Banner
Current IssueEditorial BoardOnline First ArticlesArchive
Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 53 issue 7 (july 2019) : 944-948,   Doi: 10.18805/ijar.B-3611

Quantification of Mycobacterium avium subsp. paratuberculosis from the tissues of challenged mice using SYBR Green real time PCR assay for the assessment of vaccine efficacy

R
R.K. Chaitanya
Y
Y. Krishnamohan Reddy
G
G. Dhinakar Raj
A
A. Thangavelu
1Department of Veterinary Microbiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.
Cite article:- Chaitanya R.K., Reddy Krishnamohan Y., Raj Dhinakar G., Thangavelu A. (2018). Quantification of Mycobacterium avium subsp. paratuberculosis from the tissues of challenged mice using SYBR Green real time PCR assay for the assessment of vaccine efficacy. Indian Journal of Animal Research. 53(7): 944-948. doi: 10.18805/ijar.B-3611.

ABSTRACT

A SYBR Green real time PCR assay amplifying F57 gene of Mycobacterium avium subsp. paratuberculosis (MAP) was developed to evaluate the protective efficacy of the inactivated vaccine to prevent the colonization of MAP in the tissues of BALB/c mice challenge model. Bacterial burden in the liver, spleen and intestine of vaccinated and sham immunized control mice, after intra peritoneal challenge with MAP were quantified by real time PCR assay.  A 195 bp fragment of MAP F57 sequence was cloned into TA cloning vector and the resultant recombinant plasmid was used to generate a series of quantification standards with copy number ranging from 109 to 1 copy. This absolute quantification technique is rapid and able to detect as few as 10 MAP organisms per gram of tissue and the assay has the efficiency of 0.982.

REFERENCES

  1. Bustin, S.A., Benes, V., Garson. J.A., Hallemans, J., Huggett. J., Kubista, M., Mueller, R., Nolan, T., et al. (2009). The MIQE guidelines: Minimum information for publication of quantitative Real Time PCR Experiments. Clinical Chemistry, 55: 4.
  2. Chen, L.H., Kumanan, K., Huang, C.J., McDonough, S.P., Stehman, S., Akey, B., Huntley, J., Bannantine, et al. J.P.,(2008). Immune responses in mice to Mycobacterium avium subsp. paratuberculosis following vaccination with a novel 74F recombinant polyprotein. Vaccine, 26: 1253-1262. 
  3. Cocito, C., Gilot, P., Coene, M., De Kese, M., Poupart, P., Vannuffel, P. (1994). Paratuberculosis. Clin Microbiol Rev, 7: 328-345.
  4. Herthnek, D. and Bolske, G. (2006). New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis. BMC Microbiolgy, 6: 87.
  5. Hines, M.E., Stabel, J.R., Sweeney, R.W., Griffin, F., Talaat, A.M., Bakker, D., Benedictus, G., Davis, W.C., et al (2007). Experimental challenge models for Johne’s disease: A review and proposed international guidelines. Veterinary Microbiology, 122: 197–222.
  6. Kawaji, S., Taylor, D.L., Mori, Y., Whittington, R.J. (2007). Detection of Mycobacterium avium subspecies paratuberculosisin ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture. Veterinary Microbiology, 125: 36-48.
  7. Koets, A., Hoek, A., Langelaar, M., Overdijk, M., Santema, W., Franken, P., Eden, W.V., Rutten, V. (2006) Mycobacterial 70 kD heat-    shock protein is an effective subunit vaccine against bovine paratuberculosis.Vaccine, 24: 2550–2559.
  8. Kralik, P., Nocker, A., Pavlik, I. (2010). Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment. International Journal of Food Microbiology, 141: S80-S86.
  9. Munoz, M., Garcia Marin, J.F., Garcia-Pariente, C., Reyes, L.E., Verna, A., Moreno, O., Fuertes, M., Doce, J. (2005). Efficacy of a killed vaccine (SILIRUM) in calves challenged with MAP. Proceedings of the 8th International Colloquium on Paratuberculosis, Denmark. pp.276-282.
  10. Nelli, R.K., Graham, E., Dunham, S.P., Taylor, D.J. (2008). Real-time PCR identification of Mycobacterium avium subsp. paratuberculosis in ovine and bovine tissues. Veterinary Record, 163: 422-423.
  11. Park, S.U., Kumanan, K., Mc Dough, S., Akey, B., Huntley, J., Bannantine, J.P., Chang,Y.F. (2008). Immunization with a DNA vaccine cocktail induces a Th1 response and protects mice against Mycobacterium avium subsp. paratuberculosis challenge. Vaccine, 26: 4329-4337.
  12. Poupart, P., Cone, M., Van Heuverswyn, H., Cocito, C. (1993). Preparation of a specific RNA probe for detection of Mycobacterium paratuberculosis and diagnosis of Johne’s disease. Journal of Clinical Microbiology, 31:1601-1605.
  13. Reddacliff, L., Eppleston, J., Windsor, P., Whittington, R., Jones, S. (2006). Efficacy of killed vaccine for the control of paratuberculosis in Australian sheep flocks. Veterinary Microbiology, 115: 77-90.
  14. Rosseels, V. and Huygen, K. (2008). Vaccination against paratuberculosis. Expert Reviews on Vaccines, 7: 817-832.
  15. Roupie, V., Leroy, B., Rosseels, V., Piersoel, V., Georisc, I.N., Romano, M., Govaerts, M., Letesson, M.M., Wattiez, R., Huygen, K. (2008). Immunogenicity and protective efficacy of DNA vaccines encoding MAP0586c and MAP4308c of Mycobacterium avium subsp. paratuberculosis secretome. Vaccine, 26: 4783–4794.
  16. Singh, S.V., Singh, A.V., Singh, R., Sharma, S., Shukla, N., Misra, S., Singh, P.K., Sohal, J.S., Kumar, H., et al (2008). Sero- prevalence of Johne’s disease in buffaloes and cattle population of North India using indigenous ELISA kit based on native Mycobacterium avium subspecies paratuberculosis ‘Bison type’ genotype of goat origin. Comparative Immunology Microbioogy. Infectious Diseases, 31: 419-33.
  17. Tasara, T. and Stephan, R. (2005). Development of an F57 sequence-based real-rime PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk. Applied Environmental Microbiology, 71: 5957-5968.
  18. Vansnick, E.L., De Rijk, O.P., Vercammen, F., Geysen, D., Rigouts, L., Portaels, F. (2004). Newly developed primers for the detection of Mycobacterium avium subsp. paratuberculosis. Veterinary Microbiology, 100: 197-204.
  19. Veazey, R.S., Tayor, H.W., Horohov, D.W., Krahenbuhl, J.L., Oliver, J.L., Snider, T.G. (1995). Histopathology of C57BL/6 mice inoculated orally with Mycobacterium paratuberculosis. Journal of Comparative Pathology, 113: 75-80. 
Published In
Indian Journal of Animal Research
Article Metrics
Metrics Icon
0
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Research Article

    volume 53 issue 7 (july 2019) : 944-948,   Doi: 10.18805/ijar.B-3611

    Quantification of Mycobacterium avium subsp. paratuberculosis from the tissues of challenged mice using SYBR Green real time PCR assay for the assessment of vaccine efficacy

    R
    R.K. Chaitanya
    Y
    Y. Krishnamohan Reddy
    G
    G. Dhinakar Raj
    A
    A. Thangavelu
    1Department of Veterinary Microbiology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-600 007, Tamil Nadu, India.
    Cite article:- Chaitanya R.K., Reddy Krishnamohan Y., Raj Dhinakar G., Thangavelu A. (2018). Quantification of Mycobacterium avium subsp. paratuberculosis from the tissues of challenged mice using SYBR Green real time PCR assay for the assessment of vaccine efficacy. Indian Journal of Animal Research. 53(7): 944-948. doi: 10.18805/ijar.B-3611.

    ABSTRACT

    A SYBR Green real time PCR assay amplifying F57 gene of Mycobacterium avium subsp. paratuberculosis (MAP) was developed to evaluate the protective efficacy of the inactivated vaccine to prevent the colonization of MAP in the tissues of BALB/c mice challenge model. Bacterial burden in the liver, spleen and intestine of vaccinated and sham immunized control mice, after intra peritoneal challenge with MAP were quantified by real time PCR assay.  A 195 bp fragment of MAP F57 sequence was cloned into TA cloning vector and the resultant recombinant plasmid was used to generate a series of quantification standards with copy number ranging from 109 to 1 copy. This absolute quantification technique is rapid and able to detect as few as 10 MAP organisms per gram of tissue and the assay has the efficiency of 0.982.

    REFERENCES

    1. Bustin, S.A., Benes, V., Garson. J.A., Hallemans, J., Huggett. J., Kubista, M., Mueller, R., Nolan, T., et al. (2009). The MIQE guidelines: Minimum information for publication of quantitative Real Time PCR Experiments. Clinical Chemistry, 55: 4.
    2. Chen, L.H., Kumanan, K., Huang, C.J., McDonough, S.P., Stehman, S., Akey, B., Huntley, J., Bannantine, et al. J.P.,(2008). Immune responses in mice to Mycobacterium avium subsp. paratuberculosis following vaccination with a novel 74F recombinant polyprotein. Vaccine, 26: 1253-1262. 
    3. Cocito, C., Gilot, P., Coene, M., De Kese, M., Poupart, P., Vannuffel, P. (1994). Paratuberculosis. Clin Microbiol Rev, 7: 328-345.
    4. Herthnek, D. and Bolske, G. (2006). New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis. BMC Microbiolgy, 6: 87.
    5. Hines, M.E., Stabel, J.R., Sweeney, R.W., Griffin, F., Talaat, A.M., Bakker, D., Benedictus, G., Davis, W.C., et al (2007). Experimental challenge models for Johne’s disease: A review and proposed international guidelines. Veterinary Microbiology, 122: 197–222.
    6. Kawaji, S., Taylor, D.L., Mori, Y., Whittington, R.J. (2007). Detection of Mycobacterium avium subspecies paratuberculosisin ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture. Veterinary Microbiology, 125: 36-48.
    7. Koets, A., Hoek, A., Langelaar, M., Overdijk, M., Santema, W., Franken, P., Eden, W.V., Rutten, V. (2006) Mycobacterial 70 kD heat-    shock protein is an effective subunit vaccine against bovine paratuberculosis.Vaccine, 24: 2550–2559.
    8. Kralik, P., Nocker, A., Pavlik, I. (2010). Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment. International Journal of Food Microbiology, 141: S80-S86.
    9. Munoz, M., Garcia Marin, J.F., Garcia-Pariente, C., Reyes, L.E., Verna, A., Moreno, O., Fuertes, M., Doce, J. (2005). Efficacy of a killed vaccine (SILIRUM) in calves challenged with MAP. Proceedings of the 8th International Colloquium on Paratuberculosis, Denmark. pp.276-282.
    10. Nelli, R.K., Graham, E., Dunham, S.P., Taylor, D.J. (2008). Real-time PCR identification of Mycobacterium avium subsp. paratuberculosis in ovine and bovine tissues. Veterinary Record, 163: 422-423.
    11. Park, S.U., Kumanan, K., Mc Dough, S., Akey, B., Huntley, J., Bannantine, J.P., Chang,Y.F. (2008). Immunization with a DNA vaccine cocktail induces a Th1 response and protects mice against Mycobacterium avium subsp. paratuberculosis challenge. Vaccine, 26: 4329-4337.
    12. Poupart, P., Cone, M., Van Heuverswyn, H., Cocito, C. (1993). Preparation of a specific RNA probe for detection of Mycobacterium paratuberculosis and diagnosis of Johne’s disease. Journal of Clinical Microbiology, 31:1601-1605.
    13. Reddacliff, L., Eppleston, J., Windsor, P., Whittington, R., Jones, S. (2006). Efficacy of killed vaccine for the control of paratuberculosis in Australian sheep flocks. Veterinary Microbiology, 115: 77-90.
    14. Rosseels, V. and Huygen, K. (2008). Vaccination against paratuberculosis. Expert Reviews on Vaccines, 7: 817-832.
    15. Roupie, V., Leroy, B., Rosseels, V., Piersoel, V., Georisc, I.N., Romano, M., Govaerts, M., Letesson, M.M., Wattiez, R., Huygen, K. (2008). Immunogenicity and protective efficacy of DNA vaccines encoding MAP0586c and MAP4308c of Mycobacterium avium subsp. paratuberculosis secretome. Vaccine, 26: 4783–4794.
    16. Singh, S.V., Singh, A.V., Singh, R., Sharma, S., Shukla, N., Misra, S., Singh, P.K., Sohal, J.S., Kumar, H., et al (2008). Sero- prevalence of Johne’s disease in buffaloes and cattle population of North India using indigenous ELISA kit based on native Mycobacterium avium subspecies paratuberculosis ‘Bison type’ genotype of goat origin. Comparative Immunology Microbioogy. Infectious Diseases, 31: 419-33.
    17. Tasara, T. and Stephan, R. (2005). Development of an F57 sequence-based real-rime PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in milk. Applied Environmental Microbiology, 71: 5957-5968.
    18. Vansnick, E.L., De Rijk, O.P., Vercammen, F., Geysen, D., Rigouts, L., Portaels, F. (2004). Newly developed primers for the detection of Mycobacterium avium subsp. paratuberculosis. Veterinary Microbiology, 100: 197-204.
    19. Veazey, R.S., Tayor, H.W., Horohov, D.W., Krahenbuhl, J.L., Oliver, J.L., Snider, T.G. (1995). Histopathology of C57BL/6 mice inoculated orally with Mycobacterium paratuberculosis. Journal of Comparative Pathology, 113: 75-80. 
    In this Article
      Contact us
      Follow us
      Published In
      Indian Journal of Animal Research

      Editorial Board

      View all (0)