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Longitudinal data from 3 commercial dairy herds in the northeast United States, collected from 2004 to 2011, were analyzed to determine the effect of Mycobacterium avium ssp. paratuberculosis (MAP) infection status and progression path on milk production. Disease status, as indicated by MAP test results, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues and feces at slaughter. Milk production data were collected from the Dairy Herd Information Association. Animals with positive MAP test results were categorized, based on test results over the full course of the study, as high path (at least one high-positive culture) or low path (at least one positive culture or ELISA). The cumulative numbers of positive ELISA and culture results were recorded. The effects of both MAP infection path, status, and number of positive tests on milk production were analyzed using a mixed linear model with an autocorrelation random effect structure. Low- and high-path animals produced more milk before their first positive test than always-negative animals, especially high-path animals. Although mean production decreased after a first positive test, low-path animals were shown to recover some productivity. High-path animals continued to exhibit a decrease in milk production, especially after their first high-positive fecal culture. These results show that not all animals that test positive for MAP will have long-term production losses. Milk production decreased significantly with each additional positive test. Ultimately, production loss appeared to be a function of MAP infection progression., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Salmonella enterica commonly colonizes the intestinal tract of cattle and is a leading cause of foodborne illness. A previously described investigation into the prevalence of S. enterica on a dairy farm revealed an 8-year-long asymptomatic S. enterica epidemic caused by serotypes Cerro and Kentucky in the lactating herd. To investigate the source of the S. Kentucky strains, the genomes of two S. Kentucky isolates were sequenced; one collected prior to the epidemic (2004) and one collected during the epidemic (2010). Comparative genomic analysis demonstrated significant polymorphisms between the two strains. PCR primers targeting unique and strain-specific regions were developed, and screening of the archived isolates identified the index case of the asymptomatic S. Kentucky epidemic as a heifer that was raised off-site and transported onto the study farm in 2005. Analysis of isolates collected from all heifers brought onto the farm demonstrated frequent re-introduction of clones of the epidemic strain suggesting transmission of pathogens between farms might occur repeatedly.

Salmonella isolates were recovered from bulk tank milk as part of the National Animal Health Monitoring System (NAHMS) Dairy 2002 and 2007 surveys. In-line milk filters were also tested in the 2007 survey. The objective of this study was to determine the prevalence of antimicrobial resistance among Salmonella enterica isolates from bulk milk and milk filters in the NAHMS Dairy 2002 and 2007 surveys and to further characterize resistant isolates. Susceptibilities to 15 antibiotics were determined for 176 Salmonella isolates of 26 serotypes using an automated antimicrobial susceptibility system. Resistant isolates were screened by PCR for the presence of the extended-spectrum β-lactamase (bla(CMY)) gene and class I integrons and further characterized by pulsed-field gel electrophoresis. Thirty isolates (17.0%) representing six S. enterica serotypes exhibited resistance to at least one antimicrobial agent (serotypes Newport [14 of 14 isolates exhibited resistance], Dublin [7 of 7], Typhimurium [3 of 5], Kentucky [4 of 22], Anatum [1 of 13], and Infantis [1 of 2]). Twenty isolates (11.4%), including all 14 Newport, 3 Dublin, 2 Typhimurium, and 1 Infantis isolate, displayed the typical multidrug-resistant, bla(CMY)-positive (MDR-AmpC) phenotype which included resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline, plus resistance to amoxicillin-clavulanic acid and extended-spectrum cephalosporins. Five of the MDR-AmpC isolates carried class I integrons (2.8%). Two-enzyme (XbaI and BlnI) pulsed-field gel electrophoresis discerned clades within serotypes and, together with the resistance profiles, identified strains that appeared to have persisted temporally and geographically. These results suggest that there is a low but appreciable risk of infection with MDR Salmonella from consumption of nonpasteurized milk and dairy products.

The enteric pathogen Salmonella enterica is one of the leading causes of foodborne illness in the world. The species is extremely diverse, containing more than 2,500 named serovars that are designated for their unique antigen characters and pathogenicity profiles-some are known to be virulent pathogens, while others are not. Questions regarding the evolution of pathogenicity, significance of antigen characters, diversity of clustered regularly interspaced short palindromic repeat (CRISPR) loci, among others, will remain elusive until a strong evolutionary framework is established. We present the first large-scale S. enterica subsp. enterica phylogeny inferred from a new reference-free k-mer approach of gathering single nucleotide polymorphisms (SNPs) from whole genomes. The phylogeny of 156 isolates representing 78 serovars (102 were newly sequenced) reveals two major lineages, each with many strongly supported sublineages. One of these lineages is the S. Typhi group; well nested within the phylogeny. Lineage-through-time analyses suggest there have been two instances of accelerated rates of diversification within the subspecies. We also found that antigen characters and CRISPR loci reveal different evolutionary patterns than that of the phylogeny, suggesting that a horizontal gene transfer or possibly a shared environmental acquisition might have influenced the present character distribution. Our study also shows the ability to extract reference-free SNPs from a large set of genomes and then to use these SNPs for phylogenetic reconstruction. This automated, annotation-free approach is an important step forward for bacterial disease tracking and in efficiently elucidating the evolutionary history of highly clonal organisms.

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be used as a tool in understanding the level of MAP infection in a herd or pen, it did not appear to be a sensitive diagnostic method for herd positivity in these low prevalence herds, and its use may require caution., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (Map), is a fatal disease in cattle. The objective of this study was to identify loci associated with tolerance in cows infected with Map. Tolerance was defined as a cow's fitness at a given level of Map infection intensity. Fitness was measured by Map faecal cultures, and Map infection intensity was measured by culturing four gut tissues. The quantitative phenotype of tolerance was defined by numerical indexes of cultures of peak (peak tolerance, PT) and average (average tolerance, AT) faecal and tissue Map from 245 Holstein cows. The categorical phenotype was defined as: ≥ 100 cfu Map tissue infection, and faecal shedding ≥ 75 cfu (intolerant) or <10 cfu (tolerant cows). In 94 cows, Map was identified in ≥ 1 tissue, including 44 cows with ≥ 100 Map tissue cfu and 36 with ≥ 1 faecal cfu. A genome-wide association analysis was performed after filtering, leaving genotypes for 45,789 SNPs in 90 animals for the quantitative phenotype and 16 cases and 25 controls for the categorical analysis of tolerance. rs41748405:A>C (BTA15) was associated with PT (P = 1.12 × 10(-7)) and AT (P = 2.17 × 10(-6)). Associations were identified with PT and adjacent SNPs ss61512613:A>G and ss61530518:A>G (BTA6) (P < 3.0 × 10(-5)), and with AT for ss61469568:A>G (BTA 2) (P = 3.3 × 10(-5)) and ss86284768:A>G (BTA1) (P = 3.31 × 10(-5)). For the categorical phenotype, an association was found with ss8632653:A>G (BTA6) (P < 5.0 × 10(-5)). This is the first study to identify loci associated with tolerance to Johne's disease., (© 2010 The Authors, Animal Genetics © 2010 Stichting International Foundation for Animal Genetics.)

Among the costs attributed to Mycobacterium avium ssp. paratuberculosis (MAP) infection in dairy cattle, the effects on reproduction and culling are the least documented. To estimate the cost of MAP infections and Johne's disease in a dairy herd, the rates of calving and culling were calculated for cows in each stage of MAP infection relative to uninfected cows. Data from 6 commercial dairy herds, consisting of 2,818 cows with 2,754 calvings and 1,483 cullings, were used for analysis. Every cow in each study herd was tested regularly for MAP, and herds were followed for between 4 and 7 yr. An ordinal categorical variable for Johne's disease status [test-negative, low-positive (low-shedding or ELISA-positive only), or high-shedding] was defined as a time-dependent variable for all cows with at least 1 positive test result or 2 negative test results. A Cox regression model, stratified on herd and controlling for the time-dependent infection variable, was used to analyze time to culling. Nonshedding animals were significantly less likely to be culled in comparison with animals in the low-shedding or ELISA-positive category, and high-shedding animals had nonsignificantly higher culling rates than low-shedding or ELISA-positive animals. Time to calving was analyzed using a proportional rates model, an analog to the Andersen-Gill regression model suitable for recurrent event data, stratifying on herd and weighted to adjust for the dependent censoring caused by the culling effects described above. High-shedding animals had lower calving rates in comparison with low-shedding or ELISA-positive animals, which tended to have higher calving rates than test-negative animals., (Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to assess the presence of a Listeria monocytogenes-containing biofilm in milking equipment as a potential source of bulk tank milk contamination on a dairy farm where milk contamination had been previously documented. Samples were collected from milking equipment and milking parlor premises on 4 occasions and analyzed for the presence of L. monocytogenes. Pulsed-field gel electrophoresis (PFGE) typing was conducted on L. monocytogenes isolates from the milking equipment, parlor and storage room floors, bulk tank milk, and in-line milk filters. Pieces from milk meters and rubber liners were obtained to visually assess the presence of a biofilm using scanning electron microscopy. A total of 6 (15%), 4 (25%), and 1 (6%) samples were culture-positive for L. monocytogenes in the first, second, and third sample collection, respectively. Two samples were L. monocytogenes hly PCR-positive but were culture-negative in the fourth sample collection. Combined AscI and ApaI restriction analysis yielded 6 PFGE types for 15 L. monocytogenes isolates obtained from milking equipment, parlor, bulk tank milk, and milk filters. A predominant and persistent PFGE type (PFGE type T) was observed among these L. monocytogenes isolates (9/15 isolates). Scanning electron microscopy of samples from the bottom cover of 2 milk meters showed the presence of individual and clusters of bacteria, mainly associated with surface scratches. The presence of a bacterial biofilm was observed on the bottom covers of the 2 milk meters. Prevention of the establishment of biofilms in milking equipment is a crucial step in fulfilling the requirement of safe, high-quality milk., (2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

This study investigated the effect of somatic cell count (SCC) in goat milk on yield, free fatty acid (FFA) profile, and sensory quality of semisoft cheese. Sixty Alpine goats without evidence of clinical mastitis were assigned to 3 groups with milk SCC level of <500,000 (low), 500,000 to 1,000,000 (medium), and 1,000,000 to 1,500,000 (high) cells/mL. Thirty kilograms of goat milk with mean SCC levels of 410,000 (low), 770,000 (medium), and 1,250,000 (high) cells/mL was obtained for the manufacture of semisoft cheese for 2 consecutive weeks in 3 lactation stages. The composition of milk was analyzed and cheese yield was recorded on d 1. Cheese samples on d 1, 60, and 120 were analyzed for total sensory scores, flavor, and body and texture by a panel of 3 expert judges and were also analyzed for FFA. Results indicated that milk composition did not change when milk SCC varied from 214,000 to 1,450,000 cells/mL. Milk with higher SCC had a lower standard plate count, whereas coliform count and psychrotrophic bacteria count were not affected. However, milk components (fat, protein, lactose, casein, and total solids) among the 3 groups were similar. As a result, no significant differences in the yield of semisoft goat cheeses were detected. However, total sensory scores and body and texture scores for cheeses made from the high SCC milk were lower than those for cheeses made from the low and medium SCC milks. The difference in milk SCC levels also resulted in diverse changes in cheese texture (hardness, springiness, and so on) and FFA profiles. Individual and total FFA increased significantly during ripening, regardless the SCC levels. It is concluded that SCC in goat milk did not affect the yield of semisoft cheese but did result in inferior sensory quality of aged cheeses., (Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The purpose of this study was to identify loci associated with Mycobacterium avium subspecies paratuberculosis (Map) infection status in US Holsteins using the Illumina BovineSNP50 BeadChip whole genome single nucleotide polymorphism (SNP) assay. Two hundred forty-five cows from dairies in New York, Pennsylvania and Vermont enrolled in longitudinal herd studies between January 1999 and November 2007 were assessed for the presence of Map in both faecal and tissue samples. An animal was considered tissue infected if any sample contained at least one colony forming unit of Map per gram of tissue (CFU/g) and the same definition was employed for faecal samples. Each animal was genotyped with the Illumina BovineSNP50 BeadChip and after quality assurance filtering, 218 animals and 45 683 SNPs remained. We sought to identify loci associated with four different case/control classifications: presence of Map in the tissue, presence of Map in faeces, presence of Map in both tissue and faeces and presence of Map in tissue but not faeces. A case-control genome wide association study was conducted to test the four different classifications of Map infection status (cases) when compared with a Map-negative control group (control). Regions on chromosomes 1, 5, 7, 8, 16, 21 and 23 were identified with moderate significance (P < 5 x 10(-5)). Two regions, one on chromosome 3 (near EDN2) and another on chromosome 9 (no positional gene candidates), were identified with a high level of association to the presence of Map in tissue and both tissue and faeces respectively (P < 5 x 10(-7), genome-wide Bonferonni P < 0.05).

Longitudinal data from 3 commercial dairy herds in the northeast United States were collected from 2004 to 2007. Johne's disease status, as indicated by Mycobacterium avium ssp. paratuberculosis infection levels, was determined through quarterly ELISA serum testing, biannual fecal culture, and culture of tissues at slaughter. Milk production data were collected from the Dairy Herd Improvement Association. The effect of Johne's disease status on milk production was analyzed using a mixed linear model with an autocorrelation random effect structure. Infected animals produced more milk than uninfected cows before they began shedding M. avium ssp. paratuberculosis. Cows infected with M. avium ssp. paratuberculosis had monthly decreases of 0.05 to 1 kg in daily milk production relative to uninfected animals, with greater decreases in progressive disease categories. Animals with fecal culture results of >30 cfu/g produced approximately 4 kg less milk per day compared with uninfected cows. These results will be valuable in calculating the economic effect of Johne's disease.

Endemic infectious diseases in dairy cattle are of significant concern to the industry as well as for public health because of their potential impact on animal and human health, milk and meat production, food safety, and economics. We sought to provide insight into the dynamics of important endemic infectious diseases in 3 northeastern US dairy herds. Fecal samples from individual cows and various environmental samples from these farms were tested for the presence of major zoonotic pathogens (i.e., Salmonella, Campylobacter, and Listeria) as well as commensal bacteria Escherichia coli and enterococci. Additionally, the presence of Mycobacterium avium ssp. paratuberculosis was tested in fecal and serum samples from individual cows. Test results and health and reproductive records were maintained in a database, and fecal, plasma, DNA, and tissue samples were kept in a biobank. All bacteria of interest were detected on these farms and their presence was variable both within and between farms. The prevalence of Listeria spp. and L. monocytogenes in individual fecal samples within farm A ranged from 0 to 68.2% and 0 to 25.5%, respectively, over a period of 3 yr. Within farm B, continuous fecal shedding of Salmonella spp. was observed with a prevalence ranging from 8 to 88%; Salmonella Cerro was the predominant serotype. Farm C appeared less contaminated with Salmonella and Listeria, although in the summer of 2005, 50 and 19.2% of fecal samples were positive for Listeria and L. monocytogenes, respectively. The high prevalence of E. coli (89 to 100%), Enterococcus (75 to 100%), and Campylobacter (0 to 81%) in feces suggested they were ubiquitous throughout the farm environment. Fecal culture and ELISA results indicated a low prevalence of Mycobacterium avium ssp. paratuberculosis infection in these farms (0 to 13.6% and 0 to 4.9% for culture-positive and ELISA-positive, respectively), although the occasional presence of high shedders was observed. Results have major implications for food safety and epidemiology by providing a better understanding of infectious disease dynamics on dairy farms. Comprehensive understanding of these infections may lead to better farm management practices and pathogen reduction programs to control and reduce the on-farm contamination of these pathogens and to prevent their further entry into the food-chain.

The objective of the study was to screen a large number of herd management practices and herd characteristics from US dairies to identify herd-level risk factors associated with the presence of Listeria monocytogenes in bulk tank milk (BTM). A total of 71 variables was univariately evaluated for their association with the presence of L. monocytogenes in BTM. Results from the univariate analysis indicated that using automatic take offs and having an open herd management increased the risk of BTM contamination with L. monocytogenes, while storing manure in outside pens not accessible to cattle decreased the risk. These variables, however, were not sustained in the multivariable model, which indicated that the presence of L. monocytogenes in BTM was significantly associated with region of the country (farms in the southeast and northeast were six and four times more likely respectively, to have BTM contamination than farms in the west) and number of milking cows (herds with >500 milking cows were five times more likely to have BTM contamination than herds with <100 milking cows). In conclusion, our results suggest that risk factors associated with BTM contamination are different depending on the geographical region and herd size of the operation.

Although dairy cattle are known reservoirs for salmonellae, cattle that are shedding this organism are often asymptomatic and difficult to identify. A dairy herd that was experiencing a sustained, subclinical outbreak of Salmonella enterica subsp. enterica Cerro was monitored for 2 years. Fecal samples from the lactating cows were collected every 6 to 8 weeks and tested for the presence of Salmonella. Fecal prevalence of Salmonella fluctuated throughout the observation period and ranged from 8 to 88%. Manure composites and water trough samples were collected along with the fecal samples, and bulk milk and milk filters were cultured for the presence of Salmonella on a weekly basis. Over 90% of the manure composites--representing high-animal-traffic areas-were positive at each sampling. Salmonella was detected in 11% of milk samples and in 66% of the milk filters. Results of weekly bulk milk quality testing (i.e., bulk tank somatic cell score, standard plate count, preliminary incubation count) were typically well within acceptable ranges. Milk quality variables had low correlations with herd Salmonella fecal prevalence. When observed over time, sampling period average prevalence of Salmonella in milk filters closely paralleled fecal prevalence of Salmonella in the herd. Based on results of this study, milk filters appear to be an effective method for monitoring shedding prevalence at the herd level. In-line filter testing is also a more sensitive measure of Salmonella, and perhaps other pathogens, in raw milk than testing the milk alone.

Johne's disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection and results in economic losses in the dairy industry. To control MAP transmission in herds, test-based culling has been recommended and immediate culling of high shedding animals is typically implemented. In this study, we quantified the effects of MAP control in US dairy herds, using the basic reproduction ratio R(0). The effectiveness of culling strategies was evaluated for good and poor herd management (low- and high-transmission rates, respectively) by a phase diagram approach. To establish a quantitative relationship between culling rates and test properties, we defined the average detection times for low and high shedding animals. The effects of various culling strategies and test characteristics, such as test sensitivity, test turnaround time, and testing interval, were analyzed. To understand the overall effect of model parameters on R(0), we performed global uncertainty and sensitivity analyses. We also evaluated the effectiveness of culling only high shedding animals by comparing three test methods (fecal culture, fecal polymerase chain reaction, PCR, and enzyme-linked immunosorbent assay, ELISA). Our study shows that, in the case of good herd management, culling of only high shedding animals may be effective in controlling MAP transmission. However, in the case of poor management, in addition to immediate culling of high shedding animals, culling of low shedding animals (based on the fecal culture test) will be necessary. Culling of low shedding animals may be delayed 6-12 months, however, if a shorter testing interval is applied. This study suggests that if farmers prefer culling only high shedding animals, faster MAP detection tests (such as the fecal PCR and ELISA) of higher sensitivity should be applied with high testing frequency, particularly on farms with poor management. Culling of infectious animals with a longer testing interval is generally not effective to control MAP.

We developed a mathematical model of the transmission dynamics of salmonella to describe an outbreak of S. Cerro infection that occurred in a Pennsylvania dairy herd. The data were collected as part of a cooperative research project between the Regional Dairy Quality Management Alliance and the Agricultural Research Service. After the initial detection of a high prevalence of S. Cerro infection in the herd, a frequent and intensive sampling was conducted and the outbreak was followed for 1 year. The data showed a persistent presence of S. Cerro with a high prevalence of infection in the herd. The dynamics of host and pathogen were modelled using a set of nonlinear differential equations. A more realistically distributed (gamma-distributed) infectious period using multiple stages of infection was considered. The basic reproduction number was calculated and relevance to the intervention strategies is discussed.

Salmonellae are a major group of foodborne pathogens known to affect both humans and animals. Dairy cattle are a known reservoir of these bacteria and human Salmonella infections have been associated with the consumption of improperly processed or contaminated dairy products. Many of the over 2500 known serotypes of Salmonella are known to infect cattle, resulting in asymptomatic to fatal salmonellosis. This study describes the course of a Salmonella outbreak and subsequent endemic infection on a dairy farm in Pennsylvania. The outbreak was initially detected when a few cows with clinical symptoms and one fatality were found to be infected with Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagan. Based upon sampling of the farm environment, Salmonella Typhimurium var. Copenhagan was succeeded within 3 months by Salmonella enterica subsp. enterica serovar Kentucky. Salmonella enterica subsp. enterica serovar Cerro ultimately supplanted Typhimurium var. Copenhagan and Kentucky in individual animals and environmental samples and persisted in the herd at high prevalence for almost 2 years. Since there were no obvious clinical consequences of the Salmonella Cerro infection, these data suggest that some serotypes of S. enterica subsp. enterica can behave as commensal organisms in dairy cattle and illustrate the difficulties of controlling Salmonella in milk production systems. The consistent finding of Salmonella in the environment reinforces the potential for human exposure to this pathogen and the need to understand the dynamics and ecology of Salmonella in dairy production settings.

Aims: To compare survival of Escherichia coli and faecal coliforms (FC) in bovine faeces deposited in a pasture or incubated in a controlled laboratory environment at temperatures within the same range., Methods and Results: Faecal samples from three cow herds were deposited as shaded and nonshaded cowpats in a field and incubated in a laboratory for one month at 21.1, 26.7 and 32.2 degrees C. Both FC and E. coli concentrations increased as much as 1.5 orders of magnitude both in the field and in the laboratory during the 1st week and subsequently decreased. In shaded cowpats, the die-off of E. coli and FC was significantly slower, and the proportion of E. coli in FC was significantly larger as compared with nonshaded cowpats. The die-off was faster in the field than in the laboratory at similar temperatures., Conclusions: FC and E. coli die-off rates were substantially lower in laboratory conditions than in the field within the same range of temperatures., Significance and Impact of the Study: This study underscores the importance of field data on survival of manure-borne FC and E. coli, and indicates that laboratory die-off rates have to be corrected to be used for field condition simulations.

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5 degrees C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (gamma-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA+, gamma-tir+, stx2+, rfbE+, fliC+, hlyA+) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable gamma-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk.

Aim: To test the hypothesis that Escherichia coli and enterococci bacteria have similar release rates and transport characteristics after being released from land-applied manure., Methods and Results: Turfgrass soil sod was placed into 200 cm long boxes that had the top two 25 cm sections separated to monitor the release and infiltration of bacteria, which affected bacteria transport in the rest of the box. Dairy manure with added KBr was broadcast on the top two sections. Boxes with either live or dead grass stand were placed under a rainfall simulator for 90 min. Runoff and infiltration samples were collected and analysed for Br, E. coli, enterococci and turbidity. Significant differences in release kinetics of E. coli and enterococci were found. A change from first-order release kinetics to zero-order kinetics after 1 h of rainfall simulation was observed., Conclusion: Differences in release rates but not in the subsequent transport were observed for E. coli and enterococci., Significance and Impact of the Study: Because both E. coli and enterococci are currently used as indicator organisms for manure-borne pathogens, the differences in their release rates may affect the efficiency of using these organisms as indicators.

Sixty-one Listeria monocytogenes strains from raw milk were analyzed with an automated repetitive element-based PCR (rep-PCR) system to examine the utility of this system for serotype grouping and to determine whether specific regional relationships could be identified. Results of the similarity analysis revealed two primary clusters of L. monocytogenes isolates. Cluster 2 exclusively contained serogroup 1/2a isolates; however, two 1/2a isolates were also found in cluster 1. Isolates of serogroups 1/2b, 4b, 3b, and 4c were also in cluster 1. Clusters 1 and 2 were separated at a relative similarity of 86%. Listeria species other than L. monocytogenes (L. ivanovii, L. seeligeri, L. welshimeri, L. grayi, and L. innocua) had similarity scores of less than 80% in pairwise comparisons with the L. monocytogenes isolates. Thus, this method may be useful for species identification once an isolate is characterized as Listeria. When rep-PCR fingerprints of the L. monocytogenes 1/2a isolates were compared, there was no apparent regional grouping. However, discrimination between isolates suggests that the rep-PCR assay might be useful for tracking L. monocytogenes 1/2a and for tracking isolates across regions or within smaller ecological niches. The automated rep-PCR method could not discriminate between serotypes 1/2b and 4b but may be useful for discriminating between 1/2a and other serotypes and for tracking isolates within serotype 1/2a.

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring System Dairy 2002 survey, were analyzed for the presence of Salmonella enterica using a commercially available real-time polymerase chain reaction (PCR) kit. Samples from 854 farms in 21 states were collected and enriched in tetrathionate broth to amplify any salmonellae present, and DNA was isolated from the resulting biomass. One hundred one samples (11.8%) were shown to contain Salmonella enterica using the real-time PCR assay, whereas conventional culture techniques detected the pathogen in only 22 (2.6%) of the samples. A conventional PCR assay targeting a different gene from Salmonella enterica confirmed the presence of the organism in 94 of the real-time PCR-positive samples. Thus, assay of milk samples by real-time PCR indicates that the prevalence of Salmonella enterica in US bulk tank milk is substantially higher than previously reported.

The objective of this study was to determine the prevalence of Salmonella, Listeria monocytogenes, and fecal coliforms in bulk tank milk in the United States. As part of the NAHMS Dairy 2002 survey, 861 bulk tank milk samples were collected from farms in 21 states. Milk was directly plated on selective agars for direct bacterial enumeration and was enriched in selective broths to increase detection sensitivity. Somatic cell counts (SCC) and standard plate counts (SPC) were also determined. Coliforms were detected in 95% (818 of 860) of the samples, and the average SCC was 295,000 cells/mL. Twenty-two samples (2.6%) were culture-positive for Salmonella, and 9 serotypes were identified: Montevideo (n = 7), Newport (n = 4), Muenster (n = 2), Meleagridis (n = 2), Cerro (n = 2), 44:Z36 (Z38) (n = 2), Dublin (n = 1), Anatum (n = 1), and 9, 12:nonmo-tile (n = 1). Listeria monocytogenes was isolated from 56 (6.5%) samples, and serotyping of these isolates yielded 5 serotypes (1/2a, 1/2b, 3b, 4b, and 4c). Of the L. monocytogenes isolates, 93% were serotypes 1/2a, 1/2b, and 4b, the most common human clinical isolates. Regional differences in L. monocytogenes and Salmonella prevalence were observed, but more studies are needed to determine the validity of these differences. There were no apparent relationships between SCC or SPC and incidence of Salmonella or L. monocytogenes. Although the prevalence of L. monocytogenes and Salmonella was low, these pathogens represent a potential risk to consumers of raw milk and raw milk products.

The purpose of this study was to determine the efficacy of a portable real-time PCR system in detecting Salmonella spp. in raw milk. The 200 bulk milk samples chosen for this study constituted a subset of the samples for a larger study; this subset contained 24 samples that were culture positive for Salmonella and 176 that were culture negative. Milk was both plated directly on selective agar and plated after enrichment in selective media. Presumptive Salmonella colonies were isolated by direct culturing of five samples, while Salmonella was isolated from the remaining 19 positive samples only after enrichment. Presumptive Salmonella isolates were serotyped, and isolates from 22 samples were confirmed to be Salmonella isolates. PCR assays of culture-positive milk prior to enrichment yielded no evidence of Salmonella. DNA extracts of bacterial pellets from the enriched samples were analyzed for Salmonella by real-time PCR with the Ruggedized Advanced Pathogen Identification Device (RAPID). Fifty-four samples from the enrichment pellets tested positive for Salmonella by real-time PCR. Two samples that tested positive for Salmonella by culture and serotyping tested Salmonella negative by real-time PCR. Serotyping identified isolates from these samples as Salmonella Montevideo. All DNA extracts of Salmonella Montevideo isolates tested positive for Salmonella by real-time PCR. Thirty-three samples tested negative by culture and positive by real-time PCR. These results indicate that the portable real-time PCR system appears to be a useful tool for detecting Salmonella in raw milk. Additionally, the combination of enrichment and real-time PCR techniques used in this study can yield results in 24 h, compared with the 48 to 72 h required for traditional culture.

Forty crossbred steers were used to determine the effects of carbohydrate supply site on the indigenous bacteria of the gastrointestinal tract. Steers were fitted with ruminal and abomasal infusion catheters and assigned randomly to one of eight groups in a complete randomized block design. The experimental period was 36 d. Treatments included: 1) a pelleted basal diet fed at 0.163 Mcal ME x (kg BW(0.75)) x 1 x d(-1) (LE); 2) the basal diet fed at 0.215 Mcal ME x (kg BW(0.75)) (-1) x d(-1) (HE); 3) the basal diet fed at 0.163 Mcal ME x (kg BW(0.75))(-1) x d(-1) with ruminal infusion of starch hydrolysate (SH) (RSH); 4) the basal diet fed at 0.163 Mcal ME x (kg BW(0.75))(-1) x d(-1) with abomasal infusion of SH (ASH); and 5) the basal diet fed at 0.163 Mcal ME x (kg BW(0.75))(-1) x d(-1) with abomasal infusion of glucose (AG). The total volume ofinfusate (5 kg x site(-1) x d(-1)) was equalized across treatments and infusion sites by infusion of water. Glucose and SH were infused at rates of 14.35 and 12.64 g x (kg BW(0.75)) x d(-1), respectively. Ruminal, cecal, and fecal samples were obtained on d 36. Ruminal pH was low (5.79) in LE steers and unaffected (P > 0.10) by increased energy intake or carbohydrate infusion. Cecal and fecal pH were 6.93 and 7.00, respectively, for LE steers. Increasing energy intake (P < 0.10) and the rate of carbohydrate infusion (P < 0.01) significantly decreased cecal and fecal pH compared with LE. Ruminal counts of anaerobic bacteria in LE steers were 8.99 log10 cells/g and abomasal carbohydrate infusion had no affect (P > 0.10) on these numbers. However, ASH and AG steers had approximately 1.5 log10 cells/g more (P < 0.01) cecal and fecal anaerobic populations. Ruminal, cecal, and fecal aerobic bacterial counts were 40, 22, and 23%, respectively, lower than anaerobic counts. Generally, aerobic counts responded similarly to the anaerobic counts. Less than 1% of the anaerobic bacteria enumerated in the rumen, cecum, and feces were coliforms, and 97% of the coliforms were Escherichia coli. Carbohydrate infusions resulted in only numerical increases in fecal coliform and E. coli concentrations (P > 0.10). Fecal E. coli were highly acid sensitive in all steers, with less than 1% surviving a 1-h exposure to low pH (2.0). This suggests that cecal or fecal pH is not a good indicator of acid resistance, and it supports the concept that there are other factors that may induce acid resistance.

The objective of this study was to investigate the feasibility of using near-infrared reflectance spectroscopy (NIRS) to determine nutrient concentrations in dairy manures. We assayed diverse dairy manures (n = 107), collected from dairy farms in the northeastern United States (CT, MD, NY, PA, and VA) by conventional means and NIRS for total C, total N, NH3-N, moisture, P, K, and pH. Samples were scanned from 400 to 2498 nm in polyethylene bags on a FOSS-NIR-Systems Model 6500 scanning monochromator equipped with a sample transport device. We developed calibrations using a one-out cross validation procedure under partial least-squares regression. Preliminary results showed that eight samples were outliers either because of inaccurate conventional analysis or because they were uncharacteristic (i.e., two samples had moisture content below 72%, while all others were above 78%). These outliers were removed from further consideration. Final calibration results with the remaining 99 samples demonstrated that NIRS can accurately determine the moisture (r2 = 0.945, root mean squared deviation or RMSD = 1.0%), total C (r2 = 0.950, RMSD = 0.40%), total N (r2 = 0.956, RMSD = 0.030%), and NH3-N (r2 = 0.967, RMSD = 0.013%) concentrations, but not P or K concentrations in dairy manures. In conclusion, NIRS was shown to be a viable alternative to conventional analysis procedures for determining moisture, total C, total N, and NH3-N in a very diverse set of dairy manures.

Manure nutrient analyses performed rapidly on the farm could be useful for nutrient management programs. The objective of this experiment was to evaluate six quick tests for their accuracy in estimating total manure N or NH4+-N. The quick tests included the hydrometer, electrical conductivity meter and pen, reflectometer, Agros N Meter, and Quantofix-N-Volumeter. The hydrometer was used to estimate total N, while the remaining tests were used to estimate NH4+-N. Samples (107) were collected from dairy farms in five northeastern states. Samples were analyzed for total N and NH4+-N by traditional laboratory methods and using each of the quick tests. Manure compositions ranged from 1.4 to 38.6% dry matter (DM), 0.9 to 9.5 kg/m3 total N, and 0.3 to 4.7 kg/m3 NH4+-N. The estimated concentration of total N or NH4+-N determined by each quick test was regressed against laboratory-determined values. The hydrometer did not estimate total N accurately. The strongest relationship for estimation of NH4+-N was with the Quantofix-N-Volumeter followed by the Agros N Meter, the reflectometer, and the electrical conductivity meter and pen. When the samples were split into high (>12%) and low (< or =12%) DM groups, in all cases the r2 for the regression equation was higher for the low DM group than for the high DM group. The Agros N Meter, the reflectometer, and the conductivity meter and pen did not perform well for the high DM group. These data indicate that several quick tests are viable options for measuring NH4+-N concentrations in dairy slurries and solids.

When mixed ruminal bacteria were starved in vitro for 24 h, cellular ATP decreased, but there was little change in cell protein. Starved ruminal bacteria derived most of their ATP from cellular polysaccharide. Because polysaccharide declined at a first-order rate of 23%/h, it was possible to estimate the endogenous polysaccharide utilization rate at various stages of starvation by multiplying the amount of utilizable polysaccharide remaining at each time point by 0.23. The bacteria initially had a rate of soluble carbohydrate fermentation that was > 717 micrograms of hexose equivalent/mg of protein per h. Starvation had little impact on the rate of soluble carbohydrate fermentation until 8 to 12 h, and the endogenous polysaccharide utilization rate was < 10 micrograms of hexose/mg of protein per h. The bacteria digested ball-milled cellulose at a rate of 24 micrograms of hexose/mg of protein per h for 8 to 12 h. Even bacteria that had been starved for 24 h fermented cellulose at a rate of 16 micrograms of hexose/mg of protein per h. The rate of methane production was initially 70 nmol of methane/mg of protein per min. Short periods of starvation (< 12 h) had little impact on methane production, but longer times caused an almost complete inhibition of methanogenesis. The rate of amino acid deamination was initially 31 nmol of ammonia/mg of protein per min, and the critical phase of starvation was again 8 to 12 h. Ruminal bacteria that were harvested at 24 h after feeding had 10-fold less polysaccharide than did bacteria that were harvested at 2 h after feeding, but this polysaccharide supported high rates of soluble carbohydrate and cellulose fermentation, deamination, and methane production.

The predominant ruminal bacteria that were obtained from a 10(8) dilution of ruminal fluid could be maintained as a mixed population for long periods as long as the bacteria were provided with a complex mixture of carbohydrates. Growth of predominant ruminal bacteria in carbohydrate-limited, ammonia-excess, continuous cultures (0.07/h) had a low requirement for maintenance energy, but the nongrowth energy dissipation of ammonia-limited, carbohydrate-excess, predominant ruminal bacteria was approximately 10-fold higher (0.96 vs. 0.09 mg of hexose equivalent/mg of protein per h, respectively). Mathematical derivations indicated that this additional nongrowth energy dissipation could be accommodated by an energy spilling function that was independent of the growth rate. Peptides and amino acids had little impact on the yield of carbohydrate-limited, ammonia-excess, continuous cultures (0.07/ h), but amino N greatly increased the growth rate and yield of excess-energy batch cultures. The change in growth rate and yield that was dependent on amino N indicated that the energy-excess batch cultures had the same capacity to spill energy as did the ammonia-limited, carbohydrate-excess, predominant ruminal bacteria (0.80 vs. 0.86 mg of hexose equivalent/mg of protein per h, respectively). When the energy-excess batch cultures were provided with amino N, the growth rate increased, the difference in anabolic and catabolic rates was smaller, and less energy was spilled.

A steady-state model of the production, absorption, passage, and concentration of ruminal VFA and pH is developed from published literature data and is structured to use the feed descriptions and inputs from the net carbohydrate and protein system. Included are the effects of pH on growth rate and yield of structural and non-structural carbohydrate-fermenting bacteria; production of acetate, propionate, butyrate, lactate, and methane; conversion of lactate to VFA; ruminal absorption of acids; and prediction of ruminal pH from dietary measures and from ruminal buffering and acidity. The root mean square error of predicted total VFA concentration was 12 mM. Individual VFA fractions were inadequately predicted. In a review of literature data, effective NDF (eNDF) provided a better correlation with ruminal pH than forage or NDF. Digestion rate of NDF remained at normal levels above pH 6.2, which corresponds to a minimum eNDF of 20% of dietary DM. Further research is needed to determine the individual VFA produced from carbohydrate fractions at various pH, the appropriateness of partitioning the starch and pectin carbohydrate pool into slowly and rapidly degraded fractions, and the effect on microbial yield, total tract digestibility, and predicted energy values of feeds.

Strain SR, a monensin-sensitive, ammonia-producing ruminal bacterium, grew rapidly on arginine and lysine, but only if sodium was present. Arginine transport could be driven by either an electrical potential or a chemical gradient of sodium. Arginine was converted to ornithine, and it appeared that ornithine efflux created a sodium gradient which in turn drove arginine transport. There was a linear decline in arginine transport as pH was decreased from 7.5 to 5.5, and the cells did not grow at a pH less than 6.0. The Eadie-Hofstee plot was biphasic, and arginine could also be taken by a high-capacity diffusion mechanism. Because arginine was a strong inhibitor of lysine transport and lysine was a weak inhibitor of arginine transport, it appeared that both lysine and arginine were taken up by an arginine-lysine carrier which had a preference for arginine. The rate of lysine fermentation was always proportional to the extracellular lysine concentration, and facilitated diffusion was the dominant mechanism of lysine transport. When SR was grown in continuous culture on arginine or lysine, the theoretical maximal growth yield was similar (13 g of cells per mol of ATP), but the apparent maintenance energy requirement for arginine was greater than lysine (9.4 versus 4.4 mmol of ATP per g of cells per h). On the basis of differences in yield and maintenance energy, it appeared that active arginine transport accounted for approximately 40% of the total ATP.