Pharmacokinetics and pharmacodynamics of two in-feed chlortetracycline regimens provided to beef cattle.

Plasma chlortetracycline (CTC) concentration data were subjected to Monte Carlo simulation of area under the concentration curve (AUC) values related to bovine respiratory disease pathogen MIC distributions to evaluate target attainment rates. Crossbred Hereford heifers were randomly assigned into two treatment groups. Treatment group (A) received chlortetracycline (CTC) at a target dose of 22 mg/kg of bodyweight daily for 5 consecutive days (n = 8) and group (B) received CTC at 350 mg/head per day (1.5 ± 0.2 mg/kg based on actual bodyweights) for seven consecutive days (n = 8). Non-compartmental analysis was used to calculate plasma-free drug CTC area under the concentration curves. The mean observed (±SD) free drug AUC values were 4.18 (±1.72) µg × h/mL and 0.30 (±0.06) µg × h/mL for treatment groups A and B, respectively. The probability of target attainment for AUC24/MIC values of 25 and 12.5 was modeled using Monte Carlo simulations. Treatment group A achieved >90% target attainment (AUC24/MIC of 25) at an MIC of 0.06 µg/mL, whereas treatment group B displayed only 12.6% target attainment (AUC24/MIC of 12.5) at the lowest MIC evaluated (0.015 µg/mL). Both in-feed CTC regimens failed to obtain a reasonable target attainment rate in light of expected MIC distributions of potential pathogens.

Animal drug shortages limit veterinary therapeutic options and introduce artifacts in antimicrobial sales reporting.

Supply chain issues disrupt veterinary care and cause downstream consequences that alter the practice of veterinary medicine. Antimicrobials are just 1 class of pharmaceuticals that have been impacted by supply chain issues over the last couple of years. Since February 2021, 2 sponsors/manufacturers of penicillin products have reported shortages in the active pharmaceutical ingredient. With the release of the 2021 Summary Report on Antimicrobials Sold or Distributed for Use in Food-Producing Animals by the FDA, a key finding was a 19% decrease in penicillin sales and distribution from 2020 to 2021. Herein, we provide our clinicians' professional perspective regarding how drug shortages, specifically that of penicillin, might contribute to misconstrued patterns in antimicrobial use and what can be done by veterinarians and the FDA to minimize the impact of an antimicrobial drug shortage on animal health and well-being.

On-farm antimicrobial usage in commercial turkey production in the United States, 2013-2021.

A key component of antimicrobial stewardship is the ability to collect antimicrobial use data and ultimately use this information to ensure that administrations are necessary and effective. National antimicrobial sales data cannot help in this capacity because the data lack context, for example, details concerning target species and disease indication. The objective of this study was to continue the development of a system for collecting flock-level on-farm antimicrobial use data from the U.S. turkey industry and to have it be representative of national turkey production in the U.S. This study utilized a public-private partnership to enable collection and protection of sensitive flock-level data from an extremely large industry while releasing deidentified and aggregated information regarding the details of antimicrobial use on U.S. turkey farms over time. Participation was voluntary. Data were collected for the period 2013 through 2021 and are reported on a calendar year basis. Using production statistics from USDA:NASS as a denominator, the data supplied by participating companies represented approximately 67.3% of turkey production in the U.S. in 2013, approximately 69.1% in 2017, and approximately 71.4% in 2021. The data that were submitted for 2021 are based on approximately 149,000,192 turkeys slaughtered and 4,929,773,506 pounds liveweight produced. Detailed prescription records representing approximately 60-70% of the birds were available for the 2018-2021 dataset. The estimated percentage of turkey poults placed that received hatchery antimicrobials decreased from 96.9% in 2013 to 40.5% in 2021. The use of in-feed antimicrobials was practically eliminated, with in-feed tetracycline being the only medically important antimicrobial used in 2021. Use of in-feed tetracyclines decreased approximately 80% between 2013 and 2021. Water-soluble antimicrobial use declined over the study period. Between 2013 and 2021, water-soluble penicillin use decreased approximately 41% but water-soluble tetracycline use increased approximately 22%. Key diseases that were treated with water-soluble antimicrobials included bacterial poult enteritis and clostridial dermatitis. Efforts to reduce the incidence of these diseases would reduce the need for antimicrobial therapy, thereby enabling continued decreases in antimicrobial use without sacrificing animal welfare. However, this will require an investment in research to find efficacious and cost-effective mitigation strategies.

Antimicrobial usage in broiler chicken production in the United States, 2013-2021.

Although efforts to improve antimicrobial stewardship should include the collection of antimicrobial use data, most antimicrobial datasets collected at the national level consist of antimicrobial sales data which cannot inform stewardship. These data lack context, such as information regarding target species, disease indication, and regimen specifics like dose, route and duration. Therefore, the goal of this study was to develop a system for collecting data on the use of antimicrobials in the U.S. broiler chicken industry. This study utilized a public-private partnership to enable collection and protection of sensitive data from an extremely large industry while releasing deidentified and aggregated information regarding the details of antimicrobial use on U.S. broiler chicken farms over time. Participation was voluntary. Data were collected for the period 2013 through 2021 and are reported on a calendar year basis. Using production statistics from USDA:NASS as a denominator, the data supplied by participating companies represented approximately 82.1% of broiler chicken production in the U.S. in 2013, approximately 88.6% in 2017, and approximately 85.0% in 2021. The data that were submitted for 2021 are based on approximately 7,826,121,178 chickens slaughtered and 50,550,817,859 pounds liveweight produced. Granular flock-level treatment records were available for 75-90% of the birds represented in the 2018-2021 dataset. There was no use of antimicrobials in the hatchery for the years 2020 and 2021. Medically important in-feed antimicrobial use decreased substantially, with all in-feed tetracycline use being eliminated by 2020, and the use of virginiamycin being reduced by more than 97% since 2013. Medically important water-soluble antimicrobials are used for the treatment of disease in broiler production. Use decreased substantially for most water-soluble antimicrobials. The most important diseases necessitating treatment were necrotic enteritis and gangrenous dermatitis as well as E. coli-related disease. A focus on reducing the incidence of these diseases would reduce the need for antimicrobial therapy but will require an investment in research to find efficacious and cost-effective interventions for these diseases.

Antimicrobial use in 20 U.S. beef feedyards: 2018-2019.

The objective of this study was to report antimicrobial use in a convenience sample of U.S. beef feedyards for the years 2018 and 2019. In addition to antimicrobial use metrics, also reported are the indications for antimicrobial use and outcomes related to these indications. Antimicrobial use is characterized at the study and feedyard levels for a total of 1,141,846 head of cattle in 20 U.S. feedyards. Antimicrobial use is reported as milligrams of active antimicrobial ingredient per kilogram of liveweight sold (mg/kg-LW) and regimens of antimicrobials per animal year (Reg/AY). Regimens are described by antimicrobial class within use category as characterized by mg of active antimicrobial product per regimen (mg/Reg) and calendar days of administration per regimen (CDoA/Reg). A total of 1,128,515 regimens of medically important antimicrobials were captured from records. The number of regimens/100 head-in (Reg/100 head-in) are described in a subset of 10 feedyards with adequate data granularity to directly determine indications for antimicrobial administration. For the indications of bovine respiratory disease (BRD), Lameness (Lame), Liver Abscess Control (LAC), and Other (e.g., central nervous system disease, cellulitis) the Reg/100 head-in study-level values are 37.1, 0.8, 98.4, and 0.7, respectively, for 2018, with similar values for 2019. The regimens for BRD are further categorized in these 10 feedyards by the use categories in-feed, control of BRD, and individual animal therapy, yielding study level values of 4.6, 19.6, and 12.9 Reg/100 head-in, respectively, for 2018, with similar values for 2019. Outcomes of therapy for individual animal treatment of BRD, Lame, and Other are reported as treatment success, retreatment, or mortality by 30 days after the initial therapy of an animal for a disease. Treatment success rates (no treatment or mortality in the next 30 days) for 2018 in the 10 feedyards with sufficient data granularity are 76.5, 86.5, and 83.0% for BRD, Lame, and Other, respectively. The comparison of these results with other reports of antimicrobial use in North American feedyards highlights how differing approaches in calculating metric values may result in substantially different conclusions regarding antimicrobial use, especially in relation to long-duration uses.

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.

The association of multiple metrics for evaluating antimicrobial use in U.S. beef feedyards.

In order to accurately portray antimicrobial use in food animals, the need for standardized metrics, and an understanding of the characteristics of different metrics, has long been recognized. Fourteen U.S. feedyards were used to evaluate the effects of using centralized constants such as defined daily dose (DDD) and defined course dose (DCD) applied to the weight of medically important antimicrobials by class (mg) as opposed to using electronic individual animal treatment records and lot level in-feed antimicrobial records obtained from the same population. Three numerators were calculated directly from recorded data for each drug product: the number of antimicrobial regimens associated with indication (Reg), milligrams of drug administered per regimen (mg), and calendar days of administration for each regimen (CDoA). There were four use indications to which numerators were assigned: liver abscess control (LAC), bovine respiratory disease (BRD), lameness (lame), or all other indications combined (other). Three denominators were also calculated directly from the data, these being the number of days animals were present (head days), number of cattle received (head in), and kilograms of live weight sold (kg-LW). Numerators and denominators were calculated at the lot level. The use of DDD or DCD was explored to determine how their use would affect interpretation of comparisons between lots or feedyards. At the lot level across both study years, the lot estimate of nDDD differed from the CDoA value by >25% in 49.2% of the lots. The number of Defined Course Doses (nDCD) was then compared to the number of Regimens (Reg). Comparing nDCD to Reg at the lot level across both study years, the lot estimate of nDCD differed from the Reg value by >25% in 46.4% of lots. Both year and metric were also shown to affect numerical feedyard ranking by antimicrobial use according to seven different metrics. The analysis reported here adds to the body of literature reporting substantial effects of metric choice on the conclusions drawn from comparing antimicrobial use across multiple production sites.

Using telehealth clinical case vignettes to enhance clinical confidence and competence in veterinary students.

Veterinarians contribute substantially to the health of their patients and enhance the communities in which they live. Delivery of veterinary curricula continues to evolve to ensure that veterinary graduates are prepared to meet their professional obligations on Day One of their careers. In this study, veterinary practitioners were recruited to deliver telehealth case rounds to veterinary students at Kansas State University and Texas A&M University. Case discussions were hosted virtually once per month in the 2020-2021 and 2021-2022 academic years for a total of 16 sessions. Each presenting practitioner was instructed to develop a brief presentation for a case routinely seen in their practice, and to discuss important clinical decision points in diagnosis, treatment and management. Cases could also highlight important ethical or communication issues encountered in veterinary medicine. The overall goals of this project were to increase the quantity and diversity of clinical cases to which veterinary students were exposed during their professional training and to evaluate the feasibility and acceptability of telehealth technology as a teaching strategy. Student participants were surveyed to determine the effectiveness of telehealth sessions in increasing overall confidence and competence in case management, and veterinary presenters were surveyed to determine motivations for participating in the project and perceived value of the telehealth sessions. More than 95% of students indicated that participation in telehealth sessions increased their clinical confidence and competence. Presenting practitioners unanimously indicated that they would participate in similar instruction in the future. Recommendations are provided to improve the educational experience for future adopters of telehealth teaching sessions.

Detection of bovine inflammatory cytokines IL-1ß, IL-6, and TNF-α with a multiplex electrochemiluminescent assay platform.

Commercially available bovine-specific assays are limited in number, and multiplex assays for this species are rare. Our objective was to develop a multiplex assay for the bovine inflammatory cytokines IL-1ß, IL-6, and TNF-α using the Meso Scale Discovery U-PLEX platform. "Do-It-Yourself" ELISA kits that contained polyclonal antibodies, both unlabeled and biotinylated, and the specific recombinant bovine cytokine standard, were purchased for each of these three cytokines. The biotinylated antibodies were coupled to linkers that bind to specific locations within each well of the U-PLEX plate. Unique linkers were used for each of the cytokines. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to performing an optimization on the multiplex assay containing reagents for all three cytokines. To calculate cytokine concentrations, standard curves were developed using the recombinant cytokines and were run concurrently on each plate. Standard curves for IL-1ß and TNF-α were run at concentrations ranging from 0 to 50,000 pg/mL, and for IL-6 from 0 to 10,000 pg/mL. The average lowest level of detection concentration measured by the standard curves were 5.3 pg/mL, 0.92 pg/mL, and 22.34 pg/mL for IL-1ß, IL-6, and TNF-α respectively, as determined by data from seven plates containing bovine plasma samples from a combination of healthy and diseased cattle. The U-PLEX platform was a viable means to develop custom analyte- and species-specific multiplex assays using privately developed or purchased sets of commercially available reagents.

Cytokine and Haptoglobin Profiles From Shipping Through Sickness and Recovery in Metaphylaxis- or Un-Treated Cattle.

Fifty-six head of cattle, 28 animals with bovine respiratory disease complex (BRDC), and 28 healthy animals that were matched by treatment, sale barn of origin, day, and interactions among these variables, were identified from a population of 180 animals (60 each purchased at three sale barns located in Missouri, Tennessee, and Kentucky) enrolled in a study comparing animals receiving metaphylaxis to saline-treated controls. Cattle were transported to a feedlot in KS and assigned to treatment group. Blood samples were collected at Day 0 (at sale barn), Day 1, Day 9, and Day 28 (at KS feedlot), and transported to the US Meat Animal Research Center in Clay Center, NE where plasma was harvested and stored at -80°C until assayed for the cytokines IFN-γ, IL-1ß, IL-6, and TNF-α, and the acute stress protein haptoglobin (HPT). Our objectives were to determine if cytokine and haptoglobin profiles differed between control and metaphylaxis treatment groups over time, and if profiles differed between animals presenting with BRDC and those that remained healthy. There was no difference between the treated animals and their non-treated counterparts for any of the analytes measured. Sale barn of origin tended to affect TNF-α concentration. Differences for all analytes changed over days, and on specific days was associated with state of origin and treatment. The Treatment by Day by Case interaction was significant for HPT. The analyte most associated with BRDC was HPT on D9, possibly indicating that many of the cattle were not exposed to respiratory pathogens prior to entering the feedlot.

Estimates of on-farm antimicrobial usage in broiler chicken production in the United States, 2013-2017.

Antimicrobial use is a key selective force behind the emergence of resistant bacteria. Therefore, optimizing strategies for more efficacious and targeted antimicrobial use is an essential component of efforts to combat antimicrobial resistance. To bolster stewardship programmes in animal agriculture, processes are needed for the systematic collection of on-farm antimicrobial use data. The objective of this study was to develop a system for collecting on-farm antimicrobial use data from the US broiler industry and to have it be representative of the largest commercial broiler producers in the United States that comprise the vast majority of national broiler production. Participation was voluntary. Data were collected for the period 2013 through 2017 and are reported on a calendar year basis. Using statistics from USDA:NASS as a denominator, the data supplied by participating companies accounted for approximately 81.7% of broiler production in the United States in 2013 and increased to approximately 87.2% in 2017. The data that were submitted for 2017 are based on approximately 7,897,339,357 chicks placed, 7,541,449,430 chickens slaughtered and 48,225,124,865 pounds liveweight produced. The use of antimicrobials in the hatchery decreased substantially between 2013 and 2017; the approximate percentage of broiler chicks placed that received hatchery antimicrobials decreased from 93% in 2013 to 17% in 2017. Medically important in-feed antimicrobial use decreased substantially. For example, in-feed tetracycline use decreased approximately 95% between 2013 and 2017. Medically important water-soluble antimicrobial use decreased substantially for most antimicrobials. Between 2013 and 2017, water-soluble penicillin use decreased approximately 21%, water-soluble tetracycline use decreased approximately 47%, and water-soluble lincomycin use decreased approximately 28%. While a reduction in antimicrobial amounts used may be an important indicator of improved stewardship, reducing the need for antimicrobials through improved disease prevention should be considered a more important objective and a better indicator of overall flock health and optimal antimicrobial use.

Antimicrobial use in 22 U.S. beef feedyards: 2016-2017.

This study describes antimicrobial use in 22 U.S. beef feedyards by use of two metrics: milligrams of antimicrobial per kilogram of liveweight sold (mg/kg-LW) and antimicrobial regimens per animal year (Reg/AY). The primary objectives were to determine the feasibility of collecting antimicrobial use data at the level of the production system and to identify factors unique to feedyard systems which may confound interpretation of summarized antimicrobial use measures. Records were accessed directly from feedyards or through a data intermediary and then subjected to a process of standardization and quality assurance. Use is reported at the study level with all individual cattle lots from all feedyards combined, as well as at the feedyard level where values were first calculated for each feedyard and then expressed as means, standard deviations and median values. Use at the study level is first reported by antimicrobial class and then by antimicrobial class within the use categories of in-feed, control of bovine respiratory disease (BRD) and individual animal treatment. Due to wide variations of antimicrobial class within use category, feedyard level antimicrobial use is reported only by antimicrobial class. Use values for medically important and not medically important antimicrobial classes are reported separately. Regimens are described for each antimicrobial class within use category by milligrams per regimen and by timeframe from first to last administration. The authors' selection of measures reported here was driven by a desire to minimize necessary assumptions, resulting in transparent reporting of values which can be directly related back to the factors within feedyard systems which may have influenced calculations. Reporting the number of regimens stratified by antimicrobial class within use category and then describing the characteristics of the reported regimens (milligrams per regimen and timeframe) optimize these objectives for reporting.

Estimates of on-farm antimicrobial usage in turkey production in the United States, 2013-2017.

With increasing concern about the emergence and spread of resistant bacteria, there is an increasing motivation to optimize antimicrobial use administrations in animal agriculture. A key component of antimicrobial stewardship is the ability to collect antimicrobial use data and ultimately use this information to assess that administrations are necessary and effective. The objective of this study was to develop a system for collecting on-farm antimicrobial use data from the US turkey industry and to have it be representative of the largest commercial turkey producers in the United States that comprise the vast majority of national turkey production. Participation was voluntary. Data were collected for the period 2013 through 2017 and are reported on a calendar year basis. Using statistics from USDA:NASS as a denominator, the data supplied by participating companies represented approximately 67.3% of turkey production in the United States in 2013 and increased to approximately 69.8% in 2017. The data that were submitted for 2017 are based on approximately 187,016,604 poults placed, 164,081,335 turkeys slaughtered, and 5,178,431,422 pounds liveweight produced. The estimated percentage of turkey poults placed that received hatchery antimicrobials decreased from 96% in 2013 to 41% in 2017. Medically important in-feed antimicrobial use decreased substantially. For example, in-feed tetracycline use decreased approximately 67% between 2013 and 2017. Medically important water-soluble antimicrobial use decreased substantially for most antimicrobials. Between 2013 and 2017, water-soluble penicillin use decreased approximately 42%, water-soluble tetracycline use decreased approximately 28%, and water-soluble lincomycin use decreased approximately 46%. Reducing the total amounts of antimicrobials used might be a crude indicator for mitigating the selection of antimicrobial resistance. Reducing the need for such use and verifying that treatment regimens deliver beneficial outcomes to animal health are more meaningful objectives.

Comparison of surveys and use records for quantifying medically important antimicrobial use in 18 U.S. Beef Feedyards.

The objective of this study was to evaluate agreement between medically important antimicrobial use metrics derived from in-person surveys of feedyard management as opposed to metrics derived from production unit-level antimicrobial use records. Survey respondents were asked to estimate values which would allow calculation of the metrics of regimens per animal year (Reg/AY) and milligrams of antimicrobial per kilogram of liveweight sold (mg/kg-LW). At the study level, values were calculated by antimicrobial class within the use categories of in-feed use, control of bovine respiratory disease (BRD) and individual animal treatment. At the feedyard level, values were calculated by total overall use and total use within use category. Feedyard level correlation coefficients between survey and record values for total use were 0.76 (p = .0004) and 0.73 (p = .0009) for Reg/AY and mg/kg-LW, respectively. Correlation coefficients for use category within metric ranged from 0.25 (p = .3224) to 0.65 (p = .0061). Comparing feedyard level survey and record values for total Reg/AY and mg/kg-LW using a Wilcoxon signed-rank test resulted in p-values (95% CI) of 0.3247 (-1.06, 0.25) and 0.7019 (-14.49, 10.387), respectively. Evaluation of comparative rankings as total use by metric indicated that for Reg/AY, only two of the five top-ranked feedyards were consistent between the two data sources. The relationship for mg/kg-LW demonstrated the lowest two and highest three values were consistent between sources; however, the ranking similarities appear to markedly decline in the middle ranks. This report demonstrates that survey-based antimicrobial use data may closely reflect summary values determined from records across multiple beef feedyards. However, individual feedyard relationships between their record and survey values vary widely and ranking by survey may lead to different conclusions as to highest and lowest use than ranking by data collected from use records.

Antimicrobial use quantification in adult dairy cows - Part 3 - Use measured by standardized regimens and grams on 29 dairies in the United States.

This study describes antimicrobial use in terms of standardized regimens per cow year (REG/CY) and grams per cow year (GMS/CY) for 29 dairies in the United States during the years 2016 and 2017. To explore potential priorities for antimicrobial stewardship programs, these measures were stratified by both disease syndrome and antimicrobial class. Potential confounders of use measurements are discussed and challenges for measure interpretation are identified. When measured as REG/CY, the results indicate that mastitis is the disease syndrome with the greatest contribution to overall antimicrobial use. However, when GMS/CY is measured, metritis, lameness and unknown disease syndromes are also significant contributors. When use is stratified by antimicrobial class, measures of REG/CY indicate the greatest magnitude of use is the cephalosporin class. However, when measures of GMS/CY are stratified by drug class, use within the penicillin class contributes more than any other single class. These differences highlight the need for a more complete understanding of the relationship between antimicrobial use measures and their relationship to antimicrobial resistance selection pressure.

Antimicrobial use quantification in adult dairy cows - Part 1 - Standardized regimens as a method for describing antimicrobial use.

This study describes a process to acquire and convert farm treatment records into a standardized regimen format. Multiple sources of on-farm data were utilized to convert the original treatment records to standardized regimens, enabling the generation of objective, granular descriptions of antimicrobial use. These standard regimen descriptions allow antimicrobial use data to be qualitatively stratified by active substance, disease syndrome treated, use category, and route of administration. Quantitative distributions are available for the grams of active substance per administration, the grams of active substance per regimen, the number of administrations, number of days of therapy and the time frame between the first and last administration. Granular quantification by this method informs future research, surveillance and policy.

Antimicrobial use quantification in adult dairy cows - Part 2 - Developing a foundation for pharmacoepidemiology by comparing measurement methods.

As stewardship programmes seek to influence therapeutic choices and reduce resistance selection pressure, it is essential to understand the multiple factors which may influence any given antimicrobial use measure. This article compares 9 measures of antimicrobial use pertaining to adult cows in US dairy systems based on treatment records standardized to a single standardized regimen (SReg) format. There are 3 fundamental parameters underlying the nine measures: amount of active substance (AMOUNT), number of SRegs (ANIMALS) and treatment time (TIME). Additionally, three use categories were considered in the analysis: control of mastitis (Dry Cow), treatment of detected mastitis (Clinical Mastitis) and treatment of other diseases (Other Treatment). The assumptions necessary for calculation of each of the nine measures are identified. The influence of measure choice on farm rank was explored at the farm level both within and across fundamental parameters. Across all use categories, when measures of TIME and ANIMALS were compared, correlations between measures were greater than 0.91. However, when measures of AMOUNT were compared to either measures of TIME or ANIMALS, the correlation between measures was more variable across use categories (R = 0.31-0.91). These comparisons demonstrate that the selection of antimicrobial use metric can affect which dairies are considered the highest users of antimicrobials. Measurement selection also influences which use category has the greatest potential for use reduction. This indicates that measure selection can influence which use category is prioritized as a target for reduction of antimicrobial use. Utilization of this information may allow those developing dairy antimicrobial stewardship programmes to better understand the potential effects of measurement selection on driving changes in antimicrobial use.

Differences between predicted outer membrane proteins of genotype 1 and 2 Mannheimia haemolytica.

BACKGROUND: Mannheimia haemolytica strains isolated from North American cattle have been classified into two genotypes (1 and 2). Although members of both genotypes have been isolated from the upper and lower respiratory tracts of cattle with or without bovine respiratory disease (BRD), genotype 2 strains are much more frequently isolated from diseased lungs than genotype 1 strains. The mechanisms behind the increased association of genotype 2 M. haemolytica with BRD are not fully understood. To address that, and to search for interventions against genotype 2 M. haemolytica, complete, closed chromosome assemblies for 35 genotype 1 and 34 genotype 2 strains were generated and compared. Searches were conducted for the pan genome, core genes shared between the genotypes, and for genes specific to either genotype. Additionally, genes encoding outer membrane proteins (OMPs) specific to genotype 2 M. haemolytica were identified, and the diversity of their protein isoforms was characterized with predominantly unassembled, short-read genomic sequences for up to 1075 additional strains. RESULTS: The pan genome of the 69 sequenced M. haemolytica strains consisted of 3111 genes, of which 1880 comprised a shared core between the genotypes. A core of 112 and 179 genes or gene variants were specific to genotype 1 and 2, respectively. Seven genes encoding predicted OMPs; a peptidase S6, a ligand-gated channel, an autotransporter outer membrane beta-barrel domain-containing protein (AOMB-BD-CP), a porin, and three different trimeric autotransporter adhesins were specific to genotype 2 as their genotype 1 homologs were either pseudogenes, or not detected. The AOMB-BD-CP gene, however, appeared to be truncated across all examined genotype 2 strains and to likely encode dysfunctional protein. Homologous gene sequences from additional M. haemolytica strains confirmed the specificity of the remaining six genotype 2 OMP genes and revealed they encoded low isoform diversity at the population level. CONCLUSION: Genotype 2 M. haemolytica possess genes encoding conserved OMPs not found intact in more commensally prone genotype 1 strains. Some of the genotype 2 specific genes identified in this study are likely to have important biological roles in the pathogenicity of genotype 2 M. haemolytica, which is the primary bacterial cause of BRD.

Classification of 16S rRNA reads is improved using a niche-specific database constructed by near-full length sequencing.

Surveys of microbial populations in environmental niches of interest often utilize sequence variation in the gene encoding the ribosomal small subunit (the 16S rRNA gene). Generally, these surveys target the 16S genes using semi-degenerate primers to amplify portions of a subset of bacterial species, sequence the amplicons in bulk, and assign to putative taxonomic categories by comparison to databases purporting to connect specific sequences in the main variable regions of the gene to specific organisms. Due to sequence length constraints of the most popular bulk sequencing platforms, the primers selected amplify one to three of the nine variable regions, and taxonomic assignment is based on relatively short stretches of sequence (150-500 bases). We demonstrate that taxonomic assignment is improved through reduced unassigned reads by including a survey of near-full-length sequences specific to the target environment, using a niche of interest represented by the upper respiratory tract (URT) of cattle. We created a custom Bovine URT database from these longer sequences for assignment of shorter, less expensive reads in comparisons of the upper respiratory tract among individual animals. This process improves the ability to detect changes in the microbial populations of a given environment, and the accuracy of defining the content of that environment at increasingly higher taxonomic resolution.