2.9 Viral challenge assay in cattle
At 90 dpv, cattle were challenged by aerosol exposition as described
previously (Romera et al., 2014; Quattrocchi et al., 2017) with a total
of 5 mL in each nostril of 1x106.81TCID50/mL with BoHV-1 LA strain. At 0, 3, 5, 6, 7, 8,
and 12 dpc, calves were clinically examined, and rectal temperature was
recorded. Clinical score after viral challenge was established according
to: Grade 0 = normal; grade 1 = slight rhinitis with serous mucus with
or without mild serous conjunctivitis; grade 2 = moderate/heavy rhinitis
with fibrinous serous mucus with or without moderate serous
conjunctivitis; grade 3 = fibrinopurulent mucus with moderate or severe
conjunctivitis; grade 4 = rhinotracheitis with or without conjunctivitis
(Romera et al., 2014).
To measure viral excretion, nasal swabs were obtained at 0, 3, 5, 6, 7,
8, and 12 dpc by inserting tampons into each nostril and dipping them in
MEM containing 5,000 IU/mL penicillin, 2,500 µg/mL streptomycin, and 10
µg/mL amphotericin B. For virus titration in nasal swabs, samples were
serially diluted and inoculated onto MDBK cell monolayers, which were
inspected for cytopathic effect (cpe). Virus titration was performed by
the end point dilution method (Reed and Muench, 1938).