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Research Article

volume 54 issue 8 (august 2020) : 1006-1011,   Doi: 10.18805/ijar.B-3774

Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep

J
Jyoti Kumar
G
G.G. Sonawane
F
Fateh Singh
S
S. Jegaveera Pandian
R
Rajiv Kumar
1Division of Animal Health ICAR-Central Sheep and Wool Research Institute, Avikanagar-304 501, Rajasthan, India.
Cite article:- Kumar Jyoti, Sonawane G.G., Singh Fateh, Pandian Jegaveera S., Kumar Rajiv (2020). Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep. Indian Journal of Animal Research. 54(8): 1006-1011. doi: 10.18805/ijar.B-3774.

ABSTRACT

Pasteurella multocida is one of the bacterial species involved in cases of ovine respiratory complex that has been implicated to cause significant economic losses in sheep production system worldwide. The present study was undertaken with the aim of evaluating a SYBR Green dye based real time PCR assay targeting KMT1 gene for the detection of P. multocida. The analytical specificity and sensitivity of the PCR primers were evaluated. The test showed ten-fold more sensitivity than conventional PCR and detected down to 275.5 fg/ µl of genomic DNA concentration, equivalent to 100 copies of KMT1 gene of P. multocida. The real-time PCR was found to be specific for KMT1 gene of P. multocida, as no cross reactivity was detected with a variety of known bacterial isolates. A total of 52 ovine lung tissue samples were screened for P. multocida, which showed improved level of detection as compared to conventional PCR. It is concluded that, this assay may be used as a valuable diagnostic tool for the rapid and specific detection of P. multocida. By virtue of its high throughput format and its ability to accurately identify as well as quantify the bacterial DNA, the method may be useful in large scale epidemiological studies and clarification of pathogenesis.

REFERENCES

  1. Bhimani, M., Bhanderi, B. and Roy, A. (2015). Loop-mediated isothermal amplification assay (LAMP) based detection of Pasteurella multocida in cases of haemorrhagic septicaemia and fowl cholera. Vet. Ital. 51: 115-121.
  2. Broeders, S., Huber, I., Grohmann, L., Berben, G., Taverniers I., Mazzara, M., Roosens, N. and Morisset, D. (2014). Guidelines for validation of qualitative real-time PCR methods. Trends Food Sci Tech. 37 (2):115-126.
  3. Cameron, C. M., Engelbrecht, M. M., and Vermueulen, A. S. M. (1978). The serological and immunological relationship of type strains A and D of Pasteurella multocida to field isolates from sheep. Onderstepoort J Vet. 45: 215-220.
  4. Chandrasekharan, S., Hizat, K., Saad., Z., Johara, M. Y. and Yeap, P. C. (1991). Evaluation of combined pasteurella vaccines in control of sheep pneumonia. Brit Vet J. 147: 437-443.
  5. Davies, R. L., Watson, P. and Caffrey, B. (2003). Comparative analyses of Pasteurella multocida strains associated with ovine pneumonia and ovine vaginal tract. Vet Rec. 152: 7-10. 
  6. Department of Animal Husbandry, Dairying and Fisheries (DADF). Annual Report (2017-2018). Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture and Farmers Welfare, Government of India. 1st ed. New Delhi, India: DADF; 2018.
  7. Diker, K. S., Akan, M. and Haziroglu, R. (1994). Antimicrobial susceptibility of Pasteurella haemolytica and Pasteurella multocida isolated from pneumonic ovine lungs. Vet Rec. 134: 597-598. 
  8. Dimri, U., Lonkar, P.S., Srivastaya, C. P. and Bhagwan, P. S. K. (1994). An outbreak of pneumonia due to Pasteurella multocida in sheep. Indian Vet J. 71: 1163-1167. 
  9. Donachie, W. (2007). Pasteurellosis. In: Diseases of Sheep. 4th edn. [Ed Aitken I. D]. Blackwell Science. pp 224-231.
  10. Food and Agriculture Organization of the United Nations (FAO). Livestock. In: Gennari P, editor. FAO Statistical Pocket Book, World Food and Agriculture. Food and Agriculture Organization of the United Nations, Rome: FAO Statistics Division; 2015. pp. 30.
  11. Gilmour, N. J. L. and Gilmour, J. S. (1989). Pasteurellosis of sheep. In Pasteurella and Pasteurellosis. [Eds Adlam, C., Rutter, J. M., Academic Press. London, pp 223-262. 
  12. Goodwin, K. A., Jackson, R., Brown, C., Davies, P.R., Morris, R.S. and Perkins, N. R. (2004). Pneumonic lesions in lambs in New Zealand: patterns of prevalence and effects on production. New Zeal Vet J. 52: 175-179. 
  13. Hancock, R. D., Fallavena, L. C. and Rebeiro, L. A. O. (1991). Pneumonic pasteurellosis due to Pasteurella multocida in a flock of lambs in Brazil. Vet Rec. 128: 154-155.
  14. Hassan, G. M., El-Feky, Z. A., Eissa, E. A. and Teleb, A. A. (2016). Rapid diagnosis of virulent Pasteurella multocida isolated from farm animals with clinical manifestation of pneumonia respiratory infection using 16S rDNA and KMT1 gene. Asian Pac J Trop Dis. 6: 21-26.
  15. Jensen, R., Pierson, R. E., Weibel, J. L., Tucker, J. O. and Swift, B. L. (1982). Middle ear infection in feedlot lambs. J Am Vet Med A. 181: 805-807.
  16. Jonathan, L. and Arora, A. K. (2012). Comparison of detection threshold of different Pasteurella multocida specific PCRs. Indian. J. Anim Res. 46(1):28-33. 
  17. Liu, D., Lawrence, M.L. and Austin, F.W., (2004). Specific PCR identification of Pasteurella multocida based on putative transcriptional regulator genes. J. Microbiol. Methods. 58: 263-267.
  18. Lung, O., Ohene-Adjei, S., Buchanan, C., Joseph, T., King, R., Erickson, A., Detmer, S. and Ambagala, A. (2017). Multiplex PCR and microarray for detection of swine respiratory pathogen. Transbound. Emerg. Dis. 64(3): 834-848.
  19. Marois, C., Cariolet, R., Morvan, H. and Kobisch, M. (2008). Transmission of pathogenic respiratory bacteria to specific pathogen free pigs at slaughter. Vet. Microbiol. 129: 325-332.
  20. McPherson, M.J. and Moller, S. G. (2000). Polymerase Chain Reaction. BIOS Scientific Publishers Ltd.; Oxford, 1st ed., United Kingdom. pp 1-18.
  21. Miflin, J.K. and Blackall, P.J. (2001). Development of a 23S rRNA-    based PCR assay for the identification of Pasteurella multocida. Lett. Appl. Microbiol. 33: 216-221.
  22. Odugbo, M. O., Odama, L. E., Umoh, J. U. and Lamorde, A. G. (2006). Pasteurella multocida pneumonic infection in sheep: prevalence, clinical and pathological studies. Small Ruminant Res. 66: 273- 277.
  23. Petridou E.J., Gianniki Z., Giadinis N.D., Filioussis G., Dovas C.I. and Psychas V. (2011). Outbreak of polyarthritis in lambs attributed to Pasteurella multocida. Vet Rec. 168 (2): 50. 
  24. Rad, M., Movassaghi, A. R., Sharifi, K., Naseri, Z. and Seifi, H. A. (2011). Two outbreaks of Pasteurella multocida septicemia in neonatal lambs. Comp Clin Path. 20(1): 57-59. 
  25. Register, K. B. and de Jong, K. D. (2006). Analytical verification of a multiplex PCR for identification of Bordetella bronchiseptica and Pasteurella multocida from swine. Vet. Microbiol. 117: 201-210.
  26. Sabiel, Y.A., Musa, M.T. and Ann, V.Z. (2012). Identification of Mannhemia haemolytica and Pasteurella multocida by Polymerase Chain Reaction and Random Amplification of Polymorphic DNA. Sudan J. Vet. Res. 27: 55-59.
  27. Sahay, S., Shome, R., Sankarasubramanian, J., Vishnu, U. S., Prajapati, A., Natesan, K., et al (2018). Insights into the genome sequence of ovine Pasteurella multocida type a strain associated with pneumonic pasteurellosis. Small Ruminant Res. 169: 167-175.
  28. Singh, F., Sonawane, G. G. and Meena, R. K. (2018). Molecular detection of virulent Mannheimia haemolytica and Pasteurella multocida in lung tissues of pneumonic sheep from semiarid tropics, Rajasthan, India. Turk. J. Vet. Anim. Sci. 42: 556-561. 
  29. Svec, D., Tichopad, A., Novosadova, V., Pfaffl, M. W. and Kubista, M. (2015). How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments. Biomolecular Detection and Quantification. 3: 9-16. 
  30. Townsend, K. M., Frost, A. J., Lee, C. W., Papadimitrious, J. M. and Dawkins, H. J. S. (1998). Development of PCR assays for species and type specific identification of Pasteurella multocida isolates. J. Clin. Mirobiol. 36: 1096 1100.
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Indian Journal of Animal Research
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    Research Article

    volume 54 issue 8 (august 2020) : 1006-1011,   Doi: 10.18805/ijar.B-3774

    Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep

    J
    Jyoti Kumar
    G
    G.G. Sonawane
    F
    Fateh Singh
    S
    S. Jegaveera Pandian
    R
    Rajiv Kumar
    1Division of Animal Health ICAR-Central Sheep and Wool Research Institute, Avikanagar-304 501, Rajasthan, India.
    Cite article:- Kumar Jyoti, Sonawane G.G., Singh Fateh, Pandian Jegaveera S., Kumar Rajiv (2020). Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep. Indian Journal of Animal Research. 54(8): 1006-1011. doi: 10.18805/ijar.B-3774.

    ABSTRACT

    Pasteurella multocida is one of the bacterial species involved in cases of ovine respiratory complex that has been implicated to cause significant economic losses in sheep production system worldwide. The present study was undertaken with the aim of evaluating a SYBR Green dye based real time PCR assay targeting KMT1 gene for the detection of P. multocida. The analytical specificity and sensitivity of the PCR primers were evaluated. The test showed ten-fold more sensitivity than conventional PCR and detected down to 275.5 fg/ µl of genomic DNA concentration, equivalent to 100 copies of KMT1 gene of P. multocida. The real-time PCR was found to be specific for KMT1 gene of P. multocida, as no cross reactivity was detected with a variety of known bacterial isolates. A total of 52 ovine lung tissue samples were screened for P. multocida, which showed improved level of detection as compared to conventional PCR. It is concluded that, this assay may be used as a valuable diagnostic tool for the rapid and specific detection of P. multocida. By virtue of its high throughput format and its ability to accurately identify as well as quantify the bacterial DNA, the method may be useful in large scale epidemiological studies and clarification of pathogenesis.

    REFERENCES

    1. Bhimani, M., Bhanderi, B. and Roy, A. (2015). Loop-mediated isothermal amplification assay (LAMP) based detection of Pasteurella multocida in cases of haemorrhagic septicaemia and fowl cholera. Vet. Ital. 51: 115-121.
    2. Broeders, S., Huber, I., Grohmann, L., Berben, G., Taverniers I., Mazzara, M., Roosens, N. and Morisset, D. (2014). Guidelines for validation of qualitative real-time PCR methods. Trends Food Sci Tech. 37 (2):115-126.
    3. Cameron, C. M., Engelbrecht, M. M., and Vermueulen, A. S. M. (1978). The serological and immunological relationship of type strains A and D of Pasteurella multocida to field isolates from sheep. Onderstepoort J Vet. 45: 215-220.
    4. Chandrasekharan, S., Hizat, K., Saad., Z., Johara, M. Y. and Yeap, P. C. (1991). Evaluation of combined pasteurella vaccines in control of sheep pneumonia. Brit Vet J. 147: 437-443.
    5. Davies, R. L., Watson, P. and Caffrey, B. (2003). Comparative analyses of Pasteurella multocida strains associated with ovine pneumonia and ovine vaginal tract. Vet Rec. 152: 7-10. 
    6. Department of Animal Husbandry, Dairying and Fisheries (DADF). Annual Report (2017-2018). Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture and Farmers Welfare, Government of India. 1st ed. New Delhi, India: DADF; 2018.
    7. Diker, K. S., Akan, M. and Haziroglu, R. (1994). Antimicrobial susceptibility of Pasteurella haemolytica and Pasteurella multocida isolated from pneumonic ovine lungs. Vet Rec. 134: 597-598. 
    8. Dimri, U., Lonkar, P.S., Srivastaya, C. P. and Bhagwan, P. S. K. (1994). An outbreak of pneumonia due to Pasteurella multocida in sheep. Indian Vet J. 71: 1163-1167. 
    9. Donachie, W. (2007). Pasteurellosis. In: Diseases of Sheep. 4th edn. [Ed Aitken I. D]. Blackwell Science. pp 224-231.
    10. Food and Agriculture Organization of the United Nations (FAO). Livestock. In: Gennari P, editor. FAO Statistical Pocket Book, World Food and Agriculture. Food and Agriculture Organization of the United Nations, Rome: FAO Statistics Division; 2015. pp. 30.
    11. Gilmour, N. J. L. and Gilmour, J. S. (1989). Pasteurellosis of sheep. In Pasteurella and Pasteurellosis. [Eds Adlam, C., Rutter, J. M., Academic Press. London, pp 223-262. 
    12. Goodwin, K. A., Jackson, R., Brown, C., Davies, P.R., Morris, R.S. and Perkins, N. R. (2004). Pneumonic lesions in lambs in New Zealand: patterns of prevalence and effects on production. New Zeal Vet J. 52: 175-179. 
    13. Hancock, R. D., Fallavena, L. C. and Rebeiro, L. A. O. (1991). Pneumonic pasteurellosis due to Pasteurella multocida in a flock of lambs in Brazil. Vet Rec. 128: 154-155.
    14. Hassan, G. M., El-Feky, Z. A., Eissa, E. A. and Teleb, A. A. (2016). Rapid diagnosis of virulent Pasteurella multocida isolated from farm animals with clinical manifestation of pneumonia respiratory infection using 16S rDNA and KMT1 gene. Asian Pac J Trop Dis. 6: 21-26.
    15. Jensen, R., Pierson, R. E., Weibel, J. L., Tucker, J. O. and Swift, B. L. (1982). Middle ear infection in feedlot lambs. J Am Vet Med A. 181: 805-807.
    16. Jonathan, L. and Arora, A. K. (2012). Comparison of detection threshold of different Pasteurella multocida specific PCRs. Indian. J. Anim Res. 46(1):28-33. 
    17. Liu, D., Lawrence, M.L. and Austin, F.W., (2004). Specific PCR identification of Pasteurella multocida based on putative transcriptional regulator genes. J. Microbiol. Methods. 58: 263-267.
    18. Lung, O., Ohene-Adjei, S., Buchanan, C., Joseph, T., King, R., Erickson, A., Detmer, S. and Ambagala, A. (2017). Multiplex PCR and microarray for detection of swine respiratory pathogen. Transbound. Emerg. Dis. 64(3): 834-848.
    19. Marois, C., Cariolet, R., Morvan, H. and Kobisch, M. (2008). Transmission of pathogenic respiratory bacteria to specific pathogen free pigs at slaughter. Vet. Microbiol. 129: 325-332.
    20. McPherson, M.J. and Moller, S. G. (2000). Polymerase Chain Reaction. BIOS Scientific Publishers Ltd.; Oxford, 1st ed., United Kingdom. pp 1-18.
    21. Miflin, J.K. and Blackall, P.J. (2001). Development of a 23S rRNA-    based PCR assay for the identification of Pasteurella multocida. Lett. Appl. Microbiol. 33: 216-221.
    22. Odugbo, M. O., Odama, L. E., Umoh, J. U. and Lamorde, A. G. (2006). Pasteurella multocida pneumonic infection in sheep: prevalence, clinical and pathological studies. Small Ruminant Res. 66: 273- 277.
    23. Petridou E.J., Gianniki Z., Giadinis N.D., Filioussis G., Dovas C.I. and Psychas V. (2011). Outbreak of polyarthritis in lambs attributed to Pasteurella multocida. Vet Rec. 168 (2): 50. 
    24. Rad, M., Movassaghi, A. R., Sharifi, K., Naseri, Z. and Seifi, H. A. (2011). Two outbreaks of Pasteurella multocida septicemia in neonatal lambs. Comp Clin Path. 20(1): 57-59. 
    25. Register, K. B. and de Jong, K. D. (2006). Analytical verification of a multiplex PCR for identification of Bordetella bronchiseptica and Pasteurella multocida from swine. Vet. Microbiol. 117: 201-210.
    26. Sabiel, Y.A., Musa, M.T. and Ann, V.Z. (2012). Identification of Mannhemia haemolytica and Pasteurella multocida by Polymerase Chain Reaction and Random Amplification of Polymorphic DNA. Sudan J. Vet. Res. 27: 55-59.
    27. Sahay, S., Shome, R., Sankarasubramanian, J., Vishnu, U. S., Prajapati, A., Natesan, K., et al (2018). Insights into the genome sequence of ovine Pasteurella multocida type a strain associated with pneumonic pasteurellosis. Small Ruminant Res. 169: 167-175.
    28. Singh, F., Sonawane, G. G. and Meena, R. K. (2018). Molecular detection of virulent Mannheimia haemolytica and Pasteurella multocida in lung tissues of pneumonic sheep from semiarid tropics, Rajasthan, India. Turk. J. Vet. Anim. Sci. 42: 556-561. 
    29. Svec, D., Tichopad, A., Novosadova, V., Pfaffl, M. W. and Kubista, M. (2015). How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments. Biomolecular Detection and Quantification. 3: 9-16. 
    30. Townsend, K. M., Frost, A. J., Lee, C. W., Papadimitrious, J. M. and Dawkins, H. J. S. (1998). Development of PCR assays for species and type specific identification of Pasteurella multocida isolates. J. Clin. Mirobiol. 36: 1096 1100.
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