Fecal samples were collected from the anus with
sterilized cotton swabs at 21 days of age just before
weaning. The samples were put on ice and immediately
transported to the laboratory. One-hundred milligrams
of feces were homogenized in 10-fold (w/w) volumes
of phosphate-buffered saline (PBS; 140 mmol/L NaCl,3 mmol/L KCl, 10 mmol/L Na2
HPO4, 2 mmol/L
KH2PO4; pH 7.4). The suspensions were centrifuged at
13000 × g for 10 min at 4°C and the supernatants were
frozen at 30°C until subsequent quantification of IgA
concentration.
Blood samples were collected into vacuum tubes containing
heparin (Venoject® II; TERUMO, Tokyo, Japan)
at 28 days of age (7 days after weaning). The samples
were stored at 4°C and transported to the laboratory
within 24 h after collection. The blood samples were
used to quantify the concentration of plasma IgG and
identify the number of leukocyte subsets. Plasma samples
were recovered by centrifuging the blood at 800 × g
for 10 min at 4°C and frozen at 30°C until use