Affiliation:
1. Department of Biotechnology
2. Central JALMA Institute for Leprosy and other Mycobacterial Diseases, Taj Ganj, Agra 282001, India
3. Central Military Veterinary Laboratory, Meerut Cantt 250001, India
4. Department of Biostatistics, All India Institute of Medical Sciences, New Delhi 110029, India
Abstract
ABSTRACT
Mycobacterium tuberculosis
and
M. bovis
infect animals and humans. Their epidemiologies in developed and developing countries differ, owing to differences in the implementation of preventive measures (World Health Organization, 1999). Identification and differentiation of these closely related mycobacterial species would help to determine the source, reservoirs of infection, and disease burden due to diverse mycobacterial pathogens. The utility of the
hupB
gene (Rv2986c in
M. tuberculosis
, or
Mb3010c
in
M. bovis
) to differentiate
M. tuberculosis
and
M. bovis
was evaluated by a PCR-restriction fragment length polymorphism (RFLP) assay with 56 characterized bovine isolates (S. Prabhakar et al., J. Clin. Microbiol.
42:
2724-2732, 2004). The degree of concordance between the PCR-RFLP assay and the microbiological characterization was 99.0% (
P
< 0.001). A nested PCR (N-PCR) assay was developed, replacing the PCR-RFLP assay for direct detection of
M. tuberculosis
and
M. bovis
in bovine samples. The N-PCR products of
M. tuberculosis
and
M. bovis
corresponded to 116 and 89 bp, respectively. The detection limit of mycobacterial DNA by N-PCR was 50 fg, equivalent to five tubercle bacilli.
M. tuberculosis
and/or
M. bovis
was detected in 55.5% (105/189) of the samples by N-PCR, compared to 9.4% (18/189) by culture. The sensitivities of N-PCR and culture were 97.3 and 29.7, respectively, and their specificities were 22.2 and 77.7%, respectively. The percentages of animals or samples identified as infected with
M. tuberculosis
or
M. bovis
by N-PCR and culture reflected the clinical categorizations of the cattle (
P
of <0.05 to <0.01). Mixed infection by N-PCR was detected in 22 animals, whereas by culture mixed infection was detected in 1 animal.
Publisher
American Society for Microbiology
Reference48 articles.
1. Ayele, W. Y., S. D. Neil, J. Zinsstag, M. G. Weiss, and I. Pavlik. 2004. Bovine tuberculosis: an old disease but a new threat to Africa. Int. J. Tuberc. Lung Dis.8:924-927.
2. Barouni, A. S., C. J. Augusto, M. T. Lopes, M. S. Zanini, and C. E. Salas. 2004. A pncA polymorphism to differentiate between Mycobacterium bovis and Mycobacterium tuberculosis. Mol. Cell. Probes18:167-170.
3. Chakravorty, S., and J. S. Tyagi. 2001. Novel use of guanidinium isothiocyanate in isolation of M. tuberculosis DNA from clinical materials. FEMS Microbiol. Lett.205:113-117.
4. Utility of Universal Sample Processing Methodology, Combining Smear Microscopy, Culture, and PCR, for Diagnosis of Pulmonary Tuberculosis
5. Chimara, E., L. Ferrazoli, and S. C. Leão. 2004. Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis. Mem. Inst. Oswaldo Cruz99:745-748.