RAPID DETECTION OF BOVINE HERPESVIRUS-1 IN THE SEMEN OF INFECTED BULLS BY A NESTED POLYMERASE CHAIN-REACTION ASSAY

A nested polymerase chain reaction (PCR) assay was developed for the d etection 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 TC ID50 per 0.5 mL. In contrast, the lower limit of detection for virus i solation was 250 TCID50 of BHV-1 inoculated in 0.5 mL of extended seme n. These methods were also used to detect BHV-1 in the semen of four b ulls which were experimentally infected with BHV-1. All infected bulls demonstrated balanitis at 3 d post-inoculation (DPI) and severe balan oposthitis at 4 DPI. BHV-1 was detected in raw semen by virus isolatio n 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 D PI 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 indiv idual animals, PCR detected BHV-1 during primary infection for at leas t 1-10 d longer than virus isolation. Reactivation of BHV-1 from laten cy without the presence of visible lesions was promoted twice by two s eries of 5 d dexamethasone injections. For the first series of dexamet hasone treatments, a positive virus isolation result was obtained on t he 5th d of treatment for only one bull. In contrast, two bulls demons trated evidence of viral reactivation on this day by PCR. All bulls sh ed BHV-1 in semen on d 4 after dexamethasone treatment, as evidenced b y positive virus isolation and PCR results. One bull was still PCR pos itive 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 a fter treatment for two bulls but not from the other two bulls. In cont rast, semen samples from all bulls were PCR positive for either or bot h 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, t han did the virus isolation method.