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Recent investigations have found that Salmonella can be routinely recovered from peripheral lymph nodes (PLNs) of cattle presented for harvest. When contained within the PLNs, this foodborne pathogen is protected from currently used postharvest, inplant intervention strategies and, therefore, PLNs harboring Salmonella may be a potential contaminant of ground beef. The objective of this work was to develop a challenge model that effectively and repeatedly results in Salmonella -positive PLNs. A 10-lancet skin-allergy instrument was inoculated with Salmonella, and calves were inoculated intra- and/or transdermally by applying the device over various ventral regions of the skin. Salmonella was successfully and predictably recovered from regionspecific PLNs up to 8 days postchallenge. Furthermore, serotypes inoculated within specific regions were only recovered from the PLNs draining those regions. This model provides a method to predictably infect PLNs with Salmonella. Further, this model makes it possible to determine the duration of infection and to evaluate candidate interventions that may shorten the duration of infection.

Previous research conducted in our laboratory found a significant prevalence of multi-drug resistant (MDR) Salmonella and MDR Escherichia coli (MDR EC) in dairy calves and suggests that the MDR EC population may be an important reservoir for resistance elements that could potentially transfer to Salmonella. Therefore, the objective of the current research was to determine if resistance transfers from MDR EC to susceptible strains of inoculated Salmonella. The experiment utilized Holstein calves (approximately 3 weeks old) naturally colonized with MDR EC and fecal culture negative for Salmonella. Fecal samples were collected for culture of Salmonella and MDR EC throughout the experiment following experimental inoculation with the susceptible Salmonella strains. Results initially suggested that resistance did transfer from the MDR E. coli to the inoculated strains of Salmonella, with these stains demonstrating resistance to multiple antibiotics following in vivo exposure to MDR EC. However, serogrouping and serotyping results from a portion of the Salmonella isolates recovered from the calves post-challenge, identified two new strains of Salmonella; therefore transfer of resistance was not demonstrated under these experimental conditions.

The objective of the current research was to determine if feeding the citrus by-products(D) -limonene (DL) and citrus molasses would reduce the concentration and prevalence of Salmonella in weanling pigs experimentally infected with Salmonella Typhimurium. Twenty crossbred weanling pigs (average body weight [BW], 19.9 kg) were randomly assigned to one of four treatments: control, low-dose DL (1.5 ml/kg of BW per day), high-dose DL (3.0 ml/kg of BW per day), and citrus molasses (0.05 kg/kg of BW per day). Treatments were administered in the feed (twice daily) for 7 days, with one-half of the dose administered at each feeding. Fecal samples were collected twice daily (prior to administration of treatment) and cultured for quantitative and qualitative determination of the challenge strain of Salmonella. Upon termination of the study, pigs were euthanized and tissues from the stomach, ileum, cecum, spiral colon, and rectum, as well as luminal contents, were collected. In addition, the popliteal and ileocecal lymph nodes and liver, spleen, and tonsil tissue were collected for qualitative Salmonella culture. No significant treatment differences (P > 0.05) were observed among treatments for fecal concentration or prevalence of Salmonella throughout the 7-day collection period. Likewise, no treatment differences (P > 0.05) were observed for any of the tissue or luminal content samples collected. Salmonella was not cultured from the muscle-bound popliteal lymph node but was cultured from the mesenteric ileocecal lymph nodes. While there were no effects in the current experiment, future research may examine the effect of a lower challenge dose and/or different administration (dose or duration) of the citrus by-products.

Strategies aimed at reducing fecal shedding of Salmonella and other foodborne pathogens may be effective for limiting transmission of pathogens from food animals to humans. The objective of this study was to determine the effectiveness of gallium maltolate (GaM) against Salmonella in vitro and to determine whether oral administration of GaM would reduce fecal shedding of Salmonella in cattle. Gallium is a semimetal exhibiting antimicrobial properties against some pathogenic bacteria, including Salmonella, by exploiting their need for iron to survive and replicate. In vitro growth studies were performed in pure cultures of Salmonella and in mixed cultures from ruminal fluid. Inclusion of GaM in culture medium or in mixed cultures of ruminal fluid resulted in a significant reduction in growth of Salmonella, suggesting that GaM may be effective for limiting growth and survival in vivo. Therefore, we subsequently administered two doses of GaM to Holstein steers, experimentally infected them with Salmonella, and quantitatively and qualitatively monitored fecal shedding at 12-h intervals. Sixty hours after beginning treatment, cattle were euthanized, and luminal contents and tissue were aseptically harvested from the rumen, jejunum, spiral colon, cecum, and rectum. The luminal contents were processed for quantitative and qualitative analysis of the challenge strains of Salmonella, and tissue samples were enriched and plated for qualitative analysis. We found no significant differences between control and treated animals in quantitative levels of Salmonella in the feces or the luminal contents. Likewise, we observed no pattern between control and treated animals in the frequency of positive or negative results from enriched feces, luminal contents, or tissue samples. These results suggest that GaM was not effective for reducing Salmonella in cattle.

Aims: To determine the effect of sprinklers on faecal shedding of Escherichia coli (E. coli) O157:H7 and Salmonella in lactating dairy cattle., Methods and Results: Sprinklers were applied to lactating dairy cattle on two farms at either the feedbunk or in the holding pen prior to milking. Faecal samples were collected approx. 1 and 4 weeks following initiation of sprinkler treatments for culture of E. coli O157:H7, Salmonella and Enterococcus. No treatment differences were observed for E. coli O157:H7. Salmonella was higher (P = 0.11) in the control treatment on day-7 whereas on day-28, the bunk sprinklers increased the number of Salmonella positive cows. Salmonella prevalence decreased (P = 0.0001) on day-5 and when examined across days in cows exposed to sprinklers prior to milking. Antimicrobial susceptibility screening found very few isolates that were multi-drug resistant. All Enterococcus isolates were susceptible to vancomycin., Conclusions: This study demonstrated a significant decrease in faecal prevalence of Salmonella in lactating cattle following exposure to sprinklers administered prior to milking., Significance and Impact of the Study: Identification of current dairy management techniques that are also effective in reducing on-farm prevalence of pathogenic bacteria could have significant food safety and environmental implications.

Aim: To investigate the potential transfer of Escherichia coli O157:H7 and Salmonella from contaminated manure slurry into the tissue of tall fescue plants., Methods and Results: Tall fescue plants (n = 50) were fertilized with a manure slurry inoculated with E. coli O157:H7 and Salmonella. Soil was collected and tall fescue plants (n = 10 per day) harvested on day 1, 2, 4, 8, and 14 after manure slurry fertilization. Soil samples were positive for E. coli O157:H7 on all days and on day 1, 2, 8, and 14 for Salmonella. None of the plant tissue samples were positive for E. coli O157:H7 on day 1 or 2; however, 20%, 30% and 40% of plant tissue samples were positive for E. coli O157:H7 on day 4, 8, and 14, respectively., Conclusions: It may be possible that E. coli O157:H7 can become transmitted and internalized into tall fescue plant tissue within 4 days after exposure to an E. coli O157:H7-contaminated manure slurry. Salmonella did not appear to be transferred to tall fescue plant tissue., Significance and Impact of the Study: Faeces contaminated with E. coli O157:11H7 may be one means by which grazing ruminants spread bacterial pathogens to additional animals.

Salmonella causes an estimated 1.3 million human foodborne illnesses and more than 500 deaths each year in the United States, representing an annual estimated cost to the economy of approximately $2.4 billion. Salmonella enterica comprises more than 2,500 serotypes. With this genetic and environmental diversity, serotypes are adapted to live in a variety of hosts, which may or may not manifest with clinical illness. Thus, Salmonella presents a multifaceted threat to food production and safety. Salmonella have been isolated from all food animals and can cause morbidity and mortality in swine, cattle, sheep, and poultry. The link between human salmonellosis and host animals is most clear in poultry. During the early part of the 20th century, a successful campaign was waged to eliminate fowl typhoid caused by Salmonella Gallinarum/Pullorum. Microbial ecology is much like macroecology; environmental niches are filled by adapted and specialized species. Elimination of S. Gallinarum cleared a niche in the on-farm and intestinal microbial ecology that was quickly exploited by Salmonella Enteritidis and other serotypes that live in other hosts, such as rodents. In the years since, human salmonellosis cases linked to poultry have increased to the point that uncooked chicken and eggs are regarded as toxic in the zeitgeist. Salmonellosis caused by poultry products have increased significantly in the past 5 yr, leading to a USDA Food Safety and Inspection Service "Salmonella Attack Plan" that aims to reduce the incidence of Salmonella in chickens below the current 19%. The prevalence of Salmonella in swine and cattle is lower, but still poses a threat to food safety and production efficiency. Thus, approaches to reducing Salmonella in animals must take into consideration that the microbial ecology of the animal is a critical factor that should be accounted for when designing intervention strategies. Use of competitive exclusion, sodium chlorate, vaccination, and bacteriophage are all strategies that can reduce Salmonella in the live animal, but it is vital to understand how they function so that we do not invoke the law of unintended consequences.

The effects of the beta-agonist ractopamine, recently approved for use in feedlot cattle to improve carcass quality and performance, on fecal shedding Escherichia coli O157:H7 and Salmonella in feedlot cattle was examined. In the first study, 20 feedlot steers and heifers were randomly assigned to receive ractopamine or no ractopamine (control) by way of oral bolus for 28 days. Fecal samples were collected daily, and shedding of E. coli O157:H7 determined. When examined during the entire 28-day experimental period, ractopamine decreased (P = 0.0006) the percentage of cattle shedding E. coli O157:H7 (58% vs. 42% for control and ractopamine treatments, respectively). A second study was conducted in a commercial feedlot facility in the southwestern United States. Eighteen pens of cross-bred beef heifers (approximately 100 head/pen and 9 pens/treatment) were randomly assigned to receive either 0 (control) or 200 mg ractopamine/head x d(-1). Fresh fecal samples (30/pen) were collected off the pen floor before ractopamine supplementation and again after approximately 28 days of ractopamine supplementation (within a few days of slaughter); the samples were cultured for E. coli O157:H7 and Salmonella. The percentage of animals shedding E. coli O157:H7 was decreased when data were pooled across replicates (P = 0.05) in ractopamine-treated cattle compared with controls. The percentage of animals shedding Salmonella tended to be higher (P = 0.08) with the ractopamine treatment when data were pooled across replicates. Although further research is required to confirm these results, the potential food safety implications of this research are intriguing.

Escherichia coli O157:H7, Salmonella, and Listeria are foodborne pathogens of critical importance that often colonize cattle. E. coli O157:H7 can be specifically killed by lytic bacteriophage, and lytic bacteriophage treatment has been suggested as a pre-harvest intervention strategy to reduce foodborne pathogens in cattle. To date, no systematic approach to determine the incidence of E. coli O157:H7-infecting lytic bacteriophage has been published. Therefore, the current study was designed to determine (1) the incidence of E. coli O157, Salmonella spp., and Listeria and (2) the incidence of E. coli O157:H7-infecting bacteriophage in the feces of feedlot steers in commercial feedlots in the United States. Fecal samples (n=60) were collected from four feedlots in two Southern Great Plains states (total (n=240 fecal samples). Salmonella and E. coli O157:H7 were found in 3.8% and 11.7% of the fecal samples, respectively. Bacteriophage targeting E. coli O157:H7 were found in all four feedlots, in 15% of the individual fecal samples, and in 55% of the cattle pens. Our results indicate that such bacteriophage are widespread in feedlot cattle, suggesting that further research into the ecological role of bacteriophage in the gastrointestinal tract is needed.

To examine the effect of ionophore supplementation on fecal shedding of Escherichia coli O157:H7 and Salmonella, crossbred beef calves (n=113; mean body weight [BW], 243 kg) were fed a mineral supplement with ionophore (1.76 g lasalocid/kg) for 61 days (d). Control calves received an identical mineral supplement without lasalocid. Calves were pastured on fescue/bermudagrass paddocks and supplemented with a corn/wheat midds/soybean meal supplement (1.5% of BW/d). Upon arrival, cattle were fed a commercial receiving ration containing 1 g chlorotetracycline/kg for 10 d. Sick calves were administered one or a combination of the following: Nuflor (florfenicol), Baytril (eurofloxacin), Micotil (tilmicosin), or LA 200 (oxytetracycline). Fecal samples were collected immediately prior to ionophore supplementation, approximately midway and at the end of the experimental period (60 d total ionophore feeding) for isolation of E. coli O157:H7 and Salmonella. Putative fecal coliforms were also isolated at these sampling times and examined for antimicrobial susceptibility. The study was replicated over a two year period (year 1, n=53 head; year 2, n=60 head). Ionophore supplementation had no effect (p>0.10) on the incidence of calves shedding E. coli O157:H7 or Salmonella. The percentage of calves shedding E. coli O157:H7 varied throughout the experimental period from 0 to 30%, while Salmonella was cultured from only three calves over the 2-year experimental period. Antimicrobial susceptibility profiles of putative fecal coliforms were consistent with antibiotic treatments administered during the study (observed resistance to chlortetracycline, florfenicol, oxytetracycline), while only one treatment effect was observed. Ionophore treatment resulted in a significantly higher number of coliform isolates resistant to ampicillin compared to controls in year 1, but not year 2. A number of fecal coliform isolates demonstrated resistance to multiple antibiotics, however, this was not affected (p>0.10) by ionophore supplementation. Mineral intakes, BW gain, and the number of sick calves were similar (p>0.10) among treatments. Ionophore supplementation had no affect on fecal shedding of E. coli O157:H7 or Salmonella and a negligible impact on antimicrobial susceptibility patterns of fecal coliforms in beef calves.

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.

Heat stress and dusty conditions are common challenges for cattle during the summer, and a typical method of alleviating these problems involves sprinkling cattle and pens with water. The effect of sprinkling water on the incidence of zoonotic pathogens has not been previously studied. Four pens of heifers (n = 41) were cooled using sprinklers, and four pens (n = 43) served as controls. Heifers were crossbred Charolais, with white and red hair coats. Sprinkling was initiated when cattle were on full concentrate feed (July). Fecal samples, hide swipes, and BW were collected on d 0, 28, 63, 95, and 98. Average daily gain, DMI, and G:F were calculated, and carcass traits were collected 36 h after processing. Performance data were analyzed as a randomized complete block design, and zoonotic pathogen data were analyzed using chi2 analysis. Sprinkling tended (P = 0.054) to increase the incidence of fecal Salmonella spp. populations on d 98, but simultaneously tended to decrease (P = 0.058) the Escherichia coli O157:H7 incidence on hides on d 98. The most prevalent Salmonella serovars in this study were Kentucky, Muenster, Meleagridis, and Cerro. Performance measures and carcass traits did not differ between treatments (P > 0.10). Under our conditions, sprinkling cattle with water did not affect the incidence of zoonotic pathogens in feces or on hides.

The intestinal mucosa represents the most active defense barrier against the continuous challenge of food antigens and pathogenic microorganisms present in the intestinal lumen. Protection against harmful agents is conferred by factors such as gastric acid, peristalsis, mucus, intestinal proteolysis, and the intestinal biota. The establishment of beneficial bacterial communities and metabolites from these complex ecosystems has varying consequences for host health. This hypothesis has led to the introduction of novel therapeutic interventions based on the consumption of beneficial bacterial cultures. Mechanisms by which probiotic bacteria affect the microecology of the gastrointestinal tract are not well understood, but at least three mechanisms of action have been proposed: production/presence of antibacterial substances (e.g., bacteriocins or colicins), modulation of immune responses and specific competition for adhesion receptors to intestinal epithelium. The rapid establishment of bacterial communities has been thought to be essential for the prevention of colonization by pathogenic bacteria. Some animal models suggest that the reduction in bacterial translocation in neonatal animals could be associated with an increase in intestinal bacterial communities and bacteriocin-like inhibitory substances produced by these species. This review emphasizes the role of the intestinal microbiota in the reduction of the gastrointestinal infections and draws heavily on studies in poultry.

Mature, healthy lactating dairy cattle were sampled on two farms in the southwestern United States to examine the effects of heat stress (Experiment I) and stage of lactation (Experiment II) on the fecal shedding of E. coli O157:H7 and Salmonella. To examine the effects of heat stress, fecal samples were collected from 45 cows at 7:00 AM (coolest part of the day) and 5:00 PM (hottest part of the day) in August 2002 on a 250 cow dairy. The study was replicated one month later (n = 170 total samples). A temperature-heat index (THI) was calculated for each sampling time. In Experiment II, stage of lactation was examined by sampling lactating dairy cattle early [< 60 days in milk (DIM)] and late (> 150 DIM) in the lactation cycle in the summer of 2001. The study was replicated the following summer (60 cows/group/replicate; n = 240 total samples). For Experiment I, THI averaged 75 and 82 for the AM and PM samplings, respectively, indicating the cows were beginning to experience heat stress in the morning and by afternoon were in severe heat stress. The shedding of E. coli O157:H7 tended to be higher (p = 0.09) in the afternoon sampling of the first replicate, however was not different in the second replicate or when both replicates were pooled (p > 0.10). Salmonella shedding was not different (p > 0.10) at any sampling time with nearly 100% of the cows positive. Stage of lactation had no effect on the number of cows shedding E. coli O157:H7 (p > 0.10). Salmonella shedding tended to be higher (p = 0.09) in early lactation cows in the first replicate, while in the second replicate more late lactation cows were shedding Salmonella (p < 0.05); however, there were no differences due to stage of lactation when replicates were pooled (p > 0.10). While further research is needed, results of this research highlight the variability in pathogen shedding in healthy dairy cattle and indicate that environmental factors and/or production demands may influence shedding patterns of E. coli O157:H7 and Salmonella.

Mature dairy cattle were sampled over a 2-year period (2001-2002) on six farms in New Mexico and Texas. Fecal samples (n = 1560) were collected via rectal palpation and cultured for Salmonella, and one isolate from each positive sample was serotyped. Three isolates of each serotype, with the exception of Salmonella Newport (n = 12), were examined for susceptibility to 17 antimicrobial agents. Twenty-two different serotypes were identified from a total of 393 Salmonella isolates. Montevideo was the predominant serotype (27%) followed by Mbandaka (15%), Senftenberg (11.4%), Newport (6.4%), Anatum (4.8%), and Give (4.8%). Salmonella Typhimurium and Dublin, two frequently reported serotypes, accounted for only 1% of the observed serotypes in this study. Sixty-four percent of the serotypes were susceptible to all 17 antimicrobials, 14% were resistant to a single agent, and 22% were multiresistant (2-11 types of resistance). All isolates tested were susceptible to amikacin, apramycin, imipenem, ceftriaxone, nalidixic acid, and ciprofloxacin. The most frequent types of resistance were to sulfamethoxazole, tetracycline, streptomycin, kanamycin, chloramphenicol, and ampicillin (ranging from 8.9 to 22.4%). Serotypes demonstrating multiple resistance included Dublin and Give (resistant to three or more antibiotics), Typhimurium (resistant to five antibiotics), and Newport (four and two isolates resistant to six and nine antibiotics, respectively). Class 1 integrons were present in only two Salmonella Dublin isolates and one Salmonella Newport isolate. The most prevalent resistance patterns observed in this study were toward antimicrobial agents commonly used in cattle, while all Salmonella isolates were susceptible to ceftriaxone and ciprofloxacin, antibiotics used in human medicine.

Aims: To examine the variability in faecal shedding of Salmonella and Escherichia coli O157:H7 in healthy lactating dairy cattle and to evaluate the genetic relatedness of Salmonella isolates., Methods: Faecal samples were obtained from lactating Holstein dairy cattle on four commercial farms in the southwestern US. All farms were within an 8-km radius and were sampled in August 2001, January 2002 and August 2002 (60 cows per farm per sampling; n = 720 total samples). Samples were cultured for E. coli O157:H7 and Salmonella and a portion of the recovered Salmonella isolates were examined for genetic relatedness using pulsed-field gel electrophoresis (PFGE)., Results: Faecal shedding of E. coli O157:H7 and Salmonella varied considerably between farms and at the different sampling times. Large fluctuations in the percentage of positive animals were observed from summer to summer for both of these pathogens. Similarly, Salmonella serotype and serotype prevalence varied from farm to farm and within farm from one sampling time to another. Multiple Salmonella genotypes were detected for a number of serotypes and identical genotypes were found on different farms with one genotype of Salmonella Senftenberg identified on three of the four farms., Significance and Impact of the Study: This study demonstrated the wide variability in pathogen shedding within and among dairy farms all located in a small geographical region and highlights the complexity of pathogen control at the farm level.

Aim: To determine the prevalence of antimicrobial resistance among Salmonella isolated from a central Texas dairy calf farm that raises animals for dairy-beef production., Methods and Results: Salmonella isolates collected from 50 faecal samples were characterized for susceptibility to 20 antimicrobial agents. Seventy per cent of the faecal samples (35 of 50) tested positive for Salmonella, and high rates of resistance to the following drugs that are commonly used for treatment of bacterial enteritis in livestock were observed: ampicillin (88%), apramycin (83%), neomycin (86%), spectinomycin (91%) and oxytetracycline (90%). No resistance to the fluoroquinolone antibiotics was observed. The most prevalent Salmonella serotype was Kinshasha (22 of 35 samples), followed by Agona (4 of 35), Newport (3 of 35), Infantis (2 of 35), Montevideo (2 of 35), Lille (1 of 35) and Newington (1 of 35). The Kinshasa, Agona, Newport and Infantis serotypes all displayed resistance to ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline, and the penta-resistance phenotype was transferable to an Escherichia coli recipient strain., Significance: Multi-drug resistant Salmonella in dairy calves pose a costly animal health problem and a potential risk to the public health. This study emphasizes the need for alternative, non-antimicrobial intervention strategies for the control of zoonotic pathogens.

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).

Aims: To examine factors affecting faecal shedding of the foodborne pathogens Escherichia coli O157:H7 and Salmonella in dairy cattle and evaluate antimicrobial susceptibility of these isolates., Methods: Faecal samples were obtained in replicate from lactating (LAC; n = 60) and non-lactating (NLAC; n = 60) Holstein cattle to determine influence of heat stress, parity, lactation status (LAC vs NLAC) and stage of lactation [60 days in milk (DIM)] and cultured for E. coli O157:H7 and Salmonella. A portion of the recovered isolates were examined for antimicrobial susceptibility using the broth microdilution technique., Results: No effects of heat stress were observed. Lactating cows shed more (P < 0.01) E. coli O157:H7 than NLAC cows (43% vs 32%, respectively). Multiparous LAC cows tended to shed more (P = 0.06) Salmonella than primiparous LAC cows (39% vs 27%, respectively). Parity did not influence (P > 0.10) bacterial shedding in NLAC cows. Cows 60 DIM. Seventeen Salmonella serotypes were identified with the most prevalent being Senftenberg (18%), Newport (17%) and Anatum (15%). Seventy-nine of the Salmonella isolates were resistant to at least one of the seven antibiotics. Escherichia coli O157:H7 isolates were resistant to 11 different antibiotics with multiple resistance to nine or more antibiotics observed in five isolates., Significance and Impact of the Study: This study demonstrated differences in the shedding patterns of foodborne pathogens due to the stage of the milk production cycle and may help identify times when on-farm pathogen control would be the most effective.

Aims: To examine the effects of ionophores on Salmonella and Escherichia coli O157:H7 in pure and mixed ruminal fluid cultures., Methods and Results: Four Salmonella serotypes (Dublin, Derby, Typhimurium, and Enteriditis) and two strains of E. coli O157:H7 (ATCC 43895 and FDIU 6058) were cultured in the presence of varying concentrations of ionophores (monensin, lasalocid, laidlomycin propionate, and bambermycin) in pure and mixed ruminal fluid cultures. Bacterial growth rates in pure culture were not affected (P > 0.10) by ionophores at concentrations up to 10 times the approximate rumen ionophore concentration under normal feeding regimens. Likewise, ionophores had no effect (P > 0.10) on Salmonella or E. coli CFU plated from 24-h ruminal fluid incubations. Ionophore treatment decreased (P < 0.01) the acetate : propionate ratio in ruminal fluid cultures as expected., Conclusions: Ionophores had no effect on the foodborne pathogens Salmonella and E. coli O157:H7 in vitro., Significance and Impact of the Study: The results suggest that ionophore feeding would have little or no effect on Salmonella or E. coli populations in the ruminant.

Direct Fed Microbials (DFM) are probiotic-type compounds that are utilized in calf feeding operations as novel, non-antibiotic technologies to improve health and performance. The modes of action of most probiotics remain unknown, but their impacts on the microbial ecology of the GI tract may be responsible for positive changes in animal health and performance. Therefore, the objective of this study was to evaluate changes in the microbiome of the ileum of Holstein steer calves supplemented with a DFM comprised of a patented strain of Bacillus subtilus (CLOSTAT®; 13 g/hd/d for 35d: Kemin Industries Des Moines, IA) following an experimental, oral Salmonella challenge. Holstein steer calves (n=40) were assigned to treatments in a 2 x 2 factorial design and either fed CLOSTAT (CLO) or not (CON) and inoculated with Salmonella Typhimurium (SAL; 1.1x 106 CFU) or not (NOSAL) in the calves' milk replacer. Our lab previously reported CLO supplementation reduced Salmonella in the GI tract and reduced the febrile response in CLO calves following SAL challenge. Upon harvest in the present study, ileum tissue samples were collected and the microflora of each calf was analyzed using Ilumina MiSeq. Diversity was examined for overall richness (OTU), quantified using the Chao 1 richness estimator, and data were examined for richness and evenness distribution amongst distinct taxa using the Shannon Diversity Index. Group differences were analyzed using permutational multivariate analysis of variance. There was no difference in the number of distinct species or genera present within the microbiome between CLO and CON or the interaction of CLO supplementation and SAL inoculation (P>0.20). Inoculation of calves with Salmonella increased species/genera richness (P=0.02) in the ileum. Overall, there was no difference in the Shannon Diversity Index due to CLO (P>0.05), but the diversity was increased with SAL inoculation (P<0.01). However, there was no interaction of CLO and SAL (P>0.05) on any measure of microbial diversity. Similarly, the number of OTU's were not affected by CLO (P>0.05); however, SAL increased the number of OTU's (P=0.02) in ileal contents. While positive effects on animal health were observed in SAL calves fed CLO, there was no overall difference in the composition of the microbiome of the ileum. However, SAL administration, as expected, significantly influenced the ileal microbiome of Holstein steer calves. More research is needed to fully elucidate the effects of CLO supplementation on the microbiome of calves in the various compartments of the GI tract. [ABSTRACT FROM AUTHOR]

To evaluate the effects of a patented Bacillus subtillus probiotic (CLOSTAT®), weaned Holstein steers, (n = 200; ~90kg) were supplemented (CLO) or not (CON) with CLOSTAT® (13 g/hd/d; Kemin Industries, Des Moines, IA) in a starter ration at a calf ranch for 35 d (n = 50 head per pen; 2 pens/trt.). A subset of 40 calves was selected for an oral Salmonella challenge based on body weight, treatment records, and lack of Salmonella shedding. The calves were transported to Lubbock, TX (105 km) and assigned to 1 of 4 treatments in a 2x2 factorial design with CLO and CON calves that were orally administered Salmonella (STM) or not (CLOSTM, CLONoSTM, CONSTM, CONNoSTM). Calves assigned to receive Salmonella were challenged with 1.6 x 106 Salmonella Typhimurium (resistant to 50µg/ml nalidixic acid) in 1 L of milk replacer on d 0. Blood samples for serum and hematology were collected via jugular catheters every 8 h for 96 h, and body temperature was collected every 5 min via indwelling rectal temperature recording devices. Five calves from each treatment were harvested 48 h post-challenge, and the remaining calves were harvested 96 h post-challenge. During necropsy, tissues were collected for the isolation and quantification of the inoculated STM from various tissues. The CLO group had reduced STM concentrations in the jejunum, ileum, and transverse colon 48 h after the challenge (P ≤ 0.03) and numerically reduced STM concentrations in all gastrointestinal tissues 96 h post-challenge. There was a difference in rectal temperature (P < 0.001) in which CLOSTM calves displayed decreased rectal temperatures after the challenge when compared to CONSTM calves. White blood cells and lymphocytes were increased (P ≤ 0.05) in CLOSTM calves after the challenge in comparison to other treatments. Calves in the CLO treatment maintained greater numerical BW throughout the study; however, there was no difference (P = 0.89) in BW change during the study. In calves given STM, the CLO group had greater feed disappearance before and after the challenge (P = 0.006) in comparison to the CON group. Increased serum IL-6 and IFNγ was observed in the CONSTM group compared to other treatments. There was no difference (P = 0.42) in circulating cortisol between the treatments. Overall, CLO reduced Salmonella presence and concentrations in gastrointestinal tissues while simultaneously reducing the severity of the salmonellosis as indicated by blood parameters and the reduced febrile response. [ABSTRACT FROM AUTHOR]

To evaluate the effects of a patented Bacillus subtillus probiotic (CLOSTAT®), weaned Holstein steers, (n = 200; ~90kg) were supplemented (CLO) or not (CON) with CLOSTAT® (13 g/hd/d; Kemin Industries, Des Moines, IA) in a starter ration at a calf ranch for 35 d (n = 50 head per pen; 2 pens/ trt.). A subset of 40 calves was selected for an oral Salmonella challenge based on body weight, treatment records, and lack of Salmonella shedding. The calves were transported to Lubbock, TX (105 km) and assigned to 1 of 4 treatments in a 2x2 factorial design with CLO and CON calves that were orally administered Salmonella (STM) or not (CLOSTM, CLONoSTM, CONSTM, CONNoSTM). Calves assigned to receive Salmonella were challenged with 1.6 x 106 Salmonella Typhimurium (resistant to 50μg/ml nalidixic acid) in 1 L of milk replacer on d 0. Blood samples for serum and hematology were collected via jugular catheters every 8 h for 96 h, and body temperature was collected every 5 min via indwelling rectal temperature recording devices. Five calves from each treatment were harvested 48 h post-challenge, and the remaining calves were harvested 96 h post-challenge. During necropsy, tissues were collected for the isolation and quantification of the inoculated STM from various tissues. The CLO group had reduced STM concentrations in the jejunum, ileum, and transverse colon 48 h after the challenge (P ≤ 0.03) and numerically reduced STM concentrations in all gastrointestinal tissues 96 h post-challenge. There was a difference in rectal temperature (P < 0.001) in which CLOSTM calves displayed decreased rectal temperatures after the challenge when compared to CONSTM calves. White blood cells and lymphocytes were increased (P ≤ 0.05) in CLOSTM calves after the challenge in comparison to other treatments. Calves in the CLO treatment maintained greater numerical BW throughout the study; however, there was no difference (P = 0.89) in BW change during the study. In calves given STM, the CLO group had greater feed disappearance before and after the challenge (P = 0.006) in comparison to the CON group. Increased serum IL-6 and IFNγ was observed in the CONSTM group compared to other treatments. There was no difference (P = 0.42) in circulating cortisol between the treatments. Overall, CLO reduced Salmonella presence and concentrations in gastrointestinal tissues while simultaneously reducing the severity of the salmonellosis as indicated by blood parameters and the reduced febrile response. [ABSTRACT FROM AUTHOR]

Probiotics are gaining increased interest in calf feeding operations as some producers seek novel, non-antibiotic technologies to improve health and performance. Therefore, the objective of this study was to evaluate changes in serum metabolomic compounds of Holstein steer calves supplemented with CLOSTAT® (13g/hd/d for 35d; Kemin Industries, Des Moines, IA) following an experimental oral Salmonella challenge. Holstein steer calves (n = 40) were assigned to treatments in a 2 x 2 factorial design and either fed CLOSTAT (CLO) or not (CON) and inoculated with Salmonella Typhimurium (SAL; 1.1 x 106) or not (NOSAL) via milk replacer. Data has previously reported CLO supplementation greatly reduced Salmonella concentrations in the gastrointestinal tract of calves after experimental infection and mitigated some of the negative effects of the disease challenge. Additionally, rectal temperature was greatly reduced in calves subjected to the challenge. For this study, serum was analyzed from blood samples collected 48 h after experimental infection representing peak rectal temperature. Serum metabolites were identified and quantified using GC-MS (West Coast Metabalomics Center, UC Davis). Data were log transformed prior to analysis to achieve normality. Data were analyzed using univariate ANOVA, principal component analysis, and partial least squares-discriminate analysis using the fixed effects of metabolite, treatment, Salmonella, and their interactions using Metaboanalyst 3.0. Mean separation was determined at α = 0.05. A total of 334 compounds were quantified, and 118 were of known identity. Overall, serum metabolome was shifted more due to CLO supplementation (9.6%) as opposed to SAL inoculation (7.1%). Clostat increased the concentrations of leucine and 32 other metabolites (P < 0.05). Salmonella was responsible for increasing myristic acid, heptadecanoic acid, and other fatty acids (P < 0.05) which have known antimicrobial properties. Uric acid was increased (P < 0.05) in CLO supplemented calves and may have enhanced macrophage killing capabilities which may correlate to reduced migration, colonization, and translocation observed in the gastrointestinal tract of supplemented calves. This may partially explain the previously reported reduced rectal temperature in CLO supplemented calves. Salmonella additionally altered 25 other metabolites (P < 0.05). Only 9 interactions existed between supplementation and Salmonella infection across 334 compounds. These data suggest that supplementation of calves with CLO may alter serum metabolites and shift metabolic and immune pathways regardless of their Salmonella status which may have played a role in reducing the negative effects of a Salmonella challenge. [ABSTRACT FROM AUTHOR]

The objective of the current study was to examine the effect of pasteurization of waste milk, used to feed dairy calves, on the bacterial diversity of their lower gut. Using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing, fecal samples from dairy calves, ages 1 wk to 6 mo old and fed either pasteurized or nonpasteurized waste milk, were analyzed for bacterial diversity. Calves were maintained on 2 separate farms and, aside from how the waste milk was treated, were housed and cared for similarly. Fifteen calves were sampled from each age group (1, 2, and 4 wk, and 2, 4, and 6 mo of age; n = 90 samples per milk treatment, 180 total samples) on each farm via rectal palpation and the samples shipped and frozen before analysis. In general, bacterial diversity, as represented by the total number of different species, was greater for the calves fed pas- teurized waste milk at all ages (except 1 wk of age) and increased with increasing age in both treatments. Proteobacteria, Bacteroidetes, and Firmicutes were the predominant phyla. Differences in phyla and class were observed among treatments and age of calf but with no consistent trends. Salmonella were detected in 9 out of 14 (64%) of the 1-wk-old calves fed nonpasteurized milk. Treponema, an important beneficial bacterium in cattle rumen, was more prevalent in the pasteurized waste milk-fed animals and became higher in the older animals from this group. Escherichia-Shigella were detected among treatments at all ages, and highest at 1 wk of age, averaging approximately 21 and 20% of all bacteria for calves fed pasteurized and nonpasteurized waste milk, respectively, and decreasing as calves aged (2.6 and 1.3%). The consistent detection of Salmonella in the younger animals fed nonpasteurized milk and its absence in all other groups is an important finding related to this feeding practice. [ABSTRACT FROM AUTHOR]

A study was conducted to evaluate the effect of experimental chlorate product (ECP) feed supplementation on Salmonella Typhimurium (ST) in the crop and ceca of market-age broilers. In trial 1, 160 market-age broilers were randomly assigned to 8 treatment groups and replicated twice, with 20 broilers per pen for 1 wk. Trial 2 used the same design, but used 80 market-age broilers with 10 broilers per pen. Treatments were as follows: 1) control feed + double-distilled drinking water (dd H2O); 2) control + 18.5% experimental zeolite carrier with dd H2O; 3 to 7) control feed supplemented with 0.5, 1.0, 5.0, 10.0, or 18.5% of a feed grade ECP + dd H2O; 8) control feed + 1× ECP (0.16% w/v; containing 15 mM chlorate ion equivalent) added to dd H2O. Seven-week-old broilers were provided experimental treatments for 7 d, killed, and then ceca and crops were removed and evaluated for ST. Broilers fed 5 to 18.5% ECP or water ECP had a significantly lower (P < 0.05) incidence of ST in the crop (36 to 38% and 14%, respectively) when compared with the control (60%). Broilers fed 10% ECP or water ECP had significantly lower ST crop concentrations (1.03 log10 and 0.38 log10 ST/g, respectively) when compared with broilers fed a control diet (1.54 log10 ST/g). Crop and ceca ST incidence (32 to 48%) and concentration (1.00 to 1.82 log10 ST/g) were significantly lower in broilers fed 5 to 18.5% ECP as compared with the control (78%; 2.84 log10 ST/g). Broilers fed 5% or greater ECP had significantly higher water consumption (380 to 580 mL water/d) and litter moisture (31 to 56%) when compared with the control (370 mL water/d; 23% moisture). Only broilers fed 18.5% ECP had significantly lower 7-wk BW (2.77 kg of BW) when compared with the controls (3.09 kg of BW). Average daily gains were significantly depressed in broilers fed 10 or 18.5% ECP compared with the controls. These results indicate broilers supplemented with feed ⩽ 5% ECP or water ECP 7 d before slaughter reduced ST without affecting growth parameters. [ABSTRACT FROM AUTHOR]

Two studies were conducted to evaluate the effects of melatonin on Salmonella Enteritidis infection in experimentally challenged laying hens subjected to a forced molt. Leghorn hens (>50 wk of age) were randomly assigned to rooms, acclimated to a 16L:8D regimen, and provided ad libitum access to a nonmedicated mash layer diet and water. Birds in one room were molted (8L:16D; complete feed withdrawal), whereas birds in the second room served as nonmolted controls (CONT). Within each room, birds were randomly assigned to melatonin treatment (MEL; 12 birds/treatment), dosed orally commencing the same day as feed withdrawal for 10 d: (experiment I: 0 or 5 mg of melatonin; experiment II: 0, 10, or 20 mg of melatonin). Three days following feed withdrawal, all birds were experimentally infected with Salmonella Enteritidis, and after 10 d of feed withdrawal, all birds were killed and necropsied. In experiment I, concentrations of Salmonella Enteritidis in the cecal contents and the number of Salmonella Enteritidis-positive tissues from the crop, ceca, liver, spleen, and ovary were higher (P < 0.0001) in the MOLT compared with the CONT treatments. No differences (P > 0.10) were observed in any of the parameters examined due to MEL treatment. For experiment II, cecal concentrations of Salmonella Enteritidis were generally higher in the MOLT compared with the CONT treatment and within molted birds, cecal concentrations were higher in the MEL treatment (P < 0.05). Melatonin treatment in molted birds increased (P < 0.05) the percentage of positive crops in the MOLT+20 MEL treatment (P < 0.05). Salmonella-positive cecal tissue was increased (P < 0.001) in MOLT compared with CONT birds and was also higher in MOLT+10 MEL and MOLT+20 MEL birds compared with the MOLT-only treatment. Results from the current research suggest that dosage with high levels of melatonin may exacerbate Salmonella Enteritidis infection in layers subjected to forced molt. [ABSTRACT FROM AUTHOR]

Foodborne diseases caused by enterohemorrhagic Escherichia coli, Salmonella, and Cam pylobacter species are of public health and economic significance. Shedding of these pathogens during production and slaughter are risks for contamination of products for human consumption. Consequently, strategies are sought to prevent or reduce the carriage of these pathogens in food animals before slaughter. Experimental products containing chlorate salts have been proven efficacious in reducing concentrations of E. coli and Salmonella Typhimurium in the gut of cattle, sheep, swine, and poultry when administered as feed or water additives. Mechanistically, chlorate selectively targets bacteria expressing respiratory nitrate reductase activity, such as most members of the family Enterobacteriaceae, as this enzyme catalyzes the reduction of chlorate to lethal chlorite. Most beneficial gut bacteria lack respiratory nitrate reductase activity, and thus the technology appears compatible with many bacteria exhibiting competitive exclusion capabilities. More recently, select nitrocompounds have been investigated as potential feed additives, and although these nitrocompounds significantly reduce pathogens on their own, evidence indicates that they may most effectively be used to complement the bactericidal activity of chlorate. A particularly attractive aspect of the nitrocompound technology is that, as potent inhibitors of ruminal methanogenesis, they may allow producers the opportunity to recoup costs associated with their use. At present, neither chlorate nor the nitrocompounds have been approved as feed additives by the US Food and Drug Administration, and consequently they are not yet available for commercial use. [ABSTRACT FROM AUTHOR]