Source: IOWA STATE UNIVERSITY submitted to
MITIGATION OF FLUOROQUINOLONE-RESISTANT CAMPYLOBACTER IN CATTLE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1012068
Grant No.
2017-68003-26499
Cumulative Award Amt.
$1,199,994.00
Proposal No.
2016-09580
Multistate No.
(N/A)
Project Start Date
Jun 1, 2017
Project End Date
May 31, 2022
Grant Year
2017
Program Code
[A4171]- Effective Mitigation Strategies for Antimicrobial Resistance
Project Director
Zhang, Q.
Recipient Organization
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES,IA 50011
Performing Department
Veterinary Microbiology
Non Technical Summary
Campylobacter is a major foodborne pathogen and a leading bacterial cause of gastroenteritis in the United States and other countries. Clinical treatment of campylobacteriosis requires the use of fluoroquinolone (FQ) or macrolide antibiotics, but antibiotic-resistant Campylobacter is increasingly prevalent, compromising the efficacy of clinical treatment. Because of its significance to public health, CDC has recently identified drug-resistant Campylobacter as a serious antibiotic resistance threat in the U.S. Campylobacter is highly prevalent in food producing animals, and ruminants are important reservoirs for this pathogenic organism. Ruminant Campylobacter can be transmitted to humans via contaminated milk and water, or direct contact. Additionally, ruminants are an important part of Campylobacter ecology and may serve as a source of Campylobacter transmission to other farm animals, such as poultry. Previously, most efforts on antibiotic-resistant Campylobacter were devoted to poultry, which led to withdrawal of FQ antimicrobials from poultry production in the U.S. in 2005. However, national surveillance data indicate that FQ-resistant Campylobacter continues to persist and even shows a rising trend after FQ withdrawal in poultry, suggesting that alternative source(s) of FQ-resistant Campylobacter may exist. Our recent study on Campylobacter isolates from feedlot cattle herds in various geographic regions revealed a sharp increase in proportions of FQ-resistant Campylobacter in cattle in the U.S. during the past decade, which coincided with the approved use of fluoroquinolone antimicrobials for control and treatment of respiratory disease in cattle. To reduce the prevalence of FQ-resistant Campylobacter in the food chain, we have developed a 3-year integrated project to identify critical control points for developing mitigation strategies, disseminate science-based knowledge to cattle producers and veterinarians, and train veterinary medicine students with hands-on experience in antibiotic resistance research. Specifically, we will 1) identify critical control points for mitigating the development of FQ-resistant Campylobacter in cattle by evaluating the selective pressure imposed by different FQ treatment regimens, 2) understand the effect of FQ treatment on gut microbiota and FQ resistance by using a metagenomics approach, and 3) develop outreach and training programs to reduce FQ-resistant Campylobacter. The proposed work involves significant participation by stakeholders and directly addresses multiple priority areas of Program A4171 (Effective Mitigation Strategies for Antimicrobial Resistance). The activities will yield important outcomes that will reduce the transmission of antibiotic resistant foodborne pathogens, improve food safety, and enhance the sustainability of the U.S. cattle industry.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123440110060%
3113440118020%
7123440104020%
Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3440 - Meat, dairy cattle;

Field Of Science
1040 - Molecular biology; 1180 - Pharmacology; 1100 - Bacteriology;
Goals / Objectives
The main goal of this application is to identify critical control points and mitigation strategies for fluoroquinolone(FQ)-resistant Campylobacter in ruminants by using an integrated approach. To achieve our goal, we will pursue the following three specific objectives:1. To identify critical control points for mitigating the development of FQ-resistant Campylobacter in cattle by evaluating the selective pressure imposed by different FQ treatment regimens.2. To understand the effect of FQ treatment on gut microbiota and FQ resistance by using a metagenomics approach3. To develop outreach and training programs to reduce FQ-resistant Campylobacter.
Project Methods
Under objective 1, we will conduct laboratory animal studies using calves to evaluate the effect of different combinations of fluoroquinolone use in both healthy and sick animals, conduct detailed pharmacokinetic analysis of FQ antimicrobials in plasma and feces, and measure the development of fluoroquinolone-resistant Campylobacter. Campylobacter will be inoculated into healthy and sick calves (with experimentally induced BRD), the animals will be treated with different treatment regimens, fecal and other tissue samples will be collected for isolation of Campylobacter and pharmacokinetic analysis, and the treatment schemes that are associated with the lowest level of development of fluoroquinolone-resistant Campylobacter will be identified.For objective 2, we will conduct metagenomics and microbiological analyses to compare the intestinal microbial flora, measure fluoroquinolone-resistance genes and mutations, and quantify fluoroquinolone-resistant organisms in both healthy and diseased calves before and after different fluoroquinolone treatment regimens. Total DNA will be extracted from fecal samples of calves collected before and after treatment with fluoroquinolones. Bacterial 16S rRNA gene libraries will be constructed and pooled sequences will be determined using the Illumina MiSeq platform. Fluoroquinolone resistome in cattle gut will be determined using both culture-independent and culture-based approaches. Microbiota changes associated with FQ-resistance development will be determined.For objective 3, we will work closely with cattle producers and veterinarians for timely dissemination of knowledge gained from this project and other sources. We will educate cattle producers and veterinarians for judicial and prudent use of FQ antimicrobials. Additionally, we will take advantage of the Veterinary Medical Summer Scholars Research Program to train next generation veterinarians for appropriate usage of antimicrobials. A web based Qualtrics survey will be utilized to query feedlot managers and veterinarians on usage of antibiotics to prevent, control and treat respiratory disease in beef cattle. Research finding form this research project and FQ usage survey will be published in scientific journals, and will be summarized and posted on the eXtension website. For measuring the success and impact of our extension/outreach component, we will utilize pre- and post-extension surveys. We will use the well-established assessment instrument to determine the satisfaction and knowledge gain of the veterinary students trained by this project.

Progress 06/01/17 to 05/31/22

Outputs
Target Audience:Cattle producers, veterinarians, students, researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this project, 13 veterinary medicine students and 2 graduate students were trained for knowledge and skills in antimicrobial resistance research. How have the results been disseminated to communities of interest?Information on prudent use of animal health products including antibiotics, disease prevention, and antimicrobial resistance was presented to cattle producers and veterinarians. Also, findings from this project were published in scientific journals, and presented at multiple national and regional conferences as poster/oral presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Under Objective 1, we carried out two major studies involving 2-4 month-old commercial calves. The first study investigated the effect of single-dose danofloxacin treatment on the development of fluoroquinolone (FQ) resistance in C. jejuni in healthy and bovine respiratory disease (BRD)-induced animals. The second study was performed to investigate the effect of two different doses of enrofloxacin treatment on FQ resistance development in calves experimentally inoculated with C. jejuni. In both studies, rectal feces were collected for Campylobacter culture and microbiota analysis. Fecal and plasma were also collected for pharmacokinetic/pharmacodynamic (PK/PD) analyses. The first trial involved three separate groups (n= 10 per group) of calves purchased from a commercial source. The animals were all inoculated orally with a mixture of three FQ-susceptible (FQ-S) C. jejuni strains. After one week, the calves in one group were given intratracheally with a Mannheimia haemolytica strain to induce BRD. One week after, calves in two groups (a healthy group and the BRD-induced group) were injected s.c. with a single dose of danofloxacin at 8 mg/kg body weight, while the last group (healthy group) did not receive any antibiotic treatment and served as the control. Fecal samples were then collected for monitoring the development of FQ-resistant Campylobacter in all three groups. At necropsy, lung lesions were scored and a portion of lung from the lesion was taken for Mannheimia culture. Fecal culture results indicated that the vast majority of the calves were naturally colonized by FQ-resistant (FQ-R) C. jejuni at the beginning of the study. After inoculation with FQ-S C. jejuni, there was a notable decrease in the resistant Campylobacter population in all animals. Treatment with danofloxacin resulted in a sharp but transient increase in the relative proportion of FQ-R Campylobacter in both healthy and BRD-induced groups. There was no clear and consistent differences in Campylobacter counts (including FQ-S and FQ-R) between the healthy and BRD-induced groups. Importantly, genotyping (PFGE and MLST) indicated that the spike seen in FQ-R C. jejuni following the injection was mainly due to enrichment of preexisting FQ-R C. jejuni, rather than de novo development of FQ resistance in susceptible strains. The second study included five separate groups (healthy groups A and B, BRD-induced groups A and B, and control group; n=7 calves per group), and used two different doses (7.5 and 12.5 mg/kg; each given once s.c.) of enrofloxacin (each dose was given to one of the groups of healthy and BRD-induced calves). Different from the first trial, the vast majority of calves (30/35; 86%) were free of natural Campylobacter colonization prior to inoculation and only 4 calves carried FQ-R Campylobacter. After the inoculation, calves became stably colonized by FQ-S C. jejuni. Once enrofloxacin was administered, C. jejuni colonization declined sharply and rapidly to undetectable levels in almost all calves. However, the vast majority of animals were later recolonized by Campylobacter. Notably, no FQ-resistant C. jejuni was recovered from any of the calves that received enrofloxacin regardless of the drug dose or disease status of the animals. Together, the findings from the two separate animal trials suggest that FQ use in calves, as employed here, does not result in any measurable level of FQ-resistance development in C. jejuni. In addition to microbial profiles, PK analysis of FQs in plasma and feces from both trials conducted by Ultra High-Pressure Liquid Chromatography. The plasma PKs were similar between healthy and sick calves (with moderate BRD signs), but the fecal danofloxacin concentration was lower in the BRD group. Most PK parameters were not significantly different between healthy and sick calves in the enrofloxacin study. The dose normalized plasma and fecal danofloxacin concentrations were higher than those of enrofloxacin and its metabolite ciprofloxacin. Comparison of the plasma and fecal PK parameters show that danofloxacin had 145-fold higher concentration in feces than in plasma. Similarly, enrofloxacin and ciprofloxacin had 115- and 53-times higher concentrations in feces than in plasma, respectively. Overall, the data indicated that parenterally administered FQs expose gut microbiota and Campylobacter to high concentrations of the antibiotics (~ 20-40 µg/g for enrofloxacin/ciprofloxacin and 200-300 µg/g for danofloxacin during the 8-24 h period after the antibiotic injection). Under Objective 2, the impact of FQ treatments (danofloxacin and enrofloxacin) on gut microbiota composition and resistome development was investigated. Results showed that the fecal microbial diversity (both alpha and beta) and compositions were altered significantly in the danofloxacin treated group compared to the control group. Abundance of Campylobacter was significantly reduced after the treatment, but no significant variations occurred in the control group. Significant correlations between 21 genera and Campylobacter abundance were observed, with genera in Bacteroidetes negatively and most genera in Firmicutes & Proteobacteria positively correlated. For resistome, pooled samples from pre- and post-treatment samples were sequenced using metagenomic proximity ligation approach (ProxiMeta Hi-C). Resistance genes of tetracycline, aminoglycosides, β-lactams, macrolides, and phenicol were detected in both pre- and post-treatment samples. Firmicutes was the leading phylum associated with these ARGs, whereas Bacteroidetes appeared to acquire the ARGs following the treatment. qPCR and Hi-C had very good agreement in tracking ARG dynamics in feces. The overall results suggests that danofloxacin created an environment that might have favored the dominance of resistant strains and/or horizontal transfer of ARGs among bacteria. Similarly, microbial diversity significantly altered following the treatment with enrofloxacin irrespective of its dose. Analysis of composition of microbiome (ANCOM) showed that 35% of the bacterial families were significantly different between pre- and -post-treatment samples. A more significant number of bacterial genera were shifted in the high dose group than the low dose group. Using Hi-C, thirty-two genes conferring resistance to six classes of antibiotics were detected. Resistance determinants of tetracycline, macrolide, and aminoglycoside were highly abundant in both pre- and post-treatment pooled samples in all groups, while those of phenicol and sulfonamide were the least abundant. Furthermore, the bacterial reservoirs of each of the ARGs were identified. Overall, the resistome profiles were significantly affected in calf groups that received enrofloxacin compared to the control group. Under Objective 3 (educational training programs), we trained a total of 13 veterinary medicine students under the ISU Veterinary Summer Scholars Research Program. The students were closely mentored by the PI and co-PIs as they carried out antimicrobial resistance related studies for about 13-week. The students gained various research skills and experience including both in vitro and in vivo studies. At the end of the program, the students presented their work at the Research Day of ISU College of Veterinary Medicine as well as at a national meeting encompassing peers from other veterinary colleges nationwide. For extension, survey on antimicrobial usage practices was prepared. Extension publications on AMR and on antibiotic use in feedlot cattle were prepared. Information on prudent use of animal health products including antibiotics, disease prevention, and antimicrobial resistance was presented at multiple extension meetings (~100) in Iowa and Kansas. Meetings included Beef Quality Assurance meetings, calving schools and health management, and targeted cattle producers and veterinarians (~10,000).

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Beyi A.F., Mochel J.P., Magnin G., Hawbecker T., Slagel C., Dewell G., Dewell R., Sahin O., Coetzee J., Zhang Q., Plummer P.J. Comparisons of plasma and fecal pharmacokinetics of danofloxacin and enrofloxacin in healthy and Mannheimia haemolytica infected calves. Scientific Reports 2022, 12, 5107.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Beyi A.F., Goulart D., Hawbecker T., Hassall A., Dewell R., Dewell G., Sahin O., Zhang Q., Plummer P.J. Enrofloxacin alters gut microbiota irrespective of its dose in beef calves. Microorganisms 2021, 9(10), 2162.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Beyi A.F., Goulart D., Hawbecker T., Slagel C., Ruddell B., Hassall A., Dewell R., Dewell G., Sahin O., Zhang Q., Plummer P.J. Danofloxacin treatment alters diversity and resistome profile of gut microbiota and compositions of Campylobacter in calves. Microorganisms 2021, 9(10), 2023.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Debora Brito Goulart., Ashenafi F. Beyi, Zuowei Wu, Mehmet Adiguzel, Anastasia Schroeder, Kritika Singh, Changyu Xu, Melda Ocal, Renee Dewell, Grant Dewell, Paul Plummer, Qijing Zhang, Orhan Sahin. (2022). Effect of danofloxacin treatment on the development of fluoroquinolone resistance in Campylobacter jejuni in calves. Antibiotics, 11, 1-17.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Beyi A.F., Hawbecker T., Hassall A., Dewell R., Dewell G., Sahin O., Zhang Q., Plummer P.J. Alteration in fecal resistome due to the administration of enrofloxacin in calves. ATAVET II. International III. National Veterinary Student Congress (online), Erzurum, Turkey, December 15-16, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Beyi A.F., Hawbecker T., Hassall A., Dewell R., Dewell G., Sahin O., Zhang Q., Plummer P.J. Enrofloxacin alters gut microbial resistome irrespective of its dose in beef calves. National Institute Antimicrobial Resistance Research and Education (NIAMRRE) Quarterly Research Symposium (online), Ames, IA, October 20, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Beyi A.F., Mochel J.P., Magnin G., Hawbecker T., Slagel C., Dewell G., Dewell R., Sahin O., Coetzee J., Zhang Q., Plummer P.J. Comparisons of plasma and fecal pharmacokinetics of danofloxacin and enrofloxacin in healthy and Mannheimia haemolytica infected calves. National Institute of Antimicrobial Resistance Research and Education Annual Conference. 16-18 May 2022, Lincoln, Nebraska
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Goulart, D. B., Beyi, A., Wu, Z., Dewell, R., Dewell, G., Plummer, P., Zhang, Q., Sahin, O. (2022). Effect of danofloxacin treatment on the resistance level of fluoroquinolone resistance in Campylobacter in calves. Poster presented at the National Institute of Antimicrobial Resistance Research and Education.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Mehmet Cemal Adiguzel, Kayla Schaefer, Trevor Rodriguez, Jessica Ortiz, Orhan Sahin. 2022. Prevalence, Mechanism, Genetic Diversity, and Cross-Resistance Patterns of Methicillin-Resistant Staphylococcus Isolated from Companion Animal Clinical Samples Submitted to A Veterinary Diagnostic Laboratory in The Midwestern United States. Antibiotics 2022, 11, 609.


Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Cattle producers, veterinarians, students, researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this project, 2 graduate students and 1 veterinary student were trained for knowledge and skills in antimicrobial resistance research. How have the results been disseminated to communities of interest?Findings from this project were presented at national and regional conferences as poster/oral presentations. What do you plan to do during the next reporting period to accomplish the goals?Pharmacometrics modeling to predict the drug (between danofloxacin and enrofloxacin) and the dose (between low and high doses) that mitigates FQ resistance development in cattle will be completed. Data from this project will be submitted for peer-reviewed publication.

Impacts
What was accomplished under these goals? To assess the effect of danofloxacin treatment on gut microbiota resistome, shotgun and ProxiMeta Hi-C metagenomics were performed. Four samples (two pooled pre-treatment samples and two pooled post-treatment samples from calves that were administered danofloxacin but not challenged with Mannheimia haemolytica) were selected based on the results of 16S rRNA analysis. Samples collected right before danofloxacin injection and four days later were used for this purpose. Genes conferring resistance to tetracycline, aminoglycosides, β-lactams, macrolides, and phenicols were detected in both pre-and post-treatment samples. The abundance of tetracycline resistance determinants was the highest, with eight and nine different antimicrobial resistance gene (ARG) types detected in pre-and post-treatment pooled samples, respectively. The linkage of ARGs with their respective bacterial hosts was also established. Eight phyla associated with the ARGs were Actinobacteria, Bacteroidetes, Euryarchaeota, Firmicutes, Proteobacteria, Spirochaetes, Tenericutes, and Verrucomicrobia. Firmicutes was the leading phylum associated with these ARGs, whereas Bacteroidetes appeared to acquire the ARGs following the treatment. Most of the identified ARGs were carried by more than one clusters of bacterial taxa having ≥ 80% complete genomes. For instance, tetW was detected in 12 and 49 clusters in pre-and post-treatment samples, indicating a wide range of distribution and possible transfer among bacterial species horizontally. To assess the dynamics of antimicrobial resistance genes in feces following danofloxacin injection, we conducted qPCR to analyze selected resistance genes based on the metagenomic Hi-C results. Comparison of the treatment groups with the control group using the non-parametric pairwise Dunn test revealed significant differences in tetW, tetO and ermB abundances between the treatment groups and the control group in post-treatment pooled samples. There was overall a good agreement between the Hi-C and qPCR results in the trend of ARG changes. Similarly, shotgun and ProxiMeta Hi-C metagenomics were performed to assess the effect of enrofloxacin treatment on gut microbiota resistome. Thirty-two ARGs belonging to six classes of antibiotics were detected in the samples. Tetracycline, macrolide, and aminoglycoside resistance genes were highly abundant in both pre-and post-treatment pooled samples in all groups, whereas phenicol and sulfonamide resistance determinants were least abundant. The copy numbers and the host ranges of the ARGs were dynamic in both the control and the treatment groups; however, these variations were more prominent in the treatment groups. Generally, there was no remarkable difference between the two high-dose groups. In contrast, it appears that high dose enrofloxacin increased copy number and host ranges, while the low dose reduced the number of hits and host ranges of ARGs. The Hi-C method enabled us to know the bacterial taxa that carry each of the identified ARG based on the proximity ligations. Most of the ARGs were carried by species in Bacteroidetes, Firmicutes, Clostridium, Prevotella, and Treponema. In addition, pooled fecal samples were used to compare pre-and post-treatment levels of the ARGs using the qPCR. Results showed that tetW and ermB had significant differences between the treatment groups and the control group, but the other ARGs did not show a significant difference between the pre-and post-treatment pooled samples among different groups. Comparison of the pattern of ARG quantitative changes between the qPCR method and the Hi-C method showed an overall good agreement in indicating the direction of ARG changes. These findings provide new insights into the abundance and dissemination of ARGs in cattle gut microbiota and how antibiotic treatments affect their dynamic changes. Under educational training programs, we trained a veterinary medicine student during this reporting period under the ISU Veterinary Summer Scholars Research Program (about 13-week long). At the end of the program, the student remotely presented her work at the virtual Research Day of ISU College of Veterinary Medicine. In addition, 45 extension meetings with 3000 producers took place. Meetings included Beef Quality Assurance meetings, calving schools and health management. Prudent use of animal health products, disease prevention, and antimicrobial resistance were discussed at each of these meetings.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Debora Goulart, Ashenafi Beyi, Zuowei Wu, Renee Dewell, Grant Dewell, Paul Plummer, Qijing Zhang, Orhan Sahin. Effects of enrofloxacin treatment on the selection of fluoroquinolone-resistant Campylobacter in cattle. Poster presented at the National Institute of Antimicrobial Resistance Research and Education Virtual Poster Session, April 20-21, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Debora Goulart, Ashenafi Beyi, Zuowei Wu, Renee Dewell, Grant Dewell, Paul Plummer, Qijing Zhang, Orhan Sahin. Genetic diversity of Campylobacter jejuni. Poster presented at the Iowa State University Research Day, March 23-25, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Debora Goulart, Ashenafi Beyi, Zuowei Wu, Renee Dewell, Grant Dewell, Paul Plummer, Qijing Zhang, Orhan Sahin. Influence of enrofloxacin and danofloxacin treatments on fluoroquinolone-resistant Campylobacter jejuni isolated from feces of dairy calves challenged with Mannheimia haemolytica. Poster presented at the Iowa State University Research Day, March 23-25, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Debora Goulart, Ashenafi Beyi, Samantha Wilson, Anastasia Schroeder, Melda Meral, Mehmet Adiguzel, Zuowei Wu, Kritika Singh, Renee Dewell, Grant Dewell, Paul Plummer, Qijing Zhang, Orhan Sahin. Effect of different fluoroquinolone (FQ) treatments and health status on the development of FQ resistance in calves colonized with either FQ-resistant or FQ-susceptible Campylobacter jejuni. Poster presented at the College of Veterinary Medicine Research Day Virtual Poster Session, Iowa State University, August 7, 2020, Ames, IA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Debora Goulart, Ashenafi Beyi, Samantha Wilson, Anastasia Schroeder, Melda Meral, Mehmet Adiguzel, Zuowei Wu, Kritika Singh, Renee Dewell, Grant Dewell, Paul Plummer, Orhan Sahin, Qijing Zhang. Effect of different fluoroquinolone treatments on the dynamics of fluoroquinolone resistance in Campylobacter jejuni in calves. Poster presentation at the ASM Microbe Online, July, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ashenafi F Beyi, Tyler Hawbecker, Alan Hassall, Renee Dewell, Grant Dewell, Orhan Sahin, Qijing Zhang, Paul J Plummer. A single injection of enrofloxacin alters gut microbial diversities irrespective of its dose in beef calves. CRWAD 2020 virtual conference meeting, oral presentation, December 5-8, 2020.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Mehmet Cemal Adiguzel, Debora Brito Goulart, Lei Dai, Zuowei Wu, Jinji Pang, Seyda Cengiz, Qijing Zhang, and Orhan Sahin. 2021. Distribution of CRISPR types in fluoroquinolone resistant Campylobacter jejuni isolates. Pathogens 2021, 10(3), 345; https://doi.org/10.3390/pathogens10030345.


Progress 06/01/19 to 05/31/20

Outputs
Target Audience:Veterinarinas, students, cattle producers and extension personnel, researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Under this project, 4 veterinary medicine students and 2 graduate students were trained for knowledge and skills in antimicrobial resistance research. How have the results been disseminated to communities of interest?Information on prudent use of animal health products including antibiotics, disease prevention, and antimicrobial resistance has been presented to cattle producers and veterinarians. Also, findings from this project were presented at national and regional conferences as poster/oral presentations. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will continue to analyze the samples from all animal trials performed, complete PK/PD and metagenomics analysis from both trials, and build PK/PD models to predict the impacts of the antibiotics on the emergence of antimicrobial resistance. We will also be quantifying the number of antibiotic determinants using real time quantitative PCR. Educational and outreach activities will also be performed. Data from this projects will be submitted for peer-reviewed publication.

Impacts
What was accomplished under these goals? Under Objective 1, we conducted a second animal trial involving 2-3 months old calves. The purpose of the trial was to investigate the effect of multiple-dose enrofloxacin treatments in calves experimentally inoculated with C. jejuni on the development of FQ-resistant (FQ-R) Campylobacter in healthy and diseased animals. The trial involved five separate groups (healthy group A and B, BRD-induced group A and B, and control group; n=7 per group) of calves purchased from a commercial source. The animals in all groups were tested for Campylobacter status via culturing of rectal feces and inoculated orally with a three-strain cocktail of FQ-susceptible (FQ-S) C. jejuni. After one week, the calves in BRD-induced groups were administered intratracheally with a Mannheimia haemolytica strain to induce BRD. One week after, calves in the healthy group A and BRD-induced group A were injected s.c. with a single low dose (7.5 mg/kg body weight) of enrofloxacin (Baytril), and calves in the healthy group B and BRD-induced group B were injected s.c. with a single high dose (12.5 mg/kg body weight) of enrofloxacin while the last group (healthy group) did not receive any antibiotic treatment and served as the control. Fecal samples were periodically collected for Campylobacter culture and microbiota analysis as well as pharmacokinetic/pharmacodynamic (PK/PD) analyses. At necropsy, lung lesions were scored and a portion of lung from the lesion was taken for Mannheimia culture. Two Campylobacter-like colonies (grown on selective plates without ciprofloxacin) from each animal on each sampling day were selected. The bacterial isolates were identified by MALDI-TOF mass spectrometry to the species level. Prior to experimental inoculation with the laboratory strains, culture results from feces indicated that the vast majority of calves were Campylobacter-negative and only 3 calves in the control group carried FQ-resistant (FQ-R) C. jejuni. After inoculation with the laboratory strains of FQ-susceptible (FQ-S) C. jejuni, almost all of the calves became colonized with the inoculated C. jejuni within a few days. Also, there was a notable decrease in the FQ-resistant Campylobacter population in the control group, suggesting that the inoculated FQ-S Campylobacter displaced the pre-existing FQ-R Campylobacter in the absence of antibiotic selection pressure. Enrofloxacin injection did not appear to induce development of FQ resistance from the inoculated FQ-S C. jejuni strains in any of the groups throughout the study regardless of the disease status or dose of the antibiotic used in this study. Also, Campylobacter counts between the healthy and BRD-induced groups were comparable; however, clinical signs and lung lesion scores were only mild. Antimicrobial susceptibility testing of 218 C. jejuni isolates collected throughout the study indicated that they were mostly susceptible to the majority of antibiotics tested except for ciprofloxacin (9% resistant), nalidixic acid (23% resistant) and tetracycline (77% resistant).The FQ-R Campylobacter isolates were mostly from animals of the control group that were pre-colonized with Campylobacter before oral inoculation. Together, the results indicate that a single treatment with enrofloxacin does not appear toinduce development of de novo FQ-resistance from FQ-S C. jejuni strains in cattle. Under Objective 2, we continued to analyze the effect of danofloxacin treatment on the gut microbiota composition (based on 16S rRNA based metagenomics) in rectal feces (collected at four sampling points before and three sampling points after danofloxacin injection) from calves. Specifically, the effect of treatment on the relative abundance of Campylobacter was examined in more detail. Analysis indicated that Campylobacter abundance dropped sharply at 24 h following the antibiotic injection, but it returned to the pre-treatment level at 96 h post injection. When the abundance of Campylobacter was compared with other genera, we identified 21 (most of the genera in Firmicutes and Proteobacteria) with a positive correlation, and 10 with a negative correlation (seven of the eight Bacteroidetes genera) in all fecal samples. Furthermore, antibiotic resistance genes (ARGs) were determined before and after danofloxacin injection using ProxiMeta Hi-C metagenomics. Genes conferring resistance to tetracycline, aminoglycosides and erythromycin were detected in the samples. Four groups of ARGs (tetO, sat, ermB, and ermF) were detected in the pre-treatment samples whereas four new antibiotic genes (tetW, tetL, tetX, and aph3) were detected only in the post-treatment samples. tetO and sat genes increased in the post-treatment samples while ermB decreased and ermF was absent in the post-treatment samples. The linkage of ARGs with their respective bacterial hosts was also established. Order Clostridiales hosted the largest number of ARGs followed by class Clostridia. This findings indicate that danofloxacin resulted in enrichment of antibiotic resistant determinants in gut microbiota. In addition, we examined the effect of two different s.c. doses of enrofloxacin on the gut microbial diversities in groups of calves in the second study (done in a similar design to danofloxacin treatment described above). Preliminary analyses indicated that alpha diversities (species richness and diversity) decreased significantly after the treatment, and the beta diversities (variation in microbial diversity) were significantly different between pre- and post-treatment communities (Bray Curtis, q=0.001). The two different antibiotic doses used did not affect the microbial diversities differently; the metrics of both alpha and beta diversities did not show significant variation between the low and the high dose cohorts (q>0.05). In contrast, the microbial diversities significantly shifted in both the low and the high dose compared to the control group (q<0.05 for both alpha and beta diversities). These results indicated that subcutaneous administration of a single therapeutic dose of enrofloxacin changes the diversities of gut microbiota in calves irrespective of its dose. Pharmacokinetic (PK) analysis indicated that danofloxacin concentration in plasma quickly rose after injection (detectable as early as within 0.25 h), peaked between 2-4 hours, and significantly decreased after 4 hours (not detected at 36 and 48 h post-injection). The antibiotic concentrations in fecal samples also rose quickly (detected as early as 1 h s.c. injection), peaked at 12 h post injection and remained at detectable levels until the last testing done at 48 h post injection. Interestingly, the drug concentrations were much higher in feces than plasma for most of the testing points in the majority of animals. Currently, concentrations of enrofloxacin in both plasma and feces remain to be analyzed. Under Objective 3 (educational training programs), we trained 4 veterinary medicine students during this reporting period under the ISU Veterinary Summer Scholars Research Program. The students were closely mentored by the PI and co-PIs as they carried out antimicrobial resistance related studies for about 13-week. The students gained various research skills and experience including both in vitro and in vivo studies. At the end of the program, the students presented their work at the Research Day of ISU College of Veterinary Medicine as well as at a national meeting encompassing peers from other veterinary colleges nationwide. In addition, information on managing herd health, prudent use of animal health products including antibiotics, disease prevention, and antimicrobial resistance was presented at multiple extension meetings. These included 38 meetings in Iowa and 20 in Kansas (such as Beef Quality Assurance, calving schools, and health management) that reached approximately 3,550 producers and veterinarians.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Samantha Wilson, Debora Goulart, Ashenafi Beyi, Renee Dewell, Grant Dewell, Orhan Sahin, Paul Plummer, Qijing Zhang. Effect of danofloxacin treatment on the development of fluoroquinolone resistance in Campylobacter jejuni in cattle. Poster presentation at the College of Veterinary Medicine Research Day, Iowa State University, August 9, 2019, Ames, IA and at the National Veterinary Scholars Symposium, Tufts University, July 24-27, 2019, Medford, MA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Jessica Ortiz, Mehmet Cemal Adiguzel, Debora Brito-Goulart, Qijing Zhang, Paul Plummer, Grant Dewell and Orhan Sahin. Genetic Diversity and mechanism of methicillin resistance in Staphylococcus species from companion animals. Poster presentation at CVM Summer Scholars Research Day, Iowa State University, August 9, 2019, Ames, IA; and at the National Veterinary Scholars Symposium, Tufts University, July 24-27, 2019, Medford, MA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Tyler J. Hawbecker, Ashenafi F. Beyi, Clare Slagel, Brandon Ruddell, Alan J. Hassall, Renee Dewell, Grant Dewell, Orhan Sahin, Qijing Zhang, and Paul J. Plummer. Impact of fluoroquinolone treatment on the gastrointestinal microbiome and antibiotic resistance in calves. Poster presentation at CVM Summer Scholars Research Day, Iowa State University, August 9, 2019, Ames, IA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Debora Goulart, Ashenafi Beyi, Samantha Wilson, Renee Dewell, Grant Dewell, Paul Plummer, Melda Ocal, Zuowei Wu, Kritika Singh, Lei Dai, Changyun Xu, Jing Xia, Brandon Ruddell, Qijing Zhang, and Orhan Sahin. Effect of danofloxacin treatment on the development of fluoroquinolone resistance in Campylobacter jejuni in cattle. Oral presentation at Conference of Research Workers in Animal Diseases (CRWAD) Meeting, November 2-5, 2019, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ashenafi Beyi, Tyler Hawbecker, Clare Slagel, Brandon Ruddell, Alan Hassall, Renee Dewell, Grant Dewell, Orhan Sahin, Qijing Zhang, Paul J Plummer. Effect of danofloxacin treatment on the development of fluoroquinolone resistance in Campylobacter jejuni in cattle. Oral presentation at Conference of Research Workers in Animal Diseases (CRWAD) Meeting, November 2-5, 2019, Chicago, IL.
  • Type: Other Status: Published Year Published: 2020 Citation: Ashenafi Beyi, Tyler Hawbecker, Clare Slagel, Brandon Ruddell, Alan Hassall, Renee Dewell, Grant Dewell, Orhan Sahin, Qijing Zhang, Paul J Plummer. Treatment of bovine respiratory disease with danofloxacin altered diversities and resistome profiles of gut microbiota and relative abundance of Campylobacter in beef calves. Oral presentation at Microbiome Bioinformatics with QIIME 2 workshop at NIH, Bethesda, Maryland, January 8-10, 2020.
  • Type: Other Status: Published Year Published: 2020 Citation: Ashenafi Beyi, Tyler Hawbecker, Clare Slagel, Brandon Ruddell, Alan Hassall, Renee Dewell, Grant Dewell, Orhan Sahin, Qijing Zhang, Paul J Plummer. Treatment of bovine respiratory disease with danofloxacin changed diversity and resistome profile of gut microbiota and abundance of Campylobacter in beef calves. iPoster presentation at ISU Presidential Interdisciplinary Research Initiative AMR Consortium, May 30, 2020.
  • Type: Other Status: Published Year Published: 2020 Citation: Ashenafi Beyi, Tyler Hawbecker, Clare Slagel, Brandon Ruddell, Alan Hassall, Renee Dewell, Grant Dewell, Orhan Sahin, Qijing Zhang, Paul J Plummer. A single subcutaneous injection of enrofloxacin alters gut microbial diversities irrespective of its dose in beef calves. iPoster presentation at ISU Presidential Interdisciplinary Research Initiative AMR Consortium, May 30, 2020.


Progress 06/01/18 to 05/31/19

Outputs
Target Audience:Veterinarinas, students, cattle producers and extension personnel, researchers Changes/Problems:None. What opportunities for training and professional development has the project provided?Under this project, 4 veterinary medicine students, 2 graduate students and 1 undergraduate student were trained for knowledge and skills in antimicrobial resistance research. How have the results been disseminated to communities of interest?Information on antimicrobial resistance and judicious use of antibiotics has been presented to cattle producers, and findings from this project were presented at national and regional conferences. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we will continue to analyze the samples from the second animal trial, complete PK/PD and metagenomics analysis from both trials, and further conduct training of veterinary students and outreach efforts to producers.

Impacts
What was accomplished under these goals? Under Objective 1, we conducted two major animal studies involving ~2-3 months old calves. The first study investigated the effect of single-dose danofloxacin treatment on the development of fluoroquinolone (FQ) resistance in Campylobacter jejuni in heathy and bovine respiratory disease (BRD)-induced animals. The second study was performed with the same purpose and similar design but investigated the effects of multiple-dose enrofloxacin treatments in calves experimentally inoculated with C. jejuni. In both studies, rectal feces were collected for Campylobacter culture and microbiota analysis. Additionally, fecal and blood samples were also collected for pharmacokinetic/pharmacodynamic (PK/PD) analyses of the antibiotic in these animals. The first trial involved three separate groups (n= 10 per group) of calves purchased from a commercial source. The animals were all inoculated orally with a mixture of three C. jejuni strains. After one week, the calves in one group were administered intratracheally with a Mannheimia haemolytica strain to induce BRD. One week after, calves in two groups (a healthy group and the BRD-induced group) were injected s.c. with a single dose of danofloxacin (Advocin; a FQ antibiotic) at 8 mg/kg body weight, while the last group (healthy group) did not receive any antibiotic treatment and served as the control. Fecal samples were then collected for a week for monitoring the development of FQ-resistant Campylobacter in all three groups of calves. At necropsy, lung lesions were scored and a portion of lung from the lesion (when observed) was taken for Mannheimia culture. Two Campylobacter-like colonies (grown on plates without ciprofloxacin) from each animal on each sampling day were selected. The bacterial isolates were identified by MALDI-TOF mass spectrometry at the species level. Culture results from feces indicated that the vast majority of the calves were naturally colonized by FQ-resistant C. jejuni at about log10 4-6 CFU/g feces prior to experimental inoculation with the laboratory strains. After inoculation with the laboratory strains of FQ-susceptible C. jejuni, there was a notable decrease in the resistant Campylobacter population in all groups of animals, suggesting that the inoculated FQ-susceptible Campylobacter displaced the pre-existing FQ-R Campylobacter in the absence of antibiotic selection pressure. Treatment with danofloxacin resulted in an increase in FQ-resistant Campylobacter populations in both the healthy and BRD-induced groups, while FQ-S population in the control group (not treated with antibiotic) remained dominant until the end of the experiment. There appeared to be no clear and consistent differences in Campylobacter counts (including FQ-resistant and total populations) between the healthy and BRD-induced groups; however, clinical signs and lung lesion scores were only mild, suggesting a very low grade of BRD in calves inoculated with Mannheimia. Genotyping via pulsed-field gel electrophoresis (PFGE) of a subset of isolates indicated an overall high genetic diversity. One strain was chosen from each PFGE cluster for multilocus sequence typing (MLST) analysis. A total of 4 different sequence types (STs) were identified, which included ST- 982 (46%), ST-8 (23%), ST-61 (15%), and ST-929 (8%). ST-982 (not included in the inoculum) remained FQ-R while ST-8 (included in the inoculum) remained susceptible to ciprofloxacin throughout the study. Antimicrobial susceptibility testing of 187 C. jejuni isolates so far indicated that they were mostly susceptible to the majority of antibiotics tested (azithromycin, erythromycin, florfenicol, gentamicin, and clindamycin) except for ciprofloxacin (57% resistant), nalidixic acid (58% resistant) and tetracycline (88% resistant). Additionally, preliminary PK analysis indicated that danofloxacin concentration in plasma quickly rose after injection, peaked between 2-4 hours, and significantly decreased after 4 hours. The antibiotic concentrations in fecal samples remain to be determined. In summary, the results from the first animal study suggest that treatment with danofloxacin enriches pre-existing FQ-resistant C. jejuni populations rather thaninducing development of de novo FQ-resistance from FQ-susceptible C. jejuni strains in cattle. Samples from the second animal trial are being tested and analyzed, and the results will be presented in the next report. Under Objective 2, the effect of danofloxacin treatment on gut microbiota composition (based on 16S rRNA based metagenomics) was examined on rectal feces (collected at four sampling points before and three sampling points after danofloxacin injection) from all three groups of calves. Overall, eleven bacterial phyla with relative abundance of ≥ 0.1% were identified; which included Firmicutes (55.88%), Bacteroidetes (31.62%), Verrucomicrobia (3.41%), Proteobacteria (2.24%), Spirochaetes (1.84%), Tenericutes (1.81%), Cyanobacteria (0.93%), Actinobateria (0.88%), Euryachaeota (0.72%), Elusimicrobia (0.33%), and Planctomycetes (0.26%). Significant differences (p < 0.05) at the class level comparison of many of these taxa were observed between the control group (did not receive antibiotic) and both treatment groups. For instances, Methanobacteria, Holophagae, Betaproteobacteria, Planctomycetia, and Mollicutes significantly increased in the treatment groups, whereas Actinobacteria, Bacilli, Epsilonproteobacteria, and Synergistia decreased. Comparisons between the two treatment groups (BRD-induced Group and healthy Group) showed significant differences for certain bacterial classes such as Actinobacteria, Planctomycetia, Betaproteobacteria, and Tenericutes- RF3. When bacterial compositions were analyzed pre- and post-danofloxacin injection in the two treatment groups, a shift in the composition was observed. Methanobacteria, Cyanobacteria-4C0d-2, Bacilli, Erysipelotrichi, Spirochaetes, and other significantly decreased while Planctomycetia, Gammaproteobacteria, and Verruco-5 significantly increased following the antibiotic injection. In addition, comparison of bacterial genera by sampling time points for all three calf groups indicated that Bacillus, Campylobacter, Treponema, and Streptococcus were found to be affected significantly by danofloxacin treatment. In summary, these data indicate that the use of a FQ antibiotic significantly alters the composition of the intestinal microbiota in calves and that certain bacteria with public health importance such as Campylobacter increases in abundance following danofloxacin treatment. Under Objective 3 (educational training programs), we have trained 4 veterinary medicine students during this reporting period under the ISU Veterinary Medical Summer Scholars Research Program in antimicrobial resistance. These students were closely mentored by the PI and co-PIs as they carried out a project on antimicrobial resistance, applied microbiology, pharmacology and metagenomics for about 13-week and exposed to various research areas including both in vitro and in vivo studies. At the end of the program, the students presented their findings at the Research Day of ISU College of Veterinary Medicine as well as at a national meeting encompassing peers from other veterinary colleges nationwide. In addition, information on antimicrobial resistance and judicious use of antibiotics has been presented to 7,748 producers at a total of 40 different meetings in Kansas and Iowa.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Anastasia Schroeder, Debora Brito-Goulart, Ashenafi Beyi, Zuowei Wu, Kritika Singh, Orhan Sahin, Paul Plummer, Grant Dewell, Renee Dewell, Qijing Zhang. Effect of Enrofloxacin treatment on the prevalence of fluoroquinolone resistant Campylobacter in cattle. Poster presentation at CVM Summer Scholars Research Day, Iowa State University, August 10, 2018, Ames, IA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Tyler J. Hawbecker, Linda D. Zeller, Ronald W. Griffith, Kelly M. Still Brooks, Paul J. Plummer. Evaluation of antimicrobial resistance to Chlortetracycline in Mannheimia haemolytica and Pasteurella multocida. Poster presentation at CVM Summer Scholars Research Day, Iowa State University, August 10, 2018, Ames, IA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Trevor Rodriguez, Melda Meral Ocal, Kritika Singh, Adam Krull, Qijing Zhang, and Orhan Sahin. Mechanism and cross-resistance patterns of methicillin resistance in Staphylococcus from companion animals. Poster presentation at CVM Summer Scholars Research Day, Iowa State University, August 10, 2018, Ames, IA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Goulart D., A. Schroeder, A. Beyi, M. Meral, Z. Wu, K. Singh, O. Sahin, P. Plummer, G. Dewell, R. Dewell, and Q. Zhang. 2018. Effect of enrofloxacin treatment on the prevalence of fluoroquinolone resistant Campylobacter in cattle. Abstract of the 99th Annual Conference of Research Workers in Animal Diseases, Dec. 1-4, 2018, Chicago, IL, USA


Progress 06/01/17 to 05/31/18

Outputs
Target Audience:Veterinarians, producers, researchers, Extension personnel Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has proved training and learning opportunities for 4 veterinary medicine students, 2 graduate students, and 2 undergraduate students. How have the results been disseminated to communities of interest?The study is still in the early stage and the results have not been disseminated. What do you plan to do during the next reporting period to accomplish the goals?During the next period, we will continue to perform animal studies to evaluate the effect of different FQ antibiotic treatment regimens on the development of FQ-resistant Campylobacter in healthy vs. BRD-induced calves. PK/PD analysis in addition to the bacteriology work will be conducted, metagenomics analysis will be initiated, training of veterinary scholars will be continued, and extension materials will be disseminated.

Impacts
What was accomplished under these goals? We conducted a pilot trial using ~3-4 month old calves. These animals were purchased from a commercial farm, divided into two groups (n = 5 per group), tested for Campylobacter status via culturing of rectal feces, and inoculated orally with a mix of C. jejuni strains. After one week, the calves in one groups were administered intratracheally with a Mannheimia haemolytica strain to induce bovine respiratory disease (BRD). A week after, calves in both groups were injected s.c. with a single dose of enrofloxacin (a fluoroquinolone [FQ] antibiotic) at 12 mg/kg body weight. Fecal samples were then collected for a week for monitoring the development of FQ-resistant Campylobacter in both groups of calves. At necropsy, lung lesions were scored and a portion of lung from the lesion was taken for Mannheimia culture. Culture results from feces indicated that all but one calves were colonized by C. jejuni at about log10 5 CFU/g feces prior to experimental inoculation with the laboratory strains. The majority of the C. jejuni colonies were FQ-resistant. Interestingly, following oral inoculation with a mixture of C. jejuni strains (all FQ-susceptible), the level of FQ-resistant C. jejuni populations dropped dramatically (even to below the detectable level in several animals) starting from DPI 5 and lasted until DPI 14, suggesting that the inoculated FQ-susceptible Campylobacter strains reduced the endogenous FQ-resistant Campylobacter in the animals. However, as soon as 24 h following the enrofloxacin treatment, the C. jejuni populations reverted to FQ-resistant state and remained such for at least another 4 days. This observation suggested that the antibiotic treatment pressure enriched FQ-resistant C. jejuni. Comparison of the Campylobacter loads (including FQ-resistant and total population) in the healthy and BRD-induced groups of calves indicated that there was no statistically significant differences between the groups during the study period. For characterization of the isolates, two Campylobacter-like colonies (grown on plates without ciprofloxacin) from each animal on each sampling day were selected. All Campylobacter colonies were identified as C. jejuni by MALDI-TOF MS. Genotyping of selected isolates via pulsed-field gel electrophoresis and MLST indicated the cattle were pre-colonized with genetic diverse C. jejuni. MIC testing of about 40 selected C. jejuni isolates indicates that 56% were resistant to ciprofloxacin and 59% were resistant to tetracycline. For the educational program, we trained 4 veterinary medicine students and additional 4 are currently in the course of training in the area of antimicrobial resistance. These students worked closely with the PI and co-PIs as they carry out a project on antimicrobial resistance, gained hand-on experience by performing in vitro and in vivo studies, and developed extension materials. Additionally, the students presented their findings at the Research Day of ISU College of Veterinary Medicine and at the national symposium for veterinary scholars. For extension, survey on antimicrobial usage practices has been prepared. Extension publications on AMR and on antibiotic use in feedlot cattle were drafted and are currently under review.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Sarah Muirhead, Yizhi Tang, Nada Pavlovic, Qijing Zhang. 2017. Identification of a predominant Campylobacter coli clone in feedlot cattle in the United States. Poster presentation at the National Veterinary Scholars Symposium, August 3-6, 2017, Washington, D.C.