Buffalo Bulletin (June 2009) Vol.28 No.2 SEROPREVALENCE OF ...

Buffalo Bulletin (June 2009) Vol.28 No.2SEROPREVALENCE OF BRUCELLA SPP. IN BUFFALOES IN THE CENTRAL GUJARATREGION OF INDIAM.N. Brahmabhatt, R.N. Varasada, C.D. Bhong and J.B. NayakABSTRACTBrucellosis remains a major threat from thezoonotic as well as the economic point of view. Theprevalence of brucellosis in the central Gujarat wasstudied using the rose bengal presipitation test(RBPT), standard tube agglutination test (STAT) andindirect enzyme-linked immunosorbent assay (i-ELISA). The seroprevalence was found to be 12.75percent, 11.16 percent and 19.12 percent by RBPT,STAT and i-ELISA, respectively.Keywords: buffalo, brucellosis, seroprevalenceINTRODUCTIONBrucellosis has long been a major threat tolivestock. It is mainly a disease of domestic animalscaused by various strains of Brucella spp. Bovinebrucellosis is found worldwide, but it has beeneradicated from many countries. The rate of infectionvaries greatly from one country to another andbetween regions within a country. The diseasecauses heavy economic losses due to abortion,premature births, decreased milk yield and repeatbreeding leading to temporary or permanent infertilityin infected livestock (Yagupsky, 1999).The diagnosis of the disease can bechallenging and is frequently delayed or missedbecause the clinical picture may mimic otherinfectious and noninfectious conditions (Araj, 1999;Yagupsky, 1999). Diagnosis can be established bylaboratory methods such as serology and bloodcultures. Prolonged incubation periods, specialgrowth media, and subcultures are required for theisolation of these fastidious, slow growing bacteria.However, cultures are not always positive whenother tests are positive (Romero et al., 1995).Automated systems have been reported to detectmore than 95 % of Brucella melitensis-positivecultures within seven days of incubation (Yagupsky,1999). The technology is lacking in developingcountries or rural areas where the disease isprevalent and diagnoses rely mainly on serology.Many serological tests have been used for thediagnosis of human brucellosis, such as agglutinationtests, indirect immunofluorescence, rose bengal platetest (RBPT), standard tube agglutination Test(STAT), and ELISA specially Dot-ELISA &Indirect-ELISA. The most commonly used tests arethe serum agglutination test, Coombs anti-Brucellatest, rose bengal test, and complement fixation(Orduna,∼2000).Buffalo milk and milk products are anappreciable source of income of farmers of India,that is why buffaloes are integral part of theeconomy of rural people. Hence, this study wascarried out to find out the seroprevalence ofBrucella spp. in central Gujarat, Anand, Kaira,Ahmedabad and Vadodara districts of Gujarat(India). These districts are one of the biggest pocketsof milk production in India and also the location of aworld famous dairy co-operative-AMUL.MATERIALS AND METHODSA total of 251 (230 female and 21 male) serasamples from buffaloes were collected from variousplaces of four districts in Central Gujarat, viz., Anand(78), Kaira (60), Ahmedabad (86) and Vadodara(27). Collected sera samples were subjected to rosebengal plate test. (RBPT), standard tubeDepartment of Veterinary Public Health, College of Veterinary Science & Animal Husbandry, AnandAgricultural University, Anand Gujarat - 388 001, India73

Buffalo Bulletin (June 2009) Vol.28 No.2agglutination Test (STAT) and indirect-enzyme linkedimmunosorbant assay (i-ELISA). The RBPT antigenand Brucella abortus agglutinating antigen for STATwas procured from the Division of BiologicalProducts, Indian Veterinary Research Institute(I.V.R.I.); Izatnagar, Uttar Pradesh (India). The testswere conducted as per manufacturer’s instructions.For i-ELISA, smooth lipopolysaccharide (S-LPS)based (A-B-ELISA) kits supplied by the All IndiaCoordinated Research Project (AICRP) on AnimalDisease Monitoring and Surveillance (ADMAS),Bangalore, was used. The test was performed asper the manufacture’s instructions.i-ELISA was compared with RBPT andSTAT, considering i-ELISA as the gold standard testas per Hobbs (1985) and Nielsen et al. (1996), todetermine the relative sensitivity and specificity ofRBPT and STAT.RESULTS AND DISCUSSIONOut of 251 sera tested during present studywith RBPT, 32 (12.75 %) were found positive. WithSTAT, 28 (11.16 %) gave positive while 48(19.12 %) reacted as positive when tested with i-ELISA. The highest (23.26 %) prevalence was foundin Ahemdabad district, while prevalences were20.51 %, 7.40 % and 16.67 % in Anand, Vadodaraand Kaira districts, respectively (Table 1).Out of 230 females and 21 males tested, 43(18.70 %), 29 (12.61 %) and 25 (10.87 %) werefound positive by the i-ELISA, RBPT, and STATtests, respectively, in females while 5 (23.81 %), 3(14.29 %), and 3 (14.29 %) were found positive inbulls by the respective tests.The prevalence of brucellosis was 19.12 %.These findings were comparable to the results ofSharma and Saini (1995), who found 14.61 %prevalence in Punjab, India. This finding alsosupported Chatterjee et al. (1984) who found 19.6percent prevalence.Lower seroprevalences were reported byIsloor et al. (1998), 1.8 %; Mishra et al. (2005),4.18 percent; Bhattacharya et al. (2005), 11.94 %;and Agarwal et al. (2007), 4.6 %, while theprevalence found in the present study was lowerthan that observed by Chauhan et al. (2000),38.9 % in North Gujarat region of India,Chandramohan et al. (1992) 21.74 %.The seroprevalences determined by varioustests differed from one another. This could be dueto variation in the numbers of false positives andfalse negatives detected by various tests. Similarfindings were reported by Rao et al. (1999) andSingh et al. (2004).In the present study, RBPT shows 64.58 %sensitivity and 99.50 % specificity when comparedwith i-ELISA. This is in agreement with Uzal et al.(1995) and Saravi et al. (1995), who reported 98.9% and 99.7 % specificity, respectively. PrahladKumar et al. (1999) showed 33.33 % sensitivity;this was lower than the present findings.Table 1. Geographical distribution of brucellosis antibodies.i-ELISA RBPT STATDistrictNumer ofsamples testedNo. of samples+ve (%)No. of samples+ve (%)No. of samples+ve (%)Anand 78 16 (20.51) 11 (14.10) 9 (11.53)Vadodara 27 2 (7.40) 2 (7.40) 2 (7.40)Ahemdabad 86 20 (23.26) 13 (15.11) 12 (13.95)Kaira 60 10 (16.67) 6 (10.00) 5 (8.34)TOTAL 251 48 (19.12) 32 (12.74) 28 (11.16)74

Buffalo Bulletin (June 2009) Vol.28 No.2STAT showed 56.25 % sensitivity and99.50 % specificity when compared with i-ELISA.Higher sensitivity (81.81 %) was observed byAgrawal and Batra (1999). Prahlad Kumar et al.(1999) reported more than 90 % specificity. Therelative sensitivity of RBPT was 64.58 % and thatof STAT was 56.25 %. The relative specificityobserved was more than 99.00 % in the above case.Thus, i-ELISA test in conjunction with otherserological tests can give more reliable diagnosis.REFERENCESAgarwal, R., M. Kumar and J.L. Singh. 2007.Seroprevalence of brucellosis in Uttranchal.Indian Vet. J., 84: 204-205.Agrawal, G.S. and H.V. Batra. 1999. Comparisionof an inhibition enzyme linked immunosorbentassay with other serological tests for detectionof antibodies to Brucella. Indian Vet. J., 76:10-12.Araj, G.F. 1999. Human brucellosis: a classicalinfectious disease with persistent diagnosticchallenges. Clin Lab Sci., 12: 207-212.Bhattacharya, D.K., K. Ahmed and H. Rahman.2005. Studies on seroprevalence of bovinebrucellosis by different tests. J. Vet. Pub.Hlth., 3: 131-133.Chandramohan, C.P., P. Ramdass and N. Raghavan.1992. Studies on bovine brucellosis in anendemic area. Indian Vet. J., 69: 581-583.Chatterjee, B.N., J. Bidyanata, M. Chakraborty, P.Mondal and G.P. Sen. 1984. Seroepidemiologicalstudies on bovine brucellosisin organized herds in West Bengal. Indian J.Anim. Sci., 55: 249-252.Chauhan, H.C., B.S. Chandel and N.M. Shah. 2000.Seroprevalence of brucellosis in buffaloes ofGujarat. Indian Vet. J., 77: 1105-1106.Hobbs, I.F. 1985. Comparision of indirect enzymelinked immunosorbent assay (ELISA) with thecomplement fixation test (CFT) forserodiagnosis of bovine brucellosis. N.Z. Vet.J., 33: 112-116.Isloor, S., G.J. Renukaradhya and M. Rajshekhar.1998. A serological survey of bovinebrucellosis in India. Rev. Sci. Tech., 17: 781-785.Nielsen, K., P. Smith, D. Gall, Perez, C. Cosma, P.Mueller, J. Trottier and J. Bosse. 1996.Development and validation of an indirectenzyme linked immunosorbent assay fordetection for detection of antibody to Brucellaabortus in milk. Vet. Microbiol., 52: 165-173.Orduna,∼A., A. Almaraz and A. Prado. 2000.Evaluation of an immunocapture-agglutinationtest (Brucellacapt) for serodiagnosis of humanbrucellosis. J. Clin. Microbiol., 38: 4000-4005.Prahlad Kumar, D.K. Singh and S.B. Barbuddhe.1999. Seroprevalence of brucellosis andcomparision of serological test to diagnosis itin buffaloes. Buff. J., 15: 361-370.Rao, S.T., V. Rama Devi, R. Madhu Babu AndA.V.C. Narsinha Rao. 1999. Comparision ofrapid plate agglutination, standard tubeagglutination and dot-ELISA tests fordetection of antibodies to Brucella in bovines.Indian Vet. J., 76: 255-256.Romero, C., C. Gamazo, M. Pardo and I. Lo’pezgon.1995. Specific detection of BrucellaDNA by PCR. J. Clin. Microbiol., 33: 615-617.Saravi, M.A., P.P. Wright, R.J. Gregort and D.E.Gall. 1995. Comparative performance of theenzyme linked immunosorbent assay (ELISA)and conventional assays in the diagnosis ofbovine brucellosis in Argentina. Vet. Immunol.Immunopathol., 47: 93-99.Sharma, J.K. and S.S. Saini. 1995. Seroprevalenceof brucellosis among farm animals of Punjab.Indian Vet. J., 72: 881-882.Singh, G., D.R. Sharma and N.K. Dhand. 2004.Seroprevalence of bovine brucellosis inPunjab. Indian Vet. J., 81: 620-623.Uzal, F.A., A.E. Carrasco, S. Echaide, K. Nielsenand C.A. Robles. 1995. Evaluation of indirectELISA for the diagnosis of bovine brucellosis.J. Vet. Digan. Invest., 7: 473-475.Yagupsky, P. 1999. Detection of Brucella in bloodcultures. J. Clin. Microbiol., 31: 1927-1931.75