Uji Efikasi Mikrokapsul IgG Anti Avian Influenza H5N1: Studi In-ovo Menggunakan Telur Ayam Berembrio

Abstract

Penelitian ini bertujuan untuk mengevaluasi kemampuan mikrokapsul IgG anti AI H5N1 dalam menetralkan virus Avian Influenza (AI) H5N1 isolat Indonesia melalui studi in-ovo menggunakan telur ayam berembrio (TAB). Sebanyak 6 ekor induk sapi Friesian Holstein bunting trimester akhir, dibagi menjadi kelompok kontrol (n=3; tidak divaksin) dan kelompok vaksin (n=3; divaksin). Induk sapi kelompok vaksin diinjeksi vaksin Avian Influenza (AI) H5N1 komersial (killed vaccine) sub-kutan, sebanyak 5 dosis/ekor (106 EID50/dosis) dua kali dengan interval antar vaksinasi 2 minggu, induk sapi diberi imunomodulator 0,1 mg/kg bb peroral selama 3 hari berturut-turut sebelum vaksinasi kemudian dilakukan priming dengan menyuntikkan antigen AI H5N1 in-aktif tanpa adjuvant (intravena) 3 hari berturut-turut (dosis 10 × 29 HAU/ekor). Sampel darah dikoleksi melalui vena coccygea sebelum priming (pre vaksinasi) dan 2 minggu post vaksinasi I serta II untuk dianalisis konsentrasi total protein, albumin, globulin, rasio albumin/globulin (rasio A/G) dan titer IgG anti AI H5N1. Sampel kolostrum dikoleksi segera setelah induk sapi melahirkan selama 2 hari berturut-turut dan dipreparasi melalui teknik defatting, pengendapan amonium sulfat dan dialisis untuk dikoleksi IgG anti AI H5N1 serta diukur titernya. Suspensi IgG anti AI H5N1 tersebut dimikroenkapsulasi menggunakan penyalut alginat dan kitosan dan diuji efikasinya secara in-ovo (melalui uji netralisasi metode alpha). Telur ayam berembrio dibagi menjadi 3 kelompok inokulan yaitu TAB diinokulasi virus AI H5N1 pengenceran 10-4 sampai 10-8 (n=5/pengenceran), TAB diinokulasi virus AI H5N1 pengenceran 10-4 sampai 10-8 yang ditambahkan mikrokapsul tanpa IgG anti AI H5N1 (n=5/pengenceran), TAB diinokulasi virus AI H5N1 pengenceran 10-4 sampai 10-8 yang ditambahkan mikrokapsul IgG anti AI H5N1 (n=5/pengenceran). Selama 4 hari dilakukan pengamatan terhadap TAB dan koleksi cairan alantois (hari ke-5) untuk uji aglutinasi cepat, penghitungan titer infektif 50% (EID50), indeks netralisasi (IN) dan konfirmasi keberadaan virus melalui uji reverse transcriptase polymerase chain reaction (RT-PCR). Hasil pengamatan menunjukkan tidak terdapat perbedaan yang signifikan pada konsentrasi total protein, albumin, globulin dan rasio A/G baik antar kelompok maupun antar waktu pengamatan (p>0,05). Hiperimunisasi induk sapi bunting dengan vaksin AI H5N1 berhasil menginduksi pembentukan antibodi anti AI H5N1 dalam darah dan kolostrum (titer 27,3 pada 100% induk sapi). Hasil uji netralisasi pada mikrokapsul IgG anti AI H5N1 dapat menetralisasi virus AI H5N1 titer 107,83 EID50/mL dengan IN 7,83 dan pada mikrokapsul tanpa IgG anti AI H5N1 dengan IN sebesar 1,09. Hasil deteksi keberadaan gen virus AI H5N1 dengan RT-PCR dari cairan alantois TAB yang diinokulasi virus AI H5N1 dan ditambahkan mikrokapsul IgG anti AI H5N1 ditemukan adanya gen H5 yang menunjukkan bahwa virus AI H5N1 masih berada dalam cairan alantois namun tidak bereplikasi, yang diindikasikan dari hasil negatif uji aglutinasi cepat. Dapat disimpulkan bahwa mikrokapsul yang berisi IgG anti AI H5N1 mampu menetralisasi virus AI H5N1 secara sempurna.

This study aims to evaluate the ability of the anti AI H5N1 IgG microcapsule in neutralizing the Indonesian isolate Avian Influenza (AI) H5N1 virus through an in-ovo study using embryonated chicken eggs (ECE). A total of 6 cows of Friesian Holstein were pregnant in the final trimester, divided into the control group (n=3; not vaccinated) and the vaccine group (n=3; vaccinated). Cows in the vaccine group were injected with the commercial H5N1 Avian Influenza (AI) vaccine (killed vaccine), 5 doses/head (106 EID50 /dose) twice with 2 week intervals between vaccinations, the cows were given immunomodulators 0.1 mg/kg bw orally for 3 consecutive days before vaccination, then priming was done by injecting in-active AI H5N1 antigen without adjuvant (intravenously) for 3 consecutive days (dose 10 × 29 HAU/head). Blood samples were collected through the coccygeal vein before priming (pre vaccination) and 2 weeks post vaccination I and II to analyze the total concentration of protein, albumin, globulin, albumin/globulin (A/G) ratio and IgG anti AI H5N1 titers. Colostrum samples were collected immediately after the cows gave birth for 2 consecutive days and prepared through defatting techniques, ammonium sulfate deposition and dialysis to collect anti AI H5N1 IgG and measured its titer. The anti AI H5N1 IgG suspension was micro-encapsulated using alginate and chitosan coatings and tested for its efficacy in-ovo (through the alpha method neutralization test). Embryonated chicken eggs were divided into 3 groups of inoculants, namely ECE inoculated with AI H5N1 virus, 10-4 to 10-8 dilution (n=5/dilution), ECE inoculated with AI H5N1 virus, 10-4 to 10-8, which were added with microcapsules without anti-AI IgG H5N1 (n=5/dilution), ECE was inoculated with AI H5N1 virus, 10-4 to 10-8 dilutions were added with anti AI H5N1 IgG microcapsules (n=5/dilution). For 4 days the ECE was observed (candling) and the allantoic fluid was collected (day 5) for rapid agglutination test, calculated the infective titer 50% (EID50), neutralization index (NI) and tested reverse transcriptase polymerase chain reaction (RT-PCR) to confirm the presence of the virus. The results of the observations showed that there were no significant differences in the concentration of total protein, albumin, globulin and A/G ratio between groups and between observation times (p> 0.05). Hyperimmunization of pregnant cows with AI H5N1 vaccine was successful in inducing the formation of anti-AI H5N1 antibodies in blood and colostrum (titer 27.3 in 100% of cows). Neutralization test results on anti AI H5N1 IgG microcapsules can neutralize AI H5N1 virus titer 107.83 EID50/mL with NI 7.83 and on microcapsules without IgG anti AI H5N1 with an NI of 1.09. The results of the detection of the presence of the AI H5N1 virus gene by RT-PCR from the ECE allantoic fluid inoculated with the AI H5N1 virus and added with anti AI H5N1 IgG microcapsules, found the H5 gene, indicating that the AI H5N1 virus was still in the allantoic fluid but did not replicate, as indicated by the results. Negative rapid agglutination test. It can be concluded that the microcapsules containing anti AI H5N1 IgG were able to completely neutralize the AI H5N1 virus.