J . Vet. Med.

B 43, 163-166 (1996)

01996 Blackwell Wissenschafts - Verlag, Berlin
ISSN 0931-1793

Institute of Veterinary Pathology, University of Zurich, Switzerland

Immunohistochemical Detection of Bovine Viral Diarrhea

Virus in Skin Biopsies: a Reliable and Fast Diagnostic Tool
B. THUR, K. ZLINSZKY
and F. EHRENSPERGER
Address of authors: F. EHRENSPERGER,
Institute of Veterinary Pathology Winterthurerstrage
268, CH-8057 Zurich, Switzerland

With 1 figure and 1 table

(Received for publication September 21, 1 9 9 j )

Summary
Skin biopsies from 184 cattle were immunohistochemically tested for bovine viral diarrhea
virus (BVDV) infection. BVDV infection was detected sensitively and specifically by visceral
organ immunohistochemistry or by standard virological methods.

Introduction
In cattle persistently infected with bovine viral diarrhea virus (BVDV), o r suffering
from mucosal disease, BVDV can be detected in many visceral organs by immunohistochemical methods (BIELEFELDT
OHMANN,1982, 1988; WILHELMSEN
et a]., 1991 ;
WOHRMANN
et al., 1992). It has also been reported that BVDV is present in skin samples
(BIELEFELDT
OHMANN,
1988), but systematic studies investigating the diagnostic value
of skin biopsies had not yet been carried out.
The aim of this study was therefore to determine the prevalence of BVDV-positive
skin samples in animals persistently infected with BVDV. An immunohistochemical
assay, using a panel of monoclonal antibodies on cryostat sections of skin, proved to
be a fast and reliable test and an alternative to conventional virological methods for
the diagnosis of BVDV infection.
Material and Methods
Animals
A total of 184 cattle were tested for the presence of BVDV antigen in the skin. Seventy of
these animals displayed clinical signs suggestive of BVDV infection, 74 showed no overt
symptoms but originated from two herds with a history of repetitive BVDV infections, and 40
animals had neither typical symptoms nor a history of BVDV infection.
Tissue samples
Skin biopsies from the lateral aspect of the neck were obtained from 144 animals using a
6-mm biopsy punch (Stiefel Laboratories, Winterthur, Switzerland) after local anaesthesia.
Corresponding skin samples were taken post mortem from 40 animals. Fourteen cattle previously
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164

THUR,ZLINSZKY
and EHRENSPERGER
Table 1. Immunohistological diagnosis of BVD infections in skin samples

No. of animals examined by immunohistology

positive
negative
No. of animals tested in parallel by immunohistology and Ag-ELISA or virus
isolation
positive
negative
No. of animals with multiple body-site skin testing
positive
negative
No. of animals examined postmortem (parallel testing of skin and visceral organs)
positive
negative

184
37
147
124
20
104
27
18
9
54
29
25

tested by skin biopsy were also sampled post mortem. From all 54 animals examined post mortem,
tissue samples from organs such as thyroid gland, oral mucosa, esophagus and abomasum
were immunohistochemically tested for the presence of BVDV. To rule out the possibility of
topographical differences in antigen load, multiple skin samples were taken from 2-5 different
sites of the body (neck, head, trunk, legs) from 27 post mortem cases.

Blood samples

EDTA blood was drawn simultaneously with the skin biopsy from 118 live animals to test
the buffy-coat fraction for the presence of BVDV.

Virus detection
Tissue samples for immunohistochemistry were snap frozen in liquid nitrogen. Cryostat
sections of 6-8 pm were mounted on silane-coated glass slides and fixed for 10 min in -20C
cold aceton. Specific binding of monoclonal antibodies to BVDV (mab's C16, CA3 and CA34)
was detected after 60 min of incubation at 37 "C by using a peroxidase-labelled streptavidinbiotin test kit (LSAB-Kit, Dako A/S, Glostrup, Denmark).
Buffy-coat cells were analysed for the presence of BVDV using an antigen-capture ELISA
(Ag-ELISA; GOITSCHALK,
1991; STRASSERet al., 1995). For virus isolation, performed in 10
cases, samples from visceral organs were processed, incubated for 5 days on susceptible cell
cultures, and tested for the presence of BVDV, as described previously (THUR,1993).

Results
From a total of 184 cattle, 37 were found t o be positive by immunohistochemistry
(Table 1). From these positive animals 20 were tested in parallel by other methods:
nine cases b y virus isolation and 15 cases by Ag-ELISA. The results of immunohistochemistry and virus isolation were all identical. T h e Ag-ELISA was positive in 13
out of the 15 cases investigated. I n the t w o ELISA-negative but immunohistochernically
positive cases, virus isolation on cell culture was successful.
Negative immunohistochemical results were compared to Ag-ELISA in 104 cases.
No BVDV could be detected in the buffy coat in any of these cattle.
In 27 animals (18 BVDV positive and 9 negative), skin samples from 2-5 different

Detection of Bovine Viral Diarrhea Virus

165

Fig. 1. Skin. LSAB, Cl6. BVDV-specific intracytoplasmic reaction in epithelial cells of the
epidermis and of hair follicles (arrow), as well as in mesenchymal cells (arrowhead; bar = 30 pm)

body sites were examined. The results were identical, irrespective of the location. The
pattern of antigen distribution in a skin biopsy is shown in Figure 1 .
In the 54 cases (29 BVDV positive and 25 negative), where skin samples as well
as visceral organs were tested by immunohistochemistry, the results were consistent
in all cases.

Discussion
This study demonstrates that immunohistochemical examination of skin samples
is a useful method for identification of BVDV carriers. The results thus obtained were
reliable when compared to virus isolation, immunohistochemistry of visceral organs,
or Ag-ELISA of the buffy-coat fraction of blood. In two cases, immunohistochemistry
and virus isolation were more sensitive than the Ag-ELISA of buffy-coat cells. The
Ag-ELISA used in this study has been previously demonstrated to sensitively detect
1991 ; STRASSER
et al., 1995). Systematic
persistently infected animals (GOTTSCHALK,
studies evaluating the sensitivity of BVDV detection by Ag-ELISA in transiently
infected cattle have not been carried out. However, virus isolation is generally considered to be more sensitive than Ag-ELISA (STEFFEN1993).
No difference in immunohistochemical reactivity was found in biopsies compared
with post mortem skin samples, and no site predilection of BVDV was observed.
These findings correspond with the theory that BVDV is disseminated throughout the

THUR, ZLINSZKY
and EHRENSPERGER

166

body of persistently infected animals (BIELEFELDT

OHMANN,
1982, 1988). H o w e v e r ,
the immunocytological detection of BVDV antigen in epithelial swabs (nasal, conjunctival, vaginal etc.), as well as in buffy-coat cells, is inconsistent or inadequate d u e
to non-specific reactions (B. THURet al., unpubl. data).
In conclusion, this s t u d y has s h o w n that B V D V can reliably be detected by skin
biopsy using immunohistochernical methods. The speed and the relative ease of the
m e t h o d make it a n attractive tool for t h e detection of virus carriers both ante a n d post
mortem.

Acknowledgements
The authors would like to thank the members of the Bovine Medical Clinic, University of
Zurich, as well as of the Institute of Veterinary Virology, University of Berne, for their
cooperation. The authors thank their colleagues Patrick CAPLAZI
and Denise MYERSfor critical
reading of the manuscript. Thanks go also to Dr. V. MOENNIG(Institute of Virology, Veterinary
School, Hannover, Germany) and to Dr. BOMMELI
(Liebefeld-Bern, Switzerland) for supplying
BVDV antibodies.

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