Genome-Wide Analysis of Escherichia coli Isolated from Dairy Animals Identifies Virulence Factors and Genes Enriched in Multidrug-Resistant Strains.

The gastrointestinal tracts of dairy calves and cows are reservoirs of antimicrobial-resistant bacteria (ARB), which are present regardless of previous antimicrobial therapy. Young calves harbor a greater abundance of resistant bacteria than older cows, but the factors driving this high abundance are unknown. Here, we aimed to fully characterize the genomes of multidrug-resistant (MDR) and antimicrobial-susceptible Escherichia coli strains isolated from pre-weaned calves, post-weaned calves, dry cows, and lactating cows and to identify the accessory genes that are associated with the MDR genotype to discover genetic targets that can be exploited to mitigate antimicrobial resistance in dairy farms. Results indicated that both susceptible and resistant E. coli isolates recovered from animals on commercial dairy operations were highly diverse and encoded a large pool of virulence factors. In total, 838 transferrable antimicrobial resistance genes (ARGs) were detected, with genes conferring resistance to aminoglycosides being the most common. Multiple sequence types (STs) associated with mild to severe human gastrointestinal and extraintestinal infections were identified. A Fisher's Exact Test identified 619 genes (ARGs and non-ARGs) that were significantly enriched in MDR isolates and 147 genes that were significantly enriched in susceptible isolates. Significantly enriched genes in MDR isolates included the iron scavenging aerobactin synthesis and receptor genes (iucABCD-iutA) and the sitABCD system, as well as the P fimbriae pap genes, myo-inositol catabolism (iolABCDEG-iatA), and ascorbate transport genes (ulaABC). The results of this study demonstrate a highly diverse population of E. coli in commercial dairy operations, some of which encode virulence genes responsible for severe human infections and resistance to antibiotics of human health significance. Further, the enriched accessory genes in MDR isolates (aerobactin, sit, P fimbriae, and myo-inositol catabolism and ascorbate transport genes) represent potential targets for reducing colonization of antimicrobial-resistant bacteria in the calf gut.

Characterization of Antimicrobial Resistance Genes and Virulence Factors in the Genomes of Escherichia coli ST69 Isolates from Preweaned Dairy Calves and Their Phylogenetic Relationship with Poultry and Human Clinical Strains.

Escherichia coli sequence type 69 (ST69) are common causative agents of extraintestinal infections occurring in the bloodstream, cerebrospinal fluid, surgical sites, and, most frequently, the urinary tract. The objective of this study was to analyze the genomic characteristics of 45 antimicrobial-resistant Escherichia coli ST69 strains that were isolated from 28 calves on eight dairy farms in Pennsylvania, USA. The genomes were sequenced and the antimicrobial resistance genes (ARGs), virulence factors (VFs), and plasmid replicons were identified in silico. A phylogenetic analysis was conducted to compare these calf isolate genomes with poultry and human clinical E. coli ST69 genomes. In total, 23 ARGs, 45 VFs, and 15 plasmid replicons were identified. The majority of genomes (n = 36, 80%) had a multidrug-resistant (MDR) genotype and carried genes conferring resistance to antibiotics of human health significance. Phylogenetic analysis based on the core genomes revealed that calf isolates were nested within clades that included human and poultry isolates, indicating that they are not phylogenetically distinct. Results suggest that dairy calves are a reservoir of MDR E. coli ST69 strains with diverse ARG and VF profiles. This information will be helpful in assessing public health risks associated with E. coli ST69 in commercial dairy production systems.

Genomic diversity of antimicrobial-resistant and Shiga toxin gene-harboring non-O157 Escherichia coli from dairy calves.

OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) are globally significant foodborne pathogens. Dairy calves are a known reservoir of both O157 and non-O157 STEC. The objective of this study was to comprehensively evaluate the genomic attributes, diversity, virulence factors, and antimicrobial resistance gene (ARG) profiles of the STEC from preweaned and postweaned dairy calves in commercial dairy herds. METHODS: In total, 31 non-O157 STEC were identified as part of a larger study focused on the pangenome of >1000 E. coli isolates from the faeces of preweaned and postweaned dairy calves on commercial dairy farms. These 31 genomes were sequenced on an Illumina NextSeq500 platform. RESULTS: Based on the phylogenetic analyses, the STEC isolates were determined to be polyphyletic, with at least three phylogroups: A (32%), B1 (58%), and G (3%). These phylogroups represented at least 16 sequence types and 11 serogroups, including two of the 'big six' serogroups, O103 and O111. Several Shiga toxin gene subtypes were identified in the genomes, including stx1a, stx2a, stx2c, stx2d, and stx2g. Using the ResFinder database, the majority of the isolates (>50%) were determined to be multidrug-resistant strains because they harboured genes conferring resistance to three or more classes of antimicrobials, including some of human health significance (e.g., ß-lactams, macrolides, and fosfomycin). Additionally, non-O157 STEC strain persistence and transmission within a farm was observed. CONCLUSION: Dairy calves are a reservoir of phylogenomically diverse multidrug-resistant non-O157 STEC. Information from this study may inform assessments of public health risk and guide preharvest prevention strategies focusing on STEC reservoirs.

Development and Validation of Indirect Enzyme-Linked Immunosorbent Assays for Detecting Antibodies to SARS-CoV-2 in Cattle, Swine, and Chicken.

Multiple domestic and wild animal species are susceptible to SARS-CoV-2 infection. Cattle and swine are susceptible to experimental SARS-CoV-2 infection. The unchecked transmission of SARS-CoV-2 in animal hosts could lead to virus adaptation and the emergence of novel variants. In addition, the spillover and subsequent adaptation of SARS-CoV-2 in livestock could significantly impact food security as well as animal and public health. Therefore, it is essential to monitor livestock species for SARS-CoV-2 spillover. We developed and optimized species-specific indirect ELISAs (iELISAs) to detect anti-SARS-CoV-2 antibodies in cattle, swine, and chickens using the spike protein receptor-binding domain (RBD) antigen. Serum samples collected prior to the COVID-19 pandemic were used to determine the cut-off threshold. RBD hyperimmunized sera from cattle (n = 3), swine (n = 6), and chicken (n = 3) were used as the positive controls. The iELISAs were evaluated compared to a live virus neutralization test using cattle (n = 150), swine (n = 150), and chicken (n = 150) serum samples collected during the COVID-19 pandemic. The iELISAs for cattle, swine, and chicken were found to have 100% sensitivity and specificity. These tools facilitate the surveillance that is necessary to quickly identify spillovers into the three most important agricultural species worldwide.

Virulome and genome analyses identify associations between antimicrobial resistance genes and virulence factors in highly drug-resistant Escherichia coli isolated from veal calves.

Food animals are known reservoirs of multidrug-resistant (MDR) Escherichia coli, but information regarding the factors influencing colonization by these organisms is lacking. Here we report the genomic analysis of 66 MDR E. coli isolates from non-redundant veal calf fecal samples. Genes conferring resistance to aminoglycosides, ß-lactams, sulfonamides, and tetracyclines were the most frequent antimicrobial resistance genes (ARGs) detected and included those that confer resistance to clinically significant antibiotics (blaCMY-2, blaCTX-M, mph(A), erm(B), aac(6')Ib-cr, and qnrS1). Co-occurrence analyses indicated that multiple ARGs significantly co-occurred with each other, and with metal and biocide resistance genes (MRGs and BRGs). Genomic analysis also indicated that the MDR E. coli isolated from veal calves were highly diverse. The most frequently detected genotype was phylogroup A-ST Cplx 10. A high percentage of isolates (50%) were identified as sequence types that are the causative agents of extra-intestinal infections (ExPECs), such as ST69, ST410, ST117, ST88, ST617, ST648, ST10, ST58, and ST167, and an appreciable number of these isolates encoded virulence factors involved in the colonization and infection of the human urinary tract. There was a significant difference in the presence of multiple accessory virulence factors (VFs) between MDR and susceptible strains. VFs associated with enterohemorrhagic infections, such as stx, tir, and eae, were more likely to be harbored by antimicrobial-susceptible strains, while factors associated with extraintestinal infections such as the sit system, aerobactin, and pap fimbriae genes were more likely to be encoded in resistant strains. A comparative analysis of SNPs between strains indicated that several closely related strains were recovered from animals on different farms indicating the potential for resistant strains to circulate among farms. These results indicate that veal calves are a reservoir for a diverse group of MDR E. coli that harbor various resistance genes and virulence factors associated with human infections. Evidence of co-occurrence of ARGs with MRGs, BRGs, and iron-scavenging genes (sit and aerobactin) may lead to management strategies for reducing colonization of resistant bacteria in the calf gut.

Differences in the Microbial Community and Resistome Structures of Feces from Preweaned Calves and Lactating Dairy Cows in Commercial Dairy Herds.

Preweaned dairy calves and lactating dairy cows are known reservoirs of antibiotic-resistant bacteria. To further understand the differences in the resistomes and microbial communities between the two, we sequenced the metagenomes of fecal composite samples from preweaned dairy calves and lactating dairy cows on 17 commercial dairy farms (n = 34 samples). Results indicated significant differences in the structures of the microbial communities (analysis of similarities [ANOSIM] R = 0.81, p = 0.001) and resistomes (ANOSIM R = 0.93 to 0.96, p = 0.001) between the two age groups. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the predominant members of the communities, but when the groups were compared, Bacteroidetes and Verrumicrobia were significantly more abundant in calf fecal composite samples, whereas Firmicutes, Spirochaetes, Deinococcus-Thermus, Lentisphaerae, Planctomycetes, Chlorofexi, and Saccharibacteria-(TM7) were more abundant in lactating cow samples. Diverse suites of antibiotic resistance genes (ARGs) were identified in all samples, with the most frequently detected being assigned to tetracycline and aminoglycoside resistance. When the two groups were compared, ARGs were significantly more abundant in composite fecal samples from calves than those from lactating cows (calf median ARG abundance = 1.8 × 100 ARG/16S ribosomal RNA [rRNA], cow median ARG abundance = 1.7 × 10-1 ARG/16S rRNA) and at the antibiotic resistance class level, the relative abundance of tetracycline, trimethoprim, aminoglycoside, macrolide-lincosamide-streptogramin B, ß-lactam, and phenicol resistance genes was significantly higher in calf samples than in cow samples. Results of this study indicate that composite feces from preweaned calves harbor different bacterial communities and resistomes than composite feces from lactating cows, with a greater abundance of resistance genes detected in preweaned calf feces.

Metagenomic Analysis of the Microbial Communities and Resistomes of Veal Calf Feces.

Antimicrobial resistance (AMR) is a major public health concern, and dairy calves, including veal calves, are known reservoirs of resistant bacteria. To investigate AMR in the fecal microbial communities of veal calves, we conducted metagenomic sequencing of feces collected from individual animals on four commercial veal operations in Pennsylvania. Fecal samples from three randomly selected calves on each farm were collected soon after the calves were brought onto the farms (n = 12), and again, just before the calves from the same cohorts were ready for slaughter (n = 12). Results indicated that the most frequently identified phyla were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Fecal microbial communities in samples collected from the calves at the early and late stages of production were significantly different at the genus level (analysis of similarities [ANOSIM] on Bray-Curtis distances, R = 0.37, p < 0.05), but not at the phylum level. Variances among microbial communities in the feces of the younger calves were significantly higher than those from the feces of calves at the late stage of production (betadisper F = 8.25, p < 0.05). Additionally, our analyses identified a diverse set of mobile antimicrobial resistance genes (ARGs) in the veal calf feces. The fecal resistomes mostly consisted of ARGs that confer resistance to aminoglycosides, tetracyclines, and macrolide-lincosamide-streptogramin B (MLS), and these ARGs represented more than 70% of the fecal resistomes. Factors that are responsible for selection and persistence of resistant bacteria in the veal calf gut need to be identified to implement novel control points and interrupt detrimental AMR occurrence and shedding.

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

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

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

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

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

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

Antimicrobial Resistance in Fecal Escherichia coli and Salmonella enterica from Dairy Calves: A Systematic Review.

The discovery of antibiotics brought with it many advances in the health and well-being of humans and animals; however, in recent years development of antimicrobial resistance (AMR) has increasingly become a concern. Much of the antibiotic use on dairy farms is for disease management in mature cattle, and AMR in fecal organisms is relatively rare in this group. However, young dairy calves often carry high levels of AMR in their fecal Escherichia coli and Salmonella enterica, which could provide a potential reservoir of AMR genes on dairy farms. To develop practical and effective antibiotic stewardship policies for dairy calf rearing, it is vital to have a solid understanding of the current state of knowledge regarding AMR in these animals. A systematic review process was used to summarize the current scientific literature regarding AMR in fecal S. enterica and E. coli and associations between management practices and AMR prevalence in dairy calves in the United States and Canada. Seven online databases were searched for literature published from 1997 to 2018. Multiple studies indicated an association between preweaned calves and increased risk of fecal shedding of resistant bacteria, compared to other animal groups on dairy farms. There also was evidence, although less consistent, of an impact of antibiotic treatment, antibiotic-containing milk replacer feeding, and feeding nonsalable or waste milk (WM) on the presence of AMR bacteria. Overall, the research summarized in this systematic review highlights the need for continued research on the impact of management practices, including antibiotic use, WM feeding, and disease prevention practices in reducing AMR in E. coli and S. enterica in dairy calves. In addition, few data were available on physiological and microbiological factors that may contribute to the high relative populations of resistant bacteria in young calves, suggesting another valuable area of future research.

Erratum for Byukusenge et al., "Complete Genome Sequences of Four Bovine Coronavirus Isolates from Pennsylvania".

[This corrects the article DOI: 10.1128/genomeA.00467-18.].

Complete Genome Sequences of Four Bovine Coronavirus Isolates from Pennsylvania.

We report four full-genome sequences of bovine coronavirus (BCoV) isolates from dairy calves in Pennsylvania obtained in 2016 and 2017. BCoV is a pathogen of great importance to cattle health, and this is the first report of full-genome sequences of BCoV from PA cattle.

Mycobacterium avium subsp. paratuberculosis antibody response, fecal shedding, and antibody cross-reactivity to Mycobacterium bovis in M. avium subsp. paratuberculosis-infected cattle herds vaccinated against Johne's disease.

Vaccination for Johne's disease with killed inactivated vaccine in cattle herds has shown variable success. The vaccine delays the onset of disease but does not afford complete protection. Johne's disease vaccination has also been reported to interfere with measurements of cell-mediated immune responses for the detection of bovine tuberculosis. Temporal antibody responses and fecal shedding of Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease, were measured in 2 dairy cattle herds using Johne's disease vaccine (Mycopar) over a period of 7 years. Vaccination against Johne's disease resulted in positive serum M. avium subsp. paratuberculosis antibody responses in both herds, and the responses persisted in vaccinated cattle up to 7 years of age. Some vaccinated animals (29.4% in herd A and 36.2% in herd B) showed no serological reactivity to M. avium subsp. paratuberculosis. M. avium subsp. paratuberculosis-specific antibody responses were also detected in milk from Johne's disease-vaccinated animals, but fewer animals (39.3% in herd A and 49.4% in herd B) had positive results with milk than with serum samples. With vaccination against M. avium subsp. paratuberculosis, fecal shedding in both dairy herds was reduced significantly (P < 0.001). In addition, when selected Johne's disease-vaccinated and -infected animals were investigated for serological cross-reactivity to Mycobacterium bovis, no cross-reactivity was observed.

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

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

Persistence of Mycobacterium avium subsp. paratuberculosis in soil, crops, and ensiled feed following manure spreading on infected dairy farms.

The goal of this study was to determine the persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in soil, crops, and ensiled feeds following manure spreading. This bacterium was often found in soil samples, but less frequently in harvested feeds and silage. Spreading of manure on fields used for crop harvest is preferred to spreading on grazing pastures.

Persistance deMycobacterium aviumssp.paratuberculosisdans le sol, les récoltes et l'ensilage après l'épandage de fumier dans des fermes laitières infectées. Le but de cette étude était de déterminer la persistance de Mycobacterium avium ssp. paratuberculosis (MAP) dans le sol, les récoltes et l'ensilage après l'épandage de fumier. Cette bactérie se trouvait souvent dans des échantillons de sol, mais moins fréquemment dans les récoltes d'aliments pour le bétail et l'ensilage. L'épandage de fumier dans les champs utilisés pour la récolte des cultures est préféré à l'épandage dans les pâturages.(Traduit par Isabelle Vallières).

Regional distribution of two dairy-associated Salmonella enterica serotypes.

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

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

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

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

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

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.