Monoject

One blood sample was collected from each calf at six-time points on days 0, 127, 140, 154, 168, and 182 via jugular venipuncture into a 10 mL evacuated tube without additive (Monoject, Covidien, Mansfield, MA, USA). Blood samples were stored in an insulated cooler with ice packs. Samples were allowed to clot at room temperature and then centrifuged at 2100× g for 20 min at 4 °C. Post centrifugation, serum was extracted and transferred to 2 mL microtubes and stored at −20 °C until serological analysis.
Serology was performed at the Oklahoma Animal Disease Diagnostic Laboratory (Stillwater, Oklahoma). A modified microtiter virus neutralization test measured antibody titers against bovine viral diarrhea virus type 1 (BVDV-1) and BRSV [14 (link)]. Briefly, serum samples were diluted 2-fold using Dulbecco’s minimum essential medium (DMEM) in 96-well microtiter plates. An equal volume (25 µL) of virus diluted in DMEM to contain about 100 TCID50/25 µL was added to all sample wells. After incubating the serum/virus mixtures for 1 h, a cell suspension of MDBK cells containing about 10⁴ cells in DMEM containing 10% fetal bovine serum was added to each well. Plates were incubated for 3 days at 37 °C, and wells were examined for virus-specific cytopathic effects (CPE). Titers were expressed as the reciprocal of the highest dilution of serum that completely neutralized the virus.

Whole blood samples were collected from 10 AGMs naturally infected with T. trichiura [28 (link),29 ] as part of other studies and transferred to 5 mL vacutainers (Covidien Monoject, Massachusetts, USA). Sera were separated immediately by centrifugation at 2,000 g for 15 min at 4°C and stored at -80°C until thawed at room temperature and pooled for analysis as below.