New Brucella abortus S19 Mutant to Improve Distinction Between Infected and Vaccinated Animals

Document Type : Brief Report


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

2 Department of Bioscience and Biotechnology, Malek-Ashtar University of Technology, Tehran, Iran

3 Department of Brucellosis, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran


Background: Using Brucella abortus Strain 19 (S19) to control bovine brucellosis is restricted due to induce antibodies to the O-side chain of the smooth lipopolysaccharide (LPS) which may be difficult to differentiate vaccinated and infected animals. Furthermore, it is virulent for humans and can induce abortion to cattle.
Objectives: The aim of this study was to employ gene knockout B. abortus S19 for the first time to eliminate diagnostic defects and obtain the attenuated mutant strain.
Material and Methods: The wbkA gene, which is one of the LPS O-chain coding genes, was knocked out in vaccinal Brucella abortus S19. The proliferative response and immunoglobulin M production were analyzed in wbkA deletion strain-infected BALB/c mice.
Results: The loss of wbkA gene function resulted in induction of the splenocyte proliferative response in mice infected by the mutant S19 strain compare to those induced by parental S19 and RB51 strains. Moreover, wbkA mutant did not induce any IgM antibody response using the enzyme-linked immunosorbent assay.
Conclusions: As a result, the new mutant S19 strain had deficiency in its LPS O-chain structure, besides cannot induce IgM response then, reduce mistakes to discriminate between vaccinated and infected animal, and also can be considered as a new vaccine candidate.


Main Subjects

1.           Godfroid J, Cloeckaert A, Liautard JP, Kohler S, Fretin D, Walravens K, 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. doi: 10.1051/vetres:2005003 pmid: 15845228
2.           Adams LG. The pathology of brucellosis reflects the outcome of the battle between the host genome and the Brucella genome. Vet Microbiol. 2002;90(1-4):553-561. doi: 10.1016/s0378-1135(02)00235-3 pmid: 12414171
3.           Cardoso PG, Macedo GC, Azevedo V, Oliveira SC. Brucella spp noncanonical LPS: structure, biosynthesis, and interaction with host immune system. Microb Cell Fact. 2006;5:13. doi: 10.1186/1475-2859-5-13 pmid: 16556309
4.           Conde-Alvarez R, Arce-Gorvel V, Iriarte M, Mancek-Keber M, Barquero-Calvo E, Palacios-Chaves L, et al. The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition. PLoS Pathog. 2012;8(5):e1002675. doi: 10.1371/journal.ppat.1002675 pmid: 22589715
5.           Zygmunt MS, Blasco JM, Letesson JJ, Cloeckaert A, Moriyon I. DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers. BMC Microbiol. 2009;9:92. doi: 10.1186/1471-2180-9-92 pmid: 19439075
6.           Olsen SC. Recent developments in livestock and wildlife brucellosis vaccination. Rev Sci Tech. 2013;32(1):207-217. doi: 10.20506/rst.32.1.2201 pmid: 23837378
7.           Crasta OR, Folkerts O, Fei Z, Mane SP, Evans C, Martino-Catt S, et al. Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes. PLoS One. 2008;3(5):e2193. doi: 10.1371/journal.pone.0002193 pmid: 18478107
8.           Zhu J, Larson CB, Ramaker MA, Quandt K, Wendte JM, Ku KP, et al. Characterization of recombinant B. abortus strain RB51SOD toward understanding the uncorrelated innate and adaptive immune responses induced by RB51SOD compared to its parent vaccine strain RB51. Front Cell Infect Microbiol. 2011;1:10. doi: 10.3389/fcimb.2011.00010 pmid: 22919576
9.           Vemulapalli R, He Y, Buccolo LS, Boyle SM, Sriranganathan N, Schurig GG. Complementation of Brucella abortus RB51 with a functional wboA gene results in O-antigen synthesis and enhanced vaccine efficacy but no change in rough phenotype and attenuation. Infect Immun. 2000;68(7):3927-3932. doi: 10.1128/iai.68.7.3927-3932.2000 pmid: 10858205
10.        Caporale V, Bonfini B, Di Giannatale E, Di Provvido A, Forcella S, Giovannini A, et al. Efficacy of Brucella abortus vaccine strain RB51 compared to the reference vaccine Brucella abortus strain 19 in water buffalo. Vet Ital. 2010;46(1):13-19, 15-11. pmid: 20391363
11.        Monreal D, Grillo MJ, Gonzalez D, Marin CM, De Miguel MJ, Lopez-Goni I, et al. Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model. Infect Immun. 2003;71(6):3261-3271. doi: 10.1128/iai.71.6.3261-3271.2003 pmid: 12761107
12.        Nayeri Fasaei B, Zahraei Salehi T, Naserli S, Saeedinia AR, Behroozikhah AM. Site-directed mutagenesis in Brucella abortus S19 by overlap extension PCR-based procedure. Journal of the Hellenic Veterinary Medical Society. 2018;68(3):273. doi: 10.12681/jhvms.15468
13.        Arenas-Gamboa AM, Ficht TA, Kahl-McDonagh MM, Rice-Ficht AC. Immunization with a single dose of a microencapsulated Brucella melitensis mutant enhances protection against wild-type challenge. Infect Immun. 2008;76(6):2448-2455. doi: 10.1128/IAI.00767-07 pmid: 18362129
14.        Barbier T, Nicolas C, Letesson JJ. Brucella adaptation and survival at the crossroad of metabolism and virulence. FEBS Lett. 2011;585(19):2929-2934. doi: 10.1016/j.febslet.2011.08.011 pmid: 21864534
15.        Avila-Calderon ED, Lopez-Merino A, Sriranganathan N, Boyle SM, Contreras-Rodriguez A. A history of the development of Brucella vaccines. Biomed Res Int. 2013;2013:743509. doi: 10.1155/2013/743509 pmid: 23862154
16.        Corbel MJ. Brucellosis in humans and animals. Geneva: World Health Organization; 2006.
17.        Li ZQ, Gui D, Sun ZH, Zhang JB, Zhang WZ, Zhang H, et al. Immunization of BALB/c mice with Brucella abortus 2308DeltawbkA confers protection against wild-type infection. J Vet Sci. 2015;16(4):467-473. doi: 10.4142/jvs.2015.16.4.467 pmid: 26040616
18.        Grillo MJ, Manterola L, de Miguel MJ, Munoz PM, Blasco JM, Moriyon I, et al. Increases of efficacy as vaccine against Brucella abortus infection in mice by simultaneous inoculation with avirulent smooth bvrS/bvrR and rough wbkA mutants. Vaccine. 2006;24(15):2910-2916. doi: 10.1016/j.vaccine.2005.12.038 pmid: 16439039
19.        Lacerda TL, Cardoso PG, Augusto de Almeida L, Camargo IL, Afonso DA, Trant CC, et al. Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice. Vaccine. 2010;28(34):5627-5634. doi: 10.1016/j.vaccine.2010.06.023 pmid: 20580469
20.        Lalsiamthara J, Gogia N, Goswami TK, Singh RK, Chaudhuri P. Intermediate rough Brucella abortus S19Deltaper mutant is DIVA enable, safe to pregnant guinea pigs and confers protection to mice. Vaccine. 2015;33(22):2577-2583. doi: 10.1016/j.vaccine.20 15.04.004 pmid: 25869887