Plasmodium, Marchiafava & Celli, 1885

Charpentier, M. J. E., Boundenga, L., Beaulieu, M., Dibakou, S. E., Arnathau, C., Sidobre, C., Willaume, E., Mercier-Delarue, S., Simon, F., Rougeron, V. & Prugnolle, F., 2019, A longitudinal molecular study of the ecology of malaria infections in freeranging mandrills, International Journal for Parasitology: Parasites and Wildlife 10, pp. 241-251 : 243

2.4. Plasmodium diagnosis and parasitaemia estimate

Infections were determined for 215 blood samples collected throughout the years on 53 males and 47 females (1–7 samples per individual). Total DNA was extracted from approximately 200 μl of red blood cells using the DNeasy Blood and Tissue Kit (Qiagen, France) and used as templates for the detection of Plasmodium species according to previously described protocols (Prugnolle et al., 2010). The amplification of the portion of cytochrome b (cyt-b) gene is based on a nested PCR with two sets of primers (DW2-DW4: (Perkins and Schall, 2002); Cytb1 and Cytb2: (Schwöbel et al., 2003)). All amplified products (10 μl) were run on 1.5% agarose gels in Tris-acetate-EDTA (TAE) buffer. The PCR-amplified products were used as templates for sequencing (Eurofin MWG). Only two haemosporidian species were detected in the samples: P. mandrilli and P. gonderi .

We then developed and used a qPCR for detecting co-infections within each sample and quantifying each parasite species (i.e. parasitaemia). Primers for qPCR amplification were designed to amplify two different fragments located in the cytochrome b gene, one specific of P. mandrilli (106bp) and the other of P. gonderi (188bp). Primer sequences for amplification of specific P. mandrilli fragment (106 bp) were F 5′-CATACGTCACACCAATACAG-3′ and R 5′-GTTAAAACAATTAATAAA CCTGCA-3'. Primers sequences for amplification of specific P. gonderi fragment (188 bp) were F 5′-GTTATTGGGGTGCAACTGTT-3′ and R 5′-GCTACCATGTAAATGTAAAAAGAAA-3'. qPCR amplifications were performed in a Light Cycler 96 (Roche). Reaction mixtures were prepared in a 10 μl final volume containing 1.5 μl of template DNA, 2 μl of 5X Hot Firepol Evagreen qPCR Mix Plus and 5 pmol of each primer. The qPCR conditions consisted of an initial melting cycle at 95 ̊C for 12min, followed by 40 cycles of amplification at 95 ̊C for 15 s, 56 ̊C ( P. mandrilli ) or 57 ̊C ( P. gonderi ) for 20 s and 72 ̊C for 20 s. Dissociation curves were generated after the final amplification cycle by denaturing the amplicons at 95 ̊C for 15 s, 56 ̊C for 1 min and 95 ̊C for 1 s. Dissociation curves were used to estimate the specific melting temperature of both amplicon. To evaluate the efficiency and quantify parasitaemia, standard curves were generated for each species from 10- fold serial dilutions of synthesized fragments.

2.5. SIV-STLV

SIV status was determined for 209 sera collected on 53 males and 46 females (1–6 samples per individual) and by identifying specific antibodies against SIVmnd-1 and SIVmnd-2 as previously reported (Souquiere et al., 2001). Briefly, we determined the antibody reactivity against the SIV V3 loop Env protein by a specific SIVmnd peptide-based immunoassay. Positive samples were further tested by PCR and pol sequenced for phylogenetic analyses aiming to differentiate between both SIV types (Souquiere et al., 2001). Positive sera were further confirmed by western blotting and primary infection was determined according to a composite criterion including seroconversion patterns between serial samples or the increasing immunoassay reactivities combined to Western blot profiles (Onanga et al., 2006) . STLV status was determined using a commercial immunoassay HTVL-1-based (ARCHITECT rHTLV-I/II immunoassay Abbott, Chigaco Ill) according to the antigenic homology between both HTLV-1 and STLV-1 (Mahieux et al., 1998). The study mandrills were exclusively infected by SIVmnd- 1 and STLV-1 as expected given their range. For further analyses, we only considered males (aged 6 yrs and older) because only two adult females were found SIV-positive and one adult female was STLV-positive (unpub. data). In addition, prevalence of both SIV and STLV in young animals (≤5 and ≤7 yrs resp.; unpub. data) was null.