Heat treatment of serum from calves PI with BVDV to remove interference of colostrum-derived specific antibodies with antigen detection by ELISA

Objectives

Bovine viral diarrhoea (BVD) is one of the most prevalent and economically significant viral diseases of cattle worldwide. A range of diagnostic tools are available for the detection of both viral antigen. The antigen ELISA is a rapid, inexpensive and robust test that successfully identifies persistently infected (PI) cattle. However, the antigen ELISA is subject to interference from colostrum-derived specific antibodies in serum of neonatal and young PI calves. This study investigated methods to remove specific antibodies from serum samples and, therefore, reduce interference.

Materials and Methods

Serum samples containing BVDV antigen and/or specific antibodies were treated by methods adapted from More and Copeman (Trop Med Parasitol 1991;42:91-94): treatment with heat (boiling) with or without the addition of an equal volume 0.1M EDTA pH 4.5, 5.5, 6.5 or 7.5. Treated samples were centrifuged and the supernatant recovered. All treated and untreated samples were tested for detectable BVD viral antigen and specific antibodies by ELISA (IDEXX BVDV Serum/Ag Plus ELISA and IDEXX BVDV Total Ab ELISA, respectively). The optimal treatment was identified and applied to serum samples collected longitudinally from three PI and seven non-PI calves from the day of birth to five weeks of age. As above, all treated and untreated samples were tested for detectable BVD viral antigen and specific antibodies by ELISA.

Results

All treatments resulted in an increase in antigen signal and a decrease in detectable antibody levels. Optimal treatment was identified as heat treatment with the addition of an equal volume of 0.1M EDTA pH 5 ± 0.5, resulting in antigen signal recovery in excess of 90%. When applied to field samples collected longitudinally from three PI calves from birth until five weeks of age, all treated samples tested positive for antigen at all time points. By comparison, the same samples when untreated returned negative antigen results for between six days and four weeks post-colostrum ingestion. The vast majority (98.6%) of samples from non-PI calves collected after the day of birth tested negative for BVDV antigen. On the day of birth, four of seven non-PI calves returned (low) positive results.

Conclusions

Heat and EDTA treatment of serum samples presents an opportunity to improve performance of BVDV antigen ELISAs for testing of colostrum fed calves. Diagnostic sensitivity was improved, however, diagnostic specificity was decreased substantially on the day of birth. The data indicates that adjustment of the positivity threshold is appropriate and will result in excellent diagnostic performance. Further study on larger numbers of calves is warranted, but the results of this study indicate that treatment of serum samples has the potential to eliminate interference of colostrum-derived specific antibodies with BVDV antigen detection.

Comments

This work has been accepted for publication by the Journal of Veterinary Diagnostic Investigation.

Current address for Prof. M. P. Reichel: School of Veterinary Medicine, City University of Hong Kong