Fta elute card

A total of 605 G. coprotheres from 24 localities, across the South African distribution of the species, were sampled for this study (Supplementary Table 8). Based on sample localities, samples were grouped into three geographic regions: Western (n = 18), Middle (n = 462) and Northern (n = 125). In addition to the regional grouping, the 266 samples collected from six breeding colonies (Fig. 1) were analysed separately. South African representatives of G. barbatus (n = 54), N. monachus (n = 54), and G. africanus (n = 68) were also included and genotyped using the same microsatellite loci, to allow for direct comparison of genetic diversity values.
Samples consisted of feather samples, collected opportunistically from feeding sites, sites of electrocutions, poisoning events and below nests at six main breeding colonies^{18 (link)}. Blood samples were also collected when vultures were captured and fitted with global positioning system/global system for mobile transmitters^{14 (link)}. Bloods were stored on Whatman FTA® Elute cards (Sanford, USA). Archival museum samples (dried skin snips) were sourced from local South African museums (Supplementary Table 8). Ethical approval was obtained for this study from the University of KwaZulu-Natal Animal Ethics subcommittee (Reference number: 045/15/Animal) and all experiments were performed in accordance with relevant guidelines and regulations.

Genomic DNA from bacterial cultures was extracted based on the method developed by Murray and Thompson (1980)[22 (link)]. Bacterial DNA was isolated from infected plant tissue either by diffusion of bacteria, centrifugation at 4.000 rpm for 10 min in 10°C and application of the previously mentioned protocol, or as crude DNA extract using Whatman FTA Elute Cards (USA). Samples were collected daily after inoculation.

5 d after infection, the animals were monitored for any signs of clinical disease and bled 5 μl from a prick made with a lancet in the marginal ear vein to measure daily parasitemia. Parasitemia was determined using a thick blood smear stained with Giemsa as described in the Earle and Perez (1932) technique (102 (link)). Blood samples were also collected at regular intervals from the femoral vein to assess humoral immune responses against P. vivax blood stage proteins, for complete blood count and blood chemistry (liver and renal panel), for collection of parasite DNA on FTA Elute cards (Whatman), and for RNA in TRIzol solution (Invitrogen) for molecular biology studies. The animals were treated with mefloquine (MQ) at 25 mg/kg orally by gastric intubation to end the experiment.

Whole blood was collected from animals by jugular vein puncture using Vacutainer tubes® with or without anti-coagulant. Blood with EDTA were used for preparation of blood films, for hematological evaluation and for DNA extractions using FTA® Elute cards (Whatman Cat. No. WB120410). Blood smears were stained with Giemsa stain for 15 min and examined by analyzing twenty five fields in each thin blood film at a 1000× magnification [21 (link)]. The indirect immunofluorescent slides of T. equi and B. caballi, as well as the control equine sera were kindly donated by VMRD Inc. (Pullman, WA, USA). The IFAT was performed using equine sera at a 1:50 dilution.

Total DNA was extracted from B. caballi-infected blood using FTA^® elute cards and purification reagent (Whatman) following the manufacturer’s instructions. DNA was eluted and PCR was performed for amplification of the B. caballi sbp4 according to Mahmoud et al. [6 (link)] using primers Bcsbp4-For (5′-ATG GCT GCC TTC TCG ACC CGC TCC-′) and Bcsbp4-Rev (5′-CTC AGA CTT TTC GGC GGC TTC AGC-3′). The sequences of the primers were derived from the sbp4 gene identified in the B. caballi genome. Babesia caballi DNA positive control for PCR was kindly donated from the OIE equine piroplasmosis reference laboratory located in Pullman, WA, USA. The PCR amplicons were electrophoresed on 1.5% agarose gel and stained with SYBR Safe (Invitrogen, Waltham, USA). The length of the amplified products was estimated using a 1 Kbp DNA ladder (Invitrogen) and the amplified products were visualized with an UV trans-illuminator (Bachofer, Germany), and photographed using a gel documentation system (BioDocAnalyze-Biometra Analytik GmbH, Göttingen, Germany).

P. falciparum strains 3D7, Dd2, 7G8, and K1 were used in this study. The parasite was incubated at 37 °C, 5% CO₂ in a flask containing 10 ml of complete medium (see Supplementary Table 7 for culture recipe) with a starting parasitemia of 0.5% for 4 days. The parasite solution was then collected by 600 × g centrifugation at room temperature for 6 minutes. Clinical samples were obtained from 64 parasite-positive patients visiting a clinic at Sam Ratulangi University in Manado, Sulawesi, Indonesia, with informed consent and approved by the Ethical Committee of Sam Ratulangi University. Ten samples were preserved with PAXGene DNA Blood tubes (PreAnalytix) and 54 samples were preserved with FTA Elute cards (Whatman). Additional 11 and 5 parasite-positive samples were obtained from Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand and National Institute of Hygiene and Epidemiology, Hanoi, Vietnam, respectively. For Complexity of Infection (COI) experiment, we mixed laboratory culture of 3D7 and Dd2 strains with parasitemia ratio of 1:9, 2:8, 3:7, 5:5, 7:3, 8:1, and 9:1. All experiments were performed in accordance with the relevant guidelines and regulations.

Plasmodium falciparum 3D7 parasites were cultured in vitro according to previously published protocols [14 (link)] utilizing O⁺ human red blood cells purchased from Interstate Blood Bank (Memphis, TN). Parasitaemia was determined by routine Giemsa staining of blood smears. Final parasitaemia was measured and the number of parasitized red blood cells (pRBCs) per µL was obtained. Isolated ring stage P. falciparum parasites from culture were spiked into whole blood and serially diluted. For spiked sample analysis, gDNA was extracted from 1 mL of whole blood at each parasite dilution. Alternately, approximately 100–200 µL of blood was utilized from capillary collection tubes collected from asymptomatic blood smear negative adults from Ghana. For both spiked and Ghanaian blood samples, lysis of RBCs was induced by adding saponin (Sigma, MO, USA) to a final concentration of 0.15%. Remaining cellular material was sedimented by centrifugation at 16,000×g for 10 min and washed three times with 1× PBS. gDNA from the pellet was obtained using the QIAmp DNA Blood Mini Kit according to the manufacturer’s instructions and eluted in 60 µL ultrapure water. gDNA extracts from dried blood samples spotted onto Whatman^® FTA Elute cards (were prepared according to the manufacturer’s instructions.