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Koyun ve farelerde kist hidatik proteinlerinin karşılaştırmalı analizi ve antijenik proteinlerde glikoprotein varlığının değerlendirilmesi

Year 2014, Volume: 61 Issue: 4, 243 - 248, 01.12.2014

Serap Ünübol Aypak Hamdi Uysal

https://doi.org/10.1501/Vetfak_0000002637

Abstract

Bu çalışmada koyun ve farelerde kist hidatik proteinlerinin, antijenik proteinler ve antijenik glikoproteinler yönünden karşılaştırılması amaçlandı. Bu amaçla 20’şer adet enfekte ve 10’ar adet sağlıklı koyun ve fare kullanıldı. Hidatidozisli koyunlardan ve deneysel enfekte farelerden elde edilen kist sıvısı, kist membranı ve protoskolekslere ait proteinler Sodyum dodesil sülfat poliakrilamid jel elektroforez (SDS-PAGE) yöntemi ile ayırt edildi. Ayırt edilen proteinlerden hangilerinin hastalığa spesifik antijenler içerdiği immunblotting tekniği ile belirlendi. Koyun kist sıvısı, membran ve protoskolekslerde 116 kDa ile 26 kDa arasında, fare kist sıvısı ve membranlarında 106 ile 30 kDa arasında değişik sayılarda antijenik bantlar tespit edildi. Koyun kist sıvısı ile fare kist sıvısı arasındaki ortak antijenik bantların 40 kDa ve 26 kDa, koyun kist mebranı ile fare kist mebranı arasındaki ortak antijenik bantların 48 kDa ve 40 kDa olduğu belirlendi. Belirlenen antijenik proteinlerden, glikoprotein yapısında olanlar ve bunların hangi şekerleri bulundurdukları glikan kitleri ile belirlendi. Koyunlarda her üç kist materyalinde de (kist sıvısı, kist membranı, protoskoleks) 116 kDa, 68kDa, 48 kDa’luk proteinlerin, farelerde her iki kist materyalinde de (kist sıvısı, kist membranı) 106 kDa, 48, 40 kDa’luk proteinlerin ortak glikoproteinler olduğu tespit edildi. Bu glikoproteinlerin mannoz, galaktoz, N-asetil galaktozamin, sialik asit gibi şekerlerden bir ya da bir kaçını içerdikleri belirlendi. Koyun ve farelerin kist materyalleri arasında yapılan karşılaştırmada ortak bantların, molekül büyüklüğü, glikoprotein varlığı ve şeker yapıları yönünden benzerlikler ve farklılıklar gösterdiği görüldü

Keywords

Antijenik protein fare glikoprotein kist hidatik koyun

References

  • Akyol ÇV (2001): Hidatidoz ve halk sağlığı yönünden önemi. J. Fac. Vet. Med., 20, 137- 142.
  • Al-Yaman FM, Knobloch J. (1989): Isolation and partial characterisation of species specific and cross reactive antigens of Echinococcus granulosus cyst fluid. Mol. Biochem. Parasitol, 37, 101-108.
  • Burgu A, Doganay A, Gonenç B, Sarimehmetoglu HO, Kalinbacak F (2000): Analysis of fluids of hydatid cyst from sheep by SDS-PAGE and determination of specific antigens in protein structure by western blotting. Türk. J. Vet. Anim. Sci, 24, 493-500.
  • Burnie JP, Holland M, Matthews RC, Lees W (1987): Role immunoblotting in the diagnosis of culture negative and enterococcal endocarditis. J. Clin. Pathol, 40, 1149- 1158.
  • Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri, GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004): Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis , 25, 1327-1333.
  • Carmena D, Sánchez-Serrano LP, Barbero-Martínez I (2008): Echinococcus granulosus infection in Spain. Zoonoses Public Health, 55, 156–165.
  • Craig PS, Budke CM, Schantz PM (2007): Human echinococcosis: A neglected disease? Tropical Medicine and Healt. 35, 283-292.
  • Craig PS, Mcmanus DP, Lightowlers MW, Chabalgoity JA, Garcia HH, Gavidia CM, Gilman RH, Gonzalez AE, Lorca M, Naquira C, Nieto A, Shantz PM (2007): Prevention and control of cystic echinococcosis. Lancet Infect Dis, 7, 385-94.
  • Díaz A, Fontana EC, Todeschini AR, Soulé S, González H, Casaravilla C, Portela M, Mohana-Borges R, Mendonça-Previato L, Previato JO, Ferreira F (2009): theEchinococcus granulosus cyst: mucin-type O-glycans decorated by novel galactose-based structures. Biochemistry, 48, 11678-11691.
  • Díaz A, Casaravilla C, Allen JE, Sim RB, Ferreira AM (2011): Understanding the laminated layer of larval Echinococcus II: immunology. Trends Parasitol, 7, 264-73.
  • Doganay A, Burgu A, Gonenç B, Sarimehmetoglu HO (2000): Koyun kist hidatik protoskolekslerinin protein yapısının analizi ve spesifik antijenlerin saptanması. Ankara. Vet. Fak. Derg, 47, 63-71.
  • Ghorui SK, Prasad RL, Pal AK, Sahai BN (1990): Proteins and glycoproteins distribution in cyst wall and germinal layer of hydatid cyst. Indian Vet. J, 67, 802-804.
  • Gonzalez G, Spinelli P, Lorenzo C, Hellman U, Nieto A, Willis A, Salinas G (2000): Molecular characterization of P-29, a metacestode-specific component of Echinococcus granulosus which is immunologically related to, but distinct from, antigen 5. Mol Biochem Parasitol, 105, 177- 184.
  • Irabuena O, Nıeto , Ferreıra AM, Battistoni J, Ferragut G. (2000): Characterization and optimization of Bovine Echinococcus granulosus cyst fluid to be used ın immundiagnosis of hydatid disease by ELISA Rev. Inst. Med. Trop. S. Paulo, 42, 255-262.
  • Kanvar JR, Kaushık SP, Sawhney M.S, Kambo MS, Mehta S, Vinayak VK. (1992): Spesific antibodies in serum of patients with hydatidosis recognised by immunoblotting. J.Med.Microbiol, 16, 46-51.
  • Kanvar JR, Vinayak VK. (1993): Isolation& immunochemical characterization of diagnostically relevant antigens of Echinococcus granulosus. Indian J. Med. Res, 97, 75-82.
  • Karaman U, Atambay M, Aycan OM, Daldal N (2002): İndirekt hemaglütinasyon tekniğinde (IHA) insan, inek ve koyun antijenlerinin karşılaştırılması. T. Parazitol. Derg, 26, 251-253.
  • Khoo KK, Nıeto A, Morris HR, Dell A. (1997): Structural characterisation of the N-glycans from Echinococcus granulosus hydatid cyst membrane and protoscoleces. Mol. Biochem. Parasitol, 86, 237-248.
  • Köksal F, Serin MS, Kekeç Y, Sadri YE (1995): İnsan ve hayvan kökenli kist hidatik sıvılarının SDS-PAGE metoduyla analizi ve westernblot metodunun klinik önemi. T. Parazitol. Derg, 19, 221-229.
  • Laemmli K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.
  • Leggatt GR, Yong W, Mcmanus DP. (1992): Serological evaluation of the 12 kDa subunit of antigen B in E. granulosus cyst fluid by immunblot analysis. Trans. R. Soc. Trop. Med. Hyg, 86, 189-92.
  • Lisowska E. (2002). The role of glycosylation in protein antigenic properties. Cell. Mol. Life Sci, 59, 445-455.
  • Lowry OH, Rosebrough NJ, Farr AL, And Randall RJ (1951): Protein measurement with the folin phenol reagent. J. Biol. Chem, 193, 265-275.
  • Maddison SE, Slemenda SB, Schwantz PM, Fried JA, Wilson M, Tsang VCW. (1989): A Specific diagnostic antigen of Echinococcus Granulosus with an apparent molecular weight of 8 kda. Am. J. Trop. Hyg , 40, 377- 383.
  • March F, Enrich C, Mercader M, Sánchez F, Muñoz C, Coll P, Prats G (1991): Echinococcus granulosus: antigen characterization by chemical treatment and enzymatic deglycosylation. Exp. Parasitol, 73, 433-439.
  • Menezes da Silva A (2010): Human echinococcosis: a neglected disease. Gastroenterology Research and Practice., doi: 10.1155/2010/583297.
  • Richard MD, Ligtowlers MW (1986). Immundiagnosis of hydatid disease. In thompson RCA (Editor). The Biology of Echinococcus and Hydatid Disease. George Allen and Unwin, London: 217-249.
  • Rudd PM, Ellıott T, Cresswell P,Wılson IA, Dwek RA (2001): Glycosylation and the İmmun system. Science, 291, 2370-2376.
  • Sarimehmetoglu OH, Kubar A, Tanyuksel M, Gun H. (1998): Koyun karaciğer kist sıvısından 8 kDa’luk antijenin purifikasyonu. T Parazitol Derg, 22, 137-141.
  • Sbihi Y, Jansen D, Osuna A (1996): Serologic Rcognition of Hydatid cyst antigens using different purification methods. Diagn. Microbiol. Infect. Dis, 24, 205-211.
  • Shepherd JC, Mcmanus, DP (1987): Specific and cross- reactive antigens of Echinococcus granulosus Hydatid cyst fluid. Mol. Biochem Parasitol, 25, 143-154.
  • Siracusano A, İoppolo S, Notargiacomo S, Ortona E, Teggi A, De Rose F, Vicari G. (1991): Detection of antibodies against E. granulosus major antigens and their subunits by immunoblotting. Trans. of Royal. Soc. Trop. Med. Hyg, 85, 239-243.
  • Siracusano A, Delunardo F, Teggi A, OrtonaE (2012): Host-Parasite Relationship in Cystic Echinococcosis: An Evolving Story. Clin. Dev. Immunol. doi:10.1155/2012/639362.
  • Sasmaz E, Hashempoor GR, Bahar İH, Yulug N. (1995): Comparative antigenic analysis in Echinocaccus granulosus. T. Parasitol Derg, 19, 83-87.
  • Simşek S, Ütük AE, Babur C, Koroglu E. (2004): Ekinokok Antijenleri. Akciğer arşivi, 5, 158-161.
  • Walsh G, Jefferis R. (2006): Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol, 24, 1241-1252.
  • Varkı A, Cummıngs R, Esko J, Freeze H, Har, G, Marth J (1999): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. New York.
  • Zhang XL (2006): Roles of glycans and glycopeptides in immune system and immune-related diseases. Curr Med Chem, 13, 1141-7.
  • Zingales B. (1984): Analysis of proteins by dodecyl sulfate-polyacrylamide gel electrophoresis. In: Genes and Antigenes of Parasites. C.M. Marel Ed., Fiocruz, Rio de Janeiro, pp. 357-363.

Comparative analysis of sheep and mice hydatid cyst proteins and evaluation of the glycoproteinoccurence in the antigenic proteins

Year 2014, Volume: 61 Issue: 4, 243 - 248, 01.12.2014

Serap Ünübol Aypak Hamdi Uysal

https://doi.org/10.1501/Vetfak_0000002637

Abstract

The aim of this study is to compare the hydatid cyst proteins of mice and sheep in terms of antigenic proteins and antigenic glycoproteins. For this reason 20 each infected sheep and mice and 10 each healthy sheep and mice were used. Cyst fluid, cyst membrane and proteins from protoscolex which were derived from hydatidosis infected sheeps and experimentaly infected mice were separated by SDS-PAGE. Antigenic proteins were determined by immunblotting technique. Moleculer weights of antigenic proteins from cyst fluid, cyst membrane and protoscolex of sheep were determined in size between 116 kDa and 26 kDa by SDSPAGE. Cyst fluid and cyst membrane of mice, molecular weights of antigenic proteins were found between 106 kDa and 30kDa. Common antigenic bands of hydatid fluid from sheep and mice were found to be 40 kDa and 26 kDa. Common antigenic bands of hydatid membrane from sheep and mice found to be 48 kDa and 40 kDa. Sugars of antigenic proteins in glycoprotein structure were determined by glycan kits. Glycan analysis of samples showed that 116 kDa, 68 kDa and 48 kDa proteins in each three cyst material (cyst fluid, cyst membrane and protoscolex) obtained from sheep were found to be common glycoproteins. In addition to this, 106 kDa, 48 kDa and 40 kDa proteins in each two cyst material (cyst fluid, cyst membrane) obtained from mice were also found to be common glycoproteins. It was determined that these glycoproteins include one or more sugar structures such as mannose, galactose, N-acetyl galactosamine and sialic acid. In the context of sheep and mice cyst material comparison, it was determined that common proteins shows similarities and dissimilarities in molecular weight, glycoprotein and sugar structures in general

Keywords

Antigenic protein cyst hydatid glycoprotein mice sheep

References

  • Akyol ÇV (2001): Hidatidoz ve halk sağlığı yönünden önemi. J. Fac. Vet. Med., 20, 137- 142.
  • Al-Yaman FM, Knobloch J. (1989): Isolation and partial characterisation of species specific and cross reactive antigens of Echinococcus granulosus cyst fluid. Mol. Biochem. Parasitol, 37, 101-108.
  • Burgu A, Doganay A, Gonenç B, Sarimehmetoglu HO, Kalinbacak F (2000): Analysis of fluids of hydatid cyst from sheep by SDS-PAGE and determination of specific antigens in protein structure by western blotting. Türk. J. Vet. Anim. Sci, 24, 493-500.
  • Burnie JP, Holland M, Matthews RC, Lees W (1987): Role immunoblotting in the diagnosis of culture negative and enterococcal endocarditis. J. Clin. Pathol, 40, 1149- 1158.
  • Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri, GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004): Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis , 25, 1327-1333.
  • Carmena D, Sánchez-Serrano LP, Barbero-Martínez I (2008): Echinococcus granulosus infection in Spain. Zoonoses Public Health, 55, 156–165.
  • Craig PS, Budke CM, Schantz PM (2007): Human echinococcosis: A neglected disease? Tropical Medicine and Healt. 35, 283-292.
  • Craig PS, Mcmanus DP, Lightowlers MW, Chabalgoity JA, Garcia HH, Gavidia CM, Gilman RH, Gonzalez AE, Lorca M, Naquira C, Nieto A, Shantz PM (2007): Prevention and control of cystic echinococcosis. Lancet Infect Dis, 7, 385-94.
  • Díaz A, Fontana EC, Todeschini AR, Soulé S, González H, Casaravilla C, Portela M, Mohana-Borges R, Mendonça-Previato L, Previato JO, Ferreira F (2009): theEchinococcus granulosus cyst: mucin-type O-glycans decorated by novel galactose-based structures. Biochemistry, 48, 11678-11691.
  • Díaz A, Casaravilla C, Allen JE, Sim RB, Ferreira AM (2011): Understanding the laminated layer of larval Echinococcus II: immunology. Trends Parasitol, 7, 264-73.
  • Doganay A, Burgu A, Gonenç B, Sarimehmetoglu HO (2000): Koyun kist hidatik protoskolekslerinin protein yapısının analizi ve spesifik antijenlerin saptanması. Ankara. Vet. Fak. Derg, 47, 63-71.
  • Ghorui SK, Prasad RL, Pal AK, Sahai BN (1990): Proteins and glycoproteins distribution in cyst wall and germinal layer of hydatid cyst. Indian Vet. J, 67, 802-804.
  • Gonzalez G, Spinelli P, Lorenzo C, Hellman U, Nieto A, Willis A, Salinas G (2000): Molecular characterization of P-29, a metacestode-specific component of Echinococcus granulosus which is immunologically related to, but distinct from, antigen 5. Mol Biochem Parasitol, 105, 177- 184.
  • Irabuena O, Nıeto , Ferreıra AM, Battistoni J, Ferragut G. (2000): Characterization and optimization of Bovine Echinococcus granulosus cyst fluid to be used ın immundiagnosis of hydatid disease by ELISA Rev. Inst. Med. Trop. S. Paulo, 42, 255-262.
  • Kanvar JR, Kaushık SP, Sawhney M.S, Kambo MS, Mehta S, Vinayak VK. (1992): Spesific antibodies in serum of patients with hydatidosis recognised by immunoblotting. J.Med.Microbiol, 16, 46-51.
  • Kanvar JR, Vinayak VK. (1993): Isolation& immunochemical characterization of diagnostically relevant antigens of Echinococcus granulosus. Indian J. Med. Res, 97, 75-82.
  • Karaman U, Atambay M, Aycan OM, Daldal N (2002): İndirekt hemaglütinasyon tekniğinde (IHA) insan, inek ve koyun antijenlerinin karşılaştırılması. T. Parazitol. Derg, 26, 251-253.
  • Khoo KK, Nıeto A, Morris HR, Dell A. (1997): Structural characterisation of the N-glycans from Echinococcus granulosus hydatid cyst membrane and protoscoleces. Mol. Biochem. Parasitol, 86, 237-248.
  • Köksal F, Serin MS, Kekeç Y, Sadri YE (1995): İnsan ve hayvan kökenli kist hidatik sıvılarının SDS-PAGE metoduyla analizi ve westernblot metodunun klinik önemi. T. Parazitol. Derg, 19, 221-229.
  • Laemmli K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685.
  • Leggatt GR, Yong W, Mcmanus DP. (1992): Serological evaluation of the 12 kDa subunit of antigen B in E. granulosus cyst fluid by immunblot analysis. Trans. R. Soc. Trop. Med. Hyg, 86, 189-92.
  • Lisowska E. (2002). The role of glycosylation in protein antigenic properties. Cell. Mol. Life Sci, 59, 445-455.
  • Lowry OH, Rosebrough NJ, Farr AL, And Randall RJ (1951): Protein measurement with the folin phenol reagent. J. Biol. Chem, 193, 265-275.
  • Maddison SE, Slemenda SB, Schwantz PM, Fried JA, Wilson M, Tsang VCW. (1989): A Specific diagnostic antigen of Echinococcus Granulosus with an apparent molecular weight of 8 kda. Am. J. Trop. Hyg , 40, 377- 383.
  • March F, Enrich C, Mercader M, Sánchez F, Muñoz C, Coll P, Prats G (1991): Echinococcus granulosus: antigen characterization by chemical treatment and enzymatic deglycosylation. Exp. Parasitol, 73, 433-439.
  • Menezes da Silva A (2010): Human echinococcosis: a neglected disease. Gastroenterology Research and Practice., doi: 10.1155/2010/583297.
  • Richard MD, Ligtowlers MW (1986). Immundiagnosis of hydatid disease. In thompson RCA (Editor). The Biology of Echinococcus and Hydatid Disease. George Allen and Unwin, London: 217-249.
  • Rudd PM, Ellıott T, Cresswell P,Wılson IA, Dwek RA (2001): Glycosylation and the İmmun system. Science, 291, 2370-2376.
  • Sarimehmetoglu OH, Kubar A, Tanyuksel M, Gun H. (1998): Koyun karaciğer kist sıvısından 8 kDa’luk antijenin purifikasyonu. T Parazitol Derg, 22, 137-141.
  • Sbihi Y, Jansen D, Osuna A (1996): Serologic Rcognition of Hydatid cyst antigens using different purification methods. Diagn. Microbiol. Infect. Dis, 24, 205-211.
  • Shepherd JC, Mcmanus, DP (1987): Specific and cross- reactive antigens of Echinococcus granulosus Hydatid cyst fluid. Mol. Biochem Parasitol, 25, 143-154.
  • Siracusano A, İoppolo S, Notargiacomo S, Ortona E, Teggi A, De Rose F, Vicari G. (1991): Detection of antibodies against E. granulosus major antigens and their subunits by immunoblotting. Trans. of Royal. Soc. Trop. Med. Hyg, 85, 239-243.
  • Siracusano A, Delunardo F, Teggi A, OrtonaE (2012): Host-Parasite Relationship in Cystic Echinococcosis: An Evolving Story. Clin. Dev. Immunol. doi:10.1155/2012/639362.
  • Sasmaz E, Hashempoor GR, Bahar İH, Yulug N. (1995): Comparative antigenic analysis in Echinocaccus granulosus. T. Parasitol Derg, 19, 83-87.
  • Simşek S, Ütük AE, Babur C, Koroglu E. (2004): Ekinokok Antijenleri. Akciğer arşivi, 5, 158-161.
  • Walsh G, Jefferis R. (2006): Post-translational modifications in the context of therapeutic proteins. Nat. Biotechnol, 24, 1241-1252.
  • Varkı A, Cummıngs R, Esko J, Freeze H, Har, G, Marth J (1999): Essentials of Glycobiology. Cold Spring Harbor Laboratory Press. New York.
  • Zhang XL (2006): Roles of glycans and glycopeptides in immune system and immune-related diseases. Curr Med Chem, 13, 1141-7.
  • Zingales B. (1984): Analysis of proteins by dodecyl sulfate-polyacrylamide gel electrophoresis. In: Genes and Antigenes of Parasites. C.M. Marel Ed., Fiocruz, Rio de Janeiro, pp. 357-363.
There are 39 citations in total.

Details

Primary Language English
Subjects Veterinary Surgery
Other ID JA28NA27CH
Journal Section Research Article
Authors

Serap Ünübol Aypak

Hamdi Uysal

Publication Date December 1, 2014
Published in Issue Year 2014Volume: 61 Issue: 4

Cite

APA Ünübol Aypak, S., & Uysal, H. (2014). Comparative analysis of sheep and mice hydatid cyst proteins and evaluation of the glycoproteinoccurence in the antigenic proteins. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 61(4), 243-248. https://doi.org/10.1501/Vetfak_0000002637
AMA Ünübol Aypak S, Uysal H. Comparative analysis of sheep and mice hydatid cyst proteins and evaluation of the glycoproteinoccurence in the antigenic proteins. Ankara Univ Vet Fak Derg. December 2014;61(4):243-248. doi:10.1501/Vetfak_0000002637
Chicago Ünübol Aypak, Serap, and Hamdi Uysal. “Comparative Analysis of Sheep and Mice Hydatid Cyst Proteins and Evaluation of the Glycoproteinoccurence in the Antigenic Proteins”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 61, no. 4 (December 2014): 243-48. https://doi.org/10.1501/Vetfak_0000002637.
EndNote Ünübol Aypak S, Uysal H (December 1, 2014) Comparative analysis of sheep and mice hydatid cyst proteins and evaluation of the glycoproteinoccurence in the antigenic proteins. Ankara Üniversitesi Veteriner Fakültesi Dergisi 61 4 243–248.
IEEE S. Ünübol Aypak and H. Uysal, “Comparative analysis of sheep and mice hydatid cyst proteins and evaluation of the glycoproteinoccurence in the antigenic proteins”, Ankara Univ Vet Fak Derg, vol. 61, no. 4, pp. 243–248, 2014, doi: 10.1501/Vetfak_0000002637.
ISNAD Ünübol Aypak, Serap - Uysal, Hamdi. “Comparative Analysis of Sheep and Mice Hydatid Cyst Proteins and Evaluation of the Glycoproteinoccurence in the Antigenic Proteins”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 61/4 (December 2014), 243-248. https://doi.org/10.1501/Vetfak_0000002637.
JAMA Ünübol Aypak S, Uysal H. Comparative analysis of sheep and mice hydatid cyst proteins and evaluation of the glycoproteinoccurence in the antigenic proteins. Ankara Univ Vet Fak Derg. 2014;61:243–248.
MLA Ünübol Aypak, Serap and Hamdi Uysal. “Comparative Analysis of Sheep and Mice Hydatid Cyst Proteins and Evaluation of the Glycoproteinoccurence in the Antigenic Proteins”. Ankara Üniversitesi Veteriner Fakültesi Dergisi, vol. 61, no. 4, 2014, pp. 243-8, doi:10.1501/Vetfak_0000002637.
Vancouver Ünübol Aypak S, Uysal H. Comparative analysis of sheep and mice hydatid cyst proteins and evaluation of the glycoproteinoccurence in the antigenic proteins. Ankara Univ Vet Fak Derg. 2014;61(4):243-8.