Our project under this funding began January 2016 and this report includes progress from January 2016 until July 31, 2017 (end of project). Our overall objective of this project is to increase the capability to identify infected animals with brucellosis in the Greater Yellowstone Area (GYA). We are tackling this issue by the creation and validation of a novel molecular assay (qPCR) for B. abortus, B. suis, and B. melitensis. To date, we have identified novel primer/probe regions for this assay, which are currently being validated on. We have run a blinded in parallel assay with tissues that were collected from Yellowstone National Park bison. Tissues were split and half went to United States Department of Agriculture – National Veterinary Services Laboratory (USDA-NVSL), and half stayed at the University of Wyoming / Wyoming State Veterinary Laboratory for qPCR testing. Of the animals cultured thus far, our qPCR assay has detected two times times as many positives as culture (n=63). Incorporated with this project was a producer/veterinary educational extension effort to educate stakeholders on brucellosis. With additional funding from the Institute for Infectious Animal Diseases – United States Department of Homeland Security and the Wyoming Department of Agriculture, we completed meetings with stakeholders in Pinedale, Cody, Greybull, and Sheridan, Wyoming in the summer of 2016. In total over 120 participants were present at these meetings. A panel of experts presented information on brucellosis as well as engage in a discussion on the disease. The panel included two previously quarantined producers, the Wyoming State Veterinarian, Wyoming Game and Fish Department, Wyoming Extension Office, members from the University of Wyoming/Wyoming State Veterinary Laboratory, an agricultural economist, and an veterinary student extern. We additionally collected 127 brucella negative animals from outside of the endemic area. These samples were tested on our candidate sets for real-time qPCR and no amplification was detected, indicating that the assay was 100% specific.
Our first objective is to identify and validate a novel quantitative polymerase chain reaction assay for that is highly sensitive and specific for brucellosis in the GYA. Ideally, this assay can be used ante-mortem, but will first be validated in post-mortem tissues.
Our second objective is to host producer/veterinarian meetings on brucellosis. These meeting will consist of a panel of brucellosis/animal disease experts and will include a discussion on brucellosis. Included in this meeting will be a pre and post knowledge test for the stakeholders. Factsheets will be provided for reference.
An in-silico analysis was completed on 103 Brucella genomes acquired from United States Department of Agriculture – National Veterinary Services Laboratory and National Institutes of Health. Descriptive regions were isolated and primers and/or probes were ordered for testing of a novel molecular diagnostic.
Primers and/or probes were optimized on various parameters. Animal sample extracted DNA from serologically positive, culture positive animals were tested on the candidate sets.
In parallel, producer meeting were planed in four locations through out Wyoming (as determined by UW Extension Office). These meeting would be advertised in pertinent media outlets to increase attendance. Discussions were hold with experts in the room on the background of brucellosis, testing that is currently done, future of testing, regulatory implications, and lessons learned from quarantined producers.
The molecular diagnostics portion of this project was completed. We were able to acquire enough samples to validate this assay and the statistics are soon to be reported in the peer-reviewed literature. Comparisons between the gold-standard of bacteriologic culture, and serology are pending.
In all there were 120 producers that attended the four meeting in the state of Wyoming. All of the meetings went longer than expected due to the participation of the producers and various other stakeholders that attended the meeting. By request from the University of Wyoming – College of Agriculture and Natural Resources, we have a plan to continue another round of these meetings in the winter of 2017/18 due to the excellent feedback.
Overall, this novel diagnostic assay will make inroads into replacing current non-optimal diagnostics that are in place. This will allow practitioners to more readily identify infected herds and will provide faster turn around times based on currently used methodologies.
The producer meetings were very helpful in educating stakeholders about the current issues with brucellosis. This disease and regulatory implications are ever-changing and this forum allowed the stakeholders to be better informed and to have transparency in the regulatory process.
Educational & Outreach Activities
Four meetings were held in the state of Wyoming (Pinedale, Cody, Greybull, and Sheridan). These meetings brought together academic researchers, members of the Wyoming State Veterinary Laboratory, the State Veterinarian, the Assistant State Veterinarian, Wyoming Game & Fish Department, UW Extension, and affected producers. There were 120 participants across all the meetings.
We are in the midst of preparing a manuscript detailing our qPCR assay in comparison to culture data from USDA-NVSL on the Yellowstone bison. We will target the Proceedings of the National Academy of Sciences (PNAS) for this publication.
In total, we had 120 participants at the meetings. The goal was to educate producers on brucellosis, risk factors for the disease, testing that can be accomplished, and what happen if an animal is positive. We had two producers that have recently been affected by quarantines and were able to give their perspective of the process. Since these meetings, the Wyoming Livestock Board has seen a marked increase in the number of risk assessments it has been asked to provide to producers. Additionally, there has been an increase in the number of voluntary sampling done on herd outside of our Designated Surveillance Area.
To date, we have made numerous accomplishments on this project. We identified eight primer/probe sequences, which are being validated against culture-positive animal tissues. These eight candidates have been trimmed down to the top three; one that identifies all Brucella abortus, one that only identifies RB51 vaccine and one that identifies S19 vaccine strain. We have reached our sample size for bison samples, as we were able to utilize tissues from the Yellowstone National Park bison slaughter. We are still seeking reactor cattle to boast our sample size in cattle. We have reached out sample size in negative elk tissues with cooperation of the National Park Service and Wind Cave National Park. We are seeking negative cattle and bison samples to validate our specificity.
We have run blinded assays with the Yellowstone bison and preliminary data has shown that our assay detects 3.5 times more positive animals than culture. Per previous research, only 46% of serologically positive bison will isolated on culture. This leaves the question of the 54% “gap” animals, and their status. Currently, the Yellowstone bison are showing a culture proportion of 20% while our assay is detecting 70% as being positive. To ensure that our amplifications were true Brucella field strain, amplicons from a subset of these positives were sent for sequencing. Sequencing confirmed that we were amplifying Brucella abortus. We continue to validate these primer/probe sets as we strive to replace culture with a molecular assay.
We conducted extension outreach efforts to stakeholders in the GYA. We completed four meetings for these stakeholders. Only July 18, a meeting in Cody (Park County, WY) at the EOC room of the Park County Courthouse. On July 19, a meeting in Pinedale, WY (Sublette County, WY) at the Sublette County Library Lovatt Room. On July 25, a meeting in Sheridan, WY (Sheridan County, WY) at the Watt Building room 131. Finally, on July 26, a meeting in Greybull, WY (Big Horn County, WY) at the Weed and Pest Extension office. All meetings began at 6PM and a light dinner was provided in order to increase attendance. The schedule was as follows:
6:00 pm – Arrive, Dinner, & Introductions – Barton Stam
6:15 – 6:25 pm – “Basics and Background Information about Brucellosis,” presented by Chrissy Casey
6:25 – 6:50 pm – “Vaccination, Quarantine, and Regulations,” presented by Jim Logan
6:50 – 7:00 pm – “Current and Future Diagnostics,” presented by Brant Schumaker and Noah Hull
7:00 – 7:15 pm – “Wildlife Reservoirs and Management,” presented by a local WGFD officer
7:15 – 7:45 pm – “Personal experiences with Brucellosis Quarantines,” Producers
7:45 – 8:00 pm – “The Cost of Quarantining, an Agricultural Economist’s Perspective.” Dannele Peck
8:00 – 8:30pm – Conclusion & Discussion
Moderator: University of Wyoming Extension Educator – Barton Stam
Panelists: University of Georgia DVM candidate – Chrissy Casey
Wyoming State Veterinarian – Dr. Jim Logan
Veterinary Epidemiologist – Dr. Brant Schumaker
University of Wyoming PhD candidate – Noah Hull
Wyoming Game and Fish Department – Regional Game and Fish Officer
Affected Producers (2) – Sublette County (quarantined) and Park County (previously quarantined)
University of Wyoming Agricultural Economist – Dr. Dannele Peck