The devil you know and the devil you don't: current status and challenges of bovine tuberculosis eradication in the United States.

Having entered into its second century, the eradication program for bovine tuberculosis (bTB, caused by Mycobacterium bovis) in the United States of America occupies a position both enviable and daunting. Excepting four counties in Michigan comprising only 6109 km2 (0.06% of US land area) classified as Modified Accredited, as of April 2022 the entire country was considered Accredited Free of bTB by the US Department of Agriculture for cattle and bison. On the surface, the now well-described circumstances of endemic bTB in Michigan, where white-tailed deer (Odocoileus virginianus) serve as a free-ranging wildlife maintenance host, may appear to be the principal remaining barrier to national eradication. However, the situation there is unique in the U.S., and far-removed from the broader issues of bTB control in the remainder of the country. In Michigan, extensive surveillance for bTB in deer over the last quarter century, and regulatory measures to maximize the harvest of publicly-owned wildlife, have been implemented and sustained. Prevalence of bTB in deer has remained at a low level, although not sufficiently low to eliminate cattle herd infections. Public attitudes towards bTB, cattle and deer, and their relative importance, have been more influential in the management of the disease than any limitations of biological science. However, profound changes in the demographics and social attitudes of Michigan's human population are underway, changes which are likely to force a critical reevaluation of the bTB control strategies thus far considered integral. In the rest of the U.S. where bTB is not self-sustaining in wildlife, changes in the scale of cattle production, coupled with both technical and non-technical issues have created their own substantial challenges. It is against this diverse backdrop that the evolution of whole genome sequencing of M. bovis has revolutionized understanding of the history and ecology of bTB in Michigan, resolved previously undiscernible epidemiological puzzles, provided insights into zoonotic transmission, and unified eradication efforts across species and agencies. We describe the current status of bTB eradication in the U.S., how circumstances and management have changed, what has been learned, and what remains more elusive than ever.

Improving farm-level antimicrobial stewardship benchmarks by reporting antimicrobial use within the context of both the magnitude of disease pressure and the outcome of therapy.

This manuscript explores a method of benchmarking antimicrobial use within the context of farm level therapeutic incidence (a proxy for disease incidence), and the outcome of that therapy. This is reported both within the same farm over time (2016-2019), as well as evaluated across participating farms. Reporting antimicrobial use in this format addresses multiple primary questions necessary for evaluating on farm antimicrobial stewardship: How much disease is recorded? How much antimicrobial use is recorded? How often are antimicrobials included in therapy for each disease? What is the outcome of therapy? The three primary metrics reported are: therapeutic events per 100 cow years (TE/100CY), antimicrobial regimens per 100 cow years (REG/100CY), and the percent therapeutic success (% Success). Success was defined as: the cow remained in the herd and had no further TE recorded within 30 days of the end of the TE being evaluated. These measures identify opportunities for change on an individual farm, such as improvement in disease prevention, or a change in choices about when to include an antimicrobial in the treatment protocol. Therapeutic outcomes provide additional context, in some instances demonstrating differences in recording practices and case definitions, while in other cases serving to safeguard animal welfare as efforts are made to decrease antimicrobial use in the future. Although developed for farm level reporting, the metrics may also be more broadly summarized to meet future reporting requirements for marketing chain or national level antimicrobial use reports. The process outlined here serves as a prototype to be considered when developing antimicrobial use reporting systems where farm level antimicrobial stewardship is the primary objective.

Enhanced detection of Giardia duodenalis mixed assemblage infections in pre-weaned dairy calves using next generation sequencing.

Giardia duodenalis is one of the most common parasitic causes of gastrointestinal illness in humans worldwide with widespread infections in mammalian hosts. It frequently infects cattle, producing a high number of cysts. Cattle can harbor both host-adapted assemblage E and human pathogenic assemblages A and B. Previous studies have demonstrated that conventional molecular methods lack the sensitivity required for detecting mixed infections and that the occurrence of mixed infections in cattle are likely underestimated. To investigate the presence of mixed assemblage infections in cattle, 2539 pre-weaned dairy calves from the United Stated were screened for the presence of G. duodenalis using molecular tools. Next generation amplicon sequencing (NGS) was then performed for a subset of around 30% of positive samples (n = 314) and the ability of NGS and Sanger sequencing to detect mixed assemblage infections was compared. The overall prevalence of G. duodenalis in pre-weaned dairy calves in the sample using PCR was high (1013/2539; 39.9%). Molecular genotyping identified only assemblage A and E, with assemblage E as the predominant assemblage. Out of the 314 samples examined by both Sanger and NGS, 9 samples (2.9%) were identified as mixed A/E infections by Sanger while NGS identified 56 samples (17.8%), which was six-times more mixed infections compared with Sanger sequencing. NGS demonstrated superior sensitivity to Sanger in detecting assemblages present in low abundances. The percentage of mixed A/E infections found in the sampled dairy calves was higher than was hypothesized using values from the literature. This underestimation could be present in the wider cattle population as well, though further exploration would be needed to verify that claim. These findings highlight the advantages of NGS application in molecular epidemiological studies of Giardia. To better understand Giardia epidemiology, establish routes of transmission, and assess the potential role of cattle and other animals as a source of environmental contamination with zoonotic assemblages it is necessary to uncover mixed assemblage infections.

Human-to-Cattle Mycobacterium tuberculosis Complex Transmission in the United States.

The Mycobacterium tuberculosis complex (MTBC) species includes both M. tuberculosis, the primary cause of human tuberculosis (TB), and M. bovis, the primary cause of bovine tuberculosis (bTB), as well as other closely related Mycobacterium species. Zoonotic transmission of M. bovis from cattle to humans was recognized more than a century ago, but transmission of MTBC species from humans to cattle is less often recognized. Within the last decade, multiple published reports from around the world describe human-to-cattle transmission of MTBC. Three probable cases of human-to-cattle MTBC transmission have occurred in the United States since 2013. In the first case, detection of active TB disease (M. bovis) in a dairy employee in North Dakota prompted testing and ultimate detection of bTB infection in the dairy herd. Whole genome sequencing (WGS) demonstrated a match between the bTB strain in the employee and an infected cow. North Dakota animal and public health officials concluded that the employee's infection was the most likely source of disease introduction in the dairy. The second case involved a Wisconsin dairy herd with an employee diagnosed with TB disease in 2015. Subsequently, the herd was tested twice with no disease detected. Three years later, a cow originating from this herd was detected with bTB at slaughter. The strain in the slaughter case matched that of the past employee based on WGS. The third case was a 4-month-old heifer calf born in New Mexico and transported to Texas. The calf was TB tested per Texas entry requirements and found to have M. tuberculosis. Humans are the suspected source of M. tuberculosis in cattle; however, public health authorities were not able to identify an infected human associated with the cattle operation. These three cases provide strong evidence of human-to-cattle transmission of MTBC organisms and highlight human infection as a potential source of introduction of MTBC into dairy herds in the United States. To better understand and address the issue, a multisectoral One Health approach is needed, where industry, public health, and animal health work together to better understand the epidemiology and identify preventive measures to protect human and animal health.

The National Animal Health Monitoring System's perspective on respiratory disease in dairy cattle.

In 1991, USDA's National Animal Health Monitoring System (NAHMS) conducted its first dairy study, a baseline assessment that focused on the health and management of heifer calves. During the study, producers ranked respiratory disease among the top two most common health problems affecting dairy calves. Over the last 25 years, U.S. dairy producers have participated in six NAHMS studies, yielding a rich repository of information that has helped identify needs for research, extension, and education in the dairy industry. NAHMS' most recent dairy study, conducted in 2014, provides the latest estimates on dairy cattle health and includes another in-depth look at heifer-calf health. While overall mortality in calves has decreased, bovine respiratory disease (BRD) remains an important cause of morbidity in calves. This raises the concern that BRD mitigation may be at a standstill on dairies. Research and on-farm experience have done much to elucidate the challenges associated with detection and perceived impacts of this complex disease. Continued development and implementation of new methods for monitoring health and detecting disease will provide additional tools to upend stalemating factors associated with BRD control, helping the dairy industry 'turn a corner' on this important disease.

Colostrum Management for Dairy Calves.

Colostrum management is the single most important management factor in determining calf health and survival. Additional benefits of good colostrum management include improved rate of gain and future productivity. Successful colostrum management requires producers to provide calves with a sufficient volume of clean, high-quality colostrum within the first few hours of life. This article reviews the process of colostrogenesis and colostrum composition, and discusses key components in developing a successful colostrum management program. In addition, the article discusses approaches for monitoring and proposes new goals for passive immunity in dairy herds.

Zoonotic and genetically diverse subtypes of Blastocystis in US pre-weaned dairy heifer calves.

Blastocystis is an emerging zoonotic pathogen with global distribution. However, limited data exist on the prevalence and genetic diversity of Blastocystis in the USA and in food animals. We conducted the first large-scale molecular investigation of Blastocystis in the USA by testing 2539 fecal samples from dairy heifer calves from 13 states. Blastocystis was detected in 73 (2.9%) fecal samples and in 10 of the 13 tested states. Molecular characterization showed a wide diversity of subtypes. Eleven subtypes were identified, seven previously reported (ST-3, ST-4, ST-5, ST-10, ST-14, ST-17, and ST-21) and four potentially novel subtypes (named ST-23 to ST-26). Zoonotic subtypes 3, 4, and 5 were found in 67% (49) of the positive specimens in this population. Our results suggest that cattle could serve as reservoirs of infection for humans and other domestic animals highlighting the potential risk of zoonotic transmission for Blastocystis.

Genetic diversity and virulence profiles of Listeria monocytogenes recovered from bulk tank milk, milk filters, and milking equipment from dairies in the United States (2002 to 2014).

Unpasteurized dairy products are known to occasionally harbor Listeria monocytogenes and have been implicated in recent listeriosis outbreaks and numerous sporadic cases of listeriosis. However, the diversity and virulence profiles of L. monocytogenes isolates recovered from these products have not been fully described. Here we report a genomic analysis of 121 L. monocytogenes isolates recovered from milk, milk filters, and milking equipment collected from bovine dairy farms in 19 states over a 12-year period. In a multi-virulence-locus sequence typing (MVLST) analysis, 59 Virulence Types (VT) were identified, of which 25% were Epidemic Clones I, II, V, VI, VII, VIII, IX, or X, and 31 were novel VT. In a multi-locus sequence typing (MLST) analysis, 60 Sequence Types (ST) of 56 Clonal Complexes (CC) were identified. Within lineage I, CC5 and CC1 were among the most abundant, and within lineage II, CC7 and CC37 were the most abundant. Multiple CCs previously associated with central nervous system and maternal-neonatal infections were identified. A genomic analysis identified variable distribution of virulence markers, Listeria pathogenicity islands (LIPI) -1, -3, and -4, and stress survival island-1 (SSI-1). Of these, 14 virulence markers, including LIPI-3 and -4 were more frequently detected in one lineage (I or II) than the other. LIPI-3 and LIPI-4 were identified in 68% and 28% of lineage I CCs, respectively. Results of this analysis indicate that there is a high level of genetic diversity among the L. monocytogenes present in bulk tank milk in the United States with some strains being more frequently detected than others, and some being similar to those that have been isolated from previous non-dairy related outbreaks. Results of this study also demonstrate significant number of strains isolated from dairy farms encode virulence markers associated with severe human disease.

A comparison of a novel time-based summary measure of dairy cow health against cumulative disease frequency.

BACKGROUND: There is an increasing push for dairy production to be scientifically grounded and ethically responsible in the oversight of animal health and well-being. Addressing underlying challenges affecting the quality and length of productive life necessitates novel assessment and accountability metrics. Human medical epidemiologists developed the Disability-Adjusted Life Year metric as a summary measure of health addressing the complementary nature of disease and death. The goal of this project was to develop and implement a dairy Disease-Adjusted Lactation (DALact) summary measure of health, as a comparison against cumulative disease frequency. METHODS: A total of 5694 cows were enrolled at freshening from January 1st, 2014 through May 26th, 2015 on 3 similarly managed U.S. Midwestern Plains' region dairies. Eleven health categories of interest were tracked from enrollment until culling, death, or the study's completion date. The DALact accounted for the days of life lost due to illness, forced removal, and death relative to the average lactation length across the participating farms. RESULTS: The DALact consistently identified mastitis as the primary disease of concern on all 3 dairies (19,007-23,955 days lost). Secondary issues included musculoskeletal injuries (19,559 days), pneumonia (11,034 days), or lameness (8858 days). By comparison, cumulative frequency measures pointed to mastitis (31-50%) and lameness (25-54%) as the 2 most frequent diseases. Notably, the DALact provided a robust accounting of health events such as musculoskeletal injuries (5010-19,559 days) and calving trauma (2952-5868 days) otherwise overlooked by frequency measures (0-3%). CONCLUSIONS: The DALact provides a time-based method for assessing the overall burden of disease on dairies. It is important to emphasize that a summary measure of dairy health goes beyond simply linking morbidity to culling and mortality in a standardized fashion. A summary measure speaks to the burden of disease on both the well-being and productivity of individuals and populations. When framed as lost days, years, or lactations the various health issues on a farm are more comprehensible than they may be by frequency measures alone. Such an alternative accounting of disease highlights the lost opportunity costs of production as well as the burden of disease on life as a whole.

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.

Dairy cow disability weights.

Over the past 175 years, data related to human disease and death have progressed to a summary measure of population health, the Disability-Adjusted Life Year (DALY). As dairies have intensified there has been no equivalent measure of the impact of disease on the productive life and well-being of animals. The development of a disease-adjusted metric requires a consistent set of disability weights that reflect the relative severity of important diseases. The objective of this study was to use an international survey of dairy authorities to derive disability weights for primary disease categories recorded on dairies. National and international dairy health and management authorities were contacted through professional organizations, dairy industry publications and conferences, and industry contacts. Estimates of minimum, most likely, and maximum disability weights were derived for 12 common dairy cow diseases. Survey participants were asked to estimate the impact of each disease on overall health and milk production. Diseases were classified from 1 (minimal adverse effects) to 10 (death). The data was modelled using BetaPERT distributions to demonstrate the variation in these dynamic disease processes, and to identify the most likely aggregated disability weights for each disease classification. A single disability weight was assigned to each disease using the average of the combined medians for the minimum, most likely, and maximum severity scores. A total of 96 respondents provided estimates of disability weights. The final disability weight values resulted in the following order from least to most severe: retained placenta, diarrhea, ketosis, metritis, mastitis, milk fever, lame (hoof only), calving trauma, left displaced abomasum, pneumonia, musculoskeletal injury (leg, hip, back), and right displaced abomasum. The peaks of the probability density functions indicated that for certain disease states such as retained placenta there was a relatively narrow range of expected impact whereas other diseases elicited a wider breadth of impact. This was particularly apparent with respect to calving trauma, lameness and musculoskeletal injury, all of which could be redefined using gradients of severity or accounting for sequelae. These disability weight distributions serve as an initial step in the development of the disease-adjusted lactation (DALact) metric. They will be used to assess the time lost due to dynamic phases of dairy cow diseases and injuries. Prioritizing health interventions based on time expands the discussion of animal health to view profits and losses in light of the quality and length of life.

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.

Summary of BRD data from the 2011 NAHMS feedlot and dairy heifer studies.

The USDA:APHIS National Animal Health Monitoring System collects data on health and health management in livestock and poultry populations throughout the USA in order to provide stakeholders with population estimates to use as benchmarks for comparison, to guide policy development, and to identify research needs and prioritize education efforts. Recent studies of both the beef cattle feedlot industry and dairy heifer rearing operations provided information about BRD occurrence as well as information about prevention and treatment practices used on these operations. While a great deal of effort is dedicated to BRD prevention, there are still opportunities to improve the strategies used. Despite efforts to prevent the disease, BRD continues to be widespread on both of these types of operations.

The impact of movements and animal density on continental scale cattle disease outbreaks in the United States.

Globalization has increased the potential for the introduction and spread of novel pathogens over large spatial scales necessitating continental-scale disease models to guide emergency preparedness. Livestock disease spread models, such as those for the 2001 foot-and-mouth disease (FMD) epidemic in the United Kingdom, represent some of the best case studies of large-scale disease spread. However, generalization of these models to explore disease outcomes in other systems, such as the United States's cattle industry, has been hampered by differences in system size and complexity and the absence of suitable livestock movement data. Here, a unique database of US cattle shipments allows estimation of synthetic movement networks that inform a near-continental scale disease model of a potential FMD-like (i.e., rapidly spreading) epidemic in US cattle. The largest epidemics may affect over one-third of the US and 120,000 cattle premises, but cattle movement restrictions from infected counties, as opposed to national movement moratoriums, are found to effectively contain outbreaks. Slow detection or weak compliance may necessitate more severe state-level bans for similar control. Such results highlight the role of large-scale disease models in emergency preparedness, particularly for systems lacking comprehensive movement and outbreak data, and the need to rapidly implement multi-scale contingency plans during a potential US outbreak.

A national-scale picture of U.S. cattle movements obtained from Interstate Certificate of Veterinary Inspection data.

We present the first comprehensive description of how shipments of cattle connect the geographic extent and production diversity of the United States cattle industry. We built a network of cattle movement from a state-stratified 10% systematic sample of calendar year 2009 Interstate Certificates of Veterinary Inspection (ICVI) data. ICVIs are required to certify the apparent health of cattle moving across state borders and allow us to examine cattle movements at the county scale. The majority of the ICVI sample consisted of small shipments (<20 head) moved for feeding and beef production. Geographically, the central plains states had the most connections, correlated to feeding infrastructure. The entire nation was closely connected when interstate movements were summarized at the state level. At the county-level, the U.S. is still well connected geographically, but significant heterogeneities in the location and identity of counties central to the network emerge. Overall, the network of interstate movements is described by a hub structure, with a few counties sending or receiving extremely large numbers of shipments and many counties sending and receiving few shipments. The county-level network also has a very low proportion of reciprocal movements, indicating that high-order network properties may be better at describing a county's importance than simple summaries of the number of shipments or animals sent and received. We suggest that summarizing cattle movements at the state level homogenizes the network and a county level approach is most appropriate for examining processes influenced by cattle shipments, such as economic analyses and disease outbreaks.

Farm-level associations with the shedding of Salmonella and antimicrobial-resistant Salmonella in U.S. dairy cattle.

Salmonella enterica is the leading cause of foodborne-related deaths and hospitalizations within the United States. Infections caused by antimicrobial-resistant (AMR) strains are associated with higher hospital costs and case fatality. The objective for this study was to determine the association of management practices with the recovery of Salmonella and AMR Salmonella on dairy herds. Individual adult cow fecal samples and/or composite fecal samples were collected from 265 dairy herds in 17 states. Samples were cultured for Salmonella, and the MIC was determined for 15 antimicrobials. Herds were classified as Salmonella positive if at least one isolate was recovered, and AMR Salmonella positive if at least one resistant isolate was recovered. Questionnaires regarding management practices were administered to herd operators, and a subset of practices was selected based on subject knowledge and prior research. Data on preventive and therapeutic antimicrobial usage were included in the analysis. Logistic regression models were used to determine which practices were significantly (p<0.05) associated with each herd classification. A total of 124 and 25 herds were classified as Salmonella positive and AMR Salmonella positive, respectively. Variables significantly associated with Salmonella-positive herds included using sprinklers or misters for heat abatement (OR=2.8; CI: 1.6-4.9), feeding anionic salts to cows (OR=1.9; CI: 1.1-3.5), and feeding ionophores to cows (OR=2.1; CI: 1.2-3.7). Herds that used a broadcast/solid spread had lower odds (OR=0.26; CI: 0.11-0.63) of being Salmonella positive. Herds with at least one resistant isolate were more likely to have used composted/dried manure for bedding relative to herds with only susceptible isolates (OR=3.6; CI: 1.2-11.0). These results can be useful to focus additional research aimed at decreasing the prevalence of Salmonella and AMR Salmonella on U.S. dairy herds.

Epidemiology and economics of paratuberculosis.

Johne's disease is the clinical manifestation of Mycobacterium avium subsp. paratuberculosis (MAP) infection and has become widespread since it was first observed in the United States in the early 1900s. MAP is primarily spread through the fecal-oral route, and herds generally become infected by unknowingly purchasing infected animals. The economic losses from the disease are primarily due to decreased milk production, decreased weaning weights in nursing young stock, increased replacement costs, and decreased slaughter value.

Consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis in ruminants.

The Standards for Reporting of Diagnostic Accuracy (STARD) statement (www.stard-statement.org) was developed to encourage complete and transparent reporting of key elements of test accuracy studies in human medicine. The statement was motivated by widespread evidence of bias in test accuracy studies and the finding that incomplete or absent reporting of items in the STARD checklist was associated with overly optimistic estimates of test performance characteristics. Although STARD principles apply broadly, specific guidelines do not exist to account for unique considerations in livestock studies such as herd tests, potential use of experimental challenge studies, a more diverse group of testing purposes and sampling designs, and the widespread lack of an ante-mortem reference standard with high sensitivity and specificity. The objective of the present study was to develop a modified version of STARD relevant to paratuberculosis (Johne's disease) in ruminants. Examples and elaborations for each of the 25 items were developed by a panel of experts using a consensus-based approach to explain the items and underlying concepts. The new guidelines, termed STRADAS-paraTB (Standards for Reporting of Animal Diagnostic Accuracy Studies for paratuberculosis), should facilitate improved quality of reporting of the design, conduct and results of paratuberculosis test accuracy studies which were identified as "poor" in a review published in 2008 in Veterinary Microbiology.

Prevalence of Salmonella enterica, Listeria monocytogenes, and Escherichia coli virulence factors in bulk tank milk and in-line filters from U.S. dairies.

The zoonotic bacteria Salmonella enterica, Listeria monocytogenes, and Escherichia coli are known to infect dairy cows while not always causing clinical signs of disease. These pathogens are sometimes found in raw milk, and human disease outbreaks due to these organisms have been associated with the consumption of raw milk or raw milk products. Bulk tank milk (BTM) samples (536) and in-line milk filters (519) collected from dairy farms across the United States during the National Animal Health Monitoring System's Dairy 2007 study were analyzed by real-time PCR for the presence of S. enterica and pathogenic forms of E. coli and by culture techniques for the presence of L. monocytogenes. S. enterica was detected in samples from 28.1% of the dairy operations, primarily in milk filters. Salmonella was isolated from 36 of 75 PCR-positive BTM samples and 105 of 174 PCR-positive filter samples, and the isolates were serotyped. Cerro, Kentucky, Muenster, Anatum, and Newport were the most common serotypes. L. monocytogenes was isolated from 7.1% of the dairy operations, and the 1/2a complex was the most common serotype, followed by 1/2b and 4b (lineage 1). Shiga toxin genes were detected in enrichments from 15.2% of the BTM samples and from 51.0% of the filters by real-time PCR. In most cases, the cycle threshold values for the PCR indicated that toxigenic strains were not a major part of the enrichment populations. These data confirm those from earlier studies showing significant contamination of BTM by zoonotic bacterial pathogens and that the consumption of raw milk and raw milk products presents a health risk.

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.