PSIX-16 Relationship between gastrointestinal nematodes and milk somatic cells in Katahdin ewes.

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Abstract During late pregnancy and lactation, ewes undergo a rise in the fecal egg counts, which marks an increase in the number and activity of gastrointestinal nematodes (GIN). An increase in GIN reduces nutrient availability and challenges the ewes’ immune system. Previous research associated the rise of GIN with cases of mastitis. However, the relationship between GIN infection and the mammary gland immune cell population and activity has not been established. This project aims to study the correlation between the GIN levels in lactating ewes and their somatic cell counts, differential cell count, and expression of cytokines. Twenty-four primiparous Katahdin ewes were used in this preliminary exploratory study. Fecal and milk samples were collected 7 days post-lambing. Fecal egg count (FEC) was performed using the fecal samples. Total somatic cells (SC) were determined in the milk. Two 50 mL milk aliquots were centrifuged to extract the cell pellets. One of the pellets was smeared in a glass slide and stained with panoptic cell staining. Slides were evaluated to determine the proportion of lymphocytes, macrophages, neutrophils, and eosinophils. The second pellet was resuspended in RNA shield and preserved at -80°C. RNA was extracted and used to synthesize cDNA. qPCR for 40 cytokine-related genes (interleukins, chemokines, TNF, and TGF) were performed using Qiagen RT2 Primer Assay Plates. Theil-Sen Siegel regression analysis was used to evaluate the cause-effect relationship between FEC and SC traits. For the correlation analysis, the ewes were divided between HIGH and LOW GIN load based on the FEC median (1925 eggs/g of feces). The correlation was performed using Spearman’s correlation coefficient (ρ). Statistical significance was considered at p < 0.05, and coefficients ρ > 0.70 were deemed strong and biologically relevant. Regression analysis showed that FEC was positively related (p < 0.05) with the percentage of lymphocytes, eosinophils, and four chemokines (C5, CCL16, CCL17, and CCL19). FEC were negatively related to chemokines (CCL1, CCL5, CCL8), interferon (IFNG), interleukins (IL1A and IL6), Tumor Necrosis Factor (TNF), and TNF receptors (LTA, LTB, CD40, and TNFSF10). Correlation analysis in HIGH ewes showed a strong negative correlation between FEC and CD4 gene (ρ = -0.71). Meanwhile, in LOW sheep, FEC negatively correlated with IFNG (ρ = -0.70) and IFN1 (ρ = -0.72) and positively correlated with C5 (ρ = 0.73). These results suggest that FEC is related to greater lymphocytes and eosinophils in the SC; however, FEC was negatively associated with the expression of multiple cytokines. The data support GIN’s capability of reducing the activity of the SC in the mammary gland and the failure of these cells to protect the gland against mastitis. Further studies should aim to extend the research to multiparous ewes and experimentally controlled GIN infections.

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