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Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 53 issue 6 (june 2019) : 821-826,   Doi: 10.18805/ijar.B-3584

Single-tube duplex-PCR for specific detection and differentiation of Brucella abortus S19 vaccine strain from other Brucella spp

A
Aparajita Das
B
Bablu Kumar
S
Soumendu Chakravarti
K
Karam Pal Singh
A
Abhishek
G
Garima Shrinet
1Division of Biological Products, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh-243 122, India.
Cite article:- Das Aparajita, Bablu Kumar, Chakravarti Soumendu, Singh Pal Karam, Abhishek, Shrinet Garima (2018). Single-tube duplex-PCR for specific detection and differentiation of Brucella abortus S19 vaccine strain from other Brucella spp. Indian Journal of Animal Research. 53(6): 821-826. doi: 10.18805/ijar.B-3584.

ABSTRACT

A simple single-tube duplex-PCR assay was optimized for rapid and sensitive detection and differentiation of Brucella abortus (B.abortus) S19 vaccine strain from other Brucella spp.(B.abortus 544,B abortus S99,B.melitensis 16M,B.suis).This assay was optimized using two primer pairs that were designed, one targeting genus specific multicopy IS711 and another eryC for the development of the duplex-PCR assay. Specificity of the assay was assessed using DNA templates from various Brucella species (B.abortus, B.melitensis, B.suis) and non Brucella bacteria like Staphylococcus aureus, Proteus vulgaris, Pasteurella multocida, Pseudomonas aeruginosa, Citrobacter freundii, E. coli, Salmonella enteritidis, Campylobacter jejuni and Listeria monocytogenes. The assay was also evaluated on spiked milk samples with known B.abortus cultures. The assay was able to detect up to 2.47 x 103CFU of B. abortus S99 organism in spiked milk sample. The assay was further validated in 53 clinical specimens (aborted foetal stomach content). Results confirm that Single tube duplex-PCR assay was useful for early and rapid detection and differentiation of B. abortus vaccine strain S19 from other Brucella spp in milk as well as in clinical specimen. 

REFERENCES

  1. Alvarez, L.P., Marcellino, R.B., Martinez, A. and Robles, C.A.(2017). Duplex PCR for the diagnosis of Brucella melitensis and its differentiation from the REV-1 vaccine strain. Small Rumin Res., 146:1-4.
  2. Arenas-Gamboa, A.M., Ficht, T.A., Kahl-McDonagh, M.M., Gomez, G. and Rice-Ficht, A.C.(2009) .The Brucella abortus S19 ÄvjbR live vaccine candidate is safer than S19 and confers protection against wild-type challenge in BALB/c mice when delivered in a sustained-release vehicle. Infect Immun., 77:877-884.
  3. Arora, S., Agarwal, R. K., and Bist, B. (2006).Comparison of ELISA and PCR vis-à-vis cultural methods for detecting Aeromonas spp. in foods of animal origin. Int. J Food Microbiol., 106: 177-183.
  4. Bricker, B. J. and Halling, S. M. (1995). Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J Clin. Microbiol .,33: 1640–1642.
  5. Corbel, M. J. (2006).Brucellosis in humans and animals. World Health Organization.
  6. Cordes, D.O. and Carter, M.E.(1979). Persistence of Brucella abortus infection in six herds of cattle under brucellosis eradication. N Z Vet J., 27:255-259.
  7. Dorneles, E.M., Lima, G.K., Teixeira-Carvalho, A., Araújo, M.S., Martins-Filho, O.A.,et al (2015a). Immune response of calves vaccinated with Brucella abortus S19 or RB51 and revaccinated with RB51. PloS one., 10:e0136696.
  8. Dorneles, E. M. S., Sriranganathan, N. and Lage, A. P. (2015b). Recent advances in Brucella abortus vaccines. Vet Res., 46:76.
  9. Espinosa, I., Báez, M., Percedo, M. I., and Martinez, S. (2013).Evaluation of simplified DNA extraction methods for Streptococcus suis typing. Rev. Salud Anim., 35: 59-63.
  10. Garcia-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. Kanani, A.N., Jain, L., Patel, T.J., Rank, D.N., Joshi, C.G. and Purohit, J.H.(2008) Detection of Brucella DNA in semen using the polymerase chain reaction assay’. Indian J. Anim. Res., 42:222-224.
  12. Kaur, P., Sharma, N.S. and Arora, A.K.(2017).Investigation of brucellosis in cattle and buffaloes by conventional and molecular assays. Indian J. Anim. Res., doi:10.18805/ijar.B-3375.
  13. Kang, S.I., Her, M., Kim, J.W., Kim, J.Y., Ko, K.Y., Ha, Y.M. and Jung, S.C.(2011). Advanced multiplex PCR assay for differentiation of Brucella species. Appl. Environ. Microbiol., 77:6726-6728.
  14. Kumar, A., Kumar, A., Sadish, S., Latha, C., Kumar, K. and Kumar, A.(2010). Epidemiology of Brucellosis in Occupationally Exposed Human Beings. Indian J. Anim. Res., 44:188-192.
  15. Kumar, V., Maan, S., Kumar, A., Batra, K., Chaudhary, D., Dalal, A., Gupta, A.K., Bansal, N., Sheoran, N. and Maan, N.S. (2017). Real-time PCR assay for differentiation of Brucella abortus and Brucella melitensis. Indian J. Anim. Res., doi:10.18805/ijar.    v0iOF.8464.52: 1037-1042
  16. Lopez-Goni, I., Garcia-Yoldi, D., Marin, C.M., De Miguel, M.J., Munoz, 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.
  17. Luna-Martýinez, J. E. and Mejýia-Teran, C. (2002). Brucellosis in Mexico: current status and trends.Vet Microbiol .,90:19–30.
  18. McDiarmid,A.(1957). The degree and duration of immunity in cattle resulting from vaccination with S. 19 Br. abortus vaccine and its implication in the future control and eventual eradication of Brucellosis.Vet Rec., 69:877–879.
  19. Morales-Estrada, A.I., Castillo-Salto, J., Lopez-Merino, A., Morales-Garcia, M.R., Valle-Valdez, J.G. and Contreras-Rodriguez, A.(2012). Characterization of Brucella species in Mexico by Bruce-Ladder polymerase chain reaction (PCR). Afr J Microbiol Res., 6: 2793-2796.
  20. Mothershed, E.A. and Whitney, A.M.(2006). Nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory. Clin. Chim. Acta., 363:206-220.
  21. Moussa, I.M., Omnia, M.E., Amin, A.S. and Selim, S.A., (2011). Evaluation of the currently used polymerase chain reaction assays for molecular detection of Brucella species. Afr. J. Microbiol. Res., 5:1511-1520.
  22. Nan, W., Tan, P., Wang, Y., Xu, Z., Mao, K., Peng, D. and Chen, Y.( 2014). Duplex PCR for differentiation of the vaccine strain Brucella suis S2 and B. suis biovar 1 from other strains of Brucella spp. Vet J., 201:427-428.
  23. Olsen, S.C. and Stoffregen, W.S.(2005). Essential role of vaccines in brucellosis control and eradication programs for livestock. Expert Rev. Vaccines., 4:915-928.
  24. Perry, B. and Grace, D. (2009). The impacts of livestock diseases and their control on growth and development processes that are pro-    poor. Philos Trans R Soc Lond B Biol Sci., 364:2643–2655.
  25. Probert, W.S., Schrader, K.N., Khuong, N.Y., Bystrom, S.L. and Graves, M.H. (2004). Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J. Clin. Microbiol., 42:1290-1293.
  26. Romero, C. and Lopez-Goñi, I. (1999). Improved method for purification of bacterial DNA from bovine milk for detection of Brucella spp. by PCR. Appl Environ Microbiol., 65:3735–3737.
  27. Sharifi Yazdi, H., Khazraiinia, P., Zahraei Salehi, T., and Behroozikhah, A. M. (2008). Development of a multiplex polymerase chain reaction assay for differentiation of field strain isolates and vaccine strains S19 and RB51 of Brucella in Iran. Iran J Vet. Res., 9: 19-24.
  28. Tabit, F. T. (2016). Advantages and limitations of potential methods for the analysis of bacteria in milk: a review. J Food Sci. Technol .,53:42–49.
  29. Wallach, J.C., Ferrero, M.C., Delpino, M.V., Fossati, C.A. and Baldi, P.C.(2008). Occupational infection due to Brucella abortus S19 among workers involved in vaccine production in Argentina. Clin Microbiol Infect., 14:805-807.
  30. Weiner, M., Iwaniak, W. and Szulowski, K. (2011). Comparison of PCR-based AMOS, Bruce-Ladder and MLVA assays for typing of Brucella species. Bull Vet Inst., 55:625–630.
  31. Whatmore, A.M., Shankster, S.J., Perrett, L.L., Murphy, T.J., Brew, S.D., Thirlwall, R.E., Cutler, S.J. and MacMillan, A.P.(2006). Identification and characterization of variable-number tandem-repeat markers for typing of Brucella spp. J Clinical Microbiol., 44:1982-1993. 
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Indian Journal of Animal Research
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    Research Article

    volume 53 issue 6 (june 2019) : 821-826,   Doi: 10.18805/ijar.B-3584

    Single-tube duplex-PCR for specific detection and differentiation of Brucella abortus S19 vaccine strain from other Brucella spp

    A
    Aparajita Das
    B
    Bablu Kumar
    S
    Soumendu Chakravarti
    K
    Karam Pal Singh
    A
    Abhishek
    G
    Garima Shrinet
    1Division of Biological Products, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh-243 122, India.
    Cite article:- Das Aparajita, Bablu Kumar, Chakravarti Soumendu, Singh Pal Karam, Abhishek, Shrinet Garima (2018). Single-tube duplex-PCR for specific detection and differentiation of Brucella abortus S19 vaccine strain from other Brucella spp. Indian Journal of Animal Research. 53(6): 821-826. doi: 10.18805/ijar.B-3584.

    ABSTRACT

    A simple single-tube duplex-PCR assay was optimized for rapid and sensitive detection and differentiation of Brucella abortus (B.abortus) S19 vaccine strain from other Brucella spp.(B.abortus 544,B abortus S99,B.melitensis 16M,B.suis).This assay was optimized using two primer pairs that were designed, one targeting genus specific multicopy IS711 and another eryC for the development of the duplex-PCR assay. Specificity of the assay was assessed using DNA templates from various Brucella species (B.abortus, B.melitensis, B.suis) and non Brucella bacteria like Staphylococcus aureus, Proteus vulgaris, Pasteurella multocida, Pseudomonas aeruginosa, Citrobacter freundii, E. coli, Salmonella enteritidis, Campylobacter jejuni and Listeria monocytogenes. The assay was also evaluated on spiked milk samples with known B.abortus cultures. The assay was able to detect up to 2.47 x 103CFU of B. abortus S99 organism in spiked milk sample. The assay was further validated in 53 clinical specimens (aborted foetal stomach content). Results confirm that Single tube duplex-PCR assay was useful for early and rapid detection and differentiation of B. abortus vaccine strain S19 from other Brucella spp in milk as well as in clinical specimen. 

    REFERENCES

    1. Alvarez, L.P., Marcellino, R.B., Martinez, A. and Robles, C.A.(2017). Duplex PCR for the diagnosis of Brucella melitensis and its differentiation from the REV-1 vaccine strain. Small Rumin Res., 146:1-4.
    2. Arenas-Gamboa, A.M., Ficht, T.A., Kahl-McDonagh, M.M., Gomez, G. and Rice-Ficht, A.C.(2009) .The Brucella abortus S19 ÄvjbR live vaccine candidate is safer than S19 and confers protection against wild-type challenge in BALB/c mice when delivered in a sustained-release vehicle. Infect Immun., 77:877-884.
    3. Arora, S., Agarwal, R. K., and Bist, B. (2006).Comparison of ELISA and PCR vis-à-vis cultural methods for detecting Aeromonas spp. in foods of animal origin. Int. J Food Microbiol., 106: 177-183.
    4. Bricker, B. J. and Halling, S. M. (1995). Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J Clin. Microbiol .,33: 1640–1642.
    5. Corbel, M. J. (2006).Brucellosis in humans and animals. World Health Organization.
    6. Cordes, D.O. and Carter, M.E.(1979). Persistence of Brucella abortus infection in six herds of cattle under brucellosis eradication. N Z Vet J., 27:255-259.
    7. Dorneles, E.M., Lima, G.K., Teixeira-Carvalho, A., Araújo, M.S., Martins-Filho, O.A.,et al (2015a). Immune response of calves vaccinated with Brucella abortus S19 or RB51 and revaccinated with RB51. PloS one., 10:e0136696.
    8. Dorneles, E. M. S., Sriranganathan, N. and Lage, A. P. (2015b). Recent advances in Brucella abortus vaccines. Vet Res., 46:76.
    9. Espinosa, I., Báez, M., Percedo, M. I., and Martinez, S. (2013).Evaluation of simplified DNA extraction methods for Streptococcus suis typing. Rev. Salud Anim., 35: 59-63.
    10. Garcia-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. Kanani, A.N., Jain, L., Patel, T.J., Rank, D.N., Joshi, C.G. and Purohit, J.H.(2008) Detection of Brucella DNA in semen using the polymerase chain reaction assay’. Indian J. Anim. Res., 42:222-224.
    12. Kaur, P., Sharma, N.S. and Arora, A.K.(2017).Investigation of brucellosis in cattle and buffaloes by conventional and molecular assays. Indian J. Anim. Res., doi:10.18805/ijar.B-3375.
    13. Kang, S.I., Her, M., Kim, J.W., Kim, J.Y., Ko, K.Y., Ha, Y.M. and Jung, S.C.(2011). Advanced multiplex PCR assay for differentiation of Brucella species. Appl. Environ. Microbiol., 77:6726-6728.
    14. Kumar, A., Kumar, A., Sadish, S., Latha, C., Kumar, K. and Kumar, A.(2010). Epidemiology of Brucellosis in Occupationally Exposed Human Beings. Indian J. Anim. Res., 44:188-192.
    15. Kumar, V., Maan, S., Kumar, A., Batra, K., Chaudhary, D., Dalal, A., Gupta, A.K., Bansal, N., Sheoran, N. and Maan, N.S. (2017). Real-time PCR assay for differentiation of Brucella abortus and Brucella melitensis. Indian J. Anim. Res., doi:10.18805/ijar.    v0iOF.8464.52: 1037-1042
    16. Lopez-Goni, I., Garcia-Yoldi, D., Marin, C.M., De Miguel, M.J., Munoz, 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.
    17. Luna-Martýinez, J. E. and Mejýia-Teran, C. (2002). Brucellosis in Mexico: current status and trends.Vet Microbiol .,90:19–30.
    18. McDiarmid,A.(1957). The degree and duration of immunity in cattle resulting from vaccination with S. 19 Br. abortus vaccine and its implication in the future control and eventual eradication of Brucellosis.Vet Rec., 69:877–879.
    19. Morales-Estrada, A.I., Castillo-Salto, J., Lopez-Merino, A., Morales-Garcia, M.R., Valle-Valdez, J.G. and Contreras-Rodriguez, A.(2012). Characterization of Brucella species in Mexico by Bruce-Ladder polymerase chain reaction (PCR). Afr J Microbiol Res., 6: 2793-2796.
    20. Mothershed, E.A. and Whitney, A.M.(2006). Nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory. Clin. Chim. Acta., 363:206-220.
    21. Moussa, I.M., Omnia, M.E., Amin, A.S. and Selim, S.A., (2011). Evaluation of the currently used polymerase chain reaction assays for molecular detection of Brucella species. Afr. J. Microbiol. Res., 5:1511-1520.
    22. Nan, W., Tan, P., Wang, Y., Xu, Z., Mao, K., Peng, D. and Chen, Y.( 2014). Duplex PCR for differentiation of the vaccine strain Brucella suis S2 and B. suis biovar 1 from other strains of Brucella spp. Vet J., 201:427-428.
    23. Olsen, S.C. and Stoffregen, W.S.(2005). Essential role of vaccines in brucellosis control and eradication programs for livestock. Expert Rev. Vaccines., 4:915-928.
    24. Perry, B. and Grace, D. (2009). The impacts of livestock diseases and their control on growth and development processes that are pro-    poor. Philos Trans R Soc Lond B Biol Sci., 364:2643–2655.
    25. Probert, W.S., Schrader, K.N., Khuong, N.Y., Bystrom, S.L. and Graves, M.H. (2004). Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J. Clin. Microbiol., 42:1290-1293.
    26. Romero, C. and Lopez-Goñi, I. (1999). Improved method for purification of bacterial DNA from bovine milk for detection of Brucella spp. by PCR. Appl Environ Microbiol., 65:3735–3737.
    27. Sharifi Yazdi, H., Khazraiinia, P., Zahraei Salehi, T., and Behroozikhah, A. M. (2008). Development of a multiplex polymerase chain reaction assay for differentiation of field strain isolates and vaccine strains S19 and RB51 of Brucella in Iran. Iran J Vet. Res., 9: 19-24.
    28. Tabit, F. T. (2016). Advantages and limitations of potential methods for the analysis of bacteria in milk: a review. J Food Sci. Technol .,53:42–49.
    29. Wallach, J.C., Ferrero, M.C., Delpino, M.V., Fossati, C.A. and Baldi, P.C.(2008). Occupational infection due to Brucella abortus S19 among workers involved in vaccine production in Argentina. Clin Microbiol Infect., 14:805-807.
    30. Weiner, M., Iwaniak, W. and Szulowski, K. (2011). Comparison of PCR-based AMOS, Bruce-Ladder and MLVA assays for typing of Brucella species. Bull Vet Inst., 55:625–630.
    31. Whatmore, A.M., Shankster, S.J., Perrett, L.L., Murphy, T.J., Brew, S.D., Thirlwall, R.E., Cutler, S.J. and MacMillan, A.P.(2006). Identification and characterization of variable-number tandem-repeat markers for typing of Brucella spp. J Clinical Microbiol., 44:1982-1993. 
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