Uji Efikasi Mikrokapsul IgG Anti Avian Influenza H5N1: Studi In-ovo Menggunakan Telur Ayam Berembrio
Date
2021Author
Wulandari, Safitria
Esfandiari, Anita
Murtini, Sri
Wulansari, Retno
Metadata
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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.
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- MT - Veterinary Science [909]