Tuberculosis in wild animals in India.

India is renowned for its complex megadiverse ecosystems and abundant biodiversity. Bovine tuberculosis (bTB) often remains synonymous with Mycobacterium bovis infection in cattle. The domain of tuberculosis (TB) among wild animals, induced by members of the Mycobacterium tuberculosis complex organisms (MTBC), is often underexplored and underreported in India. Within this context, instances of wild animal tuberculosis (wTB) have manifested across both captive and free-roaming animals. The sources contributing to wTB in animals can be human, animal, or environmental factors, thus illuminating the complex transmission pathways. The diagnosis of wTB continues to pose a formidable challenge, a consequence of the expansive taxonomic diversity in both the host and the pathogen. Complications inherent in acquiring samples from wildlife, the absence of standardized diagnostic protocols, limited insights into infection prevalence, and resource constraints compound diagnosis. Amidst these, adopting the comprehensive One Health paradigm surfaces as an imperative, accentuating the interconnectedness bridging human, animal, and environmental health. Recognizing key stakeholders and fostering intersectoral collaboration to provide enhanced diagnostic techniques driven by skilled personnel and advanced infrastructure play pivotal roles in a comprehensive strategy. Additionally, leveraging vaccination efforts contributes to effective control. A national wTB surveillance program is a cornerstone, ensuring an integrated and holistic approach to disease management. Through this review, we delve into the current landscape of wTB in India, unveiling its multifaceted challenges, and further explore the multifarious strategies that the One Health approach proffers in this dynamic endeavor.

Genomic Characterization of IS6110 Insertions in Mycobacterium orygis.

Mycobacterium orygis, a subspecies of the Mycobacterium tuberculosis complex (MTBC), has emerged as a significant concern in the context of One Health, with implications for zoonosis or zooanthroponosis or both. MTBC strains are characterized by the unique insertion element IS6110, which is widely used as a diagnostic marker. IS6110 transposition drives genetic modifications in MTBC, imparting genome plasticity and profound biological consequences. While IS6110 insertions are customarily found in the MTBC genomes, the evolutionary trajectory of strains seems to correlate with the number of IS6110 copies, indicating enhanced adaptability with increasing copy numbers. Here, we present a comprehensive analysis of IS6110 insertions in the M. orygis genome, utilizing ISMapper, and elucidate their genetic consequences in promoting successful host adaptation. Our study encompasses a panel of 67 paired-end reads, comprising 11 isolates from our laboratory and 56 sequences downloaded from public databases. Among these sequences, 91% exhibited high-copy, 4.5% low-copy, and 4.5% lacked IS6110 insertions. We identified 255 insertion loci, including 141 intragenic and 114 intergenic insertions. Most of these loci were either unique or shared among a limited number of isolates, potentially influencing strain behavior. Furthermore, we conducted gene ontology and pathway analysis, using eggNOG-mapper 5.0, on the protein sequences disrupted by IS6110 insertions, revealing 63 genes involved in diverse functions of Gene Ontology and 45 genes participating in various KEGG pathways. Our findings offer novel insights into IS6110 insertions, their preferential insertion regions, and their impact on metabolic processes and pathways, providing valuable knowledge on the genetic changes underpinning IS6110 transposition in M. orygis.

Bovine tuberculosis in India: The need for One Health approach and the way forward.

Bovine tuberculosis (bTB) is a chronic granulomatous infectious illness in cattle. The etiological agent of bTB is Mycobacterium bovis. However, other members belonging to the Mycobacterium tuberculosis complex, like M. tuberculosis, M. africanum, M. caprae, M. orygis, and M. microti are known to cause bTB in cattle. There are 303.76 million bovines in India, and it is the largest producer of milk and the second largest producer of meat worldwide. The prevalence of bTB among farm and dairy cattle in India is estimated to be around 7.3%, which makes it a country with one of the largest infected herds in the world. While bTB control programs have had considerable success in reducing the prevalence of the disease in many developed countries, they have yet to be formulated or implemented in India. Bovine TB also has a zoonotic and reverse component, which means that the disease can spread from cattle to human and from human to cattle. In a country like India, which contributes to nearly one-fourth of the global TB burden, the zoonotic aspect must be addressed so that the disease can be curbed. While cattle are the primary reservoir host to bTB, animals like goats, deer, bison, pigs, dogs, badgers, possums, and primates are also susceptible to the disease. This review talks about the burden of bTB in India and the necessity of One Health approach to combat the disease.

Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis: A Review and Update.

Pyrazinamide (PZA) has remained a keystone of tuberculosis (TB) therapy, and it possesses high imperative sterilizing action that can facilitate reduction in the present chemotherapy regimen. The combination of PZA works both with first- and second-line TB drugs, notably fluoroquinolones, clofazimine, bedaquiline, delamanid and pretomanid. Pyrazinamide inhibits various targets that are involved in different cellular processes like energy production (pncA), trans-translation (rpsA) and pantothenate/coenzyme A (panD) which are required for persistence of the pathogen. It is well known that pncA gene encoding pyrazinamidase is involved in the transition of PZA into the active form of pyrazinoic acid, which implies that mutation in the pncA gene can develop PZA resistance in Mycobacterium tuberculosis (M. tuberculosis) strain leading to a major clinical and public health concern. Therefore, it is very crucial to understand its resistance mechanism and to detect it precisely to help in the management of the disease. Scope of this review is to have a deep understanding of molecular mechanism of PZA resistance with its multiple targets which would help study the association of mutations and its resistance in M. tuberculosis. This will in turn help learn about the resistance of PZA and develop more accurate molecular diagnostic tool for drug-resistant TB in future TB therapy.

COVID-19 in animals: A need for One Health approach.

BACKGROUND: SARS-CoV-2 has been identified as the cause of the COVID-19, which caused a global pandemic. It is a pathogen that causes respiratory disease and can easily navigate the interspecies barrier. A significant number of COVID-19 cases in animals have been reported worldwide, including but not limited to animals in farms, captivity, and household pets. Thus, assessing the affected population and anticipating 'at risk' population becomes essential. OBJECTIVES: This article aims to emphasize the zoonotic potential of SARS- CoV-2 and discuss the One Health aspects of the disease. CONTENT: This is a narrative review of recently published studies on animals infected with SARS-CoV-2, both experimental and natural. The elucidation of the mechanism of infection by binding SARS-CoV-2 spike protein to the ACE-2 receptor cells in humans has led to bioinformatic analysis that has identified a few other susceptible species in silico. While infections in animals have been extensively reported, no intermediary host has yet been identified for this disease. The articles collected in this review have been grouped into four categories; experimental inoculations, infection in wild animals, infection in farm animals and infection in pet animals, along with a review of literature in each category. The risk of infection transmission between humans and animals and vice versa and the importance of the One Health approach has been discussed at length in this article.

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

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-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 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.

Role of a Putative Alkylhydroperoxidase Rv2159c in the Oxidative Stress Response and Virulence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, which causes tuberculosis, is one of the leading infectious agents worldwide with a high rate of mortality. Following aerosol inhalation, M. tuberculosis primarily infects the alveolar macrophages, which results in a host immune response that gradually activates various antimicrobial mechanisms, including the production of reactive oxygen species (ROS), within the phagocytes to neutralize the bacteria. OxyR is the master regulator of oxidative stress response in several bacterial species. However, due to the absence of a functional oxyR locus in M. tuberculosis, the peroxidase stress is controlled by alkylhydroperoxidases. M. tuberculosis expresses alkylhydroperoxide reductase to counteract the toxic effects of ROS. In the current study, we report the functional characterization of an orthologue of alkylhydroperoxidase family member, Rv2159c, a conserved protein with putative peroxidase activity, during stress response and virulence of M. tuberculosis. We generated a gene knockout mutant of M. tuberculosis Rv2159c (MtbΔ2159) by specialized transduction. The MtbΔ2159 was sensitive to oxidative stress and exposure to toxic transition metals. In a human monocyte (THP-1) cell infection model, MtbΔ2159 showed reduced uptake and intracellular survival and increased expression of pro-inflammatory molecules, including IL-1ß, IP-10, and MIP-1α, compared to the wild type M. tuberculosis and Rv2159c-complemented MtbΔ2159 strains. Similarly, in a guinea pig model of pulmonary infection, MtbΔ2159 displayed growth attenuation in the lungs, compared to the wild type M. tuberculosis and Rv2159c-complemented MtbΔ2159 strains. Our study suggests that Rv2159c has a significant role in maintaining the cellular homeostasis during stress and virulence of M. tuberculosis.

A putative short-chain dehydrogenase Rv0148 of Mycobacterium tuberculosis affects bacterial survival and virulence.

During infection, Mycobacterium tuberculosis combats the stress generated by the host cells through the action of short-chain dehydrogenases/reductases (SDRs). Rv0148 belongs to the oxidoreductase family with the SDRs domain, which regulates the homeostasis of M. tuberculosis. In our earlier studyusing knockout mutant strain (∆0148), we reported that Rv0148 is involved in intermediary metabolism, drug resistance and cell homeostasis of M. tuberculosis. In the current study, we explored the functional role of Rv0148 using gene knockout mutant in-vitro and in-vivo models of infection. We report the ∆0148 is attenuated for virulence of M. tuberculosis. During human monocyte (THP-1) cell line infection, M. tuberculosis Δ0148 displayed reduced intracellular survival compared to the wild type at successive time points. Similarly, in a guinea pig animal model of aerosol infection, Δ0148 displayed a growth attenuation at 5- and 10-week post-infection in the lungs and spleen compared to the wild-type M. tuberculosis and Rv0148-complemented Δ0148 strains. Our study suggest that Rv0148 has a distinct role in the intracellular virulence of M. tuberculosis.

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

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.

Double deletion of PknI/DacB2 leads to attenuation of Mycobacterium tuberculosis for growth and virulence.

Serine/Threonine Protein Kinases (STPKs) phosphorylates target proteins thereby regulates various important cellular signal transduction pathways such as cell division and cell wall synthesis. It has been demonstrated that the STPKs regulate peptidoglycan biosynthesis by phosphorylating penicillin binding proteins (PBPs). We extensively characterized both PknI (STPK) and DacB2 (PBP) roles individually as well as combining by genetic knockout and phenotypic characterization studies. In the present study, we analyzed the role of PknI and DacB2 in cell division and virulence. The double knockout (DKO) strain growth was reduced under stress conditions like acidic pH, nutrient depletion media and low oxygen availability conditions. We also found that the DKO growth was significantly reduced in macrophage cell line and it was hypersensitive to oxidative and nitrosative stress condition. The DKO strain significantly attenuated in guinea pig model which was measured by reduced bacillary load, gross pathological and histopathological damages. Overall, these results clearly demonstrated that both PknI and DacB2 together play an important role in cell division under stress conditions, the DKO strain significantly attenuated both in vitro and in vivo models.

Protein-protein interaction of Rv0148 with Htdy and its predicted role towards drug resistance in Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis resides inside host macrophages during infection and adapts to resilient stresses generated by the host immune system. As a response, M. tuberculosis codes for short-chain dehydrogenases/reductases (SDRs). These SDRs are nicotinamide adenine dinucleotide-reliant oxidoreductases involved in cell homeostasis. The precise function of oxidoreductases in bacteria especially M. tuberculosis were not fully explored. This study aimed to know the detail functional role of one of the oxidoreductase Rv0148 in M. tuberculosis. RESULTS: In silico analysis revealed that Rv0148 interacts with Htdy (Rv3389) and the protein interactions were confirmed using far western blot. Gene knockout mutant of Rv0148 in M. tuberculosis was constructed by specialized transduction. Macrophage cell line infection with this knockout mutant showed increased expression of pro-inflammatory cytokines. This knockout mutant is sensitive to oxidative, nitrogen, redox and electron transport inhibitor stress agents. Drug susceptibility testing of the deletion mutant showed resistance to first-line drugs such as streptomycin and ethambutol and second-line aminoglycosides such as amikacin and kanamycin. Based on interactorme analysis for Rv0148 using STRING database, we identified 220 most probable interacting partners for Htdy protein. In the Rv0148 knockout mutants, high expression of htdy was observed and we hypothesize that this would have perturbed the interactome thus resulting in drug resistance. Finally, we propose that Rv0148 and Htdy are functionally interconnected and involved in drug resistance and cell homeostasis of M. tuberculosis. CONCLUSIONS: Our study suggests that Rv0148 plays a significant role in various functional aspects such as intermediatory metabolism, stress, homeostasis and also in drug resistance.

A review on bovine tuberculosis in India.

Bovine tuberculosis (bTB) is a chronic illness in animals, especially in cattle, leading to loss in the productivity and signifies a crucial public health risk. Regardless of the zoonotic threat and significant economic costs associated with the disease, precise estimates of bTB prevalence are deficient in many countries, including India, where national control programs are yet to be instigated. The true burden of the disease remains unknown due to lack of routine surveillance data from most of the developing countries. India is progressing well towards attaining the End TB goal, yet bTB continues to remain largely hidden. Moreover, the paucity of literature on bTB in India might lead to undue complacency and hence has to be scrupulously guarded and prevented from gaining any misconceptions in the minds of the common people. Preventing and controlling bTB at the animal interface is pivotal to evade transmission to human, increase food safety and guard the livelihood of the people. To attain this goal, implementation of strategies based on international norms and a multi-sectoral approach will empower enhanced surveillance and diagnosis of disease in animals and subsequently reduce the risk for humans. As an initiative, we step forward to address this review which briefly summarizes the available data in the literature from early 20th century to date to assess the status of bTB in India. We have discussed in detail, the epidemiology, transmission and diagnosis pertaining to bTB. The review also focuses on the interconnection between the health of people and animal, discuss the preventions and control strategies and recommend the use of vaccination in cattle to reduce the spread of infection among other animals and humans. Implementing One Health approach in India, which recognizes the interdependence of the health of people and animals will help the nation in the fight against TB.

Isolation and comparative genomics of Mycobacterium tuberculosis isolates from cattle and their attendants in South India.

The major human pathogen Mycobacterium tuberculosis is rarely reported to cause disease in other animals. Cases in livestock are thought to occur through contact with infected handlers, but previous studies evaluating putative livestock-human transmission used typing techniques with limited resolution. Here, we undertook cross-sectional surveillance for tuberculosis in 271 livestock handlers and 167 cattle on three farms in Chennai, India and defined the relatedness of cultured isolates using whole genome sequencing. Humans and livestock were screened for active mycobacterial infection, and opportunistic post-mortem examination was performed on comparative intradermal test-positive cattle that died. Four cattle and 6 handlers on two farms were culture-positive for M. tuberculosis; M. bovis was not isolated. All 10 isolates (one from each case) belonged to Lineage 1. Pairwise genome comparisons of single nucleotide polymorphism (SNP) differences ranged from 1 to 600 SNPs, but 3 isolate pairs were less than 5 SNPs different. Two pairs were from handlers and the third pair were from two cattle on the same farm. The minimum pairwise SNP difference between a cattle and human isolate was >250 SNPs. Our study confirms the presence of M. tuberculosis infection in cattle in India, sequencing of which characterised relatedness between human and cattle-derived isolates.

Whole-Genome Sequencing of a Mycobacterium orygis Strain Isolated from Cattle in Chennai, India.

Here, we report the isolation of Mycobacterium orygis from dairy cattle in Chennai, India. Spoligotyping assigned the isolate to spoligotype 587 (ST587), which belongs to M. orygis This species was confirmed as M. orygis using whole-genome sequencing.

Characterization of FtsY, its interaction with Ffh, and proteomic identification of their potential substrates in Mycobacterium tuberculosis.

The universally conserved signal recognition particle (SRP) pathway that mediates co-translational targeting of membrane and secretory proteins is essential for eukaryotic and prokaryotic cells. The Mycobacterium tuberculosis SRP pathway consists of 2 proteins, Ffh and FtsY, and a 4.5S RNA molecule. Although the Escherichia coli SRP pathway is well studied, understanding of the M. tuberculosis SRP pathway components is very limited. In this study, we have overexpressed and characterized the M. tuberculosis SRP receptor (SR) FtsY as a GTP binding protein. Further, we established the direct protein-protein interaction between Ffh and FtsY. The Ffh-FtsY complex formation resulted in mutual stimulation of their GTP hydrolysis activity. We also attempted to biochemically characterize the SRP components by constructing the antisense gene knockdown strains of ffh and ftsY in M. tuberculosis. Loss of ffh and ftsY resulted in a decreased in vitro growth rate of the antisense ffh strain as compared with the antisense ftsY strain. Finally, 2-D gel electrophoresis of antisense depleted ffh and ftsY strains identified differential expression of 14 proteins.

Molecular characterization of AmiC, a positive regulator in acetamidase operon of Mycobacterium smegmatis.

Mycobacterium smegmatis, a rapidly growing non-pathogenic mycobacterium, is currently used as a model organism to study mycobacterial genetics. Acetamidase of M. smegmatis is the highly inducible enzyme of Mycobacteria, which utilizes several amide compounds as sole carbon and nitrogen sources. The acetamidase operon has a complex regulatory mechanism, which involves three regulatory proteins, four promoters, and three operator elements. In our previous study, we showed that over-expression of AmiA leads to a negative regulation of acetamidase by blocking the P2 promoter. In this study, we have identified a new positive regulatory protein, AmiC that interacts with AmiA through protein-protein interaction. Gel mobility shift assay showed that AmiC protein inhibits AmiA from binding to the P2 promoter. Interaction of AmiC with cis-acting elements identified its binding ability to multiple regulatory regions of the operon such as P3, OP3, and P1 promoter/operator. Consequently, the addition of inducer acetamide to AmiC complexe trips the complexes, causing AmiC to appear to be the sensory protein for the amides. Homology modeling and molecular docking studies suggest AmiC as a member of Periplasmic binding proteins, which preferentially bind to the inducers and not to the suppressor. Over-expression of AmiC leads to down-regulation of the negative regulator, amiA, and constitutive up-regulation of acetamidase. Based on these findings, we conclude that AmiC positively regulates the acetamidase operon.

Mycobacterium tuberculosis Whole Genome Sequences From Southern India Suggest Novel Resistance Mechanisms and the Need for Region-Specific Diagnostics.

BACKGROUND.: India is home to 25% of all tuberculosis cases and the second highest number of multidrug resistant cases worldwide. However, little is known about the genetic diversity and resistance determinants of Indian Mycobacterium tuberculosis, particularly for the primary lineages found in India, lineages 1 and 3. METHODS.: We whole genome sequenced 223 randomly selected M. tuberculosis strains from 196 patients within the Tiruvallur and Madurai districts of Tamil Nadu in Southern India. Using comparative genomics, we examined genetic diversity, transmission patterns, and evolution of resistance. RESULTS.: Genomic analyses revealed (11) prevalence of strains from lineages 1 and 3, (11) recent transmission of strains among patients from the same treatment centers, (11) emergence of drug resistance within patients over time, (11) resistance gained in an order typical of strains from different lineages and geographies, (11) underperformance of known resistance-conferring mutations to explain phenotypic resistance in Indian strains relative to studies focused on other geographies, and (11) the possibility that resistance arose through mutations not previously implicated in resistance, or through infections with multiple strains that confound genotype-based prediction of resistance. CONCLUSIONS.: In addition to substantially expanding the genomic perspectives of lineages 1 and 3, sequencing and analysis of M. tuberculosis whole genomes from Southern India highlight challenges of infection control and rapid diagnosis of resistant tuberculosis using current technologies. Further studies are needed to fully explore the complement of diversity and resistance determinants within endemic M. tuberculosis populations.

Functional Characterization of PknI-Rv2159c Interaction in Redox Homeostasis of Mycobacterium tuberculosis.

Mycobacterium tuberculosis adapts to stress conditions by responding to the signals from its external environment. M. tuberculosis genome encodes 11 eukaryotic like serine/threonine protein kinases (STPK) and their importance in regulating the physiology and virulence of the bacteria are being explored. Previous study from our lab identified the M. tuberculosis STPK, PknI interacts with two peroxidase proteins such as Rv2159c and Rv0148. In this study, we have characterized the biological function behind the PknI-Rv2159c interaction in M. tuberculosis. Point mutation of Ala-Gly-Trp motif identified that only Ala49 and Gly50 amino acids of Rv2159c are responsible for interaction and there is no phosphorylation involved in the PknI-Rv2159c interaction. Rv2159c is a member from the carboxymuconolactone decarboxylase family with peroxidase activity. Enzymatic assays with catalytic site point mutants showed that Cys84 of Rv2159c was responsible for its alkylhydroperoxidase activity. Interestingly, interaction with PknI increased its peroxidase activity by several folds. Gene knockdown of Rv2159c in M. tuberculosis showed increased sensitivity to peroxides such as cumene hydroperoxide and hydrogen peroxide. Proteomic analysis of differentially expressing Rv2159c strains by 2D gel electrophoresis and mass spectrometry revealed the differential abundance of 21 proteins. The total absence of oxidoreductase, GuaB1 suggests the essential role of Rv2159c in redox maintenance. Our findings provide new insights on signaling mechanisms of PknI in maintaining the redox homeostasis during oxidative stresses.

Lipoprotein LpqS deficient M. tuberculosis mutant is attenuated for virulence in vivo and shows protective efficacy better than BCG in guinea pigs.

Bacterial lipoproteins are a functionally diverse class of membrane anchored proteins. Lipoproteins constitute nearly 2.5% of the Mycobacterium tuberculosis proteome. Inactivation of genes coding for individual lipoproteins results in attenuated phenotype of the mutants. LpqS is a lipoprotein highly conserved among slow growing pathogenic mycobacteria. Our previous study has shown that the lpqS gene deletion mutant of M. tuberculosis (MtbΔlpqS) poorly replicates in THP1-(human acute monocytic leukemia cell line) derived macrophagic cell line. In addition, guinea pigs, when infected with the mutant strain exhibited significantly reduced bacterial burden and pathological damage in the infected tissues in comparison with the parental strain infected group. Subsequently, we evaluated the protective efficacy of the mutant by immunization of guinea pigs through aerosol and subcutaneous routes. We observed that immunization of guinea pigs with MtbΔlpqS offered superior protection in lungs as compared to BCG. In addition, MtbΔlpqS also prevented the haematogenous spread of the disease which was evident from the significantly reduced splenic bacillary load compared to saline vaccinated animals. The gross pathological observations and the histopathological observations well corroborated the bacterial findings. We also observed that aerogenic route of immunization imparts superior protection compared to subcutaneous route of immunization. These findings well establishes the efficacy of M. tuberculosis mutant in imparting protection against pulmonary TB.