Demonstration of Antibody to Bovine Leukosis Virus in Milk by the Elisa Test

H 0 fir e k B. , Mar taG ran' t 0 v , : Demonstration of Antibody to Bovine Leukosis Virus by the ELISA Test. Acta vet. Bma, 57, 1988: 133-146. The ELISA test and the iDlDunodiffusion test were compared for their suitability to detect antibody to bovine leukosis virus (BVL) in milk of dairy cows. Sensitivity of the immunodiffusion test in the determination of BLV antibodies in milk proved to be low: positive response was obtained only in 37.7% of the dairy cows in which the respective serum antibodies were demonstrated. On the other hand, the ELISA method aaployed under the same conditions, yielded 96.3-100 % identical results in serum and milk. A highly positive correlation between the antibody titre in milk and serum was demonstrated. !he antibody titre in milk compared with that in serum was approximately 25. times lower. It is concluded that the determination of BVL antibodies in cow' s milk is a suitable method of detection of leukosis reagents. Enzootic bovine leukosis, antibody,serum, milk. A widespread survey of enzootic bovine leukosis (EBL) incidence carried out in Czechoslovakia in the years 1982-1984 yielded a fairly good overview as to the infection of herds with this serious disease and enabled to set up a program of eradication (B end a 1984). Eradication of EBL in foci of its occurrence is based upon timely identification of serologically positively reacting animals and their iDlDediate culling. Therefore, two serological methods, iDlDunodiffusion (ID) and ELISA tests have been introduced into the routine practice along with other tests. Results obtained by these methods in the EBL eradication in large-scale cattle operations (H 0 fir e k et a1. 1985) were pUblished; repeated serological examination and elimination of the reagents proved to be an effective control measure. The demonstration of BLV antibodies is usually carried out in serum of examined animals. However, in the eradication program these blood samplings represent a hard and dangerous labour. Furthermore, blood is a potential source of infection and contamination of the environment especially during mass blood sampling. Therefore, less hazardous surveillance methods have been looked for, e.g. antibody detection in cow s milk: ( A 1 tan e.r 134 et al. 1982; Tom a et al. 1984; Z a j a c et al. 1984; F ran c et al. 1984). In this work, detection of BLV antibodies in milk was attempted with particular attention paid to comparison of the ELISA and immunodiffusion test sensitivities. Using the ELISA method we also compared the onset of antibody production induced by experimental and natural contact infection, the phase of lactation and milk yield being taken into consideration as well. The final aim of our study was to evaluate the reliability of ~LV antibody detection in milk from the viewpoint of routine diagnosis and eradication of EBL in its occurrence foci. Materials and Methods In a herd infected with EBL for a long time we examined 87 dairy cows, 45 out of them showing a positive reaction when tested for specific BLV antibodies. Three serological methods were used: serum neutralization test (PsNT) , (Z a v a d a et al. 1978), ELISA test and immunodiffusion test (ID) • Milk samples of the 45 positive animals were examined first by the ID test, antibodies to BLV being determined in fresh milk, in whey obtained by artificial coagulation by Lactochym and in whey obtained by natural coagulation of milk. In the second experiment in the same group of 45 cows a parallel ,examination of serum and milk by the ELISA method was carried out and siorultaneously the titre of BLV antibodies was determined. The procedure was repeated using the ID test. Fourteen animals of this herd (they were housed together in a smaller cow-house) were selected for repeated examination for the presence of antibodies to BLV in serum and milk. These animals were also examined by repeated serum neutralization test. Out of them 8 were positive (Table 5) and 6 negative (Table 7) (3 of them were infected experimentally by i.p. inoculation of 1 ml of blood of a virus-positive donor). The remaining three cows were negative and used as controls, being, however, in contact with positive and experimentally infected animals. The interval of examination ranged from 7 to 14 days, the entire experimental period lasted 67 days. A total of 122 parallel milk and blood samples were taken from the 14 animals. The illll1lllIiodiffusion test was carried out in agarose gel using a micromethod. A cOlllllercial antigen Agebion (Bioveta, Nitra, CSSR) was used. The indirect immunoperoxidase test ELISA was carried out using the ELISA sets for diagnosis of EBL (Bioveta, Ivanovice na Bane, CSSR). The results were evaluated spectrophotometrically on a Titertek Multiscan apparatus. For the serum neutralization test (PsNT) the pseudotype virus of vesicular stomatitis and virus of bovine leukosis was used (Z a v a d a et al. 1978). The virus detection in the donor whose lymphocytes were used for experimental infection was carried out using the syncytial test (F err e r and Dig 1 i