Gusti Ayu Yuniati Kencana
Fakultas Kedokteran Hewan, Universitas Udayana, Bali

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Peran Coding dan Non-Coding Region dari Gen Polimerase Kompleks Dalam Adaptasi Virus Avian Influenza

Buletin Veteriner Udayana Vol. 4 No.1 Pebruari 2012
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Avian influenza (AI) is an avian diseases that can cause deadly humaninfection. The disease is caused by Orthomyxoviridae virus, genus influenzavirus type A,subtype H5N1. Pathogenicity of AI virus is polygenic, which means AI virus isdetermined by all the genes. Here the role of coding region (CR) and non-coding region(NCR) of polymerase gene complex is critically reviewed. The coding region of thepolymerase genes can be explained as follows. The amino acid position 627 PB2 gene isa factor adaptation of AI viruses in mammals. Polymerase basic-1 (PB1) protein acts as acentral activity of the enzyme catalyzing the viral polymerase. Polymerase Basic-1 bindsto the terminal end of the vRNA and the cRNA and shows endo-nuclease activity. Theactivity has been identified on the E508, E519, and D522. Polymerase Acid (PA) proteinsplay a role in supporting the biological activity of the polymerase gene complex, but itsmechanism of action in transcription, replication has not been disclosed as clear. Anotherrole of the PA protein is forming a complex of RNA polymerase and express anproteolytic role of cell proteins that suppress cell division. Polymerase acid gene has alsoserine protease activity has been identified at position S624. The non-coding regionmight also play role in the pathogenecity of influenza virus. The results of sequenceanalysis of non-coding region (NCR) at 5’-end of polymerase gene complex of AI virussubtype H5N1 from poultry and pigs in Indonesia showed that the NCR genes PB2, PB1and PA are homogeneous, whereas there are variants of the PB1 gene isolates from ducksA / Duck / Badung, 2006. Occurrence of deletions, insertions, and mutations in the NCRand CR polymerase complex genes may likely lead to genetic changes in viruses whichpotentially also change the nature of biology of AI virus. The study on the 3’-end of thegenes needs to be carried out.

Vaksin Gumboro Menyebabkan Imunosupresif pada Respons Primer Vaksin Penyakit Tetelo Ayam Pedaging

Jurnal Veteriner Vol 12, No 4 (2011)
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The variety of Gumboro live vaccine strains (hot, intermediate, and mild) are available inIndonesia. The immunosuppresive effect of these vaccines under field conditions is not known.This research was conducted to determine this devastating effect of such vaccine strains on theimmune response of chickens vaccinated againts Newcastle disease (ND). Sixty chickens werekept separately in five groups (i.e. V1, V2, V3, V4, and K). At the age of seven days, group V1, V2,and V3 were given hot, intermediate, and mild strains of Gumboro live vaccine respectively whilethe other two groups recieved no Gumboro vaccine (V4 and K). At the age of 14 days, all groups,except group K which were kept as a negative control, were vaccinated against ND. The level ofantibody produced in response to ND vaccination was measured in sera collected at day 0, 7, 14,and 21 post ND vaccination using a standard micro-haemaglutination inhibition test. Data of theantibody titers were analyzed using analysis of variance followed by Duncan’s multiple range test.The results showed that all Gumboro vaccine strains still retain its immunosuppressive nature onhumoral immune response in chickens that later vaccinated against ND. The geometric meantiter (GMT) of anti-NDV antibody of group V4 (unvaccinated againts Gumboro) was significantlyhigher than that of group V1, V2, and V3, i.e. groups of chickens that had been given varietystrains of Gumboro vaccines, at the first and second week after ND vaccination (p<0.05). Thedifference of this immunosuppressivenes among variety of Gumboro vaccine strains need furtherclarification.

Seroprevalensi Penyakit Avian Influenza Pada Itik Di Kabupaten Klungkung

Buletin Veteriner Udayana Vol. 5 No. 2 Agustus 2013
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Itik memiliki peran penting dalam penyebaran virus Avian Influenza subtipe  H5N1 karena merupakan reservoir alami virus dan infeksinya bersifat subklinis. Pendistribusian itik terjadi dari pasar unggas ke peternakan itik atau sebaliknya. Penelitian ini bertujuan untuk mengetahui perbedaan tingkat seroprevalensi virus Avian Influenza di Pasar Unggas Galiran dan peternakan itik di kabupaten  Klungkung pada saat yang bersamaan.  Sampel penelitian adalah serum dari itik yang tidak divaksin yang diambil dari pasar dan peternakan di kabupaten Klungkung.  Pengambilan  sampel dilakukan setiap bulan mulai bulan Maret sampai dengan bulan Agustus 2012.  Sampel serum selanjutnya  diuji dengan uji Hambatan Hemaglutinasi. Hasil penelitian menunjukkan seroprevalensi AI di Pasar Unggas Galiran dan peternakan itik di Kabupaten Klungkung adalah sebesar 81.4%,  perbedaan yang signifikan terjadi pada Juni dan Agustus tetapi tidak signifikan pada bulan Maret, April, Mei, dan Juli.  Seroprevalensi virus AI di Pasar Unggas Galiran adalah sebesar 76.2% dan di peternakan sebesar 86.7% dan secara statistik berbeda sangat nyata. Monitoring terhadap virus Avian Influenza berkelanjutan perlu dilakukan baik di peternakan maupun  di pasar unggas di Klungkung.

Penyebaran Virus Vaksin ND Pada Sekelompok Ayam Pedaging Yang Tidak Divaksinasi dan dipelihara bersama ayam yang divaksinasi

Buletin Veteriner Udayana Vol. 4 No.2 Agustus 2012
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Telah dilakukan penelitian untuk mengetahui daya sebar vaksin ND aktif galurlentogenik (La Sota) dan respons immune ayam yang tidak divaksin yang dipeliharabersama ayam yang divaksin secara intramuskuler. Penelitian ini menggunakan rancanganacak lengkap pola berjenjang (split time) dengan faktor utama perlakukan vaksinasi (TO:0% divaksin dan 100% tidak divaksin , T1: divaksin 50 % dan 50 tidak divaksin dan T2:divaksin 75% dan 25% tidak divaksin) dengan sembilan kali ulangan. Faktor tambahanadalah waktu pengambilan serum (minggu ke-0, ke-1, ke-2 dan ke-3) sehingga jumlahsampel adalah 3x9x4= 108 sampel serum. Ayam umur 3 hari divaksinasi ND secara tetesmata kemudian dilakukan vaksinasi intramuskuler pada umur 21 hari sesuai perlakuan.Titer antibodi ND pada ayam perlakuan diuji dengan uji hambatanhemaglutinasi/hemagglutination inhibition (HI) satu hari sebelum vaksinasi, serta satuminggu, dua minggu, dan tiga minggu setelah vaksinasi. Data tentang titer antibodi (GMTHI)terhadap ND ditransformasi dengan akar X+1, dianalisis dengan sidik ragam dandilanjutkan dengan uji jarak berganda Duncan. Hasil penelitian menunjukkan bahwa titerantibodi terhadap ND pada ayam yang tidak divaksin dipengaruhi oleh persentase ayamyang divaksin. Antibodi HI unit terhadap virus ND pada ayam yang tidak divaksinasimulai teramati pada minggu ke-2 dan ke-3 setelah vaksinasi. Titer antibodi ayam yangtidak divaksinasi pada kelompok ayam yang hanya divaksin 75% mempunyai titer antibodiyang nyata lebih tinggi dibandingkan dengan kelompok ayam yang divaksin 50% dankontrol (P<0,05). Pada kelompok ayam yang divaksin 50%, titer antibody ND pada ayamyang tidak divaksin secara statistik berbeda tidak nyata dibandingkan dengan kelompokyang divaksin 0% (P>0,05). Pada minggu ke tiga, titer antibody ND ayam yang tidakdivaksinasi pada kelompok ayam yang divaksin 75% nyata lebih tinggi dibandingkandengan pada kelompok ayam yang divaksin 50% (P,0,05). Vaksin ND aktif lentogeik LaSota dapat menyebar dari ayam yang divaksin secara intramuskuler kea yam yang tidakdivaksin

Pelacakan Kasus Flu Burung pada Ayam dengan Reverse Transcriptase Polymerase Chain Reaction* (DETECTION OF AVIAN INFLUENZA IN CHICKENS BY REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION)

Jurnal Veteriner Vol 13, No 3 (2012)
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Avian Influenza (AI) or Bird Flu is a fatal zoonotic disease caused by highly pathogenic avian influenza(HPAI) virus of H5N1 sub-type. The disease is still endemic in Indonesia. This study was conducted toinvestigate AI cases in chickens in Bali. Virus isolation was performed in 9 day-old embryonated chickeneggs, and then followed by serologic testing by haemaglutination (HA) and Haemaglutination Inhibition(HI) assay using standard microtiter procedure. All of the samples were further tested with reversetrancriptasepolymerase chain reaction (RT-PCR). All work has been done in the Biomedical and MolecularBiology Laboratory, Faculty of Veterinary Medicine, Udayana University, Denpasar, during the period2009-2011. A total of ten samples were examined A total of ten chicken samples consisting of 6 fieldsamples and 4 meat samples have been confirmed to be AIV H5N1. All field cases showed clinical signsand gross pathology that were typical to the infection of avian influenza. The result indicates that AI casesare still prevalent among chickens in Bali.

ASSESSMENT OF VIRAL CONTENT IN NEWCASTLE DISEASE VACCINE OBTAINED FROM TWO DIFFERENT POULTRY SHOPS USING PRIMARY CHICKEN FIBROBLAST CELL CULTURE

Buletin Veteriner Udayana Vol. 5 No. 2 Agustus 2013
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Newcastle Disease (ND) is still endemic in Indonesia, characterized by its your-round occurrence. Many measures have been adopted by the government to prevent the spread of the disease including vaccination using both active and inactive vaccine. The quality of vaccine is influenced by its viral content which determines the success of ND vaccination in chicken flock. The viral content in ND vaccine can be determined by measuring its eggs lethal dose-50 (ELD50) or Tissue Culture Infective Dose-50 (TCID50) and the minimum viral content considered to be appropriate for active ND vaccine is 66,5/single dose. This study was conducted to find out the viral content of ND vaccine marketed in some poultry shops. ND vaccine of  LaSota strain were obtained from 2 different poultry shops and the viral content was determined in Chicken embryo fibroblast (CEF). The vaccines were reconstituted vaccine diluent and diluted serially in 10-fold diluted. Each dilution was inoculated into 4 wells of confluent CEF cultured in 96 well microplate. The TCID50 was then calculated by Reed and Muench method. The TCID50 of each vaccine was determined 4 times (4 replications). The result showed that the titer of the virus in the vaccine were 66,7and 67 TCID50/per dose which mean that both vaccines were still above the minimum standard of viral content recommended by some workers.

Kepekaan Telur Spesific Pathogen Free dan Clean Egg Terhadap Virus Flu Burung (SENSITIVITY OF SPESIFIC PATHOGEN FREE EGGS AND CLEAN EGG TO THE AVIAN INFLUENZA VIRUSES SUBTYPE H5N1)

Jurnal Veteriner Vol 15, No 1 (2014)
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Avian Influenza which is  in Indonesia  known as Flu Burung  is caused by the avian influenza virussubtype H5N1 (AIV-H5N1). Vaccination is one of the major strategies for preventing and eradicatingAIV-H5N1 in Indonesia. Several factors can affect the potential vaccine such as viral content and mediaused for the propagation of the virus. One of the media commonly used to propagate  the virus is pathogenspecific free (SPF) embryonated chicken eggs. However, as the SPF eggs production is limited and expensive,the use of clean embryonated chicken eggs as an alternative need to examined. This study aimed todetermine the sensitivity of SPF and clean embryonated chicken eggs to the AIV-H5N1. The virus usedwas seed avian influenza virus (A/ Chicken/West Java (Subang)/29/2007)  which haa previously werepropagated  in SPF eggs and the Clean Eggs. The virus titer was determined as Embryo infective Dose 50%(EID50) using Reed and Muench method. Sensitivity of SPF eggs and Clean Egg to the VAI-H5N1 wascompared using  Chi-square statistical analysis. The titers of Avian Influenza Virus subtype H5N1 were106.83EID50/0.1ml in SPF eggs and 106.17EID50/0.1 ml in the Clean Eggs. Statistical analysis showed that,the sensitivity of SPF Eggs and Egg Clean  for the propagation of the VAI-H5N1 was not significantlydifferent.

Respons Antibodi terhadap Penyakit Tetelo pada Ayam yang Divaksin Tetelo dan Tetelo-Flu Burung (NEWCASTLE DISEASE/ND ANTIBODY RESPONSE OF CHICKENS VACCINATED WITH ND SINGLE AND COMBINED ND AND AVIAN INFLUENZA VACCINES)

Jurnal Veteriner Vol 16, No 2 (2015)
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The aim of this study was to investigate antibody response of specific pathogen-free (SPF) chickens vaccinatedwith single inactivated Newcastle disease (ND) vaccine and combined inactive ND and avian influenza(AI) vaccines and to known the efficacy of both vaccines. The vaccines used were killed ND vaccine andkilled ND-AI vaccine produced by PT. Sanbio Laboratories Bogor, West Java. SPF chickens were vaccinatedwith 3 different doses. Antibody titer of SPF chickens against ND virus were determined byhaemagglutination inhibition (HI) test. As many as 130 two week old SPF chickens were used and theywere divided into 2 groups (A and B) consisting of 60 chickens and 10 chickens were used as control withoutvaccine. Group A chickens were vaccinated with ND-K vaccine and group B were vaccinated with combinedkilled ND-AI vaccines. Each group was further divided into 3 subsgroups (1, 2 and 3) consisting 20 chickens.Subgroups 1, 2 and 3 were vaccinated intramuscularly respectively with intramuskular 1, 1/10 and 1/100doses of each vaccines. Antibody response of chickens against ND virus was examined before vaccinationand every three week after vaccination and was expresses as geometric mean titre (GMT) HI units. Theresult showed that the titre antibody against ND increased at the second week following the vaccination.The antibody titer against ND virus of chickens vaccinated single killed ND at the second week in eachdose were 6.05 GMT HI unit, 4.05 GMT HI unit, and 0.9 GMT HI unit. The antibody titre at the third week were 7.90 GMT HI unit ,5.40 GMT HI unit and 2.20 GMT HI unit. The antibody titre against ND virus ofchickens vaccinated with combined ND-AI vaccine at the second week were 6.30 GMT HI unit , 4.15 GMTHI unit , and 2.05 GMT HI unit. At the third week, the antibody titre against ND virus of chickensvaccinated with combined ND-AI vaccine in each subgroup were 7.45 GMT HI unit, 5.60 GMT HI unit , and2.40 GMT HI unit . It showed that the antibody titers at single doses of killed ND vaccine (7.90 GMT HIunit) and combined ND-AI vaccine (7.45 GMT HI unit) at the third week after vaccination were both stilleffective as both titres were above standard protective titre.

Seroprevalensi Penyakit Tetelo pada Peternakan Itik dan Pasar Galiran di Kabupaten Klungkung, Bali (NEWCASTLE DISEASE SEROPREVALENCE IN LIVESTOCK DUCK AND MARKETS GALIRAN OF KLUNGKUNG RECIDENCE, BALI)

Jurnal Veteriner Vol 16, No 3 (2015)
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Ducks are one of the birds that play a role in the spread and transmission of Newcastle disease virus.Newcastle disease infection in ducks are rarely accompanied by clinical symptoms, so the ducks couldpotentially spread the disease to other birds. In general, the distribution of ducks is going from farm toMarket Poultry and vice versa. The aim of this study was to determine the seroprevalence Newcastledisease infection in ducks reared on farms and sold at market Galiran Klungkung regency. The sampleused in this study was serum unvaccinated ducks ND. The samples used were 420 samples taken from thefarm and from Klungkung Galiran Market from March to August 2012.Serum samples were tested for thepresence of NDV antibody by using hemagglutinationtest(Haemaglutination Inhibition Test / HI test) atthe Biomedical Laboratory of the Faculty of Veterinary Medicine Udayana University. The results showedthat the seroprevalence Newcastle disease virus in ducks in Galiran Market were 33.3% and in farmsamounted to 46.2%. There are differences in seroprevalence Newcastle disease (p <0.05) in March, June,and August. Overall seroprevalence in Galiran Market and Newcastle disease in livestock in Klungkungregency equal to 39.8%. It can be concluded that Newcastle disease seroprevalence in Klungkung regency isquite high and might be potentially transmit the virus to other poultry. Therefore, periodic monitoring isnecessary as an effort to early prevention.

The Production and Use of Monoclonal Antibodies for the Detection of Avian Influenza Antigen in the in Infected Chickens

Jurnal Veteriner Vol 13, No 3 (2012)
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A safe and appropriate diagnostic method for avian influenza virus (AIV) infection in chickens wasestablished using monoclonal antibodies (mAbs) against the virus. The virus used for the production of themonoclonal antibodies was an Indonesian AIV-H5N1 isolate. Immortal mouse myeloma cells were fusedwith the lymphocytes derived from the spleen of mice immunized with the virus. The mAbs were tested fortheir specificity by enzyme linked immunosorbent assay (ELISA) and western blotting using formaldehydeinactivated virus and normal allantoic fluid as antigens. Twelve mAbs specific against AIV were isolatedand 8 mAbs were used for immunodetection of AIV antigen in chicken’s tissues. By indirect ELISA, themAbs were able to detect AIV antigen in allantoic fluid at the titre as low as 2-2 to 2-4 HA units per 0.1 ml.By immunoperoxidase staining AIV–antigen was detected in paraffin embedded tissues of AIV-infectedchickens. AIV antigen was not detected in chickens which were confirmed to be AIV negative. In theinfected chickens, high intensity of AIV antigen was detected in proventricle gland and small intestine.The AIV antigen with a lesser intensity was detected in lungs and spleen but hardly detected in muscle,brain and several other tissues. This study show clear evidences that mAbs produced in this study areapplicable for use in the detection of AIV antigen in infected chickens.