An Investigation of the Innate Immune Response in Bovine Mammary Epithelial Cells Challenged by Prototheca zopfii

Prototheca zopfii is an important bovine mastitis pathogen, which could result in severe mammary infection. However, the innate immune response in bovine mastitis associated with P. zopfii was not clear. Therefore, the aim of this study is to investigate in vitro innate immune responses implicated by P. zopfii. Bovine mammary epithelial cells (bMECs) were infected with 5.0 × 104 cells/ml P. zopfii genotypes I and II independently, and the mRNA expression of TLR-2, TLR-4, TNF-α, IL-1β, IL-8, NOD-1, NOD-2 and β-defensin-5 was determined by real-time polymerase chain reaction (RT-PCR) over a time course of 1, 3, 6, 12 and 24 h. The detection of the NF-κB p65 protein in nucleus and cytoplasm of infected bMECs over the same time course was evaluated. Results showed that P. zopfii genotype II has ability to up-regulate the expression of TLR-2, TLR-4, TNF-α, IL-1β, IL-8, NOD-1, NOD-2 and β-defensin-5 'more strongly than genotype I. Western blot results showed that when bMECs were challenged by P. zopfii genotype II, the expression of NF-κB p65 protein in the nucleus was up-regulated, while in cytoplasm it appeared to be repressed, which indicated that bMECs partly regulate the innate immune responses and inflammation by the NF-κB signaling pathway while being infected by P. zopfii genotype II. It was concluded that adhesion of genotype II was stronger than genotype I, and therefore the genotype II regulatory ability is more robust than that of the genotype I, which causes inflammation of bovine mammary tissue.