The rapid detection and differentiation of Mycobacterium tuberculosis complex members from cattle and water buffaloes in the delta area of Egypt, using a combination of real-time and conventional PCR
Mycobacterium tuberculosis complex (MTBC) has the potential to cause infections in animals and human beings. The combination of real-time PCR targeting atpE or lpqT and RD1, and conventional PCR targeting regions of difference (RD) was rigorously evaluated as a descriptive molecular epidemiology tool. A total of 2100 cattle and buffaloes from the Menoufia, Sharkia, Gharbia, Dakahlia, Elbuhaira, and Cairo Governorates were tested by single intradermal comparative cervical tuberculin test (SICCT). The frequency was 74/2100 (3.5%); thereafter, on post-mortem examination (PM), 49/74 (66.21%) showed visible lesions, while only 25/74 (33.78%) were non-visible with a significant difference of (p < .0001). Real-time PCR using atpE or lpqT and RD1 similarly detected the frequency of infection, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, which represented 73/74 (98.65%), 98.65, 100, 100, 90.91, and 98.81%, respectively. Multiplex conventional PCR targeting RD1, 4, 9, and 12 confirmed that 49/74 (66.21%) were M.bovis, while the simplex conventional PCR targeting RD4 and RD9 confirmed mycobacteria in 71/74 (95.94%) samples, which included 61/74 (82.4%) M.bovis and 2/74 (2.7%) M.tuberculosis. Additionally, 8/74 (10.8%) exhibited mixed patterns of M.bovis and M.tuberculosis, and 3/74 (4.05%) were negative. There was a significant difference between the results of simplex and multiplex conventional PCR (p < .0001). Moreover, simplex conventional PCR targeting RD4 and RD9 proved higher sensitivity, specificity, positive predictive value, negative predictive value, and accuracy, which were 95.95, 100, 100, 76.92, and 96.43%, respectively, when compared with the values of multiplex conventional PCR targeting RD1,4,9, and 12 which were 66.22, 100, 100, 28.57, and 70.24%, respectively. The repeatability results of real-time PCR using atpE or lpqT and RD1, and simplex conventional PCR targeting RD4 and RD9 were acceptable. In conclusion, a combination of real-time PCR using atpE or lpqT and RD1 as the first step with simplex conventional PCR targeting RD4 and RD9 as the second step was reliable as a diagnostic tool.
KeywordsReal-time Conventional PCR RD region Mycobacterium tuberculosis complex (MTC)
The authors would like to acknowledge the Culture Affair and Mission Sector in Egypt for supporting the short-term scholarship during which this study was completed. The authors acknowledge Prof. Essam Amin Nasser at the Veterinary Serum and Vaccine Research Institute Egypt for supporting them with some isolates.
MSE, AA: Conceptualization, MSE, AA: Data curation, MSE, AA: Formal analysis, MSE: Investigation, MSE: Methodology, MSE: Project administration, MSE: Resources, MSE: Writing original draft, MSE, AA: Writing, review, and editing. All authors have read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that there are no competing interests.
The protocol was approved by the institutional animal care and use committee (IACUC) at the Faculty of Veterinary Medicine, University of Sadat City. Furthermore, our manuscript reporting adheres to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The authors obtained written informed consent to use the animals in their study from the owner(s) of the animals. The infected cases were slaughtered and the Egyptian Law number 517 in 1986 for the routine work of meat inspection at the slaughterhouse was applied. The carcasses were judged to pass for human consumption if no there were no granulomas or were condemned either partially or totally according to the number and site of the granulomas found. The owners received financial compensation for their tuberculin positive and slaughtered cases.
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