Microdiversity of Salmonella Kentucky During Long-Term Colonization of a Dairy Herd.

Salmonella enterica subsp. enterica serovar Kentucky was repeatedly isolated from a commercial dairy herd that was enrolled in a longitudinal study where feces of asymptomatic dairy cattle were sampled intensively over an 8-year period. The genomes of 5 Salmonella Kentucky isolates recovered from the farm 2 years before the onset of the long-term colonization event and 13 isolates collected during the period of endemicity were sequenced. A phylogenetic analysis inferred that the Salmonella Kentucky strains from the farm were distinct from poultry strains collected from the same region, and three subclades (K, A1, and A2) were identified among the farm isolates, each appearing at different times during the study. Based on the phylogenetic analysis, three separate lineages of highly similar Salmonella Kentucky were present in succession on the farm. Genomic heterogeneity between the clades helped identify regions, most notably transcriptional regulators, of the Salmonella Kentucky genome that may be involved in competition among highly similar strains. Notably, a region annotated as a hemolysin expression modulating protein (Hha) was identified in a putative plasmid region of strains that colonized a large portion of cows in the herd, suggesting that it may play a role in asymptomatic persistence within the bovine intestine. A cell culture assay of isolates from the three clades with bovine epithelial cells demonstrated a trend of decreased invasiveness of Salmonella Kentucky isolates over time, suggesting that clade-specific interactions with the animals on the farm may have played a role in the dynamics of strain succession. Results of this analysis further demonstrate an underappreciated level of genomic diversity within strains of the same Salmonella serovar, particularly those isolated during a long-term period of asymptomatic colonization within a single dairy herd.

Diversity of Extended-Spectrum Cephalosporin-Resistant Escherichia coli in Feces from Calves and Cows on Pennsylvania Dairy Farms.

The global incidence of human infections associated with extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli is increasing. Dairy animals are reservoirs of ESBL-producing E. coli, especially, third-generation cephalosporin (3GC)-resistant strains. To further understand the diversity of 3GC-resistant E. coli across animals of different age groups (e.g., pre- and postweaned calves, lactating cows, and dry cows) and farms, we used pulsed-field gel electrophoresis (PFGE) to characterize 70 fecal isolates from 14 dairy farms located in nine Pennsylvania counties. Results of this analysis indicated that 3GC-resistant E. coli were highly diverse and grouped into 27 PFGE clades (80% similarity cutoff) and 24 unique antimicrobial resistance patterns were observed among the isolates. For eight farms, clonal E. coli with the same resistance patterns were isolated from two or more age groups, indicating that strains were carried in both the calves and adult cows within the same herd. However, there were also several isolates with the same resistance pattern that were distributed to different clades, including isolates from different animal age groups on the same farm, suggesting different strains of E. coli within a farm harbored the same resistance-conferring elements. Results of this analysis indicated that 3GC-resistant E. coli were highly diverse, associated with multidrug resistance, and circulated through different (noncommingled) animal groups on individual farms.

Antimicrobial Resistance Among Escherichia coli Isolated from Veal Calf Operations in Pennsylvania.

Antimicrobial resistance (AR) is a pressing public health concern, and agricultural operations such as dairy and beef cattle production have been implicated as potential sources of resistant bacteria or genetic elements. This study aimed to determine the prevalence of antimicrobial-resistant Escherichia coli from calf pens in 6 auction houses (56 manure composite samples) and 12 veal calf operations (240 fecal samples in 2 visits: after the calves arrived at the farm and shortly before the animals were sent to slaughter) in the Commonwealth of Pennsylvania. A total of 1567 generic E. coli were isolated and screened for resistance phenotypes. Resistant E. coli were isolated from all auction houses and farms sampled. Based on nonparametric Kruskal-Wallis tests, incremental prevalence of E. coli resistant to ampicillin, azithromycin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, streptomycin, sulfisoxazole, trimethoprim-sulfamethoxazole, and tetracycline in the samples from auction houses and the first and second farm visits was observed (χ2 6.98-15.91, p < 0.05). Multidrug-resistant E. coli (resistant to more than three antimicrobial classes) were identified in 76.8%, 90.8%, and 100% of samples collected from the auction houses, first farm visits, and second farm visits, respectively. The presence of blaCTX-M-E. coli in 11 of the 12 farms presents the possibility of veal production environments being a reservoir for resistant genetic materials that may pose a risk to human health if they are transferred to human pathogens. Additional research on the impact of various management strategies in veal calf rearing is needed for a complete scenario of AR in these production environments.

Age-Associated Distribution of Antimicrobial-Resistant Salmonella enterica and Escherichia coli Isolated from Dairy Herds in Pennsylvania, 2013-2015.

Antimicrobial resistance has become a major global public health concern, and agricultural operations are often implicated as a source of resistant bacteria. This study characterized the prevalence of antimicrobial-resistant Salmonella enterica and Escherichia coli from a total of 443 manure composite samples from preweaned calves, postweaned calves, dry cows, and lactating cows from 80 dairy operations in Pennsylvania. A total of 1095 S. enterica and 2370 E. coli isolates were screened and tested for resistance to 14 antimicrobials on the National Antimicrobial Resistance Monitoring System Gram-negative (NARMS GN) panel. Salmonellae were isolated from 67% of dairy operations, and 99% of the isolates were pan-susceptible. Salmonella were isolated more frequently from lactating and dry cow samples than from pre- and postweaned calf samples. Overall, the most prevalent serotypes were Cerro, Montevideo, Kentucky, and Newport. E. coli were isolated from all the manure composite samples, and isolates were commonly resistant to tetracyclines, sulfonamides, and aminoglycosides. Resistance was detected more frequently in the E. coli isolates from pre- and postweaned calf samples than in isolates from dry and lactating cow samples (p < 0.05). Multidrug-resistant E. coli (i.e., resistant to >3 antimicrobial classes) were isolated from 66 farms (83%) with significantly greater prevalence in preweaned calves (p < 0.05) than in the older age groups. The blaCTX-M and blaCMY genes were detected in the cephalosporin-resistant E. coli from 4% and 35% of the farms, respectively. These findings indicate that dairy animals, especially the calf population, serve as significant reservoirs for antimicrobial-resistant bacteria. Additional research on the colonization and persistence of resistant E. coli in calves is warranted to identify potential avenues for mitigation.

Prevalence of Salmonella enterica, Listeria monocytogenes, and pathogenic Escherichia coli in bulk tank milk and milk filters from US dairy operations in the National Animal Health Monitoring System Dairy 2014 study.

The dairy farm environment is a well-documented reservoir for zoonotic pathogens such as Salmonella enterica, Shiga-toxigenic Escherichia coli, and Listeria monocytogenes, and humans may be exposed to these pathogens via consumption of unpasteurized milk and dairy products. As part of the National Animal Health Monitoring System Dairy 2014 study, bulk tank milk (BTM, n = 234) and milk filters (n = 254) were collected from a total of 234 dairy operations in 17 major dairy states and analyzed for the presence of these pathogens. The invA gene was detected in samples from 18.5% of operations and Salmonella enterica was isolated from 18.0% of operations. Salmonella Dublin was detected in 0.7% of operations. Sixteen Salmonella serotypes were isolated, and the most common serotypes were Cerro, Montevideo, and Newport. Representative Salmonella isolates (n = 137) were tested against a panel of 14 antimicrobials. Most (85%) were pansusceptible; the remaining were resistant to 1 to 9 antimicrobials, and within the resistant strains the most common profile was resistance to ampicillin/clavulanic acid, ampicillin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline. Listeria spp. were isolated from 19.9% of operations, and L. monocytogenes was isolated from 3.0% of operations. Serogroups 1/2a and 1/2b were the most common, followed by 4b and 4a. One or more E. coli virulence genes were detected in the BTM from 30.5% of operations and in the filters from 75.3% of operations. A combination of stx2, eaeA, and γ-tir genes was detected in the BTM from 0.5% of operations and in the filters from 6.6% of operations. The results of this study indicate an appreciable prevalence of bacterial pathogens in BTM and filters, including serovars known to infect humans.

Prevalence, antimicrobial resistance, and molecular characterization of Campylobacter spp. in bulk tank milk and milk filters from US dairies.

Campylobacter spp. are frequently isolated from dairy cows as commensal organisms. Sporadic Campylobacter infections in humans in the United States are generally attributed to poultry, but outbreaks are also commonly associated with dairy products, particularly unpasteurized or raw milk. Bulk tank milk samples and milk filters from US dairy operations were collected during the National Animal Health Monitoring System Dairy 2014 study and analyzed using real-time PCR and traditional culture techniques for the presence of thermophilic Campylobacter species. The weighted prevalence of operations from which we detected Campylobacter spp. in either bulk tank milk or milk filters was 24.9%. We detected Campylobacter spp. in a higher percentage of operations with 100-499 cows (42.8%) and 500 or more cows (47.5%) than in operations with 30-99 cows (6.5%). Campylobacter spp. were also more frequently detected in operations in the west than the east (45.9 and 22.6%, respectively). We isolated Campylobacter spp. from approximately half of PCR-positive samples, representing 12.5% (weighted prevalence) of operations. The majority (91.8%) of isolates were C. jejuni, but C. lari and C. coli were also isolated. We detected resistance to tetracycline in 68.4% of C. jejuni isolates, and resistance to ciprofloxacin and nalidixic acid in 13.2% of C. jejuni isolates. Based on pulsed-field gel electrophoresis, we found that dairy-associated C. jejuni were genotypically diverse, although clonal strains were isolated from different geographic regions. These results suggest that bulk tank milk can be contaminated with pathogenic Campylobacter spp., and that the consumption of unpasteurized or raw milk presents a potential human health risk.

Irrigation waters and pipe-based biofilms as sources for antibiotic-resistant bacteria.

The presence of antibiotic-resistant bacteria in environmental surface waters has gained recent attention. Wastewater and drinking water distribution systems are known to disseminate antibiotic-resistant bacteria, with the biofilms that form on the inner-surfaces of the pipeline as a hot spot for proliferation and gene exchange. Pipe-based irrigation systems that utilize surface waters may contribute to the dissemination of antibiotic-resistant bacteria in a similar manner. We conducted irrigation events at a perennial stream on a weekly basis for 1 month, and the concentrations of total heterotrophic bacteria, total coliforms, and fecal coliforms, as well as the concentrations of these bacterial groups that were resistant to ampicillin and tetracycline, were monitored at the intake water. Prior to each of the latter three events, residual pipe water was sampled and 6-in. sections of pipeline (coupons) were detached from the system, and biofilm from the inner-wall was removed and analyzed for total protein content and the above bacteria. Isolates of biofilm-associated bacteria were screened for resistance to a panel of seven antibiotics, representing five antibiotic classes. All of the monitored bacteria grew substantially in the residual water between irrigation events, and the biomass of the biofilm steadily increased from week to week. The percentages of biofilm-associated isolates that were resistant to antibiotics on the panel sometimes increased between events. Multiple-drug resistance was observed for all bacterial groups, most often for fecal coliforms, and the distributions of the numbers of antibiotics that the total coliforms and fecal coliforms were resistant to were subject to change from week to week. Results from this study highlight irrigation waters as a potential source for antibiotic-resistant bacteria, which can subsequently become incorporated into and proliferate within irrigation pipe-based biofilms.

Dynamics of Escherichia coli Virulence Factors in Dairy Herds and Farm Environments in a Longitudinal Study in the United States.

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

Diversity of Listeria monocytogenes within a U.S. dairy herd, 2004-2010.

Listeria monocytogenes, the causative agent of listeriosis, is frequently isolated from the environment. Dairy cows and dairy farm environments are reservoirs of this pathogen, where fecal shedding contributes to its environmental dispersal and contamination of milk, dairy products, and meat. The molecular diversity of 40 L. monocytogenes isolates representing 3 serogroups (1/2a, 1/2b, and 4b) collected between 2004 and 2010 from the feces of dairy cattle on a single dairy farm was assessed using a multivirulence locus sequence typing (MVLST) assay. The dairy farm L. monocytogenes MVLST patterns were compared to those from 138 strains isolated globally from clinical cases, foods, and the environment. Results of the study demonstrated that several distantly related L. monocytogenes strains persisted among members of the herd over the course of the study while other strains were transient. Furthermore, some strains isolated during this study appear to be distantly related to previously isolated L. monocytogenes while others are closely related to Epidemic Clones associated with human illness. This work demonstrates that dairy cows can be reservoirs of a diverse population of potentially human pathogenic L. monocytogenes that represents a risk to consumers of milk, dairy products, and meat.

Stochastic modeling of imperfect Salmonella vaccines in an adult dairy herd.

Salmonella is a major cause of bacterial foodborne disease. Human salmonellosis results in significant public health concerns and a considerable economic burden. Dairy cattle are recognized as a key source of several Salmonella serovars that are a threat to human health. To lower the risk of Salmonella infection, reduction of Salmonella prevalence in dairy cattle is important. Vaccination as a control measure has been applied for reduction of preharvest Salmonella prevalence on dairy farms. Salmonella vaccines are usually imperfect (i.e., vaccines may provide a partial protection for susceptible animals, reduce the infectiousness and shedding level, shorten the infectious period of infected animals, and/or curb the number of clinical cases), and evaluation of the potential impacts of imperfect Salmonella vaccines at the farm level is valuable to design effective intervention strategies. The objective of this study was to investigate the impact of imperfect Salmonella vaccines on the stochastic transmission dynamics in an adult dairy herd. To this end, we developed a semi-stochastic and individual-based continuous time Markov chain (CTMC) vaccination model with both direct and indirect transmission, and applied the CTMC vaccination model to Salmonella Cerro transmission in an adult dairy herd. Our results show that vaccines shortening the infectious period are most effective in reducing prevalence, and vaccines decreasing host susceptibility are most effective in reducing the outbreak size. Vaccines with multiple moderate efficacies may have the same effectiveness as vaccines with a single high efficacy in reducing prevalence, time to extinction, and outbreak size. Although the environment component has negligible contributions to the prevalence, time to extinction, and outbreak size for Salmonella Cerro in the herd, the relative importance of environment component was not assessed. This study indicates that an effective vaccination program against Salmonella Cerro spread in the herd can be designed with (1) vaccines with a single high efficacy in reducing either the infectious period or susceptibility of the host, or (2) if such single high efficacy vaccines are not available, vaccines with multiple moderate efficacies may be considered instead. These findings are also of general value for designing vaccination program for Salmonella serotypes in livestock.

Invasion and transmission of Salmonella Kentucky in an adult dairy herd using approximate Bayesian computation.

BACKGROUND: An outbreak of Salmonella Kentucky followed by a high level of sustained endemic prevalence was recently observed in a US adult dairy herd enrolled in a longitudinal study involving intensive fecal sampling. To understand the invasion ability and transmission dynamics of Salmonella Kentucky in dairy cattle, accurate estimation of the key epidemiological parameters from longitudinal field data is necessary. The approximate Bayesian computation technique was applied for estimating the transmission rate (ß), the recovery rate (γ) and shape (n) parameters of the gamma distribution for the infectious (shedding) period, and the basic reproduction ratio (R0), given a susceptible-infectious-recovered-susceptible (SIRS) compartment model with a gamma distribution for the infectious period. RESULTS: The results report that the mean transmission rate (ß) is 0.417 month-1 (median: 0.417, 95% credible interval [0.406, 0.429]), the average infectious period (γ-1) is 7.95 months (median: 7.95, 95% credible interval [7.70, 8.22]), the mean shape parameter (n) of the gamma distribution for the infectious period is 242 (median: 182, 95% credible interval [16, 482]), and the mean basic reproduction ratio (R0) is 2.91 (median: 2.91, 95% credible interval [2.83, 3.00]). CONCLUSIONS: This study shows that Salmonella Kentucky in this herd was of mild infectiousness and had a long infectious period, which together provide an explanation for the observed prevalence pattern after invasion. The transmission rate and the recovery rate parameters are inferred with better accuracy than the shape parameter, therefore these two parameters are more sensitive to the model and the observed data. The estimated shape parameter (n) has large variability with a minimal value greater than one, indicating that the infectious period of Salmonella Kentucky in dairy cattle does not follow the conventionally assumed exponential distribution.

Regional distribution of two dairy-associated Salmonella enterica serotypes.

Salmonella enterica is a zoonotic pathogen that is often associated with dairy farms. The organism can cause disease in cows but is also frequently shed in large numbers by dairy cows that are asymptomatic. Long-term asymptomatic infections with serotypes Cerro and Kentucky were previously identified in cows on a 100-head dairy farm in Pennsylvania, United States (focal dairy). Milk filters were collected from farms within 30 miles of the focal dairy to determine whether the infections by Cerro and Kentucky were limited to the focal dairy or whether the infection might be more regional in nature. Analysis of milk filters showed that Cerro and Kentucky were widespread in the surrounding region with 16 of 39 farms (41%) positive for one or both serotypes. Pulsed-field gel electrophoresis showed that the milk filter Kentucky strains shared >90% similarity with strains from the focal dairy and from local streams. Although there was more variation between Cerro strains (>80% similarity), most milk filter Cerro isolates from most milk filters were highly similar (>90%) to strains isolated from the focal dairy and local streams. In this intensely dairy-farmed region, Salmonella infection of dairy cows appears to be regional in nature, a fact that will impact efforts to control these pathogens.

Dynamics of Salmonella serotype shifts in an endemically infected dairy herd.

Salmonella is a leading cause of foodborne illness in the United States. It is a zoonotic pathogen found in many species of food animals, and contamination of foodstuffs by strains of Salmonella found on farms is an important source of human exposure. Here we describe a long-term (2004-2010) study of Salmonella colonization on a typical dairy farm in the Northeastern United States. The fecal shedding prevalence in the herd ranged from 8% to 97%, and greater than 50% of the herd was shedding Salmonella for more than two-thirds of the study period. Salmonella enterica serotype Cerro was first detected in September 2004, after a small and very short-lived outbreak of Salmonella Kentucky. Cerro persisted within the herd for over 3 years, with no clinical signs of salmonellosis in the animals. In the winter of 2006, Kentucky was again detected within the herd, and over a 2-year period, Kentucky gradually supplanted Cerro. Kentucky was the only serotype detected from March 2008 until September 2009, when Cerro was again detected in 15% of the cows on the farm. Since September 2009, Kentucky and Cerro have coexisted within the herd, which continues to harbor these serotypes at high prevalence. Pulsed-field gel electrophoresis (PFGE) could not discern differences between Cerro strains isolated during this study, but it did suggest that the strain of Kentucky that seemed to behave as a commensal in these dairy cows is distinct from the transient strain isolated in 2004. Understanding the dynamics of competition between these two serotypes that seem to behave as commensal colonizers of dairy cows may provide insights into the mechanisms by which Salmonella establishes infection in the lower gut of dairy cows and may lead to the development of measures to prevent or limit Salmonella colonization of dairy cows.

Effects of temperature on bacterial transport and destruction in bioretention media: field and laboratory evaluations.

Microbial activities are significantly influenced by temperature. This study investigated the effects of temperature on the capture and destruction of bacteria from urban stormwater runoff in bioretention media using 2-year field evaluations coupled with controlled laboratory column studies. Field data from two bioretention cells show that the concentration of indicator bacteria (fecal coliforms and Escherichia coli) was reduced during most storm events, and that the probability of meeting specific water quality criteria in the discharge was increased. Indicator bacteria concentration in the input flow typically increased with higher daily temperature. Although bacterial removal efficiency was independent of temperature in the field and laboratory, column tests showed that bacterial decay coefficients in conventional bioretention media (CBM) increase exponentially with elevated temperature. Increases in levels of protozoa and heterotrophic bacteria associated with increasing temperature appear to contribute to faster die-off of trapped E. coli in CBM via predation and competition.

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis in a longitudinal study of three dairy herds.

The objective of this study was to evaluate whether cows that were low shedders of Mycobacterium avium subsp. paratuberculosis were passively shedding or truly infected with M. avium subsp. paratuberculosis. We also investigated whether it is possible that these M. avium subsp. paratuberculosis-infected animals could have been infected as adults by contemporary high-shedding animals (supershedders). The M. avium subsp. paratuberculosis isolates were obtained from a longitudinal study of three dairy herds in the northeastern United States. Isolates were selected from fecal samples and tissues at slaughter from all animals that were culture positive at the same time that supershedders were present in the herds. Shedding levels (CFU of M. avium subsp. paratuberculosis/g of feces) for the animals at each culture-positive occasion were determined. Using a multilocus short-sequence-repeat technique, we found 15 different strains of M. avium subsp. paratuberculosis from a total of 142 isolates analyzed. Results indicated herd-specific infection patterns; there was a clonal infection in herd C, with 89% of isolates from animals sharing the same strain, whereas herds A and B showed several different strains infecting the animals at the same time. Tissues from 80% of cows with at least one positive fecal culture (other than supershedders) were culture positive, indicating a true M. avium subsp. paratuberculosis infection. The results of M. avium subsp. paratuberculosis strain typing and observed shedding levels showed that at least 50% of low shedders have the same strain as that of a contemporary supershedder. Results of this study suggest that in a dairy herd, more of the low-shedding cows are truly infected with M. avium subsp. paratuberculosis than are passively shedding M. avium subsp. paratuberculosis. The sharing of strains between low shedders and the contemporary supershedders suggests that low shedders may have been infected by environmental exposure of M. avium subsp. paratuberculosis.

Increased in vitro adherence and on-farm persistence of predominant and persistent Listeria monocytogenes strains in the milking system.

Dairy farms are a reservoir for Listeria monocytogenes, and the reduction of this pathogen at the farm level is important for reducing human exposure. The objectives of this research were to study the diversity of L. monocytogenes strains on a single dairy farm, assess strain dynamics within the farm, identify potential sources of L. monocytogenes in bulk tank milk and milk filters, and assess the adherence abilities of representative strains. A total of 248 L. monocytogenes isolates were analyzed by pulsed-field gel electrophoresis (PFGE). Combined AscI and ApaI restriction analysis yielded 40 PFGE types (strains). The most predominant strains were T (28.6%), D (22.6%), and F (14.9%). A high level of heterogeneity of strains among isolates from fecal (Simpson's index of diversity [SID] = 0.96) and environmental (SID = 0.96) samples was observed. A higher homogeneity of strains was observed among isolates from milk filters (SID = 0.71) and bulk tank milk (SID = 0.65). Six of 17 L. monocytogenes isolates (35.3%) were classified in an in vitro assay as having a "low adherence ability," 9 (52.9%) were classified as having a "medium adherence ability," and 2 (11.8%) were classified as having a "high adherence ability." The L. monocytogenes strains that were predominant and persistent showed significantly better adherence than did strains that were only sporadic, predominant, or persistent (P = 0.0006). Our results suggest that the milking system was exposed to several L. monocytogenes strains from different sources. Only 3 strains, however, were successful in persisting within the milking system, suggesting that some strains are more suitable to that particular ecological environment than others.

Correlation between Herrold egg yolk medium culture and real-time quantitative polymerase chain reaction results for Mycobacterium avium subspecies paratuberculosis in pooled fecal and environmental samples.

Real-time quantitative polymerase chain reaction (qPCR) testing for Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples is a rapid alternative to culture on Herrold egg yolk medium (HEYM), the traditional antemortem reference test for MAP. Although the sensitivity and specificity of these 2 tests have been estimated based on dichotomized test results, the correlation between real-time qPCR threshold cycle (Ct) values and colony-forming units (CFU) on HEYM for fresh and thawed samples has not been evaluated. The objectives of the present study were to estimate the correlation and association between Ct and CFU in fresh and thawed pooled fecal and environmental samples. Results of HEYM culture of 1,997 pooled fecal samples from cows in 14 herds, and 802 environmental samples from 109 dairies nationwide were negatively (inversely) correlated with their respective real-time qPCR results. The Spearman's rank correlation between Ct and CFU was good (-0.66) in fresh and thawed pooled fecal samples, and excellent (-0.76) and good (-0.61) in fresh and thawed environmental samples, respectively. The correlation varied from good (-0.53) to excellent (-0.90) depending on the number of samples in a fecal pool. Truncated regression models indicated a significant negative association between Ct and CFU in fecal pools and environmental samples. The use of real-time qPCR instead of HEYM can yield rapid, quantitative estimates of MAP load and allow for incorporation of real-time qPCR results of pooled and environmental samples in testing strategies to identify dairy cow groups with the highest MAP shedding.

The capture and destruction of Escherichia coli from simulated urban runoff using conventional bioretention media and iron oxide-coated sand.

The performance, sustainability, and mechanisms of bacterial removal from stormwater runoff by bioretention systems are poorly understood. The potential for removal of microorganisms in bioretention systems was evaluated using column studies and simulated urban stormwater runoff. Conventional bioretention media (CBM) removed 82% of Escherichia coli O157:H7 strain B6914 cells; iron-oxide coated sand (IOCS) significantly enhanced capture, with 99% efficiency. This improvement possibly was because of the greater positive surface charge and roughness, of the IOCS. Trapped strain B6914 cells decayed more rapidly in CBM, however, with more than 99.98% die-off within one week compared with the IOCS in which approximately 48% of trapped cells survived. Predation and competition from native microorganisms in CBM were verified to play a dominant role in rapid destruction of trapped strain B6914. In particular, protozoan grazing appeared to play an important role, with the die-off of trapped B6914 increasing with increasing concentrations of protozoa.

Molecular ecology of Listeria monocytogenes: evidence for a reservoir in milking equipment on a dairy farm.

A longitudinal study aimed to detect Listeria monocytogenes on a New York State dairy farm was conducted between February 2004 and July 2007. Fecal samples were collected every 6 months from all lactating cows. Approximately 20 environmental samples were obtained every 3 months. Bulk tank milk samples and in-line milk filter samples were obtained weekly. Samples from milking equipment and the milking parlor environment were obtained in May 2007. Fifty-one of 715 fecal samples (7.1%) and 22 of 303 environmental samples (7.3%) were positive for L. monocytogenes. A total of 73 of 108 in-line milk filter samples (67.6%) and 34 of 172 bulk tank milk samples (19.7%) were positive for L. monocytogenes. Listeria monocytogenes was isolated from 6 of 40 (15%) sampling sites in the milking parlor and milking equipment. In-line milk filter samples had a greater proportion of L. monocytogenes than did bulk tank milk samples (P<0.05) and samples from other sources (P<0.05). The proportion of L. monocytogenes-positive samples was greater among bulk tank milk samples than among fecal or environmental samples (P<0.05). Analysis of 60 isolates by pulsed-field gel electrophoresis (PFGE) yielded 23 PFGE types after digestion with AscI and ApaI endonucleases. Three PFGE types of L. monocytogenes were repeatedly found in longitudinally collected samples from bulk tank milk and in-line milk filters.

Integrating waveguide biosensor.

The Integrating Waveguide Biosensor was developed for rapid and sensitive detection of bacterial cells, spores, and toxins. A sandwich format of immunoassay was employed using Salmonella as model. The analyte was immunocaptured on the inner surface of the waveguide and then detected by the antibody conjugated with fluorescent dye. The waveguide was illuminated by an excitation light at a 90 degrees angle. The emitted light from fluorescent labels on the surface of the waveguide was efficiently collected and channeled to a detector at the end of the waveguide, while minimizing interference from the excitation light. Utilizing fluorescent dye Cy5, a 635-nm diode laser for excitation, and a photomultiplier tube detector, the Integrating Waveguide Sensor System was able to detect approximately ten captured cells of Salmonella.