METAPHYLAXIS FOR RESPIRATORY DISEASE IN HIGH-RISK STOCKER CATTLE: IMPACTS ON MANNHEIMIA HAEMOLYTICA, THE MICROBIOME, AND THE RESISTOME

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1018463
• Grant No.: 2019-67017-29111
• Cumulative Award Amt.: $499,845.00
• Proposal No.: 2018-06228
• Project Start Date: Apr 1, 2019
• Project End Date: Mar 31, 2024
• Grant Year: 2019
• Program Code: [A1362]- Understanding Antimicrobial Resistance

Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762

Performing Department
CVM Pathobiology/Population Me

Non Technical Summary
Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in beef cattle, and mass antimicrobial administration (metaphylaxis) is the most effective prevention. Metaphylaxis can also be life-saving in other livestock and humans, but the practice may be compromised by spread of multidrug resistant (MDR) pathogens. MDR isolates of the BRD pathogen Mannheimia haemolytica can be found in 90% of some cattle 14 days after metaphylaxis. The mechanisms leading to MDR after metaphylaxis are unknown, but rapid transfer of mobile genetic elements (MGE) encoding antimicrobial resistance (AMR) is likely. Using a randomized field trial assessing treated and untreated conventionally managed cattle, we will 1) compare prevalence of nasopharyngeal MDR M. haemolytica isolates, AMR genes, and MGE, using culture, susceptibility testing, and whole genome sequencing; 2) compare nasopharyngeal metagenomes, using 16S amplicon sequencing; 3) compare the nasopharyngeal resistome, using target-enriched sequencing of AMR and MGE gene sequences; and 4) use target-enriched sequencing to compare absolute abundance of M. haemolytica and sequences specific to other BRD pathogens. This approach will provide unprecedented resolution of the ecology of respiratory AMR in cattle receiving metaphylaxis, revealing targets for mitigating AMR. This research supports sustainability of U.S. agriculture with evidence-based strategies to ensure livestock health and limit AMR, by improving understanding of the emergence and transmission of MGE encoding AMR to medically important AM, and developing tools to track AMR genes in animal environments.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3310	1100	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1100 - Bacteriology;

Keywords

bacteria

beef cattle

bovine

macrolides

respiratory

Goals / Objectives
Goal 1: To compare the prevalence of antimicrobial resistance genes and mobile genetic elements in multidrug resistant M. haemolytica isolated from nasopharyngeal swabs (NPS) of cattle given tulathromycin metaphylaxis or not, and/or other antimicrobial treatment.Goal 2: To characterize and compare the nasopharyngeal microbiome, using metagenomic 16S rRNA gene sequencing of DNA isolated from NPS of cattle given metaphylaxis or not, and/or other antimicrobial treatment.Goal 3: To characterize and compare all known antimicrobial resistance genes and mobile genetic elements (i.e. the nasopharyngeal metagenomic resistome) in cattle given metaphylaxis or not, and/or other antimicrobial treatment; and to compare the metagenomic resistome to antimicrobial resistance profiles of M. haemolytica isolated from the same cattle (completed in Goal 1).Goal 4: To describe genomes or genetic elements of known bacterial bovine respiratory disease pathogens in the nasopharyngeal metagenome of cattle given metaphylaxis or not, and/or other antimicrobial treatment.Goal 5: To describe bovine respiratory disease morbidity, mortality, and weight gain over a 21-day backgrounding period in cattle sampled for Goals 1 through 4.ActivitiesConduct research trials to test the effect of metaphylaxis or other antimicrobial treatment on the antimicrobial resistance genes, resistome, and microbiome of cattle, to accomplish our goals outlined above.Present the results of research to microbiologists, geneticists, veterinarians, and cattle producers at national and international conferences.Make available to other scientists the data describing the microbiome, resistome, and the whole genome sequences of Mannheimia haemolytica that will be obtained in the research undertaken to accomplish our goals.

Project Methods
We and others have shown a remarkable increase in the prevalence of a multidrug resistant (MDR) pathogen, M. haemolytica, over a short period of time in cattle given antimicrobial (AM) metaphylaxis (META) to control BRD. However, that work did not allow us to determine whether a) META alone induced high prevalence MDR, b) subsequent therapy with additional AM (AM TX) for clinical BRD in some of the cattle induced high prevalence MDR, or c) high prevalence MDR would have occurred even without metaphylaxis or BRD therapy, because of other factors, such as co-mingling of cattle from multiple origins. This proposal addresses those important questions.To determine mechanisms by which MDR can propagate rapidly in a population, we need to assess not only the MDR agent we know about (M. haemolytica) but also the thousands of other genera that may serve as unexpected reservoirs for antimicrobial resistance (AMR) genes, or MGE that help AMR genes move rapidly between bacteria. We propose to do this by comparing the resistome and microbiome among cattle exposed to AM drugs for META, AM TX, or both.In the proposed research, we will use guarded nasopharyngeal swabs (NPS) to characterize respiratory bacteria by culture and metagenomic sequencing. Most research investigating the microbiology of BRD in cattle populations in recent years has used NPS, so our approach will be consistent. Members of our team have shown that NPS represent microbes in the bovine lower respiratory with good to excellent agreement, indicating that our findings will be relevant to bacteria that cause BRD. In the proposed research, we will culture NPS from each individual calf enrolled, but for metagenomic sequencing we will pool NPS from treatment groups: META and NO META on d. 0, and META, META + AM TX, NO META, and NO META + AM TX on d. 21.The absolute number of pathogenic bacteria that develop AMR in a population given mass medication may be the factor that most importantly influences outcomes such as rates of morbidity, mortality, and treatment failure in that population. Alternatively, the specific genetic variants of pathogenic species that develop AMR may most importantly influence these outcomes. With the target-enriched metagenomic sequencing approach we will develop in the proposed research, we expect to be able to identify the absolute number of M. haemolytica genomes in the DNA we purify from NPS; we also expect to be able to identify sequence variants in the M. haemolytica genomes we identify. This will allow us to determine which measure of increased prevalence is most closely related to outcomes in the sampled cattle, and in future trials.Metagenomic sequencing can provide a complete picture of the bacteria community (microbiome) in a respiratory or other sample. However, the 16S gene shotgun sequencing commonly used cannot reliably identify bacteria at the species level. Here we propose to overcome this limitation with the novel target-enriched metagenomic sequencing approach we will develop to specifically identify M. haemolytica and other BRD pathogens. We will develop baits to target the entire genome of M. haemolytica, the primary focus of this study. We will also develop baits for specific sequences of the 3 other bacteria that most commonly also contribute to BRD: Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. We have shown this approach to accurately and sensitively identify specific gene sequences.To discover mechanisms that lead to rapid dissemination of MDR in populations given META, we need to track specific bacteria that harbor and share AMR genes and mobile genetic elements (MGE) encoding AMR from the beginning to the end of the period of AM exposure, and beyond. The novel target-enriched metagenomic sequencing approach we will develop should allow us to identify specific sequences or single nucleotide polymorphisms (SNPs) that uniquely identify bacteria that also carry AMR genes and/or MGE, as we have shown previously. If successful, this application of the tool could allow specific AMR bacteria to be tracked, and their interactions with other bacteria to be monitored, in populations or ecosystems. This would provide unprecedented resolution of the bacterial interactions that lead to dissemination of AMR and MDR, providing a powerful opportunity to block or modify relevant pathways. For example, if unexpected bacterial genera are found to harbor AMR genes or MGE that later spread to BRD bacteria, future research could test targeted mitigation of these genera, possibly through gene editing strategies mediated by phage therapy. We may also apply different doses or durations of therapy in future research to modify dissemination of AMR genes or MGE.The efforts to change knowledge, actions and conditions will include: 1) presentation of information resulting from the research at national and international conferences of microbiologists, geneticists, veterinarians, animal scientists, and cattle producers; 2) dissemination of databases including the nasopharyngeal microbiome, resistome, and M. haemolytica whole genome sequences, along with the metadata including cattle health and growth data; 3) dissemination of the methodology for using the novel target-enriched shotgun sequencing method and the expanded library of baits that can be specifically used to identify genetic determinants for BRD pathogens as well as genetic determinants encoding antimicrobial resistance.

Progress 04/01/19 to 03/31/24

Outputs
Target Audience:Veterinarians in cattle practice and researchers studying antimicrobial resistance in BRD. Changes/Problems:The data resulting from the targeted-enriched pull-down assay to identify Mannheimia haemolytica genomes in metagenomes (Objective 4) was more challenging to evaluate than expected. As of August 2024 we have solved the problems that delayed completion of Objective 4, and we reported the results in a poster (Doster et al.) presented at the 2024 Bovine Respiratory Disease Symposium in Denver, CO August 7-8, 2024. Completion of this evaluation will allow us to complete the final two publications describing work from the project that included assessment using this method. What opportunities for training and professional development has the project provided?This project supported the PhD training of a DVM, Dr. William B. Crosby. Dr. Crosby obtained his PhD in May 2023 as a result of this work. Veterinary and undergraduate students who assisted with sample collection during the field trials also obtained research experience. How have the results been disseminated to communities of interest?Abstracts of the research were presented at the Conference for Research Workers in Animal Disease, the American Association of Bovine Practitioners, the Annual Forum of the American College of Veterinary Internal Medicine, and at the World Buiatrics Congress in Madrid, Spain in 2022, and in Cancun, Mexico in May 2024. Also, a pubication describing a portion of the work was published in Frontiers in Veterinary Science in the fall of 2023. Two more publications related to the work at in progress and expect to be published in late 2024 or early 2025. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Major activities completed: We collected nasopharyngeal swabs from 331 crossbred beef cattle on arrival (day 0), day 21, and day 70, with half of the cattle having received metaphylaxis (META) and the other half not (NO META). We checked the cattle daily from day 0 to day 21 and administered antimicrobial treatment for bovine respiratory disease (BRD) or other health problems as needed, based on standardized scoring systems established a priori. We completed culture of nasopharyngeal swabs to isolate M. haemolytica, and characterized the antmicrobial susceptibility of the isolates. We completed whole genome sequencing of the M. haemolytica isolates, and we identified antimicrobial resistance genes (ARG) and evidence of mobile genetic elements (integrative conjugative elements, ICE) in the genomes. We evaluated the relationship between metaphylaxis, isolation of M. haemolytica, and phenotypic or genotypic resistance and health outcomes in the cattle. Specific objectives met: We completed objective 1, 2, 3, and 5. We are still working on Objective 4. Significant results achieved: Animals in the META group had higher odds of isolation of MDR M hemolytica at 3 weeks [OR (95% CI) = 13.08 (5-30.9), p < 0.0001] and 10 weeks [OR (95% CI) = 5.92 (1.34-26.14), p = 0.019] after arrival. There was no difference in risk of isolation of any M haemolytica (resistant or susceptible) between META and NO META groups at all timepoints. Animals in the NO META group had 3 times higher odds of being treated for BRD [WK3: OR (95% CI) = 3.07 (1.70-5.52), p = 0.0002; WK10: OR (95% CI) = 2.76 (1.59-4.80), p = 0.0002]. Antimicrobial resistance genes found within isolates were associated with integrative conjugative element (ICE) genes. Tulathromycin metaphylaxis increased risk of isolation of MDR M haemolytica and in this population, the increase in MDR M haemolytica appeared to be associated with ICE containing antimicrobial resistance genes for multiple antimicrobial classes. There was no effect of tulathromycin on richness or diversity of the microbiome on WK3. However, Shannon's diversity index decreased at WK3 compared to arrival, and there was a difference in microbial community structure in all TxGroups at WK3 compared to arrival, due to an increase in Mycoplasmataceae at WK3 (50-80%) compared to arrival (25-50 %). At WK3 there was an increase in richness and diversity of ARGs compared to arrival in animals that received metaphylaxis, and an increase in diversity of ARGs in animals that received no antimicrobials. Aminoglycoside ARGs were the only class with differences in relative abundance among TxGroups at WK3. Although this has not yet been published, we used the new target-enriched sequencing method we developed to show that multiple variants of M haemolytica can be present in the same animal. This is completely new information which shows us that M haemolytica, a leading cause of bovine respiratory disease in cattle, is more complex than previously understood. In the past it was believed that only one variant of M haemolytica was present in a cow or calf, but now we have learned that multiple variants are present at the same time. This indicates that this organism is more variable than we previously understood. Key outcomes: For cattle treated with tulathromycin metaphylaxis, M haemolytica isolated from their respiratory tract had significantly higher rates of phenotypic antimicrobial resistance, and multidrug resistance, than cattle not receiving metaphylaxis. Resistance to the macrolide class of antimicrobials, which includes tulathromycin, was particularly high. However, the evaluation of the genetic markers of resistance in the metagenomes from DNA isolated from pooled nasopharyngeal swabs did not reveal significantly more genes for macrolide resistance in cattle receiving metaphylaxis. This was surprising, and it indicates that evaluation of the live bacteria isolated from the cattle yielded different information than we obtained from evaluating the sequences of bacterial DNA isolated from respiratory tract of the cattle. This is an important finding, because it shows us that two different methods that are commonly used to evaluate antimicrobial resistance yielded quite different answers. This tells us that, as we go forward to try to identify ways to track and understand antimicrobial resistance, we should continue to isolate bacteria to evaluate, and not rely only on genetic sequencing of bacterial DNA. Another important finding was that, although bacteria isolated from cattle that received metaphylaxis were more likely to display antimicrobial resistance than bacteria isolated from cattle that did not receive metaphylaxis, the cattle were less likely to become sick with BRD, and they gained significantly more weight than cattle that did not receive metaphylaxis. This is an ethical dilemma: cattle that received metaphylaxis stayed more healthy, even though antimicrobial resistant bacteria were more likely to be isolated from them! So now we need to figure out ways to keep cattle as healthy as they stay when they receive metaphylaxis, while not increasing antimicrobial resistance.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Crosby WB, Karisch B, Loy JD, Hiott L, Pittman A, Austin FW, Epperson W, Blanton Jr J, Morley PS, Capik SF, Jackson CR, Frye J, Woolums A. The effect of tulathromycin on morbidity and prevalence of Mannheimia haemolytica genotype 2 in 84 stocker heifers. Proceedings of the Conference for Research Workers in Animal Disease 2020. Virtual conference. Abstract #153.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Crosby WB, Richeson JT, Loy JD, Gow SP, Capik SF, Padilla N, Woolums A, Morley PS. Comparison of 3 sampling methods for recovery of Mannheimia haemolytica from feedlot cattle. Conference of Research Workers in Animal Diseases (CRWAD). Virtual (online only) meeting. December 5-8, 2020. Abstract #246.
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Crosby WB, Richeson JT, Loy JD, Gow SP, Seo KS, Capik SF, Padilla N, Woolums AR, Morley PS. Comparison of sampling methods and diagnostic techniques for recovery of Mannheimia haemolytica from feedlot cattle. Conference of the American Association of Bovine Practitioners (AABP). Salt Lake City, UT. October 7-9, 2021
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Crosby WB, R Richeson JT, Loy JD, Gow SP, Seo KS, Capik SF, Woolums AR, Morley PS. Comparison of sampling and diagnostic techniques for recovery of Mannheimia haemolytica from feedlot cattle. Conference for Research Workers in Animal Disease (CRWAD). Chicago IL. December 5-7, 2021.
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Crosby WB, Pittman A, Karisch BB, Hiott LM, Loy JD, Epperson WB, Capik SF, Morley PS, Frye JG, Jackson CR, Woolums AR. Tulathromycin metaphylaxis increases prevalence of multidrug resistant Mannheimia haemolytica while improving health in stocker calves. American Association of Bovine Practitioners (AABP) Conference Proceedings. Long Beach CA. September 22-24, 2022.
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Crosby WB, Pinnell LJ, Richeson JT, Wolfe C, Castle J, Loy JD, Gow SP, Seo KS, Capik SF, Woolums AR, Morley PS. Comparison of sampling and diagnostic techniques for recovery of Mannheimia haemolytica from feedlot cattle. 31st World Buiatrics Congress. Madrid, Spain. September 4-8, 2022. #ID-59
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Crosby WB, Pittman A, Karisch BB, Loy DJ, Epperson WB, Capik SF, Morley PS, Frye JG, Jackson CR, Woolums AR. Effect of tulathromycin metaphylaxis on Mannheimia haemolytica isolation and health outcomes in stocker heifers. American College of Veterinary Internal Medicine Annual Forum. Dallas, TX. June 22, 2022.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Woolums A. Effect of tulathromycin metaphylaxis on the nasopharyngeal microbiome, resistome, and Mannheimia haemolytica isolation in cattle. 32nd World Buiatrics Congress, Canc�n, Mexico, May 20 - 24, 2024.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Crosby WB, Karisch BB, Hiott LM, Pinnell LJ, Doster E, Wolfe C, PIttman A, Frye JG, Jackson CR, Loy JD, Epperson WB, Blanton Jr. J, Capik S, Morley PS, Woolums AR. Effect of metaphylaxis on the nasopharyngeal microbiome, resistome, and Mannheimia haemolytica in stocker heifers. Conference for Research Workers in Animal Disease (CRWAD). Chicago IL. January 20-23, 2024. #P002.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Doster E, Wolfe C, Crosby WB, Clawson ML, Woolums AR, PInnell LJ. Morley PS. Enriching without culture: target-enriched metagenomics allows for strain-level characterization of M. haemolytica. Conference for Research Workers in Animal Disease (CRWAD). Chicago IL. January 20-23, 2024. #195.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Doster E, Pinnell LJ, Wolfe CA, Crosby WB, Valeris-Chacin R, Richeson JT, Noyes NR, Clawson ML, Woolums AR, Morley PS. Culture independent target-enriched sequencing enables characterization of genetic variants of Mannheimnia haemolytica in metagenomic samples. 2024 Bovine Respiratory Disease Symposium. Denver CO. August 7-8, 2024.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Crosby WB, Karisch BB, Hiott LM, Pinnell LJ, Pittman A, Frye JG, Jackson CR, Loy JD, Epperson WB, Blanton J Jr, Capik SF, Morley PS. Woolums AR. Tulathromycin metaphylaxis increases nasopharyngeal isolation of multidrug resistant Mannheinia haemolytica in stocker heifers. Front Vet Sci. 2023. Nov 20;10:1256997, doi:10.3389/fvets.2023.1256997. PMID: 38053814.

Progress 04/01/22 to 03/31/23

Outputs
Target Audience:Veterinarians in cattle practice and researchers studying antimicrobial resistance in BRD. Changes/Problems:The data resulting from the targeted-enriched pull-down assay to identify Mannheimia haemolytica genomes in metagenomes (Objective 4) has proven to be challenging to evaluate. Although the project has ended, we will continue to work on this, because the approach will be powerful if it is ultimately successful. What opportunities for training and professional development has the project provided?This project supported the PhD training of a DVM, Dr. William B. Crosby. Dr. Crosby obtained his PhD in May 2023 as a result of this work. Veterinary and undergraduate students who assisted with sample collection during the field trials also obtained research experience. How have the results been disseminated to communities of interest?Abstracts of the research were presented at the Conference for Research Workers in Animal Disease in January 2024, and at the World Buiatrics Congress in May 2024. Also, a pubication describing a portion of the work was published in Frontiers in Veterinary Science in the fall of 2023. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Major activities completed: We completed the analysis of the metagenome and resistome data for all cattle for all years. Specific objectives met: We have completed objective 1, 2, 3, and 5. We are still working on Objective 4. Significant results achieved: For all 4 trials, META calves had higher odds of isolation of MDR MH at 3 weeks (OR (95% CI)=13.08 (5-30.9), P<0.0001) after arrival. There was no difference in risk of isolation of any MH (resistant or susceptible) between META and NO META groups at all timepoints. Animals in the NO META group had 3 times higher odds of being treated for BRD (WK3: OR (95% CI)=3.07 (1.70-5.52), P=0.0002; WK10: OR (95% CI)=2.76 (1.59-4.80), P=0.0002). Antimicrobial resistance genes found within MH were associated with integrative conjugative element (ICE) genes. There was no effect of tulathromycin on richness or diversity of the microbiome on WK3. However, Shannon's diversity index decreased at WK3 compared to arrival, and there was a difference in microbial community structure in all TxGroups at WK3 compared to arrival, due to an increase in Mycoplasmataceae at WK3 (50-80%) compared to arrival (25-50 %). At WK3 there was an increase in richness and diversity of ARGs compared to arrival in animals that received metaphylaxis, and an increase in diversity of ARGs in animals that received no antimicrobials. Aminoglycoside ARGs were the only class with differences in relative abundance among TxGroups at WK3. Key outcomes: For cattle treated with tulathromycin metaphylaxis, Mannheimia haemolytica isolated from their respiratory tract had significantly higher rates of phenotypic antimicrobial resistance, and multidrug resistance, than cattle not receiving metaphylaxis. Resistance to the macrolide class of antimicrobials, which includes tulathromycin, was particularly high. However, the evaluation of the genetic markers of resistance in the metagenomes from DNA isolated from pooled nasopharyngeal swabs did not reveal significantly more genes for macrolide resistance in cattle receiving metaphylaxis. This was surprising, and it indicates that evaluation of the live bacteria isolated from the cattle yielded different information than we obtained from evaluating the sequences of bacterial DNA isolated from respiratory tract of the cattle. This is an important finding, because it shows us that two different methods that are commonly used to evaluate antimicrobial resistance yielded quite different answers. This tells us that, as we go forward to try to identify ways to track and understand antimicrobial resistance, we should continue to isolate bacteria to evaluate, and not rely only on genetic sequencing of bacterial DNA. Another important finding was that, although bacteria isolated from cattle that received metaphylaxis were more likely to display antimicrobial resistance than bacteria isolated from cattle that did not receive metaphylaxis, the cattle were less likely to become sick with BRD, and they gained significantly more weight than cattle that did not receive metaphylaxis. This is an ethical dilemma: cattle that received metaphylaxis stayed more healthy, even though antimicrobial resistant bacteria were more likely to be isolated from them! So now we need to figure out ways to keep cattle as healthy as they stay when they receive metaphylaxis, while not increasing antimicrobial resistance.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Woolums A. Effect of tulathromycin metaphylaxis on the nasopharyngeal microbiome, resistome, and Mannheimia haemolytica isolation in cattle. 32nd World Buiatrics Congress, Canc�n, Mexico
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Crosby WB, Karisch BB, Hiott LM, Pinnell LJ, Doster E, Wolfe C, PIttman A, Frye JG, Jackson CR, Loy JD, Epperson WB, Blanton Jr. J, Capik S, Morley PS, Woolums AR. Effect of metaphylaxis on the nasopharyngeal microbiome, resistome, and Mannheimia haemolytica in stocker heifers. Conference for Research Workers in Animal Disease (CRWAD). Chicago, IL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Doster E, Wolfe C, Crosby WB, Clawson ML, Woolums AR, PInnell LJ. Morley PS. Enriching without culture: target-enriched metagenomics allows for strain-level characterization of M. haemolytica. Conference for Research Workers in Animal Disease (CRWAD). Chicago IL.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Crosby WB, Karisch BB, Hiott LM, Pinnell LJ, Pittman A, Frye JG, Jackson CR, Loy JD, Epperson WB, Blanton J Jr, Capik SF, Morley PS. Woolums AR. Tulathromycin metaphylaxis increases nasopharyngeal isolation of multidrug resistant Mannheinia haemolytica in stocker heifers. Front Vet Sci. 2023. Nov 20;10:1256997, doi:10.3389/fvets.2023.1256997. PMID: 38053814.

Progress 04/01/21 to 03/31/22

Outputs
Target Audience:Veterinarians in cattle practice and researchers studying antimicrobial resistance in BRD. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project is supporting a DVM pursuing a PhD, Dr. Will Crosby. Dr. Crosby leads organization of all trials and sample collection, he also conducts Mannheimia haemoltyica isolation and coordinates with collaborators as needed to ship or receive materials to complete objectives at other sites. Dr. Crosby will also be conducting the bioinformatical analysis to determine the effect of metaphylaxis on the metagenome, the resistome, and on the identification of M.haemolytica genomes in DNA isolated from nasopharyngeal swabs from the cattle in the study. How have the results been disseminated to communities of interest?No research has been disseminated in the reporting period described here, but Dr. Crosby will be presenting abstracts describing the results of the research at 3 national meetings and 1 international meeting in the next reporting period. What do you plan to do during the next reporting period to accomplish the goals?We will conduct the metagenomic sequencing and targeted pull-down to detect M. haemolytica genomes using DNA isolated from nasopharyngeal swabs in trials 1-4, in order to determine results needed to complete objectives 1 - 4.

Impacts
What was accomplished under these goals? Major activities completed: We collected nasopharyngeal swabs on day 70 from calves in the third of 4 planned trials, and we completed the fourth planned trial, and collected nasopharyngeal swabs from the 84 enrolled cattle at arrival (day 0), day 21, and day 70. Importantly, the antimicrobial susceptibility profiles for all M. haemolytica isolates from all 4 trials have been determined. We have also completed isolation of DNA from nasopharyngeal swabs of all cattle in all 4 trials in preparation to begin metagenomic sequencing. We have completed development of the novel targeted pulldown assay to allow identification and sequencing of M. haemolytica genomes in total DNA isolated from bovine nasopharyngeal swabs. Specific objectives met: We have completed objective 5, and we have collected all of the samples necessary to complete objectives 1-4. Significant results achieved: Naturally occurring BRD occurred in the cattle in trial 4 at rates comparable to that seen in trials 1-3. The day 21 BRD morbidity rates in trial 4 was 16%; for META cattle the BRD morbidity was 2% and for NO META cattle the BRD morbidity was 29%. For all trials together, the BRD morbidity rate at day 21 was 20%; for META cattle across all trials the day 21 BRD morbidity rate was 12% and for NO META cattle it was 28%. For all trials together, the BRD morbidity rate at day 70 was 22%; for META cattle the day 70 BRD morbidity rate was 14% and for NO META cattle the day 70 BRD morbidity rate was 30% Regarding Mannheimia haemolytica isolation from the cattle: in trial 3, at day 70, M. haemolytica isolation from nasopharyngeal swabs for META and NO META cattle was 21% and 13%. For trial 4 cattle, the M. haemolytica isolation rate for META and NO META cattle on day 0, 21, and 40 was, respectively, 45% and 41%, 41% and 52%, and 15% and 10%. Regarding antimicrobial resistance in M. haemolytica isolated from META and NO META cattle at d 0, 21, and 70: when multidrug resistance (MDR) was defined as resistance to antimicrobials in 3 or more classes, the percent of M. haemolytica isolated that were MDR on day 0 for META and NO META cattle was 21% and 27%. The percent of isolates that were MDR on day 21 was 69% for META cattle, and 9% for NO META cattle (P = 0.0002). For META and NO META cattle that required additional antimicrobials after arrival (META-TX and NO META-TX), the percent of M. haemolytica isolates that were MDR was 100% for META-TX cattle and 53% for NO META-TX cattle. ?Key outcomes: This project is in progress, and so outcomes for objectives 1 - 4 have not yet been determined. However, for objective 5, we showed that, as previously reported, administration of long acting macrolide metaphylaxis at arrival was associated with decreased BRD morbidity. The odds of treatment for BRD was 3 times greater for NO META than META cattle (P = 0.0002). However, the proportion of M. haemolytica isolated from cattle that received metaphylaxis that were MDR was significantly higher than the proportion of MDR M. haemolytica isolates from NO META cattle (P = 0.0002). Therefore an important finding from this study is that metaphylaxis continues to be associated with decreased BRD morbidity, but it is also associated with significant increase in the proportion of M. haemolytica isolated from cattle receiving metaphylaxis that are MDR, compared to control cattle not receiving metaphylaxis. The increase in proportion of MDR M. haemolytica in populations of cattle receiving metaphylaxis may ultimately lead to decreased response to treatment for BRD when cattle require that after metaphylaxis. However, the number of cattle that required treatment for BRD following metaphylaxis was too low to identify that effect in this study.

Publications

Progress 04/01/20 to 03/31/21

Outputs
Target Audience:Veterinarians in cattle practice and researchers studying antimicrobial resistance in BRD. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project is supporting a DVM pursuing a PhD, Dr. Will Crosby. Dr. Crosby leads organization of all trials and sample collection, he also conducts Mannheimia haemoltyica isolation and coordinates with collabortors to ship samples to sites for MALDI or for DNA isolation. Dr. Crosby presented the abstracts described above. How have the results been disseminated to communities of interest?The work was presented in abstracts at the 2020 Mississippi State University College of Veterinary Medicine Research Day, and at 2020 CRWAD, which was a virtual meeting. What do you plan to do during the next reporting period to accomplish the goals?We plan to conduct trial 4, in which 84 stocker cattle will be sampled as described above, after allocation to treatment groups as planned. We will test isolates by Sensititer to confirm the antimicrobial susceptibility of all M. haemolytica isolates for trials 1-4. We will begin isolation of DNA from nasopharyngeal swabs collected in all 4 trials, in preparation for the metagenomic sequencing and targeted pull-down to detect M. haemolytica genomes in isolated DNA. The graduate student Will Crosby DVM will begin to learn the techniques in bioinformatics that will be necessary to describe the microbiome and the resistome in all treatment groups.

Impacts
What was accomplished under these goals? Major activities completed: We have completed 3 of 4 planned trials. We have enrolled, managed, and sampled on time 84 stocker heifers in each of the 3 trials. Cattle were sampled by nasopharyngeal swabbing at arrival, day 20 or 21, and day 70 for trials 1 and 2 (for trial 3 day 70 has not yet arrived). Mannheimia haemolytica were isolated by culture with identification and genotype confirmed by MALDI-TOF We have continued to work to develop the novel targeted pulldown assay to identify M. haemolytica genomes in total DNA isolated from bovine nasopharyngeal swabs. We will wait until samples are collected from all 4 trials before we begin the metagenomic sequencing and the assessment of the presence of M. haemolytica genomes using the pulldown assay. Specific objectives met: We have completed three-quarters of the work for objective 5, and we have collected three-quarters of the samples needed to complete objectives 1 - 4. Significant results achieved: Naturally occurring BRD occurred in all three groups at rates typical of high risk stocker cattle, as expected. The BRD morbidity rates in trials 1, 2, and 3 were 20%, 19%, and 26%. The mortality rates for trials 1, 2, and 3 were 0%, 4%, and 6%. These mortality rates are within the range expected for high risk stocker cattle. The BRD morbidity for cattle treated with metaphylaxis was sometimes lower than for cattle not treated with metaphylaxis; the BRD morbidity rates for META and NO META cattle for trials 1, 2 and 3 were, respectively: 22% and 17%; 14% and 24%, and 10% and 44%. Regarding Mannheimia haemolytica isolation from the cattle at day 0 and day 21, there are differences between the groups that are sometimes unexpected. For example, in some trials, more calves in one group vs the other shed M.haemolytica at arrival, in spite of the fact that the calves were randomly allocated to groups, and should not have differed. Also, in some trials, a larger percentage of META calves shed M. haemolytica on day 21, when it might be expected that the long acting antimicrobial given to META calves at arrival might suppress M. haemolytica colonization. For each of the three trials, the percent of META and NO META calves positive for M. haemolytica on nasopharyngeal swabbing on days 0, 21, and 70 was, respectively, for trial 1: 26% and 5%, 49% and 29%, and 5% and 5%; for trial 2: 19% and 21%, 57% and 49%, and 14% and 5%; and for trial 3, 5% and 12%, and 34% and 15% (day 70 results for trial 3 pending). Results of testing for antimicrobial sensitivity by Sensititer of M. haemolytica isolates are pending. Key outcomes: This project is in progress, and all outcomes at this point are thus preliminary. However, it was important to find that our commercially sourced cattle developed BRD and shed Mannheimia haemolytrica at expected rates. This indicates that we should be able to test the proposed hypothesis related to prevalence of antimicrobial resistance genetic elements in cattle receiving metaphylaxis or cattle not receiving metaphylaxis.

Publications

• Type: Other Status: Other Year Published: 2020 Citation: Abstract: Crosby WB, Karisch B, Loy JD, Hiott L, Pittman A, Austin FW, Epperson W, Blanton Jr J, Morley PS, Capik SF, Jackson CR, Frye J, Woolums A. The effect of tulathromycin on morbidity and prevalence of Mannheimia haemolytica genotype 2 in 84 stocker heifers. Proceedings of the Conference for Research Workers in Animal Disease 2020. Virtual conference. Abstract #153.

Progress 04/01/19 to 03/31/20

Outputs
Target Audience:Veterinarians in cattle practice and researchers studying antimicrobial resistance in BRD. Changes/Problems:Because of the COVID-19 pandemic, trial 2, which was scheduled to occur in the spring of 2020, wasmoved to fall 2020. Fortunately,we were able to complete the trial on schedule in fall 2020. Assessment of antimicrobial resistance in the Mannheimia haemolytica isolated in trials 1 and 2 has been delayed because the laboratory of our collaborators at USDA ARS was closed for several weeks but we expect those to be completed in the next reporting period. What opportunities for training and professional development has the project provided?This project is supporting a DVM pursuing a PhD, Dr. Will Crosby. Dr. Crosby leads organization of all trials and sample collection, he also conducts Mannheimia haemoltyica isolation and coordinates with collabortors to ship samples to sites for MALDI or for DNA isolation. Dr. Crosby presented the abstracts described above. How have the results been disseminated to communities of interest?The work was presented in abstracts at the 2020 Mississippi State University College of Veterinary Medicine Research Day, and at 2020 CRWAD, which was a virtual meeting. What do you plan to do during the next reporting period to accomplish the goals?We plan to conduct trials 3 and 4, in which 84 stocker cattle will be sampled as described above, after allocation to treatment groups as planned. We will begin the metagenomic sequencing of DNA isolated from nasopharyngeal swabs in trials 1 and 2, and the graduate student Will Crosby DVM will begin to learn the techniques in bioinformatics that will be necessary to describe the microbiome and the resistome in all treatment groups. We will begin testing the novel target-enriched bait pulldown method for identifying Mannheimia haemolytica in the metagenomes, using first positive control samples and, when the technique is verified to be functioning properly, using samples from trials 1 and 2. We also expect testing of Mannheimia hemolytica isolates from trials 1 and 2 for antimicrobial resistance of

Impacts
What was accomplished under these goals? Major activities completed: We have completed 2 of 4 planned trials. We have enrolled, managed, and sampled on time 84 stocker heifers in each of the 2 trials. Cattle were sampled by nasopharyngeal swabbing at arrival, day 20 or 21, and day 70 (for trial 1, day 70 for trial 2 has not yet arrived). Mannheimia haemolytica were isolated by culture with identification and genotype confirmed by MALDI-TOF. Results of testing for antimicrobial sensitivity by Sensititer are pending. Naturally occurring BRD occurred in both groups at rates typical of high risk stocker cattle, as expected, with morbidity rates of 25% - 35% in both groups, and an overall mortality of 2%. We have also completed isolation of DNA from nasopharyngeal swabs of all cattle in preparation to begin metagenomic sequencing. We have also completed development of baits for the novel targeted pulldown assay and will be testing the pulldown assay in positive control samples in the coming weeks. Specific objectives met: We have completed half of the work for objective 5, and we have collected half of the samples needed to complete objectives 1 - 4. Significant results achieved: In trial 1, 14% of cattle were positive for Mannheimia haemolytica. Despite random allocation of cattle to groups, the number of cattle positive in the metaphylaxis (META) group (11 out of 42) was significantly higher (P= 0.0158) than the NO META group (2 out of 42). At arrival, as expected, 83% of isolates were genotype 1. On day 20, 40% of calves were positive (META = 20, NO META = 12), and all were genotype 2. In trial 1, there was no association between metaphylaxis and rate of Mannheimia isolation or genotype. Key outcomes: This project is in progress, and all outcomes at this point are thus preliminary. However, it was important to find that our commercially sourced cattle developed BRD and shed Mannheimia haemolytrica at expected rates. This indicates that we should be able to test the proposed hypothesis related to prevalence of antimicrobial resistance genetic elements in cattle receiving metaphylaxis or cattle not receiving metaphylaxis.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Crosby WB, Karisch B, Loy JD, Hiott L, Pittman A, Austin FW, Epperson W, Blanton Jr J, Morley PS, Capik SF, Jackson CR, Frye J, Woolums A. The effect of tulathromycin on morbidity and prevalence of Mannheimia haemolytica genotype 2 in 84 stocker heifers. Proceedings of the Conference for Research Workers in Animal Disease 2020. Virtual conference. Abstract #153.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: We presented a research abstract at Research Day at the College of Veterinary Medicine at Mississippi State University in August 2020.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: We presented a research abstract at the Conference for Research Workers in Animal Disease (CRWAD) in December 2020.