Molecular identification of Brucella bacteria in clinical samples of Sistan region by multiplex polymerase chain reaction

Document Type : Original Article

Authors

1 MS.c graduate of genetic, Department of Biology, Faculty of Sciences, University of Zabol, Zabol, Iran

2 Assistant Professor, Department of Biology, Faculty of Sciences, University of Zabol, Zabol, Iran

3 Associated professor, Department of pathobiology, Faculty of veterinary medicine, University of Zabol, Zabol, Iran

Abstract

Brucellosis or malt fever (Mediterranean fever) occurs due to the growth and multiplication of the gram-negative coccobacillus Brucella in mammalian cells, and this disease is important as a common disease between humans and animals. Several serological tests have been investigated to identify this bacterium, however, these methods have limitations in the accurate identification of the bacterium. The present study was conducted with the aim of investigating and molecular identification of Brucella bacteria in samples collected from infected sheep in Sistan region using multiplex PCR technique. At first, blood and milk samples were collected from infected sheep. Confirmation of bacterial infection was initially done using blood samples and Rose Bengal serology method. Next, DNA was extracted from the collected milk and molecular analysis was performed using multiplex PCR and amplification of Omp31 and BLS genes, in order to amplify fragments with sizes of 347 bp and 256 bp respectively. Out of the 40 clinical samples, all of them showed a positive Rose Bengal test, but molecular analysis using multiplex PCR showed 5 false positive samples confirmed with the Rose Bengal test. Due to the higher sensitivity and accuracy and short time of identification the molecular methods can be used as a suitable option for identifying pathogens.

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References

1- Maleknejad P, Peeri-DoGaheh H, AmirZargar A, Jafari S, Fatollahzadeh B. Diagnosis of brucellosis by use of BACTEC blood culture and confirmation by PCR. J Vet Res. 2007; 62(4): 83-6.
2- Adams, L G. The pathology of brucellosis reflects the outcome of the battle between the host genome and the Brucella genome. Veterinary microbiology. 2002; 90(1-4): 553-561.‏
3- Ghasemi A, et al. Immune reactivity of Brucella melitensis-vaccinated rabbit serum with recombinant Omp31 and DnaK proteins. Iranian Journal of Microbiology. 2013; 5: 19–23.
4- Zhang K, Wang H, Guo F, Yuan L, Zhang W, Wang Y, et al. OMP31 of Brucella melitensis 16M impairs the apoptosis of macrophages triggered by TNF-α. Experimental and therapeutic medicine. 2016; 12(4): 2783-2789.
5- Sha T, Li Z, Zhang C, Zhao X, Chen Z, Zhang F, et al. Bioinformatics analysis of candidate proteins Omp2b, P39 and BLS for Brucella multivalent epitope vaccines. Microbial Pathogenesis. 2020; 147: 104318.
6- Talebzadeh S, Zaker Bostanabad S, Nazari R. Study and Comparison of Diagnostic Methods (Culture–Molecular) for Brucella in the Samples Isolated from Patients Suspected to Brucellosis in Different Regions of Tehran. NCMBJ. 2016; 6 (23): 105-110.
7- Zhang H, Fan Q, et al. CD14 gene silencing alters the microRNA expression profile of RAW264.7 cells stimulated by Brucella melitensis infection. Innate Immun. 2017; 23(5): 424–431.
8- Refai, M. Incidence and control of brucellosis in the Near East region. Veterinary microbiology. 2002; 90(1-4): 81-110.
9- Yagupsky, P. Detection of Brucellae in blood cultures. Journal of clinical microbiology. 1999; 37(11): 3437-42.
10- Elfaki, M.G, Uz-Zaman, T., Al-Hokail,A.A. and Nakeeb, S.M. Detection of Brucella DNA in sera from patients with brucellosis by polymerase chain reaction. Diagnostic microbiology and infectious disease. 2005; 53(1): 1-7
11- Gamazo C, Vitas A, Lopez-Goni I, Diaz R, Moriyon I. Factors affecting detection of Brucella melitensis by BACTEC NR730, a nonradiometric system for hemocultures. Journal of clinical microbiology. 1993; 31(12): 3200-3.
12- Queipo-Ortuno M, Garcia-Ordonez, M, Colmenero J, Morata P. Hydrogen peroxide improves the efficiency of a peripheral blood PCR assay for diagnosis of human brucellosis. Biotechniques. 1999; 2 7(2): 248-52.
13- Satei E, Mirshahabi H, Zeighami H, Gholoobi A, Sadeghi H. Molecular survey of BCSP31 and IS711 using PCR assays in detection of Brucella spp. in raw milk. Meta Gene. 2020; 24: 1006-1018.
14- Tantillo G, Di Pinto A, Vergara A, Buonavoglia C. Polymerase chain reaction for the direct detection of Brucella spp. in milk and cheese. Journal of food protection. 2001; 64(2): 164-167.
15- Mahzounieh M, Salimi M. “Short article” Serological and molecular survey on camel brucellosis in Najaf Abad. Biological Journal of Microorganism. 2015; 4(14): 167-174.
New Findings in Veterinary Microbiology
Volume 6, Issue 2
February 2024
Pages 20-27

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History

  • Receive Date: 20 December 2022
  • Revise Date: 18 January 2023
  • Accept Date: 19 April 2023

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