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  • TNP is an evergreen rainforest located in the south-west of Côte d’Ivoire (0°15’– 6°07’N, 7°25’– 7°54’W). The climate of TNP is sub-equatorial with two rainy seasons (major: August-October, minor: March-June) and a total average annual rainfall of 1800 mm. While the Upper Guinea Forest belt once stretched from Ghana to Sierra Leone, TNP is the largest remaining section today, covering an area of 3300 km2, and is surrounded by a 200 km2 buffer zone. Almost 1000 species of vertebrates have been described in the TNP ecosystem, and the park was awarded UNESCO Natural World Heritage status in 1982 [19,20]. This work has been performed in the research area of the Taï Chimpanzee Project that has studied the local habituated chimpanzee groups since 1979 [19]. Samples of red colobus monkeys, black-and-white-colobus monkeys and sooty mangabeys were collected between 2006 and 2015, resulting in a total of 41 monkey samples (S1 Table). Monkeys were anesthetized using a combination of ketamine (5mg/kg) and medetomidine (0.05mg/kg), administered intramuscularly via blowpipe (Telinject GmbH, Dudenhofen, Germany) or dart gun (Dan- Inject, Borkop, Denmark). Induction took 5–10 min and anesthesia lasted for 30–40 min. Blood samples were collected in EDTA coated tubes. After antagonization with an intramuscular injection of 0.25mg/kg atipamezole, recovery took 60–120 min. Animals were not left before they could climb without observable difficulties and returned to their social group. Blood samples were centrifuged at 3000 rpm for 10 min upon return to our forest laboratory, separated into plasma, buffy coat and erythrocytes and subsequently stored in liquid nitrogen. Samples were transported on dry ice and conserved at -80°C for long-term storage. Sampling of Maxwell’s duikers was performed in 2013 and 2016 (S1 Table). A total of nine duikers were trapped using the night-time net capture technique described by Newing [21], which is based on the animals freezing when stunned with a strong flashlight. Following capture with the net capture technique, animals were initially anesthetized using medetomidine (0.1 mg/kg), ketamine (4.0 mg/kg) and midazolam (0.1 mg/kg). Because the level of anesthesia reached with this combination of anesthetics was rather deep for our needs, dosages were adapted to 0.07 mg/kg medetomidine and 2.5 mg/kg ketamine with slightly increased midazolam (0.17 mg/kg). This combination yielded appropriate anesthetic depth for our sampling needs. Induction took 5–10 min and animals were anesthetized for 30–50 min. The same sampling protocol described above for monkeys was used for duikers. After antagonization with atipamezole, animals fully recovered within 20–90 minutes. A large well-trained team is needed for this method for capturing duikers and further optimization might be possible (e.g., combining dazzling with a flashlight with GPS marked darts for anesthesia). One chimpanzee serum sample taken under anesthesia during an emergency surgery in a chimpanzee with air-sacculitis was available from 2009 [22]. A further eight whole blood samples were obtained during necropsies of freshly deceased animals that died in outbreaks of respiratory disease in 2004, 2006 and 2009 that tested negative for Bcbva in qPCR (S1 Table) [23,24]. All wildlife samples were collected with permission of the research ministries of Côte d’Ivoire and ethical approval of the Ivorian Office of National Parks, which reviewed the study design (permits Nr. 048/MESRS/DGRSIT/KCS/TM and 90/MESRS/DGRSIT/mo). Samples have been exported with the required CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) permits. The study was approved by the Centre Suisse de Recherche Scientifique en Côte d’Ivoire and the Laboratoire National de la Pathologie Animale, Bingerville, Côte d’Ivoire. All chimpanzee samples originated from free-ranging chimpanzees. Samples were collected from chimpanzees that had died of natural reason in outbreaks of respiratory disease by our team of veterinarians routinely investigating wildlife mortality in TNP [23,24]. In one case samples were obtained from an individual on whom surgery had to be performed due to a life threatening infection [22]. No chimpanzee was anesthetized or touched for the sole purpose of sample collection. Human sera used as controls in this study were donated by the authors of the study themselves and were anonymized immediately after sample donation. Humans were not vaccinated against anthrax to serve as controls in this study, but had received anthrax vaccinations in the past due to their work in anthrax endemic areas. All human sera were donated by adults after giving written informed consent, and the use of human sera in this study was approved by the ethics committee of Charité-Universitätsmedizin Berlin. No standardized approaches are available to investigate anthrax seroprevalence in wildlife. Thus, all samples were tested for antibodies against the anthrax protective antigen (PA, Quadratech Diagnostics, Surrey, UK) using an in-house ELISA and an in-house Western Blot and for antibodies against anthrax lethal factor (LF, Quadratech Diagnostics) using an in-house Western Blot. Assays are described in detail below. Testing for PA and LF does not allow for a discrimination between classical B. anthracis and Bcbva, as both pathogens produce the typical anthrax toxins [25]. However, during our extensive carcass monitoring over the last 15 years, each of the 81 anthrax cases that were detected in TNP was caused by Bcbva. This was shown by qPCR screening for the Bcbva specific genomic island IV, isolation and subsequent whole-genome sequencing [8]. Therefore, in the TNP ecosystem antibodies generated against PA and LF likely originate exclusively from exposure to Bcbva. Human positive and negative controls were used for all monkey and chimpanzee assays as no species-specific controls were available. Negative controls were selected from a set of available human sera of unvaccinated donors, which were unreactive in Western Blot against PA and LF. Two of these sera that were representative of the range for PA-negative human sera in PA ELISA were chosen as negative controls and included on each ELISA test-plate under the same conditions as the samples for inter-plate comparison. A positive control serum from a hyper-immunized human donor was included as an 8-step log2 serial dilution curve (starting concentration: 1 in 4000) with repetitious reactivity and accurate results on every test-plate. For duiker assays, a negative control was available from a red forest duiker (Cephalophus natalensis, courtesy of Berlin zoo), which was unreactive in PA and LF Western Blot. A pool of goats vaccinated with the B. anthracis Sterne spore live vaccine [26] (courtesy of Dr. W. Beyer) was used as a positive control in the same fashion as stated for the positive human control (starting concentration: 1 in 1000). No specific conjugated antibodies were available for any of the species tested. For primate samples, polyvalent goat anti-human horseradish peroxidase (HRP) labeled conjugate (Dianova, Hamburg, Germany) was used, as described previously [27,28]. For duikers, we tested the reactivity of duiker serum with different commercially available conjugates from the Bovidae family (sheep, cow, goat) in a comparative dot blot approach with logarithmic duiker serum dilutions starting at 1:10. We found that polyvalent rabbit anti-goat HRP labeled conjugate (Dianova, Hamburg, Germany) was the most suitable commercially available conjugate for duiker samples. PA ELISA was performed as described by Hahn et al., with slight modifications [29,30]. Briefly, each well of high-binding microtiter plates (Maxisorp Nunc, Sigma Aldrich, Munich, Germany) was coated with 0.1 μg of recombinant PA in PBS at 4°C overnight. Wells were washed with phosphate-buffered saline containing 0.02% (v/v) Tween 20 (PBS-Tween) and blocked with 5% skimmed milk powder in PBS-Tween. Samples and negative controls were diluted 1:500 in blocking solution and incubated in duplicate together with the positive and negative controls for 2 hours at room temperature. Secondary antibodies were used in a concentration of 1:10000 and 1:4000 (previously evaluated and adjusted) for humanoid and duiker assays, respectively. Plates were developed in the dark with 100 μl of TMB SeramunBlau fast (Seramun Diagnostics GmbH, Heidesee, Germany) substrate per well for 10 min and stopped with 100 μl H2SO4 (2M). Absorbance was measured at 450nm (reference wavelength 620nm) using a Tecan Sunrise 96-well-reader (Tecan Group Ltd., Männedorf, Switzerland). The mean of the negative controls plus two times their standard deviation (SD) was set as the cut-off value for each plate (S1 Table). For the PA and LF Western Blot assay, 380 ng of purified recombinant PA or LF diluted in 125 μl of phosphate-buffered saline (PBS) were blotted onto an Immobilion-P PVDF-Membrane (Merck, Darmstadt, Germany) after running in a 12% agarose gel. Then 3 mm stripes (approx. 25 per gel) were cut from the membrane and samples and controls were added in a dilution of 1:1000 in the dilution buffer containing tris-buffered saline with 0.05% Tween (TBS-Tween) and 3% powdered milk. Samples were incubated at room temperature for one hour. Goat anti-human HRP conjugate was added to primate samples and human controls in a 1:10000 dilution in the dilution buffer (1:8000 for LF Western Blot). For duiker samples and goat/duiker controls, rabbit anti-goat HRP conjugate was diluted 1:4000 in the dilution buffer (1:8000 for LF Western Blot). The conjugate was left to incubate for one hour. Reactions were detected with precipitating peroxidase substrate TMB SeramunBlau prec (Seramun Diagnostics GmbH, Heidesee, Germany) after 10 min of incubation.
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