Use of milk samples and monoclonal antibodies directed against BLV-p24 to identify cattle infected with bovine leukemia virus (BLV).

An indirect double-antibody sandwich (IDAS) enzyme-linked immunosorbent assay (ELISA) using milk samples was developed to identify cows infected with bovine leukemia virus (BLV). Two monoclonal antibodies (McAbs) were used. One, which was directed against BLV core protein p24, was used to coat ELISA plates; the other was used to prepare a horseradish peroxidase (HRP) conjugate directed against bovine immunoglobulin. The IDAS-ELISA detected antibodies directed against BLV-p24 in 97% of the milk samples collected from known seropositive cows identified by the agar gel precipitation test (AGTP). Even when milk samples were diluted 1:50, 93% of the seropositive cows were identified. Only 0.43% of the 4000 milk samples collected in The Netherlands reacted nonspecifically. Nonspecific binding disappeared, however, when these samples were diluted 50 times in BLV-negative milk. In a comparative evaluation of BLV test-kits in various European laboratories, our IDAS-ELISA using McAb directed against p24 was one of the most sensitive.

Protective efficacy of Marek's disease virus (MDV) CVI-988 CEF65 clone C against challenge infection with three very virulent MDV strains.

Comparative 50% protective dose (PD50) assays were performed using a plaque-purified preparation of Marek's disease virus (MDV) strain CVI-988 at the 65th chicken embryo fibroblast (CEF) passage level (MDV CVI-988 CEF65 clone C) and three commercial MD vaccines: herpesvirus of turkeys (HVT) FC126, MDV CVI-988 CEF35, and a bivalent vaccine composed of HVT FC126 and MDV SB-1. In addition, comparative PD50 assays were performed in groups of chickens with maternal antibody to each of the three vaccines. Three representatives of the newly emerged biovariant very virulent (vv) MDV strains-RB/1B, Tun, and Md5-were employed as challenge virus. The experiments made feasible the differentiation between virulent MDV and vvMDV strains, within serotype 1. Vaccination with CVI-988 clone C vaccine resulted in PD50 estimates of about 5 plaque-forming units (PFUs) against challenge infection with each of the three vvMDV strains. The PD50 estimate of CVI-988 clone C vaccine was 12-fold below the PD50 of HVT FC126. The protective synergism of bivalent vaccine, composed of HVT and SB-1, was confirmed by groups given the lowest vaccine doses. The bivalent vaccine, however, resulted in incomplete protection in groups given the highest vaccine doses. Homologous maternal antibodies to serotype 1 caused a fivefold increase in the PD50 estimate of CVI-988 clone C. Heterologous maternal antibodies against HVT did not interfere with efficacy of CVI-988 clone C vaccination. However, the combination of maternal antibodies against both HVT and SB-1 (serotypes 2 and 3) showed a strong adverse effect on CVI-988 clone C vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of avian leukosis virus shedding hens. Comparison of ALV gs-antigen levels in various test samples.

Various materials from two White Leghorn basic breeder flocks were tested by the enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT) for detection of avian leukosis/sarcoma virus (ALV) group-specific (gs) antigens and the phenotypic mixing test (PMT) to compare the applicability of these techniques for the identification of hens which congenitally excrete exogenous ALV. The predictive value of each test was evaluated on the basis of association between ALV gs-antigen detection and ALV recovery and congenital virus transmission demonstrable in the hens. In materials from two flocks under study a close correlation was observed between congenital transmission of exogenous ALV to embryos and ALV gs-antigen detection in albumen samples. Results of examinations of vaginal/cloaca swabs and meconium samples were poorly associated with congenital virus excretion. ALV gs-antigen levels were generally higher in embryos than in albumen samples. Considerable variations in gs-antigen levels were observed in embryos and albumen samples from eggs of individual ALV shedding hens and also within a series of embryos from eggs of one hen.

The use of ELISA for detection of exogenous and endogenous avian leukosis viral antigens in basic breeding flocks.

An enzyme-linked immunosorbent assay (ELISA) for avian leukosis/ sarcoma virus (ALV) group-specific (gs) antigens was used to study the identification of hens which congenitally excrete exogenous ALV. The sensitivity of this assay was compared with that of the phenotypic mixing test (PMT) and the direct complement fixation test (CFT) by testing limiting dilutions of purified avian myeloblastosis virus (AMV), embryo homogenates and albumens. About 0.4 ng/ml of purified AMV protein could be detected by ELISA and 23.8 ng/ml of AMV protein was demonstrable by the CFT. The lowest levels of gs-antigen detection corresponded with about 100 median tissue culture infectious doses (TCID50) of infectious ALV. Albumens and embryos of three White Leghorn flocks and one White Plymouth Rock flock were tested for the presence of avian leukosis virus (ALV) gs-antigens by the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA) and exogenous ALV employing the phenotypic mixing test (PMT). The highest number of ALV-gs antigen positive samples of egg albumens was obtained by the ELISA. In embryo homogenates prepared from eggs of the four flocks under study 100% scores for gs-antigens were obtained by ELISA. A differentiation between gs-antigens of endogenous and exogenous ALV was made by testing of supernatant fluids after one chick embryo fibroblast passage of the C/E phenotype. Endogenous viral (ev) genes leading to the expression of endogenous ALV gs-antigens were apparently present in practically all chickens of the four flocks under study. Complete endogenous ALV (subgroup E) was detected in embryos (41%) from the White Plymouth Rock grandparent flock, but was not found in embryos of two White Leghorn basic breeder flocks. The results indicate that both flocks of White Leghorn chickens are endowed with ev 3 genes. Circumstantial evidence was obtained for the prevalence of endogenous ALV gs-antigens in albumen samples of eggs from flocks with gs+chf+ and V-E+ phenotype respectively.