Development of an In House Enzyme-Linked Immunosorbent Assay (ELISA) Prototype Using Different Anti- gens for Serological Diagnosis of Brucella ovis

Öz

In this study, it was aimed to develop an Enzyme-Linked Immunosorbent Assay (ELISA) prototype to evalua- te humoral immune response against Brucella ovis by using different antigenic fractions from bacteria comparatively. For this purpose, four different antigens, namely Hot saline extract, Rough Lipopolysaccharide, Phage lysate 1, and Phage lysate 2 were used to test 84 sheep serum samples in ELISA. Seropositivity rates were, 14.3%, 13.1%, 10.7%, and 15.5%, respectively. Although the highest seropositivity rate was obtained by using the Phage lysate 2 antigen, it did not reach the level of statistical significance (χ2=0.89; P>0.05) difference between these antigens. It was concluded that in future studies, there would be a need for comparing the results of more serum samples tested in ELISA using Phage lysate 2 with the results of standard serological tests such as complement fixation test and agar gel immunodif- fusion test or available commercial ELISA kit. Thus, the potential possibility of commercialization of a new in-house ELISA prototype will be evaluated more realistically. Given the seropositivity rate of Brucella ovis infection obtained by using a small number of serum samples, a large-scale serological study with greater serum samples from different parts of Turkey will reveal the real disease situation in our country.

Anahtar Kelimeler

• sheep

Brucella ovis’in Serolojik Tanısı Amacıyla Farklı Antijenlerin Kullanıldığı Bir In House Enzyme- Linked Immunosorbent Assay (ELISA) Prototipinin Geliştirilmesi

Öz

Bu çalışmada, Brucella ovis’e karşı humoral yanıtın değerlendirilmesinde kullanılmak üzere, bu bakterinin farklı antijenik fraksiyonlarından hazırlanan antijenlerin karşılaştırılmasıyla bir Enzyme -Linked Immunosorbent Assay (ELISA) prototipinin geliştirilmesi amaçlandı. Bu amaçla, dört farklı antijen (Hot salin ekstrakt, Rough Lipopolisakkarit, Faj lizat 1 ve Faj lizat 2), 84 koyun serum örneği ile test edildi. Brucella ovis açısından ELISA seropozitiflikleri, kullanı- lan antijenlere göre sırasıyla %14.3, %13.1, %10.7 ve %15.5 olarak saptandı. En yüksek pozitiflik oranı Faj lizat 2 ile elde edildi, ancak, Brucella ovis’e karşı oluşan antikorların ELISA ile belirlenmesi için kullanılan 4 farklı antijenin test sonuçları arasındaki farklılık, istatistiki açıdan önemsiz (χ2=0.89; P>0.05) bulundu. Sonuç olarak, gelecekteki çalışma- larda, Faj lizat 2 antijeni kullanılarak ELISA ile test edilen çok daha fazla serum örneği için elde edilen sonuçların, komplement fikzasyon testi ve agar jel immunodifüzyon testi gibi standart testler ve temin edilebilecek ticari kitlerin sonuçları ile karşılaştırılmasına ihtiyaç duyulacağı ve böylece in house ELISA prototipinin ticarileşme potansiyelinin daha sağlıklı olarak değerlendirilebileceği kanısına varıldı. Ayrıca, az sayıda serum örneğiyle elde edilen seropozitiflik oranı göz önünde bulundurulduğunda, Türkiye'nin farklı bölgelerini kapsayan daha fazla sayıda serum örneği ile geniş ölçekli bir serolojik çalışmanın yapılması ile ülkemizdeki gerçek hastalık durumunun ortaya çıkarılabileceği düşünül- mektedir.

Anahtar Kelimeler

• Brucella ovis
• ELISA
• koyun
• sheep

Kaynakça

1. Alton GG, Jones LM, Angus RD, Verger JM. Brucella canis. In: Techniques for the Brucellosis Labora- tory. Paris: Institut National de la Recherche Ag- ronomique, 1988; pp. 169-74.
2. Barrouin-Melo SM, Poester FP, Riberio MB, Alcanta- ra AC, Aguiar PHP, Nascimento IL, Schaer RE, Nascimento RM, Freire SM. Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis. Res Vet Sci 2007; 83: 340-6.
3. Blasco JM. Brucella ovis. Nielsen K, Duncan JR. eds. In: Animal Brucellosis. Florida: CRC Press, 1990; pp. 351-78.
4. Chiarenza G, Villari S, Galluzzo P, Alfano M, Pilato V, Vicari D, Stancanelli A. Evaluation of Brucella ovis seroprevalance in Sicilian farms (Italy). Lucr. ştiinţ. - Inst. Agron. "Nicolae Bălcescu", Ser C Med vet 2017; 3: 16-9.
5. Chiarenza G, Villari S, Galluzzo P, Brigano S, Alfano M, Tagliarini A, Pilato V, Guercio A, Stancanelli A. Brucella ovis presence in Sicilian farms (Italy). Int J Infect Dis 2018; 73: 385-6.
6. Cho HJ, Nilo L. Diagnostic sensitivity, and specificity of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams. Can J Vet Res 1987; 51 (1): 99-103.
7. Corbel MJ. Recent advance in the study of Brucella antigen and their serological cross reaction. The Vet Bull 1985; 55: 927-72.
8. Elderbrook M, Schumaker B, Cornish T, Peck D, Sondgeroth K. Seroprevalence and risk factors of Brucella ovis in domestic sheep in Wyoming, USA. BMC Vet Res 2019; 15(246): 1-12.

9. Elderbrook MJ, Schumaker BA, Ueti MW, Almeida MB, Vieara TSWJ, Vieara RFC, Sondgeroth KS. Comparison of 2 ELISAs for detecting exposure to Brucella ovis. J Vet Diagn 2020; 32(5): 700-5.
10. Erdenliğ Gürbilek S, Keskin O, Tel OY. The first re- port of Brucella ovis infection in a ram in Turkey. İkinci Uluslararası Veteriner Mikrobiyoloji Kongre- si, 16-19 Ekim, 2018; Antalya-Türkiye.
11. Escobar GI, Boeri EJ, Ayala SM, Lucero NE. The feasibility of using antigens prepared with rough Brucella strains for diagnosis of canine brucello- sis. Rev Argent Microbiol 2010; 42: 35-40.
12. Estein SM, Baldi PC, Bowden RA. Comparison of serological tests based on outer membrane or internal antigens for detecting antibodies to Bru- cella ovis in infected flocks. J Vet Diagn Invest 2002; 14: 407-11.
13. França SA, Mol JPS, Costa EA, Silva APC, Xavier MN, Tsolis RM, Reis JKP, Paixão TA, Santos RL. Indirect ELISA for diagnosis of Brucella ovis infec- tion in rams. Arq Bras Med Vet Zootec 2014; 66 (6): 1695-702.
14. Gall D, Nielsen K, Vigliocco A, Smith P, Perez B, Rojas, X, Robles C. Evaluation of an indirect enzyme-linked immunoassay for presumptive serodiagnosis of Brucella ovis in sheep. Small Rumin Res 2003; 48: 173-9.
15. Lopez G, Ayala SM, Escobar GI, Lucero NE. Use of Brucella canis antigen for detection of ovine se- rum antibodies against Brucella ovis. Vet Micro- biol 2005; 105: 181-7.
16. Lopez G, Escobar GI, Ayala SM, Lucero NE. Detec- tion of antibodies to Brucella ovis in sheep milk using B. ovis and B. canis antigen. Vet Microbiol 2006; 116: 232-8.
17. Moreno E, Jones LM, Berman DT. Immunochemical characterization of Rough Brucella lipopoly- saccharides. Infect Immun 1984; 43(3): 779-82.
18. Myers DM, Jones LM, Varela-Diaz VM. Studies of antigen for complement fixation and gel diffusion test in the diagnosis of infections caused by Brucella ovis and other Brucella. Appl Microbiol 1972; 23 (5): 894-902.
19. Nielsen K, Smith P, Conde S, Dragide Benitez G, Gall D, Halbert G, Kenny K, Massengill C, Mu- enks Q, Rojas X, Perez B, Samartino L, Silva P, Tollersrud T, Lolley M. Rough lipopolysaccharide of Brucella abortus RB51 as a common antigen for serological detection of B. ovis, B. canis, and B. abortus RB51 exposure using indirect enzyme immunoassay and fluorescence polarization as- say. J Immunoass Immunoch 2004; 25 (2): 171- 82.
20. Nielsen K, Smith P, Yu WL, Rojas X, Perez B, Conde S, Samartino L, Robles C. Detection of ovine antibody to Brucella ovis by indirect enzyme immuno- assay. J Immunoass Immunoch 2007; 28: 243-50.
21. OIE. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2021 Chapter 3.8.7. Ovine epididymitis (Brucella ovis). https://www.oie.int/ fileadmin/Home/eng/Health_standards/ tahm/3.08.07_OVINE_EPID.pdf; Accessed Date: 15.12. 2021.
22. De Oliveira MZ, Vale V, Keid L, Freire SM, Meyer R, Portela RW, Barrouin-Melo SM. Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis. Res Vet Sci 2011; 90(3): 425-31.
23. Praud A, Champion JL, Corde Y, Drapeau A, Meyer L, Garin-Bastuji B. Assessment of the diagnostic sensitivity and specificity of an indirect ELISA kit for the diagnosis of Brucella ovis infection in rams. BMC Vet Res 2012; 8(68): 1-7.
24. Saxena HM, Raj S. A novel immunotherapy of Bru- cellosis in cows monitored non invasively through a specific biomarker. PLoS Negl Trop Dis 2018; 12(4): e0006393.
25. Savić S, Stošić MZ, Pušić I, Polaček V, Grgić Z, Marčić D, Dačić M, Bugarski D. Seroprevalence and spreading of Brucella ovis in South Bačka and Srem district. Vet Arh 2018; 11(2): 89-101.
26. Tel OY, Gürbilek SE, Keskin O. The evaluation of indirect enzyme-linked immunosorbent assay using antigens prepared from Brucella abortus RB51 and Brucella canis M (-) variant strains for serologic diagnosis of Brucella ovis infection. Kaf- kas Univ Vet Fak Derg 2016; 22(11): 63-7.
27. Türütoğlu H. Detection of Brucella ovis infection in rams in the Konya region by microcomplement fixation tests. Veterinarium 1992; 3(2): 3-6.
28. Uçan US, Aras Z. Seroprevalence of Brucella ovis infection in rams from some flocks in the provin- ces Konya and Sivas. Eurasian J Vet Sci 2007; 23: 35-8.
29. Worthington RW, Weddell W, Penrose ME. A compa- rison of three serological tests for the diagnosis of Brucella ovis infection in rams. N Z Vet J 1984; 32: 58-60.
30. Yi, EC, Hackett M. Rapid isolation method for lipo- polysaccharide and lipid A from gram-negative bacteria. Analyst Apr 2000; 125(4): 651-6.
31. Zagelbaum NK, Sayegh GP, Vernatter JN. First case of Brucella ovis in human. New York Chapter ACP, Annual Scientific Meeting, Medical Student Clinical Vignette, Poster Presentation. June 3, 2017; New York-The USA.
32. Zoha SJ, Carmicheal LE. Properties of cell wall anti- gens of virulent Brucella canis and a less mucoid variant of reduced pathogenicity. Am J Vet Res 1982; 43: 171-4.