Ten healthy calves (6 to 9-month-old) were obtained from a calve farm that was negative for BVDV infections. All animals were confirmed to be free of BVDV,IBRV,BPI3, Mycoplasma bovis infections using enzyme-linked immunosorbent assay (ELISA) kits for the detection of antibodies against BVDV, IBRV, BPI3, or M. bovis (Idexx Labs Inc., American) and by reverse transcription-PCR (RT-PCR) or PCR for viral nucleic acid detection [
26‐
28]. All animal work and experimental procedures were conducted with an approval of Institutional Animal Care and Use Committee of Jinlin University, China. The 10 calves were divided randomly into treatment group and control group, with 5 animals in each group. Each calve of the treatment group was inoculated intranasally (IN) with ~6×10
7.0 tissue culture infective doses (TCID
50) of BVDV JL-1 virus. The animals in control group were inoculated IN with DMEM. All animals were monitored daily for clinical signs as described previously [
29]. Clinical assessments were made at the same time each morning by investigators who were blinded to the treatment groups. Clinical signs included depression, nasal discharge, diarrhea, coughing and rectal temperatures. EDTA-blood samples from calves were collected at days−2 to 0 prior to inoculation and days 2, 4, 6, 8, 10, 12, 14 post-inoculation (dpi) and used to count white blood cells. Additionally, at day 0, 2, 4, 6, 8, 10, 12, 14 post-inoculation (dpi), heparin blood was taken for buffy-coat preparations to test for viremia in the challenged calves. Deep nasal swab specimens were obtained from one day prior to challenge through 14 days post inoculation. The procedure to isolate BVDV from buffy-coat samples was conducted as described previously [
30]. Sera obtained before inoculation with BVDV JL-1 and on day 14, Serum sample were tested for neutralizing antibodies to BVDV JL-1 using standard microtitration procedures [
31].Two calves out from each group were necropsied at 14 DPI, and their tissue samples were collected and fixed in 10% neutral buffered formalin and processed for histopathological examination following hematoxylin and eosin (H&E) staining and immunohistochemistry.
The two groups (treatment and control) were analyzed and compared with respect to the primary clinical signs including rectal temperature, nasal and ocular discharges,diarrhea, leukopenia and virus shedding, using GraphPad Prism (version 4.0) software. The level of statistically significant difference was set at p<0.05.