Pet 101 d topo vectors

The recombinant antigens were prepared as previously described [11 (link)]. Briefly, the genes that encode antigens EtsC, OmpA, OmpT, and TraT were PCR amplified from χ7122 (etsC, ompT, traT) and RS218 (ompA) strains using primers found in S1 Table, and then cloned into pET-101/D-TOPO^® vectors (Invitrogen). The sequence and the orientation of the genes in the plasmids were verified by sequencing. Recombinant proteins were produced in E. coli BL21, purified via His-tag using ProBond Ni-NTA resin columns (Invitrogen), and then endotoxin was removed using Pierce^™ endotoxin removal spin columns (Thermo Scientific).

Ninety-six-well plates were coated with 2.0 μg/ml of lipopolysaccharide (LPS, Salmonella enterica serovar Typhimurium, Sigma), salmochelin receptor (IroN), aerobactin receptor (IutA), or 0.25 μg/ml unlabeled chicken IgA (i.e., total IgA; H+L, Thermo Fisher Scientific) overnight at 4°C. LPS is common in gram-negative bacteria, and IroN and IutA are virulence factors involved in iron acquisition. Recombinant IroN and IutA proteins were purified from culture of E. coli BL21 containing the pET-101/D-TOPO vectors (Invitrogen) carrying iroN or iutA genes as previously described (Mellata et al., 2016 (link)). SISs were diluted 1:1 in SEA blocking buffer (Thermo Fisher Scientific), serially diluted 1:2, and incubated for 1 h at room temperature. Goat-anti-chicken-IgA-AP (H+L, Thermo Fisher Scientific) was added, followed by PNPP substrate (Thermo Fisher Scientific), and absorbance was measured at 405 nm. To measure antibody titer, the reciprocal of the highest dilution values doubling the control value (i.e., CON birds) were considered positive. ELISAs were done in duplicate per individual bird and independently replicated twice.