Extensive Serological Survey of Multiple African Nonhuman Primate Species Reveals Low Prevalence of Immunoglobulin G Antibodies to 4 Ebola Virus Species

Extensive Serological Survey of Multiple African Nonhuman Primate Species Reveals Low Prevalence... Abstract Bats are considered a reservoir species for Ebola viruses, but nonhuman primates (NHPs) have represented a source of infection in several outbreaks in humans. Here we report serological screening of blood or fecal samples from monkeys (n = 2322) and apes (n = 2327). Thirty-six NHP species from Cameroon, Democratic Republic of the Congo, and Ivory Coast were tested with a sensitive and specific Luminex-based assay for immunoglobulin G antibodies to 4 Ebola virus species. Using the simultaneous presence of antibodies to nucleoproteins and glycoproteins to define positivity, we showed that specific Ebola virus antibodies are not widespread among NHPs. Only 1 mustached monkey (Cercopithecus cephus) from Cameroon was positive for Sudan ebolavirus. These observations support that NHPs are most likely intermediate hosts for Ebola viruses. With the increasing frequency of Ebola outbreaks, it is crucial to identify the animal reservoir and understand the ecology of Ebola viruses to inform disease control. monkey, ape, Ebola, Africa Ebola virus disease (EVD) is a complex zoonosis, and each reported outbreak is most likely the result of an independent zoonotic event [1]. Today it is believed that bats constitute a reservoir and that they infect humans directly or via intermediate hosts, such as nonhuman primates (NHPs), duikers, or other mammals [2, 3]. Although without direct evidence, exposure to bat bushmeat is suggested in the 2007 outbreak in Luebo, Democratic Republic of the Congo (DRC), and bats are also suspected to be at the origin of the major EVD outbreak in West Africa in 2014 [4, 5]. For several outbreaks, recent contact with blood of NHPs through hunting or butchering of carcasses by the index individual was reported [1, 6]. Apes represented a source of infection in humans in Gabon, Republic of Congo, and Ivory Coast. Moreover, these human outbreaks coincided temporally and geographically with EVD outbreaks in apes, associated with high mortality rates [7–10]. Contact with monkeys is suspected in at least 2 outbreaks; in Boende, DRC in 2014, and in the Republic of the Congo in 2003 [11, 12]. Interestingly, in the EVD outbreak among chimpanzees in the Tai forest in Ivory Coast in 1994, an association was also observed between the consumption of monkeys (western red colobus) by chimpanzees and their Ebola virus infection rates [10]. Between the different EVD outbreaks of 1994 and 2003 in Gabon, 35 mortality and morbidity episodes were reported in wild animals in areas where previous EVD epidemics occurred, involving a wide diversity of NHP species (gorilla, chimpanzee, greater spot-nosed and mustached monkeys, black colobus, mandrill), but also bush pigs, sitatungas, and duikers [13]. Unfortunately, no laboratory tests were performed to identify the causes of death. Today, the role of reservoir and intermediate species in EVD outbreaks is still unclear and better knowledge on circulation of Ebola viruses in different wildlife species is thus necessary. Despite high mortality rates of Ebola in apes, antibodies have been observed in several wild-born but captive NHPs including chimpanzees, gorillas, mandrills, drills, De Brazza monkeys, and baboons [14, 15]. These observations suggest that Ebola could be widespread among NHPs and that nonlethal and asymptomatic or pauci-symptomatic infections occur in certain NHP species, as seen in humans [16, 17]. Moreover, a recent study showed that 10% of gorillas from an area with a high lethal EVD outbreak possess antibodies, suggesting that some infected animals can survive from the disease. More importantly, these observations also suggest that Ebola viruses circulate in areas where no outbreaks have yet been reported, such as Cameroon or Kenya [14, 15]. This is also the case for bats where antibodies have been detected in animals from West Africa (Ghana, Nigeria), West-Central Africa (Cameroon), East Africa (Zambia), and Asia (Bangladesh, China) [18–22]. One difficulty regarding interpretation and comparison of data from the different studies on NHPs, bats, or other wildlife species is the diversity of antibody assays employed, criteria used to define positivity, and the Ebola viruses that they target. For example, among the 5 studies that reported today on Ebola in NHPs, antibody detection was done using immunofluorescence, enzyme-linked immunosorbent assay (ELISA), or Western blotting [14, 15, 23, 24]. To date, 4 different Ebola virus species have been reported in humans in Africa, initially apparently limited to certain geographic regions: Zaire ebolavirus (EBOV) in West-Central Africa (western part of DRC, Gabon, and Republic of Congo); Sudan ebolavirus (SUDV) in Sudan and Uganda; Bundibugyo ebolavirus (BDBV) in East Africa (DRC and Uganda); and Tai ebolavirus in West Africa (Ivory Coast) [1]. However, the recent EVD outbreak in West Africa was confirmed as EBOV, showing thus a wider geographical spread of EBOV in Africa. EBOV was also identified in the last outbreak in eastern DRC (North Kivu), although previous outbreaks in this area were due to BDBV and SUDV [1] (Mbala et al, unpublished data). The majority of existing data on wildlife report only on EBOV, but in analogy to EBOV it cannot be excluded that the other Ebola viruses have a larger geographical spread than actually observed. Therefore, we developed a high-throughput Luminex-based assay that included antigens from EBOV, SUDV, and BDBV from Africa as well as from Reston ebolavirus (RESTV), which has to date only been reported in macaques and pigs from Asia and is apparently not harmful to humans [25]. The frequency of EVD outbreaks seems to have recently increased; for example, between May 2017 and July 2018, 3 independent EVD outbreaks occurred in 3 different provinces in DRC, and 2 outbreaks reached densely populated cities [26]. Therefore, studies on the ecology and animal reservoir of Ebola viruses are now urgently needed to quantify risks for future outbreaks and implement prevention measures. Although NHPs have been a source of infection in several outbreaks, only limited and disparate information is available on Ebola in NHPs, especially in monkeys. Here, we focused on the potential role of NHPs and studied to what extent antibodies to 4 different Ebola species can be detected in NHPs from DRC and Ivory Coast, 2 countries that have experienced EVD outbreaks, and from Cameroon, considered to be at high risk for future outbreaks [1]. MATERIALS AND METHODS Samples From Monkeys and Apes Samples were collected from bushmeat and pet monkeys between 1999 and 2016 as part of previous and ongoing studies on retroviral infections [27–30]. Bushmeat samples were collected at 14 different forest sites in southern Cameroon and DRC (Figure 1). Samples from pets were collected at 18 different sites in Cameroon (Supplementary Figure 1). Whole blood was collected from monkey bushmeat, either by intracardiac puncture and subsequent storage at –20°C, or by whole blood collection at the points of hunting injury and spotting, as a dried blood spot (DBS) on Whatman 903 filter paper (GE Healthcare) as described previously [28, 29]. Blood was drawn on ethylenediaminetetraacetic acid tubes from pet monkeys by venipuncture after tranquilization with ketamine [27]. Species were visually identified in the field and confirmed on a subset of samples by sequence analysis, as previously described [27–30]. Figure 1. View largeDownload slide Collection sites. Sites where samples from nonhuman primates were collected are highlighted with circles on the maps, as follows: yellow indicates sites where bushmeat samples from monkeys were collected; green, sites where fecal samples from apes were collected; yellow and green, sites where bushmeat samples from monkeys and fecal samples from apes were collected. The samples from Tai forest in Ivory Coast are not shown. Maps are adapted from Pigott et al [50]; areas closer to dark red are estimated at highest risk for Ebola virus spillover events, and areas in light yellow are least at risk. Abbreviations: BP, Bipindi; BQ, north of Dja; CP, Campo; DJ, Djoum; EB, Eboumetoum; EK, Ekom; EW, Ebolowa; GM, Goma; KL, Kole; LA, Lomako-Yokokala; MB, Mambele for green dot, Mbandaka for yellow dot; ML, Malabo; MM, Mengame; MN, Mindourou; MS, Messok; MT, Mintom; MZ, Manzana; ND, Nditam; WK, Walikale; YD, Yaoundé. Figure 1. View largeDownload slide Collection sites. Sites where samples from nonhuman primates were collected are highlighted with circles on the maps, as follows: yellow indicates sites where bushmeat samples from monkeys were collected; green, sites where fecal samples from apes were collected; yellow and green, sites where bushmeat samples from monkeys and fecal samples from apes were collected. The samples from Tai forest in Ivory Coast are not shown. Maps are adapted from Pigott et al [50]; areas closer to dark red are estimated at highest risk for Ebola virus spillover events, and areas in light yellow are least at risk. Abbreviations: BP, Bipindi; BQ, north of Dja; CP, Campo; DJ, Djoum; EB, Eboumetoum; EK, Ekom; EW, Ebolowa; GM, Goma; KL, Kole; LA, Lomako-Yokokala; MB, Mambele for green dot, Mbandaka for yellow dot; ML, Malabo; MM, Mengame; MN, Mindourou; MS, Messok; MT, Mintom; MZ, Manzana; ND, Nditam; WK, Walikale; YD, Yaoundé. Fecal samples were collected between 2005 and 2017 from wild ape populations at 11 different sites in Cameroon and DRC as part of previous studies on retroviral infections [31–33] (Figure 1). Samples were collected from central chimpanzees (Pan troglodytes troglodytes), western lowland gorillas (Gorilla gorilla gorilla), and bonobos (Pan paniscus). Most samples were collected around night nests and feeding sites, but also opportunistically. Samples were stored in RNAlater (Ambion), kept at ambient temperature in the field for a maximum of 3 weeks, and then stored at –20°C or –80°C. In the framework of a long-term veterinary follow-up program approved by the competent local authorities, fecal samples were obtained in the research area of the Tai National Park (Côte d’Ivoire) in 2001 and blood samples between 2004 and 2015 from either immobilized or necropsied NHPs that died of natural causes. The study was approved by the respective ministries of environment, research, and/or health and the national ethics committees. Screening for Ebola Virus Antibodies All samples were tested using our previously described serological assay based on Luminex technology [34]. Recombinant of nucleoprotein (NP), viral protein 40 (VP40), and/or glycoprotein (GP) for different Ebola virus species (EBOV, SUDV, BDBV, and RESTV) are used in this assay [34]. Whole blood, plasma, and DBS samples were tested at a final dilution of 1:1000 in assay buffer, taking into account the hematocrit of 50% in reconstituted plasma from DBS or whole blood. For all fecal samples, RNAlater-precipitated immunoglobulins were resolubilized by diluting the fecal/RNAlater mixture (2 mL) with phosphate-buffered saline (PBS)–Tween 20 (7 mL), followed by incubation for 1 hour at 60°C, centrifugation (3900g for 10 minutes) to clarify the solution, and dialysis against PBS overnight at 4°C [31–33]. The reconstituted extracts were then tested in the Luminex assay as previously described [34]. In brief, tests were performed in 96-well flat-bottomed filter plates (Millipore), and 100 μL of samples (final plasma dilution 1:1000; final fecal sample dilution 3:4) was incubated with 50 µL of beads for 16 hours at 4°C in the dark on a plate shaker at 300 rpm/minute. After washing with assay buffer, 50 µL of antihuman immunoglobulin G (IgG) biotin labeled (BD Pharmingen) was added at a concentration of 4 µg/mL in each well and incubated for 30 minutes in the dark while shaking at 300 rpm. After washing, 50 µL streptavidin-R-phycoerythrin (Fisher Scientific/Life Technologies) at 4 µg/mL was added per well and incubated for 10 minutes while shaking at 300 rpm. Antigen/antibody reactions were subsequently read on BioPlex-200 equipment (Bio-Rad); at least 100 events were read for each bead set, and the results were expressed as median fluorescence intensity (MFI) per 100 beads. In the absence of positive control samples for NHPs, we analyzed our data obtained from plasma and DBS samples with different statistical methods to determine MFI cutoff values for each antigen as reported in our previous study on bats [21, 35, 36]. We used a change-point analysis [37] with the R package “changepoint” [38] and calculated 1 single shift in the arithmetic mean with the AMOC (at most 1 change) method [39]. In analogy with other studies on Ebola virus serology in bats or wildlife, we also fitted univariate distributions to our data and defined the cutoff based on a 0.001 risk of error [40]. The set of candidate distributions was reduced with a bootstrapped skewness-kurtosis analysis [41]. Maximum likelihood estimation was performed to select the best-fit distribution based on the Akaike information criterion using the R library “fitdistrplus” [42]. The best-fit distribution was the negative binomial, but the negative exponential distribution was also used as in other studies on serology in wildlife [40]. Data were bootstrapped 10 000 times and averaged for each antigen. Analyses were done with R software version 3.3.2. We then compared the cutoff values identified by the 3 different methods and calculated their mean as a consensus cutoff that we used in this study (Supplementary Table 1). We considered a sample antigen reactive if MFI was above the cutoff value. Reactivity to both GP and NP proteins indicated specific EBOV and SUDV positivity [34]. For fecal samples, we first evaluated to what extent Ebola virus antibodies can be detected in feces from EBOV survivors from the Postebogui cohort in Guinea [43]. We also spiked different dilutions of EBOV survivors’ plasma samples in gorilla fecal dialysates to test the persistence of reactivity in this media. We compared MFI values in paired plasma and fecal samples and adapted the cutoff values in accordance with MFI values observed in feces as compared to plasma. RESULTS Diversity of NHP Species Tested A total of 4649 samples from 36 different NHP species were analyzed: 2322 were from monkeys and 2327 from apes. The species in sampling sites reflects the NHP distribution according to the biogeographic areas. The numbers of each monkey species collected at the different sites, illustrated in Figure 1, are shown in Table 1. In Cameroon, 1614 samples were tested from 17 different monkey species. The predominant species were Cercopithecuscephus (34.9%) and Cercopithecus nictitans (27.3%), followed by Cercopithecus pogonias (11.5%), Cercocebus agilis (8.1%), and Lophocebus albigena (6.4%). Among the 644 samples from DRC, the predominant monkey species was Cercopithecus ascanius (37.4%), followed by Piliocolobus tholloni (13.4%), Cercopithecus wolfi (11.2%), Cercopithecus mitis (7.9%), Allenopithecus nigroviridis (7.3%), and Cercopithecus lhoesti (5.8%). In Ivory Coast, among the 64 monkey samples, Piliocolobus badius (35.9%), Cercocebus atys (32.8%), and Colobus polykomos (17.2%) predominated. Table 1. Number of Samples Collected for Each Species at the Different Collection Sites in Cameroon, Democratic Republic of the Congo, and Ivory Coast Genus Species Common Name CM CM CM CM CM CM CM CM CM DRC DRC DRC DRC DRC DRC IC No. of Samples Pets BP ND YD BQ EW EB MS MN MB ML MK KL WK GM TAI Allenopithecus nigroviridis Allen swamp monkey … … … … … … … … … 44 … 2 1 … … … 47 Cercocebus agilis Agile mangabey 14 … … 1 9 … 39 13 55 3 … … … … … … 134 atys Sooty mangabey … … … … … … … … … … … … … … … 21 21 torquatus Red-capped mangabey 2 … … … … 4 … … … … … … … … … … 6 Colobus angolensis Angolan colobus … … … … … … … … … … … 4 21 … … … 25 guereza Mantled guereza 1 … 15 8 … … 2 … 8 1 … … 1 … … … 36 polykomos King’s colobus … … … … … … … … … … … … … … … 11 11 satanus Black colobus … … … … … 8 … … … … … … … … … … 8 Piliocolobus badius Western red colobus … … … … … … … … … … … … … … … 23 23 tholloni Tsuapa red colobus … … … … … … … … … … … 2 84 … … … 86 Procolobus verus Olive colobus … … … … … … … … … … … … … … … 2 2 Cercopithecus ascanius Red-tailed monkey … … … … … … … … … 41 42 12 106 7 33 … 241 campbelli Campbell's monkey … … … … … … … … … … … … … … … 1 1 cephus Mustached monkey 29 6 10 32 25 122 197 14 129 … … … … … … … 564 diana Diana monkey … … … … … … … … … … … … … … … 3 3 hamlyni Hamlyn’s monkey … … … … … … … … … … … … … … 6 … 6 lhoesti l’Hoest monkey … … … … … … … … … … … … … 6 31 … 37 mitis Blue monkey … … … … … … … … … … … … … 20 31 … 51 mona Mona monkey 9 … … 1 … … … … … … … … … … … … 10 neglectus De Brazza monkey 5 … … 4 8 2 13 … 2 18 2 1 6 … … … 61 nictitans Greater spot-nosed monkey 42 10 22 66 … 71 35 36 158 9 … … … … … … 449 petaurista Lesser spot-nosed monkey … … … … … … … … … … … … … … … 3 3 pogonias Crested mona monkey 5 2 11 19 11 24 31 17 66 … … … … 1 1 … 188 preussi Preuss monkey 1 … … … … … … … … … … … … … … … 1 wolfi Wolf’s monkey … … … … … … … … … 21 1 5 31 6 8 … 72 Chlorocebus tantalus Tantalus monkey 15 … … … … … … … … … … … … … … … 15 Erythrocebus patas Patas monkey 17 … … … … … … … … … … … … … … … 17 Lophocebus albigena Gray-cheeked mangabey 7 … 10 … … … 2 16 68 … … 1 … … 2 … 106 aterrimus Black mangabey … … … … … … … … … … … 2 31 … … … 33 Mandrillus leucophaeus Drill 1 … … … … … … … … … … … … … … … 1 sphinx Mandrill 16 1 … … … 10 … … … … … … … … … … 27 Miopithecus talapoin Northern talapoin 8 3 … 1 … 8 … … … … … … … … … … 20 Papio anubis Olive baboon 16 … … … … 1 … … … … … … … … … … 17 Total 188 22 68 132 53 250 319 96 486 137 45 29 281 40 112 64 2322 Genus Species Common Name CM CM CM CM CM CM CM CM CM DRC DRC DRC DRC DRC DRC IC No. of Samples Pets BP ND YD BQ EW EB MS MN MB ML MK KL WK GM TAI Allenopithecus nigroviridis Allen swamp monkey … … … … … … … … … 44 … 2 1 … … … 47 Cercocebus agilis Agile mangabey 14 … … 1 9 … 39 13 55 3 … … … … … … 134 atys Sooty mangabey … … … … … … … … … … … … … … … 21 21 torquatus Red-capped mangabey 2 … … … … 4 … … … … … … … … … … 6 Colobus angolensis Angolan colobus … … … … … … … … … … … 4 21 … … … 25 guereza Mantled guereza 1 … 15 8 … … 2 … 8 1 … … 1 … … … 36 polykomos King’s colobus … … … … … … … … … … … … … … … 11 11 satanus Black colobus … … … … … 8 … … … … … … … … … … 8 Piliocolobus badius Western red colobus … … … … … … … … … … … … … … … 23 23 tholloni Tsuapa red colobus … … … … … … … … … … … 2 84 … … … 86 Procolobus verus Olive colobus … … … … … … … … … … … … … … … 2 2 Cercopithecus ascanius Red-tailed monkey … … … … … … … … … 41 42 12 106 7 33 … 241 campbelli Campbell's monkey … … … … … … … … … … … … … … … 1 1 cephus Mustached monkey 29 6 10 32 25 122 197 14 129 … … … … … … … 564 diana Diana monkey … … … … … … … … … … … … … … … 3 3 hamlyni Hamlyn’s monkey … … … … … … … … … … … … … … 6 … 6 lhoesti l’Hoest monkey … … … … … … … … … … … … … 6 31 … 37 mitis Blue monkey … … … … … … … … … … … … … 20 31 … 51 mona Mona monkey 9 … … 1 … … … … … … … … … … … … 10 neglectus De Brazza monkey 5 … … 4 8 2 13 … 2 18 2 1 6 … … … 61 nictitans Greater spot-nosed monkey 42 10 22 66 … 71 35 36 158 9 … … … … … … 449 petaurista Lesser spot-nosed monkey … … … … … … … … … … … … … … … 3 3 pogonias Crested mona monkey 5 2 11 19 11 24 31 17 66 … … … … 1 1 … 188 preussi Preuss monkey 1 … … … … … … … … … … … … … … … 1 wolfi Wolf’s monkey … … … … … … … … … 21 1 5 31 6 8 … 72 Chlorocebus tantalus Tantalus monkey 15 … … … … … … … … … … … … … … … 15 Erythrocebus patas Patas monkey 17 … … … … … … … … … … … … … … … 17 Lophocebus albigena Gray-cheeked mangabey 7 … 10 … … … 2 16 68 … … 1 … … 2 … 106 aterrimus Black mangabey … … … … … … … … … … … 2 31 … … … 33 Mandrillus leucophaeus Drill 1 … … … … … … … … … … … … … … … 1 sphinx Mandrill 16 1 … … … 10 … … … … … … … … … … 27 Miopithecus talapoin Northern talapoin 8 3 … 1 … 8 … … … … … … … … … … 20 Papio anubis Olive baboon 16 … … … … 1 … … … … … … … … … … 17 Total 188 22 68 132 53 250 319 96 486 137 45 29 281 40 112 64 2322 Collection sites are shown in Figure 1. Abbreviations: BP, Bipindi; BQ, North Dja; CM, Cameroon; DRC, Democratic Republic of the Congo; EB, Eboumetoum; EW, Ebolowa; GM, Goma; IC, Ivory Coast; KL, Kole; MB, Mbandaka; MK, Monkoto; ML, Malebo; MN, Mindourou; MS, Messok; ND, Nditam; TAI, Tai National Park; YD, Yaoundé. View Large Table 1. Number of Samples Collected for Each Species at the Different Collection Sites in Cameroon, Democratic Republic of the Congo, and Ivory Coast Genus Species Common Name CM CM CM CM CM CM CM CM CM DRC DRC DRC DRC DRC DRC IC No. of Samples Pets BP ND YD BQ EW EB MS MN MB ML MK KL WK GM TAI Allenopithecus nigroviridis Allen swamp monkey … … … … … … … … … 44 … 2 1 … … … 47 Cercocebus agilis Agile mangabey 14 … … 1 9 … 39 13 55 3 … … … … … … 134 atys Sooty mangabey … … … … … … … … … … … … … … … 21 21 torquatus Red-capped mangabey 2 … … … … 4 … … … … … … … … … … 6 Colobus angolensis Angolan colobus … … … … … … … … … … … 4 21 … … … 25 guereza Mantled guereza 1 … 15 8 … … 2 … 8 1 … … 1 … … … 36 polykomos King’s colobus … … … … … … … … … … … … … … … 11 11 satanus Black colobus … … … … … 8 … … … … … … … … … … 8 Piliocolobus badius Western red colobus … … … … … … … … … … … … … … … 23 23 tholloni Tsuapa red colobus … … … … … … … … … … … 2 84 … … … 86 Procolobus verus Olive colobus … … … … … … … … … … … … … … … 2 2 Cercopithecus ascanius Red-tailed monkey … … … … … … … … … 41 42 12 106 7 33 … 241 campbelli Campbell's monkey … … … … … … … … … … … … … … … 1 1 cephus Mustached monkey 29 6 10 32 25 122 197 14 129 … … … … … … … 564 diana Diana monkey … … … … … … … … … … … … … … … 3 3 hamlyni Hamlyn’s monkey … … … … … … … … … … … … … … 6 … 6 lhoesti l’Hoest monkey … … … … … … … … … … … … … 6 31 … 37 mitis Blue monkey … … … … … … … … … … … … … 20 31 … 51 mona Mona monkey 9 … … 1 … … … … … … … … … … … … 10 neglectus De Brazza monkey 5 … … 4 8 2 13 … 2 18 2 1 6 … … … 61 nictitans Greater spot-nosed monkey 42 10 22 66 … 71 35 36 158 9 … … … … … … 449 petaurista Lesser spot-nosed monkey … … … … … … … … … … … … … … … 3 3 pogonias Crested mona monkey 5 2 11 19 11 24 31 17 66 … … … … 1 1 … 188 preussi Preuss monkey 1 … … … … … … … … … … … … … … … 1 wolfi Wolf’s monkey … … … … … … … … … 21 1 5 31 6 8 … 72 Chlorocebus tantalus Tantalus monkey 15 … … … … … … … … … … … … … … … 15 Erythrocebus patas Patas monkey 17 … … … … … … … … … … … … … … … 17 Lophocebus albigena Gray-cheeked mangabey 7 … 10 … … … 2 16 68 … … 1 … … 2 … 106 aterrimus Black mangabey … … … … … … … … … … … 2 31 … … … 33 Mandrillus leucophaeus Drill 1 … … … … … … … … … … … … … … … 1 sphinx Mandrill 16 1 … … … 10 … … … … … … … … … … 27 Miopithecus talapoin Northern talapoin 8 3 … 1 … 8 … … … … … … … … … … 20 Papio anubis Olive baboon 16 … … … … 1 … … … … … … … … … … 17 Total 188 22 68 132 53 250 319 96 486 137 45 29 281 40 112 64 2322 Genus Species Common Name CM CM CM CM CM CM CM CM CM DRC DRC DRC DRC DRC DRC IC No. of Samples Pets BP ND YD BQ EW EB MS MN MB ML MK KL WK GM TAI Allenopithecus nigroviridis Allen swamp monkey … … … … … … … … … 44 … 2 1 … … … 47 Cercocebus agilis Agile mangabey 14 … … 1 9 … 39 13 55 3 … … … … … … 134 atys Sooty mangabey … … … … … … … … … … … … … … … 21 21 torquatus Red-capped mangabey 2 … … … … 4 … … … … … … … … … … 6 Colobus angolensis Angolan colobus … … … … … … … … … … … 4 21 … … … 25 guereza Mantled guereza 1 … 15 8 … … 2 … 8 1 … … 1 … … … 36 polykomos King’s colobus … … … … … … … … … … … … … … … 11 11 satanus Black colobus … … … … … 8 … … … … … … … … … … 8 Piliocolobus badius Western red colobus … … … … … … … … … … … … … … … 23 23 tholloni Tsuapa red colobus … … … … … … … … … … … 2 84 … … … 86 Procolobus verus Olive colobus … … … … … … … … … … … … … … … 2 2 Cercopithecus ascanius Red-tailed monkey … … … … … … … … … 41 42 12 106 7 33 … 241 campbelli Campbell's monkey … … … … … … … … … … … … … … … 1 1 cephus Mustached monkey 29 6 10 32 25 122 197 14 129 … … … … … … … 564 diana Diana monkey … … … … … … … … … … … … … … … 3 3 hamlyni Hamlyn’s monkey … … … … … … … … … … … … … … 6 … 6 lhoesti l’Hoest monkey … … … … … … … … … … … … … 6 31 … 37 mitis Blue monkey … … … … … … … … … … … … … 20 31 … 51 mona Mona monkey 9 … … 1 … … … … … … … … … … … … 10 neglectus De Brazza monkey 5 … … 4 8 2 13 … 2 18 2 1 6 … … … 61 nictitans Greater spot-nosed monkey 42 10 22 66 … 71 35 36 158 9 … … … … … … 449 petaurista Lesser spot-nosed monkey … … … … … … … … … … … … … … … 3 3 pogonias Crested mona monkey 5 2 11 19 11 24 31 17 66 … … … … 1 1 … 188 preussi Preuss monkey 1 … … … … … … … … … … … … … … … 1 wolfi Wolf’s monkey … … … … … … … … … 21 1 5 31 6 8 … 72 Chlorocebus tantalus Tantalus monkey 15 … … … … … … … … … … … … … … … 15 Erythrocebus patas Patas monkey 17 … … … … … … … … … … … … … … … 17 Lophocebus albigena Gray-cheeked mangabey 7 … 10 … … … 2 16 68 … … 1 … … 2 … 106 aterrimus Black mangabey … … … … … … … … … … … 2 31 … … … 33 Mandrillus leucophaeus Drill 1 … … … … … … … … … … … … … … … 1 sphinx Mandrill 16 1 … … … 10 … … … … … … … … … … 27 Miopithecus talapoin Northern talapoin 8 3 … 1 … 8 … … … … … … … … … … 20 Papio anubis Olive baboon 16 … … … … 1 … … … … … … … … … … 17 Total 188 22 68 132 53 250 319 96 486 137 45 29 281 40 112 64 2322 Collection sites are shown in Figure 1. Abbreviations: BP, Bipindi; BQ, North Dja; CM, Cameroon; DRC, Democratic Republic of the Congo; EB, Eboumetoum; EW, Ebolowa; GM, Goma; IC, Ivory Coast; KL, Kole; MB, Mbandaka; MK, Monkoto; ML, Malebo; MN, Mindourou; MS, Messok; ND, Nditam; TAI, Tai National Park; YD, Yaoundé. View Large Among the 2327 samples from apes, 1182 (51%) were from western lowland gorillas (G. g. gorilla) in Cameroon, 353 (15%) from bonobos (P. paniscus) in DRC, and 792 (34%) from chimpanzees across Africa including samples from the western (Pan troglodytes verus, n = 57) and central chimpanzee (P. t. troglodytes, n = 735). Table 2 displays for each ape species or subspecies numbers collected at each site, illustrated in Figure 1. Table 2. Number of Samples Collected for Each Ape Species, by Site Species Country and Site No. Pan troglodytes verus Ivory Coast  Tai 57  Subtotal 57 Pan troglodytes troglodytes Cameroon  BQ 120  CP 114  DJ 45  EK 120  MB 316  MT 20  Subtotal 735 Gorilla gorilla gorilla Cameroon  BP 143  BQ 239  CP 289  DJ 167  EK 72  MB 161  MT 22  MM 89  Subtotal 1182 Pan paniscus DRC  LA 137  MZ 166  ML 50  Subtotal 353 Total 2327 Species Country and Site No. Pan troglodytes verus Ivory Coast  Tai 57  Subtotal 57 Pan troglodytes troglodytes Cameroon  BQ 120  CP 114  DJ 45  EK 120  MB 316  MT 20  Subtotal 735 Gorilla gorilla gorilla Cameroon  BP 143  BQ 239  CP 289  DJ 167  EK 72  MB 161  MT 22  MM 89  Subtotal 1182 Pan paniscus DRC  LA 137  MZ 166  ML 50  Subtotal 353 Total 2327 Collection sites are shown in Figure 1. Abbreviations: BP, Bipindi; BQ, North Dja; CP, Campo; DJ, Djoum; DRC, Democratic Republic of Congo; EK, Ekom; LA, Lomako-Yokokala; MB, Mambele; ML, Malebo; MM, Mengame; MT, Mintom; MZ, Manzana. View Large Table 2. Number of Samples Collected for Each Ape Species, by Site Species Country and Site No. Pan troglodytes verus Ivory Coast  Tai 57  Subtotal 57 Pan troglodytes troglodytes Cameroon  BQ 120  CP 114  DJ 45  EK 120  MB 316  MT 20  Subtotal 735 Gorilla gorilla gorilla Cameroon  BP 143  BQ 239  CP 289  DJ 167  EK 72  MB 161  MT 22  MM 89  Subtotal 1182 Pan paniscus DRC  LA 137  MZ 166  ML 50  Subtotal 353 Total 2327 Species Country and Site No. Pan troglodytes verus Ivory Coast  Tai 57  Subtotal 57 Pan troglodytes troglodytes Cameroon  BQ 120  CP 114  DJ 45  EK 120  MB 316  MT 20  Subtotal 735 Gorilla gorilla gorilla Cameroon  BP 143  BQ 239  CP 289  DJ 167  EK 72  MB 161  MT 22  MM 89  Subtotal 1182 Pan paniscus DRC  LA 137  MZ 166  ML 50  Subtotal 353 Total 2327 Collection sites are shown in Figure 1. Abbreviations: BP, Bipindi; BQ, North Dja; CP, Campo; DJ, Djoum; DRC, Democratic Republic of Congo; EK, Ekom; LA, Lomako-Yokokala; MB, Mambele; ML, Malebo; MM, Mengame; MT, Mintom; MZ, Manzana. View Large Low Prevalence of Ebola Virus IgG Antibodies in Monkeys The 2322 monkey samples were tested for Ebola antibodies with the multiplex antibody assay. The results are summarized for each antigen and for each Ebola virus species in Tables 3 and 4. No sample reacted simultaneously with GP and NP proteins from EBOV, and we therefore considered that none of the samples had specific antibodies to EBOV. Only a single sample, derived from a mustached monkey (C. cephus) from Cameroon, had antibodies to both NP and GP proteins from SUDV. Reactivity to another combination of 2 antigens was also low: 1 baboon (Papio anubis) was reactive with VP and GP proteins from EBOV, SUDV, and BDVB and 1 greater spot-nosed monkey (C. nictitans) with VP and GP from BDBV. All the other samples reacted only with 1 antigen: 0.6% for NP and 0.5%–1% reactivity for the different VP40 proteins. Highest reactivity was observed with GP proteins, and ranged from 2.2% to 2.6% with GP proteins derived from the African Ebola virus species (EBOV, SUDV, and BDBV) and was 1.5% for the Asian Ebola virus species (RESTV). Almost all GP-reactive samples were simultaneously reactive to GP proteins from >1 Ebola virus species. Highest reactivities (>2%) to GP proteins were observed in the following species: C. nictitans, C. cephus, C. agilis, Chlorocebus tantalus, and Colobus polykomos. Table 3. Number and Percentage of Samples for Each Species Reactive With the Different Antigens Used in the Luminex Assay for Zaire ebolavirus Genus Species Country No. Tested NP GP GP VP40 NP + GP EBOV EBOV-K EBOV-M EBOV EBOV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 4 (3.0; 1.1–7.4) 0 (0; .0–2.8) 0 (0; .0–2.8) atys IC 21 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) (0; .0–13.3) 1 (4.0; .7–19.5) 0 (0; .0–13.3) guereza Cam, DRC 36 1 (2.8; .5–14.2) 0 (0; .0–9.6) 0 (0; .0–9.6) 1 (2.8; .5–14.2) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 1 (9.1; 1.6–37.7) 1 (9.1; 1.6–37.7) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 Cercopithecus ascanius DRC 241 0 (0; .0–1.6) 1 (0.4; .07–2.3) 2 (0.8; .2–2.9) 2 (0.8; .2–2.9) 0 (0; .0–1.6) campbelli IC 1a 0 0 0 0 0 cephus Cam, DRC 565 1 (0.2; .03–1.0) 29 (5.1; 3.6–7.3) 34 (6.0; 4.3–8.3) 3 (0.5; .2–1.6) 0 (0; .0–.7) diana IC 3a 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 lhoesti DRC 37 1 (2.8; .5–13.8) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 0 (0; .0–5.9) 0 (0; .0–5.9) nictitans Cam, DRC 448 7 (1.6; .7–3.2) 17 (3.8; 2.4–6.0) 16 (3.6; 2.2–5.7) 3 (0.7; .2–1.9) 0 (0; .0–.9) petaurista IC 3a 0 3 3 0 0 pogonias Cam, DRC 188 1 (0.5; .1–2.9) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 1 (0.9; .2–5.2) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) Papio anubis Cam 17 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) Total 2322 15 (0.6; .4–1.1) 58 (2.5; 1.9–3.2) 61 (2.6; 2.1–3.4) 12 (0.5; .3–.9) 0 (0; .0–.2) Genus Species Country No. Tested NP GP GP VP40 NP + GP EBOV EBOV-K EBOV-M EBOV EBOV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 4 (3.0; 1.1–7.4) 0 (0; .0–2.8) 0 (0; .0–2.8) atys IC 21 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) (0; .0–13.3) 1 (4.0; .7–19.5) 0 (0; .0–13.3) guereza Cam, DRC 36 1 (2.8; .5–14.2) 0 (0; .0–9.6) 0 (0; .0–9.6) 1 (2.8; .5–14.2) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 1 (9.1; 1.6–37.7) 1 (9.1; 1.6–37.7) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 Cercopithecus ascanius DRC 241 0 (0; .0–1.6) 1 (0.4; .07–2.3) 2 (0.8; .2–2.9) 2 (0.8; .2–2.9) 0 (0; .0–1.6) campbelli IC 1a 0 0 0 0 0 cephus Cam, DRC 565 1 (0.2; .03–1.0) 29 (5.1; 3.6–7.3) 34 (6.0; 4.3–8.3) 3 (0.5; .2–1.6) 0 (0; .0–.7) diana IC 3a 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 lhoesti DRC 37 1 (2.8; .5–13.8) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 0 (0; .0–5.9) 0 (0; .0–5.9) nictitans Cam, DRC 448 7 (1.6; .7–3.2) 17 (3.8; 2.4–6.0) 16 (3.6; 2.2–5.7) 3 (0.7; .2–1.9) 0 (0; .0–.9) petaurista IC 3a 0 3 3 0 0 pogonias Cam, DRC 188 1 (0.5; .1–2.9) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 1 (0.9; .2–5.2) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) Papio anubis Cam 17 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) Total 2322 15 (0.6; .4–1.1) 58 (2.5; 1.9–3.2) 61 (2.6; 2.1–3.4) 12 (0.5; .3–.9) 0 (0; .0–.2) Data are presented as No. positive (%; 95% confidence interval) unless otherwise indicated. Abbreviations: Cam, Cameroon; DRC, Democratic Republic of the Congo; EBOV, Zaire ebolavirus; GP, glycoprotein; IC, Ivory Coast; K, Kissoudougou strain; M, Mayinga strain; NP, nucleoprotein; VP40, viral protein 40. aPercentages were not calculated when number of samples tested was <10. View Large Table 3. Number and Percentage of Samples for Each Species Reactive With the Different Antigens Used in the Luminex Assay for Zaire ebolavirus Genus Species Country No. Tested NP GP GP VP40 NP + GP EBOV EBOV-K EBOV-M EBOV EBOV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 4 (3.0; 1.1–7.4) 0 (0; .0–2.8) 0 (0; .0–2.8) atys IC 21 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) (0; .0–13.3) 1 (4.0; .7–19.5) 0 (0; .0–13.3) guereza Cam, DRC 36 1 (2.8; .5–14.2) 0 (0; .0–9.6) 0 (0; .0–9.6) 1 (2.8; .5–14.2) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 1 (9.1; 1.6–37.7) 1 (9.1; 1.6–37.7) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 Cercopithecus ascanius DRC 241 0 (0; .0–1.6) 1 (0.4; .07–2.3) 2 (0.8; .2–2.9) 2 (0.8; .2–2.9) 0 (0; .0–1.6) campbelli IC 1a 0 0 0 0 0 cephus Cam, DRC 565 1 (0.2; .03–1.0) 29 (5.1; 3.6–7.3) 34 (6.0; 4.3–8.3) 3 (0.5; .2–1.6) 0 (0; .0–.7) diana IC 3a 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 lhoesti DRC 37 1 (2.8; .5–13.8) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 0 (0; .0–5.9) 0 (0; .0–5.9) nictitans Cam, DRC 448 7 (1.6; .7–3.2) 17 (3.8; 2.4–6.0) 16 (3.6; 2.2–5.7) 3 (0.7; .2–1.9) 0 (0; .0–.9) petaurista IC 3a 0 3 3 0 0 pogonias Cam, DRC 188 1 (0.5; .1–2.9) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 1 (0.9; .2–5.2) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) Papio anubis Cam 17 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) Total 2322 15 (0.6; .4–1.1) 58 (2.5; 1.9–3.2) 61 (2.6; 2.1–3.4) 12 (0.5; .3–.9) 0 (0; .0–.2) Genus Species Country No. Tested NP GP GP VP40 NP + GP EBOV EBOV-K EBOV-M EBOV EBOV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 4 (3.0; 1.1–7.4) 0 (0; .0–2.8) 0 (0; .0–2.8) atys IC 21 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) (0; .0–13.3) 1 (4.0; .7–19.5) 0 (0; .0–13.3) guereza Cam, DRC 36 1 (2.8; .5–14.2) 0 (0; .0–9.6) 0 (0; .0–9.6) 1 (2.8; .5–14.2) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 1 (9.1; 1.6–37.7) 1 (9.1; 1.6–37.7) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 Cercopithecus ascanius DRC 241 0 (0; .0–1.6) 1 (0.4; .07–2.3) 2 (0.8; .2–2.9) 2 (0.8; .2–2.9) 0 (0; .0–1.6) campbelli IC 1a 0 0 0 0 0 cephus Cam, DRC 565 1 (0.2; .03–1.0) 29 (5.1; 3.6–7.3) 34 (6.0; 4.3–8.3) 3 (0.5; .2–1.6) 0 (0; .0–.7) diana IC 3a 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 lhoesti DRC 37 1 (2.8; .5–13.8) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 0 (0; .0–5.9) 0 (0; .0–5.9) nictitans Cam, DRC 448 7 (1.6; .7–3.2) 17 (3.8; 2.4–6.0) 16 (3.6; 2.2–5.7) 3 (0.7; .2–1.9) 0 (0; .0–.9) petaurista IC 3a 0 3 3 0 0 pogonias Cam, DRC 188 1 (0.5; .1–2.9) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 1 (0.9; .2–5.2) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) Papio anubis Cam 17 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) Total 2322 15 (0.6; .4–1.1) 58 (2.5; 1.9–3.2) 61 (2.6; 2.1–3.4) 12 (0.5; .3–.9) 0 (0; .0–.2) Data are presented as No. positive (%; 95% confidence interval) unless otherwise indicated. Abbreviations: Cam, Cameroon; DRC, Democratic Republic of the Congo; EBOV, Zaire ebolavirus; GP, glycoprotein; IC, Ivory Coast; K, Kissoudougou strain; M, Mayinga strain; NP, nucleoprotein; VP40, viral protein 40. aPercentages were not calculated when number of samples tested was <10. View Large Table 4. Number and Percentage of Samples for Each Species Reactive With the Different Antigens Used in the Luminex Assay for Sudan ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus Genus Species Country No. Tested NP GP VP40 NP + GP GP VP40 GP SUDV SUDV SUDV SUDV BDBV BDBV RESTV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; 0–7.5) 0 (0; .0–7.5) 1 (2.1; .4–11.1) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 2 (1.5; .4–5.3) 0 (0; .0–2.8) 5 (3.7; 1.6–8.4) 2 (1.5; .4–5.3) 4 (3.0; 1.1–7.4) atys IC 21 2 (9.5; 2.6–28.9) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) guereza Cam, DRC 36 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 1 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 0 0 Cercopithecus ascanius DRC 241 2 (0.8; .2–2.9) 1 (0.4; .07–2.3) 1 (0.4; .07–2.3) 0 (0; .0–1.6) 2 (0.8; .2–2.9) 0 (0; .0–1.6) 1 (0.4; .07–2.3) campbelli IC 1a 0 0 0 0 0 0 0 cephus Cam, DRC 565 5 (0.9; .4–2.1) 34 (6.0; 4.3–8.3) 4 (0.7; .3–1.8) 1 (0.2; .03–1.0) 30 (5.4; 3.7–7.5) 6 (1.1; .5–2.3) 19 (3.4; 2.2–5.2) diana IC 3a 0 0 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 0 0 lhoesti DRC 37 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 0 (0; .0–5.9) 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) nictitans Cam, DRC 448 2 (0.5; .1–1.6) 8 (1.8; .9–3.5) 5 (1.1; .5–2.6) 0 (0; .0–.9) 13 (2.9; 1.7–4.9) 5 (1.1; .5–2.6) 6 (1.3; .6–2.9) petaurista IC 3a 0 1 0 0 3 0 0 pogonias Cam, DRC 188 0 (0; .0–2.0) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 1 (1.4; .2–7.5) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 2 (1.9; .5–6.6) 0 (0; .0–3.5) 3 (2.9; .9–8.0) 0 (0; .0–3.5) 0 (0; .0–3.5) 1 (0.9; .2–5.2) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 1 (3.7; .6–18.3) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 3 (15.0; 5.2–36.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 2 (10.0; 2.7–30.1) 0 (0; .0–16.1) Papio anubis Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 2 (11.8; 3.3–34.3) 2 (11.8; 3.3–34.3) Total 2322 14 (0.6; .4–1.0) 51 (2.2; 1.7–2.9) 23 (1.0; .7–1.5) 1 (0.04; 0–.3) 56 (2.4; 1.9–3.1) 20 (0.9; .6–1.3) 35 (1.5; 1.1–2.1) Genus Species Country No. Tested NP GP VP40 NP + GP GP VP40 GP SUDV SUDV SUDV SUDV BDBV BDBV RESTV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; 0–7.5) 0 (0; .0–7.5) 1 (2.1; .4–11.1) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 2 (1.5; .4–5.3) 0 (0; .0–2.8) 5 (3.7; 1.6–8.4) 2 (1.5; .4–5.3) 4 (3.0; 1.1–7.4) atys IC 21 2 (9.5; 2.6–28.9) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) guereza Cam, DRC 36 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 1 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 0 0 Cercopithecus ascanius DRC 241 2 (0.8; .2–2.9) 1 (0.4; .07–2.3) 1 (0.4; .07–2.3) 0 (0; .0–1.6) 2 (0.8; .2–2.9) 0 (0; .0–1.6) 1 (0.4; .07–2.3) campbelli IC 1a 0 0 0 0 0 0 0 cephus Cam, DRC 565 5 (0.9; .4–2.1) 34 (6.0; 4.3–8.3) 4 (0.7; .3–1.8) 1 (0.2; .03–1.0) 30 (5.4; 3.7–7.5) 6 (1.1; .5–2.3) 19 (3.4; 2.2–5.2) diana IC 3a 0 0 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 0 0 lhoesti DRC 37 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 0 (0; .0–5.9) 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) nictitans Cam, DRC 448 2 (0.5; .1–1.6) 8 (1.8; .9–3.5) 5 (1.1; .5–2.6) 0 (0; .0–.9) 13 (2.9; 1.7–4.9) 5 (1.1; .5–2.6) 6 (1.3; .6–2.9) petaurista IC 3a 0 1 0 0 3 0 0 pogonias Cam, DRC 188 0 (0; .0–2.0) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 1 (1.4; .2–7.5) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 2 (1.9; .5–6.6) 0 (0; .0–3.5) 3 (2.9; .9–8.0) 0 (0; .0–3.5) 0 (0; .0–3.5) 1 (0.9; .2–5.2) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 1 (3.7; .6–18.3) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 3 (15.0; 5.2–36.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 2 (10.0; 2.7–30.1) 0 (0; .0–16.1) Papio anubis Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 2 (11.8; 3.3–34.3) 2 (11.8; 3.3–34.3) Total 2322 14 (0.6; .4–1.0) 51 (2.2; 1.7–2.9) 23 (1.0; .7–1.5) 1 (0.04; 0–.3) 56 (2.4; 1.9–3.1) 20 (0.9; .6–1.3) 35 (1.5; 1.1–2.1) Data are presented as No. positive (%; 95% confidence interval) unless otherwise indicated. Abbreviations: BDBV, Bundibugyo ebolavirus; Cam, Cameroon; DRC, Democratic Republic of the Congo; GP, glycoprotein; IC, Ivory Coast; NP, nucleoprotein; RESTV, Reston ebolavirus; SUDV, Sudan ebolavirus; VP40, viral protein 40. aPercentages were not calculated when the number of samples tested was <10. View Large Table 4. Number and Percentage of Samples for Each Species Reactive With the Different Antigens Used in the Luminex Assay for Sudan ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus Genus Species Country No. Tested NP GP VP40 NP + GP GP VP40 GP SUDV SUDV SUDV SUDV BDBV BDBV RESTV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; 0–7.5) 0 (0; .0–7.5) 1 (2.1; .4–11.1) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 2 (1.5; .4–5.3) 0 (0; .0–2.8) 5 (3.7; 1.6–8.4) 2 (1.5; .4–5.3) 4 (3.0; 1.1–7.4) atys IC 21 2 (9.5; 2.6–28.9) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) guereza Cam, DRC 36 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 1 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 0 0 Cercopithecus ascanius DRC 241 2 (0.8; .2–2.9) 1 (0.4; .07–2.3) 1 (0.4; .07–2.3) 0 (0; .0–1.6) 2 (0.8; .2–2.9) 0 (0; .0–1.6) 1 (0.4; .07–2.3) campbelli IC 1a 0 0 0 0 0 0 0 cephus Cam, DRC 565 5 (0.9; .4–2.1) 34 (6.0; 4.3–8.3) 4 (0.7; .3–1.8) 1 (0.2; .03–1.0) 30 (5.4; 3.7–7.5) 6 (1.1; .5–2.3) 19 (3.4; 2.2–5.2) diana IC 3a 0 0 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 0 0 lhoesti DRC 37 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 0 (0; .0–5.9) 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) nictitans Cam, DRC 448 2 (0.5; .1–1.6) 8 (1.8; .9–3.5) 5 (1.1; .5–2.6) 0 (0; .0–.9) 13 (2.9; 1.7–4.9) 5 (1.1; .5–2.6) 6 (1.3; .6–2.9) petaurista IC 3a 0 1 0 0 3 0 0 pogonias Cam, DRC 188 0 (0; .0–2.0) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 1 (1.4; .2–7.5) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 2 (1.9; .5–6.6) 0 (0; .0–3.5) 3 (2.9; .9–8.0) 0 (0; .0–3.5) 0 (0; .0–3.5) 1 (0.9; .2–5.2) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 1 (3.7; .6–18.3) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 3 (15.0; 5.2–36.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 2 (10.0; 2.7–30.1) 0 (0; .0–16.1) Papio anubis Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 2 (11.8; 3.3–34.3) 2 (11.8; 3.3–34.3) Total 2322 14 (0.6; .4–1.0) 51 (2.2; 1.7–2.9) 23 (1.0; .7–1.5) 1 (0.04; 0–.3) 56 (2.4; 1.9–3.1) 20 (0.9; .6–1.3) 35 (1.5; 1.1–2.1) Genus Species Country No. Tested NP GP VP40 NP + GP GP VP40 GP SUDV SUDV SUDV SUDV BDBV BDBV RESTV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; 0–7.5) 0 (0; .0–7.5) 1 (2.1; .4–11.1) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 2 (1.5; .4–5.3) 0 (0; .0–2.8) 5 (3.7; 1.6–8.4) 2 (1.5; .4–5.3) 4 (3.0; 1.1–7.4) atys IC 21 2 (9.5; 2.6–28.9) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) guereza Cam, DRC 36 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 1 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 0 0 Cercopithecus ascanius DRC 241 2 (0.8; .2–2.9) 1 (0.4; .07–2.3) 1 (0.4; .07–2.3) 0 (0; .0–1.6) 2 (0.8; .2–2.9) 0 (0; .0–1.6) 1 (0.4; .07–2.3) campbelli IC 1a 0 0 0 0 0 0 0 cephus Cam, DRC 565 5 (0.9; .4–2.1) 34 (6.0; 4.3–8.3) 4 (0.7; .3–1.8) 1 (0.2; .03–1.0) 30 (5.4; 3.7–7.5) 6 (1.1; .5–2.3) 19 (3.4; 2.2–5.2) diana IC 3a 0 0 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 0 0 lhoesti DRC 37 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 0 (0; .0–5.9) 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) nictitans Cam, DRC 448 2 (0.5; .1–1.6) 8 (1.8; .9–3.5) 5 (1.1; .5–2.6) 0 (0; .0–.9) 13 (2.9; 1.7–4.9) 5 (1.1; .5–2.6) 6 (1.3; .6–2.9) petaurista IC 3a 0 1 0 0 3 0 0 pogonias Cam, DRC 188 0 (0; .0–2.0) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 1 (1.4; .2–7.5) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 2 (1.9; .5–6.6) 0 (0; .0–3.5) 3 (2.9; .9–8.0) 0 (0; .0–3.5) 0 (0; .0–3.5) 1 (0.9; .2–5.2) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 1 (3.7; .6–18.3) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 3 (15.0; 5.2–36.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 2 (10.0; 2.7–30.1) 0 (0; .0–16.1) Papio anubis Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 2 (11.8; 3.3–34.3) 2 (11.8; 3.3–34.3) Total 2322 14 (0.6; .4–1.0) 51 (2.2; 1.7–2.9) 23 (1.0; .7–1.5) 1 (0.04; 0–.3) 56 (2.4; 1.9–3.1) 20 (0.9; .6–1.3) 35 (1.5; 1.1–2.1) Data are presented as No. positive (%; 95% confidence interval) unless otherwise indicated. Abbreviations: BDBV, Bundibugyo ebolavirus; Cam, Cameroon; DRC, Democratic Republic of the Congo; GP, glycoprotein; IC, Ivory Coast; NP, nucleoprotein; RESTV, Reston ebolavirus; SUDV, Sudan ebolavirus; VP40, viral protein 40. aPercentages were not calculated when the number of samples tested was <10. View Large No Evidence of IgG Ebola Virus Antibodies in Apes As previously observed for human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) cross-reactive antibody detection in fecal samples from apes, sensitivity is lower and interpretation criteria needed to be adapted [31–33]. Therefore, we first tested 57 paired feces (preserved in RNAlater) and plasma samples from EBOV survivors to evaluate the feasibility and sensitivity of Ebola virus antibody detection in fecal samples. In contrast with the antibody profile observed in plasma (ie, simultaneous positivity against NP + GP or NP + GP + VP), antibody reactivity in feces was more frequently directed against a single protein (ie, 57/57 [100%] in plasma vs 18/57 [31.6%] in fecal samples; Supplementary Table 2). However, 29 of 57 (50.9%) of fecal samples were reactive with at least 1 antigen, but a mean decrease of 0.67 to 1.37 log10 of MFI values was observed compared to plasma (Supplementary Table 2). Thus, we adapted the interpretation criteria in fecal samples to antibody reactivity with a single antigen and decreased the above used cutoff values with 1 log10 value. The lower sensitivity in fecal samples is comparable to that observed for HIV/SIV cross-reactive antibody detection in fecal samples from apes. Using these adapted criteria, none of the 2316 fecal samples reacted with any of the recombinant proteins in the Luminex assay. In addition, the 11 blood samples from western chimpanzees from the Tai forest in Ivory Coast were also negative for all antigens. DISCUSSION Although there is evidence that NHPs, and especially apes, play a role in zoonotic transmission of EVD outbreaks, few data are available on Ebola infection in NHPs. Here we report an extensive serological survey to 4 different Ebola virus species in 4649 NHP samples representing 36 different species. We applied a Luminex-based sensitive and specific antibody assay for the simultaneous detection of antibodies to 4 of the 5 species previously described in humans [34]. To identify specific Ebola virus antibodies, we used the same algorithm to NHPs as that developed for human EBOV survivors (ie, simultaneous positivity to NP and GP recombinant proteins); this was deemed appropriate because a recent study in cynomolgus macaques (Macaca fascicularis) naturally infected with RESTV revealed also that convalescent macaques having cleared the virus presented antibodies to NP and GP proteins, both by ELISA and immunofluorescence [44]. As such, we observed only 1 mustached monkey (C. cephus) from southern Cameroon with antibodies to GP and NP from SUDV. Only 2 other samples had antibodies to 2 different antigens from the same Ebola virus species; 1 P. anubis sample was reactive to VP40 and GP proteins from EBOV, SUDV, and BDBV, suggesting nonspecific or cross-reactivity, and 1 C. nictitans sample from Cameroon was reactive with GP and VP40 from BDBV. This is the first study to have evaluated a large number of monkeys using the same assay and interpretation criteria. We tested 2322 monkeys compared to the previous total of 930 from studies in Cameroon (n = 177), Gabon (n = 318), DRC (n = 251), and Kenya (n = 184) using a wide diversity of antibody detection assays [14, 15, 23, 24]. In these previous studies, EBOV-positive samples were seen in 3 of 184 captive baboons in a primate center in Kenya and in captive monkeys from Cameroon: in 1 of 8 De Brazza monkeys, 1 of 25 baboons, 1 of 18 mandrills, and 5 of 34 drills [14, 15]. In our study, the 27 mandrills and 61 De Brazza monkeys were all negative and 1 of 17 baboon samples was reactive with VP40 and GP proteins of different Ebola virus species. The different results may be due to the low numbers tested per species, the different geographic areas, and the different tests used. Antibody detection in fecal samples has been validated and used for Ebola using Western blot in gorillas that live in areas in Republic of Congo with previous EBOV outbreaks, showing that 8 of 80 (10%) had EBOV antibodies [16]. In our study, no evidence for Ebola virus antibodies was observed in 2316 fecal samples, corresponding to around 1362 apes taken into account from previous resampling estimates [31–33]. Our samples are mainly from areas without previously documented outbreaks; however, the majority were obtained in areas considered at risk for outbreaks [1]. Moreover, some sites like Djoum, Mambele, Mintom, or Mengame (Figure 1), which account for >650 samples in southern Cameroon, are located within a 100- to 300-km flight distance from areas of previous outbreaks in Gabon. Moreover, the samples from Ivory Coast were from the Tai National Park where an Ebola outbreak was documented among chimpanzees in 1994 and all fecal samples were collected in 2001 in the specific outbreak area. In contrast, a previous study using blood samples from wild-born but captive chimpanzees and gorillas revealed high EBOV antibody levels in apes: 21 of 119 (17.7%), 3 of 71 (4.2%), and 5 of 35 (14.3%) chimpanzees from Cameroon, Gabon, and Republic of the Congo, respectively, and 2 of 17 (11.2%) gorillas from Cameroon [15]. If EBOV prevalence in apes in Cameroon is as high as suggested in the previous study, we would have expected some reactive samples. As our assay has been shown to have a >95% sensitivity on human samples [34] and a 50% sensitivity on fecal samples, we would expect to have observed some reactivity in at least 100 fecal samples from chimpanzees or gorillas. Reactivity to a single Ebola virus antigen, especially GP, in NHPs has to be further explored, for example with neutralization assays, to ascertain whether it corresponds to nonspecific antibody reactivity or cross-reactivity with another pathogen, or different kinetics of antibodies to the different Ebola proteins. Recent studies have shown that bats can be infected with Ebola viruses that are different from the species known to infect humans, which could induce cross-reactivity with GP antigens [45, 46]. NHPs and bats share habitats and fruits in their diet. Virus transmission from bats to NHPs is suggested to occur when primates come in contact with fruit that is contaminated with feces, urine, or saliva from infected bats [2, 3]. Interestingly, a recent study reported that Cercopithecus species hunt roosting bats for consumption, which could be another, and a particularly efficient, route for Ebola virus transmission [47]. Preying on bats has been reported in C. ascanius and C. mitis species in East Africa, and also in bonobos in DRC [47, 48]. It is also possible that different modes of exposure to Ebola virus could lead to different antibody profiles, that is, contaminated fruit vs contact with infected bats during hunting. Whereas the majority of the outbreaks have been limited in terms of geographic spread and number of people infected, the 2014–2015 EVD outbreak in West Africa clearly illustrates the epidemic potential of a single zoonotic transmission in the presence of certain factors in favor of epidemic spread [49]. The recent EVD outbreaks in 2018 in the Equateur and Kivu provinces of DRC also illustrate that the virus can reach urban centers, even in a context of weak mobility infrastructure [26]. In conclusion, combining results on NHPs from our (4649 samples) and previous studies (~1300 samples), it is clear that Ebola virus antibodies are not widespread among NHPs, which confirms that NHPs are not reservoir species and that if Ebola virus infection in NHPs occurs, few animals survive. More samples from NHPs, bats, and other animal species from different regions across Africa should be studied to define which animals play a role in EVD outbreaks by acting as a reservoir species or as an amplifying host species. Nevertheless, with the increasing frequency of Ebola virus outbreaks (3 outbreaks in a 1-year period in DRC), it becomes extremely urgent to identify the animal reservoir(s) and to understand the ecology of Ebola viruses. It is estimated that transmission from animals to humans is possible in 23 countries across Central and West Africa with a total of >300 million inhabitants, and that at least 22 million of these people live in high-risk areas [50]. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. We thank the staff and SIV team from Projet PRESICA (Innocent Ndong Bass, Aime Mebenga, Joseph Moudindo, and Thomas Atemkem) and Donald Mbohli from Projet Grand Singes for the collection of samples and logistical support in Cameroon. In addition, we thank the field staff from DRC (Mubonga Mukulumanya, Lunguya-Metila Octavie, and Mbenzo-Abokome Valentin); Dr Mazongo, Dr Abanda, Dr Jonnhy, and all the local staff in Equateur and Nord-Kivu provinces for their collaboration and participation in this study; the staff of the World Wildlife Fund for Nature/DRC; the Institut National de Recherches Biomédicales (Kinshasa, DRC); the Bonobo Conservation Initiative; and Vie Sauvage, Didier Mazongo, Octavie Lunguya, Muriel Aloni, and Valentin Mbenzo-Abokome for field work in DRC. We thank the Ivorian authorities, especially the Ministry of the Environment and Forests, as well as the Ministry of Research; the Swiss Research Centre for Scientific Research; the directorship of the Taï National Park; and the Tai Chimpanzee Projects direction (Dr Wittig), the veterinarians (mainly A. Düx, K. Nowak, A. Lang), and the field assistants for continuous support. Financial support. This work was supported in part by grants from Institut national de la santé et de la recherche médicale/Ebola Task Force; REACTing; the US National Institutes of Health (grant number RO1 AI 50529); Agence Nationale de Recherches sur le SIDA (grant numbers ANRS 12125, 12182, and 12325); the Christophe Mérieux Prize 2015 awarded to J.-J. M.-T. M.; and the Capacity building and surveillance for Ebola Virus Disease (EBO-SURSY) project funded by the European Union, International Mixt Laboratory “PreVIHMI” of the Institut de Recherche pour le Developpement (IRD). C.-J. V. A. was supported by a fellowship from IRD and the Labex EpiGenMed, via the National Research Agency, Programme for Future Investment (ANR-10-LABX-12-01), and University of Montpellier. A. K. K. was supported by a fellowship from Montpellier Université d’excellence (MUSE) (I-Site MUSE, ANR-16-IDEX-0006). This work has also benefited from sample collections performed in the course of the German Research Foundation (DFG) projects (grant number FOR2136/LE1813/10–1). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: Ninth International Symposium on Filoviruses, Marburg, Germany, 13–16 September 2017. References 1. Pigott DM , Golding N , Mylne A , et al. Mapping the zoonotic niche of Ebola virus disease in Africa . Elife 2014 ; 3 : e04395 . Google Scholar Crossref Search ADS PubMed 2. Leroy EM , Kumulungui B , Pourrut X , et al. Fruit bats as reservoirs of Ebola virus . Nature 2005 ; 438 : 575 – 6 . Google Scholar Crossref Search ADS PubMed 3. Pourrut X , Souris M , Towner JS , et al. Large serological survey showing cocirculation of Ebola and Marburg viruses in Gabonese bat populations, and a high seroprevalence of both viruses in Rousettus aegyptiacus . BMC Infect Dis 2009 ; 9 : 159 . Google Scholar Crossref Search ADS PubMed 4. Leroy EM , Epelboin A , Mondonge V , et al. Human Ebola outbreak resulting from direct exposure to fruit bats in Luebo, Democratic Republic of Congo, 2007 . Vector Borne Zoonotic Dis 2009 ; 9 : 723 – 8 . Google Scholar Crossref Search ADS PubMed 5. Marí Saéz A , Weiss S , Nowak K , et al. Investigating the zoonotic origin of the West African Ebola epidemic . EMBO Mol Med 2015 ; 7 : 17 – 23 . Google Scholar Crossref Search ADS PubMed 6. Pourrut X , Kumulungui B , Wittmann T , et al. The natural history of Ebola virus in Africa . Microbes Infect 2005 ; 7 : 1005 – 14 . Google Scholar Crossref Search ADS PubMed 7. Rouquet P , Froment JM , Bermejo M , et al. Wild animal mortality monitoring and human Ebola outbreaks, Gabon and Republic of Congo, 2001-2003 . Emerg Infect Dis 2005 ; 11 : 283 – 90 . Google Scholar Crossref Search ADS PubMed 8. Bermejo M , Rodríguez-Teijeiro JD , Illera G , Barroso A , Vilà C , Walsh PD . Ebola outbreak killed 5000 gorillas . Science 2006 ; 314 : 1564 . Google Scholar Crossref Search ADS PubMed 9. Walsh PD , Abernethy KA , Bermejo M , et al. Catastrophic ape decline in western equatorial Africa . Nature 2003 ; 422 : 611 – 4 . Google Scholar Crossref Search ADS PubMed 10. Formenty P , Boesch C , Wyers M , et al. Ebola virus outbreak among wild chimpanzees living in a rain forest of Côte d’Ivoire . J Infect Dis 1999 ; 179 ( Suppl 1 ): S120 – 6 . Google Scholar Crossref Search ADS PubMed 11. Maganga GD , Kapetshi J , Berthet N , et al. Ebola virus disease in the Democratic Republic of Congo . N Engl J Med 2014 ; 371 : 2083 – 91 . Google Scholar Crossref Search ADS PubMed 12. Boumandouki P , Formenty P , Epelboin A , et al. Clinical management of patients and deceased during the Ebola outbreak from October to December 2003 in Republic of Congo . Bull Soc Pathol Exot 2005 ; 98 : 218 – 23 . Google Scholar PubMed 13. Lahm SA , Kombila M , Swanepoel R , Barnes RF . Morbidity and mortality of wild animals in relation to outbreaks of Ebola haemorrhagic fever in Gabon, 1994-2003 . Trans R Soc Trop Med Hyg 2007 ; 101 : 64 – 78 . Google Scholar Crossref Search ADS PubMed 14. Johnson BK , Gitau LG , Gichogo A , et al. Marburg, Ebola and Rift Valley Fever virus antibodies in East African primates . Trans R Soc Trop Med Hyg 1982 ; 76 : 307 – 10 . Google Scholar Crossref Search ADS PubMed 15. Leroy EM , Telfer P , Kumulungui B , et al. A serological survey of Ebola virus infection in Central African nonhuman primates . J Infect Dis 2004 ; 190 : 1895 – 9 . Google Scholar Crossref Search ADS PubMed 16. Reed PE , Mulangu S , Cameron KN , et al. A new approach for monitoring ebolavirus in wild great apes . PLoS Negl Trop Dis 2014 ; 8 : e3143 . Google Scholar Crossref Search ADS PubMed 17. Glynn JR , Bower H , Johnson S , et al. Asymptomatic infection and unrecognised Ebola virus disease in Ebola-affected households in Sierra Leone: a cross-sectional study using a new non-invasive assay for antibodies to Ebola virus . Lancet Infect Dis 2017 ; 17 : 645 – 53 . Google Scholar Crossref Search ADS PubMed 18. Hayman DT , Yu M , Crameri G , et al. Ebola virus antibodies in fruit bats, Ghana, West Africa . Emerg Infect Dis 2012 ; 18 : 1207 – 9 . Google Scholar Crossref Search ADS PubMed 19. Ogawa H , Miyamoto H , Nakayama E , et al. Seroepidemiological prevalence of multiple species of filoviruses in fruit bats (Eidolon helvum) migrating in Africa . J Infect Dis 2015 ; 212 ( Suppl 2 ): S101 – 8 . Google Scholar Crossref Search ADS PubMed 20. Olival KJ , Islam A , Yu M , et al. Ebola virus antibodies in fruit bats, Bangladesh . Emerg Infect Dis 2013 ; 19 : 270 – 3 . Google Scholar Crossref Search ADS PubMed 21. Yuan J , Zhang Y , Li J , Zhang Y , Wang LF , Shi Z . Serological evidence of ebolavirus infection in bats, China . Virol J 2012 ; 9 : 236 . Google Scholar Crossref Search ADS PubMed 22. De Nys H , Mbala Kingebeni P , Keita A , et al. Large serological and molecular survey of Ebola viruses in a wide diversity of frugivorous and insectivorous bat species in Guinea, Cameroon and the Democratic Republic of Congo . Emerg Infect Dis 2018 ; 24:2228–2240. 23. Leirs H , Mills JN , Krebs JW , et al. Search for the Ebola virus reservoir in Kikwit, Democratic Republic of the Congo: reflections on a vertebrate collection . J Infect Dis 1999 ; 179 ( Suppl 1 ): S155 – 63 . Google Scholar Crossref Search ADS PubMed 24. Breman JG , Johnson KM , van der Groen G , et al. A search for Ebola virus in animals in the Democratic Republic of the Congo and Cameroon: ecologic, virologic, and serologic surveys, 1979–1980. Ebola Virus Study Teams . J Infect Dis 1999 ; 179 ( Suppl 1 ): S139 – 47 . Google Scholar Crossref Search ADS PubMed 25. Cantoni D , Hamlet A , Michaelis M , Wass MN , Rossman JS . Risks posed by Reston, the forgotten Ebola virus . mSphere 2016 ; 1 . doi:10.1128/mSphere.00322-16. 26. Centers for Disease Control and Prevention . Ebola (Ebola virus disease). Outbreaks . https://www.cdc.gov/vhf/ebola/outbreaks/index-2018.html. Accessed 20 October 2018. 27. Peeters M , Courgnaud V , Abela B , et al. Risk to human health from a plethora of simian immunodeficiency viruses in primate bushmeat . Emerg Infect Dis 2002 ; 8 : 451 – 7 . Google Scholar Crossref Search ADS PubMed 28. Aghokeng AF , Ayouba A , Mpoudi-Ngole E , et al. Extensive survey on the prevalence and genetic diversity of SIVs in primate bushmeat provides insights into risks for potential new cross-species transmissions . Infect Genet Evol 2010 ; 10 : 386 – 96 . Google Scholar Crossref Search ADS PubMed 29. Ahuka-Mundeke S , Ayouba A , Mbala-Kingebeni P , et al. Erratum to: high prevalences and a wide genetic diversity of simian retroviruses in non-human primate bushmeat in rural areas of the Democratic Republic of Congo . Ecohealth 2017 ; 14 : 115 . Google Scholar Crossref Search ADS PubMed 30. Ahuka-Mundeke S , Ayouba A , Mbala-Kingebeni P , et al. Novel multiplexed HIV/simian immunodeficiency virus antibody detection assay . Emerg Infect Dis 2011 ; 17 : 2277 – 86 . Google Scholar Crossref Search ADS PubMed 31. Van Heuverswyn F , Li Y , Bailes E , et al. Genetic diversity and phylogeographic clustering of SIVcpzPtt in wild chimpanzees in Cameroon . Virology 2007 ; 368 : 155 – 71 . Google Scholar Crossref Search ADS PubMed 32. D’arc M , Ayouba A , Esteban A , et al. Origin of the HIV-1 group O epidemic in western lowland gorillas . Proc Natl Acad Sci U S A 2015 ; 112 : E1343 – 52 . Google Scholar Crossref Search ADS PubMed 33. Li Y , Ndjango JB , Learn GH , et al. Eastern chimpanzees, but not bonobos, represent a simian immunodeficiency virus reservoir . J Virol 2012 ; 86 : 10776 – 91 . Google Scholar Crossref Search ADS PubMed 34. Ayouba A , Touré A , Butel C , et al. Development of a sensitive and specific serological assay based on Luminex technology for detection of antibodies to zaire Ebola virus . J Clin Microbiol 2017 ; 55 : 165 – 76 . Google Scholar Crossref Search ADS PubMed 35. Peel AJ , McKinley TJ , Baker KS , et al. Use of cross-reactive serological assays for detecting novel pathogens in wildlife: assessing an appropriate cutoff for henipavirus assays in African bats . J Virol Methods 2013 ; 193 : 295 – 303 . Google Scholar Crossref Search ADS PubMed 36. Gilbert AT , Fooks AR , Hayman DT , et al. Deciphering serology to understand the ecology of infectious diseases in wildlife . Ecohealth 2013 ; 10 : 298 – 313 . Google Scholar Crossref Search ADS PubMed 37. Lardeux F , Torrico G , Aliaga C . Calculation of the ELISA’s cut-off based on the change-point analysis method for detection of Trypanosoma cruzi infection in Bolivian dogs in the absence of controls . Mem Inst Oswaldo Cruz 2016 ; 111 : 501 – 4 . Google Scholar Crossref Search ADS PubMed 38. Killick R , Eckley IA . Changepoint: an R package for changepoint analysis . J Stat Soft 2014 ; 58 : 1 – 19 . Google Scholar Crossref Search ADS 39. Hinkley DV . Inference about the change-point in a sequence of random variables . Biometrika 1970 ; 57 : 1 – 17 . Google Scholar Crossref Search ADS 40. Laing ED , Mendenhall IH , Linster M , et al. Serologic evidence of fruit bat exposure to filoviruses, Singapore, 2011–2016 . Emerg Infect Dis 2018 ; 24 : 114 – 7 . Google Scholar Crossref Search ADS PubMed 41. Cullen AC , Frey HC. Probabilistic techniques in exposure assessment . New York, NY : Plenum Press , 1999 : 81 – 159 . 42. Delignette-Muller ML , Dutang C . Fitdistrplus: an R package for fitting distributions . J Stat Soft 2015 ; 64 : 1 – 34 . Google Scholar Crossref Search ADS 43. Etard JF , Sow MS , Leroy S , et al. Postebogui Study Group . Multidisciplinary assessment of post-Ebola sequelae in Guinea (Postebogui): an observational cohort study . Lancet Infect Dis 2017 ; 17 : 545 – 52 . Google Scholar Crossref Search ADS PubMed 44. Taniguchi S , Sayama Y , Nagata N , et al. Analysis of the humoral immune responses among cynomolgus macaque naturally infected with Reston virus during the 1996 outbreak in the Philippines . BMC Vet Res 2012 ; 8 : 189 . Google Scholar Crossref Search ADS PubMed 45. Yang XL , Zhang YZ , Jiang RD , et al. Genetically diverse filoviruses in Rousettus and Eonycteris spp. bats, China, 2009 and 2015 . Emerg Infect Dis 2017 ; 23 : 482 – 6 . Google Scholar Crossref Search ADS PubMed 46. Goldstein T , Anthony SJ , Gbakima A , et al. Author correction: the discovery of Bombali virus adds further support for bats as hosts of ebolaviruses . Nat Microbiol 2018 ; 3 : 1486 . Google Scholar Crossref Search ADS PubMed 47. Tapanes E , Detwiler KM , Cords M . Bat predation by Cercopithecus monkeys: implications for zoonotic disease transmission . Ecohealth 2016 ; 13 : 405 – 9 . Google Scholar Crossref Search ADS PubMed 48. Bermejo M , Illera G , Sabater P . Animals and mushrooms consumed by bonobos (Pan paniscus): new records from Lilungu (Ikele), Zaire . Int J Primatol 1994 ; 15 : 879 – 98 . Google Scholar Crossref Search ADS 49. Alexander KA , Sanderson CE , Marathe M , et al. What factors might have led to the emergence of Ebola in West Africa? PLoS Negl Trop Dis 2015 ; 9 : e0003652 . Google Scholar Crossref Search ADS PubMed 50. Pigott DM , Millear AI , Earl L , Morozoff C . Updates to the zoonotic niche map of Ebola virus disease in Africa . eLife 2016 ; 5 : 16412 . Google Scholar Crossref Search ADS © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Loading next page...
 
/lp/oxford-university-press/extensive-serological-survey-of-multiple-african-nonhuman-primate-VI3Zxih4CN
Publisher
Oxford University Press
Copyright
© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
ISSN
0022-1899
eISSN
1537-6613
D.O.I.
10.1093/infdis/jiz006
Publisher site
See Article on Publisher Site

Abstract

Abstract Bats are considered a reservoir species for Ebola viruses, but nonhuman primates (NHPs) have represented a source of infection in several outbreaks in humans. Here we report serological screening of blood or fecal samples from monkeys (n = 2322) and apes (n = 2327). Thirty-six NHP species from Cameroon, Democratic Republic of the Congo, and Ivory Coast were tested with a sensitive and specific Luminex-based assay for immunoglobulin G antibodies to 4 Ebola virus species. Using the simultaneous presence of antibodies to nucleoproteins and glycoproteins to define positivity, we showed that specific Ebola virus antibodies are not widespread among NHPs. Only 1 mustached monkey (Cercopithecus cephus) from Cameroon was positive for Sudan ebolavirus. These observations support that NHPs are most likely intermediate hosts for Ebola viruses. With the increasing frequency of Ebola outbreaks, it is crucial to identify the animal reservoir and understand the ecology of Ebola viruses to inform disease control. monkey, ape, Ebola, Africa Ebola virus disease (EVD) is a complex zoonosis, and each reported outbreak is most likely the result of an independent zoonotic event [1]. Today it is believed that bats constitute a reservoir and that they infect humans directly or via intermediate hosts, such as nonhuman primates (NHPs), duikers, or other mammals [2, 3]. Although without direct evidence, exposure to bat bushmeat is suggested in the 2007 outbreak in Luebo, Democratic Republic of the Congo (DRC), and bats are also suspected to be at the origin of the major EVD outbreak in West Africa in 2014 [4, 5]. For several outbreaks, recent contact with blood of NHPs through hunting or butchering of carcasses by the index individual was reported [1, 6]. Apes represented a source of infection in humans in Gabon, Republic of Congo, and Ivory Coast. Moreover, these human outbreaks coincided temporally and geographically with EVD outbreaks in apes, associated with high mortality rates [7–10]. Contact with monkeys is suspected in at least 2 outbreaks; in Boende, DRC in 2014, and in the Republic of the Congo in 2003 [11, 12]. Interestingly, in the EVD outbreak among chimpanzees in the Tai forest in Ivory Coast in 1994, an association was also observed between the consumption of monkeys (western red colobus) by chimpanzees and their Ebola virus infection rates [10]. Between the different EVD outbreaks of 1994 and 2003 in Gabon, 35 mortality and morbidity episodes were reported in wild animals in areas where previous EVD epidemics occurred, involving a wide diversity of NHP species (gorilla, chimpanzee, greater spot-nosed and mustached monkeys, black colobus, mandrill), but also bush pigs, sitatungas, and duikers [13]. Unfortunately, no laboratory tests were performed to identify the causes of death. Today, the role of reservoir and intermediate species in EVD outbreaks is still unclear and better knowledge on circulation of Ebola viruses in different wildlife species is thus necessary. Despite high mortality rates of Ebola in apes, antibodies have been observed in several wild-born but captive NHPs including chimpanzees, gorillas, mandrills, drills, De Brazza monkeys, and baboons [14, 15]. These observations suggest that Ebola could be widespread among NHPs and that nonlethal and asymptomatic or pauci-symptomatic infections occur in certain NHP species, as seen in humans [16, 17]. Moreover, a recent study showed that 10% of gorillas from an area with a high lethal EVD outbreak possess antibodies, suggesting that some infected animals can survive from the disease. More importantly, these observations also suggest that Ebola viruses circulate in areas where no outbreaks have yet been reported, such as Cameroon or Kenya [14, 15]. This is also the case for bats where antibodies have been detected in animals from West Africa (Ghana, Nigeria), West-Central Africa (Cameroon), East Africa (Zambia), and Asia (Bangladesh, China) [18–22]. One difficulty regarding interpretation and comparison of data from the different studies on NHPs, bats, or other wildlife species is the diversity of antibody assays employed, criteria used to define positivity, and the Ebola viruses that they target. For example, among the 5 studies that reported today on Ebola in NHPs, antibody detection was done using immunofluorescence, enzyme-linked immunosorbent assay (ELISA), or Western blotting [14, 15, 23, 24]. To date, 4 different Ebola virus species have been reported in humans in Africa, initially apparently limited to certain geographic regions: Zaire ebolavirus (EBOV) in West-Central Africa (western part of DRC, Gabon, and Republic of Congo); Sudan ebolavirus (SUDV) in Sudan and Uganda; Bundibugyo ebolavirus (BDBV) in East Africa (DRC and Uganda); and Tai ebolavirus in West Africa (Ivory Coast) [1]. However, the recent EVD outbreak in West Africa was confirmed as EBOV, showing thus a wider geographical spread of EBOV in Africa. EBOV was also identified in the last outbreak in eastern DRC (North Kivu), although previous outbreaks in this area were due to BDBV and SUDV [1] (Mbala et al, unpublished data). The majority of existing data on wildlife report only on EBOV, but in analogy to EBOV it cannot be excluded that the other Ebola viruses have a larger geographical spread than actually observed. Therefore, we developed a high-throughput Luminex-based assay that included antigens from EBOV, SUDV, and BDBV from Africa as well as from Reston ebolavirus (RESTV), which has to date only been reported in macaques and pigs from Asia and is apparently not harmful to humans [25]. The frequency of EVD outbreaks seems to have recently increased; for example, between May 2017 and July 2018, 3 independent EVD outbreaks occurred in 3 different provinces in DRC, and 2 outbreaks reached densely populated cities [26]. Therefore, studies on the ecology and animal reservoir of Ebola viruses are now urgently needed to quantify risks for future outbreaks and implement prevention measures. Although NHPs have been a source of infection in several outbreaks, only limited and disparate information is available on Ebola in NHPs, especially in monkeys. Here, we focused on the potential role of NHPs and studied to what extent antibodies to 4 different Ebola species can be detected in NHPs from DRC and Ivory Coast, 2 countries that have experienced EVD outbreaks, and from Cameroon, considered to be at high risk for future outbreaks [1]. MATERIALS AND METHODS Samples From Monkeys and Apes Samples were collected from bushmeat and pet monkeys between 1999 and 2016 as part of previous and ongoing studies on retroviral infections [27–30]. Bushmeat samples were collected at 14 different forest sites in southern Cameroon and DRC (Figure 1). Samples from pets were collected at 18 different sites in Cameroon (Supplementary Figure 1). Whole blood was collected from monkey bushmeat, either by intracardiac puncture and subsequent storage at –20°C, or by whole blood collection at the points of hunting injury and spotting, as a dried blood spot (DBS) on Whatman 903 filter paper (GE Healthcare) as described previously [28, 29]. Blood was drawn on ethylenediaminetetraacetic acid tubes from pet monkeys by venipuncture after tranquilization with ketamine [27]. Species were visually identified in the field and confirmed on a subset of samples by sequence analysis, as previously described [27–30]. Figure 1. View largeDownload slide Collection sites. Sites where samples from nonhuman primates were collected are highlighted with circles on the maps, as follows: yellow indicates sites where bushmeat samples from monkeys were collected; green, sites where fecal samples from apes were collected; yellow and green, sites where bushmeat samples from monkeys and fecal samples from apes were collected. The samples from Tai forest in Ivory Coast are not shown. Maps are adapted from Pigott et al [50]; areas closer to dark red are estimated at highest risk for Ebola virus spillover events, and areas in light yellow are least at risk. Abbreviations: BP, Bipindi; BQ, north of Dja; CP, Campo; DJ, Djoum; EB, Eboumetoum; EK, Ekom; EW, Ebolowa; GM, Goma; KL, Kole; LA, Lomako-Yokokala; MB, Mambele for green dot, Mbandaka for yellow dot; ML, Malabo; MM, Mengame; MN, Mindourou; MS, Messok; MT, Mintom; MZ, Manzana; ND, Nditam; WK, Walikale; YD, Yaoundé. Figure 1. View largeDownload slide Collection sites. Sites where samples from nonhuman primates were collected are highlighted with circles on the maps, as follows: yellow indicates sites where bushmeat samples from monkeys were collected; green, sites where fecal samples from apes were collected; yellow and green, sites where bushmeat samples from monkeys and fecal samples from apes were collected. The samples from Tai forest in Ivory Coast are not shown. Maps are adapted from Pigott et al [50]; areas closer to dark red are estimated at highest risk for Ebola virus spillover events, and areas in light yellow are least at risk. Abbreviations: BP, Bipindi; BQ, north of Dja; CP, Campo; DJ, Djoum; EB, Eboumetoum; EK, Ekom; EW, Ebolowa; GM, Goma; KL, Kole; LA, Lomako-Yokokala; MB, Mambele for green dot, Mbandaka for yellow dot; ML, Malabo; MM, Mengame; MN, Mindourou; MS, Messok; MT, Mintom; MZ, Manzana; ND, Nditam; WK, Walikale; YD, Yaoundé. Fecal samples were collected between 2005 and 2017 from wild ape populations at 11 different sites in Cameroon and DRC as part of previous studies on retroviral infections [31–33] (Figure 1). Samples were collected from central chimpanzees (Pan troglodytes troglodytes), western lowland gorillas (Gorilla gorilla gorilla), and bonobos (Pan paniscus). Most samples were collected around night nests and feeding sites, but also opportunistically. Samples were stored in RNAlater (Ambion), kept at ambient temperature in the field for a maximum of 3 weeks, and then stored at –20°C or –80°C. In the framework of a long-term veterinary follow-up program approved by the competent local authorities, fecal samples were obtained in the research area of the Tai National Park (Côte d’Ivoire) in 2001 and blood samples between 2004 and 2015 from either immobilized or necropsied NHPs that died of natural causes. The study was approved by the respective ministries of environment, research, and/or health and the national ethics committees. Screening for Ebola Virus Antibodies All samples were tested using our previously described serological assay based on Luminex technology [34]. Recombinant of nucleoprotein (NP), viral protein 40 (VP40), and/or glycoprotein (GP) for different Ebola virus species (EBOV, SUDV, BDBV, and RESTV) are used in this assay [34]. Whole blood, plasma, and DBS samples were tested at a final dilution of 1:1000 in assay buffer, taking into account the hematocrit of 50% in reconstituted plasma from DBS or whole blood. For all fecal samples, RNAlater-precipitated immunoglobulins were resolubilized by diluting the fecal/RNAlater mixture (2 mL) with phosphate-buffered saline (PBS)–Tween 20 (7 mL), followed by incubation for 1 hour at 60°C, centrifugation (3900g for 10 minutes) to clarify the solution, and dialysis against PBS overnight at 4°C [31–33]. The reconstituted extracts were then tested in the Luminex assay as previously described [34]. In brief, tests were performed in 96-well flat-bottomed filter plates (Millipore), and 100 μL of samples (final plasma dilution 1:1000; final fecal sample dilution 3:4) was incubated with 50 µL of beads for 16 hours at 4°C in the dark on a plate shaker at 300 rpm/minute. After washing with assay buffer, 50 µL of antihuman immunoglobulin G (IgG) biotin labeled (BD Pharmingen) was added at a concentration of 4 µg/mL in each well and incubated for 30 minutes in the dark while shaking at 300 rpm. After washing, 50 µL streptavidin-R-phycoerythrin (Fisher Scientific/Life Technologies) at 4 µg/mL was added per well and incubated for 10 minutes while shaking at 300 rpm. Antigen/antibody reactions were subsequently read on BioPlex-200 equipment (Bio-Rad); at least 100 events were read for each bead set, and the results were expressed as median fluorescence intensity (MFI) per 100 beads. In the absence of positive control samples for NHPs, we analyzed our data obtained from plasma and DBS samples with different statistical methods to determine MFI cutoff values for each antigen as reported in our previous study on bats [21, 35, 36]. We used a change-point analysis [37] with the R package “changepoint” [38] and calculated 1 single shift in the arithmetic mean with the AMOC (at most 1 change) method [39]. In analogy with other studies on Ebola virus serology in bats or wildlife, we also fitted univariate distributions to our data and defined the cutoff based on a 0.001 risk of error [40]. The set of candidate distributions was reduced with a bootstrapped skewness-kurtosis analysis [41]. Maximum likelihood estimation was performed to select the best-fit distribution based on the Akaike information criterion using the R library “fitdistrplus” [42]. The best-fit distribution was the negative binomial, but the negative exponential distribution was also used as in other studies on serology in wildlife [40]. Data were bootstrapped 10 000 times and averaged for each antigen. Analyses were done with R software version 3.3.2. We then compared the cutoff values identified by the 3 different methods and calculated their mean as a consensus cutoff that we used in this study (Supplementary Table 1). We considered a sample antigen reactive if MFI was above the cutoff value. Reactivity to both GP and NP proteins indicated specific EBOV and SUDV positivity [34]. For fecal samples, we first evaluated to what extent Ebola virus antibodies can be detected in feces from EBOV survivors from the Postebogui cohort in Guinea [43]. We also spiked different dilutions of EBOV survivors’ plasma samples in gorilla fecal dialysates to test the persistence of reactivity in this media. We compared MFI values in paired plasma and fecal samples and adapted the cutoff values in accordance with MFI values observed in feces as compared to plasma. RESULTS Diversity of NHP Species Tested A total of 4649 samples from 36 different NHP species were analyzed: 2322 were from monkeys and 2327 from apes. The species in sampling sites reflects the NHP distribution according to the biogeographic areas. The numbers of each monkey species collected at the different sites, illustrated in Figure 1, are shown in Table 1. In Cameroon, 1614 samples were tested from 17 different monkey species. The predominant species were Cercopithecuscephus (34.9%) and Cercopithecus nictitans (27.3%), followed by Cercopithecus pogonias (11.5%), Cercocebus agilis (8.1%), and Lophocebus albigena (6.4%). Among the 644 samples from DRC, the predominant monkey species was Cercopithecus ascanius (37.4%), followed by Piliocolobus tholloni (13.4%), Cercopithecus wolfi (11.2%), Cercopithecus mitis (7.9%), Allenopithecus nigroviridis (7.3%), and Cercopithecus lhoesti (5.8%). In Ivory Coast, among the 64 monkey samples, Piliocolobus badius (35.9%), Cercocebus atys (32.8%), and Colobus polykomos (17.2%) predominated. Table 1. Number of Samples Collected for Each Species at the Different Collection Sites in Cameroon, Democratic Republic of the Congo, and Ivory Coast Genus Species Common Name CM CM CM CM CM CM CM CM CM DRC DRC DRC DRC DRC DRC IC No. of Samples Pets BP ND YD BQ EW EB MS MN MB ML MK KL WK GM TAI Allenopithecus nigroviridis Allen swamp monkey … … … … … … … … … 44 … 2 1 … … … 47 Cercocebus agilis Agile mangabey 14 … … 1 9 … 39 13 55 3 … … … … … … 134 atys Sooty mangabey … … … … … … … … … … … … … … … 21 21 torquatus Red-capped mangabey 2 … … … … 4 … … … … … … … … … … 6 Colobus angolensis Angolan colobus … … … … … … … … … … … 4 21 … … … 25 guereza Mantled guereza 1 … 15 8 … … 2 … 8 1 … … 1 … … … 36 polykomos King’s colobus … … … … … … … … … … … … … … … 11 11 satanus Black colobus … … … … … 8 … … … … … … … … … … 8 Piliocolobus badius Western red colobus … … … … … … … … … … … … … … … 23 23 tholloni Tsuapa red colobus … … … … … … … … … … … 2 84 … … … 86 Procolobus verus Olive colobus … … … … … … … … … … … … … … … 2 2 Cercopithecus ascanius Red-tailed monkey … … … … … … … … … 41 42 12 106 7 33 … 241 campbelli Campbell's monkey … … … … … … … … … … … … … … … 1 1 cephus Mustached monkey 29 6 10 32 25 122 197 14 129 … … … … … … … 564 diana Diana monkey … … … … … … … … … … … … … … … 3 3 hamlyni Hamlyn’s monkey … … … … … … … … … … … … … … 6 … 6 lhoesti l’Hoest monkey … … … … … … … … … … … … … 6 31 … 37 mitis Blue monkey … … … … … … … … … … … … … 20 31 … 51 mona Mona monkey 9 … … 1 … … … … … … … … … … … … 10 neglectus De Brazza monkey 5 … … 4 8 2 13 … 2 18 2 1 6 … … … 61 nictitans Greater spot-nosed monkey 42 10 22 66 … 71 35 36 158 9 … … … … … … 449 petaurista Lesser spot-nosed monkey … … … … … … … … … … … … … … … 3 3 pogonias Crested mona monkey 5 2 11 19 11 24 31 17 66 … … … … 1 1 … 188 preussi Preuss monkey 1 … … … … … … … … … … … … … … … 1 wolfi Wolf’s monkey … … … … … … … … … 21 1 5 31 6 8 … 72 Chlorocebus tantalus Tantalus monkey 15 … … … … … … … … … … … … … … … 15 Erythrocebus patas Patas monkey 17 … … … … … … … … … … … … … … … 17 Lophocebus albigena Gray-cheeked mangabey 7 … 10 … … … 2 16 68 … … 1 … … 2 … 106 aterrimus Black mangabey … … … … … … … … … … … 2 31 … … … 33 Mandrillus leucophaeus Drill 1 … … … … … … … … … … … … … … … 1 sphinx Mandrill 16 1 … … … 10 … … … … … … … … … … 27 Miopithecus talapoin Northern talapoin 8 3 … 1 … 8 … … … … … … … … … … 20 Papio anubis Olive baboon 16 … … … … 1 … … … … … … … … … … 17 Total 188 22 68 132 53 250 319 96 486 137 45 29 281 40 112 64 2322 Genus Species Common Name CM CM CM CM CM CM CM CM CM DRC DRC DRC DRC DRC DRC IC No. of Samples Pets BP ND YD BQ EW EB MS MN MB ML MK KL WK GM TAI Allenopithecus nigroviridis Allen swamp monkey … … … … … … … … … 44 … 2 1 … … … 47 Cercocebus agilis Agile mangabey 14 … … 1 9 … 39 13 55 3 … … … … … … 134 atys Sooty mangabey … … … … … … … … … … … … … … … 21 21 torquatus Red-capped mangabey 2 … … … … 4 … … … … … … … … … … 6 Colobus angolensis Angolan colobus … … … … … … … … … … … 4 21 … … … 25 guereza Mantled guereza 1 … 15 8 … … 2 … 8 1 … … 1 … … … 36 polykomos King’s colobus … … … … … … … … … … … … … … … 11 11 satanus Black colobus … … … … … 8 … … … … … … … … … … 8 Piliocolobus badius Western red colobus … … … … … … … … … … … … … … … 23 23 tholloni Tsuapa red colobus … … … … … … … … … … … 2 84 … … … 86 Procolobus verus Olive colobus … … … … … … … … … … … … … … … 2 2 Cercopithecus ascanius Red-tailed monkey … … … … … … … … … 41 42 12 106 7 33 … 241 campbelli Campbell's monkey … … … … … … … … … … … … … … … 1 1 cephus Mustached monkey 29 6 10 32 25 122 197 14 129 … … … … … … … 564 diana Diana monkey … … … … … … … … … … … … … … … 3 3 hamlyni Hamlyn’s monkey … … … … … … … … … … … … … … 6 … 6 lhoesti l’Hoest monkey … … … … … … … … … … … … … 6 31 … 37 mitis Blue monkey … … … … … … … … … … … … … 20 31 … 51 mona Mona monkey 9 … … 1 … … … … … … … … … … … … 10 neglectus De Brazza monkey 5 … … 4 8 2 13 … 2 18 2 1 6 … … … 61 nictitans Greater spot-nosed monkey 42 10 22 66 … 71 35 36 158 9 … … … … … … 449 petaurista Lesser spot-nosed monkey … … … … … … … … … … … … … … … 3 3 pogonias Crested mona monkey 5 2 11 19 11 24 31 17 66 … … … … 1 1 … 188 preussi Preuss monkey 1 … … … … … … … … … … … … … … … 1 wolfi Wolf’s monkey … … … … … … … … … 21 1 5 31 6 8 … 72 Chlorocebus tantalus Tantalus monkey 15 … … … … … … … … … … … … … … … 15 Erythrocebus patas Patas monkey 17 … … … … … … … … … … … … … … … 17 Lophocebus albigena Gray-cheeked mangabey 7 … 10 … … … 2 16 68 … … 1 … … 2 … 106 aterrimus Black mangabey … … … … … … … … … … … 2 31 … … … 33 Mandrillus leucophaeus Drill 1 … … … … … … … … … … … … … … … 1 sphinx Mandrill 16 1 … … … 10 … … … … … … … … … … 27 Miopithecus talapoin Northern talapoin 8 3 … 1 … 8 … … … … … … … … … … 20 Papio anubis Olive baboon 16 … … … … 1 … … … … … … … … … … 17 Total 188 22 68 132 53 250 319 96 486 137 45 29 281 40 112 64 2322 Collection sites are shown in Figure 1. Abbreviations: BP, Bipindi; BQ, North Dja; CM, Cameroon; DRC, Democratic Republic of the Congo; EB, Eboumetoum; EW, Ebolowa; GM, Goma; IC, Ivory Coast; KL, Kole; MB, Mbandaka; MK, Monkoto; ML, Malebo; MN, Mindourou; MS, Messok; ND, Nditam; TAI, Tai National Park; YD, Yaoundé. View Large Table 1. Number of Samples Collected for Each Species at the Different Collection Sites in Cameroon, Democratic Republic of the Congo, and Ivory Coast Genus Species Common Name CM CM CM CM CM CM CM CM CM DRC DRC DRC DRC DRC DRC IC No. of Samples Pets BP ND YD BQ EW EB MS MN MB ML MK KL WK GM TAI Allenopithecus nigroviridis Allen swamp monkey … … … … … … … … … 44 … 2 1 … … … 47 Cercocebus agilis Agile mangabey 14 … … 1 9 … 39 13 55 3 … … … … … … 134 atys Sooty mangabey … … … … … … … … … … … … … … … 21 21 torquatus Red-capped mangabey 2 … … … … 4 … … … … … … … … … … 6 Colobus angolensis Angolan colobus … … … … … … … … … … … 4 21 … … … 25 guereza Mantled guereza 1 … 15 8 … … 2 … 8 1 … … 1 … … … 36 polykomos King’s colobus … … … … … … … … … … … … … … … 11 11 satanus Black colobus … … … … … 8 … … … … … … … … … … 8 Piliocolobus badius Western red colobus … … … … … … … … … … … … … … … 23 23 tholloni Tsuapa red colobus … … … … … … … … … … … 2 84 … … … 86 Procolobus verus Olive colobus … … … … … … … … … … … … … … … 2 2 Cercopithecus ascanius Red-tailed monkey … … … … … … … … … 41 42 12 106 7 33 … 241 campbelli Campbell's monkey … … … … … … … … … … … … … … … 1 1 cephus Mustached monkey 29 6 10 32 25 122 197 14 129 … … … … … … … 564 diana Diana monkey … … … … … … … … … … … … … … … 3 3 hamlyni Hamlyn’s monkey … … … … … … … … … … … … … … 6 … 6 lhoesti l’Hoest monkey … … … … … … … … … … … … … 6 31 … 37 mitis Blue monkey … … … … … … … … … … … … … 20 31 … 51 mona Mona monkey 9 … … 1 … … … … … … … … … … … … 10 neglectus De Brazza monkey 5 … … 4 8 2 13 … 2 18 2 1 6 … … … 61 nictitans Greater spot-nosed monkey 42 10 22 66 … 71 35 36 158 9 … … … … … … 449 petaurista Lesser spot-nosed monkey … … … … … … … … … … … … … … … 3 3 pogonias Crested mona monkey 5 2 11 19 11 24 31 17 66 … … … … 1 1 … 188 preussi Preuss monkey 1 … … … … … … … … … … … … … … … 1 wolfi Wolf’s monkey … … … … … … … … … 21 1 5 31 6 8 … 72 Chlorocebus tantalus Tantalus monkey 15 … … … … … … … … … … … … … … … 15 Erythrocebus patas Patas monkey 17 … … … … … … … … … … … … … … … 17 Lophocebus albigena Gray-cheeked mangabey 7 … 10 … … … 2 16 68 … … 1 … … 2 … 106 aterrimus Black mangabey … … … … … … … … … … … 2 31 … … … 33 Mandrillus leucophaeus Drill 1 … … … … … … … … … … … … … … … 1 sphinx Mandrill 16 1 … … … 10 … … … … … … … … … … 27 Miopithecus talapoin Northern talapoin 8 3 … 1 … 8 … … … … … … … … … … 20 Papio anubis Olive baboon 16 … … … … 1 … … … … … … … … … … 17 Total 188 22 68 132 53 250 319 96 486 137 45 29 281 40 112 64 2322 Genus Species Common Name CM CM CM CM CM CM CM CM CM DRC DRC DRC DRC DRC DRC IC No. of Samples Pets BP ND YD BQ EW EB MS MN MB ML MK KL WK GM TAI Allenopithecus nigroviridis Allen swamp monkey … … … … … … … … … 44 … 2 1 … … … 47 Cercocebus agilis Agile mangabey 14 … … 1 9 … 39 13 55 3 … … … … … … 134 atys Sooty mangabey … … … … … … … … … … … … … … … 21 21 torquatus Red-capped mangabey 2 … … … … 4 … … … … … … … … … … 6 Colobus angolensis Angolan colobus … … … … … … … … … … … 4 21 … … … 25 guereza Mantled guereza 1 … 15 8 … … 2 … 8 1 … … 1 … … … 36 polykomos King’s colobus … … … … … … … … … … … … … … … 11 11 satanus Black colobus … … … … … 8 … … … … … … … … … … 8 Piliocolobus badius Western red colobus … … … … … … … … … … … … … … … 23 23 tholloni Tsuapa red colobus … … … … … … … … … … … 2 84 … … … 86 Procolobus verus Olive colobus … … … … … … … … … … … … … … … 2 2 Cercopithecus ascanius Red-tailed monkey … … … … … … … … … 41 42 12 106 7 33 … 241 campbelli Campbell's monkey … … … … … … … … … … … … … … … 1 1 cephus Mustached monkey 29 6 10 32 25 122 197 14 129 … … … … … … … 564 diana Diana monkey … … … … … … … … … … … … … … … 3 3 hamlyni Hamlyn’s monkey … … … … … … … … … … … … … … 6 … 6 lhoesti l’Hoest monkey … … … … … … … … … … … … … 6 31 … 37 mitis Blue monkey … … … … … … … … … … … … … 20 31 … 51 mona Mona monkey 9 … … 1 … … … … … … … … … … … … 10 neglectus De Brazza monkey 5 … … 4 8 2 13 … 2 18 2 1 6 … … … 61 nictitans Greater spot-nosed monkey 42 10 22 66 … 71 35 36 158 9 … … … … … … 449 petaurista Lesser spot-nosed monkey … … … … … … … … … … … … … … … 3 3 pogonias Crested mona monkey 5 2 11 19 11 24 31 17 66 … … … … 1 1 … 188 preussi Preuss monkey 1 … … … … … … … … … … … … … … … 1 wolfi Wolf’s monkey … … … … … … … … … 21 1 5 31 6 8 … 72 Chlorocebus tantalus Tantalus monkey 15 … … … … … … … … … … … … … … … 15 Erythrocebus patas Patas monkey 17 … … … … … … … … … … … … … … … 17 Lophocebus albigena Gray-cheeked mangabey 7 … 10 … … … 2 16 68 … … 1 … … 2 … 106 aterrimus Black mangabey … … … … … … … … … … … 2 31 … … … 33 Mandrillus leucophaeus Drill 1 … … … … … … … … … … … … … … … 1 sphinx Mandrill 16 1 … … … 10 … … … … … … … … … … 27 Miopithecus talapoin Northern talapoin 8 3 … 1 … 8 … … … … … … … … … … 20 Papio anubis Olive baboon 16 … … … … 1 … … … … … … … … … … 17 Total 188 22 68 132 53 250 319 96 486 137 45 29 281 40 112 64 2322 Collection sites are shown in Figure 1. Abbreviations: BP, Bipindi; BQ, North Dja; CM, Cameroon; DRC, Democratic Republic of the Congo; EB, Eboumetoum; EW, Ebolowa; GM, Goma; IC, Ivory Coast; KL, Kole; MB, Mbandaka; MK, Monkoto; ML, Malebo; MN, Mindourou; MS, Messok; ND, Nditam; TAI, Tai National Park; YD, Yaoundé. View Large Among the 2327 samples from apes, 1182 (51%) were from western lowland gorillas (G. g. gorilla) in Cameroon, 353 (15%) from bonobos (P. paniscus) in DRC, and 792 (34%) from chimpanzees across Africa including samples from the western (Pan troglodytes verus, n = 57) and central chimpanzee (P. t. troglodytes, n = 735). Table 2 displays for each ape species or subspecies numbers collected at each site, illustrated in Figure 1. Table 2. Number of Samples Collected for Each Ape Species, by Site Species Country and Site No. Pan troglodytes verus Ivory Coast  Tai 57  Subtotal 57 Pan troglodytes troglodytes Cameroon  BQ 120  CP 114  DJ 45  EK 120  MB 316  MT 20  Subtotal 735 Gorilla gorilla gorilla Cameroon  BP 143  BQ 239  CP 289  DJ 167  EK 72  MB 161  MT 22  MM 89  Subtotal 1182 Pan paniscus DRC  LA 137  MZ 166  ML 50  Subtotal 353 Total 2327 Species Country and Site No. Pan troglodytes verus Ivory Coast  Tai 57  Subtotal 57 Pan troglodytes troglodytes Cameroon  BQ 120  CP 114  DJ 45  EK 120  MB 316  MT 20  Subtotal 735 Gorilla gorilla gorilla Cameroon  BP 143  BQ 239  CP 289  DJ 167  EK 72  MB 161  MT 22  MM 89  Subtotal 1182 Pan paniscus DRC  LA 137  MZ 166  ML 50  Subtotal 353 Total 2327 Collection sites are shown in Figure 1. Abbreviations: BP, Bipindi; BQ, North Dja; CP, Campo; DJ, Djoum; DRC, Democratic Republic of Congo; EK, Ekom; LA, Lomako-Yokokala; MB, Mambele; ML, Malebo; MM, Mengame; MT, Mintom; MZ, Manzana. View Large Table 2. Number of Samples Collected for Each Ape Species, by Site Species Country and Site No. Pan troglodytes verus Ivory Coast  Tai 57  Subtotal 57 Pan troglodytes troglodytes Cameroon  BQ 120  CP 114  DJ 45  EK 120  MB 316  MT 20  Subtotal 735 Gorilla gorilla gorilla Cameroon  BP 143  BQ 239  CP 289  DJ 167  EK 72  MB 161  MT 22  MM 89  Subtotal 1182 Pan paniscus DRC  LA 137  MZ 166  ML 50  Subtotal 353 Total 2327 Species Country and Site No. Pan troglodytes verus Ivory Coast  Tai 57  Subtotal 57 Pan troglodytes troglodytes Cameroon  BQ 120  CP 114  DJ 45  EK 120  MB 316  MT 20  Subtotal 735 Gorilla gorilla gorilla Cameroon  BP 143  BQ 239  CP 289  DJ 167  EK 72  MB 161  MT 22  MM 89  Subtotal 1182 Pan paniscus DRC  LA 137  MZ 166  ML 50  Subtotal 353 Total 2327 Collection sites are shown in Figure 1. Abbreviations: BP, Bipindi; BQ, North Dja; CP, Campo; DJ, Djoum; DRC, Democratic Republic of Congo; EK, Ekom; LA, Lomako-Yokokala; MB, Mambele; ML, Malebo; MM, Mengame; MT, Mintom; MZ, Manzana. View Large Low Prevalence of Ebola Virus IgG Antibodies in Monkeys The 2322 monkey samples were tested for Ebola antibodies with the multiplex antibody assay. The results are summarized for each antigen and for each Ebola virus species in Tables 3 and 4. No sample reacted simultaneously with GP and NP proteins from EBOV, and we therefore considered that none of the samples had specific antibodies to EBOV. Only a single sample, derived from a mustached monkey (C. cephus) from Cameroon, had antibodies to both NP and GP proteins from SUDV. Reactivity to another combination of 2 antigens was also low: 1 baboon (Papio anubis) was reactive with VP and GP proteins from EBOV, SUDV, and BDVB and 1 greater spot-nosed monkey (C. nictitans) with VP and GP from BDBV. All the other samples reacted only with 1 antigen: 0.6% for NP and 0.5%–1% reactivity for the different VP40 proteins. Highest reactivity was observed with GP proteins, and ranged from 2.2% to 2.6% with GP proteins derived from the African Ebola virus species (EBOV, SUDV, and BDBV) and was 1.5% for the Asian Ebola virus species (RESTV). Almost all GP-reactive samples were simultaneously reactive to GP proteins from >1 Ebola virus species. Highest reactivities (>2%) to GP proteins were observed in the following species: C. nictitans, C. cephus, C. agilis, Chlorocebus tantalus, and Colobus polykomos. Table 3. Number and Percentage of Samples for Each Species Reactive With the Different Antigens Used in the Luminex Assay for Zaire ebolavirus Genus Species Country No. Tested NP GP GP VP40 NP + GP EBOV EBOV-K EBOV-M EBOV EBOV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 4 (3.0; 1.1–7.4) 0 (0; .0–2.8) 0 (0; .0–2.8) atys IC 21 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) (0; .0–13.3) 1 (4.0; .7–19.5) 0 (0; .0–13.3) guereza Cam, DRC 36 1 (2.8; .5–14.2) 0 (0; .0–9.6) 0 (0; .0–9.6) 1 (2.8; .5–14.2) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 1 (9.1; 1.6–37.7) 1 (9.1; 1.6–37.7) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 Cercopithecus ascanius DRC 241 0 (0; .0–1.6) 1 (0.4; .07–2.3) 2 (0.8; .2–2.9) 2 (0.8; .2–2.9) 0 (0; .0–1.6) campbelli IC 1a 0 0 0 0 0 cephus Cam, DRC 565 1 (0.2; .03–1.0) 29 (5.1; 3.6–7.3) 34 (6.0; 4.3–8.3) 3 (0.5; .2–1.6) 0 (0; .0–.7) diana IC 3a 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 lhoesti DRC 37 1 (2.8; .5–13.8) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 0 (0; .0–5.9) 0 (0; .0–5.9) nictitans Cam, DRC 448 7 (1.6; .7–3.2) 17 (3.8; 2.4–6.0) 16 (3.6; 2.2–5.7) 3 (0.7; .2–1.9) 0 (0; .0–.9) petaurista IC 3a 0 3 3 0 0 pogonias Cam, DRC 188 1 (0.5; .1–2.9) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 1 (0.9; .2–5.2) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) Papio anubis Cam 17 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) Total 2322 15 (0.6; .4–1.1) 58 (2.5; 1.9–3.2) 61 (2.6; 2.1–3.4) 12 (0.5; .3–.9) 0 (0; .0–.2) Genus Species Country No. Tested NP GP GP VP40 NP + GP EBOV EBOV-K EBOV-M EBOV EBOV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 4 (3.0; 1.1–7.4) 0 (0; .0–2.8) 0 (0; .0–2.8) atys IC 21 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) (0; .0–13.3) 1 (4.0; .7–19.5) 0 (0; .0–13.3) guereza Cam, DRC 36 1 (2.8; .5–14.2) 0 (0; .0–9.6) 0 (0; .0–9.6) 1 (2.8; .5–14.2) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 1 (9.1; 1.6–37.7) 1 (9.1; 1.6–37.7) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 Cercopithecus ascanius DRC 241 0 (0; .0–1.6) 1 (0.4; .07–2.3) 2 (0.8; .2–2.9) 2 (0.8; .2–2.9) 0 (0; .0–1.6) campbelli IC 1a 0 0 0 0 0 cephus Cam, DRC 565 1 (0.2; .03–1.0) 29 (5.1; 3.6–7.3) 34 (6.0; 4.3–8.3) 3 (0.5; .2–1.6) 0 (0; .0–.7) diana IC 3a 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 lhoesti DRC 37 1 (2.8; .5–13.8) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 0 (0; .0–5.9) 0 (0; .0–5.9) nictitans Cam, DRC 448 7 (1.6; .7–3.2) 17 (3.8; 2.4–6.0) 16 (3.6; 2.2–5.7) 3 (0.7; .2–1.9) 0 (0; .0–.9) petaurista IC 3a 0 3 3 0 0 pogonias Cam, DRC 188 1 (0.5; .1–2.9) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 1 (0.9; .2–5.2) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) Papio anubis Cam 17 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) Total 2322 15 (0.6; .4–1.1) 58 (2.5; 1.9–3.2) 61 (2.6; 2.1–3.4) 12 (0.5; .3–.9) 0 (0; .0–.2) Data are presented as No. positive (%; 95% confidence interval) unless otherwise indicated. Abbreviations: Cam, Cameroon; DRC, Democratic Republic of the Congo; EBOV, Zaire ebolavirus; GP, glycoprotein; IC, Ivory Coast; K, Kissoudougou strain; M, Mayinga strain; NP, nucleoprotein; VP40, viral protein 40. aPercentages were not calculated when number of samples tested was <10. View Large Table 3. Number and Percentage of Samples for Each Species Reactive With the Different Antigens Used in the Luminex Assay for Zaire ebolavirus Genus Species Country No. Tested NP GP GP VP40 NP + GP EBOV EBOV-K EBOV-M EBOV EBOV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 4 (3.0; 1.1–7.4) 0 (0; .0–2.8) 0 (0; .0–2.8) atys IC 21 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) (0; .0–13.3) 1 (4.0; .7–19.5) 0 (0; .0–13.3) guereza Cam, DRC 36 1 (2.8; .5–14.2) 0 (0; .0–9.6) 0 (0; .0–9.6) 1 (2.8; .5–14.2) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 1 (9.1; 1.6–37.7) 1 (9.1; 1.6–37.7) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 Cercopithecus ascanius DRC 241 0 (0; .0–1.6) 1 (0.4; .07–2.3) 2 (0.8; .2–2.9) 2 (0.8; .2–2.9) 0 (0; .0–1.6) campbelli IC 1a 0 0 0 0 0 cephus Cam, DRC 565 1 (0.2; .03–1.0) 29 (5.1; 3.6–7.3) 34 (6.0; 4.3–8.3) 3 (0.5; .2–1.6) 0 (0; .0–.7) diana IC 3a 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 lhoesti DRC 37 1 (2.8; .5–13.8) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 0 (0; .0–5.9) 0 (0; .0–5.9) nictitans Cam, DRC 448 7 (1.6; .7–3.2) 17 (3.8; 2.4–6.0) 16 (3.6; 2.2–5.7) 3 (0.7; .2–1.9) 0 (0; .0–.9) petaurista IC 3a 0 3 3 0 0 pogonias Cam, DRC 188 1 (0.5; .1–2.9) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 1 (0.9; .2–5.2) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) Papio anubis Cam 17 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) Total 2322 15 (0.6; .4–1.1) 58 (2.5; 1.9–3.2) 61 (2.6; 2.1–3.4) 12 (0.5; .3–.9) 0 (0; .0–.2) Genus Species Country No. Tested NP GP GP VP40 NP + GP EBOV EBOV-K EBOV-M EBOV EBOV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 4 (3.0; 1.1–7.4) 0 (0; .0–2.8) 0 (0; .0–2.8) atys IC 21 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) (0; .0–13.3) 1 (4.0; .7–19.5) 0 (0; .0–13.3) guereza Cam, DRC 36 1 (2.8; .5–14.2) 0 (0; .0–9.6) 0 (0; .0–9.6) 1 (2.8; .5–14.2) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 1 (9.1; 1.6–37.7) 1 (9.1; 1.6–37.7) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 Cercopithecus ascanius DRC 241 0 (0; .0–1.6) 1 (0.4; .07–2.3) 2 (0.8; .2–2.9) 2 (0.8; .2–2.9) 0 (0; .0–1.6) campbelli IC 1a 0 0 0 0 0 cephus Cam, DRC 565 1 (0.2; .03–1.0) 29 (5.1; 3.6–7.3) 34 (6.0; 4.3–8.3) 3 (0.5; .2–1.6) 0 (0; .0–.7) diana IC 3a 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 lhoesti DRC 37 1 (2.8; .5–13.8) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 0 (0; .0–5.9) 0 (0; .0–5.9) nictitans Cam, DRC 448 7 (1.6; .7–3.2) 17 (3.8; 2.4–6.0) 16 (3.6; 2.2–5.7) 3 (0.7; .2–1.9) 0 (0; .0–.9) petaurista IC 3a 0 3 3 0 0 pogonias Cam, DRC 188 1 (0.5; .1–2.9) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) 0 (0; .5–.1) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 1 (0.9; .2–5.2) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 0 (0; .0–16.1) Papio anubis Cam 17 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) Total 2322 15 (0.6; .4–1.1) 58 (2.5; 1.9–3.2) 61 (2.6; 2.1–3.4) 12 (0.5; .3–.9) 0 (0; .0–.2) Data are presented as No. positive (%; 95% confidence interval) unless otherwise indicated. Abbreviations: Cam, Cameroon; DRC, Democratic Republic of the Congo; EBOV, Zaire ebolavirus; GP, glycoprotein; IC, Ivory Coast; K, Kissoudougou strain; M, Mayinga strain; NP, nucleoprotein; VP40, viral protein 40. aPercentages were not calculated when number of samples tested was <10. View Large Table 4. Number and Percentage of Samples for Each Species Reactive With the Different Antigens Used in the Luminex Assay for Sudan ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus Genus Species Country No. Tested NP GP VP40 NP + GP GP VP40 GP SUDV SUDV SUDV SUDV BDBV BDBV RESTV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; 0–7.5) 0 (0; .0–7.5) 1 (2.1; .4–11.1) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 2 (1.5; .4–5.3) 0 (0; .0–2.8) 5 (3.7; 1.6–8.4) 2 (1.5; .4–5.3) 4 (3.0; 1.1–7.4) atys IC 21 2 (9.5; 2.6–28.9) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) guereza Cam, DRC 36 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 1 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 0 0 Cercopithecus ascanius DRC 241 2 (0.8; .2–2.9) 1 (0.4; .07–2.3) 1 (0.4; .07–2.3) 0 (0; .0–1.6) 2 (0.8; .2–2.9) 0 (0; .0–1.6) 1 (0.4; .07–2.3) campbelli IC 1a 0 0 0 0 0 0 0 cephus Cam, DRC 565 5 (0.9; .4–2.1) 34 (6.0; 4.3–8.3) 4 (0.7; .3–1.8) 1 (0.2; .03–1.0) 30 (5.4; 3.7–7.5) 6 (1.1; .5–2.3) 19 (3.4; 2.2–5.2) diana IC 3a 0 0 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 0 0 lhoesti DRC 37 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 0 (0; .0–5.9) 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) nictitans Cam, DRC 448 2 (0.5; .1–1.6) 8 (1.8; .9–3.5) 5 (1.1; .5–2.6) 0 (0; .0–.9) 13 (2.9; 1.7–4.9) 5 (1.1; .5–2.6) 6 (1.3; .6–2.9) petaurista IC 3a 0 1 0 0 3 0 0 pogonias Cam, DRC 188 0 (0; .0–2.0) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 1 (1.4; .2–7.5) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 2 (1.9; .5–6.6) 0 (0; .0–3.5) 3 (2.9; .9–8.0) 0 (0; .0–3.5) 0 (0; .0–3.5) 1 (0.9; .2–5.2) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 1 (3.7; .6–18.3) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 3 (15.0; 5.2–36.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 2 (10.0; 2.7–30.1) 0 (0; .0–16.1) Papio anubis Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 2 (11.8; 3.3–34.3) 2 (11.8; 3.3–34.3) Total 2322 14 (0.6; .4–1.0) 51 (2.2; 1.7–2.9) 23 (1.0; .7–1.5) 1 (0.04; 0–.3) 56 (2.4; 1.9–3.1) 20 (0.9; .6–1.3) 35 (1.5; 1.1–2.1) Genus Species Country No. Tested NP GP VP40 NP + GP GP VP40 GP SUDV SUDV SUDV SUDV BDBV BDBV RESTV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; 0–7.5) 0 (0; .0–7.5) 1 (2.1; .4–11.1) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 2 (1.5; .4–5.3) 0 (0; .0–2.8) 5 (3.7; 1.6–8.4) 2 (1.5; .4–5.3) 4 (3.0; 1.1–7.4) atys IC 21 2 (9.5; 2.6–28.9) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) guereza Cam, DRC 36 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 1 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 0 0 Cercopithecus ascanius DRC 241 2 (0.8; .2–2.9) 1 (0.4; .07–2.3) 1 (0.4; .07–2.3) 0 (0; .0–1.6) 2 (0.8; .2–2.9) 0 (0; .0–1.6) 1 (0.4; .07–2.3) campbelli IC 1a 0 0 0 0 0 0 0 cephus Cam, DRC 565 5 (0.9; .4–2.1) 34 (6.0; 4.3–8.3) 4 (0.7; .3–1.8) 1 (0.2; .03–1.0) 30 (5.4; 3.7–7.5) 6 (1.1; .5–2.3) 19 (3.4; 2.2–5.2) diana IC 3a 0 0 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 0 0 lhoesti DRC 37 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 0 (0; .0–5.9) 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) nictitans Cam, DRC 448 2 (0.5; .1–1.6) 8 (1.8; .9–3.5) 5 (1.1; .5–2.6) 0 (0; .0–.9) 13 (2.9; 1.7–4.9) 5 (1.1; .5–2.6) 6 (1.3; .6–2.9) petaurista IC 3a 0 1 0 0 3 0 0 pogonias Cam, DRC 188 0 (0; .0–2.0) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 1 (1.4; .2–7.5) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 2 (1.9; .5–6.6) 0 (0; .0–3.5) 3 (2.9; .9–8.0) 0 (0; .0–3.5) 0 (0; .0–3.5) 1 (0.9; .2–5.2) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 1 (3.7; .6–18.3) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 3 (15.0; 5.2–36.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 2 (10.0; 2.7–30.1) 0 (0; .0–16.1) Papio anubis Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 2 (11.8; 3.3–34.3) 2 (11.8; 3.3–34.3) Total 2322 14 (0.6; .4–1.0) 51 (2.2; 1.7–2.9) 23 (1.0; .7–1.5) 1 (0.04; 0–.3) 56 (2.4; 1.9–3.1) 20 (0.9; .6–1.3) 35 (1.5; 1.1–2.1) Data are presented as No. positive (%; 95% confidence interval) unless otherwise indicated. Abbreviations: BDBV, Bundibugyo ebolavirus; Cam, Cameroon; DRC, Democratic Republic of the Congo; GP, glycoprotein; IC, Ivory Coast; NP, nucleoprotein; RESTV, Reston ebolavirus; SUDV, Sudan ebolavirus; VP40, viral protein 40. aPercentages were not calculated when the number of samples tested was <10. View Large Table 4. Number and Percentage of Samples for Each Species Reactive With the Different Antigens Used in the Luminex Assay for Sudan ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus Genus Species Country No. Tested NP GP VP40 NP + GP GP VP40 GP SUDV SUDV SUDV SUDV BDBV BDBV RESTV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; 0–7.5) 0 (0; .0–7.5) 1 (2.1; .4–11.1) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 2 (1.5; .4–5.3) 0 (0; .0–2.8) 5 (3.7; 1.6–8.4) 2 (1.5; .4–5.3) 4 (3.0; 1.1–7.4) atys IC 21 2 (9.5; 2.6–28.9) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) guereza Cam, DRC 36 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 1 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 0 0 Cercopithecus ascanius DRC 241 2 (0.8; .2–2.9) 1 (0.4; .07–2.3) 1 (0.4; .07–2.3) 0 (0; .0–1.6) 2 (0.8; .2–2.9) 0 (0; .0–1.6) 1 (0.4; .07–2.3) campbelli IC 1a 0 0 0 0 0 0 0 cephus Cam, DRC 565 5 (0.9; .4–2.1) 34 (6.0; 4.3–8.3) 4 (0.7; .3–1.8) 1 (0.2; .03–1.0) 30 (5.4; 3.7–7.5) 6 (1.1; .5–2.3) 19 (3.4; 2.2–5.2) diana IC 3a 0 0 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 0 0 lhoesti DRC 37 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 0 (0; .0–5.9) 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) nictitans Cam, DRC 448 2 (0.5; .1–1.6) 8 (1.8; .9–3.5) 5 (1.1; .5–2.6) 0 (0; .0–.9) 13 (2.9; 1.7–4.9) 5 (1.1; .5–2.6) 6 (1.3; .6–2.9) petaurista IC 3a 0 1 0 0 3 0 0 pogonias Cam, DRC 188 0 (0; .0–2.0) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 1 (1.4; .2–7.5) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 2 (1.9; .5–6.6) 0 (0; .0–3.5) 3 (2.9; .9–8.0) 0 (0; .0–3.5) 0 (0; .0–3.5) 1 (0.9; .2–5.2) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 1 (3.7; .6–18.3) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 3 (15.0; 5.2–36.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 2 (10.0; 2.7–30.1) 0 (0; .0–16.1) Papio anubis Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 2 (11.8; 3.3–34.3) 2 (11.8; 3.3–34.3) Total 2322 14 (0.6; .4–1.0) 51 (2.2; 1.7–2.9) 23 (1.0; .7–1.5) 1 (0.04; 0–.3) 56 (2.4; 1.9–3.1) 20 (0.9; .6–1.3) 35 (1.5; 1.1–2.1) Genus Species Country No. Tested NP GP VP40 NP + GP GP VP40 GP SUDV SUDV SUDV SUDV BDBV BDBV RESTV Allenopithecus nigrovidis DRC 47 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; .0–7.5) 0 (0; 0–7.5) 0 (0; .0–7.5) 1 (2.1; .4–11.1) Cercocebus agilis Cam, DRC 134 0 (0; .0–2.8) 4 (3.0; 1.1–7.4) 2 (1.5; .4–5.3) 0 (0; .0–2.8) 5 (3.7; 1.6–8.4) 2 (1.5; .4–5.3) 4 (3.0; 1.1–7.4) atys IC 21 2 (9.5; 2.6–28.9) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) 0 (0; .0–15.5) torquatus Cam 6a 0 0 0 0 0 0 0 Colobus angolensis DRC 25 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) 0 (0; .0–13.3) guereza Cam, DRC 36 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) 0 (0; .0–9.6) polykomos IC 11 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) 0 (0; .0–25.9) satanus Cam 8a 0 1 0 0 0 0 0 Piliocolobus badius IC 23 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) 0 (0; .0–14.3) tholloni DRC 86 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) 0 (0; .0–4.3) Procolobus verus IC 2a 0 0 0 0 0 0 0 Cercopithecus ascanius DRC 241 2 (0.8; .2–2.9) 1 (0.4; .07–2.3) 1 (0.4; .07–2.3) 0 (0; .0–1.6) 2 (0.8; .2–2.9) 0 (0; .0–1.6) 1 (0.4; .07–2.3) campbelli IC 1a 0 0 0 0 0 0 0 cephus Cam, DRC 565 5 (0.9; .4–2.1) 34 (6.0; 4.3–8.3) 4 (0.7; .3–1.8) 1 (0.2; .03–1.0) 30 (5.4; 3.7–7.5) 6 (1.1; .5–2.3) 19 (3.4; 2.2–5.2) diana IC 3a 0 0 0 0 0 0 0 hamlyni DRC 6a 0 0 0 0 0 0 0 lhoesti DRC 37 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) 0 (0; .0–9.4) mitis DRC 51 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) 0 (0; .0–7.0) mona Cam 10 1 (10.0; 1.8–40.4) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) 0 (0; .0–27.8) neglectus Cam, DRC 61 0 (0; .0–5.9) 1 (1.6; .3–8.7) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) 0 (0; .0–5.9) 1 (1.6; .3–8.7) nictitans Cam, DRC 448 2 (0.5; .1–1.6) 8 (1.8; .9–3.5) 5 (1.1; .5–2.6) 0 (0; .0–.9) 13 (2.9; 1.7–4.9) 5 (1.1; .5–2.6) 6 (1.3; .6–2.9) petaurista IC 3a 0 1 0 0 3 0 0 pogonias Cam, DRC 188 0 (0; .0–2.0) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 1 (0.5; .1–2.9) 0 (0; .0–2.0) 0 (0; .0–2.0) preussi Cam 1a 0 0 0 0 0 0 0 wolfi DRC 72 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 0 (0; .0–5.1) 1 (1.4; .2–7.5) 0 (0; .0–5.1) Chlorocebus tantalus Cam 15 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) 0 (0; .0–20.4) 1 (6.7; 1.2–29.8) Erythrocebus patas Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.0–26.9) 0 (0; .0–18.4) Lophocebus albigena Cam 106 2 (1.9; .5–6.6) 0 (0; .0–3.5) 3 (2.9; .9–8.0) 0 (0; .0–3.5) 0 (0; .0–3.5) 1 (0.9; .2–5.2) 0 (0; .0–3.5) aterrimus DRC 33 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) 0 (0; .0–10.4) Mandrillus leucophaeus Cam 1a 0 0 0 0 0 0 0 sphinx Cam 27 0 (0; .0–12.5) 0 (0; .0–12.5) 1 (3.7; .6–18.3) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) 0 (0; .0–12.5) Miopithecus talapoin Cam 20 0 (0; .0–16.1) 0 (0; .0–16.1) 3 (15.0; 5.2–36.1) 0 (0; .0–16.1) 0 (0; .0–16.1) 2 (10.0; 2.7–30.1) 0 (0; .0–16.1) Papio anubis Cam 17 0 (0; .0–18.4) 0 (0; .0–18.4) 1 (5.9; 1.4–26.9) 0 (0; .0–18.4) 0 (0; .0–18.4) 2 (11.8; 3.3–34.3) 2 (11.8; 3.3–34.3) Total 2322 14 (0.6; .4–1.0) 51 (2.2; 1.7–2.9) 23 (1.0; .7–1.5) 1 (0.04; 0–.3) 56 (2.4; 1.9–3.1) 20 (0.9; .6–1.3) 35 (1.5; 1.1–2.1) Data are presented as No. positive (%; 95% confidence interval) unless otherwise indicated. Abbreviations: BDBV, Bundibugyo ebolavirus; Cam, Cameroon; DRC, Democratic Republic of the Congo; GP, glycoprotein; IC, Ivory Coast; NP, nucleoprotein; RESTV, Reston ebolavirus; SUDV, Sudan ebolavirus; VP40, viral protein 40. aPercentages were not calculated when the number of samples tested was <10. View Large No Evidence of IgG Ebola Virus Antibodies in Apes As previously observed for human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) cross-reactive antibody detection in fecal samples from apes, sensitivity is lower and interpretation criteria needed to be adapted [31–33]. Therefore, we first tested 57 paired feces (preserved in RNAlater) and plasma samples from EBOV survivors to evaluate the feasibility and sensitivity of Ebola virus antibody detection in fecal samples. In contrast with the antibody profile observed in plasma (ie, simultaneous positivity against NP + GP or NP + GP + VP), antibody reactivity in feces was more frequently directed against a single protein (ie, 57/57 [100%] in plasma vs 18/57 [31.6%] in fecal samples; Supplementary Table 2). However, 29 of 57 (50.9%) of fecal samples were reactive with at least 1 antigen, but a mean decrease of 0.67 to 1.37 log10 of MFI values was observed compared to plasma (Supplementary Table 2). Thus, we adapted the interpretation criteria in fecal samples to antibody reactivity with a single antigen and decreased the above used cutoff values with 1 log10 value. The lower sensitivity in fecal samples is comparable to that observed for HIV/SIV cross-reactive antibody detection in fecal samples from apes. Using these adapted criteria, none of the 2316 fecal samples reacted with any of the recombinant proteins in the Luminex assay. In addition, the 11 blood samples from western chimpanzees from the Tai forest in Ivory Coast were also negative for all antigens. DISCUSSION Although there is evidence that NHPs, and especially apes, play a role in zoonotic transmission of EVD outbreaks, few data are available on Ebola infection in NHPs. Here we report an extensive serological survey to 4 different Ebola virus species in 4649 NHP samples representing 36 different species. We applied a Luminex-based sensitive and specific antibody assay for the simultaneous detection of antibodies to 4 of the 5 species previously described in humans [34]. To identify specific Ebola virus antibodies, we used the same algorithm to NHPs as that developed for human EBOV survivors (ie, simultaneous positivity to NP and GP recombinant proteins); this was deemed appropriate because a recent study in cynomolgus macaques (Macaca fascicularis) naturally infected with RESTV revealed also that convalescent macaques having cleared the virus presented antibodies to NP and GP proteins, both by ELISA and immunofluorescence [44]. As such, we observed only 1 mustached monkey (C. cephus) from southern Cameroon with antibodies to GP and NP from SUDV. Only 2 other samples had antibodies to 2 different antigens from the same Ebola virus species; 1 P. anubis sample was reactive to VP40 and GP proteins from EBOV, SUDV, and BDBV, suggesting nonspecific or cross-reactivity, and 1 C. nictitans sample from Cameroon was reactive with GP and VP40 from BDBV. This is the first study to have evaluated a large number of monkeys using the same assay and interpretation criteria. We tested 2322 monkeys compared to the previous total of 930 from studies in Cameroon (n = 177), Gabon (n = 318), DRC (n = 251), and Kenya (n = 184) using a wide diversity of antibody detection assays [14, 15, 23, 24]. In these previous studies, EBOV-positive samples were seen in 3 of 184 captive baboons in a primate center in Kenya and in captive monkeys from Cameroon: in 1 of 8 De Brazza monkeys, 1 of 25 baboons, 1 of 18 mandrills, and 5 of 34 drills [14, 15]. In our study, the 27 mandrills and 61 De Brazza monkeys were all negative and 1 of 17 baboon samples was reactive with VP40 and GP proteins of different Ebola virus species. The different results may be due to the low numbers tested per species, the different geographic areas, and the different tests used. Antibody detection in fecal samples has been validated and used for Ebola using Western blot in gorillas that live in areas in Republic of Congo with previous EBOV outbreaks, showing that 8 of 80 (10%) had EBOV antibodies [16]. In our study, no evidence for Ebola virus antibodies was observed in 2316 fecal samples, corresponding to around 1362 apes taken into account from previous resampling estimates [31–33]. Our samples are mainly from areas without previously documented outbreaks; however, the majority were obtained in areas considered at risk for outbreaks [1]. Moreover, some sites like Djoum, Mambele, Mintom, or Mengame (Figure 1), which account for >650 samples in southern Cameroon, are located within a 100- to 300-km flight distance from areas of previous outbreaks in Gabon. Moreover, the samples from Ivory Coast were from the Tai National Park where an Ebola outbreak was documented among chimpanzees in 1994 and all fecal samples were collected in 2001 in the specific outbreak area. In contrast, a previous study using blood samples from wild-born but captive chimpanzees and gorillas revealed high EBOV antibody levels in apes: 21 of 119 (17.7%), 3 of 71 (4.2%), and 5 of 35 (14.3%) chimpanzees from Cameroon, Gabon, and Republic of the Congo, respectively, and 2 of 17 (11.2%) gorillas from Cameroon [15]. If EBOV prevalence in apes in Cameroon is as high as suggested in the previous study, we would have expected some reactive samples. As our assay has been shown to have a >95% sensitivity on human samples [34] and a 50% sensitivity on fecal samples, we would expect to have observed some reactivity in at least 100 fecal samples from chimpanzees or gorillas. Reactivity to a single Ebola virus antigen, especially GP, in NHPs has to be further explored, for example with neutralization assays, to ascertain whether it corresponds to nonspecific antibody reactivity or cross-reactivity with another pathogen, or different kinetics of antibodies to the different Ebola proteins. Recent studies have shown that bats can be infected with Ebola viruses that are different from the species known to infect humans, which could induce cross-reactivity with GP antigens [45, 46]. NHPs and bats share habitats and fruits in their diet. Virus transmission from bats to NHPs is suggested to occur when primates come in contact with fruit that is contaminated with feces, urine, or saliva from infected bats [2, 3]. Interestingly, a recent study reported that Cercopithecus species hunt roosting bats for consumption, which could be another, and a particularly efficient, route for Ebola virus transmission [47]. Preying on bats has been reported in C. ascanius and C. mitis species in East Africa, and also in bonobos in DRC [47, 48]. It is also possible that different modes of exposure to Ebola virus could lead to different antibody profiles, that is, contaminated fruit vs contact with infected bats during hunting. Whereas the majority of the outbreaks have been limited in terms of geographic spread and number of people infected, the 2014–2015 EVD outbreak in West Africa clearly illustrates the epidemic potential of a single zoonotic transmission in the presence of certain factors in favor of epidemic spread [49]. The recent EVD outbreaks in 2018 in the Equateur and Kivu provinces of DRC also illustrate that the virus can reach urban centers, even in a context of weak mobility infrastructure [26]. In conclusion, combining results on NHPs from our (4649 samples) and previous studies (~1300 samples), it is clear that Ebola virus antibodies are not widespread among NHPs, which confirms that NHPs are not reservoir species and that if Ebola virus infection in NHPs occurs, few animals survive. More samples from NHPs, bats, and other animal species from different regions across Africa should be studied to define which animals play a role in EVD outbreaks by acting as a reservoir species or as an amplifying host species. Nevertheless, with the increasing frequency of Ebola virus outbreaks (3 outbreaks in a 1-year period in DRC), it becomes extremely urgent to identify the animal reservoir(s) and to understand the ecology of Ebola viruses. It is estimated that transmission from animals to humans is possible in 23 countries across Central and West Africa with a total of >300 million inhabitants, and that at least 22 million of these people live in high-risk areas [50]. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Acknowledgments. We thank the staff and SIV team from Projet PRESICA (Innocent Ndong Bass, Aime Mebenga, Joseph Moudindo, and Thomas Atemkem) and Donald Mbohli from Projet Grand Singes for the collection of samples and logistical support in Cameroon. In addition, we thank the field staff from DRC (Mubonga Mukulumanya, Lunguya-Metila Octavie, and Mbenzo-Abokome Valentin); Dr Mazongo, Dr Abanda, Dr Jonnhy, and all the local staff in Equateur and Nord-Kivu provinces for their collaboration and participation in this study; the staff of the World Wildlife Fund for Nature/DRC; the Institut National de Recherches Biomédicales (Kinshasa, DRC); the Bonobo Conservation Initiative; and Vie Sauvage, Didier Mazongo, Octavie Lunguya, Muriel Aloni, and Valentin Mbenzo-Abokome for field work in DRC. We thank the Ivorian authorities, especially the Ministry of the Environment and Forests, as well as the Ministry of Research; the Swiss Research Centre for Scientific Research; the directorship of the Taï National Park; and the Tai Chimpanzee Projects direction (Dr Wittig), the veterinarians (mainly A. Düx, K. Nowak, A. Lang), and the field assistants for continuous support. Financial support. This work was supported in part by grants from Institut national de la santé et de la recherche médicale/Ebola Task Force; REACTing; the US National Institutes of Health (grant number RO1 AI 50529); Agence Nationale de Recherches sur le SIDA (grant numbers ANRS 12125, 12182, and 12325); the Christophe Mérieux Prize 2015 awarded to J.-J. M.-T. M.; and the Capacity building and surveillance for Ebola Virus Disease (EBO-SURSY) project funded by the European Union, International Mixt Laboratory “PreVIHMI” of the Institut de Recherche pour le Developpement (IRD). C.-J. V. A. was supported by a fellowship from IRD and the Labex EpiGenMed, via the National Research Agency, Programme for Future Investment (ANR-10-LABX-12-01), and University of Montpellier. A. K. K. was supported by a fellowship from Montpellier Université d’excellence (MUSE) (I-Site MUSE, ANR-16-IDEX-0006). This work has also benefited from sample collections performed in the course of the German Research Foundation (DFG) projects (grant number FOR2136/LE1813/10–1). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Presented in part: Ninth International Symposium on Filoviruses, Marburg, Germany, 13–16 September 2017. References 1. Pigott DM , Golding N , Mylne A , et al. Mapping the zoonotic niche of Ebola virus disease in Africa . Elife 2014 ; 3 : e04395 . Google Scholar Crossref Search ADS PubMed 2. Leroy EM , Kumulungui B , Pourrut X , et al. Fruit bats as reservoirs of Ebola virus . Nature 2005 ; 438 : 575 – 6 . Google Scholar Crossref Search ADS PubMed 3. Pourrut X , Souris M , Towner JS , et al. Large serological survey showing cocirculation of Ebola and Marburg viruses in Gabonese bat populations, and a high seroprevalence of both viruses in Rousettus aegyptiacus . BMC Infect Dis 2009 ; 9 : 159 . Google Scholar Crossref Search ADS PubMed 4. Leroy EM , Epelboin A , Mondonge V , et al. Human Ebola outbreak resulting from direct exposure to fruit bats in Luebo, Democratic Republic of Congo, 2007 . Vector Borne Zoonotic Dis 2009 ; 9 : 723 – 8 . Google Scholar Crossref Search ADS PubMed 5. Marí Saéz A , Weiss S , Nowak K , et al. Investigating the zoonotic origin of the West African Ebola epidemic . EMBO Mol Med 2015 ; 7 : 17 – 23 . Google Scholar Crossref Search ADS PubMed 6. Pourrut X , Kumulungui B , Wittmann T , et al. The natural history of Ebola virus in Africa . Microbes Infect 2005 ; 7 : 1005 – 14 . Google Scholar Crossref Search ADS PubMed 7. Rouquet P , Froment JM , Bermejo M , et al. Wild animal mortality monitoring and human Ebola outbreaks, Gabon and Republic of Congo, 2001-2003 . Emerg Infect Dis 2005 ; 11 : 283 – 90 . Google Scholar Crossref Search ADS PubMed 8. Bermejo M , Rodríguez-Teijeiro JD , Illera G , Barroso A , Vilà C , Walsh PD . Ebola outbreak killed 5000 gorillas . Science 2006 ; 314 : 1564 . Google Scholar Crossref Search ADS PubMed 9. Walsh PD , Abernethy KA , Bermejo M , et al. Catastrophic ape decline in western equatorial Africa . Nature 2003 ; 422 : 611 – 4 . Google Scholar Crossref Search ADS PubMed 10. Formenty P , Boesch C , Wyers M , et al. Ebola virus outbreak among wild chimpanzees living in a rain forest of Côte d’Ivoire . J Infect Dis 1999 ; 179 ( Suppl 1 ): S120 – 6 . Google Scholar Crossref Search ADS PubMed 11. Maganga GD , Kapetshi J , Berthet N , et al. Ebola virus disease in the Democratic Republic of Congo . N Engl J Med 2014 ; 371 : 2083 – 91 . Google Scholar Crossref Search ADS PubMed 12. Boumandouki P , Formenty P , Epelboin A , et al. Clinical management of patients and deceased during the Ebola outbreak from October to December 2003 in Republic of Congo . Bull Soc Pathol Exot 2005 ; 98 : 218 – 23 . Google Scholar PubMed 13. Lahm SA , Kombila M , Swanepoel R , Barnes RF . Morbidity and mortality of wild animals in relation to outbreaks of Ebola haemorrhagic fever in Gabon, 1994-2003 . Trans R Soc Trop Med Hyg 2007 ; 101 : 64 – 78 . Google Scholar Crossref Search ADS PubMed 14. Johnson BK , Gitau LG , Gichogo A , et al. Marburg, Ebola and Rift Valley Fever virus antibodies in East African primates . Trans R Soc Trop Med Hyg 1982 ; 76 : 307 – 10 . Google Scholar Crossref Search ADS PubMed 15. Leroy EM , Telfer P , Kumulungui B , et al. A serological survey of Ebola virus infection in Central African nonhuman primates . J Infect Dis 2004 ; 190 : 1895 – 9 . Google Scholar Crossref Search ADS PubMed 16. Reed PE , Mulangu S , Cameron KN , et al. A new approach for monitoring ebolavirus in wild great apes . PLoS Negl Trop Dis 2014 ; 8 : e3143 . Google Scholar Crossref Search ADS PubMed 17. Glynn JR , Bower H , Johnson S , et al. Asymptomatic infection and unrecognised Ebola virus disease in Ebola-affected households in Sierra Leone: a cross-sectional study using a new non-invasive assay for antibodies to Ebola virus . Lancet Infect Dis 2017 ; 17 : 645 – 53 . Google Scholar Crossref Search ADS PubMed 18. Hayman DT , Yu M , Crameri G , et al. Ebola virus antibodies in fruit bats, Ghana, West Africa . Emerg Infect Dis 2012 ; 18 : 1207 – 9 . Google Scholar Crossref Search ADS PubMed 19. Ogawa H , Miyamoto H , Nakayama E , et al. Seroepidemiological prevalence of multiple species of filoviruses in fruit bats (Eidolon helvum) migrating in Africa . J Infect Dis 2015 ; 212 ( Suppl 2 ): S101 – 8 . Google Scholar Crossref Search ADS PubMed 20. Olival KJ , Islam A , Yu M , et al. Ebola virus antibodies in fruit bats, Bangladesh . Emerg Infect Dis 2013 ; 19 : 270 – 3 . Google Scholar Crossref Search ADS PubMed 21. Yuan J , Zhang Y , Li J , Zhang Y , Wang LF , Shi Z . Serological evidence of ebolavirus infection in bats, China . Virol J 2012 ; 9 : 236 . Google Scholar Crossref Search ADS PubMed 22. De Nys H , Mbala Kingebeni P , Keita A , et al. Large serological and molecular survey of Ebola viruses in a wide diversity of frugivorous and insectivorous bat species in Guinea, Cameroon and the Democratic Republic of Congo . Emerg Infect Dis 2018 ; 24:2228–2240. 23. Leirs H , Mills JN , Krebs JW , et al. Search for the Ebola virus reservoir in Kikwit, Democratic Republic of the Congo: reflections on a vertebrate collection . J Infect Dis 1999 ; 179 ( Suppl 1 ): S155 – 63 . Google Scholar Crossref Search ADS PubMed 24. Breman JG , Johnson KM , van der Groen G , et al. A search for Ebola virus in animals in the Democratic Republic of the Congo and Cameroon: ecologic, virologic, and serologic surveys, 1979–1980. Ebola Virus Study Teams . J Infect Dis 1999 ; 179 ( Suppl 1 ): S139 – 47 . Google Scholar Crossref Search ADS PubMed 25. Cantoni D , Hamlet A , Michaelis M , Wass MN , Rossman JS . Risks posed by Reston, the forgotten Ebola virus . mSphere 2016 ; 1 . doi:10.1128/mSphere.00322-16. 26. Centers for Disease Control and Prevention . Ebola (Ebola virus disease). Outbreaks . https://www.cdc.gov/vhf/ebola/outbreaks/index-2018.html. Accessed 20 October 2018. 27. Peeters M , Courgnaud V , Abela B , et al. Risk to human health from a plethora of simian immunodeficiency viruses in primate bushmeat . Emerg Infect Dis 2002 ; 8 : 451 – 7 . Google Scholar Crossref Search ADS PubMed 28. Aghokeng AF , Ayouba A , Mpoudi-Ngole E , et al. Extensive survey on the prevalence and genetic diversity of SIVs in primate bushmeat provides insights into risks for potential new cross-species transmissions . Infect Genet Evol 2010 ; 10 : 386 – 96 . Google Scholar Crossref Search ADS PubMed 29. Ahuka-Mundeke S , Ayouba A , Mbala-Kingebeni P , et al. Erratum to: high prevalences and a wide genetic diversity of simian retroviruses in non-human primate bushmeat in rural areas of the Democratic Republic of Congo . Ecohealth 2017 ; 14 : 115 . Google Scholar Crossref Search ADS PubMed 30. Ahuka-Mundeke S , Ayouba A , Mbala-Kingebeni P , et al. Novel multiplexed HIV/simian immunodeficiency virus antibody detection assay . Emerg Infect Dis 2011 ; 17 : 2277 – 86 . Google Scholar Crossref Search ADS PubMed 31. Van Heuverswyn F , Li Y , Bailes E , et al. Genetic diversity and phylogeographic clustering of SIVcpzPtt in wild chimpanzees in Cameroon . Virology 2007 ; 368 : 155 – 71 . Google Scholar Crossref Search ADS PubMed 32. D’arc M , Ayouba A , Esteban A , et al. Origin of the HIV-1 group O epidemic in western lowland gorillas . Proc Natl Acad Sci U S A 2015 ; 112 : E1343 – 52 . Google Scholar Crossref Search ADS PubMed 33. Li Y , Ndjango JB , Learn GH , et al. Eastern chimpanzees, but not bonobos, represent a simian immunodeficiency virus reservoir . J Virol 2012 ; 86 : 10776 – 91 . Google Scholar Crossref Search ADS PubMed 34. Ayouba A , Touré A , Butel C , et al. Development of a sensitive and specific serological assay based on Luminex technology for detection of antibodies to zaire Ebola virus . J Clin Microbiol 2017 ; 55 : 165 – 76 . Google Scholar Crossref Search ADS PubMed 35. Peel AJ , McKinley TJ , Baker KS , et al. Use of cross-reactive serological assays for detecting novel pathogens in wildlife: assessing an appropriate cutoff for henipavirus assays in African bats . J Virol Methods 2013 ; 193 : 295 – 303 . Google Scholar Crossref Search ADS PubMed 36. Gilbert AT , Fooks AR , Hayman DT , et al. Deciphering serology to understand the ecology of infectious diseases in wildlife . Ecohealth 2013 ; 10 : 298 – 313 . Google Scholar Crossref Search ADS PubMed 37. Lardeux F , Torrico G , Aliaga C . Calculation of the ELISA’s cut-off based on the change-point analysis method for detection of Trypanosoma cruzi infection in Bolivian dogs in the absence of controls . Mem Inst Oswaldo Cruz 2016 ; 111 : 501 – 4 . Google Scholar Crossref Search ADS PubMed 38. Killick R , Eckley IA . Changepoint: an R package for changepoint analysis . J Stat Soft 2014 ; 58 : 1 – 19 . Google Scholar Crossref Search ADS 39. Hinkley DV . Inference about the change-point in a sequence of random variables . Biometrika 1970 ; 57 : 1 – 17 . Google Scholar Crossref Search ADS 40. Laing ED , Mendenhall IH , Linster M , et al. Serologic evidence of fruit bat exposure to filoviruses, Singapore, 2011–2016 . Emerg Infect Dis 2018 ; 24 : 114 – 7 . Google Scholar Crossref Search ADS PubMed 41. Cullen AC , Frey HC. Probabilistic techniques in exposure assessment . New York, NY : Plenum Press , 1999 : 81 – 159 . 42. Delignette-Muller ML , Dutang C . Fitdistrplus: an R package for fitting distributions . J Stat Soft 2015 ; 64 : 1 – 34 . Google Scholar Crossref Search ADS 43. Etard JF , Sow MS , Leroy S , et al. Postebogui Study Group . Multidisciplinary assessment of post-Ebola sequelae in Guinea (Postebogui): an observational cohort study . Lancet Infect Dis 2017 ; 17 : 545 – 52 . Google Scholar Crossref Search ADS PubMed 44. Taniguchi S , Sayama Y , Nagata N , et al. Analysis of the humoral immune responses among cynomolgus macaque naturally infected with Reston virus during the 1996 outbreak in the Philippines . BMC Vet Res 2012 ; 8 : 189 . Google Scholar Crossref Search ADS PubMed 45. Yang XL , Zhang YZ , Jiang RD , et al. Genetically diverse filoviruses in Rousettus and Eonycteris spp. bats, China, 2009 and 2015 . Emerg Infect Dis 2017 ; 23 : 482 – 6 . Google Scholar Crossref Search ADS PubMed 46. Goldstein T , Anthony SJ , Gbakima A , et al. Author correction: the discovery of Bombali virus adds further support for bats as hosts of ebolaviruses . Nat Microbiol 2018 ; 3 : 1486 . Google Scholar Crossref Search ADS PubMed 47. Tapanes E , Detwiler KM , Cords M . Bat predation by Cercopithecus monkeys: implications for zoonotic disease transmission . Ecohealth 2016 ; 13 : 405 – 9 . Google Scholar Crossref Search ADS PubMed 48. Bermejo M , Illera G , Sabater P . Animals and mushrooms consumed by bonobos (Pan paniscus): new records from Lilungu (Ikele), Zaire . Int J Primatol 1994 ; 15 : 879 – 98 . Google Scholar Crossref Search ADS 49. Alexander KA , Sanderson CE , Marathe M , et al. What factors might have led to the emergence of Ebola in West Africa? PLoS Negl Trop Dis 2015 ; 9 : e0003652 . Google Scholar Crossref Search ADS PubMed 50. Pigott DM , Millear AI , Earl L , Morozoff C . Updates to the zoonotic niche map of Ebola virus disease in Africa . eLife 2016 ; 5 : 16412 . Google Scholar Crossref Search ADS © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

The Journal of Infectious DiseasesOxford University Press

Published: Jan 18, 2019

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create folders to
organize your research

Export folders, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off