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

ABSTRACT: 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.

ORGANISM(S): Bos taurus

PROVIDER: GSE159283 | GEO | 2020/12/07

REPOSITORIES: GEO

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Project description:Methods: During the last trimester of the gestation, 12 heifers were immunized using intramuscular injection with heat-killed E. coli P4 emulsified in Montanide ISA 61VG® oil adjuvant (SEPPIC), as recommended by the manufacturer. Two months later, half of the heifers (IM group, n=6) got a second injection as a booster. Others received 50 µg of protein concentrate prepared from E. coli P4 supernate in two quarters (IMM group, n=6). Cows of the control group (CONT, n=6) were injected intramuscularly with adjuvant only at the same dates. Cows were then challenged on average at 49 days in milk in one healthy quarter by infusion of a E. coli P4 bacterial suspension (10e3 bacteria). Blood was collected before experiment, and seven days after the first and the second injection (immunization phase), and then at 0, 12 and 40 hours after infection (challenge phase). Results : Whole blood transcriptome through RNA-seq identifies the main biological functions altered during an E. coli mastitis. Systemic and mammary routes of E. coli immunization elicit different gene expression profiles that correlated with the levels of protection Conclusions: Intramuscular immunization is correlated with a high systemic Th1 response that is not as protective as the local route of immunization. During the last trimester of the gestation, 12 heifers were immunized using intramuscular injection with heat-killed E. coli P4 emulsified in Montanide ISA 61VG® oil adjuvant (SEPPIC), as recommended by the manufacturer. Two months later, half of the heifers (IM group, n=6) got a second injection as a booster. Others received 50 µg of protein concentrate prepared from E. coli P4 supernate in two quarters (IMM group, n=6). Cows of the control group (CONT, n=6) were injected intramuscularly with adjuvant only at the same dates. Cows were then challenged on average at 49 days in milk in one healthy quarter by infusion of a E. coli P4 bacterial suspension (10e3 bacteria). Blood was collected before experiment, and seven days after the first and the second injection (immunization phase), and then at 0, 12 and 40 hours after infection (challenge phase).

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

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