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For decades, Salmonella Typhimurium and Salmonella Enteritidis have prevailed in several countries as agents of salmonellosis outbreaks. In Brazil, the largest exporter of poultry meat, relatively little attention has been paid to infrequent serovars. Here, we report the emergence and characterization of rare serovars isolated from food and related sources collected between 2014 and 2016 in Brazil. Twenty-two Salmonella enterica isolates were analyzed through the use of whole-genome sequencing (WGS) and clustered regularly interspaced short palindromic repeats (CRISPR) genotyping. These isolates were classified into 10 infrequent serovars, including S. Abony, S. Isangi, S. Rochdale, S. Saphra, S. Orion, S. Ouakam, S. Grumpensis, S. Carrau, S. Abaetetuba, and S. Idikan. The presence of six antimicrobial resistance (AMR) genes, qnrB19, bla CMY-2 , tetA, aac(6')-Iaa, sul2 and fosA7, which encode resistance to quinolones, third-generation cephalosporin, tetracycline, aminoglycoside, sulfonamide and fosfomycin, respectively, were confirmed by WGS. All S. Isangi harbored qnrB19 with conserved genomic context across strains, while S. Abony harbored bla CMY-2 . Twelve (54.5%) strains displayed chromosomal mutations in parC (Thr57→Ser). Most serovars were classified as independent lineages, except S. Abony and S. Abaetetuba, which phylogenetically nested with Salmonella strains from different countries. CRISPR analysis revealed that the spacer content was strongly correlated with serovar and multi-locus sequence type for all strains, independently confirming the observed phylogenetic patterns, and highlighting the value of CRISPR-based genotyping for Salmonella. These findings add valuable information to the epidemiology of S. enterica in Brazil, where the emergency of antibiotic-resistant Salmonella continues to evolve., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The housing of laying hens is important for social, industrial, and regulatory aspects. Many studies have compared hen housing systems on the research farm, but few have fully examined commercial housing systems and management strategies. The current study compared hens housed in commercial cage-free aviary, conventional cage, and enriched colony cage systems. Environmental and eggshell pool samples were collected from selected cages/segments of the housing systems throughout the production cycle and monitored for Salmonella and Campylobacter prevalence. At 77 wk of age, 120 hens per housing system were examined for Salmonella and Campylobacter colonization in the: adrenal glands, spleen, ceca, follicles, and upper reproductive tract. All isolates detected from environmental swabs, eggshell pools, and tissues were identified for serotype. Two predominant Salmonella were detected in all samples:S.Braenderup andS.Kentucky.Campylobacter coli and C. jejuni were the only Campylobacter detected in the flocks. Across all housing systems, approximately 7% of hens were colonized with Salmonella, whereas >90% were colonized with Campylobacter Salmonella Braenderup was the isolate most frequently detected in environmental swabs (P<0.0001) and housing system impacted Salmonella spp. shedding (P<0.0001).Campylobacter jejuni was the isolate most frequently found in environmental swabs (P<0.01), while housing system impacted the prevalence of C. coli and jejuniin ceca (P<0.0001). The results of this study provide a greater understanding of the impact of hen housing systems on hen health and product safety. Additionally, producers and academia can utilize the findings to make informed decisions on hen housing and management strategies to enhance hen health and food safety., (Published by Oxford University Press on behalf of Poultry Science Association 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Hen housing for commercial egg production continues to be a societal and regulatory concern. Controlled studies have examined various aspects of egg safety, but a comprehensive assessment of commercial hen housing systems in the US has not been conducted. The current study is part of a holistic, multidisciplinary comparison of the diverse aspects of commercial conventional cage, enriched colony cage, and cage-free aviary housing systems and focuses on environmental and egg microbiology. Environmental swabs and eggshell pools were collected from all housing systems during 4 production periods. Total aerobes and coliforms were enumerated, and the prevalence of Salmonella and Campylobacter spp. was determined. Environmental aerobic and coliform counts were highest for aviary drag swabs (7.5 and 4.0 log cfu/mL, respectively) and enriched colony cage scratch pad swabs (6.8 and 3.8 log cfu/mL, respectively). Aviary floor and system wire shell pools had the greatest levels of aerobic contamination for all eggshell pools (4.9 and 4.1 log cfu/mL, respectively). Hens from all housing systems were shedding Salmonella spp. (89-100% of manure belt scraper blade swabs). The dry belt litter removal processes for all housing systems appear to affect Campylobacter spp. detection (0-41% of manure belt scraper blade swabs) considering detection of Campylobacter spp. was much higher for other environmental samples. Aviary forage area drag swabs were 100% contaminated with Campylobacter spp., whereas enriched colony cage scratch pads had a 93% positive rate. There were no differences in pathogen detection in the shell pools from the 3 housing systems. Results indicate egg safety is enhanced when hens in alternative housing systems use nest boxes. Additionally, current outcomes indicate the use of scratch pads in hen housing systems needs to be more thoroughly investigated for effects on hen health and egg safety., (© The Author 2015. Published by Oxford University Press on behalf of Poultry Science Association.)

Postchill neck skin maceration (NSM) and whole-carcass rinsing (WCR) are frequently used methods to detect salmonellae from processed broilers. These are practical, nondestructive methods, but they are insensitive and may result in false negatives (20 to 40%). Neck skin samples comprise only 4% of the skin from the broiler carcass by weight, while WCR will not detect firmly attached Salmonella organisms and only 7.5% of the rinsate is utilized. Whole-carcass enrichment (WCE) involves incubation of the whole carcass overnight in a preenrichment broth and can recover as few as 8 inoculated Salmonella cells per carcass. The objective of this study was to use NSM, WCR, and WCE sampling to detect naturally occurring Salmonella from the same commercially processed broiler either prechill or postchill. Ten carcasses were obtained prechill and another 10 postchill on each of two replicate days from each of two commercial processing plants. From each carcass, 8.3 g of neck skin was sampled, and then the carcass was rinsed with 400 ml of 1% buffered peptone water. Thirty milliliters was removed and incubated (WCR), and the remaining 370 ml of broth and the carcass were incubated at 37°C for 24 h (WCE). Overall, Salmonella organisms were detected on 21, 24, and 32 of 40 prechill carcasses by NSM, WCR, and WCE, respectively, while 2, 2, and 19 of 40 postchill carcasses were positive by the respective methods. Prechill carcasses were 64% (77 of 120) positive for Salmonella, while postchill carcasses were 19% (23 of 120) positive. Commercial processing reduced the positive-sample prevalence by 45%. Salmonella organisms were detected on 20% (24 of 120) of the samples from plant 1 and 63% (76 of 120) of the carcasses from plant 2. This study demonstrates significant differences in the results for Salmonella prevalence among sampling methods both before and after immersion chilling, as well as between processing plants on days that samples were taken.

Campylobacter is an important human pathogen, and consumption of undercooked poultry has been linked to significant human illnesses. To reduce human illness, intervention strategies targeting Campylobacter reduction in poultry are in development. For more than a decade, there has been an ongoing national and international controversy about whether Campylobacter can pass from one generation of poultry to the next via the fertile egg. We recognize that there are numerous sources of Campylobacter entry into flocks of commercial poultry (including egg transmission), yet the environment is often cited as the only source. There has been an abundance of published research globally that refutes this contention, and this article lists and discusses many of them, along with other studies that support environment as the sole or primary source. One must remember that egg passage can mean more than vertical, transovarian transmission. Fecal bacteria, including Campylobacter, can contaminate the shell, shell membranes, and albumen of freshly laid fertile eggs. This contamination is drawn through the shell by temperature differential, aided by the presence of moisture (the "sweating" of the egg); then, when the chick emerges from the egg, it can ingest bacteria such as Campylobacter, become colonized, and spread this contamination to flock mates in the grow house. Improvements in cultural laboratory methods continue to advance our knowledge of the ecology of Campylobacter, and in the not-so-distant future, egg passage will not be a subject continuously debated but will be embraced, thus allowing the development and implementation of more effective intervention strategies.

The objectives of this study were to estimate the prevalence of Salmonella for individual, pooled, and composite fecal samples and to compare culture results from each sample type for determining herd Salmonella infection status and identifying Salmonella serovar(s). During the U.S. Department of Agriculture National Animal Health Monitoring System Dairy 2007 study, data and samples were collected from dairy operations in 17 major dairy states. As part of the study, composite fecal samples (six per operation) were collected from cow areas, such as holding pens, alleyways, and lagoons, where manure accumulates. Fecal samples also were collected from individual cows (35 per operation), and fecal sample pools were created by combining samples from 5 cows (7 per operation). A total of 1,541 composite fecal samples were collected from 260 operations in 17 states, and 406 (26.3%) of these samples were culture positive for Salmonella. Among the 116 operations for which all three sample types were obtained, 41.4% (48 operations) were Salmonella culture positive based on individual samples, 39.7% (46 operations) were positive based on pooled samples, and 49.1% (57 operations) were positive based on composite fecal samples. Relative to individual samples, the sensitivity of composite fecal samples for determining herd infection status was 85.4% and the sensitivity of pooled fecal samples was 91.7%. On 33.6% of operations (39 of 116), Salmonella was cultured from all three fecal sample types (individual, pooled, and composite), and 20 (51.3%) of these operations had exactly the same serovar in all three sample types. Use of composite fecal samples is less costly and time-consuming than use of individual or pooled samples and provides similar results for detecting the presence and identifying serovars of Salmonella in dairy herds. Therefore, composite sampling may be an appropriate alternative to culture of individual samples when assessing Salmonella status in dairy herds.

Concern about Salmonella contamination of food is compounded by fear that antimicrobials traditionally used to combat the infection will become useless due to rising antibiotic resistance. Livestock, in particular swine, often are blamed for illnesses caused by Salmonella and for increasing antibiotic resistance due to use of antibiotics in pigs. As part of the National Animal Health Monitoring System Swine 2000 and 2006 studies, swine fecal samples were cultured for Salmonella. These samples were collected from 123 operations in 17 states in 2000 and from 135 operations in 17 states in 2006. At each operation, 50 and 60 fecal samples were collected from late finisher pig pens in 2000 and 2006, respectively. Salmonella isolates were characterized to determine serogroup and serotype and were tested for susceptibility to a panel of 17 and 15 antimicrobial drugs in 2000 and 2006, respectively. A total of 5,470 and 7,788 samples were cultured for Salmonella in 2000 and 2006, respectively. Overall, 6.2% of the samples and 34.2% of the farms were positive for Salmonella in 2000. In 2006, 7.2% of the samples and 52.6% of the farms were positive. Salmonella Derby, Salmonella Typhimurium var. 5- (formerly Salmonella Typhimurium var. Copenhagen), and Salmonella Agona were the three serotypes most often recovered in both study years. The most common antimicrobial resistance pattern for Salmonella Derby in the two study years was resistance to streptomycin, sulfisoxazole, and tetracycline. Most isolates were resistant to tetracycline, sulfisoxazole, and streptomycin in both study years. The proportion of Salmonella isolates that were susceptible to all antimicrobials (pansusceptible) was 38.1% in 2000 and 20.4% in 2006. The proportion of Salmonella isolates that were resistant to three or more antimicrobials (multidrug resistant) was similar in 2000 and in 2006 (52.8 and 57.7%, respectively).

Two isolation methods were compared for isolation of Clostridium difficile from food animal feces. The single alcohol shock method (SS) used selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by alcohol shock and isolation on tryptic soy agar supplemented with 5% sheep blood, and cycloserine-cefoxitin fructose agar. The double alcohol shock method (DS) used alcohol shock prior to and after selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate, followed by isolation on tryptic soy agar supplemented with 5% sheep blood and cycloserine-cefoxitin fructose agar. A total of 55 (15.9%, n = 345) swine fecal samples, 32 (2.4%, n = 1,325) dairy cattle fecal samples, and 188 (6.3%, n = 2,965) beef cattle fecal samples were positive for C. difficile by either method. However, the DS was significantly better than the SS for the recovery of C. difficile from swine feces, while the SS was significantly better than the DS for the recovery of C. difficile from beef cattle feces. There was no significant difference between methods for the recovery of C. difficile from dairy cattle feces. This study suggests that food animals might harbor C. difficile and it provides critical information that isolation methods might not have universal application across animal species.

Sampling protocols for detecting Salmonella on poultry differ among various countries. In the United States, the U.S. Department of Agriculture Food Safety and Inspection Service dictates that whole broiler carcasses should be rinsed with 400 ml of 1% buffered peptone water, whereas in the European Union 25-g samples composed of neck skin from three carcasses are evaluated. The purpose of this study was to evaluate a whole carcass rinse (WCR) and a neck skin excision (NS) procedure for Salmonella and Escherichia coli isolation from the same broiler carcass. Carcasses were obtained from three broiler processing plants. The skin around the neck area was aseptically removed and bagged separately from the carcass, and microbiological analysis was performed. The corresponding carcass was bagged and a WCR sample was evaluated. No significant difference (alpha

Campylobacter spp. are frequently present in the intestinal tract and internal tissues of broiler breeder and broiler chickens. Campylobacter spp. ecology in commercial Leghorn laying hens has not been extensively studied. The objectives of the current study were to determine 1) Campylobacter spp. presence in the reproductive tract, lymphoid organs, liver-gallbladder, and ceca of commercial Leghorn laying hens; 2) species of Campylobacter present; and 3) antimicrobial resistance pattern of Campylobacter isolates. In study 1, three flocks ranging from 94 to 105 wk of age were sampled from a commercial laying complex. In study 2, two flocks, 82 and 84 wk of age, were sampled from a separate complex. Hens were killed, defeathered, aseptically necropsied, and the spleen, liver-gallbladder, ovarian follicles, and upper (infundibulum, magnum, and isthmus) and lower (shell gland and vagina) reproductive tracts were aseptically removed before the ceca. Samples were packed on ice and transported to the laboratory for evaluation. For speciation, a standard BAX real-time PCR method was used while susceptibility testing was performed using US National Antimicrobial Resistance Monitoring System (NARMS) standards and recommended quality control organisms. Isolates were examined for susceptibility using a semi-automated testing system (Sensititer) to the following 9 antimicrobials: azithromycin, clindamycin, ciprofloxacin, erythromycin, florfenicol, gentamicin, nalidixic acid, telithromycin, and tetracycline. In study 1, the isolation rate was 13, 67, 53, 3, 13, and 57% from the ovarian follicles, lower reproductive tract, upper reproductive tract, spleen, liver-gallbladder, and ceca, respectively. In study 2, the isolation rate was 17, 43, 33, 20, 17, and 73% from the ovarian follicles, lower reproductive tract, upper reproductive tract, spleen, liver-gallbladder, and ceca, respectively. Overall, 50% of isolates were Campylobacter jejuni, 49% Campylobacter coli, and 1% Campylobacter lari. In study 1, all of the isolates were pan-susceptible. In study 2, thirty-seven percent of the isolates were resistant to tetracycline. Commercial table egg laying hens housed in colony cages on wire floors had diverse Campylobacter spp. recovered from different tissues and these isolates were not resistant to a broad range of antimicrobials.

The objective of this study was to measure the effect of broiler processing on the prevalence, serotype, and antimicrobial resistance profiles of salmonellae. Twenty U.S. commercial processing plants representing eight integrators in 13 states were included in the survey. In each of four replications, 10 carcasses from one flock were collected at rehang and 10 more carcasses were collected at postchill; each carcass was sampled by whole-carcass rinse. Salmonella organisms were isolated from carcass rinses by standard cultural techniques, serotypes were determined, and the resistance to 15 antimicrobials was measured. Overall, Salmonella was detected on 72% of carcasses at rehang (ranging from 35 to 97%) and on 20% of carcasses postchill (ranging from 2.5 to 60%). In every instance, a significant (P < 0.05) decrease in Salmonella prevalence was noted between rehang and postchill. The four most common serotypes, accounting for 64% of all Salmonella isolates, were Kentucky, Heidelberg, Typhimurium, and Typhimurium var. 5-; most isolates of Kentucky (52%), Heidelberg (79%), and Typhimurium (54%) serotypes were susceptible to all antimicrobial drugs tested. However, only 15% of the Typhimurium var. 5- isolates were pansusceptible; more than one-half of the isolates of this serotype were resistant to three or more drugs. No isolate of any serotype exhibited resistance to amikacin, ceftriaxone, ciprofloxacin, or trimethoprim-sulfamethoxazole. These data demonstrate that although processing lessens carcass contamination with Salmonella, antimicrobial-resistant isolates may still be present.

The purpose of this study was to compare a conventional culture broth method (Bolton enrichment), a newly developed proprietary broth method (TECRA Campylobacter enrichment), and direct plating for recovery of Campylobacter spp. from chicken carcass rinsates. Whole carcass rinses were taken from 140 carcasses at rehang (immediately after defeathering but before evisceration) and from 140 carcasses at postchill from eight different processing plants in the United States. The rinsate samples were packed in ice and shipped overnight to the laboratory. Aliquots of the rinsate were transferred into Bolton and TECRA enrichment broths and were direct plated. Standard laboratory procedures with Campy-cefex plates were followed for recovery of Campylobacter spp. For rehang carcasses, 94% were positive for Campylobacter spp. with the TECRA enrichment broth and 74% were positive with the Bolton enrichment broth. For postchill carcasses, 74% were positive for Campylobacter spp. with the TECRA enrichment broth and 71% were positive with the Bolton enrichment broth. Compared with the Bolton enrichment broth, TECRA enrichment broth significantly suppressed non-Campylobacter microflora (P < 0.05). Overall, TECRA enrichment broth yielded an 11% higher total number of Campylobacter-positive samples compared with the Bolton enrichment broth. Campylobacter spp. detection in postchill samples was significantly greater (P < 0.05) by enrichment (84%) than by direct plating (19%). The high number of Campylobacter-positive samples obtained with all procedures indicated that 99% of the carcass rinsates obtained at rehang and 84% obtained at postchill contained Campylobacter spp.

The objective in this study was to assess breed effects in fecal prevalence of Escherichia coli O157:H7 in heifers on a development program in Florida and in their steer half siblings in stocker and feedlot phases in Oklahoma. A secondary objective was to characterize fecal shedding of Campylobacter and Salmonella in subsets of the same samples. After weaning, heifers (n = 501; purebreds and F1 crosses of Angus, Brahman, and Romosinuano) were preconditioned and placed in a local development program. Steers (n = 481) were transported to Oklahoma, where they grazed wheat for 6 months and then were placed in feedlot pens. Fecal samples were obtained at least every 28 days for 12 months on most animals. None of the 10,982 samples tested positive for E. coli O157:H7. Overall fecal prevalences of Campylobacter and Salmonella in heifers were 1.7 and 0.04%, respectively. Corresponding overall prevalences in steer samples were 27.2 and 0.6%. Campylobacter isolates were mostly C. jejuni and were tetracycline resistant. Eight Salmonella isolates were Salmonella Typhimurium that were either quad or penta resistant, most often to ampicillin, chloramphenicol, sulfamexathole, and tetracycline. Feedlot steers had greater odds of positive detection of Campylobacter (odds ratio, 8.5; confidence interval, 3.7, 19.5) than when grazing winter wheat. No breed effect was detected for fecal prevalence of these pathogens.

Although enterococci are considered opportunistic nosocomial pathogens, their contribution to foodborne illnesses via dissemination through retail food remains undefined. In this study, prevalence and association of antimicrobial resistance and virulence factors of 80 Enterococcus faecalis isolates from retail food items were investigated. The highest rates of resistance were observed for lincomycin (73 of 80 isolates, 91%) and bacitracin (57 of 80 isolates, 71%), and lower rates of resistance (< or =40%) were found for chloramphenicol, ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, nitrofurantoin, penicillin, and tylosin. Overall resistance to antimicrobials was low for most isolates tested. Of the virulence factors tested, the majority of isolates were positive for ccf (78 of 80 isolates, 98%), efaAfs (77 of 80, 96%), and cpd (74 of 80, 93%). Isolates also commonly contained cob (72 of 80 isolates, 90%) and gelE (68 of 80, 85%). Very few isolates contained cylMBA (12 of 80 isolates [15%] for cylM and 9 of 80 isolates [11%] for both cylB and cylA) and efaAfm (2 of 80 isolates, 3%). Positive statistical associations (significance level of 0.05) were found between agg and tetracycline resistance, cylM and erythromycin resistance, and gelE and efaAfs and lincomycin resistance. The presence of the cylB and cylA alleles also was positively correlated with bacitracin and tetracycline resistance. Negative correlations were observed between many of the virulence attributes and resistance to ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, and tylosin. These data suggest that both positive and negative associations exist between antimicrobial resistance genes and virulence factors in E. faecalis isolates from foods commonly purchased from grocery stores.

The use of antimicrobials in food animal production, particularly those commonly used to treat infections in humans, has become a source of debate in recent years. However, limited data are available regarding the development of resistance following the subtherapeutic or therapeutic administration of antimicrobials in animal production. The objective of this study was to evaluate the effect of the administration of therapeutic and subtherapeutic concentrations of tylosin on the erythromycin susceptibility of Campylobacter jejuni and Campylobacter coli isolated from the ceca of treated broilers. In three replicated studies, day-of-hatch chicks were exposed to macrolide-susceptible C. jejuni or C. coli. At 2 weeks of age, tylosin was administered at subtherapeutic (22 ppm, continuously in the diet) or therapeutic concentrations (529 ppm, in the drinking water for 5 days). Broilers were sacrificed weekly. Total and erythromycin-resistant Campylobacter spp. were enumerated from individual ceca plus cecal contents. Overall erythromycin resistance was observed at a higher frequency (P < 0.01) among C. coli isolates (70.8%) than among C. jejuni isolates (36.8%) following tylosin administration. Across Campylobacter species, erythromycin resistance was observed at a higher frequency (P < 0.001) when tylosin was administered at subtherapeutic (62.7%) than at therapeutic (11.4%) concentrations. Subtherapeutic administration resulted in the recovery of 83.3 and 56.1% erythromycin-resistant isolates compared with only 33.3 and 7.9% of the isolates expressing erythromycin resistance following the administration of therapeutic concentrations for C. coli and C. jejuni, respectively. Further studies are needed to determine the factors involved in the apparent difference in the acquisition of macrolide resistance in C. coli compared with C. jejuni.

Campylobacter is a human pathogen associated with chicken and chicken meat products. This study was designed to examine the prevalence and number of Campylobacter on broiler chicken carcasses in commercial processing plants in the United States. Carcass samples were collected from each of 20 U.S. plants four times, roughly approximating the four seasons of 2005. At each plant on each sample day, 10 carcasses were collected at rehang (prior to evisceration), and 10 carcasses from the same flock were collected postchill. A total of 800 carcasses were collected at rehang and another 800 were collected postchill. All carcasses were subjected to a whole-carcass rinse, and the rinse diluent was cultured for Campylobacter. The overall mean number of Campylobacter detected on carcasses at rehang was 2.66 log CFU per ml of carcass rinse. In each plant, the Campylobacter numbers were significantly reduced by broiler processing; the mean concentration after chill was 0.43 log CFU/ml. Overall prevalence was also reduced by processing from a mean of > or =30 of 40 carcasses at rehang to > or =14 of 40 carcasses at postchill. Seven different on-line reprocessing techniques were applied in the test plants, and all techniques resulted in <1 log CFU/ml after chilling. Use of a chlorinated carcass wash before evisceration did not affect the postchill Campylobacter numbers. However, use of chlorine in the chill tank was related to lower numbers on postchill carcasses. Overall, U.S. commercial poultry slaughter operations are successful in significantly lowering the prevalence and number of Campylobacter on broiler carcasses during processing.

Tylosin phosphate is an antimicrobial drug approved for use in broiler feed at subtherapeutic levels for growth promotion. Erythromycin is often the drug of choice for treating humans with campylobacteriosis. Both tylosin and erythromycin are classified as macrolide drugs and cross-resistance between these antimicrobials occurs. Commercial broiler chicks were placed in isolation grow-out chambers and colonized with Campylobacter jejuni. From 14 d of age through grow-out, broilers were fed ad libitim a diet that included 22 ppm of tylosin phosphate (20 g/ton). Control broilers received the same diet without tylosin phosphate. At 42 d of age, broilers were processed in a pilot plant with equipment that closely modeled commercial conditions. Carcass rinses were collected after feather removal, after inside and outside washing, and after immersion chilling. Campylobacter numbers recovered from carcasses after feather removal did not differ according to feed type (3.53 log cfu/mL of rinse for control carcasses, and 3.60 log cfu/mL of rinse for those fed medicated feed). Likewise, medicated feed did not affect Campylobacter numbers on carcasses after inside-outside washing (3.11 and 3.07 log cfu/mL of rinse). However, carcasses of broilers fed tylosin phosphate had lower numbers of Campylobacter after chilling (1.45 log cfu/mL of rinse) than control carcasses (2.31 log cfu/mL of rinse). No Campylobacter isolated from control carcasses were resistant to erythromycin; all Campylobacter recovered from carcasses fed tylosin phosphate were resistant to erythromycin. Application of tylosin phosphate in feed results in lower numbers of Campylobacter on chilled carcasses; however, the Campylobacter that do remain are resistant to erythromycin.

Fecal samples collected from healthy pigs from 13 of the top 17 swine-producing states were tested for Escherichia coli O157:H7 as part of the National Animal Health Monitoring System Swine 2000 study. Serogroup O157 strains were isolated from 106 of 2,526 fecal samples. None of the isolates were positive by PCR for the fliCh7 (H7 flagellin) gene or for the hly933 (hemolysin) gene; however, one isolate was positive for the stxl gene (Shiga toxin 1), an additional four isolates were positive for the stx2 gene (Shiga toxin 2), and three isolates possessed the eae gene (intimin).

The U.S. Food Safety and Inspection Service (FSIS) tests for Salmonella in meat, poultry, and egg products through three regulatory testing programs: the Pathogen Reduction-Hazard Analysis and Critical Control Point (PR-HACCP) program, the ready-to-eat program for meat and poultry products, and the pasteurized egg products program. From 1998 through 2003, 293,938 samples collected for these testing programs were analyzed for the presence of Salmonella enterica serotypes. Of these samples, 12,699 (4.3%) were positive for Salmonella, and 167 (1.3%) of the positive samples (0.06% of all samples) contained Salmonella Enteritidis. The highest incidence of Salmonella Enteritidis was observed in ground chicken PR-HACCP samples (8 of 1,722 samples, 0.46%), and the lowest was found in steer-heifer PR-HACCP samples (0 of 12,835 samples). Salmonella Enteritidis isolates were characterized by phage type, pulsed-field gel electrophoretic pattern, and antimicrobial susceptibility. Phage typing of 94 Salmonella Enteritidis isolates identified PT13 (39 isolates) and PT8 (36 isolates) as the most common types. One isolate from a ready-to-eat ham product was characterized as PT4. Electrophoretic analysis of 148 Salmonella Enteritidis isolates indicated genetic diversity among the isolates, with 28 unique XbaI electrophoretic patterns identified. Of these 148 isolates, 136 (92%) were susceptible to each of 16 antimicrobials tested. Two isolates were resistant to ampicillin alone, and 10 isolates were resistant to two or more antimicrobials. Isolation of Salmonella Enteritidis from FSIS-regulated products emphasizes the need for continued consumer education on proper food handling and cooking practices and continued work to decrease the prevalence of Salmonella in meat, poultry, and pasteurized egg products.

Although enterococci are considered opportunistic pathogens, they can be reservoirs of antimicrobial resistance. Antimicrobial resistance is increasingly important because of foodborne illnesses from meat and infections from produce. From 2000 through 2001, food items (vegetables, fruits, and meats) were obtained from grocery store chains in northern Georgia and cultured for the presence of enterococci; 47.7% (189 of 396) of these samples were positive for enterococci. For the fruits and vegetables, enterococci were cultured most often from tomatoes (9 of 27 samples, 33%) and radishes (10 of 11 samples, 91%), respectively. Among the meat items tested, enterococci were isolated from 95% (21 of 22) of the chicken samples, 73% (16 of 22) of the beef samples, 95% (20 of 21) of the turkey samples, and 68% (15 of 22) of the pork samples. The predominant species identified was Enterococcus faecalis (n = 80) from meat and Enterococcus casseliflavus (n = 66) from fruits and vegetables. Although high numbers of isolates were resistant to lincomycin (176 of 185 isolates, 95.1%) and bacitracin (150 of 185 isolates, 81.1%), very few isolates were resistant to salinomycin (2 isolates, 1.1%), penicillin (3 isolates, 1.6%), or nitrofurantoin (9 isolates, 4.9%). None of the isolates were resistant to linezolid or vancomycin. These data suggest that foods commonly purchased from grocery stores are a source of enterococci; however, overall resistance to antimicrobials is relatively low.

The development of antimicrobial resistance in bacteria has become a global problem. Isolates of Salmonella and Escherichia coli recovered from shell egg samples, collected at 3 commercial plants, were analyzed for resistance to 16 antimicrobial agents (n=990). Eggs were sampled by rinsing in a saline solution. Pooled samples were preenriched in buffered peptone water and then selectively isolated using standard broths and agars. Salmonella-positive isolates were serogrouped immunologically before being serotyped. Enterobacteriaceae were enumerated from individual samples using violet red bile glucose agar plates. Escherichia coli were identified biochemically from presumptive Enterobacteriaceae isolates. Salmonella and generic E. coli antimicrobial-susceptibility testing was conducted using a semiautomated broth microdilution system. More resistance was observed in the Salmonella isolates (n=41) than in the E. coli isolates (n=194). Salmonella Typhimurium was the most prevalent (69.0%) serotype and demonstrated the greatest multiple resistance. Salmonella Kentucky, the least prevalent (5.0%) serotype recovered, was the most susceptible. Although 34.1% of the Salmonella serotypes were susceptible to all antimicrobial agents, 60.1% were resistant to 11 or more compounds. Many Salmonella isolates exhibited resistance to tetracycline (63.4%), nalidixic acid (63.4%), and streptomycin (61.0%). Most E. coli isolates (73.2%) were susceptible to all antimicrobial drugs. Many E. coli isolates exhibited resistance to tetracycline (29.9%), streptomycin (6.2%), and gentamicin (3.1%). Only 1% of the E. coli isolates were resistant to 4 antimicrobial agents. These data indicate that shell eggs can harbor resistant foodborne and commensal bacteria; among Salmonella isolates, resistance was serotype-dependent.

Broiler carcasses (n=325) were sampled at three sites along the processing line (prescalding, prechilling, and postchilling) in a commercial poultry processing plant during five plant visits from August to October 2004. Pulsed-field gel electrophoresis (PFGE) was used to determine the genomic fingerprints of Camospylobacter coli (n=27), Campylobacter jejuni (n=188), Arcobacter butzleri (n=138), Arcobacter cryaerophilus 1A (n=4), and A. cryaerophilus 1B (n=31) with the restriction enzymes SmaI and KpnI for Campylobacter and Arcobacter, respectively. Campylobacter species were subtyped by the Centers for Disease Control and Prevention PulseNet 24-h standardized protocol for C. jejuni. A modification of this protocol with a different restriction endonuclease (KpnI) and different electrophoresis running conditions produced the best separation of restriction fragment patterns for Arcobacter species. Both unique and common PFGE types of Arcobacter and Campylobacter strains were identified. A total of 32.8% (57 of 174) of the Arcobacter isolates had unique PFGE profiles, whereas only 2.3% (5 of 215) of the Campylobacter isolates belonged to this category. The remaining Arcobacter strains were distributed among 25 common PFGE types; only eight common Campylobacter PFGE types were observed. Cluster analysis showed no associations among the common PFGE types for either genus. Each of the eight common Campylobacter types consisted entirely of isolates from one sampling day, whereas more than half of the common Arcobacter types contained isolates from different sampling days. Our results demonstrate far greater genetic diversity for Arcobacter than for Campylobacter and suggest that the Campylobacter types are specific to individual flocks of birds processed on each sampling day.

Salmonella serotypes are important foodborne pathogens of humans that can be acquired through consumption of contaminated meat and dairy products. Salmonella infection also can be a significant animal health issue. As part of a national study of U.S. dairy operations conducted between March and September 2002, fecal samples were collected from representative cows in 97 dairy herds in 21 states and were cultured to determine the prevalence of Salmonella shedding. Salmonella was recovered from the feces of at least one cow in 30.9% of the herds. Overall, 7.3% of fecal samples were culture positive for Salmonella. The three most frequently recovered serotypes were Salmonella Meleagridis (24.1%), Salmonella Montevideo (11.9%), and Salmonella Typhimurium (9.9%). The susceptibilities of Salmonella isolates recovered were determined using a panel of 16 antimicrobial drugs. Salmonella isolates recovered from dairy cows had relatively little resistance to these antimicrobial agents; 83.0% of the isolates were susceptible to all antimicrobials tested. This study provides updated information on the prevalence and susceptibility patterns of Salmonella in dairy herds and on cow and herd characteristics. These data contribute to our understanding of the ecology of Salmonella in the dairy farm environment.

Our laboratory has developed a bacterial competitive-exclusion (CE) culture against enteropathogens (which are considered human foodborne pathogens) for use in swine. In this article, we document the effects of this CE culture, PCF1, on cecal colonization by and fecal shedding of Salmonella Choleraesuis in neonatal and weaned pigs and its effects on the horizontal transmission of this pathogen between weaned penmates. Piglets treated with the PCF1 culture twice within their first day of life and challenged with Salmonella 48 h after birth shed Salmonella at a significantly (P < 0.05) lower rate than did control pigs in experiment 1. Significant reductions of the pathogen were also observed in the cecum, the cecal contents, the ileocolic junction, and the colon contents (P < 0.05). In experiment 2, culture of the cecal contents and lymph nodes revealed a significant reduction in Salmonella isolated from PCF1-treated pigs (P < 0.05). Pigs in experiment 3 were treated as pigs in experiments 1 and 2 were: however, they were followed through day 10 postweaning. Significant reductions in shedding were noted for treated groups both pre- and postweaning (P < 0.05). Experiments 4 and 5 assessed the effects of PCF1 treatment on the horizontal transmission of Salmonella between littermates that were followed through day 14 postweaning. In these experiments, litters were divided into untreated contacts (UC), untreated seeders (US), treated contacts (TC), and treated seeders (TS). Overall, TC in experiment 4 shed Salmonella at a significantly lower rate than UC and US did (P < 0.05). In experiment 5, the transmission of Salmonella was significantly reduced for litters in which TS or TC were present, as evidenced by reduced shedding of Salmonella by both treated and untreated animals within these litters (P < 0.05). TS shed less often than US did, resulting in reduced levels of Salmonella shedding by both treated and untreated contacts (P < 0.05). Litters containing both TC and UC or both TC and US also shed Salmonella at lower rates than did litters in which only UC and US were present (P < 0.05).

The subtyping and identification of bacterial pathogens throughout food processing and production chains is useful to the new hazard analysis critical control point-based food safety plans. Traditional manual serotyping remains the primary means of subtyping Salmonella isolates. Molecular biology techniques, however, offer the promise of more rapid and sensitive subtyping of Salmonella. This study evaluates the potential of restriction enzyme PvuII, followed by probing with the rRNA operon from Escherichia coli, to generate serotype-specific DNA fingerprints. A total of 32 identified serotypes were found with an overall agreement in 208 of the 259 (80%) isolates tested between U.S. Department of Agriculture serotype identification and riboprint serotype identification. Many of the isolates that did not correlate were serotype identified as Salmonella Montevideo, which indicates that for this serotype, there are multiple ribotypes. When Salmonella Montevideo isolates were not included, the ribotype identification agreed with serotyping in 207 of the 231 (90%) isolates. The primary outcome of any ribotyping procedure is to give distinct ribotype patterns. This extensive poultry epidemiological study demonstrates that, in addition to ribotype patterns, the identification of isolates to known serotypes provides the investigator with additional information that can be more useful than traditional epidemiology and isolate identification studies.

Surveillance data of cattle and human isolates of Salmonella enterica serovar Typhimurium DT104 indicate that this pathogen emerged worldwide in the 1980s, particularly in cattle. Studies were conducted to determine the prevalence of Salmonella Typhimurium DT104 in ground beef. Samples were also tested for the presence of generic Escherichia coli. A total of 404 fresh ground beef samples obtained at retail stores from New York, San Francisco, Philadelphia, Denver, Atlanta, Houston, and Chicago were shipped overnight to Georgia for processing. Salmonella spp. were isolated from 14 (3.5%) samples. Eight different serotypes were identified among the isolates, including Salmonella Typhimurium (5), Salmonella Lille (3), Salmonella Montevideo (1), Salmonella Hadar (1), Salmonella Meleagridis (1), Salmonella Cerro (1), Salmonella Kentucky (1), and Salmonella Muenster (1). Antibiotic resistance profiles indicated that all five Salmonella Typhimurium isolates were resistant to ampicillin, streptomycin, sulfamethoxazole, ticarcillin, and tetracycline but that they were sensitive to chloramphenicol. Phage typing revealed that all five Salmonella Typhimurium isolates were DT104A, a subtype of DT104. All five Salmonella Typhimurium DT104A isolates were obtained from ground beef sampled from retail outlets in San Francisco. Pulsed-field gel electrophoresis (PFGE) genomic DNA profiles of the five Salmonella Typhimurium DT104A isolates from ground beef were indistinguishable from those of four control Salmonella Typhimurium DT104 penta-resistant isolates from cattle that were used for comparison. A total of 102 generic E. coli isolates were obtained, only three of which were multiresistant to antibiotics. In addition, three E. coli isolates were recovered from samples that were positive for Salmonella Typhimurium DT104A. No correlation of antibiotic resistance profiles was observed between Salmonella Typhimurium DT104A and generic E. coli, as two of the three E. coli isolates were susceptible to all of the antibiotics tested, and the third isolate was resistant only to cephalothin. These data indicate that Salmonella Typhimurium DT104A can be isolated from retail ground beef, and because there was little overlap in antibiotic resistance patterns between Salmonella Typhimurium DT104A and E. coli isolates from the same ground beef samples, these limited data suggest that the transfer of antibiotic resistance genes among enteric bacteria in ground beef may not be common. This latter observation is further supported by the limited isolation of multiantibiotic-resistant E. coli from retail ground beef.

The prevalence of an antibiotic-resistant strain of Salmonella Typhimurium definitive phage type 104 (DT104) has increased dramatically in recent years resulting in increased morbidity and mortality in both animals and humans. Colonization and shedding of Salmonella Typhimurium DT104 was studied in broiler chickens in two trials. In trial 1, 180 day-of-hatch chicks (n = 60 per group, n = 30 per replicate) were challenged with 10(6) CFU DT104 (wild-type isolate from poultry) or were commingled with a seeder chick challenged with 10(6) CFU DT104. In trial 2, 360 day-of-hatch chicks (n = 120 per treatment, n = 30 per rep) were divided into three groups. Chicks in the susceptible group were commingled with two seeder chicks that were orally challenged with 10(7) CFU/bird of a pan-sensitive strain of Salmonella Typhimurium DT104. Chicks in the resistant group were commingled with two seeder chicks that were orally challenged with 10(7) CFU/bird DT104 used in trial 1. For both trials, a control group was not exposed to DT104, composite fecal samples were evaluated twice weekly for levels of Salmonella shedding and 20 chicks per group were necropsied weekly and their cecal contents were cultured. At hatch all groups were colonized with naturally occurring Salmonella Senftenberg and Salmonella Mbandaka (trial 1) or Salmonella Senftenberg and Salmonella Ohio (trial 2) prior to exposure to DT104. Throughout the study, the level of Salmonella spp. shedding in feces (trial 1 means 3.1, 2.9, and 3.0 log10 CFU per g feces for challenged, seeder, and control groups, respectively) or ceca (trial 2 means 2.9. 2.9. and 2.5 log10 CFU per g ceca for resistant, susceptible, and control groups, respectively) did not differ among groups. In trial 1, colonization of DT104 remained constant at higher levels in the challenged group (mean 87%, P < 0.01), increased over time in the seeder group (10 to 50%, P < 0.02) and was not recovered from the control chicks. Salmonella Mbandaka colonization remained steady within each group with challenge and seeder groups maintaining higher levels of colonization than the control group. Salmonella Senftenberg colonization levels tended to decline (P = .058) over time in the challenged group (20 to 0%) and significantly decreased (P < 0.01) over time for both the seeder (80 to 0%) and control chicks (85 to 10%). In trial 2, the percentage of chicks colonized with susceptible DT104 declined (r = 0.90, P < 0.05) over the course of the trial from 45 to 0%, while recovery of the resistant DT104 persisted at a mean percentage of 27%. DT104 was not recovered from the control chicks. Salmonella Ohio colonization levels tended to decline (r = 0.79, P > 0.05) over time in the control group (75 to 20%) and significantly decreased (P < 0.05) over time in both susceptible and resistant groups (40 to 10%, r = 0.82 and 55 to 5%, r = 0.85, respectively). Salmonella Senftenberg was recovered from the control group at low frequency throughout the trial and was not recovered from the other groups. For either trial, no apparent affect on morbidity or mortality was observed. Introduction of DT104 by commingling may induce colonization resulting in persistent high levels of shedding in flocks simultaneously with other Salmonella species.

Many serotypes of Salmonella survive well in the environment. Conversely, it is believed that Salmonella Choleraesuis, the host-adapted serotype of swine, does not survive well outside the host. We examined the survival capability of Salmonella Choleraesuis in swine feces. Six pigs were infected with Salmonella Choleraesuis and feces were collected and pooled on days 2, 4, 7, and 10 postinoculation (PI). Feces were stored in a wet and a dry form, and survival was measured over 13 months. Salmonella Choleraesuis was recovered from wet feces through 3 months of storage. In a desiccated (dry) form, Salmonella Choleraesuis was recovered from at least 13 months. Salmonella Choleraesuis shed from swine prior to 4 days PI did not survive as well as that shed 4 days PI or later. We also examined the infectivity of Salmonella Choleraesuis resident in dry feces. Six- or 13-week-old pigs were inoculated with dry feces that had been stored either 2 months or 4 months, respectively. Pigs were inoculated either intranasally or by mixing dry feces with the swine ration. Although clinical signs were mild, Salmonella Choleraesuis was widely disseminated among the tissues of all the pigs inoculated. This study demonstrates that Salmonella Choleraesuis remains viable and infective in the environment. Therefore, contaminated fecal matter can serve as a reservoir for Salmonella Choleraesuis as well as other Salmonella spp. Control measures must consider this environmental reservoir as a source of new infections.

As part of a national study of the U.S. dairy cow population, fecal samples were collected from representative cows on 91 dairies and 97 cull dairy cow markets in 19 states. Salmonella spp. were recovered from 5.4% of milk cows, 18.1% of milk cows expected to be culled within 7 days, and 14.9% of culled dairy cows at markets. On a premise basis, Salmonella shedding in milk cows was detected on 21.1% of dairies and 66% of cull dairy cow markets. The percentage of herds with at least one cow with detectable Salmonella fecal shedding was higher during the sampling period from May through July, in herds with at least 100 milk cows, and in herds in the South region. The most common Salmonella serogroups isolated were E (30.8% of isolates) and C1 (28.6%); the most common serotypes isolated were Salmonella Montevideo (21.5% of isolates), Salmonella Cerro (13.3%), and Salmonella Kentucky (8.5%). Fecal shedding of Salmonella Typhimurium or Salmonella Typhimurium var. copenhagen was infrequent (2.8% of isolates). Most isolates (88.9%) were susceptible to all 17 antimicrobials evaluated; multiple resistance was an infrequent occurrence. This study provides information describing the distribution of Salmonella fecal shedding from dairy cows on farm and at markets and will serve as a baseline for future studies.

Salmonella prevalence on cow-calf operations was studied as a part of a national study of health and management of the U.S. beef cow-calf industry and was conducted as part of the National Animal Health Monitoring System. Within this study, the prevalence of Salmonella spp. shed in feces was determined. A total of 5,049 fecal samples were collected from 187 beef cow-calf operations each visited on a single occasion. The number of fecal samples collected per operation was predetermined based on herd size. Salmonellae were recovered from 1 or more fecal samples collected on 11.2% (21 of 187) of the operations. Overall 78 salmonellae representing 22 serotypes were recovered from 1.4% (70 of 5,049) of samples. Multiple serotypes were recovered from eight samples from a single operation. The five most common serotypes were Salmonella Oranienburg (21.8% of isolates), and Salmonella Cerro (21.8%), followed by Salmonella Anatum (10.3%), Salmonella Bredeney (9.0%), and Salmonella Mbandaka (5.1%). The most common serogroups identified were C1 (33.3%), K (21.8%), B (16.7%), and E (15.4%). Even though the recovery rate of salmonellae from fecal samples was very low, 43.6% (34 of 78) and 38.5% (30 of 78) of the isolates were among the 10 most common serotypes from cattle with clinical signs of disease or isolated from humans, respectively. The majority of the isolates (50 of 78; 64.1%) were recovered from fecal samples from two operations. All isolates were screened for resistance to a panel of 17 antimicrobics, and 87.2% (68 of 78) were susceptible to all of the antimicrobics. The resistant isolates were most commonly resistant to streptomycin (n = 9) and/or sulfamethoxazole (n = 9). Nine isolates showed multiple (> or =2 antimicrobics) resistance most commonly to streptomycin and sulfamethoxazole (n = 6).

A mucosal competitive exclusion culture has been shown to reduce or eliminate Salmonella spp. in poultry. Using similar techniques, a mucosal competitive exclusion culture from swine (MCES) was produced from the cecum of a 6-week-old pig. Suckling pigs were inoculated with 5 ml of MCES by oral gavage within 6 h postfarrowing (PF) and again at 24 h PE All pigs were challenged with 10(3) CFU of Salmonella Choleraesuis at 48 h PF by intranasal instillation, including pigs from two sows that had not been given MCES. Clinical signs and rectal swabs were monitored daily, and pigs were allowed to suckle throughout the experiment. All pigs underwent necropsy on day 7 PF, and presence of Salmonella was determined in both qualitative (10 tissues) and quantitative (two tissues) samples. Clinical signs were inapparent in all pigs throughout the experiment. Recovery of Salmonella from rectal swabs was variable. However, 28% of the gut tissues were positive from the MCES-treated pigs versus 79% from the control pigs. A 2- to 5-log10 reduction of Salmonella in the cecal contents or ileocolic junction was observed in the MCES-treated pigs when compared with the controls. These data indicate that use of MCES may be a useful approach for control of Salmonella.

A national study of health and management of cattle in feedlots was conducted. Within this study, the prevalence of Salmonella spp. in fecal samples was determined. Fifty fecal samples were collected from each of 100 feedlots. Within each feedlot, 25 fresh fecal samples were collected from the floor of the pens of cattle which had been on feed the shortest and 25 from those on feed the longest periods of time. The total number of samples collected was 4,977; 2,484 and 2,495 from pens of cattle on feed the shortest and longest times, respectively. Salmonella spp. were recovered from 38% (38 of 100) of the feedlots. Salmonella spp. were recovered from 5.5% (273 of 4,977) of all samples and from 3.5% (88 of 2,484) and 7.4% (185 of 2,495) of samples from pens of cattle shortest and longest on feed, respectively. The most common serotype recovered was S. anatum (27.9%), followed by S. montevideo (12.9%), S. muenster (11.8%), S. kentucky (8.2%), and S. newington (4.3%). The most common serogroups identified were E1 (39.6%), C1 (20.7%), and B (10.4%). Shedding of the serotypes most commonly associated with human illness occurred infrequently (13 of 273: 4.8%). This study provides information on the status of Salmonella spp. from cattle in feedlots and may serve as baseline information for future studies.