Hayvanlardan izole edilen Bordetella bronchiseptica suşlarının virulens faktörleri ve bağışıklama gücünün saptanması

Bu calismada, kopek, kedi, domuz ve deney hayvanlari gibi evcil ve vahsi memeli hayvanlarda agir seyidi solunum yolu infeksiyonlan olusturan Bordetella bronchiseptica suslannin sahip oldugu virulens aktorleri, bu suslardaki bazi antijenlerin bakteri tarafindan sentezlenme durundan ve bu antijenlerin bagisikliktaki rolleri arastirildi. Farkli kaynaklardan saglanan 15 adet Bordetella bronchiseptica susunda, adenilat siklaz-hemolizin (AC-Hly)'in uremenin erken doneminde sentezlenmeye basladigi ve genel olarak suslarda yuksek degerlere (9,2-17,8 AC unitesi) ulastigi, filamentoz hemaglutinin (FHA) proteini sentezinin ise daha gec donemde ortaya ciktigi ve dusuk duzeyde uretildigi (1/2-1/8) belirlendi. B. bronchiseptica suslannin cogunda gozlenen guclu letal etki ile AC- Hly sentezi arasmda iliski oldugu goruldu. B. bronchiseptica suslannin bakteri lizati ve ure-isi-ekstrakte proteini profilleri SDS- PAGE analizinde karsilastirmali olarak incelendiginde, suslann cogunda patojenitede onemli olan FHA, AC-Hly, pertaktin, Fim D, 35 kDa protein ve fimbriya proteinlerinin ana bantlar olarak yer aldigi gozlendi. Farelerde tum hucre asisi ile yapilan bagisiklamasi sonucu olusan antikor yanitinin ure-isi ekstrakte proteinleri ile yapilan western-blotting analizinde, FHA, AC-Hly, pertaktin, Fim D, 35 kDa protein ve fimbriya proteinlerine karsi genel olarak belirgin derecede antikor yaniti olustugu belirlendi. Bu bulgularin, butun suslara ait inaktif hucre asilan ile bagisiklanan farelerde olusan yuksek ve capraz aglutinin duzeyi (1:512- 1:8192) ile virulent sus F415 epruvasyonuna karsi iyi duzeyde (% 80-100) saglanan korunma ile de uyumlu oldugu belirlendi. Ure-isi-ekstrakte proteinden yuksek basinc altinda Superdex 75 jel filtrasyon kolonunda tek asamada saflastinlan FimD-35 kDa protein-Fim uclu protein grubunun B. bronchiseptica suslannin bagisik yanit olusturmasinda onemli oldugu belirlendi. Bu protein grubu ile farelerde yapilan bagisiklama sonrasinda, serum mikroaglutinasyon turelerinin 1/32-1/256 gibi dusuk degerlerde olmasina karsin, 4 LD50 periton ici epruvasyona karsi bazi suslarda yuksek oranda (% 100) koruma sagladigi goruldu. Sonucta, incelenen B. bronchiseptica suslan arasinda onemli antijenik farkliliklarin bulundugu, suslardan hazirlanan tum hucre asilarinin iyi bir koruma saglamalarina karsin, ozellikle saflastinlan fimbriya grubu ile yapilan bagisiklama calismalan sonrasinda korunma duzeylerinin susa ve doza bagli degistigi saptandi.AbstractIn this study, virulence factors of different strains of B. bronchiseptica which is the causative agent of important respiratory diseases in domestic and wild mammalian animals such as dogs, cats, pigs and experimental animals, the synthesis level of some of the antigens by the bacteria and the roles of these antigens in immunity were investigated. Generally FHA was synthesized in later stages and continued at low levels (1/2-1/8) throughout the culture, however, AC-Hly secretion was initiated at early stages of the liquid culture and reached high levels (9.2-17.8 AC unit) at 15 strains of Bordetella bronchiseptica from different origins. Besides, a correlation between the strong letality of most strains in mice and AC-Hly production was determined. When bacteria lysates and urea-heat-extracted protein profiles of B. bronchiseptica strains were analysed by SDS-PAGE, FHA, AC-Hly, pertactin, Fim D, 38 kDa porin protein and fimbriae proteins were shown as major bands at most of the strains. The profile of the antibody response of the mice after immunization with whole-cell bacteria was analysed by western-blotting with urea-heat-extracted proteins and significantly similar antibody response to FHA, AC-Hly, pertactin, Fim D, 35 kDa porin protein and fimbriae proteins occurred at most of the strains. This situation was compatible with the high-cross microaglutination titres (1:512-1:8192) of the sera from mice immunized with inactive whole cell of all strains and also the high level (80-100%) protection against challenge with the virulent strain F415. The triple protein group consisting of Fim D-35kDa protein-Fimbriae which was purified from those urea-heat-extracted proteins of the strains in a single step by using Superdex 75 gel filtration column under high pressure was determined to be important in the immune response of B. bronchiseptica. Although the serum microaglutination titres after immunization with this triple protein group were at low values (1:32-1:256), this protein group of some of the strains provided a high protection (100%) against intraperitoneal challenge in mice with 4 LD50 of the virulent strain F415. This protective immune response was found to be associated with the sufficient quantity of each protein present in FimD-35 kDa protein-Fimbriae group. In conclusion, whole-cell vaccines prepared from the strains provided high level protection, however, protection levels after immunization with the purified fimbriae group were determined to be related with the strain and the dose of the vaccine because of the major antigenic differences observed among B. bronchiseptica strains.