SELEKSI DAN KARAKTERISASI BAKTERI ASAM LAKTAT (BAL) UNTUK PENANGGULANGAN PENYAKIT VIBRIOSIS PADA IKAN KERAPU MACAN (Epinephelus fuscoguttatus)

NURSYIRWANI, Prof. drh. Widya Asmara, SU, Ph.D.

2013 | Disertasi | S3 Sain Veteriner

Vibriosis merupakan salah satu penyakit bakterial yang sering ditemukan pada budidaya ikan kerapu, terutama disebabkan oleh jenis Vibrio alginolyticus. Untuk mengatasi vibriosis, maka perlu dicari cara yang relatif aman, antara lain dengan menggunakan probiotik seperti bakteri asam laktat (BAL). Penelitian ini bertujuan untuk mengisolasi, menseleksi dan mengkarakterisasi BAL untuk mengatasi vibriosis pada budidaya ikan kerapu macan (Epinephelus fuscoguttatus). Bakteri asam laktat diisolasi dari usus ikan dan diidentifikasi berdasarkan morfologi koloni dan sel, sifat fisiologi dan uji biokimia. Isolat BAL kemudian diseleksi berdasarkan antagonisme terhadap V. alginolyticus, toleransi terhadap pH dan garam empedu, uji ko-kultur, adesi dan patogenisitas pada ikan kerapu macan. Tiga isolat BAL terpilih masing-masing diberikan kepada ikan melalui pakan pada konsentrasi 108 sel/g selama 28 hari, kemudian pemberian BAL dihentikan dan ikan diinfeksi dengan V.alginolyticus secara intraperitoneal (IP) pada konsentrasi 109 sel/mL sebanyak 0,1 mL. Setiap tujuh hari selama periode pemeliharaan dan setelah diinfeksi dilakukan pengamatan terhadap jumlah total BAL dan Vibrio pada usus, respon imun nonspesifik (kadar hematokrit, total dan diferensiasi leukosit dan aktifitas fagositosis). Perubahan histopatologi, pertumbuhan, sintasan dan kualitas air wadah pemeliharaan ikan juga diamati. Dari 21 isolat BAL yang didapat, 20 isolat mampu menghambat pertumbuhan V. alginolyticus dan toleran terhadap pH 3, 4, 5 dan 6, dan 19 isolat dapat tumbuh pada garam empedu 0,2, 0,3, 0,4 dan 0,5 %. Semua isolat tidak bersifat patogen pada ikan kerapu macan. Tiga isolat BAL (KSBU 12C, KSBU 5Da dan KSBU 9) dapat menekan pertumbuhan V. alginolyticus pada uji ko-kultur, dan mempunyai kemampuan adesi yang lebih tinggi daripada V. alginolyticus. Selama pemberian pakan dengan suplementasi BAL, BAL dijumpai pada usus ikan kerapu macan mulai hari ke-7 sampai hari-35. Pemberian BAL dapat mengurangi jumlah total Vibrio pada usus. Respon imun nonspesifik ikan yang diberi pakan BAL meningkat dibandingkan ikan kontrol. Kadar hematokrit, jumlah leukosit dan persentase heterofil tertinggi didapatkan pada ikan yang diberi isolat KSBU 12C. Persentase monosit dan aktifitas fagositosis tertinggi dijumpai pada ikan yang diberi isolat KSBU 9 dan isolat KSBU 5Da. Namun dari pengamatan histopatologi, ketiga isolat BAL ini tidak dapat mencegah terjadinya infeksi vibriosis pada ikan kerapu macan. Dari sifat fenotip dan genotip, ketiga isolat BAL tersebut mempunyai kesamaan dengan Enterococcus hirae dengan tingkat homologi 99 %.

Vibriosis is a bacterial disease frequently found in grouper culture, mainly due to Vibrio alginolyticus. A relatively safe method is required to control the disease, one of which by using probiotic such as lactic acid bacteria (LAB). The objectives of this research were to isolate, select and characterize LAB to control vibriosis in tiger grouper (Epinephelus fuscoguttus) The lactic acid bacteria was isolated from tiger grouper intestine, and identified based on morphology of colony and cells, physiological and biochemical properties. The LAB isolates were selected as probiotic based on antagonism toward V. alginolyticus, tolerances to low pH and bile salts, co-culture, adhesion to intestinal epithelial cells and pathogenicity to tiger grouper. Each of three selected LAB isolates was supplemented to fish diet at density of 108 cells/g for 28 days, then LAB administration was ceased and the fish were infected intraperitoneally (IP) with 0,1 mL suspension of V. alginolyticus secara intraperitoneal (IP) at density of 109 cells/mL. Total LAB and Vibrio counts, nonspecific immune responses (i.e. hematocrit value, total and differential leucocyte and phagocytic activity) were observed every seven days during 28 day-LAB feeding and seven days after the cessation of LAB feeding. In addition, histophatological changes, fish growth and survival, and water quality of rearing media were also examined at the final observation. Twenty of 21 LAB isolates were able to inhibit the growth of V. alginolyticus and were tolerance to pH 3, 4, 5 dan 6, and 19 isolates could grow at bile salts of 0.2, 0.3, 0.4 and 0.5 %. All isolates were not pathogenic to tiger grouper. In co-culture test, three LAB isolates (KSBU 12C, KSBU 5Da and KSBU 9) were able to suppress the growth of V. alginolyticus, and had higher ability to adhere to intestinal epithelial cells than that of V. alginolyticus. During the LAB supplementation, LAB was found in fish intestines from days 7 until days 35. LAB administration reduced total Vibrio in the intestines, and enhanced the nonspecific immune responses in comparison to fish fed control diet. The highest hematocrit value, total leucocyte and heterophil percentage were obtained in fish fed with KSBU 12C isolate. The highest percentages of monocyte and phagocytic activity were obtained from fish fed with isolates KSBU 9 and KSBU 5Da, respectively. However, histopatological observation indicated that the LAB isolates could not prevent vibriosis infection in tiger grouper. The phenotype and genotype properties of the three LAB isolates were similar to Enterococcus hirae at 99 % homology level.

Kata Kunci : Seleksi, karakterisasi, bakteri asam laktat, vibriosis, kerapu macan.