¹Trakia University, Faculty of Veterinary Medicine, Department of Veterinary Microbiology, Infectious and Parasitic Diseases, 6000 Stara Zagora, BULGARIA
²University of Forestry, Faculty of Veterinary Medicine, 10 Kliment Ohridski Str. 1756 Sofia, BULGARIA
³Medical University, Department of Medical Microbiology, 2, Zdrave Str. 1431 Sofia, BULGARIA
⁴National Reference Laboratory “Enzootic Bovine Leukosis” (2013-2015), Department of Virology and Viral Diseases, National Diagnostic and Research Veterinary Medical Institute, 15, Pencho Slaveykov Blvd., 1606 Sofia, BULGARIA DOI : 10.9775/kvfd.2021.26505 The aim of this study was to compare the diagnostic capabilities of the agar gel immunodiff usion test (AGID) and two types of PCR, nested PCR (with nucleic acid extraction from blood) and direct blood nested PCR (db nested PCR) - without extraction, vs. enzyme-linked immunosorbent assay (ELISA) as the gold standard in the routine diagnostics of this disease. A total of 409 blood samples were obtained from cattle 18 mo. to 5 yrs. of age, and all the samples were analyzed using the four assays. Following the initial testing, the samples were stored at -20°С and re-tested using all the four techniques after a month of freeze storage to determine the reproducibility of the results. ELISA detected 57 animals as positive (13.9%) versus 33 ones using AGID and 56 using the two types of nested PCR. AGID showed low sensitivity of 57.9% and moderate agreement compared to ELISA. In addition, AGID did not show consistency in the results from the two independent measurements. The two types of nested PCR showed nearly full agreement with ELISA with a kappa value of 0.99. Since AGID showed lower sensitivity and lack of reproducibility in the results for 22 samples as compared to the other techniques used in this study, we suggest that the future application of this test for the diagnosis of Enzootic bovine leucosis in blood samples should be reconsidered. On the other hand, db nested PCR demonstrated very good sensitivity and reproducibility of results, it also requires less sample processing. All this makes it potentially suitable for routine diagnostics. Keywords : AGID, comparison, db nested PCR, ELISA, Enzootic bovine leukosis, Nested PCR