Trich Laboratory Testing Standardization

1. Trich Laboratory Testing Standardization Trichomonas foetus DNA testing Proper & Confidential

2. Trich Laboratory Testing Standardization Objectives • To build confidence in Trich testing • To increase consistency in test results • To minimize sample quality influencevariables • To minimize laboratory testing variables • To reduce the burden and cost to man and beast Proper & Confidential

3. Trich Laboratory Testing Standardization Opportunities • Veterinarian sample collection • Veterinarian sample handling & shipment recommended by Laboratory • Laboratory sample preparation method • Laboratory extract volume used • LaboratoryTaq enzyme type used • Laboratory analysis threshold level • Laboratory use of IPC (Internal Positive Control) • Laboratory use of USDA licensed kits • Laboratory pooling of samples Proper & Confidential

4. Pooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection of Tritrichomonas foetus–colonized bulls Lee Effinger Lalitha Peddireddi Marilyn Simunich Richard Oberst Catherine O’Connell Ivan Leyva-Baca Oregon Department of Agriculture, Animal Health and Identification Division, Animal Health Laboratory, Salem, OR (Effinger) Department of Diagnostic Medicine/Pathobiology (Peddireddi), Kansas State University, Manhattan, KS Kansas State Veterinary Diagnostic Laboratory (Oberst), Kansas State University, Manhattan, KS Animal Health Laboratory, Idaho State Department of Agriculture, Boise, ID (Simunich) Animal Health and Food Safety Group at Life Technologies, Austin, TX (Leyva-Baca, O’Connell) JVDI, 2014, Vol. 26(1) 72-87 Proper & Confidential

5. Study Background 2010 AAVLD parasitology committee • Proposed a study to determine whether T. foetus samples can be pooled in order to reduce the costs for testing • Lee Effinger from Oregon State Department of Agriculture led Experimental Design for the project • Marilyn Simunich Idaho State Department of Agriculture served as Study Coordinator & Data Keeper • The Life Technologies Animal Health & Food Safety Group agreed to support the study Proper & Confidential

6. Study Objectives • Determine the effect of pooling a single positive sample having various CT ranges with four negative samples (1:5). If a negative effect was seen, a 1:3 pooling study would then be conducted • Compare different sample preparation systems and various real-time PCR (feeder lab workflows) with the 5X MagMAXTM-pathogen RNA/DNA purification kit and amplification with VetMAXTMT. foetus reagents (Life Technologies workflow) • Assess the specificity of the VetMAXTMT. foetus reagents by sequencing all positive samples with CT values less than 38 and suspect sample CT values between 38 and less than 40 cycles Proper & Confidential

7. Materials and Methods • Sample collection (Cultured Smegma Samples) • 5 Feeder labs provided 803 samples • 1 on the West Coast • 1 in the Southwest • 1 in the Central States • 2 in the South • Each feeder lab ran their own protocol including sample preparation system and real-time PCR • 1 Central Study lab (KSVDL) • Sample preparation with MagMAXTM • Real-time PCR with VetMAXTMT. foetus reagents Proper & Confidential

8. Cultured smegma samples provided by feeder labs * As reported by the feeder labs Proper & Confidential

9. Real Time PCR Parameters for Feeder Laboratories Proper & Confidential

10. Results: Individual Sample Testing Study Lab Results vs. Feeder Lab Results Order of the call = KSVDL Study Lab / Feeder Laboratory Pos = positive, Neg = negative, Inc = inconclusive, PresPos= presumptive positive Proper & Confidential

11. Conclusions Individual Testing • 803 smegma samples were provided by feeder labs (FL) • All the samples were tested by study laboratory with Life Technologies workflow systems: • MagMAXTM • VetMAXTMT. foetus reagents • Agreement of 95.6% was reached with 768/803 samples between feeder labs and study lab • Interestingly, Lab F reached almost 100% agreement using a different sample prep system and a modified McMillen’s assay • Study laboratory (KSVDL) with LT protocol identified 24 more positives than the feeder laboratories. On retesting, one of the feeder labs missed 9 samples reported as positives. Proper & Confidential

12. Pooling Study Positives, presumptive positive and negative samples used from each lab for pooling Proper & Confidential

13. Pooling results Effect of pooling for T. foetus samples with CT>35 after individual testing *WFA: Suspect workflow A; ** samples confirmed T. foetus by sequencing Proper & Confidential

14. Pooling results 1:5 Pools • 1:5 pooling of positive samples with a CT of 35 and below were all detected • Only 3 of 9 positive samples with CTs between 36-39.9 were detected in 1:5 pools • Pooling at 1:5 missed 4% (7/176) of T. foetus positive samples 1:3 Pools • Only 8 of 15 positive samples with CTs between 36-39.9 were detected in the 1:3 pools • 1:3 pooling missed 3.5% (6/176) of T. foetus positive samples Proper & Confidential

15. Sequencing primer design for nested PCR (M13-I-F-TFSM-Primer) (O-F-TFSM-Primer) TTAGCTTTCTTT GCGA T. foetus TTAGCTAACAAT GCGA S. moskowitzi (R-TFSM-primer) (M-13-R-TFSM-primer) GenBank: GQ254636.1 Simplicimonas moskowitzi GenBank: AY349189.1 Tritrichomonas foetus Proper & Confidential

16. Sequencing results for 175 T. foetus positives 175/176 T. foetus positive samples, including three late risers, were confirmed T. foetus by DNA sequencing Proper & Confidential

17. Sensitivity, specificity, & predictive values of positive & negative results for all cultured smegma samples * Calculations made after qPCR and sequence confirmation Proper & Confidential

18. Sequencing Results • 175/176 positive samples by qPCR were able to be sequenced • 1 sample (A-7-25) with a CT 33.95 was not able to be sequenced. • It is possible that there are point mutations in this positive sample in the sequencing primer regions, which were designed based on a few T. foetus and a single S. moskowitzi sequences from GenBank • Most importantly, none of the samples reported S. moskowitzi DNA sequences Proper & Confidential

19. Overall Study Results • 95.6 % agreement was reached between Study Lab (KSVDL) using Life technologies MagMAXTM and VetMAXTMT. foetus reagents and the feeder laboratories • 1:5 Pooling it is likely to miss 4% of the positives • 1:3 Pooling it is likely to miss 3.5% of the positives • DNA sequencing • 175/176 positive samples were confirmed to be T. foetus, the 176th sample could not be sequenced with the primers designed for this study Proper & Confidential

20. Acknowledgements Lalitha Peddireddi, KSVDL – performed the study at KSVDL Lee Effinger, ODA-Animal Health Laboratory Marilyn Simunich, Idaho State Dept. of Agriculture Cate O’Connell, Life Technologies Mangkey Bounpheng, Texas Veterinary Medical Diagnostic Laboratory Dawn Bueschel, NMDAVeterinaryDiagnostic Services MuthuChengappa, Kansas State Veterinary Diagnostic Laboratory Alfonso Clavijo, Texas Veterinary Medical Diagnostic Laboratory Kris A. Clothier, California Animal Health & Food Safety Lab System Hemant K. Naikare, Texas Veterinary Medical Diagnostics Laboratory Jeff Zinza, Life Technologies Mary Anne Williams, Life Technologies Proper & Confidential

21. Trich Laboratory Testing Standardization Objectives • To build confidence in Trich testing • To increase consistency in test results • To minimize sample quality influencevariables • To minimize laboratory testing variables • To reduce the burden and cost to man and beast Proper & Confidential

22. Trich Laboratory Testing Standardization Trichomonas foetus DNA testing Proper & Confidential