Veterinary Microbiology, 33 ( 1992 ) 129-142 Elsevier Science Publishers B.V., Amsterdam
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African horse sickness in Spain M. Rodriguez a, H. Hooghuisb and Ma. Casta~o a ~Departamento de Patologia Animal - IL Facultad de Veterinaria, Universidad Complutense, Madrid, Spain bLaboratorio de Sanidad y Produccidn Animal Algete, Ministerio de Agricultura, Pescay Alimentacidn, Madrid, Spain (Accepted 26 June 1992)
ABSTRACT Rodriguez, M., Hooghuis, H. and Casta~o, Ma., 1992. African horse sickness in Spain. Vet. Microbiol., 33: 129-142. The aetiology, pathogenesis and epizootiology of African horse sickness (AHS) are reviewed with special reference to recent outbreaks in the Iberian peninsula. AHS is a highly fatal insect-borne viral disease of Equidae. It is caused by an Orbivirus (family Reoviridae) and nine serotypes are recognised. Outbreaks occurred in central Spain in 1987 and in southern regions of the Iberian peninsula in 1988, 1989 and 1990. All were associated with serotype 4 of the virus, whereas other occurrences of AHS outside Africa have all been caused by serotype 9. The clinical picture in the outbreaks was mainly of the acute (pulmonary) form except in 1988 when the subacute (cardiac) form of disease predominated. Several hundred horses died or were destroyed as a result of the outbreaks. Further spread was contained by a combination of slaughter of sick animals, movement controls, and vaccination which was extended over an increasingly wide area in successive years. The 1987 outbreak is believed to be associated with infected zebras imported from Africa. Possible explanations for the recurrence of disease in Spain in successive years are considered to include (a) the climatic conditions in Southern Spain, which could permit continuous vector activity, (b) the relative clinical resistance of mules and donkeys, which may permit subclinical circulation of the virus, (c) incomplete population immunity among horses due to possible gaps in the vaccination strategy.
INTRODUCTION
African horse sickness (AHS) is a viral disease which affects the Equidae family (horses, mules, donkeys and zebras) (Buxton and Fraser, 1977; Mohanty and Dutta, 1981 ), and is transmitted by haematophagous arthropods of the genus Culicoides (Wetzel et al., 1970 ). Together with Venezuelan equine encephalitis, it is the viral disease which causes the greatest mortality rate in these animals (Fenner et al., 1987 ). The incidence of the disease is seasonal Correspondence to." M. Rodriguez, Dept. de Patologia Animal-II, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain.
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and it usually occurs towards the end of the summer in warm, humid areas, with the prevalence rate being directly related to the number of vectors present (Dardiri, 1986). In horses, AHS takes on an acute or subacute clinical form whose symptomatolology includes severe respiratory and circulatory functional alterations, and a mortality rate which is close to 90%. Mules and donkeys are less susceptible to AHS; the death rate in these animals reaches approximately 50% (Blood et al., 1989).
Geographical distribution The virus responsible for AHS is enzootic on the African continent south of the Sahara but has occasionally appeared in the Middle East and in North Africa. Outbreaks have also occurred in Iran and in other Persian Gulf countries, and in Afghanistan and Pakistan between 1959 and 1964 (French and Geering, 1978). In 1960 the disease spread to India, Turkey and the eastern Mediterranean, and in 1965 appeared in the Maghreb countries, specifically Tunisia, Algeria and Morocco. More than 300,000 horses, mules and donkeys died as a result of that epidemic (Fenner et al., 1987). In 1966 an outbreak occurred in Spain (Diaz-Montilla and Patios, 1967) and 21 years later, in 1987, another outbreak occurred in the province of Madrid (Rodriguez et al., 1987).
African horse sickness in Spain: an historical rOsum~ In 1966 the disease appeared in Spain in the area around Gibraltar, and as a result, according to official statistics, a total of 637 animals died or were slaughtered (Diaz-Montilla and Patios, 1967). In September of 1987 we diagnosed AHS in a Safari Park near Madrid (Aldea del Fresno). The disease spread throught the Alberche and Perales river basins, affecting a strip of land 100 km long and 50 km wide. The number of Equidae which died or had to be destroyed rose to 146 until official eradication in December 1987. In the interim a total of 38,000 animals were vaccinated using a polyvalent attenuated vaccine (Bulletin O.I.E., 1987; Diaz Yubero, 1987 ). Almost one year later, at the beginning of October 1988, and almost 600 km away from the previous outbreak, horses began to die on a farm and in an equine complex (Sotogrande) in the province of Cadiz. Laboratory tests confirmed a new outbreak of AHS on October 16. The disease affected various municipalities in the south of the provinces of Cadiz and M~ilaga, and was responsible for a total of 156 deaths, including those animals which died of AHS and those which were slaughtered. The last death was reported in December of 1988 (Rodriguez et al., 1989). About 18,000 animals were vaccinated, at first with an attenuated polyvalent vaccine and later with a Type 4 monovalent vaccine.
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On July 3 1, 1989, another horse with suspicious symptomatology died in the same horse complex cited previously, and laboratory findings confirmed a new outbreak of the disease on August 11 (Bulletin O.I.E., 1989a). This outbreak was the most extensive and serious of all those to date, with disease detected in the provinces of Badajoz, Cadiz, Huelva, Cordoba and Sevilla, even affecting some internationally famous stud farms in this last province. Simultaneously, cases of AHS were reported in September of 1988 in the Portuguese Algarve (Bulletin O.I.E., 1989b), and Morocco declared the presence of the disease in the north of the country, near the Straits of Gibraltar, in October (Bulletin O.I.E., 1989v, Anon, 1990b). In Spain, 110 animals died directly as a result of the disease and more than 900 were destroyed. Approximately one hundred outbreaks were seen in the five provinces affected before official eradication in January of 1990. A monovalent, Type 4 modified live vaccine was administered to 242,000 susceptible animals in 12 provinces, thus establishing a buffer zone which in many cases extended over more than 250 km from the most peripheral outbreaks detected (Anon, 1990a) (Fig. 1). It is interesting to note that in August of 1989 AHS was also reported in the region of Abha in Saudi Arabia, but due in this case to a Type 9 AHS virus (Mellor et al., 1990).
Fig. 1. AHS in Spain, Portugal and Morocco 1989.
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At the beginning of September of 1990, after nine months of epizootic silence in Spain, a new case of AHS was confirmed, through laboratory isolation and typing, in the Guadalhorce river valey, in the province of M~ilaga. The disease appeared within the territorial limits of 12 towns of this province, including Torremolinos and Mijas, and caused 66 deaths. The last positive case was diagnosed in November of that year (Anon, 1990a). After this outbreak, mandatory vaccination of the entire equine population of Andalusia was carried out. During the same time-period new cases of AHS were reported in northern Morocco, once again due to the Type 4 AHS virus, but the disease did not reappear in Portugal.
A etiopathogenesis AHS is caused by an RNA double-stranded, icosahedral viscerotropic virus belonging to the Reoviridae family, genus Orbivirus, similar to the Bluetongue and Ibaraki viruses (Oellerman et al., 1970). Neutralization and crossimmunity tests have identified nine different serotypes, all of which share common group-specific antigens (Dardiri, 1986), although antigenic variability phenomena may occur due to serial passages in susceptible Equidae (Buxton and Fraser, 1977 ). The 1966 outbreak in Spain was caused by serotype 9, the same serotype which prevailed at that time in North Africa (Diaz-Montilla and Patios, 1967 ). Except for the latest outbreaks in Spain, Portugal and Morocco, caused by the Type 4 AHS virus, all the epizootics reported outside the African continent have been due to the Type 9 virus, including the cases diagnosed in Saudi Arabia in 1989 (Mellor et al., 1990). AHS and Bluetongue in sheep share certain clinical signs, so the pathogenesis of both diseases is probably similar. Viral replication occurs in a local lymph node after the bite of an arthropod carrier. The viral particles then undergo a primary dissemination, reaching tissues and organs of the reticuioendothelial system, and finally a secondary viraemia occurs. The exact manner in which the disease is produced is unknown, but it is probable that the first step involves an inflammatory response in small and middle-sized vessels (Fenner et al., 1987). AHS is, therefore, a disease which affects the vascular endothelium of many organs (Maurer and McCully, 1963; Dardiri, 1984).
Symptomatology Three main forms of the disease are recognized: the acute (pulmonary) form, the subacute (cardiac) form and the febrile form ("horse sickness fever") (Dardiri, 1986).
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Acute (pulmonary)form This form is characterized by a short clinical course, and a high mortality rate. There is sudden fever up to 41 ° C, followed by pulmonary oedema, with increasingly severe dyspnoea, attacks of paroxysmal coughing and frothy nasal discharge. The sick animals adopt postures which favour pulmonary ventilation, separating the forelimbs and dilating the nostrils. The animals may die just a few hours after the first onset of the clinical signs. A mixed clinical syndrome characterized the 1987 outbreak in Madrid and the following outbreaks in 1988, 1989 and 1990, although the pulmonary form appeared in some of the cases (Rodriguez et al., 1989). In Madrid, the process began with a fever of 39-39.5°C which lasted 2-4 days, with no other apparent clinical signs except a slight congestion of the mucous membranes and petechiae on the ventral surface of the tongue. Functional respiratory alterations appeared subsequently, these included tachypnoea, convulsive productive cough, and increasing difficulty in breathing, along with frothy nasal discharge. Some animals were seen emitting abundant frothy liquid from the nasal cavities on lowering their heads or following a slight exertion. Pulmonary auscultation detected crepitant, moist rales and diminished pulmonary sounds, together with a lengthening of the expiration period. Cardiac examination detected intense tachycardia, a weak arterial pulse and a diminished apex beat. In general terms, subcutaneous oedemae were infrequent, although oedema was observed at the supraorbital fossae (Fig. 2). In the final phase of the disease the animals were weak, displayed an unsteady gait and depression, and death was caused by terminal cardio-respiratory insufficiency with pulmonary oedema. Hypothermia (35.5-36°C) was noted and foamy liquid was often expelled from the nostrils before death occurred.
Subacute (cardiac)form In these cases the course of the illness is somewhat longer. The head and neck usually present oedematous inflammation, and there is a characteristic protrusion of the supraorbital fossae. Frequently oedema of the eyelids, lips and tongue and even of the vental thorax and abdomen are also seen. There is a progressive increase in the respiratory rate and breathing becomes more abdominal. Sick animals tend to remain standing, except in cases of colic pain. As the disease progresses the animals become recumbent and death is usually preceeded by a sudden increase of the respiratory rate, muscle tremors and sweating. The horses infected during the Andalusian outbreak of 1988 presented a mixed clinical picture in which cardiac signs prevailed (Rodriguez et al., 1989). The process began with an elevation of body temperature up to 3940°C which lasted for 3 to 10 days. The clinical signs began to appear in a
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Fig. 2. Palpebral and supraorbital fossaeoedema. slow and progressive fashion and were fundamentally due to functional circulatory disorders. As the tachycardia increased, the arterial pulse became weaker, and liquid in the pericardium was detected on auscultation. As the disease progressed, the oedemae of the subcutaneous connective tissue became more pronounced, and particularly involved the head, neck and anterior sternal region. The most notable oedemae were located in the supraorbital fossae and the eyelids, often accounting for blepharitis and conjunctivitis with chemosis (Fig. 3 ). Oedema of the frontal region and of the lips was also frequently seen. Almost all the sick animals presented petechiae on the ventral surface of the tongue. These animals displayed increasingly superficial and dyspnoeic breathing, and progressive tachypnoea, but rarely presented frothy nasal discharge. As the course of the illness advanced, crepitant and subcrepitant moist rales could be detected on auscultation. The sick animals frequently suffered abdominal pain, although this was not accompanied by a noticeable loss of appetite. At times the horses adopted a stance of urinating, with the penis somewhat relaxed. Death was brought about by cardio-respiratory failure with progressive pulmonary oedema.
Febrileform ("horse sicknessfever") This is the least severe, often subclinical form of the disease, and is typical of endemic areas. The animal may have a rectal temperature of over 40°C for
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Fig. 3. Palpebral,supraorbital fossae and conjunctival oedema. 1 or 2 days, although occasionally this will last up to 5 days. There may be a slight dyspnoea, along with an increase of the heart rate and a slight conjunctivitis. The Equidae which suffer this form of the disease recover quickly and without sequelae (Dardiri, 1986). Lesions
In the 1987 Madrid outbreak we observed the lesions typical of a mixed clinical picture with prevalence of pulmonary damage (Rodriguez et al., 1987). These lesions coincided with those which appeared in Andalusia in 1989 (Rodriguez et al., 1989)and 1990. The animals displayed a good external appearance, but there was congestive-cyanotic mucous membranes, petechiae on the underside of the tongue, and almost invariably whitish or yellow-reddish foam in the nostrils. There was also oedema of the supraorbital fossae. The yellowish, gelatinous oedema of the subcutaneous connective tissue, especially in the intercostal muscles, was a notable feature observed in the necropsies. On opening the abdominal cavity, we observed a slight ascites, focal subcapsular splenic petechiae and slightly hypertrophic mesenteric lymph nodes, moist on opening and reddish or greyish in colour.
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All the animals necropsied displayed pronounced submucosal congestion of the fundus of the stomach (Fig. 4), subserous and submucous petechiae in certain areas of the small intestine and, in some cases, diffuse congestion of the mucous membrane of the large intestine. No other abdominal cavity organs displayed any notable macroscopic alterations. The lesions seen in the thoracic cavity of some of the animals consisted of a small accumulation of liquid, hypertrophic lungs with interstitial and alveolar oedemae and frothy liquid in the trachea, with petechiae and oedema of the mucous and serous membranes. All 23 of the animals necropsied in 1987 displayed hydropericardium: between 500 and 2000 ml of yellowish liquid was present in each case. Petechiae were seen on the coronary sulcus and there was evidence of subendocardial haemorrhage, although both of these lesions were unobtrusive. The mediastinal lymph nodes were slightly hypertrophic, moist on opening and reddish in colour. In the Andalusian outbreak of 1988, the lesions corresponded with those of a mixed clinical picture, although cardiac involvement predominated (Rodriguez et al., 1989). On external inspection the animals displayed a good general appearance, but on closer inspection congestive-cyanotic mucous membranes and prominence of the supraorbital fossae were evident although to a lesser degree than in live animals. There was marked blepharitis and pro-
Fig. 4. Congestion o f the fundus in the stomach.
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nounced oedema of the palpebral conjunctiva, often accompanied by chemosis. These lesions were not seen as frequently in the earlier outbreaks. The necropsies disclosed the presence of a yellowish gelatinous oedema of the subcutaneous connective tissue, more severe than that of the previously described lesional picture, located principally in the frontal, cervical and sternal regions and the ventral portion of the abdomen, and especially pronounced in the muscular fasciae. There was abundant ascites and hydrothorax, and hydropericardium was seen in all cases, while the other lesions were very similar to those found in Madrid. Histopathological study of the samples obtained during the outbreaks demonstrated the presence of lesions indicative of an acute inflammatory selective and unspecific process, marked congestion and oedema but few inflammatory cells.
Diagnosis The clinical signs, the pathological lesions and the epizootic nature of the disease permit a presumptive early diagnosis of AHS although the clinical signs and lesions of AHS are not pathognomonic (Dardiri, 1984, 1986). The confirmation of clinical diagnosis is made by isolation of the virus from samples of heparinized blood obtained in the febrile phase or from spleen samples taken during necropsy (Buxton and Fraser, 1977; Dardiri, 1986). The diagnosis of the outbreak cannot be made from serological tests, because the animals with clinical signs do not generate an immune response until at least 14 to 21 days after infection, during which time most of them die (B.J. Erasmus, personal communication, 1988 ). Two methods are generally used for viral diagnosis: inoculation of cell culture (VERO and MS) and inoculation of suckling mouse brain. When inoculation of cell culture is carried out, there is usually a cytopathic effect during the second or the third passage in case of positive samples. Virus neutralization tests are then used for subsequent identification and typing of the virus. In positive cases, the suckling mice present nervous clinical signs (incoordination, tremors, convulsions, ataxia) (Callis et al., 1982). A homogenate of the infected brains is then obtained for inoculation in cell culture and the subsequent typing, or simply for identification of the virus by complement fixation test (Dardiri, 1986). A third method of aetiological diagnosis involves the inoculation of suspicious samples (blood or spleen macerate) into susceptible Equidae (Callis et al., 1982). However, this option is rarely used due to its high cost and the risk of spreading the disease. Nowadays, an indirect, group specific ELISA sandwich test is available for the detection of AHS virus and viral antigens. This test involves the capture of viral particles by means of rabbit hyperimmune anti-AHS virus serum adsorbed to ELISA microtitre plates. Purified hyperimmune Anti-AHS virus serum, obtained in
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guinea pigs, is added for viral detection, and the reaction is quantified colorimetrically (Hamblin et al., 1991a). This method has the enormous advantage of its speed, as the results are available within a few hours.
Epizootiological hypotheses The majority of the theories relating to the 1966 outbreak indicate that the AHS virus reached the area around Gibraltar by means of arthropod vectors carried by the wind or by boat. This supposition is logical, as at that time there was an outbreak of AHS due to AHS serotype 9 in Morocco and the serotype for the Spanish outbreak was also type 9. Once AHS was detected in Spain, all Equidae within an area 15 km around the primary outbreak were destroyed. At the same time a preventive vaccination programme was initiated and approximately 60000 Equidae were vaccinated. The last death occurred in October of 1966 and the disease did not reappear in Spain until July 1987, in Madrid. The most plausible hypothesis for the origin of the 1987 outbreak is that it was related to the entry on June 26 of several zebra from Namibia into a Safari Park in the outskirts of Madrid. The first Equidae with suspicious clinical signs died in that same location on July 22 and 23 although those deaths were initially attributed to enterotoxaemia. At the beginning of September of the same year horses also began to die outside the initial outbreak area. The animal health authorities were alerted and on September 11 laboratory tests confirmed the presence of AHS caused by the type 4 virus. One hundred and forty six animals died during this outbreak and 38000 Equidae were vaccinated in a few days. The last death occurred in the middle of October of the same year. The first outbreak in the south of the Iberian peninsula was detected almost 12 months afterwards, in 1988, in the province of Cadiz, and the same serotype was once again involved. On this occasion there was no clear explanation concerning the origin of the problem, and several hypotheses have been suggested. Among these is the possible arrival in Spain of sick equidae from Africa or the arrival of infected arthropod vectors from the same continent. Neither of these theories is very plausible, as during this period the presence of AHS had not been declared by any Maghreb country and due to its dramatic nature this disease is nearly impossible to conceal. A possible reversion to virulence of the vaccine virus was also considered, but this possibility is also improbable, not only because of the stability of vaccine strains, but also because of the time-lapse subsequent to vaccination. The possible role of mules and donkeys as sources of the AHS virus during the period between both outbreaks was also taken into consideration. These animals present a longer viraemia than horses and are also more resistant to the disease. The result is a clinical syndrome which attracts less attention and may even occasionally go
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unnoticed. These facts, together with the diminished vector population during the winter season, and the longer lifespan of the arthropods due to the lower temperatures, may result in an apparent epizootic silence. The problem flares up again when the vector population increases in a largely susceptible equine population. Nevertheless, more than 600 km separated the two outbreaks, and it would have been very difficult for the death of horses to have gone unnoticed during this interval. The most worrying possibility is that this outbreak was a recrudescence of the disease. However, all investigation on this subject indicate that carrier Equidae do not exist (Dardiri, 1984), and that the virus cannot be transmitted from one generation of vectors to the next (B.J. Erasmus, personal communication, 1988 ). Taking the three Andalusian outbreaks (1988, 1989, 1990) as a whole, if the i m m u n e response of the vaccinated Equidae, which survived infection does not permit persistence of the virus, and if transovarial transmission through the vector population does not exist, then no clear explanation as to the reappearance of the disease exists. Nevertheless, several factors must be taken into account. Firstly, the climate in the south of the Iberian peninsula permits the arthropod vectors to be active throughout the year, that is to say, there is no interruption of their life-cycle. Secondly, it is possible that the official vaccination campaign did not cover the entire equine population. For example, in the marshes of the Guadalquivir river there is a 75000 ha wildlife park with groups of wild horses whose control is almost impossible. Also, various horse owners have systematically avoided vaccinating their animals due to the possibility of commercial repercussions through a ban on the movement of serpositive animals. Finally, the equine population of the area is large and very dispersed, which makes an effective immunization programme difficult. The most likely hypothesis to explain the reappearance of AHS in Andalusia during the last three years is the lack of any true epizootic silence between the outbreaks, that is to say, during this period animals were infected and died without AHS being recognized as the cause. Once again, the possible role of mules and donkeys as sources of virus during these intervals must be taken into account, with the disease only showing its true virulence during period of m a x i m u m vector activity. A second problem to account for is the spread of the disease once it has been declared. In 1989 and 1990, AHS reappeared in an equine population in which the majority of animals had been vaccinated. The spread of the disease under such conditions might be due to a phenomenon of partial immunity in which viral replication is not impeded but clinical signs are attenuated. In this context, about 10% of the Equidae vaccinated do not present a measurable serological reaction, although this does not necessarily imply a lack of protection (Hamblin et al., 1991b). Finally, another factor which must be
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considered is the causal agent itself: the type 4 AHS virus is the only virus which does not cross-reac with other serotypes (B.J. Erasmus, personal communication, 1988), which may imply a distinctive behavior on its part. Control m e a s u r e s
In order to control the spread of AHS, strong preventive measures must be taken in the area where the disease is detected, and the widest possible buffer zone surrounding the outbreak area must be established (Fig. 5). The chief measures which should be taken are: • ban on the movements of all susceptible animals • protection of all Equidae against the vectors: use of insecticides, stabling at night, elimination of vectors' breeding sites, use of insect repellents, etc. • slaughter or immediate isolation of sick animals which might act as sources of virus • mandatory immediate and complete vaccination of the entire equine population, and identification of vaccinated animals preferably by means of branding. In Spain, the effective control and elimination of AHS from the country requires a coordinated effort by the equine sector and by the central and auto-
Fig. 5. AHS in Spain 1990. Vaccination and surveillance area.
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n o m i c g o v e r n m e n t s ; this is t h e o n l y s u r e w a y t o s a f e g u a r d t h e S p a n i s h e q u i n e i n d u s t r y a n d its g e n e t i c w e a l t h .
REFERENCES Anon, 1990a. Informe sobre la aparici6n de un brote de peste equina en M~ilaga. Subdirecci6n General de Sanidad Animal, Ministerio de Agricultura, Pesca y Alimentaci6n, Espatia. Anon, 199019. Peste Equina. In: Informe de la Comisi6n de la Fiebre Aftosa y otras Epizootias, 58 Sesi6n General de la Oficina Internacional de Epizootias, Paris 14-18 May, pp. 9-13. Blood, D.C., Radostits, O.M. and Henderson, J.A., 1989. Veterinary Medicine, a textbook of diseases ofacttle, sheep, pigs, goats, and horses, 7th Ed., Bailliere-Tindall, London, pp. 788. Bulletin de l'Office International des Epizooties, 1987.99: 46-48. Bulletin de l'Office International des Epizooties, 1989a. 101: 496. Bulletin de l'Office International des Epizooties, 1989b. I 01: 550. Buxton, A. and Fraser, G., 1977. Animal Microbiology, Vol. 2. Blackwell Scientific Publications Ltd, Oxford, pp. 632-635. Callis, I.J., Dardiri, A.H., Ferris, D.H., Gay, J., Mason, J. and Wilder, F.W., 1982. Illustrated manual for the recognition and diagnosis of certain animal diseases. Mexico-United States Commission for the prevention of foot and mouth disease, pp. 46-48. Dardiri, A.H., 1984. African Horse Sickness. In: Foreign Animal Diseases. Reference Manual. Vol. 1. USDA, A.P.H.I.S., NVSL, Ames, Iowa, pp. 10-19. Dardiri, A.H., 1986. Peste Equina Africana. In: Enfermedades ex6ticas de los animales. Su prevencion, diagn6stico y control. Comit6 de enfermedades ex6ticas de la Asociaci6n de Sanidad Animal de los Estados Unidos. Comisi6n Mexico-Americana para la prevenci6n de la fiebre aftosa. Mexico, D.F., pp. 302-311. Diaz-Montilla, R. and Patios Marti, P., 1967. Epizootiologia de la Peste Equina en Espatia. Bull. Office Int. Epizoot., 68: 705-714. Diaz Yubero, M.A., 1987. Informe Peste Equina en Espatia. lnformaci6n epizootiol6gica, no. Esp. 87/5/142, Office Int. Epizoot., Paris. Fenner, F., Bachmann, P.A., Gibbs, E.P.J., Murphy, F.A., Studdert, M.J. and White, D.O., 1987. Veterinary Virology, Academic Press, Orlando, pp. 587-590. French, A. and Geering, W.A., 1978. Exotic Diseases of Animals. A manual for diagnosis. Australian Government Publishing Service. Canberra, pp. 41-45. Hamblin, C., Graham, S.D., Anderson, E.C. and Crowther, J.C., 1991a. A serogroup Specific Enzyme-Linked-lmmunosorbent-Assay for the detection and identification of African Horse Sickness viruses. J. Virol. Methods, 31: 285-292. Hamblin, C., Mellor, P.S., Graham, S.D., Hooghuis, H., Montejano, R.C., Cubillo, M.A. and Boned, J., 1991b. Antibodies in horses, mules and donkeys following monovalent vaccination against African Horse Sickness. Epidemiol. Infect., 106: 365-371. Maurer, F.D. and McCully, R.M., 1963. African Horse Sickness-with emphasis on Pathology. Am. J. Vet. Res., 24: 235-266. Mellor, P.S., Hamblin, C. and Graham, S.D., 1990. African Horse Sickness in Saudi Arabia. Vet. Rec., 127: 41-42. Mohanty, S.B. and Dutta, S.K., 1981. Veterinary Virology. Lea and Febiger, Philadelphia, pp. 172-174. Oellermann, R.A., Els, H.J. and Erasmus, B.J., 1970. Characterization of African Horse sickness viruses. Archiv Ges. virusforsch., 29:163-174. Rodriguez, M., Castatio, M., Escolar, E., Flores, J., Toni, P., Gonzalez, M., Jimenez, F., Gon-
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zalez, J.L. and Montoya, J.A., 1987. Peste Equina Africana: descripci6n del brote en Espafia 1987. Med. Vet., 4: 537-557. Rodriguez, M., Castaf~o, M. and Garcia, I., 1989. Peste Equina Africana en Espa~a: Focos de 1987, 1988 y 1989. Proceedings, III Jornadas T6cnicas sobre cl caballo. Expoaviga 89, pp. 25-30. Wetzel, H., Nevill, E.M. and Erasmus, B.J., 1970. Studies on the transmission of African Horse Sickness. Onderstepoort J. Vet. Res., 37:165-168.
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African horse sickness in Spain M. Rodriguez a, H. Hooghuisb and Ma. Casta~o a ~Departamento de Patologia Animal - IL Facultad de Veterinaria, Universidad Complutense, Madrid, Spain bLaboratorio de Sanidad y Produccidn Animal Algete, Ministerio de Agricultura, Pescay Alimentacidn, Madrid, Spain (Accepted 26 June 1992)
ABSTRACT Rodriguez, M., Hooghuis, H. and Casta~o, Ma., 1992. African horse sickness in Spain. Vet. Microbiol., 33: 129-142. The aetiology, pathogenesis and epizootiology of African horse sickness (AHS) are reviewed with special reference to recent outbreaks in the Iberian peninsula. AHS is a highly fatal insect-borne viral disease of Equidae. It is caused by an Orbivirus (family Reoviridae) and nine serotypes are recognised. Outbreaks occurred in central Spain in 1987 and in southern regions of the Iberian peninsula in 1988, 1989 and 1990. All were associated with serotype 4 of the virus, whereas other occurrences of AHS outside Africa have all been caused by serotype 9. The clinical picture in the outbreaks was mainly of the acute (pulmonary) form except in 1988 when the subacute (cardiac) form of disease predominated. Several hundred horses died or were destroyed as a result of the outbreaks. Further spread was contained by a combination of slaughter of sick animals, movement controls, and vaccination which was extended over an increasingly wide area in successive years. The 1987 outbreak is believed to be associated with infected zebras imported from Africa. Possible explanations for the recurrence of disease in Spain in successive years are considered to include (a) the climatic conditions in Southern Spain, which could permit continuous vector activity, (b) the relative clinical resistance of mules and donkeys, which may permit subclinical circulation of the virus, (c) incomplete population immunity among horses due to possible gaps in the vaccination strategy.
INTRODUCTION
African horse sickness (AHS) is a viral disease which affects the Equidae family (horses, mules, donkeys and zebras) (Buxton and Fraser, 1977; Mohanty and Dutta, 1981 ), and is transmitted by haematophagous arthropods of the genus Culicoides (Wetzel et al., 1970 ). Together with Venezuelan equine encephalitis, it is the viral disease which causes the greatest mortality rate in these animals (Fenner et al., 1987 ). The incidence of the disease is seasonal Correspondence to." M. Rodriguez, Dept. de Patologia Animal-II, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain.
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and it usually occurs towards the end of the summer in warm, humid areas, with the prevalence rate being directly related to the number of vectors present (Dardiri, 1986). In horses, AHS takes on an acute or subacute clinical form whose symptomatolology includes severe respiratory and circulatory functional alterations, and a mortality rate which is close to 90%. Mules and donkeys are less susceptible to AHS; the death rate in these animals reaches approximately 50% (Blood et al., 1989).
Geographical distribution The virus responsible for AHS is enzootic on the African continent south of the Sahara but has occasionally appeared in the Middle East and in North Africa. Outbreaks have also occurred in Iran and in other Persian Gulf countries, and in Afghanistan and Pakistan between 1959 and 1964 (French and Geering, 1978). In 1960 the disease spread to India, Turkey and the eastern Mediterranean, and in 1965 appeared in the Maghreb countries, specifically Tunisia, Algeria and Morocco. More than 300,000 horses, mules and donkeys died as a result of that epidemic (Fenner et al., 1987). In 1966 an outbreak occurred in Spain (Diaz-Montilla and Patios, 1967) and 21 years later, in 1987, another outbreak occurred in the province of Madrid (Rodriguez et al., 1987).
African horse sickness in Spain: an historical rOsum~ In 1966 the disease appeared in Spain in the area around Gibraltar, and as a result, according to official statistics, a total of 637 animals died or were slaughtered (Diaz-Montilla and Patios, 1967). In September of 1987 we diagnosed AHS in a Safari Park near Madrid (Aldea del Fresno). The disease spread throught the Alberche and Perales river basins, affecting a strip of land 100 km long and 50 km wide. The number of Equidae which died or had to be destroyed rose to 146 until official eradication in December 1987. In the interim a total of 38,000 animals were vaccinated using a polyvalent attenuated vaccine (Bulletin O.I.E., 1987; Diaz Yubero, 1987 ). Almost one year later, at the beginning of October 1988, and almost 600 km away from the previous outbreak, horses began to die on a farm and in an equine complex (Sotogrande) in the province of Cadiz. Laboratory tests confirmed a new outbreak of AHS on October 16. The disease affected various municipalities in the south of the provinces of Cadiz and M~ilaga, and was responsible for a total of 156 deaths, including those animals which died of AHS and those which were slaughtered. The last death was reported in December of 1988 (Rodriguez et al., 1989). About 18,000 animals were vaccinated, at first with an attenuated polyvalent vaccine and later with a Type 4 monovalent vaccine.
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On July 3 1, 1989, another horse with suspicious symptomatology died in the same horse complex cited previously, and laboratory findings confirmed a new outbreak of the disease on August 11 (Bulletin O.I.E., 1989a). This outbreak was the most extensive and serious of all those to date, with disease detected in the provinces of Badajoz, Cadiz, Huelva, Cordoba and Sevilla, even affecting some internationally famous stud farms in this last province. Simultaneously, cases of AHS were reported in September of 1988 in the Portuguese Algarve (Bulletin O.I.E., 1989b), and Morocco declared the presence of the disease in the north of the country, near the Straits of Gibraltar, in October (Bulletin O.I.E., 1989v, Anon, 1990b). In Spain, 110 animals died directly as a result of the disease and more than 900 were destroyed. Approximately one hundred outbreaks were seen in the five provinces affected before official eradication in January of 1990. A monovalent, Type 4 modified live vaccine was administered to 242,000 susceptible animals in 12 provinces, thus establishing a buffer zone which in many cases extended over more than 250 km from the most peripheral outbreaks detected (Anon, 1990a) (Fig. 1). It is interesting to note that in August of 1989 AHS was also reported in the region of Abha in Saudi Arabia, but due in this case to a Type 9 AHS virus (Mellor et al., 1990).
Fig. 1. AHS in Spain, Portugal and Morocco 1989.
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At the beginning of September of 1990, after nine months of epizootic silence in Spain, a new case of AHS was confirmed, through laboratory isolation and typing, in the Guadalhorce river valey, in the province of M~ilaga. The disease appeared within the territorial limits of 12 towns of this province, including Torremolinos and Mijas, and caused 66 deaths. The last positive case was diagnosed in November of that year (Anon, 1990a). After this outbreak, mandatory vaccination of the entire equine population of Andalusia was carried out. During the same time-period new cases of AHS were reported in northern Morocco, once again due to the Type 4 AHS virus, but the disease did not reappear in Portugal.
A etiopathogenesis AHS is caused by an RNA double-stranded, icosahedral viscerotropic virus belonging to the Reoviridae family, genus Orbivirus, similar to the Bluetongue and Ibaraki viruses (Oellerman et al., 1970). Neutralization and crossimmunity tests have identified nine different serotypes, all of which share common group-specific antigens (Dardiri, 1986), although antigenic variability phenomena may occur due to serial passages in susceptible Equidae (Buxton and Fraser, 1977 ). The 1966 outbreak in Spain was caused by serotype 9, the same serotype which prevailed at that time in North Africa (Diaz-Montilla and Patios, 1967 ). Except for the latest outbreaks in Spain, Portugal and Morocco, caused by the Type 4 AHS virus, all the epizootics reported outside the African continent have been due to the Type 9 virus, including the cases diagnosed in Saudi Arabia in 1989 (Mellor et al., 1990). AHS and Bluetongue in sheep share certain clinical signs, so the pathogenesis of both diseases is probably similar. Viral replication occurs in a local lymph node after the bite of an arthropod carrier. The viral particles then undergo a primary dissemination, reaching tissues and organs of the reticuioendothelial system, and finally a secondary viraemia occurs. The exact manner in which the disease is produced is unknown, but it is probable that the first step involves an inflammatory response in small and middle-sized vessels (Fenner et al., 1987). AHS is, therefore, a disease which affects the vascular endothelium of many organs (Maurer and McCully, 1963; Dardiri, 1984).
Symptomatology Three main forms of the disease are recognized: the acute (pulmonary) form, the subacute (cardiac) form and the febrile form ("horse sickness fever") (Dardiri, 1986).
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Acute (pulmonary)form This form is characterized by a short clinical course, and a high mortality rate. There is sudden fever up to 41 ° C, followed by pulmonary oedema, with increasingly severe dyspnoea, attacks of paroxysmal coughing and frothy nasal discharge. The sick animals adopt postures which favour pulmonary ventilation, separating the forelimbs and dilating the nostrils. The animals may die just a few hours after the first onset of the clinical signs. A mixed clinical syndrome characterized the 1987 outbreak in Madrid and the following outbreaks in 1988, 1989 and 1990, although the pulmonary form appeared in some of the cases (Rodriguez et al., 1989). In Madrid, the process began with a fever of 39-39.5°C which lasted 2-4 days, with no other apparent clinical signs except a slight congestion of the mucous membranes and petechiae on the ventral surface of the tongue. Functional respiratory alterations appeared subsequently, these included tachypnoea, convulsive productive cough, and increasing difficulty in breathing, along with frothy nasal discharge. Some animals were seen emitting abundant frothy liquid from the nasal cavities on lowering their heads or following a slight exertion. Pulmonary auscultation detected crepitant, moist rales and diminished pulmonary sounds, together with a lengthening of the expiration period. Cardiac examination detected intense tachycardia, a weak arterial pulse and a diminished apex beat. In general terms, subcutaneous oedemae were infrequent, although oedema was observed at the supraorbital fossae (Fig. 2). In the final phase of the disease the animals were weak, displayed an unsteady gait and depression, and death was caused by terminal cardio-respiratory insufficiency with pulmonary oedema. Hypothermia (35.5-36°C) was noted and foamy liquid was often expelled from the nostrils before death occurred.
Subacute (cardiac)form In these cases the course of the illness is somewhat longer. The head and neck usually present oedematous inflammation, and there is a characteristic protrusion of the supraorbital fossae. Frequently oedema of the eyelids, lips and tongue and even of the vental thorax and abdomen are also seen. There is a progressive increase in the respiratory rate and breathing becomes more abdominal. Sick animals tend to remain standing, except in cases of colic pain. As the disease progresses the animals become recumbent and death is usually preceeded by a sudden increase of the respiratory rate, muscle tremors and sweating. The horses infected during the Andalusian outbreak of 1988 presented a mixed clinical picture in which cardiac signs prevailed (Rodriguez et al., 1989). The process began with an elevation of body temperature up to 3940°C which lasted for 3 to 10 days. The clinical signs began to appear in a
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Fig. 2. Palpebral and supraorbital fossaeoedema. slow and progressive fashion and were fundamentally due to functional circulatory disorders. As the tachycardia increased, the arterial pulse became weaker, and liquid in the pericardium was detected on auscultation. As the disease progressed, the oedemae of the subcutaneous connective tissue became more pronounced, and particularly involved the head, neck and anterior sternal region. The most notable oedemae were located in the supraorbital fossae and the eyelids, often accounting for blepharitis and conjunctivitis with chemosis (Fig. 3 ). Oedema of the frontal region and of the lips was also frequently seen. Almost all the sick animals presented petechiae on the ventral surface of the tongue. These animals displayed increasingly superficial and dyspnoeic breathing, and progressive tachypnoea, but rarely presented frothy nasal discharge. As the course of the illness advanced, crepitant and subcrepitant moist rales could be detected on auscultation. The sick animals frequently suffered abdominal pain, although this was not accompanied by a noticeable loss of appetite. At times the horses adopted a stance of urinating, with the penis somewhat relaxed. Death was brought about by cardio-respiratory failure with progressive pulmonary oedema.
Febrileform ("horse sicknessfever") This is the least severe, often subclinical form of the disease, and is typical of endemic areas. The animal may have a rectal temperature of over 40°C for
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Fig. 3. Palpebral,supraorbital fossae and conjunctival oedema. 1 or 2 days, although occasionally this will last up to 5 days. There may be a slight dyspnoea, along with an increase of the heart rate and a slight conjunctivitis. The Equidae which suffer this form of the disease recover quickly and without sequelae (Dardiri, 1986). Lesions
In the 1987 Madrid outbreak we observed the lesions typical of a mixed clinical picture with prevalence of pulmonary damage (Rodriguez et al., 1987). These lesions coincided with those which appeared in Andalusia in 1989 (Rodriguez et al., 1989)and 1990. The animals displayed a good external appearance, but there was congestive-cyanotic mucous membranes, petechiae on the underside of the tongue, and almost invariably whitish or yellow-reddish foam in the nostrils. There was also oedema of the supraorbital fossae. The yellowish, gelatinous oedema of the subcutaneous connective tissue, especially in the intercostal muscles, was a notable feature observed in the necropsies. On opening the abdominal cavity, we observed a slight ascites, focal subcapsular splenic petechiae and slightly hypertrophic mesenteric lymph nodes, moist on opening and reddish or greyish in colour.
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All the animals necropsied displayed pronounced submucosal congestion of the fundus of the stomach (Fig. 4), subserous and submucous petechiae in certain areas of the small intestine and, in some cases, diffuse congestion of the mucous membrane of the large intestine. No other abdominal cavity organs displayed any notable macroscopic alterations. The lesions seen in the thoracic cavity of some of the animals consisted of a small accumulation of liquid, hypertrophic lungs with interstitial and alveolar oedemae and frothy liquid in the trachea, with petechiae and oedema of the mucous and serous membranes. All 23 of the animals necropsied in 1987 displayed hydropericardium: between 500 and 2000 ml of yellowish liquid was present in each case. Petechiae were seen on the coronary sulcus and there was evidence of subendocardial haemorrhage, although both of these lesions were unobtrusive. The mediastinal lymph nodes were slightly hypertrophic, moist on opening and reddish in colour. In the Andalusian outbreak of 1988, the lesions corresponded with those of a mixed clinical picture, although cardiac involvement predominated (Rodriguez et al., 1989). On external inspection the animals displayed a good general appearance, but on closer inspection congestive-cyanotic mucous membranes and prominence of the supraorbital fossae were evident although to a lesser degree than in live animals. There was marked blepharitis and pro-
Fig. 4. Congestion o f the fundus in the stomach.
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nounced oedema of the palpebral conjunctiva, often accompanied by chemosis. These lesions were not seen as frequently in the earlier outbreaks. The necropsies disclosed the presence of a yellowish gelatinous oedema of the subcutaneous connective tissue, more severe than that of the previously described lesional picture, located principally in the frontal, cervical and sternal regions and the ventral portion of the abdomen, and especially pronounced in the muscular fasciae. There was abundant ascites and hydrothorax, and hydropericardium was seen in all cases, while the other lesions were very similar to those found in Madrid. Histopathological study of the samples obtained during the outbreaks demonstrated the presence of lesions indicative of an acute inflammatory selective and unspecific process, marked congestion and oedema but few inflammatory cells.
Diagnosis The clinical signs, the pathological lesions and the epizootic nature of the disease permit a presumptive early diagnosis of AHS although the clinical signs and lesions of AHS are not pathognomonic (Dardiri, 1984, 1986). The confirmation of clinical diagnosis is made by isolation of the virus from samples of heparinized blood obtained in the febrile phase or from spleen samples taken during necropsy (Buxton and Fraser, 1977; Dardiri, 1986). The diagnosis of the outbreak cannot be made from serological tests, because the animals with clinical signs do not generate an immune response until at least 14 to 21 days after infection, during which time most of them die (B.J. Erasmus, personal communication, 1988 ). Two methods are generally used for viral diagnosis: inoculation of cell culture (VERO and MS) and inoculation of suckling mouse brain. When inoculation of cell culture is carried out, there is usually a cytopathic effect during the second or the third passage in case of positive samples. Virus neutralization tests are then used for subsequent identification and typing of the virus. In positive cases, the suckling mice present nervous clinical signs (incoordination, tremors, convulsions, ataxia) (Callis et al., 1982). A homogenate of the infected brains is then obtained for inoculation in cell culture and the subsequent typing, or simply for identification of the virus by complement fixation test (Dardiri, 1986). A third method of aetiological diagnosis involves the inoculation of suspicious samples (blood or spleen macerate) into susceptible Equidae (Callis et al., 1982). However, this option is rarely used due to its high cost and the risk of spreading the disease. Nowadays, an indirect, group specific ELISA sandwich test is available for the detection of AHS virus and viral antigens. This test involves the capture of viral particles by means of rabbit hyperimmune anti-AHS virus serum adsorbed to ELISA microtitre plates. Purified hyperimmune Anti-AHS virus serum, obtained in
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guinea pigs, is added for viral detection, and the reaction is quantified colorimetrically (Hamblin et al., 1991a). This method has the enormous advantage of its speed, as the results are available within a few hours.
Epizootiological hypotheses The majority of the theories relating to the 1966 outbreak indicate that the AHS virus reached the area around Gibraltar by means of arthropod vectors carried by the wind or by boat. This supposition is logical, as at that time there was an outbreak of AHS due to AHS serotype 9 in Morocco and the serotype for the Spanish outbreak was also type 9. Once AHS was detected in Spain, all Equidae within an area 15 km around the primary outbreak were destroyed. At the same time a preventive vaccination programme was initiated and approximately 60000 Equidae were vaccinated. The last death occurred in October of 1966 and the disease did not reappear in Spain until July 1987, in Madrid. The most plausible hypothesis for the origin of the 1987 outbreak is that it was related to the entry on June 26 of several zebra from Namibia into a Safari Park in the outskirts of Madrid. The first Equidae with suspicious clinical signs died in that same location on July 22 and 23 although those deaths were initially attributed to enterotoxaemia. At the beginning of September of the same year horses also began to die outside the initial outbreak area. The animal health authorities were alerted and on September 11 laboratory tests confirmed the presence of AHS caused by the type 4 virus. One hundred and forty six animals died during this outbreak and 38000 Equidae were vaccinated in a few days. The last death occurred in the middle of October of the same year. The first outbreak in the south of the Iberian peninsula was detected almost 12 months afterwards, in 1988, in the province of Cadiz, and the same serotype was once again involved. On this occasion there was no clear explanation concerning the origin of the problem, and several hypotheses have been suggested. Among these is the possible arrival in Spain of sick equidae from Africa or the arrival of infected arthropod vectors from the same continent. Neither of these theories is very plausible, as during this period the presence of AHS had not been declared by any Maghreb country and due to its dramatic nature this disease is nearly impossible to conceal. A possible reversion to virulence of the vaccine virus was also considered, but this possibility is also improbable, not only because of the stability of vaccine strains, but also because of the time-lapse subsequent to vaccination. The possible role of mules and donkeys as sources of the AHS virus during the period between both outbreaks was also taken into consideration. These animals present a longer viraemia than horses and are also more resistant to the disease. The result is a clinical syndrome which attracts less attention and may even occasionally go
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unnoticed. These facts, together with the diminished vector population during the winter season, and the longer lifespan of the arthropods due to the lower temperatures, may result in an apparent epizootic silence. The problem flares up again when the vector population increases in a largely susceptible equine population. Nevertheless, more than 600 km separated the two outbreaks, and it would have been very difficult for the death of horses to have gone unnoticed during this interval. The most worrying possibility is that this outbreak was a recrudescence of the disease. However, all investigation on this subject indicate that carrier Equidae do not exist (Dardiri, 1984), and that the virus cannot be transmitted from one generation of vectors to the next (B.J. Erasmus, personal communication, 1988 ). Taking the three Andalusian outbreaks (1988, 1989, 1990) as a whole, if the i m m u n e response of the vaccinated Equidae, which survived infection does not permit persistence of the virus, and if transovarial transmission through the vector population does not exist, then no clear explanation as to the reappearance of the disease exists. Nevertheless, several factors must be taken into account. Firstly, the climate in the south of the Iberian peninsula permits the arthropod vectors to be active throughout the year, that is to say, there is no interruption of their life-cycle. Secondly, it is possible that the official vaccination campaign did not cover the entire equine population. For example, in the marshes of the Guadalquivir river there is a 75000 ha wildlife park with groups of wild horses whose control is almost impossible. Also, various horse owners have systematically avoided vaccinating their animals due to the possibility of commercial repercussions through a ban on the movement of serpositive animals. Finally, the equine population of the area is large and very dispersed, which makes an effective immunization programme difficult. The most likely hypothesis to explain the reappearance of AHS in Andalusia during the last three years is the lack of any true epizootic silence between the outbreaks, that is to say, during this period animals were infected and died without AHS being recognized as the cause. Once again, the possible role of mules and donkeys as sources of virus during these intervals must be taken into account, with the disease only showing its true virulence during period of m a x i m u m vector activity. A second problem to account for is the spread of the disease once it has been declared. In 1989 and 1990, AHS reappeared in an equine population in which the majority of animals had been vaccinated. The spread of the disease under such conditions might be due to a phenomenon of partial immunity in which viral replication is not impeded but clinical signs are attenuated. In this context, about 10% of the Equidae vaccinated do not present a measurable serological reaction, although this does not necessarily imply a lack of protection (Hamblin et al., 1991b). Finally, another factor which must be
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considered is the causal agent itself: the type 4 AHS virus is the only virus which does not cross-reac with other serotypes (B.J. Erasmus, personal communication, 1988), which may imply a distinctive behavior on its part. Control m e a s u r e s
In order to control the spread of AHS, strong preventive measures must be taken in the area where the disease is detected, and the widest possible buffer zone surrounding the outbreak area must be established (Fig. 5). The chief measures which should be taken are: • ban on the movements of all susceptible animals • protection of all Equidae against the vectors: use of insecticides, stabling at night, elimination of vectors' breeding sites, use of insect repellents, etc. • slaughter or immediate isolation of sick animals which might act as sources of virus • mandatory immediate and complete vaccination of the entire equine population, and identification of vaccinated animals preferably by means of branding. In Spain, the effective control and elimination of AHS from the country requires a coordinated effort by the equine sector and by the central and auto-
Fig. 5. AHS in Spain 1990. Vaccination and surveillance area.
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n o m i c g o v e r n m e n t s ; this is t h e o n l y s u r e w a y t o s a f e g u a r d t h e S p a n i s h e q u i n e i n d u s t r y a n d its g e n e t i c w e a l t h .
REFERENCES Anon, 1990a. Informe sobre la aparici6n de un brote de peste equina en M~ilaga. Subdirecci6n General de Sanidad Animal, Ministerio de Agricultura, Pesca y Alimentaci6n, Espatia. Anon, 199019. Peste Equina. In: Informe de la Comisi6n de la Fiebre Aftosa y otras Epizootias, 58 Sesi6n General de la Oficina Internacional de Epizootias, Paris 14-18 May, pp. 9-13. Blood, D.C., Radostits, O.M. and Henderson, J.A., 1989. Veterinary Medicine, a textbook of diseases ofacttle, sheep, pigs, goats, and horses, 7th Ed., Bailliere-Tindall, London, pp. 788. Bulletin de l'Office International des Epizooties, 1987.99: 46-48. Bulletin de l'Office International des Epizooties, 1989a. 101: 496. Bulletin de l'Office International des Epizooties, 1989b. I 01: 550. Buxton, A. and Fraser, G., 1977. Animal Microbiology, Vol. 2. Blackwell Scientific Publications Ltd, Oxford, pp. 632-635. Callis, I.J., Dardiri, A.H., Ferris, D.H., Gay, J., Mason, J. and Wilder, F.W., 1982. Illustrated manual for the recognition and diagnosis of certain animal diseases. Mexico-United States Commission for the prevention of foot and mouth disease, pp. 46-48. Dardiri, A.H., 1984. African Horse Sickness. In: Foreign Animal Diseases. Reference Manual. Vol. 1. USDA, A.P.H.I.S., NVSL, Ames, Iowa, pp. 10-19. Dardiri, A.H., 1986. Peste Equina Africana. In: Enfermedades ex6ticas de los animales. Su prevencion, diagn6stico y control. Comit6 de enfermedades ex6ticas de la Asociaci6n de Sanidad Animal de los Estados Unidos. Comisi6n Mexico-Americana para la prevenci6n de la fiebre aftosa. Mexico, D.F., pp. 302-311. Diaz-Montilla, R. and Patios Marti, P., 1967. Epizootiologia de la Peste Equina en Espatia. Bull. Office Int. Epizoot., 68: 705-714. Diaz Yubero, M.A., 1987. Informe Peste Equina en Espatia. lnformaci6n epizootiol6gica, no. Esp. 87/5/142, Office Int. Epizoot., Paris. Fenner, F., Bachmann, P.A., Gibbs, E.P.J., Murphy, F.A., Studdert, M.J. and White, D.O., 1987. Veterinary Virology, Academic Press, Orlando, pp. 587-590. French, A. and Geering, W.A., 1978. Exotic Diseases of Animals. A manual for diagnosis. Australian Government Publishing Service. Canberra, pp. 41-45. Hamblin, C., Graham, S.D., Anderson, E.C. and Crowther, J.C., 1991a. A serogroup Specific Enzyme-Linked-lmmunosorbent-Assay for the detection and identification of African Horse Sickness viruses. J. Virol. Methods, 31: 285-292. Hamblin, C., Mellor, P.S., Graham, S.D., Hooghuis, H., Montejano, R.C., Cubillo, M.A. and Boned, J., 1991b. Antibodies in horses, mules and donkeys following monovalent vaccination against African Horse Sickness. Epidemiol. Infect., 106: 365-371. Maurer, F.D. and McCully, R.M., 1963. African Horse Sickness-with emphasis on Pathology. Am. J. Vet. Res., 24: 235-266. Mellor, P.S., Hamblin, C. and Graham, S.D., 1990. African Horse Sickness in Saudi Arabia. Vet. Rec., 127: 41-42. Mohanty, S.B. and Dutta, S.K., 1981. Veterinary Virology. Lea and Febiger, Philadelphia, pp. 172-174. Oellermann, R.A., Els, H.J. and Erasmus, B.J., 1970. Characterization of African Horse sickness viruses. Archiv Ges. virusforsch., 29:163-174. Rodriguez, M., Castatio, M., Escolar, E., Flores, J., Toni, P., Gonzalez, M., Jimenez, F., Gon-
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zalez, J.L. and Montoya, J.A., 1987. Peste Equina Africana: descripci6n del brote en Espafia 1987. Med. Vet., 4: 537-557. Rodriguez, M., Castaf~o, M. and Garcia, I., 1989. Peste Equina Africana en Espa~a: Focos de 1987, 1988 y 1989. Proceedings, III Jornadas T6cnicas sobre cl caballo. Expoaviga 89, pp. 25-30. Wetzel, H., Nevill, E.M. and Erasmus, B.J., 1970. Studies on the transmission of African Horse Sickness. Onderstepoort J. Vet. Res., 37:165-168.
