Leukocyte concentration
A 20μl blood filled capillary was released in 1ml of a kit solution (LEUKO-TIC “blue”, Bioanalytic, Germany). This solution allows a microscopic count of leukocytes after the lysis of the erythrocytes and the fixation of the leukocyte nucleus stained in light blue. The counts were done at x100 enlargement using a Malassez counting chamber by a single observer (C.R.). Only the leukocytes entirely located inside the four 1mm² corner squares (total volume of 4*1mm2*0.2mm = 0.8µl) were counted. All determinations of leukocyte concentration were done within 24 hours after blood collection.
Between 2013 and 2015, the leukocyte concentration was determined for 79 samples from 34 dominant individuals sampled between two and three times. 23 individuals had two samples in different years and 11 individuals had three. In this dataset, the age of the individuals varied from 3 to 12 years with an average age at 6.2 years (Figure S1A).
Leukocyte profile
Immediately upon blood collection, a drop of blood was smeared onto a slide, later stained with Giemsa stains using an aerospray (Aerospray Hematology Slide/Cytocentrifuge 7150, Wescor, France). Neutrophils, lymphocytes, monocytes, eosinophils and basophils were counted (observer: CR) up to 100 leukocytes, at 100x enlargement, according to Hawkey and Dennett’s criteria (Hawkey & Dennett, 1989).
In Mammals, lymphocytes and neutrophils make up the majority (80%) of the leukocytes (Jain 1993). Lymphocytes play a central role in adaptive immunity: they are involved in immunoglobulin (antibodies) production, in the modulation of immune defence and in the production of memory cells (Jain, 1993; Roitt et al., 2001). Neutrophils are involved in the innate immune response as the primary phagocytic leukocytes, and circulating phagocytes proliferate in response to infections, inflammation and stress (Jain, 1993). Monocytes are long-lived phagocytic cells associated with innate defences against infections and bacteria (Roitt et al., 2001). They differentiate into macrophages in tissues and they are also involved in antigen presentation and cytokine production. Eosinophils play a role in the inflammation process and are associated with defence against internal parasites (Jain, 1993). Basophils, which are rare, play a key role against macroparasites and are also involved in the inflammation process (Karasuyama et al., 2011). The relative number of basophils was low for nearly all individuals (min = 0, max = 49, median = 0) and therefore excluded in the subsequent analyses.
Between 2011 and 2015, the leukocyte profiles were determined for 169 blood smears from 52 dominant individuals sampled between two and five times. 18 individuals had two samples in different years, 12 had three, 13 had four, and 9 individuals had five. 33 of them were both measured for leukocyte concentration and leukocyte counts (which corresponds to 75 measurements). In this dataset, the age of the individuals varied from 3 to 12 years with an average age at 5.8 years (Figure S1B).