Sample Handling and Transport in Bovine Trichomoniasis SurveillanceAnthony Smith, Ph.D.

BioMed Diagnostics, Inc. ● White City, Oregon 97503 USA

www.biomeddiagnostics.com

2014 NIAA/USAHA JOINT FORUM ON TRICHOMONIASIS STANDARDS

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’

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

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

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

What’s 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; 23:337-41Mukhufhi et al. Theriogenology 2003; 60:1269-78Hammond and Bartlett. Am J of Vet Res 1943; 4:143-49 Madden. Univ. Wyoming Vet Sciences M.S. Thesis. 2007 (via ProQuest)

What’s 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)

The most important component?

~4 mL collection media

~0.5-1.0 mL preputial smegma

The most important component is T. foetus

~4 mL collection media

~0.5-1.0 mL preputial smegma

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

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)

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

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

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

Trends in practice:Informal VDL Survey, February-March 2014:

2013 T

F T

ests

(est.

)

Trends in practice:Informal VDL Survey, February-March 2014:

n = 32

Trends in practice:Informal VDL Survey, February-March 2014:

Incubation @ practitioners lab

15 States(24-48h at 37°C)

Others don’t 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

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. Ringer’s/saline

4. Rule communication (web/print/telephone, etc.)

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.

2. 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)

3. Some states will issue technical reccomndations by telephone

yet have no written sample handling & submission rules

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. Don’t ‘paint yourself into a corner’

Tritrichomonas foetus