Bacterial dissemination in Mycobacterium tuberculosis by CD+ T-cells & proinflammatory cytokines

Background & objectives: As CD4+ and CD8+ T lymphocyte numbers decline, the conventional, localized forms of tuberculosis shift to the atypical, disseminated forms. Variations in lymphocyte and immune cell expression levels affect how tuberculosis manifests in disseminated forms. Understanding the relationship between lymphocyte counts (CD4+ and CD8+) and pro-inflammatory cytokines such as tumour necrosis factor-alpha, interleukin-12 and interferon, we may therefore be able to shed light on how infections spread and suggest potential biomarkers for these immune factors. Methods: In this study, 15 guinea pigs were infected with Mycobacterium tuberculosis (M.tb) H37Rv strain and grouped into three groups of five each for further investigation. Serum samples and bronchoalveolar lavage (BAL) fluid were examined for the expression of pro-inflammatory cytokines and T-cell subsets in guinea pigs infected with pulmonary tuberculosis and disseminated tuberculosis. Results: We found that M.tb escapes macrophages due to pro-inflammatory cytokine dysregulation. Despite the protective immunity created by T-cells and cytokines, M.tb bacilli may spread to other organs due to inflammation induced by these immune components. A high number of T-cells and stimulated cytokine production are involved in triggering inflammation after necrotic tissue develops and tuberculosis spreads. Interpretation & conclusions: Our findings imply that increased bacilli in the spleen at the 8th wk of infection may be caused by the overexpression of CD4+ T-cell lymphocyte subsets and cytokines that generated inflammation during the 4th wk of infection. This is a pilot study with a small sample size and less assertive inference. Larger studies would be helpful to validate the results of the present investigation.

Omental desmoplastic small round cell tumor with metastasis

Desmoplastic small round cell tumor (DSRCT) is a very rare diagnosis with about 200 cases reported in literature. DSRCT is a recently described histopathological entity by Gerald and Rosai in 1989. Abdominopelvic cavity especially peritoneum is the most common site. We report a case of a huge omental DSRCT with lymph node metastasis which was initially misdiagnosed as gastrointestinal stromal tumor on radiology. A 26-year-old male presented with complaints of upper abdominal swelling associated with constant dull pain. On examination there was a large 15 × 12 cm intraabdominal mass in the epigastric and umbilical region. Imaging studies were suggestive of neoplastic mesenchymal etiology. Image-guided fine-needle aspiration cytology (FNAC) was suggestive of mesenchymal neoplastic etiology. On laparotomy, there was a huge 20 × 15 cm mass arising from omentum with multiple omental and mesenteric seedlings and mesenteric, peripancreatic and perigastric lymphadenopathy. The patient underwent debulking surgery with uneventful post-operative recovery. Histopathological examination with immunohistochemistry revealed a diagnosis of DSRCT of omentum and small bowel mesentery with lymph node metastasis. Patient then received adjuvant chemotherapy with multiple chemotherapeutic drugs as per P6 protocol and has stable disease at 1 year follow up.

Rapid detection of ethambutol-resistant Mycobacterium tuberculosis in clinical specimens by real-time polymerase chain reaction hybridisation probe method

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens. Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa. Materials and Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein–Jenson (L–J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB. Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L–J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L–J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing. Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.

Genetic diversity of Mycobacterium tuberculosis isolates from central India.

Background & objectives: There is a paucity of data available on genetic biodiversity of Mycobacterium tuberculosis isolates from central India. The present study was carried out on isolates of M. tuberculosis cultured from diagnostic clinical samples of patients from Bhopal, central India, using spoligotyping as a method of molecular typing. Methods: DNA was extracted from 340 isolates of M. tuberculosis from culture, confirmed as M. tuberculosis by molecular and biochemical methods and subjected to spoligotyping. The results were compared with the international SITVIT2 database. Results: Sixty five different spoligo international type (SIT) patterns were observed. A total of 239 (70.3%) isolates could be clustered into 25 SITs. The Central Asian (CAS) and East African Indian (EAI) families were found to be the two major circulating families in this region. SIT26/CAS1_DEL was identified as the most predominant type, followed by SIT11/EAI3_IND and SIT288/CAS2. Forty (11.8%) unique (non-clustered) and 61 (17.9%) orphan isolates were identified in the study. There was no significant association of clustering with clinical and demographic characteristics of patients. Interpretation & conclusions: Well established SITs were found to be predominant in our study. SIT26/CAS1_DEL was the most predominant type. However, the occurrence of a substantial number of orphan isolates may indicate the presence of active spatial and temporal evolutionary dynamics within the isolates of M. tuberculosis.

Comparative Study of Real-Time PCR with Culture in the Diagnosis of Tubercular Lymphadenopathy.

One-third of the world’s population is estimated to be infected with M.tuberculosis. Timely and accurate diagnosis is pivotal in the management of the disease. Conventional tests, although accurate, are time consuming as compared to the more promising latest molecular techniques like real-time PCR which are rapid, reproducible and reliable. Objective : The aim of the study is to compare the real-time PCR with culture method in the diagnosis of tubercular lymphadenopathy. Material and Methods: Present study included 40 patients belonging to the age group 0-40 years and presenting as cervical lymphadenopathy, attending the indoor and out-patient of Department of TB & Chest Diseases, S.N. Medical College, Agra (U.P.) from Jan’2009 to June’2010. All patients were subjected to routine investigations, e.g. hemogram, montoux test, Chest skiagram, AFB staining, and sputum examination. Lymph node aspirate were subjected to decontamination, DNA isolation and Real time PCR( q PCR). Also specimen were simultaneously put to culture on L.J. Media. Results of both modalities compared. Results: Conventional culture method was able to detect M.tuberculosis in 75% of the cases as compared to real-time PCR which was positive in 77.5% with comparable sensitivity (100% vs 96.7%) and specificity (100% vs 87.5%). Conclusion: Conventional culture method is gold standard in the diagnosis of tuberculosis since long but recent molecular assays like Real-time PCR is one of the latest addenda to the armamentarium for the rapid and accurate diagnosis of M.tuberculosis. Needless to say, early diagnosis is advantageous to the management of tuberculosis.

Clonal Diversity and Drug Resistance in Mycobacterium tuberculosis Isolated from Extra-pulmonary Samples in Central India - a Pilot Study.

In India, extrapulmonary tuberculosis (EPTB) accounts for 10 - 15% of all types of tuberculosis. To identify and compare predominant spoligotypes and drug‑resistance patterns in strains of Mycobacterium tuberculosis isolated from extrapulmonary and pulmonary specimens in central India, drug susceptibility testing and spoligotyping were carried out. Spoligotyping data was analyzed using SITVIT2 database. ST11/EAI3_Ind with 33% isolates among extrapulmonary specimens and ST26/ CAS1_DEL with 28% isolates among pulmonary specimens were the most predominant lineages. Multidrug resistance was found in 5.5% of the strains isolated from extrapulmonary specimens in contrast to 17% isolated from pulmonary specimens.

A pilot study to determine genetic polymorphism in Mycobacterium tuberculosis isolates in Central India.

This study was carried out to identify predominant spoligotypes responsible for transmission and prevalence of tuberculosis in central India since there is no data available about the genetic biodiversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in this region. 35 strains of Mycobacterium tuberculosis were subjected to spoligotyping according to the standard protocol. A total of 25 strains out of the 35 (71.42%) could be grouped in to 6 clusters. The largest cluster comprised 8 isolates. Unique (Non-clustered) spoligotypes were seen in 10 isolates, Nine strains did not match the data base (Spol DB-4 data base). The results indicate that there may be a number of orphan strains unique to this geographical area. Further studies on a larger sample size derived from this area would help us delineate the epidemiology of Mycobacterium tuberculosis infection in this area.

Comparison of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide tube method with the conventional method and real-time polymerase chain reaction for the detection of rifampicin resistance in Mycobacterium tuberculosis.

Colorimetric methods are cheap, reproducible, and rapid methods of detecting drug resistance in Mycobacterium tuberculosis. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) method is one such technique that has been established in our laboratory to detect rifampicin resistance. The present study compared the results of the MTT method with those of the proportion method and real-time polymerase chain reaction (RTPCR) in order to establish sensitivity and specificity of MTT. The mutations for rifampicin resistance occur in rpoB gene, and the commonest reported are in codons 526 and 531. Therefore, RTPCR was targeted at these two codons. The concordance of MTT with the proportion method and RTPCR was 94 and 72.77%, respectively, and that of RTPCR with the proportion method was 77.77%. While the study confirmed that the MTT method is a good method for detecting rifampicin resistance, it also brought out the fact that RTPCR when targeted for limited mutations is not a good tool. Either the genotypic method used should target the total 81-bp rpoB genome or methods such as DNA sequencing should be used. For resource-constraint laboratories, the MTT method can be considered as a better choice.

Mycobacterium indicus pranii as stand-alone or adjunct immunotherapeutic in treatment of experimental animal tuberculosis.

Background & objectives: Mycobacterium w (M.w) is a saprophytic cultivable mycobacterium and shares several antigens with M. tuberculosis. It has shown good immunomodulation in leprosy patients. Hence in the present study, the efficacy of M.w immunotherapy, alone or in combination with multi drug chemotherapeutic regimens was investigated against drug sensitive M. tuberculosis H37Rv and three clinical isolates with variable degree of drug resistance in mice. Methods: BALB/c mice were infected with M. tuberculosis H37Rv (susceptible to all first and second line drugs) and three clinical isolates taken from the epository of the Institute. The dose of 200 bacilli was used for infection via respiratory route in an aerosol chamber. Chemotherapy (5 days/wk) was given one month after infection and the vaccinated group was given a dose of 1×107 bacilli by subcutaneous route. Bacterial load was measured at 4 and 6 wk after initiation of chemotherapy. Results: M.w when given along with chemotherapy (4 and 6 wk) led to a greater reduction in the bacterial load in lungs and other organs of TB infected animals compared to. However, the reduction was significantly (P<0.05) more in terms of colony forming units (cfu) in both organs (lungs and spleen). Conclusion: M.w (as immunomodulator) has beneficial therapeutic effect as an adjunct to chemotherapy.

Molecular typing of Mycobacterium leprae strains from northern India using short tandem repeats.

Background & objectives: Due to the inability to cultivate Mycobacterium leprae in vitro and most cases being paucibacillary, it has been difficult to apply classical genotyping methods to this organism. The objective of this study was therefore, to analyze the diversity among M. leprae strains from Uttar Pradesh, north India, by targeting ten short tandem repeats (STRs) as molecular markers. Methods: Ninety specimens including 20 biopsies and 70 slit scrappings were collected in TE buffer from leprosy patients, who attended the OPD of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, and from villages of Model Rural Health Research Unit (MRHRU) at Ghatampur, Kanpur, Uttar Pradesh. DNA was extracted from these specimens and ten STRs loci were amplified by using published and in-house designed primers. The copy numbers were determined by electrophoretic mobility as well as sequence analysis. Phylogenetic analysis was done on variable number of tandem repeats (VNTRs) data sets using start software. Results: Diversity was observed in the cross-sectional survey of isolates obtained from 90 patients. Allelic index for different loci was found to vary from 0.7 to 0.8 except for rpoT for which allelic index was 0.186. Similarity in fingerprinting profiles observed in specimens from the cases from same house or nearby locations indicated a possible common source of infection. Such analysis was also found to be useful in discriminating the relapse from possible reinfection. Interpretation & conclusions: This study led to identification of STRs eliciting polymorphism in north Indian strains of M. leprae. The data suggest that these STRs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci.

Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates.

Background & objectives In drug resistant, especially multi-drug resistant (MDR) tuberculosis, fluoroquinolones (FQs) are used as second line drugs. However, the incidence of FQ-resistant Mycobacterium tuberculosis is rapidly increasing which may be due to extensive use of FQs in the treatment of various other diseases. The most important known mechanism i.e., gyrA mutation in FQ resistance is not observed in a significant proportion of FQ resistant M. tuberculosis isolates suggesting that the resistance may be because of other mechanisms such as an active drug efflux pump. In this study we evaluated the role of the efflux pumps in quinolone resistance by using various inhibitors such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophenol (DNP) and verapamil, in clinical isolates of M. tuberculosis. Methods A total of 55 M. tuberculosis clinical isolates [45 ofloxacin (OFL) resistant and 10 ofloxacin sensitive] were tested by Resazurin microtitre assay (REMA) to observe the changes in ofloxacin minimum inhibitory concentration (MIC) levels in presence of efflux inhibitors as compared to control (without efflux inhibitor). Results The MIC levels of OFL showed 2-8 folds reduction in presence of CCCP (16/45; 35.5%), verapamil (24/45; 53.3%) and DNP (21/45; 46.6%) while in case of isolates identified as OFL sensitive these did not show any effect on ofloxacin MICs. In 11 of 45 (24.5%) isolates change in MIC levels was observed with all the three inhibitors. Overall 30 (66.6%) isolates had reduction in OFL MIC after treatment with these inhibitors. A total of eight isolates were sequenced for gyrA gene, of which, seven (87.5%) showed known mutations. Of the eight sequenced isolates, seven (87.5%) showed 2 to 8 fold change in MIC in presence of efflux inhibitors. Interpretation & conclusions Our findings suggest the involvement of active efflux pumps of both Major Facilitator Super Family (MFS) family (inhibited by CCCP and DNP) and ATP Binding Cassette (ABC) transporters (inhibited by verapamil) in the development of OFL resistance in M. tuberculosis isolates. Epidemiological significance of these findings needs to be determined in prospective studies with appropriate number of samples / isolates.

jefA (Rv2459), a drug efflux gene in Mycobacterium tuberculosis confers resistance to isoniazid & ethambutol.

Background & objectives: Drug efflux pumps have been contributing factor(s) in the development of multidrug resistance in various clinically relevant bacteria. During efflux pump gene expression studies on mycobacteria, we have found a previously uncharacterized open reading frame (ORF) Rv2459 to be overexpressed in drug stressed conditions. The objective of the present study was to investigate the role of this ORF as a drug efflux pump, which might add new information in our understanding about the alternative mechanisms of drug resistance in mycobacteria. Methods: The open reading frame Rv2459 of Mycobacterium tuberculosis encoding a probable drug efflux protein has been cloned using pSD5 E.coli-Mycobacterium shuttle vector and overexpressed in M. tuberculosis H37Rv. This ORF was named as jefA. Overexpression of this gene in clones has been verified by real-time reverse transcription PCR. Minimum inhibitory concentrations (MICs) of recombinant as well as non-recombinant clones were determined by resazurin microtitre assay plate method (REMA) with and without efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil. Results: In recombinant strains of M. tuberculosis, the overexpression of this gene led to an increase in MIC of anti-tubercular drugs isoniazid and ethambutol when tested by REMA. In the presence of CCCP and verapamil, the recombinant strains showed decrease in MIC for these drugs. Bioinformatic analysis has shown a close relation of JefA protein with drug efflux pumps of other clinically relevant bacteria. In homology derived structure prepared from nearest available model, it was observed that amino acids forming TMH 1, 8 and 11 participated in ethambutol specificity and those forming TMH 2, 7 and 10 participated in isoniazid specificity in JefA. Interpretation & conclusion: The increased transcription of jefA leads to increased resistance to ethambutol and isoniazid in M. tuberculosis via efflux pump like mechanism and contributes in the development of resistance to these drugs. JefA amino acid sequence is well conserved among clinically important bacterial genera, which further provides evidence of being a potent drug efflux pump. The involvement in drug resistance and very little homology with any of the human proteins makes JefA important to be included in the list of potential drug targets.

Comparative growth pattern of multi drug resistance versus susceptible isolates of Mycobacterium tuberculosis in mice lungs.

Background & objectives: Rise in prevalence of multi-drug resistance (MDR) in tubercle bacilli is a serious cause of concern. As mutations with two house keeping genes rpoB and katG are associated with resistance to two important anti-tubercular drugs rifampicin and isoniazid respectively, there is a need to understand the growth kinetics of organisms with such mutated genes in experimental animals. This study was undertaken to study the growth kinetics of susceptible as well multi-drug resistance Mycobacterium tuberculosis isolates in mice. Methods: Two MDR (having mutations in rpoB and catG) and two drug susceptible isolates of M. tuberculosis along with H37Rv were grown in mice after aerogenic infection. Results: The MDR isolates grew slowly up to 3 wk though the growth was significantly different from sensitive strains. However, after 3 wk, the growth in sensitive as well MDR strains was similar, suggesting that even the mutations in the MDR strains did not have any impact on the growth kinetics. Interpretation & conclusions: The effect of mutations in other parts of these genes need to be studied. Retention of property of MDR strains to establish infection after aerogenic infection has epidemiological significance in terms of the transmission of MDR tuberculosis.

In vitro effect of fl uoroquinolones against Mycobacterium tuberculosis isolates from Agra & Kanpur region of north India.

Background & objectives: Fluoroquinolones (FQs) are important drugs used for treatment of drug resistant tuberculosis and are also now being considered as fi rst line drugs to shorten the duration of treatment of tuberculosis (TB). In order to fi nd out useful FQs for treatment of tuberculosis, the comparative effi cacy of fi ve FQs, namely, ofl oxacin (OFL), ciprofl oxacin (CIP), sparfl oxacin (SPX), gatifl oxacin (GAT) and levofl oxacin (LEVX) was studied against Mycobacterium tuberculosis (MTB) isolates obtained from both treated and untreated patients from Agra and Kanpur regions of north India. Methods: A total of 162 MTB isolates [including 110 MTB isolates obtained from untreated patients (Cat-I) and 52 isolates from treated patients (Cat-II)] were tested for their susceptibilities to FQs using standard minimum inhibitory concentration (MIC) method on Löwenstein-Jensen medium. Results: Keeping in view the therapeutically achievable drug levels, it was found that in Cat-I 97.2 per cent (107/110) isolates were sensitive to GAT, 89 per cent (98/110) to LEVX at 1 μg/ml whereas 92.7 per cent (102/110) isolates were inhibited by OFL at 2 μg/ml and 73.6 per cent (81/110) to SPX at 0.5 μg/ml. Only 63.6 per cent (70/110) isolates were found to be sensitive to CIP at 2 μg/ml which increased to 89 per cent (98/110) at 4 μg/ml (higher than achievable peak serum level). On the other hand, among 52 isolates for Cat-II, 37 (71.2%) were found to be sensitive to GAT and 33 (63.5%) to LEVX at 1 μg/ml concentration, 28 (53.8%) to SPX at 0.5 μg/ml whereas 33 (63.5%) and 24 (46.2%) isolates were found to be sensitive to OFL and CIP at 2 μg/ml, respectively. Interpretation & conclusions: It appears that GAT has higher activity against MTB isolates followed by OFL, LEVX and SPX whereas CIP showed the lowest activity. GAT was also found to be the most effective FQ against multi-drug resistant (MDR) isolates both from Cat-I and Cat-II patients. Thus, except CIP, other FQs showed potential to be included in the treatment regimens of tuberculosis including MDR-TB.

Identification of environmental mycobacteria isolated from Agra, north India by conventional & molecular approaches.

Background & objectives: Several environmental mycobacteria have been shown to be important human pathogens linked to immunomodulation especially in relation to effect on vaccination. Hence identification of mycobacteria to the species level is not only relevant to patient management but also to understand epidemiology of mycobacterial diseases and effect on vaccination. We undertook this study to assess the usefulness of various conventional and molecular methods in identification of environmental mycobacterial species from Agra, north India. Methods: One hundred nineteen isolates of environmental mycobacteria were grown from 291 (116 soil and 175 water) samples. These isolates were identified by standard biochemical tests, and a simple, rapid and cost-effective in-house developed gene amplification restriction analysis targeting 16S-23S rRNA spacer and flanking region and 16S rRNA sequencing. Results: Biochemical tests could clearly identify only 68.1 per cent (81/119) of isolates to species level. An in-house developed gene amplification - restriction analysis method could confirm the identity of 102 of 119 (85.7%) isolates and the remaining 17 isolates (14.3%) were confirmed by 16S rRNA sequencing also. These 119 environmental mycobacterial isolates, included several potentially pathogenic species such as M. fortuitum, M. chelonae, M. avium, M. marinum, M. manitobense, M. kansasii and others belonged to nonpathogenic species, M. terrae, M. smegmatis and M. flavescens. M. chelonae was isolated from water samples only whereas M. fortuitum was isolated from both water as well as soil samples. Interpretation & conclusion: The in-house developed gene amplification restriction analysis method though failed to accurately identify 14.3 per cent of isolates, facilitated rapid differentiation of most of environmental mycobacteria including potential pathogens from this area and thus would have diagnostic potential in cases with NTM infections. This combination strategy using PCR-RFLP and 16S rRNA sequencing may be useful for characterization of mycobacteria from similar environmental settings from other parts of world.

Serological diagnosis of tuberculosis.

OBJECTIVE: To evaluate the efficacy of ELISA for the detection of IgG antibodies against antigen 85 complex (Ag 85 complex) of Mycobacterium tuberculosis. METHODS: Children of either sex, 0-18 years of age, attending the outpatient department and admitted in the casualty and wards of the Department of Pediatrics, S.N. Medical College, Agra, were included in present study. The study was carried out on children with pulmonary and CNS tuberculosis along with matching controls (83 cases and 32 controls). Informed consents of their parents or guardians were taken. They were subjected to clinical examination, relevant laboratory investigations, tuberculin test and chest radiograph. Relevant body fluids were subjected to bacteriological tests; ELISA was applied to serum samples for detection of IgG antibodies against antigen 85 complex (Ag85). The result of ELISA was compared with bacteriological tests [Ziehl Neelson (ZN) staining for acid-fast bacilli, culture on Lowenstein Jensen (LJ) medium and culture on BacT/Alert 3D system]. RESULTS: ELISA tests showed a significantly higher sensitivity (59.1%) as compared with LJ medium culture method (19.3%), BacT/Alert 3D system (24.1%) and ZN staining (16.9%) in all patients (p<0.001). Specificity of ELISA test was 71.9%. CONCLUSION: In view of the convenience, low cost and good sensitivity, ELISA tests have a promising future in the diagnosis of childhood tuberculosis.

Isolation of Mycobacterium bovis & M. tuberculosis from cattle of some farms in north India--possible relevance in human health.

BACKGROUND & OBJECTIVE: Infection due to Mycobacterium bovis typically occurs in cattle and animals transmit infection to each other. The choice of appropriate clinical specimen is very important for isolation of M. bovis and M. tuberculosis from cattle. The present study reports the isolation of M. tuberculosis and M. bovis from different types of specimens from cattle suspected to be suffering from tuberculosis in certain organized cattle farms in north India. METHODS: A total of 768 specimens (heparinized or EDTA containing blood (162), fine needle aspirates from prescapular lymph gland (PSLG,160), milk (154), pharyngeal swab (PhS, 98), rectal pinch (RP, 97) and faecal sample (97) from 161 cattle of organized cattle farms in north India suspected to be suffering from tuberculosis were analyzed. After decontamination by modified Petroff's method isolation of M.tuberculosis complex was done on Lowenstein-Jensen medium (with and without pyruvate). The culture isolates were identified as M. tuberculosis and M. bovis on the basis of biochemical tests. RESULTS: A total of 54 M. tuberculosis complex isolates were obtained, of them 40 were identified as M.bovis and 14 as M. tuberculosis. M.bovis were isolated from 12 of 38 animals in group A (Tuberculin +ve with signs of tuberculosis), 7 of 37 animals in group B (Tuberculin +ve and apparently healthy), 9 of 21 group C animals in (Tuberculin -ve with clinical signs of tuberculosis), 4 of 26 animals in group D (Tuberculin -ve and apparently healthy), 4 of 27 group E animals (having non-mycobacterial infection) and 4 of 12 animals in group F (having clinical signs such as debilitated condition, cough, decreasing milk production, etc). Maximum number of M. bovis (19/40, 47.5%) and M. tuberculosis (5/14, 35.7%) isolates were grown from prescapular lymph gland biopsy (PSLG) followed by blood from which 9/40 (22.5%) M. bovis and 4/14 (28.5%) M. tuberculosis were isolated. M. bovis [6/40(15%)] and M. tuberculosis [4/14(28.5%)] were also isolated from milk. Only 3/40 (7.5%) isolates of M.bovis could be isolated from 97 rectal pinch followed by 98 pharyngeal swab 2/40 (5%) and 97 fecal samples 1/40 (2.5%) while 1/14 (7.1%) M.tuberculosis isolates were obtained from pharyngeal swab. INTERPRETATION & CONCLUSION: Among the samples analyzed, PSLG was found to be most suitable specimen for isolation of M. tuberculosis complex from cattle and is thus of diagnostic importance. M. bovis in milk indicates the need to investigate the transmission to human in such settings. Isolation of M. bovis and/or M. tuberculosis from apparently healthy cattle indicates sub-clinical infection in the herd. Further, isolation of a significant number of M. tuberculosis from cattle suggests possible human-to-cattle transmission which need to be confirmed by prospective studies including tools like DNA fingerprinting.