魚 病 研 究 Fish Pathology,33(4),165-180,1998.10

Strategies for the Control of Viral Diseases of in the Americas

Donald V. Lightner and R. M. Redman

Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona 85721 U.S.A.

(Received February 6, 1998)

Viral diseases have severely impacted many of the penaeid industries of the world causing significant production and economic losses. Nearly 20 distinct , 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 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 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

among which the viruses that infect penaeid shrimp are Introduction especially well represented. The penaeid shrimp culture industries of the world In 1974 the first known shrimp virus was described developed from their experimental beginnings three de and named Baculovirus penaei (BP = PVSNPV) (Couch, cades ago into major industries providing millions of a, 1974b; Bonami et al., 1995). By 1996,1974 the list jobs and billions of U. S. dollars annually in export rev of penaeid shrimp viruses had grown to include at least enue (Weidner and Rosenberry, 1992;Wang et al., 1995; 20 viruses or unique strains of viruses representing seven Rosenberry, 1996). Concomitant with the rapid growth virus families (Tables 1-2), with all but two or three of of the shrimp culture industries has been the recognition these 20 viruses having been described in penaeid of the ever increasing importance of pathogenic agents shrimp from aquaculture settings (Lightner, 1996a, b). While some of the known1996 penaeid shrimp viruses seem to be of little economic importance, others FAX: 1520621-4899 E-mail: [email protected] can cause serious disease in their penaeid shrimp hosts 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 et al., 1995; Fraser and Owens, 1996). Some well culture them. Devastating epizootics due to various known examples of major virus caused disease epizoot virus pathogens of penaeid shrimp have caused signifi ics in Asian shrimp include 's 1987-1988 epi cant, and sometimes catastrophic economic losses, in zootic when production fell from 100,000t to 30,000t commercial penaeid shrimp culture (Boonyaratpalin et (Chen, 1995). In 1992-1993 the Chinese shrimp al., 1993; Brock, 1991; Lightner, 1988, 1992; Stern, aquaculture industry saw their production collapse from 1995; Takahashi et al., 1994; Fraser and Owens, 1996; 220,000t in 1991 to 30,000 t in 1993 (Wang et al., 1995; Flegel, 1997). Rosenberry, 1994a, 1994b;Chamberlain, 1994). Like Viral pathogens have been implicated in the collapse wise, recent major epizootics in , , of important shrimp aquaculture industries in Asia and Japan, Taiwan, and India have been accompanied by the Indopacific region. Even Australia, with its rela significant losses that, in some instances, reached 50 to tively small penaeid culture industry, has experi 90% of expected production for the year (Flegel et al., enced serious epizootics due to viral pathogens (Spann b; Winamo 1995; Flegel, 1997; Lo et al., 1996).1995 Control of shrimp viral diseases in the Americas167

Table 2. Viruses reported from Eastern (Asian, Australian , Table 3. Major shrimp producing countries in the Western European, and African) and Western Hemisphere Hemisphere: production and number of farms in (the Americas and Hawaii) of penaeid shrimp (com 1996 (Modified from Rosenberry 1996) piled from Lightner 1996a)

ducer of farmed shrimp after Thailand with its approxi mately 160,000t of production in 1996. After , other large shrimp farming countries in the Americas (and their approximate 1996 productions) include (12,000 t), (10,000 t), (5,000 t), (3,000 t), (2,000 t), (2,000 t), and the United States (1,300 t), and a few other coun tries like , , , and various The shrimp culture industries of the Americas have Caribbean island countries that collectively produced also been adversely affected by serious epizootics due another —16,000t in 1996 (Rosenberry, 1996). to viral pathogens. Infectious hypodermal and hemato In terms of significant production, only two penaeid poietic necrosis virus (IHHNV) (Brock and Lightner, shrimp species are farmed in the Americas. Of these, 1990; Lightner, 1993, 1996a, 1996b; Lightner et al., Penaeus vannamei (the Pacific white shrimp) accounts a, 1992b) and Taura syndrome virus (TSV) (Cham1992 for 80 to 90% of the total production. P. stylirostris, berlain, 1994; Brock et al., 1995; Hasson et al., 1995; the Pacific blue shrimp is next in importance accounting Lightner et al., 1995; Lightner, 1996a, 1996b; Bonami for 10 to 20% of the production, and its importance is et al., 1997) have had enormous negative impacts on the increasing rapidly because it is more resistant to TSV continents' developing aquaculture industries and, in one infection and disease than is P. vannamei. Other spe instance for IHHNV, on a commercial fishery as well cies such as P. setiferus and P. schmitti (the Gulf of (Moore and Brand, 1993; Lightner, 1996b; Pantoja et Mexico and Caribbean white shrimp, respectively) and al., "in press"). This paper reviews the published P. californiensis (the Pacific brown shrimp), are impor literature and as yet unpublished (but available anecdot tant in some locations, but they account for less than a ally) recent information on BP, IHHNV, and TSV in few percent of the total shrimp aquaculture production penaeid shrimp aquaculture in the Western Hemisphere in the Americas (Rosenberry, 1996). and the strategies used by the aquaculture industry to The methods of culture employed by shrimp farmers manage the diseases caused by these pathogens. are similar throughout the Americas. The principal differences among the producers is the source of "seed"

Western Hemisphere Shrimp Aquaculture Industries (PLs or postlarvae) used to stock the farms. Most of the grow-out shrimp farms in the Americas are semi The shrimp farming industry of the Western Hemi intensive and consist of large ponds of 1 to 20 ha with sphere produced approximately 25% of the world's earthen dikes. Stocking densities in most semi-inten farmed shrimp in 1996 (Table 3). This amounted to sive shrimp farms in the Americas range from- less than 172,300 metric tons (t). Ecuador accounted for 70% 5 per m2to 50 per m2. There are a few intensive farms of this total, which ranks it as the second largest pro in the Americas. These farms typically have pond sizes 168D. V. Lightner and R. M. Redman of 0.25 ha to 5 ha, mechanical aeration, and stocking The Major Viruses in the Americas rates that range from 35 to 200 shrimp per m2. Some regions of the Americas (Venezuela, Brazil, the Although nearly 10 of the known shrimp viruses have Caribbean island nations, and the United States) rely been found in cultured penaeid shrimp in the Americas almost entirely on hatcheries and domesticated brood (Table 2), only three of these have caused panzootic stock for the production of "seed". However, the disease in the Western Hemisphere (Lightner, 1996a, major shrimp producers in the Western Hemisphere rely b). These three viruses, or perhaps1996 more correctly on the use of wild broodstock or wild seed (PLs) for groups of different strains of the same virus group their stock. In Ecuador for example, wild PLs are include BP, IHHNV, and TSV (Table 4). available for much of the year (and nearly, but not, every year) in the coastal zone and estuaries. Nearly BP-Type Baculoviruses and Disease 100,000 people in Ecuador make a living collecting BP disease: BP (Baculovirus penaei or PVSNBV) wild PLs with special fine mesh push nets (called has been reported to cause significant disease in the lar chinchorros') from beaches and estuaries, and 'selling val, postlarval, and early juvenile stages of several the wild PLs that they collect to farms. When wild PLs penaeid species in the Americas (Couch, 1974a, 1974b; are not abundant, the approximately 300 penaeid shrimp Lightner et al., 1985; Johnson and Lightner, 1988; hatcheries in Ecuador (which are called "laboratories" Overstreet et al., 1988; LeBlanc and Overstreet, 1990; in Ecuador as well as in most of the Americas) collect Bueno et al., 1990; Krol et al., 1990; Bonami et al., wild adult shrimp from the coastal fishery, spawn them, 1995; Lightner, 1996a). BP is known to cause signifi and use the resultant larvae to produce the PLs needed cant disease in larval and postlarval stages of P. aztecus, to stock farms. The hatcheries may produce PLs from P. stylirostris and P. vannamei (Couch, 1991; Johnson "wild nauplii" (which are produced from wild-caught, and Lightner, 1988; Lightner and Redman, 1991, 1992; fertilized females within 1-2 days of capture) or from LeBlanc and Overstreet, 1990, 1991; LeBlanc et al., "maturation nauplii/seed" (which are produced from 1991). BP has been a sporadically occurring, but captive wild males and female adults that are matured often serious, hatchery disease of the larval stages of and mated in specialized "maturation" units in the hatch P. vannamei in many of the commercial hatcheries on eries) (Rosenberry, 1996). the Pacific Coast side of Central and South America, The dependence of the shrimp aquaculture industry including Peru, Ecuador, Columbia, Panama, Costa on wild broodstock and wild PLs as the principal source Rica, Honduras, and on the Atlantic side of the conti of its stock has limited the industry's options for disease nent in Brazil, and in the United States management and prevention. This dependence on wild (Bueno et al., 1990; Lightner and Redman, 1991, 1992; stock has left the industry vulnerable to major viral Lightner, 1996a). In Mexico, BP has caused serious disease outbreaks like the panzootic due to IHHNV in epizootics in cultured larval and post larval P. stylirostris the 1970's and 1980's and the more recent panzootic (Lightner et al., 1989). A morphologically and geneti due to TSV that began in Ecuador in 1991-1992 and cally unique strain of BP occurs in wild P. marginatus spread throughout the Americas (Lightner, 1996b; in Hawaii (Lightner et al., 1985, 1994; Brock et al., Hasson et al., 1997a; Brock et al., 1997). 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 Control of shrimp viral diseases in the Americas169

BP infects only the hepatopancreas and midgut BMN (Momoyama, 1988; Lightner, 1996a). epithelial cells, and it is transmitted from shrimp to Diagnosis and detection of BP: Classical micro shrimp exclusively per os. The typical route of infec scopic, histological methods, and molecular methods are tion of shrimp larvae in hatcheries is via fecal (from BP available for detection of BP virus and the diagnosis of infected adult spawners) contamination of spawned the disease it causes (Table 5). Patent infections by BP eggs (Johnson and Lightner, 1988; Lightner, 1996a). are easily diagnosed by the demonstration of prominent Additional routes of horizontal transmission of BP (and tetrahedral occlusion bodies in unstained squash prepa other gut-infecting baculoviruses) in larval culture sys rations of hepatopancreas, midgut or feces, or in appro tems results from fecal-oral contamination (via feces priate histological sections from infected animals. In from infected larvae) or from cannibalism of diseased histological sections, these occlusion bodies are demon larvae (Sano et al., 1985; Momoyama, 1988; Overstreet strated to occur as single or multiple, eosinophilic, usu et al., 1988; Momoyama and Sano, 1989; LeBlanc and ally triangular, inclusion bodies within hypertrophied Overstreet, 1990, 1991; Sano and Momoyama, 1992). nuclei of hepatopancreatic tubule epithelial cells or less Experimental direct transmission of BP virus from frequently in midgut epithelial cells (Lightner, 1996a). shrimp to shrimp has demonstrated that it has an incu Molecular detection methods have been developed for bation period of about 24 h (Overstreet et al., 1988), BP. In situ hybridization assays of Davidson's pre which is similar to incubation periods for MBV and 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) 170D. V. Lightner and R. M. Redman

labeled probes and PCR methods have been developed virus: IHHNV is the smallest of the known penaeid and found to be more sensitive for detection of BP in shrimp viruses (Bonami et al., 1990; Adams and infected tissues than the classical methods of direct mi Bonami, 1991). The IHHN virion is a non-enveloped croscopy, histopathology, and serology with polyclonal icosahedron averaging 22 nm in diameter; it has a den antibodies (Lewis, 1986; Bruce et al., 1991, 1993, sity of 1.40 g/ml in CsCl, contains linear ssDNA with an a, 1994b; Lightner et al., 1992c; Bonami et1994 al., estimated size of 4.1 kb, and its contains four 1995). Polymerase Chain Reaction (PCR) methods polypeptides with molecular weights of 74, 47, 39, and have been developed for the detection of BP, and the 37.5 kDa. Because of these characteristics, IHHNV method has been successfully applied to the detection of has been classifiedas a member of the family Parvoviridae BP in feces (Lightner et al., 1994; Lightner, 1996a; (Bonami et al., 1990). Wang et al., 1996). This application of PCR may IHHN disease: Infection by IHHNV causes serious eventually be used as a non-destructive method to test disease in P. stylirostris, and acute catastrophic epizoot valuable broodstock individually for their BP infection ics in semi-intensively or intensively cultured juveniles status. of that species (Lightner et al., 1983; Bell and Lightner, Morphometric comparisons of BP virions and nucleo 1987; Brock and Lightner, 1990; Lightner, 1996a). In from different geographic regions of North, other penaeids like P. vannamei, IHHNV was recog Central, and South America and Hawaii suggest that two nized soon after its discovery in P. stylirostris to infect or three distinct strains of BP probably occur (Lightner and cause disease. However, infected P. vannamei was et al., 1985; Bonami et al., 1995). In morphometric considered to be highly resistant to IHHN disease rela studies, BP virion nucleocapsids in Ecuadorian P. tive to P. stylirostris (Bell and Lightner, 1984). How vannamei were found to be slightly larger than BP ever, despite its relative resistance to IHHN disease, "runt nucleocapsids in Gulf of Mexico P. aztecus and P. deformity syndrome" (RDS) in cultured P. vannamei duorarum, while both were considerably larger than the was linked by epizootiological data to infection by BP nucleocapsids found in Hawaiian P. marginatus IHHNV (Kalagayan et al., 1991; Browdy et al., 1993; (Couch, 1974a, 1974b; Lightner et al., 1985; Brock et Castille et al., 1993). Affected shrimp with RDS are al., 1986). Bruce et al. (1993) compared Ecuadorian characterized by variable, often greatly reduced growth isolates of BP with other geographic strains using a panel rates and by a variety of cuticular deformities affecting of five probes developed to the Ecuadorian strain of BP. the rostrum (resulting in a "bent rostrum"), antennae, The probes distinguished at least three different strains and other thoracic and abdominal areas of the exoskel of BP virus (Table 6). eton (Brock and Main, 1994; Kalagayan et al., 1991; Lightner, 1992, 1996a). RDS is an economically sig IHHN Virus and Disease nificant disease of cultured P. vannamei, which has been Infectious hypodermal and hematopoietic necrosis 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* Control of shrimp viral diseases in the Americas171 which the species is cultured. Cultured populations Distribution of IHHN: The virus has a world-wide affected by RDS may contain up to 30% runts, and con distribution and wide host range in cultured penaeid sequently a wide distribution of size ("count" or the shrimp, but its original distribution in wild penaeids re number of shrimp per pound) classes. Because small-size mains unknown (Lightner and Redman, 1991; Lightner, (runts) shrimp have a lower market value than a, 1996b). However, the occurrence in Southeast1996 unaffected shrimp, RDS significantly reduces the Asia (Singapore, Malaysia, Indonesia, and the Philip market value of affected P. vannamei crops, resulting in pines) of IHHNV (or a similar agent) in shrimp culture revenue losses that can range from 10 to 50% of the facilities using only captive-wild P. monodonbroodstock, value of similar IHHNV-free (and RDS-free) crops and where Western Hemisphere American penaeids had (Kalagayan et al., 1991; Wyban, 1992). not been introduced, suggests that the region is within Diagnosis and detection of IHHN: Traditional the virus' natural geographic range, and that P. monodon methods employing histology (Brock, 1992; Brock and may be among its natural host species (Lightner, 1996a, Main, 1994; Lightner, 1993, 1996a) and molecular b). Molecular comparisons of 1996IHHNV isolates methods that use non-radioactively DIG labeled gene from various locations in the Americans and Asia using probes are the current methods of choice for diagnosis PCR and in situ hybridization with probes from regions of infection by IHHNV (Table 5) (Lightner, 1996a, of the IHHNV have not shown distinguishable b). Although monoclonal antibodies1996 have been differences among geographic isolates of IHHNV. developed for IHHNV, their use has been hampered by their cross reactivity to non-viral substances in normal Taura Syndrome Virus and Disease shrimp tissue (Poulos et al., 1994). Histological Taura syndrome virus (TSV): Taura syndrome virus demonstration of prominent Cowdry type A inclusion (TSV) is one of the most recently characterized penaeid bodies (=CAIs) provides a definitive diagnosisof IHHN. shrimp virus. TSV has been classified with the These pathognomonic IHHN inclusion bodies are intra Picornaviridae based on its morphology (a 31-32 nm nuclear, eosinophilic (when stained with H & E stains icosahedron), its cytoplasmic replication, its buoyant of tissues preserved with fixatives that contain acetic density of 1.338 g/ml, its genome consisting of a linear, acid, such as Davidson's AFA and Bouin's solution positive-sense, single stranded RNA (ssRNA) of ap {Bell and Lightner, 1988; Lightner, 1996a}) inclusion proximately 9 kb in length, and its having three major bodies, within chromatin-marginated, hypertrophied (55,40, and 24 kDa) and one minor polypeptides (58 kDa) nuclei of cells in tissues of ectodermal (epidermis, hy comprising its capsid (Hasson et al., 1995; Lightner, podermal epithelium of fore and hindgut, nerve cord and a; Bonami et al., 1997). 1996 nerve ganglia) and mesodermal origin (hematopoietic Taura syndrome disease: TSV is known to infect a organs, antennal gland, gonads, lymphoid organ, and number of penaeid shrimp species. It causes serious connective tissue) (Lightner, 1996a). disease in the PL, juvenile and adult stages of P. vannamei Non-radioactive DIG-labeled gene probes to IHHNV (Brock et al., 1995; Lightner et al., 1995; Lightner, and PCR methods for detection of the virus have been a). In larval and early PL P. vannamei, 1996infection developed (Mari et al., 1993; Lightner et al., 1992c, by TSV is apparently not expressed until about PL-11 1994; Lightner, 1996a). DIG-labeled DNA probes for or PL-12 when severe disease and mortalities have been IHHNV are now commerciallyavailable as ShrimProbeTM noted in infected populations (Lightner, 1996a). The kits from DiagXotics, Inc. (Wilton, CT, USA) in dot blot American penaeids P. stylirostris, P. schmitti, P. and in situ hybridization formats (Table 5). These setiferus and P. aztecus can also be infected by TSV, methods provide greater diagnostic sensitivity than do but serious disease resulting from infection has only more traditional methods for IHHN diagnosis that em been reported for the PL stages of P. setiferus (Overstreet ploy classic histological methods. Valuable broodstock et al., 1997). While TSV can cause serious infections shrimp may be examined for IHHNV infection with gene in P. stylirostris, serious epizootics in this species are probes by use of a non-lethal biopsy or by testing a unknown, even when the species is reared at farms where hemolymph sample. In this method, a hemolymph TSV is enzootic and causing serious epizootics in P. sample may be taken with a tuberculin syringe or an vannamei. Infectivity bioassays with the Asian appendage (a pleopod for example) may be biopsied and penaeids, P. monodon, P. japonicus and P. chinensis, used as the sample for a direct dot blot test or PCR assay suggest that P. chinensis juveniles are moderately sus for IHHNV (Lightner, 1996a). ceptible to TSV, whereas, P. monodon and P. japonicus 172D. V. Lightner and R. M . Redman

are highly resistant (Brock et al., 1997). al., 1995; Lightner, 1996a, 1996b).

Transmission of the virus by cannibalism, passive A cDNA probe has been developed recently for TSV

transmission by insects and birds, and vertical transmis and it has been shown to provide excellent diagnostic

sion are likely for TSV. The salinity-tolerant water sensitivity when used as a non-radioactive DIG-labeled

boatman, Trichocorixa reticulata (Corixidae), a com probe with in situ hybridization assays on fixed tissue

mon inhabitant of shrimp grow-out ponds in much of (Table 5). Intact cells within and near pathognomonic the Americas, was collected from an Ecuadorian farm TS lesions show a very strong reaction with cDNA

with an ongoing TSV epizootic and was demonstrated probes by in situ hybridization assays (Lightner, 1996a; to contain infectious TSV by bioassay with SPF juve Hasson et al., 1997a). Application of PCR for the

nile P. vannamei (Lightner, 1996b). In situ hybridiza detection of TSV has recently been accomplished using

tion assays run with histological sections of the water sequence information from cloned cDNA segments of the

boatman showed several individuals with TSV positive TSV genome (Lightner et al., 1997a, 1997b). Because gut contents, but no indication that TSV was infecting TSV's nucleic acid is ssRNA instead of DNA, the RNA

or replicating in the insect. Likewise, sea gulls (mostly template must be converted to cDNA using reverse tran

laughing gulls, Larus atricilla) have been also shown to scriptase (RT) before the target nucleic acid segment can

potentially serve as vectors of TSV. Gull feces, col be amplified by RT-PCR. Primers were chosen from lected from the levees of a TSV-infected pond in Texas TSV sequence information which amplify a small (•`200

during the 1995 epizootic, were found to contain infec bp) segment of the TSV genome. The RT-PCR method

tious TSV (Lightner, 1996b; Lightner et al., 1997c; has been successfully applied to the detection of TSV in

Garza et al., 1997). Hence, gulls and other shrimp eat hemolymph samples taken from TSV-infected shrimp

ing sea birds may transmit TSV within affected farms or in the acute, transitional, and chronic phase of the dis

to other farms within their flight range. What is not ease, and tissue homogenates following sucrose gradi

known is how long TSV remains in the gut contents of ent purification of the virus. However, the successful

gulls or other sea birds and, thus, how important these use of RT-PCR for the detection of TSV in samples pre

birds might be in spreading this disease beyond a given pared from fresh or frozen tissue homogenates has so region. far been problematic and will require further develop

Diagnosis of Taura syndrome: The current diagnostic ment. This technical limitation may restrict the appli

and detection methods for TSV include histopathology, cation of RT-PCR technique to fresh hemolymph

in situ hybridization with TSV specific complementary samples from shrimp and preclude its application to

DNA (cDNA) probes, bioassay with susceptible juve frozen or fresh whole shrimp samples, or the testing of

nile P. vannamei, and RT-PCR (Table 5). Diagnostic PLs which are too small to bleed.

histopathology for TS may be applied to diagnosis of Geographic distribution: Taura syndrome emerged

the disease in acutely affected shrimp that show gross in 1991-1993 in Ecuador as a major epizootic disease

signs of the disease. Shrimp with acute, natural, or of P. vannamei that spread rapidly throughout most of

induced TSV infections display a distinctive histopathol the shrimp growing regions of Latin America (Jimenez,

ogy that consists of multifocal areas of necrosis of the 1992; Wigglesworth, 1994; Brock et al., 1995; Hasson

cuticular epithelium and subcutis (of the general et al., 1995, 1997a, 1997b; Lightner et al., 1995;

cuticle, gills, appendages, foregut and hindgut). The Lightner, 1996a, 1996b). Although first recognized in

lesion is characterized by the presence of several to Ecuador in mid-1992(Jimenez, 1992), retrospective

extremely numerous, variably sized eosinophilic to studies have shown that TS was present in at least one

basophilic cytoplasmic inclusion bodies that give TSV shrimp farm in the Taura region of Ecuador in Septem

lesions a "peppered" or "buckshot" appearance, which ber 1991 (Hasson et al., 1997a) and a TS-like condition

is considered to be pathognomonic for the disease has been reported to have occurred even earlier in cul

(Brock et al., 1995; Hasson et al., 1995, 1997a; Lightner tured P. vannamei in (Laramore, 1995).

et al., 1995; Lightner, 1996a). A bioassay test can be During 1993 and 1994, Taura syndrome epizootics

used as a diagnostic method for TSV to demonstrate the occurred in shrimp farms throughout much of Ecuador, presence of the virus in asymptomatic carrier shrimp (or as well as in single or multiple farm sites in Peru, both

other appropriate samples). To accomplish this, SPF coasts of Colombia, western Honduras, , (specific pathogen free) juvenile P. vannamei are used , Brazil, and the United States, occurring at

as the indicator for the presence of the virus (Brock et isolated sites in Florida and Hawaii (Rosenberry, 1993, Control of shrimp viral diseases in the Americas 173

1994a, 1994b; Brock et al., 1995; Lightner et al., 1995; contaminated feces from spawning adults and by the use Lightner, 1996a, 1996b; Hasson et al., 1995, 1997a). of adequate sanitation practices (Momoyama, 1988, By mid-1996 the disease had expanded its distribution a, 1989b, 1989c, 1989d). A number of innova1989 to include virtually all of the shrimp farming regions of tive methods have been developed for reducing or elimi the Americas. Regions or countries included in its nating fecal (containing baculovirus) contamination of expansion since 1994 and with documented cases in spawned eggs. The simplest of these has been the use clude: both coasts of Mexico, Nicaragua, Belize, Costa of hatching vessels in which embryonating shrimp eggs Rica, Panama, and the U.S. states of Texas and South are rinsed with clean seawater, and in which hatched Carolina (Lightner, 1996a, 1996b;Hasson et al., 1997a). nauplii are passively rinsed by continuously flowing Laboratory challenge of the Asian penaeids, P. clean seawater and separated from contaminants and "diseased" siblings by collection using the normal monodon, P. japonicus and P. chinensis, with TSV demonstrated that juvenile P. monodon and P. japonicus phototaxic response of "healthy nauplii". Chemical are highly resistant to TSV infection and disease, while rinses of spawned eggs and collected nauplii with disin P. chinensis juveniles are only moderately susceptible fectants like chlorine, ozone, iodophores, and formalin (Overstreet et al., 1997; Brock et al., 1997). Assum are also commonly used in shrimp hatcheries in the ing that TSV is not already enzootic in Asian penaeids, Americas to help prevent BP, as well as vibriosis and the degree of resistance to TSV demonstrated by these other diseases. The use of routine sanitation and dis important Asian penaeid species suggests that the virus infection procedures for hatchery equipment and tanks may not pose a significant threat to Asian shrimp aquac after each use are also commonly used to prevent the ulture. 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 Disease Management Methods females, collect any fecal strands, and scan these using A variety of strategies have been attempted for the simple bright field microscopy for BP occlusion control of viral diseases in penaeid shrimp aquaculture. bodies. In like manner, some hatcheries sacrifice These strategies range from the use of improved culture broodstock females after spawning, excise the hepato practices (i.e. where sources of virus contamination are pancreas (HP), and examine it for the presence of BP reduced or eliminated, sanitation practices are improved, occlusion bodies using direct microscopy of tissue stocking densities are reduced, etc.) to stocking "specific squash preparations. To prevent BP infections and dis pathogen-free" (SPF) or "specific pathogen resistant" ease from occurring in the larval rearing tank systems, (SPR) species or stocks. Most recently, there have the spawns from females, found to be BP-positive from been some studies made on the use of vaccines and examination of their feces or excised HP, are discarded. immunostimulants for the prevention of viral diseases Avoidance through pathogen exclusion and develop in shrimp. In the Americas many strategies have been ment of SPF shrimp: Disease management through employed in efforts to reduce production losses due to exclusion of specific pathogens is commonplace in modern the enzootic viruses, IHHNV, BP, and TSV. agriculture. This concept of developing stocks that Prevention of BP in hatcheries: Improved hus are specific pathogen free (SPF) and rearing these stocks bandry practices have been successfully employed for in regions where the specific pathogens of concern are the control of BP, and for nearly a decade, this virus has excluded has been used in the Western Hemisphere with seldom been reported as a serious constraint to success mixed success. The successful application of the SPF ful shrimp culture. This was accomplished because concept is, of course, dependent upon the absence of the BP' s infection cycle can be interrupted with routine pathogen(s) of concern in the stocks being reared (or that hatchery management practices. BP is a gut-infecting are present), on the availability of sensitive and accurate baculovirus which is transmitted from shrimp to shrimp detection and diagnostic methods for the pathogen(s), exclusively per os (Johnson and Lightner, 1988; and the presence of an effective barrier (i.e. geographic, Overstreet et al., 1988; Overstreet, 1994). Hence, with government mandated import restrictions, etc.) to pre BP, as well as with MBV and BMN (all gut-infecting vent the introduction of the specific pathogen(s). baculoviruses of penaeid shrimp), infection from parent In the Western Hemisphere, SPF stocks of P. to off-spring in the hatchery has been prevented by elimi stylirostris and P. vannamei have been developed and nating fecal contamination of spawned eggs by virus these are being cultured successfully in some locations 174 D. V. Lightner and R. M. Redman

(Wyban, 1992; Wyban et al., 1992; Can et al., 1994; situated on the Pacific coast), and with its location on

Pruder et al., 1995; Lightner, 1996b). The ICES the Caribbean side of the continent, Belize has no natu

Guidelines (Sindermann, 1990), were followed for the rally occurring wild stocks of the penaeid species (P.

development of these stocks. The determination of vannamei and P. stylirostris) likely to serve as reservoir

which specific pathogens the selected stocks were to be hosts for TSV and IHHNV. Belize was uniquely suited

free from was based on a working lists of specific, ex to attempt to eradicate TSV and IHHNV. Seven of its

cludable pathogens (Wyban, 1992; Lotz et al., 1995). farms were depopulated of all their shrimp stocks in late

The most current working list includes eight viruses 1995; then each farm was thoroughly disinfected and

(WSSV, YHV, TSV, IHHNV, HPV, BP, MBV, and dried out to eradicate potential sources of these viruses.

BMN), certain classes of parasitic protozoa (micro The eradication program included pond disinfection

sporidians, haplospordians, and gregarines), and helm (liming pond bottoms with calcium oxide at 5,000 kg/ha inth parasites (cestodes, trematodes, and nematodes). or chlorine at 10 ppm residual for 24-48 h), pond dry-out

In the spirit of the ICES Guidelines, each "SPF candi and bottom tilling (to a depth of •`10 cm to ensure oxida

date population" of wild or cultured shrimp stocks of tion of contaminated pond-bottom detritus), farm imple

interest was identified. Samples of the stock were ment and building disinfection (with chlorine or with

taken and tested using appropriate diagnostic and patho formalin gas), spraying insecticides (to kill potential gen detection methods for the specific pathogens of reservoir hosts such as wild crabs and in supply

concern. If none were found, a founder population (F0) drainage canals), and removal of frozen shrimp from

of the "candidate SPF" stock was acquired and reared in storage from the country's packing plants (Dixon and

primary quarantine. During primary quarantine, the F0 Dorado,1997). For the 1996 and 1997 seasons, these

stock was monitored for signs of disease, sampled, and farms were stocked exclusively with SPF P. vannamei.

tested periodically for specific pathogens. If any patho TSV has not been detected in Belize since 1995, and gens of concern were detected, the stock was destroyed. IHHNV has been found only at low prevalence rates.

Those stocks that tested negative for pathogens of con In the absence of TSV, the per crop average production

cern through primary quarantine (which ran from 30 of P. vannamei in 1996 at one farm was 891 kg/ha (heads

days to as much as 1 year for some stocks) were moved on) with a stocking density of 15.5 PL/m2,and survival

to a separate secondary quarantine facility for matura to harvest has averaged 67%. In comparison, the same

tion, selection, mating, and production of a second (F1) farm's production during the TSV panzootic of 1994 was generation. The F1 stocks were maintained in quaran 390 kg/ha (stocked at 18.3 PLs/m2)with a survival of

tine for further testing for specific pathogens of concern. 36% (Dixon and Dorado, 1997). While the Belize Those that tested negative were designated as SPF and , experiment in TSV and IHHNV eradication may have

used to produce domesticated lines of SPF and "high been successful, successful duplication of its accom

health" (Wyban et al., 1992; Pruder et al., 1995). SPF plishments elsewhere in the Americas may not be fea and high health stocks of P. vannamei were used suc sible. In these regions where total stock eradication is

cessfully in U.S. shrimp farms in 1993 and 1994, and not feasible, other methods for viral disease management

resulted in nearly double the production that had been are being used.

obtained at the same farms in previous years when the Successful application of the ICES Guidelines and the

farms cultured non selected lines of P. vannamei, which, SPF concept requires that specific pathogens are exclud had been persistently affected by "runt deformity syn able. In situations where specific pathogens may not

drome" (RDS) due to chronic infection by IHHNV be excludable, the development and use of SPR stocks

(Pruder et al., 1995; Lightner, 1996a, 1996b). may be the only alternative. The fact that IHHNV and

Another interesting application of disease manage TSV have become widely distributed in the Americas ment through avoidance and the use of SPF stocks began indicates that either government or industry supported two years ago in Belize. The shrimp culture industry pathogen exclusion mechanisms and regulations must in this Central American nation is relatively small with be implemented and enforced to achieve the goals of only eight farms (Table 3). Belize was seriously im using SPF shrimp stocks, or, alternatively, that SPR pacted by the TSV panzootic in 1994 (Lightner, 1996b; stocks be developed and used.

Dixon and Dorado, 1997). The shrimp farms in Belize Farm management schemes used for virus control: are geographically isolated from other shrimp farming The management of IHHNV and TSV by the aquacul regions in adjacent countries (because most of these are ture industries of the Americas has ranged from total Control of shrimp viral diseases in the Americas 175 avoidance of the disease through the use of specific been reported and a secondary crop of marketable fish pathogen free (SPF) stocks and attempts to exclude these is obtained (Green, 1997). viruses, to modifying farm management methods to re The application of dietary immunostimulants as duce the impact of the disease, and to the use of IHHNV management tools for Taura syndrome has also been and/or TSV resistant species or stocks. In Ecuador and reported (Brock et al., 1997; Dixon and Dorado, 1997; much of Central America where Taura syndrome became Klesius and Shoemaker, 1997; D. Dugger, personal com widespread and well established before its viral etiology munication, Immunodyne, Inc, Brownsville, TX). was established in late 1994 (Hasson et al., 1995; Brock From slight improvements in survival (Klesius and et al., 1995), shrimp farmers changed farm management Shoemaker, 1997; Dixon and Dorado, 1997) to survival and operational procedures in an effort to reduce the rates of TSV-challenged P. vannamei comparable to impact of the disease (Stern, 1995; Brock et al., 1997). unchallenged control groups have been reported (Dugger, The use of wild caught PL (P. vannamei), versus hatch personal communication). ery reared PLs, gave improved crop survivals at harvest. Development and use of specific pathogen resistant Perhaps the wild PLs had either a lower prevalence of (SPR) stocks: One alternative approach to developing TSV infections when stocked than was typical of hatch SPF domesticated shrimp stocks, is to select and breed ery reared PLs, or, possibly, some increased resistance survivors of "specific pathogen-infected" (by pathogens to TS due to virus exposure and natural selection of TSV like IHHNV or TSV) stocks to develop "specific patho resistant shrimp in the wild. gen-resistant" or SPR stock. Following this scheme, Another management strategy employed to lessen the French researchers successfully developed a stock of impact of TS was the practice of stocking PLs at two or IHHNV resistant P. stylirostris in French Polynesia more times the normal stocking density for semi-inten (Weppe et al., 1992; Lightner, 1996b). This stock has sive pond culture. Farms employing this strategy would been used successfully to develop the shrimp culture experience a heavy mortality due to TSV infections early industries of Tahiti and New Caledonia. Recently, the in the culture cycle (during the late PL or early juvenile stock was introduced into an area of southwestern stages and before substantial supplemental feeding had Mexico where IHHNV is enzootic in an effort to develop begun). The survivors of the naturally occurring TSV the stock as an alternative to the slower growing P. epizootic were resistant to subsequent infection and dis vannamei which currently makes up >90% of the shrimp ease if challenged by TSV (Ligntner et al., 1997b; Lotz farmed in Mexico (Rosenberry, 1996). In view of the and Ogle, 1997). Hence, with this strategy, farms recent accidental introduction and spread of TSV in could obtain post-TSV survival rates of 10 to 40% of Mexico, the potential availability of this SPR stock, the original number stocked, and the resulting juveniles which is resistant to disease when infected by IHHNV could be managed and grown out to market size as a and TSV, may provide a viable alternative to the culture normal crop of P. vannamei (Stern, 1995). In a study of the highly TSV susceptible stocks of P. vannamei and in which different size classes of juvenile SPF P. the highly IHHNV susceptible native stocks of P. vannamei were challenged with TSV, Lotz (1997) found stylirostris, which were previously the only viable options that size alone does not affect mortality rates of P. for shrimp aquaculture development in Mexico. vannamei, and that larger shrimp size alone does not Perhaps the most promise for the control of Taura confer resistance to TSV. Related work suggests that syndrome and IHHN disease lies in the development and persistent infection by TSV in survivors of TS epizootics use of selected TSV resistant stocks of P. vannamei imparts resistance to subsequent challenge (Hasson and (Carr et al., 1997), or in the use of recently developed Lightner, unpublished data). strains of P. stylirostris which are resistant to both Polyculture of shrimp with an omnivorous fish species IHHNV and TSV (Weppe et al., 1992; Lightner, 1996b; also shows promise as a management method for reduc Brock et al., 1997). Until recently, the popularity ing the impact of TSV in some shrimp growing coun and use of the relatively IHHNV resistant species P. tries (Wang et al., 1997). With this strategy, tilapia are vannamei, in preference to the culture of the more grown in polyculture with shrimp like P. vannamei, and IHHNV susceptible P. stylirostris, was characteristic of presumably, the tilapia consume dead and dying shrimp the shrimp farming industries of the Americas. The (from TSV) keeping other shrimp in the pond from popularity of P. vannamei began to decline when TSV becoming infected with the virus by cannibalism. With emerged as a serious pathogen of this species in 1992 this method, improvements in shrimp production have and then spread to virtually all of the shrimp growing 176 D. V. Lightner and R. M. Redman regions of the Americas during the ensuing four years Selected stocks in laboratory challenge studies with TSV (Lightner, 1996b). Because P. stylirostris was found and in pond trials at farms where the virus is enzootic to be innately TSV resistant, two domesticated, geneti have shown significant survival advantages over non cally selected SPR strains of this species, which are selected stocks (Carr et al., 1997). Natural selection resistant to IHHN disease, are being developed currently for TSV resistance appears to be occurring in wild stocks and marketed in the Americas. Currently, there are two as well. In regions like Ecuador and Honduras where stocks of SPR P. stylirostris being tested or marketed in TSV has been enzootic for several years, the practice of the Americas. The first of the stocks, designated as direct stocking of farms with wild PLs is providing SPR-43, was developed by the French research agency, steadily improving survival rates, even though shrimp IFREMER, in French Polynesia and in New Caledonia. displaying classic signs of TSV infection are common This was accomplished by breeding generations of place. The same selective process for IHHNV resistance IHHN survivors in at the IFREMER stations in Tahiti seems to be occurring in some wild stocks of P. stylirostris and New Caledonia. After several generations, sur (Lightner, 1996b). Initial efforts to select and breed vival and culture performance of the stock improved. TSV resistant, domesticated strains of P. vannamei have The stock was found to carry IHHNV at low rates of resulted in improvements in harvest survivals of 20 to prevalence and severity of infection. When experi 40% (Lightner, 1996b). Similarily, the use of TSV and mentally challenged with IHHNV, the SPR-43 stock was IHHNV resistant strains of P. stylirostris have resulted found to be resistant to IHHN disease (Weppe et al., in improved harvest survival and crop values in regions 1992). The SPR-43 stock is being experimentally cul where TSV and IHHNV are enzootic. tured by one farm in Sinaloa, Mexico and it has been found to perform better than P. vannamei at the farm Acknowledgements because of its innate resistance to TSV. A second line of SPR P. stylirostris was developed in Funding for this research was provided by the Gulf Venezuela. Its development followed the same strategy Coast Research Laboratory Consortium Marine Shrimp as was used by the IFREMER in Tahiti and New Farming Program, CSREES, USDA under Grant No. 95 Caledonia. After an initial introduction of a founder 38808-1424, the National Sea Grant Program, USDC stock of P. stylirostris into a particular farm in Venezu under Grant No. NA56RG0617, the National Marine ela, the stock became IHHNV infected. Generations Fisheries Service (Saltonstall-Kennedy Act), USDC of IHHN survivors were selected and reared until that under Grant No. NA56FD0621, and a special grant from stock began to perform as well as did the normally the National Fishery Institute. IHHNV resistant stocks of P. vannamei at the same farm. When the TSV panzootic swept through the References Americas, the Venezuelan stock of P. stylirostris was Adams, J. R. and J. R. Bonami (eds. ) (1991): Atlas of inverte found to be TSV resistant. This stock possesses resis brate viruses. CRC Press, Boca Raton, FL. 684 p. tance to IHHNV and TSV, and it is being marketed as Bell T. A. and D. V. Lightner (1984): IHHN virus: Infectivity Super ShrimpTM in the Americas. 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