Protein Cell Debris dan bakterin Vibrio alginolyticus 8 sebagai vaksin dalam pengendalian penyakit vibriosis pada Kerapu Macan (Ephinephelus fuscoguttatus)

SITUMEANG, Hotnida Junita, Langkah Sembiring, M.Sc.,Ph.D

2006 | Tesis | S2 Biologi

Bakteri yang diduga sebagai V. alginolyticus 8 dikarakterisasi dan diidentifikasi untuk memastikan bahwa isolat termasuk anggota Vibrio alginolyticus . Untuk memperoleh vaksin protein cell debris (PCD) isolat dikultivasi, dan disentrifugasi pada 3000 rpm, selama 15 menit sehingga diperoleh pellet sel dan supernatan. Pellet sel disuspensikan dalam PBS yang mengandung 1 ml inhibitor protease, kemudian disonikasi pada frekuensi 159 Hz, 6x30 detik dan disentrifugasi pada 12.000 rpm, selama 15 menit. Kemudian pellet yang diperoleh diresuspensikan dalam PBS pH 7,4 kemudian disonikasi kembali pada frekuensi 159 Hz, 6x30 detik dan disentrifugasi pada 12.000 rpm, selama 15 menit, pellet yang diperoleh disebut sebagai PCD, yang kemudian didialisis selama 24 jam. PCD tersebut dipisahkan dengan cara elektroforesis (SDS PAGE), selanjutnya PCD dengan BM 30–100 kDa diisolasi dan dipurifikasi dengan elektroelusi. Untuk mendapatkan protein murni kemudian protein didialisis dan diukur konsentrasinya dengan metode Lowry. Selanjutnya PCD digunakan sebagai vaksin. Pada penelitian vaksin disuntikan sebanyak 5 μg/ikan. Untuk penentuan vaksin yang terbaik berdasarkan BM, sebanyak 75 ekor kerapu macan dibagi dalam 5 perlakuan yaitu (A) vaksin PCD dengan BM 73,434 kDa, (B) vaksin PCD dengan BM 64,136 kDa, (C) vaksin PCD dengan BM 37,846 kDa, (D) vaksin PCD dengan BM 32,009 kDa dan (K) Kontrol (disuntik 0,1 ml larutan PBS steril pH 7,4). Untuk membandingkan efek vaksin PCD dengan bakterin 45 ekor kerapu macan dibagi dalam 3 perlakuan yaitu (A) vaksin PCD dengan BM 73,434 kDa, (BKT) vaksin bakterin 8x109 sel/ ikan sebanyak 0,1 ml dan (K) kontrol (disuntik 0,1 ml PBS steril pH 7,4). Parameter yang diamati, yaitu titer antibodi, Relative Percent Survival (RPS), Rerata Waktu Kematian (RWK) dan kualitas air. Hasil penelitian menunjukkan bahwa pemberian vaksin PCD dan bakterin dapat meningkatkan titer antibodi kerapu macan. Nilai RPS ke dua perlakuan vaksin A dan BKT sama yaitu = 92,30 %, lebih besar dibanding dengan kontrol (K = 84,62 %), dan RWK ke dua perlakuan vaksin (A = 4,33 hari dan BKT = 3,00 hari). RWK ke dua perlakuan vaksin berbeda dengan K (kontrol = 3,33 hari). Dapat disimpulkan bahwa vaksin PCD V. alginolitycus 8 dengan berat molekul 73,434 kDa dan vaksin bakterin 8x109 sel/ ikan memberikan efek yang hampir sama dalam meningkatkan titer antibodi, RPS dan memperpanjang RWK kerapu macan, sehingga vaksin PCD dan bakterin mampu memberikan perlindungan pada kerapu macan terhadap serangan bakteri V. alginolitycus 8

Isolate of V. alginolyticus 8 was characterized and identified. To find out cell debris protein (CDP) isolate was cultivated and harvested by sentrifugation at 3000 rpm, for 15 minutes into pellet and supernatant. Pellet was suspended into PBS pH 7.4 and 1 ml protease inhibitor, then sonicated at 159 Hz, for 6x30 second and centrifuged at 12.000 rpm, for 15 minutes. Afterwards pellet was resuspended with PBS pH 7.4 and sonicated again at 159 Hz, for 6x30 second and sentrifuged at 12.000 rpm, for 15 minutes. The resulted pellet was called cell debris protein (CDP). For purpose of purification CDP was dialized in PBS 0.01 M pH 7.4 for 24 hours. The CDP was separated by electrophoresis (SDS PAGE), and subsequently protein with molecular weigth of 30–100 kDa were isolated and purificated by electroelution, and then sample was dialized again to obtain pure protein.Tha protein concentration of CDP was determined spectrophotometricaly by Lowry method. To find out of the best molecular weigth of CDP, to increase immune response, a number of 75 individuals kerapu macan were devided into five treatments, i.e. (A) vaccinated with CDP of molecular weight of 73.434 kDa, (B) vaccinated with CDP of molecular weight of 64.136 kDa, (C) vaccinated with CDP of molecular weight of 37.846 kDa, (D) vaccinated with CDP of molecular weight of 32.009 kDa, (K) control was injected with 0.1 ml sterile PBS (pH 7.4). The dosage of CDP was 5 μg/individual fish. To compare CDP and bacterin vaccine potencial to protect fish against V. alginolyticus 8 a number of 45 individual of kerapu macan were devided into three treatmeants, i.e. (A) vaccinated with CDP of molecular weight of 73.434 kDa at 5 μg/individual fish, (BKT) vaccinated with CDP of bacterial cell at 8x109 cell/ individual fish, and (K) control was injected by 0.1 ml sterile PBS (pH 7.4). The potencial of CDP and bacterin to protect fish was expressed as antibody titer, Relative Percent Survival (RPS), and Meant Death Time (MDT). The results of the experiment showed that the effect of both vaccine to increase immune response were found to be different from control experiment. Both vaccine could increase the antibody titer of kerapu macan after two weeks of treatment. Both vaccine were could increase the Relative Percent Survival to 92,3%, while the Meant Death Time of CDP and bacterin treated sample was found to be 4.33 days and 3.00 days, respectively. As a conclusion, the CDP vaccine with of molecular weight of 73.434 kDa at 5 μg/individual fish and bacterin vaccine at 8x109 cell/ individual fish could elicit immune response of fish as expressed by antibody titer Relative Percent Survival, and Meant Death Time for kerapu macan. Therefor, both CDP and bacterin can be used to protect fish against vibriosis caused by V. alginolyticus 8.

Kata Kunci : Ikan Kerapu Macan,Pertahanan Tubuh,Vaksin,Bakteri, Kerapu macan, V. alginolyticus 8, cell debris protein, vaccination, bacterin.