The zoonotic disease Brucellosis is a serious public health and livestock industry concern. In 50 the present study, we used bacteriological culture, Rose Bengal Test (RBT), and quantitative 51 PCR (qPCR) methods to determine the prevalence of brucellosis in serum and milk samples of 52 sheep with a history of abortion. Serum and milk samples were obtained from 100 sheep aged 53 three to five years. To determine the prevalence of brucellosis, a modified RBT was performed 54 on serum samples, Brucella was isolated from milk by bacteriological culture, and qPCR was 55 applied to detect bacterial DNA in milk. The prevalence of brucellosis using the modified RBT, 56 bacteriological culture, and qPCR was 32%, 42%, and 44%, respectively. By considering the 57 qPCR as the criterion standard, the modified RBT showed a sensitivity of 95%, a specificity of 58 100%, an accuracy of 98%, a positive predictive value (PV+) of 100%, and a negative 59 predictive value (PV-) of 97%. The sensitivity, specificity, accuracy, PV+, and PV- for 60 bacteriological culture were 77%, 100%, 90%, 100%, and 85%, respectively. The agreement 61 between qPCR and modified RBT was 0.959 (95%CI:0.896-1); between qPCR and 62 bacteriological culture was 0.792 (95%CI:0.667-0.897); and between modified RBT and 63 bacteriological culture was 0.831 (95%CI:0.709-0.38). Based on the results, bacterial isolation 64 from sheep\\\\\\\\\\\\\\\'s milk is not recommended except in specific cases due to its low sensitivity, time-65 consuming, and hazardous nature. However, the modified RBT can be used as a routine method 66 because of its cost-effectiveness, higher sensitivity, and accuracy compared to bacterial 67 3 isolation, qPCR is recommended as the gold standard test for detecting brucellosis in sheep 68 milk, especially in those with a history of abortion

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