a/G~4~5r5- VI. African Horsesickness Proc. 2nd...
a/G~4~5r5-
VI. African Horsesickness
Proc. 2nd int. Conf. Equine Infectious Diseases, Paris 1969, pp. 202-206
(Karger, Basel/München/New York 1970)
Vaccination Against African Horsesickness in
Tropical Africa : Evaluation of an Inactivated Vaccine
P. BOURDIN, J. MONNIER-CAMBON, M. R~OCHE and A. LAURENT
Institute of Breeding and Veterinary Medicine in Tropical Countries,
Alfort (France)
African horsesickness (AHS) has been known to exist in Senegal and the
Sudan since the 19th Century. The disease, especially during the rainy season,
was at that time responsible for reducing the strength of horses in the expe-
ditionary corps. Several authors reporting in that era described the disease
and identified it with a variety of names which included typhoid fever,
pernicious fever and typhomalaria. In 1925 TEPPAZ [14] reported two epizo-
otics in Saint Louis and Dakar. MORNET [12] pointed out that Senegal was
ravaged by AHS nearly every year. The intensity of ,the disease varied with the
rhythm of importation of horses from France or North Africa. Central
African countries have not been spared for C~RASSON [3] mentions the
occurrence of AHS in the Congo at Brazzaville. DOUTRE and LECLERCQ [4]
described an epizootic in Tchad at Fort Lamy due to infection of horses by
the type 9 virus and MAURICE and PROVOST [8] found serological evidence that
type 9 AHS virus was not limited in occurrence to the region around Fort
Lamy.
In 1955 our laboratory at Dakar prepared a live modified avirulent
vaccine according to the techniques described by ALEXANDER, NEITZ and
Du TOIT [Il. This vaccine was administered mainly to imported horses. Its
use reduced the incidence of the disease to a few isaolated cases in horses that
were newly imported or in local cross-bred animals.
With the occurrence of the widespread outbreak of AHS in North Africa
in 1966 and its subsequent spread into Spain, authorities became concerned
that the disease might spread into other European countries. Several French
laboratories began to study the problem of control of the disease. At Fort
Lamy, PROVOST [7] reported on hemagglutination and hemagglutination-
inhibition tests. In Dakar a supply of live modified monovalent vaccine was
BOURDIN, MONNIER-CAMBON, RI~CHE, LAURENT: Vaccination Against . . .
203
pro&& on monkey kidney ce11 lines and an inactivated monovalent vaccine
was developed from this system. We have described the results of use of this
latter vaccine [2] and will here report the final results of vaccination obtained
with horses imported from France.
The virus, furnished by the Razi Institute, is the neurotropic S2 strain
which has been modified by 102 passages in mouse brains. It is propagated on
the MS [6] line of monkey kidney ce& [9, 131 from which virus suspensions
of 10’ to 107.5rcID,,/ml are obtained. The stock virus is stored as lyophilized
virus suspension diluted 1:30 with a lactose-peptone-tris buffer at -20°C.
The MS ce11 line was grown in an Earle’s medium supplemented with 2.5%
glucose, 0.5% lactalbumin hydrolysate, 0.01% yeast extract and 10% calf
serum. The medium was enriched by addition of glutamine (0.1 g/l), glutamic
acid (0.1 g/l), methionine (0.15 g/l), biotin (0.001 g/l), folie acid (0.001 g/l)
and arginine hydrochloride (0.04 g/l). Sodium bicarbonate 0.15% was also
added.
The virus was propagated by inoculating cells in suspension in Roux
bottles. Thus, 100 ml amounts of a ce11 suspension containing 2 x 105 cells/ml
were inoculated and incubated in a stationary manner. The ce11 layer forms in
about 30 hours, CPE is apparent usually in 48 h and 90 to 100% of the cells
are involved by 72 h. The virus is harvested at this time, centrifuged under
refrigeration and stored at 4°C. The virus titer is regularly found to be 107*1 to
107*5 in such preparations. The virus is inactivated at 37°C by addition of
1:4000 formalin and constant shaking for 48 h. The formalinized virus is then
chilled to 0°C and 0.2 g/l of betapropiolactone is added. The vaccine is held
at 0°C with frequent shaking for 24 h, the pH is adjusted to 7.0 with bicar-
bonate buffer and the completed vaccine is stored at 4°C. Two types of
adjuvant were used. The first was 1% aluminium gel and the second a minera1
oil-Arlacel preparation. The vaccines that were administered to horses
contained equal amounts of adjuvant and the inactivated virus suspension.
A serum neutralization test employing constant serum increments and
varying virus concentrations was used to assess the response to vaccination.
The results were recorded as neutralization indices expressed as loq,, values.
Serums were inactivated at 56°C for 30 min. After addition of virus, the serum-
virus mixtures incubated at 37” C for 1 h, were then used to inoculate MS cells
in suspension in tubes. The tubes were incubated for 48 h in stationary racks,
the medium was changed and the tubes were then rolled. The results were
read at 4 and 9 days after preparation of the tests,,
Horses were challenged with a mouse brain passed virulent strain of
virus from Morocco that was supplied to us by the Pasteur Institute. This virus
VI. African Horsesickness
204
was given to horses intravenously. The challenge dose was 2 ml of a 1:5
dilution of infected mouse brain suspension supernatant.
Donkeys that were vaccinated with inactivated vaccines developed
detectable neutralizing antibodies by the 10th post vaccination day. The
neutralization indices reached a plateau by the 20th day and then declined
until, by the 40th day, no neutralizing activity was present in the serums.
Administration of a booster dose of vaccine on the 50th day resulted in a
rapid increase in antibodies. The titer reached its maximum on the 8th day
after the booster dose and persisted for at least 100 days [Z].
Six horses imported from France were used to test the two adjuvanted
preparations. Three were giventhe aluminium gel and three the oily adjuvanted
vaccine. No untoward reactions were observed in these animals. The develop-
ment of the neutralization index in horses is illustrated in figure 1. The animals
were given 5 ml of inactivated vaccine as a primary dose. With either adjuvant
antibody was found from the 8th day onward, the maximum titers were
Neutralization index
5
3 -
-
L
2-
l-
Time in months
0
i
;
3
1:
Injection A
0
Fig. 1. The development of serum neutralizing antibodies (neutralization indices,
log*O) of horses vaccinated with an inactived African horsesickness virus vaccine, (A =
booster injection; B = challenge with virulent virus).
BOURDIN, M ONNIER-CAMBON, RI~CHE, LAURENT: Vaccination Against . . .
205
reached on the 15th day and the titers decreased progressively until the 30th
day. The administration of a booster dose resulted in a rapid response with
a maximum titer value being detected from the 13th to 20th day after vacci-
nation. These titers persisted for 12 months, decreasing thereafter.
The first challenge was done in the Spring, on the 45th post vaccination
day, for 2 animals. It produced no disease. The second challenge of 2 horses
was carried out 8 months after the first vaccination and after the rainy season
with the same results. The third group of 2 horses was challenged at 15
months during the hot season. No detectable reaction to challenge occurred.
The latter two animals were kept in the open for 3 months in the period
between vaccination and challenge and it should be pointed out that they
could possibly have been exposed to the virus by mosquitoes which were
abundant at the time.
MIRCHAMSY
and TASLIMI [9] have shown that an inactivated monovalent
AHS vaccine prepared from a neurotropic strain of type 9 virus which was
grown in monkey kidney cells protects horses for at least 6 months if two
injections, administered 4 weeks apart, are given. We were unable, due to the
small number of horses available for our test, to include a control animal, but
our results suggest that protection following administration of our vaccine
lasts for a year. Horses vaccinated with our vaccine resisted severe challenge
on one hand and exposure during the rainy season on the other.
References
1.
ALEXANDER, R. A.; NEITZ, N. 0. and DUTOIT, P. J.: Horsesickness immunization
of horses and mules in the field during the season 1934-1935 with a description of the
technique of preparation of polyvalent mouse neurotropic vaccine. Onderstepoort
J. 7: 17-30 (1936).
2.
BOURDIN, P. et MONNIER-CAMBON, J.: Note préliminaire sur l’emploi d’un vaccin
inactivé contre la peste équine. Bull. Acad. vét. Fr. 40: 187-191 (1967).
3.
CURASSON, G.: Traité de pathologie exotique vétérinaire et comparée, 2me éd.,
vol. 1, pp. 170-207 (Vigot, 1942).
4.
DOUTRE, M. et LECLERCQ, A.: Existence du type T9 du virus de la peste équine au
Tchad. Rev. Elev. 15: 241-245 (1962).
5.
GILBERT, Y.: Rapport sur le fonctionnement pour l’année 1955. Laboratoire fédéral
de I’Elevage de Dakar, 67-70 (1955).
6.
KANDA, Y. and M ELNICK, J. L.: In vitro differentiation of virulent and attenuated
polioviruses by their growth characteristics on MS cells. J. exp. Med. 109: 9-24 (1959).
7.
M AURICE, Y. et PROVOST, A.: Les réactions d’hémagglutination et d’inhibition de
l’hémagglutination avec le virus de la peste équine. Limites de leur interprétation.
Rev. Elev. 19: 439-450 (1966).
VI. African Horsesickness
206
8. MAURICE, Y. et PROVOST, A. : La peste équine à type 9 en Afrique centrale. Enquête
sérologique. Rev. Elev. 20: 5-20 (1967).
9. MIRCHAMSY, H. and TASLIMI, H.: Visualization of African horsesickness virus
by the fluorescent antibody technique. Immunol. 7: 213-218 (1964).
10. MIRCHAMSY, H. and TANMI, H.: Attempts to vaccinate foals with living tissue
culture-adapted horsesickness virus. Bull. Off. Int. Epiz. 62: 91 l-921 (1964).
II. MICHAMSY, H. and TASLIMI, H.: Inactivated African horsesickness cell culture
vaccine. Immunol. 14: 81-88 (1968).
12. MORNET, P.: Sur une évolution atypique de la peste équine particulière à 1’A.O.F.
Rev. Elev. 3: 101-103 (1949).
13. OZAWA, Y. and HAZRATI, A.: Growth of African horsesickness live virus tissue
culture vaccine. Amer. J. vet. Res. 26: 154 (1965).
14. TEPPAZ, L.: Contribution à l’étude de la typho-malaria du cheval (horsesickness)
au Sénégal (Editions Médicales, Paris 1925).
Authors’ address: Dr. P. BOURDIN; Dr. J. MONNIER-CAMBON; Dr. M. RI~CHE and
Dr. A. LAURENT, Laboratoire National de 1’Élevage et de Recherches Vétérinaires, B. P.
2057, Dakar (Senegal).
VI. African Horsesickness
Proc. 2nd int. Conf. Equine Infectious Diseases, Paris 1969, pp. 202-206
(Karger, Basel/München/New York 1970)
Vaccination Against African Horsesickness in
Tropical Africa : Evaluation of an Inactivated Vaccine
P. BOURDIN, J. MONNIER-CAMBON, M. R~OCHE and A. LAURENT
Institute of Breeding and Veterinary Medicine in Tropical Countries,
Alfort (France)
African horsesickness (AHS) has been known to exist in Senegal and the
Sudan since the 19th Century. The disease, especially during the rainy season,
was at that time responsible for reducing the strength of horses in the expe-
ditionary corps. Several authors reporting in that era described the disease
and identified it with a variety of names which included typhoid fever,
pernicious fever and typhomalaria. In 1925 TEPPAZ [14] reported two epizo-
otics in Saint Louis and Dakar. MORNET [12] pointed out that Senegal was
ravaged by AHS nearly every year. The intensity of ,the disease varied with the
rhythm of importation of horses from France or North Africa. Central
African countries have not been spared for C~RASSON [3] mentions the
occurrence of AHS in the Congo at Brazzaville. DOUTRE and LECLERCQ [4]
described an epizootic in Tchad at Fort Lamy due to infection of horses by
the type 9 virus and MAURICE and PROVOST [8] found serological evidence that
type 9 AHS virus was not limited in occurrence to the region around Fort
Lamy.
In 1955 our laboratory at Dakar prepared a live modified avirulent
vaccine according to the techniques described by ALEXANDER, NEITZ and
Du TOIT [Il. This vaccine was administered mainly to imported horses. Its
use reduced the incidence of the disease to a few isaolated cases in horses that
were newly imported or in local cross-bred animals.
With the occurrence of the widespread outbreak of AHS in North Africa
in 1966 and its subsequent spread into Spain, authorities became concerned
that the disease might spread into other European countries. Several French
laboratories began to study the problem of control of the disease. At Fort
Lamy, PROVOST [7] reported on hemagglutination and hemagglutination-
inhibition tests. In Dakar a supply of live modified monovalent vaccine was
BOURDIN, MONNIER-CAMBON, RI~CHE, LAURENT: Vaccination Against . . .
203
pro&& on monkey kidney ce11 lines and an inactivated monovalent vaccine
was developed from this system. We have described the results of use of this
latter vaccine [2] and will here report the final results of vaccination obtained
with horses imported from France.
The virus, furnished by the Razi Institute, is the neurotropic S2 strain
which has been modified by 102 passages in mouse brains. It is propagated on
the MS [6] line of monkey kidney ce& [9, 131 from which virus suspensions
of 10’ to 107.5rcID,,/ml are obtained. The stock virus is stored as lyophilized
virus suspension diluted 1:30 with a lactose-peptone-tris buffer at -20°C.
The MS ce11 line was grown in an Earle’s medium supplemented with 2.5%
glucose, 0.5% lactalbumin hydrolysate, 0.01% yeast extract and 10% calf
serum. The medium was enriched by addition of glutamine (0.1 g/l), glutamic
acid (0.1 g/l), methionine (0.15 g/l), biotin (0.001 g/l), folie acid (0.001 g/l)
and arginine hydrochloride (0.04 g/l). Sodium bicarbonate 0.15% was also
added.
The virus was propagated by inoculating cells in suspension in Roux
bottles. Thus, 100 ml amounts of a ce11 suspension containing 2 x 105 cells/ml
were inoculated and incubated in a stationary manner. The ce11 layer forms in
about 30 hours, CPE is apparent usually in 48 h and 90 to 100% of the cells
are involved by 72 h. The virus is harvested at this time, centrifuged under
refrigeration and stored at 4°C. The virus titer is regularly found to be 107*1 to
107*5 in such preparations. The virus is inactivated at 37°C by addition of
1:4000 formalin and constant shaking for 48 h. The formalinized virus is then
chilled to 0°C and 0.2 g/l of betapropiolactone is added. The vaccine is held
at 0°C with frequent shaking for 24 h, the pH is adjusted to 7.0 with bicar-
bonate buffer and the completed vaccine is stored at 4°C. Two types of
adjuvant were used. The first was 1% aluminium gel and the second a minera1
oil-Arlacel preparation. The vaccines that were administered to horses
contained equal amounts of adjuvant and the inactivated virus suspension.
A serum neutralization test employing constant serum increments and
varying virus concentrations was used to assess the response to vaccination.
The results were recorded as neutralization indices expressed as loq,, values.
Serums were inactivated at 56°C for 30 min. After addition of virus, the serum-
virus mixtures incubated at 37” C for 1 h, were then used to inoculate MS cells
in suspension in tubes. The tubes were incubated for 48 h in stationary racks,
the medium was changed and the tubes were then rolled. The results were
read at 4 and 9 days after preparation of the tests,,
Horses were challenged with a mouse brain passed virulent strain of
virus from Morocco that was supplied to us by the Pasteur Institute. This virus
VI. African Horsesickness
204
was given to horses intravenously. The challenge dose was 2 ml of a 1:5
dilution of infected mouse brain suspension supernatant.
Donkeys that were vaccinated with inactivated vaccines developed
detectable neutralizing antibodies by the 10th post vaccination day. The
neutralization indices reached a plateau by the 20th day and then declined
until, by the 40th day, no neutralizing activity was present in the serums.
Administration of a booster dose of vaccine on the 50th day resulted in a
rapid increase in antibodies. The titer reached its maximum on the 8th day
after the booster dose and persisted for at least 100 days [Z].
Six horses imported from France were used to test the two adjuvanted
preparations. Three were giventhe aluminium gel and three the oily adjuvanted
vaccine. No untoward reactions were observed in these animals. The develop-
ment of the neutralization index in horses is illustrated in figure 1. The animals
were given 5 ml of inactivated vaccine as a primary dose. With either adjuvant
antibody was found from the 8th day onward, the maximum titers were
Neutralization index
5
3 -
-
L
2-
l-
Time in months
0
i
;
3
1:
Injection A
0
Fig. 1. The development of serum neutralizing antibodies (neutralization indices,
log*O) of horses vaccinated with an inactived African horsesickness virus vaccine, (A =
booster injection; B = challenge with virulent virus).
BOURDIN, M ONNIER-CAMBON, RI~CHE, LAURENT: Vaccination Against . . .
205
reached on the 15th day and the titers decreased progressively until the 30th
day. The administration of a booster dose resulted in a rapid response with
a maximum titer value being detected from the 13th to 20th day after vacci-
nation. These titers persisted for 12 months, decreasing thereafter.
The first challenge was done in the Spring, on the 45th post vaccination
day, for 2 animals. It produced no disease. The second challenge of 2 horses
was carried out 8 months after the first vaccination and after the rainy season
with the same results. The third group of 2 horses was challenged at 15
months during the hot season. No detectable reaction to challenge occurred.
The latter two animals were kept in the open for 3 months in the period
between vaccination and challenge and it should be pointed out that they
could possibly have been exposed to the virus by mosquitoes which were
abundant at the time.
MIRCHAMSY
and TASLIMI [9] have shown that an inactivated monovalent
AHS vaccine prepared from a neurotropic strain of type 9 virus which was
grown in monkey kidney cells protects horses for at least 6 months if two
injections, administered 4 weeks apart, are given. We were unable, due to the
small number of horses available for our test, to include a control animal, but
our results suggest that protection following administration of our vaccine
lasts for a year. Horses vaccinated with our vaccine resisted severe challenge
on one hand and exposure during the rainy season on the other.
References
1.
ALEXANDER, R. A.; NEITZ, N. 0. and DUTOIT, P. J.: Horsesickness immunization
of horses and mules in the field during the season 1934-1935 with a description of the
technique of preparation of polyvalent mouse neurotropic vaccine. Onderstepoort
J. 7: 17-30 (1936).
2.
BOURDIN, P. et MONNIER-CAMBON, J.: Note préliminaire sur l’emploi d’un vaccin
inactivé contre la peste équine. Bull. Acad. vét. Fr. 40: 187-191 (1967).
3.
CURASSON, G.: Traité de pathologie exotique vétérinaire et comparée, 2me éd.,
vol. 1, pp. 170-207 (Vigot, 1942).
4.
DOUTRE, M. et LECLERCQ, A.: Existence du type T9 du virus de la peste équine au
Tchad. Rev. Elev. 15: 241-245 (1962).
5.
GILBERT, Y.: Rapport sur le fonctionnement pour l’année 1955. Laboratoire fédéral
de I’Elevage de Dakar, 67-70 (1955).
6.
KANDA, Y. and M ELNICK, J. L.: In vitro differentiation of virulent and attenuated
polioviruses by their growth characteristics on MS cells. J. exp. Med. 109: 9-24 (1959).
7.
M AURICE, Y. et PROVOST, A.: Les réactions d’hémagglutination et d’inhibition de
l’hémagglutination avec le virus de la peste équine. Limites de leur interprétation.
Rev. Elev. 19: 439-450 (1966).
VI. African Horsesickness
206
8. MAURICE, Y. et PROVOST, A. : La peste équine à type 9 en Afrique centrale. Enquête
sérologique. Rev. Elev. 20: 5-20 (1967).
9. MIRCHAMSY, H. and TASLIMI, H.: Visualization of African horsesickness virus
by the fluorescent antibody technique. Immunol. 7: 213-218 (1964).
10. MIRCHAMSY, H. and TANMI, H.: Attempts to vaccinate foals with living tissue
culture-adapted horsesickness virus. Bull. Off. Int. Epiz. 62: 91 l-921 (1964).
II. MICHAMSY, H. and TASLIMI, H.: Inactivated African horsesickness cell culture
vaccine. Immunol. 14: 81-88 (1968).
12. MORNET, P.: Sur une évolution atypique de la peste équine particulière à 1’A.O.F.
Rev. Elev. 3: 101-103 (1949).
13. OZAWA, Y. and HAZRATI, A.: Growth of African horsesickness live virus tissue
culture vaccine. Amer. J. vet. Res. 26: 154 (1965).
14. TEPPAZ, L.: Contribution à l’étude de la typho-malaria du cheval (horsesickness)
au Sénégal (Editions Médicales, Paris 1925).
Authors’ address: Dr. P. BOURDIN; Dr. J. MONNIER-CAMBON; Dr. M. RI~CHE and
Dr. A. LAURENT, Laboratoire National de 1’Élevage et de Recherches Vétérinaires, B. P.
2057, Dakar (Senegal).