Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle.
OBJECTIVE
To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV.


METHODS
Serum samples and pooled bulk-tank milk samples from cattle.


METHODS
The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome.


RESULTS
Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR.


CONCLUSIONS
This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.
Gutiérrez
S E
SE
Departamento de Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, Argentina.
Dolcini
G L
GL
Arroyo
G H
GH
Rodriguez Dubra
C
C
Ferrer
J F
JF
Esteban
E N
EN
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000302:isolation & purification; D002417:Cattle; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D016583:Enzootic Bovine Leukosis / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D005260:Female; D001909:Leukemia Virus, Bovine / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008892:Milk / Q000821:virology; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012372:ROC Curve; D015531:Radioimmunoprecipitation Assay / Q000662:veterinary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000914:Antibodies, Viral; D000956:Antigens, Viral; D004279:DNA, Viral