Rapid separation and concentration of food-borne pathogens in food samples prior to quantification by viable-cell counting and real-time PCR

Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low- and high-speed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10(1) to 10(3) CFU/g using the RTi-qPCR assay, and amounts as small as 10(0) to 10(1) CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10(1) to 10(2) CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.     

Detection of Small Numbers of Campylobacter jejuni and Campylobacter coli Cells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as ≤3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.     

Purification of Xenopus laevis embryo and liver nuclei using metrizamide.

While the nuclei of many diverse types of tissue can be purified by centrifugation through dense sucrose solutions, Xenopus laevis embryo and liver nuclei present special purification problems due to the presence of large numbers of melanosomes in embryo and liver cells. These melanosomes were removed by isopycnic centrifugation in Metrizamide gradients. In Metrizamide, embryo nuclei banded at an average buoyant density of ρ_5°C = 1.288 ± 0.003 g/cm³. Liver nuclei separated into two peaks, one containing nonparenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.274 ± 0.003 g/cm³ and the other peak containing parenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.284 ± 0.001 g/cm³.     

Effects of Lipooligosaccharide Inner Core Truncation on Bile Resistance and Chick Colonization by Campylobacter jejuni

Campylobacter jejuni is the most common bacterium that causes diarrhea worldwide, and chickens are considered the main reservoir of this pathogen. This study investigated the effects of serial truncation of lipooligosaccharide (LOS), a major component of the outer membrane of C. jejuni, on its bile resistance and intestinal colonization ability in chickens. Genes encoding manno-heptose synthetases or glycosyltransferases were inactivated to generate isogenic mutants. Serial truncation of the LOS core oligosaccharide caused a stepwise increase in susceptibilities of two C. jejuni strains, NCTC 11168 and 81-176, to bile acids. Inactivation of hldE, hldD, or waaC caused severe truncation of the core oligosaccharide, which greatly increased the susceptibility to bile acids. Both wild-type strains grew normally in chicken intestinal extracts, whereas the mutants with severe oligosaccharide truncation were not detected 12 h after inoculation. These mutants attained viable bacterial counts in the bile acid-free extracts 24 h after inoculation. The wild-type strain 11-164 was present in the cecal contents at >10⁷ CFU/g on 5 days after challenge infection and after this time period, whereas its hldD mutant was present at <10³ CFU/g throughout the experimental period. Trans-complementation of the hldD mutant with the wild-type hldD allele completely restored the in vivo colonization level to that of the wild-type strain. Mutants with a shorter LOS had higher hydrophobicities. Thus, the length of the LOS core oligosaccharide affected the surface hydrophobicity and bile resistance of C. jejuni as well as its ability to colonize chicken intestines.     

Conspicuous Growth of Intravenously Inoculated Staphylococcus aureus in Subcutaneously Established Ehrlich Ascites Tumor Tissue of Mice

Intratumoral growth of Staphylococcus aureus was compared with the intrarenal growth, to examine the usefulness of the method as a marker of its pathogenicity. When 5 × 10⁷ CFU/mouse of three derivatives from S. aureus Cowan I with different intrarenal growth were intravenously injected into Ehrlich tumor-bearing mice, they lodged in the tumor tissue at approximately 10³ CFU/0.1 g by 30 min after infection, and grew in the range of 10⁶ CFU/0.1 g to 10⁸ CFU/0.1 g by day 4, regardless of their intrarenal growth capacity. In contrast, S. saprophyticus lodged in both tissues to the same degree as S. aureus, but did not grow at all. The time course of the staphylococcal growth was different between tumor tissue and kidney, suggesting differences in the local responses against S. aureus.     

Quantitative in vitro assay to evaluate the capability of yeast cell wall fractions from Trichosporon mycotoxinivorans to selectively bind gram negative pathogens

Yeast cell wall fractions have been proposed to bind enteropathogenic bacteria. The aim of this study was to develop a quantitative assay by measuring the optical density as growth parameter of adhering bacteria. The exponential growth phase of adhering bacteria was determined by optical density reading and compared with the colony count (CFU/mL). A linear regression was compiled and the bacterial number bound to the yeast cell wall product could be determined. Further focus was the investigation of a yeast cell wall from strain Trichosporon mycotoxinivorans (MTV) for its ability to bind gram negative Salmonella, E. coli and Campylobacter strains and gram positive probiotic bacteria of the genera lactobacilli and bifidobacteria as well as gram positive Clostridium perfringens quantitatively. The gram negative probiotic strain E. coli Nissle 1917 was also investigated. Seven out of 10 S. Typhimurium and S. Enteritidis strains adhered to the cell wall product with an amount between 10³ and 10⁴ CFU/10 μg. Four out of 7 E. coli strains showed an average binding capability (10² CFU/10 µg) whereas 4 × 10³E. coli F4 cells bound per 10 μg yeast cell wall. E. coli 0149 K91, E. coli 0147 K89, C. jejuni and C. perfringens as well the genera lactobacilli and bifidobacteria did not bind to the yeast cell wall. E. coli Nissle 1917 was bound with 2 × 10² CFU/10 μg. These results demonstrate that cell wall from MTV can be used to differentially bind E. coli spp. and Salmonella spp. up to 8 × 10⁴ CFU/10 μg. Thus certain yeast cell walls may prevent enteric infections caused by selective bacteria. This methodical approach would be an accurate tool in the feed industry for quality control of yeast cell wall products.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Direct Real-Time PCR Quantification of Campylobacter jejuni in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log₁₀ CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5′-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log₁₀ were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log₁₀ CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.     

Rapid Detection Device for <Emphasis Type="Italic">Salmonella typhi</Emphasis> in Milk,Juice, Water and Calf Serum

A limit of detection of 200 CFU/mL of Salmonella typhi spiked in various sample matrices were achieved in 30 min. The sample matrices were raw/unprocessed milk, commercially available milk, juice from packed bottles, fresh juice from carts, potable water, turbid water and calf serum. The complete protocol comprised of three steps: (a) cell lysis (b) nucleic acid amplification and (c) an in situ optical detection. The cell lysis was carried out using a simple heating based protocol, while the loop-mediated isothermal amplification of DNA was carried out by an in-house designed and fabricated system. The developed system consists of an aluminum block fitted with two cartridge heaters along with a thermocouple. The system was coupled to a light source and spectrometer for a simultaneous in situ detection. Primers specific for STY2879 gene were used to amplify the nucleic acid sequence, isolated from S. typhi cells. The protocol involves 15 min of cell lysis and DNA isolation followed by 15 min for isothermal amplification and simultaneous detection. No cross-reactivity of the primers were observed at 10⁶ CFU/mL of Escherichia coli, Vibrio cholerae, Salmonella typhimurium, Salmonella paratyphi A, Pseudomonas aeruginosa, Bacillus cereus, Lysteria monocytogenes, Clostridium botulinum, Staphylococcus aureus and Salmonella havana. In addition, the system was able to detect S. typhi of 200 CFU/mL in a concoction of 10⁶ CFU/mL of E. coli, 10⁶ CFU/mL of V. cholerae, and 10⁶ CFU/mL of hepatocyte-derived cellular carcinoma HUH7 cells. The proposed rapid diagnostic system shows a promising future in the field of food and medical diagnostics.     

Isolation and characterization of microbodies and symphyomicrobodies with different buoyant densities from the fungus Blastocladiella emersonii.

Microbodies isolated from sporangia of the aquatic fungus $\text{Blastocladiella emersonii}$ have a mean buoyant density of 1.222 g/cm³ after centrifugation through a linear sucrose gradient, and contain catalase, isocitrate lyase and malate synthase. Microbodies fuse to produce one symphyomicrobody per zoospore at the time of sporogenesis. An increase in density accompanies this process. The symphyomicrobody has a mean buoyant density of 1.292 g/cm³ while the spore's single mitochondrion has a buoyant density of 1.219 g/cm³. Statistical data are also provided for both starting levels and purification of symphyomicrobody and mitochondrial enzyme markers.     

Multiplex PCR and DNA array for the detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. targeting virulence-related genes

A multiplex PCR and DNA array for quick detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. was developed using specific genetic markers derived from virulence-related genes. The genetic markers of cytK, sei, prfA, rfB, and hilA gene specifically amplified DNA fragments of 320 bp, 500 bp, 700 bp, 1.0 kb and 1.2 kb from B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., respectively. These markers are specific for the detection of the corresponding target pathogens. The sensitivity of the genetic markers was down to ～0.5 fg genomic DNA and ～10¹ CFU/ml (one bacterial cell per reaction) of bacterial culture. The combination of mPCR and DNA macroarray hybridization sensitively and specifically detected B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., in complex mixed cultures and food matrices. Thus, this mPCR and macroarray-based approach serves as rapid and reliable diagnostic tool for the detection of these five pathogens.     

Bacteriophage-Mediated Dispersal of Campylobacter jejuni Biofilms

Bacteria in their natural environments frequently exist as mixed surface-associated communities, protected by extracellular material, termed biofilms. Biofilms formed by the human pathogen Campylobacter jejuni may arise in the gastrointestinal tract of animals but also in water pipes and other industrial situations, leading to their possible transmission into the human food chain either directly or via farm animals. Bacteriophages are natural predators of bacteria that usually kill their prey by cell lysis and have potential application for the biocontrol and dispersal of target bacteria in biofilms. The effects of virulent Campylobacter specific-bacteriophages CP8 and CP30 on C. jejuni biofilms formed on glass by strains NCTC 11168 and PT14 at 37°C under microaerobic conditions were investigated. Independent bacteriophage treatments (n ≥ 3) led to 1 to 3 log₁₀ CFU/cm² reductions in the viable count 24 h postinfection compared with control levels. In contrast, bacteriophages applied under these conditions effected a reduction of less than 1 log₁₀ CFU/ml in planktonic cells. Resistance to bacteriophage in bacteria surviving bacteriophage treatment of C. jejuni NCTC 11168 biofilms was 84% and 90% for CP8 and CP30, respectively, whereas bacteriophage resistance was not found in similarly recovered C. jejuni PT14 cells. Dispersal of the biofilm matrix by bacteriophage was demonstrated by crystal violet staining and transmission electron microscopy. Bacteriophage may play an important role in the control of attachment and biofilm formation by Campylobacter in situations where biofilms occur in nature, and they have the potential for application in industrial situations leading to improvements in food safety.     

Rapid Sample Processing for Detection of Food-Borne Pathogens via Cross-Flow Microfiltration

This paper reports an approach to enable rapid concentration and recovery of bacterial cells from aqueous chicken homogenates as a preanalytical step of detection. This approach includes biochemical pretreatment and prefiltration of food samples and development of an automated cell concentration instrument based on cross-flow microfiltration. A polysulfone hollow-fiber membrane module having a nominal pore size of 0.2 μm constitutes the core of the cell concentration instrument. The aqueous chicken homogenate samples were circulated within the cross-flow system achieving 500- to 1,000-fold concentration of inoculated Salmonella enterica serovar Enteritidis and naturally occurring microbiota with 70% recovery of viable cells as determined by plate counting and quantitative PCR (qPCR) within 35 to 45 min. These steps enabled 10 CFU/ml microorganisms in chicken homogenates or 10² CFU/g chicken to be quantified. Cleaning and sterilizing the instrument and membrane module by stepwise hydraulic and chemical cleaning (sodium hydroxide and ethanol) enabled reuse of the membrane 15 times before replacement. This approach begins to address the critical need for the food industry for detecting food pathogens within 6 h or less.     

Isolation and composition of bacteriophage-like particles from kappa of killer Paramecia

Summary Phage-like particles from kappa of stock 562 of Paramecium aurelia have been isolated by CsCl density gradient centrifugation. Analyses show that the particles contain about 1.6×10¹⁶g DNA and 2.0×10^-16g protein. Their buoyant density is approximately 1.47. DNA from the particles has a buoyant density very close to that of whole kappa DNA. The presence of DNA in the particles has been confirmed by a cytochemical technique. The results support the conclusion that kappa contains a bacteriophage.     

Direct Quantification of Campylobacter jejuni and Campylobacter lanienae in Feces of Cattle by Real-Time Quantitative PCR

Campylobacter species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify Campylobacter jejuni and Campylobacter lanienae in cattle feces. For C. jejuni, the single-copy mapA gene was selected. For C. lanienae, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of C. jejuni quantification, and the limit of quantification was 19 to 25 genome copies (≈3 × 10³ CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on C. lanienae by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies (≈250 CFU/g of feces), and there was no discernible difference between the two C. lanienae secondary primer sets evaluated. Detection and quantification of C. jejuni in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect C. lanienae in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of C. lanienae, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.     

Biochemical and biophysical properties of hyphomicrobium bacteriophage hyphi30

Hyphomicrobium bacteriophage Hy30 and its nucleic acid were studied to determine some of their biochemical and biophysical properties. The molecular weight of the phage is 55.4 × 10⁶, and its buoyant density is 1.508 g/ml. The nucleic acid of Hy30 is linear, double-stranded DNA with a molecular weight of 29.7 × 10⁶. The guanine-plus-cytosine content of the DNA was 62% as determined from its melting temperature and buoyant density.     

Detection of Campylobacter jejuni in poultry samples using an enzyme-linked immunoassay coupled with an enzyme electrode

An enzyme-linked immunoassay coupled with a tyrosinase modified enzyme electrode was used for rapid detection of Campylobacter jejuni. The immunomagnetic separation (IMS) method was investigated to achieve optimal isolation of C. jejuni cells. Eight types of beads with three different sizes and function groups were coated with anti-C. jejuni to isolate C. jejuni from the sample solution. Bead size and coating methods were found to be major factors that influenced the capture efficacy. Streptavidin-labeled beads (2.8 μm) provided the greatest capture ability. Three blocking reagents were tested to minimize non-specific binding. Bovine serum albumin (BSA) showed the best blocking capability. Two IMS formats were tested. Competitive immunoassay cut the detection time to 1.5 h, but the detection limit was relatively high (10⁶ CFU/ml). This system was evaluated using C. jejuni pure culture and poultry samples inoculated with C. jejuni. This detection method for C. jejuni could be completed within 2.5 h and had a detection limit of 2.1×10⁴ CFU/ml. No significant difference was found between pure culture samples and poultry samples (P>0.01). A linear relationship was found between C. jejuni cell numbers and the peak current ratio in a range of 10²–10⁷ CFU/ml (R²=0.94).     

Laccase- and chloroperoxidase-nanotube paint composites with bactericidal and sporicidal activity

Laccase and chloroperoxidase (CPO) were separately immobilized onto multi-walled carbon nanotubes (MWNTs) and subsequently mixed with a commercial eco-friendly paint to generate biocatalytic coatings. The laccase-nanotube based paints showed >99% bactericidal activity against Escherichia coli and Staphylococcus aureus (both challenged with 10⁶ CFU/mL) within 30 min and >98% sporicidal activity against Bacillus cereus and Bacillus anthracis-ΔSterne (initially challenged with 10⁴ CFU/mL) within 120 min. The CPO-nanotube based paints also showed >99% antimicrobial activity within 30 min against E. coli and S. aureus (both challenged with 10⁶ CFU/mL). These enzyme-nanotube based formulations provide an eco-friendly route to generate biocidal compounds, which can prevent the growth of a broad spectrum of bacterial pathogens, including spores. These enzyme-containing paints may be envisioned to be applied as self-decontaminating coatings onto a wide range of surfaces, such as hospital infrastructure, medical devices and equipment, food processing and packaging, etc.; in all cases effective killing of a variety of infectious organisms is critical.     

Survival of Cold-Stressed Campylobacter jejuni on Ground Chicken and Chicken Skin during Frozen Storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4°C, freezing at −20°C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log₁₀ CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log₁₀ CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log₁₀ CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log₁₀ CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and −20°C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.     

Use of PCR for Direct Detection of Campylobacter Species in Bovine Feces

This study reports on the use of PCR to directly detect and distinguish Campylobacter species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR, Campylobacter coli, C. fetus, C. hyointestinalis, and C. jejuni were detected in all fecal samples inoculated at ≈10⁴ CFU g⁻¹, and 50 to 83% of the samples inoculated at ≈10³ CFU g⁻¹ were positive. At ≈10² CFU g⁻¹, C. fetus, C. hyointestinalis, and C. jejuni (17 to 50% of the samples) but not C. coli were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of Campylobacter were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were C. jejuni (152 isolates) and C. lanienae (42 isolates), but isolates of C. fetus subsp. fetus, Arcobacter butzleri, and A. skirrowii also were recovered (≤2 isolates per taxon). Considerable variability was observed in the frequency of isolation of campylobacters among the four media and three incubation temperatures tested. With genus-specific primers, Campylobacter DNA was detected in 75% of the fecal samples, representing an 8% increase in sensitivity relative to that obtained with microbiological isolation across the four media and three incubation temperatures tested. With nested primers, C. jejuni and C. lanienae were detected in 25 and 67% of the samples, respectively. In no instance was DNA from either C. coli, C. fetus, or C. hyointestinalis detected in uninoculated bovine feces. PCR was more sensitive than isolation on microbiological media for detecting C. lanienae (17%) but not C. jejuni. Campylobacters are a diverse and fastidious group of bacteria, and the development of direct PCR not only will increase the understanding of Campylobacter species diversity and their frequency of occurrence in feces but also will enhance the knowledge of their role in the gastrointestinal tract of livestock and of the factors that influence shedding.