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The objective of the current study was to investigate the toll-like receptors (TLR),including the soluble forms sTLR2 and sTLR4,involved in innate immune responses of dairy cows to experimentally induced Escherichia coil mastitis.Six clinically healthy Holstein dairy cows received an intramammary inoculation of E.coil Ol11:K58 between 63 and 83 d postpartum.Concentrations of sTLR2 and sTLR4,the proinflam-matory cytokines IL-6 and tumor necrosis factor-α (TNF-α),and acute phase proteins serum amyloid A (SAA) and haptoglobin (Hp) in blood were measured by ELISA.Furthermore,10 mL of milk was collected from challenged quarters immediately before inoculation and at 6,12,24,48,and 72 h after inoculation,and mRNA expression of selected genes,including TLR2,TLR4,IL-1β,IL-6,TNF-α,and IL-8,was quantified by real-time PCR.Escherichia coil intramammary infection elicited a decrease in the circulating levels of leukocytes.Rectal temperature was elevated at 6 h postinoculation (PI).Similarly,the serum concentrations of TNF-α,IL-6,and SAA increased at 6 h PI.However,serum concentrations of sTLR2,sTLR4,and Hp did not differ after challenge.The mRNA expression of TLR2,IL-1β,and IL-8 in milk somatic cells increased at 12 h PI,whereas a decreased IL-6 mRNA expression was detected from 6 to 48 h PI.In conclusion,we found that TLR2 mRNA expression increased in milk somatic cells collected from infected quarters of cows challenged with E.coli,whereas the concentrations of sTLR2 and sTLR4 remained unchanged after challenge.Thus,sTLR2 and sTLR4 may protect the host by sequestrating pathogen-associated molecular patterns during E.coli mastitis.