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Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 54 issue 10 (october 2020) : 1241-1245,   Doi: 10.18805/ijar.B-3573

Real-Time PCR Detection of Mycobacterium bovis in Blood and Lymph Node Aspirates of Bovines Positive in Tuberculosis Screening Tests

G
G.S. Sidhu
D
D. Narang
G
G. Filia
A
A. Singh
S
S.T. Singh
M
M. Chandra
N
N.S. Sharma
1Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
Cite article:- Sidhu G.S., Narang D., Filia G., Singh A., Singh S.T., Chandra M., Sharma N.S. (2020). Real-Time PCR Detection of Mycobacterium bovis in Blood and Lymph Node Aspirates of Bovines Positive in Tuberculosis Screening Tests. Indian Journal of Animal Research. 54(10): 1241-1245. doi: 10.18805/ijar.B-3573.

ABSTRACT

Bovine tuberculosis is a chronic infectious disease affecting broad range of mammalian hosts. ESAT-6 is a low molecular weight immunodominant protein coded by esxA gene located on RD1 region of genome and is responsible for virulence of Mycobacterium bovis. Out of 200 animals screened for bovine Tuberculosis (bTB), 38 animals (19%) were found positive for Comparative intradermal tuberculin test (CITT) (32 cattle, 6 buffaloes) and 41 animals were tested positive by IFN-g assay (29 cattle, 12 buffaloes). DNA extraction of blood (n=200) and lymph node aspirates (n=48) was done. Among 200 blood samples targeted for esxA (ESAT-6) gene, three samples (1.5%) whose CT was between 23-34 were considered positive by real-time PCR. Out of 48 animals (lymph node aspirates) that were positive either by CITT or IFN-g, one sample (2.08%) whose CT was between 23-34 were considered positive by real-time PCR. Remaining samples whose CT values were equal to or greater than 35 were considered negative. The sensitivity of esxA was 8 pg/ìl by real time PCR.

REFERENCES

  1. Cezar, R.D.S., Silva, N.C., Filho, A.F.B.B., Borges, J.D.M., Oliveira, P.R.F.D., Lúcio, E.C., Lima, M.A., Santana, V.L.D.A., Junior, J.W.P. (2016). Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil. BMC Veterinary Research. 12(31): 1-6.
  2. Cosivi, O., Grange, J.M., Daborn, C.J., Raviglione, M.C., Fujikura, T., Cousins, D., Robinson, R.A., Huchzermeyer, H.F.A.K., de Kantor, I., Meslin, F.X. (1998). Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerging Infectious Diseases. 4(1): 59-70.
  3. De la Rua-Domenech, R., Goodchild, A.T., Vordermeier, H.M., Hewinson, R.G., Christiansen, K.H., Clifton-Hadley, R.S. (2006). Ante mortem diagnosis of tuberculosis in cattle: a review of the tuberculin tests, gamma-interferon assay and other ancillary diagnostic techniques. Research in Veterinary Science. 81: 190-210.
  4. Dikshit, M., Sharma, R.J., Adsool, A.D., Chaphalkar, S.R. (2012). ESAT-6 and CFP-10 proteins of Mycobacterium tuberculosis in making diagnostic tool for TB. Journal of Biotechnology Letters. 3: 28-30.
  5. Gormley, E., Doyle, M.B., Fitzsimons, T., Mcgill, K., Collins, J.D. (2006). Diagnosis of M. bovis infection in cattle by use of gamma-interferon (Bovigam®) assay. Veterinary Microbiology. 112: 171-179.
  6. Monaghan, M.L., Doherty, M.L., Collins, J.D., Kazda, J.F., Quinn, P.J. (1994). The tuberculin test. Veterinary Microbiology. 40(1-2): 111-124.
  7. OIE. (2004). Manual of diagnostic tests and vaccines for terrestrial animals, 5th Edition, Chapter 2.3.3.
  8. OIE. (2009). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organisation for Animal Health, Paris, France.
  9. Parashar, D., Chauhan, D.S., Sharma, V.D., Katoch, V.M. (2006). Applications of real-time PCR technology to mycobacterial research. Indian Journal of Medical Research. 124: 385-398.
  10. Rogerson, B.J., Jung, Y.J., LaCourse, R., Ryan, L., Enright, N., North, R.J. (2006). Expression levels of Mycobacterium tuberculosis antigen encoding genes versus production levels of antigen specific T c4ells during stationary level lung infection in mice. Immunology. 118:195-201.
  11. Rothel, J.S., Jones, S.L., Corner, L.A., Cox, J.C., Wood, P.R. (1992). The gamma-interferon assay for diagnosis of bovine tuberculosis in cattle: conditions affecting the production of gamma interferon in whole blood culture. Australian Veterinary Journal. 69: 1-4.
  12. Thacker, T.C., Harris, B., Palmer, M.V., Waters, W.R. (2011). Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR. BMC Veterinary Research. 7: 50
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    Research Article

    volume 54 issue 10 (october 2020) : 1241-1245,   Doi: 10.18805/ijar.B-3573

    Real-Time PCR Detection of Mycobacterium bovis in Blood and Lymph Node Aspirates of Bovines Positive in Tuberculosis Screening Tests

    G
    G.S. Sidhu
    D
    D. Narang
    G
    G. Filia
    A
    A. Singh
    S
    S.T. Singh
    M
    M. Chandra
    N
    N.S. Sharma
    1Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India.
    Cite article:- Sidhu G.S., Narang D., Filia G., Singh A., Singh S.T., Chandra M., Sharma N.S. (2020). Real-Time PCR Detection of Mycobacterium bovis in Blood and Lymph Node Aspirates of Bovines Positive in Tuberculosis Screening Tests. Indian Journal of Animal Research. 54(10): 1241-1245. doi: 10.18805/ijar.B-3573.

    ABSTRACT

    Bovine tuberculosis is a chronic infectious disease affecting broad range of mammalian hosts. ESAT-6 is a low molecular weight immunodominant protein coded by esxA gene located on RD1 region of genome and is responsible for virulence of Mycobacterium bovis. Out of 200 animals screened for bovine Tuberculosis (bTB), 38 animals (19%) were found positive for Comparative intradermal tuberculin test (CITT) (32 cattle, 6 buffaloes) and 41 animals were tested positive by IFN-g assay (29 cattle, 12 buffaloes). DNA extraction of blood (n=200) and lymph node aspirates (n=48) was done. Among 200 blood samples targeted for esxA (ESAT-6) gene, three samples (1.5%) whose CT was between 23-34 were considered positive by real-time PCR. Out of 48 animals (lymph node aspirates) that were positive either by CITT or IFN-g, one sample (2.08%) whose CT was between 23-34 were considered positive by real-time PCR. Remaining samples whose CT values were equal to or greater than 35 were considered negative. The sensitivity of esxA was 8 pg/ìl by real time PCR.

    REFERENCES

    1. Cezar, R.D.S., Silva, N.C., Filho, A.F.B.B., Borges, J.D.M., Oliveira, P.R.F.D., Lúcio, E.C., Lima, M.A., Santana, V.L.D.A., Junior, J.W.P. (2016). Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil. BMC Veterinary Research. 12(31): 1-6.
    2. Cosivi, O., Grange, J.M., Daborn, C.J., Raviglione, M.C., Fujikura, T., Cousins, D., Robinson, R.A., Huchzermeyer, H.F.A.K., de Kantor, I., Meslin, F.X. (1998). Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerging Infectious Diseases. 4(1): 59-70.
    3. De la Rua-Domenech, R., Goodchild, A.T., Vordermeier, H.M., Hewinson, R.G., Christiansen, K.H., Clifton-Hadley, R.S. (2006). Ante mortem diagnosis of tuberculosis in cattle: a review of the tuberculin tests, gamma-interferon assay and other ancillary diagnostic techniques. Research in Veterinary Science. 81: 190-210.
    4. Dikshit, M., Sharma, R.J., Adsool, A.D., Chaphalkar, S.R. (2012). ESAT-6 and CFP-10 proteins of Mycobacterium tuberculosis in making diagnostic tool for TB. Journal of Biotechnology Letters. 3: 28-30.
    5. Gormley, E., Doyle, M.B., Fitzsimons, T., Mcgill, K., Collins, J.D. (2006). Diagnosis of M. bovis infection in cattle by use of gamma-interferon (Bovigam®) assay. Veterinary Microbiology. 112: 171-179.
    6. Monaghan, M.L., Doherty, M.L., Collins, J.D., Kazda, J.F., Quinn, P.J. (1994). The tuberculin test. Veterinary Microbiology. 40(1-2): 111-124.
    7. OIE. (2004). Manual of diagnostic tests and vaccines for terrestrial animals, 5th Edition, Chapter 2.3.3.
    8. OIE. (2009). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organisation for Animal Health, Paris, France.
    9. Parashar, D., Chauhan, D.S., Sharma, V.D., Katoch, V.M. (2006). Applications of real-time PCR technology to mycobacterial research. Indian Journal of Medical Research. 124: 385-398.
    10. Rogerson, B.J., Jung, Y.J., LaCourse, R., Ryan, L., Enright, N., North, R.J. (2006). Expression levels of Mycobacterium tuberculosis antigen encoding genes versus production levels of antigen specific T c4ells during stationary level lung infection in mice. Immunology. 118:195-201.
    11. Rothel, J.S., Jones, S.L., Corner, L.A., Cox, J.C., Wood, P.R. (1992). The gamma-interferon assay for diagnosis of bovine tuberculosis in cattle: conditions affecting the production of gamma interferon in whole blood culture. Australian Veterinary Journal. 69: 1-4.
    12. Thacker, T.C., Harris, B., Palmer, M.V., Waters, W.R. (2011). Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR. BMC Veterinary Research. 7: 50
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