Innate immune responses induced by lipopolysaccharide and lipoteichoic acid in primary goat mammary epithelial cells

Background: Innate immune responses induced by in vitro stimulation of primary mammary epithelial cells(MEC) using Gram-negative lipopolysaccharide(LPS) and Gram-positive lipoteichoic acid(LTA) bacterial cell wall components are well-characterized in bovine species. The objective of the current study was to characterize the downstream regulation of the inflammatory response induced by Toll-like receptors in primary goat MEC(pgMEC). We performed quantitative real-time RT-PCR(qPCR) to measure mRNA levels of 9 genes involved in transcriptional regulation or antibacterial activity: Toll-like receptor 2(TLR2), Toll-like receptor 4(TLR4), prostaglandin-endoperoxide synthase 2(PTGS2), interferon induced protein with tetratricopeptide repeats 3(IFIT3), interferon regulatory factor 3(IRF3), myeloid differentiation primary response 88(MYD88), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1(NFKB1), Toll interacting protein(TOLLIP), and lactoferrin(LTF). Furthermore,we analyzed 7 cytokines involved in Toll-like receptor signaling pathways: C-C motif chemokine ligand 2(CCL2), C-C motif chemokine ligand 5(CCL5), C-X-C motif chemokine ligand 6(CXCL6), interleukin 8(CXCL8), interleukin 1 beta(IL1 B), interleukin 6(IL6), and tumor necrosis factor alpha(TNF).Results: Stimulation of pgMEC with LPS for 3 h led to an increase in expression of CCL2, CXCL6, IL6, CXCL8,PTGS2, IFIT3, MYD88, NFKB1, and TLR4(P < 0.05). Except for IL6, and PTGS2, the same genes had greater expression than controls at 6 h post-LPS(P < 0.05). Expression of CCL5, PTGS2, IFIT3, NFKB1, TLR4, and TOLLIP was greater than controls after 3 h of incubation with LTA(P < 0.05). Compared to controls, stimulation with LTA for 6 h led to greater expression of PTGS2, IFIT3, NFKB1, and TOLLIP(P < 0.05) whereas the expression of CXCL6, CXCL8, and TLR4 was lower(P < 0.05). At 3 h incubation with both toxins compared to controls a greater expression(P < 0.05) of CCL2, CCL5, CXCL6, CXCL8, IL6, PTGS2, IFIT3, IRF3, MYD88, and NFKB1 was detected. After 6 h of incubation with both toxins, the expression of CCL2, CXCL6, IFIT3, MYD88, NFKB1, and TLR4 was higher than the controls(P < 0.05).Conclusions: Data indicate that in the goat MEC, LTA induces a weaker inflammatory response than LPS.This may be related to the observation that gram-positive bacteria cause chronic mastitis more often than gram-negative infections.

Bovine <i>β</i>-casein: Detection of two single nucleotide polymorphisms by bidirectional allele specific polymerase chain reaction (BAS-PCR) and monitoring of their variation

Due to the functional importance of bovine milk protein polymorphisms, their correct discrimination is of great interest both from a scientific and practical point of view. Nowadays a large number of commercial platforms are available for semiautomated or fully automated SNP geno-typing. However, in some cases the use of simple and rather cheap methods is an effective tool to be implemented within one’s own laboratory for the routine analysis of a specific SNP. The present paper describes two simple tests based on the bidirectional allele-specific polymerase chain reaction (BAS-PCR) developed for the identification of β-casein (CSN2) B and I genetic variants. The practical application of the two methods on a panel of 84 Italian Brown bulls and 100 Italian Friesian cows is also discussed, including the biological significance of the two genetic variants and the importance of taking their occurrence into account when linkage analyses are performed on milk functional properties. A combined system for analysing milk protein variants by isoelectrofocusing (IEF) and the BAS-PCR assay developed for CSN2*I is described.