Veterinary Microbiology, 10 (1985) 359--369

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

359

AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE

DETECTION OF ANTIBODIES TO BOVINE VIRAL DIARRHOEA VIRUS
(BVDV) IN CATTLE SERA

C.J. H O W A R D ,

M.C. C L A R K E

and J. B R O W N L I E

A F R C Institute for Research on Animal Diseases, Compton, Newbury, Berkshire, RG16

O N N (Gt. Britain)
(Accepted 20 December 1984)

ABSTRACT
Howard, C.J., Clarke, M.C. and Brownlie, J., 1985. An enzyme-linked immunosorbent
assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV)
in cattle sera. Vet. Microbiol., 10: 359--369.
A microtitre ELISA has been established for the quantitation of antibodies to bovine
viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody
were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of
IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization
test for detection o f antibody; comparison of an ELISA that detected IgG1 plus IgG2
antibody to BVDV with the virus neutralization test gave a correlation coefficient
(r) of 0.89 (P < 0.001 for 95 compared sera).
A l t h o u g h similar amounts of IgG1 and IgG2 antibodies were present in sera from
both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and
in the sera from young calves was predominantly IgG1. The number of adult cows with
antibody was 40 o u t o f 41 while 36 of 44 calves reared in a beef unit were found to have
produced antibody by the time they were 31.5 weeks old, an indication of the high
prevalence of BVDV in the cattle population.

INTRODUCTION
Serological evidence indicates that infection with bovine viral diarrhoea
v i r u s ( B V D V ) is v e r y c o m m o n in c a t t l e . E s t i m a t e s o f i m m u n e c a t t l e in
national herds have been reported to vary from 8 to 100% (Phillip and
D a r b y s h i r e , 1 9 7 1 ; L e i s s e t al., 1 9 7 4 ) , w h i l e S t o t t e t al. ( 1 9 8 0 ) r e p o r t e d
that about 50% of the beef calves they examined had developed serum antibody by the time they were 8 months old.
Infection of the foetus with BVDV can lead to abortion, foetal resorption
o r t e r a t o g e n i c d e f e c t s ( V a n O i r s h o t , 1 9 8 3 ) . I t c a n a l s o r e s u l t in t h e b i r t h o f
calves with a persistent viraemia, and a defective immunological capacity to
respond to BVDV; these animals produce little or no antibody detectable

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1985 Elsevier Science Publishers B.V.

360
in the neutralization test and are the ones that are at risk from subsequently
developing clinical mucosal disease (Malmquist, 1968; Leiss et al., 1974;
Steck et al., 1980; Roeder and Drew, 1984). BVDV infections o f normal
cattle are usually acute, transient and sub-clinical (Kahrs, 1971). Although
typically associated with enteric infections, BVDV has also been associated
with outbreaks of respiratory disease in calves (Phillip and Darbyshire,
1971; Stott et al., 1980).
An ELISA would have several advantages over the neutralization test
for detecting antibody. These include: e c o n o m y of labour; more rapid
availability of results; and no requirement for cells and cell culture during
the assay. However, earlier studies with BVDV (Fernelius, 1966) indicated
that most virus neutralizing activity was in the IgM fraction from bovine
sera and there was an unusually persistent IgM antibody response following
infection. Furthermore, a restricted isotype response has been reported
for certain other infections of cattle (Musoke et al., 1981). Initially, therefore, an end-point titration m e t h o d was used to examine the predominant
isotypes of BVDV antibodies. Subsequent studies compared the end-point
titration method, as the basic quantitative assay (De Savigny and Voller,
1980), with the more practical single dilution m e t h o d utilising a standard
curve. Further comparisons were made with the virus neutralization test
for detecting antibody in various bovine sera and colostrum.
MATERIALS AND METHODS

Preparation of antigen
Monolayers o f calf testis cells were prepared in 40-oz round bottles. The
cultures were inoculated with 107"s TCDs0 of BVDV (NADL strain) and
rolled for 1 h; 20 ml of cell culture medium was then added. The medium
consisted of Eagle's basal medium (Gibco Ltd.) with the addition of 2% heated (56C, 30 min) foetal bovine serum. After incubation for 18--24 h at
36 C, by which time a marked cytopathic effect was evident, the medium
was removed, and the cells suspended in 20 ml phosphate buffered saline
(Dulbecco A, PBSa) by using a silicone rubber policeman. The cells were
sedimented by centrifugation at 20C for 15 min at 500 g and 2 ml of
1% Nonidet P40 (BDH Chemicals Ltd.) added to the pellet. After mixing,
the cell suspension was incubated for 60 rain at 37C and aliquots stored
at -70C. As required, samples were thawed, centrifuged at 20C for 15
min at 500 g and the clear supernatant removed and stored at 4C for use
as antigen. Calf testis cells t h a t had not been infected with BVDV were extracted in the same way to provide control antigen.

Enzyme-linked immunosorbent assay (ELISA) for BVDV

The m e t h o d of Voller and BidweU (1976) was modified as follows: the
Nonidet-extract of BVDV infected cells contained about 20 mg protein

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ml-: by the Biorad assay with ovalbumin as a standard; the extract was diluted {usually 1/50--1/100) in distilled water and extract of non-infected cells
was diluted to give a similar protein concentration, i.e., 200--400 pg m1-1.
The o p t i m u m concentration of antigen was determined in the usual manner.
For the assay, alternate rows of wells in the same microtitre plate (flexible
polyvinyl, Falcon 3912, Becton Dickinson, Gt. Britain) were sensitised with
50 pl of diluted extract of either BVDV-infected or non-infected cells.
Plates were then incubated overnight at 37C to dry the extract. All subsequent incubations were at room temperature, about 20C; all plates were
washed 5 times with PBSa containing 0.05% Tween 20; all dilutions of test
sera, rabbit anti-bovine immunoglobulins (Ig), and enzyme-coupled goat
anti-rabbit IgG were in PBSa containing 0.05% Tween 20 and 5% normal
swine serum that was free from antibody to BVDV by the virus neutralization test.
Reagents were obtained from Miles Laboratories Ltd. (rabbit anti-bovine
IgG1, IgG2, IgA and horse-radish peroxidase (HRP) coupled goat antirabbit IgG) or Nordic Immunological Laboratories Ltd. (rabbit anti-bovine
IgM). Optimum dilutions for each reagent were determined. These were:
anti-IgG1 1/15 000; anti-IgG2 and -IgM 1/10 000; anti-IgA and HRP antirabbit Ig 1/5 000. The substrate (100-pl volumes) was 40 mg O-phenylenediamine (OPD) per 100 ml of citrate-phosphate buffer pH 5 (0.025 M
citric acid, 0.05 M Na2HPO4) containing 50 pl of 30% H202. The optical
density (OD) was read at 492 nm, after the reaction had been stopped with
25 ~1 2M H2SO4, against substrate blank on a Titerek Multiskan (Flow Laboratories).
The end-point titration assay for antibody was as follows: 200 gl of
PBSa containing 0.05% Tween and 5% swine serum (diluent) was added to all
wells of a sensitised plate; plates were incubated for 30 min and washed,
test and control antisera were diluted in 50-pl volumes in appropriate rows
of wells sensitised with BVDV or control antigen; plates were incubated
for 90 min and washed; 50 ~1 of appropriate rabbit anti-bovine Ig sera was
added per well; plates were incubated for 90 min and washed; 50 pl HRP
coupled goat anti-rabbit serum was added per well; plates were incubated
for 90 min and washed; 100 pl substrate was added per well; plates were
incubated for 20--30 min, the reaction was stopped with 25 pl 2 M H2SO4
per well and the OD read at 492 nm.
A positive control serum was included in all assays for each isotype. The
titre of this antiserum was obtained by comparison with known negative
sera, which were taken as having titres of ~ 50 (De Savigny and Voller,
1980). The number of units of antibody in this standard serum was taken
as being equal to the titre.
To determine the antibody levels in test sera, the corrected OD value was
calculated for each dilution from the following formula: (OD in wells with
BVDV antigen) -- (OD in wells with control antigen), these values were
plotted against serum dilutions and the end-point defined as the reciprocal

362
of the dilution producing the same OD value as the dilution of the standard
serum that contained 1 unit of antibody.
For the single dilution assay the same m e t h o d was used with only rabbit
anti-bovine IgG1 and IgG2 sera. Test sera were diluted 1/100 and added to
duplicate wells sensitised with BVDV or control antigen. The corrected OD
(average OD in wells with BVDV antigen -- average OD in wells with control
antigen) was calculated and the units of antibody read off the standard
curve.

Neutralization assay for antibody to BVD V

Calf kidney cells were used in a microtitre assay to measure neutralizing
antibody to BVDV (Frey and Leiss, 1971). Titres (50% end-point) were
recorded as the reciprocal of the highest dilution, obtained after addition
of virus, that inhibited cytopathogenicity due to 102 TCDs0 of strain NADL.

Bovine sera
Groups of sera from several lots of animals were examined. The first
group was from 10 calves, 5 o f which had been infected with a cytopathic
strain of BVDV, and 5 with a n o n ~ y t o p a t h i c strain. Sera were taken before
intranasal infection and 4.5 weeks after. The calves and the experimental
infection have been described previously (NuttaU et al., 1980).
The second group of sera was from 41 cows from the institute's herds
representing older animals t h a t were likely to have been exposed to BVDV
during their life. Colostrum was taken from 13 of these cows and tested.
The third group was from 21 calves aged 1--9 days that had been fed
colostrum; 13 were fed colostrum from the cows in Group 2 above.
The fourth group of sera was from 44 calves in a group on a beef-rearing
unit that collected calves from farms in southern England (Stott et al.,
1980). Calves were aged 32--52 days when first bled (time zero); they were
bled again 6, 9, 15 and 21 weeks later.
Fifty sera were taken from a group o f cattle in which some animals showed clinical mucosal disease, and 23 o f which were shown to have a BVDV
viraemia (Brownlie et al., 1984).
The standard serum used in the assay was prepared in a gnotobiotic calf
by intranasal infection with live virus (strain IRAD C2415) followed by a
subcutaneous injection of live virus (strain NADL) in Freund's complete
adjuvant (Difco Laboratories).
To examine the specificity o f the ELISA, antisera that had been prepared
by hyperimmunisation o f gnotobiotic calves with respiratory syncytial virus,
parainfluenza virus type 3, Mycoplasma dispar and Ureaplasma diversum,
and convalescent antisera from gnotobiotic calves infected with Mycoplasma
bovis or infectious bovine rhinotracheitis virus were tested.

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Fractionation o f bovine sera

T h r e e b o v i n e sera w e r e e x a m i n e d t o d e t e r m i n e w h e t h e r virus n e u t r a l i z i n g
a c t i v i t y was a s s o c i a t e d w i t h IgM, IgG1 o r I g G 2 a n t i b o d y . O n e o f t h e s e sera
was f r o m t h e g n o t o b i o t i c calf i m m u n i s e d w i t h B V D V ( S a m p l e A, T a b l e I),
t h e o t h e r 2 were a d u l t c o w sera. T h e IgM, IgG1 a n d I g G 2 f r a c t i o n s were
p r e p a r e d b y c h r o m a t o g r a p h y a n d t e s t e d b y d o u b l e d i f f u s i o n ( F e y et al.,
1976).
TABLE I
Antibody to BVDV in purified Ig fractions
Sample a

Titre by ELISA with antibody to:

Titre by
neutralization

IgG1

IgG2

IgM

A. Serum
IgG1
IgG2
IgM

5000
2400
< 10
< 10

200
350
<10
<10

<
<
<
<

50
10
10
10

3072
1024
< 3
< 3

B. Serum
IgG1
IgG2
IgM

4000
3000
< 10
20

400
30
350
10

50
< 10
< 10
10

2048
1024
512
< 3

C. Serum
IgG1
IgG2
IgM

6000
3000
< 10
20

800
10
350
< 10

< 50
10
< 10
< 10

8192
768
32
8

aSerum and immunoglobulin fractions.

RESULTS

O p t i m u m conditions for the test

P r e l i m i n a r y c h e c k e r b o a r d t i t r a t i o n s established t h e o p t i m u m c o n c e n t r a t i o n o f antigen, m e t h o d o f sensitising a n d c h o i c e o f m i c r o t i t r e plates. Addit i o n o f antigen in PBSa o r c a r b o n a t e b u f f e r p H 9.6, a n d o v e r n i g h t incubat i o n w i t h o u t d r y i n g were n o t successful in a d s o r b i n g B V D V a n t i g e n s o n t o
plates. Rigid p o l y s t y r e n e plates w e r e n o t c o m p a t i b l e w i t h t h e m e t h o d s
e m p l o y e d here, w h e n antigens w e r e a d d e d in either o f t h e 2 b u f f e r s described o r w h e n t h e antigens dried on. O p t i m u m c o n c e n t r a t i o n s o f anti-Ig
w e r e d e t e r m i n e d in t h e usual m a n n e r . N o n i d e t e x t r a c t e d antigens c o u l d b e
s t o r e d at b o t h - 7 0 C a n d 4C f o r at least 6 m o n t h s ; t h e l o n g e s t t i m e s exa m i n e d . Sensitised dried plates c o u l d also b e s t o r e d sealed w i t h S e l l o t a p e
f o r 6 m o n t h s at 4 C.

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Comparison o f the single dilution assay and the end-point titration method
In order to determine whether values obtained by the single dilution assay
were quantitative, a comparison was made between specific IgG1 and IgG2
antibody titres, determined by a single 1/100 dilution of u n k n o w n sera
with units of antibody read from a standard curve, or by the end-point
titration method. Twenty-two sera were examined. These included 10 taken
from calves 4.5 weeks after experimental infection with BVDV (Nuttall et
al., 1980), 10 sera from normal adult cows, and 2 sera from cattle with
BVDV viraemia.
For IgG1 antibody, comparison of end-point titres and number of units
of antibody gave a correlation coefficient (r) = 0.882 (P < 0.001): for IgG2
antibody r = 0.949 (P < 0.001). Thus, it was established that the single dilution m e t h o d was a satisfactory quantitative system in the assay as performed here, and it was used subsequently.

Comparison o f ELISA with virus neutralization tests

Ninety-five samples were examined for antibody to BVDV by the ELISA
and the virus neutralization test. These samples included the 10 pairs of sera
taken from calves before and 4.5 weeks after intranasal infection with
BVDV, sera from 41 adult cows, colostrum from 13 cows and sera from 21
calves taken after the feeding of colostrum. The ELISA was performed
using anti-bovine IgG1, or IgG2, or IgG1 + IgG2.
4
A
tO
v
-O

.;.

O
.El

C'4

(.9
03

!~/s

": :

Neutralising antibody titre (10 n)

Fig. 1. A comparison of ELISA and the neutralization assay for measuring antibody to
BVDV. T h e m e t h o d assayed IgG1 and IgG2 antibodies together. Ninety-five samples
were compared.

365
The correlation coefficient (r) for units of IgG1 antibody and neutralization titres was 0.86 (P < 0.001): for IgG2 antibody r = 0.007 (P > 0.1);
for IgG1 + IgG2 a n t i b o d y r = 0.89 (P < 0.001) (Fig. 1).
In a further study, the IgM, IgG1 and IgG2 fractions were prepared from
3 sera, and the virus neutralization titres and specific IgM, IgG1 and IgG2
a n t i b o d y titres of those fractions were determined by the end-point titration
m e t h o d (Table I). Both IgG1 and IgG2 fractions were detected by ELISA
and neutralization, but no BVDV antibody was convincingly demonstrated
in the IgM fractions.

Specificity o f ELISA
The hyperimmune or convalescent sera prepared in gnotobiotic calves
against parainfluenza type 3 virus, respiratory syncytial virus, infectious
bovine rhinotracheitis virus, Mycoplasma bovis, M. dispar and Ureaplasma
diversum, all contained < 50 units o f IgG1 or IgG2 antibody to BVDV per
ml. Thus, the assay appeared specific.

Comparison o f isotype response in cattle by end-point titration

To establish the predominant isotypes of bovine antibody to BVDV the
specific IgG1, IgG2, IgM and IgA antibody titres were measured in a group
of 9 sera from adult cows from the Compton herd and in 10 pairs of sera
taken from calves before and after infection with BVDV.
In all the calf sera, pre-inoculation titres of IgG1, IgG2, IgM and IgA
antibodies were ~< 25. All 10 calves had IgG1 antibodies detectable in postinoculation sera. The titres ranged from 100 to 2700 (geometric mean titre
-+ standard deviation [SD] = 102.70 ~ 0.s29) six of the 10 calves produced an
IgG2 antibody response. The titres ranged from 50 to 2400 (geometric mean
titre + SD = 101"93 0.646). Only 2 animals had detectable IgM antibodies,
the titres being 50, pre-inoculation titres were < 25 in these calves. None
of the calves had detectable IgA antibodies.
All 9 sera from adult cows contained IgG1 and IgG2 antibodies. No IgM
or IgA antibodies were detected in any o f the sera. The IgG1 antibody titres
ranged from 150 to 1100 (geometric mean titre + SD = 102.79 + 0.339) and
the IgG2 antibody titres ranged from 65 to 3400 (geometric mean titre
+ SD = 102-7s + 0.s~s).
It was concluded that assays should determine IgG1 and IgG2 antibody,
but determination o f IgM and IgA was of no extra advantage.

IgG1 and IgG2 antibodies to BVD V in cattle sera

The mean number of units o f specific IgG1 and IgG2 antibody to BVDV
in the sera o f 41 cows, colostrum from 13 of these cows, and sera from 21
calves taken after ingestion o f colostrum are shown in Table II. Also given

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are levels o f specific I g G 1 a n d I g G 2 a n t i b o d y t o B V D V in 10 pairs o f sera

t a k e n b e f o r e , a n d 4.5 w e e k s a f t e r i n t r a n a s a l i n f e c t i o n w i t h B V D V . O n l y
o n e o f t h e 41 c o w s did n o t c o n t a i n d e t e c t a b l e a n t i b o d y . B o t h IgG1 a n d
IgG2 a n t i b o d y were p r e s e n t in t h e r e m a i n d e r . A n t i b o d y t o B V D V in colost r u m a n d in sera f r o m calves t h a t h a d ingested c o l o s t r u m was p r e d o m i n a n t ly I g G 1 . B e f o r e e x p o s u r e t o B V D V all 10 e x p e r i m e n t a l l y - i n f e c t e d calves
c o n t a i n e d ~< 50 units IgG1 a n t i b o d y p e r ml o f s e r u m ; t h e s e r u m f r o m o n e
calf c o n t a i n e d 1 0 0 units o f I g G 2 a n t i b o d y while t h e o t h e r 9 sera c o n t a i n e d
50 units I g G 2 a n t i b o d y . A f t e r i n j e c t i o n b o t h I g G 1 a n d I g G 2 a n t i b o d i e s
t o B V D V were d e t e c t e d in t h e s e calf sera.
TABLE II
Comparison of levels of IgG1 and IgG2 antibody to BVDV in sera and colostrum
Sample group

No. of
animals

Geometric mean No. of

units of antibody
IgG1

Adult cows
Colostrum
Colostrum-fed-calves
Post-inoculation
ealfsera a

+ SD

IgG2

Ratio
IgG1 : IgG2
-+ SD

41
13
21

103.20 + 0.ss9
103.52 +0.310
103.33 +0.407

102.9 +0.4s2
10~4 +0.s39
102.10 +0.371

10

102.24 +_0.238

101.93 _+0.413

1.8 : 1
24 : 1
17 : 1
2

:1

apre-inoculation of calf sera contained < 101"7 units, IgG1 and IgG2 antibody except
for one calf with 102 units IgG2.

Appearance o f antibody to B V D V in sera from calves aged up to 8 m o n t h s

O u r results i n d i c a t e d t h a t in o r d e r t o m a x i m i s e t h e c h a n c e o f d e t e c t i n g
a n t i b o d y t o B V D V , IgG1 o r I g G 2 a n t i b o d y s h o u l d b e a s s a y e d or a s y s t e m
designed t h a t d e t e c t e d b o t h .
T o e x a m i n e this f u r t h e r , sera c o l l e c t e d o v e r a 2 4 - w e e k p e r i o d f r o m 44
calves, held t o g e t h e r o n a b e e f - r e a r i n g f a r m , w e r e assayed. Calves w e r e first
bled at 5--7.5 w e e k s o f age a n d 3, 6, 15 a n d 24 w e e k s later. T h e c u m u l a t i v e
n u m b e r o f calves w i t h a >~2-fold increase in t h e n u m b e r o f units o f I g G 1 ,
I g G 2 o r IgG1 + I g G 2 a n t i b o d y during t h e p e r i o d s t u d i e d was d e t e r m i n e d .
T h e c o m b i n e d s y s t e m t h a t m e a s u r e d IgG1 + I g G 2 a n t i b o d y a p p e a r e d m a r ginally s u p e r i o r , p a r t i c u l a r l y w h e n m a t e r n a l IgG1 was p r e s e n t ; it d e t e c t e d
an a n t i b o d y rise to B V D V in 2, 8, 26 a n d 36 animals, w i t h 2, 3, 27 a n d 36
rises d e t e c t e d using a n t i - I g G 1 , o r 2, 4, 18 a n d 34 using anti-IgG2. B o t h t h e
c o m b i n e d s y s t e m a n d anti-IgG1 s y s t e m d e t e c t e d 36 o f 44 ( 8 2 % ) o f calves
in this g r o u p w h i c h h a d p r o d u c e d a n t i b o d y r e s p o n s e s to B V D V b y t h e
t i m e t h e y were 2 9 - - 3 1 . 5 w e e k s old.

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Antibody in viraemic cattle

Fifty sera were examined from a herd with mucosal disease, 23 o f which
were shown later to have persistent BVDV viraemias. Antibody was measured in sera from these animals using the combined anti-IgG1 + IgG2 system.
Of the 23 viraemic cattle, 20 had < 50 units of antibody per ml of serum,
the remaining 3 sera had 102", 102.2 and 102.4 units of antibody per ml.
The range of values for the normal non-viraemic animals in the same herd
was 102"2--104"1 units; geometric mean + SD = 103.4 0.40
DISCUSSION

The end-point titration m e t h o d has been recommended as the standard

assay against which other systems can be compared (De Savigny and Voller,
1980). Although variations in antibody affinity as well as titre are known to
affect values obtained in a single-dilution assay (Butler et al., 1978), we
have shown that this test was quantitative and gave values comparable to
the end-point titration method. On the basis o f these results we used the
single dilution assay in subsequent tests.
Initial experiments to establish which Ig isotypes were produced following
BVDV infection were necessary because of the restricted isotype response
sometimes observed following infection. Examination of sera from experimentally- and naturally-infected cattle indicated antibody was predominantly IgG1 and IgG2. Low levels of IgM antibody m a y not have been detected as a result o f competitive inhibition between IgM and IgG antibodies
(Chantler and Diment, 1981). However, the results obtained with fractionated sera do n o t substantiate this view. These studies indicated both IgG1 and
IgG2 antibody had neutralizing activity for BVDV and also that the IgM
fraction did not contain a substantial proportion of the neutralizing antibody in bovine sera. The neutralizing activity of the IgM fraction of Sample
C could have been due to the presence of a small a m o u n t of IgG1 antibody
in this sample. The high and prolonged IgM response suggested by Fernelius
(1966) based on comparisons of neutralization and precipitation tests
in fractionated sera, was not observed. Subsequent studies therefore concentrated on tests designed to assay only the two IgG subclasses.
The ratio o f IgG1 : IgG2 antibody was about 2 : 1 in sera from naturallyand experimentally-infected cattle. In colostrum and sera from colostrumfed calves the ratio of IgG1 : IgG2 antibody was about 10 times higher.
This is as expected from established data on the selective transfer o f IgG1
into colostrum (Butler, 1983). It is also the obvious explanation for the poor
correlation between IgG2 antibody measured by ELISA and the neutralization assay, despite the demonstrated ability of IgG2 fractions of sera to
neutralize BVDV.
In the assays, control wells were sensitised with Nonidet extracted noninfected calf-testis cells. This enabled non-specific binding of sera to be

368
eliminated, a l t h o u g h it m a y have resulted in s o m e sera t h a t c o n t a i n e d low
levels o f a n t i b o d y and t h a t p r o d u c e d high non-specific binding being recorded as giving a negative r e a c t i o n , Pre-inoculation sera f r o m c o n v e n t i o n a l l y reared calves held in isolation t h a t were devoid o f serum a n t i b o d y b y t h e
n e u t r a l i z a t i o n test c o n t a i n e d n o a n t i b o d y d e t e c t e d b y E L I S A . Also, the
antisera p r e p a r e d in g n o t o b i o t i c calves t o o t h e r agents did n o t r e a c t in t h e
test indicating t h a t n o n - s p e c i f i c i t y was n o t a p r o b l e m .
It is c o n c l u d e d t h a t , o p t i m a l l y , assays should d e t e c t b o t h IgG1 and IgG2
a n t i b o d y a l t h o u g h an assay t h a t d e t e c t e d just IgG1 w o u l d be nearly as
effective in diagnosing i n f e c t i o n and measuring m a t e r n a l a n t i b o d y . T h e
viraemic cows had no d e t e c t a b l e , o r v e r y low a m o u n t s of, a n t i b o d y t o BVDV
and t h e assay should p r o v i d e a rapid m e t h o d f o r d e t e c t i n g i m m u n o l o g i c a l l y c o m p r o m i s e d c a t t l e at risk o f d e v e l o p i n g clinical m u c o s a l disease. It should
also be useful f o r f o l l o w i n g t h e i m m u n e response t o B V D V and d e t e c t i n g
i n f e c t i o n s with this virus. T h e high p r o p o r t i o n o f adult c a t t l e with a n t i b o d y
( 4 0 / 4 1 ) , and t h e d e t e c t i o n o f a n t i b o d y responses in calves b y t h e t i m e t h e y
were 8 m o n t h s old ( 3 6 / 4 4 ) is c o n s i s t e n t with o t h e r data, and indicates t h e
high risk o f i n f e c t i o n and high p r e v a l e n c e o f B V D V in such cattle.
ACKNOWLEDGEMENTS
We t h a n k E.J. S t o t t f o r providing antisera t o r e s p i r a t o r y s y n c y t i a l virus,
p a r a i n f l u e n z a virus t y p e 3 and i n f e c t i o u s bovine r h i n o t r a c h e i t i s virus together with sera f r o m calves e x p e r i m e n t a l l y - i n f e c t e d with BVDV a n d c l o n e d
strains N A D L and I R A D C 2 4 1 5 . We also t h a n k P.D. L u t h e r and A.J. Hayle
f o r t h e cell cultures; N.J. R o l l e y and M.A. Parsons f o r t e c h n i c a l assistance.

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