Identification and differentiation of Brucella melitensis and Brucella abortus strains isolated in Greece, directly from solid tissues by MULTIPLEX and REAL-TIME PCR methods Evridiki Boukouvala1*, Katiuscia Zilli2, Evanthia Petridou3, Erasmia Smyroglou1,3, Loukia V. Ekateriniadou1, Manuela Tittarelli2 1Hellenic Agricultural Organization DIMITRA, Veterinary Research Institute, Thermi, Greece; 2 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Teramo, Italy; 3School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece; Introduction. As a part of an EU EMIDA ERA-NET project entitled “Brucella melitensis: biotyping and differential diagnostic- Brucmel” we established a procedure to isolate and identify directly Brucella melitensis and Brucella abortus strains from solid tissues. Identification procedures of Brucella sp. are based on microbiological or/and serological methods. The main disadvantage of the serological tests is the cross-reactivity with other related bacteria (E. coli,Y. enterocolitica, etc) while cultures are time-consuming and of high risk to people handling the infected tissues. To surmount these problems a multiplex PCR as well as a Real Time PCR protocol have been developed for solid tissues and cultures. Material and Methods. The tissues (lymph nodes and spleen) for the laboratory examination were cut extensively and homogenized for 2 minutes with the addition of 1ml distilled water or PBS. The homogenization was performed by a handle cordless grinder (NIPPON Genetics, Japan). 200μl from the homogenized tissue were used for DNA extraction using the PureLink Genomic DNA Mini Kit (Life Sciences). In addition, the validity of the method was assessed using a ring test organized by IZS of Teramo. A panel of 30 spleen samples collected from cattle (10), sheep (10) and swine (10), were contaminated with a known concentration (characterized as high or low concentration) of Brucella abortus, Brucella melitensis and Brucella suis, respectively. The panel also included non-infected spleen tissue samples, as negative controls. In parallel all homogenates were cultured in blood agar base (Oxoid) supplemented with 5% equine serum and glucose 40ml of 25% concentration per liter. Moreover the Farrell’s medium used, which is prepared by the addition of six antibiotics to a basal medium. Real–time PCR was performed on genomic DNA isolated from both solid tissues and cultures. Two duplex qPCR were performed, one for the identification of Brucella spp. and Brucella melitensis and a second one for the identification Brucella spp. and Brucella abortus. As targets genes for the identification of Brucella spp., Brucella melitensis and Brucella abortus were used the multiple insertion element IS711, the BMEII0466 and BruAb2_0168 gene, respectively. The primers pairs and probes that were used are described by Hinic et al., 2008. The reaction mixtures were prepared at a final volume of 25 μl, containing 12.5μl of Platinum Quantitative qPCR Supermix-UDG (Invitrogen), 300nM of each primer, 250nM of each probe and 60-80 ng DNA. QPCRs were performed in a Chromo4TM Real-Time Detector. The cycling conditions for all the reactions consisted of an initial denaturation at 95oC for 3min, and finally 45 cycles of denaturation at 95oC for 15sec and annealing/extension at 60oC for 30sec. Multiplex PCR was performed on genomic DNA isolated from the panel of 30 spleen samples. Fifteen different primers were examined based on previous studies (Probert WS et al., 2004, Imaoka et al., 2007, Bricker and Halling, 1994, Gopaul KK et al., 2008, Scholz HC et al., 2008); the list of oligonucleotides can be send on request (boukouvala@vri.gr). We finally performed a multiplex PCR for the identification of Brucella melitensis and Brucella abortus using the primers described by Bricker and Halling, 1994. The PCR reactions were performed in 25μl final volume. Each reaction was consisted of 1X PCR buffer, 1,5mM MgCl2, 280μM of each dNTP, 400nM of each primer, 4.5U of AmpliTaq God 360 DNA polymerase (Applied Biosystems) and 100ng genomic DNA. The PCR started with an initial denaturation step at 95oC for 7min, followed by 40 cycles of denaturation at 95oC for 30sec, annealing at 60oC for 30sec and extension at 72oC for 1min and with a final extension at 72oC for 7min. Results. As for the Real Time PCR assay, detection and differentiation of Brucella species in genomic DNA isolated from cultures was achieved in all cases. On the contrary, when the Real-time PCR assay was applied on the genomic DNA isolated from the panel of 30 tissues, Brucella species were identified at very low percentage while a high percentage of false positive amplifications was observed. The genomic DNA isolated from the panel of 30 tissues was used for studing the specificity and the sensitivity of the developed multiplex PCR. As expected, in all cases of contaminated samples either by Brucella abortus or by Brucella melitensis specific fragments were amplified. On the contrary, no fragments have been amplified from the negative samples or those contaminated by Brucella suis. I II III Figure 1. Two duplex qPCR reactions performed on genomic DNA isolated directly from spleen tissues and their bacterial cultures, too. The probe for the identification of Brucella spp., is labelled with Cy5 , for Brucella melitensis with VIC and Brucella abortus with FAM. I: sample 7 (ovine –high), II: sample 8 (bovine – high), III: sample 11 (swine – high). A: the duplex qPCR for identification of Brucella melitensis applied on genomic DNA isolated from spleen tissue. B: the duplex qPCR for identification of Brucella melitensis applied on genomic DNA isolated from bacterial culture. C:the duplex qPCR for identification of Brucella abortus applied on genomic DNA isolated from spleen tissue and D: the duplex qPCR for identification of Brucella abortus applied on genomic DNA isolated from bacterial culture. 1I M 1 2 3 4 5 6 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 A B Conclusion. The procedure of the two duplex qPCR, when it was applied in cultures, has proven to be of highly specificity providing better results than the microbiological tests. The results presented here indicate that the developed multiplex PCR assay is a specific and sensitive tool for detection of Brucella spp. strains from solid tissues. For this reason, it could be used as a complementary tool in brucellosis screening programs and for detection of Brucella melitensis and Brucella abortus strains. Figure 2. Multiplex PCR reactions performed on genomic DNA extracted from spleen tissues. The reactions were electrophorised in a 2% agarose gel. M: 100bp DNA ladder (Invitrogen). 1: sample 2 - ovine negative, 2: sample 6 - bovine negative, 3: sample 12 - bovine high, 4: sample 14 - ovine high, 5: sample 24 - bovine low, 6: sample 28 - ovine low. 1: sample 1 - swine negative, 2: sample 2 - ovine negative, 3: sample 6 - bovine negative, 4: sample 12 - bovine high, 5: sample 14 - ovine high, 6: sample 11 - swine high, 7: sample 24 - bovine low, 8: sample 28 - ovine low, 9: sample 5 - swine high, 10: sample 7 - ovine high, 11: sample 20 - bovine high, 12: sample 13 - swine negative, 13: sample 15 - ovine negative, 14: sample 16 -bovine negative, 15: sample 10 - ovine low, 16: sample 18 - bovine low, 17: sample 29 - ovine high, 18: sample 8 -bovine high, 19: sample 21 - bovine negative References Bricker B.J. and Halling S.M. (1994) .Differentation of Brucella abortus bv1,2 and 4, Brucella melitensis, Brucella ovis and Brucella suis bv1 by PCR, J Clin Microbiol, 32, 2660-2666 Imaoka K., Kimura M., Suzuki M., Kamiyama T., Yamada A. (2007) Simultaneous detection of the genus Brucella by combinatorial PCR, Jpn. J. Infect. Dis., 60, 137-139 Gopaul K.K., Koylass M.S., Smith C.J., Whatmore A.M. (2008). Rapid identification of Brucella isolates to the species level by real time PCR based single nucleotide polymorphism (SNP) analysis, BMC Microbiology Probert W.S., Schrader K.N., Khuong N.Y., Bystrom S.L., 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 Scholz H.C., Pfeffer M., Witte A., Neubauer H., Al Dahouk S., Wernery U., Tomaso H. (2008). Specific detection and differentiation of Ochrobactrum anthropi, Ochrobactrum intermedium and Brucella spp. by a multi-primer PCR that targets the recA gene, J Med Microbiol, 57, 64-71 Acknowledgments The work was funded by the General Secretariat for Research & Technology (GSRT) International S&T, Cooperation Directorate – European Union Division – GREECE