On day 28 after immunization, all of the immunized calves were challenged by the IN route with a high dose of virulent BHV-1 strain Cooper (2 × 107 PFU per animal). Following challenge, calves were clinically evaluated for temperature and for the severity of nasal lesions.

The mean rectal temperature of calves in all groups showed a sharp increase after three days of challenge (Fig. 7). However, in the group vaccinated with rLaSota/gDFL virus, the temperature in two calves (R42 and R45) returned to normal by the fifth day post-challenge. These were the two calves with detectable BHV-1-neutralizing Akt inhibitor serum antibodies (Table 4). In contrast, the animals in groups immunized with rLaSota and rLaSota/gDF maintained an increased temperature over a period of eight days (Fig. 7). In

addition, whereas all of the challenged calves developed nasal lesions characteristic of BHV-1, those of calves R42 and R45 of rLaSota/gDFL group were smaller than Ku-0059436 datasheet for the other animals (data not shown). These data indicated that there was a partial protection from BHV-1 disease in two out of three calves immunized with the rLaSota/gDFL vaccine. Shedding of BHV-1 challenge virus was monitored by taking nasal swabs from day 1 to day 10 post-challenge. Infectious BHV-1 was quantified by plaque assay on MDBK cells (Fig. 8). In the control group immunized with rLaSota, the peak mean titer of challenge BHV-1 was approximately 5.0 log10/ml from days 3 to 5, after which shedding decreased but continued through day 10, the last study day. In animals immunized with rLaSota/gDF, the peak mean titer of challenge virus was approximately 5.0 log10/ml on day 3, after which it decreased to 3.0 log10/ml on days 4, 5, and 6, with shedding terminated by day 8. In animals immunized with rLaSota/gDFL, the mean titer of challenge virus did not exceed 3.0 log10/ml, and shedding terminated by day 7. These data indicated that there was partial restriction

of the BHV-1 challenge in calves immunized with either the rLaSota/gDFL or rLaSota/gDF virus, and suggested that the protective efficacy of rLaSota/gDFL virus was greater than that of the rLaSota/gDF virus. To measure MTMR9 the anamnestic response elicited in rNDV-immunized calves following BHV-1 challenge, sera were collected following challenge and analyzed by a commercial ELISA kit using purified BHV-1 virions as antigen (Fig. 9) and by the plaque reduction assay (Table 4). On day 12 post-challenge (day 40 post-immunization), the serum IgG response against BHV-1 was increased significantly in the rLaSota/gDFL and rLaSota/gDF groups compared to the rLaSota group (the average S/P ratio was 3.75, 3.16 and 2.49 in the rLaSota/gDFL, rLaSota/gDF and rLaSota group, respectively) (Fig. 9).