REVIEW OF LITERATURE

Transcription

1 REVIEW OF LITERATURE

2 2. REVIEW OF LITERATURE The past three decades have witnessed remarkable expansion, intensification and diversification of the aquaculture sector which has become enormously reliant on external inputs through movements of live aquatic animals and animal products (broodstock, eggs, fry/fingerlings, seed, and feed). Asian aquaculture advanced from a traditional practice to a science-based activity and developed into a significant food production sector, contributing to national economies and providing better livelihoods for rural and farming families. Increasing world trade liberalization and globalization as well as improved transportation efficiency contributed to a great extent for the farmer to be part of a production chain for the delivery of the safe and high quality products to the end users. The aquaculture sector has become a key supplier of aquatic food, provider of direct and indirect employment, and a great source of foreign trade earnings. However, the exponential growth of commercial shrimp farming operations has become a potential cause of many problems. Over exploitation of brood stock animals is one of the important issues. In addition, the expansion of shrimp culture is accompanied by local environmental degradation and the occurrence of diseases of both infectious and non-infectious etiologies (Lightner et al., 1992). Disease outbreaks, mainly caused by viruses and bacteria and to a lesser extent by rickettsiae, fungi and parasites, may cause losses up to 100% (Johnson, 1989; Lightner et al., 1992; Lightner and Redman, 1998). So far, about 20 viruses causing infections of shrimp have been described. White spot syndrome virus (WSSV) has had the greatest impact on shrimp culture and continues to be the most important disease problem in shrimp culture (Rosenberry, 2001). Other important viruses are infectious hypodermal and haematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), baculoviral midgut gland necrosis (BMN) virus, baculovirus penaei (BP), yellow head virus (YHV), monodon baculovirus (MBV), lymphoid organ vacuolization virus (LOVV) and Taura syndrome virus (TSV) (Lightner, 1996).

3 2.1 Viral agents of disease in shrimps Baculovirus penaei (BP) Baculovirus penaei (BP), a crustacean baculovirus was first discovered in 1974 in the pink shrimp, Penaeus duorarum (Couch 1974a, b). This virus has been reported to cause significant mortalities in the larval, postlarval and early juvenile stages of several penaeid species (Couch 1981, 1991; Johnson 1984; Lightner 1988). BP has been sporadically occurring, but serious hatchery disease of the larval stages of P. vannamei was reported in many of the commercial hatcheries on the pacific coast of Central and South America, including Peru, Ecuador, Columbia, Panama, Costa Rica and Honduras, in P. aztecus and introduced P. vannamei in hatcheries in Texas and Florida in the Gulf of Mexico Coast and in several naïve species in Brazil (Couch 1981; Lightner and Redman 1991). In Mexico, BP has caused serious epizootic in cultured larval and postlarval P. stylirostris (Lightner et al., 1989). In susceptible species, BP infection is characterized by a sudden onset of morbidity and mortality in larval and postlarval stages. The symptoms include decline in the rate of growth, cessation of feeding, lethargy and show signs of epibiont fouling (due to reduced grooming activity). The virus attacks the nuclei of hepatopancreas epithelia but can also infect midgut epithelia. Typical sources of infection of shrimp larvae include fecal contamination of spawned eggs from BP infected adult spawners (Johnson and Lightner 1988; Lightner 1996), fecal, oral contamination through feces from infected larvae or from cannibalism of diseased larvae (Overstreet et al., 1988; LeBlanc and Overstreet, 1990, 1991). BP infection can be diagnosed by the demonstration of prominent tetrahedral occlusion bodies in unstained squash preparation of hepatopancreas, midgut, or faeces or in appropriate histological sections from infected animals wherein single or multiple, eosinophilic usually triangular inclusion bodies within hypertrophoid nuclei of hepatopancreas or midgut epithelial cells are observed (Lightner et al., 1992). Polyhedral occlusion bodies occur only during advanced stages of infection (Bower et al., 1994).

4 2.1.2 Monodon Baculovius (MBV) Monodon baculovirus (MBV), a nuclear polyhedrosis virus (NPV) of the family Baculoviridae, was first reported in P. monodon shrimp in Taiwan (Lightner and Redman, 1981). As with all NPVs, it possesses a double stranded circular DNA genome of x10 6 Da with a rod shaped enveloped particle often found occluded within proteinaceous bodies. The latter is composed primarily of the protein polyhedrin (Rohrmann, 1986). MBV has been implicated in the collapse of the Taiwanese shrimp farming industry in (Lin, 1989). Similar catastrophic mortalities due to MBV have been reported in Mexico (Lightner et al., 1984), Malaysia (Nash et al., 1988) and Thailand (Thikiew, 1990). MBV like baculovirus have been described for P. monodon, P. merguiensis, P. penicillatus, P. plebejus, P. esculentus, P. semisulcatus, P. kerathurus, P. vannamei, and P. indicus (Johnson and Lightner 1988; Lightner 1988; Chen et al., 1989a; Vijayan et al., 1995; Karunasagar et al., 1998b). In Australia MBV has been reported in cultured P. monodon and wild P. merguiensis (Doubrovsky et al., 1988). Plebejus baculovirus, an MBV-like virus was described from cultured P. plebejus (Lester et al., 1987) and MBV-like virus from Metapenaeus ensis, which is cultured in Taiwan (Chen et al., 1989a, b). The target organs of MBV are hepatopancreas and anterior midgut (Lightner et al., 1983b). The presence of hypertrophoid nuclei with single or multiple spherical occlusion bodies is the principal diagnostic feature of MBV infection (Lightner et al., 1983b; Fegen et al., 1991). Direct staining with malachite green and conventional histopathology was used initially to detect MBV (Lightner and Redman, 1991). However, rapid molecular methods like PCR (Vickers et al., 1992; Chang et al., 1993; Lu et al., 1993) and genomic probes for MBV detection by DNA hybridization (either in situ or dot blot) have been developed (Vickers et al., 1993; Poulos et al., 1994b). Enzyme linked immunosorbent assay for MBV has also been described by Hsu et al. (2000). Satidkanitkul et al., (2005) recently purified polyhedrin from MBV and sequenced 25 amino acids at the N-terminus. The sequence MFDDSMMMENMDDLSGDQKMVLTLA did not correspond to the portion of the MBV polyhedrin protein reported from Taiwan (Chang et al., 1993). However, a synthetic peptide of this sequence was successfully used to produce a

5 polyclonal antibody that specifically detected MBV polyhedrin by immunohistochemistry (Satidkanitkul et al., 2005). Using the same type of purified polyhedron, 7 promising monoclonal antibodies have now been developed (Boonsanongchokying, 2005) that bind well with MBV polyhedrin in tissue sections. Since MBV is a DNA virus like white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV), a multiplex PCR method that would be capable of detecting any combination of these viruses in DNA extracts from PL have been developed. Commercial multiplex kits are currently available for several shrimp viruses from DiagXotics Co. Ltd. (Wilton, CT, USA), Farming Intelligene, Taipei (Taiwan) and the Shrimp Biotechnology Business Unit (SBBU), National Science Park, Pathumthani, Thailand (Flegel, 2006) Baculoviral Midgut Gland Necrosis Virus (BMNV) Baculoviral Midgut Gland Necrosis virus (BMNV) was first recognised by Sano et al., (1981). It is a Type C baculovirus of penaeid shrimp and is distinguished from Type-A baculovirus, of which BP and MBV are examples, by their inability to produce an occlusion body in the nuclei of infected cells (Mathews, 1982; Johnson and Lightner, 1988). This virus is reported to cause serious epizootics in hatchery reared P. japonicus in Southern Japan (Sano et al., 1981, 1984, 1985; Sano and Fukuda, 1987). A sudden onset and a high mortality rate characterize BMN disease in the larval stages of P. japonicus. The disease is reported to be most severe in the post larval stages upto about PL-9 or PL-10, by which time cumulative mortalities typically reach upto 98% of affected populations and to decrease rapidly by PL-20. The typical signs of this disease are white turbid midgut line, and PL floats inactively on the surface of water (Lightner et al., 1992). In an infectivity study carried out by Momoyama and Sano (1996), P. monodon larvae are demonstrated to have a high susceptibility to the BMNV that is nearly as high as P. japonicus. Diagnosis may be confirmed by histological demonstration of the characteristic pathology of BMN. Hepatopancreas tubule epithelial cells undergoing necrosis possess markedly hypertrophoid nuclei that are characterized by marginated chromatin, diminished nuclear chromatin, nuclear dissociation, and the absence of occlusion bodies (Sano et al., 1981, 1984, 1985; Momoyama, 1983; Sano and Fukuda, 1987). A fluorescent antibody diagnostic

6 procedure has been developed in Japan (Sano et al., 1984; Momoyama, 1988), which permits rapid diagnosis of the disease and detection of silent carriers of the virus. Sano and Momoyama (1992) have developed a technique for rinsing eggs to prevent the shrimp larvae from becoming infected with BMNV Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV) Infectious Hypodermal and Hematopoietic Necrosis virus (IHHNV) was first detected in juvenile P. stylirostris from Hawai in 1981 (Lightner et al., 1983a, b), where it caused mortalities of up to 90%. Since then, the virus has been detected in other life stages of a number of penaeids in the Americas, Oceania, East and South Asia (Lightner, 1996). IHHNV is a small, icosahedral non-enveloped virus containing a single stranded linear DNA genome approximately 4.1kb in length (Bonami et al., 1990; Mari et al., 1993). This virus has been reported to infect P. japonicus, P. chinensis, P. monodon, P. semisulcatus, P. vannamei cultured in Southeast Asian countries (Lightner and Redman, 1991; Bower et al., 1994) and in wild-caught P. stylirostris (Morales- Covarrubias et al., 1999; Pantoja et al., 1999) and shrimp species in India (Felix and Devaraj, 1993). The inadvertent introduction and establishment of IHHNV into new geographic regions by imported shrimps has been well documented. Some of the accidental introduction of IHHNV (to Hawai and Mexico) has resulted in serious negative consequences to the shrimp culture industry in those locations (Lightner et al., 1983b, 1985, 1992; Brock et al., 1983; Brock and Lightner, 1990). Based on size, morphology and biochemical structure, IHHNV is considered to be a member of the family Parvoviridae (Bonami et al., 1990). Nearly 100% of the IHHNV genomic sequence and its three large open reading frames (ORF1, 2 and 3) have been determined (Nunan et al., 2000 Genbank accession No. AF218266). IHHNV has been linked by epizootiological data to runt deformity syndrome (RDS) in cultured P. vannamei. Affected shrimp with RDS are characterized by variable, often greatly reduced growth rates and by a variety of cuticular deformities affecting the rostrum ( bent rostrum ), antennae and other thoracic and abdominal areas of the exoskeleton (Kalagayan et al., 1990, 1991; Browdy et al., 1993; Lightner, 1996a).

7 Detection is traditionally done by routine histological examination of hematoxylin and eosin stained sections of shrimp pleopods (Bell et al., 1990). Histological demonstration of prominent Cowdry type A (Cowdry, 1934) inclusion bodies (CAIs) provides a definitive diagnosis of IHHNV. Monoclonal antibody based methods have been developed but their use has been hampered by their cross reactivity with non-viral substances in normal shrimp tissue (Poulos et al., 1994a). In situ hybridization and polymerase chain reaction (PCR) provides the highest available detection sensitivity for IHHNV (Lightner et al., 1992, 1994). A real-time PCR method using a fluorogenic 5 nuclease assay and PE Applied Bio Systems Gene Amp 5700 sequence detector has been developed to detect this virus in penaeid shrimps (Tang and Lightner, 2001). Digital colour correlation method has been also developed to detect IHHNV in shrimp tissues (Alvarez-Borrego and Chavez-Sanchez, 2001). A recent research (Tang et al., 2003b) has shown that P. stylirostris persistently infected with IHHNV is markedly protected from mortality upon subsequent challenge with white spot syndrome virus (WSSV) Hepatopancreatic Parvovirus (HPV) Hepatopancreatic Parvovirus (HPV) was first reported in post larvae of P. chinensis also called as Fenneropenaeus chinensis (Lightner and Redman, 1985). HPV infected animals showed nonspecific clinical signs such as poor growth rate, anorexia, decreased preening activity, increased surface fouling and sporadic opacity of tail musculature (Lightner and Redman, 1985). In Thailand, HPV in the black tiger shrimp, P. monodon was first reported in 1992 (Flegel and Sriurairatana, 1993, 1994). HPV infects several penaeid shrimp species and is widely distributed in many parts of the world including Asia, Australia and North and South America (Paynter et al., 1985; Colorni et al., 1987; Brock and Lightner, 1990; Lightner and Redman, 1992; Lightner 1996). Currently, HPV is considered as a member of the Parvoviridae (Bonami et al., 1995); however, its position within the family still remains uncertain. Shrimp infected with HPV usually show non-specific gross signs of disease but there is anecdotal information suggesting that heavy infections can cause poor growth (Sukhumsirichart et al., 1999). High levels of HPV infections have been reported especially in early juvenile stages (Flegel et al., 1995; Lightner, 1996) and the transmission of HPV is

8 believed to be both vertical and horizontal (Lightner and Redman, 1992). The presence of HPV in hatchery -reared, early postlarvae (PL-8 to PL-10) was reported for the first time in India by Manivannan et al (2002). HPV has been isolated and characterized from P. chinensis (HPV chin) from Korea (Bonami et al., 1995) and from P. monodon (HPV mon) from Thailand (Sukhumsirichart et al., 1999). Both viruses comprise unenveloped, icosahedral particles of approximately 22-24nm diameter as seen by negative staining by TEM. The nucleic acid of both is single stranded DNA, although the genomic size of HPV chin was reported to be 4 to 4.3 kb while that of HPV mon was reported to be 5.8kb. Based on genome size it appears that HPV chin and HPV mon are quite different. Diagnostic methods to detect HPV infection include routine histological methods and transmission electron microscopy (TEM) (Lightner, 1996) and immunoassays. Monoclonal antibodies for HPV detection have been described by Rukpratanporn et al. (2005). Molecular methods such as probe hybridization and PCR are considered rapid and sensitive for the detection of HPV. Gene probes (Bonami et al., 1995b; Mari et al., 1995), PCR and PCR-ELISA (Sukhumsirichart et al., 1999, 2002; Pantoja and Lightner, 2000; Phromjai et al., 2002 ) have been developed and successfully used for diagnosis of HPV. The whole genome sequence of HPV from Thailand is now available in the GenBank under the accession number DQ (Sukhumsirichart et al., 2006). Previously, research on HPV was hampered by the lack of an experimental transmission model. However, successful experimental infections by oral challenge in post-larvae of the black tiger shrimp P. monodon have recently been reported (Catap et al., 2003). Catap and Travina (2005) have reported successful horizontal transmission of HPV in P. monodon postlarvae. These new achievements will help further research on understanding the dynamics of HPV infection in shrimp and developing suitable therapeutic measures Lymphoid Organ Parvo-like Virus (LOPV) LOPV was first detected in cultured P. monodon, P. merguiensis and P. esculentus in Australia by Owens et al. (1991). Affected shrimps exhibit multinucleated giant cell formation in their hypertrophoid lymphoid organs (Owens et al., 1991). Cells making up the giant cells

9 in the Australian shrimp displayed mild nuclear hypertrophy and marginated chromatin and formed discrete, often fibrocyte encapsulated spherical structures identical to the lymphoid organ spheroids described in P. monodon from Taiwan (Lightner et al., 1987a). Owens et al. (1991) found basophilic intranuclear inclusion bodies commonly in such giant cells. These were found to contain DNA by acridine orange staining and fluorescent microscopy. Electron microscopic studies of the lesions revealed the presence of nm diameter virus-like particles (Owens et al., 1991). The importance of LOPV is still unknown Lymphoid Organ Vacuolization Virus (LOVV) LOVV has been described as a rod shaped enveloped RNA virus that is endemic in healthy wild and cultured P. monodon in Queensland (Spann et al., 1995). The histological picture is very similar to that found in the YHV infections. However, transmission electron micrographs showed that the inclusions were cytoplasmic and consisted of amorphous material (probably in secondary phagosomes) next to hypertrophoid nuclei (Flegel et al., 1995). Histological changes include highly vacuolated cytoplasm and intracytoplasmic inclusion bodies that range from Fuelgen negative eosinophilic to poorly defined Fuelgen positive, basophilic and discreet bodies in cytoplasm of lymphoid organ cells. The nuclei of affected cells are slightly hypertrophoid with marginated chromatin; in some foci, affected cells form spheroids that lack a central vessel (Bonami et al., 1992) Yellow Head virus (YHV) Yellow Head Virus (YHV) was first discovered in Central Thailand in 1990 in pond reared black tiger prawns, P. monodon. According to Limsuwan (1991), this syndrome occurs in pond reared shrimp of 5 to 15 g in size. This virus has caused massive losses among shrimp farms in Thailand (Boonyaratpalin et al., 1993). YHV has been shown to infect and cause disease in P. vannamei and P. stylirostris (Lu et al., 1994). In 1992 in Thailand, the pond harvest losses attributed to YHV were estimated to be approximately 30 millions US dollars (Nash et al., 1995).

10 Yellow head disease is characterized by light yellow colouration of the dorsal cephalothorax area and generally pale or bleached appearance of affected prawns. The yellow colour in the cephalothorax region results from the underlying yellow hepatopancreas visible through the translucent carapace in moribund shrimp (Chantanachookin et al., 1993). In size, shape, general ultrastructural morphology and buoyant density in sucrose gradients, YHV clearly resembles the bacilliform rhabdoviruses of plants (Jackson et al., 1987; Payment and Trudel, 1993) and the rhabdo-like virus infecting the blue crab, Callinectes sapidus (Yudin and Clark, 1979). It is an RNA containing virus (Wongteerasupaya et al., 1995). YHV infection can be diagnosed histologically in moribund shrimp by the presence of intensly basophilic inclusions in many different tissues (Chantanachookin et al., 1993). For the detection of YHV, a number of rapid diagnostic procedures like simple staining (Flegel and Sriurairatana, 1993, 1994) dot blot nitrocellulose enzyme immunoassay (Lu et al., 1996; Nadala and Loh, 2000), western blot technique (Nadala et al., 1997), reverse transcriptase PCR (RT-PCR) (Wongteerasupaya et al., 1997) and gene probe (Tang and Lightner, 1999) have been developed. A probe described by Soowannayan et al. (2003) for in situ hybridization is effective in detecting both gill associated virus (GAV) as well as virulent and non-virulent forms of YHV. This 794 bp long probe was prepared by labeling a RT-PCR fragment from a virulent YHV from Thailand targeting the ORF 1b region of the viral genome. In addition to these, Cowley et al. (2000b) have published primer sequences that could be used for detection of both YHV and the related Australian lymphoid organ virus (LOV) (Spann et al., 1995) and GAV (Spann et al., 1998). The primers were designed from a 781 bp GAV cdna clone to give a 618 bp RT-PCR product. Sequencing and comparison of the 618 bp RT-PCR fragments obtained using these primers with YHV, GAV and LOV showed that that all these viruses were closely related single stranded, positive sense RNAviruses (Cowley et al., 2000a). Based on these findings, YHV, GAV and LOV have now been included in a new genus Okavirus in a new family Ronivirdae (Fauquet et al., 2004; Mayo, 2002) of the Order Nidovirales. LOV and GAV share approximately 95% DNA sequence identity and 100% amino acid identity, establishing that they are the same virus type, while GAV and YHV share approximately 85% DNA sequence

11 identity and 96% amino acid identity indicating that they are different types (Walker et al., 2001; Cowley et al., 1999). A commercial RT-PCR detection kit based on the work done in Thailand and Australia (Cowley et al., 2004) is available from Farming Intelligene of Taiwan. The kit enables differential and graded RT-PCR detection of GAV and YHV. In addition to nucleic acid-based tests for YHV group viruses, monoclonal antibody assays have also been developed (Sithigorngul et al., 2000, 2002) for diagnosis by immunohistochemistry, dot blot assay and lateral flow chromatographic assays. The latter format is particularly interesting because it is cheap and suitable for use by shrimp farmers at the pond-site (Flegel, 2006) Taura Syndrome Virus (TSV) Taura Syndrome virus (TSV) was first recognised in commercial penaeid shrimp farms located near the mouth of the Taura River in the Gulf of Guayaquil, Ecuador in mid-1992 (Jimenez, 1992). Initially, the problem was attributed to toxicity of a fungicide used in banana plantations adjacent to affected shrimp farms (Jimenez, 1992; Lightner et al., 1994; Wigglesworth, 1994). Later transmission electron microscopy studies of infected TS shrimps demonstrated the presence of putative cytoplasmic virus particles named TSV (Brock et al., 1995) and Hasson et al. (1995) demonstrated the viral etiology of TS. This viral pathogen has spread throughout South and Central America into North America in the short span of 5 years (Lightner, 1996) and has become epizootic in P. vannamei causing mass mortality upto 95% in affected post larval and juvenile populations (Lightner et al., 1995, 1997; Brock et al., 1995, 1997). However, other American species including P. setiferus, P. schmitti and P. stylirostris are less seriously affected by TSV (Hasson et al., 1995; Overstreet et al., 1997). Initially, TSV was tentatively classified as a member of the family Picorniviridae because it was an unenveloped 32 nm icosahedral virus containing a 10.2 kb ssrna genome of positive sense (Bonami et al., 1997). However, it was later assigned to the family Dicistroviridae close to the genus Cripavirus (cricket paralysis virus) (Fauquet et al., 2004; Mayo, 2005). Asian TSV outbreaks were first reported from Taiwan where P. vannamei had been imported as living fry and brooders for use in commercial aquaculture ponds (Tu et al., 1999).

12 Using molecular epidemiology, it was subsequently proposed (Robles-Sikisaka et al., 2002) that TSV was introduced to Taiwan by careless importation of stocks from Mexico. Subsequent introduction of TSV to Thailand (Nielsen et al., 2005) was also proposed to have resulted from careless importation of infected broodstock and/or post larvae from the Americas, Taiwan/China or both. It was also found that the TSV in Thailand was undergoing relatively rapid genetic change from a narrow base. A wider study of genetic variations has also been reported (Tang and Lightner, 2005). The effect of exotic TSV and its mutant variants on native crustaceans is still unknown. However, a recent study of experimental infections (Srisuvan et al., 2005) suggests that the effect of TSV on P. monodon is less serious than that with P. vannamei. TSV causes three distinct disease phases in infected shrimp. The peracute/ acute phase of the Taura syndrome (TS) disease is characterized by moribund shrimp displaying an overall pale reddish colouration caused by the expansion of the red chromatophores. Shrimp in this phase usually die during the process of moulting. If the shrimp survive through the peracute/ acute phase of TSV infection, the recovery phase begins. Multifocal, melanized cuticular lesions are the major distinguishing characteristics of the recovery phase (Lightner, 1996). In the chronic phase of TSV infection, infected shrimp appear and behave normally but remain persistently infected, perhaps for life (Hasson et al., 1997b). Rapid spread of the TSV in pond population occurs through cannibalization of infected moribund and dead shrimp by healthy members of the same population (Brock et al., 1995; Hasson et al., 1995). Shrimp with TSV infections display histological lesions characteristic of the disease which are necrosis and nuclear pyknosis of the cuticular epithelium of the general body surface, appendages, gills, mouth, esophagus, stomach and hindgut (Brock et al., 1995; Lightner et al., 1995; Lightner 1996b). The lesion is characterized by the presence of inclusion bodies that give TSV lesion a peppered or buckshot appearance which is considered to be pathognomonic for the disease (Brock et al., 1995; Hasson et al., 1995, 1997, 1999b; Lightner 1996a). The current diagnostic and detection methods for TSV include histopathology, in situ hybridization and bioassay (Lightner, 1996). More recently, reverse transcription polymerase chain reaction (RT-PCR) has been developed to detect TSV in the hemolymph of infected shrimp (Nunan et al., 1998). In situ hybridization (Hasson et al., 1997) assay employing a non-

13 radioactive digoxigenin (DIG) labelled cdna probe has been employed to detect TSV (Hasson et al., 1997) Spawner-isolated Mortality Virus (SMV) SMV was first detected in P. monodon at a research facility in Townsville, Northern Queensland and Australia in 1993 (Fraser and Owens 1996). The spawners exhibited lethargy, failure to feed, redness of the carapace and pleopods and an increased mortality rate. A reliable bioassay with 0.45 μm filtered extract of infected tissue produced mortalities approaching 100% in inoculated prawns. Excretion of red feces is a characteristic feature of this disease. Small (20nm) icosahedral virions were observed in gut cells with transmission electron microscopy and partial characterization indicated that it was a non-enveloped DNA virus, similar to parvovirus (Fraser and Owens, 1996). This virus is very similar or identical to midcrop mortality syndrome (MCMS) viral agent (Owens et al., 1998). Natural infections of the red claw crayfish Cherax quadricarinatus with SMV is recorded in Australia, but it is not known whether the SMV has transferred from shrimp to crayfish or from crayfish to shrimp (Owens and Mc Elena, 2000) Gill Associated Virus (GAV) GAV has caused stock losses to the P. monodon culture industry in Australia since 1996 (Span and Lester, 1997). Diseased P. monodon infected with GAV displayed pink to red colouration of the body and appendages and pink to yellow colouration of the gills. Other signs of disease include lethargy, lack of appetite, secondary fouling and tail rot (Spann et al., 1997). Morphologically GAV resembles yellow head virus (YHV) from Thailand (Boonyaratpalin et al., 1993). GAV is a rod-shaped, enveloped viral particle containing helical nucleocapsid which matures by budding at intracytoplasmic membranes (Spann et al., 1997). Nucleotide sequence comparisons for the putative polymerase (ORF 1b) genes have indicated that GAV and YHV are closely related but distinct viruses and are likely to be classified in the order Nidovirales, possibly in the family Coronaviridae (Cowley et al., 1999, 2000a). Nucleotide sequence comparison of regions in the putative polymerase genes of multiple GAV

14 and LOV isolates has indicated that they are genetically indistinguishable populations (Cowley et al., 2000b). Screening of wild and cultured penaeids using the sensitive RT-nested PCR test has indicated that P. monodon is the only known natural hosts of GAV in Queensland (Cowley et al., 2000b). Four species of penaeid prawns cultured in Australia (P. monodon, P. esculentus, Marsupenaeus japonicus and Fenneropenaeus merguiensis) were injected with a virulent preparation of gill-associated virus and they displayed overt signs of disease and mortalities which reached 82 to 100% within 23days of post injection (Spann et al., 2000) Mid-crop Mortality Syndrome (MCMS) associated virus Beginning from 1994, farmers in Northern Australia experienced a higher than normal mortality rate among 12-15g prawns from grow out ponds. The mortalities reached as high as 80% in some ponds. The farmers referred to this problem as mid-crop mortality syndrome (MCMS) (Owens et al., 1998). The syndrome produced no histopathognomonic lesion and this greatly hampered investigation. Early investigations showed that intramuscular injection of filtered (0.45 μm pore size), cell-free extracts of moribund prawns could kill clinically healthy prawns between 7 to 20 days post-injection (Muir, Owens and Anderson unpublished). Two distinct viral types were observed by electron microscopy in moribund prawns and observations on virogenesis suggested that at least three viruses might be involved (Owens et al., 1998). One of the viruses associated with MCMS is a parvo-like virus (Owens et al., 1998). The size of the presumptive icosahedral virus visualized by TEM was 20 to 25nm, which is consistent with the size range of parvoviruses (18-26nm) (Murphy et al., 1995). P. monodon experimentally infected with spawner-isolated mortality virus (SMV) were probe positive in exactly the same pattern as the natural and experimental MCMS infected prawns. The evidence suggested that the MCMS are either very closely related or identical to SMV (Owens et al., 1998). The MCMS-associated virus appears to be enteric and infect the midgut (Owens et al., 1998). The etiological agent of MCMS still remains to be elucidated. The virulence of this virus was enhanced by the presence of other co-infecting viruses such as an enveloped, filiform gill-associated virus (Spann et al., 1997b).

15 White Spot Syndrome virus (WSSV) White spot syndrome (WSS) continues to be one of the most serious disease problems faced by the shrimp farming industry not just in Asia but globally (Takahashi et al., 1994; Chou et al., 1995; Wongteerasupaya et al., 1995; Lo et al., 1996a, b; Flegel 1997; Karunasagar et al., 1997a; Hsu et al., 1999). WSSV was first reported in 1992 in P. japonicus cultured in North eastern Taiwan (Chou et al., 1995). Since 1992, WSSV has caused mortalities and consequent serious damage to the shrimp culture industry world wide (Inouye et al., 1994; Chou et al., 1995; Wongteerasupaya et al., 1995; Lo et al., 1996a, b; Karunasagar et al., 1997a; Lightner et al., 1997; Momoyama et al., 1997; Park et al., 1998; Jory, 2000; Hossain et al., 2001a, b). WSSV has been referred to by various other names including rod-shaped nuclear virus of P. japonicus (RV-PJ) (Inouye et al., 1994), systemic ectodermal and mesodermal baculovirus (SEMBV) (Huang et al., 1995), white spot baculovirus (WSBV) (Wang et al., 1995), P. monodon non-occluded baculovirus (PmNOB) (Chang et al., 1996) and Chinese baculovirus (CBV) (Nadala et al., 1997). Recent analysis of the WSSV DNA revealed the presence of putative genes for the large and the small subunit of ribonucleotide reductase (RR1 and RR2) by van Hulten et al., (2000a) who surmise that it belongs to the eukaryotic branch of an unrooted parsimonius tree. The two virion protein genes of WSSV showed no homology to baculovirus structural proteins, suggesting together with the lack of DNA sequence homology to other viruses that WSSV may be a representative of a new virus family and proposed the name Whispoviridae (van Hulten et al., 2000b). The principal clinical sign of WSSV is the presence of white spots on the exoskeleton and epidermis, ranging from 0.5-3mm in diameter. Affected shrimp present lethargy, anorexia loose cuticle and go off their feed. In shrimp ponds, they congregate in the shallows along the edges of the pond and in culture tanks they sink inactively to the bottom where they are frequently attacked and cannibalized by the healthier shrimp (Chou et al., 1995, 1998; Nakano et al., 1994; Durand et al., 1997; Karunasagar et al., 1997a; Otta et al., 1999). WSSV is found to infect most tissues originating from both ectoderm and mesoderm. These include the subcuticular epithelium, gills, lymphoid organ, antennal gland, hematopoietic

16 tissues, connetive tissue, ovary and the ventral nerve cord (Wongteerasupaya et al., 1995; Lightner, 1996; Wang et al., 1999; Mohan et al., 1998). WSSV is known to affect most commercially important species of penaeid shrimp including P. monodon, P. japonicus, P. indicus, P. chinensis, P. merguiensis, P. aztecus, P. stylirostris, P. vannamei, P. duorarum and P. setiferus (Lightner, 1996). Wild marine shrimp such as P. semisulcatus, Metapenaeus dobsoni, M. monoceros, M. elegans, Heterocarpus sp., Aristeus sp., Parapenaeopsis stylifera, Solenocera indica, Squilla mantis and freshwater cultured species, Macrobrachium rosenbergii have also been found to harbour this virus (Lo et al., 1996a; Rajendran et al., 1999; Hossain et al., 2001a ; Chakraborty et al., 2002). This virus have been also detected in many captured and cultured crustaceans and other arthropods including crabs (Charybdis feriatus, C. annulata, C. lucifera, C. hoplites, C. cruciata, Macrophthalmus sulcatus, Gelasimus marionis, Metopograpsus messor, Scylla serrata, Sesarma oceanica, Matuta planipes, Helice tridens, Pseudograpsus intermedius ), pest prawn Acetes sp. small pest palaeomonidae prawn, larvae of Ephydridae insect and Artemia (Lo et al., 1996a ; Maeda et al., 1998; Otta et al., 1999; Chen et al., 2000; Hossain et al., 2001a, b; Chakraborty et al., 2002). There has been some concern that polychaetes (Perinereis spp.) in shrimp ponds and in natural environment that were found positive by PCR specific for WSSV (Ruangsri and Supamattaya, 1999; Tandavanitj and Kaowtapee, 2000) could transmit the virus to the broodstocks and subsequently to the postlarvae (Withyachumnarnkul, 1999). The studies of Supak Laoaroon et al. (2005) revealed that P. monodon that feed on or in co-habitation with PCR-positive polychaete Pereneis nuntia did not develop WSD and it strongly suggest that WSSV cannot be transmitted from P. nuntia to P. monodon. WSSV infection of shrimp can be confirmed by microscopic examination of stained squashed or impression smears of sub-cuticular epithelial tissue, connective tissue and gills, for the presence of hypertrophoid nuclei containing marginated chromatin and basophilic central inclusions (Chou et al., 1995; Lightner, 1996). Rapid molecular methods such as gene probes (Durand et al., 1996; Lo et al., 1996a; Wongteerasupaya et al., 1996; Nunan and Lightner, 1997) and polymerase chain reaction (Takahashi et al., 1996; Lo et al., 1996b) have been found useful in diagnosing WSSV infection. Other molecular and immunological methods such as in situ hybridization (Durand et al., 1996; Wongteerasupaya et al., 1996; Chang et al., 1996,

17 1998a; Lo et al., 1997; Nunan and Lightner, 1997; Wang et al., 1998; Chen et al., 2000), dot blot nitrocellulose enzyme immunoassay (Nadala and Loh, 2000), ELISA (Nadala et al., 1997; Sahul Hameed et al., 1998) and Western blotting (Nadala et al., 1997; Magbanua et al., 2000) have also been applied to detect WSSV in shrimp and carrier species. Recently, a competitive PCR assay has been developed for quantification of WSSV (Tang and Lightner, 2000). In addition, the major WSSV structural proteins have been characterized and the complete genome sequence has been determined (Tsai et al., 2000; van Hulten et al., 2000; 2001b; 2002). In addition to PCR tests, immunological tests have also been described (Poulos et al., 2001; Liu et al., 2002; Anil et al., 2002; Dai et al., 2003; Okumura et al., 2004) and lateral flow chromatographic detection strips are now available from both Japan ( and Thailand ( In contrast to the well-studied effect of microbial immunostimulants on the immune system, there is limited information about the immune response to viral infections. Pan et al. (2000) reported the presence of viral inhibitors in tissue extracts from crab, shrimp and crayfish against a variety of viruses. It was found that a 440 kda molecule was able to non-specifically inhibit infection of 6 types of both RNA and DNA viruses. Furthermore, an up-regulation of the lipopolysaccharide and β-1, 3-glucan binding protein gene was observed upon infection with WSSV (Roux et al., 2001). Also, up-regulation of protease inhibitors, apoptotic peptides and tumor related proteins has been observed upon WSSV infection (Rojtinnakorn et al., 2002). However, the recent reports on the possible presence of adaptive immunity in crustaceans have spurred fresh research interests. In vivo experiments with P. japonicus demonstrated the presence of a quasi immune response after re-challenging survivors of both natural and experimental infection with WSSV (Venegas et al., 2000). Wu et al. (2002) observed the presence of WSSV neutralizing activity in plasma of infected shrimp from 20 days up to well over 2 months after infection. These results suggest the induction of antiviral responses and suggest that vaccination of shrimp against WSSV may be possible. Several researchers have used whole virions or recombinant proteins for protection against WSSV infection with considerable success. Namikoshi et al. (2004) studied efficacy of vaccines made of inactivated WSSV with and without immunostimulants (β-1,3 glucan or killed Vibrio penaeicida) and of recombinant proteins of WSSV (rvp26, rvp28) and tested

18 these by intramuscular vaccination followed by intramuscular challenge of kuruma shrimp P. japonicus with WSSV. Their results indicated that VP26 and VP28 are protective antigens which can evoke protection of shrimp by vaccination upto 30 days post vaccination. Witteveldt et al. (2004b) evaluated the usefulness of intramuscularly injected WSSV envelope proteins VP19 and VP28 individually as well as in combination to vaccinate P. monodon against WSSV and found that vaccination with VP19 or VP19+VP28 resulted in significant protection against WSSV challenge. Vaseeharan et al. (2006) conducted vaccination trial in shrimps by intramascular injection of purified VP292 protein of WSSV. P. monodon were fed food pellets coated with inactivated bacteria over expressing two WSSV envelope proteins Witteveldt et al. (2004b), VP19 and VP28. In a recent study, two structural WSSV proteins (VP28 and VP19) were N- terminally fused to the maltose binding proteins (MBP) and purified after expression in bacteria. Shrimps were vaccinated by intramuscular injection of purified WSSV proteins and challenged 2 and 25 days after vaccination to assess the onset and duration of protection. The results showed that protection could be generated in shrimps against WSSV using its structural proteins as a subunit vaccine. The same subunit protein was also attempted as oral vaccine and showed the protection after 3 and 7 days post vaccination. This suggests that the shrimp immune system is able to specifically recognize and react to proteins (Witteveldt et al., 2004a, b). The most recent development in the immunization is the use of DNA vaccines encoding viral envelop proteins in P. monodon for protection against WSSV infection. Rout et al. (2007) generated recombinant constructs of four envelope proteins VP15, VP28, VP35 and VP281 into DNA vaccine vector pvax1 and immunized shrimps with these DNA constructs. The results suggested that protection was offered by the plasmids encoding VP 28 or VP281 for 7 weeks compared to the 3 week s protection offered by protein vaccination against WSSV challenge. Significantly, the immunized DNA persisted for 2 months in the muscle of shrimp. Taking cues from the success of RNA interference (RNAi) in other animals, the technique has been used as an antiviral protection mechanism in shrimp. Robalino et al. (2004 & 2005) found that in the marine shrimp Litopenaeus vannamei, the antiviral response could be induced by sequence-independent or sequence-specific double stranded RNA (dsrna) which may activate RNAi-like mechanisms. In this study, shrimp showed increased resistance to infection by two unrelated viruses, white spot syndrome virus and Taura syndrome virus.

19 According to Westenberg et al. (2005), small interfering RNA (sirna) can inhibit WSSV gene expression and replication in a sequence-independent manner. Based on these observations it could be assumed that sirna against major envelope proteins could be a potent anti-wssv mechanism to protect shrimps against infections. Kim et al. (2007) studied the effect of intramuscular injections of long dsrnas corresponding to VP28, VP281, protein kinase gene and the green fluorescence protein (GFP) gene, the last being non-specific dsrnas, in P. chinensis juveniles. All the four dsrnas showed higher survival rates against WSSV infection. Shrimp injected with dsrnas corresponding to VP28 and protein kinase showed higher survival rates than those injected with dsrnas corresponding to VP281 and GFP. Xu et al. (2007) investigated the effect of sirna using a specific 21 bp short interfering RNA against a major envelope protein gene vp28 of WSSV to induce gene silencing in vivo in P. japonicus. The transcription of vp28 was completely silenced by vp28-sirna and the synthesis of VP28 protein was totally abolished. Three doses of vp28-sirna completely eradicated WSSV from P. japonicus. The results clearly demonstrate that the vp28-sirna was capable of silencing the vp28 gene. Thus, sirna approach appears to be a promising and the efficacy and practicality of this approach need to be investigated further Infectious Myonecrosis virus (IMNV) During 2002, shrimp growers in north-east Brazil reported a disease in cultured P. vannamei characterized by focal to extensive necrotic areas in skeletal muscle tissues, primarily in the distal abdominal segments and the tail fan (Lightner et al., 2004a, b). Often the tail muscle was white and opaque in appearance. Typically, the disease progressed slowly, with low mortality rates that persisted throughout the growing season. At harvest time, cumulative mortalities in shrimp ponds reached 70% (Nunes et al., 2004). The research conducted by Poulos et al. (2006) showed that the cause of myonecrosis in P. vannamei from Brazil in 2003 is an infectious agent. The experiments revealed that the etiological agent is a 40 nm virus that possesses icosahedral symmetry, has a buoyant density of g ml -1 in CsCl and contains a monopartite dsrna genome of 7560 bp. The virus has a major capsid protein with a molecular mass of 106 kda. When virions purified from the original tissue were injected into SPF P. vannamei, the indicator shrimp exhibited the signs and

20 lesions associated with the disease, thus completing Rivers postulate for demonstration of a viral aetiology (Rivers, 1937). Based on this evidence, the disease has been named infectious myonecrosis and the aetiological agent of the disease has been designated infectious myonecrosis virus (IMNV). Tang et al. (2005) have developed a molecular probe for this virus that was used to demonstrate the presence of the agent in fixed tissue sections by in situ hybridization. The probe reacted to the histological lesions in muscle and to the lymphoid organ spheroids in the original tissue obtained from Brazilian shrimp culture facilities. The in situ hybridization results also showed that the probe reacted in the cytoplasm of infected cells, indicating that this is the most likely cellular compartment in which the virus replicates. Infection with IMNV results in a slowly progressing disease that may be influenced by conditions of temperature and salinity. Sequencing of the viral genome revealed two non-overlapping open reading frames (ORFs). The 59 ORF (ORF 1, nt ) encoded a putative RNA-binding protein and a capsid protein. The coding region of the RNA-binding protein was located in the first half of ORF 1 and contained a dsrna-binding motif in the first 60 aa. The second half of ORF 1 encoded a capsid protein, as determined by amino acid sequencing, with a molecular mass of 106 kda. The 39 ORF (ORF 2, nt ) encoded a putative RNA-dependent RNA polymerase (RdRp) with motifs characteristic of totiviruses. Phylogenetic analysis based on the RdRp clustered IMNV with Giardia lamblia virus, a member of the family Totiviridae. Based on these findings, IMNV may be a unique member of the Totiviridae or may represent a new dsrna virus family that infects invertebrate hosts (Poulos et al., 2006) Mourilyan Virus (MoV) MoV was first identified in diseased P. monodon collected from a farm near the township of Mourilyan in Northern Queensland in These prawns were also infected with high levels of gill-associated virus (GAV) (Spann et al., 1997). MoV is a newly identified virus that infects penaeid prawns P. monodon and P. japonicus. Spherical to ovoid enveloped particles ( nm diameter) possess bunyavirus-like morphology. RT-nested PCR testing has indicated that natural MoV infections occur commonly in black tiger shrimp (P. monodon)

21 and Kuruma (P. japonicus) prawns from the wild or farmed commercially in Queensland. Low levels of MoV infections were detected by in situ hybridization in vacuolated spheroid bodies within the lymphoid organ in each species. In heavily infected prawns, MoV was detected throughout the lymphoid organ and in connective tissues of other organs. In some P. japonicus, MoV has been identified in midgut and nerve tissues displaying histopathology consistent with gut-and-nerve syndrome. Preliminary studies on genome suggested that MoV genome comprises four segments of negative sense single-stranded RNA and BLAST searches identified that it was distantly related to Uukuniemi virus and other viruses within the genus Phlebovirus of the Bunyaviridae (Cowley et al., 2005). 2.2 Multiple viral infections Besides single viral pathogen, there are several reports of multiple viral infections of cultured shrimps. Chantanachookin et al. (1993) reported triple infection with YHV, HPV and MBV in farm ponds. They noted that only the presence of YHV was correlated with mortality. Yet another study by Manivannan et al. (2002) found simultaneous infection in P. monodon postlarvae by MBV, HPV and WSSV and opined that the cumulative effect of these three viruses was responsible for the observed mortality in hatchery. Cowley et al. (2005) found the presence of two viruses; MoV and GAV in P. monodon and P. japonicus in Northern Queensland. Recently Umesha et al., (2006) have reported the triple virus infection of WSSV, MBV and HPV in cultured adult P. monodon from westcoast of India. Of late, several new diseases of unknown or obscure aetiology have been reported in the shrimp culture industry such as the swollen hind gut syndrome (SHG) (Lavilla-Pitogo et al. (2002), monodon slow growth syndrome (MSGS) (Chayaburakul et al., 2004) and loose shell syndrome (Mayavu et al., 2003, Society of Aquaculture Professionals, 2004). MSGS was first observed in cultured P. monodon in Thailand and in the absence of known viral pathogens the causative agent was designated as monodon slow growth agent (MSGA) (Chayaburakul et al., 2004). Later investigations revealed the presence of a virus called Laem-Singh Virus (LSNV) (Sritunyaluksana et al., 2006). In situ hybridization tests revealed the presence of LSNV in the cytoplasm of cells in the lymphoid organ, heart and hepatopancreatic interstitial cells. Transmission electron

22 microscopy of parallel samples of the lymphoid organ tissue revealed the presence of a single type of viral like particles in the cytoplasm of lymphoid organ tubule cells in the same area of the positive in situ hybridization reaction. These were unenveloped, icosahedral particles of approximately 27 nm diameter, similar to the size of viruses in the family Luteoviridae. Tests using both in situ hybridization and RT-PCR revealed the presence of LSNV in both MSGS ponds and normal growth ponds, indicating that it was probably not the direct cause of MSGS. There still remains the possibility that the MSGS is related to the prevalence or severity of LSNV infections in a shrimp culture pond. The unusual retarded growth and wide variation in size without abnormal mortality which is similar to MSGS in Thailand, has been reported from East Africa by Anantasomboon et al. (2006). 2.3 Bacterial agents of diseases in shrimp Viral diseases are often accompanied by bacterial infestations (Lightner and Redman, 1998). Only a small number of bacterial species have been diagnosed as infectious agents in penaeid shrimp. Vibrio spp. are by far, the major bacterial pathogens and can cause severe mortalities, particularly in hatcheries. Vibriosis is often considered to be a secondary (opportunistic) infection, which usually occurs when shrimp are weakened (Johnson, 1989; Lightner et al., 1992). Primary pathogens can kill even when other environmental factors are adequate, whereas opportunistic pathogens are normally present in the natural environment of the host and only kill when other physiological or environmental factors are poor. Lavilla-Pitogo (1995) has reported eight bacterial genera that have been associated with the diseases in penaeid culture systems. Only two groups occur quite commonly: filamentous bacteria and Vibrios, with the latter being more important. Many Vibrio species have been reported in penaeids: Vibrio alginolyticus, V. anguillarum, V. cholerae (non-01), V. damsela, V. fluvialis, V. nereis, V. splendidus, V. tubiashii, V. vulnificus, V. parahaemolyticus and V. harveyii. (Lavilla-Pitogo 1995). Among the several species of vibrios, V. harveyi, V. penaecida, V. parahaemolyticus and V. vulnificus (Lightner 1996a; Ishimaru et al., 1995, Lavilla-Pitogo 1995) are the most important pathogens in shrimp. Although the taxonomy for the group is still somewhat unsettled, especially for tropical species (Suwanto et al., 1998; Bhat and Singh,