The First Prevalence Report of Direct Identification and Differentiation of B. abortus and B. melitensis using Real Time PCR in House Mouse of Iran
Brucellosis is a zoonotic disease; its symptoms and appearances are not exclusive in human and its traditional diagnosis is based on culture, serological methods and conventional PCR. For more sensitive, specific detection and differentiation of Brucella spp., the real time PCR method is recommended. This research has performed to determine the presence and prevalence of Brucella spp. and differentiation of Brucella abortus and Brucella melitensis in house mouse (Mus musculus) in west of Iran. A TaqMan analysis and single-step PCR was carried out in total 326 DNA of Mouse's spleen samples. From the total number of 326 samples, 128 (39.27%) gave positive results for Brucella spp. by conventional PCR, also 65 and 32 out of the 128 specimens were positive for B. melitensis, B. abortus, respectively. These results indicate a high presence of this pathogen in this area and that real time PCR is considerably faster than current standard methods for identification and differentiation of Brucella species. To our knowledge, this study is the first prevalence report of direct identification and differentiation of B. abortus and B. melitensis by real time PCR in mouse tissue samples in Iran.
[1] A. Al-Mariri, P. A. Tibor, X. Mertens, P. De Bolle, J. Michel, G. K.
Walravens, and J. J. Letesson, "Induction of immune response in
BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of
Brucella spp.," Infect. Immun., vol. 69, pp. 6264-6270, Apr. 2001.
[2] W. S. Probert, K. N. Schrader, N. Y. Khuong, S. L. Bystrom and M. H.
Graves, "Real-Time multiplex PCR assay for detection of Brucella spp.,
B. abortus, and B. melitensis," J. Clin. Microbiol., 2004; vol. 42, no. 3,
pp. 1290-1293, Jan. 2004.
[3] B. Kazemi, S. A. Yousefi Namin, M. Dowlatshahi, M. Bandepour, F.
Kafilzadeh, L. Gachkar, F. Mahmoudinejad, A. Samarghandi, and M.
Mardani, "Detection of Brucella by peripheral blood PCR and
comparison with culture and serological methods in suspected cases."
Iranian J. Publ. Health., vol. 37, no. 4, pp. 96-102, Agu. 2008.
[4] T. Bogdanovich, M. Skurnik, P. S. Lubeck, P. Ahrens, and J. Hoorfar,
"Validated 5' nuclease PCR assay for rapid identification of the genus
Brucella." J. Clin. Microbiol., vol. 42, no. 5, pp. 2261-2263, Des. 2004.
[5] B. J. Bricker, "PCR as a diagnostic tool for brucellosis." Vet. Microbiol.,
vol. 90, pp. 435-446, Jun. 2002.
[6] C. Romero, C. Gamazo, M. Pardo, and I. Lopez-Goni, "Specific
detection of Brucella DNA by PCR." J. Clin. Microbiol., vol. 33, pp.
615-617, Jul. 1995.
[7] T. J. Carver, K. M. Rutherford, M. Berriman, M. A. Rajandream, B. G.
Barrell, and J. Parkhill, ACT: the Artemis Comparison Tool."
Bioinformatics., vol. 21, pp. 3422-3423, Aug. 2005.
[8] E. Navarro, M. A. Casao, and J. Solera, "Diagnosis of human brucellosis
using PCR." Expert Rev. Mol. Diagn., vol. 4, pp. 115-123, Oct. 2004.
[9] B. J. Bricker, and S. M. Halling, "Differentiation of Brucella abortus bv
1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv 1 by
PCR" J. Clin. Microbiol., vol. 32, pp. 2660-2666, Feb. 1994.
[10] B. J. Bricker, "Differentiation of hard-to-type bacterial strains by RNA
mismatches cleavage." Biotechniques., vol. 27, pp. 321-324, Jul. 1999.
[11] E. Tcherneva, N. Rijpens, B. Jersek, and L. M. Herman, "Differentiation
of Brucella species by random amplified polymorphic DNA analysis.
Journal of Applied Microbiol., vol. 88, pp. 69-80, Aug. 2000.
[12] J. T. Foster, R. T. Okinaka, R. Svensson, K. Shaw, B. K. De, A. R.
Robison, W. S. Probert, L. J. Kenefic, W. D. Brown, and P. Keim,
"Real-time PCR assays of single-nucleotide polymorphisms defining the
major Brucella clades." J. Clin. Microbiol., vol. 46, pp. 296-301, Jan.
2008.
[13] J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York:
2001, 3rd edn.
[14] D. Leal-Klevezas, I.O. Martinez vazquez, A. Lopez-Merino, Martinez- J.
P. Soriano, "Single-step PCR for detection of Brucella spp. from blood
and milk of infected animals." J. Clin. Microbiol., vol. 33, no.12, pp.
3087-3090, Des. 1995.
[15] B. Osterman, and I. Moriyo'n, "International Committee on Systematics
of Prokaryotes; subcommittee on the taxonomy of Brucella: minutes of
the meeting, 17 September 2003, Pamplona, Spain." Int. J. Syst. Evol.
Microbiol., vol. 56, pp. 1173-1175, Jun. 2006.
[16] B. G. Mantur, S. K. Amarnath, and R. S. Shinde "Review of clinical and
laboratory features of human brucellosis." Indian journal of medical
microbiology., vol. 25, no. 3, pp. 188-202, Des. 2007.
[17] Y. A. Al-Eissa, "Brucellosis in Saudi Arabia: past, present and future."
Annals of Saudi medicine., vol. 19, no. 5, 401-405, May. 1999.
[18] P. A. Vershilova, G. Liamkin, V. E. Malikov, E. A. Dranovskaya, and I.
F. Taran, "Brucella strains from Mouselike Rodents in Southwestern
USSR." International Jolu Rnal of Systematbaicc Teriology., vol. 33, no.
2, pp. 399-400, Mar. 1983.
[19] C. Romero, C. Gamazo, M. Pardo, and I. Lopez-Goni, "Specific
Detection of Brucella DNA by PCR." J. Clin. Microbiol., vol. 33, no.3,
pp. 615-617, Mar. 1995.
[20] E. Navarro, M. A. Casao, and J. Solera, "Diagnosis of human brucellosis
using PCR." Expert Rev. Mol. Diagn., vol. 4, no.1, pp. 115-123, Feb.
2004.
[21] M. J. LaGier, L. A. Joseph, T. V. Passaretti, K. A. Musser, and N. M.
Cirino, "A real-time multiplexed PCR assay for rapid detection and
differentiation of Campylobacter jejuni and Campylobacter coli."
Molecular and Cellular Probes., vol. 18, pp. 275-282, Jul. 2004.
[22] L. Bounaadja, D. Albert, B. Che nais, S. Henault, M.S. Zygmunt, S.
Poliak, and B. Garin-Bastuji, "Real-time PCR for identification of
Brucella spp.: A comparative study of IS711, bcsp31 and per target
genes." Vet. Microbiol., vol. 137, pp. 156-164, Nov. 2009.
[1] A. Al-Mariri, P. A. Tibor, X. Mertens, P. De Bolle, J. Michel, G. K.
Walravens, and J. J. Letesson, "Induction of immune response in
BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of
Brucella spp.," Infect. Immun., vol. 69, pp. 6264-6270, Apr. 2001.
[2] W. S. Probert, K. N. Schrader, N. Y. Khuong, S. L. Bystrom and M. H.
Graves, "Real-Time multiplex PCR assay for detection of Brucella spp.,
B. abortus, and B. melitensis," J. Clin. Microbiol., 2004; vol. 42, no. 3,
pp. 1290-1293, Jan. 2004.
[3] B. Kazemi, S. A. Yousefi Namin, M. Dowlatshahi, M. Bandepour, F.
Kafilzadeh, L. Gachkar, F. Mahmoudinejad, A. Samarghandi, and M.
Mardani, "Detection of Brucella by peripheral blood PCR and
comparison with culture and serological methods in suspected cases."
Iranian J. Publ. Health., vol. 37, no. 4, pp. 96-102, Agu. 2008.
[4] T. Bogdanovich, M. Skurnik, P. S. Lubeck, P. Ahrens, and J. Hoorfar,
"Validated 5' nuclease PCR assay for rapid identification of the genus
Brucella." J. Clin. Microbiol., vol. 42, no. 5, pp. 2261-2263, Des. 2004.
[5] B. J. Bricker, "PCR as a diagnostic tool for brucellosis." Vet. Microbiol.,
vol. 90, pp. 435-446, Jun. 2002.
[6] C. Romero, C. Gamazo, M. Pardo, and I. Lopez-Goni, "Specific
detection of Brucella DNA by PCR." J. Clin. Microbiol., vol. 33, pp.
615-617, Jul. 1995.
[7] T. J. Carver, K. M. Rutherford, M. Berriman, M. A. Rajandream, B. G.
Barrell, and J. Parkhill, ACT: the Artemis Comparison Tool."
Bioinformatics., vol. 21, pp. 3422-3423, Aug. 2005.
[8] E. Navarro, M. A. Casao, and J. Solera, "Diagnosis of human brucellosis
using PCR." Expert Rev. Mol. Diagn., vol. 4, pp. 115-123, Oct. 2004.
[9] B. J. Bricker, and S. M. Halling, "Differentiation of Brucella abortus bv
1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv 1 by
PCR" J. Clin. Microbiol., vol. 32, pp. 2660-2666, Feb. 1994.
[10] B. J. Bricker, "Differentiation of hard-to-type bacterial strains by RNA
mismatches cleavage." Biotechniques., vol. 27, pp. 321-324, Jul. 1999.
[11] E. Tcherneva, N. Rijpens, B. Jersek, and L. M. Herman, "Differentiation
of Brucella species by random amplified polymorphic DNA analysis.
Journal of Applied Microbiol., vol. 88, pp. 69-80, Aug. 2000.
[12] J. T. Foster, R. T. Okinaka, R. Svensson, K. Shaw, B. K. De, A. R.
Robison, W. S. Probert, L. J. Kenefic, W. D. Brown, and P. Keim,
"Real-time PCR assays of single-nucleotide polymorphisms defining the
major Brucella clades." J. Clin. Microbiol., vol. 46, pp. 296-301, Jan.
2008.
[13] J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York:
2001, 3rd edn.
[14] D. Leal-Klevezas, I.O. Martinez vazquez, A. Lopez-Merino, Martinez- J.
P. Soriano, "Single-step PCR for detection of Brucella spp. from blood
and milk of infected animals." J. Clin. Microbiol., vol. 33, no.12, pp.
3087-3090, Des. 1995.
[15] B. Osterman, and I. Moriyo'n, "International Committee on Systematics
of Prokaryotes; subcommittee on the taxonomy of Brucella: minutes of
the meeting, 17 September 2003, Pamplona, Spain." Int. J. Syst. Evol.
Microbiol., vol. 56, pp. 1173-1175, Jun. 2006.
[16] B. G. Mantur, S. K. Amarnath, and R. S. Shinde "Review of clinical and
laboratory features of human brucellosis." Indian journal of medical
microbiology., vol. 25, no. 3, pp. 188-202, Des. 2007.
[17] Y. A. Al-Eissa, "Brucellosis in Saudi Arabia: past, present and future."
Annals of Saudi medicine., vol. 19, no. 5, 401-405, May. 1999.
[18] P. A. Vershilova, G. Liamkin, V. E. Malikov, E. A. Dranovskaya, and I.
F. Taran, "Brucella strains from Mouselike Rodents in Southwestern
USSR." International Jolu Rnal of Systematbaicc Teriology., vol. 33, no.
2, pp. 399-400, Mar. 1983.
[19] C. Romero, C. Gamazo, M. Pardo, and I. Lopez-Goni, "Specific
Detection of Brucella DNA by PCR." J. Clin. Microbiol., vol. 33, no.3,
pp. 615-617, Mar. 1995.
[20] E. Navarro, M. A. Casao, and J. Solera, "Diagnosis of human brucellosis
using PCR." Expert Rev. Mol. Diagn., vol. 4, no.1, pp. 115-123, Feb.
2004.
[21] M. J. LaGier, L. A. Joseph, T. V. Passaretti, K. A. Musser, and N. M.
Cirino, "A real-time multiplexed PCR assay for rapid detection and
differentiation of Campylobacter jejuni and Campylobacter coli."
Molecular and Cellular Probes., vol. 18, pp. 275-282, Jul. 2004.
[22] L. Bounaadja, D. Albert, B. Che nais, S. Henault, M.S. Zygmunt, S.
Poliak, and B. Garin-Bastuji, "Real-time PCR for identification of
Brucella spp.: A comparative study of IS711, bcsp31 and per target
genes." Vet. Microbiol., vol. 137, pp. 156-164, Nov. 2009.
@article{"International Journal of Biological, Life and Agricultural Sciences:50658", author = "A. Doosti and S. Moshkelani", title = "The First Prevalence Report of Direct Identification and Differentiation of B. abortus and B. melitensis using Real Time PCR in House Mouse of Iran", abstract = "Brucellosis is a zoonotic disease; its symptoms and appearances are not exclusive in human and its traditional diagnosis is based on culture, serological methods and conventional PCR. For more sensitive, specific detection and differentiation of Brucella spp., the real time PCR method is recommended. This research has performed to determine the presence and prevalence of Brucella spp. and differentiation of Brucella abortus and Brucella melitensis in house mouse (Mus musculus) in west of Iran. A TaqMan analysis and single-step PCR was carried out in total 326 DNA of Mouse's spleen samples. From the total number of 326 samples, 128 (39.27%) gave positive results for Brucella spp. by conventional PCR, also 65 and 32 out of the 128 specimens were positive for B. melitensis, B. abortus, respectively. These results indicate a high presence of this pathogen in this area and that real time PCR is considerably faster than current standard methods for identification and differentiation of Brucella species. To our knowledge, this study is the first prevalence report of direct identification and differentiation of B. abortus and B. melitensis by real time PCR in mouse tissue samples in Iran.
", keywords = "Differentiation, B. abortus, B. melitensis, TaqManprobe, Iran.", volume = "5", number = "2", pages = "44-4", }
