In the HI assay, 1% chicken erythrocytes and wild type NDV strain LaSota was used as the indicator virus. Serial 2-fold dilutions of heat inactivated (56 °C, 30 min) calf sera were used to inhibit 4 HA units

of the virus. Antibody responses to BHV-1 in calf sera were determined by Western blot analysis. MDBK cells were infected with BHV-1 at an MOI of 5 PFU per cell. The overlying medium was harvested after 24 h of infection. BHV-1 particles were purified from the harvested medium by sucrose gradient centrifugation. Purified BHV-1 was separated on 8% SDS-PAGE gel and blotted on to nitrocellulose membrane and incubated overnight in dilution buffer (Synbiotics, Kansas city, MO). Next day, the membranes were incubated for 2 h at room temperature with calf sera diluted 1:40 in dilution buffer. Membranes were washed with washing solution (Synbiotics, Kansas 26s Proteasome structure city, MO) four times and incubated with 1:1000 diluted HRP conjugated goat anti-bovine IgG (KPL, Gaithersburg, MD) for 1 h at room temperature. After washing four

times, gD-specific protein was detected using a chemiluminescence assay kit (GE Healthcare). Neutralizing antibodies to BHV-1 in calf sera were measured by plaque reduction neutralization assay in MDBK cells. Serial 2-fold dilutions of heat inactivated calf sera were mixed with 100 PFU of BHV-1 and incubated for 2 h at 37 °C. The residual infectious virus in the serum–virus mixture was quantified by plaque BGB324 order assay on MDBK Idoxuridine cells. The titers were expressed as the reciprocal of the highest dilution of the serum that reduced the plaque number by 60%. BHV-1 specific IgG and IgA responses were measured in serum and nasal secretions, respectively, by ELISA using the SERELISA BHV-1 total

Ab mono indirect kit (Synbiotics Corporation, Lyon, Cedex 07, France). Briefly, 1:20 dilutions of days 0–28 and 1:500 dilutions of day 41 bovine sera or 1:2 dilution of nasal secretions were incubated in duplicate on BHV-1 viral antigen coated plates for 1 h at 37 °C. Bound antibodies were detected using horseradish peroxidase-conjugated anti-bovine IgG antibodies (Kirkgaard Perry Lab.). IgG and IgA titres in serum samples and nasal secretions were expressed as sample to positive (S/P) ratio. The S/P ratio was calculated by subtracting the average normal control absorbance from each sample absorbance, then dividing the difference by the corrected positive control, which is the difference between average positive absorbance and average normal control absorbance. According to manufacturer’s protocol, a sample was considered to be positive for BHV-1 antibodies if the S/P ratio was ≥0.3. The recombinant lentogenic NDV strain LaSota containing a unique PmeI site between the P and M genes [31] was used as a vector to express the BHV-1 gD glycoprotein from an added gene.