Tülin ARASOĞLU, Medine GÜLLÜCE, Hakan ÖZKAN, Ahmet ADIGÜZEL, Fikrettin ŞAHİN

PCR detection of Brucella abortus in cow milk samples collected from Erzurum, Turkey

In this study, we designed and used 2 different type-specific polymerase chain reaction (PCR) methods for the detection of Brucella abortus. Materials and methods: There were 2 different primer sets (B4/B5 and AF/AR) selected and used for the amplification of 2 different genes that are present in all biovars of Brucella species, including bcsp31, encoding 31-kDa immunogenic cell surface proteins, and omp25, which is known to be one of the virulence factors encoding outer membrane proteins of 26–23 kDa. Results: The results showed that 273 (81.7%) and 317 (94.9%) out of 334 milk samples were positive for brucellosis, as detected by either both or one of the primer sets used in the present study, respectively. The detection limit of PCR assays for Brucella in milk samples was determined as 5 pg DNA for both of the primer sets. Conclusion: These results suggest that the use of the specific PCR assay with the primer sets used in the study is a rapid, reliable, and accurate technique in comparison to traditional and conventional methods for the detection and diagnosis of Brucella spp. in milk samples. Kew words: Brucella abortus, bovine milk, bcsp31-PCR, omp25-PCR

Anahtar Kelimeler:

Turk J Med Sci 2013; 43(4): 501-508. Full text: pdf Other articles published in the same issue: Turk J Med Sci, vol.43, iss.4.

PCR detection of Brucella abortus in cow milk samples collected from Erzurum, Turkey

Keywords:

Turk J Med Sci 2013; 43(4): 501-508. Full text: pdf Other articles published in the same issue: Turk J Med Sci, vol.43, iss.4.,

PDF

___

Ertek M, Yazgı H, Özkurt Z, Ayyıldız A, Parlak M. Comparison of the diagnostic value of the standard tube agglutination test and the ELISA IgG and IgM in patients with brucellosis. Turk J Med Sci 2006; 363: 159–63.
Motaharinia Y, Rezaee MA, Hazhir MS, Zandi F, Shapouri R, Hakhamaneshi MS et al. Evaluation of the antibacterial activity of Zataria multiflora Boiss., Rhus coriaria L. (sumac), Mentha piperita L., and Ocimum basilicum L. extracts on Brucella strains isolated from brucellosis patients. Turk J Med Sci 2012; 425: 816–22.
Bounaadja L, Albert D, Chenais B, Henault S, Zygmunt MS, Poliak S et al. Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet Microbiol 2009; 137: 156–64.
Güllüce M. Kars ve çevresinde, sığırlarda, B. abortus’a karşı oluşan antikorların ELİSA ve diğer serolojik yöntemlerle (RBPT, SAT, MRT) saptanması ve sonuçlarının karşılaştırılması. PhD, Kafkas University Veterinary Faculty, Kars, Turkey; 1993 (in Turkish).
Munir R, Farooq U, Fatima Z, Afzal M, Anwar Z, Jahangir M. Sero-prevalence of brucellosis in bovines at farms under different management conditions. Brit J Dairy Sci 2011; 2: 35–
Baddour MM, Alkhalifa DH. Evaluation of 3 PCR techniques for detection of Brucella DNA in peripheral human blood. Egyptian J Med Microbiol 2007; 16: 201–9.
Kurtaran B, Candevir, İnal AS, Kömür S, Akyıldız Ö, Saltoğlu N et al. Clinical appearance of brucellosis in adults: fourteen years of experience. Turk J Med Sci 2012; 42: 497–505.
Keid LB, Soares RM, Vasconcellos SA, Salgado VR, Megid J, Richtzenhain LJ. Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis. Vet Rec 2010; 167: 96–9.
Salehi M, Pishva E, Salehi R, Rahmani M. Isolation of Brucella abortus using PCR-RFLP analysis. Iranian J Publ Health 2006; 35: 22–7.
Çalık Ş, Gökengin AD. Human brucellosis in Turkey: a review of the literature between 1990 and 2009. Turk J Med Sci 2011; 41: 549–5.
Cutler SJ. Brucellosis, the most common bacterial zoonosis? Biomed Sci 2006; 63: 336–41.
Abdoel TH, Smits HL. Rapid latex agglutination test for the serodiagnosis of human brucellosis. Diagn Micr Infec Dis 2007; 57: 123 –8.
Mukherjee F, Jain J, Patel V, Nair M. Multiple genus-specific markers in PCR assays improve the specificity and sensitivity of diagnosis of brucellosis in field animals. J Med Microbiol 2007; 56: 1309–16.
Leal-Klevezas DS, Martínez-Vázquez IO, García-Cantş J, López-Merino A, Martínez-Soriano JP. Use of polymerase chain reaction to detect Brucella abortus biovar 1 in infected goats. Vet Microbiol 2000; 75: 91–7.
Gupta VK, Verma DK, Rout PK, Singh SV, Vihan VS. Polymerase chain reaction for detection of Brucella melitensis in goat milk. Small Ruminant Res 2006; 65: 79–84.
Ilhan Z, Aksakal A, Ekin IH, Gülhan T, Solmaz H, Erdenlig S. Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep. Lett Appl Microbiol 2008; 46: 301–6.
Bricker BJ. Diagnostic strategies used for the identification of Brucella. Vet Microbiol 2002; 90: 433–4.
Güler L, Gündüz K, Ok Ü. Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples. Vet Microbiol 2003; 93: 53–61.
O’Leary S, Sheahan M, Sweeney T. Brucella abortus detection by PCR assay in blood, milk and lymph tissue of serologically positive cows. Res Vet Sci 2006; 81: 170–6.
Adıgüzel A. Bazı termal tesislerden alınan su örneklerinden izole edilen termofilik bakterilerin moleküler karakterizasyonu. PhD, Atatürk University Institute of Sciences, Erzurum, Turkey; 2006 (in Turkish).
Arasoğlu T. Erzurum İli inek sütlerinde PZR yöntemi ile Brucella abortus ve Brucella melitensis türlerinin tanilanması. PhD, Atatürk University Graduate School of Natural and Applied Sciences, Erzurum, Turkey; 2010 (in Turkish).
Whatmore AM, Perrett L, MacMillan AP. Characterisation of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol 2007; 7: 1–15.
Elfaki MG, Uz-Zaman T, Al-Hokail AA, Nakeeb SM. Detection of Brucella DNA in sera from patients With brucellosis by polymerase chain reaction. Diagn Microbiol Infect Dis 2005; 53: 1–7.
Navarro E, Escribano J, Fernandez JA, Solera J. Comparison of three different PCR methods for detection of Brucella spp. in human blood samples. FEMS Immunol Med Microbiol 2002; 34: 147–51.
Romero C, Lopez-Goni I. Improved method for purification of bacterial DNA from bovine milk for detection of Brucella spp. by PCR. Appl Environ Microbiol 1999; 65: 3735–7.
Fredricks DN, Relman DA. Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J Clin Microbiol 1998; 36: 2810–6.
Matrone M, Keid LB, Rocha VCM, Vejarano MP, Ikuta CY, Rodriguez CAR et al. Evaluation of DNA extraction protocols for Brucella abortus PCR detection in aborted fetuses or calves born from cows experimentally infected with strain 2308. Braz J Microbiol 2009; 40: 480–9.
Matar GM, Khneisser IA, Abdelnoor AM. Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-kilodalton Brucella antigen DNA. J Clin Microbiol 1996; 34: 477–8.
Leal-Klevezas DS, Martínez-Vázquez IO, López-Merino A, Martínez-Soriano JP. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J Clin Microbiol 1995; 33: 3087–90.
Rijpens PN, Jannes G, Asbroeck MV, Rossau R, Herman LMF. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. Appl Environ Microbiol 1996; 62: 1683–8.