309 (2010) 1–14

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Aquaculture

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Review Molecular studies, disease status and prophylactic measures in aquaculture: Economic importance, diseases and immunology

Ramasamy Harikrishnan a,⁎, Chellam Balasundaram b, Moon-Soo Heo a,⁎ a Marine Applied Microbes and Aquatic Organism Disease Control Lab, Department of Aquatic Biomedical Sciences, School of Marine Biomedical Sciences, College of Ocean Sciences & Marine and Environmental Research Institute, Jeju National University, Jeju 690-756, South Korea b MarineDepartment of Science, Bharathidasan University, Tiruchirapalli-620024, India article info abstract

Article history: are potentially important and economically valuable aquaculture in Southeast Asian Received 22 August 2010 countries. Among sport and artisanal fishers is a prized catch demanding lucrative price. Groupers belong to Received in revised form 11 September 2010 the subfamily Epinephelinae, which include 159 species under 15 genera; they are widely distributed in the Accepted 13 September 2010 tropical and sub-tropical waters of Atlantic Ocean and Mediterranean . Grouper culture was first introduced in the early 1970s in Singapore, Malaysia, Hong Kong, , and Taiwan, and is now widely Keywords: practiced throughout Southeast Asia. At present, its production is essentially in the hands of small-scale Diagnosis Disease farmers; however the interest in its larger scale production in offshore systems is gaining momentum. In Economic loss recent years the global production of groupers has increased tremendously due to its escalated demand with Grouper 60,774, 99,378, 163,093, and 198,690 mt in 1990, 2000, 2005, and 2007 respectively. Despite the wholesale Production premium price of up to US$100/kg groupers fetch in the Chinese live-fish markets in Hong Kong and South its culture is ravaged by various diseases; for instance the large-scale seed production of groupers continues to encounter increasing difficulties, especially with a of infectious diseases including various viral, bacterial, and parasitic pathogens and non-infectious (environment, management, and nutritional) disease agents and a number of undiagnosed diseases of unknown origin. Apart from some documented viral problems in Southeast Asia in groupers, little is known about the impact of major diseases that may go beyond direct mortalities and production losses. Immunization with formalin-killed viral and parasitic vaccines increased survival rates and dietary administration of DHA/EPA, L-ascorbic acid, vitamin C and E, sodium alginate, κ-carrageenan yeast, probiotics, and herbals significantly increased the specific and non-specific immune response and protect from diseases. In this review will discuss the available information on infectious diseases and its immune status in grouper culture. Published by Elsevier B.V.

Contents

1. Introduction ...... 2 1.1. and geographical distribution ...... 2 1.2. Economic importance of the groupers ...... 2 1.3. Grouper culture ...... 2 1.4. Production and cost ...... 3 1.5. Grouper seed hatcheries ...... 3 2. Disease status in grouper aquaculture ...... 3 2.1. Diseases ...... 3 2.2. Bacterial diseases ...... 3 2.3. Parasitic diseases ...... 5 2.4. Viral diseases ...... 6 2.4.1. Sleepy grouper disease (SGD) ...... 6 2.4.2. Grouper iridovirus (GIV) ...... 6 2.4.3. Singapore grouper iridovirus (SGIV) ...... 7 2.4.4. Megalocystiviruses ...... 7

⁎ Corresponding authors. Tel.: +82 64 754 3473; fax: +82 64 756 3493. E-mail addresses: [email protected] (R. Harikrishnan), [email protected] (M.-S. Heo).

0044-8486/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.aquaculture.2010.09.011 2 R. Harikrishnan et al. / Aquaculture 309 (2010) 1–14

2.4.5. Nodavirus and Betanodaviruses ...... 7 2.4.6. Grouper nervous necrosis virus (GNNV) ...... 7 2.4.7. Viral nervous necrosis virus (VNNV) ...... 7 3. Diagnostic features ...... 8 3.1. Techniques ...... 8 3.2. Antibodies...... 9 3.3. Cell lines ...... 9 4. Immunology ...... 9 5. Conclusions ...... 10 Acknowledgements ...... 10 References ...... 10

1. Introduction costae, E. haifensis, and E. marginatus. Besides two species of E. tauvina and E. coioides are also present in the Mediterranean sea of Indo- Groupers are popular carnivorous fish with a high market demand Pacific. Another grouper, Lessepsian migrants entered the Mediterra- in many parts of the world, such as in Kuwait, Indonesia, Singapore, nean via the Suez Canal from the Red Sea (Ben-Tuvia and Lourie, Malaysia, Thailand, Philippines, Hong Kong, Taiwan, China, Mexico, 1969; Heemstra and Golani, 1993; Parenti and Bressi, 2000; Golani Japan, and the USA (INFOFISH, 1989; Brais, 1987). Groupers are ideal et al., 2002). The (Gill, 1962), is represented by candidate species for intensive aquaculture particularly in the Asia– 15 species which usually live on coral reefs and rocky bottoms. In the Pacific region because of high consumer demand, desirable taste, eastern Atlantic, the genus is represented by two species: Mycter- hardiness in a crowded environment, fast growth, efficient feed operca rubra, ranging from the Mediterranean Sea to the Bay of Biscay conversion, and rapid growth (Sim et al., 2005; Chen and Tsai, and along the west coast of South Africa to Angola, and M. fusca found 1994; Millamena, 2002; Boonyaratpalin, 1997; Fukuhara, 1989; Chen, in Madeira, the Azores and the Canary and Cape Verde islands 1990; Kuo, 1995; Kohno et al., 1993). Besides due to overexploitation (Baldwin and Johnson, 1993). M. rubra was originally described as E. of wild stock grouper culture is emerging as a viable venture in many ruber (Bloch, 1973). Jordan and Eigenmann (1988) synonimized E. countries. However despite its advantages as an ideal candidate ruber with M. rubra based on the number of fin rays, projecting lower species culture is still in infancy with problems like disease jaw, pointed snout and the general features regarding the shape of the management, feed, and seed production; as a new species preferred body, fins, head, and teeth. for intensive culture there is a paucity of molecular studies for many grouper species. Taxonomy, economic importance, culture, produc- 1.2. Economic importance of the groupers tion, infectious diseases and its immune status in groupers belonging to the genera , Cromileptes, and will be Groupers are economically valuable, making up an important part summarized in this review. of the catch of sport and artisanal fishers throughout their distribution (Seng, 1998). About 40 species of groupers occur in the Philippines, where they are caught by small-scale fishermen with hook and line, 1.1. Taxonomy and geographical distribution bamboo traps, or dip net from estuaries and coral reefs (Kohno et al., 1988). Global grouper production increased dramatically in recent The and subfamily Epinephelinae, commonly years, with 60,774, 99,378, 163,093, and 198,690 mt in 1990, 2000, known as groupers, includes 159 species that come under 15 genera, 2005, and 2007, respectively (FAO, 2005a,b, 2009). Historically the which are distributed world-wide in the tropical and sub-tropical , E. morio constituted the most important finfish fishery in waters; species of the genus Epinephelus, , Mycter- the Mexican territorial waters within the Gulf of Mexico (Albanez- operca, and are present in the Atlantic Ocean and in the Lucero and Arreguín-Sánchez, 2009). Growth of the fishery has been Mediterranean sea (Maggio et al., 2005; Heemstra and Randall, 1993; observed from 1947 to 1972, with the highest yield of about Zhu and Yue, 2008; Boglione et al., 2009).Smith (1971) has given the 21,000 mt in the year 2000s (Burgos-Rosas and Pérez-Pérez, 2006). detailed account of the groupers whereas Heemstra (1991) has Since then, the wild stock appears to have been depleted; by 2004, the completed a taxonomic revision of fourteen species of groupers that yields were less than 6000 mt. Present stocks are about a third of occur in the eastern Atlantic Ocean and Mediterranean Sea. Randall those estimated in the early 70 s (Doi et al., 1981). In 1995, feral and Heemstra (1991) have revised the Indo-Pacific groupers consist- production of groupers reached 27,359 mt from the Philippines, ing of 110 species. Johnson (1983, 1988), Kendall (1984) and Leis Malaysia, Taiwan, and Thailand; while the total grouper production (1986) have been reported the composition and phylogenetic from the entire area yielded 1348 mt from brackish relationships of the family Serranidae. The FAO catalogue is most water aquaculture and 771 mt from mariculture (SEAFDEC, 1997). comprehensive guide to the worldwide distribution of groupers However there is an insatiable demand for groupers as luxury protein. (Heemstra and Randall, 1993). For instance in Spain, dusky grouper is highly appreciated by The RAPD, Polymerase Chain Reaction-Restriction Fragment consumers owing to the excellent properties of the meat. This species Length Polymorphism (PCR-RFLP), and Polymerase Chain Reaction- is frequently sold around 30–40 €/kg in the market place (Asensio Single Strand Conformation Polymorphism (PCR-SSCP) assays have et al., 2009). been used for identification of grouper species and taxonomy (Parenrengi et al., 2001; Govindaraju and Jayasankar, 2004; Asensio et al., 2001). Richardson and Gold (1997) have been developed 1.3. Grouper culture Mitochondrial DNA variations assay for grouper species identification. Further microsatellite studies also carried out for identification of Grouper culture was first introduced in the early 1970s in grouper populations (Stevenson et al., 1998; Nugroho et al., 1998; Singapore, Malaysia, Hong Kong, Thailand, and Taiwan, and now Sola et al., 1999; De-Innocentiis et al., 2001). occurs throughout Southeast Asia (Seng, 1998). In Thailand, grouper Five species of Epinephelus endemic to Eastern Atlantic are present farming appears to have begun in the 1980s in Southern Thailand, in the Mediterranean Sea; they are Epinephelus aeneus, E. caninus, E. although a small number of producers have been raising grouper for R. Harikrishnan et al. / Aquaculture 309 (2010) 1–14 3 up to 30 years. Globally there are about 20 species of groupers, which hatcheries in Taiwan (Chen, 1990). In Taiwan grouper aquaculture has are being commercially cultured mainly in Hong Kong, Taiwan, and been developing rapidly since 1991. The grouper industry in Taiwan the Southeast Asian region, usually by stocking juveniles collected consists of three interrelated sectors: fry production, grow out farm from the wild. At present, grouper production is essentially in the production, and marketing. The total farming area for grow out hands of small-scale producers, although there is interest in the production in Taiwan was 562 ha with an annual production of development of larger scale production like in the offshore systems in 4912 mt valued at 1,350,458,000 TD in 1998 (FACA, 1999). the region (Kongkeo and Phillips, 2002). Among 159 species of groupers distributed world-wide, the orange- 2. Disease status in grouper aquaculture spotted grouper/green grouper (E. coioides), (E. tauvina), malabia/ (E. malabaricus), brown-marbled/tiger grou- 2.1. Diseases per (E. fuscoguttatus), (E. lanceolatus), polka dot/ (Cromileptes altivelis), (E. aeneus), In the aquaculture industry grouper is widely used for intensive yellow/banded grouper (E. awoara), (E. merra), red farming (Boonyaratpalin, 1997; Heemstra and Randall, 1993). However, grouper (E. morio), red spotted grouper, (E. akaara), dusky grouper the intensive culture of grouper leads to the incidences of infectious and (E. marginatus), leopard grouper (), nassau non-infectious diseases (Chua et al., 1994; Fukuda et al., 1999)that grouper (E. striatus), and (E. tukula) are commonly result in serious economic losses (Lee, 1995; Yeh et al., 2008)bothin cultured around the world predominantly in net pens, cages, and ponds; farms and hatcheries. For example with the rapid development of the E. of these the majority culture systems are cage based, with the other awoara culture industry, infectious diseases caused by bacteria, viruses, systems employed to a lesser degree. and parasites are becoming more and more severe (Wang and Wu, 2007). Vibriosis is a common problem in the intensive culture farms of 1.4. Production and cost grouper (Rasheed, 1989; Liao et al., 1996). Viral pathogenic nodaviruses and iridoviruses are the causative agents of viral nervous necrosis and Perfection of the techniques for seed production and culture are sleepy disease in hatchery-reared larvae and juvenile groupers, essential for cost effective aquaculture and stock management (Yeh respectively (Chua et al., 1994; Fukuda et al., 1999). During larviculture, et al., 2003). It is estimated that the global grouper production was intracohort cannibalism usually results in mass mortality (Fukuda et al., about 10,000 mt in 2000 (Lupatsch et al., 2005). In Taiwan production 1999; Lim, 1993; Watanabe et al., 1996). Large scale seed production is of farmed grouper has increased more than two fold from 5052 mt in still encountering increasing difficulties, especially due to various 2001 to 12,103 mt in 2004 (Cheng et al., 2007). Among the orange- infectious diseases caused by virus, bacteria, and parasites. spotted grouper species, E. coioides is an important mariculture species in Asian countries including Southern mainland of China, Hong Kong, 2.2. Bacterial diseases and Taiwan. In Hong Kong mariculture of humpback grouper C. altivelis is at an experimental scale (Liu and Sadovy de Mitcheson). Groupers Grouper culture industry has been threatened by various diseases fetch a premium price of up to US$100/kg on wholesale in the Chinese due to intensive farming (Lee, 1995; Rasheed, 1989; Nash et al., 1987; live-fish markets of Hong Kong and Southern China (Laining et al., Wong and Leong, 1990; Mori et al., 1991; Leong and Wong, 1993; Chi 2003). Trash fish costs 15–20 Taiwan dollar (TD)/kg and market size et al., 1997; Lee et al., 2002). A number of bacterial diseases in grouper grouper command a farmgate price of 420–530 TD/kg in 1988 (Chen, aquaculture have been reported in the Asian Pacific region (Table 1). 1990). The orange-spotted grouper is an important marine fish cultured in southern China with an annual production of about Table 1 10,000 mt (Yin et al., 2006). In Eastern Asia improved hatchery Bacterial diseases in different countries reported in the groupers (Original). technology and a more reliable supply of hatchery-reared fry has Species Countries Host References resulted in a 10 fold expansion in grouper culture since the mid-1990's, with an annual production of more than 52,000 mt (FAO, 2005a,b). Vibrios Malaysia Epinephelus Palanisamy et al. (1999) Trash fish is presently the main food source for rearing grouper in the spp. ""E. salmoides Ong (1988) region but its decreasing supply, increasing cost and downstream ""E. malabaricus Leong and Wong (1988) environmental impacts pose a major constraint (Beveridge, 1996; """Nash et al. (1987) New, 1996; Williams, 2002). Even temperate countries like Japan and " China E. coioides Sun et al. (2009) Korea realizing the economic importance of groupers have expended " Philippines E. suillus Lavilla-Pitogo et al. (1992) " Italy E. marginatus Mesa et al. (2008) much effort to produce artificial feeds (Fukuhara, 1989). V. alginolyticus Korea E. malabaricus Lee (1995) " Thailand " Danayadol et al. (1995) 1.5. Grouper seed hatcheries V. carchariae Korea " Chi et al. (1997) " Taiwan Epinephelus Lee et al. (1999) Hatchery based large-scale commercial production of grouper seed sp. ""E. coioides Yii et al. (1997) is currently limited to Taiwan, where about 300 private hatcheries V. harveyi Kuwaiit E. tauvina Saeed (1995) annually produce nearly 20 million fry of E. coioides and E. malabaricus " India " Punitha et al. (2008); (Chu, 1996). Vagaries in production due to unpredictable and the low Chinabut (1996) level of survival often hike the sale price of fingerlings (Rimmer, 1999). V. parahaemolyticus Thailand E. malabaricus Danayadol et al. (1995) Pseudomonas spp. China E. coioides Sun et al. (2009) Further as yet, small-scale fry production methods for most groupers are " Malaysia E. tauvina Nash et al. (1987) not available (Sadovy, 2000), although techniques for the intensive " Italy E. marginatus Mesa et al. (2008) production is available for some groupers like the humpback grouper Acinetobacter spp. China E. coioides Sun et al. (2009) developed at the Gondol Institute for Mariculture, Bali (Sugama et al., Flexibacter spp. Thailand E. malabaricus Danayadol et al. (1995) 2001). The technology has also been successfully applied to the " India E. tauvina Punitha et al. (2008) " Malaysia " Nash et al. (1987) production of and is reportedly now being used in more Aeromonas spp. Italy E. marginatus Mesa et al. (2008) than 60 hatcheries in Indonesia, the majority of which are ‘back-yard’ Streptococcus spp. Thailand E. malabaricus Danayadol et al. (1995) facilities (Sugama, 2003). The annual market for grouper larvae is worth Photobacterimun dameselae Malaysia E. tauvina Mesa et al. (2008) more than US$300 million (Wang et al., 2010). In 1988, moderate subsp. piscicida " Japan E. akaara Muroga (1995) success has been reported in a number of commercial grouper 4 R. Harikrishnan et al. / Aquaculture 309 (2010) 1–14

Table 2 Protozoan, trematodes, nematodes, and parasites diseases in different countries reported in the groupers (Original).

Species Countries Host References

Protozoa Brooklynella sp. China Epinephelus sp. Chen (1996) Cryptobia sp. " " Chen (1996) irritans Indonesia E. bontoides Koesharyani et al. (1998, 1999) ""E. coioides Koesharyani et al. (1998, 1999) ""E. malabaricus Koesharyani et al. (1998, 1999) " Thailand Wong and Leong (1990); Leong and Yong (1988); Ruangpan and Tubkaew (1993); Danayadol (1999) " Malaysia " Wong and Leong (1990); Leong and Yong (1988) " China Epinephelus sp. Chen (1996); Luo et al. (2007) " Indonesia C. altivelis Koesharyani et al. (1998, 1999); Yuasa et al. (1999) " Kuwait E. tauvina Rasheed (1989) Riboscyphidia sp. Philippines E. coioides Cruz-Lacierda et al. (1999a,b) Scyphidia sp. Thailand E. malabaricus Chinabut (1996) Trichodina sp. Philippines E. coioides Cruz-Lacierda et al. (1999a,b) " Indonesia E. bontoides Koesharyani et al. (1999) " Thailand E. malabaricus Chinabut (1996) " Malaysia " Wong and Leong (1990); Leong and Yong (1988) Vorticella sp. Philippines E. coioides Cruz-Lacierda et al. (1999a,b)

Myxozoa Myxosoma sp. China Epinephelus sp. Chen (1996) Ceratomyxa sp. " " Chen (1996) Sphaerospora sp. Thailand E. malabaricus Chinabut (1996)

Microspora Pleistophora sp. China Epinephelus sp. Chen (1996)

Monogenea Benedenia sp. Indonesia E. malabaricus Koesharyani et al. (1998, 1999) " Thailand " Chinabut (1996) " Myanmar Epinephelus spp. Si Si Hla Bu (1999) " Kuwait E. tauvina Al-Marzouq and Al-Rifae (1994) " Singapore " Chua et al. (1993) B. epinepheli Japan E. akaara Ogawa et al. (1995a,b) ""E. moara Ogawa et al. (1995a,b) ""E. septemfasciatus Ogawa et al. (1995a,b) Cycloplectanum epinepheli Thailand E. malabaricus Chinabut (1996) sp. Philippines E. coioides Cruz-Lacierda et al. (1999a,b) " Thailand E. malabaricus Danayadol (1999); Danayadol and Direkbusarakom (1987) Gyrodactylus sp. " " Chinabut (1996) Haliotrema sp. Indonesia C. altivelis Koesharyani et al. (1998, 1999) Neobenedenia girellae Japan E. akaara Ogawa et al. (1995a,b) ""E. cyanopodus Ogawa et al. (1995a,b) " Indonesia E. bontoides Koesharyani et al. (1998, 1999) ""E. coioides Koesharyani et al. (1998, 1999) ""E. malabaricus Koesharyani et al. (1998, 1999) ""P. leopardus Koesharyani et al. (1998, 1999) " Myanmar Epinephelus spp. Si Si Hla Bu (1999) Megacotyloides epenepheli Malaysia E. malabaricus Leong and Yong (1988) Megacotyloides sp. Myanmar Epinephelus spp. Si Si Hla Bu (1999) Tareenia sp. " " Si Si Hla Bu (1999) Diplectanum sp. " " Si Si Hla Bu (1999) Pseudorhabdosynochus sp. Indonesia C. altivelis Koesharyani et al. (1999) " Myanmar Epinephelus spp. Si Si Hla Bu (1999) Pseudorhabdosynochus Malaysia E. malabaricus Leong and Yong (1988) epinepheli Thailand " Leong and Yong (1988) " Philippines " Leong and Yong (1988) F. Dactylogyridae " E. coioides Erazo-Pagador (1999) F. "" Erazo-Pagador (1999)

Trematoda Allopodocotyle sp. Malaysia E. tauvina Kolandasamy and Shaharom-Harrison (1999) Allopodocotyle serrani " E. malabaricus Leong and Yong (1988) Cardicola sp. Thailand " Chinabut (1996); Leong and Yong (1988) " Malaysia " Leong and Yong (1988) Ectenurus sp. Malaysia " Leong and Yong (1988) Gonapodasmius sp. Thailand " Chinabut (1996) Helicometrina nimia Malaysia " Leong and Yong (1988) Lecithochirium Thailand " Chinabut (1996) neopacificum Malaysia " Leong and Yong (1988) " Philippines " Leong and Yong (1988) Pearsonellum sp. Myanmar Epinephelus spp. Si Si Hla Bu (1999) Prosorhynchus pacificus Thailand E. malabaricus Chinabut (1996); Leong and Yong (1988) R. Harikrishnan et al. / Aquaculture 309 (2010) 1–14 5

Table 2 (continued) Species Countries Host References

Trematoda " Malaysia " Leong and Yong (1988) " Philippines " Leong and Yong (1988) Prosorhynchus sp. Malaysia E. tauvina Kolandasamy and Shaharom-Harrison, 1999 Prosorhynchus sp. Myanmar Epinephelus spp. Chinabut, 1996 " Malaysia E. malabaricus Leong and Yong (1988) Pseudopecoeloides spp. Thailand " Chinabut, 1996 " Malaysia " Leong and Yong (1988) Stephanostomum sp. " " Leong and Yong (1988) F. Didymozidae Philippines E. coioides Cruz-Lacierda et al. (1999a,b) " Myanmar Epinephelus spp. Si Si Hla Bu (1999)

Crustacea Caligus sp. Indonesia C. altivelis Koesharyani et al. (1999) " Thailand E. malabaricus Chinabut (1996) " Malaysia " Leong and Yong (1988) Ergasilus borneoensis " Leong and Yong (1988) Gnathia sp. Thailand " Chinabut (1996) Lepeoptheirus sp. Indonesia E. coioides Koesharyani et al. (1998, 1999) ""E. malabaricus Koesharyani et al. (1998, 1999) ""E. fuscoguttatus Koesharyani et al. (1998, 1999) ""P. leopardus Koesharyani et al. (1998, 1999) Thebius sp. Thailand E. malabaricus Chinabut (1996)

Nematoda Contracaecum sp. Malaysia " Leong and Yong (1988) Echinocephalus sp. " " Leong and Yong (1988) Raphidascaris sp. () Thailand " Chinabut (1996); Leong and Yong (1988)

Cestoda Tetraphyllidae Thailand " Chinabut (1996) " Malaysia " Leong and Yong (1988) Cestoda gen sp. Thailand " Leong and Yong (1988) (metacestode) Philippines " Leong and Yong (1988)

Acanthocephala Acanthocephalus sp. Malaysia " Leong and Yong (1988) " Thailand " Leong and Yong (1988)

Vibriosis, a causal agent of Vibrio carchariae and V. alginolyticus, is one attracting world-wide attention because of the damage it inflicts to of the most serious diseases and a common problem in various stages aquaculture. The parasite has been taxonomically reassigned a new mariculture including grouper culture (Yin et al., 2006; Egidius, 1987; family name Cryptocaryonidae (Wright and Colorni, 2002). Aquarium Li and Woo, 2003). Saeed (1995) reported the association of V. harveyi fishes, wild fishes and cultured species are included in the list of a wide with mass mortalities of greasy groupers. Extracellular proteases range of hosts of C. irritans. In addition, the fish hosts that could be produced by pathogenic Vibrio species isolated from diseased grouper infected by C. irritans inhabit bodies of water distributed world-wide, have been described as important virulent factors (Lee, 1995; Lee such as the Indo-Pacific, the Atlantic, the , the Red Sea and et al., 1999). Various Vibrio species have been demonstrated to be the Carribean Sea (Colorni and Burgess, 1997). Different strains or causative agents of disease in cultured marine fish in Japan (Kusuda isolates of C. irritans have been reported in groupers (Diggles and and Salati, 1993) and Taiwan (Liao et al., 1996). V. carchariae Adlard, 1997; Yambot et al., 2003). Recently, a low salinity variant of C. ( of V. harveyi) has been identified from diseased cultured irritans was identified infecting seabream Sparus sarba cultured in grouper (Lee and Yii, 1996) and has been characterized as a causative 5 ppt brackish water pond in Taiwan causing high mortality (Yambot agent of gastroenteritis in groupers (Yii et al., 1997). The pathogens V. et al., 2003). This finding has expanded the parasite habitat and the alginolyticus and V. carchariae are causative agents of vibriosis already broad range of hosts of C. irritans. The high value grouper like E. associated with a gastroenteritis syndrome (swollen intestine con- coioides and several other marine fish species grown commercially in taining yellow fluid) (Lee, 1995; Rasheed, 1989; Liao et al., 1996; Yii Taiwan are not spared from infection by C. irritans (Yambot and Song, et al., 1997). Recently, V. carchariae has been isolated and character- 2006). The parasite has become a frequent pest in commercial fish ized as a causative agent of gastroenteritis in groupers (Yii et al., farms and is considered as one of the most devastating parasites of 1997), but the pathogen caused mass mortality only after challenge marine fishes cultured in temperate and tropical (Wright and dosage. The pathogenic bacteria V. alginolyticus (Lee, 1995), V. Colorni, 2002; Colorni, 1987). Rasheed (1989) reported 50% mortality carchariae (Yii et al., 1997), Pseudomonas sp. (Nash et al., 1987), and of tank cultured juvenile brown-spotted grouper in Kuwait. Other Flexibacter sp. (Arthur and Ogawa, 1996) have also been reported in records of C. irritans infection include of a range of wild fishes in infected groupers. In addition, Arthur and Ogawa (1996) in an (Diggles and Lester, 1996) and cage cultured fishes in Japan overview of the disease problems in marine finfish culture in East and (Yoshinaga and Nakazoe, 1997). There have been reports that trash Southeast Asia showed that Streptococcus sp. is the causative agent of fish can be a source of infection (Yashiro et al., 1999), and that some red boil disease called as streptococcosis in grouper. diseases result from deficient nutrition involving opportunistic pathogens. Leong and Wong (1988) reported that the trematodes, 2.3. Parasitic diseases cestodes, nematodes, acanthocephalans, etc. recovered from cultured grouper were most probably transmitted through trash fish. A number The ciliate parasite Cryptocaryon irritans invades the skin, eyes, and of parasitic diseases have been reported in the grouper aquaculture in gills and causes white spot disease in marine fishes. The parasite is Asian Pacific region (Table 2). 6 R. Harikrishnan et al. / Aquaculture 309 (2010) 1–14

2.4. Viral diseases 2.4.2. Grouper iridovirus (GIV) Grouper iridovirus (GIV) is a large icosahedral cytoplasmic DNA In recent years, with rapidly developing marine farming activities, virus, of the family Iridoviridae, which includes five genera: Iridovirus, outbreaks of viral diseases have affected grouper aquaculture industry Chloriridovirus, Ranavirus, Lymphocystisvirus, and Megalocytivirus causing heavy economic losses (Wei et al., 2010). Correspondingly the (Lai et al., 2000; Williams et al., 2000, 2005; Chinchar et al., 2005; Tsai number of viral diseases in Asian Pacific region has increased steadily et al., 2005); these are associated with severe diseases, high with expansion and intensification of grouper aquaculture and trade mortalities, and heavy economic loss in ornamental fish and farmed (Table 3). Since 1994, viral nervous necrosis (VNN) disease has been food fish including grouper in Southeast Asia (Chua et al., 1994; Qin continuously reported in cultured groupers, and the highest mor- et al., 2002; Chang et al., 2002; Villena, 2003; Gibson-Kueh et al., 2004; talities (80–100%) always occur among 1-month and 2-month-old Kasornchandra and Khongpradit, 1995; Chou et al., 1998). World- larvae (Chi et al., 1997, 2001, 2003). Iridovirus and nervous necrosis wide Iridoviral pathogens have been identified as one of the most virus (nodavirus) are the two newly emerging viral pathogens important pathogens infecting more than 30 grouper species in the isolated and identified as the most important pathogen infecting last decade (Lü et al., 2001; Qin et al., 2003; Chou et al., 1998; Ahne grouper in the last decade. Singapore grouper iridovirus (SGIV), a et al., 1989; Hyatt et al., 2000; Piaskoski and Plumb, 1997; Wang et al., novel virus that comes under the genus Ranavirus, is one of the major 2007). The hosts include farmed grouper species, such as the cultured pathogens that have inflicted significant economic losses in grouper E. malabaricus (Danayadol et al., 1996) and E. tauvina (Chua et al., culture (Qin et al., 2003; Chang et al., 2002). The defense mechanisms, 1994) and several other important cultured fish like Pagrus major, particularly those against viruses are still poorly understood in Opelegnathus fasciatus, Caranx delicatissimus, and Seriola quinqeradia- groupers (Wei et al., 2010). tata (Nakai, 1996; Inouye et al., 1992; Oshima et al., 1996). Recent reports suggest that diseases in hatchery-reared groupers are due to viral infection (Chou et al., 1994; Chi et al., 2001, 2003; Chou et al., 2.4.1. Sleepy grouper disease (SGD) 1998). The virus caused more than 90% mortality in grouper (Qin There is a critical phase in grouper rearing between weaning and et al., 2003). Cases of iridovirus infection in farmed groupers were metamorphosis when the larvae are vulnerable to viral infection reported in Taiwan (Chou et al., 1994; Chi et al., 2003; Chou et al., adduced to insufficient nutrient ingestion (Chi et al., 1999a,b; Feng 1998; Chou et al., 1998). mortality due to iridovirus infections et al., 2008). Outbreak of a novel viral disease called ‘sleepy grouper ranged from 30% (adult) to 100% (fry). Members of the iridovirus disease (SGD)’ was first reported in the brown-spotted grouper in family can also infect a diverse range of invertebrates and poikilo- Singapore during 1994 (Chua et al., 1994). Recently SGD has caused thermic vertebrate hosts including insects, fish, amphibians, and significant economic losses in some marine net-cage farms in reptiles, causing a wide range of diseases (Williams, 1993). Systemic Singapore (Fukuda et al., 1999). iridovirus infections have also been reported in a wide variety of

Table 3 Viral diseases in different countries reported in the groupers (Original).

Diseases Countries Host References

Sleepy grouper disease Singapore E. tauvina Chua et al. (1994) Iridovirus-like infection Thailand E. malabaricus Danayadol et al. (1996) Singapore grouper iridovirus China E. akaara Huang et al. (2004, 2008, 2009) ""E. coioides Teng et al. (2008) " Singapore E. tauvina Shi et al. (2003); Chua et al. (1995) ""E. malabaricus Chua et al. (1995); Huang et al. (2004) Grouper iridovirus Taiwan E. coioides Yeh et al. (2008) ""E. awoara Lai et al. (2000, 2003, 2008) ""Epinephelus sp. Chu (1996); Chua et al. (1994) " Singapore E. coioides Qin et al. (2006); Xia et al. (2010) ""E. tauvina Qin et al. (2001) ""E. coioides Lü et al. (2001) " China " Lü et al. (2001) ""E. fuscoguttatus Wei et al. (2010) " Thailand " Khongpradit et al. (1999) Nervous necrosis virus Japan E. akaara Mori et al. (1991) ""E. moara Nakai et al. (1994); Muroga (1995) ""E. septemfasciatus Burgos-Rosas et al. (2006) " Thailand E. malabaricus Danayadol et al. (1995) " South Korea E. septemfasciatus Sohn and Park (1998) Grouper nervous necrosis virus Singapore E. coioides Chi et al. (1999a) ""E. tauvina Hegde et al. (2002) " Taiwan E. coioides Kai et al. (2010); Lin et al. (2006, 2007) " Philippines E. fuscogutattus Pakingking et al. (2010) " Japan E. septemfasticatus Chew-Lim et al. (1992); Fukuda et al. (1999) Reovirus Singapore Plectropomus maculates Chew-Lim et al. (1992) Ranavirus " E. tauvina Qin et al. (2002, 2003) Nodavirus " E. coioides Qin et al. (2006) " Taiwan " Cheng et al. (2007) " " Wen et al. (2008); Kai and Chi (2008) Megalocytivirus " " Wen et al. (2008) Infectious pancreatic necrosis " Epinephelus sp Lee et al. (1999) SJNNV Singapore E. tauvina Chew-Lim et al. (1992) Astro-like virus Sumatra " Arthur and Ogawa (1996) Lymphocystis Malaysia E. fuscogutattus Oseko et al. (1999) Herpes virus China E. awoara Chen (1996) R. Harikrishnan et al. / Aquaculture 309 (2010) 1–14 7 freshwater and marine food fish (Gibson-Kueh et al., 2003; He et al., 2.4.6. Grouper nervous necrosis virus (GNNV) 2000; Jung and Oh, 2000; Murali et al., 2002). Grouper nervous necrosis virus (GNNV) causes mass mortality, near 100% in larvae and juveniles, which has great economic impact 2.4.3. Singapore grouper iridovirus (SGIV) on the aquaculture of the marine fish. However, VNN has been a Singapore grouper iridovirus (SGIV) is a major pathogen resulting serious problem in the hatcheries and aquaculture of these grouper. in heavy economic loss to grouper Aquaculture (Xia et al., 2010). Chi et al. (1999a,b) showed, grouper nervous necrosis virus (GNNV) Singapore grouper iridovirus (SGIV), a novel Ranavirus, was isolated caused 100% mortality within 3 days in grouper larvae. from diseased grouper in Singapore. The virus has been isolated and characterized as a novel member of the family iridoviridae based on morphological, biochemical, and genetic properties, and tentatively 2.4.7. Viral nervous necrosis virus (VNNV) named as SGIV (Qin et al., 2001; Chang et al., 2002). The definitive The annual production of grouper was about 10,000 mt from years symptom of iridoviral infection is the formation of inclusion body- 2000 to 2004 and it is on the top-ten highest yielding aquaculture bearing cells in infected organs. They are icosahedral double stranded species in Taiwan (Liao, 2005). However in recent years, mass DNA viruses and the virions consist of an envelope, a capsid, and an mortalities of hatchery-reared grouper larvae and juveniles of grouper internal lipid membrane. The capsid is isometric and has a diameter of in Taiwan and other countries have frequently occurred due to 125–300 nm and consists of probably 72 capsomers (ICTVdB nervous necrosis viral (NNV) disease, and these have produced severe Management, 2006; www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/). Piscine economic impacts on the aquaculture industry (Wang et al., 2010). iridoviruses are classified either under genus Lymphocystivirus, However, the industry also faces heavy losses due to various disease Ranavirus, or Megalocytivirus. Nakajima and Kurita (2005) further problems including bacterial, viral, and parasitic diseases (Wong and classified megalocytiviruses into three major groups by phylogenic Leong, 1990; Lee and Yii, 1996; Yii et al., 1997; Freedman, 1991). analysis of the major capsid protein (MCP): red sea bream iridoviruses Viral nervous necrosis (VNN) caused by nervous necrosis virus (RSIV), infectious spleen and kidney necrosis iridoviruses (ISKNV), (NNV) is one of the most important viral diseases that cause mass and turbot reddish body iridoviruses (TRBIV). Iridoviruses have been mortality in more than 39 marine fish species in 10 families (Chua isolated and identified from at least 50 fish species resulted in et al., 1994; Chi et al., 1997, 1999a,b; Lai et al., 2003; Hegde et al., significant economic losses in grouper aquaculture in China and 2002; Munday et al., 2002; Mori et al., 1992; Liao, 2005; Freedman, Southeast Asian countries (Wang et al., 2007). SGIV was first isolated 1991; Munday and Nakai, 1997; Nakai et al., 2009; Renault et al., and identified from the brown-spotted grouper and malabar grouper, 1991a,b; Grotmol et al., 1997; Bovo et al., 1999; Cutrín et al., 2007; and was characterized as a novel ranavirus (Qin et al., 2001, 2006). In Aranguren et al., 2002; Comps et al., 1994). Recently mass mortality of Taiwan, epizootic iridovirus outbreaks have occurred in several VNN in hatchery-reared grouper larvae and juveniles has been important farmed fish such as grouper (Epinephelus spp.), giant reported in Taiwan (Oshima et al., 1996). VNN otherwise known as seaperch (Lates calcarifer), and red seabream P. major resulting in viral encephalopathy and retinopathy (VER) is one of the most significant economic losses (Lai et al., 2000; Chou et al., 1998; Chao important viral diseases in aquaculture in many countries. VER exhibit et al., 2002; Wang et al., 2003). an array of neuropathological signs characterized by necrosis and extensive cellular vacuolation of the brain, spinal cord, and retina, 2.4.4. Megalocystiviruses abnormal swimming behavior, and dark body coloring (Chi et al., Megalocystiviruses are well-known causative agents of many 1997; Mori et al., 1992; Comps et al., 1996). For the last two decades serious systemic diseases that have been reported in many econom- the disease continued to inflict serious economic losses among high ically important freshwater and marine farmed and feral fishes world- value marine fish species primarily at the larval and juvenile stages wide. infected by the megalocystivirus include brown-spot (Chi et al., 1997; Weng et al., 2002; Munday et al., 2002; Munday and grouper (Chua et al., 1993), malabar grouper (Danayadol et al., 1996), Nakai, 1997; Nakai et al., 2009; Renault et al., 1991a,b; Grotmol et al., grouper, Epinephelus sp. (Chou et al., 1998), and other cultured fin 1997; Weng et al., 2002; Jung and Oh, 2000; Chen et al., 2007; Shi fishes like red seabream, P. major (Inouye et al., 1992); seabass, et al., 2004; Mori et al., 1992; Liao, 2005; Freedman, 1991; Comps Lateolabrax sp. (Nakajima and Sorimachi, 1995); angelfish, Pterophyl- et al., 1994, 1996; Cutrín et al., 2007; Aranguren et al., 2002; Shieh and lum scalare (Rodge et al., 1997); tilapia, Oreochromis niloticus Chi, 2005; Muroga, 2001; Yoshikoshi and Inoue, 1990; Bloch et al., (McGrogan et al., 1998); mandarin fish, Siniperca chuatsi (He et al., 1991; Breuil et al., 1991; Nakai et al., 1995; LeBreton et al., 1997). 2000); African lamp eye, Aplocheilichthys normani (Sudthongkong The causal pathogen of VNN disease has been characterized as a et al., 2002); dwarf gourami, Colisa lalia (Sudthongkong et al., 2001); small, non-enveloped, bi-segmented, single-stranded, positive sense red drum, Sciaenop socellata (Weng et al., 2002); rockbream, RNA virus with a diameter of 25±30 nm (Chi et al., 2001; Mori et al., Oplegnathus fasciatus (Jung and Oh, 2000); large yellow croaker, 1992; Comps et al., 1994) belonging to the genus Betanodavirus, Larimichthys crocea (Chen et al., 2007); and turbot, Scophthalmus family Nodaviridae (Mori et al., 1992). Betanodaviruses are small, maximus (Shi et al., 2004). spherical, nonenveloped viruses with a genome consisting of two single-stranded RNA segments with a diameter of 25±30 nm: RNA1 2.4.5. Nodavirus and Betanodaviruses (3.1 kb), which encodes the RNA-dependent RNA polymerase Piscine nodavirus, a member of the Betanodavirdae family, is the responsible for the replication of the genome, and RNA 2 (1.4 kb), causative agent of viral nervous necrosis or fish encephalitis that which encodes the capsid protein that builds up the icosahedral cause severe epizootic outbreaks resulting in mass mortality and capsid into which the genome is packaged (Chi et al., 2001; Comps economic losses in farmed marine fishes in Taiwan as well as other et al., 1994; Schneemann et al., 2004). Moreover, a third RNA segment, Southeast Asian countries (Chen et al., 2007). Betanodaviruses RNA3 (0.4 kb), sub-genomically transcribed from RNA1, encodes a including nodavirus are neuropathogenic and inflict conspicuous polypeptide of unknown function. RNA3 is present only in the damage characterized by the formation of vacuoles and degeneration infected cells and is not packaged into the viral particles (Mori et al., of neurons throughout the central nervous system (Chen et al., 2007). 1992; Shieh and Chi, 2005; Renault et al., 1991a,b; Grotmol et al., Nodavirus is unenveloped with a 25–30 nm diameter icosahedral 1997; Cutrín et al., 2007; Aranguren et al., 2002; Munday et al., 2002; capsid (25–30 nm in diameter). This viral infection produces high Muroga, 2001; Yoshikoshi and Inoue, 1990; Bloch et al., 1991; Breuil mortalities in hatchery-reared larvae and juvenile marine fish (Munday et al., 1991; Nakai et al., 1995; LeBreton et al., 1997; Schneemann et al., 2002). The genome is composed of bipartite, single-stranded, et al., 2004; Nishizawa et al., 1995; Sommerset and Nerland, 2004; and positive sense RNA molecules (Mori et al., 1992). Iwamoto et al., 2005). 8 R. Harikrishnan et al. / Aquaculture 309 (2010) 1–14

The VNN threatens hatchery production of larvae and juveniles of preliminary results of in vivo temperature effect on GNNV infection marine fish world-wide. In addition, the number of fish species affected also indicated that, within the permissive temperatures for viral by VNN disease has rapidly increased (Mori et al., 1991; Nakai et al., infection, lower temperature will delay the outbreak of mass 1994; Fukuda et al., 1996; Munday et al., 2002; Renault et al., 1991a,b; mortality. It is widely recognized that a high mortality rate attributed LeBreton et al., 1997; Nguyen et al., 1994). Grouper (Epinephelus spp.) to VNN disease usually occurs in summer (Chi et al., 1997; Fukuda are commercially highly valued fish species in Asian aquaculture. Due to et al., 1996; LeBreton et al., 1997; Arimoto et al., 1994). their large size (30–60 kg per fish) and the high market value (≥USD Studies published to date indicate that NNV viral production in 30,000 per fish) of brood fish in some species of groupers, it is very vitro is poor or ablated at temperatures above 36 °C or below 20 °C; challenging to ask the farmers to segregate or eliminate the NNV carrier- GNNV propagated in GBC1 cells between 15 °C and 37 °C. Thus, both spawners. To date, NNV-induced heavy mortalities have resulted in host cell and viral genotype appear to influence the optimal complete destruction of fish population and reoccurrence of VNN at the temperature for viral propagation. Oh et al. (2006) reported that fatal early stage of groupers (Chi et al., 2003; Munday et al., 2002). The fish iridovirus infection occurred among turbots (Psetta maximus) reared survived VNN acute infection, usually become persistently infected, and between 22 °C and 25 °C, whereas no infection was observed among the viral persistence can extend as long as 1 year (Johansen et al., 2004; turbots reared between 17 °C and 20 °C. GSIV-K1 propagated at the Chi et al., 2005). temperatures, between 15 and 30 °C in GBC4, lower than GIV which All the NNVs can be classified into one of the four following proliferate in GB at the temperatures between 24 and 32 °C (Lai et al., genotypes based on partial sequences of the coat protein genes: 2003). Although there is some variation in temperature requirement SJNNV in striped jack NNV, RGNNV in red spotted grouper NNV, (Lai et al., 2003; Wang et al., 2003; Jung and Oh, 2000), iridovirus TPNNV in tiger puffer NNV, and BFNNV in barfin flounder NNV propagation and disease outbreak appear optimal between 25 °C (Nishizawa et al., 1997). The NNVs discovered in Taiwan, including and 30 °C. grouper NNV (GNNV) and NNV (YGNNV) have the RGNNV genotype (Lai et al, 2003; Chi et al., 2003). The virions were 3. Diagnostic features isolated and identified by RT-PCR as a fish nodavirus, designated GNNV (Chi et al., 1997). In order to amplify GNNV in vitro, the cell line 3.1. Techniques GF-1 was developed from the fin tissue of a grouper, E. coioides (Chi et al., 1999a), and was used to study the biochemical and biophysical Diagnostics and research should provide services to farmers to properties of GNNV (Chi et al., 2001). The SSN-1 cell line was derived assist in disease management to solving aquatic animal health from the whole fry tissue of Southeast Asian striped snakehead problems so as to sustain farm productivity. Farmers should be (Ophicephalus striatus) and proved to be persistently infected with a trained to understand the importance and value of diagnostic details C-type retrovirus, snakehead retrovirus (SnRV) (Frerichs et al., 1991). in determining the course or nature of a disease outbreak, providing Notably, this cell line is permissive to NNV infection and has been accurate and rapid diagnosis, and enabling sensible management employed for NNV isolation and amplification (Frerichs et al., 1996). decisions for intervention and control. Fish nodaviruses amplified in SSN-1 cells typically have a high titre. Conventional methods for pathogen screening and disease Thus, SnRV may perform a significant role in fish nodavirus replication diagnosis include tissue culture and electron miscroscopy for viruses, in SSN-1 cells (Iwamoto et al., 2000). isolation, and serology for bacteria, necropsy using light microscopy Outbreaks of VNN in fish hatcheries have apparently declined over for parasites, and histopathology for understanding the mechanism of the past years with the advent of control procedures such as disease. Considerable progress has been made in the development of elimination of virus-carrying brood fish and disinfection of fertilized immunoassays and DNA-based diagnostic methods, such as immu- and rearing water (Bloch et al., 1991; Breuil et al., 1991; Renault nohistochemistry (IHC) (LeBreton et al., 1997), indirect enzyme- et al., 1991a,b; Nakai et al., 1995; LeBreton et al., 1997; Schneemann linked immunosorbent assay (ELISA) (Shieh and Chi, 2005; Arimoto et al., 2004; Nishizawa et al., 1995; Sommerset and Nerland, 2004; et al., 1992), in situ hybridization (Comps et al., 1996), reverse Iwamoto et al., 2005; Nakai et al., 1994; Nguyen et al., 1994; Johansen transcription-polymerase chain reaction (RT-PCR) (Nishizawa et al., 2004; Chi et al., 2005; Nishizawa et al., 1997; Frerichs et al., et al., 1995; Dalla Valle et al., 2005), fish cell line (Chi et al., 1991, 1996; Iwamoto et al., 2000; Mushiake et al., 1994; Mori et al., 1999a,b), and the microfluidic chip method, nucleic acid sequence 1998; Watanabe et al., 2000; Grotmol and Totland, 2000; Tsuchihashi amplification (NASBA) (Starkey et al., 2004). Most of these methods are et al., 2002). Serious outbreaks of VNN reported in grouper, red time-consuming and require expensive instruments and reagents. Thus, spotted grouper, orange-spotted grouper, and brown-marbled and an effective affordable diagnostic method is required for disease other marine fish species such as sea bass during the net-cage culture prevention and control. The current “gold standard method” to detect in the open sea (Mori et al., 1991; Fukuda et al., 1996; LeBreton et al., NNV uses conventional RT-PCR (Nishizawa et al., 1995) but there are 1997). The exact transmission mode of betanodaviruses in fish at the still some disadvantages, such as the need to use a thermal cycler for the grow out stages remains vague although wild fishes have been reaction, the multiple and complex operation process and low speculated as reservoirs or carriers of the virus (Gomez et al., 2004; amplification efficiency (Starkey et al., 2004; Tomita et al., 2008). The Barker et al., 2002). VNN occurs in the larvae of many other cultured vertical transmission may be controlled or even prevented by screening fish species, such as the Japanese parrotfish, O. fasciatus (Yoshikoshi of infected spawners by either ELISA (Breuil et al., 2001; Arimoto et al., and Inoue, 1990), , L. calcarifer (Glazebrook et al., 1990), 1992) or PCR (Mushiake et al., 1994). European seabass, Dicentrarchus labrax (Breuil et al., 2001), and Betanodavirus has been detected in the gonad and the eggs of turbot, S. maximus (Bloch et al., 1991). striped jack Pseudocaranx dentex spawners by RT-PCR (Mushiake Mass mortalities associated with VNN have been reported during et al., 1994; Arimoto et al., 1992) and by fluorescent antibodies summer in several species of grouper and seabass, and water (Nguyen et al., 1994). However, betanodaviruses are not always temperature during mass mortalities in these reports ranged from detectable in the gonad, but can become detectable after stressful 25 to 28 °C; however, the mortality ceased when the temperature procedures of repeated spawning (Arimoto et al., 1992). Similar mode decreased to 23 °C or lower (Mori et al., 1991; Fukuda et al., 1996; of transmission has been detected in seven band grouper E LeBreton et al., 1997). In Taiwan, VNN disease repeatedly occurred septemfasciatus, Atlantic halibut Hippoglossus hippoglossus, barfin from April to September, and the peak of mortalities was recorded in flounder Verasper moseri, and also European seabass Dicentrarchus summer (June to August) when the average temperature was around labrax (Bloch et al., 1991; Renault et al., 1991a,b; Nakai et al., 1995; 30–32 °C; mortalities have gradually decreased since October. The Iwamoto et al., 2005). R. Harikrishnan et al. / Aquaculture 309 (2010) 1–14 9

Use of DNA-based methods for diagnosis of important diseases and interferon I and Mx (Lin et al., 2006; Lu et al., 2008; Wu and Chi, 2006). development of a PCR based technology to identify viral encephalop- Wu and Chi (2006) revealed that Mx is expressed in the cell line isolated athy and retinopathy or VER is recommended (Walker and Sub- from barramundi brain (BB), which displays persistent NNV infection; asinghe, 2000). In Japan, PCR is used for detecting VNN infection in whereas the NNV-free cell line cBB lacks Mx expression. Lin et al. (2006) spawners and eggs of striped jack. In Korea, it is used for confirming showed that resistance to NNV was partially recovered after transfection VNN infection of sevenband grouper. Other countries such as of Mx gene into GB3 cells, which are NNV-permissive and express low Thailand, Chinese Taipei, and Singapore use tissue culture methods levels of Mx. In addition, Chi et al. (1999a) demonstrated that GF-1 cells and electron microscopy for diagnosis of viral infection. Rapid were GNNV resistant until transformed. It is possible that these cells lose detection and diagnosis are required for important diseases such as the ability to express Mx upon transformation. In this regard, Chen et al. those currently affecting grouper. Early detection of disease is (2008) demonstrated that Mx gene-transfected GF-1 cells have limited important so that rational decisions for management, intervention viral propagation, due to the observed binding of Mx to the viral coat or control can be made. protein. Mx expression was shown to decrease viral titers by

approximately 2 log TCID50 (Lin et al., 2006; Chen et al., 2008). 3.2. Antibodies However, it is possible that antiviral mechanisms other than Mx expression may function in GBC4 cells because viral propagation Rabbit polyclonal antibodies sensitive and specific to SGIV and in these cells is even lower than in those of Mx constitutively expressing GNNV were produced for grouper (Table 4). The antigenic properties cells. of SGIV were first characterized using various immunoassays. The grouper iridovirus antigens could be rapidly and specifically detected 4. Immunology from infected cell cultures and virus challenged fish. The techniques and results described the identification and classification of the viral The nonspecific immune system is the first line of defense against pathogen will also facilitate further development of diagnostic invading organisms while specific immune system second line of techniques and effective vaccines. immune system and also affords protection re-exposure to the same pathogen. These immune systems are able to resist the infectious 3.3. Cell lines agents. Since groupers have received recent attention as candidate species for aquaculture disease management strategies are in infancy. Isolation of GNNV from moribund grouper larvae in the GF-1 cell Chemotherapeutics and antibiotics are less favored due to bioaccu- line and the effect of temperature on GNNV infection in that cell line mulation and emergence of resistant strains in the environment and and in grouper larvae were studied; purification of virus from several restrictions imposed on imported food against the residues moribund grouper (E. fuscogutatus and E. akaara) juveniles, was like formalin and malachite green (Harikrishnan et al., 2009). Some of identified as fish nodavirus, and subsequently named as GNNV 9410 the chemicals are being established to evoke positive immune strain (Chi et al., 1997). Almost all the viral agents belong to the response (Harikrishnan et al., 2010). In groupers there are a few groups of RSIV or ISKNV (unpublished data; personal communication studies on vaccines. Seven days post-vaccinated larvae of E. coioides with Dr. Chao Institute for Animal Disease Prevention and Control, had specific immune response after challenge with anti-NNV VP Kaohsiung, Taiwan); only a few are ranaviruses (Tsai et al., 2005; specific antibodies and achieved a relative percentage survival (RPS) Murali et al., 2002). Although many fish cell lines have been of 64.2% and 69.5%, respectively (Lin et al., 2007). RPS in E. coioides established, relatively few have the ability to propagate betanoda- after bath immunization for thirty days with binary ethyllenimine or viruses or iridoviruses; a few cell lines (Lai et al., 2003; Qin et al., 2006; formalin-inactivated betanodavirus was 79–95% and 39–43%, respec- Chi et al., 1999a; Lai et al., 2003; Imajoh et al., 2007; Nakajima et al., tively (Lu et al., 2008). E. coioides acquired protective immunity 1997; Chi et al., 1999b) are capable of viral propagation but produce through intraperitoneal injection with formalin-killed C. irritans the viruses slowly and yield low viral titers. Thus it has become crucial theronts at 100 μg/fish; after three weeks no mortalities were to develop suitable cell lines not only for vaccine development but observed while 40% mortality was recorded at a dose of 10 μg/fish also for isolation and study of the viruses. with higher antibody titres in the mucus (Yambot and Song, 2006). In The snakehead cell line SSN-1 was the first fish cell line used to E. lanceolatus after immunization with virus-like particles of GNNV for isolate, cultivate, and characterize betanodaviruses (Frerichs et al., 3 weeks resulted in maximum antibody titres by second booster with 1996). However, SSN-1 is persistently infected with a C-type retrovirus. a RPS of 38% at 10 μg dose and 29% at 100 μg(Liu et al., 2006). The To study VNN disease, the NNV-permissive cell lines GF-1, GB3, GS, and highest neutralizing antibody titres were found in E. coioides with GB were established from the fin, brain, and spleen of orange-spotted intramuscular immunization with NNV vaccine after 5 months (Kai grouper and from the brain of yellow grouper (Lai et al., 2000; Chi et al., et al., 2010). In E. fuscoguttatus immunization with formalin-killed 1999a; Lin et al., 2006). Unfortunately, all of these cell lines produced VNN vaccine significantly enhanced the survival rate and resulted in a 9 −1 low viral titers (no more than 10 TCID50 ml ), with the exception of 3 fold drop in neutralizing antibody titers of after 15 and 75 days post- the E-11 clone of SSN-1 that has been shown to produce titers of up to vaccination (Pakingking et al., 2010). −1 109.6 TCID50 ml . Therefore, the GBC1 cell line, which yielded a GNNV In E. malabaricus dietary supplementation with high DHA/EPA 10 −1 titer above 10 TCID50 ml , provides improved efficiency and ratio significantly enhanced the phagocytic and respiratory burst economy for viral production. Propagation of GNNV in GBC4 cells was activities of heat-kidney leucocytes when compared to the diet with poor and typically generated persistent infection without any obvious low ratio (Wu et al., 2003). E. awoara immunized with formalin-killed CPE. Persistent infection with NNV results from the induction of both V. vulnificus vaccine after treatment with vitamin C at doses of 1500 and 2000 mg/kg for 20 weeks afforded effectively protection ensuring 100% survival rate (Qin et al., 2000). In juvenile leopard grouper, Table 4 Mycteroperca rosacea fed with diet containing live yeast of Debar- Development of antibodies for grouper viral diseases (original). yomyces hansenii after 4 weeks promoted a high survival rate and Disease Host References significantly increased IgM as well as antioxidant enzymes such as SGIV E. tauvina and E. malabaricus Shi et al. (2003) SOD and CAT (Martha et al., 2008). In E. malabaricus diet with NCuHSe " Epinephelus sp. Qin et al. (2002) was enhanced the leucocytes respiratory burst activity and plasma " E. coioides Chang et al. (2002) total immunoglobulin concentration than diet fed with NCuNSe GNNV In vitro Lai et al. (2002) containing diet for 8 weeks (Lin and Shiau, 2007). In E. malabaricus 10 R. Harikrishnan et al. / Aquaculture 309 (2010) 1–14 feeding diet enriched with L-ascorbic acid at 288 mg/kg for 8 weeks the financial assistance through KOSEF Postdoctoral Fellowship and had higher respiratory burst activity and alternate complement BK 21 program of the Ministry of Education, South Korea, which made activity inducing disease resistance to V. carchariae (Lin and Shiau, this work possible. CB is grateful to UGC-SAP and DST-FIST for the 2005). E. malabaricus fed with vitamin-E enriched diet had higher facilities made available and the invitation to visit Marine Biomedical WBC, leucocytes respiratory burst activity, plasma lysozyme, and Laboratory, Jeju National University, South Korea under BK21 alternate complementary activity after 2 weeks (Lin and Shiau, 2005). program for discussions. In E. coioides fed with diet containing L. plantarum when challenged with Streptococcus sp. and iridovirus suffered minimum mortality of 20.0% and 36.7%, respectively; the challenged group also had References significantly higher complement, lysozyme, and glutathione activities Ahne, W.H.J., Schlotfeldt, H.J., Thomsen, I., 1989. Fish viruses: isolation of an icosahedral after 4 weeks (Chen et al., 2008). E. coioides fed with diet enriched cytoplasmic deoxyribovirus from sheatfish (Silurus glanis). J. Vet. Med. 36, with sodium alginate 20 mg/kg for 24 and 72 h and subsequently 333–336. received iota-carrageenan enriched diet after 72 and 120 h showed Albanez-Lucero, M.O., Arreguín-Sánchez, A., 2009. Modelling the spatial distribution of fi red grouper (Epinephelus morio) at Campeche Bank, México, with respect substrate. signi cantly increased respiratory burst SOD, and phagocytic activi- Ecol. 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(Eds.), lin-killed viral and parasitic vaccines increased survival rates; dietary Instituto Nacional de la Pesca, Secretaria de Agricultura, Ganadería, Desarrollo administration of DHA/EPA, L-ascorbic acid, vitamin C and E, sodium Rural, Pesca y Alimentación, México, pp. 503–522. alginate, κ-carrageenan, yeast, probiotics, and herbals also signifi- Chang, S.F., Ngoh-Lim, G.H., Kueh, L.F.S., Qin, Q.W., Seng, E.K., Sin, Y.M., 2002. initial fi cantly increased the specific and non-specific immune response investigations into two viruses isolated from marine food sh in Singapore. Vet. Rec. 150, 15–16. affording protection from diseases. This review could be useful in the Chao, C.B., Yang, S.C., Tsai, H.Y., Chen, C.Y., Lin, C.S., Huang, H.T., 2002. A nested PCR for treatment of different fish diseases. 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