Oral fluid testing:
Field applications and work in progress
J Zimmerman, J Prickett.
Iowa State University

Big Picture

Morrison et al. 2008.
Regional control of
PRRS in Minnesota

= field data
Morrison et al. 2008.
Regional control of
PRRS in Minnesota

… and production
management
= field data
Morrison et al. 2008.
Regional control of
PRRS in Minnesota

Example 1. Predict
outbreaks
Given the same virus,
increasing population size
results in …
1. Endemicity (no herd immunity)
2. Continual cases w/ periodic
outbreaks
3. Shorter inter-epidemic intervals
4. Larger outbreaks at more
regular intervals
Haggett. 2000. Geographical
Structure of Epidemics

Example 2. Predict performance
Age- and source-matched PRRSV = non-uniform size
negative controls. Highly
uniform body size

Example 2. Predict performance
450
400
PRRSV uneven
350 and 2 weeks late
300
positive
Pounds
250
negative
200
Difference
150
100
50
0
Day Day Day Day Day Day Day Day Day Day Day Day
42 56 70 84 98 112 119 133 147 161 175 189
Days post-infection

… and production
management
= field data
Morrison et al. 2008.
Regional control of
PRRS in Minnesota

Is the current infrastructure working?
• Wean-finish mortality 0.2% per year 1996-2005
• Global trend. Not linked w/ PCV2.
• Concurrent increase in health costs
Mortality Rates
Inching Upward. Nov
15, 2006

Is the current infrastructure working?
• We are big and complex …
Wayne Spencer. Network
analysis of the role of pig
movements in area spread
of PRRSV. 2007 Internat’l
PRRS Symposium
Morrison et al. 2008.
Regional control of PRRS in
Minnesota

Is the current infrastructure working?
Images from WHO/CDS/2005.28

… and production
management
= field data
= YES!!
Morrison et al. 2008.
Regional control of
PRRS in Minnesota

… and production
management
= field data
= producer driven, cheap, efficient
Morrison et al. 2008.
Regional control of
PRRS in Minnesota

… and production
management
= field data
= it is up to us
Morrison et al. 2008.
Regional control of
PRRS in Minnesota

The real story
on 2009 H1N1
…
How different would things
have been IF we had our
own spokesman and our
own data on 2009
H1N1?
– Dollar-wise?
– Public relations-wise?

Big Picture Conclusions
To control disease, optimize productivity,
and protect our industry, we need cheap,
plentiful, current data on infectious agents

Oral fluid testing:
Field applications - human

Field applications
• 1988. Rational programme for screening travelers
for antibodies to hepatitis A. Lancet 1(8600):1447.
• 1992. Comparison of saliva and serum for HIV
surveillance in developing countries. Lancet 340: 1496
• 1994. HIV surveillance by testing saliva from
injecting drug users: a national study in New
Zealand. J Epidemiol Community Health 48:55
• 1994. The use of oral fluid for hepatitis C antibody
screening. Am J Gastroenterol 89:2025.

Field applications
• 1997. Surveillance of measles in England and
Wales: implications of a national saliva testing
program. Bull World Health Organ 75:515
• 2001. Has oral fluid the potential to replace serum
for the evaluation of population immunity levels? A
study of measles, rubella, and hepatitis B in rural
Ethiopia. Bull World Health Organ 79:588.

Field applications
• 2004. A population-based seroprevalence study of
hepatitis A virus using oral fluid in England and Wales.
Am J Epidemiol 159:786. Mail-in population survey.
5,500 samples collected.
• 2007. Participant-collected, mail-delivered oral fluid
specimens can replace traditional serosurvey: a
demonstration-of-feasibility survey of hepatitis A virus-
specific antibodies in adults. Can J Public Health 98:37
• 2009. Feasibility of collecting oral fluid samples in the
home to determine seroprevalence of infections. Epidemiol
Infect 137:211. Mailed in samples received from 11,698
children.

Field applications
• 2005 - 10 NY STD clinics implement oral fluid
testing = 40% increase in number tested
• Specificity 99.8%
• Sensitivity ~99%
• March 2005 – May 2008
– 166,058 oral fluid HIV tests
– 442 (0.27%) false positive rate
2009. Ann Emerg Med 53:151
OraQuick® Advance Rapid
HIV-1/2 Antibody Test

Summary and conclusions
• Lots of research on oral fluid diagnostics in humans
beginning as early as 1909
• Focus primarily on antibody detection
• Widely used in surveillance
– Especially HIV
– Large mail-in surveys. Self-collected samples
sent via mail = lots of cheap data
• Tests need to be optimized to the specimen
– Some tests better than others

Oral fluid testing:
Field applications – wean-to-finish pigs

Summary of PCR field data
10 sites x 6 pens per site (n = 600)
1100 head wean-finish barns

Our experience in the field …
- Samples collected by site personnel
- 10 sites x 6 pens x 10 samples per site = 600 samples
- Supplies stored on-site
Rope, coolers, felt-tipped pens, address
labels, etc.
- “How to Collect” poster displayed at each site
- Just one set of samples from one barn arrived late
OF sampling in 1,100
head wean-finish
barns

Summary of field data: 10 sites x 6 pens per site

Summary of field data: 10 sites x 6 pens per site
Sites show different patterns of infection

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids

10 wean-finish sites monitored using oral fluids
Torque teno virus PCR testing [work in progress]
Among 600 oral fluid samples,
- 25 of 121 (21%) PCR-positive for TTV-1
- 97 of 121 (80%) PCR-positive for TTV-2
- 23 of 25 TTV-1 samples also positive for TTV-2

10 wean-finish sites monitored using oral fluids
FACT:
Impossible to reproduce this study or these
interactions under experimental conditions

10 wean-finish sites monitored using oral fluids
Observations …. This stuff is not in the books
- Pigs arrive at 3 weeks of age shedding PCV2, PRRSV, SIV

10 wean-finish sites monitored using oral fluids
Observations …. This stuff is not in the books
- Pigs arrive at 3 weeks of age shedding PCV2, PRRSV, SIV
- Sites are in the same system, but very different patterns
• Duration of PCV2, PRRSV, and SIV shedding highly
variable • Repeat episodes of virus circulation
(PRRSV and/or SIV)
• Prolonged PCV2 circulation post-vaccination

10 wean-finish sites monitored using oral fluids
Observations …. This stuff is not in the books
- Pigs arrive at 3 weeks of age shedding PCV2, PRRSV, SIV
- Sites are in the same system, but very different patterns
• Duration of PCV2, PRRSV, and SIV shedding highly
variable • Repeat episodes of virus circulation
(PRRSV and/or SIV)
• Prolonged PCV2 circulation post-vaccination
- Some PCV2 vaccination protocols work better than others!

10 wean-finish sites monitored using oral fluids
Observations …. This stuff is not in the books
- Pigs arrive at 3 weeks of age shedding PCV2, PRRSV, SIV
- Sites are in the same system, but very different patterns
• Duration of PCV2, PRRSV, and SIV shedding highly
variable • Repeat episodes of virus circulation
(PRRSV and/or SIV)
• Prolonged PCV2 circulation post-vaccination
- Some PCV2 vaccination protocols work better than others!
- Economic / productivity effects? [work in progress]

10 wean-finish sites monitored using oral fluids
Applications?
- Monitor negative status populations (esp boar studs, sow herds)
- Evaluate vaccine protocols and vaccine efficacy
- Monitor vaccine compliance
- Monitor controlled exposure
- Monitor antibiotic withdrawal [idea in progress]
- Predict disease losses
- Predict growth rates and uniformity
- Plan, time, and test interventions

Sample number and frequency?

Sample number and frequency?

What’s in the pipeline?

Research in progress
• Antibody test development for oral fluids
– KSU (PCV2)
– ISU (PCV2, PRRSV, SIV, M hyo)
– SDSU (PRRSV)
• PCR optimization (ISU)
• Oral fluid sampling
from sows and
boars (PIC, UM, ISU)

Research in progress
• Sample collection from boars expands the
possible applications to studs and sow
herds
– Most boars are cooperators
– Sample recovery is easy
– And more welfare-friendly than bleeding

Summary

Surveillance of commercial swine units using oral fluid samples
Quick,
easy,
cheap
to collect►
▼ PCR detection of SIV, PCV2, PRRSV. Others?
▼ PRRSV and PCV2 ELISA antibody assays.
Others in progress ..
Prickett et al., 2008. Detection of PRRSV infection in porcine oral fluid samples. J Vet Diagn Invest 20:156-163.
Prickett et al., 2008. Oral-fluid samples for surveillance of commercial growing pigs for PRRSV and PCV2 infections. JSHAP 16(2):86-91.
Hoffman P, Prickett J, et al., 2008. Implementation and validation of swine oral fluid collection in a commercial system. 38th Annual Meeting
of the AASV. San Diego, California, pp. 301-302.

ORAL FLUID
PROS CONS
• Lower cost than serum • Barn/site level testing
• Lower tech than serum (not pig level) - will
• Lower sample numbers require a paradigm shift
than serum • PCRs are too expensive
• Some tests available • Antibody tests need to
and additional tests be optimized
should be able in 2010 • Much data missing
(research in progress) • New and scary

Acknowledgements and Thanks
• John Prickett, Apisit • Pat Hoffmann, Ann and Ernie
Kittawornratt, Jeremy Johnson, Kurtz - Murphy-Brown LLC
Trevor Schwartz, Dan Western Operations
• Drs. Rodger Main, John • Drs. Michael Roof & Reid
Johnson, Sheela Philips - Boehringer Ingelhem
Ramamoorthy, Erin Strait, K-J Vetmedica, Inc.
Yoon, Wayne Chittick, Kent • Drs. Steve Sornsen & Ron
Schwartz, Chong Wang - Iowa White - Pfizer, Inc.
State University • Dr. Bob Rowland - Kansas
• Drs. Bob Morrison, Mike State University
Murtaugh - University of • USDA PRRS CAP
Minnesota
• Dr. Ramon Molina - ITSON,
Sonora Mexico
• Dr. Mark Engle - PIC