Document Type

Thesis - University Access Only

Award Date

2001

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Bovine leukemia virus (BL V) is one of the most common and economically damaging pathogens in dairy and beef cattle. BL V is the causative agent of enzootic bovine leukosis (EBL), persistent lymphocytosis (PL) and lymphosarcoma. Since BLV is a retrovirus, there is concern over its transmission to humans through contaminated milk. Transmission of BLV occurs primarily through blood and milk, and in utero. The virus is highly cell-associated and is labile outside its host cell, the B lymphocyte. The antibody ELISA is the most common method for detecting the presence of BLV in an infected animal. ELISA testing is quick and easy, but depends on an immune response, that may take as long as 14 weeks to develop. The BLV antibody ELISA also has difficulties assaying milk, an easily collectable specimen for BL V screening. The polymerase chain reaction (PCR) provides a valuable adjunct to BL V antibody ELISA testing. PCR can identify BLV infected animals before BLV antibodies are detectable, and it can be used to screen blood and milk specimens. The amplification of BLV proviral DNA from infected B lymphocytes would be the most sensitive method of BLV detection. In this project, a BL V PCR assay was designed to test blood and milk samples. Nested PCR primers were chosen from three different regions: gag, env, and pol, of the BLV genome. PCR was performed on genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) and milk lymphocytes from 101 dairy cattle, from five different geographical locations: Kentucky, Minnesota, Oklahoma, South Dakota, and Wisconsin. The results of this assay were compared with a BLV antibody ELISA used on plasma. Using nested PCR, primers from the pol region identified the largest number of seropositive cattle in blood (98%) and milk (65%) and detected BLV in three seronegative cattle. Primers from the gag and env regions worked less effectively than the pol primers in identifying BLV infected animals from blood (80% and 90%, respectively) and milk (9% and 28%, respectively). Freshening dates for most cattle were also collected, but there was no correlation between BLV PCR detection in milk and stage of lactation. In addition to nested PCR, real-time PCR with molecular beacons was used to screen blood and milk specimens for BLV proviral DNA. Using an ABI 7700 Sequence Detection System, all the specimens were retested using the molecular beacons. Realtime PCR with molecular beacons identified seropositive cattle in both blood (94%) and milk (59%), and detected BLV in four seronegative animals. A fluorogenic probe-based product enhanced reverse transcriptase (PERT) assay was also used on samples from BLV infected animals. This assay detects the reverse transcriptase (RT) enzyme found in all retroviruses. The activity of this enzyme was detected by amplifying the DNA product that it transcribed from an RNA template. This product was amplified by real-time PCR using a fluorescently labeled Taqman ® probe, on an ABI 7700 Sequence Detection System. Serum and lymphocytes from eight cattle were tested for RT activity using this PERT assay. The fluorogenic probe-based PERT assay did not detect RT activity in BLV infected serum or lymphocyte cultures, however, RT was detected from bovine immunodeficiency virus (BIV) and avian myeloblastoma virus (AMV). This project was successful in detecting BLV in blood and milk specimens using both nested PCR and real-time PCR with molecular beacons. This test can be used for the early detection of BLV, before an immune response has occurred. It can also be used to screen milk specimens, where a large sample is easily collected. The PERT assay was successful in detecting AMV and BIV, but not BLV. This is likely because BLV produces defective RT molecules and the enzyme is highly cell-associated. Although the BLV antibody ELISA is the standard in BLV detection, a PCR based assay can provide a sensitive adjunct for producers of transgenic cattle, exporters, and owners of valuable breeding stock. The ability of PCR to detect BLV before an antibody ELISA also allows the earlier implementation of control measures to prevent transmission of infectious virus.

Library of Congress Subject Headings

Bovine leukemia virus -- Diagnosis
Retroviruses
Polymerase chain reaction -- Diagnostic use

Format

application/pdf

Number of Pages

100

Publisher

South Dakota State University

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