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Incidence of brucellosis in Sri Lanka: an overview
Veterinary Microbiology 90 (2002) 197±207
Incidence of brucellosis in Sri Lanka: an overview A.B. Bandaraa,*, M.B. Mahipalab a
Center for Molecular Medicine and Infectious Diseases, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA b Department of Animal Science, University of Peradeniya, Peradeniya, Sri Lanka
Abstract Infection by Brucella abortus seems to be a major cause of abortions among cattle and buffaloes in Sri Lanka. The incidence of this disease is more prominent among the animals in the Dry zone of the country raised under extensive management systems. The present low incidence of this disease and the small size of the country may facilitate launching of an effective disease control scheme. The milk ring test (MRT) has proven to be usable in testing milk for the infection at farm level. An ELISA technique could be employed to test the seroprevalence of infection among MRT-positive animals. A program to purchase the diseased animals by the state for slaughter, and a countrywide vaccination program with B. abortus strain RB51 would enable the country's livestock industry to eventually eradicate this disease. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Sri Lanka; Brucellosis; Brucella abortus; Milk ring test; RB51
1. Introduction Brucellosis has been an emerging disease since the discovery of B. melitensis by Bruce in 1887. Subsequently, an increasingly complex pattern of strains has emerged with the identi®cation of B. abortus, B. suis, B. neotomae, B. ovis, B. canis and, more recently, types infecting marine mammals. Worldwide, brucellosis remains a major source of disease in humans and domesticated animals (FAO-WHO-OIE, 1994). Each Brucella species has a preferred natural host that serves as a reservoir of infection (Quinn et al., 1994). Although reported incidence and prevalence of the disease vary widely from country to country, bovine brucellosis caused mainly by B. abortus is still the most * Corresponding author. Tel.: 1-540-231-4002; fax: 1-540-231-3426. E-mail address: [email protected] (A.B. Bandara).
0378-1135/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 2 5 4 - 7
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widespread form (FAO-WHO-OIE, 1994). Bovine brucellosis is essentially a disease of the sexually mature animals with a predilection for ungulate placentas, fetal ¯uids and testes of male animals. B. abortus is excreted in bovine milk and can remain viable in milk, water and damp soil for up to 4 months. The infection is transmitted by direct or indirect contact with infective excretes (often via ingestion and also via venereal routes). The mostcommon clinical feature of brucellosis in cattle and buffaloes is abortion, in addition, it can cause calving and breeding-associated problems such as repeat breeding, retained placenta, metritis, weak calves and low milk production. Infected females usually abort only once after which a degree of immunity develops and the animals remain infected. Large numbers of Brucellae can be excreted in fetal ¯uids in such bovids at subsequent parturitions. The spread of the disease to man results from ingestion of raw or unpasteurized infected milk or dairy products, inhalation of contaminated dust, and contact with infected uterine contents, discharges and infected carcasses (Blood and Radostits, 1989; Nielsen, 1997). The diagnosis, prevention and eradication of brucellosis in cattle and buffaloes are important because of public-health implications. In this paper, we review the status of the incidence of brucellosis in Sri Lanka, and discuss the potential for undertaking effective brucellosis control measures. 2. Physical and climatic features of Sri Lanka Sri Lanka is an Island located between 6 and 108 latitude north of equator, at the southern tip of the Indian sub-continent. A total of 6.56 million hectare of land makes up this island. In Sri Lanka the most important weather parameter that has the greatest impact on agriculture and animal production is the amount of rainfall. The country is under the in¯uence of two monsoons, namely south±west and north±east, which occur alternatively bringing rain to south west part and north east part of the island respectively. In addition, conventional rain occurs during inter-monsoonal periods. Based on the amount of rainfall received annually, Sri Lanka is divided into three climatic zones, namely, the Wet zone, the Intermediate zone and the Dry zone, which cover 23, 13 and 64% land area, respectively. These three climatic zones are further divided into seven agro-ecological zones (as shown in Table 1) based on the altitude from the sea level and the annual rainfall (Land and Water Use Division, Department of Agriculture, 1979). The Wet zone receives a year around supply of rainfall suf®cient for satisfactory pasture growth through out the year. However, the Intermediate and the Dry zones receive rain only during two seasons (March±May and October±December). During the non-rainy seasons in these two zones, pasture availability is seriously affected leading to poor nutrition of farm animals. 3. Animal production in Sri Lanka A bovid population of 2,319,700 was reported in Sri Lanka according to the latest census and statistics published in the year 1998 (Table 2; Census and Statistical Department, 1998). Most of Sri lanka's animal farmers are small-holders keeping 1±5 cattle and/or
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Table 1 Environmental parameters of major agro ecological regions in Sri Lanka Agro ecological region
Elevation (m)
Mean temperature (8C)
Mean rainfall (mm)
Wet zone Up country Mid country Low country
1000±2000 500±1000 0±300
10±15 15±20 20±25
2500±5000 2000±3000 2000±3000
Intermediate zone Up country Mid country Low country
1000±1500 350±500 0±300
15±22 24±26 25±29
1500±2250 1500±2250 2000±2200
28±30
900±1000
Dry zone Low country
0±300
Source: Map of agro ecological regions of Sri Lanka. Land and Water Use Division, Department of Agriculture, 1979.
buffaloes per farm. There are a few large-scale farms each with 20±200 cows where cattle and buffaloes are raised as dairy animals. High productive temperate dairy cattle breeds like Friesian and Jersey are raised in the Wet zone areas without any major constrain. Jersey, Indian high productive cattle breeds like Sindhi and Tharpakar, and their crosses are popular among farmers in the Intermediate zone. The low productive indigenous cattle types and their crosses with Indian breeds are abundant in the Dry zone. High productive buffalo breeds like Murrah, Niliravi and Surthi are popular among farmers in the Table 2 Livestock production in Sri Lanka in 1998 Livestock type
Number of animals
Neat cattle Milk cows Other cows Bulls Calves
682,700 305,600 254,400 356,300
Buffaloes Milk cows Other cows Bulls Calves
227,300 187,500 151,800 154,100
Goats Sheep Pigs
519,300 11,800 76,300
Average monthly production (l) Cow milk Buffalo milk
21,361,400 7,087,000
Source: Statistical Abstract. Department of Census and Statistics, Colombo, Sri Lanka, 1998.
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Intermediate zone. Indigenous water buffalo and their crosses are raised in the Dry zone. Sheep are raised in a few parts of the Intermediate zone whereas goats are popular in the Intermediate and Dry zones. In the Dry and Intermediate zones, the cattle and buffaloes are mostly managed under the extensive system. In this system, the animals are allowed to graze freely during daytime and kept in open paddocks during the night. Some farmers in these zones practice semi-intensive management system in which the animals are tethered on pasture at daytime and are tethered in a stall in the night. Under the intensive system, which is commonly practiced in the Wet zone, the animals are kept inside and managed both day and night. Sri Lanka is divided into 24 administrative districts, and each district is again divided into three to 20 administrative divisions. A veterinary surgeon is allocated for each administrative division to take care of animal health issues. Nearly, 50 veterinary surgeons are graduated each year from the Faculty of Veterinary Medicine in the University of Peradeniya. The national Veterinary Research Institute in Kandy (VRI) investigates and monitors the animal disease situation in the country, and provides necessary advise to the ®eld veterinary surgeons. 4. Diagnosis of bovine brucellosis in Sri Lanka Except in a few large-scale farms, the tests are not usually employed for diagnosis of bovine brucellosis. However, a number of conventional and non-conventional tests are being employed by the researchers for diagnosis of this disease. These tests include. 4.1. Milk ring test (MRT) This test is based on agglutination of antibodies present in the milk sample with an antigen employed (Alton et al., 1975). Extensive studies at VRI have proved that by ®ner modi®cation of the original method, MRT could be adopted even to test milk of individual cows in a herd. Souring of milk in transit particularly in the tropics imposes a severe limitation on the usefulness of the test, but this can be circumvented by preserving the milk samples with Merthiolate. It has also been determined that the incubation of the test sample for agglutination reaction at room temperature for 2 h, gives the same results as at 37 8C for 1 h within an incubator. An added advantage of this test is that it could be undertaken at the ®eld level with minimum facilities (Wickramasuriya and Kumaraswamy, 1982). 4.2. Rose Bengal plate test (RBPT) The original test method was described by Alton et al. (1975). The antigen used in the test is prepared at VRI, using a bacterial culture originally imported from the United States of America. Rose Bengal Dye is used for staining and could be used at the ®eld level (Wickramasuriya and Kumaraswamy, 1982).
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4.3. Standard agglutination test (SAT) The antigen used in the test is prepared at VRI, from a culture originally imported from the United Kingdom and is standardised against International anti-Brucella abortus serum level (Wickramasuriya and Kumaraswamy, 1982). The test is carried out as described by Alton et al. (1975). 4.4. Coombs test (CT) The test determines enhancement of titres above the level obtained in the SAT. The ordinary SAT test is used as the starting point for the CT. The test is done as originally described by Alton et al. (1975). If there is enhancement of the titre to the level normally considered positive on the SAT, the serum sample is regarded as positive (Wickramasuriya and Kumaraswamy, 1982). 4.5. Complement fixation test (CFT) The test is done as originally described by Hill (1963). The antigen used in this test is the standardised CFT antigen obtained from Central Veterinary Laboratory at Weybridge in the United Kingdom (Wickramasuriya and Kumaraswamy, 1982). 4.6. Enzyme-linked immunosorbent assay (ELISA) Brucella eradication schemes have relied upon serological tests to detect seropositive animals. Dif®culties may occur when bovids with long-standing infection remain serologically negative as a result of antibody turnover. Such bovids become serologically positive during or after pregnancy (Sutherland and Mackenzie, 1983). Therefore, lack of serum antibodies does not necessarily indicate non-exposure to the organism because, it may be dif®cult to detect this disease by serological means alone in herds with longstanding infection (Sutherland and Mackenzie, 1983). Therefore, seroprevelance may indicate currently infected animals or a successful immune response by the host to a previous infection (Sutherland, 1984). ELISA positively detects animals that test negative with the above conventional tests. Therefore, this assay can detect bovids with persisting low titres (Sutherland, 1985) and provides an extra criterion in the selection of seropositive bovids (Hornitzky and Searson, 1986). In a recent countrywide study, the indirect ELISA technique was successfully used to estimate the seroprevalence of B. abortus in bovids in Sri Lanka (details to follow). 5. Incidence of brucellosis in Sri Lanka The incidence of B. abortus infections in Sri Lanka has been reported higher than that of the other neighboring countries of the South Asia (Table 3). Incidences of B. melitensis and B. ovis also have been reported from Sri Lanka, but there are no reports about B. suis infections from this island (FAO-WHO-OIE, 1994).
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Table 3 Brucellosis among animals in South Asian countries, 1994 Country
Bovine (B. abortus)
Ovine/caprine (B. melitensis)
Porcine (B. suis)
Ovine (B. ovis)
Afghanistan Bangladesh Bhutan India Sri Lanka
ND ND
?
ND ND ND
( ): Not present; (): low sporadic incidence; (): high incidence; (?): presence uncertain; ND: no data. Source: FAO-WHO-OIE (1994).
From the available data, it has not been possible to state whether brucellosis had existed in the indigenous stock of this country in early times or not. It is quite probable that this disease could have been introduced into Sri Lanka during the second world war in the early forties, from the animals imported for the British Army. The biotype 3 of B. abortus was reported as the causative organism (Kumaraswamy, 1971). Bovine brucellosis was ®rst reported in Sri Lanka in 1956 by the Livestock Research Station in the Polonnaruwa district that maintained Sindhi and Tharpakar breeds of cattle (Pillai and Kumaraswamy, 1957). From this farm, the infection probably spread to the other areas due to transfer of animals between farms (Kumaraswamy, 1971). Wickramasuriya and Kumaraswamy (1982) surveyed the incidence of brucellosis in dairy herds in the districts of Colombo and Gampaha using MRT. The incidences were con®rmed by quantitative tests like SAT, CFT, and CT on suspected sera. In all, 305 herds involving 1478 dairy cows were tested. A herd infection of 1.6% and an individual animal infection of 0.9% were reported. Wickramasuriya et al. (1983) tested the bulk milk received at the milk collection centres in the districts of Polonnaruwa, Batticaloa and Ampara for brucellosis, using MRT. The herd incidence in the Polonnaruwa, Batticaloa and Ampara districts were 7.7, 12.0 and 14.0%, respectively (Table 4). In Polonnaruwa district, the incidence of reactor milking cows decreased with the size of the herd (Table 5). This could be attributed to the management systems practised in the district. The spread of the infection is easier in small herds that are normally con®ned in a limited space. In the case of large herds, animals are kept in the large tracts of grasslands and if an abortion occurs in the ®eld, the foetus is subjected to intense sunlight and heat,
Table 4 Incidence of reactor milking cows in MRT-positive herds in Polonnaruwa, Batticaloa and Ampara districts District
Number of cows tested
Positive reactors (%)
Polonnaruwa Batticaloa Ampara
430 191 121
7.7 12.0 14.0
Source: Wickramasuriya et al. (1983).
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Table 5 Incidence of reactor milking cows in MRT-positive herds classified according to herd size in Polonnaruwa district Number of milking cows in herd
Number of cows tested
Reactors (%)
1±5 6±10 11±20 21±30 31±50
31 43 232 22 164
32.3 11.6 11.2 9.1 4.9
Source: Wickramasuriya et al. (1983).
which would destroy the Brucella organisms thereby minimising the risk of the spread of the infection (Wickramasuriya et al., 1983). In a countrywide study carried out by Silva et al. (2000), an indirect-ELISA technique was employed to estimate the seroprevalence of B. abortus antibodies in cattle and buffaloes. In this study, the bovids in privately owned herds that had never been vaccinated, were used; therefore, the seropositivity reported indicated the natural exposure to B. abortus. The overall seroprevalence of B. abortus in bovids was reported as 4.6% (4.7% in cattle and 4.2% in buffaloes; 3.6% in male animals and 4.9% in female animals). Female bovids 3 years or older were two-times more likely of being seropositive compared to those that were below 3 years (Table 6). This was expected because brucellosis is essentially a disease of mature animals. The seroprevalence rates were not different between cattle and buffaloes (Silva et al., 2000). Bovids from the Dry zone were at approximately six-times higher risk of being seropositive compared to those from the other zones, after adjusting for the possible confounding effects of age and management methods practiced. Female bovids from the Dry zone were approximately at ®ve-times higher risk of being seropositive compared to Table 6 Seroprevalence of B. abortus antibodies in bovids in Sri Lanka grouped according to potential risk factors (1992±1995) Potential risk factor
Cattle
Buffaloes
Number tested
Seropositive (%)
Number tested
Seropositive (%)
Age >3 years <3 years
1643 1433
6 3
388 452
5 3
Ecological zone Dry zone Other zones
832 2244
4 5
333 507
8 2
Management system Extensive Other
1886 1190
6 3
541 299
4 4
Source: Silva et al., 2000.
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Table 7 Clinical signs recorded and the seropositivity in female bovids over 3 years of age in Sri Lanka (1992±1995) Clinical signs
Seropositivity
Cattle (n 1470)
Buffaloes (n 312)
Aborted at least once
Positive Negative
29 30
9 16
Repeat breeding
Positive Negative
0 25
0 2
Other signsa
Positive Negative
4 15
1 3
Source: Silva et al. (2000). a Retained placenta, still birth, and weak calf.
those from the other zones. The prevalence in bovids in the Dry zone managed extensively was 11% compared to 1.9% in other management systems. Female bovids reared under extensive management system were at approximately two-times higher risk of being seropositive compared to those that were reared under other management systems (Silva et al., 2000). Hot weather (Table 1) and poor nutrition likely contributed for high seroprevalence among bovids in the Dry zone. Extensive management systems facilitate unrestricted animal to animal contact, leading easy and quick spread of the disease. The association between the recorded clinical signs and seropositivity in females over 3 years of age recorded in this study is shown in the Table 7 (Silva et al., 2000). Bovids with a history of abortion were at higher risk of being seropositive compared to those without a history of abortion. Only 49% of the cattle and 36% of buffaloes experiencing abortion were seropositive. Among the seropositive females of over 3 years old, 25% of the cattle and 40% of the buffaloes had aborted at least once. None of the sampled bovids with a history of repeat breeding (25 cattle and 2 buffaloes) had detectable levels of antibodies. Twenty-three bovids were reported to have shown other clinical signs suggestive of brucellosis, and only ®ve of them were seropositive (Silva et al., 2000). These ®ndings suggest that Brucella could be responsible for a substantial proportion of abortions in bovids in Sri Lanka. Nonetheless, other causes of abortions in cattle and buffaloes in Sri Lanka should be investigated to identify their etiologies. All repeat breeders n 27 and a substantial number of bovids with other clinical signs suggestive of brucellosis (18 of 23) were seronegative. Therefore, it would be of interest to identify and quantify the causes for repeat breeding and retained placenta in bovids in Sri Lanka. 6. Brucellosis in human and other animals in Sri Lanka There have been isolated incidences of ovine brucellosis due to B. ovis infection and ovine/caprine brucellosis due to B. melitensis (FAO-WHO-OIE, 1994). Although human brucellosis in Sri Lanka is not common, the disease was recognized in Sri Lanka during the early 1960s in studies of unknown fevers (Pyrexia's of unknown origin, PUO). The acute fever in humans may progress to a more chronic form with involvement of the musculoskeletal system and other organs, including orchitis (in¯ammation of the testicles) in
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males (de Alwis, 2002). Among humans, the most vulnerable populations are veterinarians, livestock farm and abattoir workers and others closely associated with susceptible animals. Infection usually occurs through skin abrasions or through the conjunctiva. Infection through milk is virtually non-existent since the consumption of raw, unprocessed milk is not a practice in Sri Lanka (de Alwis, 2002). 7. Control of brucellosis in Sri Lanka So far, a large-scale control program has not been adopted in Sri Lanka. Serological testing, separation and occasional elimination of positive reactors have been successful in reducing the incidence of brucellosis in state-owned cattle farms (Peiris, 1981). Vaccination with reduced dose (1/20th of the dose) of B. abortus S19 has been used in a few state-owned farms (Peiris et al., 1983). Inadequate laboratory facilities and manpower and ®nancial restrictions are constraints to a control program for brucellosis in Sri Lanka. Private owners are very reluctant to adopt control measures particularly those requiring the slaughtering of infected animals. Generally, when farmers are informed of a positive result of a diagnostic test, they tend to sell their animals to a different farmer; this avoids religious and cultural taboos, and minimizes their economic loss. This practice alone contributes to spread of infection. Therefore, if a test-and-slaughter policy were to be implemented as a control measure, many farmers would be noncooperative. Infection in animals with B. abortus can be prevented to some extent by the use of appropriate vaccines. Live vaccines as well as inactivated ones have been developed. However, live vaccines have proven to induce better protective immunity (Schurig et al., 1995). Live vaccine B. abortus strain 19 has been used extensively in other parts of the world, to protect cattle against infection. Peiris et al. (1983) showed that vaccination with B. abortus strain 19 signi®cantly lower the disease incidence. In this study, the animals in a number of government cattle farms in Sri Lanka were immunised with one-twentieth the recommended dose of the vaccine. Subsequently, at different stages, the antibody levels in the serum samples were determined by the SAT. It was observed that at 2 weeks after vaccination, 90.9% of the calves and 92.5% of the adults had very high antibody titres of 1 in 320 or over, re¯ecting the effectiveness of the vaccine at low dosage. At 22 weeks after vaccination, 93.1% of the calves and 75.05%, of the adults had negative titres of 1 in 20 or less. These negative and low antibody titres indicated minimum trouble with residual titres. No abortions were reported when the cows in various stages of pregnancy were immunised at the same dosage of vaccine. This absence of post-vaccinal abortion proved that vaccination of whole herds was practicable. However, the strain 19 vaccine has several undesirable characteristics. These include: its ability to induce serum and milk antibodies if given in inappropriate dosages; and, the vaccine strain is relatively virulent and can induce abortion as well as persistent infection of some of the vaccinated animals (Schurig et al., 1995). B. abortus strain RB51 is a rough attenuated and stable mutant, which appears to have characteristics of an ideal Brucella vaccine. This strain can be used live to vaccinate cattle without inducing positive Brucella serology regardless of the age of the vaccinated animal,
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the route of inoculation, or the frequency of vaccinations. The strain is able to induce protective immunity in cattle against B. abortus infection (Schurig et al., 1995). 8. Necessity of an effective brucellosis control scheme for Sri Lanka According to the limited survey ®ndings, it is clear that the incidence of bovine brucellosis is low in Sri Lanka (Wickramasuriya and Kumaraswamy, 1982; Wickramasuriya et al., 1983; Silva et al., 2000). Experience in other countries has shown that control and eradication are far more practicable when the percentage of infection is low. Being a small country, in Sri Lanka, the eradication of brucellosis could be achieved quite easily provided the government could allocate adequate resources and enforce necessary measures. An effective milk-testing program at the farm level using MRT would be the ®rst step of a brucellosis eradication program. Subsequently, the animals positive to MRT should be tested by ELISA for the seroprevalence of antibodies to Brucellae. Since many farmers are reluctant to dispose their infected animals mostly due to economic reasons, a scheme for compulsory purchase of such animals by state for slaughter is necessary. In parallel, a countrywide vaccination scheme against Brucella is necessary and an attenuated vaccine strain B. abortus RB51 would be an ideal choice for this task. References Alton, G.G., Lois, M.J., Pietz, D.E., 1975. Laboratory Techniques in Brucellosis. Monograph Series No. 55, World Health Organisation, Geneva. Blood, D.C., Radostits, O.M., 1989. Diseases caused by Brucella spp. Veterinary Medicine, 7th ed. Bailliere Tindall, London, pp. 677±690. Census and Statistical Department,1998. Statistical Abstract. Colombo, Sri Lanka. de Alwis, M.C.L., 2002. Animal healthÐpublic health concerns. The Daily News, The Associated Newspapers of Ceylon Ltd., 19 January 2002. FAO-WHO-OIE Animal Health Yearbook, 1994. FAO Rome, 1964. Hill, W.K.W., 1963. Bull. OIT. Int. Epizoot. 60, 401. Hornitzky, M., Searson, J., 1986. The relationship between the isolation of Brucella abortus and serological status of infected, non-vaccinated cattle. Aust. Vet. J. 63, 172±175. Kumaraswamy, S., 1971. The significance of brucellosis in Ceylon. Ceylon Vet. J. 4, 119±123. Land and Water Use Division. Department of Agriculture, Peradeniya, Sri Lanka, 1979. The map of the agroecological zones of Sri Lanka. Nielsen, K., 1997. Brucellosis: development and success using ELISAs for diagnosis. In: Proceedings of the International Symposium on Diagnosis and Control of Livestock Diseases using Nuclear and Related Techniques, Vienna, Austria, 7±11 April. Peiris, G.S., 1981. Reproductive disorders of livestock. Sri Lanka Vet. J. 29, 25. Peiris, G.S., Wickramasuriya, U.G.J.S., Karunasena, S.B., Karunadasa, W.M., 1983. Control of brucellosis: reduced dose whole-herd vaccination with Brucella abortus strain 19. Sri Lanka Vet. J. 31, 49±50. Pillai, C.P., Kumaraswamy, S., 1957. Infertility studies among dairy animals in Ceylon. Ceylon Vet. J. 5, 8±18. Quinn, P.J., Carter, M.E., Markey, B., Carter, G.R., 1994. Brucella species. Clinical Veterinary Microbiology. Wolfe Publishing, Spain, pp. 261±267. Schurig, G., Boyle, S., Sriranganathan, N., 1995. Brucella abortus vaccine strain RB51: a brief review. Arch. Med. Vet. XXVII, 19±22.
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Silva, I., Dangolla, A., Kulachelvy, K., 2000. Seroepidemiology of Brucella abortus infection in bovids in Sri Lanka. Prev. Vet. Med. 46 (1), 51±59. Sutherland, S.S., 1984. Evaluation of the enzyme linked immunosorbent assay in the detection of cattle infected with Brucella abortus. Vet. Microbiol. 10, 23±32. Sutherland, S.S., 1985. Comparison of enzyme linked immunosorbent assay and complement fixation test for the detection of specific antibody in cattle vaccinated and challenged with B. abortus. J. Clin. Microbiol. 22, 44±47. Sutherland, S.S., Mackenzie, R.M., 1983. Applied serology in the latter stages of the eradication of bovine brucellosis. Aust. Vet. J. 60, 240±242. Wickramasuriya, U.G.J.S., Kumaraswamy, S., 1982. The incidence of brucellosis in the district of Colombo. J. Natl. Sci. Council Sri Lanka 10, 99±105. Wickramasuriya, U.G.J.S., Peiris, G.S., Kendaragama, K.M.T., Karunadasa, W.M., 1983. A survey on the incidence of bovine brucellosis in three districts in Sri Lanka. Sri Lanka Vet. J. 31, 27±31.
Incidence of brucellosis in Sri Lanka: an overview A.B. Bandaraa,*, M.B. Mahipalab a
Center for Molecular Medicine and Infectious Diseases, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA b Department of Animal Science, University of Peradeniya, Peradeniya, Sri Lanka
Abstract Infection by Brucella abortus seems to be a major cause of abortions among cattle and buffaloes in Sri Lanka. The incidence of this disease is more prominent among the animals in the Dry zone of the country raised under extensive management systems. The present low incidence of this disease and the small size of the country may facilitate launching of an effective disease control scheme. The milk ring test (MRT) has proven to be usable in testing milk for the infection at farm level. An ELISA technique could be employed to test the seroprevalence of infection among MRT-positive animals. A program to purchase the diseased animals by the state for slaughter, and a countrywide vaccination program with B. abortus strain RB51 would enable the country's livestock industry to eventually eradicate this disease. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Sri Lanka; Brucellosis; Brucella abortus; Milk ring test; RB51
1. Introduction Brucellosis has been an emerging disease since the discovery of B. melitensis by Bruce in 1887. Subsequently, an increasingly complex pattern of strains has emerged with the identi®cation of B. abortus, B. suis, B. neotomae, B. ovis, B. canis and, more recently, types infecting marine mammals. Worldwide, brucellosis remains a major source of disease in humans and domesticated animals (FAO-WHO-OIE, 1994). Each Brucella species has a preferred natural host that serves as a reservoir of infection (Quinn et al., 1994). Although reported incidence and prevalence of the disease vary widely from country to country, bovine brucellosis caused mainly by B. abortus is still the most * Corresponding author. Tel.: 1-540-231-4002; fax: 1-540-231-3426. E-mail address: [email protected] (A.B. Bandara).
0378-1135/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 2 ) 0 0 2 5 4 - 7
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widespread form (FAO-WHO-OIE, 1994). Bovine brucellosis is essentially a disease of the sexually mature animals with a predilection for ungulate placentas, fetal ¯uids and testes of male animals. B. abortus is excreted in bovine milk and can remain viable in milk, water and damp soil for up to 4 months. The infection is transmitted by direct or indirect contact with infective excretes (often via ingestion and also via venereal routes). The mostcommon clinical feature of brucellosis in cattle and buffaloes is abortion, in addition, it can cause calving and breeding-associated problems such as repeat breeding, retained placenta, metritis, weak calves and low milk production. Infected females usually abort only once after which a degree of immunity develops and the animals remain infected. Large numbers of Brucellae can be excreted in fetal ¯uids in such bovids at subsequent parturitions. The spread of the disease to man results from ingestion of raw or unpasteurized infected milk or dairy products, inhalation of contaminated dust, and contact with infected uterine contents, discharges and infected carcasses (Blood and Radostits, 1989; Nielsen, 1997). The diagnosis, prevention and eradication of brucellosis in cattle and buffaloes are important because of public-health implications. In this paper, we review the status of the incidence of brucellosis in Sri Lanka, and discuss the potential for undertaking effective brucellosis control measures. 2. Physical and climatic features of Sri Lanka Sri Lanka is an Island located between 6 and 108 latitude north of equator, at the southern tip of the Indian sub-continent. A total of 6.56 million hectare of land makes up this island. In Sri Lanka the most important weather parameter that has the greatest impact on agriculture and animal production is the amount of rainfall. The country is under the in¯uence of two monsoons, namely south±west and north±east, which occur alternatively bringing rain to south west part and north east part of the island respectively. In addition, conventional rain occurs during inter-monsoonal periods. Based on the amount of rainfall received annually, Sri Lanka is divided into three climatic zones, namely, the Wet zone, the Intermediate zone and the Dry zone, which cover 23, 13 and 64% land area, respectively. These three climatic zones are further divided into seven agro-ecological zones (as shown in Table 1) based on the altitude from the sea level and the annual rainfall (Land and Water Use Division, Department of Agriculture, 1979). The Wet zone receives a year around supply of rainfall suf®cient for satisfactory pasture growth through out the year. However, the Intermediate and the Dry zones receive rain only during two seasons (March±May and October±December). During the non-rainy seasons in these two zones, pasture availability is seriously affected leading to poor nutrition of farm animals. 3. Animal production in Sri Lanka A bovid population of 2,319,700 was reported in Sri Lanka according to the latest census and statistics published in the year 1998 (Table 2; Census and Statistical Department, 1998). Most of Sri lanka's animal farmers are small-holders keeping 1±5 cattle and/or
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Table 1 Environmental parameters of major agro ecological regions in Sri Lanka Agro ecological region
Elevation (m)
Mean temperature (8C)
Mean rainfall (mm)
Wet zone Up country Mid country Low country
1000±2000 500±1000 0±300
10±15 15±20 20±25
2500±5000 2000±3000 2000±3000
Intermediate zone Up country Mid country Low country
1000±1500 350±500 0±300
15±22 24±26 25±29
1500±2250 1500±2250 2000±2200
28±30
900±1000
Dry zone Low country
0±300
Source: Map of agro ecological regions of Sri Lanka. Land and Water Use Division, Department of Agriculture, 1979.
buffaloes per farm. There are a few large-scale farms each with 20±200 cows where cattle and buffaloes are raised as dairy animals. High productive temperate dairy cattle breeds like Friesian and Jersey are raised in the Wet zone areas without any major constrain. Jersey, Indian high productive cattle breeds like Sindhi and Tharpakar, and their crosses are popular among farmers in the Intermediate zone. The low productive indigenous cattle types and their crosses with Indian breeds are abundant in the Dry zone. High productive buffalo breeds like Murrah, Niliravi and Surthi are popular among farmers in the Table 2 Livestock production in Sri Lanka in 1998 Livestock type
Number of animals
Neat cattle Milk cows Other cows Bulls Calves
682,700 305,600 254,400 356,300
Buffaloes Milk cows Other cows Bulls Calves
227,300 187,500 151,800 154,100
Goats Sheep Pigs
519,300 11,800 76,300
Average monthly production (l) Cow milk Buffalo milk
21,361,400 7,087,000
Source: Statistical Abstract. Department of Census and Statistics, Colombo, Sri Lanka, 1998.
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Intermediate zone. Indigenous water buffalo and their crosses are raised in the Dry zone. Sheep are raised in a few parts of the Intermediate zone whereas goats are popular in the Intermediate and Dry zones. In the Dry and Intermediate zones, the cattle and buffaloes are mostly managed under the extensive system. In this system, the animals are allowed to graze freely during daytime and kept in open paddocks during the night. Some farmers in these zones practice semi-intensive management system in which the animals are tethered on pasture at daytime and are tethered in a stall in the night. Under the intensive system, which is commonly practiced in the Wet zone, the animals are kept inside and managed both day and night. Sri Lanka is divided into 24 administrative districts, and each district is again divided into three to 20 administrative divisions. A veterinary surgeon is allocated for each administrative division to take care of animal health issues. Nearly, 50 veterinary surgeons are graduated each year from the Faculty of Veterinary Medicine in the University of Peradeniya. The national Veterinary Research Institute in Kandy (VRI) investigates and monitors the animal disease situation in the country, and provides necessary advise to the ®eld veterinary surgeons. 4. Diagnosis of bovine brucellosis in Sri Lanka Except in a few large-scale farms, the tests are not usually employed for diagnosis of bovine brucellosis. However, a number of conventional and non-conventional tests are being employed by the researchers for diagnosis of this disease. These tests include. 4.1. Milk ring test (MRT) This test is based on agglutination of antibodies present in the milk sample with an antigen employed (Alton et al., 1975). Extensive studies at VRI have proved that by ®ner modi®cation of the original method, MRT could be adopted even to test milk of individual cows in a herd. Souring of milk in transit particularly in the tropics imposes a severe limitation on the usefulness of the test, but this can be circumvented by preserving the milk samples with Merthiolate. It has also been determined that the incubation of the test sample for agglutination reaction at room temperature for 2 h, gives the same results as at 37 8C for 1 h within an incubator. An added advantage of this test is that it could be undertaken at the ®eld level with minimum facilities (Wickramasuriya and Kumaraswamy, 1982). 4.2. Rose Bengal plate test (RBPT) The original test method was described by Alton et al. (1975). The antigen used in the test is prepared at VRI, using a bacterial culture originally imported from the United States of America. Rose Bengal Dye is used for staining and could be used at the ®eld level (Wickramasuriya and Kumaraswamy, 1982).
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4.3. Standard agglutination test (SAT) The antigen used in the test is prepared at VRI, from a culture originally imported from the United Kingdom and is standardised against International anti-Brucella abortus serum level (Wickramasuriya and Kumaraswamy, 1982). The test is carried out as described by Alton et al. (1975). 4.4. Coombs test (CT) The test determines enhancement of titres above the level obtained in the SAT. The ordinary SAT test is used as the starting point for the CT. The test is done as originally described by Alton et al. (1975). If there is enhancement of the titre to the level normally considered positive on the SAT, the serum sample is regarded as positive (Wickramasuriya and Kumaraswamy, 1982). 4.5. Complement fixation test (CFT) The test is done as originally described by Hill (1963). The antigen used in this test is the standardised CFT antigen obtained from Central Veterinary Laboratory at Weybridge in the United Kingdom (Wickramasuriya and Kumaraswamy, 1982). 4.6. Enzyme-linked immunosorbent assay (ELISA) Brucella eradication schemes have relied upon serological tests to detect seropositive animals. Dif®culties may occur when bovids with long-standing infection remain serologically negative as a result of antibody turnover. Such bovids become serologically positive during or after pregnancy (Sutherland and Mackenzie, 1983). Therefore, lack of serum antibodies does not necessarily indicate non-exposure to the organism because, it may be dif®cult to detect this disease by serological means alone in herds with longstanding infection (Sutherland and Mackenzie, 1983). Therefore, seroprevelance may indicate currently infected animals or a successful immune response by the host to a previous infection (Sutherland, 1984). ELISA positively detects animals that test negative with the above conventional tests. Therefore, this assay can detect bovids with persisting low titres (Sutherland, 1985) and provides an extra criterion in the selection of seropositive bovids (Hornitzky and Searson, 1986). In a recent countrywide study, the indirect ELISA technique was successfully used to estimate the seroprevalence of B. abortus in bovids in Sri Lanka (details to follow). 5. Incidence of brucellosis in Sri Lanka The incidence of B. abortus infections in Sri Lanka has been reported higher than that of the other neighboring countries of the South Asia (Table 3). Incidences of B. melitensis and B. ovis also have been reported from Sri Lanka, but there are no reports about B. suis infections from this island (FAO-WHO-OIE, 1994).
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Table 3 Brucellosis among animals in South Asian countries, 1994 Country
Bovine (B. abortus)
Ovine/caprine (B. melitensis)
Porcine (B. suis)
Ovine (B. ovis)
Afghanistan Bangladesh Bhutan India Sri Lanka
ND ND
?
ND ND ND
( ): Not present; (): low sporadic incidence; (): high incidence; (?): presence uncertain; ND: no data. Source: FAO-WHO-OIE (1994).
From the available data, it has not been possible to state whether brucellosis had existed in the indigenous stock of this country in early times or not. It is quite probable that this disease could have been introduced into Sri Lanka during the second world war in the early forties, from the animals imported for the British Army. The biotype 3 of B. abortus was reported as the causative organism (Kumaraswamy, 1971). Bovine brucellosis was ®rst reported in Sri Lanka in 1956 by the Livestock Research Station in the Polonnaruwa district that maintained Sindhi and Tharpakar breeds of cattle (Pillai and Kumaraswamy, 1957). From this farm, the infection probably spread to the other areas due to transfer of animals between farms (Kumaraswamy, 1971). Wickramasuriya and Kumaraswamy (1982) surveyed the incidence of brucellosis in dairy herds in the districts of Colombo and Gampaha using MRT. The incidences were con®rmed by quantitative tests like SAT, CFT, and CT on suspected sera. In all, 305 herds involving 1478 dairy cows were tested. A herd infection of 1.6% and an individual animal infection of 0.9% were reported. Wickramasuriya et al. (1983) tested the bulk milk received at the milk collection centres in the districts of Polonnaruwa, Batticaloa and Ampara for brucellosis, using MRT. The herd incidence in the Polonnaruwa, Batticaloa and Ampara districts were 7.7, 12.0 and 14.0%, respectively (Table 4). In Polonnaruwa district, the incidence of reactor milking cows decreased with the size of the herd (Table 5). This could be attributed to the management systems practised in the district. The spread of the infection is easier in small herds that are normally con®ned in a limited space. In the case of large herds, animals are kept in the large tracts of grasslands and if an abortion occurs in the ®eld, the foetus is subjected to intense sunlight and heat,
Table 4 Incidence of reactor milking cows in MRT-positive herds in Polonnaruwa, Batticaloa and Ampara districts District
Number of cows tested
Positive reactors (%)
Polonnaruwa Batticaloa Ampara
430 191 121
7.7 12.0 14.0
Source: Wickramasuriya et al. (1983).
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Table 5 Incidence of reactor milking cows in MRT-positive herds classified according to herd size in Polonnaruwa district Number of milking cows in herd
Number of cows tested
Reactors (%)
1±5 6±10 11±20 21±30 31±50
31 43 232 22 164
32.3 11.6 11.2 9.1 4.9
Source: Wickramasuriya et al. (1983).
which would destroy the Brucella organisms thereby minimising the risk of the spread of the infection (Wickramasuriya et al., 1983). In a countrywide study carried out by Silva et al. (2000), an indirect-ELISA technique was employed to estimate the seroprevalence of B. abortus antibodies in cattle and buffaloes. In this study, the bovids in privately owned herds that had never been vaccinated, were used; therefore, the seropositivity reported indicated the natural exposure to B. abortus. The overall seroprevalence of B. abortus in bovids was reported as 4.6% (4.7% in cattle and 4.2% in buffaloes; 3.6% in male animals and 4.9% in female animals). Female bovids 3 years or older were two-times more likely of being seropositive compared to those that were below 3 years (Table 6). This was expected because brucellosis is essentially a disease of mature animals. The seroprevalence rates were not different between cattle and buffaloes (Silva et al., 2000). Bovids from the Dry zone were at approximately six-times higher risk of being seropositive compared to those from the other zones, after adjusting for the possible confounding effects of age and management methods practiced. Female bovids from the Dry zone were approximately at ®ve-times higher risk of being seropositive compared to Table 6 Seroprevalence of B. abortus antibodies in bovids in Sri Lanka grouped according to potential risk factors (1992±1995) Potential risk factor
Cattle
Buffaloes
Number tested
Seropositive (%)
Number tested
Seropositive (%)
Age >3 years <3 years
1643 1433
6 3
388 452
5 3
Ecological zone Dry zone Other zones
832 2244
4 5
333 507
8 2
Management system Extensive Other
1886 1190
6 3
541 299
4 4
Source: Silva et al., 2000.
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Table 7 Clinical signs recorded and the seropositivity in female bovids over 3 years of age in Sri Lanka (1992±1995) Clinical signs
Seropositivity
Cattle (n 1470)
Buffaloes (n 312)
Aborted at least once
Positive Negative
29 30
9 16
Repeat breeding
Positive Negative
0 25
0 2
Other signsa
Positive Negative
4 15
1 3
Source: Silva et al. (2000). a Retained placenta, still birth, and weak calf.
those from the other zones. The prevalence in bovids in the Dry zone managed extensively was 11% compared to 1.9% in other management systems. Female bovids reared under extensive management system were at approximately two-times higher risk of being seropositive compared to those that were reared under other management systems (Silva et al., 2000). Hot weather (Table 1) and poor nutrition likely contributed for high seroprevalence among bovids in the Dry zone. Extensive management systems facilitate unrestricted animal to animal contact, leading easy and quick spread of the disease. The association between the recorded clinical signs and seropositivity in females over 3 years of age recorded in this study is shown in the Table 7 (Silva et al., 2000). Bovids with a history of abortion were at higher risk of being seropositive compared to those without a history of abortion. Only 49% of the cattle and 36% of buffaloes experiencing abortion were seropositive. Among the seropositive females of over 3 years old, 25% of the cattle and 40% of the buffaloes had aborted at least once. None of the sampled bovids with a history of repeat breeding (25 cattle and 2 buffaloes) had detectable levels of antibodies. Twenty-three bovids were reported to have shown other clinical signs suggestive of brucellosis, and only ®ve of them were seropositive (Silva et al., 2000). These ®ndings suggest that Brucella could be responsible for a substantial proportion of abortions in bovids in Sri Lanka. Nonetheless, other causes of abortions in cattle and buffaloes in Sri Lanka should be investigated to identify their etiologies. All repeat breeders n 27 and a substantial number of bovids with other clinical signs suggestive of brucellosis (18 of 23) were seronegative. Therefore, it would be of interest to identify and quantify the causes for repeat breeding and retained placenta in bovids in Sri Lanka. 6. Brucellosis in human and other animals in Sri Lanka There have been isolated incidences of ovine brucellosis due to B. ovis infection and ovine/caprine brucellosis due to B. melitensis (FAO-WHO-OIE, 1994). Although human brucellosis in Sri Lanka is not common, the disease was recognized in Sri Lanka during the early 1960s in studies of unknown fevers (Pyrexia's of unknown origin, PUO). The acute fever in humans may progress to a more chronic form with involvement of the musculoskeletal system and other organs, including orchitis (in¯ammation of the testicles) in
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males (de Alwis, 2002). Among humans, the most vulnerable populations are veterinarians, livestock farm and abattoir workers and others closely associated with susceptible animals. Infection usually occurs through skin abrasions or through the conjunctiva. Infection through milk is virtually non-existent since the consumption of raw, unprocessed milk is not a practice in Sri Lanka (de Alwis, 2002). 7. Control of brucellosis in Sri Lanka So far, a large-scale control program has not been adopted in Sri Lanka. Serological testing, separation and occasional elimination of positive reactors have been successful in reducing the incidence of brucellosis in state-owned cattle farms (Peiris, 1981). Vaccination with reduced dose (1/20th of the dose) of B. abortus S19 has been used in a few state-owned farms (Peiris et al., 1983). Inadequate laboratory facilities and manpower and ®nancial restrictions are constraints to a control program for brucellosis in Sri Lanka. Private owners are very reluctant to adopt control measures particularly those requiring the slaughtering of infected animals. Generally, when farmers are informed of a positive result of a diagnostic test, they tend to sell their animals to a different farmer; this avoids religious and cultural taboos, and minimizes their economic loss. This practice alone contributes to spread of infection. Therefore, if a test-and-slaughter policy were to be implemented as a control measure, many farmers would be noncooperative. Infection in animals with B. abortus can be prevented to some extent by the use of appropriate vaccines. Live vaccines as well as inactivated ones have been developed. However, live vaccines have proven to induce better protective immunity (Schurig et al., 1995). Live vaccine B. abortus strain 19 has been used extensively in other parts of the world, to protect cattle against infection. Peiris et al. (1983) showed that vaccination with B. abortus strain 19 signi®cantly lower the disease incidence. In this study, the animals in a number of government cattle farms in Sri Lanka were immunised with one-twentieth the recommended dose of the vaccine. Subsequently, at different stages, the antibody levels in the serum samples were determined by the SAT. It was observed that at 2 weeks after vaccination, 90.9% of the calves and 92.5% of the adults had very high antibody titres of 1 in 320 or over, re¯ecting the effectiveness of the vaccine at low dosage. At 22 weeks after vaccination, 93.1% of the calves and 75.05%, of the adults had negative titres of 1 in 20 or less. These negative and low antibody titres indicated minimum trouble with residual titres. No abortions were reported when the cows in various stages of pregnancy were immunised at the same dosage of vaccine. This absence of post-vaccinal abortion proved that vaccination of whole herds was practicable. However, the strain 19 vaccine has several undesirable characteristics. These include: its ability to induce serum and milk antibodies if given in inappropriate dosages; and, the vaccine strain is relatively virulent and can induce abortion as well as persistent infection of some of the vaccinated animals (Schurig et al., 1995). B. abortus strain RB51 is a rough attenuated and stable mutant, which appears to have characteristics of an ideal Brucella vaccine. This strain can be used live to vaccinate cattle without inducing positive Brucella serology regardless of the age of the vaccinated animal,
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the route of inoculation, or the frequency of vaccinations. The strain is able to induce protective immunity in cattle against B. abortus infection (Schurig et al., 1995). 8. Necessity of an effective brucellosis control scheme for Sri Lanka According to the limited survey ®ndings, it is clear that the incidence of bovine brucellosis is low in Sri Lanka (Wickramasuriya and Kumaraswamy, 1982; Wickramasuriya et al., 1983; Silva et al., 2000). Experience in other countries has shown that control and eradication are far more practicable when the percentage of infection is low. Being a small country, in Sri Lanka, the eradication of brucellosis could be achieved quite easily provided the government could allocate adequate resources and enforce necessary measures. An effective milk-testing program at the farm level using MRT would be the ®rst step of a brucellosis eradication program. Subsequently, the animals positive to MRT should be tested by ELISA for the seroprevalence of antibodies to Brucellae. Since many farmers are reluctant to dispose their infected animals mostly due to economic reasons, a scheme for compulsory purchase of such animals by state for slaughter is necessary. In parallel, a countrywide vaccination scheme against Brucella is necessary and an attenuated vaccine strain B. abortus RB51 would be an ideal choice for this task. References Alton, G.G., Lois, M.J., Pietz, D.E., 1975. Laboratory Techniques in Brucellosis. Monograph Series No. 55, World Health Organisation, Geneva. Blood, D.C., Radostits, O.M., 1989. Diseases caused by Brucella spp. Veterinary Medicine, 7th ed. Bailliere Tindall, London, pp. 677±690. Census and Statistical Department,1998. Statistical Abstract. Colombo, Sri Lanka. de Alwis, M.C.L., 2002. Animal healthÐpublic health concerns. The Daily News, The Associated Newspapers of Ceylon Ltd., 19 January 2002. FAO-WHO-OIE Animal Health Yearbook, 1994. FAO Rome, 1964. Hill, W.K.W., 1963. Bull. OIT. Int. Epizoot. 60, 401. Hornitzky, M., Searson, J., 1986. The relationship between the isolation of Brucella abortus and serological status of infected, non-vaccinated cattle. Aust. Vet. J. 63, 172±175. Kumaraswamy, S., 1971. The significance of brucellosis in Ceylon. Ceylon Vet. J. 4, 119±123. Land and Water Use Division. Department of Agriculture, Peradeniya, Sri Lanka, 1979. The map of the agroecological zones of Sri Lanka. Nielsen, K., 1997. Brucellosis: development and success using ELISAs for diagnosis. In: Proceedings of the International Symposium on Diagnosis and Control of Livestock Diseases using Nuclear and Related Techniques, Vienna, Austria, 7±11 April. Peiris, G.S., 1981. Reproductive disorders of livestock. Sri Lanka Vet. J. 29, 25. Peiris, G.S., Wickramasuriya, U.G.J.S., Karunasena, S.B., Karunadasa, W.M., 1983. Control of brucellosis: reduced dose whole-herd vaccination with Brucella abortus strain 19. Sri Lanka Vet. J. 31, 49±50. Pillai, C.P., Kumaraswamy, S., 1957. Infertility studies among dairy animals in Ceylon. Ceylon Vet. J. 5, 8±18. Quinn, P.J., Carter, M.E., Markey, B., Carter, G.R., 1994. Brucella species. Clinical Veterinary Microbiology. Wolfe Publishing, Spain, pp. 261±267. Schurig, G., Boyle, S., Sriranganathan, N., 1995. Brucella abortus vaccine strain RB51: a brief review. Arch. Med. Vet. XXVII, 19±22.
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Silva, I., Dangolla, A., Kulachelvy, K., 2000. Seroepidemiology of Brucella abortus infection in bovids in Sri Lanka. Prev. Vet. Med. 46 (1), 51±59. Sutherland, S.S., 1984. Evaluation of the enzyme linked immunosorbent assay in the detection of cattle infected with Brucella abortus. Vet. Microbiol. 10, 23±32. Sutherland, S.S., 1985. Comparison of enzyme linked immunosorbent assay and complement fixation test for the detection of specific antibody in cattle vaccinated and challenged with B. abortus. J. Clin. Microbiol. 22, 44±47. Sutherland, S.S., Mackenzie, R.M., 1983. Applied serology in the latter stages of the eradication of bovine brucellosis. Aust. Vet. J. 60, 240±242. Wickramasuriya, U.G.J.S., Kumaraswamy, S., 1982. The incidence of brucellosis in the district of Colombo. J. Natl. Sci. Council Sri Lanka 10, 99±105. Wickramasuriya, U.G.J.S., Peiris, G.S., Kendaragama, K.M.T., Karunadasa, W.M., 1983. A survey on the incidence of bovine brucellosis in three districts in Sri Lanka. Sri Lanka Vet. J. 31, 27±31.