Identification of biovars of Brucella abortus in aborted cattle and buffaloes herd in Sri Lanka

Bovine brucellosis is an endemic disease in Sri Lanka, caused by Brucella abortus and had been reported all part of the country for last six decades. Since available biovar is still unknown, the objective of the study was to identify the biovar of B.abortus from sporadically aborted cattle and buffaloes. Samples were collected from 18 aborted herds out of 19 herds of Cattle and Buffaloes in the year 2010. Rose Bengal plate test and Compliment Fixation test were carried out. Milk, vaginal swabs, placental contents and aborted fetus were collected and cultured by conventional bacteriological methods. The detection of biovars were based on growth on Thionin and Bacto fuschin,CO requirement, H S 2 2 production, serum agglutination with Brucella negative, A,M and R reference antiserum. Eighteen herds investigated out of 19 herds reported, 11 herds were serologically positive for brucellosis (61.11%) and only Brucella abortus were isolated from 8 individuals from six herds. All were identified as Biovar 3 of Brucella abortus.


Introduction
which Brucella abortus survives in the environment (Qiunn et al, 1994).Bovine brucellosis, caused by bacterium Brucella abortus is an important zoonotic disease in Materials and methods many developing countries (Smits, H.L. and Kadri, Eighteen herds out of 19 herds in the Sri Lanka S.M.,2005).The disease has been reported in different were investigated and samples were collected from provinces in India, Pakistan and Bangladesh cattle and buffaloes with the history of abortion (Abubakar, M. et al, 2010., Mantur, B.G., and Amaranth, nationwide.Information were received from regional S.K., 2008., Rahman,et al, 2006).Brucellosis is Government Veterinary offices when sporadic considered an threatening disease in Sri Lanka abortions continuing in particular regions, is the (Priyantha et al,2008) and had been introduced routine disease reporting system established in the through importation of cattle during the Second World country.Samples were collected 1-14 days from day War (De Alwis,1993) Brucella agar (Oxoid CMO169) was used as base for negative anti-serum and A,M and R reference antisera.both selective and non selective medium with 4%

Results
sheep blood.The OXOID CMO 169 selective supplement was added only for selective media to Eighteen aborted herd were investigated (Out of inhibit contamination.The isolated organisms were 19 reported in 2010), 11 herds were identified as tested by biochemical tests and serum agglutination serologically positive for brucellosis (61.11%).The test with Brucella reference antiserum brought from seropositive animals were detected in herds at Dambulla, International reference laboratory, France.Milk Anamaduwa, Mihintale, Sippukulam, Buttala, samples were collected directly to sterile bottles (20 Mannar and Bingiriya and their Complement Fixation ml each) from each teat of aborted cows aseptically.Test (CFT) titers were varied from 1:16 to 1:2018.The samples were centrifuged at 6000 g for 15 There were nine Brucella abortus organism isolated minutes.Both deposited and cream layer were from six different herds and biovar 3 was isolated from cultured on selective and non selective media, were milk, aborted fetus and vaginal swabs.incubated at 10% CO and without CO as described by 2 2

Discussion
Alton et al, 1988.Sterile, 10 cm long swabs were used for this purpose.Samples were (Five samples from Brucellosis is an endemic disease specially in each and pooled) collected directly from the vaginal dry zone (Priyantha et al,2008 for long time, (Priyantha et al, 2008, Kumaraswami, al, 1993).However, those in this study were remain 1971).negative for serologically even at calving and excreting The isolation of Brucella abortus were from nine organism with milk and reproductive secretion, which individuals, representing six different herds (Table -3).needs to be study further.

Most isolates were from indigenous and extensive or
The causative bacteria, Brucella abortus has semi extensive herds of dry zone and only one isolate been isolated from at least nine species of domestic from Bingiriya.The rearing indigenous animals are livestock (Nielsen &Duncan, 1990).All isolated were found in dry zone (Priyantha et al, 2008).Those herds as biovar 3 by biochemical and agglutination tests are sent to jungle or pasture land around big tanks in described by Alton et al, 1988, (Table -3) out of 7 the region, shearing same pasture by different herd and biovars identified from B.abortus (OIE, 2009).
Brucella abortus biovar 3, only biovar was given cross contamination is common (Priyantha et al, positive for CO requirement for growth, H S production, 2008).The oral contamination which is the most

2 2 common 2 infection(
method of transmission in an extensive herd growth on Thionine and basic fuschin reagent.It was (Abubakar, M.et al, 2010.Nielsen &Duncan, 1990).given positive results for Brucella type A antisera and Higher stocking density has been cited as an important negative for M and R antisera.All isolates required factor influencing transmission and persistence of CO for growth on artificial media and produced H S, 2 Nielsen &Duncan,1990).described as biovar1,2, 3,4 or 9 of B.abortus.Also all The important finding of this study was nine Isolates were grown on Thionin and basic fuchin detection of latent carriers of Brucella abortus.There given concentration by Alton et al,1988 and were three animals detected at Mundalama and one categorized under biovar 3 after the agglutination test detected at Pannala.They were excreted bacteria of A.M and R. Biovar 1 of Brucella abortus is through milk and reproductive secretion although not appeared as most common and widely distributed in detected by RBP test.The latent carrier may cause the world (Nielsen &Duncan, 1990).The distribution detrimental results in control program (Nielsen is varied with one country to the other and Only 1.2 &Duncan, 1990., Dolan L.A.et al, 1980), reported by and 4 are reported in USA while biovar 3 and 7 are not different authors in different hosts (Huurne et al, yet reported in England (Nielsen &Duncan, 1990).In 1993.,Verma et al, 2000.,Dolan,L.A.,1980).Meanwhile the literature published, Only biovar 1,3,5 of Brucella calves infected by B.abortus in utero or after ingestion abortus have been reported in India, which is of infected milk may acquire a persistent infection, considered as the source of brucellosis in Sri Lanka remain negative until calving or abortion (Huurne et during the period of second world war (Perumal

,
De Alwis et al,1989), wall, cultured on Brucella specific medium and where the highest density of cattle reported in the 0 country.The serological brucellosis in local herds has incubated at 37 C with and without 10% CO incubation 2 been studied at regional level, although surveillance as described by Alton et al,1988.The aborted fetus is on brucellosis is still a subject area of priority.It was considered as one of the best sample to isolates apparent that brucellosis is the main cause of sporadic Brucella in cattle and Buffalo (Nielsen & Duncan,1990).abortion in cattle at last trimester of pregnancy and Inoculations were made from aborted fetuses specially 61.11% of aborted herds examined were found positive from stomach contents, lungs, spleen, liver, on serologically.Similar information was published by Brucella specific and nonspecific medium as Priyantha et al in 2010, indicated that Brucella abortus described by Alton et al, 1988.However, collection of was the main cause of abortion in water buffaloes.fetuses from extensive herds were difficult as fetuses Furthermore, Brucellosis is considered major were eaten by other animals.reproductive disease of cattle in Dry zone of Sri Lanka The identification of biovar of Brucella was