PENGARUH APLIKASI dsRNA VP-15 IN VITRO DAN IN VIVO TERHADAP SINTASAN DAN RESPONS IMUN UDANG WINDU Penaeus monodon

Teknologi RNA interference (RNAi) merupakan salah satu pendekatan yang digunakan untuk meningkatkan resistensi udang windu terhadap infeksi patogen termasuk WSSV. Pengembangan teknologi RNAi melalui aplikasi untai ganda RNA (dsRNA) yang berasal dari gen pengkode viral protein (VP) dari WSSV telah mulai dikembangkan pada udang. Penelitian ini bertujuan untuk mengkaji sintasan dan respons imun udang windu yang diberi VP-15 pasca uji tantang dengan WSSV. Udang windu (panjang 15,21 ± 1,19 cm dan bobot 32,5 ± 1,83 g) diinjeksi dengan 0,2 µg/ekor dsRNA in vitro (A), dsRNA in vivo (B), dan larutan garam/kontrol (C). Setelah tiga hari vaksinasi, udang windu ditantang dengan WSSV dengan dosis 50 µL/ekor. Pengamatan sintasan dilakukan setiap hari, sedangkan respons imun (THC dan aktivitas proPO) dilakukan pada awal dan hari ke-1, ke-3, dan ke-5 pasca uji tantang, serta analisis ekspresi gen antivirus dan histopatologi hepatopankreas dilakukan pada akhir penelitian. Hasil penelitian menunjukkan bahwa aplikasi dsRNA berpengaruh nyata (P<0,05) terhadap sintasan, THC, dan proPO. Sintasan udang windu yang diberi dsRNA VP-15 in vitro dan in vivo memberikan sintasan yang lebih tinggi 75% dibandingkan dengan kontrol. Nilai proPO tertinggi didapatkan pada dsRNA in vivo (0,138); kemudian dsRNA in vitro (0,093); dan terendah kontrol (0,061); sedangkan THC tertinggi (5.704 x 104 sel/mL) pada dsRNA in vivo, kemudian dsRNA in vitro (3.516 x 104 sel/mL) dan terendah pada perlakuan kontrol (3.322 x 104 sel/mL). Ekspresi gen antivirus semakin meningkat dengan semakin lamanya udang windu terpapar dengan WSSV. Jaringan hepatopankreas udang windu pada perlakuan kontrol (tanpa dsRNA) menunjukkan adanya kerusakan sel akibat infeksi virus.RNA interference (RNAi) technology is one of the approaches used to improve tiger shrimp Penaeus monodon resistance against WSSV infection. The development of RNAi technology through double-stranded RNA (dsRNA) isolated from gene encoding viral protein (VP) of WSSV has been applied to shrimp. This study was aimed to assess the survival rate and immune response of injected-VP-15 WSSV tiger shrimp after a challenge with WSSV. The tiger shrimp (15.21 ± 1.19 cm in length and 32.5 ± 1.83 g in weight) were injected with 0.02 µg/shrimp of in vitro dsRNA (A), in vivo dsRNA (b) and saline solution (C). After three days of vaccination, the tiger shrimp were challenged with WSSV using a dosage of 50 µL/shrimp. The survival rate was observed daily. Analyses of immune responses (hemocyte total and PO activity) were performed in several stages: before the challenge test and day-1, day-3, and day-5 post-challenge test. The expression of the antivirus gene and hepatopancreas histophatology were was observed at the end of the experiment. The results showed that the application of dsRNA significantly influenced the shrimp survival rate, THC, and proPO. Tiger shrimp injected with dsRNA VP-15 of in vitro and in vivo exhibited a higher 75% survival rate than the control (P<0.05). The highest proPO activity (0.138) was obtained at dsRNA in vivo, followed by dsRNA in vitro (0.093) and the lowest (0.061) in the control. The highest THC (5,704 x 104 cell/mL) was in vivo dsRNA, then in vitro dsRNA (3,516 x 104 cell/mL), and the lowest in the control (3,322 x 104 cell/mL). The longer the exposure with WSSV, the higher the antivirus gene expression. Histopathology analysis showed some damages to the hepatopancreas cells in the control shrimp (without dsRNA) caused by the virus infection.

Download Full-text

Screening of probiotics bacteria from coral reef using co-culture method for controlling vibriosis in tiger shrimp (Penaeus monodon) larvae

Jurnal Akuakultur Indonesia ◽

10.19027/jai.10.174-182 ◽

2015 ◽

Vol 10 (2) ◽

pp. 174

Author(s):

Ade Dwi Sasanti ◽

Widanarni . ◽

Sukenda .

Keyword(s):

Coral Reef ◽

Survival Rate ◽

16S Rrna ◽

Vibrio Harveyi ◽

Penaeus Monodon ◽

Culture Method ◽

In Vivo Test ◽

Tiger Shrimp

ABSTRACT This study was carried out to obtain bacteria isolates from coral reef using co-culture method which potentially inhibit Vibrio harveyi growth. A total of 110 isolates were isolated from Acropora sp., Merulina sp., Hystrix sp., Poecillophora sp., Porites sp., and Haliophora sp., and were screened for their antagonistic activity against V. harveyi in in vitro and in vivo test. Five candidate probiotics (5H1 candidate probiotics isolated from Acropora sp., 11I and 11G isolates isolated from Hystrix sp. and 13B and 13G1 isolates isolated from Poecillophora sp., was able to inhibit growth of V. harveyi MR5339 RFR up to 101‒102 cfu/mL. Two isolates (13B and 13G1) were not pathogenic at concentration 106 cfu/mL bacteria and could increase of survival rate of tiger shrimp (Penaeus monodon) larvae in in vivo test. Survival rate of tiger shrimp larvae that treatment with 13B and 13G1 were 86,67% and 88,33%, and have a significant different with positive control (61,67%). Partial sequencing of 16S-rRNA showed that 13G1 isolate was similar to V. alginolyticus. Keywords: vibriosis, Vibrio harveyi, tiger shrimp, probiotic, coral reef ABSTRAK Penelitian ini bertujuan untuk mendapatkan bakteri probiotik asal terumbu karang dengan metode kultur bersama untuk pengendalian penyakit vibriosis pada larva udang windu (Penaeus monodon). Sebanyak 110 isolat berhasil diisolasi dari Acropora sp., Merulina sp., Hystrix sp., Poecillophora sp., Porites sp., dan Heliophora sp. dan dilakukan penapisan untuk melihat aktivitas kemampuannya melawan Vibrio harveyi MR 5339 RfR dalam uji in vitro dan in vivo. Sebanyak 56 isolat menghasilkan daya hambat terhadap V. harveyi MR5339 RfR pada metode kultur bersama. Lima isolat kandidat probiotik (isolate 5H1 diisolasi dari Acropora sp., isolat 11I dan 11G diisolasi dari Hystrix sp., serta isolat 13B dan 13G1 yang diisolasi dari Poecillophora sp.), mampu menekan pertumbuhan V. harveyi MR5339 RfR hingga 101–102 cfu/mL. Kedua isolat (13B dan 13G1) terbukti tidak bersifat patogen pada konsentrasi 106 cfu/mL dan mampu meningkatkan sintasan larva udang windu pada uji in vivo. Nilai sintasan larva pada perlakuan yang diberi kandidat probiotik 13B dan 13G1 berturut-turut adalah 86,67% dan 88,33%, namun berbeda nyata dengan kontrol positif (61,67%). Hasil analisis sekuen sebagian gen 16S-rRNA menunjukkan bahwa isolat 13G1 termasuk spesies V. algynoliticus. Kata kunci: vibriosis, Vibrio harveyi, udang windu, probiotik, terumbu karang

Download Full-text

The Effect Hydroquinone Extracted from Sonneratia caseolaris Fruit to Control Vibrio harveyi Artificial Infection on Tiger Shrimp, Penaeus monodon Fab.

Jurnal Akuakultur Indonesia ◽

10.19027/jai.3.29-35 ◽

2007 ◽

Vol 3 (1) ◽

pp. 29

Author(s):

. Arifuddin ◽

. Sukenda ◽

D. Dana

Keyword(s):

Survival Rate ◽

Minimum Inhibitory Concentration ◽

Inhibitory Concentration ◽

Vibrio Harveyi ◽

Penaeus Monodon ◽

Challenge Test ◽

Total Count ◽

Sonneratia Caseolaris

The role of hydroquinone extracted from Soneratia caseolaris fruit to control Vibrio harveyi infection on tiger prawn was carried out. In vitro experiment was conducted using disc diffusion and MIC {minimum inhibitory concentration) methods to know the sensitivity of V. harveyi to hydroquinone. Two kinds of in vivo experiments were (1) hydroquinone was injected into shrimps muscle and a week later the shrimps were challenged with V. harveyi (2) the shrimps were challenged with V. harveyi and one day later hidroquinone was injected. Total count of live V. harveyi on the shrimps and survival rate were observed after challenge test. Hydroquinone showed antibacterial activity with MIC at 3000 ppm. Hydroquinone injected shrimp showed higher survival rate compared with control (100% vs 50%). Total count of V. harveyi from injected shrimp, either before or after challenged, decreased by 2,61xl04 cfu/g and l,61xl04 cfu/g, respectively. These findings indicated that crude hydroquinone have anti-bacterial effect to control V. harveyi infection. Key words: hydroquinone, Sonneratia caseolaris, Vibrio harveyi, Penaeus monodon ABSTRAK Telaah peran hidrokuinon yang diekstraksi dari buah Soneratia caseolaris untuk mengontrol infeksi Vibrio harveyi pada udang windu dilakukan. Percobaan in vitro dilakukan menggunakan metode difusi cakram dan MIC {minimum inhibitory concentration) untuk mengetahui sensitivitas V. harveyi terhadap hidrokuinon. Percobaan in vivo dilakukan dengan dua cara (1) hidrokuinon disuntikkan pada otot udang dan seminggu kemudian udang diuji tantang dengan V. harveyi (2) udang ditantang terlebih dahulu dengan V. harveyi dan sehari kemudian hidrokuinon disuntikkan. Jumlah total bakteri V. harveyi hidup pada udang dan kelangsungan hidup udang diamati setelah uji tantang. Hidrokuinon menunjukkan aktivitas antibakteri dengan MIC 3000 ppm. Udang yang diinjeksi dengan hidrokuinon mempunyai kelangsungan hidup yang lebih tinggi dibandingkan kontrol (100% vs 50%). Jumlah total V. harveyi pada udang yang diinfeksi, baik sebelum maupun sesudah, masing-masing turun sampai 2,61 x 104 cfu/g dan 1,61 x 104 cfu/g. Hasil ini menunjukkan bahwa hidrokuinon mempunyai efek anti-bakterial untuk mengontrol infeksi V. harveyi. Kata kunci: hidrokuinon, Sonneratia caseolaris, Vibrio harveyi, Penaeus monodon

Download Full-text

Benefits of Tiwai Onion (Eleutherine americana) Extract as Phytopharmaceutical Plant to Inhibit the Growth of Vibrio harveyi Through in-Vitro and in-Vivo

Jurnal Ilmiah Perikanan dan Kelautan ◽

10.20473/jipk.v12i1.12826 ◽

2020 ◽

Vol 12 (1) ◽

pp. 105

Author(s):

Azis Azis ◽

Jimmy Cahyadi

Keyword(s):

Penaeus Monodon ◽

Challenge Test ◽

Inhibition Zone ◽

In Vivo Test ◽

Average Value ◽

Tiger Shrimp ◽

In Vivo Tests ◽

Eleutherine Americana

HighlightsEleutherine americana was a plant that was known to contain antibacterial alkaloids, steroids, phenolics, and flavonoidsEleutherine americana extract was able to inhibit the development of V. harveyi both through in vitro and in vivo tests on tiger shrimp (Penaeus monodon).The use of vibriosis antibiotics in tiger shrimp was often not controlled so the results obtained were not effective AbstractThe use of vibriosis antibiotics in tiger shrimp was often not controlled, so the results obtained were not effective. The addition of antibiotics would cause resistance to V. harveyi. Eleutherine americana is a plant that is known to contain antibacterial alkaloids, steroids, phenols, and flavonoids. This study aimed to determine the effectiveness of inhibitory effects of E. americana extract against V. harveyi through in-vitro and in-vivo tests on tiger shrimp larvae. In-vitro testing consisted of 7 treatments and 3 replications, namely treatments A (0.1%), B (0.2%), C (0.3%), D (0.4%), E (0, 5%), F ethanol 70% (K-), and G chloramphenicol 0.01% (K +) treatment. The largest inhibition zone diameter of E. americana extract was shown in treatment C (0.3%), with an average value of the inhibition zone produced of 7.5 mm. Challenge test with V. harveyi concentration of 107 CFU / ml in the in-vivo test consisted of 5 treatments and four replications namely; A treatment without E. americana extract, B extract 6 ppm, C extract 12 ppm, D extract 18 ppm, and treatment E without extract and V. harveyi. The results of the challenge test with V. harveyi bacteria were significantly different in control (chloramphenicol 0.01%), where the highest survival rate was in the treatment of 12 ppm extract (43.34%). E. americana extract could inhibit the development of V. harveyi bacteria both through in-vitro and in-vivo tests on tiger shrimp (Penaeus monodon).

Download Full-text

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

Diseases of Aquatic Organisms ◽

10.3354/dao03475 ◽

2020 ◽

Vol 139 ◽

pp. 153-160

Author(s):

S Peeralil ◽

TC Joseph ◽

V Murugadas ◽

PG Akhilnath ◽

VN Sreejith ◽

...

Keyword(s):

Gene Expression ◽

Virulence Genes ◽

Vibrio Harveyi ◽

Penaeus Monodon ◽

Challenge Test ◽

Virulence Gene ◽

Economic Losses ◽

Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

Download Full-text

The Effectiveness of Candidate Probiotic Bacteria to Control Vibriosis Disease

Aquatic Science and Technology ◽

10.5296/ast.v5i2.11594 ◽

2017 ◽

Vol 5 (2) ◽

pp. 1

Author(s):

Mulyati Mulyati ◽

Suryati Suryati ◽

Irfani Baga

Keyword(s):

Survival Rate ◽

Sea Water ◽

Challenge Test ◽

Probiotic Bacteria ◽

Pathogenicity Test ◽

Inhibitory Ability ◽

Significant Difference ◽

Portunid Crab

The study aims to isolate, characterize, and examine probiotic bacteria's inhibitory ability against Vibrio harveyi bacteria, both in-vitro and in vivo. Methods used in the study consist of 1) An Isolation of Candidate Probiotic Bacteria, 2) An Antagonistic Test of Candidate Probiotic Bacteria in vitro, 3) An Identification of Bacteria, 4) A Pathogenicity Test of Candidate Probiotic Bacteria, 5) An Antagonistic Test of Candidate Probiotic Bacteria against V. harveyi in vivo. According to the isolation of candidate probiotic bacteria, there are 18 isolated candidate probiotic. After being tested for its inhibitory ability in vitro, there are 8 isolates with zone of inhibition as follows: isolate MM 7 from intestine (22 mm), isolate MM 6 from intestine (12 mm), isolate MM 10 from sea water (10 mm), isolate MM 5 from intestine (9 mm), isolate MM 4 from intestine (8 mm), isolate MM 3 from intestine (7 mm), isolate MM 2.2 from intestine (7 mm), isolate MM 2.1 from intestine (7 mm). Eight genera of the candidate probiotic bacteria is derived from Portunid crab, they are Staphylococcus, Streptococcus, bacillus, vibrio, Alcaligenes, Lactobacillus, micrococcus. Before proceeding the V. harveyi bacterial challenge test in vivo, three potential isolates consisting of MM6, MM7 and MM10 as the probiotic bacteria are pathogenicity-tested against V. harveyi. The survival rate of Portunid crab on pathogenicity test using MM6, MM7 and MM10 generates 91.11-100%, while the control generates 100% survival rate. Variance analysis result through post-hoc Tukey's Honest Significant Difference (HSD) test at 95% confidence interval indicates that isolate MM7 and MM10 are significantly able to increase hatchling Portunid crab's survival rate.

Download Full-text

PENGKLONAN GEN PENYANDI VIRAL PROTEIN 15 (VP-15) WSSV DAN APLIKASINYA SEBAGAI VAKSIN REKOMBINAN PADA UDANG WINDU

Jurnal Riset Akuakultur ◽

10.15578/jra.13.1.2018.57-65 ◽

2018 ◽

Vol 13 (1) ◽

pp. 57

Author(s):

Andi Parenrengi ◽

Sri Redjeki Hesti Mulyaningrum ◽

Andi Tenriulo ◽

Agus Nawang

Keyword(s):

Nucleotide Sequence ◽

Viral Protein ◽

White Spot Syndrome Virus ◽

Penaeus Monodon ◽

Recombinant Vaccine ◽

White Spot ◽

Tiger Shrimp ◽

White Spot Syndrome ◽

Insert Region

Infeksi white spot syndrome virus (WSSV) dapat menyebabkan kematian massal pada budidaya udang windu Penaeus monodon di Indonesia. Infeksi yang terjadi secara sistematis tersebut disebabkan oleh peran gen nucleocapsid viral protein (VP-15). Upaya pengembangan gen VP-15 WSSV untuk menginduksi respons imun dan menetralisasi terhadap infeksi WSSV pada udang windu perlu dilakukan. Penelitian ini bertujuan untuk mengisolasi dan merekombinasikan gen penyandi VP-15 WSSV sebagai vaksin dsRNA, serta menganalisis aplikasinya pada udang windu. Gen VP-15 diisolasi dari udang windu yang terinfeksi WSSV, dikloning ke dalam suatu vektor dan ditransformasikan ke sel kompeten (bakteri Escheria coli DH5a). Plasmid diisolasi untuk mengonfirmasi insert region gen VP-15 melalui sekuensing nukleotida. Pembuatan vaksin rekombinan dilakukan secara in-vitro menggunakan kit MEGAscript RNAi dan diaplikasikan ke udang windu melalui metode injeksi dengan dosis tunggal 0,2 µg dan kontrol (tanpa injeksi vaksin). Hewan uji yang digunakan berukuran panjang 14,75±3,17 g dan bobot 11,64±0,76 cm; serta dipelihara pada wadah bak fiber volume 250 L dengan kepadatan 10 ekor/bak. Hasil penelitian menunjukkan bahwa gen penyandi VP-15 telah diisolasi dari udang windu dan vaksin rekombinan telah dihasilkan secara in-vitro. Analisis sekuens nukleotida memperlihatkan bahwa sisipan gen DNA VP-15 sebesar 253 bp dan menunjukkan kemiripan yang tinggi (99%) pada GenBank. Penggunaan vaksin rekombinan dsRNA dengan dosis 0,2 µg memperlihatkan sintasan udang windu yang dapat mencapai 40,0% dibandingkan dengan kontrol hanya 3,3% (peningkatan 36,7%). Gambaran histopatologi pada jaringan hepatopankreas udang windu pada perlakuan kontrol menunjukkan adanya kerusakan inti sel, akibat infeksi WSSV. Gene VP-15 berpotensi sebagai bahan vaksin rekombinan dsRNA dalam mencegah infeksi WSSV.Infection of white spot syndrome virus (WSSV) causes bulk mortalities of tiger shrimp Penaeus monodon cultured in Indonesia. The nucleocapsid viral protein-15 (VP-15) is strongly suspected to be responsible for the systemic infection of WSSV. The development of VP-15 WSSV gene for inducing the immune response to and neutralize WSSV infection of tiger shrimp is vitally needed. The aim of this study was to isolate and clone the gene encoding VP-15 WSSV as dsRNA vaccine and assess the vaccine application to tiger shrimp. VP-15 gene was isolated from the genomic DNA of infected tiger shrimps, cloned into the vector, and transformed into competent cells (Escheria coli DH5a). The plasmid was isolated to confirm the insert region gene of VP-15 by the nucleotide sequence. Production of dsRNA vaccine was performed by in-vitro using MEGAscript RNAi kit and applied to tiger shrimp through muscular injection at a single dosage of 0.2 µg and without dsRNA as a control treatment. The average size of tiger shrimps used was 14.75±3.17 g in weight and 11.64±0.76 cm in length and stocked in 250 L fiber tank at 10 ind./tank. The results of the study showed the VP-15 gene was successfully isolated from the tiger shrimps and the recombinant vaccine was produced by in-vitro. The analysis of nucleotide sequence showed that the inserted DNA was 253 bp and showed a high similarity (99%) with VP-15 gene deposited in the GenBank. The application of dsRNA vaccine showed that the dosage of 0.2 ¼g resulted in the survival rate of 40.0% compared with without dsRNA (control) of 3.3% (36.7% increment). Hepatopancreas histology indicated obvious damages to cell nucleus in the un-vaccinated tiger shrimp caused by the virus infection. We suggest that the VP-15 gene is a very promising dsRNA recombinant vaccine against WSSV infection.

Download Full-text

RESPONS IMUN UDANG WINDU Penaeus monodon TERHADAP VAKSIN dsRNA VP-24 PADA DOSIS BERBEDA

Jurnal Riset Akuakultur ◽

10.15578/jra.13.1.2018.77-84 ◽

2018 ◽

Vol 13 (1) ◽

pp. 77

Author(s):

Sri Redjeki Hesti Mulyaningrum ◽

Andi Parenrengi ◽

Bunga Rante Tampangallo ◽

Ike Trismawanti

Keyword(s):

Immune Response ◽

Survival Rate ◽

Saline Solution ◽

Penaeus Monodon ◽

Challenge Test ◽

Injection Method ◽

Tiger Shrimp ◽

Total Hemocyte Count ◽

Hemocyte Count

Peningkatan produksi udang windu Penaeus monodon terus diupayakan, salah satunya dengan peningkatan respons imun udang terhadap infeksi penyakit WSSV. Penelitian ini bertujuan untuk mengetahui respons imun udang terhadap pemberian vaksin dsRNA VP-24 pada berbagai dosis. Konstruksi vaksin dsRNA VP-24 dilakukan menggunakan Megascript kit dengan DNA genom VP-24 sebagai template. Vaksinasi dilakukan dengan metode injeksi pada udang windu yang berukuran rata-rata 15,88 ± 3,50 g; dosis vaksin yang diujikan adalah 0,02 µg; 0,2 µg; 2 µg; dan sebagai kontrol adalah udang yang tidak diberi vaksin. Penelitian terdiri atas empat perlakuan dosis vaksin dengan masing-masing dua ulangan dan dipelihara selama lima hari. Uji tantang dilakukan selama enam hari dengan menginjeksi virus WSSV dalam saline solution (1:3 v/v). Pengamatan terhadap sintasan udang windu dilakukan setiap hari, sedangkan penghitungan total hemocyte (THC) dan ProPO diamati pada hari I, III, dan VI setelah diinfeksi WSSV. Pada akhir pengujian dilakukan pengambilan jaringan hepatopankreas untuk analisis histopatologi. Analisis data dilakukan secara statistik dengan analisis ragam (ANOVA). Hasil yang diperoleh memperlihatkan bahwa injeksi vaksin dsRNA VP-24 dengan dosis 0,2 µg memiliki pengaruh yang signifikan terhadap sintasan dan respons imun udang windu (P<0,05). Vaksin dsRNA VP-24 dengan dosis 0,2 µg mampu memberikan sintasan udang windu P. monodon sebesar 65% dan meningkatkan respons imun udang dengan THC (1.550 x 10t sel/mL) dan ProPO (0,042 Abs).One of the efforts to increase the production of tiger shrimp Penaeus monodon is increasing the immune response against WSSV disease. This study aims to evaluate shrimp immune response to dsRNA VP-24 vaccination at various doses. The construction of dsRNA VP-24 vaccine was performed using Megascript kit with the VP-24 DNA genome as a template. The vaccination was done by injection method on shrimp sized 15.88 ± 3.50 g. The tested vaccine doses (treatments) were 0.02 ¼g; 0.2 ¼g; 2 ¼g; and unvaccinated shrimp as the control. The study consisted of four treatments of vaccine doses with two replicates for each treatment. The challenge test was performed by injecting the WSSV virus in saline solution (1:3 v/v). The observation on shrimp survival rate was done daily, while the total hemocyte count (THC) and ProPO observation were performed on the 1st day, 3rd day, and 6th day after WSSV infection. At the end of the experiment, samplings of hepatopancreas for analysis were performed. Data were statistically analyzed using ANOVA. The present study indicated that the injection of 0.2 ¼ g dsRNA VP-24 vaccine had a significant effect on the survival rate and immune response of shrimp (P<0.05). The dose of 0.2 ¼g dsRNA VP-24 had resulted in 65% of survival rate and increased immune response of P. monodon with THC (1,550 x 10t cell/mL) and ProPO (0.042 Abs).

Download Full-text

Effect of Heating at Various Temperatures for 30 Minutes on Pathogenicity of White Spot Syndrome Virus (WSSV) in Tiger Prawn (Penaeus monodon Fabr.)

Jurnal Akuakultur Indonesia ◽

10.19027/jai.5.5-12 ◽

2007 ◽

Vol 5 (1) ◽

pp. 5

Author(s):

D. Priatni ◽

M. Alifuddin ◽

D. Djokosetiyanto

Keyword(s):

Survival Rate ◽

White Spot Syndrome Virus ◽

Penaeus Monodon ◽

Challenge Test ◽

White Spot ◽

Tiger Shrimp ◽

Pathogenic Virus ◽

Short Period ◽

Tiger Prawn ◽

White Spot Syndrome

White spot syndrome virus (WSSV) is a strong pathogenic virus which spread very rapidly and can cause tiger shrimp mass mortality within a short period. Enhancement of shrimp immunity by infecting inactivated WSSV is one of the efforts to overcome WSSV infection in shrimp. In this study, inactivated WSSV were prepared by heating them with various temperatures namely 45, 50, 55 and 60oC for 30 minutes. The results shows that infection with heating inactivated WSSV at 45°C and 60°C for 30 min on PL-15 could increase their immunities. The survival rate of inactivated WSSV-infected shrimp after challenge test with WSSV virulent reached 77%, while no survive shrimp was observed in control. This suggests that shrimp immunity could be improved by vaccination using WSSV virus inactivated by heating. Keywords: WSSV virus, pathogen, tiger shrimp, heating ABSTRAK White Spot Syndrome Virus (WSSV) merupakan virus yang sangat ganas bagi udang windu, dengan penularan yang sangat cepat dan menyebabkan kematian dalam waktu yang cepat. Peningkatan imunitas udang dengan meenginfeksikan WSSV inaktif merupakan salah satu upaya untuk menanggulangi infeksi WSSV pada udang. Pada penelitian ini, inaktivasi WSSV dilakukan menggunakan pemanasan pada suhu berbeda, yaitu 45, 50, 55 dan 60oC selama 30 menit. Hasil penelitian menunjukkan bahwa infeksi PL-15 menggunakan virus WSSV yang telah diinaktivasi dengan pemanasan pada suhu 45oC dan 60oC dapat meningkatkan daya tahan udang. Kelangsungan hidup udang yang telah diinfeksi dengan WSSV hasil inaktivasi sebelum uji tantang dengan WSSV virulen mencapai 77%, sementara udang yang tidak diinfeksi dengan WSSV hasil inaktivasi adalah semua mati. Dengan demikian dapat disimpulkan bahwa daya tahan udang dapat ditingkatkan melalui vaksinasi menggunakan WSSV yang telah diinaktivasi dengan pemanasan. Kata kunci: virus WSSV, patogen, udang windu, pemanasan

Download Full-text

Inhibitory Mechanism of Robiotic Bacteria on The Growth of Vibrio harveyi in Tiger Shrimp (Penaeus monodon) Larvae

Jurnal Akuakultur Indonesia ◽

10.19027/jai.7.179-188 ◽

2008 ◽

Vol 7 (2) ◽

pp. 179

Author(s):

. Widanarni ◽

E. Ayuzar ◽

. Sukenda

Keyword(s):

Survival Rate ◽

Vibrio Harveyi ◽

Penaeus Monodon ◽

Vibrio Alginolyticus ◽

Probiotic Bacteria ◽

Rrna Gene ◽

Inhibitory Mechanism ◽

Water Culture ◽

Tiger Shrimp

Three probiotics named SKT-b, 1Ub, and Ua had inhibitory activity against the growth of Vibrio harveyi. These strains were mutated by rifampicin resistant. The inhibitory effect of SKT-b,1Ub, and Ua on the growth of V. harveyi was investigated by concomitant incubation of the two bacteria in a culture shrimp larvae. Colony forming unit of V. harveyi, probiotic and total of bacteria in dead, live larvae and water culture was monitored, and survival rate of larvae was investigated. Shrimp inoculated probiotic previously had survival rate higher than control (without probiotic). Number of V. harveyi in treatment without probiotic inoculation also higher compared to treatment with probiotic inoculation in dead, live larvae and water culture. It demonstrated possible inhibition of probiotic bacteria on V. harveyi through competition for adherence sites or nutrition source. Partial sequencing of 16S-rRNA gene showed that 1Ub was similar to Pseudoalteromonas piscicida, whereas SKT-b and Ua were similar to Vibrio alginolyticus. Keywords: probiotic bacteria, inhibitory mechanism, V. harveyi, tiger shrimp ABSTRAK Tiga isolat bakteri probiotik yaitu 1Ub, SKT-b dan Ua telah diuji memiliki aktivitas penghambatan terhadap pertumbuhan V. harveyi secara in vitro. Ketiga isolat ini kemudian diberi penanda resisten rifampisin (RfR) melalui mutasi spontan untuk mempelajari mekanisme penghambatannya pada larva udang windu. Efek penghambatan dari 1Ub, SKT-b dan Ua terhadap pertumbuhan V. harveyi diamati melalui pemberian secara bersamaan antara bakteri probiotik dan V. harveyi tersebut dalam air pemeliharaan larva udang. Jumlah sel bakteri probiotik, V. harveyi dan total bakteri baik pada larva mati, larva hidup dan air pemeliharaan diamati dan kelangsungan hidup larva dihitung. Nilai kelangsungan hidup udang pada perlakuan yang diinokulasi bakteri probiotik lebih tinggi daripada kontrol (tanpa penambahan bakteri probiotik). Jumlah sel V. harveyi pada perlakuan tanpa penambahan probiotik juga lebih tinggi, dibanding pada perlakuan dengan penambahan probiotik baik pada larva mati, larva hidup maupun air media pemeliharaan. Hal ini menunjukkan adanya penghambatan bakteri probiotik terhadap V. harveyi yang kemungkinan melalui kompetisi tempat pelekatan atau sumber nutrisi. Hasil analisis sekuen sebagian gen 16-rRNA menunjukkan bahwa isolat 1Ub termasuk spesies Pseudoalteromonas piscicida, sedangkan SKT-b dan Ua termasuk spesies Vibrio alginolyticus. Kata kunci: bakteri probiotik, mekanisme penghambatan, V. harveyi, udang windu

Download Full-text

Selection of lactic acid bacteria as a probiotic and evaluated its performance on gnotobiotic catfish Clarias sp.

Jurnal Akuakultur Indonesia ◽

10.19027/jai.17.1.68-80 ◽

2018 ◽

Vol 17 (1) ◽

pp. 68

Author(s):

Enita Romasni Turnip ◽

Widanarni, Widanarni ◽

Anja Meryandini

Keyword(s):

Growth Rate ◽

Lactic Acid ◽

Survival Rate ◽

Lactic Acid Bacteria ◽

Aeromonas Hydrophila ◽

Pathogenic Bacteria ◽

Challenge Test ◽

Feed Conversion

ABSTRACT This study aimed to select lactic acid bacteria (LAB) as a potential probiotic that producing anti‒microbial compounds in order to treat motile aeromonads septicemia diseases caused by Aeromonas hydrophila on catfish Clarias sp. and evaluated its performance on gnotobiotic catfish. The in vitro assay was done to select several LAB isolates based on antagonistic activity against pathogenic bacteria. The selected isolate was tested in vivo to observe their ability to improve growth performances of catfish. The study was conducted with five treatments consists of K‒ (normal catfish without addition probiotic, without challenge test), K+ (normal catfish without addition of probiotic, with challenge test), Np (normal catfish with addition of probiotic and challenge test), G (gnoto catfish without addition of probiotic, with challenge test), and Gp (gnoto catfish with addition of probiotic and challenge test). The results showed that the addition of Pediococcus pentosaceus E2211 as selected probiotic could increase survival rate, specific growth rate, and immune response towards infection of A. hydrophila. The best survival rate after challenge test was obtained in Np and Gp treatments (88.46%), followed by G treatment (65.38%), while the K+ was only 53.84%. The conclusion of this study was P. pentosaceus E2211 potentially used as a probiotic candidate for normal and gnotobiotic catfish. The presence of normal microflora with P. pentosaceus E2211 in Np treatment showed the best probiotic performance with daily growth rate 3.28%, feed conversion ratio 1.79, and total intestinal bacteria reached 108 CFU/mL significantly different from other treatments (P<0.05).Keywords: Aeromonas hydrophila, catfish, LAB, probiotic, screening ABSTRAKTujuan penelitian ini adalah menyeleksi bakteri asam laktat (BAL) sebagai probiotik potensial penghasil senyawa antimikrob guna menanggulangi penyakit motile aeromonad septicemia akibat Aeromonas hydrophila pada ikan lele Clarias sp. dan evaluasi kinerjanya pada ikan lele gnotobiotik. Pengujian in vitro dilakukan untuk menyeleksi beberapa isolat BAL sebagai kandidat probiotik berdasarkan aktivitas antagonis terhadap bakteri patogen. Isolat terpilih kemudian diuji in vivo untuk mengetahui kemampuannya dalam meningkatkan performa tumbuh ikan lele. Penelitian ini menggunakan rancangan acak lengkap dengan lima perlakuan, yaitu: K‒ (lele normal tanpa probiotik dan tanpa tanpa diuji tantang), K+ (lele normal tanpa probiotik dan diuji tantang), Np (lele normal diberi probiotik dan diuji tantang), G (lele gnoto tanpa probiotik dan diuji tantang), dan Gp (lele gnoto diberi probiotik dan diuji tantang). Hasil penelitian menunjukkan pemberian probiotik terpilih BAL Pediococcus pentosaceus E2211 mampu meningkatkan sintasan, laju pertumbuhan, dan respons imun ikan lele terhadap infeksi A. hydrophila. Sintasan terbaik pascauji tantang diperoleh pada perlakuan Np dan Gp yaitu sebesar 88,46%, diikuti perlakuan G sebesar 65,38%, sementara pada K+ hanya mencapai 53,84%. Kesimpulan dari penelitian ini ialah isolat BAL terpilih P. pentosaceus E2211 berpotensi sebagai kandidat probiotik untuk ikan lele normal dan lele gnotobiotik Clarias sp. Keberadaan mikroflora normal yang berasosiasi dengan P. pentosaceus E2211 pada perlakuan Np menunjukkan kinerja probiotik terbaik dengan nilai laju pertubuhan harian 3,28%, rasio konversi pakan 1,79 dan total bakteri usus mencapai 108 CFU/mL yang berbeda signifikan dibanding perlakuan lainnya (P<0,05).Kata kunci: A. hydrophila, BAL, ikan lele, probiotik, seleksi

Download Full-text