Edta containing vacutainer tubes

Nasal and ocular swabs, and whole blood samples were obtained from randomly selected live goats with clinical presentation suggestive of PPR. Whole blood was collected from the jugular vein using EDTA-containing vacutainer tubes (BD Biosciences, Franklin Lakes, NJ, USA). In addition, lungs, intestines, lymph nodes, whole blood and, ocular and nasal swabs were obtained from five goats in Mvomero district and four goats in Ngorongoro district that died naturally. Buffy coat from whole blood was obtained by centrifuging 500 μl of histopaque (Sigma-Aldrich, St. Louis, MO, USA) layered with 1 ml of whole blood at 400 g for 30 min at 4°C. The upper layer was discarded while the opaque interface containing the buffy coat was carefully collected. Nasal and ocular swabs were collected and placed in universal viral transport medium (BD Biosciences) followed by vortexing to dislodge cells from the swabs and centrifugation at 8000 g for 5 min at room temperature. Portions of lungs, intestines and lymph nodes from the same animal were pooled before they were homogenized in F-12 basal cell culture medium (Invitrogen, Carlsbad, CA, USA) to obtain a 10% tissue suspension. The tissue supernatant was obtained by centrifugation of tissue suspensions at 8000 g for 5 min at room temperature. All samples and the buffy coat were stored at −80°C until RNA extraction was performed.

Whole blood was collected in EDTA-containing vacutainer tubes (Becton Dickinson Co., Rutherford, NJ, USA); genomic DNA was extracted from peripheral blood mononuclear cells (PBMC) using a standard phenol/chloroform procedure. The DNA amount for each sample was determined by measuring the optical density at 260 nm wavelengths using a spectrophotometer (SmartSpec Plus, Bio-Rad, Irvine, CA, USA). The DNA samples were stored at −20 °C until use.

To study neuro-endocrine parameters, whole blood was collected in vacutainer tubes (Becton Dickinson and Co., Rutherford, NJ, USA) at baseline (T0) and after intervention (T1). The blood samples were centrifuged at 3000 rpm for 10 min to separate sera. Serum was stored at −80 °C until use.
Plasma was obtained from 10 milliliters of whole blood collected in EDTA-containing vacutainer tubes (Becton Dickinson and Co., Rutherford, NJ, USA), centrifuged at 3000 rpm for 10 min, and stored at −80 °C until use.

Peripheral blood samples were collected in EDTA-containing vacutainer tubes (Becton Dickinson and Co., Rutherford, NJ, USA), and were centrifuged at 2000× g for 10 min to obtain plasma and stored at −80 °C until testing.

Whole blood was collected in 3 mL EDTA-containing vacutainer tubes (Becton Dickinson Co., Rutherford, NY, USA); genomic DNA was extracted from blood using standard phenol/chloroform procedures. Phenol chloroform extraction was performed by inducing cell lysis and DNA release with sodium dodecylsulfate (SDS) and proteinase K. Next, a phenol/chloroform/isoamyl alcohol mixture was added to the cell lysate to separate the proteins from the DNA [30 ]. The amount of DNA present in each sample was determined by measuring the optical density at 260 nm wavelengths using a Nanovue spectrophotometer (GE Healthcare, Chicago, IL, USA). DNA samples were stored at −20 °C until use.