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.

Direct detection and identification of Mycobacterium tuberculosis and Mycobacterium bovis in bovine samples by a novel nested PCR assay: correlation with conventional techniques.

Mycobacterium tuberculosis and M. bovis infect animals and humans. Their epidemiology in developed and developing countries differs, 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. 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.

Bovine tuberculosis in India: potential basis for zoonosis.

Our laboratory has designed a specific nested-PCR (N-PCR) assay, based on the hupB gene of Mycobacterium tuberculosis (Rv2986c) and Mycobacterium bovis (Mb3010c) as a method to differentiate these closely related species. The present paper deciphers the utility of this assay for identification of pathogenic Mycobacteria in clinical samples. Extra-pulmonary clinical samples obtained from cattle and humans were investigated. Pre-dominance of M. tuberculosis (15.7%) and M. bovis (26.8%) was seen in humans and cattle, respectively. However, more importantly, both mycobacterial pathogens (mixed infection) were identified in a number of samples. In humans 8.7% of the samples and 35.7% in cattle were classified as mixed infection. The detection of mixed infection with the mycobacterial pathogenic duo in humans and bovines denotes the prospect of potential transmission of these pathogens from humans to cattle (zoonosis) and vice versa (reverse zoonosis).

Use of the hupB gene encoding a histone-like protein of Mycobacterium tuberculosis as a target for detection and differentiation of M. tuberculosis and M. bovis.

The gene for histone-like protein (hupB [Rv2986c]) of Mycobacterium tuberculosis has been identified as a singular target which allows differentiation of two closely related mycobacterial species, namely, M. tuberculosis and M. bovis of the MTB complex, by a PCR assay. The N and S primer-generated PCR amplicons differed in M. tuberculosis and M. bovis; these amplicons were determined to be 645 and 618 bp, respectively. This difference was localized to the C-terminal part of the gene by using primers M and S. The C-terminal PCR amplicons of M. tuberculosis and M. bovis were determined to be 318 and 291 bp, respectively. The differences in the C-terminal portion of the gene were confirmed by restriction fragment length polymorphism analysis and sequencing. Sequence analysis indicated that in M. bovis there was a deletion of 27 bp (9 amino acids) in frame after codon 128 in the C-terminal part of the hupB gene. In the present study 104 mycobacterial strains and 11 nonmycobacterial species were analyzed for hupB gene sequences. Of the 104 mycobacterial strains included, 62 belonged to the MTB complex and 42 were non-MTB complex strains and species. Neither the hupB gene-specific primers (N and S) nor the C-terminal primers (M and S) amplify DNA from any other mycobacteria, making the assay suitable for distinguishing members of the MTB complex from other mycobacterial species, as well as for differentiating between members of the MTB complex, namely, M. tuberculosis and M. bovis.

Evaluation of the immunocontraceptive potential of Escherichia coli-expressed recombinant dog ZP2 and ZP3 in a homologous animal model.

Dog zona pellucida glycoprotein 2 (dZP2), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned and expressed as a polyhistidine fusion protein in Escherichia coli to evaluate the immunocontraceptive efficacy of ZP glycoproteins. The recombinant dZP2 (rec-dZP2) revealed a 70 kDa band corresponding to the full length transcript, as well as several low molecular mass fragments in western blot analysis. In addition to rec-dZP2, E. coli expressed recombinant dog ZP glycoprotein 3 (rec-dZP3), which has also been evaluated for its efficacy to block fertility in a homologous system. Three groups of female dogs (n = 4 per group) were immunized with rec-dZP2 conjugated to diphtheria toxoid (rec-dZP2-DT), rec-dZP3 conjugated to DT (rec-dZP3-DT) and DT alone. Immunization of female dogs with rec-dZP2-DT and rec-dZP3-DT led to generation of antibodies against the respective ZP proteins as well as to DT. Subsequent to mating, the four female dogs immunized with rec-dZP2-DT all conceived, which is indicative of failure of the anti-rec-dZP2 antibodies to block fertility. In the group of dogs immunized with rec-dZP3-DT, three of four animals did not conceive when mated with males of proven fertility. The block in fertility was associated with anti-dZP3 antibody titres. Ovarian histopathology revealed that the block in fertility in the group immunized with rec-dZP3-DT is probably manifested by inhibition in the development of follicles and is due to atretic changes in the zona pellucida. These results, although preliminary, indicate that immunization with dZP3 may be a feasible proposition to control dog populations provided that adequate antibody titres are achieved.