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Kanatlılarda Kronik Solunum Yolu Hastalığının (CRD) Serolojik Tanısı İçin in-House Enzyme- Linked Immunosorbent Assay (ELISA) Geliştirilmesi

Yıl 2023, Cilt: 34 Sayı: 2, 198 - 204, 28.12.2023
https://doi.org/10.35864/evmd.1330808

Öz

Tavukların üst solunum yollarında Mycoplasma gallisepticum (MG)’un oluşturduğu kronik solunum sistemi hastalığı (CRD), dünyanın birçok ülkesinde kanatlı sağlığı ve hayvansal üretim üzerinde büyük etkileri olan, ciddi ekonomik kayıplara sebebiyet veren bulaşıcı bir infeksiyondur. Hem hastalığın teşhisinde hem de antikor titresinin belirlenmesinde yaygın olarak kullanılan Enzyme-Linked Immunosorbent Assay (ELISA) sensitivite ve spesifitesi yüksek olan bir testtir. Bu çalışmada, CRD infeksiyonuna karşı gelişen antikorları saptamak için farklı suşların antijen olarak kullanıldığı “in house” bir ELISA prototipi geliştirilmesi amaçlanmıştır. Bu amaçla 1 standart suş (MG S6), 1 aşı suşu (MG 6/85) ve 3 saha suşu (MG HMA 9, MG HMA 79, MG HMA 120) olmak üzere 5 suş ile hazırlanan ELISA’ların referans serumlar (125 gerçek pozitif ve 44 gerçek negatif) kullanılarak duyarlılığı ve özgüllüğü saptanmıştır. Buna göre MG 6/85, MG HMA 9, MG HMA 79, MG HMA 120 ve MG S6 suşlarından hazırlanan ELISA’ların duyarlılığı sırası ile % 92, %94, %95, % 96,8 ve % 92 olmuştur. Testlerin özgüllüğü aynı sıra ile, % 95,6 %95,6, %95,6, %96,7 ve % 93,6 olarak hesaplanmıştır. Özgüllüğü ve duyarlılığı diğerlerine göre daha yüksek olan MG HMA 120 suşundan hazırlanan ELISA prototipi ticari üretim için seçilmiştir. M. gallisepticum ile infekte bir sürüden alınan 35 serumun MG HMA 120 ELISA ile test edilmesi sonucunda serumların 28 adedi pozitif, 5 adedi şüpheli ve 2 adedi negatif olarak saptanmıştır. OD değerlerine bağlı olarak aynı serumların ticari MG ELISA ile test edilmesi ile alınan sonuçlar yerli MG HMA 120 ELISA ile alınan sonuçlar karşılaştırılmıştır. İki kit arasındaki yüksek bir korelasyon katsayısı bulunmuş (Pearson’s korelasyon katsayısı (r)=0,78, p=0,01) ve yerli bir suştan yeni geliştirilen in house ELISA’ nın M. gallisepticum’un neden olduğu CRD vakalarında güvenle kullanılabilecek, ticari muadillerine göre benzer özgüllük ve duyarlılık gösteren ve saha kullanımı için pratik ve ucuz bir alternatif olduğunu göstermektedir.

Destekleyen Kurum

Harran Üniversitesi Bilimsel Araştırma Koordinasyon Kurulu Başkanlığı

Proje Numarası

21304

Teşekkür

Pendik Veteriner Kontrol Enstitüsü, WOAH Mycoplasma Referens Laboratuvarı ve MSD Hayvan Sağlığı’na teşekkür ederiz.

Kaynakça

  • Akan M. (2008) Tavuklarda Mikoplazma İnfeksiyonları: Koruma ve Kontrol. Veteriner Tavukçuluk Derneği Mektup Ankara. 6, 21-24.
  • Ali Z, Sultana S, Karim R, Hassan Z, Yousuf A, Hossen A, Samad MA, Giasuddin M, Rahman M. (2017) Compared the effect of indirect ELISA and serum plate agglutination (SPA) test fort he detection of Mycoplasma gallisepticum in chicken. Int J Health Animal Sci Food Saf, 4, 59-66. https://doi.org/10.13130/2283-3927/8975
  • Elyazeed HA, Al-Atfeehy NM, Abotaleb R, Sayed R, Marouf S. (2020) Preparation of ELISA and Lateral Flow Kits for rapid Diagnosis of Mycoplasma gallisepticum in Poultry. Sci Rep, 10, 9056. https://doi.org/10.1038/s41598-020-65848-7
  • Esendal ÖM. (2002) Mikoplazma Enfeksiyonları “Kanatlı Hayvan Hastalıkları”. Ed; Müjgan İzgür ve Mehmet Akan, 1. baskı, 79-94. Medisan, Ankara, Türkiye. Evans JD, Leigh SA, Branton SL, Collier SD, Pharr GT, Bearson SMD. (2005) Mycoplasma gallisepticum: current and devoloping means to control the avian pathogen. Poult Sci. 14, 757-763. https://doi.org/10.1093/japr/14.4.757
  • Frey ML, Hanson RP, Anderson DP. (1968) A Medium for the Isolation of Avian Mycoplasmas. Am J Vet Res. 29, 2163-2171. Haghighi-Khoshkhoo P, Akbariazad G, Roohi M, Inanlo J, Masoumi M, Sami-Yousefi P. (2011) Seroprevalence of Mycoplasma gallisepticum and Mycoplasma synoviae infection in the commercial layer flocks of the centernorth of Iran. Afr J Microbiol Res, 5(18), 2834-2837. https://doi.org/10.5897/AJMR11.647
  • Hosseini S, Rito-Palomares M, Vazquez-Villegas P, Martinez-Chapa SO. (2018) Enzyme-linked Immunosorbent Assay From A to Z. Springer, India. https://doi.org/10.1007/978-981-10-6766-2 IBM Corp. (2020) IBM SPSS Statistics for Windows (Version 16.0) [Computer software]. IBM Corp.
  • Kapetanov M, Orlić D, Potkonjak D, Velhner M, Stojanov I, Milanov D, Stojanovic D. (2010) Mycoplasma in poultry flocks in the year 2009 compared to the year 2000 and significance of the control measures. Lucr. ştiinţ. - Univ. Agron. "Ion Ionescu de la Brad" Iaşi, Ser. zooteh.-med. vet., 43(1), 249-253.
  • Kleven SH. (1998) Mycoplasmosis. In: A laboratory Manual for the Isolation and Identification of Avian Pathogens. 4th ed. DE Swayne, JR Glisson, MW Jackwood, JE Pearson and WM Reed (eds). American Association of Avian Pathologists, University of Pennsylvania, New Bolton Center, Kennett Square, PA. 74-80.
  • Kleven SH. (2008) Control of Avian Mycoplasma Infections in Commercial Poultry. Avian Dis. 52, 367-374. https://doi.org/10.1637/8323-041808-Review.1
  • Koca S. (2017) Türkiye’de Broiler Et Üretimi: Hedefler ve Potansiyel Problemler. Veteriner Tavukculuk Derneği Mektup Ankara. 15(1), 4-10.
  • Malik M, Sohail M, Sajid M, Hammidullah, Bibi F, Shoaib M. (2019) Seroprevalence of Mycoplasma gallisepticum in non Vaccinated broiler flocks in Abbottabad Khyberpakhtunkhwa, Pakistan. Research in: Agricultural & Veterinary Sciences, 3(1), 36-42.
  • Miao D, Zhang P, Gong Y, Yamaguchi T, Iritani Y, Blackall PJ (2000) The development and application of a blocking ELISA kit for the diagnosis of infectious coryza. Avian Pathol. 29: 219-225. https://doi.org/10.1080/03079450050045477
  • Noormohammadi AH, Browning GF, Cowling PJ, O’Rourke D, Whithear WG, Markham PF (2002) Detection of antibodies to Mycoplasma gallisepticum vaccine ts-11 by an autologous pMGA enzyme-linked immunosorbent assay. Avian Dis. 46, 405-411. https://doi.org/10.1637/0005-2086(2002)046[0405:DOATMG]2.0.CO;2
  • Osman KM, Aly MM, Amin ZMS, Hasan BS. (2009): Mycoplasma gallisepticum: an emerging challenge to the poultry industry in Egypt. Rev Sci tech Off int Epiz. 28 (3), 1015-1023. http://dx.doi.org/10.20506/rst.28.3.1940
  • Özdemir Ö. (2013) Tavukçuluk Sektöründe Devam Eden Sorun: Mycoplasma gallisepticum (MG) İnfeksiyonuna Genel Bir Bakış. Veteriner Tavukçuluk Derneği Mektup Ankara. 11(2), 5-10.
  • Özgün MR, Türkyılmaz S. (2016) The Determination of Mycoplasma gallisepticum and Mycoplasma synoviae antibodies in poultry blood sera with ELISA. Kocatepe Vet J. 9(1), 19-23. DOI: 10.5578/kvj.10870
  • Peng P, Liu C, Li Z, Xue X, Mao P, Hu J, Xu F, Yao C, You M. (2022) Emerging ELISA derived technologies for in vitro diagnostics. Trens Anal Chem, 152, 116605. https://doi.org/10.1016/j.trac.2022.116605
  • Rasool A, Anjum AA, Rabbani M, Lateef M, Nawaz M, Akhtar F, Kanwal A, Sattar S. (2017) Preparation of Mycoplasma synoviae antigens and evaluation by rapid slide agglutination and enzyme linked immunosorbent assay. The J Anim Plant Sci. 27(3), 841-847.
  • Sato S. (1996) Avian mycoplasmosis in Asia. Rev Sci tech Off int Epiz. 15(4), 1555-1567. https://doi.org/10.20506/rst.15.4.984
  • Swayne DE. (2013) Diseases of Poultry, 13th Edt. John Wiley & Sons, Inc. UK. Wanasawaeng W, Chaichote S, Chansiripornchai N. (2015) Development of ELISA and Serum Plate Agglutination for Detecting Antibodies of Mycoplasma gallisepticum using Strain of Thai Isolate. Thai. J Vet Med, 45(4), 499-507.
  • WOAH Terresterial Manual. (2021) Avian mycoplasmosis. Manual of diagnostic tests and vaccines for terrestrial animals, Chapter 3.3.5.

Preparation of In-House Enzyme- Linked Immunosorbent Assay (ELISA for serologic diagnosis of chronic respiratory disease (CRD) in poultry

Yıl 2023, Cilt: 34 Sayı: 2, 198 - 204, 28.12.2023
https://doi.org/10.35864/evmd.1330808

Öz

Chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum (MG) in the upper respiratory tract of chickens, is a contagious infection that causes severe economic losses in many countries of the world with a great impact on poultry health and animal production. Enzyme-Linked Immunosorbent Assay (ELISA) has high sensitivity and specificity, which is widely used for both diagnosis of the disease and antibody titer determination. In this study, it was aimed to develop an "in house" ELISA prototype in which different strains are used as antigens to detect antibodies to CRD infection. For this purpose, the sensitivity and specificity of ELISAs prepared with a total of 5 strains, including 1 standard strain (MG S6), 1 vaccine strain (MG 6/85 and 3 field strains (MG HMA 9, MG HMA 79, MG HMA 120), were determined using 125 true positive and 44 true negative reference sera. Sentitivity rates were found as 92%, 94%, 95%, 96.8% and 92%, respectively. Specifities were found as 95.6%, 95.6% ,95.6%, 96.7% and 93.6%, respectively. According to the results, MG HMA 120 local strain based in house ELISA was chosen for commercial production for later stage. A total of 35 sera from M. gallisepticum infected flock were tested by commercial ELISA and our in house MG HMA120 ELISA for comparison based on OD values. 28 out of 35 were found positive, 5 suspected and 2 negative by using MG HMA 120 ELISA. A high correlation coefficient (r)=0.78, p=0.01) was determined between commercial and in house ELISA. It was concluded that newly developed in house ELISA using local whole cell antigen could be used safely, giving similar sensitivity and specificity to commercial ELISA kit, for serologic diagnosis of M. gallisepticum infections and it is practical and cheap alternative for field use.

Proje Numarası

21304

Kaynakça

  • Akan M. (2008) Tavuklarda Mikoplazma İnfeksiyonları: Koruma ve Kontrol. Veteriner Tavukçuluk Derneği Mektup Ankara. 6, 21-24.
  • Ali Z, Sultana S, Karim R, Hassan Z, Yousuf A, Hossen A, Samad MA, Giasuddin M, Rahman M. (2017) Compared the effect of indirect ELISA and serum plate agglutination (SPA) test fort he detection of Mycoplasma gallisepticum in chicken. Int J Health Animal Sci Food Saf, 4, 59-66. https://doi.org/10.13130/2283-3927/8975
  • Elyazeed HA, Al-Atfeehy NM, Abotaleb R, Sayed R, Marouf S. (2020) Preparation of ELISA and Lateral Flow Kits for rapid Diagnosis of Mycoplasma gallisepticum in Poultry. Sci Rep, 10, 9056. https://doi.org/10.1038/s41598-020-65848-7
  • Esendal ÖM. (2002) Mikoplazma Enfeksiyonları “Kanatlı Hayvan Hastalıkları”. Ed; Müjgan İzgür ve Mehmet Akan, 1. baskı, 79-94. Medisan, Ankara, Türkiye. Evans JD, Leigh SA, Branton SL, Collier SD, Pharr GT, Bearson SMD. (2005) Mycoplasma gallisepticum: current and devoloping means to control the avian pathogen. Poult Sci. 14, 757-763. https://doi.org/10.1093/japr/14.4.757
  • Frey ML, Hanson RP, Anderson DP. (1968) A Medium for the Isolation of Avian Mycoplasmas. Am J Vet Res. 29, 2163-2171. Haghighi-Khoshkhoo P, Akbariazad G, Roohi M, Inanlo J, Masoumi M, Sami-Yousefi P. (2011) Seroprevalence of Mycoplasma gallisepticum and Mycoplasma synoviae infection in the commercial layer flocks of the centernorth of Iran. Afr J Microbiol Res, 5(18), 2834-2837. https://doi.org/10.5897/AJMR11.647
  • Hosseini S, Rito-Palomares M, Vazquez-Villegas P, Martinez-Chapa SO. (2018) Enzyme-linked Immunosorbent Assay From A to Z. Springer, India. https://doi.org/10.1007/978-981-10-6766-2 IBM Corp. (2020) IBM SPSS Statistics for Windows (Version 16.0) [Computer software]. IBM Corp.
  • Kapetanov M, Orlić D, Potkonjak D, Velhner M, Stojanov I, Milanov D, Stojanovic D. (2010) Mycoplasma in poultry flocks in the year 2009 compared to the year 2000 and significance of the control measures. Lucr. ştiinţ. - Univ. Agron. "Ion Ionescu de la Brad" Iaşi, Ser. zooteh.-med. vet., 43(1), 249-253.
  • Kleven SH. (1998) Mycoplasmosis. In: A laboratory Manual for the Isolation and Identification of Avian Pathogens. 4th ed. DE Swayne, JR Glisson, MW Jackwood, JE Pearson and WM Reed (eds). American Association of Avian Pathologists, University of Pennsylvania, New Bolton Center, Kennett Square, PA. 74-80.
  • Kleven SH. (2008) Control of Avian Mycoplasma Infections in Commercial Poultry. Avian Dis. 52, 367-374. https://doi.org/10.1637/8323-041808-Review.1
  • Koca S. (2017) Türkiye’de Broiler Et Üretimi: Hedefler ve Potansiyel Problemler. Veteriner Tavukculuk Derneği Mektup Ankara. 15(1), 4-10.
  • Malik M, Sohail M, Sajid M, Hammidullah, Bibi F, Shoaib M. (2019) Seroprevalence of Mycoplasma gallisepticum in non Vaccinated broiler flocks in Abbottabad Khyberpakhtunkhwa, Pakistan. Research in: Agricultural & Veterinary Sciences, 3(1), 36-42.
  • Miao D, Zhang P, Gong Y, Yamaguchi T, Iritani Y, Blackall PJ (2000) The development and application of a blocking ELISA kit for the diagnosis of infectious coryza. Avian Pathol. 29: 219-225. https://doi.org/10.1080/03079450050045477
  • Noormohammadi AH, Browning GF, Cowling PJ, O’Rourke D, Whithear WG, Markham PF (2002) Detection of antibodies to Mycoplasma gallisepticum vaccine ts-11 by an autologous pMGA enzyme-linked immunosorbent assay. Avian Dis. 46, 405-411. https://doi.org/10.1637/0005-2086(2002)046[0405:DOATMG]2.0.CO;2
  • Osman KM, Aly MM, Amin ZMS, Hasan BS. (2009): Mycoplasma gallisepticum: an emerging challenge to the poultry industry in Egypt. Rev Sci tech Off int Epiz. 28 (3), 1015-1023. http://dx.doi.org/10.20506/rst.28.3.1940
  • Özdemir Ö. (2013) Tavukçuluk Sektöründe Devam Eden Sorun: Mycoplasma gallisepticum (MG) İnfeksiyonuna Genel Bir Bakış. Veteriner Tavukçuluk Derneği Mektup Ankara. 11(2), 5-10.
  • Özgün MR, Türkyılmaz S. (2016) The Determination of Mycoplasma gallisepticum and Mycoplasma synoviae antibodies in poultry blood sera with ELISA. Kocatepe Vet J. 9(1), 19-23. DOI: 10.5578/kvj.10870
  • Peng P, Liu C, Li Z, Xue X, Mao P, Hu J, Xu F, Yao C, You M. (2022) Emerging ELISA derived technologies for in vitro diagnostics. Trens Anal Chem, 152, 116605. https://doi.org/10.1016/j.trac.2022.116605
  • Rasool A, Anjum AA, Rabbani M, Lateef M, Nawaz M, Akhtar F, Kanwal A, Sattar S. (2017) Preparation of Mycoplasma synoviae antigens and evaluation by rapid slide agglutination and enzyme linked immunosorbent assay. The J Anim Plant Sci. 27(3), 841-847.
  • Sato S. (1996) Avian mycoplasmosis in Asia. Rev Sci tech Off int Epiz. 15(4), 1555-1567. https://doi.org/10.20506/rst.15.4.984
  • Swayne DE. (2013) Diseases of Poultry, 13th Edt. John Wiley & Sons, Inc. UK. Wanasawaeng W, Chaichote S, Chansiripornchai N. (2015) Development of ELISA and Serum Plate Agglutination for Detecting Antibodies of Mycoplasma gallisepticum using Strain of Thai Isolate. Thai. J Vet Med, 45(4), 499-507.
  • WOAH Terresterial Manual. (2021) Avian mycoplasmosis. Manual of diagnostic tests and vaccines for terrestrial animals, Chapter 3.3.5.
Toplam 21 adet kaynakça vardır.

Ayrıntılar

Birincil Dil Türkçe
Konular Veteriner Mikrobiyolojisi
Bölüm Araştırma Makaleleri
Yazarlar

Sevil Erdenlig Gürbilek 0000-0002-0377-2650

Ayfer Güllü Yücetepe 0000-0002-9842-3305

Ahmet Murat Saytekin 0000-0001-7486-8054

Oktay Keskin 0000-0002-5977-7872

Yaşar Tel 0000-0001-7848-3899

Proje Numarası 21304
Yayımlanma Tarihi 28 Aralık 2023
Gönderilme Tarihi 21 Temmuz 2023
Yayımlandığı Sayı Yıl 2023 Cilt: 34 Sayı: 2

Kaynak Göster

APA Erdenlig Gürbilek, S., Güllü Yücetepe, A., Saytekin, A. M., Keskin, O., vd. (2023). Kanatlılarda Kronik Solunum Yolu Hastalığının (CRD) Serolojik Tanısı İçin in-House Enzyme- Linked Immunosorbent Assay (ELISA) Geliştirilmesi. Etlik Veteriner Mikrobiyoloji Dergisi, 34(2), 198-204. https://doi.org/10.35864/evmd.1330808


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