Wild Animal Mortality Monitoring and Human Ebola Outbreaks, Gabon and Republic of Congo, 2001–2003

An animal mortality monitoring network in Gabon and the Republic of Congo has demonstrated potential to predict and possibly prevent human Ebola outbreaks.

E bola virus, a member of the Filoviridae family, causes severe hemorrhagic fever in humans and nonhuman primates. The human case-fatality rate ranged from 50% to 89%, according to the viral subtype, from the first outbreaks in Zaire and Sudan in 1976 to the 2003 outbreaks in the Republic of Congo (1)(2)(3)(4). No effective therapy or prophylaxis exists, and Ebola is a major public health concern. The first recorded human Ebola outbreaks (Yambuku Zaire 1976;Nzara, Sudan, 1976 and1979;Tandala, Zaire, 1977) occurred abruptly, from an unidentified source, with sub-sequent person-to-person spread (1,2,5,6). No trace of the virus was initially found in wild animals close to the outbreaks (7)(8)(9). In 1989, for the first time, a nonhuman primate outbreak due to a new subtype of Ebola virus, Ebola subtype Reston, occurred in a colony of Macaca fascicularis in a quarantine facility in Reston, Virginia, USA, after the introduction of monkeys from the Philippines (10). Ebola Reston caused severe hemorrhagic fever in monkeys, but no clinical cases of human infection were identified, even though anti-filovirus antibodies were found in quarantine facility personnel (11). Later, in 1994, Ebolaspecific immunohistochemical staining was positive on necropsy specimens from 1 of 12 chimpanzees that died in the Tai forest of Côte d'Ivoire (12). During this outbreak, an ethnologist was infected while performing an autopsy on a chimpanzee carcass; this was the first documented case of human infection transmitted by a nonhuman primate (13). During the 1996 outbreak in Mayibout (Gabon), an epidemiologic survey showed that the index casepatients had been infected by contact with a chimpanzee carcass. Concurrently, many nonhuman primate carcasses were reported in the area close to the outbreak, but none was recovered (14,15). Recently, we showed that all the human Ebola virus outbreaks that occurred in the past 3 years in Gabon and the Republic of Congo resulted from multiple introductions of the virus from different infected animal carcasses (16). We describe the development, testing, and evaluation of an Animal Mortality Monitoring Network (AMMN) in northeastern Gabon and northwestern Republic of Congo designed to alert human and animal health authorities on emerging epidemics.  Figure 1).
All information on human cases of viral hemorrhagic syndrome or on the presence of dead animals in affected areas was centralized by a Viral Hemorrhagic Fever Committee (VHFC), composed of representatives of the Ministries of Health, Forestry, and Environment, the World Health Organization (WHO), wildlife agencies, and the Centre International de Recherches Médicales de Franceville (CIRMF). VHFC was also charged with sending specialized CIRMF teams to sample animal carcasses for diagnostic purposes. CIRMF is the regional reference laboratory for viral hemorrhagic fevers, and communicates its results to the ministries of health, forestry, and environment and to WHO.

Ebola Outbreak Investigation: Human Case Data
The Gabonese and Congolese Ministries of Health, in close collaboration with WHO and its partners in the Global Outbreak Alert and Response Network (GOARN), were in charge of human epidemiologic investigations. A case of Ebola hemorrhagic fever was defined as any probable or laboratory-confirmed case, based on internationally recognized criteria (definition available from http://www.who.int/emc/diseases/ebola/ebola7.html).  (17,18).

Carcass Detection
Local hunters (primarily adult and adolescent men of the Bakota, Bakola, Mboko, Mongom, and Pygmy tribes) were the main sources of information regarding the location of carcasses. Their reported sightings were confirmed by ECOFAC monitoring teams who recorded both the global positioning system (GPS) position on a Cyber Traker field computer (available from http://www.cybertracker.co.za/) and carcass status before alerting VHFC. and its distance from CIRMF, a base camp was established nearby. GPS location of the carcasses, and the information provided on their state of decomposition, allowed the autopsy team to sample only the freshest carcasses.

Wild animal Carcass Sampling
Ideally, the carcass sampling teams comprised a minimum of 5 persons (3 porters and 2 persons to perform the autopsy). One of the porters was charged with disinfection procedures. Digital photographs were taken. Necropsy was performed with high-level precautions, including watertight clothes (Pro-Tech "C," Tyvek, Contern, Luxembourg) equipped with air filtration equipment and Proflow Automask Litehood face shields (Delta Protection, Lyon, France) (Figure 2), and disposable lancets and forceps. A 2% chlorine spray was used to disinfect reusable equipment (masks and filtration apparatus), as well as the autopsy site and carcass remnants. Hermetic 60-L containers equipped with safety tops were used to transport reusable equipment and waste. Waste was returned to the main camp for incineration.
The nature of the samples taken depended on the state of the carcasses. When the carcasses were in good condition, 0.5-cm 3 specimens of liver, spleen, muscle, and skin were taken. Half of the samples were placed in Nunc CryoTube vials (Nalge International, Rochester, New York, USA), which were placed in a small liquid nitrogen dry-shipper container (5.4 L) for cryopreservation (-196°C). The other samples were placed in Nunc CryoTube vials containing 10% formalin, for immunohistochemical testing. Bones were placed in hermetic containers. At the main camp, the dry-shipper contents were transferred into larger dry-shipper containers (20.3 L), which were then forwarded to the CIRMF laboratory at the end of the mission.

Sample Preparation
Potentially infected specimens were collected and manipulated according to WHO guidelines on viral hemorrhagic fever agents in Africa (19). Muscle and skin tissue were fragmented and homogenized in phosphate-buffered saline, and the final supernatant was filtered for antigen detection and RNA amplification. Bones were cut, and internal tissue was scraped. Bone marrow or internal bone tissue was prepared in the same way as muscle and skin.

Testing
Muscle and skin tissue samples were tested by polymerase chain reaction (PCR), antigen detection, and, in some cases, immunohistochemical staining. Bone marrow and internal bone tissue were tested by PCR only.

Antigen Detection
Samples were used for antigen detection as previously described (20). Briefly, Maxisorp (Nalge International) plates were coated with a cocktail of 7 monoclonal antibodies against Ebola virus Zaire antigens; control plates were coated with normal mouse ascitic fluid produced from a parent myeloma cell line. Sample extracts (see above) were then added to the wells, followed by hyperimmune rabbit Ebola polyvalent antiserum and then peroxidase-conjugated goat antibodies against rabbit immunoglobulin G (IgG). The TMB detector system (Dynex Technologies, Issy-les Molineaux, France) was used to measure optical density.

Immunohistochemical Staining
Formalin-fixed specimens were sent to the Centers for Disease Control and Prevention (Atlanta, Georgia, USA) for immunohistochemical staining as previously described (22).   (Figure 3). Only 6% of carcasses sampled were in good condition (entire body); 57% were in poor condition (partial carcasses with muscles or skin); and 38% were in bad condition (bones only). Two peaks of animal deaths were observed (Figure 4)

Laboratory Findings
An animal carcass was considered infected by Ebola virus if >1 of the 3 laboratory tests (antigen detection, DNA amplification, and immunohistochemical staining) was positive. When possible, DNA amplification was confirmed by sequencing the PCR products. Twenty-one gorilla, chimpanzee, and duiker carcasses were sampled in the wild and analyzed in the CIRMF biosafety level 4 (BSL-4) laboratory. Fourteen of these carcasses tested positive for Ebola virus, 6 in 2 or 3 tests and 8 in only 1 test (Table). Eight positive samples were muscles, and 6 were bones or bone marrow. All the muscle and skin tissue samples were tested by both PCR and antigen detection. In total, 10 gorillas, 3 chimpanzees, and 1 duiker tested positive. All the relatively well-preserved gorilla and chimpanzee carcasses tested positive. In contrast, well-preserved samples taken from carcasses of C. cephus, Genetta sp., and Tragelaphus sp. were negative.

Discussion
We describe the successful implementation of a surveillance network of Ebola outbreaks in wild large   mammals. We often identified wild animal outbreaks before human Ebola outbreaks. Twice this enabled us to alert the health authorities of Republic of Congo and Gabon to an imminent risk for human outbreaks, after the discovery of carcasses of Ebola virus-infected animals. Human Ebola outbreaks in this region have always occurred in remote areas, raising major logistic problems. Roads are often barely passable, and means of communication are frequently nonexistent. The carcass detection and investigation network therefore had to rely on teams already present in these forest zones, and notably those possessing radios or satellite telephones. Conservation organizations such as ECOFAC, WCS, and WWF were thus the ideal partners. ECOFAC monitoring teams played a critical role by exploring remote forest zones, capitalizing on the information provided by villagers and hunters.
Performing an autopsy on high-risk animal carcasses requires heavy equipment, highly qualified personnel, and experienced veterinarians, as illustrated by the case of the Swiss anthropologist who was infected after examining a chimpanzee carcass without adequate protective measures in the Tai forest (13). Carcasses decompose very rapidly in the equatorial forest: an adult male gorilla carcass (≈150 kg) takes only 10 days to decompose entirely, i.e., be reduced to a heap of bones and hair ( Figure 5). Carcasses observed 3-4 days after death bear few signs of scavenger activity but are covered with fly eggs and maggots. Maggots consume the entire flesh within 5 to 10 days, while scavengers (mainly mongoose) take pieces and disseminate them around the site. Thus, after ≈3weeks, only a few bones bearing small-mammal gnaw marks remain.
Although the PCR technique used by CIRMF can detect Ebola virus genetic material in carcasses 3-4 weeks old, the material is often degraded and incomplete. Often, only a small sequence of the L-gene (RNA polymerase) can be analyzed, and this cannot be used for strain identification. Furthermore, degraded samples increase the false-negative rate (25). Rapid sampling is therefore crucial for successful diagnosis, and the availability of a small aeroplane was particularly helpful in certain cases. The presence of the CIRMF BSL4 laboratory relatively close to the outbreak area was a considerable advantage.
Using a combination of 3 laboratory techniques (PCR, immunohistochemical staining, and antigen capture), we showed for the first time that wild gorillas and chimpanzees can be decimated by Ebola. Bones of a Cephalophus dorsalis carcass also tested positive for Ebola virus by reverse transcription (RT)-PCR, indicating that a third wild species may be naturally susceptible. In Africa, only chimpanzees had previously been diagnosed as positive for Ebola virus, by immunohistochemical testing, in the Tai forest of Côte d'Ivoire, and were considered the cause of the human outbreak in Mayibout (Gabon) (12,14,15). The large number of carcasses found in this region, together with the results of animal population censuses conducted in the Lossi reserve before and after outbreaks, indicates that great apes are affected massively and duikers to a lesser extent (16,26). The lowland gorilla population density in this region (<6 times as high as the chimpanzee population density) is among the highest in the world (<10 gorillas/km 2 ) (27), which likely explains why more gorilla carcasses than chimpanzee carcasses were found. High population density can amplify outbreaks but cannot alone explain their severity. Small monkeys, although abundant in this area, do not seem to be affected. Only 1 carcass of Cercopithecus cephus was found; it was in good condition but was negative by RT-PCR and antigen capture (Table). Some Potamochoerus porcus carcasses were reported by hunters but none could be sampled. Carcasses of large animals are more likely to be found than those of small animals, because the time taken for a carcass to decompose depends on its size.
The source of gorilla infection is unknown, but several lines of evidence point to direct infection by >1 natural hosts. First, the detection of different strains of Ebola virus in gorilla carcasses located only a few kilometers apart argues against a major role of gorilla-to-gorilla transmission. Indeed, Ebola virus remains genetically stable during a given outbreak, from the first to the last case (28,29), whereas we obtained 4 different glycoprotein sequences (E.M. Leroy, P. Rouquet, unpub. data) from samples of gorillas and chimps located in the Lossi sanctuary. The large distance separating positive carcasses found during a short period, and the existence of physical barriers such as roads and rivers, also supports direct transmission from a natural host. Finally, the occurrence of simultaneous outbreaks in 2 or 3 different species that display little interspecies contact (30) provides further evidence that gorillas and chimpanzees are directly infected by >1 natural hosts. However, cases of gorilla-to-gorilla transmission cannot be ruled out, especially within a given group. Indeed, 5 gorilla carcasses belonging to the same group were found in a close area in the Lossi sanctuary. Ebola outbreaks in gorilla groups may result in their rapid dissolution, especially if the dominant male is rapidly affected, which forces possibly infected females to integrate into another group. However, this type of intergroup transmission appears to be marginal.
Chimpanzees are probably infected by the same mechanisms as gorillas. During the Tai outbreak in Côte d'Ivoire, carnivorous behavior (especially consumption of Colobus monkeys) was the suspected source of infection (12), but this notion is challenged by the infection of gorillas, which are almost exclusively herbivorous. However, chimpanzees are considered to be the primate species whose behavior (mainly fighting, social grooming, sexual activities, and predation) carry the highest risk for both intra-and interspecies pathogen transmission (30). This idea is supported by the detection of the infected carcasses of a mother and her 1-year-old offspring. Repeated contact between young individuals and their mothers is known to be a significant risk factor for Ebola virus transmission (2,6).
Duikers represent a special case. Although they are the most common large-mammal species in this region, few carcasses were found. This circumstance may be due to the lack of interactions among individuals, as duikers generally live alone or in pairs. Some duikers, despite being herbivorous, eat the flesh of decomposing carcasses (K. Abernethy, unpub. data). Thus, in addition to being directly infected by the natural host(s), duikers might also become infected by licking or eating fresh carcasses of  Humans do not seem to be at a major risk for infection by the unidentified natural host(s). Large outbreaks among wild animals can amplify human outbreaks by increasing the number of index transmission events. Therefore, reducing contacts between humans and dead wildlife can reduce the risks for transmission.
Epidemiologic surveillance of animal mortality rates can thus help prevent the emergence of the disease in human populations (Figure 6). At the time of the Kéllé (Republic of Congo) outbreak, our network detected infected gorilla carcasses (Lossi, December 6, 2002) 3 weeks before the disease emerged in humans (December 25, 2002), showing active Ebola virus propagation in this area. We were thus able to warn health authorities of an imminent human outbreak in the region. Nonetheless, a human outbreak occurred. In June 2003, we issued a new alert on a risk for human outbreaks after the discovery of an infected gorilla carcass near the village of Mbanza (Republic of Congo). An outbreak occurred in this village in November 2003. These failures suggest that human and animal health authorities need to work together more closely. In the future, health authorities need to educate local populations on the risk for infection through contact with carcasses at all times. During expected disease outbreaks, health authorities need to be able to respond immediately by sending teams to affected areas (24). The early successes of the network in this area warrant its extension to all countries with known outbreaks of hemorrhagic fevers. The participation of new frontline partners, such as foresters, would be invaluable to expend logistical existing capacity provided largely by field conservationists. Finally, as the capacity of such a system to react rapidly is crucial for its success, sampling teams should be created to collect material and obtain virologic testing results with a minimum of delay in other countries harboring hemorrhagic viruses. An efficient animal mortality monitoring network backed up by a rapid reaction system would allow public health authorities to predict and possibly prevent human Ebola outbreaks. Three animal species are known to be sensitive to Ebola virus and to act as sources of human outbreaks, gorillas, chimpanzees, and duikers. Person-to-person transmission from hunters to their family and then to hospital workers (e). The wild animal mortality surveillance network can predict and might prevent human outbreaks. Medical surveillance can prevent Ebola virus propagation in the human population.
CIRMF is supported by the Government of Gabon, Total-Fina-Elf Gabon, and Ministère de la Coopération Française. This work was also supported by a Fonds de Solidarité Prioritaire and a Fonds d'Aide et de Coopération, grants from the Ministère des Affaires Etrangères de la France (FSP no. 2002005700 and FAC no. 1999-49).
Dr. Rouquet is the head of the Primate Center at CIRMF. He is experienced in the treatment and evaluation of simian immunodeficiency virus and simian/HIV transmission in different primate models of HIV infection (pathogenic and nonpathogenic). Since 1995, he has been involved in hemorrhagic fever research, particularly Ebola.