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Research Article

volume 52 issue 10 (october 2018) : 1482-1487,   Doi: 10.18805/ijar.B-3375

Investigation of brucellosis in cattle and buffaloes by conventional and molecular assays

P
Paviter Kaur
N
N.S Sharma
A
A.K Arora
D
Deepti
1Department of Veterinary Microbiology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India
Cite article:- Kaur Paviter, Sharma N.S, Arora A.K, Deepti (2017). Investigation of brucellosis in cattle and buffaloes by conventional and molecular assays. Indian Journal of Animal Research. 52(10): 1482-1487. doi: 10.18805/ijar.B-3375.

ABSTRACT

The present study was carried out to evaluate the conventional and molecular techniques for diagnosis of bovine brucellosis. A total of four isolates of Brucella abortus obtained from 100 clinical samples of foetal stomach contents, vaginal mucus and uterine discharges were characterized biochemically. The isolates were confirmed as Brucella spp. by PCR using B4/B5 primer pair and as B. abortus by Bruce Ladder multiplex PCR. By Hinic Real-time PCR, all the four isolates were confirmed as Brucella spp. with Ct values between 14-16. With DNA extracted from 40 clinical samples of foetal stomach contents, vaginal mucus and uterine discharges, Real time PCR appeared most sensitive of the three molecular and conventional techniques as it detected maximum number of positive samples.

REFERENCES

  1. Abbas, B. A. and Talei, A. B. (2010). Isolation, identification and biotyping of Brucella spp. from milk product at Basrah Province. Bas J.Vet.Res., 9: 152-162.
  2. Al-Saadi, J. A. A., Al-Rodh, M. A. and Najum, A. A. (2012). Clinical, Serological, Hormonal, Bacteriological and Molecular Detection of Brucellosis in Aborted Cows and Buffalos. International Conference on Applied Life Sciences (ICALS2012), Turkey, September 10-12, pp. 327-36.
  3. Baily, G. C., Kraahn, J. B., Drasar, B. S. and Stokeer, N. G. (1992). Detection of Brucella melitensis and Brucella abortus by DNA amplification. J. Trop. Med. Hyg., 95: 271-275.
  4. Bounaadja, L., Albert, D., Chénais, B., Hénault, S., Zygmunt, M. S., Poliak, S. and Garin-Bastuji, B. (2009). Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet. Microbiol., 137(1-2):156-164.
  5. Bricker, B. J. (2002). PCR as diagnostic tool for brucellosis. Vet. Microbiol., 90: 435-446.
  6. Bricker, B. J. and Halling, S. M. (1994). Differentiation of Brucella abortus bv. 1, 2 and 4, Brucella melitensis, Brucella ovis and Brucella suis bv 1 by PCR. J. Clin. Microbiol., 32: 2660-2666.
  7. Corbel, M. J. and Brinley-Morgan, W. J. (1984). Genus Brucella Meyer and Shaw 1920, 173AL, p. 377-388. In: Krieg N R et al. (ed.), Bergey’s Manual of Systemic Bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore, MD.
  8. Doosti, A. and Ghasemi, D. P. (2011). Application of real-time PCR for identification and differentiation of Brucella abortus and Brucella melitensis in cattle. Bulgarica J. Vet. Med., 14(2): 109™115.
  9. Ewalt, D. R. and Bricker, B. J. (2000). Validation of the Abbreviated Brucella AMOS PCR as a Rapid Screening Method for Differentiation of Brucella abortus Field Strain Isolates and the Vaccine Strains, 19 and RB51. J. Clin.Microbiol.,38(8): 3085-3086. 
  10. García-Yoldi, D., Marin, C. M., de Miguel, M. J., Munoz, P. M., Vizmanos, J. L. and Lopez-Goni, I. (2006). Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin.Chem., 52: 779-781.
  11. Geresu, M. A., Amaeni, G., Wibete, A., Gamboa, A. and Kassa, G. M. (2016). Isolation and identification of Brucella spp. from dairy cattle by biochemical tests: The 1st report from Ethiopia. World’s Vet. J., 6 (2): 80-88.
  12. Ghodasra, S. N., Roy, A. and Bhanderi, B. B. (2010). Comparison of Rose Bengal Plate agglutination, standard tube agglutination and Indirect ELISA tests for detection of Brucella antibodies in cows and buffaloes. Vet.World., 3(2): 61-64.
  13. Godfroid, J., Nielsen, K. and Saegerman, C. (2010). Diagnosis of brucellosis in livestock and wildlife. Croatian Med. J.,51(4): 296-305. 
  14. Goñi, L., García, Y. D., Marín, C. M., Miguel, M. J., Muñoz, P. M., Blasco, J. M., et al., (2008). Evaluation of a Multiplex PCR Assay (Bruce-ladder) for Molecular Typing of All Brucella Species, Including the Vaccine Strains. J. Clin.Microbiol., 46: 3484- 3487.
  15. Güler, L., Gündüz, K. and Ok, Ü. (2003). Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples. Vet. Microbiol. 93: 53–61.
  16. Hinic, V., Brodard, I., Thomann, A., Cvetnic, Z., Makaya, P. V., Frey, J. and Abril, C. (2008). Novel identification and differentiation of Brucella melitensis, B.abortus, B.suis, B.ovis, B. canis and B. neotomae suitable for both conventional and real time PCR systems. J. Microbiol. Methods., 75: 375-378.
  17. Hinic. V., Brodard, I., Thomann, A., Holub, M., Miserez, R. and Abril, C. (2009). IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology. BMC Vet. Res., 5: 22.
  18. Ica, T., Aydin, F., Gumussoy, K. S., Percin, D., Sumerkan, A. B., Ocak, F., Abay, S., et al., (2012). Conventional and molecular biotyping of Brucella strains isolated from cattle, sheep and human. Veteriner-Fakultesi-Dergisi,-Ankara-Universitesi., 59: 259-264.
  19. Jabbar, A. A., Saaidi, A. L., Mohsen, A., Al, R. and Ali, A. N. (2012). Clinical, serological, hormonal, bacteriological and molecular detection of brucellosis in aborted cows and buffaloes. International Conference on Applied Life Sciences. Turkey, Sep. 10-    12, 2012.
  20. Jain, A., Sharma, N. S., Paviter, Kaur. and Arora, A. K. (2013). Isolation, biotyping and antibiogram evaluation of Brucella abortus from cattle and buffaloes from Punjab (India). Applied Biol.Res., 15(1): 27-31.
  21. Leal, K. D. S., Martinez, V. I. O., Lopez, M. A. and Martinez, S. J. P. (1995). Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J. Clin. Microbiol., 3: 3087-90.
  22. Lopez, Goni. I., Garcia, Y. D., Marin, C. M., De Miguel, M. J., Barquero, C. E., et al.,(2011). New Bruce ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of B. suis and B. canis. Vet. Microbiol., 154: 152-155.
  23. Mayer-Scholl, A., Draeger, A., Gollner, C., Holger, C., Scholz. and Nockler, K. (2010). Advancement of a multiplex PCR for the differentiation of all currently described Brucella species. Journal of Microbiological Methods 80: 112–14.
  24. Megid J, Albert D, Fagliari J J, Paes A C, Listoni F P, Pinto M R A, Ribeiro M G, Thiebaud M, Ueno T and Garin Bastuji B. 2005. Isolation of Brucella abortus from cattle and buffalo in Brazil. Vet. Rec., 156(5): 147-148.
  25. Mirnejad, R., Doust, R. H., Kachuei, R., Mortazavi, S. M., Khoobdel, M. and Ahamadi, A. (2013). Simultaneous detection and differentiation of Brucella abortus and Brucella melitensis by combinatorial PCR. Asian Pac. J. Trop. Med., 5(1): 24-28.
  26. Osterman, B. and Moriyon, I. (2006). International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Brucella. Int. J. Syst. Evol. Microbiol., 56: 1173-1175.
  27. Priyantha, M. A. R. (2011). Identification of biovars of Brucella abortus in aborted cattle and buffaloes herd in Sri Lanka. Vet. World., 19(4): 542-545.
  28. Quinn, P. J., Carter, M. E., Markey, B. R. and Carter, G. R. (1994). Clinical Veterinary Microbiology pp 268-72. Wolfe Publishing, an imprint of Mosby year book Europe Limited, Spain.
  29. Richtzenhain, L. J., Cortez, A., Heinemann, M. B., Soares, R. M., Sakamoto, S. M., et al., (2002). A multiplex PCR for the detection of Brucella sp. and Leptospira spp. DNA from aborted bovine fetuses. Vet. Microbiol., 87: 139-147.
  30. Sanjrani, S. N., Mirbahar, K. B., Soomro, H. and Sahito, H A. (2013). Prevalence of abortion in Kundhi buffalo in district Hyderabad, Sindh – Pakistan. Herald J. Agri. Food Sci. Res., 2: 70-77.
  31. Shahzad, A., Qurban, A., Falk, M., Iahtasham, K., Shamim, A., Heinrich, N. and Syed, J. (2014). Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR. Trop. Anim. Health. Prod., 46: 73.
  32. Surucuoglu, S., El, S., Ural, S., Gazi, H., Kurutepe, S., Taskiran, P. and Yurtsever, S. G. (2009). Evaluation of real-time PCR method for rapid diagnosis of brucellosis with different clinical manifestations. Polish J. Microbiol.,58: 15-19.
  33. Yu, W. L. and Nielsen, K. (2010). Review of detection of Brucella spp. by polymerase chain reaction. Croatian Med. J.51: 306-313.
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Indian Journal of Animal Research
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    Research Article

    volume 52 issue 10 (october 2018) : 1482-1487,   Doi: 10.18805/ijar.B-3375

    Investigation of brucellosis in cattle and buffaloes by conventional and molecular assays

    P
    Paviter Kaur
    N
    N.S Sharma
    A
    A.K Arora
    D
    Deepti
    1Department of Veterinary Microbiology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India
    Cite article:- Kaur Paviter, Sharma N.S, Arora A.K, Deepti (2017). Investigation of brucellosis in cattle and buffaloes by conventional and molecular assays. Indian Journal of Animal Research. 52(10): 1482-1487. doi: 10.18805/ijar.B-3375.

    ABSTRACT

    The present study was carried out to evaluate the conventional and molecular techniques for diagnosis of bovine brucellosis. A total of four isolates of Brucella abortus obtained from 100 clinical samples of foetal stomach contents, vaginal mucus and uterine discharges were characterized biochemically. The isolates were confirmed as Brucella spp. by PCR using B4/B5 primer pair and as B. abortus by Bruce Ladder multiplex PCR. By Hinic Real-time PCR, all the four isolates were confirmed as Brucella spp. with Ct values between 14-16. With DNA extracted from 40 clinical samples of foetal stomach contents, vaginal mucus and uterine discharges, Real time PCR appeared most sensitive of the three molecular and conventional techniques as it detected maximum number of positive samples.

    REFERENCES

    1. Abbas, B. A. and Talei, A. B. (2010). Isolation, identification and biotyping of Brucella spp. from milk product at Basrah Province. Bas J.Vet.Res., 9: 152-162.
    2. Al-Saadi, J. A. A., Al-Rodh, M. A. and Najum, A. A. (2012). Clinical, Serological, Hormonal, Bacteriological and Molecular Detection of Brucellosis in Aborted Cows and Buffalos. International Conference on Applied Life Sciences (ICALS2012), Turkey, September 10-12, pp. 327-36.
    3. Baily, G. C., Kraahn, J. B., Drasar, B. S. and Stokeer, N. G. (1992). Detection of Brucella melitensis and Brucella abortus by DNA amplification. J. Trop. Med. Hyg., 95: 271-275.
    4. Bounaadja, L., Albert, D., Chénais, B., Hénault, S., Zygmunt, M. S., Poliak, S. and Garin-Bastuji, B. (2009). Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet. Microbiol., 137(1-2):156-164.
    5. Bricker, B. J. (2002). PCR as diagnostic tool for brucellosis. Vet. Microbiol., 90: 435-446.
    6. Bricker, B. J. and Halling, S. M. (1994). Differentiation of Brucella abortus bv. 1, 2 and 4, Brucella melitensis, Brucella ovis and Brucella suis bv 1 by PCR. J. Clin. Microbiol., 32: 2660-2666.
    7. Corbel, M. J. and Brinley-Morgan, W. J. (1984). Genus Brucella Meyer and Shaw 1920, 173AL, p. 377-388. In: Krieg N R et al. (ed.), Bergey’s Manual of Systemic Bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore, MD.
    8. Doosti, A. and Ghasemi, D. P. (2011). Application of real-time PCR for identification and differentiation of Brucella abortus and Brucella melitensis in cattle. Bulgarica J. Vet. Med., 14(2): 109™115.
    9. Ewalt, D. R. and Bricker, B. J. (2000). Validation of the Abbreviated Brucella AMOS PCR as a Rapid Screening Method for Differentiation of Brucella abortus Field Strain Isolates and the Vaccine Strains, 19 and RB51. J. Clin.Microbiol.,38(8): 3085-3086. 
    10. García-Yoldi, D., Marin, C. M., de Miguel, M. J., Munoz, P. M., Vizmanos, J. L. and Lopez-Goni, I. (2006). Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin.Chem., 52: 779-781.
    11. Geresu, M. A., Amaeni, G., Wibete, A., Gamboa, A. and Kassa, G. M. (2016). Isolation and identification of Brucella spp. from dairy cattle by biochemical tests: The 1st report from Ethiopia. World’s Vet. J., 6 (2): 80-88.
    12. Ghodasra, S. N., Roy, A. and Bhanderi, B. B. (2010). Comparison of Rose Bengal Plate agglutination, standard tube agglutination and Indirect ELISA tests for detection of Brucella antibodies in cows and buffaloes. Vet.World., 3(2): 61-64.
    13. Godfroid, J., Nielsen, K. and Saegerman, C. (2010). Diagnosis of brucellosis in livestock and wildlife. Croatian Med. J.,51(4): 296-305. 
    14. Goñi, L., García, Y. D., Marín, C. M., Miguel, M. J., Muñoz, P. M., Blasco, J. M., et al., (2008). Evaluation of a Multiplex PCR Assay (Bruce-ladder) for Molecular Typing of All Brucella Species, Including the Vaccine Strains. J. Clin.Microbiol., 46: 3484- 3487.
    15. Güler, L., Gündüz, K. and Ok, Ü. (2003). Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples. Vet. Microbiol. 93: 53–61.
    16. Hinic, V., Brodard, I., Thomann, A., Cvetnic, Z., Makaya, P. V., Frey, J. and Abril, C. (2008). Novel identification and differentiation of Brucella melitensis, B.abortus, B.suis, B.ovis, B. canis and B. neotomae suitable for both conventional and real time PCR systems. J. Microbiol. Methods., 75: 375-378.
    17. Hinic. V., Brodard, I., Thomann, A., Holub, M., Miserez, R. and Abril, C. (2009). IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology. BMC Vet. Res., 5: 22.
    18. Ica, T., Aydin, F., Gumussoy, K. S., Percin, D., Sumerkan, A. B., Ocak, F., Abay, S., et al., (2012). Conventional and molecular biotyping of Brucella strains isolated from cattle, sheep and human. Veteriner-Fakultesi-Dergisi,-Ankara-Universitesi., 59: 259-264.
    19. Jabbar, A. A., Saaidi, A. L., Mohsen, A., Al, R. and Ali, A. N. (2012). Clinical, serological, hormonal, bacteriological and molecular detection of brucellosis in aborted cows and buffaloes. International Conference on Applied Life Sciences. Turkey, Sep. 10-    12, 2012.
    20. Jain, A., Sharma, N. S., Paviter, Kaur. and Arora, A. K. (2013). Isolation, biotyping and antibiogram evaluation of Brucella abortus from cattle and buffaloes from Punjab (India). Applied Biol.Res., 15(1): 27-31.
    21. Leal, K. D. S., Martinez, V. I. O., Lopez, M. A. and Martinez, S. J. P. (1995). Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J. Clin. Microbiol., 3: 3087-90.
    22. Lopez, Goni. I., Garcia, Y. D., Marin, C. M., De Miguel, M. J., Barquero, C. E., et al.,(2011). New Bruce ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of B. suis and B. canis. Vet. Microbiol., 154: 152-155.
    23. Mayer-Scholl, A., Draeger, A., Gollner, C., Holger, C., Scholz. and Nockler, K. (2010). Advancement of a multiplex PCR for the differentiation of all currently described Brucella species. Journal of Microbiological Methods 80: 112–14.
    24. Megid J, Albert D, Fagliari J J, Paes A C, Listoni F P, Pinto M R A, Ribeiro M G, Thiebaud M, Ueno T and Garin Bastuji B. 2005. Isolation of Brucella abortus from cattle and buffalo in Brazil. Vet. Rec., 156(5): 147-148.
    25. Mirnejad, R., Doust, R. H., Kachuei, R., Mortazavi, S. M., Khoobdel, M. and Ahamadi, A. (2013). Simultaneous detection and differentiation of Brucella abortus and Brucella melitensis by combinatorial PCR. Asian Pac. J. Trop. Med., 5(1): 24-28.
    26. Osterman, B. and Moriyon, I. (2006). International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Brucella. Int. J. Syst. Evol. Microbiol., 56: 1173-1175.
    27. Priyantha, M. A. R. (2011). Identification of biovars of Brucella abortus in aborted cattle and buffaloes herd in Sri Lanka. Vet. World., 19(4): 542-545.
    28. Quinn, P. J., Carter, M. E., Markey, B. R. and Carter, G. R. (1994). Clinical Veterinary Microbiology pp 268-72. Wolfe Publishing, an imprint of Mosby year book Europe Limited, Spain.
    29. Richtzenhain, L. J., Cortez, A., Heinemann, M. B., Soares, R. M., Sakamoto, S. M., et al., (2002). A multiplex PCR for the detection of Brucella sp. and Leptospira spp. DNA from aborted bovine fetuses. Vet. Microbiol., 87: 139-147.
    30. Sanjrani, S. N., Mirbahar, K. B., Soomro, H. and Sahito, H A. (2013). Prevalence of abortion in Kundhi buffalo in district Hyderabad, Sindh – Pakistan. Herald J. Agri. Food Sci. Res., 2: 70-77.
    31. Shahzad, A., Qurban, A., Falk, M., Iahtasham, K., Shamim, A., Heinrich, N. and Syed, J. (2014). Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR. Trop. Anim. Health. Prod., 46: 73.
    32. Surucuoglu, S., El, S., Ural, S., Gazi, H., Kurutepe, S., Taskiran, P. and Yurtsever, S. G. (2009). Evaluation of real-time PCR method for rapid diagnosis of brucellosis with different clinical manifestations. Polish J. Microbiol.,58: 15-19.
    33. Yu, W. L. and Nielsen, K. (2010). Review of detection of Brucella spp. by polymerase chain reaction. Croatian Med. J.51: 306-313.
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