ENTERIC DISEASES OF FOOD ANIMALS: ENHANCED PREVENTION, CONTROL AND FOOD SAFETY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1003414
• Project No.: WYO-520-14
• Multistate No.: NC-_old1202
• Project Start Date: Jun 30, 2014
• Project End Date: Sep 30, 2017

Project Director
Bisha, BL, .

Recipient Organization
UNIVERSITY OF WYOMING
1000 E UNIVERSITY AVE DEPARTMENT 3434
LARAMIE,WY 82071-2000

Performing Department
Animal Science

Non Technical Summary
The long-term goal of this collaborative project is to prevent and control enteric diseases of cattle, swine and chickens with a mandate to decrease food and waterborne illness in the USA. Foodborne illness has been a prominent public health concern for over two decades yet the Centers for Disease Control (CDC) still list many enteric foodborne pathogens as leading causes of morbidity and mortality in the US. Despite many concerted efforts to use hygiene and sanitation measures to control these pathogens in food animals pre- and postharvest, incidence of many food and waterborne pathogens remains high and some are increasing. Nevertheless, a broad range of educational, scientific and practical controls have succeeded in decreasing the incidence of five key foodborne pathogens. Over the last 10 years, our enteric diseases group has been a part of that effort and we are dedicated to prevent and control animal and human disease due to enteric pathogens. Our collaborative efforts harmonize with national efforts established this year under the FDA Food Safety Modernization Act to ensure the US food supply is safe by shifting the focus of federal regulators from responding to contamination to preventing it. A main avenue for prevention is decreasing carriage and disease due to enteric pathogens in food animals. The research group at the University of Wyoming will focus on the development of high throughput identification and typing methods for antimicrobial resistant (AMR) bacteria from cattle operations and wildlife, methods for capture of viruses from bioaerosols in agricultural environments, and development of field-based and rapid diagnostics for microbial pathogens.

Animal Health Component

10%

Research Effort Categories

Basic

25%

Applied

45%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
722	4010	1040	25%
712	3599	1100	25%
712	3310	1100	25%
311	3299	1070	25%

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

Subject Of Investigation
3299 - Poultry, general/other; 3599 - Swine, general/other; 4010 - Bacteria; 3310 - Beef cattle, live animal;

Field Of Science
1070 - Ecology; 1100 - Bacteriology; 1040 - Molecular biology;

Keywords

food safety

diagnostics

sample preparation

typing

Goals / Objectives
Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals. Focus on preventions and interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and foodborne and waterborne pathogens.

Project Methods
We will 1) develop MALDI-TOF MS protocols for identification of antimicrobial resistant bacteria from several cattle feedlots, avian and mammalian wildilife, and agricultural environments using formic acid/ethanol extractions for sample preparation, and 2) examine prevalence of these AMR bacteria to identify potential intervention practices. We will evaluate a multitude of sample preparation techniques to aid in enhancing subtyping capabilities of the developed method.Solvent systems, detergent extractions, enzymatic treatments, sample fractionation, and multiple MALDI matrices will be evaluated using panels of well characterized bacteria from environmental, human, and food sources, with specific focus on E. coli, Enterococcus spp., and Staphylococcus spp. Statistical tools (multivariate analysis and hierarchical and correlational clustering) within the Bruker Daltonics Biotyper software package will then be used for mass spectral comparisons of the isolates to evaluate isolate-to-isolate similarity. Representative panels of the confirmed isolates will also be subjected to pulsed-field gel electrophoresis (PFGE) and antimicrobial sensitivity testing for comparative evaluations of MALDI-TOF MS typing efficacy. The discriminatory mass spectral data, determined through extensive bioinformatic interpretations and by indirect comparisons to PFGE/antimicrobial sensitivity profiles, will be used to build reference mass spectral libraries. To understand the epidemiology of the AMR bacteria, we will examine prevalence via analysis of isolates originating from cattle feces, feed, water, as well as mammalian and avian wildlife then analyze the isolates via PFGE, 16s sequencing, antimicrobial susceptibility tests and compare them to MALDI-TOF MS results to elucidate the epidemiology of these isolates. In addition, we will also develop 3) an enhanced method for capture and concentration of viruses from agricultural environments for subsequent molecular detection which builds upon already existing bioaerosol sampling equipment with modification aimed at increasing sensitivity and simplicity of the procedure in a streamlined process. Laboratory studies will evaluate the modified devices' ability to capture viruses (including multiple variants of influenza viruses) using a bioaerosol chamber. The laboratory evaluations will also determine the minimal concentrations of virus that can be detected in bioaerosols with this method. qRT-PCR will then be used for detection/quantification. Following the laboratory tests, evaluations of the best performing bioaerosol sampling device will be conducted in animal facilities. In addition to RT-PCR testing for the viruses in animal facilities, we will utilize next-generation sequencing to provide a pilot assessment of the total microbiota captured with the bioaerosol samplers, as our proposed method should capture a wide array of microorganisms of relevance to the livestock industry. The work will be performed on bacterial isolates and viral particles and not on live animals, thus not requiring IACUC approval.

Progress 06/30/14 to 09/30/17

Outputs
Target Audience:Target audiences included undergraduate and graduate students, microbiologists, food safety specialists, extension professionals, mass spectrometrists, biotechnology and animal health industry, food industry, veterinarians, academic personnel, consumers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported opportunities for training of three graduate students, four undergraduate students, one postdoctoral fellow, and one research scientist. How have the results been disseminated to communities of interest?Results have been disseminated via peer reviewed publications, conference presentations, teaching, and extension publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals. We have focused on elucidating the ecology and dissemination of antibiotic resistant (AMR) bacteria with special emphasis on the role of wildlife in the maintenance and transmission of AMR to livestock operations. This work has involved extensive characterization of isolates via phenotypic methods (culture-based, susceptibility testing, MALDI-TOF MS) and genotypic (PCR, whole genome sequencing). Thousands of isolates of AMR Escherichia coli and Enterococcus spp. (cephalosporin and fluoroquinolone resistant E. coli, and macrolide resistant Enterococcus spp.,) have been fully characterized phenotypically for resistance to 18 and 13 major antibiotics, respectively. These isolates were obtained from approximately 36 concentrated animal feeding (CAFOs) facilities, associated wildlife (mammalian and avian), and environment (feed and water). For enterococci, high numbers of resistant isolates were observed especially for erythromycin, tetracycline, doxycycline, rifampin and ciprofloxacin. Cephalosporin-resistant E. coli displayed especially high resistance to ampicillin, cefazolin, aztreonam, cefotaxime, piperacillin, ceftazidime, and tetracycline, while fluoroquinolone-resistant isolates were observed to be often resistant to ampicillin, nalidixic acid, ciprofloxacin, piperacillin, trimethoprim/sulfamethoxazole, chloramphenicol, and tetracycline. In E. coli, predominant class A ß-lactamase genes blaCTX-M, blaTEM and CIT-type AmpCs were commonly associated with important resistance phenotypes. Focus on preventions and interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and foodborne and waterborne pathogens. We have focused on the development of Matrix- Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)-based methods for rapid identification and characterization of AMR bacteria. Following phenotypic testing of subsets of the isolates for antimicrobial susceptibility; peak alignments and correlative analyses were performed, leading to the identification of specific biomarkers which were predictive of antimicrobial susceptibility within this dataset, with some biomarkers indicative of antimicrobial susceptibility to multiple antibiotics. For E. coli, predictive biomarkers were identified among others for ampicillin, amoxicillin, aztreonam, ciprofloxacin, and tetracycline. For enterococci, predictive biomarkers were identified for nitrofurantoin, quinupristin-dalfoprisin, rifampin, chloramphenicol, and doxycycline among others. Furthermore, improved methods to capture and detect viruses from bioaerosols have been developed. Initially, we showed that modification of SKC BioSamplers for capture of viruses from bioaerosols improved detection sensitivity by 8.5x and 2x for the virus surrogates MS2 and Φ6 bacteriophages, respectively. Later, we applied the anion-exchange resin method to concentrate influenza viruses impinged within the liquid matrix of the widely used impinger, the BioSampler (SKC Inc., Eighty Four, PA). The anion-exchange resin allowed for the improved qRT-PCR detection of type A influenza viruses and type B influenza viruses within BioSamplers, compared to direct testing of the liquid matrix. On average, the resin improved detection by 6.77× and 3.33× for type A and type B influenza viruses, respectively. Lastly, the focus of diagnostics development has been on microfluidic devices (primarily paper-based) for colorimetric or electrochemical detection of pathogenic or indicator bacteria, with several prototypes developed, including one capable of both colorimetric and electrochemical detection.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Chandler JC, Schaeffer JW, Davidson M, Magzamen SL, P�rez-M�ndez A, Reynolds SJ, Goodridge LD, Volckens J, Franklin AB, Shriner SA, Bisha B. A method for the improved detection of aerosolized influenza viruses and the male-specific (F+) RNA coliphage MS2. J Virol Methods. 2017 Aug;246:38-41. doi: 10.1016/j.jviromet.2017.04.004.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Adkins JA, Boehle K, Friend C, Chamberlain B, Bisha B, Henry CS. Colorimetric and Electrochemical Bacteria Detection Using Printed Paper- and Transparency-Based Analytic Devices. Anal Chem. 2017 Mar 21;89(6):3613-3621. doi: 10.1021/acs.analchem.6b05009.
• Type: Other Status: Published Year Published: 2017 Citation: Bisha B. Cutting-edge tech traces food contamination to its sources. Reflections Magazine (University of Wyoming College of Agriculture and Natural Resources). 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Chandler J, Schaeffer J, Davidson M, Magzamen S, Perez-Mendez A, Reynolds S, Goodridge L, Volckens J, Franklin A, Shriner S, Bisha B. A method for the improved detection of aerosolized influenza viruses using impingers that incorporate anion exchange resin. International Association for Food Protection Annual Meeting. July 9- July 12, 2015, Tampa, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Aljasir S, Chandler J, Hamidi A, Sylejmani D, Wang B, Schwam K, Bisha B. A survey of antimicrobial resistance among dairy cattle in Kosovo. International Association for Food Protection Annual Meeting. July 9- July 12, 2015, Tampa, FL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Bisha B. Small Things Considered: From bioaerosols to microfluidics. Iowa State University, Department of Food Science and Human Nutrition Seminar Series, March 29, 2017 (invited talk).
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Bisha B. Exploring alternative tools for diagnostics and characterization of foodborne pathogens. Colorado State University, Department of Food Science and Human Nutrition Seminar Series, November 9, 2017 (invited talk).

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Target audiences have included livestock producers, federal and state governmental public health officials, graduate and undergraduate students in food microbiology/animal science/veterinary science, food safety and quality professionals, and extension educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training opportunities for two graduate students and one undergraduate student. How have the results been disseminated to communities of interest?The results have been disseminated via journal publications, and conference presentations and abstracts. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) Objective 1: Focus on emerging diseases We have continued to focus on antibiotic resistant (AMR) bacteria with special emphasis on the role of wildlife in the dissemination of AMR to livestock operations. A subset of approximately 1,370 isolates of AMR Escherichia coli and Enterococcus spp. was fully characterized phenotypically for resistance to 18 and 13 major antibiotics, respectively. These isolates originated from a pool of approximately 3,000 presumptive cephalosporin and fluoroquinolone resistant E. coli, macrolide resistant Enterococcus spp., and methicillin resistant Staphylococcus spp. isolates collected from 40 concentrated animal feeding (CAFOs) facilities, associated wildlife (mammalian and avian), and environment (feed and water). For enterococci, high numbers of resistant isolates were observed especially for erythromycin, tetracycline, doxycycline, rifampin and ciprofloxacin. For E. coli, depending on whether initial isolation procedures involved selection for cephalosporin or fluoroquinolone resistance, different resistance profiles were observed. Cephalosporin-resistant E. coli displayed especially high resistance to ampicillin, cefazolin, aztreonam, cefotaxime, piperacillin, ceftazidime, and tetracycline, while fluoroquinolone-resistant isolates were observed to be often resistant to ampicillin, nalidixic acid, ciprofloxacin, piperacillin, trimethoprim/sulfamethoxazole, chloramphenicol, and tetracycline. Molecular characterization of representative isolates is under way. In addition, 145 Oceanobacillus spp. were also analyzed which originated from the feces of cattle, rodents, raccoons, coyotes, birds, and water samples. Analyses of partial 16S rDNA revealed a diversity of Oceanobacillus spp. never before recognized within a single environment, with isolates belonging to ten genotypes. Multiple drug resistances (MDR) were identified in representative isolates for all but two of the genotypes. 2) Objective 2: Focus on preventions and interventions We have emphasized the development of Matrix- Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)-based methods for rapid identification and characterization of AMR bacteria. We have developed MALDI-TOF MS protocols for identification and typing of antimicrobial resistant bacteria. Following phenotypic testing of subsets of the isolates for antimicrobial susceptibility; peak alignments and correlative analyses were performed, leading to the identification of specific biomarkers were predictive of antimicrobial susceptibility within this dataset, with some biomarkers were indicative of antimicrobial susceptibly to multiple antibiotics. For E. coli, predictive biomarkers were identified among others for ampicillin, amoxicillin, aztreonam, ciprofloxacin, and tetracycline. For enterococci, predictive biomarkers were identified for nitrofurantoin, quinupristin-dalfoprisin, rifampin, chloramphenicol, and doxycycline among others. In addition, improved methods to capture and detect viruses from bioaerosols have been developed. In previous work we showed that modification of SKC BioSamplers for capture of viruses from bioaerosols improved detection sensitivity by 8.5x and 2x for the virus surrogates MS2 and Φ6 bacteriophages, respectively. Here we further tested the ability of anion-exchange resin to concentrate influenza viruses impinged within the liquid matrix of the widely used impinger, the BioSampler (SKC Inc., Eighty Four, PA). Thus, the anion-exchange resin serves as a second virus concentration step within the overall sampling strategy. Influenza viruses are reported to have negative charges and have been used in the evaluation of impingers. Influenza viruses were obtained from the FluMist Quadrivalent vaccine (FluMist) (2015-2016 formulation; MedImmune, Gaithersburg, MD) which contains four vaccine strains: A/Bolivia/559/2013 (H1N1), A/New Caledonia/71/2014 (H3N2), B/Phuket/3073/2013 (B/Yamagata/16/88 lineage), and B/Brisbane/60/2008 (B/Victoria/2/87 lineage). The anion-exchange resin allowed for the improved qRT-PCR detection of type A influenza viruses and type B influenza viruses within BioSamplers, compared to direct testing of the liquid matrix. On average, the resin improved detection by 6.77× and 3.33× for type A and type B influenza viruses, respectively.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Chandler J, Franklin A, Shriner S, Root J, Mooers N, Bentler K, Ellis J, LeJeune J, Goodridge L, Stahl R, Russell M, Robeson M, and Bisha B. Oceanobacillus spp., Antimicrobial Resistant Bacteria at the Wildlife-Livestock Interface. Scientific Reports (submitted).
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Chandler J, P�rez-M�ndez A, Paar J, Doolittle M, Bisha B, Goodridge L. Field-based evaluation of a male-specific (F+) RNA coliphage concentration method.J Virol Methods. 2016 Oct 21;239:9-16. doi: 10.1016/j.jviromet.2016.10.007.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Harris L, Lieberman V, Mashiana R, Atwill E, Yang M, Chandler J, Bisha B, Jones T.Prevalence and Amounts of Salmonella Found on Raw California Inshell Pistachios.J Food Prot. 2016 Aug;79(8):1304-15. doi: 10.4315/0362-028X.JFP-16-054.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Maus A, Anders J, Bisha B, and Basile F. Differentiation of Bacteria at the Strain Level by MALDI-MS of Proteins >15kDa. 64th American society for Mass Spectrometry Annual Conference. June 5-9, San Antonio, TX.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Chandler C, Franklin A, Shriner S, Root J, Anders J, Wang B, and Bisha B. Synanthropic Wildlife Associated with Livestock Production as Carriers of High Priority Antimicrobial Resistances. International Association for Food Protection Annual Meeting. July 31- August 3, 2016, St. Louis, MO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Anders J, Wang B, Chandler C, Prenni J, Franklin A, Carlson J, LeJeune J, and Bisha B. MALDI-TOF MS Biotyping in the Characterization of Antimicrobial resistant Enterococcus spp. from Wildlife Associated with Concentrated Animal Feeding Operations. International Association for Food Protection Annual Meeting. July 31- August 3, 2016, St. Louis, MO.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target audiences have included food safety professionals, extension educators, graduate and undergraduate students in food microbiology, federal and state governmental public health officials, and producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training and professional development opportunities for one graduate student, one postdoctoral fellow, and one research scientist. How have the results been disseminated to communities of interest?Results have been disseminated via peer-reviewed publications, conference presentations and abstracts, and panel discussions. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) Objective 1: Focus on emerging diseases The focus has been on methods to mitigate the spread of antibiotic resistant (AMR) bacteria to livestock. We have developed MALDI-TOF MS protocols for identification and typing of antimicrobial resistant bacteria. Approximately 3,000 presumptive cephalosporin and fluoroquinolone resistant E. coli, macrolide resistant Enterococcus spp., and methicillin resistant Staphylococcus spp. isolates collected from 40 cattle facilities and associated wildlife were initially identified by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS): PCA-type analyses which consider the entirety of the data set were able to distinguish phenotypes below the species level, as indicated by the clustering patterns which show that isolates can be differentiated below the taxonomic level of species. That differentiation does not extend to specific antimicrobial susceptibility. Subsets of the isolates were tested phenotypically for antimicrobial susceptibility via phenotypic methods; then through peak alignment and correlative analyses, specific biomarkers were predictive of antimicrobial susceptibility within this dataset. Certain biomarkers were indicative of antimicrobial susceptibly to multiple antibiotics. 2) Objective 2: Focus on preventions and interventions The focus has been on developing improved methods to capture and detect viruses from bioaerosols. Modification of SKC BioSamplers for capture of viruses from bioaerosols improved detection sensitivity by 8.5x and 2x for the virus surrogates MS2 and Φ6 bacteriophages, respectively. The modifications to the SKC BioSampler are straight forward and add minimum cost, allowing for simplification of sampling procedures and addition of Sample buffer incorporated protectants.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Chandler JC, Manley WA, P�rez-M�ndez A, Bisha B, Adkins JA, Henry CS, Prenni JE, Goodridge LD. 2015. Molecular and phenotypic properties of cantaloupe-associated Listeria monocytogenes. Food Microbiology (submitted).
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Johnson DC, Bzdek JP, Fahrenbruck CR, Chandler JC, Bisha B, Goodridge LD, Hybertson BM. 2015. An innovative non-thermal plasma reactor to eliminate microorganisms in water. Desalination and Water Treatment. DOI: 10.1080/19443994.2015.1024752.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Bisha B, Brehm-Stecher BF. 2015. Flow cytometry for rapid detection of Salmonella spp. in seed sprouts. ScienceOpen Research. doi: 10.14293/S2199-1006.1.SOR-LIFE.AJ19WR.v1.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: J Anders, B Wang, J Chandler, J Prenni, A Franklin, J Carlson, J LeJeune, B Bisha. MALDI-TOF MS Biotyping for Characterization of Antimicrobial-resistant Escherichia coli from Concentrated Animal Feeding Operations and Associated Wildlife. International Association for Food Protection Annual Meeting. July 25- July 28, 2015, Portland, OR
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S Coleman, H Kessler, X He, J Avens, J Chandler, B Bisha, L Goodridge, M Bunning. Evaluation of Consumer Washing Techniques to Reduce Natural Microbiota on the Surface of Whole Cantaloupes. International Association for Food Protection Annual Meeting. July 25- July 28, 2015, Portland, OR.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: B Bisha. Antimicrobial Resistance: Food Safety. Microbial Ecology in a Changing World: Emerging Issues in Antimicrobial Resistance. The 6th Annual Public Health Symposium at Colorado State University. April 15, 2015, Fort Collins, CO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: J Chandler, M Davidson, J Schaeffer, A Perez-Mendez, J Volckens, S Magzamen, L Goodridge, S Reynolds, B Bisha. Development of an Improved Sampling Method for Concentrating Viruses from Bioaerosols. International Association for Food Protection Annual Meeting. July 25- July 28, 2015, Portland, OR.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: A Franklin, S Shriner, J Root, B Bisha, J Chandler. The Role of Wildlife in Disseminating Antibiotic-resistant Bacteria to and from Livestock Facilities. 3d International Symposium on the Environmental Dimension of Antibiotic Resistance. May 17 - May 21, 2015, Wernigerode, Germany.

Progress 06/30/14 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided training for one graduate student and one postdoctoral fellow. How have the results been disseminated to communities of interest? Results have been disseminated via peer-reviewed journal publications and conference presentations and abstracts. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1) Objective 1: Focus on emerging diseases The focus has been on methods to mitigate the spread of antibiotic resistant (AMR) bacteria to livestock. We have developed MALDI-TOF MS protocols for identification and typing of antimicrobial resistant bacteria. A total of 1,177 presumptive AMR Escherichia coli (ciprofloxacin and cefotaxime resistant), Staphylococcus spp. (cefoxitin resistant), and Enterococcus spp. (erythromycin resistant) isolates from wildlife, feed, and water associated with concentrated animal feeding operations were analyzed. Detailed analyses of mass spectral profiles identified numerous spectral peaks, which may be useful for subtyping applications, including differentiation of AMR phenotypes. 2) Objective 2: Focus on preventions and interventions An improved method to capture and detect viruses from bioaerosols has been developed. Experiments have been performed in a bioaerosol chamber to evaluate the capture of enteric viruses by using MS2 and ?6 bacteriophages as surrogates at different inoculum concentrations. Various bioaerosol samplers were tested including SKC BioSamplers, sorbent tubes, and BC-251 personal samplers which contained IRA-900 and multiple filter types (including PVC, PTFE, and polycarbonate) integrated into SKC Button Samplers and BC-251 personal samplers. The SKC BioSampler modified with IRA-900 anionic exchange resin allowed for over one log increase in detection sensitivity when coupled with molecular detection.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: JL Edwards, B Bisha, S Fisher, H Van Campen, LV Kendall. Development of a Multiplex Bead-Based Array Protocol for Determination of Positive Sera for Select Feline Viruses. P174. American Association for Laboratory Animal Science (AALAS) National Meeting. October 19- October 23, 2014, San Antonio, TX.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: A Perez-Mendez, J Chandler, B Bisha, S Coleman, L Goodridge. Rapid Resin-based Method for Concentration of Rotavirus, Hepatitis A Virus and Adenovirus 40 from Tap Water. P1-643. International Association for Food Protection Annual Meeting. July 31- August 3, 2014, Indianapolis, IN.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: JC Chandler, B Wang, JE. Anders, AB Franklin, JT LeJeune, JE Prenni, JC Carlson, B Bisha. Application of MALDI-TOF MS for the identification and characterization of AMR bacteria from wildlife associated with concentrated animal feeding operations. Conference of Research Workers in Animal Diseases (CRWAD). December 7-9, 2014, Chicago, IL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: L Krause, S Reynolds, J Schaeffer, A VanDyke, M Davidson, B Bisha. Evaluation of Bacterial Preservation During Air Sampling of Culturable Bioaerosols. The American Industrial Hygiene Conference & Exposition (AIHce). May 31- June 5, 2014, San Antonio, TX.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: B Saeed, K Mills, D OToole, B Schumaker, B Bisha, W Laegreid. An Improved Primer Set for the Genotyping of Clostridium perfringens. American Association of Veterinary Laboratory Diagnosticians (AAVLD) 57th Annual Meeting. October 16- October 22, 2014, Kansas City, MO.