Karakterisasi Molekuler Avibacterium paragallinarum dari Sampel Sinus Infraorbitalis Ayam Petelur yang Menunjukan Gejala Infectious Coryza / Snot

Fadhli Nanda Putra, Prof. Dr. drh. A.E.T.H. Wahyuni, M.Si.; Dr. drh. Bambang Sutrisno, M.P.

2023 | Tesis | MAGISTER SAINS VETERINER

Infectious coryza / snot merupakan penyakit unggas yang sangat merugikan peternakan ayam petelur yang disebabkan oleh bakteri Avibacterium paragallinarum. Berbagai metode diagnostik telah dikembangkan untuk mendeteksi penyakit ini, termasuk isolasi dan identifikasi melalui kultur bakteri dan uji biokemis. Isolasi dan identifikasi secara konvensional masih relatif sulit untuk dilakukan karena karakter bakteri membutuhkan faktor pertumbuhan tertentu. Penelitian ini bertujuan untuk melakukan isolasi dan identifikasi A. paragallinarum secara konvesional melalui kultur bakteri dan uji biokemis serta karakterisasi molekuler A. paragallinarum dari sampel klinis sinus infraorbitalis. Sampel sinus infraorbitalis dilakukan isolasi bakteri menggunakan plat agar cokelat (PAC) dan diinkubasi secara mikroaerofilik selama 24 jam pada suhu 37^oC. Koloni dugaan dilakukan konfirmasi pengecatan Gram untuk melihat morfologi sel bakteri. Bakteri dilakukan proses pemurnian dan di kultur pada media agar miring cokelat. Biak murni dilakukan identifikasi dengan melalui uji biokemis, seperti: katalase, oksidase, urease, indol, fermentasi karbohidrat (glukosa, laktosa, sukrosa, maltose, dan mannitol). Sampel yang sama dilakukan deteksi molekuler gen pyrG secara parsial (285 bp) melalui primer HPG-2 (target amplikon 500 bp) dengan polymerase chain reaction (PCR) dan sekuensing. Hasil sekuensing dilakukan analisis bioinformatika meliputi pemotongan, penyejajaran nukleotida, prediksi asam amino, dan konstruksi filogenetik. Hasil penelitian menunjukkan 15 sampel terisolasi dan teridentifikasi A. paragallinarum melalui uji biokemis. Empat dari 15 sampel (26,6%) terdeteksi A. paragallinarum menggunakan metode PCR dengan gen target HPG-2 (500 bp). Sekuen nukleotida dari 4 sampel positif PCR melalui basic local alignment search tool (BLAST) dengan data sekuen yang terdapat pada National Center for Biotechnology Information (NCBI) menunjukkan persen identik 98,64%-100% A. paragallinarum. Analisis parsial gen pyrG melalui konstruksi filogenetik nukleotida dan asam amino menunjukkan A. paragallinarum yang terdeteksi berada pada satu klaster yang sama, terpisah dari klaster Pasteurella multocida, Avibacterium sp., dan Rodentibacter pneumotropicus. Analisis parsial gen pyrG pada sampel LP9 menunjukkan perubahan susunan nukleotida pada empat titik, yaitu: T-A pada urutan 1373, G-T pada urutan 1379, T-G pada urutan 1381 serta G-T pada ururan 1382 tanpa adanya perubahan dalam translasi ke asam amino. Hasil penelitian disimpulkan bahwa 15 sampel terisolasi dan teridentifikasi A. paragallinarum melalui uji biokemis. Kesimpulan berdasarkan karakterisasi molekuler menunjukkan 4 sampel teridentifikasi A. paragallinarum dengan adanya silent mutation pada gen pyrG pada sampel LP9. Sekuen parsial pyrG pada sampel LT-12, LT-7, dan V5 identik dengan strain MODESTO sebagai strain referen.

Infectious coryza / snot is a poultry disease that is very detrimental to laying hens farms caused by the bacterium Avibacterium paragallinarum. Various diagnostic methods have been developed to detect this disease, including isolation and identification through bacterial culture and biochemical tests. Conventional isolation and identification are still relatively difficult because the character of the bacteria requires certain growth factors. This study aims to isolate and identify A. paragallinarum conventionally through bacterial culture and biochemical tests as well as molecular characterization of A. paragallinarum from clinical samples of infraorbital sinus. Infraorbital sinus samples were isolated by bacteria using a chocolate agar plate (PAC) and incubated microaerophilically for 24 hours at 37^oC. Presumed colonies were confirmed by Gram staining to see the morphology of the bacterial cells. The bacteria were purified and cultured on chocolate oblique agar media. Pure cultures were identified by means of biochemical tests, such as: catalase, oxidase, urease, indole, and carbohydrate fermentation test (glucose, lactose, sucrose, maltose, and mannitol). Molecular detection of the pyrG gene (285 bp) was carried out for the same sample using HPG-2 primer (500 bp amplicon target) by polymerase chain reaction (PCR) and sequencing. The sequencing results were subjected to bioinformatics analysis including trimming, nucleotide alignment, amino acid prediction, and phylogenetic construction. The results showed that 15 samples were isolated and identified as A. paragallinarum through biochemical tests. Four of the 15 samples (26.6%) detected A. paragallinarum using the PCR method with the target gene HPG-2 (500 bp). Nucleotide sequences from 4 PCR positive samples using the basic local alignment search tool (BLAST) with sequence data available at the National Center for Biotechnology Information (NCBI) show a 98.64-100% identical percentage of A. paragallinarum. Partial analysis of the pyrG gene through nucleotide and amino acid phylogenetic construction showed that the detected A. paragallinarum was in the same cluster, separate from the Pasteurella multocida, Avibacterium sp., and Rodentibacter pneumotropicus clusters. Partial analysis of the pyrG gene in the LP9 sample showed 4 SNPs (T-A at sequence 1373, G-T at sequence 1379, T-G at sequence 1381 and G-T at sequence 1382) without any change in translation to amino acids. The results of the study concluded that 15 samples were isolated and identified as A. paragallinarum through biochemical tests. The conclusion based on molecular characterization showed that 4 samples identified A. paragallinarum with silent mutations in the pyrG gene in LP9 samples. The sequence of partial pyrG gene in LT-12, LT-7, and V5 samples was identical to strain MODESTO as Reference strain.

Kata Kunci : Infectious coryza, snot, Avibacterium paragallinarum, molekuler, PCR, HPG-2.