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.

Characterization by single strand conformation polymorphism of mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis in strains from Vancouver, Mexico City and New Delhi.

OBJECTIVE: The study was conducted to evaluate usefulness of single strand conformation polymorphism (SSCP) over DNA sequencing in the diagnosis of rifampicin (Rif)-resistant tuberculosis. METHODS: Forty seven isolates of Mycobacterium tuberculosis (MTB) Rif-resistant and 25 Rif-sensitive were obtained from Vancouver, Mexico city and New Delhi and were analyzed by polymerase chain reaction (PCR) amplification of rpoB gene and the mutations were identified by DNA sequencing and SSCP. RESULTS: The mutations observed by DNA sequencing in 47 RIF-resistant isolates showed that the most common mutation among Vancouver isolates was in codon 526, Hist-->Arg and in Mexico isolates was in codon 531, Ser-Leu and New Delhi isolates was in codon 516, Asp-->Val. Using fluorescence based PCR-SSCP, it was possible to distinguish Rif-resistant isolates from Rif-sensitive isolates. CONCLUSION: DNA sequencing is a highly accurate method for the detection of mutations associated with drug resistance in tuberculosis but is more expensive and requires special equipment and personnel. SSCP is a simple, accurate method and suitable for analysis of large number of samples and the results are available in less than 72 hours.

Flowcytometric assessment of intracellular interferon-γ production in human CD4 + ve T-cells on mycobacterial antigen stimulation.

Interferon- (IFN-γ ) has been considered to be a critical protective immunomodulatory component against Mycobacterium tuberculosis (M. tb.) infection. In this study T-cell proliferation and IFN-γ production upon stimulation with M. tb. were assessed in patients of pulmonary tuberculosis and healthy contacts. The studies were based on lymphocyte transformation test and detection of intracellular IFN-γ production by CD4 + ve T-cells by flowcytometry. Patients showed lower levels of proliferation, the stimulation index being in the range of 2·17 1·1 (mean + SD) compared to the contacts (SI = 4·59± 1·6) (P < 0·01). The kinetics of intracellular induction of IFN-γ on M. tb. stimulation showed a proportional increase in the CD4 + ve T-cell population. The increase was maximal between 96-120 h of culture. In healthy contacts the number of IFN-γ expressing CD + ve T-cells increased to 2·5 to 41 × 104 cells/ml in M. tb. stimulated cultures compared to control cultures (0·1 —15 × 104). In contrast patients showed no/marginal increase in CD4 + ve T-cell population expressing intracellular IFN- γ Thus the lack of induction of IFN in CD4 + ve T-cells in patients could be a critical shortcoming in their ability to combat tubercle bacilli infection.