Vacutainer needle

Blood samples were collected prior to morning feeding from the jugular vein using a 20-gauge BD vacutainer needles (Becton Dickinson, Franklin Lakes, NJ). Samples were collected into evacuated tubes (10 mL, BD Vacutainer^®, Becton Dickinson, Franklin Lakes, NJ) containing either serum clot activator or sodium heparin within 30 minutes after the morning feeding starting from the 1 d after calving (i.e., 0 wk) and subsequent samples were collected weekly until wk 5. After blood collection, tubes with sodium heparin were placed on ice (4°C), and tubes with clot activator were kept at 21°C (~30 min) until centrifugation. Serum and plasma were obtained by centrifugation of clot activator and sodium heparin tubes, respectively, at 1,200 × g for 15 minutes. Aliquots of serum and plasma were frozen (-80°C) until further analysis.

In each of the 22 selected villages, a list of all households keeping pigs was generated in collaboration with the local government. From that list, households were randomly selected and invited to participate. Per household, one animal was selected for blood collection if it was aged three months or older, not weak or emaciated, not pregnant, and did not have a litter less than two months old.
Pigs were restrained using a snare and bled from the cranial vena cava using BD Vacutainer® needles and BD Vacutainer® plain tubes. The blood samples were kept standing in an ice box at 4°C to avoid haemolysis. At the field laboratory, blood was centrifuged and serum harvested into barcoded vials that were stored at -20°C until processed at Makerere University in Kampala, Uganda. Prior to shipment to Friedrich-Loeffler-Institute, Greifswald, Germany, the samples were heat-inactivated at 56°C for 20 minutes.

Blood samples were collected from the jugular vein using 20-gauge BD Vacutainer needles before colostrum (baseline), 48 h after colostrum, and then at 2, 3, and 6 weeks of age. Samples were collected into evacuated serum tubes (BD Vacutainer, Becton Dickinson, Franklin Lakes, NJ, USA) containing either clot activator or lithium heparin for serum or plasma, respectively. After blood collection, tubes with sodium heparin were placed on ice, and tubes with clot activator were kept at room temperature until centrifugation (~30 min) at 1300× g for 15 min at 4 °C and 21 °C, respectively. Aliquots of serum and plasma were frozen (−80 °C) until further analysis for determination of blood metabolites, oxidative stress biomarkers, and acute-phase proteins (APP). Fecal samples were collected by finger stimulation with a sterile glove within 48 h post-colostrum feeding and then at 1 and 3 weeks of age. Fecal debris was removed from the perianal region with a paper towel before fecal sampling, and the first portion of the sample was discarded to avoid cross-contamination with the environment. The samples were placed in a cryovial and immediately frozen in liquid nitrogen at −80 °C until further microbiota analysis.

Samples were taken from participants who met the eligibility and inclusion criteria set by the National AIDs/STI Control Programme. The closed system of blood draw (BD Vacutainer needles and evacuated 5 ml K₃EDTA tubes) was used in obtaining approximately 3.5–4 ml of whole blood. Samples that were not tested on the same day were stored at 2–8°C and tested before the end of 72 hours.