Susceptibility of West African Dwarf Sheep to the Indigenous Wesselsbron Virus

Br. vel.}. ( 1980 ), 136,5 7

SUSCEPTIBILITY OF WEST AFRICAN DWARF SHEEP TO THE INDIGENOUS WESSELSBRON VIRUS BY

A. H. FAGBAMI

Virw Research Laboratory, Universit.y of1badan, Jbadan, Nigeria

SUMMARY

West African Dwarf sheep infected with Wesselsbron virus developed clinical disease characterized by fever anorexia, mild leucopenia and abortion. All inoculated animals developed viraemia lasting three to four days. Haemmagglutination inhibiting and neutralizing antibodies to Wesselsbron and related Aavi\'iruses were detected in all infected animals.

INTRODUCTION

Wesselsbron virus, a Aavivirus, has been responsible for severe disease outbreaks in sheep in South Africa (Weiss, Haig & Alexander, 1956: Belonje, C. W. A., 1957, unpublished data). Clinical infection in sheep is characterized by high fever and severe leucopenia in adults, abortion in pregnant ewes and high mortality in lambs. Wcsselsbron virus was first isolated in Nigeria from a camel during a routine virus surveil lance in Northern Nigeria in 1968 ( Kempel al., 1973). Although immunity surveys have shown that antibodies to Wesselsbron virus and other Aaviviruses are prevalent in domestic an imal sera (Fagbami, A. H ., unpublished data), no clinical ep isode of Wcssclsbron disease has been described in Nigeria. In the present study, West Af'rican Dwarf sheep were infected with Wesselsbron virus in order to determine the pathogenicity of rhe virus for this breed .

MATERIALS AND METHODS

Viruses The Nigerian isolate of Wesselsbron virus I b-AN 31956 isolated in 1968 from the blood of a camel was used in the present studies. It had undergone four intracerebral (i.e.) passages in mouse brain . Ten per cent infected suckling mouse .brain suspension prepared in Eagle's minimal essential medium (MEM) and centrifuged at 10 000 rev./min served as stock virus. Titre of stock virus was 8 ·5 1og 10 LD 5 0 /0·02 mi. Other Aaviviruses used in serological tests are shown in Table I.

58

BRITISH VETERINARY JOURNAL, 136, I TABLE I VIRUSES USED IN HAEMAGGLUTINATION INHIBITION AND NEUTRALIZATION TESTS

Virus

Passages level

Wesselsbron Ib-AN 31956 West Nile !b-AN 4067 Yellow fever Jb- H 43913 Dengue t)•pc I Ib - H 28328 Dakar bat I b-An 8646

4th

5th lOth 64th

1/aemngglutillalicmlitre 128 256 512 32

5th

Experimental procedure Four seronegative adult West African Dwarf sheep were infected with Wesselsbron virus. Two were infected subcutaneously (s .c.) with 5 x 105 suckling mouse intracerebral (SMIC) LD 50 . Two others, a ram and a pregnant ewe, were inoculated intravenously (i .v.) with 5 x !05 SMIC LD 50 of the virus . The control animal received 2 ml of 10% normal suckling mouse brain in MEM. Infected animals were examined daily for signs of clinical disease and daily rectal temperatures were taken . Blood samples were obtained for viraemia and total leucocyte counts. Sera were obtained on days 0, 8, 23 and 90 days post inoculation (p.i.) and tested by haemagglutination inhibition (HI) and neutralization tests for antibodies against Wesselsbron and other related Aaviviruses. Viraemia Serial blood samples were titrated and screened by i.e. inoculation of two to fourday-old suckling mice. LD 50 end points were calculated by the method of Reed &: Muench (1938). Serology Neutraliz.ation tests. Tests for neutralizing (N) antibody in infected animal sera were carried out in suckling mice using the constant virus -constanr serum technique . 0· I ml of undiluted unactivated serum was added to 0· I ml of virus suspension previously calculated to contain 100 LD 50 of virus . Virus-serum mixtures were incubated at 37°C for one hour and then inoculated i.e. or intraperitoneally (i.p.) into suckling mice. Actual challenge dose ranged from 40 t:o 120 LD 50 . Survival ratios of 6/6, 5/6 and 5/5 were considered positive, 4/5 and 4/6 partial positive and others negative. H aemagglutination-inhibition test. Wesselsbron (WSL), dengue type I (DEN -I), yellow fever (YF) and West Nile (WN) virus antigens were prepared by sucrose acewne extraction of infected suckling mouse brains as described by Clarke &: Casals ( 1958). HI tests were performed on kaolin-treated sera according to the method of Clarke&: Casals (I 958) adapted to micro titre plates. RESULTS

Clinical response All four infected sheep developed clinical disease characterized by high fever, anorexia, mild leucopenia and abortion. In animals inoculated i.v., a febrile response

WESSELSBRON VIRUS IN SHEEP

59

6--Alnlravenaus e--einoculo1ion I

I Subcutaneous

tr---6 inoculation

o-- ---o Control 42

41

p 0

s:

"'~

0"

"'0.E

2! 0

u

"'

0::

I

I

2

3456789

I

I

I

I

I

I

Days post i noculo t ion

Fig. I. Rect a l te mp e ratures o fWest Afri ca n dwarf sh eep infected with Wcssclsbron virus.

was observed 48 h p.i . which lasted six days (Fig. 1). Peak rectal temperatures of 41 ·5°C and 41· 7°C were obtained on day 5 p.i . in the two i.v.-inoculated sheep . A slight depression of the total leucocyte count (4000 to 5600/mml) was observed. There was loss of appetite at the onset of fever, although this improved gradually on day 4 p.i. On day 19 p .i. abortion occurred in the pregnant ewe. Both sheep inoculated s.c. developed fever from day 3 to day 7 p .i. with peak of febrile response on day 5 or 6 p .i. A depression of leucocyte count (3300 to 5000 mml) was observed on days 3 and 4 p.i .

Pathology No gross pathological changes were found m the liver and brain of the aborted fo etus or an infected sheep killed on day 7 p.i . Viraemia Table I I shows the pattern of viraemia in the four sheep infected with Wesselsbron virus . Virus was detected in the blood ofi.v.-infected sheep within 24 h after infection; in sheep inoculated s.c., viraemia was detected 48 h p.i. There was no marked difference in the levels of viraemia . Antibody conversions The antibody response of sheep infected with Wesselsbron virus is shown in Table Ill. All inoculated animals developed HI antibodies to Wesselsbron and other

BRITISH VETERINARY JOURNAL, I36 , I

60

TABLE II VI RAEM lA PATTERN IN WEST AFRICAN DWARF SHEEP INFECTED WITH WESSELSBRON VIRUS

Days post inoculation Titre in log 10 Animal

Route of inoculation

2 3 4 5

Intravenous I nrravenous Subcutaneous Subcutaneous Comrol

2

2·5 26

+ +

J ..1

I6

}

30 2·8 2·5 28

1

2·5 2·5 25 2·4

TABLE III HAEMAGGLUTINATION

INHIBITIO N TEST ON CONVALESCENT SERA COLLECTED WESSELSBRON-1 N FECTED S HEEl'

FROM

Day of samjJie

WSL

W.l\1

l'F

DEN-I

0 8 23 90

< 10 808 640 80

<10 20 160 40

<10 40 160 80

<10 40 160 40

2

0 8 23 90

<10 80 640 40

<10 40 160 20

<10 40 80 10

<10 40 80 40

3

0

8 23 90

< 10 80 320 NO

< 10 20 80 NO

<10 20 80 NO

<10 20 40 NO

0 8 23 90

<10 40 160 NO

<10 10 40 NO

<10 20 40 NO

< 10 10 40 NO

Animal

4

\-\'SL Wesselsbron, WN West Nile, YF Yellow fever and DEN-I dengue t)'pe I viruses. NO nor done.

Aavivirus antigens used in the tests. HI antibody to Wesselsbron virus antigen was at least four-fold higher than to other Aavivirus antigens. Of the four serial serum samp les obtained from each infected sheep, the highest level of HI antibody was found in convalescent sera obtained on day 23 p .i. Low levels of HI antibody were still present in sera of these animals 90 days p.i. The results of screening neutralization tests performed on conva lescent sera are shown in Table IV . Neutralizing antibody to Wesselsbron and other Aaviviruses were found in sera of all infected animals 23 days p.i.

WESSELSBRON VIRUS IN SHEEP

6I

TABLE IV NEUTRALIZATION TESTS PERFORMED ON CONVALESCENT SERA COLLECTED ON DAY 23

Animal

WSL

YF

DEN -1"

DB

I 2

+ + + +

+ + + +

+ + + +

+

3

4

±

+ +

Control

+ positive,± partial positive,- negative. • Inoculation by intracerebral route. WSL Wesselsbron, YF yellow fever, DEN-I dengue type I and DB Dakar bat viruses.

DISCUSSION

The present study on the transmission of Wesselsbron virus showed that although the clinical disease has not been previously described in Nigeria the virus is pathogenic for the West African dwarf sheep. The short incubation period characteristic of natural infections (Weiss et al., 1956) was obsel\'ed in this study. Although all infected animals circulated Wesselsbron virus a low grade viraemia was detected during the febrile period of the disease . It has been shown by Tomori ( 1979) that intracerebral mouse passages could affect the level of viraemia obtained during experimental transmission . It is possible that previous passages of this virus in suckling mouse brain accounted for the low level of viraemia detected in the blood of infected sheep . Although there is serological evidence that Wesselsbron virus infection is not uncommon in Nigeria (Fagbami, A. H., unpublished data), the virus has not been encountered since the original isolation was made from the northern parts of the country in 1968 (Kemp et al., 1973). The lack of further isolations of the virus and the absence of severe natural disease in domestic animals may be due to the low level of virus surveillance in sheep and other domestic animals. The role of pre-infection Aavivirus antibodies in the modification of disease by related viruses is not clear. Henderson et al. (1970) showed experimentally that such amibodies may modif)' yellow fever infection in monkeys. However, Grossberg & Scherer ( 1959 and Fagbami & Fabiyi (1976) showed that pre-infection Aavivirus antibodies did not modify natural dengue type I infection in man. It is possible that the high prevelance of flavivirus antibodies in the West African dwarf sheep might limit the activity ofWesselsbron virus in Nigeria. REFERENCES

D. H. & CASALS,J. ( 1958). Americanjournal o)Tropical Medicine and Hygiene 7, 561 . of Tropical Medicine and Hygiene 79, 226. GROSSBERG. S. E. & SCHERER. W. F. (1959). Americanjouma/ of Hygiene 69, 60. HENDERSON. B. E., CHESHIRE. P. P., KIRYA . G. B. & LULE. M. (1970). American journal of Tropica/MedicineandHygiene 19, I 10. KEMP, G. E., CAUSEY. 0 . R., MOORE. D. L. & O'CONNOR. E. H. (1973). American journal of Veterinary Research 34, 707. CLARKE,

FAGBAMI, A. H. & FABIYI, A. ( 1976)journa/

3

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BRITISH VETERINARY JOURNAL, 136, I

REED, L. J. &: MUENCH, H. ( 1938). American journal of Tropical Medicine and H)•giene 27, 493. 0. (1979). Research in Veterinary Science. In press. WEISS, K. E., HAIG, 0. A.&: ALEXANDER, R. A . (1956) . Onderstepoortjoumal ojVeterinmy Research 27, 183.

TOMORI,

(Accepted for publication 5 july 1979)