Characterization of cytokine gene expression of the bovine mammary gland secretion in dairy cows naturally infected and normal mammary gland during various stages of lactation by real time polymerase chain reaction

The aim of this dissertation thesis was to investigate the transcriptional levels of expression of nine different cytokines in cells isolated from milk of dairy cows in different stages of the lactation cycle. Cows enrolled on the study were followed from the day they were dried off, through the first two weeks of the dry period, at calving until five days after calving. Milk samples were collected in each study point, and cells were separated and mRNA extracted for cytokine analysis through real time PCR. The levels of cytokine expression were evaluated in cows naturally infected and were compared to normal levels of cytokine expression in cows without intramammary infections. iNOS and IL8 were the most expressed cytokines in all enrolled quarters and all stages of lactation. IL17 was found to be more expressed in infected than in normal non infected quarters. The effect of the infected quarter on its contra-lateral non infected quarter was also investigated. For this matter, the levels of cytokine expression of three groups were evaluated. Infected and non infected quarters of the infected animals were compared to non infected control quarters of different animals. IL2Ra was found to be more expressed in infected and non infected quarters of the infected animals than in quarters of the control group. On the other hand, IL8 was found to me more expressed on the control group than on the quarters of the infected animals. IL17 was at found to be more expressed in infected than in control non infected quarters. However, the non infected contra-lateral quarters of infected animals did not differ from infected and from control non infected quarters, suggesting that the infected quarters affected at some intensity the levels of cytokine expression on contra lateral non infected quarters. We also compared levels of cytokine expression in late and early lactation in cows without intramammary infections. IL2Ra, IL8, IL10, IFN-gamma, iNOS and TNF-alpha were expressed in all enrolled cows on both late and early stage lactation samples. IL4, IL6 and IL17 were expressed on the majority of the samples. IL2R, IL6, IL8 and IL10 were found to be more expressed in late than early lactation samples. Although information on bovine mammary gland is available for lactating and non lactating periods, the exact immune mechanisms leading to protection of the mammary gland are still not fully understood and intramammary infections continue to cause a great economic impact to the dairy industry. Better understanding of the mechanisms triggering intramammary infections on the dry period and around parturition would help developing better therapeutics to prevent mastitis. In this study, the combined results suggest that IL17 appears to be an important cytokine involved in subclinical mastitis and could be used as an immunomodulator to be used as an alternative to antibiotic therapies to treat or prevent intramammary infections. However, further studies on IL17 need to be pursued.