Pencegahan koksidiosis pada ayam melalui pengebalan dengan antigen Ekskresi-Sekresi Sporozoit Eimeria tenella

PRASTOWO, Promotor Prof.drh. R. Wasito, M.Sc.,Ph.D

2006 | Disertasi | S3 Ilmu Pertanian (Sain Veteriner)

Penelitian ini bertujuan untuk mengidentifikasi stadium siklus hidup E. tenella yang berpotensi menimbulkan respon imun, mengidentifikasi antigen ekskresi-sekresi sporozoit yang bersifat imunogenik dan menentukan tingkat keberhasilan program vaksinasi pada ayam yang divaksin antigen ekskresi-sekresi sporozoit. Oosista E. tenella isolat Sleman, Yogyakarta diperbanyak menggunakan ayam broiler umur 3 minggu. Eksistasi sporosista dari oosista dilakukan secara mekanik dengan glass beads, sporozoit dari sporosista dieksistasi menggunakan enzim tripsin dan sodium taurokolat. Preparasi antigen ekskresi-sekresi sporozoit dilakukan dengan cara beku - cair dan sonikasi. Visualisai antigen ekskresisekresi sporozoit menggunakan SDS-PAGE, identifikasi antigen ekskresi-sekresi sporozoit dengan antibodi monoklonal menggunakan Western blot. Uji lapang terhadap antigen yang telah diisolasi dilakukan pada 60 ayam broiler yang dibagi menjadi 6 kelompok. Kelompok I divaksin dengan Immucox® dengan dosis 105 oosista, Kelompok II divaksin dengan 100 μg antigen ekskresi-sekresi sporozoit diemulsikan dalam FCA, Kelompok III divaksin dengan 100 μg antigen ekskresisekresi sporozoit yang dilarutkan dalam PBS dan Kelompok IV adalah kontrol yang tidak divaksin dan tidak ditantang. Kelompok V ditantang dan tidak divaksin, serta Kelompok VI divaksin seperti pada Kelompok II dan tidak ditantang. Vaksinasi dilakukan secara subkutan pada umur 4 hari. Uji tantang dengan pemberian 2.000 oosista secara oral pada hari ke-14. Data yang dikumpulkan adalah data gejala klinis yang dianalisis secara diskriptif, derajat lesi sekum dengan metode Rank Test, eliminasi oosista dan titer antibodi dengan Mixed Design Analysis of Variance, dilanjutkan dengan uji ortogonal kontras. Hasil visualisasi menunjukkan bahwa berat molekul antigen ekskresisekresi sporozoit mempunyai fraksi protein antara 14 KDa sampai 96 KDa dan menghasilkan 12 macam antibodi monoklonal. Hasil immunoblotting 5 sel hibridoma menunjukan reaksi yang menciri yaitu : Mabset1, Mabset2, Mabset3, Mabset4 dan Mabset5. Masing-masing Mabset tersebut mengenal epitop protein dengan berat molekul 14,7 KDa, 42 KDa, 43 KDa, 47 KDa dan 90 KDa.Vaksinasi dengan antigen ekskresi-sekresi sporozoit mampu menghilangkan gejala klinis berupa berak darah, menurunkan sekor lesi sekum, mengurangi eliminasi oosista dan meningkatkan titer antibodi.

The research was conducted to identify the life cycle stage of E. tenella that has a potential to stimulate immune response, identification of sporozoit excretory–secretory immunogenic antigen and determination of successful parameter of vaccination program in sporozoite excretory–secretory antigen vaccinated chicken. Sleman E. tenella oocyst isolate was multiplied using the 3 weeks old broiler chickens. The process of sporocyst excitation from oocyst was done mechanicaly by using glass beads, while the sporocyst excitation from sporozoit was done by adding trypsin enzyme and sodium taurokholat. The preparation of sporozoit excretory–secretory antigen protein was processed by thawing and freezing and sonication. Visualisation of sporozoite excretory–secretory protein antigen was done by using SDS–PAGE. The identification of sporozoit excretory– secretory antigen through monoclonal antibody was applied using Western blot. The field antigen challenge test was done by using 60 chickens divided into 6 groups. The first group was vaccinated with 105 oocyst dose of immucox®, the second group was vaccinated with 100 μg sporozoite excretory–secretory antigen emulsified in FCA, while the third group was vaccinated with 100 μg sporozoite excretory–secretory antigen solved in PBS and the fourth group was treated as the control group, unvaccinated and was not administered with field antigen challenge test. The fifth group was unvaccinated and administered with field antigen challenge test, while the sixth group was treated like the second group and was not administered with field antigen challenge test. Vaccination was applied to 4 days old chickens subcutaneously. The challenge test was held on day 14 orally by using 2000 of E. tenella oocyst. Data were clinical sign which were analyzed descriptively, coecum lesion degree by using Rank Test method, oocyst elimination and the antibody titer with Mixed Design Analysis of Variance continued by using the orthogonal contrast test. The visualization of sporozoite excretory–secretory antigen described that it had protein fraction with molecule weight ranging from 14 KDa to 96 KDa and resulted in 12 types of monoclonal antibodies. Immunobloting of 5 hybridomas has shown us the specific reaction, which were Mabset1, Mabset2, Mabset3, Mabset4 and Mabset5, each of which recognized epitope weight of 14.7 KDa, 42 KDa, 43 KDa, 47 KD and 90 KDa, respectivelly. Vaccination with E. tenella sporozoite excretory–secretory antigen could eliminate clinical signs, especially fecal haemorrhagi, reduce caecal lesion score, reduce oocyst elimination and elevate antibody titer.

Kata Kunci : Parasitologi Hewan, Koksidiosis, Pengendalian, Imunitas E Tenella, immune response, excretory–secretory antigen, challenge test, antibody titer.