Bovine Viral Diarrhoea (BVD) Diagnostics - Goals, Strategies, and Best Tests Lab Perspectives - Dr. Dan Givens, Associate Dean of Academic Affairs, Auburn College of Veterinary Medicine, from the 2016 NIAA Annual Conference: From Farm to Table - Food System Biosecurity for Animal Agriculture, April 4-7, 2016, Kansas City, MO, USA. More presentations at http://www.trufflemedia.com/agmedia/conference/2016_niaa_farm_table_food_system_biosecurity

1. M. Daniel Givens, DVM, PhD, DACT, DACVM
BVD Diagnostics—
Goals, Strategies and
Best Tests
BVD: An Industry Prevention
Strategies & Solutions Forum

2. Do you have BVD in your herd?
Yes NoMaybe

3. Corporate
Distribution Centers
Major Food Services
Corporate
Distribution Centers
Major Retailers
Distributor/Broker
Industry Segment
Retail Outlets/ Food Services
Rural & Urban United States
End User
Rural United States
Pre-conditioners/Backgrounders8 to 14 months 500 to 900 lbs
Feedyards
Packers
Commonly shipped to Midwest
14 to 18 months 900 to 1150 lbs
Calf age Calf weight
Birth to 8 months Birth to 500 lbsCow/Calf Producers
Typical U.S. Nationwide Beef Supply Chain

4. 1. Do you have BVD in your
herd?
Yes NoMaybe
NAHMS 2007-08 Beef Cow-calf study
15.1%
24.0%
31.5%
25.2%
4.2%
0.0% 10.0% 20.0% 30.0% 40.0%
Had not heard of before
Recognized the name
Knew some basics
Knowledgable but with no testing
Knowledgable & tested in last 3 years
BVDV Knowledge of Producers from the Southeast

5. 1. Do you have BVD in your
herd?
Yes NoMaybe
NAHMS 2007-08 Beef Cow-calf study
2.6%
8.1%
31.9%
41.8%
15.6%
0.0% 5.0% 10.0% 15.0% 20.0% 25.0% 30.0% 35.0% 40.0% 45.0%
Had not heard of before
Recognized the name
Knew some basics
Knowledgable but with no testing
Knowledgable & tested in last 3 years
BVDV Knowledge of Producers with  200 cows

6. 1. Do you have BVD in your
herd?
Yes NoMaybe
NAHMS 2007-08 Beef Cow-calf study
2.6%
8.1%
31.9%
41.8%
15.6%
0.0% 5.0% 10.0% 15.0% 20.0% 25.0% 30.0% 35.0% 40.0% 45.0%
Had not heard of before
Recognizedthe name
Knew some basics
Knowledgablebut with no testing
Knowledgable& tested in last 3 years
BVDV Knowledge of Producerswith  200 cows
The intent to test for BVDV is not the
same as testing.
(205 submitted samples of 306
ordered sample collection kits = 67%)

7. Herd Prevalence of PI calves (8 studies)
Report Reference
Data
collection
year
PI Prevalence Herd Prevalence
Houe et al. {439} 1995 7/5,481 (0.13%) 3/20 (15%)
Grooms et al. {1088} 2001 5/1,952 (0.26%)d
3/13 (23%)d
O'Connor et al. {1504} 2006 12/12,030 (0.09%)c
4/102 (4%)
Wittum et al. {1494} 1996 56/18,931 (0.3%) 3/76 (4%)
Fulton et al. {1496} 2006 26/4,407 (0.6%) 5/29 (17.2%)
Bolin et al. {1495} 1985 54/3,157 (1.7%)b
6/66 (9%)b
NAHMS {1646} 2007-2008 53/44,150 (0.12%) 18/53 (8.8%)
Lawrence & McClure {1535} 2005-07 3,489/866,602 (0.4%) 2,470/11,857 (20.83%)

8. Prevalence of PI calves (26 studies)
Report Reference
Data
collection
year
PI Prevalence
Herd
Prevalence
Hoar et al. {1500} 2004 3/900 (0.3%)
Grooms & Keilen {1145} 2000 6/332 (1.8%)
Munoz-Zanzi et al. {1190} 1999 2/434 (0.5%)
Houe et al. {439} 1995 7/5,481 (0.13%) 3/20 (15%)
Mawhinney et al. {1505} 2007 3/1,769 (0.2%)
Grooms et al. {1088} 2001 5/1,952 (0.26%)d
3/13 (23%)d
Stephenson et al. {1593} 2005 24/7,544 (0.3%)
Cornish et al. {1367} 2005 59/559 (10.5%)c
O'Connor et al. {1504} 2006 12/12,030 (0.09%)c
4/102 (4%)
Wittum et al. {1494} 1996 56/18,931 (0.3%) 3/76 (4%)
Fulton et al. {1496} 2006 26/4,407 (0.6%) 5/29 (17.2%)
Paisley et al. {309} 1996 8/1,201 (0.67%)a
Bolin et al. {1495} 1985 54/3,157 (1.7%)b
6/66 (9%)b
NAHMS {1646} 2007-2008 53/44,150 (0.12%) 18/53 (8.8%)
Report Reference
Data
collection
year
PI Prevalence
Cleveland et al. {1253} 2003 5/2,921 (0.17%)
Taylor et al. {352} 1992 51/560 (9.1%)
Taylor et al. {412} 1991 1/1,029 (<0.1%)
Loneragan et al. {1497} 1998 8/4,000 (0.2%)
Fulton et al. {1476} 2004 86/21,743 (0.4%)
Larson et al. {1478} 2004 3/938 (0.32%)
Loneragan et al. {1347} 2002-03 6/2,000 (0.3%)
Loneragan et al. {1347} 2002-03 36/1,383 (2.6%)
Loneragan et al. {1347} 2002-03 39/1,585 (2.5%)
Howard, et al. {104} 1988 12/1,538 (0.78%)
Lawrence & McClure {1535} 2005-07 3,489/866,602 (0.4%)
Yan, et al. {1686} 2006-2008 111/27,932 (0.4%)

9. From presentation by J. Lawrence at 50th AAVLD meeting October 2007.

10. Do you have BVD in your herd?
Yes NoMaybe
BVD virus is considered to not be in the herd
if the herd has high pregnancy rates, few
aborted fetuses, no birth defects, excellent
calf health and lacks diagnostic evidence for
BVD virus.

11. Do you have BVD in your herd?
Yes NoMaybe
BVD virus may be in the herd if the herd has
clinical signs consistent with BVD (e.g., lower
than expected pregnancy rates, aborted
fetuses, birth defects, low birthweight calves,
or poor calf health) but no diagnostic
confirmation of BVD virus.

12. Do you have BVD in your herd?
Yes NoMaybe
BVD virus is in your herd if virus (not
antibodies) has been detected in samples
from your herd (aborted fetuses, dead calves,
or live cattle).

13. Why test?
• Increase market value of animals
• Ensure that a herd is free of PI animals
• Identifying individual PI animals spreading
disease within a herd or group

14. Surveillance to detect
How???
1
A. Question: Is BVDV circulating in the herd?
Methods to answer the question:
Lowest Cost &
Least Reliable
1.
2.
3+2.
4+2. Submit ear notches from young calves for validated pooled PCR testing.
5+2. Submit ear notches from young calves for individual testing (ELISA or IHC).
Highest Cost &
Most Reliable
6+2.
Submit blood samples for antibody detection from unvaccinated sentinel
animals that are  7 months of age and have experienced close contact with
all other animals in the herd at least one month prior to sampling.
Submit ear notches from young calves, non-calving females and bulls for
individual testing (ELISA or IHC).
Observe for clinical signs of disease. Submit samples from all aborted and
underweight calves for BVDV testing
Observe for clinical signs of disease

15. Persistently infected (PI) cattle are the source
of ongoing exposure in herds with BVD virus,
and they need to be identified and removed
prior to the breeding season.
Will you do this?
Yes. If yes is selected, you and your veterinarian know who to test, when to
test, and how to test. If not, please review info to define this question.
Help to define:
Where do PI calves come from?
Which test should I use to detect PI cattle?
Consideration of strategy of testing for PI cattle?
No. If no is selected, you are deciding to accept the cost of continued
presence of the virus in your herd.

16. When should you test?
Which cattle should you test?

17. January
April
July
October
Calving Season
Breeding SeasonBull BSE’s
May: wean calves &
pregnancy  cows
August: strategic
summer deworming
March: castrate, dehorn,
and vaccinate calves
Intense
Biosecurity
Institute testing of calves
& introduce herd additions

18. Beef Cattle - Prebreeding Test of
Calves, Bulls, Cattle without Calves
Negative
Retain in
Herd
Positive
Sell for
Slaughter
Bulls and
Cattle without
CalvesRemove Positive
Calves and Dams
from Herd
Prebreeding
Calves
Adapted from:
Kelling,et al,The Bov Pract, 34:1 (2000) 13-22

19. Remove Positive Calves and
Dams from Herd Prebreeding
Sell positive
calves for
slaughter
Test Dams
Positive Cow -
PI calf product
of PI cow
Sell for
slaughter
Only cow may
return to herd
Negative Cow -
PI calf from
acute infection
Adapted from:
Kelling,et al,The Bov Pract, 34:1 (2000) 13-22

20. Diagnostic Test Relative cost Specimen Used for Notes
Polymerase chain
reaction (PCR)
Low to high
Serum, whole
blood, tissue
Identifying PIs and
acute infections
Rapid and sensitive on individuals.
Pooling reduces sensitivity.
Polymerase chain
reaction (PCR) of skin
Low to high
Skin-usually
taken from ear
Identifying PIs
Rapid and sensitive on individuals.
Pooling reduces sensitivity.
Immunohistochemistry
(IHC) of skin
Low
Skin-usually
taken from ear
Identifying PIs
Fresh or formalin-fixed samples.
Work closely with validated lab to
provide preferred sample.
Antigen-capture ELISA
(ACE) of serum
Low Serum Identifying PIs
Rapid results. Serum testing may
be inhibited by passive immunity,
thus not recommended for young
calves.
Antigen-capture ELISA
(ACE) of skin
Low
Skin-usually
taken from ear
Identifying PIs Rapid results.
Virus Isolation
Moderate to
high
Serum, whole
blood, tissue
samples
Identifying PIs and
acute infections
Gold standard test for detecting
BVDV; however, expensive, slow
results and requires specialized
labs.
Immunoperoxidase Assay Moderate
Serum, whole
blood
Identifying PIs
Moderate time to results and
requires specialized labs.
Virus neutralization or
antibody ELISA
Low Serum
Identification of
virus exposure-NOT
for detecting PIs
Detects immune response (titer) to
BVDV.
Which test should you use to detect PI cattle?

22. Also cost appropriate
for individual samples
Not affected by
colostral antibodies

24. If I get a positive, how sure am I that the calf is PI?
If I get a negative, how sure am I that the calf is not PI?

25. When I “remove” a PI, what am I
going to do with it?

26. Follow-up surveillance is a critical aspect to
assessing and maintaining your BVD control
program. There are several options for this:
Yes If yes is selected, you and your veterinarian know how to assess the success of your BVD control program and
ensure that no PI calves are maintained in your herd during the subsequent year.
No If no is selected, you are deciding to accept a risk of maintaining PI calves in your herd during
the subsequent year.
Please select one.
Low est Cost &
Least Reliable
1.
2.
3+2.
Highest Cost &
Most Reliable
4+2.
Complete step #2 and submit appropriate samples from all young calves for validated
pooled PCR testing.
Complete step #2 and submit appropriate samples from all young calves for validated
individual testing.
Observe for clinical signs of disease (e.g., lower than expected pregnancy rates, aborted
fetuses, birth defects, low birthweight calves, or poor calf health). Submit samples from all
aborted and underweight calves for BVDV testing
Observe for clinical signs of disease (e.g., lower than expected pregnancy rates, aborted
fetuses, birth defects, low birthweight calves, or poor calf health).

27. #1. Individual animal testing for BVDV
is tremendously rewarding to animal health and
financial productivity of a farm.
Agree DisagreeConflicted

28. #2. Individual animal testing for BVDV
is tremendously rewarding to animal health and
financial productivity of a farm
when PI cattle will be detected.
Agree DisagreeConflicted

29. A sensitive and specific screening test
at the level of the pen or farm can
appropriately motivate individual
animal testing!
Walter Coles
www.ablogabouthistory.com

30. Consumption Surface Sampling to
Detect BVDV using PCR
1 2 3 4

31. Pen #1:
Ten seronegative
calves commingled
with two PI calves
Day 0 1 2 3
sample sample sample
20 min 2h 4h 6h 23.5h
Indicates that serum, whole blood, and nasal swab
samples were collected from all calves between 20
min and 2 h after feeding to assess infection status
of all animals.
Consumption surface sampling
Validation of a non-invasive,
novel testing method
Seronegatives
only
PI’s only
Day 3 4 5 6 7 8 9 10 11
Pen #2: Only two PI calves
sample sample sample sample sample
Pen #3: Ten seronegative, exposed calves
Day 3 4 5 6 7 8 9 10 11
sample sample sample sample sample

32. Results
Consumption surface swabs obtained within 6 h
of feeding from pens containing commingled or
PI calves were consistently positive for BVDV.
Time post-feeding
Sample handling
(No. positive samples/ total samples tested)
Refrigerate 1 to 3 d Refrigerate 7 to 10 d Freeze 7 to 14 d
20 min 16/16 16/16 15/16
2 h 16/16 16/16 15/16
6 h 16/16 16/16 12/16
23.5 h 11/16 12/16 11/16

33. Conclusion
Results indicate that consumption surface
swabbing within 6 h of feed consumption is a
sensitive and non-invasive method to
determine if PI animals are present within a
group of cattle

34. Surveillance to detect
How???
1
2
Biocontainment to affect
OR
Biosecurity to protect
3
Vaccination
to keep in check

35. Factors impacting the detection of viral
RNA of BVDV from consumption surfaces
• Acute vs. Persistent Infection (PI)
• Variability in viral shedding from PI cattle
• Presence of maternal antibodies in PI calves
• Dilution of viral RNA
– Over a greater consumption surface area
– With a greater number of cattle
• Characteristics of consumption surface
• Swab used for sample collection
• Ambient temperature
• Direct sunlight
• Precipitation
• Humidity
• Time between surface contact and sample collection
• Sample handling
• Time between sample collection and assay for viral RNA

36. Factors impacting the detection of viral
RNA of BVDV from consumption surfaces
• Acute vs. Persistent Infection (PI)
– PI only
• Variability in viral shedding from PI cattle
– Detects a PI (AU #18) that exhibits:
• low serum concentrations of virus (350 to 4,600 CCID50/mL)
• neutralizing antibodies to the strain with which she is
infected
• Presence of maternal antibodies in PI calves
– Detected two PI calves (at 45 and 47 days of age)
within a group of 30 calves via 1 swab of all feed &
water buckets in 30 individual calf hutches

37. Factors impacting the detection of viral
RNA of BVDV from consumption surfaces
• Dilution of viral RNA
– Over a greater consumption surface area
– With a greater number of cattle
– Detected one PI dairy heifer in a pasture containing approximately
120, 21- to 23-month-old pregnant heifers. One swab was taken of
the 240 linear feet (73 meters) of polyethylene troughs in this pasture
to detect the presence of a PI in a large group of heifers containing
only heifers that previously tested negative by individual animal ear
notch ELISA. After a positive consumption surface swab, follow-up
testing using virus isolation from white blood cells identified the PI
heifer that produced a positive test result upon retest using individual
animal ear notch ELISA
• Characteristics of consumption surface
– Prior testing has focused on polyethylene, rubber, and concrete feed
trough and water trough surfaces. Swabs have been developed to
withstand swabbing of concrete troughs.

38. Factors impacting the detection of viral
RNA of BVDV from consumption surfaces
• Swab used for sample collection
– Optimized for durability and diagnostic sensitivity
• Ambient temperature
• Direct sunlight
– Repeated positive results achieved when swabbing PI feed troughs at
6 hours post contact at:
• -3 °C (27 °F) which were protected from exposure to sunlight
• 34 °C (93.2 °F) which were provided exposure to direct sunlight. Under these
conditions, the temperature of the black polyethylene surface of the feed
trough exceeded 50 °C (120 °F).
• Our efforts have thus far been unable to identify a minimal or maximal
ambient temperature that would prevent detection of viral RNA of BVDV from
PI cattle.
• Precipitation
– Repeated positive results achieved when swabbing PI feed troughs
during light to moderate rainfall.
• Humidity
– Repeated positive results at 44 to 92% humidity.

39. Factors impacting the detection of viral
RNA of BVDV from consumption surfaces
• Time between surface contact and sample
collection
– Research results indicate that sample collection within
6 hours of trough contact will provide repeatable and
diagnostically sensitive results.
• Sample handling
– Research results validate refrigerated storage of
diagnostic samples.
• Time between sample collection and assay for
viral RNA
– Research results validate refrigerated storage of
diagnostic samples up to 10 days after collection.