| The dairy goat industry has been the characteristic advantage industry of Shaanxi province, and it is now significant for most provinces in China dependent on its economic benefits, including Shandong, Yunnan, Henan, Northeast three provinces et al. Approximately 12 million dairy goats are bred and over 1.6 million tons of goat milk are produced in China. The same as dairy cows, milk powders are considered as the main products of dairy goats, which quality is greatly affected by fresh milk. As in previous studies, mastitis, a threat to milk production and the nutritional value of goat milk, is not only a significant hurtle to the healthy development of the dairy goat industry, but also causes considerable economic losses to dairy farmers. Therefore, prevention and control of mastitis is highlighted by dairy goat breeding, as is the case for dairy cows. However, there are limited information available for the epidemiological status of subclinical mastitis in dairy goats and limited knowledge of immune mechanisms of the mammary gland, which present an enormous challenge for the prevention and control of mastitis and result in increased therapeutic failure. Thus, an epidemiological investigation of subclinical mastitis was firstly carried out in dairy goats, and immune mechanisms of the mammary gland defense against infection were elementarily studied in the present study. The results obtained in this study expand our current understanding of subclinical mastitis in dairy goats in China and lay the foundation for immunotherapy of mastitis in dairy goats.We detected the prevalence of and pathogens involved in subclinical mastitis in dairy goats in the main breeding areas of China, by using California mastitis test(CMT) and a multiplex PCR assay, respectively. We established a mouse model of S. aureus(Staphylococcus aureus) mastitis, and T helper cell polarization and the cytokines involved in the differentiation and function of distinct T helper cell subsets were assessed in this murine model by flow cytometry and quantitative real-time PCR, respectively. Mechanisms of IL-17 and Th17 cells mediated the immune responses against S. aureus infection were also discussed in this study. Main results are as follows:1. A total of 683 dairy goats in the main breeding areas of China were selected and milk samples were collected. Out of these, 313 milk samples were detected distinct or strong positive for subclinical mastitis. The positive rate of samples from Shaanxi, Yunnan and Shandong provinces were 48.92%, 36.08% and 52.67%, respectively; and the main mastitis-causing pathogens were listed as follows: coagulase-negative staphylococci(59.52%), S. aureus(15.24%), Escherichia coli(11.43%) and Streptococcus spp.(10.95%). The results show that subclinical mastitis is a highly prevalent disease in dairy goats in China.2. After challenge with an inoculum of 4 × 106 colony-forming units of S. aureus in the mammary gland of lactating mice, pustules, tissue degeneration and alveolar atrophy were observed in the infected mammary gland, accompanied by inflammatory cells infiltration. This results show that a murine mastitis model was successfully established in this study.3. The mRNA level of IL-17 in the infected mammary gland was 1.58-fold higher; Administration of recombinant IL-17 to WT mice at the time of bacterial infection resulted in a significant increase in mRNA expression of CXCL1, CXCL2 and CXCL5(p < 0.05), and a dramatically increased neutrophils infiltration. Treatment with anti-IL-17 antibody decreased inflammatory cell infiltration in the infected mammary gland, and significantly decreased the CXCL1, CXCL2 and CXCL5 mRNA(p < 0.05). Taken together, these findings show that IL-17 mediated mastitis in mice. After infection, the proportion of Th17 cells was significantly increased(p < 0.05); Cytokines involved in Th17 differentiation, including TGF-β, IL-1β and IL-23, were greatly increased(p < 0.05). These results show that IL-17 produced by Th17 cells mediated mastitis in mice. In murine S. aureus mastitis, the proportion of Th17 cells in CD4+ T cells in spleen was significantly increased(p < 0.05), and TGF-β, IL-1β, IL-6, IL-21 and CCR6 mRNA in spleen of the infected mice were significantly increased(p < 0.05). However, IL-17 mRNA was significantly decreased(p < 0.05), and IL-23 mRNA was not markedly changed in spleen following S. aureus intramammary infection. Thus, these results suggest that the initial differentiation of Th17 cells from na?ve T cells may progress in spleen, and then the committed Th17 cells are recruited to the infected mammary gland for the generation of pathogenic effector Th17 cells.4. The increase in Th1 cells and IL-12 mRNA was significantly detected after challenge(p < 0.05). The expression level of IFN-γ was a slight but detectable increase in the infected mammary gland. These results suggest that Th1 cells may play a role in host defense against S. aureus intramammary infection via the production of IFN-γ.5. In murine S. aureus mastitis, we found that the proportion of Th2 cells in CD4+ T cells and the relative expression of its prototypical cytokine IL-4 were significantly increased(p < 0.05), which indicate that Th2 cells also involved in the immune clearance of S. aureus from the udder.6. The proportion of Treg cells and the mRNA level of IL-10 were significantly increased after challenge(p < 0.05). This show that immunity against S. aureus infection in a murine mastitis model is tightly regulated by Treg cells and IL-10.In conclusion:1. Subclinical mastitis is a highly prevalent disease in dairy goats in China, and coagulase-negative staphylococci are the predominant pathogens.2. In the adaptive immune responses, IL-17, a critical cytokine mediates mastitis, produced by effector Th17 cells in the mammary gland of mice.3. The other T helper cells, including IFN-γ+ Th1 cells and IL-4+ Th2 cells, also contribute to the mammary gland defense against bacterial infection. These responses are tightly regulated by Treg cells and anti-inflammatory cytokine IL-10 to avoid bystander damage to the tissue of the mammary gland. |
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