Zoonotic Tuberculosis - The Changing Landscape.

The study was designed to compare the efﬁcacy of cetylpyridinium chloride (CPC) and sodium chloride (NaCl) decontamination method with N-acetyl L-Cystine (NALC) and sodium hydroxide (NaOH) decontamination (the reference method) method for the recovery of Mycobacterium tuberculosis (MTB) from clinically suspected cases of pulmonary tuberculosis. To evaluate CPC-NaCl and NALC-NaOH decontamination methods, sputum specimens (n = 100) were studied (culturing on Lowenstein-Jensen medium), and the performances were compared. The CPC-NaCl decontamination method demonstrated a sensitivity, speciﬁcity, negative predictive value, and positive predictive value of 100%, 87.63%, 20%, and 100%, respectively, when compared to NALC-NaOH decontamination method. In summary, CPC-NaCl decontamination method effectively detected signiﬁcantly higher number of MTB cases (n = 15) than NALC-NaOH decontamination method (n = 3) particularly in sputum with scanty bacilli and smear-negative cases, indicating the potential of CPC-NaCl decontamination method to preserve paucibacillary cases more efﬁcient than NALC- NaOH decontamination method.

Isolation of Mycobacterium tuberculosis

Prevalence of Mycobacterium bovis in milk on dairy cattle farms: An international systematic literature review and meta-analysis.

We report the isolation of Mycobacterium orygis, a member of Mycobacterium tuberculosis complex (MTBC), from two black bucks (Antelope cervicapra) and one spotted deer (Axis axis) from the Guindy National Park forest range in Chennai, India. Lung tissue and lymph node samples collected during post-mortem examination were processed using NaOH method and cultured in solid and liquid media. DNA extracted from the cultured isolates was used to amplify the mpt64 gene by specific primers and the band visualized at 240 bps confirmed the isolates as a member of MTBC. Further examination of these isolates by spoligotyping and whole genome sequencing confirmed the isolates as M.orygis and the phylogenetic tree revealed their well clustered position with other M .orygis isolates around the globe. The deletion of RD7 to RD10, RDOryx_1, RDOryx_4, RD12Oryx, RD301 and RD315 further substantiated these isolates as M. orygis. The exact source of infection in animals was untraceable and the pairwise comparison of the genomes based on single nucleotide polymorphisms (SNP) difference did not detect any events of transmission within the affected animals. Nevertheless, it would be wise to take into account the environment where there exists a high chance of transmission due to the increased human-animal interaction. Since it is well known that the pathogen is capable of causing infection in both human and animal hosts, systematic surveillance and screening of spotted deer, black buck as well as humans in the vicinity is essential for successful implementation of the One Health approach. This article is protected by copyright. All rights reserved.

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Tuberculosis caused by Mycobacterium orygis in wild ungulates in Chennai, South India.

Bovine tuberculosis (bTB) is one of the major zoonotic concerns of the world, as milk and meat from cattle are major products for human consumption. Bovine tuberculosis not only affects the health of cattle and poses an imminent zoonotic threat, but also causes significant economic loss in both developed and developing countries. This systematic review reports the prevalence of Mycobacterium tuberculosis complex (MTBC) organisms in slaughtered cattle showing tuberculosis like lesion (TBL) with available literature worldwide. Appropriate keywords were used to search various databases to collect articles pertaining to slaughterhouse studies. Bovine TB prevalence, based on the prevalence of MTBC organisms in slaughtered cattle showing TBL by culture, microscopy, PCR and spoligotyping was assessed in each study using a random-effects model and standardized mean with 95% confidence interval (CI). Heterogeneity was assessed by the I2 statistic. Publication bias was evaluated using funnel plots. Out of 72 hits, 37 studies were selected based on title and abstract. Ten articles were excluded due to lack of desired data, and 27 studies were included in the final analysis. From the selected articles, it was found that 426 [95% CI: 302 - 560] per 1,000 slaughtered cattle with TBL were positive for the presence of MTBC organisms. The sensitivity analysis showed that no individual study alone influenced the estimation of pooled prevalence. The prevalence of MTBC organisms in slaughtered cattle showing TBL by culture, microscopy, PCR and spoligotyping was 474[95% CI: 342- 610], 385 [95% CI: 269- 515], 218 [95% CI: 132- 338], 326 [95% CI: 229-442] respectively, per 1,000 slaughtered cattle. Most of the slaughtered cattle were from the same locality as the slaughterhouse. The results obtained in this study suggest that abattoir monitoring can give an estimate of the prevalence of bTB in that locality. This study also emphasizes the need to test cattle and animal handlers who were in contact with bTB positive cattle.

Role of abattoir monitoring in determining the prevalence of bovine tuberculosis: A systematic review and meta-analysis

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 Mycobacterial 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 M. 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 (Antilope cervipara) andone gazelle (Gazella 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 differents poligotypes in different species within the family Bovidae as well as humans is discussed.

Isolation and analysis of the molecular epidemiology and zoonotic significance of Mycobacterium tuberculosis in domestic and wildlife ruminants from three states in India.

| 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 RealTime 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 micro-organism 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. RealTime 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.

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Real-Time PCRs that Discriminates Mycobacterium tuberculosis and Mycobacterium bovis Based on Single Nucleotide Polymorphism and Genome Analysis of Amplicons

Pasha Sy

Papers

Isolation and analysis of the molecular epidemiology and zoonotic significance of Mycobacterium tuberculosis in domestic and wildlife ruminants from three states in India.

Real-Time PCRs that Discriminates Mycobacterium tuberculosis and Mycobacterium bovis Based on Single Nucleotide Polymorphism and Genome Analysis of Amplicons