Evaluation of the Analytical Efficiency of Real-Time PCR in the Diagnosis of Brucellosis in Cattle and Sheep

Brucellosis is an important infectious disease that aff ects animal and public health in many developing countries, including Turkey. The control and eradication of brucellosis are contingent upon methods that provide a fast and reliable diagnosis. In this context, molecular methods which enable the enzymatic amplification of bacterial conserved gene regions are advantageous. In this study, samples of cattle and sheep blood serum sent to our laboratory from enterprises at risk of brucellosis in diff erent settlements of the Central Anatolia and the Black Sea Regions of Turkey were analyzed. In our study, we aimed to investigate brucellosis using serological methods and Real-Time PCR (RT-PCR), and to analyze these methods comparatively. To this end, RBPT, SAT and CFT tests, as well as the Brucella RT-PCR, which enables the amplification of the BCSP31 gene found in all Brucella species, were used. In the 368 serum samples analyzed, Brucella positivity was determined as 11.41%, 10.05% and 9.8% by RBPT, SAT and CFT, respectively, while the RT-PCR gave the same rate of positivity (9.8%) as CFT. When CFT is taken as a reference test, the sensitivity, specificity, positive and negative predictive value and diagnostic accuracy of the RTPCR were determined as 100%; and it was found to be perfectly compatible with CFT, with a kappa value of 1.000. The number of bacterial genomes that could be detected by the RT-PCR in the presence of DNA of the Brucella melitensis biotype 3 (Ether) reference strain was determined as 3.94x103 copies. Linear regression analysis revealed that the amplification efficiency (92.71%) of the RT-PCR was within the desired limits (90-110%) and that the RT-PCR was repeatable (CV 2.9%) and reproducible (CV 1.8%). This study indicates that the RT-PCR is a useful method for application in the diagnosis of bovine and sheep brucellosis thanks to its high analytical efficiency; it also emphasizes the importance of blood serum samples as preferable clinical materials in this context.

Sığır ve Koyunlarda Brusellozisin Tanısında Real-Time PCR’nin Analitik Yeterliliğinin Değerlendirilmesi

Brusellozis, ülkemizle birlikte gelişmekte olan birçok ülkede hayvanlarda ve halk sağlığı açısından önem arzeden infeksiyöz bir hastalıktır. Brusellozisin, kontrol ve eradikasyonunda hızlı ve güvenilir teşhis imkânı sunan yöntemlere her zaman ihtiyaç duyulmaktadır. Bu kapsamda, bakterilerin korunaklı gen bölgelerinin enzimatik amplifikasyonunu sağlayan moleküler yöntemler avantaj sağlamaktadır. Bu çalışmada, İç Anadolu ve Karadeniz Bölgelerine ait farklı yerleşim birimlerindeki brusellozis riski taşıyan işletmelerden laboratuvarımıza gönderilen sığır ve koyunlara ait kan serum örnekleri incelenmiştir. Çalışmamızda, brusellozisin serolojik yöntemler ve Real-Time PCR (RT-PCR) ile araştırılması ve bu yöntemlerin karşılaştırmalı analizi amaçlanmıştır. Bu amaçla, RBPT, SAT ve KFT testleri ile birlikte tüm Brucella türlerinde bulunan BCSP31 geninin amplifikasyonunu sağlayan Brusella RT-PCR metodu kullanılmıştır. İncelenen 368 serum örneğinde RBPT, SAT ve KFT ile Brucella pozitifl iği sırayla %11.41, %10.05 ve %9.8 belirlenirken, RT-PCR analizi sonucu, KFT ile aynı oranda (%9.8) pozitifl ik elde edilmiştir. KFT referans test olarak dikkate alındığında, RT-PCR’nin sensitivite, spesifite, pozitif ve negatif prediktif değerleri ve tanı doğruluğu %100 ve 1.000’lık kappa değeri ile KFT ile mükemmel derecede uyumlu olduğu saptanmıştır. Brucella melitensis biyotip 3 (Ether) referans suşuna ait DNA eşliğinde RT-PCR ile saptanabilen bakteriyel genom sayısı 3.94x103 kopya olarak belirlenmiştir. Gerçekleştirilen linear regresyon analizi ile RTPCR’nin amplifikasyon verimliliği, istenen sınırlar (%90-110) arasında (%92.71) yer almış ve tekrarlanabilir (CV %2.9) ve üretilebilir (CV %1.8) olduğu belirlenmiştir. Bu çalışma ile yüksek analitik verimliliği sayesinde RT-PCR’nin sığır ve koyun brusellozisin teşhisinde başvurulabilir bir yöntem olduğu öngörülmüş ve bu kapsamda kan serum örneklerinin tercih edilebilir klinik materyaller olarak önemi vurgulanmıştır.

PDF

___

1. Büyük F, Şahin M: Kars yöresinde atık yapan ineklerin çeşitli örneklerinden Brucella etkenlerinin kültürel ve moleküler yöntemlerle araştırılması ve olguların epidemiyolojik analizi. Kafkas Univ Vet Fak Derg, 17, 809-816, 2011. DOI: 10.9775/kvfd.2011.4509

2. Godfroid J: Brucellosis in livestock and wildlife: Zoonotic diseases without pandemic potential in need of innovative one health approaches. Arch Public Health, 75:34, 2017. DOI: 10.1186/s13690-017-0207-7

3. OIE Terrestial Manual: Chapter 3.1.4. Brucellosis (Brucella abortus, B. melitensis and B. suis) (Infection with B. abortus, B. melitensis and B. suis), 355-398, 2018. https://www.oie.int/fileadmin/Home/eng/Health_ standards/tahm/3.01.04_BRUCELLOSIS.pdf, Accessed: 03.01.2021.

4. Sidamonidze K, Su W, Zhgenti E, Buyuk F, Sahin M, Trapaidze N, Imnadze P, Nikolich MJ, Obiso R, Kotorashvili A: Molecular typing of Brucella species strains from Georgia and Turkey. J Bacteriol Mycol, 4 (3): 1054, 2017.

5. 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, 81, 170-176, 2006. DOI: 10.1016/j.rvsc.2005.12.001

6. Bounaadja L, Albert D, Chénais B, Hénault S, Zygmunt MS, Poliak S, Garin-Bastuji B: Real-time PCR for identification of Brucella spp.: A comparative study of IS711, bcsp31 and per target genes. Vet Microbiol, 137, 156-164, 2009. DOI: 10.1016/j.vetmic.2008.12.023

7. Probert WS, Schrader KN, Khuong NY, Bystrom SL, Graves MH: Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J Clin Microbiol, 42, 1290-1293, 2004. DOI: 10.1128/ JCM.42.3.1290-1293.2004

8. Staroscik A: Calculator for determining the number of copies of a template. URI Genomics & Sequencing Center 2004. http://cels.uri.edu/ gsc/cndna.html; Accessed: 01.02.2021.

9. Del Vecchio VG, Kapalral V, Redkar RJ, Patra G, Mujer C, Los T, Ivanova N, Anderson I, Bhattacharyya A, Lykidis A, Reznik G, Jablonski L, Larsen N, D’Souza M, Bernal A, Mazur M, Goltsman E, Selkov E, Elzer PH, Hagius S, O’Callaghan D, Letesson JJ, Haselkorn R, Kyrpides N, Overbeek R: The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci USA, 99, 443- 448, 2002. DOI:10.1073/pnas.221575398

10. Alton GG, Jones LM, Angus RD, Verger JM: Serological methods. Techniques for the Brucellosis laboratory. pp.123-130, INRA, Paris, France, 1988.

11. Republic of Turkey, Official Newspaper: Brusellosis Regulation, Issue: 27189, 2009. https://www.resmigazete.gov.tr/eskiler/2009/04/ 20090403-6.htm; Accessed: 01.02.2021.

12. MEDCALC- Easy to Use Statistical Software: Free statistical calculators, 2021. https://www.medcalc.org/calc/diagnostic_test.php; Accessed: 02.02.2021.

13. NEBio Calculator: qPCR Library Quantification. Version 1.12.0. New England Biolabs, 2021. https://nebiocalculator.neb.com/#!/qPCRlibQnt; Accessed: 02.02.2021.

14. EasyCalculation: Coefficient of Variation Calculator, 2021. https:// www.easycalculation.com/statistics/coefficient-of-variance.php; Accessed: 01.02.2021.

15. GraphPad: QuickCalcs, 2021. https://www.graphpad.com/quickcalcs/ chisquared1/; Accessed: 02.02.2021.

16. Al Dahouk S, Fleche PL, Nockler K, Jacques I, Grayon M, Scholz HC, Tomaso H, Vergnaud G, Neubauer H: Evaluation of Brucella MLVA typing for human Brucellosis. J Microbiol Methods, 69, 137-145, 2007. DOI: 10.1016/j.mimet.2006.12.015

17. Sidor IF, Dunn JL, Tsongalis GJ, Carlson J, Frasca SR: A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood and feaces from marine mammals. J Vet Diagn Invest, 25, 72-81, 2013. DOI: 10.1177/ 1040638712470945

18. D’haene B, Vandesompele J, Hellemans J: Accurate and objective copy number profiling using real-time quantitative PCR. Methods, 50, 262-270, 2010. DOI: 10.1016/j.ymeth.2009.12.007

19. Bio Rad: Real-Time PCR Applications Guide. Bio Rad Laboratories Inc., 2006. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/ Bulletin_5279.pdf; Accessed: 01.02.2021.

20. Debeaumont C, Falconnet PA, Maurin M: Real-time PCR for detection of Brucella spp. DNA in human serum samples. Eur J Clin Microbiol Infect Dis, 24, 842-845, 2005. DOI: 10.1007/s10096-005-0064-0

21. Mukherjee F, Nagmani K, Surendra KSNL, Subramanian BM, Bahekar VS, Prasad A, Rana SK, Muthappa PN, Sharma GK, Srinivasan VA: Optimization and validation of a diagnostic real-time PCR for bovine brucellosis. Adv Anim Vet Sci, 3, 577-587, 2015. DOI: 10.14737/journal. aavs/2015/3.11.577.587

22. Awwad E, Farraj M, Essawi T, Sabri I, Adwan K, Rumi I, Manasra A, Baraitareanu S, Gurau MR, Danes D: Validation of RT-qPCR technique for detection of Brucella genome in milk sheep and goat in West Bank Part of Palestine. Sci Bull Series F Biotechnologies, 20, 321-328, 2016.

23. Dal T, Kara SS, Cikman A, Balkan ÇE, Acıkgoz ZC, Zeybek H, Uslu H, Durmaz R: Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human brucellosis. J Infect Public Health, 12 (3): 337-342, 2019. DOI: 10.1016/j.jiph. 2018.11.008

24. Pappas G, Papadimitriou P: Challenges in Brucella bacteraemia. Int J Antimicrob Agents, 30 (Suppl. 1), S29-S31, 2007. DOI: 10.1016/j. ijantimicag.2007.06.011

25. Zerva L, Bourantas K, Mitka S, Kansouzidou A, Legakis NJ: Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR. J Clin Microbiol, 39, 1661-1664, 2001. DOI: 10.1128/JCM.39.4.1661- 1664.2001

26. Khan MZ, Zahoor M: An overview of Brucellosis in cattle and humans, and its serological and molecular diagnosis in control strategies. Trop Med Infect Dis, 3(2):65, 2018. DOI: 10.3390/tropicalmed3020065

27. Günaydın E, Küçükayan U, Tosun T, Ülker U, Müştak K: VKMAE 2007-2011 sığır Bruselloz seroloji verileri. Etlik Vet Mikrobiyol Derg, 26, 1-6, 2015. DOI: 10.35864/evmd.513341