Investigation of immunogenicity and protective efficiency of Bordetella bronchiseptica fimbriae in mice

Abstract

Fimbrial proteins are important for adhesion and colonization of Bordetella bronchiseptica in the upper respiratory tract. In this study, determination of immunogenicity of the fimbrial proteins isolated from different strains of B. bronchiseptica and their protective efficacy against letal challenge was aimed. For this purpose, fimbrial proteins of seven standard strains and clinical isolates of B. bronchiseptica were partially purified by gel filtration chromatography and analysed by SDSPAGE. In microagglutination test, whole cell F415 was used as the antigen and the agglutination of sera from mice immunized with the purified fimbrial proteins was observed at titers of 1:128 for strains 219 and 4617 and the isolate CA; 1:256 for strain F415 and the isolate UK7; 1:512 for the isolate UK6 and 1:4096 for strain 3036. In addition, by ELISA against F415 fimbrial antigen, the antifimbriae antibody levels of the immune mouse sera were observed at serum dilutions of 1:6400 for UK7; 1:3200 for UK6, F415 and 3036; 1:1600 for CA; 1:800 for 219 and 1:400 for 4617.The protective values of the fimbrial proteins against letal challenge with strain F415 in mice immunized with the fimbrial proteins (2x25 μg/dose) were; 100% for F415 and 3036, 84% for UK6 and 50% for UK7, but no protection was observed with the other strains and isolates. In conclusion, fimbrial antigens were determined to have important protective efficacy and anti-fimbriae antibody levels were found correlated with that protective efficacy

Keywords

• Bordetella bronchiseptica
• fimbriae
• immunogenicity
• protective efficiency

Bordetella bronchiseptica fimbriya proteinlerinin farelerde immunojenite ve koruyuculuk etkisininaraştırılmas

Abstract

Bordetella bronchiseptica’nın üst solunum yollarında adhezyonu ve kolonize olmasında fimbriya proteinleri önemlidir. Bu çalışmada, B. bronchiseptica’nın farklı suşlarından elde edilen fimbriyal proteinlerin immunojenitesi ve canlı bakteri uygulamasına karşı koruyucu etkinliğinin belirlenmesi amaçlandı. B. bronchiseptica’ya ait toplam 7 standart suş ve klinik izolata ait fimbriya proteinleri, jel filtrasyon kromatografisi yoluyla kısmi olarak saflaştırıldı ve SDS-PAGE ile analiz edildi. Saflaştırılan fimbriya proteinleri ile bağışıklanan farelerin serumlarıyla F415 suşuna karşı yapılan mikroaglutinasyon testinde; 219 ve 4617 suşları ile CA izolatı için 1:128, F415 suşu ve UK7 izolatı için 1:256, UK6 izolatı için 1:512 ve 3036 suşu için 1:4096 titrede aglutinasyon gözlendi. Ayrıca, bağışık fare serumlarında, F415 fimbriya antijeni kullanılarak yapılan ELISA’da, UK7 için 1:6400; UK6, F415 ve 3036 için 1:3200; CA için 1:1600; 219 için 1:400 ve 4617 için 1:400 titrede antikor düzeyi belirlendi. Fimbriya proteinleri ile bağışıklanan farelerde (2x25 μg/doz), F415 suşu ile yapılan canlı bakteri uygulamasında ise fimbriya proteinlerinin koruyucu değerleri; F415 ve 3036 suşları için %100, UK6 için %84 ve UK7 için %50 olarak saptandı, diğer suş ve izolatlar için herhangi bir koruyucu değer gözlenmedi. Sonuç olarak, fimbriya antijenlerinin önemli koruyucu etkinliğe sahip olduğu ve anti-fimbriya antikor düzeyi ile bu koruyucu etkinlik arasında uyumlu bir ilişki olduğu belirlendi

Keywords

• Bordetella bronchiseptica
• fimbriya
• immünojenisite
• koruyuculuk etkinliği

References

1. Bemis DA, Greisen HA, Appel MJG (1977): Pathogenesis of canine Bordetellosis. J Infect Dis, 135, 753-762.
2. Binns SH, Dawson AJ, Speakman AJ, Cuevas LE, Gaskell CJ, Hart CA, Morgan KL, Gaskell RM (1999): Prevalence and risk factors for feline Bordetella bronchiseptica infection. Vet Rec, 144, 575-580.
3. Boschwitz JS, van der Heide HGJ, Mooi FR, Relman DA (1997): Bordetella bronchiseptica expresses the fimbrial structural subunit gene fimA. J Bacteriol, 179, 7882-7885.
4. Burns EH, Norman JM, Hatcher MD, Bemis DA (1993): Fimbriae and determination of host species specificity of Bordetella bronchiseptica. J Clin Microbiol, 31, 1838-1844.
5. Büyüktanır Ö, Akan M, Özcengiz E (2005): Değişik Bordetella bronchiseptica suşlarında virulans faktörleri ve immünojenik özellikler. Türk Mikrobiol Cem Derg, 35, 248-255.
6. Cotter PA, Yuk MH, Mattoo S, Akerley BJ, Boschwitz J, Relman DA, Miller JF (1998): Filamentous hemaglutinin of Bordetella bronchiseptica is required for efficient establishment of tracheal colonization. Infect Immun, 66, 5921-5929.
7. Dworkin MS, Sullivan PS, Buskin SE, Harrington RD, Olliffe J, Mac Arthur RD, Lopez CE (1999): Bordetella bronchiseptica infection in Human Immunodeficiency Virus-infected patients. Clin Infect Dis, 28, 1095-1099.
8. Geuijen CA, Willems RJL, Bongaerts M, Top J, Gielen H, Mooi FR (1997): Role of the Bordetella pertussis minor fimbrial subunit, FimD, in colonization of the mouse respiratory tract. Infect Immun 65, 4222-4228. 9. Goodnow

9. RA (1980): Biology of Bordetella
10. bronchiseptica. Microbiol Rev, 44, 722-738.
11. Gueirard P, Guiso N. (1993): Virulence of Bordetella bronchiseptica: Role of adenylate cyclase-hemolysin. Infect Immun 61, 4072-4078.
12. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1996): Bergey’s Manual of Determinative Bacteriology. Williams and Wilkins, Ninth Edition.
13. Irons LI, Ashworth LAE, Robinson A (1985): Release and purification of fimbriae from Bordetella pertussis. Develop Biol Standard 61, 153-163.
14. Jacobs AAC, Chalmers WSK, Pasman J, van Vugt F, Cuenen LH. (1993): Feline bordetellosis: challenge and vaccine studies. Vet Rec, 133, 260–263.
15. Kania SA, Rajeev S, Burns Jr EH, Odom TF, Holloway SM, Bemis DA (2000): Characterization of fimN, a new Bordetella bronchiseptica major fimbrial subunit gene. Gene, 256, 149–155.
16. Kobisch M, Novotny N (1990): Identification of a 68- kilodalton outer membrane protein as the major protective antigen of Bordetella bronchiseptica by using specific- pathogen-free piglets. Infect Immun, 58, 352-357.
17. Kume K, Nakai T, Samejima Y, Sugimoto C (1986): Properties of dermonecrotic toxin prepared from sonic extracts of Bordetella bronchiseptica. Infect Immun, 52, 370-377.
18. Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227, 680-685.
19. Lee SW, Way AW, Osen EG (1986): Purification and subunit heterogeneity of pili of Bordetella bronchiseptica. Infect Immun, 51, 586-593.
20. Mattoo S, Miller JF, Cotter PA (2000): Role of Bordetella bronchiseptica fimbriae in tracheal colonization and development of a humoral immune response. Infect Immun, 68, 2024-2033.
21. Mattoo S, Cherry JD (2005): Molecular pathogenesis, epidemiology and clinical manifestations of respiratory infections due to Bordetella pertussis and other Bordetella subspecies. Clin Microbiol Rev, 18, 326-382.
22. Mooi FR, van der Heide HGJ, ter Avest AR, Welinder KG, Livey I, van der Zeijst BAM, Gaastra W (1987): Characterization of fimbrial subunits from Bordetella species. Microb Pathog, 2, 473-484.
23. Novotny P, Kobisch M, Cownley K, Chubb AP, Montaraz JA (1985): Evaluation of Bordetella bronchiseptica vaccines in specific-pathogen-free piglets with bacterial cell surface antigens in Enzyme-Linked Immunosorbent Assay. Infect Immun, 50, 190-198.
24. Parker CD, Payne BJ (1985): Manual of clinical microbiology. Fourth edition. ASM Chapter 34, 394-399.
25. Passerini De Rossi BN, Friedman LE, Gonzalez Flecha FL, Castello Franco MA, Rossi JP (1999): Identification of Bordetella pertussis virulence-associated outer membrane proteins. FEMS Microbiol Lett, 172, 9-13.
26. Powell HM (1951): Charcoal agar culture medium for preparing H. pertussis vaccine. Bul Hlth Rep, 66, 346-348.
27. Riboli B, Pedroni P, Cuzzoni A, Grandi G, de Ferra F (1991): Expression of Bordetella pertussis fimbrial (fim) genes in Bordetella bronchiseptica: fimX is expressed at a low level and vir-regulated. Microb Pathog, 10, 393-403.
28. Savelkoul PH, de Kerf DPG, Willems RJ, Mooi FR, van der Zeijst BAM, Gaastra W (1996): Characterization of the fim 2 and fim 3 fimbrial subunit genes of Bordetella bronchiseptica: Role of Fim 2 and Fim 3 fimbriae and flagella in adhesion. Infect Immun, 64, 5098-5105.
29. Shina A, Hart CA, Stenton MD, Dawson S, McCracken CM, Binns S.H, Gaskell RM, Winstanley C (2002): Distribution of fim3 and flaA TTGE sequence types amongst isolates of Bordetella bronchiseptica from different host animals. J Med Microbiol, 51, 557-563.
30. Towbin H, Staehelin T, Gordon J (1979):
31. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Pro Natl Acad Sci USA, 76, 4350-4354.
32. World Health Organization (WHO) Manual (1977): Manual for the production and control of vaccines, pertussis vaccine. BLG/UNDP/77.3 Rev. 1
33. Willems RJL, Geuijen C, van der Heide HGJ, Matheson M, Robinson A, Vesiuis LF, Ebberink R, Theelen J, Mooi FR (1993): Isolation of a putative fimbrial adhesin from Bordetella pertussis and the identification of its gene. Mol Microbiol, 9, 623-634.