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Detection of fish Disease Caused by Iridovirus on Grouper （Epinephelus sp.） and Pomfret Star （Trachinotus blochii） with Co-agglutination Method in Tanjungpinang, Indonesia

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Journal of Agricultural Science and Technology(B) 2016年第2期6卷 121-128页

作者： Surya Amanu Dwi Sulistiyono Inyoman Suardana Division of Microbiology Faculty of Veterinary Medicine Gadjah Mada University Yogyakarta 560862 Indonesia Tanjungpinang fish quarantine and inspection agency Tanjungpinang 29113 Indonesia

Iridovirus infection often causes death and considerable economic losses in the aquaculture industry. This research applies the co-agglutination method that is fast, cheap and accurate in confirming the diagnosis of the cause of an outbreak of illness caused by iridovirus in the field, so that remedial action can be taken quickly and appropriately to minimize the impact of wider losses. Samples were taken from the grouper and pomfret star farms that are experiencing outbreaks of infectious diseases in the months from May to August 2015, Tanjungpinang, Indonesia. The sick and allegedly attacked by iridovirus samples showed abnormal swimming clinical symptoms, weakness and the swollen spleen. The swollen spleen of sick fish created suspension in phosphate buffered saline （PBS） with pH 7.2, and then centrifuged at 8,000 rpm for I0 rain. The supernatant after centrifuge was used as the test sample. On a clean object glass, 50 μL of the supematant was treated with 50 μL kit co-agglutination pre-prepared. The positive results were shown by the agglutination reaction after 10-15 rain, while as a negative control, PBS was reacted with co-agglutination kit that looked homogeneous （no agglutination）. It was showed that the grouper （Epinepkelus sp.） on four farms and pomfret star （Thracinotus blochii） on one farm that experienced an outbreak of infectious disease in Tanjungpinang showed positively infected iridovirus. The same positive iridovirus result was also demonstrated by examination using polymerase chain reaction （PCR） at 570 bp. So, the causative agent of plague on grouper and pomfret star was iridovirus. In addition, the co-agglutination test based on serology is more quick, cheap and accurate.

关键词： Iridovirus grouper pomfret star co-agglutination and polymerase chain reaction.

Phenotypic and Genotypic Comparison of Pseudomonas stutzeri in Freshwater fish in Indonesia

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Journal of Agricultural Science and Technology(B) 2015年第4期5卷 292-296页

作者： Woro Nur Endang Sariati Kurniasih Surya Amanu Rini Widayanti fish quarantine and inspection agency Semarang Central Java 99116 Indonesia Faculty of Veterinary Medicine Gadjah Mada University Yogyakarta 55281 Indonesia

Pseudomonas stutzeri caused an outbreak of freshwater fish in Luwuk Banggai （tilapia and catfish）, Bali （tilapia）, jambi （tilapia and catfish） and Tanjung Pinang （catfish）. The study was purposed to comprehensively identify special phenotypic and genotypic characteristics of P. stutzeri isolated from several areas in Indonesia, including its morphometric and biochemical characteristics and molecular variation. Bacteria were isolated from internal organs （kidney, ulcer and eye） of fish. They were then identified using morphology and biochemical test. DNA isolates were entirely extracted, amplified and reversed on 16S rRNA region, and further then were sequenced. Phylogenetic trees of bacteria were constructed using neighbor-joining and maximum-parsimony methods. The colony were similar, such as rod shape （jambi, Tanjung Pinang, Bali）, bacil shape （Luwuk Banggai）, transparant in tryptic soy agar （TSA）（Luwuk Banggai）, creamy beige in glutamate starch phenol red （GSP）（Bali）, gram negative, motile, no reaction in the oxidative-fermentative test, positive result in catalase and oxidase test, negative in lysine decarboxylase and ornithine decarboxylase test and positive result in indole test; gelatin was degraded （only Bali）, urea was not degraded, no color change in Methyl-red and Voges-proskaeur （MR-VP） test; acid not produce from glucose, inositol or sucrose. Citrate was utilized by some isolates： positive （jambi, Tanjung Pinang） and negative （Bali, Luwuk Banggai）. Results showed us that isolates of jambi, Bali and Tanjung Pinang were monophyletic species with P. stutzeri $8 and ZH-1 comparing to gen bank. However, merely phenotypic analysis among Pseudomonas sp. was confused compared to each other.

关键词： P. stutzeri genotype phenotype.

Evaluation of Coagglutination Test Kit for Red Sea Bream Iridovirus

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Journal of Agricultural Science and Technology(B) 2015年第6期5卷 437-440页

作者： Surya Amanu Achmad Bahtiar Rifai Lina Yulianah Pramudya Dwiwahyu Department of Microbiology Faculty of Veterinary Medicine Gadjah Mada University Yogyakarta 560862 Indonesia fish quarantine and inspection agency Batam 29464 Indonesia

Coagulation test, in principle, is an immunodiagnostics technique, in which immunoglobuline G of antibody is bound to protein A from Staphylococcus aureus. The aim of study is to develop a rapid test kit for detecting iridovirus infection in fish. Method was summarized as follows：（1） vaccine of iridovirus was injected to rabbit four times with a dosage as 0.5 mL, 1 mL, 2mL, 3 mL each week. Serum was collected at the fifth week as a coagglutination test kit; （2） through the positif polymerase chain reaction （PCR） test, the kidney and spleen sample infected with iridovirus are homogenized by using the phosphate buffered saline （PBS） solution of pH 7.2 with ratio 1：2 （WN）; （3） the supernatant material is collected after centrifugation at 8,000 rpm for 15 min; （4） filtrate/supernatant from sample was dropped on a slide an added with coagglutination test kit with the same volume （l：l）; （5） the agglutination observation is done after the 30, 60 and 90 min incubate at room temperature. The coagglutination test gave positive result in 25% of the test samples.

关键词： Diagnostics coagglutination test kit iridovirus.

Phenotypic and Genotypic Comparison of Vibrio in Seawater fish from Batam and Mataram, Indonesia

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Journal of Agricultural Science and Technology(B) 2015年第8期5卷 561-566页

作者： Rio Aditya Kumiawan Kumiasih Ministry of Marine Affairs and fisheries Republic of Indonesia Fish Quarantine and Inspection Agency Jayapura Papua 99352Indonesia Faculty of Veterinary Medicine Gadjah Mada University Yogyakarta 55281 Indonesia

Grouper and snapper are the potential fishery commodity in Indonesia with a high economic value, as well as an export commodity. A common disease in grouper and snapper aquaculture is vibriosis. Vibriosis is a disease caused by bacteria of the genus Vibrio. The aim of study was to compare between phenotypic and genotypic identification of Vibrio isolated from Batam and Mataram, Indonesia. Bacteria were isolated from anterior kidney and eye of fish, then grown in thiosulfate-citrate-bile salts-sucrose （TCBS） and incubated in room temperature （25-28 ~C） for 24 h, and identified using morphology and biochemical test. Bacterial isolates were extracted, amplified and sequenced on 16S rRNA region. Phylogenetic tree of bacteria was constructed using neighbor-joining and maximum-parsimony methods. The phenotypic identification was found six isolates of Vibrio from Batam, such as K alginolyticus, V. carchariae, K damselae, V. fluvialis, V. furnissii and K parahaemolyticus. Three isolates were found from Mataram, such as 1I. alginolyticus, V. carchariae and V. fluvialis. Blast analysis showed isolates of V. alginolyticus_btm and V. carchariae_btm homolog to V. parahaemolyticus strain DAHMV3; isolates of V. damselae_btm and K alginolyticus_mtr homolog to V. neocaledonicus strain MS1; isolates of V. parahaemolyticus__btm and V. furnisii_btm homolog with Photobacterium damselae subsp, damselae strain： 04Ya311 and isolate of K fluvialis_mtr homolog to V. azureus strain MMRF532, respectively. All phenotypic identification was not supported by molecular identification on 16S rRNA region. It was suggested that phenotypic identification should be supported by molecular examination, especially in identification of Vibrio species.

关键词： Vibrio phenotype genotype 16S rRNA.

Phylogeny and Histopathology of Streptococcus iniae from Indonesia

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Journal of Agricultural Science and Technology(B) 2015年第2期5卷 135-140页

作者： Titis Candra Dewi Kumiasih Surya Amanu Rini Widayati fish quarantine and inspection agency Semarang 50129 Indonesia Department of Pathology Faculty of Veterinary Medicine Gadjah Mada University Yogyakarta 560862 Indonesia Department of Microbiology Faculty of Veterinary Medicine Gadjah Mada University Yogyakarta 560862 Indonesia Department of Biochemistry Facul(y of Veterinary Medicine Gadjah Mada University Yogyakarta 560862 Indonesia

Streptococcus iniae has been detected in several regions in Indonesia, such as Bali, jambi and Papua. Outbreak of meningoencephalitis and septicemia was still often found in Papua. The aim of study was to compare phenotype, genotype and histopathology of S. iniae from Papua. Bacteria was identified using morphological and biochemical tests. It was also extracted, amplified and sequenced to see genotypic characters. The primer used were 5＇-AGAGTTTGATCCTGGCT-3＇（24F） and 5＇-AAGGGAGGTGATCCAGCCGCA-3＇（1540R） in 16S rRNA region. Result of sequencing was analyzed by the neighbor joining and maximum parsimony methods. The identification result of isolate from Papua was S. iniae. The amplification result was a sharp band--l,500 bp band in 16S rRNA region. The phylogenetic tree showed that isolate from Papua was closely related to S. iniae strains CMS005 from Guangxi China. Bacterial clumps of S. iniae was firstly found in the blood vessel of liver at the 3rd day after infection and then caused the inflammation to spleen, heart, brain, kidney and gut at the 7th day post infection.

关键词： Phenotype genotype phylogeny.