Detection of bovine herpes virus-1 (BHV-1) infection in breeding bulls by serological and molecular methods and its characterization by sequencing of PCR products
Jain, Lata (2006) Detection of bovine herpes virus-1 (BHV-1) infection in breeding bulls by serological and molecular methods and its characterization by sequencing of PCR products. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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Bovine herpes virus 1 (BHV-1), the causative agent of infectious bovine rhinotracheitis (IBR), is considered to be the most common viral pathogen found in bovine semen. BHV-1 is associated with several clinical conditions including infectious bovine rhinotracheitis, infectious pustular vulvovaginitis, balanoposthitis, conjunctivitis and generalized disease in newborn calves causing great economical loss to the livestock industry. The present study was under taken to detect the seroprevalence of BHV-1 and to detect the presence of BHV-1 in semen of breeding bulls of Gujarat. The seroprevalence study was done by M-ELISA where as indirect FAT was applied only on few of serum samples to detect its efficacy as compared to M-ELISA. To detect the presence of viral antigen and viral genome in semen, respectively, direct FAT and PCR using two sets of primers were applied and comparison among them was made to detect their relative sensitivity and specificity. A comparison was also made between M-ELISA and PCR to detect the presence of antibody and viral genome in serum and semen, respectively, of the same bulls. A total of 89 serum samples were screened for presence of BHV-1 antibodies and 26 sera were found to be positive by M-ELISA revealing an overall seroprevalence of BHV-1 antibodies in 29.21% of breeding bulls of Gujarat. The species-wise seroprevalence was found to be 34.21% and 25.49% in cattle and buffalo bulls, respectively. In comparison of indirect FAT with that of M-ELISA, only 8 sera (6 M-ELISA positive and 2 M-ELISA negative) could be tested resulting in to almost cent per cent correlation. For detection viral antigen in semen, a total of 101 samples were screened by applying direct FAT of which 33 (32.67%) were found to be positive for BHV-1 antigen with an equal distribution between cattle and buffalo bulls. In the process of standardization of methods for DNA extraction from semen QIAamp Mini Kit protocol was found most suitable among all the four methods tried. Similarly, in the process of standardization of methods for PCR using DNA extraction from semen the result was only obtained while using the Hot start Master Mix out of the five methods tried. The viral genome in 101 semen samples was detected by employing PCR using two sets of primers viz., gB1/gB2 and gC1/gC2 from gB and gC genes coding for viral envelope. In gB gene based PCR, 47 (46.53%) semen samples were found to be positive producing an amplified PCR product of size 478 bp. While in gC gene based PCR, 43 (42.57%) were found to be positive for presence of viral genome producing an amplified PCR product of size 173 bp. In comparison of efficacy of both the primers, the results of these two primers were in agreement for 95 samples out of the 101 tested samples. Thus, both the primers can be applied effectively in detection of BHV-1 DNA in semen. When the comparison between direct FAT and gB-PCR was made the relative sensitivity and specificity of direct FAT compared to gB-PCR was found 68.08% and 98.15%, respectively. The overall agreement between these two methods was found to be 84.15%. Similarly when the comparison between direct FAT and gC-PCR was done the relative sensitivity and specificity of direct FAT compared to gC-PCR was found to be 73.17% and 91.67%, respectively. The overall agreement between these two tests was found to be 84.15%. While comparing M-ELISA and gB gene based PCR, 11 bulls revealed the presence of antibodies and viral genome both, where as 25 bulls did not reveal presence of both. Further, Further, 10 seronegative bulls revealed presence of viral genome in semen, where as 4 seropositive bulls could not reveal viral genome in semen. Thus, there was not always correlation between presence of antibody in serum and viral DNA in semen. In sequencing of gB-gene based PCR products a consensus sequence of 459 bp was obtained. The sequences of field isolate matches completely with the sequence of BHV-1.1. Finally, the study revealed presence of BHV-1 antibody in serum and presence of BHV-1 in the semen of breeding bulls of Gujarat. Simultaneously the seronegative bulls also revealed the presence of virus and vice versa. Thus, under Sexual Health Control Programme proper measures must be taken at State level for controlling BHV-1 infection. All breeding bulls must be tested periodically for detection of both BHV-1 antibody in serum and presence of BHV-1 in semen. The bulls must be free from BHV-1 infection prior to use.
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