Aquaculture Extension Manual No. 44 July 2010 Prevention and control measures against viral nervous necrosis (VNN) in marine fish hatcheries Leobert D. de la Peña Aquaculture Department Southeast Asian Fisheries Development Center www.seafdec.org.ph Government of Japan Trust Fund Aquaculture Extension Manual No. 44 July 2010 Prevention and control measures against viral nervous necrosis (VNN) in marine fish hatcheries Leobert D. de la Peña Aquaculture Department Southeast Asian Fisheries Development Center www.seafdec.org.ph Government of Japan Trust Fund Aquaculture Extension Manual No. 44 Prevention and control measures against viral nervous necrosis (VNN) in marine fish hatcheries July 2010 ISSN 0115-5369 Published and printed by: Aquaculture Department Southeast Asian Fisheries Development Center (SEAFDEC) Tigbauan, Iloilo, Philippines Copyright © 2010 Aquaculture Department Southeast Asian Fisheries Development Center Tigbauan, Iloilo, Philippines All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system without the permission in writing from the publisher. For your comments/suggestions, please contact: SEAFDEC Aquaculture Department Tel (63-33) 511-9170, 511-9171 Fax (63-33) 511-9070, 511-8709 Email aqdchief@seafdec.org.ph AQD website http://www.seafdec.org.ph Foreword The emergence of fish diseases caused by viruses, bacteria, and other parasites is one of the downsides of the booming aquaculture industry in Southeast Asia. These diseases can result in massive economic losses if left unmanaged. One of these diseases, viral nervous necrosis or VNN, is considered as one of the most devastating in cultured marine fish. SEAFDEC/AQD is in the forefront of developing prevention and control measures against fish diseases. Hence, SEAFDEC/AQD is implementing the Regional Fish Disease Project, which is being funded by the Government of Japan-Trust Fund (GOJ-TF). The project has developed a surveillance system for diseases of aquatic animals in Southeast Asia through the establishment of resources and facilities for fish health diagnosis, human capacity building, and conduct research on prevention and control of diseases. This manual aims to provide necessary information about VNN, as well as its detection and preventive methods especially in the hatchery phase. We hope that the information in this manual would prove invaluable to researchers, hatchery operators and managers, and other stakeholders to help manage the disease, thus reducing mortalities and improving seed production. Joebert D. Toledo, D. Agri. Chief SEAFDEC Aquaculture Department Preface Fish disease is one of the main constraints in aquaculture production. Viral diseases such as viral nervous necrosis (VNN) can severely affect the production of economically important marine fishes from the hatchery to grow-out phases. Because of the threats posed by VNN and other diseases, SEAFDEC Aquaculture Department (SEAFDEC/AQD)has been conducting studies toward the development of preventive and control measures against some of the most important fish diseases. Some of these studies have been supported by the Government of Japan-Trust Fund through the Regional Fish Disease Project. Results and data from some of the studies make their way into this manual. Included are information on the clinical signs, affected fish species, and geographic distribution of VNN. Methods in detecting the presence of VNN in marine fish using advanced technologies, as well as practical guides to follow in preventing and controlling the disease in the hatchery, are also contained in this manual. We hope that our concerned stakeholders will find this manual a useful guide in aquaculture health management of marine fish. Teruo Azuma, Ph.D. Deputy Chief SEAFDEC Aquaculture Department Table of Contents Foreword Preface Acknowledgement Introduction................................................................................................... 1 Clinical signs............................................................................................1 Behavioral changes...................................................................................2 Affected species........................................................................................2 Causative agent........................................................................................6 Geographical distribution.........................................................................6 VNN infection in the Philippines..............................................................7 Detection methods for VNN..........................................................................8 P. resumptive diagnosis..............................................................................8 Macroscopic detection.........................................................................8 Microscopic detection.........................................................................8 Cell culture...............................................................................................8 Immunological......................................................................................... 9 Molecular................................................................................................. 9 Commercial kit.........................................................................................11 Prevention and control of VNN in the hatchery.............................................12 Screening of broodstock using PCR..........................................................12 Feeding of broodstock using commercially available diet.........................16 Disinfection of eggs using various disinfectants........................................18 Larval sampling for screening/diagnosis...................................................18 Removal of weak and dead fish from the rearing tanks............................18 Disinfection of rearing tanks and other paraphernalia for seed production.....................................................................................22 Reducing stress factors.............................................................................22 Vaccination.............................................................................................. 23 References...................................................................................................... 24 Glossary Appendices Acknowledgment My heartfelt gratitude to the Government of Japan (GOJ)-Trust Fund through SEAFDEC/AQD for the financial support to the VNN research projects and for the publication of this manual. My sincere thanks to our Trust Fund CoManagers: Drs. Yasuo Inui, Kazuya Nagasawa, Koichi Okuzawa, Hiroshi Ogata and Teruo Azuma for their continued encouragement to publish this manual. Thanks are also due to Drs. Joebert D. Toledo, Clarissa M. Marte, Evelyn Grace D. Ayson, Gilda Lio-Po, Celia R. Lavilla-Pitogo, Erlinda C. Lacierda, Gerald F. Quinitio, Arnil C. Emata and Mr. Deny Chavez for providing their valuable advice as well as fish samples. Technical assistance was provided by Marica Baguisi, Jane Frances Napa, Christopher Sombito, Geimbo Capulos, Reynalyn Hechanova, Vonie Suarnaba and Mary Nia Santos. I also acknowledge our colleagues at the Fish Health Section Diagnostic Services: Milagros Paner, Remia Traviña, Fely Torreta, Maila Peniero; and Nursery and Big Hatchery staff for their technical assistance in the preparation and processing of samples. I appreciate the Publications Review Committee for the critical review, you have greatly helped me improve this manual. Thank you to the staff of Development Communication Section, Mila Castaños, Rommel Guarin and Basil Baylon for editing and layouting this manual. Introduction Viral diseases are considered as limiting factors in the rapid development of marine fish aquaculture. High mortality rates associated with viral infection have caused major losses in aquaculture industries worldwide. Among these viral diseases, viral nervous necrosis (VNN) is considered as one of the most devastating diseases in a variety of cultured marine fish. The disease is also known as encephalomyelitis (Bloch et al., 1991) and vacuolating encephalopathy and retinopathy (VER) (Munday et al., 1992). Clinical signs Larvae and juveniles are the most affected stages, although significant mortalities are also observed in older fish (up to harvest size) such as in European sea bass, grouper and Atlantic halibut (Munday and Nakai, 1997). The disease is characterized by: • Enlargement of swimbladder in larvae (Figure 1) and broodstock (Figure 2) • Change in coloration - Dark (i.e. grouper, adult seabass) - Pale (i.e. larval seabass) • Lesions in the brain and retina - anterior brain is more severely affected than the posterior brain and spinal cord • Vacuolation of the central nervous tissues (brain, spinal cord) and the retina (Figure 3) • Other lesions include pyknosis, shrinkage and basophilia of affected cells (Yoshikoshi Figure 1. Orange-spotted grouper and Inoue, 1990; Arimoto et al., 2003) (Epinephelus coioides) larvae infected with VNN. Note the bloated appearance of the stomach due to hyperinflation of the swimbladder Figure 2. A. VNN infected orange-spotted grouper broodstock showing bloated stomach B. Hyperinflated swimbladder (arrow) Behavioral changes • Abnormal swimming behavior - Corkscrew swimming (some fish sink to the bottom then float to surface again) - Darting - Whirling swim pattern, rotating, spinning, horizontal looping • Loss of equilibrium (belly up at rest) • Lethargy Figure 3. Conspicuous vacuolation • Loss of appetite, thinness from anorexia (arrow) in the retina of VNN- infected grouper larvae (Photo courtesy of Dr. Y. Maeno) Affected species VNN was first reported in hatchery-reared larvae and juveniles of Oplegnathus fasciatus (Japanese parrotfish) between 1986-1987 (Yashikoshi and Inoue, 1990). Since then, the virus has been reported in 39 species (see Table 1) belonging to 22 families and 8 orders (Nakai et al., 2009) of marine fish distributed worldwide. Among the affected species, the disease is prevalent in groupers, sea bass and flatfish (Munday et al., 2002). Cultured grouper species susceptible to VNN include Epinephelus akaara (red-spotted grouper), E. coioides (orange-spotted grouper), E. tauvina (greasy grouper), E. fuscoguttatus (black-spotted grouper), E. septemfasciatus (sevenband grouper), E. malabricus (brown-spotted grouper), and Cromileptes altivelis (humpback grouper). Other fish species susceptible to VNN are listed in Table 1. The susceptibility of fish species to VNN is age-dependent (Munday et al., 2002). There are variations in the age at which VNN is first observed and the period over which mortality occurs (Table 2). In general, the earlier the signs of disease occur, the greater is the rate of mortality. In some species, disease occurrence at juvenile stages is very rare, while mass mortalities often occur at juvenile to young stages in other fish species, usually not reaching 100% (Munday et al., 2002). Mortalities have been reported in production-size European sea bass and grouper, but even in these cases mortalities were greatest in younger fish. 2 Prevention and control measures against VNN in marine fish hatcheries Table 1. Fish species affected by VNN Fish Species Sturgeon (Acipenser gueldenstaedti) European eel (Anguilla anguilla) Guppy (Poecilia reticulata) Country/Region Greece Taiwan Singapore Atlantic cod (Gadus morhua) Haddock (Melanogrammus aeglefinus) Golden grey mullet (Liza auratus) Grey mullet (Mugil cephalus) Barramundi/Asian seabass (Lates calcarifer) Japanese seabass (Lateolabrax japonicus) European seabass (Dicentrarchus labrax) White grouper (Epinephelus aeneus) Red-spotted grouper (E. akaara) Yellow grouper (E. awoara) Orange-spotted grouper (E. coioides) Black-spotted grouper (E. fuscoguttatus) Dusky grouper (E. marginatus) Kelp grouper (E. moara) Sevenband grouper (E. septemfasciatus) Brown-spotted grouper (E. malabaricus) Greasy grouper (E. tauvina) United Kingdom, Canada, USA, Norway Canada Iran Israel Australia, China, Indonesia, Israel, Malaysia, Philippines, Singapore, Tahiti, Taiwan, Thailand, India Japan Caribbean, France, Greece, Italy, Malta, Portugal, Spain, Israel Israel Japan, Taiwan Taiwan Philippines, Taiwan Taiwan Mediterranean Japan Japan, Korea Thailand Malaysia, Philippines, Singapore Source of sample/s grow-out wild ornamental fish farming wild and farmed; hatchery hatchery wild grow-out (mariculture) hatchery; mariculture hatchery hatchery; mariculture grow-out (mariculture) hatchery hatchery hatchery hatchery grow-out hatchery hatchery hatchery hatchery de la Peña 3 cont. Table 1 Humpback grouper (Cromileptes altivelis) Dragon grouper (E. lanceolatus) Striped trumpeter (Latris lineate) Striped jack (Pseudocaranx dentex) Purplish amberjack (Seriola dumerili) Pompano (Trachinotus blochii) Yellow-wax pompano (Trachinotus falcatus) Gilthead sea bream (Sparus aurata) Shi drum (Umbrina cirrosa) Red drum (Sciaenops ocellatus) White seabass (Atractoscion nobilis) Japanese parrotfish (Oplegnathus fasciatus) Rock porgy (O. punctatus) Sleepy cod (Oxyeleotris lineolatus) Cobia (Rachycentron canadum) Japanese tilefish (Branchiostegus japonicus) Firespot snapper (Lutjanus erythropterus) Bluefin tuna (Thunnus thynnus) Barfin flounder (Verasper moseri) Halibut (Hippoglossus hippoglossus) Japanese flounder (Paralichthys olivaceus) Turbot (Scophthalmus maximus) Indonesia, Taiwan Taiwan Australia Japan Japan Taiwan Taiwan France, Italy France, Italy Korea, Israel USA Japan Japan Australia Taiwan Japan Taiwan Japan Norway, United Kingdom Japan Norway hatchery hatchery hatchery hatchery hatchery hatchery hatchery hatchery hatchery grow-out (mariculture) hatchery hatchery hatchery wild hatchery hatchery hatchery hatchery hatchery hatchery 4 Prevention and control measures against VNN in marine fish hatcheries cont. Table 1 Dover sole (Solea solea) United Kingdom wild and farmed Senegalese sole (S. senegalensis) Spain and Portugal grow-out Chinese catfish (Parasilurus asotus) Taiwan hatchery Australian catfish (Tandanus tandanus) Australia wild Japanese puffer fish (Takifugu rubripes) Japan hatchery (Modified from Nakai et al., 2009, Maltese and Bovo, 2007 and Manual of Diagnostic Tests for Aquatic Animals 2009 (www.baphiq.gov.tw) Table 2. Important features of VNN of larval and juvenile fish (OIE, 2003) Fish species Asian seabass (Lates calcarifer) European seabass (Dicentrarchus labrax) Japanese parrotfish (Oplegnathus fasciatus) Red-spotted grouper (Epinephelus akaara) Brown-spotted grouper (E. malabaricus) Striped jack (Pseudocaranx dentex) Turbot (Scophthalmus maximus) Earliest occurrence of disease 9 days 10 days post-hatch 6-25 mm total length 14 days post-hatch (7-8 mm total length) - 1 day post-hatch < 21 days post-hatch Usual onset 15-18 days 25-40 days post-hatch - 9-10 mm total length 20-50 mm total length 1-4 days post-hatch - Latest occurrence of new outbreaks 24 days post-hatch body weight 400-580 g < 40 mm total length < 40 mm total length - < 20 days post- hatch (8 mm) total length body weight 50-100 mg Usual mortality rate 50-100% / month 10% / month - 80% 50-80% 100% - Highest mortality rate 100% in < 1 month up to 100% up to 100% - up to 100% de la Peña 5 Causative agent The disease is caused by a nodavirus belonging to the family Nodaviridae. There are two genera in Nodaviridae, Alphanodavirus and Betanodavirus, the hosts for the former are insects and for the latter are almost all kinds of marine fishes. Nodaviruses are microscopic, single stranded RNA viruses. It is a spherical, nonenveloped virus, about 25 nm in diameter (Mori et al., 1992; Nishizawa et al., 1994) (Figure 4). Nodaviruses generally infect the fish’s brain, eyes and spinal cord. Nodaviruses are quite common in many marine species worldwide and large adult fish can be carriers of this disease. Based on the nucleotide sequence of its coat protein gene, betanodaviruses are classified into four genotypes (Nishizawa et al., 1997): 1 SJNNV (Striped jack nervous necrosis virus) 2 TPNNV (Tiger puffer nervous necrosis virus) 3 RGNNV (Red spotted grouper nervous necrosis virus) 4 BFNNV (Barfin flounder nervous necrosis virus) Figure 4. Transmission electron micrograph of nervous necrosis virus propagated in E-11 cell line (arrow). Viral particles are shown in detail (inset). (Photo courtesy of Dr. Y. Maeno) Geographical Distribution VNN has been reported from all continents with the exception of Africa (Figure 5). These include countries in Asia (China, Taiwan, India, Indonesia, Japan, Korea, Philippines, Thailand, Singapore, Malaysia, Iran and Israel), Oceania (Australia, Tahiti), Europe (France, Greece, Italy, Malta, Portugal, Spain, United Kingdom, Norway), North America (Canada, USA) and South America (Caribbean). Majority of reports have come from those regions with intensive culture of marine species (Munday et al., 2002). 6 Prevention and control measures against VNN in marine fish hatcheries While the virus has apparently spread within the natural ranges of affected species as a result of commerce, VNN has also been reported in species in countries where they do not naturally occur and to which they have been exported. These species may have been infected by endemic strains of nodaviruses, but the epidemiological evidence suggests that the seed stock may have carried exotic strains of the viruses. Figure 5. Geographic distribution of VNN (orange dot represents affected countries) VNN infection in the Philippines In the Philippines, VNN was first reported in broodstock and larvae of hatcheryreared grouper (E. coioides) in 2001. Mortalities of 5-10% daily were observed in grouper larvae, progressing to 100% within 10 days. Clinical signs in the affected larvae included reduced feeding, darkened pigmentation, lethargy and abnormal swimming. Etiological studies using histopathology, cell culture using SSN-1 cell line, reverse transcription polymerase chain reaction (RT-PCR) and electron microscopy revealed that the mortality was caused by VNN (Maeno et al., 2002). In the same year, mass mortalities of hatchery-reared seabass (Lates calcarifer) larvae was associated with VNN. The clinical signs and pathological changes observed in affected orange-spotted grouper and seabass larvae are very similar with those described in other VNN-affected fish species. It was confirmed that a betanodavirus of the genotype RGNNV had infected and caused mortalities in larvae of sea bass and grouper E. coioides broodstock (de la Peña et al., 2008). de la Peña 7 Detection methods for VNN During the last few years, several methods for detecting betanodavirus in fish samples have been developed: 1. Presumptive Diagnosis 1.1. Macroscopic Detection In general, younger fish have more severe lesions, older fish have less extensive lesions and these may show a predilection for the retina (Munday et al., 2002). Fish material suitable for virological examination is (OIE, 2003): a. Asymptomatic fish (apparently healthy fish): whole larvae or small juveniles; brain, spinal cord, and eyes for larger size fish and/or ovarian fluid from broodstock at spawning time. b. Clinically affected fish: whole larvae or small juveniles; brain, spinal cord, and eyes for larger size fish. 1.2. Microscopic Detection (Yoshikoshi and Inoue, 1990) Presumptive diagnosis can be made on the basis of the appearance of vacuoles in the brain, spinal cord and/or retina under a light microscope. Using electron microscope, abundant numbers of the virus can be detected in the cytoplasm of affected nerve cells in infected fish. 2. Cell Culture Unlike bacteria that can be cultured in agar media, viruses require a living cell in order to propagate themselves. Thus, they are cultured in a cell line. Nodaviruses are commonly cultured in striped snakehead cells (SSN-1). E-11 cell line, derived from SSN-1, is also used. Detection of VNN using cell culture method requires that the virus are viable to subsequently infect and propagate themselves in the cell line. The cells can be grown in culture flasks using any media and serum that support rapid growth of the cells. The presence of the virus often gives rise to changes in form, size and structure of the cell. Any detectable change in the host cell due to infection is known as a cytopathic effect (CPE). CPE may consist of cell rounding, disorientation, swelling or shrinking, death, detachment from the surface, etc. (Figure 6). The CPE produced by different viruses depend on the virus and the cells on which it is grown. This can be used to aid in identification of a virus isolate. Cell culture can measure the number of infectious virus particles, but are time-consuming and complex. In SSN-1 cells, CPE is detected approximately 5-10 days after virus inoculation. 8 Prevention and control measures against VNN in marine fish hatcheries Figure 6. E-11 cell line: (A) control cell line; (B) cell line inoculated with tissue filtrate from diseased grouper. Note the conspicuous vacuolation and cell disintegration of cells due to CPE caused by VNN (arrow) 3. Immunological Methods Immunological methods are based on cellular immune system mechanism and responses. Enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT) are commonly used in detection of betanodaviruses. ELISA is a biochemical technique to detect the presence of an antibody or an antigen in a sample. IFAT is the detection of antibodies to specific antigenic material in the substrate using fluorescent microscopy. Betanodaviruses can be detected using IFAT (Munday and Nakai, 1997; Munday et al., 1994; Totland et al., 1999; de la Peña et al., 2008) and ELISA (Arimoto et al., 1992). These are routinely used for confirmative diagnosis of the disease. However, these immunological methods are laborious, timeconsuming and sometimes useful only in cases of active outbreak (Dalla Valle et al., 2005). 4. Molecular Methods The Polymerase Chain Reaction (PCR) as a tool for the amplification of specific nucleic acid sequences was introduced by Saiki et al. in 1985. With this technique, one can rapidly detect a virus and rapidly synthesize, clone and sequence any segment of DNA. In aquaculture, PCR is a valuable tool for the prevention, control and management of various diseases. For fish and shrimp farmers, it permits fast, widespread and sensitive screening of virus carriers, and is also ideal for early or light infections. The test can be carried out on body fragments, blood or feces from the fish or shrimp being tested. PCR can be used to screen both broodstock and larvae before stocking. Reverse transcription-polymerase chain reaction (RT-PCR) assays have been developed as a powerful diagnostic tool alone (Nishizawa et al., 1994), or in combination with cell culture (Iwamoto et al., 2000). RT-PCR can detect the virus not only from diseased fish but also from asymptomatic or carrier-stage fish (Nakai, 2002). Sensitivity is further enhanced using the nested step PCR. de la Peña 9 These PCR protocols have greatly improved test sensitivity, allowing better control of VNN infection. A positive result in the first step implies a serious infection, whereas, when a positive result is obtained in the second amplification step (nested PCR) only, a latent or carrier state infection is indicated (Figure 7). However, since only the presence or absence of the viral nucleic acid is being tested, the results could not tell whether the virus is still viable or not. The real-time PCR assays are more sensitive than the one step RT-PCR. Realtime PCR offers not only speed, sensitivity and a reduced risk of carry-over contamination, but also the possibility of an accurate quantification. In realtime PCR the actual copy number of the viral template in samples can be determined. A SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus has been developed by Dalla Valle et al. (2005). 1 23 4 5 Figure 7. Agarose gel electrophoresis of nested PCR amplification products of VNN. Lanes: (1) 100 bp DNA marker; (2) positive control showing 280 bp PCR product; (3) Sample 1-positive for VNN also showing 280 bp product; (4) Sample 2-negative for VNN (5) negative control 10 Prevention and control measures against VNN in marine fish hatcheries 5. Commercial Kit At present, the only commercially kit available for VNN detection is IQ2000TM (Farming IntelliGene Technology Corporation). It can detect the disease in marine fishes such as nervous necrosis viruses on striped jack (SJNNV), redspotted grouper (RGNNV), barfin flounder (BFNNV), Japanese flounder (JFNNV), and tiger puffer (TPNNV), and other species of groupers. The kit has internal control system to eliminate false negative results due to failed extraction or reaction. The detection limit is 10 copies per reaction. SEAFDEC/AQD is one of the few competent diagnostic laboratories offering services for VNN. The Fish Health Section developed a standard protocol for the detection of VNN, namely: 1 Amplification of the viral nucleic acid using RT-PCR and 2 A combination of RT-PCR and cell culture method A combination of RT-PCR and cell culture method has been developed to address the concern whether the virus that has been detected in PCR is still viable or not. In this method, samples that turn positive in the RT-PCR are homogenized and inoculated into E-11 cell line and the appearance of CPE is observed. A positive result indicates that a virus is still viable or infective. A negative result indicates that the virus is not viable or the viral load in the sample, though viable, is not enough to cause CPE in the E-11 cell line. Aliquot of the sample should be simultaneously subjected to RT-PCR and cell culture isolation. de la Peña 11 Prevention and control of VNN in the hatchery Treatment of viral diseases in aquaculture production systems is difficult to achieve. With the devastating effects of viral nervous necrosis in the hatchery and possibly in grow-out production systems of marine fish, prevention of viral infection is very important. Prevention of infection in the hatchery phase is necessary to prevent viral transmission in the grow-out phase. Disinfection of rearing water using UV lights or ozone is a must. Among the preventive measures observed in the hatchery are screening of broodstock, eggs and larvae using PCR and egg disinfection (Nakai, 2002). A full shift from trash fish feeding to full pellet diet for broodstock is recommended (Gomez et al., 2005). In a recent study (de la Peña et al., unpublished), PCR revealed that the trash fish were subclinically infected with VNN. Hence, feeding this to broodstock could be a source of viral contamination. Application of immunostimulants and vaccines are two promising fields that could potentially prevent and control VNN infection both in the hatchery and grow-out production systems. Screening of broodstock using PCR Sampling and Selection of Broodstock Screening of individual broodstock for VNN is crucial in the management of VNN infection in the hatchery because VNN-carrier broodstock can be the source of viral contamination (Arimoto et al., 1992). Selection of VNN-free broodstock is the primary step undertaken in the hatchery to prevent the horizontal and vertical transmission of the virus. Figure 8 shows the general scheme used in the screening of broodstock. Only the VNN-negative broodstock should be used. Subsequently, their eggs are further screened for the presence of the virus. Only virus-free eggs are allowed to hatch. SEAFDEC/AQD Fish Health Diagnostic Laboratory offers a diagnostic service to screen eggs for VNN. RT-PCR analysis should be carried out individually on each broodstock. A first primer set could detect any of the four genotypic variants (RGNNV, TPNNV, SJNNV and BFNNV), while the nested primer set is specific only to RGNNV genotype. It has been found that PCR-based selection of the spawner’s gonad just before spawning is effective for the prevention of VNN outbreak in seed production (Mushiake et al., 1994). In striped jack, it has been suggested that spawning should be limited to 10 times in one season to prevent the vertical transmission of SJNNV from spawners to their offspring (Mushiake et al., 1994). Since there is no specific data on other species, the spawning data on striped jack may be used instead. 12 Prevention and control measures against VNN in marine fish hatcheries broodstock screen by RT-PCR negative group (male/female) positive group (male/female) different lots before spawning screen after spawning (eggs) screen larvae screen ready for stocking positive positive positive DISCARD Figure 8. General scheme used in the screening of broodstock Non-destructive sampling can be done by collecting milt or eggs from mature broodstock and gills from immature broodstock (Figure 9) (Kiryu et al., 2007). Immediately place samples on ice and store at -80ºC until use. de la Peña 13 Pool and anesthetize fish in 200 ppm phenoxyethanol until completely sedated (completely sedated fish are not reactive to touch). Broodstock rearing tank Measure and record the total length and weight of the fish. For mature broodstock, insert a sterile polyethylene cannula (Clay Adams PE 100, inner diameter = 0.86 mm, outer diameter = 1.52 mm) into the genital pore. Check and collect the milt or eggs (minimum of 0.5 ml), and record the sex of the fish. Figure 9. General scheme used in broodstock sampling 14 Prevention and control measures against VNN in marine fish hatcheries Return the sedated fish to the rearing tank and allow to recover. Tag the fish. There are seven main categories of tagging techniques: (a) external tags (i.e. fin clip, alphanumerically coded tags), (b) external marks, (c) internal tags, (d) natural marks, (e) biotelemetrin tags, (f) genetic identifiers, and (g) chemical marks (i.e. silver nitrate marking) (Nielsen, 1992). Record the identification number. For immature broodstock, aseptically cut a portion of the gills using sterile forceps and scissors. Apply antibiotic ointment to the wound. Put tissue samples into sterile microcentrifuge tube. Place the tube on ice. de la Peña 15 Feeding of broodstock using commercially available diet Broodstock are usually fed trash fish and squid. In commercial hatcheries, trash fish and/or formulated fish diet are used. The required quantities of essential dietary components may vary according to species. A diet rich in vitamins, polyunsaturated fatty acids (n-3 PUFA) and other micronutrients is essential in obtaining viable eggs and larvae (Moretti et al., 1999). Some use both fresh food and dry feeds. Pellets are given 6 days a week supplemented with moist food twice a week and no feeding once a week. Some hatchery owners add vitamin supplements to trash fish (Moretti et al., 1999). However, trash fish could cause vertical transmission of disease. In a recent study, periodic monitoring of VNN using RT-PCR on the different species of trash fish available in the Iloilo Fishing Port and wild-caught fish in Panay Gulf was done (de la Peña, unpublished). Trash fish that were screened for VNN were Nibea sp. (croaker, abu) Auxis sp. (bullet mackerel, aloy), Sillago sp. (sillago, aso-os), Carangoides sp. (trevally, bagudlong), Priacanthus sp. (bigeye, bukaw-bukaw), Rastrelliger sp. (mackerel, bulao), Selaroides sp. (trevally, dalinu-an), Sphyraena sp. (barracuda, dubla-dubla), Scomber sp. (mackerel, hasa-hasa), Saurida sp. (lizardfish, karaho), Nemipterus sp. (threadfin bream, lagaw), Gerres sp. (mojarra, latab), Decapterus sp. (scad, marot), Selar sp. (oxeye scad, mat-an), Heniochus sp. (bannerfish, saging-saging), Parupeneus sp. (goatfish, salmonete), Leiognathus sp. (ponyfish, sap-sap), Sardinella sp. (sardine, tabagak), Decapterus sp. (scad, tamodius), Amblygaster sp. (sardinella, tuloy), and Cheilinus sp. (wrasse, upos-upos) (Figure 10). Results of the PCR screening showed that 21 species of trash fish sampled at the Iloilo Fishing Port were positive for VNN. All trash fish examined tested positive after the nested PCR except for barracuda, mackerel and lizardfish which are one-step positive, suggesting relatively higher viral loads in these samples. Similarly, almost all species of wild-caught fish were also positive for VNN after the nested PCR. These results indicate that trash fish were subclinically infected or carriers of VNN, and that the virus might have been already established in their environment. In Japan, it has been found out that large populations of wild marine fish in aquaculture areas were subclinically infected with betanodaviruses, which could serve as carriers or reservoirs of the virus (Gomez et al., 2004). These findings provide strong evidence that trash fish could be the main source of viral contamination in broodstock since they are identified as the only major input in broodstock culture systems. To minimize contamination, it is recommended to remove the head of the trash fish before feeding since the virus concentrates in the nervous tissues. Ultimately, a shift from using trash fish to commercial broodstock feeds should be done. 16 Prevention and control measures against VNN in marine fish hatcheries Nibea sp. (croaker, abo) Carangoides sp. (trevally, bagudlong) Rastrelliger sp. (mackerel, bulaw) Selaroides sp. (trevally, dalinuan) Decapterus sp. (scad, tamodius) Scomber sp. (albacore, hasa-hasa) Scomber sp. (bigeye scad, hasa-hasa) Parupeneus sp. (goatfish, salmonete) Selar sp. (oxeye scad, mat-an) Heniochus sp. (bannerfish, saging-saging) Cheilinus sp. (wrasse, upos-upos) Leiognathus sp. (ponyfish, sapsap) Saurida sp. (lizardfish, karaho) Sphyraena sp. (barracuda, dubla-dubla) Figure 10. Trash fish screened for viral nervous necrosis (VNN) de la Peña 17 Disinfection of eggs using various disinfectants Egg-washing using iodine (Figures 11 and 14) (povidone-iodine, 10% active solution), potassium peroxymonosulfate (Figure 12) and ozone (Figures 13 and 15) should be done. The optimum concentrations needed to deactivate the virus are: 25 ppm of available iodine and 1.5 ppm potassium peroxymonosulfate at an exposure time of 10 minutes. Washing the eggs with ozone-treated seawater with a residual ozone content of 0.01 ppm at an exposure time of 2.5 minutes is also effective (Table 3). Larval sampling for screening/diagnosis After spawning, larvae should be screened for virus before stocking in grow-out ponds. Bias sampling should be done, wherein weak larvae are collected and screened for virus. Only VNN-free stocks should be used for the grow-out phase. Removal of weak and dead fish from the rearing tank Weak or dead fish that could possibly serve as the source of contamination should always be removed from the larval rearing tanks. Disposal should be done by burying dead fish in the soil lined with lime. Paraphernalias used in collecting weak and dead fish should always be disinfected with 220 ppm chlorine after every use. Table 3. Egg washing and disinfection using iodine, potassium peroxymonosulfate and ozone-treated seawater Iodine Concen- Hatching tration* rate Disinfectant Potassium peroxymonosulfate Concen- Hatching tration rate Ozone Concen- Hatching tration rate PCR Result 1 step Nested Cell culture Negative control Negative 89% control Negative 89% control 94% Neg (-) Neg (-) Neg (-) 25 ppm 89% 0.5 ppm 72% 2.5 min 96% Neg (-) Pos (+) Neg (-) 50 ppm 21% 1.0 ppm 63% 5.0 min 10% Neg (-) Pos (+) Neg (-) 75 ppm 2% 1.5 ppm 84% 7.5 min 4% Neg (-) Pos (+) Neg (-) 100 ppm 3% 2.0 ppm 27% Neg (-) Pos (+) Neg (-) Positive control 70% Positive control 70% Positive control 70% Pos (+) Pos (+) Pos (+) * 1% active iodine 18 Prevention and control measures against VNN in marine fish hatcheries Figure 11. Schematic diagram of treating eggs with iodine Figure 12. Schematic diagram of treating eggs with potassium peroxymonosulfate Figure 13. Schematic diagram of treating eggs with ozonated water de la Peña 19 Collection of eggs Pooling and sampling of eggs Iodine solution for treatment of eggs Treatment of eggs with iodine, then rinsing with filtered seawater Figure 14. Washing of eggs with iodine 20 Prevention and control measures against VNN in marine fish hatcheries Collection of eggs Pooling and rinsing of eggs Ozone is made using an ozone generator. Residual ozone in the seawater is then measured. Washing eggs with ozonetreated seawater Figure 15. Washing of eggs using ozone-treated seawater de la Peña 21 Disinfection of rearing tanks and other paraphernalia for seed production Strict husbandry management in the hatchery phase is very important in the management of VNN infection. Betanodaviruses are quite resistant to some environmental parameters such as pH (2–9) (Frerichs et al., 1996) and are easily translocated via rearing water and hatchery paraphernalia. Thus, it is very important to disinfect rearing tanks and other paraphernalia used in seed production. It has been noted in an Australian barramundi hatchery that decontamination of rearing tanks after every hatching cycle was effective in preventing VNN infection (Munday and Nakai, 1997). The following are the recommended procedures for the disinfection of rearing facilities (Pitogo et al., 2000): 1 Disinfect used and infected rearing water with 220 ppm available chlorine, 2 Soak all used hatchery paraphernalia in the tank, 3 Drain the disinfectant, scrub the tank with freshly prepared disinfectant of the same concentration and 4 Rinse thoroughly with freshwater several times and allow to dry In addition, all individuals entering the production area of the hatchery should observe biosecurity measures such as the use of footbaths and boots (Figure 16) and hand disinfection. Figure 16. Installation of footbaths Reducing stress factors Reduction of identified stress factors in the hatchery system is very important. Spawning should be induced only up to 10 times because stress associated with multiple spawnings may activate the residual virus (Mushiake et al., 1994). 22 Prevention and control measures against VNN in marine fish hatcheries Reduction of larval stocking density in the tank may help reduce the possibility of viral transmission. Furthermore, rearing water temperature influences disease development. It was observed that higher mortality and earlier appearance of disease signs were observed at higher rearing water temperatures. This suggests that manipulation of rearing water temperature will help reduce disease outbreaks (Lio-Po and de la Peña, 2004). Vaccination Vaccination is done to enhance immunity. Some studies demonstrated that immunization with a recombinant viral coat protein expressed in Escherichia coli or virus-like particles expressed in a baculovirus expression system or formalininactivated virus is effective in controlling the disease (Nakai et al., 2000; Tanaka et al., 2001), but there are no commercially available vaccines at present. One study showed that primary infection with an avirulent aquabirnavirus effectively suppressed secondary betanodavirus infection (Yamashita et al., 2009), suggesting the use of the aquabirnavirus as a potential immunomodulator in vaccination. The author prepared a vaccine from DNA plasmid encoding the capsid protein of the virus. Grouper (E. coioides) weighing 8 grams intramuscularly injected with 100 ng vaccine had the highest survival rate (de la Peña et al., unpublished). A formalin-inactivated vaccine was prepared to control disease at grow-out stage. The vaccines were tested on seabass (L. calcarifer), which produced neutralizing antibodies at high titer levels from Day 10 to 116, with the highest titer at Day 60 post-vaccination. No mortalities were observed after the vaccinated fish were challenged with the red-spotted grouper nervous necrosis virus (RGNNV) SGWak97 strain (Pakingking et al., 2009). In another study, an intramuscular vaccination with formalin-inactivated Philippine strain of piscine betanodavirus (RGNNV) induces potent immune responses and long-term protection against VNN in brown-marbled grouper, E. fuscogutattus (Pakingking et al., 2010). de la Peña 23 References Arimoto M, Mori K, Nakai T, Muroga K and Furusawa I. 1993. Pathogenicity of the causative agent of viral nervous necrosis disease in striped jack, Pseudocaranx dentex (Bloch and Schneider). Journal of Fish Diseases 16:461-469 Arimoto M, Mushiake K, Mizuta Y, Nakai T, Muroga K and Furusawa I. 1992. Detection of striped jack nervous necrosis virus (SJNNV) by enzyme linked immunosorbent assay (ELISA). Fish Pathology 27, 191-195 Bloch B, Gravningen K and Larsen JL. 1991. Encephalomyelitis among turbot associated with a picornavirus-like agent. Diseases of Aquatic Organisms 10:65-70 Dalla Valle L, Toffolo V, Lamprecht M, Maltese C, Bovo G, Belvedeve P and Colombo L. 2005. Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two target real-time PCR. Veterinary Microbiology 110 (3-4):167-179 de la Peña LD, Mori K, Quinitio GF, Chavez DS, Toledo JD, Suarnaba VS, Maeno Y, Kiryu I, Nakai T. 2008. Characterization of betanodaviruses in the Philippines. Bull. Eur. Ass. Fish Pathol. 28 (6):228-235 Frerichs GN, Rodger HD and Peric Z. 1996. Cell culture isolation of piscine neuropathy nodavirus from juvenile sea bass, Dicentrarchus labrax. Journal of General Virology 77, 2067–2071 Gomez DK, Sato J, Mushiake K, Isshiki T, Okinaka Y, Nakai T. 2004. PCR-based detection of betanodaviruses from cultured and wild marine fish with no clinical signs. Journal of Fish Diseases 27:603-608 Iwamoto T, Naqkai T, Arimoto M, Furusawa I. 2000. Cloning of the fish cell line SSN-1 for piscine nodaviruses. Diseases of Aquatic Organisms 43:81-89 Lavilla-Pitogo CR, Lio-Po GD, Cruz-Lacierda ER, Alapide-Tendencia EV, de la Peña LD. 2000. Diseases of Penaeid Shrimps in the Philippines. Aquaculture Extension Manual No. 16, 2nd edition. SEAFDEC/AQD, Tigbauan, Iloilo, Philippines. 82p Lin CC Lin JHY Chen MS and Yang HL 2007. An oral nervous necrosis virus vaccine that induces protective immunity in larvae of grouper (Epinephelus coioides) Aquaculture 268, 265–273 Lio-Po GD and de la Peña LD. 2004. Viral Diseases. In: Diseases of Cultured Groupers. Nagasawa K, Lacierda EC (ed). Southeast Asian Fisheries Development Center, Aquaculture Department, Iloilo, Philippines; pp. 3-18 Maeno Y, de la Peña L, Cruz-Lacierda E. 2002. Nodavirus infection in hatcheryreared orange-spotted grouper Epinephelus coioides: first record of viral nervous necrosis in the Philippines. Fish Pathology 37 (2):87-89 Maltese C and Bovo G. 2007. Viral encephalopathy and retinopathy. Ittipatologia 4, 93-146 Manual of Diagnostic Tests for Aquatic Animals 2009 (www.bahpiq.gov.tw). Bureau of Animal and Plant Health Inspection and Quarantine ,Council of Agriculture Moretti A, Fernandez-Criado MP, Cittolin G and Guidastri R. 1999. Manual on hatchery production of seabass and gilthead bream Volume 1. Rome, FAO. 194p Mori KI, Nakai T, Muroga K, Arimoto M, Mushiake K and Furusawa I. 1992. Properties of a new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 187, 368-371 Munday BL, Kwang J and Moody N. 2002. Betanodavirus infections of teleost fish: a review. Journal of Fish Diseases 25, 127-142 Munday BL and Nakai T. 1997. Special topic review: Nodaviruses as pathogens in larval and juvenile marine finfish. World Journal of Microbiology and Biotechnology 13, 375-381 Munday BL, Nakai T and Nguyen HD. 1994. Antigenic relation-of the picorna like virus of larval barramundi, Lates calcarifer Bloch to the nodavirus of larval striped jack, Pseudocaranx dentex (Bloch and Schneider). Aust Vet J 71, 384 Munday BL, Langdon JS, Hyatt A and Humphrey JD. 1992. Mass mortality associated with a viral-induced vacoulating encephalopathy and retinopathy of larval and juvenile barramundi, Lates calcarifer Bloch. Aquaculture 103:197-211 Mushiake K, Nishizawa T, Nakai T, Furusawa I, and Muroga K. 1994. Control of VNN in striped jack: Selection of spawners based on the detection of SJNNV gene by Polymerase Chain Reaction (PCR). Fish Pathology 29(3), 177-182 Nakai T, Mori K, Sugaya T, Nishioka T, Mushiake K and Yamashita H. 2009. Current Knowledge on Viral Nervous Necrosis (VNN) and its Causative Betanodaviruses. The Israeli Journal of Aquaculture – Bamidgeh, 61(3): 198-207 Nakai T. 2002. Diagnostic and preventive practices for viral nervous necrosis (VNN). In: Disease control in Fish and Shrimp Aquaculture in Southeast Asia – Diagnosis and Husbandry Techniques. Proceedings of the SEAFDEC-OIE Seminar-Workshop on Disease Control in Fish and Shrimp Aquaculture in Southeast Asia. Inui Y, Cruz-Lacierda E (eds). Iloilo City, Philippines; pp. 80-89 Nakai T, Iwamoto T, Mori K, Arimoto M and Tanaka S. 2000. Recent Development in Identification and Control of Viral Nervous Necrosis (VNN) in Grouper. APEC FWG 02/2000 Nielsen LA. 1992. Methods of marking fish and shellfish. American Fisheries Society Special Publication 23, Bethesda, Maryland, 468 pp Nishizawa T, Mori K, Nakai T, Furusawa I, and Muroga K. 1994. Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis virus (SJNNV). Diseases of Aquatic Organisms 18:103-105 Nishizawa T, Furuhashi M, Nagai T, Nakai T and Muroga K. 1997. Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Appl. Environ. Microbiol. 63:1633-1636 OIE (Office International des Epizooties) 2003. Diagnostic Manual for Aquatic Animal Diseases. OIE, Paris. Pakingking R, Bautista NB, de Jesus-Ayson EG, and Reyes O. 2010. Protective immunity against viral nervous necrosis (VNN) in brown-marbled grouper (Epinephelus fuscogutattus) following vaccination with inactivated betanodavirus. Fish & Shellfish Immunology 28:525-533 Pakingking R, Seron R, dela Peña L, Mori K, Yamashita H and Nakai T. 2009. Immune response of Asian seabass, Lates calcarifer Bloch, against an inactivated betanodavirus vaccine. Journal of Fish Diseases 32(5), 457-463 Saiki R, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA and Arnheim N. 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle-cell anemia. Science 230:1350-1354 Tanaka S, Mori K, Arimoto M, Iwamoto T and Nakai T. 2001. Protective immunity of seven band grouper Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis. Journal of Fish Diseases. 24, 15-22 Totland GK, Grotmol S, Morita Y, Nishioka T and Nakai T. 1999. Pathogenicity of nodavirus strains from striped jack Pseudocaranx dentex and Atlantic halibut Hippoglossus hippoglossus, studied by waterborne challenge of yolk-sac larvae of both teleost species. Diseases of Aquatic Organisms 38, 169-175 Yamashita H, Mori K and Nakai T. 2009. Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Journal of Fish Disease 32(2), 201-10 Yoshikoshi K and Inoue K. 1990. Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Opelegnathus fasciatus (Temminck and Schlegel). Journal of Fish Diseases 13, 69-77 Glossary Agarose gel electrophoresis – a method used in molecular biology to separate DNA or RNA molecules by size. This is done by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones. Aqueous phase – the water portion of a system consisting of two liquid phases, one that is primarily water and a second that is a liquid immiscible (cannot undergo mixing or blending) with water. Asymptomatic – also called sub-clinical condition; it carries a disease or infection but experiences no symptoms. A condition might be asymptomatic if it fails to show the noticeable symptoms with which it is usually associated. Basophilia – a condition where the basophil (white blood cells) quantity is abnormally elevated. Cell culture – the maintenance and growth of the cells of multi-cellular organisms outside the body in specially designed containers and under precise conditions of temperature, humidity, nutrition, and free from contamination. Cell line – a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space (i.e. E-11, SSN-1) E-11 is a clonal cell line, derived from the SSN-1 cell line and is useful for qualitative and quantitative analyses of all the betanodaviruses. SSN-1 is a fish cell line, which is derived from striped snakehead Channa striatus. Cytopathic effect (CPE) – degenerative changes in cells (especially in tissue culture) associated with the multiplication of certain viruses; when, in tissue culture, spread of virus is restricted by an overlay of agar (or other suitable substance) the cytopathic effect may lead to formation of plaque. Decontamination - includes all stages of cleaning and disinfection. DEPC-treated distilled water – Diethylpyrocarbonate (DEPC), also called diethyl dicarbonate (IUPAC name), diethyl oxydiformate, ethoxyformic anhydride, or pyrocarbonic acid diethyl ester, is used in the laboratory to inactivate the RNase enzymes from water and other laboratory utensils. Disinfectant - a chemical used to destroy disease agents outside a living animal. Disinfection - the application, after thorough cleansing, of procedures intended to destroy the infectious or parasitic agents of animal diseases; applies to premises, vehicles and different objects that may have been directly or indirectly contaminated. Ethidium bromide – an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. When exposed to ultraviolet light, it will fluoresce with an orange color, intensifying almost 20-fold after binding to DNA. Homogenize – to reduce to small particles of uniform size and distribute evenly usually in a liquid Horizontal transmission – infectious agents come in contact with the hosts through the water, the feeds or through carrier animals that are in the environment. Indirect Fluorescent Antibody Technique (IFAT) - a technique in which unlabelled antibody is incubated with the antigen then overlaid with a fluorescent conjugated anti-immunoglobulin to form a sandwich. Inoculate – to implant microorganisms or infectious material into a culture medium Inverted microscope – a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up. Inverted microscopes are useful for observing living cells or organisms at the bottom of a large container (e.g. a tissue culture flask) under more natural conditions than on a glass slide, as is the case with a conventional microscope. IsoTherm – used in cooling samples effectively and consistently at –21°C or 0°C over many hours. It can be used to prevent enzymes from becoming inactive as well as for PCR sample preparation, sample transport and slow thawing of frozen samples. Necrosis – localized death of a tissue as a result of an outside agent. It may follow a wide variety of injuries, both physical (cuts, bruises) and biological (effects of disease-causing agents). The sign of necrosis is called a lesion; it is often of diagnostic value. Necrosis is brought about by intracellular enzymes that are activated upon injury and proceed to destroy damaged cells. Nested RT-PCR – a double-stage PCR process where the second round identifies a DNA sequence nested within the initial sequence thus increasing the specificity. See Polymerase chain reaction (PCR) and Reverse transcriptase- PCR (RT-PCR) Ozone-treated seawater – seawater oxygen which readily gives treated up one watiothmoozfonoexy(Oge3n), a free radical of providing a powerful oxidizing agent which is toxic to most waterborne organisms. It is a very strong, broad spectrum disinfectant. It is an effective method to inactivate harmful protozoa that form cysts. It also works well against almost all other pathogens. Ozone is made by passing oxygen through ultraviolet light or a “cold” electrical discharge. To use ozone as a disinfectant, it must be created on-site and added to the water by bubble contact. Polymerase Chain Reaction (PCR) – a laboratory technique commonly used in molecular biology to generate many copies of a DNA sequence, a process termed amplification, that can be used to detect the presence of virus DNA. See also Reverse transcriptase-PCR (RT-PCR) and Nested RT-PCR Prevalence - the proportion (or percentage) of animals in a particular population affected by a particular disease (or infection or positive antibody titre) at a given point in time. Primer – short DNA fragments containing sequences complementary to the target region that are synthesized in a laboratory. Pyknosis – or karyopyknosis, is the irreversible condensation of chromatin in the nucleus of a cell undergoing apoptosis (process of cell death that may occur in multicellular organisms). Retinopathy – refers to some form of non-inflammatory damage to the retina of the eye. In most cases, it is an ocular manifestation of systemic disease. RNA – or Ribonucleic acid consists of a long chain of nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate. In the cell, RNA is usually single-stranded, nucleotides contain ribose and has the base uracil rather than thymine that is present in DNA.RNA is transcribed from DNA by enzymes called RNA polymerases and is generally further processed by other enzymes. RNA is central to protein synthesis. RT-PCR – is a variant of polymerase chain reaction (PCR). A highly sensitive technique for the detection and quantitation of mRNA (messenger RNA) by reverse transcription to DNA followed by PCR . See Polymerase chain reaction (PCR) and Nested RT-PCR Sensitivity - the proportion of affected individuals in the tested population that are correctly identified as positive by a diagnostic test (true positive rate). Supernatant – usually clear liquid overlying material deposited by settling, precipitation, or centrifugation Surveillance - a systematic series of investigations of a given population of fish to detect the occurrence of disease for control purposes, and which may involve testing samples of a population. Susceptible - fish that can be infected with a particular disease. TBE buffer – TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA. Tissue culture infective dose (TCID) – amount of a pathogenic agent that will produce pathological change when inoculated on tissue cultures. Total length – refers to the length from the tip of the snout to the tip of the longer lobe of the caudal fin, usually measured with the lobes compressed along the midline. Vaccination - inoculation of healthy individuals with weakened or attenuated strains of disease-causing agents to provide protection from disease. Vaccine - modified strains of disease-causing agents that, when inoculated, stimulate an immune response and provide protection from disease. Vacuolation – the process of forming vacuoles (small cavity in the cytoplasm of a cell); the condition of being vacuolated. Vertical transmission – infectious agents transfer from parent to offspring. The female or male broodstock may be carriers of diseases, and transfer them to their offspring through the egg or sperm. Appendices Appendix 1. Cell culture GENERAL LIST OF MATERIALS AND EQUIPMENT NEEDED FOR CELL CULTURE -- Disposable cup (for waste media) -- Test tube rack -- 70% rubbing alcohol -- Cotton in 70% rubbing alcohol -- Paper towels -- Sterile glass pipettes -- Rubber bulbs -- Gas burner -- Clean bench -- Inverted microscope -- Incubator PREPARATION AND SUBCULTURE OF E-11 CELL LINE Materials and reagents needed -- E-11 cell line -- Versene -- Versene-trypsin -- L-15 medium (supplemented with 10% FBS) -- Cell culture flasks -- 24-well plates Procedure 1 Prepare a clean bench by spraying with 70% rubbing alcohol and wiping with paper towel. Switch on UV light for at least 30 min 2 Switch on the blower before opening the glass door of the clean bench. Turn on the gas burner and flame surfaces of the test tube rack. Flame the glass pipettes, attach the rubber bulbs and place atop the test tube rack. Different sets of pipettes should be used for each reagent. Place needed reagents inside the clean bench 3 Open the flask with the cell monolayer. Remove old medium using a sterile pipette and discard into a waste cup 4 Pipette 0.5 ml Versene into the flask and wash cells by shaking gently. Remove the washing from the flask and discard 5 Add 0.5 ml Versene-trypsin solution to the flask. Cover the flask and allow the cells to be detached from the flask surface. Tap the flask gently against the palm of one hand to allow the cells to detach completely 6 Loosen the cells by gently pipetting the Versene-trypsin solution in and out by passing solution on the cell surface to ensure the separation of the monolayer to produce a single cell suspension 7 Add 10-15 ml of the L-15 medium (supplemented with 10% FBS) to the flask and gently mix by pipetting 8 Dispense 1 ml of cell suspension into each well of the 24 well plates 9 Label, seal with parafilm, and place inside a resealable plastic bag 10 Put the plates in the incubator set at 25°C to allow the growth of the cells Note: Spray with alcohol or wipe with alcohol-soaked cotton especially near the mouth of the flasks. Glass pipettes should also be flamed before and after each use. Allow pipettes to cool down for a few seconds before using. Only the glass pipette used for removing waste and mixing of cell suspension should be inserted inside the culture flask. All other pipettes used for transferring reagents and culture media should not touch any surface of the culture flask PREPARATION OF TISSUE SAMPLES Materials and reagents needed -- Hank’s balanced salt solution (HBSS) -- Tissue samples (brain, eye) -- 1000 µL pipettor -- 1000 µL sterile pipette tips -- Sterile microcentrifuge tubes -- Plastic homogenizer -- 0.45 µm syringe filter -- 70% rubbing alcohol -- RNase AWAY -- Refrigerated centrifuge Procedure 1 Weigh 100-150 mg tissue samples in a microcentrifuge tubes 2 Add 300 µL of HBSS and homogenize using a plastic homogenizer. After using the plastic homogenizer, wipe the homogenizer with a paper towel and soak in RNase AWAY 3 Add sufficient amount of HBSS for 10-fold dilution of samples (10% w/v) 4 Centrifuge at 2,500 rpm at 4°C for 10 min 5 Filter the supernatant using a 0.45 µm syringe filter and transfer the supernatant into a new tube 6 Store at -80°C until use Note: Homogenization and filtration should be done inside the clean bench INOCULATION OF HOMOGENIZED TISSUE SAMPLES Materials and reagents needed -- L-15 medium (supplemented with 10% FBS) -- Tissue samples in HBSS (homogenized and filtered) -- 24 well plates containing cell monolayer -- Pipettors (1000 µL, 100 µL, 10 µL) -- Sterile pipette tips (1000 µL, 100 µL, 10 µL) -- Microcentrifuge tube rack Procedure 1 Check the condition of the cell in each 24-well plate. Proceed to the next step if cells are 80% (check and confirm) confluent, otherwise, allow cells to grow in the incubator. Complete monolayer should be observed within 24-48 hours after seeding 2 Gently pipette out 850-900 µL of the old culture medium from each well 3 Depending on the desired final concentration of the tissue homogenate, pipette 10 µL, 50 µL, or 100 µL of the homogenate into each well. Two to four replicates should be made. Always include positive and negative controls 4 Cover the plates and let stand for 1 hour. Carefully add 1 ml of the culture medium to each well and ensure that the cell monolayer is not disturbed 5 Seal plates with parafilm, label (include starting date of assay) and check the condition of cell monolayer under the inverted microscope. Note any disruptions in the monolayer caused by the addition of the homogenate or medium 6 Place in a resealable plastic bag and place inside the incubator set at 25°C to allow the growth of the cells 7 Check cells under the inverted microscope and observe for cytopathic effect (CPE) every 24 hours Appendix 2. Reverse transcription polymerase chain reaction (RT-PCR) RNA EXTRACTION TRI REAGENT (MRC) is a ready-to-use reagent for the isolation of total RNA from cells and tissues. The reagent is a monophasic solution of phenol and guanidine isothiacyanate. It maintains the integrity of the RNA while disrupting cells and dissolving cell components. Total RNA isolated is free of protein and DNA contamination. Materials, reagents and equipment needed -- Pipettors (1000 µL, 200 µL, 100 µL, 10 µL) -- Filtered pipette tips (1000 µL, 200 µL, 100 µL, 10 µL) -- Plastic homogenizer -- Sterile microcentrifuge tubes -- TRI REAGENT -- Chloroform (molecular biology grade) -- Isopropyl alcohol (molecular biology grade) -- Absolute ethanol -- DNAse, RNAse free distilled water or DEPC-treated distilled water -- Refrigerated centrifuge -- Vortex mixer -- Incubation oven Procedure 8 Weigh 50 - 100 mg tissue 9 Add 300 µl TRI REAGENT (Molecular Research Center, Cat. No. TR- 118) Note: TRIzol reagent is a ready-to-use, monophasic solution of phenol and guanidine isothiocynate suitable for isolation of total RNA, DNA, and proteins. It is designed for use with tissues or cells and is based upon improvements to the single-step RNA isolation method developed by Chomczynksi and Sacchi. 10 Homogenize 11 Add 700 µl TRIzol 12 Mix by inversion at least 10X 13 Leave at room temperature, 5 min (PHASE SEPARATION) 14 Add 200 µl chloroform 15 Shake vigorously, 15 sec 16 Leave at room temperature, 2 min 17 Spin at 10,800 rpm / 12,000 x g, 4oC, 15 min 18 Pipette out AQUEOUS PHASE into new tube (use filter tips) (RNA PRECIPITATION) 19 Add 500 µl isopropyl alcohol 20 Mix by inversion at least 10x 21 Leave at room temperature, 10 min 22 Spin at 10,800 rpm / 12,000 x g, 4oC, 8 min 23 Decant and discard supernatant (WASHING) 24 Add 1 ml 75% ethanol to RNA pellet 25 Vortex briefly for a few seconds 26 Spin at 8,500 rpm / 7,500 x g, 4oC, 5 min 27 Decant the ethanol-wash (SOLUBILIZATION) 28 Spin briefly and pipette out remaining ethanol-wash 29 Briefly air-dry the RNA pellet for 3-5 min Note: It is important not to completely dry the RNA pellet as this will greatly decrease its solubility. 30 Dissolve in 20 µl diethyl pyrocarbonate (DEPC)-treated distilled water 31 Incubate at 55-60oC, 10-15 min 32 Flick to mix 33 Store at –20oC or –80oC Note: Refrigerated centrifuge is Eppendorf (5417 R) using F 45-30-11 rotor. RT-PCR Materials, reagents and equipment needed -- Pipette tips (10 μL, 100 μL, 200 μL, 1000 μL) -- Pipettors (10 μL, 100 μL, 200 μL, 1000 μL) -- Sterile 0.5 ml PCR tubes -- Sterile 1.5 ml microcentrifuge tubes -- SuperScript III Reverse transcriptase -- 5X first strand buffer -- 0.1M DTT -- RNaseOUT recombinant ribonuclease inhibitor -- 2.5 mM and 10 mM dNTPs -- 5X Green Go Taq Flexi buffer --- 25 mM Go Taq FMlegxCi lD2 NA Polymerase -- F2 (forward primer) -- R3 (reverse primer) -- RGNNV-NFRG (nested forward primer) -- RGNNV-NRRG (nested reverse primer) -- DEPC-treated distilled water -- Clean bench -- Microcentrifuge tube -- Water bath -- Programmable thermal cycler -- Isotherms -- Electrophoresis machine -- TBE buffer -- Agarose gel -- Gel documentation system -- Ethidium bromide Procedure 1 To prepare RT mastermix, mix the following reagents based on the number of samples to be run. For 1 reaction, mix: • 8.1 µL SM1 • 0.5 µL R3 (reverse primer) • 0.2 µL Ribonuclease Inhibitor • 0.2 µL SuperScript III Reverse Transcriptase Components of SM1 (previously prepared pre-mix) • 60 µL 5X First Strand buffer • 30 µL 0.1M DTT • 120 µL 2.5 mM dNTPs • 36 µL DEPC-treated distilled water 2 Distribute 9 µL of RT mastermix to each PCR tubes 3 Boil RNA sample for 5 min, then chill on ice for 5 min 4 Add 1 µL RNA template to RT mastermix 5 Perform reverse transcription profile: 1 cycle 42°C 30 min 1 cycle 99°C 10 min 6 Prepare 1-step PCR mastermix: • 39 µL EM2 • 0.5 µL Forward primer • 0.5 µL Go Taq Flexi DNA Polymerase Components of EM2 (previously prepared pre-mix) • 300 µL 5X Green Go Taq Flexi buffer • • 94605µLµL25DEmPMC-MtregaCteld2 (dPirsotimlleedgaw, UatSeAr ) 7 Distribute 39 µL 1-step PCR mastermix to each tube of RT 8 Perform the 1-step PCR amplification profile: 1 cycle 95°C 2 min 30 cycles 95°C 40 s 55°C 40 s 72°C 40 s 1 cycle 42°C 5 min 4°C α 9 Electrophorese in 2% TBE-agarose gel 10 Stain in ethidium bromide 11 View gel in gel documentation system or UV transilluminator 12 Prepare the nested PCR mastermix: • 9.25 µL DEPC-treated distilled water • 5 µL Green Go Taq Flexi buffer (Promega, USA) • • 1.2 0.5 µL µL 25 10 mM mM MdNgTCPl2s (Promega, USA) (Invitrogen, USA) • 0.25 µL Nested Reverse primer • 0.25 µL Nested Forward primer • 0.25 µL Go Taq Flexi DNA Polymerase (Promega, USA) 13 Distribute 24 µL to each PCR tube previously labeled based on the total number of samples 14 Add 1 µL post 1-step PCR product to the nested PCR mastermix 15 Perform the nested PCR amplification profile: 1 cycle 95°C 5 min 30 cycles 95°C 30 s 65°C 30 s 72°C 30 s 1 cycle 42°C 5 min 4°C α 16 Electrophorese in 2% TBE-Agarose gel 17 Stain with Ethidium bromide 18 View gel in documentation system or UV transilluminator Appendix 3. List of PCR reagents/buffers STOCK SOLUTIONS 1 Tris, 2M, pH 7.4 • Distilled water (DW) • Trizma Base (Sigma, Cat. # T-6066, molecular biology grade) • Hydrochloric acid (conc.) 80 ml 24.22 g 7.5 ml (a) Mix first the two components (b) Add slowly the HCl with stirring until pH 7.4 (c) Add DW to make 100 ml (d) Autoclave 2 EDTA, 0.5M • Distilled water (DW) • Ethylenediaminetetraacetic acid (Sigma, Cat. # E-5134, molecular biology grade) (a) Mix well with stirring (b) Add 10 M NaOH until pH 8.0 (c) Add DW to make 100 ml (d) Autoclave 60 or 70 ml 18.61 g 3 NaOH, 10M • Distilled water 80 ml • Sodium hydroxide (NaOH) 40 g (a) Mix well by stirring (b) Add DW to make 100 ml 4 TE Buffer • Tris stock solution 0.5 ml • EDTA stock solution 20 ml • Distilled water (DW) 99.3 ml (a) Aliquot and autoclave ELECTROPHORESIS BUFFERS 1 TBE buffer Stock solution, 10X • Distilled water 400 ml • Tris base (Sigma, Cat. # T-6066, molecular biology grade) 54 g • Boric acid (Sigma, Cat. # B-6768, molecular biology grade) 27.5 g • EDTA (Sigma, Cat. # E-5134, molecular biology grade) 3.7 g Primer pairs: 1-step Forward: F2 (5’ to 3’) Reverse : R3 (5’ to 3’) Nested Forward: RGNNV-NFRG (5’ to 3’) Reverse: RGNNV-NRRG (5’ to 3’) Expected PCR product: 1-step: 430 bp Nested: 280 bp CgT gTC AgT CAT gTg TCg CT CgA gTC AAC ACg ggT gAA gA AAC TgA ggA gAC TAC CgC TC CAg CgA AAC CAg CCT gCA gg (a) Mix well with stirring (b) Add DW to make 500 ml Working solution, 1X • Stock solution, (10X) 40 ml • DW 360 ml Appendix 4. Disinfection of eggs using 25 ppm active iodine 1 Using the table below, add the required amount of povidone-iodine solution to the desired volume of seawater 2 Using a fine mesh net, dip the eggs into the prepared solution for 10 minutes with constant and mild agitation 3 Rinse the eggs in running seawater for about 1 minute and transfer to the hatching tank Volume of seawater (L) 5.0 10.0 15.0 Volume of povidone-iodine 10% topical solution* (ml) 12.5 25.0 37.5 * Calculations based on the 9.0% - 12.0% available iodine from a chemical complex of polyvinylpyrrolidone (povidone, PVP) and elemental iodine Appendix 5. Procedure for egg disinfection using 1.5 ppm potassium peroxymonosulfate 1 Using the table below, add the required amount of potassium peroxymonosulfate to the desired volume of seawater 2 Using a fine mesh net, dip the eggs to the prepared solution for 10 minutes with constant and mild agitation 3 Wash the eggs in running seawater for about 1 minute and transfer to the hatching tank Volume of seawater (L) 5.0 10.0 15.0 Amount of Potassium peroxymonosulfate (mg) 7.5 15.0 22.5 Recent SEAFDEC/AQD publications AQUACULTURE EXTENSION MANUALS (AEM) & STATE-OF-THE-ART SERIES (SAS) AEM 44 Prevention and control measures against viral nervous necrosis (VNN) in the hatchery phase of marine fish in the Philippines. LD de la Peña (2010). 24 pp AEM 43 Philippine Freshwater Prawns (Macrobrachium spp.) MRR Eguia et al. (2009). 50 pp AEM 42 Seed Production and Grow-out of Mud Crab (Scylla paramamosain) in Vietnam. NC Thach (2009). 26 pp AEM 41 Grow-out Culture of the Asian Catfish Clarias macrocephalus (Gunther). EB Coniza et al. (2008). 29 pp AEM 40 Breeding and Seed Production of the Asian Catfish Clarias macrocephalus (Gunther). JD Tan-Fermin et al. (2008). 28 pp AEM 39 Abalone Hatchery. AC Fermin et al. (2008). 31 pp AEM 38 Tilapia Broodstock and Hatchery Management. R Eguia, MRR Eguia (2007). 48 pp AEM 37 Giant Clam Hatchery, Ocean Nursery and Stock Enhancement. SS Mingoa-Licuanan, E Gomez (2007). 110 pp AEM 36 Tilapia Farming in Cages and Ponds. RV Eguia, MRR Eguia (2004). 40 pp (in print or CD) AEM 35 Best Management Practices for Mangrove-Friendly Shrimp Farming. DD Baliao, S Tookwinas (2002). 50 pp (Filipino version also available) AEM 34 Biology and Hatchery of Mud Crabs Scylla spp. ET Quinitio, FD Parado-Estepa (2008, 2nd ed.). 47 pp AEM 33 Induced Breeding and Seed Production of Bighead Carp. AC Gonzal et al. (2001). 40 pp AEM 32 The Farming of the Seaweed Kappaphycus. AQ Hurtado, RF Agbayani (2000). 26 pp (Filipino version also available) AEM 30 Net Cage Culture of Tilapia in Dams and Small Farm Reservoirs. DD Baliao et al. (2000). 14 pp AEM 29 Grouper Culture in Floating Net Cages. DD Baliao et al. (2000). 10 pp AEM 26 Pen Culture of Mudcrab in Mangroves. DD Baliao et al. (1999). 10 pp AEM 24 Grouper Culture in Brackishwater Ponds. DD Baliao et al. (1998). 18 pp AEM 23 Pagpapaanak ng Tilapya. RV Eguia et al. (2007). 64 pp AEM 22 Pag-aalaga ng Tilapya. RV Eguia et al. (2007). 68 pp AEM 21 Feeds and Feeding of Milkfish, Nile Tilapia, Asian Sea Bass and Tiger Shrimp. Feed Development Section (1994). 97 pp AEM 16 Diseases of Penaeid Shrimps in the Philippines. CR Lavilla-Pitogo et al. (2000) 83 pp SAS Environment-Friendly Schemes in Intensive Shrimp Farming. DD Baliao (2000). 24 pp SAS Closed Recirculating Shrimp Farming System. S Tookwinas (2000). 28 pp TEXTBOOKS, MONOGRAPHS, LABORATORY BOOKS The Malalison Experience. RF Agbayani et al. (2009). 64 pp Field Guide to Philippine Mangroves. JH Primavera. (2009). 8 pp Training Handbook on Rural Aquaculture. SEAFDEC/AQD (2009, 3rd advance reading copy). 296 pp Seaweeds of Panay. AQ Hurtado et al. (2006, 2nd ed. ). 50 pp Diseases in Farmed Mud Crabs Scylla spp.: Diagnosis, Prevention and Control. CR Lavilla-Pitogo, LD de la Peña (2004). 89 pp (in print or CD) Diseases of Cultured Groupers. K Nagasawa, ER Cruz-Lacierda (eds.) (2004). 81 pp (in print or CD) Handbook of the Mangroves of the Philippines – Panay. JH Primavera et al. (2004). 106 pp Laboratory Manual of Standardized Methods for the Analysis of Pesticide and Antibiotic Residues in Aquaculture Products. IG Borlongan, JNP Chuan (2004). 46 pp (in print or CD) Laboratory Manual of Standardized Methods for Antimicrobial Sensitivity Tests for Bacteria Isolated from Aquatic Animals and Environment. L Ruangpan, EA Tendencia (2004). 55 pp (in print or CD) Nutrition in Tropical Aquaculture. OM Millamena et al. (eds.) (2002). 221 pp Health Management in Aquaculture. GL Po et al. (eds.) (2001). 187 pp An Assessment of the Coastal Resources of Ibajay and Tangalan, Aklan. LMB Garcia (ed.) (2001). 60 pp Ecology and Farming of Milkfish. TU Bagarinao (1999). 117 pp CONFERENCE PROCEEDINGS Proceedings of the Regional Technical Consultation on Stock Enhancement. JH Primavera, ET Quinitio, MR Eguia (eds.) (2006). 150 pp Responsible Aquaculture Development in Southeast Asia. LMB Garcia (ed.) (2001). 274 pp Mangrove-Friendly Aquaculture. JH Primavera et al. (eds.) (2000). 217 pp FOR FREE DOWNLOADS, VISIT: www.seafdec.org.ph/publications Flyers, reports, newsletters, video, photos The Southeast Asian Fisheries Development Center (SEAFDEC) is a regional treaty organization established in December 1967 to promote sustainable fisheries and responsible aquaculture in the region. The member countries are Brunei Darussalam, Cambodia, Indonesia, Japan, Lao PDR, Malaysia, Myanmar, Philippines, Singapore, Thailand and Vietnam. The policy-making body of SEAFDEC is the Council of Directors, made up of representatives of the member countries. SEAFDEC has four departments that focus on different aspects of fisheries development: • Training Department (TD) in Samut Prakan, Thailand (1967) for training in marine capture fisheries • Marine Fisheries Research Department (MFRD) in Singapore (1967) for post-harvest technologies • Aquaculture Department (AQD) in Tigbauan, Iloilo, Philippines (1973) for aquaculture research and development, and • Marine Fishery Resources Development and Management Department (MFRDMD) in Kuala Terengganu, Malaysia (1992) for the development and management of fishery resources in the exclusive economic zones of SEAFDEC member countries AQD is mandated to: • Conduct scientific research to generate aquaculture technologies appropriate for Southeast Asia • Develop managerial, technical and skilled manpower for the aquaculture sector • Produce, disseminate and exchange aquaculture information AQD maintains four stations: the Tigbauan Main Station and Dumangas Brackishwater Station in Iloilo province; the Igang Marine Station in Guimaras province; and the Binangonan Freshwater Station in Rizal province. AQD also has a Manila Office in Quezon City.