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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 ß-lactamase (blaCMY) 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, blaCMY-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 avinin 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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°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 (γ-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+, γ-tir+, stx2+, rfbE+, fliC+, hlyA+) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable γ-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. [ABSTRACT FROM AUTHOR]

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 I 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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:nonmotile (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. [ABSTRACT FROM AUTHOR]

Salmonella is a prominent foodborne pathogen in the US and dairy cattle have frequently been identified as reservoirs for this organism. Here we describe a long-term outbreak of Salmonella enterica subsp. enterica Cerro that was detected in a Pennsylvania dairy herd during routine monitoring as part of a longitudinal study of several dairy herds. An initial analysis of individual fecal grab samples from the milking herd indicated a very low prevalence of Salmonella (1/102) and Salmonella was not detected in 10 environmental samples. In a subsequent sampling 6 mo later, Salmonella was isolated from 43.5% (47/108) of the cows and 40% of collected environmental samples (10/25). The Salmonella isolated in the first sampling was serovar Typhimurium var. Copenhagen, however isolates from the second herd sampling were serovars Cerro (91.5%) and Kentucky (8.5%). Sporadic environmental sampling between these initial complete milking herd samplings yielded serotypes Kentucky, Typhimurium var. Copenhagen, and Cerro and clinical salmonellosis was observed in several cows shedding Typhimurium var. Copenhagen. The herd was sampled nine additional times over the next 14 mo and fecal prevalence of Salmonella ranged from 8.4% to 89%. Prevalence remained high (58 to 69%) over the following 8 mo, showed evidence of decline over the next 3 mo, and then increased again to 89%. Cerro was the only serotype isolated during this period. Bulk milk was cultured weekly during this 22-mo period and Salmonella was isolated from 5.3% (5/94) of milk samples. For the last 14 mo of the study, milk filters were also cultured for Salmonella on a weekly basis and 65% (37/57) of the filters tested positive. There were no significant health or production concerns in the herd during this outbreak. Based on these observations we conclude that sub-clinical Salmonella enterica subsp. enterica Cerro infection can be persistent in a milking herd. [ABSTRACT FROM AUTHOR]

The objective of this research was to determine the range of colonyforming units (cfu) per gram of Mycobacterium avium paratuberculosis (MAP) in manure from cows previously classified as heavy shedders, and the role of these animals in Johne's Disease (JD) in dairy herds. Cattle are typically classified as light, moderate or heavy shedders based on the number of MAP colonies seen on Herrold's egg yolk media (HEYM). Laboratories routinely report the number of cfu up to 70 per HEYM tube. (Seventy cfu corresponds to an estimated 1,470 MAP cfu per gram of manure.) Counts above 70 are reported as 'too numerous to count' and these cows are classified as heavy shedders. Preliminary investigation found that 1:100 and 1:1,000 dilutions of feces from heavy shedders were appropriate for enumerating the actual concentration of MAP in these samples. Subsequently, more than 200 fecal samples from animals currently identified as heavy shedders were diluted and cultured on HEYM. Supershedders (SS) were defined as animals that had manure with more than 10,000 cfu/gm. Culture results on diluted samples demonstrated that a substantial proportion of heavy shedders were SS, with some animals exceeding 1,000,000 MAP cfu/gm. Whole herd cultures from 4 herds found that 10% to 15% of all culture positives were SS. Not all of these SS cattle demonstrated clinical signs of JD at the time of sampling. It was calculated that SS cattle shedding the highest concentration of MAP were shedding more cfu than 2,000 moderate or 20,000 light shedders. Subsequent culture in one herd after the removal of a SS identified up to 15 animals that were potential false positives on the previous culture. This study has demonstrated the previously undocumented presence of MAP supershedders. The results suggest that SS contribute disproportionately to the herd environmental bioburden and the exposure of uninfected animals to MAP organisms. The data also suggest that the bioburden in herds with a SS present may contribute to herdmates being incorrectly identified as culture positive. Identification and removal of SS animals is likely to be a critical component of a JD control and management program. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 highshedding 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. Highshedding 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 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.

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 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.

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.

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.

The objective of this work was to test the ability of various available quick tests to determine ammonia concentration of poultry litters. A total of 136 samples was collected from brood chambers of poultry houses. Samples were equally divided between surface samples (top 25 mm) and core samples. Samples were frozen until analysis but received no further processing. Samples were analyzed for ammonia by autoanalyzer (standard) and several quick tests (conductivity, Quantofix N-Volumeter, and Reflectoquant). In addition, samples were analyzed by near-infrared spectroscopy by scanning samples using a large-sample transport device on a FOSS-NIRSystems model 6500 (64 co-added scans from 400 to 2,498 nm). Results showed that, although ammonia could be determined with reasonable accuracy by near-infrared spectroscopy using data in the 1,100 to 2,498 nm spectral range (final calibration R2 of approximately 0.90), none of the quick tests, including near-infrared, worked as well as previously found with dairy manures. The best results were found using the Quantofix or Reflectoquant (R2 of approximately 0.75), and conductivity worked only with the core samples (R2 of approximately 0.75). It is believed that interferences due to the presence of uric acid (spectroscopy, Quantofix, and Reflectoquant) and sodium bisulfate used to treat the litter (conductivity) were the cause of the decreased accuracies as compared to results achieved previously with 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.

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.

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.