Analysis of the immunomodulatory activity of excreted and secreted products from Haemonchus placei transition infective larvae (xL3)

The excretory/secretory (E/S) products released by infective transitory larvae (xL₃) of Haemonchus placei have an important biological function in stimulating immune mechanisms during the invasive process. Our objective was to analyse the modulatory activity of 15 and 70 kDa E/S products from H. placei xL₃. Both E/S products were collected from xL₃in vitro cultures at 24 and 72 h. Proteins were confirmed by SDS-PAGE, and the corresponding spots were elicited by gel isoelectrofocusing (IEF) and characterised by mass spectrometry. Additionally, flow cytometry of CD4⁺/γδ⁺ T cells and immune gene expression were performed by proliferation assays using each E/S product to stimulate lymphocyte and peripheral blood mononuclear cells (PBMCs) from non-infected calves. The IEF results displayed two spots of 7.0 and 5.7 pI for the 15 and 70 kDa products, respectively. Additionally, 29 and 17 peptides from the 15 and 70 kDa E/S products, respectively, were identified with the hypothetical neurotransmitter and enzymatic functions necessary for larval development. The relative expression displayed upregulation of IL4, 5, 6, 8, 10, 13, IFNγ, and FCεR1A genes (from 2.0- to 17.6-fold, p < 0.05) stimulated by the 15 and 70 kDa proteins, indicating specific genes against haemonchosis. Although the percentage of median florescence intensity (MFI%) of CD4⁺/γδ⁺ T cells did not change for both E/S products compared to the negative control and concanavalin-A stimulated cells as the positive control (p > 0.05), the 15-kDa protein reduced the levels of both T cells, and the 70-kDa proteins increased the γδ⁺ cells slightly. Additionally, there was increased PBMCs proliferation by the 70 kDa proteins (p < 0.05), denoting the biological role of other immune cells. The 15 and 70 kDa protein E/S products from H. placei xL₃ showed modulation of the immune response, and although more studies are required, they indicate important functions in the host/parasite interaction.