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).