2 2. Materials and Methods A total of 200 bovine milk samples were collected from government and privately owned organized dairy farms in Quetta City and subjected to milk ring test (MRT) and indirect ELISA for the detection of antibrucella antibodies. 2.1. Milk Ring Test (MRT). A drop (30 µl) of stained brucella antigen was added to 01 ml of whole milk that has been kept at 4◦C using overnight refrigeration. The test result was read after incubation for 1 hour at 37◦C. A positive reaction was indicated by a stained cream layer over white column of milk [6] 2.2. Enzyme-Linked Immunosorbent Assay (i-ELISA). The indirect ELISA (i-ELISA) kit for the detection of antibrucella antibodies was obtained from M/S Svanova, Sweden, and the procedure was followed as per manufacturer’s procedure. All the milk samples were centrifuged at 5000 rpm for 5 minutes to remove the cream layer before use. About 100 µl of sample dilution buﬀer was added to the control wells and 4 µl of positive control serum and 4 µl of negative control serum were added, respectively, to selected precoated antigen wells. Later, 100 µl of milk samples were added to selected wells. The plate was shaken thoroughly, sealed, and incubated at 37◦C for one hour. The plate was rinsed 3 times with PBS- Tween buﬀer followed by addition of 100 µl of horse reddish peroxidase (HRP) conjugate (prepared in 11.5 ml PBS-Tween buﬀer diluted in distilled water) to each well and incubated at 37◦C for one hour. The plates were again rinsed thrice, and 100 µl substrate solutions (tetramethylbenzidine) were added to all the wells. Microtitration plate was incubated for 10– 15 minutes at room temperature. Finally, 50 µl of stopping solution was added to each well. The optical densities (OD) of the control and test-sample wells were adjusted at 405 nm wavelength using an ELISA reader (Thermo Electron, Finland). The interpretation of results was performed accordingly. PP percent positivity = Test sample OD Positive control OD 100 (1) For milk samples, a PP value equal or more than 10 was taken as positive. 3. Results and Discussion The data collected revealed high prevalence (8.5%) of the disease in the area; this prevalence is higher than previously reported (3.97%) positive cases in cattle and buﬀalo using Rose Bengal plate test (RBPT) and serum agglutination test (SAT) by FAQIR [10] who also reported the presence of brucellosis in cattle and buﬀalo in the same area after screening 680 animals. The prevalence was recorded much higher in cattle than buﬀaloes, as, out of 200 milk samples, 17 samples were found positive, all from cattle. These ﬁndings are in agreement with those of Abbas and Aldeewan [11] who also reported a 20 15 s e l p m sea 10 v i t i s o P 5 0 Veterinary Medicine International 17 6 MRT ELISA Figure 1: Graphical comparison of MRT with i-ELISA for detection of antibrucella antibodies in Milk Samples. Table 1: Prevalence of bovine brucellosis in milk samples using MRT and i-ELISA in District Quetta, Balochistan, Pakistan. Animal Cattle n = 86 Buﬀalo n = 114 Percentage n = 200 Positive samples MRT 4 (4.6%) 2 (1.7%) 6/200 (3%) i-ELISA 17 (20%) — 17/200 (8.5%) MRT: milk ring test. i-ELISA: indirect enzyme-linked immunosorbent assay. higher prevalence of brucellosis in cattle (10.5%) than that in buﬀalo (1.9%). The higher prevalence in cattle may be attributed to the specie speciﬁcity or possibly the higher fat percentage in buﬀalo milk may be the obstacle in the detection of positive cases. On farm comparison basis, the prevalence at govern- mentdairy farm (GDF) in Quetta was 14.8%, as, out of 74 samples, 11 were found positive while the private dairy Farm (PDF) exhibited 4.76% prevalence, as, out of 126 milk samples, only 6 samples were positive (Table 2). This result is in agreement with the observations of Pederson [12] who reported higher prevalence among oﬃcial herds than that of privately owned dairy farms. However, this result contradicts that of Naeem et al. [13] who reported higher prevalence of bovine brucellosis among privately owned animals. The overall prevalence in organized dairy farms was 3% by MRT, as 6 out of 200 samples were found positive while i-ELISA showed 8.5% prevalence, as 17 out of 200 milk samples were found positive (Table 1). The indirect enzyme linked immunosorbent assay (i- ELISA) detected seventeen (17) milk samples whereas only 6 milk samples were detected positive by MRT indicating high sensitivity and speciﬁcity of i-ELISA than those of milk ring test. In the present study, the milk ELISA had detected more positive cases than MRT (Figure 1). These ﬁndings corroborate with Kerkhofs et al. [14], Vanzini et al. [15], and