title: Rapid detection of bovine herpesvirus 1 in the semen of infected bulls by a nested polymerase chain reaction assay.
abstract: A nested polymerase chain reaction (PCR) assay was developed for the detection of bovine herpesvirus 1 (BHV-1) in bovine semen and compared with the virus isolation method. When extended semen, commonly used in the bovine artificial insemination industry, was inoculated with BHV-1, the PCR assay detected BHV-1 DNA in semen inoculated at 0.25-2.5 TCID50 per 0.5 mL. In contrast, the lower limit of detection for virus isolation was 250 TCID50 of BHV-1 inoculated in 0.5 mL of extended semen. These methods were also used to detect BHV-1 in the semen of four bulls which were experimentally infected with BHV-1. All infected bulls demonstrated balanitis at 3 d post-inoculation (DPI) and severe balanoposthitis at 4 DPI. BHV-1 was detected in raw semen by virus isolation and PCR at 2 DPI, before balanitis was evident. For virus isolation, the last day that BHV-1 was detected during primary infection was 7 DPI for two bulls and 9 and 11 DPI for the other two bulls. In contrast, PCR detected BHV-1 in the bulls' semen until 14 or 18 DPI. For individual animals, PCR detected BHV-1 during primary infection for at least 1-10 d longer than virus isolation. Reactivation of BHV-1 from latency without the presence of visible lesions was promoted twice by two series of 5 d dexamethasone injections. For the first series of dexamethasone treatments, a positive virus isolation result was obtained on the 5th d of treatment for only one bull. In contrast, two bulls demonstrated evidence of viral reactivation on this day by PCR. All bulls shed BHV-1 in semen on d 4 after dexamethasone treatment, as evidenced by positive virus isolation and PCR results. One bull was still PCR positive 13 d later. For the second series of dexamethasone treatments, a small amount of virus was isolated from semen collected on d 3 or 4 after treatment for two bulls but not from the other two bulls. In contrast, semen samples from all bulls were PCR positive for either or both of these 2 d. In total, from 80 semen samples, 45 were PCR positive and 26 were virus isolation positive. Thus, the PCR assay detected BHV-1 shedding in bulls earlier, more often, and for a longer duration, than did the virus isolation method.
authors.affiliation: Agriculture and Agri-Food, Canada, Animal Diseases Research Institute, Telhbridge, Alberta.
authors.forename: S A
authors.initials: SA
authors.lastname: Masri
authors.forename: W
authors.initials: W
authors.lastname: Olson
authors.forename: P T
authors.initials: PT
authors.lastname: Nguyen
authors.forename: S
authors.initials: S
authors.lastname: Prins
authors.forename: D
authors.initials: D
authors.lastname: Deregt
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002418:Cattle Diseases; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000503:physiopathology / Q000662:veterinary*; D007242:Herpesvirus 1, Bovine / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012661:Semen / Q000821:virology*; D012741:Sexual Maturation; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004279:DNA, Viral