Field investigations of brucellosis in cattle and small ruminants in Syria,

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1 Rev. sci. tech. Off. int. Epiz., 2001, 20 (3), Field investigations of brucellosis in cattle and small ruminants in Syria, M. Darwesh (1) & A. Benkirane (2) (1) Head of the Brucellosis Centre, Directorate of Animal Health, Ministry of Agriculture and Agrarian Reform, Veterinary Centre, Bab Sharki, Damascus, Syria (2) Département de Microbiologie, Immunologie et Maladies Infectieuses, Institut Agronomique et Vétérinaire Hassan II, B.P. 6202, Rabat-Instituts, Rabat, Morocco Submitted for publication: 24 August 2000 Accepted for publication: 5 April 2001 Summary The authors present the epidemiological status of brucellosis in cattle and small ruminants in Syria from 1990 to 1996, based on laboratory findings at the Brucellosis Centre, Damascus. Initial investigations using the Rose Bengal plate test, the complement fixation test and a miniaturised variant of the slow agglutination test were conducted throughout the country in 1990 and 1991, revealing an overall herd seroprevalence rate of 3.14% in cattle herds and 2.94% in small ruminant flocks. Although partially biased by previous vaccination of young female cattle with S19 vaccine, these figures indicate that brucellosis in cattle is widespread, particularly in the urban governorates (provinces) of Damascus, Aleppo and Suwaydah. Brucellosis seroprevalence in sheep and goats was relatively high in the governorates of Damascus, Aleppo and Dara a. The results of a second series of investigations, performed between 1992 and 1996, show that herd seroprevalence in cattle decreased steadily from 17.48% in 1992, to 2.59% in 1996, in the Government-owned farms, while seroprevalence increased in the private sector during the same period. The difference may be explained by the restriction of brucellosis vaccination to public farms (although this was far from systematic), combined with partial application of a test-andslaughter policy. In sheep and goats, brucellosis seroprevalence fluctuated in the two sectors, but was higher in the private sector where husbandry is principally extensive. Bacteriological investigations led to the isolation of Brucella melitensis biovars 2 and 3 in sheep and B. abortus biovar 9 in cattle. Although no specific methodology was employed, particularly with regard to sampling, this study is significant as the first international report of the distribution of brucellosis in Syria. Further, well-structured studies are required, the results of which could be used to plan an appropriate national control programme for brucellosis. Keywords Brucella abortus Brucella melitensis Brucellosis Cattle Epidemiology Small ruminants Syria. Introduction Syria is considered to be an agricultural country; a large proportion of the population, especially in the rural areas, depends on agriculture and primarily on animal production. Animal resources can be estimated at approximately twelve million sheep, one million goats, 800,000 cattle, 5,000 camels and a small number of buffalo raised in the region of Al-Ghab. Almost all veterinary services are delivered through the Government, operated under the Animal Health Directorate. A total of 458 veterinary clinics operate throughout the country; approximately half of these are mobile and deployed principally in Albadia, and the other half have a fixed location, including fourteen veterinary centres. The animal health programme is implemented by 823 veterinarians and 2,500 veterinary assistants. The programme consists principally of preventive vaccination, treatment of ecto- and endo-parasites and disease control.

2 770 Rev. sci. tech. Off. int. Epiz., 20 (3) Brucellosis causes considerable economic losses in livestock production, due to abortion, lack of fertility and reduction in milk production; in addition, the zoonotic nature of the disease has a serious impact on public health (2, 3). The disease is endemic in all countries of the Middle East and most Mediterranean countries (5). In Syria, brucellosis was first diagnosed in cattle at the beginning of the 1960s, in Al-Ghouta in the countryside near Damascus. During the years 1982, 1986 and 1988, 20,803 sheep and 5,800 cattle were serologically tested for brucellosis. The results revealed an individual seroprevalence of 1.36% in sheep and 3.4% in cattle, but these were not representative of the true distribution of the disease throughout the country, since most cattle originated from the rural areas of Damascus and Al-Ghab. In 1982, Brucella abortus S19 vaccine was used for the first time in female calves between four and six months of age in Damascus and Al-Ghab, although only in Government farms. Since 1989, a specialised National Brucellosis Centre has been established in Damascus, the mandate of which is to monitor the prevalence and distribution of the disease in the country and advise on a national strategy for the control of brucellosis. The present study was undertaken with a view to estimate the prevalence of brucellosis in cattle and small ruminants and the trend in prevalence, to accumulate valuable baseline information before embarking on a national mass vaccination programme, according to the guidelines of the Food and Agriculture Organization (FAO), World Health Organization (WHO) and Office International des Epizooties (OIE) for regional brucellosis surveillance and control in the Middle East (2). Despite the lack of a specific sampling procedure, these investigations provided, for the first time, an estimate of brucellosis herd/flock seroprevalence in the country. As yet, no vaccination has been undertaken in small ruminants, while among cattle, only those raised in some Government farms have been sporadically subjected to calf-hood vaccination with B. abortus strain 19. Therefore, the analysis of the serological results throughout the period can be assumed to be not substantially biased with regard to possible post-vaccinal serological sequelae, at least in small ruminants. A statisticallybased survey on brucellosis seroprevalence is currently being performed by the Regional Animal Disease Surveillance and Control Network for North Africa and the Near East (RADISCON) and the results of this survey, when available, will probably shed more light on epidemiological patterns of brucellosis in Syria. Materials and methods Sampling methodology Both series of investigations ( and ) used empirical sampling methodology, whereby blood samples were collected by the veterinary teams operating at the governorate level, upon the request of the National Brucellosis Centre, Damascus. No specific strategy was devised regarding the sampling frame, sampling fraction or the protocol of these investigations. Therefore, the results are not appropriate for statistical, probability-based interpretation. The bacteriological investigations were all conducted on aborted foetuses and other organs originating from Government farms which were monitored by the National Brucellosis Centre. Investigation I ( ) Blood samples were empirically collected in 4,842 flocks from 26,755 animals, including female sheep and goats over one year of age and some rams and billy-goats. Animals were branded with distinctive marks to distinguish the sheep and goats that were raised in contact with cattle (sedentary) from other sheep and goats (which were raised by nomads in Al- Badia). Data were subsequently recorded using a special code to identify males and aborted females. Similarly, 4,836 cattle herds were investigated and 12,554 cattle over eighteen months of age were sampled and submitted for serological testing. This included all aborted females, which were distinguished by a special mark. Investigation II ( ) Only herd/flock seroprevalence was considered in these investigations. In the public sector, the total number of cattle herds sampled varied between 541 (in 1995) and 1,231 (in 1994) and the total number of sheep flocks was between 985 (in 1996) and 9,689 (in 1993). In the privately-owned farms, seroprevalence varied between 1,352 cattle herds (in 1992) and 3,659 (in 1995), and between 1,230 sheep flocks (in 1993) and 1,950 (in 1992). Sampling was performed on an empirical basis, as the farms on which animals were sampled were visited on the request of the owner. Furthermore, to estimate the impact of brucellosis seropositivity on abortion, aborted cattle, sheep and goats were tested for brucellosis infection and the number of abortions associated with brucellosis were calculated and compared among governorates. Serological testing In both studies, all sera were tested using commercial antigens and reagents (Central Veterinary Laboratory, Weybridge, United Kingdom). The Rose Bengal plate test (RBPT) was used as a screening test and samples giving positive results were then tested by slow agglutination test in microplates (msat) and confirmed by the complement fixation test (CFT). The RBPT and CFT tests used and the thresholds for determining positivity were those recommended by Alton et al. (1). The SAT

3 Rev. sci. tech. Off. int. Epiz., 20 (3) 771 (Wright test) was miniaturised and adapted to microplates (Damascus Brucellosis Center, unpublished findings), in order to reduce the quantities of reagents required. All sera found to be positive to CFT were considered as positive results, as were anticomplementary sera which were positive to both RBPT and msat and which originated from a herd or flock in which at least one animal was found to be positive to CFT. Bacteriological testing Aborted foetuses from sheep (n = 104) and cattle (n = 43) encountered during the study were sent to the National Brucellosis Centre in Damascus for bacteriological testing. Using Brucella agar and trypticase soy agar, attempts were made to isolate Brucella strains from the stomach and the lungs of aborted foetuses. Samples were cultured for primary isolation on Farrell s modified serum dextrose agar containing a ready-mixed antibiotic supplement. Isolates were identified using recommended procedures (1). Biotyping of Brucella isolates was confirmed by the Bundesgesundsheitsamt (BGA), Berlin, Germany. Results Bacteriology Of the 104 ovine aborted foetuses tested, twenty-two (21.2%) were found to be positive. Similarly, of the forty-three aborted cattle foetuses, seven (16.2%) were found to be positive. The following biovars were isolated in the different regions: a) Brucella melitensis biovar 2 (six isolates), in aborted foetuses from three sheep flocks near Aleppo b) Brucella melitensis biovar 3 (sixteen isolates), in aborted foetuses from four sheep flocks near Damascus and from a ram with orchitis in Alraqa c) Brucella abortus biovar 9 (seven isolates), in foetuses from five cattle herds located in the Al-Ghab region. Serology Investigation I The results are summarised in Table I. The distribution of individual seropositivity and the herd/flock seroprevalence in the fourteen governorates of Syria are presented in Table II. The overall brucellosis seropositivity of approximately 3%, suggests that brucellosis is widespread in cattle, principally in the urban governorates of Damascus, Aleppo and Suwaydah. The same trend is demonstrated at the herd level, with Damascus and Aleppo being the governorates most heavily affected by bovine Table I Serological results of the national brucellosis survey, Animal Samples Positive RBPT Positive msat Positive CFT Cattle 12, (3.57%) 392 (3.12%) 358 (2.85%) Sheep/goats 26, (2.50%) 609 (2.28%) 484 (1.81%) Total 39,309 1,120 (2.85%) 1,001 (2.54%) 842 (2.15%) CFT : complement fixation test msat : slow agglutination test in microplates RBPT : Rose Bengal plate test Table II Distribution of seropositivity according to governorate, Cattle Sheep and goats District Individual Herds Individual Flocks Tested Positive (%) Tested Positive (%) Tested Positive (%) Tested Positive (%) Damascus 1, (7.77%) (10.38%) 2, (2.74%) (6.42%) Aleppo 1, (6.44%) (7.70%) 1, (5.49%) (5.80%) Idlib (4.12%) (1.70%) 1,021 7 (0.69%) (1.34%) Suwaydah (2.34%) (3.04%) (2.90%) (1.33%) Dara a (2.27%) (2.83%) 3, (1.84%) (6.50%) Al Rakka (2.00%) (2.68%) 1, (1.11%) (3.40%) Al-Ghab (1.96%) (2.56%) (1.45%) (2.40%) Qunaytira (1.47%) (2.29%) 2, (5.55%) (3.06%) Al Hasaka (1.38%) (1.85%) (0.15%) (0.60%) Lattakia (0.92%) (0.80%) 1, (0.90%) (1.59%) Deir el Zoor 1,431 9 (0.63%) (0.93%) 5, (0.85%) 98 1 (1.02%) Hama (0.34%) (0.64%) 4, (1.99%) 1, (1.75%) Homs (0.24%) (0.52%) (0.46%) 47 1 (2.12%) Tartus (0.00%) (0.00%) (0.00%) 82 0 (0.00%) Total 12, (2.85%) 5, (3.14%) 26, (2.10%) 4, (2.94%)

4 772 Rev. sci. tech. Off. int. Epiz., 20 (3) brucellosis. At the individual level, brucellosis seroprevalence in sheep and goats is close to 2% among the population tested. Aleppo is the most heavily affected governorate at the individual sheep/goat level and, together with Damascus and Dara a, hosts high proportions of brucellosis seropositive flocks. Brucellosis herd/flock seroprevalence was estimated at 2.94 in small ruminants and 3.14 in cattle. The comparison of brucellosis seropositive herds/flocks demonstrated that, with the exception of Tartus governorate, where animals were found to be brucellosis seronegative, abortion was associated with brucellosis seropositivity at rates varying between 14.30% and 45.50% in cattle and between 6.25% and 56% in sheep and goats (Table III). Table III Abortion associated with brucellosis seropositivity, District Aborted cattle Aborted sheep/goats Tested Seropositive Tested Seropositive Damascus Aleppo Suwaydah Al-Ghab Dara a Qunaytira Lattakia Idlib 15 7 (45.50%) (56.00%) Al Hasaka Al Rakka Hama Homs Deir el Zoor Tartus 9 7 Total (34.59%) 1, (19.09%) Investigation II The results of Investigation II, undertaken between 1992 and 1996, are presented in Table IV. In the Government-owned farms, the herd seropositivity rate declined steadily in cattle, from 17.48% in 1992 to 2.59% in 1996; in contrast, flock seropositivity fluctuated in sheep and goats, with a peak of 13.04% in Table IV Herd/flock seroprevalence rate in the public sector (Government-owned farms), Year Cattle herds Sheep flocks Tested Positive (%) Tested Positive (%) (17.48) 6, (3.57) (7.23) 9, (7.63) , (6.17) 4, (13.04) (3.51) 1, (7.00) , (2.59) (6.57) Unlike the public sector, the private sector suffered an increase in herd seroprevalence of bovine brucellosis over the five years of the study, while brucellosis flock seroprevalence remained high in sheep and goat flocks during the same period (Table V). Table V Herd/flock seroprevalence rate in the private sector, Year Cattle herds Sheep flocks Tested Positive (%) Tested Positive (%) , (3.03) 1, (9.94) , (3.90) 1, (13.90) , (9.48) 1, (12.88) , (17.48) 1, (10.25) , (16.58) 1, (12.87) Discussion Seroprevalence Differences observed in the results obtained by the various serological tests used in this study were essentially due to the varying levels of sensitivity and specificity (1, 4). The reported figures underestimate the seroprevalence of brucellosis, since sera reacting to RBPT were systematically confirmed by CFT, or by msat for cases in which CFT was not applicable. The limitations of SAT (and msat) are well documented, especially in small ruminants (1). However, despite being somewhat arbitrary, this approach was deemed to be the best compromise under the conditions present in Syria. Given the serial performance of the tests, seropositivity figures are likely to be far below the true brucellosis infection rate. The apparent discrepancies in the distribution of seropositivity in small ruminants, as presented in Tables I and II, are due to the fact that a number of sera were found to be anticomplementary and considered positive solely on the basis of the results obtained from RBPT and msat. The rate of seropositive animals is further underestimated, due to the sampling biases, including the fact that a very small number of animals were selected from each flock/herd investigated. The overall seroprevalence rate recorded in was 2.94% of the total sheep/goat flocks and 3.14% of cattle herds; this can be considered as a moderate to high prevalence (6). Furthermore, this study revealed that brucellosis seropositivity is widespread in cattle, sheep and goats in most governorates of Syria. The infection is likely to be more prevalent in and around urban areas where intensive farming is concentrated; this is due to the nature of the disease and the absence of control measures or restriction of these measures to the Government-owned farms. However, the results should be interpreted cautiously, given the abovementioned limitations.

5 Rev. sci. tech. Off. int. Epiz., 20 (3) 773 The second series of investigations, performed between 1992 and 1996, showed an apparent reduction in the seroprevalence of cattle brucellosis in public farms on which vaccination was partially practised, together with a test-and-slaughter policy, applied to a rather limited extent. Again, such findings need to be confirmed through further investigations. Data on the flock/herd seroprevalence, rather than individual rates would have been more valuable. In addition, a year-by-year comparison would have benefited from a comparable sampling for each year, which was not the case, particularly in the private sector. To date, no brucellosis vaccination has been undertaken in small ruminants in Syria. Thus, the seroprevalence figures observed in sheep and goats appear logical, despite the obvious sampling biases. Seroprevalence was maintained at similar levels both in public and private farms, but was much higher in the private sector, probably as a result of the partial test-andslaughter in Government-owned farms, as was the case in cattle herds. No statistical tests can be applied to compare the data obtained, as the test population is not representative of the target population (total ruminant population of Syria). In the districts of Idlib (cattle) and Suwaydah and Qunaytira (small ruminants) the individual seroprevalence rate exceeds herd/flock seroprevalence. This can be explained by the very small number of animals tested in each herd/flock and the clustering of brucellosis seroprevalence in a limited number of the herds/flocks. a) Cattle b) Small ruminants District 1. Aleppo 2. Al Hasaka 3. Al Rakka 4. Damascus 5. Dara a 6. Deir el Zoor 7. Hama 8. Homs 9. Idlib 10. Lattakia 11. Qunaytira 12. Suwaydah 13. Tartus Infection rate >3% 1%-3% <1% 0% Figure 1 suggests that among the governorates of Syria, seven (Idlib, Al Hasaka, Lattakia, Deir el Zoor, Hama, Homs and Tartus) are seronegative for brucellosis or have low infection rates. Most of these governorates are principally comprised of grazing lands, and extensive sheep farming is the most common type of animal husbandry. Table III suggests that approximately 35% and 20% of abortions are associated with brucellosis seropositivity in cattle and small ruminants, respectively. Aborted females found to be seronegative within a seropositive herd/flock could not be considered as brucellosis-free, as a certain number of these animal will be either undergoing brucellosis seroconversion, or will have become temporarily/definitively seronegative (a phenomenon that is well-documented in brucellosis [1, 3]). However, some of the reported abortions in small ruminants and cattle can be assumed to be due to causes other than brucellosis. The differences noted in the seroprevalence rates would suggest that, should Syria embark on a large-scale control programme, the present study should be supported by more representative findings, obtained by a statistically-based sample survey. Should the same epidemiological pattern be confirmed, some clustering (grouping of governorates having similar profiles) would be recommended, to enable the implementation of specific control measures according to the estimated infection rate at the time the programme is launched. Fig. 1 Distribution of brucellosis infection in herds/flocks by governorate Bacteriological findings The identification of Brucella isolates from Syria included isolates from sheep belonging to B. melitensis biovars 2 and 3; these biovars have already been reported in the Near East. One isolate from a ram with orchitis was identified as B. melitensis biovar 3, and those from sheep foetuses from Damascus and Aleppo were identified as B. melitensis biovar 2. The isolates obtained from cattle were typed as B. abortus biovar 9. Further investigations are required to establish the complete list of Brucella biovars in Syria and in the region.

6 774 Rev. sci. tech. Off. int. Epiz., 20 (3) Brucellose des bovins et des petits ruminants : enquêtes sur le terrain en Syrie, M. Darwesh & A. Benkirane Résumé Les auteurs présentent la situation épidémiologique de la Syrie, eu égard à la brucellose des bovins et des petits ruminants, de 1990 à 1996, à partir du résultat des examens effectués au laboratoire du Centre pour la brucellose de Damas. Les premières enquêtes ont été menées sur l ensemble du territoire en 1990 et 1991 ; les tests utilisés étaient l épreuve à l antigène tamponné, l épreuve de fixation du complément et une variante miniaturisée de l épreuve d agglutination lente. Les résultats ont révélé un taux de séroprévalence globale de 3,14 % dans les élevages bovins et de 2,94 % dans les élevages de petits ruminants. Quoique partiellement faussés par la vaccination antérieure des génisses avec la souche 19, ces chiffres indiquent que la brucellose bovine est répandue dans le pays, en particulier dans les gouvernorats (provinces) urbains de Damas, d Alep et de Suwaydah. La séroprévalence de la brucellose chez les ovins et les caprins était relativement élevée dans les gouvernorats de Damas, d Alep et de Dara a. Il ressort des résultats de la seconde série d enquêtes, effectuées entre 1992 et 1996, que la séroprévalence chez les bovins a régulièrement diminué dans les élevages détenus par l État, passant de 17,48 % en 1992 à 2,59 % en 1996, alors qu elle a augmenté dans le secteur privé pendant la même période. Cette différence s explique peut-être par le fait que la vaccination contre la brucellose n a été pratiquée que dans les élevages de l État (bien que de manière très peu systématique), en complément de l abattage sélectif des animaux reconnus atteints. Chez les ovins et les caprins, la séroprévalence de la brucellose a connu des fluctuations dans tous les élevages, mais elle a été plus forte dans les élevages du secteur privé qui sont essentiellement de type extensif. Les examens bactériologiques ont permis d isoler les biotypes 2 et 3 de Brucella melitensis chez les ovins et le biotype 9 de B. abortus chez les bovins. Bien qu aucune méthodologie spécifique n ait été appliquée, en particulier pour ce qui concerne les prélèvements, cette étude revêt une certaine importance, parce qu elle constitue le premier rapport international sur la répartition de la brucellose en Syrie. Des études plus approfondies et structurées devront suivre, permettant de mettre en œuvre un plan national de lutte contre la brucellose. Mots-clés Bovins Brucellose Épidémiologie Petits ruminants Syrie. Investigación de campo sobre brucelosis en bovinos y pequeños rumiantes en Siria, M. Darwesh & A. Benkirane Resumen Basándose en los resultados obtenidos en el laboratorio del Centro sobre la Brucelosis (Damasco), los autores exponen la situación epidemiológica de la brucelosis de bovinos y pequeños rumiantes en Siria entre 1990 y En 1990 y 1991 se realizaron las primeras investigaciones en todo el país, utilizando las

7 Rev. sci. tech. Off. int. Epiz., 20 (3) 775 pruebas de rosa de Bengala y de fijación del complemento, así como una variante miniaturizada de la prueba de aglutinación lenta. Esas investigaciones revelaron una tasa global de seroprevalencia por rebaños del 3,14% para los bovinos y del 2,94% para los pequeños rumiantes. Tales cifras, aunque parcialmente sesgadas por la aplicación previa de vacunas S19 a terneras, indicaban la abundante presencia de brucelosis en el ganado vacuno, especialmente en las provincias urbanas de Damasco, Aleppo y Suwaydah. La seroprevalencia de la enfermedad en ovejas y cabras resultó relativamente alta en las provincias de Damasco, Aleppo y Dara a. Los resultados de una segunda tanda de investigaciones efectuadas entre 1992 y 1996 indican que la seroprevalencia por rebaños registró una caída constante entre los bovinos de las granjas de propiedad pública (del 17,48% en 1992 al 2,59% en 1996), a la par que aumentaba en el sector privado durante el mismo periodo. Esa diferencia puede explicarse por la decisión de administrar vacunas únicamente en granjas públicas (aunque esa disposición distara mucho de aplicarse sistemáticamente), combinada con la aplicación parcial de medidas de sacrificio sanitario selectivo. En el caso de ovejas y cabras, y aunque los valores de seroprevalencia resultaron fluctuantes tanto en el sector público como en el privado, se observaron valores más altos en este último, caracterizado por la aplicación de técnicas agropecuarias básicamente extensivas. Las investigaciones bacteriológicas llevaron al aislamiento de los biovares 2 y 3 de Brucella melitensis en la oveja y el biovar 9 de B. abortus en los bovinos. La importancia de este estudio, pese a su falta de metodología específica (sobre todo en cuanto a procedimientos de muestreo), radica en su condición de primer informe internacional sobre la distribución de la brucelosis en Siria. Es necesario proceder a nuevos estudios bien estructurados, cuyos resultados podrían utilizarse para elaborar un adecuado programa nacional de lucha contra la brucelosis. Palabras clave Bovinos Brucelosis Epidemiología Pequeños rumiantes Siria. References 1. Alton G.G., Jones L.M., Angus R.D. & Verger J.M. (1988). Techniques for the brucellosis laboratory. Institut National de Recherche Agronomique, Paris, 190 pp. 2. Anon. (1997). Regional brucellosis control programme for the Middle East. In Food and Agriculture Organization (FAO), World Health Organization, Office International des Epizooties (OIE) guidelines prepared at the workshop by Drs Mustafa and Nicoletti, FAO consultants, February 1993, Amman, Jordan, amended at the round table meeting, September 1995, Maisons-Alfort. OIE, Paris, 4 pp. 3. Garin-Bastuji B., Blasco J.M., Grayon M. & Verger J.M. (1998). B. melitensis infection in sheep: present and future. Vet. Res., 29, MacMillan A.P. & McGiven J. (2000). Immunological aspects of brucellosis diagnosis. In Brucellosis 2000, including the 53rd Brucellosis research conference, Scientific Program and Abstracts, 7-9 September, Nîmes (J.-J. Letesson, ed.). Nîmes, Manes G. (1984). Epidemiological situation of brucellosis in Mediterranean countries. Dev. biol. Standard., 56, Robinson R.A. (2001). Guidelines for co-ordinated human and animal brucellosis surveillance. Food and Agriculture Organization (FAO)/World Health Organization (WHO), Rome/Geneva (in press).