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Meeting of the scientific group on yellow fever research in east Africa, Geneva, 29-31 October 1963: report to the Director-General

December 31, 1964 World Health Organization

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WORLD HEALTH ORGANIZATION MEETING OP THE SCIENTIFIC GROUP ON YELLOW FEVER RESEARCH IN EAST AFRICA Geneva, 29-31 October 1963

ORGANISATION MONDIALE DE LA SANTÉ ORIGINAL: ENGLISH

REPORT TO THE DIRECTOR-GENERAL

MH0/PA/30.f^

CONTENTS

L i s t of p a r t i c i p a n t s P r o v i s i o n a l agenda Introduction Report on surveys c a r r i e d out i n E t h i o p i a The studies at the Manera f i e l d s t a t i o n Discussion . . . . . ., .

on the findings a t the Manera f i e l d s t a t i o n , 1962-63

R e f r i g e r a t i o n of specimens f o r transport Yellow fever i n the Sudan Experiments with three yellow-fevef* vaccines by s c a r i f i c a t i o n Mass vaccination i n E t h i o p i a Quarantine problems i n E t h i o p i a Report of a possible new outbreak Suggestions f o r the 1963-64 studies i n E t h i o p i a Recommendations Annex I - Epidemiological and s e r o l o g i c a l study, . . . ... .

1962-1963 campaign Annex I I - S e r o l o g i c a l study

Annex I I I - Entomological study Annex TV - Programme f o r the 1963-64 campaign Annex V - Vaccine studies .

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LIST OF PAjmciPAwrs

Dr P . B r e s , I n s t i t u t Pasteur de Dakar, 36 Avenue Pasteur, Dakar, Senegal Dr F . B . Hylander, Senior A d v i s e r , M i n i s t r y of Public H e a l t h , P . O . Box 1240, Addis Ababa, E t h i o p i a Dr R. P a n t h i e r , I n s t i t u t Pasteur, 25 rue du Do-jteur Ro'jx, P a r i s , Prance (Chairman) Dr M. H . S a t t i , D i r e c t o r of Research, î h e StacK Medical Research L a b o r a t o r i e s , P.O. Box 287, Khartoum, Sudan Dr C h . S é r i é , I n s t i t u t Pasteur d ' E t h i o p i e , P .O. Box 1242, Addis Ababa, E t h i o p i a Dr M. C . 'Williams, East A f r i c a V i r u s Research I n s t i t u t e , Entebbe, Ugeinda (Rapporteur) P . O . Box 49,

Secretsiriat Dr W. Chas. Cockburn, C h i e f , V i r u s Diseases, W H O ( J o i n t Secretary) Dr A . C . Saenz, Medical O f f i c e r , V i r u s Diseases, W H O (Joint Secretary)

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PROVISIONAL AGENDA

1. 2. 3 .

Opening address by representative o f the Director-General Appointment of Chairman Appointment o f Rapporteur

4. ^ Report on the studies c a r r i e d out i n E t h i o p i a since the l a s t meeting of the S c i e n t i f i c Group 5. Report on entomological, v i r o l o g i c a l and s e r o l o g i c a l t e s t s c a r r i e d out by c o l l a b o r a t i n g l a b o r a t o r i e s

(Dr Sérié)

(Dr Panthier, Dr Williams, Dr Bres)

6. 7. 8. 9. 10.

Discussion o f items 4 and 5 Yellow-fever studies i n Sudan Tests o f experimental vaccines (Dr S a t t i ) (Dr Sérié)

Proposed future studies Other business

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INIRODUCTION

•-

•'

The S c i e n t i f i c Group on Yellow Fever Research i n East A f r i c a met f o r the t h i r d time m Geneva from 29-31 'October 19^3• The s e r o l o g i c a l and entomological surveys

c a r r i e d out i n s e v e r a l areas of E t h i o p i a and the i n t e n s i v e e p i d e m i o l o g i c a l and e c o l o g i c a l s t u d i e s c a r r i e d out i n Manera were reviewed and d i s c u s s e d . f u t u r e s t u d i e s t o be made i n 1963-64 were d i s c u s s e d . \ Dr C. Raska, D i r e c t o r of the D i v i s i o n of Communicable Diseases, welcomed t h e Plans f o r

p a r t i c i p a n t s on behalf of the D i r e c t o r - G e n e r a l , s t r e s s i n g the importance of these s t u d i e s i n E t h i o p i a f o r the c l a r i f i c a t i o n of e p i d e m i o l o g i c a l and e c o l o g i c a l problems i n yellow f e v e r as v/ell as i n other arthropod-borne v i r u s i n f e c t i o n s . Dr R. Panthier was e l e c t e d Chairman and Dr M. C. Williams was e l e c t e d R a p p o r t e u r ; The p r o v i s i o n a l agenda was adopted. Dr F. B. Hylander conveyed to the members of the Group the g r e e t i n g s o f the M i n i s t e r of Health of E t h i o p i a . REPORT ON SURVEYS CARRIED OUT IN ETHIOPIA D e t a i l s of the geographical, e p i d e m i o l o g i c a l , s e r o l o g i c a l and e n t o m o l o g i c a l s t u d i e s can be seen i n annexes 1, 2 and 3» Dr Sérié s t a r t e d h i s p r e s e n t a t i o n of these papers by reviewing previous work and the h i s t o r i c a l background of the e p i d e m i o l o g i c a l work on yellow f e v e r i n E t h i o p i a . In a n a l y s i n g the present s i t u a t i o n , he pointed out that i n Assab Group B v i r u s a n t i b o d i e s were present, but Ae'des aegypti and other Aedes mosquitos were absent. Culex simiens was the only common mosquito. I n the Awash v a l l e y Group A and Group B a n t i b o d i e s were found but a n t i b o d i e s to y e l l o w - f e v e r v i r u s d i d not occur. I n the Blue N i l e V a l l e y , i n the upper r e g i o n , the only a n t i b o d i e s detected s i g n i f i c a n t numbers were those of Ekinyamwera v i r u s , but i n the lower p a r t of the v a l l e y , near the Sudan f r o n t i e r and the confluence of the Didessa r i v e r , both Group A and Group B a n t i b o d i e s were found. of yellow f e v e r . Nothing, however, suggested the presence in

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In Sidamo-Borena, i n the south, mass v a c c i n a t i o n had r e c e n t l y been c a r r i e d out. The s e r o l o g i c a l t e s t s confirmed the success of t h i s programme. At Gandima, where

v a c c i n a t i o n had not been done, i t was thought t h a t yellow f e v e r had been a c t i v e . In the Ogaden area where previous work by Smlthburn & Mahaffi had found s e v e r a l y e l l o w - f e v e r p o s i t i v e sera i n Dagabour, only one p o s i t i v e serum was found a t t h i s p l a c e , but the sera from the area as a whole contained very few arbovirus a n t i b o d i e s . Dr Sérié then analysed the r e s u l t s of the work of d i f f e r e n t l a b o r a t o r i e s on sera c o l l e c t e d i n I96I-62, which had a l l been brought together f o r the f i r s t time. pointed out the agreement of r e s u l t s from d i f f e r e n t l a b o r a t o r i e s using the same a n t i g e n , but when d i f f e r e n t s t r a i n s of y e l l o w - f e v e r v i r u s were used an obvious d i f f e r e n c e occurred between the epidemic area from which v i r u s s t r a i n s had been i s o l a t e d and the areas where epidemics had not been recorded. The Couma s t r a i n of He

yellow f e v e r appeared t o be more e a s i l y n e u t r a l i z e d than the standard French neurotropic yellow-fever v i r u s . In d i s c u s s i o n i t was decided t o take the Assab r e g i o n f i r s t and t o review the other regions from the periphery t o the epidemic area. The consensus of o p i n i o n

f o r Assab was t h a t a Group B v i r u s other than yellow f e v e r was r e s p o n s i b l e f o r the Group B a n t i b o d i e s . The problem of i s o l a t i n g v i r u s i n t h i s r a t h e r remote area was ,

d i s c u s s e d , and i t seemed t h a t the short p e r i o d during and a f t e r the r a i n s would be the best time f o r such work. In the Awash area i t was f e l t that the h i g h l e v e l of Group A and Group B v i r u s e s i n d i c a t e d a r i c h arbovirus a c t i v i t y , but that yellow f e v e r d i d not occur. In the Blue N i l e the absence of arbovirus a c t i v i t y i n the upper reaches w i t h the occurrence of Bunyamwera was remarkable, but might have been a s s o c i a t e d w i t h I n the lower area there was no evidence of yellow f e v e r ,

the h e i g h t (I8OO-I5OO m).

which was s u r p r i s i n g as i n the Sudan the 1959 epidemic had been confirmed by n e u t r a l i z a t i o n t e s t s and h i s t o p a t h o l o g i c a l s t u d i e s . I n E t h i o p i a the epidemic was apparently

confined t o the lower areas bordering the Sudan but d i d not reach the h i g h a l t i t u d e s .

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Dr S a t t i mentioned the r e s u l t s of a study by Dr T h e i l e r on s e r a from an outbreak of p a r a l y s i s which occurred 200 miles north of the Blue N i l e i n the Sudan a t Kakoom.

The s e r a showed a h i g h incidence of West N i l e and Z i k a v i r u s a n t i b o d i e s , but o n l y one serum showed a p a t t e r n of y e l l o w - f e v e r i n f e c t i o n . I t appeared t h e r e f o r e t h a t

y e l l o w f e v e r had not occurred i n the area to a s i g n i f i c a n t degree. In the v a l l e y s of the Baro and G h i l o where no cases of yellow f e v e r had the s e r o l o g i c a l r e s u l t s were d i f f e r e n t from the epidemic area. occurred

The d i f f e r e n c e s

between the r e s u l t s w i t h Couma and French neurotropic s t r a i n s of y e l l o w - f e v e r v i r u s were discussed and i t was f und impossible to decide whether yellow f e v e r occurred i n the area or not. I t was f e l t that these d i f f e r e n c e s v.'ere important and that s p e c i a l i z e d l a b o r a t o r i e s should be asked t o c a r r y out f u r t h e r s t u d i e s . The lower Didessa V a l l e y r e s u l t s were discussed. They were thought t o be •

s i m i l a r t o the lower Blue N i l e V a l l e y r e s u l t s , but i n Amalta and adjacent v i l l a g e s , higher up the v a l l e y , i t was thought that d e f i n i t e y e l l o w - f e v e r p o s i t i v e s e r a had V a l l e y was considered.

been found and the p o s s i b i l i t y of spread from the Omo The Sldomo-Borena area was reviewed

i n three p a r t s , f i r s t l y the area on the S e r o l o g i c a l l y the success

Kenya border, v/here mass v a c c i n a t i o n had been made. of the programme was confirmed. occurred.

To the east no yellow fever or a r b o v i r u s a c t i v i t y

In the west i n Gandima and another v i l l a g e , both of which had not been

v a c c i n a t e d , there was a very h i g h p r o p o r t i o n of y e l l o w - f e v e r p o s i t i v e s e r a , and i t was thought t h a t t h i s i n d i c a t e d an outbreak of yellow f e v e r . About the Ogaden area there was l i t t l e d i s c u s s i o n and no e x p l a n a t i o n was g i v e n f o r the p o s i t i v e y e l l o w - f e v e r sera from Dagabour. Dr Sérié s a i d that i n the epidemic zone of the Omo from 1961 to 1962, approximately 200 000 ^ Basin where there had been, deaths due to

cases and more than 30 000

y e l l o w f e v e r , the serum r e s u l t s showed t h a t Z i k a v i r u s was present as w e l l as y e l l o w fever v i r u s . I n d i s c u s s i o n the question of where the epidemic o r i g i n a t e d was considered

I t was expressed

t h a t some evidence pointed to the area near Akobo t o the west, but t h a t there were no d e f i n i t e i n d i c a t i o n s about t h i s .

the general o p i n i o n was

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In answer t o a question Dr Sérié explained t h a t the s e r a from the epidemic area were c o l l e c t e d from cases or from the f a m i l y of a person who had d i e d . I n the non-

epidemic areas e f f o r t s were made t o c o l l e c t sera from c h i l d r e n and young a d u l t s , but i n some areas l o c a l d i f f i c u l t i e s prevented t h i s . THE STUDIES AT THE MANERA FIELD STATION Dr Sérié o u t l i n e d the c o n s t r u c t i o n of the f i e l d s t a t i o n a t Manera, the place where previous work had l e d t o the i s o l a t i o n of a number o f y e l l o w - f e v e r s t r a i n s from man and Aëdes simpsoni. An a i r - l i f t was necessary t o get e v e r y t h i n g t o t h i s i s o l a t e d area. The

geographical featiares were mentioned and i t was noted t h a t some v i l l a g e s had high y e l l o w - f e v e r antibody r a t e s whereas others had no y e l l o w - f e v e r a n t i b o d i e s . Zika

a n t i b o d i e s were found, but they d i d not c o r r e l a t e w i t h y e l l o w - f e v e r a n t i b o d i e s . The reasons f o r t h i s are unknown. V i r u s i s o l a t i o n d u r i n g the year from l62 pools l e d t o the i s o l a t i o n of one s t r a i n of y e l l o w - f e v e r v i r u s from a pool of Aëdes a f r i c a n u s , two s t r a i n s of an as-yet u n i d e n t i f i e d v i r u s from two pools of Eretmapodites s p e c i e s , two s t r a i n s r e c e n t l y i s o l a t e d from Aëdes dentatus, and one from Culex s p e c i e s . of presumed v i r u s I s o l a t i o n were mentioned. D i f f i c u l t i e s i n adaptation

I n i t i a l l y , v i r u s i s o l a t i o n s were made To overcome t h i s

i n mice o l d e r than one day due t o the d i f f i c u l t i e s of t r a n s p o r t .

problem methods were found t o keep the mosquitos a l i v e f o r up t o a week, when I s o l a t i o n c o u l d be attempted i n Addis Ababa. The passage h i s t o r y o f a s t r a i n o f y e l l o w - f e v e r

v i r u s from a f o u r - t o - f i v e - w e e k s - o l d colobos monkey was presented. Next, the entomology o f the area was o u t l i n e d . forest. Studies were confined t o the The use

Taxonomic, e c o l o g i c a l and b i o l o g i c a l problems were mentioned.

of t r e e platforms w i t h catches a t d i f f e r e n t l e v e l s g i v e s a wealth of i n f o r m a t i o n . A t o t a l of 51 570 mosquitos were c o l l e c t e d belonging t o 53 s p e c i e s . P a r t i c u l a r l y i n t e r e s t i n g r e s u l t s have been obtained from s e r i a l blood samples from the Manera p o p u l a t i o n , showing t h a t 9 / 4 5 converted t o yellow f e v e r p o s i t i v e I t i s interesting

but only one case of mild I l l n e s s i n one person had been seen. t h a t t h i s person had albuminuria.

. 8 s t u d i e s had been s t a r t e d w i t h Aëdes simpsoni a r t i f i G i a l l y i n f e c t e d w i t h y e l l o w ^ f e v e r v i r u s t o see i f there was a d i f f e r e n c e i n development of the v i r u s when t h e mosquitos were kept i n the h i g h l y " p o s i t i v e " area of Manera and i n the " n e g a t i v e " area of Boga s i t u a t e d about 30O m higher. DISCUSSION ON THE FINDINGS AT THE MANERA FIELD STATION, 1962-I963 Dr Sérié was congratulated on the success and extent of h i s s t u d i e s under d i f f i c u l t conditions. I t was considered t h a t the i s o l a t i o n of the y e l l o w - f e v e r v i r u s s t r a i n from Aëdes a f r i c a n u s c o l l e c t e d at the height of 10-29 m on 2-4 September was thus showing f o r the f i r s t time i n t h i s area a spread to the f o r e s t . I t was reported t h a t v/ork at Entebbe had shown the two s t r a i n s of v i r u s from Eretmapodites species to be c l o s e l y r e l a t e d , but t h a t no r e l a t i o n s h i p had been found valid;

to y e l l o w - f e v e r v i r u s or 36 other group B arboviruses known t o occur i n A f r i c a . The y e l l o w - f e v e r s t r a i n d e r i v e d from the foior- t o five-weeks-old colobus monkey was discussed and the unusual passage h i s t o r y through guinea-pigs and many passages i n mice i s noted. As t h i s i s o l a t i o n d i d not f u l f i l the requirements normally was was as

accepted f o r p u b l i c a t i o n i n a s c i e n t i f i c paper the v a l i d i t y of t h i s i s o l a t i o n discvissed a t some l e n g t h . not due to contamination.

A l l a v a i l a b l e evidence suggested t h a t the i s o l a t i o n

Elsewhere i n East A f r i c a colobus have been c o n s i d e r e d I t was recognized t h a t inadequate

important to the w i l d y e l l o w - f e v e r c y c l e .

l a b o r a t o r y f a c i l i t i e s hampered Dr Sérié'3 i s o l a t i o n work. I t was noted t h a t the o v e r - a l l r e s u l t s were very s i m i l a r t o those found i n Uganda. important. REFRIGERATION OF SPECIMENS FOR TRANSPORT During the meeting Dr T. Guthe, Chief of the Venereal Diseases and Treponematoses U n i t , k i n d l y gave, at the request of the Group, a short paper on the t r a n s p o r t and maintenance of micro-organisms at cryogenic temperatures. I t appears t h a t apparatus u s i n g l i q u i d n i t r o g e n i s a v a i l a b l e t o overcome the d i f f i c i i l t i e s of t r a n s p o r t of deep-frozen m a t e r i a l under t r o p i c a l c o n d i t i o n s . for the p r o d u c t i o n of l i q u i d n i t r o g e n are a v a i l a b l e ; Small machines The presence of Aëdes aegypti not b i t i n g man i n the area was considered

although cheap t o run they are

c o m p a r a t i v e l y expensive t o i n s t a l l .

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YEIIDW FEVER IN THE SUDAN Dr S a t t i o u t l i n e d the h i s t o r i c a l background of yellow fever In the Sudan, where i t has been known f o r many years. He mentioned survey work on an e n c e p h a l i t i c

disease known l o c a l l y as "weal" d i s e a s e , o c c u r r i n g i n the upper N i l e a t Zaraf I s l a n d . S e r o l o g i c a l t e s t s w i t h arboviruses c a r r i e d out at Entebbe showed a h i g h l e v e l of a r b o v i r u s a c t i v i t y i n the area, but no evidence of yellow f e v e r . There has been no recrudescence of the 1959 epidemic which appeared t o have a The only l i k e l y v e c t o r

l a r g e p r o p o r t i o n of a t y p i c a l i n f l u e n z a - l i k e I n f e c t i o n s . was Aëdes v i t t a t u s .

EXPERINENTS WITH THREE YELLOW-FEVER VACCINES BY SCARIFICATION 'Detailed r e s u l t s may be"seen i n Annex 5. v a c c i n e s , Dakar, Yaba and Bogota, were used. The work was done i n a d u l t s . Three

The conversion r a t e s a f t e r v a c c i n a t i o n

were 100 per c e n t , w i t h Dakar, 54 per c e n t , w i t h Yaba and 8o per cent, w i t h Bogota. The haemagglutination i n h i b i t i o n and the n e u t r a l i z a t i o n t e s t s were i n agreement. No f e b r i l e r e a c t i o n occurred w i t h Yaba and Bogota v a c c i n e s ; f e b r i l e r e a c t i o n s occurred w i t h the Dakar s t r a i n . MASS VACCINATION IN ETHIOPIA - - -' - " ft• ; about 15 per cent, of

Dr Hylander s a i d that more than 1 000 000 v a c c i n a t i o n s have been completed i n areas p o t e n t i a l l y a t r i s k . The great m a j o r i t y were made w i t h Dakar vaccine and

smallpox vaccine at the same time.

Dr Sérié s a i d t h a t one t o two per cent, of Very

f e b r i l e r e a c t i o n s occurred w i t h the v a c c i n e , but no deaths were recorded. few c h i l d r e n under 10 years were v a c c i n a t e d .

I t was n o t i c e d t h a t l e s s r e a c t i o n

occurred i f smallpox and yellow f e v e r vaccines were placed on d i f f e r e n t arms. Dr S a t t i added t h a t 120 000 people were vaccinated by j e t i n j e c t i o n w i t h 17D vaccine

I n the Sudan and only four a l l e r g i c r e a c t i o n s occurred which responded t o a n t i h i s t a m i n e s . QUARANTINE PROBLEMS IN ETHIOPIA Dr Hylander s a i d that the i n c l u s i o n of Addis Ababa i n the y e l l o w - f e v e r endemic zone l e d to considerable d i f f i c u l t i e s and was u n j u s t i f i e d because of i t s geographical position. Dr P. J . Delon of the I n t e r n a t i o n a l Quarantine U n i t thought that the matter i n f o r m a t i o n from the Health A d m i n i s t r a t i o n

c o u l d be reviewed on r e c e i p t of the necessary of Ethiopia.

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REPORT OF A POSSIBLE NEW OUTBREAK During the session Dr Sérié read a telegram received from E t h i o p i a which s a i d that an outbreak thought to be, on c l i n i c a l grounds, y e l l o w fever was the Soddu area i n the Omo v a l l e y . SUGGESTIONS FOR THE .occurring i n ; .

196>64

STUDIES I N ETHIOPIA

Dr Sérié suggested that the next year 's work should include the f o l l o w i n g . (1) A completion of the s e r o l o g i c a l survey of E t h i o p i a by studying the

Tessene area i n the north west, and the area to the south, i n c l u d i n g the Wabi Sïxabel r i v e r (2) basin. I n the entomological f i e l d ,

The work at Manera should be continued.

study should move on to the behaviour of Aëdes africanus and Aëdes aeg3rpti. Virus i s o l a t i o n should be i n t e n s i f i e d from mosquitos and where p o s s i b l e from man and animals.

Studies on the v i r u s r e s e r v o i r should include rodents, bats, monkeys and l i z a r d s as w e l l as domestic animals and man. Capture and recapture

techniques would be used as w e l l as s e n t i n e l animals. Discvission centred on the l o g i s t i c s of the i d e n t i f i c a t i o n o f v i r u s e s and the cooperation of the Interested l a b o r a t o r i e s . The absence of a representative from the

New York l a b o r a t o r i e s o f the R o c k e f e l l e r Foundation made i t d i f f i c u l t to decide on the d i s t r i b u t i o n of séra. The Pasteur I n s t i t u t e s i n P a r i s and Dakar and the I n s t i t u t e

at Entebbe offered to continue t h e i r a s s i s t a n c e . RECONDVENDATIONS 1. The s e r o l o g i c a l surveys i n Tessene and the Wabi S h a b e l r i v e r b a s i n should be • ' suggested

c a r r i e d out. 2. 3. 4. 5.

Intensive study i n Manera on the yellow-fever cycle should be continued as

As other arboviruses w i l l be found i n the Manera area they should a l s o be s t u d i e d , I f other outbreaks of yellow fever occur they should be Investigated. A study of the l o c a l Aëdes aegypti i s considered of great i n t e r e s t .

- 11 -

Laboratory s t u d i e s should be undertaken on the Couma s t r a i n and perhaps on other

l o c a l s t r a i n s t o compare t h e i r a n t i g e n i c p r o p e r t i e s w i t h c l a s s i c a l y e l l o w - f e v e r v i r u s strains. 7. The co-operation which has been going on between the v i r u s l a b o r a t o r i e s should

be continued as i t has been so f r u i t f u l .

- 12 ANNEX 1

EPIDEMIOLOGICAL

AND SEROLOGICAL STUDY

1962-1963 Campaign

I n a c c o r d a n c e w i t h t h e recommendations made a t t h e p r e v i o u s m e e t i n g h e l d i n Geneva i n May 1962, t h e f o l l o w i n g a r e a s were s u r v e y e d f r o m November I962 t o March (a) the Assab r e g i o n ( p o r t o f E r i t r e a ) where a s m a l l p r o p o r t i o n o f p o s i t i v e surveys;

I963:

s e r o l o g i c a l t e s t s had been found d u r i n g p r e v i o u s (b)

t h e Awash V a l l e y ( r i v e r s k i r t i n g t h e Choa p l a t e a u and d r a i n i n g the

f u n n e l d e l i m i t e d by t h e h i g h p l a t e a u x o f t h e Choa and t h e A r o u s s i s and t e r m i n a t i n g t h e l a k e f a u l t towards t h e n o r t h e a s t ) ; (c) t h e s o u t h o f E t h i o p i a , e s s e n t i a l l y t h e p r o v i n c e s o f t h e Sidamo -Borena

( f r o n t i e r o f Kenya); (d) t h e Upper V a l l e y o f t h e B l u e N i l e , one o f whose t r i b u t a r i e s , the year;

D i d e s s a R i v e r , had been s u r v e y e d t h e p r e v i o u s (e)

the p r o v i n c e o f Ogaden a s f a r a s t h e Wabi S h e b e l r i v e r i n eastern

E t h i o p i a n e a r t h e S o m a l i f r o n t i e r , an a r e a s u r v e y e d i n 1942 by Smithbum & M a h a f f i , where p o s i t i v e s e r o - p r o t e c t i o n t e s t s v/ere o b t a i n e d a t Dagabur.

The aim o f a l l t h i s was t o a s c e r t a i n t h e p o s i t i o n i n t h e s e r e g i o n s , apparently u n t o u c h e d by y e l l o w f e v e r , and t o p l a n t h e n e c e s s a r y accordance w i t h the r e s u l t s . The p u r p o s e o f t h i s s u r v e y was t o draw up a map o f a r b o v i r u s e s unstudied 1. ASSAB Two surveys were c a r r i e d o u t a t A s s a b u s i n g a Land R o v e r . regions. i n these previoxisly p r o p h y l a c t i c measures i n

13 Annex 1

(a)

An entomologleal survey i n September I962 (6-17 September) The pericxi chosen was one when the scanty annual r a i n s make t h e i r

appearance (from January to September 1962 there was 1.1 m m of r a i n ) . o The minimum temperature was 25.9 C. o ' The maximum temperature was 42 C. The r e l a t i v e humidity was 45 per cent. No Anopheles or Aëdes were found i n e i t h e r the new or the o l d town. The only mosquito encountered was Culex simiens which a t t a c k s man w i t h v o r a c i t y d u r i n g the n i g h t (400 a d u l t s were captured). Two batches of 200 a d u l t s each were used f o r i n o c u l a t i n g baby mice but no v i r u s was isolated.

Search f o r l a r v a l breeding places l e d t o the d e t e c t i o n of l a r v a e of Anopheles c u l i c i f a c i e s , v a r i e t y adenensis, of Culex s i t i e n s and of Anopheles d ' t h a l i . C a r e f u l i n v e s t i g a t i o n s a l l over the town d i d not lead to the i d e n t i f i c a t i o n of any Aëdes l a r v a e . Information given by the quarantine s e r v i c e i n d i c a t e d t h a t d u r i n g the l a s t three years no Aëdes had been found e i t h e r i n the town or i n the p o r t of Assab. (b) An e p i d e m i o l o g i c a l survey i n November 1962 (15--21 November) Previous knowledge concerning Assab and i t s surroundings was G e o g r a p h i c a l l y , the Assab r e g i o n i s a desert zone. confirmed. .. •

I n t e r r o g a t i o n d i d not r e v e a l any abnormal morbidity or m o r t a l i t y . One hundred and f i f t y - s i x blood samples were taken i n s c h o o l s . The

subjects were c h i l d r e n or adolescents and a few rare a d u l t s who had always l i v e d a t Assab (were born there and had never l e f t the town).

- Ik. Annex 1

We now have the r e s u l t s of a p a r t i a l study of these s e r a , as f o l l o w s . Three per cent, of the sera examined i n New York show the presence of Chikungunya and S e m l i k i a n t i b o d i e s . On the other hand, study of Group B i s of great i n t e r e s t and has revealed

seven per c e n t , of West N i l e a n t i b o d i e s among the sera studied a t Dakar, as w e l l as 25 per cent, of Murray V a l l e y a n t i b o d i e s and three per c e n t , of N'Taya a n t i b o d i e s among the sera studied i n P a r i s . I t should be noted that three per c e n t , of y e l l o w - f e v e r p o s i t i v e s e r a were found at Dakar and New Comments The presence of Group B arbovirus antibodies i n the sera examined a t P a r i s might e x p l a i n the presence of the three per cent, p o s i t i v e sera c o n s t a n t l y found i n the s e r o l o g i c a l t e s t s c a r r i e d out a t Assab during previous surveys, f o r a g g l u t i n i n s , heterologous to y e l l o w - f e v e r v i r u s , were i n f a c t found i n the sera t e s t e d i n P a r i s . to s e r o York.

Moreover, the r e s u l t s from Dakar, where c e r t a i n sera were submitted

p r o t e c t i o n t e s t s vis-à-vis the Couma and y e l l o w - f e v e r v i r u s e s , gave 17 per c e n t , p o s i t i v e s e r o - p r o t e c t i o n t e s t s against Couma and three per cent, against y e l l o w fever. Here again, the Couma and Murray V a l l e y v i r u s e s seem to have a c e r t a i n cormion antigenicity. This a f f o r d s considerable support f o r the theory of the n o n - s p e c i f i c i t y

of the y e l l o w - f e v e r s e r o - p r o t e c t i o n t e s t s i n t h i s area. Thus, the problem r a i s e d by Assab has been p a r t l y solved. While a w a i t i n g

i s o l a t i o n of the v i r u s s t r a i n , the theory p r e v i o u s l y advsmced would seem t o be a valid 2. one. THE AWASH VALLEY (Survey c a r r i e d out by Land Rover and Unimog) This survey took place from 20 October t o 1 November I962. v a l l e y of the Awash was The middle and lower

i n v e s t i g a t e d ( t h i s part s t r e t c h e s from the Awash s t a t i o n a t

Gawani and the Tendao-Assaita r e g i o n ) . G e o g r a p h i c a l l y , the Av/ash r i v e r , from Awash S t a t i o n onwards, flows from south t.

n o r t h and t r a v e r s e s the Dankali d e s e r t , r e c e i v i n g i t s t r i b u t a r i e s on tlie l e f t bank, from

- 15 Annex 1

the Choa p l a t e a u .

I t i r r i g a t e s a serai-desert p l a i n w i t h a s c a t t e r e d nomadic • • •

population (Dankalis).

From the e p i d e m i o l o g i c a l viewpoint, t h i s r e g i o n would seem t o be protected from any a r b o v i r u s i n f e c t i o n . I t would appear impossible f o r any i n f e c t i o n to spread i n

t h i s area, where the few p o p u l a t i o n groups are concentrated t e m p o r a r i l y around water sources, without any l i n k betv/een them. Twelve v i l l a g e s were surveyed and a t o t a l of 20? blood samples taken. Larvae of the f o l l o w i n g were found at Tandao: Anopheles gambiae.

Anopheles pharoensis, Culex p l p i e n s and Ae'des u n i l i n e a t u s . At Combolcia ( C h i a f f a v a l l e y ) were found l a r v a e of Aëdes m e t a l l i c u s and Aëdes gambiae. Study of the p a r t i a l r e s u l t s of the s e r o l o g i c a l survey i n t h i s r e g i o n showed t h a t , c o n t r a r y to what we had thought, arboviruses were c e r t a i n l y present and yellow f e v e r completely absent. . '— ' . .• ' • —'-• • t e s t e d the

Group A i s w i d e l y represented by S i n d b i s .

P a r i s and New York, who

s e r a w i t h t h i s a n t i g e n , confirm i t s presence (20 per cent, p o s i t i v e sera i n P a r i s and 25 per cent, i n New York).

As concerns Group B, i n t e r p r e t a t i o n of the r e s u l t s i s more d i f f i c u l t because each I n s t i t u t e d i d not employ the same antigens. Nevertheless, the presence of West N i l e seems c e r t a i n : and 20 per cent, a t New York. • P a r i s i n d i c a t e s 27 per cent, of Murray V a l l e y a n t i b o d i e s and 13 per cent, of N'taya a n t i b o d i e s , while Dakar r e p o r t s 24 per cent, of Uganda S a n t i b o d i e s . I n a c t u a l f a c t , the presence of heterologous a g g l u t i n i n s seems probable, each of these v i r u s e s being present i n much smaller p r o p o r t i o n s . An important f a c t i s the p o s i t i v i t y of 26 per c e n t , of the sera towards the Couma s t r a i n , whereas there i s no trace of yellow f e v e r i n t h i s r e g i o n . This would seem 32 per cent, at Dakar

t o show the a n t i g e n i c p l u r a l i t y of t h i s s t r a i n , thus c o n f i r m i n g the theory put forward a t Assab, where Couma and the arboviruses i n i t s group (West N i l e , N'taya, Uganda S and Mtirray V a l l e y ) are thought to have common antigens.

- 16 Annex 1

UPPER VALLEY OF IHE BLUE NILE This t r i p was c a r r i e d out by h e l i c o p t e r on 10-16 December 1962 (700 km from

Bahar Dahr t o Sc i o g a l i ) . G e o g r a p h i c a l l y , t h i s region can be d i v i d e d i n t o three p a r t s . (a) From Bahar Dahr to the f a l l s of the N i l e The r i v e r , on l e a v i n g Lake Tana, flows across high f e r t i l e p l a i n s (1850 m) surrounded i n the f a r distance by s t i l l higher h i l l s . s t r e t c h e s f o r 60 (b) km. Didessa This area

From the f a l l s of the N i l e to the j u n c t i o n w i t h the

The mountains c l o s e i n and the r i v e r flows through a canyon from which i t emerges 550 km f u r t h e r on. • m above sea l e v e l . .•

At the Nlle-Didessa j u n c t i o n the r i v e r i s 700 (c) From the j u n c t i o n of the Didessa to S c i o g a l i

The N i l e enters the v a l l e y , which i s g e o g r a p h i c a l l y Sudanese. The p l a i n s s t r e t c h f u r t h e r and f u r t h e r and the Sudanese savannah spreads out on e i t h e r side of the r i v e r . These areas have a s c a t t e r e d p o p u l a t i o n . The middle r e g i o n between populated. m above

the f a l l s of the N i l e and the j u n c t i o n w i t h the Didessa i s densely

Information c o l l e c t e d shows t h a t the v i l l a g e of Barkassa, s i t u a t e d 700

sea l e v e l , w i t h a p o p u l a t i o n belonging to the Sessé race, was attacked e i g h t months p r e v i o u s l y (60 i n h a b i t a n t s out of 15O are s a i d to have died) by a disease which

f o l l o w e d i t s course i n two or three days. I t seems t h a t t h i s v i l l a g e i s the most advanced p o i n t of y e l l o w - f e v e r pénétrât The r e s u l t s of the s e r o l o g i c a l survey w i l l enable us to throw l i g h t on the epidemicl o g i c a l data. However t h i s may survey. be, no p a t i e n t was found and no y e l l o w - f e v e r focus

detected throughout the

- 17 Annex 1 T.;enty-six v i l l a ~ e s were surveyed along the l e n g t h of the Elue,.Nile and a t o t a l of 322 serum samples taken. The r e s u l t s show that arboviruses are p r a c t i c a l l y absent beyond the v i l l a g e of Orsa Karsa. Downstream, ths f i n d i n g s were as f o l l o w s : Chikungunya: 10 per cent, i n New York, three per c e n t , i n Dakar, cent.)

f i v e per cent, a t P a r i s , w i t h a few S i n d b i s - p o s i t i v e sera (tv;o per

As concerns Group B, an i n i t i a l f a c t to be noted i s the absence of yellow f e v e r , s i n c e none of the sera was H.I. p o s i t i v e i n any of the d i f f e r e n t i n s t i t u t e s . Moreover, West N i l e , which i s the only v i r u s noted i n a d d i t i o n to Z i k a , would seem to be present only downstream from the Didessa jmction.

I t appears, t h e r e f o r e , t h a t the Didessa has been i n f e c t e d by an epidemic descending the course of the r i v e r and the i n f e c t i o n eomes perhaps from the upper v a l l e y of the Omo ( t h i s i s only a t h e o r y ) .

However t h i s may be, an important f a c t i s the p o s i t i v i t y vis-à-vis Couma (S.P.) i n the lower E t h i o p i a n v a l l e y of the Blue N i l e . This confirms the c o r r e l a t i o n s

noted elsewhere between the Couma s t r a i n and the other a r b o v i r u s e s . 4, STDAMO-BORENA (Erom 2-27 Unimog (2234 km) ) January I963) (Survey c a r r i e d out by Land Rover and

G e o g r a p h i c a l l y , the study of t h i s r e g i o n , which borders on the f r o n t i e r of Kenya to the south and Gamo Goffa (epidemic focus) t o the west, i s p a r t i c u l a r l y i n t e r e s t i n g . The f o l l o w i n g can be d i s t i n g u i s h e d : (a) the lake f a u l t , whose v e g e t a t i o n i s l i m i t e d t o poor savannah w i t h

spiny p l a n t s ; (b) the r i c h Sidamo area, to the west of Lake Marguerite, and the Shamo

area w i t h i t s c o f f e e p l a n t a t i o n s ;

- 18 Annex 1

(c)

an a r i d area which f o l l o w s the r i v e r Canale D o r i a up t o the

Kenya f r o n t i e r ; (d) f i n a l l y , the very populous and h i g h l y c u l t i v a t e d area of the Konso

country t o the north-east o f t h i s province, beyond the Sagan r i v e r , which s t r e t c h e s t o the f o o t - h i l l s o f the Gamo Goffa mountains. The whole of t h i s area ranges frora 13OO-I7OO m i n a l t i t u d e . From the e p i d e m i o l o g i c a l viewpoint, no t r a c e o f epidemic was d e t e c t e d throughout

t h i s a r e a , apart from the Konso country where an abnormal m o r t a l i t y was noted i n the v i l l a g e of Gandima. The entomological f i n d i n g s were as f o l l o w s : larvae o f : Culex Culex Culex Culex Culex Culex Culex Aëdes (S) Anopheles (M) Anopheles (M) 1 adult Aëdes (A) (Neoculex) (Neoculex) (Culex) (Culex) (Culex) (Culex) (Lutzia) shoae rubinotiis simpsoni univittatus trifilatus ethiopicus trigipes vittatus cinerus funestus arabiensis The reason why so few a d u l t s were, encountered

Only a s i n g l e a d u l t was captured.

i s t h a t the survey took place d u r i n g the dry season.

19 ' Anhë:c" 1

Thirteen v i l l a g e s were siarveyed and a t o t a l of 213 blood samples taken. S e r o l o g i c a l examination i n the v i l l a g e of Gandima. The o v e r - a l l percentages c a l c u l a t e d f o r the region as a whole i n d i c a t e , shov;ed the presence of yellow-.fever v i r u s - p o s i t i v e sera

r e s p e c t i v e l y , f i v e per cent, and 10 per cent, of p o s i t i v e sera (five per cent. P a r i s and 10 per cent. New York). S i m i l a r l y , a t Mega we fo-ond two subjects with p o s i t i v e s e r o l o g i c a l tests among the non-vaccinated population. I t may be concluded that yellow fever i s present i n the Konso country and i n Sidamo-Borena province. I t should be noted that t h i s western region, i r r i g a t e d by focus.

the Sagan r i v e r , borders on the I96I epidemic

Furthermore, three per cent, of sera were N' taya-positive. In regard t o antibodies against Group A v i r u s e s , they are represented by Sindbis and four per cent. Chikungtinya found i n New York. A large part of the population of t h i s region was vaccinated i n an e f f o r t to prevent any spread o f the epidemic 5. frora Gamo Goffa towards the south and south-west,

PROVINCE OF OGADEN - WABI SHEBEL VALLEY This t r i p was c a r r i e d out by Land Rover and Unimog from 20 February to 13 March 1963

(2700 km). The aim of t h i s siarvey was to study the f o l l o w i n g , (1) The Ogaden, the Dagabur area i n p a r t i c u l a r , where i n 1942 Smlthburn & Mahaffi

had found sera g i v i n g a p o s i t i v e sero-protection t e s t . (2) The Wabi Shebel v a l l e y .

The team was unfortunately unable t o undertake t h i s part of the survey i n view of the state of the roads and the lack of co-operation on the part of the l o c a l population.

- 20 Annex 1

Geographically speaking, Ogaden province i s a large semi-desert p l a t e a u g r a d u a l l y ' s l o p i n g from the northern heights towards the south, I6OO m a t G l g i g a and 650 a t Vardene. The lowest regions are along the Wabi Shebel r i v e r , the only permanent s t r e a j n i n the whole area, which i s not more than 300 m h i g h a t i ^ s t a h i l . Ihe Somali t r i b e s who i n h a b i t these regions are nomadic and very No t r a c e of epidemic was found. uncooperative m

The i n f o r m a t i o n c o l l e c t e d i n the v a r i o u s

d i s p e n s a r i e s encountered was completely negative. One hundred and seventy-three serum samples were taken i n 13 towns and The town of Dagabur was v i s i t e d w i t h s p e c i a l I n t e r e s t . were taken; population. The p a r t i a l r e s u l t s which we have so f a r have not revealed any p o s i t i v e serum Indeed, t h i s whole semi-desert r e g i o n proved t o be f r e e from a l l a n t i b o d y . The absence of y e l l o w f e v e r i s an Important i n i t i a l datum. As regards Group B, at most two per cent, of Z i k a was found by New York as as two per cent, of S i n d b i s belonging to Group A . Uhfortunately, the v a l l e y of the Wabi Shebel proper could not be i n v e s t i g a t e d because of the d i f f i c u l t i e s of a l l kinds encountered. well villages

Twenty-two serum sairple-^

i t was not p o s s i b l e t o o b t a i n more because of the h o s t i l i t y o f t h e

Such a survey should be made e i t h e r by road, coming from the west and d e s c e n d i n g the v a l l e y , or by h e l i c o p t e r , which would c e r t a i n l y be the b e t t e r s o l u t i o n i n t h i s rather hostile region.

DISTRIBUTION OF ARBOVIRUSES - GROUP A

DISTRIBUTION OF ARBOVIRUSES

GROUP

DISTRIBUTION OF ARBOVIRUSES • GROUPS A

- 21 kmEX 2 SEROLOGICAL STUDY

Our aim i n t h i s d i s c u s s i o n i s to compare the r e s u l t s as a whole obtained so f a r . F i r s t l y , we s h a l l consider the p a r t i a l r e s u l t s obtained during the campaign. Secondly, we s h a l l r e t u r n to the r e s u l t s of the I96I-62 campaign, part of which was published i n the preliminary note; from these we s h a l l be able to draw more

1962-63

d e f i n i t e conclusions and give an opinion on c e r t a i n points of v i r o l o g i c a l and epidemiological interest. '

RESULTS OF THE

1962-63 CAMPAIGN

As planned, the sera c o l l e c t e d were d i v i d e d betv/een the d i f f e r e n t i n s t i t u t e s m equitable proportions. The f o l l o w i n g samples were taken: 1 5 6 sera a t Assab 2 0 7 sera i n the Awash Valley 3 2 2 i n the Blue N i l e V a l l e y 213 i n the Sidamo-Borena 1 7 3 i n the Ogaden ^ - :rv ; = • u.vv • . ,. ; , • •

totalling

1 0 7 1 f o r the epidemiological survey proper, to which should be added: 4 o 8 sera frora Manera, t o be studied l a t e r on 287 sera from domestic and wild animals, a l s o t o be s t u d i e d l a t e r on . ; .

Total

6 9 5 sera i . e . a t o t a l o f 1766 sera f o r the

1962-63 campaign.

- 22 Annex 2

These sera were d i s t r i b u t e d as follows: Ethiopia: New York: Entebbe : Dakar: Paris : two ampoules of each serum, i . e . 422

I766

569 512 251

The sera were d i s t r i b u t e d i n such a way that each i n s t i t u t e has specimens f r o m each of the regions studied. Itoreover, a l l the serum samples from Manera were s e n t

to Entebbe, a l l the animal sera to Dakar, and a l l the sera from regions not p r e v i o i i s l y surveyed to New York and P a r i s . So f a r we are unable to submit the r e s u l t s from Addis Ababa. be examined there i n the coming months. The following general comments should be made: not reached us; so f a r the Entebbe r e s u l t s have These s e r a will

the p l u r a l i t y of the antigens used f o r haemagglutination i n h i b i t i o n on the whole,

makes i t d i f f i c u l t to compare the r e s u l t s from d i f f e r e n t i n s t i t u t e s ;

however, the method adopted gave valuable information, as we s h a l l see l a t e r on. In a l l , and to date, tests at the various i n s t i t u t e s have been c a r r i e d out the following viruses : Group A: Chikungunya Sindbis Semliki O'Nyong-Nyong Bunyamwera ^ , with

- 23 Annex 2

Group B:

Yellow fever Dengue West Nile Zika Uganda 3 Murray V a l l e y N'taya SAH ' • •

. . .

" '

• '••

336 ^

Wesselsbron

Group C:

Manituba Oriboca Carapuru

F i n a l l y , the Tahyna and Bwambae groups. As shown by the Figures below, the f o l l o w i n g comments can be made. 1, Group A The p o s i t i v i t y of Sindbis j r.

The number of p o s i t i v e sera i s r e l a t i v e l y small. (Awash) and Chikungunya (Blue N i l e ) should be noted. 2. Group B

Hie t e s t s i n d i c a t e : (a) The presence of yellow fever i n Sidamo-Borena and more e s p e c i a l l y

i n the west of that province i n the areas bordering on the epidemic zone.

- 24 Annex 2

(b)

The unsuspected r i c h n e s s of the Awash V a l l e y In a r b o v i r u s e s : Murray V a l l e y N'taya West N i l e Uganda S '

In a l l p r o b a b i l i t y , heterologous

a g g l u t i n i n s e x i s t and the c a r r y i n g out of s e r o -

p r o t e c t i o n t e s t s f o r each of the v i r u s e s t e s t e d would be of great i n t e r e s t , (G ) The presence of Murray V a l l e y at Assab i s very Important and serves t o

e x p l a i n the r e s u l t s of previous s e r o - p r o t e c t i o n t e s t s , (d) The survey of the Blue N i l e V a l l e y was d i s a p p o i n t i n g i n view of the few

p o s i t i v e r e s u l t s obtained.

The absence of yellow fever i n the Lov;er N i l e V a l l e y i s i t because the Didessa V a l l e y

pc^es an important e p i d e m i o l o g i c a l problem: has not been i n f e c t e d from the Omo Only West N i l e are found, (c) Valley?

In the Ogaden, the r e s u l t s so f a r obtained show the poorness of t h i s

area i n arboviruses and I t should be p a r t i c u l a r l y noted t h a t no t r a c e of y e l l o w f e v e r was found at Dagabur In the s e r o l o g i c a l record of the persons t e s t e d . Tnese are the c o n s i d e r a t i o n s from which the known o v e r - a l l e p i d e m i o l o g i c a l results follow. .

F i n a l l y , other s p e c i a l l y important problems may be examined and we s h a l l e n c o u n t e r them again i n the 196I-62 survey which we s h a l l review once more, making use of the Figures. (i) Examination of F i g . 1 shows t h a t the r e s u l t s are concordant f o r the whole This does not h o i " t r u e , however, f o r the non-epidemic This was

of tho epidemic area.

area (the percentages given were c a l c u l a t e d on the r e s u l t s as a whole).

done w i t h the aim of showing that there i s no doubt as to the y e l l o w - f e v e r epidemic i n the Omo Valley.

Outside the epidemic a r e a , we are confronted w i t h a d i f f e r e n t problem w h i c h v/e s h a l l d e a l w i t h l a t e r on.

FIG. 1 T O T A L POSITIVE SEROLOGICAL RESULTS CONCERNING YELLOW FEVER CONCORDANT AND NON - CONCORDANT RESULTS IN THE AREAS STUDIED

90 80 70 60 50 40 30 20 10 0 1 2 3 Epidemic 4 area

Results concordant with Y . F . Results not concordant witti Y . F .

6 7 Mon-epidemic area

COMPARATIVE STUDY OF THE DIFFERENT SEROLOGICAL TECHNIQUES! YELLOW FEVER (F.N. STRAIN) | S.P. (Entebbe) H.I. (New York) C F . (Paris)

Epidemic

area

6 7 Non- epidemic

FIG. 3 SERO PROTECTION TESTS ( + ) Y . F .

FIG. 6 H.I. OF THE ARBOVIRUSES ( NEW YORK)

Group A Chikungunya

! l ® ^ * ^ ^, Germistonl

Bunyamwera Group

Non-epidemic areo

FIG. 5 H.I. OF THE ARBOVIRUSES (NEW YORK) GROUP B Yellow Fever Wesselbron Zika West Nile Ntaya

E p i d e m Ic

area

Non'epidemic area

- 25 Annex 2

(ii)

We f e l t t h a t i t would be u s e f u l to show at the same time ( P i g . 2)

the r e s u l t s obtained i n r e l a t i o n to the d i f f e r e n t t e s t s used. These r e s u l t s confirm the u s u a l l y accepted view t h a t the complement-fixation t e s t i s more s e n s i t i v e than the others. The haemagglutination i n h i b i t i o n t e s t r e s u l t s f o l l o w a curve which i s about the same but w i t h a lower percentage. epidemic area. (iii) Turning now t o P i g , 3 which shows the r e s u l t s of the s e r o - p r o t e c t i o n These curves d i v e r g e i n the non-

t e s t u s i n g the two antigens, Couma and y e l l o w f e v e r , we f i n d a p a r a l l e l i s m of the r e s u l t s i n the epidemic area and a very marked divergence i n the nonepidemic area. (iv) On the other hand, c o n s i d e r i n g F i g . 4, which g i v e s the haemagglutination

i n h i b i t i o n r e s u l t s alone (as i f these t e s t s had been c a r r i e d out s o l e l y f o r y e l l o w f e v e r ) , we observe a very d i s t i n c t concordance between the two a n t i g e n s . • Comparison of P i g s . 3 and 4 shows t h a t the Couma s t r a i n has a haemagglutin a t i n g power a p p r e c i a b l y the same as the F.N. s t r a i n but t h a t , on the other hand, the gmtigenic power of the Couma s t r a i n i s much more e x t e n s i v e . the s e r a from the non-epidemic Furthermore,

area have a n e u t r a l i z i n g power f o r the Couma one.

s t r a i n which they do not have f o r the F.N.

I n view of the r e s u l t s already submitted, a problem now a r i s e s , namely, : the d i f f e r e n c e between the Couma and F.N. s t r a i n s . As we s h a l l see, the

. e x i s t e n c e of t h i s problem i s confirmed when the arboviruses t e s t e d are s t u d i e d . (v) P i g , 5» which covers a l l the r e s u l t s obtained f o r the arboviruses (study

based e s s e n t i a l l y on the New York r e s u l t s ) shows the presence of N'taya and e s p e c i a l l y of West N i l e i n the non-epidemic area. affected

We f e e l i t cam be concluded t h a t the sera from t h i s a r e a , undoubtedly

by these v i r u s e s , n e u t r a l i z e the Couma s t r a i n because the l a t t e r possesses analogour antigens, Couma thus covers not only the y e l l o w - f e v e r epidemic area but a l s o the

areas where West N i l e and N'taya are present.

- 26 Annex 2

Turning now t o the r e s u l t s obtained d u r i n g the 1962-63 survey, we see t h a t they have been confirmed f o r these two v i r u s e s , p a r t i c u l a r l y i n the Awash V a l l e y

and t h a t , moreover, the p r o t e c t i o n extends a l s o t o the Uganda S and Murray V a l l e y viruses. Ihe Couma s t r a i n i s t h e r e f o r e one w i t h p o l y v a l e n t a n t i g e n i c power c o v e r i n g a wide range o f the B Group arboviruses so that our theory of a "grandfather" s t r a i n seems t o be confirmed. A p o i n t t o be noted, however, i s t h a t the New York r e s u l t s do not appear t o agree w i t h t h i s viewpoint ( c f . 1962 B u l l e t i n ) . Has there been y e l l o w f e v e r i n the Baro and G h i l o V a l l e y s ? This q u e s t i o n

remains debatable, although i t may be f e l t t h a t i t has been present, a t l e a s t a l o n g the Baro. New York supports t h i s i d e a , while the s e r o - p r o t e c t i o n t e s t s c a r r i e d

out against the P.N. v i r u s by Entebbe and P a r i s a l s o seem t o confirm i t . F i n a l l y , F i g . 6 shows the presence of A Group and Bunyamwera a n t i g e n s . Chikungunya i s found p r a c t i c a l l y everywhere i n a l l the t e r r i t o r i e s surveyed. ' As

regards the Bunyamwera group, i t i s much more Important i n the non-epidemic a r e a . As regards Group C, the antigens tested by New York showed no p o s i t i v i t y vis-à.-v the sera from the regions surveyed.

INSTALLATION OF THE MANERA EXPERIMENTAL STATION F o l l o w i n g the recommendations o f the Geneva S c i e n t i f i c Committee i n May I962, an experimental s t a t i o n was set up i n the v i l l a g e o f Manera i n the Shushuma V a l l e y , 1. INSTALLATION OF THE CAMP The i n i t i a l i n s t a l l a t i o n work commenced on 11 October 1962 and the p e r i o d o f i n s t a l l a t i o n l a s t e d from l 4 October t o 15 November I962. commenced on 15 November. The t o t a l absence of any means o f communication between Manera and the n e a r e s t t< Gimma, o b l i g e d us t o b r i n g i n a l l the equipment by a i r : more than 10 tons o f The s c i e n t i f i c work

equipment was parachuted by the E t h i o p i a n A i r Force, w h i l e the s t a f f was f l o w n i n by h e l i c o p t e r .

- 27

Annex ,2

Because of the cost of the h e l i c o p t e r , we c l e a r e d a l a n d i n g s t r i p 35 minutes away from the camp i n order t o reduce transport expenses and f a c i l i t a t e communication w i t h the s t a t i o n . t h i s purpose. By 15 November 1962, (a) the camp was i n running order and comprised: m each, one s e r v i n g as a l a b o r a t o r y "dispensary" For the past month Cessna and P i p e r Cub planes have been used f o r

two l a r g e t e n t s measuring 25

and l i v i n g - r o o m and the other as animal q u a r t e r s , store and

(the p l a n of these tents had been drawn up by us and they had been made at N a i r o b i ) ; (b) (c) (d) two Manyara s l e e p i n g tents of B r i t i s h o r i g i n ; one ordinary tent f o r personnel; one k i t c h e n .

The whole compound has an area of one hectare and i n c l u d e s a k i t c h e n garden and helicopter-port. A l l t h i s work was c a r r i e d out by personnel from the Pasteur I n s t i t u t e of E t h i o p i a and l o c a l l y r e c r u i t e d personnel. The camp was equipped w i t h one e l e c t r i c generating s e t , one r e f r i g e r a t o r working

on p a r a f f i n , one r a d i o r e c e i v i n g and t r a n s m i t t i n g set f o r communication w i t h the Pasteur I n s t i t u t e a t Addis Ababa, and running water f o r domestic and laboratory needs. 2. PERSONNEL ^' " "

The personnel c o n s i s t e d of Dr Sérié, Dr Andral and Mr N e r i , as w e l l as l a b o r a t o r y t e c h n i c i a n s Ato Petros H a i l e , Ato A l l Mohamed and Ato Gabre. The personnel have been r e g u l a r l y r e l i e v e d since 15 November 1962, by means of a r o t a system. So f a r , Mr N e r i , the entomologist, has spent most of h i s time at

the experimental s t a t i o n . A r e g u l a r monthly f l i g h t by h e l i c o p t e r has been i n s t i t u t e d f o r the r e l i e f of personnel.

- 28 Annex 2

A weekly mule s e r v i c e has a l s o been set up between the camp and Gimma f o r the r e g u l a r d i s p a t c h of mail and equipment. Twenty employees were r e c r u i t e d l o c a l l y and t r a i n e d f o r v a r i o u s t a s k s . They

cohiprise a team of nine f o r c a p t u r i n g i n s e c t s and two employed f o r c a p t u r i n g w i l d animals, w h i l e the remaining workers have been assigned various Jobs. 3. SCIENTIFIC INSTALIATIONS During November and December I962 and January I963 the f o l l o w i n g work was c a r r i e d out: (a) (b) c l e a r i n g of paths i n the Manera f o r e s t which a t f i r s t was q u i t e c o n s t r u c t i o n of platforms i n t r e e s . impassable;

At present three t r e e s are equipped These p l a t f o r m s

w i t h three platforms each and two w i t h two platforms each. are s i t u a t e d 10-20

and 30 m above the ground i n the case of the t r e e s w i t h

three of them and at heights of 15 and 25 m, r e s p e c t i v e l y , f o r the t r e e s w i t h two p l a t f o r m s ; (c) s e t t i n g up of a generating u n i t i n the f o r e s t f o r n i g h t captures (originally

e l e c t r i c b a t t e r i e s were used but these proved too expensive); (d) i n s t a l l a t i o n of three m e t e o r o l o g i c a l s t a t i o n s , one a t the Manera camp, . their • ..

the second i n the v i l l a g e of Boga and the t h i r d i n the f o r e s t ; (e)

topographical survey of a l l the huts i n the v i l l a g e of Manera and

numbering so as to check the s a n i t a r y c o n d i t i o n of the v i l l a g e .

EXPERIMENTAL STATION OF MANERA 1. A. VIROLOGICAL STUDY I s o l a t i o n of v i r u s from mosquitos • , '

Our main e f f o r t s were d i r e c t e d to the i s o l a t i o n of v i r u s from pools of mosquitos. From November I962 to September 1963» l62 i n o c u l a t i o n s using mosqiiito pools were c a r r i e d out as f o l l o w s :

- 29 Annex 2

56 batches of Eretmapodites ( a l l s p e c i e s ) corresponding t o 9 batches of Culex ( a i l s p e c i e s ) corresponding t o 23 batches of Aëdes (A) p h y l l o l a b i s - t a r s a l i s corresponding t o 15 batches of Aëdes ( A ) cumminsi corresponding t o 5 batches of Aëdes ( A ) s t o k e s i corresponding t o 6 batches of Aëdes (A) dentatus corresponding t o 7 batches of Aëdes (B) c i r c u m l u t e o l u s 1 batch o f Aëdes (D) f u r c i p e s

9 673 a d u l t s 750 a d u l t s 8 5

182 a d u l t s 792 a d u l t s 90 a d u l t s

2 727 a d u l t s ; 257 a d u l t s

:-:•„;:'• 5 a d u l t s 710 a d u l t s " . . : ' "

5 batches of Aëdes ( S ) simpsoni 33 batches of Aëdes ( S ) a f r i c a n u s 2 batches of Aëdes ( S ) luteocephalus

2.623 a d u l t s 25 a d u l t s

T o t a l 162 batches comprising

234 a d u l t s

In a d d i t i o n , 20 l i t t e r s of baby mice were exposed i n sentry cages. and animal s e r a were a l s o used f o r i n o c u l a t i o n s . So f a r (25 September I963) i t may be s a i d t h a t : (1) two s t r a i n s have been i s o l a t e d from Eretmapodites p o o l s .

Ten human

These are

s t r a i n 124, comprising 204 Eretmapodites belonging to the v a r i o u s groups, and s t r a i n l 4 7 , c o n s i s t i n g of I3I Eretmapodites belonging t o the v a r i o u s groups. These two f r e e z e - d r i e d s t r a i n s were sent t o New York, Entebbe and Dakar. < = " have not r ^ c e l v e d ariy r e p l y concerning t h e i r i d e n t i f i c a t i o n . So f a r w (2) Moreover, one s t r a i n from an Aëdes a f r i c a n u s batch has J u s t been i s o l a t e d

and seems t o be p a r t i c u l a r l y v i r u l e n t , w i t h an i n c u b a t i o n period of f o u r days. We give below a r e c a p i t u l a t i v e t a b l e of the batches used f o r passages.

- 30 Ann. :: 2

Mo.

Origin

bate". 1 1 ' . : 117 12'r

Date . . isolated 27.12.62 4. 3.63 11. 3*63 19. 3.63 30. 3.63 29. 4.63 17. 5-63 19. 5.63 19. 5.63 24. 5.63 16. 6.63 2. 7.63 25.7.63 25. 7.63 25. 7.63 1 1

Number of passages lOth passage lOth passage j 31st passage

Aëdes a f r i c a n u s (16) Eretmapodites (112) Ere tmapcd1te s (205) Aëdes a f r i c a n u s (22) Eretmapodites (131) Aëdes a f r i c a n u s (40) Al'des a f r i c a n u s (I87) Aëdes a f r i c a n u s (134) Aëdes p h y l l o l a b i s - t a r s a l i s (105) Aodes cumminsi (476) Aïdes s t o k e s i (21) Acdes (B) c i r c u m l u t e o l u s (42) Âëdes (A) s t o k e s i (27) Aëdes a f r i c a n u s (159) Aëdes (B) c i r c u m l u t e o l u s (31) Aëdes (A) s t o k e s i (18) Aëdes (A) p h y l l o l a b i s - t a r s a l i s (521)

15: 14 r 174 176 179 lOi 103 19': 204

8th passage 26th passage 7th passage 3 ^ passage 3rd passage 4th passage 3rd passage 2nd passage 3rd passage 3rd passage 2nd passage 4th passage 2nd passage 1st passage 5th passage

20u 207

20G 209 229 . c.r:. ( ,

Aëdes (A) p h y l l o l a b i s - t a r s a l i s (580) 25. 7.63 Pëdes (A) p h y l l o l a b i s - t a r s a l i s (302) Aëdes a f r i c a n u s (46) 1. 9.63 5. 9.63

- 31 -

Annex 2

This work of I s o l a t i o n c a l l s f o r c e r t a i n comments. (a) A large number of the pools inoculated gave r i s e to passages, but these The virulence seems to have decreased and f i n a l l y disappeared. of course, led us to use two-day-old mice f o r outset

passages died out.

This f a c t , a f a m i l i a r one,

our passages as soon as p o s s i b l e , but t h i s could not be done at the of the work. The mice were inoculated by three routes:

i n t r a c e r e b r a l , subcutaneous

and i n t r a p e r i t o n e a l . Moreover, we very much f e l t the need f o r a deep-freezer (-60°C) which

would have made i t easy to keep part of the brains used f o r passages, but unfortunately t h i s was impossible. . It i s well-

I t i s true that freeze-drying could have been c a r r i e d out.

known that the arboviruses w i l l w i t h s t a n d f r e o z o - d r y i n g hut i ^xporionce shov.'od us that there was a f a l l i n antigenic power on the freeze-drying of e a r l y passages.

In the case of a h i g h l y v i r u l e n t s t r a i n , however. Immediate freeze-drying i s p o s s i b l e , but t h i s does not apply to s t r a i n s of low v i r u l e n c e . (b) The mosquitos used f o r the pools were k i l l e d with ether. have had some detrimental effect. centriPossibly the

ether may (c) fuge.

Centrifugation was

c a r r i e d out i n an ordinary, non-refrigerated

So as to Increase the niAmber of pools and shorten the number of passages

at the s t a t i o n , which necessitated the presence o f a large niJmber of mice, we contemplate using next year l i q u i d nitrogen deep-freezers mosquitos to Addis Ababa. f o r transporting one,

This s o l u t i o n c e r t a i n l y seems to be the best

since weekly transport by airplane w^uld be p o s s i b l e .

I s o l a t i o n of a yellow-fever s t r a i n from a colobus monkey One s t r a i n i s o l a t e d from the b r a i n of a yomg colobus monkey was identified by

the R o c k e f e l l e r Foundation, New being a yellow-fever s t r a i n .

York, and the Virus Research I n s t i t u t e , Entebbe, as

- 32 Atinex2

This young animal, aged about three to four weeks, was captured i n Manera f o r e s t cn 25 January 1962 a f t e r i t s mother had been k i l l e d so as to take a blood sample storage

(the s e r o l o g i c a l t e s t s unfortunately could not be c a r r i e d out because of poor of the serum c o l l e c t e d ) .

The young monkey was taken to Addis Ababa on 2? January and fed w i t h a f e e d i n g bottle. Unfortunately i t developed d i a r r h o e a on 29 January and a l l treatment I t died on 3 February without having shown any other morbid symptoms. after

proved u s e l e s s .

I t s b r a i n was removed immediately

death and h i s t o l o g i c a l and b i o l o g i c a l t e s t s had

c a r r i e d out t o e l i m i n a t e any s u s p i c i o n of r a b i e s i n f e c t i o n i n the persons who cared f o r the animal.

Passages were obtained, f i r s t l y on young guinea-pigs, then on 21-day mice, and as from the e i g h t h passage a s t r a i n became s t a b i l i z e d . The f o l l o w i n g t a b l e

g i v e s an o u t l i n e of the main s e r i e s of passages c a r r i e d out t o achieve the f i x a t i o n of t h i s s t r a i n . Comments In other words, we are confronted w i t h a colobus monkey aged three to f o u r weeks which d i e d from a d i a r r h o e a l syndrome e i g h t days a f t e r capture, the c l i n i c a l having l a s t e d three days. A y e l l o w - f e v e r s t r a i n was i s o l a t e d from i t s b r a i n . drawn from t h i s ? One f a c t i s c e r t a i n : SToffered

disease

What conclusions can

t h a t the colobus was a y e l l o w - f e v e r c a r r i e r . The answer would seem to be no, c l i n i c a l l y

Had i t speaking. Is i t u s u a l

from y e l l o w f e v e r ?

This monkey d i e d , i n a l l p r o b a b i l i t y , from I n f a n t i l e g a s t r o - e n t e r i t l s . f o r the v i r u s t o be found i n the b r a i n ? i s known t o occur.

C e r t a i n l y not, although c e r e b r a l involvement

I t i s i n t e r e s t i n g t o report i n t h i s connexion the r e s u l t s of the of the Ammon's horn, which was d i s s e c t e d out both f o r i n o c u reasons.

p a t h o l o g i c a l examination

l a t i o n purposes and f o r h i s t o l o g i c a l

- 33 -

Annex 2

B.D.2389-(ROO IÏO) Monkey - Ammon's horn O r i g i n of tho s t r a i n Isolated

This small fragment of c e r e b r a l matter, which includes the Ammon's horn, does not show any c e l l u l a r inflammator;^ r e a c t i o n : no area o f oedema or haemorrhage. However, i n the inner part of the c e l l u l a r l a y e r of the Ammon's horn, the nerve c e l l s have been g r e a t l y decreased by n e c r o s i s and the remaining c e l l s sor-îonsly a f f e c t e d , with disappearance of the n u c l e i . of the cytoplasm and more or l e s s pronounced pyknosis t h i s part of the c e l l u l a r layer seems very transparent no " c u f f i n g " , no e n c e p h a l i t i c nodules,

Consequently,

as compared with the outer part of the same l a y e r . Mann s t a i n i n g of these sections gave only i n d e c i s i v e r e s u l t s . "Mann Addis Ababa" s t a i n i n g showed elements coloured red i n the cytoplasmic r e s t s but which seemed t o be more a r t e f a c t s than l e s l o n a l Guinea-pig b r a i n lesions. These sections were likewise submitted to Dr Atanasiu, who also reported necrosis o f the Inner part of the c e l l s of the Ammon's horn, altho^agh t h i s could not be i n t e r p r e t e d as a v i r a l l e s i o n . The lesions must be i n the l i v e r ; viraemia? elements.

( f i r s t passage) d i d not show any appreciable inflammatory

At the time of capture t h i s monkey showed a l l the e x t e r n a l signs of normality, i . e . a week before i t s death. I f i t i s considered to have died.of yellow fever T h i s , of

then the disease must have become apparent three days a f t e r capture. course, i s p o s s i b l e but very u n l i k e l y .

Was the animal a healthy c a r r i e r or a monkey which had contracted the disease? I t should be noted that the young monkey was immune as i t was being suckled by the mother (immunity due to maternal antibodies i f the mother h e r s e l f was protected). These are the data on the basis of which a d i s c u s s i o n of t h i s case can be opened. We are faced with the i s o l a t i o n o f a f o r e s t yellow-fever s t r a i n from a monkey b r a i n and we s h a l l retui'n to t h i s problem i n futvire work.

- 34 ANNEX 3 ENTOMOLOGICAL STUDY

Tîiis study w i l l d e a l e s s e n t i a l l y w i t h the work c a r r i e d out a t the Manera Experimental S t a t i o n from November 1962 u n t i l August 1963. We s h a l l consider i n succession ( l ) taxonomic s t u d i e s , (2) e c o l o g i c a l s t u d i e s , and (3) b i o l o g i c a l s t u d i e s . 1, (A) TAXONOMIC STUDIES Figttres f o r a l l mosquitos captured (a) Manera f o r e s t We give here the t o t a l captures d u r i n g the 10 months which t h i s work l a s t e d . During t h i s p e r i o d , 36 751 mosquitos i n a l l were captured, d i s t r i b u t e d over 40 d i f f e r e n t s p e c i e s . The f o l l o w i n g t a b l e g i v e s a breakdown of these captures and the number per month f o r each of them. Furthermore, 15 species were i d e n t i f i e d from a small number o f specimens. These species do not appear i n the t a b l e and are as f o l l o w s : 1 - Culex (Culex) t h e i l e r i 2 - Culex (Culex) decens ,1 ; '

3 - Culex (Culex) u n i v i t t a t u s VAR. naevei 4 - Culex (Culex) ingrami 5 - Culex ( L u t z i a ) t i g r i p e s

6 - Culex (Culiciomya) nebulosus 7 - Culex (Culiciomya) m a c f i e i 8 - Culex (Neoculex) wigglesworthi

9 - Harpagomyia t a e n i a r o s t r i s 10 - Ifranotaenia ornata

- 35"Annex 3

TABLE 1.

FIGURES FOR ALL MOSQUITOS CAPTURED .

Species Aëdes (s) africanus Aëdes (s) luteocephalus Aëdes (s) simpsoni Aëdes (s) aegypti Aëdes (S) ?

November

December

January

February

March

April

May

June

July

August

Totals

1962 25 1

1962 18

1963

1963 31 0

1963 140

1963 73 1 1

1963 604 44 3

1963 571 < ^ 48

1963 579 19 3 0 2 2 990 1 192 2 80 0 0 0 0 117 0 0 5 0 1

1963 479 12 3 0 2 2 2 124

2 512 126 15 0

1 .,....--,2... , Q . ... . .

0 ....... 3 0 0

0 0 0 160 i _ _ _

0 0 1 5 113 2 371 1 81 0 0 0 0 166 0 j 0 16 0 4

0 ..a,..,-,. 609

0 0

0 ,0

1 0 56 0 0 4

0 200

Aëdes (A) t a r s a l i s group) Aëdes (A) phyllolabis ) Aëdes ( A ) cumminsi . i

280 95 0

1 101

18 12

15 651 9 228 2 313 325

-zc Q n j 0 Q Q i o 0 i o 0

109 0

38 1

2 536 2 47 0 0 0 0 126 c 0

843

Aëdes (A) dentatus Aëdes (A) stokesi Aëdes (A) tricholabis Aëdes ( A ) caliginosus — — Aëdes ( A ) marshalli _

| ;

0 4

2 308 89 0 0 0 • 152 196 0 13 8 0 0 0

7 1 n 0 . 0

5 1 I i

9 0

,-—

~—• Aëdes (A) quasiunivittatus Aëdes ( B ) circumluteolus Aëdes ( B ) lineatopennis Aëdes ( B ) albothorax Aëdes ( D ) f u r c i f e r Aëdes (F) pulchrithorax Aëdes ( F ) ingram.i Aëdes (Mucidus) ? sp. n .

' i j .—.—^

1 0

0 0

0 1 0 1 0 0 1 0 0 0 [

1 1

j i " j : ' !

0 l i i i 0 n

0 0

i .

0 0

j i

0 0

152 608

1 ' f

— —

— — — — — — — — — 0 i

0 0

^ 1 i 1 7 1 ;

0 0 ,

0 0

° 0 0 0

2'^ 0

1 i 1

; i

0 0

j 1

i1.

° ,, . 0

2 ^ i '

- 36 Annex 3

TABLE 1.

FIGURES FOR ALL MOSQUITOS CAPTURED (continued)

Species

November

December

January

February

March

April

May

June

July

, August

Totals

1962 Taenlorhynchus a f r i c a n u s Taenlorhynchus uniforrais 3

1962 0

1963 0 0

1963 0 0 0 0 2 730 l4i 0 0 4 0 0 0 0 0 0 0 0 3 050 ' •

1963 0 0 0 0 3 344 18 0 0 3 1 0 1 0 0 0 0 0 3 545

1963 1 0 0 0 2 104 437 302 11 0 0 2, 0 1 0 0 0 0 '

1963 1 0 0 0 1 i

1963 < i 2 0 0 0 1 700 733 117 51 0 0 53 0 0 12 16 0 1 0 ; ^

1963 2 0 0 1 2 944 523 159 26 0 0 107 0 0 17 4 0 0 0 1 8 766 11 2 1

1963 4 0 0 0 023 125 104

13 1 1

1 1

Taenlcrhynchus m e t a l l i c u s Taenlorhynchus _ ——— aureus

0 0 962 685 0 1 6 0 j j 1

0 0 1 249

0 165 176 0 2 i .—-

Eretmapodites Gr. chrysogaster ) Isretmapodltes Gr. quinquevittatus) Genus lEretmapodites Gr. inornatus ) |Culex (genus) iHieobaldla v— iAnopheles 1 ^ 'iAnopheles — IAnopheles ? (A) coustani VAR. tenebrosus (A) Implexus (A) paludis

470 930 348 • :

10 2 4 : 12 19: 1 03L 1 822 4 . • 3

627 0 0 1 0 0 0 0 0

26 1 0 6 0 1 19 16 0 0 2 0

61 3

0 99 0 0 20 8 1

.>T

287

iAnopheles (c) squamosus iAnopheles (M) funestus Anopheles Anopheles L Anopheles Anopheles Anopheles (M) gambiae (M) macullpalpis — (M) m a r s h a l l i (M) p r e t o r i e n s i s (M) n a t a l e n s i s VAR. m u l t l c i n o t u s TOTALS

0 0 0

T73 44 •

—"

1 . ,

0 0 0 0

2 1 -

0 •

[ 0 ]

0 0 2 256

0 0

2 1

0 2 4o6

° 11 057

581

002

412

676

5675

- 37 Annex 3

11 _ Toxorhynchites b r e v i p a l p i s 12 - Toxorhynchites n a l r o b i e n s i s 13 _ .Eretmapodites dracaenae 14 _ Eretmapodites semisimplices

15 _ Eretmapodites grahaml (b) V i l l a g e of Manera During the 10 months of the S'orvey the f o l l o w i n g species were captured i n the v i l l a g e of Manera i t s e l f : Aëdes (Stegomyia) simpsoni (throughout the year) ' 1.. . . .

Eretmapodites chrysogaster (captured i n J u l y and August I963) Uranotaenia ornata Harpagomyia t a e n i a r o s t r i s ) ) throughout the year , . .

Toxorhynchites b r e v i p a l p i s ) (B) D e s c r i p t i o n of a p a r t i c u l a r species

During the numerous day-time hunts c a r r i e d out, a l a r g e number of Aëdes (A) p h y l l o l a b i s were captured. From the eggs l a i d by the captured a d u l t s we

obtained l a r v a e which, i n the f o u r t h stage, showed appreciable d i f f e r e n c e s i n the number of spines on the comb of the syphon and i n i t s l e n g t h , as w e l l as i n the number of f r i n g e d s c a l e s on the comb of the e i g h t h segment. Numerous l a r v a e found i n the marshes and i n holes i n the ground had the same c h a r a c t e r i s t i c s and f o r t h i s reason we regrouped these types of Aëdes p h y l l o l a b i s p r o v i s i o n a l l y i n a group which we designated "manerae". F o l l o w i n g a d e f i n i t i v e i d e n t i f i c a t i o n by Mme Van Someren, t h i s group of a d u l t s has been included i n the t a r s a l i s group.

However, i t should be noted t h a t so f a r , i n the Manera F o r e s t a l l the Aëdes p h y l l o l a b i s l a r v a e c o l l e c t e d , without e x c e p t i o n , show t h i s Important v a r i a t i o n and t h a t we have never found a specimen of the t y p i c a l l a r v a e as d e s c r i b e d i n the 1952 "Hopkins".

-38Annex 3

2. (A)

ECOLOGICAL STUDIES L i s t of captures i n r e l a t i o n to the time when made During the past 10 months, 209 morning hunts were c a r r i e d out, from 9 a.m. to

11 a.m.,

and I78 evening hunts, from 6 p.m.

to 8

p.m.

The d i s t r i b u t i o n of the d i f f e r e n t species was as shown i n Tables 2 and 3. (B) Graphic representation We f e l t that the r e s u l t s would be c l e a r e r i f these tables were presented i n the 3. (A) form of graphs. BIOLOGICAL STUDIES Study of Aëdes (S) africanus ' -

Two f a c t s were recently noted i n regard to the biology o f t h i s species, (a) The number of Aëdes africanus i n r e l a t i o n to temperature In our 6 p.m. to 8 p.m. hunts, c a r r i e d out simultaneously on three

platforms and on the ground, we observed that very small temperature d i f f e r e n c e s may have a considerable e f f e c t on attack by Aëdes a f r i c a n u s . This i s so i n v a r i a b l e , with a few rare exceptions, that i t i s possible to give a rough estimate of the number of Aëdes africanus captured merely by observing the temperature at the beginning of the hunt. This temperature

s e n s i t i v i t y of Aëdes africanus i n the Manera f o r e s t confirms previous observations by Haddov; i n Uganda. We f e l t that i t would be i n t e r e s t i n g to draw up a graph showing the number of Aëdes africanus captured from 6 p.m. temperature measured at 6 p.m. to 8 p.m. i n r e l a t i o n t o the

. The graph based on 30 hunts i s as f o l l o w s .

- 39 -

Annex 3 TABLE 2. RESULTS OF MORNING HUNTS PROM 9 a.m. TO 11 a.m.

November Species Aëdes (s) a f r i c a n u s Aëdes (s) simpsonl Aëdes (s) aegypti Aëdes (s) c h a u s s i e r l Aëdes (S) aedlmorphus (without d i s t i n c t i o n o f t v p p l 'Aëdes (A) s t o k e s i Aëdes (A) Gr. t a r s a l i s p h y l l o l a b i s Aëdes (A) cummensi • Aëdes (A) dentatus Aëdes (A) m a r s h a l l i Aëdes ( A ) q u a s i u n i v i t t a t u s Aëdes (B) c i r c u m l u t e o l u s Theo, Aëdes (B) albothorax Aëdes (F) barnardi Taenlorhynchus (M) a f r i c a n u s Theo Culex (without d i s t i n c t i o n of type) Anopheles ( A ) implexus Theo. Anopheles (M) funestus 'An^rheles (M) gambiae ; .... . ._

a December 1962

January 1963

February 1963

March 1963 6 1 0 0 • 30 5 0 0 0 0

April 1963 0 1 0 0 0 9 55 0 0 X

May 1963 13 3 1 0 0 47 4 155 2 229 0 0 0 122 0 2 0 25 28 0 0 0

June 1963 17 2 0 0 0 79 4 980 2 260 0 0 0 161 0 2 1 49 38 0 0 2

July 1963 29 1 0 0 0 77 2 923 1

August 1963 13

Totals

1962

0 0

2 0

1 0 0 0 375 3 0 0 0 0 0 0 0 0 0 627

3 0 0 0 139 4 0 0 0 0 0 0 0 0 0 137

84 9 1 2

1 0 2 0

0 0 153 0

0 0 704 7

84 ——— 2 082 2 779 2 308 0 152 189 13

0 0 0

0 0 0 0

1 1 I 14 -

401 315 195

147 0 0 0 114 0 1 2 25 97 2

8 415

2 308 1 152 587 13 5

j | •

o ^ 0 0

0 0

0 0 0 0 0 17 1 0 0 0

0 1 0 0 0 5 1 0 0 0

j j

0 0

0 174 3 0 0 0 681

9 1 786 212 2 1 4

46 35 0 0 0

6 0 0 1

1 0 0 0

2 0 0 0

1 1

.^norheles (M) coustarà VAR. t l p i c u s

- 40 Annex 3 TABLE 2. RESULTS OF MORNIIU HUNTS PROM 9 a.m. TO 11 a.m. (continued)

Species Eretmapodites (without d i s t i n c t i o n of t7/De) Eretmapodites Gr. chrysogaster Eretmapodites Gr. quinquevittatus Eretmapodites Gr. inornatus Toxorynchites Tabanidae TOTALS

November 1962 162 0 0 0 0 0 495

December 1962 947 0 0 0 0 0 2 348

January 1963 1 249 0 0 0 0 0 2 256

February 1963 2 666 0 0 0 0 0 2 951

March 1963 3 188 0 0 0 0 81 3 329

April 1963 0 1 965 434 301 1 0 2 774

May 1963 0 1 354 926 345 0 0 9 250

June 1963 0 1 580 731 117 0 0 10 019

July 1963 0 2 748 520 154 0 0 7 842

August 1963 0 1 903 1 113 103 0 0 9 865

Totals

8 212 9 550 3 724 1 020 1 81 51 129

- 41 Annex 3

TABLE 3 .

RESULTS OF EVENIM} HUOTS FROM 6 p.m. TO 8 p.m.

Species

November 1962 25 1 0 0 0 0 39

December 1962

January 19C3

February 1963 28 0 0 0 0 0 0 î î 5

March 19^3 134 0 2 0 0 i 0

April 1963 73 1 0 1 0 0 0

May 1963 591

June 1963 554 47 0 0 0 0 0

July 1963

August 1963

Totals

1Aëdes.(s) a f r i c a n u s 'Aëdes (S) luteocephalus 1 Aëdes (S) simpsonl Aëdes (s) aegypti Aëdes (s) c h a u s s i e r l Aëdes (s) pseudo-africanus Aëdes (S) aedlmorphus (without d i s t i n c t i o n o f tvne^ Aëdes (A) s t o k e s i Aëdes (A) Gr. t a r s a l i s p h y l l o l a b i s Aëdes (A) cumminsi Aëdes (A) dentatus —• ~ -

15 1 2 0 0 0

/ / / 0 0 0

542 19 1 0 0 0 0 3 77 45 0 3 5 0 0 1 0 1

466 12 2 0 0 0 0 5 42

2 429 125 7 1 0 1 53 8 298 527 2 16 56 4 1 5 1 36

44 0 0 0 0 0

0 0 0 1 , 0 0 , — 0 0 0 type) 2

0 1

0 ,.

1 0 0 0 1 0 0 0 0 6

45 307 0 4 26 0 1 1 0 1

133 111 0 0 16 2 0 1 0 2

0 0 0 0 0 1 0 0 0 4

0 0 0 0 0 1 0 0 0 1

0 0 1 0 0 0 0 1 4

0 0 0 0 0 0 0 0 0

64 2 7 8 0 0 2

Aëdes (B) c i r c u m l u t e o l u s Theo. Aëdes (D) f u r c i f e r Aëdes (M) emboensis Aëdes (M) sp. n. Taenlorhynjhus (M) a f r i c a n u s Theo. Taenimhynohus (C) m e t a l l i c u s Culex (wlth->ut d i s t i n c t i o n i f . --

- 42 -

Annex 3 TABLE 3. PESULTS OP EVENING HUNTS FROM 6 p.m. TO 8 p.m. (aontinued)

Species Anopheles (A) implexus Theo. Anopheles (A, n a t a l e n s i s VAR. m u l t i c i n c t u s Anopheles (M) funestus Anopheles (M) gambiae Anopheles Anopheles coustani paludis

November 1962

December 1962 0

January 1963 0 0 0 0 0 0 0 0 0 0 0 0 0

February 1963 2 0 0 0 0 0 64

March 1963 2 0 1 0 0 1 156 0 0 0 2 2 303

April 1963 1 0 0 0 0 0 0 139 3 1 0 0 227

May 1963 33 0 19 16 0 0 0 126 4 3 0 0 1 221

June 1963 0 0 0 16 0 0 0 114 0 0 1 0 997

July 1963 10 0 16 1 0 0 0 196 3 ,

August 1963 64

Totals

115 1 60 41 2 1 r

0 4 0 2 0

0 0 0 0 0 15

1 20 8 0 0 15 120 12

Eretmapodites (without d i s t i n c t i o n of type) Eretmapodites Gr. chrysogaster Eretmapodites Eretmapodites Tabanidae Genus aedomyia TOTALS quinquevittatus inornatus 0 0 0 0 0 80

253 695 22 10

0 0 0 0 0 54

0 0 0 1 0 100

——1 5 0 0 928 1 0 0 866

4^ 2 4 776

FIG. 1 T O T A L MOSQUITOS C A P T U R E D FROM NOVEMBER 1962 TO AUGUST 1963

Rainfall

WHO

4252

FIG. 2 CAPTURES BY SPECIES GENUS: Culex Aedes ( A ) cumminsi

Number of inosquitos Rainfall WHO '+253

FIG. 3 VERTICAL DISTRIBUTION OF SPECIES ( Evening hunts 6 p.m. -8 p.m. ) A. (S)africanus A. (S) luteocephalus A . (D) furcifer G. Eretmapodites

Number of mosquitos *H0 425'»

FIG. 4 HORIZONTAL DISTRIBUTION OF SPECIES ( 30 morning hunts 9 a.m. -11 a.m. from 17.8 to 18.9 1963 ) Manera forest

Eaux chaudes

Aéroport

3500

zone A

I I J i

g 3000 2 500 2000 1500 1000 500 0 zone B A.(A) dentatus zone B A. ( A ) cumminsi zone A A.(A) G. tarsalis zone A Eretmapodites WHO

zone B

WÎS

- 43 Annex "3

(b)

L a r v a l breeding places of Aëdes africanus-••• •

' ^ bi

An i n t e r e s t i n g p o i n t i s the d i f f e r e n c e between the places frequented the a d u l t s and the d i s t r i b u t i o n of the l a r v a l f o c i . In a d d i t i o n to the normal f o r e s t breeding places we detected numerous breeding places i n the few small t r e e s i n the v i l l a g e of Manera, about a kilometre away from the f o r e s t . However, despite numerous hunts organized i n the v i l l a g e from 6 p.m. 8 p.m. not a s i n g l e Aëdes a f r i c a n u s was captured.

S i m i l a r l y , i n the v i l l a g e

of Boga, about two km from the f o r e s t and 300 m higher up, we found Ae'des a f r i c a n u s breeding places i n holes i n the only two trees i n the v i l l a g e . P o s s i b l y we s h a l l arrange evening hunts l a t e r on to see whether a d u l t s : can be captured.

The theory that Aëdes a f r i c a n u s females go a considerable d i s t a n c e outside the f o r e s t to l a y t h e i r eggs and that the a d u l t s from these eggs r e t u r n to the f o r e s t f o r feeding seems to us a strange one, to say the l e a s t . • With the commencement of hunts throughout the 24 hours and w i t h surveilT of the l a r v a l breeding places we s h a l l perhaps be able to f i n d an explp"**^^°" f p r these unusual f a c t s . (B) Study of Aëdes (S) aegypti During numerous i n s p e c t i o n s of l a r v a l breeding pi-^es c a r r i e d out a t Manera we found a l a r g e number of Aëdes aegypti b r e e d i ^ places i n holes i n trees a t the edge of and i n s i d e the f o r e s t , as w e l l s-'5-n o l d earthenware u t e n s i l s i n the v i l l a g e of Manera and i n our camp, i n w a t " taken from the l i d s of p e t r o l drums. Holes i n t r e e s seem ^ o be the most numerous breeding p l a c e s . On the other hand,

throughout the 10 months of morning and evening hunts we captured only two a d u l t s i n the f o r e s t and none i n the v i l l a g e . The almost complete absence of Aëdes aegypti considered zoophilie.

a d u l t s i n the Shushuma v a l l e y encourages the theory t h a t t h i s s p e c i e s ,

to be domestic and a n t h r o p o p h i l i c , i s , on the c o n t r a r y , f o r e s t - d w e l l i n g and

- 44 Annex 3

(c )

Study of the genus Eretrnapori i tes (a) L a r v a l breeding places

. . .

Ihese have important r e p e r ç a s s i o n s on d i f f e r e n t groups found and e x p l a i n t h e i r uneven d i s t r i b u t i o n : .

Chrysogaster group: d r i e d leaves which have f a l l e n t o the ground and form a h o l l o w ; the i n s i d e of f i g s which have f a l l e n to the ground; Quinquevittatus group: Inornatus group: (b) Dracaena; pineapples; (rare).

C o l o c a s i a e s c u l e n t a , hollows formed by leaves

The above a f f o r d s an explanation of the d i s t r i b u t i o n of the v a r i o u s -

species i n r e l a t i o n to r a i n f a l l . 3. SEROLOGICAL STUDIES AT MANERA

During the 1962-63 campaign we c a r r i e d out a s e r o l o g i c a l survey which may w e l l provide v a l u a b l e i n f o r m a t i o n . (A) Human These •

-, Pour hundred and e i g h t serum samples were taken i n the v i l l a g e of Manera. specimens were taJcen from one person per hut and the huts were numbered. Ihe blood samples were taken i n November: the f l i s t f o l l o w - u p was i n A p r i l ,

the second i n J u l y , while there i s to be a t h i r d i n October. J

Thus each subject i s r e g u l a r l y f o l l o w e d up every three months. are not yet a v a i l a b l e .

Ihe r e s u l t s

•ihe aim of the experiment i s to keep track of s e r o l o g i c a l developments i n a v i l l a g e a f f e c t e d by the y e l l o w - f e v e r epidemic a year ago and to d e t e c t the appearance,

r i s e or f a l l of y e l l o w - f e v e r antibody i n p a r t i c u l a r and of arbovirus a n t i b o d i e s i n general. The r e s u l t s of the f i r s t haemagglutination i n h i b i t i o n t e s t s c a r r i e d out show t h a t the sera from nine out of 45 persons have converted, between March and J u l y I963, f r o m n é g a t i v e to p o s i t i v e against yellow f e v e r . In a d d i t i o n , a 40 y e a r - o l d womem was i l l Serological tests

d u r i n g the month of J u l y w i t h a f e b r i l e syndrome w i t h a l b u m i n u r i a .

have shown t h a t t h i s was a mild attack of yellow f e v e r , from which she recovered i n a few d a y s .

FIG. 6 DISTRIBUTION OF DIFFERENT SPECIES IN RELATION TO RAINFALL 3500 U

Total Eretmapodites Er. Gr. chrysogaster Er. Gr. quinquevittatus Ef. Gr. inornatus Rainfall W H O t257

FIG. 7 PERCENTAGE GENUS AEDES AND GENUS ERETMAPODITES' MORNING HUNTS FROM 9 A.M. TO 11 A.M.

Annex 3

A l l t h i s presents evidence t h a t y e l l o w - f e v e r v i r u s i s s t i l l c i r c u l a t i n g i n Manera one and a h a l f years a f t e r the o r i g i n a l outbreak. (B) Animal As we mentioned l a s t year at the previous Expert Meeting, we considered the problem of domestic animals during the 1962-63 campaign. We f e l t that one way of studying t h e i r s e r o l o g i c a l behaviour would be to vaccinate them against yellow fever and note the s e r o l o g i c a l changes o c c u r r i n g i n r e l a t i o n to time. . The animals were d i v i d e d i n t o tvio c a t e g o r i e s : one comprising animàls a l i v e epidemic.

d u r i n g the epidemic p e r i o d , and another comprising animals born a f t e r the The experimental d e t a i l s were as follows.

The domestic animals were bled by j u g u l a r puncture, branded on the outside of the l e f t ear, and then vaccinated (Dakar No. 33 vaccine) i n the median sulcus of the i n s i d e of the r i g h t ear by making s e v e r a l 1-1.5 For c a l v e s , 15-20 cm s c a r i f i c a t i o n s w i t h a v a c c l n o s t y l e .

s c a r i f i c a t i o n s were made through three drops of vaccine, while s c a r i f i c a t i o n s were used w i t h two drops of vaccine. In the

f o r sheep and goats 10-15

Samples were taken on the e i g h t h and f i f t e e n t h day a f t e r v a c c i n a t i o n . case of 55 calves and 14 goats and sheep, three samples were taken.

So f a r the r e s u l t s have not yet reached us ; the t e s t s have been entrusted to the Dakar Pasteur I n s t i t u t e . 4. EXPERIMENTS AT BOGA AND MANERA As reported during the l a s t meeting i n 1962, i t was found t h a t epidemic incidence of the disease i n the v i l l a g e s i n the v a l l e y during the I96I Shushuma v a l l e y epidemic was dependent on t h e i r geographical p o s i t i o n . These f a c t s were confirmed by a more thorough study of these v i l l a g e s . The absence of yellow f e v e r i n c e r t a i n v i l l a g e s , p a r t i c u l a r l y i n Boga, s i t u a t e d two km away from Manera i n a s t r a i g h t l i n e and 8OO ra from the f o r e s t , loosed a problem, s i n c e a l l the c o n d i t i o n s f o r the outbreak of the epidemic were present.

- 46 Annex 3

Why had t h i s v i l l a g e been spared?

The theory t o the e f f e c t t h a t the epidemic

had not y e t a r r i v e d i n the v i l l a g e proved groundless, since the epidemic ended a y e a r ago and the v i l l a g e has remained untouched. I n view of ( l ) the presence of vectors both a t Manera and a t Boga, (2) the presence of s i m i l a r v e g e t a t i o n , (3) the population of the same race, and (4) the i d e n t i c a l h a b i t a t , we f e l t that only c l i m a t i c c o n d i t i o n s could be i n v o l v e d , Manera being s i t u a t e d a t 1200 m and Boga a t 1500 m a l t i t u d e . We e s t a b l i s h e d a m e t e o r o l o g i c a

s t a t i o n and d u r i n g the l a s t 10 months i t has been found t h a t the r a i n f a l l i s s i m i l a r , the humidity i s d i s t i n c t l y l e s s a t Boga than a t Manera (the f i g u r e w i l l be g i v e n later

on), and the minimal temperatures are lower (these f i g u r e s too w i l l be g i v e n l a t e r on) In agreement w i t h the experts of the group, we have commenced experiments w i t h the aim of d e t e c t i n g the p o s s i b l e Influence of c l i m a t i c c o n d i t i o n s on the development of the y e l l o w - f e v e r v i r u s d u r i n g i t s latency i n the v e c t o r mosquito. For t h i s purpose two batches of mosquitos were i n f e c t e d under the same e x p e r i mental c o n d i t i o n s . Immediately a f t e r i n f e c t i o n one of them was taken t o Boga w h i l e

the other was kept a t Manera. This experiment i s now under way and w i l l be completed by the middle of November. Experimental procedure

A batch o f Aëdes simpsonl was bred :from eggs coming from mosquitos captured at Manera. This batch was bred a t the Manera s t a t i o n i n an insectarlum constructed f o r the purpose. The mosquitos were I n f e c t e d w i t h the Couma s t r a i n through the intermediary o f an " a r t i f i c i a l mouse" (see summary), the idea of which was communicated t o us by Dr W i l l i a m s .

- 47 Annex 3

The i n f e c t e d mosquitos were d i v i d e d i n t o two l o t s , one remaining a t iVIanera w h i l e the other was taken t o Boga. The mosquitos w i l l be placed i n cages and f e d on mice, i n c l u d i n g baby mice. l a t t e r , a f t e r they have been bi\.ten, w i l l be kept under o b s e r v a t i o n . w i l l continue u n t i l the death of the mosquitos. I t should be noted that a t Manera we f i r s t of a l l s u c c e s s f u l l y I n f e c t e d mosquitos by means of the " a r t i f i c i a l mouse" and then t r a n s m i t t e d the v i r u s from these mosquitos, a f t e r g r i n d i n g up, t o week-old mice. The

The experiment

- 48 ANNEX 4

TROGRAMME FOR THE 1963-64 CAMPAIGN

We f e e l t h a t before t a c k l i n g the future programme I t Is advisable t o draw up a balance sheet o f the work done so f a r . 1. A s e r o l o g i c a l survey covering 3500 sera has enabled us t o * (a) draw up a map f o r yellow f e v e r and arboviruses i n general covering

two-thirds of E t h i o p i a ; (b) t o r e a l i z e the importance o f c e r t a i n regions because o f t h e i r r i c h n e s s

In arbovirtises; (c) 2. t o e x p l a i n c e r t a i n p a r a d o x i c a l r e s u l t s , such as those a t Assab.

The v i r o l o g i c a l , e n t o m o l o g l j a l and e p i d e m i o l o g i c a l s t u d i e s have enabled us t o : (a) (b) (c) a c c u r a t e l y l o c a l i z e the epidemic zone; dlagnoze yellow f e v e r ; I s o l a t e the v i r u s from man and show the a n t i g e n i c importance o f the

"ouma s t r a i n ; (d) f i n d the man/man v e c t o r In the E t h i o p i a n epidemic and I s o l a t e 15

y e l l o w - f e v e r s t r a i n s from t h i s v e c t o r ; (e) I s o l a t e a s t r a i n from a colobus monkey and demonstrate f o r the f i r s t

time the presence o f y e l l o w - f e v e r v i r u s i n w i l d animals; (f) (g) year; (h) (l) undertake a study o f domestic animals; I s o l a t e two v i r u s s t r a i n s from Eretmapodites,- (so f a r s t i l l unidentified);

draw up a l i s t of v e c t o r fauna (mosquitos) covering the 12 months of the

c a r r y out a f i e l d experiment on the t r a n s m i s s i b i l i t y of the v i r u s i n by a d i s t a n c e of two km and a d i f f e r e n c e i n

two d i f f e r e n t places separated h e i g h t o f 300 m.

- 49 -

Annex 4

To complete the work undertaken i t s t i l l (A)

remains:

TO survey the n o r t h of E t h i o p i a , i . e . the western p a r t of E r i t r e a .

Headquarters should be e s t a b l i s h e d a t Tessene on the Asmara-Khartoum road. The f o l l o w i n g i n v e s t i g a t i o n s could be based on t h i s town: (a) survey of the Tessene r e g i o n i t s e l f along the Gash r i v e r ; this

r e g i o n i s a l l the more i n t e r e s t i n g i n t h a t i t i s opposite the importaint town o f K a s s a l a i n the Sudan; (b) survey o f the area south of the S e t i t r i v e r which enters the Sudan upstream;

w i t h the Tekeza r i v e r (c)

survey o f the area n o r t h of the Borea r i v e r which flows towards

Tokar i n the Sudan. A study o f t h i s r e g i o n would complete the survey of the E t h i o p i a n Sudanese f r o n t i e r t o the n o r t h and, i n f a c t , p r a c t i c a l l y the whole of E t h i o p i a l i a b l e t o be a f f e c t e d by yellow f e v e r . helicopter. Furthermore, we i n t e n d t o study the Wabi Shebel, s t a r t i n g from the A r u s s i s mountain and proceeding towards Imi. because o f p o l i t i c a l c o n d i t i o n s . from bases a t Ghimir and Goba. a l s o be i n v e s t i g a t e d . (B) TO continue work a t the Manera s t a t i o n The past year has been p a r t i c u l a r l y d i f f i c u l t f o r i t was necessary t o c a r r y on a t the same time the work o f f i t t i n g up the s t a t i o n under very d i f f i c u l t c o n d i t i o n s , t h e - t r a i n i n g of l o c a l personnel f o r entomological hunts and, moreover, •the s c i e n t i f i c work, which commenced immediately. This survey has so f a r not been p o s s i b l e This survey would be c a r r i e d out by

I t could a l s o be c a r r i e d out by h e l i c o p t e r The upper v a l l e y of the Canale D o r i a might

. - 50 -

We now have at our d i s p o s a l a properly equipped s t a t i o n with p e r s o n n e l used to the work with which they are entrusted; i s commencing to show worthwhile i n other words, the s t a t i o n

r e s u l t s and during the 1963-64 campaign the

f r u i t of the work c a r r i e d out t h i s year w i l l be harvested. The goal we hope to achieve i s a t r i p l e (a) Entomological Continue and complete the survey i n t h i s f i e l d ; (i) (ii) by s y s t e m a t i c a l l y studying l a r v a l breeding p l a c e s ; by carrying out hunts throughout the 24 hours; this was one.

not p o s s i b l e during the past year and w i l l perhaps enable us to resolve c e r t a i n problems such as, f o r example, that of Aëdes aegypti. (b) Virological We also hope to continue work on the i s o l a t i o n of v i r u s s t r a i n s pooled mosquitos. We s h a l l have deep-freeze f a c i l i t i e s f o r the d i s p a t c h of from

(i)

mosquitos. (ii) We s h a l l also be able to breed s u f f i c i e n t mice to provide

an adeqviate number of l i t t e r s f o r i n o c u l a t i o n s . With the means at our d i s p o s a l we hope to double or t r e b l e the number of pools made t h i s year. (c) Study of the v i r u s r e s e r v o i r I h l s item was included i n the

I962-63 programme.

I t was not p o s s i b l e

to implement i t f o r reasons of time, personnel, equipment and knowledge. At present we have the f o l l o w i n g at our d i s p o s a l : (i) p r e c i s e information from Entebbe and Nairobi concerning the

capture of r a t s and b i r d s ;

- 51 Annex 4,

(li)

p o s s i b i l i t y of i d e n t i f i c a t i o n thanks to the kindness of

Mr Guggisberg of N a i r o b i ; (ill) nets f o r the capture of b i r d s , thanks t o the kindness of

Dr Dovms (we r e c e i v e d them on 20 September l a s t ) ; (iv) an a i r r i f l e w i t h an anaesthetic charge f o r c a p t u r i n g

monkeys. A l l t h i s m a t e r i a l w i l l enable us t o : (1) Set 300 cage traps f o r r a t s i n the Manera f o r e s t and capture This w i l l make p o s s i b l e a

an average of one to two r a t s d a i l y .

v a l i d s e r o l o g i c a l I n v e s t i g a t i o n of an adequate number of rodents. A f t e r t a k i n g the blood sample the animals w i l l be ringed and set (2) free. I t w i l l be p o s s i b l e t o capture b i r d s w i t h the n e t s . We

intend to c a r r y out a s e r o l o g i c a l study using them. (3) F i n a l l y , thanks t o the r i f l e w i t h an anaesthetic p r o j e c t i l e

we hope t o be able t o organize a systematic hunt of "gorezas" and baboons. I s o l a t i o n of a v i r u s s t r a i n from the b r a i n of one of them encourages us to continue along these l i n e s . Our plan i s to

place the anaesthetized monkeys i n sacks and take them t o the camp. An autopsy w i l l be c a r r i e d out on a c e r t a i n number of

them (removal of the b r a i n and l i v e r f o r i n o c u l a t i o n purposes). The specimens w i l l be placed i n tubes, and transported to Addis Ababa i n l i q u i d n i t r o g e n deep-freezers. In some cases, only a blood specimen w i l l be taken and the animals w i l l then be r i n g e d and s e t f r e e . (4) F i n a l l y , we f e e l t h a t the stixiy of l i z a r d s i s of i n t e r e s t .

We i n t e n d to undertake t h i s i n v e s t i g a t i o n s i n c e we have observed t h a t these r e p t i l e s are b i t t e n by mosquitos.

- 52 Annex 4

Furthermore, we need h a r d l y say t h a t we s h a l l continue our i n v e s t i g a t i o n s i n Manera v i l l a g e . The s e r o l o g i c a l r e s u l t s obtained during the 1962-63 campaign w i l l

enable us to d e t e c t immunological changes i n the sera of subjects there and t o observe the appearance of a n t i b o d i e s corresponding to c e r t a i n types of a r b o v i r u s . be able t o attempt t h e i r i s o l a t i o n d u r i n g the coming year. F i n a l l y , the r e s u l t s given by the survey of domestic animals w i l l serve as a guide f o r a more thorough study i n t h i s field. We shall

Such i s the programme f o r the 1963-64 campaign and v/e f e e l t h a t i t can be c a r r i e d out. yellow fever. I t w i l l round o f f and terminate the I n i t i a l stage of t h i s work on

- 53 ANNEX 5

VACCINE STUDIES Study of the conversion rate In Ethiopian subjects vaccinated with Dakar, Yaba, and Bogota vaccine

The experiment comprised two

stages.

The f i r s t stage was c a r r i e d out during November I961 and the second during J u l y 1962. The f i r s t s e r i e s of vaccinations was performed at the Wonji Sugar Works, while the second s e r i e s of vaccinées included the Ethiopian s t a f f of the French Embassy and the s t a f f of the Pasteur I n s t i t u t e of E t h i o p i a . Out of a t o t a l of nearly 500 persons vaccinated and from whom a blood sample was taken on the day of v a c c i n a t i o n , we were able to c a r r y out follow-up t e s t s on

only l40 persons. 1. (A) ^ Method of using the d i f f e r e n t vaccines Dakar; (batches M32-37 i n November I96I and batch M67 i n J u l y 1962) It

The Dakar vaccine was employed i n accordance with the c l a s s i c procedure. temperatures.

v/as f i r s t kept f o r a week at normal

Three drops of vaccine were placed on the arm and three scratches made through each drop. (B) Yaba; .(batch M34/59) The Yaba vaccine was used i n the same way as the Dakar vaccine. constantly i n the r e f r i g e r a t o r and vised immediately. (C) Bogota ; (batch 283) Two drops were I t was kept

Ihe vaccine was kept i n the r e f r i g e r a t o r u n t i l the time of use.

used, each drop being the amount of vaccine required to covet^ each face of the vacclnostyle.

- 54 Annex 5 (B) A f t e r use, each of the vaccines was t i t r a t e d One i.e. ampoule from the same batches, kept under the same c o n d i t i o n s as the vaccine,

l e f t on the laboratory bench, was t e s t e d . The f i r s t t i t r a t i o n , c a r r i e d out on 28 November I96I, gave the f o l l o w i n g r e s u l t s : Dakar (batches r452-37) Bogota (batch 283) 10 4 lO"

4 40 Yaba (batch r434/59) ' 10~ *

Second t i t r a t i o n , on 28 J u l y 1962: Dakar (batches M32/37) lO"''^*^^ -4 00 10 * 10'^*°° • With the Dakar v a c c i n e ,

Bogota (batch 283) Yaba (batch ?) Vaccinal reactions

No r e a c t i o n was noted to the Yaba and Bogota" vaccines.

there was a f e v e r i s h r e a c t i o n w i t h headache on the t h i r d day, but only i n 10 c a s e s . S e r o l o g i c a l study ( c a r r i e d out at the R o c k e f e l l e r Foundation, New York, by Dr C a s a l s and Dr T h e i l e r ) A haemagglutination i n h i b i t i o n t e s t was c a r r i e d out s y s t e m a t i c a l l y w i t h a l l s e r a , using as s t r a i n s : yellow f e v e r . West N i l e , Z l k a , Wesselsbron, SAE 336, Tahnya, Bunyamwera, I l e s h a and Bwamba. (a) A l l the p o s t - v a c c i n a l s e r a were t e s t e d , a f t e r d i l u t i o n to I/20 and l/40, l i s t e d above. t e s t e d i n the d i l u t i o n range 1/20 to 1/1280, as w e l l as against Chikungunya,

e i g h t l i n i t s of the antigens

Every p o s i t i v e serum was the p r e - v a c c i n a l serum.

- 55 Annex 5

(b)

A l l negative post-vaceinal sera as w e l l as t h e i r pre-vaccinal homologues were re-

tested against yellow-fever, Zika and West N i l e antigens by the mixture d i l u t i o n method with the aim of detecting any conversion at a t i t r e below 1/20 t i n a t i o n Inhibition test. (c) (d) (e) The s e r o l o g i c a l t e s t s were used to check 4 l sera. To serve as an i n d i c a t i o n , these sera were a l l tested f o r Group A and Bunyamwera. Only yellow fever was taken i n t o consideration i n the r e s u l t s . i n the normal haemagglu-

. 56 GRAPHICAL REPRESENTATION OP THE RESULTS OBTAINED BY NEW YORK

Graph 1 shows the percentage of sera converted

as ordinates and the p o s i t i v i t y

t i t r e s as abscissae, f o r the d i f f e r e n t vaccines. 2. We also f e l t i t would be i n t e r e s t i n g to give at the same time the h a e m a g g l u t i n a t i o n

i n h i b i t i o n and sero-protection r e s u l t s , expressing the o v e r - a l l r e s u l t s obtained as abscissae and the number of cases as ordinates (Graph 2 ) .

FIG. 1 P E R C E N T A G E S O F POSITIVITY O F HI A N D SP

DAKAR

Number of persons v a c c i n a t e d

Number of negative p o s t - v a c c i n a t i o n sera B Number of p o s t - v o c c i n o t i o n converted sera C

B C 100 %

VTO

1/it

i/w

'/»

i/w

1/=

•/«

1/1»

YABA

1/X)

1/x

i/«o

\lK

t/»

t/m

I'M

BOGOTA

80 ?^

1/30

1/»

1*)

Vî»

V'MO

WHO

4250

FIG. 2 COMPARISON B E T W E E N R E S U L T S O B T A I N E D WITH HI A N D SP T E S T S

Key facts

Organisation World Health Organization (WHO)

Document type Technical Documents

Adoption date December 31, 1964

Source World Health Organization