DISTRIBUTION ANALYSIS OF ENLARGED CELLS DERIVED FROM GROUPER SLEEPY DISEASE IRIDOVIRUS (GSDIV) INFECTED HUMPBACK GROUPER Cromileptes altivelis

Characteristic of Megalocytivirus infection has been known to produce formation of inclusion body bearing cells (IBCs) on internals organs of fish predominantly on spleen and kidney. Megalocytivirus that infected grouper is known as Grouper Sleepy Disease Iridovirus (GSDIV). This study was conducted to answer the effect of entry sites of GSDIV on distribution of enlarged cells formed on the internal organs of humpback grouper Cromileptes altivelis. Enlarged cells were observed histologically under the light microscope on spleen, head kidney, trunk kidney, liver, gill, heart, stomach, intestine, muscle and brain. Entry sites were designated to intramuscularly injection, intraperitoneally injection, dipped gill and inoculum added feed. Enlarged cells were formed on spleen, head kidney, trunk kidney, liver, gill, heart, stomach, muscle, except on intestine and brain. All the entry sites resulted in formation of enlarged cells on spleen, head kidney, trunk kidney, liver, heart. Spleen and head kidney were the most frequent observed organ. These results suggested that distribution of enlarged cells were not affected by the entry site of GSDIV

THE EFFECTS OF CRUDE RECOMBINANT VIRAL PROTEIN VACCINES AGAINST GROUPER SLEEPY DISEASE IRIDOVIRUS (GSDIV) ON HUMPBACK GROUPER (Cromileptes altivelis)

Infection of Megalocytivirus cause serious mass mortality in marine fish in South East Asian countries. The aim of this study was to produce recombinant of GSDIV capsid protein and its protection to humpback grouper Cromileptes altivelis against grouper sleepy disease iridovirus (GSDIV). A major capsid protein (MCP) was selected for use as a crude subunit vaccines. This gene target (MCP) was inserted to the protein expression system vector of pET SUMO and cloned in cells bacteria Escherichia coli strain BL-21. The MCP was succeded to be induced using 1 mM of IPTG. Results of protein analysis using MALDI TOF-TOF indicated that the MCP has measurement of 49.566 kDa with PI index of 6.00, and contained 453 amino acids. BLAST homology analysis exhibited that the amino acid sequence of the MCP showed high similarity with MCP of Red Sea Bream Iridovirus (RSIV). E. coli expressing MCP protein was inactivated using 0.03% formalin overnight and washed using PBS. The inactivated E. coli as a crude subunit vaccine was then injected intramuscularly to humpback grouper juveniles. Subsequently, the juveniles were challenged tested with GSDIV. The juveniles vaccinated with the MCP recombinant bacteria showed significantly higher survival rates than control those vaccinated with PBS. Thus, the MCP fusion protein is considered as a potential vaccine against GSDIV infections in grouper

Ketahanan Ikan Kerapu Hibrida Cantang (Epinephelus fuscoguttatus>

Ikan kerapu hibrida cantang memiliki ketahanan yang baik terhadap patogen. Penelitian ini menguji ketahanan ikan kerapu cantang terhadap infeksi GSDIV (grouper sleepy disease iridovirus), SBIV (sea bass iridovirus), VNN (viral nervous necrosis) dan bakteri Vibrio sp. Sejumlah 150 ekor ikan kerapu hibrida cantang (rata-rata panjang total 11,01±1,6 cm dan berat 14,67±1,09 g) dibagi dalam 5 perlakuan dengan 3 ulangan bak. Setiap ikan diinjeksi secara intramuskular dengan 200 µl: PBS (A), inokulum GSDIV (B), inokulum SBIV (C), inokulum VNN (D) dan 1010 cfu/ml suspensi Vibrio sp. (E). Parameter meliputi gejala klinis, mortalitas ikan, hematokrit pada saat dimulai gejala dan penyembuhan, serta histologi organ. Pengamatan dimulai setelah  infeksi buatan hingga kematian terhenti sampai 14 hari. Gejala klinis dimulai 5 hari pasca infeksi (HPI) yaitu perubahan warna menjadi hitam atau pucat, berkurangnya nafsu makan dan berenang lemah. Gejala ini ditunjukkan oleh ikan yang diijeksi patogen (B, C, D dan E). Sementara kematian dimulai saat 6 HPI pada ikan yang diinjeksi dengan patogen yang berupa virus (B, C, dan D). Kadar hematokrit ikan normal (A) meningkat hingga 7,5% pada akhir pengamatan. Hal ini terjadi juga pada ikan yang diinfeksi dengan GSDIV (B;19,5%) dan VNN (D;4%). Sementara penurunan kadar hematokrit terjadi pada ikan yang diinjeksi dengan SBIV (C;4,16%) dan Vibrio sp. (E;9,34%). Gambaran histologi menunjukkan bahwa ikan yang diinjeksi patogen mengalami kerusakan jaringan terutama pada limpa dan ginjal depan (B, C, D, E). Ikan kerapu hibrida cantang tahan terhadap infeksi bakteri Vibrio sp., meskipun menunjukkan gejala klinis yang sama dengan infeksi virus, namun infeksi virus dapat menyebabkan kematian setelah memperlihatkan gejala klinis

PERKEMBANGAN JUMLAH EKTOPARASIT Pseudorhabdosynochus spp. PADA INSANG KERAPU HIBRIDA CANTIK (Epinephelus fuscoguttatus x E. polyphekadion) MELALUI METODE KOHABITASI

Genus Pseudorhabdosynocus merupakan Monogenea yang sering menginfeksi ikan kerapu. Pengaruh perbedaan jarak antara ikan sakit dengan ikan sehat terhadap perkembangan jumlah Pseudorhabdosynochus spp. pada ikan kerapu hibrida “cantik” dipelajari dalam penelitian ini melalui metode kohabitasi. Kohabitasi dilakukan dengan dua metode yaitu (a) menempatkan lima ekor ikan sakit ke dalam keranjang dengan jarak 25 cm dari dasar bak, dan (b) menempatkan lima ekor ikan sakit ke dalam keranjang dengan jarak 10 cm dari dasar bak. Kedua keranjang tersebut diapungkan ke dalam bak plastik berbeda dengan volume 100 L air laut (33 ppt) yang masing-masing telah berisi 30 ekor ikan kerapu hibrida “cantik” sehat. Masing-masing lima ekor ikan dari kedua metode kohabitasi diambil pada hari ke-2, 4, 6, 8, 10, dan 15 pemeliharaan. Hasil penelitian menunjukkan bahwa infeksi buatan menggunakan metode kohabitasi (b) lebih cepat menyebarkan Pseudorhabdosynochus spp. dari ikan sakit ke ikan sehat dibandingkan dengan metode kohabitasi (a). Perkembangan populasi Pseudorhabdosynochus spp. dan telurnya pada metode kohabitasi (b) lebih tinggi yaitu 1.495 ± 206,3 ekor/ikan dan 18,6 ± 3,8 telur/ikan dibandingkan dengan metode kohabitasi (a) yaitu 163,2 ± 16,3 ekor/ikan dan 3,8 ± 0,7 telur/ikan pasca 15 hari kohabitasi. Secara histopatologi, lamela insang yang terinfeksi Pseudorhabdosynochus spp. menunjukkan adanya hyperplasia epitel sel filamen insang yang menimbulkan fusi filamen. Kerusakan filamen di hampir semua lamela insang menyebabkan terganggunya sistem pernapasan ikan kerapu. Hasil tersebut dapat disimpulkan bahwa penyebaran Pseudorhabdosynochus spp. semakin cepat dengan semakin dekat jarak kontak antara ikan sakit dengan ikan sehat.Pseudorhabdosynocus is a genus of Monogenea that frequently infect grouper fish. This study aimed to observe the changes of density patterns of Pseudorhabdosynochus spp. in hybrid grouper gill through cohabitation. Two cohabitation methods were applied to understand the effects of distance between sick and healthy fish in terms of parasite infection. The cohabitation methos were arranged as follows: (a) five fish infected with Pseudorhabdosynochus spp. were placed into a basket at a distance of 25 cm from the bottom of the tank, and (b) five fish infected with Pseudorhabdosynochus spp. were placed into a basket at a distance of 10 cm from the bottom of the tank. The two baskets were floated into different plastic tanks of 100 L of seawater (33 ppt), each of which contained 30 healthy hybrid groupers. Each of the five fish from the two cohabitation methods was sampled on day 2, 4, 6, 8, 10, and 15 after cohabitation. The results showed that the spread of Pseudorhabdosynochus spp. from sick fish to healthy fish with the cohabitation method b was faster than the cohabitation method a. The development number of Pseudorhabdosynochus spp. and its eggs in the cohabitation method b were higher, reaching 1,495 ± 206.3 parasite/fish and 18.6 ± 3.8 eggs/fish than the cohabitation method a, 163.2 ± 16.3 parasite/fish and 3.8 ± 0.7 eggs/fish after 15 days of cohabitation. Histopathologically, gill lamella infected with Pseudorhabdosynochus spp. showed the presence of epithelial hyperplasia of gill filament cells causing fusion. Damage of the gill filament in all of gill lamella has caused disruption of the grouper breathing system. From these findings, it can be concluded that the spread of Pseudorhabdosynochus spp. was faster if the distance of direct contact between sick and healthy fish was closer

IDENTIFICATION AND LIFE CYCLE OF MARINE LEECH ISOLATED FROM CULTURED HYBRID GROUPER IN THE NORTHERN BALI WATERS OF INDONESIA

The aims of this study were to identify and to determine life cycle of marine leech isolated from cultured hybrid grouper “cantik” (Epinephelus fuscoguttatus fx E. polyphekadion m) in the northern Bali waters of Indonesia under laboratory conditions. Observation of the life cycle of the marine leech was done using petri-dishes (9 cm in diameter) arranged into two groups. In group-1, a petri-dish was filled with sterile seawater (with water exchange of 50%-60% every two days) and in group-2, a petri-dish was filled with continuous running water. DNA sequence was aligned with the sequences from GenBank by BLAST program. Results of similarity index with GenBank sequence exhibited that the nucleic acid of the marine leech isolated from the hybrid grouper “cantik” showed high similarity (99%) with Zeylanicobdella arugamensis. One adult leech could produce 1-63 eggs. The eggs were developed into morula, blastula, and gastrula within five days. The early phase of the embryo with daily water exchange treatment started on day-6 and hatched into larvae on day-10. The eggs incubated with continuous running water had hatched faster (eight days). However, not all eggs hatched at the same time. Some of the eggs hatched 1-3 days after the first one. Hatching rate of eggs varied from 2.70% to 100%. The newly hatched Z. arugamensis larva has transparent color and length of 1.0-1.5 mm. On day-6, Z. arugamensis larvae were already seen attaching to the body of the fish. The size of the Z. arugamensis larvae ranged between 3-11 mm on day-9. In that stage, they were able to produce eggs. Therefore, we argue that Z. arugamensis only requires 17 to 22 days to develop into the adult stage

ADDITION OF ADJUVANTS IN RECOMBINANT SUBUNIT VACCINES FOR THE PREVENTION OF GROUPER SLEEPY DISEASE IRIDOVIRUS (GSDIV) INFECTION IN HUMPBACK GROUPER, Cromileptes altivelis

Infection of grouper sleepy disease iridovirus (GSDIV) which is a member of Megalocytivirus causes mass mortalities in marine fish in Indonesia. This study was conducted to know the effectiveness of recombinant subunit vaccine of GSDIV with an addition of adjuvants against GSDIV infection. Inactive bacteria Eschericia coli containing recombinant MCP-GSDIV protein was added with montanide ISA adjuvant at a ratio of 3:7. The vaccine was administered to humpback grouper, Cromileptes altivelis by intramuscular and intraperitoneal injection at a dose of 0.1 mL/fish. Test of the vaccine in humpback grouper was performed in four replicates (four trials). Results of the vaccination showed that the recombinant protein vaccine added with the adjuvant increased immunity of humpback grouper, indicated by higher relative percent survival (RPS= 77.78%) compared to negative control (PBS) and 50% higher compared to protein control (pET Sumo CAT) at two weeks post vaccination. The RPS values of the recombinant protein vaccine were still higher (53.57%-72.73%) than those of the control vaccine and 25%-53.33% of the protein control in the 4th week post vaccination. GSDIV detection by PCR showed that MCP-GSDIV-DNA and pET Sumo CAT-DNA were not detected in the vaccinated fish after one, two, three, and four weeks post vaccination. The fish died in both of vaccinated and control groups after experimental challenge with GSDIV were found to be infected with GSDIV. It can be stated that recombinant subunit vaccine of GSDIV with the addition of montanide ISA adjuvant could be used to prevent and diminish mortalities of grouper against GSDIV infection

Pengaruh Modal, Harga Dan Volume Penjualan Terhadap Pendapatan Pedagang Pengecer Kelapa Di Pasar Langsa Kota

The coconut retailer run the business to get a worth profit to fulfill their family needs and develop their business also. This research used a survey method. To define the sample used ad full sampling. The coconut retailer sample was 29 person and analyzed used multiple linear regression. The result showed that stock, price and selling volume effected the coconut retailer profit in Langsa City Market

INTENSITAS PARASIT INSANG (TREMATODA MONOGENEA: Pseudorhabdosynochus sp.) PADA IKAN KERAPU HIBRIDA MELALUI INFEKSI BUATAN

Infeksi trematoda monogenea: Pseudorhabdosynochus sp. dapat menyebabkan kematian massal pada ikan kerapu. Prevalensi infeksi trematoda ini mencapai 100%, namun intensitasnya hingga menimbulkan gejala klinis dan kematian ikan belum diketahui. Tujuan dari penelitian ini adalah untuk mengetahui intensitas parasit insang (Pseudorhabdosynochus sp.) pada ikan kerapu hibrida “cantik” melalui infeksi buatan. Ikan uji direndam dalam air tawar dengan 100 mg/L formalin selama satu jam sebelum digunakan. Infeksi buatan dilakukan melalui (A) kohabitasi antara ikan sehat dengan ikan sakit dan (B) penempelan potongan lamella insang ikan yang terinfeksi parasit ke lamella insang ikan sehat. Pada perlakuan (A) sebanyak lima ekor ikan uji dipelihara bersama dengan dua ekor ikan sakit selama 3-4 hari, sedangkan perlakuan (B) penempelan lamella insang ikan sakit (1 g lamella insang/ikan) dilakukan pada lima ekor ikan uji selama lima menit. Masing-masing perlakuan diulang tiga kali. Pada Kontrol kepadatan ikan uji sehat sebanyak enam ekor dan lima ekor. Wadah yang digunakan berupa bak plastik volume 100 L. Pengamatan intensitas parasit dan telurnya pada setiap lamella insang bagian kanan dan kiri dari lima ekor ikan uji dilakukan selama tiga minggu dengan interval waktu satu minggu. Hasil pengamatan menunjukkan bahwa kohabitasi antara ikan sehat dan ikan sakit menyebabkan intensitas parasit Pseudorhabdosynochus sp. dan telurnya lebih tinggi dibandingkan dengan penempelan lamella insang. Pada kontrol, Pseudorhabdosynochus sp. maupun telurnya tidak ditemukan selama tiga minggu pemeliharaan. Hasil ini menunjukkan penyebaran Pseudorhabdosynochus sp. dari ikan sakit ke ikan sehat lebih cepat dibandingkan dengan penempelan dari lamella insang ikan sakit.Monogenetic trematode (Pseudorhabdosynochus sp.) infection often leads to mass mortality in groupers, with prevalency reaching 100%. However, the intensity of this parasite to infect grouper fish has yet to be reported. The purpose of this research was to study the intensity of Pseudorhabdosynochus sp. in hybrid grouper “cantik” through experimental infection. The test fish were soaked with 100 mg/L of formalin in fresh water before used. The experimental infections were performed through: (A) cohabitation between healthy fish with sick fish and (B) infection with attached of gills lamella of sick fish to gill lamella of healthy fish. In treatment (A), a total of five test fish were cohabited with two sick fish for 3-4 days, while in treatment (B), attachment of gills lamella (1 g/fish) was done on five test fish for five minutes. Each treatment was repeated for three times. Six and five test fish were maintened as controls. The containers used were 100 L plastic tanks volume. Observation of intensity of the parasite infection and its egg production in each lamella along the right and left gills of five fish were conducted for three weeks with one week intervals. The result showed that the cohabitation between healthy and sick fish caused an increase in total of Pseudorhabdosynochus sp. and its eggs compared with the attachment of chopped-gills. In the control group, Pseudorhabdosynochus sp. and its eggs were not found for three weeks. These results indicate the spread of Pseudorhabdosynochus sp. from the sick fish to the healthy is fish faster than the attachment of gill lamella from the sick fish

EFFECTIVENESS OF A RECOMBINANT VACCINE BASED ON RNA2 CAPSID PROTEIN AGAINST NERVOUS NECROSIS VIRUS IN HYBRID GROUPER

Nervous Necrosis Virus (NNV) is a devastating viral disease in marine aquaculture, causing significant economic losses worldwide, including in Indonesia. The virus mainly infects larvae and juveniles of marine fishes. This study aimed to determine the effectiveness of a recombinant vaccine from betanodavirus coat proteins expressed in Escherichia coli fish against NNV infection in hybrid grouper. An RNA2 capsid protein was selected and used as the recombinant vaccine. NNV-RNA2 gene was inserted into the protein expression system vector of pET SUMO and cloned in cells of bacteria Escherichia coli strain BL-21. The results of blast homology analysis exhibited that the amino acid sequence of the NNV-RNA2 showed high similarity with Lates calcarifer encephalitis viral coat protein gene. E. coli expressing NNV-RNA2 protein was inactivated using 0.03% formalin and mechanically inactivated by freeze-thaw and sonication methods. The inactivated recombinant E. coli vaccine was then injected intramuscularly into hybrid grouper juveniles (single vaccine). Subsequently, the juveniles were challenged with NNV at 7, 14, and 21 dpv (days post-vaccination). Injection of 0.1 mL sterile PBS served as the control. Single vaccine applications using formalin-inactivated vaccines resulted in higher antibody titers than those of mechanically-inactivated vaccines. Both vaccines were only able to increase antibody titer up to 7 dpv. Therefore, re-vaccination (booster vaccine) was done on day-10 after the first vaccination using a formalin-inactivated vaccine. The booster vaccine could protect hybrid grouper against NNV (P<0.05) at four weeks post-vaccination. However, the mortality of vaccinated and control fish was not significantly different (P>0.05) after challenged with NNV for six weeks after. This recombinant vaccine has the potential to be developed into a polyvalent vaccine by combining viral and other bacterial vaccines in future research

Kematian Ikan Kerapu Hibrida Cantik (Kerapu Macan >< Kerapu Batik) dan Cantang (Kerapu Macan >< Kerapu Kertang) di Keramba Jaring Apung di Teluk Kaping, Buleleng-Bali Akibat Infeksi Ektoparasit

Several constraints in aquaculture are seasonal dependent especially for parasitic disease. Natural condition of aquatic environment are affecting pathogen and fish along season. This study observed parasitic infection causing mass mortality of hybrid grouper juveniles thruoghout the year. This study was conducted in floating net cage at Kaping Bay North Bali in 2016. The juveniles were stocked in May, July and August. Parasitic infection were observed in Juli-August. Two ectoparasite were found, they are gill worms (Pseudorhabdosynochus sp.) and sea leeches (Zeylanicobdella arugamensis). They caused appetite decreasing, swimming on the surface, dark body color, thiny body andt decreased the population about 44.9% - 47,53%. Gill worm infected the fish at the initial with intensity of 54-187 individu/gill, While sea leeches infected the fish later in August with intensity about 40-50 individu/fish. The record of water quality showed that temperature and salinity were increased in July. This conditions were coincide with seasonal change in 2016. This result suggested that seasonal change was unfavourable conditions for fish health moreover for stocking time.