article information

Jundishapur Journal of Microbiology: Vol.6, issue 6; e7197
published online: August 1, 2013
article type: Research Article
received: July 11, 2012
revised: December 31, 2012
accepted: January 27, 2013
DOI: https://doi.org/10.5812/jjm.7197
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Molecular Typing of Brucella melitensis and B. abortus From Human Blood Samples Using PCR-RFLP Method

authors:

avatar Reza Mirnejad 1 , avatar Mozafar Mohammadi 1 , avatar Ali Majdi 2 , avatar Niloufar Taghizoghi 3 , avatar Vahhab Piranfar 4 , *

Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran
Young Researchers Club, Tonekabon Branch, Islamic Azad University, Tonekabon, IR Iran
Bounty house, Stowage, Bellerbys College, London, United Kingdom
Department of Biology, Tonekabon Branch, Islamic Azad university of Tonekabon, Tonekabon, IR Iran

how to cite: Mirnejad R, Mohammadi M, Majdi A, Taghizoghi N, Piranfar V. Molecular Typing of Brucella melitensis and B. abortus From Human Blood Samples Using PCR-RFLP Method. Jundishapur J Microbiol. 2013;6(6):e7197. https://doi.org/10.5812/jjm.7197.

Abstract

Background:

Brucella is an intracellular parasite of the disease brucellosis throughout the world. Although several molecular typing methods are introduced to find DNA polymorphism that is able to identify Brucella species and biovars, but among these methods, detection of polymorphisms by PCR–RFLP has several advantages including the easy implementation, interpretation and the ease of use for large quantities of samples.

Objectives:

In the current study, the technique was used for molecular typing of Brucellaabortus and B. melitensis that was isolated from human blood samples.

Materials and Methods:

Blood samples of 160 patients were transferred to Kerman clinical centers with chief complain of acute brucellosis and showed high blood serum level (about 1.80). Their DNAs were extracted by Phenol chloroform method, and the PCR was optimized by using the fragments of designed primers for omp2a and omp2b. Therefore PCR products were restricted by restriction endonuclease as PstI and Hinf1. Finally they were electrophoresed for analyzing the digestion results on agarose gels (2%).

Results:

In 160 blood samples that were studied with PCR technique, 52 cases obtained bands of 1100 bp for omp2a locus and 1200 bp for omp2b locus from within 52 positive samples by PCR–RFLP method 25 cases (48%) were positive out of which 56% were B. melitensis biovar1 and 44% were B. abortus biovars of 3, 5, 6 or 9.

Conclusions:

The results of this study showed that PCR–RFLP technique was a fast and applicable method especially for separation, detection and differentiation between species of B. melitensis and B. abortus biovars in blood sample. Also the presented data showed that B. melitensis biovar 1 was the prevalence biovar.

1. Background

Brucellosis is a bacterial disease caused by Brucella members and it is one of the important common diseases in human and animals and an important reason of abortion in animals (1-4). This disease is endemically found all around the world especially in Asian, African, North American and Mediterranean countries (5, 6). Annually so many people in the World are afflicted by this disease, hence, that is an important health problem in developing countries and its control in these areas is very important (5, 6). Several studies have shown that to control and prevent the disease, it is very important to have the epidemiological information. The microbiologists all over the world use various molecular and non-molecular methods to obtain this information (7).

The genus of Brucella includes several species and biovars. The difference between their species and biovars is mainly based on phenotypic characteristics of LPS antigens, sensitivity to colors, need to CO2, H2S production, metabolic features and phage typing (8, 9). But these methods are very difficult and their sensitivity is too low. On the other hand, since DNA homology for all species in the genus of Brucella is more than 90%, so the attempt is providing accurate identification of Brucella species and biovars, by molecular methods (8, 10).

Several molecular typing methods are introduced to find DNA polymorphism that is able to identify the Brucella species and biovars, among which detection of polymorphisms by PCR - RFLP has several advantages including the easy implementation, interpretation and use for large quantities of samples (11-13). In this method, by omp25, omp2 and omp31 loci and all Brucella species can be differentiated and their biovars identified (14). Several studies use these genes to differentiate Brucella species and biovars performed around the World (12, 15-17), but in Iran, no study was done especially on clinical samples by this method, and it is the first time that a study is done on molecular typing of B. melitensis and B. abortus isolated from blood samples.

2. Objectives

The aims of this study were Molecular typing and Molecular Epidemiology of B. melitensis and B. abortus from human blood samples based on PCR assay to find prevalence biovars in human blood.

3. Materials and Methods

3.1. Blood Samples Collection

160 Clinical specimens were received from patients who referred to Kerman clinical centers with Brucellosis detection as they had measurable antibody titers 1/80 during the last two years. 5 ml blood was taken from patients and the blood samples were transferred to laboratory and were kept in -20°C.

3.2. DNA Extraction

A modification of the method was described by Queipo-Ortuño et al. (18). Briefly, 0.5 ml of blood samples with 1 ml of erythrocyte lyses’ solution (320 mM saccharose, 5 mM MgCl2, 1% Triton X-100, 10 mM Tris HCl [pH 7.5]) were mixed and centrifuged at 15000×g for 2 min. The supernatant was discarded, and the above steps were repeated for four times until the pellet lost all reddish coloring. Four hundred microliters of nucleic lyses’ buffer(1% SDS ‚10 mM Tris-Hcl‚ 10mM EDTA‚ 10 mM Sodium Acetate [pH 8]) containing proteinase K (10 mg/ml) were mixed and incubated for 30 min at 55°C in shaker incubator. Then, 100 ml of ammonium acetate (7.5 M) was added and centrifuged at 15,000 ×g for 10 min. To the supernatant, two volumes of absolute ethanol were added, and after centrifuging at 15,000 ×g for 10 min, the pellets were dissolved in 25 µl of TE buffer (pH 8.0) and stored at 4°C for PCR or at -20°C for long-term storage.

3.3. Amplification of omp2a and omp2b Fragments

Cloackaert et al. ( 13 ) designed primers for amplification of omp2a and omp2b fragments and their sequences are shown in Table 1. Each PCR reaction mixture contained; 1X PCR buffer, 2 mM MgCl 2 , 1 μl template DNA (0.5 μg) , 0.15 mMdNTP, 2.5 U Taq DNA polymerase, 20 pmol of each forward and reverse primers and sterile distilled water up to 50 μl. According to the following program, PCR for omp2a was performed in a GenAmp PCR system (Eppendorf, Germany): pre-denaturation for 5 min at 94°C followed by 35 cycles each containing denaturation at 94°C for 60 sec, annealing at 50°C for 120 sec and extension at 72°C for 180 sec, followed by final extension at 72°C for 7 min. Also PCR for omp2b was performed according to the following program: Pre-denaturation for 5 min at 94°C followed by 35 cycles each containing denaturation at 94°C for 45 sec, annealing at 58°C for 60 sec and extension at 72°C for 60 sec, followed by final extension at 72°C for 7 min. Then, The PCR products were analyzed using the electrophoresis technique on 2% agarose gel for 1 hour at 25 mA, stained by SYBR-Green and visualized under UV transilluminator (Figure 1). Finally, amplification products were further evaluated restriction digestion procedures.

Table 1.

Primers for Amplification of omp2a and omp2b Fragments

PrimerSequence
omp2a F5’-GGCTATTCAAAATTCTGGCG-3’
omp2a R5’-ATCGATTCTCACGCTTTCGT-3’
omp2b F5’-CCTTCAGCCAAATCAGAATG-3’
omp2b R5’-GGTCAGCATAAAAAGCAAGC-3’
Agarose Gel Electrophoresis of PCR Amplified Products Generated From DNA Samples
Lane 1 shows DNA size marker (100bp DNA ladder). Lane 2 is negative control. Lanes3 and 4 show amplified locus of omp2b. Lane 6 and 7 show amplified locus of omp2a, Lane 5 and 8 is positive control.

Lane 1 shows DNA size marker (100bp DNA ladder). Lane 2 is negative control. Lanes3 and 4 show amplified locus of omp2b. Lane 6 and 7 show amplified locus of omp2a, Lane 5 and 8 is positive control.

Figure 1.

Agarose Gel Electrophoresis of PCR Amplified Products Generated From DNA Samples

3.4. Enzymatic Digestion

To identify polymorphisms, the amplified products were subject to restriction enzymes. For enzymatic digestion or PCR-RFLP, the following 15 μl reactions were prepared for positive samples. 6 μl free nuclease sterile water, 1.7 μl enzyme buffers, 0.3 μl restriction enzyme (10-20 U), and 7 μl of PCR product. After providing the above reaction, micro tubes were put in the water bath for 2 hrs at 37°C. After 2 hours, products of RFLP were electrophoresed on 2% gel agarose. Finally the size of the fragments which were resulted from enzymatic digestion of all positive PCR samples compared with each other and according to the results of Cloackaert et al. (15), the biovars were identified.

3.5. Statistical Analysis

Statistical analysis was conducted to determine how many samples were positive for each bacterium, as well as those positive for two bacterial species. Perspective analyses were performed and data rounded numerical values (percentage) were documented.

4. Results

In 160 blood samples studied by PCR technique, 52 cases obtained bands of 1100 bp foromp2alocus and 1200 bp for omp2b locus which indicates that the samples are positive for Brucella (Figure 1).

According to Cloackaert et al. ( 13 ) regarding enzymatic digestion of the amplified fragments, and comparing the results of the present study with their obtained patterns, for 52 cases from digestion of omp2a fragment by Pst1 and Hinf1 enzymes, P3 and P2 Patterns were obtained respectively, and for omp2b, P1 and P1 patterns were resulted, which indicates the B. melitensis biovar 1 (Figure 2) ( 15 ),and for 33 cases (44.5%) from digestion of omp2a fragment by Pst1 and Hinf1 enzymes, Patterns of P2 and P2 and for omp2b patterns of P1 and P1 were resulted respectively, which indicates the B. abortus one of the biovars 3, 5, 6 or 9 (Figure 3). As a final conclusion, the used primers and enzymes in the current study showed that they were able to differentiate between B. melitensis biovars, but they cannot separate all of the B. abortus biovars from each other.

The Resulted Patterns of Enzymatic Digestion for B. melitensis Samples
Lane 1 is DNA size marker (100bp DNA ladder), Lanes 2 and 3 are digestion of omp2a fragment by Pst1 and Hinf1 and Lanes 4 and 5 are digestion of omp2b fragment by Pst1 and Hinf1.

Lane 1 is DNA size marker (100bp DNA ladder), Lanes 2 and 3 are digestion of omp2a fragment by Pst1 and Hinf1 and Lanes 4 and 5 are digestion of omp2b fragment by Pst1 and Hinf1.

Figure 2.

The Resulted Patterns of Enzymatic Digestion for B. melitensis Samples

The Resulted Patterns of Enzymatic Digestion for B. abortus Samples
Lane 1 is DNA size marker (100 bp DNA ladder). Lanes 2 and 3 are digestion of omp2a fragment by Pst1 and Hinf1. Lanes 4 and 5 are digestion of omp2b fragment by Pst1 and Hinf1.

Lane 1 is DNA size marker (100 bp DNA ladder). Lanes 2 and 3 are digestion of omp2a fragment by Pst1 and Hinf1. Lanes 4 and 5 are digestion of omp2b fragment by Pst1 and Hinf1.

Figure 3.

The Resulted Patterns of Enzymatic Digestion for B. abortus Samples

5. Discussion

Due to differences in the pathogenicity of Brucella species and biovars, with a view to epidemiology of brucellosis, recognition of Brucella biovars is important and hence typing of various strains is the main task of control centers of brucellosis and must be performed continuously (9, 13, 15). Distinction between the Brucella species and biovars in the past was mainly based on phenotypic characteristics. Today, with advancement of molecular techniques, several molecular typing methods to find DNA polymorphism are used, which are capable to identify Brucella biovars and species. The PBR assay is a technique used for typing (9, 10, 19). The current study used this technique to determine the species and biovars of B. abortus and B. melitensis isolated from clinical specimens in Iran.

Different genetic fragments such as omp31, 16srDNA, omp2 and so on are used in different studies, to identify and differentiate Brucella species and biovars, which could not show ability to differentiate Brucella biovars completely (14, 20, 21). Therefore, in subsequent studies especially in Cloackaert et al. study, omp2a and omp2b fragments and several restriction enzymes were used (15). These primers and enzymes were used in different parts of the world by different people, also in central parts of Iran they were used by Salehi et al. for molecular typing of common Brucella biovars (15-17, 22).

Results of all these studies, like those of the current study showed that the primers and enzymes are able to differentiate B. melitensis biovars but could not distinguish all of the B. abortus biovars from each other. However, present study differs from most of the studies in type of sample; in the current study human clinical samples were used whereas the others used animal samples. Also compared with the studies in Iran; there were differences, such as sample types, the enzymes used, and the sample figure. Salehi et al. by one or two pairs of primers and an enzyme identified Brucella isolated from animal samples (22). But in the current study, isolated Brucella from clinical samples were typed with two pairs of primers and two restriction enzymes. They were not able to differentiate between other B. melitensis biovars and biovars of B. abortus, but results of the current study showed that two species can be differentiated from each other and one can determine their biovars.

The results of this study like others in different parts of Iran showed that B. melitensis biovar 1 is the predominant form of these species in Iran (23, 24). Results of the current study showed that one of the B. abortus biovars 3, 5, 6 and 9 is the dominant form of these species, according to the previous studies in Iran, the biovar 3 was more likely (23, 24).

Same as the results of other studies in other parts of the world, the current study (15-17) showed that the PCR-RFLP techniques, by the primers and enzymes, were able to differentiate species and biovars of B. abortus and B. melitensis in clinical samples well. So they can be used to molecular typing of biovars isolated from different samples. Also comparing the obtained results with those of the other studies showed that the dominant pattern which causes the disease of brucellosis, especially B. melitensis, was not different in Iran, so, control and preventive measures of relevant organizations were appropriate and this trend should continue.

Acknowledgements

References

  • 1.

    Cutler SJ, Whatmore AM, Commander NJ. Brucellosis--new aspects of an old disease. J Appl Microbiol. 2005;98(6):1270-81. [PubMed ID: 15916641]. https://doi.org/10.1111/j.1365-2672.2005.02622.x.

  • 2.

    Christopher S, Umapathy BL, Ravikumar KL. Brucellosis: review on the recent trends in pathogenicity and laboratory diagnosis. J Lab Physicians. 2010;2(2):55-60. [PubMed ID: 21346896]. https://doi.org/10.4103/0974-2727.72149.

  • 3.

    Guerra H. The brucellae and their success as pathogens. Crit Rev Microbiol. 2007;33(4):325-31. [PubMed ID: 18033597]. https://doi.org/10.1080/10408410701647644.

  • 4.

    Davar SS, Reza AM, Sahar K, Mehdi SS, Arfa M. Biological and immunological characteristics of Brucella abortus S99 major outer membrane proteins. Jundishapur J Microbiol. 2012;4(1).

  • 5.

    Shapouri R, Rahnema M. Evaluation of antimicrobial effect of hops extracts on intramacrophages Brucella abortus and B. melitensis. Jundishapur J Microbiol. 2011;4(2):S51-S58.

  • 6.

    Seimenis A, Morelli D, Mantovani A. Zoonoses in the Mediterranean region. Ann Ist Super Sanita. 2006;42(4):437-45. [PubMed ID: 17361068].

  • 7.

    Pappas G, Memish ZA. Brucellosis in the middle East: a persistent medical, socioeconomic and political issue. J Chemother. 2007;19(3):243-8. [PubMed ID: 17594917].

  • 8.

    Li W, Raoult D, Fournier PE. Bacterial strain typing in the genomic era. FEMS Microbiol Rev. 2009;33(5):892-916. [PubMed ID: 19453749]. https://doi.org/10.1111/j.1574-6976.2009.00182.x.

  • 9.

    Nagalingam M, Shome R, Balamurugan V, Shome BR, NarayanaRao K, et al. Molecular typing of Brucella species isolates from livestock and human. Trop Anim Health Prod. 2012;44(1):5-9. [PubMed ID: 21647774]. https://doi.org/10.1007/s11250-011-9886-1.

  • 10.

    Cerekci A, Kilic S, Bayraktar M, Uyanik MH, Yasar E, Esen B. [Comparison of conventional methods and real-time multiplex polymerase chain reaction for identification and typing of Brucella isolates of human origin]. Mikrobiyol Bul. 2011;45(3):392-400. [PubMed ID: 21935772].

  • 11.

    Wattiau P, Whatmore AM, Van Hessche M, Godfroid J, Fretin D. Nucleotide polymorphism-based single-tube test for robust molecular identification of all currently described Brucella species. Appl Environ Microbiol. 2011;77(18):6674-9. [PubMed ID: 21803907]. https://doi.org/10.1128/AEM.00767-11.

  • 12.

    Garcia-Yoldi D, Marin CM, Lopez-Goni I. Restriction site polymorphisms in the genes encoding new members of group 3 outer membrane protein family of Brucella spp. FEMS Microbiol Lett. 2005;245(1):79-84. [PubMed ID: 15796983]. https://doi.org/10.1016/j.femsle.2005.02.026.

  • 13.

    Lopez-Goni I, Garcia-Yoldi D, Marin CM, de Miguel MJ, Munoz PM, Blasco JM, et al. Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. J Clin Microbiol. 2008;46(10):3484-7. [PubMed ID: 18716225]. https://doi.org/10.1128/JCM.00837-08.

  • 14.

    Cloeckaert A, Vizcaino N, Paquet JY, Bowden RA, Elzer PH. Major outer membrane proteins of Brucella spp.: past, present and future. Vet Microbiol. 2002;90(1-4):229-47. [PubMed ID: 12414146].

  • 15.

    Cloeckaert A, Verger JM, Grayon M, Grepinet O. Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella. Microbiology. 1995;141 ( Pt 9):2111-21. [PubMed ID: 7496522].

  • 16.

    Al Dahouk S, Tomaso H, Prenger-Berninghoff E, Splettstoesser WD, Scholz HC, Neubauer H. Identification of brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Crit Rev Microbiol. 2005;31(4):191-6. [PubMed ID: 16417200]. https://doi.org/10.1080/10408410500304041.

  • 17.

    Ancora M, De Santis P, Di Giannatale E, Luciani M, Alessiani A. Molecular typing of Brucella field strains isolated in Italy. Vet Ital. 2005;41(1):51-5. [PubMed ID: 20437371].

  • 18.

    Queipo-Ortuno MI, Morata P, Ocon P, Manchado P, Colmenero JD. Rapid diagnosis of human brucellosis by peripheral-blood PCR assay. J Clin Microbiol. 1997;35(11):2927-30. [PubMed ID: 9350761].

  • 19.

    Mirnejad R, Doust RH, Kachuei R, Mortazavi SM, Khoobdel M, Ahamadi A. Simultaneous detection and differentiates of Brucella abortus and Brucella melitensis by combinatorial PCR. Asian Pac J Trop Med. 2012;5(1):24-8. [PubMed ID: 22182638]. https://doi.org/10.1016/S1995-7645(11)60239-3.

  • 20.

    Vizcaino N, Kittelberger R, Cloeckaert A, Marin CM, Fernandez-Lago L. Minor nucleotide substitutions in the omp31 gene of Brucella ovis result in antigenic differences in the major outer membrane protein that it encodes compared to those of the other Brucella species. Infect Immun. 2001;69(11):7020-8. [PubMed ID: 11598077]. https://doi.org/10.1128/IAI.69.11.7020-7028.2001.

  • 21.

    Da Costa M, Guillou JP, Garin-Bastuji B, Thiebaud M, Dubray G. Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification. J Appl Bacteriol. 1996;81(3):267-75. [PubMed ID: 8810054].

  • 22.

    Salehi M, Pishva E, Salehi R, Rahmani R. Isolation of Brucella abortus using PCR-RFLP analysis. Iran J Pub Health. 2006;35(4).

  • 23.

    Najafi N, Ghassemian R, Davoody AR, Tayebi A. An unusual complication of a common endemic disease: clinical and laboratory aspects of patients with brucella epididymoorchitis in the north of Iran. BMC Res Notes. 2011;4:286. [PubMed ID: 21834966]. https://doi.org/10.1186/1756-0500-4-286.

  • 24.

    Shareef JM. A review of serological investigations of brucellosis among farm animals and humans in northern provinces of iraq (1974-2004). J Vet Med B Infect Dis Vet Public Health. 2006;53 Suppl 1:38-40. [PubMed ID: 17123367]. https://doi.org/10.1111/j.1439-0450.2006.01021.x.