The majority of tuberculosis cases in ruminants are caused by Mycobacterium bovis (MB). However, in this study, the authors reported the isolation of Mycobacterium tuberculosis (MT) from bovine milk, nasal swabs and post-mortem tissue samples (n = 841) collected from cattle and buffaloes in the states of Telangana, Maharashtra and Gujarat in India in the period from 2010 to 2015. The isolates (n = 7) were confirmed as Mycobacterium due to their growth characteristics and colony morphology in a commercial liquid medium Mycobacteria Growth Indicator Tube (MGIT)™ employing the BD BACTEC™ MGIT™ 960 system and the Lowenstein Jensen (LJ) medium supplemented with glycerol but not with sodium pyruvate, and BD-DIFCO™ Middlebrook 7H10 agar containing oleic albumin dextrose catalase (OADC). These isolates were initially identified as members of the Mycobacterium tuberculosis complex (MTC) using a commercial nested polymerase chain reaction (PCR) kit based on the IS6110 MTC specific nucleotide sequence. The isolates were confirmed as MT using three commercial line probe assay kits, were further genotyped, and the spoligotypes identified were of East African Indian (EAI) 3_IND, EAI5, Central-Asian (CAS) 1_DELHI, U and T1 lineages. Two MT isolates from one antelope (Antelope cervipara) and one gazelle (Gazelle bennettii) from Gujarat, which were identified previously, were spoligotyped during this study and identified as belonging to EAI3_IND and EAI5 lineages, respectively. The epidemiological significance and zoonotic implications of regional presence and documentation of the same or two different spoligotypes in different species within the family Bovidae as well as humans is discussed.
Accurate identification of Mycobacterium strains is necessary from a diagnostic standpoint. Differences in gyrA 95 and gyrB (1410) genome due to specific single nucleotide polymorphism (SNP) were used to design Real-Time PCRs that can discriminate Mycobacterium tuberculosis (MT) from Mycobacterium bovis (MB) belonging to the Mycobacterium Tuberculosis Complex (MTBC). The Real-Time PCR employing the VIC labeled gyrA 95 probe specifically detected the ATCC reference strain of MT H37Rv and eight field strains as MT, but not MB or other members of MTBC and other microorganism tested. The VIC labeled gyrA 95 assay could detect up to 450 fg of MT. The FAM labeled gyrB (1410) specifically detected MB, but not MT or other members of MTBC and other microorganism tested. The assay could detect up to 600 fg of MB, but it could not detect MB from any field isolate. Both assays were completed in 78 minutes. The results of both Real-Time PCR assays did not differ significantly from culture by Student's t-test. The analytical sensitivity of gyrA 95 assays determined by Receiver Operating Characteristic curve (ROC) was 100% at 95% Confidence Intervals (CI) |66.4-100.0|, and analytical specificity 100% at 95% CI |75.3-100.0|. The repeatability and reproducibility of both above assays tested by Bland-Altman Plot indicated that the mean Cq values were within acceptable statistical limits of + 1.96 SD. Comparison of partial genome sequences of gyrA 95 and gyrB (1410) of field strains of MT with MT H37Rv by Phylogenetic Tree and Disparity Index tests indicated that eight field strains were homologous to MT H37Rv, but one was homologous to Mycobacterium intracellulare. Real-Time PCR assays were found rapid, specific, sensitive, repeatable and reproducible. Further studies are necessary for conversion of these assays to quantitative formats and determine parameters of diagnostic estimates before their use under clinical settings.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.