Aplikasi Sinbiotik dengan Dosis Prebiotik Inulin Berbeda untuk Pencegahan Penyakit Edwardsiellosis pada Ikan Patin

Abstract

Usaha budidaya ikan patin (Pangasius sp.) di Indonesia hingga saat ini masih menghadapi hambatan berupa penyakit edwardsiellosis yang dipicu oleh infeksi Edwardsiella tarda. Pengendalian penyakit dengan antibiotik yang dilakukan secara berulang menimbulkan sejumlah persoalan, antara lain penyebaran sifat resistansi bakteri, akumulasi residu antibiotik pada produk budidaya, serta dampak pencemaran lingkungan. Kondisi tersebut mendorong perlunya alternatif yang lebih aman dan efektif, salah satunya melalui pemanfaatan sinbiotik. Penelitian ini bertujuan untuk mengevaluasi aplikasi sinbiotik dengan dosis prebiotik inulin berbeda dalam meningkatkan sistem imun pada ikan patin untuk pencegahan penyakit edwardsiellosis. Sinbiotik yang diaplikasikan merupakan kombinasi probiotik Bacillus cereus BR2 dan prebiotik berupa inulin komersial. Benih ikan patin berbobot rata-rata 6,22±0,34 g dipelihara dengan kepadatan 25 ekor per akuarium. Percobaan disusun menggunakan Rancangan Acak Lengkap (RAL) yang terdiri atas lima perlakuan dan empat ulangan, yaitu P1 (1% BR2 + 0,1% inulin), P2 (1% BR2 + 0,2% inulin), P3 (1% BR2 + 0,3% inulin), K+ (kontrol positif), serta K- (kontrol negatif). Pakan perlakuan diberikan selama 30 hari, kemudian pada hari ke-31 ikan diuji tantang melalui injeksi intraperitoneal menggunakan suspensi E. tarda dengan kepadatan 107 CFU mL?¹, dan pengamatan dilanjutkan selama 14 hari setelah uji tantang. Produksi biomassa probiotik Bacillus cereus BR2 OTCR diawali dengan inokulasi satu ose kultur bakteri ke dalam media cair Tryptic Soy Broth (TSB). Kultur tersebut kemudian dihomogenkan menggunakan vortex dan diinkubasi pada shaker berkecepatan 1400 rpm selama 24 jam. Indikasi pertumbuhan bakteri ditunjukkan oleh perubahan media menjadi keruh. Setelah inkubasi selesai, dilakukan penghitungan jumlah bakteri melalui metode Total Plate Count (TPC). Sebanyak 1 mL kultur B. cereus BR2 OTCR dimasukkan ke dalam mikrotube Eppendorf, lalu disentrifugasi pada kecepatan 10000 rpm selama 10 menit guna memisahkan pelet dan supernatan. Pelet yang diperoleh dicuci dua kali menggunakan larutan phosphate buffered saline (PBS), kemudian ditambahkan kembali PBS hingga mencapai volume akhir 1.000 µL. Suspensi bakteri selanjutnya diencerkan secara bertingkat (serial dilution). Setiap hasil pengenceran ditumbuhkan pada media Tryptic Soy Agar (TSA) dengan teknik sebar (spread plate), kemudian diinkubasi selama 24 jam untuk menentukan jumlah koloni. Berdasarkan hasil penghitungan, diperoleh kepadatan bakteri sebesar 108 CFU mL?¹. Kultur bakteri tersebut dimanfaatkan sebagai bahan formulasi pakan. Penelitian ini menggunakan pakan komersial dengan kandungan protein 31-33% (kode 781-1) sebagai pakan dasar. Probiotik Bacillus cereus BR2 OTCR dan prebiotik inulin dicampurkan ke dalam pakan sesuai dosis pada masing-masing perlakuan, dengan penambahan putih telur sebanyak 2% sebagai bahan perekat. Proses pelapisan pakan dilakukan dengan mencampurkan probiotik B. cereus BR2 OTCR dan inulin sesuai takaran yang telah ditentukan, kemudian campuran tersebut dimasukkan ke dalam alat semprot (sprayer). Larutan selanjutnya disemprotkan secara merata ke permukaan pakan sambil diaduk hingga homogen, lalu dikeringanginkan selama kurang lebih 15 menit. Coating pakan dilakukan setiap dua hari sekali. Setelah pakan diberikan kepada ikan, sisa pakan perlakuan disimpan dalam lemari pendingin pada suhu 4°C hingga jadwal pemberian berikutnya. Parameter yang diamati dalam penelitian ini mencakup performa pertumbuhan, meliputi laju pertumbuhan spesifik, rasio konversi pakan, dan tingkat kelangsungan hidup. Selain itu, dianalisis pula aspek hematologi dan respons imun yang terdiri atas jumlah total eritrosit dan leukosit, nilai hematokrit, kadar hemoglobin, aktivitas fagositosis, serta respiratory burst. Pengamatan lainnya meliputi aktivitas enzim pencernaan (amilase, protease, dan lipase), histologi usus, keragaman mikrobiota saluran pencernaan, uji mikrobiologi, histopatologi hati, serta evaluasi gejala klinis dan tingkat infeksi. Hasil penelitian memperlihatkan bahwa perlakuan P3 memberikan laju pertumbuhan spesifik tertinggi sekaligus rasio konversi pakan terendah. Perlakuan tersebut juga menunjukkan peningkatan nyata pada aktivitas enzim pencernaan, perbaikan struktur vili usus, serta penguatan parameter imun. Aplikasi sinbiotik berkontribusi terhadap perubahan komposisi dan peningkatan keanekaragaman mikrobiota di saluran pencernaan ikan patin. Seiring dengan bertambahnya dosis inulin, viabilitas dan kolonisasi probiotik Bacillus cereus BR2 di usus meningkat secara signifikan. Lebih lanjut, perlakuan P3 menghasilkan jumlah E. tarda terendah pada organ hati dan tingkat kelangsungan hidup yang lebih tinggi dibandingkan kontrol positif (K+). Berdasarkan hasil pengamatan histopatologi, ikan patin yang diuji tantang dengan E. tarda memperlihatkan perubahan struktur jaringan hati yang tidak normal pada perlakuan K+, P1, P2, dan P3. Kelainan yang teridentifikasi meliputi piknosis dan kariolisis, hemoragi, degenerasi lemak, degenerasi hidropis, serta fibrosis hati. Derajat kerusakan jaringan hati pada kelompok yang memperoleh suplementasi sinbiotik tampak lebih ringan dibandingkan kontrol positif (K+), dengan perbedaan yang signifikan (p<0,05). Penilaian berdasarkan sistem skoring menunjukkan variasi tingkat kerusakan. Tingkat kerusakan organ hati pada perlakuan sinbiotik lebih rendah dan berbeda nyata (p<0,05) dibandingkan K+. Selain itu, kelompok yang memperoleh perlakuan sinbiotik menunjukkan indeks keragaman dan jumlah operational taxonomic units (OTU) lebih tinggi dibandingkan kelompok kontrol. Analisis hierarchical clustering memperlihatkan adanya pola pengelompokan yang tegas antarperlakuan berdasarkan kemiripan komposisi mikrobiota, khususnya pada P2 dan P3. Hasil Principal Coordinate Analysis (PCoA) juga menunjukkan bahwa sampel P3 berada sangat dekat dengan P2. Analisis pada tingkat kelas mengungkapkan bahwa perlakuan sinbiotik menghasilkan proporsi Bacilli tertinggi dibandingkan perlakuan kontrol. Secara umum, pemberian sinbiotik terbukti mampu memperbaiki performa pertumbuhan, meningkatkan aktivitas enzim pencernaan, memperbaiki struktur vili usus, memperkaya keragaman mikrobiota saluran cerna, memperkuat respons imun, serta meningkatkan populasi probiotik Bacillus cereus BR2 pada organ usus. Selain itu, perlakuan ini juga meningkatkan ketahanan ikan patin terhadap infeksi edwardsiellosis, dengan dosis terbaik yaitu dosis 0,3% prebiotik inulin.

Striped catfish (Pangasius sp.) culture in Indonesia continues to face significant constraints due to edwardsiellosis caused by Edwardsiella tarda. Repeated use of antibiotics for disease control has raised several concerns, including the spreading of antibiotic resistant bacteria, accumulation of antibiotic residues in aquaculture products, and potential environmental contamination. These challenges highlight the need for safer and more sustainable disease management strategies, among which synbiotic supplementation represents a promising alternative. Therefore, this study aimed to evaluate the application of synbiotics containing different inclusion levels of inulin as a prebiotic in enhancing the immune response of striped catfish for the prevention of edwardsiellosis. The synbiotic formulation consisted of the probiotic Bacillus cereus BR2 combined with commercial inulin as the prebiotic component. Striped catfish juveniles with an average initial weight of 6.22±0.34 g were stocked at a density of 25 fish per aquarium. The experiment was arranged in a completely randomized design (CRD) with five treatments and four replicates: P1 (1% BR2 + 0.1% inulin), P2 (1% BR2 + 0.2% inulin), P3 (1% BR2 + 0.3% inulin), positive control (K+), and negative control (K-). Experimental diets were administered for 30 days. On day 31, fish were challenged via intraperitoneal injection with Edwardsiella tarda at a concentration of 107 CFU mL?¹, and post-challenge observations were conducted for 14 days. Biomass production of the probiotic Bacillus cereus BR2 OTCR was initiated by inoculating a single loopful of bacterial culture into Tryptic Soy Broth (TSB). The culture was homogenized using a vortex mixer and incubated on a shaker at 1400 rpm for 24 h. Bacterial growth was indicated by turbidity in the medium. Following incubation, bacterial density was quantified using the Total Plate Count (TPC) method. An aliquot of 1 mL of B. cereus BR2 OTCR culture was transferred into an Eppendorf microtube and centrifuged at 10.000 rpm for 10 min to separate the pellet from the supernatant. The resulting pellet was washed twice with phosphate buffered saline (PBS), resuspended in PBS to a final volume of 1.000 µL, and subjected to serial dilution. Each dilution was spread onto Tryptic Soy Agar (TSA) plates using the spread plate technique and incubated for 24 h to determine colony-forming units. The final bacterial density obtained was 108 CFU mL?¹. The prepared bacterial suspension was subsequently incorporated into the experimental diet formulation. A commercial diet containing 31-33% crude protein (code 781-1) was used as the basal feed in this study. The probiotic Bacillus cereus BR2 OTCR and the prebiotic inulin were incorporated into the feed according to the respective treatment dosages, with 2% egg white added as a binder. Feed coating was performed by first preparing a mixture of B. cereus BR2 OTCR and inulin at predetermined concentrations. The mixture was then transferred into a spray bottle and uniformly applied onto the feed pellets while continuously mixing to ensure homogeneity. The coated feed was air-dried for approximately 15 minutes. The coating process was conducted every two days. After feeding, any remaining treated feed was stored at 4°C until the next feeding schedule. The evaluated parameters included growth performance indicators, namely specific growth rate (SGR), feed conversion ratio (FCR), and survival rate (SR). Hematological and immune responses were also assessed, including total erythrocyte and leukocyte counts, hematocrit value, hemoglobin concentration, phagocytic activity, and respiratory burst activity. Additional observations comprised digestive enzyme activities (amylase, protease, and lipase), intestinal histology, gut microbiota diversity, microbiological analysis, liver histopathology, as well as the assessment of clinical signs and infection intensity. The findings demonstrated that treatment P3 yielded the highest specific growth rate (SGR) and the lowest feed conversion ratio (FCR). This treatment also resulted in significant enhancements in digestive enzyme activities, improved intestinal villi morphology, and strengthened immune-related parameters. Synbiotic supplementation contributed to shifts in microbial composition and increased gut microbiota diversity in striped catfish. Increasing dietary inulin levels significantly enhanced the viability and intestinal colonization of B. cereus BR2, along with the total bacterial population. Furthermore, fish in the P3 group exhibited the lowest E. tarda load in the liver and achieved a higher survival rate compared to the positive control (K+). Histopathological observations revealed that striped catfish challenged with E. tarda exhibited abnormal hepatic tissue structures in the K+, P1, P2, and P3 groups. The identified lesions included pyknosis and karyolysis, hemorrhage, fatty degeneration, hydropic degeneration, and hepatic fibrosis. However, the severity of liver tissue damage in synbiotic-supplemented groups was markedly lower than in the positive control (K+), with significant differences observed (p<0.05). Scoring-based evaluation further confirmed variations in the degree of tissue damage among treatments. Fish receiving synbiotic supplementation demonstrated significantly reduced hepatic lesion scores compared to K+ (p<0.05). Furthermore, the synbiotic-treated groups exhibited a higher number of diversity index and Operational Taxonomic Units (OTUs) compared to the control group. Hierarchical clustering analysis revealed a clear separation pattern among treatments based on similarities in gut microbiota composition, particularly between P2 and P3. Principal Coordinate Analysis (PCoA) further demonstrated that samples from P3 clustered closely with those of P2, indicating comparable microbial community structures. At the class level, microbiota profiling indicated that synbiotic supplementation resulted in a higher relative abundance of Bacilli compared to the control treatments. Overall, synbiotic supplementation significantly improved growth performance, enhanced digestive enzyme activities, promoted better intestinal morphology, increased gut microbiota diversity, strengthened immune responses, and elevated the intestinal population of Bacillus cereus BR2. Moreover, this dietary intervention enhanced the resistance of striped catfish against edwardsiellosis. Among the evaluated treatments, 0.3% inulin was identified as the most effective prebiotic dosage.