REVIEW OF LITERATURE

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).

2.1 Viral agents of disease in shrimps

2.1.1 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).

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 80 - 100x106

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

1987-88 (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

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).

2.1.3 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

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.

2.1.4 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).

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).

2.1.5 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

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 DQ002873 (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.

2.1.6 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”

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 25-30 nm diameter virus-like

particles (Owens et al., 1991). The importance of LOPV is still unknown.

2.1.7 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).

2.1.8 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).

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

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).

2.1.9 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).

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-

radioactive digoxigenin (DIG) labelled cDNA probe has been employed to detect TSV (Hasson

et al., 1997).

2.1.10 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 mid-

crop 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).

2.1.11 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

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).

2.1.12 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).

2.1.13 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

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,

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 (http://enbiotec.co.jp/en/)

and Thailand (http://www.shrimpbiotec.com).

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

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.

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.

2.1.14 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

1.366 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

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 136-4953) 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 5241-7451) 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).

2.1.15 Mourilyan Virus (MoV)

MoV was first identified in diseased P. monodon collected from a farm near the

township of Mourilyan in Northern Queensland in 1996. 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 (85×100 nm diameter) possess bunyavirus-like morphology. RT-nested PCR testing

has indicated that natural MoV infections occur commonly in black tiger shrimp (P. monodon)

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

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,

1998), molecular methods using polymerase chain reaction (PCR) technology are now

becoming available for rapid and precise identification of species (Genmoto et al., 1996;

Rojlorsakul et al., 1998; Karunasagar et al., 1997b; Saulnier et al., 2000). Harris and Owens

(1999) have shown that virulent strains of V. harveyi are capable of producing potent toxic

proteins and that these may vary from strain to strain. Goarant et al. (2000) have shown that V.

penaecida also produces potent toxic substances in broth culture. Knowledge of the nature and

relationship amongst these toxins and their mode of action on shrimp are needed. It is clear that

strains incapable of producing these toxins are harmless to shrimp. Thus intervention strategies

may be available to protect the shrimp that need not require attempted elimination (almost

impossible of relevant bacteria from the shrimp rearing environment (Flegel, 2002). Oxley et al

(2002) reported on the composition of the gut bacterial species in the gut of the penaeid prawn

P. merguiensis. The bacterial composition was compared, both qualitatively and quantitatively,

in the four distinct regions of the gut in both cultured prawns and those gathered from a natural

environment.

The recent publication by Misciattelli et al. (1998) indicates the potential that may

eventually be exploited in the direction of inactivating the toxin production. Minute quantities

of algal products were apparently capable of inactivating Vibrio toxin production even though

their growth was not inhibited (actually enhanced). Perhaps this is the real potential for

‘probiotics’ and perhaps it can explain how a small number of one microbe can have a

significant effect on a larger population of another species in the shrimp rearing environment.

Rengpipat et al., (1998) reported better yield and good control over disease in P. monodon

when they used Bacillus strain S11 as a probiotic. Much more work is needed in this area of

environmental microbiology and the focus should be altered from the myopic quest for species

that are capable of inhibiting the growth of Vibrio species. Clearly the Vibrio activity is more

important than species identity or number of cells per ml (Flegel, 2002). Karunasagar et al.

(2005) also screened several Bacillus spp. and found that several strains of B. megaterium, B.

licheniformis, B. coagulans and B. circularis isolated from shrimp farm environments have

anti-vibrio activity.

Techniques from the field of environmental microbiology have shown convincingly that

only a small number of the microbes in the environment are culturable and that the dominant

species often are not (Head et al., 1998). This may also be true for the shrimp rearing

environment in hatcheries and rearing ponds and even for shrimp pathogens (Flegel, 2002).

Flegel et al. (2005) from their recent study suggested that two quite different

bacteriophages, one from the family Myoviridae and the other from the family Siphoviridae,

can change the phenotype of V. harveyi isolates from non-virulent to virulent strain and thus act

as mobile genetic elements influencing the virulence factors of pathogenic vibrios. Karunasagar

et al. (2005) have reported that bacteriophages can be used as biocontrol agents against

pathogenic Vibrio spp. in aquaculture.

2.3.1 Bacterial white spot syndrome (BWSS)

BWSS was first detected from a shrimp farm in Malaysia in 1998 (Wang et al., 1999,

2000). BWSS showed similar gross clinical signs of white spots which caused confusion during

PCR-based screening for white spot disease since shrimp with apparent white spot disease virus

clinical signs gave negative results. Later it was suggested that the bacterium Bacillus subtilis

was the possible causative agent due to its association with the white spots (Wang et al., 2000)

but no causal relationship has been demonstrated, nor have infectivity studies been conducted.

The clinical signs observed due to this infection are dull white spots appeared on the

carapace and all over the body. The white spots are rounded and not as dense as those seen in

white spot disease. Wet mount microscopy reveals the spots as opaque brownish lichen-like

lesions with a crenellated margin. The spot center is often eroded and even perforated. Delayed

moulting, reduced growth and low mortalities have been reported in severely infected shrimp

(Wang et al., 2000).

Less well understood are the intracellular bacterial pathogens of shrimp. The presence

of some of these is apparent by gross signs and /or histopathology by the light microscope. For

example, the recently described mollicute from China causes reddening of the shrimp gut

(Jifang, 1998) and rickettsial infections and necrotizing hepatopancreatitis (Lightner, 1996a)

can be seen histologically. Others like the Mycoplasma described by Ghadersohi and Owens

(1998) may be more difficult to detect and may require the use of molecular techniques. As

with the non-symptomatic viruses, the availability of appropriate probes will be critical for

determining prevalence and impact. If these pathogens are spread by carriers, the molecular

probes (Ghadersohi and Owens, 1998; Loy et al., 1996) will also be important for discovering

the reservoir species. Much more work is needed on this group of organisms.

Storch et al. (1984) studied the early effects of nutritional stress on the liver of milkfish

Chanos chanos and on the hepatopancreas of the tiger prawn P. monodon. Baticados et al.

(1986) conducted field survey of prawn ponds in the island of Panay (Philippines) and showed

that occurrence of soft-shelled prawns could be predicted with 98% accuracy under poor soil

and water conditions in the ponds and some management practices were highly correlated with

the soft-shell syndrome.

2.4 Viral Diseases of Macrobrachium rosenbergii

The giant freshwater prawn Macrobrachium rosenbergii is an economically important

farmed crustacean. The culture of this species was mostly developed in Southeast Asian

countries and to a lesser extent in the Carribean (Northern South America and The West

Indies).

Except for two reports of viruses viz, parvo-like virus (Anderson et al., 1990) and

Macrobrachium muscle virus (MMV) in Macrobrachium rosenbergii (Tung et al., 1999), no

serious viral diseases have been reported to date in this economically important farmed species.

Although a third virus (WSSV) was reported in cultivated M. rosenbergii (Lo et al 1996, Peng

et al., 1998) and the disease experimentally induced in this species (Chang et al., 1998, Wang

et al., 1998). WSSV should not be considered as a viral disease of M. rosenbergii but rather, as

a penaeid shrimp virus capable of developing in a large number of crustacean hosts that include

M. rosenbergii.

M. rosenbergii nodavirus (MrNV) is a recently reported viral pathogen of this

crustacean (Arcier et al., 1999). This virus is an agent of white tail disease (WTD). The disease

was first reported in Guadeloupe in 1997 (Arcier et al., 1999), and then in Martinique (French

West Indies) (unpublished results) and subsequently in The People’s Republic of China, in

Zhejiang, Jiangsu, Guangdong and Shanghai provinces (Qian et al., 2003). Similar clinical

signs associated with small viral particles of the same size (25–30 nm) were reported in

diseased M. rosenbergii from Taiwan (Tung et al., 1999). More recently this disease was

observed in many freshwater prawn hatcheries and nursery ponds in several parts in India

causing high mortalities and huge economic losses (Sahul Hameed et al., 2004a).

Earlier the disease that occurred in French West Indies was attributed to noda-like virus

called MrNV. Subsequently a second virus-like particle unusually small and named “extra

small virus” (XSV) (Qian et al., 2003, Sri Widada and Bonami, 2004) was found to be

associated with the noda-like virus. It is interesting to note that these two types of particles

were found in WTD-infected M. rosenbergii from the French West Indies, as well as in

diseased animals sampled in China. The two types of particles are icosahedral in shape, un-

enveloped, and located in the cytoplasm of infected target cells, particularly connective tissue

cells (Qian et al., 2003; Sahul Hameed et al., 2004b). Detection methods for MrNV are

available: a double antibody sandwich enzyme-linked immunosorbent assay (DS-ELISA)

(Romestand & Bonami 2003) and a viral genome-based detection method, i.e. dot blot and in

situ hybridization and amplification (RT-PCR) (Sri Widada et al., 2003). Genome-based

detection methods, i.e. dot-blot hybridization and RT-PCR, are also available for XSV (Sri

Widada et al., 2004). The RT-PCR assay has proven to be the most sensitive among the

available methods to detect these two viruses (Sri Widada et al., 2003, 2004; Sahul Hameed et

al., 2004a) and to avoid the necessity of carrying out two separate RT-PCR reactions,

Yoganandan et al. (2005) have developed a modified method for simultaneous detection of

MrNV and XSV in a single tube, one-step multiplex RT-PCR. Bonami et al., (2005) have

characterized these two viruses by physically separating and purifying them.