Veterinary Research Forum
Vet Res Forum

Genetic diversity of Brucella melitensis isolates from sheep and goat milk in Iran

Document Type : Original Article

Authors

Heidar Rahimi 1
Amir Tukmechi 1
Ehsan Rashidian 2

1 Department of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

2 Department of Microbiology, Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Iran

https://doi.org/10.30466/vrf.2023.1988859.3768
Abstract
The genetic diversity of Brucella strains has not been fully understood. To investigate this, the genetic characteristics of 64 isolates of Brucella melitensis from sheep and goats’ milk were studied using random fragment length polymorphism (RFLP) and multiple locus variable-number tandem repeat analysis (MLVA-16) methods developed in Orsay, France (MLVA-16Orsay). The RFLP analysis revealed that all 64 isolates were of biovar one. The MLVA-typing showed that one sample was simultaneously infected with two strains of B. melitensis and the genotype of 65 isolate was analyzed. Four genotypes (47, 42, 43, and 63) were identified using MLVA-8 (panel 1), whereas six genotypes (138, 125, 116, 108, and two unknown genotypes) were identified using MLVA11 (panels 1 and 2A). From the review of MLVA-16 (panels 1, 2A, and 2B), panel 2B showed a very high discriminatory power. Two loci of Bruc04 and Bruc30 from this panel had diversity index values higher than 0.71 and the average diversity index was 0.619. So MLVA-16Orsay 34 showed the genotype indicating a low genetic homogeneity among the isolates. The findings of MLVA genotyping of the isolates suggest that strains of B. melitensis isolated from the milk of small ruminants in Iran are most closely related to the isolates from neighboring countries of the Eastern Mediterranean group. To the best of our knowledge, this is the first study to indicate the potential use of MLVA genotyping for simultaneous detection of specimen contamination using two different B. melitensis biovars.

Keywords

Brucella melitensis
Goat
Milk
MLVA
Sheep

Subjects

  1. Moreno E, Cloeckaert A, Moriyón I. Brucella evolution and taxonomy. Vet Microbiol 2002; 90(1-4): 209-227.
  2. Godfroid J, Cloeckaert A, Liautard JP, et al. From the discovery of the Malta fever’s agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Vet Res 2005; 36(3): 313-326.
  3. Corbel MJ. Brucellosis: an overview. Emerg Infect Dis 1997; 3(2): 213-221.
  4. Seleem MN, Boyle SM, Sriranganathan N. Brucellosis: a re-emerging zoonosis. Vet Microbiol 2010; 140(3-4): 392-398.
  5. Blasco JM, Molina-Flores B. Control and eradication of Brucella melitensis infection in sheep and goats. Vet Clin North Am Food Anim Pract 2011; 27(1): 95-104.
  6. Dadar M, Brangsch H, Alamian A, et al. Whole-genome sequencing for genetic diversity analysis of Iranian Brucella isolated from humans and livestock. One Health 2023; 16: 100483. doi: 10.1016/j.onehlt. 2023.100483.
  7. Zowghi E, Ebadi A, Yarahmadi M. Isolation and identification of Brucella organisms in Iran. Arch Clin Infect Dis 2008; 3(4): 185-188.
  8. Behroozikhah AM, Bagheri Nejad R, Amiri K, et al. Identification at biovar level of Brucella isolates causing abortion in small ruminants of Iran. J Pathog 2012; 2012: 357235. doi: 10.1155/2012/357235.
  9. Ashrafganjooyi SH, Saedadeli N, Alamian S, et al. Isolation and biotyping of Brucella from sheep and goats raw milk in southeastern Iran. Trop Biomed 2017; 34(3): 507-511.
  10. Golshani M, Buozari S. A review of brucellosis in Iran: epidemiology, risk factors, diagnosis, control, and prevention. Iran Biomed J 2010; 21(6): 349-359.
  11. Ebrahimi M, BagheriNejad R, Alamian S, et Safety and efficacy of reduced doses of Brucella melitensis strain Rev.1 vaccine in pregnant Iranian fat-tailed ewes. Vet Ital 2012; 48(4): 405-412.
  12. Blasco JM. Existing and future vaccines against brucellosis in small ruminants. Small Rumin Res 2006; 62(1): 33-37.
  13. Bahmani N, Hashemi SH, Arabestani MR, et al. Molecular typing of Brucella species isolated from humans and animals using polymerase chain reaction-restriction fragment length polymorphism technique. Arch Clin Infect Dis 2018; 13(2): e59305. doi: 10.5812/archcid.59305.
  14. Dadar M, Alamian S, Behrozikhah AM, et al. Molecular identification of Brucellaspecies and biovars associated with animal and human infection in Iran. Vet Res Forum 2019; 10(4): 315-321.
  15. Cloeckaert A, Verger JM, Grayon M, et al. Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella. Microbiology (Reading) 2015; 141(Pt 9): 2111-2121.
  16. Le Flèche P, Jacques I, Grayon M, et al. Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol 2006; 6: 9. doi: 1186/1471-2180-6-9.
  17. Al Dahouk S, Flèche PL, Nöckler K, et al. Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Methods 2007; 69(1): 137-145.
  18. Marianelli C, Graziani C, Santangelo C, et al. Molecular epidemiological and antibiotic susceptibility characterization of Brucella isolates from humans in Sicily, Italy. J Clin Microbiol 2007; 45(9): 2923-2928.
  19. Kang SI, Her M, Erdenebaataar J, et al. Molecular epidemiological investigation of Brucella melitensis circulating in Mongolia by MLVA16. Comp Immunol Microbiol Infect Dis 2017; 50: 16-22.
  20. Shevtsova E,Vergnaud G, Shevtsov A, et al. Genetic diversity of Brucella melitensis in Kazakhstan in relation to world-wide diversity. Front Microbiol 2019; 10: 1897. doi: 10.3389/fmicb.2019.01897.
  21. Alvarez LP, Ruiz-Villalobos N, Suárez-Esquivel M, et al. Molecular characterization of Brucella ovis in Argentina. Vet Microbiol 2020; 245: 108703. doi: 10.1016/j.vetmic.2020.108703.
  22. Pelerito A, Nunes A, Grilo T, et al. Genetic characterization of Brucella whole genome sequencing-based approach for the determination of multiple locus variable number tandem repeat profiles. Front Microbiol 2021; 12: 740068. doi: 10.3389/ fmicb.2021.740068.
  23. Georgi E, Walter MC, Pfalzgraf MT, et al. Whole genome sequencing of Brucella melitensis isolated from 57 patients in Germany reveals high diversity in strains from Middle East. PLoS One 2017; 12(4): e0175425. doi: 10.1371/journal.pone.0175425.
  24. Moradkasani S, Jazi FM, Sadeghifard N, et al. Multiple-locus variable-number tandem-repeat analysis genotyping of Brucella isolates from Iran. Clin Lab 2020; 66(10). doi: 10.7754/Clin.Lab.2020.200119.
  25. Mirkalantari S, Masjedian F, Fateme A. Determination of investigation of the link between human and animal Brucella isolates in Iran using multiple-locus variable number tandem repeat method comprising 16 loci (MLVA-16). Braz J Infect Dis 2021; 25(1): 101043. doi: 10.1016/j.bjid.2020.11.008.
  26. Alton GG. Control of Brucella melitensis infection in sheep and goats - - a review. Trop Anim Health Prod 1987; 19(2): 65-74.
  27. Baily GG, Krahn JB, Drasar BS, et al. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 1992; 95(4): 271-275.
  28. Bricker BJ, Halling SM. Differentiation of Brucella abortus 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin Microbiol 1994; 32(11): 2660-2666.
  29. Hunter PR, Gaston MA. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol 1988; 26(11): 2465-2466.
  30. Rahimi H, Tukmechi A, Rashidian E. Use of touch-down polymerase chain reaction to enhance the sensitivity of Brucella melitensis detection in raw milk. Anim Biotechnol 2022; 33(1): 104-109.
  31. Kattar MM, Jaafar RF, Araj GF, et al. Evaluation of a multilocus variable-number tandem-repeat analysis scheme for typing human Brucella isolates in a region of brucellosis endemicity. J Clin Microbiol 2008; 46(12): 3935-3940.
  32. Mirnejad R, Mohammadi M, Majdi A, et al. Molecular typing of Brucella melitensis and abortus from human blood samples using PCR-RFLP method. Jundishapur J Microbiol 2013; 6(6): e7197. doi: 10.5812/jjm.7197.
  33. Pishva E, Salehi R, Hoseini A, et al. Molecular typing of Brucella species isolates from human and livestock bloods in Isfahan province. Adv Biomed Res 2015; 4: 104. doi: 10.4103/2277-9175.157798.
  34. García-Yoldi D, Le Fleche P, Marín CM, et al. Assessment of genetic stability of Brucella melitensis Rev 1 vaccine strain by multiple-locus variable-number tandem repeat analysis. Vaccine 2007; 25(15): 2858-2862.
  35. Ferreira AC, Chambel L, Tenreiro T, et MLVA16 typing of Portuguese human and animal Brucella melitensis and Brucella abortus isolates. PLoS One 2012; 7(8): e42514. doi: 10.1371/journal.pone.0042514.
  36. Vergnaud G, Hauck Y, Christiany D, et al. Genotypic expansion within the population structure of classical Brucella species revealed by MLVA16 typing of 1404 Brucella isolates from different animal and geographic origins, 1974–2006. Front Microbiol 2018; 9: 1545. doi: 10.3389/fmicb.2018.01545.
  37. Kiliç S, Ivanov IN, Durmaz R, et al. Multiple-locus variable-number tandem-repeat analysis genotyping of human Brucella isolates from Turkey. J Clin Microbiol 2011; 49(9): 3276-3283.
  38. Bodur H, Balaban N, Aksaray S, et al. Biotypes and antimicrobial susceptibilities of Brucella isolates. Scand J Infect Dis 2003; 35(5): 337-338.
  39. Jiang H, Fan M, Chen J, et al. MLVA genotyping of Chinese human Brucella melitensis biovar 1, 2 and 3 isolates. BMC Microbiol 2011; 11: 256. doi: 10.1186/ 1471-2180-11-256.
Veterinary Research Forum
Volume 14, Issue 12
December 2023
Pages 649-657

  • Receive Date 31 January 2023
  • Revise Date 25 April 2023
  • Accept Date 20 May 2023

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