Iranian Veterinary Journal

Iranian Veterinary Journal

Evaluation of the cross-reactive antibodies among Brucella strains: validation of serological detection tests of Brucellosis

Document Type : Research Paper

Authors
Farangis Nargesi 1
Mohammad Khosravi 2
Darioush Gharibi 3
Mahdi Pourmahdi Borujeni 4
Masoud Ghorbanpoor 5
1 PhD Student in Bacteriology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
2 Associate Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
3 Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
4 Professor, Department of Food hygiene, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran
5 Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran
10.22055/ivj.2025.531580.2809
Abstract
    The present study aimed to investigate the antibody cross-reactions in sheep serum against vaccine strains and pathogenic strains of Brucella, as well as validation of the agglutination results with ELISA using both vaccine and wild strains of Brucella. The used vaccine and wild strains were RB51, Rev1, B. melitensis, and B. abortus, all of which were confirmed by PCR test. A total of 81 sheep serum samples were included in the study, encompassing both positive and negative reactions in the agglutination tests. Following the characterization of the sera, indirect ELISA tests utilizing the lipopolysaccharide of the bacteria were employed to validate the agglutination results and to statistically assess the serum cross-reactions against the vaccine and wild strains of Brucella. Out of 81 evaluated suspected serum samples, the Rose-Bengal test yielded 61 positive and 20 negative results. The Wright and Wright-2ME tests revealed 38 and 36 positive, and 43 and 45 negative results, respectively. According to the ROC curve analysis, the highest area under the curve was 84% for IRIBA, 75% for Rev1, 70% for B. melitensis, and 68% for B. abortus. Statistical analysis indicated that the strain of antigen in ELISA tests had a significant effect on S/P values. The mean and standard error of the S/P values were as Rev1 vaccine 0.54 ± 0.03, B. melitensis 0.82 ± 0.02, IRIBA vaccine 0.82 ± 0.04, and B. abortus 0.98 ± 0.04. The highest sensitivity and specificity were achieved using B. melitensis and IRIBA antigens, respectively. The indirect ELISA utilizing both vaccine and wild strains of Brucella demonstrated appropriate sensitivity and specificity, suggesting its cross-reactions and potential as a reliable diagnostic method for vaccinated and infected sheep.
Keywords
Brucella
Sheep
Vaccine
Cross-reaction
ELISA

Subjects

Abdelgawad, H. A., Lian, Z., Yin, Y., Fang, T., Tian, M., & Yu, S. (2023). Characterization of Brucella abortus Mutant A19mut2, a Potential DIVA Vaccine Candidate with a Modification on Lipopolysaccharide. Vaccines, 11(7), 1273.
Banyasz, B., Antal, J., & Denes, B. (2025). Ghostbuster—A Western Blot-Based Panel Method to Resolve False-Positive Brucellosis Serology Test Results. Microorganisms, 13(3), 574.
Berhanu, G., & Pal, M. (2020). Brucellosis: A highly infectious zoonosis of public health and economic importance. Journal of Emerging Environmental Technologies and Health Protection, 3, 5-9.‏
Cai, G. H., Guo, C. Y., Guo, K. X., Zhang, J. D., Chen, H. C., & Liu, Z. F. (2025). Development of a competitive ELISA based on Brucella neotomae lipopolysaccharide for detecting brucellosis in livestock. Analytical Biochemistry, 115880.‏
 Copin, R., Vitry, M. A., Hanot Mambres, D., Machelart, A., De Trez, C., Vanderwinden, J. M., ... & Muraille, E. (2012). In situ microscopy analysis reveals local innate immune response developed around Brucella infected cells in resistant and susceptible mice. PLoS pathogens, 8(3), e1002575.‏
Daro, B. S., Deneke, Y. A., & Negasa, J. G. (2024). Bovine Brucellosis: A Zoonotic Disease of Public Health and Economic Concern in Ethiopia. Journal of Clinical Case Studies Reviews & Reports, 2 (2), 1-8.
Davis Jr, M. R., & Goldberg, J. B. (2012). Purification and visualization of lipopolysaccharide from Gram-negative bacteria by hot aqueous-phenol extraction. Journal of visualized experiments: JoVE, (63), 3916.‏
Dorneles, E. M., Lima, G. K., Teixeira-Carvalho, A., Araújo, M. S., Martins-Filho, O. A., Sriranganathan, N., ... & Lage, A. P. (2015). Immune response of calves vaccinated with Brucella abortus S19 or RB51 and revaccinated with RB51. PloS one, 10(9), e0136696.
Falenski, A., Mayer-Scholl, A., Filter, M., Göllner, C., Appel, B., & Nöckler, K. (2011). Survival of Brucella spp. in mineral water, milk and yogurt. International Journal of Food Microbiology, 145(1), 326-330.‏
Galinska, E. M., & Zagórski, J. (2013). Brucellosis in humans-etiology, diagnostics, clinical forms. Annals of agricultural and environmental medicine, 20(2), 233-238.
Godfroid, J., Nielsen, K., & Saegerman, C. (2010). Diagnosis of brucellosis in livestock and wildlife. Croatian medical journal, 51(4), 296-305.‏
Golding, B., Scott, D. E., Scharf, O., Huang, L. Y., Zaitseva, M., Lapham, C., ... & Golding, H. (2001). Immunity and protection against Brucella abortus. Microbes and infection, 3(1), 43-48.
Golshani, M., & Buozari, S. (2017). A review of brucellosis in Iran: epidemiology, risk factors, diagnosis, control, and prevention. Iranian biomedical journal, 21(6), 349.‏
Haine, V., Sinon, A., Van Steen, F., Rousseau, S., Dozot, M., Lestrate, P., ... & De Bolle, X. (2005). Systematic Targeted Mutagenesis of Brucella melitensis 16M Reveals a Major Role for GntR Regulators in the Control of Virulence. Infection and immunity, 73(9), 5578-5586.‏
Heidary, M., Dashtbin, S., Ghanavati, R., Mahdizade Ari, M., Bostanghadiri, N., Darbandi, A., ... & Talebi, M. (2022). Evaluation of brucellosis vaccines: a comprehensive review. Frontiers in veterinary science, 9, 925773.
Haro, A. D. G., Samaniego, M. Y. B., Moreno-Caballeros, P., Burbano-Enríquez, A., Torres, V. A. S., Mulki, M. C. G., ... & Saegerman, C. (2025). Bayesian Estimation of the True Bovine Brucellosis Prevalence in Vaccinated and Non-Vaccinated Ecuadorian Cattle Populations, and the Sensitivity and Specificity of a Competitive and Indirect ELISA Using a New Synthetic Antigen. Microorganisms, 13(1), 69.‏
Ibarra, M., Campos, M., Hernan, B., Loor-Giler, A., Chamorro, A., & Nuñez, L. (2023). Comparison of diagnostic tests for detecting bovine brucellosis in animals vaccinated with S19 and RB51 strain vaccines. Veterinary World, 16(10), 2080.‏
Ibarra, M., Campos, M., Ibarra, C., Gladys, U., Huera, D., Gutierrez, M., Nunez, L. (2023). Financial losses associated with bovine brucellosis (Brucella abortus) in carchi-ecuador. Open Journal of Animal Sciences, 13(2), 205-216.
Idrees, H., Abbas, J., Raza, A., Qamar, M. H., Waheed, M. A., Arshad, M. B., ... & Raheem, A. (2024). A systematic overview of bovine brucellosis and its implications for public health: Bovine Brucellosis: A Zoonotic Threat. Letters In Animal Biology, 04(1), 01-09.
Islam, M. S., Habib, M. A., Tonu, N. S., Haque, M. S., & Rahman, M. M. (2025). Beyond Serology: A Meta‐Analysis of Advancements in Molecular Detection of Brucella spp. in Seronegative Animals and Biological Samples. Veterinary Medicine and Science, 11(1), e70200.
Jiao, H., Zhou, Z., Li, B., Xiao, Y., Li, M., Zeng, H., ... & Gu, G. (2021). The mechanism of facultative intracellular parasitism of Brucella. International journal of molecular sciences, 22(7), 3673.
Keyvanfar, A., Sali, S., & Zamani, A. (2021). Epidemiology, clinical and laboratory manifestations, and outcomes of brucellosis among 104 patients in referral hospitals of Tehran, Iran. Archives of Clinical Infectious Diseases, 16(2), e111546.‏
Khosravi, Mohammad. Ghorbanpour, Masoud. Fatemi Tabatabaei, Seyed Reza. Mohammad Alipour, Yasman. (2018). The effect of oral administration of betaine on the humoral immune response of mice to Brucella abortus. Iranian Veterinary Journal, 14(1), 2018.
Kiros, A., Asgedom, H., & Abdi, R. D. (2016). A review on bovine brucellosis: Epidemiology, diagnosis and control options. ARC Journal of Animal and Veterinary Sciences (AJAVS), 2(3), 8-21.‏
Lalsiamthara, J., Gogia, N., Goswami, T. K., Singh, R. K., & Chaudhuri, P. (2015). Intermediate rough Brucella abortus S19Δper mutant is DIVA enable, safe to pregnant guinea pigs and confers protection to mice. Vaccine, 33(22), 2577-2583.‏
Legesse, A., Mekuriaw, A., Gelaye, E., Abayneh, T., Getachew, B., Weldemedhin, W., Tesgera, T., Deresse, G. and Birhanu, K. (2023). Comparative evaluation of RBPT, I-ELISA, and CFT for the diagnosis of brucellosis and PCR detection of Brucella species from Ethiopian sheep, goats, and cattle sera. BMC microbiology, 23(1), 216.
Li, Z. Q., Shi, J. X., Fu, W. D., Zhang, Y., Zhang, J., Wang, Z., ... & Zhang, H. (2015). A Brucella melitensis M5-90 wboA deletion strain is attenuated and enhances vaccine efficacy. Molecular Immunology, 66(2), 276-283.‏
Lopez-Goni, I., Garcia-Yoldi, D., Marin, C. M., De Miguel, M. J., Munoz, P. M., Blasco, J. M., & Garin-Bastuji, B. (2008). Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. Journal of clinical microbiology, 46(10), 3484-3487.
Markey, B., Leonard, F., Archambault, M., Culliname, A., and Maguire, D. (2013). Brucella species. Clinical Veterinary Microbiology. 2nd Ed. Elsevier, Toronto, Ontario, 325-334.
Martirosyan, A., & Gorvel, J. P. (2013). Brucella evasion of adaptive immunity. Future microbiology, 8(2), 147-154.‏
Pal, M., Gizaw, F., Fekadu, G., Alemayehu, G., & Kandi, V. (2017). Public health and economic importance of bovine Brucellosis: an overview. Am J Epidemiol, 5(2), 27-34.‏
Pandya, A. J., & Ghodke, K. M. (2007). Goat and sheep milk products other than cheeses and yoghurt. Small Ruminant Research, 68(1-2), 193-206.‏
Perez, R. O., Mata, R. A., & Dominguez, G. A. (2024). Retrospective Brucellosis Study in Mexico. International Journal of Veterinary Medicine, 3(1), 1-6.
Poester, F. P., Gonçalves, V. S., Paixao, T. A., Santos, R. L., Olsen, S. C., Schurig, G. G., & Lage, A. P. (2006). Efficacy of strain RB51 vaccine in heifers against experimental brucellosis. Vaccine, 24(25), 5327-5334.‏
Poester, F. P., Nielsen, K., Samartino, L. E., & Yu, W. L. (2010). Diagnosis of brucellosis. Open Veterinary Science Journal, 4(1), 46-60.‏
Priyantha, M. A. R. (2021). An overview of human brucellosis: a neglected zoonotic disease in livestock. Sri Lanka Veterinary Journal, 68(1-2), 27-37.
Rehman, S., Ullah, S., Kholik, K., Munawaroh, M., Sukri, A., Malik, M. I. U., ... & Sucipto, T. H. (2025). A detailed review of bovine brucellosis. Open Veterinary Journal, 15(4), 1520-1520.‏
Sadaqat, M. H. (2024). Pathogenesis, clinical manifestations, treatment and prevention of brucellosis in humans and animals. Afghanistan Journal of Basic Medical Science, 1(1), 85-90.‏
Sadeghifard, N., & Pakzad, R. (2023). Investigation of epidemiological features of brucellosis in Iran: A systematic review and meta-analysis study. Technology and Research Information System, 5(4), 0-0.
Smirnova, E. A., Vasin, A. V., Sandybaev, N. T., Klotchenko, S. A., Plotnikova, M. A., Chervyakova, O. V., ... & Kiselev, O. I. (2013). Current methods of human and animal brucellosis diagnostics. Advances in Infectious Diseases, 3(3), 177-184.‏
Sofian, M., Aghakhani, A., Velayati, A. A., Banifazl, M., Eslamifar, A., & Ramezani, A. (2008). Risk factors for human brucellosis in Iran: a case–control study. International journal of infectious diseases, 12(2), 157-161.‏
Stranahan, L. W., & Arenas-Gamboa, A. M. (2021). When the going gets rough: The significance of Brucella lipopolysaccharide phenotype in host–pathogen interactions. Frontiers in Microbiology, 12, 713157.‏
Whatmore, A. M. (2009). Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infection, Genetics and Evolution, 9(6), 1168-1184.‏
World Organization for Animal Health (OIE), (2021) Chapter 2.4.3; Brucellosis (Brucella abortus, B. melitensis and B. suis) (infection with B. abortus, B. melitensis and B. suis) (version adopted in May 2016). In: manual of diagnostic tests and vaccines for terrestrial animals 2021, 355-398.
Yaeger, M. J., Holler, L. D. (2007). Bacterial causes of bovine infertility and abortion. In Current therapy in large animal theriogenology (2nd ed) Chapter 49, Elsevier, St. Louis, 389-399.
Yang, Z., Chai, Z., Wang, X., Zhang, Z., Zhang, F., Kang, F., ... & Yue, J. (2024). Comparative genomic analysis provides insights into the genetic diversity and pathogenicity of the genus Brucella. Frontiers in Microbiology, 15, 1389859.‏
Yousefinejad, S., Khosravi, M., Gharibi, D., Mayahi, M., & Seyfi Abad Shapouri, M. R. (2024). Tracking the antibody level against Ornithobacterium rhinotracheal in the serum of slaughtered turkeys in Khuzestan province using In-house ELISA. Iranian Veterinary Journal, 20(2), 128-138.
Zhang, H., et al. (2025). Establishment and application of a qPCR method for differential detection of Brucella S2 vaccine strain. BMC Veterinary Research, 21(1), 238.