KAJIAN EPIDEMIOLOGI RABIES DI BALI DALAM MENINGKATKAN PROGRAM PENGENDALIANNYA

Drh. I Nyoman Dibia, Prof. Dr. drh. Bambang Sumiarto, S.U., M.Sc.; drh. Heru Susetya, M.P., Ph.D.; drh. Anak Agung Gde Putra, S.H., M.Sc., Ph.D.

2014 | Disertasi | S3 Sain Veteriner

Rabies di Bali dikonfirmasi pertama kali pada November 2008. Penyakit ini memunculkan masalah kesehatan masyarakat yang serius di seluruh kabupaten/kota di Provinsi Bali. Anjing merupakan sumber utama penularan rabies. Mayoritas masyarakat Bali memelihara anjing secara dilepas. Hal ini menegaskan pentingnya mengembangkan program pengendalian rabies yang efektif dengan mempertimbangkan aspek sosial budaya dan lingkungan masyarakat Bali. Pengetahuan tentang epidemiologi infeksi rabies pada hewan akan sangat membantu mencegah penularannya pada manusia. Tujuan penelitian ini adalah menyidik faktor-faktor risiko yang berasosiasi terhadap rabies pada anjing di provinsi Bali, mendeteksi penanda molekuler dan menentukan hubungan kekerabatan terhadap isolat-isolat virus rabies Bali, serta mengidentifikasi jalur penularan virus rabies berdasarkan lalulintas hewan penular rabies (HPR) yang masuk ke Bali. Kajian kasus kontrol telah dilakukan untuk menyidik faktor risiko yang berasosiasi dengan kejadian rabies. Difinisi kasus adalah anjing yang telah dikonfirmasi rabies dengan direct flourescent antibody test (dFAT). Sebanyak 51 anjing kasus dan 102 anjing sehat sebagai kontrol telah digunakan pada kajian ini. Faktor-faktor yang disidik diperoleh dengan menggunakan kuesioner. Data yang diperoleh dianalisis menggunakan Chi-square (X2) dan Odds Ratio (OR). Selanjutnya, data dianalisis dengan regresi logistik (logistic regression) untuk memperoleh model infeksi. Dua puluh sembilan sampel otak hewan telah digunakan untuk menemukan penanda molekuler dan menentukan hubungan kekerabatan isolat-isolat virus rabies Bali. Sampel-sampel yang dikoleksi dari hewan-hewan yang terinfeksi rabies tahun 2008-2010 (22 anjing, 3 sapi, 2 kambing, 1 kucing, dan 1 babi) diperoleh dari beberapa daerah di Indonesia (Bali, Sumatera, Jawa, Kalimantan, Sulawesi, dan Flores). Ribonucleic acid (RNA) virus rabies diekstraksi dengan TRIzol. Fragmen gen penyandi nukleoprotein diamplifikasi menggunakan metoda one-step reverse transcription and polymerase chain reaction (RT-PCR) dengan pasangan primer yang didesain secara spesifik. Selanjutnya, fragmen gen tersebut disekuensing menggunakan ABI automatic sequencer. Multiple alignment dari sekuen-sekuen nukleotida yang diperoleh pada penelitian ini menerapkan metode Clustal-W. Konstruksi pohon filogenetik dilakukan dengan metode Neighbour-joining dan jarak genetik dianalisis dengan mengaplikasikan model Kimura’s two-parameter dalam program MEGA 4.0. Untuk mengidentifikasi jalur masuknya rabies ke Bali melalui lalu lintas hewan penular rabies telah diwawancarai 22 nelayan antar pulau dan 22 petugas karantina hewan yang bertugas mengawasi lalu lintas hewan di masing-masing pelabuhan resmi di Bali. Responden diambil secara random sederhana. Data yang diperoleh melalui wawancara dengan kuesioner, selanjutnya dianalisis menggunakan statistik deskriptif. Hasil analisis hubungan dan kekuatan asosiasi menunjukkan bahwa faktor-faktor yang berasosiasi dengan infeksi rabies pada anjing adalah status vaksinasi rabies (P= 0,000; OR= 19,133), kontak dengan anjing lain (P= 0,000; OR= 12,551), kondisi fisik anjing (P= 0,002; OR= 3,019), jumlah anjing yang dipelihara (P= 0,002; OR= 2,962), dan pemeriksaan kesehatan anjing (P= 0,022; OR= 2,444). Analisis model menunjukkan bahwa faktor yang berpengaruh bermakna (P<0,05) terhadap kejadian rabies pada anjing berturut-turut yaitu status vaksinasi rabies (+3,919) dan kontak dengan anjing lain (+3,457). Model ini memiliki akurasi 85% untuk memprediksi peluang terjadinya kasus rabies pada anjing di provinsi Bali. Kajian molekuler gen nukleoprotein menemukan bahwa isolat-isolat virus rabies Bali mempunyai asam amino spesifik Isoleusin pada posisi 308, sedangkan semua virus rabies di Indonesia dan negara-negara lain di dunia termasuk virus rabies strain vaksin memiliki asam amino Valin pada posisi yang sama, sehingga asam amino yang spesifik tersebut dapat digunakan sebagai penanda molekuler virus rabies Bali. Temuan ini menjadi kajian pertama tentang penanda molekuler virus rabies di Indonesia. Penelitian ini juga menunjukkan bahwa seluruh isolat dari Indonesia termasuk dalam Lyssavirus genotipe 1, yang merupakan genotipe virus rabies klasik, dan mempertegas bahwa virus rabies Bali berada dalam kelompok virus rabies Indonesia. Berdasarkan sekuen nukleotida fragmen gen N (1125 bp), isolat-isolat virus rabies Bali mempunyai homologi 99,7-100%. Hal ini mengindikasikan bahwa virus rabies yang masuk ke Bali merupakan introduksi tunggal. Virus rabies Bali mempunyai hubungan kekerabatan yang sangat dekat dengan virus-virus yang diisolasi di Sulawesi, Kalimantan, dan Flores dengan kesamaan secara berturut-turut 98,5-98,6%, 98,2%, dan 98,2%. Sementara itu, isolat-isolat dari pulau Jawa dan Sumatera adalah 97,3-97,5%, dan 97,2- 97,6%. Sebanyak 77,3% (17/22) petugas karantina yang diwawancarai menyatakan pernah menahan HPR (anjing, kucing, monyet, musang, luwak, dan kelelawar) di pelabuhan resmi dan seluruh HPR tersebut berasal dari Jawa. Jalur masuknya HPR ke Bali melalui pelabuhan tradisional pada penelitian ini ditemukan 4,5% (1/22) nelayan antar pulau melalulintaskan anjing dari Sulawesi dengan alasan untuk keamanan dalam pelayaran. Penelitian ini juga menunjukkan 33,3% (3/9) petugas pernah menemukan anjing yang dilalulintaskan ke Bali melalui pelabuhan tradisional oleh pelaut-pelaut dari Pulau Kalimantan dan Sulawesi. Dari hasil penelitian ini dapat disimpulkan bahwa terjadi pergerakan anjing pada pelayaran tradisional ke Bali. Berdasarkan analisis filogenetik dan penelusuran lalu lintas HPR menunjukkan bahwa wabah rabies Bali diakibatkan karena introduksi tunggal dari Pulau Sulawesi, yang paling mungkin adalah melalui anjing akibat intervensi pelaut.

Rabies in Bali was first confirmed laboratorically in November 2008. The disease gives rise to a serious public health problem in all districts of Bali Province. Among reservoir animals, dogs constituted the most important sources of rabies infection. Therefore, it is essential to develop an effective rabies control program based on the social-cultural aspects and environment of the Balinese community. The knowledge of the epidemiology of the rabies infection in animals is therefore extremely helpful in preventing the spread of the disease to humans. The objectives of this study were to investigate the risk factors associated with rabid dogs in Bali, to find out the molecular marker and to derive the phylogenetic relationship of Bali’s rabies virus (BRV) isolates, as well as to identify the pathway of introduction of the rabies virus from reservoir animals traffic entering Bali. A case-control study was conducted to investigate the risk factors associated with rabid dogs in Bali. Cases were dogs confirmed having rabies by direct fluorescent antibody test (dFAT). The determination of amount of samples in each district was calculated proportionally and they were taken by simple random method. As many as fifty-one rabid dog cases between 2010 and 2011 and one hundred and two healthy dogs as the control were used in this study. A number of putative factors were obtained by means of questionnaires. The data were subsequently analyzed using chi-square (X2) and odds-ratio (OR), which were ultimately analyzed by means of logistic regression to build up a model. Twenty nine animal brain samples were used in order to find out the molecular marker and to derive the phylogenetic relationship of Bali’s rabies virus (BRV) isolates. The brain samples collected from rabid animals (22 dogs, 3 cattle, 2 goats, 1 cat and 1 swine) were obtained from parts of Indonesia (Bali, Sumatra, Java, Kalimantan, Sulawesi and Flores). The dFAT was used to detect the presence of rabies viral antigen. Ribonucleic acid (RNA) of rabies viruses was extracted with TRIzol reagent. Fragments of N gene were amplified using one-step reverse transcription and polymerase chain reaction (RT-PCR) method with specifically designed primer pairs and sequenced using ABI automatic sequencer. Multiple alignment of nucleotide and deduced amino acid sequences of nucleoprotein (N) gene fragments were analyzed using Clustal W method. The construction of a phylogenetic tree was undertaken by using Neighbour-joining method and the genetic distance was performed by using Kimura’s two-parameter model in MEGA 4.0 program. To ascertain the pathway of introduction of the rabies virus from reservoir animals traffic entering Bali, 22 inter-island fishermen in Bukit Peninsula were interviewed. In addition, interviews with 22 animal quarantine officers in official harbours all over Bali were carried out. All respondents were taken by applying simple random sampling. Data were obtained by means of questionnaires and subsequently analyzed descriptively. This study revealed that factors associated with rabid dog were the status of rabies vaccination (P= 0.000; OR= 19.133), contact with other dog (P= 0.000; OR= 12.551), condition of dog (P= 0.002; OR= 3.019), number of raised dog (P= 0.002; OR= 2.962), and veterinary care (P= 0.022; OR= 2.444). An appropriate logit model was found to estimate the probability of rabid dog events in Bali as follows : Logit Pr (rabies=1| x) = - 4.413 + 3.919 (status of rabies vaccination) + 3.457 (contact with other dog). This model had accuracy of 85% in predicting the probability of rabid dogs occurance in Bali. Bali’s rabies virus isolates have conserved amino acid Isoleucine alterations at position 308. Isoleucine distinguished between all Bali’s isolates and all isolates from other areas of Indonesia and other parts of the world. Therefore, it can be proposed to be a molecular marker which is believed to be the first study of molecular marker of rabies virus in Indonesia. The result of the phylogenetic analysis showed that Indonesian rabies virus isolates were confirmed as genotype 1 of lyssavirus and Bali’s rabies viruses were within an Indonesian cluster. The similarities among all Bali’s isolates ranged from 99,7 to 100%. It indicated that the rabies virus entered Bali as a single introduction. The Bali’s rabies viruses were closely related to rabies isolates in Sulawesi, Kalimantan, and Flores islands with 98.5–98.6%, 98.2%, and 98.2% of the similarity, respectively. Meanwhile, Java and Sumatra islands were 97.3–97.5%, and 97.2–97.6%, respectively. Reservoir animals traffic entering Bali through official harbours showed that 77.3% (17/22) quarantine officers interviewed, ever detained rabies transmitter animals namely; dogs, cats, monkeys, marten, civet cat, and bats. All officers declared that the detained ones were from Java. In this study, reservoir animals pathway entering Bali through the traditional port of Bukit Peninsula showed that 4.5% (1/22) of inter-island fisherman were found to carry out dog traffic from Sulawesi for the sake of safe sailing. This study also suggested that 33.3% (3/9) quarantine officers ever found dogs traffic to Bali through traditional port by inter-island sailors from Kalimantan and Sulawesi islands. It was clearly shown that dogs movements take places in traditional sailing by sailors from Sulawesi into Bali. Integrated results of phylogenetic and reservoir animals traffic showed that the rabies outbreak in Bali was due to a single introduction from Sulawesi, most likely through dog by sailor intervention.

Kata Kunci : Rabies, Bali, faktor risiko, penanda molekuler, filogenetik, lalu lintas HPR.