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Objectives: To identify and characterize a novel cfr variant that recently emerged and confers multidrug resistance in Campylobacter , a major foodborne pathogen.Methods: WGS was initially used to identify the cfr (C) gene in Campylobacter isolates and its function was further verified by cloning into an antibiotic-susceptible Campylobacter jejuni strain. Distribution of cfr (C) in various Campylobacter isolates was determined by PCR analysis. Genotyping of cfr (C)-positive strains was done by PFGE and MLST.Results: The cfr (C) gene is predicted to encode a protein that shares 55.1% and 54.9% identity with Cfr and Cfr(B), respectively. cfr (C) was located on a conjugative plasmid of ∼48 kb. Cloning of cfr (C) into C. jejuni NCTC 11168 and conjugative transfer of the cfr (C)-containing plasmid confirmed its role in conferring resistance to phenicols, lincosamides, pleuromutilins and oxazolidinones, and resulted in an 8-256-fold increase in their MICs in both C. jejuni and Campylobacter coli . The cfr (C) gene was detected in multiple C. coli (34 of 344; 10%) isolates derived from different cattle farms in different states, and molecular typing of the cfr (C)-positive C. coli isolates revealed its spread mainly via clonal expansion.Conclusions: These results identify cfr (C) as a new multidrug resistance mechanism in Campylobacter and suggest the potential transmission of this mechanism via the foodborne route, warranting enhanced efforts to monitor its spread in Campylobacter and other foodborne pathogens. [ABSTRACT FROM AUTHOR]

Aims: European starlings ( Sturnus vulgaris) are an invasive species in the United States and are considered a nuisance pest to agriculture. The goal of this study was to determine the potential for these birds to be reservoirs and/or vectors for the human pathogen Escherichia coli O157:H7. Materials and Results: Under biosecurity confinement, starlings were challenged with various doses of E. coli O157:H7 to determine a minimum infectious dose, the magnitude and duration of pathogen shedding, and the potential of pathogen transmission among starlings and between starlings and cattle. Birds transiently excreted E. coli O157:H7 following low-dose inoculation; however, exposure to greater than 105.5 colony-forming units (CFUs) resulted in shedding for more than 3 days in 50% of the birds. Colonized birds typically excreted greater than 103 CFU g−1 of faeces, and the pathogen was detected for as long as 14 days postinoculation. Cohabitating E. coli O157:H7-positive starlings with culture-negative birds or 12-week-old calves resulted in intra- and interspecies pathogen transmission within 24 h. Likewise, E. coli O157:H7 was recovered from previously culture-negative starlings following 24-h cohabitation with calves shedding E. coli O157:H7. Conclusions: European starlings may be a suitable reservoir and vector of E. coli O157:H7. Significance and Impact of the Study: Given the duration and magnitude of E. coli O157:H7 shedding by European starlings, European starlings should be considered a public health hazard. Measures aimed at controlling environmental contamination with starling excrement, on the farm and in public venues, may decrease food-producing animal and human exposure to this pathogen. [ABSTRACT FROM AUTHOR]

The number of bacterial genomes deposited each year in public databases is growing exponentially. However, efforts to use these genomes to track trends in antimicrobial resistance (AMR) have been limited thus far. We used 22,102 genomes from public databases to track AMR trends in nontyphoidal Salmonella in food animals in the United States. In 2018, genomes deposited in public databases carried genes conferring resistance, on average, to 2.08 antimicrobial classes in poultry, 1.74 in bovines, and 1.28 in swine. This represents a decline in AMR of over 70% compared to the levels in 2000 in bovines and swine, and an increase of 13% for poultry. Trends in resistance inferred from genomic data showed good agreement with U.S. phenotypic surveillance data (weighted mean absolute difference ± standard deviation, 5.86% ± 8.11%). In 2018, resistance to 3rd-generation cephalosporins in bovines, swine, and poultry decreased to 9.97% on average, whereas in quinolones and 4th-generation cephalosporins, resistance increased to 12.53% and 3.87%, respectively. This was concomitant with a decrease of bla CMY-2 but an increase in bla CTX-M-65 and gyrA D87Y (encoding a change of D to Y at position 87). Core genome single-nucleotide polymorphism (SNP) phylogenies show that resistance to these antimicrobial classes was predominantly associated with Salmonella enterica serovar Infantis and, to a lesser extent, S. enterica serovar Typhimurium and its monophasic variant I 4,[5],12:i:-, whereas quinolone resistance was also associated with S. enterica serovar Dublin. Between 2000 and 2018, trends in serovar prevalence showed a composition shift where S. Next-generation sequencing has led to an exponential increase in the number of genomes deposited in public repositories. This growing volume of information presents opportunities to track the prevalence of genes conferring antimicrobial resistance (AMR), a growing threat to the health of humans and animals. Using 22,102 public genomes, we estimated that the prevalence of multidrug resistance (MDR) in the United States decreased in nontyphoidal Salmonella isolates recovered from bovines and swine between 2000 and 2018, whereas it increased in poultry. These trends are consistent with those detected by national surveillance systems that monitor resistance using phenotypic testing. However, using genomes, we identified that genes conferring resistance to critically important antimicrobials were associated with specific MDR serovars that could be the focus for future interventions. Our analysis illustrates the growing potential of public repositories to monitor AMR trends and shows that similar efforts could soon be carried out in other regions where genomic surveillance is increasing.S. Infantis increased. Our findings illustrate the growing potential of using genomes in public databases to track AMR in regions where sequencing capacities are currently expanding. IMPORTANCE Next-generation sequencing has led to an exponential increase in the number of genomes deposited in public repositories. This growing volume of information presents opportunities to track the prevalence of genes conferring antimicrobial resistance (AMR), a growing threat to the health of humans and animals. Using 22,102 public genomes, we estimated that the prevalence of multidrug resistance (MDR) in the United States decreased in nontyphoidal Salmonella isolates recovered from bovines and swine between 2000 and 2018, whereas it increased in poultry. These trends are consistent with those detected by national surveillance systems that monitor resistance using phenotypic testing. However, using genomes, we identified that genes conferring resistance to critically important antimicrobials were associated with specific MDR serovars that could be the focus for future interventions. Our analysis illustrates the growing potential of public repositories to monitor AMR trends and shows that similar efforts could soon be carried out in other regions where genomic surveillance is increasing.

Campylobacter cause gastroenteritis in humans and may be shed in the feces of livestock and poultry species, including cattle, chicken, turkey, and swine. However, a synthesis of the prevalence on farms in the United States and Canada is currently lacking. Thus, our objective was to conduct a systematic review and meta-analysis to estimate the prevalence of Campylobacter coli , Campylobacter jejuni , and Campylobacter spp. on livestock and poultry farms operated under commercial conditions in the United States and Canada. The relevant literature was identified and examined for eligibility based on a priori inclusion and exclusion criteria. Relevant data were extracted, and a meta-analysis was performed. The data were transformed using the Freeman-Tukey arcsine transformation to stabilize the variance. A separate meta-analysis was performed for each animal species, level of sampling (individual versus pooled), and species of Campylobacter , for a total of 29 meta-analyses. C. jejuni and Campylobacter spp. were present in all livestock and poultry species of interest, whereas C. coli was found in all species of interest with the exception of chickens. Furthermore, substantial heterogeneity was observed in most meta-analyses. In an attempt to account for this, subgroup analyses were performed on potential moderators. However, with the exception of beef cattle, where studies in feedlot cattle reported a consistently higher prevalence compared with adult cattle on pasture, significant heterogeneity remained in the majority of meta-analyses after accounting for potential moderators. The results of this review can be used to inform future risk assessment, consumer and producer awareness, and resource allocation, and identify gaps for future research.

Salmonella Infantis carrying extended spectrum β-lactamase bla on a pESI-like megaplasmid has recently emerged in United States poultry. In order to determine the carriage rate and gene content variability of this plasmid in U.S. CTX-M-65 on a pESI-like megaplasmid has recently emerged in United States poultry. In order to determine the carriage rate and gene content variability of this plasmid in U.S. Salmonella Infantis, whole genome sequences of Salmonella isolates from humans and animals in the U.S. and internationally containing the pESI-like plasmid were analyzed. The U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) identified 654 product sampling isolates containing pESI-like plasmids through hazard analysis and critical control point (HACCP) verification testing in 2017 and 2018. The Centers for Disease Control and Prevention identified 55 isolates with pESI-like plasmids in 2016-2018 through the National Antimicrobial Resistance Monitoring System. Approximately 49% of pESI-like plasmids from FSIS verification isolates and 71% from CDC NARMS contained bla CTX-M-65 . Pan-plasmid genome analysis was also performed. All plasmids contained traN and more than 95% contained 172 other conserved genes; 61% contained bla CTX-M-65 . In a hierarchical clustering analysis, some plasmids from U.S. animal sources clustered together and some plasmids from South America clustered together, possibly indicating multiple plasmid lineages. However, most plasmids contained similar genes regardless of origin. Carriage of the pESI-like plasmid in U.S. appears to be limited to Salmonella Infantis and carriage rates increased from 2017 to 2018.

Genomic analyses were performed on florfenicol-resistant (FFN r ) Campylobacter coli isolates recovered from cattle, and the cfr (C) gene-associated multidrug resistance (MDR) plasmid was characterized. Sixteen FFN r C. coli isolates recovered between 2013 and 2018 from beef cattle were sequenced using MiSeq. Genomes and plasmids were found to be closed for three of the isolates using the PacBio system. Single nucleotide polymorphisms (SNPs) across the genome and the structures of MDR plasmids were investigated. Conjugation experiments were performed to determine the transferability of cfr (C)-associated MDR plasmids. The spectrum of resistance encoded by the cfr (C) gene was further investigated by agar dilution antimicrobial susceptibility testing. All 16 FFN r isolates were MDR and exhibited coresistance to ciprofloxacin, nalidixic acid, clindamycin, and tetracycline. All isolates shared the same resistance genotype, carrying aph (3')-III , hph , ΔaadE (truncated), bla OXA-61 , cfr (C), and tet (O) genes plus a mutation of GyrA (T86I). The cfr (C), aph (3')-III , hph , ΔaadE , and tet (O) genes were colocated on transferable MDR plasmids ranging in size from 48 to 50 kb. These plasmids showed high sequence homology with the pTet plasmid and carried several Campylobacter virulence genes, including virB2 , virB4 , virB5 , VirB6 , virB7 , virB8 , virb9 , virB10 , virB11 , and virD4 The cfr is a leading cause of foodborne diarrheal illness worldwide, with more than one million cases each year in the United States alone. The global emergence of antimicrobial resistance in this pathogen has become a growing public health concern. Florfenicol-resistant (FFN cfr (C) gene located in the conjugative pTet MDR/virulence plasmid is present in diverse strains, where it confers high levels of resistance to several antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. This report highlights the power of genomic antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies. IMPORTANCE has been very rare in the United States. In this study, we employed whole-genome sequencing to characterize 16 multidrug-resistant Campylobacter isolates recovered from cattle in the United States. A gene [ r ) Campylobacter has been very rare in the United States. In this study, we employed whole-genome sequencing to characterize 16 multidrug-resistant Campylobacter coli isolates recovered from cattle in the United States. A gene [ cfr (C)] was found to be responsible for resistance not only to florfenicol but also to several other antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. The results showed that cfr (C) is located in a conjugative pTet MDR/virulence plasmid. This report highlights the power of antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies.

Disease incidence is intimately associated with an animal's commensal bacteria populations (microbiome), as microbes that are involved with morbidity and mortality are commonly found in animals with no sign of disease. An understanding of the animal's resident respiratory pathogens, in the upper nasal cavity prior to weaning, may help us to understand the impact of these pathogens on incidence of respiratory disease. For this research, the overall goal was to characterize bacterial populations associated with calves at an early age and through time periods prior to weaning in 3 herds at the U.S. Meat Animal Research Center. Nasal swabs from the upper nasal cavity were collected at initial vaccination (approximately 40 d of age), preconditioning (approximately 130 d of age), and weaning (approximately 150 d of age) in 2015 and 2016. DNA was extracted from nasal swabs and combined into 2 pools of 10 animals for each sampling time point, in each herd, for a total of 6 pools at each sampling time point and 18 pools for all sampling time points within each year. To evaluate and compare the microbiome of each pooled sample, hypervariable regions 1 through 3 along the 16S ribosomal RNA (rRNA) gene were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for identification of the bacterial taxa present. Alpha and beta diversity were also measured. Overall, microbial communities were different between combinations of sampling year, herd location, and sampling time prior to weaning as shown by beta diversity. Analysis of these specific respiratory pathogens prior to weaning will present a clearer picture of the distribution of microbial populations in animals prior to weaning and not exhibiting clinical signs of respiratory disease. Therefore, evaluation of the animal's resident bacterial populations in the upper nasal cavity during different phases of the beef production system may help us to understand the impact of the microbiome on incidence of respiratory disease in cattle., (Published by Oxford University Press on behalf of the American Society of Animal Science 2019.)

Objectives: To sequence the genomes and determine the genetic mechanisms for linezolid resistance identified in three strains of Enterococcus isolated from cattle and swine caecal contents as part of the US National Antimicrobial Resistance Monitoring System (NARMS) surveillance programme., Methods: Broth microdilution was used for in vitro antimicrobial susceptibility testing to assess linezolid resistance. Resistance mechanisms and plasmid types were identified from data generated by WGS on Illumina® and PacBio® platforms. Conjugation experiments were performed to determine whether identified mechanisms were transmissible., Results: Linezolid resistance plasmids containing optrA were identified in two Enterococcus faecalis isolates and one Enterococcus faecium. The E. faecium isolate also carried the linezolid resistance gene cfr on the same plasmid as optrA. The linezolid resistance plasmids had various combinations of additional resistance genes conferring resistance to phenicols (fexA), aminoglycosides [spc and aph(3')-III] and macrolides [erm(A) and erm(B)]. One of the plasmids was confirmed to be transmissible by conjugation, resulting in linezolid resistance in the transconjugant., Conclusions: To the best of our knowledge, this is the first identification of linezolid resistance in the USA in bacteria isolated from food animals. The oxazolidinone class of antibiotics is not used in food animals in the USA, but the genes responsible for resistance were identified on plasmids with other resistance markers, indicating that there may be co-selection for these plasmids due to the use of different antimicrobials. The transmissibility of one of the plasmids demonstrated the potential for linezolid resistance to spread horizontally. Additional surveillance is necessary to determine whether similar plasmids are present in human strains of Enterococcus.

Cattle hides are an important source of enterohaemorrhagic Escherichia coli (EHEC) carcass contamination at slaughter. Seven EHEC serogroups are adulterants in raw, non-intact beef: EHEC O26, O45, O103, O111, O121, O145 and O157. The objective of this study was to estimate the probability for hide contamination with EHEC among US market beef cows at slaughter and to test the effects of season and geographic region on prevalence of hide contamination. Hides (n = 800) of market cows were swabbed at slaughter immediately after exsanguination, prior to hide removal. Cows were sampled from two geographically distinct beef packing plants during four seasons of 2015. Cattle source was categorized by northern or southern region. Samples were tested for EHEC by a molecular screening assay. The effects of region, season and their interaction on the probability of hide contamination by each EHEC serogroup were tested in separate multilevel multivariable logistic regression models, accounting for the random effect of clustering by plant. Statistical significance was set α = .05. Of 800 total samples, at least one EHEC was detected on 630 (79%) hides. Enterohaemorrhagic E. coli O26 was detected on 129 (16%) of all hides sampled, EHEC O45 on 437 (55%), EHEC O103 on 289 (36%), EHEC O111 on 189 (24%), EHEC O121 on 140 (18%), EHEC O145 on 171 (21%) and EHEC O157 on 89 (11%). Detection of EHEC O26 and EHEC O121 was associated with season. Season and region were associated with detecting EHEC O45 and EHEC O157. Season-by-region interactions were associated with the outcome of detecting EHEC O103, EHEC O111 and EHEC O145. Season, region of origin and the interaction of these factors affect hide contamination of market beef cattle at slaughter by EHEC, and each serogroup responds to these factors uniquely., (© 2018 Blackwell Verlag GmbH.)

Transmission of antimicrobial resistance (AMR) from animal production systems to humans through the food supply is a public health concern. Currently, little is known about the prevalence of AMR among veal calves in the United States. Therefore, the objective of this prospective cohort study was to estimate the prevalence of AMR and multidrug resistance (MDR) among Escherichia coli within a vertically integrated production system. In addition, this study aimed to identify genes associated with phenotypic resistance to third- and fourth-generation cephalosporins (3GC and 4GC). Calves from four veal cohorts were randomly sampled resulting in a total of 166 farm fecal samples, 159 harvest fecal swabs, 164 preevisceration swabs, and 122 final carcass swabs. The prevalence of MDR among random-pick E. coli isolates recovered from the respective samples was 97% (161/166), 35% (55/159), 61% (51/84), and 24% (5/21). A selective isolation protocol found cefotaxime (a 3GC)-resistant isolates in 91% (127/140) of farm fecal samples, 34% (55/164) of preevisceration swabs, and 19% (23/122) of final carcass swabs tested. Isolates resistant to cefepime, a 4GC, were found among 24% (33/140), 6.7% (11/164), and 0.8% (1/122) of the same, respective samples. Isolates resistant to ciprofloxacin, a fluoroquinolone, were recovered from 75% (73/98) of farm fecal samples, 23% (38/164) of preevisceration swabs, and 6.6% (8/122) of final carcass swabs. The bla CMY-2 and bla CTX-M resistance genes were found in 89% (93/105) and 100% (42/42) of tested subsets of 3GC- and 4GC-resistant isolates, respectively. Pulsed-field gel electrophoresis (PFGE) analysis conducted on 3GC- and fluoroquinolone-resistant isolates showed three indistinguishable PFGE patterns from cefotaxime-resistant isolates recovered at farm and from two preevisceration carcass swabs. Although the prevalence of resistance declined between initial farm fecal samples and final carcass swabs, resistant bacteria recovered from carcasses illustrate the potential transmission of AMR to the human food supply.

Objectives: To identify and characterize a novel cfr variant that recently emerged and confers multidrug resistance in Campylobacter , a major foodborne pathogen., Methods: WGS was initially used to identify the cfr (C) gene in Campylobacter isolates and its function was further verified by cloning into an antibiotic-susceptible Campylobacter jejuni strain. Distribution of cfr (C) in various Campylobacter isolates was determined by PCR analysis. Genotyping of cfr (C)-positive strains was done by PFGE and MLST., Results: The cfr (C) gene is predicted to encode a protein that shares 55.1% and 54.9% identity with Cfr and Cfr(B), respectively. cfr (C) was located on a conjugative plasmid of ∼48 kb. Cloning of cfr (C) into C. jejuni NCTC 11168 and conjugative transfer of the cfr (C)-containing plasmid confirmed its role in conferring resistance to phenicols, lincosamides, pleuromutilins and oxazolidinones, and resulted in an 8-256-fold increase in their MICs in both C. jejuni and Campylobacter coli . The cfr (C) gene was detected in multiple C. coli (34 of 344; 10%) isolates derived from different cattle farms in different states, and molecular typing of the cfr (C)-positive C. coli isolates revealed its spread mainly via clonal expansion., Conclusions: These results identify cfr (C) as a new multidrug resistance mechanism in Campylobacter and suggest the potential transmission of this mechanism via the foodborne route, warranting enhanced efforts to monitor its spread in Campylobacter and other foodborne pathogens., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The United States Department of Agriculture Food Safety and Inspection Service has declared seven Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, nonintact beef products. The objective of this study was to determine the prevalence of these seven serogroups and the associated virulence genes (Shiga toxin [stx1, stx2], and intimin [eae]) in cattle feces during summer (June-August 2013) and winter (January-March 2014) months. Twenty-four pen floor fecal samples were collected from each of 24 cattle pens, in both summer and winter months, at a commercial feedlot in the United States. Samples were subjected to culture-based detection methods that included enrichment, serogroup-specific immunomagnetic separation and plating on selective media, followed by a multiplex polymerase chain reaction for serogroup confirmation and virulence gene detection. A sample was considered STEC positive if a recovered isolate harbored an O gene, stx1, and/or stx2, and eae genes. All O serogroups of interest were detected in summer months, and model-adjusted prevalence estimates are as follows: O26 (17.8%), O45 (14.6%), O103 (59.9%), O111 (0.2%), O121 (2.0%), O145 (2.7%), and O157 (41.6%); however, most non-O157 isolates did not harbor virulence genes. The cumulative model-adjusted sample-level prevalence estimates of STEC O26, O103, O145, and O157 during summer (n=576) were 1.0, 1.6, 0.8, and 41.4%, respectively; STEC O45, O111, and O121 were not detected during summer months. In winter, serogroups O26 (0.9%), O45 (1.5%), O103 (40.2%), and O121 (0.2%) were isolated; however, no virulence genes were detected in isolates from cattle feces collected during winter (n=576). Statistically significant seasonal differences in prevalence were identified for STEC O103 and O157 (p<0.05), but data on other STEC were sparse. The results of this study indicate that although non-O157 serogroups were present, non-O157 STEC were rarely detected in feces from the feedlot cattle populations tested in summer and winter months.

Pathogenic Escherichia coli or its associated virulence factors have been frequently detected in dairy cow manure, milk, and dairy farm environments. However, it is unclear what the long-term dynamics of E. coli virulence factors are and which farm compartments act as reservoirs. This study assessed the occurrence and dynamics of four E. coli virulence factors (eae, stx1, stx2, and the gamma allele of the tir gene [γ-tir]) on three U.S. dairy farms. Fecal, manure, water, feed, milk, and milk filter samples were collected from 2004 to 2012. Virulence factors were measured by postenrichment quantitative PCR (qPCR). All factors were detected in most compartments on all farms. Fecal and manure samples showed the highest prevalence, up to 53% for stx and 21% for γ-tir in fecal samples and up to 84% for stx and 44% for γ-tir in manure. Prevalence was low in milk (up to 1.9% for stx and 0.7% for γ-tir). However, 35% of milk filters were positive for stx and 20% were positive for γ-tir. All factors were detected in feed and water. Factor prevalence and levels, expressed as qPCR cycle threshold categories, fluctuated significantly over time, with no clear seasonal signal independent from year-to-year variability. Levels were correlated between fecal and manure samples, and in some cases autocorrelated, but not between manure and milk filters. Shiga toxins were nearly ubiquitous, and 10 to 18% of the lactating cows were potential shedders of E. coli O157 at least once during their time in the herds. E. coli virulence factors appear to persist in many areas of the farms and therefore contribute to transmission dynamics., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Q fever is a worldwide zoonosis historically associated with exposure to infected livestock. This study summarizes cases of Q fever, a notifiable disease in the United States, reported to the Centers for Disease Control and Prevention through two national surveillance systems with onset during 2000-2012. The overall incidence rate during this time was 0.38 cases per million persons per year. The reported case fatality rate was 2.0%, and the reported hospitalization rate was 62%. Most cases (61%) did not report exposure to cattle, goats, or sheep, suggesting that clinicians should consider Q fever even in the absence of livestock exposure. The prevalence of drinking raw milk among reported cases of Q fever (8.4%) was more than twice the national prevalence for the practice. Passive surveillance systems for Q fever are likely impacted by underreporting and underdiagnosis because of the nonspecific presentation of Q fever., (© The American Society of Tropical Medicine and Hygiene.)

Data indicate that prevalence of specific serovars of Salmonella enterica in human foodborne illness is not correlated with their prevalence in feed. Given that feed is a suboptimal environment for S. enterica, it appears that survival in poultry feed may be an independent factor unrelated to virulence of specific serovars of Salmonella. Additionally, S. enterica serovars appear to have different host specificity and the ability to cause disease in those hosts is also serovar dependent. These differences among the serovars may be related to gene presence or absence and expression levels of those genes. With a better understanding of serovar specificity, mitigation methods can be implemented to control Salmonella at preharvest and postharvest levels.

Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are the leading cause of STEC-associated infections in humans in the United States. In the United States, these organisms are considered adulterants in raw nonintact beef products and in intact beef destined to be made into or used in nonintact raw beef products. The objective of this study was to provide an estimate of the burden of the six serogroups of non-O157 STEC in ground beef obtained from retail stores across the United States. A convenience sample of commercial ground beef products (n = 1,129) were purchased from retail stores in 24 states from October 2011 to May 2012. The samples had various lean/fat proportions, muscle group of origin (chuck, round, sirloin, or not specified), and packaging types. For each ground beef sample, 25 g was inoculated in 225 ml of modified tryptic soy broth, stomached for 1 min, and then incubated at 41°C for 18 ± 2 h. These enrichment cultures were then screened for stx, eae, and O group genes using a commercially available, closed-platform PCR-based method. The potential positive samples were subjected to immunomagnetic separation and plated on modified Rainbow agar. Morphologically typical colonies were subjected to latex agglutination and PCR determination of stx and eae genes. Nine (0.8%) of the ground beef samples were potentially positive for at least one STEC serogroup after PCR screening. The serogroups detected by PCR assay were O26 (four samples), O103 (four samples), O145 (three samples), O45 (two samples), and O121 (one sample). No STEC isolates belonging to these serogroups were recovered from the sample cultures. The current research provides updated surveillance data for non-O157 STEC isolates among commercial ground beef products and information regarding the potential sources of contamination from different parts of beef trims destined for ground beef production.

Bovine peripheral lymph nodes (LNs), including subiliac LNs, have been identified as a potential source of human exposure to Salmonella enterica, when adipose trim containing these nodes is incorporated into ground beef. In order to gain a better understanding of the burden of S. enterica in peripheral LNs of feedlot and cull cattle, a cross-sectional study was undertaken in which 3327 subiliac LNs were collected from cattle at harvest in seven plants, located in three geographically distinct regions of the United States. Samples were collected in three seasons: Fall 2010, Winter/Spring 2011, and Summer/Fall 2011. A convenience sample of 76 LNs per day, 2 days per season (approximately 1 month apart), was collected per plant, from carcasses held in the cooler for no less than 24 h. Every 10(th) carcass half on a rail was sampled, in an attempt to avoid oversampling any single cohort of cattle. Median point estimates of S. enterica contamination were generally low (1.3%); however, median Salmonella prevalence was found to be greater in subiliac LNs of feedlot cattle (11.8%) compared to those of cull cattle (0.65%). Enumeration analysis of a subset of 618 feedlot cattle LNs showed that 67% of those harboring S. enterica (97 of 144) did so at concentrations ranging from <0.1 to 1.8 log10 CFU/g, while 33% carried a higher burden of S. enterica, with levels ranging from 1.9 to >3.8 log10 CFU/g. Serotyping of S. enterica isolated identified 24 serotypes, with the majority being Montevideo (44.0%) and Anatum (24.8%). Antimicrobial susceptibility phenotypes were determined for all isolates, and the majority (86.1%) were pansusceptible; however, multidrug-resistant isolates (8.3%) were also occasionally observed. As Salmonella contained within LNs are protected from carcass interventions, research is needed to define opportunities for mitigating the risk of Salmonella contamination in LNs of apparently healthy cattle.

The incidence of Clostridium difficile infection has recently increased in North American and European countries. This pathogen has been isolated from retail pork, turkey, and beef products and reported associated with human illness. This increase in infections has been attributed to the emergence of a toxigenic strain designated North America pulsed-field gel electrophoresis type 1 (NAP1). The NAP1 strain has been isolated from calves as well as ground meat products, leading to speculation of illness from consumption of contaminated meat products. However, information on C. difficile associated with beef cattle during processing and commercially produced ground beef is limited. To address this data gap, samples from various steps during beef production were collected. Samples from hides (n = 525), preevisceration carcasses (n = 475), postintervention carcasses (n = 471), and 956 commercial ground beef samples were collected from across the United States. The prevalence of C. difficile spores on hides was 3.2%. C. difficile spores were not detected on preevisceration and postintervention carcasses or in commercially produced ground beef. Phenotypic and genetic characterizations were carried out for all 18 isolates collected from hide samples. Twenty-two percent of the isolates were nontoxigenic strains, while 78% of the isolates were toxigenic. Toxinotyping and PCR ribotyping patterns revealed that 6 and 33% of the isolates were identified as NAP1 and NAP7 strains, respectively. This article evidences that the prevalence of C. difficile, specifically pathogenic strains, in the U.S. beef production chain is low.

The disinfectant and antibiotic susceptibility profiles of 344 Escherichia coli O157:H7 strains from cattle carcasses, feces, and hides and ground beef from the United States were determined. A low prevalence of antibiotic resistance was observed (14%). The highest prevalences of resistance were to sulfisoxazole (10.5%), tetracycline (9.9%), streptomycin (7%), and chloramphenicol (4.9%). Four strains were resistant to eight antibiotics (two strains from ground beef and one strain each from hide and preevisceration carcass swabs of cull cattle at harvest). Pulsed-field gel electrophoresis analysis of the E. coli O157:H7 strains revealed two major groups (designated 1 and 2) composed of 17 and 20 clusters, respectively. Clusters 1A, 1B, 1C, and 1G.1 were associated with multidrug-resistant strains. There was no observed correlation between disinfectant resistance and antibiotic resistance. Sixty-nine (20%) of the 344 strains were resistant to chlorhexidine or benzalkonium chloride or the MICs of benzyldimethyldodecylammonium chloride were elevated. Inducible resistance was observed at elevated concentrations of antibiotics (1.4%) and disinfectants (6.1%). The highest rate of disinfectant inducible resistance was to OdoBan, quaternary ammonium chlorides, and the surface disinfectants F25, FS512, and MG, which are used in dairies, restaurants, and food processing plants. High MICs (1,024 to 4,096 m g/ml) of acetic, lactic, and citric acids were found. The decreasing order of acid potency based on molar MICs (MICs(molar)) was acetic, citric, and lactic acid. The correlation of the concentration of dissociated organic acids and MICs(molar) strongly suggests that the observed inhibition of E. coli O157:H7 was primarily due to dissociated forms of the acids.

Salmonella enterica is the leading cause of foodborne-related deaths and hospitalizations within the United States. Infections caused by antimicrobial-resistant (AMR) strains are associated with higher hospital costs and case fatality. The objective for this study was to determine the association of management practices with the recovery of Salmonella and AMR Salmonella on dairy herds. Individual adult cow fecal samples and/or composite fecal samples were collected from 265 dairy herds in 17 states. Samples were cultured for Salmonella, and the MIC was determined for 15 antimicrobials. Herds were classified as Salmonella positive if at least one isolate was recovered, and AMR Salmonella positive if at least one resistant isolate was recovered. Questionnaires regarding management practices were administered to herd operators, and a subset of practices was selected based on subject knowledge and prior research. Data on preventive and therapeutic antimicrobial usage were included in the analysis. Logistic regression models were used to determine which practices were significantly (p<0.05) associated with each herd classification. A total of 124 and 25 herds were classified as Salmonella positive and AMR Salmonella positive, respectively. Variables significantly associated with Salmonella-positive herds included using sprinklers or misters for heat abatement (OR=2.8; CI: 1.6-4.9), feeding anionic salts to cows (OR=1.9; CI: 1.1-3.5), and feeding ionophores to cows (OR=2.1; CI: 1.2-3.7). Herds that used a broadcast/solid spread had lower odds (OR=0.26; CI: 0.11-0.63) of being Salmonella positive. Herds with at least one resistant isolate were more likely to have used composted/dried manure for bedding relative to herds with only susceptible isolates (OR=3.6; CI: 1.2-11.0). These results can be useful to focus additional research aimed at decreasing the prevalence of Salmonella and AMR Salmonella on U.S. dairy herds.

To assess the potential for food contamination with Clostridium difficile from food animals, we conducted a cross-sectional fecal prevalence study in 944 randomly selected cattle harvested at seven commercial meat processing plants, representing four distant regions (median distance of 1,500 km) of the United States. In all, 944 animals were sampled in the summer of 2008. C. difficile was isolated from 1.8% (17 of 944) of cattle, with median fecal shedding concentration of 2.2 log CFU/g (range = 1.6 to 4.8, 95% confidence interval = 1.6, 4.3). Toxigenic C. difficile isolates were recovered from only four (0.4%) cattle. One of these isolates was emerging PCR ribotype 078/toxinotype V. The remaining toxigenic isolates were toxinotype 0, one of which was an isolate with resistance to linezolid, clindamycin, and moxifloxacin (by the E-test). All isolates were susceptible to vancomycin, metronidazole, and tigecycline, but the MICs against linezolid were as high as the highest reported values for human-derived isolates. The source of the linezolid-clindamycin-moxifloxacin resistance in a toxigenic C. difficile isolate from cattle is uncertain. However, since the use of these three antimicrobial agents in cattle is not allowed in North America, it is possible that resistance originated from an environmental source, from other species where those antimicrobial agents are used, or transferred from other intestinal bacteria. This study confirms that commercial cattle can carry epidemiologically relevant C. difficile strains at the time of harvest, but the prevalence at the time they enter the food chain is low.

Staphylococcus aureus is a major agent of bovine mastitis. The concomitant emergence of pig-associated methicillin-resistant S. aureus (MRSA) in human carriage and infection requires a reexamination of the host range and specificity of human- and cow-associated S. aureus strains, something which has not been systematically studied previously. The genetic relatedness of 500 S. aureus isolates from bovine mastitis cases, 57 isolates from nasal carriage of farmers, and 133 isolates from nonfarmers was determined by amplified fragment length polymorphism (AFLP) analysis and spa typing. Multilocus sequence typing (MLST) was conducted on a subset of isolates to match AFLP clusters with MLST clonal complexes (CCs). This data set allowed us to study host range and host specificity and to estimate the extent of bovine-to-human transmission. The genotype compositions of S. aureus isolates from farmers and nonfarmers were very similar, while the mastitis isolates were quite distinct. Overall, transmission was low, but specific genotypes did show increased cow-to-human transmission. Unexpectedly, more than one-third of mastitis isolates belonged to CC8, a lineage which has not been considered to be bovine mastitis associated, but it is well known from human carriage and infection (i.e., USA300). Despite the fact that we did detect some transmission of other genotypes from cows to farmers, no transmission of CC8 isolates to farmers was detected, except for one tentative case. This was despite the close genetic relatedness of mastitis CC8 strains to nonfarmer carriage strains. These results suggest that the emergence of the new bovine-adapted genotype was due to a recent host shift from humans to cows concurrent with a loss of the ability to colonize humans. More broadly, our results indicate that host specificity is a lineage-specific trait that can rapidly evolve.

Since cattle are a major source of food and the cattle industry engages people from farms to processing plants and meat markets, it is conceivable that beef-products contaminated with Campylobacter spp. would pose a significant public health concern. To better understand the epidemiology of cattle-associated Campylobacter spp. in the USA, we characterized the prevalence, genotypic and phenotypic properties of these pathogens. Campylobacter were detected in 181 (19.2%) out of 944 fecal samples. Specifically, 71 C. jejuni, 132 C. coli, and 10 other Campylobacter spp. were identified. The prevalence of Campylobacter varied regionally and was significantly (P<0.05) higher in fecal samples collected from the South (32.8%) as compared to those from the North (14.8%), Midwest (15.83%), and East (12%). Pulsed Field Gel Electrophoresis (PFGE) analysis showed that C. jejuni and C. coli isolates were genotypically diverse and certain genotypes were shared across two or more of the geographic locations. In addition, 13 new C. jejuni and two C. coli sequence types (STs) were detected by Multi Locus Sequence Typing (MLST). C. jejuni associated with clinically human health important sequence type, ST-61 which was not previously reported in the USA, was identified in the present study. Most frequently observed clonal complexes (CC) were CC ST-21, CC ST-42, and CC ST-61, which are also common in humans. Further, the cattle associated C. jejuni strains showed varying invasion and intracellular survival capacity; however, C. coli strains showed a lower invasion and intracellular survival potential compared to C. jejuni strains. Furthermore, many cattle associated Campylobacter isolates showed resistance to several antimicrobials including ciprofloxacin, erythromycin, and gentamicin. Taken together, our results highlight the importance of cattle as a potential reservoir for clinically important Campylobacter.

Contamination can occur at a number of stages during farm-to-fork processing. Preharvest intervention is an ongoing area of interest in reduction of risk of foodborne illness. This study examined risk factors associated with detection of Salmonella from cattle in U.S. feedlots. During two visits to 73 feedlots, 25 fresh fecal samples were collected from each of three pen floors. Associations between management and demographic factors and culture status were evaluated using logistic regression. Factors positively associated with culture-positive status included brewers' grains (odds ratio [OR] = 26.35; confidence interval [CI], 10.33-67.20), corn gluten (OR = 10.35; CI, 5.98-17.91), or cottonseed hulls (OR = 8.34; CI, 3.58-19.42) in the ration, and sourcing animals in a pen from multiple herds of origin (OR = 5.17; CI, 2.32-11.51). Factors negatively associated with positive culture status included urea (OR = 0.27; CI, 0.16-0.44), alfalfa, clover, or sorghum silage (OR = 0.31; CI, 0.12-0.79), and antimicrobials of the tetracycline class in the ration (within 2 weeks before sampling, OR = 0.04 and CI, 0.02-0.09; more than 2 weeks before sampling, OR = 0.23 and CI, 0.06-0.80). Since 18.3% of positive samples were on a single operation, a second model was constructed after excluding data from this operation. Three additional variables were retained in the second model, including grain-processing method (OR for dry roll, cracked, or unprocessed grain = 2.99; CI, 1.55-5.75), soybean meal (OR = 2.74; CI, 1.58-4.75), and use of a coccidiostat in the ration (OR for no coccidiostat = 4.50; CI, 2.03-10.01). Considering the increasing use of by-products of the biofuel industry as feeds, further investigation of the association between feeding brewers' grains and corn gluten and Salmonella recovery is warranted.

Third-generation cephalosporin resistance of Salmonella and commensal Escherichia coli isolates from cattle in the United States is predominantly conferred by the cephamycinase CMY-2, which inactivates beta-lactam antimicrobial drugs used to treat a wide variety of infections, including pediatric salmonellosis. The emergence and dissemination of bla(CMY-2)(-)-bearing plasmids followed and may in part be the result of selection pressure imposed by the widespread utilization of ceftiofur, a third-generation veterinary cephalosporin. This study assessed the potential effects of ceftiofur on bla(CMY-2) transfer and dissemination by (i) an in vivo experimental study in which calves were inoculated with competent bla(CMY-2)-bearing plasmid donors and susceptible recipients and then subjected to ceftiofur selection and (ii) an observational study to determine whether ceftiofur use in dairy herds is associated with the occurrence and frequency of cephalosporin resistance in Salmonella and commensal E. coli. The first study revealed bla(CMY-2) plasmid transfer in both ceftiofur-treated and untreated calves but detected no enhancement of plasmid transfer associated with ceftiofur treatment. The second study detected no association (P = 0.22) between ceftiofur use and either the occurrence of ceftiofur-resistant salmonellosis or the frequency of cephalosporin resistance in commensal E. coli. However, herds with a history of salmonellosis (including both ceftiofur-resistant and ceftiofur-susceptible Salmonella isolates) used more ceftiofur than herds with no history of salmonellosis (P = 0.03) These findings fail to support a major role for ceftiofur use in the maintenance and dissemination of bla(CMY-2)-bearing plasmid mediated cephalosporin resistance in commensal E. coli and in pathogenic Salmonella in these dairy cattle populations.

A significant portion (15 to 20%) of beef in the United States is produced in small beef processing plants that harvest fewer than 1000 cattle per day. However, there are little data on the prevalence and levels of Escherichia coli O157:H7 and Salmonella in these processing plants. To address this lack of data, hides (n=1995) and carcasses (n=1995) of cattle at seven small processing plants located across the United States were analyzed for E. coli O157:H7 and Salmonella. Across all plants, hide prevalence of E. coli O157:H7 and Salmonella was 71 and 91%, respectively. Twelve percent of hides had E. coli O157:H7 at enumerable levels (> or =40 CFU/100 cm2), while 36% of hides had Salmonella at enumerable levels. Across all plants, the prevalence of E. coli O157:H7 on preevisceration carcasses was 33%, with 2% at an enumerable level (> or = 0.8 CFU/ 100 cm2). Across all plants, Salmonella prevalence on preevisceration carcasses was 58%, with 8% at an enumerable level. Significant plant-to-plant variations in levels and prevalence of pathogens on carcasses were detected. Reduced levels of pathogens on carcasses were noted among small processors that had incorporated a hide-directed intervention. The results obtained are comparable to those observed previously for larger processors, showing that smaller beef processors face and address the same challenges as do larger beef processors.

The U.S. Department of Agriculture (USDA) requires beef abattoir operators to periodically analyze beef carcass sponge samples for levels of Escherichia coli. Additional beef carcass sponge sampling is commonly used by processors to evaluate the efficacy of beef abattoir antimicrobial intervention systems. The USDA sample preparation procedure requires that beef carcass sponge samples be mechanically stomached for 2 min before the sample fluid is squeezed out for analysis. When a large number of sponge samples must be analyzed, the stomaching step can limit throughput. In this study, we compared the USDA sample preparation procedure with repeatedly squeezing the sponge during a 10-s interval to expel the sample fluid. Separate sponge samples were obtained from each half of 100 chilled postintervention beef carcasses from a large-volume abattoir during a 4-month period. The USDA and squeezing treatments were randomly assigned to the halves of each carcass. All sponge samples were analyzed for E. coli, coliforms, Enterobacteriaceae, and aerobic mesophilic bacteria using Petrifilm methods. The sample preparation method had no significant effect (signed rank value > 0.05) on the results of any analytical test, although aerobic mesophilic bacteria counts tended to be higher after the USDA method than after manual squeezing alone. These results suggest that manual squeezing may be a simple and rapid alternative sample preparation method when gram-negative bacteria such as E. coli, coliforms, or Enterobacteriaceae are being enumerated from beef carcass sponge samples used to monitor operational abattoir hygiene.

In the 1996 U.S. Food Safety and Inspection Service's (FSIS) "Pathogen Reduction; Hazard Analysis and Critical Control Point (PR/HACCP) Systems, Final Rule," Salmonella was selected for microbiological testing and monitoring. Using data from an FSIS-sponsored survey of meat and poultry slaughter establishments, inspection results, and other establishment characteristics, potential variables affecting pathogen control, as measured by Salmonella test results, were investigated. The analysis data sets included 153 federally inspected young chicken slaughter establishments, of which 111 exceeded half the Salmonella performance standard at least once from 2003 through 2005, and 121 federally inspected pork slaughter establishments, of which 28 exceeded half the Salmonella performance standard. Logistic regression results for young chicken slaughter establishments indicate they were more likely to exceed half the standard if they had higher inspection noncompliance rates (P = 0.10) and older production space (P = 0.10), and were less likely to exceed it if they used a higher percentage of raw poultry inputs purchased from outside sources (P = 0.10). Results for pork slaughter establishments indicate they were more likely to exceed half the standard if they had a higher rate of voluntary microbiological testing (P = 0.08), and were less likely to exceed it if they were larger (P = 0.08) and used a higher percentage of raw pork inputs purchased from outside sources (P = 0.02). In general, indicators of plant characteristics, food safety practices, and management philosophy are associated with different levels of pathogen control performance that vary by species slaughtered.

Optical maps for five representative clinical, food-borne and bovine-derived isolates from the 2006 Escherichia coli O157 : H7 outbreak linked to fresh spinach in the United States showed a common set of 14 distinct chromosomal markers that define the outbreak strain. Partial 454 DNA sequencing was used to characterize the optically mapped chromosomal markers. The markers included insertions, deletions, substitutions and a simple single nucleotide polymorphism creating a BamHI site. The Shiga toxin gene profile of the spinach-associated outbreak isolates (stx1(-) stx2(+) stx2c(+)) correlated with prophage insertions different from those in the prototypical EDL933 and Sakai reference strains (stx1(+) stx2(+) stx2c(-)). The prophage occupying the yehV chromosomal position in the spinach-associated outbreak isolates was similar to the stx1(+) EDL933 cryptic prophage V, but it lacked the stx1 gene. In EDL933, the stx2 genes are within prophage BP933-W at the wrbA chromosomal locus; this locus was unoccupied in the spinach outbreak isolates. Instead, the stx2 genes were found within a chimeric BP933-W-like prophage with a different integrase, inserted at the argW locus in the outbreak isolates. An extra set of Shiga toxin genes, stx2c, was found in the outbreak isolates within a prophage integrated at the sbcB locus. The optical maps of two additional clinical isolates from the outbreak showed a single, different prophage variation in each, suggesting that changes occurred in the source strain during the course of this widespread, multi-state outbreak.

To develop a process for predicting the likelihood of Salmonella contamination on beef carcasses, we evaluated the influence of several possible causative factors (i.e., year, abattoir, day of week, month, and intervention system components) on the risk of Salmonella and indicator organism contamination. Hide and carcass sponge samples were collected in 2005 to 2006 in six steps at three abattoirs in the East (A), Midwest (B), and Southwest (C) United States. Each abattoir used the same intervention system. Samples were analyzed for aerobic plate counts (APCs; n = 18,990) and Enterobacteriaceae counts (EBCs; n = 18,989) and the presence or absence of Salmonella (n = 5,355). Our results demonstrated that many factors play a significant role in the level of microbial contamination of beef carcasses. Overall, Salmonella prevalence and EBC levels were significantly higher in 2006 than in 2005. APCs and EBCs were highest in abattoirs A (3.57 log CFU/100 cm2) and B (1.31 log CFU/100 cm2). The odds of detecting a positive Salmonella isolate were greatest in abattoir C and lowest in abattoir A. Across the three abattoirs, the overall intervention process effectively reduced microbiological contamination. Salmonella prevalence fell from 45% (preevisceration) to 0.47% (postchilled-lactic acid), and there were APC and EBC reductions of 5.43 and 5.28 log CFU/100 cm2, respectively, from hide-on to postchilled-lactic acid samples. At each abattoir, composites of three individual EBC-negative carcass samples yielded Salmonella-negative results 97 to 99% of the time. These results suggest the possibility of using indicator test results to accurately predict the absence of Salmonella in a beef carcass sample.

Listeria monocytogenes, the causative agent of epidemic and sporadic listeriosis, is routinely isolated from many sources, including cattle, yet information on the prevalence of Listeria in beef processing plants in the United States is minimal. From July 2005 through April 2006, four commercial cow and bull processing plants were sampled in the United States to determine the prevalence of Listeria and the serovar diversity of L. monocytogenes. Samples were collected during the summer, fall, winter, and spring. Listeria prevalence on hides was consistently higher during cooler weather (28 to 92% of samples) than during warmer weather (6 and 77% of samples). The Listeria prevalence data collected from preevisceration carcass ranged from undetectable in some warm season samples to as high as 71% during cooler weather. Listeria on postintervention carcasses in the chill cooler was normally undetectable, with the exception of summer and spring samples from one plant where > 19% of the carcasses were positive for Listeria. On hides, L. monocytogenes serovar 1/2a was the predominant serovar observed, with serovars 1/2b and 4b present 2.5 times less often and serovar 1/2c not detected on any hides sampled. L. monocytogenes serovars 1/2a, 1/2c, and 4b were found on postintervention carcasses. This prevalence study demonstrates that Listeria species are more prevalent on hides during the winter and spring and that interventions being used in cow and bull processing plants appear to be effective in reducing or eliminating Listeria contamination on carcasses.

A total of 60 Salmonella enterica serovar Agona isolates (25 pan-susceptible isolates and 35 isolates resistant to five or more antimicrobials) submitted to the National Antimicrobial Resistance Monitoring System-Enteric Bacteria (NARMS) from 1997 through 2003 were examined for plasmids and class 1 integrons. Samples originated from cattle, turkey, chicken, and swine presented at federally inspected slaughter and processing plants. Large plasmids (33-291 kb) were present in 83% of the isolates resistant to five or more antimicrobials; however, 16% of the pan-susceptible isolates also had large plasmids. The presence of large plasmids did not correspond to the isolate source or the year the isolate was recovered but did appear to correspond to XbaI pulsed-field gel electrophoresis (PFGE) patterns. Two sizes of large plasmids appeared most often: 145.4 kb and 97 kb. Class 1 integrons were not detected on plasmids but were detected on the chromosome of 8% (2/25) of the pan-susceptible isolates and 49% (17/35) of the isolates with multiple drug resistance. Expression of multiple drug resistance among S. Agona isolates occurred regardless of the presence of class 1 integrons, suggesting that plasmids play an equally important role in the development of resistant S. Agona. More research is needed to understand better the mechanisms by which S. Agona acquires, harbors, and transfers resistance determinants.

Phenotypic traits of coexisting epidemic and nonepidemic strains of Salmonella enterica serovars Typhimurium and Newport were compared. Different stress conditions were relatively more or less favorable for the epidemic strains. Transcriptional analysis identified specific upregulated genes during defined stress conditions, but there were no common traits shared by epidemic serovars.

The U.S. Food Safety and Inspection Service (FSIS) tests sets of samples of selected raw meat and poultry products for Salmonella to ensure that federally inspected establishments meet performance standards defined in the pathogen reduction-hazard analysis and critical control point system (PR-HACCP) final rule. In the present report, sample set results are described and associations between set failure and set and establishment characteristics are identified for 4,607 sample sets collected from 1998 through 2003. Sample sets were obtained from seven product classes: broiler chicken carcasses (n = 1,010), cow and bull carcasses (n = 240), market hog carcasses (n = 560), steer and heifer carcasses (n = 123), ground beef (n = 2,527), ground chicken (n = 31), and ground turkey (n = 116). Of these 4,607 sample sets, 92% (4,255) were collected as part of random testing efforts (A sets), and 93% (4,166) passed. However, the percentage of positive samples relative to the maximum number of positive results allowable in a set increased over time for broilers but decreased or stayed the same for the other product classes. Three factors associated with set failure were identified: establishment size, product class, and year. Set failures were more likely early in the testing program (relative to 2003). Small and very small establishments were more likely to fail than large ones. Set failure was less likely in ground beef than in other product classes. Despite an overall decline in set failures through 2003, these results highlight the need for continued vigilance to reduce Salmonella contamination in broiler chicken and continued implementation of programs designed to assist small and very small establishments with PR-HACCP compliance issues.

Three hundred sixty-one quinupristin-dalfopristin (Q-D)-resistant Enterococcus faecium (QDREF) isolates were isolated from humans, turkeys, chickens, swine, dairy and beef cattle from farms, chicken carcasses, and ground pork from grocery stores in the United States from 1995 to 2003. These isolates were evaluated by pulsed-field gel electrophoresis (PFGE) to determine possible commonality between QDREF isolates from human and animal sources. PCR was performed to detect the streptogramin resistance genes vatD, vatE, and vgbA and the macrolide resistance gene ermB to determine the genetic mechanism of resistance in these isolates. QDREF from humans did not have PFGE patterns similar to those from animal sources. vatE was found in 35%, 26%, and 2% of QDREF isolates from turkeys, chickens, and humans, respectively, and was not found in QDREF isolates from other sources. ermB was commonly found in QDREF isolates from all sources. Known streptogramin resistance genes were absent in the majority of isolates, suggesting the presence of other, as-yet-undetermined, mechanisms of Q-D resistance.

Fecal specimens were obtained from 12-24-month-old dairy heifers on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. PCR positive specimens for Enterocytozoon bieneusi were found in 131 of 571 heifers examined (23%) and on all the farms visited. The prevalence of E. bieneusi varied considerably across farms, with the lowest prevalence (4.7%) on MD-2 and the highest prevalence (37.8%) on NY-2. All PCR positive specimens that amplified the ITS region as well as a portion of the flanking large and small subunit ribosomal RNA genes were sequenced to determine the genotype(s) of the E. bieneusi present and six genotypes were identified. Most were identified as cattle-specific genotypes, previously reported from cattle as BEB1, BEB2, BEB3, and BEB4. Two isolates were genetically identical or similar to E. bienesusi reported as the human pathogens Peru 6 and Peru 9 (or D) genotypes. Although our data demonstrate the presence of zoonotic genotypes in cattle, most genotypes found in cattle were host specific.

Salmonella is one of the most serious foodborne pathogenic bacteria in the United States, causing an estimated 1.3 million human illnesses each year. Dairy cows can be reservoirs of foodborne pathogenic bacteria, including Salmonella spp.; it is estimated that from 27 to 31% of dairy herds across the United States are colonized by Salmonella. The present study was designed to examine the occurrence of Salmonella spp. on dairies and to examine the serotypic diversity of Salmonella isolates on sampled dairies from across the United States. Fecal samples (n = 60 per dairy) were collected from 4 dairies in each of 4 states for a total of 960 fecal samples representing a total population of 13,200 dairy cattle. In the present study, 93 of 960 samples (9.96%) collected were culture-positive for Salmonella enterica. At least one Salmonella fecal-shedding cow was found in 9 of the 16 herds (56%) and the within-herd prevalence varied in our study from 0% in 7 herds to a maximum of 37% in 2 herds, with a mean prevalence among Salmonella-positive herds of 17%. Seventeen different serotypes were isolated, representing 7 different Salmonella serogroups. There were 2 or more different serogroups and serotypes present on 7 of the 9 Salmonella-positive farms. Serotypes Montevideo and Muenster were the most frequent and widespread. From our data, it appears that subclinical colonization with Salmonella enterica is relatively common on dairy farms and is represented by diverse serotypes on US dairy farms.

Background: A fundamental question that arises during epidemiological investigations of bacterial disease outbreaks is whether the outbreak strain is genetically related to a proposed index strain. Highly discriminating genetic markers for characterizing bacterial strains can help in clarifying the genetic relationships among strains. Under the auspices of the European Society of Clinical Microbiology and Infectious Diseases, the European Study Group for Epidemiological Markers (ESGEM) established guidelines for evaluating the performance of typing systems based of a number of criteria. Recently, HOOF-Print genotype analysis, a new method for typing Brucella abortus strains based on hypervariability at eight tandem repeat loci, was described. This paper evaluates the HOOF-Print assay by four of the criteria set out by the ESGEM: typeability, reproducibility, power of discrimination, and concordance with other typing methods., Results: The HOOF-Print Assay was evaluated with a test population composed of 97 unrelated field isolates and 6 common laboratory strains of B. abortus. Both typeability and reproducibility of the assay were excellent. Allele diversity and frequency varied widely among the eight loci, ranging from 1 to 13 alleles. The power of discrimination, measured by the Hunter-Gaston discrimination index (HGDI), varied by locus ranging from 0 to 0.89, where a maximal value of 1.0 indicates discrimination of all strains. The HGDI values calculated for subgroups sorted by biovar were similar to the values determined for the whole population. None of the individual loci achieved the recommended HGDI threshold of 0.95, but the HGDI of the composite profiles was 0.99 (93 unique genotypes from 97 field strains evaluated), well above the recommended threshold. By comparison, the HGDI value for biovar typing was 0.61 in a test population biased with disproportionate numbers of the less common biovars. Cluster analysis based on HOOF-Print genotypes assembled the strains into hierarchical groups with no apparent association with the time or location of strain isolation. Likewise, these hierarchical groups were not homogeneous with regard to biotype. In one extreme case, two field isolates with identical fingerprints were identified as different biovars by conventional methods., Conclusion: The main purpose of this study was to assess the ability of HOOF-Print genotyping to discriminate unrelated field strains of B. abortus, and whether the assay met established requirements for bacterial strain typing methods. The discriminatory power of the assay was remarkable, considering the genetic homogeneity found among species within the genus. The assay met or exceeded all of the recommended levels for the performance criteria of typeability, reproducibility, and power of discrimination, however some inconsistencies with conventional biovar typing were observed. Nevertheless, the results indicate that with cautious interpretation, multilocus genotyping of polymorphic tandem repeats by HOOF-Print analysis could be a valuable complement to routine epidemiological investigations into localized B. abortus outbreaks.

For two large beef processing plants, one located in the southern United States (plant A) and one located in the northern United States (plant B), prevalence of Escherichia coli O157:H7, Listeria spp., Listeria monocytogenes, and Salmonella was determined for hide, carcass, and facility environmental samples over the course of 5 months. The prevalence of E. coli O157:H7 (68.1 versus 55.9%) and Salmonella (91.8 versus 50.3%) was higher (P < 0.05), and the prevalence of Listeria spp. (37.7 versus 75.5%) and L. monocytogenes (0.8 versus 18.7%) was lower (P < 0.05) for the hides of cattle slaughtered at plant A versus plant B. Similarly, the prevalence of Salmonella (52.0 versus 25.3%) was higher (P < 0.05) and the prevalence of Listeria spp. (12.0 versus 40.0%) and L. monocytogenes (1.3 versus 14.7%) was lower (P < 0.05) for the fence panels of the holding pens of plant A versus plant B. The prevalence of E. coli O157:H7 (3.1 versus 10.9%), Listeria spp. (4.5 versus 14.6%), and L. monocytogenes (0.0 versus 1.1%) was lower (P < 0.05) for preevisceration carcasses sampled at plant A versus plant B. Salmonella (both plants), Listeria spp. (plant B), and L. monocytogenes (plant B) were detected on fabrication floor conveyor belts (product contact surfaces) late during the production day. For plant B, 21 of 148 (14.2%) late-operational fabrication floor conveyor belt samples were L. monocytogenes positive. For plant B, E. coli O157:H7 and L. monocytogenes were detected in preoperational fabrication floor conveyor belt samples. Overall results suggest that there are regional differences in the prevalence of pathogens on the hides of cattle presented for harvest at commercial beef processing plants. While hide data may reflect the regional prevalence, the carcass data is indicative of differences in harvest practices and procedures in these plants.

The majority of interactions between microorganisms and animals are based on convenient relations for both of them. Symbiotic microorganisms, like intestinal microbiota, produce important vitamins for animals and protects them from putative pathogens. In general, for monogastric animals, the main contribution of intestinal microorganisms is to supply with growth factors the animal diet, and in some cases they are responsible for providing essential vitamins (e.g. vitamin K). Some particular and relatively few microbes like viruses, bacteria, fungi, protozoa and algae are responsible for animal illness. Because microorganisms are easily dispersed, display physiological diversity, and tolerate extreme conditions, they are ubiquitous and may contaminate and grow in many products, including food and raw materials. Foodborne diseases are caused by consumption of contaminated food or beverages. Many different disease-causing pathogens can contaminate food, so there are many different foodborne infections. In addition, poisonous chemicals and biological toxins can cause disease if they are present in food. To know how a particular disease is spreading is an important matter to take appropriate steps to stop it. For example Escherichia coli O157:H7 infections can spread through contaminated food (meat, vegetables, cheese, etc.), contaminated drinking water or juices, contaminated swimming water and from person to person. Among foodborne pathogens, the most frequently detected are bacteria, but also parasitic protozoa and worms, viruses, natural toxins and other pathogenic agents like prions are important agents for foodborne diseases. Particular pathogenic types of E. coli, classified by their specific pathogenic mechanisms (toxins, adhesins, invasiveness, etc.) are actually known as E. coli virotypes. Enterohaemorrhagic E. coli (EHEC), which constitute the main part of this review, were also named verotoxigenic E. coli (VTEC) or Shiga toxigenic E. coli (STEC). EHEC strains cause haemorrhagic colitis (HC), haemolytic uremic syndrome (HUS) and thrombotic thrombocytopaenic purpura (TP) in humans. They synthetize shigatoxins (verotoxins) which are potent cytotoxic substances, adherence factors and enterohaemolysin. EHEC are responsible for many outbreaks of bloody diarrhoea caused by contaminated foods: beef, milk, fruits, juice, water, etc. The most important serogroups among EHEC are O26, O111 and O157, being O157:H7 the most relevant serotype in foodborne outbreaks. The normal intestinal microflora of cattle was found to be the most relevant reservoir of EHEC strains.

Campylobacter jejuni, Campylobacter coli, and Arcobacter spp. were detected in feces of healthy dairy cows by highly specific multiplex-PCR assays. For C. jejuni, at this one-time sampling, cows from 80.6% of farm operations (n = 31) and 37.7% of individual dairy cattle fecal samples (n = 2,085) were positive. Farm management factors were correlated with prevalence in herds in which >25% of cows were positive for C. jejuni. Statistical significance was set at a P of 0.20. Using these criteria, application of manure with broadcast spreaders (P = 0.17), feeding of whole cottonseed or hulls (P = 0.17) or alfalfa (P = 0.15), and accessibility of feed to birds (P = 0.17) were identified as possible risk factors for C. jejuni infection. C. coli was detected in at least one animal in 19.4% of operations and 1.8% of individual cows (n = 2,085). At the herd level, use of broadcaster spreaders was not a risk factor for C. coli infection. For Arcobacter, cows from 71% of dairy operations (n = 31) and 14.3% of individual dairy cattle fecal samples (n = 1,682) were positive. At the herd level, for Arcobacter spp., feeding of alfalfa (P = 0.11) and use of individual waterers (P = 0.19) were protective. This is the first description of Arcobacter spp. in clinically healthy dairy cattle and the first attempt to correlate their presence with C. jejuni.

In the 1996 U.S. Meat and Poultry Inspection Regulations, Escherichia coli biotype I counts were included as "performance criteria" of the slaughtering process. The criteria were based on a three-class attributes sampling plan applied in a moving window. The values for m and M and c and n were set at 5 and 100 CFU/cm2, and 3 and 13 samples, respectively, for beef carcasses after overnight chilling following slaughter. In this study, beef carcasses were analyzed for counts of E. coli, and the results were expressed according to the above criteria. Furthermore, probabilities of passing E. coli performance criteria were determined. Carcasses were sampled in seven slaughtering plants (four steer and heifer; three cow and bull), during two seasons, and at three plant locations (pre-evisceration, after final carcass washing, and after 24 h of carcass chilling). Each entire carcass sample (100 cm2 from the brisket, flank, and rump) was analyzed individually for E. coli counts. Compared with the regulation, which set the value of m and the acceptable range based on the 80th percentile of E. coli contamination data from U. S. Food Safety and Inspection Service nationwide baseline studies, our results showed that, on the average and depending on plant and season, 84.2 to 100% of the chilled carcass samples were in the acceptable range. The average percentages of chilled samples in the unacceptable range, set at the 98th percentile, were 0 to 6.7%. Depending on plant and season, the overall probabilities of chilled carcasses passing the regulatory requirement were 0.597 to 1.0 (brisket), 0.471 to 1.0 (flank), and 0.485 to 1.0 (rump). The results indicated substantial variation among plants and between seasons in ability to meet the E. coli performance criteria.

Increased consumer awareness and concern about microbial foodborne diseases has resulted in intensified efforts to reduce contamination of raw meat, as evidenced by new meat and poultry inspection regulations being implemented in the United States. In addition to requiring operation of meat and poultry slaughtering and processing plants under the principles of the hazard analysis critical control point (HACCP) system, the new regulations have established microbiological testing criteria for Escherichia coli and Salmonella, as a means of evaluating plant performance. These developments have renewed and intensified interest in the development and commercial application of meat and poultry decontamination procedures. Technologies developed and evaluated for decontamination include live animal cleaning/washing, chemical dehairing, carcass knife-trimming to remove physical contaminants, steam/hot water-vacuuming for spot-cleaning/decontamination of carcasses, spray washing/rinsing of carcasses with water of low or high pressures and temperatures or chemical solutions, and exposure of carcass sides to pressurized steam. Under appropriate conditions, the technologies applied to carcasses may reduce mean microbiological counts by approximately one-three log colony forming units (cfu)/cm2, and some of them have been approved and are employed in commercial applications (i.e., steam-vacuuming; carcass spray-washing with water, chlorine, organic acid or trisodium phosphate solutions; hot water deluging/spraying/rinsing, and pressurized steam). The contribution of these decontamination technologies to the enhancement of food safety will be determined over the long term, as surveillance data on microbial foodborne illness are collected. This review examines carcass decontamination technologies, other than organic acids, with emphasis placed on recent advances and commercial applications.

The genomic fingerprints of 26 Escherichia coli O157:H7 isolates from calves on 20 farms in 16 states were determined by pulsed-field gel electrophoresis (PFGE). Digestion of genomic DNAs with the restriction enzymes SfiI and XbaI yielded 14 and 18 restriction endonuclease digestion profiles (REDP), respectively. Seventeen farms (85%) had E. coli O157:H7 with a unique REDP, and when more than one calf tested positive on a farm, the isolates displayed identical REDP. Isolates from different farms within the same state displayed distinct REDP, as did most isolates from farms in different states. The exceptions were three farms in New York, Ohio, and Washington that had calves harboring E. coli O157:H7 with the same REDP. In addition to REDP, the toxin profiles of all 26 isolates were determined using oligonucleotide probes to Shiga-like toxins (SLT) I and II. Nineteen (73%) of the E. coli O157:H7 isolates harbored the genes for both SLT and II, while the remaining seven isolates (27%) had the gene for SLT II only. Also, isolates with the same REDP had the same toxin profile. The genomic relatedness among the E. coli O157:H7 isolates was also determined by principal component analysis of Dice similarity indices of REDP. Three clusters were identified, but none of these were associated with a geographic region or toxin profile.

The prevalence of Escherichia coli O157:H7 in dairy herds is poorly understood, even though young dairy animals have been reported to be a host. From February to May 1993, 662 fecal samples from 50 control herds in 14 states, and from June to August 1993, 303 fecal samples from 14 case herds in 11 states were collected for isolation of E. coli O157:H7. Case herds were those in which E. coli O157:H7 was isolated from preweaned calves in a previous U.S. Department of Agriculture study, whereas control herds from which E. coli O157:H7 had not been isolated previously were randomly selected from the same states as case herds. Among the control herds, E. coli O157:H7 was isolated from 6 of 399 calves (1.5%) that were between 24 h old and the age of weaning and from 13 of 263 calves (4.9%) that were between the ages of weaning and 4 months. Eleven of 50 control herds (22%) were positive. Among the case herds, E. coli O157:H7 was isolated from 5 of 171 calves (2.9%) that were between 24 h old and the age of weaning and from 7 of 132 calves (5.3%) that were between the ages of weaning and 4 months. Seven of 14 case herds (50%) were positive. Sixteen of 31 isolates were obtained by direct plating, with populations ranging from 10(3) to 10(5) CFU/g. Fifteen of 31 isolates were isolated by enrichment only. Nineteen of the isolates produced both verocytotoxin 1 (VT-1) and VT-2, whereas 12 produced VT-2 only.

Transmissible mink encephalopathy (TME) is a rare foodborne disease of ranch-raised mink produced by an as yet unidentified contaminated feed ingredient. Because of the clinicopathologic similarities to scrapie and the indistinguishable physicochemical properties of their transmissible agents, it was initially assumed that TME was caused by feeding mink scrapie-infected sheep. However, subsequent studies testing the oral susceptibility of mink to scrapie were unsuccessful. Epidemiologic investigations of individual incidents of TME have not identified an association between the occurrence of disease and the feeding of any particular ingredient. However, there are two incidents in which the rancher was confident that sheep were not fed. The most recent of these was in Stetsonville, Wisconsin in 1985 where the meat portion of the diet was composed almost exclusively of downer dairy cows. To examine the possibility that cattle may have been the source of infection on the Stetsonville ranch, mink brain was experimentally inoculated intracerebrally into two Holstein steers. Both of these animals developed fatal spongiform encephalopathies 18 and 19 months after inoculation. These findings are compatible with the Stetsonville incident of TME being caused by feeding mink infected cattle tissue and they suggest the presence of an unrecognized BSE-like disease in the United States. Further experimental studies on the Stetsonville source of TME have identified two distinct strains of the transmissible agent in Syrian hamsters. These strains vary in length of incubation period, clinical signs, endstage brain infectivity titre, and pathogenicity for mink.(ABSTRACT TRUNCATED AT 250 WORDS)

Beginning in 1973, all available laboratory and field strains of bluetongue virus (BTV) from the United States were serotyped. Of the viral strains serotyped, 27 were collected from 1953 through 1972; 173 were collected from 1973 through 1977. Although 20 BTV serotypes have been found worldwide, only BTV serotypes 10, 11, 13, and 17 have been found in the United States. Since 1973, serotypes 11 and 17 have been the prevalent serotypes. Samples were collected over a 24-year period in the United States and represent a wide geographic area and diverse host sources (sheep, cattle, wild ruminants, and insect vectors). The collection was not a statistical sampling.

Newborn calves from dams free of staphyloccocal udder infection were assigned to treatment groups in two experiments. Following colostrum feeding for 2 days, a culture of Staphylococcus aureus was added to pasteurized milk fed to one group twice weekly for a total of nine feedings. A control group received only pasteurized milk. Bull calves were in a short experiment to determine whether the organism was established in body tissues, and a second experiment was to determine the effect on incidence of mastitis at calving. No staphylococcus aureus was isolated from any body tissue or surfaces of bull calves necropsied at about 7 wk of age. Moreover, there was no difference in incidence of staphylococcal udder infection at first calving between heifers exposed to the organism as calves and controls. There appears little reason for concern about detrimental effects of feeding mastitic milk to calves under conditions where they are maintained in individual pens.

Members of the genus Salmonella isolated from chickens, turkeys, cattle, and swine and submitted to the National Veterinary Services Laboratories for serotyping during October 1981 through September 1982 were tested for sensitivity to 12 antibacterial drugs. A total of 3,500 isolates was tested. A high rate of drug resistance was observed. Three cultures were resistant to each of the drugs and 30% were resistant to each drug except chloramphenicol, cephalothin, and gentamicin. Multiple resistance patterns were observed for 80% of the cultures with an even higher percentage in cultures from swine.

There are 5 known serotypes of bluetongue (BT) virus (BTV) in the US: 2, 10, 11, 13 and 17. The most recently discovered US serotype, BTV 2, was isolated from blood collected from cattle at Ona, Florida in 1982. Isolations were made from the September through November bleedings and from 1 pool of Culicoides insignis Lutz collected in October. Seventeen viral isolates were obtained by injecting the samples into embryonated chicken eggs (ECE) followed by serial passage onto BHK-21 cells. A single isolate was made from inoculation of pooled bovine blood into sheep. The isolates were shown to be Orbivirus-like particles by electron microscopy and were identified as BTV by indirect immunofluorescence tests. All the isolates were found to be serotype 2, however genome analysis using polyacrylamide gel electrophoresis (PAGE) identified 2 distinct electropherotypes. There were major differences between the 2 electropherotypes at least in genome segments 1, 5, 7, 8 and 9. The electropherotypes of viral isolates from bovine blood collected in September, 5 of those from the October bleedings, the insect isolate, and that from pooled blood were identical by PAGE and were designated Ona A. This electropherotype was also found to be indistinguishable from the prototype of the African serotype 2. The 2nd electropherotype, Ona B, included 3 of the viral isolates from the October bleedings and the single isolate from November. Ona A appeared to be closely related to the African prototype while Ona B may have resulted as a variant of Ona A after being subjected to a different environment. Both electropherotypes, however, appear to be stable forms of BTV serotype 2.