivo artificial rumen apparatus revealed that the protozoal contribution to rumen fluid ATP pool size was much more substantial than was the bacterial contribution. The rumen fluid ATP concentration was greater in cattle with protozoa than in those that were defaunated. Differences in ATP concentration due to size differences of ciliate protozoa were observed. Due to the unbalanced distribution of ATP in rumen microbes, ATP appears to be an unsuitable indicator of rumen microbial biomass.

Effects of lasalocid or monensin on legume or grain (feedlot) bloat.

Doses of .66 to .99 mg monensin/kg body weight reduced legume bloat in cattle about 66% when compared with pretreatment bloat scores. Similar doses of lasalocid reduced legume bloat about 26%. A dose of 44 mg poloxalene/kg body weight (recommended dose for field use) reduced legume bloat 100%. Monensin or lasalocid combined with 25 or 50% of the recommended dose of poloxalene reduced bloat under that of the antibiotics alone, but did not achieve 100% reduction. The antibiotic thiopeptin provided no preventive effect on legume bloat. Lasalocid, monensin or an experimental polyether antibiotic (X-14,547 A) at a dose of 1.32 mg/kg body weight when tested on cattle bloated on high grain diets reduced bloat by 92, 64 and 25%, respectively. Lasalocid at .66 mg/kg effectively prevented bloat from developing when given to animals before the feeding of high grain diets; however, a 1.32-mg dose was required to control bloat in cattle that were already bloating before they were given lasalocid. A dose of 1.32 mg salinomycin was ineffective in controlling grain bloat.

Effect of lasalocid, monensin or thiopeptin on lactic acidosis in cattle.

Lasalocid, monensin or thiopeptin was administered intraruminally each at .33, .65 or 1.3 mg/kg body weight and evaluated for its effectiveness in preventing experimentally induced lactic acidosis in cattle. Four rumen-fistulated cattle were used for each dosage level and the design was a 4 x 4 Latin square with each animal receiving lasalocid, monensin, thiopeptin or no antibiotic. Acidosis was induced by intraruminal administration of glucose (12.5 g/kg body weight). Control cattle exhibited the typical drop in rumen pH and concurrent increases in L(+) and D(-) lactate concentrations commonly observed in cases of lactic acidosis. Alkali reserves were depleted in the control cattle as evidenced by a decrease in blood bicarbonate and a negative shift in base excess. In all three trials, cattle given lasalocid had higher rumen pH and lower lactate concentrations than did control cattle or cattle given monensin or thiopeptin. Cattle given monensin had a significantly higher rumen pH and a lower lactate concentration than the controls only at the .65 and 1.3 mg/kg body weight dosages, whereas thiopeptin was effective only at the 1.3-mg dosage. Concentrations of total VFA in rumen fluid decreased in the controls but remained unchanged in cattle given antibiotics. A significant reduction in the molar proportion of acetate and an increase in the molar proportion of propionate were observed in the rumen fluid of the cattle given antibiotics. Colony counts of Streptococcus bovis and Lactobacillus were significantly reduced in rumen fluid of cattle given 1.3 mg antibiotic/kg body weight. Counts of lactate-utilizing bacteria increased in both control cattle and cattle given antibiotics. Cattle given antibiotics showed no evidence of lacticacidemia, hemoconcentration or change in acid-base balance.