Original Scientific Article
Effects of lyophilization on diagnostic potential of Brucella antigens
Lubna F. Farahat ,
Waleed S. Shell ,
Marwa S. Diab ,
Azza M. Mohamed ,
Maha A. Elshazly *

Mac Vet Rev 2026; 49 (1): i - xii

10.2478/macvetrev-2026-0016

Received: 30 January 2025

Received in revised form: 24 November 2025

Accepted: 18 December 2025

Available Online First: 06 March 2026

Published on: 15 March 2026

Correspondence: Maha A. Elshazly, dr.mahaahmed2006@gmail.com
PDF HTML

Abstract

Brucella is the causative agent of brucellosis, a serious zoonotic bacterial illness that affects a variety of mammals worldwide. Brucellosis control and eradication programs mainly depend on effective and safe vaccination programs and accurate diagnostic tools. Animals are typically tested for bovine brucellosis using the complement fixation test (CFT), indirect ELISA (I-ELISA), and slide agglutination assays. The aim of the work is to produce freeze-dried slide agglutination antigens to be a strategic store for emergency outbreaks and to evaluate these antigens based on their diagnostic performance characteristics. In-house freeze-dried and liquid Rose Bengal antigens and freeze-dried and Liquid Buffered Acidified Plate Agglutination antigens were developed. I-ELISA with in-house produced antigen titrated and coated with smooth lipopolysaccharide was prepared and validated. The diagnostic performance parameters for each test were estimated using CFT as a gold standard test. The diagnostic performance characteristics for slide agglutination tests using different antigen preparations were estimated using CFT as a reference test. The study included 95 bovine sera tested for brucellosis. The Rose Bengal plate test was positive in 68 and 67 of sera by both LRB and FDRB, respectively. BAPA was positive in 72, 71, and 58 of sera when tested by LBAPA, FDBAPA, and I-ELISA respectively, while 48 sera were positive when confirmed by CFT. It was concluded that the newly prepared freeze-dried slide agglutination antigens were approximately as efficient as traditionally prepared antigens in terms of diagnostic performance items.

Keywords: Brucella, bovine brucellosis, S-LPS, Rose Bengal test, slide agglutination

References

  1. González-Barrientos, R., Hernández-Mora, G. (2023). Chapter 83-marine brucellosis. Fowler's Zoo Wild Anim Med Curr Ther. 10, 573-580. https://doi.org/10.1016/B978-0-323-82852-9.00083-6 
  2. Scholz, C., Heckers, K.O., Appelt, S., Geier-Dömling, D., Schlegel, P., Wattam, A.R. (2023). Isolation of Brucella inopinata from a White's tree frog (Litoria caerulea): pose exotic frogs a potential risk to human health? Front Microbiol. 14, 1173252. https://doi.org/10.3389/fmicb.2023.1173252 PMid:37362939 PMCid:PMC10285381
  3. Hubálek, Z., Křivanová, A., Nesvadbová, J., Rudolf, I. (2023). Zoonotic potential of Brucella microti. Vector Borne Zoonotic Dis. 23(8): 437-439. https://doi.org/10.1089/vbz.2022.0085 PMid:37367199
  4. Freddi, L., De la Garza-García, J.A., Al Dahouk, S., Occhialini, A., Köhler, S. (2023). Brucella spp. are facultative anaerobic bacteria under denitrifying conditions. Microbiol Spectr. 11(6): e0276723. https://doi.org/10.1128/spectrum.02767-23 PMid:37882559 PMCid:PMC10714718
  5. Mohamed, N.S., Stephen, M.B., Nammalwar S. (2010). Brucellosis: a re-emerging zoonosis, Vet Microbiol. 140(3-4): 392-398. https://doi.org/10.1016/j.vetmic.2009.06.021 PMid:19604656
  6. El Jaouhari, M., Striha, M., Edjoc, R., Bonti-Ankomah, S. (2022). Laboratory-acquired infections in Canada from 2016 to 2021. Can Commun Dis Rep. 48(7/8): 303-307. https://doi.org/10.14745/ccdr.v48i78a02 PMid:37334256 PMCid:PMC10275617
  7. Erazo, V., Ramon, R., Moreira, J.M., Morejón, F.B. (2024). Study of the prevalence of bovine brucellosis (Brucella abortus) in general Proaño Parish: evaluation using Rose Bengal test and confirmation through competitive ELISA. J Pure Appl Microbiol. 18(3): 2113-2120. https://doi.org/10.22207/JPAM.18.3.60 
  8. OIE (2018): Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees). http://www.oie.int/conferences-events/other-oie-key-events/2008/ 
  9. Blasco, J.M., Garin-Bastuji, B., Marin, C.M., Gerbier, G., Fanlo, J., Jiménez de Bagués, M.P., Cau, C. (1994). Efficacy of different Rose Bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats. Vet Rec. 134(16): 415-420. https://doi.org/10.1136/vr.134.16.415 PMid:8036772
  10. Alton, G., Jones, L.M., Angus, R.D., Verger, J.M. (1988). Techniques for the Brucellosis Laboratory. Cedex: INRA Publications , 192 pp.
  11. Angus, R.D. (1984). An evaluation of the stability of Brucella abortus strain 19 reduced dosage lyophilized vaccines produced by different Dev Biol Stand. 56, 659-578.
  12. British Pharmacopoeia (2012). Published by The Stationery Office on behalf of the Medicines and Healthcare products Regulatory Agency (MHRA), 4: (pp. 3631-3632). London.
  13. Hasannia, E., Soleimani, S., Alamian, S., Behrozikhah, A., Emadi, A., Dostdari, S. (2015). Stability study of Iriba brucellosis full-dose and reduced-dose vaccine produced by Razi Institute in Iran. Arch Razi Inst. 70(1): 37-44.
  14. Plackett, P., Cottew, G.S., Best, S.J. (1976). An indirect haemolysis test (IHLT) for bovine brucellosis. Aust Vet J. 52(3):136-140. https://doi.org/10.1111/j.1751-0813.1976.tb05448.x PMid:985227
  15. Hennager, S.G. (2004). Reagent production protocol-guinea pig complement preparation for the complement fixation test. USDA, APHIS, National Veterinary Services Laboratories (NVSL), Ames, IA, USA.
  16. Hennager, S.G. (2015). SOP - complement fixation test for detection of antibodies to Brucella abortus and Brucella suis. USDA, APHIS, National Veterinary Services Laboratories (NVSL), Ames, IA, USA.
  17. Roushdy, C.M., Moustafa, A.M.M., Abdelwahab, M.G., Ibrahim, K., El-bauomy, E.M. (2021). Latex agglutination: a rapid, specific immunoassay for diagnosis of ruminant brucellosis. Adv Anim Vet Sci. 9(9): 1292-1301. https://doi.org/10.17582/journal.aavs/2021/9.9.1292.1301 
  18. Blasco, J.M., Marín, C., Jiménez de Bagués, M., Barberán, M., Hernández, A., Molina, L., Velasco, J., Díaz, R., Moriyón, I. (1994). Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep. J Clin Microbiol. 32(8): 1835-1840. https://doi.org/10.1128/jcm.32.8.1835-1840.1994 PMid:7989528 PMCid:PMC263887
  19. Mikolon, A.B., Gardner, I.A., Hietala, S.K., Hernandez de Anda, J., Chamizo Pestaña, E., Hennager, G., Edmondson, A.J. (1998). Evaluation of North American antibody detection tests for diagnosis of brucellosis in goats. J Clin Microbiol. 36(6): 1716-1722. https://doi.org/10.1128/JCM.36.6.1716-1722.1998 PMid:9620406 PMCid:PMC104906
  20. Erganiş, O., Hadimli, H., Solmaz, H., Corlu, M. (2005). Comparison of Rose Bengal plate test antigens prepared from Brucella abortus, Brucella melitensis and Brucella suis. Bull Vet Inst Pulawy. 49, 165-167.
  21. MacMillan, A. (1990). Conventional serological tests. In: K. Nielsen, J.R. Duncan, (Eds.). Animal Brucellosis (1st Ed.) (pp. 153-198). Boca Raton: CRC Press
  22. World Health Organization (1986). Joint FAO/WHO Expert Committee on Brucellosis: Sixth Report. WHO Technical Reports Series, No. 740, WHO,
  23. Garin-Bastuji, B., Blasco, J.M. (1997). Caprine and ovine brucellosis (excluding B. ovis infection). In: Manual of standards for diagnostic tests and vaccines (Third ed.), (pp. 350-368). Paris:OIE.
  24. Athira, K., Shyma, V.H., Justin, K.D., Vijayakumar, K., Jayakumar, C., Greeshma, J. (2023). Diagnosis of canine brucellosis using Rose Bengal plate test. J Vet Anim Sci. 54(2): 544-551. https://doi.org/10.51966/jvas.2023.54.2.544-551 
  25. Loubet, P., Magnan, C., Salipante, F., Pastre, T., Keriel, A., O'Callaghan, D., Sotto, A., Lavigne, J.P. (2024). Diagnosis of brucellosis: combining tests to improve performance. PLoS Negl Trop Dis. 18(9): e0012442. https://doi.org/10.1371/journal.pntd.0012442 PMid:39236075 PMCid:PMC11407618
  26. Legesse, A., Mekuriaw, A., Gelaye, E., Abayneh, T., Getachew, B., Weldemedhin, W., Tesgera, T., Deresse, G., 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 Microbiol. 23(1): 216. https://doi.org/10.1186/s12866-023-02962-2 PMid:37563597 PMCid:PMC10413706
  27. Šerić-Haračić, S., Velić, L., Šaljić, E., Čengić, B., Tandir, F., Hadžimusić, N. (2022). Agreement among Rose Bengal, complement fixation test, and iELISA in diagnostic discrimination of sheep and goat brucellosis (Brucella melitensis). Acta Vet Eurasia. 48(1): 30-34. https://doi.org/10.5152/actavet.2022.21050
  28. Schofield, T.L. (2009). Vaccine stability study design and analysis to support product licensure. Biologicals. 37(6): 387-396. https://doi.org/10.1016/j.biologicals.2009.08.009 PMid:19717312
  29. Wang, W. (2000). Lyophilization and development of solid protein Int J Pharm. 203(1- 2): 1-60. https://doi.org/10.1016/S0378-5173(00)00423-3 PMid:10967427
  30. Knezevic, I. (2009). Stability evaluation of vaccines: WHO approach. Biologicals. 37(6): 357-359. https://doi.org/10.1016/j.biologicals.2009.08.004 PMid:19729320

Copyright

©2026 Farahat L.F. This is an open-access article published under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Conflict of Interest Statement

The authors declared that they have no financial or non-financial conflict of interest regarding authorship and publication of this article.

Citation Information

Macedonian Veterinary Review. Volume 49, Issue 1, Pages i-xii, e-ISSN 1857-7415, p-ISSN 1409-7621, DOI:  https://doi.org/10.2478/macvetrev-2026-0016

The all content of the Macedonian Veterinary Review, except where otherwise noted, is licensed under a Creative Commons Attribution License.