THE JOURNAL OF INFECTIOUS DISEASES. VOL. 142. NO.3. SEPTEMBER 1980© 1980 by The University of Chicago. 0022-1899/80/4203-0009$00.75

Ebola Hemorrhagic Fever: Tandala, Zaire, 1977-1978

D. L. Heymann, J. S. Weisfeld, P. A. Webb,K. M. Johnson, T. Cairns, and H. Berquist

From the Bureaus of Smallpox Eradication andLaboratories, Center for Disease Control,

Atlanta, Georgia; and the Tandala Mission Hospital,Tandala, Zaire

Ebola virus was recovered from a nine-year-old girl who died of acute hemorrhagic feverin June 1977 at Tandala Hospital in northwestern Zaire, in the first reported recognizedcase of this disease since the discovery epidemics of 1976 in Zaire and Sudan. Investiga­tions undertaken in the Tandala region revealed that two previous clinical infectionswith Ebola virus had occurred in 1972 and that about 7% of the residents had im­munofluorescent antibodies to the virus. Females younger than 30 years of age had ahigher prevalence of antibodies than males of comparable age, but above the age of 30years there was no sex difference. No other clues to the still-mysterious natural reservoirof Ebola virus were uncovered.

During the summer of 1976, reports of a disease ofhigh mortality that spread rapidly among hospitalstaffs in southern Sudan and northern Zaire led tothe investigation and discovery of a previouslyunknown virus, morphologically related to Mar­burg virus but antigenically different [1-3]. Thisvirus, named Ebola virus, was responsible formore than 430 deaths in the two epidemic zones[4, 5].

Clinical symptoms of Ebola virus-relateddisease included fever, progressive sore throat,maculopapular rash, intractable abdominal pain,and bleeding from multiple sites (principally thegastrointestinal tract), with progression in mostpatients to death. Recovery, when it occurred, wascomplete. The case-fatality rate in Sudan wasestimated at 51070 and in Zaire at 88070 . Theepidemics were found to be associated with eitheran injection at hospitals in the epidemic zones(presumably via unsterile needles) or with closecontact with another case. Both epidemics wererapidly terminated following institution of basicisolation techniques and improvement in handlingof potentially contaminated materials.

The spectrum of Ebola virus-related diseaseranged from fatal hemorrhagic fever to mild

Received for publication July 16, 1979, and in revised formApril 14, 1980.

Dr. Heymann was assigned to the Organisation de Coordina­tion pour la Lutte contre les Endernies en Afrique Centrale,B.P. 228, United Republic of Cameroon, during this investiga­tion.

Please address requests for reprints to Dr. J. S. Weisfeld,Research and Evaluation Branch, Bureau of Smallpox Eradica­tion, Center for Disease Control, Atlanta, Georgia 30333.

372

disease, indistinguishable from other febrile illnessin the area. In the Sudan, 19070 of contacts of per­sons with the disease and no history of illnessthemselves had antibodies to Ebola virus detectedby the indirect immunofluorescent (IIF) method.In Zaire, 1070 of persons surveyed in villages out­side the epidemic zone who had neither contactwith patients nor symptoms of disease were foundto have antibodies to Ebola virus [4]. No furtherEbola virus-like disease was reported in Sudan orZaire after these two epidemics terminated in late1976. We report here the isolation of Ebola virusfrom a patient with fatal, acute hemorrhagic feverin June 1977 at Tandala, Zaire, together with theresults of an investigation undertaken in thisregion, which is 325 km west of the original Ebolaepicenter at Yambuku in northern Zaire (figure 1).

Description of the Area

Bonduni, the native village of the child who diedof Ebola virus disease in June 1977, and the Tan­dala Mission Hospital are located in the transitionzone of tropical rain forest to savannah in north­western Zaire's Congo basin. The principalagricultural crops of the area are coffee, ground­nuts, cotton, and palm oil. Endemic diseases in­clude measles, malaria, onchocerciasis, goiter,cretinism, and intestinal parasitism. Sleepingsickness and yellow fever are sporadic; in 1971there was an epidemic of >50 serologically con­firmed cases of yellow fever. The economy of thearea is based on agriculture, hunting, and fishing.Contact with nature is intimate, with villages

at Penn State University (Paterno L

ib) on August 12, 2014

http://jid.oxfordjournals.org/D

ownloaded from

Ebola Hemorrhagic Fever

Central Afr ican Republic

Zambia

Figure 1. Map of Zaire, showing Tandala and Yam­buku, 1978.

located in clearings of the dense rain forest oralong the rivers of the savannah.

Materials and Methods

The postmortem blood specimen from a nine-year­old female was inoculated into tube cultures ofVero cells maintained in Eagle's minimal essen­tial medium containing 20/0 heat-inactivated fetalcalf serum, 100 units of penicillin, 50 lAg of strep­tomycin, and 21lg of amphotericin B/ml. A 0.2-mlsample of whole blood was inoculated ip into eachof four Hartley strain guinea pigs. Selected organsfrom one of these animals, which died four dayslater, were ground in a mortar and pestle and mixedwith diluent (phosphate-buffered saline [pH 7.2],1.5% bovine albumin, and the above antibiotics)to make a 10% (vol/vol) suspension. After clarifi­cation by centrifugation at 1,200 g for 15 min, thesupernatant was used as the inoculum and titratedin Vero cell cultures.

Isolates of Ebola virus were identified by an IIFtest described previously [6]. This technique wasused to test blood or serum specimens for anti­bodies to Ebola virus with use of virus-infectedVero cells on Teflon" -templated glass slides as thesource of antigen.

Blood specimens for antibody testing were ob­tained by capillary puncture using filter paper

373

disks according to the method of Mathews et al.[7]. Disks were air-dried overnight, packed with adessicant, and frozen at - 20 C. Solutionsrepresenting an estimated 1:4 dilution of serumwere prepared by soaking the disks in sterilephosphate-buffered saline (pH 7.2) overnight andthen squeezing out the resulting solution. Thesesera were screened for antibodies to Ebola virus ata 1:4 dilution, and those positive for virus weretitrated to end point, which was defined as thehighest dilution giving weak but specific stainingof characteristic large cytoplasmic Ebola virus an­tigen inclusions. A titer of ~I:16 was taken asevidence of Ebola virus-specific antibodies.

Results

Confirmed Ebota virus disease, June 1977. Anine-year-old girl was admitted to the TandalaMission Hospital in June 1977, with a three-dayhistory of fever, abdominal pain, and hema­temesis. The child lived with her family in Bon­duni, a village 20 km from the Mission. She hadnot traveled outside this area before her illness andhad previously been in good health. No otherfamily member or villager in Bonduni was ill withsimilar disease during the four weeks precedingthe child's hospitalization, and none became illafter the child's illness and death.

Upon admission to the hospital, the child'stemperature was 39.5 C and rose to a maximum of41 C 2 hr later. Physical examination disclosedmidabdominal pain with guarding and hepato­megaly; bowel sounds were hyperactive, and noscleral icterus was noted. The clinical diagnosis ofhemorrhagic fever was made, and the patient wasplaced in an isolation ward. All waste materialsand soiled bedding were burned, and all needlesand syringes were sterilized. Epistaxis and melenawere present throughout hospitalization. Twenty­eight hours after admission the child lost con­sciousness and died, at which time a postmortemblood specimen was obtained. It arrived at theCenter for Disease Control (Atlanta, Ga.) ninedays later without refrigeration.

Three attempts to isolate virus in Vero cellsfrom this material were unsuccessful because ofheavy bacterial contamination. One of fourguinea pigs receiving the original blood died sud­denly four days after inoculation and was frozenentire at -70 C. However, sera from two of the

at Penn State University (Paterno L

ib) on August 12, 2014

http://jid.oxfordjournals.org/D

ownloaded from

374

three surviving animals obtained 40 days after in­oculation had IIF titers to Ebola virus of 1:32.Liver, spleen, and kidney tissues from the deadanimal were ground and titrated in Vero cells, andEbola virus was identified in all of these organs intiters ranging from IOu to 101.2. Ebola virus wassuccessfully reisolated (titer, 10"'1.7) from the orig­inal blood specimen by inoculation of guinea pigsand blind harvest seven days later, using Vero cellsto detect and identify the agent.

None of the household members of this girl'sfamily had antibody to Ebola virus.

Possible Ebola virus disease, February 1977­February 1978. Possible Ebola virus disease wasdefined for review of Tandala Hospital charts asany illness with fever, headache, nausea, and ab­dominal pain of at least three days' duration withor without hemorrhage and no apparent responseto antibiotic or antimalarial therapy.

Two such persons were identified during asearch of hospital records dating from February1977 to February 1978; both had died after severaldays of hospitalization. One was a 12-year-oldfemale from Bowabili, 30 km south of Tandala,who died of a febrile hemorrhagic disease inNovember 1977. No serum sample or liver biopsyspecimen was obtained from this patient, anddiagnosis remained unconfirmed. The other was a45-year-old male from Bogbadono, 5 km east ofthe hospital, who died in December 1977 with theclinical diagnosis of yellow fever that proved to besevere chronic aggre ssive hepatitis on liver biopsy.

An interview with the family of the 12-year-oldgirl from Bowabili who died of hemorrhagicdisease revealed that her six-year-old sister hadbeen ill at the same time with high fever for morethan seven days and with headache, abdominalpain, vomiting, and conjunctivitis. No otherhousehold member had any illness at this time,and it could not be ascertained by detailed ques­tioning which child had become ill first, orwhether they had fallen ill at the same time. Serumobtained from the six-year-old girl at the time ofthe investigation was positive for antibodies toEbola virus. No other household members had an­tibodies.

Ebola virus disease, 1972. After Ebola viruswas recovered from the patient of June 1977,serum specimens from 5) missionaries working innorthwestern Zaire, including a few staff membersof Tandala Hospital, which had been collected in

Heymann et al.

February 1977, were examined for antibodies toEbola virus. One specimen, donated by a TandalaHospital physician, was positive (titer, 1:64). InFebruary 1978, sera were obtained from 72 Zairoisand expatriate staff members of this institution.Again only a single positive serum was found (thesame physician).

It was learned that this physician had per­formed an autopsy on a Zairois bible school stu­dent who died in May 1972 of a hemorrhagic illnessdiagnosed clinically as yellow fever. He lacerated afinger during this procedure and became sick 12days later. His hospital record was reviewed. Itdisclosed a severe illness with sore throat,headache, backache, generalized myalgia, andtemperature to 41 C. Vomiting, mild diarrhea, andconjunctivitis were present. Ulcers appeared onthe soft palate and buccal mucosa, and anassociated macular rash appeared on the face,back, arms, and legs during the fifth day of illnessand lasted for three days . The intensity of therash varied directly with temperature. On day 6,decreased hearing and tinnitus were noted in theleft ear. There were no bleeding manifestations,and the fever resolved by lysis after 10 days. Thepatient lost 18 kg of body weight, and about sixweeks were required for restoration of energy andnormal auditory function . Leukopenia to 1,450cells/nun' and moderate albuminuria were ob­served during the acute febrile phase of illness.

Antibodies among residents of the Tandalaarea. Historic and serologic surveys were done ineight villages within a 40-km radius of Tandala(figure 2). Three of these towns where patientswith proven or possible Ebola virus disease hadlived; another, Botuto, was the site of the funeralof the nine-year-old Ebola virus disease patient,

BOGBAZELE

Fjgure 2. Map of the villages within a radius of 40 kmof Tandala, Zaire, 1978.

at Penn State University (Paterno L

ib) on August 12, 2014

http://jid.oxfordjournals.org/D

ownloaded from

Ebo/a Hemorrhagic Fever 375

Table 1. Distribution of indirect immunofluorescent antibodies to Ebola virus among residents of Tandala, Zaire,in 1978, by age and sex.

Age (years)

";45-9

10-1920-29

30-3940-4950-59>60

Total

NOTE. Titer of at least 1:16 by indirect immunofluorescence test indicated the presence of antibody to Ebola virus.

and four were selected at random. Filter paperblood samples were obtained from 1,096 personsin these villages.

Although no person gave a history compatiblewith clinically severe Ebola virus disease, 79 in­dividuals (7070) were found to have IIF antibodiesto Ebola virus. The prevalence of such antibodiesby age and sex is given in table 1. Values rangedfrom 1% among young male children to 21%among men >60 years of age. Overall, however,females had a higher rate (9%) than males (5%).It was also noteworthy that females <30 years ofage had a significantly higher frequency of an­tibody than males of the same age (9% vs. 3%;P < 0.01, X2

) . The rate for males <30 years of agewas also statistically lower (P <0.001, X2

) than thatof the remainder of the population tested. Abovethe age of 29 years there was no sex difference inantibody prevalence.

As shown in table 2, antibodies to Ebola viruswere found in residents of all of the villages sam­pled, with prevalences ranging from 4% to 10%.

Discussion

sion of a virus presumptively introduced fromsouthwestern Sudan by unidentified persons [8].

This case and two others that were retrospec­tively uncovered at Tandala Hospital did notresult in nosocomial outbreaks of infection nor insecondary transmission of virus among familymembers. Basic measures, consisting of patientisolation, precautions for enteric-type diseases,and rigorous adherence to aseptic techniques,probably were responsible for containment of thevirus and lend further credence to the concept thataerosols are rarely responsible for person-to­person spread of Ebola virus. Where good basictechniques were not observed, however, transmis­sion to hospital staff had been as high as 81% [9].

That antibody to Ebola virus was prevalentamong 7% of the residents of the Tandala regionstrongly suggests that severe hemorrhagic fevermay represent an unusual human response to in­fection with this agent, a concept that has been ad-

Table 2. Distribution of indirect immunofluorescentantibodies to Ebola virus by village, in the Tandala areaof Zaire, 1978.

Isolation of Ebola virus from a child with hemor­rhagic fever in northern Zaire, who had no overtrelationship to the original epidemic that precededthis case by more than six months, firmly estab­lishes that Ebola virus is endemic and presumablyenzootic in the Zaire River basin. This conclusioncould not be reached in 1976 at the time of thelarge epidemic near Yambuku, which was thoughtto be caused by direct person-to-person transmis-

Village

BogbaseleBonduniBoyazalaBotutuBoyakotiBowabiliBogbadonoBozambali

No. of personssurveyed

21255

27985

12611789

133

No. of personswith antibody (070)

16 (8)4 (7)

20 (7)4 (5)9 (7)9 (8)4 (4)

13 (10)

at Penn State University (Paterno L

ib) on August 12, 2014

http://jid.oxfordjournals.org/D

ownloaded from

376

vanced previously [8, 10]. A final conclusion on thispoint must be reserved until another method formeasuring antibodies to Ebola virus becomesavailable, since occasional false-positive reactionsat a serum dilution of 1:16 may occur in the IIFtest. One such serum specimen was found among200 specimens from Cona Indians of Panama,who are unlikely to have been exposed to thevirus. In support of our selection of this level asindicative of specific antibody, however, is thefact that all eight sera from eight persons infectedwith the virus in Zaire during 1976 had such an­tibodies from one to three years later, with a tem­poral trend toward lower titers in the rangeof 1:16-1 :64.

A striking finding was the fact that males <30years of age had a lower prevalence of antibodiesto Ebola virus than women in this age group orthan all other persons tested. During the 1976epidemic in Zaire, women between 15 and 29 yearsof age experienced the highest infection rate, in partrelated to attendance at a prenatal clinic, wherethey received injections with (presumptively)virus-contaminated needles [4]. No such evidenceexists in the Tandala region. A study of socialhabits, especially those that might relate to ageand sex differences in degree of contact with anunknown enzootic virus cycle, should be includedin future work on the epidemiology of Ebolavirus.

Finally, the circumstances of isolation of Ebolavirus in this instance were remarkable. Ebola viruscontains RNA and has essential lipids in itsenvelope, properties usually associated with ratherhigh thermolability. We suspect that proteins inwhole blood provided the protection required forsurvival of at least a small minority of the virionscontained in this serum specimen, and it was clearthat animal inoculation, as opposed to use of cellcultures, resolved the attendant problem ofbacterial contamination promoted by the longshipping time under conditions highly unfavorableto viral survival. It is evident, therefore, that isola­tion attempts should be made in cases of severe

Heymann et al.

hemorrhagic fever in Africa even where resourcesfor freezing specimens are not available. It is alsoclear that such materials must be scrupulouslypackaged and handled as highly infectious at alltimes.

References

1. Johnson, K. M., Webb, P. A., Lange, J. V., Murphy,F. A. Isolation and partial characterization of a newvirus causing acute haemorrhagic fever in Zaire. Lancet1:569-571, 1977.

2. Murphy, F. A., Van der Groen, G., Whitfield, S. G.,Lange, J. V. Ebola and Marburg virus morphology andtaxonomy. In S. R. Pattyn [ed.], Ebola virus haemor­rhagic fever. Elsevier/North-Holland, Amsterdam,1978, p. 61-82.

3. Webb, P. A., Johnson, K. M., Wulff, H., Lange, J. V.Some therapeutic observations on the properties ofEbola viruses. In S. R. Pattyn [ed.]. Ebola virushaemorrhagic fever. Elsevier/North-Holland, Amster­dam, 1978, p. 91-94.

4. Ebola haemorrhagic fever in Zaire, 1976; report of an in­ternational commission. Bull. W.H.O. 56:271-293,1978.

5. Ebola haemorrhagic fever in Sudan, 1976. Bull. W.H.O.56:247-270, 1978.

6. Wulff, H., Lange, J. V. Indirect immunofluorescence forthe diagnosis of Lassa fever infection. Bull. W.H.O.52:429-436, 1975.

7. Mathews, H. M., Fisher, G. V., Kagan, I. G. Persistenceof malaria antibody in Tobago, West Indies, follow­ing' eradication as measured by the indirect hemaggluti­nation test. Am. J. Trop. Med. Hyg. 19:581-585, 1970.

8. Breman, J. G., Piot, P., Johnson, K. M., White, M. K.,Mbuyi, M., Sureau, P., Heymann, D. L., VanNieuwenhove, S., McCormick, J. B., Ruppol, J. P., Kin­toki, V., Isaacson, M., Van der Groen, G., Webb,P. A., Ngvete, K. The epidemiology of Ebola haemor­rhagic fever in Zaire, 1976. In S. R. Pattyn [ed.]. Ebolavirus haemorrhagic fever. Elsevier /North-Holland,Amsterdam, 1978, p. 103-124.

9. Francis, D. P., Smith, D. H., Highton, R. B., Simpson,D. I. H., Lolik, P., Deng, I. M., Gillo, A. L., Idris,A. A., Tahir, B. R. Ebola fever in the Sudan, 1976: epi­demiological aspects of the disease. In S. R. Pattyn [ed.].Ebola virus haemorrhagic fever. Elsevier/North-Holland,Amsterdam, 1978, p. 129-135.

10. Johnson, K. M., Webb, P. A., Heymann, D. L. Evalua­tion of the plasmapheresis program in Zaire. In S. R.Pattyn [ed.]. Ebola virus haemorrhagic fever.Elsevier/North-Holland, Amsterdam, 1978, p. 219-222.

at Penn State University (Paterno L

ib) on August 12, 2014

http://jid.oxfordjournals.org/D

ownloaded from