Veterinaria Italiana, 2011, 47 (1), 59‐64 © Istituto G. Caporale 2011 www.izs.it/vet_italiana Vol. 47 (1), Vet Ital 59 Standardisation of an indirect enzyme‐linked immunosorbent assay for the detection of Brucella antibodies in milk from water buffalo (Bubalus bubalis) in Italy Manuela Tittarelli, Barbara Bonfini, Fabrizio De Massis, Armando Giovannini & Massimo Scacchia Summary An indirect enzyme‐linked immunosorbent assay (ELISA) was evaluated for the detection of Brucella antibodies in milk from water buffalo (Bubalus bubalis Linnaeus, 1758). The test accuracy was evaluated on milk samples from the Campania Region in Italy. A total of 100 negative samples were collected from 10 officially brucellosis‐free herds in Salerno Province, while 30 positive samples were collected from 3 herds in Caserta Province, where animals gave positive results to the official tests and it was here that Brucella abortus biovar 1 had been isolated. Test sensitivity was 100%, with a confidence interval (CI) of 90.8%‐100%, while specificity was 98% (CI 93%‐99.4%) on individual milk samples. To simulate bulk milk samples from herds infected at various levels of infection, dilutions from 1:10 to 1:100 of positive milk samples in negative milk were also used. The probability of detecting antibodies in positive milk samples was higher than 50% up to a dilution of 1:50 in negative milk. Considering the average national water buffalo herd size, the probability of identifying infection in a water buffalo herd by bulk milk testing is 50% (CI 33.1%‐66.9%) in the worst case scenario of a single infected animal contributing to the bulk milk. Keywords Brucella abortus, Brucellosis, Control, ELISA, Enzyme‐linked immunosorbent assay, Italy, Milk, Water buffalo. Introduction The regulations in force for the eradication of brucellosis in buffalo and bovines in Italy (7) are based on serological testing that need to be conducted twice a year in individual blood samples using the Rose Bengal test (RBT) and complement fixation test (CFT). For water buffalo (Bubalus bubalis Linnaeus, 1758), special control measures applied in Italian regions with high prevalence of animal brucellosis (i.e. Calabria, Campania, Apulia and Sicily) include the use of milk testing as a complement to serological tests (8). Milk testing should be performed periodically, in accordance with the guidelines of the National Brucellosis Task Force, so as to increase the sensitivity of the control system in place. In this context, the use of an indirect enzyme‐ linked immunosorbent assay (ELISA) for the detection of Brucella antibodies on water buffalo bulk milk (m‐ELISA) can increase the sensitivity of the diagnostic system. Moreover, besides the intrinsic high sensitivity of ELISA tests, the simplicity of milk ELISAs also means Istituto Zo o p ro fila ttic o Sp e rim e nta le d e ll‘ Ab ruzzo & d e l Mo lise ‘ G . C a p o ra le ’ , Via C a m p o Bo a rio , 64100 Te ra m o , Ita ly m .titta re lli@ izs.it