DEVELOPMENT AND VALIDATION OF A POLYMERASE CHAIN REACTION TEST FOR DIAGNOSIS OF BRUCELLA ABORTUS INFECTIONS IN LIVESTOCK IN WYOMING

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1003044
• Project No.: WYO-525-14
• Project Start Date: May 20, 2014
• Project End Date: Sep 30, 2016

Project Director
Schumaker, BR, A..

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

Performing Department
Veterinary Sciences

Non Technical Summary
Brucellosis is one of the world's most widespread zoonoses but ranks as one of the seven most neglected, according to the World Health Organization. There are over 500,000 new human cases of brucellosis annually, which is believed to be an underestimate due to underreporting. In the US, the State-Federal Cooperative Brucellosis Eradication Plan, initiated in 1934, was successful in eradicating brucellosis from livestock populations. However, the disease still has a reservoir in elk (Cervus canadensis) and bison (Bison bison) in the greater Yellowstone area (GYA) with multiple instances of spillover into Wyoming livestock in the last decade. Positive cases in livestock lead to costly quarantine and culling in addition to time consuming diagnostictesting. The Wyoming Game and Fish Department uses up to six tests over a period of weeks to reach a conclusion about the status of an animal. Therefore, having reliable testing and a short diagnostic lag time is crucial to eliminating the disease from the Wyoming and any other possible areas affected by the disease.The goal of our project is to design and validate a molecular assay using PCR techniques and DNA isolation kits that are specific for different tissue types, as well as employing sequence specific technologies that will enrich reactions and lead to higher sensitivities. It is our hope that by developing this assay, it will aid in diagnosing brucellosis cases at the species level and differentiating animals that have been previously vaccinated. The success of this project will bring us closer to eradicating brucellosis in the United States while providing stability to livestock producers in Wyoming.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
308	4010	1170	100%

Knowledge Area
308 - Improved Animal Products (Before Harvest);

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1170 - Epidemiology;

Keywords

brucellosis

molecular diagnostics

polymerase chain reaction

Goals / Objectives
The major goal of this project is to develop, validate and implement a polymerase-chain-reaction (PCR) test for Brucella abortus. It is our central hypothesis that the development of a PCR diagnostic test will have increased sensitivity over culture. We have defined three specific objectives:Using Brucella abortus isolates from domestic livestock, test validity of proposed DNA primers.Optimize DNA extraction and purification, primer composition, reaction conditions, and reagent mixture.Perform bench and field validation of the PCR assay.

Project Methods
Brucellosis diagnostic testing and eradication can be challenging due to the deficiencies of culture as a true gold standard test. Lack of sensitivity, potential for bacterial overgrowth, and prolonged incubation times make culture unreliable. PCR development has been attempted by a number of laboratories that have described difficulties with DNA isolation from a multitude of tissue samples. Phenol Chloroform has historically been used, however low yields, the added dangers of these chemicals, and increase of coextraction PCR inhibitors does not make it an ideal method. Instead, we propose extraction with readily available commercial kits.In November 2013, research team members visited the Antlers Ranch and collected over 400 whole blood samples for use in developing the molecular assay. In addition, our Producer Project Partner has agreed to sell us sero-positive bison to acquire a full suite of diagnostic tissues. We will extract DNA from whole blood, tissue samples, and primary bacterial isolates. Kits have been selected for use on each tissue sample to allow the highest yield possible of genomic DNA. Several groups pioneered multiplex PCR techniques as a diagnostic test for brucellosis. However, for the Wyoming problem we are only interested in Brucella abortus as well as all possible vaccine strains to differentiate active infection from previous vaccination. In addition, multiplex PCR suffers from lower sensitivity and specificity due to conflicts between the primer melting temperatures and annealing temperatures. We propose to develop a single-sample multilane PCR.Three additional sample lanes will test for the presence of B. abortus field and vaccine-specific genomic DNA. The first sample lane will test for Brucella abortus genomic DNA using the hypothetical protein gene BRA0907 that consists of 466 amino acids. This is a highly conserved region that is found in Brucella abortus and B. suis samples. Primers were designed using NCBI Primer-Blast with the intention of having amplicons of differing sizes between lanes to readily identify a positive test result. The amplicon product of BRA0907 is 1.6 kbp in size. In order to identify animals that have been previously vaccinated we will run up to two additional lanes, dependent on species. For bovine samples we will run a lane that uses previously described primers for the RB51 vaccine, these primers will produce an amplicon of 1682 bp. Previously described primers for S19 vaccine will be used and will produce an amplicon of 587 bp. Similar sized amplicons for B. abortus field strain and RB51 create a problem in multiplex PCRs however, since each lane will have specific primers this will not be an issue for our test. After the test has been bench validated, we will calculate test performance characteristics including sensitivity, specificity, likelihood ratios, positive and negative predictive values at various prevalence levels, as well as 95% confidence intervals for all parameters. All statistical analyses will be performed using R version 3.0.1 (Vienna, Austria). One limitation of PCR is that it does not indicate whether isolated genomic DNA is from live infectious bacteria or from non-viable or immunologically cleared bacteria. This is of only minimal concern since, from a regulatory perspective, all brucellosis exposures are currently considered infections for life. In the future, this can be addressed by retesting a sample from a subsequent time point. While we anticipate that no Wyoming livestock samples will have been exposed to S19 vaccine for B. abortus, we will still have primers identified should we believe there is the possibility of vaccination or exposure to this strain. If the amplicons for B.abortus field strain and RB51 are too close in size we can create a probe to differentiate the PCR products to differentiate vaccinated from infected individuals. Finally, if there are any issues with DNA extraction or concentration, this can be addressed by developing magnetic beads (Dynabeads®) with bacterial genomic primers to immunoconcentrate samples and increase sensitivity further.

Progress 05/20/14 to 09/30/16

Outputs
Target Audience:The project's target audience were producers, veterinarians, and other stakeholders that would benefit from current information pertaining to brucellosis in Greater Yellowstone Area. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project directly contributed to the graduate training of PhD student, Noah Hull. Additionally, we trained high school student, John Lopez in laboratory techniques related to our project and Jonathan Miller, Robert West, Sierra Amundson, Corinne Vaughan, Sam Jacobs as undergraduate research fellows. We also trained a veterinary extern, Chrissy Casey. How have the results been disseminated to communities of interest?We performed an extension project coordinated by the University of Wyoming research laboratory and the University of Wyoming's Extension Office. A panel of subject matter experts provided a brief overview covering different aspects relevant to brucellosis. Following these brief presentations, questions were encouraged and an open discussion for all attending was facilitated. Speakers on the panel include one quarantined and one previously quarantined producer, the Wyoming State Veterinarian, an agricultural economist, veterinary researchers, and a WGFD representative. The objective of this project was to produce a mutually beneficial learning environment for all attending the meetings and create open lines of communication between scientists, producers, and other stakeholders. Two meetings were held within the Wyoming DSA and two meetings were held outside the DSA in the newly established Area of Concern in Wyoming. Informational material, handouts, and contact information for subject matter experts were provided at each meeting. Additionally, multiple presentations were made to the Wyoming Brucellosis Coordination Team. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Overall, we have succeeded in developing a candidate molecular assay to detect bovine brucellosis and completed preliminary validation.During the project period we collected field samples from seroreactor bison and cattlefrom currently quarantined domestic herds in Wyoming. We evaluated the optimum DNA extraction and purification protocol using spiked tissues, reporting on these results at multiple professional meetings. We acquired over 90 bacterial whole-genome sequences in order to develop DNA primers for bacterial detection. Eight primer sets passed initial optimization and moved on to full validation. Preliminary validation has been completed and the assay shows promise at replacing bacterial culture.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Vaughan, C., N. Hull, J. Miller, and B. SCHUMAKER. The optimization of a novel qPCR assay for brucellosis. Undergraduate Research Day. April 30, 2016, Laramie, WY.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Miller, J., N. Hull, S. Amundson, C. Vaughan, S. Jacobs, and B. SCHUMAKER. Development and evaluation of real time PCR for detection and differentiation of Brucella abortus and vaccine strains. Poster presented at Undergraduate Research Day. April 30, 2016, Laramie, WY.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Hull, N. and B. SCHUMAKER. Development and validation of a molecular assay for diagnosis of brucellosis. Consortium for the Advancement of Brucellosis Science. May 24, 2016, Denver, CO.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Hull, N. and B. SCHUMAKER. Producer meetings. Current and future brucellosis diagnostics. WY producer meetings. July 18, 2016, Cody, WY.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Hull, N. and B. SCHUMAKER. Producer meetings. Current and future brucellosis diagnostics. WY producer meetings. July 19, 2016, Pinedale, WY.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Hull, N. and B. SCHUMAKER. Producer meetings. Current and future brucellosis diagnostics. WY producer meetings. July 25, 2016, Sheridan, WY.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Hull, N. and B. SCHUMAKER. Producer meetings. Current and future brucellosis diagnostics. WY producer meetings. July 26, 2016, Greybull, WY.

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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project directly contributed to the graduate training of PhD student, Noah Hull. Additionally, we trained high school student, John Lopez in laboratory techniques related to our project and Jonathan Miller, Robert West, Sierra Amundson, andCorinne Vaughan as undergraduate research fellows. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are actively determining primer and probe targets on Chromosome 2 for evaluation.During this process we evaluate the best primer composition, reaction conditions, and reagent mixture. We plan to bench validate our PCR assay during this period and expect to begin the field validation of the assay.

Impacts
What was accomplished under these goals? During the reporting period we collected field samples from seroreactor bison and cattle from currently quarantined domestic herds in Wyoming. We acquired over 90 bacterial whole-genome sequences in order to propose DNA primers for bacterial detection. In total,we have acquired five bison from our producer partner and six cattle from newly found cases. All animals have been necropsied with samples banked for test validation. Of these, three bison and one cow were sero-reactors and one was culture positive both here at the WSVL and at NVSL. Culture results are pending on five of the remaining cattle. All culture positive animals had DNA extracted from all tissues collected and other whole tissues were inactivated in order to be within compliance of Select Agent rules. In association with USDA/NVSL, we have secured over 90 whole genome sequences of B. abortus for use in our in-silico analysis. These isolates represent all known biovars in North America. Additionally, we have been granted access to whole genome sequences of B. canis, ovis, suis, and melitensis. We completed the assemblies and alignments on all the B. abortus isolates, and are continuing to search for biovar specific primers. Once completed this will be the most robust in-silico analysis ever done for Brucella and should provide numerous novel primer/probe targets that will be used to validate a novel PCR assay. Thus far we have tested 45 pairs of primers and one set of primer probes. Three primer sets and one primer probe will be moving forward for full validation. We have completed the in-silico analysis on Chromosome 2 and are working on Chromosome 1.

Publications

Progress 05/20/14 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 directly contributed to the graduate training of PhD student, Noah Hull. Additionally, we trained high school student, John Lopez in laboratory techniques related to our project. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We are actively aligning our whole genome sequences to develop primer targets and plan to test these primers during the next reporting period. During this process we evaluate the bestprimer composition, reaction conditions, and reagent mixture. We plan to bench validate our PCR assay during this period and expect to begin the field validation of the assay.

Impacts
What was accomplished under these goals? During the reporting period we collected field samples from seroreactor bison from the currently quarantined domestic herd in Wyoming. We evaluated the optimum DNA extraction and purification protocol using spiked tissues, reporting on these results at multiple professional meetings. We acquired over 90 bacterial whole-genome sequences in order to propose DNA primers for bacterial detection.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lopez, J., N.C. Hull, J. Miller, D.J. Berry, and B.A. SCHUMAKER. Evaluation of commercial DNA extraction kits for Brucella abortus Strain 19. Poster presented at the National Science Foundation Experimental Program to Stimulate Competitive Research (EPSCoR) Summer Research Apprenticeship Program symposium. July 18, 2014, Laramie, WY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lopez, J., N.C. Hull, J. Miller, D.J. Berry, and B.A. SCHUMAKER. Evaluation of commercial DNA extraction kits for Brucella abortus Strain 19. National Science Foundation Experimental Program to Stimulate Competitive Research (EPSCoR) Summer Research Apprenticeship Program symposium. July 18, 2014, Laramie, WY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Hull, N.C. G. Andrews, J. Gigley, W. Laegreid, M. Miller, and B.A. SCHUMAKER. Development of Brucella PCR assay and deployment in East Africa. Poster presented at the Department of Homeland Security  Zoonotic Animal Disease Defense conference. September 9, 2014, Nashville, TN.