Transcriptome analysis of mammary CD45+ CD4+ lymphocytes after immunization against E. coli P4 strain and homologous challenge

Methods: During the last trimester of the gestation, three heifers were immunized using intramuscular injections of heat-killed E. coli P4 emulsified in Montanide ISA 61VG® adjuvant (SEPPIC). Two months later, they received a mammary ductal injection of 50 µg protein from concentrated E. coli supernate as a booster. Control cows were injected intramuscularly with adjuvant only at the same dates. Cows were then challenged on average at 140 days in milk in one healthy quarter by infusion of a E. coli P4 bacterial suspension (10e3 bacteria in total). Cows were then euthanized 16 hours after inoculation for mammary tissue collection. After collagenase and DNase treatment and Percoll-gradient separation, CD45+ CD4+ mononuclear cells were sorted by flow cytometry. RNA was extracted and sequenced using Illumina protocol. Results : Mammary Th17-polarized CD4 T cells predominate in the mammary tissue of cows immunized through the local route, and are activated after challenge with E. coli. Conclusions: Local immunization promotes the development of TH17 immunity that correlated with protection. Overall design: During the last trimester of the gestation, three heifers were immunized using intramuscular injections of heat-killed E. coli P4 emulsified in Montanide ISA 61VG® adjuvant (SEPPIC). Two months later, they received a mammary ductal injection of 50 µg protein from concentrated E. coli supernate as a booster. Control cows were injected intramuscularly with adjuvant only at the same dates. Cows were then challenged on average at 140 days in milk in one healthy quarter by infusion of a E. coli P4 bacterial suspension (10e3 bacteria in total). Cows were then euthanized 16 hours after inoculation for mammary tissue collection. After collagenase and DNase treatment and Percoll-gradient separation, CD45+ CD4+ mononuclear cells were sorted by flow cytometry. RNA was extracted and sequenced using Illumina protocol.