Feyzullah BEYAZ, Anıl İÇA, Ayhan ATASEVER, Latife BEYAZ

Immunohistochemical studies on S100 reactivity in chicks experimentally ınfected with Eimeria tenella I: the localization of S100 protein and ıts subunits a and $beta$ in stages of Eimeria tenella

Bu araştırmada, intrasellüler protozoon olan Eimeria tenella’da bir kalsiyum bağlayıcı protein olan S100 ve onun α ve β alt ünitelerinin ekspresyonu incelendi. Bu amaç için, E. tenella ile deneysel olarak enfekte edilen civcivlerin sekum, ileum, kolon, rektum ve bursa Fabricius’larından alınan parafin kesitlere anti-S100 whole molekül poliklonal antikoru ile anti-S100α ve anti-S100β monoklonal antikorları uygulandı. Araştırmada, 2-3 günlük 175 adet spesifik patojen free (SPF) civciv kullanıldı. Toplam 175 adet civciv, deney ve kontrol olmak üzere sırasıyla 94 ve 81 civciv içeren 2 gruba ayrıldı. Deney grubundaki tüm civcivlere, tek dozda 0,5 ml inokulum içerisinde 10.000 sporlu oosist verildi. Deney süresi 18 günle sınırlandırıldı. Toplam 175 civciv inokulasyondan sonraki 1. saat ile 432. saat arasında servikal dislokasyon yöntemiyle öldürüldü. Kullanılan antikorların immunoreaktivitelerini göstermek amacıyla Streptavidin-biotin-kompleks (StreptABC) yöntemi kullanıldı. Elde edilen sonuçlar, sekum, ileum, kolon, rektum ve bursa Fabricius’da, E. tenella’nın şizont, mikrogamet, makrogamet ve oosistlerinde S100 whole molekülü ve α ve β alt ünitelerinin her biri için pozitif immunoreaksiyon olduğunu gösterdi. Bu sonuçlar, S100 proteinin hem α hem de β alt ünitelerinin E. tenella’nın tüm endojen safhalarında bulunduğunu ortaya koymuştur. Bu proteinler, muhtemelen, E. tenella’nın konakçı hücreleri içerisindeki parazitik gelişimine aracılık etmektedir.

Civcivlerde deneysel Eimeria tenalla enfeksiyonunda S100 reaktivitesi üzerine ımmunohistokimyasal çalışmalar I: Eimeria tenella'da S100 proteini ve a ile B alt ünitelerinin immunohistokimyasal lokalizasyonu

The expression of the calcium-binding protein S100 and its subunits α and β in the intracellular protozoon Eimeria tenella was investigated. For this purpose, anti-S100 whole molecule polyclonal antibody and anti-S100α and anti-S100β monoclonal antibodies were used on paraffin sections collected from the cecum, ileum, colon, rectum, and bursa of Fabricius of chicks experimentally infected with E. tenella. One hundred seventy-five 2- to 3-day-old specific pathogen-free chicks were used. The chicks were divided into experiment and control groups, to include 94 and 81 chicks, respectively. All the chicks in the experimental group received 10,000 spored oocytes in 0.5 ml inoculum in a single dose. The duration of the experiment was limited to 18 days. All 175 chicks were sacrificed by cervical dislocation between hours 1 and 432 following inoculation. The streptavidin-biotin complex method was used to detect the immunoreactivity of the antibodies used. The results revealed a positive immune reaction for the S100 whole molecule and for α and β subunits in the schizonts, microgametes, macrogametes, and oocytes of the E. tenella in the cecum, ileum, colon, rectum, and bursa of Fabricius. These results show that both α and β subunits of the S100 protein existed in all the endogenous phases of E. tenella. These proteins are likely to mediate the parasitic development of E. tenella within host cells.

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1. Fanò, G., Biocca, S., Fulle, S., Mariggiò, M.A., Belia, S., Calissano, P.: The S100: a protein family in search of a function. Prog. Neurobiol., 1995; 46: 71-82.
2. Heizmann, C.W., Cox, J.A.: New perspectives on S100 proteins: a multi-functional Ca2+, Zn2+ and Cu2+ binding protein family. Biometals, 1998; 11: 383-397.
3. Donato, R.: Functional roles of S100 proteins, calcium-binding proteins of the EF-hand type. Biochim. Biophys. Acta, 1999; 1450: 191-231.
4. Donato, R.: S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles. Int. J. Biochem. Cell Biol., 2001; 33: 637-668.
5. Donato R.: Intracellular and extracellular roles of S100 proteins. Microsc. Res. Tech., 2003; 60: 540-551.
6. Heizmann, C.W., Fritz, G., Schäfer, B.W.: S100 proteins: structure, functions and pathology. Front. Biosci., 2002; 7: 1356- 1368.
7. Haimoto, H., Hosoda, S., Kato, K.: Differential distribution of immunoreactive S100-α and S100-β proteins in normal nonnervous human tissues. Lab. Invest., 1987; 57: 489-498.
8. Zimmer, D.B., Cornwall, E.H., Landar, A., Song, W.: The S100 protein family: history, function and expression. Brain Res. Bull., 1995; 37: 417-429.
9. Zimmer, D.B, Wright Sadosky, P., Weber, D.J.: Molecular mechanism of S100-target protein interactions. Microsc. Res. Tech., 2003; 60: 552-559.
10. Kato, K., Kimura, S.: S100ao(αα) protein is mainly located in heart and striated muscles. Biochim. Biophys. Acta, 1985; 842: 146-151.
11. Semba, R., Kato, K., Isobe, T.: S-100 antigen in rat kidney. Neurochem. Res., 1984; 9: 1125-1126.
12. Parsons, M., Valentine, M., Carter, V.: Protein kinases in divergent eukaryotes: identification of protein kinase activities regulated during trypanosome development. Proc. Natl. Acad. Sci. USA, 1993; 90: 2656-2660.
13. Bejon, P.A., Bannister, L.H., Fowler, R.E., Fookes, R.E., Webb, S.E., Wright, A., Mitchell, G.M.: A role for microtubules in Plasmodium falciparum merozoite invasion. Parasitology, 1997; 114: 1-6.
14. del Cacho, E., López-Bernad, F., Gallego, M., Quílez, J., Sánchez- Acedo, C.: Expression and localization of an S100 protein-like molecule in Eimeria tenella. J. Parasitol., 1998; 84: 1174-1177.
15. López-Bernad, F., del Cacho, E., Gallego, M., Quílez, J., Sánchez- Acedo, C.: Immunohistochemical study of S100-like protein in Eimeria brunetti and Eimeria acervulina. Vet. Parasitol., 2000; 88: 1-6.
16. Long, P.L., Millard, B.J., Joyner, L.P., Norton, C.C.: A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet. Lat., 1976; 6: 201-217.
17. Read, L.K., Mikkelsen, R.B.: Cyclic AMP- and Ca2+-dependent protein kinases in Plasmodium falciparum. Exp. Parasitol., 1990; 71: 39-48.
18. Bumstead J.M., Dunn, P.P.J., Tomley, F.M.: Nitrocellulose immunoblotting for identification and molecular gene cloning of Eimeria maxima antigens that stimulate lymphocyte proliferation. Clin. Diagn. Lab. Immunol., 1995; 2: 524-530.
19. Dunn, P.P.J., Bumstead, J.M., Tomley, F.M.: Sequence, expression and localization of calmodulin-domain protein kinases in Eimeria tenella and Eimeria maxima. Parasitology, 1996; 113: 439-448.
20. McNutt, N.S.: The S100 family of multipurpose calcium-binding proteins. J. Cutan. Pathol., 1998; 25: 521-529.
21. Santamaria-Kisiel, L., Rintala-Dempsey, A.C., Shaw, G.S.: Calcium-dependent and -independent interactions of the S100 protein family. Biochem. J., 2006; 396: 201-214.
22. Lillehoj, H.S., Trout, J.M.: Avian gut-associated lymphoid tissues and intestinal immune responses to Eimeria parasites. Clin. Microbiol. Rev., 1996; 9: 349-360.
23. López-Bernad, F., del Cacho, E., Gallego, M., Quílez, J., Sánchez- Acedo, C.: Identification of a fibronectin-like molecule on Eimeria tenella. Parasitology, 1996; 113: 505-510.