Strategies for the Control of Viral Diseases

Transcription

1 Strategies for the Control of Viral Diseases of Shrimp in the Americas Donald V. Lightner and R. M. Redman Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona U.S.A. (Received February 6, 1998) Viral diseases have severely impacted many of the penaeid shrimp farming industries of the world causing significant production and economic losses. Nearly 20 distinct viruses, or groups of viruses, are known to infect penaeid shrimp. Viruses belonging to the WSSV, MBV, BMN, HPV, IHHNV, and YHV groups have been important pathogens of cultured shrimp in Asia and the Indo-Pacific regions, while TSV, IHHNV, and BP have been the principal viruses of concern in the Americas. Numerous strategies have been attempted for the control of viral diseases in penaeid shrimp aquaculture. These strategies range from the use of improved culture prac tices (i.e. where sources of virus contamination are reduced or eliminated, sanitation practices are improved, stocking densities are reduced, etc.) to stocking "specific pathogen-free" (SPF) or "specific pathogen resistant" (SPR) species or stocks. In the Americas many strategies have been employed in efforts to reduce production losses due to the enzootic viruses IHHNV, BP, and TSV. Improved husbandry practices have been successfully employed for the control of BP, and for nearly a decade, this virus has seldomly been reported as an economic constraint to successful shrimp culture. Until recently, the popularity and use of the relatively IHHNV resistant species Penaeus vannamei, in prefer ence to the culture of the more IHHNV susceptible P. stylirostris, was characteristic of the shrimp farming indus tries of the Americas. The popularity of P. vannamei began to decline when TSV emerged as a very serious pathogen of this species in 1992 and then spread to virtually all of the shrimp growing regions of the Americas during the ensuing four years. Because P. stylirostris was found to be innately TSV resistant, two domesticated, genetically selected SPR strains of this species, which are resistant to IHHN disease, are currently being devel oped and marketed in the Americas. In some regions, these SPR stocks of TSV and IHHNV resistant P. stylirostris are replacing P. vannamei stocks in culture. Other shrimp farming interests are using wild or domesticated stocks of P. vannamei that show improved resistance to TSV. While resistance to TSV was used as a selection criteria for the domesticated stocks of P. vannamei, natural selection for TSV resistance appears to be occurring in wild stocks where TSV has been enzootic for several years. The same selective process for IHHNV resistance seems to be occurring in some wild stocks of P. stylirostris. Key words: Penaeus, aquaculture, shrimp viruses, IHHNV, TSV, BP, HPV, SPF, SPR Introduction The penaeid shrimp culture industries of the world developed from their experimental beginnings three de cades ago into major industries providing millions of jobs and billions of U. S. dollars annually in export rev enue (Weidner and Rosenberry, 1992; Wang et al., 1995; Rosenberry, 1996). Concomitant with the rapid growth of the shrimp culture industries has been the recognition of the ever increasing importance of pathogenic agents FAX: dvl@u.arizona.edu among which the viruses that infect penaeid shrimp are especially well represented. In 1974 the first known shrimp virus was described and named Baculovirus penaei (BP = PVSNPV) (Couch, a, 1974b; Bonami et al., 1995). By 1996, 1974 the list of penaeid shrimp viruses had grown to include at least 20 viruses or unique strains of viruses representing seven virus families (Tables 1-2), with all but two or three of these 20 viruses having been described in penaeid shrimp from aquaculture settings (Lightner, 1996a, b). While some of the known 1996 penaeid shrimp viruses seem to be of little economic importance, others can cause serious disease in their penaeid shrimp hosts

2 166D. V. Lightner and R. M. Redman Table 1. Viruses of penaeid shrimp (as of October, 1997; modified from Lightner 1996a except as indicated*) and significant economic losses to the industries which culture them. Devastating epizootics due to various virus pathogens of penaeid shrimp have caused signifi cant, and sometimes catastrophic economic losses, in commercial penaeid shrimp culture (Boonyaratpalin et al., 1993; Brock, 1991; Lightner, 1988, 1992; Stern, 1995; Takahashi et al., 1994; Fraser and Owens, 1996; Flegel, 1997). Viral pathogens have been implicated in the collapse of important shrimp aquaculture industries in Asia and the Indopacific region. Even Australia, with its rela tively small penaeid prawn culture industry, has experi enced serious epizootics due to viral pathogens (Spann et al., 1995; Fraser and Owens, 1996). Some well known examples of major virus caused disease epizoot ics in Asian shrimp include Taiwan's epi zootic when production fell from 100,000t to 30,000t (Chen, 1995). In the Chinese shrimp aquaculture industry saw their production collapse from 220,000t in 1991 to 30,000 t in 1993 (Wang et al., 1995; Rosenberry, 1994a, 1994b; Chamberlain, 1994). Like wise, recent major epizootics in Thailand, Indonesia, Japan, Taiwan, and India have been accompanied by significant losses that, in some instances, reached 50 to 90% of expected production for the year (Flegel et al., b; Winamo 1995; Flegel, 1997; Lo et al., 1996). 1995

3 Control of shrimp viral diseases in the Americas167 Table 2. Viruses reported from Eastern (Asian, Australian, European, and African) and Western Hemisphere (the Americas and Hawaii) of penaeid shrimp (com piled from Lightner 1996a) Table 3. Major shrimp producing countries in the Western Hemisphere: production and number of farms in 1996 (Modified from Rosenberry 1996) The shrimp culture industries of the Americas have also been adversely affected by serious epizootics due to viral pathogens. Infectious hypodermal and hemato poietic necrosis virus (IHHNV) (Brock and Lightner, 1990; Lightner, 1993, 1996a, 1996b; Lightner et al., a, 1992b) and Taura syndrome virus (TSV)(Cham 1992 berlain, 1994; Brock et al., 1995; Hasson et al., 1995; Lightner et al., 1995; Lightner, 1996a, 1996b; Bonami et al., 1997) have had enormous negative impacts on the continents' developing aquaculture industries and, in one instance for IHHNV, on a commercial fishery as well (Moore and Brand, 1993; Lightner, 1996b; Pantoja et al., "in press"). This paper reviews the published literature and as yet unpublished (but available anecdot ally) recent information on BP, IHHNV, and TSV in penaeid shrimp aquaculture in the Western Hemisphere and the strategies used by the aquaculture industry to manage the diseases caused by these pathogens. Western Hemisphere Shrimp Aquaculture Industries The shrimp farming industry of the Western Hemi sphere produced approximately 25% of the world's farmed shrimp in 1996 (Table 3). This amounted to 172,300 metric tons (t). Ecuador accounted for 70% of this total, which ranks it as the second largest pro ducer of farmed shrimp after Thailand with its approxi mately 160,000 t of production in After Ecuador, other large shrimp farming countries in the Americas (and their approximate 1996 productions) include Mexico (12,000 t), Honduras (10,000 t), Peru (5,000 t), Nicaragua (3,000 t), Belize (2,000 t), Venezuela (2,000 t), and the United States (1,300 t), and a few other coun tries like Brazil, Costa Rica, Panama, and various Caribbean island countries that collectively produced another 16,000 t in 1996 (Rosenberry, 1996). In terms of significant production, only two penaeid shrimp species are farmed in the Americas. Of these, Penaeus vannamei (the Pacific white shrimp) accounts for 80 to 90% of the total production. P. stylirostris, the Pacific blue shrimp is next in importance accounting for 10 to 20% of the production, and its importance is increasing rapidly because it is more resistant to TSV infection and disease than is P. vannamei. Other spe cies such as P. setiferus and P. schmitti (the Gulf of Mexico and Caribbean white shrimp, respectively) and P. californiensis (the Pacific brown shrimp), are impor tant in some locations, but they account for less than a few percent of the total shrimp aquaculture production in the Americas (Rosenberry, 1996). The methods of culture employed by shrimp farmers are similar throughout the Americas. The principal differences among the producers is the source of "seed" (PLs or postlarvae) used to stock the farms. Most of the grow-out shrimp farms in the Americas are semi intensive and consist of large ponds of 1 to 20 ha with earthen dikes. Stocking densities in most semi-inten sive shrimp farms in the Americas range from - less than 5 per m<sup>2<sup> to 50 per m<sup>2<sup>. There are a few intensive farms in the Americas. These farms typically have pond sizes

4 168D. V. Lightner and R. M. Redman of 0.25 ha to 5 ha, mechanical aeration, and stocking rates that range from 35 to 200 shrimp per m<sup>2<sup>. Some regions of the Americas (Venezuela, Brazil, the Caribbean island nations, and the United States) rely almost entirely on hatcheries and domesticated brood stock for the production of "seed". However, the major shrimp producers in the Western Hemisphere rely on the use of wild broodstock or wild seed (PLs) for their stock. In Ecuador for example, wild PLs are available for much of the year (and nearly, but not, every year) in the coastal zone and estuaries. Nearly 100,000 people in Ecuador make a living collecting wild PLs with special fine mesh push nets (called chinchorros') from beaches and estuaries, and ' selling the wild PLs that they collect to farms. When wild PLs are not abundant, the approximately 300 penaeid shrimp hatcheries in Ecuador (which are called "laboratories" in Ecuador as well as in most of the Americas) collect wild adult shrimp from the coastal fishery, spawn them, and use the resultant larvae to produce the PLs needed to stock farms. The hatcheries may produce PLs from "wild nauplii" (which are produced from wild-caught, fertilized females within 1-2 days of capture) or from "maturation nauplii/seed" (which are produced from captive wild males and female adults that are matured and mated in specialized "maturation" units in the hatch eries) (Rosenberry, 1996). The dependence of the shrimp aquaculture industry on wild broodstock and wild PLs as the principal source of its stock has limited the industry's options for disease management and prevention. This dependence on wild stock has left the industry vulnerable to major viral disease outbreaks like the panzootic due to IHHNV in the 1970's and 1980's and the more recent panzootic due to TSV that began in Ecuador in and spread throughout the Americas (Lightner, 1996b; Hasson et al., 1997a; Brock et al., 1997). The Major Viruses in the Americas Although nearly 10 of the known shrimp viruses have been found in cultured penaeid shrimp in the Americas (Table 2), only three of these have caused panzootic disease in the Western Hemisphere (Lightner, 1996a, b). These three viruses, or perhaps 1996more correctly groups of different strains of the same virus group include BP, IHHNV, and TSV (Table 4). BP-Type Baculoviruses and Disease BP disease: BP (Baculovirus penaei or PVSNBV) has been reported to cause significant disease in the lar val, postlarval, and early juvenile stages of several penaeid species in the Americas (Couch, 1974a, 1974b; Lightner et al., 1985; Johnson and Lightner, 1988; Overstreet et al., 1988; LeBlanc and Overstreet, 1990; Bueno et al., 1990; Krol et al., 1990; Bonami et al., 1995; Lightner, 1996a). BP is known to cause signifi cant disease in larval and postlarval stages of P. aztecus, P. stylirostris and P. vannamei (Couch, 1991; Johnson and Lightner, 1988; Lightner and Redman, 1991, 1992; LeBlanc and Overstreet, 1990, 1991; LeBlanc et al., 1991). BP has been a sporadically occurring, but often serious, hatchery disease of the larval stages of P. vannamei in many of the commercial hatcheries on the Pacific Coast side of Central and South America, including Peru, Ecuador, Columbia, Panama, Costa Rica, Honduras, and on the Atlantic side of the conti nent in Brazil, Florida and Texas in the United States (Bueno et al., 1990; Lightner and Redman, 1991, 1992; Lightner, 1996a). In Mexico, BP has caused serious epizootics in cultured larval and post larval P. stylirostris (Lightner et al., 1989). A morphologically and geneti cally unique strain of BP occurs in wild P. marginatus in Hawaii (Lightner et al., 1985, 1994; Brock et al., 1986). BP has not been reported outside of the Ameri cas and Hawaii (Lightner and Redman, 1992; Overstreet, 1994). Table 4. The major viruses of Western Hemisphere penaeid shrimp

5 Control of shrimp viral diseases in the Americas169 BP infects only the hepatopancreas and midgut epithelial cells, and it is transmitted from shrimp to shrimp exclusively per os. The typical route of infec tion of shrimp larvae in hatcheries is via fecal (from BP infected adult spawners) contamination of spawned eggs (Johnson and Lightner, 1988; Lightner, 1996a). Additional routes of horizontal transmission of BP (and other gut-infecting baculoviruses) in larval culture sys tems results from fecal-oral contamination (via feces from infected larvae) or from cannibalism of diseased larvae (Sano et al., 1985; Momoyama, 1988; Overstreet et al., 1988; Momoyama and Sano, 1989; LeBlanc and Overstreet, 1990, 1991; Sano and Momoyama, 1992). Experimental direct transmission of BP virus from shrimp to shrimp has demonstrated that it has an incu bation period of about 24 h (Overstreet et al., 1988), which is similar to incubation periods for MBV and BMN (Momoyama, 1988; Lightner, 1996a). Diagnosis and detection of BP: Classical micro scopic, histological methods, and molecular methods are available for detection of BP virus and the diagnosis of the disease it causes (Table 5). Patent infections by BP are easily diagnosed by the demonstration of prominent tetrahedral occlusion bodies in unstained squash prepa rations of hepatopancreas, midgut or feces, or in appro priate histological sections from infected animals. In histological sections, these occlusion bodies are demon strated to occur as single or multiple, eosinophilic, usu ally triangular, inclusion bodies within hypertrophied nuclei of hepatopancreatic tubule epithelial cells or less frequently in midgut epithelial cells (Lightner, 1996a). Molecular detection methods have been developed for BP. In situ hybridization assays of Davidson's pre served tissue sections with digoxigenin-11 dutp (DIG) Table 5. Summary of diagnostic and detection methods for the major viruses of concern to the shrimp culture industry (modified from Lightner 1996a)

6 170 D. V. Lightner and R. M. Redman labeled probes and PCR methods have been developed and found to be more sensitive for detection of BP in infected tissues than the classical methods of direct mi croscopy, histopathology, and serology with polyclonal antibodies (Lewis, 1986; Bruce et al., 1991, 1993, a, 1994b; Lightner et al., 1992c; Bonami et 1994 al., 1995). Polymerase Chain Reaction (PCR) methods have been developed for the detection of BP, and the method has been successfully applied to the detection of BP in feces (Lightner et al., 1994; Lightner, 1996a; Wang et al., 1996). This application of PCR may eventually be used as a non-destructive method to test valuable broodstock individually for their BP infection status. Morphometric comparisons of BP virions and nucleo capsids from different geographic regions of North, Central, and South America and Hawaii suggest that two or three distinct strains of BP probably occur (Lightner et al., 1985; Bonami et al., 1995). In morphometric studies, BP virion nucleocapsids in Ecuadorian P. vannamei were found to be slightly larger than BP nucleocapsids in Gulf of Mexico P. aztecus and P. duorarum, while both were considerably larger than the BP nucleocapsids found in Hawaiian P. marginatus (Couch, 1974a, 1974b; Lightner et al., 1985; Brock et al., 1986). Bruce et al. (1993) compared Ecuadorian isolates of BP with other geographic strains using a panel of five probes developed to the Ecuadorian strain of BP. The probes distinguished at least three different strains of BP virus (Table 6). IHHN Virus and Disease Infectious hypodermal and hematopoietic necrosis virus: IHHNV is the smallest of the known penaeid shrimp viruses (Bonami et al., 1990; Adams and Bonami, 1991). The IHHN virion is a non-enveloped icosahedron averaging 22 nm in diameter; it has a den sity of 1.40 g/ml in CsCl, contains linear ssdna with an estimated size of 4.1 kb, and its capsid contains four polypeptides with molecular weights of 74, 47, 39, and 37.5 kda. Because of these characteristics, IHHNV has been classified as a member of the family Parvoviridae (Bonami et al., 1990). IHHN disease: Infection by IHHNV causes serious disease in P. stylirostris, and acute catastrophic epizoot ics in semi-intensively or intensively cultured juveniles of that species (Lightner et al., 1983; Bell and Lightner, 1987; Brock and Lightner, 1990; Lightner, 1996a). In other penaeids like P. vannamei, IHHNV was recog nized soon after its discovery in P. stylirostris to infect and cause disease. However, infected P. vannamei was considered to be highly resistant to IHHN disease rela tive to P. stylirostris (Bell and Lightner, 1984). How ever, despite its relative resistance to IHHN disease, "runt deformity syndrome" (RDS) in cultured P. vannamei was linked by epizootiological data to infection by IHHNV (Kalagayan et al., 1991; Browdy et al., 1993; Castille et al., 1993). Affected shrimp with RDS are characterized by variable, often greatly reduced growth rates and by a variety of cuticular deformities affecting the rostrum (resulting in a "bent rostrum"), antennae, and other thoracic and abdominal areas of the exoskel eton (Brock and Main, 1994; Kalagayan et al., 1991; Lightner, 1992, 1996a). RDS is an economically sig nificant disease of cultured P. vannamei, which has been observed in virtually every country in the Americas in Table 6. Strains of BP in the Western Hemisphere: morphology and reaction with specific DIG-labeled probes in situ with histological sections of BP infected shrimp from different geographic regions*

7 Control of shrimp viral diseases in the Americas171 which the species is cultured. Cultured populations affected by RDS may contain up to 30% runts, and con sequently a wide distribution of size ("count" or the number of shrimp per pound) classes. Because small-size (runts) shrimp have a lower market value than unaffected shrimp, RDS significantly reduces the market value of affected P. vannamei crops, resulting in revenue losses that can range from 10 to 50% of the value of similar IHHNV-free (and RDS-free) crops (Kalagayan et al., 1991; Wyban, 1992). Diagnosis and detection of IHHN: Traditional methods employing histology (Brock, 1992; Brock and Main, 1994; Lightner, 1993, 1996a) and molecular methods that use non-radioactively DIG labeled gene probes are the current methods of choice for diagnosis of infection by IHHNV (Table 5) (Lightner, 1996a, b). Although monoclonal antibodies 1996 have been developed for IHHNV, their use has been hampered by their cross reactivity to non-viral substances in normal shrimp tissue (Poulos et al., 1994). Histological demonstration of prominent Cowdry type A inclusion bodies (=CAIs) provides a definitive diagnosis of IHHN. These pathognomonic IHHN inclusion bodies are intra nuclear, eosinophilic (when stained with H & E stains of tissues preserved with fixatives that contain acetic acid, such as Davidson's AFA and Bouin's solution { Bell and Lightner, 1988; Lightner, 1996a}) inclusion bodies, within chromatin-marginated, hypertrophied nuclei of cells in tissues of ectodermal (epidermis, hy podermal epithelium of fore and hindgut, nerve cord and nerve ganglia) and mesodermal origin (hematopoietic organs, antennal gland, gonads, lymphoid organ, and connective tissue) (Lightner, 1996a). Non-radioactive DIG-labeled gene probes to IHHNV and PCR methods for detection of the virus have been developed (Mari et al., 1993; Lightner et al., 1992c, 1994; Lightner, 1996a). DIG-labeled DNA probes for IHHNV are now commercially available as ShrimProbeTM kits from DiagXotics, Inc. (Wilton, CT, USA) in dot blot and in situ hybridization formats (Table 5). These methods provide greater diagnostic sensitivity than do more traditional methods for IHHN diagnosis that em ploy classic histological methods. Valuable broodstock shrimp may be examined for IHHNV infection with gene probes by use of a non-lethal biopsy or by testing a hemolymph sample. In this method, a hemolymph sample may be taken with a tuberculin syringe or an appendage (a pleopod for example) may be biopsied and used as the sample for a direct dot blot test or PCR assay for IHHNV (Lightner, 1996a). Distribution of IHHN: The virus has a world-wide distribution and wide host range in cultured penaeid shrimp, but its original distribution in wild penaeids re mains unknown (Lightner and Redman, 1991; Lightner, a, 1996b). However, the occurrence in Southeast 1996 Asia (Singapore, Malaysia, Indonesia, and the Philip pines) of IHHNV (or a similar agent) in shrimp culture facilities using only captive-wild P. monodon broodstock, and where Western Hemisphere American penaeids had not been introduced, suggests that the region is within the virus' natural geographic range, and that P. monodon may be among its natural host species (Lightner, 1996a, b). Molecular comparisons of 1996 IHHNV isolates from various locations in the Americans and Asia using PCR and in situ hybridization with probes from regions of the IHHNV genome have not shown distinguishable differences among geographic isolates of IHHNV. Taura Syndrome Virus and Disease Taura syndrome virus (TSV): Taura syndrome virus (TSV) is one of the most recently characterized penaeid shrimp virus. TSV has been classified with the Picornaviridae based on its morphology (a nm icosahedron), its cytoplasmic replication, its buoyant density of g/ml, its genome consisting of a linear, positive-sense, single stranded RNA (ssrna) of ap proximately 9 kb in length, and its having three major (55, 40, and 24 kda) and one minor polypeptides (58 kda) comprising its capsid (Hasson et al., 1995; Lightner, a; Bonami et al., 1997) Taura syndrome disease: TSV is known to infect a number of penaeid shrimp species. It causes serious disease in the PL, juvenile and adult stages of P. vannamei (Brock et al., 1995; Lightner et al., 1995; Lightner, a). In larval and early PL P. vannamei, 1996 infection by TSV is apparently not expressed until about PL-11 or PL-12 when severe disease and mortalities have been noted in infected populations (Lightner, 1996a). The American penaeids P. stylirostris, P. schmitti, P. setiferus and P. aztecus can also be infected by TSV, but serious disease resulting from infection has only been reported for the PL stages of P. setiferus (Overstreet et al., 1997). While TSV can cause serious infections in P. stylirostris, serious epizootics in this species are unknown, even when the species is reared at farms where TSV is enzootic and causing serious epizootics in P. vannamei. Infectivity bioassays with the Asian penaeids, P. monodon, P. japonicus and P. chinensis, suggest that P. chinensis juveniles are moderately sus ceptible to TSV, whereas, P. monodon and P. japonicus

8 172 D. V. Lightner and R. M. Redman are highly resistant (Brock et al., 1997). Transmission of the virus by cannibalism, passive transmission by insects and birds, and vertical transmis sion are likely for TSV. The salinity-tolerant water boatman, Trichocorixa reticulata (Corixidae), a com mon inhabitant of shrimp grow-out ponds in much of the Americas, was collected from an Ecuadorian farm with an ongoing TSV epizootic and was demonstrated to contain infectious TSV by bioassay with SPF juve nile P. vannamei (Lightner, 1996b). In situ hybridiza tion assays run with histological sections of the water boatman showed several individuals with TSV positive gut contents, but no indication that TSV was infecting or replicating in the insect. Likewise, sea gulls (mostly laughing gulls, Larus atricilla) have been also shown to potentially serve as vectors of TSV. Gull feces, col lected from the levees of a TSV-infected pond in Texas during the 1995 epizootic, were found to contain infec tious TSV (Lightner, 1996b; Lightner et al., 1997c; Garza et al., 1997). Hence, gulls and other shrimp eat ing sea birds may transmit TSV within affected farms or to other farms within their flight range. What is not known is how long TSV remains in the gut contents of gulls or other sea birds and, thus, how important these birds might be in spreading this disease beyond a given region. Diagnosis of Taura syndrome: The current diagnostic and detection methods for TSV include histopathology, in situ hybridization with TSV specific complementary DNA (cdna) probes, bioassay with susceptible juve nile P. vannamei, and RT-PCR (Table 5). Diagnostic histopathology for TS may be applied to diagnosis of the disease in acutely affected shrimp that show gross signs of the disease. Shrimp with acute, natural, or induced TSV infections display a distinctive histopathol ogy that consists of multifocal areas of necrosis of the cuticular epithelium and subcutis (of the general cuticle, gills, appendages, foregut and hindgut). The lesion is characterized by the presence of several to extremely numerous, variably sized eosinophilic to basophilic cytoplasmic inclusion bodies that give TSV lesions a "peppered" or "buckshot" appearance, which is considered to be pathognomonic for the disease (Brock et al., 1995; Hasson et al., 1995, 1997a; Lightner et al., 1995; Lightner, 1996a). A bioassay test can be used as a diagnostic method for TSV to demonstrate the presence of the virus in asymptomatic carrier shrimp (or other appropriate samples). To accomplish this, SPF (specific pathogen free) juvenile P. vannamei are used as the indicator for the presence of the virus (Brock et al., 1995; Lightner, 1996a, 1996b). A cdna probe has been developed recently for TSV and it has been shown to provide excellent diagnostic sensitivity when used as a non-radioactive DIG-labeled probe with in situ hybridization assays on fixed tissue (Table 5). Intact cells within and near pathognomonic TS lesions show a very strong reaction with cdna probes by in situ hybridization assays (Lightner, 1996a; Hasson et al., 1997a). Application of PCR for the detection of TSV has recently been accomplished using sequence information from cloned cdna segments of the TSV genome (Lightner et al., 1997a, 1997b). Because TSV's nucleic acid is ssrna instead of DNA, the RNA template must be converted to cdna using reverse tran scriptase (RT) before the target nucleic acid segment can be amplified by RT-PCR. Primers were chosen from TSV sequence information which amplify a small ( `200 bp) segment of the TSV genome. The RT-PCR method has been successfully applied to the detection of TSV in hemolymph samples taken from TSV-infected shrimp in the acute, transitional, and chronic phase of the dis ease, and tissue homogenates following sucrose gradi ent purification of the virus. However, the successful use of RT-PCR for the detection of TSV in samples pre pared from fresh or frozen tissue homogenates has so far been problematic and will require further develop ment. This technical limitation may restrict the appli cation of RT-PCR technique to fresh hemolymph samples from shrimp and preclude its application to frozen or fresh whole shrimp samples, or the testing of PLs which are too small to bleed. Geographic distribution: Taura syndrome emerged in in Ecuador as a major epizootic disease of P. vannamei that spread rapidly throughout most of the shrimp growing regions of Latin America (Jimenez, 1992; Wigglesworth, 1994; Brock et al., 1995; Hasson et al., 1995, 1997a, 1997b; Lightner et al., 1995; Lightner, 1996a, 1996b). Although first recognized in Ecuador in mid-1992(jimenez, 1992), retrospective studies have shown that TS was present in at least one shrimp farm in the Taura region of Ecuador in Septem ber 1991 (Hasson et al., 1997a) and a TS-like condition has been reported to have occurred even earlier in cul tured P. vannamei in Colombia (Laramore, 1995). During 1993 and 1994, Taura syndrome epizootics occurred in shrimp farms throughout much of Ecuador, as well as in single or multiple farm sites in Peru, both coasts of Colombia, western Honduras, El Salvador, Guatemala, Brazil, and the United States, occurring at isolated sites in Florida and Hawaii (Rosenberry, 1993,

9 Control of shrimp viral diseases in the Americas a, 1994b; Brock et al., 1995; Lightner et al., 1995; Lightner, 1996a, 1996b; Hasson et al., 1995, 1997a). By mid-1996 the disease had expanded its distribution to include virtually all of the shrimp farming regions of the Americas. Regions or countries included in its expansion since 1994 and with documented cases in clude: both coasts of Mexico, Nicaragua, Belize, Costa Rica, Panama, and the U.S. states of Texas and South Carolina (Lightner, 1996a, 1996b; Hasson et al., 1997a). Laboratory challenge of the Asian penaeids, P. monodon, P. japonicus and P. chinensis, with TSV demonstrated that juvenile P. monodon and P. japonicus are highly resistant to TSV infection and disease, while P. chinensis juveniles are only moderately susceptible (Overstreet et al., 1997; Brock et al., 1997). Assum ing that TSV is not already enzootic in Asian penaeids, the degree of resistance to TSV demonstrated by these important Asian penaeid species suggests that the virus may not pose a significant threat to Asian shrimp aquac ulture. Disease Management Methods A variety of strategies have been attempted for the control of viral diseases in penaeid shrimp aquaculture. These strategies range from the use of improved culture practices (i.e. where sources of virus contamination are reduced or eliminated, sanitation practices are improved, stocking densities are reduced, etc.) to stocking "specific pathogen-free" (SPF) or "specific pathogen resistant" (SPR) species or stocks. Most recently, there have been some studies made on the use of vaccines and immunostimulants for the prevention of viral diseases in shrimp. In the Americas many strategies have been employed in efforts to reduce production losses due to the enzootic viruses, IHHNV, BP, and TSV. Prevention of BP in hatcheries: Improved hus bandry practices have been successfully employed for the control of BP, and for nearly a decade, this virus has seldom been reported as a serious constraint to success ful shrimp culture. This was accomplished because BP' s infection cycle can be interrupted with routine hatchery management practices. BP is a gut-infecting baculovirus which is transmitted from shrimp to shrimp exclusively per os (Johnson and Lightner, 1988; Overstreet et al., 1988; Overstreet, 1994). Hence, with BP, as well as with MBV and BMN (all gut-infecting baculoviruses of penaeid shrimp), infection from parent to off-spring in the hatchery has been prevented by elimi nating fecal contamination of spawned eggs by virus contaminated feces from spawning adults and by the use of adequate sanitation practices (Momoyama, 1988, a, 1989b, 1989c, 1989d). A number of innova 1989 tive methods have been developed for reducing or elimi nating fecal (containing baculovirus) contamination of spawned eggs. The simplest of these has been the use of hatching vessels in which embryonating shrimp eggs are rinsed with clean seawater, and in which hatched nauplii are passively rinsed by continuously flowing clean seawater and separated from contaminants and "diseased" siblings by collection using the normal phototaxic response of "healthy nauplii". Chemical rinses of spawned eggs and collected nauplii with disin fectants like chlorine, ozone, iodophores, and formalin are also commonly used in shrimp hatcheries in the Americas to help prevent BP, as well as vibriosis and other diseases. The use of routine sanitation and dis infection procedures for hatchery equipment and tanks after each use are also commonly used to prevent the occurrence of viral infections due to BP, or to limit their tank to tank spread when they do occur. Some hatcheries individually spawn their gravid females, collect any fecal strands, and scan these using simple bright field microscopy for BP occlusion bodies. In like manner, some hatcheries sacrifice broodstock females after spawning, excise the hepato pancreas (HP), and examine it for the presence of BP occlusion bodies using direct microscopy of tissue squash preparations. To prevent BP infections and dis ease from occurring in the larval rearing tank systems, the spawns from females, found to be BP-positive from examination of their feces or excised HP, are discarded. Avoidance through pathogen exclusion and develop ment of SPF shrimp: Disease management through exclusion of specific pathogens is commonplace in modern agriculture. This concept of developing stocks that are specific pathogen free (SPF) and rearing these stocks in regions where the specific pathogens of concern are excluded has been used in the Western Hemisphere with mixed success. The successful application of the SPF concept is, of course, dependent upon the absence of the pathogen(s) of concern in the stocks being reared (or that are present), on the availability of sensitive and accurate detection and diagnostic methods for the pathogen(s), and the presence of an effective barrier (i.e. geographic, government mandated import restrictions, etc.) to pre vent the introduction of the specific pathogen(s). In the Western Hemisphere, SPF stocks of P. stylirostris and P. vannamei have been developed and these are being cultured successfully in some locations

10 174 D. V. Lightner and R. M. Redman (Wyban, 1992; Wyban et al., 1992; Can et al., 1994; Pruder et al., 1995; Lightner, 1996b). The ICES Guidelines (Sindermann, 1990), were followed for the development of these stocks. The determination of which specific pathogens the selected stocks were to be free from was based on a working lists of specific, ex cludable pathogens (Wyban, 1992; Lotz et al., 1995). The most current working list includes eight viruses (WSSV, YHV, TSV, IHHNV, HPV, BP, MBV, and BMN), certain classes of parasitic protozoa (micro sporidians, haplospordians, and gregarines), and helm inth parasites (cestodes, trematodes, and nematodes). In the spirit of the ICES Guidelines, each "SPF candi date population" of wild or cultured shrimp stocks of interest was identified. Samples of the stock were taken and tested using appropriate diagnostic and patho gen detection methods for the specific pathogens of concern. If none were found, a founder population (F0) of the "candidate SPF" stock was acquired and reared in primary quarantine. During primary quarantine, the F0 stock was monitored for signs of disease, sampled, and tested periodically for specific pathogens. If any patho gens of concern were detected, the stock was destroyed. Those stocks that tested negative for pathogens of con cern through primary quarantine (which ran from 30 days to as much as 1 year for some stocks) were moved to a separate secondary quarantine facility for matura tion, selection, mating, and production of a second (F1) generation. The F1 stocks were maintained in quaran tine for further testing for specific pathogens of concern. Those that tested negative were designated as SPF and, used to produce domesticated lines of SPF and "high health" (Wyban et al., 1992; Pruder et al., 1995). SPF and high health stocks of P. vannamei were used suc cessfully in U.S. shrimp farms in 1993 and 1994, and resulted in nearly double the production that had been obtained at the same farms in previous years when the farms cultured non selected lines of P. vannamei, which, had been persistently affected by "runt deformity syn drome" (RDS) due to chronic infection by IHHNV (Pruder et al., 1995; Lightner, 1996a, 1996b). Another interesting application of disease manage ment through avoidance and the use of SPF stocks began two years ago in Belize. The shrimp culture industry in this Central American nation is relatively small with only eight farms (Table 3). Belize was seriously im pacted by the TSV panzootic in 1994 (Lightner, 1996b; Dixon and Dorado, 1997). The shrimp farms in Belize are geographically isolated from other shrimp farming regions in adjacent countries (because most of these are situated on the Pacific coast), and with its location on the Caribbean side of the continent, Belize has no natu rally occurring wild stocks of the penaeid species (P. vannamei and P. stylirostris) likely to serve as reservoir hosts for TSV and IHHNV. Belize was uniquely suited to attempt to eradicate TSV and IHHNV. Seven of its farms were depopulated of all their shrimp stocks in late 1995; then each farm was thoroughly disinfected and dried out to eradicate potential sources of these viruses. The eradication program included pond disinfection (liming pond bottoms with calcium oxide at 5,000 kg/ha or chlorine at 10 ppm residual for h), pond dry-out and bottom tilling (to a depth of `10 cm to ensure oxida tion of contaminated pond-bottom detritus), farm imple ment and building disinfection (with chlorine or with formalin gas), spraying insecticides (to kill potential reservoir hosts such as wild crabs and shrimps in supply drainage canals), and removal of frozen shrimp from storage from the country's packing plants (Dixon and Dorado,1997). For the 1996 and 1997 seasons, these farms were stocked exclusively with SPF P. vannamei. TSV has not been detected in Belize since 1995, and IHHNV has been found only at low prevalence rates. In the absence of TSV, the per crop average production of P. vannamei in 1996 at one farm was 891 kg/ha (heads on) with a stocking density of 15.5 PL/m<SUP>2<SUP>, and survival to harvest has averaged 67%. In comparison, the same farm's production during the TSV panzootic of 1994 was 390 kg/ha (stocked at 18.3 PLs/m<SUP>2<SUP>) with a survival of 36% (Dixon and Dorado, 1997). While the Belize experiment in TSV and IHHNV eradication may have been successful, successful duplication of its accom plishments elsewhere in the Americas may not be fea sible. In these regions where total stock eradication is not feasible, other methods for viral disease management are being used. Successful application of the ICES Guidelines and the SPF concept requires that specific pathogens are exclud able. In situations where specific pathogens may not be excludable, the development and use of SPR stocks may be the only alternative. The fact that IHHNV and TSV have become widely distributed in the Americas indicates that either government or industry supported pathogen exclusion mechanisms and regulations must be implemented and enforced to achieve the goals of using SPF shrimp stocks, or, alternatively, that SPR stocks be developed and used. Farm management schemes used for virus control: The management of IHHNV and TSV by the aquacul ture industries of the Americas has ranged from total

11 Control of shrimp viral diseases in the Americas 175 avoidance of the disease through the use of specific pathogen free (SPF) stocks and attempts to exclude these viruses, to modifying farm management methods to re duce the impact of the disease, and to the use of IHHNV and/or TSV resistant species or stocks. In Ecuador and much of Central America where Taura syndrome became widespread and well established before its viral etiology was established in late 1994 (Hasson et al., 1995; Brock et al., 1995), shrimp farmers changed farm management and operational procedures in an effort to reduce the impact of the disease (Stern, 1995; Brock et al., 1997). The use of wild caught PL (P. vannamei), versus hatch ery reared PLs, gave improved crop survivals at harvest. Perhaps the wild PLs had either a lower prevalence of TSV infections when stocked than was typical of hatch ery reared PLs, or, possibly, some increased resistance to TS due to virus exposure and natural selection of TSV resistant shrimp in the wild. Another management strategy employed to lessen the impact of TS was the practice of stocking PLs at two or more times the normal stocking density for semi-inten sive pond culture. Farms employing this strategy would experience a heavy mortality due to TSV infections early in the culture cycle (during the late PL or early juvenile stages and before substantial supplemental feeding had begun). The survivors of the naturally occurring TSV epizootic were resistant to subsequent infection and dis ease if challenged by TSV (Ligntner et al., 1997b; Lotz and Ogle, 1997). Hence, with this strategy, farms could obtain post-tsv survival rates of 10 to 40% of the original number stocked, and the resulting juveniles could be managed and grown out to market size as a normal crop of P. vannamei (Stern, 1995). In a study in which different size classes of juvenile SPF P. vannamei were challenged with TSV, Lotz (1997) found that size alone does not affect mortality rates of P. vannamei, and that larger shrimp size alone does not confer resistance to TSV. Related work suggests that persistent infection by TSV in survivors of TS epizootics imparts resistance to subsequent challenge (Hasson and Lightner, unpublished data). Polyculture of shrimp with an omnivorous fish species also shows promise as a management method for reduc ing the impact of TSV in some shrimp growing coun tries (Wang et al., 1997). With this strategy, tilapia are grown in polyculture with shrimp like P. vannamei, and presumably, the tilapia consume dead and dying shrimp (from TSV) keeping other shrimp in the pond from becoming infected with the virus by cannibalism. With this method, improvements in shrimp production have been reported and a secondary crop of marketable fish is obtained (Green, 1997). The application of dietary immunostimulants as management tools for Taura syndrome has also been reported (Brock et al., 1997; Dixon and Dorado, 1997; Klesius and Shoemaker, 1997; D. Dugger, personal com munication, Immunodyne, Inc, Brownsville, TX). From slight improvements in survival (Klesius and Shoemaker, 1997; Dixon and Dorado, 1997) to survival rates of TSV-challenged P. vannamei comparable to unchallenged control groups have been reported (Dugger, personal communication). Development and use of specific pathogen resistant (SPR) stocks: One alternative approach to developing SPF domesticated shrimp stocks, is to select and breed survivors of "specific pathogen-infected" (by pathogens like IHHNV or TSV) stocks to develop "specific patho gen-resistant" or SPR stock. Following this scheme, French researchers successfully developed a stock of IHHNV resistant P. stylirostris in French Polynesia (Weppe et al., 1992; Lightner, 1996b). This stock has been used successfully to develop the shrimp culture industries of Tahiti and New Caledonia. Recently, the stock was introduced into an area of southwestern Mexico where IHHNV is enzootic in an effort to develop the stock as an alternative to the slower growing P. vannamei which currently makes up >90% of the shrimp farmed in Mexico (Rosenberry, 1996). In view of the recent accidental introduction and spread of TSV in Mexico, the potential availability of this SPR stock, which is resistant to disease when infected by IHHNV and TSV, may provide a viable alternative to the culture of the highly TSV susceptible stocks of P. vannamei and the highly IHHNV susceptible native stocks of P. stylirostris, which were previously the only viable options for shrimp aquaculture development in Mexico. Perhaps the most promise for the control of Taura syndrome and IHHN disease lies in the development and use of selected TSV resistant stocks of P. vannamei (Carr et al., 1997), or in the use of recently developed strains of P. stylirostris which are resistant to both IHHNV and TSV (Weppe et al., 1992; Lightner, 1996b; Brock et al., 1997). Until recently, the popularity and use of the relatively IHHNV resistant species P. vannamei, in preference to the culture of the more IHHNV susceptible P. stylirostris, was characteristic of the shrimp farming industries of the Americas. The popularity of P. vannamei began to decline when TSV emerged as a serious pathogen of this species in 1992 and then spread to virtually all of the shrimp growing

12 176 D. V. Lightner and R. M. Redman regions of the Americas during the ensuing four years (Lightner, 1996b). Because P. stylirostris was found to be innately TSV resistant, two domesticated, geneti cally selected SPR strains of this species, which are resistant to IHHN disease, are being developed currently and marketed in the Americas. Currently, there are two stocks of SPR P. stylirostris being tested or marketed in the Americas. The first of the stocks, designated as SPR-43, was developed by the French research agency, IFREMER, in French Polynesia and in New Caledonia. This was accomplished by breeding generations of IHHN survivors in at the IFREMER stations in Tahiti and New Caledonia. After several generations, sur vival and culture performance of the stock improved. The stock was found to carry IHHNV at low rates of prevalence and severity of infection. When experi mentally challenged with IHHNV, the SPR-43 stock was found to be resistant to IHHN disease (Weppe et al., 1992). The SPR-43 stock is being experimentally cul tured by one farm in Sinaloa, Mexico and it has been found to perform better than P. vannamei at the farm because of its innate resistance to TSV. A second line of SPR P. stylirostris was developed in Venezuela. Its development followed the same strategy as was used by the IFREMER in Tahiti and New Caledonia. After an initial introduction of a founder stock of P. stylirostris into a particular farm in Venezu ela, the stock became IHHNV infected. Generations of IHHN survivors were selected and reared until that stock began to perform as well as did the normally IHHNV resistant stocks of P. vannamei at the same farm. When the TSV panzootic swept through the Americas, the Venezuelan stock of P. stylirostris was found to be TSV resistant. This stock possesses resis tance to IHHNV and TSV, and it is being marketed as Super ShrimpTM in the Americas. In some regions, the SPR stocks of Super ShrimpTM (P. stylirostris) are replacing P. vannamei stocks in culture. As much as half of the shrimp farms in the Mexican states of Sonora and Sinaloa were stocked with this TSV and IHHNV resistant breed of P. stylirostris for the 1997 season. Initial harvests from the region show significant provements in production and survival rates compared to the previous season when the TSV panzootic caused mas sive losses in the P. vannamei crops at the same farms. Other shrimp farming interests are using wild or selected, domesticated SPF/SPR stocks of P. vannamei that show improved resistance to TSV. Resistance to TSV was used as a criterion for selection and develop ment of new lines of domesticated stocks of P. vannamei. im Selected stocks in laboratory challenge studies with TSV and in pond trials at farms where the virus is enzootic have shown significant survival advantages over non selected stocks (Carr et al., 1997). Natural selection for TSV resistance appears to be occurring in wild stocks as well. In regions like Ecuador and Honduras where TSV has been enzootic for several years, the practice of direct stocking of farms with wild PLs is providing steadily improving survival rates, even though shrimp displaying classic signs of TSV infection are common place. The same selective process for IHHNV resistance seems to be occurring in some wild stocks of P. stylirostris (Lightner, 1996b). Initial efforts to select and breed TSV resistant, domesticated strains of P. vannamei have resulted in improvements in harvest survivals of 20 to 40% (Lightner, 1996b). Similarily, the use of TSV and IHHNV resistant strains of P. stylirostris have resulted in improved harvest survival and crop values in regions where TSV and IHHNV are enzootic. Acknowledgements Funding for this research was provided by the Gulf Coast Research Laboratory Consortium Marine Shrimp Farming Program, CSREES, USDA under Grant No , the National Sea Grant Program, USDC under Grant No. NA56RG0617, the National Marine Fisheries Service (Saltonstall-Kennedy Act), USDC under Grant No. NA56FD0621, and a special grant from the National Fishery Institute. References Adams, J. R. and J. R. Bonami (eds. ) (1991): Atlas of inverte brate viruses. CRC Press, Boca Raton, FL. 684 p. Bell T. A. and D. V. Lightner (1984): IHHN virus: Infectivity and pathogenicity studies in Penaeus stylirostris and Penaeus vannamei. Aquaculture, 38, Bell T. A. and D. V. Lightner (1987): IHHN disease of Penaeus stylirostris: effects of shrimp size on disease expression. J. Fish Dis., 10, Bell, T. A. and D. V. Lightner (1988): A handbook of normal shrimp histology. Special Publication No. 1, World Aquac ulture Society, Baton Rouge, LA, USA, 114 p. Bonami, J. R., M. Brehelin, J. 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