BVD Diagnostics at
SDSU ADRDL: Demonstrating Persistently Infected Cattle via Antigen Detection
Russ Daly, DVM,
Extension Veterinarian
March 2007
Bovine
viral diarrhea (BVD) continues to play a significant role in the health status
of our region’s beef and dairy herds.
Diagnostic tests for BVD continue to evolve, presenting practitioners
with unprecedented tools and decisions to make regarding BVD testing in their
clients’ herds. The following is an
overview of BVD diagnostics relative to detection of persistently infected
animals (PI’s) at SDSU. Practitioners
are encouraged to consult the ADRDL
Users Guide for more information.
BVD
antigen can be detected in various ways from several sample types. Demonstration of antigen in samples is an
important step in detecting persistently infected (PI) calves. Differentiation between PI’s and transiently
infected calves is dependent on the sample, the test, and sampling time frame;
differentiation between Type I and II subtypes is only possible with PCR.
BVD Antigen Capture ELISA (Ear Notch
ELISA)
Pooled PCR Ear-Notch Screening Test for BVD
<<NEW>> BVD Serum ACE
(Antigen-capture ELISA)
BVD Outgrowth (or Microplate
VI) ELISA test
Conventional Virus Isolation from serum or whole
blood
PCR (Polymerase Chain Reaction)
What samples to submit? What tests to run? Suggestions…
BVD Antigen Capture
ELISA (Ear Notch ELISA).
This
is a recent addition to the BVD testing stable at SDSU. The test involves agitating a fresh ear notch
in saline to elute antigen from the hair follicles. The fluid is then used in a
microwell plate as for other ELISA tests, which
allows for automation in processing and interpretation.
Use: Herd screening
for BVD PI’s. This method will also
detect transiently infected animals, so a follow-up sample 4-6 weeks later
should be taken to confirm PI status. We
currently suggest that a follow-up sample consist of whole blood for VI or PCR,
not an ear notch (see discussion below).
Sample
needed: Ear notch, 1 cm x 1 cm from animal
How
to ship: Put notch in individual snap cap tube and
ship on ice. If sample will not get to
the lab the next day, freeze the sample and ship on ice to prevent thawing. Samples may be kept in freezer (without a
defrost cycle) up to one month and shipped together.
Setup
and reporting: Test results are available on the same day
the test is set up. To keep costs down,
tests are batched together and run at least every 2-3 days.
Sensitivity = 100 %, Specificity
= 98.4% (for detecting PI’s when compared to gold standard of buffy coat VI and PCR)1
Cost = $4.00
Pooled PCR
Ear-Notch Screening Test for BVD
SDSU’s Animal Disease Research and Diagnostic
Laboratory is now offering a new screening procedure
for BVD, using ear notches from calves.
Submitted ear notches will be processed much the same as for
antigen-capture ELISA testing, then the fluid from each sample will be pooled
for PCR testing.
Samples
may be pooled in groups of up to 50 samples.
Large submissions will be divided into groups of 50 plus a smaller pool,
if a remainder exists. The cost for each
pool is $60. The samples will be run
once a week (individual antigen-capture ear-notch ELISA tests are run most days
of the work week).
If
a pool tests positive, individual samples may be tested with antigen capture
ELISA to identify potential persistently infected individuals, at the regular
cost of $4 per sample. This testing will
not be done automatically but only upon request by the client; requests for
individual testing should be made within 72 hours of notification of a positive
pool.
Submission Guidelines. Fresh ear notches should be submitted in a
manner similar to that for antigen-capture ELISA:
Pooling
samples in groups of 50 substantially reduces the per-animal diagnostic cost
compared to currently available individual tests, yet maintains a reasonable
number of samples to test individually in the event a pool is positive.
It
is necessary to understand that pooled diagnostic procedures such as this
should be considered screening tests only.
In cases in which individual animals are represented as BVD PI-test
negative (e.g. purebred animals for sale), individual diagnostics utilizing the
ear notch antigen-capture ELISA, ear notch IHC, or serum ELISA should be
performed.
Pooling
procedures by their nature can result in decreased sensitivity over individual
tests. Also, PCR procedures, due to
their enhanced sensitivity, may detect transiently infected animals more often
than other tests (such as the ear-notch antigen capture ELISA). Therefore, it is possible to experience
scenarios in which a pool is PCR-positive but the individual samples are
negative on the follow-up individual ear-notch tests.
SDSU’s ADRDL is committed to providing our
practitioners with the latest and most useful tools for dealing with disease
issues in their clients’ herds. The
expertise and experience of our PCR personnel and our rigorous quality system
allow us to make this test available now.
<<NEW>>
BVD Serum ACE (Antigen-capture ELISA) (Detects BVD virus
in serum)
This
is the latest test to come online at the ADRDL.
Essentially, a serum sample (instead of an ear notch) is used in the
same antigen-capture ELISA test as is used for the ear notches. This test would be used in much the same
manner as the BVD outgrowth ELISA (see below): when serum samples are obtained
for additional testing beyond BVD-PI.
Maternal
antibodies may interfere with testing, so this test is useful only on older
calves (over 4 to 6 months old), or pre-colostral
serum samples. As with the outgrowth
ELISA, it’s possible that the test may pick up animals that have been
vaccinated within the past 3 weeks with a modified live BVD vaccine.
Sample
needed:
Serum, separated and removed from the clot.
Setup
and reporting: Test results are available on the same day
the test is set up. Tests are batched
together and run at least every 2-3 days.
Cost = $4.00
This
test involves histologic examination of a
formalin-fixed ear notch after processing and treatment with a BVD-specific immunohistochemical stain.
Use: Herd screening for BVD PI’s. This method may also detect transiently
infected animals, so a follow-up sample 4-6 weeks later should be taken to
confirm PI status. Instances have been
noted in which PI calves have become weak positives on follow-ups 3 weeks
later; accordingly, submit blood for PCR or VI for the follow-up sample
Sample
needed: Ear notch in buffered formalin.
How
to ship: Ear notch in formalin in individual
tubes. Specimens stored in formalin for
over 5 days may result in false negatives.
So when dealing with large herds, it is necessary to send samples every
day or two days to ensure proper interpretation.
Setup
and Reporting: Samples are set up all days of the week. Significant time is necessary for tissue
processing and interpretation by a pathologist. Test results are available in
10-14 days.
Sensitivity = 100 %,
Specificity = 98.8% (for detecting PI’s when compared to gold standard of buffy coat VI and PCR)1
Cost = $ 4.00 (as of
7/1/05)
BVD
Outgrowth (or Microplate VI) ELISA test
The
outgrowth ELISA test
differs from the “ear-notch” antigen-capture test. This test uses bovine cell lines in plates
similar but a little larger than serology microwell
plates. The cell lines enhance the
growth of the virus (virus isolation), which is then detected by immunologic
staining.
Use: herd screening
for PI animals. Maternal antibodies may
interfere with testing, so this test is useful only on older calves (over 6
months). It is faster than regular virus
isolation on serum or buffy coat. This procedure is often done to screen older
animals when serum is submitted simultaneously for serology, etc.
·
Acute
infections are only very rarely detected with this method.
·
This
test may pick up animals that have been vaccinated within the past 3 weeks with
a modified live BVD vaccine.
Sample
needed: Serum.
Take care to completely spin off serum from red blood cells. Do not ship on the clot. Hemolysis of the
sample may result in toxicity to the cell lines and interference with test
results.
How
to submit: Chilled on ice packs. Freeze the samples if they need to be held
longer than two days.
Setup
and Reporting: Test is set up once a week on Fridays. Samples must be received before 10 AM on
Friday. Results are read the following
Wednesday.
Specificity: 100%, Sensitivity
= 85.5% when compared to conventional virus isolation
Cost
=
$6.00 (As of 7/1/05)
Conventional Virus
Isolation from serum or whole blood
This
test relies on the growth of virus in specific cell lines and the observation
of the effect of the virus on the cells.
Use: Detects viremia in individual animals. Since PI calves by definition are viremic for an extended time, VI may be used to confirm the
PI status of animals with positive ear notches when used on whole blood buffy coats (calves).
PI calves may also be detected with pre-colostral
serum samples. Transiently infected
calves may also be detected if a single sample is submitted during the viremic phase of disease.
Sample
needed:
Serum or whole blood (purple top EDTA tube, 5 ml miniumum). Serum is
preferred for detection of PI’s, as the test is somewhat more expedient. Do not use Venoject
or PST tubes, as certain additives in those tubes may result in toxicity to the
cell lines and interfere with test results.
How
to submit:
chilled on ice packs.
Setup
and reporting: Samples are set up every day. Cultures are held two weeks before reported
as negative; positive results are occasionally available sooner.
Cost = $17.50 (2
passages: serum, buffy coat, tissues)
PCR (Polymerase
Chain Reaction)
The
current PCR test being performed at the ADRDL is a real-time PCR for screening
of BVD in serum, buffy coats, bulk tank milk, semen,
and tissues. Samples that are BVD
positive by the screening test can then be typed as to type I or II. In addition, sequencing can also be performed
if requested.
This
is an extremely sensitive test that can detect very low numbers of viral
particles. The reported sensitivity is
10 TCID 50/ml of BVD (equivalent to 1 ng/ml
BVD RNA)2
Because of this sensitivity, practitioners may utilize pooling for
diagnostic efficiency.
Sample
needed: Serum, whole blood, bulk tank milk samples,
or semen. There are advantages and disadvantages of using different samples for
PCR analysis.
Setup
and reporting: The test is performed once weekly. If STAT results are needed, these can also be
performed by contacting the Molecular Diagnostics Section at the ADRDL, or Dr.
Jane Hennings.
Cost= $25.00. The cost
per actual sample submitted may be as low
as $1.25 per sample,
depending on the # of samples that are pooled (eg.
10-20 samples pooled is equivalent to only $1.25-2.50 per sample), so this is a very economical
test to run if samples are pooled.
Editors Note: What
samples to submit? What tests to run?
Suggestions…
A. Herd or group screening of individuals: beef and dairy
calves.
®
Ear
notches, submitted fresh, chilled, or frozen for Antigen-capture ELISA (Ear notch ELISA).
Antigen-capture ELISA holds several
benefits relative to IHC: Faster
turnaround time, less subjectivity in reading of results, no formalin
necessary, more flexibility with submitting samples (no concern that samples
may sit in formalin too long). At SDSU,
there has been 100% correlation between ear notch ELISA and IHC results.
®
Or…ear
notches, submitted fresh, chilled, or frozen for Pooled PCR Ear Notch Screening.
This procedure reduces the per-animal cost
of detecting PI animals. It is important
to realize that this is a screening procedure on the group and should not be
used to represent individuals as BVD PI-free.
B. Herd or group screening of individuals: older (> 6 mo.) cattle.
®
Serum,
removed from clot, chilled or frozen for (The
new) BVD Serum ACE (Antigen-capture ELISA) or BVD Outgrowth VI ELISA.
Serum samples may be screened effectively
for PI’s on older animals by use of the new serum antigen-capture ELISA or the
outgrowth VI ELISA. These procedures may
be useful in cases where serum is being submitted for other tests, e.g. Johnes, Anaplasmosis, etc, and is
commonly used to screen ET recipients, for example. Maternal antibodies may
produce false negatives, and recent MLV BVD vaccine use may produce false
positives.
®
Or…serum
or whole blood samples that will be pooled for PCR.
Pooled groups are tested and then
individuals identified by testing individual samples within the pool. Very economical, unless a
high prevalence of pools with PI’s are present.
®
Ear
notches for antigen-capture ELISA (see above)
C.
Screening for
presence of PI’s within a population: dairy
®
Bulk
tank (or string) milk samples (50 ml, chilled, not frozen) for PCR.
®
Or…serum
or whole blood samples that will be pooled for PCR. (see above).
D.
Ear-notch positive
animals: differentiating PI’s from transiently infected calves
®
Whole
blood for Virus Isolation
(conventional)
®
Or…whole
blood for PCR.
®
Or…serum,
removed from clot, chilled or frozen for (The
new) BVD Serum ACE (Antigen-capture ELISA) or BVD Outgrowth VI ELISA. (especially animals over 6 months of
age)
A blood sample should be taken 4-6 weeks
after the initial ear notch was taken. A
second ear notch is not recommended since some transiently infected calves have been ear notch positive for two
consecutive months (Ag-capture ELISA) and three consecutive months (IHC). In fact, one transiently infected calf in the
REFERENCES:
1Cornish TE, van Olphen AL, Cavender JL, Edwards
JM, Jaeger PT, Vieyra LL, Woodard LF, Miller DR,
O'Toole D. 2005.
Comparison of ear notch immunohistochemistry, ear notch antigen-capture ELISA, and buffy coat virus isolation for detection of calves
persistently infected with bovine viral diarrhea virus. J Vet Diagn Invest. 17:110-7.
2Mahlum C., S. Haugerud, J. Shivers, K. Rossow,
S. Goyal, J. Collins, K. Faaberg.
2002. Detection of bovine viral diarrhea
virus by Taqman reverse transcription polymerase
chain reaction. J. Vet. Diagn.
Invest. 14:120-125.
Abstract: Comparison of ear notch immunohistochemistry,
ear notch antigen-capture ELISA, and buffy coat virus
isolation for detection of calves persistently infected with bovine viral
diarrhea virus
Cornish TE, van Olphen
AL, Cavender JL, Edwards JM, Jaeger PT, Vieyra LL, Woodard LF, Miller DR, O'Toole D.
Wyoming
State Veterinary Laboratory, Department of Veterinary Sciences, University of
Wyoming, Laramie, WY 82070, USA.
Two techniques performed on skin biopsy samples (ear notches), immunohistochemistry (IHC) and antigen-capture ELISA (Ear
notch ELISA, or “AgELISA”), were compared for
detection of bovine viral diarrhea virus (BVDV) persistent infection (PI) in
559 Angus calves between the ages of 1 and 5 months. The calves also were
tested for BVDV infection using virus isolation (VI) and reverse transcription
(RT)-PCR on buffy coat samples and for antibodies to
BVDV types la and 2 by serum neutralization (SN).
Sixty-seven of 559 (12.0%) calves tested positive at initial screening by IHC, AgELISA, or VI, and all 67 were kept for a minimum of 3
months and retested monthly by IHC, AgELISA, VI,
RT-PCR, and SN. Of the calves positive at initial screening, 59/67 (88.1%) were
determined PI and 8/67 (11.9%) were determined acutely infected. Both IHC and AgELISA detected 100% of PI calves; however, IHC and AgELISA also detected 6 and 8 acutely infected calves,
respectively, at initial screening. Furthermore, IHC and AgELISA
continued to detect 3 and 4 acutely infected calves, respectively, 3 months
after initial screening. Three acutely infected calves had IHC staining
indistinguishable from PI calves at initial screening. Both IHC and AgELISA are accurate at detecting BVDV-infected calves, but
veterinarians and producers should be advised that both tests detect some
calves acutely infected with BVDV in addition to PI animals. Repeat testing
using VI or RT-PCR on buffy coat samples should be
performed at 30 days after initial screening to conclusively discriminate
between acute and PI.
Editor’s
Note: This study reaffirms the value of Ear notch
ELISA and IHC for detection of BVD PI’s, as 100% of PI calves were detected
with both methods. However, both tests
also detected some transiently infected animals at time frames much longer than
many people had been accustomed to seeing.
Therefore it is currently recommended to reconfirm positive ear notch
samples with VI or PCR on blood 30 or more days later. True persistently infected animals will be VI
or PCR positive that long afterwards.
The
investigators also performed serum neutralization tests for BVD type Ia and II on all calves. Interestingly, almost all of the transiently
infected calves had extremely high titers to BVD type II (> 1:8192),
while the PI calves exhibited very low or negative titers. This is based on only 8 transiently infected
calves, but could signal an area of further research in differentiating PI from
transiently infected animals.
Practitioners
should base their recommendations on the management of these ear-notch positive
calves according to each producer’s unique situation. Unless large numbers of animals or valuable
animals are involved, immediate culling may be opted for in light of an initial
ear-notch positive case. In those cases
where valuable animals or several animals are involved, proper segregation and
isolation of positive calves should be maintained in the interval between ear
notch and confirmatory tests. When
several PI animals are present in a herd, it is not hard to imagine scenarios
in which some of those calves are in fact transiently infected due to exposure
to their PI herdmates and will be negative on the
confirmatory test.
We
now have at our disposal unprecedented knowledge and tools for aiding our
producers in dealing with and eliminating persistently infected BVD animals
from their herds. Our understanding of
these tools continues to evolve as new procedures and techniques come to
light. SDSU’s
ADRDL is committed to developing and communicating this knowledge to our
region’s veterinarians and producers.