1. Bovine Tuberculosis Molecular Epidemiology “Where are our new cases of bovine TB coming from?” Suelee Robbe-Austerman, DVM, PhD Mycobacteria Brucella Section Head National Veterinary Services Laboratories U.S. Department of Agriculture Animal and Plant Health Inspection Service Veterinary Services STAS 4/29/2015 Secretary’s Advisory Committee on Animal Health

2. Objectives Historical perspective of State-Federal Cooperative Bovine Tuberculosis Eradication Program Describe whole genome sequencing (WGS) and how USDA is using WGS to inform traces and support epidemiology. Describe the WGS characteristics of the last 132 (18 yrs. span) new US affected herds. Discuss the importance of a quality database. 2

3. 3 Percentage of U.S. Cattle Responding to the Skin Test, 1917 - 2014 Percentage Of Cattle Responding TB testing begins nationwide 4.9% U.S. declared Modified Accredited Free 0.46% ~1% today (expected fraction of false positives)

4. Affected Cattle and Cervid Herds, FY 1987-2014 4

5. TB Cases Identified Per 100,000 Cattle Imported and Number of Cattle Imported from Mexico, 1998–2014 1 1 Includes Mexican origin cases with and without official identification beginning in FY 2008. Import data not available prior to 2003. 5

6. Genotyping using WGS 2011 – First bench-top Next-Gen sequencer. 2011 – NVSL started a WGS pilot for TB and Brucella. December 2012, WGS replaced traditional lower resolution methods.

7. Impact of WGS was immediate Heiferette, no identification slaughtered in AZ, Sept 2012 Traced to cattle dealer in CA An assembled lot of 172 animals from 78 herds NVSL-ARS evaluated animal genetics –Holstein, likely bull bred Traditional bacterial genotyping –Matched several Mex isolates and a 2002 herd from CA, only 1 locus different from 100’s of isolates. This information was not enough to alter testing priorities in the field.

8. WGS – changed the field response Time Mexican Dairy Isolates 2002 CA Dairy herd 2012 Heiferette

9. WGS – changed the field response CA repriorized the their investigation and found the new herd. –Reduced the time to detection. –Reduced testing of “innocent bystander” herds.

10. WGS can reassure trading partners In 2013 a dairy herd in MI was detected outside the MAZ. The WGS results support the epidemiological investigation suggesting the introduction likely occurred when cows were purchased from the Northeastern lower peninsula in the late 1990’s prior to movement controls.

11. Complications- detection of a steer 1yr. later

12. Saginaw dairy isolates Holstein steer SNPs developed within the Saginaw dairy

13. Where are USA bTB cases coming from? –Low level circulation or residual infection occurring within the USA herd? –New introductions? 132 affected herds

14. Database of sequenced isolates 127/132 (96.2%) affected USA cattle / cervid herds sequenced since 1998 342/385 (88.9%) fed cattle (import and domestic) since 2000 306 Mexico origin 68 unknown 9 USA origin 2 Canada origin –~175 Mexican cattle - mostly dairy –77 Human

15. Affected US cattle / cervid herds 35 Strain types have been identified –Three strain types accounting for 75/130 (58%) of herds have been documented in the USA for at least 25 years. (MI and Cervid A, B) –32 strain types unexplained Unknown residual infections? New introductions?

16. Where are USA bTB cases coming from? Strain TypesOrigin No. USA herdsComment 3USA75 (57%)MI, Cervid A, Cervid B 9Unknown9 (7%)Avg distance to MRCA = 32 23Mexico48 (36%)Includes MN, CO, CA

17. CHI TAM DUN COA UNK 2012 SD Affected herd ZAC

18. Where are USA bTB cases coming from? Strain TypesOrigin No. USA herdsComment 3USA75 (57%)MI, Cervid A, Cervid B 9Unknown9 (7%)Avg distance to MRCA = 32 23Mexico48 (36%)Includes MN, CO, CA

19. 23 Strain types (36%) anchor to Mexico How are they coming to the US? Imported cattle? –Expect to find some closely related isolates from imported Mexican feeder cattle.

20. Imported cattle – 1 herd No links to imported cattle for 22/23 strains that anchor back to Mexico. −Could we be missing the cases in our slaughter surveillance program? −Is there another potential source?

21. Washington case Adult dairy cow from Washington. Investigated the herd- purchased as a heifer no other infection identified. Neighbor was a rodeo contractor.

22. With Mexico collaboration: The most closely related isolates to the Washington dairy cow are 2 human isolates from Baja CA, and Baja CA dairy cattle isolates anchor this strain to Northern Baja CA

23. Two South Dakota untraceable cows

24. The dairy cows matching the SD beef cows were identified due to a collaboration with Mexico and were from the State of Mexico (Greater Mexico City). Dairy cattle, and cattle originating from the red areas are banned from importation to the US.

25. Additional collaborations with Public Health….

26. 2009 CA Dairy

27. 2013 CA Dairy and Beef herds

28. Assisting Public Health Human isolates = Red Cattle isolates = Blue Most human isolates are from Southern CA. Most cattle isolates were from an abattoir in Tijuana. CDC notified CA of a cluster of isolates with the same genotype for investigation. Requested WGS assistance from USDA.

29. Summary WGS allows us to trace new cases more effectively. Over half (57%) of the USA affected herds can be traced to 3 strains documented to be in the USA in the early 1990’s. 36% of US herds can be linked to Mexico. None of these cases can be linked back to Mexican fed cattle. –1 case was linked to a roping steer. A surprising number of US affected herds share a most recent common ancestor with Mexican dairy cattle or human isolates.

30. Summary The database of sequenced isolates is still small and limited in scope. It is critical to work with Mexico to improve the resolution of the database to assist both countries. These results may facilitate cooperation between animal and public health officials.