CURRENT MICROBIOLOGICAL ASPECTS IN HIGH MOUNTAIN LAKE RESEARCH

Remote and normally unpolluted high mountain lakes provide habitats with no or very limited anthropogenic influences and, therefore, their hydrodynamics are mostly regulated by the natural conditions. Researches in high mountain lakes deal with measuring and modeling the response of the habitats to environmental changes especially correlated to acid deposition, pollutants influx and climatic variability. The microbial world has also become a focus in many studies of these extreme ecosystems. Despite the pressure of harsh and extreme conditions, microbial communities living in these habitats developed flexible strategies and show quick adaptation to climate oscillation. New aspects in microbiological studies in recent high mountain lake research are presented in this paper.Key words : High mountain lake, extreme environment, microbial communities, microbial diversity, psychrotolerant microbe, molecular microb

Free-Living Ice-Nucleating Active Bacteria from High Mountain Lake Habitats

We collected the culturable heterotrophic bacteria from oligotrophic high mountain lake habitats and tested their capability to induce ice formation. Direct plating was carried out using low-nutrient medium at a temperature of between 3 and 4°C. As many as 84 isolates were recovered from glacial ice and natural biofilm growing on granite rocks surface. Six out of 84 isolates were capable of expressing the ice-nucleation phenotype. After autoclaving the cell suspension at 121°C for 15 min, isolate J78 was still able to retain the ability for ice formation. Heat-stable ice nuclei produced by ice-nucleating active bacteria have potential applications in biotechnology. Characterization of INA bacteria was performed employing live-dead Gram staining and molecular methods. Universal primers for Bacteria (S-D-Bact-0008-b-S-20 and S-D-Bact-1524-a-A-18) were used for PCR to amplify almost the full length of the 16S rRNA genes of selected INA isolates. Restriction fragment length polymorphism analysis resulted in 2 unique patterns, as represented by J43 and J83, respectively. Based on DNA sequencing of 16S rRNA gene, isolate J43 (GeneBank accession no. AJ864852) was closely related to Pseudomonas mephitica (99.2% sequence similarity) and Janthinobacterium lividum (99% similarity), whereas isolate J83 (GeneBank accession no. AJ864859) showed 100% sequence identity to Pseudomonas fluorescens

Biocontrol agents in aquaculture: Production and their application

The use of biocontrol agents have become commonly accepted as a 'natural weapon' in aquaculture, either as an ecological bioremediator for low environmental quality, as well as the health promoter for cultured organisms. Biocontrol agents, which consist of beneficial microorganisms, are commonly applied as biosupplements in feeds. They are potential in replacing the use of antibiotic in inhibiting the pathogenic organisms. Therefore, their application has revealed the potential as an effective strategy to reduce the over use of antibiotics in controlling the pathogenic agents, avoid the spreading the drug resistance, or environmental deterioration of the negative effect by killing useful microorganisms. The development of suitable technology for microbial agents production, viability and stability, is a key area of research for industrial production. Production of biocontrol agents should be based on the microbial criteria, and the ability to withstand stress during processing and storage of products is crucial. This review makes an overview of biocontrol agents selection studies including techniques for isolation/identification, selection, production and its application in order to be accepted as a valuable product in aquaculture. Key words: Biocontrol agents, beneficial microorganisms, aquaculture.   ABSTRAK Pemanfaatan agen biokontrol telah dapat diterima secara luas di dunia akuakultur sebagai senjata alami, baik sebagai bioremediator ekologis untuk memperbaiki kualitas lingkungan yang rendah maupun sebagai promoter/pendukung kesehatan untuk organisme-organisme yang dibudidayakan. Agen-agen biokontrol yang terdiri atas berbagai mikroorganisme yang menguntungkan umumnya diaplikasikan sebagai biosuplemen dalam pakan. Mikroorganisme tersebut potensial dalam menggantikan penggunaan antibiotik dalam proses penghambatan terhadap organisme patogenik. Oleh karena itu, aplikasinya yang sangat potensial dapat berguna sebagai strategi efektif untuk mereduksi penggunaan berlebih dari antibiotik dalam pengendalian agen patogenik, mencegah penyebaran sifat resistensi terhadap obat-obatan, atau mencegah kerusakan lingkungan dari kematian mikroorganisme yang bermanfaat. Pengembangan teknologi yang sesuai untuk produksi, viabilitas dan stabilitas dari agen biokontrol tersebut, merupakan kunci dari penelitian untuk produksi masal pada skala industri. Proses produksi dari agen biokontrol harus berdasar pada kriteria mikrobiologis. Sifat-sifat ketahanannya terhadap stress selama pemrosesan maupun penyimpanan produk adalah penting. Pada makalah ini disajikan teknik-teknik untuk isolasi, identifikasi, produksi dan aplikasi agen biokontrol untuk dapat diterima sebagai produk yang bernilai ekonomis dalam akuakultur. Kata kunci: Agen biokontrol, mikroorganisme yang menguntungkan, akuakultur

The Use of Synbiotics to Prevent IMNV and Vibrio harveyi Co-Infection in Litopenaeus vannamei

This study evaluated the effects on viral immune responses and bacterial co-infection, of different feeding frequencies of a synbiotic supplemented diet given to Pacific white shrimp (L. vannamei). A synbiotic-supplemented diet was formulated from probiotic Vibrio alginolitycus SKT-bR and prebiotics from sweet potato (Ipomoea batatas L.) oligosaccharide. Pacific white shrimp were fed the synbiotic diet at different frequencies, i.e. daily (P1), twice a week (P2), and once a week (P3) for a 30 day pre-challenge test. After the 30 day feeding period, the shrimps were challenged by intramuscular injection of Infectious Myonecrosis Virus (IMNV) and Vibrio harveyi. The results showed that shrimp treated with a synbiotic-supplemented diet showed  significantly higher growth performance than control groups (P<0.05). Shrimp treated under regime P1 showed the highest values for phenoloxidase (PO) and respiratory burst (RB) parameters compared to shrimp given the other treatments. Following the challenge test, higher survival rate were seen in the P1 treatment group, in comparison to the positive control, and the P1 treatment group showed the highest values in total haemocyte count (THC), PO, and RB

SKRINING DAN IDENTIFIKASI BAKTERI ANTI QUORUM SENSING ASAL TAMBAK UDANG VANAME PENGHAMBAT VIRULENSI Vibrio parahaemolyticus

Vibriosis dapat dicegah dan dikendalikan dengan memanfaatkan mekanisme anti quorum sensing (AQS). Salah satu strategi anti quorum sensing dalam menghambat ekspresi faktor virulen dari Vibrio parahaemolyticus yaitu dengan mendegradasi sinyal komunikasi sel bakteri menggunakan AHL laktonase. Penelitian ini bertujuan untuk menyeleksi dan mengindentifikasi bakteri penghasil AHL laktonase yang berpotensi mampu menghambat virulensi bakteri patogen V. parahaemolyticus. Isolasi bakteri dilakukan dari sampel saluran pencernaan udang vaname, air, dan sedimen tambak. Sebanyak 18 dari 111 isolat yang diisolasi menunjukkan adanya aktivitas AQS terhadap bioindikator Chromobacterium violaceum. Hasil uji patogenitas secara in vitro pada agar darah didapatkan tiga isolat yang tidak menunjukkan aktivitas hemolisis yaitu B5, K4, dan S12. Hasil konfirmasi dan analisis gen aiiA menggunakan Blast-X menunjukkan bahwa isolat B5 dan S12 memiliki kesamaan dengan AHL laktonase pada Bacillus cereus, sedangkan K4 memiliki similaritas dengan AHL laktonase pada multispesies Bacillus sp. Hasil pensejajaran sekuen gen 16S rRNA ketiga isolat tersebut dengan data pada GenBank, teridentifikasi sebagai Bacillus siamensis (B5), Bacillus cereus (K4), dan Bacillus amyloliquefaciens (S12). Berdasarkan hasil uji antagonis dan uji kultur bersama disimpulkan bahwa isolat K4 bekerja dengan mekanisme AQS sedangkan isolat B5 dan S12 diduga berjalan dua mekanisme secara bersama yaitu antibiosis dan anti quorum sensing. Hasil penelitian ini menunjukkan bahwa ketiga isolat tersebut memiliki potensi sebagai kandidat agen biokontrol pada akuakultur sehingga perlu dilakukan uji lanjutan.Vibriosis can be prevented and controlled by utilizing the anti-quorum sensing (AQS) mechanism. One of the anti-quorum sensing mechanisms to inhibit the expression of virulent factors of Vibrio parahaemolyticus is by degrading the quorum sensing communication signals using AHL lactonase. The study aimed to select and identify AHL lactonase-producing bacteria that have the potentials to inhibit the virulence of V. parahaemolyticus. Several batches of bacteria were isolated from the digestive tract of vannamei shrimp, water, and sediment of shrimp ponds. There were 18 out of 111 isolates that showed AQS activity against Chromobacterium violaceum used as a bioindicator. In vitro pathogenicity test on blood agar showed that B5, K4, and S12 isolates showed gamma hemolysis activity. The results of confirmation and analysis of aiiA genes using Blast-X showed that B5 and S12 isolates have AHL lactonase similarities with Bacillus cereus, whereas K4 has similarities with multispecies Bacillus sp. Alignment results of the 16S rRNA gene sequences with GenBank data showed that B5, K4, and S12 isolates were identified as Bacillus siamensis, Bacillus cereus, and Bacillus amyloliquefaciens, respectively. The follow up antagonistic and coculture tests revealed that K4 uses the AQS mechanism, while B5 and S12 likely use antibiotic mechanism and anti quorum sensing to inhibit the virulent expression of V. parahaemolyticus. This study concludes that the three isolates have the potential to be used as biocontrol agents in brackishwater aquaculture. Further research is needed to determine the pathogenicity of AQS bacteria to vannamei shrimp and the effective concentration of AQS bacteria to inhibit the virulence of V. parahaemolyticus to vannamei shrimp by in vivo treatment.

Remote and Normally Unpolluted High Mountain Lakes Provide Habitats with No or Very Limited Anthropogenic Influences and, Therefore, Their Hydrodynamics Are Mostly Regulated by the Natural Conditions. Researches in High Mountain Lakes Deal with Measuring

Remote and normally unpolluted high mountain lakes provide habitats with no or very limited anthropogenic influences and, therefore, their hydrodynamics are mostly regulated by the natural conditions. Researches in high mountain lakes deal with measuring and modeling the response of the habitats to environmental changes especially correlated to acid deposition, pollutants influx and climatic variability. The microbial world has also become a focus in many studies of these extreme ecosystems. Despite the pressure of harsh and extreme conditions, microbial communities living in these habitats developed flexible strategies and show quick adaptation to climate oscillation. New aspects in microbiological studies in recent high mountain lake research are presented in this paper

BACTERIAL COMMUNITY SHIFTS OF A HIGH MOUNTAIN LAKE IN RESPONSE TO VARIABLE SIMULATED CONDITIONS: AVAILABILITY OF NUTRIENTS, LIGHT AND OXYGEN

We studied bacterial population composition shifts by exposing natural water samples to variable simulated environmental conditions. The samples were taken from Lake Jori XIII (2640 m a.s.l), an oligo-to mesotrophic cold freshwater lake, located in the eastern Swiss Alps. The Jori lakes are characterized as remote, unpolluted high mountain lakes with a long period of ice cover and typically low nutrient concentrations. Culture independent techniques (PCR-based analyses) were used for detection and molecular characterization of a large number of bacteria most of which are still uncultivable. Bacterial community shifts over three ecological conditions (nutrients, light and oxygen availability) were detected by using Temporal Temperature gradient Gel Electrophoresis (TTGE) of a PCR-amplified part of the 16S rRNA gene. The bacterial populations responded differently to the variable conditions, as revealed by TTGE pattern shifts during the experiment. Key words: Temporal Temperature gradient Gel Electrophoresis (TTGE), ARB, small subunit ribosomal RNA gene (SSU rRNA gene), alpine freshwater lake JOri XIII, PC

Existing of White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV) and Infectious Hypodermal Haematopoitic Necrosis Virus (IHHNV) in White Shrimp Litopenaeus vannamei Reared at Intensive Tambak System in Bakauheni, Lampung Selatan

Development of white shrimp, Litopenaeus vannamei culture to increase production yield should take attention in prevention of viral outbreak which  is a main factor caused harvesting failure.  Viruses that usually infect shrimp are white spot syndrome virus (WSSV), taura syndrome virus (TSV) dan infectious hypodermal hematopoietic necrosis virus (IHHNV).  To prevent virus infection and reduce risk of fail to harvest, an early warning and observation system of availability of pathogen should be taken in order to know the shrimp condition during culture process.  This study was performed to examine existing of WSSV, TSV and IHHNV in shrimp reared in tambak by PCR and histopathology methods. Samples were taken every two weeks from tambak culture at Bakauheni, Penengahan, Lampung Selatan.  Generally, WSSV, TSV, and IHHNV were found in shrimp from tambak and water around tambak.  Virus infection was first detected in shrimp derived from tambak on 66 days after crop and still exists until 107 days after crop.  Hepatopancreatic cell disorder  caused by the three viruses were found in cell nuclei that became bigger, necrosis in cytoplasm and inclusion body at nuclei.  The existing of virus in outside of tambak indicated that virus was horizontally transmitted. Keywords: WSSV, TSV, IHHNV, white shrimp   ABSTRAK Perkembangan budidaya udang vaname Litopenaeus vannamei dalam usaha meningkatkan hasil produksi harus memperhatikan keberadaan penyakit viral yang menjadi penyebab utama kegagalannya. Virus yang biasa menyerang vaname antara lain white spot syndrome virus (WSSV), taura syndrome virus (TSV) dan infectious hypodermal hematopoietic necrosis virus (IHHNV). Untuk mengantisipasi penyebaran virus dan mengurangi resiko kegagalan produksi diperlukan usaha pencegahan dengan melakukan peringatan dini (early warning) dan pemantauan tambak terhadap keberadaan patogen tersebut selama masa budidaya. Penelitian ini dilakukan untuk mengetahui keberadaan WSSV, TSV dan IHHNV yang menginfeksi udang vaname dengan analisa polymerase chain reaction (PCR) dan histopatologis. Pengambilan sampel dilakukan sebanyak delapan kali setiap dua minggu di tambak intensif udang vaname Litopenaeus vannamei di Bakauheni, Penengahan, Lampung Selatan. Secara umum, WSSV, TSV dan IHHNV telah ditemukan pada tambak serta di perairan sekitar lokasi tersebut. Infeksi virus pertama kali terdeteksi di tambak ketika udang berumur 66 hari setelah penebaran dan terdeteksi pula pada waktu pengamatan selanjutnya sampai udang berumur 107 hari setelah penebaran. Kerusakan akibat serangan ketiga jenis virus tersebut terjadi pada bagian inti sel yang mengalami pembesaran, nekrosis pada sitoplasma dan badan inklusi yang menekan inti sel. Pola transmisi virus yang terjadi adalah secara horizontal yang didukung oleh terdeteksinya virus tersebut pada udang di luar tambak.   Kata kunci : WSSV, TSV, IHHNV, udang vanam

Growth performance, immune response, and resistance of Nile tilapia fed paraprobiotic Bacillus sp. NP5 against Streptococcus agalactiae infection

ABSTRACT   This study was aimed to evaluate the effectiveness of Bacillus sp. NP5 paraprobiotic administration through commercial feed on growth performance, immune response, and resistance of Nile tilapia against Streptococcus agalactiae infection. Bacillus sp. NP5 paraprobiotic was produced through heat-inactivation at 95°C for 1 h, then performed a viability test on tryptic soy agar (TSA) media and incubated for 24 hours. Paraprobiotics could be used whether the bacteria did not grow on the TSA media. This study used a completely randomized design, containing three treatments with five replications, i.e. 1% (v/w) probiotic addition, 1% (v/w) paraprobiotic addition, and no addition of probiotic or paraprobiotic (control). The experimental fish were reared for 30 days. On day 31 of rearing, fish were challenged with S. agalactiae (107 CFU/mL) through intraperitoneal injection route, while the negative control was injected with PBS. This study results significantly improved growth performances and immune responses (P<0.05), compared to control after 30 days of probiotic and paraprobiotic Bacillus sp. NP5 administration. After challenge test, increased immune responses in probiotic and paraprobiotic of Bacillus sp. NP5 treatment had higher survival rates (P<0.05) than positive control. The administration of Bacillus sp. NP5 probiotic and paraprobiotic through commercial feed were effective in increasing growth performance, immune response, and resistance of Nile tilapia against S. agalactiae infection. Keywords: Bacillus sp. NP5, Nile tilapia, paraprobiotic, Streptococcus agalactiae   ABSTRAK   Penelitian ini bertujuan mengevaluasi efektivitas pemberian paraprobiotik Bacillus sp. NP5 melalui pakan dalam meningkatkan kinerja pertumbuhan, respons imun, dan resistansi ikan nila terhadap infeksi Streptococcus agalactiae. Proses pembuatan bakteri paraprobiotik yaitu Bacillus sp. NP5 diinaktivasi panas pada suhu 95°C selama 1 jam, dilanjutkan dengan pengujian viabilitas dengan menyebarkannya pada media tryptic soy agar kemudian diinkubasi selama 24 jam. Jika bakteri tidak tumbuh, maka paraprobiotik dapat digunakan. Penelitian ini menggunakan rancangan acak lengkap dengan tiga perlakuan dan lima ulangan, yaitu penambahan probiotik 1% (v/w), penambahan paraprobiotik 1% (v/w), dan tanpa penambahan probiotik atau paraprobiotik (kontrol). Ikan perlakuan dipelihara selama 30 hari dan pada hari ke-31, ikan diuji tantang dengan S. agalactiae (107 CFU/mL) melalui injeksi intraperitoneal, sementara perlakuan kontrol negatif diinjeksi dengan PBS. Hasil penelitian setelah 30 hari pemberian probiotik dan paraprobiotik Bacillus sp. NP5 menunjukkan kinerja pertumbuhan dan respons imun yang meningkat signifikan (P<0.05) dibandingkan dengan kontrol. Pascauji tantang, peningkatan respons imun pada perlakuan probiotik dan paraprobiotik Bacillus sp. NP5 menunjukkan tingkat kelangsungan hidup yang lebih tinggi (P<0.05) dibandingkan kontrol positif. Pemberian probiotik dan paraprobiotik Bacillus sp. NP5 melalui pakan dapat meningkatkan kinerja pertumbuhan, respons imun, dan resistansi ikan nila terhadap infeksi Streptococcus agalactiae. Kata kunci: Bacillus sp. NP5, ikan nila, paraprobiotik, Streptococcus agalactia

Existing of White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV) and Infectious Hypodermal Haematopoitic Necrosis Virus (IHHNV) in White Shrimp Litopenaeus vannamei Reared at Intensive Tambak System in Bakauheni, Lampung Selatan

Development of white shrimp, Litopenaeus vannamei culture to increase production yield should take attention in prevention of viral outbreak which  is a main factor caused harvesting failure.  Viruses that usually infect shrimp are white spot syndrome virus (WSSV), taura syndrome virus (TSV) dan infectious hypodermal hematopoietic necrosis virus (IHHNV).  To prevent virus infection and reduce risk of fail to harvest, an early warning and observation system of availability of pathogen should be taken in order to know the shrimp condition during culture process.  This study was performed to examine existing of WSSV, TSV and IHHNV in shrimp reared in tambak by PCR and histopathology methods. Samples were taken every two weeks from tambak culture at Bakauheni, Penengahan, Lampung Selatan.  Generally, WSSV, TSV, and IHHNV were found in shrimp from tambak and water around tambak.  Virus infection was first detected in shrimp derived from tambak on 66 days after crop and still exists until 107 days after crop.  Hepatopancreatic cell disorder  caused by the three viruses were found in cell nuclei that became bigger, necrosis in cytoplasm and inclusion body at nuclei.  The existing of virus in outside of tambak indicated that virus was horizontally transmitted. Keywords: WSSV, TSV, IHHNV, white shrimp   ABSTRAK Perkembangan budidaya udang vaname Litopenaeus vannamei dalam usaha meningkatkan hasil produksi harus memperhatikan keberadaan penyakit viral yang menjadi penyebab utama kegagalannya. Virus yang biasa menyerang vaname antara lain white spot syndrome virus (WSSV), taura syndrome virus (TSV) dan infectious hypodermal hematopoietic necrosis virus (IHHNV). Untuk mengantisipasi penyebaran virus dan mengurangi resiko kegagalan produksi diperlukan usaha pencegahan dengan melakukan peringatan dini (early warning) dan pemantauan tambak terhadap keberadaan patogen tersebut selama masa budidaya. Penelitian ini dilakukan untuk mengetahui keberadaan WSSV, TSV dan IHHNV yang menginfeksi udang vaname dengan analisa polymerase chain reaction (PCR) dan histopatologis. Pengambilan sampel dilakukan sebanyak delapan kali setiap dua minggu di tambak intensif udang vaname Litopenaeus vannamei di Bakauheni, Penengahan, Lampung Selatan. Secara umum, WSSV, TSV dan IHHNV telah ditemukan pada tambak serta di perairan sekitar lokasi tersebut. Infeksi virus pertama kali terdeteksi di tambak ketika udang berumur 66 hari setelah penebaran dan terdeteksi pula pada waktu pengamatan selanjutnya sampai udang berumur 107 hari setelah penebaran. Kerusakan akibat serangan ketiga jenis virus tersebut terjadi pada bagian inti sel yang mengalami pembesaran, nekrosis pada sitoplasma dan badan inklusi yang menekan inti sel. Pola transmisi virus yang terjadi adalah secara horizontal yang didukung oleh terdeteksinya virus tersebut pada udang di luar tambak.   Kata kunci : WSSV, TSV, IHHNV, udang vanam