1. Sample Handling and Transport in Bovine Trichomoniasis Surveillance Anthony Smith, Ph.D. BioMed Diagnostics, Inc. White City, Oregon 97503 USA www.biomeddiagnostics.com 2014 NIAA/USAHA JOINT FORUM ON TRICHOMONIASIS STANDARDS

3. Aims: The role of sample handling and transport in the TF testing process What is in a TF sample and what are the important characteristics? What we did: VDL Survey (32 labs!) Survey data and observations Trends in sample handling and transport practice Towards a recommended best practice

4. Trichomoniasis relevance... BioMed Diagnostics, 22 years of Trichomoniasis expertise InPouch TF Bovine since 1994 Research legacy 46 PubMed citations OSU-CVM TAMU-TVMDL UC Davis Colorado Dept. Agr. Life Technologies Relationships: cow-calf producers, practitioners, seedstock, labs, regulators USAHA, AAVLD, ASM

5. Towards harmonization... The current paradigm: 1.Sample collection-- techniques, materials and training 2.Transport-- delivering quality samples to labs 3.Analysis-- lab protocols 4.Communication-- data distribution and access

6. Sample transport issues Practitioners-Producers rural collection sites catching the samples packaging the samples what shipping rate? temperature control? Laboratories ID chain of custody integrity of analyte

7. Whats in a TF sample and why is it important? ~4 mL collection media ~0.5-1.0 mL preputial smegma Proteins & lipids Somatic cells (epithelium, leukocytes) Blood, urine, pus Bacteria & yeast (endogenous and soil borne) T. foetus (50-141 organisms) Verma et al. Vet Res Comm 1999; 23:337-41 Mukhufhi et al. Theriogenology 2003; 60:1269-78 Hammond and Bartlett. Am J of Vet Res 1943; 4:143-49 Madden. Univ. Wyoming Vet Sciences M.S. Thesis. 2007 (via ProQuest)

8. Whats in a TF sample and why is it important? ~4 mL collection media ~0.5-1.0 mL preputial smegma Proteins & lipids Somatic cells (epithelium, leukocytes) Blood, urine, pus Bacteria & yeast E. coli, Staphylococcus sp., Streptococcus sp., Klebsiella sp., Pseudomonas sp., Shigella sp., Enterobacter sp., Flavobacterium sp., Actinomyces sp., Acinetobacter sp., Bacillus sp., Mycoplasma sp., Brucella sp., Campylobacter sp., Haemophilus sp. T. foetus (50-141 organisms)

9. The most important component? ~4 mL collection media ~0.5-1.0 mL preputial smegma

10. The most important component is T. foetus ~4 mL collection media ~0.5-1.0 mL preputial smegma

11. The most important component is T. foetus ~4 mL collection media ~0.5-1.0 mL preputial smegma Culture Diagnosis– depends on visual observation of live, motile T. foetus PCR Diagnosis– depends on preservation of T. foetus DNA integrity

12. The most important component is T. foetus Culture Diagnosis– depends on visual observation of live, motile T. foetus PCR Diagnosis– depends on preservation of T. foetus DNA integrity Poor DNA integrity or PCR inhibition: Bacteria or yeast overgrowth Excess blood, urine TF necrosis (DNA destruction)

13. The most important component is T. foetus ~0.5-1.0 mL preputial smegma Culture Diagnosis– depends on visual observation of live, motile T. foetus PCR Diagnosis– depends on preservation of T. foetus DNA integrity Critical factors in sample handling and transport: time & temperature

14. Trends in practice: Informal VDL Survey, February-March 2014: Website research and telephone interviews conducted by BioMed Diagnostics Responses and data from 32 University and State Departments of Agriculture Veterinary Diagnostic Laboratories Information about sample collection, submission times, handling, accepted materials, and protocols Informal; not binding– consult your local lab for specific rules and regulations for sample submission

15. Trends in practice: Informal VDL Survey, February-March 2014: TF testing services offered Site of collection– bulls & cows Sample handling, post collection temperature time-to-lab incubate or not? shipping conditions Sample pooling? In-lab handling

16. Trends in practice: Informal VDL Survey, February-March 2014: 2013 TF Tests (est.)

17. Trends in practice: Informal VDL Survey, February-March 2014: n = 32

18. Trends in practice: Informal VDL Survey, February-March 2014: Incubation @ practitioners lab 15 States (24-48h at 37°C) Others dont specify Freeze sample and ship on ice 15 States (thawed samples may be rejected) Strictly forbidden for culture testing Pooling 3 States (Colorado, Utah, Oklahoma; Texas may be tentative) Publication of Effinger et al. (J Vet Diagn Invest. 2014; 26:72-87) may lead to wider adoption Sample handling trends for large test volume states

19. Sample handling trends pertinent to harmonization 1.Pre-lab incubation and freezing-- a big help to producers; saves time, increases sample integrity 2.Pooling samples-- benefits both producers (cost reduction) and labs (improves throughput) 3.Enrichment/selection medium vs. Ringers/saline 4.Rule communication (web/print/telephone, etc.)

20. Unlinked notes: 1.Most labs observe GLP on sample intake– may reject or issue disclaimer against regulatory use if sample is off-temp, late, visibly dirty, cloudy, etc. 1.All states recommend or specify preputial scraping as recommended sample site for bulls. Only 2 states have a recommendation for cow samples (aspirate mucus from fornix) 1.Some states will issue technical reccomndations by telephone yet have no written sample handling & submission rules

21. Towards harmonization... 1.Keep it simple a.enriched/selective medium b.48h if unincubated (protect at15-37 C), 120h if incubated and frozen 2. Make it scalable-- large and small volume testing labs 3. Dont paint yourself into a corner

22. Tritrichomonas foetus