Elsevier

Veterinary Microbiology

Volume 64, Issues 2–3, 1 January 1999, Pages 135-144
Veterinary Microbiology

A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk

https://doi.org/10.1016/S0378-1135(98)00265-XGet rights and content

Abstract

To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-structural peptide NS3 of pestiviruses. The test can be performed at high reproducibility using undiluted samples. In testing 1000 field serum samples, the ELISA showed a specificity and a sensitivity relative to the virus neutralization test of 99% and 98%, respectively. The blocking ELISA is able to detect specific antibodies in serum obtained 12 days after an acute infection and in serum of vaccinated and challenged animals. A frequency distribution diagram, obtained by testing almost 1800 random Dutch field serum samples, showed a clear separation between a negative population (maximum frequency of the % inhibition at −5%) and a positive population (maximum frequency of the % inhibition at 95%). Based on these data, the prevalence of seropositive animals was 65% (95% confidence interval: 63%–67%). By exchanging plasma- and bulk milk samples between two laboratories (The Netherlands and Denmark), a good overall agreement was found between results obtained with the Ceditest and those obtained with the Danish blocking ELISA as used in the Danish BVDV eradication programme. The results indicate that BVDV infections can reliably be diagnosed by the Ceditest ELISA and that the test is suitable for use in large scale screening and eradication programmes.

Introduction

Bovine Virus Diarrhoea Virus (BVDV) belongs to the genus Pestivirus of the family Flaviviridae. The virus has a worldwide distribution with serum antibody prevalences ranging from 50% to 90%. Acute infections in seronegative immunocompetent cattle in most cases pass subclinically or result in mild clinical signs including a transient fever and leucopenia. Viremia may last for up to 15 days during which period serum antibodies will develop. Occasionally, young animals may suffer from severe disease because of the immunosuppressive effect of the virus resulting in superinfections by opportunistic pathogens.

BVDV infection during pregnancy may result in fetal infection. Depending on the age of the fetus, infection may result in fetal resorption, abortion, mummification, congenital malformations, birth of immunotolerant persistently infected and viremic calves or birth of normal, weak or undersized calves.

Persistently infected (PI) calves, continuously shedding virus, have no or low levels of specific antibodies. Noncytopathic BVDV can be isolated from blood samples taken from such animals (Baker, 1987; Ames et al., 1990; Weiss et al., 1994; Deregt and Loewen, 1995). Acute infections of BVDV in individual animals can be monitored by antibody titration of paired serum samples (Weiss et al., 1994). Additionally, the determination of the level of BVDV-specific antibodies in pooled or randomly taken blood samples or in bulk milk can be used to monitor the infection status of an individual herd (probability of a PI or non-PI status) (Alenius et al., 1997; Bitsch et al., 1997; Waage et al., 1997). Consequently, a sensitive and specific test for the detection of BVDV specific antibodies may be a valuable tool for the diagnosis of BVDV infections in individual animals or on a herd level and useful in large scale screening and eradication programmes.

In the study presented, a simple, rapid and reliable enzyme-linked immunosorbent assay (ELISA) for the detection of BVDV-specific antibodies using undiluted serum, plasma or defatted milk samples is described.

Section snippets

Reagents used in the ELISA

The ELISA for the detection of BVDV-specific antibodies (Ceditest blocking ELISA for BVDV-Ab) is a modification of the ELISA as described by Paton et al. (1991a). Characteristics of the monoclonal antibodies "WB103" and "WB112", as used in the Ceditest ELISA, have been described earlier (Edwards et al., 1988, Edwards et al., 1991; Paton et al., 1991b). These two monoclonal antibodies recognize two different, highly conserved epitopes located on the non-structural peptide NS3 of pestiviruses

Results

The sensitivity and specificity of the ELISA was determined using the VNT as the gold standard test. The ELISA test results of one thousand field serum samples with known VNT are shown in Table 1. Based on these results, it can be calculated that the ELISA has a specificity, relative to the VNT, of 99% (=475/479 × 100%; 95% confidence interval: 98%–100%) and a relative sensitivity of 98% (=510/521 × 100%; 95% confidence interval: 96%–99%). Of the 11 ELISA-/VNT+ samples, eight had VNT titres ≤3.

To

Discussion

The Ceditest ELISA as described is a modification of the test as published by Paton et al. (1991a). The two monoclonal antibodies (WB103 and WB112) are directed against different epitopes of the highly conserved non-structural peptide NS3 of pestiviruses and do recognize most, if not all, pestivirus strains (Edwards et al., 1988, Edwards et al., 1991; Paton et al., 1991a, Paton et al., 1991b; Edwards, personal communication). Consequently, by using the monoclonal antibodies WB103 and WB112, the

Acknowledgements

The authors thank Dr. G.M. Zimmer, Animal Health Laboratory, The Netherlands for providing serum samples from vaccinated and challenged animals.

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