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Epidemiology of Ebola (Subtype Reston) Virus in the Philippines, 1996

  1. M. E. Miranda,
  2. T. G. Ksiazek,
  3. T. J. Retuya,
  4. Ali S. Khan,
  5. Anthony Sanchez,
  6. Charles F. Fulhorst*,
  7. Pierre E. Rollin,
  8. A. B. Calaor,
  9. D. L. Manalo,
  10. M. C. Roces,
  11. M. M. Dayrit and
  12. C. J. Peters
  1. Research Institute for Tropical Medicine and Field Epidemiology Training Program, Department of Health, Manila, Philippines; Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
  1. Reprints or correspondence: Dr. Thomas Ksiazek, Special Pathogens Branch, DVRD/NCID, G-14, Centers for Disease Control and Prevention, Atlanta, GA 30333.

  • ↵* Present affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas.

 
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Abstract

Ebola (subtype Reston [EBO-R]) virus infection was detected in macaques imported into the United States from the Philippines in March 1996. Studies were initiated in the Philippines to identify the source of the virus among monkey-breeding and export facilities, to establish surveillance and testing, and to assess the risk and significance of EBO-R infections in humans who work in these facilities. Over a 5-month period, acutely infected animals were found at only one facility, as determined using Ebola antigen detection. Three of 1732 monkeys and 1 of 246 animal handlers tested had detectable antibodies; all were from the same facility, which was the source of infected monkeys imported to the United States. Virus transmission, which was facilitated by poor infection-control practices, continued for several months in one facility and was stopped only when the facility was depopulated. None of the 246 employees of the facilities or 4 contacts of previously antibody-positive individuals reported an Ebola-like illness. This investigation suggests that human EBO-R infection is rare.

Ebola (EBO) (subtype Reston [EBO-R]) virus was originally discovered in the United States in 1989 in association with an outbreak of viral hemorrhagic fever among cynomolgus monkeys (Macaca fascicularis) exported from the Philippines [1]. Subsequently, 4 other episodes of EBO-R virus infection among monkeys imported from the Philippines have occurred in the United States and Italy between 1990 and 1992 [2]. In each instance, the hallmark of infection was rapid dissemination throughout the facility of a highly fatal infection in monkeys. In contrast, limited studies of humans occupationally exposed to infected monkeys or their tissues suggested that infection in humans is infrequent and asymptomatic or, at worst in these limited infections, associated with only a mild disease. There have been 4 documented asymptomatic human infections in the United States [3, 4] and 3 antibody-positive individuals identified in the Philippines [5]. Following the original episodes in 1989–1990, the United States modified the procedures used in the transportation and quarantine of nonhuman primates to include mandatory disease control requirements [6]. Similarly, in 1994, the Republic of the Philippines banned the export of wild-caught monkeys and instituted other procedures (such as a 45-day quarantine prior to export) to obviate the potential for export of EBO-R-infected monkeys [7].

In March 1996, a monkey imported from the Philippines died while in quarantine at a commercial facility in Texas; the animal was tested and found positive for EBO antigens [8]. After another animal was confirmed to be EBO virus positive by antigen virus isolation, and reverse transcription—polymerase chain reaction of EBO-R RNA, 50 animals, which constituted a quarantine cohort, of the 100 imported from the Philippines were destroyed [8]. The 8 employees in contact with the infected monkeys and the other 50 animals from the shipment were monitored but did not show any signs of illness or evidence of infection [8]. Sequence analysis of the glycoprotein gene of virus from the first EBO-R—infected monkey revealed a 98.9% nucleotide homology with the original 1989 EBO-R virus [9].

Following confirmation of the presence of EBO-R virus in these monkeys, the Philippine Department of Health placed a moratorium on the export and movement of monkeys within the Philippines, pending an investigation to explain the origin of infected animals, despite regulations that stipulate that all exported monkeys are captive-bred and must be quarantined for 45 days prior to export. Herein, we report the results of that investigation and a parallel study of the risk and health significance of EBO-R infection in occupationally exposed humans.

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Methods

Study sites

The Philippines is one of the world's major sources of purpose-bred cynomolgus monkeys for biomedical research. The five licensed breeding and export facilities provide an estimated 5000 captive-bred monkeys every year to the United States, Europe, and Japan. A small number of animals are also sold to biomedical research facilities located in the Philippines. All five of the breeding and export facilities are located in the main Philippine island group of Luzon (figure 1). Each of these facilities, depending on their size, houses between 300 and 7500 cynomolgus monkeys at any given time (table 1). Three of the facilities are actively replenishing or expanding their breeding stocks with animals collected within licensed wildlife trapping areas designated by the Philippine Department of Environment and Natural Resources on the island of Mindanao; one facility utilizes only captive-bred animals for breeding stock, and the other has suspended export operations but still has limited breeding stock. Holding facilities near wildlife trapping areas on the southern island of Mindanao serve as prequarantine stations where wild-caught monkeys selected as breeders are held prior to transfer to the breeding facilities (figure 1).

Figure 1
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Figure 1

Map showing location of primate breeding and export facilities (A) and holding facilities near trapping areas (B) in southern island of Mindanao, Philippines, where wild-caught monkeys selected as breeders are held prior to transfer to breeding facilities.

Table 1
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Table 1

Description of the five facilities of the Primate Exporters and Breeders Association of the Philippines, May 1996 (see figure 1 for map of locations).

Surveillance for EBO-R disease in monkeys

In May 1996, surveillance in monkeys was established at each of the five facilities to determine if any mortality was associated with EBO-R infection. The veterinary staff of the facilities was responsible for the collection of liver specimens from all dead monkeys (including sacrificed moribund and sick ones), regardless of the perceived cause of illness. To limit dissemination, all monkeys in the same housing unit as EBO antigen—positive animals were euthanatized. Liver and blood samples were obtained from the sacrificed monkeys in Facility A. In addition, all blood samples that were obtained from animals during the cross-sectional antibody survey (see below) in Facility A were also tested for EBO antigen. These samples were stored frozen until tested for EBO antigen, using the antigen-capture ELISA.

Survey for EBO antibodies in monkeys in primate breeding and export facilities

Blood samples were obtained from a systematic sample of animals to determine by antibody testing if past infection had occurred at each facility. The sampling plan was designed to detect a minimum antibody prevalence of 5% among defined caging groups and life stages (breeders and captive-bred growers). Once the total number of animals to be sampled within the defined groups at a facility was determined, the average number of animals within a cage or housing unit within this caging group was calculated, and the number of housing units needed to meet this requisite total sample size was then taken from evenly spaced cages or housing units. Basic demographic details (age, sex, wild-caught or captive origin) were collected on all animals in the sample. A 20% over-sampling was done, and all animals awaiting export and all newly arrived wild-caught animals were sampled at all facilities. In addition, all animals hospitalized in Facility A, the source of the infected monkeys for the Texas outbreak, were also sampled. Pregnant monkeys near term and those <1 year old were not sampled because of concerns for the trauma and stress of capture and handling.

Although an embargo on new captures in Mindanao had been initiated, collection of samples from all newly captured animals in transit at holding facilities near trapping areas in Mindanao was also initiated in an attempt to determine the source of the EBO-R.

Risk assessment on occupationally exposed humans

All employees of breeding and export facilities were interviewed. Data on possible risk factors for infection (i.e., bites or scratches from monkeys, handling of blood and other tissues) and history of any EBO-like illness (fever associated with hemorrhage, myalgias) were gathered. Blood samples were collected and tested for EBO-R IgG antibody by ELISA. Family contacts of previously identified antibody-positive persons (Miranda ME, unpublished data) were also interviewed, and blood samples were obtained for antibody testing.

Antigen and antibody assay

EBO antigen detection tests were done according to a previously published method with only slight modification [10, 11]. Blood and 10% tissue homogenates were prepared in a biosafety level 4 laboratory, gamma-irradiated with a 60Co source, and then tested at dilutions of 1:4, 1:16, 1:64, and 1:256 in both specific and nonspecific (parent myeloma ascites) coated wells. The antibody used to detect EBO virus antigen captured by monoclonal antibodies had been replaced by serum from rabbits hyperimmunized with the three known EBO virus subtypes (Zaire, Sudan, Reston) when the material was made.

Rabbit antibodies that bound to EBO antigens were detected by goat anti-rabbit—horseradish peroxidase—conjugated antibodies followed by H2O2-ABTS (Kirkegaard & Perry, Gaithersburg, MD) substrate. Adjusted optical density values (readings at 410 nm of wells coated with EBO-specific monoclonal antibody less the corresponding well coated with parent myeloma ascites) >0.1 were assigned positive status. Specimens with EBO antigen titers ⩾1:16 were considered positive.

Antibody was detected by an ELISA test, using an antigen made from basic buffer-detergent—extracted Vero E6 cells infected with the prototype isolate of EBO-R from the 1989 outbreak in Reston, Virginia [11, 12]. Sera were tested at dilutions of 1:100, 1:400, 1:1600, and 1:6400 against both the EBO antigen and a negative antigen made from mock-infected cells prepared in a similar manner. Horseradish peroxidase—conjugated anti-human IgG (Fc specific) was used for humans and primates as a detector antibody. Antibody titers were calculated in a manner similar to that for antigen except that an adjusted optical density of >0.2 at 410 nm was required for positive status for the dilution. Titers of ⩾ 1:400 were considered antibody positive.

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Results

Surveillance for monkey disease and mortality caused by EBO-R virus

Mortality was highest (13.9% based on the initial population) in Facility A; the average was 1.9% among all other breeding facilities (table 2). From May to September 1996, samples were obtained from 1051 animals and tested by antigen detection. Of these animals, 586 (56%) were dead or were sacrificed because they were sick or moribund. Of the total samples tested for EBO-R antigen, 818 were from Facility A animals, including the 287 samples from healthy animals for the cross-sectional antibody survey. Among the 353 dead and moribund monkeys collected at this facility, 113 (32%) were antigen positive, demonstrating that EBO-R virus infection was a significant cause of death. In summary, 131 (16%) of the 818 animals collected from Facility A had detectable EBO-R antigen in their blood or liver (or both). The majority of these antigen-positive monkeys were housed in gang cages. EBO virus infection among monkeys in this facility was widespread. Over a period of 5 months, 14 of 23 cage units had antigen-positive animals. All 233 samples from dead monkeys from the other facilities were negative for EBO-R antigen.

Table 2
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Table 2

Morbidity and mortality caused by EBO-R virus infection among nonhuman primates in export facilities in the Philippines, 1996.

Antibody survey among monkeys

Three of the 301 animals sampled at Facility A were IgG antibody positive, which is indicative of previous EBO-R infection. All 3 animals were resampled a week later and were again found antibody positive. These animals were resident in the hospital. Two were wild-caught, and the other was born within the facility. All of the remaining 1431 samples from the other four breeding and export facilities and all holding facilities were negative for detectable IgG antibody (table 3).

Table 3
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Table 3

Results of cross-sectional EBO-R IgG antibody survey in the nonhuman primate export facilities in the Philippines, May 1996.

Human studies

Two hundred fifty persons were included in the study. One of 18 employees in Facility A was positive for IgG antibody to EBO-R. No individuals from the other facilities had detectable antibody. The positive individual previously had been identified as positive in 1992. A blood sample that was obtained from this persons's spouse was EBO-R antibody negative. Blood samples were also obtained from 3 family members of another employee who was antibody positive in 1992 but who had no detectable EBO-R IgG antibody in 1996; the samples were antibody negative.

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Discussion

This investigation documents active serial transmission of EBO-R within a primate export facility in the Philippines. This facility was the source of the 1996 importation of EBO-R—infected monkeys into Texas, of infected monkeys from 3 of the 4 EBO-R outbreaks in the United States in 1989–1990, and of infected monkeys in Siena, Italy, in 1992 [2, 13]. The only other known source of EBO-R infected monkeys from the Philippines involved an illegal animal exporter in 1989 [6].

Results of antibody and antigen detection tests suggest that virus transmission was present only in Facility A. The constant detection of antigen-positive animals from the hospital and breeding cages confirms serial virus transmission in the facility. The serial transmission of virus was attributed to the facility's poor husbandry and infection-control practices, including reuse of needles, unrestricted movement of personnel into and out of infected housing units, damaged cages, which permitted some animals to escape and roam freely, and the high incidence of animal fighting in gang cages (due to directly connected cages housing at least 8 animals). This cage configuration was not observed in the other facilities. These same practices undoubtedly contributed to the higher overall mortality seen in Facility A, even when laboratory-confirmed EBO virus mortality is removed.

This study and the epidemiologic study of the first monkey outbreak in 1989 showed that gang-cage housing increased the risk of monkey-to-monkey transmission [13]. Close physical contact also provided the opportunity for fighting among the animals. In addition, the high viremias observed during the latter clinical stages in infected animals [14] would be sufficient to allow animal-to-animal transmission by arthropods by purely mechanical or “flying needle” means.

Following the 1989–1990 episodes, the Centers for Disease Control and Prevention modified the requirements for procedures used in the transportation and quarantine of nonhuman primates, including mandatory disease-control efforts, and they included EBO virus testing requirements [15]. This was done to strengthen infection control within quarantined primates and to reduce exposure risks to humans occupationally in contact with the primates. The containment of transmission to a single animal cohort during the 1996 outbreak in the Texas quarantine facility [8] strongly supports the effectiveness of these regulations. These recent importations of EBO-R—infected monkeys were detected while the animals were in quarantine, and, thus, the potential for transmission was minimal to facility employees and other animals in the facility. Since August 1994, the Philippines has banned the export of wild-caught monkeys and introduced additional safety measures, including a 45-day quarantine prior to export [7]. This episode suggests that there was a lapse in procedure, since the quarantine in Facility A failed to pick up infected monkeys and did not prevent their export.

Five Filipino animal handlers have been found with EBO-R antibody. During the 1989–1990 study, using the indirect fluorescent antibody test with sera considered positive if the antibody titer was ⩾ 1:256, 3 individuals were determined to be antibody positive [5]. In 1993, 2 new EBO-R antibody—positive individuals were detected, by use of the IgG antibody ELISA (Miranda ME and Ksiazek TG, unpublished data). All of these persons worked in Facility A, the source of the infected monkeys in 1989–1990 and 1992. A sample from 1 of the 3 antibody-positive persons from the 1989–1990 study was retested in the current study and found to be negative. The other positive persons were no longer working in the animal facility during the current study. In the present study, the IgG antibody ELISA was used. One of the 2 people who were EBO-R IgG—positive during the 1992 study remained positive, while the other person was no longer antibody positive. This indicates that, as determined by our current ELISA technology, some antibody-positive persons may revert to undetectable antibody status through time.

Results of this and other investigations in the Philippines and United States show that humans can be infected with the EBO-R virus [3–5]. However, transmission to humans has been infrequent and did not result in any reported illness. This suggests that infection results in a very mild or inapparent illness and that EBO-R does not pose the same public health threat as the African EBO virus subtypes.

Although we have confirmed the presence and transmission of EBO-R within Facility A, the source of the virus was not determined. It is unclear if low-level transmission of the virus had been maintained within the facility or if there was recent reintroduction of the virus from the wild. In February 1996, the deaths (retrospectively confirmed to be due to EBO-R) of 2 animals from this facility (Miranda ME and Ksiazek TG, unpublished data) demonstrated the unrecognized circulation of the virus in the facility a month before the exportation of infected primates to the United States. It is still uncertain if monkeys are the natural host of EBO-R, although mortality is high among those infected, and laboratory studies have found that experimentally infected survivors do not harbor the virus for even moderate time periods [16]. Following the 1989–1990 episodes of EBO-R associated with Facility A, all animals in the facility were reportedly destroyed [13]; however, interviews with individuals associated with the facility strongly suggest that it was not completely depopulated. This leaves open the possibility that there was some means of virus persistence within animals that continuously populated the facility, perhaps by slow serial transmission of the virus in the remaining primates and among newly introduced susceptibles, who were added as breeding stock, and their progeny or by intermittent transmission through medical fomites, such as multi-use vials of tuberculin or other biologicals regularly used within the facility. Alternatively, the virus may have been reintroduced repeatedly into the facility with newly captured wild-caught monkeys from the capture sites in Mindanao or by other sources, such as bats, rodents, insects, or other animals that may come into regular contact with the monkeys in the facility. The viral genetic data of Sanchez et al. [9] is not incompatible with any of these hypotheses for the maintenance of EBO-R.

In an attempt to contain the spread of infection, closer disease monitoring and selective euthanasia was attempted in Facility A in the weeks following this investigation. This entailed weekly monitoring and the euthanasia of all animals housed in the same cage unit as EBO-R antigen-positive animals by officers of the Philippine Department of Environment and Natural Resources and the Department of Agriculture. After the depopulation of about half of the animal population, it was noted that there was no significant improvement in infection-control practices and there was poor compliance with the monkey mortality surveillance. Additional antigen-positive animals were continuously being detected in new units. This implies that EBO-R virus transmission continued in the facility and had spread beyond the cages where the virus was originally detected. By February 1997, the complete depopulation of the facility with the eventual permanent revocation of the facility's permit to operate was executed by Philippine government authorities.

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Acknowledgments

We extend our sincere gratitude to the members of the Primate Exporters and Breeders Association of the Philippines, the Protected Areas and Wildlife Bureau, the Department of Environment and Natural Resources, and the Bureau of Animal Industry of the Department of Agriculture, Republic of the Philippines, for their assistance in the conduct of this investigation; to Mary Lane Martin, A. J. Williams, Maurice Curtis, and David Bressler for technical assistance in laboratory testing; to Laura Morgan for data management assistance; and to Kent Wagoner for the graphics.

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  1. J Infect Dis. (1999) 179 (Supplement 1): S115-S119. doi: 10.1086/514314
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