MITIGATION OF ANTIMICROBIAL RESISTANCE THROUGH ALTERNATIVE TREATMENT REGIMENS TO CONTROL BOVINE ANAPLASMOSIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1014734
• Grant No.: 2018-68003-27463
• Cumulative Award Amt.: $1,199,948.00
• Proposal No.: 2017-05600
• Project Start Date: Jan 1, 2018
• Project End Date: Dec 31, 2023
• Grant Year: 2018
• Program Code: [A4171]- Effective Mitigation Strategies for Antimicrobial Resistance

Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506

Performing Department
Diagnostic Medicine/Pathobiolo

Non Technical Summary
Understanding the ecology of antimicrobial resistance (AMR) and control of hemoparasitic diseases are national priorities for the United States Department of Agriculture (USDA) and are vital to protecting our food security and the continued sustainable development of our livestock industries. Judicious use of antimicrobials are essential to maintain susceptible bacterial populations and promote antimicrobial longevity, while minimizing development of resistant microbial populations. However, several currently Food and Drug Administration (FDA)-approved medicated livestock feed therapies utilize medically important antimicrobials with no on-label defined duration of use, such as the use of chlortetracycline (CTC)-medicated feeds for the treatment of bovine anaplasmosis (anaplasmosis) in cattle. Unintended consequences of prolonged antimicrobial exposure, especially at sub-therapeutic doses, include selection of AMR in both the target pathogen and off-target zoonotic commensals. Based on the premise that increasing exposure of bacterial populations to antimicrobial drugs increases of the risk of populations generating resistance to those drugs, the FDA, through the Federal Register, has sought information to help establish appropriate dose and duration of use policies whereby clinical therapeutic effect is maximized and risk of resistance development is minimized. The long-term goal of this program of work is to promote sustainable livestock production by reducing protracted antimicrobial exposure to control infection of the hemoparasitic rickettsial pathogen, Anaplasma marginale (Am), the etiologic agent of anaplasmosis in cattle. The guiding hypothesis of this proposal is that targeting treatment with tetracycline (TET) antimicrobials to coincide with strategic periods in the Am transmission cycle will effectively control clinical anaplasmosis in cattle while also minimizing development of AMR in Am and off-target (bovine zoonotic commensals) bacterial species. Informed by scientific data and through active, integrated cooperation between scientists, clinicians, extension, and producers, this proposal will enable livestock producers to make more efficient use of antimicrobials by aligning antimicrobial usage with the natural biological cycle of Am to optimize disease control. The primary objectives of this proposal are to: i) examine the in vivo efficacy of TET antimicrobials to control anaplasmosis in experimental and field challenges, ii) evaluate the antimicrobial susceptibility profile of target (Am) and off-target (bovine zoonotic commensals) microbial species in treated cattle; iii) determine the contribution of Am tick vector species to the ecological persistence of AMR in Am populations; and iv) develop an extension platform to provide resources to stakeholders trying to mitigate anaplasmosis and support antimicrobial stewardship.

Animal Health Component

0%

Research Effort Categories

Basic

100%

Applied

0%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4010	1040	25%
311	3310	1100	25%
311	3120	1100	25%
712	3310	1100	25%

Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3120 - Spiders, mites, ticks, and other arthropods; 3310 - Beef cattle, live animal; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

amr

anaplasma marginale

anaplasmosis

antimicrobial resistance

cattle

tetracycline

tick

Goals / Objectives
The long-term goal of this proposed program of work is to promote sustainable livestock production by reducing reliance of prolonged antimicrobial exposure to control anaplasmosis in cattle. The primary objectives of this proposal are to exmaine the in vivo effect of tetracycline antimicrobials on (1) the persistence of experimental and field A. marginale (Am)infections, (2) the antimicrobial susceptibility profile of targe (Am) and off-target (zoonotic bovine commensals) microbial species in treated cattle; (3) the ecological persistence of antimicrobial resistant Am in the tick vector; and (4) development of an extension platform to provide resources to stakeholders trying to mitigate Am infection and protect the use of medically-important antimicrobials.

Project Methods
Specific Aim 1: Compare the susceptibility phenotype, and correlating genotypic basis, of four Am isolates to multiple CTC treatment dose regimens in vivo. Selection of field isolates and isolate propagation - This study will use 2 previously characterized, Am strains that are suggested to have differential susceptibility to TET antimicrobials. We will also propogate 2 suspect-antimicrobial resistant field isolates for comparison using splenectomized calves. Infection of study calves - In this proposal we will focus on defining the susceptibility phenotype of 4 Am strains by attempting to chemosterilize persistently infected animals with different doses of continuously administered CTC. 96 spleen-intact calveswill be randomly assigned to one of 4 infection groups (n=24 calves/group) and inoculated with 1 of 4 Am strains. Once calves become persistently carriers they will be randomly assigned to CTC treatment groups. Treatment with CTC - Within each Am strain cohort, the persistenly infected calves will be randomly assigned to 1 of 4 treatment groups. Animal will be monitored for infection for 120 days post-CTC treatment initiation. Plasma drug concentration levels will be evaluated every 40 days post-CTC treatment initiation. Comparison of Am strain infection and CTC treatment regimen - Geometric mean and % coefficient of variation will be calculated. Comparisons will be made between groups infected with different Am strains using mixed procedures. A semi-parametric survival analysis will be performed to analyze the variation in time to Am strain clearance between the different treatment doses. Whole genome sequencing of new Am field strains - DNA will be extracted from blood during splenectomized calf infection and whole genome sequencing carried conducted. Genomic sequences will be assembled anannotations made using NCBI's prokaryotic genome analysis pipelines and FDA-CVM databases.The antimicrobial resistance genes and virulence genes will be analyzed. Sequencing transcriptomes from Am strains during CTC-treatment - RNA will be infected from blood and bacterial RNA enriched using the Ambion MICROBEnrich kits. For these sameles a duplex-specific thermostable nuclease normalization protocol will be applied. Normalization will be accomplished using quantiles to adjust distributions and fold changes with respect to reads per kilobase per millioncalculated. Genes with a log2 fold change will be selected for further investigation, with priority given to genes of putative efflux pumps or related to 16S transcriptional regulators.Specific Aim 2: Examine the role of the tick vector in maintaining Am antimicrobial susceptibility profiles and determine whether effect of CTC on tick acquisition and transmission of Am. Am tick acquisition feeding - The Am strains with greatest resistance phenotypewill be chosen forthis aim. On two groups of animals (control and highest CTC concentration that failed to clear Am)200 male ticks will be allowed to acquisition feed. Am tick transmission feeding - 16 calves will be randomly divided into 4 groups, half will receive CTC prophylaxis and the other half will receive no prophylaxis. 60 acquisition-fed tick will be used to challenge, via transmission feeding, prophylactically-treated or untreated calves for 7 days. All animals will be monitored for 40 dayspost-tick removal for signs of clinical anaplasmosis and ifnection.Data analysis - Geometric mean and %CV will be calculated from acquired data acquired. Am infection levels in ticks or tick tissues will be analyzed using an analysis of variance to compare mean bacterial levels in tick tissue after acquisition and transmission feeding between the different treatment groups.Specific Aim 3: determine the impact of Am treatment choice on the selection of antimicrobial resistance in off-target bovine zoonotic commensals of public health importance. The samples used for this analysis will come from animals in the infection and treatment challenge in Specific Aim 1. Evaluation of bovine gastrointestinal microbiome diversity - DNA willbe isolated from homogenized fecal samples and the V3-V4 regions of the bacterial 16S rRNA gene will be amplified by PCR, and purified PCR products paired-end sequenced on an Illumina platform. Operational taxonomic units will be clustered with a 97% similarity and sequences classified using Ribosomal Database Project classifier with a standard minimum support threshod of 80%. Microbial community structure among treatments will be explored using Permutational multivariate analysis of variance, and analysis of similarity. The significance of OTU association with treatment will be tested by analysis of variance. Isolation and identification of E. coli - E. coli from fecal samples will be isolated and species identified by MALDI-TOF bacterial ID system. E. coli antimicrobial susceptibility determination - The MIC for E. coli will be determined by broth micro-dilution method. Survival analysis will be performed for MIC data when needed after log rank assumption of normality; otherwise multilevel mixed logistic regression will be used for binary phenotype coded data.Specific Aim 4: develop a resource platform for stakeholders interested in controlling bovine anaplasmosis and mitigating the development of AMR in livestock systems. Extension demonstration of Am treatment option - One hundred cows from an Am endemic herd will be randomly assigned into one of three treatment groups. In each treatment group, half of the animals will be persistently-infected with Am and the other half uninfected. Groups will either be treated with the CTC dose determined in Specific Aim 1 to control Am infection for 30 days, 120 days or not at all (control). All animals will be monitored for signs clinical anaplasmosis and infection. An ANOVA will be used to determine whether targeted versus protractedantimicrobial treatment use equally protectedanimals. Website design - A website, linked to the Beef Cattle Institute (BCI) will be designed for those interested in fundamental concepts related to bovine anaplasmosis, bovine anaplasmosis disease management, and antimicrobial resistance management strategies. The website will: i) host a discussion forum for stakeholders to easily connect and communicate with experts; ii) a regularly-updated collection of current federal policies and FDA-approved treatment options related to bovine anaplasmosis; iii) a database of scientific articles related to the treatment and control of bovine anaplasmosis; iv) active research projects; and, v) platform for reporting suspected cases of Am antimicrobial resistance. Educational module development - Non-formal education modules will be developed and include modules on: i) understanding the basic biology of Am, including transmission mechanisms; ii) understanding anaplasmosis risk factors and management strategies; iii) understanding the basics of antimicrobial resistance; iv) understanding the importance of judicious antimicrobial use and antimicrobial stewardship; v) understanding antimicrobial resistance causes and management strategies; and, vi) application of science-based disease and resistance management practices. Biennial symposium - We will organize biennial symposium to provide interactive opportunities for producers and stakeholders to hear the most up-to-date information on bovine anaplasmosis, disease epidemiology, and disease management strategies. Evaluation of extension activities - To evaluate the effectiveness of extension educational modules and symposia, we will collaborate with the K-State Office of Educational Innovation and Evaluation (OEIE) to coordinate the formative and summative evaluation of extension activities. Methods of evaluating outcomes and impact will include collection of both qualitative and quantitative data through surveys and focus groups to access effectiveness and utility of extension activities.

Progress 01/01/18 to 12/31/23

Outputs
Target Audience:In general, and with an audience tailored approach, our communication efforts to our target audiences have focused on communicating and delivering data-driven information on bovine anaplasmosis and the role that management of this disease can have on promoting antimicrobial resistance in co-occuring off-target microbial species. Presentations to target audiences have occured as part ofboth informal (e.g. individual consulting)and formal (e.g. presentation at organized local, regional, national meetings)programs. Topic presented to target audiences have included: bovine anaplasmosis epidemiology, ecology, diagnosis, transmission, treatment, and management strategies; and, antimicrobial efficacy, mechanism of action,use practices,resistance, and broader use implications. Our target audiences have included: cattle producers, practicing large animal/mixed veterinarians, academia, federal researchers, and animal health industry. Cattle producer community: Over the course of this project, we worked with cattle producers to: i)acquire research materials (e.g. novel strains of A. marginale), ii) help evaluate efficacy of in-place chlortetracycline regimens for anaplasmosis control, iii) share research results through educational events and direct presentations, and iv) provide experitise to help advise and prioritize practices to best manage anaplasmosis in production herds. Extension agents: Through cooperating extension agents, we evaluated the efficacy of the chlortetracycline regimens for anaplasmosis control and the impact of these practices on on-target fecal microbes such as E. coli and Enterococcus spp. We also worked through extension groups to help disseminate research results to the producer and veterinarian stakeholder communities. Practicing large animal/mixed animal veterinarians: Practicing veterianrians were engaged in this research project through presentation of educational materials at local, state, and national meetings. Our research group also served this community as expert advisors to help aid and prioritize anaplasmosis control efforts for cattle production operation served by these veterinarians. Academia: Project results supported at this grant were presented at multiple scientific meetings, including local, state, and national meetings. Additionally, our Bovine Anaplasmosis Symposium faciliated multiple new collaborations on the general topic of anaplasmosis from univeristy researchers across muliple states. Federal agency researchers: During this project we developed new collaborations with some USDA-ARS units interested in anaplasmosis management. Animal health industry: Animal health industry personnel were engaged through attendance at our Bovine Anaplasmosis Symposium, and attendance during presentation of study results at local, regional, and national presentations. Changes/Problems:Due to the COVID-19 pandemic, we experienced large staffing changes/challenges over the course of this project. Because of these challenges we will still have items to report associated with this grant past it's termination (namely four to five additional peer-reviewed journal articles and one dissertationthat are currently in varyingstages of draft). What opportunities for training and professional development has the project provided?Collectively, this project has supported and/or provided primary (project basis of thesis/dissertation) training opportunities for 2PhD students and 3 Master's students. This project also provided numerous cross-training opportunities for 7 other graduate students to gain experience in a variety of animal handling protocols, biosample collection techniques, and molecular/microbiology assay protocols, team work, and data entry. In addition to graduate students, this project provided supported the training of 5 DVM students through the summer Veterinary Research Scholar Program, and 16 undergraduate students. These undergraduate and professional students gained valuable experience in several animal handing, biosample collection, and molecular/microbiology techniques, team work, and data entry. The hosted Bovine Anaplasmosis Symposium also served as a catalyst for new collaborations between faculty members at different institutions throughout the country that also led to mentorship opportunities for students at those universities (in addition to spurring new research collaborations). This project supported professional development ofstudent trainees and project faculty membersthrough opportunities to present their project results at scientific/professional meetings and stakeholderevents. Many aspects of this project also served as the foundation for student thesis and dissertation projects. For many students and faculty involved in this project, there were numerous opportunities for communicating research results with diverse stakeholder audience groups. For cattle producers, this project supported extension efforts on education producers on bovine anaplasmosis,bovine anaplasmosis management practices/strategies, and judicious antimicrobial use practices through one-on-one consultations, complementary anaplasmosis diagnostics, and through participation in cattle producer events, including the hosted Bovine Anaplasmosis Symposium. Additionally, members of the research team actively participated in trade interview requests and general audience press articles on the topics of bovine anaplasmosis, bovine anaplasmosis management, and judicious antimicrobial use. For bovine veterinarians (local,national, and international), this project supported development of educational content and materials delivered duringcontinuing education programs andlocal radio/podcast/video interviews/segments; and, through opportunities for one-on-one consultations on bovine anaplasmosis management and judicious antimicrobial use. How have the results been disseminated to communities of interest?Results of this project have been disseminated through presentations at scientific meetings (e.g. Conference for Research Workers in Animal Diseases, American Society for Rickettsiology). Project members have also communicated progress of this project through invitations to speak at stakeholder events (cattle producer meetings - local, state, national; continuing eucation programs for practicing food animal veterinarians, interview requests, etc). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1 Accomplishments: Identification of contemporary Anaplasma marginale isolates: Field surveysengagingproducers were performed and resulted in >100 strains (based on Msp1a genotyping) of Anaplasma marginaleidentified from beef cattle (samples primarily collected from KS beefherds). Propogation of contemporary and historic Anaplasma marginale isolates: 4 historic and 10novel A. marginale isolates were propogated in bovine hosts. From these hosts, stabilates of infectious blood products were prepared and stored. Characterization of contemporary A. marginale strains: The infection characteristics, antimicrobial susceptiblity, and/or tick transmissibility of 6 new contemporary A. marginalestrainswere characterizdthrough controlled cattle infection experiments. All strains were able to induce clinical anaplasmosis in bovine challenge models. Conducted large controlled comparative A. marginale infection and treatment study: A ~10 month-long, ~100 calf controlled A. marginale infection challenge and chlortetracycline (CTC) treatment experiment was conducted to compare the infection dynamics,clinical disease progression, and suscpetiblity to one of four different 120-day CTC treatment regionof two historic (VA, OK) and two new contemporary (KS-2, DR-25) A.marginalestrains. During the course of this study the following samples were collected: EDTA blood samples for A. marginale infection status and level; serum samples for analysis of seroconversion to A. marginale; plasma samples for CTC plasma level analysis; and, fecal samples for fecal microbe antimicrobial susceptibility and population analysis. Results of thisstudy demonstrated thatcontemporary A.marginalestrains were less susceptible to CTC than the historic strains, including to the experimentally-high treatment regimen. No evidence of A. marginale clearance was detected forsteers infected with a contemporary A. marginale strain whereas infection clearance was observed in some steers infected with historic A. marginale strains in a CTC-dose dependant manner (more cleared at higher CTC dosages). Objective 2 Accomplishments: Examine the antimicrobial susceptilibity profile of target microbial species (Anaplasma marginale)in CTC treated cattle: A controlled study was performed (outlined in Objective 1) to compare the susceptibility of two historic (VA, OK) and two contemporary (KS-2, DR-25) strains of A. marginale by treating steers chronically-infected with these A. marginale strains with one of 4 different CTC dosages (0, 0.5, 2.0, or 10.0 mg CTC/lb BW) daily for 120 days.Results of thisstudy demonstrated that the contemporary strains ofA. marginalewere less susceptible to CTC (had no or smaller differences in A. marginale infection levels compared to untreated group) than the historic strains. Significant decreases in A. marginale infection levels were observed for steers infected with historic A. marginale strains in a dose-dependent manner;however, a rebound in infection levels as treatment progressed indicatesthat some tolerance mechanisms may selected for in A. marginale infecting cattle treated for protracted periods of time with CTC for anaplasmosis control. No evidence ofA. marginaleclearance was detected for any steer infected with a contemporarystrains whereas infection clearance was observed in some steers infected with historicstrains in a dose-dependant manner. Examine the efficacy of chlortetracycline or oxytetracycline treatment to eliminate A. marginale infection: A study was conducted to investigate whether daily hand-fed CTC treatment for 60 days or three injections of a long-acting oxytetracycline treatment spaced 3 weeks apart would result in elimination of A. marginale infection from mature cows infected with a contemporary strain of A. marginale. Clearance of A. marginale infection was not observed in CTC nor oxytetracycline-treated catlle. This study supports that common anaplasmosis treatment and control practices are unlikely to result in clearance of A. marginale infection from chronically-infected cattle, especially cattle infected with contemporary strains of A. marginale. Examine the antimicrobial susceptilibity profile of off-target microbial species (E. coli and Enterococcus spp.)in CTC treated cattle: Isolates ofE. coli and Enterococcus from CTC-treated cattle (outlined in Objective 1) evaluated by Sensititre plate analysis were commonly resistant to tetracycline by demonstrated increased resistant to non-tetracycline antimicrobials over the course of CTC treatment. Reversion to pre-CTC treatment susceptibilities occurred for some, but not all, of observed altered antimicrobial phenotypes. Examine the microbial community from direct-collected fecal samples from CTC treated cattle: 16S rRNA sequencing was performed to investigate changes in microbial community of direct, fecal microbe populations prior to, over the course of, and after cessation of a 120-day CTC treatment regimen. Objective 3 Accomplishments: Conducted A. marginale tick transmission study using contemporary A. marginale strain DR-25: DR-25 strain found to not be tick transmissible. Conducted A. marginale tick transmission study using contemporary A. marginale strain KS-2: KS-2 strain confirmed tick-transmissible. Tick transmission of DR-25 occurred in tick-recipient cattle receiving active CTC control, albeit slightly delayed compared to tick-recipient cattle not receiving CTC. Investigate vector competence of Amblyomma americanum to transmit A. marginale: The A. americanum tick is a common tick on beef cattle. Attempted transmission of A. marginale by A. americanum failed; however, A. marginale nucleic acid was detected in a small percentage of field-collected host-seeking A. americanum. Objective 4 Accomplishments: Hosted producer-focused Bovine Anaplasmosis Symposium: >150 attendees for bovine anaplasmosis symposium. Attendees included producers, practicing large animal veterinarians, students, and industry personnel. Deliver producer-focused educational events on bovine anaplasmosis management and judicious antimicrobial use: Events included presentation at NCBA, K-State Field Days, and producer appreciation events. Deliver practicing veterinarian continuing education programs on bovine anaplasmosis management and judicious antimicrobioal use: Events included presentations at state annual VMA meetings (e.g. Missouri, Tennessee VMA), Bovine Anaplasmosis Symposium, and Academy of Vet Consultants Prepare educational materials for dispersal on bovine anaplasmosis and antimicrobial use and antimicrobial resistance Participate in stake-holder focused communication forums on the topics of bovine anaplasmosis and judicous antimicrobial use: Types of engagements included interviews for trade publications, radio shows, and podcasts. Work with extension to demonstrate the efficacy of CTC to control active anaplasmosis in a herd naturally endemic for A. marginale: Conducted studies to investigate the efficacy of various FDA-approved free-choice CTC-medicated mineralformulations to control active anaplasmosis in a endemic beef cattle herd managed by K-State Ag Extension and selected producers. In these studies, the A. marginale infection status and A. marginale strains infecting theseherds were determined prior to initiating treatment. Samples were collected prior to, throughout, and after treatment period. Although clinical anaplamsosis was not observevd in monitored cattle, A. marginale infection levels rarely fluctuated compared to untreated cattle. Directly work with producers to elucidate herd anaplasmosis status and help priortize management strategies: Over the course of this project, study personnel worked one-on-one with producers to evaluate their herd(s) anaplasmosis status and assist in priortizing anaplasmosis management practices based on individual producer goals and operation constraints(e.g. cost-conscious recommendations, working within existing management procedures, etc.).

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Lovett AC, Reppert EJ, Huser S, Robert BD, Jaeger JR, Kang Q, Anantatat T, Armstrong CL, Reif KE. Cross-sectional study of Kansas beef bulls to examine associations between chronic bovine anaplasmosis and breeding soundness. American College of Veterinary Internal Medicine  Annual Meeting. June 15-17, 2023. Philadelphia, PA. (Grad student Lovett received KSU CVM travel award to present)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bickmeier NP, Anantatat T, Beltz M, Eggers A, Weaver B, Almaraz J, Huser SM, Kang Q, Song X, Reif KE. Pregnancy success among eastern Kansas beef cows infected with Anaplasma marginale and/or Bovine Leukemia Virus. K-State Graduate Research, Arts, and Discovery Forum. March 9, 2023. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bickmeier NP, Anantatat T, Beltz M, Eggers A, Weaver B, Almaraz J, Huser SM, Kang Q, Song X, Reif KE. Pregnancy success among eastern Kansas beef cows infected with Anaplasma marginale and/or Bovine Leukemia Virus. K-State Phi Zeta Research Day. March 7, 2023. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bickmeier NP, Toillion AR, Skinner BC, Anantatat T, Amachawadi RG, Kang Q, Reppert EJ, Coetzee JF, Reif KE. Antimicrobial susceptibilities of E. coli from calves treated with chlortetracycline for anaplasmosis control. K-State Phi Zeta Research Day. March 7, 2023. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Lovett AC?, Reppert EJ, Huser S, Robert BD, Jaeger JR, Kang Q, Armstrong CL, Reif KE. Investigation of chronic Anaplasma marginale infection and breeding soundness in beef bulls. K-State Phi Zeta Research Day. March 7, 2023. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Lovett AC, Reppert EJ, Jaeger JR, Kang Q, Bickmeier NP, Anantatat T, Flowers MR, ODay SC, Armstrong CL, Reif KE. Acute anaplasmosis reduces breeding soundness in experimentally infected beef bulls. March 7, 2023. Manhattan, KS. (Grad student Lovett received 2nd Place in Applied/Clinical Science, Large Animals  Oral presentations)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Lovett AC, Reppert EJ, Huser S, Robert BD, Jaeger JR, Kang Q, Armstrong CL, Reif KE. Investigation of chronic Anaplasma marginale infection and breeding soundness in beef bulls. K-State Cattlemens Day. March 3, 2023. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Lovett AC, Reppert EJ, Jaeger JR, Kang Q, Bickmeier NP, Anantatat T, Flowers MR, ODay SC, Armstrong CL, Reif KE. Acute anaplasmosis reduces breeding soundness in experimentally infected beef bulls. K-State Cattlemens Day. March 3, 2023. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bickmeier NP, Anantatat T, Beltz M, Eggers A, Weaver B, Almaraz J, Huser SM, Kang Q, Song X, Reif KE. Pregnancy success among eastern Kansas beef cows infected with Anaplasma marginale and/or Bovine Leukemia Virus. K-State Cattlemens Day. March 3, 2023. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bickmeier NP, Toillion AR, Skinner BC, Anantatat T, Amachawadi RG, Kang Q, Reppert EJ, Coetzee JF, Reif KE. Antimicrobial susceptibilities of E. coli from calves treated with chlortetracycline for anaplasmosis control. K-State Cattlemens Day. March 3, 2023. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Lovett AC, Reppert EJ, Huser S, Robert BD, Jaeger JR, Kang Q, Anantatat T, Armstrong CL, Reif KE. Investigation of chronic Anaplasma marginale infection and breeding soundness in bulls. Conference of Research Workers in Animal Diseases  Annual Meeting. January 21-24, 2023. Chicago, IL.
• Type: Journal Articles Status: Other Year Published: 2024 Citation: Bickmeier NP, Toillion AR, Skinner BC, Anantatat T, Amachawadi RG, Kang Q, Reppert EJ, Coetzee JF, Reif KE. Antimicrobial susceptibilities of Enterococcus spp. isolated from cattle hand-fed chlortetracycline for anaplasmosis control. To be submitted to MDPI - Antibiotics (tentative year to be published - 2024)
• Type: Theses/Dissertations Status: Accepted Year Published: 2023 Citation: Administration of chlortetracycline to beef cattle: evaluation of antimicrobial activity, resistance selection, duration of detection, and efficacy.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bickmeier NP, Anantatat T, Beltz M, Eggers A, Weaver B, Almarez J, Huser SM, Kang Q, Song X, Reif KE. Pregnancy success among eastern Kansas beef cows infected with Anaplasma marginale and/or Bovine Leukemia Virus. Conference of Research Workers in Animal Diseases  Annual Meeting. January 21-24, 2023. Chicago, IL. (Grad student Bickmeier received K-State CVM Travel Award; Selected as the Society for Tropical Veterinary Medicine Best Student Presentation Award)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Bickmeier NP?, Toillion AR?, Skinner BC?, Anantatat T?, Amachawadi RG, Kang Q, Reppert EJ, Coetzee JF, Reif KE. Antimicrobial susceptibility changes in E. coli from calves treated with chlortetracycline for anaplasmosis control. Conference of Research Workers in Animal Diseases  Annual Meeting. January 21-24, 2023. Chicago, IL.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Robbins G, Rogers LR, McDermott EG, Reif KE, Krumpleman B, Galloway D, Powell JG. Serial evaluation of A. marginale positivity rate within a single herd of mature beef cows. American Society of Animal Science  Southern Section. January 21-24, 2023. Raleigh, NC.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lovett AC, Reppert EJ, Huser S, Rob�rt BD, Jaeger JR, Kang Q, Liu R, Anantatat T, Armstrong CL, Reif KE*. Kansas beef bulls with chronic anaplasmosis demonstrate satisfactory breeding soundess outcomes at breeding soundness examination. J Am Vet Med Assoc. 2023. Oct 13;262(1):53-60. doi: 10.2460/javma.23.05.0292.
• Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Bickmeier NP, Toillion AR, Skinner BC, Anantatat T, Amachawadi RG, Kang Q, Reppert EJ, Coetzee JF, Reif KE. Antimicrobial susceptibilities of Escherichia coli isolated from cattle hand-fed chlortetracycline for anaplasmosis control. Submitted to MDPI - Antibiotics (tentative year to be published - 2024)

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:In this reporting period, we have continued to deliver science-based, data driven information on bovine anaplasmosis and the possible effect this disease may have on promoting antimicrobial resistance. Our target audience includes our primary stakeholder (e.g. cattle producers),other professionalswith interest in bovine anaplasmosis and antimicrobial resistance (e.g. researchers, policy-makers), and student trainees. Forums for these communication efforts have included formal programs (e.g. professional conferences/meetings, continuing education programs), producer-focuseeducational programs/outreach/extension activities (e.g. participation in producer meetings, serving as point of contact for anaplasmosis related questions); training opportunities for undergraduate students, professional students (e.g. veterinary students seeking research opportunities), graduate students looking for a diversity of training experience, and publication of research results. Our project-related presentations at professional society meetings reach the following target audiences: academic and govenmental researchers and students intereted in bovine anaplasmosis, antimicrobial treatment and resistance, cattle health, and disease transmission. We have participated in Continuing Education events (e.g. producer educational events, KLA/K-State Ranch Management FieldDay) presenting on bovine anaplasmosis and our research efforts to better understand this disease, manage this disease with judicious antimicrobial use, and how this disease may impact other management strategies. As in previous years, we continue to interface with cattle producers and veterinarians on questions/concerns about bovine anaplasmosis within their own herds - including providing individualized disease managementrecommendations or advice. Our producer-focused events and forums, outreach, and extension activities, describing our project related data and outcomes have reached the following target audiences: beef cattle producers, practicing livestock veterinarians, livestock industry personnel, and people generally interested in cattle health, ticks, tick-borne diseases of livestock importance, and antimicrobial use/efficacy/implications as relates to bovine anaplasmosis. This project has also provided a platform for numerous student trainees, that this reporting year include graduate students (2 PhD students),undergraduate students (3 undergraduate students), and one DVM student. These students have received hands-on training in developing research skills including lab-based benchwork skills (e.g. sample handling/processing, diagnsotic assays, microbiology techniques),animal handling techniques (e.g. safely working with cattle, bio-sample collection), communication skills (e.g. participation and/or presentation in lab meetings, local/regional meetings, national professional meetings), organizational skills, and teamwork skills. Publication of research results in peer-reviewed publications also provided wider opportunities to communicate our project-related findings with various stakeholders in academia, government, industry, and other professional groups, or members of the general public interested in this work. Additionally we have generated producer-focused handout materials on the topics of bovine anaplasmosis and antimicrobial resistance. Changes/Problems:We experienced a large staffing change this year with our primary technician leaving and several students that graduated at the conclusion of last year. Additionally, we experienced signficant COVID-associated supply chain delays in receiving necessary supplies and reagents to perform key project experiments and venue challenges to host a second producer-focused bovine anaplasmosis event. Because of this, we will request an additional one-year not cost extension. What opportunities for training and professional development has the project provided?This year, this project has supported the following training and professional development opportunities. This project supported the training of one Masters andtwo PhD graduate students.This project supported research experiences and training opportunities for 1 additional master's student and 4 undergraduate students.This project supported professional developoment through student attendence and presentation at scientific meetings for 3graduate students and 1 faculty member. For regional cattle producers, this project supported extension efforts to relay information on bovine anaplasmosis and associated managment strategies thorugh one-on-one consultations, complementary anaplasmosis diagnostics, and through participation in three regional cattle producer events. Additionally, multiple interview requests were accepted to disucss the topic of bovine anaplasmosis disease and disease managment, in addition to how to distinguish anaplasmosis from theexpanding cattle disease bovine theileriosis (presents similarly to anaplasmosis; however, not susceptible to tetracyclines - this may inadvertently lead producers to think their anaplasmosis control strategies are not working). Growing concern over bovine theileriosis has also spurred a new interest in tick and the pathogens that they are capable to transmitting to livestock and increase in engagment opportunities with producers and livestock veternarians. For bovine veterinarians (both local, national), this project supported developent of educational content delivered during annual continuing education programs; as well as, opportunities for one-on-one consultations on bovine anaplasmosis management. How have the results been disseminated to communities of interest?Results of this project have been disseminated through presentations at scientific meetings and stakeholder events. Project members have also communicated progress of this project through invitations to speak at other stakeholder events (cattle producer, continuing education programs for practicing food animal veterinarians, interview requests, etc). With the Conference for Reserach Workers in Animals Diseases moving to Jan (instead of Dec), many abstracts that were generated this year for that conference will not be reported until the next reporting cycle. What do you plan to do during the next reporting period to accomplish the goals?Many of our workplans and goals for this reporting year were still impacted/delayed due to the global COVID-19 pandemic, especially as related to staffing, supply chain issues, and long cues at service centers. An additional one-year no-cost extension will allow us to address the the following in 2023: Objective 1: Complete plasma sample testing and analysis to determine CTC plasma concentration levels maintained during the CTC treatment phase.Collectively analyze molecular, serologic,microbiological (characterization of fecal microbiome), and plasma CTC concentration levels from the large controlled A. marginale infection/treatment experiment. Continue to prepare data for presentation and publication in open-access peer-reviewed journals. Objective 2:Complete processing fecal samples collected during the large controlled A. marginale infection/treatment experiment to evaluate how protracted CTC treatment affects development of antimicrobial resistance in off-target bovine gastro-intestinal Enterococcus species by Sensititre (these plates were signficantly back-ordered and we only received them at the very end of this reporting period).Submit DNA prepared from fecal samples obtained from cattle provided protracted CTC treatment for 16S microbiome analysis.Continue to prepare data for presentation and publication in open-access peer-reviewed journals. Objective 3:Continue working on A. marginale strain tick acquisition/transmission and maintenence of CTC susceptibliity phenotype data for publication. Objective 4: Continue working to develop resources for stakeholder (cattle producers and practicing food animal veterinarians) on bovine anaplasmosis and antimicrobial resistance. Plan a producer-focused anaplasmosis event relaying updated information on bovine anaplasmosis and antimicrobial based management strategies for anaplasmosis.Continue to work with producers and veterinarians on extension-based anaplasmosis studies towards developing recommendations on effective yet judicious use of medically-important antimicrobials to manage bovine anaplasmosis. Present our 2022 research data at the Conference for Reserach Workers in Animals Diseases that has now moved to Jan instead of Dec (there was no CRWAD meeting in this reporting cycle).

Impacts
What was accomplished under these goals? Many of our workplans and goals for this reporting year were still impacted/delayed due to the global COVID-19 pandemic, especially as related to staffing, supply chain issues, and long cues at service centers. Inspite of these challenges, the following was accomplished in 2022. Objective 1 Accomplishments. Continuation of sample processing and analysis from the intensive 10-month long controlled A. marginale infection and treatment experiment. Preparation of plasma samples for evaluation of chlortetracycline concentration testing (waiting in service center cue for analysis). Performed controlled A. marginale challenge experiment using adult cows treated with the current approved hand-fed dose of chlortetracycline for anaplasmosis controls (evaluated: disease progression, bacteremia, necessity of rescue treatment).Through A. marginale surveillance, identified at least 5 A. marginale strains that appear refractory to chlortetracycline treatment. Objective 2 Accomplishments. Completed antimircobial susceptibility testing of E. coli isolated from cattle treated with different concentrations of chlortetracycline for anaplasmosis control by Sensititre analysis. Completed Enterococcus isolation and mass spec species identification of isolates from cattle treated with different concentrations of chlortetracycline for anaplasmosis control. Began antimicrobial susceptibility testing of Enterococcusisolated from cattle treated with different concentrations of chlortetracycline for anaplasmosis control by Sensititre analysis (plates were significantly backordered). Prepared DNA from fecal samples derived from cattle treated withdifferent concentrations of chlortetracycline for anaplasmosis control for 16S microbiome analysis. Objective 3 Accomplishments: Continuation of data analysis and manuscript prepartion describing persistence of A. marignale chlortetracycline susceptibility phenotype after passage through the tick vector. Objective 4 Accomplishments. Development of producer-focused educational handouts on Anaplasmosis and Antibiotics.We have also continued to interface and serve as a known resource on bovine anaplasmosis management information/advice for cattle producers and veterinarians. We also helped devise individualized antimicrobial-based anaplasmosis managment strategies that support more judicious use of chlortetracycline forseveral cattle producers and are following their observed success/satisfaction with those methods. Additionally, from samples collected through producer and bovine veterinarian relationships, we continue investigaing the diversity of A. marginale strains circulating in herds with different CTC usage practicies to in the future look for genetic correlates in antimicrobial susceptbility between A. marginale starins.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Lovett AC, Reppert EJ, Jaeger JR, Kang Q, Flowers MR, Bickmeier NP, Anantatat T, ODay SC, Armstrong CL, Reif KE. Satisfactory breeding potential is transiently eliminated in beef bulls with clinical anaplasmosis. BMC Vet Res. 2022. Oct 29;18(1):381. doi: 10.1186/s12917-022-03470-7.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Toillion AR, Reppert EJ, Apley MD, Amachawadi RG, Olson KC, Kleinhenz MD, Coetzee JF, Reif KE, Kang Q. Evaluation of chlortetracycline plasma concentrations relative to anaplasmosis status of naturally endemic beef cattle following free-choice administration of CTC-medicated mineral. K-State CVM Phi Zeta Research Day. Manhattan, KS. March 1, 2022.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: ODay SC, Bickmerier NP, Schmid IJ, Anantatat T, McCann SM, Reif KE. Protective efficacy of the conditionally licensed anaplasmosis vaccine. K-State CVM Phi Zeta Research Day. Manhattan, KS. March 1, 2022.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Lovett AC, Reppert EJ, Jaeger JR, Kang Q, Bickmeier NP, Anantatat T, Flowers MR, ODay SC, Armstrong CL, Reif KE. Acute anaplasmosis reduces breeding soundness in experimentally infected beef bulls. K-State CVM Phi Zeta Research Day. Manhattan, KS. March 1, 2022.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Lovett A, Reppert EJ, Flowers M, Bickmeier N, Jaeger J, Kang Q, Reif KE. Acute anaplasmosis reduces breeding soundness in experimentally-infected beef bulls. Capital Graduate Student Research Symposium. March 29, 2022. Topeka, Kansas.

Progress 01/01/21 to 12/31/21

Outputs
Target Audience:Our efforts to communicate and deliver science-based knowledge regarding bovine anaplasmosis and the effect this disease may have on promoting antimicrobial resistance have included presentations for formal education programs (e.g.professional conferences/meetings, continuing education programs); informal educational programs/outreach/extension activities (e.g. participation in producer meetings, serving as point of contact for anaplasmosis related questions); training of undergraduate students, professional students (e.g. DVM students seeking research experience), graduate students; and publication of research results. Our project-related presentations at professional society meetings reach the following target audiences: academic and governmental researchers and students interested in bovine anaplasmosis, antimicrobial treatment and resistance, cattle health and disease transmission. We have participated in Continuing Education events (e.g. Tennessee Veterinary Medical Association Annual Meeting) presenting on bovine anaplasmosis and our research efforts to better understand and manage this disease and how this disease may impact other management strategies (e.g. use of antibiotics). This year we also were invited to presenton the threat of anaplasmosis to the Ontairo Animal Health Network through participation in their podcast series.Additionally, we continually interface with cattle producers and veterinarians on questions regarding bovine anaplasmosis - including providing an Anaplasmosis Learning Lounge presentation at the National Cattlemen'sBeef Association meeting. Our project-related presentations and participation in informal educational programs, outreach, and extension activities reach the following target audiences: beef cattle producers; practicing livestock veterinarians; livestock industry personnel; and, people generally interested in cattle health, ticks, and tick-borne diseases of livestock importance. Through participation in this project, a number of graduate and undergraduate students are presently receiving hands-on training in developing research skills (e.g. lab-based, animal handling), communication skills (e.g. via participation in local and regional meeting), organizational skills, and teamwork skills. Publication of research results in peer-reviewed publications provides wider opportunities to communicate our project-related findings with variou stakeholders in academia, government, industury, other professional groups, or members of the general public interested in this work. We also maintained wokring collaborations with some USDA-ARS units also interested in anaplasmosis management. Changes/Problems:The occurance of the global COVID-19 pandemic significantly delayed our productivity for the last two years due to COVID-19 related restrictions that the State of Kansas and Kansas State University implemented regarding research activities,student and staff on-campus activities, and hiring challenges. We will request an additional one-year no cost extension due to COVID-19 delays. What opportunities for training and professional development has the project provided?This year, this project have supported the following training and professional development opportunities. This project supported the training of one Masters students (Flowers),two PhD students (Toillion and Bickmeier), with Bickmeier joining this year - her project will focus on investigating/optimizing anaplasmosis control strategies; and a graduate student with the KSU Beef Cattle Institute that is helping to prepare educational outreach material to communication with stakeholders about anaplasmosis and judicious antimicrobial use practices. This project supported training a summer Veterinary Research Scholar Program student's project (O'Day). This project supported research experiences and training opportunities for 5 undergraduate students. This project supported cross-training of 3 other graduate/veterinary students. This project supported professional developoment through attendence and presentation at scientific meetings for 2 graduate students and 1 faculty member. For regional cattle producers, this project supported extension efforts to relay information on bovine anaplasmosis and associated managment strategies thorugh one-on-one consultations, complementary anaplasmosis diagnostics, and through participation in cattle producer events. For bovine veterinarians (both local, national, and international - Canada), this project supported developent of educational content delivered during annual continuing education programs; as well as, opportunities for one-on-one consultations on bovine anaplasmosis management. How have the results been disseminated to communities of interest?Results of this project have been disseminated through presentations at scientific meetings and stakeholder events. Project members have also communicated progress of this project through invitations to speak at other stakeholder events (cattle producer, continuing education programs for practicing food animal veterinarians, etc). What do you plan to do during the next reporting period to accomplish the goals?Because of the COVID-19 pandemic and state and university restrictions associated with the pandemic, our progress was still hampered in 2021. For the upcoming year (2022), we plan to continue working towards completing the following: Year 05, Objective 1: Complete processing and analysis of the following samples collected during the large controlled A. marginale infection/treatment experiment performed in Year 02: i) complete serum sample analysis to evaluate and compare maintenance of A. marginale-specific immune response in challenged cattle pre-CTC treatment, during CTC treatment, and post-CTC treatment; ii) complete plasma sample testing and analysis to determine CTC plasma concentration levels maintained during the CTC treatment phase; iii) prepare data for publication. Year 05, Objective 2: i) finish processing fecal samples collected during the large controlled A. marginale infection/treatment experiment performed in Year 02 to evaluate how protracted CTC treatment affects development of antimicrobial resistance in off-target bovine gastro-intestinal microbial species (E. coli and Enterococcus); ii) from a subset of samples, examine how protracted CTC treatment broadly impacts antimicrobial susceptbility - sensititer analysis of E. coli isolates; iii) submit DNA prepared from fecal samples obtained from cattle provided protracted CTC treatment for 16S microbiome analysis and analyze data; iv) prepare data for publication. Year 05, Objective 3: Prepare A. marginale strain tick acquisition/transmission and maintenence of CTC susceptibliity phenotype data for publication. Year 05, Objective 4: Continue working to develop resources for stakeholder (cattle producers and practicing food animal veterinarians) on bovine anaplasmosis and antimicrobial resistance. Begin planning the next stakeholder-focused bovine anaplasmosis symposium - to occur once pandemic restrictions have been lifted. Continue to work with producers and veterinarians on extension-based anaplasmosis studies towards developing recommendations on effective yet judicious use of medically-important antimicrobials to manage bovine anaplasmosis.

Impacts
What was accomplished under these goals? Many of our workplans and goals for Year 04, (2021) were still impacted/delayed due to the global COVID-19 pandemic and state and university restrictions associated with the pandemic. Inspite of these challenges, the following was accomplished in 2021. Year 04, Objective 1 Accomplishments. Continuation of sample processing and analysis from the intensive 10-month long controlled A. marginale infection and treatment experiment which generated over 50,000 tubes of sample material. Completion of DNA extracted and quantitative PCR on whole blood samplesfor evaluation of A. marginale infection levels. Detection of A. marginale-specific antibodies was performed from a subset of time points on serum samples. Preparation of plasma samples for evaluation of chlortetracycline concentration testing. Year 04, Objective 2 Accomplishments. A new graduate student was recruited, with Objective 2 being a portion of her projected of her graduate program. This student began evaluting the susceptibility ofE. coli isolatesgenerated from fecal samples from selected time points from the large Year 02 study. Completed E. coli and Enterococcuss isolation fromselected time points and chlortetracycline susceptibility testing.Completed DNA extraction from fecal samples for 16S microbiome analysis.Data from E. coli isolate susceptibility testing from samples collected from a field-study cattle herd that used CTC continuously for 6 months for anaplasmosis management has been published. In another field study where pastured cattle were provided chlortetracycline for 6 months, A. marginale infection status, A. marginale bacterial levels, and chlortetracycline levels were evaluated. Data from this second field study is presently being analyzed and prepared for publication. Year 04, Objective 3 Accomplishments. Data analysis and manuscript prepartion describing persistence of A. marignale chlortetracycline susceptibility phenotype after passage through the tick vector.Year 04, Objective 4 Accomplishments. We continue to interface and serve as a known resource on bovine anaplasmosis management information/advice for many cattle producers and veterinarians. We were also able to arrange enroll/garner participation from several cattle producers to perform field-evaluations of antimicrobial-based anaplasmosis management strategies. Additionally, from samples collected through producer and bovine veterinarian relationships, we continue investigaing the diversity of A. marginale strains circulating in herds with different CTC usage practicies to in the future look for genetic correlates in antimicrobial susceptbility between A. marginale starins. We also continued our multi-year antimicrobial-based anaplasmosis management in-field assessment collaboration with USDA-ARS-GRL cattle herds.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Toillion AR, Reppert EJ, Amachawadi RG, Olson KC, Coetzee JF, Kang Q, Reif KE. Effect of protracted free-choice chlortetracycline-medicated mineral for anaplasmosis control on Escherichia coli chlortetracycline resistance profile from pastured beef cattle. Microorganisms. 2021. 9(12):2495. doi: 10.3390/microorganisms9122495.
• Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Curtis AK, Kleinhenz MD, Anantatat T, Martin MS, Magnin GC, Coetzee JF, Reif KE. Failure to eliminate persistent Anaplasma marginale infection from cattle using labeled doses of chlortetracycline and oxytetracycline antimicrobials. Vet Sci. 2021. 8(11):283. doi: 10.3390/vetsci8110283.
• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Lovett AC, Reppert EJ, Jaeger JR, Kang Q, Flowers MF, Bickmeier NP, Anantatat T, O'Day S, Armstrong CL, Reif KE. Satisfactory breeding potential is transiently eliminated in beef bulls with clinical anaplasmosis.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Flowers M, Anantatat T, Reppert EJ, Reif KE. Evaluation of enrofloxacin and oxytetracycline to eliminate persistent Anaplasma marginale infection in cattle. Phi Zeta Day. March 02, 2021. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Toillion A, Reppert EJ, Amachawadi R, Olson KC, Coetzee JF, Kang Q, Reif KE. Effect of protracted free-choice CTC-medicated mineral for anaplasmosis control on the antimicrobial resistance profile of Escherichia coli in beef cattle on a pasture setting. Phi Zeta Day. March 02, 2021. Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Ward H, Thomas L, Reif KE. The University of Arkansas System collaborates with Kansas State University to conduct anaplasmosis surveillance in Arkansas beef cattle. River Valley Beef Conference. February 23, 2021. Ozark, AR.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Bickmeier NP, Flowers MR, Anantatat T, Okafor C, Whitlock B, Strickland, L, Rhinehart J, Carter C, Reif KE. Genetic diversity of Anaplasma marginale in Tennessee beef cattle herds. Conference of Research Workers in Animal Diseases. December 3-7, 2021. Chicago, Illinois.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Toillion AR, Reppert EJ, Amachawadi RG, Olson KC, Coetzee JF, Kang Q, Reif KE. Effect of antimicrobial-based anaplasmosis control on E. coli chlortetracycline susceptibility from pastured cattle. Conference of Research Workers in Animal Diseases. December 3-7, 2021. Chicago, Illinois.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Flowers M, Anantatat T, Reppert EJ, Reif KE. Evaluation of enrofloxacin and oxytetracycline to eliminate persistent Anaplasma marginale infection in cattle. Conference of Research Workers in Animal Diseases. December 3-7, 2021. Chicago, Illinois.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Krueger SA, Anantatat T, Reif KE. Evaluation of Amblyomma americanum vector competence for Anaplasma marginale. Conference of Research Workers in Animal Diseases. December 3-7, 2021. Chicago, Illinois.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: ODay S, Bickmeier NP, Schmid I, Anantatat T, Reif KE. Immune responses elicited in calves administered the conditionally approved anaplasmosis vaccine. National Veterinary Scholars Symposium. August 4-6, 2021. Virtual meeting hosted by Iowa State University.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Eggers A, Bickmeier N, Anantatat T, Beltz M, Almaraz J, Huser S, Reif K. Investigating the prevalence of Anaplasma marginale in breeding cows and the impact of chronic anaplasmosis on packed red blood cell volume and pregnancy rates. K-State Food Science Institute Undergraduate Research Symposium Fall 2021. December 14, 2021. Manhattan, KS.
• Type: Theses/Dissertations Status: Accepted Year Published: 2021 Citation: Evaluation of enrofloxacin and oxytetracycline to eliminate persistent Anaplasma marginale infection in cattle.

Progress 01/01/20 to 12/31/20

Outputs
Target Audience:Our efforts to communicate and deliver science-based knowledge regarding bovine anaplasmosis and the effect this disease may have on promoting antimicrobial resistance have included presentations for formal education programs (e.g. professional conferences/meetings, continuing education programs); informal educational programs/outreach/extension activities(e.g. participation in producer meetings, serving as point of contact for anaplasmosis related questions); training of undergraduate students, professional students (e.g. DVM students seeking research experience), graduate students; and publication of research results. Our project-related presentations at professional society meetings reach the following target audiences: academic and governmental researchers and stduents interested in bovine anaplasmosis, antimicrobial treatment and resistance, cattle health and disease transmission. We have participated in Continuing Education events (e.g. Missouri Veterinary Medical Association Annual Meeting) presenting on bovine anaplasmosis and our research efforts to better understand and manage this disease. Additionally, we continually interface with cattle producers and veterinarians on questions regarding bovine anaplasmosis. Our project-related presentations and participation in informal educational programs, outreach, and extension activities reach the following target audiences: beef cattle producers; practicing livestock veterinarians; livestock industry personnel; and, people generally interested in cattle health, ticks, and tick-borne diseases of livestock importance. Through participation in this project, anumber of graduate and undergraduate students are presently receiving hands-on training in developing research skills (e.g. lab-based, animal handling), communication skills (e.g. via participation in local and regional meeting), organizational skills, and teamwork skills. Publication of research results in peer-reviewed publications provides wider opportunities to communicate our project-related findings with variou stakeholders in academia, government, industury, other professional groups, or members of the general publicinterested in this work. We also maintained wokring collaborations with some USDA-ARS units also interested in anaplasmosis management. Changes/Problems:The occurance of the global COVID-19 pandemic significantly delayed our productivity this year due to COVID-19 related restrictions that the State of Kansas and Kansas State University implemented regarding research activities and student and staff on-campus activities. We will request a one-year no cost extension due to COVID-19 delays. What opportunities for training and professional development has the project provided?This year, this project have supported the following training and professional development opportunities. This project currently supports the training of two Masters students (Skinner and Flowers) and one PhD student (Toillion), with Toillion joining this year - her project will focus on investigating how the impact of protracted CTC treatment for anaplasmosis control on the antimicrobial susceptiblity of bovine gastro-microbial species. This porject supported training a summer Veterinary Research Scholar Program student's project (Herd); and, continuation of our 2019's VRSP student's project (Kruger). This project supported research experiences and training opportunities for 3 undergraduate students. This project supported cross-training of 3 other graduate/veterinary students. This project supported professional developoment through attendence and presentation at scientific meetings for 2 graduate students and 1 faculty member. For regional cattle producers, this project supported extension efforts to relay information on bovine anaplasmosis and associated managment strategies thorugh one-on-one consultations, complementary anaplasmosis diagnostics, and through participation in cattle producer events. For regional bovine veterinarians, this project supported developent of educational content delivered during annual continuing education programs; as well as, opportunities for one-on-one consultations on bovine anaplasmosis management. How have the results been disseminated to communities of interest?Results of this project have been disseminated through presentations at scientific meetings and stakeholder events. Project members have also communicated progress of this project through invitations to speak at other stakeholder events (cattle producer, continueing education programs for practicing food animal veterinarians, etc). Most of these events occurred vitually this year due to the COVID-19 pandemic. What do you plan to do during the next reporting period to accomplish the goals?Because of the COVID-19 pandemic and state and university restrictions associated with the pandemic, our progress was hampered in 2020. For the upcoming year (2021), we plan to continue working towards completing the following: Year 04, Objective 1: Complete processing and analysis of the following samples collected during the large controlled A. marginale infection/treatment experiment performed in Year 02: i) whole blood samples to evaluate and compare A. marginale infection kinetics pre-CTC treatment, during CTC treatment, and post-CTC treatment (1/2 of this was completed in 2020, the other half to be completed in 2021); ii) serum samples to evaluate and compare maintenance of A. marginale-specific immune response in challenged cattlepre-CTC treatment, during CTC treatment, and post-CTC treatment (a portion of this was completed in 2020, the remaining samples will be tested in 2021); iii) plasma samples to determine CTC plasma concentration levels maintained during the CTC treatment phase. Year 04, Objective 2: Continue processing fecal samples collected during thelarge controlled A. marginale infection/treatment experiment performed in Year 02 to evaluate how protracted CTC treatment affects development of antimicrobial resistance in off-target bovine gastro-intestinal microbial species, as well as how protracted CTC treatment affects the overall composition of the bovine gastro-intestinal microbiome. E. coli and Enterococcuss were isolated from 1/2 of the selected time points in 2020, the remainder will be isolated and tested in 2021. To correct a processing error that was accidentally made with evaluating the antimicrobial susceptibility of the E. coli isolates in 2020, these experiments willbe repeated in 2021. Complete DNA extraction from fecal samples for 16S microbiome analysis. Year 04, Objective 3: Finish analyzying the second contemporary A. marginale strain tick acquisition/transmission data. Prepare these results for publication. Year 04, Objective 4: Continue working to develop resources for stakeholder (cattle producers and practicing food animal veterinarians) on bovine anaplasmosis and antimicrobial resistance. Begin planning the next stakeholder-focused bovine anaplasmosis symposium - to occur once pandemic restrictions have been lifted. Continue to work with producers and veterinarians on extension-based anaplasmosis studies towards developing recommendations on effective yet judicious use of medically-important antimicrobials to manage bovine anaplasmosis.

Impacts
What was accomplished under these goals? Many of our workplans and goals for Year 03, (2020) were delayed due to the global COVID-19 pandemic and state and university restrictions associated with the pandemic. Inspite of these challenges, the following was accomplished in 2020. Year 03, Objective 1 Accomplishments. Last year, we performed the live-phase of the intensive 10-month long controlled A. marginale infection and treatment experiment which generated over 50,000 tubes of sample material. This year we've focused on processing these samples. DNA was extracted and quantitative PCR was performed on ~1/2 of the whole blood samples for evaluation of A. marginale infection levels. Detection of A. marginale-specific antibodies was performed from a subset oftime points on serum samples. Year 03, Objective 2 Accomplishments. A new graduate student was recruited, with Objective 2 being the projected focus of her graduate work. This student began isolating E. coli and Enterococcus from fecal samples from selected time points from the large Year 02 study. Concurrently, she also isolated E. coli from samples collected from a field-study cattle herd that used CTC continuously for 6 months for anaplasmosis management, and has performed antimicrobial suscpetiblity testing on these samples. She is in the process of analyzing the results and preparing a manuscript on the field study results. Year 03, Objective 3 Accomplishments. Repeated exectution of A. mraginale tick acquisition/transmission experiment on a second contemporary A. marginale strain to evaluate whether A. marginale antimicrobial susceptbility phenotype is maintained after passage throught the tick vector. This second experiment was successfully executed and data is in the process of being analyzed and prepared for publication. Year 03, Objective 4 Accomplishments. After a successfull Bovine Anaplasmosis Symposium in Year 02, we have been able to interface and serve as a known resource on bovine anaplasmosis management information/advice for many cattle producers and veterinarians. We were also able to arrange enroll/garner participation from several cattle producers to perform field-evaluations of antimicrobial-based anaplasmosis management strategies. Additionally, from samples collected through producer and bovine veterinarian relationships, we have begun investing the diversity of A. marginale strains circulating in herds with different CTC usage practicies to in the future look for genetic correlates in antimicrobial susceptbility between A. marginale starins. We also continued our multi-year antimicrobial-based anaplasmosis management in-field assessment collaboration with USDA-ARS-GRL cattle herds.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Reppert EJ, Reif KE, Montgomery SR, Magnin G, Zhang Y, Martin-Jimenez T, Olson KC, Coetzee JF. Determination of plasma chlortetracycline (CTC) concentrations in beef cattle on range being fed one of four FDA-approved free-choice CTC medicated minerals. Transl Anim Sci. 2020. 4(2):txaa048. PMID: 32705045. doi: 10.1093/tax/txaa048.
• Type: Theses/Dissertations Status: Accepted Year Published: 2020 Citation: Skinner, BC. Characterization of contemporary Anaplasma marginale strains for assessing antimicrobial-based anaplasmosis control strategies. Thesis. Kansas State University. 2020. Link: https://hdl.handle.net/2097/40615.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Toillion A, Skinner BC, Flowers MR, Anantatat T, Reppert EJ, Coetzee JF, Amachawadi R, Reif KE. Impact of chlortetracycline on the selection of antimicrobial resistance in E. coli and Enterococcus in beef cattle. 101st Annual Conference of Research Workers in Animal Diseases. December 2020. Virtual oral presentation.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Flowers MR, Anantatat T, Jaeger J, Reppert EJ, Reif KE. Investigating the occurrence of transplacental Anaplasma marginale transmission in endemic beef cattle herds. 101st Annual Conference of Research Workers in Animal Diseases. December 2020. Virtual oral presentation.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Apple GL, Thomas LR, Ward HM, Reif KE, Anantatat T, Flowers MR, White AC, Powell JG. Surveillance of Anaplasma marginale in Arkansas beef cattle herds. Virtual oral presentation. American Association of Bovine Practitioners Annual Meeting. September 2020. Virtual meeting due to COVID-19.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Herd LK?, Flowers MR, Reppert EJ, Reif KE. Efficacy of chlortetracycline to protect against clinical anaplasmosis in transiently immunosuppressed cattle. Virtual poster presentation. National VRSP Poster Session. July 2020. Held virtually due to COVID-19.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Herd LK, Flowers MR, Reppert EJ, Reif KE. Efficacy of chlortetracycline to protect against clinical anaplasmosis in transiently immunosuppressed cattle. Poster presentation. K-State CVM VRSP Poster Session. July 2020. K-State, Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Krueger S, Anantatat T, Reif KE. Evaluation of Amblyomma americanum vector competence for Anaplasma marginale. Poster presentation. K-State Phi Zeta (Sigma Chapter) Research Day. March 2020. K-State, Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Skinner BC, Reppert EJ, Anantatat T, Coetzee JF, Olson KC, Reif KE. Isolation and characterization of two Anaplasma marginale isolates from a Kansas beef cattle herd. Poster presentation. K-State Phi Zeta (Sigma Chapter) Research Day. March 2020. K-State, Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Flowers MR, Anantatat T, Jaeger J, Reppert EJ, Reif KE. Investigating the occurrence of transplacental Anaplasma marginale transmission in endemic beef cattle herds. Poster presentation. K-State Phi Zeta (Sigma Chapter) Research Day. March 2020. K-State, Manhattan, KS.
• Type: Theses/Dissertations Status: Accepted Year Published: 2020 Citation: Apple, G. Surveillance of Anaplasma marginale in Arkansas beef cattle herds. Animal Science Undergraduate Honors Theses Retrieved from https://scholarworks.uark.edu/anscuht/40
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Skinner BC, Reppert EJ, Kleinhenz MD, Coetzee JF, Anantatat T, Reif KE. Comparison of multiple chlortetracycline regimens to control diverse Anaplasma marginale strain infection. 101st Annual Conference of Research Workers in Animal Diseases. December 2020. Virtual oral presentation.

Progress 01/01/19 to 12/31/19

Outputs
Target Audience:TARGET AUDIENCE. In Year 02, the target audience that this proposal reseached included: General Cattle (Beef) Producer Community: We hosted a producer-oriented Bovine Anaplasmosis Symposium to update producers on bovine anaplasmosis epidemiology, ecology, diagnosis, treatment, and management strategies; and the Veterinary Feed Directive. Through this forum we also communicated current results from this proposal and discussed on-going study goals (the results of which will be shared during the Bovine Anaplasmosis Symposium hosted in Year 04). We also worked with producers in an extension capacity to monitor efficacy of chlortetracycline (CTC) treatment in producer-owned herds. Finally, producers were engaged during other presentations where we were invited to present updates on bovine anaplasmosis and related management strategies. Practicing Large Animal Veterinarians: Through our Bovine Anaplasmosis Symposium, we engaged with practicing large animal veterinarians and offered 5.5 hours of continuing education credit. Veterinarians were also engaged during other presentations where we were invited to present updates on bovine anaplasmosis and related management strategies. Pharmaceutical Industry: Pharmaceutical industry persons interested in the management of anaplasmosis were in attendance at the Bovine Anaplasmosis Symposium. Academia: Study results from this grant were presented at multiple scientific meetings. Through the Bovine Anaplasmosis Symposium we also developed new collaborations with researchers and extension agents from other Universities interested in anaplasmosis management. Other Federal Researchers: We maintained working collaborations with some USDA-ARS units interested in anaplasmosis managment. EFFORTS: In Year 02, project-related efforts included: Hosting the 2nd Kansas State University Symposium on Bovine Anaplasmosis (May 20, 2019). This was a stakeholder-oriented event that attracted over 150 attendees. Continued anaplasmosis management extension activities with cattle producers. Delivered project-related results through presentations at topic-related national scientific meetings (attended by other academic researchers, federal agency representatives, industry persons, etc.) Changes/Problems:For Objective 3, the initial A. marginale isolate we chose to assess the maintenance of antimicrobial susceptibility phenotype through tick transmission, turned out to not be a a tick-transmissible strain of A. marginale. This information will be prepared for publication. However, we may not be able to fully replicate this experiment for budget-related reasons. We will re-perform part of this experiment in Year 03 with another contemporary A. marginale strain. What opportunities for training and professional development has the project provided? This project currently supports the training of two Master's students (Skinner and Flowers), with Flowers joining the project this year. This project supported training a summer Veterinary Student Research Program Scholar's (Krueger) project. This project supported a second summer of research training for a previous Veterinary Student Research Program Scholar's (Lancaster). This project supported research experience and training opportunities for 5 undergraduate students. This project supported cross-training 3 other graduate/veterinary students. This project supported professional development through attendence and presentation at scientific meetings for 2 graduate students, 1 professional staff member,and 1 faculty. To the regional cattle producer community and practicing large animal veterinarians, this project supported educational and professional development opportunities through the Bovine Anaplasmosis Symposium. Through invitations of project members to speak at cattle producer and practicing large animal veterinarian events, this project has supported professional development opportunities for our key stakeholders. How have the results been disseminated to communities of interest?Results of this project have been disseminated through presentations at scientific meetings and stakeholder events. Most notably, we hosted a stakeholder-oriented Bovine Anaplasmosis Symposium to directly reach out to our stakeholder community to communicate the progress of this project. Project members have also communicated progress of this progress through invitations to speak at other stakeholder (cattle producer, practicing large animal veterinarian) events. What do you plan to do during the next reporting period to accomplish the goals?Year 03, Objective 1 Goals: Complete processing and analysis of the following samples collected during the large controlled A. marginale infection/transmission experiment in Year 02: i) whole blood samples to evaluate and compare A. marginale infection kinetics pre-CTC treatment, during CTC treatment, and post-CTC treatment; ii) serum samples to evaluate and compare maintenance of A. marginale-specific immune response in challenged cattle pre-CTC treatment, during CTC treatment, and post-CTC treatment; and, iii) plasma samples to determine the CTC plasma concentration levels maintained during the treatment phase of the experiment. Year 03, Objective 2 Goals: Process the fecal samples collected during the large controlled A. marginale infection/transmission experiment in Year 02 to evaluate how protracted CTC treatment affects development of antimicrobial resistance in off-target bovine gastro-intestinal microbial species, as well as, how protracted CTC treatment affects the overall composition of the bovine gastro-intestinal microbiome. A second goal is to further characterize the antimicrobial susceptibility phenotype of historic and contemporary A. marginale isolates/strains. Year 03, Objective 3 Goals: Repeat as much of the A. marginale tick acquisition/transmission experiment to examine antimicrobial susceptibility phenotype maintenance experimentas possible with a different contemporary A. marginale isolate/strain. Year 03, Objective 4 Goals: i)Begin developingan anaplamosis management resource website. ii) We will also begin planning for the next stakeholder-oriented Bovine Anaplasmosis Symposium to be hosted in Year 04. iii) We will continue to work with producers and veterinarians in extension-based anaplasmosis managment studies towards developing recommendations on effective yet judious use of medically-important antimicrobials to manage anaplasmosis.

Impacts
What was accomplished under these goals? Year 02, Objective 1 Accomplishments. The primary Objective 1 accomplishments this year included the following: Execution of the ~10 month long, ~100 calf controlled A. marginale infection and CTC treatment experiment. For this experiment, calves were inoculated with one of four A. marginale isolates (2 historic isolates and 2 contemporary isolates). Once animals entered the persistent phase of disease they were re-blocked into one of four CTC treatment groups (0, 0.5, 2.0 or 10.0 mg CTC/lb body weight) and treated for 120 days. Throughout this experiment the following samples were collected: i) whole blood samples to evaluate and compare A. marginale infection kinetics between different A. marginale isolates and in response to different CTC treatment regimens; ii) serum samples to evaluate and compare maintenance of A. marginale-specific immune response between animals infected with different A. marginale isolates and animals in different CTC treatment groups; iii) plasma samples to evaluate CTC blood plasma concentration in animals treated with different doses of CTC; and, iv) fecal samples to evaluate impact of CTC treatment on target and off-target microbial species (for Objective 2). From this experiment >50,000 tubes of sample material were generated, cateloged, and preserved for subsequent analysis. Processing of the collected samples has commenced and will continue to occur throughout Year 03. Additional work was performed to continue characterizing new contemporary A. marginale strains, including antimicrobial susceptibility and tick-transmissibility phenotypes. Additional work was continued to determine if certain contemporary A. marginale Msp1a genotypes are more commonly associated with clinical anaplasmosis and whether there is any correlation with antimicrobial use. Year 02, Objective 2 Accomplishments.The samples that are the basis for this objective were collected and cateloged during the successful execution of the large controlled A. marginale infection/treatment experiment. The samples for testing the impact of long-term CTC treatment on off-target (zoonotic bovine microbial commensals) included fecal sample from all study animals that were preserved in a glycerol solution or directly frozen for antimicrobial susceptibility testing and microbial community analysis, respectively. The samples for testing the impact of long-term CTC treatment on the target pathogen (A. marginale) included whole blood samples to evaluate A. marginale infection kinetics throughout CTC treatment. Processing of samples for analysis has begun and will be completed in Year 03. Year 02, Objective 3 Accomplishments. Execution of A. marginale tick acquisition/transmission experiment to evaluate whether A. marginale antimicrobial susceptibility phenotype is maintained after passage through the tick vector. This experiment was successfully executed, however, the contemporary strain chosen turned out not to be tick-transmissible. As there are no known genetic markers for tick-transmissibility in the A. marginale genome, this was not something we could have predicted. We will prepare these results for publication as the phenotype of non-tick transmissibile and antimicrobial tolerant has not previously been described. We will repeat as much of the original proposed A. marginale tick acquisition/transmission experiment as possible in Year 03 with a different contemporary A. marginale strain - one which we now have first-hand knowledge is tick-transmissible. Year 02, Objective 4 Accomplishments. We successfully hosted the 2nd Kansas State University Symposium on Bovine Anaplasmosis (Hilton Garden Inn, Manhattan, KS May 20, 2019). This was a stakeholder-focused symposium on bovine anaplasmosis, that engaged >150 attendees (~40% producers, ~40% veternarians, ~10% students, ~10% industry persons). The attendees were primarily from Kansas, however, participants from 6 other states were also in attendence. The symposium featured 9 presentations on bovine anaplasmosis by national experts, researchers and graduate students, as well as, an interactive producer panel. An optional 5.5 hours of CE credit was offered for veterinarians. Through this Symposium additional research collaborations were formed with other University faculty and extension agents, and with other Federal scientists. There was significant interest of participants in attending the Bovine Anaplasmosis Symposium to be held in Year 04 as well.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Reif KE, Anantatat T, Kleinhenz M, Reppert EJ, Coetzee JF. Comparison of chlortetracycline and oxytetracycline treatment regimens to clear bovine anaplasmosis (oral presentation). 30th Meeting of the American Society for Rickettsiology. June 2019. Santa Fe, NM.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Krueger S, Anantatat T, Reif KE. Evaluation of Amblyomma americanum vector competence for Anaplasma marginale (poster presentation). National VRSP Poster Session. July 2019. Tufts University, Medford, MA.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Reif KE, Anantatat T, Kleinhenz M, Reppert EJ, Coetzee JF. Comparison of chlortetracycline and oxytetracycline treatment regimens to clear bovine anaplasmosis (oral presentation). 100th Annual Conference of Research Workers in Animal Diseases. November 2019. Chicago, IL.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Skinner BC, Reppert EJ, Kleinhenz M, Coetzee JF, Anantatat T, Reif KE. Comparison of multiple chlortetracycline regimens to control anaplasmosis by diverse Anaplasma marginale strains (poster presentation). 100th Annual Conference of Research Workers in Animal Diseases. November 2019. Chicago, IL.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Anantatat T, Skinner BC, Reppert EJ, Reif KE. Maintenance of distinct Anaplasma marginale genotypes in different herds within the same beef cattle operation. 100th Annual Conference of Research Workers in Animal Diseases. November 2019. Chicago, IL.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Reif KE. Implications of Anaplasma marginale genetic diversity for prevention and control of bovine anaplasmosis. 18th Annual Great Plains Infectious Disease Meeting November 2019. University of Missouri, Columbia, Missouri.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Lancaster M, Peddireddi L, Anantatat T, Reif KE. Examination of Anaplasma marginale genetic diversity in Kansas beef cattle (poster presentation). K-State Phi Zeta (Sigma Chapter) Day. March 2019. K-State, Manhattan, KS.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Anantatat T, Skinner BC, Reppert EJ, Reif KE. Maintenance of distinct Anaplasma marginale genotypes in different herds within the same beef cattle operation (poster presentation). 30th Meeting of the American Society for Rickettsiology. June 2019. Santa Fe, NM.

Progress 01/01/18 to 12/31/18

Outputs
Target Audience:TARGET AUDIENCE. In Year 01, the target audience that this proposal reached included: General Cattle (Beef) Producer Community: We worked with producers to acquire A. marginale isolates for propagation and use for upcoming controlled A. marginale infection/treatment study. We worked with producers to through an extension capacity to monitor efficacy of chlortetraccycline (CTC) treatment in producer-owned herds. We began reaching out to the local and neighboring producer communities to advertise the upcoming producer-oriented Bovine Anaplasmosis Symposium. Practicing Large Animal Veternarians: In response to interest in the upcoming Bovine Anaplasmosis Symposium by practicing large animal veternarians we began advertising this event to them as well as started developing a Continuing Education option for those interested in attending the Symposium. Pharmaceutical Industry: We adverstised the upcoming Bovine Anaplasmosis Symposium to pharmaceutical companies that manufacture products used for managment of anaplasmosis. Academia: Initial study results from this project were presented at scientific meetings. We began recruiting guest speakers from other institutions for the upcoming Symposium. Federal agencies: We developed collaborations with USDA-ARS branches intersted in collaborating on project-related anaplasmosis studies. EFFORTS.In Year 01, project-related efforts included: Extension activities with cattle producers on anaplasmosis management through use of CTC. Beginning preparations (including advertising event to producers, practicing veternarians, etc) for the upcoming producer-oriented Bovine Anaplasmosis Symposium. Delivery of project-related results to presentations at topic-related national scientific meetings (attended by other academics, federal agency representatives, industry, etc) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A Master's student (Skinner) with significant cattle experience was hired for this project. This student will be a great asset in planning logistics for the upcoming large, controlled A. marginale infection/treatment study in Year 02. He spent a signficant amount of time in Year 01, identifying a cattle source, sourcing required supplies/equipment/etc, identifying student workers to help with the Year 02 study, and performing other logistical planning required for the upcoming 10 month, 100 calf study to commence in Year 02. He also helped characterize some contemporary A. marginale strains, including one that was selected for inclusion in the Year 02 controlled A. marginale infection/treatment study. The Master's student (Skinner), research assistant (Anantatat), and PD/PI (Reif) had an opportunity to present current research results at scientific meetings. A summer Veterianry Research Scholar was trained (Lancaster). Her project was determining if certain A. marginale genotypes were more associated with clinical disease. She presented her research at the National Veterinary Scholar Research meeting in at Texas A&M and also at the K-State Phi Zeta Reserach Day. Because the surgeon who was originally planned to perform the splenectomy procedures left, we had to identify and fly in a contract surgeon to perform the splenecomy procedures for the calves used to propatate the A. marginale isolates. The contractsurgeon trained Dr. Emily Reppert (Co-PI) on how to perform these surgergies providing her [Dr. Reppert] with an additional training opportunity. How have the results been disseminated to communities of interest?Results of this project have been disseminated through presentations at scientific meetings. Planning efforts are currently underway for dissemination of project results to stakeholder community in May 2019 (Project Year 02). What do you plan to do during the next reporting period to accomplish the goals?Year 02, Objective 1 Goals: Perform the large, controlled A. marginale infection/treatment experiment. This project will run ~10 months and include ~100 calves. During this experiment, calves will be infected with one of four A. marginale strains (we have two contemporary and two historic A. marginale strains). Once calves enter the persistent phase of disease they will be re-blocked into treatment groups, with each treatment group receiving a different dosage of CTC for 120 days. Samples that will be collected during this experiment include: i) whole blood samples to monitor A. marginale infection kinetics, ii) serum samples to monitor calf sero-status for A. marginale, iii) plasma samples to monitor calf CTC blood plasma concentration levels, and iv) fecal samples to assess impact of CTC treatment on bovine GI microbiome. Year 02, Objective 2 Goals: Samples (calf blood and fecal samples) will be collected during the large, controlled A. marginale infection/treatment experiment to assess the antimicrobial susceptilbiity of A. marginale (target pathogen) and bovine GI commensals (off-target microbial species/pathogens). Year 02, Objective 3 Goals: At the end of Year 02, a tick acquisition/transmission experiment will be performed to assess maintence of antimicrobial susceptibility phenotype for anA. marginale isolatethrough tick transmission. Year 02, Objective 4 Goals: A major goal will be to host the producer-oriented Bovine Anaplasmosis Symposium - one of our main platforms for disseminating results of this project to our stakeholder community. We will also continue to work with producers and veternarians in extension-based anaplamosis management studies.

Impacts
What was accomplished under these goals? Year 01, Objective 1 Accomplishments. The primary Objective 1 accomplishments this year included the following: i) Identification of contemporary Anaplasma marginale field isolates. Cattle from three different operationswere surveyed for A. marginale infection. From these herds, six unique A. marginale isolates were identified, genotyped, and isolated. Novel contemporary isolates were propagated in splenectomized calves to prepare challenge material for the upcoming A. marginale infection/treatment challenge in Year 02. Two historic A. marginale strains were also propaged in splenectomized calves to prepare challenge material for theupcoming A. marginale infection/treatment challenge in Year 02.Extensive preparations were conducted in preparation for thelarge controlled A. marginale infection/treatment study planned for Year 02. Additional work was performed to determine if certain contemporary A. marginale Msp1a genotypes are more associated with clinical anaplasmosis. Year 01, Objective 2 Accomplishments. A pilot study was conducted as an extension demonstration of the efficacy of chlortetracycline (CTC) to control active anaplasmosis in a beef cattle herd naturally endemic for A. marginale. The A. marginale, Msp1a bacterial levels and sero-status, andblood plasma CTC concentration levels were evaluated. The specific A. marginale Msp1a genotypes in this herd were identified and their susceptibility to CTC was documented. The primary experiment related to this objective will begin in project Year 02 as part of the large controlled A. marginale infection/treatment study. Year 01, Objective 3 Accomplishments. Experiments related to this objective are scheduled to start in project Year 02. Year 01, Objective 4 Accomplishments. Planning for the upcoming, producer-oriented Bovine Anaplasmosis Symposium commenced. Draft presentation schedules for the Symposium were planned, presenters and out-of-town guest speakers invited, venue secured, venue-related planning commenced, advertisement of Symposium to producer community commenced, advertisement of Symposium to practicing large animal veternarian community commenced, etc. In addition, we also engaged with a veternarian/producer in an extension capacity to help address challenges with anaplasmosis managment. We will continue to work with this veternarian/producer in project Year 02 and report the results of the chosen CTCanaplasmosis management strategies in Year 02.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Examination of Anaplasma marginale genetic diversity in Kansas beef cattle. Lancaster M, Peddireddi L, Anantatat T, Reif KE National VRSP Poster Session July 2018, Texas A&M, College Station TX
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: The current state of bovine anaplasmosis in Kansas. Reif KE CEVBD Spring National Meeting on Tick Borne Diseases Impacting Human and Animal Health May 2018, K-State, Manhattan KS
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Evaluation of multiple chlortetracycline-medicated mineral formulations to control active anaplasmosis. Reif KE, Reppert EJ, Olson KC, Ananatatat T, Coetzee JF American Society for Rickettsiology June 2018, Milwaukee, WI
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Isolation and characterization of two Anaplasma marginale isolates from a Kansas beef cattle herd. Skinner BC, Reppert EJ, Olson KC, Coetzee JF, Reif KE 99th Annual Conference of Research Workers in Animal Disease December 2018, Chicago, IL
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Implications of Anaplasma marginale genetic diversity on current bovine anaplasmosis control strategies. Reif KE, Lancaster M, Anantatat T, Peddireddi L 99th Annual Conference of Research Workers in Animal Disease December 2018, Chicago, IL